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Sample records for phagocytosis requires triacylglycerol

  1. The Breakdown of Stored Triacylglycerols Is Required during Light-Induced Stomatal Opening.

    PubMed

    McLachlan, Deirdre H; Lan, Jue; Geilfus, Christoph-Martin; Dodd, Antony N; Larson, Tony; Baker, Alison; Hõrak, Hanna; Kollist, Hannes; He, Zhesi; Graham, Ian; Mickelbart, Michael V; Hetherington, Alistair M

    2016-03-01

    Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control of water-use efficiency [2] in plants. Although the guard-cell-signaling pathway coupling blue light perception to ion channel activity is relatively well understood [3], we know less about the sources of ATP required to drive K(+) uptake [3-6]. Here, we show that triacylglycerols (TAGs), present in Arabidopsis guard cells as lipid droplets (LDs), are involved in light-induced stomatal opening. Illumination induces reductions in LD abundance, and this involves the PHOT1 and PHOT2 blue light receptors [3]. Light also induces decreases in specific TAG molecular species. We hypothesized that TAG-derived fatty acids are metabolized by peroxisomal β-oxidation to produce ATP required for stomatal opening. In silico analysis revealed that guard cells express all the genes required for β-oxidation, and we showed that light-induced stomatal opening is delayed in three TAG catabolism mutants (sdp1, pxa1, and cgi-58) and in stomata treated with a TAG breakdown inhibitor. We reasoned that, if ATP supply was delaying light-induced stomatal opening, then the activity of the plasma membrane H(+)-ATPase should be reduced at this time. Monitoring changes in apoplastic pH in the mutants showed that this was the case. Together, our results reveal a new role for TAGs in vegetative tissue and show that PHOT1 and PHOT2 are involved in reductions in LD abundance. Reductions in LD abundance in guard cells of the lycophyte Selaginella suggest that TAG breakdown may represent an evolutionarily conserved mechanism in light-induced stomatal opening. PMID:26898465

  2. The Breakdown of Stored Triacylglycerols Is Required during Light-Induced Stomatal Opening

    PubMed Central

    McLachlan, Deirdre H.; Lan, Jue; Geilfus, Christoph-Martin; Dodd, Antony N.; Larson, Tony; Baker, Alison; Hõrak, Hanna; Kollist, Hannes; He, Zhesi; Graham, Ian; Mickelbart, Michael V.; Hetherington, Alistair M.

    2016-01-01

    Summary Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control of water-use efficiency [2] in plants. Although the guard-cell-signaling pathway coupling blue light perception to ion channel activity is relatively well understood [3], we know less about the sources of ATP required to drive K+ uptake [3, 4, 5, 6]. Here, we show that triacylglycerols (TAGs), present in Arabidopsis guard cells as lipid droplets (LDs), are involved in light-induced stomatal opening. Illumination induces reductions in LD abundance, and this involves the PHOT1 and PHOT2 blue light receptors [3]. Light also induces decreases in specific TAG molecular species. We hypothesized that TAG-derived fatty acids are metabolized by peroxisomal β-oxidation to produce ATP required for stomatal opening. In silico analysis revealed that guard cells express all the genes required for β-oxidation, and we showed that light-induced stomatal opening is delayed in three TAG catabolism mutants (sdp1, pxa1, and cgi-58) and in stomata treated with a TAG breakdown inhibitor. We reasoned that, if ATP supply was delaying light-induced stomatal opening, then the activity of the plasma membrane H+-ATPase should be reduced at this time. Monitoring changes in apoplastic pH in the mutants showed that this was the case. Together, our results reveal a new role for TAGs in vegetative tissue and show that PHOT1 and PHOT2 are involved in reductions in LD abundance. Reductions in LD abundance in guard cells of the lycophyte Selaginella suggest that TAG breakdown may represent an evolutionarily conserved mechanism in light-induced stomatal opening. PMID:26898465

  3. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient

    NASA Technical Reports Server (NTRS)

    Sun, E.; Gao, Y.; Chen, J.; Roberts, A. I.; Wang, X.; Chen, Z.; Shi, Y.

    2004-01-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

  4. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient.

    PubMed

    Sun, E; Gao, Y; Chen, J; Roberts, A I; Wang, X; Chen, Z; Shi, Y

    2004-12-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance. PMID:15375386

  5. Quantitative Phagocytosis.

    ERIC Educational Resources Information Center

    McCallister, Zane Gary; McCallister, Gary Loren

    1996-01-01

    Presents a model experiment for quantifying phagocytosis using earthworm coelomocytes and determining the optimum length of time necessary to obtain maximum phagocytosis. Involves incubating coelomocytes from invertebrates with an antigen, staining the cells, counting the number of antigen particles ingested, and measuring the effect of different…

  6. Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells

    SciTech Connect

    Otsuka, Atsushi; Abe, Tadashi; Watanabe, Masami; Yagisawa, Hitoshi; Takei, Kohji; Yamada, Hiroshi

    2009-01-16

    Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

  7. Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

    PubMed Central

    Egami, Youhei; Fujii, Makoto; Kawai, Katsuhisa; Ishikawa, Yurie; Fukuda, Mitsunori; Araki, Nobukazu

    2015-01-01

    Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan. PMID:26229970

  8. Vav1 and PI3K are required for phagocytosis of beta-glucan and subsequent superoxide generation by microglia.

    PubMed

    Shah, Vaibhav B; Ozment-Skelton, Tammy R; Williams, David L; Keshvara, Lakhu

    2009-05-01

    Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. beta-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by beta-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of beta-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with beta-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to beta-glucans. PMID:19232731

  9. Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation.

    PubMed

    Lord, Caleb C; Ferguson, Daniel; Thomas, Gwynneth; Brown, Amanda L; Schugar, Rebecca C; Burrows, Amy; Gromovsky, Anthony D; Betters, Jenna; Neumann, Chase; Sacks, Jessica; Marshall, Stephanie; Watts, Russell; Schweiger, Martina; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Lathia, Justin D; Sakaguchi, Takuya F; Lehner, Richard; Haemmerle, Guenter; Zechner, Rudolf; Brown, J Mark

    2016-07-26

    Adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes. PMID:27396333

  10. Src is required for migration, phagocytosis, and interferon beta production in Toll-like receptor-engaged macrophages.

    PubMed

    Maa, Ming-Chei; Leu, Tzeng-Horng

    2016-06-01

    As an evolutionarily conserved mechanism, innate immunity controls self-nonself discrimination to protect a host from invasive pathogens. Macrophages are major participants of the innate immune system. Through the activation of diverse Toll-like receptors (TLRs), macrophages are triggered to initiate a variety of functions including locomotion, phagocytosis, and secretion of cytokines that requires the participation of tyrosine kinases. Fgr, Hck, and Lyn are myeloid-specific Src family kinases. Despite their constitutively high expression in macrophages, their absence does not impair LPS responsiveness. In contrast, Src, a barely detectable tyrosine kinase in resting macrophages, becomes greatly inducible in response to TLR engagement, implicating its role in macrophage activation. Indeed, silencing Src suppresses the activated TLR-mediated migration, phagocytosis, and interferon-beta (IFN-β) secretion in macrophages. And these physiological defects can be restored by the introduction of siRNA-resistant Src. Notably, the elevated expression and activity of Src is inducible nitric oxide synthase (iNOS)-dependent. Due to (1) iNOS being a NF-κB target, which can be induced by various TLR ligands, (2) Src can mediate NF-κB activation, therefore, there ought to exist a loop of signal amplification that regulates macrophage physiology in response to the engagement of TLRs. PMID:27514533

  11. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells

    SciTech Connect

    Zhu, Fei Yue, Wanfu; Wang, Yongxia

    2014-10-01

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages. - Highlights: • NF-κB cascade genes such as Nfκb1 and Traf1 were up-regulated by heat-inactivated S. aureus. • Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus. • NF-κB activation is required for phagocytosis of S. aureus by macrophages.

  12. Stimulation of phagocytosis by sulforaphane

    SciTech Connect

    Suganuma, Hiroyuki; Fahey, Jed W.; Bryan, Kelley E.; Healy, Zachary R.; Talalay, Paul

    2011-02-04

    Research highlights: {yields} Sulforaphane stimulates the phagocytosis of RAW 264.7 macrophages under conditions of serum deprivation. {yields} This effect does not require Nrf2-dependent induction of phase 2 genes. {yields} Inactivation of macrophage migration inhibitory factor (MIF) by sulforaphane may be involved in stimulation of phagocytosis by sulforaphane. -- Abstract: Sulforaphane, a major isothiocyanate derived from cruciferous vegetables, protects living systems against electrophile toxicity, oxidative stress, inflammation, and radiation. A major protective mechanism is the induction of a network of endogenous cytoprotective (phase 2) genes that are regulated by transcription factor Nrf2. To obtain a more detailed understanding of the anti-inflammatory and immunomodulatory effects of sulforaphane, we evaluated its effect on the phagocytosis activity of RAW 264.7 murine macrophage-like cells by measuring the uptake of 2-{mu}m diameter polystyrene beads. Sulforaphane raised the phagocytosis activity of RAW 264.7 cells but only in the absence or presence of low concentrations (1%) of fetal bovine serum. Higher serum concentrations depressed phagocytosis and abolished its stimulation by sulforaphane. This stimulation did not depend on the induction of Nrf2-regulated genes since it occurred in peritoneal macrophages of nrf2{sup -/-} mice. Moreover, a potent triterpenoid inducer of Nrf2-dependent genes did not stimulate phagocytosis, whereas sulforaphane and another isothiocyanate (benzyl isothiocyanate) had comparable inducer potencies. It has been shown recently that sulforaphane is a potent and direct inactivator of macrophage migration inhibitory factor (MIF), an inflammatory cytokine. Moreover, the addition of recombinant MIF to RAW 264.7 cells attenuated phagocytosis, but sulforaphane-inactivated MIF did not affect phagocytosis. The inactivation of MIF may therefore be involved in the phagocytosis-enhancing activity of sulforaphane.

  13. Inflammatory cytokine response to Bacillus anthracis peptidoglycan requires phagocytosis and lysosomal trafficking.

    PubMed

    Iyer, Janaki K; Khurana, Taruna; Langer, Marybeth; West, Christopher M; Ballard, Jimmy D; Metcalf, Jordan P; Merkel, Tod J; Coggeshall, K Mark

    2010-06-01

    During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease. PMID:20308305

  14. Phagocytosis of bacterial pathogens.

    PubMed

    Chung, Yoon-Suk Alexander; Kocks, Christine

    2012-01-01

    Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general. PMID:22223092

  15. Role of target geometry in phagocytosis

    PubMed Central

    Champion, Julie A.; Mitragotri, Samir

    2006-01-01

    Phagocytosis is a principal component of the body’s innate immunity in which macrophages internalize targets in an actin-dependent manner. Targets vary widely in shape and size and include particles such as pathogens and senescent cells. Despite considerable progress in understanding this complicated process, the role of target geometry in phagocytosis has remained elusive. Previous studies on phagocytosis have been performed using spherical targets, thereby overlooking the role of particle shape. Using polystyrene particles of various sizes and shapes, we studied phagocytosis by alveolar macrophages. We report a surprising finding that particle shape, not size, plays a dominant role in phagocytosis. All shapes were capable of initiating phagocytosis in at least one orientation. However, the local particle shape, measured by tangent angles, at the point of initial contact dictates whether macrophages initiate phagocytosis or simply spread on particles. The local shape determines the complexity of the actin structure that must be created to initiate phagocytosis and allow the membrane to move over the particle. Failure to create the required actin structure results in simple spreading and not internalization. Particle size primarily impacts the completion of phagocytosis in cases where particle volume exceeds the cell volume. PMID:16549762

  16. Regulation of phagocytosis by Rho GTPases

    PubMed Central

    Mao, Yingyu; Finnemann, Silvia C

    2015-01-01

    Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review. PMID:25941749

  17. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability

    PubMed Central

    Chen, Kejie; Shanmugam, Nanda Kumar N.; Pazos, Michael A.; Hurley, Bryan P.; Cherayil, Bobby J.

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  18. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

    PubMed

    Chen, Kejie; Shanmugam, Nanda Kumar N; Pazos, Michael A; Hurley, Bryan P; Cherayil, Bobby J

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  19. Ameobal Pathogen Mimivirus Infects Macrophages through Phagocytosis

    PubMed Central

    Ghigo, Eric; Kartenbeck, Jürgen; Lien, Pham; Pelkmans, Lucas; Capo, Christian; Mege, Jean-Louis; Raoult, Didier

    2008-01-01

    Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages. PMID:18551172

  20. 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

  1. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    PubMed

    Tafesse, Fikadu G; Rashidfarrokhi, Ali; Schmidt, Florian I; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L

    2015-10-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  2. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

    PubMed Central

    Schmidt, Florian I.; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L.

    2015-01-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  3. Triacylglycerol metabolism in adipose tissue

    PubMed Central

    Ahmadian, Maryam; Duncan, Robin E; Jaworski, Kathy; Sarkadi-Nagy, Eszter; Sul, Hei Sook

    2009-01-01

    Triacylglycerol (TAG) in adipose tissue serves as the major energy storage form in higher eukaryotes. Obesity, resulting from excess white adipose tissue, has increased dramatically in recent years resulting in a serious public health problem. Understanding of adipocyte-specific TAG synthesis and hydrolysis is critical to the development of strategies to treat and prevent obesity and its closely associated diseases, for example, Type 2 diabetes, hypertension and atherosclerosis. In this review, we present an overview of the major enzymes in TAG synthesis and lipolysis, including the recent discovery of a novel adipocyte TAG hydrolase. PMID:19194515

  4. JNK pathway activation is able to synchronize neuronal death and glial phagocytosis in Drosophila

    PubMed Central

    Shklover, J; Mishnaevski, K; Levy-Adam, F; Kurant, E

    2015-01-01

    Glial phagocytosis of superfluous neurons and damaged or aberrant neuronal material is crucial for normal development and maintenance of the CNS. However, the molecular mechanisms underlying the relationship between neuronal death and glial phagocytosis are poorly understood. We describe a novel mechanism that is able to synchronize neuronal cell death and glial phagocytosis of dying neurons in the Drosophila embryonic CNS. This mechanism involves c-Jun N-terminal kinase (JNK) signaling, which is required for developmental apoptosis of specific neurons during embryogenesis. We demonstrate that the dJNK pathway gain-of-function in neurons leads to dJNK signaling in glia, which results in upregulation of glial phagocytosis. Importantly, this promotion of phagocytosis is not mediated by upregulation of the glial phagocytic receptors SIMU and DRPR, but by increasing glial capacity to degrade apoptotic particles inside phagosomes. The proposed mechanism may be important for removal of damaged neurons in the developing and mature CNS. PMID:25695602

  5. Perilipin A and the control of triacylglycerol metabolism.

    PubMed

    Brasaemle, Dawn L; Subramanian, Vidya; Garcia, Anne; Marcinkiewicz, Amy; Rothenberg, Alexis

    2009-06-01

    Perilipin A is the most abundant protein associated with the lipid droplets of adipocytes and functions to control both basal and stimulated lipolysis. Under basal or fed conditions, perilipin A shields stored triacylglycerols from cytosolic lipases, thus promoting triacylglycerol storage. When catecholamines bind to cell surface receptors to initiate signals that activate cAMP-dependent protein kinase (PKA), phosphorylated perilipin A facilitates maximal lipolysis. Mutagenesis studies have revealed that central sequences of moderately hydrophobic amino acids are required to target nascent perilipin A to lipid droplets and provide an anchor into the hydrophobic environment of lipid droplets. Sequences of amino acids in the unique carboxyl terminus of perilipin A and those in amino terminal sequences flanking the first hydrophobic stretch are required for the barrier function of perilipin A in promoting triacylglycerol storage. Site-directed mutagenesis studies of serine residues within six PKA consensus sites of perilipin A reveal functions for phosphorylation of at least three of the sites. Phosphorylation of one or more of the serines within three amino terminal PKA sites is required to facilitate hormone-sensitive lipase access to lipid substrates. Phosphorylation of serines within two carboxyl terminal sites is also required for maximal lipolysis. Phosphorylation of serine 492 (site 5) triggers a massive remodeling of lipid droplets, whereby large peri-nuclear lipid droplets fragment into myriad lipid micro-droplets that scatter throughout the cytoplasm. We hypothesize that perilipin A binds accessory proteins to provide assistance in carrying out these functions. PMID:19116774

  6. Phagocytosis

    MedlinePlus Videos and Cool Tools

    Macrophages are scavenger cells that can ingest dead tissue and foreign cells. Macrophages form tentacles called pseudopods to surround an invader. Once inside the macrophage, the invader is walled off and ...

  7. Phagocytosis

    MedlinePlus

    Macrophages are scavenger cells that can ingest dead tissue and foreign cells. Macrophages form tentacles called pseudopods to surround an invader. Once inside the macrophage, the invader is walled off and then digested ...

  8. Particulate matter phagocytosis induces tissue factor in differentiating macrophages.

    PubMed

    Milano, M; Dongiovanni, P; Artoni, A; Gatti, S; Rosso, L; Colombo, F; Bollati, V; Maggioni, M; Mannucci, P M; Bertazzi, P A; Fargion, S; Valenti, L

    2016-01-01

    Airborne exposure to particulate matter with diameter < 10 mcM (PM10) has been linked to an increased risk of thromboembolic events, but the mechanisms are not completely understood. The aim of this study was to evaluate the effect of PM10 phagocytosis on the release of procoagulant molecules in human differentiating macrophages, and that of PM10 inhalation in an experimental model in rats. Human monocytes were separated from the peripheral blood by the lymphoprep method, differentiated in vitro and treated with standard PM10 or vehicle. Sprague-Dawley rats were instilled intratracheally with PM10 or vehicle alone. The outcome was expression of proinflammatory genes and of tissue factor (TF). In human differentiating macrophages, PM10 exposure upregulated inflammatory genes, but most consistently induced TF mRNA and protein levels, but not TF protein inhibitor, resulting in increased TF membrane expression and a procoagulant phenotype. Differentiation towards the anti-inflammatory M2 phenotype inhibited PM10 -mediated TF expression. TF induction required phagocytosis of PM10 , whereas phagocytosis of inert particles was less effective. PM10 phagocytosis was associated with a gene expression profile consistent with intracellular retention of iron, inducing oxidative stress. Both PM10 and iron activated the stress kinases ERK1/2 pathway, involved in the induction of TF expression. In rats, alveolar exposure to PM10 was associated with pulmonary recruitment of inflammatory cells and resulted in local, but not systemic, induction of TF expression, which was sufficient to increase circulating TF levels. In conclusion, TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure. PMID:25858758

  9. Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages.

    PubMed

    Sung, S S; Nelson, R S; Silverstein, S C

    1983-01-01

    We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 mug/ml and 10 mug/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles. PMID:6298248

  10. Hydration of Cuphea seeds containing crystallized triacylglycerols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seeds that exhibit intermediate storage behavior do not appear to survive under conventional -18C storage conditions. Cuphea wrightii, C. laminuligera, C. carthagenensis, and C. aequipetala are considered sensitive to low temperature storage. The seeds of these species have triacylglycerols (TAG) ...

  11. Photoreceptor phagocytosis is mediated by phosphoinositide signaling

    PubMed Central

    Mustafi, Debarshi; Kevany, Brian M.; Genoud, Christel; Bai, Xiaodong; Palczewski, Krzysztof

    2013-01-01

    Circadian oscillations in peripheral tissues, such as the retinal compartment of the eye, are critical to anticipating changing metabolic demands. Circadian shedding of retinal photoreceptor cell discs with subsequent phagocytosis by the neighboring retinal pigmented epithelium (RPE) is essential for removal of toxic metabolites and lifelong survival of these postmitotic neurons. Defects in photoreceptor phagocytosis can lead to severe retinal pathology, but the biochemical mechanisms remain poorly defined. By first documenting a 2.8-fold burst of photoreceptor phagocytosis events in the mouse eye in the morning compared with the afternoon by serial block face imaging, we established time points to assess transcriptional readouts by RNA sequencing (RNA-Seq). We identified 365 oscillating protein-coding transcripts that implicated the phosphoinositide lipid signaling network mediating the discrete steps of photoreceptor phagocytosis. Moreover, examination of overlapping cistromic sites by core clock transcription factors and promoter elements of these effector genes provided a functional basis for the circadian cycling of these transcripts. RNA-Seq also revealed oscillating expression of 16 long intergenic noncoding RNAs and key histone modifying enzymes critical for circadian gene expression. Our phenotypic and genotypic characterization reveals a complex global landscape of overlapping and temporally controlled networks driving the essential circadian process in the eye.—Mustafi, D., Kevany, B. M., Genoud, C., Bai, X., Palczewski, K. Photoreceptor phagocytosis is mediated by phosphoinositide signaling. PMID:23913857

  12. [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

  13. Induction of triacylglycerol production in Chlamydomonas reinhardtii: comparative analysis of different element regimes.

    PubMed

    Çakmak, Zeynep E; Ölmez, Tolga T; Çakmak, Turgay; Menemen, Yusuf; Tekinay, Turgay

    2014-03-01

    In this study, impacts of different element absence (nitrogen, sulfur, phosphorus and magnesium) and supplementation (nitrogen and zinc) on element uptake and triacylglycerol production was followed in wild type Chlamydomonas reinhardtii CC-124 strain. Macro- and microelement composition of C. reinhardtii greatly differed under element regimes studied. In particular, heavy metal quotas of the microalgae increased strikingly under zinc supplementation. Growth was suppressed, cell biovolume, carbohydrate, total neutral lipid and triacylglycerol levels increased when microalgae were incubated under these element regimes. Most of the intracellular space was occupied by lipid bodies under all nutrient starvations, as observed by confocal microscopy and transmission electron micrographs. Results suggest that sulfur, magnesium and phosphorus deprivations are superior to nitrogen deprivation for the induction triacylglycerol production in C. reinhardtii. On the other hand, FAME profiles of the nitrogen, sulfur and phosphorus deprived cells were found to meet the requirements of international standards for biodiesel. PMID:24472680

  14. Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae

    PubMed Central

    2011-01-01

    Microalgae have presented themselves as a strong candidate to replace diminishing oil reserves as a source of lipids for biofuels. Here we describe successful modifications of terrestrial plant lipid content which increase overall lipid production or shift the balance of lipid production towards lipid varieties more useful for biofuel production. Our discussion ranges from the biosynthetic pathways and rate limiting steps of triacylglycerol formation to enzymes required for the formation of triacylglycerol containing exotic lipids. Secondarily, we discuss techniques for genetic engineering and modification of various microalgae which can be combined with insights gained from research in higher plants to aid in the creation of production strains of microalgae. PMID:22047615

  15. Neutrophil-Mediated Phagocytosis of Staphylococcus aureus

    PubMed Central

    van Kessel, Kok P. M.; Bestebroer, Jovanka; van Strijp, Jos A. G.

    2014-01-01

    Initial elimination of invading Staphylococcus aureus from the body is mediated by professional phagocytes. The neutrophil is the major phagocyte of the innate immunity and plays a key role in the host defense against staphylococcal infections. Opsonization of the bacteria with immunoglobulins and complement factors enables efficient recognition by the neutrophil that subsequently leads to intracellular compartmentalization and killing. Here, we provide a review of the key processes evolved in neutrophil-mediated phagocytosis of S. aureus and briefly describe killing. As S. aureus is not helpless against the professional phagocytes, we will also highlight its immune evasion arsenal related to phagocytosis. PMID:25309547

  16. Simple method to detect triacylglycerol biosynthesis in a yeast-based recombinant system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyc...

  17. Small molecule phagocytosis inhibitors for immune cytopenias.

    PubMed

    Neschadim, Anton; Kotra, Lakshmi P; Branch, Donald R

    2016-08-01

    Immune cytopenias are conditions characterized by low blood cell counts, such as platelets in immune thrombocytopenia (ITP) and red blood cells in autoimmune hemolytic anemia (AIHA). Chronic ITP affects approximately 4 in 100,000 adults annually while AIHA is much less common. Extravascular phagocytosis and massive destruction of autoantibody-opsonized blood cells by macrophages in the spleen and liver are the hallmark of these conditions. Current treatment modalities for ITP and AIHA include the first-line use of corticosteroids; whereas, IVIg shows efficacy in ITP but not AIHA. One main mechanism of action by which IVIg treatment leads to the reduction in platelet destruction rates in ITP is thought to involve Fcγ receptor (FcγR) blockade, ultimately leading to the inhibition of extravascular platelet phagocytosis. IVIg, which is manufactured from the human plasma of thousands of donors, is a limited resource, and alternative treatments, particularly those based on bioavailable small molecules, are needed. In this review, we overview the pathophysiology of ITP, the role of Fcγ receptors, and the mechanisms of action of IVIg in treating ITP, and outline the efforts and progress towards developing novel, first-in-class inhibitors of phagocytosis as synthetic, small molecule substitutes for IVIg in ITP and other conditions where the pathobiology of the disease involves phagocytosis. PMID:27296447

  18. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes

    SciTech Connect

    Lutz, H.U.; Bussolino, F.; Flepp, R.; Fasler, S.; Stammler, P.; Kazatchkine, M.D.; Arese, P.

    1987-11-01

    Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3- antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.

