Study On The Role Of Intestinal IgA And Related Microbiome In Diarrhea-Predominant Irritable Bowel Syndrome

Irritable bowel syndrome?IBS?is a common gastrointestinal disorder in which patients are prone to low-grade inflammation of the intestinal mucosa,but the mechanism is unclear.1 Recent studies suggested that patients with diarrhea-predominant IBS?IBS-D?suffer from abnorlally hyperactive humoral immunity.2 3IgA is an important part of humoral immunity and constitutes the first immune barrier on the mucosal surface.5 The symbiotic bacteria in the intestinal flora stimulate the intestinal mucosa to produce IgA,which can also be covered by IgA itself and transformed into IgA enveloped form.The balance between the intestinal microbiome and the mucosal humoral immune system is characteristic of a healthy,gut.Previous studies have shown that IgA levels in fecal samples of patients with inflammatory bowel disease?IBD?were abnormally elevated and that IgA-coated bacteria?IgA+ bacteria?played an important role in the pathogenesis of IBD.7-11 However,in IBS-D,whether IgA and IgA-coated bacteria are changed and are involved in regulating the pathogenesis of IBS-D have not been studied.In this study,we detect the changes of IgA levels,the amount of IgA-coated bacteria and intestinal microecological structure.Moreover,the correlation between IgA level and IBS-D clinical manifestations was analyzed to explore the impact of these changes on the pathogenesis of IBS-D.Part 1 Intestinal IgA and diarrhea-predominant irritable bowelsyndromePurpose:1.To detect the IgA levels in plasma and feces,number of IgA positive cells in intestinal mucosal tissues,and the expression of IgA related genes in blood cells and intestinal mucosal tissues.2.Statistical analysis of the correlation between intestinal IgA and clinical characteristics of diarrhea-predominant irritable bowel syndrome.Materials and methods:1.Forty-four IBS-D patients and 32 healthy volunteers who were admitted to the department of gastroenterology,Shandong university Qilu hospital from 2018 to 2019 were selected as subjects for inclusion in this study.The diagnosis of IBS-D patients met the Roman IV criteria.2.The contents of IgA in the plasma were determined by ELISA.Real-time quantitative PCR was performed to detect the expression of AID,IGHA1,IGHA2,TGF-?1,BAFF-R,BCMA and TACI genes in the blood cells.3.Before the preparation of the intestines for colonoscopy,fecal samples were collected and the IgA level of feces was detected by ELISA.4.Colonoscopy was performed on the subjects to obtain the terminal ileum and cecum biopsy tissues,and immunofluorescence staining was performed on the intestinal mucosal tissues to detect IgA positive cells.Real-time quantitative PCR was used to detect expression levels of AID,IGHA1,IGHA2,TGF-?1,BAFF-R,BCMA and TACI genes in intestinal mucosal tissues.Results:1.ELISA results showed that IgA level in feces of IBS-D patients was significantly higher than that of healthy control group?p=0.0115?,while there was no significant difference in IgA level in plasma between the two groups.2.Tissue immunofluorescence test indicated that the number of IgA positive cells in the ileum terminal of IBS-D patients showed an increased trend compared with the healthy control group?p=0.0641?,while there was no significant difference in the number of IgA positive cells in the ceacal mucosa between the two groups.3.The expression level of IGHA1 in the terminal ileum of IBS-D patients was significantly higher than that of the healthy control group?p<0.0001?,while there was no significant difference in the expression level of IGHA2 between the two groups?p=0.1401?.In addition,no intergroup differences in IGHA1 and IGHA2 expression were found in the blood and cecum mucosa.4.Real-time quantitative PCR showed that in the distal ileum mucosa,the mRNA expression of AID in IBS-D group was significantly higher than that in the healthy group?p=0.0005?,but no significant difference was found in blood?p=0.3245?and ceacal mucosa?p=0.8682?between the two groups.mRNA expression of BAFF-R in the distal ileum mucosa of IBS-D patients was significantly up-regulated?p=0.0003?.However,mRNA expression of TACI showed an increasing trend?p=0.0769?,and mRNA expression of BCMA and TGF-?1 mRNA showed no statistical difference between the two groups.In addition,mRNA expression of BAFF-R,TACI,BCMA,and TGF-?1 mRNA was not different between the two groups in ceacal mucosa and blood samples.5.Defecation frequency,fecal characteristics,intensity and frequency of abdominal pain,and IBS symptom severity scale?IBS-SSS?were