Resident Peritoneal Macrophages Research Articles

Aging affects the responsiveness of rat peritoneal macrophages to GM-CSF and IL-4.

Macrophages undergo significant functional alterations during aging. The aim of the present study was to investigate changes of rat macrophage functions and response to M1/M2 polarization signals with age. Therefore, resident and thioglycollate-elicited peritoneal macrophages from young (3-month-old) and aged (18-19-month-old) rats were tested for phagocytic capacity and ability to secrete inflammatory mediators following in vitro stimulation with LPS and GM-CSF, and IL-4, prototypic stimulators for classically (M1) and alternatively activated (M2) macrophages, respectively. Aging increased the frequency of monocyte-derived (CCR7+ CD68+) and the most mature (CD163+ CD68+) macrophages within resident and thioglycollate-elicited peritoneal macrophages, respectively. The ability to phagocyte zymosan of none of these two cell subsets was affected by either LPS and GM-CSF or IL-4. The upregulated production of IL-1β, IL-6 and IL-10 and downregulated that of TGF-β was observed in response to LPS in resident and thioglycollate-elicited macrophages from rats of both ages. GM-CSF elevated production of IL-1β and IL-6 in resident macrophages from aged rats and in thioglycollate-elicited macrophages from young rats. Unexpectedly, IL-4 augmented production of proinflammatory mediators, IL-1β and IL-6, in resident macrophages from aged rats. In both resident and thioglycollate-elicited macrophages aging decreased NO/urea ratio, whereas LPS but not GM-SCF, shifted this ratio toward NO in the macrophages from animals of both ages. Conversely, IL-4 reduced NO/urea ratio in resident and thioglycollate-elicited macrophages from young rats only. In conclusion, our study showed that aging diminished GM-CSF-triggered polarization of elicited macrophages and caused paradoxical IL-4-driven polarization of resident macrophages toward proinflammatory M1 phenotype. This age-related deregulation of macrophage inflammatory mediator secretion and phagocytosis in response to M1/M2 activators may lead to the deficient control of infectious and/or inflammatory diseases in advanced age.

Angiopoietin-like protein 2 induces proinflammatory responses in peritoneal cells

Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1β, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1β, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.

Piperine metabolically regulates peritoneal resident macrophages to potentiate their functions against bacterial infection.

Pepper, a daily-used seasoning for promoting appetite, is widely used in folk medicine for treating gastrointestinal diseases. Piperine is the major alkaloid in pepper and possesses a wide range of pharmacological activities. However, the mechanism for linking metabolic and medicinal activities of piperine remains unknown. Here we report that piperine robustly boosts mTORC1 activity by recruiting more system L1 amino acid transporter (SLC7A5/SLC3A2) to the cell membrane, thus promoting amino acid metabolism. Piperine-induced increase of mTORC1 activity in resident peritoneal macrophages (pMΦs) is correlated with enhanced production of IL-6 and TNF-α upon LPS stimulation. Such an enhancement of cytokine production could be abrogated by inhibitors of the mTOR signaling pathway, indicating mTOR's action in this process. Moreover, piperine treatment protected resident pMΦs from bacterium-induced apoptosis and disappearance, and increased their bacterial phagocytic ability. Consequently, piperine administration conferred mice resistance against bacterial infection and even sepsis. Our data highlight that piperine has the capacity to metabolically reprogram peritoneal resident macrophages to fortify their innate functions against bacterial infection.

KARAKTERISASI KULTUR MAKROFAG HASIL ISOLASI Mouse Peritoneum Makrofag (MPM)

