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The essential micronutrient zinc regulates immune responses by affecting signaling pathways. In activated monocytes and macrophages, signaling networks mediate the metabolic reprogramming that meets the demands of participation in immune responses. Here, we demonstrated that cytoplasmic, bioavailable zinc was essential for promoting IL-1β production in activated human monocytes and macrophages downstream of glycolysis induced by the kinase-containing multiprotein complex mTORC1. The concentration of cytoplasmic zinc was determined by that of extracellular zinc, which was brought into cells through the zinc-specific importer Zip8. The abundance of Zip8 was increased in monocytes from patients with rheumatoid arthritis (RA), as well as in LPS-stimulated monocytes and macrophages from healthy individuals. The mTORC1-mediated phosphorylation of S6 kinase (S6K) was enhanced by zinc-mediated inhibition of PP2A, a phosphatase that targets S6K. As a result, IL-1β production was increased due to the activation of mTORC1-induced glycolysis. In monocytes of patients with RA, the expression of

Monosodium urate (MSU) crystals are an endogenous sterile particulate that has been identified as a potent damage-associated molecular pattern (DAMP). In humans, the induction of IL-1β production through MSU-induced NLRP3 inflammasome activation in monocytes/macrophages is responsible for pathogenesis of gouty arthritis. It was recently reported that in a murine model of this disease, resveratrol decreases MSU-induced recurrent attacks of gouty arthritis. Despite its demonstrated anti-inflammatory effects, the mechanisms underlying resveratrol-mediated repression of IL-1β production in MSU-activated monocytes remain poorly understood. Here, we show that resveratrol suppresses secretion of active IL-1β by human primary monocytes stimulated with MSU crystals through suppression of Syk activation. Metabolic labeling and pull-down assays to investigate de novo protein synthesis clearly demonstrated that intracellular pro-IL-1β synthesis is rapidly repressed in monocytes after resveratrol treatment due to decreased phosphorylation of Syk and p38. Resveratrol also inhibited NLRP3 inflammasome activation in MSU-stimulated monocytes by suppressing oligomerization of ASC. Furthermore, resveratrol exerted a beneficial effect by reducing IL-1β production and inhibiting neutrophil recruitment in a mouse model of MSU-mediated peritonitis. Our findings suggest that resveratrol exerts anti-inflammatory effects via post-translational regulation of IL-1β production and, thus, may prove beneficial for the treatment of MSU crystal-mediated sterile inflammation. KEY MESSAGE: Resveratrol has negative effects on pro-IL-1β synthesis through Syk and p38. Resveratrol inhibits oligomerization of ASC. Resveratrol is beneficial in a mouse model of MSU-induced peritonitis.

The essential micronutrient zinc plays regulatory roles in immune responses through its ability to affect signaling pathways. In activated monocytes/macrophages, signaling networks mediate metabolic reprogramming in order to meet the demands of participating in immune responses. Despite its known immunoregulatory roles, the effect of zinc on metabolic reprogramming in monocytes/macrophages remains unclear. Here, we demonstrate that cytoplasmic bioavailable zinc is essential for regulating IL-1β production in activated human monocytes/macrophages downstream of mTORC1-induced glycolysis. The cytoplasmic zinc level was influenced by extracellular zinc concentration through a zinc-specific importer, Zip8, which was markedly increased in monocytes of patients with rheumatoid arthritis (RA), a chronic inflammatory disease, and even in LPS-stimulated monocytes/macrophages of healthy individuals. Mechanically, phosphorylation of S6 kinase, a substrate of mTORC1, was significantly enhanced by zinc-mediated inhibition of PP2A, an S6 kinase phosphatase. As a result, IL-1β production was increased due to the activation of mTORC1-induced glycolysis. The expression of Zip8 and MT2A, a zinc-inducible gene, and the phosphorylation of S6 kinase by monocytes of RA patients was significantly enhanced compared with those of HCs and Zip8 levels positively correlated with RA clinical parameters, suggesting that Zip8-mediated zinc influx is related to inflammatory conditions. These results provide insight into the role of cytoplasmic bioavailable zinc in the metabolic reprogramming of human monocytes/macrophages which is an essential process for inflammatory responses.One Sentence SummaryCytoplasmic zinc regulates IL-1β production in monocytes/macrophages downstream of mTORC1-S6K-induced glycolysis via zinc-mediated inhibition of PP2A.

Summary IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

Autoimmune rheumatoid arthritis is characterized by chronic inflammation and hyperplasia in the synovial joints. Although the cause of rheumatoid arthritis is largely unknown, substantial evidence has supported the importance of immune cells and inflammatory cytokines in the initiation and progression of this disease. Herein, we demonstrated that the benzoxathiole derivative 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one (BOT-4-one) alleviated type II collagen-induced arthritis in a mouse model. The levels of pro-inflammatory cytokines are elevated in both human patients with rheumatoid arthritis and mice with collagen-induced arthritis. BOT-4-one treatment reduced the levels of pro-inflammatory cytokines in mice and endotoxin-stimulated macrophages. BOT-4-one treatment suppressed the polarization of Th1- and Th17-cell subsets by inhibiting the expression and production of their lineage-specific master transcription factors and cytokines, as well as activation of signal transducer and activator of transcription proteins. In addition, BOT-4-one inhibited mitogen-activated protein kinase and NF-kappaB signaling as well as the transcriptional activities and DNA-binding of transcription factors, including activator protein-1, cAMP response element-binding protein and NF-kappaB. Our results suggest that BOT-4-one may have therapeutic potential for the treatment of chronic inflammation associated with autoimmune rheumatoid arthritis.

