Apoptotic cells induce NF-?B and inflammasome negative signaling.
ABSTRACT: As they undergo phagocytosis, most early apoptotic cells negatively regulate proinflammatory signaling and were suggested as a major mechanism in the resolution of inflammation. The dextran sulfate sodium model is generally viewed as an epithelial damage model suited to investigate innate immune responses. Macrophages primed with LPS and subsequently exposed to DSS secrete high levels of IL-1? in an NLRP3-, ASC-, and caspase-1-dependent manner. The aim of this research was to test the therapeutic effect of a single dose of apoptotic cells in a DSS-colitis model and to explore possible mechanisms. Primary peritoneal macrophages, the DSS mice model, and Nlrp3-deficient mice, were used to assess the effect apoptotic cells on colitis. Immunohistochemistry, flow-cytometer, and western blots helped to explore the effect and mechanisms. Using a variety of NLRP3 triggering mechanisms, we show that apoptotic cells negatively regulate NF-?B and NLRP3 activation in primary peritoneal macrophages, at pre- and post-transcription levels, via inhibition of reactive oxygen species, lysosomal stabilization, and blocking K+ efflux. This property of apoptotic cells is demonstrated in a dramatic clinical, histological, and immunological amelioration of DSS colitis in Balb/c and B6 mice following a single administration of apoptotic cells.
Project description:Activation of cannabinoid receptor 2 (CB2R) ameliorates inflammation, but the underlying mechanism remains unclear. In the present study, we examined whether activation of CB2R could suppress the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome. In peritoneal macrophages isolated from C57BL/6 mice, LPS/DSS challenge for 24 h increased the expression of the components of NLRP3 inflammasome NLRP3, Casp-1 p20/Casp-1 p45 ratio, proIL-1? and IL-1? and also enhanced autophagy (LC3-II/LC3-I ratio, Beclin-1 and SQSTM1). Pretreatment of peritoneal macrophages with HU 308, a selective CB2R agonist, attenuated LPS/DSS-induced NLRP3 inflammasome activation, but further enhanced autophagy. In comparison with wild-type (WT) control, peritoneal macrophages from CB2R knockout (KO) mice had more robust NLRP3 inflammasome activation and attenuated autophagy upon LPS/DSS challenge. Knockdown autophagy-related gene 5 (Atg5) with a siRNA in peritoneal macrophages attenuated the inhibitory effects of HU 308 on LPS/DSS-induced NLRP3 inflammasome activation in vitro. In vivo, HU308 treatment attenuated DSS-induced colitis mice associated with reduced colon inflammation and inhibited NLRP3 inflammasome activation in wild-type mice. In CB2R KO mice, DSS-induced inflammation and NLRP3 inflammasome activation were more pronounced than those in WT control. Finally, we demonstrated that AMPK-mTOR-P70S6K signaling pathway was involved in this CB2R-mediated process. We conclude that activation of CB2R ameliorates DSS-induced colitis through enhancing autophagy that may inhibit NLRP3 inflammasome activation in macrophages.
Project description:Loss-of-function mutations in the solute carrier organic anion transporter family, member 2a1 gene (SLCO2A1), which encodes a prostaglandin (PG) transporter, have been identified as causes of chronic nonspecific multiple ulcers in the small intestine; however, the underlying mechanisms have not been revealed. We, therefore, evaluated the effects of systemic knockout of Slco2a1 (Slco2a1-/-) and conditional knockout in intestinal epithelial cells (Slco2a1?IEC) and macrophages (Slco2a1?MP) in mice with dextran sodium sulphate (DSS)-induced acute colitis. Slco2a-/- mice were more susceptible to DSS-induced colitis than wild-type (WT) mice, but did not spontaneously develop enteritis or colitis. The nucleotide-binding domain, leucine-rich repeats containing family, pyrin domain-containing-3 (NLRP3) inflammasome was more strongly upregulated in colon tissues of Slco2a-/- mice administered DSS and in macrophages isolated from Slco2a1-/- mice than in the WT counterparts. Slco2a1?MP, but not Slco2a1?IEC mice, were more susceptible to DSS-induced colitis than WT mice, partly phenocopying Slco2a-/- mice. Concentrations of PGE2 in colon tissues and macrophages from Slco2a1-/- mice were significantly higher than those of WT mice. Blockade of inflammasome activation suppressed the exacerbation of colitis. These results indicated that Slco2a1-deficiency increases the PGE2 concentration, resulting in NLRP3 inflammasome activation in macrophages, thus exacerbating intestinal inflammation.
