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Metal transporter Slc39a10 regulates susceptibility to inflammatory stimuli by controlling macrophage survival.
ABSTRACT: Zn plays a key role in controlling macrophage function during an inflammatory event. Cellular Zn homeostasis is regulated by two families of metal transporters, the SLC39A family of importers and the SLC30A family of exporters; however, the precise role of these transporters in maintaining macrophage function is poorly understood. Using macrophage-specific Slc39a10-knockout (Slc39a10fl/fl;LysM-Cre+ ) mice, we found that Slc39a10 plays an essential role in macrophage survival by mediating Zn homeostasis in response to LPS stimulation. Compared with Slc39a10fl/fl mice, Slc39a10fl/fl;LysM-Cre+ mice had significantly lower mortality following LPS stimulation as well as reduced liver damage and lower levels of circulating inflammatory cytokines. Moreover, reduced intracellular Zn concentration in Slc39a10fl/fl;LysM-Cre+ macrophages led to the stabilization of p53, which increased apoptosis upon LPS stimulation. Concomitant knockout of p53 largely rescued the phenotype of Slc39a10fl/fl;LysM-Cre+ mice. Finally, the phenotype in Slc39a10fl/fl;LysM-Cre+ mice was mimicked in wild-type mice using the Zn chelator TPEN and was reversed with Zn supplementation. Taken together, these results suggest that Slc39a10 plays a role in promoting the survival of macrophages through a Zn/p53-dependent axis in response to inflammatory stimuli.
Project description:Macrophages regulate innate immunity to maintain intestinal homeostasis and play pathological roles in intestinal inflammation. Activation of the epidermal growth factor receptor (EGFR) promotes cellular proliferation, differentiation, survival, and wound closure in several cell types. However, the impact of EGFR in macrophages remains unclear. This study was to investigate whether EGFR activation in macrophages regulates cytokine production and intestinal inflammation. We found that EGFR was activated in colonic macrophages in mice with dextran sulfate sodium (DSS)-induced colitis and in patients with ulcerative colitis. DSS-induced acute colitis was ameliorated, and recovery from colitis was promoted in Egfr(fl/fl)LysM-Cre mice with myeloid cell-specific deletion of EGFR, compared with LysM-Cre mice. DSS treatment increased IL-10 and TNF levels during the acute phase of colitis, and increased IL-10 but reduced TNF levels during the recovery phase in Egfr(fl/fl)LysM-Cre mice. An anti-IL-10 neutralizing Ab abolished these effects of macrophage-specific EGFR deletion on DSS-induced colitis in Egfr(fl/fl)LysM-Cre mice. LPS stimulated EGFR activation and inhibition of EGFR kinase activity enhanced LPS-stimulated NF-?B activation in RAW 264.7 macrophages. Furthermore, induction of IL-10 production by EGFR kinase-blocked RAW 264.7 cells, in response to LPS plus IFN-?, correlated with decreased TNF production. Thus, although selective deletion of EGFR in macrophages leads to increases in both pro- and anti-inflammatory cytokines in response to inflammatory stimuli, the increase in the IL-10 level plays a role in suppressing proinflammatory cytokine production, resulting in protection of mice from intestinal inflammation. These results reveal an integrated response of macrophages regulated by EGFR in intestinal inflammatory disorders.
Project description:Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow-derived macrophages from BVR fl/fl and LysM-Cre:BVR fl / fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVR fl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVR fl/fl mice in a model of endotoxemia. In addition, knockdown of BVR-A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a-C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization-associated chemokines, the levels of which depend in part on C5aR1 signaling.
