Microglia as Biosensors and Effectors of Neurodysfunction

DTIC Science & Technology

2010-04-01

induce or exacerbate the onset and progression of autism spectrum disorders. Dendritic spines receive the majority of excitatory synapses in the brain... autism spectrum disorders, synaptogenesis 15. NUMBER OF PAGES 24 synaptogenesis 16. PRICE CODE 17. SECURITY CLASSIFICATION OF REPORT...4/1/09-4/30/10 INTRODUCTION: Autism occurs during the post-natal period that neurons form new experience-dependent synaptic connections

Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells.

PubMed

Min, Kyoung-Jin; Choi, Kyounghwa; Kwon, Taeg Kyu

2011-08-01

Microglia are the major immune effector cells in the brain, and microglia activated by injury and infection can produce inflammatory mediators. A number of studies have reported that withaferin A has anti-inflammatory functions. However, the effects of withaferin A on the microglial inflammatory response have not been investigated. Our results show that withaferin A inhibited lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E2 (PGE(2)) production in BV2 murine microglial cells. Withaferin A had no effect on LPS-induced Akt and ERK phosphorylation, but phosphorylation of p38 and JNK was slightly decreased by withaferin A. Withaferin A significantly inhibited LPS-induced STAT1 and STAT3 phosphorylation in a dose-dependent manner. Furthermore, withaferin A inhibited nuclear translocation of STAT1 and interferon-gamma activated sequence (GAS)-promoter activity. Taken together, these results suggest that withaferin A inhibits LPS-induced PGE(2) production and COX-2 expression, at least in part, by blocking STAT1 and STAT3 activation. Copyright © 2011 Elsevier B.V. All rights reserved.

Microglia and Beyond: Innate Immune Cells As Regulators of Brain Development and Behavioral Function.

PubMed

Lenz, Kathryn M; Nelson, Lars H

2018-01-01

Innate immune cells play a well-documented role in the etiology and disease course of many brain-based conditions, including multiple sclerosis, Alzheimer's disease, traumatic brain and spinal cord injury, and brain cancers. In contrast, it is only recently becoming clear that innate immune cells, primarily brain resident macrophages called microglia, are also key regulators of brain development. This review summarizes the current state of knowledge regarding microglia in brain development, with particular emphasis on how microglia during development are distinct from microglia later in life. We also summarize the effects of early life perturbations on microglia function in the developing brain, the role that biological sex plays in microglia function, and the potential role that microglia may play in developmental brain disorders. Finally, given how new the field of developmental neuroimmunology is, we highlight what has yet to be learned about how innate immune cells shape the development of brain and behavior.

Innate immune responses in central nervous system inflammation.

PubMed

Finsen, Bente; Owens, Trevor

2011-12-01

In autoimmune diseases of the central nervous system (CNS), innate glial cell responses play a key role in determining the outcome of leukocyte infiltration. Access of leukocytes is controlled via complex interactions with glial components of the blood-brain barrier that include angiotensin II receptors on astrocytes and immunoregulatory mediators such as Type I interferons which regulate cellular traffic. Myeloid cells at the blood-brain barrier present antigen to T cells and influence cytokine effector function. Myelin-specific T cells interact with microglia and promote differentiation of oligodendrocyte precursor cells in response to axonal injury. These innate responses offer potential targets for immunomodulatory therapy. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

S100B Attenuates Microglia Activation in Gliomas: Possible Role of STAT3 Pathway

PubMed Central

Zhang, Leying; Liu, Wei; Alizadeh, Darya; Zhao, Dongchang; Farrukh, Omar; Lin, Jeffrey; Badie, Sam A.; Badie, Behnam

2010-01-01

Despite significant infiltration into tumors, the effector function of macrophages (MPs) and microglia (MG) appears to be suppressed in gliomas. Although STAT3 pathway is thought to play a role in this process, the exact mechanism by which gliomas induce STAT3 activation in MPs and MG is not known. Because activation of receptor for advanced glycation end products (RAGE) can induce STAT3, and because gliomas express high levels of S100B, a RAGE ligand, we hypothesized that MP/MG STAT3 activity may be modulated through S100B-RAGE interaction. Exposure of N9 MG and bone marrow-derived monocytes (BMM) to GL261 glioma condition medium (GCM) and low (nM) levels of S100B increased RAGE expression, induced STAT3 and suppressed MG function in vitro. Furthermore, neutralization of S100B in GCM, partially reversed IL-1β suppression in BMM, suggesting that the inhibitory effect of GCM to be in part due to S100B. Finally, blockage of S100B-RAGE interaction inhibited STAT3 activation in N9 MG and in glioma MG/MP in vivo. These findings suggest that the RAGE pathway may play an important role in STAT3 induction in glioma-associated MG/MPs, and that this process may be mediated through S100B. PMID:21264954

S100B attenuates microglia activation in gliomas: possible role of STAT3 pathway.

PubMed

Zhang, Leying; Liu, Wei; Alizadeh, Darya; Zhao, Dongchang; Farrukh, Omar; Lin, Jeffrey; Badie, Sam A; Badie, Behnam

2011-03-01

Despite significant infiltration into tumors, the effector function of macrophages (MPs) and microglia (MG) appears to be suppressed in gliomas. Although STAT3 pathway is thought to play a role in this process, the exact mechanism by which gliomas induce STAT3 activation in MPs and MG is not known. Because activation of receptor for advanced glycation end products (RAGE) can induce STAT3, and because gliomas express high levels of S100B, a RAGE ligand, we hypothesized that MP/MG STAT3 activity may be modulated through S100B-RAGE interaction. Exposure of N9 MG and bone marrow-derived monocytes (BMM) to GL261 glioma condition medium (GCM) and low (nM) levels of S100B increased RAGE expression, induced STAT3 and suppressed MG function in vitro. Furthermore, neutralization of S100B in GCM, partially reversed IL-1β suppression in BMM, suggesting that the inhibitory effect of GCM to be in part due to S100B. Finally, blockage of S100B-RAGE interaction inhibited STAT3 activation in N9 MG and in glioma MG/MP in vivo. These findings suggest that the RAGE pathway may play an important role in STAT3 induction in glioma-associated MG/MPs, and that this process may be mediated through S100B. Copyright © 2010 Wiley-Liss, Inc.

Roles of microglia in brain development, tissue maintenance and repair

PubMed Central

Michell-Robinson, Mackenzie A.; Touil, Hanane; Healy, Luke M.; Owen, David R.; Durafourt, Bryce A.; Bar-Or, Amit; Antel, Jack P.

2015-01-01

The emerging roles of microglia are currently being investigated in the healthy and diseased brain with a growing interest in their diverse functions. In recent years, it has been demonstrated that microglia are not only immunocentric, but also neurobiological and can impact neural development and the maintenance of neuronal cell function in both healthy and pathological contexts. In the disease context, there is widespread consensus that microglia are dynamic cells with a potential to contribute to both central nervous system damage and repair. Indeed, a number of studies have found that microenvironmental conditions can selectively modify unique microglia phenotypes and functions. One novel mechanism that has garnered interest involves the regulation of microglial function by microRNAs, which has therapeutic implications such as enhancing microglia-mediated suppression of brain injury and promoting repair following inflammatory injury. Furthermore, recently published articles have identified molecular signatures of myeloid cells, suggesting that microglia are a distinct cell population compared to other cells of myeloid lineage that access the central nervous system under pathological conditions. Thus, new opportunities exist to help distinguish microglia in the brain and permit the study of their unique functions in health and disease. PMID:25823474

Microglial Dynamics and Role in the Healthy and Diseased Brain

PubMed Central

Perry, V. Hugh

2015-01-01

The study of the dynamics and functions of microglia in the healthy and diseased brain is a matter of intense scientific activity. The application of new techniques and new experimental approaches has allowed the identification of novel microglial functions and the redefinition of classic ones. In this review, we propose the study of microglial functions, rather than their molecular profiles, to better understand and define the roles of these cells in the brain. We review current knowledge on the role of surveillant microglia, proliferating microglia, pruning/neuromodulatory microglia, phagocytic microglia, and inflammatory microglia and the molecular profiles that are associated with these functions. In the remodeling scenario of microglial biology, the analysis of microglial functional states will inform about the roles in health and disease and will guide us to a more precise understanding of the multifaceted roles of this never-resting cells. PMID:24722525

Immune heterogeneity in neuroinflammation: dendritic cells in the brain.

PubMed

Colton, Carol A

2013-03-01

Dendritic cells (DC) are critical to an integrated immune response and serve as the key link between the innate and adaptive arms of the immune system. Under steady state conditions, brain DC's act as sentinels, continually sampling their local environment. They share this function with macrophages derived from the same basic hemopoietic (bone marrow-derived) precursor and with parenchymal microglia that arise from a unique non-hemopoietic origin. While multiple cells may serve as antigen presenting cells (APCs), dendritic cells present both foreign and self-proteins to naïve T cells that, in turn, carry out effector functions that serve to protect or destroy. The resulting activation of the adaptive response is a critical step to resolution of injury or infection and is key to survival. In this review we will explore the critical roles that DCs play in the brain's response to neuroinflammatory disease with emphasis on how the brain's microenvironment impacts these actions.

Roles of microglia in brain development, tissue maintenance and repair.

PubMed

Michell-Robinson, Mackenzie A; Touil, Hanane; Healy, Luke M; Owen, David R; Durafourt, Bryce A; Bar-Or, Amit; Antel, Jack P; Moore, Craig S

2015-05-01

The emerging roles of microglia are currently being investigated in the healthy and diseased brain with a growing interest in their diverse functions. In recent years, it has been demonstrated that microglia are not only immunocentric, but also neurobiological and can impact neural development and the maintenance of neuronal cell function in both healthy and pathological contexts. In the disease context, there is widespread consensus that microglia are dynamic cells with a potential to contribute to both central nervous system damage and repair. Indeed, a number of studies have found that microenvironmental conditions can selectively modify unique microglia phenotypes and functions. One novel mechanism that has garnered interest involves the regulation of microglial function by microRNAs, which has therapeutic implications such as enhancing microglia-mediated suppression of brain injury and promoting repair following inflammatory injury. Furthermore, recently published articles have identified molecular signatures of myeloid cells, suggesting that microglia are a distinct cell population compared to other cells of myeloid lineage that access the central nervous system under pathological conditions. Thus, new opportunities exist to help distinguish microglia in the brain and permit the study of their unique functions in health and disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits.

PubMed

Krukowski, Karen; Feng, Xi; Paladini, Maria Serena; Chou, Austin; Sacramento, Kristen; Grue, Katherine; Riparip, Lara-Kirstie; Jones, Tamako; Campbell-Beachler, Mary; Nelson, Gregory; Rosi, Susanna

2018-05-18

Microglia are the main immune component in the brain that can regulate neuronal health and synapse function. Exposure to cosmic radiation can cause long-term cognitive impairments in rodent models thereby presenting potential obstacles for astronauts engaged in deep space travel. The mechanism/s for how cosmic radiation induces cognitive deficits are currently unknown. We find that temporary microglia depletion, one week after cosmic radiation, prevents the development of long-term memory deficits. Gene array profiling reveals that acute microglia depletion alters the late neuroinflammatory response to cosmic radiation. The repopulated microglia present a modified functional phenotype with reduced expression of scavenger receptors, lysosome membrane protein and complement receptor, all shown to be involved in microglia-synapses interaction. The lower phagocytic activity observed in the repopulated microglia is paralleled by improved synaptic protein expression. Our data provide mechanistic evidence for the role of microglia in the development of cognitive deficits after cosmic radiation exposure.

RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells.

PubMed

He, Yingbo; Yao, Xiang; Taylor, Natalie; Bai, Yuchen; Lovenberg, Timothy; Bhattacharya, Anindya

2018-05-22

Microglia play key roles in neuron-glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system. With postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGFβ signaling probed by Western blot, and direct migration by chemotaxis assay. We found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGFβ signaling and chemotaxis upon chemoattractant C5a. Collectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.

P2Y12 expression and function in alternatively activated human microglia

PubMed Central

Ase, Ariel R.; Kinsara, Angham; Rao, Vijayaraghava T.S.; Michell-Robinson, Mackenzie; Leong, Soo Yuen; Butovsky, Oleg; Ludwin, Samuel K.; Séguéla, Philippe; Bar-Or, Amit; Antel, Jack P.

2015-01-01

Objective: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation. Methods: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain. Results: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection. Conclusion: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS. PMID:25821842

Effect of NDP-α-MSH on PPAR-γ and –β Expression and Anti-Inflammatory Cytokine Release in Rat Astrocytes and Microglia

PubMed Central

Carniglia, Lila; Durand, Daniela; Caruso, Carla; Lasaga, Mercedes

2013-01-01

Brain inflammation plays a central role in numerous brain pathologies. Microglia and astrocytes are the main effector cells that become activated when an inflammatory process takes place within the central nervous system. α-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with proven anti-inflammatory properties. It binds with highest affinity to the melanocortin receptor 4 (MC4R), which is present in astrocytes and upon activation triggers anti-inflammatory pathways. The aim of this research was to identify anti-inflammatory mediators that may participate in the immunomodulatory effects of melanocortins in glial cells. Since peroxisome proliferator-activated receptors (PPARs) have recently been implicated in the modulation of inflammation, we investigated the effect of an α-MSH analog, [Nle4, D-Phe7]-α-MSH (NDP-α-MSH), on PPAR-β and PPAR-γ gene and protein expression in rat primary astrocytes and microglia. We initially demonstrated that rat primary microglia express MC4R and showed that treatment with NDP-α-MSH increases PPAR-γ protein levels and strongly decreases PPAR-β levels in both astrocytes and microglia. We also showed that extracellular signal-regulated kinase 1/2 (ERK1/2)–mediated signaling is partially involved in these effects in a cell-specific fashion. Finally, we showed that NDP-α-MSH stimulates the release of the anti-inflammatory cytokines IL-10 and TGF-β from microglia and astrocytes, respectively. The presented data suggest a role for IL-10 and TGF-β in the protective action of melanocortins and a connection between MC4R pathway and that of the nuclear receptor PPAR-γ. This is the first report providing evidence that MC4R is expressed in rat primary microglia and that melanocortins modulate PPAR levels in glial cells. Our findings provide new insights into the mechanisms underlying the activation of glial MC4R and open perspectives for new therapeutic strategies for the treatment of inflammation-mediated brain diseases. PMID:23468969

Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

PubMed

Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

2012-08-15

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures. It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning.

PubMed

Johnson, S A; Lampert-Etchells, M; Pasinetti, G M; Rozovsky, I; Finch, C E

1992-01-01

This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C1qB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and C1qB mRNA in microglia. Primary rat neuron cultures also show C1qB mRNA. In the cortex from AD brains, there were two- to threefold increases of C1qB mRNA and C4 mRNA, and increased C1qB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases of C1qB mRNA in the ipsilateral, but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity.

Regulation of Microglia Identity from an Epigenetic and Transcriptomic Point of View.

PubMed

Eggen, Bart J L; Boddeke, Erik W G M; Kooistra, Susanne M

2017-12-14

Microglia have long been recognized as the endogenous innate immune elements in the central nervous system (CNS) parenchyma. Besides fulfilling local immune-related functions, they provide cross-talk between the CNS and the immune system at large. In the adult CNS, microglia are involved in maintaining brain homeostasis, modulating synaptic transmission and clearance of apoptotic cells. During embryonic development, microglia are responsible for the removal of supernumerary synapses and neurons, and neuronal network formation. The full scale of their potential abilities has been highlighted by improvements in microglia isolation methods, the development of genetically tagged mouse models, advanced imaging technologies and the application of next-generation sequencing in recent years. Genome-wide expression analysis of relatively pure microglia populations from both mouse and human CNS tissues has thereby greatly contributed to our knowledge of their biology; what defines them under homeostatic conditions and how microglia respond to processes like aging and CNS disease? How and to what degree beneficial functions of microglia can be restored in the aged or diseased brain will be the key issue to be addressed in future research. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion

PubMed Central

2013-01-01

Background Microglia cells continuously survey the healthy brain in a ramified morphology and, in response to injury, undergo progressive morphological and functional changes that encompass microglia activation. Although ideally positioned for immediate response to ischemic stroke (IS) and reperfusion, their progressive morphological transformation into activated cells has not been quantified. In addition, it is not well understood if diverse microglia morphologies correlate to diverse microglia functions. As such, the dichotomous nature of these cells continues to confound our understanding of microglia-mediated injury after IS and reperfusion. The purpose of this study was to quantitatively characterize the spatiotemporal pattern of microglia morphology during the evolution of cerebral injury after IS and reperfusion. Methods Male C57Bl/6 mice were subjected to focal cerebral ischemia and periods of reperfusion (0, 8 and 24 h). The microglia process length/cell and number of endpoints/cell was quantified from immunofluorescent confocal images of brain regions using a skeleton analysis method developed for this study. Live cell morphology and process activity were measured from movies acquired in acute brain slices from GFP-CX3CR1 transgenic mice after IS and 24-h reperfusion. Regional CD11b and iNOS expressions were measured from confocal images and Western blot, respectively, to assess microglia proinflammatory function. Results Quantitative analysis reveals a significant spatiotemporal relationship between microglia morphology and evolving cerebral injury in the ipsilateral hemisphere after IS and reperfusion. Microglia were both hyper- and de-ramified in striatal and cortical brain regions (respectively) after 60 min of focal cerebral ischemia. However, a de-ramified morphology was prominent when ischemia was coupled to reperfusion. Live microglia were de-ramified, and, in addition, process activity was severely blunted proximal to the necrotic core after IS and 24 h of reperfusion. CD11b expression, but not iNOS expression, was increased in regions of hyper- and de-ramified microglia during the course of ischemic stroke and 24 h of reperfusion. Conclusions Our findings illustrate that microglia activation after stroke includes both increased and decreased cell ramification. Importantly, quantitative analyses of microglial morphology and activity are feasible and, in future studies, would assist in the comprehensive identification and stratification of their dichotomous contribution toward cerebral injury and recovery during IS and reperfusion. PMID:23311642

The Indispensable Roles of Microglia and Astrocytes during Brain Development

PubMed Central

Reemst, Kitty; Noctor, Stephen C.; Lucassen, Paul J.; Hol, Elly M.

2016-01-01

Glia are essential for brain functioning during development and in the adult brain. Here, we discuss the various roles of both microglia and astrocytes, and their interactions during brain development. Although both cells are fundamentally different in origin and function, they often affect the same developmental processes such as neuro-/gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis and synaptic pruning. Due to their important instructive roles in these processes, dysfunction of microglia or astrocytes during brain development could contribute to neurodevelopmental disorders and potentially even late-onset neuropathology. A better understanding of the origin, differentiation process and developmental functions of microglia and astrocytes will help to fully appreciate their role both in the developing as well as in the adult brain, in health and disease. PMID:27877121

NDRG2 promoted secreted miR-375 in microvesicles shed from M1 microglia, which induced neuron damage.

PubMed

Tang, Li-li; Wu, Yuan-bo; Fang, Chuan-qin; Qu, Ping; Gao, Zong-liang

2016-01-15

Microglia microvesicles (MVs) has shown to have significant biological functions under normal conditions. A diversity of miRNAs is involved in neuronal development, survival, function, and plasticity, but the exact functional role of NDRG2 and secreted miR-375 in MVs in neuron damage is poorly understood. We investigated the effect of NDRG2 and secreted miR-375 in MVs shed from M1 microglia on neuron damage. Expression of Nos2, Arg-1, miR-375, syntaxin-1A, NDRG2 and Pdk 1 were evaluated using RT-PCR or western blotting. Cell viability of N2A neuron was quantified by a MTT assay. Microglia can be polarized into different functional phenotypes. Expression of NDRG2 and Nos2 were significantly increased by LPS treatment on N9 cells, whereas treatment with IL-4 dramatically suppressed the expression of NDRG2 and remarkably elevated expression of Arg-1. Besides, MVs shed from LPS-treated N9 microglia significantly inhibited cell viability of N2A neurons and expression of syntaxin-1A, and NDRG2 interference reversed the up-regulated miR-375 in LPS-treated N9 microglia and MVs shed from LPS-treated N9 cells. Furthermore, NDRG2 could modulate miR-375 expression in N9 microglia and MVs. And miR-375 inhibitor remarkably elevated Pdk1 expression in N2A neurons. Finally, miR-375 inhibitor could reverse suppression effect of NDRG2 overexpression on cell viability of N2A neurons and expression of syntaxin-1A. Our results demonstrated that NDRG2 promoted secreted miR-375 in microvesicles shed from M1 microglia, which induced neuron damage. The suppression of NDRG2 and secreted miR-375 in MVs shed from M1 microglia may be potential targets for alleviation of neuron damage. Copyright © 2015 Elsevier Inc. All rights reserved.

Aging Microglia—Phenotypes, Functions and Implications for Age-Related Neurodegenerative Diseases

PubMed Central

Spittau, Björn

2017-01-01

Aging of the central nervous system (CNS) is one of the major risk factors for the development of neurodegenerative pathologies such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). The molecular mechanisms underlying the onset of AD and especially PD are not well understood. However, neuroinflammatory responses mediated by microglia as the resident immune cells of the CNS have been reported for both diseases. The unique nature and developmental origin of microglia causing microglial self-renewal and telomere shortening led to the hypothesis that these CNS-specific innate immune cells become senescent. Age-dependent and senescence-driven impairments of microglia functions and responses have been suggested to play essential roles during onset and progression of neurodegenerative diseases. This review article summarizes the current knowledge of microglia phenotypes and functions in the aging CNS and further discusses the implications of these age-dependent microglia changes for the development and progression of AD and PD as the most common neurodegenerative diseases. PMID:28659790

Microglia: new roles for the synaptic stripper.

PubMed

Kettenmann, Helmut; Kirchhoff, Frank; Verkhratsky, Alexei

2013-01-09

Any pathologic event in the brain leads to the activation of microglia, the immunocompetent cells of the central nervous system. In recent decades diverse molecular pathways have been identified by which microglial activation is controlled and by which the activated microglia affects neurons. In the normal brain microglia were considered "resting," but it has recently become evident that they constantly scan the brain environment and contact synapses. Activated microglia can remove damaged cells as well as dysfunctional synapses, a process termed "synaptic stripping." Here we summarize evidence that molecular pathways characterized in pathology are also utilized by microglia in the normal and developing brain to influence synaptic development and connectivity, and therefore should become targets of future research. Microglial dysfunction results in behavioral deficits, indicating that microglia are essential for proper brain function. This defines a new role for microglia beyond being a mere pathologic sensor. Copyright © 2013 Elsevier Inc. All rights reserved.

The molecular profile of microglia under the influence of glioma

PubMed Central

Li, Wei; Graeber, Manuel B.

2012-01-01

Microglia, which contribute substantially to the tumor mass of glioblastoma, have been shown to play an important role in glioma growth and invasion. While a large number of experimental studies on functional attributes of microglia in glioma provide evidence for their tumor-supporting roles, there also exist hints in support of their anti-tumor properties. Microglial activities during glioma progression seem multifaceted. They have been attributed to the receptors expressed on the microglia surface, to glioma-derived molecules that have an effect on microglia, and to the molecules released by microglia in response to their environment under glioma control, which can have autocrine effects. In this paper, the microglia and glioma literature is reviewed. We provide a synopsis of the molecular profile of microglia under the influence of glioma in order to help establish a rational basis for their potential therapeutic use. The ability of microglia precursors to cross the blood–brain barrier makes them an attractive target for the development of novel cell-based treatments of malignant glioma. PMID:22573310

Microglia: biology and pathology.

PubMed

Graeber, Manuel B; Streit, Wolfgang J

2010-01-01

The past 20 years have seen a gain in knowledge on microglia biology and microglia functions in disease that exceeds the expectations formulated when the microglia "immune network" was introduced. More than 10,000 articles have been published during this time. Important new research avenues of clinical importance have opened up such as the role of microglia in pain and in brain tumors. New controversies have also emerged such as the question of whether microglia are active or reactive players in neurodegenerative disease conditions, or whether they may be victims themselves. Premature commercial interests may be responsible for some of the confusion that currently surrounds microglia in both the Alzheimer and Parkinson's disease research fields. A critical review of the literature shows that the concept of "(micro)glial inflammation" is still open to interpretation, despite a prevailing slant towards a negative meaning. Perhaps the most exciting foreseeable development concerns research on the role of microglia in synaptic plasticity, which is expected to yield an answer to the question whether microglia are the brain's electricians. This review provides an analysis of the latest developments in the microglia field.

MafB antagonizes phenotypic alteration induced by GM-CSF in microglia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koshida, Ryusuke, E-mail: rkoshida-myz@umin.ac.jp; Oishi, Hisashi, E-mail: hoishi@md.tsukuba.ac.jp; Hamada, Michito

2015-07-17

Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB bymore » analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia. - Highlights: • GM-CSF alters the phenotype of microglia in vitro more potently than M-CSF. • Transcription factor MafB antagonizes the effect of GM-CSF on microglia in vitro. • MafB deficiency leads to RhoA activation in microglia in response to GM-CSF. • We show for the first time the function of MafB in microglia.« less

Microglia: An Interface between the Loss of Neuroplasticity and Depression

PubMed Central

Singhal, Gaurav; Baune, Bernhard T.

2017-01-01

Depression has been widely accepted as a major psychiatric disease affecting nearly 350 million people worldwide. Research focus is now shifting from studying the extrinsic and social factors of depression to the underlying molecular causes. Microglial activity is shown to be associated with pathological conditions, such as psychological stress, pathological aging, and chronic infections. These are primary immune effector cells in the CNS and regulate the extensive dialogue between the nervous and the immune systems in response to different immunological, physiological, and psychological stressors. Studies have suggested that during stress and pathologies, microglia play a significant role in the disruption of neuroplasticity and have detrimental effects on neuroprotection causing neuroinflammation and exacerbation of depression. After a systematic search of literature databases, relevant articles on the microglial regulation of bidirectional neuroimmune pathways affecting neuroplasticity and leading to depression were reviewed. Although, several hypotheses have been proposed for the microglial role in the onset of depression, it is clear that all molecular pathways to depression are linked through microglia-associated neuroinflammation and hippocampal degeneration. Molecular factors such as an excess of glucocorticoids and changes in gene expression of neurotrophic factors, as well as neuro active substances secreted by gut microbiota have also been shown to affect microglial morphology and phenotype resulting in depression. This review aims to critically analyze the various molecular pathways associated with the microglial role in depression. PMID:28943841

Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dannhausen, Katharina; Karlstetter, Marcus; Caramoy, Albert

Mutations in the acid sphingomyelinase (aSMase) coding gene sphingomyelin phosphodiesterase 1 (SMPD1) cause Niemann-Pick disease (NPD) type A and B. Sphingomyelin storage in cells of the mononuclear phagocyte system cause hepatosplenomegaly and severe neurodegeneration in the brain of NPD patients. However, the effects of aSMase deficiency on retinal structure and microglial behavior have not been addressed in detail yet. Here, we demonstrate that retinas of aSMase{sup −/−} mice did not display overt neuronal degeneration but showed significantly reduced scotopic and photopic responses in electroretinography. In vivo fundus imaging of aSMase{sup −/−} mice showed many hyperreflective spots and staining for the retinalmore » microglia marker Iba1 revealed massive proliferation of retinal microglia that had significantly enlarged somata. Nile red staining detected prominent phospholipid inclusions in microglia and lipid analysis showed significantly increased sphingomyelin levels in retinas of aSMase{sup −/−} mice. In conclusion, the aSMase-deficient mouse is the first example in which microglial lipid inclusions are directly related to a loss of retinal function. - Highlights: • aSMase-deficient mice show impaired retinal function and reactive microgliosis. • aSMase-deficient microglia express pro-inflammatory transcripts. • aSMase-deficient microglia proliferate and have increased cell body size. • In vivo imaging shows hyperreflective spots in the fundus of aSMase-deficient mice. • aSMase-deficient microglia accumulate sphingolipid-rich intracellular deposits.« less

Microglia immunophenotyping in gliomas

PubMed Central

Annovazzi, Laura; Mellai, Marta; Bovio, Enrica; Mazzetti, Samanta; Pollo, Bianca; Schiffer, Davide

2018-01-01

Microglia, once assimilated to peripheral macrophages, in gliomas has long been discussed and currently it is hypothesized to play a pro-tumor role in tumor progression. Uncertain between M1 and M2 polarization, it exchanges signals with glioma cells to create an immunosuppressive microenvironment and stimulates cell proliferation and migration. Four antibodies are currently used for microglia/macrophage identification in tissues that exhibit different cell forms and cell localization. The aim of the present work was to describe the distribution of the different cell forms and to deduce their significance on the basis of what is known on their function from the literature. Normal resting microglia, reactive microglia, intermediate and bumpy forms and macrophage-like cells can be distinguished by Iba1, CD68, CD16 and CD163 and further categorized by CD11b, CD45, c-MAF and CD98. The number of microglia/macrophages strongly increased from normal cortex and white matter to infiltrating and solid tumors. The ramified microglia accumulated in infiltration areas of both high- and low-grade gliomas, when hypertrophy and hyperplasia occur. In solid tumors, intermediate and bumpy forms prevailed and there is a large increase of macrophage-like cells in glioblastoma. The total number of microglia cells did not vary among the three grades of malignancy, but macrophage-like cells definitely prevailed in high-grade gliomas and frequently expressed CD45 and c-MAF. CD98+ cells were present. Microglia favors tumor progression, but many aspects suggest that the phagocytosing function is maintained. CD98+ cells can be the product of fusion, but also of phagocytosis. Microglia correlated with poorer survival in glioblastoma, when considering CD163+ cells, whereas it did not change prognosis in isocitrate dehydrogenase-mutant low grade gliomas. PMID:29399160

Resveratrol regulates microglia M1/M2 polarization via PGC-1α in conditions of neuroinflammatory injury.

PubMed

Yang, Xiaodong; Xu, Shaoqing; Qian, Yiwei; Xiao, Qin

2017-08-01

Microglia are the primary cells that exert immune function in the central nervous system (CNS), and accumulating evidence suggests that microglia act as key players in the initiation of neurodegenerative diseases. It is now well recognized that microglia have functional plasticity and dual phenotypes, proinflammatory M1 and anti-inflammatory M2 phenotypes. Inhibiting the M1 phenotype while stimulating the M2 phenotype has been suggested as a potential therapeutic approach for the treatment of neuroinflammation-related diseases. Resveratrol has been demonstrated to exert anti-inflammatory effects by suppressing M1 microglia activation. However, the role of resveratrol in regulating microglia polarization and the molecular mechanisms involved have not been fully clarified. In this study, we tested whether resveratrol could suppress microglia activation by promoting microglia polarization toward the M2 phenotype via PGC-1α by measuring M1 and M2 markers in vitro and in vivo. Our study demonstrated that resveratrol reduced inflammatory damage and promoted microglia polarization to the M2 phenotype in LPS-induced neuroinflammation. In addition, resveratrol ameliorated LPS-induced sickness behavior in mice. The promoting effects of resveratrol on M2 polarization were attenuated by knocking down PGC-1α. PGC-1α not only suppressed LPS-evoked M1 marker expression by inhibition of NF-κB activity but also increased M2 marker expression by coactivation of the STAT6 and STAT3 pathways. We propose that overexpression PGC-1α by resveratrol could be a potential therapeutic approach to suppress neuroinflammation by regulating microglia polarization. Copyright © 2017 Elsevier Inc. All rights reserved.

A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System.

PubMed

Qin, Yan; Garrison, Brian S; Ma, Wenjiang; Wang, Rui; Jiang, Aiping; Li, Jing; Mistry, Meeta; Bronson, Roderick T; Santoro, Daria; Franco, Charlotte; Robinton, Daisy A; Stevens, Beth; Rossi, Derrick J; Lu, Chafen; Springer, Timothy A

2018-06-12

Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 -/- mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 -/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 -/- microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β. Copyright © 2018 Elsevier Inc. All rights reserved.

Caveolin-1-mediated internalization of the vitamin C transporter SVCT2 in microglia triggers an inflammatory phenotype.

PubMed

Portugal, Camila C; Socodato, Renato; Canedo, Teresa; Silva, Cátia M; Martins, Tânia; Coreixas, Vivian S M; Loiola, Erick C; Gess, Burkhard; Röhr, Dominik; Santiago, Ana R; Young, Peter; Minshall, Richard D; Paes-de-Carvalho, Roberto; Ambrósio, António F; Relvas, João B

2017-03-28

Vitamin C is essential for the development and function of the central nervous system (CNS). The plasma membrane sodium-vitamin C cotransporter 2 (SVCT2) is the primary mediator of vitamin C uptake in neurons. SVCT2 specifically transports ascorbate, the reduced form of vitamin C, which acts as a reducing agent. We demonstrated that ascorbate uptake through SVCT2 was critical for the homeostasis of microglia, the resident myeloid cells of the CNS that are essential for proper functioning of the nervous tissue. We found that depletion of SVCT2 from the plasma membrane triggered a proinflammatory phenotype in microglia and resulted in microglia activation. Src-mediated phosphorylation of caveolin-1 on Tyr 14 in microglia induced the internalization of SVCT2. Ascorbate treatment, SVCT2 overexpression, or blocking SVCT2 internalization prevented the activation of microglia. Overall, our work demonstrates the importance of the ascorbate transport system for microglial homeostasis and hints that dysregulation of ascorbate transport might play a role in neurological disorders. Copyright © 2017, American Association for the Advancement of Science.

M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia

PubMed Central

2013-01-01

Background Microglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation. Methods Using biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function. Results We found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPβ, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of Aβ1-42 peptide. Conclusions We show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit. PMID:23866312

An environment-dependent transcriptional network specifies human microglia identity.

PubMed

Gosselin, David; Skola, Dylan; Coufal, Nicole G; Holtman, Inge R; Schlachetzki, Johannes C M; Sajti, Eniko; Jaeger, Baptiste N; O'Connor, Carolyn; Fitzpatrick, Conor; Pasillas, Martina P; Pena, Monique; Adair, Amy; Gonda, David D; Levy, Michael L; Ransohoff, Richard M; Gage, Fred H; Glass, Christopher K

2017-06-23

Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Lifestyle Shapes the Dialogue between Environment, Microglia, and Adult Neurogenesis.

PubMed

Valero, Jorge; Paris, Iñaki; Sierra, Amanda

2016-04-20

Lifestyle modulates brain function. Diet, stress levels, and physical exercise among other factors influence the "brain cognitive reserve", that is, the capacity of the brain to maintain a normal function when confronting neurodegenerative diseases, injury, and/or aging. This cognitive reserve relays on several cellular and molecular elements that contribute to brain plasticity allowing adaptive responses to cognitive demands, and one of its key components is the hippocampal neurogenic reserve. Hippocampal neural stem cells give rise to new neurons that integrate into the local circuitry and contribute to hippocampal functions such as memory and learning. Importantly, adult hippocampal neurogenesis is well-known to be modulated by the demands of the environment and lifestyle factors. Diet, stress, and physical exercise directly act on neural stem cells and/or their progeny, but, in addition, they may also indirectly affect neurogenesis by acting on microglia. Microglia, the guardians of the brain, rapidly sense changes in the brain milieu, and it has been recently shown that their function is affected by lifestyle factors. However, few studies have analyzed the modulatory effect of microglia on adult neurogenesis in these conditions. Here, we review the current knowledge about the dialogue maintained between microglia and the hippocampal neurogenic cascade. Understanding how the communication between microglia and hippocampal neurogenesis is affected by lifestyle choices is crucial to maintain the brain cognitive reserve and prevent the maladaptive responses that emerge during disease or injury through adulthood and aging.

Regulation of Microglia by Ionotropic Glutamatergic and GABAergic Neurotransmission

PubMed Central

Wong, Wai T.; Wang, Minhua; Li, Wei

2015-01-01

Recent studies have indicated that constitutive functions of microglia in the healthy adult CNS involve immune surveillance, synapse maintenance, and trophic support. These functions have been related to the ramified structure of “resting” microglia and the prominent motility in their processes that provide extensive coverage of the entire extracellular milleu. In this review, we examine how external signals, and in particular, ionotropic neurotransmission, regulate features of microglial morphology and process motility. Taken together, current findings indicate that microglial physiology in the healthy CNS is constitutively and reciprocally regulated by endogenous ionotropic glutamatergic and GABAergic neurotransmission. These influences do not act directly on microglial cells but indirectly via the activity-dependent release of ATP, likely through a mechanism involving pannexin channels. Microglia in the “resting” state are not only dynamically active, but are constantly engaged in ongoing communication with neuronal and macroglial components of the CNS in a functionally relevant way. PMID:22166726

Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures

PubMed Central

Bohlen, Christopher J.; Bennett, F. Chris; Tucker, Andrew F.; Collins, Hannah Y.; Mulinyawe, Sara B.; Barres, Ben A.

2017-01-01

Summary Microglia, the resident macrophages of the central nervous system (CNS), engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS. PMID:28521131

Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.

PubMed

Bohlen, Christopher J; Bennett, F Chris; Tucker, Andrew F; Collins, Hannah Y; Mulinyawe, Sara B; Barres, Ben A

2017-05-17

Microglia, the resident macrophages of the CNS, engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3.

PubMed

de Jong, Eiko K; de Haas, Alexander H; Brouwer, Nieske; van Weering, Hilmar R J; Hensens, Marjolein; Bechmann, Ingo; Pratley, Pierre; Wesseling, Evelyn; Boddeke, Hendrikus W G M; Biber, Knut

2008-06-01

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.

Activated microglia proliferate at neurites of mutant huntingtin-expressing neurons

PubMed Central

Kraft, Andrew D.; Kaltenbach, Linda S.; Lo, Donald C.; Harry, G. Jean

2011-01-01

In Huntington's disease (HD), mutated huntingtin (mhtt) causes striatal neurodegeneration which is paralleled by elevated microglia cell numbers. In vitro cortico-striatal slice and primary neuronal culture models, in which neuronal expression of mhtt fragments drives HD-like neurotoxicity, were employed to examine wild type microglia during both the initiation and progression of neuronal pathology. As neuronal pathology progressed, microglia initially localized in the vicinity of neurons expressing mhtt fragments increased in number, demonstrated morphological evidence of activation, and expressed the proliferation marker, Ki67. These microglia were positioned along irregular neurites, but did not localize with mhtt inclusions nor exacerbate mhtt fragment-induced neurotoxicity. Prior to neuronal pathology, microglia upregulated Iba1, signaling a functional shift. With neurodegeneration, interleukin-6 and complement component 1q were increased. The results suggest a stimulatory, proliferative signal for microglia present at the onset of mhtt fragment-induced neurodegeneration. Thus, microglia effect a localized inflammatory response to neuronal mhtt expression that may serve to direct microglial removal of dysfunctional neurites or aberrant synapses, as is required for reparative actions in vivo. PMID:21482444

The impact of microglial activation on blood-brain barrier in brain diseases

PubMed Central

da Fonseca, Anna Carolina Carvalho; Matias, Diana; Garcia, Celina; Amaral, Rackele; Geraldo, Luiz Henrique; Freitas, Catarina; Lima, Flavia Regina Souza

2014-01-01

The blood-brain barrier (BBB), constituted by an extensive network of endothelial cells (ECs) together with neurons and glial cells, including microglia, forms the neurovascular unit (NVU). The crosstalk between these cells guarantees a proper environment for brain function. In this context, changes in the endothelium-microglia interactions are associated with a variety of inflammation-related diseases in brain, where BBB permeability is compromised. Increasing evidences indicate that activated microglia modulate expression of tight junctions, which are essential for BBB integrity and function. On the other hand, the endothelium can regulate the state of microglial activation. Here, we review recent advances that provide insights into interactions between the microglia and the vascular system in brain diseases such as infectious/inflammatory diseases, epilepsy, ischemic stroke and neurodegenerative disorders. PMID:25404894

Microglia: An Active Player in the Regulation of Synaptic Activity

PubMed Central

Ji, Kyungmin; Miyauchi, Jeremy; Tsirka, Stella E.

2013-01-01

Synaptic plasticity is critical for elaboration and adaptation in the developing and developed brain. It is well established that astrocytes play an important role in the maintenance of what has been dubbed “the tripartite synapse”. Increasing evidence shows that a fourth cell type, microglia, is critical to this maintenance as well. Microglia are the resident macrophages of the central nervous system (CNS). Because of their well-characterized inflammatory functions, research has primarily focused on their innate immune properties. The role of microglia in the maintenance of synapses in development and in homeostasis is not as well defined. A number of significant findings have shed light on the critical role of microglia at the synapse. It is becoming increasingly clear that microglia play a seminal role in proper synaptic development and elimination. PMID:24303218

Interleukin-4 Is Essential for Microglia/Macrophage M2 Polarization and Long-Term Recovery After Cerebral Ischemia.

PubMed

Liu, Xiangrong; Liu, Jia; Zhao, Shangfeng; Zhang, Haiyue; Cai, Wei; Cai, Mengfei; Ji, Xunming; Leak, Rehana K; Gao, Yanqin; Chen, Jun; Hu, Xiaoming

2016-02-01

Interleukin-4 (IL-4) is a unique cytokine that may contribute to brain repair by regulating microglia/macrophage functions. Thus, we examined the effect of IL-4 on long-term recovery and microglia/macrophage polarization in 2 well-established stroke models. Transient middle cerebral artery occlusion or permanent distal middle cerebral artery occlusion was induced in wild-type and IL-4 knockout C57/BL6 mice. In a separate cohort of wild-type animals, IL-4 (60 ng/d for 7 days) or vehicle was infused into the cerebroventricle after transient middle cerebral artery occlusion. Behavioral outcomes were assessed by the Rotarod, corner, foot fault, and Morris water maze tests. Neuronal tissue loss was verified by 2 independent neuron markers. Markers of classically activated (M1) and alternatively activated (M2) microglia were assessed by real-time polymerase chain reaction, immunofluorescence, and flow cytometry. Loss of IL-4 exacerbated sensorimotor deficits and impaired cognitive functions ≤21 days post injury. In contrast to the delayed deterioration of neurological functions, IL-4 deficiency increased neuronal tissue loss only in the acute phase (5 days) after stroke and had no impact on neuronal tissue loss 14 or 21 days post injury. Loss of IL-4 promoted expression of M1 microglia/macrophage markers and impaired expression of M2 markers at 5 and 14 days post injury. Administration of IL-4 into the ischemic brain also enhanced long-term functional recovery. The cytokine IL-4 improves long-term neurological outcomes after stroke, perhaps through M2 phenotype induction in microglia/macrophages. These results are the first to suggest that immunomodulation with IL-4 is a promising approach to promote long-term functional recovery after stroke. © 2016 American Heart Association, Inc.

Microglia During Development and Aging

PubMed Central

Harry, G. Jean

2013-01-01

Microglia are critical nervous system-specific cells influencing brain development, maintenance of the neural environment, response to injury, and repair. They contribute to neuronal proliferation and differentiation, pruning of dying neurons, synaptic remodeling and clearance of debris and aberrant proteins. Colonization of the brain occurs during gestation with an expansion following birth with localization stimulated by programmed neuronal death, synaptic pruning, andaxonal degeneration. Changes inmicroglia phenotype relate to cellular processes including specific neurotransmitter, pattern recognition, or immune-related receptor activation. Upon activation, microglia cells have the capacity to release a number of substances, e.g., cytokines, chemokines, nitric oxide, and reactive oxygen species, which could be detrimental or beneficial to the surrounding cells. With aging, microglia shift their morphology and may display diminished capacity for normal functions related to migration, clearance, and the ability to shift from a pro-inflammatory to an anti-inflammatory state to regulate injury and repair. This shift in microgliapotentially contributes to increased susceptibility and neurodegeneration as a function of age. In the current review, information is provided on the colonization of the brain by microglia, the expression of various pattern recognition receptors to regulate migration and phagocytosis, and the shift in related functions that occur in normal aging. PMID:23644076

The Transcription Factor p53 Influences Microglial Activation Phenotype

PubMed Central

Jayadev, Suman; Nesser, Nicole K.; Hopkins, Stephanie; Myers, Scott J.; Case, Amanda; Lee, Rona J.; Seaburg, Luke A.; Uo, Takuma; Murphy, Sean P.; Morrison, Richard S.; Garden, Gwenn A.

2011-01-01

Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. PMID:21598312

Quantification of the functional expression of the Ca2+ -activated K+ channel KCa 3.1 on microglia from adult human neocortical tissue.

PubMed

Blomster, Linda V; Strøbaek, Dorte; Hougaard, Charlotte; Klein, Jessica; Pinborg, Lars H; Mikkelsen, Jens D; Christophersen, Palle

2016-12-01

The K Ca 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of K Ca 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a K Ca 3.1/K Ca 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective K Ca 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed K Ca 3.1, and the difference in current between full activation and inhibition (ΔK Ca 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial K Ca 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the K Ca 3.1 current nor the fraction of K Ca 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the K Ca 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the K Ca 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a K ir channel. The high functional expression of K Ca 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078. © 2016 Wiley Periodicals, Inc.

Microglia change from a reactive to an age-like phenotype with the time in culture.

PubMed

Caldeira, Cláudia; Oliveira, Ana F; Cunha, Carolina; Vaz, Ana R; Falcão, Ana S; Fernandes, Adelaide; Brites, Dora

2014-01-01

Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.

Microglia change from a reactive to an age-like phenotype with the time in culture

PubMed Central

Caldeira, Cláudia; Oliveira, Ana F.; Cunha, Carolina; Vaz, Ana R.; Falcão, Ana S.; Fernandes, Adelaide; Brites, Dora

2014-01-01

Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function. PMID:24917789

Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model

NASA Astrophysics Data System (ADS)

Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

2015-12-01

Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06625e

New Insights into Microglia-Neuron Interactions: A Neuron's Perspective.

PubMed

Pósfai, Balázs; Cserép, Csaba; Orsolits, Barbara; Dénes, Ádám

2018-05-19

Microglia are the primary immune cells of the central nervous system. However, recent data indicate that microglia also contribute to diverse physiological and pathophysiological processes that extend beyond immune-related functions and there is a growing interest to understand the mechanisms through which microglia interact with other cells in the brain. In particular, the molecular processes that contribute to microglia-neuron communication in the healthy brain and their role in common brain diseases have been intensively studied during the last decade. In line with this, fate-mapping studies, genetic models and novel pharmacological approaches have revealed the origin of microglial progenitors, demonstrated the role of self-maintaining microglial populations during brain development or in adulthood, and identified the unexpectedly long lifespan of microglia that may profoundly change our view about senescence and age-related human diseases. Despite the exponentially increasing knowledge about microglia, the role of these cells in health and disease is still extremely controversial and the precise molecular targets for intervention are not well defined. This is in part due to the lack of microglia-specific manipulation approaches until very recently and to the high level of complexity of the interactions between microglia and other cells in the brain that occur at different temporal and spatial scales. In this review, we briefly summarize the known physiological roles of microglia-neuron interactions in brain homeostasis and attempt to outline some major directions and challenges of future microglia research. Copyright © 2018. Published by Elsevier Ltd.

Behavioral stress alters corticolimbic microglia in a sex- and brain region-specific manner.

PubMed

Bollinger, Justin L; Collins, Kaitlyn E; Patel, Rushi; Wellman, Cara L

2017-01-01

Women are more susceptible to numerous stress-linked psychological disorders (e.g., depression) characterized by dysfunction of corticolimbic brain regions critical for emotion regulation and cognitive function. Although sparsely investigated, a number of studies indicate sex differences in stress effects on neuronal structure, function, and behaviors associated with these regions. We recently demonstrated a basal sex difference in- and differential effects of stress on- microglial activation in medial prefrontal cortex (mPFC). The resident immune cells of the brain, microglia are implicated in synaptic and dendritic plasticity, and cognitive-behavioral function. Here, we examined the effects of acute (3h/day, 1 day) and chronic (3h/day, 10 days) restraint stress on microglial density and morphology, as well as immune factor expression in orbitofrontal cortex (OFC), basolateral amygdala (BLA), and dorsal hippocampus (DHC) in male and female rats. Microglia were visualized, classified based on their morphology, and stereologically counted. Microglia-associated transcripts (CD40, iNOS, Arg1, CX3CL1, CX3CR1, CD200, and CD200R) were assessed in brain punches from each region. Expression of genes linked with cellular stress, neuroimmune state, and neuron-microglia communication varied between unstressed male and female rats in a region-specific manner. In OFC, chronic stress upregulated a wider variety of immune factors in females than in males. Acute stress increased microglia-associated transcripts in BLA in males, whereas chronic stress altered immune factor expression in BLA more broadly in females. In DHC, chronic stress increased immune factor expression in males but not females. Moreover, acute and chronic stress differentially affected microglial morphological activation state in male and female rats across all brain regions investigated. In males, chronic stress altered microglial activation in a pattern consistent with microglial involvement in stress-induced dendritic remodeling across OFC, BLA, and DHC. Together, these data suggest the potential for microglia-mediated sex differences in stress effects on neural structure, function, and behavior.

Behavioral stress alters corticolimbic microglia in a sex- and brain region-specific manner

PubMed Central

Bollinger, Justin L.; Collins, Kaitlyn E.; Patel, Rushi

2017-01-01

Women are more susceptible to numerous stress-linked psychological disorders (e.g., depression) characterized by dysfunction of corticolimbic brain regions critical for emotion regulation and cognitive function. Although sparsely investigated, a number of studies indicate sex differences in stress effects on neuronal structure, function, and behaviors associated with these regions. We recently demonstrated a basal sex difference in- and differential effects of stress on- microglial activation in medial prefrontal cortex (mPFC). The resident immune cells of the brain, microglia are implicated in synaptic and dendritic plasticity, and cognitive-behavioral function. Here, we examined the effects of acute (3h/day, 1 day) and chronic (3h/day, 10 days) restraint stress on microglial density and morphology, as well as immune factor expression in orbitofrontal cortex (OFC), basolateral amygdala (BLA), and dorsal hippocampus (DHC) in male and female rats. Microglia were visualized, classified based on their morphology, and stereologically counted. Microglia-associated transcripts (CD40, iNOS, Arg1, CX3CL1, CX3CR1, CD200, and CD200R) were assessed in brain punches from each region. Expression of genes linked with cellular stress, neuroimmune state, and neuron-microglia communication varied between unstressed male and female rats in a region-specific manner. In OFC, chronic stress upregulated a wider variety of immune factors in females than in males. Acute stress increased microglia-associated transcripts in BLA in males, whereas chronic stress altered immune factor expression in BLA more broadly in females. In DHC, chronic stress increased immune factor expression in males but not females. Moreover, acute and chronic stress differentially affected microglial morphological activation state in male and female rats across all brain regions investigated. In males, chronic stress altered microglial activation in a pattern consistent with microglial involvement in stress-induced dendritic remodeling across OFC, BLA, and DHC. Together, these data suggest the potential for microglia-mediated sex differences in stress effects on neural structure, function, and behavior. PMID:29194444

Old Maids: Aging and Its Impact on Microglia Function

PubMed Central

Koellhoffer, Edward C.; McCullough, Louise D.; Ritzel, Rodney M.

2017-01-01

Microglia are highly active and vigilant housekeepers of the central nervous system that function to promote neuronal growth and activity. With advanced age, however, dysregulated inflammatory signaling and defects in phagocytosis impede their ability to perform the most essential of homeostatic functions, including immune surveillance and debris clearance. Microglial activation is one of the hallmarks of the aging brain and coincides with age-related neurodegeneration and cognitive decline. Age-associated microglial dysfunction leads to cellular senescence and can profoundly alter the response to sterile injuries and immune diseases, often resulting in maladaptive responses, chronic inflammation, and worsened outcomes after injury. Our knowledge of microglia aging and the factors that regulate age-related microglial dysfunction remain limited, as the majority of pre-clinical studies are performed in young animals, and human brain samples are difficult to obtain quickly post-mortem or in large numbers. This review outlines the impact of normal aging on microglial function, highlights the potential mechanisms underlying age-related changes in microglia, and discusses how aging can shape the recovery process following injury. PMID:28379162

Magnetic Labeling of Activated Microglia in Experimental Gliomas1

PubMed Central

Fleige, Gerrit; Nolte, Christiane; Synowitz, Michael; Seeberger, Florian; Kettenmann, Helmut; Zimmer, Claus

2001-01-01

Abstract Microglia, as intrinsic immunoeffector cells of the central nervous system (CNS), play a very sensitive, crucial role in the response to almost any brain pathology where they are activated to a phagocytic state. Based on the characteristic features of activated microglia, we investigated whether these cells can be visualized with magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxides (USPIOs). The hypothesis of this study was that MR microglia visualization could not only reveal the extent of the tumor, but also allow for assessing the status of immunologic defense. Using USPIOs in cell culture experiments and in a rat glioma model, we showed that microglia can be labeled magnetically. Labeled microglia are detected by confocal microscopy within and around tumors in a typical border-like pattern. Quantitative in vitro studies revealed that microglia internalize amounts of USPIOs that are significantly higher than those incorporated by tumor cells and astrocytes. Labeled microglia can be detected and quantified with MRI in cell phantoms, and the extent of the tumor can be seen in glioma-bearing rats in vivo. We conclude that magnetic labeling of microglia provides a potential tool for MRI of gliomas, which reflects tumor morphology precisely. Furthermore, the results suggest that MRI may yield functional data on the immunologic reaction of the CNS. PMID:11774031

4-Hydroxy TEMPO attenuates dichlorvos induced microglial activation and apoptosis.

PubMed

Sunkaria, Aditya; Sharma, Deep Raj; Wani, Willayat Yousuf; Gill, Kiran Dip

2014-02-19

Microglial cells have been implicated in various neurodegenerative diseases. Previous studies from our lab have shown that dichlorvos (an organophosphate) could induce Parkinson's like features in rats. Recently, we have shown that dichlorvos can induce microglial activation, and if not checked in time could ultimately induce neuronal apoptosis. However, this activation does not always pose a threat to the neurons. Activated microglia also secrete various neuronal growth factors, suggesting that they have beneficial roles in CNS repair. Therefore, it is essential to control their detrimental functions selectively. Here, we tried to find out how microglial cells behave when exposed to dichlorvos in either the presence or absence of potent nitric oxide scavenger and superoxide dismutase mimetic, 4-hydroxy TEMPO (4-HT). Wistar rat pups (1 day) were used to isolate and culture primary microglial cells. We found 4-HT pretreatment successfully attenuated the dichlorvos mediated microglial activation. Moreover, 4-HT pretreatment decreased the up-regulated levels of p53 and its downstream effector, p21. The expression of various cell cycle regulators such as Chk2, CDC25a, and cyclin A remained close to their basal levels when 4-HT pretreatment was given. DNA fragmentation analysis showed significant reduction in the DNA damage of 4-HT pretreated microglia as compared to dichlorvos treated cells. In addition to this, we found 4-HT pretreatment prevented the microglial cells from undergoing apoptotic cell death even after 48 h of dichlorvos exposure. Taken together, our results showed 4-HT pretreatment could successfully ameliorate the dichlorvos induced microglial cell damage.

Methylene Blue Mitigates Acute Neuroinflammation after Spinal Cord Injury through Inhibiting NLRP3 Inflammasome Activation in Microglia.

PubMed

Lin, Zhi-Hang; Wang, Si-Yuan; Chen, Li-Li; Zhuang, Jia-Yuan; Ke, Qing-Feng; Xiao, Dan-Rui; Lin, Wen-Ping

2017-01-01

The spinal cord injury (SCI) is a detrimental neurological disease involving the primary mechanical injury and secondary inflammatory damage. Curtailing the detrimental neuroinflammation would be beneficial for spinal cord function recovery. Microglia reside in the spinal cord and actively participate in the onset, progression and perhaps resolution of post-SCI neuroinflammation. In the current study, we tested the effects of methylene blue on microglia both in vitro and in a rat SCI model. We found that methylene blue inhibited the protein levels of IL-1β and IL-18 rather than their mRNA levels in activated microglia. Further investigation indicated that methylene blue deceased the activation of NLRP3 inflammasome and NLRC4 inflammasome in microglia in vitro . Moreover, in the rat SCI model, the similar effect of methylene blue on post-SCI microglia was also observed, except that the activation of NLRC4 inflammasome was not seen. The inhibition of microglia NLRP3 inflammasome was associated with down-regulation of intracellular reactive oxygen species (ROS). The administration of methylene blue mitigated the overall post-SCI neuroinflammation, demonstrated by decreased pro-inflammatory cytokine production and leukocyte infiltrates. Consequently, the neuronal apoptosis was partially inhibited and the hind limb locomotor function was ameliorated by methylene blue treatment. Our research highlights the role of methylene blue in inhibiting post-SCI neuroinflammation, and suggests that methylene blue might be used for SCI therapy.

Methylene Blue Mitigates Acute Neuroinflammation after Spinal Cord Injury through Inhibiting NLRP3 Inflammasome Activation in Microglia

PubMed Central

Lin, Zhi-Hang; Wang, Si-Yuan; Chen, Li-Li; Zhuang, Jia-Yuan; Ke, Qing-Feng; Xiao, Dan-Rui; Lin, Wen-Ping

2017-01-01

The spinal cord injury (SCI) is a detrimental neurological disease involving the primary mechanical injury and secondary inflammatory damage. Curtailing the detrimental neuroinflammation would be beneficial for spinal cord function recovery. Microglia reside in the spinal cord and actively participate in the onset, progression and perhaps resolution of post-SCI neuroinflammation. In the current study, we tested the effects of methylene blue on microglia both in vitro and in a rat SCI model. We found that methylene blue inhibited the protein levels of IL-1β and IL-18 rather than their mRNA levels in activated microglia. Further investigation indicated that methylene blue deceased the activation of NLRP3 inflammasome and NLRC4 inflammasome in microglia in vitro. Moreover, in the rat SCI model, the similar effect of methylene blue on post-SCI microglia was also observed, except that the activation of NLRC4 inflammasome was not seen. The inhibition of microglia NLRP3 inflammasome was associated with down-regulation of intracellular reactive oxygen species (ROS). The administration of methylene blue mitigated the overall post-SCI neuroinflammation, demonstrated by decreased pro-inflammatory cytokine production and leukocyte infiltrates. Consequently, the neuronal apoptosis was partially inhibited and the hind limb locomotor function was ameliorated by methylene blue treatment. Our research highlights the role of methylene blue in inhibiting post-SCI neuroinflammation, and suggests that methylene blue might be used for SCI therapy. PMID:29311826

Interleukin-4 Ameliorates the Functional Recovery of Intracerebral Hemorrhage Through the Alternative Activation of Microglia/Macrophage.

PubMed

Yang, Jianjing; Ding, Saidan; Huang, Weilong; Hu, Jiangnan; Huang, Shengwei; Zhang, Yu; Zhuge, Qichuan

2016-01-01

Neuro-inflammation plays an important role in the recovery of brain injury after stroke. Microglia/macrophage is the major executor in the neuro-inflammation, which can be polarized into two distinct phenotypes: injurious/toxic classical activation (M1 phenotype) and protective alternative activation (M2 phenotype). Here, we investigated whether intracerebral administration of interleukin-4 (IL-4) at an early stage could affect the activation of microglia/macrophage and the corresponding outcome after intracerebral hemorrhage (ICH). The neuro-behavior was recorded between different groups in the rat ICH model. The M1 and M2 markers were then determined by qRT-PCR, western blotting, ELISA, and immunofluorescence, respectively. We observed aberrant activation of microglia/macrophage after ICH. After intracerebral injection of IL-4, M1 activation was greatly inhibited while M2 activation was enhanced, along with improving neurobehavioral recovery from deficits after ICH. Our study showed that early intracerebral injection of IL-4 potentially promotes neuro-functional recovery, probably through enhancing the alternative activation of microglia/macrophage.

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat.

PubMed

Wohleb, Eric S; Hanke, Mark L; Corona, Angela W; Powell, Nicole D; Stiner, La'Tonia M; Bailey, Michael T; Nelson, Randy J; Godbout, Jonathan P; Sheridan, John F

2011-04-27

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

β-Adrenergic Receptor Antagonism Prevents Anxiety-like Behavior and Microglial Reactivity Induced by Repeated Social Defeat

PubMed Central

Wohleb, Eric S.; Hanke, Mark L.; Corona, Angela W.; Powell, Nicole D.; Stiner, La'Tonia M.; Bailey, Michael T.; Nelson, Randy J.; Godbout, Jonathan P.; Sheridan, John F.

2011-01-01

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b+/CD45high/Ly6Chigh macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of de-ramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes (GILZ and FKBP51). The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1) after stimulation with lipopolysaccharide (LPS) compared to microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1 deficient (IL-1r1-/-) mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors. PMID:21525267

Priming of microglia in a DNA-repair deficient model of accelerated aging.

PubMed

Raj, Divya D A; Jaarsma, Dick; Holtman, Inge R; Olah, Marta; Ferreira, Filipa M; Schaafsma, Wandert; Brouwer, Nieske; Meijer, Michel M; de Waard, Monique C; van der Pluijm, Ingrid; Brandt, Renata; Kreft, Karim L; Laman, Jon D; de Haan, Gerald; Biber, Knut P H; Hoeijmakers, Jan H J; Eggen, Bart J L; Boddeke, Hendrikus W G M

2014-09-01

Aging is associated with reduced function, degenerative changes, and increased neuroinflammation of the central nervous system (CNS). Increasing evidence suggests that changes in microglia cells contribute to the age-related deterioration of the CNS. The most prominent age-related change of microglia is enhanced sensitivity to inflammatory stimuli, referred to as priming. It is unclear if priming is due to intrinsic microglia ageing or induced by the ageing neural environment. We have studied this in Ercc1 mutant mice, a DNA repair-deficient mouse model that displays features of accelerated aging in multiple tissues including the CNS. In Ercc1 mutant mice, microglia showed hallmark features of priming such as an exaggerated response to peripheral lipopolysaccharide exposure in terms of cytokine expression and phagocytosis. Specific targeting of the Ercc1 deletion to forebrain neurons resulted in a progressive priming response in microglia exemplified by phenotypic alterations. Summarizing, these data show that neuronal genotoxic stress is sufficient to switch microglia from a resting to a primed state. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential expression of MHC class II and B7 costimulatory molecules by microglia in rodent gliomas.

