Thioglycollate-elicited Peritoneal Macrophages Research Articles

High throughput screening identifies auranofin and pentamidine as potent compounds that lower IFN-γ-induced Nitric Oxide and inflammatory responses in mice: DSS-induced colitis and Salmonella Typhimurium-induced sepsis.

Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC®1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease.

PRMT5 inhibition induces pro-inflammatory macrophage polarization and increased hepatic triglyceride levels without affecting atherosclerosis in mice.

Protein arginine methyltransferase 5 (PRMT5) controls inflammation and metabolism through modulation of histone methylation and gene transcription. Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p< 0.05) and significantly increased hepatic triglyceride levels (+50%; p< 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor.

Harmine Inhibits Multiple TLR-Induced Inflammatory Expression through Modulation of NF-κB p65, JNK, and STAT1.

Harmine is a beta-carboline alkaloid present in various plants, including in the seeds of Peganum harmala L. This study aimed to investigate the anti-inflammatory activity and mechanism of harmine using macrophages stimulated with various toll-like receptor (TLR) agonists and a model of endotoxemia. The expression of inflammatory mediators induced by ligands of TLRs 2, 3, 4, and 9 were examined in thioglycollate-elicited peritoneal macrophages isolated from BALB/c and C57BL/6 mouse strains. Further, the activation of NF-κB, MAPK, AP-1, and STAT1 was explored using lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)). Finally, the liver inflammatory response during endotoxemia was examined. Harmine inhibited inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12, and other markers induced by various TLR agonists. The inhibition of NF-κB activity by harmine occurred via the modulation of p65 phosphorylation, independent of IκBα degradation. The inhibition of AP-1 activity by harmine was associated with the modulation of JNK. Harmine inhibited the LPS-induced serine and tyrosine phosphorylation of STAT1, but only affected serine phosphorylation by poly(I:C) treatment. In vivo, harmine inhibited iNOS and COX-2 expression during endotoxemia. Collectively, the results show that harmine can be effective against infectious inflammation through modulation of NF-κB, JNK, and STAT1.

2-Hydroxypropyl-beta-cyclodextrin Treatment Does Not Induce Atherosclerotic Lesion Regression in Western-Type Diet-Fed Apolipoprotein E Knockout Mice

2-Hydroxypropyl-beta-cyclodextrin (2HPβCD) is able to bind and solubilize unesterified cholesterol and may therefore be able to reverse the deposition of cholesterol in macrophages within the aortic vessel wall, a hallmark of atherosclerotic cardiovascular disease. However, conflicting results regarding the potential of 2HPβCD to induce regression of established atherosclerotic lesions have been described. In the current study, we therefore also investigated the ability of 2HPβCD to stimulate cholesterol removal from macrophage foam cells in vitro and induce the regression of established atherosclerotic lesions in apolipoprotein E knockout (APOE KO) mice. In vitro studies using murine thioglycollate-elicited peritoneal macrophages verified that 2HPβCD is able to induce cholesterol efflux from macrophages in an ATP-binding cassette transporter-independent manner. Switching Western-type-diet-fed APOE KO mice with established atherosclerotic lesions back to a chow diet was associated with a reduction in the hypercholesterolemia extent and an increase in the absolute lesion size and plaque collagen-to-macrophage ratio. Importantly, parallel subcutaneous administration of 2HPβCD was not able to prevent the diet-switch-associated lesion growth or induce atherosclerosis regression. Although in our hands, 2HPβCD does effectively stimulate cellular cholesterol efflux from macrophages, we do not consider it worthwhile to further pursue 2HPβCD as therapeutic moiety in the atherosclerosis regression context.

Lactococcus lactis subsp. cremoris C60 induces macrophages activation that enhances CD4+ T cell-based adaptive immunity.