  19. Triacylglycerol metabolism in isolated rat kidney cortex tubules.

    PubMed

    Wirthensohn, G; Guder, W G

    1980-01-15

    Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-(14)C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-beta-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-beta-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be

  20. LC3-associated phagocytosis: a crucial mechanism for antifungal host defence against Aspergillus fumigatus.

    PubMed

    Sprenkeler, Evelien G G; Gresnigt, Mark S; van de Veerdonk, Frank L

    2016-09-01

    LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway involved in the maturation of single-membrane phagosomes and subsequent killing of ingested pathogens by phagocytes. This pathway is initiated following recognition of pathogens by pattern recognition receptors and leads to the recruitment of LC3 into the phagosomal membrane. This form of phagocytosis is utilized for the antifungal host defence and is required for an efficient fungal killing. Here, we provide an overview of the LAP pathway and review the role of LAP in anti-Aspergillus host defence, as well as mechanisms induced by Aspergillus that modulate LAP to promote its survival in the host. PMID:27185357

  1. Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium

    PubMed Central

    Bloomfield, Gareth; Traynor, David; Sander, Sophia P; Veltman, Douwe M; Pachebat, Justin A; Kay, Robert R

    2015-01-01

    Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures. DOI: http://dx.doi.org/10.7554/eLife.04940.001 PMID:25815683

  2. Air-Drying of Cells, the Novel Conditions for Stimulated Synthesis of Triacylglycerol in a Green Alga, Chlorella kessleri

    PubMed Central

    Minoda, Ayumi; Tsuzuki, Mikio; Sato, Norihiro

    2013-01-01

    Triacylglycerol is used for the production of commodities including food oils and biodiesel fuel. Microalgae can accumulate triacylglycerol under adverse environmental conditions such as nitrogen-starvation. This study explored the possibility of air-drying of green algal cells as a novel and simple protocol for enhancement of their triacylglycerol content. Chlorella kessleri cells were fixed on the surface of a glass fibre filter and then subjected to air-drying with light illumination. The dry cell weight, on a filter, increased by 2.7-fold in 96 h, the corresponding chlorophyll content ranging from 1.0 to 1.3-fold the initial one. Concomitantly, the triacylglycerol content remarkably increased to 70.3 mole% of fatty acids and 15.9% (w/w), relative to total fatty acids and dry cell weight, respectively, like in cells starved of nitrogen. Reduction of the stress of air-drying by placing the glass filter on a filter paper soaked in H2O lowered the fatty acid content of triacylglycerol to 26.4 mole% as to total fatty acids. Moreover, replacement of the H2O with culture medium further decreased the fatty acid content of triacylglycerol to 12.2 mole%. It thus seemed that severe dehydration is required for full induction of triacylglycerol synthesis, and that nutritional depletion as well as dehydration are crucial environmental factors. Meanwhile, air-drying of Chlamydomonas reinhardtii cells increased the triacylglycerol content to only 37.9 mole% of fatty acids and 4.8% (w/w), relative to total fatty acids and dry cell weight, respectively, and a marked decrease in the chlorophyll content, on a filter, of 33%. Air-drying thus has an impact on triacylglycerol synthesis in C. reinhardtii also, however, the effect is considerably limited, owing probably to instability of the photosynthetic machinery. This air-drying protocol could be useful for the development of a system for industrial production of triacylglycerol with appropriate selection of the algal species. PMID

  3. The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila.

    PubMed

    Sieber, Matthew H; Thummel, Carl S

    2009-12-01

    Triacylglycerol (TAG) homeostasis is an integral part of normal physiology and essential for proper energy metabolism. Here we show that the single Drosophila ortholog of the PXR and CAR nuclear receptors, DHR96, plays an essential role in TAG homeostasis. DHR96 mutants are sensitive to starvation, have reduced levels of TAG in the fat body and midgut, and are resistant to diet-induced obesity, while DHR96 overexpression leads to starvation resistance and increased TAG levels. We show that DHR96 function is required in the midgut for the breakdown of dietary fat and that it exerts this effect through the CG5932 gastric lipase, which is essential for TAG homeostasis. This study provides insights into the regulation of dietary fat metabolism in Drosophila and demonstrates that the regulation of lipid metabolism is an ancestral function of the PXR/CAR/DHR96 nuclear receptor subfamily. PMID:19945405

  4. Phagocytosis Enhances Lysosomal and Bactericidal Properties by Activating the Transcription Factor TFEB.

    PubMed

    Gray, Matthew A; Choy, Christopher H; Dayam, Roya M; Ospina-Escobar, Erika; Somerville, Alexander; Xiao, Xuan; Ferguson, Shawn M; Botelho, Roberto J

    2016-08-01

    Macrophages internalize pathogens through phagocytosis, entrapping them into organelles called phagosomes. Phagosomes then fuse with lysosomes to mature into phagolysosomes, acquiring an acidic and hydrolytic lumen that kills the pathogens. During an ongoing infection, macrophages can internalize dozens of bacteria. Thus, we hypothesized that an initial round of phagocytosis might boost lysosome function and bactericidal ability to cope with subsequent rounds of phagocytosis. To test this hypothesis, we employed Fcγ-receptor-mediated phagocytosis and endocytosis, which internalize immunoglobulin G (IgG)-opsonized particles and polyvalent IgG immune complexes, respectively. We report that Fcγ receptor activation in macrophages enhances lysosome-based proteolysis and killing of subsequently phagocytosed E. coli compared to naive macrophages. Importantly, we show that Fcγ receptor activation causes nuclear translocation of TFEB, a transcription factor that boosts expression of lysosome genes. Indeed, Fc receptor activation is accompanied by increased expression of specific lysosomal proteins. Remarkably, TFEB silencing represses the Fcγ-receptor-mediated enhancements in degradation and bacterial killing. In addition, nuclear translocation of TFEB requires phagosome completion and fails to occur in cells silenced for MCOLN1, a lysosomal Ca(2+) channel, suggesting that lysosomal Ca(2+) released during phagosome maturation activates TFEB. Finally, we demonstrate that non-opsonic phagocytosis of E. coli also enhances lysosomal degradation in a TFEB-dependent manner, suggesting that this phenomenon is not limited to Fcγ receptors. Overall, we show that macrophages become better killers after one round of phagocytosis and suggest that phagosomes and lysosomes are capable of bi-directional signaling. PMID:27397893

  5. Triacylglycerol Metabolism, Function, and Accumulation in Plant Vegetative Tissues.

    PubMed

    Xu, Changcheng; Shanklin, John

    2016-04-29

    Oils in the form of triacylglycerols are the most abundant energy-dense storage compounds in eukaryotes, and their metabolism plays a key role in cellular energy balance, lipid homeostasis, growth, and maintenance. Plants accumulate oils primarily in seeds and fruits. Plant oils are used for food and feed and, increasingly, as feedstocks for biodiesel and industrial chemicals. Although plant vegetative tissues do not accumulate significant levels of triacylglycerols, they possess a high capacity for their synthesis, storage, and metabolism. The development of plants that accumulate oil in vegetative tissues presents an opportunity for expanded production of triacylglycerols as a renewable and sustainable bioenergy source. Here, we review recent progress in the understanding of triacylglycerol synthesis, turnover, storage, and function in leaves and discuss emerging genetic engineering strategies targeted at enhancing triacylglycerol accumulation in biomass crops. Such plants could potentially be modified to produce oleochemical feedstocks or nutraceuticals. PMID:26845499

  6. Phagocytosis in Teleosts. Implications of the New Cells Involved

    PubMed Central

    Esteban, María Ángeles; Cuesta, Alberto; Chaves-Pozo, Elena; Meseguer, José

    2015-01-01

    Phagocytosis is the process by which cells engulf some solid particles to form internal vesicles known as phagosomes. Phagocytosis is in fact a specific form of endocytosis involving the vesicular interiorization of particles. Phagocytosis is essentially a defensive reaction against infection and invasion of the body by foreign substances and, in the immune system, phagocytosis is a major mechanism used to remove pathogens and/or cell debris. For these reasons, phagocytosis in vertebrates has been recognized as a critical component of the innate and adaptive immune responses to pathogens. Furthermore, more recent studies have revealed that phagocytosis is also crucial for tissue homeostasis and remodeling. Professional phagocytes in teleosts are monocyte/macrophages, granulocytes and dendritic cells. Nevertheless, in recent years phagocytic properties have also been attributed to teleost lymphocytes and thrombocytes. The possible implications of such cells on this important biological process, new factors affecting phagocytosis, evasion of phagocytosis or new forms of phagocytosis will be considered and discussed. PMID:26690236

  7. Energy Metabolism of Human Neutrophils during Phagocytosis

    PubMed Central

    Borregaard, Niels; Herlin, Troels

    1982-01-01

    Detailed quantitative studies were performed on the generation and utilization of energy by resting and phagocytosing human neutrophils. The ATP content was 1.9 fmol/cell, was constant during rest, and was not influenced by the presence or absence of glucose in the medium. The intracellular content of phosphocreatine was less than 0.2 fmol/cell. In the presence of glucose, ATP was generated almost exclusively from lactate produced from glucose taken up from the surrounding medium. The amount of lactate produced could account for 85% of the glucose taken up by the cells, and the intracellular glycosyl store, glycogen, was not drawn upon. The rate of ATP generation as calculated from the rate of lactate production was 1.3 fmol/cell/min. During phagocytosis, there was no measurable increase in glucose consumption or lactate production, and the ATP content fell rapidly to 0.8 fmol/cell. This disappearance of ATP was apparently irreversible since no corresponding increase in ADP or AMP was observed. It therefore appears that this phagocytosis-induced fall in ATP concentration represents all the extra energy utilized in human neutrophils in the presence of glucose. In the absence of glucose, the rate of ATP generation in the resting cell was considerably smaller, 0.75 fmol/cell per min, as calculated from the rate of glycolysis, which is sustained exclusively by glycogenolysis. Under this condition, however, phagocytosis induces significant enhancement of glycogenolysis and the rate of lactate production is increased by 60%, raising the rate of ATP generation to 1.2 fmol/cell per min. Nonetheless, the ATP content drops significantly from 1.9 to 1.0 fmol/cell. Neutrophils from patients with chronic granulomatous disease have the same rate of glycolysis and the same ATP content as normal cells, thus confirming that the defective respiration of these cells does not affect their energy metabolism. PMID:7107894

  8. Pharmacologic enhancement or suppression of phagocytosis by bovine neutrophils.

    PubMed

    Paape, M J; Miller, R H; Ziv, G

    1991-02-01

    Sixty-three drugs, belonging to 10 chemical classes, were tested in vitro to determine effects on phagocytosis of 32P-labeled Staphylococcus aureus by neutrophils isolated from milk. Within each class, the number of antibiotics tested were: nonsteroidal anti-inflammatory drugs (NSAID; 8), peptolids (2), aminoglycosides (8), tetracyclines and fusidic acid (4), beta-lactam antibiotics (25), secretolytic agents (2), macrolides (5), polypeptides (2), and antibacterial quinolones (8). Percentage of phagocytosis was determined after incubating (2 hours at 37 C) 12.5 x 10(6) viable neutrophils, 200 x 10(6) 32P-labeled S aureus with antibiotics and 5% skimmed milk. Concentrations of antibiotics tested were 1,000, 500, and 10 micrograms/ml of incubation media. When compared with nonantibiotic controls at the highest drug concentration, the NSAID acetylsalicylic acid and centrophenoxine increased phagocytosis 23.2 and 8.8%, respectively, and benzydamine, indomethacin, phenylbutazone, ibuprofen, and acetominophen decreased phagocytosis 22.8, 14.2, 9.8, 27.0, and 18.2%, respectively. The peptolids novobiocin and pristinamycin decreased phagocytosis 24.5 and 22.0%, respectively. The aminoglycosides tobramycin, amikacin, and gentamicin decreased phagocytosis 21.1, 15.4, and 19.2%, respectively. For the tetracyclines and fusidic acid, minocycline and doxycycline decreased phagocytosis 39.8 and 54.2%, respectively. The beta-lactam antibiotics carfecillin, cephapirin sodium, and cephacetrile sodium decreased phagocytosis 11.2, 12.8, and 23.8%, respectively. The secretolytic agent, bromhexin, increased phagocytosis 10.8%. These data indicate that the potential for enhanced phagocytosis exists through use of some NSAID, and for depressed phagocytosis through use of aminoglycosides, peptolids, tetracyclines, and beta-lactams, as well as certain other NSAID. PMID:2012350

  9. Influenza-Specific Antibody-Dependent Phagocytosis

    PubMed Central

    Ana-Sosa-Batiz, Fernanda; Vanderven, Hillary; Jegaskanda, Sinthujan; Johnston, Angus; Rockman, Steven; Laurie, Karen; Barr, Ian; Reading, Patrick; Lichtfuss, Marit; Kent, Stephen J.

    2016-01-01

    Background Immunity to human influenza A virus (IAV) infection is only partially understood. Broadly non-neutralizing antibodies may assist in reducing disease but have not been well characterized. Methods We measured internalization of opsonized, influenza protein-coated fluorescent beads and live IAV into a monocytic cell line to study antibody-dependent phagocytosis (ADP) against multiple influenza hemagglutinin (HA) subtypes. We analyzed influenza HA-specific ADP in healthy human donors, in preparations of intravenous immunoglobulin (IVIG), and following IAV infection of humans and macaques. Results We found that both sera from healthy adults and IVIG preparations had broad ADP to multiple seasonal HA proteins and weak cross-reactive ADP to non-circulating HA proteins. The ADP in experimentally influenza-infected macaque plasma and naturally influenza-infected human sera mediated phagocytosis of both homologous and heterologous IAVs. Further, the IAV phagocytosed in an antibody-mediated manner had reduced infectivity in vitro. Conclusion We conclude that IAV infections in humans and macaques leads to the development of influenza-specific ADP that can clear IAV infection in vitro. Repeated exposure of humans to multiple IAV infections likely leads to the development of ADP that is cross-reactive to strains not previously encountered. Further analyses of the protective capacity of broadly reactive influenza-specific ADP is warranted. PMID:27124730

  10. Combining chromatography and chemometrics for the characterization and authentication of fats and oils from triacylglycerol compositional data--a review.

    PubMed

    Bosque-Sendra, Juan M; Cuadros-Rodríguez, Luis; Ruiz-Samblás, Cristina; de la Mata, A Paulina

    2012-04-29

    The characterization and authentication of fats and oils is a subject of great importance for market and health aspects. Identification and quantification of triacylglycerols in fats and oils can be excellent tools for detecting changes in their composition due to the mixtures of these products. Most of the triacylglycerol species present in either fats or oils could be analyzed and identified by chromatographic methods. However, the natural variability of these samples and the possible presence of adulterants require the application of chemometric pattern recognition methods to facilitate the interpretation of the obtained data. In view of the growing interest in this topic, this paper reviews the literature of the application of exploratory and unsupervised/supervised chemometric methods on chromatographic data, using triacylglycerol composition for the characterization and authentication of several foodstuffs such as olive oil, vegetable oils, animal fats, fish oils, milk and dairy products, cocoa and coffee. PMID:22483203

  11. A novel real time imaging platform to quantify macrophage phagocytosis.

    PubMed

    Kapellos, Theodore S; Taylor, Lewis; Lee, Heyne; Cowley, Sally A; James, William S; Iqbal, Asif J; Greaves, David R

    2016-09-15

    Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro. PMID:27475716

  12. The role of triacylglycerol in cardiac energy provision.

    PubMed

    Evans, Rhys D; Hauton, David

    2016-10-01

    Triacylglycerols (TAGs) constitute the main energy storage resource in mammals, by virtue of their high energy density. This in turn is a function of their highly reduced state and hydrophobicity. Limited water solubility, however, imposes specific requirements for delivery and uptake mechanisms on TAG-utilising tissues, including the heart, as well as intracellular disposition. TAGs constitute potentially the major energy supply for working myocardium, both through blood-borne provision and as intracellular TAG within lipid droplets, but also provide the heart with fatty acids (FAs) which the myocardium cannot itself synthesise but are required for glycerolipid derivatives with (non-energetic) functions, including membrane phospholipids and lipid signalling molecules. Furthermore they serve to buffer potentially toxic amphipathic fatty acid derivatives. Intracellular handling and disposition of TAGs and their FA and glycerolipid derivatives similarly requires dedicated mechanisms in view of their hydrophobic character. Dysregulation of utilisation can result in inadequate energy provision, accumulation of TAG and/or esterified species, and these may be responsible for significant cardiac dysfunction in a variety of disease states. This review will focus on the role of TAG in myocardial energy provision, by providing FAs from exogenous and endogenous TAG sources for mitochondrial oxidation and ATP production, and how this can change in disease and impact on cardiac function. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk. PMID:26979759

  13. Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in Triacylglycerol Biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER) localized GPAT for both of these critical metabolic pathways was recognized more...

  14. A Role for Connexin43 in Macrophage Phagocytosis and Host Survival after Bacterial Peritoneal Infection1

    PubMed Central

    Gribar, Steven C.; Richardson, Ward; Kohler, Jeff W.; Hoffman, Rosemary A.; Branca, Maria F.; Li, Jun; Shi, Xiao-Hua; Sodhi, Chhinder P.; Hackam, David J.

    2016-01-01

    The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection. PMID:19050272

  15. Lyar Is a New Ligand for Retinal Pigment Epithelial Phagocytosis.

    PubMed

    Guo, Feiye; Ding, Ying; Caberoy, Nora B; Alvarado, Gabriela; Liu, Robert; Shen, Chen; Yu, Jisu; Zhou, Yixiong; Salero, Enrique; LeBlanc, Michelle E; Wang, Weiwen; Li, Wei

    2015-10-01

    Phagocytosis is critical to tissue homeostasis, as highlighted by phagocytosis defect of retinal pigment epithelial (RPE) cells with debris accumulation, photoreceptor degeneration and blindness. Phagocytosis ligands are the key to delineating molecular mechanisms and functional roles of phagocytes, but are traditionally identified in individual cases with technical challenges. We recently developed open reading frame phage display (OPD) for phagocytosis-based functional cloning (PFC) to identify unknown ligands. One of the identified ligands was Ly-1 antibody reactive clone (Lyar) with functions poorly defined. Herein, we characterized Lyar as a new ligand to stimulate RPE phagocytosis. In contrast to its reported nucleolar expression, immunohistochemistry showed that Lyar was highly expressed in photoreceptor outer segments (POSs) of the retina. Cytoplasmic Lyar was released from apoptotic cells, and selectively bound to shed POSs and apoptotic cells, but not healthy cells. POS vesicles engulfed through Lyar-dependent pathway were targeted to phagosomes and colocalized with phagosome marker Rab7. These results suggest that Lyar is a genuine RPE phagocytosis ligand, which in turn supports the validity of OPD/PFC as the only available approach for unbiased identification of phagocytosis ligands with broad applicability to various phagocytes. PMID:25735755

  16. Epitope specificity and isotype of monoclonal anti-D antibodies dictate their ability to inhibit phagocytosis of opsonized platelets.

    PubMed

    Kjaersgaard, Mimi; Aslam, Rukhsana; Kim, Michael; Speck, Edwin R; Freedman, John; Stewart, Donald I H; Wiersma, Erik J; Semple, John W

    2007-08-15

    Rh immune globulin (WinRho SDF; Cangene, Mississauga, ON, Canada) is an effective treatment for autoimmune thrombocytopenic purpura; however, maintaining a sustained supply for its use in autoimmune thrombocytopenic purpura and its primary indication, hemolytic disease of the newborn, makes the development of alternative reagents desirable. We compared Rh immune globulin and 6 human monoclonal anti-D antibodies (MoAnti-D) with differing isotypes and specificities for their ability to opsonize erythrocytes and inhibit platelet phagocytosis in an in vitro assay. Results demonstrated that opsonization of erythrocytes with Rh immune globulin significantly (P < .001) reduced phagocytosis of fluorescently labeled opsonized platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (P < .001) inhibit platelet phagocytosis. In contrast, 2 MoAnti-D shared isotypes and differed in specificity; however, only one could significantly (P < .001) inhibit platelet phagocytosis. The results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonized platelet phagocytosis. PMID:17456719

  17. PNPLA3 mediates hepatocyte triacylglycerol remodeling.

    PubMed

    Ruhanen, Hanna; Perttilä, Julia; Hölttä-Vuori, Maarit; Zhou, You; Yki-Järvinen, Hannele; Ikonen, Elina; Käkelä, Reijo; Olkkonen, Vesa M

    2014-04-01

    The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3(I148M)) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3(WT)) and PNPLA3(I148M) on HuH7 cell lipidome after [(13)C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3(I148M) induced a net accumulation of unlabeled TAGs, but not newly synthesized total [(13)C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3(WT) and PNPLA3(I148M) induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3(WT) associated in PCA with newly synthesized [(13)C]TAGs, particularly 52:1 and 50:1, while PNPLA3(I148M) associated with similar preexisting TAGs. PNPLA3(WT) overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3(I148M). PNPLA3(I148M) localized more extensively to lipid droplets (LDs) than PNPLA3(WT), suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3(I148M). PMID:24511104

  18. Metabolic engineering Corynebacterium glutamicum to produce triacylglycerols.

    PubMed

    Plassmeier, Jens; Li, Youyuan; Rueckert, Christian; Sinskey, Anthony J

    2016-01-01

    In this study, we metabolically engineered Corynebacterium glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (iii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis), and (iv) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38 ± 0.05 g/L intracellular fatty acids and 0.64 ± 0.09 g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular fatty acids content of 17.8 ± 0.5% of the dry cell. PMID:26645801

  19. Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

    PubMed

    Chen, Xiuping; Guo, Feiye; LeBlanc, Michelle E; Ding, Ying; Zhang, Chenming; Shakya, Akhalesh; Li, Wei

    2016-08-01

    Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance. PMID:27184668

  20. Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis

    PubMed Central

    Botelho, Roberto J.; Teruel, Mary; Dierckman, Renee; Anderson, Richard; Wells, Alan; York, John D.; Meyer, Tobias; Grinstein, Sergio

    2000-01-01

    Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP2) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP2 accompanied by recruitment of type Iα phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP2 correlated with mobilization of phospholipase Cγ (PLCγ) and with the localized formation of diacylglycerol. The presence of 4,5-PIP2 and active PLCγ at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling. PMID:11134066

  1. Plasticity and constraints on fatty acid composition in the phospholipids and triacylglycerols of Arabidopsis accessions grown at different temperatures

    PubMed Central

    2013-01-01

    Background Natural selection acts on multiple traits in an organism, and the final outcome of adaptive evolution may be constrained by the interaction of physiological and functional integration of those traits. Fatty acid composition is an important determinant of seed oil quality. In plants the relative proportions of unsaturated fatty acids in phospholipids and seed triacylglycerols often increases adaptively in response to lower growing temperatures to increase fitness. Previous work produced evidence of genetic constraints between phospholipids and triacylglycerols in the widely studied Arabidopsis lines Col and Ler, but because these lines are highly inbred, the correlations might be spurious. In this study, we grew 84 wild Arabidopsis accessions at two temperatures to show that genetic correlation between the fatty acids of the two lipid types is not expected and one should not influence the other and seed oil evolution and also tested for the adaptive response of fatty acids to latitude and temperature. Results As expected no significant correlations between the two lipids classes at either growing temperature were observed. The saturated fatty acids and erucic acid of triacylglycerols followed a significant latitudinal cline, while the fatty acids in phospholipids did not respond to latitude as expected. The expected plastic response to temperature was observed for all the triacylglycerol fatty acids whereas only oleic acid showed the expected pattern in phospholipids. Considerable phenotypic variation of the fatty acids in both the lipid types was seen. Conclusion We report the first evidence supporting adaptive evolution of seed triacylglycerols in Arabidopsis on a latitudinal cline as seen in other species and also their plastic adaptive response to growing temperature. We show that as expected there is no genetic correlations between the fatty acids in triacylglycerols and phospholipids, indicating selection can act on seed triacylglycerols without

  2. The Phagocytosis and Toxicity of Amorphous Silica

    PubMed Central

    Costantini, Lindsey M.; Gilberti, Renée M.; Knecht, David A.

    2011-01-01

    cases. However, the result suggests a mechanistic difference between FcγRIIA receptor-mediated and non-opsonized silica particle phagocytosis. PMID:21311600

  3. Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis.

    PubMed

    Li, Xuefeng; He, Sisi; Zhou, Xikun; Ye, Yan; Tan, Shirui; Zhang, Shuang; Li, Rongpeng; Yu, Min; Jundt, Michael C; Hidebrand, Alec; Wang, Yongsheng; Li, Guoping; Huang, Canhua; Wu, Min

    2016-01-01

    Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection. PMID:26735693

  4. Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

    PubMed Central

    Zhou, Xikun; Ye, Yan; Tan, Shirui; Zhang, Shuang; Li, Rongpeng; Yu, Min; Jundt, Michael C.; Hidebrand, Alec; Wang, Yongsheng; Li, Guoping; Huang, Canhua; Wu, Min

    2016-01-01

    Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection. PMID:26735693

  5. A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation.

    PubMed

    Coates, Christopher J; Whalley, Tim; Wyman, Michael; Nairn, Jacqueline

    2013-11-01

    Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge. PMID:23925540

  6. Extracellular amastigotes of Trypanosoma cruzi are potent inducers of phagocytosis in mammalian cells

    PubMed Central

    Fernandes, Maria Cecilia; Flannery, Andrew R; Andrews, Norma; Mortara, Renato A

    2013-01-01

    The protozoan parasite Trypanosoma cruzi, the aetiological agent of Chagas' disease, has two infective life cycle stages, trypomastigotes and amastigotes. While trypomastigotes actively enter mammalian cells, highly infective extracellular amastigotes (type I T. cruzi) rely on actin-mediated uptake, which is generally inefficient in non-professional phagocytes. We found that extracellular amastigotes (EAs) of T. cruzi G strain (type I), but not Y strain (type II), were taken up 100-fold more efficiently than inert particles. Mammalian cell lines showed levels of parasite uptake comparable to macrophages, and extensive actin recruitment and polymerization was observed at the site of entry. EA uptake was not dependent on parasite-secreted molecules and required the same molecular machinery utilized by professional phagocytes during large particle phagocytosis. Transcriptional silencing of synaptotagmin VII and CD63 significantly inhibited EA internalization, demonstrating that delivery of supplemental lysosomal membrane to form the phagosome is involved in parasite uptake. Importantly, time-lapse live imaging using fluorescent reporters revealed phagosome-associated modulation of phosphoinositide metabolism during EA uptake that closely resembles what occurs during phagocytosis by macrophages. Collectively, our results demonstrate that T. cruzi EAs are potent inducers of phagocytosis in non-professional phagocytes, a process that may facilitate parasite persistence in infected hosts. PMID:23241026

  7. Phagocytosis of Aspergillus fumigatus conidia by primary nasal epithelial cells in vitro

    PubMed Central

    Botterel, Françoise; Gross, Karine; Ibrahim-Granet, Oumaïma; Khoufache, Khaled; Escabasse, Virginie; Coste, André; Cordonnier, Catherine; Escudier, Estelle; Bretagne, Stéphane

    2008-01-01

    Background Invasive aspergillosis, which is mainly caused by the fungus Aspergillus fumigatus, is an increasing problem in immunocompromised patients. Infection occurs by inhalation of airborne conidia, which are first encountered by airway epithelial cells. Internalization of these conidia into the epithelial cells could serve as a portal of entry for this pathogenic fungus. Results We used an in vitro model of primary cultures of human nasal epithelial cells (HNEC) at an air-liquid interface. A. fumigatus conidia were compared to Penicillium chrysogenum conidia, a mould that is rarely responsible for invasive disease. Confocal microscopy, transmission electron microscopy, and anti-LAMP1 antibody labeling studies showed that conidia of both species were phagocytosed and trafficked into a late endosomal-lysosomal compartment as early as 4 h post-infection. In double immunolabeling experiments, the mean percentage of A. fumigatus conidia undergoing phagocytosis 4 h post-infection was 21.8 ± 4.5%. Using combined staining with a fluorescence brightener and propidium iodide, the mean rate of phagocytosis was 18.7 ± 9.3% and the killing rate 16.7 ± 7.5% for A. fumigatus after 8 h. The phagocytosis rate did not differ between the two fungal species for a given primary culture. No germination of the conidia was observed until 20 h of observation. Conclusion HNEC can phagocytose fungal conidia but killing of phagocytosed conidia is low, although the spores do not germinate. This phagocytosis does not seem to be specific to A. fumigatus. Other immune cells or mechanisms are required to kill A. fumigatus conidia and to avoid further invasion. PMID:18564423

  8. HIV-1 TAT Inhibits Microglial Phagocytosis of Aβ Peptide

    PubMed Central

    Giunta, Brian; Zhou, Yuyan; Hou, Huayan; Rrapo, Elona; Fernandez, Francisco; Tan, Jun

    2008-01-01

    Human immunodeficiency virus (HIV)-associated dementia (HAD) is a subcortical neuropsychiatric syndrome that has increased in prevalence in the era of highly active antiretroviral therapy (HAART). Several studies demonstrated increased amyloidosis in brains of HIV patients and suggested that there may be a significant number of long-term HIV survivors with co-morbid Alzheimer's disease (AD) in the future. We show HIV-1 Tat protein inhibits microglial uptake of Aβ1-42 peptide, a process that is enhanced by interferon-gamma (IFN-γ) and rescued by the STAT1 inhibitor (-)-epigallocatechin-3-gallate (EGCG). It is hypothesized that reduced Aβ uptake occurs through IFN-γ mediated STAT1 activation. This process promotes a switch from a phagocytic to an antigen presenting phenotype in microglia through activation of class II transactivator (CIITA). Additionally, we show that HIV-1 Tat significantly disrupts apolipoprotein-3 (Apo-E3) promoted microglial Aβ uptake. As Tat has been shown to directly interact with the low density lipoprotein (LRP) receptor and thus inhibit the uptake of its ligands including apolipoprotein E4 (Apo-E4) and Aβ peptide in neurons, we further hypothesize that a similar inhibition of LRP may occur in microglia. Future studies will be required to fully characterize the mechanisms underlying IFN-γ enhancement of HIV-1 Tats disruption of microglial phagocytosis of Aβ and Apo-E3. PMID:18784813

  9. Identification of Characteristic Fatty Acids to Quantify Triacylglycerols in Microalgae

    PubMed Central

    Shen, Pei-Li; Wang, Hai-Tao; Pan, Yan-Fei; Meng, Ying-Ying; Wu, Pei-Chun; Xue, Song