PubMed

Badie, Behnam; Bartley, Becky; Schartner, Jill

2002-12-01

To assess the immune function of microglia and macrophages in brain tumors, the expression of MHC class II and B7 costimulatory molecules in three rodent glioma models was examined. Microglia and macrophages, which accounted for 5-12% of total cells, expressed B7.1 and MHC class II molecules in the C6 and 9L tumors, but not RG2 gliomas. Interestingly, the expression of B7.1 and MHC class II molecules by microglia and macrophage was associated with an increase in the number of tumor-infiltrating lymphocytes in C6 and 9L tumors. B7.2 expression, which was present at low levels on microglia and macrophages in normal brain, did not significantly change in tumors. Interestingly, the expression of all three surface antigens increased after microglia were isolated from intracranial C6 tumors and cultured for a short period of time. We conclude that microglia immune activity may be suppressed in gliomas and directly correlates to the immunogenecity of experimental brain tumors.

Diminished circadian rhythms in hippocampal microglia may contribute to age-related neuroinflammatory sensitization

PubMed Central

Fonken, Laura K.; Kitt, Meagan M.; Gaudet, Andrew D.; Barrientos, Ruth M.; Watkins, Linda R.; Maier, Steven F.

2016-01-01

Aged animals exhibit diminished circadian rhythms, and both aging and circadian disruption sensitize neuroinflammatory responses. Microglia –the innate immune cell of the CNS – possess endogenous timekeeping mechanisms that regulate immune responses. Here, we explored whether aging is associated with disrupted diurnal rhythms in microglia and neuroinflammatory processes. First, hippocampal microglia isolated from young rats (4 mos. F344XBN) rhythmically expressed circadian clock genes, whereas microglia isolated from the hippocampus of aged rats (25 mos.) had aberrant Per1 and Per2 rhythms. Unstimulated microglia from young rats exhibited robust rhythms of TNFα and IL-1β mRNA expression, whereas those from aged rats had flattened and tonically-elevated cytokine expression. Similarly, microglial activation markers were diurnally regulated in the hippocampus of young but not aged rats and diurnal differences in responsiveness to both ex vivo and in vivo inflammatory challenges were abolished in aged rats. Corticosterone is an entraining signal for extra-SCN circadian rhythms. Here, corticosterone stimulation elicited similar Per1 induction in aged and young microglia. Overall, these results indicate that aging dysregulates circadian regulation of neuroinflammatory functions. PMID:27568094

Microglia activation in Niemann-Pick disease, type C1 is amendable to therapeutic intervention.

PubMed

Cougnoux, Antony; Drummond, Rebecca A; Collar, Amanda L; Iben, James R; Salman, Alexander; Westgarth, Harrison; Wassif, Christopher A; Cawley, Niamh X; Farhat, Nicole Y; Ozato, Keiko; Lionakis, Michail S; Porter, Forbes D

2018-06-15

Niemann-Pick disease, type C1 (NPC1) is a neurodegenerative disorder with limited treatment options. NPC1 is associated with neuroinflammation; however, attempts to therapeutically target neuroinflammation in NPC1 have had mixed success. We show here that NPC1 neuroinflammation is characterized by an atypical microglia activation phenotype. Specifically, Npc1-/- microglia demonstrated altered morphology, reduced levels of lineage markers and a shift toward glycolytic metabolism. Treatment with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a drug currently being studied in a phase 2b/3 clinical trial, reversed all microglia-associated defects in Npc1-/- animals. In addition, impairing microglia mediated neuroinflammation by genetic deletion of IRF8 led to decreased symptoms and increased lifespan. We identified CD22 as a marker of dysregulated microglia in Npc1 mutant mice and subsequently demonstrated that elevated cerebrospinal fluid levels of CD22 in NPC1 patients responds to HPβCD administration. Collectively, these data provide the first in-depth analysis of microglia function in NPC1 and suggest possible new therapeutic approaches.

Enhanced microglial pro-inflammatory response to lipopolysaccharide correlates with brain infiltration and blood-brain barrier dysregulation in a mouse model of telomere shortening.

PubMed

Raj, Divya D A; Moser, Jill; van der Pol, Susanne M A; van Os, Ronald P; Holtman, Inge R; Brouwer, Nieske; Oeseburg, Hisko; Schaafsma, Wandert; Wesseling, Evelyn M; den Dunnen, Wilfred; Biber, Knut P H; de Vries, Helga E; Eggen, Bart J L; Boddeke, Hendrikus W G M

2015-12-01

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self-renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as 'priming'. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first-generation G1 mTerc(-/-) )- and late-generation (third-generation G3 and G4 mTerc(-/-) ) telomerase-deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late-generation mTerc(-/-) microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc(-/-) microglia are comparable with microglia derived from G1 mTerc(-/-) mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc(-/-) microglia mice show an enhanced pro-inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age-associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood-brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Alzheimer's Disease: The Role of Microglia in Brain Homeostasis and Proteopathy

PubMed Central

Clayton, Kevin A.; Van Enoo, Alicia A.; Ikezu, Tsuneya

2017-01-01

Brain aging is central to late-onset Alzheimer's disease (LOAD), although the mechanisms by which it occurs at protein or cellular levels are not fully understood. Alzheimer's disease is the most common proteopathy and is characterized by two unique pathologies: senile plaques and neurofibrillary tangles, the former accumulating earlier than the latter. Aging alters the proteostasis of amyloid-β peptides and microtubule-associated protein tau, which are regulated in both autonomous and non-autonomous manners. Microglia, the resident phagocytes of the central nervous system, play a major role in the non-autonomous clearance of protein aggregates. Their function is significantly altered by aging and neurodegeneration. This is genetically supported by the association of microglia-specific genes, TREM2 and CD33, and late onset Alzheimer's disease. Here, we propose that the functional characterization of microglia, and their contribution to proteopathy, will lead to a new therapeutic direction in Alzheimer's disease research. PMID:29311768

Microglial Lectins in Health and Neurological Diseases

PubMed Central

Siew, Jian Jing; Chern, Yijuang

2018-01-01

Microglia are the innate sentinels of the central nervous system (CNS) and are responsible for the homeostasis and immune defense of the CNS. Under the influence of the local environment and cell-cell interaction, microglia exhibit a multidimensional and context-dependent phenotypes that can be cytotoxic and neuroprotective. Recent studies suggest that microglia express multitudinous types of lectins, including galectins, Siglecs, mannose-binding lectins (MBLs) and other glycan binding proteins. Because most studies that examine lectins focus on the peripheral system, the functions of lectins have not been critically investigated in the CNS. In addition, the types of brain cells that contribute to the altered levels of lectins present in diseases are often unclear. In this review, we will discuss how galectins, Siglecs, selectins and MBLs contribute to the dynamic functions of microglia. The interacting ligands of these lectins are complex glycoconjugates, which consist of glycoproteins and glycolipids that are expressed on microglia or surrounding cells. The current understanding of the heterogeneity and functions of glycans in the brain is limited. Galectins are a group of pleotropic proteins that recognize both β-galactoside-containing glycans and non- β-galactoside-containing proteins. The function and regulation of galectins have been implicated in immunomodulation, neuroinflammation, apoptosis, phagocytosis and oxidative bursts. Most Siglecs are expressed at a low level on the plasma membrane and bind to sialic acid residues for immunosurveillance and cell-cell communication. Siglecs are classified based on their inhibitory and activatory downstream signaling properties. Inhibitory Siglecs negatively regulate microglia activation upon recognizing the intact sialic acid patterns and vice versa. MBLs are expressed upon infection in cytoplasm and can be secreted in order to recognize molecules containing terminal mannose as an innate immune defense machinery. Most importantly, multiple studies have reported dysregulation of lectins in neurological disorders. Here, we reviewed recent studies on microglial lectins and their functions in CNS health and disease, and suggest that these lectin families are novel, potent therapeutic targets for neurological diseases. PMID:29867350

Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model.

PubMed

Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

2016-01-07

Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.

Brain innate immunity regulates hypothalamic arcuate neuronal activity and feeding behavior.

PubMed

Reis, Wagner L; Yi, Chun-Xia; Gao, Yuanqing; Tschöp, Mathias H; Stern, Javier E

2015-04-01

Hypothalamic inflammation, involving microglia activation in the arcuate nucleus (ARC), is proposed as a novel underlying mechanism in obesity, insulin and leptin resistance. However, whether activated microglia affects ARC neuronal activity, and consequently basal and hormonal-induced food intake, is unknown. We show that lipopolysaccharide, an agonist of the toll-like receptor-4 (TLR4), which we found to be expressed in ARC microglia, inhibited the firing activity of the majority of orexigenic agouti gene-related protein/neuropeptide Y neurons, whereas it increased the activity of the majority of anorexigenic proopiomelanocortin neurons. Lipopolysaccharide effects in agouti gene-related protein/neuropeptide Y (but not in proopiomelanocortin) neurons were occluded by inhibiting microglia function or by blocking TLR4 receptors. Finally, we report that inhibition of hypothalamic microglia altered basal food intake, also preventing central orexigenic responses to ghrelin. Our studies support a major role for a TLR4-mediated microglia signaling pathway in the control of ARC neuronal activity and feeding behavior.

The Role of MAC1 in Diesel Exhaust Particle-induced Microglial Activation and Loss of Dopaminergic Neuron Function

PubMed Central

Levesque, Shannon; Taetzsch, Thomas; Lull, Melinda E.; Johnson, Jo Anne; McGraw, Constance; Block, Michelle L.

2013-01-01

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson’s disease-like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron-glia cultures were treated with either nanometer-sized DEP (<0.22 µM; 50µg/mL), ultrafine carbon black (ufCB, 50µg/ml), or DEP extracts (eDEP; from 50 µg/ml DEP) and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced amoeboid microglia morphology, increased H2O2 production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP-induced H2O2 production in microglia. However, pretreatment with the MAC1/CD11b inhibitor antibody blocked microglial H2O2 production in response to DEP. MAC1−/− mesencephalic neuron-glia cultures were protected from DEP-induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP-induced microglial H2O2 production and loss of DA neuron function. PMID:23470120

Temporary Depletion of Microglia during the Early Postnatal Period Induces Lasting Sex-Dependent and Sex-Independent Effects on Behavior in Rats

PubMed Central

2016-01-01

Abstract Microglia are the primary immune cells of the brain and function in multiple ways to facilitate proper brain development. However, our current understanding of how these cells influence the later expression of normal behaviors is lacking. Using the laboratory rat, we administered liposomal clodronate centrally to selectively deplete microglia in the developing postnatal brain. We then assessed a range of developmental, juvenile, and adult behaviors. Liposomal clodronate treatment on postnatal days 0, 2, and 4 depleted microglia with recovery by about 10 days of age and induced a hyperlocomotive phenotype, observable in the second postnatal week. Temporary microglia depletion also increased juvenile locomotion in the open field test and decreased anxiety-like behaviors in the open field and elevated plus maze. These same rats displayed reductions in predator odor–induced avoidance behavior, but increased their risk assessment behaviors compared with vehicle-treated controls. In adulthood, postnatal microglia depletion resulted in significant deficits in male-specific sex behaviors. Using factor analysis, we identified two underlying traits—behavioral disinhibition and locomotion—as being significantly altered by postnatal microglia depletion. These findings further implicate microglia as being critically important to the development of juvenile and adult behavior. PMID:27957532

High-frequency transcutaneous electrical nerve stimulation alleviates spasticity after spinal contusion by inhibiting activated microglia in rats.

PubMed

Hahm, Suk-Chan; Yoon, Young Wook; Kim, Junesun

2015-05-01

Transcutaneous electrical nerve stimulation (TENS) can be used as a physical therapy for spasticity, but the effects of TENS on spasticity and its underlying mechanisms remain unclear. The purpose of this study was to test the effects of TENS on spasticity and the role of activated microglia as underlying mechanisms of TENS treatment for spasticity in rats with a 50-mm contusive spinal cord injury (SCI). A spinal contusion was made at the T12 spinal segment in adult male Sprague-Dawley rats using the NYU impactor. Behavioral tests for motor function were conducted before and after SCI and before and after TENS application. To assess spasticity, the modified Ashworth scale (MAS) was used before and after SCI, high-frequency (HF)/low-frequency (LF) TENS application at 3 different intensities (motor threshold [MT], 50% and 90% MT) or minocycline administration. Immunohistochemistry for microglia was performed at the lumbar spinal segments. Motor recovery reached a plateau approximately 28 days after SCI. Spasticity was well developed and was sustained above the MAS grade of 3, beginning at 28 days after SCI. HF-TENS at 90% MT significantly alleviated spasticity. Motor function did not show any significant changes with LF- or HF-TENS treatment. HF-TENS significantly reduced the proportion of activated microglia observed after SCI. Minocycline, the microglia inhibitor, also significantly alleviated spasticity with the reduction of activated microglia expression. These results suggest that HF-TENS at 90% MT alleviates spasticity in rats with SCI by inhibiting activated microglia. © The Author(s) 2014.

Microglial Priming and Enhanced Reactivity to Secondary Insult in Aging, and Traumatic CNS injury, and Neurodegenerative Disease

PubMed Central

Norden, Diana M.; Muccigrosso, Megan M.; Godbout, Jonathan P.

2014-01-01

Glia of the central nervous system (CNS) help to maintain homeostasis in the brain and support efficient neuronal function. Microglia are innate immune cells of the brain that mediate responses to pathogens and injury. They have key roles in phagocytic clearing, surveying the local microenvironment and propagating inflammatory signals. An interruption in homeostasis induces a cascade of conserved adaptive responses in glia. This response involves biochemical, physiological and morphological changes and is associated with the production of cytokines and secondary mediators that influence synaptic plasticity, cognition and behavior. This reorganization of host priorities represents a beneficial response that is normally adaptive but may become maladaptive when the profile of microglia is compromised. For instance, microglia can develop a primed or pro-inflammatory mRNA, protein and morphological profile with aging, traumatic brain injury and neurodegenerative disease. As a result, primed microglia exhibit an exaggerated inflammatory response to secondary and sub-threshold challenges. Consequences of exaggerated inflammatory responses by microglia include the development of cognitive deficits, impaired synaptic plasticity and accelerated neurodegeneration. Moreover, impairments in regulatory systems in these circumstances may make microglia more resistant to negative feedback and important functions of glia can become compromised and dysfunctional. Overall, the purpose of this review is to discuss key concepts of microglial priming and immune-reactivity in the context of aging, traumatic CNS injury and neurodegenerative disease. PMID:25445485

AP-1 proteins in the adult brain: facts and fiction about effectors of neuroprotection and neurodegeneration.

PubMed

Herdegen, T; Waetzig, V

2001-04-30

Jun and Fos proteins are induced and activated following most physiological and pathophysiological stimuli in the brain. Only few data allow conclusions about distinct functions of AP-1 proteins in neurodegeneration and neuroregeneration, and these functions mainly refer to c-Jun and its activation by JNKs. Apoptotic functions of activated c-Jun affect hippocampal, nigral and primary cultured neurons following excitotoxic stimulation and destruction of the neuron-target-axis including withdrawal of trophic molecules. The inhibition of JNKs might exert neuroprotection by subsequent omission of c-Jun activation. Besides endogenous neuronal functions, the c-Jun/AP-1 proteins can damage the nervous system by upregulation of harmful programs in non-neuronal cells (e.g. microglia) with release of neurodegenerative molecules. In contrast, the differentiation with neurite extension and maturation of neural cells in vitro indicate physiological and potentially neuroprotective functions of c-Jun and JNKs including sensoring for alterations in the cytoskeleton. This review summarizes the multiple molecular interfunctions which are involved in the shift from the physiological role to degenerative effects of the Jun/JNK-axis such as cell type-specific expression and intracellular localization of scaffold proteins and upstream activators, antagonistic phosphatases, interaction with other kinase systems, or the activation of transcription factors competing for binding to JNK proteins and AP-1 DNA elements.

In Utero Administration of Drugs Targeting Microglia Improves the Neurodevelopmental Outcome Following Cytomegalovirus Infection of the Rat Fetal Brain

PubMed Central

Cloarec, Robin; Bauer, Sylvian; Teissier, Natacha; Schaller, Fabienne; Luche, Hervé; Courtens, Sandra; Salmi, Manal; Pauly, Vanessa; Bois, Emilie; Pallesi-Pocachard, Emilie; Buhler, Emmanuelle; Michel, François J.; Gressens, Pierre; Malissen, Marie; Stamminger, Thomas; Streblow, Daniel N.; Bruneau, Nadine; Szepetowski, Pierre

2018-01-01

Congenital cytomegalovirus (CMV) infections represent one leading cause of neurodevelopmental disorders. Recently, we reported on a rat model of CMV infection of the developing brain in utero, characterized by early and prominent infection and alteration of microglia—the brain-resident mononuclear phagocytes. Besides their canonical function against pathogens, microglia are also pivotal to brain development. Here we show that CMV infection of the rat fetal brain recapitulated key postnatal phenotypes of human congenital CMV including increased mortality, sensorimotor impairment reminiscent of cerebral palsy, hearing defects, and epileptic seizures. The possible influence of early microglia alteration on those phenotypes was then questioned by pharmacological targeting of microglia during pregnancy. One single administration of clodronate liposomes in the embryonic brains at the time of CMV injection to deplete microglia, and maternal feeding with doxycyxline throughout pregnancy to modify microglia in the litters' brains, were both associated with dramatic improvements of survival, body weight gain, sensorimotor development and with decreased risk of epileptic seizures. Improvement of microglia activation status did not persist postnatally after doxycycline discontinuation; also, active brain infection remained unchanged by doxycycline. Altogether our data indicate that early microglia alteration, rather than brain CMV load per se, is instrumental in influencing survival and the neurological outcomes of CMV-infected rats, and suggest that microglia might participate in the neurological outcome of congenital CMV in humans. Furthermore this study represents a first proof-of-principle for the design of microglia-targeted preventive strategies in the context of congenital CMV infection of the brain. PMID:29559892

Low-Fat Diet With Caloric Restriction Reduces White Matter Microglia Activation During Aging.

PubMed

Yin, Zhuoran; Raj, Divya D; Schaafsma, Wandert; van der Heijden, Roel A; Kooistra, Susanne M; Reijne, Aaffien C; Zhang, Xiaoming; Moser, Jill; Brouwer, Nieske; Heeringa, Peter; Yi, Chun-Xia; van Dijk, Gertjan; Laman, Jon D; Boddeke, Erik W G M; Eggen, Bart J L

2018-01-01

Rodent models of both aging and obesity are characterized by inflammation in specific brain regions, notably the corpus callosum, fornix, and hypothalamus. Microglia, the resident macrophages of the central nervous system, are important for brain development, neural support, and homeostasis. However, the effects of diet and lifestyle on microglia during aging are only partly understood. Here, we report alterations in microglia phenotype and functions in different brain regions of mice on a high-fat diet (HFD) or low-fat diet (LFD) during aging and in response to voluntary running wheel exercise. We compared the expression levels of genes involved in immune response, phagocytosis, and metabolism in the hypothalamus of 6-month-old HFD and LFD mice. We also compared the immune response of microglia from HFD or LFD mice to peripheral inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS). Finally, we investigated the effect of diet, physical exercise, and caloric restriction (40% reduction compared to ad libitum intake) on microglia in 24-month-old HFD and LFD mice. Changes in diet caused morphological changes in microglia, but did not change the microglia response to LPS-induced systemic inflammation. Expression of phagocytic markers (i.e., Mac-2/Lgals3, Dectin-1/Clec7a, and CD16/CD32) in the white matter microglia of 24-month-old brain was markedly decreased in calorically restricted LFD mice. In conclusion, LFD resulted in reduced activation of microglia, which might be an underlying mechanism for the protective role of caloric restriction during aging-associated decline.

Deletion of the Fractalkine Receptor, CX3CR1, Improves Endogenous Repair, Axon Sprouting, and Synaptogenesis after Spinal Cord Injury in Mice

PubMed Central

Wei, Ping; Guan, Zhen

2017-01-01

Impaired signaling via CX3CR1, the fractalkine receptor, promotes recovery after traumatic spinal contusion injury in mice, a benefit achieved in part by reducing macrophage-mediated injury at the lesion epicenter. Here, we tested the hypothesis that CX3CR1-dependent changes in microglia and macrophage functions also will enhance neuroplasticity, at and several segments below the injury epicenter. New data show that in the presence of inflammatory stimuli, CX3CR1-deficient (CX3CR1−/−) microglia and macrophages adopt a reparative phenotype and increase expression of genes that encode neurotrophic and gliogenic proteins. At the lesion epicenter (mid-thoracic spinal cord), the microenvironment created by CX3CR1−/− microglia/macrophages enhances NG2 cell responses, axon sparing, and sprouting of serotonergic axons. In lumbar spinal cord, inflammatory signaling is reduced in CX3CR1−/− microglia. This is associated with reduced dendritic pathology and improved axonal and synaptic plasticity on ventral horn motor neurons. Together, these data indicate that CX3CR1, a microglia-specific chemokine receptor, is a novel therapeutic target for enhancing neuroplasticity and recovery after SCI. Interventions that specifically target CX3CR1 could reduce the adverse effects of inflammation and augment activity-dependent plasticity and restoration of function. Indeed, limiting CX3CR1-dependent signaling could improve rehabilitation and spinal learning. SIGNIFICANCE STATEMENT Published data show that genetic deletion of CX3CR1, a microglia-specific chemokine receptor, promotes recovery after traumatic spinal cord injury in mice, a benefit achieved in part by reducing macrophage-mediated injury at the lesion epicenter. Data in the current manuscript indicate that CX3CR1 deletion changes microglia and macrophage function, creating a tissue microenvironment that enhances endogenous repair and indices of neuroplasticity, at and several segments below the injury epicenter. Interventions that specifically target CX3CR1 might be used in the future to reduce the adverse effects of intraspinal inflammation and augment activity-dependent plasticity (e.g., rehabilitation) and restoration of function. PMID:28264978

Molecular Mechanisms Modulating the Phenotype of Macrophages and Microglia

PubMed Central

Amici, Stephanie A.; Dong, Joycelyn; Guerau-de-Arellano, Mireia

2017-01-01

Macrophages and microglia play crucial roles during central nervous system development, homeostasis and acute events such as infection or injury. The diverse functions of tissue macrophages and microglia are mirrored by equally diverse phenotypes. A model of inflammatory/M1 versus a resolution phase/M2 macrophages has been widely used. However, the complexity of macrophage function can only be achieved by the existence of varied, plastic and tridimensional macrophage phenotypes. Understanding how tissue macrophages integrate environmental signals via molecular programs to define pathogen/injury inflammatory responses provides an opportunity to better understand the multilayered nature of macrophages, as well as target and modulate cellular programs to control excessive inflammation. This is particularly important in MS and other neuroinflammatory diseases, where chronic inflammatory macrophage and microglial responses may contribute to pathology. Here, we perform a comprehensive review of our current understanding of how molecular pathways modulate tissue macrophage phenotype, covering both classic pathways and the emerging role of microRNAs, receptor-tyrosine kinases and metabolism in macrophage phenotype. In addition, we discuss pathway parallels in microglia, novel markers helpful in the identification of peripheral macrophages versus microglia and markers linked to their phenotype. PMID:29176977

Lessons Learned about Neurodegeneration from Microglia and Monocyte Depletion Studies

PubMed Central

Lund, Harald; Pieber, Melanie; Harris, Robert A.

2017-01-01

While bone marrow-derived Ly6Chi monocytes can infiltrate the central nervous system (CNS) they are developmentally and functionally distinct from resident microglia. Our understanding of the relative importance of these two populations in the distinct processes of pathogenesis and resolution of inflammation during neurodegenerative disorders was limited by a lack of tools to specifically manipulate each cell type. During recent years, the development of experimental cell-specific depletion models has enabled this issue to be addressed. Herein we compare and contrast the different depletion approaches that have been used, focusing on the respective functionalities of microglia and monocyte-derived macrophages in a range of neurodegenerative disease states, and discuss their prospects for immunotherapy. PMID:28804456

Dissociation of Innate Immune Responses in Microglia Infected with Listeria monocytogenes

PubMed Central

Frande-Cabanes, Elisabet; Fernandez-Prieto, Lorena; Calderon-Gonzalez, Ricardo; Rodríguez-Del Río, Estela; Yañez-Diaz, Sonsoles; López-Fanarraga, Monica; Alvarez-Domínguez, Carmen

2014-01-01

Microglia, the innate immune cells of the brain, plays a central role in cerebral listeriosis. Here, we present evidence that microglia control Listeria infection differently than macrophages. Infection of primary microglial cultures and murine cell lines with Listeria resulted in a dual function of the two gene expression programmes involved in early and late immune responses in macrophages. Whereas the bacterial gene hly seems responsible for both transcriptional programmes in macrophages, Listeria induces in microglia only the tumor necrosis factor (TNF)-regulated transcriptional programme. Listeria also represses in microglia the late immune response gathered in two clusters, microbial degradation, and interferon (IFN)-inducible genes. The bacterial gene actA was required in microglia to induce TNF-regulated responses and to repress the late response. Isolation of microglial phagosomes revealed a phagosomal environment unable to destroy Listeria. Microglial phagosomes were also defective in several signaling and trafficking components reported as relevant for Listeria innate immune responses. This transcriptional strategy in microglia induced high levels of TNF-α and monocyte chemotactic protein-1 and low production of other neurotoxic compounds such as nitric oxide, hydrogen peroxide, and Type I IFNs. These cytokines and toxic microglial products are also released by primary microglia, and this cytokine and chemokine cocktail display a low potential to trigger neuronal apoptosis. This overall bacterial strategy strongly suggests that microglia limit Listeria inflammation pattern exclusively through TNF-mediated responses to preserve brain integrity. GLIA 2014;62:233–246 PMID:24311463

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

PubMed

Tanaka, Shigeyuki; Djamei, Armin; Presti, Libera Lo; Schipper, Kerstin; Winterberg, Sarah; Amati, Simone; Becker, Dirk; Büchner, Heike; Kumlehn, Jochen; Reissmann, Stefanie; Kahmann, Regine

2015-01-01

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery. Copyright © 2015 Elsevier GmbH. All rights reserved.

Microglial brain region-dependent diversity and selective regional sensitivities to ageing

PubMed Central

Grabert, Kathleen; Michoel, Tom; Karavolos, Michail H; Clohisey, Sara; Baillie, J Kenneth; Stevens, Mark P; Freeman, Tom C; Summers, Kim M; McColl, Barry W

2015-01-01

Microglia play critical roles in neural development, homeostasis and neuroinflammation and are increasingly implicated in age-related neurological dysfunction. Neurodegeneration often occurs in disease-specific spatially-restricted patterns, the origins of which are unknown. We performed the first genome-wide analysis of microglia from discrete brain regions across the adult lifespan of the mouse and reveal that microglia have distinct region-dependent transcriptional identities and age in a regionally variable manner. In the young adult brain, differences in bioenergetic and immunoregulatory pathways were the major sources of heterogeneity and suggested that cerebellar and hippocampal microglia exist in a more immune vigilant state. Immune function correlated with regional transcriptional patterns. Augmentation of the distinct cerebellar immunophenotype and a contrasting loss in distinction of the hippocampal phenotype among forebrain regions were key features during ageing. Microglial diversity may enable regionally localised homeostatic functions but could also underlie region-specific sensitivities to microglial dysregulation and involvement in age-related neurodegeneration. PMID:26780511

Molecular and functional interaction between GPR18 and cannabinoid CB2 G-protein-coupled receptors. Relevance in neurodegenerative diseases.

PubMed

Reyes-Resina, Irene; Navarro, Gemma; Aguinaga, David; Canela, Enric I; Schoeder, Clara T; Zaluski, Michal; Kiec-Kononowicz, Katarzyna; Saura, Carlos A; Müller, Christa E; Franco, Rafael

2018-06-02

GPR18, still considered an orphan receptor, may respond to endocannabinoids, whose canonical receptors are CB 1 and CB 2 . GPR18 and CB 2 receptors share a role in peripheral immune response regulation and are co-expressed in microglia, which are immunocompetent cells in the central nervous system (CNS). We aimed at identifying heteroreceptor complexes formed by GPR18 and CB 1 R or CB 2 R in resting and activated microglia. Receptor-receptor interaction was assessed using energy-transfer approaches, and receptor function by determining cAMP levels and ERK1/2 phosphorylation in heterologous cells and primary cultures of microglia. Heteroreceptor identification in primary cultures of microglia was achieved by in situ proximity ligation assays. Energy transfer results showed interaction of GPR18 with CB 2 R but not with CB 1 R. CB 2 -GPR18 heteroreceptor complexes displayed particular functional properties (heteromer prints) often consisting of negative cross-talk (activation of one receptor reduces signaling arising from the partner receptor) and cross-antagonism (the response of one of the receptors is blocked by a selective antagonist of the partner receptor). Activated microglia showed the heteromer print (negative cross-talk and bidirectional cross-antagonism) and increased expression of CB 2 R and GPR18. Due to the important role of CB 2 R in neuroprotection, we further investigated heteroreceptor occurrence in primary cultures of microglia from transgenic mice overexpressing human APP Sw,Ind , an Alzheimer's disease model. Microglial cells from transgenic mice showed the heteromer print and functional interactions that were similar to those found in cells from wild-type animals that were activated by treatment with lipopolysaccharide and interferon-ɤ. Our results show that GPR18 and its heteromers may play important roles in neurodegenerative processes. Copyright © 2018. Published by Elsevier Inc.

The role of MAC1 in diesel exhaust particle-induced microglial activation and loss of dopaminergic neuron function.

PubMed

Levesque, Shannon; Taetzsch, Thomas; Lull, Melinda E; Johnson, Jo Anne; McGraw, Constance; Block, Michelle L

2013-06-01

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease-like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron-glia cultures were treated with either nanometer-sized DEP (< 0.22 μM; 50 μg/mL), ultrafine carbon black (ufCB, 50 μg/mL), or DEP extracts (eDEP; from 50 μg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H2 O2 production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP-induced H2 O2 production in microglia. However, pre-treatment with the MAC1/CD11b inhibitor antibody blocked microglial H2 O2 production in response to DEP. MAC1(-/-) mesencephalic neuron-glia cultures were protected from DEP-induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP-induced microglial H2 O2 production and loss of DA neuron function. © 2013 International Society for Neurochemistry.

Selective depletion of microglial progranulin in mice is not sufficient to cause neuronal ceroid lipofuscinosis or neuroinflammation.

PubMed

Petkau, Terri L; Kosior, Natalia; de Asis, Kathleen; Connolly, Colúm; Leavitt, Blair R

2017-11-17

Progranulin deficiency due to heterozygous null mutations in the GRN gene are a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations are thought to be a rare cause of neuronal ceroid lipofuscinosis (NCL). Aged progranulin-knockout (Grn-null) mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. In the brain, progranulin is predominantly expressed in neurons and microglia, and previously, we demonstrated that neuronal-specific depletion of progranulin does not recapitulate the neuropathological phenotype of Grn-null mice. In this study, we evaluated whether selective depletion of progranulin expression in myeloid-lineage cells, including microglia, causes NCL-like neuropathology or neuroinflammation in mice. We generated mice with progranulin depleted in myeloid-lineage cells by crossing mice homozygous for a floxed progranulin allele to mice expressing Cre recombinase under control of the LyzM promotor (Lyz-cKO). Progranulin expression was reduced by approximately 50-70% in isolated microglia compared to WT levels. Lyz-cKO mice aged to 12 months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for many of these measures in Grn-null animals. To evaluate the functional effect of reduced progranulin expression in isolated microglia, primary cultures were stimulated with controlled standard endotoxin and cytokine release was measured. While Grn-null microglia display a hyper-inflammatory phenotype, Lyz-cKO and WT microglia secreted similar levels of inflammatory cytokines. We conclude that progranulin expression from either microglia or neurons is sufficient to prevent the development of NCL-like neuropathology in mice. Furthermore, microglia that are deficient for progranulin expression but isolated from a progranulin-rich environment have a normal inflammatory profile. Our results suggest that progranulin acts, at least partly, in a non-cell autonomous manner in the brain.

Suppression of inflammation with conditional deletion of the prostaglandin E2 EP2 receptor in macrophages and brain microglia.

PubMed

Johansson, Jenny U; Pradhan, Suraj; Lokteva, Ludmila A; Woodling, Nathaniel S; Ko, Novie; Brown, Holden D; Wang, Qian; Loh, Christina; Cekanaviciute, Egle; Buckwalter, Marion; Manning-Bog, Amy B; Andreasson, Katrin I

2013-10-02

Prostaglandin E2 (PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP(1-4) receptors. Here, we investigated the cell-specific in vivo function of PGE2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2(lox/lox) mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2(lox/lox) and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.

Suppression of Inflammation with Conditional Deletion of the Prostaglandin E2 EP2 Receptor in Macrophages and Brain Microglia

PubMed Central

Johansson, Jenny U.; Pradhan, Suraj; Lokteva, Ludmila A.; Woodling, Nathaniel S.; Ko, Novie; Brown, Holden D.; Wang, Qian; Loh, Christina; Cekanaviciute, Egle; Buckwalter, Marion; Manning-Boğ, Amy B.

2013-01-01

Prostaglandin E2 (PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP1–4 receptors. Here, we investigated the cell-specific in vivo function of PGE2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2lox/lox mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2lox/lox and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain. PMID:24089506

Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

PubMed

Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

2017-10-03

Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

PubMed Central

Etgen, Anne M.; Dobrenis, Kostantin; Pollard, Jeffrey W.

2011-01-01

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure. PMID:22046273

Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

PubMed

Yang, Long-Long; Zhou, Yan; Tian, Wei-Dong; Li, Hai-Juan; Kang-Chu-Li; Miao, Xia; An, Guang-Zhou; Wang, Xiao-Wu; Guo, Guo-Zhen; Ding, Gui-Rong

2016-01-01

Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1β, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1β, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process. Copyright © 2015 Elsevier Inc. All rights reserved.

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain

PubMed Central

van der Vos, Kristan E.; Abels, Erik R.; Zhang, Xuan; Lai, Charles; Carrizosa, Esteban; Oakley, Derek; Prabhakar, Shilpa; Mardini, Osama; Crommentuijn, Matheus H. W.; Skog, Johan; Krichevsky, Anna M.; Stemmer-Rachamimov, Anat; Mempel, Thorsten R.; El Khoury, Joseph; Hickman, Suzanne E.; Breakefield, Xandra O.

2016-01-01

Background To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. Methods Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. Results Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. Conclusions Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs. PMID:26433199

Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Jiamin

Arsenic is a widely distributed toxic metalloid all over the world. Inorganic arsenic species are supposed to affect astrocytic functions and to cause neuron apoptosis in CNS. Microglias are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA, we showed that Arsenic trioxide up-regulated the expression and secretion of IL-1β in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. The secretion of IL-1β caused the apoptosis of SH-SY5Y. These pro-inflammatory responses were inhibited by the STAT3 blocker, AG490 andmore » P38/JNK MAPK blockers SB202190, SP600125. Further, Arsenic trioxide exposure could induce phosphorylation and activation of STAT3, and the translocation of STAT3 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the STAT3 signaling pathway can be activated after Arsenic trioxide treatment. However, P38/JNK MAPK blockers SB202190, SP600125 also obviously attenuated STAT3 activation and transnuclear transport induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of IL-1β and STAT3 activation induced by Arsenic trioxide can be mediated by elevation of P38/JNK MAPK in HAPI microglia cells and then induced the toxicity of neurons. - Highlights: • Arsenic trioxide exposure induced expression of IL-β in HAPI microglia. • Arsenic trioxide exposure induced activation of MAPK pathways in HAPI microglia. • Arsenic trioxide exposure induced activation of STAT3 pathways in HAPI microglia. • The expression of IL-β though P38/JNK MAPK/STAT3 pathways in HAPI microglia.« less

Microglial K+ Channel Expression in Young Adult and Aged Mice

PubMed Central

Schilling, Tom; Eder, Claudia

2015-01-01

The K+ channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K+ channels and their regulation in vivo is limited. Here, we have investigated K+ currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K+ currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K+ currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K+ current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K+ currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK-type, but not IK- or SK-type, Ca2+-activated K+ channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K+ channel pattern nor K+ channel expression levels differed markedly between the three brain regions investigated. It is concluded that age-related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage-activated, but not Ca2+-activated, K+ channels. PMID:25472417

Cotinine: A Therapy for Memory Extinction in Post-traumatic Stress Disorder.

PubMed

Mendoza, Cristhian; Barreto, George E; Iarkov, Alexandre; Tarasov, Vadim V; Aliev, Gjumrakch; Echeverria, Valentina

2018-01-15

Post-traumatic stress disorder (PTSD) is a mental disorder that may develop after exposure to exceptionally threatening or unescapable horrifying events. Actual therapies fail to alleviate the emotional suffering and cognitive impairment associated with this disorder, mostly because they are ineffective in treating the failure to extinguish trauma memories in a great percentage of those affected. In this review, current behavioral, cellular, and molecular evidence supporting the use of cotinine for treating PTSD are reviewed. The role of the positive modulation by cotinine of the nicotinic acetylcholine receptors (nAChRs) and their downstream effectors, the protection of astroglia, and the inhibition of microglia in the PTSD brain are also discussed.

Functionally charged nanosize particles differentially activate BV2 microglia.

EPA Science Inventory

The effect of particle surface charge on the biological activation of immortalized mouse microglia (BV2) was examined. Nanosize (860-950 nm) spherical polystyrene microparticles (SPM) were coated with carboxyl (COOH-) or dimethyl amino (CH3)2-N- groups to give a net negative or p...

Microglia, seen from the CX3CR1 angle

PubMed Central

Wolf, Yochai; Yona, Simon; Kim, Ki-Wook; Jung, Steffen

2013-01-01

Microglial cells in brain and spinal cord are characterized by high expression of the chemokine receptor CX3CR1. Expression of the sole CX3CR1 ligand, the membrane-tethered and sheddable chemokine CX3CL1/fractalkine, is restricted in the brain parenchyma to selected neurons. Here we summarize our current understanding of the physiological role of CX3CR1 for microglia function and the CX3C axis in microglial/neuronal crosstalk in homeostasis and under challenge. Moreover, we will discuss the efforts of our laboratory and others to exploit CX3CR1 promoter activity for the visualization and genetic manipulation of microglia to probe their functional contributions in the central nerve system (CNS) context. PMID:23507975

Learned helplessness activates hippocampal microglia in rats: A potential target for the antidepressant imipramine.

PubMed

Iwata, Masaaki; Ishida, Hisahito; Kaneko, Koichi; Shirayama, Yukihiko

An accumulating body of evidence has demonstrated that inflammation is associated with the pathology of depression. We recently found that psychological stress induces inflammation in the hippocampus of the rat brain through the inflammasome, a component of the innate immune system. Microglia, the resident macrophages in the brain, play a central role in the innate immune system and express inflammasomes; thus, we hypothesized that hippocampal microglia would be key mediators in the development of depression via stress-induced inflammation. To test this hypothesis and to determine how antidepressants modulate microglial function, we used immunohistochemistry to examine the morphological changes that occur in the hippocampal microglia of rats exposed to the learned helplessness (LH) paradigm. We noted significantly increased numbers of activated microglia in the granule cell layer, hilus, CA1, and CA3 regions of the hippocampi of LH rats. Conversely, administering imipramine to LH rats for 7days produced a significant decrease in the number of activated microglia in the hilus, but not in the other examined regions. Nonetheless, there were no significant differences in the combined number of activated and non-activated microglia either in LH or LH+imipramine rats relative to control rats. In addition, treating the naïve rats with imipramine or fluvoxamine produced no discernible microglial changes. These data suggest that stress activates hippocampal microglia, while certain antidepressants decrease the number of activated microglia in the hilus, but not in other hippocampal regions. Therefore, the hilus represents a candidate target region for the antidepressant imipramine. Copyright © 2016 Elsevier Inc. All rights reserved.

Aβ-Induced Inflammatory Processes in Microglia Cells of APP23 Transgenic Mice

PubMed Central

Bornemann, Klaus D.; Wiederhold, Karl-Heinz; Pauli, Chantal; Ermini, Florian; Stalder, Martina; Schnell, Lisa; Sommer, Bernd; Jucker, Mathias; Staufenbiel, Matthias

2001-01-01

A microglial response is part of the inflammatory processes in Alzheimer’s disease (AD). We have used APP23 transgenic mice overexpressing human amyloid precursor protein with the Swedish mutation to characterize this microglia response to amyloid deposits in aged mice. Analyses with MAC-1 and F4/80 antibodies as well as in vivo labeling with bromodeoxyuridine demonstrate that microglia in the plaque vicinity are in an activated state and that proliferation contributes to their accumulation at the plaque periphery. The amyloid-induced microglia activation may be mediated by scavenger receptor A, which is generally elevated, whereas the increased immunostaining of the receptor for advanced glycation end products is more restricted. Although components of the phagocytic machinery such as macrosialin and Fc receptors are increased in activated microglia, efficient clearance of amyloid is missing seemingly because of the lack of amyloid-bound autoantibodies. Similarly, although up-regulation of major histocompatibility complex class II (IA) points toward an intact antigen-presenting function of microglia, lack of T and B lymphocytes does not indicate a cell-mediated immune response in the brains of APP23 mice. The similar characteristics of microglia in the APP23 mice and in AD render the mouse model suitable to study the role of inflammatory processes during AD pathogenesis. PMID:11141480

Exploring the role of microglia in cortical spreading depression in neurological disease

PubMed Central

Suzuki, Norihiro

2017-01-01

Microglia play a pivotal role in innate immunity in the brain. During development, they mature from myeloerythroid progenitor cells in the yolk sac and colonize the brain to establish a resident population of tissue macrophages. In the postnatal brain, they exert phagocytosis and induce inflammatory response against invading pathogens. Microglia also act as guardians of brain homeostasis by surveying the microenvironment using motile processes. Cortical spreading depression (CSD) is a slowly propagating (2–5 mm/min) wave of rapid, near-complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons. Not only has CSD been implicated in brain migraine aura, but CSD-like events have also been detected in stroke and traumatic injury. CSD causes a considerable perturbation of the ionic environment in the brain, which may be readily detected by microglia. Although CSD is known to activate microglia, the role of microglial activation in CSD-related neurological disorders remains poorly understood. In this article, we first provide an overview of microglial development and the multiple functions of microglia. Then, we review existing data on the relationship between microglia and CSD and discuss the relevance of CSD-induced microglial activation in neurological disease. PMID:28155572

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia.

PubMed

Kim, S J; Li, Jianrong

2013-07-11

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.

Thrombin-induced microglial activation impairs hippocampal neurogenesis and spatial memory ability in mice.

PubMed

Yang, Yuan; Zhang, Meikui; Kang, Xiaoni; Jiang, Chen; Zhang, Huan; Wang, Pei; Li, Jingjing

2015-09-26

To investigate the effects of microglia/macrophages activation induced by intrastriatal thrombin injection on dentate gyrus neurogenesis and spatial memory ability in mice. The male C57BL/6 mice were divided into 4 groups of 10: sham, intracerebral hemorrhage (ICH), ICH + hirudin (thrombin inhibitor), and ICH + indometacin (Indo, an anti-inflammation drug). ICH model was created by intrastriatal thrombin (1U) injection. BrdU (50 mg/kg) was administrated on the same day after surgery for 6 consecutive days. Motor functions were evaluated with rotarod and beam walking tests. The spatial memory deficit was measured with Morris water maze (MWM). Cell quantification was performed for doublecortin (DCX, immature neuron), BrdU (S-phase proliferating cell population) and CD68 (activated microglia/macrophage) immune-reactive cells. Microglia/macrophages activation induced by intrastriatal thrombin injection reduced hippocampal neurogenesis and impaired spatial memory ability, but did not affect the motor function at 3 and 5 days post-injury. Both hirudin and indometacin reduced microglia/macrophages activation, enhanced hippocampal neurogenesis, and improved spatial memory ability in mice. Microglia/macrophages activation induced by intrastriatal thrombin injection might be responsible for the spatial memory deficit. Targeting both thrombin and inflammation systems in acute phase of ICH might be important in alleviating the significant spatial memory deficits.

Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage.

PubMed

Thomas, David M; Francescutti-Verbeem, Dina M; Kuhn, Donald M

2006-03-01

Microglia are the resident antigen-presenting cells within the central nervous system (CNS), and they serve immune-like functions in protecting the brain against injury and invading pathogens. By contrast, activated microglia can secrete numerous reactants that damage neurons. The pathogenesis of various neurodegenerative diseases has been associated with microglial activation, but the signaling pathways that program a neuronally protective or destructive phenotype in microglia are not known. To increase the understanding of microglial activation, microarray analysis was used to profile the transcriptome of BV-2 microglial cells after activation. Microglia were activated by lipopolysaccharide, the HIV neurotoxic protein TAT, and dopamine quinone, each of which has been linked to dopamine neuronal damage. We identified 210 of 9882 genes whose expression was differentially regulated by all activators (116 increased and 94 decreased in expression). Gene ontology analysis assigned up-regulated genes to a number of specific biological processes and molecular functions, including immune response, inflammation, and cytokine/chemokine activity. Genes down-regulated in expression contribute to conditions that are permissive of microglial migration, lowered adhesion to matrix, lessened phagocytosis, and reduction in receptors that oppose chemotaxis and inflammation. These results elaborate a broad profile of microglial genes whose expression is altered by conditions associated with both neurodegenerative diseases and microglial activation.

Functionally Charged Polystyrene Particles Activate Immortalized Mouse Microglia (BV2): Cellular and Genomic Response

EPA Science Inventory

The effect of particle surface charge on the biological activation of immortalized mouse microglia (BV2) was examined. Same size (~850-950 nm) spherical polystyrene microparticles (SPM) with net negative (carboxyl, COOH-) or positive (dimethyl amino, CH₃)₂

Microglia show altered morphology and reduced arborization in human brain during aging and Alzheimer's disease.

PubMed

Davies, Danielle S; Ma, Jolande; Jegathees, Thuvarahan; Goldsbury, Claire

2017-11-01

Changes in microglia function are involved in Alzheimer's disease (AD) for which ageing is the major risk factor. We evaluated microglial cell process morphologies and their gray matter coverage (arborized area) during ageing and in the presence and absence of AD pathology in autopsied human neocortex. Microglial cell processes were reduced in length, showed less branching and reduced arborized area with aging (case range 52-98 years). This occurred during normal ageing and without microglia dystrophy or changes in cell density. There was a larger reduction in process length and arborized area in AD compared to aged-matched control microglia. In AD cases, on average, 49%-64% of microglia had discontinuous and/or punctate Iba1 labeled processes instead of continuous Iba1 distribution. Up to 16% of aged-matched control microglia displayed discontinuous or punctate features. There was no change in the density of microglial cell bodies in gray matter during ageing or AD. This demonstrates that human microglia show progressive cell process retraction without cell loss during ageing. Additional changes in microglia occur with AD including Iba1 protein puncta and discontinuity. We suggest that reduced microglial arborized area may be an aging-related correlate of AD in humans. These variations in microglial cells during ageing and in AD could reflect changes in neural-glial interactions which are emerging as key to mechanisms involved in ageing and neurodegenerative disease. © 2016 International Society of Neuropathology.

Overexpression of SIRT1 Induced by Resveratrol and Inhibitor of miR-204 Suppresses Activation and Proliferation of Microglia.

PubMed

Li, Lihong; Sun, Qiang; Li, Yuqian; Yang, Yang; Yang, Yanlong; Chang, Tao; Man, Minghao; Zheng, Longlong

2015-08-01

Microglia activation plays an important role in neuroinflammation. Sirtuin1 (SIRT1) has been shown to play a role in regulation of inflammation. Resveratrol, a potent SIRT1 activator, has anti-inflammation property. MicroRNA (miRNA or miR) related to inflammation pathways has been shown to be a promising therapeutic approach for septic encephalopathy (SE). The miR mediated mechanism of regulation of SIRT1 expression in encephalitis. However, the mechanism of was unknown. To address this question, we investigated whether miRNAs and resveratrol regulate the SIRT1 and the functional changes of mice microglia cell lines pre-treated with or without lipopolysaccharide (LPS). The research about direct role of miR-204 and resveratrol on expression of SIRT1 in mice microglia cell lines (N9 and BV2) pre-treated with or without LPS had been performed. Mice microglia cell lines were transfected with miR-204 mimics and inhibitors or treated with resveratrol, and the effects on cell growth, proliferation, and apoptosis of cells were assessed. LPS induced inflammation and activation of mice microglia. Through overexpression of SIRT1, resveratrol, and inhibitor of miR-204 inhibited inflammation process, proliferation of mice microglia cells and promoted its apoptosis. We identified if resveratrol and miR-204 could repress inflammation process and proliferation of mice microglia cell through promoting the expression of SIRT1.

Early Functional Deficit and Microglial Disturbances in a Mouse Model of Amyotrophic Lateral Sclerosis

PubMed Central

Rabano, Miriam; Vivanco, Maria d M; Perrin, Florence Evelyne

2012-01-01

Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by selective motoneurons degeneration. There is today no clear-cut pathogenesis sequence nor any treatment. However growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. To better characterize the time course of pathological events in an animal model that recapitulates human ALS symptoms, we investigated functional and cellular characteristics of hSOD1G93A mice. Methods and Findings We have evaluated locomotor function of hSOD1G93A mice through dynamic walking patterns and spontaneous motor activity analysis. We detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. Moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. To tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. We show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre- and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. At pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. Conclusions In conclusion, precise motor analysis updates the onset of the disease in hSOD1G93A mice and allows locomotor monitoring until the end stage of the disease. Early functional deficits coincide with alterations of neuromuscular junctions. Importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. This finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. PMID:22558300

Spinal but not cortical microglia acquire an atypical phenotype with high VEGF, galectin-3 and osteopontin, and blunted inflammatory responses in ALS rats

PubMed Central

Nikodemova, Maria; Small, Alissa L.; Smith, Stephanie M.C.; Mitchell, Gordon S.; Watters, Jyoti J.

2014-01-01

Activation of microglia, CNS resident immune cells, is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons. Despite evidence that microglia contribute to disease progression, the exact role of these cells in ALS pathology remains unknown. We immunomagnetically isolated microglia from different CNS regions of SOD1G93A rats at three different points in disease progression: presymptomatic, symptom onset and end-stage. We observed no differences in microglial number or phenotype in presymptomatic rats compared to wild-type controls. Although after disease onset there was no macrophage infiltration, there were significant increases in microglial numbers in the spinal cord, but not cortex. At disease end-stage, microglia were characterized by high expression of galectin-3, osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6, BDNF and arginase-1. Flow cytometry revealed the presence of at least two phenotypically distinct microglial populations in the spinal cord. Immunohistochemistry showed that galectin-3/osteopontin positive microglia were restricted to the ventral horns of the spinal cord, regions with severe motor neuron degeneration. End-stage SOD1G93A microglia from the cortex, a less affected region, displayed similar gene expression profiles to microglia from wild-type rats, and displayed normal responses to systemic inflammation induced by LPS. On the other hand, end-stage SOD1G93A spinal microglia had blunted responses to systemic LPS suggesting that in addition to their phenotypic changes, they may also be functionally impaired. Thus, after disease onset, microglia acquired unique characteristics that do not conform to typical M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was observed only in the most affected CNS regions, suggesting that overexpression of mutated hSOD1 is not sufficient to trigger these changes in microglia. These novel observations suggest that microglial regional and phenotypic heterogeneity may be an important consideration when designing new therapeutic strategies targeting microglia and neuroinflammation in ALS. PMID:24269728

Single-wall carbon nanohorns inhibited activation of microglia induced by lipopolysaccharide through blocking of Sirt3

NASA Astrophysics Data System (ADS)

Li, Lihong; Zhang, Jinqian; Yang, Yang; Wang, Qiang; Gao, Li; Yang, Yanlong; Chang, Tao; Zhang, Xingye; Xiang, Guoan; Cao, Yongmei; Shi, Zujin; Zhao, Ming; Gao, Guodong

2013-02-01

Single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate in cytotoxic levels within organs of various animal models and cell types, which emerge as a wide range of promising biomedical imaging. Septic encephalopathy (SE) is an early sign of sepsis and associated with an increased rate of morbidity and mortality. Microglia activation plays an important role in neuroinflammation, which contributes to neuronal damage. Inhibition of microglia activation may have therapeutic benefits, which can alleviate the progression of neurodegeneration. Therefore, we investigated the functional changes of mice microglia cell lines pre-treated with or without lipopolysaccharide (LPS) induced by SWNHs. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of microglia cell lines in mice (N9 and BV2) pre-treated with or without LPS had been performed. Our results indicate that the particle diameter of SWNHs in water is between 342 to 712 nm. The images in scanning electron microscope showed that SWNHs on polystyrene surface are individual particles. LPS induced activation of mice microglia, promoted its growth and proliferation, and inhibited its apoptosis. SWNHs inhibited proliferation, delayed mitotic entry, and promoted apoptosis of mice microglia cells. The effects followed gradually increasing cultured time and concentrations of SWNHs, especially in cells pre-treated with LPS. SWNHs induced a significantly increase in G1 phase and inhibition of S phase of mice microglia cells in a dose-manner dependent of SWNHs, especially in cells pre-treated with LPS. The transmission electron microscope images showed that individual spherical SWNH particles smaller than 100 nm in diameters were localized inside lysosomes of mice microglia cells. SWNHs inhibited mitotic entry, growth and proliferation of mice microglia cells, and promoted its apoptosis, especially in cells pre-treated with LPS. SWNHs inhibited expression of Sirt3 and energy metabolism related with Sirt3 in mice microglia cells in a dose-dependent manner, especially in cells pre-treated with LPS. The role of SWNHs on mice microglia was implicating Sirt3 and energy metabolism associated with it.

Microglial Dysfunction in Brain Aging and Alzheimer’s Disease

PubMed Central

Mosher, Kira Irving; Wyss-Coray, Tony

2014-01-01

Microglia, the immune cells of the central nervous system, have long been a subject of study in the Alzheimer’s disease (AD) field due to their dramatic responses to the pathophysiology of the disease. With several large-scale genetic studies in the past year implicating microglial molecules in AD, the potential significance of these cells has become more prominent than ever before. As a disease that is tightly linked to aging, it is perhaps not entirely surprising that microglia of the AD brain share some phenotypes with aging microglia. Yet the relative impacts of both conditions on microglia are less frequently considered in concert. Furthermore, microglial “activation” and “neuroinflammation” are commonly analyzed in studies of neurodegeneration but are somewhat ill-defined concepts that in fact encompass multiple cellular processes. In this review, we have enumerated six distinct functions of microglia and discuss the specific effects of both aging and AD. By calling attention to the commonalities of these two states, we hope to inspire new approaches for dissecting microglial mechanisms. PMID:24445162

Polysaccharides from Ganoderma lucidum attenuate microglia-mediated neuroinflammation and modulate microglial phagocytosis and behavioural response.

PubMed

Cai, Qing; Li, Yuanyuan; Pei, Gang

2017-03-24

Ganoderma lucidum (GL) has been widely used in Asian countries for hundreds of years to promote health and longevity. The pharmacological functions of which had been classified, including the activation of innate immune responses, suppression of tumour and modulation of cell proliferations. Effective fractions of Ganoderma lucidum polysaccharides (GLP) had already been reported to regulate the immune system. Nevertheless, the role of GLP in the microglia-mediated neuroinflammation has not been sufficiently elucidated. Further, GLP effect on microglial behavioural modulations in correlation with the inflammatory responses remains to be unravelled. The aim of this work was to quantitatively analyse the contributions of GLP on microglia. The BV2 microglia and primary mouse microglia were stimulated by lipopolysaccharides (LPS) and amyloid beta 42 (Aβ 42 ) oligomer, respectively. Investigation on the effect of GLP was carried by quantitative determination of the microglial pro- and anti-inflammatory cytokine expressions and behavioural modulations including migration, morphology and phagocytosis. Analysis of microglial morphology and phagocytosis modulations was confirmed in the zebrafish brain. Quantitative results revealed that GLP down-regulates LPS- or Aβ-induced pro-inflammatory cytokines and promotes anti-inflammatory cytokine expressions in BV-2 and primary microglia. In addition, GLP attenuates inflammation-related microglial migration, morphological alterations and phagocytosis probabilities. We also showed that modulations of microglial behavioural responses were associated with MCP-1 and C1q expressions. Overall, our study provides an insight into the GLP regulation of LPS- and Aβ-induced neuroinflammation and serves an implication that the neuroprotective function of GLP might be achieved through modulation of microglial inflammatory and behavioural responses.

The temporal profile of the reaction of microglia, astrocytes, and macrophages in the delayed onset paraplegia after transient spinal cord ischemia in rabbits.

PubMed

Matsumoto, Satoshi; Matsumoto, Mishiya; Yamashita, Atsuo; Ohtake, Kazunobu; Ishida, Kazuyoshi; Morimoto, Yasuhiro; Sakabe, Takefumi

2003-06-01

In the present study, we sought to elucidate the temporal profile of the reaction of microglia, astrocytes, and macrophages in the progression of delayed onset motor dysfunction after spinal cord ischemia (15 min) in rabbits. At 2, 4, 8, 12, 24, and 48 h after reperfusion (9 animals in each), hind limb motor function was assessed, and the lumbar spinal cord was histologically examined. Delayed motor dysfunction was observed in most animals at 48 h after ischemia, which could be predicted by a poor recovery of segmental spinal cord evoked potentials at 15 min of reperfusion. In the gray matter of the lumbar spinal cord, both microglia and astrocytes were activated early (2 h) after reperfusion. Microglia were diffusely activated and engulfed motor neurons irrespective of the recovery of segmental spinal cord evoked potentials. In contrast, early astrocytic activation was confined to the area where neurons started to show degeneration. Macrophages were first detected at 8 h after reperfusion and mainly surrounded the infarction area later. Although the precise roles of the activation of microglia, astrocytes, and macrophages are to be further determined, the results indicate that understanding functional changes of astrocytes may be important in the mechanism of delayed onset motor dysfunction including paraplegia. Microglia and macrophages play a role in removing tissue debris after transient spinal cord ischemia. Disturbance of astrocytic defense mechanism, breakdown of the blood-spinal cord barrier, or both seemed to be involved in the development of delayed motor dysfunction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fanarraga, M.L.; Villegas, J.C.; Carranza, G.

Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulatedmore » in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.« less

Characterization of phenotype markers and neuronotoxic potential of polarised primary microglia in vitro

PubMed Central

Chhor, Vibol; Le Charpentier, Tifenn; Lebon, Sophie; Oré, Marie-Virgine; Celador, Idoia Lara; Josserand, Julien; Degos, Vincent; Jacotot, Etienne; Hagberg, Henrik; Sävman, Karin; Mallard, Carina; Gressens, Pierre; Fleiss, Bobbi

2013-01-01

Microglia mediate multiple facets of neuroinflammation, including cytotoxicity, repair, regeneration, and immunosuppression due to their ability to acquire diverse activation states, or phenotypes. Modulation of microglial phenotype is an appealing neurotherapeutic strategy but a comprehensive study of classical and more novel microglial phenotypic markers in vitro is lacking. The aim of this study was to outline the temporal expression of a battery of phenotype markers from polarised microglia to generate an in vitro tool for screening the immunomodulatory potential of novel compounds. We characterised expression of thirty-one macrophage/microglial phenotype markers in primary microglia over time (4, 12, 36, and 72 h), using RT-qPCR or multiplex protein assay. Firstly, we selected Interleukin-4 (IL-4) and lipopolysaccharide (LPS) as the strongest M1–M2 polarising stimuli, from six stimuli tested. At each time point, markers useful to identify that microglia were M1 included iNOS, Cox-2 and IL-6 and a loss of M2a markers. Markers useful for quantifying M2b-immunomodulatory microglia included, increased IL-1RA and SOCS3 and for M2a-repair and regeneration, included increased arginase-1, and a loss of the M1 and M2b markers were discriminatory. Additional markers were regulated at fewer time points, but are still likely important to monitor when assessing the immunomodulatory potential of novel therapies. Further, to facilitate identification of how novel immunomodulatory treatments alter the functional affects of microglia, we characterised how the soluble products from polarised microglia affected the type and rate of neuronal death; M1/2b induced increasing and M2a-induced decreasing neuronal loss. We also assessed any effects of prior activation state, to provide a way to identify how a novel compound may alter phenotype depending on the stage of injury/insult progression. We identified generally that a prior M1/2b reduced the ability of microglia to switch to M2a. Altogether, we have characterised a profile of phenotype markers and a mechanism of assessing functional outcome that we can use as a reference guide for first-line screening of novel immunomodulatory therapies in vitro in the search for viable neuroprotectants. PMID:23454862

Curcumin is a potent modulator of microglial gene expression and migration

PubMed Central

2011-01-01

Background Microglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin. Methods Resting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. Results Curcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures. Conclusions Collectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders. PMID:21958395

Cellular and molecular identity of tumor-associated macrophages in glioblastoma

PubMed Central

Chen, Zhihong; Feng, Xi; Herting, Cameron J.; Garcia, Virginia Alvarez; Nie, Kai; Pong, Winnie W.; Rasmussen, Rikke; Dwivedi, Bhakti; Seby, Sandra; Wolf, Susanne A.; Gutmann, David H.; Hambardzumyan, Dolores

2017-01-01

In glioblastoma (GBM), tumor-associated macrophages (TAM) represent up to one half of the cells of the tumor mass, including both infiltrating macrophages and resident brain microglia. In an effort to delineate the temporal and spatial dynamics of TAM composition during gliomagenesis, we employed two genetically engineered mouse models where oncogenic drivers and fluorescent reporters were expressed coordinately under the control of the monocyte/microglia-selective Cx3cr1 or Ccr2 promoters, respectively. Using this approach, we demonstrated that CX3CR1LoCCR2Hi monocytes were recruited to the glioblastoma, where they transitioned to CX3CR1HiCCR2Lo macrophages and CX3CR1HiCCR2− microglia-like cells. Infiltrating macrophages/monocytes constituted ~85% of the total TAM population, with resident microglia accounting for the ~15% remaining. Bone marrow-derived infiltrating macrophages/monocytes were recruited to the tumor early during GBM initiation, where they localized preferentially to perivascular areas. In contrast, resident microglia were localized mainly to peritumoral regions. RNA-sequencing analyses revealed differential gene expression patterns unique to infiltrating and resident cells, suggesting unique functions for each TAM population. Notably, limiting monocyte infiltration via Ccl2 genetic ablation prolonged the survival of tumor-bearing mice. Our findings illuminate the unique composition and functions of infiltrating and resident myeloid cells in GBM, establishing a rationale to target infiltrating cells in this neoplasm. PMID:28235764

Microglia of the Aged Brain: Primed to be Activated and Resistant to Regulation

PubMed Central

Norden, Diana M.; Godbout, Jonathan P.

