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Sample records for murine microglial cells

  1. Activation of murine microglial N9 cells is attenuated through cannabinoid receptor CB2 signaling.

    PubMed

    Ma, Lei; Jia, Ji; Liu, Xiangyu; Bai, Fuhai; Wang, Qiang; Xiong, Lize

    2015-02-27

    Inhibition of microglial activation is effective in treating various neurological disorders. Activation of microglial cannabinoid CB2 receptor induces anti-inflammatory effects, and the mechanism, however, is still elusive. Microglia could be activated into the classic activated state (M1 state) or the alternative activated state (M2 state), the former is cytotoxic, and the latter is neurotrophic. In this study, we used lipopolysaccharide (LPS) plus interferon-γ (IFNγ) to activate N9 microglia and hypothesized the pretreatment with cannabinoid CB2 receptor agonist AM1241 attenuates microglial activation by shifting microglial M1 to M2 state. We found that pretreatment with 5 μM AM1241 at 1 h before microglia were exposed to LPS plus IFNγ decreased the expression of inducible nitric oxide synthase (iNOS) and the release of pro-inflammatory factors, increased the expression of arginase 1 (Arg-1) and the release of anti-inflammatory and neurotrophic factors in microglia. However, these effects induced by AM1241 pretreatment were significantly reversed in the presence of 10 μM cannabinoid CB2 receptor antagonist AM630 or 10 μM protein kinase C (PKC) inhibitor chelerythrine. These findings indicated that AM1241 pretreatment attenuates microglial activation by shifting M1 to M2 activated state via CB2 receptor, and the AM1241-induced anti-inflammatory effects may be mediated by PKC. PMID:25637536

  2. Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells.

    PubMed

    Malon, Jennifer T; Grlickova-Duzevik, Eliza; Vaughn, James; Beaulac, Holly; Vunk, Tyler R; Cao, Ling

    2015-02-15

    C57BL/6 (B6) mice develop peripheral neuropathy post-LP-BM5 infection, a murine model of HIV-1 infection, along with the up-regulation of select spinal cord cytokines. We investigated if calcitonin gene-related peptide (CGRP) contributed to the development of peripheral neuropathy by stimulating glial responses. An increased expression of lumbar spinal cord CGRP was observed in vivo, post-LP-BM5 infection. Consequently, in vitro CGRP co-treatments led to a microglial content-dependent attenuation of viral loads in spinal cord mixed glia infected with selected doses of LP-BM5. This inhibition was neither caused by the loss of glia nor induced via the direct inhibition of LP-BM5 by CGRP. PMID:25670002

  3. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  4. Involvement of microglial CD40 in murine retrovirus-induced peripheral neuropathy.

    PubMed

    Cao, Ling; Butler, M Brady

    2013-08-15

    B6 mice infected with LP-BM5 develop severe immunodeficiency (termed murine acquired immunodeficiency syndrome (MAIDS)) and peripheral neuropathy. To determine whether microglial CD40 is involved in LP-BM5-induced peripheral neuropathy, B6-CD40 knockout (KO) mice and B6-CD40 KO mice adoptively transferred either total leukocytes or B cells were examined for behavioral sensitivity, tissue viral loads, cytokine responses, and the development of MAIDS. All three CD40 KO groups developed MAIDS, the severity of which was correlated with peripheral cytokine responses. CD40 KO mice displayed significantly reduced mechanical hypersensitivity post-infection compared to wild-type mice regardless of cell transfer. These findings support microglial CD40 involvement in LP-BM5-induced peripheral neuropathy. PMID:23726765

  5. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  6. Anti-Neuroinflammatory Effects of the Calcium Channel Blocker Nicardipine on Microglial Cells: Implications for Neuroprotection

    PubMed Central

    Huang, Bor-Ren; Chang, Pei-Chun; Yeh, Wei-Lan; Lee, Chih-Hao; Tsai, Cheng-Fang; Lin, Chingju; Lin, Hsiao-Yun; Liu, Yu-Shu; Wu, Caren Yu-Ju; Ko, Pei-Ying; Huang, Shiang-Suo; Hsu, Horng-Chaung; Lu, Dah-Yuu

    2014-01-01

    Background/Objective Nicardipine is a calcium channel blocker that has been widely used to control blood pressure in severe hypertension following events such as ischemic stroke, traumatic brain injury, and intracerebral hemorrhage. However, accumulating evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play important roles in neurodegeneration, and the effect of nicardipine on microglial activation remains unresolved. Methodology/Principal Findings In the present study, using murine BV-2 microglia, we demonstrated that nicardipine significantly inhibits microglia-related neuroinflammatory responses. Treatment with nicardipine inhibited microglial cell migration. Nicardipine also significantly inhibited LPS plus IFN-γ-induced release of nitric oxide (NO), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, nicardipine also inhibited microglial activation by peptidoglycan, the major component of the Gram-positive bacterium cell wall. Notably, nicardipine also showed significant anti-neuroinflammatory effects on microglial activation in mice in vivo. Conclusion/Significance The present study is the first to report a novel inhibitory role of nicardipine on neuroinflammation and provides a new candidate agent for the development of therapies for inflammation-related neurodegenerative diseases. PMID:24621589

  7. Effects of Elderberry Juice from Different Genotypes on Oxidative and Inflammatory Responses in Microglial Cells

    PubMed Central

    Jiang, J.M.; Zong, Y.; Chuang, D.Y.; Lei, W.; Lu, C.-H.; Gu, Z.; Fritsche, K.L.; Thomas, A.L.; Lubahn, D.B.; Simonyi, A.; Sun, G.Y.

    2016-01-01

    Many species of berries are nutritious food and offer health benefits. However, among the different types of berries, information on health effects of American elderberries (Sambucus nigra subsp. canadensis) has been lacking and little is known about whether elderberry consumption can confer neuroprotective effects on the central nervous system. Microglial cells constitute a unique class of immune cells and exhibit characteristic properties to carry out multifunctional duties in the brain. Activation of microglial cells has been implicated in brain injury and in many types of neurodegenerative diseases. Our recent studies demonstrated the ability for endotoxin (lipopolysaccharide, LPS) and interferon gamma (IFNγ) to induce reactive oxygen species (ROS) and nitric oxide (NO) in murine microglial cells (BV-2) through activating NADPH oxidase and the MAPK pathways. In this study, BV-2 microglial cells were used to examine effects of elderberry juice obtained from different genotypes on oxidative and inflammatory responses induced by LPS and IFNγ. Results show that ‘Wyldewood’ extract demonstrated antioxidant properties by inhibiting IFNγ-induced ROS production and p-ERK1/2 expression. On the other hand, most juice extracts exerted small effects on LPS-induced NO production and some extracts showed an increase in NO production upon stimulation with IFNγ. The disparity of responses on ROS and NO production from different extracts suggests possible presence of unknown endogenous factor(s) in the extract in promoting the IFNγ-induced iNOS synthesis pathway. PMID:27158184

  8. Ethanol downregulates N-acyl phosphatidylethanolamine-phospholipase D expression in BV2 microglial cells via epigenetic mechanisms.

    PubMed

    Correa, Fernando; De Laurentiis, Andrea; Franchi, Ana María

    2016-09-01

    Excessive ethanol drinking has deleterious effects on the brain. However, the effects of alcohol on microglia, the main mediator of the brain's innate immune response remain poorly understood. On the other hand, the endocannabinoid system plays a fundamental role in regulating microglial reactivity and function. Here we studied the effects of acute ethanol exposure to murine BV2 microglial cells on N-acyl phosphatidylethanolamine-phospholipase D (NAPE-PLD), a major synthesizing enzyme of anandamide and other N-acylethanolamines. We found that ethanol downregulated microglial NAPE-PLD expression by activating cAMP/PKA and ERK1/2. These signaling pathways converged on increased phosphorylation of CREB. Moreover, ethanol induced and increase in histone acetyltransferase activity which led to higher levels of acetylation of histone H3. Taken together, our results suggest that ethanol actions on microglial NAPE-PLD expression might involve epigenetic mechanisms. PMID:27266665

  9. Effect of CCL2 on BV2 microglial cell migration: Involvement of probable signaling pathways.

    PubMed

    Bose, Shambhunath; Kim, Sunyoung; Oh, Yeonsoo; Moniruzzaman, Md; Lee, Gyeongjun; Cho, Jungsook

    2016-05-01

    Microglia, the resident macrophages of the central nervous system, play a vital role in the regulation of innate immune function and neuronal homeostasis of the brain. Currently, much interest is being generated regarding the investigation of the microglial migration that results in their accumulation at focal sites of injury. Chemokines including CCL2 are known to cause the potential induction of migration of microglial cells, although the underlying mechanisms are not well understood. In the present study, using murine neonatal BV2 microglial cells as a model, we investigate the impact of CCL2 on the migration of microglial cells and address the probable molecular events within the cellular signaling cascades mediating CCL2-induced cell migration. Our results demonstrate concentration- and time-dependent induction of BV2 cell migration by CCL2 and reveal complex mechanisms involving the activation of MEK, ERK1/2, and Akt, and their cross-talk. In addition, we demonstrate that the MEK/ERK pathway activated by CCL2 treatment mediate p90RSK activation in BV2 cells. Moreover, our findings indicate that Akt, ERK1/2, and p90RSK are the downstream effectors of PI3K in the CCL2-induced signaling. Finally, phosphorylation of the transcription factors c-jun and ATF-1 is found to be a further downstream signaling cascade in the CCL2-mediated action. Our results suggest that CCL2-induced activation of c-jun and ATF-1 is likely to be linked to the MEK/ERK and PI3K signaling pathways, respectively. Taken together, these findings contribute to a better understanding of CCL2-induced microglial migration and the probable signaling pathways involved. PMID:26878647

  10. Anti-Inflammatory and Cytoprotective Effects of TMC-256C1 from Marine-Derived Fungus Aspergillus sp. SF-6354 via up-Regulation of Heme Oxygenase-1 in Murine Hippocampal and Microglial Cell Lines

    PubMed Central

    Kim, Dong-Cheol; Cho, Kwang-Ho; Ko, Wonmin; Yoon, Chi-Su; Sohn, Jae Hak; Yim, Joung Han; Kim, Youn-Chul; Oh, Hyuncheol

    2016-01-01

    In the course of searching for bioactive secondary metabolites from marine fungi, TMC-256C1 was isolated from an ethyl acetate extract of the marine-derived fungus Aspergillus sp. SF6354. TMC-256C1 displayed anti-neuroinflammatory effect in BV2 microglial cells induced by lipopolysaccharides (LPS) as well as neuroprotective effect against glutamate-stimulated neurotoxicity in mouse hippocampal HT22 cells. TMC-256C1 was shown to develop a cellular resistance to oxidative damage caused by glutamate-induced cytotoxicity and reactive oxygen species (ROS) generation in HT22 cells, and suppress the inflammation process in LPS-stimulated BV2 cells. Furthermore, the neuroprotective and anti-neuroinflammatory activities of TMC-256C1 were associated with upregulated expression of heme oxygenase (HO)-1 and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) in HT22 and BV2 cells. We also found that TMC-256C1 activated p38 mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in HT22 and BV2 cells. These results demonstrated that TMC-256C1 activates HO-1 protein expression, probably by increasing nuclear Nrf2 levels via the activation of the p38 MAPK and PI3K/Akt pathways. PMID:27070586

  11. Anti-Inflammatory and Cytoprotective Effects of TMC-256C1 from Marine-Derived Fungus Aspergillus sp. SF-6354 via up-Regulation of Heme Oxygenase-1 in Murine Hippocampal and Microglial Cell Lines.

    PubMed

    Kim, Dong-Cheol; Cho, Kwang-Ho; Ko, Wonmin; Yoon, Chi-Su; Sohn, Jae Hak; Yim, Joung Han; Kim, Youn-Chul; Oh, Hyuncheol

    2016-01-01

    In the course of searching for bioactive secondary metabolites from marine fungi, TMC-256C1 was isolated from an ethyl acetate extract of the marine-derived fungus Aspergillus sp. SF6354. TMC-256C1 displayed anti-neuroinflammatory effect in BV2 microglial cells induced by lipopolysaccharides (LPS) as well as neuroprotective effect against glutamate-stimulated neurotoxicity in mouse hippocampal HT22 cells. TMC-256C1 was shown to develop a cellular resistance to oxidative damage caused by glutamate-induced cytotoxicity and reactive oxygen species (ROS) generation in HT22 cells, and suppress the inflammation process in LPS-stimulated BV2 cells. Furthermore, the neuroprotective and anti-neuroinflammatory activities of TMC-256C1 were associated with upregulated expression of heme oxygenase (HO)-1 and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) in HT22 and BV2 cells. We also found that TMC-256C1 activated p38 mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in HT22 and BV2 cells. These results demonstrated that TMC-256C1 activates HO-1 protein expression, probably by increasing nuclear Nrf2 levels via the activation of the p38 MAPK and PI3K/Akt pathways. PMID:27070586

  12. Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke.

    PubMed

    Fernández-López, David; Faustino, Joel; Klibanov, Alexander L; Derugin, Nikita; Blanchard, Elodie; Simon, Franziska; Leib, Stephen L; Vexler, Zinaida S

    2016-03-01

    Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFβ1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFβ1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1(GFP/+)/Ccr2(RFP/+) mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2(RFP/+) monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke. PMID:26961944

  13. (-)-Epigallocatechin-3-gallate inhibits voltage-gated proton currents in BV2 microglial cells.

    PubMed

    Jin, Sanghee; Park, Mijung; Song, Jin-Ho

    2013-01-01

    (-)-Epigallocatechin-3-gallate (EGCG), the principal constituent of green tea, protects neurons from toxic insults by suppressing the microglial secretion of neurotoxic inflammatory mediators. Voltage-gated proton channels are expressed in microglia, and are required for NADPH oxidase-dependent reactive oxygen species generation. Brain damage after ischemic stroke is dependent on proton channel activity. Accordingly, we examined whether EGCG could inhibit proton channel function in the murine microglial BV2 cells. EGCG potently inhibited proton currents with an IC(50) of 3.7 μM. Other tea catechins, (-)-epigallocatechin, (-)-epicatechin and (-)-epicatechin-3-gallate, were far less potent than EGCG. EGCG did not change the kinetics of proton currents such as the activation and the deactivation time constants, the reversal potential and the activation voltage, suggesting that the gating process of proton channels were not altered by EGCG. EGCG is known to disturb lipid rafts by sequestering cholesterol. However, neither extraction of cholesterol with methyl-β-cyclodextrin or cholesterol supplementation could reverse the EGCG inhibition of proton currents. In addition, the EGCG effect was preserved in the presence of the cytoskeletal stabilizers paclitaxel and phalloidin, phosphatase inhibitors, the antioxidant Trolox, superoxide dismutase or catalase. The proton channel inhibition can be a substantial mechanism for EGCG to suppress microglial activation and subsequent neurotoxic events. PMID:23201067

  14. JAK-STAT signaling mediates gangliosides-induced inflammatory responses in brain microglial cells.

    PubMed

    Kim, Ohn Soon; Park, Eun Jung; Joe, Eun-hye; Jou, Ilo

    2002-10-25

    Neuronal cell membranes are particularly rich in gangliosides, which play important roles in brain physiology and pathology. Previously, we reported that gangliosides could act as microglial activators and are thus likely to participate in many neuronal diseases. In the present study we provide evidence that JAK-STAT inflammatory signaling mediates gangliosides-stimulated microglial activation. Both in rat primary microglia and murine BV2 microglial cells, gangliosides stimulated nuclear factor binding to GAS/ISRE elements, which are known to be STAT-binding sites. Consistent with this, gangliosides rapidly activated JAK1 and JAK2 and induced phosphorylation of STAT1 and STAT3. In addition, gangliosides increased transcription of the inflammation-associated genes inducible nitric-oxide synthase, ICAM-1, and MCP-1, which are reported to contain STAT-binding elements in their promoter regions. AG490, a JAK inhibitor, reduced induction of these genes, nuclear factor binding activity, and activation of STAT1 and -3 in gangliosides-treated microglia. AG490 also inhibited gangliosides-induced release of nitric oxide, an inflammation hallmark. Furthermore, AG490 markedly reduced activation of ERK1/2 MAPK, indicating that ERKs act downstream of JAK-STAT signaling during microglial activation. However, AG490 did not affect activation of p38 MAPK. We also report that the sialic acid residues present on gangliosides may be one of the essential components in activation of JAK-STAT signaling. The present study indicates that JAK-STAT signaling is an early event in gangliosides-induced brain inflammatory responses. PMID:12191995

  15. Serial analysis of gene expression in a microglial cell line.

    PubMed

    Inoue, H; Sawada, M; Ryo, A; Tanahashi, H; Wakatsuki, T; Hada, A; Kondoh, N; Nakagaki, K; Takahashi, K; Suzumura, A; Yamamoto, M; Tabira, T

    1999-12-01

    We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. PMID:10559785

  16. Global gene expression changes in BV2 microglial cell line during rabies virus infection.

    PubMed

    Zhao, Pingsen; Yang, Yujiao; Feng, Hao; Zhao, Lili; Qin, Junling; Zhang, Tao; Wang, Hualei; Yang, Songtao; Xia, Xianzhu

    2013-12-01

    Microglia plays a crucial role during virus pathogenesis in the central nervous system (CNS). Infection by rabies virus (RABV) causes a fatal infection in the CNS of all warm-blooded animals. However, the microglial responses to RABV infection have been scarcely reported. To better understand microglia-RABV interactions at the transcriptional level, a genome wide gene expression profile in mouse microglial cells line BV2 was performed using microarray analysis. The global messenger RNA changes in murine microglial cell line BV2 after 12, 24 and 48 h of infection with rabies virus CVS-11 strain were investigated using DNA Microarray and quantitative real-time PCR. Infection of CVS-11 at different time points induced different gene expression signatures in BV2 cells. The expression patterns of differentially expressed genes are shown by K-means clustering in four clusters in RABV- or mock-infected microglia at 12, 24 and 48h post infection (hpi). Gene ontology and network analysis of the differentially expressed genes in responses to RABV were performed by the Ingenuity Pathway Analysis system (IPA, Ingenuity® Systems, http://www.ingenuity.com). The results revealed that 28 genes were significantly up-regulated (P<0.01) and 1 gene was significantly down-regulated (P<0.01) in microglial cells at 12hpi, 72 genes were significantly up-regulated (P<0.01) and 24 genes were significantly down-regulated (P<0.01) at 24hpi, and 671 genes were significantly up-regulated (P<0.01) and 190 genes were significantly down-regulated (P<0.01) at 48hpi. Genes in BV2 were significantly regulated (P<0.01) in response to RABV infection and they were found to be interferon stimulated genes (Isg15, Isg20, Oasl1, Oasl2, Ifit2, Irf7 and Ifi203), chemokine genes (Ccl5, Cxcl10 and Ccrl2) and the proinflammatory factor gene (Interleukin 6). The results indicated that the differentially expressed genes from microglial cells after RABV infection were mainly involved in innate immune responses

  17. A Novel Cell Line from Spontaneously Immortalized Murine Microglia

    PubMed Central

    Kulas, Joshua; Combs, Colin K.

    2014-01-01

    Background Purified microglia cultures are useful tools to study microglial behavior in vitro. Microglial cell lines serve as an attractive alternative to primary microglia culture, circumventing the costly and lengthy preparation of the latter. However, immortalization by genetic or pharmacologic manipulations may show altered physiology from primary microglia. New Method A novel microglial cell line was isolated from a primary glial culture of postnatal murine cerebral cortices. The culture contained a population of spontaneously transformed microglia that continued to divide without genetic or pharmacological manipulations. After several clones were isolated, one particular clone, SIM-A9, was analyzed for its microglial characteristics. Results SIM-A9 cells expressed macrophage/microglia-specific proteins, CD68 and Iba1. SIM-A9 cells were responsive to exogenous inflammatory stimulation with lipopolysaccharide and β-amyloid, triggering tyrosine kinase-based and NFκB signaling cascades as well as TNFα secretion. SIM-A9 cells also exhibited phagocytic uptake of fluorescent labeled β-amyloid and bacterial bioparticles. Furthermore, lipopolysaccharide increased the levels of inducible nitric oxide synthase and cyclooxygenase-2, whereas IL-4 stimulation increased arginase-1 levels demonstrating that SIM-A9 cells are capable of switching their profiles to pro- or anti-inflammatory phenotypes, respectively. Comparison with Existing Methods The use of SIM-A9 cells avoids expensive and lengthy procedures required for the preparation of primary microglia. Spontaneously immortalized SIM-A9 cells are expected to behave more comparably to primary microglia than virally transformed or pharmacologically induced microglial cell lines. Conclusions SIM-A9 cells exhibit key characteristics of cultured primary microglia and may serve as a valuable model system for the investigation of microglial behavior in vitro. PMID:24975292

  18. Layer 5 Pyramidal Neurons' Dendritic Remodeling and Increased Microglial Density in Primary Motor Cortex in a Murine Model of Facial Paralysis

    PubMed Central

    Urrego, Diana; Troncoso, Julieta; Múnera, Alejandro

    2015-01-01

    This work was aimed at characterizing structural changes in primary motor cortex layer 5 pyramidal neurons and their relationship with microglial density induced by facial nerve lesion using a murine facial paralysis model. Adult transgenic mice, expressing green fluorescent protein in microglia and yellow fluorescent protein in projecting neurons, were submitted to either unilateral section of the facial nerve or sham surgery. Injured animals were sacrificed either 1 or 3weeks after surgery. Two-photon excitation microscopy was then used for evaluating both layer 5 pyramidal neurons and microglia in vibrissal primary motor cortex (vM1). It was found that facial nerve lesion induced long-lasting changes in the dendritic morphology of vM1 layer 5 pyramidal neurons and in their surrounding microglia. Dendritic arborization of the pyramidal cells underwent overall shrinkage. Apical dendrites suffered transient shortening while basal dendrites displayed sustained shortening. Moreover, dendrites suffered transient spine pruning. Significantly higher microglial cell density was found surrounding vM1 layer 5 pyramidal neurons after facial nerve lesion with morphological bias towards the activated phenotype. These results suggest that facial nerve lesions elicit active dendrite remodeling due to pyramidal neuron and microglia interaction, which could be the pathophysiological underpinning of some neuropathic motor sequelae in humans. PMID:26064916

  19. Fractalkine Attenuates Microglial Cell Activation Induced by Prenatal Stress

    PubMed Central

    Ślusarczyk, Joanna; Trojan, Ewa; Głombik, Katarzyna; Chamera, Katarzyna; Roman, Adam; Budziszewska, Bogusława; Basta-Kaim, Agnieszka

    2016-01-01

    The potential contribution of inflammation to the development of neuropsychiatric diseases has recently received substantial attention. In the brain, the main immune cells are the microglia. As they are the main source of inflammatory factors, it is plausible that the regulation of their activation may be a potential therapeutic target. Fractalkine (CX3CL1) and its receptor CX3CR1 play a crucial role in the control of the biological activity of the microglia. In the present study, using microglial cultures we investigated whether fractalkine is able to reverse changes in microglia caused by a prenatal stress procedure. Our study found that the microglia do not express fractalkine. Prenatal stress decreases the expression of the fractalkine receptor, which in turn is enhanced by the administration of exogenous fractalkine. Moreover, treatment with fractalkine diminishes the prenatal stress-induced overproduction of proinflammatory factors such as IL-1β, IL-18, IL-6, TNF-α, CCL2, or NO in the microglial cells derived from prenatally stressed newborns. In conclusion, the present results revealed that the pathological activation of microglia in prenatally stressed newborns may be attenuated by fractalkine administration. Therefore, understanding of the role of the CX3CL1-CX3CR1 system may help to elucidate the mechanisms underlying the neuron-microglia interaction and its role in pathological conditions in the brain. PMID:27239349

  20. Microglial cells (BV-2) internalize titanium dioxide (TiO2) nanoparticles: toxicity and cellular responses.

    PubMed

    Rihane, Naima; Nury, Thomas; M'rad, Imen; El Mir, Lassaad; Sakly, Mohsen; Amara, Salem; Lizard, Gérard

    2016-05-01

    Because of their whitening and photocatalytic effects, titanium dioxide nanoparticles (TiO2-NPs) are widely used in daily life. These NPs can be found in paints, plastics, papers, sunscreens, foods, medicines (pills), toothpastes, and cosmetics. However, the biological effect of TiO2-NPs on the human body, especially on the central nervous system, is still unclear. Many studies have demonstrated that the brain is one of the target organs in acute or chronic TiO2-NPs toxicity. The present study aimed to investigate the effect of TiO2-NPs at different concentrations (0.1 to 200 μg/mL) on murine microglial cells (BV-2) to assess their activity on cell growth and viability, as well as their neurotoxicity. Different parameters were measured: cell viability, cell proliferation and DNA content (SubG1 peak), mitochondrial depolarization, overproduction of reactive oxygen species (especially superoxide anions), and ultrastructural changes. Results showed that TiO2-NPs induced some cytotoxic effects with a slight inhibition of cell growth. Thus, at high concentrations, TiO2-NPs were not only able to inhibit cell adhesion but also enhanced cytoplasmic membrane permeability to propidium iodide associated with a loss of mitochondrial transmembrane potential and an overproduction of superoxide anions. No induction of apoptosis based on the presence of a SubG1 peak was detected. The microscopic observations also indicated that small groups of nanosized particles and micron-sized aggregates were engulfed by the BV-2 cells and sequestered as intracytoplasmic aggregates after 24-h exposure to TiO2-NPs. Altogether, our data show that the accumulation TiO2-NPs in microglial BV-2 cells favors mitochondrial dysfunctions and oxidative stress. PMID:26846246

  1. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures

    PubMed Central

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K.; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. PMID:27304968

  2. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. PMID:27304968

  3. A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways

    PubMed Central

    Kim, Byung-Wook; More, Sandeep Vasant; Yun, Yo-Sep; Ko, Hyun-Myung; Kwak, Jae-Hwan; Lee, Heesoon; Suk, Kyoungho; Kim, In-Su; Choi, Dong-Kug

    2016-01-01

    Aim: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro. Methods: Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence microscopy, respectively. Results: Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). Conclusion: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells. PMID:26838070

  4. 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

  5. Microglial cell activation is a source of metalloproteinase generation during hemorrhagic transformation

    PubMed Central

    del Zoppo, Gregory J; Frankowski, Harald; Gu, Yu-Huan; Osada, Takashi; Kanazawa, Masato; Milner, Richard; Wang, Xiaoyun; Hosomi, Naohisa; Mabuchi, Takuma; Koziol, James A

    2012-01-01

    Hemorrhage and edema accompany evolving brain tissue injury after ischemic stroke. In patients, these events have been associated with metalloproteinase (MMP)-9 in plasma. Both the causes and cellular sources of MMP-9 generation in this setting have not been defined. MMP-2 and MMP-9 in nonhuman primate tissue in regions of plasma leakage, and primary murine microglia and astrocytes, were assayed by immunocytochemistry, zymography, and real-time RT-PCR. Ischemia-related hemorrhage was associated with microglial activation in vivo, and with the leakage of plasma fibronectin and vitronectin into the surrounding tissue. In strict serum-depleted primary cultures, by zymography, pro-MMP-9 was generated by primary murine microglia when exposed to vitronectin and fibronectin. Protease secretion was enhanced by experimental ischemia (oxygen-glucose deprivation, OGD). Primary astrocytes, on each matrix, generated only pro-MMP-2, which decreased during OGD. Microglia–astrocyte contact enhanced pro-MMP-9 generation in a cell density-dependent manner under normoxia and OGD. Compatible with observations in a high quality model of focal cerebral ischemia, microglia, but not astrocytes, respond to vitronectin and fibronectin, found when plasma extravasates into the injured region. Astrocytes alone do not generate pro-MMP-9. These events explain the appearance of MMP-9 antigen in association with ischemia-induced cerebral hemorrhage and edema. PMID:22354151

  6. Gypenoside Attenuates β Amyloid-Induced Inflammation in N9 Microglial Cells via SOCS1 Signaling

    PubMed Central

    Cai, Hui; Liang, Qianlei; Ge, Guanqun

    2016-01-01

    Reducing β amyloid- (Aβ-) induced microglial activation is believed to be effective in treating Alzheimer's disease (AD). Microglia can be activated into classic activated state (M1 state) or alternative activated state (M2 state), and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP) is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aβ-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1). In this study, we found that Aβ exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1) expression, IL-10, brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) releases in the Aβ-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aβ-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1. PMID:27213058

  7. Gypenoside Attenuates β Amyloid-Induced Inflammation in N9 Microglial Cells via SOCS1 Signaling.

    PubMed

    Cai, Hui; Liang, Qianlei; Ge, Guanqun

    2016-01-01

    Reducing β amyloid- (Aβ-) induced microglial activation is believed to be effective in treating Alzheimer's disease (AD). Microglia can be activated into classic activated state (M1 state) or alternative activated state (M2 state), and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP) is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aβ-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1). In this study, we found that Aβ exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1) expression, IL-10, brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) releases in the Aβ-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aβ-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1. PMID:27213058

  8. Peripheral viral infection induced microglial sensome genes and enhanced microglial cell activity in the hippocampus of neonatal piglets.

