Expression And Role Of PD-L1 In Intestinal Mucosa In Patients With Inflammatory Bowel Disease And Mice With Colitis

Background:Inflammatory bowel disease(IBD)is a group of chronic disease that causes gastrointestinal inflammation with unclear etiology and pathogenesis,including ulcerative colitis(UC)and Crohn’s disease(CD).In recent years,the incidence rate in western countries has gradually stabilized,but the number of IBD patients worldwide is still increasing,and the incidence in Asia and Africa is rising rapidly,so IBD has become a worldwide public health problem.Its pathogenesis is generally considered to be related to environmental factors,genetic background,dietary habits,intestinal flora,immune regulation,and so on,among which immune regulation is a crucial part.Programmed cell death-ligand 1(PD-L1)inhibits the proliferation of T lymphocytes and the production of some cytokines after binding with PD-1,resulting in immunologic tolerance and preventing tissue damage.As an immune checkpoint,the overexpression of PD-1/PD-L1 may cause immune escape,which may eventually lead to the incidence of different kinds of tumors.Therefore,antagonizing the PD-1/PD-L1 pathway can inhibit tumors and anti-PD-L1/PD-1 drugs are called immune checkpoint inhibitors(ICIs).With the use of ICIs,the incidence of immune-related adverse effects(Ir AEs)is increasing year by year,in which the immune-mediated colitis(IMC)occurs most.IMC is highly similar to IBD(especially UC)in clinical manifestations,pathological manifestations and endoscopic manifestations.Therefore,we speculate that there is a partial relationship between PD-1/PD-L1 pathway and the pathogenesis of IBD.In fact,some researchers believe that PD-1/PD-L1-mediated immune tolerance can guide the treatment of autoimmune diseases.Based on the above speculation,some researchers tried to use PD-L1 to treat animal colitis,but there were some disputes in different experiments.Therefore,it is necessary to clarify the therapeutic effect of PD-L1 in animal colitis firstly.So we used dextran sulfate sodium salt(DSS)to induce colitis of mice in our preliminary experiment,which is a most-common colitis modeling drug that can simulate IBD-similar symptoms like diarrhea,bloody stool and weight loss.It can be used for research on human inflammatory mechanisms,immune mechanisms,and genetics,as well as for the efficacy of anti-inflammatory drugs.According to the previous literature,protein drugs have a short half-life in plasma,however,the fusion of functional protein with Fc segment can prolong the half-life of drug in plasma and reduce the immunogenicity.Therefore,we chose PD-L1-Fc fusion protein to prolong half-life,improve molecular stability,and improve expression and detection rate.However,systemic administration may lead to the combination of PD-L1 and PD-1 in multiple parts of the body,resulting in immunosuppression and increasing the risk of infection and even tumors.Therefore,we constructed an excellent reactive oxygen species(ROS)-responsive nanomaterial to increase the targeted administration of targeted inflammatory sites,hoping to improve the efficacy and reduce side effects.In this project,we intend to clarify the role and mechanism of PD-L1 in the pathogenesis and treatment of patients with IBD and dextran sulfate sodium salt(DSS)-mediated colitis mice.Objective:1.To clarify the expression and significance of PD-L1 and related inflammatory factors in colonic mucosa of patients with IBD.2.To investigate the therapeutic effect and mechanism of PD-L1-Fc protein on DSS colitis of mice.3.To determine the therapeutic effect and side effects of PD-L1-Fc/Oxi-αCD NPs on DSS-induced colitis of mice.Methods:1.Colonic mucosa biopsy specimens were collected from IBD patients and healthy volunteers,immunohistochemistry and RT-q PCR were used to detect the expression of PD-L1 in the human colon mucosa and the expression location.Patients with UC and CD were scored on their disease activity and the correlation between PD-L1 expression and activity was assessed,and the expression levels of inflammatory factors and their correlation with PD-L1 expression were detected by RT-q PCR.2.PD-L1-Fc was injected intraperitoneally into DSS-induced colitis mice,disease activity index(DAI),colon length and histopathological score were used to evaluate the disease condition.Colonoscopy was used to observe the morphological changes of colonic mucosa in vivo and score them.The expression levels of inflammatory factors in colonic tissues of mice were detected by RT-q PCR and western blot,and the molecular changes of intestinal barrier were detected by immunofluorescence.3.A reactive oxygen species(ROS)responsive nanomaterial with good biocompatibility and biosafety: the 4-phenylboronic acid pinacol ester conjugate cyclodextrin biomaterial(Oxi-alpha CD)was constructed for load PD-L1-Fc(named as PD-L1-Fc/Oxi-alpha CD NP),the PD-L1-Fc/Oxi-αCD NP was injected intraperitoneally to intervene in DSS-induced colitis mice,the release effect of the fixed-point intestinal inflammation part was determined by in vivo imaging.The same methods were used to evaluate the disease as before and an immunosuppressive model was constructed to verify its toxic side effects.Results:1.The expression of PD-L1 in colonic mucosa of UC and CD patients was significantly higher than that of the control group,and it was mainly in the lamina propria of colonic mucosa.The pro-inflammatory and anti-inflammatory cytokines in colonic mucosa of UC patients were increased,and the increase of TGF-β1 was positively correlated with PD-L1.There is no relationship between the level of PD-L1 expression and the activity of disease at present.2.The expression of PD-L1 in colonic mucosa of DSS colitis mice was higher than that of the control mice,which is similar to IBD patients.Intraperitoneal injection of PD-L1-Fc could reduce the weight loss rate and disease activity score of colitis mice,improve colonic pathological score and colonoscopy score,promote the production of anti-inflammatory factors and inhibit the production of proinflammatory factors,reduce the loss of ZO-1,E-cadherin and MUC-2,treat DSS-induced acute colitis in mice and reduce inflammatory reaction.However,systemic administration may lead to immunosuppression and risk of infection and tumors.3.PD-L1-Fc/Oxi-αCD NP can control the accumulation and release of PD-L1-Fc at the inflammatory colonic sites,reduce the body weight loss rate and disease activity score of colitis mice,alleviate histological colon injury,improve epithelial integrity,and achieve better therapeutic effect on colitis than PD-L1-Fc.In addition,in the immunosuppressive model of Bacillus aeruginosa infection,PD-L1-Fc/Oxi-αCD NP group mice had less alveolar hemorrhage and edema than PD-L1-Fc group mice,and its specific mechanism needs to be further explored.Conclusion:1.The expression of PD-L1 increased in the intestinal mucosa of UC and CD patients,and the main expression sites were in the intestinal submucosa,lamina propria and lymphatic nodular.No clear correlation between the expression level and the severity of the disease was found at present.2.PD-L1-Fc can ameliorate DSS-induced acute colitis of mice by strengthening intestinal immune barrier,mechanical barrier and chemical barrier.3.PD-L1-Fc/Oxi-αCD NP can target the inflammatory site of colon for the treatment of DSS-induced acute colitis in mice,and can achieve better therapeutic effect and fewer side effects than PD-L1-Fc fusion protein,which is a potential target for the treatment of IBD.