2012-01-01

Innate immunity within the central nervous system (CNS) is primarily provided by resident microglia. Microglia are pivotal in immune surveillance and also facilitate the coordinated responses between the immune system and the brain. For example, microglia interpret and propagate inflammatory signals that are initiated in the periphery. This transient microglial activation helps mount the appropriate physiological and behavioral response following peripheral infection. With normal aging, however, microglia develop a more inflammatory phenotype. For instance, in several models of aging there are increased pro-inflammatory cytokines in the brain and increased expression of inflammatory receptors on microglia. This increased inflammatory status of microglia with aging is referred to as primed, reactive, or sensitized. A modest increase in the inflammatory profile of the CNS and altered microglial function in aging has behavioral and cognitive consequences. Nonetheless, there are major differences in microglial biology between young and old age when the immune system is challenged and microglia are activated. In this context, microglial activation is amplified and prolonged in the aged brain compared to adults. The cause of this amplified microglial activation may be related to impairments in several key regulatory systems with age that make it more difficult to resolve microglial activation. The consequences of impaired regulation and microglial hyper-activation following immune challenge are exaggerated neuroinflammation, sickness behavior, depressive-like behavior and cognitive deficits. Therefore the purpose of this review is to discuss the current understanding of age-associated microglial priming, consequences of priming and reactivity, and the impairments in regulatory systems that may underlie these age-related deficits. PMID:23039106

Dental X-ray exposure and Alzheimer's disease: a hypothetical etiological association.

PubMed

Rodgers, Caroline C

2011-07-01

Despite the fact that Alzheimer's disease was identified more than 100 years ago, its cause remains elusive. Although the chance of developing Alzheimer's disease increases with age, it is not a natural consequence of aging. This article proposes that dental X-rays can damage microglia telomeres - the structures at the end of chromosomes that determine how many times cells divide before they die - causing them to age prematurely. Degenerated microglia lose their neuroprotective properties, resulting in the formation of neurofibrillary tau tangles and consequently, the neuronal death that causes Alzheimer's dementia. The hypothesis that Alzheimer's is caused specifically by microglia telomere damage would explain the delay of one decade or longer between the presence of Alzheimer's brain pathology and symptoms; telomere damage would not cause any change in microglial function, it would just reset the countdown clock so that senescence and apoptosis occurred earlier than they would have without the environmental insult. Once microglia telomere damage causes premature aging and death, the adjacent neurons are deprived of the physical support, maintenance and nourishment they require to survive. This sequence of events would explain why therapies and vaccines that eliminate amyloid plaques have been unsuccessful in stopping dementia. Regardless of whether clearing plaques is beneficial or harmful - which remains a subject of debate - it does not address the failing microglia population. If microglia telomere damage is causing Alzheimer's disease, self-donated bone marrow or dental pulp stem cell transplants could give rise to new microglia populations that would maintain neuronal health while the original resident microglia population died. Copyright © 2011 Elsevier Ltd. All rights reserved.

Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge

PubMed Central

Gonzalez-Pena, Dianelys; Nixon, Scott E.; O’Connor, Jason C.; Southey, Bruce R.; Lawson, Marcus A.; McCusker, Robert H.; Borras, Tania; Machuca, Debbie; Hernandez, Alvaro G.; Dantzer, Robert; Kelley, Keith W.; Rodriguez-Zas, Sandra L.

2016-01-01

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis. PMID:26959683

Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge.

PubMed

Gonzalez-Pena, Dianelys; Nixon, Scott E; O'Connor, Jason C; Southey, Bruce R; Lawson, Marcus A; McCusker, Robert H; Borras, Tania; Machuca, Debbie; Hernandez, Alvaro G; Dantzer, Robert; Kelley, Keith W; Rodriguez-Zas, Sandra L

2016-01-01

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis.

Increased expression of T cell immunoglobulin and mucin domain 3 aggravates brain inflammation via regulation of the function of microglia/macrophages after intracerebral hemorrhage in mice.

PubMed

Xu, ChangJun; Wang, Tao; Cheng, Si; Liu, YuGuang

2013-12-01

Microglia/macrophages are known to play important roles in initiating brain inflammation after spontaneous intracerebral hemorrhage (ICH). T cell immunoglobulin and mucin domain-3 (Tim-3) have been proven to play a critical part in several inflammatory diseases through regulation of both adaptive and innate immune responses. Tim-3 can be expressed by microglia/macrophages and regulates their function in the innate immune response. However, the effect of Tim-3 on inflammatory responses following ICH is unclear. In this study, we investigated Tim-3 expression, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and brain water content in peri-hematomal brain tissue at 12 hours and at 1, 3, 5, and 7 days post-ICH in wild type (WT) ICH and Tim-3-/- ICH mice. The numbers of Tim-3 positive cells,astrocytes, neutrophils and microglia/macrophages were detected using immunofluorescence staining. Cytokines were measured by ELISA. Double immunofluorescence labeling was performed to identify the cellular source of Tim-3 expression. Mouse neurological deficit scores were assessed through animal behavior. Expression of Tim-3 increased early in mouse peri-hematomal brain tissue after autologous blood injection, peaked at day 1, and was positively correlated with the concentrations of TNF-α, IL-1β, and brain water content. Tim-3 was predominantly expressed in microglia/macrophages. Compared with WT mice, Tim-3-/- mice had reduced ICH-induced brain inflammation with decreased TNF-α and IL-1β, cerebral edema and neurological deficit scores. Moreover, Tim-/- inhibited activation of microglia/macrophages. The number of activated microglia/macrophages in Tim-3-/- ICH mice was much lower than that in WT ICH mice. Our findings demonstrate that Tim-3 plays an important role in brain inflammation after ICH, and may be a potential treatment target.

Effects of 3,3',5-triiodothyronine on microglial functions.

PubMed

Mori, Yuki; Tomonaga, Daichi; Kalashnikova, Anastasia; Furuya, Fumihiko; Akimoto, Nozomi; Ifuku, Masataka; Okuno, Yuko; Beppu, Kaoru; Fujita, Kyota; Katafuchi, Toshihiko; Shimura, Hiroki; Churilov, Leonid P; Noda, Mami

2015-05-01

L-tri-iodothyronine (3, 3', 5-triiodothyronine; T3) is an active form of the thyroid hormone (TH) essential for the development and function of the CNS. Though nongenomic effect of TH, its plasma membrane-bound receptor, and its signaling has been identified, precise function in each cell type of the CNS remained to be investigated. Clearance of cell debris and apoptotic cells by microglia phagocytosis is a critical step for the restoration of damaged neuron-glia networks. Here we report nongenomic effects of T3 on microglial functions. Exposure to T3 increased migration, membrane ruffling and phagocytosis of primary cultured mouse microglia. Injection of T3 together with stab wound attracted more microglia to the lesion site in vivo. Blocking TH transporters and receptors (TRs) or TRα-knock-out (KO) suppressed T3-induced microglial migration and morphological change. The T3-induced microglial migration or membrane ruffling was attenuated by inhibiting Gi /o -protein as well as NO synthase, and subsequent signaling such as phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). Inhibitors for Na(+) /K(+) -ATPase, reverse mode of Na(+) /Ca(2+) exchanger (NCX), and small-conductance Ca(2+) -dependent K(+) (SK) channel also attenuated microglial migration or phagocytosis. Interestingly, T3-induced microglial migration, but not phagocytosis, was dependent on GABAA and GABAB receptors, though GABA itself did not affect migratory aptitude. Our results demonstrate that T3 modulates multiple functional responses of microglia via multiple complex mechanisms, which may contribute to physiological and/or pathophysiological functions of the CNS. © 2015 Wiley Periodicals, Inc.

Microglial Fc Receptors Mediate Physiological Changes Resulting From Antibody Cross-Linking of Myelin Oligodendrocyte Glycoprotein

PubMed Central

Marta, Cecilia B.; Bansal, Rashmi; Pfeiffer, Steven E.

2009-01-01

Antibodies to myelin oligodendrocyte glycoprotein (MOG) have been implicated in Multiple Sclerosis demyelination through activation of complement and/or macrophage-effector processes. We presented a novel mechanism, whereby MOG on oligodendrocytes, when cross-linked with anti-MOG and secondary antibody resulted in its repartitioning into lipid rafts, and changes in protein phosphorylation and morphology. Here, we show that similar events occur when anti-MOG is cross-linked with Fc receptors (FcRs) present on microglia but not with complement. These results indicate that FcRs are endogenous antigen/antibody cross-linkers in vitro, suggesting that FcRs could be physiologically relevant in vivo and possible targets for therapy in Multiple Sclerosis. PMID:18406472

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

PubMed Central

Caldeira, Cláudia; Cunha, Carolina; Vaz, Ana R.; Falcão, Ana S.; Barateiro, Andreia; Seixas, Elsa; Fernandes, Adelaide; Brites, Dora

2017-01-01

Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages. PMID:28912710

Effector CD4 cell tolerization is mediated through functional inactivation and involves preferential impairment of TNF-α and IFN-γ expression potentials

PubMed Central

Long, Meixiao; Higgins, Amy D.; Mihalyo, Marianne A.; Adler, Adam J.

2010-01-01

It has recently been shown that effector/memory T cells can undergo peripheral tolerization in response to self-antigen. In the present study, we found that within 24 h self-antigen profoundly impairs the ability of CD4 effectors to express TNF-α (and to a lesser extent IFN-γ); however, several days of self-antigen exposure is required to impair non-effector functions such as IL-2 expression and proliferation. Since only half of the initial effector CD4 cell population expresses effector cytokines following brief antigenic stimulation, tolerization might have been mediated either through functional inactivation of effector-competent cells, or alternatively by the selective deletion of competent and expansion of non-competent cells. When briefly stimulated effectors were fractionated based on their expression of IFN-γ, the IFN-γ− sub-population was able to express IFN-γ following secondary stimulation, indicating that all effector CD4 cells are functionally competent. Furthermore, both IFN-γ+ and IFN-γ− sub-populations underwent tolerization in response to self-HA (although the former was slightly more prone to deletion at later time points). Thus, effector CD4 cell tolerization is mediated primarily through the functional inactivation of effector-competent cells. PMID:14609577

Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

PubMed

Mizoguchi, Yoshito; Kato, Takahiro A; Seki, Yoshihiro; Ohgidani, Masahiro; Sagata, Noriaki; Horikawa, Hideki; Yamauchi, Yusuke; Sato-Kasai, Mina; Hayakawa, Kohei; Inoue, Ryuji; Kanba, Shigenobu; Monji, Akira

2014-06-27

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Microglial cell dysregulation in brain aging and neurodegeneration

PubMed Central

von Bernhardi, Rommy; Eugenín-von Bernhardi, Laura; Eugenín, Jaime

2015-01-01

Aging is the main risk factor for neurodegenerative diseases. In aging, microglia undergoes phenotypic changes compatible with their activation. Glial activation can lead to neuroinflammation, which is increasingly accepted as part of the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD). We hypothesize that in aging, aberrant microglia activation leads to a deleterious environment and neurodegeneration. In aged mice, microglia exhibit an increased expression of cytokines and an exacerbated inflammatory response to pathological changes. Whereas LPS increases nitric oxide (NO) secretion in microglia from young mice, induction of reactive oxygen species (ROS) predominates in older mice. Furthermore, there is accumulation of DNA oxidative damage in mitochondria of microglia during aging, and also an increased intracellular ROS production. Increased ROS activates the redox-sensitive nuclear factor kappa B, which promotes more neuroinflammation, and can be translated in functional deficits, such as cognitive impairment. Mitochondria-derived ROS and cathepsin B, are also necessary for the microglial cell production of interleukin-1β, a key inflammatory cytokine. Interestingly, whereas the regulatory cytokine TGFβ1 is also increased in the aged brain, neuroinflammation persists. Assessing this apparent contradiction, we have reported that TGFβ1 induction and activation of Smad3 signaling after inflammatory stimulation are reduced in adult mice. Other protective functions, such as phagocytosis, although observed in aged animals, become not inducible by inflammatory stimuli and TGFβ1. Here, we discuss data suggesting that mitochondrial and endolysosomal dysfunction could at least partially mediate age-associated microglial cell changes, and, together with the impairment of the TGFβ1-Smad3 pathway, could result in the reduction of protective activation and the facilitation of cytotoxic activation of microglia, resulting in the promotion of neurodegenerative diseases. PMID:26257642

An immortalized microglial cell line (Mocha) derived from rat cochlea.

PubMed

Seigel, G M; Manohar, S; Bai, Y Y; Ding, D; Salvi, R

2017-12-01

Microglia are glial-immune cells that are essential for the function and survival of the central nervous system. Microglia not only protect neural tissues from immunological insults, but also play a critical role in neural development and repair. However, little is known about the biology of microglia in the cochlea, the auditory portion of the inner ear. In this study, we detected TMEM119+, CD11b+, CD45+ and Iba1+ populations of cells in the rat cochlea, particularly in Rosenthal's canal, inner sulcus and stria vascularis. Next, we isolated and enriched the population of CD11b+ cells from the cochlea and immortalized these cells with the 12S E1A gene of adenovirus in a replication-incompetent retroviral vector to derive a novel microglial cell line, designated Mocha (microglia of the cochlea). The resulting Mocha cells express a number of markers consistent with microglia and respond to lipopolysaccharide (LPS) stimulation by upregulation of genes (Cox2, ICAM-1, Il6r, Ccl2, Il13Ra and Il15Ra) as well as releasing cytokines (IL-1beta, IL-12, IL-13 and RANTES). As evidence of microglial function, Mocha cells phagocytose fluorescent beads at 37°C, but not at 4°C. The expression pattern of microglial markers in Mocha cells suggests that immortalization leads to a more primitive phenotype, a common phenomenon in immortalized cell lines. In summary, Mocha cells display key characteristics of microglia and are now available as a useful model system for the study of cochlear microglial behavior, both in vitro and in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of glia phenotype modulators based on select glial function regulatory signaling pathways.

PubMed

Lee, Sun-Hwa; Suk, Kyoungho

2018-04-20

Despite the considerable social and economic burden on the healthcare system worldwide due to neurodegenerative diseases, there are currently few disease-altering treatment options for many of these conditions. Therefore, new approaches for both prevention and intervention for neurodegenerative diseases are urgently required. Microglia-mediated neurotoxicity is one of the pathologic hallmarks common to Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Current therapeutic approaches to target microglia-mediated neurotoxicity are focused on the identification of glia phenotype modulators (GPMs), which can inhibit the 'classical' pro-inflammatory and neurotoxic phenotypes of microglia. Areas covered: This article reviews selected microglial molecular targets and pathways involved in either neurotoxicity or neuroprotection and how their identification. Expert opinion: Microglial activation and their signaling pathways have important implications in the neurotoxicity and brain disorders. Pharmacological modulation of microglial activation may serve as a potential therapeutic approach for targeting microglia-mediated neurotoxicity. However, given that microglia change their activation states depending on the timing, stage, and severity of disease, and even aging, the appropriate window should be considered for this approach to be clinically effective. In the future, the identification of unknown extracellular signals and intracellular molecular switches that control phenotypic shifts may facilitate the development of novel therapeutics targeting microglia-mediated neurotoxicity.

Are microglia minding us? Digging up the unconscious mind-brain relationship from a neuropsychoanalytic approach

PubMed Central

Kato, Takahiro A.; Kanba, Shigenobu

2013-01-01

The unconscious mind-brain relationship remains unresolved. From the perspective of neuroscience, neuronal networks including synapses have been dominantly believed to play crucial roles in human mental activities, while glial contribution to mental activities has long been ignored. Recently, it has been suggested that microglia, glial cells with immunological/inflammatory functions, play important roles in psychiatric disorders. Newly revealed microglial roles, such as constant direct contact with synapses even in the normal brain, have defied the common traditional belief that microglia do not contribute to neuronal networks. Recent human neuroeconomic investigations with healthy volunteers using minocycline, an antibiotic with inhibitory effects on microglial activation, suggest that microglia may unconsciously modulate human social behaviors as “noise.” We herein propose a novel unconscious mind structural system in the brain centering on microglia from a neuropsychoanalytic approach. At least to some extent, microglial activation in the brain may activate unconscious drives as “psychological immune memory/reaction” in the mind, and result in various emotions, traumatic reactions, psychiatric symptoms including suicidal behaviors, and (psychoanalytic) transference during interpersonal relationships. Microglia have the potential to bridge the huge gap between neuroscience, biological psychiatry, psychology and psychoanalysis as a key player to connect the conscious and the unconscious world. PMID:23443737

Activation of microglia and astrocytes in the nucleus tractus solitarius during ventilatory acclimatization to 10% hypoxia in unanesthetized mice.

PubMed

Tadmouri, A; Champagnat, J; Morin-Surun, M P

2014-05-01

Nucleus tractus solitarius (NTS) is the integrative sensory relay of autonomic functions in the brainstem. To explore the nonneuronal cellular basis of central chemosensitivity during the first 24 hr of ventilatory acclimatization to hypoxia (VHA), we have investigated glial activation markers in the NTS. Conscious mice (C57/BL6) were placed in a hermetic hypoxia chamber containing a plethysmograph to record ventilation. After 4 days of habituation to the normoxic environment, mice were subjected to physiological hypoxia (10% O2 ) for 1, 6, or 24 hr. To dissociate interactions between microglia and astrocytes, another group received daily minocycline, a microglia activation blocker. By immunochemical localization of astrocytes (GFAP), activated microglia (Cd11b), and total microglia (Iba-1), we identified an oxygen-sensing glial layer in the NTS, in which astrocytes are first activated after 1-6 hr of hypoxia, followed by microglia after 6-24 hr of hypoxia. Minocycline administration suppressed microglial activation and decreased astrocyte activation at 6 hr and VHA at 24 hr of hypoxia. These results suggest that astrocytes contribute to the neuronal response during the first hour of hypoxia, whereas microglial cells, via cross-talk with astrocytes, are involved in the VHA during the first 24 hr of acclimatization. Copyright © 2014 Wiley Periodicals, Inc.

Resveratrol induces dynamic changes to the microglia transcriptome, inhibiting inflammatory pathways and protecting against microglia-mediated photoreceptor apoptosis.

PubMed

Wiedemann, Johanna; Rashid, Khalid; Langmann, Thomas

2018-06-18

Microglia activation is central to the pathophysiology of retinal degenerative disorders. Resveratrol, a naturally occurring non-flavonoid phenolic compound present in red wine has potent anti-inflammatory and immunomodulatory properties. However, molecular mechanisms by which resveratrol influences microglial inflammatory pathways and housekeeping functions remain unclear. Here, we first studied the immuno-modulatory effects of resveratrol on BV-2 microglial cells at the transcriptome level using DNA-microarrays and selected qRT-PCR analyses. We then analyzed resveratrol effects on microglia morphology, phagocytosis and migration and estimated their neurotoxicity on 661 W photoreceptors by quantification of caspase 3/7 levels. We found that resveratrol effectively blocked gene expression of a broad spectrum of lipopolysaccharide (LPS)-induced pro-inflammatory molecules, including cytokines and complement proteins. These transcriptomic changes were accompanied by potent inhibition of LPS-induced nitric oxide secretion and reduced microglia-mediated apoptosis of 661 W photoreceptor cultures. Our findings highlight novel targets involved in the anti-inflammatory and neuroprotective action of resveratrol against neuroinflammatory responses. Copyright © 2018 Elsevier Inc. All rights reserved.

SMAD4 is Involved in the Development of Endotoxin Tolerance in Microglia.

PubMed

Liu, Xiaorong; Qin, Yongwei; Dai, Aihua; Zhang, Yu; Xue, Huaqing; Ni, Haidan; Han, Lijian; Zhu, Liang; Yuan, Debin; Tao, Tao; Cao, Maohong

2016-07-01

Initial exposure of macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Smad4 plays important roles in the induction of LPS tolerance. However, the function of Smad4 in microglia remains unknown. Here we show that expression of Smad4 was highly up-regulated in LPS-tolerized mouse cerebral cortex. Smad4 was mostly colocalized with microglia, rarely with neurons. Using a microglia cell line, BV2, we find that LPS activates endogenous Smad4, inducing its migration into the nucleus and increasing its expression. Smad4 significantly suppressed TLR-triggered production of proinflammatory cytokines (IL-6), increased anti-inflammatory cytokine in LPS-tolerized microglia. Moreover, IL-6 concentrations in culture supernatants after second LPS challenge are higher in SMAD4 small interfering RNA (siRNA) BV2 cells than control siRNA BV2 cells, indicating failure to induce tolerance in absence of Smad4 signaling. In our study, we conclude that both in vivo and in vitro, Smad4 signaling is required for maximal induction of endotoxin tolerance.

Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1

PubMed Central

Zhu, Wen; Carney, Karen E.; Pigott, Victoria M.; Falgoust, Lindsay M.; Clark, Paul A.; Kuo, John S.; Sun, Dandan

2016-01-01

Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na+/H+ exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia–glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1+ microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma–microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation. PMID:27287871

Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

PubMed

Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

2015-12-01

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

Microglia and macrophages in malignant gliomas: recent discoveries and implications for promising therapies.

PubMed

da Fonseca, Anna Carolina Carvalho; Badie, Behnam

2013-01-01

Malignant gliomas are the most common primary brain tumors. Their deadliest manifestation, glioblastoma multiforme (GBM), accounts for 15% of all primary brain tumors and is associated with a median survival of only 15 months even after multimodal therapy. There is substantial presence of microglia and macrophages within and surrounding brain tumors. These immune cells acquire an alternatively activated phenotype with potent tumor-tropic functions that contribute to glioma growth and invasion. In this review, we briefly summarize recent data that has been reported on the interaction of microglia/macrophages with brain tumors and discuss potential application of these findings to the development of future antiglioma therapies.

Microglial ablation and lipopolysaccharide preconditioning affects pilocarpine-induced seizures in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mirrione, M.M.; Mirrione, M.M.; Konomosa, D.K.

2010-04-01

Activated microglia have been associated with neurodegeneration in patients and in animal models of Temporal Lobe Epilepsy (TLE), however their precise functions as neurotoxic or neuroprotective is a topic of significant investigation. To explore this, we examined the effects of pilocarpine-induced seizures in transgenic mice where microglia/macrophages were conditionally ablated. We found that unilateral ablation of microglia from the dorsal hippocampus did not alter acute seizure sensitivity. However, when this procedure was coupled with lipopolysaccharide (LPS) preconditioning (1 mg/kg given 24 h prior to acute seizure), we observed a significant pro-convulsant phenomenon. This effect was associated with lower metabolic activationmore » in the ipsilateral hippocampus during acute seizures, and could be attributed to activity in the mossy fiber pathway. These findings reveal that preconditioning with LPS 24 h prior to seizure induction may have a protective effect which is abolished by unilateral hippocampal microglia/macrophage ablation.« less

Light-induced migration of retinal microglia into the subretinal space.

PubMed

Ng, T F; Streilein, J W

2001-12-01

To explore the effects of light exposure and deprivation on the distribution and function of microglia in the subretinal space of mice. Using a monoclonal antibody, 5D4, that identifies resting, ramified microglia, the distribution and density of microglia in the retina, and the subretinal space were determined by confocal microscopy and by immunohistochemistry of cryopreserved sections of eyes of albino and pigmented mice exposed to diverse levels of light, ranging from complete darkness to intense brightness. Axotomized retinal ganglion cells were retrograde labeled by fluorescent tracer to determine whether the marker colocalizes to 5D4+ cells. Electron microscopy was used to evaluate microglia for evidence of phagocytosis. 5D4+ microglia in pigmented eyes were limited to the inner retinal layers, but in albino eyes 5D4+ cells were found in the outer retinal layers and subretinal space as well. The subretinal space of eyes of albino mice raised from birth in complete darkness contained few 5D4+ cells, but exposure to light caused the rapid accumulation of 5D4+ cells at this site. 5D4+ cell density in the subretinal space correlated directly with intensity of ambient light. Retrograde labeling of axotomized ganglion cells resulted in 5D4+ cells in the subretinal space that contained the retrograde label. Subretinal microglia contained phagocytized rod outer segment discs. On intense light exposure, 5D4+ cells adopted an active morphology, but failed to express class II major histocompatibility complex (MHC) molecules. Light exposure induced retinal microglia migration into the subretinal space in albino mice. Subretinal microglia appeared to augment through phagocytosis the capacity of pigment epithelium to take up the photoreceptor debris of light toxicity. The unexpected presence of these cells in the subretinal space raises questions concerning their potential contribution to immune privilege in this space and to the fate of retinal transplants.

Microglia Morphological Categorization in a Rat Model of Neuroinflammation by Hierarchical Cluster and Principal Components Analysis.

PubMed

Fernández-Arjona, María Del Mar; Grondona, Jesús M; Granados-Durán, Pablo; Fernández-Llebrez, Pedro; López-Ávalos, María D

2017-01-01

It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main points Microglia undergo a quantifiable morphological change upon neuraminidase induced inflammation.Hierarchical cluster and principal components analysis allow morphological classification of microglia.Brain location of microglia is a relevant factor.

Microglia Morphological Categorization in a Rat Model of Neuroinflammation by Hierarchical Cluster and Principal Components Analysis

PubMed Central

Fernández-Arjona, María del Mar; Grondona, Jesús M.; Granados-Durán, Pablo; Fernández-Llebrez, Pedro; López-Ávalos, María D.

2017-01-01

It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main points Microglia undergo a quantifiable morphological change upon neuraminidase induced inflammation.Hierarchical cluster and principal components analysis allow morphological classification of microglia.Brain location of microglia is a relevant factor. PMID:28848398

Neuron-derived IgG protects dopaminergic neurons from insult by 6-OHDA and activates microglia through the FcγR I and TLR4 pathways.

PubMed

Zhang, Jie; Niu, Na; Wang, Mingyu; McNutt, Michael A; Zhang, Donghong; Zhang, Baogang; Lu, Shijun; Liu, Yuqing; Liu, Zhihui

2013-08-01

Oxidative and immune attacks from the environment or microglia have been implicated in the loss of dopaminergic neurons of Parkinson's disease. The role of IgG which is an important immunologic molecule in the process of Parkinson's disease has been unclear. Evidence suggests that IgG can be produced by neurons in addition to its traditionally recognized source B lymphocytes, but its function in neurons is poorly understood. In this study, extensive expression of neuron-derived IgG was demonstrated in dopaminergic neurons of human and rat mesencephalon. With an in vitro Parkinson's disease model, we found that neuron-derived IgG can improve the survival and reduce apoptosis of dopaminergic neurons induced by 6-hydroxydopamine toxicity, and also depress the release of NO from microglia triggered by 6-hydroxydopamine. Expression of TNF-α and IL-10 in microglia was elevated to protective levels by neuron-derived IgG at a physiologic level via the FcγR I and TLR4 pathways and microglial activation could be attenuated by IgG blocking. All these data suggested that neuron-derived IgG may exert a self-protective function by activating microglia properly, and IgG may be involved in maintaining immunity homeostasis in the central nervous system and serve as an active factor under pathological conditions such as Parkinson's disease. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Therapeutic concentration of lithium stimulates complement C3 production in dendritic cells and microglia via GSK-3 inhibition.

PubMed

Yu, Zhiqian; Ono, Chiaki; Aiba, Setsuya; Kikuchi, Yoshie; Sora, Ichiro; Matsuoka, Hiroo; Tomita, Hiroaki

2015-02-01

Evidence indicates that widely prescribed mood stabilizer, lithium (Li), mediates cellular functions of differentiated monocytic cells, including microglial migration, monocyte-derived dendritic cell (MoDC) differentiation, and amelioration of monocytic malfunctions observed in neuropsychiatric diseases. Here, we surveyed molecules which take major roles in regulating these monocytic cellular functions. MoDCs treated with 1 and 5 mM Li, and microglia separated from Li-treated mice were subjected to microarray-based comprehensive gene expression analyses. Findings were validated using multiple experiments, including quantitative PCR, ELISA and immunostaining studies. Differing effects of Li on the two cell types were observed. Inflammation- and chemotaxis-relevant genes were significantly over-represented among Li-induced genes in MoDCs, whereas no specific category of genes was over-represented in microglia. The third component of complement (C3) was the only gene which was significantly induced by a therapeutic concentration of Li in both MoDCs and microglia. C3 production was increased by Li via GSK-3 inhibition. Li-induced C3 production was seen only in differentiated monocytic cells, but not in circulating monocytes. Our findings highlight a link between Li treatment and C3 production in differentiated monocytic cells, and reveal a regulatory role of GSK-3 in C3 production. Induction of microglial C3 production might be a novel neuroprotective mechanism of Li via regulating interactions between microglia and neurons. GLIA 2015;63:257-270. © 2014 Wiley Periodicals, Inc.

Deficiency of leptin receptor in myeloid cells disrupts hypothalamic metabolic circuits and causes body weight increase.

PubMed

Gao, Yuanqing; Vidal-Itriago, Andrés; Milanova, Irina; Korpel, Nikita L; Kalsbeek, Martin J; Tom, Robby Zachariah; Kalsbeek, Andries; Hofmann, Susanna M; Yi, Chun-Xia

2018-01-01

Leptin is a cytokine produced by adipose tissue that acts mainly on the hypothalamus to regulate appetite and energy homeostasis. Previous studies revealed that the leptin receptor is expressed not only in neurons, but also in glial cells. Microglia are resident immune cells in the brain that play an essential role in immune defense and neural network development. Previously we reported that microglial morphology and cytokine production are changed in the leptin receptor deficient db/db mouse, suggesting that leptin's central effects on metabolic control might involve signaling through microglia. In the current study, we aimed to uncover the role of leptin signaling in microglia in systemic metabolic control. We generated a mouse model with leptin receptor deficiency, specifically in the myeloid cells, to determine the role of microglial leptin signaling in the development of metabolic disease and to investigate microglial functions. We discovered that these mice have increased body weight with hyperphagia. In the hypothalamus, pro-opiomelanocortin neuron numbers in the arcuate nucleus (ARC) and α-MSH projections from the ARC to the paraventricular nucleus (PVN) decreased, which was accompanied by the presence of less ramified microglia with impaired phagocytic capacity in the PVN. Myeloid cell leptin receptor deficient mice partially replicate the db/db phenotype. Leptin signaling in hypothalamic microglia is important for microglial function and a correct formation of the hypothalamic neuronal circuit regulating metabolism. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Microglial Function across the Spectrum of Age and Gender

PubMed Central

Nissen, Jillian C.

2017-01-01

Microglia constitute the resident immunocompetent cells of the central nervous system. Although much work has focused on their ability to mount an inflammatory response in reaction to pathology, recent studies have delved into their role in maintaining homeostasis in the healthy brain. It is important to note that the function of these cells is more complex than originally conceived, as there is increasing evidence that microglial responses can vary greatly among individuals. Here, this review will describe the changing behavior of microglia from development and birth through to the aged brain. Further, it is not only age that impacts the state of the neuroimmune milieu, as microglia have been shown to play a central role in the sexual differentiation of the brain. Finally, this review will discuss the implications this has for the differences in the incidence of neurodegenerative disorders between males and females, and between the young and old. PMID:28273860

Microglial SMAD4 regulated by microRNA-146a promotes migration of microglia which support tumor progression in a glioma environment

PubMed Central

Karthikeyan, Aparna; Gupta, Neelima; Tang, Carol; Mallilankaraman, Karthik; Silambarasan, Maskomani; Shi, Meng; Lu, Lei; Ang, Beng Ti; Ling, Eng-Ang; Dheen, S. Thameem

2018-01-01

Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-β (TGFβ) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGFβ level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell line-based model increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGFβ signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.

Receptor-heteromer mediated regulation of endocannabinoid signaling in activated microglia. Role of CB1 and CB2 receptors and relevance for Alzheimer's disease and levodopa-induced dyskinesia.

PubMed

Navarro, Gemma; Borroto-Escuela, Dasiel; Angelats, Edgar; Etayo, Íñigo; Reyes-Resina, Irene; Pulido-Salgado, Marta; Rodríguez-Pérez, Ana I; Canela, Enric I; Saura, Josep; Lanciego, José Luis; Labandeira-García, José Luis; Saura, Carlos A; Fuxe, Kjell; Franco, Rafael

2018-01-01

Endocannabinoids are important regulators of neurotransmission and, acting on activated microglia, they are postulated as neuroprotective agents. Endocannabinoid action is mediated by CB 1 and CB 2 receptors, which may form heteromeric complexes (CB 1 -CB 2 Hets) with unknown function in microglia. We aimed at establishing the expression and signaling properties of cannabinoid receptors in resting and LPS/IFN-γ-activated microglia. In activated microglia mRNA transcripts increased (2 fold for CB 1 and circa 20 fold for CB 2 ), whereas receptor levels were similar for CB 1 and markedly upregulated for CB 2 ; CB 1 -CB 2 Hets were also upregulated. Unlike in resting cells, CB 2 receptors became robustly coupled to G i in activated cells, in which CB 1 -CB 2 Hets mediated a potentiation effect. Hence, resting cells were refractory while activated cells were highly responsive to cannabinoids. Interestingly, similar results were obtained in cultures treated with ß-amyloid (Aß 1-42 ). Microglial activation markers were detected in the striatum of a Parkinson's disease (PD) model and, remarkably, in primary microglia cultures from the hippocampus of mutant β-amyloid precursor protein (APP Sw,Ind ) mice, a transgenic Alzheimer's disease (AD) model. Also of note was the similar cannabinoid receptor signaling found in primary cultures of microglia from APP Sw,Ind and in cells from control animals activated using LPS plus IFN-γ. Expression of CB 1 -CB 2 Hets was increased in the striatum from rats rendered dyskinetic by chronic levodopa treatment. In summary, our results showed sensitivity of activated microglial cells to cannabinoids, increased CB 1 -CB 2 Het expression in activated microglia and in microglia from the hippocampus of an AD model, and a correlation between levodopa-induced dyskinesia and striatal microglial activation in a PD model. Cannabinoid receptors and the CB 1 -CB 2 heteroreceptor complex in activated microglia have potential as targets in the treatment of neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Repeat-containing protein effectors of plant-associated organisms

PubMed Central

Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

Repeat-containing protein effectors of plant-associated organisms.

PubMed

Mesarich, Carl H; Bowen, Joanna K; Hamiaux, Cyril; Templeton, Matthew D

2015-01-01

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

PubMed Central

Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

RIPK3/MLKL-Mediated Neuronal Necroptosis Modulates the M1/M2 Polarization of Microglia/Macrophages in the Ischemic Cortex

PubMed Central

Yang, Jiping; Zhao, Youyi; Zhang, Li; Fan, Hong; Qi, Chuchu; Zhang, Kun; Liu, Xinyu; Fei, Lin; Chen, Siwei; Wang, Mengmeng; Kuang, Fang; Wang, Yazhou; Wu, Shengxi

2018-01-01

Abstract Cell death and subsequent inflammation are 2 key pathological changes occurring in cerebral ischemia. Active microglia/macrophages play a double-edged role depending on the balance of their M1/M2 phenotypes. Necrosis is the predominant type of cell death following ischemia. However, how necrotic cells modulate the M1/M2 polarization of microglia/macrophages remains poorly investigated. Here, we reported that ischemia induces a rapid RIPK3/MLKL-mediated neuron-dominated necroptosis, a type of programmed necrosis. Ablating RIPK3 or MLKL could switch the activation of microglia/macrophages from M1 to the M2 type in the ischemic cortex. Conditioned medium of oxygen-glucose deprivation (OGD)-treated wild-type (WT) neurons induced M1 polarization, while that of RIPK3−/− neurons favored M2 polarization. OGD treatment induces proinflammatory IL-18 and TNFα in WT but not in RIPK3−/− neurons, which in turn upregulate anti-inflammatory IL-4 and IL-10. Furthermore, the expression of Myd88—a common downstream adaptor of toll-like receptors—is significantly upregulated in the microglia/macrophages of ischemic WT but not of RIPK3−/− or MLKL−/− cortices. Antagonizing the function of Myd88 could phenocopy the effects of RIPK3/MLKL-knockout on the polarization of microglia/macrophages and was neuroprotective. Our data revealed a novel role of necroptotic neurons in modulating the M1/M2 balance of microglia/macrophages in the ischemic cortex, possibly through Myd88 signaling. PMID:29746630

Aging-like Changes in the Transcriptome of Irradiated Microglia

PubMed Central

Li, Matthew D.; Burns, Terry C.; Kumar, Sunny; Morgan, Alexander A.; Sloan, Steven A.; Palmer, Theo D.

2014-01-01

Whole brain irradiation remains important in the management of brain tumors. Although necessary for improving survival outcomes, cranial irradiation also results in cognitive decline in long-term survivors. A chronic inflammatory state characterized by microglial activation has been implicated in radiation-induced brain injury. We here provide the first comprehensive transcriptional profile of irradiated microglia. Fluorescence-activated cell sorting (FACS) was used to isolate CD11b+ microglia from the hippocampi of C57BL/6 and Balb/c mice 1 month after 10Gy cranial irradiation. Affymetrix gene expression profiles were evaluated using linear modeling, rank product analyses. One month after irradiation, a conserved irradiation signature across strains was identified, comprising 448 and 85 differentially up- and down-regulated genes, respectively. Gene set enrichment analysis (GSEA) demonstrated enrichment for inflammation, including M1 macrophage-associated genes, but also an unexpected enrichment for extracellular matrix and blood coagulation-related gene sets, in contrast previously described microglial states. Weighted gene co-expression network analysis (WGCNA) confirmed these findings and further revealed alterations in mitochondrial function. The RNA-seq transcriptome of microglia 24h post-radiation proved similar to the 1-month transcriptome, but additionally featured alterations in apoptotic and lysosomal gene expression. Re-analysis of published aging mouse microglia transcriptome data demonstrated striking similarity to the 1 month irradiated microglia transcriptome, suggesting that shared mechanisms may underlie aging and chronic irradiation-induced cognitive decline. PMID:25690519

Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury.

PubMed

Qu, Wen-Sheng; Tian, Dai-Shi; Guo, Zhi-Bao; Fang, Jun; Zhang, Qiang; Yu, Zhi-Yuan; Xie, Min-Jie; Zhang, Hua-Qiu; Lü, Jia-Gao; Wang, Wei

2012-07-23

Emerging evidence indicates that reactive microglia-initiated inflammatory responses are responsible for secondary damage after primary traumatic spinal cord injury (SCI); epidermal growth factor receptor (EGFR) signaling may be involved in cell activation. In this report, we investigate the influence of EGFR signaling inhibition on microglia activation, proinflammatory cytokine production, and the neuronal microenvironment after SCI. Lipopolysaccharide-treated primary microglia/BV2 line cells and SCI rats were used as model systems. Both C225 and AG1478 were used to inhibit EGFR signaling activation. Cell activation and EGFR phosphorylation were observed after fluorescent staining and western blot. Production of interleukin-1 beta (IL-1 β) and tumor necrosis factor alpha (TNF α) was tested by reverse transcription PCR and ELISA. Western blot was performed to semi-quantify the expression of EGFR/phospho-EGFR, and phosphorylation of Erk, JNK and p38 mitogen-activated protein kinases (MAPK). Wet-dry weight was compared to show tissue edema. Finally, axonal tracing and functional scoring were performed to show recovery of rats. EGFR phosphorylation was found to parallel microglia activation, while EGFR blockade inhibited activation-associated cell morphological changes and production of IL-1 β and TNF α. EGFR blockade significantly downregulated the elevated MAPK activation after cell activation; selective MAPK inhibitors depressed production of cytokines to a certain degree, suggesting that MAPK mediates the depression of microglia activation brought about by EGFR inhibitors. Subsequently, seven-day continual infusion of C225 or AG1478 in rats: reduced the expression of phospho-EGFR, phosphorylation of Erk and p38 MAPK, and production of IL-1 β and TNF α; lessened neuroinflammation-associated secondary damage, like microglia/astrocyte activation, tissue edema and glial scar/cavity formation; and enhanced axonal outgrowth and functional recovery. These findings indicate that inhibition of EGFR/MAPK suppresses microglia activation and associated cytokine production; reduces neuroinflammation-associated secondary damage, thus provides neuroprotection to SCI rats, suggesting that EGFR may be a therapeutic target, and C225 and AG1478 have potential for use in SCI treatment.

Microglia and Macrophages in Malignant Gliomas: Recent Discoveries and Implications for Promising Therapies

PubMed Central

Carvalho da Fonseca, Anna Carolina; Badie, Behnam

2013-01-01

Malignant gliomas are the most common primary brain tumors. Their deadliest manifestation, glioblastoma multiforme (GBM), accounts for 15% of all primary brain tumors and is associated with a median survival of only 15 months even after multimodal therapy. There is substantial presence of microglia and macrophages within and surrounding brain tumors. These immune cells acquire an alternatively activated phenotype with potent tumor-tropic functions that contribute to glioma growth and invasion. In this review, we briefly summarize recent data that has been reported on the interaction of microglia/macrophages with brain tumors and discuss potential application of these findings to the development of future antiglioma therapies. PMID:23864876

Evidence of Tau Hyperphosphorylation and Dystrophic Microglia in the Common Marmoset.

PubMed

Rodriguez-Callejas, Juan D; Fuchs, Eberhard; Perez-Cruz, Claudia

2016-01-01

Common marmosets ( Callithrix jacchus ) have recently gained popularity in biomedical research as models of aging research. Basically, they confer advantages from other non-human primates due to their shorter lifespan with onset of appearance of aging at 8 years. Old marmosets present some markers linked to neurodegeneration in the brain such as amyloid beta (Aβ) 1-42 and Aβ 1-40 . However, there are no studies exploring other cellular markers associated with neurodegenerative diseases in this non-human primate. Using immunohistochemistry, we analyzed brains of male adolescent, adult, old, and aged marmosets. We observed accumulation of Aβ 1-40 and Aβ 1-42 in the cortex of aged subjects. Tau hyperphosphorylation was already detected in the brain of adolescent animals and increased with aging in a more fibrillary form. Microglia activation was also observed in the aging process, while a dystrophic phenotype accumulates in aged subjects. Interestingly, dystrophic microglia contained hyperphosphorylated tau, but active microglia did not. These results support previous findings regarding microglia dysfunctionality in aging and neurodegenerative diseases as Alzheimer's disease. Further studies should explore the functional consequences of these findings to position this non-human primate as animal model of aging and neurodegeneration.

Evidence of Tau Hyperphosphorylation and Dystrophic Microglia in the Common Marmoset

PubMed Central

Rodriguez-Callejas, Juan D.; Fuchs, Eberhard; Perez-Cruz, Claudia

2016-01-01

Common marmosets (Callithrix jacchus) have recently gained popularity in biomedical research as models of aging research. Basically, they confer advantages from other non-human primates due to their shorter lifespan with onset of appearance of aging at 8 years. Old marmosets present some markers linked to neurodegeneration in the brain such as amyloid beta (Aβ)1-42 and Aβ1-40. However, there are no studies exploring other cellular markers associated with neurodegenerative diseases in this non-human primate. Using immunohistochemistry, we analyzed brains of male adolescent, adult, old, and aged marmosets. We observed accumulation of Aβ1-40 and Aβ1-42 in the cortex of aged subjects. Tau hyperphosphorylation was already detected in the brain of adolescent animals and increased with aging in a more fibrillary form. Microglia activation was also observed in the aging process, while a dystrophic phenotype accumulates in aged subjects. Interestingly, dystrophic microglia contained hyperphosphorylated tau, but active microglia did not. These results support previous findings regarding microglia dysfunctionality in aging and neurodegenerative diseases as Alzheimer’s disease. Further studies should explore the functional consequences of these findings to position this non-human primate as animal model of aging and neurodegeneration. PMID:28066237

Progranulin Is a Chemoattractant for Microglia and Stimulates Their Endocytic Activity

PubMed Central

Pickford, Fiona; Marcus, Jacob; Camargo, Luiz Miguel; Xiao, Qiurong; Graham, Danielle; Mo, Jan-Rung; Burkhardt, Matthew; Kulkarni, Vinayak; Crispino, Jamie; Hering, Heike; Hutton, Michael

2011-01-01

Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid β1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid β. PMID:21224065

E3 Ubiquitin Ligase c-cbl Inhibits Microglia Activation After Chronic Constriction Injury.

PubMed

Xue, Pengfei; Liu, Xiaojuan; Shen, Yiming; Ju, Yuanyuan; Lu, Xiongsong; Zhang, Jinlong; Xu, Guanhua; Sun, Yuyu; Chen, Jiajia; Gu, Haiyan; Cui, Zhiming; Bao, Guofeng

2018-06-22

E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1β and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.

Immune dysregulation and cognitive vulnerability in the aging brain: Interactions of microglia, IL-1β, BDNF and synaptic plasticity.

PubMed

Patterson, Susan L

2015-09-01

Older individuals often experience declines in cognitive function after events (e.g. infection, or injury) that trigger activation of the immune system. This occurs at least in part because aging sensitizes the response of microglia (the brain's resident immune cells) to signals triggered by an immune challenge. In the aging brain, microglia respond to these signals by producing more pro-inflammatory cytokines (e.g. interleukin-1beta or IL-1β) and producing them for longer than microglia in younger brains. This exaggerated inflammatory response can compromise processes critical for optimal cognitive functioning. Interleukin-1β is central to the inflammatory response and is a key mediator and modulator of an array of associated biological functions; thus its production and release is usually very tightly regulated. This review will focus on the impact of dysregulated production of IL-1β on hippocampus dependent-memory systems and associated synaptic plasticity processes. The neurotrophin brain-derived neurotrophic factor (BNDF) helps to protect neurons from damage caused by infection or injury, and it plays a critical role in many of the same memory and hippocampal plasticity processes compromised by dysregulated production of IL-1β. This suggests that an exaggerated brain inflammatory response, arising from aging and a secondary immune challenge, may erode the capacity to provide the BDNF needed for memory-related plasticity processes at hippocampal synapses. This article is part of a Special Issue entitled 'Neuroimmunology and Synaptic Function'. Copyright © 2014 Elsevier Ltd. All rights reserved.

A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

PubMed Central

Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

2016-01-01

Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

Glial response during cuprizone-induced de- and remyelination in the CNS: lessons learned

PubMed Central

Gudi, Viktoria; Gingele, Stefan; Skripuletz, Thomas; Stangel, Martin

2014-01-01

Although astrogliosis and microglia activation are characteristic features of multiple sclerosis (MS) and other central nervous system (CNS) lesions the exact functions of these events are not fully understood. Animal models help to understand the complex interplay between the different cell types of the CNS and uncover general mechanisms of damage and repair of myelin sheaths. The so called cuprizone model is a toxic model of demyelination in the CNS white and gray matter, which lacks an autoimmune component. Cuprizone induces apoptosis of mature oligodendrocytes that leads to a robust demyelination and profound activation of both astrocytes and microglia with regional heterogeneity between different white and gray matter regions. Although not suitable to study autoimmune mediated demyelination, this model is extremely helpful to elucidate basic cellular and molecular mechanisms during de- and particularly remyelination independently of interactions with peripheral immune cells. Phagocytosis and removal of damaged myelin seems to be one of the major roles of microglia in this model and it is well known that removal of myelin debris is a prerequisite of successful remyelination. Furthermore, microglia provide several signals that support remyelination. The role of astrocytes during de- and remyelination is not well defined. Both supportive and destructive functions have been suggested. Using the cuprizone model we could demonstrate that there is an important crosstalk between astrocytes and microglia. In this review we focus on the role of glial reactions and interaction in the cuprizone model. Advantages and limitations of as well as its potential therapeutic relevance for the human disease MS are critically discussed in comparison to other animal models. PMID:24659953

Plasticity of Subventricular Zone Neuroprogenitors in MPTP (1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine) Mouse Model of Parkinson’s Disease Involves Cross Talk between Inflammatory and Wnt/β-Catenin Signaling Pathways: Functional Consequences for Neuroprotection and Repair

PubMed Central

L’Episcopo, Francesca; Tirolo, Cataldo; Testa, Nunzio; Caniglia, Salvatore; Morale, Maria C.; Deleidi, Michela; Serapide, Maria F.; Pluchino, Stefano; Marchetti, Bianca

2013-01-01

In Parkinson’s disease (PD), neurogenesis is impaired in the subventricular zone (SVZ) of postmortem human PD brains, in primate nonhuman and rodent models of PD. The vital role of Wingless-type MMTV integration site (Wnt)/β-catenin signaling in the modulation of neurogenesis, neuroprotection, and synaptic plasticity coupled to our recent findings uncovering an active role for inflammation and Wnt/β-catenin signaling in MPTP-induced loss and repair of nigrostriatal dopaminergic (DAergic) neurons prompted us to study the impact of neuroinflammation and the Wnt/β-catenin pathway in the response of SVZ neuroprogenitors (NPCs) in MPTP-treated mice. In vivo experiments, using bromodeoxyuridine and cell-specific markers, and ex vivo time course analyses documented an inverse correlation between the reduced proliferation of NPCs and the generation of new neuroblasts with the phase of maximal exacerbation of microglia reaction, whereas a shift in the microglia proinflammatory phenotype correlated with a progressive NPC recovery. Ex vivo and in vitro experiments using microglia–NPC coculture paradigms pointed to NADPH-oxidase (gpPHOX91), a major source of microglial ROS, and reactive nitrogen species as candidate inhibitors of NPC neurogenic potential via the activation of glycogen synthase 3 (pGSK-3βTyr216), leading to loss of β-catenin, a chief downstream transcriptional effector. Accordingly, MPTP/MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) caused β-catenin downregulation and pGSK-3βTyr216 overexpression, whereas manipulation of Wnt/β-catenin signaling with RNA interference-mediated GSK-3β knockdown or GSK-3β antagonism reversed MPTP-induced neurogenic impairment ex vivo/in vitro or in vivo. Reciprocally, pharmacological modulation of inflammation prevented β-catenin downregulation and restored neurogenesis, suggesting the possibility to modulate this endogenous system with potential consequences for DAergic neuroprotection and self-repair. PMID:22323720

Ligand-gated purinergic receptors regulate HIV-1 Tat and morphine related neurotoxicity in primary mouse striatal neuron-glia co-cultures.

PubMed

Sorrell, Mary E; Hauser, Kurt F

2014-03-01

Emerging evidence suggests that opioid drugs, such as morphine and heroin, can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that opioid drugs acting via μ opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. Key aspects of microglial function are regulated by the P2X family of ATP activated ligand-gated ion channels. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, which prompted us to investigate whether the cation-permeable P2X receptors contribute to the neurotoxic effects of HIV and morphine. To address this question, neuron survival, as well as other endpoints including changes in dendritic length, extracellular ATP levels, and intracellular calcium levels, were assayed in primary neuron-glia co-cultures from mouse striatum. Treatment with TNP-ATP, a non-selective P2X antagonist, prevented the neurotoxic effects of exposure to morphine and/or HIV Tat, or ATP alone, suggesting P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. Although P2X7, and perhaps P2X1, receptor activation decreases neuron survival, neither P2X1, P2X3, nor P2X7 selective receptor antagonists prevented Tat and/or morphine-induced neurotoxicity. These and other experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and provide circumstantial evidence implicating P2X4 receptors in particular. Our findings reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompanies neuroAIDS and opiate abuse.

Microglia in the developing brain: a potential target with lifetime effects

PubMed Central

Harry, G. Jean; Kraft, Andrew D.

2012-01-01

Microglia are a heterogeneous group of monocyte-derived cells serving multiple roles within the brain, many of which are associated with immune and macrophage like properties. These cells are known to serve a critical role during brain injury and to maintain homeostasis; yet, their defined roles during development have yet to be elucidated. Microglial actions appear to influence events associated with neuronal proliferation and differentiation during development, as well as, contribute to processes associated with the removal of dying neurons or cellular debris and management of synaptic connections. These long-lived cells display changes during injury and with aging that are critical to the maintenance of the neuronal environment over the lifespan of the organism. These processes may be altered by changes in the colonization of the brain or by inflammatory events during development. This review addresses the role of microglia during brain development, both structurally and functionally, as well as the inherent vulnerability of the developing nervous system. A framework is presented considering microglia as a critical nervous system-specific cell that can influence multiple aspects of brain development (e.g., vascularization, synaptogenesis, and myelination) and have a long term impact on the functional vulnerability of the nervous system to a subsequent insult, whether environmental, physical, age-related, or disease-related. PMID:22322212

Development and maintenance of the brain's immune toolkit: Microglia and non-parenchymal brain macrophages.

PubMed

Lopez-Atalaya, Jose P; Askew, Katharine E; Sierra, Amanda; Gomez-Nicola, Diego

2018-06-01

Microglia and non-parenchymal macrophages located in the perivascular space, the meninges and the choroid plexus are independent immune populations that play vital roles in brain development, homeostasis, and tissue healing. Resident macrophages account for a significant proportion of cells in the brain and their density remains stable throughout the lifespan thanks to constant turnover. Microglia develop from yolk sac progenitors, later evolving through intermediate progenitors in a fine-tuned process in which intrinsic factors and external stimuli combine to progressively sculpt their cell type-specific transcriptional profiles. Recent evidence demonstrates that non-parenchymal macrophages are also generated during early embryonic development. In recent years, the development of powerful fate mapping approaches combined with novel genomic and transcriptomic methodologies have greatly expanded our understanding of how brain macrophages develop and acquire specialized functions, and how cell population dynamics are regulated. Here, we review the transcription factors, epigenetic remodeling, and signaling pathways orchestrating the embryonic development of microglia and non-parenchymal macrophages. Next, we describe the dynamics of the macrophage populations of the brain and discuss the role of progenitor cells, to gain a better understanding of their functions in the healthy and diseased brain. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 561-579, 2018. © 2017 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.

Microglia ablation alleviates myelin-associated catatonic signs in mice

PubMed Central

Janova, Hana; Arinrad, Sahab; Balmuth, Evan; Mitjans, Marina; Bittner, Robert A.; Pan, Hong; Goebbels, Sandra; Begemann, Martin; Gerwig, Ulrike C.; Langner, Sönke; Werner, Hauke B.; Davatzikos, Christos; Völzke, Henry; West, Brian L.; Reif, Andreas; Grabe, Hans Jörgen; Nave, Klaus-Armin

2017-01-01

The underlying cellular mechanisms of catatonia, an executive “psychomotor” syndrome that is observed across neuropsychiatric diseases, have remained obscure. In humans and mice, reduced expression of the structural myelin protein CNP is associated with catatonic signs in an age-dependent manner, pointing to the involvement of myelin-producing oligodendrocytes. Here, we showed that the underlying cause of catatonic signs is the low-grade inflammation of white matter tracts, which marks a final common pathway in Cnp-deficient and other mutant mice with minor myelin abnormalities. The inhibitor of CSF1 receptor kinase signaling PLX5622 depleted microglia and alleviated the catatonic symptoms of Cnp mutants. Thus, microglia and low-grade inflammation of myelinated tracts emerged as the trigger of a previously unexplained mental condition. We observed a very high (25%) prevalence of individuals with catatonic signs in a deeply phenotyped schizophrenia sample (n = 1095). Additionally, we found the loss-of-function allele of a myelin-specific gene (CNP rs2070106-AA) associated with catatonia in 2 independent schizophrenia cohorts and also associated with white matter hyperintensities in a general population sample. Since the catatonic syndrome is likely a surrogate marker for other executive function defects, we suggest that microglia-directed therapies may be considered in psychiatric disorders associated with myelin abnormalities. PMID:29252214

Microglia Gone Rogue: Impacts on Psychiatric Disorders across the Lifespan

PubMed Central

Tay, Tuan Leng; Béchade, Catherine; D’Andrea, Ivana; St-Pierre, Marie-Kim; Henry, Mathilde S.; Roumier, Anne; Tremblay, Marie-Eve

2018-01-01

Microglia are the predominant immune response cells and professional phagocytes of the central nervous system (CNS) that have been shown to be important for brain development and homeostasis. These cells present a broad spectrum of phenotypes across stages of the lifespan and especially in CNS diseases. Their prevalence in all neurological pathologies makes it pertinent to reexamine their distinct roles during steady-state and disease conditions. A major question in the field is determining whether the clustering and phenotypical transformation of microglial cells are leading causes of pathogenesis, or potentially neuroprotective responses to the onset of disease. The recent explosive growth in our understanding of the origin and homeostasis of microglia, uncovering their roles in shaping of the neural circuitry and synaptic plasticity, allows us to discuss their emerging functions in the contexts of cognitive control and psychiatric disorders. The distinct mesodermal origin and genetic signature of microglia in contrast to other neuroglial cells also make them an interesting target for the development of therapeutics. Here, we review the physiological roles of microglia, their contribution to the effects of environmental risk factors (e.g., maternal infection, early-life stress, dietary imbalance), and their impact on psychiatric disorders initiated during development (e.g., Nasu-Hakola disease (NHD), hereditary diffuse leukoencephaly with spheroids, Rett syndrome, autism spectrum disorders (ASDs), and obsessive-compulsive disorder (OCD)) or adulthood (e.g., alcohol and drug abuse, major depressive disorder (MDD), bipolar disorder (BD), schizophrenia, eating disorders and sleep disorders). Furthermore, we discuss the changes in microglial functions in the context of cognitive aging, and review their implication in neurodegenerative diseases of the aged adult (e.g., Alzheimer’s and Parkinson’s). Taking into account the recent identification of microglia-specific markers, and the availability of compounds that target these cells selectively in vivo, we consider the prospect of disease intervention via the microglial route. PMID:29354029

Flow cytometric characterization of tumor-associated macrophages in experimental gliomas.

PubMed

Badie, B; Schartner, J M

2000-04-01

Although microglia have been suggested to be a component of the inflammatory reaction to tumors of the central nervous system, their role in glioma biology remains unknown. One obstacle to studying the function of microglia is the inability to effectively separate them from macrophages. Because flow cytometry can effectively discern immune cells with similar surface antigens, we evaluated its role in characterizing the mononuclear cell infiltration in experimental gliomas. Freshly prepared rat C6, 9L, and RG-2 tumor specimens were labeled ex vivo with monoclonal antibodies against CD11b/c, CD45, and CD8a antigens and analyzed by flow cytometry. The extent of microglia (CD11b/c(high), CD45(low)), macrophage (CD11b/c(high), CD45(high)), and lymphocyte (CD11b/c(negative), CD45(high)) infiltration into tumors, tumor periphery, and contralateral tumor-free hemispheres was measured for each glioma type. Microglia, which accounted for 13 to 34% of viable cells, were distributed throughout the central nervous system and were present in the tumors, tumor periphery, and contralateral tumor-free hemispheres. In contrast, macrophages were less prominent within the tumors and tumor periphery (4.2-12%) and were scarce in the contralateral tumor-free hemispheres (0.9-1.1%). Among the tumor types, RG-2 gliomas had the least microglia/macrophage infiltration. The frequency and the distribution pattern of lymphocytes also varied among tumor models. Whereas lymphocytes accounted for more than one-third of the cells in C6 and 9L tumors, they represented only 1% of cells in RG-2 gliomas. More abundant than macrophages and scattered throughout the central nervous system, microglia account for a significant component of the inflammatory response to experimental gliomas. A better understanding of microglial function in gliomas may be important in the development of immunotherapy strategies.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

PubMed

Sperschneider, Jana; Gardiner, Donald M; Dodds, Peter N; Tini, Francesco; Covarelli, Lorenzo; Singh, Karam B; Manners, John M; Taylor, Jennifer M

2016-04-01

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au. © 2015 CSIRO New Phytologist © 2015 New Phytologist Trust.

Microglial aging in the healthy CNS: phenotypes, drivers, and rejuvenation

PubMed Central

Wong, Wai T.