    PubMed

    Ji, Peng; Schachtschneider, Kyle M; Schook, Lawrence B; Walker, Frederick R; Johnson, Rodney W

    2016-05-01

    Although poorly understood, early-life infection is predicted to affect brain microglial cells, making them hypersensitive to subsequent stimuli. To investigate this, we assessed gene expression in hippocampal tissue obtained from a previously published study reporting increased microglial cell activity and reduced hippocampal-dependent learning in neonatal piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus that induces interstitial pneumonia. Infection altered expression of 455 genes, of which 334 were up-regulated and 121 were down-regulated. Functional annotation revealed that immune function genes were enriched among the up-regulated differentially expressed genes (DEGs), whereas calcium binding and synaptic vesicle genes were enriched among the down-regulated DEGs. Twenty-six genes encoding part of the microglia sensory apparatus (i.e., the sensome) were up-regulated (e.g., IL1R1, TLR2, and TLR4), whereas 15 genes associated with the synaptosome and synaptic receptors (e.g., NPTX2, GABRA2, and SLC5A7) were down-regulated. As the sensome may foretell microglia reactivity, we next inoculated piglets with culture medium or PRRSV at PD 7 and assessed hippocampal microglia morphology and function at PD 28 when signs of infection were waning. Consistent with amplification of the sensome, microglia from PRRSV piglets had enhanced responsiveness to chemoattractants, increased phagocytic activity, and secreted more TNFα in response to lipopolysaccharide and Poly I:C. Immunohistochemical staining indicated PRRSV infection increased microglia soma length and length-to-width ratio. Bipolar rod-like microglia not evident in hippocampus of control piglets, were present in infected piglets. Collectively, this study suggests early-life infection alters the microglia sensome as well as microglial cell morphology and function. PMID:26872419

  9. Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease

    PubMed Central

    Ghosh, Soumitra; Geahlen, Robert L.

    2015-01-01

    Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1–42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells. PMID:26870803

  10. Microglial Cells as a Link between Cannabinoids and the Immune Hypothesis of Psychiatric Disorders.

    PubMed

    Lisboa, Sabrina F; Gomes, Felipe V; Guimaraes, Francisco S; Campos, Alline C

    2016-01-01

    Psychiatric disorders are one of the leading causes of disability worldwide. Although several therapeutic options are available, the exact mechanisms responsible for the genesis of these disorders remain to be fully elucidated. In the last decade, a body of evidence has supported the involvement of the immune system in the pathophysiology of these conditions. Microglial cells play a significant role in maintaining brain homeostasis and surveillance. Dysregulation of microglial functions has been associated with several psychiatric conditions. Cannabinoids regulate the brain-immune axis and inhibit microglial cell activation. Here, we summarized evidence supporting the hypothesis that microglial cells could be a target for cannabinoid influence on psychiatric disorders, such as anxiety, depression, schizophrenia, and stress-related disorders. PMID:26858686

  11. Microglial Cells as a Link between Cannabinoids and the Immune Hypothesis of Psychiatric Disorders

    PubMed Central

    Lisboa, Sabrina F.; Gomes, Felipe V.; Guimaraes, Francisco S.; Campos, Alline C.

    2016-01-01

    Psychiatric disorders are one of the leading causes of disability worldwide. Although several therapeutic options are available, the exact mechanisms responsible for the genesis of these disorders remain to be fully elucidated. In the last decade, a body of evidence has supported the involvement of the immune system in the pathophysiology of these conditions. Microglial cells play a significant role in maintaining brain homeostasis and surveillance. Dysregulation of microglial functions has been associated with several psychiatric conditions. Cannabinoids regulate the brain–immune axis and inhibit microglial cell activation. Here, we summarized evidence supporting the hypothesis that microglial cells could be a target for cannabinoid influence on psychiatric disorders, such as anxiety, depression, schizophrenia, and stress-related disorders. PMID:26858686

  12. Role of endoplasmic reticulum (ER) stress in cocaine-induced microglial cell death.

    PubMed

    Costa, Blaise Mathias; Yao, Honghong; Yang, Lu; Buch, Shilpa

    2013-06-01

    While it has been well-documented that drugs of abuse such as cocaine can enhance progression of human immunodeficiency virus (HIV)-associated neuropathological disorders, the underlying mechanisms mediating these effects remain poorly understood. The present study was undertaken to examine the effects of cocaine on microglial viability. Herein we demonstrate that exposure of microglial cell line-BV2 or rat primary microglia to exogenous cocaine resulted in decreased cell viability as determined by MTS and TUNEL assays. Microglial toxicity of cocaine was accompanied by an increase in the expression of cleaved caspase-3 as demonstrated by western blot assays. Furthermore, increased microglial toxicity was also associated with a concomitant increase in the production of intracellular reactive oxygen species, an effect that was ameliorated in cells pretreated with NADPH oxidase inhibitor apocynin, thus emphasizing the role of oxidative stress in this process. A novel finding of this study was the involvement of endoplasmic reticulum (ER) signaling mediators such as PERK, Elf2α, and CHOP, which were up regulated in cells exposed to cocaine. Reciprocally, blocking CHOP expression using siRNA ameliorated cocaine-mediated cell death. In conclusion these findings underscore the importance of ER stress in modulating cocaine induced microglial toxicity. Understanding the link between ER stress, oxidative stress and apoptosis could lead to the development of therapeutic strategies targeting cocaine-mediated microglial death/dysfunction. PMID:23404095

  13. NitroDIGE analysis reveals inhibition of protein S-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells

    PubMed Central

    2014-01-01

    Background Nitric oxide (NO) is a signaling molecule regulating numerous cellular functions in development and disease. In the brain, neuronal injury or neuroinflammation can lead to microglial activation, which induces NO production. NO can react with critical cysteine thiols of target proteins forming S-nitroso-proteins. This modification, known as S-nitrosylation, is an evolutionarily conserved redox-based post-translational modification (PTM) of specific proteins analogous to phosphorylation. In this study, we describe a protocol for analyzing S-nitrosylation of proteins using a gel-based proteomic approach and use it to investigate the modes of action of a botanical compound found in green tea, epigallocatechin-3-gallate (EGCG), on protein S-nitrosylation after microglial activation. Methods/Results To globally and quantitatively analyze NO-induced protein S-nitrosylation, the sensitive gel-based proteomic method, termed NitroDIGE, was developed by combining two-dimensional differential in-gel electrophoresis (2-D DIGE) with the modified biotin switch technique (BST) using fluorescence-tagged CyDye™ thiol reactive agents to label S-nitrosothiols. The NitroDIGE method showed high specificity and sensitivity in detecting S-nitrosylated proteins (SNO-proteins). Using this approach, we identified a subset of SNO-proteins ex vivo by exposing immortalized murine BV-2 microglial cells to a physiological NO donor, or in vivo by exposing BV-2 cells to endotoxin lipopolysaccharides (LPS) to induce a proinflammatory response. Moreover, EGCG was shown to attenuate S-nitrosylation of proteins after LPS-induced activation of microglial cells primarily by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response. Conclusions These results demonstrate that NitroDIGE is an effective proteomic strategy for “top-down” quantitative analysis of protein S-nitrosylation in multi-group samples in response to nitrosative stress due

  14. Suppression of LPS-induced inflammatory responses by inflexanin B in BV2 microglial cells.

    PubMed

    Lim, Ji-Youn; Sul, Donggeun; Hwang, Bang Yeon; Hwang, Kwang Woo; Yoo, Ki-Yeol; Park, So-Young

    2013-02-01

    Microglia are a type of resident macrophage that functions as an inflammation modulator in the central nervous system. Over-activation of microglia by a range of stimuli disrupts the physiological homeostasis of the brain, and induces inflammatory response and degenerative processes, such as those implicated in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, we investigated the possible anti-inflammatory mechanisms of inflexanin B in murine microglial BV2 cells. Lipopolysaccharide (LPS) activated BV2 cells and induced the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (interleukins-1β and -6, and tumour necrosis factor α). The LPS-induced production of pro-inflammatory mediators was associated with the enhancement of nuclear factor-kappaB (NF-κB) nuclear translocation and the activation of mitogen-activated protein kinase (MAPK) including ERK1/2 and JNK. Conversely, pretreatment of cells with inflexanin B (10 and 20 μg/mL) significantly reduced the production of pro-inflammatory mediators. This was accompanied with the reduced nuclear translocation of NF-κB and reduced activation of MAPKs. These results suggest that inflexanin B attenuated the LPS-induced inflammatory process by inhibiting the activation of NF-κB and MAPKs. PMID:23458198

  15. Automatic Counting of Microglial Cells in Healthy and Glaucomatous Mouse Retinas

    PubMed Central

    Rojas, Blanca; Ramírez, Ana I.; de Hoz, Rosa; Salazar, Juan J.; Triviño, Alberto; Ramírez, José M.

    2015-01-01

    Proliferation of microglial cells has been considered a sign of glial activation and a hallmark of ongoing neurodegenerative diseases. Microglia activation is analyzed in animal models of different eye diseases. Numerous retinal samples are required for each of these studies to obtain relevant data of statistical significance. Because manual quantification of microglial cells is time consuming, the aim of this study was develop an algorithm for automatic identification of retinal microglia. Two groups of adult male Swiss mice were used: age-matched controls (naïve, n = 6) and mice subjected to unilateral laser-induced ocular hypertension (lasered; n = 9). In the latter group, both hypertensive eyes and contralateral untreated retinas were analyzed. Retinal whole mounts were immunostained with anti Iba-1 for detecting microglial cell populations. A new algorithm was developed in MATLAB for microglial quantification; it enabled the quantification of microglial cells in the inner and outer plexiform layers and evaluates the area of the retina occupied by Iba-1+ microglia in the nerve fiber-ganglion cell layer. The automatic method was applied to a set of 6,000 images. To validate the algorithm, mouse retinas were evaluated both manually and computationally; the program correctly assessed the number of cells (Pearson correlation R = 0.94 and R = 0.98 for the inner and outer plexiform layers respectively). Statistically significant differences in glial cell number were found between naïve, lasered eyes and contralateral eyes (P<0.05, naïve versus contralateral eyes; P<0.001, naïve versus lasered eyes and contralateral versus lasered eyes). The algorithm developed is a reliable and fast tool that can evaluate the number of microglial cells in naïve mouse retinas and in retinas exhibiting proliferation. The implementation of this new automatic method can enable faster quantification of microglial cells in retinal pathologies. PMID:26580208

  16. BJ-1103, 6-aminopyridin-3-ol skeletal compound, modulates neuroprotective and anti-neuroinflammatory effects in murine hippocampal and microglial cells via Nrf2-mediated heme oxygenase-1 expression.

    PubMed

    Lee, Dong-Sung; Nam, Tae-Gyu; Jeong, Byeong-Seon; Jeong, Gil-Saeng

    2016-08-01

    BJ-1103, as a 6-aminopyridin-3-ol skeletal compound, was originally developed as an antioxidant against free radicals and oxidative stress was prepared from pyridoxine·HCl by the reported procedure. In the present study, we examined the effect of BJ-1103 on neuroprotection and neuroinflammation. Our data showed that BJ-1103 can protect HT22 cells against glutamate-induced cell cytotoxicity. And, BJ-1103 also inhibited LPS-induced inflammatory action. In addition, BJ-1103-induced heme oxygenase-1 (HO-1) expression and elevated HO-1 activities in the two cell lines studied. Additionally, BJ-1103 treatment induced nuclear transcription factor erythroid-2 related factor 2 (Nrf2) and increased the promoter activity of antioxidant response elements (AREs). We have demonstrated using the Nrf2 siRNA, HO inhibitor or HO-1 siRNA that BJ-1103 suppressed neurotoxicity and neuroinflammation through the Nrf2-mediated HO-1 expression. These results demonstrated that BJ-1103 may have good therapeutic agent against neurodegenerative diseases that are induced by oxidative stress and neuroinflammation. PMID:27238747

  17. Constitutive and functional expression of YB-1 in microglial cells.

    PubMed

    Keilhoff, G; Titze, M; Esser, T; Langnaese, K; Ebmeyer, U

    2015-08-20

    Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS

  18. Microglial cell death induced by glycated bovine serum albumin: nitric oxide involvement.

    PubMed

    Khazaei, Mohammad R; Habibi-Rezaei, Mehran; Karimzadeh, Fereshteh; Moosavi-Movahedi, Ali Akbar; Sarrafnejhad, Abdo Alfattah; Sabouni, Farzaneh; Bakhti, Mostafa

    2008-08-01

    Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE-BSA. PMID:18463114

  19. Radiation-Induced c-Jun Activation Depends on MEK1-ERK1/2 Signaling Pathway in Microglial Cells

    PubMed Central

    Deng, Zhiyong; Sui, Guangchao; Rosa, Paulo Mottin; Zhao, Weiling

    2012-01-01

    Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation. PMID:22606284

  20. Phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by carbon nanotube agglomerates.

    PubMed

    Shigemoto-Mogami, Yukari; Hoshikawa, Kazue; Hirose, Akihiko; Sato, Kaoru

    2016-01-01

    Although carbon nanotubes (CNTs) are used in many fields, including energy, healthcare, environmental technology, materials, and electronics, the adverse effects of CNTs in the brain are poorly understood. In this study, we investigated the effects of CNTs on cultured microglia, as microglia are the first responders to foreign materials. We compared the effects of sonicated suspensions of 5 kinds of CNTs and their flow-through filtered with a 0.22 µm membrane filter on microglial viability. We found that sonicated suspensions caused microglial cell damage, but their flow-through did not. The number of microglial aggregates was well correlated with the extent of the damage. We also determined that the CNT agglomerates consisted of two groups: one was phagocytosed by microglia and caused microglial cell damage, and the other caused cell damage without phagocytosis. These results suggest that phagocytosis-dependent and independent mechanisms underlie the microglial cell damage caused by CNT agglomerates and it is important to conduct studies about the relationships between physical properties of nanomaterial-agglomerates and cell damage. PMID:27432236

  1. Microglial activation mediates host neuronal survival induced by neural stem cells.

    PubMed

    Wu, Hui-Mei; Zhang, Li-Feng; Ding, Pei-Shang; Liu, Ya-Jing; Wu, Xu; Zhou, Jiang-Ning

    2014-07-01

    The rational of neural stem cells (NSCs) in the therapy of neurological disease is either to replace dead neurons or to improve host neuronal survival, the latter of which has got less attention and the underlying mechanism is as yet little known. Using a transwell co-culture system, we reported that, in organotypic brain slice cultures, NSCs significantly improved host neuronal viability. Interestingly, this beneficial effect of NSCs was abrogated by a microglial inhibitor minocycline, while it was mimicked by a microglial agonist, Toll-like receptor 9 (TLR9) ligand CpG-ODN, which supports the pro-vital mediation by microglia on this NSCs-improved neuronal survival. Moreover, we showed that NSCs significantly induced host microglial movement and higher expression of a microglial marker IBA-1, the latter of which was positively correlated with TLR9 or extracellular-regulated protein kinases 1/2 (ERK1/2) activation. Real-time PCR revealed that NSCs inhibited the expression of pro-inflammatory molecules, but significantly increased the expression of molecules associated with a neuroprotective phenotype such as CX3CR1, triggering receptor expressed on myeloid cells-2 (TREM2) and insulin growth factor 1 (IGF-1). Similarly, in the microglia cells, NSCs induced the same microglial response as that in the slices. Further treatment with TLR9 ligand CpG-ODN, TLR9 inhibitor chloroquine (CQ) or ERK1/2 inhibitor U0126 demonstrated that TLR9-ERK1/2 pathway was involved in the NSCs-induced microglial activation. Collectively, this study indicated that NSCs improve host neuronal survival by switching microglia from a detrimental to a neuroprotective phenotype in adult mouse brain, and the microglial TLR9-ERK1/2 pathway seems to participate in this NSCs-mediated rescue action. PMID:24725889

  2. Fine-tuning the central nervous system: microglial modelling of cells and synapses

    PubMed Central

    Xavier, Anna L.; Menezes, João R. L.; Goldman, Steven A.; Nedergaard, Maiken

    2014-01-01

    Microglia constitute as much as 10–15% of all cells in the mammalian central nervous system (CNS) and are the only glial cells that do not arise from the neuroectoderm. As the principal CNS immune cells, microglial cells represent the first line of defence in response to exogenous threats. Past studies have largely been dedicated to defining the complex immune functions of microglial cells. However, our understanding of the roles of microglia has expanded radically over the past years. It is now clear that microglia are critically involved in shaping neural circuits in both the developing and adult CNS, and in modulating synaptic transmission in the adult brain. Intriguingly, microglial cells appear to use the same sets of tools, including cytokine and chemokine release as well as phagocytosis, whether modulating neural function or mediating the brain's innate immune responses. This review will discuss recent developments that have broadened our views of neuro-glial signalling to include the contribution of microglial cells. PMID:25225087

  3. Recognition of Betaine as an Inhibitor of Lipopolysaccharide-Induced Nitric Oxide Production in Activated Microglial Cells

    PubMed Central

    Amiraslani, Banafsheh; Sabouni, Farzaneh; Abbasi, Shahsanam; Nazem, Habiballah; Sabet, Mohammadsadegh

    2012-01-01

    Background: Neuroinflammation, as a major outcome of microglia activation, is an important factor for progression of neurodegenerative disorders including Alzheimer's disease and Parkinson's disease. Microglial cells, as the first-line defense in the central nervous system, act as a source of neurotoxic factors such as nitric oxide (NO), a free radical which is involved in neuronal cell death. The aim of this study was to inhibit production of NO in activated microglial cells in order to decrease neurological damages that threat the central nervous system. Methods: An in vitro model of a newborn rat brain cell culture was used to examine the effect of betaine on the release of NO induced by lipopolysaccharide (LPS). Briefly, primary microglial cells were stimulated by LPS and after 2 minutes, they were treated by different concentrations of betaine. The production of NO was assessed by the Griess assay while cell viability was determined by the MTT assay. Results: Our investigations indicated that LPS-induced NO release was attenuated by betaine, suggesting that this compound might inhibit NO release. The effects of betaine on NO production in activated microglial cells after 24 h were "dose-dependent". It means that microglial cells which were treated with higher concentrations of betaine, released lower amounts of NO. Also our observations showed that betaine compound has no toxic effect on microglial cells. Conclusion: Betaine has an inhibitory effect on NO release in activated microglial cells and may be an effective therapeutic component to control neurological disorders. PMID:22801281

  4. 1950 MHz IMT-2000 field does not activate microglial cells in vitro.

    PubMed

    Hirose, Hideki; Sasaki, Atsushi; Ishii, Nana; Sekijima, Masaru; Iyama, Takahiro; Nojima, Toshio; Ugawa, Yoshikazu

    2010-02-01

    Given the widespread use of the cellular phone today, investigation of potential biological effects of radiofrequency (RF) fields has become increasingly important. In particular, much research has been conducted on RF effects on brain function. To examine any biological effects on the central nervous system (CNS) induced by 1950 MHz modulation signals, which are controlled by the International Mobile Telecommunication-2000 (IMT-2000) cellular system, we investigated the effect of RF fields on microglial cells in the brain. We assessed functional changes in microglial cells by examining changes in immune reaction-related molecule expression and cytokine production after exposure to a 1950 MHz Wideband Code Division Multiple Access (W-CDMA) RF field, at specific absorption rates (SARs) of 0.2, 0.8, and 2.0 W/kg. Primary microglial cell cultures prepared from neonatal rats were subjected to an RF or sham field for 2 h. Assay samples obtained 24 and 72 h after exposure were processed in a blind manner. Results showed that the percentage of cells positive for major histocompatibility complex (MHC) class II, which is the most common marker for activated microglial cells, was similar between cells exposed to W-CDMA radiation and sham-exposed controls. No statistically significant differences were observed between any of the RF field exposure groups and the sham-exposed controls in percentage of MHC class II positive cells. Further, no remarkable differences in the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) were observed between the test groups exposed to W-CDMA signal and the sham-exposed negative controls. These findings suggest that exposure to RF fields up to 2 W/kg does not activate microglial cells in vitro. PMID:19650078

  5. Anti-inflammatory effects of saponins derived from the roots of Platycodon grandiflorus in lipopolysaccharide‑stimulated BV2 microglial cells.

    PubMed

    Jang, Kyung-Jun; Kim, Hong Ki; Han, Min Ho; Oh, You Na; Yoon, Hyun-Min; Chung, Yoon Ho; Kim, Gi Young; Hwang, Hye Jin; Kim, Byung Woo; Choi, Yung Hyun

    2013-06-01

    Radix platycodi is the root of Platycodon grandiflorus A. DC, which has been widely used as a food material and for the treatment of a number of chronic inflammatory diseases in traditional oriental medicine. In this study, the anti‑inflammatory effects of the saponins isolated from radix platycodi (PGS) on the production of inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated BV2 murine microglial cells were examined. We also investigated the effects of PGS on LPS‑induced nuclear factor‑κB (NF-κB) activation and phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) signaling pathways. Following stimulation with LPS, elevated nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokine production was detected in the BV2 microglial cells. However, PGS significantly inhibited the excessive production of NO, PGE2 and pro‑inflammatory cytokines, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner without causing any cytotoxic effects. In addition, PGS suppressed NF-κB translocation and inhibited the LPS-induced phosphorylation of AKT and MAPKs. Our results indicate that the inhibitory effect of PGS on LPS-stimulated inflammatory response in BV2 microglial cells is associated with the suppression of NF-κB activation and the PI3K/AKT and MAPK signaling pathways. Therefore, these findings suggest that PGS may be useful in the treatment of neurodegenerative diseases by inhibiting inflammatory responses in activated microglial cells. PMID:23563392

  6. Docosahexaenoic acid modulates inflammatory and antineurogenic functions of activated microglial cells.

    PubMed

    Antonietta Ajmone-Cat, Maria; Lavinia Salvatori, Maria; De Simone, Roberta; Mancini, Melissa; Biagioni, Stefano; Bernardo, Antonietta; Cacci, Emanuele; Minghetti, Luisa

    2012-03-01

    The complex process of microglial activation encompasses several functional activation states associated either with neurotoxic/antineurogenic or with neurotrophic/proneurogenic properties, depending mainly on the extent of activation and the nature of the activating stimuli. Several studies have demonstrated that acute exposure to the prototypical activating agent lipopolysaccharide (LPS) confers antineurogenic properties upon microglial cells. Acutely activated microglia ortheir conditioned media (CM) reduce neural stem progenitor cell (NPC) survival and prevent NPC differentiation into neurons. The present study tested the hypothesis that docosahexaenoic acid (DHA), a long-chain polyunsatured fatty acid (L-PUFA) with potent immunomodulatory properties, could dampen microglial proinflammatory functions and modulate their antineurogenic effect. We demonstrate that DHA dose dependently inhibits the synthesis of inflammatory products in activated microglia without inducing an alternative antiinflammatory phenotype. Among the possible DHA mechanisms of action, we propose the inhibition of p38 MAPK phosphorylation and the activation of the nuclear receptor peroxisome proliferator activated receptor (PPAR)-γ. The attenuation of M1 proinflammatory phenotype has relevant consequences for the survival and differentiation of NPC, because DHA reverses the antineurogenic activities of conditioned media from LPS-activated microglia. Our study identifies new relevant potentially protective and proneurogenic functions of DHA, exerted through the modulation of microglial functions, that could be exploited to sustain or promote neuroregenerative processes in damaged/aged brain. PMID:22057807

  7. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

    PubMed

    Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

    2016-06-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032. PMID:27054189

  8. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA)

    PubMed Central

    Narayan, Malathi; Seeley, Kent W.; Jinwal, Umesh K.

    2016-01-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032. PMID:27054189

  9. Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells.

    PubMed

    Taka, Equar; Mazzio, Elizabeth A; Goodman, Carl B; Redmon, Natalie; Flores-Rozas, Hernan; Reams, Renee; Darling-Reed, Selina; Soliman, Karam F A

    2015-09-15

    Thymoquinone (TQ), the main pharmacological active ingredient within the black cumin seed (Nigella sativa) is believed to be responsible for the therapeutic effects on chronic inflammatory conditions such as arthritis, asthma and neurodegeneration. In this study, we evaluated the potential anti-inflammatory role of TQ in lipopolysaccharide (LPS)-stimulated BV-2 murine microglia cells. The results obtained indicate that TQ was effective in reducing NO2(-) with an IC50 of 5.04μM, relative to selective iNOS inhibitor LNIL-l-N6-(1-iminoethyl)lysine (IC50 4.09μM). TQ mediated reduction in NO2(-) was found to parallel the decline of iNOS protein expression as confirmed by immunocytochemistry. In addition, we evaluated the anti-inflammatory effects of TQ on ninety-six (96) cytokines using a RayBio AAM-CYT-3 and 4 cytokine antibody protein array. Data obtained establish a baseline protein expression profile characteristic of resting BV-2 cells in the order of osteopontin>MIP-1alpha>MIP-1g>IGF-1 and MCP-I. In the presence of LPS [1ug/ml], activated BV-2 cells produced a sharp rise in specific pro-inflammatory cytokines/chemokine's IL-6, IL-12p40/70, CCL12 /MCP-5, CCL2/MCP-1, and G-CSF which were attenuated by the addition of TQ (10μM). The TQ mediated attenuation of MCP-5, MCP-1 and IL-6 protein in supernatants from activated BV-2 cells were corroborated by independent ELISA. Moreover, the data obtained from the RT(2) PCR demonstrated a similar pattern where the LPS mediated elevation of mRNA for IL-6, CCL12/MCP-5, CCL2/MCP-1 were significantly attenuated by TQ (10μM). Also, in this study, consistent data were obtained for both protein antibody array densitometry and ELISA assays. In addition, TQ was found to reduce LPS mediated elevation in gene expression of Cxcl10 and a number of other cytokines in the panel. These findings demonstrate the significant anti-inflammatory properties of TQ in LPS activated microglial cells. Therefore, the obtained results might indicate

  10. Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation

    PubMed Central

    Cloarec, Robin; Bauer, Sylvian; Luche, Hervé; Buhler, Emmanuelle; Pallesi-Pocachard, Emilie; Salmi, Manal; Courtens, Sandra; Massacrier, Annick; Grenot, Pierre; Teissier, Natacha; Watrin, Françoise; Schaller, Fabienne; Adle-Biassette, Homa; Gressens, Pierre; Malissen, Marie; Stamminger, Thomas; Streblow, Daniel N.; Bruneau, Nadine; Szepetowski, Pierre

    2016-01-01

    Background Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. Objectives and Methods In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. Results Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b– lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. Conclusion In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which

  11. Nitrated Alpha Synuclein Induced Alterations in Microglial Immunity is Regulated by CD4+ T Cell Subsets1

    PubMed Central

    Reynolds, Ashley D.; Stone, David K.; Mosley, R. Lee; Gendelman, Howard E.

    2009-01-01

    Microglial inflammatory neuroregulatory activities affect the tempo of nigrostriatal degeneration during Parkinson's disease (PD). Such activities are induced, in part, by misfolded, nitrated alpha-synuclein (N-α-syn) within Lewy bodies released from dying or dead dopaminergic neurons. Such pathobiologic events initiate innate and adaptive immune responses affecting neurodegeneration. We posit that the neurobiological activities of activated microglia are affected by cell-protein and cell-cell contacts, in that microglial interactions with N-α-syn and CD4+ T cells substantively alter the microglial proteome. This leads to alterations in cell homeostatic functions and disease. CD4+CD25+ regulatory T cells (Treg) suppress N-α-syn microglial induced reactive oxygen species and nuclear factor kappa B activation by modulating redox-active enzymes, cell migration, phagocytosis, and bioenergetic protein expression and cell function. In contrast, CD4+CD25− effector T cells exacerbate microglial inflammation and induce “putative” neurotoxic responses. These data support the importance of adaptive immunity in the regulation of PD-associated microglial inflammation. PMID:19299711

  12. Entry and distribution of microglial cells in human embryonic and fetal cerebral cortex.

    PubMed

    Monier, Anne; Adle-Biassette, Homa; Delezoide, Anne-Lise; Evrard, Philippe; Gressens, Pierre; Verney, Catherine

    2007-05-01

    Microglial cells penetrate into and scatter throughout the human cortical grey and white matter according to a specific spatiotemporal pattern during the first 2 trimesters of gestation. Routes of entry were quantitatively and qualitatively different from those identified in the diencephalon. Starting at 4.5 gestational weeks, amoeboid microglial cells, characterized by different antibodies as Iba1, CD68, CD45, and MHC-II, entered the cerebral wall from the ventricular lumen and the leptomeninges. Migration was mainly radial and tangential toward the immature white matter, subplate layer, and cortical plate, whereas pial cells populated the prospective layer I. The intraparenchymal vascular route of entry was detectable only from 12 gestational weeks. Interestingly, microglial cells accumulated in restricted laminar bands particularly at 19 to 24 gestational weeks among the corona radiata fibers rostrally, extending caudally in the immature white matter to reach the visual radiations. This accumulation of proliferating MIB1-positive microglia (as shown by MIB1-Iba1 double immunolabeling) was located at the site of white matter injury in premature neonates. The spatiotemporal organization of microglia in the immature white and grey matter suggests that these cells may play active roles in developmental processes and in injury to the developing brain. PMID:17483694

  13. Levo-Tetrahydropalmatine Attenuates Bone Cancer Pain by Inhibiting Microglial Cells Activation

    PubMed Central

    Zhang, Mao-yin; Liu, Yue-peng; Zhang, Lian-yi; Yue, Dong-mei; Qi, Dun-yi; Liu, Gong-jian; Liu, Su

    2015-01-01

    Objective. The present study is to investigate the analgesic roles of L-THP in rats with bone cancer pain caused by tumor cell implantation (TCI). Methods. Thermal hyperalgesia and mechanical allodynia were measured at different time points before and after operation. L-THP (20, 40, and 60 mg/kg) were administrated intragastrically at early phase of postoperation (before pain appearance) and later phase of postoperation (after pain appearance), respectively. The concentrations of TNF-α, IL-1β, and IL-18 in spinal cord were measured by enzyme-linked immunosorbent assay. Western blot was used to test the activation of astrocytes and microglial cells in spinal cord after TCI treatment. Results. TCI treatment induced significant thermal hyperalgesia and mechanical allodynia. Administration of L-THP at high doses significantly prevented and/or reversed bone cancer-related pain behaviors. Besides, TCI-induced activation of microglial cells and the increased levels of TNF-α and IL-18 were inhibited by L-THP administration. However, L-THP failed to affect TCI-induced astrocytes activation and IL-1β increase. Conclusion. This study suggests the possible clinical utility of L-THP in the treatment of bone cancer pain. The analgesic effects of L-THP on bone cancer pain maybe underlying the inhibition of microglial cells activation and proinflammatory cytokines increase. PMID:26819501

  14. c-Src function is necessary and sufficient for triggering microglial cell activation.

