| Background:Inflammatory bowel disease(IBD)mainly including Crohn’s disease(CD)and Ulcerative colitis(UC).is a chronic,non-specific intestinal inflammatory disease.IBD patients have many clinical symptoms such as abdominal pain,diarrhea,rectal bleeding,malaise,and weight loss.Therefore,the life quality of IBD patients is severely affected.Among CD and UC,UC is one of the most common causes of colorectal cancer(CR).The occurrence of IBD is related to the dysfunction of the body’s immune system,the intestinal microenvironment such as the intestinal flora,and the body’s hereditary factors,Nevertheless,the mechanisms of IBD is still not fully-understood..The circadian rhythm is important for maintaining most of the physiological functions.The biological clock oscillates continuously in a 24-hour period,and it also keeps timing through external environmental stimuli(such as light,temperature and food intake).The maintenance of mammalian biological rhythms mainly depends on a set of transcription-translation feedback loops,which are driven by specific circadian clock proteins(Bmall,Clock,Per1,Per2,Per3.Cry).The main clock that regulates the circadian rhythm is located in the central system of the suprachiasmatic nucleus of the hypothalamus.In addition,there are many sub-clocks located in peripheral organs,such as the intestine.The Intestinal rhythm disorder destroys intestinal homeostasis,depending on the complex interactions between the microbiota,intestinal epithelial cells and the host immune system.Once the balance of these interactions is broken,it will promote the occurrence of the IBD.However,the role and regulation mechanism of circadian rhythm in IBD is still unclear.Aims:(1)To uncover the relationship between the circadian clock rhythm and IBD.(2)The circadian clock gene Bmall knockout(Bmall-/-)mouse model was used to identify the subpopulations of intestinal intraepithelial lymphocytes especially intestinal intraepithelial B cells mainly regulated by the biological rhythm.(3)The circadian clock gene Bmall knockout(Bmall-/-)mouse model was used to study the role of Bmall in regulation of intestinal intraepithelial B lymphocytes in colitis and colitis associated colorectal cancer(CR).(4)To study the molecular mechanism and clinical significance of this kind of intestinal intraepithelial B lymphocytes regulated by Bmall in colitis and colitis associated colorectal cancer(CR).Methods:Three kinds of mouse models for biological rhythm disorders,including chronic social jet lag(Chronic Jet Lag,CJ),a mouse model with the core circadian clock gene Bmall deficiency(Bmall-/-),and a core circadian clock gene Perl and Per2 gene deficiency(Per1-/-Per2-/-)mouse models,were used to study the effects of circadian clock disorders on IBD.To mimic clinical ulcerative colitis,dextran sodium sulfate(DSS)were used to induce colitis mouse model.Colitis associated colorectal cancer(CAC)mouse model was induced by combination administration of DSS and azoxymethane(AOM).The colorectal length in mice,the weight change of the mice during the induction period,and the damage of the mouse intestinal epithelium,were evaluated.Flow cytometry analysis and cell sorting,in vivo cell adoptive experiment and other methods,were used to analyze the roles of subpopulations of the intestinal intraepithelial lymphocytes or other immune cells regulated by circadian clock in colitis and CAC.Flow cytometry sorting,western blotting,RT-PCR,luciferase reporter assay,chromatin immunoprecipitation assay,etc,were used to study the molecular mechanism of the circadian clock in regulating intestinal intraepithelial lymphocytes in colitis and CAC.Semi-quantitative analysis of immunohistochemistry was used to determine the expression and distribution of circadian clock gene-positive lymphocytes in various organs and tissues,and correlation analysis were used to determine the clinical significance of the intestinal intraepithelial lymphocyte groups regulated by circadian clock gene.Results:1.CJ mouse with light-induced rhythm disorders