Project description:Colorectal cancer (CRC) is one of the most common malignancies worldwide. Inflammation contributes to cancer development and inflammatory bowel disease is an important risk factor for CRC. The aim of this study is to assess whether a widely used probiotic Enterococcus faecalis can modulate the NLRP3 inflammasome and protect against colitis and colitis-associated CRC. We studied the effect of heat-killed cells of E. faecalis on NLRP3 inflammasome activation in THP-1-derived macrophages. Pretreatment of E. faecalis or NLRP3 siRNA can inhibit NLRP3 inflammasome activation in macrophages in response to fecal content or commensal microbes, P. mirabilis or E. coli, according to the reduction of caspase-1 activation and IL-1β maturation. Mechanistically, E. faecalis attenuates the phagocytosis that is required for the full activation of the NLRP3 inflammasome. In in vivo mouse experiments, E. faecalis can ameliorate the severity of intestinal inflammation and thereby protect mice from dextran sodium sulfate (DSS)-induced colitis and the formation of CRC in wild type mice. On the other hand, E. faecalis cannot prevent DSS-induced colitis in NLRP3 knockout mice. Our findings indicate that application of the inactivated probiotic, E. faecalis, may be a useful and safe strategy for attenuation of NLRP3-mediated colitis and inflammation-associated colon carcinogenesis.
Project description:Epimorphin (Epim), a member of the syntaxin family of membrane-bound, intracellular vesicle-docking proteins, is expressed in intestinal myofibroblasts and macrophages. We demonstrated previously that Epimorphin(-/-)(Epim(-/-)) mice are protected, in part, from dextran sodium sulfate (DSS)-induced colitis. Although interleukin (IL)-6/p-Stat3 signaling has been implicated in the pathogenesis of colitis, the myofibroblast contribution to IL-6 signaling in colitis remains unexplored. Our aim was to investigate the IL-6 pathway in Epim(-/-) mice in the DSS colitis model. Whole colonic tissue, epithelium, and stroma of WT and congenic Epim(-/-) mice treated with 5% DSS for 7 days were analyzed for IL-6 and a downstream effector, p-Stat3, by immunostaining and immunoblot. Colonic myofibroblast and peritoneal macrophage IL-6 secretion were evaluated by enzyme-linked immunosorbent assay. IL-6 and p-Stat3 expression were decreased in Epim(-/-) vs WT colon. A relative increase in stromal vs epithelial p-Stat3 expression was observed in WT mice but not in Epim(-/-) mice. Epim deletion abrogates IL-6 secretion from colonic myofibroblasts treated with IL-1? and decreases IL-6 secretion from peritoneal macrophages in a subset of DSS-treated mice. Epim deletion inhibits IL-6 secretion most profoundly from colonic myofibroblasts. Distribution of Stat3 activation is altered in DSS-treated Epim(-/-) mice. Our findings support the notion that myofibroblasts modulate IL-6/p-Stat3 signaling in DSS-treated Epim(-/-) mice.