    PubMed

    Socodato, Renato; Portugal, Camila C; Domith, Ivan; Oliveira, Nádia A; Coreixas, Vivian S M; Loiola, Erick C; Martins, Tânia; Santiago, Ana Raquel; Paes-de-Carvalho, Roberto; Ambrósio, António F; Relvas, João B

    2015-03-01

    Microglial cells are the resident macrophages of the central nervous system. Their function is essential for neuronal tissue homeostasis. After inflammatory stimuli, microglial cells become activated changing from a resting and highly ramified cell shape to an amoeboid-like morphology. These morphological changes are associated with the release of proinflammatory cytokines and glutamate, as well as with high phagocytic activity. The acquisition of such phenotype has been associated with activation of cytoplasmic tyrosine kinases, including those of the Src family (SFKs). In this study, using both in vivo and in vitro inflammation models coupled to FRET-based time-lapse microscopy, lentiviruses-mediated shRNA delivery and genetic gain-of-function experiments, we demonstrate that among SFKs c-Src function is necessary and sufficient for triggering microglia proinflammatory signature, glutamate release, microglia-induced neuronal loss, and phagocytosis. c-Src inhibition in retinal neuroinflammation experimental paradigms consisting of intravitreal injection of LPS or ischemia-reperfusion injury significantly reduced microglia activation changing their morphology to a more resting phenotype and prevented neuronal apoptosis. Our data demonstrate an essential role for c-Src in microglial cell activation. PMID:25421817

  15. Anti-inflammatory effect of ginsenoside Rg5 in lipopolysaccharide-stimulated BV2 microglial cells.

    PubMed

    Lee, Yu Young; Park, Jin-Sun; Jung, Ji-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2013-01-01

    Microglia are resident immune cells in the central nervous system. They play a role in normal brain development and neuronal recovery. However, overactivation of microglia causes neuronal death, which is associated with neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Therefore, controlling microglial activation has been suggested as an important target for treatment of neurodegenerative diseases. In the present study, we investigated the anti-inflammatory effect of ginsenoside Rg5 in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. The data showed that Rg5 suppressed LPS-induced nitric oxide (NO) production and proinflammatory TNF-α secretion. In addition, Rg5 inhibited the mRNA expressions of iNOS, TNF-α, IL-1b, COX-2 and MMP-9 induced by LPS. Further mechanistic studies revealed that Rg5 inhibited the phophorylations of PI3K/Akt and MAPKs and the DNA binding activities of NF-kB and AP-1, which are upstream molecules controlling inflammatory reactions. Moreover, Rg5 suppressed ROS production with upregulation of hemeoxygenase-1 (HO-1) expression in LPS-stimulated BV2 cells. Overall, microglial inactivation by ginsenoside Rg5 may provide a therapeutic potential for various neuroinflammatory disorders. PMID:23698769

  16. Microglial Activation & Chronic Neurodegeneration

    PubMed Central

    Lull, Melinda E.; Block, Michelle L.

    2010-01-01

    Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurodegenerative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including TNFα, NO, IL1-β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (ex. LPS or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss over time. While the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson’s Disease. Here, we review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype. PMID:20880500

  17. Are Microglial Cells the Regulators of Lymphocyte Responses in the CNS?

    PubMed Central

    Almolda, Beatriz; González, Berta; Castellano, Bernardo

    2015-01-01

    The infiltration of immune cells in the central nervous system is a common hallmark in different neuroinflammatory conditions. Accumulating evidence indicates that resident glial cells can establish a cross-talk with infiltrated immune cells, including T-cells, regulating their recruitment, activation and function within the CNS. Although the healthy CNS has been thought to be devoid of professional dendritic cells (DCs), numerous studies have reported the presence of a population of DCs in specific locations such as the meninges, choroid plexuses and the perivascular space. Moreover, the infiltration of DC precursors during neuroinflammatory situations has been proposed, suggesting a putative role of these cells in the regulation of lymphocyte activity within the CNS. On the other hand, under specific circumstances, microglial cells are able to acquire a phenotype of DC expressing a wide range of molecules that equip these cells with all the necessary machinery for communication with T-cells. In this review, we summarize the current knowledge on the expression of molecules involved in the cross-talk with T-cells in both microglial cells and DCs and discuss the potential contribution of each of these cell populations on the control of lymphocyte function within the CNS. PMID:26635525

  18. Critical role of microglial CD40 in the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain.

    PubMed

    Cao, Ling; Palmer, Christopher D; Malon, Jennifer T; De Leo, Joyce A

    2009-12-01

    We recently demonstrated a contributing role of spinal cord infiltrating CD4+ T lymphocytes in the maintenance of mechanical hypersensitivity in a rodent model of neuropathic pain, spinal nerve L5 transection (L5Tx). It has been demonstrated that microglia play a role in the etiology of pain states. We hypothesized that infiltrating CD4+ T lymphocytes communicate with microglia via a CD40-CD154 interaction. Here, we investigated the role of CD40 in the development of mechanical hypersensitivity post-L5Tx. CD40 KO mice displayed significantly decreased mechanical sensitivity compared with WT mice starting from day 5 post-L5Tx. Using bone marrow chimeric mice, we further identified a pro-nociceptive role of CNS microglial CD40 rather than the peripheral leukocytic CD40. Flow cytometric analysis determined a significant increase of CD40+ microglia in the ipsilateral side of lumbar spinal cord post-L5Tx. Further, spinal cord proinflammatory cytokine (IL-1beta, IL-6, IL-12, and TNF-alpha) profiling demonstrated an induction of IL-6 in both WT and CD40 KO mice post-L5Tx prior to the increase of microglial CD40 expression, indicating a CD40-independent induction of IL-6 following L5Tx. These data establish a novel role of microglial CD40 in the maintenance of nerve injury-induced behavioral hypersensitivity, a behavioral sign of neuropathic pain. PMID:19750482

  19. Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

    PubMed

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

    2015-01-01

    Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions. PMID:25859655

  20. Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells.

    PubMed

    Sun, Grace Y; Li, Runting; Cui, Jiankun; Hannink, Mark; Gu, Zezong; Fritsche, Kevin L; Lubahn, Dennis B; Simonyi, Agnes

    2016-09-01

    Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8 h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways. PMID:27209361

  1. Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal Models of Parkinson's Disease

    PubMed Central

    Panicker, Nikhil; Saminathan, Hariharan; Jin, Huajun; Neal, Matthew; Harischandra, Dilshan S.; Gordon, Richard; Kanthasamy, Kavin; Lawana, Vivek; Sarkar, Souvarish; Luo, Jie; Anantharam, Vellareddy; Kanthasamy, Anumantha G.

    2015-01-01

    Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson's disease (PD), but the key molecular signaling events regulating persistent microglial activation have yet to be clearly defined. In the present study, we examined the role of Fyn, a non-receptor tyrosine kinase, in microglial activation and neuroinflammatory mechanisms in cell culture and animal models of PD. The well-characterized inflammogens LPS and TNFα rapidly activated Fyn kinase in microglia. Immunocytochemical studies revealed that activated Fyn preferentially localized to the microglial plasma membrane periphery and the nucleus. Furthermore, activated Fyn phosphorylated PKCδ at tyrosine residue 311, contributing to an inflammogen-induced increase in its kinase activity. Notably, the Fyn-PKCδ signaling axis further activated the LPS- and TNFα-induced MAP kinase phosphorylation and activation of the NFκB pathway, implying that Fyn is a major upstream regulator of proinflammatory signaling. Functional studies in microglia isolated from wild-type (Fyn+/+) and Fyn knock-out (Fyn−/−) mice revealed that Fyn is required for proinflammatory responses, including cytokine release as well as iNOS activation. Interestingly, a prolonged inflammatory insult induced Fyn transcript and protein expression, indicating that Fyn is upregulated during chronic inflammatory conditions. Importantly, in vivo studies using MPTP, LPS, or 6-OHDA models revealed a greater attenuation of neuroinflammatory responses in Fyn−/− and PKCδ −/− mice compared with wild-type mice. Collectively, our data demonstrate that Fyn is a major upstream signaling mediator of microglial neuroinflammatory processes in PD. SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a complex multifactorial disease characterized by the progressive loss of midbrain dopamine neurons. Sustained microglia-mediated neuroinflammation has

  2. Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6

    PubMed Central

    2014-01-01

    Microglia are the primary immunocompetent cells in brain tissue and microglia-mediated inflammation is associated with the pathogenesis of various neuronal disorders. Recently, many studies have shown that mesenchymal stem cells (MSCs) display a remarkable ability to modulate inflammatory and immune responses through the release of a variety of bioactive molecules, thereby protecting the central nervous system. Previously, we reported that MSCs have the ability to modulate inflammatory responses in a traumatic brain injury model and that the potential mechanisms may be partially attributed to upregulated TNF-α stimulated gene/protein 6 (TSG-6) expression. However, whether TSG-6 exerts an anti-inflammatory effect by affecting microglia is not fully understood. In this study, we investigated the anti-inflammatory effects of MSCs and TSG-6 in an in vitro lipopolysaccharide (LPS)-induced BV2 microglial activation model. We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia. However, MSC effects on microglia were attenuated when TSG-6 expression was silenced. In addition, we found that the activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6. Furthermore, we found that the presence of CD44 in BV2 microglial cells was essential for MSC- and TSG-6-mediated inhibition of pro-inflammatory gene expression and of NF-κB and MAPK activation in BV2 microglial cells. The results of this study suggest that MSCs can modulate microglia activation through TSG-6 and that TSG-6 attenuates the inflammatory cascade in activated microglia. Our study indicates that novel mechanisms are responsible for the immunomodulatory effect of MSCs on microglia and that MSCs, as well as TSG-6, might be promising therapeutic agents for the treatment of neurotraumatic injuries or neuroinflammatory diseases associated with

  3. The non-psychoactive plant cannabinoid, cannabidiol affects cholesterol metabolism-related genes in microglial cells.

    PubMed

    Rimmerman, Neta; Juknat, Ana; Kozela, Ewa; Levy, Rivka; Bradshaw, Heather B; Vogel, Zvi

    2011-08-01

    Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that is clinically used in a 1:1 mixture with the psychoactive cannabinoid Δ(9)-tetrahydrocannabinol (THC) for the treatment of neuropathic pain and spasticity in multiple sclerosis. Our group previously reported that CBD exerts anti-inflammatory effects on microglial cells. In addition, we found that CBD treatment increases the accumulation of the endocannabinoid N-arachidonoyl ethanolamine (AEA), thus enhancing endocannabinoid signaling. Here we proceeded to investigate the effects of CBD on the modulation of lipid-related genes in microglial cells. Cell viability was tested using FACS analysis, AEA levels were measured using LC/MS/MS, gene array analysis was validated with real-time qPCR, and cytokine release was measured using ELISA. We report that CBD significantly upregulated the mRNAs of the enzymes sterol-O-acyl transferase (Soat2), which synthesizes cholesteryl esters, and of sterol 27-hydroxylase (Cyp27a1). In addition, CBD increased the mRNA of the lipid droplet-associated protein, perilipin2 (Plin2). Moreover, we found that pretreatment of the cells with the cholesterol chelating agent, methyl-β-cyclodextrin (MBCD), reversed the CBD-induced increase in Soat2 mRNA but not in Plin2 mRNA. Incubation with AEA increased the level of Plin2, but not of Soat2 mRNA. Furthermore, MBCD treatment did not affect the reduction by CBD of the LPS-induced release of the proinflammatory cytokine IL-1β. CBD treatment modulates cholesterol homeostasis in microglial cells, and pretreatment with MBCD reverses this effect without interfering with CBD's anti-inflammatory effects. The effects of the CBD-induced increase in AEA accumulation on lipid-gene expression are discussed. PMID:21533611

  4. Characterization of putative Japanese encephalitis virus receptor molecules on microglial cells.

    PubMed

    Thongtan, Thananya; Wikan, Nitwara; Wintachai, Phitchayapak; Rattanarungsan, Chutima; Srisomsap, Chantragan; Cheepsunthorn, Poonlarp; Smith, Duncan R

    2012-04-01

    Japanese encephalitis virus (JEV) a mosquito-borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein-binding assay (VOPBA) and liquid chromatography-mass spectrometry (LC/MS/MS) identified the 37/67 kDa high-affinity laminin receptor protein and nucleolin as a potential JEV-binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV-2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti-laminin receptor and anti-CD4 antibodies significantly reduced virus entry while anti-Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down-regulation of CD4 and moderate down-regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role. PMID:22337301

  5. Delayed activation of human microglial cells by high dose ionizing radiation.

    PubMed

    Chen, Hongxin; Chong, Zhao Zhong; De Toledo, Sonia M; Azzam, Edouard I; Elkabes, Stella; Souayah, Nizar

    2016-09-01

    Recent studies have shown that microglia affects the fate of neural stem cells in response to ionizing radiation, which suggests a role for microglia in radiation-induced degenerative outcomes. We therefore investigated the effects of γ-irradiation on cell survival, proliferation, and activation of microglia and explored associated mechanisms. Specifically, we evaluated cellular and molecular changes associated with exposure of human microglial cells (CHME5) to low and high doses of acute cesium-137 γ rays. Twenty-four hours after irradiation, cell cycle analyses revealed dose-dependent decreases in the fraction of cells in S and G2/M phase, which correlated with significant oxidative stress. By one week after irradiation, 20-30% of the cells exposed to high doses of γ rays underwent apoptosis, which correlated with significant concomitant decrease in metabolic activity as assessed by the MTT assay, and microglial activation as judged by both morphological changes and increased expression of Glut-5 and CR43. These changes were associated with increases in the mRNA levels for IL-1α, IL-10 and TNFα. Together, the results show that human CHME5 microglia are relatively resistant to low and moderate doses of γ rays, but are sensitive to acute high doses, and that CHME5 cells are a useful tool for in vitro study of human microglia. PMID:27265419

  6. Effects of aspirin on expression of iron transport and storage proteins in BV-2 microglial cells.

    PubMed

    Xu, Yan Xin; Du, Fang; Jiang, Li Rong; Gong, Jing; Zhou, Yu-Fu; Luo, Qian Qian; Qian, Zhong Ming; Ke, Ya

    2015-12-01

    In the light of recent studies, we hypothesized that aspirin might have the functions to regulate the expression of iron transport proteins and then affect cellular iron levels. To test this hypothesis, we investigated the effects of aspirin on expression of iron uptake protein transferrin receptor 1 (TfR1), iron release protein ferroportin 1 (Fpn1) and iron storage protein ferritin using Western blot analysis and on tumor necrosis factor (TNF)-αlpha, interleukin (IL)-6, interleukin (IL)-10 and hepcidin using quantitative real-time PCR in BV-2 microglial cells treated with lipopolysaccharides (LPS). We found that aspirin significantly down-regulated TfR1, while also up-regulated Fpn1 and ferritin expressions in BV-2 microglial cells in vitro. We also showed that TfR1 and Fpn1 expressions were significantly higher, while ferritin contents, IL-6, TNF-alpha and hepcidin mRNA levels were lower in cells treated with aspirin plus LPS than those in cells treated with LPS only. We concluded that aspirin has a negative effect on cell iron contents under 'normal' conditions and could partly reverse LPS-induced-disruption in cell iron balance under in vitro inflammatory conditions. Our findings also suggested that hepcidin might play a dominant role in the control of TfR1 expression by aspirin in the cells treated with LPS. PMID:26522688

  7. Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

    PubMed Central

    2010-01-01

    Background Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state. Results Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists. Conclusions The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with

  8. Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

    PubMed Central

    Lee, Jee Youn

    2014-01-01

    Background Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis. Methods Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed. Results Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells. Conclusion Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone. PMID:25309797

  9. Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells

    PubMed Central

    Guo, Jia-Zhi; Zhang, Wei; He, Ying; Song, Rui; Wang, Wen-Min; Xiao, Chun-Jie; Lu, Di

    2012-01-01

    Background Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells. Methodology/Principal Findings BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). Conclusion and Implications This study indicates that resveratrol inhibited LPS-induced proinflammatory

  10. [The immunomodulatory role of retinal microglial cells in age-related macular degeneration].

    PubMed

    Zhang, P F; Sun, X D

    2016-05-11

    Age-related macular degeneration (AMD) is one of the major causes of visual impairment in the elder population. Recent studies have revealed that retinal microgliacytes may play an important role in the pathogenesis of AMD, and the activation of retinal microglia could regulate the progress of AMD. The immunomodulatory role of retinal microglial cells is reviewed in this article, so as to investigate the mechanism and provide new insight for prevention and treatment of AMD.(Chin J Ophthalmol, 2016, 52: 386-390). PMID:27220713

  11. Anti-inflammatory effects and antioxidant activity of dihydroasparagusic acid in lipopolysaccharide-activated microglial cells.

    PubMed

    Salemme, Adele; Togna, Anna Rita; Mastrofrancesco, Arianna; Cammisotto, Vittoria; Ottaviani, Monica; Bianco, Armandodoriano; Venditti, Alessandro

    2016-01-01

    The activation of microglia and subsequent release of toxic pro-inflammatory factors are crucially associated with neurodegenerative disease, characterized by increased oxidative stress and neuroinflammation, including Alzheimer and Parkinson diseases and multiple sclerosis. Dihydroasparagusic acid is the reduced form of asparagusic acid, a sulfur-containing flavor component produced by Asparagus plants. It has two thiolic functions able to coordinate the metal ions, and a carboxylic moiety, a polar function, which may enhance excretion of the complexes. Thiol functions are also present in several biomolecules with important physiological antioxidant role as glutathione. The aim of this study is to evaluate the anti-inflammatory and antioxidant potential effect of dihydroasparagusic acid on microglial activation in an in vitro model of neuroinflammation. We have used lipopolysaccharide to induce an inflammatory response in primary rat microglial cultures. Our results suggest that dihydroasparagusic acid significantly prevented lipopolysaccharide-induced production of pro-inflammatory and neurotoxic mediators such as nitric oxide, tumor necrosis factor-α, prostaglandin E2, as well as inducible nitric oxide synthase and cyclooxygenase-2 protein expression and lipoxygenase activity in microglia cells. Moreover it effectively suppressed the level of reactive oxygen species and affected lipopolysaccharide-stimulated activation of mitogen activated protein kinase, including p38, and nuclear factor-kB pathway. These results suggest that dihydroasparagusic acid's neuroprotective properties may be due to its ability to dampen induction of microglial activation. It is a compound that can effectively inhibit inflammatory and oxidative processes that are important factors of the etiopathogenesis of neurodegenerative diseases. PMID:26592472

  12. Effects of chronic low dose rotenone treatment on human microglial cells

    PubMed Central

    2009-01-01

    Background Exposure to toxins/chemicals is considered to be a significant risk factor in the pathogenesis of Parkinson's disease (PD); one putative chemical is the naturally occurring herbicide rotenone that is now used widely in establishing PD models. We, and others, have shown that chronic low dose rotenone treatment induces excessive accumulation of Reactive Oxygen Species (ROS), inclusion body formation and apoptosis in dopaminergic neurons of animal and human origin. Some studies have also suggested that microglia enhance the rotenone induced neurotoxicity. While the effects of rotenone on neurons are well established, there is little or no information available on the effect of rotenone on microglial cells, and especially cells of human origin. The aim of the present study was to investigate the effects of chronic low dose rotenone treatment on human microglial CHME-5 cells. Methods We have shown previously that rotenone induced inclusion body formation in human dopaminergic SH-SY5Y cells and therefore used these cells as a control for inclusion body formation in this study. SH-SY5Y and CHME-5 cells were treated with 5 nM rotenone for four weeks. At the end of week 4, both cell types were analysed for the presence of inclusion bodies, superoxide dismutases and cell activation (only in CHME-5 cells) using Haematoxylin and Eosin staining, immunocytochemical and western blotting methods. Levels of active caspases and ROS (both extra and intra cellular) were measured using biochemical methods. Conclusion The results suggest that chronic low dose rotenone treatment activates human microglia (cell line) in a manner similar to microglia of animal origin as shown by others. However human microglia release excessive amounts of ROS extracellularly, do not show excessive amounts of intracellular ROS and active caspases and most importantly do not show any protein aggregation or inclusion body formation. Human microglia appear to be resistant to rotenone (chronic, low

  13. Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells

    PubMed Central

    Kim, Dong-Cheol; Yoon, Chi-Su; Quang, Tran Hong; Ko, Wonmin; Kim, Jong-Su; Oh, Hyuncheol; Kim, Youn-Chul

    2016-01-01

    In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE2 in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE2 in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation. PMID:26907256

  14. Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells.

    PubMed

    Kim, Dong-Cheol; Yoon, Chi-Su; Quang, Tran Hong; Ko, Wonmin; Kim, Jong-Su; Oh, Hyuncheol; Kim, Youn-Chul

    2016-01-01

    In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE₂ in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE₂ in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation. PMID:26907256

  15. Regulation of Macrophage, Dendritic Cell, and Microglial Phenotype and Function by the SOCS Proteins

    PubMed Central

    McCormick, Sarah M.; Heller, Nicola M.

    2015-01-01

    Macrophages are innate immune cells of dynamic phenotype that rapidly respond to external stimuli in the microenvironment by altering their phenotype to respond to and to direct the immune response. The ability to dynamically change phenotype must be carefully regulated to prevent uncontrolled inflammatory responses and subsequently to promote resolution of inflammation. The suppressor of cytokine signaling (SOCS) proteins play a key role in regulating macrophage phenotype. In this review, we summarize research to date from mouse and human studies on the role of the SOCS proteins in determining the phenotype and function of macrophages. We will also touch on the influence of the SOCS on dendritic cell (DC) and microglial phenotype and function. The molecular mechanisms of SOCS function in macrophages and DCs are discussed, along with how dysregulation of SOCS expression or function can lead to alterations in macrophage/DC/microglial phenotype and function and to disease. Regulation of SOCS expression by microRNA is discussed. Novel therapies and unanswered questions with regard to SOCS regulation of monocyte–macrophage phenotype and function are highlighted. PMID:26579124

  16. Anti-inflammatory effects of cordycepin via suppression of inflammatory mediators in BV2 microglial cells.

    PubMed

    Jeong, Jin-Woo; Jin, Cheng-Yun; Kim, Gi-Young; Lee, Jae-Dong; Park, Cheol; Kim, Gun-Do; Kim, Wun-Jae; Jung, Won-Kyo; Seo, Su Kil; Choi, Il-Whan; Choi, Yung Hyun

    2010-12-01

    Cordyceps militaris, a traditional medicinal mushroom, produces the bioactive compound cordycepin (3'-deoxyadenosine). Although cordycepin has been shown to have pharmacological, immunological stimulating, anti-cancer, and anti-inflammatory activities, its activities and cellular mechanisms during microglial activation have yet to be elucidated. Thus, we evaluated the anti-inflammatory effect of cordycepin on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We also investigated the effects of cordycepin on LPS-induced nuclear factor-kappaB (NF-κB) activation and on phosphorylation of mitogen-activated protein kinases (MAPKs). After LPS stimulation, nitric oxide (NO), prostaglandin E₂ (PGE₂), and pro-inflammatory cytokine production was detected in BV2 microglia. However, we found that cordycepin significantly inhibited the excessive production of NO, PGE₂, and pro-inflammatory cytokines in a concentration-dependent manner without causing cytotoxicity. In addition, cordycepin suppressed NF-κB translocation by blocking IkappaB-α (IκB-α) degradation and inhibited the phosphorylation of Akt, ERK-1/2, JNK, and p38 kinase. Our results indicate that the inhibitory effect of cordycepin on LPS-stimulated inflammatory mediator production in BV2 microglia is associated with the suppression of the NF-κB, Akt, and MAPK signaling pathways. Therefore, cordycepin may be useful in treating neurodegenerative diseases by inhibiting inflammatory mediator production in activated microglia. PMID:20937401

  17. In vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma

    PubMed Central

    Bosco, Alejandra; Romero, Cesar R; Ambati, Balamurali K; Vetter, Monica L

    2015-01-01

    SHORT ABSTRACT Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma. LONG ABSTRACT Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders. This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1-GFPGFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200–300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in

  18. Anti-neuro-inflammatory effects of Nardostachys chinensis in lipopolysaccharide-and lipoteichoic acid-stimulated microglial cells.

    PubMed

    Park, Sun Young; Kim, Young Hun; Park, Geuntae

    2016-05-01

    Excessive microglial cell activation is related to the progression of chronic neuro-inflammatory disorders. Heme oxygenase-1 (HO-1) expression mediated by the NFE2-related factor (Nrf-2) pathway is a key regulator of neuro-inflammation. Nardostachys chinensis is used as an anti-malarial, anti-nociceptive, and neurotrophic treatment in traditional Asian medicines. In the present study, we examined the effects of an ethyl acetate extract of N. chinensis (EN) on the anti-neuro-inflammatory effects mediated by HO-1 up-regulation in Salmonella lipopolysaccharide (LPS)- or Staphylococcus aureus lipoteichoic acid (LTA)-stimulated BV2 microglial cells. Our results indicated that EN suppressed pro-inflammatory cytokine production and induced HO-1 transcription and translation through Nrf-2/antioxidant response element (ARE) signaling. EN markedly inhibited LPS- and LTA-induced activation of nuclear factor-kappa B (NF-κB) as well as phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT). Furthermore, EN protected hippocampal HT22 cells from indirect neuronal toxicity mediated by LPS- and LTA-treated microglial cells. These results suggested that EN impairs LPS- and LTA-induced neuro-inflammatory responses in microglial cells and confers protection against indirect neuronal damage to HT22 cells. In conclusion, our findings indicate that EN could be used as a natural anti-neuro-inflammatory and neuroprotective agent. PMID:27478097

  19. Diversity and plasticity of microglial cells in psychiatric and neurological disorders.

    PubMed

    Nakagawa, Yutaka; Chiba, Kenji

    2015-10-01

    Recent advanced immunological analyses have revealed that the diversity and plasticity of macrophages lead to the identification of functional polarization states (classically activated M1 type and alternatively activated M2 type) which are dependent on the extracellular environment. M1 and M2 polarization states of macrophages play an important role in controlling the balance between pro-inflammatory and anti-inflammatory conditions. Microglial cells are resident mononuclear phagocytes in the central nervous system (CNS), express several macrophage-associated markers, and appear to display functional polarization states similar to macrophages. Like M1 macrophages, M1 polarized microglia can produce pro-inflammatory cytokines and mediators such as interleukin (IL) 1β, IL-6, tumor necrosis factor-α, CC-chemokine ligand 2, nitric oxide, and reactive oxygen species, suggesting that these molecules contribute to dysfunction of neural network in the CNS. On the other hand, M2 polarized microglia can produce anti-inflammatory cytokine, IL-10 and express several receptors that are implicated in inhibiting inflammation and restoring homeostasis. In this review, we summarize the diversity, plasticity, and immunoregulatory functions of M1 and M2 microglia in psychiatric and neurological disorders. Based on these aspects, we propose a contribution of imbalance between M1 and M2 polarization of microglia in bipolar disorder, obesity, amyotrophic lateral sclerosis, and Rett syndrome. Consequently, molecules that normalize the imbalance between M1 and M2 microglial polarization states may provide a beneficial therapeutic target for the treatment of these disorders. PMID:26129625

  20. Regulatory Effects of Caffeic Acid Phenethyl Ester on Neuroinflammation in Microglial Cells

    PubMed Central

    Tsai, Cheng-Fang; Kuo, Yueh-Hsiung; Yeh, Wei-Lan; Wu, Caren Yu-Ju; Lin, Hsiao-Yun; Lai, Sheng-Wei; Liu, Yu-Shu; Wu, Ling-Hsuan; Lu, Jheng-Kun; Lu, Dah-Yuu

    2015-01-01

    Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells. PMID:25768341

  1. Regulatory effects of caffeic acid phenethyl ester on neuroinflammation in microglial cells.

    PubMed

    Tsai, Cheng-Fang; Kuo, Yueh-Hsiung; Yeh, Wei-Lan; Wu, Caren Yu-Ju; Lin, Hsiao-Yun; Lai, Sheng-Wei; Liu, Yu-Shu; Wu, Ling-Hsuan; Lu, Jheng-Kun; Lu, Dah-Yuu

    2015-01-01

    Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells. PMID:25768341

  2. Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells

    PubMed Central

    Kiebala, Michelle; Maggirwar, Sanjay B.

