Role of Myeloid Tet Methylcytosine Dioxygenase 2 in Pulmonary and Peritoneal Inflammation Induced by Lipopolysaccharide and Peritonitis Induced by Escherichia coli.
ABSTRACT: Tet methylcytosine dioxygenase 2 (Tet2) mediates demethylation of DNA. We here sought to determine the expression and function of Tet2 in macrophages upon exposure to lipopolysaccharide (LPS), and in the host response to LPS induced lung and peritoneal inflammation, and during Escherichia (E.) coli induced peritonitis. LPS induced Tet2 expression in mouse macrophages and human monocytes in vitro, as well as in human alveolar macrophages after bronchial instillation in vivo. Bone marrow-derived macrophages from myeloid Tet2 deficient (Tet2fl/flLysMCre) mice displayed enhanced production of IL-1β, IL-6 and CXCL1 upon stimulation with several Toll-like receptor agonists; similar results were obtained with LPS stimulated alveolar and peritoneal macrophages. Histone deacetylation was involved in the effect of Tet2 on IL-6 production, whilst methylation at the Il6 promoter was not altered by Tet2 deficiency. Tet2fl/flLysMCre mice showed higher IL-6 and TNF levels in bronchoalveolar and peritoneal lavage fluid after intranasal and intraperitoneal LPS administration, respectively, whilst other inflammatory responses were unaltered. E. coli induced stronger production of IL-1β and IL-6 by Tet2 deficient peritoneal macrophages but not in peritoneal lavage fluid of Tet2fl/flLysMCre mice after in vivo intraperitoneal infection. Tet2fl/flLysMCre mice displayed enhanced bacterial growth during E. coli peritonitis, which was associated with a reduced capacity of Tet2fl/flLysMCre peritoneal macrophages to inhibit the growth of E. coli in vitro. Collectively, these data suggest that Tet2 is involved in the regulation of macrophage functions triggered by LPS and during E. coli infection.
Project description:EGF receptor (EGFR) in tumor cells serves as a tumor promoter. However, information about EGFR activation in macrophages in regulating M2 polarization and tumor development is limited. This study aimed to investigate the effects of EGFR activation in macrophages on M2 polarization and development of gastrointestinal tumors. IL-4, a cytokine to elicit M2 polarization, stimulated release of an EGFR ligand, HB-EGF, and transactivation and down-regulation of EGFR in Raw 264.7 cells and peritoneal macrophages from WT mice. Knockdown of HB-EGF in macrophages inhibited EGFR transactivation by IL-4. IL-4-stimulated STAT6 activation, Arg1 and YM1 gene expression, and HB-EGF production were further enhanced by inhibition of EGFR activity in Raw 264.7 cells using an EGFR kinase inhibitor and in peritoneal macrophages from Egfr(wa5) mice with kinase inactive EGFR and by knockdown of EGFR in peritoneal macrophages from Egfr(fl/fl) LysM-Cre mice with myeloid cell-specific EGFR deletion. Chitin induced a higher level of M2 polarization in peritoneal macrophages in Egfr(fl/fl) LysM-Cre mice than that in Egfr(fl/fl) mice. Accordingly, IL-4-conditioned medium stimulated growth and epithelial-to-mesenchymal transition in gastric epithelial and colonic tumor cells, which were suppressed by that from Raw 264.7 cells with HB-EGF knockdown but promoted by that from Egfr(wa5) and Egfr(fl/fl) LysM-Cre peritoneal macrophages. Clinical assessment revealed that the number of macrophages with EGFR expression became less, indicating decreased inhibitory effects on M2 polarization, in late stage of human gastric cancers. Thus, IL-4-stimulated HB-EGF-dependent transactivation of EGFR in macrophages may mediate inhibitory feedback for M2 polarization and HB-EGF production, thereby inhibiting gastrointestinal tumor growth.
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: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:Akirin2 is an evolutionally conserved nuclear protein involved in the regulation of a set of inflammatory gene expression in various cell types. We used microarrays to examine the effect of Akirin2 deficiency in LPS-inducible gene expression in macrophages Peritoneal macrophages from wild-type and LysM-Cre+;Akirin2fl/fl mice were stimulated with LPS for 0, 2 and 4 hours, followed by RNA extraction and microarray analysis.
