Peritoneal Mφs Research Articles

Cholinergic macrophages promote the resolution of peritoneal inflammation

The non-neural cholinergic system plays a critical role in regulating immune equilibrium and tissue homeostasis. While the expression of choline acetyltransferase (ChAT), the enzyme catalyzing acetylcholine biosynthesis, has been well documented in lymphocytes, its role in the myeloid compartment is less understood. Here, we identify a significant population of macrophages (Mϕs) expressing ChAT and synthesizing acetylcholine in the resolution phase of acute peritonitis. Using Chat-GFP reporter mice, we observed marked upregulation of ChAT in monocyte-derived small peritoneal Mϕs (SmPMs) in response to Toll-like receptor agonists and bacterial infections. These SmPMs, phenotypically and transcriptionally distinct from tissue-resident large peritoneal macrophages, up-regulated ChAT expression through a MyD88-dependent pathway involving MAPK signaling. Notably, this process was attenuated by the TRIF-dependent TLR signaling pathway, and our tests with a range of neurotransmitters and cytokines failed to induce a similar response. Functionally, Chat deficiency in Mϕs led to significantly decreased peritoneal acetylcholine levels, reduced efferocytosis of apoptotic neutrophils, and a delayed resolution of peritonitis, which were reversible with exogenous ACh supplementation. Intriguingly, despite B lymphocytes being a notable ChAT-expressing population within the peritoneal cavity, Chat deletion in B cells did not significantly alter the resolution process. Collectively, these findings underscore the crucial role of Mϕ-derived acetylcholine in the resolution of inflammation and highlight the importance of the non-neuronal cholinergic system in immune regulation.

C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages.

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 -/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 -/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 -/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 -/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 -/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 -/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 -/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 -/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our invitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Pharmacological Mechanism of Pingxiao Formula against Colorectal Cancer.

Colorectal cancer (CRC) is the most common cancer worldwide and develops due to a broad range of causative factors. Pingxiao (PX) formula and Xihuang (XH) formula are two commonly used drugs to treat CRC, especially as an alternative therapy for those patients who could not suffer surgery, chemotherapy, or immunotherapy, namely, elder or advanced CRC patients. However, the pertinent pharmacological mechanisms are still elusive. The investigation was designed to explain the pharmacological mechanisms of the PX formula. A murine model of CRC was established by injecting CT26.WT cells into the caecum of 4-week-old male Balb/c mice, following PX or XH treatment for 30 days. Network pharmacology analysis combined with weighted gene coexpression network analysis (WGCNA) predicted the pharmacological mechanisms and therapeutic value. High-throughput 16S rRNA sequencing determined the alterations in the gut microbiota communities. Western blotting, immunofluorescence, and flow cytometry examined the influence of PX on the tumor microenvironment (TME). Injection of CT26.WT-induced CRC in Balb/c mice was markedly attenuated by PX treatment. Compared with XH administration, PX exhibited a stronger antitumor effect, such as smaller tumor volume, lower interleukin 17 (IL-17), IL-6 and tumor necrosis factor-alpha (TNFα) serum levels, and higher interferon-gamma (IFN-γ) concentration. Network pharmacology analysis demonstrated that both PX and XH targets were enriched in cancers and inflammatory responses. RNA sequencing confirmed that PX treatment induced cancer cell apoptosis and inhibited inflammatory reactions within the tumor. Moreover, the PX formula considerably restored homeostasis of the gut microbiota, which was not observed in the XH group. PX targets, those associated with the survival probability of CRC patients, correlated with macrophage (Mφ) infiltration, which presented an independent risk factor for the CRC outcome. PX treatment promoted the transition of alternatively activated Mφs (M2 Mφs) to classically activated Mφs (M1 Mφs). Moreover, the peritoneal Mφs from the PX group inhibited the migration of CW26.WT cells, as evidenced by the wound healing experiment and transwell assay, which was consistent with the decreased expression of the vascular endothelial growth factor (VEGF). Furthermore, the coculturing system confirmed that PX-treated Mφs suppressed colorectal tumor-derived organoid proliferation. PX formula exhibits a potential antitumor effect against CRC by suppressing the colonization of pathological microorganisms, reshaping Mφ effector functions and hence inhibiting cancer cell proliferation.