<p><em>The </em><em>aims </em><em>of t</em><em>his study </em><em>is </em><em>to determine the MPM isolation techniques</em><em>, </em><em>results of </em><em>the </em><em>isolation and identification, so it can be used as a reference for the studies using macrophage cell cultures. The most appropriate source for culturing macrophages are the primary culture of MPM. Resident peritoneal macrophages (RPM) is a free-living phagocytic cells in the peritoneal cavity. Millions of resident macrophages can be taken from the mice. The peritoneal cavity is an easy place to harvest the resident macrophages.</em><em> I</em><em>dentification of</em><em> macrophages was conducted using flow</em><em>cy</em><em>tometr</em><em>y</em><em> </em><em>with</em><em> specific marker CD 11B. While the morphology of macrophages in culture was observed microscopically</em><em> The results </em><em>of the study </em><em>showed the average number of macrophages 1.03 x 106 cells / ml were isolated from peritoneal cavity of mice. The result of identification with flow</em><em>cy</em><em>tometr</em><em>y</em><em> with marker CD 11 B shows positive results macrophages. While the observation of morphological characteristics of macrophages cultures show the same characteristics as the result of research conducted by Kim in 1997 and Wang et al, 2010 so that it can be concluded that macrophages can be isolated from the peritoneal cavity and can be used as a source of primary cultures of macrophages with good results and can be applied to studies related to the function and activation of macrophages in vitro.</em></p> <em>Keyword; Kultur Makrofag, Isolasi Mouse Peritoneum Makrofag (Mpm)</em>

CD73 Activity is Dispensable for the Polarization of M2 Macrophages.

The ectoenzyme CD73 catalyzes the hydrolysis of AMP, and is one of the most important producers of extracellular adenosine. On regulatory T cells, CD73 is necessary for immunosuppressive functions, and on Th17 cells CD73-generated adenosine exerts anti-inflammatory effects. However, the expression and function of CD73 in pro-inflammatory M1 and in immunosuppressive M2 macrophages is largely unknown. Here we show that CD73 expression and enzyme activity were induced in in vitro polarized pro-inflammatory human M(LPS+TNF) monocytes/macrophages, while CD73 was absent from immunosuppressive M(IL-4+M-CSF)-polarized macrophages. Inhibition of CD73 activity with the inhibitor AMPCP did not affect the polarization of human monocytes. In mice, CD73 was present on resident peritoneal macrophages. In striking contrast, elicited peritoneal macrophages remained CD73 negative regardless of their polarization towards either a pro-inflammatory M(LPS) or anti-inflammatory M(IL-4c) direction. Finally, the ability of peritoneal macrophages to polarize to pro- and anti-inflammatory cells was perfectly normal in CD73-deficient mice in vivo. These data indicate that, in contrast to other major leukocyte subpopulations, CD73 activity on macrophages does not play a major role in their polarization and that in mice host CD73 on any cell type is not required in vivo for peritoneal macrophage polarization towards either a pro- or an anti-inflammatory direction.

The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K.

Specific Contributions of CSF-1 and GM-CSF to the Dynamics of the Mononuclear Phagocyte System

M-CSF (or CSF-1) and GM-CSF can regulate the development and function of the mononuclear phagocyte system (MPS). To address some of the outstanding and sometimes conflicting issues surrounding this biology, we undertook a comparative analysis of the effects of neutralizing mAbs to these CSFs on murine MPS populations in the steady-state and during acute inflammatory reactions. CSF-1 neutralization, but not of GM-CSF, in normal mice rapidly reduced the numbers of more mature Ly6C(-) monocytes in blood and bone marrow, without any effect on proliferating precursors, and also the numbers of the resident peritoneal macrophages, observations consistent with CSF-1 signaling being essential only at a relatively late state in steady-state MPS development; in contrast, GM-CSF neutralization had no effect on the numbers of these particular populations. In Ag-induced peritonitis (AIP), thioglycolate-induced peritonitis, and LPS-induced lung inflammation, CSF-1 neutralization lowered inflammatory macrophage number; in the AIP model, this reduced number was not due to suppressed proliferation. More detailed studies with the convenient AIP model indicated that CSF-1 neutralization led to a relatively uniform reduction in all inflammatory cell populations; GM-CSF neutralization, in contrast, was more selective, resulting in the preferential loss among the MPS populations of a cycling, monocyte-derived inflammatory dendritic cell population. Some mechanistic options for the specific CSF-dependent biologies enumerated are discussed.

