Aryl Hydrocarbon Receptor Activation Involved In Ameliorating Inflammatory Bowel Disease And The Regulation Of Intestinal Epithelial Derived IL-7

BackgroundInflammatory bowel disease(IBD) is a kind of chronic intestinal inflammatory diseases including crohn’s disease(CD) and ulcerative colitis(UC). The main symptoms of IBD are abdominal pain, diarrhea and bloody stool. IBD was common diseases in developed countries, but as the growth of economic and cultural level, the number of cases of IBD is increasing in China.Although the precise etiology of colitis is only partly understood, it features the dysregulation of tightly regulated intestinal immune system, in which chronic, unsolved, robust but aseptic inflammation is continually activated. For better understanding of pathology of colitis in humans, scientists developed a lot of experimental models of colitis. Over two decades,dextran sulfate sodium(DSS) induced colitis has been proved to be one of the most commonly used models. DSS-induced colitis is simple to induce and it has the same clinical features(such as diarrhea, bloody stools, weight loss and morphological features) with human. Furthermore, mice data come from DSS-induced colitis can be translated to human disease.6-formylindolo(3,2-b) carbazole(Ficz) is one of ligands of aryl hydrocarbon receptor(AHR), which is known as a dioxin receptor and a member of the basic helix-loop-helix /Per-ARNT-Sim(PAS) homology superfamily. AHR is present in the cytosol in an inactive form. After binding with its ligands, AHR migrates in the nucleus and binds AHR nuclear translocator(ARNT) to initiate the transcription of a variety of genes. Evidences have suggested that AHR is involved in inflammation inhibition. TCDD which is the best known ligand of AHR has been proved to increase probability to infection and suppresses both cellular and humoral immune responses. More recent studies have shown that Ficz induced signals ug-regulate IL-22 production and inhibit colitis in the gastrointestinal tract of mice. Besides, studies proved that AHR plays a crucial role in maintaining IELs.Interleukin-7(IL-7) plays a critical role in T and B cell development. A dramatic reduction in cellularity of the thymus and arrested B cell development of the bone marrow was observed in IL-7-deficient mice. Recently, it is proved that intestinal epithelial derived IL-7 can lead to the development of chronic intestinal inflammation by activing the mucosal immune responses. Previous studies have also found that epithelial derived IL-7 is an important cytokine involved in alteration of intestinal IEL phenotype.IEL are one of the main immunological compartments involved in regulating intestinal mucosal immune responses. IELs maintain epithelial barrier function and initiate the early response to infection, but also regulate the activation of immune cells. The majority of IELs are characterized “partial activation” state, suggesting that the activated part of IELs is capable of rapidly responding to stimuli, and therefore, IELs must be held in tight check to prevent unwanted reactions.Since AHR is involved in inflammation inhibition, whereas IL-7 is proved to active the mucosal immune responses that lead to the development of chronic intestinal inflammation and both play an important role in IEL, we investigated the effect of Ficz, the ligand of AHR, on the expression of epithelial derived IL-7, the phenotype of IEL and the following inflammation change.Methods1、Established DSS-induced colitis. Sacrificed the mice and collected colon tissues. RNA analysis, Western blot, immunofluorescence were to detect the expression of AHR and IL-7. Flow cytometry technique was to detect colon IEL phenotype.2、LoVo cells was purchased. The expression of AHR and IL-7 was detected by immunofluorescence and RT-PCR after administration of Ficz; si-RNA was used to interference AHR, then same methods were used to detect the expression of AHR and IL-7.3、AHR agonist Ficz was used. Mice were killed and colon tissues were gathered. The expression of AHR and IL-7, the phenotype of IELs were detected again. The inflammation of DSS-induced colitis was assessed by the length of colons, H&E staining, weight of mice and expression of common inflammatory factor.Results1. AHR expression was significantly decreased while IL- 7 expression was significantly increased in mice with DSS-induced colitis compared with the wild-type mice. After administration of Ficz for 5 days, mice with DSS-induced colitis showed more AHR expression but less IL-7 expression. Protein and mRNA levels of sham, DSS, DSS + Ficz group were quantified, results were the same as IF.(*P<0.05 compared with sham group, n = 5 per group).2.Ficz- treated LoVo cells presented more expression of AHR, but less expression of IL-7. But after AHR was silented by si-RNA,Ficz-treated LoVo cells can not presented less expression of IL-7 compared with normal LoVo cells.3. The wild-type mice were gaining weight but DSS-treated mice showed weight loss from the 3~4 days of DSS treatment. Administration of ficz greatly decelerated the weight loss(Data indicate the cumulative mean weight data ± SD of 5 experiments.*p<0.05). The average colon lengths of the control, DSS-treated, Ficz+DSS-treated group were 8cm, 5cm, 6cm(P<0.05, n=5). H&E staining of sections were from the middle colon of the 3 groups of mice, Histologic examination of colonic tissues showed that more injury was observed in DSS group than Ficz+DSS group, while none in sham group.4. Data were obtained from CD45-positive cells. Increased percentage of CD8αβ+IEL subpopulations was found in DSS-treated mice compared with wild-type mice, but Ficz decreased it in Ficz+DSS-treated mice. The percentage of CD8+ and CD4+IEL subpopulations were increased in mice with DSS-induced colitis compared with wild-type mice, but they were found decreased in Ficz+DSS-treated mice. Different from CD8αβ+, CD4+, CD8+IEL subpopulations, TCRγδ+IEL subpopulation was decreased in mice with DSS-induced colitis. However, administration of Ficz led to increased percentage of TCRγδ+IEL subpopulation.(4~5 mice per group, every experiment was repeated 3 times. P<0.05)5. Data were gated on CD8 and CD4 positive cells. Figures showed that compared with wild-type mice, mice with DSS-induced colitis had a much higher level of IEL activation, as measured using CD69 positive cells. After administration of Ficz, decreased percentage of CD69+IEL subpopulation was observed.(4~5 mice per group, every experiment was repeated 2. AHR activation by Ficz ameliorates DSS-induced colitis. 3. AHR activation by Ficz alters intraepithelial lymphocyte phenotype in mice colon. 4. Ficz reduced the percentage of activated CD4+ and CD8+IEL subpopulations. 5. AHR and Ficz may have a possible benefit for the therapy of IBD.