NLRP3 activated macrophages promote endometrial stromal cells migration in endometriosis

Endometriosis (EMs) is a common gynecological disease whose pathogenesis remains unclear. Immunological factors have been a key hotspot in recent years. Peritoneal fluid samples from women with EMs show defectively activated macrophages (MΦs) and strong NOD-like receptor family pyrin domain containing 3 (NLRP3) expression. Activated MΦs secrete interleukin 1β, which stimulates migration of endometrial stromal cells (ESCs) and promotes accumulation of extracellular matrix. Levels of interleukin 1β in peritoneal fluid were significantly higher in patients with stage III-IV EMs compared with stage I-II EMs. We also found that the size and weight of endometrial lesions in NLRP3-/- mice were significantly lower than those of wild-type mice, and this phenomenon was reversed by intraperitoneally injecting peritoneal MΦs derived from wild-type mice. Moreover, we observed that the NLRP3 inflammasome was activated in MΦs by crosstalk between MΦs and ESCs. Targeted inhibition of NLRP3 significantly reduced lesion development in vivo and suppressed the migration ability of ESCs in vitro. Collectively, these findings suggest that the occurrence of EMs may be associated with the interaction between MΦs and ESCs.

Quantitative In-Depth Transcriptome Analysis Implicates Peritoneal Macrophages as Important Players in the Complement and Coagulation Systems.

To obtain a more detailed picture of macrophage (MΦ) biology, in the current study, we analyzed the transcriptome of mouse peritoneal MΦs by RNA-seq and PCR-based transcriptomics. The results show that peritoneal MΦs, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-β1 and TGF-β2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that MΦs are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the MΦs, altogether indicating that MΦs are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal MΦs but that there were also many major differences. Similar to the mouse peritoneal MΦs, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal MΦs, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal MΦs showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major MΦ subpopulation in the mouse peritoneum and the large peritoneal MΦs and places the transcriptome profile of the peritoneal MΦs in a broader context, including a comparison of the peritoneal MΦ transcriptome with that of human peripheral blood monocytes and the liver.

Towards understanding the cell surface phenotype, metabolic properties and immune functions of resident macrophages of the peritoneal cavity and splenic red pulp using high resolution quantitative proteomics

Background:Resident macrophages (Mϕs) are distributed throughout the body and are important for maintaining tissue homeostasis and for defence against infections. Tissue Mϕs are highly adapted to their microenvironment and thought to mediate tissue-specific functions involving metabolism and immune defence that are not fully elucidated. Methods:We have used high resolution quantitative proteomics to gain insights into the functions of two types of resident tissue Mϕs: peritoneal cavity Mϕs and splenic red pulp Mϕs. The cellular expression levels of many proteins were validated by flow cytometry and were consistently in agreement with the proteomics data.Results:Peritoneal and splenic red pulp macrophages displayed major differences in cell surface phenotype reflecting their adaptation to different tissue microenvironments and tissue-specific functions. Peritoneal Mϕs were shown to be enriched in a number of key enzymes and metabolic pathways normally associated with the liver, such as metabolism of fructose, detoxification, nitrogen homeostasis and the urea cycle. Supporting these observations, we show that peritoneal Mϕs are able to utilise glutamine and glutamate which are rich in peritoneum for urea generation. In comparison, splenic red pulp Mϕs were enriched in proteins important for adaptive immunity such as antigen presenting MHC molecules, in addition to proteins required for erythrocyte homeostasis and iron turnover. We also show that these tissue Mϕs may utilise carbon and nitrogen substrates for different metabolic fates to support distinct tissue-specific roles.Conclusions:This study provides new insights into the functions of tissue Mϕs in immunity and homeostasis. The comprehensive proteomics data sets are a valuable resource for biologists and immunologists.