Project description:Dysregulation of immune responses in the gut often associates with inflammatory bowel diseases (IBD). Mouse CD1d1, an ortholog of human CD1d mainly participating in lipid-antigen presentation to NKT cells, is able to generate intrinsic signals upon stimulation. Mice with macrophage-specific CD1d1 deficiency (LymCD1d1-/- ) acquire resistance to dextran sodium sulfate (DSS)-induced colitis, attributing to the transcriptional inhibition of NLRP3 inflammasome components. The hyperactivation of NLRP3 inflammasome accounts for gut epithelial proliferation and intestine-blood barrier integrity. Mechanistically, occupancy by the natural ligand glycosphingolipid iGb3, CD1d1 responds with intracellular Ser330 dephosphorylation thus to reduce the Peroxiredoxin 1 (PRDX1)-associated AKT-STAT1 phosphorylation and subsequent NF-?B activation, eventually causing transcriptional down-regulation of Nlrp3 and its immediate substrates Il1b and Il18 in macrophages. Therefore, the counterbalancing role of CD1d1 in macrophages appears to determine severity of DSS-mediated colitis in mice. These findings propose new intervention strategies for treating IBD and other inflammatory disorders.
Project description:Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. MuAstV is found predominantly in the gastrointestinal tract of mice. Human and turkey astroviruses have been shown to disrupt tight junctions in the intestinal epithelium. The potential of MuAstV to alter research results was tested in a well-established dextran sodium sulfate (DSS)-induced colitis model in Nod-like receptor 3 (NLRP3)-deficient mice. This model offers a direct approach to determine whether MuAstV, as a component of the mouse microbiome, contributes to the issue of poor reproducibility in murine inflammatory bowel disease research. In this model, defective inflammasome activation causes loss of epithelial integrity, resulting in leakage of intestinal bacteria and colitis. Our goal was to determine whether MuAstV, which also may affect intestinal permeability, altered the onset or severity of colitis. Male and female mice (age, 8 to 12 wk) homozygous or heterozygous for an NLRP3 mutation were inoculated orally with MuAstV or mock-inoculated with media 3 or 20 d prior to being exposed to 2% DSS in their drinking water for 9 d. MuAstV infection alone did not cause clinical signs or histopathologic changes in NLRP3-/- or NLRP3+/- mice. No significant difference was seen in weight loss, clinical disease, intestinal inflammation, edema, hyperplasia, or mucosal ulceration between MuAstV- infected and mock-infected mice that received 2% DSS for 9 d. Therefore, MuAstV does not appear to be a confounding variable in the DSS colitis model in NLRP3 mice.
Project description:Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD), which is a chronic, relapsing condition associated with the disorder of gut microbial communities. A previous study reported that levels of Roseburia intestinalis (R.I), a butyrate?producing bacterium, are significantly decreased in patients with IBD and exert an anti?inflammatory function in dextran sulfate sodium (DSS)?induced colitis. However, the role of R.I flagellin in UC and its underlying molecular mechanism are not yet fully understood. Therefore, a DSS?induced colitis model in C57Bl/6 mice and the LPS/ATP?induced THP?1 macrophages were treated with R.I flagellin, which were used to investigate the anti?inflammatory effects of R.I flagellin. The results demonstrated that R.I flagellin decreased colitis?associated disease activity index, colonic shortening and the pathological damage of the colon tissues in murine colitis models. Furthermore, R.I flagellin decreased the serum levels of proinflammatory cytokines and inhibited activation of the nucleotide?binding oligomerization segment?like receptor family 3 (NLRP3) inflammasome in murine colitis. R.I flagellin was also demonstrated to decrease the Gasdermin D to yield the N?terminal fragment membrane pore and inhibit inflammasome?triggered pyroptosis. In vitro analysis indicated that microRNA (miR)?223?3p was involved in the regulation of R.I flagellin on NLRP3 inflammasome activation. Taken together, the results of the present study demonstrated that R.I flagellin inhibited activation of the NLRP3 inflammasome and pyroptosis via miR?223?3p/NLRP3 signaling in macrophages, suggesting that R.I flagellin may be used as a novel probiotic product for the treatment of UC.
