Study On The Immunological Mechanism Of Hypoxia Affecting The Development Of Experimental Colitis In Mice

[Background]In recent years,gastrointestinal diseases caused by environmental factors have gradually attracted attention,especially in the remote areas at high altitude,the harm of gastrointestinal diseases and parasitic diseases is still quite serious.The most important feature of plateau environment is hypoxia.As the most basic pathological link of diseases,hypoxia can appear in the occurrence and development of a variety of diseases,causing a series of physiological changes.There are also many researches related to the effects of hypoxia exposure on respiratory and circulatory systems,but the effects of high altitude hypoxia on intestinal immune system has not yet received sufficient attention.Therefore,explore the immune mechanism related to intestinal health of the plateau population can provide health protection for the population entering the plateau,and also provide medical support for the economy and national defense of the Qinghai Tibet plateau.[Objective]Gastrointestinal diseases,such as diarrhea,are one of the infectious diseases that seriously endanger human health.Enteropathogenic e.coli(EPEC)and enterohemorrhagic e.coli(EHEC)are the main pathogens of bacterial diarrhea.Due to the immunosuppression caused by hypoxia in high altitude area,diarrhea caused by EPEC and EHEC often leads to serious fatal diseases.Therefore,it is of great significance for the prevention and treatment of high altitude infectious diseases to study the immunological regulation mechanism of hypoxia affecting the anti-infection ability.Based on the successful establishment of the C.rodentium infection model,the hypothetical hypobaric oxygen chamber was used to simulate the altitude of 5000 meter as the experimental condition.The purpose of our study was to explore the effect of hypoxia on intestinal mucosal immunity,and to further clarify the changes of differentiation subtypes and mature activation state of dendritic cells(DC)under hypoxia,revealing the immune response mechanism of hypoxia on the body's anti-infection.[Methods]1.To explore the effects of hypoxia on immune response to gastrointestinal pathogenic bacteria infection,the following research methods were adopt:firstly,we established a hypoxic colitis mouse model.32 female,8-week-old BALB/c mice(18-20 g)were randomly divided into normal group and hypoxia group,each group was divided into control group and C.rodentium infection group.Namely divided into four groups(n=8),control group(Control),C.rodentium infection group(C.rodentium),hypoxia group(Hypoxia)and hypoxia C.rodentium infection group(Hypoxia+C.rodentium).Mice in the C.rodentium infection groups were induced by C.rodentium(5×10⁸ CFU/mouse).Mice in normal group were fed in the IVC cages(2260 m,atmospheric pressure 582 mm Hg,PO₂ 121.6 mm Hg,equivalent to 16%O₂ at sea level,Xining,China).Mice in hypoxia group were fed in a hypobaric oxygen chamber,with simulated an altitude of 5000 meters(atmospheric pressure 405 mm Hg,PO₂ 84.7mm Hg,equivalent to 11%O₂ at sea level).During the modeling period,the body weight and fecal excretion of mice in each group were monitored every other day.The pulmonary artery pressure(PAP)and heart rate(HR)of mice in the Normal and Hypoxia group were measured and compared,and the changes of blood biochemical parameters,including hemoglobin content(Hb),Hematocrit(Hct)value and red blood cell count(Rbc)were analyzed.Colonic H.E.staining was used to observe the morphological changes of colonic inflammation,and the expression of colonic antibacterial peptides and inflammatory factors was detected by q RT-PCR.Enzyme-linked immunosorbent assay(Elisa)and flow cytometry(FACS)were used to detect the type of CD4⁺T cells and the expression of related cytokines in mesenteric lymph nodes(MLN)and spleen,and to detect serum-related immunoglobulin levels.Meanwhile,q RT-PCR was used to detect the expression of HIF-1?,IL-12,IL-23p19and Th cells differentiation upstream proteins(T-bet,ROR?t)in colon tissues.In addition,in order to determine the changes in the mucosal barrier in C.rodentium colitis,q RT-PCR was used to detect the tight junction proteins(Occludin,ZO-1),MUC2 and i NOS in colon tissue.2.To investigate the effects of hypoxia on the phenotype and function of mucosal antigen-presenting cells,the following research methods were adopt:experimental animals,groups,model establishment and treatment are consistent with the first part.The MLN and Peyer's patches of mice were collected after C.rodentium infection 2weeks.FACS was used to detect the expression of different differentiation subtypes of dendritic cells(DC),including myeloid DC(MDC,CD11c⁺CD11b⁺CD8?^-)and lymphoid DC(LDC,CD11c⁺CD11b^-CD8?⁺).The levels of IL-12 and IFN-?were detected.Furthermore,the surface maturation marker factors of CD11c⁺DC(MHC-II,CD80 and CD86)were detected by FACS to analyze the effects of hypoxia on the maturation and activation status of DC.[Results]After 2 weeks of hypoxia treatment,PAP,Hct and Hb in Hypoxia group were significantly higher than those in Normal group.The mice in Hypoxia+C.rodentium group were lost more weight than those in C.rodentium group,and the fecal bacteria excretion was increased.Pathological examination in Hypoxia+C.rodentium group showed severe inflammatory changes,also accompanied by a significant increase in pathological score.Compared with C.rodentium group,the antibacterial peptides Reg3?,IL-17 and IL-22 were decreased,and accompanied by the inflammatory factors such as TNF-?,IL-6 and COX-2 were increased in Hypoxia+C.rodentium group.Th response-related cytokines showed that the levels of IFN-?,IL-17 and Ig G2a were increased in MLN and Spleen in the C.rodentium group,while were significantly reduced in the Hypoxia+C.rodentium group,indicating that the Th1 and Th17responses were partially down regulated under hypoxia.Meanwhile,compared with C.rodentium group,IL-12 and IL-23p19 were significantly reduced,and the expressions of T-bet and ROR?t were decreased in Hypoxia+C.rodentium group.In addition,the TLR4,NF-?B,TNF-?,IL-6 and COX-2 were significant increase in Hypoxia+C.rodentium group.Mucosal barrier-related factors showed that the Occludin,ZO-1 and MUC2 in Hypoxia+C.rodentium group were lower than that of the C.rodentium group,while with an increase in i NOS.The state and phenotype of dendritic cells under hypoxia were detected,results showed that,compared with the C.rodentium group,the level of LDC(CD11c⁺CD11b^-CD8?⁺)was decreased in Hypoxia+C.rodentium group,and with the levels of IL-12 and IFN-?were decreased.Further,FACS analysis of CD11c⁺DC surface marker maturation factor showed that,compared with the C.rodentium group,the expression of MHC-II and CD86 on the surface of DC was significantly decreased in Hypoxia+C.rodentium group.[Conclusions]In conclusion,we established a mouse model of C.rodentium colitis under hypoxia to study the immunological mechanism of the effect of hypoxia on the occurrence and development of colitis.The results showed that Th1 and Th17immune responses were significantly down-regulated under hypoxia,accompanied by the destruction of intestinal mucosal barrier under hypoxia,which ultimately aggravated the hypoxic C.rodentium colitis.Further study on the phenotype and function of dendritic cells showed that hypoxia affects the maturation and differentiation of dendritic cells,and further affects the proliferation and differentiation of CD4⁺T cells,and ultimately affects the function of T cells in intestinal immune response.