A Preliminary Study On The Effect Of PD-1 Inhibitor On Gut Microbiota And Intestinal Barrier In Mice With TNBS-induced Enteritis

Background and purpose Inflammatory bowel disease?IBD?is a kind of etiology-unknown chronic nonspecific intestinal inflammatory diseases,including Ulcerative colitis?UC?and Crohn's disease?CD?.It is generally believed that the disease is caused by genetic,immune,intestinal barrier and gut microbiota imbalance.Abdominal pain,diarrhea,and bloody stools are the main clinical manifestations.With the change of environment,diet,work pressure and so on,the morbidity is rising year by year and has become a global disease.Due to the chronic repeaded course,IBD also has a significant impact on social economy.Intestinal epithelial barrier dysfunction is an important factor in mediating the development of IBD,and tight junctions are considered as the structural basis of the intestinal epithelial barrier.The tight junctions are mainly composed of the peritonin ZO,the transmembrane protein Occludin,Claudin,and regulatory proteins.Destruction of this structural basis will lead to increased permeability of the intestinal epithelium,triggering a disorder of the mucosal immune response and chronic enteritis.Physiologically,there are large amounts of bacteria and their metabolites in human intestine,relying on the adjustment by the intestinal epithelial mechanical barrier,mucosal immune system and mucous barrier to form stable gut environment.With more studies indicate,intestinal mucosal immunity plays an important role in regulating gut microbiota.The regulation of gut microbiota can alleviate the patients' symptom and reduce mucosal inflammation.The application of immunosuppressive agents is also an important treatment for patients with hormone-dependent IBD.Programmed death receptor 1?PD-1?is a kind of important immune negative regulatory molecules?also known as "immune checkpoints"?.Through binding the PDL1 ligand on the surface of cells,it can activate the negative control signal and reduce the activation of immune cells.In recent years,anti-PD-1 and anti-PD-L1 antibodies have become a hot topic in tumor immunotherapy research.However,the efficacy of PD-1/PD-L1 antibody was strongly influenced by the tumor microenvironment and gut microbiota.Such as Bifidobacterium may promote the efficacy of PD-1/PD-L1 monoclonal antibody drugs,etc.However,there is few study about PD-1/PDL-1 in the field of IBD,and still unknown for the efficacy in IBD.Therefore,we constructed TNBS-induced mice enteritis model and used PD-1 inhibitor?PD-1 inhibitor?to specifically block the PD-1/PD-L1 pathway,and observed the effects of PD-1 antibodies on IBD as well as the effects of gut microbiota and intestinal mucosal barrier.We hope to provide certain experimental data and theoretical basis for the diagnosis and treatment of IBD.Methods Clinical research: We Collected intestinal mucosal tissues of clinical UC and CD patients and healthy volunteers,and detected the expression of PD-L1 protein by immunohistochemistry.Animal experiment:48 BALB/c mice were divided into Normal control group,TNBS model group,TNBS+5-ASA group,TNBS+ PD-1 inhibitor group.The TNBS model group,TNBS+ 5-ASA group,TNBS+ PD-1 inhibitor group were anesthetized in the same day and given a TNBS ethanol solution enema.The Normal control group was given 50% ethanol solution enema.After 24 h,TNBS+5-ASA group was given 5-amino-salicylic acid suspension,and TNBS+ PD-1 inhibitor group was given PD-1 inhibitor by intraperitoneal injection,and the Normal control group and TNBS model group were gavaged by PBS?Once-daily for all groups?.DAI scores were recorded daily on the weight,stool property and occult blood of the mice.After execution,we paraffin-embedded colon tissues and stained with HE to observe inflammation;Tissue immunofluorescence and immunohistochemistry were used to observe the changes of intestinal tight junction proteins.ELISA was used to detect the expression levels of related cytokines in blood and intestinal tissues.Before the end of the experiment,we collected the feces and analyzed the changes of fecal microbiota of mice by using 16 s high throughput sequencing of microorganism group.Finally,the mice were hocussed,ligated colon,given a 30-min enema with fitc-dextran and collected serum for testing fluorescence for assessing intestinal wall permeability.At last,we analyzed the whole relevant indicators statistically.Results 1.Increased expression of PD-L1 protein in intestinal tissue in patients with IBD We selected 10 samples from UC,CD and healthy volunteers respectively to detect PD-L1 expression by immunohistochemical.The results showed that compared with healthy volunteers,PD-L1 protein in intestinal mucosa of UC patients and CD patients increased.2.PD-1 inhibitors may help to improve the general condition and disease activity of TNBS mice Disease activity index?DAI?: The fifth day,TNBS model groups' DAI increased significantly.In the end of the experiment,TNBS+5-ASA,TNBS+ PD-1 inhibitor group and Normal control group were lower than TNBS model group.Colon general situation: Colon length: TNBS model group vs Normal control group shortened,TNBS+5-ASA group,TNBS+ PD-1 inhibitor group