High-fat diet (HFD) leads to systemic low-grade inflammation, which has been involved in the pathogenesis of diverse metabolic and inflammatory diseases. Colon is thought to be the first organ suffering from inflammation under HFD conditions due to the pro-inflammatory macrophages infiltration, however, the mechanisms concerning the induction of pro-inflammatory phenotype of colonic macrophages remains unclear. In this study, we show that HFD increased the percentage of gram-positive bacteria, especially genus Clostridium, and resulted in the significant increment of fecal deoxycholic acid (DCA), a gut microbial metabolite produced by bacteria mainly restricted to genus Clostridium. Notably, reducing gram-positive bacteria with vancomycin diminished fecal DCA and profoundly alleviated pro-inflammatory macrophage infiltration in colon, whereas DCA-supplemented feedings to vancomycin-treated mice provoked obvious pro-inflammatory macrophage infiltration and colonic inflammation. Meanwhile, intra-peritoneal administration of DCA also elicited considerable recruitment of macrophages with pro-inflammatory phenotype. Mechanistically, DCA dose-dependently promoted M1 macrophage polarization and pro-inflammatory cytokines production at least partially through toll-like receptor 2 (TLR2) transactivated by M2 muscarinic acetylcholine receptor (M2-mAchR)/Src pathway. In addition, M2-mAchR mediated increase of TLR2 transcription was mainly achieved via targeting AP-1 transcription factor. Moreover, NF-κB/ERK/JNK signalings downstream of TLR2 are involved in the DCA-induced macrophage polarization. In conclusion, our findings revealed that high level DCA induced by HFD may serve as an initiator to activate macrophages and drive colonic inflammation, thus offer a mechanistic basis that modulation of gut microbiota or intervening specific bile acid receptor signaling could be potential therapeutic approaches for HFD-related inflammatory diseases.

We recently have proved that excessive fecal DCA caused by high-fat diet may serve as an endogenous danger-associated molecular pattern to activate NLRP3 inflammasome and thus contributes to the development of inflammatory bowel disease (IBD). Moreover, the effect of DCA on inflammasome activation is mainly mediated through bile acid receptor sphingosine-1-phosphate receptor 2 (S1PR2); however, the intermediate process remains unclear. Here, we sought to explore the detailed molecular mechanism involved and examine the effect of S1PR2 blockage in a colitis mouse model. In this study, we found that DCA could dose dependently upregulate S1PR2 expression. Meanwhile, DCA-induced NLRP3 inflammasome activation is at least partially achieved through stimulating extracellular regulated protein kinases (ERK) signaling pathway downstream of S1PR2 followed by promoting of lysosomal cathepsin B release. DCA enema significantly aggravated DSS-induced colitis in mice and S1PR2 inhibitor as well as inflammasome inhibition by cathepsin B antagonist substantially reducing the mature IL-1β production and alleviated colonic inflammation superimposed by DCA. Therefore, our findings suggest that S1PR2/ERK1/2/cathepsin B signaling plays a critical role in triggering inflammasome activation by DCA and S1PR2 may represent a new potential therapeutic target for the management of intestinal inflammation in individuals on a high-fat diet.

Background: Dysfunctional p53 signaling is one of the major causes of hepatocellular carcinoma (HCC) tumorigenesis and development, but the mechanisms underlying p53 inactivation in HCC have not been fully clarified. The role of Krüppel-associated box (KRAB)-type zinc-finger protein ZNF498 in tumorigenesis and the underlying mechanisms are poorly understood. Methods: Clinical HCC samples were used to assess the association of ZNF498 expression with clinicopathological characteristics and patient outcomes. A mouse model in which HCC was induced by diethylnitrosamine (DEN) was used to explore the role of ZNF498 in HCC initiation and progression. ZNF498 overexpression and knockdown HCC cell lines were employed to examine the effects of ZNF498 on cellular proliferation, apoptosis, ferroptosis and tumor growth. Western blotting, immunoprecipitation, qPCR, luciferase assays and flow cytometry were also conducted to determine the underlying mechanisms related to ZNF498 function. Results: ZNF498 was found to be highly expressed in HCC, and increased ZNF498 expression was positively correlated with advanced pathological grade and poor survival in HCC patients. Furthermore, ZNF498 promoted DEN-induced hepatocarcinogenesis and progression in mice. Mechanistically, ZNF498 directly interacted with p53 and suppressed p53 transcriptional activation by inhibiting p53 Ser46 phosphorylation. ZNF498 competed with p53INP1 for p53 binding and suppressed PKCδ- and p53INP1-mediated p53 Ser46 phosphorylation. In addition, functional assays revealed that ZNF498 promoted liver cancer cell growth in vivo and in vitro in a p53-dependent manner. Moreover, ZNF498 inhibited p53-mediated apoptosis and ferroptosis by attenuating p53 Ser46 phosphorylation. Conclusions: Our results strongly suggest that ZNF498 suppresses apoptosis and ferroptosis by attenuating p53 Ser46 phosphorylation in hepatocellular carcinogenesis, revealing a novel ZNF498-PKCδ-p53INP1-p53 axis in HCC cells that would enrich the non-mutation p53-inactivating mechanisms in HCC.