Abstract 168: The Acceleration of Foam Cell Formation by Chlamydia Pneumoniae Requires Nlrp3 Inflammasome Activation and Il-1 Signaling

Background & Objective: Foam cell formation (FCF) due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Chlamydia pneumoniae (Cp) promotes FCF in the presence of oxLDL, but the exact molecular mechanisms are still not completely delineated. Recent data indicates that the Nlrp3 inflammasome plays an important role in the formation of atherosclerotic plaques. Here we investigated the role of the Nlrp3 inflammasome during the acceleration of FCF by Cp infection. Methods and Results: In order to determine if the NLRP3 inflammasome played a role in Cp infection induced acceleration of FCF, we treated resident peritoneal macrophages exposed to oxLDL and Cp with the IL-1R antagonist, Anakinra, to block IL-1 signaling. Treatment with Anakinra resulted in a significant reduction in FCF. Nlrp3-/-, Casp1-/-, and WT macrophages were also treated with live Cp in the presence or absence of oxLDL. We found that Nlrp3-/- and Casp1-/- macrophages had significantly less FCF compared with WT cells. Interestingly, both ABCA1 (cholesterol efflux transporter) and its transcription factor, liver X receptor (LXR-α), were increased in Nlrp3-/- and Casp1-/- macrophages compared with WT cells. Addition of rIL-1β to Nlrp3-/- macrophages led to a decrease in ABCA1 expression and greater FCF. Importantly, Il1r-/- macrophages also had greater ABCA1 expression and reduced FCF when exposed to oxLDL and Cp infection. Conclusion: These data suggest that Cp infection facilitates foam cell formation in the presence of oxLDL by producing NLRP3 dependent IL-1 cytokines, which then feed back on the macrophages and interferes with cholesterol efflux by negatively regulating ABCA1. In the absence of IL-1 signaling, the expression of ABCA1 is upregulated leading to greater cholesterol efflux and reduced FCF. Thus we have identified a novel regulatory loop controlling FCF. Understanding these interacting pathways will lead to new therapeutic strategies against atherosclerosis.

Role of plasma dyslipidemia in regulation of macrophage K + channels: distinct role of oxLDL and acLDL

Our earlier studies have shown that an increase in cellular cholesterol results in suppressing the activity of inwardly-rectifying K+ (Kir) channels in endothelial cells. Here, we extend these studies to determine the impact of dyslipidemia on Kir channels in macrophages. Our new data show that acLDL and oxLDL have opposite effects on the activity of Kir channels in Bone-Marrow Derived macrophages (BMDMs). Specifically, we show that loading the cells with cholesterol using either MbCD-cholesterol complex or acetylated LDL (acLDL) suppressed macrophage Kir-channel activity but didn't affect Kv-channels. In contrast, exposure to oxLDL activated both types of channels, an effect opposite to that of acLDL and similar to cholesterol depletion. oxLDL-induced activation of both Kir and Kv was fully reversed by supplying surplus of cholesterol. Furthermore, we show that monocytes isolated from the bone marrow of dyslipidemic ApoE-/- mice have significantly lower currents than those isolated from the wild type (WT) mice. This effect is observed only immediately after the isolation and is dissipated within 24-48 hours suggesting that this difference should be attributed to the dyslipidemic environment rather than the loss of ApoE gene per se. Interestingly, an effect of dyslipidemia on resident peritoneal macrophages isolated from ApoE-/- is highly variable with some mice having currents significantly lower than WT controls and some mice showing significantly higher currents. We propose that this variability may be due to variability in the oxidation status of the peritoneal macrophages between mice.

Targeting myeloid cells in the tumor microenvironment enhances vaccine efficacy in murine epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages, and is associated with a high relapse rate. Patients in remission are ideal candidates for immunotherapy aimed at cure or prolonging disease-free periods. However, immunosuppressive pathways in the tumor microenvironment are obstacles to durable anti-tumor immunity. In a metastatic syngeneic mouse model of EOC, immunosuppressive macrophages and myeloid-derived suppressor cells (MDSCs) accumulate in the local tumor environment. In addition, resident peritoneal macrophages from non-tumor-bearing mice were highly immunosuppressive, abrogating stimulated T cell proliferation in a cell contact-dependent manner. Immunization with microparticles containing TLR9 and NOD-2 ligands (MIS416) significantly prolonged survival in tumor-bearing mice. The strategy of MIS416 immunization followed by anti-CD11b administration further delayed tumor progression, thereby establishing the proof of principle that myeloid depletion can enhance vaccine efficacy. In patients with advanced EOC, ascites analysis showed substantial heterogeneity in the relative proportions of myeloid subsets and their immunosuppressive properties. Together, these findings point to immunosuppressive myeloid cells in the EOC microenvironment as targets to enhance vaccination. Further studies of myeloid cell accumulation and functional phenotypes in the EOC microenvironment may identify patients who are likely to benefit from vaccination combined with approaches that deplete tumor-associated myeloid cells.