and normal control group showed no significant difference.3.PD-1 inhibitors may help to relieve TNBS-induced mice in intestinal inflammation Colonic HE staining TNBS model group showed mucosal ulcer,edema,hyperplasia,and extensive inflammatory cell infiltration.TNBS+5-ASA group and TNBS+ PD-1 inhibitor group had different degrees of ulcer,inflammatory cell infiltration,mucosal hyperplasia and edema,but the degree was significantly improved compared with TNBS model group.Serum TNF-? level: TNBS model group'serum TNF-? was higher than the Normal control group.TNBS model group vs TNBS+ PD-1 inhibitor group significantly decreased,the difference was statistically significant?P < 0.05?.4.PD-1 inhibitors may help to improve TNBS-induced intestinal bacterial disturbance in mice,adjust bacterial structure,up-regulate the production of short-chain fatty acid bacteria and mucosal immune-related bacterial abundance 1)Alpha diversity analysis: In TNBS model group,there was a significant decrease in alpha diversity vs Normal control group.Compared with TNBS model group,TNBS+5-ASA group and TNBS+ PD-1 inhibitor group' alpha diversity increased significantly.2)Beta diversity analysis: PCA analysis and Uni Frac multi-sample similarity tree analysis showed that the gut microbiota structure of the normal control group and TNBS model group was significantly different while TNBS+5-ASA group and TNBS+ PD-1 inhibitor group was similar;The abundance of Firmicutes increased in TNBS mice after PD-1 inhibitor intervention,and the abundance of Proteobacteria and Bacteroidetes decreased.3)Bacterial community in the phylum level: Firmicutes and Deferribacteres were increased after PD-1 inhibitor intervention,and Proteobacteria,Actinobacteria,Tenericutes and Bacteroidetes were decreased.Bacterial community in the Class level: After PD-1 inhibitor intervention,Clostridia were increased,Bacteroidia,Mollicutes,Erysipelotrichia,Alphaproteobacteria,Bacilli and unidentified Antinobacteria were decreased.Bacterial community in the Order level: After PD-1 inhibitor intervention,Clostridiales increased,Bacteroidales,Erysipelotrichales,Pseudomonadales,Rhodospirillales,Bifidobacteriales and Mollicutes-RF9 decreased.Bacterial community in the Family level:After PD-1 inhibitor intervention,Rikenellaceae,Lachnospiraceae,Clostridiales-vadin BB60-group,Peptococcaceae and Ruminococcaceae increased,Christensenellaceae,Prevotellaceae,Aerococcaceae,Rhodospirllaceae,Streptococcaceae,Moraxellaceae,Bacteroidaceae,Family-XII,Clostridiaceae and Erysipelotrichaceae were decreased.Bacterial community in the Genus level:After PD-1 inhibitor intervention,Butyricicoccus,Ruminiclostridum,Ruminiclostridum-9,Oscillibacter,Ruminiclostridum-5,Rikenella,Roseburia,Ruminococcaceae,Anaerotruncus and unidentified Lachnospiraceae were increased,Acinetobacter,Bacteroides,Parabacteroides and Alloprevotella were decreased.Bacterial community in the Species level: After PD-1 inhibitor intervention,Escherichia-coli,Bacteroides acidifaciens,Lachnospiraceae-COE1 and Clostridiumleptum were increased,and Acinetobacter-baumannii and Bacteroides-sartorii were decreased.Among them: Lachnospiraceae,Rikenellaceae,Ruminococcaceae,Butyricicoccus and Roseburia are important bacteria producing short-chain fatty acids in intestinal commensal bacteria;Bacteroides acidifaciens and Clostridium leptum are involved in the formation of intestinal mucosal immune tolerance.4)Biomarker biomarkers and Bacterial community in OTU level: Biomarker biomarkers showed that PD-1 inhibitor could down-regulate the relative abundance of Alloprevotella bacteria in TNBS mice.According to Meta Stat method analysis,the relative abundance of Deferribacteres,clostridium_stricto₁,Empedobacter and Mucispirillum was increased after PD-1 inhibitor intervention.Among them,Mucispirillum and Candidatus_Arthromitus showed significant correlation with T celldependent Ig A secretion.5.PD-1 inhibitor can relieve TNBS-induced barrier defects to some extent Immunofluorescence: The expression of ZO-1 in the colon was decreased after TNBS modeling,and the expression of ZO-1 in colon was up-regulated after 5-ASA and PD-1 inhibitor intervention.Immunohistochemistry:The expression of ZO-1 and Occludin in the colon were decreased after TNBS modeling,and the expression of ZO-1 and Occludin in colon were up-regulated after 5-ASA and PD-1 inhibitor intervention.Serum FITC fluorescence intensity: the serum fluorescence intensity of TNBS model group was higher than the Normal control group,the TNBS+ 5-ASA group and TNBS+ PD-1 inhibitor group were lower than the TNBS model group,and there was no significant difference among the groups.Conclusion 1.Expression of PD-L1 protein in intestinal mucosa of IBD patients was increased.2.PD-1 inhibitors may help to alleviate intestinal inflammation and disease activity in mice with TNBS-induced enteritis;3.PD-1 inhibitors may help to improve the gut microbiota of TNBS-induced enteritis mice,upregulate the diversity of gut microbiota,adjust the bacterial structure,increase the production of short-chain fatty acid bacteria and mucosal immune-related bacterial abundance;4.PD-1 inhibitors may help to improve barrier defects in TNBS-induced enteritis mice.