Project description:NLRP3 inflammasome is a novel therapeutic target for inflammatory bowel disease (IBD). The aim of this study was to investigate the anti-inflammatory effect of a bioactive flavonoid-oroxylin A on the treatment of dextran sulfate sodium (DSS)-induced murine colitis via targeting NLRP3 inflammasome. In this study, we found that oroxylin A attenuated experimental colitis in mice, including loss of body weights, shortening of the colon lengths and infiltration of inflammatory cells. The production of IL-1?, IL-6 and TNF-? in colon was also markedly reduced by oroxylin A. Moreover, oroxylin A significantly decreased the expression of NLRP3 in intestinal mucosal tissue. In addition, NLRP3-/- mice were observably protected from DSS-induced acute colitis, and oroxylin A treatment had no effects on attenuating inflammation in NLRP3-/- mice. Further study found that the activation of NLRP3 inflammasome was dose-dependently inhibited by oroxylin A in both THP-Ms and BMDMs, followed by decrease in the cleavage of caspase-1 and secretion of IL-1?. This inhibitory effect of oroxylin A was due to restraint of the NLRP3 protein expression and the inflammasome formation in macrophages. Furthermore, the reduction of NLRP3 protein expression by oroxylin A was dependent on the inhibition of NF-?B p65 expression and nuclear translocation. Besides, oroxylin A directly suppressed the ASC speck formation and the inflammasome assembly which in turn restrained the activation of NLRP3 inflammasome. Our findings demonstrated that oroxylin A inhibited NLRP3 inflammasome activation and could potentially be used for the treatment of IBD.
Project description:Niacin, as an antidyslipidemic drug, elicits a strong flushing response by release of prostaglandin (PG) D2 However, whether niacin is beneficial for inflammatory bowel disease (IBD) remains unclear. Here, we observed niacin administration-enhanced PGD2 production in colon tissues in dextran sulfate sodium (DSS)-challenged mice, and protected mice against DSS or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in D prostanoid receptor 1 (DP1)-dependent manner. Specific ablation of DP1 receptor in vascular endothelial cells, colonic epithelium, and myeloid cells augmented DSS/TNBS-induced colitis in mice through increasing vascular permeability, promoting apoptosis of epithelial cells, and stimulating pro-inflammatory cytokine secretion of macrophages, respectively. Niacin treatment improved vascular permeability, reduced apoptotic epithelial cells, promoted epithelial cell update, and suppressed pro-inflammatory gene expression of macrophages. Moreover, treatment with niacin-containing retention enema effectively promoted UC clinical remission and mucosal healing in patients with moderately active disease. Therefore, niacin displayed multiple beneficial effects on DSS/TNBS-induced colitis in mice by activation of PGD2/DP1 axis. The potential efficacy of niacin in management of IBD warrants further investigation.
Project description:The roles of Rev-erb? and circadian clock in colonic inflammation remain unclarified. Here we show colon clock genes (including Rev-erb?) are dysregulated in mice with DSS-induced colitis. In turn, disruption of the circadian clock exacerbates experimental colitis. Rev-erb?-deficient mice are more sensitive to DSS-induced colitis, supporting a critical role of Rev-erb? in disease development. Further, Rev-erb? ablation causes activation of Nlrp3 inflammasome in mice. Cell-based experiments reveal Rev-erb? inactivates Nlrp3 inflammasome mainly at the priming stage. Rev-erb? directly represses Nlrp3 transcription through specific binding to the promoter region. Additionally, Rev-erb? represses p65 transcription and indirectly repressed Nlrp3 via the NF-?B pathway. Interestingly, Rev-erb? activation in wild-type mice by SR9009 attenuates DSS-induced colitis, whereas the protective effects are lost in Nlrp3-/- and Rev-erb?-/- mice. Taken together, Rev-erb? regulates experimental colitis through its repressive action on the NF-?B/Nlrp3 axis. Targeting Rev-erb? may represent a promising approach for prevention and management of colitis.