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:<h4>Background</h4>Pain is a predominant symptom in rheumatoid arthritis (RA) patients that results from joint inflammation and is augmented by central sensitization. Regulator of G-protein signaling 12 (RGS12) is the largest protein in the RGS protein family and plays a key role in the development of inflammation. This study investigated the regulation of RGS12 in inflammatory pain and explored the underlying mechanisms and potential RA pain targets.<h4>Methods</h4>Macrophage-specific RGS12-deficient (LysM-Cre<sup>+</sup>;RGS12<sup>fl/fl</sup>) mice were generated by mating RGS12<sup>fl/fl</sup> mice with LysM-Cre<sup>+</sup> transgenic mice. Collagen antibody-induced arthritis (CAIA) models were induced in LysM-Cre<sup>+</sup>;RGS12<sup>fl/fl</sup> mice by the administration of a cocktail of five monoclonal antibodies and LPS. Mouse nociception was examined using the von Frey and heat plate tests. Primary macrophages and RAW264.7 cells were used to analyze the regulatory function and mechanism of RGS12 <i>in vitro</i>. The expression and function of RGS12 and COX2 (cyclooxygenase 2) were determined by real-time PCR, ELISA, and luciferase assays.<h4>Results</h4>Ablation of RGS12 in macrophages decreased pain-related phenotypes, such as paw swelling, the clinical score, and the inflammatory score, in the CAIA model. LysM-Cre<sup>+</sup>;RGS12<sup>fl/fl</sup> mice displayed increased resistance to thermal and mechanical stimulation from day 3 to day 9 during CAIA, indicating the inhibition of inflammatory pain. Overexpression of COX2 and PGE2 in macrophages enhanced RGS12 expression, and PGE2 regulated RGS12 expression through the G-protein-coupled receptors EP2 and EP4. Furthermore, RGS12 or the RGS12 PTB domain strengthened the transcriptional regulation of COX2 by NF-κB, whereas inhibiting NF-κB suppressed RGS12-mediated regulation of COX2 in macrophages.<h4>Conclusions</h4>Our results demonstrate that the deletion of RGS12 in macrophages attenuates inflammatory pain, which is likely due to impaired regulation of the COX2/PGE2 signaling pathway.
Project description: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 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:Conditional and inducible Cre-loxP systems are used to target gene deletion to specific cell lineages and tissues through promoter-restricted expression of the bacterial DNA recombinase, Cre. Although Cre-loxP systems are widely used to target gene deletion in lung macrophages, limited data are published on the specificity and efficiency of "macrophage targeting" Cre lines. Using R26-stop<sup>fl/fl</sup>-TdTomato and tetOn-GFP reporter lines, we assessed the specificity and efficiency of four commercially available Cre driver lines that are often considered "macrophage specific." We evaluated two conditional (Csf1r-Cre and LysM-Cre) and two inducible [CX<sub>3</sub>CR1-estrogen receptor-Cre (ERCre) and CD68-rtTA] lines. We assessed Cre activation in six resident lung myeloid populations, as well as activation in lung leukocytes, lung epithelial and endothelial cells, peripheral blood leukocytes, and tissue macrophages of the spleen, bone marrow, and peritoneal cavity. Although Csf1r-Cre and LysM-Cre target resident alveolar macrophages (ResAM) and interstitial macrophages (IM) with high efficiency, neither line is specific for macrophages. Csf1r-Cre targets all leukocyte populations, while LysM-Cre targets dendritic cell, neutrophils, monocytes, and a quarter of lung epithelial cells. CX<sub>3</sub>CR1-ERCre and CD68-rtTA both target IM, but do not target ResAM. Further, although neither line is specific for macrophages, a pulse-wait administration of tamoxifen or doxycycline can be used to significantly improve IM specificity in these inducible lines. In summary, while Cre-loxP remains a powerful tool to study macrophage function, numerous pitfalls exist. Herein, we document strengths and weaknesses of Csf1r-Cre, LysM-Cre, CX<sub>3</sub>CR1-ERCre, and CD68-rtTA systems for targeting specific macrophage populations in the lungs and provide data that will aid investigators in selecting the proper strain.