Lactococcus lactis subsp. cremoris C60 is a probiotic strain that induces diverse functional modifications in immune cells. In this report, as a novel effect of C60 on myeloid lineage cells, we show that C60 enhances the immunological function of macrophages that consequently promotes CD4+ T cell activity in an antigen-dependent manner. Heat-killed (HK) C60 induced the production of pro-inflammatory cytokines in thioglycolate-elicited peritoneal macrophages (TPMs) much stronger than Toll-like receptor (TLR) ligand stimulation. The HK-C60 treatment also augmented the expression of antigen-presenting and co-stimulatory molecules, such as major histocompatibility complex (MHC) class II, CD80, and CD86, as well as antigen uptake in TPMs. These HK-C60-mediated functional upregulations in TPMs resulted in the promotion of CD4+ T cell activation in an antigen-dependent manner. Interestingly, the TPMs that originated from the mice fed the HK-C60 diet showed pre-activated characteristics, which was confirmed by the upregulation of cytokine production and antigen presentation-related molecule expression under lipopolysaccharide (LPS) stimulation. Furthermore, the antigen-dependent CD4+ T cell activation was also enhanced by the TPMs. This implied that antigen presentation activity was enhanced in the TPMs that originated from the HK-C60 diet mice. Thus, C60 effectively upregulates the immunological function of macrophages that directly connects to CD4+ T cell-based adaptive immunity.

Development and validation of a method to deliver vitamin A to macrophages.

Macrophages are critical players in the development of atherosclerotic lesions, where they promote local and systemic inflammation. Macrophages engulf lipoproteins and cell debris upon entry into the arterial wall, becoming lipid-laden foam cells. While most lipids found in foam cells are triglyceride and cholesterol, these cells accumulate several other lipids with bioactive properties, such as vitamin A and carotenoids. Vitamin A has strong immunomodulatory actions in macrophages and other immune cells. For example, macrophages release vitamin A as retinoic acid to modulate T cell differentiation, but the implication of intracellular vitamin A stores in this process remains elusive due to the lack of an adequate experimental model to load vitamin A into macrophages. The purpose of this study was to develop a reliable method to deliver vitamin A to cultured murine macrophages. Our results show that thioglycolate-elicited peritoneal macrophages fail to take up significant levels of vitamin A when provided as free retinol. Cultured macrophages and macrophages in the peritoneal cavity can take up retinyl esters, either as retinyl ester-loaded serum or retinyl esters infused directly into the peritoneal cavity. HPLC analyses in macrophage lysates revealed that the intraperitoneal injection method results in a fourfold greater vitamin A loading efficiency than retinyl ester-loaded serum added to cultured cells. These two alternative methods provide an efficient and reliable methodology to load macrophages with vitamin A for downstream applications such as studies of gene regulation trafficking of intracellular vitamin A, and vitamin A release from macrophages.

Loss of Mir146b with aging contributes to inflammation and mitochondrial dysfunction in thioglycollate-elicited peritoneal macrophages.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

Induction of Pro‐Inflammatory Cytokines TNF, IL‐6, and HMGB1 by SARS‐CoV‐2 Spike Proteins in Mice and in Murine Macrophage Cultures

The pandemic COVID‐19 disease with severe acute respiratory syndrome (SARS) is mediated by the coronavirus SARS‐CoV‐2. The highly conserved surface spike glycoprotein of the SARS‐coronavirus is critical for viral entry into cells, a central step in pathogenesis. To further understand and characterize viral spike proteins in host cell immune responses, we generated two species of spike proteins: a recombinant receptor binding domain (RBD) and a truncated part of RBD, the receptor binding motif (RBM). In cell cultures with murine macrophage‐like RAW 264.7 cells or with thioglycollate‐elicited peritoneal primary mouse macrophages, recombinant RBD and RBM dose‐dependently induced the release of TNF and HMGB1, pro‐inflammatory molecules observed in increased systemic levels in COVID‐19 patients (Chen R et al, Heliyon 6, 2020, e05672). Intratracheal administration of RBD (100 µg/mouse) or RBM (100 µg/mouse) also induced HMGB1 release in bronchoalveolar lung (BAL) fluid at 24 hours post‐instillation (HMGB1 levels = 71 ± 30 ng/mL in PBS group vs. 253 ± 19* ng/mL in RBD group and 284 ± 42* ng/mL in RBM group; n=3‐4 mice/group; *: p<0.05 vs. vehicle PBS group). Furthermore, intratracheal administration of recombinant RBM induced TNF, IL‐6 and IL‐1β release in bronchoalveolar lavage fluid at 24 hours post‐instillation (n=13 per group, p<0.05 vs.PBS group). Taken together, our studies reveal that recombinant RBD and RBM induce pro‐inflammatory cytokine release in vivo and in cultured murine macrophages.