    2011-01-01

    Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorders (HAND) occur, in part, due to the inflammatory response to viral proteins, such as the HIV-1 transactivator of transcription (Tat), in the central nervous system (CNS). Given the need for novel adjunctive therapies for HAND, we hypothesized that ibudilast would inhibit Tat-induced excess production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα) in microglial cells. Ibudilast is a non-selective cyclic AMP phosphodiesterase inhibitor that has recently shown promise as a treatment for neuropathic pain via its ability to attenuate glial cell activation. Accordingly, here we demonstrate that pre-treatment of both human and mouse microglial cells with increasing doses of ibudilast inhibited Tat-induced synthesis of TNFα by microglial cells in a manner dependent on serine/threonine protein phosphatase activity. Ibudilast had no effect on Tat-induced p38 MAP kinase activation, and blockade of adenosine A2A receptor activation did not reverse ibudilast's inhibition of Tat-induced TNFα production. Interestingly, ibudilast reduced Tat-mediated transcription of TNFα, via modulation of nuclear factor-kappa B (NF-κB) signaling, as shown by transcriptional activity of NF-κB and analysis of inhibitor of kappa B alpha (IκBα) stability. Together, our findings shed light on the mechanism of ibudilast's inhibition of Tat-induced TNFα production in microglial cells and may implicate ibudilast as a potential novel adjunctive therapy for the management of HAND. PMID:21494611

  3. Prostaglandin signaling suppresses beneficial microglial function in Alzheimer's disease models.

    PubMed

    Johansson, Jenny U; Woodling, Nathaniel S; Wang, Qian; Panchal, Maharshi; Liang, Xibin; Trueba-Saiz, Angel; Brown, Holden D; Mhatre, Siddhita D; Loui, Taylor; Andreasson, Katrin I

    2015-01-01

    Microglia, the innate immune cells of the CNS, perform critical inflammatory and noninflammatory functions that maintain normal neural function. For example, microglia clear misfolded proteins, elaborate trophic factors, and regulate and terminate toxic inflammation. In Alzheimer's disease (AD), however, beneficial microglial functions become impaired, accelerating synaptic and neuronal loss. Better understanding of the molecular mechanisms that contribute to microglial dysfunction is an important objective for identifying potential strategies to delay progression to AD. The inflammatory cyclooxygenase/prostaglandin E2 (COX/PGE2) pathway has been implicated in preclinical AD development, both in human epidemiology studies and in transgenic rodent models of AD. Here, we evaluated murine models that recapitulate microglial responses to Aβ peptides and determined that microglia-specific deletion of the gene encoding the PGE2 receptor EP2 restores microglial chemotaxis and Aβ clearance, suppresses toxic inflammation, increases cytoprotective insulin-like growth factor 1 (IGF1) signaling, and prevents synaptic injury and memory deficits. Our findings indicate that EP2 signaling suppresses beneficial microglia functions that falter during AD development and suggest that inhibition of the COX/PGE2/EP2 immune pathway has potential as a strategy to restore healthy microglial function and prevent progression to AD. PMID:25485684

  4. Quercetin Attenuates Inflammatory Responses in BV-2 Microglial Cells: Role of MAPKs on the Nrf2 Pathway and Induction of Heme Oxygenase-1

    PubMed Central

    Sun, Grace Y.; Chen, Zihong; Jasmer, Kimberly J.; Chuang, Dennis Y.; Gu, Zezong; Hannink, Mark; Simonyi, Agnes

    2015-01-01

    A large group of flavonoids found in fruits and vegetables have been suggested to elicit health benefits due mainly to their anti-oxidative and anti-inflammatory properties. Recent studies with immune cells have demonstrated inhibition of these inflammatory responses through down-regulation of the pro-inflammatory pathway involving NF-κB and up-regulation of the anti-oxidative pathway involving Nrf2. In the present study, the murine BV-2 microglial cells were used to compare anti-inflammatory activity of quercetin and cyanidin, two flavonoids differing by their alpha, beta keto carbonyl group. Quercetin was 10 folds more potent than cyanidin in inhibition of lipopolysaccharide (LPS)-induced NO production as well as stimulation of Nrf2-induced heme-oxygenase-1 (HO-1) protein expression. In addition, quercetin demonstrated enhanced ability to stimulate HO-1 protein expression when cells were treated with LPS. In an attempt to unveil mechanism(s) for quercetin to enhance Nrf2/HO-1 activity under endotoxic stress, results pointed to an increase in phospho-p38MAPK expression upon addition of quercetin to LPS. In addition, pharmacological inhibitors for phospho-p38MAPK and MEK1/2 for ERK1/2 further showed that these MAPKs target different sites of the Nrf2 pathway that regulates HO-1 expression. However, inhibition of LPS-induced NO by quercetin was not fully reversed by TinPPIX, a specific inhibitor for HO-1 activity. Taken together, results suggest an important role of quercetin to regulate inflammatory responses in microglial cells and its ability to upregulate HO-1 against endotoxic stress through involvement of MAPKs. PMID:26505893

  5. Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia

    PubMed Central

    2013-01-01

    Background Brain-derived neurotrophic factor (BDNF) has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs) control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation. Methods Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL) in the absence or in the presence of the A2AR antagonist, SCH58261 (50 nM), as well as other modulators of A2AR signaling. The BDNF cellular content and secretion were quantified by Western blotting and ELISA, A2AR density was probed by Western blotting and immunocytochemistry and cell proliferation was assessed by BrdU incorporation. Additionally, the A2AR modulation of LPS-driven cell proliferation was also tested in primary cultures of mouse microglia. Results LPS induced time-dependent changes of the intra- and extracellular levels of BDNF and increased microglial proliferation. The maximal LPS-induced BDNF release was time-coincident with an LPS-induced increase of the A2AR density. Notably, removing endogenous extracellular adenosine or blocking A2AR prevented the LPS-mediated increase of both BDNF secretion and proliferation, as well as exogenous BDNF-induced proliferation. Conclusions We conclude that A2AR activation plays a mandatory role controlling the release of BDNF from activated microglia, as well as the autocrine/paracrine proliferative role of BDNF. PMID:23363775

  6. Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

    PubMed

    Komm, Barbara; Beyreis, Marlena; Kittl, Michael; Jakab, Martin; Ritter, Markus; Kerschbaum, Hubert H

    2014-08-01

    Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs. PMID:24760586

  7. Myeloid/Microglial Driven Autologous Hematopoietic Stem Cell Gene Therapy Corrects a Neuronopathic Lysosomal Disease

    PubMed Central

    Sergijenko, Ana; Langford-Smith, Alexander; Liao, Ai Y; Pickford, Claire E; McDermott, John; Nowinski, Gabriel; Langford-Smith, Kia J; Merry, Catherine LR; Jones, Simon A; Wraith, J Edmond; Wynn, Robert F; Wilkinson, Fiona L; Bigger, Brian W

    2013-01-01

    Mucopolysaccharidosis type IIIA (MPSIIIA) is a lysosomal storage disorder caused by mutations in N-sulfoglucosamine sulfohydrolase (SGSH), resulting in heparan sulfate (HS) accumulation and progressive neurodegeneration. There are no treatments. We previously demonstrated improved neuropathology in MPSIIIA mice using lentiviral vectors (LVs) overexpressing SGSH in wild-type (WT) hematopoietic stem cell (HSC) transplants (HSCTs), achieved via donor monocyte/microglial engraftment in the brain. However, neurological disease was not corrected using LVs in autologous MPSIIIA HSCTs. To improve brain expression via monocyte/microglial specificity, LVs expressing enhanced green fluorescent protein (eGFP) under ubiquitous phosphoglycerate kinase (PGK) or myeloid-specific promoters were compared in transplanted HSCs. LV-CD11b-GFP gave significantly higher monocyte/B-cell eGFP expression than LV-PGK-GFP or LV-CD18-GFP after 6 months. Subsequently, autologous MPSIIIA HSCs were transduced with either LV-PGK-coSGSH or LV-CD11b-coSGSH vectors expressing codon-optimized SGSH and transplanted into MPSIIIA mice. Eight months after HSCT, LV-PGK-coSGSH vectors produced bone marrow SGSH (576% normal activity) similar to LV-CD11b-coSGSH (473%), but LV-CD11b-coSGSH had significantly higher brain expression (11 versus 7%), demonstrating improved brain specificity. LV-CD11b-coSGSH normalized MPSIIIA behavior, brain HS, GM2 ganglioside, and neuroinflammation to WT levels, whereas LV-PGK-coSGSH partly corrected neuropathology but not behavior. We demonstrate compelling evidence of neurological disease correction using autologous myeloid driven lentiviral-HSC gene therapy in MPSIIIA mice. PMID:23748415

  8. Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.

    PubMed

    Ricard, Clément; Tchoghandjian, Aurélie; Luche, Hervé; Grenot, Pierre; Figarella-Branger, Dominique; Rougon, Geneviève; Malissen, Marie; Debarbieux, Franck

    2016-01-01

    Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP(+) cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP(+) cells. Fluorescent invading cells were conventional XCR1(+) and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP(+)/EYFP(+) monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response. PMID:27193333

  9. Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice

    PubMed Central

    Ricard, Clément; Tchoghandjian, Aurélie; Luche, Hervé; Grenot, Pierre; Figarella-Branger, Dominique; Rougon, Geneviève; Malissen, Marie; Debarbieux, Franck

    2016-01-01

    Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP+ cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP+ cells. Fluorescent invading cells were conventional XCR1+ and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP+/EYFP+ monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response. PMID:27193333

  10. Molecular mechanisms of microglial activation.

    PubMed

    Zielasek, J; Hartung, H P

    1996-01-01

    Microglial cells are brain macrophages which serve specific functions in the defense of the central nervous system (CNS) against microorganisms, the removal of tissue debris in neurodegenerative diseases or during normal development, and in autoimmune inflammatory disorders of the brain. In cultured microglial cells, several soluble inflammatory mediators such as cytokines and bacterial products like lipopolysaccharide (LPS) were demonstrated to induce a wide range of microglial activities, e.g. increased phagocytosis, chemotaxis, secretion of cytokines, activation of the respiratory burst and induction of nitric oxide synthase. Since heightened microglial activation was shown to play a role in the pathogenesis of experimental inflammatory CNS disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies for neurodegenerative disorders, multiple sclerosis and bacterial or viral infections of the nervous system. PMID:8876774

  11. Dual RNA Sequencing Reveals the Expression of Unique Transcriptomic Signatures in Lipopolysaccharide-Induced BV-2 Microglial Cells

    PubMed Central

    Kim, Sun Hwa; Park, Kyoung Sun; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2015-01-01

    Microglial cells become rapidly activated through interactions with pathogens, and the persistent activation of these cells is associated with various neurodegenerative diseases. Previous studies have investigated the transcriptomic signatures in microglia or macrophages using microarray technologies. However, this method has numerous restrictions, such as spatial biases, uneven probe properties, low sensitivity, and dependency on the probes spotted. To overcome this limitation and identify novel transcribed genes in response to LPS, we used RNA Sequencing (RNA-Seq) to determine the novel transcriptomic signatures in BV-2 microglial cells. Sequencing assessment and quality evaluation showed that approximately 263 and 319 genes (≥ 1.5 log2-fold), such as cytokines and chemokines, were strongly induced after 2 and 4 h, respectively, and the induction of several genes with unknown immunological functions was also observed. Importantly, we observed that previously unidentified transcription factors (TFs) (irf1, irf7, and irf9), histone demethylases (kdm4a) and DNA methyltransferases (dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. The gene expression levels, transcription start sites (TSS), isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with LPS. In addition, gene ontology, molecular networks and pathway analyses identified the top significantly regulated functional classification, canonical pathways and network functions at each activation status. Moreover, we further analyzed differentially expressed genes to identify transcription factor (TF) motifs (−950 to +50 bp of the 5’ upstream promoters) and epigenetic mechanisms. Furthermore, we confirmed that the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This

  12. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis

    PubMed Central

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  13. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis.

    PubMed

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  14. In situ Synthesis of Fluorescent Gold Nanoclusters by Nontumorigenic Microglial Cells.

    PubMed

    West, Abby L; Schaeublin, Nicole M; Griep, Mark H; Maurer-Gardner, Elizabeth I; Cole, Daniel P; Fakner, Alexis M; Hussain, Saber M; Karna, Shashi P

    2016-08-24

    To date, the directed in situ synthesis of fluorescent gold nanoclusters (AuNCs) has only been demonstrated in cancerous cells, with the theorized synthesis mechanism prohibiting AuNC formation in nontumorigenic cell lines. This limitation hinders potential biostabilized AuNC-based technology in healthy cells involving both chemical and mechanical analysis, such as the direct sensing of protein function and the elucidation of local mechanical environments. Thus, new synthesis strategies are required to expand the application space of AuNCs beyond cancer-focused cellular studies. In this contribution, we have developed the methodology and demonstrated the direct in situ synthesis of AuNCs in the nontumorigenic neuronal microglial line, C8B4. The as-synthesized AuNCs form in situ and are stabilized by cellular proteins. The clusters exhibit bright green fluorescence and demonstrate low (<10%) toxicity. Interestingly, elevated ROS levels were not required for the in situ formation of AuNCs, although intracellular reductants such as glutamate were required for the synthesis of AuNCs in C8B4 cells. To our knowledge, this is the first-ever demonstration of AuNC synthesis in nontumorigenic cells and, as such, it considerably expands the application space of biostabilized fluorescent AuNCs. PMID:27328035

  15. TREM2 regulates microglial cell activation in response to demyelination in vivo

    PubMed Central

    Cantoni, Claudia; Bollman, Bryan; Licastro, Danilo; Xie, Mingqiang; Mikesell, Robert; Schmidt, Robert; Yuede, Carla M.; Galimberti, Daniela; Olivecrona, Gunilla; Klein, Robyn S.; Cross, Anne H.; Otero, Karel; Piccio, Laura

    2015-01-01

    Microglia are phagocytic cells that survey the brain and perform neuroprotective functions in response to tissue damage, but their activating receptors are largely unknown. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial immunoreceptor whose loss-of-function mutations in humans cause presenile dementia, while genetic variants are associated with increased risk of neurodegenerative diseases. In myeloid cells, TREM2 has been involved in the regulation of phagocytosis, cell proliferation and inflammatory responses in vitro. However, it is unknown how TREM2 contributes to microglia function in vivo. Here, we identify a critical role for TREM2 in the activation and function of microglia during cuprizone (CPZ)-induced demyelination. TREM2-deficient (TREM2−/−) mice had defective clearance of myelin debris and more axonal pathology, resulting in impaired clinical performances compared to wild-type (WT) mice. TREM2−/− microglia proliferated less in areas of demyelination and were less activated, displaying a more resting morphology and decreased expression of the activation markers MHC II and inducible nitric oxide synthase as compared to WT. Mechanistically, gene expression and ultrastructural analysis of microglia suggested a defect in myelin degradation and phagosome processing during CPZ intoxication in TREM2−/− microglia. These findings place TREM2 as a key regulator of microglia activation in vivo in response to tissue damage. PMID:25631124

  16. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells.

    PubMed

    Kambhampati, Siva P; Mishra, Manoj K; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A; Kannan, Rangaramanujam M

    2015-09-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (∼ 21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ∼ 100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  17. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

    PubMed Central

    Kambhampati, Siva P.; Mishra, Manoj K.; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A.; Kannan, Rangaramanujam M.

    2016-01-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (~21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ~100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  18. NG2 expression in microglial cells affects the expression of neurotrophic and proinflammatory factors by regulating FAK phosphorylation

    PubMed Central

    Zhu, Lie; Su, Qing; Jie, Xiang; Liu, Antang; Wang, Hui; He, Beiping; Jiang, Hua

    2016-01-01

    Neural/glial antigen 2 (NG2), a chondroitin sulfate proteoglycan, is significantly upregulated in a subset of glial cells in the facial motor nucleus (FMN) following CNS injury. NG2 is reported to promote the resulting inflammatory reaction, however, the mechanism by which NG2 mediates these effects is yet to be determined. In this study, we examined the changes in NG2 expressing microglial cells in the FMN in response to facial nerve axotomy (FNA) in mice. Our findings indicated that NG2 expression was progressively induced and upregulated specifically in the ipsilateral facial nucleus following FNA. To further investigate the effects of NG2 expression, in vivo studies in NG2-knockout mice and in vitro studies in rat microglial cells transfected with NG2 shRNAs were performed. Abolition of NG2 expression both in vitro and in vivo resulted in increased expression of neurotrophic factors (nerve growth factor and glial derived neurotrophic factor), decreased expression of inflammatory mediators (tumor necrosis factor-α and interleukin-1β) and decreased apoptosis in the ipsilateral facial nucleus in response to FNA. Furthermore, we demonstrated the role of FAK in these NG2-induced effects. Taken together, our findings suggest that NG2 expression mediates inflammatory reactions and neurodegeneration in microglial cells in response to CNS injury, potentially by regulating FAK phosphorylation. PMID:27306838

  19. SENP1 inhibits the IH-induced apoptosis and nitric oxide production in BV2 microglial cells.

    PubMed

    Liu, Song; Wang, Zhong-hua; Xu, Bo; Chen, Kui; Sun, Jin-yuan; Ren, Lian-ping

    2015-11-27

    To reveal SUMOylation and the roles of Sentrin-specific proteases (SENP)s in microglial cells under Intermittent hypoxia (IH) condition would provide more intensive view of understanding the mechanisms of IH-induced central nervous system (CNS) damage. Hence, in the present study, we detected the expression levels of SENPs in microglial cells under IH and normoxia conditions via RT-PCR assay. We found that SENP1 was significantly down-regulated in cells exposure to IH. Subsequently, the effect of IH for the activation of microglia and the potential roles of SENP1 in the SENP1-overexpressing cell lines were investigated via Western blotting, RT-PCR and Griess assay. The present study demonstrated the apoptosis-inducing and activating role of IH on microglia. In addition, we revealed that the effect of IH on BV-2 including apoptosis, nitric oxide synthase (iNOS) expression and nitric oxide (NO) induction can be attenuated by SENP1 overexpression. The results of the present study are of both theoretical and therapeutic significance to explore the potential roles of SENP1 under IH condition and elucidated the mechanisms underlying microglial survival and activation. PMID:26499079

  20. Maternal immune activation evoked by polyinosinic:polycytidylic acid does not evoke microglial cell activation in the embryo

    PubMed Central

    Smolders, Silke; Smolders, Sophie M. T.; Swinnen, Nina; Gärtner, Annette; Rigo, Jean-Michel; Legendre, Pascal; Brône, Bert

    2015-01-01

    Several studies have indicated that inflammation during pregnancy increases the risk for the development of neuropsychiatric disorders in the offspring. Morphological brain abnormalities combined with deviations in the inflammatory status of the brain can be observed in patients of both autism and schizophrenia. It was shown that acute infection can induce changes in maternal cytokine levels which in turn are suggested to affect fetal brain development and increase the risk on the development of neuropsychiatric disorders in the offspring. Animal models of maternal immune activation reproduce the etiology of neurodevelopmental disorders such as schizophrenia and autism. In this study the poly (I:C) model was used to mimic viral immune activation in pregnant mice in order to assess the activation status of fetal microglia in these developmental disorders. Because microglia are the resident immune cells of the brain they were expected to be activated due to the inflammatory stimulus. Microglial cell density and activation level in the fetal cortex and hippocampus were determined. Despite the presence of a systemic inflammation in the pregnant mice, there was no significant difference in fetal microglial cell density or immunohistochemically determined activation level between the control and inflammation group. These data indicate that activation of the fetal microglial cells is not likely to be responsible for the inflammation induced deficits in the offspring in this model. PMID:26300736

  1. Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex.

    PubMed

    Bollinger, Justin L; Bergeon Burns, Christine M; Wellman, Cara L

    2016-02-01

    Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1-CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females. PMID:26441134

  2. Differential Effects of Stress on Microglial Cell Activation in Male and Female Medial Prefrontal Cortex

    PubMed Central

    Bollinger, Justin L.; Bergeon Burns, Christine M.; Wellman, Cara L.

    2016-01-01

    Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1-CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females. PMID:26441134

  3. Vaccinium bracteatum Thunb. Exerts Anti-Inflammatory Activity by Inhibiting NF-κB Activation in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Ko, Yong-Hyun; Seo, Jee-Yeon; Lee, Bo-Ram; Lee, Taek Hwan; Kim, Sun Yeou; Lee, Seok-Yong; Jang, Choon-Gon

    2016-01-01

    This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-κB p65). VBME significantly inhibited LPS-induced production of NO and PGE2 and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-κB p65 translocation by blocking IκB-α phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-κB signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells. PMID:27169820

  4. Thioxanthenes, chlorprothixene and flupentixol inhibit proton currents in BV2 microglial cells.

    PubMed

    Kim, Jiwon; Song, Jin-Ho

    2016-05-15

    The thioxanthene antipsychotic drugs chlorprothixene and flupentixol have anti-inflammatory and antioxidant properties. The reactive oxygen species produced by NADPH oxidase during microglia-mediated inflammatory responses cause neuronal damage, thereby contributing to various neurodegenerative diseases. Voltage-gated proton channels sustain the NADPH oxidase activity, and inhibition of the channels' activity reduces the production of reactive oxygen species. Herein, the effects of chlorprothixene and flupentixol on proton currents were investigated in BV2 microglial cells using the whole-cell patch-clamp method. Both drugs inhibited the proton currents in a concentration-dependent manner (IC50=1.7μM and 6.6μM, respectively). Chlorprothixene at 3μM slightly shifted the activation voltage toward depolarization. Both the activation and the deactivation kinetics of the proton currents were slowed by chlorprothixene 1.2- and 3.5-fold, respectively. Thus, the inhibition of proton currents may be partly responsible for the antioxidant effects of thioxanthene antipsychotic drugs. PMID:26945819

  5. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue.

    PubMed

    Wang, Cong; Tao, Sijue; Fang, Yukun; Guo, Jing; Zhu, Lirui; Zhang, Shengxiang

    2016-01-01

    Transplantation of embryonic cortical tissue is considered as a promising therapy for brain injury. Grafted neurons can reestablish neuronal network and improve cortical function of the host brain. Microglia is a key player in regulating neuronal survival and plasticity, but its activation and dynamics in grafted cortical tissue remain unknown. Using two-photon intravital imaging and parabiotic model, here we investigated the proliferation and source of microglia in the donor region by transplanting embryonic cortical tissue into adult cortex. Live imaging showed that the endogenous microglia of the grafted tissue were rapidly lost after transplantation. Instead, host-derived microglia infiltrated and colonized the graft. Parabiotic model suggested that the main source of infiltrating cells is the parenchyma of the host brain. Colonized microglia proliferated and experienced an extensive morphological transition and eventually differentiated into resting ramified morphology. Collectively, these results demonstrated that donor tissue has little contribution to the activated microglia and host brain controls the microglial population in the graft. PMID:27615195

  6. Glucocorticoid receptors in murine erythroleukaemic cells

    SciTech Connect

    Hammond, K.D.; Torrance, J.M.; DiDomenico, M.

    1987-01-01

    Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation. Specific binding of dexamethasone was measured. A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified. Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 +/- 8.2 pmol/g protein) than in untreated controls (87.9 +/- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM). Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.

  7. Cannabinoids and neuronal damage: differential effects of THC, AEA and 2-AG on activated microglial cells and degenerating neurons in excitotoxically lesioned rat organotypic hippocampal slice cultures.

    PubMed

    Kreutz, Susanne; Koch, Marco; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2007-01-01

    Cannabinoids (CBs) are attributed neuroprotective effects in vivo. Here, we determined the neuroprotective potential of CBs during neuronal damage in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). OHSCs are the best characterized in vitro model to investigate the function of microglial cells in neuronal damage since blood-borne monocytes and T-lymphocytes are absent and microglial cells represent the only immunocompetent cell type. Excitotoxic neuronal damage was induced by NMDA (50 microM) application for 4 h. Neuroprotective properties of 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), N-arachidonoylethanolamide (AEA) or 2-arachidonoylglycerol (2-AG) in different concentrations were determined after co-application with NMDA by counting degenerating neurons identified by propidium iodide labeling (PI(+)) and microglial cells labeled by isolectin B(4) (IB(4)(+)). All three CBs used significantly decreased the number of IB(4)(+) microglial cells in the dentate gyrus but the number of PI(+) neurons was reduced only after 2-AG treatment. Application of AM630, antagonizing CB2 receptors highly expressed by activated microglial cells, did not counteract neuroprotective effects of 2-AG, but affected THC-mediated reduction of IB(4)(+) microglial cells. Our results indicate that (1) only 2-AG exerts neuroprotective effects in OHSCs; (2) reduction of IB(4)(+) microglial cells is not a neuroprotective event per se and involves other CB receptors than the CB2 receptor; (3) the discrepancy in the neuroprotective effects of CBs observed in vivo and in our in vitro model system may underline the functional relevance of invading monocytes and T-lymphocytes that are absent in OHSCs. PMID:17010339

  8. Cannabinoid CB2 Receptor Mediates Nicotine-Induced Anti-Inflammation in N9 Microglial Cells Exposed to β Amyloid via Protein Kinase C

    PubMed Central

    Jia, Ji; Peng, Jie; Li, Zhaoju; Wu, Youping; Wu, Qunlin; Tu, Weifeng; Wu, Mingchun

    2016-01-01

    Background. Reducing β amyloid- (Aβ-) induced microglial activation is considered to be effective in treating Alzheimer's disease (AD). Nicotine attenuates Aβ-induced microglial activation; the mechanism, however, is still elusive. Microglia could be activated into classic activated state (M1 state) or alternative activated state (M2 state); the former is cytotoxic and the latter is neurotrophic. In this investigation, we hypothesized that nicotine attenuates Aβ-induced microglial activation by shifting microglial M1 to M2 state, and cannabinoid CB2 receptor and protein kinase C mediate the process. Methods. We used Aβ1–42 to activate N9 microglial cells and observed nicotine-induced effects on microglial M1 and M2 biomarkers by using western blot, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA). Results. We found that nicotine reduced the levels of M1 state markers, including inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor α (TNF-α) and interleukin- (IL-) 6 releases; meanwhile, it increased the levels of M2 state markers, including arginase-1 (Arg-1) expression and brain-derived neurotrophic factor (BDNF) release, in the Aβ-stimulated microglia. Coadministration of cannabinoid CB2 receptor antagonist or protein kinase C (PKC) inhibitor partially abolished the nicotine-induced effects. Conclusion. These findings indicated that cannabinoid CB2 receptor mediates nicotine-induced anti-inflammation in microglia exposed to Aβ via PKC. PMID:26884647

  9. LPS-induced iNOS expression in N9 microglial cells is suppressed by geniposide via ERK, p38 and nuclear factor-κB signaling pathways.

    PubMed

    Zhang, Gu; He, Jun-Lin; Xie, Xiao-Yan; Yu, Chao

    2012-09-01

    Activated microglia producing reactive nitrogen species, inflammatory factors, reactive oxygen species (ROS) and other neurovirulent factors, can lead to the development of neurodegenerative diseases. Certain compounds can inhibit the activation of microglia. However, the mechanisms remain unclear. In the present study, we investigated the inhibitory effect of geniposide on the production of ROS and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated N9 murine microglial cells through the p38, ERK1/2 and nuclear factor-κB (NF-κB) signaling pathways. After the N9 cells were pre-treated with the vehicle or geniposide and exposed to LPS for the time indicated, the MTT conversion test was used to assess cell viability. Suitable concentrations were chosen and adjusted according to the experiments. Extracellular nitric oxide (NO) release was measured by Griess reaction. The formation of ROS and intracellular NO was evaluated by fluorescence imaging. NOS activities were determined using commercially available kits. The morphology of the N9 cells was examined by hematoxylin and eosin staining. The expression of iNOS mRNA was examined by RT-PCR. The protein levels of iNOS, p38 mitogen-activated protein kinase (MAPK), ERK1/2 and NF-κB, inhibitory factor-κB-α (IκB-α) were determined by western blot analysis. The results showed that geniposide attenuated the activation of N9 cells and inhibited the overproduction of NO, intracellular ROS and the expression of iNOS induced by LPS in the cells. In addition, geniposide blocked the phosphorylation of p38, ERK1/2 and inhibited the drop-off of IκB induced by LPS in the cells. These data indicate that geniposide has therapeutic potential for the treatment of neurodegenerative diseases, and that it exerts its effects by inhibiting inflammation. PMID:22710392

  10. Microglial Dysregulation in Psychiatric Disease

    PubMed Central

    Frick, Luciana Romina; Williams, Kyle

    2013-01-01

    Microglia, the brain's resident immune cells, are phagocytes of the macrophage lineage that have a key role in responding to inflammation and immune challenge in the brain. More recently, they have been shown to have a number of important roles beyond immune surveillance and response, including synaptic pruning during development and the support of adult neurogenesis. Microglial abnormalities have been found in several neuropsychiatric conditions, though in most cases it remains unclear whether these are causative or are a reaction to some other underlying pathophysiology. Here we summarize postmortem, animal, neuroimaging, and other evidence for microglial pathology in major depression, schizophrenia, autism, obsessive-compulsive disorder, and Tourette syndrome. We identify gaps in the existing literature and important areas for future research. If microglial pathology proves to be an important causative factor in these or other neuropsychiatric diseases, modulators of microglial function may represent a novel therapeutic strategy. PMID:23690824

  11. Olfactory Sensory Activity Modulates Microglial-Neuronal Interactions during Dopaminergic Cell Loss in the Olfactory Bulb.

    PubMed

    Grier, Bryce D; Belluscio, Leonardo; Cheetham, Claire E J

    2016-01-01

    The mammalian olfactory bulb (OB) displays robust activity-dependent plasticity throughout life. Dopaminergic (DA) neurons in the glomerular layer (GL) of the OB are particularly plastic, with loss of sensory input rapidly reducing tyrosine hydroxylase (TH) expression and dopamine production, followed by a substantial reduction in DA neuron number. Here, we asked whether microglia participate in activity-dependent elimination of DA neurons in the mouse OB. Interestingly, we found a significant reduction in the number of both DA neurons and their synapses in the OB ipsilateral to the occluded naris (occluded OB) within just 7 days of sensory deprivation. Concomitantly, the volume of the occluded OB decreased, resulting in an increase in microglial density. Microglia in the occluded OB also adopted morphologies consistent with activation. Using in vivo 2-photon imaging and histological analysis we then showed that loss of olfactory input markedly altered microglial-neuronal interactions during the time that DA neurons are being eliminated: both microglial process motility and the frequency of wrapping of DA neuron somata by activated microglia increased significantly in the occluded OB. Furthermore, we found microglia in the occluded OB that had completely engulfed components of DA neurons. Together, our data provide evidence that loss of olfactory input modulates microglial-DA neuron interactions in the OB, thereby suggesting an important role for microglia in the activity-dependent elimination of DA neurons and their synapses. PMID:27471450

  12. Olfactory Sensory Activity Modulates Microglial-Neuronal Interactions during Dopaminergic Cell Loss in the Olfactory Bulb

    PubMed Central

    Grier, Bryce D.; Belluscio, Leonardo; Cheetham, Claire E. J.