    2016-01-01

    The fatty acid profiles of lipids from microalgae are unique. Polyunsaturated fatty acids are generally enriched in polar lipids, whereas saturated and monounsaturated fatty acids constitute the majority of fatty acids in triacylglycerols (TAG). Each species has characteristic fatty acids, and their content is positively or negatively correlated with TAGs. The marine oleaginous diatom Phaeodactylum tricornutum was used as the paradigm to determine the quantitative relationship between TAG and characteristic fatty acid content. Fatty acid profiles and TAG content of Phaeodactylum tricornutum were determined in a time course. C16:0/C16:1 and eicosapentaenoic acid (EPA, C20:5n3) were identified as characteristic fatty acids in TAGs and polar lipids, respectively. The percentage of those characteristic fatty acids in total fatty acids had a significant linear relationship with TAG content, and thus, the correlation coefficient presenting r2 were 0.96, 0.94, and 0.97, respectively. The fatty acid-based method for TAG quantification could also be applied to other microalgae such as Nannochloropsis oceanica in which the r2 of C16:0 and EPA were 0.94 and 0.97, respectively, and in Chlorella pyrenoidosa r2-values for C18:1 and C18:3 with TAG content were 0.91 and 0.99, repectively. This characteristic fatty acid-based method provided a distinct way to quantify TAGs in microalgae, by which TAGs could be measured precisely by immediate transesterification from wet biomass rather than using conventional methods. This procedure simplified the operation and required smaller samples than conventional methods. PMID:26941747

  10. Identification of Characteristic Fatty Acids to Quantify Triacylglycerols in Microalgae.

    PubMed

    Shen, Pei-Li; Wang, Hai-Tao; Pan, Yan-Fei; Meng, Ying-Ying; Wu, Pei-Chun; Xue, Song

    2016-01-01

    The fatty acid profiles of lipids from microalgae are unique. Polyunsaturated fatty acids are generally enriched in polar lipids, whereas saturated and monounsaturated fatty acids constitute the majority of fatty acids in triacylglycerols (TAG). Each species has characteristic fatty acids, and their content is positively or negatively correlated with TAGs. The marine oleaginous diatom Phaeodactylum tricornutum was used as the paradigm to determine the quantitative relationship between TAG and characteristic fatty acid content. Fatty acid profiles and TAG content of Phaeodactylum tricornutum were determined in a time course. C16:0/C16:1 and eicosapentaenoic acid (EPA, C20:5n3) were identified as characteristic fatty acids in TAGs and polar lipids, respectively. The percentage of those characteristic fatty acids in total fatty acids had a significant linear relationship with TAG content, and thus, the correlation coefficient presenting r (2) were 0.96, 0.94, and 0.97, respectively. The fatty acid-based method for TAG quantification could also be applied to other microalgae such as Nannochloropsis oceanica in which the r (2) of C16:0 and EPA were 0.94 and 0.97, respectively, and in Chlorella pyrenoidosa r (2)-values for C18:1 and C18:3 with TAG content were 0.91 and 0.99, repectively. This characteristic fatty acid-based method provided a distinct way to quantify TAGs in microalgae, by which TAGs could be measured precisely by immediate transesterification from wet biomass rather than using conventional methods. This procedure simplified the operation and required smaller samples than conventional methods. PMID:26941747

  11. Melanization and phagocytosis: implications for age related macular degeneration.

    PubMed

    Sarangarajan, Rangaprasad; Apte, Shireesh P

    2005-01-01

    Signaling pathways that upregulate melanization in the retinal pigment epithelium (RPE) may also be implicated in the downregulation of rod outer segment (ROS) phagocytosis by the RPE. Melanization activating pathways may also modulate oxygen consumption by the photoreceptors, apolipoprotein E4 levels, and the rate of photoisomerization events such that the net effect may be a reduction in drusen and/or lipofuscin accumulation. An increase in melanin at the apical microvilli of the RPE may shield ROS from light thereby contributing in part to the decrease in the rate of ROS phagocytosis. This decrease in ROS phagocytosis by the RPE may serve to maintain a balance between ingestion and degradation/recycling thereby avoiding an increase to its already substantial metabolic load. Several experimental drugs for age related macular degeneration (ARMD) coincidentally are also capable of decreasing the rate of ROS phagocytosis. This review attempts to identify the signaling pathways that may link the upregulation of melanization to the downregulation of ROS phagocytosis. Phagocytic pathways that are modulated by melanization need to be studied in isolation to determine what role, if any, they possess in ameliorating the onset and progression of ARMD. Many more empirical studies are needed to unravel specific pathways and mechanisms that seem to link melanization with ARMD. PMID:16030499

  12. Host-pathogen interactions: leukocyte phagocytosis and associated sequelae.

    PubMed

    Voyich, Jovanka M; DeLeo, Frank R

    2002-01-01

    Polymorphonuclear leukocytes (PMNs) are a critical component of the human innate immune response and are the first line of defense against invading microorganisms. Phagocytosis of invading microbes induces production of reactive oxygen species (ROS) by PMNs, which facilitates bactericidal activity. In addition to eliminating microorganisms, phagocytosis also accelerates PMN apoptosis, a process critical for resolution of inflammation. Inasmuch as leukocyte phagocytosis and ROS production are key components of the innate immune response, we developed flow cytometric methods to evaluate these processes in human PMNs. In contrast to traditional microscopy-based analyses, the methods described herein provide objective and high throughput measures of host cell-pathogen interactions. Importantly, they can be adapted for use with a number of fluorometric probes, and bacterium and host cell of choice, and each is based upon a common phagocytosis assay system. We also describe methods to measure phagocytosis-induced PMN apoptosis with this assay system. These methods entail detecting surface-exposed phosphatidylserine (early apoptosis), and measuring PMN chromatin condensation and DNA fragmentation (late apoptosis). Taken together, these assays provide rapid and accurate assessment of critical PMN processes. PMID:12815296

  13. Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

    PubMed Central

    Coleman, R A; Haynes, E B

    1984-01-01

    The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta. PMID:6696738

  14. Critical ratios for structural analysis of triacylglycerols using mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent developments have finally allowed fragment behaviors using APCI-MS to be elucidated after twenty years of literature reports. Critical Ratios have been defined that correspond to various aspects of triacylglycerol (TAG) analysis, from overall degree of unsaturation to localization of fatty ac...

  15. Investigations of biomechanical activity of macrophages during phagocytosis

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel; Curtis, Jennifer

    2012-02-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways that trigger the process and lead to the deformation of the cell as it engulfs a target. Physical changes in the cell include rearrangement and polymerization of actin in the phagocytic cup, large membrane deformations, increased membrane area via exocytosis, and closure of the phagocytic cup through membrane fusion. Hence, phagocytosis is a fine-tuned balance between biophysical cellular events and chemical signaling, which are responsible for driving these materials and mechanical changes. We present a series of assays designed to probe the physical/mechanical parameters that govern a cell during phagocytosis. Custom built micropipette manipulators are used to manipulate individual cells, facilitating high-resolution microscopy of individual phagocytic events. This work has been supported by NSF PoLS #0848797.

  16. Inhibition of phagocytosis by high molecular weight hyaluronate.

    PubMed Central

    Forrester, J V; Balazs, E A

    1980-01-01

    The effect of sodium hyaluronate on phagocytosis was studied using a sensitive polystyrene latex sphere assay in mouse peritoneal macrophage monolayers. Viscous solutions of high molecular weight hyaluronate (4.6 X 10(5)--2.8 X 10(6)) caused a dose-dependent inhibition of phagocytosis, but low molecular weight hyaluronate (9.0 X 10(4)) was not inhibitory at equivalent viscosity. The inhibitory effect of high molecular weight hyaluronate did not appear to be mediated by the polyanionic charge of the molecule since sulphated glycosaminoglycans with greater charge density (heparin and chondroitin sulphate) were ineffective. In addition, competitive inhibition studies indicated that a direct effect on possible cell surface membrane receptors was unlikely. Instead, physical factors such as steric hindrance by the continuous polymeric network, were considered of more importance. Alternatively, the hydrophilic polysaccharide may have inhibited phagocytosis by providing an unsuitable surface for adhesive contact between the latex beads and the cell surface. PMID:7429537

  17. Altered microglial phagocytosis in GPR34-deficient mice.

    PubMed

    Preissler, Julia; Grosche, Antje; Lede, Vera; Le Duc, Diana; Krügel, Katja; Matyash, Vitali; Szulzewsky, Frank; Kallendrusch, Sonja; Immig, Kerstin; Kettenmann, Helmut; Bechmann, Ingo; Schöneberg, Torsten; Schulz, Angela

    2015-02-01

    GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis. PMID:25142016

  18. CD13 mediates phagocytosis in human monocytic cells.

    PubMed

    Licona-Limón, Ileana; Garay-Canales, Claudia A; Muñoz-Paleta, Ofelia; Ortega, Enrique

    2015-07-01

    CD13 is a membrane-bound ectopeptidase, highly expressed on monocytes, macrophages, and dendritic cells. CD13 is involved in diverse functions, including degradation of peptide mediators, cellular adhesion, migration, viral endocytosis, signaling, and positive modulation of phagocytosis mediated by FcγRs and other phagocytic receptors. In this work, we explored whether besides acting as an accessory receptor, CD13 by itself is a primary phagocytic receptor. We found that hCD13 mediates efficient phagocytosis of large particles (erythrocytes) modified so as to interact with the cell only through CD13 in human macrophages and THP-1 monocytic cells. The extent of this phagocytosis is comparable with the phagocytosis mediated through the canonical phagocytic receptor FcγRI. Furthermore, we demonstrated that hCD13 expression in the nonphagocytic cell line HEK293 is sufficient to enable these cells to internalize particles bound through hCD13. CD13-mediated phagocytosis is independent of other phagocytic receptors, as it occurs in the absence of FcγRs, CR3, and most phagocytic receptors. Phagocytosis through CD13 is independent of its enzymatic activity but is dependent on actin rearrangement and activation of PI3K and is partially dependent on Syk activation. Moreover, the cross-linking of CD13 with antibodies rapidly induced pSyk in human macrophages. Finally, we observed that antibody-mediated cross-linking of hCD13, expressed in the murine macrophage-like J774 cell line, induces production of ROS. These results demonstrate that CD13 is a fully competent phagocytic receptor capable of mediating internalization of large particles. PMID:25934926

  19. Identification of a Chemoattractant G-Protein-Coupled Receptor for Folic Acid that Controls Both Chemotaxis and Phagocytosis.

    PubMed

    Pan, Miao; Xu, Xuehua; Chen, Yong; Jin, Tian

    2016-02-22

    Eukaryotic phagocytes search and destroy invading microorganisms via chemotaxis and phagocytosis. The social amoeba Dictyostelium discoideum is a professional phagocyte that chases bacteria through chemotaxis and engulfs them as food via phagocytosis. G-protein-coupled receptors (GPCRs) are known for detecting chemoattractants and directing cell migration, but their roles in phagocytosis are not clear. Here, we developed a quantitative phosphoproteomic technique to discover signaling components. Using this approach, we discovered the long sought after folic acid receptor, fAR1, in D. discoideum. We showed that the seven-transmembrane receptor fAR1 is required for folic acid-mediated signaling events. Significantly, we discovered that fAR1 is essential for both chemotaxis and phagocytosis of bacteria, thereby representing a chemoattractant GPCR that mediates not only chasing but also ingesting bacteria. We revealed that a phagocyte is able to internalize particles via a chemoattractant-mediated engulfment process. We propose that mammalian phagocytes may also use this mechanism to engulf and ingest bacterial pathogens. PMID:26906738

  20. Target-specific mechanics of phagocytosis: protrusive neutrophil response to zymosan differs from the uptake of antibody-tagged pathogens

    PubMed Central

    Lee, Cheng-Yuk; Herant, Marc; Heinrich, Volkmar

    2011-01-01

    The physical mechanisms that control target-specific responses of human neutrophils to distinct immune threats are poorly understood. Using dual-micropipette manipulation, we have quantified and compared the time courses of neutrophil phagocytosis of two different targets: zymosan (a prominent model of fungal infection), and antibody-coated (Fc) particles. Our single-live-cell/single-target approach exposes surprising differences between these two forms of phagocytosis. Unlike the efficient uptake of 3-μm Fc targets (within ~66 seconds), the engulfment of similarly sized zymosan is slow (~167 seconds), mainly due to the formation of a characteristic pedestal that initially pushes the particle outwards by ~1 μm. Despite a roughly twofold difference in maximum cortical tensions, the top ‘pull-in’ speeds of zymosan and Fc targets are indistinguishable at ~33 nm/second. Drug inhibition shows that both actin as well as myosin II partake in the regulation of neutrophil cortical tension and cytoplasmic viscosity; other than that, myosin II appears to play a minor role in both forms of phagocytosis. Remarkably, an intact actin cytoskeleton is required to suppress, in antibody-mediated phagocytosis, the initially protrusive deformation that distinguishes the neutrophil response to zymosan. PMID:21385838

  1. Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes

    PubMed Central

    Rengarajan, Michelle; Hayer, Arnold; Theriot, Julie A.

    2016-01-01

    Vascular endothelial cells act as gatekeepers that protect underlying tissue from blood-borne toxins and pathogens. Nevertheless, endothelial cells are able to internalize large fibrin clots and apoptotic debris from the bloodstream, although the precise mechanism of such phagocytosis-like uptake is unknown. We show that cultured primary human endothelial cells (HUVEC) internalize both pathogenic and non-pathogenic Listeria bacteria comparably, in a phagocytosis-like process. In contrast with previously studied host cell types, including intestinal epithelial cells and hepatocytes, we find that endothelial internalization of Listeria is independent of all known pathogenic bacterial surface proteins. Consequently, we exploited the internalization and intracellular replication of L. monocytogenes to identify distinct host cell factors that regulate phagocytosis-like uptake in HUVEC. Using siRNA screening and subsequent genetic and pharmacologic perturbations, we determined that endothelial infectivity was modulated by cytoskeletal proteins that normally modulate global architectural changes, including phosphoinositide-3-kinase, focal adhesions, and the small GTPase Rho. We found that Rho kinase (ROCK) is acutely necessary for adhesion of Listeria to endothelial cells, whereas the actin-nucleating formins FHOD1 and FMNL3 specifically regulate internalization of bacteria as well as inert beads, demonstrating that formins regulate endothelial phagocytosis-like uptake independent of the specific cargo. Finally, we found that neither ROCK nor formins were required for macrophage phagocytosis of L. monocytogenes, suggesting that endothelial cells have distinct requirements for bacterial internalization from those of classical professional phagocytes. Our results identify a novel pathway for L. monocytogenes uptake by human host cells, indicating that this wily pathogen can invade a variety of tissues by using a surprisingly diverse suite of distinct uptake mechanisms that

  2. Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes.

    PubMed

    Rengarajan, Michelle; Hayer, Arnold; Theriot, Julie A

    2016-05-01

    Vascular endothelial cells act as gatekeepers that protect underlying tissue from blood-borne toxins and pathogens. Nevertheless, endothelial cells are able to internalize large fibrin clots and apoptotic debris from the bloodstream, although the precise mechanism of such phagocytosis-like uptake is unknown. We show that cultured primary human endothelial cells (HUVEC) internalize both pathogenic and non-pathogenic Listeria bacteria comparably, in a phagocytosis-like process. In contrast with previously studied host cell types, including intestinal epithelial cells and hepatocytes, we find that endothelial internalization of Listeria is independent of all known pathogenic bacterial surface proteins. Consequently, we exploited the internalization and intracellular replication of L. monocytogenes to identify distinct host cell factors that regulate phagocytosis-like uptake in HUVEC. Using siRNA screening and subsequent genetic and pharmacologic perturbations, we determined that endothelial infectivity was modulated by cytoskeletal proteins that normally modulate global architectural changes, including phosphoinositide-3-kinase, focal adhesions, and the small GTPase Rho. We found that Rho kinase (ROCK) is acutely necessary for adhesion of Listeria to endothelial cells, whereas the actin-nucleating formins FHOD1 and FMNL3 specifically regulate internalization of bacteria as well as inert beads, demonstrating that formins regulate endothelial phagocytosis-like uptake independent of the specific cargo. Finally, we found that neither ROCK nor formins were required for macrophage phagocytosis of L. monocytogenes, suggesting that endothelial cells have distinct requirements for bacterial internalization from those of classical professional phagocytes. Our results identify a novel pathway for L. monocytogenes uptake by human host cells, indicating that this wily pathogen can invade a variety of tissues by using a surprisingly diverse suite of distinct uptake mechanisms that

  3. Failure of Immune Sera to Enhance Significantly Phagocytosis of Staphylococus aureus: Nonspecific Adsorption of Phagocytosis-Promoting Factors

    PubMed Central

    Shayegani, Mehdi

    1970-01-01

    Serum from rabbits immunized with either heat-killed or live nonencapsulated Staphylococcus aureus failed further to enhance phagocytosis and intracellular killing of the homologous organism by either normal rabbit polymorphonuclear leukocytes or monocytes, when compared with normal rabbit serum. These immune sera did, however, show an increase in agglutinating and precipitating antibody level. Adsorption of normal human serum with some gram-positive and gram-negative bacteria, yeast, and some inert particles significantly reduced the phagocytosis-promoting factors of the serum. It would seem, then, that nonencapsulated S. aureus differs from other pathogenic bacteria in that the humoral antibacterial factors promoting its phagocytosis and intracellular killing are not significantly enhanced by infection or immunization. PMID:16557910

  4. Structured triacylglycerol containing behenic and oleic acids suppresses triacylglycerol absorption and prevents obesity in rats

    PubMed Central

    2010-01-01

    Background Dietary 1(3)-behenoyl-2,3(1)-dioleoyl-rac-glycerol (BOO) has been reported to inhibit pancreatic lipase activity in vitro and suppress postprandial hypertriacylglycerolemia in humans. In the present study, the anti-obesity activities of BOO and its inhibitory effects on lymphatic triacylglycerol (TAG) absorption were investigated in rats. Methods In Experiment 1, rats were fed either BOO or soybean oil (SO) diet for 6 weeks. In the BOO diet, 20% of SO was replaced with an experimental oil rich in BOO. In Experiments 2 and 3, rats cannulated in the thoracic duct were administered an emulsions containing trioleoylglycerol (OOO) or an oil mixture (OOO:BOO, 9:1). Tri[1-14C]oleoylglycerol (14C-OOO) was added to the emulsions administered in Experiment 3. Results No observable differences were detected in food intake or body weight gain between the BOO and SO groups in Experiment 1. Plasma and liver TAG concentrations and visceral fat weights were significantly lower in the BOO group than in the SO group. The apparent absorption rate of fat was significantly lower in the BOO group than in the SO group. In Experiment 2, the lymphatic recovery of oleic and behenic acids was significantly lower at 5 and 6 h after BOO administration than after OOO administration. In Experiment 3, the lymphatic recovery of 14C-OOO was significantly lower at 5 and 6 h after BOO administration than after OOO administration. Conclusions These results suggest that BOO prevents deposition of visceral fat and hepatic TAG by lowering and delaying intestinal absorption of TAG. PMID:20653972

  5. Red palm oil-supplemented and biofortified gari on the carotenoid and retinyl palmitate concentrations of triacylglycerol-rich plasma of women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Boiled biofortified cassava containing ß-carotene (BC) can increase retinyl palmitate (RP) in triacylglycerol (TAG)-rich plasma. Thus, it might alleviate vitamin A deficiency. Cassava requires extensive preparation to decrease its level of cyanogenic glucosides, which can be fatal. Garification ...

  6. ACTIVATED NEUTROPHILS INHIBIT PHAGOCYTOSIS BY HUMAN MONOCYTE CELLS IN VITRO

    EPA Science Inventory

    We have previously reported the correlation of decreased phagocytosis of opsonized zymosan by sputum monocytic cells with the increase in sputum neutrophils in volunteers 6h after inhalation of endotoxin (20,000 EU) (Alexis, et al. JACI, 2003;112:353). To define whether an intrin...

  7. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

    PubMed Central

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition. PMID:25904329

  8. Bovine-associated CNS species resist phagocytosis differently

    PubMed Central

    2013-01-01

    Background Coagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only comprising slight changes in the appearance of milk and possibly slight swelling. However, clinical mastitis with severe signs has also been reported. The reasons for the differences in clinical expression are largely unknown. Macrophages play an important role in the innate immunity of the udder. This study examined phagocytosis and killing by mouse macrophage cells of three CNS species: Staphylococcus chromogenes (15 isolates), Staphylococcus agnetis (6 isolates) and Staphylococcus simulans (15 isolates). Staphylococcus aureus (7 isolates) was also included as a control. Results All the studied CNS species were phagocytosed by macrophages, but S. simulans resisted phagocytosis more effectively than the other CNS species. Only S. chromogenes was substantially killed by macrophages. Significant variations between isolates were seen in both phagocytosis and killing by macrophages and were more common in the killing assays. Significant differences between single CNS species and S. aureus were observed in both assays. Conclusion This study demonstrated that differences in the phagocytosis and killing of mastitis-causing staphylococci by macrophages exist at both the species and isolate level. PMID:24207012

  9. Triacylglycerol profiling of marine microalgae by mass spectrometry.

    PubMed

    Danielewicz, Megan A; Anderson, Lisa A; Franz, Annaliese K

    2011-11-01

    We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and ¹H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels. PMID:21840867

  10. Synthesis and tandem mass spectrometry of chlorinated triacylglycerols.

    PubMed

    Lefsay, Abir M; Guy, Robert D; Chatt, Amares; White, Robert L

    2013-09-01

    The incorporation of 9,10-dichlorooctadecanoyl groups using enzyme-catalyzed acylation and protecting group strategies yielded specific regioisomers of di- and tetrachlorinated triacylglycerols. Hexachloro- and hexabromotriacylglycerols were synthesized by addition of chlorine or bromine to tri-(cis-9-octadecenoyl)glycerol. Upon electrospray ionization and tandem mass spectrometry, the sodium adduct ions of all compounds containing a 9,10-dichlorooctadecanoyl group readily lost two molecules of HCl when subjected to collision-induced dissociation. A mechanism describing sequential HCl losses and the formation of a conjugated diene is proposed for the loss of both vicinal chlorine atoms from an alkyl chain. This characteristic fragmentation behavior and the availability of characterized standards will facilitate the development of quantitative analytical methods for the determination of chlorinated triacylglycerols in lipid mixtures isolated from marine and other biological sources. PMID:23872189

  11. Isolation and characterization of a mucosal triacylglycerol pool undergoing hydrolysis

    SciTech Connect

    Tipton AD IV; Frase, S.; Mansbach, C.M. II )

    1989-12-01

    Absorbed and processed mucosal neutral lipid has been shown to be composed of at least two pools of triacylglycerol. One is likely to subserve chylomicron formation, and the other appears to be transported from the intestine via a nonlymphatic route. In the present study, 50 +/- 5% of the mucosal lipid pellets was centrifuged at 75,000 g.min (low-speed pellet (LSP)). Discontinuous sucrose density gradient centrifugation of LSP showed that 61 +/- 7% of the lipid banded at the 0.25-0.86 M sucrose interface. Neutral lipid analysis showed that this subfraction was only 58% triacylglycerol, suggesting it was undergoing hydrolysis. Active lipolytic activity in vitro was found on incubation. The lipase had an alkaline pH optimum (pH 8.5) and persisted despite pancreatic ductular diversion. Lipolysis in vivo in a LSP fraction was shown by infusing (14C)glyceryltrioleate for 3.5 h followed by (3H)glyceryltrioleate for 30 min. Discontinuous sucrose density centrifugation of the LSP followed by an analysis of the lipids at the 0.25-0.86 M sucrose interface showed that 14C-neutral lipids were only 70 +/- 6% triacylglycerol, whereas 3H-neutral lipids were 88 +/- 2% triacylglycerol. 3H entered LSP slowly compared with the floating lipid in the same centrifuge tube. These studies suggest both in vivo and in vitro mucosal lipolysis by a specific, alkaline-active lipase. The turnover rate of LSP is likely to be slow by comparison with neutral lipid floating to the top of the centrifuge tube.

  12. Anion Exchanger 2 Regulates Dectin-1-Dependent Phagocytosis and Killing of Candida albicans.

    PubMed

    Urso, Katia; Charles, Julia F; Shull, Gary E; Aliprantis, Antonios O; Balestrieri, Barbara

    2016-01-01

    Anion exchanger 2 (Ae2; gene symbol, Slc4a2) is a plasma membrane Cl-/HCO3- exchanger expressed in the gastrointestinal tract, kidney and bone. We have previously shown that Ae2 is required for the function of osteoclasts, bone resorbing cells of the macrophage lineage, to maintain homeostatic cytoplasmic pH and electroneutrality during acid secretion. Macrophages require endosomal acidification for pathogen killing during the process known as phagocytosis. Chloride is thought to be the principal ion responsible for maintaining electroneutrality during organelle acidification, but whether Cl-/HCO3- exchangers such as Ae2 contribute to macrophage function is not known. In this study we investigated the role of Ae2 in primary macrophages during phagocytosis. We find that Ae2 is expressed in macrophages where it regulates intracellular pH and the binding of Zymosan, a fungal cell wall derivative. Surprisingly, the transcription and surface expression of Dectin-1, the major phagocytic receptor for Candida albicans (C. albicans) and Zymosan, is reduced in the absence of Ae2. As a consequence, Zymosan-induced Tnfα expression is also impaired in Ae2-deficient macrophages. Similar to Ae2 deficiency, pharmacological alkalinization of lysosomal pH with bafilomycin A decreases both Dectin-1 mRNA and cell surface expression. Finally, Ae2-deficient macrophages demonstrate defective phagocytosis and killing of the human pathogenic fungus C. albicans. Our results strongly suggest that Ae2 is a critical factor in the innate response to C. albicans. This study represents an important contribution to a better understanding of how Dectin-1 expression and fungal clearance is regulated. PMID:27391897

  13. Anion Exchanger 2 Regulates Dectin-1-Dependent Phagocytosis and Killing of Candida albicans

    PubMed Central

    Urso, Katia; Charles, Julia F.; Shull, Gary E.; Aliprantis, Antonios O.; Balestrieri, Barbara

    2016-01-01

    Anion exchanger 2 (Ae2; gene symbol, Slc4a2) is a plasma membrane Cl-/HCO3- exchanger expressed in the gastrointestinal tract, kidney and bone. We have previously shown that Ae2 is required for the function of osteoclasts, bone resorbing cells of the macrophage lineage, to maintain homeostatic cytoplasmic pH and electroneutrality during acid secretion. Macrophages require endosomal acidification for pathogen killing during the process known as phagocytosis. Chloride is thought to be the principal ion responsible for maintaining electroneutrality during organelle acidification, but whether Cl-/HCO3- exchangers such as Ae2 contribute to macrophage function is not known. In this study we investigated the role of Ae2 in primary macrophages during phagocytosis. We find that Ae2 is expressed in macrophages where it regulates intracellular pH and the binding of Zymosan, a fungal cell wall derivative. Surprisingly, the transcription and surface expression of Dectin-1, the major phagocytic receptor for Candida albicans (C. albicans) and Zymosan, is reduced in the absence of Ae2. As a consequence, Zymosan-induced Tnfα expression is also impaired in Ae2-deficient macrophages. Similar to Ae2 deficiency, pharmacological alkalinization of lysosomal pH with bafilomycin A decreases both Dectin-1 mRNA and cell surface expression. Finally, Ae2-deficient macrophages demonstrate defective phagocytosis and killing of the human pathogenic fungus C. albicans. Our results strongly suggest that Ae2 is a critical factor in the innate response to C. albicans. This study represents an important contribution to a better understanding of how Dectin-1 expression and fungal clearance is regulated. PMID:27391897

  14. [Change of leukocytic phagocytosis during repeat hemoperfusion with cross-linked agar beads entrapped attapulgite clay].