Frontline Science: CD40 signaling restricts RNA virus replication in Mϕs, leading to rapid innate immune control of acute virus infection.

Many acute viral infections target tissue Mϕs, yet the mechanisms of Mϕ-mediated control of viruses are poorly understood. Here, we report that CD40 expressed by peritoneal Mϕs restricts early infection of a broad range of RNA viruses. Loss of CD40 expression enhanced virus replication as early as 12-24h of infection and, conversely, stimulation of CD40 signaling with an agonistic Ab blocked infection. With peritoneal cell populations infected with the filovirus, wild-type (WT) Ebola virus (EBOV), or a BSL2 model virus, recombinant vesicular stomatitis virus encoding Ebola virus glycoprotein (rVSV/EBOV GP), we examined the mechanism conferring protection. Here, we demonstrate that restricted virus replication in Mϕs required CD154/CD40 interactions that stimulated IL-12 production through TRAF6-dependent signaling. In turn, IL-12 production resulted in IFN-γ production, which induced proinflammatory polarization of Mϕs, protecting the cells from infection. These CD40-dependent events protected mice against virus challenge. CD40-/- mice were exquisitely sensitive to intraperitoneal challenge with a dose of rVSV/EBOV GP that was sublethal to CD40+/+ mice, exhibiting viremia within 12h of infection and rapidly succumbing to infection. This study identifies a previously unappreciated role for Mϕ-intrinsic CD40 signaling in controlling acute virus infection.

Inflammatory macrophages switch to CCL17-expressing phenotype and promote peritoneal fibrosis.

Peritoneal fibrosis remains a problem in kidney failure patients treated with peritoneal dialysis. Severe peritoneal fibrosis with encapsulation or encapsulating peritoneal sclerosis is devastating and life-threatening. Although submesothelial fibroblasts as the major precursor of scar-producing myofibroblasts in animal models and M2 macrophage (Mϕ)-derived chemokines in peritoneal effluents of patients before diagnosis of encapsulating peritoneal sclerosis have been identified, attenuation of peritoneal fibrosis is an unmet medical need partly because the mechanism for cross talk between Mϕs and fibroblasts remains unclear. We use a sodium hypochlorite-induced mouse model akin to clinical encapsulated peritoneal sclerosis to study how the peritoneal Mϕs activate fibroblasts and fibrosis. Sodium hypochlorite induces the disappearance of CD11bhigh F4/80high resident Mϕs but accumulation of CD11bint F4/80int inflammatory Mϕs (InfMϕs) through recruiting blood monocytes and activating local cell proliferation. InfMϕs switch to express chemokine (C-C motif) ligand 17 (CCL17), CCL22, and arginase-1 from day 2 after hypochlorite injury. More than 75% of InfMϕs undergo genetic recombination by Csf1r-driven Cre recombinase, providing the possibility to reduce myofibroblasts and fibrosis by diphtheria toxin-induced Mϕ ablation from day 2 after injury. Furthermore, administration of antibody against CCL17 can reduce Mϕs, myofibroblasts, fibrosis, and improve peritoneal function after injury. Mechanistically, CCL17 stimulates migration and collagen production of submesothelial fibroblasts in culture. By breeding mice that are induced to express red fluorescent protein in Mϕs and green fluorescence protein (GFP) in Col1a1-expressing cells, we confirmed that Mϕs do not produce collagen in peritoneum before and after injury. However, small numbers of fibrocytes are found in fibrotic peritoneum of chimeric mice with bone marrow from Col1a1-GFP reporter mice, but they do not contribute to myofibroblasts. These data demonstrate that InfMϕs switch to pro-fibrotic phenotype and activate peritoneal fibroblasts through CCL17 after injury. CCL17 blockade in patients with peritoneal fibrosis may provide a novel therapy. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Systemic vasoprotection by inhaled carbon monoxide is mediated through prolonged alterations in monocyte/macrophage function

