The Function And Mechanism Of Long-chain Fatty Acid Receptor FFAR4 In Immune Colitis

Dietary fatty acids regulate several physiological functions: they are used as energy sources and constitute biological membranes.In addition,they act as signaling molecules by binding with their receptors,which are located on the surface of the cell membrane.Epidemiologic studies have implicated dietary fatty acid intake in the etiopathogenesis of Inflammatory bowel disease(IBD),and excessive or imbalanced intake of fatty acids increases the risk of IBD.FFAR4 is the receptor of the most common dietary fatty acids,mediating the roles of fatty acids in regulating metabolism and immune homeostasis.However,whether FFAR4 is involved in the occurrence and development of IBD has yet to be defined.Therefore,our study explored the role of FFAR4 in IBD and investigated the possible mechanism of its action.The major findings of this study are the following:First,we constructed mice with deletion of FFAR4 and established dextran sulfate sodium(DSS)-or dinitrobenzenesulfonic acid(DNBS)-or oxazolone-induced immune colitis in mice.Pathological indicators of experimental colitis were used as references,and we confirmed the effect of FFAR4 deficiency on colitis.The results showed that compared with wild-type mice,FFAR4-deficient mice had lower body loss and lower disease activity index(DAI)scores.Additionally,systemic FFAR4 deficiency significantly improved colitis parameters such as colon length,spleen weight,colonic inflammation and pathological score.This finding demonstrates that FFAR4 deficiency alleviates colitis in mice.Furthermore,we constructed intestinal FFAR4-deficient and FFAR4-overexpressing mice by using the Cre-Lox P system and modelled the mice with DSS.These results indicate that intestinal FFAR4 deficiency relieves colitis and that intestinal FFAR4 overexpression aggravates colitis.Second,we used a transcriptomic method to detect and analyse colonic gene expression changes and biological function changes in intestinal FFAR4-deficient mice.The results of biological function analysis showed that the item “Negative regulation of adaptive immune response” was enriched among the upregulated colonic genes in the intestinal FFAR4-deficient mice,and this result suggests that the colonic immune response is suppressed in the DSS-induced intestinal FFAR4-deficient mice.Subsequent Treg cell detection was performed using flow cytometry,and the results showed that compared with those in the control genotype mice(the Treg cell proportions in the spleen,mesenteric lymph nodes and colon were 11.5±0.5%,16.3±0.4% and 2.5±0.3%,respectively),Treg cells were significantly increased in the intestinal FFAR4-deficient mice(the Treg cell proportions in the spleen,mesenteric lymph nodes and colon were 15±0.5%,22±0.9% and 5.7±0.3%,respectively).In addition,Treg cell deletion aggravated colitis in intestinal FFAR4-deficient mice.In addition,the severity of colitis was unchanged in the control genotype mice.These results demonstrate that Treg cells are a key contributor to colitis remission.Furthermore,we investigated the mechanism by which intestinal FFAR4 deficiency relieves colitis via Treg cells from an IL33 perspective.The results showed that colonic IL33 m RNA was significantly increased in intestinal FFAR4-deficient mice(37-fold increase).Silencing colonic IL33 aggravated colitis in intestinal FFAR4-deficient mice.In addition,flow cytometry results showed that compared with those in the control mice(the Treg cell proportions in the spleen,mesenteric lymph nodes and colon were 12.9±0.5%,23.2±0.5%and 5.4±0.4%,respectively),silencing colonic IL33 significantly decreased the Treg cell proportions in the intestinal FFAR4-deficient mice(the Treg cell proportions in the spleen,mesenteric lymph nodes and colon were 10±0.7%,16.9±0.6% and 2.3±0.3%,respectively).These results demonstrate that intestinal FFAR4 deficiency relieves colitis by upregulating IL33,which in turn stimulates Treg cell proliferation.Finally,we investigated how FFAR4 regulates IL33 transcription by using the transcriptomic method and the dual luciferase reporter assay.The results showed that compared with those in the control cells(HT29-sh Concrol cells),ZBED6(a transcription factor)showed a marked upregulation in the FFAR4 knockdown cells(HT29-sh FFAR4 cells).Silencing FFAR4 resulted in a significant decrease in IL33 m RNA levels in both HT29-sh Concrol cells and HT29-sh FFAR4 cells.In addition,the results of the dual luciferase reporter assay showed that ZBED6 led to transcriptional induction of IL33(the relative fluorescence intensity displayed an upregulation of 5.8-fold).This result further indicates that ZBED6 is the transcription factor of IL33.In conclusion,our study found that systemic FFAR4 deficiency relieves colitis and revealed that intestinal epithelial FFAR4 deficiency is the key contributor to colitis remission.Furthermore,we identified intestinal epithelial cell deletion of FFAR4 by upregulating ZBED6,which in turn induces IL33 transcription,and IL33 elevates the number of Tregs,which ameliorates colitis.To a certain degree,this study furthers our understanding of the mechanism of the FFAR4 and dietary fatty acids in IBD,and provides a theoretical basis for the diagnosis and treatment and personalized nutritional interventions of IBD.