Study On The Mechanism Of Bifidobacterium Infantis Mediated PD-1/PD-L1 Pathway Inhibiting Immune Response In Inflammatory Bowel Disease

Objective: The pathogenesis of Inflammatory bowel disease(IBD)is related to immunity,infection,environment and heredity.Abnormal immune response or impaired normal immune tolerance is the central pathogenesis of IBD.It has been confirmed that the intestinal inflammatory response of IBD originates from the abnormally hyperactive T cell response of intestinal immune system to symbiotic bacteria.Therefore,inhibiting hyperactivity of T cells may play a key role in the treatment of IBD.Regulatory T cells(Tregs)can inhibit autoreactive effector T cells.Tregs mainly secrete cytokines such as IL-10,TGF-beta 1 and IL-35,maintain autoimmune tolerance,and play a key role in the immune regulation.Studies have shown that the decrease of Tregs number,defective expression of surface molecules and impaired inhibition function may be related to the occurrence of IBD.Forkheadbox protein 3(Foxp3)is a transcription factor of Tregs,which plays an irreplaceable role in the immunosuppressive function and cell stability of Tregs,and can be used as a specific marker of functional Tregs.In recent years,it has been found that PD-1/PD-L1 pathway participates in the occurrence and development of IBD.Animal experiments have proved that PD-L1 has a significant therapeutic effect on DSS-induced IBD mice,reducing the proportion of CD4+T cells secreting inflammatory factor IL-17 A and the expression of inflammatory factors IL-1,IL-6 and TNF-alpha.When CD4+PD-1+T cells were injected into IBD mice,the inflammatory response of chronic colitis mice was inhibited.It has been found that PD-L1 may induce the differentiation of Tregs and enhance its immunosuppressive function.Animal experiments showed that adoptive transfer of immature CD4+T cells to wild-type Rag-/-mice and PD-L1-/-Rag-/-mice showed that Tregs decreased significantly in PD-L1-/-Rag-/-mice,suggesting that PD-L1 played an important role in Tregs induced differentiation.The more Tregs in tumor microenvironment,the more IL-10 and TGF-beta produced by tumor cells.The differentiation of Tregs depends on the signal pathway mediated by PD-L1.The higher the level of PD-L1 expressed by hepatic dendritic cells,the more Tregs can be induced,thus maintaining the tolerance of transplanted organs.At present,the application of probiotics or adjuvant therapy for IBD has become a new research hotspot.Bifidobacterium infantis(B.infantis)can promote the proliferation of Tregs,promote the secretion of inflammatory factors such as IL-10,TGF-beta 1,and inhibit inflammatory factors such as IFN-gamma,IL-2,IL-17.Although a large number of clinical and basic experiments have confirmed that B.infantis has obvious therapeutic effect on IBD,its specific therapeutic mechanism still needs to be further studied.PI3K-Akt-m TOR signaling pathway is involved in mediating cell growth,differentiation,apoptosis and other important signal transduction.Activating PI3K-Akt-m TOR signaling pathway can inhibit the differentiation of Tregs.Inhibiting PI3K-Akt-m TOR signaling pathway will promote the expression of Foxp3 and affect the content of Tregs cells in animals.Francisco LM et al.found that the antigen presenting cells with negative PD-L1 induced the transformation of CD4+T cells to Foxp3+Treg to a minimum extent.This project intends to study the protective effect of B.infantis on IBD by using experimental IBD animal models and cell experiments.It also proves that B.infantis inhibits intestinal immune response through PD-1/PD-L1 signaling pathway,and further studies the mechanism of B.infantis 's abnormal immune response to inflammatory bowel disease,so as to provide new theoretical basis and potential molecular targets for the treatment of IBD.Methods: 1.Protective effect and molecular mechanism of B.infantis on IBD mice.Eight-week-old BALB/c mice were randomly divided into five groups: Control group,DSS model group,high concentration B.infantis treatment group,medium concentration B.infantis treatment group and low concentration B.infantis treatment group.The control group mice drank water freely,while the other four groups drank water containing 5% DSS freely.The control group and DSS model group were fed with NS400?L daily;the content of B.infantis in high concentration treatment group,medium concentration treatment group and low concentration treatment group were 1X109 CFU,1x108 CFU and 1x107 CFU respectively.DAI scores were performed daily and body weight was recorded.On the 8th day,the mice were killed,the length of intestinal tract was recorded and histological scores were made.The expression of intestinal proteins PD-L1,PD-1,Foxp3,IL-10 and TGF-beta 1 were detected by immunohistochemistry and Western blotting,and the protein localization was observed.2.Mechanisms of B.infantis regulating intestinal PD-1/PD-L1 pathway and abnormal immune response in IBD mice.Eight-week-old BALB/c mice were randomly divided into five groups: control group,DSS model group,DSS+ B.infantis group,DSS+ B.infantis +anti-PD-L1 group,DSS+anti-PD-L1 group,control group mice drinking water freely,the other four groups mice drinking water containing 5% DSS freely.The control group,DSS model group and DSS+anti-PD-L1 group were given NS400 ?L per day by gastric lavage;the DSS+ B.infantis group and DSS+ B.infantis +anti-PD-L1 group were given NS400 mu L per day and 1X109 CFU of B.infantis