The Role And Mechanism Of Intestinal Flora In Graves' Disease

1 IntroductionGraves' disease(GD)is a typical organ-specific autoimmune disease which occurs frequently in young women.One of the main pathological features of GD is the presence of various antithyroid antibodies,such as thyrotropin receptor antibody(TRAb),thyroid peroxidase antibodies(TPOAb)and thyroglobulin antibody(TGAb).Among them,TRAb is the characteristic autoantibody of GD.Especially,the presence of thyroid stimulating antibody(TSAb),one type of the TRAb,is the unique characteristic of GD patients.TSAb is directly responsible for the elevated level of thyroxine and GD development by stimulating the TSH receptor.The pathogenesis of GD is very complicated,and genetic factors are currently considered to play an important role.However,the prevalence of autoimmune thyroid disease(AITD)represented by GD is only 3-9%in fraternal twins and only 20-40%in identical twins,which indicates that non-genetic factors,including the environment,also play key roles in the onset of GD.However,the exact mechanism involved in these processes has not yet been fully elucidated.Regulatory T cells(Treg)are an important subset of T helper cells and play a key role in maintaining the body's normal immune homeostasis and immune tolerance.In various autoimmune diseases such as systemic lupus erythematosus,inflammatory bowel disease,and multiple sclerosis,the function and/or number of Treg cells are significantly abnormal.As a typical autoimmune disease,Treg cells also play a key role in the occurrence and development of GD.The removal of Treg cells significantly increases the incidence and severity of autoimmune diseases including GD and autoimmune thyroiditis in mice.The number of Treg cells in the peripheral blood and thyroid of patients with GD were markedly reduced,and their functions were also significantly abnormal.Th17 cells are another important subset of T helper cells which can secrete pro-inflammatory cytokines of IL-17.Th17 cells are closely related to the development of many autoimmune diseases such as multiple sclerosis,rheumatoid arthritis and type 1 diabetes.However,the relationship between Th17 cells and GD still needs further researches to elucidate.Th17 and Treg cells are closely related and interaction with each other.The balance between them plays an important role in immune diseases.However,the molecular mechanism of abnormality in Th17 cell number and function of GD patients is still poorly understood.In order to elucidate the mechanism of Treg and Th17 cell abnormalities in GD patients,more in-depth research is needed.As an important part of the body,the intestinal flora has profound effects on the body's health and is closely related to many diseases.In recent years,a large number of studies have shown that the intestinal flora,as an important environmental factor,plays an important role in the development and function of the immune system.The intestinal flora can regulate a variety of immunoinflammatory cells including Treg and Th17 cells in the intestinal mucosa and gut-associated lymphoid tissue(GALT).Not only that,a variety of intestinal bacteria and their metabolites or constituents(such as peptide polysaccharides)can also enter the blood circulation and regulate Treg,Th17 cells and immune inflammation in extra-intestinal tissues/organs.Immune cells in the intestine such as Treg and Th17,which are affected by the flora,can also enter the blood,thereby regulating the immune inflammation of remote tissues and organs.For example,short-chain fatty acids(SCFAs)produced by intestinal microorganisms like Clostridiosis and Bacteroides can directly or indirectly regulate the size and function of Treg and Th17 cell pools in the colon and circulation.Obviously,the intestinal flora is closely related to immune tolerance,and the abnormalities of the intestinal flora play a key role in the occurrence and development of autoimmune diseases.For example,autoimmune diseases of gastrointestinal tract including Crohn's disease and ulcerative colitis are closely related to the increases in the abundances of pro-inflammatory bacteria(Escherichia coli strains)and the decreases in the abundances of anti-inflammatory bacteria(Faecalibacterium prausnitzii)in gut.In addition,a variety of parenteral autoimmune diseases like systemic lupus erythematosus,type 1 diabetes,rheumatoid arthritis,and multiple sclerosis are also closely related to gut dysbiosis.For example,Bifidobacteria and Bacteroides fragilis are significantly reduced and Prevotella copri is significantly increased in the intestine of patients with early rheumatoid arthritis.Clostridiosis and Bacteroides fragilis have protective effects on experimental autoimmune encephalomyelitis(EAE)mice.These findings indicate that the imbalance in gut bacteria composition plays an important role in autoimmune diseases,especially in Treg and Th17 cell abnormalities.Gastrointestinal dysfunction such as diarrhea is one of the important clinical features of GD,which often leads to intestinal pathogenic bacteria infection such as Yersinia enterocolitica.Many studies have shown that Yersinia enterocolitica is related to GD,but mice fed only with Yersinia enterocolitica did not develop GD.Recently,a study reported the compositional abnormalities in intestinal flora of GD patients and speculated the relationship between gut flora and GD development.However,this descriptive study did not explore the mechanisms underlying the association between GD and intestinal flora.Therefore,considering the close relationship between intestinal flora and inflammation and/or autoimmunity,very interesting questions arise as to whether gut dysbiosis is associated with abnormal immune regulation(such as the abnormalities in Treg and Th17 cells)in GD patients and what the precise mechanism underlying this association is.Therefore,more research is needed to answer the