    2016-01-01

    The mammalian olfactory bulb (OB) displays robust activity-dependent plasticity throughout life. Dopaminergic (DA) neurons in the glomerular layer (GL) of the OB are particularly plastic, with loss of sensory input rapidly reducing tyrosine hydroxylase (TH) expression and dopamine production, followed by a substantial reduction in DA neuron number. Here, we asked whether microglia participate in activity-dependent elimination of DA neurons in the mouse OB. Interestingly, we found a significant reduction in the number of both DA neurons and their synapses in the OB ipsilateral to the occluded naris (occluded OB) within just 7 days of sensory deprivation. Concomitantly, the volume of the occluded OB decreased, resulting in an increase in microglial density. Microglia in the occluded OB also adopted morphologies consistent with activation. Using in vivo 2-photon imaging and histological analysis we then showed that loss of olfactory input markedly altered microglial-neuronal interactions during the time that DA neurons are being eliminated: both microglial process motility and the frequency of wrapping of DA neuron somata by activated microglia increased significantly in the occluded OB. Furthermore, we found microglia in the occluded OB that had completely engulfed components of DA neurons. Together, our data provide evidence that loss of olfactory input modulates microglial-DA neuron interactions in the OB, thereby suggesting an important role for microglia in the activity-dependent elimination of DA neurons and their synapses. PMID:27471450

  13. Lysosomal iron liberation is responsible for the vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes.

    PubMed

    Petters, Charlotte; Thiel, Karsten; Dringen, Ralf

    2016-01-01

    Iron oxide nanoparticles (IONPs) are used for various biomedical and neurobiological applications. Thus, detailed knowledge on the accumulation and toxic potential of IONPs for the different types of brain cells is highly warranted. Literature data suggest that microglial cells are more vulnerable towards IONP exposure than other types of brain cells. To investigate the mechanisms involved in IONP-induced microglial toxicity, we applied fluorescent dimercaptosuccinate-coated IONPs to primary cultures of microglial cells. Exposure to IONPs for 6 h caused a strong concentration-dependent increase in the microglial iron content which was accompanied by a substantial generation of reactive oxygen species (ROS) and by cell toxicity. In contrast, hardly any ROS staining and no loss in cell viability were observed for cultured primary astrocytes and neurons although these cultures accumulated similar specific amounts of IONPs than microglia. Co-localization studies with lysotracker revealed that after 6 h of incubation in microglial cells, but not in astrocytes and neurons, most IONP fluorescence was localized in lysosomes. ROS formation and toxicity in IONP-treated microglial cultures were prevented by neutralizing lysosomal pH by the application of NH4Cl or Bafilomycin A1 and by the presence of the iron chelator 2,2'-bipyridyl. These data demonstrate that rapid iron liberation from IONPs at acidic pH and iron-catalyzed ROS generation are involved in the IONP-induced toxicity of microglia and suggest that the relative resistance of astrocytes and neurons against acute IONP toxicity is a consequence of a slow mobilization of iron from IONPs in the lysosomal degradation pathway. PMID:26287375

  14. COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

    PubMed Central

    Marban, Céline; Redel, Laetitia; Suzanne, Stella; Van Lint, Carine; Lecestre, Dominique; Chasserot-Golaz, Sylvette; Leid, Mark; Aunis, Dominique; Schaeffer, Evelyne; Rohr, Olivier

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region. PMID:15849318

  15. Isolation of Murine Embryonic Hemogenic Endothelial Cells.

    PubMed

    Fang, Jennifer S; Gritz, Emily C; Marcelo, Kathrina L; Hirschi, Karen K

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  16. Isolation of Murine Embryonic Hemogenic Endothelial Cells

    PubMed Central

    Marcelo, Kathrina L.; Hirschi, Karen K.

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  17. Proteomic Analysis of the Effects of Aged Garlic Extract and Its FruArg Component on Lipopolysaccharide-Induced Neuroinflammatory Response in Microglial Cells

    PubMed Central

    Mossine, Valeri V.; Nknolise, Dineo L.; Li, Jilong; Chen, Zhenzhou; Cheng, Jianlin; Greenlief, C. Michael; Mawhinney, Thomas P.; Brown, Paula N.; Fritsche, Kevin L.; Hannink, Mark; Lubahn, Dennis B.; Sun, Grace Y.; Gu, Zezong

    2014-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress. PMID:25420111

  18. Proteomic analysis of the effects of aged garlic extract and its FruArg component on lipopolysaccharide-induced neuroinflammatory response in microglial cells.

    PubMed

    Zhou, Hui; Qu, Zhe; Mossine, Valeri V; Nknolise, Dineo L; Li, Jilong; Chen, Zhenzhou; Cheng, Jianlin; Greenlief, C Michael; Mawhinney, Thomas P; Brown, Paula N; Fritsche, Kevin L; Hannink, Mark; Lubahn, Dennis B; Sun, Grace Y; Gu, Zezong

    2014-01-01

    Aged garlic extract (AGE) is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (FruArg). The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS)-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO) production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE) with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress. PMID:25420111

  19. Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells.

    PubMed

    Ajmone-Cat, Maria Antonietta; D'Urso, Maria Cristina; di Blasio, Giorgia; Brignone, Maria Stefania; De Simone, Roberta; Minghetti, Luisa

    2016-07-01

    Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1β, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/β-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies. PMID:26593276

  20. A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.

    PubMed

    Fukano, Yasufumi; Okino, Nozomu; Furuya, Shigeki; Ito, Makoto

    2016-09-16

    We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi. PMID:27480930

  1. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells

    PubMed Central

    Yang, Ju; Shen, Ting-ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer’s disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1–42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases. PMID:26824354

  2. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells.

    PubMed

    He, Gen-Lin; Luo, Zhen; Yang, Ju; Shen, Ting-Ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer's disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1-42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases. PMID:26824354

  3. Anthocyanin-rich acai (Euterpe oleracea mart.) fruit pulp fractions attenuate inflammatory stress signaling in mouse brain BV-2 microglial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Age-related increases in oxidative stress and inflammation are associated with loss of cognitive and motor functions. Previous research has shown that supplementation with berry fruits can modulate signaling in primary hippocampal neurons or BV-2 mouse microglial cells. Because of the high polypheno...

  4. Follicular dendritic cell function and murine AIDS.

    PubMed Central

    Masuda, A; Burton, G F; Fuchs, B A; Bhogal, B S; Rupper, R; Szakal, A K; Tew, J G

    1994-01-01

    Infection of mice with LP-BM5 elicits an immunodeficiency state referred to as murine acquired immune deficiency syndrome (MAIDS). Shortly after infection, retrovirus particles become associated with follicular dendritic cells (FDC) and this study was undertaken to determine whether retroviruses alter FDC functions. The FDC functions examined included the ability to: (1) retain antigen (Ag) trapped prior to infection; (2) trap new Ag after infection; (3) maintain specific IgG responses; and (4) provide co-stimulatory signals to B cells. Mice were infected with LP-BM5 and the ability of their FDC to trap and retain 125I-Ag (HSA) was assessed. Serum anti-HSA levels were monitored and FDC co-stimulatory activity was indicated by increased B-cell proliferation. HSA trapped on FDC prior to infection began to disappear by 3 weeks and was practically gone by 6 weeks. Serum anti-HSA titres were maintained normally for about 3 weeks after infection and then declined precipitously. The ability of FDC to trap new Ag began to disappear around the second and third week of infection and was markedly depressed by the fourth week. However, FDC recovered from infected mice retained their ability to co-stimulate anti-mu- and interleukin-4 (IL-4)-activated B cells throughout a 5-week period. In short, the ability of FDC to trap and retain specific Ag and maintain specific antibody levels was markedly depressed after retrovirus infection. However, FDC from infected mice continued to provide co-stimulatory signals and these signals may contribute to the lymphadenopathy and splenomegaly characteristic of MAIDS. Images Figure 4 PMID:8132218

  5. Acute HCV/HIV Coinfection Is Associated with Cognitive Dysfunction and Cerebral Metabolite Disturbance, but Not Increased Microglial Cell Activation

    PubMed Central

    Garvey, Lucy J.; Pavese, Nicola; Ramlackhansingh, Anil; Thomson, Emma; Allsop, Joanna M.; Politis, Marios; Kulasegaram, Ranjababu; Main, Janice; Brooks, David J.; Taylor-Robinson, Simon D.; Winston, Alan

    2012-01-01

    Background Microglial cell activation and cerebral function impairment are described in both chronic hepatitis C viral (HCV) and Human-Immune-Deficiency viral (HIV) infections. The aim of this study was to investigate the effect of acute HCV infection upon cerebral function and microglial cell activation in HIV-infected individuals. Methods A case-control study was conducted. Subjects with acute HCV and chronic HIV coinfection (aHCV) were compared to matched controls with chronic HIV monoinfection (HIVmono). aHCV was defined as a new positive plasma HCV RNA within 12 months of a negative RNA test. Subjects underwent neuro-cognitive testing (NCT), cerebral proton magnetic resonance spectroscopy (1H-MRS) and positron emission tomography (PET) using a 11C-radiolabeled ligand (PK11195), which is highly specific for translocator protein 18 kDA receptors on activated microglial cells. Differences between cases and controls were assessed using linear regression modelling. Results Twenty-four aHCV cases completed NCT and 1H-MRS, 8 underwent PET. Of 57 HIVmono controls completing NCT, 12 underwent 1H-MRS and 8 PET. Subjects with aHCV demonstrated on NCT, significantly poorer executive function (mean (SD) error rate 26.50(17.87) versus 19.09(8.12), p = 0.001) and on 1H-MRS increased myo-inositol/creatine ratios (mI/Cr, a marker of cerebral inflammation) in the basal ganglia (ratio of 0.71(0.22) versus 0.55(0.23), p = 0.03), compared to subjects with HIVmono. On PET imaging, no difference in 11C-PK11195 binding potential (BP) was observed between study groups (p>0.10 all cerebral locations), however lower BPs were associated with combination antiretroviral therapy (cART) use in the parietal (p = 0.01) and frontal (p = 0.03) cerebral locations. Discussion Poorer cognitive performance and disturbance of cerebral metabolites are observed in subjects with aHC,V compared to subjects with HIVmono. Higher 11C-PK11195 BP was not observed in subjects with aHCV, but was

  6. Redefining Myeloid Cell Subsets in Murine Spleen

    PubMed Central

    Hey, Ying-Ying; Tan, Jonathan K. H.; O’Neill, Helen C.

    2016-01-01

    Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed “L-DC” in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11bhiCD11cloMHCII−Ly6C−Ly6G− subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−Ly6C−Ly6G− cells, which are CD43+, Siglec-F− and CD115−. Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types. PMID:26793192

  7. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  8. Depression as a microglial disease.

    PubMed

    Yirmiya, Raz; Rimmerman, Neta; Reshef, Ronen

    2015-10-01

    Despite decades of intensive research, the biological mechanisms that causally underlie depression are still unclear, and therefore the development of novel effective antidepressant treatments is hindered. Recent studies indicate that impairment of the normal structure and function of microglia, caused by either intense inflammatory activation (e.g., following infections, trauma, stroke, short-term stress, autoimmune or neurodegenerative diseases) or by decline and senescence of these cells (e.g., during aging, Alzheimer's disease, or chronic unpredictable stress exposure), can lead to depression and associated impairments in neuroplasticity and neurogenesis. Accordingly, some forms of depression can be considered as a microglial disease (microgliopathy), which should be treated by a personalized medical approach using microglial inhibitors or stimulators depending on the microglial status of the depressed patient. PMID:26442697

  9. Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS.

    PubMed

    Hu, Zhuqin; Yu, Fengxiang; Gong, Ping; Qiu, Yu; Zhou, Wei; Cui, Yongyao; Li, Juan; Chen, Hongzhuan

    2014-04-15

    Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxic concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IкB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation. PMID

  10. Telomere sister chromatid exchange in telomerase deficient murine cells

    SciTech Connect

    Wang, Yisong; Giannone, Richard J; Liu, Yie

    2005-01-01

    We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

  11. Microglial P2Y12 receptors regulate microglial activation and surveillance during neuropathic pain.

    PubMed

    Gu, Nan; Eyo, Ukpong B; Murugan, Madhuvika; Peng, Jiyun; Matta, Sanjana; Dong, Hailong; Wu, Long-Jun

    2016-07-01

    Microglial cells are critical in the pathogenesis of neuropathic pain and several microglial receptors have been proposed to mediate this process. Of these receptors, the P2Y12 receptor is a unique purinergic receptor that is exclusively expressed by microglia in the central nervous system (CNS). In this study, we set forth to investigate the role of P2Y12 receptors in microglial electrophysiological and morphological (static and dynamic) activation during spinal nerve transection (SNT)-induced neuropathic pain in mice. First, we found that a genetic deficiency of the P2Y12 receptor (P2Y12(-/-) mice) ameliorated pain hypersensitivities during the initiation phase of neuropathic pain. Next, we characterised both the electrophysiological and morphological properties of microglia in the superficial spinal cord dorsal horn following SNT injury. We show dramatic alterations including a peak at 3days post injury in microglial electrophysiology while high resolution two-photon imaging revealed significant changes of both static and dynamic microglial morphological properties by 7days post injury. Finally, in P2Y12(-/-) mice, these electrophysiological and morphological changes were ameliorated suggesting roles for P2Y12 receptors in SNT-induced microglial activation. Our results therefore indicate that P2Y12 receptors regulate microglial electrophysiological as well as static and dynamic microglial properties after peripheral nerve injury, suggesting that the microglial P2Y12 receptor could be a potential therapeutic target for the treatment of neuropathic pain. PMID:26576724

  12. Differential expression of miRNA-146a-regulated inflammatory genes in human primary neural, astroglial and microglial cells

    PubMed Central

    Li, Yuan Yuan; Cui, Jian Guo; Dua, Prerna; Pogue, Aileen I.; Bhattacharjee, Surjyadipta; Lukiw, Walter J.

    2013-01-01

    Micro RNA-146a (miRNA-146a) is an inducible, 22 nucleotide, small RNA over-expressed in Alzheimer’s disease (AD) brain. Up-regulated miRNA-146a targets several inflammation-related and membrane-associated messenger RNAs (mRNAs), including those encoding complement factor-H (CFH) and the interleukin-1 receptor associated kinase-1 (IRAK-1), resulting in significant decreases in their expression (p < 0.05, ANOVA). In this study we assayed miRNA-146a, CFH, IRAK-1 and tetraspanin-12 (TSPAN12), abundances in primary human neuronal-glial (HNG) co-cultures, in human astroglial (HAG) and microglial (HMG) cells stressed with Aβ42 peptide and tumor necrosis factor alpha (TNFα). The results indicate a consistent inverse relationship between miRNA-146a and CFH, IRAK-1 and TSPAN12 expression levels, and indicate that HNG, HAG and HMG cell types each respond differently to Aβ42-peptide + TNFα-triggered stress. While the strongest miRNA-146a-IRAK-1 response was found in HAG cells, the largest miRNA-146a-TSPAN12 response was found in HNG cells, and the most significant miRNA-146a-CFH changes were found in HMG cells, the ‘resident scavenging macrophages’ of the brain. PMID:21640790

  13. Down-regulation of BNIP3 by olomoucine, a CDK inhibitor, reduces LPS- and NO-induced cell death in BV2 microglial cells.

    PubMed

    Tsou, Yu-Chi; Wang, Hsiao-Hsien; Hsieh, Chii-Cheng; Sun, Kuang-Hui; Sun, Guang-Huan; Jhou, Ren-Shiang; Lin, Tz-I; Lu, Shou-Yun; Liu, Huan-Yun; Tang, Shye-Jye

    2016-08-15

    Proinflammatory responses eliciting the microglial production of cytokines and nitric oxide (NO) have been reported to play a crucial role in the acute and chronic pathogenic effects of neurodegeneration. Chemical inhibitors of cyclin-dependent kinases (CDKs) may prevent the progression of neurodegeneration by both limiting cell proliferation and reducing cell death. However, the mechanism underlying the protective effect of CDK inhibitors on microglia remains unexplored. In this study, we found that olomoucine, a CDK inhibitor, alleviated lipopolysaccharide (LPS)-induced BV2 microglial cell death by reducing the generation of NO and inhibiting the gene expression of proinflammatory cytokines. In addition, olomoucine reduced inducible NO synthase promoter activity and alleviated NF-κB- and E2F-mediated transcriptional activation. NO-induced cell death involved mitochondrial disruptions such as cytochrome c release and loss of mitochondrial membrane potential, and pretreatment with olomoucine prior to NO exposure reduced these disruptions. Microarray analysis revealed that olomoucine treatment induced prominent down-regulation of Bcl2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-apoptotic Bcl-2 family protein that is involved in mitochondrial disruption. As BNIP3 knock-down significantly increased the viability of LPS- and NO-treated BV2 cells, we conclude that olomoucine may protect cells by limiting proinflammatory responses, thereby reducing NO generation. Simultaneously, down-regulation of BNIP3 prevents NO stimulation from inducing mitochondrial disruption. PMID:27345388

  14. Neurotoxicity effects of atrazine-induced SH-SY5Y human dopaminergic neuroblastoma cells via microglial activation.

    PubMed

    Ma, Kun; Wu, Hao-Yu; Zhang, Bo; He, Xi; Li, Bai-Xiang

    2015-11-01

    Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine; ATR) is a broad-spectrum herbicide with a wide range of applications worldwide. However, ATR is neurotoxic; it reduces dopamine levels in the substantia nigra and corpus striatum in the midbrain, affects the absorption of synaptic vesicles and synaptic bodies, and interferes with dopamine storage and uptake in synaptic vesicles, leading to neurodegenerative disorders. Microglia are resident immunocompetent and phagocytic cells that regulate and participate in the microenvironment in the central nervous system. They demonstrate macrophage characteristics after activation by releasing inflammatory cytokines and neurotoxic substances to increase the inflammatory response, and are thus involved in neurodegeneration. The aim of this study was to investigate the neurotoxic effects of ATR-activated microglia-mediated neuronal damage in terms of human dopaminergic neuroblastoma SH-SY5Y cell death. ATR was administered to BV-2 microglial cells at 12.5, 25, and 50 μM for 1, 6, 12, 24 and 48 h, respectively. ATR increased activated-microglia-induced overexpression of reactive oxygen species, inducible nitric oxide synthase, nitric oxide, gp91(phox), p47(phox), and the inflammatory cytokines tumor necrosis factor α and interleukin-1β, thus reducing SH-SY5Y cell viability. These results suggest that activated microglia may play a critical role in inflammation-mediated dopaminergic neuronal death, and provide the basis for further studies on the mechanisms of ATR-induced dopaminergic system toxicity. PMID:26256823

  15. Attenuation of microglial RANTES by NEMO-binding domain peptide inhibits the infiltration of CD8(+) T cells in the nigra of hemiparkinsonian monkey.

    PubMed

    Roy, A; Mondal, S; Kordower, J H; Pahan, K

    2015-08-27

    Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Despite intense investigations, little is known about its pathological mediators. Here, we report the marked upregulation of RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in the serum of hemiparkinsonian monkeys. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), a Parkinsonian toxin, increased the expression of RANTES and eotaxin in mouse microglial cells. The presence of NF-κB binding sites in promoters of RANTES and eotaxin and down-regulation of these genes by NEMO-binding domain (NBD) peptide, selective inhibitor of induced NF-κB activation, in MPP(+)-stimulated microglial cells suggest that the activation of NF-κB plays an important role in the upregulation of these two chemokines. Consistently, serum enzyme-linked immuno assay (ELISA) and nigral immunohistochemistry further confirmed that these chemokines were strongly upregulated in MPTP-induced hemiparkinsonian monkeys and that treatment with NBD peptides effectively inhibited the level of these chemokines. Furthermore, the microglial upregulation of RANTES in the nigra of hemiparkinsonian monkeys could be involved in the altered adaptive immune response in the brain as we observed greater infiltration of CD8(+) T cells around the perivascular niche and deep brain parenchyma of hemiparkinsonian monkeys as compared to control. The treatment of hemiparkinsonian monkeys with NBD peptides decreased the microglial expression of RANTES and attenuated the infiltration of CD8(+) T cells in nigra. These results indicate the possible involvement of chemokine-dependent adaptive immune response in Parkinsonism. PMID:25783477

  16. Fasudil inhibits LPS-induced migration of retinal microglial cells via regulating p38-MAPK signaling pathway

    PubMed Central

    Xu, Fan; Xu, Yue; Zhu, Liqiong; Rao, Pinhong; Wen, Jiamin; Sang, Yunyun; Shang, Fu

    2016-01-01

    Purpose To investigate the effect and possible molecular mechanisms of fasudil on retinal microglial (RMG) cell migration. Methods Primary cultured RMG cells were incubated with lipopolysaccharide (LPS), fasudil, and/or SB203580 (a p38 inhibitor). RMG cell motility was determined with the scratch wound assay and the Transwell migration assay. The phosphorylation of p38 and levels of matrix metalloproteinase 2 (MMP-2) and MMP-9 were measured with western blot. Results In the scratch-induced migration assay, as well as in the Transwell migration assay, the results indicated that LPS stimulated the migratory potential of RMG cells and fasudil significantly reduced LPS-stimulated RMG cell migration in a concentration-dependent manner. However, fasudil had no effect on RMG cell migration in the absence of LPS stimulation. Moreover, fasudil reduced the level of phosphor-p38 mitogen-activated protein kinase (p-p38-MAPK) in a concentration-dependent manner, without effects on the levels of phospho-p44/42 (p-ERK1/2) and phospho-c-Jun N-terminal kinase (p-JNK). Cotreatment with SB203580 (a p38 inhibitor) and fasudil resulted in the synergistic reduction of MMP-2, MMP-9, and p-p38-MAPK, as well as a reduction in the LPS-stimulated migration capabilities of the RMG cells, suggesting fasudil suppresses the LPS-stimulated migration of RMG cells via directly downregulating the p38-MAPK signaling pathway. Conclusions Our studies indicated that fasudil inhibited LPS-stimulated RMG cell migration via suppression of the p38-MAPK signaling pathway. PMID:27441000

  17. Microglial Derived Tumor Necrosis Factor-α Drives Alzheimer’s Disease-Related Neuronal Cell Cycle Events

    PubMed Central

    Bhaskar, Kiran; Maphis, Nicole; Xu, Guixiang; Varvel, Nicholas H.; Kokiko-Cochran, Olga N; Weick, Jason P.; Staugaitis, Susan M.; Cardona, Astrid; Ransohoff, Richard M.; Herrup, Karl; Lamb, Bruce T.

    2013-01-01

    Massive neuronal loss is a key pathological hallmark of Alzheimer’s disease (AD). However, the mechanisms are still unclear. Here we demonstrate that neuroinflammation, cell autonomous to microglia, is capable of inducing neuronal cell cycle events (CCEs), which are toxic for terminally differentiated neurons. First, oligomeric amyloid-beta peptide (ApO)-mediated microglial activation induced neuronal CCEs via the tumor-necrosis factor-α (TNFα) and the c-Jun Kinase (JNK) signaling pathway. Second, adoptive transfer of CD11b+ microglia from AD transgenic mice (R1.40) induced neuronal cyclin D1 expression via TNFα signaling pathway. Third, genetic deficiency of TNFα in R1.40 mice (R1 .40-Tnfα−/−) iled to induce neuronal CCEs. Finally, the mitotically active neurons spatially co-exist with F4/80+ activated microglia in the human AD brain and that a portion of these neurons are apoptotic. Together our data suggest a cell-autonomous role of microglia, and identify TNFα as the responsible cytokine, in promoting neuronal CCEs in the pathogenesis of AD. PMID:24141019

  18. Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells

    SciTech Connect

    Park, Sun Young; Kim, Ji-Hee; Lee, Sang Joon; Kim, YoungHee

    2013-04-01

    Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E{sub 2} (PGE{sub 2}), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, a component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates

  19. Hypoxia induced amoeboid microglial cell activation in postnatal rat brain is mediated by ATP receptor P2X4

    PubMed Central

    2011-01-01

    Background Activation of amoeboid microglial cells (AMC) and its related inflammatory response have been linked to the periventricular white matter damage after hypoxia in neonatal brain. Hypoxia increases free ATP in the brain and then induces various effects through ATP receptors. The present study explored the possible mechanism in ATP induced AMC activation in hypoxia. Results We first examined the immunoexpression of P2X4, P2X7 and P2Y12 in the corpus callosum (CC) and subependyma associated with the lateral ventricles where both areas are rich in AMC. Among the three purinergic receptors, P2X4 was most intensely expressed. By double immunofluorescence, P2X4 was specifically localized in AMC (from P0 to P7) but the immunofluorescence in AMC was progressively diminished with advancing age (P14). It was further shown that P2X4 expression was noticeably enhanced in P0 day rats subjected to hypoxia and killed at 4, 24, 72 h and 7 d versus their matching controls by double labeling and western blotting analysis. P2X4 expression was most intense at 7 d whence the inflammatory response was drastic after hypoxia. We then studied the association of P2X4 with cytokine release in AMC after hypoxic exposure. In primary microglial cells exposed to hypoxia, IL-1β and TNF-α protein levels were up-regulated. Blockade of P2X4 receptor with 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate, a selective P2X1-7 blocker resulted in partial suppression of IL-1β (24% vs hypoxic group) and TNF-α expression (40% vs hypoxic group). However, pyridoxal phosphate-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate, a selective P2X1-3, 5-7 blocker did not exert any significant effect on the cytokine expression. Conclusions It is concluded that P2X4 which is constitutively expressed by AMC in postnatal rats was enhanced in hypoxia. Hypoxia induced increase in IL-1β and TNF-α expression was reversed by 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate

  20. Differential transcriptional profiles mediated by exposure to the cannabinoids cannabidiol and Δ9-tetrahydrocannabinol in BV-2 microglial cells

    PubMed Central

    Juknat, Ana; Pietr, Maciej; Kozela, Ewa; Rimmerman, Neta; Levy, Rivka; Coppola, Giovanni; Geschwind, Daniel; Vogel, Zvi

    2012-01-01

    BACKGROUND AND PURPOSE Apart from their effects on mood and reward, cannabinoids exert beneficial actions such as neuroprotection and attenuation of inflammation. The immunosuppressive activity of cannabinoids has been well established. However, the underlying mechanisms are largely unknown. We previously showed that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signalling pathways. EXPERIMENTAL APPROACH To characterize the transcriptional effects of CBD and THC, we treated BV-2 microglial cells with these compounds and performed comparative microarray analysis using the Illumina MouseRef-8 BeadChip platform. Ingenuity Pathway Analysis was performed to identify functional subsets of genes and networks regulated by CBD and/or THC. KEY RESULTS Overall, CBD altered the expression of many more genes; from the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD and 58 by THC, and 524 gene products were down-regulated by CBD and only 36 by THC. CBD-specific gene expression profile showed changes associated with oxidative stress and glutathione depletion, normally occurring under nutrient limiting conditions or proteasome inhibition and involving the GCN2/eIF2α/p8/ATF4/CHOP-TRIB3 pathway. Furthermore, CBD-stimulated genes were shown to be controlled by nuclear factors known to be involved in the regulation of stress response and inflammation, mainly via the (EpRE/ARE)-Nrf2/ATF4 system and the Nrf2/Hmox1 axis. CONCLUSIONS AND IMPLICATIONS These observations indicated that CBD, but much less than THC, induced a cellular stress response in microglial cells and suggested that this effect could underlie its anti-inflammatory activity. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012

  1. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  2. Negative feedback between prostaglandin and alpha- and beta-chemokine synthesis in human microglial cells and astrocytes.

    PubMed

    Janabi, N; Hau, I; Tardieu, M

    1999-02-01

    The understanding of immune surveillance and inflammation regulation in cerebral tissue is essential in the therapy of neuroimmunological disorders. We demonstrate here that primary human glial cells were able to produce alpha- and beta-chemokines (IL-8 > growth related protein alpha (GROalpha) > RANTES > microphage inflammatory protein (MIP)-1alpha and MIP-1beta) in parallel to PGs (PGE2 and PGF2alpha) after proinflammatory cytokine stimulation: TNF-alpha + IL-1beta induced all except RANTES, which was induced by TNF-alpha + IFN-gamma. Purified cultures of astrocytes and microglia were also induced by the same combination of cytokines, to produce all these mediators except MIP-1alpha and MIP-1beta, which were produced predominantly by astrocytes. The inhibition of PG production by indomethacin led to a 37-60% increase in RANTES, MIP-1alpha, and MIP-1beta but not in GROalpha and IL-8 secretion. In contrast, inhibition of IL-8 and GRO activities using neutralizing Abs resulted in a specific 6-fold increase in PGE2 but not in PGF2alpha production by stimulated microglial cells and astrocytes, whereas Abs to beta-chemokines had no effect. Thus, the production of PGs in human glial cells down-regulates their beta-chemokine secretion, whereas alpha-chemokine production in these cells controls PG secretion level. These data suggest that under inflammatory conditions, the intraparenchymal production of PGs could control chemotactic gradient of beta-chemokines for an appropriate effector cell recruitment or activation. Conversely, the elevated intracerebral alpha-chemokine levels could reduce PG secretion, preventing the exacerbation of inflammation and neurotoxicity. PMID:9973432

  3. Phlorofucofuroeckol B suppresses inflammatory responses by down-regulating nuclear factor κB activation via Akt, ERK, and JNK in LPS-stimulated microglial cells.