Project description:Neuron-microglia interactions have a crucial role in maintaining the neuroimmune system. The balance of neuroimmune system has emerged as an important process in the pathophysiology of depression. However, how neuron-microglia interactions contribute to major depressive disorders has been poorly understood. Herein, we demonstrated that microglia-derived synaptic changes induced antidepressive-like behavior by using microglia-specific signal transducer and activator of transcription 3 (STAT3) knockout (KO) (STAT3fl/fl;LysM-Cre+/-) mice. We found that microglia-specific STAT3 KO mice showed antidepressive-like behavior in the forced swim, tail suspension, sucrose preference, and open-field tests. Surprisingly, the secretion of macrophage colony-stimulating factor (M-CSF) was increased from neuronal cells in the brains of STAT3fl/fl;LysM-Cre+/- mice. Moreover, the phosphorylation of antidepressant-targeting mediators and brain-derived neurotrophic factor expression were increased in the brains of STAT3fl/fl;LysM-Cre+/- mice as well as in neuronal cells in response to M-CSF stimulation. Importantly, the miniature excitatory postsynaptic current frequency in the medial prefrontal cortex was increased in STAT3fl/fl;LysM-Cre+/- mice and in the M-CSF treatment group. Collectively, microglial STAT3 regulates depression-related behaviors via neuronal M-CSF-mediated synaptic activity, suggesting that inhibition of microglial STAT3 might be a new therapeutic strategy for depression.
Project description:Macrophages form an important component of the innate immune system and serve as first responders against invading pathogens. While pathways critical for initiation of inflammatory responses between macrophages and other LysM+ myeloid cells are largely similar, it remains unknown whether a specific pathway has differential effects on inflammatory responses mediated between these cells. Recent studies demonstrated that depletion of SAG (Sensitive to Apoptosis Gene), an E3 ubiquitin ligase, blocked inflammatory responses generated by macrophages and dendritic cells in response to LPS in cell culture settings. However, the in vivo role of Sag on modulation of macrophages and neutrophil is not known. Here we generated LysM-Cre/Sag fl/fl mice with selective Sag deletion in myeloid lineage, and found that in contrast to in vitro observations, LysM-Cre/Sag fl/fl mice showed increased serum levels of proinflammatory cytokines and enhanced mortality in response to LPS. Interestingly, while Sag -/- macrophages released less proinflammatory cytokines, Sag -/- neutrophils released more. Mechanistically, expression of a list of genes response to LPS was significantly altered in bone marrow cells from LysM-Cre +/Sag fl/fl mice after LPS challenge. Specifically, induction by LPS of myeloperoxidase (Mpo), a key neutrophil enzyme, and Elane, neutrophil expressed elastase, was significantly decreased upon Sag depletion. Collectively, our study revealed that Sag plays a differential role in the activation of macrophages and neutrophils.
Project description:Although uncontrolled inflammatory response plays a central role in the pathogenesis of acute lung injury (ALI), the precise molecular mechanisms underlying the development of this disorder remain poorly understood. SOCS3 is an important negative regulator of IL-6-type cytokine signaling. SOCS3 is induced in lung during LPS-induced lung injury, suggesting that generation of SOCS3 may represent a regulatory product during ALI. In the current study, we created mice lacking SOCS3 expression in macrophages and neutrophils (LysM-cre SOCS3(fl/fl)). We evaluated the lung inflammatory response to LPS in both LysM-cre SOCS3(fl/fl) mice and the wild-type (WT) mice (SOCS3(fl/fl)). LysM-cre SOCS3(fl/fl) mice displayed significant increase of the lung permeability index (lung vascular leak of albumin), neutrophils, lung neutrophil accumulation (myeloperoxidase activity), and proinflammatory cytokines/chemokines in bronchial alveolar lavage fluids compared to WT mice. These phenotypes were consistent with morphological evaluation of lung, which showed enhanced inflammatory cell influx and intra-alveolar hemorrhage. We further identify the transcription factor, CCAAT/enhancer-binding protein (C/EBP) ? as a critical downstream target of SOCS3 in LPS-induced ALI. These results indicate that SOCS3 has a protective role in LPS-induced ALI by suppressing C/EBP? activity in the lung. Elucidating the function of SOCS3 would represent prospective targets for a new generation of drugs needed to treat ALI.
Project description:Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNF? using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNF? although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.