were susceptible to colitis.The expression of clock genes Bmall,Perl and Per2 in the intestinal intraepithelial lymphocytes of CJ mouse had abnormal rhythm fluctuations.Bmall is one of the key components of core clock genes,we further studied the role of biological rhythm regulated by clock genes in colitis using Bmall knockout(Bmall-/-)mice and found that Bmall-/-mouse was susceptible to colitis.2.A type of Breg cells expressing B220+CDld+CD5+ in intestinal epithelium of Bmal1-/-,Perl-/-Per2-/-and CJ mouse,was significantly lower than that in WT(Wild-Type)mice under physiological conditions.In the experimental colitis mouse model induced by DSS,the proportion of B220+CD1d+CD5+ cells in the intestinal epithelium of wild-type mouse exhibited rhythmic fluctuations,and was significantly lower than that in untreated WT mouse.However,the proportion of B220+CD1d+CD5+cells in the intestinal epithelium of Bmall-/-mouse lost rhythmic fluctuations and did not affected after DSS treatment.To further identify the role of B220+CDld+CD5+cells regulated by the clock gene Bmall in colitis,we used an adoptive cell transfer strategy by transferring B cells isolated from Bmall-/-mice or WT mice to Bmall-/-mice or WT mice We found that adoptive cell transfer treatment using B cells isolated from WT mice successfully alleviated colitis in Bmall-/-mice while adoptive cell transfer treatment using B cells from Bmal1-/-mice greatly accelerates the progress of colitis in WT mice.Notably,regulatory B(Breg)cells highly expressing PD-L1 in intestinal intraepithelial lymphocytes(IEL)helped to alleviate the severity of colitis after DSS treatment and was dysregulated in DSS-treated Bmall-/-mice.3.To find the key factors involved in Bmall-regulated PD-L1+B220+CD1d+CD5+cells,we examined the messenger RNA(mRNA)expression of inflammatory factors including IL-4,IL-6.IL-10,IL-21,IL-33,transforming growth factor-β(TGF-β).and monocyte chemoattractant protein-1(MCP-1)in IEL from DSS-treated or-untreated Bmall knockout and WT mice.We found that IL33 in the intestinal microenvironment is key for Bmall-regulated PD-L1+B220+CD1d+CD5+ cells.In vitro experiments further confirmed that an increase of IL-33 mainly promoted the expression of PD-L1 on wild-type B cells,but had little effect on the expression of PD-L1 on Bmall-/-B cells.Furthermore,we found that Bmall protein could bind to the E-box site in the promoter region of IL-33,promoted the transcription of IL-33 and the expression of PD-L1.4.The number of CD4+ T cells in Bmall-/-mice was approximately half of that in WT mice,while the number of CD8+ T cells in Bmall-/-mice was approximately 82%of that in WT mice.The late apoptosis index of CD4+ T cells of IEL in Bmall-/-mice were significantly higher than that in WT mice.PD-L1+ B cells induce cell death of activated CD4+ T cells in DSS-treated Bmall-/-mice.5.CJ,Bmall-/-,Perl-/-Per2-/-mice,were susceptible to develop CAC.Circadian clock disorders characterized as decreased numbers of PD-L1+B220+CDld+CD5+ cells and dysfunction of CD4+T cells promotes colitis-associated CRC.The number of CD4+cells in Bmall-/-and Perl-/-Per2-/-mice with internal circadian rhythm disorder or CJ mice with external circadian rhythm disorder were significantly lower than that in WT mice.Among these CD4+ cells in mice with abnormal biological clocks,especially in Perl-/-Per2-/-mice,the number of immunosuppressive CD4+ cells,such as PD-1+CD4+cells,was higher than that in WT mice.The number of effective CD4+ cells such as IFN-γ+CD4+ and Granzyme B+CD4+ in mice with internal rhythm disorder were both significantly lower than those in WT mice..Conclusion:We identified the role of the biological clock,or biological clock genes regulated a class of PD-L1+B220+CDld+CD5+ cells in experimental IBD and IBD-associated CRC in mice.We found that circadian clock disorders led to high risks for IBD and IBD-associated CRC by decreasing the number of PD-L1+B220+CD1d+CD5+ cells and dysfunction of CD4+T cells.. |
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