Carbon monoxide (CO) is anti-inflammatory and protective in models of disease. Its actions in vitro are short-lived but are sustained in vivo. We hypothesize that systemic CO can mediate prolonged phenotype changes in vivo, with a focus on macrophages (Mφs). Mφs isolated from CO treated rats responded to lipopolysaccharide (LPS) with increased IL6, IL10 and iNOS expression but decreased TNF. Conditioned media (CM) collected from peritoneal Mφs isolated from CO treated rats stimulated endothelial cell (EC) proliferation versus CM from Mφs from air treated rats. This effect was mediated by Mφ released VEGF and HMGB1. Inhaled CO reduced LPS induced Mφ M1 inflammatory phenotype for up to 5 days. Mitochondrial oxygen consumption in LPS treated Mφs from CO treated mice was preserved compared to LPS treated Mφs from control mice. Finally, transient reduction of inflammatory cells at the time of inhaled CO treatment eliminated the vasoprotective effect of CO in a rodent carotid injury model. Thus, inhaled CO induces a prolonged mixed phenotype change in Mφs, and potentially other inflammatory cells, that contribute to vasoprotection. These findings demonstrate the ability of inhaled CO to modify Mφs in a sustained manner to mediate its therapeutic actions, supporting the translational potential of inhaled CO.

GPR37 regulates macrophage phagocytosis and resolution of inflammatory pain.

The mechanisms of pain induction by inflammation have been extensively studied. However, the mechanisms of pain resolution are not fully understood. Here, we report that GPR37, expressed by macrophages (MΦs) but not microglia, contributes to the resolution of inflammatory pain. Neuroprotectin D1 (NPD1) and prosaptide TX14 increase intracellular Ca2+ (iCa2+) levels in GPR37-transfected HEK293 cells. NPD1 and TX14 also bind to GPR37 and cause GPR37-dependent iCa2+ increases in peritoneal MΦs. Activation of GPR37 by NPD1 and TX14 triggers MΦ phagocytosis of zymosan particles via calcium signaling. Hind paw injection of pH-sensitive zymosan particles not only induces inflammatory pain and infiltration of neutrophils and MΦs, but also causes GPR37 upregulation in MΦs, phagocytosis of zymosan particles and neutrophils by MΦs in inflamed paws, and resolution of inflammatory pain in WT mice. Mice lacking Gpr37 display deficits in MΦ phagocytic activity and delayed resolution of inflammatory pain. Gpr37-deficient MΦs also show dysregulations of proinflammatory and antiinflammatory cytokines. MΦ depletion delays the resolution of inflammatory pain. Adoptive transfer of WT but not Gpr37-deficient MΦs promotes the resolution of inflammatory pain. Our findings reveal a previously unrecognized role of GPR37 in regulating MΦ phagocytosis and inflammatory pain resolution.

Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow-derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-β was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (Pam3CSK4), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1β, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2-related factor 2 (NRF2) but not on nuclear liver X receptors α and β (LXRα,β), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.

