Therapeutic Effects Of IEC-derived HIF-1? In Mouse Colitis

Background:Inflammatory bowel disease?IBD?,mainly including crohn's disease and ulcerative colitis,is a kind of special intestinal inflammatory diseases often damaging the ileum,rectum and colon.Patients with IBD often present with diarrhea,abdominal pain,and perianal bleeding.The pathogenesis of IBD is not yet clear,but it has been confirmed that the IBD affected bowel area is chronically hypoxic due to vascular damage and increased metabolic demands.Therefore,there is increasing evidence that hypoxia-inducible factor-1?HIF-1?may be a key regulator of intestinal inflammation.HIF-1 is a transcription factor that plays an important role in hypoxia response and consists of two subunits,HIF-1?and HIF-1?.As a gene regulatory protein,HIF-1?can promote the expression of various related genes and mediate various anoxia-related adaptive responses,such as weakening intestinal inflammation,protecting mucosal barrier,hypoxic tissue angiogenesis,erythropoiesis,promoting glycolysis,inducing or inhibiting apoptosis and so on.Previous studies have reported that HIF-1?helps maintain intestinal homeostasis and defend against intestinal pathogens,protects intestinal mucosal cells from ischemia/reperfusion injury after mitochondrial injury,and is central to maintaining tight junctions between intestinal epithelial cells.However,the specific molecular mechanisms by which HIF-1?regulates intestinal inflammation remain unclear.Recently,there is increasing evidence that HIF-1?plays an important role in the function and development of immune cells.Studies have reported that intestinal dendritic cell-derived HIF-1?can affect the number and function of regulatory T cells,and the development of regulatory T cells also depends on the internal expression of HIF-1?.Intestinal immune homeostasis depends on the close regulation between symbiotic bacteria,mucosal immune cells and intestinal epithelial cells,while the destruction of intestinal homeostasis will lead to intestinal dysfunction and pathological changes,such as IBD.It has been demonstrated that intestinal epithelial cells?IEC?-derived cytokines are involved in the regulation of innate and acquired intestinal immunity.It has been reported that epithelial-derived IL-7,IL-15,and IL-18 can affect the formation of intestinal intraepithelial lymphocytes?IELs?subgroups,and Thymic stromal lymphopoietin?TSLP?and Transforming growth factor?Transforming growth factor-?,TGF-??secreted by IEC can regulate the functions of dendritic cells and mast cells.However,it is not clear how IEC-derived HIF-1?affects peripheral immune cells.So this study will adopt qRT-PCR,cell flow analysis technology,through the establishment of dextran sodium sulfate?DSS?induced experimental colitis,to explore the role of IEC-derived HIF-1?in IBD,as well as the influence on the surrounding lymphocytes,in terms of cell number and function,which will partly expounds IEC-related molecular mechanisms involved in the regulation of lymphocytes,and provide new theoretical basis for the maintenance of intestinal immune steady-state.Methods:1.The mouse model of ulcerative colitis was established by dextran sulfate sodium treatment for 7 days.The expression of intestinal HIF-1?protein in physiological condition and the changes induced by DSS were detected by immunofluorescence technology.2.Experimental colitis models were established by using mice with specific knockout of HIF-1?in IEC(HIF-1?^IEC)and its sibling HIF-1?⁺f/+ff/+f mice.The weight,excrement and the presence of blood stools of mice were observed daily.The change of colon length and disease activity index?DAI?were measured after 7 days.HE staining was used to observe the severity of colon inflammation injury,qRT-PCR was adopted to detect the expression changes of cytokines associated with inflammation;3.After the mice were sacrificed,the small intestine and colon were separated,and IEL and lamina propria lymphocytes?LPLs?were extracted.The subsets and surface antigen of IELs were examined by flow cytometry.Also,the percentage of Regulatory T cells?Tregs?in lamina propria was detected.Results:1.Immunofluorescence results showed that HIF-1?was expressed in small amounts in the intestinal tract under normal conditions,and the expression of HIF-1?was significantly up-regulated after the establishment of an experimental ulcerative colitis model with DSS.2.Observation showed that DSS treatment resulted in significant weight loss in mice,especially on the sixth and seventh day.The colon length was significantly shortened.Meanwhile,the weight loss,colon shortening and disease activity index increasing were more significantly obvious in the HIF-1?^IEC+DSS group than in the HIF-1?⁺f/+ff/+f group.Also,HE staining showed that the colon structure was significantly damaged under the condition of colitis,and HIF-1?^+f/+f group was more serious;qRT-PCR results showed that the mRNA expressions of IL-6,Lipocalin2 and F4/80 were obviously up-regulated under colitis,which was more obvious in knockout mice.3.Flow cytometry results showed that:under normal conditions,there was no significant difference in the proportion of Tregs in the lamina propria between the HIF-1?⁺f/+ff/+f group and the HIF-1?^IECEC group;After administration of DSS,the proportion of Tregs cells in lamina propria was significantly up-regulated in CD3⁺cells,but the increasing of Tregs in HIF-1?^IEC+DSS group was blocked.Meanwhile,qRT-PCR showed that the expression of IL-10 and Aldh1a3 increased after DSS exposure,and the Aldh1a3 was higher in control group.4.Flow cytometry results showed that compared with the control group,the IEL subsets changed in HIF-1?^IEC mice with higher rates of CD8??⁺cells and lower rates of CD8??⁺,TCR??⁺cells.In addition,BrdU-positive CD8??⁺cells decreased,suggesting a low proliferation rate;there is a higher apoptosis rate of CD8??⁺and CD8??⁺cells;The proportion of CD103 positive CD8??⁺cells decreased,suggesting lower homing and attachment ability.Under DSS conditions,CD8??⁺and TCR??⁺cells also decreased slightly.Conclusions:1.Under physiological conditions,HIF-1?was slightly expressed in the intestinal tract.After DSS treatment,HIF-1?was activated and the expression was significantly up-regulated,indicating that intestinal hypoxia was aggravated.2.HIF-1?specific knockout in IECs can significantly aggravate colon shortening,intestinal mucosal structure destruction,inflammatory factor secretion and inflammatory cell infiltration caused by DSS,suggesting that IEC-derived HIF-1?plays a protective role in DSS-induced colitis models,and HIF-1?may become a new target for intervention in the pathological progression of inflammatory bowel disease.3.HIF-1?specific knockout in IECs inhibits the DSS-induced up-regulation of Tregs cells in the lamina propria,and Tregs play a protective role in IBD,suggesting that HIF-1?may play an indirectly protecting role through Tregs.Meanwhile,the expression of Aldh1a3in IEC was affected by HIF-1?,which implied that HIF-1?may regulate the induction of Tregs through RA.4.That HIF-1?specific knockout in IECs can affect the subsets,multiplication capacity,apoptosis ability,and surface functional antigen of IELs which play an important role in the maintaining stability of intestinal barrier suggests that IEC-derived HIF-1?may relieve DSS-induced inflammatory injury and barrier damage through regulation of neighboring lymphocytes.