    PubMed

    Yu, Dong-Kyung; Lee, Bonggi; Kwon, Misung; Yoon, Nayoung; Shin, Taisun; Kim, Nam-Gil; Choi, Jae-Sue; Kim, Hyeung-Rak

    2015-10-01

    Microglial activation has been implicated in many neurological disorders for its inflammatory and neurotrophic effects. In this study, we investigated the effects of phlorofucofuroeckol B (PFF-B) isolated from Ecklonia stolonifera, on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated microglia. PFF-B decreased secretion of pro-inflammatory cytokines including tumor necrosis factor α, interleukin (IL)-1β, and IL-6 and the expression of pro-inflammatory proteins such as cyclooxygenase-2 and inducible nitric oxide synthase in LPS-stimulated BV-2 cells. Profoundly, PFF-B inhibited activation of nuclear factor kappaB (NF-κB) by preventing the degradation of inhibitor κB-α (IκB-α), which led to prevent the nuclear translocation of p65 NF-κB subunit. Moreover, PFF-B inhibited the phosphorylation of Akt, ERK, and JNK. These results indicate that the anti-inflammatory effect of PFF-B on LPS-stimulated microglial cells is mainly regulated by the inhibition of IκB-α/NF-κB and Akt/ERK/JNK pathways. Our study suggests that PFF-B can be considered as a therapeutic agent against neuroinflammation by inhibiting microglial activation. PMID:26341413

  4. Autologous bone marrow mononuclear cells therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury

    PubMed Central

    Bedi, Supinder S.; Walker, Peter A.; Shah, Shinil K.; Jimenez, Fernando; Thomas, Chelsea P.; Smith, Philippa; Hetz, Robert A.; Xue, Hasen; Pati, Shibani; Dash, Pramod K.; Cox, Charles S.

    2014-01-01

    Background Autologous bone marrow-derived mononuclear cells (AMNC) have shown therapeutic promise for central nervous system insults such as stroke and traumatic brain injury (TBI). We hypothesized that intravenous injection of AMNC provides neuroprotection which leads to cognitive improvement after TBI. Methods A controlled cortical impact (CCI) rodent traumatic brain injury (TBI) model was used to examine blood-brain barrier permeability (BBB), neuronal and glial apoptosis and cognitive behavior. Two groups of rats underwent CCI with (CCI-Autologous) or without AMNC treatment (CCI-Alone), consisting of 2 million AMNC/kilogram body weight harvested from the tibia and intravenously injected 72 hr after injury. CCI-Alone animals underwent sham harvests and received vehicle injections. Results 96 hr after injury, AMNC significantly reduced the BBB permeability in injured animals, and there was an increase in apoptosis of pro-inflammatory activated microglia in the ipsilateral hippocampus. At 4 weeks after injury, we examined changes in spatial memory after TBI due to AMNC treatment. There was a significant improvement in probe testing of CCI-Autologous group in comparison to CCI-Alone in the Morris Water Maze paradigm. Conclusions Our data demonstrate that the intravenous injection of AMNC after TBI leads to neuroprotection by preserving early BBB integrity and increasing activated microglial apoptosis. In addition, AMNC also improves cognitive function. PMID:23928737

  5. Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system

    PubMed Central

    Dilger, Ryan N.; Johnson, Rodney W.

    2008-01-01

    Recent studies suggest that activation of the peripheral immune system elicits a discordant central (i.e., in the brain) inflammatory response in aged but otherwise healthy subjects compared with younger cohorts. A fundamental difference in the reactive state of microglial cells in the aged brain has been suggested as the basis for this discordant inflammatory response. Thus, the aging process appears to serve as a “priming” stimulus for microglia, and upon secondary stimulation with a triggering stimulus (i.e., peripheral signals communicating infection), these primed microglia release excessive quantities of proinflammatory cytokines. Subsequently, this exaggerated cytokine release elicits exaggerated behavioral changes including anorexia, hypersomnia, lethargy, decreased social interaction, and deficits in cognitive and motor function (collectively known as the sickness behavior syndrome). Whereas this reorganization of host priorities is normally adaptive in young subjects, there is a propensity for this response to be maladaptive in aged subjects, resulting in greater severity and duration of the sickness behavior syndrome. Consequently, acute bouts of cognitive impairment in elderly subjects increase the likelihood of poor self-care behaviors (i.e., anorexia, weight loss, noncompliance), which ultimately leads to higher rates of hospitalization and mortality. PMID:18495785

  6. Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces human immunodeficiency virus (HIV) transcription in human microglial cells

    PubMed Central

    Wires, Emily S.; Alvarez, David; Dobrowolski, Curtis; Wang, Yun; Morales, Marisela; Karn, Jonathan; Harvey, Brandon K.

    2012-01-01

    Human immunodeficiency virus (HIV) primarily infects glial cells in the central nervous system (CNS). Recent evidence suggests that HIV-infected individuals who abuse drugs such as methamphetamine (METH) have higher viral loads and experience more severe neurological complications than HIV-infected individuals who do not abuse drugs. The aim of this study was to determine the effect of METH on HIV expression from the HIV long terminal repeats (LTR) promoter and on an HIV integrated provirus in microglial cells, the primary host cells for HIV in the CNS. Primary human microglial cells immortalized with SV40 T-antigen (CHME-5 cells) were co-transfected with an HIV LTR reporter and the HIV Tat gene, a key regulator of viral replication and gene expression, and exposed to METH. Our results demonstrate that METH treatment induced LTR activation, an effect potentiated in the presence of Tat. We also found that METH increased the nuclear translocation of the nuclear factor kappa B (NF-κB), a key cellular transcriptional regulator of the LTR promoter, and the activity of an NF-κB-specific reporter plasmid in CHME-5 cells. The presence of a dominant-negative regulator of NF-κB blocked METH-related activation of the HIV LTR. Furthermore, treatment of HIV-latently infected CHME-5 (CHME-5/HIV) cells with METH induced HIV expression in a dose-dependent manner, and nuclear translocation of the p65 subunit of NF-κB. These results suggest that METH can stimulate HIV gene expression in microglia cells through activation of the NF-κB signaling pathway. This mechanism may outline the initial biochemical events leading to the observed increased neurodegeneration in HIV-positive individuals who use METH. PMID:22618514

  7. Intravenous Multipotent Adult Progenitor Cell Therapy Attenuates Activated Microglial/Macrophage Response and Improves Spatial Learning After Traumatic Brain Injury

    PubMed Central

    Bedi, Supinder S.; Hetz, Robert; Thomas, Chelsea; Smith, Philippa; Olsen, Alex B.; Williams, Stephen; Xue, Hasen; Aroom, Kevin; Uray, Karen; Hamilton, Jason; Mays, Robert W.

    2013-01-01

    We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood-brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long-term behavioral and anti-inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI-2) or 10 million cells per kilogram (CCI-10). CCI-10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI-10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI-10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI-10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long-term improvements in spatial learning as well as attenuation of neuroinflammation. PMID:24191266

  8. Diphtheria toxin-based recombinant murine IL-2 fusion toxin for depleting murine regulatory T cells in vivo.

    PubMed

    Wei, Min; Marino, Jose; Trowell, Aaron; Zhang, Huiping; Stromp Peraino, Jaclyn; Rajasekera, Priyani V; Madsen, Joren C; Sachs, David H; Huang, Christene A; Benichou, Gilles; Wang, Zhirui

    2014-09-01

    Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells which suppress immune responses of effector cells and are known to play a very important role in protection against autoimmune disease development, induction of transplantation tolerance and suppression of effective immune response against tumor cells. An effective in vivo Treg depletion agent would facilitate Treg-associated studies across many research areas. In this study, we have developed diphtheria toxin-based monovalent and bivalent murine IL-2 fusion toxins for depleting murine IL-2 receptor positive cells including CD25(+) Treg in vivo. Their potencies were assessed by in vitro protein synthesis inhibition and cell proliferation inhibition assays using a murine CD25(+) CTLL-2 cell line. Surprisingly, in contrast to our previously developed recombinant fusion toxins, the monovalent isoform (DT390-mIL-2) was approximately 4-fold more potent than its bivalent counterpart (DT390-bi-mIL-2). Binding analysis by flow cytometry demonstrated that the monovalent isoform bound stronger than the bivalent version. In vivo Treg depletion with the monovalent murine IL-2 fusion toxin was performed using C57BL/6J (B6) mice. Spleen Treg were significantly depleted with a maximum reduction of ∼70% and detectable as early as 12 h after the last injection. The spleen Treg numbers were reduced until Day 3 and returned to control levels by Day 7. We believe that this monovalent murine IL-2 fusion toxin will be an effective in vivo murine Treg depleter. PMID:25147093

  9. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  10. Reticulocalbin-1 Facilitates Microglial Phagocytosis

    PubMed Central

    Ding, Ying; Caberoy, Nora B.; Guo, Feiye; LeBlanc, Michelle E.; Zhang, Chenming; Wang, Weiwen; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis is critical to the clearance of apoptotic cells, cellular debris and deleterious metabolic products for tissue homeostasis. Phagocytosis ligands directly recognizing deleterious cargos are the key to defining the functional roles of phagocytes, but are traditionally identified on a case-by-case basis with technical challenges. As a result, extrinsic regulation of phagocytosis is poorly defined. Here we demonstrate that microglial phagocytosis ligands can be systematically identified by a new approach of functional screening. One of the identified ligands is reticulocalbin-1 (Rcn1), which was originally reported as a Ca2+-binding protein with a strict expression in the endoplasmic reticulum. Our results showed that Rcn1 can be secreted from healthy cells and that secreted Rcn1 selectively bound to the surface of apoptotic neurons, but not healthy neurons. Independent characterization revealed that Rcn1 stimulated microglial phagocytosis of apoptotic but not healthy neurons. Ingested apoptotic cells were targeted to phagosomes and co-localized with phagosome marker Rab7. These data suggest that Rcn1 is a genuine phagocytosis ligand. The new approach described in this study will enable systematic identification of microglial phagocytosis ligands with broad applicability to many other phagocytes. PMID:25992960

  11. Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells

    PubMed Central

    Chessell, I P; Michel, A D; Humphrey, P P A

    1997-01-01

    We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X7 (P2Z) receptor in the NTW8 mouse microglial cell line. At room temperature, in an extracellular solution containing 2 mM Ca2+ and 1 mM Mg2+, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate (Bz-ATP; 300 μM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of −90 mV, of 549±191 and 644±198 pA, respectively. Current-voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification. In an extracellular solution containing zero Mg2+ and 500 μM Ca2+ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909±138 and 1818±218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15–20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-effect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC50 values of 58.3, 298 and 505 μM, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl- 2′, 4′-disulphonic acid (PPADS; 30 μM) but not suramin (100 μM). In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 μM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 μM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32°C, current augmentation also

  12. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

  13. High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells.

    PubMed

    Goode, Angela E; Gonzalez Carter, Daniel A; Motskin, Michael; Pienaar, Ilse S; Chen, Shu; Hu, Sheng; Ruenraroengsak, Pakatip; Ryan, Mary P; Shaffer, Milo S P; Dexter, David T; Porter, Alexandra E

    2015-11-01

    Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNTs, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 h exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 h. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic

  14. Miglustat improves purkinje cell survival and alters microglial phenotype in feline Niemann-Pick disease type C.

    PubMed

    Stein, Veronika M; Crooks, Alexandra; Ding, Wenge; Prociuk, Maria; O'Donnell, Patricia; Bryan, Caroline; Sikora, Tracey; Dingemanse, Jasper; Vanier, Marie T; Walkley, Steven U; Vite, Charles H

    2012-05-01

    Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid-trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form of human NPC disease received daily miglustat orally beginning at 3 weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 μg/mL, and 104.1 ± 16.6 μg hours/mL, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains of treated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats. PMID:22487861

  15. Miglustat Improves Purkinje Cell Survival and Alters Microglial Phenotype in Feline Niemann-Pick Disease Type C

    PubMed Central

    Stein, Veronika M.; Crooks, Alexandra; Ding, Wenge; Prociuk, Maria; O’Donnell, Patricia; Bryan, Caroline; Sikora, Tracey; Dingemanse, Jasper; Vanier, Marie T.; Walkley, Steven U.; Vite, Charles H.

    2012-01-01

    Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form of human NPC disease received daily miglustat orally beginning at 3 weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 μg/ml, and 104.1 ± 16.6 μg hours/ml, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats, and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains of treated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats. PMID:22487861

  16. High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells

    PubMed Central

    Gonzalez Carter, Daniel A.; Motskin, Michael; Pienaar, Ilse S.; Chen, Shu; Hu, Sheng; Ruenraroengsak, Pakatip; Ryan, Mary P.; Shaffer, Milo S. P.; Dexter, David T.

    2016-01-01

    Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNT, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 hrs exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 hours. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic

  17. Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis

    PubMed Central

    2012-01-01

    Background In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand. Methods Recombinant HmC1q (rHmC1q) was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques. Results rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia. Conclusions A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech. PMID:22356764

  18. The role of the JAK2-STAT3 pathway in pro-inflammatory responses of EMF-stimulated N9 microglial cells

    PubMed Central

    2010-01-01

    Background In several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inflammatory responses in more detail than the previous study. Methods N9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor (Pyridone 6, P6) of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA). Results EMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure. Conclusions EMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that

  19. Isolation of Murine Lymph Node Stromal Cells

    PubMed Central

    Lagarde, Nadège; Rossi, Simona W.

    2014-01-01

    Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed. PMID:25178108

  20. Development of the microglial phenotype in culture.

    PubMed

    Szabo, M; Gulya, K

    2013-06-25

    Selected morphological, molecular and functional aspects of various microglial cell populations were characterized in cell cultures established from the forebrains of E18 rat embryos. The mixed primary cortical cultures were maintained for up to 28days using routine culturing techniques when the microglial cells in the culture were not stimulated or immunologically challenged. During culturing, expansion of the microglial cell populations was observed, as evidenced by quantitative assessment of selected monocyte/macrophage/microglial cell-specific markers (human leukocyte antigen (HLA) DP, DQ, DR, CD11b/c and Iba1) via immunocyto- and histochemistry and Western blot analysis. The Iba1 immunoreactivity in Western blots steadily increased about 750-fold, and the number of Iba1-immunoreactive cells rose at least 67-fold between one day in vitro (DIV1) and DIV28. Morphometric analysis on binary (digital) silhouettes of the microglia revealed their evolving morphology during culturing. Microglial cells were mainly ameboid in the early stages of in vitro differentiation, while mixed populations of ameboid and ramified cell morphologies were characteristic of older cultures as the average transformation index (TI) increased from 1.96 (DIV1) to 15.17 (DIV28). Multiple immunofluorescence labeling of selected biomarkers revealed different microglial phenotypes during culturing. For example, while HLA DP, DQ, DR immunoreactivity was present exclusively in ameboid microglia (TI<3) between DIV1 and DIV10, CD11b/c- and Iba1-positive microglial cells were moderately (TI<13) and progressively (TI<81) more ramified, respectively, and always present throughout culturing. Regardless of the age of the cultures, proliferating microglia were Ki67-positive and characterized by low TI values (TI<3). The microglial function was assessed by an in vitro phagocytosis assay. Unstimulated microglia with low TI values were significantly more active in phagocytosing fluorescent microspheres than

  1. Flow Cytometric Analysis of Immune Cells Within Murine Aorta.

    PubMed

    Gjurich, Breanne N; Taghavie-Moghadam, Parésa L; Galkina, Elena V

    2015-01-01

    The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis. PMID:26445788

  2. Nanoelectroablation therapy for murine basal cell carcinoma.

    PubMed

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S; Epstein, Ervin H; Tang, Jean Y

    2012-08-01

    When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1(+/-)K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology [2,20] and in response to drug therapy [19]. We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 ± 5 (SEM) mm(3) shrunk by 76 ± 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses. PMID:22771794

  3. Nanoelectroablation Therapy for Murine Basal Cell Carcinoma

    PubMed Central

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S.; Epstein, Ervin H.; Tang, Jean Y.

    2012-01-01

    When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally as effective in the treatment of autochthonous BCC tumors in Ptch1+/−K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology [2;20] and in response to drug therapy [19]. We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5–7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 ± 5 (SEM) mm3 shrunk by 76 ± 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 minute-long treatment of 2700 pulses. PMID:22771794

  4. Maternal separation activates microglial cells and induces an inflammatory response in the hippocampus of male rat pups, independently of hypothalamic and peripheral cytokine levels.

    PubMed

    Roque, Angélica; Ochoa-Zarzosa, Alejandra; Torner, Luz

    2016-07-01

    Adult animals subjected to chronic stress show an inflammatory response in the hippocampus which has been related to cognitive dysfunction and psychopathology. However the immediate consequences of early life stress on hippocampal glial cells have not been studied. Here we analyzed the effects of maternal separation (MS) on astrocyte and microglial cell morphology in the hippocampal hilus, compared the expression of cytokines in the hippocampus and hypothalamus, and the peripheral response of cytokines, on postnatal day (PD) 15. Male rat pups of MS (3h/day, PD1-PD14) and Control (CONT) pups showed similar microglial cell densities in the hilus, but MS pups presented more activated microglia. MS decreased astrocyte density and the number of processes in the hilus. Cytokine mRNA expression (qPCR) was analyzed in MS and CONT groups, sacrificed (i) under basal (B) conditions or (ii) after a single stress event (SS) at PN15. In hippocampal extracts, MS increased IL-1β mRNA, under B and SS conditions while IL-6 and TNF-α did not change. In hypothalamic tissue, MS increased TNF-α and IL-6 mRNA, but not IL-1b, after SS. Peripheral concentrations of IL-1β were decreased under B and SS conditions in MS; IL-6 concentration increased after SS in MS pups, and TNF-α concentration was unchanged. In conclusion, MS activates microglial cells and decreases astrocyte density in the hippocampus. A differential cytokine expression is observed in the hippocampus and the hypothalamus after MS, and after SS. Also, MS triggers an independent response of peripheral cytokines. These specific responses together could contribute to decrease hippocampal neurogenesis and alter the neuroendocrine axis. PMID:26431692

  5. Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS

    SciTech Connect

    Hu, Zhuqin; Yu, Fengxiang; Gong, Ping; Qiu, Yu; Zhou, Wei; Cui, Yongyao; Li, Juan Chen, Hongzhuan

    2014-04-15

    Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxic concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IkB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation

  6. The antiviral drug ganciclovir does not inhibit microglial proliferation and activation

    PubMed Central

    Skripuletz, Thomas; Salinas Tejedor, Laura; Prajeeth, Chittappen K.; Hansmann, Florian; Chhatbar, Chintan; Kucman, Valeria; Zhang, Ning; Raddatz, Barbara B.; Detje, Claudia N.; Sühs, Kurt-Wolfram; Pul, Refik; Gudi, Viktoria; Kalinke, Ulrich; Baumgärtner, Wolfgang; Stangel, Martin

    2015-01-01

    Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS. PMID:26447351

  7. The antiviral drug ganciclovir does not inhibit microglial proliferation and activation.

    PubMed

    Skripuletz, Thomas; Salinas Tejedor, Laura; Prajeeth, Chittappen K; Hansmann, Florian; Chhatbar, Chintan; Kucman, Valeria; Zhang, Ning; Raddatz, Barbara B; Detje, Claudia N; Sühs, Kurt-Wolfram; Pul, Refik; Gudi, Viktoria; Kalinke, Ulrich; Baumgärtner, Wolfgang; Stangel, Martin

    2015-01-01

    Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS. PMID:26447351

  8. Murine trabecular meshwork cells in tissue culture.

    PubMed

    Begley, C G; Yue, B Y; Hendricks, R L

    1991-11-01

    Trabecular meshwork cells from an inbred strain of mice (A/J) were established in tissue culture. Within 1 hour of enucleation, tissue containing the cornea and the chamber angle was excised and placed in tissue culture. Two to five days later, three cell types grew from the explants. Two of these cell types, corneal endothelium and fibroblasts, grew together, with the fibroblasts preferentially spreading on top of the endothelial cells. The trabecular meshwork cells extended from the explant as a distinct morphological type. The corneal endothelium and its associated fibroblasts were then removed from the culture flask with a sterile cotton swab, leaving a monolayer of pure trabecular meshwork cells. These cells required 3-4 weeks to reach confluency and could be passaged five times. They were actively phagocytic in culture and exhibited immunoreactivity to antibodies against two extracellular matrix components, laminin and collagen type IV. Mouse trabecular meshwork cells also expressed receptors for acetylated low-density lipoprotein, a property shared by trabecular meshwork cells derived from other species. The availability of trabecular meshwork cells from an inbred strain of mice will facilitate future in vivo functional studies of these cells in a syngeneic system, as well as investigations of potential immunoregulatory properties of the trabecular meshwork. PMID:1782800

  9. Nanoelectroablation therapy for murine basal cell carcinoma

    SciTech Connect

    Nuccitelli, Richard; Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela; Chang, Kris S.; Epstein, Ervin H.; Tang, Jean Y.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  10. Lonicera japonica THUNB. Extract Inhibits Lipopolysaccharide-Stimulated Inflammatory Responses by Suppressing NF-κB Signaling in BV-2 Microglial Cells.

    PubMed

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hong, Sa-Ik; Lee, Seok-Yong; Jang, Choon-Gon

    2015-07-01

    In the current study, we evaluated the anti-inflammatory effects of Lonicera japonica THUNB. (LJ) and its underlying molecular mechanism in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Our results indicated that LJ significantly inhibits LPS-stimulated production of nitric oxide (NO) and prostaglandin E2 (PGE2). In addition, LJ inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the protein and mRNA levels. In LPS-stimulated BV-2 microglial cells, LJ inhibited proinflammatory cytokines and chemokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) enzymatic activities, and/or mRNA expression, as well as reactive oxygen species (ROS) production. LJ significantly suppressed activation of nuclear factor-κB (NF-κB) and its translocation from the cytosol to the nucleus and suppressed the DNA-binding activity of NF-κB. Furthermore, LJ significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinases (PI3K)/Akt, and Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT)1/3. Collectively, our findings indicated that the antineuroinflammatory properties of LJ in LPS-induced BV-2 microglial cells is due to downregulation of proinflammatory cytokines and chemokines downstream of inhibition of NF-κB activation. PMID:25897683

  11. Modulation of Lipopolysaccharide Stimulated Nuclear Factor kappa B Mediated iNOS/NO Production by Bromelain in Rat Primary Microglial Cells

    PubMed Central

    Abbasi Habashi, Soraya; Sabouni, Farzaneh; Moghimi, Ali; Ansari Majd, Saeed

    2016-01-01

    Background: Microglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation. Methods: Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student's t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain. Results: Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-κB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death. Conclusion: Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases. PMID:26459398

  12. Extrathymic development of murine T cells after bone marrow transplantation

    PubMed Central

    Holland, Amanda M.; Zakrzewski, Johannes L.; Tsai, Jennifer J.; Hanash, Alan M.; Dudakov, Jarrod A.; Smith, Odette M.; West, Mallory L.; Singer, Natalie V.; Brill, Jessie; Sun, Joseph C.; van den Brink, Marcel R.M.

    2012-01-01

    Restoring T cell competence is a significant clinical challenge in patients whose thymic function is severely compromised due to age or cytoreductive conditioning. Here, we demonstrate in mice that mesenteric LNs (MLNs) support extrathymic T cell development in euthymic and athymic recipients of bone marrow transplantation (BMT). Furthermore, in aged murine BMT recipients, the contribution of the MLNs to the generation of T cells was maintained, while the contribution of the thymus was significantly impaired. Thymic impairment resulted in a proportional increase in extrathymic-derived T cell progenitors. Extrathymic development in athymic recipients generated conventional naive TCRαβ T cells with a broad Vβ repertoire and intact functional and proliferative potential. Moreover, in the absence of a functional thymus, immunity against known pathogens could be augmented using engineered precursor T cells with viral specificity. These findings demonstrate the potential of extrathymic T cell development for T cell reconstitution in patients with limited thymic function. PMID:23160195

  13. Changes in phagocytosis and expression of microglial cells in craniocerebral injury mice models.

    PubMed

    Guo, F Y; Liu, T; Chen, J J; Gao, W; Yang, F; Zhou, X Y; Liao, Y L

    2016-01-01

    The objective of this study was to investigate the changes in phagocytic function and expression quantities of CD11b and tumor necrosis factor-α (TNF-α) among microglia cells of craniocerebral injury mice. Modified Feeney method was used to establish the craniocerebral injury mice models. Twenty-one male SPF mice were divided into a control group and a trauma group. The scalp was incised and a bone window was opened in the control group without cerebral injury. In the trauma group, the mice were sacrificed after the craniocerebral injury at 1, 3, 6, 12, 24 and 48 h to make frozen sections of cerebral tissues. The phagocytic rate of microglia cells was observed by using fluorescent microsphere. The changes in the expression quantities of CD11b and TNF-α were detected by enzyme-linked immuno sorbent assay (ELISA). The phagocytic ability of the microglia cells after the craniocerebral injury increased at 1 h after injury compared with that of the control group (P less than 0.01). The expression of surface antigen CD11b of the microglia cells and the expression of TNF-α increased at 1, 3, 6, 12, 24 and 48 h after the injury compared with those of the control group (P less than 0.01). The phagocytic ability of the microglia cells increased. The expressions of CD11b and TNF-α were also gradually enhanced in the acute phase after craniocerebral injury, and then gradually decreased to the normal level. The expressions of CD11b and TNF-α indicated a high consistency with the changing trend of the phagocytic ability, suggesting that the microglia cells may participate in the regulation of the inflammatory process of the central nervous system through absorbing apoptotic cells and increasing and secreting inflammatory and anti-inflammatory factors. PMID:27358141

  14. Non-cell-autonomous Neurotoxicity of α-synuclein Through Microglial Toll-like Receptor 2

    PubMed Central

    Kim, Changyoun; Lee, He-Jin; Masliah, Eliezer

    2016-01-01

    Synucleinopathies are a collection of neurological diseases that are characterized by deposition of α-synuclein aggregates in neurons and glia. These diseases include Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. Although it has been increasingly clear that α-synuclein is implicated in the pathogenesis of PD and other synucleinopathies, the precise mechanism underlying the disease process remains to be unraveled. The past studies on how α-synuclein exerts pathogenic actions have focused on its direct, cell-autonomous neurotoxic effects. However, recent findings suggested that there might be indirect, non-cell-autonomous pathways, perhaps through the changes in glial cells, for the pathogenic actions of this protein. Here, we present evidence that α-synuclein can cause neurodegeneration through a non-cell-autonomous manner. We show that α-synuclein can be secreted from neurons and induces inflammatory responses in microglia, which in turn secreted neurotoxic agents into the media causing neurodegeneration. The neurotoxic response of microglia was mediated by activation of toll-like receptor 2 (TLR2), a receptor for neuron-derived α-synuclein. This work suggests that TLR2 is the key molecule that mediates non-cell-autonomous neurotoxic effects of α-synuclein, hence a candidate for the therapeutic target. PMID:27358579

  15. Small-Ruminant Lentivirus Enhances PrP-Sc Accumulation in Cultured Sheep Microglial Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scrapie is the prototype member of the family of transmissible spongiform encephalopathies, fatal neurodegenerative diseases associated with conversion and accumulation of prion proteins in a number of neural and extraneural cell types. Although scrapie has been the focus of research investigations...

  16. Cynandione A from Cynanchum wilfordii attenuates the production of inflammatory mediators in LPS-induced BV-2 microglial cells via NF-κB inactivation.