Interferon regulatory factor (IRF)-1 is a master regulator of the cross talk between macrophages and L929 fibrosarcoma cells for nitric oxide dependent tumoricidal activity.

Macrophage tumoricidal activity relies, mainly, on the release of Tumor Necrosis Factor alpha (TNFα) and/or on reactive oxygen or nitrogen intermediates. In the present work, we investigated the cytotoxic activity of resident peritoneal macrophages against L929 fibrosarcoma cell line in vitro and in vivo. Resident macrophages lysed L929 cells in a mechanism independent of TNFα and cell-to-cell contact. The cytotoxic activity was largely dependent on nitric oxide (NO) release since treatment with L-NAME (NOS inhibitor) inhibited L929 cells killing. Macrophages from mice with targeted deletion of inducible NO synthase (iNOS) together with L929 cells produced less NO and displayed lower, but still significant, tumoricidal activity. Notably, NO production and tumor lysis were abolished in co-cultures with macrophages deficient in Interferon Regulatory Factor, IRF-1. Importantly, the in vitro findings were reproduced in vivo as IRF-1 deficient animals inoculated i.p with L929 cells were extremely susceptible to tumor growth and their macrophages did not produce NO, while WT mice killed L929 tumor cells and their macrophages produced high levels of NO. Our results indicate that IRF-1 is a master regulator of bi-directional interaction between macrophages and tumor cells. Overall, IRF-1 was essential for NO production by co-cultures and macrophage tumoricidal activity in vitro as well as for the control of tumor growth in vivo.

Cyclophilin D disruption attenuates lipopolysaccharide-induced inflammatory response in primary mouse macrophages.

According to recent results, various mitochondrial processes can actively regulate the immune response. In the present report, we studied whether mitochondrial permeability transition (mPT) has such a role. To this end, we compared bacterial lipopolysaccharide (LPS)-induced inflammatory response in cyclophilin D (CypD) knock-out and wild-type mouse resident peritoneal macrophages. CypD is a regulator of mPT; therefore, mPT is damaged in CypD(-/-) cells. We chose this genetic modification-based model because the mPT inhibitor cyclosporine A regulates inflammatory processes by several pathways unrelated to the mitochondria. The LPS increased mitochondrial depolarisation, cellular and mitochondrial reactive oxygen species production, nuclear factor-κB activation, and nitrite- and tumour necrosis factor α accumulation in wild-type cells, but these changes were diminished or absent in the CypD-deficient macrophages. Additionally, LPS enhanced Akt phosphorylation/activation as well as FOXO1 and FOXO3a phosphorylation/inactivation both in wild-type and CypD(-/-) cells. However, Akt and FOXO phosphorylation was significantly more pronounced in CypD-deficient compared to wild-type macrophages. These results provide the first pieces of experimental evidence for the functional regulatory role of mPT in the LPS-induced early inflammatory response of macrophages.

Differential modulation of macrophage response elicited by glycoinositolphospholipids and lipophosphoglycan from Leishmania (Viannia) shawi

In this work, some aspects of the glycobiology of Leishmania shawi were examined, as it is a causative agent of cutaneous leishmaniasis in the New World. Additionally, the interaction of L. shawi's main glycoconjugates [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] with macrophages was evaluated in vitro. L. shawi LPG was devoid of side-chains in its repeat units, whereas monosaccharide analysis showed that GIPLs were suggestive of mannose-rich (type I or hybrid). In order to evaluate the biological roles of those molecules, BALB/c resident peritoneal macrophages were incubated with these glycoconjugates for 24h, and the levels of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-12p70 and IL-10, were determined. In general, the GIPLs exhibited a greater proinflammatory role than the LPGs did. However, for the first time, the GIPLs from this species were able to trigger the production of IL-10, an anti-inflammatory cytokine. In conclusion, L. shawi glycoconjugates were able to interact with the innate immune compartment. These data reinforce the role of parasite glycoconjugates during parasite and host cell interactions.