    PubMed

    Huang, Wei; Ma, Yu; Yang, Xiaolan; Tang, Xianjue; Shu, Changda

    2003-06-01

    The leukocytic phagocytosis rate and the index of phagocytosis of rats on cross-linked agar beads entrapped attapulgite clay (CAA) hemoperfusion were studied. The results revealed that the leukocytic phagocytosis rate and the index of phagocytosis descended significantly after 1 hour and rose gradually after 6 hours. Finally it reached the normal level after 48 hours. Hemoperfusion repeated two times gave similar results. In conclusion, the function of leukocytic phagocytosis declined temporarily during CAA hemoperfusion. Many times hemoperfusion will not notably affect the body's defense system of rats. PMID:12856604

  15. Mechanisms of glucocorticoid induced suppression of phagocytosis in murine peritoneal macrophage cultures

    SciTech Connect

    Becker, J.L.

    1986-01-01

    Glucocorticoids suppress phagocytosis of heat killed Saccharomyces cerevisiae in macrophage cultures. In order to determine the mechanisms by which this response occurs, this investigation was initiated to examine whether the suppression of phagocytosis is mediated by a steroid induced phagocytosis inhibitory protein (PIP). Furthermore, it is postulated that these suppressive effects may be associated with alterations in macrophage phospholipid metabolism. To assess the association between phospholipid metabolism and phagocytosis, control and 1 ..mu..M dexamethasone treated macrophages were exposed to the phospholipase inhibitor bromophenacylbromide. The enzyme inhibitor suppressed phagocytosis in a time and dose dependent manner. However, supplying dexamethasone treated cultures with arachidonate did not reverse the steroid induced suppression of phagocytosis, whether the arachidonate was supplied alone or together with indomethacin and nordihydroguaiaretic acid. Control cells, prelabeled with /sup 3/H-arachidonate, exhibited an increased percentage of the radiolabeled fatty acid in neutral lipids following phagocytosis, with a corresponding decrease in the percentage associated with phosphatidylcholine.

  16. Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet phagocytosis by macrophages.

    PubMed

    Wan, Shu-Wen; Yang, Yi-Wen; Chu, Ya-Ting; Lin, Chiou-Feng; Chang, Chih-Peng; Yeh, Trai-Ming; Anderson, Robert; Lin, Yee-Shin

    2016-03-01

    Thrombocytopenia is an important clinical manifestation of dengue disease. The hypotheses concerning the pathogenesis of thrombocytopenia include decreased production and increased destruction or consumption of platelets. We previously suggested a mechanism of molecular mimicry in which antibodies (Abs) directed against dengue virus (DENV) nonstructural protein 1 (NS1) cross-react with platelets. Furthermore, several lines of evidence show activation of endothelial cells (ECs) and macrophages are related to dengue disease severity. Previous studies also suggested that Ab-opsonised platelets facilitate the engulfment of platelets by macrophages. Here we show that TNF-α-activated ECs upregulate adhesion molecule expression to enhance the binding of platelets and macrophages and lead to anti-DENV NS1 Ab-mediated platelet phagocytosis. We further demonstrate that the interaction between macrophages and TNF-α-activated ECs requires binding of FcγR with the Fc region of platelet-bound anti-DENV NS1 Abs. Importantly, the binding of anti-DENV NS1 Abs to platelets did not interfere with platelet adhesion to ECs. The adhesion molecules ICAM-1 and β3 integrin expressed on ECs as well as the FcγR expressed on macrophages were critical in anti-DENV NS1 Ab-mediated platelet phagocytosis on activated ECs. Moreover, anti-DENV NS1 Abs dramatically enhanced platelet engulfment by macrophages in a murine model of DENV infection. Our study provides evidence for a novel role for anti-DENV NS1 Abs in the pathogenesis of thrombocytopenia in dengue disease by enhancing platelet phagocytosis by macrophages. PMID:26632672

  17. Lipopolysaccharide O-Antigen Prevents Phagocytosis of Vibrio anguillarum by Rainbow Trout (Oncorhynchus mykiss) Skin Epithelial Cells

    PubMed Central

    Lindell, Kristoffer; Fahlgren, Anna; Hjerde, Erik; Willassen, Nils-Peder; Fällman, Maria; Milton, Debra L.

    2012-01-01

    Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues. PMID:22662189

  18. [The phagocytosis of polymorphonuclear neutrophilic granulocytes in progressive periodontitis].

    PubMed

    Konopka, T; Zietek, M

    1995-01-01

    The aim of this paper was the evaluation of the phagocytic activity of neutrophils in blood and in gingival pocket fluid in patients suffering from rapidly progressive periodontitis (RPP) and postjuvenile periodontitis (PJP). Prior to periodontal treatment the authors evaluated the capacity to phagocytose latex particles of peripheral blood neutrophils from 21 patients with RPP, 51 with PJP and 59 healthy subjects (control group) as well as the phagocytic activity of neutrophils in pocket fluid from 21 patients with RPP, 14 with PJP and from 20 healthy subjects. This phagocytic activity was significantly lower in all examined groups in comparison with the control group. A similar evaluation executed 3 months after treatment revealed normal phagocytosis of blood neutrophils from patients with RPP. In patients receiving complementary pharmacotherapy (spiramycine combined with metronidazol), a better improvement of phagocytosis was noted, than that observed in patients treated only surgically. PMID:7481699

  19. Phagocytosis in pup and adult harbour, grey and harp seals.

    PubMed

    Frouin, Héloïse; Lebeuf, Michel; Hammill, Mike; Fournier, Michel

    2010-04-15

    Knowledge on pinniped immunology is still in its infancy. For instance, age-related and developmental aspects of the immune system in pinnipeds need to be better described. The present study examined the phagocytic activity and efficiency of harbour, grey and harp seal leukocytes. In the first part of the study, peripheral blood was collected from captive female harbour seals of various ages. Data showed an age-related decrease in phagocytosis in female harbour seals from sub-adult to adulthood. In the second part of the study, changes in phagocytosis were quantified during lactation in wild newborn harbour, grey and harp seals and in their mothers (harp and grey seals). In newborns of the same age, leukocytes of harbour and harp seals phagocytosed less than those of grey seal pups. The phagocytic activity and efficiency increased significantly from early to mid-lactation in newborn harbour seals, and from early to late lactation in newborn grey seals, which could suggest that the transfer of phagocytosis-promoting factor(s) in colostrum is an important feature of temporary protection for pups. In contrast, no changes in phagocytic activity and efficiency were observed in lactating females of the two seal species, harp and grey, examined. At late lactation, phagocytic activity in both grey and harp seal pups and phagocytic efficiency in grey seal pups were significantly higher than in their mothers. These results could reflect either the capacity of phagocytes of the newborn harp and grey seals to respond to pathogens. Results from this study suggest that the phagocytosis of the seal species examined is not fully developed at birth as it generally increases in pups during lactation. Thereafter, the phagocytic activity of seals appears to decrease throughout adulthood. PMID:19766324

  20. Control of triacylglycerol biosynthesis in plants

    SciTech Connect

    Not Available

    1993-01-31

    Seeds of most species of the Umbelliferae (Apiaciae), Araliaceae, and Garryaceae families are characterized by their high content of the unusual C[sub 18] monounsaturated fatty acid petroselinic acid (18:l[Delta][sup 6cis]). Prior to a recent report of this lab, little was known of the biosynthetic origin of the cis[Delta][sup 6] double bond of petroselinic acid. Such knowledge may be of both biochemical and biotechnological significance. Because petroselinic acid is potentially the product of a novel desaturase, information regarding its synthesis may contribute to an understanding of fatty acid desaturation mechanisms in plants. Through chemical cleavage at its double bond, petroselinic acid can be used as a precursor of lauric acid (12:0), a component of detergents and surfactants, and adipic acid (6:0 dicarboxylic), the monomeric component of nylon 6,6. Therefore, the development of an agronomic source of an oil rich in petroselinic acid is of biotechnological interest. As such, studies of petroselinic acid biosynthesis may provide basic information required for any attempt to genetically engineer the production and accumulation of this fatty acid in an existing oilseed.

  1. Involvement of protein kinase C in phagocytosis of human retinal pigment epithelial cells and induction of matrix metalloproteinase secretion.

    PubMed

    Irschick, Eveline U; Haas, Gertrud; Troger, Josef; Ueberall, Florian; Huemer, Hartwig P

    2009-10-01

    Protein kinase C (PKC) is involved in cell activation. We investigated PKC-mediated pathways and secretion of matrix metalloproteinases (MMPs) in phagocytosis by human retinal pigment epithelial cells (RPE). We used time-resolved fluorometry for europium-labeled microsphere uptake and gel zymography to assay the influence of PKC modulators. PKC inhibitors blocked phagocytosis by RPE. ARPE-19, a human RPE-cell line, showed reduced secretion of MMP-2, although MMP-9 secretion by PKC activation was conserved in both cell types, namely in the primary RPEs and in the RPE-cell line. Particle uptake by RPE cells requires activation of PKC; the use of PKC inhibitors as new anticancer drugs may possibly cause ocular side-effects. PMID:18641922

  2. Hyposialylated α1-acid glycoprotein inhibits phagocytosis of feline neutrophils.

    PubMed

    Rossi, G; Capitani, L; Ceciliani, F; Restelli, L; Paltrinieri, S

    2013-10-01

    Feline α1-acid glycoprotein (fAGP) modifies both its serum concentration and its glycan moiety during diseases. fAGP is hyposialylated in cats with feline infectious peritonitis (FIP), but not in clinically healthy cats or in cats with other diseases. This study was aimed to determine whether hyposialylated fAGP influences phagocytosis. A flow cytometric method based on ingestion of fluoresceinated bacteria and adapted to feline blood was used to assess phagocytosis of leukocytes incubated with 'non-pathological' fAGP (purified from sera with normal concentrations of AGP) and 'pathological' fAGP (purified from sera with >1.5mg/mL hyposialylated AGP). The flow cytometric method provided repeatable results for neutrophils (coefficients of variations, CVs <15%) but not for monocytes (CVs>20%) which had also a high individual variability. Compared with saline solution and with non-pathological fAGP, pathological fAGP significantly decreased phagocytosis in neutrophils and monocytes. This study demonstrated that hyposialylated fAGP down-regulates the phagocytic activity of feline neutrophils. PMID:23726663

  3. It is all about fluidity: Fatty acids and macrophage phagocytosis.

    PubMed

    Schumann, Julia

    2016-08-15

    Phagocytosis is an early and fundamental step for the effective clearance of disease causing agents. The ability to engulf and kill pathogens is considered as a major effector function of macrophages. In their phagocytic role macrophages are part of the first line of innate immune defense. A number of studies investigating fatty acid effects on macrophage phagocytosis have been conducted over many years. In vitro-data consistently report that alterations in macrophage membrane fatty acid composition are linked to an altered phagocytic capacity, i.e. an increase in membrane unsaturated fatty acid content is associated with an increase in engulfment and killing rate. The mode of action of fatty acids seems to be the modulation of the physical nature of the macrophage plasma membrane. It appears that the saturated-to-unsaturated fatty acid ratio of macrophage membrane phospholipids is of importance in determining macrophage phagocytic capacity. Available in vivo-data are less clear. At present, there is a lack of systematic studies elucidating key factors such as fatty acid efficacy, effective dose or dosing intervals. Without this knowledge the targeted modulation of macrophage phagocytosis in vivo by fatty acids is still a distant possibility. PMID:25987422

  4. Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors

    PubMed Central

    Qi, Yi-xiang; Huang, Jia; Li, Meng-qi; Wu, Ya-su; Xia, Ren-ying; Ye, Gong-yin

    2016-01-01

    Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution. DOI: http://dx.doi.org/10.7554/eLife.12241.001 PMID:26974346

  5. In vitro induction of lysosomal enzymes by phagocytosis.

    PubMed

    Axline, S G; Cohn, Z A

    1970-06-01

    The in vitro induction of lysosomal enzymes by phagocytosis was demonstrated in cultivated mouse peritoneal macrophages. The contribution of each of several steps in the endocytic process to enzyme induction was examined. The enzymatic response after the uptake of equal numbers of erythrocytes (RBC) and nondigestible particles were compared. Phagocytosis of RBC produced a marked increase in the levels of acid phosphatase, beta-glucuronidase, and cathepsin D. Puromycin (1 microg/ml) inhibited the enzyme response. In contrast, phagocytosis of polyvinyl toluene, polystyrene, and insoluble starch particles produced no increase in macrophage lysosomal enzymes, although fusion of phagosomes with preexisting lysosomes occurred normally. The endocytic stimulus to synthesis of inducible lysosomal enzymes, therefore, occurred at or beyond the stage of digestion. Purified protein (bovine gamma globulin) aggregates and homopolymer coacervates of poly-l-glutamic acid: poly-l-lysine were effective inducers of lysosomal acid phosphatase, beta-glucuronidase, and cathepsin D, whereas homopolymers of the same D-amino acids were ineffective as inducers. Both the quantity of phagocytized substrate and its rate of enzymatic hydrolysis appear to control the level and persistance of lysosomal hydrolases. PMID:4911552

  6. Buoyant triacylglycerol-filled green algae and methods therefor

    DOEpatents

    Goodenough, Ursula; Goodson, Carrie

    2015-04-14

    Cultures of Chlamydomonas are disclosed comprising greater than 340 mg/l triacylglycerols (TAG). The cultures can include buoyant Chlamydomonas. Methods of forming the cultures are also disclosed. In some embodiments, these methods comprise providing Chlamydomonas growing in log phase in a first culture medium comprising a nitrogen source and acetate, replacing the first culture medium with a second medium comprising acetate but no nitrogen source, and subsequently supplementing the second medium with additional acetate. In some embodiments, a culture can comprise at least 1,300 mg/l triacyglycerols. In some embodiments, cultures can be used to produce a biofuel such as biodiesel.

  7. Blood triacylglycerols: a lipidomic window on diet and disease.

    PubMed

    Sanders, Francis; McNally, Ben; Griffin, Julian L

    2016-04-15

    Although the measurement of triacylglycerols (TAGs) by clinical chemistry has been used in the diagnosis of a range of metabolic diseases, such approaches ignore the different species of TAGs that contribute to the total concentration. With the advent of LC and direct infusion forms of MS it is now possible to profile the individual TAGs in blood plasma or tissue extracts. This mini review surveys the information that is obtainable from the lipidomic profiling of TAGs in following metabolic diseases such as type 2 diabetes (T2DM), cardiovascular disease (CVD) and non-alcoholic fatty liver disease, as well as the development of insulin resistance and obesity. PMID:27068982

  8. Group V secreted phospholipase A2 is upregulated by IL-4 in human macrophages and mediates phagocytosis via hydrolysis of ethanolamine phospholipids.

    PubMed

    Rubio, Julio M; Rodríguez, Juan P; Gil-de-Gómez, Luis; Guijas, Carlos; Balboa, María A; Balsinde, Jesús

    2015-04-01

    Studies on the heterogeneity and plasticity of macrophage populations led to the identification of two major polarization states: classically activated macrophages or M1, induced by IFN-γ plus LPS, and alternatively activated macrophages, induced by IL-4. We studied the expression of multiple phospholipase A2 enzymes in human macrophages and the effect that polarization of the cells has on their levels. At least 11 phospholipase A2 genes were found at significant levels in human macrophages, as detected by quantitative PCR. None of these exhibited marked changes after treating the cells with IFN-γ plus LPS. However, macrophage treatment with IL-4 led to strong upregulation of the secreted group V phospholipase A2 (sPLA2-V), both at the mRNA and protein levels. In parallel with increasing sPLA2-V expression levels, IL-4-treated macrophages exhibited increased phagocytosis of yeast-derived zymosan and bacteria, and we show that both events are causally related, because cells deficient in sPLA2-V exhibited decreased phagocytosis, and cells overexpressing the enzyme manifested higher rates of phagocytosis. Mass spectrometry analyses of lipid changes in the IL-4-treated macrophages suggest that ethanolamine lysophospholipid (LPE) is an sPLA2-V-derived product that may be involved in regulating phagocytosis. Cellular levels of LPE are selectively maintained by sPLA2-V. By supplementing sPLA2-V-deficient cells with LPE, phagocytosis of zymosan or bacteria was fully restored in IL-4-treated cells. Collectively, our results show that sPLA2-V is required for efficient phagocytosis by IL-4-treated human macrophages and provide evidence that sPLA2-V-derived LPE is involved in the process. PMID:25725101

  9. Contribution of Ion Channels in Calcium Signaling Regulating Phagocytosis: MaxiK, Cav1.3 and Bestrophin-1.

    PubMed

    Strauß, Olaf; Reichhart, Nadine; Gomez, Nestor Mas; Müller, Claudia

    2016-01-01

    Mutations in the BEST1 gene lead to a variety of retinal degenerations including Best's vitelliforme macular degeneration. The BEST1 gene product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE). It is likely that mutant bestrophin-1 impairs functions of the RPE which support photoreceptor function and will thus lead to retinal degeneration. However, the RPE function which is influenced by bestrophin-1 is so far not identified. Previously we showed that bestrophin-1 interacts with L-type Ca²⁺ channels of the CaV1.3 subtype and that the endogenously expressed bestrophin-1 is required for intracellular Ca²⁺ regulation. A hallmark of Best's disease is the fast lipofuscin accumulation occurring already at young ages. Therefore, we addressed the hypothesis that bestrophin-1 might influence phagocytosis of photoreceptor outer segments (POS) by the RPE. Here, siRNA knock-down of bestrophin-1 expression as well as inhibition of L-type Ca²⁺ channel activity modulated the POS phagocytosis in vitro. In vivo CaV1.3 expression appeared to be diurnal regulated with a higher expression rate in the afternoon. Compared to wild-type littermates, Ca V 1.3 (-/-) mice showed a shift in the circadian POS phagocytosis with an increased activity in the afternoon. Thus we suggest that mutant bestrophin-1 leads to an impaired regulation of the POS phagocytosis by the RPE which would explain the fast lipofuscin accumulation in Best patients. PMID:26427483

  10. Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins

    PubMed Central

    Schlam, Daniel; Bagshaw, Richard D.; Freeman, Spencer A.; Collins, Richard F.; Pawson, Tony; Fairn, Gregory D.; Grinstein, Sergio

    2015-01-01

    Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles. Through a screen of 62 RhoGAP-family members, we demonstrate that ARHGAP12, ARHGAP25 and SH3BP1 are responsible for GTPase inactivation. Silencing these RhoGAPs impairs phagocytosis of large targets. The GAPs are recruited to large—but not small—phagocytic cups by products of PI3K, where they synergistically inactivate Rac and Cdc42. Remarkably, the prominent accumulation of phosphatidylinositol 3,4,5-trisphosphate characteristic of large-phagosome formation is less evident during phagocytosis of small targets, accounting for the contrasting RhoGAP distribution and the differential requirement for PI3K during phagocytosis of dissimilarly sized particles. PMID:26465210

  11. Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

    PubMed Central

    García Gil, Merche; Alonso, Fernando; Alvarez Chiva, Vicente; Sánchez Crespo, Mariano; Mato, José M.

    1982-01-01

    We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-14C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process. PMID:6181780

  12. Diffusion Barriers, Mechanical Forces, and the Biophysics of Phagocytosis.

    PubMed

    Ostrowski, Philip P; Grinstein, Sergio; Freeman, Spencer A

    2016-07-25

    Phagocytes recognize and eliminate pathogens, alert other tissues of impending threats, and provide a link between innate and adaptive immunity. They also maintain tissue homeostasis, consuming dead cells without causing alarm. The receptor engagement, signal transduction, and cytoskeletal rearrangements underlying phagocytosis are paradigmatic of other immune responses and bear similarities to macropinocytosis and cell migration. We discuss how the glycocalyx restricts access to phagocytic receptors, the processes that enable receptor engagement and clustering, and the remodeling of the actin cytoskeleton that controls the mobility of membrane proteins and lipids and provides the mechanical force propelling the phagocyte membrane toward and around the phagocytic prey. PMID:27459066

  13. Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen.

    PubMed Central

    Brown, E J; Bohnsack, J F; Gresham, H D

    1988-01-01

    We have investigated the effects of the monoclonal antibodies against the cell surface molecule Mac-1 on C3bi-mediated rosetting and IgG-mediated rosetting and phagocytosis by human peripheral blood monocytes. Highly purified M1/70 F(ab')2, used in the fluid phase, inhibited both monocyte functions. Half-maximal C3bi rosette inhibition occurred at a concentration of 2 nM F(ab')2 M1/70. An equivalent decrease in IgG-mediated rosetting required 10 nM M1/70 F(ab')2, and 50% inhibition of IgG-mediated phagocytosis required 7 nM antibody. Mo-1 F(ab')2 inhibited EC3bi binding with an ID50 of 0.3 nM, whereas 50% decrease in IgG-mediated rosetting required 70 nM of this antibody. OKM1 did not inhibit rosettes of sheep erythrocytes opsonized with IgG antibody (EA) at all. F(ab')2 M1/70 did not affect the binding of monomeric human IgG to monocytes, but did substantially decrease the binding of IgG aggregates. Half-maximal inhibition of aggregated IgG binding at 0 degrees C occurred at 8 nM F(ab')2 M1/70, very close to the concentration that caused equivalent inhibition of IgG-mediated phagocytosis. Aggregated IgG inhibited the binding of radiolabeled M1/70 to monocytes by approximately 40%, suggesting that some, but not all Mac-1 molecules were associated with IgG receptors under these conditions. When cells were allowed to adhere to surfaces coated with M1/70 or Mo-1 F(ab')2, C3bi-mediated rosetting was inhibited, but IgG mediated-phagocytosis was unaffected. Moreover, the dose response of inhibition of phagocytosis by fluid-phase F(ab')2, of anti-Mac-1 monoclonals was similar on monocytes adherent to albumin-coated and antibody-coated surfaces. Kinetic experiments showed that even prolonged incubation of monocytes on M1/70 coated surfaces did not lead to inhibition of EA binding nor did these incubations alter the dose response for inhibition of EA binding by fluid-phase M1/70 F(ab')2. This suggested that not all molecules recognized by M1/70 are freely mobile in the

  14. Authentication of Coffea arabica according to Triacylglycerol Stereospecific Composition.

    PubMed

    Cossignani, L; Montesano, D; Simonetti, M S; Blasi, F

    2016-01-01

    Stereospecific analysis is an important tool for the characterization of lipid fraction of food products. In the present research, an approach to characterize arabica and robusta varieties by structural analysis of the triacylglycerol (TAG) fraction is reported. The lipids were Soxhlet extracted from ground roasted coffee beans with petroleum ether, and the fatty acids (FA) were determined as their corresponding methyl esters. The results of a chemical-enzymatic-chromatographic method were elaborated by a chemometric procedure, Linear Discriminant Analysis (LDA). According to the total and intrapositional FA composition of TAG fraction, the obtained results were able to characterize roasted pure coffee samples and coffee mixtures with 10% robusta coffee added to arabica coffee. Totally correct classified samples were obtained when the TAG stereospecific results of the considered coffee mixture (90 : 10 arabica/robusta) were elaborated by LDA procedure. PMID:27547482

  15. Authentication of Coffea arabica according to Triacylglycerol Stereospecific Composition

    PubMed Central

    Cossignani, L.; Simonetti, M. S.; Blasi, F.

    2016-01-01

    Stereospecific analysis is an important tool for the characterization of lipid fraction of food products. In the present research, an approach to characterize arabica and robusta varieties by structural analysis of the triacylglycerol (TAG) fraction is reported. The lipids were Soxhlet extracted from ground roasted coffee beans with petroleum ether, and the fatty acids (FA) were determined as their corresponding methyl esters. The results of a chemical-enzymatic-chromatographic method were elaborated by a chemometric procedure, Linear Discriminant Analysis (LDA). According to the total and intrapositional FA composition of TAG fraction, the obtained results were able to characterize roasted pure coffee samples and coffee mixtures with 10% robusta coffee added to arabica coffee. Totally correct classified samples were obtained when the TAG stereospecific results of the considered coffee mixture (90 : 10 arabica/robusta) were elaborated by LDA procedure. PMID:27547482

  16. Controlled One-on-One Encounters between Immune Cells and Microbes Reveal Mechanisms of Phagocytosis.

    PubMed

    Heinrich, Volkmar

    2015-08-01

    Among many challenges facing the battle against infectious disease, one quandary stands out. On the one hand, it is often unclear how well animal models and cell lines mimic human immune behavior. On the other hand, many core methods of cell and molecular biology cannot be applied to human subjects. For example, the profound susceptibility of neutropenic patients to infection marks neutrophils (the most abundant white blood cells in humans) as vital immune defenders. Yet because these cells cannot be cultured or genetically manipulated, there are gaps in our understanding of the behavior of human neutrophils. Here, we discuss an alternative, interdisciplinary strategy to dissect fundamental mechanisms of immune-cell interactions with bacteria and fungi. We show how biophysical analyses of single-live-cell/single-target encounters are revealing universal principles of immune-cell phagocytosis, while also dispelling misconceptions about the minimum required mechanistic determinants of this process. PMID:26244729

  17. Effects of endogenous antidiuretic hormone (ADH) on macrophage phagocytosis

    SciTech Connect

    Fernandez-Repollet, E.; Opava-Stitzer, S.; Tiffany, S.; Schwartz, A.

    1983-07-01

    Although several studies have indicated that antidiuretic hormone (ADH) enhances the phagocytic function of the reticuloendothelial system (RES) in shock syndromes, it remains unknown what influence ADH exerts upon the individual phagocytic components of this system. The present investigation was designed to evaluate the effects of endogenous ADH on the phagocytic activity of peritoneal macrophage cells. As a phagocytic stimuli, fluorescent methacrylate microbeads were injected intraperitoneally into Brattleboro (ADH deficient) and normal Long Evans rats in the presence and absence of exogenous ADH. Peritoneal cells were harvested 19-22 hr after the administration of the microbeads and the percent phagocytosis was determined in macrophage cells using a fluorescence-activated cell sorter (FACS II). Our results indicate that the percentage of peritoneal macrophages ingesting the fluorescent methacrylate microbeads was significantly reduced in the absence of ADH (Brattleboro rats: 5.4 +/- 0.6% versus Long Evans rats: 16.8 +/- 2.3%; p less than 0.001). In addition, our data demonstrate that exogenous administration of ADH significantly enhanced macrophage phagocytosis in Brattleboro (14.7 +/- 2.2%) and normal Long Evans (49.6 +/- 4.5%) rats. These data suggest, for the first time, that endogenous ADH might play a modulatory role in the phagocytic activity of a specific component of the RES, namely, the macrophage cell.

  18. Functional characterization of macrophage receptors for in vitro phagocytosis of unopsonized Pseudomonas aeruginosa.

    PubMed Central

    Speert, D P; Wright, S D; Silverstein, S C; Mah, B

    1988-01-01

    The phagocytic receptor for unopsonized Pseudomonas aeruginosa was characterized functionally using human monocyte-derived macrophages. Freshly isolated human peripheral blood monocytes were unable to ingest unopsonized P. aeruginosa; ingestion did not occur until the cells had been in culture for 2 d and it became maximal after 4 d. Macrophages plated on coverslips derivatized with anti-BSA IgG or with human gamma-globulin lost the capacity to phagocytose unopsonized P. aeruginosa, unopsonized zymosan, and EIgG but bound C3bi-coated erythrocytes normally. Each of the four human IgG subclasses and Fc fragments of anti-BSA IgG inhibited phagocytosis of both unopsonized P. aeruginosa and EIgG. Phagocytosis of P. aeruginosa and zymosan was markedly impaired and EIgG minimally inhibited if macrophages were plated on coverslips derivatized with mannan or when mannan was added to the phagocytosis buffer. Phagocytosis of P. aeruginosa and zymosan, and binding of EC3bi was dependent on the presence of divalent cations, but phagocytosis of EIgG was not. The macrophage phagocytic receptor for unopsonized P. aeruginosa was inactivated by proteolytic enzymes. Phagocytosis of P. aeruginosa was inhibited by D-mannose, L-fucose, and alpha methyl mannoside, but not by L-mannose, D-fucose, or D-glucose. The same sugars inhibited phagocytosis of unopsonized zymosan. We conclude that phagocytosis of unopsonized P. aeruginosa by human monocyte-derived macrophages is facilitated by mannose receptors. Images PMID:3138287

  19. Bactericidal/permeability-increasing protein promotes complement activation for neutrophil-mediated phagocytosis on bacterial surface

    PubMed Central

    Nishimura, H; Gogami, A; Miyagawa, Y; Nanbo, A; Murakami, Y; Baba, T; Nagasawa, S

    2001-01-01

    The neutrophil bactericidal/permeability-increasing protein (BPI) has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Flow cytometric analysis indicated that fluorescein-labelled E. coli treated with BPI were phagocytosed in the presence of serum at two- to five-fold higher levels than phagocytosis of the bacteria without the treatment. In contrast, phagocytosis of the fluoresceined bacteria with or without treatment by BPI did not occur at all in the absence of serum. The phagocytosis stimulated by BPI and serum was dose-dependent. The effect of BPI on phagocytosis in the presence of serum was not observed on Gram-positive bacteria (Staphylococcus aureus). Interestingly, the complement C3b/iC3b fragments were deposited onto the bacterial surface also as a function of the BPI concentration under conditions similar to those for phagocytosis. Furthermore, the BPI-promoted phagocytosis was blocked completely by anti-C3 F(ab′)2 and partially by anti-complement receptor (CR) type 1 and/or anti-CR type 3. These findings suggest that BPI accelerates complement activation to opsonize bacteria with complement-derived fragments, leading to stimulation of phagocytosis by neutrophils via CR(s). PMID:11529944

  20. Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis.