2013-01-01

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and age-related macular degeneration (AMD), share two characteristics in common: (1) a disease prevalence that increases markedly with advancing age, and (2) neuroinflammatory changes in which microglia, the primary resident immune cell of the CNS, feature prominently. These characteristics have led to the hypothesis that pathogenic mechanisms underlying age-related neurodegenerative disease involve aging changes in microglia. If correct, targeting features of microglial senescence may constitute a feasible therapeutic strategy. This review explores this hypothesis and its implications by considering the current knowledge on how microglia undergo change during aging and how the emergence of these aging phenotypes relate to significant alterations in microglial function. Evidence and theories on cellular mechanisms implicated in driving senescence in microglia are reviewed, as are “rejuvenative” measures and strategies that aim to reverse or ameliorate the aging microglial phenotype. Understanding and controlling microglial aging may represent an opportunity for elucidating disease mechanisms and for formulating novel therapies. PMID:23493481

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D.

1995-05-01

Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) havemore » been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.« less

Production of proinflammatory mediators in activated microglia is synergistically regulated by Notch-1, glycogen synthase kinase (GSK-3β) and NF-κB/p65 signalling.

PubMed

Cao, Qiong; Karthikeyan, Aparna; Dheen, S Thameem; Kaur, Charanjit; Ling, Eng-Ang

2017-01-01

Microglia activation and associated inflammatory response are involved in the pathogenesis of different neurodegenerative diseases. We have reported that Notch-1 and NF-κB/p65 signalling pathways operate in synergy in regulating the production of proinflammatory mediators in activated microglia. In the latter, there is also evidence by others that glycogen synthase kinase 3β (GSK-3β) mediates the release of proinflammatory cytokines but the interrelationships between the three signalling pathways have not been fully clarified. This is an important issue as activated microglia are potential therapeutic target for amelioration of microglia mediated neuroinflammation. Here we show that blocking of Notch-1 with N-[(3,5-Difluorophenyl) acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT) in LPS activated BV-2 microglia not only suppressed Notch intracellular domain (NICD) and Hes-1 protein expression, but also that of GSK-3β. Conversely, blocking of the latter with lithium chloride (LiCl) decreased NICD expression in a dose-dependent manner; moreover, Hes-1 immunofluorescence was attenuated. Along with this, the protein expression level of p-GSK-3β and p-AKT protein expression was significantly increased. Furthermore, DAPT and LiCl decreased production of IL-1β, TNF-α, IL-6, iNOS, Cox2 and MCP-1; however, IL-10 expression was increased notably in LiCl treated cells. The effects of DAPT and LiCl on changes of the above-mentioned biomarkers were confirmed by immunofluorescence in both BV-2 and primary microglia. Additionally, NF-κB/p65 immunofluorescence was attenuated by DAPT and LiCl; as opposed to this, IκBα protein expression was increased. Taken together, it is suggested that Notch-1, NF-κB/p65 and GSK-3β operate in synergy to inhibit microglia activation. This may be effected via increased expression of phospho-GSK-3β (p-GSK-3β), phospho-protein kinase B (PKB) (p-AKT) and IκBα. It is concluded that the three signalling pathways are functionally interlinked in regulating microglia activation.

Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

PubMed Central

Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

2013-01-01

Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library Jstacs, and is freely available as a web-application and a command line program. PMID:23526890

Chronic ethanol intake induces partial microglial activation that is not reversed by long-term ethanol withdrawal in the rat hippocampal formation.

PubMed

Cruz, Catarina; Meireles, Manuela; Silva, Susana M

2017-05-01

Neuroinflammation has been implicated in the pathogenesis of several disorders. Activation of microglia leads to the release of pro-inflammatory mediators and microglial-mediated neuroinflammation has been proposed as one of the alcohol-induced neuropathological mechanisms. The present study aimed to examine the effect of chronic ethanol exposure and long-term withdrawal on microglial activation and neuroinflammation in the hippocampal formation. Male rats were submitted to 6 months of ethanol treatment followed by a 2-month withdrawal period. Stereological methods were applied to estimate the total number of microglia and activated microglia detected by CD11b immunohistochemistry in the hippocampal formation. The expression levels of the pro-inflammatory cytokines TNF-α, COX-2 and IL-15 were measured by qRT-PCR. Alcohol consumption was associated with an increase in the total number of activated microglia but morphological assessment indicated that microglia did not exhibit a full activation phenotype. These data were supported by functional evidence since chronic alcohol consumption produced no changes in the expression of TNF-α or COX-2. The levels of IL-15 a cytokine whose expression is increased upon activation of both astrocytes and microglia, was induced by chronic alcohol treatment. Importantly, the partial activation of microglia induced by ethanol was not reversed by long-term withdrawal. This study suggests that chronic alcohol exposure induces a microglial phenotype consistent with partial activation without significant increase in classical cytokine markers of neuroinflammation in the hippocampal formation. Furthermore, long-term cessation of alcohol intake is not sufficient to alter the microglial partial activation phenotype induced by ethanol. Copyright © 2017 Elsevier B.V. All rights reserved.

Hierarchical Cluster Analysis of Three-Dimensional Reconstructions of Unbiased Sampled Microglia Shows not Continuous Morphological Changes from Stage 1 to 2 after Multiple Dengue Infections in Callithrix penicillata

PubMed Central

Diniz, Daniel G.; Silva, Geane O.; Naves, Thaís B.; Fernandes, Taiany N.; Araújo, Sanderson C.; Diniz, José A. P.; de Farias, Luis H. S.; Sosthenes, Marcia C. K.; Diniz, Cristovam G.; Anthony, Daniel C.; da Costa Vasconcelos, Pedro F.; Picanço Diniz, Cristovam W.

2016-01-01

It is known that microglial morphology and function are related, but few studies have explored the subtleties of microglial morphological changes in response to specific pathogens. In the present report we quantitated microglia morphological changes in a monkey model of dengue disease with virus CNS invasion. To mimic multiple infections that usually occur in endemic areas, where higher dengue infection incidence and abundant mosquito vectors carrying different serotypes coexist, subjects received once a week subcutaneous injections of DENV3 (genotype III)-infected culture supernatant followed 24 h later by an injection of anti-DENV2 antibody. Control animals received either weekly anti-DENV2 antibodies, or no injections. Brain sections were immunolabeled for DENV3 antigens and IBA-1. Random and systematic microglial samples were taken from the polymorphic layer of dentate gyrus for 3-D reconstructions, where we found intense immunostaining for TNFα and DENV3 virus antigens. We submitted all bi- or multimodal morphological parameters of microglia to hierarchical cluster analysis and found two major morphological phenotypes designated types I and II. Compared to type I (stage 1), type II microglia were more complex; displaying higher number of nodes, processes and trees and larger surface area and volumes (stage 2). Type II microglia were found only in infected monkeys, whereas type I microglia was found in both control and infected subjects. Hierarchical cluster analysis of morphological parameters of 3-D reconstructions of random and systematic selected samples in control and ADE dengue infected monkeys suggests that microglia morphological changes from stage 1 to stage 2 may not be continuous. PMID:27047345

Features of Microglia and Neuroinflammation Relevant to Environmental Exposure and Neurotoxicity

PubMed Central

Kraft, Andrew D.; Harry, G. Jean

2011-01-01

Microglia are resident cells of the brain involved in regulatory processes critical for development, maintenance of the neural environment, injury and repair. They belong to the monocytic-macrophage lineage and serve as brain immune cells to orchestrate innate immune responses; however, they are distinct from other tissue macrophages due to their relatively quiescent phenotype and tight regulation by the CNS microenvironment. Microglia actively survey the surrounding parenchyma and respond rapidly to changes such that any disruption to neural architecture or function can contribute to the loss in regulation of the microglia phenotype. In many models of neurodegeneration and neurotoxicity, early events of synaptic degeneration and neuronal loss are accompanied by an inflammatory response including activation of microglia, perivascular monocytes, and recruitment of leukocytes. In culture, microglia have been shown to be capable of releasing several potentially cytotoxic substances, such as reactive oxygen intermediates, nitric oxide, proteases, arachidonic acid derivatives, excitatory amino acids, and cytokines; however, they also produce various neurotrophic factors and quench damage from free radicals and excitotoxins. As the primary source for pro-inflammatory cytokines, microglia are implicated as pivotal mediators of neuroinflammation and can induce or modulate a broad spectrum of cellular responses. Neuroinflammation should be considered as a balanced network of processes whereby subtle modifications can shift the cells toward disparate outcomes. For any evaluation of neuroinflammation and microglial responses, within the framework of neurotoxicity or degeneration, one key question in determining the consequence of neuroinflammation is whether the response is an initiating event or the consequence of tissue damage. As examples of environmental exposure-related neuroinflammation in the literature, we provide an evaluation of data on manganese and diesel exhaust particles. PMID:21845170

Hierarchical Cluster Analysis of Three-Dimensional Reconstructions of Unbiased Sampled Microglia Shows not Continuous Morphological Changes from Stage 1 to 2 after Multiple Dengue Infections in Callithrix penicillata.

PubMed

Diniz, Daniel G; Silva, Geane O; Naves, Thaís B; Fernandes, Taiany N; Araújo, Sanderson C; Diniz, José A P; de Farias, Luis H S; Sosthenes, Marcia C K; Diniz, Cristovam G; Anthony, Daniel C; da Costa Vasconcelos, Pedro F; Picanço Diniz, Cristovam W

2016-01-01

It is known that microglial morphology and function are related, but few studies have explored the subtleties of microglial morphological changes in response to specific pathogens. In the present report we quantitated microglia morphological changes in a monkey model of dengue disease with virus CNS invasion. To mimic multiple infections that usually occur in endemic areas, where higher dengue infection incidence and abundant mosquito vectors carrying different serotypes coexist, subjects received once a week subcutaneous injections of DENV3 (genotype III)-infected culture supernatant followed 24 h later by an injection of anti-DENV2 antibody. Control animals received either weekly anti-DENV2 antibodies, or no injections. Brain sections were immunolabeled for DENV3 antigens and IBA-1. Random and systematic microglial samples were taken from the polymorphic layer of dentate gyrus for 3-D reconstructions, where we found intense immunostaining for TNFα and DENV3 virus antigens. We submitted all bi- or multimodal morphological parameters of microglia to hierarchical cluster analysis and found two major morphological phenotypes designated types I and II. Compared to type I (stage 1), type II microglia were more complex; displaying higher number of nodes, processes and trees and larger surface area and volumes (stage 2). Type II microglia were found only in infected monkeys, whereas type I microglia was found in both control and infected subjects. Hierarchical cluster analysis of morphological parameters of 3-D reconstructions of random and systematic selected samples in control and ADE dengue infected monkeys suggests that microglia morphological changes from stage 1 to stage 2 may not be continuous.

Anti-inflammatory and immunomodulatory mechanisms of atorvastatin in a murine model of traumatic brain injury.

PubMed

Xu, Xin; Gao, Weiwei; Cheng, Shiqi; Yin, Dongpei; Li, Fei; Wu, Yingang; Sun, Dongdong; Zhou, Shuai; Wang, Dong; Zhang, Yongqiang; Jiang, Rongcai; Zhang, Jianning

2017-08-23

Neuroinflammation is an important secondary injury mechanism that has dual beneficial and detrimental roles in the pathophysiology of traumatic brain injury (TBI). Compelling data indicate that statins, a group of lipid-lowering drugs, also have extensive immunomodulatory and anti-inflammatory properties. Among statins, atorvastatin has been demonstrated as a neuroprotective agent in experimental TBI; however, there is a lack of evidence regarding its effects on neuroinflammation during the acute phase of TBI. The current study aimed to evaluate the effects of atorvastatin therapy on modulating the immune reaction, and to explore the possible involvement of peripheral leukocyte invasion and microglia/macrophage polarization in the acute period post-TBI. C57BL/6 mice were subjected to TBI using a controlled cortical impact (CCI) device. Either atorvastatin or vehicle saline was administered orally starting 1 h post-TBI for three consecutive days. Short-term neurological deficits were evaluated using the modified neurological severity score (mNSS) and Rota-rod. Brain-invading leukocyte subpopulations were analyzed by flow cytometry and immunohistochemistry. Pro- and anti-inflammatory cytokines and chemokines were examined using enzyme-linked immunosorbent assay (ELISA). Markers of classically activated (M1) and alternatively activated (M2) microglia/macrophages were then determined by quantitative real-time PCR (qRT-PCR) and flow cytometry. Neuronal apoptosis was identified by double staining of terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) staining and immunofluorescence labeling for neuronal nuclei (NeuN). Acute treatment with atorvastatin at doses of 1 mg/kg/day significantly reduced neuronal apoptosis and improved behavioral deficits. Invasions of T cells, neutrophils and natural killer (NK) cells were attenuated profoundly after atorvastatin therapy, as was the production of pro-inflammatory cytokines (IFN-γ and IL-6) and chemokines (RANTES and IP-10). Notably, atorvastatin treatment significantly increased the proportion of regulatory T cells (Tregs) in both the peripheral spleen and brain, and at the same time, increased their main effector cytokines IL-10 and TGF-β1. We also found that atorvastatin significantly attenuated total microglia/macrophage activation but augmented the M2/M1 ratio by both inhibiting M1 polarization and enhancing M2 polarization. Our data demonstrated that acute atorvastatin administration could modulate post-TBI neuroinflammation effectively, via a mechanism that involves altering peripheral leukocyte invasion and the alternative polarization of microglia/macrophages.

(Putative) Sex differences in neuroimmune modulation of memory

PubMed Central

Tronson, Natalie C.; Collette, Katie M.

2016-01-01

The neuroimmune system is significantly sexually dimorphic, with sex differences evident in the number and activation states of microglia, in the activation of astrocytes, and in cytokine release and function. Neuroimmune cells and signaling are now recognized as critical for many neural functions throughout the lifespan, including synaptic plasticity and memory function. Here we address the question of how cytokines, astrocytes, and microglia contribute to memory, and specifically how neuroimmune modulation of memory differentially affects males and females. Understanding sex differences in both normal memory processes and dysregulation of memory in psychiatric and neurological disorders is critical for developing treatment and preventive strategies for memory disorders that are effective for both men and women. PMID:27870428

Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines.

PubMed

Ghosh, Mousumi; Xu, Yong; Pearse, Damien D

2016-01-13

Microglia and macrophages play a central role in neuroinflammation. Pro-inflammatory cytokines trigger their conversion to a classically activated (M1) phenotype, sustaining inflammation and producing a cytotoxic environment. Conversely, anti-inflammatory cytokines polarize the cells towards an alternatively activated (M2), tissue reparative phenotype. Elucidation of the signal transduction pathways involved in M1 to M2 phenotypic conversion may provide insight into how the innate immune response can be harnessed during distinct phases of disease or injury to mediate neuroprotection and neurorepair. Microglial cells (cell line and primary) were subjected to combined cyclic adenosine monophosphate (cyclic AMP) and IL-4, or either alone, in the presence of pro-inflammatory mediators, lipopolysaccharide (LPS), or tumor necrosis factor-α (TNF-α). Their effects on the expression of characteristic markers for M1 and M2 microglia were assessed. Similarly, the M1 and M2 phenotypes of microglia and macrophages within the lesion site were then evaluated following a contusive spinal cord injury (SCI) to the thoracic (T8) spinal cord of rats and mice when the agents were administered systemically. It was demonstrated that cyclic AMP functions synergistically with IL-4 to promote M1 to M2 conversion of microglia in culture. The combination of cyclic AMP and IL-4, but neither alone, induced an Arg-1(+)/iNOS(-)cell phenotype with concomitant expression of other M2-specific markers including TG2 and RELM-α. M2-converted microglia showed ameliorated production of pro-inflammatory cytokines (TNF-α and IP-10) and reactive oxygen species, with no alteration in phagocytic properties. M2a conversion required protein kinase A (PKA), but not the exchange protein directly activated by cyclic AMP (EPAC). Systemic delivery of cyclic AMP and IL-4 after experimental SCI also promoted a significant M1 to M2a phenotypic change in microglia and macrophage population dynamics in the lesion. Using primary microglia, microglial cell lines, and experimental models of CNS injury, we demonstrate that cyclic AMP levels are a critical determinant in M1-M2 polarization. High levels of cyclic AMP promoted an Arg-1(+) M2a phenotype when microglia were activated with pro-inflammatory stimuli and Th2 cytokines. Th2 cytokines or cyclic AMP independently did not promote these changes. Phenotypic conversion of microglia provides a powerful new therapeutic approach for altering the balance of cytotoxic to reparative microglia in a diversity of neurological diseases and injury.

Chronic caffeine ingestion causes microglia activation, but not proliferation in the healthy brain

PubMed Central

Steger, Rob; Kamal, Arifa; Lutchman, Sara; Intrabartolo, Liliana; Sohail, Rabia; Brumberg, Joshua C.

2014-01-01

Caffeine is the most popular psychoactive drug in the world which contributes to behavioral and metabolic changes when ingested. Within the central nervous system (CNS), caffeine has a high affinity for A1 and A2a adenosine receptors. Serving as an antagonist, caffeine affects the ability for adenosine to bind to these receptors. Caffeine has been shown to alter neuronal functioning through increasing spontaneous firing. However, the effects of caffeine on non-neuronal cells in the CNS has been not been studied extensively. Microglia are one phenotype of non-neuronal glia within the CNS. Acting as phagocytes, they contribute to the immune defense system of the brain and express A1 and A2a adenosine receptors. Caffeine, therefore, may affect microglia. In order to test this hypothesis, CD-1 mice were randomly placed into one of three groups: control, low caffeine (0.3g/L water) and high caffeine (1.0g/L water) and were allowed to drink freely for 30 days. Following 30 days, brain sections were stained to reveal microglia. Morphological reconstructions and density measurements were examined in cortical and subcortical areas including the primary sensory cortex, primary motor cortex and striatum. Results indicate that microglial density throughout the brain is decreased in the caffeine groups as compared to the control. Caffeine also impacted microglia morphology shortening process length and decreasing branching. These results suggest that chronic caffeine ingestion has a systemic impact on microglia density and their activation. PMID:24881873

Microglia-Neuron Communication in Epilepsy.

PubMed

Eyo, Ukpong B; Murugan, Madhuvika; Wu, Long-Jun

2017-01-01

Epilepsy has remained a significant social concern and financial burden globally. Current therapeutic strategies are based primarily on neurocentric mechanisms that have not proven successful in at least a third of patients, raising the need for novel alternative and complementary approaches. Recent evidence implicates glial cells and neuroinflammation in the pathogenesis of epilepsy with the promise of targeting these cells to complement existing strategies. Specifically, microglial involvement, as a major inflammatory cell in the epileptic brain, has been poorly studied. In this review, we highlight microglial reaction to experimental seizures, discuss microglial control of neuronal activities, and propose the functions of microglia during acute epileptic phenotypes, delayed neurodegeneration, and aberrant neurogenesis. Future research that would help fill in the current gaps in our knowledge includes epilepsy-induced alterations in basic microglial functions, neuro-microglial interactions during chronic epilepsy, and microglial contribution to developmental seizures. Studying the role of microglia in epilepsy could inform therapies to better alleviate the disease. GLIA 2016;65:5-18. © 2016 Wiley Periodicals, Inc.

Genomic background-related activation of microglia and reduced β-amyloidosis in a mouse model of Alzheimer's disease.

PubMed

Fröhlich, Christina; Paarmann, Kristin; Steffen, Johannes; Stenzel, Jan; Krohn, Markus; Heinze, Hans-Jochen; Pahnke, Jens

2013-03-01

Alzheimer's disease (AD) is by far the most common neurodegenerative disease. AD is histologically characterized not only by extracellular senile plaques and vascular deposits consisting of β-amyloid (Aβ) but also by accompanying neuroinflammatory processes involving the brain's microglia. The importance of the microglia is still in controversial discussion, which currently favors a protective function in disease progression. Recent findings by different research groups highlighted the importance of strain-specific and mitochondria-specific genomic variations in mouse models of cerebral β-amyloidosis. Here, we want to summarize our previously presented data and add new results that draw attention towards the consideration of strain-specific genomic alterations in the setting of APP transgenes. We present data from APP-transgenic mice in commonly used C57Bl/6J and FVB/N genomic backgrounds and show a direct influence on the kinetics of Aβ deposition and the activity of resident microglia. Plaque size, plaque deposition rate and the total amount of Aβ are highest in C57Bl/6J mice as compared to the FVB/N genomic background, which can be explained at least partially by a reduced microglia activity towards amyloid deposits in the C57BL/6J strain.

Suppression of Brain Mast Cells Degranulation Inhibits Microglial Activation and Central Nervous System Inflammation.

PubMed

Dong, Hongquan; Zhang, Xiang; Wang, Yiming; Zhou, Xiqiao; Qian, Yanning; Zhang, Shu

2017-03-01

Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the "first responder" in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the "mast cell degranulator" compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. "Mast cell stabilizer" disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (H 1 R), histamine receptor 4 (H 4 R), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient Kit W-sh/W-sh mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.

Redox Regulation of NF-κB p50 and M1 Polarization in Microglia

PubMed Central

Taetzsch, Thomas; Levesque, Shannon; McGraw, Constance; Brookins, Savannah; Luqa, Rafy; Bonini, Marcelo G.; Mason, Ronald P.; Oh, Unsong; Block, Michelle L.

2014-01-01

Redox-signaling is implicated in deleterious microglial activation underlying CNS disease, but how ROS program aberrant microglial function is unknown. Here, the oxidation of NF-κB p50 to a free radical intermediate is identified as a marker of dysfunctional M1 (pro-inflammatory) polarization in microglia. Microglia exposed to steady fluxes of H2O2 showed altered NF-κB p50 protein-protein interactions, decreased NF-κB p50 DNA binding, and augmented late-stage TNFα expression, indicating that H2O2 impairs NF-κB p50 function and prolongs amplified M1 activation. NF-κB p50−/− mice and cultures exhibited a disrupted M2 (alternative) response and impaired resolution of the M1 response. Persistent neuroinflammation continued 1 week after LPS (1mg/kg, IP) administration in the NF-κB p50−/− mice. However, peripheral inflammation had already resolved in both strains of mice. Treatment with the spin-trap DMPO mildly reduced LPS-induced 22 h TNFα in the brain in NF-κB p50+/+ mice. Interestingly, DMPO failed to reduce and strongly augmented brain TNFα production in NF-κB p50−/− mice, implicating a fundamental role for NF-κB p50 in the regulation of chronic neuroinflammation by free radicals. These data identify NF-κB p50 as a key redox-signaling mechanism regulating the M1/M2 balance in microglia, where loss of function leads to a CNS-specific vulnerability to chronic inflammation. PMID:25331559

Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging

PubMed Central

Plaza-Zabala, Ainhoa; Sierra-Torre, Virginia; Sierra, Amanda

2017-01-01

Autophagy is emerging as a core regulator of Central Nervous System (CNS) aging and neurodegeneration. In the brain, it has mostly been studied in neurons, where the delivery of toxic molecules and organelles to the lysosome by autophagy is crucial for neuronal health and survival. However, we propose that the (dys)regulation of autophagy in microglia also affects innate immune functions such as phagocytosis and inflammation, which in turn contribute to the pathophysiology of aging and neurodegenerative diseases. Herein, we first describe the basic concepts of autophagy and its regulation, discuss key aspects for its accurate monitoring at the experimental level, and summarize the evidence linking autophagy impairment to CNS senescence and disease. We focus on acute, chronic, and autoimmunity-mediated neurodegeneration, including ischemia/stroke, Alzheimer’s, Parkinson’s, and Huntington’s diseases, and multiple sclerosis. Next, we describe the actual and potential impact of autophagy on microglial phagocytic and inflammatory function. Thus, we provide evidence of how autophagy may affect microglial phagocytosis of apoptotic cells, amyloid-β, synaptic material, and myelin debris, and regulate the progression of age-associated neurodegenerative diseases. We also discuss data linking autophagy to the regulation of the microglial inflammatory phenotype, which is known to contribute to age-related brain dysfunction. Overall, we update the current knowledge of autophagy and microglia, and highlight as yet unexplored mechanisms whereby autophagy in microglia may contribute to CNS disease and senescence. PMID:28282924

Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging.

PubMed

Plaza-Zabala, Ainhoa; Sierra-Torre, Virginia; Sierra, Amanda

2017-03-09

Autophagy is emerging as a core regulator of Central Nervous System (CNS) aging and neurodegeneration. In the brain, it has mostly been studied in neurons, where the delivery of toxic molecules and organelles to the lysosome by autophagy is crucial for neuronal health and survival. However, we propose that the (dys)regulation of autophagy in microglia also affects innate immune functions such as phagocytosis and inflammation, which in turn contribute to the pathophysiology of aging and neurodegenerative diseases. Herein, we first describe the basic concepts of autophagy and its regulation, discuss key aspects for its accurate monitoring at the experimental level, and summarize the evidence linking autophagy impairment to CNS senescence and disease. We focus on acute, chronic, and autoimmunity-mediated neurodegeneration, including ischemia/stroke, Alzheimer's, Parkinson's, and Huntington's diseases, and multiple sclerosis. Next, we describe the actual and potential impact of autophagy on microglial phagocytic and inflammatory function. Thus, we provide evidence of how autophagy may affect microglial phagocytosis of apoptotic cells, amyloid-β, synaptic material, and myelin debris, and regulate the progression of age-associated neurodegenerative diseases. We also discuss data linking autophagy to the regulation of the microglial inflammatory phenotype, which is known to contribute to age-related brain dysfunction. Overall, we update the current knowledge of autophagy and microglia, and highlight as yet unexplored mechanisms whereby autophagy in microglia may contribute to CNS disease and senescence.

Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

PubMed Central

Bozic, Iva; Savic, Danijela; Jovanovic, Marija; Bjelobaba, Ivana; Laketa, Danijela; Nedeljkovic, Nadezda; Stojiljkovic, Mirjana; Pekovic, Sanja; Lavrnja, Irena

2015-01-01

Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation. PMID:26413464

Surface functionality affects the biodistribution and microglia-targeting of intra-amniotically delivered dendrimers.

PubMed

Zhang, Fan; Nance, Elizabeth; Zhang, Zhi; Jasty, Venkatasai; Kambhampati, Siva P; Mishra, Manoj K; Burd, Irina; Romero, Roberto; Kannan, Sujatha; Kannan, Rangaramanujam M

2016-09-10

Cerebral Palsy (CP) is a chronic childhood disorder with limited therapeutic options. Maternal intrauterine inflammation/infection is a major risk factor in the pathogenesis of CP. In pre-clinical models, dendrimer-based therapies are viable in postnatal period, attenuating inflammation and improving motor function in vivo. However, treatment to the mother, in the prenatal period, may provide the possibility of preventing/resolving inflammation at early stages. Towards this goal, we used a maternal intrauterine inflammation-induced rabbit model of CP to study fetal-maternal transport and neuroinflammation targeting of intra-amniotically administrated dendrimers with neutral/anionic surface functionality. Our study suggested both hydroxyl-terminated 'neutral' (D-OH) and carboxyl-terminated 'anionic' (D-COOH) Polyamidoamine (PAMAM) dendrimers were absorbed by fetuses and demonstrated bi-directional transport between fetuses and mother. D-OH was more effective in crossing the fetal blood-brain barrier, and targeting activated microglia. The cell-specific targeting was associated with the extent of microglia activation. This study demonstrated intra-amniotically administered hydroxyl PAMAM dendrimers could be an effective drug delivery vehicle for targeting fetal inflammation and preventing subsequent neurologic injury associated with chorioamnionitis. Copyright © 2016 Elsevier B.V. All rights reserved.

Surface functionality affects the biodistribution and microglia-targeting of intra-amniotically delivered dendrimers☆

PubMed Central

Zhang, Fan; Nance, Elizabeth; Zhang, Zhi; Jasty, Venkatasai; Kambhampati, Siva P.; Mishra, Manoj K.; Burd, Irina; Romero, Roberto; Kannan, Sujatha; Kannan, Rangaramanujam M.

2017-01-01

Cerebral Palsy (CP) is a chronic childhood disorder with limited therapeutic options. Maternal intrauterine inflammation/infection is a major risk factor in the pathogenesis of CP. In pre-clinical models, dendrimer-based therapies are viable in postnatal period, attenuating inflammation and improving motor function in vivo. However, treatment to the mother, in the prenatal period, may provide the possibility of preventing/resolving inflammation at early stages. Towards this goal, we used a maternal intrauterine inflammation-induced rabbit model of CP to study fetal-maternal transport and neuroinflammation targeting of intra-amniotically administrated dendrimers with neutral/anionic surface functionality. Our study suggested both hydroxyl-terminated ‘neutral’ (D-OH) and carboxyl-terminated ‘anionic’ (D-COOH) Polyamidoamine (PAMAM) dendrimers were absorbed by fetuses and demonstrated bi-directional transport between fetuses and mother. D-OH was more effective in crossing the fetal blood-brain barrier, and targeting activated microglia. The cell-specific targeting was associated with the extent of microglia activation. This study demonstrated intra-amniotically administered hydroxyl PAMAM dendrimers could be an effective drug delivery vehicle for targeting fetal inflammation and preventing subsequent neurologic injury associated with chorioamnionitis. PMID:27378700

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

PubMed

Urbanus, Malene L; Quaile, Andrew T; Stogios, Peter J; Morar, Mariya; Rao, Chitong; Di Leo, Rosa; Evdokimova, Elena; Lam, Mandy; Oatway, Christina; Cuff, Marianne E; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw P; Taipale, Mikko; Savchenko, Alexei; Ensminger, Alexander W

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

PubMed Central

Catz, Sergio Daniel

2013-01-01

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection[OPEN

PubMed Central

Henry, Elizabeth; Jauneau, Alain; Deslandes, Laurent

2017-01-01

To cause disease, diverse pathogens deliver effector proteins into host cells. Pathogen effectors can inhibit defense responses, alter host physiology, and represent important cellular probes to investigate plant biology. However, effector function and localization have primarily been investigated after overexpression in planta. Visualizing effector delivery during infection is challenging due to the plant cell wall, autofluorescence, and low effector abundance. Here, we used a GFP strand system to directly visualize bacterial effectors delivered into plant cells through the type III secretion system. GFP is a beta barrel that can be divided into 11 strands. We generated transgenic Arabidopsis thaliana plants expressing GFP1-10 (strands 1 to 10). Multiple bacterial effectors tagged with the complementary strand 11 epitope retained their biological function in Arabidopsis and tomato (Solanum lycopersicum). Infection of plants expressing GFP1-10 with bacteria delivering GFP11-tagged effectors enabled direct effector detection in planta. We investigated the temporal and spatial delivery of GFP11-tagged effectors during infection with the foliar pathogen Pseudomonas syringae and the vascular pathogen Ralstonia solanacearum. Thus, the GFP strand system can be broadly used to investigate effector biology in planta. PMID:28600390

Early Microglia Activation Precedes Photoreceptor Degeneration in a Mouse Model of CNGB1-Linked Retinitis Pigmentosa.

PubMed

Blank, Thomas; Goldmann, Tobias; Koch, Mirja; Amann, Lukas; Schön, Christian; Bonin, Michael; Pang, Shengru; Prinz, Marco; Burnet, Michael; Wagner, Johanna E; Biel, Martin; Michalakis, Stylianos

2017-01-01

Retinitis pigmentosa (RP) denotes a family of inherited blinding eye diseases characterized by progressive degeneration of rod and cone photoreceptors in the retina. In most cases, a rod-specific genetic defect results in early functional loss and degeneration of rods, which is followed by degeneration of cones and loss of daylight vision at later stages. Microglial cells, the immune cells of the central nervous system, are activated in retinas of RP patients and in several RP mouse models. However, it is still a matter of debate whether activated microglial cells may be responsible for the amplification of the typical degenerative processes. Here, we used Cngb1 -/- mice, which represent a slow degenerative mouse model of RP, to investigate the extent of microglia activation in retinal degeneration. With a combination of FACS analysis, immunohistochemistry and gene expression analysis we established that microglia in the Cngb1 -/- retina were already activated in an early, predegenerative stage of the disease. The evidence available so far suggests that early retinal microglia activation represents a first step in RP, which might initiate or accelerate photoreceptor degeneration.

Norepinephrine enhances the LPS-induced expression of COX-2 and secretion of PGE2 in primary rat microglia

PubMed Central

2010-01-01

Background Recent studies suggest an important role for neurotransmitters as modulators of inflammation. Neuroinflammatory mediators such as cytokines and molecules of the arachidonic acid pathway are generated and released by microglia. The monoamine norepinephrine reduces the production of cytokines by activated microglia in vitro. However, little is known about the effects of norepinephrine on prostanoid synthesis. In the present study, we investigate the role of norepinephrine on cyclooxygenase- (COX-)2 expression/synthesis and prostaglandin (PG)E2 production in rat primary microglia. Results Interestingly, norepinephrine increased COX-2 mRNA, but not protein expression. Norepinephrine strongly enhanced COX-2 expression and PGE2 production induced by lipopolysaccharide (LPS). This effect is likely to be mediated by β-adrenoreceptors, since β-, but not α-adrenoreceptor agonists produced similar results. Furthermore, β-adrenoreceptor antagonists blocked the enhancement of COX-2 levels induced by norepinephrine and β-adrenoreceptor agonists. Conclusions Considering that PGE2 displays different roles in neuroinflammatory and neurodegenerative disorders, norepinephrine may play an important function in the modulation of these processes in pathophysiological conditions. PMID:20064241

Novel celecoxib analogues inhibit glial production of prostaglandin E2, nitric oxide, and oxygen radicals reverting the neuroinflammatory responses induced by misfolded prion protein fragment 90-231 or lipopolysaccharide.

PubMed

Villa, Valentina; Thellung, Stefano; Bajetto, Adriana; Gatta, Elena; Robello, Mauro; Novelli, Federica; Tasso, Bruno; Tonelli, Michele; Florio, Tullio

2016-11-01

We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E 2 (PGE 2 ) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE 2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Behind the lines–actions of bacterial type III effector proteins in plant cells

PubMed Central

Büttner, Daniela

2016-01-01

Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

Kv1.3 inhibition as a potential microglia-targeted therapy for Alzheimer's disease: preclinical proof of concept.

PubMed

Maezawa, Izumi; Nguyen, Hai M; Di Lucente, Jacopo; Jenkins, David Paul; Singh, Vikrant; Hilt, Silvia; Kim, Kyoungmi; Rangaraju, Srikant; Levey, Allan I; Wulff, Heike; Jin, Lee-Way

2018-02-01

Microglia significantly contribute to the pathophysiology of Alzheimer's disease but an effective microglia-targeted therapeutic approach is not yet available clinically. The potassium channels Kv1.3 and Kir2.1 play important roles in regulating immune cell functions and have been implicated by in vitro studies in the 'M1-like pro-inflammatory' or 'M2-like anti-inflammatory' state of microglia, respectively. We here found that amyloid-β oligomer-induced expression of Kv1.3 and Kir2.1 in cultured primary microglia. Likewise, ex vivo microglia acutely isolated from the Alzheimer's model 5xFAD mice co-expressed Kv1.3 and Kir2.1 as well as markers traditionally associated with M1 and M2 activation suggesting that amyloid-β oligomer induces a microglial activation state that is more complex than previously thought. Using the orally available, brain penetrant small molecule Kv1.3 blocker PAP-1 as a tool, we showed that pro-inflammatory and neurotoxic microglial responses induced by amyloid-β oligomer required Kv1.3 activity in vitro and in hippocampal slices. Since we further observed that Kv1.3 was highly expressed in microglia of transgenic Alzheimer's mouse models and human Alzheimer's disease brains, we hypothesized that pharmacological Kv1.3 inhibition could mitigate the pathology induced by amyloid-β aggregates. Indeed, treating APP/PS1 transgenic mice with a 5-month oral regimen of PAP-1, starting at 9 months of age, when the animals already manifest cognitive deficits and amyloid pathology, reduced neuroinflammation, decreased cerebral amyloid load, enhanced hippocampal neuronal plasticity, and improved behavioural deficits. The observed decrease in cerebral amyloid deposition was consistent with the in vitro finding that PAP-1 enhanced amyloid-β uptake by microglia. Collectively, these results provide proof-of-concept data to advance Kv1.3 blockers to Alzheimer's disease clinical trials. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Runx1t1 (Runt-Related Transcription Factor 1; Translocated to, 1) Epigenetically Regulates the Proliferation and Nitric Oxide Production of Microglia

PubMed Central

Baby, Nimmi; Li, Yali; Ling, Eng-Ang; Lu, Jia; Dheen, S. Thameem

2014-01-01

Background Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions. Methods and Findings siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2. Conclusion/Interpretation In conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs. PMID:24586690

Regulation of Therapeutic Hypothermia on Inflammatory Cytokines, Microglia Polarization, Migration and Functional Recovery after Ischemic Stroke in Mice

PubMed Central

Lee, Jin Hwan; Wei, Zheng Z; Cao, Wenyuan; Won, Soonmi; Gu, Xiaohuan; Winter, Megan; Dix, Thomas A.; Wei, Ling; Yu, Shan Ping

2016-01-01

Stroke is a leading threat to human life and health in the US and around the globe, while very few effective treatments are available for stroke patients. Preclinical and clinical studies have shown that therapeutic hypothermia (TH) is a potential treatment for stroke. Using novel neurotensin receptor 1 (NTR1) agonists, we have demonstrated pharmacologically induced hypothermia and protective effects against brain damages after ischemic stroke, hemorrhage stroke, and traumatic brain injury (TBI) in rodent models. To further characterize the mechanism of TH-induced brain protection, we examined the effect of TH (at ±33°C for 6 hrs) induced by the NTR1 agonist HPI-201 or physical (ice/cold air) cooling on inflammatory responses after ischemic stroke in mice and oxygen glucose deprivation (OGD) in cortical neuronal cultures. Seven days after focal cortical ischemia, microglia activation in the penumbra reached a peak level, which was significantly attenuated by TH treatments commenced 30 min after stroke. The TH treatment decreased the expression of M1 type reactive factors including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-12, IL-23, and inducible nitric oxide synthase (iNOS) measured by RT-PCR and Western blot analyses. Meanwhile, TH treatments increased the expression of M2 type reactive factors including IL-10, Fizz1, Ym1, and arginase-1. In the ischemic brain and in cortical neuronal/BV2 microglia cultures subjected to OGD, TH attenuated the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), two key chemokines in the regulation of microglia activation and infiltration. Consistently, physical cooling during OGD significantly decreased microglia migration 16 hrs after OGD. Finally, TH improved functional recovery at 1, 3, and 7 days after stroke. This study reveals the first evidence for hypothermia mediated regulation on inflammatory factor expression, microglia polarization, migration and indicates that the anti-inflammatory effect is an important mechanism underlying the brain protective effects of a TH therapy. PMID:27659107

Classical and alternative complement activation on photoreceptor outer segments drives monocyte-dependent retinal atrophy.

PubMed

Katschke, Kenneth J; Xi, Hongkang; Cox, Christian; Truong, Tom; Malato, Yann; Lee, Wyne P; McKenzie, Brent; Arceo, Rommel; Tao, Jianhua; Rangell, Linda; Reichelt, Mike; Diehl, Lauri; Elstrott, Justin; Weimer, Robby M; Campagne, Menno van Lookeren

2018-05-09

Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.

Phytophthora suppressor of RNA silencing 2 is a conserved RxLR effector that promotes infection in soybean and Arabidopsis thaliana.

PubMed

Xiong, Qin; Ye, Wenwu; Choi, Duseok; Wong, James; Qiao, Yongli; Tao, Kai; Wang, Yuanchao; Ma, Wenbo

2014-12-01

The genus Phytophthora consists of notorious and emerging pathogens of economically important crops. Each Phytophthora genome encodes several hundreds of cytoplasmic effectors, which are believed to manipulate plant immune response inside the host cells. However, the majority of Phytophthora effectors remain functionally uncharacterized. We recently discovered two effectors from the soybean stem and root rot pathogen Phytophthora sojae with the activity to suppress RNA silencing in plants. These effectors are designated Phytophthora suppressor of RNA silencing (PSRs). Here, we report that the P. sojae PSR2 (PsPSR2) belongs to a conserved and widespread effector family in Phytophthora. A PsPSR2-like effector produced by P. infestans (PiPSR2) can also suppress RNA silencing in plants and promote Phytophthora infection, suggesting that the PSR2 family effectors have conserved functions in plant hosts. Using Agrobacterium rhizogenes-mediated hairy roots induction, we demonstrated that the expression of PsPSR2 rendered hypersusceptibility of soybean to P. sojae. Enhanced susceptibility was also observed in PsPSR2-expressing Arabidopsis thaliana plants during Phytophthora but not bacterial infection. These experiments provide strong evidence that PSR2 is a conserved Phytophthora effector family that performs important virulence functions specifically during Phytophthora infection of various plant hosts.

Macroglia-Microglia Interactions via TSPO Signaling Regulates Microglial Activation in the Mouse Retina

PubMed Central

Wang, Minhua; Wang, Xu; Zhao, Lian; Ma, Wenxin; Rodriguez, Ignacio R.; Fariss, Robert N.

2014-01-01

Chronic retinal inflammation in the form of activated microglia and macrophages are implicated in the etiology of neurodegenerative diseases of the retina, including age-related macular degeneration, diabetic retinopathy, and glaucoma. However, molecular biomarkers and targeted therapies for immune cell activation in these disorders are currently lacking. To address this, we investigated the involvement and role of translocator protein (TSPO), a biomarker of microglial and astrocyte gliosis in brain degeneration, in the context of retinal inflammation. Here, we find that TSPO is acutely and specifically upregulated in retinal microglia in separate mouse models of retinal inflammation and injury. Concomitantly, its endogenous ligand, diazepam-binding inhibitor (DBI), is upregulated in the macroglia of the mouse retina such as astrocytes and Müller cells. In addition, we discover that TSPO-mediated signaling in microglia via DBI-derived ligands negatively regulates features of microglial activation, including reactive oxygen species production, TNF-α expression and secretion, and microglial proliferation. The inducibility and effects of DBI-TSPO signaling in the retina reveal a mechanism of coordinated macroglia-microglia interactions, the function of which is to limit the magnitude of inflammatory responses after their initiation, facilitating a return to baseline quiescence. Our results indicate that TSPO is a promising molecular marker for imaging inflammatory cell activation in the retina and highlight DBI-TSPO signaling as a potential target for immodulatory therapies. PMID:24599476

Feeding the beast: can microglia in the senescent brain be regulated by diet?

PubMed

Johnson, Rodney W

2015-01-01

Microglial cells, resident macrophages in the central nervous system (CNS), are relatively quiescent but can respond to signals from the peripheral immune system and induce neuroinflammation. In aging, microglia tend to transition to the M1 pro-inflammatory state and become hypersensitive to messages emerging from immune-to-brain signaling pathways. Thus, whereas in younger individuals where microglia respond to signals from the peripheral immune system and induce a well-controlled neuroinflammatory response that is adaptive (e.g., when well controlled, fever and sickness behavior facilitate recovery from infection), in older individuals with an infection, microglia overreact and produce excessive levels of inflammatory cytokines causing behavioral pathology including cognitive dysfunction. Importantly, recent studies indicate a number of naturally occurring bioactive compounds present in certain foods have anti-inflammatory properties and are capable of mitigating brain microglial cells. These include, e.g., flavonoid and non-flavonoid compounds in fruits and vegetables, and n-3 polyunsaturated fatty acids (PUFA) in oily fish. Thus, dietary bioactives have potential to restore the population of microglial cells in the senescent brain to a more quiescent state. The pragmatic concept to constrain microglia through dietary intervention is significant because neuroinflammation and cognitive deficits are co-morbid factors in many chronic inflammatory diseases. Controlling microglial cell reactivity has important consequences for preserving adult neurogenesis, neuronal structure and function, and cognition. Copyright © 2014 Elsevier Inc. All rights reserved.

Microglia used as vehicles for both inducible thymidine kinase gene therapy and MRI contrast agents for glioma therapy.

PubMed

Ribot, E; Bouzier-Sore, A-K; Bouchaud, V; Miraux, S; Delville, M-H; Franconi, J-M; Voisin, P

2007-08-01

Microglia are phagocytic cells that are chemoattracted by brain tumors and can represent up to 70% of the tumor cell population. To get insight into gene therapy against glioma, we decided to take advantage of those microglia properties and to use those cells as vehicles to transport simultaneously a suicide gene (under the control of a heat-sensitive promoter) and contrast agents to localize them by magnetic resonance imaging before applying any therapeutic treatment. Thymidine kinase (TK) expression and its functionality after gancyclovir administration were investigated. After the heat shock (44 degrees C and 20 min), TK was expressed in 50% of the cells. However, after gancyclovir treatment, 90% of the cells died by apoptosis, showing an important bystander effect. Then, the cells were incubated with new lanthanide contrast agents to check both their potential toxicity and their MR properties. Results indicate that the nanoparticles did not induce any cell toxicity and yield a hypersignal on MR images at 4.7 T. These in vitro experiments indicate that microglia are good candidates as vectors in gene therapy against brain tumors. Finally, microglia containing gadolinium-grafted nanoparticles were injected in the close vicinity of C6 tumor, in a mouse. The hyperintensive signal obtained on in vivo images as well as its retention time show the potential of the novel contrast agents for cellular imaging.

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro.

PubMed

Peake, Kyle B; Campenot, Robert B; Vance, Dennis E; Vance, Jean E

2011-09-01

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1(-/-) mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1(+/+) and Npc1(-/-) microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1(-/-) microglia is ~60% lower than in Npc1(+/+) microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1(-/-) and Npc1(+/+) neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1(-/-) microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease. Copyright © 2011 Elsevier B.V. All rights reserved.

Monocyte trafficking to the brain with stress and inflammation: a novel axis of immune-to-brain communication that influences mood and behavior

PubMed Central

Wohleb, Eric S.; McKim, Daniel B.; Sheridan, John F.; Godbout, Jonathan P.

2015-01-01

HIGHLIGHTS Psychological stress activates neuroendocrine pathways that alter immune responses.Stress-induced alterations in microglia phenotype and monocyte priming leads to aberrant peripheral and central inflammation.Elevated pro-inflammatory cytokine levels caused by microglia activation and recruitment of monocytes to the brain contribute to development and persistent anxiety-like behavior.Mechanisms that mediate interactions between microglia, endothelial cells, and macrophages and how these contribute to changes in behavior are discussed.Sensitization of microglia and re-distribution of primed monocytes are implicated in re-establishment of anxiety-like behavior. Psychological stress causes physiological, immunological, and behavioral alterations in humans and rodents that can be maladaptive and negatively affect quality of life. Several lines of evidence indicate that psychological stress disrupts key functional interactions between the immune system and brain that ultimately affects mood and behavior. For example, activation of microglia, the resident innate immune cells of the brain, has been implicated as a key regulator of mood and behavior in the context of prolonged exposure to psychological stress. Emerging evidence implicates a novel neuroimmune circuit involving microglia activation and sympathetic outflow to the peripheral immune system that further reinforces stress-related behaviors by facilitating the recruitment of inflammatory monocytes to the brain. Evidence from various rodent models, including repeated social defeat (RSD), revealed that trafficking of monocytes to the brain promoted the establishment of anxiety-like behaviors following prolonged stress exposure. In addition, new evidence implicates monocyte trafficking from the spleen to the brain as key regulator of recurring anxiety following exposure to prolonged stress. The purpose of this review is to discuss mechanisms that cause stress-induced monocyte re-distribution in the brain and how dynamic interactions between microglia, endothelial cells, and brain macrophages lead to maladaptive behavioral responses. PMID:25653581

Microglial Morphology and Dynamic Behavior Is Regulated by Ionotropic Glutamatergic and GABAergic Neurotransmission

PubMed Central

Fontainhas, Aurora M.; Wang, Minhua; Liang, Katharine J.; Chen, Shan; Mettu, Pradeep; Damani, Mausam; Fariss, Robert N.; Li, Wei; Wong, Wai T.

2011-01-01

Purpose Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior. Methods We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia. Results Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels. Conclusions Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity. PMID:21283568

Sleep and immune function: glial contributions and consequences of aging

PubMed Central

Ingiosi, Ashley M.; Opp, Mark R.; Krueger, James M.

2013-01-01

The reciprocal interactions between sleep and immune function are well-studied. Insufficient sleep induces innate immune responses as evidenced by increased expression of pro-inflammatory mediators in the brain and periphery. Conversely, immune challenges upregulate immunomodulator expression, which alters central nervous system-mediated processes and behaviors, including sleep. Recent studies indicate that glial cells, namely microglia and astrocytes, are active contributors to sleep and immune system interactions. Evidence suggests glial regulation of these interactions is mediated, in part, by adenosine and adenosine 5′-triphosphate actions at purinergic type 1 and type 2 receptors. Furthermore, microglia and astrocytes may modulate declines in sleep-wake behavior and immunity observed in aging. PMID:23452941

Sleep and immune function: glial contributions and consequences of aging.

PubMed

Ingiosi, Ashley M; Opp, Mark R; Krueger, James M

2013-10-01

The reciprocal interactions between sleep and immune function are well-studied. Insufficient sleep induces innate immune responses as evidenced by increased expression of pro-inflammatory mediators in the brain and periphery. Conversely, immune challenges upregulate immunomodulator expression, which alters central nervous system-mediated processes and behaviors, including sleep. Recent studies indicate that glial cells, namely microglia and astrocytes, are active contributors to sleep and immune system interactions. Evidence suggests glial regulation of these interactions is mediated, in part, by adenosine and adenosine 5'-triphosphate actions at purinergic type 1 and type 2 receptors. Furthermore, microglia and astrocytes may modulate declines in sleep-wake behavior and immunity observed in aging. Copyright © 2013. Published by Elsevier Ltd.

Space-based multifunctional end effector systems functional requirements and proposed designs

NASA Technical Reports Server (NTRS)

Mishkin, A. H.; Jau, B. M.

1988-01-01

The end effector is an essential element of teleoperator and telerobot systems to be employed in space in the next decade. The report defines functional requirements for end effector systems to perform operations that are currently only feasible through Extra-Vehicular Activity (EVA). Specific tasks and functions that the end effectors must be capable of performing are delineated. Required capabilities for forces and torques, clearances, compliance, and sensing are described, using current EVA requirements as guidelines where feasible. The implications of these functional requirements on the elements of potential end effector systems are discussed. The systems issues that must be considered in the design of space-based manipulator systems are identified; including impacts on subsystems tightly coupled to the end effector, i.e., control station, information processing, manipulator arm, tool and equipment stowage. Possible end effector designs are divided into three categories: single degree-of-freedom end effectors, multiple degree of freedom end effectors, and anthropomorphic hands. Specific design alternatives are suggested and analyzed within the individual categories. Two evaluations are performed: the first considers how well the individual end effectors could substitute for EVA; the second compares how manipulator systems composed of the top performers from the first evaluation would improve the space shuttle Remote Manipulator System (RMS) capabilities. The analysis concludes that the anthropomorphic hand is best-suited for EVA tasks. A left- and right-handed anthropomorphic manipulator arm configuration is suggested as appropriate to be affixed to the RMS, but could also be used as part of the Smart Front End for the Orbital Maneuvering Vehicle (OMV). The technical feasibility of the anthropomorphic hand and its control are demonstrated. An evolutionary development approach is proposed and approximate scheduling provided for implementing the suggested manipulator systems in time for space stations operations in the early 1990s.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

PubMed Central

Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

PubMed Central

Charpentier, Xavier; Gabay, Joëlle E.; Reyes, Moraima; Zhu, Jing W.; Weiss, Arthur; Shuman, Howard A.

2009-01-01

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions. PMID:19578436

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

PubMed Central

Shames, Stephanie R.; Liu, Luying; Havey, James C.; Schofield, Whitman B.; Goodman, Andrew L.; Roy, Craig R.

2017-01-01

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis. PMID:29133401

RNA-Seq Analysis of Microglia Reveals Time-Dependent Activation of Specific Genetic Programs following Spinal Cord Injury

PubMed Central

Noristani, Harun N.; Gerber, Yannick N.; Sabourin, Jean-Charles; Le Corre, Marine; Lonjon, Nicolas; Mestre-Frances, Nadine; Hirbec, Hélène E.; Perrin, Florence E.

2017-01-01

Neurons have inherent competence to regrow following injury, although not spontaneously. Spinal cord injury (SCI) induces a pronounced neuroinflammation driven by resident microglia and infiltrating peripheral macrophages. Microglia are the first reactive glial population after SCI and participate in recruitment of monocyte-derived macrophages to the lesion site. Both positive and negative influence of microglia and macrophages on axonal regeneration had been reported after SCI, raising the issue whether their response depends on time post-lesion or different lesion severity. We analyzed molecular alterations in microglia at several time-points after different SCI severities using RNA-sequencing. We demonstrate that activation of microglia is time-dependent post-injury but is independent of lesion severity. Early transcriptomic response of microglia after SCI involves proliferation and neuroprotection, which is then switched to neuroinflammation at later stages. Moreover, SCI induces an autologous microglial expression of astrocytic markers with over 6% of microglia expressing glial fibrillary acidic protein and vimentin from as early as 72 h post-lesion and up to 6 weeks after injury. We also identified the potential involvement of DNA damage and in particular tumor suppressor gene breast cancer susceptibility gene 1 (Brca1) in microglia after SCI. Finally, we established that BRCA1 protein is specifically expressed in non-human primate spinal microglia and is upregulated after SCI. Our data provide the first transcriptomic analysis of microglia at multiple stages after different SCI severities. Injury-induced microglia expression of astrocytic markers at RNA and protein levels demonstrates novel insights into microglia plasticity. Finally, increased microglia expression of BRCA1 in rodents and non-human primate model of SCI, suggests the involvement of oncogenic proteins after CNS lesion. PMID:28420963

Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation.

PubMed

Huang, Zijing; Zhou, Tian; Sun, Xiaowei; Zheng, Yingfeng; Cheng, Bing; Li, Mei; Liu, Xialin; He, Chang

2018-01-01

Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases.

Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury.

PubMed

Fan, H; Tang, H-B; Kang, J; Shan, L; Song, H; Zhu, K; Wang, J; Ju, G; Wang, Y-Z

2015-12-17

Microglia/macrophages play a crucial role in inflammation after spinal cord injury (SCI). Although extensive studies have been performed on the mechanisms of microglia/macrophage activation and recruitment, how microglia/macrophages are eliminated remains unclear. In the present study, we observed a high-level expression of mixed lineage kinase domain-like protein (MLKL), a key molecule in the execution of necroptosis, in microglia/macrophages after SCI in mice. In vivo PI-labeling and Necrostatin-1 treatment confirmed the necroptosis of microglia/macrophages. Interestingly, our electronic microscopic (EM) study revealed that MLKL localized not only at the membrane but also on the endoplasmic reticulum (ER) of necroptotic microglia/macrophages. Furthermore, receptor-interacting protein 3 (RIP3), another necrosome component, was also found on the ER of necroptotic microglia/macrophages. And Glucose-regulated protein 78 (GRP78), an ER stress sensor, was up-regulated in MLKL-positive microglia/macrophages after SCI, suggesting a possible link between necroptosis and ER stress. In vitro, oxygen-glucose deprivation (OGD) stress induced ER stress and necroptosis in microglia. Inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly blocked the OGD-induced necroptosis of microglia. In the end, our data showed that, GRP78 and phosphorylated MLKL were co-expressed by the microglia/macrophages in the injured human spinal cord. Taken together, these results suggested that microglia/macrophages undergo an ER-stress involved necroptosis after SCI, implying that ER stress and necroptosis could be manipulated for modulating inflammation post-SCI. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation

PubMed Central

Huang, Zijing; Zhou, Tian; Sun, Xiaowei; Zheng, Yingfeng; Cheng, Bing; Li, Mei; Liu, Xialin; He, Chang

2018-01-01

Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases. PMID:28885615

Cross talk between AT1 receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus.

PubMed

Biancardi, Vinicia Campana; Stranahan, Alexis M; Krause, Eric G; de Kloet, Annette D; Stern, Javier E

2016-02-01

ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (∼65% increase, P < 0.001), an effect that was blunted in the absence of functional TLR4. ANG II increased ROS production, as indicated by dihydroethidium fluorescence, within the PVN of rats and mice (P < 0.0001 in both cases), effects that were also dependent on the presence of functional TLR4. The microglial inhibitor minocycline attenuated ANG II-mediated ROS production, yet ANG II effects persisted in PVN single-minded 1-AT1a knockout mice, supporting the contribution of a non-neuronal source (likely microglia) to ANG II-driven ROS production in the PVN. Taken together, these results support functional interactions between AT1 receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN. Copyright © 2016 the American Physiological Society.

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia.

PubMed

Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R; Aranda, Jacob; Grant, Maria B; Chaqour, Brahim

2015-09-18

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3'-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia*

PubMed Central

Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R.; Aranda, Jacob; Grant, Maria B.; Chaqour, Brahim

2015-01-01

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair. PMID:26242736

Cellular players that shape evolving pathology and neurodegeneration following traumatic brain injury.

PubMed

Puntambekar, Shweta S; Saber, Maha; Lamb, Bruce T; Kokiko-Cochran, Olga N

2018-03-27

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide, and has emerged as a critical risk factor for multiple neurodegenerative diseases, particularly Alzheimer's disease (AD). How the inflammatory cascade resulting from mechanical stress, axonal shearing and the loss of neurons and glia following initial impact in TBI, contributes to the development of AD-like disease is unclear. Neuroinflammation, characterized by blood-brain barrier (BBB) dysfunction and activation of brain-resident microglia and astrocytes, resulting in secretion of inflammatory mediators and subsequent recruitment of peripheral immune cells has been the focus of extensive research in attempts to identify drug-targets towards improving functional outcomes post TBI. While knowledge of intricate cellular interactions that shape lesion pathophysiology is incomplete, a major limitation in the field is the lack of understanding of how distinct cell types differentially alter TBI pathology. The aim of this review is to highlight functional differences between populations of bone marrow derived, infiltrating monocytes/macrophages and brain-resident microglia based on differential expression of the chemokine receptors CCR2 and CX 3 CR1. This review will focus on how unique subsets of mononuclear phagocytes shape TBI pathophysiology, neurotoxicity and BBB function, in a disease-stage dependent manner. Additionally, this review summarizes the role of multiple microglia and macrophage receptors, namely CCR2, CX 3 CR1 and Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) in pathological neuroinflammation and neurodegeneration vs. recovery following TBI. TREM2 has been implicated in mediating AD-related pathology, and variants in TREM2 are particularly important due to their correlation with exacerbated neurodegeneration. Finally, this review highlights behavioral outcomes associated with microglial vs. macrophage variances, the need for novel treatment strategies that target unique subpopulations of peripheral macrophages, and the importance of development of therapeutics to modulate inflammatory functions of brain-resident microglia at specific stages of TBI. Copyright © 2018 Elsevier Inc. All rights reserved.

18β-glycyrrhetinic acid suppresses experimental autoimmune encephalomyelitis through inhibition of microglia activation and promotion of remyelination.

PubMed

Zhou, Jieru; Cai, Wei; Jin, Min; Xu, Jingwei; Wang, Yanan; Xiao, Yichuan; Hao, Li; Wang, Bei; Zhang, Yanyun; Han, Jie; Huang, Rui

2015-09-02

Microglia are intrinsic immune cells in the central nervous system (CNS). The under controlled microglia activation plays important roles in inflammatory demyelination diseases, such as multiple sclerosis (MS). However, the means to modulate microglia activation as a therapeutic modality and the underlying mechanisms remain elusive. Here we show that administration of 18β-glycyrrhetinic acid (GRA), by using both preventive and therapeutic treatment protocols, significantly suppresses disease severity of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The treatment effect of GRA on EAE is attributed to its regulatory effect on microglia. GRA-modulated microglia significantly decreased pro-inflammatory profile in the CNS through suppression of MAPK signal pathway. The ameliorated CNS pro-inflammatory profile prevented the recruitment of encephalitogenic T cells into the CNS, which alleviated inflammation-induced demyelination. In addition, GRA treatment promoted remyelination in the CNS of EAE mice. The induced remyelination can be mediated by the overcome of inflammation-induced blockade of brain-derived neurotrophic factor expression in microglia, as well as enhancing oligodendrocyte precursor cell proliferation. Collectively, our results demonstrate that GRA-modulated microglia suppresses EAE through inhibiting microglia activation-mediated CNS inflammation, and promoting neuroprotective effect of microglia, which represents a potential therapeutic strategy for MS and maybe other neuroinflammatory diseases associated with microglia activation.

High immunosuppressive burden in advanced hepatocellular carcinoma patients: Can effector functions be restored?

PubMed

Lugade, Amit A; Kalathil, Suresh; Miller, Austin; Iyer, Renuka; Thanavala, Yasmin

2013-07-01

The accumulation of immunosuppressive cells and exhausted effector T cells highlight an important immune dysfunction in advanced stage hepatocellular carcinoma (HCC) patients. These cells significantly hamper the efficacy immunotherapies and facilitate HCC progression. We have recently demonstrated that the multipronged depletion of immunosuppressive cells potentially restores effector T-cell function in HCC.

Identification of legionella effectors using bioinformatic approaches.

PubMed

Segal, Gil

2013-01-01

Legionella pneumophila the causative agent of Legionnaires' disease, actively manipulates host cell processes to establish a replication niche inside host cells. The establishment of its replication niche requires a functional Icm/Dot type IV secretion system which translocates about 300 effector proteins into host cells during infection. Many of these effectors were first identified as effector candidates by several bioinformatic approaches, and these predicted effectors were later examined experimentally for translocation and a large number of which were validated as effector proteins. Here, I summarized the bioinformatic approaches that were used to identify these effectors.

Glucose pathways adaptation supports acquisition of activated microglia phenotype.