Tet2 restrains inflammatory gene expression in macrophages

Tet methylcytosine dioxygenase 2 (TET2) is one of the earliest and most frequently mutated genes in clonal hematopoiesis of indeterminate potential (CHIP) and myeloid cancers, including myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). TET2 catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, leading to DNA demethylation, and also affects transcription by recruiting histone modifiers. Inactivating TET2 mutations cause epigenetic dysregulation, clonal hematopoietic stem cell (HSC) dominance, and monocytic lineage skewing. Here, we found that Tet2 was the most highly expressed Tet enzyme in murine macrophage (MΦ) differentiation. Tet2 transcription was further induced by lipopolysaccharide (LPS), but not interleukin (IL)-4, stimulation, potentially in a nuclear factor κβ-dependent manner. Tet2 loss did not affect early LPS gene responses in vitro, but increased Il-1b, Il-6, and Arginase 1 (Arg1) mRNA expression at later stages of stimulation in bone-marrow-derived MΦs (BMMΦs). Tet2-deficient peritoneal MΦs, however, demonstrated profound, constitutive expression of LPS-induced genes associated with an inflammatory state in vivo. In contrast, Tet2 deficiency did not affect alternative MΦ gene expression significantly in response to IL-4. These results suggested impaired resolution of inflammation in the absence of Tet2 both in vitro and in vivo. For the first time, we also detected TET2 mutations in BMMΦs from MDS and CMML patients and assayed their effects on LPS responses, including their potential influence on human IL-6 expression. Our results show that Tet2 restrains inflammation in murine MΦs and mice, raising the possibility that loss of TET2 function in MΦs may alter the immune environment in the large elderly population with TET2-mutant CHIP and in TET2-mutant myeloid cancer patients.

Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages

Mesenchymal stromal cells (MSCs) are multipotent cells with abilities to exert immunosuppressive response promoting tissue repair. Studies have shown that MSCs can secrete extracellular vesicles (MVs-MSCs) with similar regulatory functions to the parental cells. Furthermore, strong evidence suggesting that MVs-MSCs can modulate several immune cells (i.e., Th1, Th17, and Foxp3+ T cells). However, their precise effect on macrophages (Mϕs) remains unexplored. We investigated the immunoregulatory effect of MVs-MSCs on activated M1-Mϕs in vitro and in vivo using differentiated bone marrow Mϕs and an acute experimental model of thioglycollate-induced peritonitis, respectively. We observed that MVs-MSCs shared surface molecules with MSCs (CD44, CD105, CD90, CD73) and expressed classical microvesicle markers (Annexin V and CD9). The in vitro treatment with MVs-MSCs exerted a regulatory-like phenotype in M1-Mϕs, which showed higher CD206 level and reduced CCR7 expression. This was associated with decreased levels of inflammatory molecules (IL-1β, IL-6, nitric oxide) and increased immunoregulatory markers (IL-10 and Arginase) in M1-Mϕs. In addition, we detected that MVs-MSCs promoted the downregulation of inflammatory miRNAs (miR-155 and miR-21), as well as, upregulated its predicted target gene SOCS3 in activated M1-Mϕs. In vivo MVs-MSCs treatment reduced the Mϕs infiltrate in the peritoneal cavity inducing a M2-like regulatory phenotype in peritoneal Mϕs (higher arginase activity and reduced expression of CD86, iNOS, IFN-γ, IL-1β, TNF-α, IL-1α, and IL-6 molecules). This in vivo immunomodulatory effect of MVs-MSCs on M1-Mϕs was partially associated with the upregulation of CX3CR1 in F4/80+/Ly6C+/CCR2+ Mϕs subsets. In summary, our findings indicate that MVs-MSCs can modulate an internal program in activated Mϕs establishing an alternative regulatory-like phenotype.

MMGL receptor plays an important role to macrophage activation during Trypanosoma cruzi infection