    PubMed

    Shockey, Jay; Regmi, Anushobha; Cotton, Kimberly; Adhikari, Neil; Browse, John; Bates, Philip D

    2016-01-01

    The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. PMID:26586834

  1. Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis1[OPEN

    PubMed Central

    Browse, John

    2016-01-01

    The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. PMID:26586834

  2. Mycobacterium marinum MgtC Plays a Role in Phagocytosis but is Dispensable for Intracellular Multiplication

    PubMed Central

    Belon, Claudine; Gannoun-Zaki, Laïla; Lutfalla, Georges; Kremer, Laurent; Blanc-Potard, Anne-Béatrice

    2014-01-01

    MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg2+ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg2+ deprivation and is required for optimal growth in Mg2+-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg2+ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen's lifestyles. PMID:25545682

  3. Impact of Association Colloids on Lipid Oxidation in Triacylglycerols and Fatty Acid Ethyl Esters.

    PubMed

    Homma, Rika; Suzuki, Karin; Cui, Leqi; McClements, David Julian; Decker, Eric A

    2015-11-25

    The impact of association colloids on lipid oxidation in triacylglycerols and fatty acid ethyl esters was investigated. Association colloids did not affect lipid oxidation of high oleic safflower and high linoleic safflower triacylglycerols, but were prooxidative in fish triacylglycerols. Association colloids retarded aldehyde formation in stripped ethyl oleate, linoleate, and fish oil ethyl esters. Interfacial tension revealed that lipid hydroperoxides were surface active in the presence of the surfactants found in association colloids. The lipid hydroperoxides from ethyl esters were less surface active than triacylglycerol hydroperoxides. Stripping decreased iron and copper concentrations in all oils, but more so in fatty acid ethyl esters. The combination of lower hydroperoxide surface activity and low metal concentrations could explain why association colloids inhibited lipid oxidation in fatty acid ethyl esters. This research suggests that association colloids could be used as an antioxidant technology in fatty acid ethyl esters. PMID:26506263

  4. Fast comprehensive analysis of vitamin D and triacylglycerols in dietary supplements using multiple parallel mass spectrometers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New, faster methods have been developed for analysis of vitamin D and triacylglycerols that eliminate hours of wet chemistry and preparative chromatography, while providing more information than classical methods for analysis. Unprecedented detail is provided by combining liquid chromatography with ...

  5. Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

    2013-12-13

    Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source. PMID:24187129

  6. Physics of phagocytosis of foreign versus self-tolerance

    NASA Astrophysics Data System (ADS)

    Tsai, Richard; Rodriguez, Pia; Discher, Dennis

    2009-03-01

    The first cells to `attack' an implanted or injected foreign material or microbe are phagocytic cells of the innate immune system. These cells actively and rapidly phagocytose foreign cells, surfaces, or particles, but the process that is inefficient when faced with ``self'' cells. We have examined the biochemistry and some of the physics of this decision to eat or not eat. One particular protein on all animal cell membranes, called CD47, seems to engage phagocytic cell couter-receptors, and deactivate the force-generating myosin machinery that otherwise makes phagocytosis efficient. We will map the phagocytic synapse between phagocytes and particles or cells and describe the physicochemical dynamics that mediate this key decision in compatability.

  7. A novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosis.

    PubMed

    Liu, Ning; Lan, Jiang-Feng; Sun, Jie-Jie; Jia, Wen-Ming; Zhao, Xiao-Fan; Wang, Jin-Xing

    2015-04-01

    Crustins are cationic cysteine-rich antimicrobial peptides (AMPs) that contain multiple domains (glycine-rich, cysteine-rich, or proline-rich) at the N-terminus and whey acidic protein (WAP) domains at the C-terminus. Crustins have multiple functions, including protease inhibition and antimicrobial activity. Other functions of crustins need to be clarified. In this study, a novel crustin with a cysteine-rich region, and a single WAP domain, belonging to type I crustins, was identified in Marsupenaeus japonicus and designated as MjCru I-1. MjCru I-1 was expressed in various tissues. The expression of MjCru I-1 was upregulated in the hemocytes of shrimp challenged with bacteria. MjCru I-1 could bind to bacteria by binding to the cell wall molecules of the bacteria, such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoteichoic acid (LTA). The synthesized WAP domain of MjCru I-1 but not synthesized Cys-rich domain has antibacterial and agglutinative activities. Scanning electron microscope assay showed that the bacterial cells treated with sMjCru I-1 appeared to be disrupted and cracked compared with those of the control samples. The knockdown of MjCru I-1 could reduce bacterial clearance and injection of MjCru I-1 could significantly increase the survival rate of shrimp infected with Vibrio anguillarum and Staphylococcus aureus compared with those of the control samples. Further study discovered that MjCru I-1 could increase the hemocyte phagocytosis against V. anguillarum and S. aureus. These results suggest that MjCru I-1 has dual functions, bactericidal and phagocytosis promoting activities, in the antibacterial immunity of shrimp. PMID:25479014

  8. PKC-ε pseudosubstrate and catalytic activity are necessary for membrane delivery during IgG-mediated phagocytosis

    PubMed Central

    Wood, Tiffany R.; Chow, Rachel Y.; Hanes, Cheryl M.; Zhang, Xuexin; Kashiwagi, Kaori; Shirai, Yasuhito; Trebak, Mohamed; Loegering, Daniel J.; Saito, Naoaki; Lennartz, Michelle R.

    2013-01-01

    In RAW 264.7 cells [1], PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-ε−/− cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-ε−/− macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-ε−/− macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-ε−/− BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension. PMID:23670290

  9. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

    PubMed Central

    Hornik, Tamara C.; Vilalta, Anna; Brown, Guy C.

    2016-01-01

    ABSTRACT Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis. PMID:26567213

  10. Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis

    PubMed Central

    Sierra, Amanda; Abiega, Oihane; Shahraz, Anahita; Neumann, Harald

    2012-01-01

    Microglia are the resident brain macrophages and they have been traditionally studied as orchestrators of the brain inflammatory response during infections and disease. In addition, microglia has a more benign, less explored role as the brain professional phagocytes. Phagocytosis is a term coined from the Greek to describe the receptor-mediated engulfment and degradation of dead cells and microbes. In addition, microglia phagocytoses brain-specific cargo, such as axonal and myelin debris in spinal cord injury or multiple sclerosis, amyloid-β deposits in Alzheimer's disease, and supernumerary synapses in postnatal development. Common mechanisms of recognition, engulfment, and degradation of the different types of cargo are assumed, but very little is known about the shared and specific molecules involved in the phagocytosis of each target by microglia. More importantly, the functional consequences of microglial phagocytosis remain largely unexplored. Overall, phagocytosis is considered a beneficial phenomenon, since it eliminates dead cells and induces an anti-inflammatory response. However, phagocytosis can also activate the respiratory burst, which produces toxic reactive oxygen species (ROS). Phagocytosis has been traditionally studied in pathological conditions, leading to the assumption that microglia have to be activated in order to become efficient phagocytes. Recent data, however, has shown that unchallenged microglia phagocytose apoptotic cells during development and in adult neurogenic niches, suggesting an overlooked role in brain remodeling throughout the normal lifespan. The present review will summarize the current state of the literature regarding the role of microglial phagocytosis in maintaining tissue homeostasis in health as in disease. PMID:23386811

  11. Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte-derived dendritic cells.

    PubMed

    Argueta-Donohué, Jesús; Wilkins-Rodríguez, Arturo A; Aguirre-García, Magdalena; Gutiérrez-Kobeh, Laila

    2016-06-01

    Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C-type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte-derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC-SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC-SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes. PMID:26399218

  12. Microalgal Triacylglycerols as Feedstocks for Biofuel Production: Perspectives and Advances

    SciTech Connect

    Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M; Seibert, M.; Darzins, A.

    2008-01-01

    Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.

  13. Characterization of key triacylglycerol biosynthesis processes in rhodococci

    PubMed Central

    Amara, Sawsan; Seghezzi, Nicolas; Otani, Hiroshi; Diaz-Salazar, Carlos; Liu, Jie; Eltis, Lindsay D.

    2016-01-01

    Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production. PMID:27126051

  14. Triacylglycerol Accumulation in Photosynthetic Cells in Plants and Algae.

    PubMed

    Du, Zhi-Yan; Benning, Christoph

    2016-01-01

    Plant and algal oils are some of the most energy-dense renewable compounds provided by nature. Triacylglycerols (TAGs) are the major constituent of plant oils, which can be converted into fatty acid methyl esters commonly known as biodiesel. As one of the most efficient producers of TAGs, photosynthetic microalgae have attracted substantial interest for renewable fuel production. Currently, the big challenge of microalgae based TAGs for biofuels is their high cost compared to fossil fuels. A conundrum is that microalgae accumulate large amounts of TAGs only during stress conditions such as nutrient deprivation and temperature stress, which inevitably will inhibit growth. Thus, a better understanding of why and how microalgae induce TAG biosynthesis under stress conditions would allow the development of engineered microalgae with increased TAG production during conditions optimal for growth. Land plants also synthesize TAGs during stresses and we will compare new findings on environmental stress-induced TAG accumulation in plants and microalgae especially in the well-characterized model alga Chlamydomonas reinhardtii and a biotechnologically relevant genus Nannochloropsis. PMID:27023236

  15. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.

    PubMed

    Yen, Chi-Liang Eric; Nelson, David W; Yen, Mei-I

    2015-03-01

    The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation. PMID:25231105

  16. Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum.

    PubMed

    Barka, Frederik; Angstenberger, Max; Ahrendt, Tilman; Lorenzen, Wolfram; Bode, Helge B; Büchel, Claudia

    2016-03-01

    Diatoms accumulate triacylglycerols (TAGs) as storage lipids, but the knowledge about the molecular mechanisms of lipid metabolism is still sparse. Starting from a partial sequence for a putative TAG-lipase of the diatom Phaeodactylum tricornutum retrieved from the data bases, we have identified the full length coding sequence, tgl1. The gene encodes an 813 amino acid sequence that shows distinct motifs for so called "true" TAG-lipases [EC 3.1.1.3] that have been functionally characterized in model organisms like Arabidopsis thaliana and Saccharomyces cerevisiae. These lipases mediate the first initial step of TAG breakdown from storage lipids. To test whether Tgl1 can act as a TAG-lipase, a His-tagged version was overexpressed in Escherichia coli and the protein indeed showed esterase activity. To identify the TAG degrading function of Tgl1 in P. tricornutum, knock-down mutant strains were created using an antisense RNA approach. In the mutant cell lines the relative tgl1-mRNA-level was reduced up to 20% of that of the wild type, accompanied by a strong increase of TAG in the lipid extracts. In spite of the TAG accumulation, the polar lipid species pattern appeared to be unchanged, confirming the TAG-lipase function of Tgl1. PMID:26747649

  17. Relative oxidative stability of diacylglycerol and triacylglycerol oils.

    PubMed

    Qi, Jin F; Wang, Xiang Y; Shin, Jung-Ah; Lee, Young-Hwa; Jang, Young-Seok; Lee, Jeung Hee; Hong, Soon-Taek; Lee, Ki-Teak

    2015-03-01

    To compare the oxidative stability between diacylglycerol (DAG) oil and conventional triacylglycerol (TAG) oil (that is, soybean oil), the prepared stripped diacylglycerol oil (SDO) and soybean oil (SSBO) were stored at 60 °C in the dark for 144 h. During storage peroxide values (POVs), contents of aldehydes, unsaturated fatty acids were measured to evaluate the oxidative stabilities of the 2 oils. The results showed the content of C18:2, C18:3, and total unsaturated fatty acid decreased faster in DAG oil than in soybean oil, whereas the decreased rate of C18:1 was similar in 2 oils. Also, both rate constants (K1 and K2) obtained from POV (K1 ) and total aldehydes (K2 ) indicated that DAG oil (K1 = 3.22 mmol/mol FA h(-1) , K2 = 0.023 h(-1)) was oxidized more rapidly than soybean oil (K1 = 2.56 mmol/mol FA h(-1) , K2 = 0.021 h(-1)), which was mainly due to the difference of acylglycerol composition of the 2 oils along with higher C18:3 (9.6%) in SDO than SSBO (5.7%). It is concluded that DAG was more easily oxidized than soybean oil at 60 °C in the dark for 144 h. PMID:25678328

  18. Enhancing Cardiac Triacylglycerol Metabolism Improves Recovery From Ischemic Stress

    PubMed Central

    Liu, Li; Goldberg, Ira J.

    2015-01-01

    Elevated cardiac triacylglycerol (TAG) content is traditionally equated with cardiolipotoxicity and suggested to be a culprit in cardiac dysfunction. However, previous work demonstrated that myosin heavy-chain–mediated cardiac-specific overexpression of diacylglycerol transferase 1 (MHC-DGAT1), the primary enzyme for TAG synthesis, preserved cardiac function in two lipotoxic mouse models despite maintaining high TAG content. Therefore, we examined whether increased cardiomyocyte TAG levels due to DGAT1 overexpression led to changes in cardiac TAG turnover rates under normoxia and ischemia-reperfusion conditions. MHC-DGAT1 mice had elevated TAG content and synthesis rates, which did not alter cardiac function, substrate oxidation, or myocardial energetics. MHC-DGAT1 hearts had ischemia-induced lipolysis; however, when a physiologic mixture of long-chain fatty acids was provided, enhanced TAG turnover rates were associated with improved functional recovery from low-flow ischemia. Conversely, exogenous supply of palmitate during reperfusion suppressed elevated TAG turnover rates and impaired recovery from ischemia in MHC-DGAT1 hearts. Collectively, this study shows that elevated TAG content, accompanied by enhanced turnover, does not adversely affect cardiac function and, in fact, provides cardioprotection from ischemic stress. In addition, the results highlight the importance of exogenous supply of fatty acids when assessing cardiac lipid metabolism and its relationship with cardiac function. PMID:25858561

  19. Triacylglycerol kinetics in endotoxic rats with suppressed lipoprotein lipase activity

    SciTech Connect

    Bagby, G.J.; Corll, C.B.; Martinez, R.R.

    1987-07-01

    Hypertriglyceridemia observed in animals after bacterial endotoxin administration and some forms of sepsis can result from increased hepatic triacylglycerol (TG) output or decreased TG clearance by extrahepatic tissues. To differentiate between these two possibilities, TG and free fatty acid (FFA) kinetics were determined in control and endotoxin-injected rats 18 h after treatment. Plasma TG and FFA kinetics were assessed by a constant intravenous infusion with (9,10-/sup 3/H)palmitate-labeled very low-density lipoprotein and (1-/sup 14/C)palmitate bound to albumin, respectively. In addition, lipoprotein lipase (LPL) activity was determined in heart, skeletal muscle, and adipose tissue as well as in postheparin plasma of functionally hepatectomized, adrenalectomized, and gonadectomized rats. Plasma FFA acid concentrations were slightly increased in endotoxin-treated rats but their turnover did not differ from control. Endotoxin-treated rats had a threefold increase in plasma TG concentrations and decreased heart, skeletal muscle, and post-heparin plasma LPL activity. Plasma TG turnover was decreased, indicating that hypertriglyceridemia was not due to an increased TG output by the liver. Instead, the endotoxin-induced increase in plasma TG concentration was consequence of the 80% reduction in TG metabolic clearance rate. Thus, suppression of LPL activity in endotoxic animals impairs TG clearance resulting in hypertriglyceridemia. Furthermore, endotoxin administration reduced the delivery of TG-FFA to extrahepatic tissues because hepatic synthesis and secretion of TG from plasma FFA was decreased and LPL activity was suppressed.

  20. Characterization of key triacylglycerol biosynthesis processes in rhodococci

    DOE PAGESBeta

    Amara, Sawsan; Seghezzi, Nicolas; Otani, Hiroshi; Diaz-Salazar, Carlos; Liu, Jie; Eltis, Lindsay D.

    2016-04-29

    In this study, oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcriptsmore » were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.« less

  1. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism

    PubMed Central

    Yen, Chi-Liang Eric; Nelson, David W.; Yen, Mei-I

    2015-01-01

    The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation. PMID:25231105

  2. In vivo Reconstitution of Algal Triacylglycerol Production in Saccharomyces cerevisiae

    PubMed Central

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-01-01

    The current fascination with algal biofuel production stems from a high lipid biosynthetic capacity and little conflict with land plant cultivation. However, the mechanisms which enable algae to accumulate massive oil remain elusive. An enzyme for triacylglycerol (TAG) biosynthesis in Chlamydomonas reinhardtii, CrDGTT2, can produce a large amount of TAG when expressed in yeast or higher plants, suggesting a unique ability of CrDGTT2 to enhance oil production in a heterologous system. Here, we performed metabolic engineering in Saccharomyces cerevisiae by taking advantage of CrDGTT2. We suppressed membrane phospholipid biosynthesis at the log phase by mutating OPI3, enhanced TAG biosynthetic pathway at the stationary phase by overexpressing PAH1 and CrDGTT2, and suppressed TAG hydrolysis on growth resumption from the stationary phase by knocking out DGK1. The resulting engineered yeast cells accumulated about 70-fold of TAG compared with wild type cells. Moreover, TAG production was sustainable. Our results demonstrated the enhanced and sustainable TAG production in the yeast synthetic platform. PMID:26913021

  3. Enhancing Cardiac Triacylglycerol Metabolism Improves Recovery From Ischemic Stress.

    PubMed

    Kolwicz, Stephen C; Liu, Li; Goldberg, Ira J; Tian, Rong

    2015-08-01

    Elevated cardiac triacylglycerol (TAG) content is traditionally equated with cardiolipotoxicity and suggested to be a culprit in cardiac dysfunction. However, previous work demonstrated that myosin heavy-chain-mediated cardiac-specific overexpression of diacylglycerol transferase 1 (MHC-DGAT1), the primary enzyme for TAG synthesis, preserved cardiac function in two lipotoxic mouse models despite maintaining high TAG content. Therefore, we examined whether increased cardiomyocyte TAG levels due to DGAT1 overexpression led to changes in cardiac TAG turnover rates under normoxia and ischemia-reperfusion conditions. MHC-DGAT1 mice had elevated TAG content and synthesis rates, which did not alter cardiac function, substrate oxidation, or myocardial energetics. MHC-DGAT1 hearts had ischemia-induced lipolysis; however, when a physiologic mixture of long-chain fatty acids was provided, enhanced TAG turnover rates were associated with improved functional recovery from low-flow ischemia. Conversely, exogenous supply of palmitate during reperfusion suppressed elevated TAG turnover rates and impaired recovery from ischemia in MHC-DGAT1 hearts. Collectively, this study shows that elevated TAG content, accompanied by enhanced turnover, does not adversely affect cardiac function and, in fact, provides cardioprotection from ischemic stress. In addition, the results highlight the importance of exogenous supply of fatty acids when assessing cardiac lipid metabolism and its relationship with cardiac function. PMID:25858561

  4. Phagocytosis of gram-negative bacteria by a unique CD14-dependent mechanism.

    PubMed

    Schiff, D E; Kline, L; Soldau, K; Lee, J D; Pugin, J; Tobias, P S; Ulevitch, R J

    1997-12-01

    THP-1-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI)-anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated)-opsonized Escherichia coli (opEc). Both the GPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coli opsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolyl-maleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab')2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 beta-chain) or anti-Fcgamma receptor mAb. PMID:9400820

  5. Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.

    PubMed

    Tartaro, Karrie; VanVolkenburg, Maria; Wilkie, Dean; Coskran, Timothy M; Kreeger, John M; Kawabata, Thomas T; Casinghino, Sandra

    2015-01-01

    The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the

  6. Identification of Diacylglycerols and Triacylglycerols Containing Trihydroxy Fatty Acids in Castor Oil and the Regiospecific Identification of Triacylglycerols by Mass SpecTrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ricinoleate, a monohydroxy fatty acid in castor oil, has many industrial uses. Dihydroxy and trihydroxy fatty acids can also be used in industry. We report here the identification of diacylglycerols and triacylglycerols containing trihydroxy fatty acids in castor oil. The C18 HPLC fractions of casto...

  7. Interaction of non-esterified fatty acid and insulin in control of triacylglycerol secretion by Hep G2 cells.

    PubMed Central

    Byrne, C D; Brindle, N P; Wang, T W; Hales, C N

    1991-01-01

    The role of insulin in the regulation of plasma triacylglycerol is poorly understood. Conflicting actions of insulin on rat liver cells have been reported, insulin inhibiting triacylglycerol secretion in short incubations (less than 24 h) and stimulating triacylglycerol secretion in longer incubations (48-72 h). The present study was undertaken to examine regulation of triacylglycerol secretion by insulin and investigate the interaction between insulin and non-esterified fatty acid over 72 h in human hepatoblastoma (Hep G2) cells. Insulin inhibited triacylglycerol secretion throughout the 72 h period. The inhibition increased from 66% in the first 24 h to 88% in the final 24 h. Increasing the initial concentration of oleic acid from 200 microM to 1000 microM resulted in a 358% increase in triacylglycerol secretion and a 712% increase in accumulation over 24 h. Oleic acid uptake by the cells was rapid, with only 2.4% of the initial concentration (500 microM) remaining after 24 h. Supplementation of the medium with oleic acid to maintain the concentration between 750 microM and 1000 microM throughout a 5 h period resulted in a 350% increase in triacylglycerol secretion. Supplementation also decreased the insulin-induced inhibition of triacylglycerol secretion (18.2 to 7.8%; P less than 0.001). These results demonstrate that there is not a biphasic action of insulin on triacylglycerol secretion by Hep G2 cells. Experiments of this nature have not previously taken into account the rapid uptake of non-esterified fatty acid by hepatocytes and have consequently underestimated the effect of a sustained concentration on triacylglycerol metabolism. Oleic acid is therefore an even more potent stimulus to triacylglycerol synthesis and secretion than has previously been recognized. In addition, in the presence of a sustained increase in oleic acid concentration, there is a decrease in the action of insulin to inhibit triacylglycerol secretion. PMID:1660268

  8. Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols

    PubMed Central

    Bomar, Lindsey; Brugger, Silvio D.; Yost, Brian H.; Davies, Sean S.

    2016-01-01

    ABSTRACT Bacterial interspecies interactions play clinically important roles in shaping microbial community composition. We observed that Corynebacterium spp. are overrepresented in children free of Streptococcus pneumoniae (pneumococcus), a common pediatric nasal colonizer and an important infectious agent. Corynebacterium accolens, a benign lipid-requiring species, inhibits pneumococcal growth during in vitro cocultivation on medium supplemented with human skin surface triacylglycerols (TAGs) that are likely present in the nostrils. This inhibition depends on LipS1, a TAG lipase necessary for C. accolens growth on TAGs such as triolein. We determined that C. accolens hydrolysis of triolein releases oleic acid, which inhibits pneumococcus, as do other free fatty acids (FFAs) that might be released by LipS1 from human skin surface TAGs. Our results support a model in which C. accolens hydrolyzes skin surface TAGS in vivo releasing antipneumococcal FFAs. These data indicate that C. accolens may play a beneficial role in sculpting the human microbiome. PMID:26733066

  9. Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae.

    PubMed

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Sone, Toshiyuki; Era, Atsuko; Miyagishima, Shin-Ya; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2015-10-01

    Most microalgae produce triacylglycerol (TAG) under stress conditions such as nitrogen depletion, but the underlying molecular mechanism remains unclear. In this study, we focused on the role of target of rapamycin (TOR) in TAG accumulation. TOR is a serine/threonine protein kinase that is highly conserved and plays pivotal roles in nitrogen and other signaling pathways in eukaryotes. We previously constructed a rapamycin-susceptible Cyanidioschyzon merolae, a unicellular red alga, by expressing yeast FKBP12 protein to evaluate the results of TOR inhibition (Imamura et al. in Biochem Biophys Res Commun 439:264-269, 2013). By using this strain, we here report that rapamycin-induced TOR inhibition results in accumulation of cytoplasmic lipid droplets containing TAG. Transcripts for TAG synthesis-related genes, such as glycerol-3-phosphate acyltransferase and acyl-CoA:diacylglycerol acyltransferase (DGAT), were increased by rapamycin treatment. We also found that fatty acid synthase-dependent de novo fatty acid synthesis was required for the accumulation of lipid droplets. Induction of TAG and up-regulation of DGAT gene expression by rapamycin were similarly observed in the unicellular green alga, Chlamydomonas reinhardtii. These results suggest the general involvement of TOR signaling in TAG accumulation in divergent microalgae. PMID:26350402

  10. Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas.

    PubMed

    Solaiman, D K; Ashby, R D; Foglia, T A

    2001-09-01

    Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates. PMID:11601611

  11. Tracking synthesis and turnover of triacylglycerol in leaves

    PubMed Central

    Tjellström, Henrik; Strawsine, Merissa; Ohlrogge, John B.