PubMed

Gimeno-Bayón, J; López-López, A; Rodríguez, M J; Mahy, N

2014-06-01

With its capacity to survey the environment and phagocyte debris, microglia assume a diversity of phenotypes to respond specifically through neurotrophic and toxic effects. Although these roles are well accepted, the underlying energetic mechanisms associated with microglial activation remain largely unclear. This study investigates microglia metabolic adaptation to ATP, NADPH, H(+) , and reactive oxygen species production. To this end, in vitro studies were performed with BV-2 cells before and after activation with lipopolysaccharide + interferon-γ. Nitric oxide (NO) was measured as a marker of cell activation. Our results show that microglial activation triggers a metabolic reprogramming based on an increased glucose uptake and a strengthening of anaerobic glycolysis, as well as of the pentose pathway oxidative branch, while retaining the mitochondrial activity. Based on this energy commitment, microglial defense capacity increases rapidly as well as ribose-5-phosphate and nucleic acid formation for gene transcription, essential to ensure the newly acquired functions demanded by central nervous system signaling. We also review the role of NO in this microglial energy commitment that positions cytotoxic microglia within the energetics of the astrocyte-neuron lactate shuttle. Copyright © 2014 Wiley Periodicals, Inc.

Significance and In Vivo Detection of Iron-Laden Microglia in White Matter Multiple Sclerosis Lesions

PubMed Central

Gillen, Kelly M.; Mubarak, Mayyan; Nguyen, Thanh D.; Pitt, David

2018-01-01

Microglia are resident immune cells that fulfill protective and homeostatic functions in the central nervous system (CNS) but may also promote neurotoxicity in the aged brain and in chronic disease. In multiple sclerosis (MS), an autoimmune demyelinating disease of the CNS, microglia and macrophages contribute to the development of white matter lesions through myelin phagocytosis, and possibly to disease progression through diffuse activation throughout myelinated white matter. In this review, we discuss an additional compartment of myeloid cell activation in MS, i.e., the rim and normal adjacent white matter of chronic active lesions. In chronic active lesions, microglia and macrophages may contain high amounts of iron, express markers of proinflammatory polarization, are activated for an extended period of time (years), and drive chronic tissue damage. Iron-positive myeloid cells can be visualized and quantified with quantitative susceptibility mapping (QSM), a magnetic resonance imaging technique. Thus, QSM has potential as an in vivo biomarker for chronic inflammatory activity in established white matter MS lesions. Reducing chronic inflammation associated with iron accumulation using existing or novel MS therapies may impact disease severity and progression. PMID:29515576

TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke.

PubMed

Doyle, Kristian P; Cekanaviciute, Egle; Mamer, Lauren E; Buckwalter, Marion S

2010-10-11

TGFβ is both neuroprotective and a key immune system modulator and is likely to be an important target for future stroke therapy. The precise function of increased TGF-β1 after stroke is unknown and its pleiotropic nature means that it may convey a neuroprotective signal, orchestrate glial scarring or function as an important immune system regulator. We therefore investigated the time course and cell-specificity of TGFβ signaling after stroke, and whether its signaling pattern is altered by gender and aging. We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To determine which cell type is the source of increased TGFβ production after stroke, brain sections were stained with an anti-TGFβ antibody, colocalized with markers for reactive astrocytes, neurons, and activated microglia. To determine which cells are responding to TGFβ after stroke, brain sections were double-labelled with anti-pSmad2, a marker of TGFβ signaling, and markers of neurons, oligodendrocytes, endothelial cells, astrocytes and microglia. TGFβ signaling increased 2 fold after stroke, beginning on day 1 and peaking on day 7. This pattern of increase was preserved in old animals and absolute TGFβ signaling in the brain increased with age. Activated microglia and macrophages were the predominant source of increased TGFβ after stroke and astrocytes and activated microglia and macrophages demonstrated dramatic upregulation of TGFβ signaling after stroke. TGFβ signaling in neurons and oligodendrocytes did not undergo marked changes. We found that TGFβ signaling increases with age and that astrocytes and activated microglia and macrophages are the main cell types that undergo increased TGFβ signaling in response to post-stroke increases in TGFβ. Therefore increased TGFβ after stroke likely regulates glial scar formation and the immune response to stroke.

CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

PubMed Central

Hu, Shuxian; Sheng, Wen S.; Rock, R. Bryan

2013-01-01

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB1/CB2 agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB2 receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety of ways, acting principally through the CB2 receptors and microglia. PMID:24147028

Neurotransmitter receptors on microglia

PubMed Central

Liu, Huan; Leak, Rehana K; Hu, Xiaoming

2016-01-01

As the resident immune cells in the central nervous system, microglia have long been hypothesised to promote neuroinflammation and exacerbate neurotoxicity. However, this traditional view has undergone recent revision as evidence has accumulated that microglia exert beneficial and detrimental effects depending on activation status, polarisation phenotype and cellular context. A variety of neurotransmitter receptors are expressed on microglia and help mediate the bidirectional communication between neurons and microglia. Here we review data supporting the importance of neurotransmitter receptors on microglia, with a special emphasis on glutamate, γ-aminobutyric acid (GABA), norepinephrine, cannabinoid and acetylcholine receptors. We summarise evidence favouring a significant role for neurotransmitter receptors in modulating microglial activation, phagocytic clearance and phenotypic polarisation. Elucidating the effects of neurotransmitter receptors on microglia and dissecting the underlying mechanisms may help accelerate the discovery of novel drugs that tap the therapeutic potential of microglia. PMID:28959464

Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

USDA-ARS?s Scientific Manuscript database

Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

QueTAL: a suite of tools to classify and compare TAL effectors functionally and phylogenetically

PubMed Central

Pérez-Quintero, Alvaro L.; Lamy, Léo; Gordon, Jonathan L.; Escalon, Aline; Cunnac, Sébastien; Szurek, Boris; Gagnevin, Lionel

2015-01-01

Transcription Activator-Like (TAL) effectors from Xanthomonas plant pathogenic bacteria can bind to the promoter region of plant genes and induce their expression. DNA-binding specificity is governed by a central domain made of nearly identical repeats, each determining the recognition of one base pair via two amino acid residues (a.k.a. Repeat Variable Di-residue, or RVD). Knowing how TAL effectors differ from each other within and between strains would be useful to infer functional and evolutionary relationships, but their repetitive nature precludes reliable use of traditional alignment methods. The suite QueTAL was therefore developed to offer tailored tools for comparison of TAL effector genes. The program DisTAL considers each repeat as a unit, transforms a TAL effector sequence into a sequence of coded repeats and makes pair-wise alignments between these coded sequences to construct trees. The program FuncTAL is aimed at finding TAL effectors with similar DNA-binding capabilities. It calculates correlations between position weight matrices of potential target DNA sequence predicted from the RVD sequence, and builds trees based on these correlations. The programs accurately represented phylogenetic and functional relationships between TAL effectors using either simulated or literature-curated data. When using the programs on a large set of TAL effector sequences, the DisTAL tree largely reflected the expected species phylogeny. In contrast, FuncTAL showed that TAL effectors with similar binding capabilities can be found between phylogenetically distant taxa. This suite will help users to rapidly analyse any TAL effector genes of interest and compare them to other available TAL genes and should improve our understanding of TAL effectors evolution. It is available at http://bioinfo-web.mpl.ird.fr/cgi-bin2/quetal/quetal.cgi. PMID:26284082

Innate immune activation in neurodegenerative disease.

PubMed

Heneka, Michael T; Kummer, Markus P; Latz, Eicke

2014-07-01

The triggering of innate immune mechanisms is emerging as a crucial component of major neurodegenerative diseases. Microglia and other cell types in the brain can be activated in response to misfolded proteins or aberrantly localized nucleic acids. This diverts microglia from their physiological and beneficial functions, and leads to their sustained release of pro-inflammatory mediators. In this Review, we discuss how the activation of innate immune signalling pathways - in particular, the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome - by aberrant host proteins may be a common step in the development of diverse neurodegenerative disorders. During chronic activation of microglia, the sustained exposure of neurons to pro-inflammatory mediators can cause neuronal dysfunction and contribute to cell death. As chronic neuroinflammation is observed at relatively early stages of neurodegenerative disease, targeting the mechanisms that drive this process may be useful for diagnostic and therapeutic purposes.

Microglia and neuroprotection: implications for Alzheimer's disease.

PubMed

Streit, Wolfgang J

2005-04-01

The first part of this paper summarizes some of the key observations from experimental work in animals that support a role of microglia as neuroprotective cells after acute neuronal injury. These studies point towards an important role of neuronal-microglial crosstalk in the facilitation of neuroprotection. Conceptually, injured neurons are thought to generate rescue signals that trigger microglial activation and, in turn, activated microglia produce trophic or other factors that help damaged neurons recover from injury. Against this background, the second part of this paper summarizes recent work from postmortem studies conducted in humans that have revealed the occurrence of senescent, or dystrophic, microglial cells in the aged and Alzheimer's disease brain. These findings suggest that microglial cells become increasingly dysfunctional with advancing age and that a loss of microglial cell function may involve a loss of neuroprotective properties that could contribute to the development of aging-related neurodegeneration.

Microglial activation in white matter lesions and nonlesional white matter of ageing brains.

PubMed

Simpson, J E; Ince, P G; Higham, C E; Gelsthorpe, C H; Fernando, M S; Matthews, F; Forster, G; O'Brien, J T; Barber, R; Kalaria, R N; Brayne, C; Shaw, P J; Stoeber, K; Williams, G H; Lewis, C E; Wharton, S B

2007-12-01

White matter lesions (WML), a common feature in brain ageing, are classified as periventricular (PVL) or deep subcortical (DSCL), depending on their anatomical location. Microglial activation is implicated in a number of neurodegenerative diseases, but the microglial response in WML is poorly characterized and its role in pathogenesis unknown. We have characterized the microglial response in WML and control white matter using immunohistochemistry to markers of microglial activation and of proliferation. WML of brains from an unbiased population-based autopsy cohort (Medical Research Council's Cognitive Function and Ageing Study) were identified by post mortem magnetic resonance imaging and sampled for histology. PVL contain significantly more activated microglia, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules B7-2 and CD40, than either control white matter (WM) or DSCL. Furthermore, we show that significantly more microglia express the replication licensing protein minichromosome maintenance protein 2 within PVL, suggesting this is a more proliferation-permissive environment than DSCL. Although microglial activation occurs in both PVL and DSCL, our findings suggest a difference in pathogenesis between these lesion-types: the ramified, activated microglia associated with PVL may reflect immune activation resulting from disruption of the blood brain barrier, while the microglia within DSCL may reflect an innate, amoeboid phagocytic phenotype. We also show that microglia in control WM from lesional cases express significantly more MHC II than control WM from nonlesional ageing brain, suggesting that WML occur in a 'field-effect' of abnormal WM.

Interferon-γ and Tumor Necrosis Factor-α Regulate Amyloid-β Plaque Deposition and β-Secretase Expression in Swedish Mutant APP Transgenic Mice

PubMed Central

Yamamoto, Masaru; Kiyota, Tomomi; Horiba, Masahide; Buescher, James L.; Walsh, Shannon M.; Gendelman, Howard E.; Ikezu, Tsuneya

2007-01-01

Reactive astrocytes and microglia in Alzheimer’s disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-β peptide (Aβ) accumulation is poorly understood. Thus, we generated a novel Swedish β-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-γ receptor type I was knocked out (APP/GRKO). IFN-γ signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated Aβ induced IFN-γ production from co-culture of astrocytes and microglia, and IFN-γ elicited tumor necrosis factor (TNF)-α secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-γ and TNF-α enhanced Aβ production from APP-expressing astrocytes and cortical neurons. TNF-α directly stimulated β-site APP-cleaving enzyme (BACE1) expression and enhanced β-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-γ and TNF-α activation of WT microglia suppressed Aβ degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-γ and TNF-α enhance Aβ deposition through BACE1 expression and suppression of Aβ clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer’s disease pathogenesis. PMID:17255335

Ubiquitin Ligases and Deubiquitinating Enzymes in CD4+ T Cell Effector Fate Choice and Function.

PubMed

Layman, Awo A K; Oliver, Paula M

2016-05-15

The human body is exposed to potentially pathogenic microorganisms at barrier sites such as the skin, lungs, and gastrointestinal tract. To mount an effective response against these pathogens, the immune system must recruit the right cells with effector responses that are appropriate for the task at hand. Several types of CD4(+) T cells can be recruited, including Th cells (Th1, Th2, and Th17), T follicular helper cells, and regulatory T cells. These cells help to maintain normal immune homeostasis in the face of constantly changing microbes in the environment. Because these cells differentiate from a common progenitor, the composition of their intracellular milieu of proteins changes to appropriately guide their effector function. One underappreciated process that impacts the levels and functions of effector fate-determining factors is ubiquitylation. This review details our current understanding of how ubiquitylation regulates CD4(+) T cell effector identity and function. Copyright © 2016 by The American Association of Immunologists, Inc.

Nitric Oxide-Mediated Tumoricidal Activity of Murine Microglial Cells12

PubMed Central

Brantley, Emily C; Guo, Lixia; Zhang, Chenyu; Lin, Qingtang; Yokoi, Kenji; Langley, Robert R; Kruzel, Ewa; Maya, Marva; Kim, Seung Wook; Kim, Sun-Jin; Fan, Dominic; Fidler, Isaiah J

2010-01-01

Experimental metastases in the brain of mice are infiltrated by microglia, and parabiosis experiments of green fluorescent protein (GFP+) and GFP- mice revealed that these microglia are derived from circulating monocytes (GFP+, F4/80+, and CD68+). These findings raised the question as to whether microglia (specialized macrophages) possess tumoricidal activity. C8-B4 murine microglia cells were incubated in vitro in medium (control) or in medium containing both lipopolysaccharide and interferon-γ. Control microglia were not tumoricidal against a number of murine and human tumor cells, whereas lipopolysaccharide/interferon-γ-activated microglia lysed murine and human tumor cells by release of nitric oxide. Parallel experiments with murine peritoneal macrophages produced identical results. Neither activated microglia nor activated macrophages lysed nontumorigenic murine or human cells. Collectively, these data demonstrate that brain metastasis-associated microglia are derived from circulating mononuclear cells and exhibit selective and specific tumoricidal activity. PMID:21151477

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

PubMed Central

Schroeder, Gunnar N.

2018-01-01

The defective in organelle trafficking/intracellular multiplication (Dot/Icm) Type IVb secretion system (T4SS) is the essential virulence factor for the intracellular life style and pathogenicity of Legionella species. Screens demonstrated that an individual L. pneumophila strain can use the Dot/Icm T4SS to translocate an unprecedented number of more than 300 proteins into host cells, where these, so called Icm/Dot-translocated substrates (IDTS) or effectors, manipulate host cell functions to the benefit of the bacteria. Bioinformatic analysis of the pan-genus genome predicts at least 608 orthologous groups of putative effectors. Deciphering the function of these effectors is key to understanding Legionella pathogenesis; however, the analysis is challenging. Substantial functional redundancy renders classical, phenotypic screening of single gene deletion mutants mostly ineffective. Here, I review experimental approaches that were successfully used to identify, validate and functionally characterize T4SS effectors and highlight new methods, which promise to facilitate unlocking the secrets of Legionella's extraordinary weapons arsenal. PMID:29354599

Electroacupuncture Improves Cognitive Function and Hippocampal Neurogenesis after Brain Irradiation.

PubMed

Fan, Xing-Wen; Liu, Huan-Huan; Wang, Hong-Bing; Chen, Fu; Yang, Yu; Chen, Yan; Guan, Shi-Kuo; Wu, Kai-Liang

2017-06-01

Cognitive impairments after brain irradiation seriously affect quality of life for patients, and there is currently no effective treatment. In this study using an irradiated rat model, the role of electroacupuncture was investigated for treatment of radiation-induced brain injury. Animals received 10 Gy exposure to the entire brain, and electroacupuncture was administered 3 days before irradiation as well as up to 2 weeks postirradiation. Behavioral tests were performed one month postirradiation, and rats were then sacrificed for histology or molecular studies. Electroacupuncture markedly improved animal performance in the novel place recognition test. In the emotion test, electroacupuncture reduced defecation during the open-field test, and latency to consumption of food in the novelty suppressed feeding test. Brain irradiation inhibited the generation of immature neurons, but did not cause neural stem cell loss. Electroacupuncture partially restored hippocampal neurogenesis. Electroacupuncture decreased the amount of activated microglia and increased resting microglia in the hippocampus after irradiation. In addition, electroacupuncture promoted mRNA and protein expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. In conclusion, electroacupuncture could improve cognitive function and hippocampal neurogenesis after irradiation, and the protective effect of electroacupuncture was associated with the modulation of microglia and upregulation of BDNF in the hippocampus.

Human endogenous retrovirus W env increases nitric oxide production and enhances the migration ability of microglia by regulating the expression of inducible nitric oxide synthase.

PubMed

Xiao, Ran; Li, Shan; Cao, Qian; Wang, Xiuling; Yan, Qiujin; Tu, Xiaoning; Zhu, Ying; Zhu, Fan

2017-06-01

Human endogenous retrovirus W env (HERV-W env) plays a critical role in many neuropsychological diseases such as schizophrenia and multiple sclerosis (MS). These diseases are accompanied by immunological reactions in the central nervous system (CNS). Microglia are important immunocytes in brain inflammation that can produce a gasotransmitter-nitric oxide (NO). NO not only plays a role in the function of neuronal cells but also participates in the pathogenesis of various neuropsychological diseases. In this study, we reported increased NO production in CHME-5 microglia cells after they were transfected with HERV-W env. Moreover, HERV-W env increased the expression and function of human inducible nitric oxide synthase (hiNOS) and enhanced the promoter activity of hiNOS. Microglial migration was also enhanced. These data revealed that HERV-W env might contribute to increase NO production and microglial migration ability in neuropsychological disorders by regulating the expression of inducible NOS. Results from this study might lead to the identification of novel targets for the treatment of neuropsychological diseases, including neuroinflammatory diseases, stroke, and neurodegenerative diseases.

Microglial pathology.

PubMed

Streit, Wolfgang J; Xue, Qing-Shan; Tischer, Jasmin; Bechmann, Ingo

2014-09-26

This paper summarizes pathological changes that affect microglial cells in the human brain during aging and in aging-related neurodegenerative diseases, primarily Alzheimer's disease (AD). It also provides examples of microglial changes that have been observed in laboratory animals during aging and in some experimentally induced lesions and disease models. Dissimilarities and similarities between humans and rodents are discussed in an attempt to generate a current understanding of microglial pathology and its significance during aging and in the pathogenesis of Alzheimer dementia (AD). The identification of dystrophic (senescent) microglia has created an ostensible conflict with prior work claiming a role for activated microglia and neuroinflammation during normal aging and in AD, and this has raised a basic question: does the brain's immune system become hyperactive (inflamed) or does it become weakened (senescent) in elderly and demented people, and what is the impact on neuronal function and cognition? Here we strive to reconcile these seemingly contradictory notions by arguing that both low-grade neuroinflammation and microglial senescence are the result of aging-associated free radical injury. Both processes are damaging for microglia as they synergistically exhaust this essential cell population to the point where the brain's immune system is effete and unable to support neuronal function.

Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity

PubMed Central

Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C.; Gilroy, Eleanor M.; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R. J.; Brunner, Frédéric

2014-01-01

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans. PMID:24763622

Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

PubMed Central

McBean, Gethin; Cindric, Marina; Egea, Javier; López, Manuela G.; Rada, Patricia; Zarkovic, Neven

2014-01-01

Abstract Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain. Antioxid. Redox Signal. 21, 1766–1801. PMID:24597893

Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina.

PubMed

White, David T; Sengupta, Sumitra; Saxena, Meera T; Xu, Qingguo; Hanes, Justin; Ding, Ding; Ji, Hongkai; Mumm, Jeff S

2017-05-02

Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration-i.e., selective cell-loss paradigms akin to degenerative disease-are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of ( i ) rod cell clearance, ( ii ) MG/progenitor cell proliferation, and ( iii ) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.

Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina

PubMed Central

White, David T.; Sengupta, Sumitra; Saxena, Meera T.; Xu, Qingguo; Hanes, Justin; Ding, Ding; Ji, Hongkai

2017-01-01

Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration—i.e., selective cell-loss paradigms akin to degenerative disease—are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions. PMID:28416692

Ventral tegmental area/substantia nigra and prefrontal cortex rodent organotypic brain slices as an integrated model to study the cellular changes induced by oxygen/glucose deprivation and reperfusion: effect of neuroprotective agents.

PubMed

Colombo, Laura; Parravicini, Chiara; Lecca, Davide; Dossi, Elena; Heine, Claudia; Cimino, Mauro; Wanke, Enzo; Illes, Peter; Franke, Heike; Abbracchio, Maria P

2014-01-01

Unveiling the roles of distinct cell types in brain response to insults is a partially unsolved challenge and a key issue for new neuroreparative approaches. In vivo models are not able to dissect the contribution of residential microglia and infiltrating blood-borne monocytes/macrophages, which are fundamentally undistinguishable; conversely, cultured cells lack original tissue anatomical and functional complexity, which profoundly alters reactivity. Here, we tested whether rodent organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra and prefrontal cortex (VTA/SN-PFC) represent a suitable model to study changes induced by oxygen/glucose deprivation and reperfusion (OGD/R). OGD/R induced cytotoxicity to both VTA/SN and PFC slices, with higher VTA/SN susceptibility. Neurons were highly affected, with astrocytes and oligodendrocytes undergoing very mild damage. Marked reactive astrogliosis was also evident. Notably, OGD/R triggered the activation of CD68-expressing microglia and increased expression of Ym1 and Arg1, two markers of "alternatively" activated beneficial microglia. Treatment with two well-known neuroprotective drugs, the anticonvulsant agent valproic acid and the purinergic P2-antagonist PPADS, prevented neuronal damage. Thus, VTA/SN-PFC cultures are an integrated model to investigate OGD/R-induced effects on distinct cells and easily screen neuroprotective agents. The model is particularly adequate to dissect the microglia phenotypic shift in the lack of a functional vascular compartment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapamycin protects against neuronal death and improves neurological function with modulation of microglia after experimental intracerebral hemorrhage in rats.

PubMed

Li, D; Liu, F; Yang, T; Jin, T; Zhang, H; Luo, X; Wang, M

2016-09-30

Intracerebral hemorrhage (ICH) results in a devastating brain disorder with high mortality and poor prognosis and effective therapeutic intervention for the disease remains a challenge at present. The present study investigated the neuroprotective effects of rapamycin on ICH-induced brain damage and the possible involvement of activated microglia. ICH was induced in rats by injection of type IV collagenase into striatum. Different dose of rapamycin was systemically administrated by intraperitoneal injection beginning at 1 h after ICH induction. Western blot analysis showed that ICH led to a long-lasting increase of phosphorylated mTOR and this hyperactivation of mTOR was reduced by systemic administration of rapamycin. Rapamycin treatment significantly improved the sensorimotor deficits induced by ICH, and attenuated ICH-induced brain edema formation as well as lesion volume. Nissl and Fluoro-Jade C staining demonstrated that administration with rapamycin remarkably decreased neuronal death surrounding the hematoma at 7 d after ICH insult. ELISA and real-time quantitative PCR demonstrated that rapamycin inhibited ICH-induced excessive expression of TNF-α and IL-1β in ipsilateral hemisphere. Furthermore, activation of microglia induced by ICH was significantly suppressed by rapamycin administration. These data indicated that treatment of rapamycin following ICH decreased the brain injuries and neuronal death at the peri-hematoma striatum, and increased neurological function, which associated with reduced the levels of proinflammatory cytokines and activated microglia. The results provide novel insight into the neuroprotective therapeutic strategy of rapamycin for ICH insult, which possibly involving the regulation of microglial activation.

Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides.

PubMed

Salomon, Dor; Guo, Yirui; Kinch, Lisa N; Grishin, Nick V; Gardner, Kevin H; Orth, Kim

2013-01-01

Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells.

Neuronal CCL2 is upregulated during hepatic encephalopathy and contributes to microglia activation and neurological decline

PubMed Central

2014-01-01

Background Acute liver failure leads to systemic complications with one of the most dangerous being a decline in neurological function, termed hepatic encephalopathy. Neurological dysfunction is exacerbated by an increase of toxic metabolites in the brain that lead to neuroinflammation. Following various liver diseases, hepatic and circulating chemokines, such as chemokine ligand 2 (CCL2), are elevated, though their effects on the brain following acute liver injury and subsequent hepatic encephalopathy are unknown. CCL2 is known to activate microglia in other neuropathies, leading to a proinflammatory response. However, the effects of CCL2 on microglia activation and the pathogenesis of hepatic encephalopathy following acute liver injury remain to be determined. Methods Hepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) in the presence or absence of INCB 3284 dimesylate (INCB), a chemokine receptor 2 inhibitor, or C 021 dihydrochloride (C021), a chemokine receptor 4 inhibitor. Mice were monitored for neurological decline and time to coma (loss of all reflexes) was recorded. Tissue was collected at coma and used for real-time PCR, immunoblots, ELISA, or immunostaining analyses to assess the activation of microglia and consequences on pro-inflammatory cytokine expression. Results Following AOM administration, microglia activation was significantly increased in AOM-treated mice compared to controls. Concentrations of CCL2 in the liver, serum, and cortex were significantly elevated in AOM-treated mice compared to controls. Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry. Administration of INCB or C021 significantly improved the neurological outcomes of AOM-treated mice, reduced microglia activation, reduced phosphorylation of ERK1/2, and alleviated AOM-induced cytokine upregulation. Conclusions These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy. Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury. PMID:25012628

Neuronal CCL2 is upregulated during hepatic encephalopathy and contributes to microglia activation and neurological decline.

PubMed

McMillin, Matthew; Frampton, Gabriel; Thompson, Michelle; Galindo, Cheryl; Standeford, Holly; Whittington, Eric; Alpini, Gianfranco; DeMorrow, Sharon

2014-07-10

Acute liver failure leads to systemic complications with one of the most dangerous being a decline in neurological function, termed hepatic encephalopathy. Neurological dysfunction is exacerbated by an increase of toxic metabolites in the brain that lead to neuroinflammation. Following various liver diseases, hepatic and circulating chemokines, such as chemokine ligand 2 (CCL2), are elevated, though their effects on the brain following acute liver injury and subsequent hepatic encephalopathy are unknown. CCL2 is known to activate microglia in other neuropathies, leading to a proinflammatory response. However, the effects of CCL2 on microglia activation and the pathogenesis of hepatic encephalopathy following acute liver injury remain to be determined. Hepatic encephalopathy was induced in mice via injection of azoxymethane (AOM) in the presence or absence of INCB 3284 dimesylate (INCB), a chemokine receptor 2 inhibitor, or C 021 dihydrochloride (C021), a chemokine receptor 4 inhibitor. Mice were monitored for neurological decline and time to coma (loss of all reflexes) was recorded. Tissue was collected at coma and used for real-time PCR, immunoblots, ELISA, or immunostaining analyses to assess the activation of microglia and consequences on pro-inflammatory cytokine expression. Following AOM administration, microglia activation was significantly increased in AOM-treated mice compared to controls. Concentrations of CCL2 in the liver, serum, and cortex were significantly elevated in AOM-treated mice compared to controls. Systemic administration of INCB or C021 reduced liver damage as assessed by serum liver enzyme biochemistry. Administration of INCB or C021 significantly improved the neurological outcomes of AOM-treated mice, reduced microglia activation, reduced phosphorylation of ERK1/2, and alleviated AOM-induced cytokine upregulation. These findings suggest that CCL2 is elevated systemically following acute liver injury and that CCL2 is involved in both the microglia activation and neurological decline associated with hepatic encephalopathy. Methods used to modulate CCL2 levels and/or reduce CCR2/CCR4 activity may be potential therapeutic targets for the management of hepatic encephalopathy due to acute liver injury.

Glial and Neuroimmune Mechanisms as Critical Modulators of Drug Use and Abuse.

PubMed

Lacagnina, Michael J; Rivera, Phillip D; Bilbo, Staci D

2017-01-01

Drugs of abuse cause persistent alterations in synaptic plasticity that may underlie addiction behaviors. Evidence suggests glial cells have an essential and underappreciated role in the development and maintenance of drug abuse by influencing neuronal and synaptic functions in multifaceted ways. Microglia and astrocytes perform critical functions in synapse formation and refinement in the developing brain, and there is growing evidence that disruptions in glial function may be implicated in numerous neurological disorders throughout the lifespan. Linking evidence of function in health and under pathological conditions, this review will outline the glial and neuroimmune mechanisms that may contribute to drug-abuse liability, exploring evidence from opioids, alcohol, and psychostimulants. Drugs of abuse can activate microglia and astrocytes through signaling at innate immune receptors, which in turn influence neuronal function not only through secretion of soluble factors (eg, cytokines and chemokines) but also potentially through direct remodeling of the synapses. In sum, this review will argue that neural-glial interactions represent an important avenue for advancing our understanding of substance abuse disorders.

CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

PubMed Central

Yao, Shuyu; Huang, Dan; Chen, Crystal Y.; Halliday, Lisa; Wang, Richard C.; Chen, Zheng W.

2014-01-01

The possibility that CD4+ T cells can act as “innate-like” cells to contain very-early M. tuberculosis (Mtb) dissemination and function as master helpers to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes during development of adaptive immunity against primary tuberculosis(TB) has not been demonstrated. We showed that pulmonary Mtb infection of CD4-depleted macaques surprisingly led to very-early extrathoracic Mtb dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during Mtb infection revealed the ability of CD8+ T cells to compensate and rapidly differentiate to Th17-like/Th1-like, and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. While CD3-negative non-T lymphocytes in presence of CD4+ T cells developed predominant Th22-like and NK-like (perforin production) responses to Mtb infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3-negative lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint of more severe TB, but presented pulmonary IL-4+ T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22+ and perforin+ cells despite presence of IL-17+ and IL-4+ cells. These results implicate previously-unknown “innate-like” ability of CD4+ T cells to contain extrathoracic Mtb dissemination at very early stage. Data also suggest that CD4+ T cells are required to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes and to prevent rapid TB progression during Mtb infection of nonhuman primates. PMID:24489088

Microglia, the missing link in maternal immune activation and fetal neurodevelopment; and a possible link in preeclampsia and disturbed neurodevelopment?

PubMed

Prins, Jelmer R; Eskandar, Sharon; Eggen, Bart J L; Scherjon, Sicco A

2018-04-01

Disturbances in fetal neurodevelopment have extensively been related to neurodevelopmental disorders in early and later life. Fetal neurodevelopment is dependent on adequate functioning of the fetal immune system. During pregnancy, the maternal immune system is challenged to both tolerate the semi-allogenic fetus and to protect the mother and fetus from microbes. The fetal immune system is influenced by maternal immune disturbances; therefore, perturbations in maternal immunity likely do not only alter pregnancy outcome but also alter fetal neurodevelopment. A possible common pathway could be modulating the functioning of tissue macrophages in the placenta and brain. Maternal immune tolerance towards the fetus involves several complex adaptations. In this active maternal immune state, the fetus develops its own immunity. As cytokines and other players of the immune system -which can pass the placenta- are involved in neurodevelopment, disruptions in immune balance influence fetal neurodevelopment. Several studies reported an association between maternal immune activation, complications of pregnancy as preeclampsia, and altered neonatal neurodevelopment. A possible pathway involves dysfunctioning of microglia cells, the immune cells of the brain. Functionality of microglia cells during normal pregnancy is, however, poorly understood. The recent outbreak of ZIKA virus (ZKV), but also the literature on virus infections in general and its consequences on microglial cell function and fetal neurodevelopment show the devastating effects a virus infection during pregnancy can have. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Bacterial virulence effectors and their activities.

PubMed

Hann, Dagmar R; Gimenez-Ibanez, Selena; Rathjen, John P

2010-08-01

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes. Copyright 2010 Elsevier Ltd. All rights reserved.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

PubMed Central

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility

PubMed Central

Hutin, Mathilde; Pérez-Quintero, Alvaro L.; Lopez, Camilo; Szurek, Boris

2015-01-01

Many plant-pathogenic xanthomonads rely on Transcription Activator-Like (TAL) effectors to colonize their host. This particular family of type III effectors functions as specific plant transcription factors via a programmable DNA-binding domain. Upon binding to the promoters of plant disease susceptibility genes in a sequence-specific manner, the expression of these host genes is induced. However, plants have evolved specific strategies to counter the action of TAL effectors and confer resistance. One mechanism is to avoid the binding of TAL effectors by mutations of their DNA binding sites, resulting in resistance by loss-of-susceptibility. This article reviews our current knowledge of the susceptibility hubs targeted by Xanthomonas TAL effectors, possible evolutionary scenarios for plants to combat the pathogen with loss-of-function alleles, and how this knowledge can be used overall to develop new pathogen-informed breeding strategies and improve crop resistance. PMID:26236326

In vivo analysis of the time and spatial activation pattern of microglia in the retina following laser-induced choroidal neovascularization.

PubMed

Crespo-Garcia, Sergio; Reichhart, Nadine; Hernandez-Matas, Carlos; Zabulis, Xenophon; Kociok, Norbert; Brockmann, Claudia; Joussen, Antonia M; Strauss, Olaf

2015-10-01

Microglia play a major role in retinal neovascularization and degeneration and are thus potential targets for therapeutic intervention. In vivo assessment of microglia behavior in disease models can provide important information to understand patho-mechanisms and develop therapeutic strategies. Although scanning laser ophthalmoscope (SLO) permits the monitoring of microglia in transgenic mice with microglia-specific GFP expression, there are fundamental limitations in reliable identification and quantification of activated cells. Therefore, we aimed to improve the SLO-based analysis of microglia using enhanced image processing with subsequent testing in laser-induced neovascularization (CNV). CNV was induced by argon laser in MacGreen mice. Microglia was visualized in vivo by SLO in the fundus auto-fluorescence (FAF) mode and verified ex vivo using retinal preparations. Three image processing algorithms based on different analysis of sequences of images were tested. The amount of recorded frames was limiting the effectiveness of the different algorithms. Best results from short recordings were obtained with a pixel averaging algorithm, further used to quantify spatial and temporal distribution of activated microglia in CNV. Morphologically, different microglia populations were detected in the inner and outer retinal layers. In CNV, the peak of microglia activation occurred in the inner layer at day 4 after laser, lacking an acute reaction. Besides, the spatial distribution of the activation changed by the time over the inner retina. No significant time and spatial changes were observed in the outer layer. An increase in laser power did not increase number of activated microglia. The SLO, in conjunction with enhanced image processing, is suitable for in vivo quantification of microglia activation. This surprisingly revealed that laser damage at the outer retina led to more reactive microglia in the inner retina, shedding light upon a new perspective to approach the immune response in the retina in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Role of microglia in ethanol’s apoptotic action on hypothalamic neuronal cells in primary cultures

PubMed Central

Boyadjieva, Nadka I.; Sarkar, Dipak K.

2010-01-01

Background Microglia are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. In this study, we determined the role of microglia in ethanol’s apoptotic action on neuronal cells obtained from the mediobasal hypothalamus and maintained in primary cultures. We also tested the effect of cAMP, a signaling molecule critically involved in hypothalamic neuronal survival, on microglia-mediated ethanol’s neurotoxic action. Methods Ethanol’s neurotoxic action was determined on enriched fetal mediobasal hypothalamic neuronal cells with or without microglia cells or ethanol-activated microglia conditioned media. Ethanol’s apoptotic action was determined using nucleosome assay. Microglia activation was determined using OX6 histochemistry and by measuring inflammatory cytokines secretion from microglia in cultures using enzyme-linked immunosorbent assay (ELISA). An immunoneutralization study was conducted to identify the role of a cytokine involved in ethanol’s apoptotic action. Results We show here that ethanol at a dose range of 50 and 100 mM induces neuronal death by an apoptotic process. Ethanol’s ability to induce an apoptotic death of neurons is increased by the presence of ethanol-activated microglia conditioned media. In the presence of ethanol, microglia showed elevated secretion of various inflammatory cytokines, of which TNF-α shows significant apoptotic action on mediobasal hypothalamic neuronal cells. Ethanol’s neurotoxic action was completely prevented by cAMP. The cell-signaling molecule also prevented ethanol-activated microglial production of TNF-α. Immunoneutralization of TNF-α prevented microglia-derived media’s ability to induce neuronal death. Conclusions These results suggest that ethanol’s apoptotic action on hypothalamic neuronal cells might be mediated via microglia, possibly via increased production of TNF-α. Furthermore, cAMP reduces TNF-α production from microglia to prevent ethanol’s neurotoxic action. PMID:20662807

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

PubMed

Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Caspase inhibitors protect neurons by enabling selective necroptosis of inflamed microglia.

PubMed

Fricker, Michael; Vilalta, Anna; Tolkovsky, Aviva M; Brown, Guy C

2013-03-29

Microglia are resident brain macrophages, which can cause neuronal loss when activated in infectious, ischemic, traumatic, and neurodegenerative diseases. Caspase-8 has both prodeath and prosurvival roles, mediating apoptosis and/or preventing RIPK1-mediated necroptosis depending on cell type and stimulus. We found that inflammatory stimuli (LPS, lipoteichoic acid, or TNF-α) caused an increase in caspase-8 IETDase activity in primary rat microglia without inducing apoptosis. Inhibition of caspase-8 with either Z-VAD-fmk or IETD-fmk resulted in necrosis of activated microglia. Inhibition of caspases with Z-VAD-fmk did not kill non-activated microglia, or astrocytes and neurons in any condition. Necrostatin-1, a specific inhibitor of RIPK1, prevented microglial caspase inhibition-induced death, indicating death was by necroptosis. In mixed cerebellar cultures of primary neurons, astrocytes, and microglia, LPS induced neuronal loss that was prevented by inhibition of caspase-8 (resulting in microglial necroptosis), and neuronal death was restored by rescue of microglia with necrostatin-1. We conclude that the activation of caspase-8 in inflamed microglia prevents their death by necroptosis, and thus, caspase-8 inhibitors may protect neurons in the inflamed brain by selectively killing activated microglia.

Effector prediction in host-pathogen interaction based on a Markov model of a ubiquitous EPIYA motif

PubMed Central

2010-01-01

Background Effector secretion is a common strategy of pathogen in mediating host-pathogen interaction. Eight EPIYA-motif containing effectors have recently been discovered in six pathogens. Once these effectors enter host cells through type III/IV secretion systems (T3SS/T4SS), tyrosine in the EPIYA motif is phosphorylated, which triggers effectors binding other proteins to manipulate host-cell functions. The objectives of this study are to evaluate the distribution pattern of EPIYA motif in broad biological species, to predict potential effectors with EPIYA motif, and to suggest roles and biological functions of potential effectors in host-pathogen interactions. Results A hidden Markov model (HMM) of five amino acids was built for the EPIYA-motif based on the eight known effectors. Using this HMM to search the non-redundant protein database containing 9,216,047 sequences, we obtained 107,231 sequences with at least one EPIYA motif occurrence and 3115 sequences with multiple repeats of the EPIYA motif. Although the EPIYA motif exists among broad species, it is significantly over-represented in some particular groups of species. For those proteins containing at least four copies of EPIYA motif, most of them are from intracellular bacteria, extracellular bacteria with T3SS or T4SS or intracellular protozoan parasites. By combining the EPIYA motif and the adjacent SH2 binding motifs (KK, R4, Tarp and Tir), we built HMMs of nine amino acids and predicted many potential effectors in bacteria and protista by the HMMs. Some potential effectors for pathogens (such as Lawsonia intracellularis, Plasmodium falciparum and Leishmania major) are suggested. Conclusions Our study indicates that the EPIYA motif may be a ubiquitous functional site for effectors that play an important pathogenicity role in mediating host-pathogen interactions. We suggest that some intracellular protozoan parasites could secrete EPIYA-motif containing effectors through secretion systems similar to the T3SS/T4SS in bacteria. Our predicted effectors provide useful hypotheses for further studies. PMID:21143776

TAL effectors and the executor R genes

PubMed Central

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized—recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance. PMID:26347759

TAL effectors and the executor R genes.

PubMed

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized-recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

Maternal inflammation induces immune activation of fetal microglia and leads to disrupted microglia immune responses, behavior, and learning performance in adulthood.

PubMed

Schaafsma, Wandert; Basterra, Laura Bozal; Jacobs, Sabrina; Brouwer, Nieske; Meerlo, Peter; Schaafsma, Anne; Boddeke, Erik W G M; Eggen, Bart J L

2017-10-01

Maternal inflammation during pregnancy can have detrimental effects on embryonic development that persist during adulthood. However, the underlying mechanisms and insights in the responsible cell types are still largely unknown. Here we report the effect of maternal inflammation on fetal microglia, the innate immune cells of the central nervous system (CNS). In mice, a challenge with LPS during late gestation stages (days 15-16-17) induced a pro-inflammatory response in fetal microglia. Adult whole brain microglia of mice that were exposed to LPS during embryonic development displayed a persistent reduction in pro-inflammatory activation in response to a re-challenge with LPS. In contrast, hippocampal microglia of these mice displayed an increased inflammatory response to an LPS re-challenge. In addition, a reduced expression of brain-derived neurotrophic factor (BDNF) was observed in hippocampal microglia of LPS-offspring. Microglia-derived BDNF has been shown to be important for learning and memory processes. In line with these observations, behavioral- and learning tasks with mice that were exposed to maternal inflammation revealed reduced home cage activity, reduced anxiety and reduced learning performance in a T-maze. These data show that exposure to maternal inflammation during late gestation results in long term changes in microglia responsiveness during adulthood, which is different in nature in hippocampus compared to total brain microglia. Copyright © 2017 Elsevier Inc. All rights reserved.

Neuroinflammation increases GABAergic tone and impairs cognitive and motor function in hyperammonemia by increasing GAT-3 membrane expression. Reversal by sulforaphane by promoting M2 polarization of microglia.

PubMed

Hernandez-Rabaza, Vicente; Cabrera-Pastor, Andrea; Taoro-Gonzalez, Lucas; Gonzalez-Usano, Alba; Agusti, Ana; Balzano, Tiziano; Llansola, Marta; Felipo, Vicente

2016-04-18

Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: (a) Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. (b) Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. (c) Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them. We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3. Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination. Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.

Stat3 inhibition activates tumor macrophages and abrogates glioma growth in mice.

PubMed

Zhang, Leying; Alizadeh, Darya; Van Handel, Michelle; Kortylewski, Marcin; Yu, Hua; Badie, Behnam

2009-10-01

As the main effector-cell population of the central nervous system, microglia (MG) are considered to play an important immunoregulatory function in a number of pathological conditions such as inflammation, trauma, degenerative disease, and brain tumors. Recent studies, however, have suggested that the anti-neoplastic function of MG may be suppressed in malignant brain tumors. Considering the proposed suppressive role of signal transducers and activators of transcription 3 (Stat3) in antitumor immunity, we evaluated the role of Stat3 inhibition on MG and macrophage (MP) activation and tumor growth in a murine glioma model. N9 MG cells were exposed to GL261 glioma conditioned medium (GL261-CM) and evaluated for Stat3 activity and cytokine expression. Furthermore, the role of Stat3 inhibition on MG and MP activation was studied both in vitro and in vivo. Finally, the effect of Stat3 inhibition on tumor growth was assessed in intracranial GL261 gliomas. GL261-CM increased Stat3 activity in N9 cells in vitro and resulted in overexpression of IL-10 and IL-6, and downregulation of IL1-beta, a pro-inflammatory cytokine. Inhibition of Stat3 by CPA-7 or siRNA reversed glioma-induced cytokine expression profile in N9 cells. Furthermore, inactivation of Stat3 in intracranial GL261 tumors by siRNA resulted in MG/MP activation and tumor growth inhibition. Glioma-induced MG and MP suppression may be mediated thorough Stat3. Inhibition of Stat3 function in tumor MG/MP may result in their activation and can potentially be used as an adjunct immunotherapy approach for gliomas.

An assay for entry of secreted fungal effectors into plant cells.

PubMed

Lo Presti, Libera; Zechmann, Bernd; Kumlehn, Jochen; Liang, Liang; Lanver, Daniel; Tanaka, Shigeyuki; Bock, Ralph; Kahmann, Regine

2017-01-01

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity.

PubMed

Ma, Ka-Wai; Ma, Wenbo

2016-12-01

Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Noninvasive Quantification of Retinal Microglia Using Widefield Autofluorescence Imaging.

PubMed

Kokona, Despina; Schneider, Nadia; Giannakaki-Zimmermann, Helena; Jovanovic, Joel; Ebneter, Andreas; Zinkernagel, Martin

2017-04-01

To validate widefield autofluorescence (AF) in vivo imaging of the retina in mice expressing green fluorescent protein (gfp) in microglia, and to monitor retinal microglia reconstitution in vivo after lethal irradiation and bone marrow transplantation. Transgenic Cx3cr1gfp/gfp and wildtype Balb/c mice were used in this study. A confocal scanning laser ophthalmoscope was used for AF imaging with a 55° and a widefield 102° lens. Intrasession reproducibility was assessed for each lens. To investigate reconstitution in vivo, bone marrow from Cx3cr1gfp/gfp mice was used to rescue lethally irradiated wildtype mice. Data were compared to confocal microscopy of retinal flat mounts. Both the 55° and the 102° lens produced high resolution images of retinal microglia with similar microglia density. However, compared to the 55° lens, the widefield 102° lens captured approximately 3.6 times more microglia cells (1515 ± 123 cells versus 445 ± 76 cells [mean ± SD], for 102° and 55°, respectively, P < 0.001). No statistical difference in the number of gfp positive cells within corresponding areas was observed within the same imaging session. Imaging of microglia reconstitution showed a similar time course compared to flat mount preparations with an excellent correlation between microglia cell numbers in AF and gfp-stained flat mounts (R = 0.92, P < 0.0001). Widefield AF imaging of mice with gfp expressing microglia can be used to quantify retinal microglia. In vivo microglia counts corresponded very well with ex vivo counts on retinal flat mounts. As such, AF imaging can largely replace ex vivo quantification.

Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

NASA Astrophysics Data System (ADS)

Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

2003-06-01

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain

PubMed Central

Anandasabapathy, Niroshana; Victora, Gabriel D.; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L.; Nussenzweig, Michel C.; Steinman, Ralph M.

2011-01-01

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5–7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia. PMID:21788405

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain.

PubMed

Anandasabapathy, Niroshana; Victora, Gabriel D; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L; Nussenzweig, Michel C; Steinman, Ralph M; Liu, Kang

2011-08-01

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5-7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia.

Time-dependent effects of CX3CR1 in a mouse model of mild traumatic brain injury.

PubMed

Febinger, Heidi Y; Thomasy, Hannah E; Pavlova, Maria N; Ringgold, Kristyn M; Barf, Paulien R; George, Amrita M; Grillo, Jenna N; Bachstetter, Adam D; Garcia, Jenny A; Cardona, Astrid E; Opp, Mark R; Gemma, Carmelina

2015-09-02

Neuroinflammation is an important secondary mechanism that is a key mediator of the long-term consequences of neuronal injury that occur in traumatic brain injury (TBI). Microglia are highly plastic cells with dual roles in neuronal injury and recovery. Recent studies suggest that the chemokine fractalkine (CX3CL1, FKN) mediates neural/microglial interactions via its sole receptor CX3CR1. CX3CL1/CX3CR1 signaling modulates microglia activation, and depending upon the type and time of injury, either protects or exacerbates neurological diseases. In this study, mice deficient in CX3CR1 were subjected to mild controlled cortical impact injury (CCI), a model of TBI. We evaluated the effects of genetic deletion of CX3CR1 on histopathology, cell death/survival, microglia activation, and cognitive function for 30 days post-injury. During the acute post-injury period (24 h-15 days), motor deficits, cell death, and neuronal cell loss were more profound in injured wild-type than in CX3CR1(-/-) mice. In contrast, during the chronic period of 30 days post-TBI, injured CX3CR1(-/-) mice exhibited greater cognitive dysfunction and increased neuronal death than wild-type mice. The protective and deleterious effects of CX3CR1 were associated with changes in microglia phenotypes; during the acute phase CX3CR1(-/-) mice showed a predominant anti-inflammatory M2 microglial response, with increased expression of Ym1, CD206, and TGFβ. In contrast, increased M1 phenotypic microglia markers, Marco, and CD68 were predominant at 30 days post-TBI. Collectively, these novel data demonstrate a time-dependent role for CX3CL1/CX3CR1 signaling after TBI and suggest that the acute and chronic responses to mild TBI are modulated in part by distinct microglia phenotypes.

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

PubMed Central

2016-01-01

SUMMARY Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. PMID:27784797

Effector biology of plant-associated organisms: concepts and perspectives.

PubMed

Win, J; Chaparro-Garcia, A; Belhaj, K; Saunders, D G O; Yoshida, K; Dong, S; Schornack, S; Zipfel, C; Robatzek, S; Hogenhout, S A; Kamoun, S

2012-01-01

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

DOE PAGES

Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; ...

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

Putative terminator and/or effector functions of Arf GAPs in the trafficking of clathrin-coated vesicles.

PubMed

Kon, Shunsuke; Funaki, Tomo; Satake, Masanobu

2011-05-01

The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection.

PubMed

Jaouannet, Maëlle; Rosso, Marie-Noëlle

2013-09-01

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

PubMed

Lee, Chang-Han; Romain, Gabrielle; Yan, Wupeng; Watanabe, Makiko; Charab, Wissam; Todorova, Biliana; Lee, Jiwon; Triplett, Kendra; Donkor, Moses; Lungu, Oana I; Lux, Anja; Marshall, Nicholas; Lindorfer, Margaret A; Goff, Odile Richard-Le; Balbino, Bianca; Kang, Tae Hyun; Tanno, Hidetaka; Delidakis, George; Alford, Corrine; Taylor, Ronald P; Nimmerjahn, Falk; Varadarajan, Navin; Bruhns, Pierre; Zhang, Yan Jessie; Georgiou, George

2017-08-01

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance.

PubMed

Bolmont, Tristan; Haiss, Florent; Eicke, Daniel; Radde, Rebecca; Mathis, Chester A; Klunk, William E; Kohsaka, Shinichi; Jucker, Mathias; Calhoun, Michael E

2008-04-16

Microglial cells aggregate around amyloid plaques in Alzheimer's disease, but, despite their therapeutic potential, various aspects of their reactive kinetics and role in plaque pathogenesis remain hypothetical. Through use of in vivo imaging and quantitative morphological measures in transgenic mice, we demonstrate that local resident microglia rapidly react to plaque formation by extending processes and subsequently migrating toward plaques, in which individual transformed microglia somata remain spatially stable for weeks. The number of plaque-associated microglia increased at a rate of almost three per plaque per month, independent of plaque volume. Larger plaques were surrounded by larger microglia, and a subset of plaques changed in size over time, with an increase or decrease related to the volume of associated microglia. Far from adopting a more static role, plaque-associated microglia retained rapid process and membrane movement at the plaque/glia interface. Microglia internalized systemically injected amyloid-binding dye at a much higher rate in the vicinity of plaques. These results indicate a role for microglia in plaque maintenance and provide a model with multiple targets for therapeutic intervention.

Dynamics of the Microglial/Amyloid Interaction Indicate a Role in Plaque Maintenance

PubMed Central

Bolmont, Tristan; Haiss, Florent; Eicke, Daniel; Radde, Rebecca; Mathis, Chester A.; Klunk, William E.; Kohsaka, Shinichi; Jucker, Mathias

2008-01-01

Microglial cells aggregate around amyloid plaques in Alzheimer's disease, but, despite their therapeutic potential, various aspects of their reactive kinetics and role in plaque pathogenesis remain hypothetical. Through use of in vivo imaging and quantitative morphological measures in transgenic mice, we demonstrate that local resident microglia rapidly react to plaque formation by extending processes and subsequently migrating toward plaques, in which individual transformed microglia somata remain spatially stable for weeks. The number of plaque-associated microglia increased at a rate of almost three per plaque per month, independent of plaque volume. Larger plaques were surrounded by larger microglia, and a subset of plaques changed in size over time, with an increase or decrease related to the volume of associated microglia. Far from adopting a more static role, plaque-associated microglia retained rapid process and membrane movement at the plaque/glia interface. Microglia internalized systemically injected amyloid-binding dye at a much higher rate in the vicinity of plaques. These results indicate a role for microglia in plaque maintenance and provide a model with multiple targets for therapeutic intervention. PMID:18417708

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

PubMed Central

Cianfanelli, Francesca R.; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J.

2016-01-01

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery. PMID:27352036

Transcription Factor Networks Directing the Development, Function, and Evolution of Innate Lymphoid Effectors

PubMed Central

Kang, Joonsoo; Malhotra, Nidhi

2015-01-01

Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity. PMID:25650177

Identification and functional analysis of secreted effectors from phytoparasitic nematodes.

PubMed

Rehman, Sajid; Gupta, Vijai K; Goyal, Aakash K

2016-03-21

Plant parasitic nematodes develop an intimate and long-term feeding relationship with their host plants. They induce a multi-nucleate feeding site close to the vascular bundle in the roots of their host plant and remain sessile for the rest of their life. Nematode secretions, produced in the oesophageal glands and secreted through a hollow stylet into the host plant cytoplasm, are believed to play key role in pathogenesis. To combat these persistent pathogens, the identity and functional analysis of secreted effectors can serve as a key to devise durable control measures. In this review, we will recapitulate the knowledge over the identification and functional characterization of secreted nematode effector repertoire from phytoparasitic nematodes. Despite considerable efforts, the identity of genes encoding nematode secreted proteins has long been severely hampered because of their microscopic size, long generation time and obligate biotrophic nature. The methodologies such as bioinformatics, protein structure modeling, in situ hybridization microscopy, and protein-protein interaction have been used to identify and to attribute functions to the effectors. In addition, RNA interference (RNAi) has been instrumental to decipher the role of the genes encoding secreted effectors necessary for parasitism and genes attributed to normal development. Recent comparative and functional genomic approaches have accelerated the identification of effectors from phytoparasitic nematodes and offers opportunities to control these pathogens. Plant parasitic nematodes pose a serious threat to global food security of various economically important crops. There is a wealth of genomic and transcriptomic information available on plant parasitic nematodes and comparative genomics has identified many effectors. Bioengineering crops with dsRNA of phytonematode genes can disrupt the life cycle of parasitic nematodes and therefore holds great promise to develop resistant crops against plant-parasitic nematodes.

NADPH oxidase 2-derived reactive oxygen species in spinal cord microglia contribute to peripheral nerve injury-induced neuropathic pain

PubMed Central

Kim, Donghoon; You, Byunghyun; Jo, Eun-Kyeong; Han, Sang-Kyou; Simon, Melvin I.; Lee, Sung Joong

2010-01-01

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain. PMID:20679217

Nutrients, Microglia Aging, and Brain Aging.

PubMed

Wu, Zhou; Yu, Janchun; Zhu, Aiqin; Nakanishi, Hiroshi

2016-01-01

As the life expectancy continues to increase, the cognitive decline associated with Alzheimer's disease (AD) becomes a big major issue in the world. After cellular activation upon systemic inflammation, microglia, the resident immune cells in the brain, start to release proinflammatory mediators to trigger neuroinflammation. We have found that chronic systemic inflammatory challenges induce differential age-dependent microglial responses, which are in line with the impairment of learning and memory, even in middle-aged animals. We thus raise the concept of "microglia aging." This concept is based on the fact that microglia are the key contributor to the acceleration of cognitive decline, which is the major sign of brain aging. On the other hand, inflammation induces oxidative stress and DNA damage, which leads to the overproduction of reactive oxygen species by the numerous types of cells, including macrophages and microglia. Oxidative stress-damaged cells successively produce larger amounts of inflammatory mediators to promote microglia aging. Nutrients are necessary for maintaining general health, including the health of brain. The intake of antioxidant nutrients reduces both systemic inflammation and neuroinflammation and thus reduces cognitive decline during aging. We herein review our microglia aging concept and discuss systemic inflammation and microglia aging. We propose that a nutritional approach to controlling microglia aging will open a new window for healthy brain aging.

Nutrients, Microglia Aging, and Brain Aging

PubMed Central

Wu, Zhou; Yu, Janchun; Zhu, Aiqin; Nakanishi, Hiroshi

2016-01-01

As the life expectancy continues to increase, the cognitive decline associated with Alzheimer's disease (AD) becomes a big major issue in the world. After cellular activation upon systemic inflammation, microglia, the resident immune cells in the brain, start to release proinflammatory mediators to trigger neuroinflammation. We have found that chronic systemic inflammatory challenges induce differential age-dependent microglial responses, which are in line with the impairment of learning and memory, even in middle-aged animals. We thus raise the concept of “microglia aging.” This concept is based on the fact that microglia are the key contributor to the acceleration of cognitive decline, which is the major sign of brain aging. On the other hand, inflammation induces oxidative stress and DNA damage, which leads to the overproduction of reactive oxygen species by the numerous types of cells, including macrophages and microglia. Oxidative stress-damaged cells successively produce larger amounts of inflammatory mediators to promote microglia aging. Nutrients are necessary for maintaining general health, including the health of brain. The intake of antioxidant nutrients reduces both systemic inflammation and neuroinflammation and thus reduces cognitive decline during aging. We herein review our microglia aging concept and discuss systemic inflammation and microglia aging. We propose that a nutritional approach to controlling microglia aging will open a new window for healthy brain aging. PMID:26941889

Disease-related microglia heterogeneity in the hippocampus of Alzheimer's disease, dementia with Lewy bodies, and hippocampal sclerosis of aging.

PubMed

Bachstetter, Adam D; Van Eldik, Linda J; Schmitt, Frederick A; Neltner, Janna H; Ighodaro, Eseosa T; Webster, Scott J; Patel, Ela; Abner, Erin L; Kryscio, Richard J; Nelson, Peter T

2015-05-23

Neuropathological, genetic, and biochemical studies have provided support for the hypothesis that microglia participate in Alzheimer's disease (AD) pathogenesis. Despite the extensive characterization of AD microglia, there are still many unanswered questions, and little is known about microglial morphology in other common forms of age-related dementia: particularly, dementia with Lewy bodies (DLB) and hippocampal sclerosis of aging (HS-Aging). In addition, no prior studies have attempted to compare and contrast the microglia morphology in the hippocampus of various neurodegenerative conditions. Here we studied cases with pathologically-confirmed AD (n = 7), HS-Aging (n = 7), AD + HS-aging (n = 4), DLB (n = 12), and normal (cognitively intact) controls (NC) (n = 9) from the University of Kentucky Alzheimer's Disease Center autopsy cohort. We defined five microglia morphological phenotypes in the autopsy samples: ramified, hypertrophic, dystrophic, rod-shaped, and amoeboid. The Aperio ScanScope digital neuropathological tool was used along with two well-known microglial markers: IBA1 (a marker for both resting and activated microglia) and CD68 (a lysosomal marker in macrophages/microglia associated with phagocytic cells). Hippocampal staining analyses included studies of subregions within the hippocampal formation and nearby white matter. Using these tools and methods, we describe variation in microglial characteristics that show some degree of disease specificity, including, (1) increased microglia density and number in HS-aging and AD + HS-aging; (2) low microglia density in DLB; (3) increased number of dystrophic microglia in HS-aging; and (4) increased proportion of dystrophic to all microglia in DLB. We conclude that variations in morphologies among microglial cells, and cells of macrophage lineage, can help guide future work connecting neuroinflammatory mechanisms with specific neurodegenerative disease subtypes.

Interferon-gamma induces apoptosis and augments the expression of Fas and Fas ligand by microglia in vitro.

PubMed

Badie, B; Schartner, J; Vorpahl, J; Preston, K

2000-04-01

Activation of microglia by interferon-gamma (IFN-gamma) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-gamma has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-gamma-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-gamma (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-gamma concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-gamma also potentiated the expression of Fas and FasL in a similar dose-response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-gamma may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-gamma modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes. Copyright 2000 Academic Press.

Nanoscale laminin coating modulates cortical scarring response around implanted silicon microelectrode arrays

NASA Astrophysics Data System (ADS)

He, Wei; McConnell, George C.; Bellamkonda, Ravi V.

2006-12-01

Neural electrodes could significantly enhance the quality of life for patients with sensory and/or motor deficits as well as improve our understanding of brain functions. However, long-term electrical connectivity between neural tissue and recording sites is compromised by the development of astroglial scar around the recording probes. In this study we investigate the effect of a nanoscale laminin (LN) coating on Si-based neural probes on chronic cortical tissue reaction in a rat model. Tissue reaction was evaluated after 1 day, 1 week, and 4 weeks post-implant for coated and uncoated probes using immunohistochemical techniques to evaluate activated microglia/macrophages (ED-1), astrocytes (GFAP) and neurons (NeuN). The coating did not have an observable effect on neuronal density or proximity to the electrode surface. However, the response of microglia/macrophages and astrocytes was altered by the coating. One day post-implant, we observed an ~60% increase in ED-1 expression near LN-coated probe sites compared with control uncoated probe sites. Four weeks post-implant, we observed an ~20% reduction in ED-1 expression along with an ~50% reduction in GFAP expression at coated relative to uncoated probe sites. These results suggest that LN has a stimulatory effect on early microglia activation, accelerating the phagocytic function of these cells. This hypothesis is further supported by the increased mRNA expression of several pro-inflammatory cytokines (TNF-α, IL-1 and IL-6) in cultured microglia on LN-bound Si substrates. LN immunostaining of coated probes immediately after insertion and retrieval demonstrates that the coating integrity is not compromised by the shear force during insertion. We speculate, based on these encouraging results, that LN coating of Si neural probes could potentially improve chronic neural recordings through dispersion of the astroglial scar.