Abstract The C-type lectin receptor (MGL) is exclusively expressed on macrophages (MΦs) and dendritic cells (DCs), recognizes glycosylated antigens, particularly galactose (Gal) and N-acetylgalactosamine (GalNAc), that are present in tumors, fungi and viruses. However, little is known the role of MGL in the interaction with parasites. We evaluated the role of murine MGL (mMGL) in the activation and functionality of MΦs in Trypanosoma cruzi (T. cruzi) infection, a parasite that has molecules glycosylated rich in Gal and GalNAc in its membrane. Peritoneal MΦs from C57BL/6 WT and mMGL nockout (mMGL−/−) mice were infected in vitro with epimastigotes of T. cruz,i Qro strain (10000 parasites). The percentage of infected MΦs and the number of internalized parasites/MΦs was analyzed by microcopy. Cytokines TNF-α, IL-12 and IL-10 were determine in supernatants by ELISA. The trypanocidal capacity of MΦs was determined by the incorporation of tritiated thymidine ([3H] TdR). Expression of TLR-4, TLR-2 and mannose receptor (MR) in MΦs stimulated in vitro with T. cruzi antigen was analyzed by flow cytometry. mMGL−/− MΦs were higher infected with more parasites per MΦ and had a reduced capacity to eliminate the T. cruzi, these were associated to lower levels of TNF-α, TLR-4 and MR expression compared to WT MΦs. These results demonstrate that absence of mMGL favors susceptibility to infection with T. cruzi, suggesting a regulatory role of mMGL on the expression of other important receptors in the immune response to T. cruzi infection.

Myeloid-derived NF-κB negative regulation of PU.1 and c/EBP-β-driven pro-inflammatory cytokine production restrains LPS-induced shock.

Sepsis is a life-threatening event predominantly caused by Gram-negative bacteria. Bacterial infection causes a pronounced macrophage (MΦ) and dendritic cell activation that leads to excessive pro-inflammatory cytokine IL-1β, IL-6 and TNF-α production (cytokine storm), resulting in endotoxic shock. Previous experimental studies have revealed that inhibiting NF-κB signaling ameliorates disease symptoms; however, the contribution of myeloid p65 in endotoxic shock remains elusive. In this study, we demonstrate increased mortality in mice lacking p65 in the myeloid lineage (p65Δmye) compared with wild type mice upon ultra-pure LPS challenge. We show that increased susceptibility to LPS-induced shock was associated with elevated serum level of IL-1β and IL-6. Mechanistic analyses revealed that LPS-induced pro-inflammatory cytokine production was ameliorated in p65-deficient bone marrow-derived MΦs; however, p65-deficient 'activated' peritoneal MΦs exhibited elevated IL-1β and IL-6. We show that the elevated pro-inflammatory cytokine secretion was due, in part, to increased accumulation of IL-1β mRNA and protein in activated inflammatory MΦs. The increased IL-1β was linked with heightened binding of PU.1 and CCAAT/enhancer binding protein-β to Il1b and Il6 promoters in activated inflammatory MΦs. Our data provide insight into a role for NF-κB in the negative regulation of pro-inflammatory cytokines in myeloid cells.

Tet2 Deficiency Leads to an Increased Inflammatory Phenotype in Murine Macrophages