Background: Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal disease in premature neonates with high mortality and morbidity, while the underlining mechanism of intestinal injury and profound neurological dysfunction remains unclear. Here, we aimed to investigate the involvement of NLPR3 inflammasome activation in NEC-related enterocolitis and neuroinflammation, especially long-term cognitive impairment, meanwhile, explore the protective effect of NLRP3 inhibitor MCC950 on NEC in mice. Methods: NLRP3 inflammasome activation in the intestine and brain was assessed in the NEC mouse model, and NLRP3 inhibitor MCC950 was administrated during the development of NEC. Survival rate, histopathological injury of the intestine and brain, and expression of mature IL-1β and other pro-inflammatory cytokines were analyzed. Long-term cognitive impairment was evaluated by behavioral test. Results: The expression of NLRP3 and mature IL-1β in the intestine and brain was greatly upregulated in NEC mice compared to the controls. MCC950 treatment efficiently improved NEC survival rate, reduced intestinal and brain inflammation, and ameliorated the severity of pathological damage in both organs. Additionally, in vivo blockage of NLRP3 inflammasome with MCC950 in early life of NEC pups potently protected against NEC-associated long-term cognitive impairment. Conclusions: Our findings suggest that NLRP3 inflammasome activation participates in NEC-induced intestinal and brain injury, and early intervention with NLRP3 inhibitor may provide beneficial therapeutic effect on NEC infants.

Background: High-fat diet (HFD) is closely associated with the increased prevalence of inflammatory bowel disease (IBD). Excessive gut microbial metabolite deoxycholic acid (DCA) caused by HFD plays significant roles in eliciting intestinal inflammation, however, the mechanism underlining the induction of inflammatory response by DCA has not been fully elucidated. The purpose of this study was to investigate the role of DCA in the triggering of inflammation via affecting CD4⁺ T cell differentiation. Results: Murine CD4⁺T cells were cultured under Th1, Th2 or Th17-polarizing conditions treated with or without different dosage of DCA, and flowcytometry was conducted to detect the effect of DCA on CD4⁺ T cell differentiation. Alteration of gene expression in CD4⁺ T cells upon DCA treatment was determined by RNA-sequencing and qRT-PCR. Bioinformatic analysis, cholesterol metabolic profiling, ChIP assay and immuno-fluorescent staining were further applied to explore the DCA-regulated pathway that involved in CD4⁺T cell differentiation. The results showed that DCA could dose-dependently promote the differentiation of CD4⁺ T cell into Th17 linage with pathogenic signature. Mechanistically, DCA stimulated the expression of cholesterol biosynthetic enzymes CYP51 and led to the increased generation of endogenous RORγt agonists, including zymosterol and desmosterol, therefore facilitating Th17 differentiation. Up-regulation of CYP51 by DCA was largely mediated via targeting transcription factor SREBP2 and at least partially through bile acid receptor TGR5. In addition, DCA-supplemented diet significantly increased intestinal Th17 cell infiltration and exacerbated TNBS-induced colitis. Administration of cholestyramine to eliminate fecal bile acid obviously alleviated colonic inflammation accompanied by decreased Th17 cells in HFD-fed mice. Conclusions: Our data establish a link between DCA-induced cholesterol biosynthesis in immune cells and gut inflammation. Modulation of bile acid level or targeting cholesterol metabolic pathway may be potential therapeutic measurements for HFD-related colitis.