The p38 MAPK inhibitor SB203580 differentially modulates LPS-induced interleukin 6 expression in macrophages.

The p38 mitogen-activated protein kinase (MAPK) plays a key role in lipopolysaccharide (LPS)-induced signal transduction pathways that lead to inflammatory cytokine synthesis in macrophages; however, whether the inhibition of p38 MAPK regulates LPS-induced inflammatory cytokine expression in different types of macrophages remains the subject of debate. Herein, we assessed whether the inhibition of p38 MAPK by SB203580 regulates LPS-induced expression of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in RAW264.7 and resident peritoneal macrophages. Lipopolysaccharide stimulation of RAW264.7 macrophages or mouse resident peritoneal macrophages significantly increased TNF-α and IL-6 production. The addition of SB203580 to cultures dramatically blocked LPS-induced TNF-α production in RAW264.7 and mouse resident peritoneal macrophages, and dramatically blocked LPS-induced IL-6 production in RAW264.7 macrophages, but not in mouse resident peritoneal macrophages. Additionally, high concentrations of SB203580 resulted in increased IL-6 production. However, LPS-stimulation significantly up-regulated the mRNA transcript levels of TNF-α and IL-6 in RAW264.7 and mouse resident peritoneal macrophages, whereas pretreatment with SB203580 dramatically down-regulated LPS-induced mRNA transcript levels of TNF-α and IL-6 in these cells. Our data show that SB203580 differentially modulates LPS-induced production of the inflammatory cytokine IL-6 in two different sources of macrophages, and that this course of regulation occurs at the IL-6 mRNA post-transcriptional stage.

Atf3 negatively regulates Ptgs2/Cox2 expression during acute inflammation

By generating prostaglandins, cyclooxygenase-2 (Cox-2/Ptgs2) plays a critical role in regulating inflammatory responses. While several inflammatory stimuli have been shown to increase Ptgs2 expression, less is known about how the transcription of this gene is terminated. Here we show that stimulation of macrophages with yeast zymosan, a TLR2/6 and dectin-1 agonist, causes a transient increase in the expression of Ptgs2 accompanied by a simultaneous increase in the expression of the transcriptional repressor, activating transcription factor-3 (Atf3). The expression of Ptgs2 was significantly higher in resident peritoneal macrophages isolated from Atf3−/− mice than that from Atf3+/+ mice and was associated with higher prostaglandin production upon stimulation with zymosan. In activated macrophages, Atf3 accumulated in the nucleus and chromatin-immunoprecipitation analysis showed that Atf3 is recruited to the Ptgs2 promoter region. In acute peritonitis and in cutaneous wounds, there was increased leukocyte accumulation and higher levels of prostaglandins (PGE2/PGD2) in inflammatory exudates of Atf3−/− mice compared with WT mice. Collectively, these results demonstrate that during acute inflammation Atf3 negatively regulates Ptgs2 and therefore dysregulation of this axis could potentially contribute to aberrant Ptgs2 expression in chronic inflammatory diseases. Moreover, this axis could be a new therapeutic target for suppressing Ptgs2 expression and the resultant inflammatory responses.

Exposure of Mouse Peritoneal Resident Macrophages to Pristine MWCNT Promote Nitric Oxide Synthase Production and Acute Cytokine Response

The potential biomedical applications of carbon nanotubesare greatly limited due to the growing evidences of their toxic effects on consumers. Recently, the cells of the monocyte-macrophage-system were observed to be the main structures of accumulation and toxicity of these nanomaterials.This study aimed to determine the changes in the inflammatory status and apoptosissuffered by macrophages following exposure to pristine multi-walled carbon nanotubes (MWCNT). Macrophages were isolated from the mouse peritoneum and cultivated in vitro following a protocol previously described by Zhang et al.(2008). The immunocytochemicalexpression of iNOs, IL-6 and apoptosis was measured by fluorometry usingConfocal laser scanning microscopy (CLSM). Exposure of peritoneal macrophages to MWCNT promotesiNOs production and IL-6 expression in a dose-related matter. The exposure to 10 and 25 I¼g/dl MWCNTdoes not induce apoptosis in macrophage culture, although a significant increase in numbers of cellsundergoing apoptosis is observed for the group exposed at 50I¼g/dl MWCNT. In the present research we demonstrated that exposure of mouse macrophages to pristine MWCNTpromote nitric oxide synthase production and an acute cytokine responsecharacterized by the elevation of IL-6 levels. Apoptosis is increased only at high levels of MWCNT exposure, being in respect of our results, a late expression of toxicity.