Introduction:In the context of MDS and CMML, little is known about the underlying causes of aberrant immune modulation, particularly with respect to the contribution of recurrently mutated genes. Inactivatingmutations in Tet methylcytosine dioxygenase 2 (TET2) cause loss of hydroxymethylation and a corresponding enrichment of 5-methylcytosine marks, changes which are thought to precipitate clonal dominance and monocytic skewing. Currently, the impact of TET2 loss on the properties of disease-relevant monocytes/macrophages (MΦs) is poorly understood. Therefore, our goals were to (1) characterize Tet2 expression during MΦ LPS and interferon gamma (IFNγ) treatment, (2) determine the effect of Tet2-deficiency on LPS signaling in these cells, and (3) explore how the demethylating agent 5-azacytidine (AZA) impacts abnormally expressed genes in Tet2-knockout MΦs.Methods:Peritoneal (PMΦ) and bone marrow-derived (BMMΦ) MΦs were obtained from Vav1-Cre-driven Tet2 knockout (Tet2-/-) mice in accordance with Queen’s Animal Care protocols. Gene expression profiling was performed using the NanoString nCounter Mouse Immunology Gene Expression CodeSet plus 30 custom targets (591 candidate genes in total).Results:Previously, our group reported that Tet2 expression was induced 3h after LPS treatment in both primary PMΦ and BMMΦ cultures as well as RAW264.7 monocytic cells (Cull et al. Blood Abstract 2015: 646). To further understand the signalling pathways underpinning this induction, RAW264.7 cells were treated for 3h with 100ng/mL LPS alone, 10ng/mL IFNγ alone or a combination of LPS and IFNγ, as IFNγ is known to potentiate LPS signalling. As expected, LPS alone caused Tet2 mRNA levels to increase by 4- to 6-fold. The combined treatment of LPS and IFNγ lead to a 5- to 8-fold induction whereas IFNγ alone failed to increase Tet2 expression, suggesting that Tet2 induction is mainly IFNγ-independent.To evaluate relevant TLR4 signalling pathways, RAW264.7 cells were pretreated with the inhibitor compounds SP600125, BAY11-7082 and PD184352 prior to 3h LPS stimulation. Tet2 induction was abolished in cells pretreated with BAY 11-7082, an NF-κB inhibitor. Mining human ChIP-seq data from the ENCODE database indicated a number of NF-κB (p65) binding sites within the putative TET2 promoter and regulatory regions, some of which are conserved in the murine locus. ChIP studies are currently underway to evaluate binding sites of interest.We have previously reported that untreated Tet2-/- PMΦs constitutively overexpress a variety of genes involved in LPS-mediated inflammatory signalling (Cull et al. Blood Abstract 2015: 646). Based on these findings, we used NanoString gene expression analysis to evaluate the status of Tet2-/- versus Tet2f/f BMMΦs (n=3/genotype). We found gene expression in Tet2-/- BMMΦs to be very similar to control cells. In addition, early (3h) LPS gene expression profiles did not differ appreciably between Tet2-/- and Tet2f/f BMMΦs (n=3/genotype). However, at 12-24h following LPS treatment, Il1b, Il6 and Arg1 mRNA expression were significantly elevated in Tet2-/- BMMΦs. Given that IL-1β and IL-6 are both potent pro-inflammatory cytokines whereas Arg1 is associated with anti-inflammatory alternatively activated MΦfunctions (AAMΦ), we hypothesize that Tet2-/- BMMΦs are unable to resolve inflammation and compensate through overexpression of anti-inflammatory genes such as Arg1.Finally, we determined the effect that the hypomethylating agent AZA had on the mRNA expression of Il1b, Il6 and Arg1 in BMMΦs. In a pilot experiment, pooled Tet2-/- BMMΦs (n=3) were treated with 5μM AZA for 24h prior to 12h LPS stimulation. Compared to LPS alone, AZA pretreatment and subsequent LPS stimulation lead to a reduction in Arg1 (0.47-fold) and Il6 (0.65-fold) levels in Tet2-/- BMMΦs, whereas Il1b expression remained similar (0.97-fold). Based on these initial results, we hypothesize that AZA treatment leads to demethylation of genomic regions that have been enriched in methylation marks due to Tet2 loss, leading to the repression of promoters such as Arg1 and Il6. Further studies are underway to address these questions.Conclusions:In summary, we have demonstrated that Tet2 loss in MΦs leads to overexpression of genes involved in LPS signalling and LPS-related inflammation, suggesting that these cells may contribute to the abnormal immune environment found in myeloid cancers. DisclosuresNo relevant conflicts of interest to declare.

IMMUNOSUPPRESIVE EFFECTS OF THE METHANOLIC EXTRACT OF CHRYSOPHYLLUM CAINITO LEAVES ON MACROPHAGE FUNCTIONS.