ADAM17 Boosts Cholesterol Efflux and Downstream Effects of High-Density Lipoprotein on Inflammatory Pathways in Macrophages.

[Figure: see text].

Enhanced Ca2+ handling in thioglycolate-elicited peritoneal macrophages

In macrophage biology, resident peritoneal macrophages (RPMs) and thioglycolate-elicited peritoneal macrophages (TGPMs) have been traditionally utilized as primary cultured models. RPMs and TGPMs exhibit distinct morphological, functional and metabolic characteristics, although it remains unclear how cellular Ca2+ handling differs between them. In our Fura-2 Ca2+ imaging, TGPMs displayed elevated resting Ca2+ levels, increased store Ca2+ contents and facilitated store-operated Ca2+ entry (SOCE) compared with RPMs. The intensified intracellular Ca2+ stores were enriched with major luminal Ca2+-binding proteins inducibly expressed in TGPMs. The elevated resting Ca2+ level was predominantly maintained by constitutive Ca2+ influx, probably through the transient receptor potential (TRP) family members TRPP2, TRPM7 and TRPA1. These TRP family channels seemed to be largely activated in a manner dependent on phospholipase C activity, and together with Orai channels, contributed to SOCE. Moreover, Ca2+-dependent K+ channels efficiently facilitated SOCE by enhancing the Ca2+ driving force in TGPMs. The consolidated cellular Ca2+ handling described may underlie the specialized cell-physiological features of TGPMs, such as vital proliferation, active migration and avid phagocytosis.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits Tcell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in Tcells remains enigmatic. Here weinvestigated how PD-1 affects transcriptome changes induced by Tcell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of Tcell populations.

Peritoneal macrophages have an impaired immune response in obesity which can be reversed by subsequent weight loss

IntroductionObesity is recognized as a risk factor for various microbial infections. The immune system, which is affected by obesity, plays an important role in the pathophysiology of these infections and...

A distinct transcriptional profile in response to endothelial monocyte activating polypeptide II is partially mediated by JAK-STAT3 in murine macrophages.

Macrophages are important responders to environmental changes such as secreted factors. Among the secreted factors in injured tissues, the highly conserved endothelial monocyte activating polypeptide II (EMAP II) has been characterized to limit vessel formation, to be locally expressed near sites of injury labeling it a "find-me" signal, and to recruit macrophages and neutrophils. The molecular mechanisms mediated by EMAP II within macrophages once they are recruited are unknown. In this study, using a model of partially activated, recruited thioglycollate-elicited peritoneal macrophages, a transient, transcription profile of key functional genes in macrophages exposed to EMAP II was characterized. We found that EMAP II-mediated changes were elicited mainly through signal transducer and activator of transcription 3 (STAT3) as evidenced by increased Y705 phosphorylation and changes in activity and upstream of it, Janus associated kinase (JAK)1/2 upstream. Both inhibition of JAK1/2 and knockdown of Stat3 abrogated a subset of genes that are upregulated by EMAP II. Our results identify a rapid EMAP II-mediated STAT3 activation that coincides with altered pro- and anti-inflammatory gene expression in macrophages.

Abstract B197: Translational control in macrophages during inflammatory response

Abstract Translational control has been increasingly appreciated as a critical gene-regulatory mechanism during immune responses. Previous studies suggested that translation of select cytokines could be regulated by mRNA poly(A)-tail lengths in memory T-cells and macrophages. We carried out transcriptome-wide analysis with mRNA sequencing, ribosome-footprinting profiling as well as poly(A)-tail length profiling in both RAW264.7 cells and mouse thioglycollate-elicited peritoneal macrophages. No coupling between poly(A)-tail length and translational efficiency (TE) was observed in either cell type, arguing against a global translational-regulatory regime mediated by poly(A)-tail lengths. Immune activation with the treatment of lipopolysaccharides (LPS) to these cells did not change the uncoupled regime. Moreover, gene expression landscape in LPS-treated macrophages was dominated by mRNA level changes, with little contribution from TE changes. The up- or down-regulation of TE was not accompanied by concerted lengthening or shortening of poly(A) tails, implying existence of yet unclear translational control mechanisms in these immune cells. Finally, our studies in other biologic systems, including HeLa cells, Xenopus oocytes and yeast suggest that saturating levels of poly(A)-binding proteins might suppress poly(A) tail length-mediated translational control in various uncoupled systems, including immune cells. Citation Format: Kehui Xiang, David P. Bartel. Translational control in macrophages during inflammatory response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B197.

Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal mouse Macrophages by Flow Cytometry.

Internalization of cell surface Toll Like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry.

Rhodanthpyrone A and B play an anti-inflammatory role by suppressing the nuclear factor-κB pathway in macrophages.

Macrophage-associated inflammation is crucial for the pathogenesis of diverse diseases including metabolic disorders. Rhodanthpyrone (Rho) is an active component of Gentiana rhodantha, which has been used in traditional Chinese medicine to treat inflammation. Although synthesis procedures of RhoA and RhoB were reported, the biological effects of the specific compounds have never been explored. In this study, the anti-inflammatory activity and mechanisms of action of RhoA and RhoB were studied in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment with RhoA and RhoB decreased inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW 264.7 cells and in thioglycollate-elicited mouse peritoneal macrophages. In addition, it downregulated transcript levels of several inflammatory genes in LPS-stimulated RAW 264.7 cells, including inflammatory cytokines/chemokines (Tnfa, Il6, and Ccl2) and inflammatory mediators (Nos2 and Ptgs2). Macrophage chemotaxis was also inhibited by treatment with the compounds. Mechanistic studies revealed that RhoA and RhoB suppressed the nuclear factor (NF)-κB pathway, but not the canonical mitogen activated protein kinase pathway, in LPS-stimulated condition. Moreover, the inhibitory effect of RhoA and RhoB on inflammatory gene expressions was attenuated by treatment with an NF-κB inhibitor. Our findings suggest that RhoA and RhoB play an anti-inflammatory role at least in part by suppressing the NF-κB pathway during macrophage-mediated inflammation.

MTOR- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration through the iNOS/Src/FAK Axis.

Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43+/- mice compared with TGEMs from Cx43+/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CM-induced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80+CD11b+ macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.

Apoptotic cell induction of miR-10b in macrophages contributes to advanced atherosclerosis progression in ApoE-/- mice.

We previously found that miR-10b inhibits cholesterol efflux from thioglycollate-elicited mouse peritoneal macrophages through repressing ATP binding cassette transporter (ABCA1). Herein, we deciphered the mechanism underlying macrophage miR-10b expression and the role of miR-10b in atherosclerosis. MiR-10b expression was increased in the arteries with advanced but not early atherosclerotic plaques of ApoE-/- mice. Free cholesterol-induced macrophage apoptotic cells (FC-AM) promoted miR-10b expression in mouse resident peritoneal macrophages (RPM) by up-regulation of Twist1/2 in a Mer receptor tyrosine kinase dependent manner. Surprisingly, antagomiR-10b de-repressed ABCA1 in RPM engulfing FC-AM but not in RPM alone or RPM-derived foam cells; systemic delivery of antagomiR-10b enhanced reverse cholesterol transport from RPM engulfing FC-AM but not from RPM or the foam cells in ApoE-/- mice. Mechanistically, RPM engulfing FC-AM possessed sufficient miR-10b functionally repressing ABCA1 expression, whereas RPM and foam cells had little miR-10b incompetently repressing ABCA1 expression. Notably, antagomiR-10b administration reduced advanced plaque size and also enhanced plaque stability in ApoE-/-mice, which were associated with increased plaque macrophage ABCA1 expression and reduced plaque apoptosis and inflammation. However, antagomiR-10b administration did not affect early atherosclerotic plaque formation in ApoE-/- mice. These data suggest that apoptotic cell induction of miR-10b in macrophages is important in advanced atherosclerosis progression.

