Study On The Role Of Immune Response To PFOR In Dysbiosis Of Patients With Ulcerative Colitis

Background and Aims:Inflammatory bowel disease(IBD),including Crohn's disease(CD)and ulcerative colitis(UC)is a chronic and recurrent gastrointestinal disease that has a gradual increase in global incidence.The etiology and pathogenesis of UC are still not fully elucidated so far,and it is suspected to arise from the interaction between the host's genetic background,environment,mucosal immunity,and the resident bacterial flora.In humans,the gastrointestinal tract represents a large microbial ecosystem,housing several trillion microbial cells.Numerous observational studies have reported dysbiosis,an imbalance between protective and harmful bacteria,to be relevant to the etiology and pathogenesis of UC.Most importantly,a decrease in the proportion of Firmicutes and in particular of Faecalibacterium prausnitzii(F.prausnitzii)has been described in UC patients.Recently,many researches provide further evidence that F.prausnitzii is an anti-inflammatory bacterium with therapeutic potential for UC patients.As the most abundant class of antibody found in the intestinal lumen of humans and most other mammals,secretory IgA(SIgA)has long been recognized as the first line of defense in protecting the intestinal epithelium from enteric pathogens and toxins.Antibodies generated in response to microbial colonization of the gut can shape the composition of the microbiota.Using flow-cytometry-based bacterial cell sorting and 16S sequencing,researchers characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A(IgA-SEQ),and show that F.prausnitzii was highly coated in UC.Thus,we aim to clarify whether highly IgA coating would induce the reduction of F.prausnitzii and explore specific bacterial antigen recognized by the immune system.Methods:1.Clinical data collection:We enrolled diagnosed UC patients and healthy controls,collected their results of laboratory tests and calculated the mayo score.Then the stool samples were obtained for the 16S rRNA pyrosequencing.2.Fecal IgA detection of clinical sample:ELISA was done to test total IgA and F.prausnitzii specific IgA from fecal supernatant.The specific proteins with strong IgA reactivity was identified using Western Blot.3.Antigen identification and confirmation:The IgA reactive antigen of F.prausnitzii were extracted from the gels stained with Coomassie brilliant blue R250,and were further analyzed using a NanoLC-ESI-MS/MS system.The immunoreactivity of recombinant protein was detected by Western blot and purified using a Ni-NTA agarose column.After screening,the pyruvate:ferredoxin(flavodoxin)oxidoreductase(PFOR)was identified as potential antigen.4.PBMCs culturation and PFOR stimulation:Peripheral blood mononuclear cells(PBMCs)were isolated by Ficoll-Paque density gradient centrifugation.Cells were cultured and stimulated with PFOR and inhibitor NTZ.Then the mRNA level of Il1b,1l6Il,17a,Tnfa and Ifnr were detected using qPCR and IFN-? ELISpot assays were performed.5.F.prausnitzii immunization model construction:New Zealand White Rabbits were randomly divided into three groups:PBS,QuilA,QuilA+FP.We recorded the body weight change and collected stool samples for 16S rRNA sequencing.Tissues were fixed,embedded,sectioned,stained with H&E,and evaluated for evidence of inflammation and epithelial damage.We tested secretory IgA by ELISA and the mRNA levels of pIgR,Ifnr,116,Il1b,1122 and Cox2 were detected by qPCR.6.PFOR immunization model construction:New Zealand White Rabbits were randomly divided into two groups:QuilA,QuilA+ PFOR.Body weight change,16S rRNA sequencing,histological evaluation,IgA level,and tissue mRNA levels were evaluated.7.We analyzed the sequence data to identify other bacteria which contained POFR,and performed homology analysis and phylogenetic tree construction.Furtherly,the abundance of selected bacterium was analyzed.Results:1.UC patients showed a lower microbiota ?-diversity and significantly lower abundance of F.prausnitzii.2.The total IgA and anti-F.prausnitzii IgA were increased significantly in fecal supernatant of UC patients.Western blot of bacterial lysates showed strong IgA reactivity at 130 kDa and 85 kDa.We identified the pyruvate:ferredoxin(flavodoxin)oxidoreductase(PFOR)as potential antigen.The anti-PFOR IgA level was decreased in UC patients.3.PFOR was able to stimulate PBMCs to generate robust IFN-y and can be inhibited by NTZ4.F.prausnitzii immunization caused transiently weight loss and increased mucosal inflammation in ileum and cecum.The level of fecal total IgA,F.prausnitzii specific IgA and PFOR IgA increased after F.prausnitzii immunization.,which caused gut microbiota change and the abundance of PFOR gene decreased.The expression of Ifnr was higher in colon after immunization.5.Rabbits in PFOR immunization group lost weight transiently and gained body weight lately and was higher than that in QuilA group.Although there was no difference of histological score between the two groups,the Ifnr expression was higher in PFOR immunization group.PFOR immunization caused the structure of gut microbiota changed and the abundance of PFOR gene decreased.Conclusions:1.There are significant dysbiosis and lower abundance of F.prausnitzii in UC patients.The fecal total IgA,anti-F.prausnitzii IgA and anti-PFOR IgA are increased.2.The antigen PFOR exists in many bacteria and is relatively conservative in evolution.PFOR stimulate IFN-y expression of PBMCs3.Animal studies show that F.prausnitzii and PFOR immunization change body weight,induce mucosal inflammation,higher expression of tissue Ifnr,gut microbiota dysbiosis and lower fecal PFOR abundance.