Background:The aim of this work was to evaluate the immunomodulatory effect of the methanol extract (MeOH) from Chrysophyllum cainito leaves on the MΦs functions.Material and Methods:Peritoneal murine MΦs isolated from Balb/c mice were treated with the MeOH extract and stimulated with LPS. The effect on the phagocytosis was evaluated by flow cytometry assay. The nitric oxide (NO) and hydrogen peroxide (H2O2) production was measured by the Griess reagent and phenol red reaction, respectively. Levels of IL-6 and TNF-α was measured using an ELISA kit. Viability of MΦs and Vero cells was determined by the MTT method.Results:The MeOH extract of C. cainito leaves inhibited significantly the phagocytosis, and decreased IL-6 and TNF-α production as well as NO and H2O2 released by the MΦs, in a concentration-dependent manner. In addition, MeOH extract of C. cainito showed low cytotoxicity effect against the cells.Conclusion:These results suggest that MeOH extract of C. cainito leaves has an immunosuppressive effect on murine MΦs, without effects on cell viability. GC-MS chromatogram analysis of MeOH extract showed that lupeol acetate and alpha-amyrin acetate are the principal compounds.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up‐regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin‐1 receptor antagonist‐deficient mice

The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10μg/mL of Aggregatibacter actinomycetemcomitans LPS for 24h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. The levels of IL-1α, IL-1β, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A.actinomycetemcomitans LPS were significantly increased by approximately 4- (p<0.05), 5- (p<0.05), 1.3- (p<0.05) and 6- (p<0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p<0.05), 1.6- (p<0.05), 2.5- (p<0.05), 1.6- (p<0.05) and 1.9- (p<0.05) fold, respectively, in IL-1Ra KO mice stimulated with A.actinomycetemcomitans LPS compared with WT mice. IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Transcriptional Regulation of IL-10 Expression by Peroxisome Proliferator-Activated Receptor (Pparγ) in Human Intestinal Epithelial Cells

IL-10 is an important immunomodulatory cytokine in the regulation of intestinal inflammation in inflammatory bowel disease (IBD). PPARγ is a nuclear receptor known to modulate transcriptional activities of many cytokines and is highly expressed in intestinal epithelial cells (IECs). Human IECs have been shown to express IL-10, but how its expression is regulated remains obscure especially in the setting of inflammation. We have demonstrated that intestinal 15d-PGJ2 (an endogenous PPARγ ligand) is predominantly induced by infiltrated macrophages (Mφs) through toll-like receptor 4 (TLR4)-mediated Cox-2 induction during colitis. In this study, we addressed whether PPARγ activation regulates expression of IL-10 in IECs. Human IEC cell line SW480-APC expressing TLR4 (top) and mouse peritoneal Mφs (bottom) were co-cultured in transwells. 15d-PGJ2 production and IL-10 mRNA expression were measured by enzyme linked assay and real-time PCR in the presence or absence of lipopolysaccharide (LPS). In order to examine whether the IL-10 promoter has PPARγ responsive elements, a 2,000 bp of IL-10 promoter was cloned from SW480-APC genomic DNA into the luciferase reporter vector. Expression and activity of PPARγ were blocked by siRNA and a specific inhibitor SR202, respectively. The IL-10 promoter activity was analyzed by luciferase reporter assay. PPARγ activity was confirmed using a reporter vector PPRE X3-TK-luc. Co-culturing SW480-APC with Mφs increased IL-10 expression in IECs within 24 hours, which was significantly enhanced by LPS stimulation in later time point (72 hours). LPS stimulation of SW480-APC resulted in PPARγ activation peaked at 48 hours. During SW480-APC/Mφ co-culture, LPS stimulation induced 15d-PGJ2 production, which is abolished when TLR4-deficient Mφs were used. Knocking down the expression of PPARγ suppressed IL-10 promoter activity and mRNA expression in SW480-APC in baseline as well as after LPS stimulation. These results suggest that PPARγ positively regulates IL-10 expression in IECs at the transcriptional level. During inflammation, mucosal Mφs induce PPARγ ligand (15d-PGJ2) in a TLR4-dependent manner that contributes to PPARγ activation in IECs and thus IL-10 expression. This may be an intrinsic immunoregulatory program that is mediated through host-bacterial interactions, which dampens on-going inflammation during intestinal inflammation and thus can be a novel therapeutic target for IBD.