    PubMed

    Yang, Seung Bo; Lee, Sang Min; Park, Ji-Hae; Lee, Tae Hoon; Baek, Nam-In; Park, Hi-Joon; Lee, Hyejung; Kim, Jiyoung

    2014-01-01

    Cynanchum wilfordii is one of most widely used medicinal plants in Oriental medicine for the treatment of various conditions. In the present study, we isolated cynandione A (CA) from an extract of Cynanchum wilfordii roots (CWE) and investigated the effects of CA on the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced BV-2 microglial cells. CWE and CA significantly decreased LPS-induced nitric oxide production and the expression of iNOS in a concentration-dependent manner, while they (CWE up to 500 µg/mL and CA up to 80 µM) did not exhibit cytotoxic activity. Results from reverse transcription-polymerase chain reaction (RT-PCR) analysis and enzyme-linked immunosorbent assay (ELISA) showed that CA significantly attenuated the expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β in LPS-stimulated BV-2 cells. Furthermore, CA inhibited the phosphorylation of inhibitor kappa B-alpha (IκB-α) and translocation of nuclear factor-kappa B (NF-κB) to the BV-2 cell nucleus, indicating that CWE and CA may have effective anti-inflammatory activities via NF-κB inactivation in stimulated microglial cells. PMID:25087960

  17. Amyloid β25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

    PubMed

    Part, Kristin; Künnis-Beres, Kai; Poska, Helen; Land, Tiit; Shimmo, Ruth; Zetterström Fernaeus, Sandra

    2015-12-10

    Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aβ) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aβ through ROS and metals are mainly studied in neuronal cells using full-length Aβ1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aβ25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aβ25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aβ25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aβ25-35 induced ROS. PMID:26505916

  18. Microglial TIR-domain-containing adapter-inducing interferon-β (TRIF) deficiency promotes retinal ganglion cell survival and axon regeneration via nuclear factor-κB

    PubMed Central

    2012-01-01

    Background TIR-domain-containing adapter-inducing interferon-β (TRIF) is the sole downstream adaptor of Toll-like receptor (TLR)3, which is one of the major signaling pathways in immune cells leading to neuroinflammation in the central nervous system. Overexpression of TRIF may lead to activation of inflammatory responses, and contribute to pathophysiological progression in both acute and chronic neurodegenerative retinal diseases. In the present study, was aimed to elucidate the contributions of TRIF to optic nerve (ON) regeneration and retinal ganglion cell (RGC) survival following injury to the ON, a widely studied model of central nervous system injury and of degenerative diseases such as glaucoma. Methods We used retrograde labeling with a fluorochrome, hydroxystilbamidine (Fluorogold) to evaluate RGC survival, and immunostaining with growth-associated protein-43 to evaluate axon regeneration in an ON crush model. Changes in microglial cytokines following RGC injury was examined with ELISA and real-time PCR. In vivo studies were carried out in wild-type and trif-/- mice. A Transwell co-culture system and migration test were used to mimic the crosstalk between microglia and RGCs. TRIF-associated downstream adaptors were determined by western blotting. Results Compared with wild-type (WT) mice, TRIF knockout (KO) mice displayed a robust ability to regenerate axons 3 or 7 days after nerve injury. In addition, RGC survival was considerably higher in trif-/- than in WT mice. ON lesion induced less microglial activation in trif-/- than in WT mice. and more WT microglia distorted and migrated toward the foramen opticum. In the transwell system, few trif-/- microglia migrated through the membrane when stimulated by the performed lesion on RGC axons in a transwell system. Inactivation of microglial cells in trif-/- mice was associated with reduced production of inflammatory cytokines, as detected with real-time RT-PCR and ELISA. Furthermore western blot analysis

  19. Directed Ig class switch recombination in activated murine B cells.

    PubMed Central

    Winter, E; Krawinkel, U; Radbruch, A

    1987-01-01

    Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination. Images Fig. 1. Fig. 2. Fig. 6. PMID:3038529

  20. Mesenchymal Stem Cells Reduce Murine Atherosclerosis Development

    PubMed Central

    Frodermann, Vanessa; van Duijn, Janine; van Pel, Melissa; van Santbrink, Peter J.; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C. A.

    2015-01-01

    Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies. PMID:26490642

  1. Safflower Yellow regulates microglial polarization and inhibits inflammatory response in LPS-stimulated Bv2 cells.

    PubMed

    Yang, Xing-Wang; Li, Yan-Hua; Zhang, Hui; Zhao, Yong-Fei; Ding, Zhi-Bin; Yu, Jie-Zhong; Liu, Chun-Yun; Liu, Jian-Chun; Jiang, Wei-Jia; Feng, Qian-Jin; Xiao, Bao-Guo; Ma, Cun-Gen

    2016-03-01

    Activated microglia, especially polarized M1 cells, produce pro-inflammatory cytokines and free radicals, thereby contributing directly to neuroinflammation and various brain disorders. Given that excessive or chronic neuroinflammation within the central nervous system (CNS) exacerbates neuronal damage, molecules that modulate neuroinflammation are candidates as neuroprotective agents. In this study, we provide evidence that Safflor yellow (SY), the main active component in the traditional Chinese medicine safflower, modulates inflammatory responses by acting directly on BV2 microglia. LPS stimulated BV2 cells to upregulate expression of TLR4-Myd88 and MAPK-NF-κB signaling pathways and to release IL-1β, IL-6, TNF-α, and COX-2. However, SY treatment inhibited expression of TLR4-Myd88 and p-38/p-JNK-NF-κB, downregulated expression of iNOS, CD16/32, and IL-12, and upregulated CD206 and IL-10. In conclusion, our results demonstrate that SY exerts an anti-inflammatory effect on BV2 microglia, possibly through TLR-4/p-38/p-JNK/NF-κB signaling pathways and the conversion of microglia from inflammatory M1 to an anti-inflammatory M2 phenotype. PMID:26634402

  2. Anti-inflammatory effects of three-dimensional graphene foams cultured with microglial cells.

    PubMed

    Song, Qin; Jiang, Ziyun; Li, Ning; Liu, Ping; Liu, Liwei; Tang, Mingliang; Cheng, Guosheng

    2014-08-01

    One of the key goals in nerve tissue engineering is to develop new materials which cause less or no neuroinflammation. Despite the rapid advances of using graphene as a neural interface material, it still remains unknown whether graphene could provoke neuroinflammation or not, and whether and how the topographical features of graphene influence the neuroinflammation induction. By immunofluorescence, Elisa technique, western blot, scanning electron microscope (SEM) methods, we investigated the pro- and/or anti-inflammatory responses of microglia in the graphene films (2D-graphene) or graphene foams (3D-graphene) culturing systems. Furthermore, the growth situations of the neural stem cells (NSCs) in the conditioned culture medium produced in the graphene substrates were evaluated. The results show that: 1) neither 2D nor 3D graphene induced distinct neuroinflammation when compared to the tissue culture polystyrene (TCPS) substrates; 2) the topographical structures of the graphene might affect the material/cell interactions, leading to disparate effects on lipopolysaccharide (LPS)-induced neuroinflammation; 3) 3D graphene exhibited a remarkable capability of rescuing LPS-induced neuroinflammation probably through the restriction of microglia morphological transformation by the unique topographical features on the surface, showing the ability of anti-inflammation against external insults, while 2D graphene failed to. These results provide insights into the diverse biological effects of the material's topographical structures and open new opportunity for the applications of graphene in neuroscience. PMID:24875763

  3. Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro.

    PubMed

    Rey, C; Nadjar, A; Buaud, B; Vaysse, C; Aubert, A; Pallet, V; Layé, S; Joffre, C

    2016-07-01

    Sustained inflammation in the brain together with microglia activation can lead to neuronal damage. Hence limiting brain inflammation and activation of microglia is a real therapeutic strategy for inflammatory disease. Resolvin D1 (RvD1) and resolvin E1 (RvE1) derived from n-3 long chain polyunsaturated fatty acids are promising therapeutic compounds since they actively turn off the systemic inflammatory response. We thus evaluated the anti-inflammatory activities of RvD1 and RvE1 in microglia cells in vitro. BV2 cells were pre-incubated with RvD1 or RvE1 before lipopolysaccharide (LPS) treatment. RvD1 and RvE1 both decreased LPS-induced proinflammatory cytokines (TNF-α, IL-6 and IL-1β) gene expression, suggesting their proresolutive activity in microglia. However, the mechanisms involved are distinct as RvE1 regulates NFκB signaling pathway and RvD1 regulates miRNAs expression. Overall, our findings support that pro-resolving lipids are involved in the resolution of brain inflammation and can be considered as promising therapeutic agents for brain inflammation. PMID:26718448

  4. Inhibition of Nitric Oxide Production in BV2 Microglial Cells by Triterpenes from Tetrapanax papyriferus.

    PubMed

    Cho, Namki; Moon, Eun Hye; Kim, Hyun Woo; Hong, Jaewoo; Beutler, John A; Sung, Sang Hyun

    2016-01-01

    It is well known that activated microglia produce nitric oxide (NO), which has an important role in the pathophysiology of several neurodegenerative diseases such as Alzheimer's disease. In the course of searching for novel therapeutic agents from medicinal plants against neuroinflammatory diseases, the methanolic extract of Tetrapanax papyriferus was found to have significant NO inhibitory activity in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. Nine oleanane-type triterpenes, including two new compounds, epipapyriogenin C-3-O-β-d-glucopyranoside (6) and 11-O-butylpapyrioside LIIc (9), were isolated from the leaves and stems of Tetrapanax papyriferus. The structures of these compounds were elucidated with 1D- and 2D-NMR and MS data. Among these Δ(11,13) oleanane-type triterpenes, compound 3 showed significant NO inhibitory activity in BV-2 cells, reducing the LPS-induced expression of COX-2 and pro-inflammatory cytokines such as TNF-α and IL-6. Compounds 7 and 9 also showed NO inhibitory activities among the Δ(12) oleanane-type triterpene saponins. These results show that oleanane-type triterpenes isolated from T. papyriferus could be a potential natural resource of NO inhibitors used in the treatment of neurodegenerative disorders. PMID:27070561

  5. Dye-mediated photosensitization of murine neuroblastoma cells

    SciTech Connect

    Sieber, F.; Sieber-Blum, M.

    1986-04-01

    The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

  6. Exposure to electromagnetic field attenuates oxygen-glucose deprivation-induced microglial cell death by reducing intracellular Ca(2+) and ROS.

    PubMed

    Duong, Cao Nguyen; Kim, Jae Young

    2016-01-01

    Purpose The aim of this research was to demonstrate the protective effects of electromagnetic field (EMF) exposure on the human microglial cell line, HMO6, against ischemic cell death induced by in vitro oxygen-glucose deprivation (OGD). Materials and methods HMO6 cells were cultured for 4 h under OGD with or without exposure to EMF with different combinations of frequencies and intensities (10, 50, or 100 Hz/1 mT and 50 Hz/0.01, 0.1, or 1 mT). Cell survival, intracellular calcium and reactive oxygen species (ROS) levels were measured. Results OGD caused significant HMO6 cell death as well as elevation of intracellular Ca(2+) and ROS levels. Among different combinations of EMF frequencies and intensities, 50 Hz/1 mT EMF was the most potent to attenuate OGD-induced cell death and intracellular Ca(2+) and ROS levels. A significant but less potent protective effect was also found at 10 Hz/1 mT, whereas no protective effect was found at other combinations of EMF. A xanthine oxidase inhibitor reversed OGD-induced ROS production and cell death, while NADPH oxidase and mitochondrial respiration chain complex II inhibitors did not affect cell death. Conclusions 50 Hz/1 mT EMF protects human microglial cells from OGD-induced cell death by interfering with OGD-induced elevation of intracellular Ca(2+) and ROS levels, and xanthine oxidase is one of the main mediators involved in OGD-induced HMO6 cell death. Non-invasive treatment of EMF radiation may be clinically useful to attenuate hypoxic-ischemic brain injury. PMID:26882219

  7. [Microglial Phagocytosis in the Neurodegenerative Diseases].

    PubMed

    Cao, Sheng-nan; Bao, Xiu-qi; Sun, Hua; Zhang, Dan

    2016-04-01

    Microglia are the resident innate immune cells in the brain. Under endogenous or exogenous stimulates, they become activated and play an important role in the neurodegenerative diseases. Microglial phagocytosis is a process of receptor-mediated engulfment and degradation of apoptotic cells. In addition, microglia can phagocyte brain-specific cargo, such as myelin debris and abnormal protein aggregation. However, recent studies have shown that microglia can also phagocyte stressed-but-viable neurons, causing loss of neurons in the brain. Thus, whether microglial phagocytosis is beneficial or not in neurodegenerative disease remains controversial. This article reviews microglial phagocytosis related mechanisms and its potential roles in neurodegenerative diseases, with an attempt to provide new insights in the treatment of neurodegenerative diseases. PMID:27181903

  8. Involvement of mitogen-activated protein kinase and NF-κB signaling pathways in perfluorooctane sulfonic acid-induced inflammatory reaction in BV2 microglial cells.

    PubMed

    Zhu, Jingying; Qian, Wenyi; Wang, Yixin; Gao, Rong; Wang, Jun; Xiao, Hang

    2015-12-01

    Microglial activation is closely related to the pathogenesis of neurodegenerative diseases by producing proinflammatory cytokines. Perfluorooctane sulfonic acid (PFOS), known as an emerging persistent organic pollutant, is reported to disturb human immune homeostasis; however, whether it affects cytokine production or the immune response in the central nervous system remains unclear. The present study was aimed to explore whether PFOS contributed to inflammatory action and to investigate the corresponding mechanisms in BV2 microglia. PFOS-mediated morphologic changes, cytokine responses and signaling events were examined by light microscopy, real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot assays. Our results indicated that PFOS increased BV2 cells activation and simultaneously increased tumor necrosis factor alpha and interleukin-6 expression. In addition, the c-Jun N-terminal protein kinase inhibitor (SP600125), as well as ERK1/2 blocker (PD98059), transcriptionally at least, displayed anti-inflammatory properties on PFOS-elicited cytokine responses. Moreover, the inflammatory transcription factor NF-κB was specifically activated by PFOS as well. These results, taken together, suggested that PFOS exerts its functional effects on the response of microglial cell activation via, in part, the c-Jun N-terminal protein kinase, ERK and NF-κB signaling pathways with its subsequent influence on proinflammatory action. PMID:25677194

  9. β-chemokine production by neural and glial progenitor cells is enhanced by HIV-1 Tat: Effects on microglial migration

    PubMed Central

    Hahn, Yun Kyung; Vo, Phu; Fitting, Sylvia; Block, Michelle L.; Hauser, Kurt F.; Knapp, Pamela E.

    2010-01-01

    HIV-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins Tat or gp120. In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/RANTES, CCL3/MIP-1α, and CCL4/MIP-1β were increased by 12 h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pretreatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous. PMID:20403075

  10. Proinflammatory-activated glioma cells induce a switch in microglial polarization and activation status, from a predominant M2b phenotype to a mixture of M1 and M2a/B polarized cells

    PubMed Central

    Lisi, Lucia; Stigliano, Egidio; Lauriola, Libero; Navarra, Pierluigi; Russo, Cinzia Dello

    2014-01-01

    Malignant gliomas are primary brain tumors characterized by morphological and genetic complexities, as well as diffuse infiltration into normal brain parenchyma. Within gliomas, microglia/macrophages represent the largest tumor-infiltrating cell population, contributing by at least one-third to the total tumor mass. Bi-directional interactions between glioma cells and microglia may therefore play an important role on tumor growth and biology. In the present study, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. We found that microglial cells undergo a different pattern of activation depending on the stimulus; in the presence of activated glioma-derived factors, i.e. a condition mimicking the late stage of pathology, microglia presents as a mixture of polarization phenotypes (M1 and M2a/b), with up-regulation of iNOS (inducible nitric oxide synthase), ARG (arginase) and IL (interleukine)-10. At variance, microglia exposed to basal glioma-derived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2b polarization status and up-regulation of IL-10 only. As far as viability and cell proliferation are concerned, both LI-CM [LPS (lipopolysaccharide)–IFNγ (interferon γ) conditioned media] and C-CM (control-conditioned media) induce similar effects on microglial morphology. Finally, in human glioma tissue obtained from surgical resection of patients with IV grade glioblastoma, we detected a significant amount of CD68 positive cells, which is a marker of macrophage/microglial phagocytic activity, suggesting that in vitro findings presented here might have a relevance in the human pathology as well. PMID:24689533

  11. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

  12. Murine Norovirus Transcytosis across an In Vitro Polarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells

    PubMed Central

    Gonzalez-Hernandez, Mariam B.; Liu, Thomas; Blanco, Luz P.; Auble, Heather; Payne, Hilary C.

    2013-01-01

    Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host. PMID:24049163

  13. Murine norovirus transcytosis across an in vitro polarized murine intestinal epithelial monolayer is mediated by M-like cells.

    PubMed

    Gonzalez-Hernandez, Mariam B; Liu, Thomas; Blanco, Luz P; Auble, Heather; Payne, Hilary C; Wobus, Christiane E

    2013-12-01

    Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host. PMID:24049163

  14. The dynamics of murine mammary stem/progenitor cells

    PubMed Central

    DONG, Qiaoxiang; SUN, Lu-Zhe

    2014-01-01

    The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups. PMID:25580105

  15. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  16. Permissive and restricted virus infection of murine embryonic stem cells.

    PubMed

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey; Kellam, Paul

    2012-10-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  17. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  18. Armeniacae semen extract suppresses lipopolysaccharide-induced expressions of cyclooxygenase [correction of cycloosygenase]-2 and inducible nitric oxide synthase in mouse BV2 microglial cells.

    PubMed

    Chang, Hyun-Kyung; Yang, Hye-Young; Lee, Taeck-Hyun; Shin, Min-Chul; Lee, Myoung-Hwa; Shin, Mal-Soon; Kim, Chang-Ju; Kim, Ok-Jin; Hong, Seon-Pyo; Cho, Sonhae

    2005-03-01

    Armeniacae semen is the seed of Prunus armeniaca L. var. ansu MAXIM which is classified into Rosaceae. In traditional oriental medicine, Armeniacae semen has been used for the treatment of pain and inflammatory diseases. In this study, the effect of Armeniacae semen extract on lipopolysaccharide-induced inflammation was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot, prostaglandin E2 immunoassay, and nitric oxide detection on mouse BV2 microglial cells. In the present results, Armeniacae semen extract suppressed prostaglandin E2 synthesis and nitric oxide production by inhibiting the lipopolysaccharide-stimulated enhancement of cyclooxygenase-2 and inducible nitric oxide synthase mRNA expression in BV2 cells. These results show that Armeniacae semen exerts anti-inflammatory and analgesic effects probably by suppression of cyclooxygenase-2 and inducible nitric oxide synthase expressions. PMID:15744067

  19. Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway

    PubMed Central

    Kim, Su Jung; Hong, Seung Bok; Park, Jin-Kyu

    2016-01-01

    Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway. PMID:27563334

  20. Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway.

    PubMed

    Im, Eun Ju; Kim, Su Jung; Hong, Seung Bok; Park, Jin-Kyu; Rhee, Man Hee

    2016-01-01

    Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway. PMID:27563334

  1. Resistance to cyclopentenylcytosine in murine leukemia L1210 cells.

    PubMed

    Zhang, H; Cooney, D A; Zhang, M H; Ahluwalia, G; Ford, H; Johns, D G

    1993-12-01

    Cyclopentenyl cytosine (CPEC) exhibits oncological activity in murine and human tumor cells and has now entered Phase I clinical trials. Its mode of action as an antitumor agent appears to be inhibition by its triphosphate (CPEC-TP) of CTP synthase, the enzyme which converts UTP to CTP. In an attempt to elucidate the mechanism of resistance to CPEC, a murine leukemia cell line resistant to CPEC (L1210/CPEC) was developed by N-methyl-N-nitro-N-nitrosoguanidine-induced mutagenesis and subsequent selection by cultivation of the L1210 cells in the presence of 2 microM CPEC. Resistant clones were maintained in CPEC-free medium for 6 generations before biochemical studies were performed. The resistant clone selected for further studies was approximately 13,000-fold less sensitive to growth inhibition by CPEC than the parental cells, and the concentration of CPEC required to deplete CTP in the resistant cells was 50-fold higher than in the sensitive cells. A comparison of the kinetic properties of CTP synthase from sensitive and resistant cells indicated alteration in the properties of the enzyme from the latter; the median inhibitory concentration for CPEC-TP increased from 2 to 14 microM, Km for UTP decreased from 126 to 50 microM, and Vmax increased 12-fold from 0.2 to 2.3 nmol/mg/min. Northern blot analyses of polyadenylated RNA from the resistant and sensitive cells indicated a 3-fold increase in transcripts of the CTP synthase gene in the resistant line. Consistent with these alterations in the properties of the enzyme, the resistant cells exhibited significantly expanded CTP and dCTP pools (4- 5-fold) when compared with the sensitive cells. No change was observed, however, in the properties of uridine-cytidine kinase, the enzyme responsible for the initial phosphorylation of CPEC; despite this, however, cellular uptake of CPEC was greatly decreased, and phosphorylation of CPEC and its incorporation into RNA were 10-fold less than in the parental cells. These latter

  2. Effects of trichostatins on differentiation of murine erythroleukemia cells

    SciTech Connect

    Yoshida, M.; Nomura, S.; Beppu, T.

    1987-07-15

    The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation but not by treatment with dimethyl sulfoxide. This enhanced activity was completely inhibited by adding cycloheximide to the culture medium 2 h after exposure to TS, suggesting that TS are dimethyl sulfoxide-type inducers of erythroid differentiation. No inhibitory effect of TS was observed on macromolecular synthesis in cultured cells.

  3. Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension.

    PubMed

    Trost, A; Motloch, K; Bruckner, D; Schroedl, F; Bogner, B; Kaser-Eichberger, A; Runge, C; Strohmaier, C; Klein, B; Aigner, L; Reitsamer, H A

    2015-07-01

    Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology

  4. Isolation of Primary Murine Brain Microvascular Endothelial Cells

    PubMed Central

    Ruck, Tobias; Bittner, Stefan; Epping, Lisa; Herrmann, Alexander M.; Meuth, Sven G.

    2014-01-01

    The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies. This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed. PMID:25489873

  5. Nicotine contributes to the neural stem cells fate against toxicity of microglial-derived factors induced by Aβ via the Wnt/β-catenin pathway.

    PubMed

    Jiang, De-Qi; Wei, Mei-Dan; Wang, Ke-Wan; Lan, Yan-Xian; Zhu, Ning; Wang, Yong

    2016-01-01

    Recent studies have demonstrated that the molecules secreted from microglias play important roles in the cell fate determination of neural stem cells (NSCs), and nicotinic acetylcholine receptor agonist treatment could reduce neuroinflammation in some neurodegenerative disease models, such as Alzheimer's disease (AD). However, it is not clear how nicotine plays a neuroprotective role in inflammation-mediated central nervous diseases, and its possible mechanisms in the process remain largely elusive. The aim of this study is to improve the survival microenvironment of NSCs co-cultured with microglias in vitro by weakening inflammation that mediated by accumulation of β-amyloid peptide (Aβ). The viability, proliferation, differentiation, apoptosis of NSCs and underlying mechanisms associated with Wnt signaling pathway were investigated. The results showed that Aβ could directly damage NSCs. Furthermore, concomitant to elevated levels of TNF-α, IL-1β derived from microglias, the NSCs had been damaged more severely with the upregulation of Axin 2, p-β-catenin and the downregulation of β-catenin, p-GSK-3β, microtubule-associated protein-2, choline acetyltransferase. However, addition of 10 μmol/L nicotine before microglias treated with Aβ was beneficial to protect the NSCs against neurotoxicity of microglial-derived factors induced by Aβ, which partially rescued proliferation, differentiation and inhibited apoptosis of NSCs via activation of Wnt/β-catenin pathway. Taken together, these data imply that low concentration nicotine attenuates NSCs injury induced by microglial-derived factors via Wnt signaling pathway. Thus, treatment with nicotinic acetylcholine receptor agonist provides a promising research field for neural stem cell fate and therapeutic intervention in neuroinflammation diseases. PMID:26001208

  6. Glucocerebrosidase 1 deficient Danio rerio mirror key pathological aspects of human Gaucher disease and provide evidence of early microglial activation preceding alpha-synuclein-independent neuronal cell death

    PubMed Central

    Keatinge, Marcus; Bui, Hai; Menke, Aswin; Chen, Yu-Chia; Sokol, Anna M.; Bai, Qing; Ellett, Felix; Da Costa, Marc; Burke, Derek; Gegg, Matthew; Trollope, Lisa; Payne, Thomas; McTighe, Aimee; Mortiboys, Heather; de Jager, Sarah; Nuthall, Hugh; Kuo, Ming-Shang; Fleming, Angeleen; Schapira, Anthony H.V.; Renshaw, Stephen A.; Highley, J. Robin; Chacinska, Agnieszka; Panula, Pertti; Burton, Edward A.; O'Neill, Michael J.; Bandmann, Oliver

    2015-01-01

    Autosomal recessively inherited glucocerebrosidase 1 (GBA1) mutations cause the lysosomal storage disorder Gaucher's disease (GD). Heterozygous GBA1 mutations (GBA1+/−) are the most common risk factor for Parkinson's disease (PD). Previous studies typically focused on the interaction between the reduction of glucocerebrosidase (enzymatic) activity in GBA1+/− carriers and alpha-synuclein-mediated neurotoxicity. However, it is unclear whether other mechanisms also contribute to the increased risk of PD in GBA1+/− carriers. The zebrafish genome does not contain alpha-synuclein (SNCA), thus providing a unique opportunity to study pathogenic mechanisms unrelated to alpha-synuclein toxicity. Here we describe a mutant zebrafish line created by TALEN genome editing carrying a 23 bp deletion in gba1 (gba1c.1276_1298del), the zebrafish orthologue of human GBA1. Marked sphingolipid accumulation was already detected at 5 days post-fertilization with accompanying microglial activation and early, sustained up-regulation of miR-155, a master regulator of inflammation. gba1c.1276_1298del mutant zebrafish developed a rapidly worsening phenotype from 8 weeks onwards with striking reduction in motor activity by 12 weeks. Histopathologically, we observed marked Gaucher cell invasion of the brain and other organs. Dopaminergic neuronal cell count was normal through development but reduced by >30% at 12 weeks in the presence of ubiquitin-positive, intra-neuronal inclusions. This gba1c.1276_1298del zebrafish line is the first viable vertebrate model sharing key pathological features of GD in both neuronal and non-neuronal tissue. Our study also provides evidence for early microglial activation prior to alpha-synuclein-independent neuronal cell death in GBA1 deficiency and suggests upregulation of miR-155 as a common denominator across different neurodegenerative disorders. PMID:26376862

  7. FLOW CYTOMETRIC COMPARISON OF THE EFFECTS OF TRIALKYTING ON THE MURINE ERYTHROLEUKEMIC CELL

    EPA Science Inventory

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) ...

  8. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  9. Anti-inflammatory mechanism of α-viniferin regulates lipopolysaccharide-induced release of proinflammatory mediators in BV2 microglial cells.

    PubMed

    Dilshara, Matharage Gayani; Lee, Kyoung-Tae; Kim, Hee Ju; Lee, Hak-Ju; Choi, Yung Hyun; Lee, Chang-Min; Kim, Lark Kyun; Kim, Gi-Young

    2014-07-01

    α-Viniferin is an oligostilbene of trimeric resveratrol and has anticancer activity; however, the molecular mechanism underlying the anti-inflammatory effects of α-viniferin has not been completely elucidated thus far. Therefore, we determined the mechanism by which α-viniferin regulates lipopolysaccharide (LPS)-induced expression of proinflammatory mediators in BV2 microglial cells. Treatment with α-viniferin isolated from Clematis mandshurica decreased LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). α-Viniferin also downregulated the LPS-induced expression of proinflammatory genes such as iNOS and COX-2 by suppressing the activity of nuclear factor kappa B (NF-κB) via dephosphorylation of Akt/PI3K. Treatment with a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), indirectly showed that NF-κB is a crucial transcription factor for expression of these genes in the early stage of inflammation. Additionally, our results indicated that α-viniferin suppresses NO and PGE2 production in the late stage of inflammation through induction of heme oxygenase-1 (HO-1) regulated by nuclear factor erythroid 2-related factor (Nrf2). Taken together, our data indicate that α-viniferin suppresses the expression of proinflammatory genes iNOS and COX-2 in the early stage of inflammation by inhibiting the Akt/PI3K-dependent NF-κB activation and inhibits the production of proinflammatory mediators NO and PGE2 in the late stage by stimulating Nrf2-mediated HO-1 signaling pathway in LPS-stimulated BV2 microglial cells. These results suggest that α-viniferin may be a potential candidate to regulate LPS-induced inflammation. PMID:24859013

  10. Kaempferol acts through mitogen-activated protein kinases and protein kinase B/AKT to elicit protection in a model of neuroinflammation in BV2 microglial cells

    PubMed Central

    Park, SE; Sapkota, K; Kim, S; Kim, H; Kim, SJ

    2011-01-01

    BACKGROUND AND PURPOSE Kaempferol, a dietary flavonoid and phyto-oestrogen, is known to have anti-inflammatory properties. Microglial activation has been implicated in various neurodegenerative diseases. Anti-inflammatory effects of kaempferol and the underlying mechanisms were investigated by using LPS-stimulated microglial BV2 cells. EXPERIMENTAL APPROACH Cell viability was measured using MTT and neutral red assays. elisa, Western blot, immunocytochemistry and electrophoretic mobility-shift assay were used to analyse NO, PGE2, TNF-α and IL-1β production, inducible NOS (iNOS), COX-2 expression and the involvement of signalling pathways such as toll-like receptor-4 (TLR4), MAPK cascades, PKB (AKT) and NF-κB. Accumulation of reaction oxygen species (ROS) was measured by nitroblue tetrazolium and 2′7′-dichlorofluorescein diacetate assay. Matrix metalloproteinase activity was investigated by zymography and immunoblot assay. Phagocytotic activity was assessed by use of latex beads. KEY RESULTS Kaempferol significantly attenuated LPS-induced NO, PGE2, TNF-α, IL-1β and ROS production and phagocytosis in a concentration-dependent manner. Kaempferol suppressed the expression of iNOS, COX-2, MMP-3 and blocked the TLR4 activation. Moreover, kaempferol inhibited LPS-induced NF-κB activation and p38 MAPK, JNK and AKT phosphorylation. CONCLUSION AND IMPLICATIONS Kaempferol was able to reduce LPS-induced inflammatory mediators through the down-regulation of TLR4, NF-κB, p38 MAPK, JNK and AKT suggesting that kaempferol has therapeutic potential for the treatment of neuroinflammatory diseases. PMID:21449918

  11. The Anti-Inflammatory Activity of Eucommia ulmoides Oliv. Bark. Involves NF-κB Suppression and Nrf2-Dependent HO-1 Induction in BV-2 Microglial Cells.

