Characteristics Of Gut Microbiota Enterotype And Their Effect On Immune System And Prognosis In Patients With Autoimmune Diseases

Objective:1.To compare the diversity,abundance,metabolic pathway and enterotype characteristics of intestinal flora between patients with autoimmune disease(AID)and healthy controls(HC).2.To explore the differences of peripheral lymphocyte subsets and cytokines between AID and HC.3.To analyze the correlation between intestinal flora,lymphocyte subsets and cytokines for AID patients,and further discover the relationship between intestinal flora and AID disease activity by constructing enterotype and its characteristic E-score.4.To explore the influence of different enterotypes and E-score on joint inflammation characters,and further explore the possible mechanisms by detecting the metabolism and cytokine levels in different microbiota rodents.Methods:1.High-throughput second-generation sequencing was used to detect 16 S r RNA intestinal flora amplicons of 1102 AID and 475 HC.Microbiota Process and bray-curtis distances were applied to compare differences of alpha and beta diversity between AID and HC.PICRUSt2 was used to predict functional abundance based on 16 S r RNA sequences.STEMP was used to compare differences in species and metabolic pathways among groups.Dirichlet polynomials mixed with unsupervised clustering were used to describe the variability of microbiome data,and the intestinal flora of the subjects was classified to mine the characteristics of the intestinal flora of the subjects.2.The peripheral blood of 13937 AID patients and 267 HC was collected.Total T(CD45+ CD3+),B(CD45+ CD3-CD19 +),CD4+T(CD45+ CD3+ CD4+),CD8+T(CD45+ CD3+ CD8+)and NK(CD45+ CD3-CD16 +CD56),Th1(CD4+ INFγ+),Th2(CD4+ IL-4+),Th17(CD4+ IL-17+),Treg(CD4+ CD25+ Foxp3+)were detected by flow cytometry(FCM).The levels of peripheral cytokines such as IL-2,IL-4,IL-6,IL-10,INF-γ and TNF-α were detected by CBA.The overall difference between AID and HC was determined by independent sample T test,and ANOVA analysis of variance was performed between various AID diseases and HC.3.Pearson correlation was used to analyze the correlation between different bacteria and lymphocyte subsets and cytokines.Dirichlet polynomial and unsupervised clustering were used to describe the variation of microbiome data and mining AID intestinal type characteristics.E-score of intestinal type was constructed according to the dominant bacteria of different intestinal types to evaluate the ability of intestinal type and E-score to evaluate the response to methotrexate treatment.4.In order to reshape intestinal type,resistant starch intake was given to CIA mice.The changes of intestinal type and E-score were compared between normal diet and resistant starch intake.The arthritic index,pathological inflammatory characteristics and changes of bone mass of CIA mice with different intestinal type and E-score were also compared.Serum levels of short-chain fatty acids and cytokines in CIA mice with different intestinal types and E-score were also analyzed.Results:1.The alpha diversity of AID patients was insufficient.Detailly,the Observed,Chao1,ACE,Shannon,Simpson and Puelou’s Evenness J in AID were significantly lower than those of HC(P<0.001).The abundance and evenness of rheumatoid arthritis(RA),systemic lupus erythematosus(SLE),primary Sjogren’s syndrome(p SS),undifferentiated spondyloarthropathy(USPA),ankylosing spondylitis(AS),Behcet’s syndrome(BD)decreased significantly compared with HC(P<0.05).The evenness of polymyositis/dermatomyositis(DM/PM),mixed connective tissue disease(MCTD)and vasculitis decreased significantly(P<0.05),but there was no significant difference in the reduction of richness(P>0.05).The alpha diversity of psoriatic arthritis(Ps A)was lower,but there was no statistical difference(P>0.05).2.The beta diversity of RA,SLE,p SS,USPA,AS,BD,DM/PM,MCTD and vasculitis was different from that of HC(P<0.05),the beta diversity of Ps A and Gout was different from that of normal subjects,but there was no statistical difference(P>0.05).The difference in beta diversity between AID patients and HC was masked by differences in beta diversity between AID patients(P>0.05).3.The main bacteria at the phylum level in AID and HC feces included Firmicutes,Bacteroidetes,Proteobacteria,actinobacteria,Verrucomicrophyla,Fusobacteria,Desulphuricobacteria,Cyanobacteria,Sycomitobacteria,Archaea and Campylobacter.At the genus level,there were