Project description:Hereditary hemochromatosis and iron imbalance are associated with susceptibility to bacterial infection; however, the underlying mechanisms are poorly understood. Here, we performed in vivo bacterial infection screening using several mouse models of hemochromatosis, including Hfe (Hfe-/- ), hemojuvelin (Hjv-/- ), and macrophage-specific ferroportin-1 (Fpn1fl/fl ;LysM-Cre+ ) knockout mice. We found that Hjv-/- mice, but not Hfe-/- or Fpn1fl/fl ;LysM-Cre+ mice, are highly susceptible to peritoneal infection by both Gram-negative and Gram-positive bacteria. Interestingly, phagocytic cells in the peritoneum of Hjv-/- mice have reduced bacterial clearance, IFN-? secretion, and nitric oxide production; in contrast, both cell migration and phagocytosis are normal. Expressing Hjv in RAW264.7 cells increased the level of phosphorylated Stat1 and nitric oxide production. Moreover, macrophage-specific Hjv knockout mice are susceptible to bacterial infection. Finally, we found that Hjv facilitates the secretion of IFN-? via the IL-12/Jak2/Stat4 signaling pathway. Together, these findings reveal a novel protective role of Hjv in the early stages of antimicrobial defense.
Project description:Macrophages were derived from the bone-marrow of 3 x fl/+ Dicer LysCre +/- (wild-type) and 3 x fl/fl Dicer LysCre +/- mice and stimulated with IL-4 (50ng/mL) for 72h. Total RNA was isolated and analyzed by gene array. In this experiment, we derived Dicer deficient bone-marrow macrophages using Dicer fl/+ LysM-Cre by Dicer fl/+ crossed mice to obtain Dicer fl/fl LysM-cre progeny (and Dicer deficient macrophages). Next, we studied the effects of IL-4 stimulation in macrophage with a deficiency in Dicer/microRNAs.
Project description:Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas, altered inflammatory responses, and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. Cre pos LPL fl/fl ) and S100A8 Cre (S100A8.Cre pos LPL fl/fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast, infected CD11c.Cre pos LPL fl/fl mice did not display elevated susceptibility to infection, implicating the myeloid L-plastin-sufficient cells (e.g., macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically, the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa, augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively, our data highlight the importance of neutrophils in controlling ocular paucibacteriality, reveal molecular and cellular events involved in the process, and suggest a link between commensal exposure and resistance to infection.
Project description:Macrophage secretion of proinflammatory cytokines contributes to the pathogenesis of obesity-related insulin resistance. An important regulator of inflammation is the suppressor of cytokine signaling-1 (SOCS1), which inhibits the JAK-STAT and toll-like receptor-4 (TLR4) pathways. Despite the reported role of SOCS1 in inhibiting insulin signaling, it is surprising that a SOCS1 polymorphism that increases SOCS1 promoter activity is associated with enhanced insulin sensitivity despite obesity. In the current study, we investigated the physiological role of myeloid and lymphoid cell SOCS1 in regulating inflammation and insulin sensitivity.We used mice generated by crossing SOCS1 floxed mice with mice expressing Cre recombinase under the control of the LysM-Cre promoter (SOCS1 LysM-Cre). These mice have deletion of SOCS1 in macrophages and lymphocytes. We assessed macrophage inflammation using flow cytometry and serum cytokine levels using Bioplex assays. We then measured insulin sensitivity using glucose tolerance tests and the euglycemic-hyperinsulinemic clamp. Using bone marrow-derived macrophages, we tested the effects of SOCS1 deletion in regulating responses to the TLR4 ligands: lipopolysaccharide (LPS) and palmitic acid.SOCS1 LysM-Cre mice had increased macrophage expression of CD11c, enhanced sensitivity to LPS, and palmitic acid and increased serum concentrations of tumor necrosis factor-?, interleukin-6, and monocyte chemoattractant protein. Increased inflammation was associated with impaired glucose tolerance and hyperinsulinemia as a result of reduced hepatic but not skeletal muscle insulin sensitivity.The expression of SOCS1 in hematopoietic cells protects mice against systemic inflammation and hepatic insulin resistance potentially by inhibiting LPS and palmitate-induced TLR4 signaling in macrophages.