    2015-01-01

    Triacylglycerol (TAG), typically represents <1% of leaf glycerolipids but can accumulate under stress and other conditions or if leaves are supplied with fatty acids, or in plants transformed with regulators or enzymes of lipid metabolism. To better understand the metabolism of TAG in leaves, pulse–chase radiolabelling experiments were designed to probe its synthesis and turnover. When Arabidopsis leaves were incubated with [14C]lauric acid (12:0), a major initial product was [14C]TAG. Thus, despite low steady-state levels, leaves possess substantial TAG biosynthetic capacity. The contributions of diacylglycerol acyltransferase1 and phospholipid:diacylglycerol acyltransferase1 to leaf TAG synthesis were examined by labelling of dgat1 and pdat1 mutants. The dgat1 mutant displayed a major (76%) reduction in [14C]TAG accumulation whereas pdat1 TAG labelling was only slightly reduced. Thus, DGAT1 has a principal role in TAG biosynthesis in young leaves. During a 4h chase period, radioactivity in TAG declined 70%, whereas the turnover of [14C]acyl chains of phosphatidylcholine (PC) and other polar lipids was much lower. Sixty percent of [14C]12:0 was directly incorporated into glycerolipids without modification, whereas 40% was elongated and desaturated to 16:0 and 18:1 by plastids. The unmodified [14C]12:0 and the plastid products of [14C]12:0 metabolism entered different pathways. Although plastid-modified 14C-labelled products accumulated in monogalactosyldiacylglycerol, PC, phosphatidylethanolamine, and diacylglcerol (DAG), there was almost no accumulation of [14C]16:0 and [14C]18:1 in TAG. Because DAG and acyl-CoA are direct precursors of TAG, the differential labelling of polar glycerolipids and TAG by [14C]12:0 and its plastid-modified products provides evidence for multiple subcellular pools of both acyl-CoA and DAG. PMID:25609824

  12. Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

    PubMed

    Allmann, Stefan; Mazet, Muriel; Ziebart, Nicole; Bouyssou, Guillaume; Fouillen, Laetitia; Dupuy, Jean-William; Bonneu, Marc; Moreau, Patrick; Bringaud, Frédéric; Boshart, Michael

    2014-01-01

    Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse. PMID:25493940

  13. Gut Microbial Fatty Acid Metabolites Reduce Triacylglycerol Levels in Hepatocytes.

    PubMed

    Nanthirudjanar, Tharnath; Furumoto, Hidehiro; Zheng, Jiawen; Kim, Young-Il; Goto, Tsuyoshi; Takahashi, Nobuyuki; Kawada, Teruo; Park, Si-Bum; Hirata, Akiko; Kitamura, Nahoko; Kishino, Shigenobu; Ogawa, Jun; Hirata, Takashi; Sugawara, Tatsuya

    2015-11-01

    Hydroxy and oxo fatty acids were recently found to be produced as intermediates during gut microbial fatty acid metabolism. Lactobacillus plantarum produces these fatty acids from unsaturated fatty acids such as linoleic acid. In this study, we investigated the effects of these gut microbial fatty acid metabolites on the lipogenesis in liver cells. We screened their effect on sterol regulatory element binding protein-1c (SREBP-1c) expression in HepG2 cells treated with a synthetic liver X receptor α (LXRα) agonist (T0901317). The results showed that 10-hydroxy-12(Z)-octadecenoic acid (18:1) (HYA), 10-hydroxy-6(Z),12(Z)-octadecadienoic acid (18:2) (γHYA), 10-oxo-12(Z)-18:1 (KetoA), and 10-oxo-6(Z),12(Z)-18:2 (γKetoA) significantly decreased SREBP-1c mRNA expression induced by T0901317. These fatty acids also downregulated the mRNA expression of lipogenic genes by suppressing LXRα activity and inhibiting SREBP-1 maturation. Oral administration of KetoA, which effectively reduced triacylglycerol accumulation and acetyl-CoA carboxylase 2 (ACC2) expression in HepG2 cells, for 2 weeks significantly decreased Srebp-1c, Scd-1, and Acc2 expression in the liver of mice fed a high-sucrose diet. Our findings suggest that the hypolipidemic effect of the fatty acid metabolites produced by L. plantarum can be exploited in the treatment of cardiovascular diseases or dyslipidemia. PMID:26399511

  14. Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei

    PubMed Central

    Allmann, Stefan; Mazet, Muriel; Ziebart, Nicole; Bouyssou, Guillaume; Fouillen, Laetitia; Dupuy, Jean-William; Bonneu, Marc; Moreau, Patrick; Bringaud, Frédéric; Boshart, Michael

    2014-01-01

    Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse. PMID:25493940

  15. Deprivation and repletion of androgen in vivo modifies triacylglycerol synthesis by rat hepatocytes.

    PubMed

    Elam, M B; Umstot, E S; Andersen, R N; Solomon, S S; Heimberg, M

    1987-10-17

    Given the same quantity of fatty acid, livers from male rats esterify less fatty acid and secrete less triacylglycerol in very-low-density lipoprotein than do livers from female animals. To elucidate the role of testosterone in maintenance of this male pattern, conversion of [1-14C]oleic acid into triacylglycerol was assessed in vitro by rat hepatocytes (male) following gonadectomy and replacement with testosterone. Following castration, incorporation of fatty acid into triacylglycerol was increased. In contrast, esterification of exogenous fatty acid into phospholipid, cholesteryl esters, and diacylglycerol was unchanged. Treatment with testosterone (75 micrograms/day) reduced incorporation of exogenous fatty acid into triacylglycerol. Higher doses of testosterone (200 or 100 micrograms/day) modified the effect, such that inhibition was observed only at low oleate (0.5 mM) concentrations. At higher substrate concentrations (1.0-2.0 mM) the inhibitory effect was no longer observed. Further, a similar dose-dependent effect of testosterone was observed following in vivo treatment of castrate females with testosterone. These data support the concept of a regulatory role of testosterone in hepatic triacylglycerol synthesis. These findings also demonstrate a biphasic effect of testosterone, an effect that is dependent not only upon the dose of testosterone administered, but also on the concentration of fatty acid to which the hepatocyte is exposed in vitro. PMID:3663694

  16. Triacylglycerol profile in cocoa liquors using MALDI-TOF and LC-ESI tandem mass spectrometry.

    PubMed

    Bono, Luca; Seraglia, Roberta; Roverso, Marco; Di Carro, Marina; Magi, Emanuele

    2014-09-01

    Triacylglycerols are responsible for chocolate's peculiar melting behavior: the type and position of fatty acids on the glycerol molecule strongly affect the melting range of cocoa butter. For this reason, the characterization of triglyceride composition in cocoa products is particularly important. In this work, triacylglycerols extracted from cocoa liquor samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry (MS/MS) coupled to liquid chromatography. Extracted samples were initially analyzed by direct injection in MS to obtain information on triglyceride molecular weights; relevant MS parameters were optimized, and the possible formation of the adducts [M + Na](+) and [M + NH(4)](+) was studied. Tandem mass experiments (both with triple quadrupole and TOF/TOF) were performed to study the fragmentation pathways (in particular, the loss of palmitic, stearic and oleic acid) and identify the triacylglycerols in cocoa liquors. Some signals of the spectra obtained with both MS techniques could indicate the presence of diacylglycerols in the cocoa extract, but different experimental evidences demonstrated that they were generated by the in-source fragmentation of triglycerides. A nonaqueous reversed-phase chromatographic separation was also developed and used to support the identification of the analytes; nine triacylglycerols were recognized in the cocoa liquor extracts. The three different batches of Ecuador cocoa liquor did not show significant differences in the triacylglycerol profile. PMID:25230186

  17. KIM-1-mediated phagocytosis reduces acute injury to the kidney.

    PubMed

    Yang, Li; Brooks, Craig R; Xiao, Sheng; Sabbisetti, Venkata; Yeung, Melissa Y; Hsiao, Li-Li; Ichimura, Takaharu; Kuchroo, Vijay; Bonventre, Joseph V

    2015-04-01

    Kidney injury molecule 1 (KIM-1, also known as TIM-1) is markedly upregulated in the proximal tubule after injury and is maladaptive when chronically expressed. Here, we determined that early in the injury process, however, KIM-1 expression is antiinflammatory due to its mediation of phagocytic processes in tubule cells. Using various models of acute kidney injury (AKI) and mice expressing mutant forms of KIM-1, we demonstrated a mucin domain-dependent protective effect of epithelial KIM-1 expression that involves downregulation of innate immunity. Deletion of the mucin domain markedly impaired KIM-1-mediated phagocytic function, resulting in increased proinflammatory cytokine production, decreased antiinflammatory growth factor secretion by proximal epithelial cells, and a subsequent increase in tissue macrophages. Mice expressing KIM-1Δmucin had greater functional impairment, inflammatory responses, and mortality in response to ischemia- and cisplatin-induced AKI. Compared with primary renal proximal tubule cells isolated from KIM-1Δmucin mice, those from WT mice had reduced proinflammatory cytokine secretion and impaired macrophage activation. The antiinflammatory effect of KIM-1 expression was due to the interaction of KIM-1 with p85 and subsequent PI3K-dependent downmodulation of NF-κB. Hence, KIM-1-mediated epithelial cell phagocytosis of apoptotic cells protects the kidney after acute injury by downregulating innate immunity and inflammation. PMID:25751064

  18. Aggregation of human polymorphonuclear leucocytes during phagocytosis of bacteria.

    PubMed Central

    Henricks, P A; van der Tol, M E; Verhoef, J

    1984-01-01

    The process of aggregation of human polymorphonuclear leucocytes (PMN) during the uptake of bacteria was studied. Radiolabelled S. aureus were opsonized in different sera, washed, resuspended in buffer and added to the PMN. Uptake of the bacteria and aggregation of the PMN were measured simultaneously. Maximal aggregation occurred within 6 min, when 5 X 10(6) PMN had phagocytosed 2.5 X 10(8) S. aureus. Also the effects of serum concentrations and different sera for opsonization of the bacteria on PMN aggregation were studied. Despite normal uptake, aggregation of PMN was low when bacteria were opsonized in complement-deficient sera. Furthermore when PMN were treated with pronase to inactivate complement receptors on the cell surface of the PMN, and bacteria preopsonized in immune serum were added, no change in uptake occurred, although the degree of aggregation halved compared to control PMN. So, interaction between the bacteria and the complement receptor of the PMN cell membrane is needed for triggering the process of aggregation. By using dansylcadaverin and diphenylamine to modulate lysosomal enzyme release, azide or PMN from a chronic granulomatous disease patient to study the effect of the formation of oxygen species, and theophylline, DB-cAMP or 8 Br-cAMP to increase cAMP levels, it was concluded that aggregation of PMN during phagocytosis was not dependent on oxygen metabolism, degranulation or cAMP levels of PMN. PMID:6086503

  19. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    NASA Technical Reports Server (NTRS)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  20. Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages.

    PubMed

    Hodge, S; Hodge, G; Brozyna, S; Jersmann, H; Holmes, M; Reynolds, P N

    2006-09-01

    Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng x mL(-1)) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, alphavbeta3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease. PMID:16737992

  1. P2Y6 Receptor-Mediated Microglial Phagocytosis in Radiation-Induced Brain Injury.

    PubMed

    Xu, Yongteng; Hu, Weihan; Liu, Yimin; Xu, Pengfei; Li, Zichen; Wu, Rong; Shi, Xiaolei; Tang, Yamei

    2016-08-01

    Microglia are the resident immune cells and the professional phagocytic cells of the CNS, showing a multitude of cellular responses after activation. However, how microglial phagocytosis changes and whether it is involved in radiation-induced brain injury remain unknown. In the current study, we found that microglia were activated and microglial phagocytosis was increased by radiation exposure both in cultured microglia in vitro and in mice in vivo. Radiation increased the protein expression of the purinergic receptor P2Y6 receptor (P2Y6R) located on microglia. The selective P2Y6 receptor antagonist MRS2578 suppressed microglial phagocytosis after radiation exposure. Inhibition of microglial phagocytosis increased inhibitory factor Nogo-A and exacerbated radiation-induced neuronal apoptosis and demyelination. We also found that the levels of protein expression for phosphorylated Ras-related C3 botulinum toxin substrate 1 (Rac1) and myosin light chain kinase (MLCK) were elevated, indicating that radiation exposure activated Rac1 and MLCK. The Rac1 inhibitor NSC23766 suppressed expression of MLCK, indicating that the Rac1-MLCK pathway was involved in microglial phagocytosis. Taken together, these findings suggest that the P2Y6 receptor plays a critical role in mediating microglial phagocytosis in radiation-induced brain injury, which might be a potential strategy for therapeutic intervention to alleviate radiation-induced brain injury. PMID:26099306

  2. Investigation of in vitro interactions between different polymeric surfaces and blood proteins via phagocytosis phenomena.

    PubMed

    Ayhan, F; Rad, A Yousefi; Ayhan, H

    2003-01-01

    The effect of various polymeric materials on blood components and their in vitro phagocytosis was the object of the present research. Polystyrene- (PS) and polymethylmetacrylate- (PMMA) based microspheres were produced by phase-inversion polymerization and chemically modified to obtain different surface hydrophilicities. The interactions between blood proteins and chemically- and biologically-modified surfaces were investigated and compared to plain microspheres. Adsorption properties of albumin, fibrinogen and total immunoglobulines on microspheres were tested. Hydrophilic surfaces have high ability for human serum albumin (HSA) adsorption, which also leads to less phagocytosis of microspheres in vitro. In the case of activated PMMA(PVA) microspheres, both protein adsorption and phagocytosis were significant. Interaction of blood proteins with microspheres did not cause any change in phagocytosis by leukocytes and monocytes. BSA adsorption on microspheres with different hydrophilicities showed the same blood protein adsorption results and phagocytosis was not detected. On the other hand, the highest level of phagocytosis was found with fibronectin-modified microspheres. The changes occurring in intrinsic and extrinsic coagulation mechanisms were determined by measuring the activated partial tromboplastin time (APTT) and the prothrombin time (PT). PT values of blood samples did not increase by treatment with microspheres, except for PS/HEMA, while chemical modification caused important prolongation in APTT. PMID:14870945

  3. Innate phagocytosis by peripheral blood monocytes is altered in Alzheimer's disease.

    PubMed

    Gu, Ben J; Huang, Xin; Ou, Amber; Rembach, Alan; Fowler, Christopher; Avula, Pavan K; Horton, Adam; Doecke, James D; Villemagne, Victor L; Macaulay, S Lance; Maruff, Paul; Fletcher, Erica L; Guymer, Robyn; Wiley, James S; Masters, Colin L

    2016-09-01

    Sporadic Alzheimer's disease (AD) is characterised by the deposition and accumulation of specific protein aggregates. Failure of clearance could underlie this process, and recent genetic association studies point towards involvement of the phagocytosis and autophagy pathways. We developed a real-time tri-color flow cytometry method to quantitate the phagocytic function of human peripheral blood monocyte subsets including non-classic CD14(dim)CD16(+), intermediate CD14(+)CD16(+) and classic CD14(+)CD16(-) monocytes. Using this method, we have measured the phagocytic ability of fresh monocytes in a study of preclinical, prodromal and clinical AD, matched with cognitively normal healthy control subjects. Basal levels of phagocytosis in all three subsets of monocytes were similar between healthy controls and AD patients, while a significant increase of basal phagocytosis was found in subjects with high Aβ-amyloid burden as assessed by PET scans. Pre-treating cells with Copaxone (CPX, to stimulate phagocytosis) or ATP (an inhibitor of P2X7-mediated phagocytosis) showed a differential response depending on clinical or Aβ-burden status, indicating a relative functional deficit. Overall the results are consistent with a perturbation of basal and stimulated innate phagocytosis in sporadic AD. PMID:27411339

  4. Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

    PubMed Central

    Dransfield, I; Zagórska, A; Lew, E D; Michail, K; Lemke, G

    2015-01-01

    Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization. PMID:25695599

  5. O-glycosylation in cell wall proteins in Scedosporium prolificans is critical for phagocytosis and inflammatory cytokines production by macrophages.

    PubMed

    Xisto, Mariana I D S; Bittencourt, Vera C B; Liporagi-Lopes, Livia Cristina; Haido, Rosa M T; Mendonça, Morena S A; Sassaki, Guilherme; Figueiredo, Rodrigo T; Romanos, Maria Teresa V; Barreto-Bergter, Eliana

    2015-01-01

    In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines. PMID:25875427

  6. Methods to monitor monocytes-mediated amyloid-beta uptake and phagocytosis in the context of adjuvanted immunotherapies.

    PubMed

    Hallé, Maxime; Tribout-Jover, Pascale; Lanteigne, Anne-Marie; Boulais, Jonathan; St-Jean, Julien R; Jodoin, Rachel; Girouard, Marie-Pier; Constantin, Florin; Migneault, Annik; Renaud, Frédéric; Didierlaurent, Arnaud M; Mallett, Corey P; Burkhart, David; Pilorget, Anthony; Palmantier, Rémi; Larocque, Daniel

    2015-09-01

    Antibody-mediated capture of amyloid-beta (Aβ) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aβ was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aβ, and to examine how two immunological devices--an immunostimulatory Adjuvant System and different amyloid specific antibodies--may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aβ in their plasma. In contrast, the proportion of blood monocytes containing Aβ tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aβ uptake capacity, particularly in the presence of anti-Aβ antibodies. Biochemical experiments demonstrated that cells achieved Aβ uptake and internalization followed by Aβ degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aβ-specific monoclonal antibodies and plasma from Aβ-immunized mice enhanced the phagocytosis of 1 μm Aβ-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aβ uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon. PMID:26002154

  7. O-Glycosylation in Cell Wall Proteins in Scedosporium prolificans Is Critical for Phagocytosis and Inflammatory Cytokines Production by Macrophages

    PubMed Central

    Xisto, Mariana I. D. S.; Bittencourt, Vera C. B.; Liporagi-Lopes, Livia Cristina; Haido, Rosa M. T.; Mendonça, Morena S. A.; Sassaki, Guilherme; Figueiredo, Rodrigo T.; Romanos, Maria Teresa V.; Barreto-Bergter, Eliana

    2015-01-01

    In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines. PMID:25875427

  8. Multigene Engineering of Triacylglycerol Metabolism Boosts Seed Oil Content in Arabidopsis1[W][OPEN

    PubMed Central

    van Erp, Harrie; Kelly, Amélie A.; Menard, Guillaume; Eastmond, Peter J.

    2014-01-01

    Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased. PMID:24696520

  9. ZAP1-mediated modulation of triacylglycerol levels in yeast by transcriptional control of mitochondrial fatty acid biosynthesis.

    PubMed

    Singh, Neelima; Yadav, Kamlesh Kumar; Rajasekharan, Ram

    2016-04-01

    The transcriptional activator Zap1p maintains zinc homeostasis in Saccharomyces cerevisiae. In this study, we examined the role of Zap1p in triacylglycerol (TAG) metabolism. The expression of ETR1 is reduced in zap1Δ. The altered expression of ETR1 results in reduced mitochondrial fatty acid biosynthesis and reduction in lipoic acid content in zap1Δ. The transcription factor Zap1 positively regulates ETR1 expression. Deletion of ETR1 also causes the accumulation of TAG, and the introduction of ETR1 in zap1Δ strain rescues the TAG level. These results demonstrated that the compromised mitochondrial fatty acid biosynthesis causes a reduction in lipoic acid and loss of mitochondrial function in zap1Δ. Functional mitochondria are required for the ATP production and defect in mitochondria slow down the process which may channeled carbon towards lipid biosynthesis and stored in the form of TAG. PMID:26711224

  10. Dietary saturated triacylglycerols suppress hepatic low density lipoprotein receptor activity in the hamster.

    PubMed Central

    Spady, D K; Dietschy, J M

    1985-01-01

    The liver plays a key role in the regulation of circulating levels of low density lipoproteins (LDL) because it is both the site for the production of and the major organ for the degradation of this class of lipoproteins. In this study, the effects of feeding polyunsaturated or saturated triacylglycerols on receptor-dependent and receptor-independent hepatic LDL uptake were measured in vivo in the hamster. In control animals, receptor-dependent LDL transport manifested an apparent Km value of 85 mg/dl (plasma LDL-cholesterol concentration) and reached a maximum transport velocity of 131 micrograms of LDL-cholesterol/hr per g, whereas receptor-independent uptake increased as a linear function of plasma LDL levels. Thus, at normal plasma LDL-cholesterol concentrations, the hepatic clearance rate of LDL equaled 120 and 9 microliter/hr per g by receptor-dependent and receptor-independent mechanisms, respectively. As the plasma LDL-cholesterol was increased, the receptor-dependent (but not the receptor-independent) component declined. When cholesterol (0.12%) alone or in combination with polyunsaturated triacylglycerols was fed for 30 days, receptor-dependent clearance was reduced to 36-42 microliter/hr per g, whereas feeding of cholesterol plus saturated triacylglycerols essentially abolished receptor-dependent LDL uptake (5 microliter/hr per g). When compared to the appropriate kinetic curves, these findings indicated that receptor-mediated LDL transport was suppressed approximately equal to 30% by cholesterol feeding alone and this was unaffected by the addition of polyunsaturated triacylglycerols to the diet. In contrast, receptor-dependent uptake was suppressed approximately equal to 90% by the intake of saturated triacylglycerols. As compared to polyunsaturated triacylglycerols, the intake of saturated lipids was also associated with significantly higher plasma LDL-cholesterol concentrations and lower levels of cholesteryl esters in the liver. Images PMID:2989830

  11. Regulation of Mammary and Adipose Tissue Lipoprotein Lipase and Blood Triacylglycerol in Rats during Late Pregnancy

    PubMed Central

    Spooner, Peter M.; Garrison, Mary M.; Scow, Robert O.

    1977-01-01

    The effects of several prostaglandins on lipoprotein lipase activity of mammary gland and adipose tissue and serum triacylglycerol were studied during late pregnancy in rats. Prostaglandins were injected twice daily for 2 days before and once on the day of analysis. In rats pregnant 20 days, prostaglandin F2α (PGF2α) increased the activity of lipoprotein lipase in mammary gland fourfold, reduced the activity in adipose tissue about 60%, and decreased serum concentration of triacylglycerol 50%. PGF2α also reduced serum concentration of progesterone 90% and increased that of prolactin fivefold, but had no effect on serum concentrations of either immuno-reactive insulin or 17β-estradiol. Injections of 13,14-dihydro-15-keto PGF2α, a metabolite of PGF2α, had similar effects in rats pregnant 20 days, whereas prostaglandins E1 and E2 did not. In rats pregnant 16 days, PGF2α did not affect lipoprotein lipase activity in the tissues or the concentration of triacylglycerol and prolactin in serum, although it decreased serum progesterone 80%. 2-Br-α-ergocryptine prevented the increase in serum prolactin in response to PGF2α, but did not alter the effect of PGF2α on lipoprotein lipase activity or serum triacylglycerol. Progesterone completely blocked the effects of PGF2α on lipoprotein lipase activity and serum triacylglycerol and prolactin concentrations. These findings indicate that the changes in lipoprotein lipase activity and serum triacylglycerol in PGF2α-treated rats are probably related to the inhibitory action of PGF2α on progesterone secretion. They also suggest that endogenous F prostaglandins may play a role in the regulation of lipoprotein lipase activity in mammary gland and adipose tissue near parturition. PMID:893673

  12. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

  13. MiR-146a activates WAVE2 expression and enhances phagocytosis in lipopolysaccharide-stimulated RAW264.7 macrophages.

    PubMed

    Cao, Zhongwei; Yao, Qunyan; Zhang, Shuncai

    2015-01-01

    MiR-146a has been shown to play a critical role in cell immunity and phagocytosis, processes that require rearrangement of the cytoskeleton. However, the detailed mechanism by which miR-146a regulates these events remains elusive. Here, we used luciferase reporter and protein assays to show that the cytoskeleton-regulatingprotein verprolin-homologous protein 2 (WAVE2), is a direct target of miR-146a. MiR-146a overexpression resulted in a decrease in WAVE2 protein expression under endotoxin-free culture conditions. Unexpectedly, however, miR-146a activated rather than repressed the expression of WAVE2 in macrophage RAW264.7 cells when cultured continuously in the presence of endotoxin. Furthermore, we demonstrated that miR-146a induced WAVE2 expression and enhanced phagocytosis in lipopolysaccharide-stimulated RAW264.7 macrophages. Our study suggests that lipopolysaccharide- induced miR146a indirectly activates WAVE2 expression; thus, facilitating cytoskeletal reorganization and phagocytosis in lipopolysaccharide-stimulated macrophages. PMID:26396677

  14. MiR-146a activates WAVE2 expression and enhances phagocytosis in lipopolysaccharide-stimulated RAW264.7 macrophages

    PubMed Central

    Cao, Zhongwei; Yao, Qunyan; Zhang, Shuncai

    2015-01-01

    MiR-146a has been shown to play a critical role in cell immunity and phagocytosis, processes that require rearrangement of the cytoskeleton. However, the detailed mechanism by which miR-146a regulates these events remains elusive. Here, we used luciferase reporter and protein assays to show that the cytoskeleton-regulatingprotein verprolin-homologous protein 2 (WAVE2), is a direct target of miR-146a. MiR-146a overexpression resulted in a decrease in WAVE2 protein expression under endotoxin-free culture conditions. Unexpectedly, however, miR-146a activated rather than repressed the expression of WAVE2 in macrophage RAW264.7 cells when cultured continuously in the presence of endotoxin. Furthermore, we demonstrated that miR-146a induced WAVE2 expression and enhanced phagocytosis in lipopolysaccharide-stimulated RAW264.7 macrophages. Our study suggests that lipopolysaccharide- induced miR146a indirectly activates WAVE2 expression; thus, facilitating cytoskeletal reorganization and phagocytosis in lipopolysaccharide-stimulated macrophages. PMID:26396677

  15. Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations.

    PubMed Central

    Lasunción, M A; Herrera, E

    1983-01-01

    Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme. PMID:6870799

  16. Recurrent phagocytosis-induced apoptosis in the cyclical generation change of the compound ascidian Botryllus schlosseri.