Abstract 2337: Loss of the LIM-only protein FHL2 enhances TGF-β expression and fibrogenesis

Abstract The four and a half LIM-only protein 2 (FHL2) is a multifunctional protein involved in many biological and physiopathological processes. We previously showed that enhanced expression of FHL2 in hepatocytes increases both cell proliferation and apoptosis, and promotes liver tumorigenesis associated with activation of the Wnt/β-catenin signaling (Nouët et al, J Hepatol., 2012) and that deletion of FHL2 suppresses in vitro and in vivo an array of NF-κB-mediated effects including cytokine expression, hepatocyte proliferation, and DEN-induced hepatocarcinogenesis (Dahan et al, Mol. Cell. Biol., 2013). Here we report that FHL2-deficient mice developed more severe hepatic fibrosis compared to wild type (wt) counterparts after bile duct ligation operation or administration of chemical agents thioacetamide and carbon tetrachloride, as demonstrated by Sirius red staining and mRNA expression of α-smooth muscle actin, collagen-α1 and transforming growth factor (TGF)-β1. Moreover, in the absence of any treatment, the resident hepatic macrophages (Kupffer cells) and peritoneal macrophages in FHL2-deficient mice produce elevated levels of the profibrotic cytokine TGF-β1. Because FHL2 exerts dual functions as a transcription co-activator or co-repressor, we searched for a direct effect of FHL2 on the TGF-β1 promoter. Co-transfection of murine TGF-β1 promoter-luciferase reporter constructs with FHL2 into 293T cells did not evidence significant repressive effects of FHL2 overexpression on the TGF-β1 promoter. Remarkably however, the TGF-β1 promoter activity was drastically increased in FHL2-/- mouse embryonic fibroblasts (MEFs) compared to wt MEFs. The hypothesis that FHL2 may play a part in chromatin remodeling is currently investigated by analyzing the role of FHL2 on histone modifications including acetylation, phosphorylation and methylation. Of note, we have recently shown that FHL2 activates TGF-β signaling by increasing the activity of Arkadia, a positive regulator of the pathway (Xia et al, J Biol. Chem. 2013). All together, these data suggest a complex regulation of TGF-β signaling by FHL2. Finally, in line with recently published data by Huss et al (BMC Gastroenterology, 2013) and Alnajar et al (PLoS ONE, 2013), we also observed increased renal fibrogenesis in FHL2-mutant mice following Leptospira infection, implicating FHL2 in the control of fibrogenesis in multiple tissues. Note: This abstract was not presented at the meeting. Citation Format: Jennifer Dahan, Florence Levillayer, Catherine Werts, Grégory Jouvion, Yann Nouët, Minou Adib-Conquy, Anne-Marie Cassard-Doulcier, Tian Xia, Ju Chen, Thierry Tordjmann, Marie-Annick Buendia, Yu Wei. Loss of the LIM-only protein FHL2 enhances TGF-β expression and fibrogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2014-2337

SIGNR1-mediated phagocytosis, but not SIGNR1-mediated endocytosis or cell adhesion, suppresses LPS-induced secretion of IL-6 from murine macrophages