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

PubMed

Thellung, Stefano; Villa, Valentina; Corsaro, Alessandro; Pellistri, Francesca; Venezia, Valentina; Russo, Claudio; Aceto, Antonio; Robello, Mauro; Florio, Tullio

2007-11-01

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

The brain parenchyma has a type I interferon response that can limit virus spread.

PubMed

Drokhlyansky, Eugene; Göz Aytürk, Didem; Soh, Timothy K; Chrenek, Ryan; O'Loughlin, Elaine; Madore, Charlotte; Butovsky, Oleg; Cepko, Constance L

2017-01-03

The brain has a tightly regulated environment that protects neurons and limits inflammation, designated "immune privilege." However, there is not an absolute lack of an immune response. We tested the ability of the brain to initiate an innate immune response to a virus, which was directly injected into the brain parenchyma, and to determine whether this response could limit viral spread. We injected vesicular stomatitis virus (VSV), a transsynaptic tracer, or naturally occurring VSV-derived defective interfering particles (DIPs), into the caudate-putamen (CP) and scored for an innate immune response and inhibition of virus spread. We found that the brain parenchyma has a functional type I interferon (IFN) response that can limit VSV spread at both the inoculation site and among synaptically connected neurons. Furthermore, we characterized the response of microglia to VSV infection and found that infected microglia produced type I IFN and uninfected microglia induced an innate immune response following virus injection.

Gamma frequency entrainment attenuates amyloid load and modifies microglia

PubMed Central

Iaccarino, Hannah F.; Singer, Annabelle C.; Martorell, Anthony J.; Rudenko, Andrii; Gao, Fan; Gillingham, Tyler Z.; Mathys, Hansruedi; Seo, Jinsoo; Kritskiy, Oleg; Abdurrob, Fatema; Adaikkan, Chinnakkaruppan; Canter, Rebecca G.; Rueda, Richard; Brown, Emery N.; Boyden, Edward S.; Tsai, Li-Huei

2017-01-01

Changes in gamma oscillations (20-50 Hz) have been observed in several neurological disorders. However, the relationship between gamma and cellular pathologies is unclear. Here, we show reduced behaviorally-driven gamma before the onset of plaque formation or cognitive decline in a mouse model of Alzheimer's disease (AD). Optogenetically driving FS-PV-interneurons at gamma (40 Hz), but not other frequencies, reduced levels of amyloid-β (A β)1-40 and A β1-42 isoforms. Gene expression profiling revealed induction of genes associated with morphological transformation of microglia and histological analysis confirmed increased microglia co-localization with A β. Subsequently, we designed a non-invasive 40 Hz light-flickering paradigm that reduced A β1-40 and A β1-42 levels in visual cortex of pre-depositing mice and mitigated plaque load in aged, depositing mice. Our findings uncover a previously unappreciated function of gamma rhythms in recruiting both neuronal and glial responses to attenuate AD-associated pathology. PMID:27929004

Estrogens, Neuroinflammation, and Neurodegeneration

PubMed Central

Villa, Alessandro; Vegeto, Elisabetta; Poletti, Angelo

2016-01-01

Inflammatory activation of microglia is a hallmark of several disorders of the central nervous system. In addition to protecting the brain against inflammatory insults, microglia are neuroprotective and play a significant role in maintaining neuronal connectivity, but the prolongation of an inflammatory status may limit the beneficial functions of these immune cells. The finding that estrogen receptors are present in monocyte-derived cells and that estrogens prevent and control the inflammatory response raise the question of the role that this sex steroid plays in the manifestation and progression of pathologies that have a clear sex difference in prevalence, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. The present review aims to provide a critical review of the current literature on the actions of estrogen in microglia and on the involvement of estrogen receptors in the manifestation of selected neurological disorders. This current understanding highlights a research area that should be expanded to identify appropriate replacement therapies to slow the progression of such diseases. PMID:27196727

Age-related cognitive impairment is associated with long-term neuroinflammation and oxidative stress in a mouse model of episodic systemic inflammation.

PubMed

d'Avila, Joana Costa; Siqueira, Luciana Domett; Mazeraud, Aurélien; Azevedo, Estefania Pereira; Foguel, Debora; Castro-Faria-Neto, Hugo Caire; Sharshar, Tarek; Chrétien, Fabrice; Bozza, Fernando Augusto

2018-01-30

Microglia function is essential to maintain the brain homeostasis. Evidence shows that aged microglia are primed and show exaggerated response to acute inflammatory challenge. Systemic inflammation signals to the brain inducing changes that impact cognitive function. However, the mechanisms involved in age-related cognitive decline associated to episodic systemic inflammation are not completely understood. The aim of this study was to identify neuropathological features associated to age-related cognitive decline in a mouse model of episodic systemic inflammation. Young and aged Swiss mice were injected with low doses of LPS once a week for 6 weeks to induce episodic systemic inflammation. Sickness behavior, inflammatory markers, and neuroinflammation were assessed in different phases of systemic inflammation in young and aged mice. Behavior was evaluated long term after episodic systemic inflammation by open field, forced swimming, object recognition, and water maze tests. Episodic systemic inflammation induced systemic inflammation and sickness behavior mainly in aged mice. Systemic inflammation induced depressive-like behavior in both young and aged mice. Memory and learning were significantly affected in aged mice that presented lower exploratory activity and deficits in episodic and spatial memories, compared to aged controls and to young after episodic systemic inflammation. Systemic inflammation induced acute microglia activation in young mice that returned to base levels long term after episodic systemic inflammation. Aged mice presented dystrophic microglia in the hippocampus and entorhinal cortex at basal level and did not change morphology in the acute response to SI. Regardless of their dystrophic microglia, aged mice produced higher levels of pro-inflammatory (IL-1β and IL-6) as well as pro-resolution (IL-10 and IL-4) cytokines in the brain. Also, higher levels of Nox2 expression, oxidized proteins and lower antioxidant defenses were found in the aged brains compared to the young after episodic systemic inflammation. Our data show that aged mice have increased susceptibility to episodic systemic inflammation. Aged mice that showed cognitive impairments also presented higher oxidative stress and abnormal production of cytokines in their brains. These results indicate that a neuroinflammation and oxidative stress are pathophysiological mechanisms of age-related cognitive impairments.

Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora.

PubMed

Siamer, Sabrina; Gaubert, Stéphane; Boureau, Tristan; Brisset, Marie-Noëlle; Barny, Marie-Anne

2013-05-01

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Functional versus effector-specific organization of the human posterior parietal cortex: revisited

PubMed Central

Leone, Frank T. M.; Medendorp, W. Pieter

2016-01-01

It has been proposed that the posterior parietal cortex (PPC) is characterized by an effector-specific organization. However, strikingly similar functional MRI (fMRI) activation patterns have been found in the PPC for hand and foot movements. Because the fMRI signal is related to average neuronal activity, similar activation levels may result either from effector-unspecific neurons or from intermingled subsets of effector-specific neurons within a voxel. We distinguished between these possibilities using fMRI repetition suppression (RS). Participants made delayed, goal-directed eye, hand, and foot movements to visual targets. In each trial, the instructed effector was identical or different to that of the previous trial. RS effects indicated an attenuation of the fMRI signal in repeat trials. The caudal PPC was active during the delay but did not show RS, suggesting that its planning activity was effector independent. Hand and foot-specific RS effects were evident in the anterior superior parietal lobule (SPL), extending to the premotor cortex, with limb overlap in the anterior SPL. Connectivity analysis suggested information flow between the caudal PPC to limb-specific anterior SPL regions and between the limb-unspecific anterior SPL toward limb-specific motor regions. These results underline that both function and effector specificity should be integrated into a concept of PPC action representation not only on a regional but also on a fine-grained, subvoxel level. PMID:27466132

Gene duplication and fragment recombination drive functional diversification of a superfamily of cytoplasmic effectors in Phytophthora sojae.

PubMed

Shen, Danyu; Liu, Tingli; Ye, Wenwu; Liu, Li; Liu, Peihan; Wu, Yuren; Wang, Yuanchao; Dou, Daolong

2013-01-01

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.

TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.

PubMed

Ji, Rui; Tian, Shifu; Lu, Helen J; Lu, Qingjun; Zheng, Yan; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

2013-12-15

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Two Faces of Chondroitin Sulfate Proteoglycan in Spinal Cord Repair: A Role in Microglia/Macrophage Activation

PubMed Central

London, Anat; Segev, Yifat; Jacob-Hirsch, Jasmin; Amariglio, Ninette; Rechavi, Gidon; Schwartz, Michal

2008-01-01

Background Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study. Methods and Findings We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-α) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells. Conclusions Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair. PMID:18715114

Impact of end effector technology on telemanipulation performance

NASA Technical Reports Server (NTRS)

Bejczy, A. K.; Szakaly, Z.; Ohm, T.

1990-01-01

Generic requirements for end effector design are briefly summarized as derived from generic functional and operational requirements. Included is a brief summary of terms and definitions related to end effector technology. The second part contains a brief overview of end effector technology work as JPL during the past ten years, with emphasis on the evolution of new mechanical, sensing and control capabilities of end effectors. The third and major part is devoted to the description of current end effector technology. The ongoing work addresses mechanical, sensing and control details with emphasis on mechanical ruggedness, increased resolution in sensing, and close electronic and control integration with overall telemanipulator control system.

Space Station end effector strategy study

NASA Technical Reports Server (NTRS)

Katzberg, Stephen J.; Jensen, Robert L.; Willshire, Kelli F.; Satterthwaite, Robert E.

1987-01-01

The results of a study are presented for terminology definition, identification of functional requirements, technolgy assessment, and proposed end effector development strategies for the Space Station Program. The study is composed of a survey of available or under-developed end effector technology, identification of requirements from baselined Space Station documents, a comparative assessment of the match between technology and requirements, and recommended strategies for end effector development for the Space Station Program.

Mutant alpha-synuclein overexpression mediates early proinflammatory activity.

PubMed

Su, Xiaomin; Federoff, Howard J; Maguire-Zeiss, Kathleen A

2009-10-01

Microglia provide immune surveillance for the brain through both the removal of cellular debris and protection against infection by microorganisms and "foreign" molecules. Upon activation, microglia display an altered morphology and increased expression of proinflammatory molecules. Increased numbers of activated microglia have been identified in a number of neurodegenerative diseases including Parkinson's disease (PD). What remains to be determined is whether activated microglia result from ongoing cell death or are involved in disease initiation and progression. To address this question we utilized a transgenic mouse model that expresses a mutated form of a key protein involved in Parkinson's disease, alpha-synuclein. Herein, we report an increase in activated microglia and proinflammatory molecules in 1-month-old transgenic mice well before cell death occurs in this model. Frank microglial activation is resolved by 6 months of age while a subset of proinflammatory molecules remain elevated for 12 months. Both tyrosine hydroxylase mRNA expression and alpha-synuclein protein are decreased in the striatum of older animals evidence of dystrophic neuritic projections. To determine whether mutated alpha-synuclein could directly activate microglia primary microglia-enriched cell cultures were treated with exogenous mutated alpha-synuclein. The data reveal an increase in activated microglia and proinflammatory molecules due to direct interaction with mutated alpha-synuclein. Together, these data demonstrate that mutated alpha-synuclein mediates a proinflammatory response in microglia and this activity may participate in PD pathogenesis.

Microglia recapitulate a hematopoietic master regulator network in the aging human frontal cortex

PubMed Central

Wehrspaun, Claudia C.; Haerty, Wilfried; Ponting, Chris P.

2015-01-01

Microglia form the immune system of the brain. Previous studies in cell cultures and animal models suggest altered activation states and cellular senescence in the aged brain. Instead, we analyzed 3 transcriptome data sets from the postmortem frontal cortex of 381 control individuals to show that microglia gene markers assemble into a transcriptional module in a gene coexpression network. These markers predominantly represented M1 and M1/M2b activation phenotypes. Expression of genes in this module generally declines over the adult life span. This decrease was more pronounced in microglia surface receptors for microglia and/or neuron crosstalk than in markers for activation state phenotypes. In addition to these receptors for exogenous signals, microglia are controlled by brain-expressed regulatory factors. We identified a subnetwork of transcription factors, including RUNX1, IRF8, PU.1, and TAL1, which are master regulators (MRs) for the age-dependent microglia module. The causal contributions of these MRs on the microglia module were verified using publicly available ChIP-Seq data. Interactions of these key MRs were preserved in a protein-protein interaction network. Importantly, these MRs appear to be essential for regulating microglia homeostasis in the adult human frontal cortex in addition to their crucial roles in hematopoiesis and myeloid cell-fate decisions during embryogenesis. PMID:26002684

Sex differences in the phagocytic and migratory activity of microglia and their impairment by palmitic acid.

PubMed

Yanguas-Casás, Natalia; Crespo-Castrillo, Andrea; de Ceballos, Maria L; Chowen, Julie A; Azcoitia, Iñigo; Arevalo, Maria Angeles; Garcia-Segura, Luis M

2018-03-01

Sex differences in the incidence, clinical manifestation, disease course, and prognosis of neurological diseases, such as autism spectrum disorders or Alzheimer's disease, have been reported. Obesity has been postulated as a risk factor for cognitive decline and Alzheimer's disease and, during pregnancy, increases the risk of autism spectrum disorders in the offspring. Obesity is associated with increased serum and brain levels of free fatty acids, such as palmitic acid, which activate microglial cells triggering a potent inflammatory cascade. In this study, we have determined the effect of palmitic acid in the inflammatory profile, motility, and phagocytosis of primary male and female microglia, both in basal conditions and in the presence of a pro-inflammatory stimulus (interferon-γ). Male microglia in vitro showed higher migration than female microglia under basal and stimulated conditions. In contrast, female microglia had higher basal and stimulated phagocytic activity than male microglia. Palmitic acid did not affect basal migration or phagocytosis, but abolished the migration and phagocytic activity of male and female microglia in response to interferon-γ. These findings extend previous observations of sex differences in microglia and suggest that palmitic acid impairs the protective responses of these cells. © 2017 Wiley Periodicals, Inc.

Enrichment increases hippocampal neurogenesis independent of blood monocyte-derived microglia presence following high-dose total body irradiation.

PubMed

Ruitenberg, Marc J; Wells, Julia; Bartlett, Perry F; Harvey, Alan R; Vukovic, Jana

2017-06-01

Birth of new neurons in the hippocampus persists in the brain of adult mammals and critically underpins optimal learning and memory. The process of adult neurogenesis is significantly reduced following brain irradiation and this correlates with impaired cognitive function. In this study, we aimed to compare the long-term effects of two environmental paradigms (i.e. enriched environment and exercise) on adult neurogenesis following high-dose (10Gy) total body irradiation. When housed in standard (sedentary) conditions, irradiated mice revealed a long-lasting (up to 4 months) deficit in neurogenesis in the granule cell layer of the dentate gyrus, the region that harbors the neurogenic niche. This depressive effect of total body irradiation on adult neurogenesis was partially alleviated by exposure to enriched environment but not voluntary exercise, where mice were single-housed with unlimited access to a running wheel. Exposure to voluntary exercise, but not enriched environment, did lead to significant increases in microglia density in the granule cell layer of the hippocampus; our study shows that these changes result from local microglia proliferation rather than recruitment and infiltration of circulating Cx 3 cr1 +/gfp blood monocytes that subsequently differentiate into microglia-like cells. In summary, latent neural precursor cells remain present in the neurogenic niche of the adult hippocampus up to 8 weeks following high-dose total body irradiation. Environmental enrichment can partially restore the adult neurogenic process in this part of the brain following high-dose irradiation, and this was found to be independent of blood monocyte-derived microglia presence. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Phosphorylation of p53 by LRRK2 induces microglial tumor necrosis factor α-mediated neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, Dong Hwan, E-mail: ethan2887@gmail.com; Seol, Wongi; Eun, Jin Hwan

Leucine-rich repeat kinase (LRRK2), a major causal gene of Parkinson's disease (PD), functions as a kinase. The most prevalent mutation of LRRK2 is G2019S. It exhibits increased kinase activity compared to the wildtype LRRK2. Previous studies have shown that LRRK2 can phosphorylate p53 at T304 and T377 of threonine-X-arginine (TXR) motif in neurons. Reduction of LRRK2 expression or inhibition of LRRK2 kinase activity has been shown to be able to alleviate LPS-induced neuroinflammation in microglia cells. In this study, we found that LRRK2 could also phosphorylate p53 in microglia model BV2 cells. Transfection of BV2 with phosphomimetic p53 T304/377D significantlymore » increased the secretion of pro-inflammatory cytokine TNFα compared to BV2 transfected with p53 wild type after LPS treatment. In addition, conditioned media from these transfected cells increased the death of dopaminergic neuronal SN4741 cells. Moreover, such neurotoxic effect was rescued by co-treatment with the conditioned media and etanercept, a TNFα blocking antibody. Furthermore, TNFα secretion was significantly increased in primary microglia derived from G2019S transgenic mice treated with LPS compared to that in cells derived from their littermates. These results suggest that LRRK2 kinase activity in microglia can contribute to neuroinflammation in PD via phosphorylating p53 at T304 and T377 site. - Highlights: • LPS stimulates LRRK2-mediated p53 phosphorylation and its nuclear localization. • Phosphorylation of p53 by LRRK2 in microglia enhances TNFα expression. • Microglial TNFα via LRRK2-induced p53 phosphorylation decreases neuronal survival.« less

Cortical neuroinflammation contributes to long-term cognitive dysfunctions following adolescent delta-9-tetrahydrocannabinol treatment in female rats.

PubMed

Zamberletti, Erica; Gabaglio, Marina; Prini, Pamela; Rubino, Tiziana; Parolaro, Daniela

2015-12-01

Over 180 million people consume cannabis globally. Cannabis use peaks during adolescence with a trend for continued consumption by adults. Notably, several studies have shown that long-term and heavy cannabis use during adolescence can impair brain maturation and predispose to neurodevelopmental disorders, although the neurobiological mechanisms underlying this association remain largely unknown. In this study, we evaluated whether, in female rats, chronic administration of increasing doses of the psychotropic plant-derived cannabis constituent, delta-9-tetrahydrocannabinol (THC), during adolescence (PND 35-45) could affect microglia function in the long-term. Furthermore, we explored a possible contribution of microglia to the development of THC-induced alterations in mood and cognition in adult female rats. Present data indicate that adolescent THC administration induces a persistent neuroinflammatory state specifically localized within the adult prefrontal cortex (PFC), characterized by increased expression of the pro-inflammatory markers, TNF-α, iNOS and COX-2, and reduction of the anti-inflammatory cytokine, IL-10. This neuroinflammatory phenotype is associated with down-regulation of CB1 receptor on neuronal cells and up-regulation of CB2 on microglia cells, conversely. Interestingly, blocking microglia activation with ibudilast during THC treatment significantly attenuates short-term memory impairments in adulthood, simultaneously preventing the increases in TNF-α, iNOS, COX-2 levels as well as the up-regulation of CB2 receptors on microglia cells. In contrast, THC-induced depressive-like behaviors were unaffected by ibudilast treatment. Our findings demonstrate that adolescent THC administration is associated with persistent neuroinflammation within the PFC and provide evidence for a causal association between microglial activation and the development long-term cognitive deficits induced by adolescent THC treatment. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

PubMed

Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes

PubMed Central

Majumdar, Amitabha; Capetillo-Zarate, Estibaliz; Cruz, Dana; Gouras, Gunnar K.; Maxfield, Frederick R.

2011-01-01

Incomplete lysosomal acidification in microglia inhibits the degradation of fibrillar forms of Alzheimer's amyloid β peptide (fAβ). Here we show that in primary microglia a chloride transporter, ClC-7, is not delivered efficiently to lysosomes, causing incomplete lysosomal acidification. ClC-7 protein is synthesized by microglia but it is mistargeted and appears to be degraded by an endoplasmic reticulum–associated degradation pathway. Activation of microglia with macrophage colony-stimulating factor induces trafficking of ClC-7 to lysosomes, leading to lysosomal acidification and increased fAβ degradation. ClC-7 associates with another protein, Ostm1, which plays an important role in its correct lysosomal targeting. Expression of both ClC-7 and Ostm1 is increased in activated microglia, which can account for the increased delivery of ClC-7 to lysosomes. Our findings suggest a novel mechanism of lysosomal pH regulation in activated microglia that is required for fAβ degradation. PMID:21441306

Microglia constitute a barrier that prevents neurotoxic protofibrillar Aβ42 hotspots around plaques

PubMed Central

Condello, Carlo; Yuan, Peng; Schain, Aaron; Grutzendler, Jaime

2014-01-01

In Alzheimer’s disease (AD), β-amyloid (Aβ) plaques are tightly enveloped by microglia processes, but the significance of this phenomenon is unknown. Here we show that microglia constitute a barrier with profound impact on plaque composition and toxicity. Using high-resolution confocal and in vivo two-photon imaging in AD mouse models, we demonstrate that this barrier prevents outward plaque expansion and leads to compact plaque microregions with low Aβ42 affinity. Areas uncovered by microglia are less compact but have high Aβ42 affinity, leading to formation of protofibrillar Aβ42 hotspots that are associated with more severe axonal dystrophy. In aging, microglia coverage is reduced, leading to enlarged protofibrillar Aβ42 hotspots and more severe neuritic dystrophy. CX3CR1 gene deletion or anti-Aβ immunotherapy causes expansion of microglia coverage and reduced neuritic dystrophy. Failure of the microglia barrier and the accumulation of neurotoxic protofibrillar Aβ hotspots may constitute novel therapeutic and clinical imaging targets for AD. PMID:25630253

MCP-1-mediated activation of microglia promotes white matter lesions and cognitive deficits by chronic cerebral hypoperfusion in mice.

PubMed

Yuan, Bangqing; Shi, Hui; Zheng, Kuang; Su, Zulu; Su, Hai; Zhong, Ming; He, Xuenong; Zhou, Changlong; Chen, Hao; Xiong, Qijiang; Zhang, Yi; Yang, Zhao

2017-01-01

Microglia activation played a vital role in the pathogenesis of white matter lesions (WMLs) by chronic cerebral hypoperfusion. In addition, hypoxia induced up-regulated expression of MCP-1, promotes the activation of microglia. However, the role of MCP-1-mediated microglia activation in chronic cerebral ischemia is still unknown. To explore that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in mice. The activation of microglia and the related signal pathway p38MAPK/PKC in white matter, and working memory of mice were observed. We found that stenosis of common carotid arteries could induce MCP-1-mediated activation of microglia through p38MAPK/PKC pathway and white matter lesions. Taken together, our findings represent a novel mechanism of MCP-1 involved in activation of microglia and provide a novel therapeutical strategy for chronic cerebral hypoperfusion. Copyright © 2016 Elsevier Inc. All rights reserved.

The Role of Microglia and Matrix Metalloproteinases Involvement in Neuroinflammation and Gliomas

PubMed Central

Könnecke, Helen; Bechmann, Ingo

2013-01-01

Matrix metalloproteinases (MMPs) are involved in the pathogenesis of neuroinflammatory diseases (such as multiple sclerosis) as well as in the expansion of malignant gliomas because they facilitate penetration of anatomical barriers (such as the glia limitans) and migration within the neuropil. This review elucidates pathomechanisms and summarizes the current knowledge of the involvement of MMPs in neuroinflammation and glioma, invasion highlighting microglia as major sources of MMPs. The induction of expression, suppression, and multiple pathways of function of MMPs in these scenarios will also be discussed. Understanding the induction and action of MMPs might provide valuable information and reveal attractive targets for future therapeutic strategies. PMID:24023566

Deletion of Aryl Hydrocarbon Receptor AHR in Mice Leads to Subretinal Accumulation of Microglia and RPE Atrophy

PubMed Central

Kim, Soo-Young; Yang, Hyun-Jin; Chang, Yi-Sheng; Kim, Jung-Woong; Brooks, Matthew; Chew, Emily Y.; Wong, Wai T.; Fariss, Robert N.; Rachel, Rivka A.; Cogliati, Tiziana; Qian, Haohua; Swaroop, Anand

2014-01-01

Purpose. The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis. Methods. RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr−/− mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours. Results. In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr−/− mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr−/− retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr−/− mice displayed reduced ERG c-wave amplitudes. Conclusions. The Ahr−/− mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr−/− mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention. PMID:25159211

The critical role of Nramp1 in degrading α-synuclein oligomers in microglia under iron overload condition.

PubMed

Wu, Kuo-Chen; Liou, Horng-Huei; Kao, Yu-Han; Lee, Chih-Yu; Lin, Chun-Jung

2017-08-01

Oligomeric α-synuclein is a key mediator in the pathogenesis of Parkinson's disease (PD) and is mainly cleared by autophagy-lysosomal pathway, whose dysfunction results in the accumulation and cell-to-cell transmission of α-synuclein. In this study, concomitant with the accumulation of iron and oligomeric α-synuclein, higher expression of a lysosomal iron transporter, natural resistance-associated macrophage protein-1 (Nramp1), was observed in microglia in post-mortem striatum of sporadic PD patients. Using Nramp1-deficient macrophage (RAW264.7) and microglial (BV-2) cells as in-vitro models, iron exposure significantly reduced the degradation rate of the administered human α-synuclein oligomers, which can be restored by the expression of the wild-type, but not mutant (D543N), Nramp1. Likewise, under iron overload condition, mice with functional Nramp1 (DBA/2 and C57BL/6 congenic mice carrying functional Nramp1) had a better ability to degrade infused human α-synuclein oligomers than mice with nonfunctional Nramp1 (C57BL/6) in the brain and microglia. The interplay between iron and Nramp1 exhibited parallel effects on the clearance of α-synuclein and the activity of lysosomal cathepsin D in vitro and in vivo. Collectively, these findings suggest that the function of Nramp1 contributes to microglial degradation of oligomeric α-synuclein under iron overload condition and may be implicated in the pathogenesis of PD. Copyright © 2017 Elsevier Inc. All rights reserved.

LOCALIZER: subcellular localization prediction of both plant and effector proteins in the plant cell

PubMed Central

Sperschneider, Jana; Catanzariti, Ann-Maree; DeBoer, Kathleen; Petre, Benjamin; Gardiner, Donald M.; Singh, Karam B.; Dodds, Peter N.; Taylor, Jennifer M.

2017-01-01

Pathogens secrete effector proteins and many operate inside plant cells to enable infection. Some effectors have been found to enter subcellular compartments by mimicking host targeting sequences. Although many computational methods exist to predict plant protein subcellular localization, they perform poorly for effectors. We introduce LOCALIZER for predicting plant and effector protein localization to chloroplasts, mitochondria, and nuclei. LOCALIZER shows greater prediction accuracy for chloroplast and mitochondrial targeting compared to other methods for 652 plant proteins. For 107 eukaryotic effectors, LOCALIZER outperforms other methods and predicts a previously unrecognized chloroplast transit peptide for the ToxA effector, which we show translocates into tobacco chloroplasts. Secretome-wide predictions and confocal microscopy reveal that rust fungi might have evolved multiple effectors that target chloroplasts or nuclei. LOCALIZER is the first method for predicting effector localisation in plants and is a valuable tool for prioritizing effector candidates for functional investigations. LOCALIZER is available at http://localizer.csiro.au/. PMID:28300209

The YopJ superfamily of type III efforts in plant-associated bacteria

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defense responses or innate immunity. However, some effector proteins may be recognized by...

Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

PubMed

Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Frickel, Eva-Maria; Ploegh, Hidde L

2016-11-01

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. © 2016 The Authors.

High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia.

PubMed

Baufeld, Caroline; Osterloh, Anja; Prokop, Stefan; Miller, Kelly R; Heppner, Frank L

2016-09-01

Diets high in fat (HFD) are known to cause an immune response in the periphery as well as the central nervous system. In peripheral adipose tissue, this immune response is primarily mediated by macrophages that are recruited to the tissue. Similarly, reactivity of microglia, the innate immune cells of the brain, has been shown to occur in the hypothalamus of mice fed a high-fat diet. To characterize the nature of the microglial response to diets high in fat in a temporal fashion, we studied the phenotypic spectrum of hypothalamic microglia of mice fed high-fat diet for 3 days and 8 weeks by assessing their tissue reaction and inflammatory signature. While we observed a significant increase in Iba1+ myeloid cells and a reaction of GFAP+ astrocytes in the hypothalamus after 8 weeks of HFD feeding, we found the hypothalamic myeloid cell reaction to be limited to endogenous microglia and not mediated by infiltrating myeloid cells. Moreover, obese humans were found to present with signs of hypothalamic gliosis and exacerbated microglia dystrophy, suggesting a targeted microglia response to diet in humans as well. Notably, the glial reaction occurring in the mouse hypothalamus was not accompanied by an increase in pro-inflammatory cytokines, but rather by an anti-inflammatory reaction. Gene expression analyses of isolated microglia not only confirmed this observation, but also revealed a downregulation of microglia genes important for sensing signals in the microenvironment. Finally, we demonstrate that long-term exposure of microglia to HFD in vivo does not impair the cell's ability to respond to additional stimuli, like lipopolysaccharide. Taken together, our findings support the notion that microglia react to diets high in fat in a region-specific manner in rodents as well as in humans; however, this response changes over time as it is not exclusively pro-inflammatory nor does exposure to HFD prime microglia in the hypothalamus.

Systemic Tolerance Mediated by Melanoma Brain Tumors is Reversible by Radiotherapy and Vaccination

PubMed Central

Jackson, Christopher M.; Kochel, Christina M.; Nirschl, Christopher J.; Durham, Nicholas M.; Ruzevick, Jacob; Alme, Angela; Francica, Brian J.; Elias, Jimmy; Daniels, Andrew; Dubensky, Thomas W.; Lauer, Peter; Brockstedt, Dirk G.; Baxi, Emily G.; Calabresi, Peter A.; Taube, Janis M.; Pardo, Carlos A.; Brem, Henry; Pardoll, Drew M.; Lim, Michael; Drake, Charles G.

2016-01-01

Purpose Immune responses to antigens originating in the CNS are generally attenuated, since collateral damage can have devastating consequences. The significance of this finding for the efficacy of tumor-targeted immunotherapies is largely unknown. Experimental Design The B16 murine melanoma model was used to compare cytotoxic responses against established tumors in the CNS and in the periphery. Cytokine analysis of tissues from brain tumor-bearing mice detected elevated TGF-β secretion from microglia and in the serum and TGF-β signaling blockade reversed tolerance of tumor antigen-directed CD8 T cells. Additionally, a treatment regimen using focal radiation therapy and recombinant Listeria monocytogenes was evaluated for immunologic activity and efficacy in this model. Results CNS melanomas were more tolerogenic than equivalently progressed tumors outside the CNS as antigen-specific CD8 T cells were deleted and exhibited impaired cytotoxicity. Tumor-bearing mice had elevated serum levels of TGF-β; however, blocking TGF-β signaling with a small molecule inhibitor or a monoclonal antibody did not improve survival. Conversely, tumor antigen-specific vaccination in combination with focal radiation therapy reversed tolerance and improved survival. This treatment regimen was associated with increased polyfunctionality of CD8 T cells, elevated T effector to T regulatory cell ratios and decreased TGF-β secretion from microglia. Conclusions These data suggest that CNS tumors may impair systemic antitumor immunity and consequently accelerate cancer progression locally as well as outside the CNS while antitumor immunity may be restored by combining vaccination with radiation therapy. These findings are hypothesis-generating and warrant further study in more contemporary melanoma models as well as human trials. PMID:26490306

Emerging concepts in effector biology of plant-associated organisms.

PubMed

Hogenhout, Saskia A; Van der Hoorn, Renier A L; Terauchi, Ryohei; Kamoun, Sophien

2009-02-01

Plant-associated organisms secrete proteins and other molecules to modulate plant defense circuitry and enable colonization of plant tissue. Understanding the molecular function of these secreted molecules, collectively known as effectors, became widely accepted as essential for a mechanistic understanding of the processes underlying plant colonization. This review summarizes recent findings in the field of effector biology and highlights the common concepts that have emerged from the study of cellular plant pathogen effectors.

Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

PubMed

Ham, Jong Hyun; Majerczak, Doris R; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L

2009-06-01

The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well.

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

PubMed

Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

2017-01-01

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

Involvement of the CD200 receptor complex in microglia activation in experimental glaucoma

PubMed Central

Taylor, Sarah; Calder, Claudia J.; Albon, Julie; Erichsen, Jonathan T.; Boulton, Micheal E.; Morgan, James E.

2013-01-01

The interaction of the myeloid restricted molecule CD200R with its widely expressed ligand CD200 is involved in the down-regulation of microglia activation. In the present study, we examined the involvement of CD200R in microglia activation in experimental ocular hypertension to determine the role of microglia activation in retinal ganglion cell (RGC) death, the key pathological event in glaucoma. Experimental glaucoma was induced in adult Brown Norway rats by sclerosis of the episcleral veins with the injection of hypertonic saline. Immunohistochemical methods were used to determine the involvement of microglia using GFAP, CD45, OX42 and OX41 and the involvement of CD200 and CD200R in the optic nerve head. Our data demonstrate the increased presence of microglia within the optic nerve head during ocular hypertension, identified by positive staining with OX42 and OX41. The peak of microglia correlates with peak in RGC death at days 20–27 (T3) post OHT induction. In addition, CD200 and CD200R positive cells were increased in ocular hypertensive eyes. Increased expression of CD200 was detected in the early phase (days 1–7; T1) of OHT and decreased over time, whilst the expression of CD200R was detected in the middle phase (days 20–27; T3) of OHT, correlating with the increase in microglia markers. Changes in the expression of CD200R/CD200 occur early in experimental glaucoma and precede the peak in microglia infiltration and RGC death, suggesting that CD200R-positive microglia play an important role in the initiation of RGC death during OHT, indicating a potential area for therapeutic intervention in treating glaucoma. PMID:21296076

Inhibition of JAK2 attenuates the increase in inflammatory markers in microglia from APP/PS1 mice.

PubMed

Jones, Raasay S; Minogue, Aedín M; Fitzpatrick, Orla; Lynch, Marina A

2015-10-01

There is a wealth of evidence indicating that macrophages adopt distinct phenotypes when exposed to specific stimuli and, in the past few years, accumulating data suggest that microglia behave somewhat similarly. Therefore, microglia can adopt the so-called M1 or M2 phenotypes in response to interferon-γ (IFNγ) and interleukin-4, respectively. Although it has yet to be unequivocally proven in the context of microglia, acutely activated M1 cells are probably protective, although a persistent M1 state is likely to be damaging, whereas M2 cells may be reparative and restorative. In this case, particularly because the current evidence suggests the development of a predominantly M1 state with age and in neurodegenerative diseases, it is important to identify mechanisms by which polarization of microglia can be modulated. The present findings indicate that exposure of cultured microglia to IFNγ increased expressions of the archetypal markers of the M1 phenotype, tumour necrosis factor-α, and inducible nitric oxide synthase, and preexposure of cells to amyloid-β (Aβ) sensitized microglia to subsequent stimulation with IFNγ. Importantly, this synergy was also evident in microglia prepared from the brains of transgenic mice that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1, APP/PS1 mice) and are exposed to a combination of increasing concentrations of endogenous Aβ from 4 or 5 months of age and an age-related increase in IFNγ. Significantly, the JAK2 inhibitor, TG101209, attenuated the IFNγ-induced changes in cultured microglia and in isolated microglia prepared from APP/PS1 mice. These findings suggest that targeting JAK2 may be a potential strategy for reducing neuroinflammation in Alzheimer's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulation of Dynamic Behavior of Retinal Microglia by CX3CR1 Signaling

PubMed Central

Liang, Katharine J.; Lee, Jung Eun; Wang, Yunqing D.; Ma, Wenxin; Fontainhas, Aurora M.; Fariss, Robert N.; Wong, Wai T.

2009-01-01

PURPOSE Microglia in the central nervous system display a marked structural dynamism in their processes in the resting state. This dynamic behavior, which may play a constitutive surveying role in the uninjured neural parenchyma, is also highly responsive to tissue injury. The role of CX3CR1, a chemokine receptor expressed in microglia, in regulating microglia morphology and dynamic behavior in the resting state and after laser-induced focal injury was examined. METHODS Time-lapse confocal imaging of retinal explants was used to evaluate the dynamic behavior of retinal microglia labeled with green fluorescent protein (GFP). Transgenic mice in which CX3CR1 signaling was ablated (CX3CR1GFP/GFP/CX3CR1−/−) and preserved (CX3CR1+/GFP/CX3CR1+/−) were used. RESULTS Retinal microglial density, distribution, cellular morphology, and overall retinal tissue anatomy were not altered in young CX3CR1−/− animals. In the absence of CX3CR1, retinal microglia continued to exhibit dynamic motility in their processes. However, rates of process movement were significantly decreased, both under resting conditions and in response to tissue injury. In addition, microglia migration occurring in response to focal laser injury was also significantly slowed in microglia lacking CX3CR1. CONCLUSIONS CX3CR1 signaling in retinal microglia, though not absolutely required for the presence of microglial dynamism, plays a role in potentiating the rate of retinal microglial process dynamism and cellular migration. CX3CL1 signaling from retinal neurons and endothelial cells likely modulates dynamic microglia behavior so as to influence the level of microglial surveillance under basal conditions and the rate of dynamic behavior in response to tissue injury. PMID:19443728

Phytophthora parasitica Effector PpRxLR2 Suppresses Nicotiana benthamiana Immunity.

PubMed

Dalio, R J D; Maximo, H J; Oliveira, T S; Dias, R O; Breton, M C; Felizatti, H; Machado, M

2018-04-01

Phytophthora species secrete several classes of effector proteins during interaction with their hosts. These proteins can have multiple functions including modulation of host physiology and immunity. The RxLR effectors have the ability to enter plant cells using the plant machinery. Some of these effectors have been characterized as immunity suppressors; however, very little is known about their functions in the interaction between Phytophthora parasitica and its hosts. Using a bioinformatics pipeline, we have identified 172 candidate RxLR effectors (CREs) in the isolate IAC 01_95 of P. parasitica. Of these 172 CREs, 93 were found to be also present in eight other genomes of P. parasitica, isolated from different hosts and continents. After transcriptomics and gene expression analysis, we have found five CREs to be up-regulated in in-vitro and in-planta samples. Subsequently, we selected three CREs for functional characterization in the model plant Nicotiana benthamiana. We show that PpRxLR2 is able to completely suppress INF-1-induced cell death, whereas PpRxLR3 and PpRxLR5 moderately suppressed N. benthamiana immunity in a less-extensive manner. Moreover, we confirmed the effector-triggered susceptibility activity of these proteins after transient transformation and infection of N. benthamiana plants. All three CREs enhanced virulence of P. parasitica during the interaction with N. benthamiana. These effectors, in particular PpRxLR2, can be targeted for the development of biotechnology-based control strategies of P. parasitica diseases.

Inhibitory effects of palm α-, γ- and δ-tocotrienol on lipopolysaccharide-induced nitric oxide production in BV2 microglia.

PubMed

Tan, Shi Wei; Ramasamy, Rajesh; Abdullah, Maha; Vidyadaran, Sharmili

2011-01-01

Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment (p<.05). None of the tocotrienol doses tested affected microglia viability. Copyright © 2011 Elsevier Inc. All rights reserved.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity.

PubMed

Mehrabadi, Abbas Rezaeian; Korolainen, Minna A; Odero, Gary; Miller, Donald W; Kauppinen, Tiina M

2017-09-01

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target. Copyright © 2017 Elsevier Ltd. All rights reserved.

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Expanded functions for a family of plant intracellular immune receptors beyond specific recognition of pathogen effectors

PubMed Central

Bonardi, Vera; Tang, Saijun; Stallmann, Anna; Roberts, Melinda; Cherkis, Karen; Dangl, Jeffery L.

2011-01-01

Plants and animals deploy intracellular immune receptors that perceive specific pathogen effector proteins and microbial products delivered into the host cell. We demonstrate that the ADR1 family of Arabidopsis nucleotide-binding leucine-rich repeat (NB-LRR) receptors regulates accumulation of the defense hormone salicylic acid during three different types of immune response: (i) ADRs are required as “helper NB-LRRs” to transduce signals downstream of specific NB-LRR receptor activation during effector-triggered immunity; (ii) ADRs are required for basal defense against virulent pathogens; and (iii) ADRs regulate microbial-associated molecular pattern-dependent salicylic acid accumulation induced by infection with a disarmed pathogen. Remarkably, these functions do not require an intact P-loop motif for at least one ADR1 family member. Our results suggest that some NB-LRR proteins can serve additional functions beyond canonical, P-loop–dependent activation by specific virulence effectors, extending analogies between intracellular innate immune receptor function from plants and animals. PMID:21911370

Legionella and Coxiella effectors: strength in diversity and activity.

PubMed

Qiu, Jiazhang; Luo, Zhao-Qing

2017-10-01

Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular pathogens that use the Dot/Icm type IV secretion system to translocate effectors into host cells. These effectors are essential for the establishment of membrane-bound compartments known as replication vacuoles, which enable the survival and replication of bacteria inside host cells. The effectors interfere with diverse signalling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, to promote pathogen survival. In this Review, we explore Dot/Icm effectors from L. pneumophila and C. burnetii as key virulence factors, and we examine the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence.

Sulforaphane induces Nrf2 target genes and attenuates inflammatory gene expression in microglia from brain of young adult and aged mice.

PubMed

Townsend, Brigitte E; Johnson, Rodney W

2016-01-01

Increased neuroinflammation and oxidative stress resulting from heightened microglial activation are associated with age-related cognitive impairment. The objectives of this study were to examine the effects of the bioactive sulforaphane (SFN) on the nuclear factor E2-related factor 2 (Nrf2) pathway in BV2 microglia and primary microglia, and to evaluate proinflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated primary microglia from adult and aged mice. BV2 microglia and primary microglia isolated from young adult and aged mice were treated with SFN and LPS. Changes in Nrf2 activity, expression of Nrf2 target genes, and levels of proinflammatory markers were assessed by quantitative PCR and immunoassay. SFN increased Nrf2 DNA-binding activity and upregulated Nrf2 target genes in BV2 microglia, while reducing LPS-induced interleukin (IL-)1β, IL-6, and inducible nitric oxide synthase (iNOS). In primary microglia from adult and aged mice, SFN increased expression of Nrf2 target genes and attenuated IL-1β, IL-6, and iNOS induced by LPS. These data indicate that SFN is a potential beneficial supplement that may be useful for reducing microglial mediated neuroinflammation and oxidative stress associated with aging. Copyright © 2015 Elsevier Inc. All rights reserved.

Crosstalk between Activated Microglia and Neurons in the Spinal Dorsal Horn Contributes to Stress-induced Hyperalgesia

PubMed Central

Qi, Jian; Chen, Chen; Meng, Qing-Xi; Wu, Yan; Wu, Haitao; Zhao, Ting-Bao

2016-01-01

Stress has been shown to enhance pain sensitivity resulting in stress-induced hyperalgesia. However, the underlying mechanisms have yet to be elucidated. Using single-prolonged stress combined with Complete Freund’s Adjuvant injection model, we explored the reciprocal regulatory relationship between neurons and microglia, which is critical for the maintenance of posttraumatic stress disorder (PTSD)-induced hyperalgesia. In our assay, significant mechanical allodynia was observed. Additionally, activated neurons in spinal dorsal horn were observed by analysis of Fos expression. And, microglia were also significantly activated with the presence of increased Iba-1 expression. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or minocycline (a specific microglia inhibitor) attenuated mechanical allodynia. Moreover, intrathecal administration of c-fos ASO significantly suppressed the activation of neurons and microglia. Interestingly, inhibition of microglia activation by minocycline significantly suppressed the activation of both neurons and microglia in spinal dorsal horn. P38 inhibitor SB203580 suppressed IL-6 production, and inhibition of IL-6 receptor (IL-6R) activation by tocilizumab suppressed Fos expression. Together, our data suggest that the presence of a “crosstalk” between activated microglia and neurons in the spinal dorsal horn, which might contribute to the stress-induced hyperactivated state, leading to an increased pain sensitivity. PMID:27995982

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

PubMed

Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

2017-01-01

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

Absence of cytotoxicity towards microglia of iron oxide (α-Fe 2O 3) nanorhombohedra

DOE PAGES

Crystal S. Lewis; Wong, Stanislaus S.; Torres, Luisa; ...

2016-02-26

Understanding the nature of interactions between nanomaterials, such as commercially ubiquitous hematite (α-Fe 2O 3) nanorhombohedra (N-Rhomb) and biological systems is of critical importance for gaining insight into the practical applicability of nanomaterials. Microglia represent the first line of defense in the central nervous system (CNS) during severe injury or disease such as Parkinson's and Alzheimer's disease as illustrative examples. Hence, to analyze the potential cytotoxic effect of N-Rhomb exposure in the presence of microglia, we have synthesized Rhodamine B (RhB)-labeled α-Fe 2O 3 N-Rhomb, with lengths of 47 ± 10 nm and widths of 35 ± 8 nm. Internalizationmore » of RhB-labeled α-Fe 2O 3 N-Rhomb by microglia in the mouse brain was observed, and a dose-dependent increase in the cellular iron content as probed by cellular fluorescence was detected in cultured microglia after nanoparticle exposure. The cells maintained clear functional viability, exhibiting little to no cytotoxic effects after 24 and 48 hours at acceptable, physiological concentrations. Importantly, the nanoparticle exposure did not induce microglial cells to produce either tumor necrosis factor alpha (TNFα) or interleukin 1-beta (IL1β), two pro-inflammatory cytokines, nor did exposure stimulate the production of nitrites and reactive oxygen species (ROS), which are common indicators for the onset of inflammation. Finally, we propose that under the conditions of our experiments, i.e. in the presence of RhB labeled-α-Fe 2O 3 N-Rhomb maintaining concentrations of up to 100 μg mL–1 after 48 hours of incubation, the in vitro and in vivo internalization of RhB-labeled α-Fe 2O 3 N-Rhomb are likely to be clathrin-dependent, which represents a conventional mechanistic uptake route for most cells. Furthermore, given the crucial role that microglia play in many neurological disorders, understanding the potential cytotoxic effects of these nanostructures is of fundamental importance if they are to be used in a therapeutic setting.« less

Unique inflammatory RNA profiles of microglia in Creutzfeldt-Jakob disease

NASA Astrophysics Data System (ADS)

Baker, Christopher A.; Manuelidis, Laura

2003-01-01

Previous studies in Creutzfeldt-Jakob disease (CJD) have shown that myeloid cells in the periphery as well as derivative microglial cells in the brain are infectious. Microglia can show an activated phenotype before prion protein (PrP) pathology is detectable in brain, and isolated infectious microglia contain very little PrP. To find whether a set of inflammatory genes are significantly induced or suppressed with infection, we analyzed RNA from isolated microglia with relevant cDNA arrays, and identified 30 transcripts not previously examined in any transmissible spongiform encephalopathy. This CJD expression profile contrasted with that of uninfected microglia exposed to prototypic inflammatory stimuli such as lipopolysaccharide and IFN-, as well as PrP amyloid. These findings underscore inflammatory pathways evoked by the infectious agent in brain. Transcript profiles unique for CJD microglia and other myeloid cells provide opportunities for more sensitive preclinical diagnoses of infectious and noninfectious neurodegenerative diseases.

Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme

PubMed Central

Lucca, Liliana E.; Lerner, Benjamin A.; Gunel, Murat; Raddassi, Khadir; Coric, Vlad; Hafler, David A.; Love, J. Christopher

2017-01-01

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM), the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25—CD127+Foxp3—effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1—CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1—CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. PMID:28880903

Multi-walled carbon nanotube (MWCNT) synthesis, preparation, labeling, and functionalization.

PubMed

Kateb, Babak; Yamamoto, Vicky; Alizadeh, Darya; Zhang, Leying; Manohara, Harish M; Bronikowski, Michael J; Badie, Behnam

2010-01-01

Nanomedicine is a growing field with a great potential for introducing new generation of targeted and personalized drug. Amongst new generation of nano-vectors are carbon nanotubes (CNTs), which can be produced as single or multi-walled. Multi-walled carbon nanotubes (MWCNTs) can be fabricated as biocompatible nanostructures (cylindrical bulky tubes). These structures are currently under investigation for their application in nanomedicine as viable and safe nanovectors for gene and drug delivery. In this chapter, we will provide you with the necessary information to understand the synthesis of MWCNTs, functionalization, PKH26 labeling, RNAi, and DNA loading for in vitro experimentation and in vivo implantation of labeled MWCNT in mice as well as materials used in this experimentation. We used this technique to manipulate microglia as part of a novel application for the brain cancer immunotherapy. Our published data show this is a promising technique for labeling, and gene and drug delivery into microglia.

The G protein-coupled receptor GPR34 - The past 20 years of a grownup.

PubMed

Schöneberg, Torsten; Meister, Jaroslawna; Knierim, Alexander Bernd; Schulz, Angela

2018-04-22

Research on GPR34, which was discovered in 1999 as an orphan G protein-coupled receptor of the rhodopsin-like class, disclosed its physiologic relevance only piece by piece. Being present in all recent vertebrate genomes analyzed so far it seems to improve the fitness of species although it is not essential for life and reproduction as GPR34-deficient mice demonstrate. However, closer inspection of macrophages and microglia, where it is mainly expressed, revealed its relevance in immune cell function. Recent data clearly demonstrate that GPR34 function is required to arrest microglia in the M0 homeostatic non-phagocytic phenotype. Herein, we summarize the current knowledge on its evolution, genomic and structural organization, physiology, pharmacology and relevance in human diseases including neurodegenerative diseases and cancer, which accumulated over the last 20 years. Copyright © 2018 Elsevier Inc. All rights reserved.

A bio-inspired approach for the design of a multifunctional robotic end-effector customized for automated maintenance of a reconfigurable vibrating screen.

PubMed

Makinde, O A; Mpofu, K; Vrabic, R; Ramatsetse, B I

2017-01-01

The development of a robotic-driven maintenance solution capable of automatically maintaining reconfigurable vibrating screen (RVS) machine when utilized in dangerous and hazardous underground mining environment has called for the design of a multifunctional robotic end-effector capable of carrying out all the maintenance tasks on the RVS machine. In view of this, the paper presents a bio-inspired approach which unfolds the design of a novel multifunctional robotic end-effector embedded with mechanical and control mechanisms capable of automatically maintaining the RVS machine. To achieve this, therblig and morphological methodologies (which classifies the motions as well as the actions required by the robotic end-effector in carrying out RVS machine maintenance tasks), obtained from a detailed analogy of how human being (i.e. a machine maintenance manager) will carry out different maintenance tasks on the RVS machine, were used to obtain the maintenance objective functions or goals of the multifunctional robotic end-effector as well as the maintenance activity constraints of the RVS machine that must be adhered to by the multifunctional robotic end-effector during the machine maintenance. The results of the therblig and morphological analyses of five (5) different maintenance tasks capture and classify one hundred and thirty-four (134) repetitive motions and fifty-four (54) functions required in automating the maintenance tasks of the RVS machine. Based on these findings, a worm-gear mechanism embedded with fingers extruded with a hexagonal shaped heads capable of carrying out the "gripping and ungrasping" and "loosening and bolting" functions of the robotic end-effector and an electric cylinder actuator module capable of carrying out "unpinning and hammering" functions of the robotic end-effector were integrated together to produce the customized multifunctional robotic end-effector capable of automatically maintaining the RVS machine. The axial forces ([Formula: see text] and [Formula: see text]), normal forces ([Formula: see text]) and total load [Formula: see text] acting on the teeth of the worm-gear module of the multifunctional robotic end-effector during the gripping of worn-out or new RVS machine subsystems, which are 978.547, 1245.06 and 1016.406 N, respectively, were satisfactory. The nominal bending and torsional stresses acting on the shoulder of the socket module of the multifunctional robotic end-effector during the loosing and tightening of bolts, which are 1450.72 and 179.523 MPa, respectively, were satisfactory. The hammering and unpinning forces utilized by the electric cylinder actuator module of the multifunctional robotic end-effector during the unpinning and hammering of screen panel pins out of and into the screen panels were satisfactory.

Kinematic functions for the 7 DOF robotics research arm

NASA Technical Reports Server (NTRS)

Kreutz, K.; Long, M.; Seraji, Homayoun

1989-01-01

The Robotics Research Model K-1207 manipulator is a redundant 7R serial link arm with offsets at all joints. To uniquely determine joint angles for a given end-effector configuration, the redundancy is parameterized by a scalar variable which corresponds to the angle between the manipulator elbow plane and the vertical plane. The forward kinematic mappings from joint-space to end-effector configuration and elbow angle, and the augmented Jacobian matrix which gives end-effector and elbow angle rates as a function of joint rates, are also derived.

Differential Pro-Inflammatory Responses of Astrocytes and Microglia Involve STAT3 Activation in Response to 1800 MHz Radiofrequency Fields

PubMed Central

Lu, Yonghui; He, Mindi; Zhang, Yang; Xu, Shangcheng; Zhang, Lei; He, Yue; Chen, Chunhai; Liu, Chuan; Pi, Huifeng; Yu, Zhengping; Zhou, Zhou

2014-01-01

Microglia and astrocytes play important role in maintaining the homeostasis of central nervous system (CNS). Several CNS impacts have been postulated to be associated with radiofrequency (RF) electromagnetic fields exposure. Given the important role of inflammation in neural physiopathologic processes, we investigated the pro-inflammatory responses of microglia and astrocytes and the involved mechanism in response to RF fields. Microglial N9 and astroglial C8-D1A cells were exposed to 1800 MHz RF for different time with or without pretreatment with STAT3 inhibitor. Microglia and astrocytes were activated by RF exposure indicated by up-regulated CD11b and glial fibrillary acidic protein (GFAP). However, RF exposure induced differential pro-inflammatory responses in astrocytes and microglia, characterized by different expression and release profiles of IL-1β, TNF-α, IL-6, PGE2, nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Moreover, the RF exposure activated STAT3 in microglia but not in astrocytes. Furthermore, the STAT3 inhibitor Stattic ameliorated the RF-induced release of pro-inflammatory cytokines in microglia but not in astrocytes. Our results demonstrated that RF exposure differentially induced pro-inflammatory responses in microglia and astrocytes, which involved differential activation of STAT3 in microglia and astrocytes. Our data provide novel insights into the potential mechanisms of the reported CNS impacts associated with mobile phone use and present STAT3 as a promising target to protect humans against increasing RF exposure. PMID:25275372

Differential pro-inflammatory responses of astrocytes and microglia involve STAT3 activation in response to 1800 MHz radiofrequency fields.

PubMed

Lu, Yonghui; He, Mindi; Zhang, Yang; Xu, Shangcheng; Zhang, Lei; He, Yue; Chen, Chunhai; Liu, Chuan; Pi, Huifeng; Yu, Zhengping; Zhou, Zhou

2014-01-01

Microglia and astrocytes play important role in maintaining the homeostasis of central nervous system (CNS). Several CNS impacts have been postulated to be associated with radiofrequency (RF) electromagnetic fields exposure. Given the important role of inflammation in neural physiopathologic processes, we investigated the pro-inflammatory responses of microglia and astrocytes and the involved mechanism in response to RF fields. Microglial N9 and astroglial C8-D1A cells were exposed to 1800 MHz RF for different time with or without pretreatment with STAT3 inhibitor. Microglia and astrocytes were activated by RF exposure indicated by up-regulated CD11b and glial fibrillary acidic protein (GFAP). However, RF exposure induced differential pro-inflammatory responses in astrocytes and microglia, characterized by different expression and release profiles of IL-1β, TNF-α, IL-6, PGE2, nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Moreover, the RF exposure activated STAT3 in microglia but not in astrocytes. Furthermore, the STAT3 inhibitor Stattic ameliorated the RF-induced release of pro-inflammatory cytokines in microglia but not in astrocytes. Our results demonstrated that RF exposure differentially induced pro-inflammatory responses in microglia and astrocytes, which involved differential activation of STAT3 in microglia and astrocytes. Our data provide novel insights into the potential mechanisms of the reported CNS impacts associated with mobile phone use and present STAT3 as a promising target to protect humans against increasing RF exposure.

TRPM2 contributes to LPS/IFNγ-induced production of nitric oxide via the p38/JNK pathway in microglia.

PubMed

Miyake, Takahito; Shirakawa, Hisashi; Kusano, Ayaka; Sakimoto, Shinya; Konno, Masakazu; Nakagawa, Takayuki; Mori, Yasuo; Kaneko, Shuji

2014-02-07

Microglia are immune cells that maintain brain homeostasis at a resting state by surveying the environment and engulfing debris. However, in some pathological conditions, microglia can produce neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO) that lead to neuronal degeneration. Inflammation-induced calcium (Ca(2+)) signaling is thought to underlie this abnormal activation of microglia, but the mechanisms are still obscure. We previously showed that combined application of lipopolysaccharide and interferon γ (LPS/IFNγ) induced-production of NO in microglia from wild-type (WT) mice is significantly reduced in microglia from transient receptor potential melastatin 2 (TRPM2)-knockout (KO) mice. Here, we found that LPS/IFNγ produced a late-onset Ca(2+) signaling in WT microglia, which was abolished by application of the NADPH oxidase inhibitor diphenylene iodonium (DPI) and ML-171. In addition, pharmacological blockade or gene deletion of TRPM2 channel in microglia did not show this Ca(2+) signaling. Furthermore, pharmacological manipulation and Western blotting revealed that Ca(2+) mobilization, the proline-rich tyrosine kinase 2 (Pyk2), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) contributed to TRPM2-mediated LPS/IFNγ-induced activation, while the extracellular signal-regulated protein kinase (ERK) did not. These results suggest that LPS/IFNγ activates TRPM2-mediated Ca(2+) signaling, which in turn increases downstream p38 MAPK and JNK signaling and results in increased NO production in microglia. Copyright © 2014 Elsevier Inc. All rights reserved.

Electroporation of Functional Bacterial Effectors into Mammalian Cells

DOE PAGES

Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

2015-01-19

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less

Deciphering Interplay between Salmonella Invasion Effectors

PubMed Central

Koronakis, Vassilis

2008-01-01

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction. PMID:18389058

Curcumin Delays Retinal Degeneration by Regulating Microglia Activation in the Retina of rd1 Mice.

PubMed

Wang, Yanhe; Yin, Zhiyuan; Gao, Lixiong; Sun, Dayu; Hu, Xisu; Xue, Langyue; Dai, Jiaman; Zeng, YuXiao; Chen, Siyu; Pan, Boju; Chen, Min; Xie, Jing; Xu, Haiwei

2017-01-01

Retinitis pigmentosa (RP) is characterized by degeneration of photoreceptors, and there are currently no effective treatments for this disease. However, curcumin has shown neuroprotectant efficacy in a RP rat and swine model, and thus, may have neuroprotective effects in this disease. Immunofluorescence staining, electroretinogram recordings, and behavioral tests were used to analyze the effects of curcumin and the underlying mechanism in retinal degeneration 1 (rd1) mice. The number of apoptotic cells in the retina of rd1 mice at postnatal day 14 significantly decreased with curcumin treatment and visual function was improved. The activation of microglia and secretion of chemokines and matrix metalloproteinases in the retina were inhibited by curcumin. These effects were also observed in a co-culture of BV2 microglial cells and retina-derived 661W cells. Curcumin delayed retinal degeneration by suppressing microglia activation in the retina of rd1 mice. Thus, it may be an effective treatment for neurodegenerative disorders such as RP. © 2017 The Author(s). Published by S. Karger AG, Basel.

Functionalized Iron Oxide Nanoparticles for Controlling the Movement of Immune Cells

PubMed Central

White, Ethan E; Pai, Alex; Weng, Yiming; Suresh, Anil K.; Van Haute, Desiree; Pailevanian, Torkom; Alizadeh, Darya; Hajimiri, Ali; Badie, Behnam; Berlin, Jacob M.

2015-01-01

Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed “cell box” was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain. PMID:25848983

Functionalized iron oxide nanoparticles for controlling the movement of immune cells.

PubMed

White, Ethan E; Pai, Alex; Weng, Yiming; Suresh, Anil K; Van Haute, Desiree; Pailevanian, Torkom; Alizadeh, Darya; Hajimiri, Ali; Badie, Behnam; Berlin, Jacob M

2015-05-07

Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed "cell box" was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain.

Anti-Inflammatory Effects of Omega-3 Fatty Acids in the Brain: Physiological Mechanisms and Relevance to Pharmacology.

PubMed

Layé, Sophie; Nadjar, Agnès; Joffre, Corinne; Bazinet, Richard P

2018-01-01

Classically, polyunsaturated fatty acids (PUFA) were largely thought to be relatively inert structural components of brain, largely important for the formation of cellular membranes. Over the past 10 years, a host of bioactive lipid mediators that are enzymatically derived from arachidonic acid, the main n-6 PUFA, and docosahexaenoic acid, the main n-3 PUFA in the brain, known to regulate peripheral immune function, have been detected in the brain and shown to regulate microglia activation. Recent advances have focused on how PUFA regulate the molecular signaling of microglia, especially in the context of neuroinflammation and behavior. Several active drugs regulate brain lipid signaling and provide proof of concept for targeting the brain. Because brain lipid metabolism relies on a complex integration of diet, peripheral metabolism, including the liver and blood, which supply the brain with PUFAs that can be altered by genetics, sex, and aging, there are many pathways that can be disrupted, leading to altered brain lipid homeostasis. Brain lipid signaling pathways are altered in neurologic disorders and may be viable targets for the development of novel therapeutics. In this study, we discuss in particular how n-3 PUFAs and their metabolites regulate microglia phenotype and function to exert their anti-inflammatory and proresolving activities in the brain. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

A Combination of Ex vivo Diffusion MRI and Multiphoton to Study Microglia/Monocytes Alterations after Spinal Cord Injury

PubMed Central

Noristani, Harun N.; Boukhaddaoui, Hassan; Saint-Martin, Guillaume; Auzer, Pauline; Sidiboulenouar, Rahima; Lonjon, Nicolas; Alibert, Eric; Tricaud, Nicolas; Goze-Bac, Christophe; Coillot, Christophe; Perrin, Florence E.

