Correlation Of Intestinal Flora Disorders With Memory T/B Cell Subsets In RA Patients

Objective1.To clarify the changes of intestinal flora in RA patients in the active and remission of the disease;2.To clarify the changes of Treg,memory Tfh,memory B cells and related cytokines in RA patients in the active and remission of the disease.3.To clarify the correlation between intestinal flora disorders and Treg,memory Tfh, memory B cells,related cytokines and clinical indices in RA patients.4.To explore the role of intestinal flora disorders on the regulation of Treg,memory Tfh,memory B cells and related cytokines in RA patients,in order to explore the pathogenesis of RA and identify new therapeutic targets.Methods60 patients diagnosed with RA in the Department of Rheumatology and Immunology,General Hospital of Ningxia Medical University since March 2021 were selected,and all patients met the 2010 ACR/EULAR classification criteria for RA,excluding those with comorbidities of other connective tissue diseases,severe infections,tumors,nerve damage during pregnancy and lactation.The RA patients were divided into two groups according to the DAS28-ESR score.DAS28-ESR≧2.6 for the active disease group(Active RA)and DAS28-ESR < 2.6 for the remission disease group(Remission RA).And 30 healthy people with no significant abnormalities in blood routine,liver and kidney function,urine routine,blood glucose and blood lipids were selected from the health checkup center of Ningxia Medical University General Hospital.Blood samples and stool samples were collected from all RA patients and the healthy population.The abundance and diversity of intestinal flora in RA patients’ disease activity group,disease remission group and healthy population and the differences in the composition of intestinal flora were analyzed by 16 Sr RNA gene sequencing technique;the number of Treg cells,memory Tfh cells and memory B cell subpopulations in peripheral blood of RA patients’ disease activity group,disease remission group and healthy population and their ability to secrete The number of Treg cells,memory Tfh cells and memory B-cell subpopulations and their ability to secrete cytokines were analyzed by flow cytometry;the serum levels of cytokines(IL-15,IL-21,IL-33 and IL-35)in the peripheral blood of RA patients in the active,remission and healthy groups were measured by ELISA assay;Spearman’s analysis of intestinal flora and clinical indices,Treg cells,memory Tfh cells and memory B-cell subpopulations and related cytokines correlation.All experimental data were analyzed using SPSS version 26.0 and Prism version 9.0.Results1.Alteration of intestinal microorganisms in RA patients with active and remission disease groupsThe diversity of intestinal microbiota in RA patients with active disease and remission stage was significantly different from those in healthy controls(HC).Chao1,observed species,Shannon and Faith’s PD indices were reduced in RA patients during both active and remission phases of the disease compared to HC.At the phylum level,the relative abundance of Bacteroidetes and Actinobacteria was decreased in RA patients with remission stage,while the relative abundance of Fusobacteria was increased in RA patients with active disease compared to HC.At the genus level,the relative abundance of Ruminococcaceae_Ruminococcus and Bacteroides was decreased,while the abundance of Megamonas was increased in RA patients with active disease and remission stage compared to HC.2.Alterations in Treg,memory Tfh and memory B cell subsets in RA patients with active and remitting disease groupsCompared with healthy controls,CD4+CD25+Fox P3+Treg cells,CD4+CD25high CD127 lowTreg cells,CD4+CD45RO+CCR7+CXCR5+central memory Tfh cells and CD4+CD45RO+CCR7-CXCR5+ effector memory Tfh in the peripheral blood of RA patient cells were significantly increased in the disease activity group.In contrast,Treg cells,central memory Tfh cells and effector memory Tfh cells were significantly reduced in RA patients in the disease remission group compared with the disease activity group.Compared with healthy controls,CD19+CD27+memory B cells were reduced in RA patients in disease remission,while CD19+CD27+Ig M+memory B cells were significantl y reduced in RA patients in disease activity.Compared with healthy controls,CD19+ CD27+Ig D+pre-conversion memory B cells were decreased in both disease activity and remission,while CD19+CD27-Ig D-double negative memory B cells were significantly in creased in both disease activity and remission.3.Changes in cytokines in RA patients in the disease activity and disease remission groupsCompared with healthy controls,the levels of cytokines IL-17 A,IL-10,IFN-γ and TNF-α secreted by CD4+ T cells were increased in RA patients in the active and remission groups of the disease.And the serum levels of IL-21 and IL-33 in peripheral blood were increased,while IL-35 was decreased.4.Correlation of intestinal flora with clinical indicators,immune cells and cytokines in RA patients with the active phase of the diseaseAt the phylum level,Actinobacteria were positively correlated with IL-17 A a nd Fusobacteria were positively correlated with IL-21.At the genus level,Ruminococc aceae_Ruminococcus bacteria were positively correlated with CD19+CD27+Ig M+memory B cells,while they were negatively correlated with IL-17 A,IL-10,and TNF-α.And Lachnospiraceae_Clostridium bacteria were positively correlated with CD4+CD45R O+CCR7+CXCR5+central memory Tfh cells.And Ruminococcaceae_Ruminococcus and Megamonas bacteria were positively correlated with IL-33.5.Correlation of intestinal flora with clinical indicators,immune cells and cytokines in RA patients in remission stageAt the level of the phylum,Actinobacteria were positively correlated with CD19+CD27+memory B cells and IL-6,and Bacteroidetes were positively correlated with clinical indicators Ig G,Ig A,and Ig M.At the genus level,Ruminococcaceae_Ruminococcus bacteria were negatively correlated with cytokine IL-17A;Bacteroides bacteria were negatively correlated with CD4+CD45RO+CCR7+CXCR5+ central memory Tfh cells,and with CD19+CD27+Ig D+pre-switched memory B cells,CD19+CD27+Ig M+ memory B cells and clinical indicators Ig G,Ig A,and Ig M were positively correlated.Megamonas bacteria were positively correlated with CD19+CD27-Ig D-double negative memory B cells.Ruminococcaceae_Ruminococcus bacteria and Bacteroides bacteria were both negatively correlated with clinical indicator CRP.Conclusion1.Reduced abundance and diversity of intestinal flora in RA patients during active disease versus disease remission.2.Imbalance of Treg,memory Tfh and memory B cell expression in RA patients during active and remission disease.3.There is a correlation between gut microbial disorders and lymphocyte subpopulations,cytokines and clinical indicators;alterations of gut microbial composition can lead to imbalance of Treg cells,memory Tfh cells and cytokines and thus affect disease activity in RA patients.4.Intestinal flora disorders may be involved in the pathogenesis of RA and play a role in the development of the disease.5.Bacteroides,Ruminococcaceae_Ruminococcus,Megamonas and Lachnospiraceae_Clo stridium may be predictive indicators of improvement in clinical symptoms and cli nical disease activity in RA patients.