C-type lectin receptors (CLRs) serve as phagocytosis receptors for pathogens and also function as adhesion molecules and in the recognition and endocytosis of glycosylated self-antigens. In the present study, we demonstrated that phagocytosis mediated by a mouse mannose-binding CLR, SIGNR1 significantly suppressed the LPS-induced secretion of the specific pro-inflammatory cytokines from the resident peritoneal macrophages and the mouse macrophage-like cells that express SIGNR1 (RAW-SIGNR1). LPS-induced secretion of IL-6 from peritoneal macrophages suppressed in response to uptake of oligomannose-coated liposomes (OMLs), and the suppression was partly inhibited by treatment with an anti-SIGNR1 antibody. LPS-induced secretion of IL-6 from RAW-SIGNR1 cells was also clearly inhibited by treatment of the cells with OMLs >0.4μm in diameter, but treatment with OMLs <0.4μm in diameter did not affect the IL-6 secretion. In contrast, LPS-induced TNF-α secretion from the cells was not affected on treatment of the cells with OMLs. Suppression of the IL-6 secretion was not observed following treatment with oligomannose-containing soluble polymers or when cells were bound to an oligomannose-coated solid phase. Phagocytosis of oligomannose-coated liposomes did not interfere with the transcription of IL-6 mRNA, but did affect IL-6 mRNA stability, leading to suppression of IL-6 secretion. Interestingly, treatment of the cells with Ly290042, a PI3 kinase inhibitor, partly blocked the suppression of LPS-induced secretion of IL-6 by OML. Thus, we conclude that SIGNR1-mediated phagocytosis but not SIGNR1-mediated endocytosis and cell adhesion, suppresses the TLR4-mediated production of specific proinflammatory cytokines via PI3 kinase signaling.

95: Sex-differences in classical monocyte recruitment during inflammation

Background Certain human cardiovascular diseases are subject to gender bias with more prevalence in men. Sexual dimorphisms in murine leukocyte responses during inflammation have previously been described, with males recruiting more neutrophils [1] . This study aimed to determine sex-differences in storage, trafficking and responses of classical monocytes (CMs), in an acute inflammatory model. Materials and methods Peritonitis was induced by intraperitoneal administration of zymosan (1 mg) to CX3 CR 1 + / gfp mice. Leukocytes were isolated from blood, peritoneal cavity, bone marrow (BM) and spleen. CMs were identified as CX3CR1+ Gr1+ by flow cytometry. Results Equal basal circulating, peritoneal and BM CM numbers were observed between the sexes, however males exhibited a greater splenic storage pool. Expression of key chemokine receptors was similar on splenic CMs from both sexes, as was the basal splenic chemokine environment. In response to zymosan, male mice demonstrated 2-fold greater classical monocytosis at 3 h post-administration than females, which translated into greater recruitment to the peritoneum. Reduction in BM cells at 3 h indicated mobilization; this was equal in both sexes. Equivalent spleen CM numbers at 3 h suggest greater outward flux in males than females. Female resident peritoneal macrophages were more numerous however expression of zymosan-sensing TLR2–TLR6 was equal to males and no sex-differences were seen in the basal or inflamed peritoneal cytokine environments. Conclusions These data indicate that male mice have greater splenic stores of CMs and furthermore recruit more CMs to the tissue during peritonitis. Sex-differences in classical monocyte trafficking during inflammation may translate to monocyte/macrophage-dependant pathologies more prevalent in males, like atherosclerosis, and may provide a more personalised method of targeting such diseases.

Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival.

The transcription factor Gata6 regulates proliferation and differentiation of epithelial and endocrine cells and cancers. Among hematopoietic cells, Gata6 is expressed selectively in resident peritoneal macrophages. We thus examined whether the loss of Gata6 in the macrophage compartment affected peritoneal macrophages, using Lyz2-Cre x Gata6(flox/flox) mice to tackle this issue. In Lyz2-Cre x Gata6(flox/flox) mice, the resident peritoneal macrophage compartment, but not macrophages in other organs, was contracted, with only a third the normal number of macrophages remaining. Heightened rates of death explained the marked decrease in peritoneal macrophage observed. The metabolism of the remaining macrophages was skewed to favor oxidative phosphorylation and alternative activation markers were spontaneously and selectively induced in Gata6-deficient macrophages. Gene expression profiling revealed perturbed metabolic regulators, including aspartoacylase (Aspa), which facilitates generation of acetyl CoA. Mutant mice lacking functional Aspa phenocopied the higher propensity to death and led to a contraction of resident peritoneal macrophages. Thus, Gata6 regulates differentiation, metabolism, and survival of resident peritoneal macrophages.