2017-01-01

Central nervous system (CNS) injury has been observed to lead to microglia activation and monocytes infiltration at the lesion site. Ex vivo diffusion magnetic resonance imaging (diffusion MRI or DWI) allows detailed examination of CNS tissues, and recent advances in clearing procedures allow detailed imaging of fluorescent-labeled cells at high resolution. No study has yet combined ex vivo diffusion MRI and clearing procedures to establish a possible link between microglia/monocytes response and diffusion coefficient in the context of spinal cord injury (SCI). We carried out ex vivo MRI of the spinal cord at different time-points after spinal cord transection followed by tetrahydrofuran based clearing and examined the density and morphology of microglia/monocytes using two-photon microscopy. Quantitative analysis revealed an early marked increase in microglial/monocytes density that is associated with an increase in the extension of the lesion measured using diffusion MRI. Morphological examination of microglia/monocytes somata at the lesion site revealed a significant increase in their surface area and volume as early as 72 hours post-injury. Time-course analysis showed differential microglial/monocytes response rostral and caudal to the lesion site. Microglia/monocytes showed a decrease in reactivity over time caudal to the lesion site, but an increase was observed rostrally. Direct comparison of microglia/monocytes morphology, obtained through multiphoton, and the longitudinal apparent diffusion coefficient (ADC), measured with diffusion MRI, highlighted that axonal integrity does not correlate with the density of microglia/monocytes or their somata morphology. We emphasize that differential microglial/monocytes reactivity rostral and caudal to the lesion site may thus coincide, at least partially, with reported temporal differences in debris clearance. Our study demonstrates that the combination of ex vivo diffusion MRI and two-photon microscopy may be used to follow structural tissue alteration. Lesion extension coincides with microglia/monocytes density; however, a direct relationship between ADC and microglia/monocytes density and morphology was not observed. We highlighted a differential rostro-caudal microglia/monocytes reactivity that may correspond to a temporal difference in debris clearance and axonal integrity. Thus, potential therapeutic strategies targeting microglia/monocytes after SCI may need to be adjusted not only with the time after injury but also relative to the location to the lesion site. PMID:28769787

Regulation of Effector Treg Cells in Murine Lupus.

PubMed

Chandrasekaran, Uma; Yi, Woelsung; Gupta, Sanjay; Weng, Chien-Huan; Giannopoulou, Eugenia; Chinenov, Yurii; Jessberger, Rolf; Weaver, Casey T; Bhagat, Govind; Pernis, Alessandra B

2016-06-01

Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could limit disease development. © 2016, American College of Rheumatology.

Translational Advancement of Somatostatin Gene Delivery for Disease Modification and Cognitive Sparing in Intractable Epilepsy

DTIC Science & Technology

2015-09-01

morphological analysis revealed significantly more activated (p < 0.001) and highly activated ( phagocytic ; p < 0.05) microglia in the dentate gyri of...process with small cell bodies (Figure 10A). Activated microglia exhibit less ramified processes and larger nuclei (Figure 10B) and phagocytic microglia...significantly increases in the dentate gyri of kindled rats along with the total number of highly activated or phagocytic microglia. We are currently

Phenelzine (monoamine oxidase inhibitor) increases production of nitric oxide and proinflammatory cytokines via the NF-κB pathway in lipopolysaccharide-activated microglia cells.

PubMed

Chung, Hwan-Suck; Kim, Hyunseong; Bae, Hyunsu

2012-10-01

Phenelzine is a potent monoamine oxidase inhibitor that is used in patients with depression. It is also well known that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, which suggests that NO is involved in the pathogenesis of depression. The purpose of this study was to determine if phenelzine affects the production of NO and tumor necrosis factor-alpha (TNF-α) in activated microglia cells. BV-2 microglia cells and primary microglia cells were cultured in DMEM and DMEM/F12 and then cells were treated with LPS or LPS plus phenelzine for 24 h. The culture medium was collected for determination of NO, TNF-α, and IL-6 and cells were harvested by lysis buffer for Western blot analysis. Phenelzine increased the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS), as well as the release of TNF-α and IL-6 in BV-2 microglia cells. It is also confirmed that phenelzine increased the levels of NO, TNF-α and IL-6 in LPS-activated primary microglia cells. Phenelzine increased nuclear translocation of NF-κB by phosphorylation of IκB-α in LPS-activated microglia cells. These findings suggest that high doses of phenelzine could aggravate inflammatory responses in microglia cells that are mediated by NO and TNF-α.

Impaired capacity for upregulation of MHC class II in tumor-associated microglia.

PubMed

Schartner, Jill M; Hagar, Aaron R; Van Handel, Michelle; Zhang, Leying; Nadkarni, Nivedita; Badie, Behnam

2005-09-01

Immunotherapy for malignant gliomas is being studied as a possible adjunctive therapy for this highly fatal disease. Thus far, inadequate understanding of brain tumor immunology has hindered the design of such therapies. For instance, the role of microglia and macrophages, which comprise a significant proportion of tumor-infiltrating inflammatory cells, in the regulation of the local anti-tumor immune response is poorly understood. To study the response of microglia and macrophages to known activators in brain tumors, we injected CpG oligodeoxynucleotide (ODN), interferon-gamma (IFN-gamma), and IFN-gamma/LPS into normal and intracranial RG2 glioma-bearing rodents. Microglia/macrophage infiltration and their surface expression of MHC class II B7.1 and B7.2 was examined by flow cytometry. Each agent evaluated yielded a distinct microglia/macrophage response: CpG ODN was the most potent inducer of microglia/macrophage infiltration and B7.1 expression, while IFN-gamma resulted in the highest MHC-II expression in both normal and tumors. Regardless of the agent injected, however, MHC-II induction was significantly muted in tumor microglia/macrophage as compared with normal brain. These data suggest that microglia/macrophage responsiveness to activators can vary in brain tumors when compared with normal brain. Understanding the mechanism of these differences may be critical in the development of novel immunotherapies for malignant glioma. (c) 2005 Wiley-Liss, Inc.

IFNβ secreted by microglia mediates clearance of myelin debris in CNS autoimmunity.

PubMed

Kocur, Magdalena; Schneider, Reiner; Pulm, Ann-Kathrin; Bauer, Jens; Kropp, Sonja; Gliem, Michael; Ingwersen, Jens; Goebels, Norbert; Alferink, Judith; Prozorovski, Timour; Aktas, Orhan; Scheu, Stefanie

2015-04-03

Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological disability. Interferon β (IFNβ) represents a standard treatment for relapsing-remitting MS and exogenous administration of IFNβ exhibits protective effects in experimentally induced CNS autoimmunity. Also, genetic deletion of IFNβ in mice leads to an aggravation of disease symptoms in the MS model of experimental autoimmune encephalomyelitis (EAE). However, neither the underlying mechanisms mediating the beneficial effects nor the cellular source of IFNβ have been fully elucidated. In this report, a subpopulation of activated microglia was identified as the major producers of IFNβ in the CNS at the peak of EAE using an IFNβ-fluorescence reporter mouse model. These IFNβ expressing microglia specifically localized to active CNS lesions and were associated with myelin debris in demyelinated cerebellar organotypic slice cultures (OSCs). In response to IFNβ microglia showed an enhanced capacity to phagocytose myelin in vitro and up-regulated the expression of phagocytosis-associated genes. IFNβ treatment was further sufficient to stimulate association of microglia with myelin debris in OSCs. Moreover, IFNβ-producing microglia mediated an enhanced removal of myelin debris when co-transplanted onto demyelinated OSCs as compared to IFNβ non-producing microglia. These data identify activated microglia as the major producers of protective IFNβ at the peak of EAE and as orchestrators of IFNβ-induced clearance of myelin debris.

Gap junctions and hemichannels composed of connexins: potential therapeutic targets for neurodegenerative diseases

PubMed Central

Takeuchi, Hideyuki; Suzumura, Akio

2014-01-01

Microglia are macrophage-like resident immune cells that contribute to the maintenance of homeostasis in the central nervous system (CNS). Abnormal activation of microglia can cause damage in the CNS, and accumulation of activated microglia is a characteristic pathological observation in neurologic conditions such as trauma, stroke, inflammation, epilepsy, and neurodegenerative diseases. Activated microglia secrete high levels of glutamate, which damages CNS cells and has been implicated as a major cause of neurodegeneration in these conditions. Glutamate-receptor blockers and microglia inhibitors (e.g., minocycline) have been examined as therapeutic candidates for several neurodegenerative diseases; however, these compounds exerted little therapeutic benefit because they either perturbed physiological glutamate signals or suppressed the actions of protective microglia. The ideal therapeutic approach would hamper the deleterious roles of activated microglia without diminishing their protective effects. We recently found that abnormally activated microglia secrete glutamate via gap-junction hemichannels on the cell surface. Moreover, administration of gap-junction inhibitors significantly suppressed excessive microglial glutamate release and improved disease symptoms in animal models of neurologic conditions such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. Recent evidence also suggests that neuronal and glial communication via gap junctions amplifies neuroinflammation and neurodegeneration. Elucidation of the precise pathologic roles of gap junctions and hemichannels may lead to a novel therapeutic strategies that can slow and halt the progression of neurodegenerative diseases. PMID:25228858

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3more » in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.« less

In vitro proliferation of axotomized rat facial nucleus-derived activated microglia in an autocrine fashion.

PubMed

Nakajima, Kazuyuki; Graeber, Manuel B; Sonoda, Maya; Tohyama, Yoko; Kohsaka, Shinichi; Kurihara, Tadashi

2006-08-01

Transection of rat adult facial nerve leads to an increase in the number of activated microglia in the facial nucleus (FN), with a peak in proliferation 3 days after transection. To investigate the characteristics of these activated microglia, we isolated the cells with high purity from axotomized FN (axFN) 3 days after transection according to the previously reported procedure for explant culture. The isolated microglia exhibited immunocytochemical properties similar to those in vivo, and their numbers increased approximately five- to sevenfold over a period of 10 days without the addition of any mitogens, suggesting that self-reproduction was occurring. Actually, the microglia actively incorporated bromodeoxyuridine (BrdU) and strongly expressed an S-phase-specific protein marker, proliferating cell nuclear antigen (PCNA). To examine the mechanism underlying this proliferation, the expression of the mitogens and specific receptors of the microglia were analyzed in conditioned medium (CM) and cells. Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-CSF (GM-CSF) were detected in the CM as well as in the cells. Their specific receptor proteins, c-Fms and GMCSFRalpha, were also detected in the cell homogenate. These proliferating microglia were not found to produce deleterious factors for neurons. In summary, the microglia isolated from the axFN were found to be proliferative in an autocrine fashion and to have some cellular properties in common with those observed in vivo.

Activation of microglia induces symptoms of Parkinson’s disease in wild-type, but not in IL-1 knockout mice

PubMed Central

2013-01-01

Background Parkinson’s disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice. Methods Lipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection. Results LPS treatment induced the activation of microglia, as demonstrated by production of IL-1β and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice. Conclusions The subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment. PMID:24289537

Mild Traumatic Brain Injury Produces Neuron Loss That Can Be Rescued by Modulating Microglial Activation Using a CB2 Receptor Inverse Agonist

PubMed Central

Bu, Wei; Ren, Huiling; Deng, Yunping; Del Mar, Nobel; Guley, Natalie M.; Moore, Bob M.; Honig, Marcia G.; Reiner, Anton

2016-01-01

We have previously reported that mild TBI created by focal left-side cranial blast in mice produces widespread axonal injury, microglial activation, and a variety of functional deficits. We have also shown that these functional deficits are reduced by targeting microglia through their cannabinoid type-2 (CB2) receptors using 2-week daily administration of the CB2 inverse agonist SMM-189. CB2 inverse agonists stabilize the G-protein coupled CB2 receptor in an inactive conformation, leading to increased phosphorylation and nuclear translocation of the cAMP response element binding protein (CREB), and thus bias activated microglia from a pro-inflammatory M1 to a pro-healing M2 state. In the present study, we showed that SMM-189 boosts nuclear pCREB levels in microglia in several brain regions by 3 days after TBI, by using pCREB/CD68 double immunofluorescent labeling. Next, to better understand the basis of motor deficits and increased fearfulness after TBI, we used unbiased stereological methods to characterize neuronal loss in cortex, striatum, and basolateral amygdala (BLA) and assessed how neuronal loss was affected by SMM-189 treatment. Our stereological neuron counts revealed a 20% reduction in cortical and 30% reduction in striatal neurons bilaterally at 2–3 months post blast, with SMM-189 yielding about 50% rescue. Loss of BLA neurons was restricted to the blast side, with 33% of Thy1+ fear-suppressing pyramidal neurons and 47% of fear-suppressing parvalbuminergic (PARV) interneurons lost, and Thy1-negative fear-promoting pyramidal neurons not significantly affected. SMM-189 yielded 50–60% rescue of Thy1+ and PARV neuron loss in BLA. Thus, fearfulness after mild TBI may result from the loss of fear-suppressing neuron types in BLA, and SMM-189 may reduce fearfulness by their rescue. Overall, our findings indicate that SMM-189 rescues damaged neurons and thereby alleviates functional deficits resulting from TBI, apparently by selectively modulating microglia to the beneficial M2 state. CB2 inverse agonists thus represent a promising therapeutic approach for mitigating neuroinflammation and neurodegeneration. PMID:27766068

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

PubMed Central

Zhou, Yan; Dong, Na; Hu, Liyan; Shao, Feng

2013-01-01

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells. PMID:23469023

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

PubMed

Sharma, Shailendra; Sharma, Shiveta; Hirabuchi, Akiko; Yoshida, Kentaro; Fujisaki, Koki; Ito, Akiko; Uemura, Aiko; Terauchi, Ryohei; Kamoun, Sophien; Sohn, Kee Hoon; Jones, Jonathan D G; Saitoh, Hiromasa

2013-05-01

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A homologous form of human interleukin 16 is implicated in microglia recruitment following nervous system injury in leech Hirudo medicinalis.

PubMed

Croq, Françoise; Vizioli, Jacopo; Tuzova, Marina; Tahtouh, Muriel; Sautiere, Pierre-Eric; Van Camp, Christelle; Salzet, Michel; Cruikshank, William W; Pestel, Joel; Lefebvre, Christophe

2010-11-01

In contrast to mammals, the medicinal leech Hirudo medicinalis can completely repair its central nervous system (CNS) after injury. This invertebrate model offers unique opportunities to study the molecular and cellular basis of the CNS repair processes. When the leech CNS is injured, microglial cells migrate and accumulate at the site of lesion, a phenomenon known to be essential for the usual sprouting of injured axons. In the present study, we demonstrate that a new molecule, designated HmIL-16, having functional homologies with human interleukin-16 (IL-16), has chemotactic activity on leech microglial cells as observed using a gradient of human IL-16. Preincubation of microglial cells either with an anti-human IL-16 antibody or with anti-HmIL-16 antibody significantly reduced microglia migration induced by leech-conditioned medium. Functional homology was demonstrated further by the ability of HmIL-16 to promote human CD4+ T cell migration which was inhibited by antibody against human IL-16, an IL-16 antagonist peptide or soluble CD4. Immunohistochemistry of leech CNS indicates that HmIL-16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells to the injured axons. To our knowledge, this is the first identification of a functional interleukin-16 homologue in invertebrate CNS. The ability of HmIL-16 to recruit microglial cells to sites of CNS injury suggests a role for HmIL-16 in the crosstalk between neurons and microglia in the leech CNS repair.

Effects of Moderate Prenatal Alcohol Exposure during Early Gestation in Rats on Inflammation across the Maternal-Fetal-Immune Interface and Later-Life Immune Function in the Offspring

PubMed Central

Terasaki, Laurne S.; Schwarz, Jaclyn M.

2017-01-01

During early brain development, microglial activation can negatively impact long-term neuroimmune and cognitive outcomes. It is well-known that significant alcohol exposure during early gestation results in a number of cognitive deficits associated with fetal alcohol spectrum disorders (FASD). Additionally, microglia are activated following high levels of alcohol exposure in rodent models of FASD. We sought to examine whether moderate prenatal alcohol exposure (70 mg/dL blood alcohol concentration) activates microglia in the fetal rat brain, and whether moderate fetal alcohol exposure has long-term negative consequences for immune function and cognitive function in the rat. We also measured inflammation within the placenta and maternal serum following moderate alcohol exposure to determine whether either could be a source of cytokine production in the fetus. One week of moderate prenatal alcohol exposure produced a sex-specific increase in cytokines and chemokines within the fetal brain. Cytokines were also increased within the placenta, regardless of the sex of the fetus, and independent of the low levels of circulating cytokines within the maternal serum. Adult offspring exposed to alcohol prenatally had exaggerated cytokine production in the brain and periphery in response to lipopolysaccharide (25 μg/kg), as well as significant memory deficits precipitated by this low-level of inflammation. Thus the immune system, including microglia, may be a key link to understanding the etiology of fetal alcohol spectrum disorders and other unexplored cognitive or health risks associated with even low levels of fetal alcohol exposure. PMID:27318824

Potential roles of microglial cell progranulin in HIV-associated CNS pathologies and neurocognitive impairment

PubMed Central

Suh, Hyeon-Sook; Gelman, Benjamin B.; Lee, Sunhee C.

2013-01-01

Progranulin (PGRN) is a highly unusual molecule with both neuronal and microglial expression with two seemingly unrelated functions, i.e., as a neuronal growth factor and a modulator of neuroinflammation. Haploinsufficiency due to loss of function mutations lead to a fatal presenile dementing illness (frontotemporal lobar degeneration), indicating that adequate expression of PGRN is essential for successful aging. PGRN might be a particularly relevant factor in the pathogenesis of HIV encephalitis (HIVE) and HIV-associated neurocognitive disorders (HAND). We present emerging data and a review of the literature which show that cells of myeloid lineage such as macrophages and microglia are the primary sources of PGRN and that PGRN expression contributes to pathogenesis of CNS diseases. We also present evidence that PGRN is a macrophage antiviral cytokine. For example, PGRN mRNA and protein expression are significantly upregulated in brain specimens with HIVE, and in HIV-infected microglia in vitro. Paradoxically, our preliminary CHARTER data analyses indicate that lower PGRN levels in CSF trended towards an association with HAND, particularly in those without detectable virus. Based upon these findings, we introduce the hypothesis that PGRN plays dual roles in modulating antiviral immunity and neuronal dysfunction in the context of HIV infection. In the presence of active viral replication, PGRN expression is increased functioning as an anti-viral factor as well as a neuroprotectant. In the absence of active HIV replication, ongoing inflammation or other stressors suppress PGRN production from macrophages/microglia contributing to neurocognitive dysfunction. We propose CSF PGRN as a candidate surrogate marker for HAND. PMID:23959579

Polymorphisms in the microglial marker molecule CX3CR1 affect the blood volume of the human brain.

PubMed

Sakai, Mai; Takeuchi, Hikaru; Yu, Zhiqian; Kikuchi, Yoshie; Ono, Chiaki; Takahashi, Yuta; Ito, Fumiaki; Matsuoka, Hiroo; Tanabe, Osamu; Yasuda, Jun; Taki, Yasuyuki; Kawashima, Ryuta; Tomita, Hiroaki

2018-06-01

CX3CR1, a G-protein-coupled receptor, is involved in various inflammatory processes. Two non-synonymous single nucleotide polymorphisms, V249I (rs3732379) and T280M (rs3732378), are located in the sixth and seventh transmembrane domains of the CX3CR1 protein, respectively. Previous studies have indicated significant associations between T280M and leukocyte functional characteristics, including adhesion, signaling, and chemotaxis, while the function of V249I is unclear. In the brain, microglia are the only proven and widely accepted CX3CR1-expressing cells. This study aimed to specify whether there were specific brain regions on which these two single nucleotide polymorphisms exert their biological impacts through their functional effects on microglia. Associations between the single nucleotide polymorphisms and brain characteristics, including gray and white matter volumes, white matter integrity, resting arterial blood volume, and cerebral blood flow, were evaluated among 1300 healthy Japanese individuals. The major allele carriers (V249 and T280) were significantly associated with an increased total arterial blood volume of the whole brain, especially around the bilateral precuneus, left posterior cingulate cortex, and left posterior parietal cortex. There were no significant associations between the genotypes and other brain structural indicators. This finding suggests that the CX3CR1 variants may affect arterial structures in the brain, possibly via interactions between microglia and brain microvascular endothelial cells. © 2018 The Authors. Psychiatry and Clinical Neurosciences © 2018 Japanese Society of Psychiatry and Neurology.

Mitochondrial Ca2+ and membrane potential, an alternative pathway for Interleukin 6 to regulate CD4 cell effector function

PubMed Central

Yang, Rui; Lirussi, Dario; Thornton, Tina M; Jelley-Gibbs, Dawn M; Diehl, Sean A; Case, Laure K; Madesh, Muniswamy; Taatjes, Douglas J; Teuscher, Cory; Haynes, Laura; Rincón, Mercedes

2015-01-01

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca2+ late during activation of CD4 cells. Increased levels of mitochondrial Ca2+ in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca2+ is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases. DOI: http://dx.doi.org/10.7554/eLife.06376.001 PMID:25974216

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells

PubMed Central

Carrette, Florent; Henriquez, Monique L.; Fujita, Yu

2018-01-01

T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus–specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7−/− effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7−/− CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells. PMID:29491007

[Effects of electromagnetic pulse exposure on the morphological change and excretion function of BV-2 cells and possible mechanism].

PubMed

Yang, Long-long; Zhou, Yan; Li, Hai-juan; Guo, Juan; Zhang, Yan-jun; Ding, Gui-rong; Guo, Guo-zhen

2012-03-01

To study the effects of electromagnetic pulse (EMP) exposure on the morphological change and excretion functions of mouse microglia (BV-2) cells and possible mechanism. BV-2 cells were divided into two groups: the group exposed to EMP at 200 kV/m for 200 pulses and sham exposure group. At 1, 6, 12 and 24 hour after exposure the cells and culture supernatant were collected. Cellular morphological change was observed under invert microscope, the levels of TNF-α, IL-1β and IL-10 in culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA), nitric oxide (NO) and reactive oxygen species (ROS) were detected by nitrate reductase method and DCFH-DA probe, respectively. The protein and phosphorylation levels of ERK, JNK and p38 were measured by Western Blot method. After the cells pre-treated with the inhibitor of p38 (SB203580) were exposed to EMP, the levels of NO and ROS in culture supernatant were detected. It was found that the large ameboid shape appeared in some microglia cells exposed to EMP for 1, 6 and 12 h. Moreover, the number of microglia cells with ameboid shape increased significantly at 1 h, 6 h and 12 h after EMP exposure compared with sham group (P < 0.05). The levels of cytokines, such as TNF-α, IL-1β and IL-10, in culture supernatant did not change obviously after EMP exposure. The levels of NO and ROS increased significantly at 1h after EMP exposure, reached the peak at 6 h, began to recover at 12 h and recovered to sham group level at 24 h (P < 0.05). Western blot results showed that the protein and protein phosphorylation levels of ERK and JNK did not change significantly after EMP exposure, however, the protein and protein phosphorylation levels of p38 increased obviously at 1 h and 6 h after EMP exposure, compared with sham group (P < 0.05). In addition, the pretreatment of p38 inhibitor (SB203580) significantly decreased NO and ROS production induced by EMP. EMP exposure may activate microglia cells and promote the production of NO and ROS in mouse microglia cells, and p38 pathway is involved in this process.

System xC- is a mediator of microglial function and its deletion slows symptoms in amyotrophic lateral sclerosis mice.

PubMed

Mesci, Pinar; Zaïdi, Sakina; Lobsiger, Christian S; Millecamps, Stéphanie; Escartin, Carole; Seilhean, Danielle; Sato, Hideyo; Mallat, Michel; Boillée, Séverine

2015-01-01

Amyotrophic lateral sclerosis is the most common adult-onset motor neuron disease and evidence from mice expressing amyotrophic lateral sclerosis-causing SOD1 mutations suggest that neurodegeneration is a non-cell autonomous process where microglial cells influence disease progression. However, microglial-derived neurotoxic factors still remain largely unidentified in amyotrophic lateral sclerosis. With excitotoxicity being a major mechanism proposed to cause motor neuron death in amyotrophic lateral sclerosis, our hypothesis was that excessive glutamate release by activated microglia through their system [Formula: see text] (a cystine/glutamate antiporter with the specific subunit xCT/Slc7a11) could contribute to neurodegeneration. Here we show that xCT expression is enriched in microglia compared to total mouse spinal cord and absent from motor neurons. Activated microglia induced xCT expression and during disease, xCT levels were increased in both spinal cord and isolated microglia from mutant SOD1 amyotrophic lateral sclerosis mice. Expression of xCT was also detectable in spinal cord post-mortem tissues of patients with amyotrophic lateral sclerosis and correlated with increased inflammation. Genetic deletion of xCT in mice demonstrated that activated microglia released glutamate mainly through system [Formula: see text]. Interestingly, xCT deletion also led to decreased production of specific microglial pro-inflammatory/neurotoxic factors including nitric oxide, TNFa and IL6, whereas expression of anti-inflammatory/neuroprotective markers such as Ym1/Chil3 were increased, indicating that xCT regulates microglial functions. In amyotrophic lateral sclerosis mice, xCT deletion surprisingly led to earlier symptom onset but, importantly, this was followed by a significantly slowed progressive disease phase, which resulted in more surviving motor neurons. These results are consistent with a deleterious contribution of microglial-derived glutamate during symptomatic disease. Therefore, we show that system [Formula: see text] participates in microglial reactivity and modulates amyotrophic lateral sclerosis motor neuron degeneration, revealing system [Formula: see text] inactivation, as a potential approach to slow amyotrophic lateral sclerosis disease progression after onset of clinical symptoms. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana.

PubMed

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium -mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa , Psa -NZ V13 and Psa -NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana . Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium -mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1 , NDR1 , or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana . In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta .

Pseudomonas syringae pv. actinidiae Type III Effectors Localized at Multiple Cellular Compartments Activate or Suppress Innate Immune Responses in Nicotiana benthamiana

PubMed Central

Choi, Sera; Jayaraman, Jay; Segonzac, Cécile; Park, Hye-Jee; Park, Hanbi; Han, Sang-Wook; Sohn, Kee Hoon

2017-01-01

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta. PMID:29326748

Respiratory Viral Infection in Neonatal Piglets Causes Marked Microglia Activation in the Hippocampus and Deficits in Spatial Learning

PubMed Central

Elmore, Monica R. P.; Burton, Michael D.; Conrad, Matthew S.; Rytych, Jennifer L.; Van Alstine, William G.

2014-01-01

Environmental insults during sensitive periods can affect hippocampal development and function, but little is known about peripheral infection, especially in humans and other animals whose brain is gyrencephalic and experiences major perinatal growth. Using a piglet model, the present study showed that inoculation on postnatal day 7 with the porcine reproductive and respiratory syndrome virus (PRRSV) caused microglial activation within the hippocampus with 82% and 43% of isolated microglia being MHC II+ 13 and 20 d after inoculation, respectively. In control piglets, <5% of microglia isolated from the hippocampus were MHC II+. PRRSV piglets were febrile (p < 0.0001), anorectic (p < 0.0001), and weighed less at the end of the study (p = 0.002) compared with control piglets. Increased inflammatory gene expression (e.g., IL-1β, IL-6, TNF-α, and IFN-γ) was seen across multiple brain regions, including the hippocampus, whereas reductions in CD200, NGF, and MBP were evident. In a test of spatial learning, PRRSV piglets took longer to acquire the task, had a longer latency to choice, and had a higher total distance moved. Overall, these data demonstrate that viral respiratory infection is associated with a marked increase in activated microglia in the hippocampus, neuroinflammation, and impaired performance in a spatial cognitive task. As respiratory infections are common in human neonates and infants, approaches to regulate microglial cell activity are likely to be important. PMID:24501353

Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells.

PubMed

Quentin, Michaëel; Abad, Pierre; Favery, Bruno

2013-01-01

Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells (FCs). Effectors synthesized in the esophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized FCs requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defense responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalized within FC nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesized roles in the unique feeding behavior of these pests.

Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens.

PubMed

Vleeshouwers, Vivianne G A A; Oliver, Richard P

2014-03-01

One of most important challenges in plant breeding is improving resistance to the plethora of pathogens that threaten our crops. The ever-growing world population, changing pathogen populations, and fungicide resistance issues have increased the urgency of this task. In addition to a vital inflow of novel resistance sources into breeding programs, the functional characterization and deployment of resistance also needs improvement. Therefore, plant breeders need to adopt new strategies and techniques. In modern resistance breeding, effectors are emerging as tools to accelerate and improve the identification, functional characterization, and deployment of resistance genes. Since genome-wide catalogues of effectors have become available for various pathogens, including biotrophs as well as necrotrophs, effector-assisted breeding has been shown to be successful for various crops. "Effectoromics" has contributed to classical resistance breeding as well as for genetically modified approaches. Here, we present an overview of how effector-assisted breeding and deployment is being exploited for various pathosystems.

Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Cheng; Nie, Xiaoke; Zhang, Yan

2015-10-15

Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remain largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NOmore » and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis. - Highlights: • PFOS exposure induced expression of iNOS and production of NO in HAPI microglia. • PFOS induced the production of ROS in HAPI microglia. • ERK/JNK MAPK pathways were activated following PFOS exposure in HAPI microglia. • NO released by HAPI microglia participated in the apoptosis of SH-SY5Y cells.« less

Neurotensin stimulates sortilin and mTOR in human microglia inhibitable by methoxyluteolin, a potential therapeutic target for autism.

PubMed

Patel, Arti B; Tsilioni, Irene; Leeman, Susan E; Theoharides, Theoharis C

2016-09-23

We had reported elevated serum levels of the peptide neurotensin (NT) in children with autism spectrum disorders (ASD). Here, we show that NT stimulates primary human microglia, the resident immune cells of the brain, and the immortalized cell line of human microglia-SV40. NT (10 nM) increases the gene expression and release (P < 0.001) of the proinflammatory cytokine IL-1β and chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 from human microglia. NT also stimulates proliferation (P < 0.05) of microglia-SV40. Microglia express only the receptor 3 (NTR3)/sortilin and not the NTR1 or NTR2. The use of siRNA to target sortilin reduces (P < 0.001) the NT-stimulated cytokine and chemokine gene expression and release from human microglia. Stimulation with NT (10 nM) increases the gene expression of sortilin (P < 0.0001) and causes the receptor to be translocated from the cytoplasm to the cell surface, and to be secreted extracellularly. Our findings also show increased levels of sortilin (P < 0.0001) in the serum from children with ASD (n = 36), compared with healthy controls (n = 20). NT stimulation of microglia-SV40 causes activation of the mammalian target of rapamycin (mTOR) signaling kinase, as shown by phosphorylation of its substrates and inhibition of these responses by drugs that prevent mTOR activation. NT-stimulated responses are inhibited by the flavonoid methoxyluteolin (0.1-1 μM). The data provide a link between sortilin and the pathological findings of microglia and inflammation of the brain in ASD. Thus, inhibition of this pathway using methoxyluteolin could provide an effective treatment of ASD.

Activated Microglia Desialylate and Phagocytose Cells via Neuraminidase, Galectin-3, and Mer Tyrosine Kinase

PubMed Central

Nomura, Koji; Vilalta, Anna; Allendorf, David H.; Hornik, Tamara C.

2017-01-01

Activated microglia can phagocytose dying, stressed, or excess neurons and synapses via the phagocytic receptor Mer tyrosine kinase (MerTK). Galectin-3 (Gal-3) can cross-link surface glycoproteins by binding galactose residues that are normally hidden below terminal sialic acid residues. Gal-3 was recently reported to opsonize cells via activating MerTK. We found that LPS-activated BV-2 microglia rapidly released Gal-3, which was blocked by calcineurin inhibitors. Gal-3 bound to MerTK on microglia and to stressed PC12 (neuron-like) cells, and it increased microglial phagocytosis of PC12 cells or primary neurons, which was blocked by inhibition of MerTK. LPS-activated microglia exhibited a sialidase activity that desialylated PC12 cells and could be inhibited by Tamiflu, a neuraminidase (sialidase) inhibitor. Sialidase treatment of PC12 cells enabled Gal-3 to bind and opsonize the live cells for phagocytosis by microglia. LPS-induced microglial phagocytosis of PC12 was prevented by small interfering RNA knockdown of Gal-3 in microglia, lactose inhibition of Gal-3 binding, inhibition of neuraminidase with Tamiflu, or inhibition of MerTK by UNC569. LPS-induced phagocytosis of primary neurons by primary microglia was also blocked by inhibition of MerTK. We conclude that activated microglia release Gal-3 and a neuraminidase that desialylates microglial and PC12 surfaces, enabling Gal-3 binding to PC12 cells and their phagocytosis via MerTK. Thus, Gal-3 acts as an opsonin of desialylated surfaces, and inflammatory loss of neurons or synapses may potentially be blocked by inhibiting neuraminidases, Gal-3, or MerTK. PMID:28500071

DCPIB, a potent volume-regulated anion channel antagonist, attenuates microglia-mediated inflammatory response and neuronal injury following focal cerebral ischemia.

PubMed

Han, Qingdong; Liu, Shengwen; Li, Zhengwei; Hu, Feng; Zhang, Qiang; Zhou, Min; Chen, Jingcao; Lei, Ting; Zhang, Huaqiu

2014-01-13

Accumulating evidence indicates that extensive microglia activation-mediated local inflammation contributes to neuronal injury in cerebral ischemia. We have previously shown that 4-(2-butyl-6, 7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), a potent volume-regulated anion channel (VRAC) inhibitor, suppresses pathological glutamate release and excitatory neurotoxicity in reversible middle cerebral artery occlusion (rMCAO) model in vivo. In the present study, we sought to determine whether DCPIB also attenuates microglia activation that could contribute to neuronal injury in the cerebral ischemia/reperfusion pathology. We show that oxygen-glucose deprivation (OGD) induced microglia proliferation, migration, and secretion of cytokines and all these pathological changes were effectively inhibited by DCPIB in vitro. In the microglia/neuron co-cultures, OGD induced neuronal damage was reduced markedly in the presence of DCPIB. In rat rMCAO animal model, DCPIB significantly attenuated microglia activation and neuronal death. Activation of mitogen-activated protein kinase (MAPK) signaling pathway is known to be a critical signaling pathway for microglia activation. We further explored a potential involvement of DCPIB in this pathway by western blot analysis. Under the conditions that MAPK pathway was activated either by lipopolysaccharides (LPS) or OGD, the levels of phosphorylated ERK1/2, JNK and p38 were reduced significantly in the presence of DCPIB. Altogether, our study demonstrated that DCPIB inhibits microglia activation potently under ischemic conditions both in vitro and in vivo. The DCPIB effect is likely attributable to both direct inhibition VRAC and indirect inhibition of MAPK pathway in microglia that are beneficial for the survival of neurons in cerebral ischemic conditions. © 2013 Elsevier B.V. All rights reserved.

Increased White Matter Inflammation in Aging- and Alzheimer's Disease Brain.

PubMed

Raj, Divya; Yin, Zhuoran; Breur, Marjolein; Doorduin, Janine; Holtman, Inge R; Olah, Marta; Mantingh-Otter, Ietje J; Van Dam, Debby; De Deyn, Peter P; den Dunnen, Wilfred; Eggen, Bart J L; Amor, Sandra; Boddeke, Erik

2017-01-01

Chronic neuroinflammation, which is primarily mediated by microglia, plays an essential role in aging and neurodegeneration. It is still unclear whether this microglia-induced neuroinflammation occurs globally or is confined to distinct brain regions. In this study, we investigated microglia activity in various brain regions upon healthy aging and Alzheimer's disease (AD)-related pathology in both human and mouse samples. In purified microglia isolated from aging mouse brains, we found a profound gene expression pattern related to pro-inflammatory processes, phagocytosis, and lipid homeostasis. Particularly in white matter microglia of 24-month-old mice, abundant expression of phagocytic markers including Mac-2, Axl, CD16/32, Dectin1, CD11c, and CD36 was detected. Interestingly, in white matter of human brain tissue the first signs of inflammatory activity were already detected during middle age. Thus quantification of microglial proteins, such as CD68 (commonly associated with phagocytosis) and HLA-DR (associated with antigen presentation), in postmortem human white matter brain tissue showed an age-dependent increase in immunoreactivity already in middle-aged people (53.2 ± 2.0 years). This early inflammation was also detectable by non-invasive positron emission tomography imaging using [ 11 C]-(R)-PK11195, a ligand that binds to activated microglia. Increased microglia activity was also prominently present in the white matter of human postmortem early-onset AD (EOAD) brain tissue. Interestingly, microglia activity in the white matter of late-onset AD (LOAD) CNS was similar to that of the aged clinically silent AD cases. These data indicate that microglia-induced neuroinflammation is predominant in the white matter of aging mice and humans as well as in EOAD brains. This white matter inflammation may contribute to the progression of neurodegeneration, and have prognostic value for detecting the onset and progression of aging and neurodegeneration.

Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function

USDA-ARS?s Scientific Manuscript database

The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization via activation of Arp2/3 and plays a role in the dynamics of the immunological synapse. How these events influence subsequent gene expression and effector function is unclear. We studied the role of WASp in CD4+ T cell effe...

Neural control of the kidney: functionally specific renal sympathetic nerve fibers.

PubMed

DiBona, G F

2000-11-01

The sympathetic nervous system provides differentiated regulation of the functions of various organs. This differentiated regulation occurs via mechanisms that operate at multiple sites within the classic reflex arc: peripherally at the level of afferent input stimuli to various reflex pathways, centrally at the level of interconnections between various central neuron pools, and peripherally at the level of efferent fibers targeted to various effectors within the organ. In the kidney, increased renal sympathetic nerve activity regulates the functions of the intrarenal effectors: the tubules, the blood vessels, and the juxtaglomerular granular cells. This enables a physiologically appropriate coordination between the circulatory, filtration, reabsorptive, excretory, and renin secretory contributions to overall renal function. Anatomically, each of these effectors has a dual pattern of innervation consisting of a specific and selective innervation by unmyelinated slowly conducting C-type renal sympathetic nerve fibers in addition to an innervation that is shared among all the effectors. This arrangement permits the maximum flexibility in the coordination of physiologically appropriate responses of the tubules, the blood vessels, and the juxtaglomerular granular cells to a variety of homeostatic requirements.

Functionally specific renal sympathetic nerve fibers: role in cardiovascular regulation.

PubMed

DiBona, G F

2001-06-01

The sympathetic nervous system provides differentiated regulation of the functions of various organs. This differentiated regulation occurs through mechanisms that operate at multiple sites within the classic reflex arc: peripherally at the level of afferent input stimuli to various reflex pathways, centrally at the level of interconnections between various central neuron pools, and peripherally at the level of efferent fibers targeted to various effectors within the organ. In the kidney, increased renal sympathetic nerve activity regulates the functions of the intrarenal effectors: the tubules, the blood vessels, and the juxtaglomerular granular cells. This enables a physiologically appropriate coordination between the circulatory, filtration, reabsorptive, excretory, and renin secretory contributions to overall renal function. Anatomically, each of these effectors has a dual pattern of innervation consisting of a specific and selective innervation by unmyelinated slowly conducting C-type renal sympathetic nerve fibers and an innervation that is shared among all the effectors. This arrangement facilitates maximum flexibility in the coordination of the tubules, the blood vessels, and the juxtaglomerular granular cells so as to produce physiologically appropriate responses to a variety of homeostatic requirements.

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins.

PubMed

Hewezi, Tarek

2015-10-01

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. © 2015 American Society of Plant Biologists. All Rights Reserved.

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

PubMed Central

Selin, Carrie; de Kievit, Teresa R.; Belmonte, Mark F.; Fernando, W. G. Dilantha

2016-01-01

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. PMID:27199930

Calcineurin A beta deficiency ameliorates HFD-induced hypothalamic astrocytosis in mice.

PubMed

Pfuhlmann, Katrin; Schriever, Sonja C; Legutko, Beata; Baumann, Peter; Harrison, Luke; Kabra, Dhiraj G; Baumgart, Emily Violette; Tschöp, Matthias H; Garcia-Caceres, Cristina; Pfluger, Paul T

2018-02-08

ᅟ: Astrocytosis is a reactive process involving cellular, molecular, and functional changes to facilitate neuronal survival, myelin preservation, blood brain barrier function and protective glial scar formation upon brain insult. The overall pro- or anti-inflammatory impact of reactive astrocytes appears to be driven in a context- and disease-driven manner by modulation of astrocytic Ca 2+ homeostasis and activation of Ca 2+ /calmodulin-activated serine/threonine phosphatase calcineurin. Here, we aimed to assess whether calcineurin is dispensable for astrocytosis in the hypothalamus driven by prolonged high fat diet (HFD) feeding. Global deletion of calcineurin A beta (gene name: Ppp3cb) led to a decrease of glial fibrillary acidic protein (GFAP)-positive cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) of mice exposed chronically to HFD. The concomitant decrease in Iba1-positive microglia in the VMH further suggests a modest impact of Ppp3cb deletion on microgliosis. Pharmacological inhibition of calcineurin activity by Fk506 had no impact on IBA1-positive microglia in hypothalami of mice acutely exposed to HFD for 1 week. However, Fk506-treated mice displayed a decrease in GFAP levels in the ARC. In vivo effects could not be replicated in cell culture, where calcineurin inhibition by Fk506 had no effect on astrocytic morphology, astrocytic cell death, GFAP, and vimentin protein levels or microglia numbers in primary hypothalamic astrocytes and microglia co-cultures. Further, adenoviral overexpression of calcineurin subunit Ppp3r1 in primary glia culture did not lead to an increase in GFAP fluorescence intensity. Overall, our results point to a prominent role of calcineurin in mediating hypothalamic astrocytosis as response to acute and chronic HFD exposure. Moreover, discrepant findings in vivo and in cell culture indicate the necessity of studying astrocytes in their "natural" environment, i.e., preserving an intact hypothalamic microenvironment with neurons and non-neuronal cells in close proximity.

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

PubMed Central

Liu, Yunxiao; Lan, Xia; Song, Shiren; Yin, Ling; Dry, Ian B.; Qu, Junjie; Xiang, Jiang; Lu, Jiang

2018-01-01

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria. PMID:29706971

Microglia energy metabolism in metabolic disorder.

PubMed

Kalsbeek, Martin J T; Mulder, Laurie; Yi, Chun-Xia

2016-12-15

Microglia are the resident macrophages of the CNS, and are in charge of maintaining a healthy microenvironment to ensure neuronal survival. Microglia carry out a non-stop patrol of the CNS, make contact with neurons and look for abnormalities, all of which requires a vast amount of energy. This non-signaling energy demand increases after activation by pathogens, neuronal damage or other kinds of stimulation. Of the three major energy substrates - glucose, fatty acids and glutamine - glucose is crucial for microglia survival and several glucose transporters are expressed to supply sufficient glucose influx. Fatty acids are another source of energy for microglia and have also been shown to strongly influence microglial immune activity. Glutamine, although possibly suitable for use as an energy substrate by microglia, has been shown to have neurotoxic effects when overloaded. Microglial fuel metabolism might be associated with microglial reactivity under different pathophysiological conditions and a microglial fuel switch may thus be the underlying cause of hypothalamic dysregulation, which is associated with obesity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody

PubMed Central

Luo, Wenjie; Liu, Wencheng; Hu, Xiaoyan; Hanna, Mary; Caravaca, April; Paul, Steven M.

2015-01-01

Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer’s disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies. PMID:26057852

Modulation of hemoglobin dynamics by an allosteric effector

DOE PAGES

Lal, Jyotsana; Maccarini, Marco; Fouquet, Peter; ...

2016-12-15

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb andmore » HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo (NSE) measurements accompanied by wideangle x-ray scattering (WAXS) to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. Furthermore, these observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.« less

SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual function protein that facilitates infection while protecting from wheat-produced chitinases

USDA-ARS?s Scientific Manuscript database

All fungal plant pathogens produce effectors to manipulate the plant immune system to colonize and gain nutrients from the plant cell. Much is known about how fungal pathogens classified as biotrophs use effectors to interact with their hosts and how the host responds, however, less is known about ...

A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat

USDA-ARS?s Scientific Manuscript database

It has now been established that the wheat pathogen Stagonospora nodorum causes disease on wheat in an inverse gene-for-gene manner through the interaction of pathogen effector proteins and corresponding dominant susceptibility host genes. One such effector, SnToxA, interacts with the Tsn1 gene to c...

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

Gene Therapy for Neuropathic Pain through siRNA-IRF5 Gene Delivery with Homing Peptides to Microglia.

PubMed

Terashima, Tomoya; Ogawa, Nobuhiro; Nakae, Yuki; Sato, Toshiyuki; Katagi, Miwako; Okano, Junko; Maegawa, Hiroshi; Kojima, Hideto

2018-06-01

Astrocyte- and microglia-targeting peptides were identified and isolated using phage display technology. A series of procedures, including three cycles of both in vivo and in vitro biopanning, was performed separately in astrocytes and in M1 or M2 microglia, yielding 50-58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocytes (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely, astrocytes, M1 microglia, or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between peptide MG1 and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Germane, Katherine L.; Spiller, Benjamin W.

2011-09-20

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

PubMed

Suzuki, Godai; Wang, Yang; Kubo, Karen; Hirata, Eri; Ohnuki, Shinsuke; Ohya, Yoshikazu

2018-02-20

The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

PubMed

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Nuclear processes associated with plant immunity and pathogen susceptibility

PubMed Central

Motion, Graham B.; Amaro, Tiago M.M.M.; Kulagina, Natalja

2015-01-01

Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant–microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants. PMID:25846755

Nuclear processes associated with plant immunity and pathogen susceptibility.

PubMed

Motion, Graham B; Amaro, Tiago M M M; Kulagina, Natalja; Huitema, Edgar

2015-07-01

Plants are sessile organisms that have evolved exquisite and sophisticated mechanisms to adapt to their biotic and abiotic environment. Plants deploy receptors and vast signalling networks to detect, transmit and respond to a given biotic threat by inducing properly dosed defence responses. Genetic analyses and, more recently, next-generation -omics approaches have allowed unprecedented insights into the mechanisms that drive immunity. Similarly, functional genomics and the emergence of pathogen genomes have allowed reciprocal studies on the mechanisms governing pathogen virulence and host susceptibility, collectively allowing more comprehensive views on the processes that govern disease and resistance. Among others, the identification of secreted pathogen molecules (effectors) that modify immunity-associated processes has changed the plant-microbe interactions conceptual landscape. Effectors are now considered both important factors facilitating disease and novel probes, suited to study immunity in plants. In this review, we will describe the various mechanisms and processes that take place in the nucleus and help regulate immune responses in plants. Based on the premise that any process required for immunity could be targeted by pathogen effectors, we highlight and describe a number of functional assays that should help determine effector functions and their impact on immune-related processes. The identification of new effector functions that modify nuclear processes will help dissect nuclear signalling further and assist us in our bid to bolster immunity in crop plants. © The Author 2015. Published by Oxford University Press.

Functionalized iron oxide nanoparticles for controlling the movement of immune cells

NASA Astrophysics Data System (ADS)

White, Ethan E.; Pai, Alex; Weng, Yiming; Suresh, Anil K.; van Haute, Desiree; Pailevanian, Torkom; Alizadeh, Darya; Hajimiri, Ali; Badie, Behnam; Berlin, Jacob M.

2015-04-01

Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed ``cell box'' was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain.Immunotherapy is currently being investigated for the treatment of many diseases, including cancer. The ability to control the location of immune cells during or following activation would represent a powerful new technique for this field. Targeted magnetic delivery is emerging as a technique for controlling cell movement and localization. Here we show that this technique can be extended to microglia, the primary phagocytic immune cells in the central nervous system. The magnetized microglia were generated by loading the cells with iron oxide nanoparticles functionalized with CpG oligonucleotides, serving as a proof of principle that nanoparticles can be used to both deliver an immunostimulatory cargo to cells and to control the movement of the cells. The nanoparticle-oligonucleotide conjugates are efficiently internalized, non-toxic, and immunostimulatory. We demonstrate that the in vitro migration of the adherent, loaded microglia can be controlled by an external magnetic field and that magnetically-induced migration is non-cytotoxic. In order to capture video of this magnetically-induced migration of loaded cells, a novel 3D-printed ``cell box'' was designed to facilitate our imaging application. Analysis of cell movement velocities clearly demonstrate increased cell velocities toward the magnet. These studies represent the initial step towards our final goal of using nanoparticles to both activate immune cells and to control their trafficking within the diseased brain. Electronic supplementary information (ESI) available: Transmission electron microscopy images of the particles, additional independent experiments for the NFκB activity and exocytosis assays, TEM images for the SPION untreated cells, bright field microscopy images of the cells alone in the presence and absence of magnet, images of the magnetic movement experiments at higher doses of SPION, full uncropped images of the post-migration LIVE/DEAD assay, and a video file of cell movement. See DOI: 10.1039/c3nr04421a

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

New insights into Blimp-1 in T lymphocytes: a divergent regulator of cell destiny and effector function.

PubMed

Fu, Shin-Huei; Yeh, Li-Tzu; Chu, Chin-Chen; Yen, B Lin-Ju; Sytwu, Huey-Kang

2017-07-21

B lymphocyte-induced maturation protein-1 (Blimp-1) serves as a master regulator of the development and function of antibody-producing B cells. Given that its function in T lymphocytes has been identified within the past decade, we review recent findings with emphasis on its role in coordinated control of gene expression during the development, differentiation, and function of T cells. Expression of Blimp-1 is mainly confined to activated T cells and is essential for the production of interleukin (IL)-10 by a subset of forkhead box (Fox)p3 + regulatory T cells with an effector phenotype. Blimp-1 is also required to induce cell elimination in the thymus and critically modulates peripheral T cell activation and proliferation. In addition, Blimp-1 promotes T helper (Th) 2 lineage commitment and limits Th1, Th17 and follicular helper T cell differentiation. Furthermore, Blimp-1 coordinates with other transcription factors to regulate expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. In CD8 + T cells, Blimp-1 expression is distinct in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation when they encounter antigens. Moreover, Blimp-1 plays a fundamental role in coordinating cytokine receptor signaling networks and transcriptional programs to regulate diverse aspects of the formation and function of effector and memory CD8 + T cells and their exhaustion. Blimp-1 also functions as a gatekeeper of T cell activation and suppression to prevent or dampen autoimmune disease, antiviral responses and antitumor immunity. In this review, we discuss the emerging roles of Blimp-1 in the complex regulation of gene networks that regulate the destiny and effector function of T cells and provide a Blimp-1-dominated transcriptional framework for T lymphocyte homeostasis.

In vitro modeling of HIV proviral activity in microglia.

PubMed

Campbell, Lee A; Richie, Christopher T; Zhang, Yajun; Heathward, Emily J; Coke, Lamarque M; Park, Emily Y; Harvey, Brandon K

2017-12-01

Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-κB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Gestational Exposure to Air Pollution Alters Cortical Volume, Microglial Morphology, and Microglia-Neuron Interactions in a Sex-Specific Manner.

PubMed

Bolton, Jessica L; Marinero, Steven; Hassanzadeh, Tania; Natesan, Divya; Le, Dominic; Belliveau, Christine; Mason, S N; Auten, Richard L; Bilbo, Staci D

2017-01-01

Microglia are the resident immune cells of the brain, important for normal neural development in addition to host defense in response to inflammatory stimuli. Air pollution is one of the most pervasive and harmful environmental toxicants in the modern world, and several large scale epidemiological studies have recently linked prenatal air pollution exposure with an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD). Diesel exhaust particles (DEP) are a primary toxic component of air pollution, and markedly activate microglia in vitro and in vivo in adult rodents. We have demonstrated that prenatal exposure to DEP in mice, i.e., to the pregnant dams throughout gestation, results in a persistent vulnerability to behavioral deficits in adult offspring, especially in males, which is intriguing given the greater incidence of ASD in males to females (∼4:1). Moreover, there is a striking upregulation of toll-like receptor (TLR) 4 gene expression within the brains of the same mice, and this expression is primarily in microglia. Here we explored the impact of gestational exposure to DEP or vehicle on microglial morphology in the developing brains of male and female mice. DEP exposure increased inflammatory cytokine protein and altered the morphology of microglia, consistent with activation or a delay in maturation, only within the embryonic brains of male mice; and these effects were dependent on TLR4. DEP exposure also increased cortical volume at embryonic day (E)18, which switched to decreased volume by post-natal day (P)30 in males, suggesting an impact on the developing neural stem cell niche. Consistent with this hypothesis, we found increased microglial-neuronal interactions in male offspring that received DEP compared to all other groups. Taken together, these data suggest a mechanism by which prenatal exposure to environmental toxins may affect microglial development and long-term function, and thereby contribute to the risk of neurodevelopmental disorders.

Dexamethasone-induced abolition of the inflammatory response in an experimental glioma model: a flow cytometry study.

PubMed

Badie, B; Schartner, J M; Paul, J; Bartley, B A; Vorpahl, J; Preston, J K

2000-10-01

Commonly used for management of cerebral edema in patients with brain tumors, steroid medications also have immunosuppressive functions. To characterize the effects of steroids on the central nervous system's response to tumors more clearly, flow cytometry was used to quantify the extent of inflammatory cell infiltration in an immunogenic rat glioma model. Freshly prepared 11-day-old intracranial C6 tumors that had been excised from dexamethasone-treated and untreated rats were labeled ex vivo with monoclonal antibodies against CD 11b/c, CD45, and CD8a antigens. The extent of microglia (CD11b/c-highly positive, CD45-slightly positive cell), macrophage (CD11b/c-highly positive, CD45-highly positive cell), lymphocyte (CD11b/c-negative, CD45-highly positive cell), and cytotoxic T-cell (CD8a-positive cell) infiltration into each rat's tumor, tumor periphery, and contralateral tumor-free hemisphere was analyzed using flow cytometry. Microglia and lymphocytes constituted a significant component of infiltrating cells in this model, comprising 23 +/- 3% and 33 +/- 5% of viable cells, respectively. Macrophages, on the other hand, accounted for only 9 +/- 1% of infiltrating cells. Treatment of rats with a 7-day course of low-dose dexamethasone (0.1 mg/kg/day) resulted in a greater than 50% inhibition of microglia (p = 0.03) and lymphocyte (p = 0.001) infiltration into tumors. Increasing the dexamethasone dose to 1 mg/kg/day further abolished lymphocyte infiltration (89% inhibition, p = 0.001) but had no additional inhibitory effect on microglia invasion. Macrophage infiltration of tumors was not inhibited at the dexamethasone doses used in this study (p = 0.42). Flow cytometry is a valuable technique for characterizing tumor-associated inflammatory cells in gliomas. Even at low doses, dexamethasone was found to inhibit significantly the infiltration of brain tumors by lymphocytes and microglia. These findings should be considered when experimental immunotherapeutic strategies are evaluated for clinical application.

Gestational Exposure to Air Pollution Alters Cortical Volume, Microglial Morphology, and Microglia-Neuron Interactions in a Sex-Specific Manner

PubMed Central

Bolton, Jessica L.; Marinero, Steven; Hassanzadeh, Tania; Natesan, Divya; Le, Dominic; Belliveau, Christine; Mason, S. N.; Auten, Richard L.; Bilbo, Staci D.

2017-01-01

Microglia are the resident immune cells of the brain, important for normal neural development in addition to host defense in response to inflammatory stimuli. Air pollution is one of the most pervasive and harmful environmental toxicants in the modern world, and several large scale epidemiological studies have recently linked prenatal air pollution exposure with an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD). Diesel exhaust particles (DEP) are a primary toxic component of air pollution, and markedly activate microglia in vitro and in vivo in adult rodents. We have demonstrated that prenatal exposure to DEP in mice, i.e., to the pregnant dams throughout gestation, results in a persistent vulnerability to behavioral deficits in adult offspring, especially in males, which is intriguing given the greater incidence of ASD in males to females (∼4:1). Moreover, there is a striking upregulation of toll-like receptor (TLR) 4 gene expression within the brains of the same mice, and this expression is primarily in microglia. Here we explored the impact of gestational exposure to DEP or vehicle on microglial morphology in the developing brains of male and female mice. DEP exposure increased inflammatory cytokine protein and altered the morphology of microglia, consistent with activation or a delay in maturation, only within the embryonic brains of male mice; and these effects were dependent on TLR4. DEP exposure also increased cortical volume at embryonic day (E)18, which switched to decreased volume by post-natal day (P)30 in males, suggesting an impact on the developing neural stem cell niche. Consistent with this hypothesis, we found increased microglial-neuronal interactions in male offspring that received DEP compared to all other groups. Taken together, these data suggest a mechanism by which prenatal exposure to environmental toxins may affect microglial development and long-term function, and thereby contribute to the risk of neurodevelopmental disorders. PMID:28620294

Activation of mixed glia by Abeta-specific Th1 and Th17 cells and its regulation by Th2 cells.

PubMed

McQuillan, K; Lynch, Marina A; Mills, Kingston H G

2010-05-01

Microglia are innate immune cells of the CNS, that act as antigen-presenting cells (APC) for antigen-specific T cells and respond to inflammatory stimuli, such as amyloid-beta (Abeta), resulting in the release of neurotoxic factors and pro-inflammatory cytokines. Astrocytes can also act as APC and modulate the function of microglia. However, the role of distinct T cell subtypes, in particular Th17 cells, in glial activation and subsequent modulatory effects of Th2 cells are poorly understood. Here, we generated Abeta-specific Th1, Th2, and Th17 cells and examined their role in modulating Abeta-induced activation of microglia in a mixed glial culture, a preparation which mimics the complex APC types in the brain. We demonstrated that mixed glia acted as an effective APC for Abeta-specific Th1 and Th17 cells. Addition of Abeta-specific Th2 cells suppressed the Abeta-induced IFN-gamma production by Th1 cells and IL-17 production by Th17 cells with glia as the APC. Co-culture of Abeta-specific Th1 or Th17 cells with glia markedly enhanced Abeta-induced pro-inflammatory cytokine production and expression of MHC class II and co-stimulatory molecules on the microglia. Addition of Abeta-specific Th2 cells inhibited Th17 cell-induced IL-1beta and IL-6 production by mixed glia and attenuated Th1 cell-induced CD86 and CD40 expression on microglia. The modest enhancement of MHC class II and CD86 expression on astrocytes by Abeta-specific Th1 and Th17 was not attenuated by Th2 cells. These data indicate that Abeta-specific Th1 and Th17 cells induce inflammatory activation of glia, and that this is in part regulated by Th2 cells. Copyright 2010 Elsevier Inc. All rights reserved.

Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, Akt, NF-kappaB, and p300 pathway in microglia.

PubMed

Tang, Chih-Hsin; Lu, Da-Yuu; Yang, Rong-Sen; Tsai, Huei-Yann; Kao, Ming-Ching; Fu, Wen-Mei; Chen, Yuh-Fung

2007-07-15

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-kappaB inhibitor (pyrrolidine dithiocarbamate), IkappaB protease inhibitor (L-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IkappaBalpha phosphorylation inhibitor (Bay 117082), or NF-kappaB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IkappaB kinase alpha/IkappaB kinase beta, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Leptin-mediated an increase of IkappaB kinase alpha/IkappaB kinase beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-kappaB and p300 signaling pathway.

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors.

PubMed

Wei, Hai-Lei; Collmer, Alan

2017-12-25

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

PubMed

Genga, Ryan M; Kearns, Nicola A; Maehr, René

2016-05-15

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of glucocorticoid, interleukin-1β, and antioxidants in prenatal stress effects on embryonic microglia.

PubMed

Bittle, Jada; Stevens, Hanna E

2018-02-16

Maternal stress during pregnancy is associated with an increased risk of psychopathology in offspring. Resident immune cells of the brain, microglia, may be mediators of prenatal stress and altered neurodevelopment. Here, we demonstrate that neither the exogenous pro-inflammatory cytokine, interleukin-1β (IL-1β), nor the glucocorticoid hormone, corticosterone, recapitulated the full effects of prenatal stress on the morphology of microglial cells in the cortical plate of embryonic mice; IL-1β effects showed greater similarity to prenatal stress effects on microglia. Unexpectedly, oil vehicle alone, which has antioxidant properties, moderated the effects of prenatal stress on microglia. Microglia changes with prenatal stress were also sensitive to the antioxidant, N-acetylcysteine, suggesting redox dysregulation as a mechanism of prenatal stress.

In vitro cytokines profile and ultrastructural changes of microglia and macrophages following interaction with Leishmania.