mainly Bacteroides,Clostridium tender,Escherichia coli,Prevotella,Roche,Acetobacter,Brucella,Microbacter,Rare micrococcus,Parabacteroides and bifidobacteria,with different relative abundance at phylum and genus level(P<0.05).4.The metabolism of the intestinal flora of AID and HC was significantly different.Compared with HC,AID patients had higher levels of lipoic acid metabolism,propanoate metabolism,taurine and low taurine metabolism,lipopolysaccharide biosynthesis,glutathione metabolism and other functions(P<0.01);but a lower level of metabolism of histidine and RNA polymerase(P<0.001).5.According to the intestinal flora characteristics of AID and HC,both AID and HC can be divided into two intestinal types(E1 and E2).At the phylum level,firmicutes dominant in E1,while and proteobacteria were dominant E2.At the genus level,E1 had more prevotella while E2 enriched in Bacteroidetes.Alpha diversity of AID and HC(P<0.001),beta diversity(P<0.001),the composition,distribution,abundance and metabolism of bacteria were significantly different between E1 and E2(P<0.05).6.Compared with HC,the numbers of T,NK and Treg cell in AID were decreased(P<0.001),Th1 and Th17 were increased(P<0.001).7.All kinds of AID patients had a lower level of Tregs,including RA,SLE,p SS,USPA,AS,BD,DM/PM,MCTD,vasculitis significantly lower than that of HC(P<0.05).8.The ratio of effector cells(Teff)to Treg showed a higher trend in AID.Compared with HC,all Teff/Treg ratios(T/Treg,B/Treg,NK/Treg,CD4T/Treg,CD8T/Treg,Th1/Treg,Th2/Treg,Th17/Treg)of AID were significantly higher than those of HC(P<0.001).The ratio of Teff/Treg in AID patients was higher than that in HC(P<0.05).9.There were differences in immune cells among different AID diseases.SLE and myositis showed a decrease in universal lymphocyte subsets,while AS,Ps A and gout mainly show an increase in Teff and a decrease in Treg.10.AID patients had higher levels of IL-2,IL-4,IL-6,IL-10,IL-17,INF-γ and TNF-α compared with those of HC(P<0.001),the levels of IL-2,IL-6,IL-17 and TNF-αwere highest in RA(P<0.001),USPA,AS and Ps A showed extremely high levels of several cytokines(P<0.001).11.Whether at the phylum level or the genus level,the intestinal bacteria in AID patients and their predicted metabolic function were correlated with the numbers of T,B,CD4+T,CD8+T,NK,Th1,Th2,Th17,Treg cells and the levels of IL-2,IL-4,IL-6,IL-10,INF-γ,TNF-α and other cytokines(P<0.05).12.E-score of intestinal type was constructed by the characteristic flora of intestinal type,which could distinguish E1 and E2.ACU under ROC curve was 93.3%(95%CI:90.2-96.3%).E-score of E1 was significantly lower than that of E2(P<0.001).13.E-score was able to effectively evaluate the response rate of MTX: the response rate of RA patients was 21.4% in high E-score group,compared with 69.2% in low E-Score group.The E-score of non-response group(NR)was significantly higher than that of response group(R)(P<0.05).14.The E-score of CIA mice on resistant starch diet was lower than that on normal diet(P<0.01),suggesting resistant starch intake induced intestinal E1 transformation.15.Compared with E2 type in CIA mice,E1 had lower paw inflammation scores,less degree of bone destruction in knee histopathology,higher bone substance content,lower ratio of bone surface area to bone volume(BS/BV),higher relative bone volume or bone volume fraction(BV/TV)(P<0.05).All these parameters were closely correlated with E-score(P<0.05).16.E1 CIA mice had higher levels of serum SCFA,propionic acid,isobutyric acid compared with those of E2(P<0.05),which was closely correlated with E-score(P<0.05).17.The serum IL-10 in E1 CIA mice was higher than E2(P<0.01).E-score was negatively correlated with IL-10(R =-0.680,P = 0.014).Conclusion:AID patients had a disturbance of intestinal flora,disorders of peripheral lymphocytes and cytokines.The disturbed flora was closely related lymphocytes and the cytokines.The intestinal flora of AID patients has inherent characteristics,which can be divided into E1 and E2 intestinal types.The E-Score can be used to evaluate the response to methotrexate treatment.High diet reduced E-Score score,induced intestinal type conversion to E1,and further improved the inflammatory symptoms of joints in CIA mice.The mechanism may be that intestinal flora with low E-score or E1 type secretes more short-chain fatty acids,stimulated the secretion of anti-inflammatory IL-10 cytokines,and finally induced the autoimmune tolerance of mice.