    PubMed

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hwang, Ji-Young; Ko, Yong-Hyun; Seo, Ji-Yeon; Lee, Bo-Ram; Lee, Seok-Yong; Jang, Choon-Gon

    2016-05-01

    In the present study, we investigated the anti-inflammatory properties of Eucommia ulmoides Oliv. Bark. (EUE) in lipopolysaccharide (LPS)-stimulated microglial BV-2 cells and found that EUE inhibited LPS-mediated up-regulation of pro-inflammatory response factors. In addition, EUE inhibited the elevated production of pro-inflammatory cytokines, mediators, and reactive oxygen species (ROS) in LPS-stimulated BV-2 microglial cells. Subsequent mechanistic studies revealed that EUE suppressed LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/Akt, glycogen synthase kinase-3β (GSK-3β), and their downstream transcription factor, nuclear factor-kappa B (NF-κB). EUE also blocked the nuclear translocation of NF-κB and inhibited its binding to DNA. We next demonstrated that EUE induced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated heme oxygenase-1 (HO-1) expression. We determined that the significant up-regulation of HO-1 expression by EUE was a consequence of Nrf2 nuclear translocation; furthermore, EUE increased the DNA binding of Nrf2. In contrast, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, blocked the ability of EUE to inhibit NO and PGE₂ production, indicating the vital role of HO-1. Overall, our results indicate that EUE inhibits pro-inflammatory responses by modulating MAPKs, PI3K/Akt, and GSK-3β, consequently suppressing NF-κB activation and inducing Nrf2-dependent HO-1 activation. PMID:27068259

  12. Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells

    PubMed Central

    Chuang, Dennis Y.; Cui, Jiankun; Simonyi, Agnes; Engel, Victoria A.; Chen, Shanyan; Fritsche, Kevin L.; Thomas, Andrew L.; Applequist, Wendy L.; Folk, William R.; Lubahn, Dennis B.; Sun, Albert Y.; Sun, Grace Y.

    2014-01-01

    Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R. PMID:25324465

  13. The Anti-Inflammatory Activity of Eucommia ulmoides Oliv. Bark. Involves NF-κB Suppression and Nrf2-Dependent HO-1 Induction in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hwang, Ji-Young; Ko, Yong-Hyun; Seo, Ji-Yeon; Lee, Bo-Ram; Lee, Seok-Yong; Jang, Choon-Gon

    2016-01-01

    In the present study, we investigated the anti-inflammatory properties of Eucommia ulmoides Oliv. Bark. (EUE) in lipopolysaccharide (LPS)-stimulated microglial BV-2 cells and found that EUE inhibited LPS-mediated up-regulation of pro-inflammatory response factors. In addition, EUE inhibited the elevated production of pro-inflammatory cytokines, mediators, and reactive oxygen species (ROS) in LPS-stimulated BV-2 microglial cells. Subsequent mechanistic studies revealed that EUE suppressed LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/Akt, glycogen synthase kinase-3β (GSK-3β), and their downstream transcription factor, nuclear factor-kappa B (NF-κB). EUE also blocked the nuclear translocation of NF-κB and inhibited its binding to DNA. We next demonstrated that EUE induced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated heme oxygenase-1 (HO-1) expression. We determined that the significant up-regulation of HO-1 expression by EUE was a consequence of Nrf2 nuclear translocation; furthermore, EUE increased the DNA binding of Nrf2. In contrast, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, blocked the ability of EUE to inhibit NO and PGE2 production, indicating the vital role of HO-1. Overall, our results indicate that EUE inhibits pro-inflammatory responses by modulating MAPKs, PI3K/Akt, and GSK-3β, consequently suppressing NF-κB activation and inducing Nrf2-dependent HO-1 activation. PMID:27068259

  14. Detection of human myeloid progenitor cells in a murine background.

    PubMed

    Carow, C E; Harrington, M A; Broxmeyer, H E

    1993-01-01

    Cell-mixing experiments were performed to determine whether human (hu) peripheral blood plasma would select for the growth of hu myeloid progenitor cells in vitro. Mixtures of hu male umbilical cord blood and murine (mu) female bone marrow (100% hu, 100% mu, 1.0% hu or 10% hu and 50% hu) were plated in methylcellulose cultures that contained either hu plasma or fetal bovine serum (FBS). Cultures were supplemented with recombinant (r) hu erythropoietin (Epo) alone or in combination with rhu granulocyte-macrophage colony stimulating factor (GM-CSF), rmuGM-CSF or rhu steel factor (SLF). DNA was extracted from day 14 colonies and clusters, and the polymerase chain reaction (PCR) was used to detect the hu Y-chromosome satellite DNA sequence. Results of these studies revealed that hu plasma used in combination with hu growth factors selected for the growth of hu progenitor cells. Mu cells grew in hu plasma only at high cell-plating concentrations. This selective effect was due to a heat labile factor or factors, since mu cells grew equally well in heat-inactivated hu plasma and FBS. Cells in individual progenitor cell colonies and clusters cultured in hu plasma contained hu Y-chromosome-specific DNA sequences that were detectable after PCR-mediated amplification, thus eliminating the need for time-consuming Southern transfer. This study describes a method whereby hu/immune-deficient mice can be screened rapidly for hu myeloid engraftment. These results also indicate that the hu identity of colonies and clusters cultured in hu plasma must be genetically confirmed, especially when hu cells may represent a low percentage of the total cells plated. PMID:7678088

  15. Microenvironment influences vascular differentiation of murine cardiovascular progenitor cells.

    PubMed

    Gluck, Jessica M; Delman, Connor; Chyu, Jennifer; MacLellan, W Robb; Shemin, Richard J; Heydarkhan-Hagvall, Sepideh

    2014-11-01

    We examined the effects of the microenvironment on vascular differentiation of murine cardiovascular progenitor cells (CPCs). We isolated CPCs and seeded them in culture exposed to the various extracellular matrix (ECM) proteins in both two-dimensional (2D) and 3D culture systems. To better understand the contribution of the microenvironment to vascular differentiation, we analyzed endothelial and smooth muscle cell differentiation at both day 7 and day 14. We found that laminin and vitronectin enhanced vascular endothelial cell differentiation while fibronectin enhanced vascular smooth muscle cell differentiation. We also observed that the effects of the 3D electrospun scaffolds were delayed and not noticeable until the later time point (day 14), which may be due to the amount of time necessary for the cells to migrate to the interior of the scaffold. The study characterized the contributions of both ECM proteins and the addition of a 3D culture system to continued vascular differentiation. Additionally, we demonstrated the capability bioengineer a CPC-derived vascular graft. PMID:24687591

  16. Replication of the Moloney murine sarcoma-leukemia virus in XC cells.

    PubMed

    Trowbridge, S T; Benyesh-Melnick, M; Biswal, N

    1973-01-01

    The XC rat cell line was found to support the replication of a strain of the Moloney murine sarcoma-leukemia virus. In growth curve experiments cytopathology was paralleled by the production of murine sarcoma virus and leukemia virus progeny having the biologic, antigenic, and biophysical properties of the infecting virus. PMID:4346280

  17. Chinese medicinal herbs inhibit growth of murine renal cell carcinoma.

    PubMed

    Lau, B H; Ruckle, H C; Botolazzo, T; Lui, P D

    1994-01-01

    Tumors are known to produce factors suppressing immune functions. We previously showed that a murine renal cell carcinoma (Renca) suppressed macrophage function in vitro and that this suppression was abolished by co-incubation with extracts of two Chinese medicinal herbs. We now report that these phytochemicals are capable of inhibiting growth of Renca in vivo. BALB/c mice were transplanted intraperitoneally (IP) with 1-2 x 10(5) Renca cells. One day after tumor transplant, mice were randomized into two groups. One group was treated IP, daily for 10 days, with 100 microliters of phytochemicals containing 500 micrograms each of Astragalus membranaceus and Ligustrum lucidum, while the other group received saline as controls. A cure rate of 57% was obtained with these phytochemicals when the initial tumor load was 2 x 10(5), and 100% when the initial tumor load was 1 x 10(5). Additional experiments were performed to investigate the mechanisms involved in this protection. Splenic macrophages from tumor-bearing mice were shown to have depressed chemiluminescent oxidative burst activity, and this depression was restored with phytochemical treatment. Splenocytes from mice transplanted with Renca responded less favorably to interleukin-2 (IL-2) in generating lymphokine-activated killer (LAK) cells; again this depression was restored with phytochemical treatment. Our data suggest that these phytochemicals may have exerted their antitumor effects via augmentation of phagocyte and LAK cell activities. PMID:7812364

  18. High-dimensional analysis of the murine myeloid cell system.

    PubMed

    Becher, Burkhard; Schlitzer, Andreas; Chen, Jinmiao; Mair, Florian; Sumatoh, Hermi R; Teng, Karen Wei Weng; Low, Donovan; Ruedl, Christiane; Riccardi-Castagnoli, Paola; Poidinger, Michael; Greter, Melanie; Ginhoux, Florent; Newell, Evan W

    2014-12-01

    Advances in cell-fate mapping have revealed the complexity in phenotype, ontogeny and tissue distribution of the mammalian myeloid system. To capture this phenotypic diversity, we developed a 38-antibody panel for mass cytometry and used dimensionality reduction with machine learning-aided cluster analysis to build a composite of murine (mouse) myeloid cells in the steady state across lymphoid and nonlymphoid tissues. In addition to identifying all previously described myeloid populations, higher-order analysis allowed objective delineation of otherwise ambiguous subsets, including monocyte-macrophage intermediates and an array of granulocyte variants. Using mice that cannot sense granulocyte macrophage-colony stimulating factor GM-CSF (Csf2rb(-/-)), which have discrete alterations in myeloid development, we confirmed differences in barrier tissue dendritic cells, lung macrophages and eosinophils. The methodology further identified variations in the monocyte and innate lymphoid cell compartment that were unexpected, which confirmed that this approach is a powerful tool for unambiguous and unbiased characterization of the myeloid system. PMID:25306126

  19. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

  20. 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

  1. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

  2. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB. PMID:10865941

  3. TAM receptors regulate multiple features of microglial physiology.

    PubMed

    Fourgeaud, Lawrence; Través, Paqui G; Tufail, Yusuf; Leal-Bailey, Humberto; Lew, Erin D; Burrola, Patrick G; Callaway, Perri; Zagórska, Anna; Rothlin, Carla V; Nimmerjahn, Axel; Lemke, Greg

    2016-04-14

    Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis is normally driven by both TAM receptor ligands Gas6 and protein S. Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson's disease. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease. PMID:27049947

  4. Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

    PubMed Central

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S.; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS. PMID:23675538

  5. New compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton from Clematis mandshurica: Anti-inflammatory effects in lipopolysaccharide-stimulated BV2 microglial cells.

    PubMed

    Dilshara, Matharage Gayani; Lee, Kyoung-Tae; Lee, Chang-Min; Choi, Yung Hyun; Lee, Hak-Ju; Choi, Il-Whan; Kim, Gi-Young

    2015-01-01

    Microglia are main immune cells to exacerbate neural disorders in persistent overactivating. Therefore, it is a good strategy to regulate microglia for the treatment of neural disorders. In the present study, we isolated and characterized a novel compound, 5-O-isoferuloyl-2-deoxy-D-ribono-γ-lacton (5-DRL) from Clematis mandshurica, and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-treated BV2 microglial cells. 5-DRL inhibited the expression of LPS-stimulated proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), as well as their regulatory genes inducible NO syntheses (iNOS) and cyclooxygenase-2 (COX-2). 5-DRL also downregulated the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) through suppression of the nuclear translocation of the NF-κB subunits, p65 and p50. Consistent with the inhibition of iNOS and COX-2 via NF-κB activity with 5-DRL, an inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), also led to the suppression of LPS-induced iNOS and COX-2 expression. Additionally, 5-DRL corresponding with antioxidants, N-acetylcysteine (NAC) and glutathione (GSH), remarkably inhibited reactive oxygen species (ROS) generation. Both NAC and GSH, thus attenuated the expression of iNOS and COX-2 by suppressing NF-κB activation, indicating that 5-DRL suppresses LPS-induced iNOS and COX-2 expression through downregulation of the ROS-dependent NF-κB signaling pathway. The present study also indicated that 5-DRL suppresses NO and PGE2 production by inducing heme oxygenase-1 (HO-1) via nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, the present data indicate that 5-DRL attenuates the production of proinflammatory mediators such as NO and PGE2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting ROS-dependent NF-κB activation and stimulating the Nrf2/HO-1 signal pathway. These data may be implicated in the application of 5-DRL in LPS

  6. Inhibitory effect of ent-Sauchinone on amyloidogenesis via inhibition of STAT3-mediated NF-κB activation in cultured astrocytes and microglial BV-2 cells

    PubMed Central

    2014-01-01

    Background ent-Sauchinone is a polyphenolic compound found in plants belonging to the lignan family. ent-Sauchinone has been shown to modulate the expression of inflammatory factors through the nuclear factor-kappa B (NF-κB) signaling pathway. It is well known that neuroinflammation is associated with amyloidogenesis. Thus, in the present study, we investigated whether ent-Sauchinone could have anti-amyloidogenic effects through the inhibition of NF-κB pathways via its anti-inflammatory property. Methods To investigate the potential effect of ent-Sauchinone on anti-neuroinflammation and anti-amyloidogenesis in in vitro studies, we used microglial BV-2 cells and cultured astrocytes treated with ent-Sauchinone (1, 5, and 10 μM) for 24 hours. For the detection of anti-neuro-inflammatory responses, reative oxygen species (ROS) and Nitric oxide (NO) generation and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were measured with assay kits and western blotting. β-secretase and β-secretase activities and β-amyloid levels were determined for measuring the anti-amyloidogenic effects of ent-Sauchinone by enzyme assay kits. NF-κB and STAT3 signals were detected with electromobility shift assay (EMSA) to study the related signaling pathways. The binding of ent-Sauchinone to STAT3 was evaluated by a pull-down assay and by a docking model using Autodock VINA software (Hoover’s Inc., Texas, United states). Results ent-Sauchinone (1, 5, and 10 μM) effectively decreased lipopolysaccharide (LPS)-(1 μg/ml) induced inflammatory responses through the reduction of ROS and NO generations and iNOS and COX-2 expressions in cultured astrocytes and microglial BV-2 cells. ent-Sauchinone also inhibited LPS-induced amyloidogenesis through the inhibition of β-secretase and β-secretase activity. NF- κB amyloid and STAT3, critical transcriptional factors regulating not only inflammation but also amyloidogenesis, were also inhibited in a

  7. Role of Cytosolic Calcium Diffusion in Murine Cardiac Purkinje Cells

    PubMed Central

    Limbu, Bijay; Shah, Kushal; Weinberg, Seth H.; Deo, Makarand

    2016-01-01

    Cardiac Purkinje cells (PCs) are morphologically and electrophysiologically different from ventricular myocytes and, importantly, exhibit distinct calcium (Ca2+) homeostasis. Recent studies suggest that PCs are more susceptible to action potential (AP) abnormalities than ventricular myocytes; however, the exact mechanisms are poorly understood. In this study, we utilized a detailed biophysical mathematical model of a murine PC to systematically examine the role of cytosolic Ca2+ diffusion in shaping the AP in PCs. A biphasic spatiotemporal Ca2+ diffusion process, as recorded experimentally, was implemented in the model. In this study, we investigated the role of cytosolic Ca2+ dynamics on AP and ionic current properties by varying the effective Ca2+ diffusion rate. It was observed that AP morphology, specifically the plateau, was affected due to changes in the intracellular Ca2+ dynamics. Elevated Ca2+ concentration in the sarcolemmal region activated inward sodium–Ca2+ exchanger (NCX) current, resulting in a prolongation of the AP plateau at faster diffusion rates. Artificially clamping the NCX current to control values completely reversed the alterations in the AP plateau, thus confirming the role of NCX in modifying the AP morphology. Our results demonstrate that cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights in the increased arrhythmogeneity of PCs. PMID:27478391

  8. Glial Cell Line-Derived Neurotrophic Factor Family Members Reduce Microglial Activation via Inhibiting p38MAPKs-Mediated Inflammatory Responses

    PubMed Central

    Rickert, Uta; Grampp, Steffen; Wilms, Henrik; Spreu, Jessica; Knerlich-Lukoschus, Friederike; Held-Feindt, Janka; Lucius, Ralph

    2014-01-01

    Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. However, little is known about direct influences of the GFL on microglia function, which are known to express part of the GDNF receptor system. Using RT-PCR and immunohistochemistrym we investigated the expression of the GDNF family receptor alpha 1 (GFR alpha) and the coreceptor transmembrane receptor tyrosine kinase (RET) in rat microglia in vitro as well as the effect of GFL on the expression of proinflammatory molecules in LPS activated microglia. We could show that GFL are able to regulate microglia functions and suggest that part of the well known neuroprotective action may be related to the suppression of microglial activation. We further elucidated the functional significance and pathophysiological implications of these findings and demonstrate that microglia are target cells of members of the GFL (GDNF and the structurally related neurotrophic factors neurturin (NRTN), artemin (ARTN), and persephin (PSPN)). PMID:26317008

  9. Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells

    PubMed Central

    Kumari, Bharti; Jain, Pratistha; Das, Shaoli; Ghosal, Suman; Hazra, Bibhabasu; Trivedi, Ashish Chandra; Basu, Anirban; Chakrabarti, Jayprokas; Vrati, Sudhanshu; Banerjee, Arup

    2016-01-01

    Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation. PMID:26838068

  10. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed Central

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-01-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells. PMID:8751945

  11. New findings about iron oxide nanoparticles and their different effects on murine primary brain cells

    PubMed Central

    Neubert, Jenni; Wagner, Susanne; Kiwit, Jürgen; Bräuer, Anja U; Glumm, Jana

    2015-01-01

    The physicochemical properties of superparamagnetic iron oxide nanoparticles (SPIOs) enable their application in the diagnostics and therapy of central nervous system diseases. However, since crucial information regarding side effects of particle–cell interactions within the central nervous system is still lacking, we investigated the influence of novel very small iron oxide particles or the clinically approved ferucarbotran or ferumoxytol on the vitality and morphology of brain cells. We exposed primary cell cultures of microglia and hippocampal neurons, as well as neuron–glia cocultures to varying concentrations of SPIOs for 6 and/or 24 hours, respectively. Here, we show that SPIO accumulation by microglia and subsequent morphological alterations strongly depend on the respective nanoparticle type. Microglial viability was severely compromised by high SPIO concentrations, except in the case of ferumoxytol. While ferumoxytol did not cause immediate microglial death, it induced severe morphological alterations and increased degeneration of primary neurons. Additionally, primary neurons clearly degenerated after very small iron oxide particle and ferucarbotran exposure. In neuron–glia cocultures, SPIOs rather stimulated the outgrowth of neuronal processes in a concentration- and particle-dependent manner. We conclude that the influence of SPIOs on brain cells not only depends on the particle type but also on the physiological system they are applied to. PMID:25792834

  12. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    PubMed

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. PMID:25782915

  13. Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells

    PubMed Central

    Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, Denis

    2015-01-01

    Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used. PMID:26498381

  14. Adenosine augments interleukin-10 production by microglial cells through an A2B adenosine receptor-mediated process

    PubMed Central

    Koscsó, Balázs; Csóka, Balázs; Selmeczy, Zsolt; Himer, Leonóra; Pacher, Pál; Virág, László; Haskó, György

    2011-01-01

    Microglia are activated by pathogen-associated molecular patterns and produce pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and is a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. Here, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of pro-inflammatory cytokines. Since the order of potency of selective AR agonists in inducing IL-10 production was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) ≥ 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS21680), and the A2BAR antagonist MRS-1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered shRNA blocked the enhancing effect of adenosine on IL-10 production confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia. PMID:22116830

  15. Generation and characterization of regulatory dendritic cells derived from murine induced pluripotent stem cells

    PubMed Central

    Zhang, Qi; Fujino, Masayuki; Iwasaki, Shizue; Hirano, Hiroshi; Cai, Songjie; Kitajima, Yuya; Xu, Jinhua; Li, Xiao-Kang

    2014-01-01

    Regulatory dendritic cells (DCregs) represent a potential therapeutic tool for assessing a variety of immune overreaction conditions; however, current approaches for generating DCregs for therapeutic purposes are limited. We attempted to generate and characterize DCregs from murine induced pluripotent stem (iPS) cells. The iPS cells co-cultured with OP9 cells displayed mesodermally differentiated flat colonies. GM-CSF drove most of the colonies exhibiting a differentiated morphology. Thereafter, cells became morphologically heterologous under the effects of TGF-β and IL-10. Most of the floating cells developed an irregular shape with areas of protrusion. The generated iPS-DCregs demonstrated high CD11b/c and low CD40, CD80, CD86 and MHC-II expressions with a high antigen uptake ability and poor T-cell stimulatory function. Importantly, iPS-DCregs showed immune responsiveness regulation effects both in vitro and in vivo and the ability to generate regulatory T-cells in vitro. Our result illustrates a feasible approach for generating functional DCregs from murine iPS cells. PMID:24496181

  16. In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells.

    PubMed

    Haase, Ingo; Knaup, Renate; Wartenberg, Maria; Sauer, Heinrich; Hescheler, Jürgen; Mahrle, Gustav

    2007-12-01

    Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments. PMID:17716780

  17. Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

    PubMed

    Ray, N B; Ewalt, L C; Lodmell, D L

    1995-02-01

    To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the

  18. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    SciTech Connect

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling; Liu, Wei-Wei; Xu, Qian; Xia, Ming-Yu; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2015-02-20

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.

  19. Microglial action in glioma: a boon turns bane.

    PubMed

    Ghosh, Anirban; Chaudhuri, Swapna

    2010-06-15

    Microglia has the potential to shape the neuroimmune defense with vast array of functional attributes. The cells prime infiltrated lymphocytes to retain their effector functions, play crucial role in controlling microenvironmental milieu and significantly participate in glioma. Reports demonstrate microglial accumulation in glioma and predict their assistance in glioma growth and spreading. Clarification of the 'double-edged' appearance of microglia is necessary to unfold its role in glioma biology. In this article the interpretation of microglial activities has been attempted to reveal their actual function in glioma. Contrary to the trendy acceptance of its glioma promoting infamy, accumulated evidences make an effort to view the state of affairs in favor of the cell. Critical scrutiny indicates that microglial immune assaults are intended to demolish the neoplastic cells in brain. But the weaponry of microglia has been tactically utilized by glioma in their favor as the survival strategy. Hence the defender appears as enemy in advanced glioma. PMID:20338195

  20. Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung.

    PubMed

    Aguilar, Susana; Nye, Emma; Chan, Jerry; Loebinger, Michael; Spencer-Dene, Bradley; Fisk, Nick; Stamp, Gordon; Bonnet, Dominique; Janes, Sam M

    2007-06-01

    Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well-differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies. Disclosure of potential conflicts of interest is found at the end of this article. PMID:17363552

  1. Developmental changes in microglial mobilization are independent of apoptosis in the neonatal mouse hippocampus.

    PubMed

    Eyo, Ukpong B; Miner, Samuel A; Weiner, Joshua A; Dailey, Michael E

    2016-07-01

    During CNS development, microglia transform from highly mobile amoeboid-like cells to primitive ramified forms and, finally, to highly branched but relatively stationary cells in maturity. The factors that control developmental changes in microglia are largely unknown. Because microglia detect and clear apoptotic cells, developmental changes in microglia may be controlled by neuronal apoptosis. Here, we assessed the extent to which microglial cell density, morphology, motility, and migration are regulated by developmental apoptosis, focusing on the first postnatal week in the mouse hippocampus when the density of apoptotic bodies peaks at postnatal day 4 and declines sharply thereafter. Analysis of microglial form and distribution in situ over the first postnatal week showed that, although there was little change in the number of primary microglial branches, microglial cell density increased significantly, and microglia were often seen near or engulfing apoptotic bodies. Time-lapse imaging in hippocampal slices harvested at different times over the first postnatal week showed differences in microglial motility and migration that correlated with the density of apoptotic bodies. The extent to which these changes in microglia are driven by developmental neuronal apoptosis was assessed in tissues from BAX null mice lacking apoptosis. We found that apoptosis can lead to local microglial accumulation near apoptotic neurons in the pyramidal cell body layer but, unexpectedly, loss of apoptosis did not alter overall microglial cell density in vivo or microglial motility and migration in ex vivo tissue slices. These results demonstrate that developmental changes in microglial form, distribution, motility, and migration occur essentially normally in the absence of developmental apoptosis, indicating that factors other than neuronal apoptosis regulate these features of microglial development. PMID:26576723

  2. Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation

    PubMed Central

    Liu, Jie; Huang, Dongping; Xu, Jing; Tong, Jiabin; Wang, Zishan; Huang, Li; Yang, Yufang; Bai, Xiaochen; Wang, Pan; Suo, Haiyun; Ma, Yuanyuan; Yu, Mei; Fei, Jian; Huang, Fang

    2015-01-01

    Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson’s disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD. PMID:26499517

  3. Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen.

    PubMed

    Xu, Zhengzhong; Meng, Chuang; Qiang, Bin; Gu, Hongyan; Sun, Lin; Yin, Yuelan; Pan, Zhiming; Chen, Xiang; Jiao, Xinan

    2015-01-01

    Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation. PMID:26473844

  4. Retinoic acid receptor agonist Am80 inhibits CXCL2 production from microglial BV-2 cells via attenuation of NF-κB signaling.

    PubMed

    Takaoka, Yuichiro; Takahashi, Moeka; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2016-09-01

    Accumulating lines of evidence suggest that retinoic acid receptor agonists such as Am80 exerts anti-inflammatory actions in the central nervous system, although detailed mechanisms of the action remain largely unknown. Our previous findings suggest that Am80 provides therapeutic effect on intracerebral hemorrhage in mice via suppression of expression of chemokine (C-X-C motif) ligand 2 (CXCL2). Here we investigated the mechanisms of inhibitory action of Am80 on expression of CXCL2 and other pro-inflammatory factors in microglial BV-2 cells. Pretreatment with Am80 markedly suppressed lipopolysaccharide (LPS)-induced expression of CXCL2 mRNA and release of CXCL2 protein. Am80 had no effect on LPS-induced activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. On the other hand, Am80 prevented LPS-induced nuclear translocation of p65 subunit of NF-κB complex. In addition, total expression levels of p65 and IκBα proteins, as well as of mRNAs encoding p65 and IκBα, were lowered by Am80. Dependence of CXCL2 expression on NF-κB was confirmed by the effect of an NF-κB inhibitor caffeic acid phenethyl ester that abolished LPS-induced CXCL2 expression. Caffeic acid phenethyl ester also abolished LPS-induced expression of inducible nitric oxide synthase, interleukin-1β and tumor necrosis factor α, which may be relevant to the inhibitory effect of Am80 on expression of these pro-inflammatory factors. We additionally found that Am80 attenuated LPS-induced up-regulation of CD14, a co-receptor for Toll-like receptor 4 (TLR4). These results suggest that inhibitory effect on TLR4 signaling mediated by NF-κB pathway underlies the anti-inflammatory action of retinoic acid receptor agonists in microglia. PMID:27351827

  5. Recruitment of Tat to Heterochromatin Protein HP1 via Interaction with CTIP2 Inhibits Human Immunodeficiency Virus Type 1 Replication in Microglial Cells

    PubMed Central

    Rohr, Olivier; Lecestre, Dominique; Chasserot-Golaz, Sylvette; Marban, Céline; Avram, Dorina; Aunis, Dominique; Leid, Mark; Schaeffer, Evelyne

    2003-01-01

    The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1α. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1α associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1α complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis. PMID:12692243

  6. Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.

    PubMed

    Feng, Zipei; Jensen, Shawn M; Messenheimer, David J; Farhad, Mohammed; Neuberger, Michael; Bifulco, Carlo B; Fox, Bernard A

    2016-05-01

    Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3(+)CD4(+) and CD3(+)CD8(+) T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution. PMID:26994219

  7. Multispectral Imaging of T and B Cells in Murine Spleen and Tumor

    PubMed Central

    Feng, Zipei; Jensen, Shawn M.; Messenheimer, David J.; Farhad, Mohammed; Neuberger, Michael; Bifulco, Carlo B.

    2016-01-01

    Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3+CD4+ and CD3+CD8+ T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution. PMID:26994219

  8. Compound C induces the ramification of murine microglia in an AMPK-independent and small rhogtpase-dependent manner.

    PubMed

    Huang, C; Lu, X; Wang, J L; Tong, L J; Ling, Y; Jiang, B; Yang, R R; Zhang, W

    2016-09-01

    Microglial cells are the pivotal immune cells of the central nervous system. Adult microglia cells under physiological conditions are in a ramification state with extensively branched processes. Upon disease stimulation, they retract their processes and become activated. Induction of ramification is an attracting strategy to terminate the excessive activation of microglia. Here, we investigated the influence of compound C (CC) on microglial shape. Results showed that CC reversibly induced a ramification of murine microglia in both basal and inflammatory conditions. These pro-ramification effects were independent of adenosine 5'-monophosphate-activated protein kinase (AMPK) inhibition as both AMPKα1 and AMPKα2 silence failed to induce microglial ramification. The ramification state of microglia induced by CC was associated with a decrease in pro-inflammatory factors and an increase in brain-derived neurotrophic factors (BDNF) protein and phagocytic activity. Mechanistic studies confirmed that the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signal, extracellular signal-regulated kinase 1/2 (ERK1/2) or small RhoGTPase activation mediated the effect of CC on microglial shape change based on the following observations: (i) CC induced a significant activation of the small RhoGTPase Rac1 and Cdc42; (ii) CC promoted the phosphorylation of ERK1/2 and Akt; (iii) inhibition of Rac1, Cdc42, ERK1/2, or the PI3K-Akt signal abolished the effect of CC on microglial shape change. These signal mechanisms were also ascertained in primary microglia. Our results explore a potential agent that promotes microglial ramification, and provide an alternative explanation for the neuroprotective effects of CC in various disease models such as brain ischemia and subarachnoid hemorrhage. PMID:27318303

  9. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

    PubMed

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  10. Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

    PubMed Central

    Lieberman, H B; Lin, P F; Yeh, D B; Ruddle, F H

    1988-01-01

    We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed. Images PMID:3244356