positively correlated with IgA level in feces and mRNA expression of ileum terminal AID and IGHA1 gene.Conclusions:Compared with the healthy control group,IBS-D patients showed a significant increase in intestinal IgA,mainly reflected in the increased expression of IgAl subtype at the terminal ileum.In addition,the activity of the IgA class switch recombination?CSR?regulatory gene at the terminal ileum was enhanced,indicating that the class switch recombination of IgA antibody at the terminal ileum was enhanced.The mRNA expression levels of fecal IgA and ileum terminal AID and IGHA1 genes were positively correlated with the defecation frequency,fecal characteristics,intensity and frequency of abdominal pain,IBS-SSS and other clinical indicators.This study confirmed that intestinal IgA was closely related to IBS-D,providing a theoretical basis for further exploration of the pathogenesis and diagnosis and treatment of IBS-D.Part 2 Changes of IgA-coated bacteria in diarrhea-predominant irritable bowel syndromePurpose:1.To test the percentage of IgA+bacteria in feces of the subjects.2.Statistical analysis of the correlation between IgA bacteria and clinical characteristics of diarrhea-predominant irritable bowel syndrome.Materials and methods:1.Fecal samples were collected before subjects prepared their intestines for colonoscopy.2.Mpbio Fastprep Lysing Matrix D tube was used for fecal homogenization,and after staining with anti-human IgA-PE antibody,flow cytometry analysis was conducted to obtain the proportions of IgA-coated bacteria in fecal bacteria.Results:1.Compared with the healthy control group,the proportion of IgA+bacteria in the feces of patients was significantly increased?p=0.0024?.2.The proportion of IgA-coated bacteria in feces was positively correlated with the frequency of defecation,fecal characteristics,intensity and frequency of abdominal pain and IBS-SSS.Conclusions:This study suggested that IgA coated intestinal flora was closely related to diarrhea-predominant irritable bowel syndrome.The proportion of IgA+bacteria in the feces of IBS-D patients was significantly higher than that of the healthy control group,and was positively correlated with the frequency of defecation,fecal characteristics,intensity and frequency of abdominal pain,and IBS-SSS.Part 3 The role of IgA related intestinal microbiome in diarrhea-predominant irritable bowel syndromePurpose:1.To separate IgA+ bacteria and IgA-bacteria in feces.2.Sequencing was performed to analyze the flora composition of total fecal bacteria,IgA+ bacteria and IgA-bacteria.Materials and methods:1.Fecal samples were collected before intestinal preparation.2.Fecal samples stained with Anti-PE MicroBeads and QuadroMACS starter kits against anti-human IgA-PE antibody were used for bacterial sorting.After magnetic activated cell sorting,fecal bacteria were divided into IgA negative and IgA positive parts.3.16S rRNA gene sequencing analysis of IgA negative bacteria and IgA positive bacteria.Results:1.There were 17 bacterial species in the intestines of IBS-D patients,which were significantly different from the healthy control group.2.The composition of IgA+ bacteria and IgA-bacteria in IBS-D patients was different.Compared with IgA-bacteria,IgA+bacteria showed higher relative abundance of Escherichia-Shigella,Romboutsia,Streptococcus,Tyzzerella₄,Klebsiella,Intestinibacter,Granulicatella and Haemophilus.3.17 genera different from the healthy control group intersected with the highly IgA-coated genera,including three taxa Escherichia-Shigella,Granulicatella and Haemophilus.4.The relative abundance of Escherichia-Shigella was positively correlated with the frequency of defecation,fecal characteristics,intensity and frequency of abdominal pain and IBS-SSS.Escherichia-Shigella ICI score was positively correlated with anxiety score and depression score,respectively.Conclusions:Compared with the healthy control group,the composition of intestinal flora in IBS-D patients was changed.And the IgA+ bacteria of IBS-D patients was different IgA-bacteria.There was an intersection of the two ranges,including Escherichia-Shigella,Granulicatella and Haemophilus.Among them,Escherichia-Shigella was positively correlated with the frequency of defecation,fecal characteristics,intensity and frequency of abdominal pain,IBS-SSS,and anxiety and depression.This indicates that intestinal flora may be closely related to diarrhea-predominant irritable bowel syndrome by inducing the generation of IgA in the local immune system of the body,and Escherichia-Shigella plays an important role that cannot be ignored.