by gastric lavage;the DSS+ B.infantis +anti-PD-L1 group and DSS+anti-PD-L1 group were given 200 UG of PD-L1 blocker intraperitoneally at 0,3,5 and 7 days,respectively,and the control group,DSS+anti-PD-L1 group and DSS+ B.infantis group were given intraperitoneal injection of PBS of equal volume.The changes of protein and gene expression of PD-L1,PD-1,Foxp3,IL-10 and TGF-beta 1 were observed.Flow cytometry was used to observe the changes of CD4+CD25+Foxp3+cells in blood and spleen.3.B.infantis inhibits PI3K-Akt-m TOR signaling pathway through PD-L1 to study Foxp 3 in colon cells.The effects of B.infantis supernatant on the signal pathways of PD-L1,PD-1,Foxp3 and PI3K-Akt-m TOR in colon cells were observed by culturing HCT-116.We also observe the effect of blocking PD-L1 on PD-1,Foxp3 protein and m RNA,and PI3K-Akt-m TOR signaling pathway protein.Results: 1.From the 4th day of model building,the weight loss of mice in DSS model group was significantly higher than that of control group(P < 0.05);from the 5th day of model building,the weight of mice in high concentration B.infantis treatment group,medium concentration B.infantis treatment group and low concentration B.infantis treatment group changed successively,and increased significantly compared with DSS model group(P < 0.05).After DSS intervention,mice showed signs of emaciation,loose stool,hematochezia and other diseases.On the third day,DAI scores began to increase significantly,and peaked on the seventh day.There was a statistical difference between the two groups(P < 0.01).Compared with DSS model group,the DAI scores ofBifidobacterium infants in each concentration group were significantly lower(P < 0.05).The colon of DSS model group was shorter than that of control group(P < 0.01),suggesting that the model was successful.The colon of the three treatment groups with B.infantis intervention was significantly longer than that of the DSS model group(P < 0.01).In DSS model group,mucosal structure was obviously damaged,including disorder of glandular duct arrangement,extensive crypt destruction,deformation and even atrophy,goblet cell reduction,epithelial atrophy,necrosis,and even disappearance of epithelial cells.A large number of inflammatory cells could be seen,involving the lamina propria and submucosa,crypt inflammation and crypt abscess could be seen,ulcers could be seen in some specimens,and histological damage score was significantly higher than that of Control.In group B(P < 0.01),the histological damage of B.infantis group was significantly reduced(P < 0.05).Compared with group B,the histological damage score of low concentration B.infantis treatment group was significantly higher than that of high concentration B.infantis treatment group and medium concentration B.infantis treatment group(P < 0.05).Immunohistochemical results showed that both PD-L1 and PD-1 existed in cell membrane and cytoplasm,and PD-L1 was expressed in intestinal epithelial cells and lamina propria,while PD-1 was only expressed in lamina propria;Foxp3 was expressed in the nucleus of lymphocyte in lamina propria;IL-10 and TGF-beta 1 were secretory proteins,which were widely expressed in mucosa and lamina propria.B.infantis significantly increased the expression of PD-L1,PD-1,Foxp3,IL-10 and TGF-beta 1.There was a concentration gradient effect on the changes of PD-L1 and Foxp3.2.The expression of PD-L1,PD-1,Foxp3,IL-10 and TGF-beta 1 in intestinal tract of DSS mice was significantly decreased(P < 0.05),and the proportion of CD4+CD25+Foxp3+cells in spleen and blood was decreased(P < 0.05).B.infantis could promote the expression of PD-L1,PD-1,Foxp3,IL-10 and TGF-beta 1(P < 0.05)and increase the proportion of CD4+CD25+Foxp3+cells in spleen and blood(P < 0.05).After blocking PD-L1,the promotion of protein and gene expression of Foxp3,IL-10 and TGF-beta 1 by B.infantis was inhibited(P < 0.05),and the proliferation of CD4+CD25+Foxp3+ cells in spleen and blood was also inhibited(P < 0.05).3.The supernatant of B.infantis could up-regulate the expression of PD-L1,Foxp3 protein and m RNA,and down-regulate the phosphorylation protein expression of PI3 K,Akt and m TOR(P < 0.05),but there was no change in the total protein expression of the three.After blocking PD-L1,the promoting effect of B.infantis supernatant on Foxp3 was weakened,and the inhibitory effect on phosphorylated proteins of PI3 K,Akt and m TOR was also weakened.Conclusion: 1.B.infantis can increase the expression of PD-L1 and PD-1 in colon tissue of mice,promote the expression of Tregs nuclear transcription factor Foxp3,and increase the expression of anti-inflammatory factors IL-10 and TGF-beta 1.B.infantis can significantly improve the disease activity index score and histological damage of DSS-induced acute enteritis mice.B.infantis can alleviate the damage of intestinal epithelium and protect mice with inflammatory bowel disease.2.B.infantis may promote the proliferation of CD4+CD25+Foxp3+T cells in spleen and peripheral blood by activating the PD-1/PD-L1 pathway and the expression of Foxp3 in intestinal tract.It also promotes the expression of IL-10 and TGFbeta 1 in intestinal tract,thereby alleviating the intestinal immune inflammation and has certain therapeutic effect on IBD mice.3.B.infantis may inhibit PI3K-Akt-m TOR signaling pathway and promote Foxp 3 expression through PD-L1,thus exerting immunosuppressive effect,which may be a potential target for B.infantis to exert immunosuppressive effect.