Distinct Transcriptional Profile Activated by SCYE1 Through JAK‐mediated STAT3 in Partially Activated Macrophages

Small inducible cytokine subfamily E, member 1, SCYE1 (also known as EMAP II) is a secreted factor that is locally expressed near sites of injury, labeled as a ‘find‐me’ signal, and recruits macrophages and neutrophils. Our previous studies identified that macrophages sustained protein expression of SCYE1. The transcriptional events that occur within the recruited macrophages in response to SCYE1 are unknown. The objective was to identify the transcription factor activated in partially activated thioglycollate macrophages upon exposure to SCYE1.Partially activated murine thioglycollate‐elicited macrophages (TEPMs) were exposed to SCYE1. Following 2 hours of exposure to either vehicle or SCYE1, fractionated nuclear proteins from TEPMs were used for a luminescence based transcription factor activation screen. The acute SCYE1‐mediated transcription factor activation was assessed by immunoblotting and gene expression by quantitative PCR. Secreted cytokines were measured in supernatants of TEPMs exposed to either vehicle or SCYE1 while pre‐treated with +/− JAK1/2 inhibitor, Ruxolitinib.In this study, we identify STAT3 (Signal Transducer and Activator of Transcription 3) as the most activated transcription factor mediated by SCYE1 in a model of partially activated, recruited thioglycollate‐elicited peritoneal macrophages. The acute and dose‐dependent phosphorylation of STAT3 (Y705) occurs between 5 and 15 minutes in response to SCYE1. Its activation is SCYE1‐specific and could be prevented by inhibition of Janus Associated Kinase (JAK) 1/2.We profiled not only anti‐ and pro‐inflammatory genes but also that of STAT3 target genes upon exposure to SCYE1. STAT3 target genes Arg1 and Socs3, commonly activated in anti‐inflammatory macrophages, are maximal by 1 to 2 hours of exposure to SCYE1. Yet Il10 expression decreased by 1 hour suggesting that any persisting STAT3 activation was not due to Il10. There was also rapid increase of chemokine STAT3 target genes Ccl5 along with other genes activated that are typically found in pro‐inflammatory macrophages.Unlike activated transcription, neither secreted IL‐6 nor IL‐10 was elevated by SCYE1. Consistent with a JAK‐STAT signature, both IL‐1a and G‐CSF secretion by SCYE1 was elevated and could be prevented by JAK inhibition.Our results identify a rapid SCYE1‐mediated STAT3 activation that coincides with both pro‐ and anti‐inflammatory gene expression in partially activated macrophages.Support or Funding InformationFunding: This publication was made possible in part by 5R01HL114977 (MAS) from NIH and the Lilly Endowment, Inc. Physician Scientist Initiative (MAS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Frontline Science: Specialized proresolving lipid mediators inhibit the priming and activation of the macrophage NLRP3 inflammasome.

The prototypic proinflammatory cytokine IL-1β plays a central role in innate immunity and inflammatory disorders. The formation of mature IL-1β from an inactive pro-IL-1β precursor is produced via nonconventional multiprotein complexes called the inflammasomes, of which the most common is the nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3) inflammasome composed by NLRP3, (ASC) apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD), and caspase-1. Specialized proresolving mediators (SPMs) promote resolution of inflammation, which is an essential process to maintain host health. SPMs prevent excessive inflammation by terminating the inflammatory response and returning to tissue homeostasis without immunosupression. This study tested the hypothesis that modulation of the NLRP3 inflammasome in macrophages is one mechanism involved in the SPM-regulated processes during resolution. Our findings demonstrate that the SPM resolvin D2 (RvD2) suppressed the expression of pro-IL-1β and reduced the secretion of mature IL-1β in bone marrow-derived macrophages challenged with LPS+ATP (classical NLRP3 inflammasome model) or LPS+palmitate (lipotoxic model). Similar findings were observed in thioglycolate-elicited peritoneal macrophages, in which RvD2 remarkably reduced ASC oligomerization, inflammasome assembly, and caspase-1 activity. In vivo, in a self-resolving zymosan A-induced peritonitis model, RvD2 blocked the NLRP3 inflammasome leading to reduced release of IL-1β into the exudates, repression of osteopontin, and MCP-1 expression and induction of M2 markers of resolution (i.e., CD206 and arginase-1) in peritoneal macrophages. RvD2 inhibitory actions were receptor mediated and were abrogated by a selective GPR18 antagonist. Together, these findings support the hypothesis that SPMs have the ability to inhibit the priming and to expedite the deactivation of the NLRP3 inflammasome in macrophages during the resolution process.