    PubMed

    Franchi, Nicola; Ballin, Francesca; Manni, Lucia; Schiavon, Filippo; Basso, Giuseppe; Ballarin, Loriano

    2016-09-01

    Colonies of the marine, filter-feeding ascidian Botryllus schlosseri undergo cyclical generation changes or takeovers. These events are characterised by the progressive resorption of adult zooids and their replacement by their buds that grow to adult size, open their siphons and start filtering. During the take-over, tissues of adult zooids undergo extensive apoptosis; circulating, spreading phagocytes enter the effete tissues, ingest dying cells acquiring a giant size and a round morphology. Then, phagocytes re-enter the circulation where they represent a considerable fraction (more than 20%) of circulating haemocytes. In this study, we evidence that most of these circulating phagocytes show morphological and biochemical signs of apoptosis. Accordingly, these phagocytes express transcripts of orthologues of the apoptosis-related genes Bax, AIF1 and PARP1. Electron microscopy shows that giant phagocytes contain apoptotic phagocytes inside their own phagocytic vacuole. The transcript of the orthologues of the anti-apoptotic gene IAP7 was detected only in spreading phagocytes, mostly abundant in phases far from the take-over. Therefore, the presented data suggest that, at take-over, phagocytes undergo phagocytosis-induced apoptosis (PIA). In mammals, PIA is assumed to be a process assuring the killing and the complete elimination of microbes, by promoting the disposal of terminally differentiated phagocytes and the resolution of infection. In B. schlosseri, PIA assumes a so far undescribed role, being required for the control of asexual development and colony homeostasis. PMID:27106705

  17. Aspergillus Cell Wall Melanin Blocks LC3-Associated Phagocytosis to Promote Pathogenicity.

    PubMed

    Akoumianaki, Tonia; Kyrmizi, Irene; Valsecchi, Isabel; Gresnigt, Mark S; Samonis, George; Drakos, Elias; Boumpas, Dimitrios; Muszkieta, Laetitia; Prevost, Marie-Christine; Kontoyiannis, Dimitrios P; Chavakis, Triantafyllos; Netea, Mihai G; van de Veerdonk, Frank L; Brakhage, Axel A; El-Benna, Jamel; Beauvais, Anne; Latge, Jean-Paul; Chamilos, Georgios

    2016-01-13

    Concealing pathogen-associated molecular patterns (PAMPs) is a principal strategy used by fungi to avoid immune recognition. Surface exposure of PAMPs during germination can leave the pathogen vulnerable. Accordingly, β-glucan surface exposure during Aspergillus fumigatus germination activates an Atg5-dependent autophagy pathway termed LC3-associated phagocytosis (LAP), which promotes fungal killing. We found that LAP activation also requires the genetic, biochemical or biological (germination) removal of A. fumigatus cell wall melanin. The attenuated virulence of melanin-deficient A. fumigatus is restored in Atg5-deficient macrophages and in mice upon conditional inactivation of Atg5 in hematopoietic cells. Mechanistically, Aspergillus melanin inhibits NADPH oxidase-dependent activation of LAP by excluding the p22phox subunit from the phagosome. Thus, two events that occur concomitantly during germination of airborne fungi, surface exposure of PAMPs and melanin removal, are necessary for LAP activation and fungal killing. LAP blockade is a general property of melanin pigments, a finding with broad physiological implications. PMID:26749442

  18. Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

    PubMed Central

    Lefkir, Yaya; Malbouyres, Marilyne; Gotthardt, Daniel; Ozinsky, Adrian; Cornillon, Sophie; Bruckert, Franz; Aderem, Alan A.; Soldati, Thierry; Cosson, Pierre; Letourneur, François

    2004-01-01

    The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1- cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1- cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1- cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation. PMID:14617812

  19. 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

  20. Neuroendocrine mediators in the modulation of phagocytosis by exercise: physiological implications.

    PubMed

    Ortega, Eduardo

    2003-01-01

    Neuroendocrine mediation of the effects of exercise on macrophage and neutrophil phagocytosis, the organism's first line of defense against external aggression, is reviewed. Exercise modulates the immune system via the actions of "stress hormones". Although stress had long been regarded as generally immunosuppressive, it is now accepted that this is not always true. Indeed exercise-induced stress stimulates the "phagocytic process" of phagocytes. One of the new physiological interpretations emerging from recent studies is that the general stimulation of phagocytosis and other innate mechanisms during strenuous physical activity may counterbalance the decreased lymphoid activity, preventing the entry and survival of microorganisms in situations where the specific responses are depressed. In some cases this behaviour is also medicated by "stress hormones", unlike in lymphocytes in which glucocorticoids and catecholamines both are immunosuppressive. The mediatory role of glucocorticoids in macrophages may also differ between the non-specific functions, like chemotaxis and phagocytosis, and the more specific ones, like antigen-presentation. Neutrophils and monocytes may be stimulated by catecholamines or sympathetic signals, and variations in phagocytosis and catecholamines have been proposed as a good "neuroimmuno-endocrinological marker" in athletes. Other hormones (prolactin, GH, endorphins, thyroid hormones) in general also contribute to the effects of exercise-stress on phagocytosis. This review focuses on a physiological interpretation of the immune response to exercise which differs markedly from the classical immunosuppression-centered view. More studies on in vivo variations of stress hormones during exercise are needed. PMID:14686096

  1. Analysis of the dynamic energy flow associated with phagocytosis of bacteria.

    PubMed

    Okpala, Paul; Omenyi, Sam; Ozoegwu, Godwin; Achebe, Chinonso

    2015-09-01

    This paper treats the phenomenon of phagocytosis from the flow of energy point of view. Considerable efforts have been made towards elucidating the subject of phagocytosis in other fields of learning, but little has been said about the mechanical work that is done during phagocytosis. Phagocytosis without doubt is an interaction that involves the flow of energy. Energy equation model of phagocytosis is then presented in this paper to analyze the mechanical energy that is involved in the build-up of the engulfment of bacteria by the phagocytes. Data of the E Coli bacteria from published work was then applied to the solution of the energy equation. A borderline contact angle [Formula: see text] of [Formula: see text] between the phagocyte and the bacteria at [Formula: see text] was deduced in this work. It was shown that when [Formula: see text], [Formula: see text], engulfment is favoured and when [Formula: see text], [Formula: see text], engulfment is not favoured for E-coli. This condition is conceptually in line with ΔFNET approach reported in the literature. Data of four different bacterial species were also used to plot the graphs of the engulfment parameter [Formula: see text] against contact angle [Formula: see text] which revealed that the more hydrophobic bacteria are easily phagocytized than the more hydrophilic ones. PMID:27441215

  2. MAP1S Protein Regulates the Phagocytosis of Bacteria and Toll-like Receptor (TLR) Signaling.

    PubMed

    Shi, Ming; Zhang, Yifan; Liu, Leyuan; Zhang, Tingting; Han, Fang; Cleveland, Joseph; Wang, Fen; McKeehan, Wallace L; Li, Yu; Zhang, Dekai

    2016-01-15

    Phagocytosis is a critical cellular process for innate immune defense against microbial infection. The regulation of phagocytosis process is complex and has not been well defined. An intracellular molecule might regulate cell surface-initiated phagocytosis, but the underlying molecular mechanism is poorly understood (1). In this study, we found that microtubule-associated protein 1S (MAP1S), a protein identified recently that is involved in autophagy (2), is expressed primarily in macrophages. MAP1S-deficient macrophages are impaired in the phagocytosis of bacteria. Furthermore, we demonstrate that MAP1S interacts directly with MyD88, a key adaptor of Toll-like receptors (TLRs), upon TLR activation and affects the TLR signaling pathway. Intriguingly, we also observe that, upon TLR activation, MyD88 participates in autophagy processing in a MAP1S-dependent manner by co-localizing with MAP1 light chain 3 (MAP1-LC3 or LC3). Therefore, we reveal that an intracellular autophagy-related molecule of MAP1S controls bacterial phagocytosis through TLR signaling. PMID:26565030

  3. Quantitative analysis of positional isomers of triacylglycerols via electrospray ionization tandem mass spectrometry of sodiated adducts.

    PubMed

    Herrera, Lisandra Cubero; Potvin, Michael A; Melanson, Jeremy E

    2010-09-01

    Herein we report a reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC/MS/MS) method for the analysis of positional isomers of triacylglycerols (TAGs) in vegetable oils. The fragmentation behavior of [M + X](+) ions (X = NH(4), Li, Na or Ag) was studied on a quadrupole-time-of-flight (Q-TOF) mass spectrometer under low-energy collision-induced dissociation (CID) conditions. Mass spectra that were dependent on the X(+) ion and the nature and position of the acyl substituents were observed for four pairs of 'AAB/ABA'-type TAGs, namely PPO/POP, OOP/OPO, LLO/LOL and OOL/OLO (where P is 16:0, palmitic acid; O is 18:1, oleic acid; and L is 18:2, linoleic acid). For the majority of [M + X](+) adducts, the loss of the fatty acid in the outer positions (sn-1 or sn-3) was favored over the loss in the central position (sn-2), which enabled the determination of the fractional abundance of the isomers. Ratios of the intensity of fragment ions at various AAB/ABA compositions produced linear calibration curves with positive slopes, comparable to those obtained traditionally by ESI-MS/MS of [M + NH(4)](+) adducts. The only exceptions were the [M + Ag](+) adducts of the PPO/POP system, which produced calibration curves with negative slopes. Sodium adducts provided the most consistent level of isomeric discrimination for the TAGs studied and also offered the most convenience in that they required no additive to the mobile phase. Therefore, calibration curve data derived from [M + Na](+) adducts were applied to the quantification of TAG regioisomers in sunflower and olive oils. The regiospecific analysis showed that palmitic acid was typically located at positions sn-1 or sn-3, whereas unsaturated fatty acids, oleic and linoleic acids were mostly found at the sn-2 position. PMID:20814981

  4. CALORIMETRIC PROPERTIES OF WATER AND TRIACYLGLYCEROLS IN FERN SPORES RELATING TO STORAGE AT CRYOGENIC TEMPERATURES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects of triacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at -15 degrees C or liquid nitrogen temperatures. We us...

  5. BIODEGRADATION KINETICS AND TOXICITY OF VEGETABLE OIL TRIACYLGLYCEROLS UNDER AEROBIC CONDITIONS

    EPA Science Inventory

    The aerobic biodegradation of five triacylglycerols (TAGs), three liquids [triolein (OOO), trilinolein (LLL), and trilinolenin (LnLnLn)] and two solids [tripalmitin (PPP) and tristearin (SSS)] was studied in water. Respirometry tests were designed and conducted to determine the b...

  6. Molecular species of diacylglycerols and triacylglycerols containing dihydroxy fatty acids in castor oil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The identification of four molecular species of diacylglycerols and eight molecular species of triacylglycerols containing dihydroxy fatty acids in castor oil was reported. The structures of the three dihydroxy fatty acids were proposed as 11,12-dihydroxy-9-octadecenoic acid, 11,12-dihydroxy-9,13-oc...

  7. Enhanced bioavailability of EPA from emulsified fish oil preparations versus capsular triacylglycerol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pre-emulsified fish oil supplements, an alternative to capsular triacylglycerol, may enhance the uptake of LCn3 fatty acids it contains. A randomized, Latin-square crossover design was used to compare the effects of four fish oil supplement preparations on phospholipid (PLFA) and chylomicron fatty ...

  8. Ratios of regioisomers of triacylglycerols containing dihydroxy fatty acids in castor oil by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The triacylglycerols (TAG) containing dihydroxy fatty acids have been recently identified by mass spectrometry in castor oil. These new dihydroxy fatty acids were proposed earlier as 11,12-dihydroxy-9-octadecenoic acid (diOH18:1), 11,12-dihydroxy-9,13-octadecadienoic acid (diOH18:2) and 11,12-dihydr...

  9. Analysis of regiospecific triacylglycerols by electrospray ionization-mass spectrometry 3 of lithiated adducts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some regiospecific triacylglycerol standards containing normal fatty acids, e.g., 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) and 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), were analyzed by the electrospray ionization MS3 of their lithiated adducts. The fragment ions of the MS3 from the loss of alpha,...

  10. Water-triacylglycerol interactions affect oil body structure and seed viability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are investigating interactions between water and triacylglycerols (TAG) that appear to affect oil body stability and viability of seeds. Dried seeds are usually stored at freezer temperatures (-20oC) for long-term conservation of genetic resources. This globally accepted genebanking practice is...

  11. An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The polyunsaturated fatty acids (PUFAs), linoleic (18:2) and '-linolenic (18:3), in triacylglycerols (TAG) are major factors that affect the quality of plant oils for human health, as well as for biofuels and other renewable applications. These PUFAs are essential fatty acids for animals and plants,...

  12. Analysis of regiospecific triacylglycerols in vegetable oils and animal fats by electrospray mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method of regiospecific analysis of triacylglycerols (TAG) in vegetable oils and animal fats is reported here using the electrospray ionization MS3 of TAG lithiated adducts. The fragment ions of the MS3 from the loss of fatty acids at the sn-2 position as alpha, Beta-unsaturated fatty acids were u...

  13. Acylglycerols Containing Trihydroxy Fatty Acids in Castor Oil and the Regiospecific Quantification of Triacylglycerols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ricinoleate, a monohydroxy fatty acid in castor oil, has many industrial uses. Dihydroxy and trihydroxy fatty acids can also be used in industry. We report here the identification of diacylglycerols and triacylglycerols containing trihydroxy fatty acids in castor oil. The C18 HPLC fractions of casto...

  14. Quadruple parallel mass Spectrometry for analysis of vitamin D and triacylglycerols in a dietary supplement

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A ‘dilute-and-shoot’ method for vitamin D and triacylglycerols is demonstrated that employed four mass spectrometers, operating in different ionization modes, for a ‘quadruple parallel mass spectrometry’ analysis, plus three other detectors, for seven detectors overall. Sets of five samples of diet...

  15. Regiospecific Quantification of Triacylglycerols Containing Dihydroxy Fatty Acids in Castor Oil by Mass Spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The triacylglycerols (TAG) containing dihydroxy fatty acids have been recently identified by mass spectrometry in castor oil. These new dihydroxy fatty acids were proposed earlier as 11,12-dihydroxy-9-octadecenoic acid (diOH18:1), 11,12-dihydroxy-9,13-octadecadienoic acid (diOH18:2) and 11,12-dihydr...

  16. Ratios of the molecular species of triacylglycerols in lesquerella (Physaria fendleri) oil estimated by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ratios of regioisomers of 72 molecular species of triacylglycerols (TAG) in lesquerella oil were estimated using the electrospray ionization mass spectrometry of the lithium adducts of TAG in the HPLC fractions of lesquerella oil. The ratios of ion signal intensities (or relative abundances) of ...

  17. Ratios of the molecular species of triacylglycerols in lesquerella (Physaria fendleri) oil estimated by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ratios of regioisomers of 74 molecular species of triacylglycerols (TAG) in lesquerella oil were estimated using HPLC and the electrospray ionization mass spectrometry of the lithium adducts of TAG in the HPLC fractions of lequerella oil. The ratios of relative abundances of the fragment ions fr...

  18. An fMLP receptor is involved in activation of phagocytosis by hemocytes from specific insect species.

    PubMed

    García-García, Erick; García-García, Patricia Lucero; Rosales, Carlos

    2009-06-01

    In mammalian phagocytes, the bacterial formylated peptide fMLP functions both as a potent enhancer of phagocytosis and chemoattractant. fMLP has been reported to be chemotactic for hemocytes of two marine invertebrates, and of the insect Manduca sexta (Lepidoptera). Whether fMLP is also able to activate phagocytosis has not been explored in hemocytes of any invertebrate. To determine the effect of fMLP on insect hemocyte phagocytosis, in vitro phagocytosis assays were performed with hemocytes from the insects: Gromphadorhina portentosa (Blattodea), Acheta domesticus (Orthoptera), Zophobas morio (Coleoptera), and Galleria mellonella (Lepidoptera). Phagocytosis of latex, zymosan (yeast), Gram-positive and Gram-negative bacteria was measured by flow cytometry, in the presence of increasing fMLP concentrations. G. portentosa hemocytes showed no enhancement of phagocytosis by fMLP. A. domesticus hemocytes had increased phagocytosis of latex and Gram-negative bacteria in the presence of fMLP. Z. morio hemocytes increased phagocytosis of latex, yeast, and Gram-negative bacteria after fMLP stimulation. Galleria mellonella hemocytes increased phagocytosis of latex after fMLP stimulation. Treating hemocytes with Pertussis toxin, a known inhibitor of the signaling pathway initiated by the mammalian fMLP receptor, returned phagocytosis to basal levels. Also, hemocytes from all insect species tested presented a similar chemotactic response to fMLP. These data suggest that, whereas the ability of hemocytes to chemotactically-respond to fMLP is conserved in insects ranging from Blattodea to Lepidoptera, the ability to respond to fMLP by activating phagocytosis is restricted to specific insect species. PMID:19166874

  19. Earthworm coelomocyte phagocytosis: An in vitro assay for terrestrial toxicity identification evaluation

    SciTech Connect

    Burch, S.W.; Goven, A.J.; Fitzpatrick, L.C.; Venables, B.J.; Callahan, C.A.

    1995-12-31

    An in vitro assay has been developed for rapid (48 h) evaluation of cytotoxic effects of exposure (24 h) of earthworm coelomocytes. The assay, inhibition of phagocytosis (24 h) of stained yeast cells and cell viability, links a traditional soil bioassay organism (Lumbricus terrestris) with a laboratory protocol for use in evaluating physical/chemical fractions resulting from terrestrial TIE manipulations. The assay was developed using copper sulfate as a reference toxicant. Copper exposures as low as 2--4 pg/ml. resulted in 20--60% inhibition of phagocytosis without significant decrease in cell viability. Exposures above 10 pg/ml resulted in reduced cell viability and inhibition of phagocytosis. The assay was successfully applied to terrestrial TIE fractions derived from extractions of soil from a PCP contaminated wood treatment site.

  20. Enhanced phagocytosis of group A streptococci M type 6 by oleic acid

    SciTech Connect

    Speert, D.P.; Quie, P.G.; Wannamaker, L.W.

    1981-04-01

    M protein, located on the surface fimbriae of group A streptococci, is antiphagocytic by unknown means. It is known that oleic acid kills group A streptococci and distorts the fimbriae. The effect of oleic acid on phagocytosis of group A streptococci was examined. Phagocytosis of a strain possessing M protein (M+) and its M- variant was assessed by uptake of radiolabeled bacteria and by chemiluminescence. The M- but not the M+ streptococci were well phagocytized and induced chemiluminescence. Oleic acid-killed and heat-killed streptococci (both M+ and M-) were readily phagocytized and induced sustained chemiluminescence. M+ streptococci killed by ultraviolet irradiation were inefficiently phagocytized and did not induce chemiluminescence. Oleic acid-killed M+ streptococci absorbed type-specific antibody. An extract of M protein reduced the bactericidal capacity of oleic acid. It is proposed that oleic acid may bind to and alter the M protein of group A streptococci and thereby enhance phagocytosis.

  1. Primary Phagocytosis of Neurons by Inflamed Microglia: Potential Roles in Neurodegeneration

    PubMed Central

    Neher, Jonas J.; Neniskyte, Urte; Brown, Guy C.

    2012-01-01

    Microglial phagocytosis of dead or dying neurons can be beneficial by preventing the release of damaging and/or pro-inflammatory intracellular components. However, there is now evidence that under certain conditions, such as inflammation, microglia can also phagocytose viable neurons, thus executing their death. Such phagocytic cell death may result from exposure of phosphatidylserine (PS) or other eat-me signals on otherwise viable neurons as a result of physiological activation or sub-toxic insult, and neuronal phagocytosis by activated microglia. In this review, we discuss the mechanisms of phagocytic cell death and its potential roles in Alzheimer’s Disease, Parkinson’s Disease, and Frontotemporal Dementia. PMID:22403545

  2. Role of arachidonic acid metabolites in the action of a beta adrenergic agonist on human monocyte phagocytosis.

    PubMed

    Borda, E S; Tenenbaum, A; Sales, M E; Rumi, L; Sterin-Borda, L

    1998-02-01

    The mechanisms by which beta adrenergic stimulation regulates phagocytosis of Candida albicans by human peripheral monocytes (HPM) are characterized. Isoproterenol (ISO) inhibits phagocytosis in a concentration-dependent manner. This effect was blunted by propranolol, inhibitors of phospholipase A2 (PLA2), cyclooxygenase and verapamil, pointing to a participation of arachidonic acid (AA) metabolites and calcium in the phenomenon. Prostaglandin E2 (PGE2) and dibutyryl cyclic AMP (db-cAMP) also exerted the same inhibitory effect on phagocytosis. ISO interacts with beta adrenergic receptors of HPM increasing PGE2 and cAMP. We conclude that the mechanisms by which beta adrenergic stimulation regulates phagocytosis of Candida albicans by HPM appear to be secondary to beta adrenoceptor-mediated hydrolysis of AA accompanied by an increase in PGE2 generation and cAMP production. Both PGE2 and cAMP could act as mediators of the inhibitory action of beta agonists on the HPM-phagocytosis process. PMID:9578144

  3. Specific non-coplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure.

    PubMed

    Levin, Milton; Morsey, Brenda; Mori, Chiharu; Guise, Sylvain

    2004-10-01

    Contaminant-induced immunosuppression by organochlorines (OC), particularly polychlorinated biphenyls (PCBs), has been suspected as a cofactor in the deaths of thousands of marine mammals. One important innate defense mechanism is phagocytosis, the ability of cells to ingest extracellular macromolecules. The present study was aimed at characterizing the immunomodulatory potential of representative OCs on phagocytosis in bottlenose dolphins and beluga whales. The ability of peripheral blood leukocytes to engulf fluorescent microspheres was evaluated using flow cytometry. The immunomodulatory effects of three non-coplanar PCB congeners, 138, 153, and 180, one coplanar PCB, 169, and 2,3,7,8-TCDD and all possible mixtures (26) were tested upon in vitro exposure. In both species, all mixtures containing at least two non-coplanar PCBs significantly reduced both neutrophil and monocyte phagocytosis, with effects more marked in dolphins than in belugas. Coplanar OCs, on their own or when added to non-coplanar congeners, did not further modulate phagocytosis, suggesting an Ah receptor-independent mechanism. Concentration-response experiments with individual congeners further demonstrated a non-coplanar PCB-induced suppression of phagocytosis, while coplanar congeners produced no consistent effects. Our results suggest simple additive interactions of chemicals in a mixture. However, calculation of toxic equivalency (TEQs) failed to predict the experimentally induced immunomodulatory effects of OCs on dolphin and beluga phagocytosis, confirming the Ah receptor-independent nature of the effects on phagocytosis. Overall, our results suggest that non-AhR mechanisms may explain one facet of immunotoxicity (phagocytosis), something that is not captured using the TEQ approach. This is the first report demonstrating the immunomodulatory effects of OCs on dolphin and beluga phagocytosis, and the first overall demonstration of immunomodulatory effects on phagocytosis mediated

  4. Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

    PubMed Central

    Cox, D; Wessels, D; Soll, D R; Hartwig, J; Condeelis, J

    1996-01-01

    The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120. Images PMID:8744952

  5. A Fluorescently Tagged C-Terminal Fragment of p47phox Detects NADPH Oxidase Dynamics during Phagocytosis

    PubMed Central

    Li, Xing Jun; Tian, Wei; Stull, Natalie D.; Grinstein, Sergio; Atkinson, Simon

    2009-01-01

    The assembly of cytosolic p47phox and p67phox with flavocytochrome b558 at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47phox and p67phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47phox PRR. This fragment was recruited in a flavocytochrome b558-dependent, p67phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67phox SH3b domain and suggest that the p47phox/p67phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes. PMID:19129478

  6. Effect of complement and of the carbohydrate components of sputum on phagocytosis by human polymorphonuclear leucocytes

    PubMed Central

    Brogan, T. D.

    1964-01-01

    The phagocytic activity of human polymorphonuclear leucocyte preparations, which were free from plasma, has been estimated by direct determination under phase contrast of the number of living cells containing test particles. Spores of Aspergillus fumigatus were phagocytosed in the absence of added serum but phagocytosis of paraffin wax particles occurred only in the presence of serum containing the heat-labile and C′4 components of complement. In view of the unreactive nature of the paraffin hydrocarbons, it was considered unlikely that natural antibody played any part in the phenomenon. Although no phagocytosis of wax particles occurred in the absence of serum, almost 100 per cent of cells were phagocytic in preparations containing adequate concentrations of serum. It was therefore possible to determine the serum concentration necessary for 50 per cent of the polymorphs to phagocytose wax particles. By this means it was demonstrated that the addition of the carbohydrate components of sputum had a small but significant inhibitory effect on phagocytosis and that dextran had no such effect. The sputum mucoprotein depressed the complement titre of serum and this might have accounted for the reduction in the ability of a serum to promote phagocytosis when this complex was added. The sputum mucopolysaccharide had no such effect on the complement titre of serum and must have exerted its inhibitory action in some other way. ImagesFIG. 2 PMID:14239838

  7. Optical tracking of phagocytosis with an activatable profluorophore metabolically incorporated into bacterial peptidoglycan.

    PubMed

    Tian, Yunpeng; Yu, Mingzu; Li, Zhu; Han, Jiahuai; Yang, Liu; Han, Shoufa

    2015-08-18

    Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by "always-on" signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report "turn-on" fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan. E. coli and S. aureus become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficient in situ distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis. PMID:26202911

  8. Rediae of echinostomatid and heterophyid trematodes suppress phagocytosis of haemocytes in Littorina littorea (Gastropoda: Prosobranchia).

    PubMed

    Iakovleva, Nadya V; Shaposhnikova, Tania G; Gorbushin, Alexander M

    2006-05-01

    A modulation of the phagocytic activity of hemocytes from the common periwinkle Littorina littorea by secretory-excretory products (SEP) released by trematode rediae during axenic in vitro cultivation was studied. The SEP released by the parasites Himasthla elongata (Echinostomatidae) and Cryptocotyle lingua (Heterophyidae) were found to inhibit the phagocytosis of zymozan particles by periwinkle hemocytes. The specificity of SEP effects was assessed: SEP of Himasthla militaris and Cryptocotyle concavum, two trematodes belonging to the same genera but infecting another closely related prosobranch snail Hydrobia ulvae, were also shown to be able to suppress L. littorea hemocytes phagocytic activity. However, no decrease in phagocytosis rate was observed when SEP of H. elongata and C. lingua were applied to monolayers of hemocytes from the bivalve mollusc Mytilus edulis. SEP from H. elongata was fractionated; only those fractions containing proteins of molecular weight more than 50 kDa were shown to possess inhibitory activity. Different H. elongata SEP concentrations were tested in for their ability to suppress phagocytosis by L. littorea hemocytes. Even very low SEP concentrations were shown to retain their ability to decrease phagocytosis rate, the inhibitory effect being dose-dependent. Hemocytes derived from snails naturally infected with H. elongata were also found to have lower phagocytic ability as compared to healthy individuals. PMID:16438967

  9. Effects of cyclic strain on the morphology and phagocytosis of macrophages.

    PubMed

    Miyazaki, H; Hayashi, K

    2001-01-01

    Morphology and phagocytosis of macrophages were studied after cultured under cyclic strain. Peritoneal macrophages obtained from 6-week old female C57BL/6J mice were attached to silicone rubber sheets, preincubated for 2 hours, and then cultured for 24 and 48 hours under cyclic strain of 5% amplitude and 1 Hz frequency (strained group) or no strain (non-strained group). Control data were obtained from 0-hour cultured cells (control group). Cell morphology was observed with optical and scanning electron microscopes, and shape index (SI) of each cell was determined from the perimeter and adhesion area. Phagocytotic activity was evaluated from the uptake of latex particles 1.5 microm in diameter. The morphology and phagocytosis of macrophages were affected by cyclic strain, although the percentage of differentiated cells was not the case. SI in the strained group was smaller than that in the non-strained group, and had a tendency of decreasing with time. Cyclic strain suppressed the phagocytosis of macrophages for latex particles, and there was a significant difference in the phagocytosis between the strained and the non-strained groups after 24-hour culture. PMID:11790862

  10. A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis

    PubMed Central

    Le Pabic, Hélène; Germain-Amiot, Noëlla; Bordeau, Valérie; Felden, Brice

    2015-01-01

    Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation. PMID:26240382

  11. Immune phagocytosis of Plasmodium yoelii-infected erythrocytes by macrophages and eosinophils.

    PubMed Central

    Tosta, C E; Wedderburn, N

    1980-01-01

    Unstimulated peritoneal cells from C57Bl mice were allowed to phagocytose in vitro different mixtures of Percoll-separated parasitized and non-parasitized erythrocytes (PE and NPE) from the blood of mice infected with Plasmodium yoelii in the presence of immune and normal serum. Immune serum caused a significant enhancement of phagocytosis, and both the number of PE adhering to and/or ingested by 100 macrophages and the number of the latter cells engaged in phagocytosis was increased. The effect of immune serum was more marked when the ratio of PE/macrophages was 5--40/1, but was less at a ratio of 80/1, when considerable phagocytosis of PE occurred in the presence of normal serum. From 83--100% of the phagocytosed cells were parasitized erythrocytes, even when the ratio of PE/NPE was as low as 1/15. In the system used, macrophages were unable to discriminate between non-parasitized erythrocytes from infected mice and normal erythrocytes. Eosinophils were also observed to engage in phagocytosis of parasitized erythrocytes. Their activity was entirely dependent on immune serum and was never directed against non-parasitized red blood cells. PMID:7460387

  12. A simple microscopy assay to teach the processes of phagocytosis and exocytosis.

    PubMed

    Gray, Ross; Gray, Andrew; Fite, Jessica L; Jordan, Renée; Stark, Sarah; Naylor, Kari

    2012-01-01

    Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to organelle renewal and maintenance, we felt that we needed to do a better job of teaching these types of processes. Thus, we developed a classroom-based protocol to simultaneously study phagocytosis and exocytosis in Tetrahymena pyriformis. In this paper, we present our results demonstrating that our undergraduate classroom experiment delivers results comparable with those acquired in a professional research laboratory. In addition, students performing the experiment do learn the mechanisms of phagocytosis and exocytosis. Finally, we demonstrate a mathematical exercise to help the students apply their data to the cell. Ultimately, this assay sets the stage for future inquiry-based experiments, in which the students develop their own experimental questions and delve deeper into the mechanisms of phagocytosis and exocytosis. PMID:22665590

  13. Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

    PubMed

    Kurucz, Eva; Márkus, Róbert; Zsámboki, János; Folkl-Medzihradszky, Katalin; Darula, Zsuzsanna; Vilmos, Péter; Udvardy, Andor; Krausz, Ildikó; Lukacsovich, Tamás; Gateff, Elisabeth; Zettervall, Carl-Johan; Hultmark, Dan; Andó, István

    2007-04-01

    The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome. PMID:17363253

  14. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells

    PubMed Central

    2016-01-01

    Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB) infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect. PMID:27413759

  15. High Fc Density Particles Result in Binary Complement Activation but Tunable Macrophage Phagocytosis

    NASA Astrophysics Data System (ADS)

    Sulchek, Todd; Pacheco, Patricia; White, David

    2014-03-01

    Macrophage phagocytosis and complement system activation represent two key components of the immune system and both can be activated through the presentation of multiple Fc domains of IgG antibodies. We have created functionalized micro- and nanoparticles with various densities of Fc domains to understand the modulation of the immune system for eventual use as a novel immunomodulation platform. Phagocytosis assays were carried out by adding functionalized particles to macrophage cells and quantitatively determined using fluorescent microscopy and flow cytometry. Complement system activation by the functionalized particles in human serum was quantified with an enzyme immunoassay. Our phagocytosis assay revealed a strong dependence on particle size and Fc density. For small particles, as the Fc density increased, the number of particles phagocytosed also increased. Large particles were phagocytosed at significantly lower levels and showed no dependency on Fc density. Complement was successfully activated at levels comparable to positive controls for small particles at high Fc densities. However at low Fc densities, there is a significant decrease in complement activation. This result suggests a binary response for complement system activation with a threshold density for successful activation. Therefore, varying the Fc density on micro/nanoparticles resulted in a tunable response in macrophage phagocytosis while a more binary response for complement activation.