PubMed

Ramos, Patricia Karla Santos; Brito, Maysa de Vasconcelos; Silveira, Fernando Tobias; Salgado, Cláudio Guedes; De Souza, Wanderley; Picanço-Diniz, Cristovam Wanderley; Picanço-Diniz, José Antonio Junior

2014-07-01

In the present study, we assessed morphological changes and cytokine production after in vitro interaction with causative agents of American cutaneous leishmaniasis and compared the microglia and macrophage immune responses. Cultures of microglia and macrophages infected with stationary-phase promastigotes of Leishmania (Viannia) shawi, Leishmania (Viannia) braziliensis or Leishmania (Leishmania) amazonensis were evaluated 24, 48 and 72 h after interaction. Macrophages only presented the classical phagocytic process while microglia also displayed large cytoplasmic projections similar to the ruffles described in macropinocytosis. In the macrophage cultures, the percentage of infected cells increased over time, in a fashion that was dependent on the parasite species. In contrast, in microglial cells as the culture time progressed, there was a significant reduction in the percentage of infected cells independent of parasite species. Measurements of cytokines in macrophage cultures 48 h after interactions revealed distinct expression patterns for different parasites, whereas in microglial cultures they were similar for all Leishmania tested species. Taken together, our results suggest that microglia may have a higher phagocytic ability and cytotoxic potential than macrophages for all investigated species. The robust response of microglia against all parasite species may suggest microglia have an important role in the defence against cerebral leishmaniasis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai

Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment. Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with differentmore » specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.« less

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Dystrophic microglia in the aging human brain.

PubMed

Streit, Wolfgang J; Sammons, Nicole W; Kuhns, Amanda J; Sparks, D Larry

2004-01-15

We have studied microglial morphology in the human cerebral cortex of two nondemented subjects using high-resolution LN-3 immunohistochemistry. Several abnormalities in microglial cytoplasmic structure, including deramification, spheroid formation, gnarling, and fragmentation of processes, were identified. These changes were determined to be different from the morphological changes that occur during microglial activation and they were designated collectively as microglial dystrophy. Quantitative evaluation of dystrophic changes in microglia revealed that these were much more prevalent in the older subject (68-year-old) than in the younger one (38-year-old). Thus, we conclude that microglial dystrophy is a sign of microglial cell senescence. We hypothesize that microglial senescence could be important for understanding age-related declines in cognitive function. Copyright 2003 Wiley-Liss, Inc.

The role of microglia and macrophages in glioma maintenance and progression

PubMed Central

Hambardzumyan, Dolores; Gutmann, David H; Kettenmann, Helmut

2016-01-01

There is a growing recognition that gliomas are complex tumors composed of neoplastic and non-neoplastic cells, which each individually contribute to cancer formation, progression and response to treatment. The majority of the non-neoplastic cells are tumor-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, that create a supportive stroma for neoplastic cell expansion and invasion. TAMs are recruited to the glioma environment, have immune functions, and can release a wide array of growth factors and cytokines in response to those factors produced by cancer cells. In this manner, TAMs facilitate tumor proliferation, survival and migration. Through such iterative interactions, a unique tumor ecosystem is established, which offers new opportunities for therapeutic targeting. PMID:26713745

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

PubMed

Sanders, Anne M; Stehle, John R; Blanks, Michael J; Riedlinger, Gregory; Kim-Shapiro, Jung W; Monjazeb, Arta M; Adams, Jonathan M; Willingham, Mark C; Cui, Zheng

2010-03-31

Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

PubMed

Mukhtar, M Shahid; Carvunis, Anne-Ruxandra; Dreze, Matija; Epple, Petra; Steinbrenner, Jens; Moore, Jonathan; Tasan, Murat; Galli, Mary; Hao, Tong; Nishimura, Marc T; Pevzner, Samuel J; Donovan, Susan E; Ghamsari, Lila; Santhanam, Balaji; Romero, Viviana; Poulin, Matthew M; Gebreab, Fana; Gutierrez, Bryan J; Tam, Stanley; Monachello, Dario; Boxem, Mike; Harbort, Christopher J; McDonald, Nathan; Gai, Lantian; Chen, Huaming; He, Yijian; Vandenhaute, Jean; Roth, Frederick P; Hill, David E; Ecker, Joseph R; Vidal, Marc; Beynon, Jim; Braun, Pascal; Dangl, Jeffery L

2011-07-29

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.

Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants.

PubMed

Anderson, Ryan G; Casady, Megan S; Fee, Rachel A; Vaughan, Martha M; Deb, Devdutta; Fedkenheuer, Kevin; Huffaker, Alisa; Schmelz, Eric A; Tyler, Brett M; McDowell, John M

2012-12-01

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR-like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non-pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's host. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Julie Anne Roden, Branids Belt, Jason Barzel Ross, Thomas Tachibana, Joe Vargas, Mary Beth Mudgett

2004-11-23

The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resultedmore » in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.« less

α-Iso-cubebene exerts neuroprotective effects in amyloid beta stimulated microglia activation.

PubMed

Park, Sun Young; Park, Se Jin; Park, Nan Jeong; Joo, Woo Hong; Lee, Sang-Joon; Choi, Young-Whan

2013-10-25

Schisandra chinensis is commonly used for food and as a traditional remedy for the treatment of neuronal disorders. However, it is unclear which component of S. chinensis is responsible for its neuropharmacological effects. To answer this question, we isolated α-iso-cubebene, a dibenzocyclooctadiene lignin, from S. chinensis and determined if it has any anti-neuroinflammatory and neuroprotective properties against amyloid β-induced neuroinflammation in microglia. Microglia that are stimulated by amyloid β increased their production of pro-inflammatory cytokines and chemokines, prostaglandin E2 (PGE2), nitric oxide (NO) and reactive oxygen species (ROS) and the enzymatic activity of matrix metalloproteinase 9 (MMP-9). We found this was all inhibited by α-iso-cubebene. Consistent with these results, α-iso-cubebene inhibited the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2) and MMP-9 in amyloid β-stimulated microglia. Subsequent mechanistic studies revealed that α-iso-cubebene inhibited the phosphorylation and degradation of IκB-α, the phosphorylation and transactivity of NF-κB, and the phosphorylation of MAPK in amyloid β-stimulated microglia. These results suggest that α-iso-cubebene impairs the amyloid β-induced neuroinflammatory response of microglia by inhibiting the NF-κB and MAPK signaling pathways. Importantly, α-iso-cubebene can provide critical neuroprotection for primary cortical neurons against amyloid β-stimulated microglia-mediated neurotoxicity. To the best of our knowledge, this is the first report showing that α-iso-cubebene can provide neuroprotection against, and influence neuroinflammation triggered by, amyloid β activation of microglia. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Hv1 proton channel facilitates production of ROS and pro-inflammatory cytokines in microglia and enhances oligodendrocyte progenitor cells damage from oxygen-glucose deprivation in vitro.

PubMed

Yu, Ying; Yu, Zhiyuan; Xie, Minjie; Wang, Wei; Luo, Xiang

2018-03-25

The contribution of microglial activation to oligodendrocyte precursor cell (OPC) damage in the brain is considered to be a principal pathophysiological feature of periventricular leukomalacia (PVL). Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-dependent reactive oxygen species (ROS) produced in microglia has been shown to be significantly toxic to OPCs. The voltage-gated proton channel Hv1 is selectively expressed in microglia and is essential for NOX-dependent ROS production in the central nervous system. This study aimed to investigate the effects of microglial Hv1 deficiency on the protection of OPCs from oxygen-glucose deprivation (OGD)-induced injury in vitro. In the present study, the levels of OGD-induced ROS and pro-inflammatory cytokine production were dramatically lower in Hv1-deficient microglia (Hv1 -/- ) than in wild-type (WT) microglia. Following OGD, OPCs co-cultured with WT microglia had increased apoptosis and decreased proliferation and maturation, while those co-cultured with Hv1 -/- microglia had attenuated apoptosis and greater proliferation and differentiation. Furthermore, the attenuated damage and enhanced regeneration of OPCs were associated with decreases in extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase phosphorylation. These results indicate that the protective effects of Hv1 deficiency on OPCs are due to the suppression of ROS and pro-inflammatory cytokine production in microglia. We thus suggest that the microglial proton channel Hv1 may be a potential therapeutic target in PVL. Copyright © 2017 Elsevier Inc. All rights reserved.

Prominent microglial activation in cortical white matter is selectively associated with cortical atrophy in primary progressive aphasia.

PubMed

Ohm, D T; Kim, G; Gefen, T; Rademaker, A; Weintraub, S; Bigio, E H; Mesulam, M-M; Rogalski, E; Geula, C

2018-04-21

Primary progressive aphasia (PPA) is a clinical syndrome characterized by selective language impairments associated with focal cortical atrophy favouring the language dominant hemisphere. PPA is associated with Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) and significant accumulation of activated microglia. Activated microglia can initiate an inflammatory cascade that may contribute to neurodegeneration, but their quantitative distribution in cortical white matter and their relationship with cortical atrophy remain unknown. We investigated white matter activated microglia and their association with grey matter atrophy in 10 PPA cases with either AD or FTLD-TDP pathology. Activated microglia were quantified with optical density measures of HLA-DR immunoreactivity in two regions with peak cortical atrophy, and one nonatrophied region within the language dominant hemisphere of each PPA case. Nonatrophied contralateral homologues of the language dominant regions were examined for hemispheric asymmetry. Qualitatively, greater densities of activated microglia were observed in cortical white matter when compared to grey matter. Quantitative analyses revealed significantly greater densities of activated microglia in the white matter of atrophied regions compared to nonatrophied regions in the language dominant hemisphere (P < 0.05). Atrophied regions of the language dominant hemisphere also showed significantly more activated microglia compared to contralateral homologues (P < 0.05). White matter activated microglia accumulate more in atrophied regions in the language dominant hemisphere of PPA. While microglial activation may constitute a response to neurodegenerative processes in white matter, the resultant inflammatory processes may also exacerbate disease progression and contribute to cortical atrophy. © 2018 British Neuropathological Society.

MicroRNA-101a regulates microglial morphology and inflammation.

PubMed

Saika, Reiko; Sakuma, Hiroshi; Noto, Daisuke; Yamaguchi, Shuhei; Yamamura, Takashi; Miyake, Sachiko

2017-05-30

Microglia, as well as other tissue-resident macrophages, arise from yolk sac progenitors. Thus, it is likely that the central nervous system environment is critical for the acquisition of a distinct microglial phenotype. Several microRNAs that are enriched in the brain play crucial roles in brain development and may also play a role in the differentiation of microglia. To track the differentiation of hematopoietic cells into microglia, lineage-negative bone marrow cells were co-cultured with astrocytes in the absence or presence of microRNAs or their inhibitors. Microglia-like cells were identified as small, round cells that were immunopositive for CD11b, Iba1, CX3CR1, and triggering receptor expressed on myeloid cells (TREM)-2. Five microRNAs (miR-101a, miR-139-3p, miR-214 * , miR-218, and miR-1186) were identified as modifiers of the differentiation of bone marrow-derived microglia-like cells. Among them, miR-101a facilitated the differentiation of bone marrow cells into microglia-like cells most potently. Small, round cells expressing CD11b, Iba1, CX3CR1, and TREM-2 were predominant in cells treated by miR-101a. miR-101a was abundantly expressed in non-microglial brain cells. Transfection of miR-101a into microglia significantly increased the production of IL-6 in response to LPS. Finally, miR-101a downregulated the expression of MAPK phosphatase-1. miR-101a, which is enriched in the brain, promotes the differentiation of bone marrow cells into microglia-like cells.

The effector candidate repertoire of the arbuscular mycorrhizal fungus Rhizophagus clarus.

PubMed

Sędzielewska Toro, Kinga; Brachmann, Andreas

2016-02-09

Arbuscular mycorrhizal fungi (AMF) form an ecologically important symbiosis with more than two thirds of studied land plants. Recent studies of plant-pathogen interactions showed that effector proteins play a key role in host colonization by controlling the plant immune system. We hypothesise that also for symbiotic-plant interactions the secreted effectome of the fungus is a major component of communication and the conservation level of effector proteins between AMF species may be indicative whether they play a fundamental role. In this study, we used a bioinformatics pipeline to predict and compare the effector candidate repertoire of the two AMF species, Rhizophagus irregularis and Rhizophagus clarus. Our in silico pipeline revealed a list of 220 R. irregularis candidate effector genes that create a valuable information source to elucidate the mechanism of plant infection and colonization by fungi during AMF symbiotic interaction. While most of the candidate effectors show no homologies to known domains or proteins, the candidates with homologies point to potential roles in signal transduction, cell wall modification or transcription regulation. A remarkable aspect of our work is presence of a large portion of the effector proteins involved in symbiosis, which are not unique to each fungi or plant species, but shared along the Glomeromycota phylum. For 95% of R. irregularis candidates we found homologs in a R. clarus genome draft generated by Illumina high-throughput sequencing. Interestingly, 9% of the predicted effectors are at least as conserved between the two Rhizophagus species as proteins with housekeeping functions (similarity > 90%). Therefore, we state that this group of highly conserved effector proteins between AMF species may play a fundamental role during fungus-plant interaction. We hypothesise that in symbiotic interactions the secreted effectome of the fungus might be an important component of communication. Identification and functional characterization of the primary AMF effectors that regulate symbiotic development will help in understanding the mechanisms of fungus-plant interaction.

A novel phosphodiesterase-5 Inhibitor: Yonkenafil modulates neurogenesis, gliosis to improve cognitive function and ameliorates amyloid burden in an APP/PS1 transgenic mice model.

PubMed

Zhu, Lei; Yang, Jing-yu; Xue, Xue; Dong, Ying-xu; Liu, Yang; Miao, Feng-rong; Wang, Yong-feng; Xue, Hong; Wu, Chun-fu

2015-09-01

In Alzheimer's disease (AD), activated microglia invade and surround β-amyloid plaques, possibly contributing to the aggregation of amyloid β (Aβ), which affect the survival of neurons and lead to memory loss. Phosphodiesterase-5 (PDE-5) inhibitors have recently been shown a potential therapeutic effect on AD. In this study, the effects of yonkenafil (yonk), a novel PDE-5 inhibitor, on cognitive behaviors as well as the pathological features in transgenic AD mice were investigated. Seven-month-old APP/PS1 transgenic mice were treated with yonk (2, 6, or 18 mg/kg, intraperitoneal injection (i.p.)) or sildenafil (sild) (6 mg/kg, i.p.) daily for 3 months and then behavioral tests were performed. The results demonstrated that yonk improved nesting-building ability, ameliorated working memory deficits in the Y-maze tasks, and significantly improved learning and memory function in the Morris water maze (MWM) tasks. In addition, yonk reduced the area of Aβ plaques, and inhibited over-activation of microglia and astrocytes. Furthermore, yonk increased neurogenesis in the dentate granule brain region of APP/PS1 mice, indicated by increased BrdU(+)/NeuN(+) and BrdU(+)/DCX(+) cells compared to vehicle-treated transgenic mice. These results suggest that yonk could rescue cognitive deficits by ameliorated amyloid burden through regulating APP processing, inhibited the over-activation of microglia and astrocytes as well as restored neurogenesis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The upregulation of specific interleukin (IL) receptor antagonists and paradoxical enhancement of neuronal apoptosis due to electrode induced strain and brain micromotion.

PubMed

Karumbaiah, Lohitash; Norman, Sharon E; Rajan, Nithish B; Anand, Sanjay; Saxena, Tarun; Betancur, Martha; Patkar, Radhika; Bellamkonda, Ravi V

2012-09-01

The high mechanical mismatch between stiffness of silicon and metal microelectrodes and soft cortical tissue, induces strain at the neural interface which likely contributes to failure of the neural interface. However, little is known about the molecular outcomes of electrode induced low-magnitude strain (1-5%) on primary astrocytes, microglia and neurons. In this study we simulated brain micromotion at the electrode-brain interface by subjecting astrocytes, microglia and primary cortical neurons to low-magnitude cyclical strain using a biaxial stretch device, and investigated the molecular outcomes of induced strain in vitro. In addition, we explored the functional consequence of astrocytic and microglial strain on neural health, when they are themselves subjected to strain. Quantitative real-time PCR array (qRT-PCR Array) analysis of stretched astrocytes and microglia showed strain specific upregulation of an Interleukin receptor antagonist - IL-36Ra (previously IL-1F5), to ≈ 1018 and ≈ 236 fold respectively. Further, IL-36Ra gene expression remained unchanged in astrocytes and microglia treated with bacterial lipopolysaccharide (LPS) indicating that the observed upregulation in stretched astrocytes and microglia is potentially strain specific. Zymogram and western blot analysis revealed that mechanically strained astrocytes and microglia upregulated matrix metalloproteinases (MMPs) 2 and 9, and other markers of reactive gliosis such as glial fibrillary acidic protein (GFAP) and neurocan when compared to controls. Primary cortical neurons when stretched with and without IL-36Ra, showed a ≈ 400 fold downregulation of tumor necrosis factor receptor superfamily, member 11b (TNFRSF11b). Significant upregulation of members of the caspase cysteine proteinase family and other pro-apoptotic genes was also observed in the presence of IL-36Ra than in the absence of IL-36Ra. Adult rats when implanted with microwire electrodes showed upregulation of IL-36Ra (≈ 20 fold) and IL-1Ra (≈ 1500 fold) 3 days post-implantation (3 DPI), corroborating in vitro results, although these transcripts were drastically down regulated by ≈ 20 fold and ≈ 1488 fold relative to expression levels 3 DPI, at the end of 12 weeks post-implantation (12 WPI). These results demonstrate that IL receptor antagonists may be negatively contributing to neuronal health at acute time-points post-electrode implantation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of CTL Recognized Epitopes on Human Breast Tumors

DTIC Science & Technology

1996-09-01

maturation and effector function of cellular immune cytotoxic effectors such as CTL (11). (c) The epitopes defined on tumor Ag are self-peptides of...have been reported to be expressed in breast and ovarian cancer cells (18), and they apparently function by maintaining the undifferentiated state...Body of the Report The purpose of the present work continues to be the characterization of the functional significance of the CTL epitopes as potential

Peptides released by ameboid microglia regulate astroglial proliferation

PubMed Central

1985-01-01

Peptides that stimulate astroglial proliferation are produced in traumatized adult rat brain by 10 d after injury. These same peptides are released by ameboid microglia activated in vitro. Our findings suggest that astroglial scarring is regulated in part by the release of factors from ameboid microglia near the site of brain injury. PMID:4066764

Rotenone and Paraquat do not Directly Activate Microglia or Induce Inflammatory Cytokine Release

PubMed Central

Klintworth, Heather; Garden, Gwenn; Xia, Zhengui

2009-01-01

Both epidemiological and pathological data suggest an inflammatory response including microglia activation and neuro-inflammation in the Parkinsonian brain. Treatments with lipopolysacchride (LPS), rotenone and paraquat have been used as models for Parkinson’s disease, as they cause dopaminergic neuron degeneration in culture and in animals. Recent studies have suggested that rotenone and paraquat induce neuro-inflammation, however, it is not known if they can directly activate microglia. Here, we use primary cultured microglia to address this question. Microglia activation was analyzed by morphological changes and release of nitric oxide and inflammatory cytokines. Treatment with LPS was used as a positive control. While LPS induced morphological changes characteristic of microglial activation and release of nitric oxide and inflammatory cytokines, rotenone and paraquat did not. Our results suggest that paraquat and rotenone do not act directly on microglia and that neuro-inflammation and microglial activation in animals treated with these agents is likely non-cell autonomous, and may occur as a result of dopaminergic neuron damage or factors released by neurons and other cells. PMID:19559752

A Starring Role for Microglia in Brain Sex Differences

PubMed Central

Lenz, Kathryn M.; McCarthy, Margaret M.

2017-01-01

Microglia, the resident innate immune cells in the brain, have long been understood to be crucial to maintenance in the nervous system, by clearing debris, monitoring for infiltration of infectious agents, and mediating the brain’s inflammatory and repair response to traumatic injury, stroke, or neurodegeneration. A wave of new research has shown that microglia are also active players in many basic processes in the healthy brain, including cell proliferation, synaptic connectivity, and physiology. Microglia, both in their capacity as phagocytic cells and via secretion of many neuroactive molecules, including cytokines and growth factors, play a central role in early brain development, including sexual differentiation of the brain. In this review, we present the vast roles microglia play in normal brain development and how perturbations in the normal neuroimmune environment during development may contribute to the etiology of brain-based disorders. There are notable differences between microglia and neuroimmune signaling in the male and female brain throughout the life span, and these differences may contribute to the vast differences in the incidence of neuropsychiatric and neurological disorders between males and females. PMID:24871624

A starring role for microglia in brain sex differences.

PubMed

Lenz, Kathryn M; McCarthy, Margaret M

2015-06-01

Microglia, the resident innate immune cells in the brain, have long been understood to be crucial to maintenance in the nervous system, by clearing debris, monitoring for infiltration of infectious agents, and mediating the brain's inflammatory and repair response to traumatic injury, stroke, or neurodegeneration. A wave of new research has shown that microglia are also active players in many basic processes in the healthy brain, including cell proliferation, synaptic connectivity, and physiology. Microglia, both in their capacity as phagocytic cells and via secretion of many neuroactive molecules, including cytokines and growth factors, play a central role in early brain development, including sexual differentiation of the brain. In this review, we present the vast roles microglia play in normal brain development and how perturbations in the normal neuroimmune environment during development may contribute to the etiology of brain-based disorders. There are notable differences between microglia and neuroimmune signaling in the male and female brain throughout the life span, and these differences may contribute to the vast differences in the incidence of neuropsychiatric and neurological disorders between males and females. © The Author(s) 2014.

The Genome Biology of Effector Gene Evolution in Filamentous Plant Pathogens.

PubMed

Sánchez-Vallet, Andrea; Fouché, Simone; Fudal, Isabelle; Hartmann, Fanny E; Soyer, Jessica L; Tellier, Aurélien; Croll, Daniel

2018-05-16

Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease

PubMed Central

Manocha, Gunjan D.; Floden, Angela M.; Rausch, Keiko; Kulas, Joshua A.; McGregor, Brett A.; Rojanathammanee, Lalida; Puig, Kelley R.; Puig, Kendra L.; Karki, Sanjib; Nichols, Michael R.; Darland, Diane C.; Porter, James E.

2016-01-01

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP−/− mice compared with wild-type mice. The mAPP−/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains. PMID:27511018

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

PubMed

Gupta, Bhawna; Iancu, Emanuela M; Gannon, Philippe O; Wieckowski, Sébastien; Baitsch, Lukas; Speiser, Daniel E; Rufer, Nathalie

2012-07-01

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Neurogenesis-Promoting Natural Product α-Asarone Modulates Morphological Dynamics of Activated Microglia

PubMed Central

Cai, Qing; Li, Yuanyuan; Mao, Jianxin; Pei, Gang

2016-01-01

α-Asarone is an active constituent of Acori Tatarinowii, one of the widely used traditional Chinese Medicine to treat cognitive defect, and recently is shown to promote neurogenesis. Here, we demonstrated that low level (3 μM) of α-asarone attenuated LPS-induced BV2 cell bipolar elongated morphological change, with no significant effect on the LPS-induced pro-inflammatory cytokine expressions. In addition, time-lapse analysis also revealed that α-asarone modulated LPS-induced BV2 morphological dynamics. Consistently a significant reduction in the LPS-induced Monocyte Chemoattractant Protein (MCP-1) mRNA and protein levels was also detected along with the morphological change. Mechanistic study showed that the attenuation effect to the LPS-resulted morphological modulation was also detected in the presence of MCP-1 antibodies or a CCR2 antagonist. This result has also been confirmed in primary cultured microglia. The in vivo investigation provided further evidence that α-asarone reduced the proportion of activated microglia, and reduced microglial tip number and maintained the velocity. Our study thus reveals α-asarone effectively modulates microglial morphological dynamics, and implies this effect of α-asarone may functionally relate to its influence on neurogenesis. PMID:28018174

G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

PubMed

Bouchard, Caroline; Pagé, Julie; Bédard, Andréanne; Tremblay, Pierrot; Vallières, Luc

2007-06-01

G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

CCL11 enhances excitotoxic neuronal death by producing reactive oxygen species in microglia.

PubMed

Parajuli, Bijay; Horiuchi, Hiroshi; Mizuno, Tetsuya; Takeuchi, Hideyuki; Suzumura, Akio

2015-12-01

The chemokine CCL11 (also known as eotaxin-1) is a potent eosinophil chemoattractant that mediates allergic diseases such as asthma, atopic dermatitis, and inflammatory bowel diseases. Previous studies demonstrated that concentrations of CCL11 are elevated in the sera and cerebrospinal fluids (CSF) of patients with neuroinflammatory disorders, including multiple sclerosis. Moreover, the levels of CCL11 in plasma and CSF increase with age, and CCL11 suppresses adult neurogenesis in the central nervous system (CNS), resulting in memory impairment. However, the precise source and function of CCL11 in the CNS are not fully understood. In this study, we found that activated astrocytes release CCL11, whereas microglia predominantly express the CCL11 receptor. CCL11 significantly promoted the migration of microglia, and induced microglial production of reactive oxygen species by upregulating nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), thereby promoting excitotoxic neuronal death. These effects were reversed by inhibition of NOX1. Our findings suggest that CCL11 released from activated astrocytes triggers oxidative stress via microglial NOX1 activation and potentiates glutamate-mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders. © 2015 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kakita, Hiroki; Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601; Aoyama, Mineyoshi, E-mail: ao.mine@med.nagoya-cu.ac.jp

Influenza-associated encephalopathy (IAE) is a central nervous system complication with a high mortality rate, which is increased significantly by the non-steroidal anti-inflammatory drug diclofenac sodium (DCF). In the present study, we investigated the effects of DCF on brain immune cells (i.e. microglia) stimulated with three proinflammatory cytokines, namely tumor necrosis factor-α, interleukin-1β, and interferon-γ. Similar to previous findings in astrocytes, all three cytokines induced the expression of inducible NO synthase (iNOS), as well as NO production, in microglia. The addition of DCF to the culture system augmented iNOS expression and NO production. Immunocytochemical analysis and the phagocytosis assay revealed thatmore » cytokine treatment induced morphological changes to and phagocytosis by the microglia. The addition of DCF to the culture system enhanced microglial activation, as well as the phagocytic activity of cytokine-stimulated microglia. Inhibitors of nuclear factor (NF)-κB inhibited iNOS gene expression in cytokine-stimulated microglia with or without DCF, suggesting that the NF-κB pathway is one of the main signaling pathways involved. The iNOS inhibitor N{sup G}-monomethyl-L-arginine (L-NMMA) reduced both cytokine-induced phagocytosis and phagocytosis induced by the combination of cytokines plus DCF. Furthermore, the NO donor sodium nitroprusside induced phagocytosis, indicating that NO production is a key regulator of microglial phagocytosis. In conclusion, DCF acts synergistically with proinflammatory cytokines to increase the production of NO in microglia, leading to phagocytic activity of the activated microglia. These findings, together with previous observations regarding astrocytes, may explain the significant increase in mortality of IAE patients treated with DCF. - Highlights: ► Influenza-associated encephalopathy (IAE) is associated with a high mortality rate. ► Hyperimmunization in the brain is believed to be responsible for IAE. ► The use of diclofenac sodium (DCF) increases the mortality of IAE. ► DCF enhances the cytokine-induced phagocytosis of microglia, brain immune cells. ► DCF-enhanced activation of microglia may explain the greater mortality rate of IAE.« less

Increased White Matter Inflammation in Aging- and Alzheimer’s Disease Brain

PubMed Central

Raj, Divya; Yin, Zhuoran; Breur, Marjolein; Doorduin, Janine; Holtman, Inge R.; Olah, Marta; Mantingh-Otter, Ietje J.; Van Dam, Debby; De Deyn, Peter P.; den Dunnen, Wilfred; Eggen, Bart J. L.; Amor, Sandra; Boddeke, Erik

2017-01-01

Chronic neuroinflammation, which is primarily mediated by microglia, plays an essential role in aging and neurodegeneration. It is still unclear whether this microglia-induced neuroinflammation occurs globally or is confined to distinct brain regions. In this study, we investigated microglia activity in various brain regions upon healthy aging and Alzheimer’s disease (AD)-related pathology in both human and mouse samples. In purified microglia isolated from aging mouse brains, we found a profound gene expression pattern related to pro-inflammatory processes, phagocytosis, and lipid homeostasis. Particularly in white matter microglia of 24-month-old mice, abundant expression of phagocytic markers including Mac-2, Axl, CD16/32, Dectin1, CD11c, and CD36 was detected. Interestingly, in white matter of human brain tissue the first signs of inflammatory activity were already detected during middle age. Thus quantification of microglial proteins, such as CD68 (commonly associated with phagocytosis) and HLA-DR (associated with antigen presentation), in postmortem human white matter brain tissue showed an age-dependent increase in immunoreactivity already in middle-aged people (53.2 ± 2.0 years). This early inflammation was also detectable by non-invasive positron emission tomography imaging using [11C]-(R)-PK11195, a ligand that binds to activated microglia. Increased microglia activity was also prominently present in the white matter of human postmortem early-onset AD (EOAD) brain tissue. Interestingly, microglia activity in the white matter of late-onset AD (LOAD) CNS was similar to that of the aged clinically silent AD cases. These data indicate that microglia-induced neuroinflammation is predominant in the white matter of aging mice and humans as well as in EOAD brains. This white matter inflammation may contribute to the progression of neurodegeneration, and have prognostic value for detecting the onset and progression of aging and neurodegeneration. PMID:28713239

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

PubMed

Kale, Shiv D; Gu, Biao; Capelluto, Daniel G S; Dou, Daolong; Feldman, Emily; Rumore, Amanda; Arredondo, Felipe D; Hanlon, Regina; Fudal, Isabelle; Rouxel, Thierry; Lawrence, Christopher B; Shan, Weixing; Tyler, Brett M

2010-07-23

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues. Copyright 2010 Elsevier Inc. All rights reserved.

Critical data-based re-evaluation of minocycline as a putative specific microglia inhibitor.

PubMed

Möller, Thomas; Bard, Frédérique; Bhattacharya, Anindya; Biber, Knut; Campbell, Brian; Dale, Elena; Eder, Claudia; Gan, Li; Garden, Gwenn A; Hughes, Zoë A; Pearse, Damien D; Staal, Roland G W; Sayed, Faten A; Wes, Paul D; Boddeke, Hendrikus W G M

2016-10-01

Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of microglia. It is, however, unclear how this "inhibition" is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788-1794. © 2016 Wiley Periodicals, Inc.

Upregulated TLR3 Promotes Neuropathic Pain by Regulating Autophagy in Rat With L5 Spinal Nerve Ligation Model.

PubMed

Chen, Weijia; Lu, Zhijun

2017-02-01

Microglia, rapidly activated following peripheral nerve injury (PNI), accumulate within the spinal cord and adopt inflammation that contributes to development and maintenance of neuropathic pain. Microglia express functional Toll-like receptors (TLRs), which play pivotal roles in regulating inflammatory processes. However, little is known about the role of TLR3 in regulating neuropathic pain after PNI. Here TLR3 expression and autophagy activation was assayed in dorsal root ganglions and in microglia following PNI by using realtime PCR, western blot and immunohistochemistry. The role of TLR3/autophagy signaling in regulating tactile allodynia was evaluated by assaying paw mechanical withdrawal threshold and cold allodynia after intrathecal administration of Poly (I:C) and 3-methyladenine (3-MA). We found that L5 spinal nerve ligation (SNL) induces the expression of TLR3 in dorsal root ganglions and in primary rat microglia at the mRNA and protein level. Meanwhile, L5 SNL results in an increased activation of autophagy, which contributes to microglial activation and subsequent inflammatory response. Intrathecal administration of Poly (I:C), a TLR3 agonist, significantly increases the activation of microglial autophagy, whereas TLR3 knockdown markedly inhibits L5 SNL-induced microglial autophagy. Poly (I:C) treatment promotes the expression of proinflammatory mediators, whereas 3-MA (a specific inhibitor of autophagy) suppresses Poly (I:C)-induced secretion of proinflammatory cytokines. Autophagy inhibition further inhibits TLR3-mediated mechanical and cold hypersensitivity following SNL. These results suggest that inhibition of TLR3/autophagy signaling contributes to alleviate neurophathic pain triggered by SNL.

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

PubMed Central

Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

2015-01-01

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

PubMed Central

Lippé, Roger; Miaczynska, Marta; Rybin, Vladimir; Runge, Anja; Zerial, Marino

2001-01-01

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases. PMID:11452015

Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

PubMed Central

Boesch, Austin W.; Osei-Owusu, Nana Yaw; Crowley, Andrew R.; Chu, Thach H.; Chan, Ying N.; Weiner, Joshua A.; Bharadwaj, Pranay; Hards, Rufus; Adamo, Mark E.; Gerber, Scott A.; Cocklin, Sarah L.; Schmitz, Joern E.; Miles, Adam R.; Eckman, Joshua W.; Belli, Aaron J.; Reimann, Keith A.; Ackerman, Margaret E.

2016-01-01

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies. PMID:28018355

Hippocampal activation of microglia may underlie the shared neurobiology of comorbid posttraumatic stress disorder and chronic pain.

PubMed

Sun, Rao; Zhang, Zuoxia; Lei, Yishan; Liu, Yue; Lu, Cui'e; Rong, Hui; Sun, Yu'e; Zhang, Wei; Ma, Zhengliang; Gu, Xiaoping

2016-01-01

The high comorbidity rates of posttraumatic stress disorder and chronic pain have been widely reported, but the underlying mechanisms remain unclear. Emerging evidence suggested that an excess of inflammatory immune activities in the hippocampus involved in the progression of both posttraumatic stress disorder and chronic pain. Considering that microglia are substrates underlying the initiation and propagation of the neuroimmune response, we hypothesized that stress-induced activation of hippocampal microglia may contribute to the pathogenesis of posttraumatic stress disorder-pain comorbidity. We showed that rats exposed to single prolonged stress, an established posttraumatic stress disorder model, exhibited persistent mechanical allodynia and anxiety-like behavior, which were accompanied by increased activation of microglia and secretion of pro-inflammatory cytokines in the hippocampus. Correlation analyses showed that hippocampal activation of microglia was significantly correlated with mechanical allodynia and anxiety-like behavior. Our data also showed that both intraperitoneal and intra-hippocampal injection of minocycline suppressed single prolonged stress-induced microglia activation and inflammatory cytokines accumulation in the hippocampus, and attenuated both single prolonged stress-induced mechanical allodynia and anxiety-like behavior. Taken together, the present study suggests that stress-induced microglia activation in the hippocampus may serve as a critical mechanistic link in the comorbid relationship between posttraumatic stress disorder and chronic pain. The novel concept introduces the possibility of cotreating chronic pain and posttraumatic stress disorder. © The Author(s) 2016.

Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase.

PubMed

Block, M L; Wu, X; Pei, Z; Li, G; Wang, T; Qin, L; Wilson, B; Yang, J; Hong, J S; Veronesi, B

2004-10-01

The contributing role of environmental factors to the development of Parkinson's disease has become increasingly evident. We report that mesencephalic neuron-glia cultures treated with diesel exhaust particles (DEP; 0.22 microM) (5-50 microg/ml) resulted in a dose-dependent decrease in dopaminergic (DA) neurons, as determined by DA-uptake assay and tyrosine-hydroxylase immunocytochemistry (ICC). The selective toxicity of DEP for DA neurons was demonstrated by the lack of DEP effect on both GABA uptake and Neu-N immunoreactive cell number. The critical role of microglia was demonstrated by the failure of neuron-enriched cultures to exhibit DEP-induced DA neurotoxicity, where DEP-induced DA neuron death was reinstated with the addition of microglia to neuron-enriched cultures. OX-42 ICC staining of DEP treated neuron-glia cultures revealed changes in microglia morphology indicative of activation. Intracellular reactive oxygen species and superoxide were produced from enriched-microglia cultures in response to DEP. Neuron-glia cultures from NADPH oxidase deficient (PHOX-/-) mice were insensitive to DEP neurotoxicity when compared with control mice (PHOX+/+). Cytochalasin D inhibited DEP-induced superoxide production in enriched-microglia cultures, implying that DEP must be phagocytized by microglia to produce superoxide. Together, these in vitro data indicate that DEP selectively damages DA neurons through the phagocytic activation of microglial NADPH oxidase and consequent oxidative insult.

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

PubMed Central

Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

Advanced Aerodynamic Design of Passive Porosity Control Effectors

NASA Technical Reports Server (NTRS)

Hunter, Craig A.; Viken, Sally A.; Wood, Richard M.; Bauer, Steven X. S.

2001-01-01

This paper describes aerodynamic design work aimed at developing a passive porosity control effector system for a generic tailless fighter aircraft. As part of this work, a computational design tool was developed and used to layout passive porosity effector systems for longitudinal and lateral-directional control at a low-speed, high angle of attack condition. Aerodynamic analysis was conducted using the NASA Langley computational fluid dynamics code USM3D, in conjunction with a newly formulated surface boundary condition for passive porosity. Results indicate that passive porosity effectors can provide maneuver control increments that equal and exceed those of conventional aerodynamic effectors for low-speed, high-alpha flight, with control levels that are a linear function of porous area. This work demonstrates the tremendous potential of passive porosity to yield simple control effector systems that have no external moving parts and will preserve an aircraft's fixed outer mold line.

Immunosenescence of microglia and macrophages: impact on the ageing central nervous system.

PubMed

Rawji, Khalil S; Mishra, Manoj K; Michaels, Nathan J; Rivest, Serge; Stys, Peter K; Yong, V Wee

2016-03-01

Ageing of the central nervous system results in a loss of both grey and white matter, leading to cognitive decline. Additional injury to both the grey and white matter is documented in many neurological disorders with ageing, including Alzheimer's disease, traumatic brain and spinal cord injury, stroke, and multiple sclerosis. Accompanying neuronal and glial damage is an inflammatory response consisting of activated macrophages and microglia, innate immune cells demonstrated to be both beneficial and detrimental in neurological repair. This article will propose the following: (i) infiltrating macrophages age differently from central nervous system-intrinsic microglia; (ii) several mechanisms underlie the differential ageing process of these two distinct cell types; and (iii) therapeutic strategies that selectively target these diverse mechanisms may rejuvenate macrophages and microglia for repair in the ageing central nervous system. Most responses of macrophages are diminished with senescence, but activated microglia increase their expression of pro-inflammatory cytokines while diminishing chemotactic and phagocytic activities. The senescence of macrophages and microglia has a negative impact on several neurological diseases, and the mechanisms underlying their age-dependent phenotypic changes vary from extrinsic microenvironmental changes to intrinsic changes in genomic integrity. We discuss the negative effects of age on neurological diseases, examine the response of senescent macrophages and microglia in these conditions, and propose a theoretical framework of therapeutic strategies that target the different mechanisms contributing to the ageing phenotype in these two distinct cell types. Rejuvenation of ageing macrophage/microglia may preserve neurological integrity and promote regeneration in the ageing central nervous system. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

PubMed

Qin, Sisi; Yang, Canhong; Huang, Weihua; Du, Shuhua; Mai, Hantao; Xiao, Jijie; Lü, Tianming

2018-01-31

Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1β, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Injury-stimulated Sonic hedgehog expression in microglia contributes to neuroinflammatory response in the MPTP model of Parkinson's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeong Hwi; Department of Biochemistry & Molecular Biology, School of Medicine Kyung Hee University, Seoul 130-701; Chung, Young Cheul

Parkinson's disease (PD) is a progressive neurodegenerative disorder in which dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) region are selectively destroyed. Sonic hedgehog (Shh) has been well known to play a key role in a variety of processes such as embryogenesis, cell proliferation and protection, and tissue repair during inflammation. However, the evidences for the innate role of Shh in adult brain injury are presently lacking and studies have been needed to unveil the importance of Shh in the process of neurodegeneration. Here, we investigated the role of Shh in the pathologic progress of Parkinson's disease inmore » MPTP-induced animal model system. Interestingly, we observed that Shh expression was gradually increased in MPTP affected SNpc region. Activated microglia exclusively expressed SHH in vivo and we could recapitulate Shh induction in activated cultured primary microglia cells. Using the SHH responsive Cre-loxP binary genetic reporter transgenic mouse system, we also found that most of the cell types except for oligodendrocyte in the SNpc region reacted to the SHH by MPTP injection. Taken together, activated microglia induced Shh expression and most neural cells except oligodendrocyte responded to microglia-derived SHH in MPTP-treated SN. These results suggest that SHH in activated microglia by MPTP-injection might be involved in the innate processes of recovery from neurotoxin induced injury in the PD animal model system. - Highlights: • Sonic hedgehog (Shh) was induced by MPTP neurotoxin at the Substantia Nigra (SN) in vivo. • Activated microglia are major cell type for SHH expression in vivo and in vitro. • Different types of cells in the brain, except oligodendrocyte, respond to microglia-derived SHH in SN region.« less

DHA inhibited AGEs-induced retinal microglia activation via suppression of the PPARγ/NFκB pathway and reduction of signal transducers in the AGEs/RAGE axis recruitment into lipid rafts.

PubMed

Wang, Li; Chen, Ka; Liu, Kai; Zhou, Yong; Zhang, Ting; Wang, Bin; Mi, Mantian

2015-04-01

Recent studies revealed that dietary intake of docosahexaenoic acid (DHA) prevented diabetic retinopathy (DR), but the underlying mechanism was not fully understood. Retinal microglia are a specialized population of macrophages in retina. Considerable evidence has shown that microglia activation may trigger neuronal death and vascular dysfunction in DR. The aim of this study was to investigate the effects of DHA on advanced glycation end products (AGEs)-induced microglia activation using an in vitro microglia culture system, and concurrently to explore the mediating mechanisms. DHA inhibited AGEs-induced microglia activation and tumor necrosis factor α (TNFα) secretion. These effects of DHA were directly linked with suppression of nuclear factor-kappa B (NFκB) activity, as evident by the reduction of p-IκBα expression, p-NFκB p65 nucleus translocation, NFκB DNA binding activity, and the regulation of gene transcription (TNFα, IL-1β, ICAM-1, and RAGE mRNA). Furthermore, DHA significantly increased phosphorylation of peroxisome proliferator-activated receptor-gamma (PPARγ), and combined with PPARγ stealth RNAi oligonucleotide, we confirmed that DHA inhibition of AGEs-induced microglia activation was partially through the PPARγ/NFκB pathway. Moreover, although AGEs incubation dramatically elevated expression of the cell surface receptor for AGEs (RAGE), DHA significantly inhibited RAGE and Src recruitment into lipid rafts. The AGEs-RAGE axis downstream signal transducers increased mitogen-activated protein kinase (p38 and JNK) phosphorylation. Taken together, DHA might inhibit AGEs-induced microglia activation via suppression of the PPARγ/NFκB pathway, and reduction of RAGE and AGEs/RAGE transducer recruitment into lipid rafts. These results provide a novel potential mechanism for the anti-inflammatory effects of DHA in DR prevention.

Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway

PubMed Central

Rolón-Reyes, Kimberleve; Kucheryavykh, Yuriy V.; Cubano, Luis A.; Inyushin, Mikhail; Skatchkov, Serguei N.; Eaton, Misty J.; Harrison, Jeffrey K.; Kucheryavykh, Lilia Y.

2015-01-01

Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells. PMID:26098895

Automated Morphological Analysis of Microglia After Stroke.

PubMed

Heindl, Steffanie; Gesierich, Benno; Benakis, Corinne; Llovera, Gemma; Duering, Marco; Liesz, Arthur

2018-01-01

Microglia are the resident immune cells of the brain and react quickly to changes in their environment with transcriptional regulation and morphological changes. Brain tissue injury such as ischemic stroke induces a local inflammatory response encompassing microglial activation. The change in activation status of a microglia is reflected in its gradual morphological transformation from a highly ramified into a less ramified or amoeboid cell shape. For this reason, the morphological changes of microglia are widely utilized to quantify microglial activation and studying their involvement in virtually all brain diseases. However, the currently available methods, which are mainly based on manual rating of immunofluorescent microscopic images, are often inaccurate, rater biased, and highly time consuming. To address these issues, we created a fully automated image analysis tool, which enables the analysis of microglia morphology from a confocal Z-stack and providing up to 59 morphological features. We developed the algorithm on an exploratory dataset of microglial cells from a stroke mouse model and validated the findings on an independent data set. In both datasets, we could demonstrate the ability of the algorithm to sensitively discriminate between the microglia morphology in the peri-infarct and the contralateral, unaffected cortex. Dimensionality reduction by principal component analysis allowed to generate a highly sensitive compound score for microglial shape analysis. Finally, we tested for concordance of results between the novel automated analysis tool and the conventional manual analysis and found a high degree of correlation. In conclusion, our novel method for the fully automatized analysis of microglia morphology shows excellent accuracy and time efficacy compared to traditional analysis methods. This tool, which we make openly available, could find application to study microglia morphology using fluorescence imaging in a wide range of brain disease models.

Depletion of microglia and inhibition of exosome synthesis halt tau propagation

PubMed Central

Asai, Hirohide; Ikezu, Seiko; Tsunoda, Satoshi; Medalla, Maria; Luebke, Jennifer; Haydar, Tarik; Wolozin, Benjamin; Butovsky, Oleg; Kügler, Sebastian; Ikezu, Tsuneya

2015-01-01

Accumulation of pathological tau protein is a major hallmark of Alzheimer’s disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. PMID:26436904

Estrogen anti-inflammatory activity in brain: a therapeutic opportunity for menopause and neurodegenerative diseases

PubMed Central

Vegeto, Elisabetta; Benedusi, Valeria; Maggi, Adriana

2008-01-01

Recent studies highlight the prominent role played by estrogens in protecting the central nervous system (CNS) against the noxious consequences of a chronic inflammatory reaction. The neurodegenerative process of several CNS diseases, including Multiple Sclerosis, Alzheimer’s and Parkinson’s Diseases, is associated with the activation of microglia cells, which drive the resident inflammatory response. Chronically stimulated during neurodegeneration, microglia cells are thought to provide detrimental effects on surrounding neurons. The inhibitory activity of estrogens on neuroinflammation and specifically on microglia might thus be considered as a beneficial therapeutic opportunity for delaying the onset or progression of neurodegenerative diseases; in addition, understanding the peculiar activity of this female hormone on inflammatory signalling pathways will possibly lead to the development of selected anti-inflammatory molecules. This review summarises the evidence for the involvement of microglia in neuroinflammation and the anti-inflammatory activity played by estrogens specifically in microglia. PMID:18522863

Protective effects of nicergoline against neuronal cell death induced by activated microglia and astrocytes.

PubMed

Mizuno, Tetsuya; Kuno, Reiko; Nitta, Atsumi; Nabeshima, Toshitaka; Zhang, Guiqin; Kawanokuchi, Jun; Wang, Jinyan; Jin, Shijie; Takeuchi, Hideyuki; Suzumura, Akio

2005-12-20

We examined the neuroprotective role of nicergoline in neuron-microglia or neuron-astrocytes co-cultures. Nicergoline, an ergoline derivative, significantly suppressed the neuronal cell death induced by co-culture with activated microglia or astrocytes stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma. To elucidate the mechanism by which nicergoline exerts a neuroprotective effect, we examined the production of inflammatory mediators and neurotrophic factors in activated microglia and astrocytes following nicergoline treatment. In microglia stimulated with LPS and IFN-gamma, nicergoline suppressed the production of superoxide anions, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha in a dose-dependent manner. In astrocytes, nicergoline also suppressed the production of proinflammatory cytokines and enhanced brain-derived neurotrophic factor (BDNF). Thus, nicergoline-mediated neuroprotection resulted primarily from the inhibition of inflammatory mediators and the upregulation of neurotrophic factors by glial cells.

Microglial migration and interactions with dendrimer nanoparticles are altered in the presence of neuroinflammation.

PubMed

Zhang, Fan; Nance, Elizabeth; Alnasser, Yossef; Kannan, Rangaramanujam; Kannan, Sujatha

2016-03-22

Microglial cells have been implicated in neuroinflammation-mediated injury in the brain, including neurodevelopmental disorders such as cerebral palsy (CP) and autism. Pro-inflammatory activation of microglial cells results in the impairment of their neuroprotective functions, leading to an exaggerated, ongoing immune dysregulation that can persist long after the initial insult. We have previously shown that dendrimer-mediated delivery of an anti-inflammatory agent can attenuate inflammation in a rabbit model of maternal inflammation-induced CP and significantly improve the motor phenotype, due to the ability of the dendrimer to selectively localize in activated microglia. To elucidate the interactions between dendrimers and microglia, we created an organotypic whole-hemisphere brain slice culture model from newborn rabbits with and without exposure to inflammation in utero. We then used this model to analyze the dynamics of microglial migration and their interactions with dendrimers in the presence of neuroinflammation. Microglial cells in animals with CP had an amoeboid morphology and impaired cell migration, demonstrated by decreased migration distance and velocity when compared to cells in healthy, age-matched controls. However, this decreased migration was associated with a greater, more rapid dendrimer uptake compared to microglial cells from healthy controls. This study demonstrates that maternal intrauterine inflammation is associated with impaired microglial function and movement in the newborn brain. This microglial impairment may play a role in the development of ongoing brain injury and CP in the offspring. Increased uptake of dendrimers by the "impaired" microglia can be exploited to deliver drugs specifically to these cells and modulate their functions. Host tissue and target cell characteristics are important aspects to be considered in the design and evaluation of targeted dendrimer-based nanotherapeutics for improved and sustained efficacy. This ex vivo model also provides a rapid screening tool for evaluation of the effects of various therapies on microglial function.

Inflammation is detrimental for neurogenesis in adult brain

NASA Astrophysics Data System (ADS)

Ekdahl, Christine T.; Claasen, Jan-Hendrik; Bonde, Sara; Kokaia, Zaal; Lindvall, Olle

2003-11-01

New hippocampal neurons are continuously generated in the adult brain. Here, we demonstrate that lipopolysaccharide-induced inflammation, which gives rise to microglia activation in the area where the new neurons are born, strongly impairs basal hippocampal neurogenesis in rats. The increased neurogenesis triggered by a brain insult is also attenuated if it is associated with microglia activation caused by tissue damage or lipopolysaccharide infusion. The impaired neurogenesis in inflammation is restored by systemic administration of minocycline, which inhibits microglia activation. Our data raise the possibility that suppression of hippocampal neurogenesis by activated microglia contributes to cognitive dysfunction in aging, dementia, epilepsy, and other conditions leading to brain inflammation.

Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β.

PubMed

Jana, Malabendu; Pahan, Kalipada

2012-08-01

Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug, in primary human microglia. Bacterial lipopolysachharides (LPS) induced the expression of various proinflammatory molecules and upregulated the expression of microglial surface marker CD11b in human microglia. However, gemfibrozil markedly suppressed proinflammatory molecules and CD11b in LPS-stimulated microglia. Human microglia expressed PPAR-β and -γ, but not PPAR-α. Interestingly, either antisense knockdown of PPAR-β or antagonism of PPAR-β by a specific chemical antagonist abrogated gemfibrozil-mediated inhibition of microglial activation. On the other hand, blocking of PPAR-α and -γ had no effect on gemfibrozil-mediated anti-inflammatory effect in microglia. These results highlight the fact that gemfibrozil regulates microglial activation by inhibiting inflammatory gene expression in a PPAR-β dependent pathway and further reinforce its therapeutic application in several neuroinflammatory and neurodegenerative diseases.

Microglia P2Y₆ receptors mediate nitric oxide release and astrocyte apoptosis.

PubMed

Quintas, Clara; Pinho, Diana; Pereira, Clara; Saraiva, Lucília; Gonçalves, Jorge; Queiroz, Glória

2014-09-03

During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis. Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining. UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia. In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

Salt-Inducible Kinase 1 (SIK1) is Induced by Alcohol and Suppresses Microglia Inflammation via NF-κB Signaling.

PubMed

Zhang, Yu; Gao, Weida; Yang, Kongbin; Tao, Haiquan; Yang, Haicheng

2018-06-19

Alcohol consumption has been shown to cause neuroinflammation and increase a variety of immune-related signaling processes. Microglia are a crucial part of alcohol-induced neuroinflammation and undergo apoptosis. Even though the importance of these inflammatory processes in the effects of alcohol-related neurodegeneration have been established, the mechanism of alcohol-induced microglia apoptosis is unknown. In prior research, we discovered that alcohol increases expression of salt-inducible kinase 1 (SIK1) in rodent brain tissue. In this study, we sought to determine what role SIK1 expression plays in alcohol-induced neuroinflammation as well as whether and by what mechanism it regulates microglia apoptosis. Adult C57BL/6 mice were divided into four groups and for 3 weeks treated with either 0%, 5%, 10%, or 15% alcohol during 3 hour periods. The mice were sacrificed and their brains excised for analysis. Additionally, primary microglia were isolated from neonatal mice. SIK1 expression in alcohol-treated brain tissue and microglia was analyzed via RT-PCR and western blotting. TUNEL staining, caspase-3, and caspase-9 activity assays were performed to evaluate microglial apoptosis. Cell fluorescence staining and NF-κB luciferase activity assays were used to evaluate the effects of SIK1 expression on the NF-κB signaling pathway. SIK1 expression was increased in the brains of mice that consumed alcohol, and this effect was seen in mouse primary microglia. SIK1 knockdown in microglia increased alcohol-induced apoptosis in these cells. Furthermore, SIK1 reduced NF-κB signaling pathway factors, and SIK1 knockdown in microglia promoted alcohol-induced NF-κB activity. TUNEL staining, caspase-3, and caspase-9 activity assays consistently revealed that alcohol-induced microglial apoptosis was inhibited by depletion of p65. Finally, we determined that NF-κB signaling is required for alcohol-induced, SIK1-mediated apoptosis in microglia. This study establishes for the first time not only that SIK1 is crucial to regulating alcohol-induced microglial apoptosis, but also that the NF-κB signaling pathway is required for its activity. Overall, our results help elucidate mechanisms of alcohol-induced neuroinflammation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

PubMed

Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae.

PubMed

Verdier, Valérie; Triplett, Lindsay R; Hummel, Aaron W; Corral, Rene; Cernadas, R Andres; Schmidt, Clarice L; Bogdanove, Adam J; Leach, Jan E

2012-12-01

Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood. Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties. Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence. SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

PubMed

Zhang, Lei; Davies, Laura J; Elling, Axel A

2015-01-01

Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Functional classification of memory CD8(+) T cells by CX3CR1 expression.

PubMed

Böttcher, Jan P; Beyer, Marc; Meissner, Felix; Abdullah, Zeinab; Sander, Jil; Höchst, Bastian; Eickhoff, Sarah; Rieckmann, Jan C; Russo, Caroline; Bauer, Tanja; Flecken, Tobias; Giesen, Dominik; Engel, Daniel; Jung, Steffen; Busch, Dirk H; Protzer, Ulrike; Thimme, Robert; Mann, Matthias; Kurts, Christian; Schultze, Joachim L; Kastenmüller, Wolfgang; Knolle, Percy A

2015-09-25

Localization of memory CD8(+) T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX3CR1 distinguishes memory CD8(+) T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX3CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8(+) T cells with effector function. We find CD62L(hi)CX3CR1(+) memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX3CR1(+) memory CD8(+) T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3CR1-based functional classification will help to resolve the principles of protective CD8(+) T-cell memory.

Transcription Factor Foxo1 Is a Negative Regulator of NK Cell Maturation and Function

PubMed Central

Deng, Youcai; Kerdiles, Yann; Chu, Jianhong; Yuan, Shunzong; Wang, Youwei; Chen, Xilin; Mao, Hsiaoyin; Zhang, Lingling; Zhang, Jianying; Hughes, Tiffany; Deng, Yafei; Zhang, Qi; Wang, Fangjie; Zou, Xianghong; Liu, Chang-Gong; Freud, Aharon G.; Li, Xiaohui; Caligiuri, Michael A; Vivier, Eric; Yu, Jianhua

2015-01-01

SUMMARY Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes through upregulating CD62L expression, and impaired late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21+/− mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions. PMID:25769609

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P.

PubMed

Zhang, Xiaoxiao; Farah, Nadya; Rolston, Laura; Ericsson, Daniel J; Catanzariti, Ann-Maree; Bernoux, Maud; Ve, Thomas; Bendak, Katerina; Chen, Chunhong; Mackay, Joel P; Lawrence, Gregory J; Hardham, Adrienne; Ellis, Jeffrey G; Williams, Simon J; Dodds, Peter N; Jones, David A; Kobe, Bostjan

2018-05-01

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

PubMed

Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

2014-01-01

Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Celecoxib Inhibits Prion Protein 90-231-Mediated Pro-inflammatory Responses in Microglial Cells.

PubMed

Villa, Valentina; Thellung, Stefano; Corsaro, Alessandro; Novelli, Federica; Tasso, Bruno; Colucci-D'Amato, Luca; Gatta, Elena; Tonelli, Michele; Florio, Tullio

2016-01-01

Activation of microglia is a central event in the atypical inflammatory response occurring during prion encephalopathies. We report that the prion protein fragment encompassing amino acids 90-231 (PrP90-231), a model of the neurotoxic activity of the pathogenic prion protein (PrP(Sc)), causes activation of both primary microglia cultures and N9 microglial cells in vitro. This effect was characterized by cell proliferation arrest and induction of a secretory phenotype, releasing prostaglandin E2 (PGE2) and nitric oxide (NO). Conditioned medium from PrP90-231-treated microglia induced in vitro cytotoxicity of A1 mesencephalic neurons, supporting the notion that soluble mediators released by activated microglia contributes to the neurodegeneration during prion diseases. The neuroinflammatory role of COX activity, and its potential targeting for anti-prion therapies, was tested measuring the effects of ketoprofen and celecoxib (preferential inhibitors of COX1 and COX2, respectively) on PrP90-231-induced microglial activation. Celecoxib, but not ketoprofen significantly reverted the growth arrest as well as NO and PGE2 secretion induced by PrP90-231, indicating that PrP90-231 pro-inflammatory response in microglia is mainly dependent on COX2 activation. Taken together, these data outline the importance of microglia in the neurotoxicity occurring during prion diseases and highlight the potentiality of COX2-selective inhibitors to revert microglia as adjunctive pharmacological approach to contrast the neuroinflammation-dependent neurotoxicity.

Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3β/PTEN axis.

PubMed

Zhou, Kai; Zhong, Qi; Wang, Yan-Chun; Xiong, Xiao-Yi; Meng, Zhao-You; Zhao, Ting; Zhu, Wen-Yao; Liao, Mao-Fan; Wu, Li-Rong; Yang, Yuan-Rui; Liu, Juan; Duan, Chun-Mei; Li, Jie; Gong, Qiu-Wen; Liu, Liang; Yang, Mei-Hua; Xiong, Ao; Wang, Jian; Yang, Qing-Wu

2017-03-01

Inflammation mediated by the peripheral infiltration of inflammatory cells plays an important role in intracerebral hemorrhage (ICH) induced secondary injury. Previous studies have indicated that regulatory T lymphocytes (Tregs) might reduce ICH-induced inflammation, but the precise mechanisms that contribute to ICH-induced inflammatory injury remain unclear. Our results show that the number of Tregs in the brain increases after ICH. Inducing Tregs deletion using a CD25 antibody or Foxp3 DTR -mice increased neurological deficient scores (NDS), the level of inflammatory factors, hematoma volumes, and neuronal degeneration. Meanwhile, boosting Tregs using a CD28 super-agonist antibody reduced the inflammatory injury. Furthermore, Tregs depletion shifted microglia/macrophage polarization toward the M1 phenotype while boosting Tregs shifted this transition toward the M2 phenotype. In vitro, a transwell co-culture model of microglia and Tregs indicated that Tregs changed the polarization of microglia, decreased the expression of MHC-II, IL-6, and TNF-α and increased CD206 expression. IL-10 originating from Tregs mediated the microglia polarization by increasing the expression of Glycogen Synthase Kinase 3 beta (GSK3β), which phosphorylates and inactivates Phosphatase and Tensin homologue (PTEN) in microglia, TGF-β did not participate in this conversion. Thus, Tregs ameliorated ICH-induced inflammatory injury by modulating microglia/macrophage polarization toward the M2 phenotype through the IL-10/GSK3β/PTEN axis.

Molecular Targets for PET Imaging of Activated Microglia: The Current Situation and Future Expectations.

PubMed

Tronel, Claire; Largeau, Bérenger; Santiago Ribeiro, Maria Joao; Guilloteau, Denis; Dupont, Anne-Claire; Arlicot, Nicolas

2017-04-11

Microglia, as cellular mediators of neuroinflammation, are implicated in the pathogenesis of a wide range of neurodegenerative diseases. Positron emission tomography (PET) imaging of microglia has matured over the last 20 years, through the development of radiopharmaceuticals targeting several molecular biomarkers of microglial activation and, among these, mainly the translocator protein-18 kDa (TSPO). Nevertheless, current limitations of TSPO as a PET microglial biomarker exist, such as low brain density, even in a neurodegenerative setting, expression by other cells than the microglia (astrocytes, peripheral macrophages in the case of blood brain barrier breakdown), genetic polymorphism, inducing a variation for most of TSPO PET radiopharmaceuticals' binding affinity, or similar expression in activated microglia regardless of its polarization (pro- or anti-inflammatory state), and these limitations narrow its potential interest. We overview alternative molecular targets, for which dedicated radiopharmaceuticals have been proposed, including receptors (purinergic receptors P2X7, cannabinoid receptors, α7 and α4β2 nicotinic acetylcholine receptors, adenosine 2A receptor, folate receptor β) and enzymes (cyclooxygenase, nitric oxide synthase, matrix metalloproteinase, β-glucuronidase, and enzymes of the kynurenine pathway), with a particular focus on their respective contribution for the understanding of microglial involvement in neurodegenerative diseases. We discuss opportunities for these potential molecular targets for PET imaging regarding their selectivity for microglia expression and polarization, in relation to the mechanisms by which microglia actively participate in both toxic and neuroprotective actions in brain diseases, and then take into account current clinicians' expectations.