B7-H3, a newly identified B7 family member, has functional duality as a co-stimulator and co-inhibitor that fine-tunes T cell-mediated immune responses. Given that B7-H3 expression on human monocytes and dendritic cells is enhanced by inflammatory cytokines, its potential inmmunoregulatory role at sites of inflammation has been suggested. Further, monocytes play crucial roles in the pathophysiology of various inflammatory disorders including autoimmune diseases; however, the immunological role of B7-H3 in rheumatoid arthritis (RA) has not been defined. Thus, we aimed to investigate the possible roles of monocyte B7-H3 in the pathogenesis of RA. Synovial monocytes, but not peripheral monocytes, in RA patients predominantly express surface B7-H3. The 4Ig isoform of B7-H3 is exclusively induced on the cell surface, whereas the 2Ig B7-H3 isoform is constitutively expressed in the intracytoplasmic region of both peripheral and synovial monocytes. B7-H3 knockdown experiments reveal that surface B7-H3 has an inhibitory effect on IFN-γ production in CD4 memory cells. Moreover, surface B7-H3 expression on synovial monocytes inversely correlates with RA clinical parameters. Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune responses and immunomodulatory roles affecting RA pathogenesis.

Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-β expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-β promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-β cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-β expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 .

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14+CD16+, but not CD14dimCD16+, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14+ monocytes was clearly induced by TGF-β, although co-treatment with IL-1β, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14+CD16+ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-β. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.

Background B7-H3 (B7-H3), newly identified B7 family member, has functional duality as a T-cell costimulator or coinhibitor for fine-tuning T-cell mediated immune responses. Given that B7-H3 expression is enhanced in human monocyte and dendritic cells by inflammatory cytokines, it has been suggested their potential inmmunoregulatory function at sites of inflammation. Monocytes play a crucial role in the pathophysiology of various inflammatory disorders including autoimmune diseases. However, the immunological role of B7-H3 in rheumatoid arthritis (RA) has not been defined. Objectives We aimed to investigate the possible roles of B7-H3 on monocyte in pathogenesis of RA. Methods Synovial fluid and peripheral blood from 33 RA patients and peripheral blood from 19 healthy controls (HCs) were obtained. The expression of B7-H3 on monocytes in RA patients and HCs were analyzed using flow cytometic analysis and quantitative RT-PCR. Purified synovial and peripheral monocytes were used for immunoblotting and intracellular flow cytometic analysis to investigate the distinct expression and localization of two isoforms of B7-H3. B7-H3-expressing THP-1 cells were transfected with B7-H3-specific siRNA and subjected to coculture assay with CD4 T cells. Proliferation and cytokine production of the cocultured T cells were assessed by CFSE dilution and ELISA, respectively. Clinical relevance of enhanced B7-H3 expression in RA was tested by comparison with clinical parameters and disease severity in the patients and with synovial cytokine levels measured by multiplex cytokine assay. Results Synovial monocytes but not peripheral monocytes in RA patients predominantly induce surface B7-H3 expression. The induced B7-H3 is exclusively 4Ig isoform, whereas 2Ig B7-H3 isoform constitutively express the intracytoplasmic region both peripheral and synovial monocytes. The B7-H3 knockdown experiments provided the evidence that 4Ig B7-H3 isoform has an inhibitory effect on interferon-gamma (IFN-γ) production of CD4 memory cells. Moreover, the expression level of 4Ig B7-H3 on synovial monocytes was inversely correlated with clinical parameters such as C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and Disease Activity Score in 28 joints (DAS28). Conclusions Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune response and immunomodulatory role affecting pathogenesis of RA. Disclosure of Interest None declared

B7-H3, a newly identified B7 family member, has functional duality as a T-cell costimulator and coinhibitor that fine-tunes T-cell mediated immune responses. Given that B7-H3 expression on human monocytes and dendritic cells is enhanced by inflammatory cytokines, its potential inmmunoregulatory role at sites of inflammation has been suggested. Monocytes also play crucial roles in the pathophysiology of various autoimmune diseases. However, the immunological role of B7-H3 in RA has not been defined. We aimed to investigate the possible roles of monocyte B7-H3 in the pathogenesis of RA. Synovial monocytes, but not peripheral monocytes, in RA patients predominantly express surface B7-H3. The 4Ig isoform of B7-H3 is exclusively induced on the cell surface, whereas the 2Ig B7-H3 isoform is constitutively expressed in the intracytoplasmic region of both peripheral and synovial monocytes. B7-H3 knockdown experiments provide evidence that surface B7-H3 has an inhibitory effect on IFN-γ production in CD4 memory cells. Moreover, the expression level of 4Ig B7-H3 on synovial monocytes inversely correlates with clinical parameters such as CRP levels, ESR, and DAS28. Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune responses and immunomodulatory roles thereby affecting the pathogenesis of RA.