  16. Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

    PubMed Central

    Matte, Christine; Casgrain, Pierre-André; Séguin, Olivier; Moradin, Neda; Hong, Wan Jin; Descoteaux, Albert

    2016-01-01

    The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery. PMID:27280768

  17. Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.

    PubMed

    Matte, Christine; Casgrain, Pierre-André; Séguin, Olivier; Moradin, Neda; Hong, Wan Jin; Descoteaux, Albert

    2016-06-01

    The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery. PMID:27280768

  18. SCAVENGER RECEPTORS FOUND ON CHICKEN HETEROPHILS CONTRIBUTE TO THE PHAGOCYTOSIS OF SALMONELLA ENTERITIDIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pattern recognition receptors (PRR) are a critical component of the innate immune response and the hosts¿ ability to recognize self from infectious non-self. Scavenger receptors (SR), a type of PRR, are cell surface glycoproteins involved in receptor-mediated phagocytosis of polyanionic ligands. H...

  19. DEVELOPMENTAL EXPOSURE TO A THYROID DISRUPTING CHEMICAL STIMULATES PHAGOCYTOSIS IN JUVENILE SPRAGUE-DAWLEY RATS

    EPA Science Inventory

    Developmental Exposure to a Thyroid Disrupting Chemical Stimulates Phagocytosis in Juvenile Sprague-Dawley Rats.
    AA Rooney1, R Matulka2, and R Luebke3. 1NCSU/US EPA CVM, Department of Anatomy, Physiological Sciences and Radiology, Raleigh, NC;2UNC Department of Toxicology, Cha...

  20. A Simple Microscopy Assay to Teach the Processes of Phagocytosis and Exocytosis

    ERIC Educational Resources Information Center

    Gray, Ross; Gray, Andrew; Fite, Jessica L.; Jordan, Renee; Stark, Sarah; Naylor, Kari

    2012-01-01

    Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to…

  1. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.

    PubMed

    Rämet, Mika; Manfruelli, Pascal; Pearson, Alan; Mathey-Prevot, Bernard; Ezekowitz, R Alan B

    2002-04-11

    The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis. PMID:11912489

  2. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

    PubMed Central

    2012-01-01

    Background Human Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat

  3. Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production

    SciTech Connect

    Kakita, Hiroki; Aoyama, Mineyoshi; Nagaya, Yoshiaki; Asai, Hayato; Hussein, Mohamed Hamed; Suzuki, Mieko; Kato, Shin; Saitoh, Shinji; Asai, Kiyofumi

    2013-04-15

    Influenza-associated encephalopathy (IAE) is a central nervous system complication with a high mortality rate, which is increased significantly by the non-steroidal anti-inflammatory drug diclofenac sodium (DCF). In the present study, we investigated the effects of DCF on brain immune cells (i.e. microglia) stimulated with three proinflammatory cytokines, namely tumor necrosis factor-α, interleukin-1β, and interferon-γ. Similar to previous findings in astrocytes, all three cytokines induced the expression of inducible NO synthase (iNOS), as well as NO production, in microglia. The addition of DCF to the culture system augmented iNOS expression and NO production. Immunocytochemical analysis and the phagocytosis assay revealed that cytokine treatment induced morphological changes to and phagocytosis by the microglia. The addition of DCF to the culture system enhanced microglial activation, as well as the phagocytic activity of cytokine-stimulated microglia. Inhibitors of nuclear factor (NF)-κB inhibited iNOS gene expression in cytokine-stimulated microglia with or without DCF, suggesting that the NF-κB pathway is one of the main signaling pathways involved. The iNOS inhibitor N{sup G}-monomethyl-L-arginine (L-NMMA) reduced both cytokine-induced phagocytosis and phagocytosis induced by the combination of cytokines plus DCF. Furthermore, the NO donor sodium nitroprusside induced phagocytosis, indicating that NO production is a key regulator of microglial phagocytosis. In conclusion, DCF acts synergistically with proinflammatory cytokines to increase the production of NO in microglia, leading to phagocytic activity of the activated microglia. These findings, together with previous observations regarding astrocytes, may explain the significant increase in mortality of IAE patients treated with DCF. - Highlights: ► Influenza-associated encephalopathy (IAE) is associated with a high mortality rate. ► Hyperimmunization in the brain is believed to be responsible for

  4. Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells

    PubMed Central

    Fuma, Shinichiro; Murase, Hiromi; Kuse, Yoshiki; Tsuruma, Kazuhiro; Shimazawa, Masamitsu

    2015-01-01

    Purpose Photobiomodulation is the treatment with light in the far-red to near-infrared region of the spectrum and has been reported to have beneficial effects in various animal models of disease, including an age-related macular degeneration (AMD) mouse model. Previous reports have suggested that phagocytosis is reduced by age-related increased oxidative stress in AMD. Therefore, we investigated whether photobiomodulation improves phagocytosis caused by oxidative stress in the human retinal pigment epithelial (ARPE-19) cell line. Methods ARPE-19 cells and human primary retinal pigment epithelium (hRPE) cells were incubated and irradiated with near-infrared light (670 nm LED light, 2,500 lx, twice a day, 250 s/per time) for 4 d. Next, hydrogen peroxide (H2O2) and photoreceptor outer segments (POS) labeled using a pH-sensitive fluorescent dye were added to the cell culture, and phagocytosis was evaluated by measuring the fluorescence intensity. Furthermore, cell death was observed by double staining with Hoechst33342 and propidium iodide after photobiomodulation. CM-H2DCFDA, JC-1 dye, and CCK-8 were added to the cell culture to investigate the reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell viability, respectively. We also investigated the expression of phagocytosis-related proteins, such as focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK). Results Oxidative stress inhibited phagocytosis, and photobiomodulation increased the oxidative stress-induced hypoactivity of phagocytosis in ARPE-19 cells and hRPE cells. Furthermore, H2O2 and photobiomodulation did not affect cell death in this experimental condition. Photobiomodulation reduced ROS production but did not affect cell viability or mitochondrial membrane potential. The expression of phosphorylated MerTK increased, but phosphorylated FAK was not affected by photobiomodulation. Conclusions These findings indicate that near-infrared light photobiomodulation (670 nm) may

  5. Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase

    SciTech Connect

    Dousset, N.; Negre, A.; Salvayre, R.; Rogalle, P.; Dang, Q.Q.; Douste-Blazy, L.

    1988-06-01

    A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate.

  6. Cannabidiol enhances microglial phagocytosis via transient receptor potential (TRP) channel activation

    PubMed Central

    Hassan, Samia; Eldeeb, Khalil; Millns, Paul J; Bennett, Andrew J; Alexander, Stephen P H; Kendall, David A

    2014-01-01

    Background and Purpose Microglial cells are important mediators of the immune response in the CNS. The phytocannabinoid, cannabidiol (CBD), has been shown to have central anti-inflammatory properties, and the purpose of the present study was to investigate the effects of CBD and other phytocannabinoids on microglial phagocytosis. Experimental Approach Phagocytosis was assessed by measuring ingestion of fluorescently labelled latex beads by cultured microglial cells. Drug effects were probed using single-cell Ca2+ imaging and expression of mediator proteins by immunoblotting and immunocytochemistry. Key Results CBD (10 μM) enhanced bead phagocytosis to 175 ± 7% control. Other phytocannabinoids, synthetic and endogenous cannabinoids were without effect. The enhancement was dependent upon Ca2+ influx and was abolished in the presence of EGTA, the Ca2+ channel inhibitor SKF96365, the transient receptor potential (TRP) channel blocker ruthenium red, and the TRPV1 antagonists capsazepine and AMG9810. CBD produced a sustained increase in intracellular Ca2+ concentration in BV-2 microglia and this was abolished by ruthenium red. CBD rapidly increased the expression of TRPV2 and TRPV1 proteins and caused a translocation of TRPV2 to the cell membrane. Wortmannin blocked CBD enhancement of BV-2 cell phagocytosis, suggesting that it is mediated by PI3K signalling downstream of the Ca2+ influx. Conclusions and Implications The TRPV-dependent phagocytosis-enhancing effect of CBD suggests that pharmacological modification of TRPV channel activity could be a rational approach to treating neuroinflammatory disorders involving changes in microglial function and that CBD is a potential starting point for future development of novel therapeutics acting on the TRPV receptor family. PMID:24641282

  7. Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis.

    PubMed

    Nainu, Firzan; Tanaka, Yumiko; Shiratsuchi, Akiko; Nakanishi, Yoshinobu

    2015-12-15

    We investigated whether phagocytosis participates in the protection of insects from viral infection using the natural host-virus interaction between Drosophila melanogaster and Drosophila C virus (DCV). Drosophila S2 cells were induced to undergo apoptotic cell death upon DCV infection. However, UV-inactivated virus was unable to cause apoptosis, indicating the need for productive infection for apoptosis induction. S2 cells became susceptible to phagocytosis by hemocyte-derived l(2)mbn cells after viral infection, and the presence of phagocytes in S2 cell cultures reduced viral proliferation. Phagocytosis depended, in part, on caspase activity in S2 cells, as well as the engulfment receptors Draper and integrin βν in phagocytes. To validate the in vivo situation, adult flies were abdominally infected with DCV, followed by the analysis of fly death and viral growth. DCV infection killed flies in a dose-responding manner, and the activation of effector caspases was evident, as revealed by the cleavage of a target protein ectopically expressed in flies. Furthermore, hemocytes isolated from infected flies contained DCV-infected cells, and preinjection of latex beads to inhibit the phagocytic activity of hemocytes accelerated fly death after viral infection. Likewise, viral virulence was exaggerated in flies lacking the engulfment receptors, and was accompanied by the augmented proliferation of virus. Finally, phagocytosis of DCV-infected cells in vitro was inhibited by phosphatidylserine-containing liposome, and virus-infected flies died early when a phosphatidylserine-binding protein was ectopically expressed. Collectively, our study demonstrates that the apoptosis-dependent, phosphatidylserine-mediated phagocytosis of virus-infected cells plays an important role in innate immune responses against viral infection in Drosophila. PMID:26546607

  8. Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

    PubMed

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki; Fukuzawa, Hideya

    2015-06-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  9. Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency1[OPEN

    PubMed Central

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki

    2015-01-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  10. Effect of triacylglycerols containing medium- and long-chain fatty acids on serum triacylglycerol levels and body fat in college athletes.

    PubMed

    Takeuchi, Hiroyuki; Kasai, Michio; Taguchi, Nobuo; Tsuji, Hiroaki; Suzuki, Masashige

    2002-04-01

    Triacylglycerols containing medium- and long-chain fatty acids (TML) have medium- and long-chain fatty acids in the same molecule. The effects of dietary TML on serum lipid levels and body fat were studied in six young men belonging to a university rowing club. A double-blind crossover study was performed in which for 3 wk the subjects ingested a liquid diet containing 20 g/d of soybean oil or TML in addition to their regular diets. Throughout the study, they were asked to keep their usual lifestyle, including diet and physical activity. The body composition of the subjects was measured weekly. Blood samples were taken at 0, 2, and 3 wk of each treatment period. There was no significant difference in energy intake between the soybean oil diet period and the TML diet period. The rate of variation of serum triacylglycerol concentration was significantly lower after a consumption of the TML liquid diet for 3 wk compared with the soybean oil liquid diet. The rate of variation of body fat mass was also significantly lower after a consumption of the TML liquid diet for 3 wk compared with the soybean oil liquid diet. However, the serum cholesterol concentration did not change significantly during either dietary treatment. These results suggest that TML, compared with soybean oil, may have the potential to prevent hypertriglyceridemia and obesity caused by consumption of a high-fat diet. PMID:12171430

  11. Sesamol treatment reduces plasma cholesterol and triacylglycerol levels in mouse models of acute and chronic hyperlipidemia.

    PubMed

    Kumar, Nitesh; Mudgal, Jayesh; Parihar, Vipan K; Nayak, Pawan G; Kutty, N Gopalan; Rao, C Mallikarjuna

    2013-06-01

    The active constituents of Sesamum indicum, sesamin and sesamolin, have already been explored for hypolipidemic action. In this study we have explored the anti-dyslipidemic activity of another active component and metabolite of sesamolin (sesamol), by using acute models of hyperlipidemia viz., a fat tolerance test, a tyloxapol-induced hyperlipidemia model and a chronic model of hyperlipidemia viz., a high-fat diet-induced hyperlipidemia model in Swiss albino mice. Sesamol (100 and 200 mg/kg) significantly (P < 0.05) decreased triacylglycerol absorption in the fat tolerance test by showing a dose-dependent decrease in triacylglycerol levels. The hypolipidemic effect of sesamol at 200 mg/kg was equivalent to 10 mg/kg of orlistat. In the tyloxapol-induced hyperlipidemia model, Sesamol at 200 mg/kg reversed the elevated levels of cholesterol and triacylglycerol compared with the tyloxapol group at 12 and 24 h, which indicates its probable effect on cholesterol synthesis. Chronic hyperlipidemia in mice was produced by feeding a high-diet, a mixture of cholesterol (2 % w/w), cholic acid (1 % w/w) and coconut oil 30 % (v/w) with standard powdered standard animal chow (up to 100 g). Niacin (100 mg/kg) and sesamol (100 mg/kg) significantly (P < 0.05) reduced the elevated body weight compared with the high fat diet control group. Elevated levels of cholesterol and triacylglycerol were significantly (P < 0.05) reversed by the sesamol (50 and 100 mg/kg), implying that it might reduce the absorption and increase the excretion of cholesterol as well. PMID:23504268

  12. 76 FR 55264 - Lipase, Triacylglycerol; Exemption From the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-07

    ... February 25, 2011 (76 FR 1058) (FRL- 8863-4), EPA issued a notice pursuant to section 408 of FFDCA, 21 U.S... review under Executive Order 12866, entitled Regulatory Planning and Review (58 FR 51735, October 4, 1993... Supply, Distribution, or Use (66 FR 28355, May 22, 2001) or Executive Order 13045, entitled Protection...

  13. An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy

    PubMed Central

    Daniel, Jaiyanth; Sirakova, Tatiana; Kolattukudy, Pappachan

    2014-01-01

    Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids. PMID:25490545

  14. Inulin stimulates phagocytosis of PMA-treated THP-1 macrophages by involvement of PI3-kinases and MAP kinases.

    PubMed

    Nagahara, Yukitoshi; Nagamori, Taome; Tamegai, Hidekazu; Hitokuwada, Mami; Yoshimi, Yoji; Ikekita, Masahiko; Shinomiya, Takahisa

    2011-01-01

    Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation. PMID:22038771

  15. Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor

    PubMed Central

    2013-01-01

    Background Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. Results We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. Conclusions The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single

  16. 1α,25-dihydroxyvitamin D3 and resolvin D1 retune the balance between amyloid-β phagocytosis and inflammation in Alzheimer's disease patients.

    PubMed

    Mizwicki, Mathew T; Liu, Guanghao; Fiala, Milan; Magpantay, Larry; Sayre, James; Siani, Avi; Mahanian, Michelle; Weitzman, Rachel; Hayden, Eric Y; Rosenthal, Mark J; Nemere, Ilka; Ringman, John; Teplow, David B

    2013-01-01

    As immune defects in amyloid-β (Aβ) phagocytosis and degradation underlie Aβ deposition and inflammation in Alzheimer's disease (AD) brain, better understanding of the relation between Aβ phagocytosis and inflammation could lead to promising preventive strategies. We tested two immune modulators in peripheral blood mononuclear cells (PBMCs) of AD patients and controls: 1α,25(OH)2-vitamin D3 (1,25D3) and resolvin D1 (RvD1). Both 1,25D3 and RvD1 improved phagocytosis of FAM-Aβ by AD macrophages and inhibited fibrillar Aβ-induced apoptosis. The action of 1,25D3 depended on the nuclear vitamin D and the protein disulfide isomerase A3 receptors, whereas RvD1 required the chemokine receptor, GPR32. The activities of 1,25D3 and RvD1 commonly required intracellular calcium, MEK1/2, PKA, and PI3K signaling; however, the effect of RvD1 was more sensitive to pertussis toxin. In this case study, the AD patients: a) showed significant transcriptional up regulation of IL1RN, ITGB2, and NFκB; and b) revealed two distinct groups when compared to controls: group 1 decreased and group 2 increased transcription of TLRs, IL-1, IL1R1 and chemokines. In the PBMCs/macrophages of both groups, soluble Aβ (sAβ) increased the transcription/secretion of cytokines (e.g., IL1 and IL6) and chemokines (e.g., CCLs and CXCLs) and 1,25D3/RvD1 reversed most of the sAβ effects. However, they both further increased the expression of IL1 in the group 1, sβ-treated cells. We conclude that in vitro, 1,25D3 and RvD1 rebalance inflammation to promote Aβ phagocytosis, and suggest that low vitamin D3 and docosahexaenoic acid intake and/or poor anabolic production of 1,25D3/RvD1 in PBMCs could contribute to AD onset/pathology. PMID:23186989

  17. Mammalian Septins Are Required for Phagosome Formation

    PubMed Central

    Huang, Yi-Wei; Yan, Ming; Collins, Richard F.; DiCiccio, Jessica E.; Grinstein, Sergio

    2008-01-01

    Septins are members of a highly conserved family of filamentous proteins that are required in many organisms for the completion of cytokinesis. In addition, septins have been implicated in a number of important cellular processes and have been suggested to have roles in regulating membrane traffic. Given the proposed role of septins in cell membrane dynamics, we investigated the function of septins during FcγR-mediated phagocytosis. We show that several septins are expressed in RAW264.7 and J774 mouse macrophage cell lines and that SEPT2 and SEPT11 are colocalized with submembranous actin-rich structures during the early stages of FcγR-mediated phagocytosis. In addition, SEPT2 accumulation is seen in primary human neutrophils and in nonprofessional phagocytes. The time course of septin accumulation mirrors actin accumulation and is inhibited by latrunculin and genistein, but not other inhibitors of phagocytosis. Inhibition of septin function by transient expression of the BD3 domain of BORG3, known to cause septin aggregation, or depletion of SEPT2 or SEPT11 by RNAi, significantly inhibited FcγR-mediated phagocytosis of IgG-coated latex beads. Interestingly, this occurred without affecting the accumulation of actin or the actin-associated protein coronin-1. These observations show that, although not necessary for actin recruitment, septins are required for efficient FcγR-mediated phagocytosis. PMID:18272790

  18. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization.

    PubMed

    Fornetti, J; Flanders, K C; Henson, P M; Tan, A-C; Borges, V F; Schedin, P

    2016-02-01

    After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and

  19. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization

    PubMed Central

    Fornetti, J; Flanders, K C; Henson, P M; Tan, A-C; Borges, V F; Schedin, P

    2016-01-01

    After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and

  20. Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis, Lipid Droplet Formation, and Host Invasion in Metarhizium robertsii

    PubMed Central

    Gao, Qiang; Shang, Yanfang; Huang, Wei

    2013-01-01

    Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation. PMID:24077712

  1. Direct Analysis of Triacylglycerols from Crude Lipid Mixtures by Gold Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Son, Jeongjin; Lee, Gwangbin; Cha, Sangwon

    2014-05-01

    Triacylglycerols (TAGs), essential energy storage lipids, are easily detected by conventional MALDI MS when occurring on their own. However, their signals are easily overwhelmed by other lipids, mainly phosphatidylcholines (PCs) and, therefore, require purification. In order to profile TAGs from crude lipid mixtures without prefractionation, we investigated alternative matrixes that can suppress phospholipid ion signals and enhance cationization of TAGs. We found that an aqueous solution of citrate-capped gold nanoparticles (AuNPs) with a diameter of 12 nm is a superior matrix for the laser desorption/ionization mass spectrometry (LDI MS) of TAGs in crude lipid mixtures. The AuNP matrix effectively suppressed other lipid signals such as phospholipids and also provided 100 times lower detection limit for TAGs than 2,5-dihydroxybenzoic acid (DHB), the best conventional MALDI matrix for TAGs. The AuNP-assisted LDI MS enabled us to obtain detailed TAG profiles including minor species directly from crude beef lipid extracts without phospholipid interference. In addition, we could detect TAGs at a trace level from a total brain lipid extract.

  2. Differential effects of gram-positive and gram-negative bacterial products on morphine induced inhibition of phagocytosis

    PubMed Central

    Jana, Ninkovic; Vidhu, Anand; Raini, Dutta; Zhang, Li; Saluja, Anuj; Meng, Jingjing; Lisa, Koodie; Santanu, Banerjee; Sabita, Roy

    2016-01-01

    Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (−) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (−) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (−) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers. PMID:26891899

  3. POLY(1):POLY(C)-ENHANCED ALVEOLAR AND PERITONEAL MACROPHAGE PHAGOCYTOSIS: QUANTIFICATION BY A NEW METHOD UTILIZING FLUOROESCENT BEADS

    EPA Science Inventory

    A technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed utilizing fluorescent polystyrene beads. When incorporated into inhalation studies, the technique can be used to determine whether the toxic effect of...

  4. Phagocytosis and Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils.

    PubMed

    Kobayashi, Scott D; Porter, Adeline R; Dorward, David W; Brinkworth, Amanda J; Chen, Liang; Kreiswirth, Barry N; DeLeo, Frank R

    2016-05-15

    Carbapenem-resistantKlebsiella pneumoniaestrains classified as multilocus sequence type 258 (ST258) are among the most widespread multidrug-resistant hospital-acquired pathogens. Treatment of infections caused by these organisms is difficult, and mortality is high. The basis for the success of ST258, outside of antibiotic resistance, remains incompletely determined. Here we tested the hypothesis that ST258K. pneumoniaehas enhanced capacity to circumvent killing by human neutrophils, the primary cellular defense against bacterial infections. There was limited binding and uptake of ST258 by human neutrophils, and correspondingly, there was limited killing of bacteria. On the other hand, transmission electron microscopy revealed that any ingested organisms were degraded readily within neutrophil phagosomes, thus indicating that survival in the neutrophil assays is due to limited phagocytosis, rather than to microbicide resistance after uptake. Our findings suggest that enhancing neutrophil phagocytosis is a potential therapeutic approach for treatment of infection caused by carbapenem-resistant ST258K. pneumoniae. PMID:26768252

  5. Phagocytosis of sperm by follicle cells of the carnivorous sponge Asbestopluma occidentalis (Porifera, Demospongiae).

    PubMed

    Riesgo, Ana

    2010-06-01

    During spermatogenesis of the carnivorous sponge Asbestopluma occidentalis, follicle cells that lined the spermatocysts phagocytosed unreleased mature sperm. Such follicle cells are part of the complex envelope that limits spermatocysts of A. occidentalis, which is also comprised of a collagen layer, a thick layer of intertwined cells, and spicules. Follicle cells showed vesicles containing single phagocytosed spermatozoa within their cytoplasm. Additionally, lipids and other inclusions were observed within the cytoplasm of follicle cells. It is likely that follicle cells recapture nutrients by phagocytosing spermatozoa and use them to form lipids and other inclusions. Such sperm phagocytosis is usually performed in higher invertebrates and vertebrates by Sertoli cells that are located in the testis wall. While Sertoli cells develop a wide range of functions such as creating a blood-testis barrier, providing crucial factors to ensure correct progression of spermatogenesis, and phagocytosis of aberrant, degenerating, and unreleased sperm cells, sponge follicle cells may only display phagocytotic activity on spermatogenic cells. PMID:20409567

  6. The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis.

    PubMed

    Wijeyesakere, Sanjeeva Joseph; Bedi, Sukhmani Kaur; Huynh, David; Raghavan, Malini

    2016-05-01

    Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum. Calreticulin is detectable on the surface of apoptotic cells under some apoptosis-inducing conditions, where it promotes the phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains: a globular domain, an extended arm-like P-domain, and a C-terminal acidic region containing multiple low-affinity calcium binding sites. We show that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared with other phospholipids and that this interaction is calcium dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher-affinity calcium-dependent mode that is acidic region dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower-affinity P-domain and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that cooperates with other PS-binding factors to promote the phagocytosis of apoptotic cells. PMID:27036911

  7. A Sequential Model of Host Cell Killing and Phagocytosis by Entamoeba histolytica

    PubMed Central

    Sateriale, Adam; Huston, Christopher D.

    2011-01-01

    The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model. PMID:21331284

  8. Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis.

    PubMed

    Alsharif, Khalaf F; Thomas, Mark R; Judge, Heather M; Khan, Haroon; Prince, Lynne R; Sabroe, Ian; Ridger, Victoria C; Storey, Robert F

    2015-08-01

    In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10(-8)M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7%±4.4 vs. control 22.6%±2.4; p<0.01) by acting on the high-affinity A1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10(-8)M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6±6.6 vs. 28.0±6.6; p=0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection. PMID:25869515

  9. Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

    PubMed

    Huizinga, Ruth; van der Star, Baukje J; Kipp, Markus; Jong, Rosa; Gerritsen, Wouter; Clarner, Tim; Puentes, Fabiola; Dijkstra, Christine D; van der Valk, Paul; Amor, Sandra

    2012-03-01

    Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood-borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well-characterized antibodies to axonal antigens, that axonal damage is associated with HLA-DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA-DR(+) cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF-L), present in white and gray matter, was observed in human microglia. The number of NF-L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF-L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF-L is taken up and degraded by the cell. In vivo, NF-L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA-DR(+) cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens. PMID:22161990

  10. MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells.

    PubMed

    Healy, Luke M; Perron, Gabrielle; Won, So-Yoon; Michell-Robinson, Mackenzie A; Rezk, Ayman; Ludwin, Samuel K; Moore, Craig S; Hall, Jeffery A; Bar-Or, Amit; Antel, Jack P

    2016-04-15

    Multifocal inflammatory lesions featuring destruction of lipid-rich myelin are pathologic hallmarks of multiple sclerosis. Lesion activity is assessed by the extent and composition of myelin uptake by myeloid cells present in such lesions. In the inflamed CNS, myeloid cells are comprised of brain-resident microglia, an endogenous cell population, and monocyte-derived macrophages, which infiltrate from the systemic compartment. Using microglia isolated from the adult human brain, we demonstrate that myelin phagocytosis is dependent on the polarization state of the cells. Myelin ingestion is significantly enhanced in cells exposed to TGF-β compared with resting basal conditions and markedly reduced in classically activated polarized cells. Transcriptional analysis indicated that TGF-β-treated microglia closely resembled M0 cells. The tyrosine kinase phagocytic receptor MerTK was one of the most upregulated among a select number of differentially expressed genes in TGF-β-treated microglia. In contrast, MerTK and its known ligands, growth arrest-specific 6 and Protein S, were downregulated in classically activated cells. MerTK expression and myelin phagocytosis were higher in CNS-derived microglia than observed in monocyte-derived macrophages, both basally and under all tested polarization conditions. Specific MerTK inhibitors reduced myelin phagocytosis and the resultant anti-inflammatory biased cytokine responses for both cell types. Defining and modulating the mechanisms that regulate myelin phagocytosis has the potential to impact lesion and disease evolution in multiple sclerosis. Relevant effects would include enhancing myelin clearance, increasing anti-inflammatory molecule production by myeloid cells, and thereby permitting subsequent tissue repair. PMID:26962228