above-mentioned questionsConsidering the importance of intestinal flora in the regulation of immune function and the pathogenesis of autoimmune diseases,in order to clarify the above problems,we studied the changes in the gut microbiome of GD patients and the relationship with immunity.In this study,we first detected the changes in the ratio of Treg and Th17 cells in the peripheral blood of GD patients and their relationship with intestinal immunity Then,we used 16S rRNA gene second-generation sequencing technology and targeted/non-targeted metabolomic techniques.Upon integrating with clinical data,we discovered the association between immune dysfunctions and abnormalities in the composition and metabolism of intestinal flora in GD patients.We found that the composition of the intestinal flora and its metabolic profile of GD patients were significantly abnormal.The contents of Bacteroides fragilis and propionic acid in the intestine are significantly negatively correlated with the characteristic clinical indicator of GD(serum TRAb),which indicated the close relationship between abnormal intestinal flora and GD2 Objectives(1)Using 16S rRNA sequencing technology to identify the changes of intestinal flora composition in GD patients(2)Using targeted/non-targeted metabolomics techniques to identify the changes of intestinal metabolites profiling in GD patients.(3)Screening the abnormal intestinal bacteria and metabolite which regulate Treg and Th17 cells in GD patients.3 Methods3.1 Collection of clinical samples:In this study,58 initially untreated GD patients(GD)and 63 healthy individuals(Control)were collected.Their stool,serum and whole blood samples were collected in the morning after fasting for 8 hours.The stool samples were used for intestinal flora detection.The serum samples were used for cytokine and clinical indicators detection,and whole blood samples were used for peripheral blood mononuclear cells(PBMCs)and Treg/Th17 detection.3.2 Detection of intestinal flora:The cetyl trimethylammonium bromide(CTAB)method was used to extract fecal sample DNA.The second-generation sequencing technology was used to sequence the 16S rRNA gene sequence(V1-V2)of the intestinal flora.The composition and changes of the intestinal flora were obtained throughbiological analysis software such as QIIME1,USearch,and Calypso.3.3 Intestinal metabolite detection:The non-targeted metabolomics technology was used to detect the composition and changes of metabolites in the intestine by liquid chromatography-mass spectrometry(LC-MS).Changes in the content of SCFAs in the intestine were detected by targeted metabolomics technology using gaschromatography-mass spectrometry(GC-MS).3.4 Multi-omics data integration analysis:The factor analysis,feature selection,WGCNA,and multivariate analysis tool were used to perform the integration analysis for the 16S rRNA sequencing data,metabolomics data,and clinical indicators data of GD.We did the above integration analysis to find the intestinal bacteria and corresponding metabolites closely related to GD.3.5 Flow cytometric analysis of immune cells:Flow cytometry was used to detect changes in the proportion of Treg and Th17 cells in peripheral blood.The CCR9positive Treg and Th17 cells in peripheral blood were also detected.3.6 Enzyme-Linked ImmunoSorbent Assay(ELISA):The cytokines TGF-?,IL-10,sCD14,sCD25,IL-2,IL-17A,IL-1?,IL-6,IL-12,and IL-18 in serum were detected byELISA.3.7 Real-time quantitative polymerase chain reaction(RT-PCR):The relative abundance of Firmicutes and Bacteroidetes,the changes of SCFAs-produced bacteria in gut and the content of key enzyme genes(ButA,LcdA,PduP,and MmdA)which produce SCFAs in the intestine were analyzed by RT-PCR with the bacterial 16S rRNA gene as the internal control.3.8 Nucleic acid gel electrophoresis:Detection of Yersinia enterocolitica infection inhealthy controls,GD patients with diarrhea,and GD patients without diarrhea.3.9 Statistical analysis:The statistical analysis was executed with software SPSS 25.0 and GraphPadPrism 8.0.First,we used the Shapiro-Wilk normality test to test the data for normality distribution.For normally distributed data,which were presented as mean±SD,t-test was used for comparison between two groups,while one-way ANOVA test with post hoc test(Turky or Dunnett)for multiple testing correction was used for multi-group comparison.For data not distributed normally,which were presented as median±IQR,Wilcoxon rank sum test was used for comparison between two groups,while Kruskal-Wallis test with post hoc test(Dunn or Steel-Dwass)for multiple testing correction was used for multi-group comparison.When performing multiple comparison or statistical analysis on metabonomics data,16S rRNA gene sequencing data and other omics data,t-test or Wilcoxon rank sum test adjusted by Benjamini-Hochberg procedure with FDR<5%was used.For the statistics of categorical variables,chi-square test or Fisher's exact test is used.P<0.05 or adjusted P<0.05 was considered statistically significant.4 Result4.1 The immune and inflammatory state of the GD patients were significantly changed and these changes were associated with the intestine.We measured the Treg and Thl7 cells in the peripheral blood of healthy controls and GD patients by flow cytometry,and found that Treg cells in peripheral blood of GD patients were significantly reduced while Th17 cells were significantly increased which led to the imbalance of Treg and Th17 in GD patients(P<0.05).CCR9 is one of the most important intestinal homing markers.CCR9 positive cells indicate that they are derived from the intestine or closely related to the intestine.The changes of CCR9-positive Treg and Thl7 cells in peripheral blood are consistent with the Treg and Th17 cells(P<0.05).Furthermore,compared to healthy individuals,the levels of serum anti-inflammatory cytokines(TGF-1 and IL-10)were markedly decreased,while the levels of serum pro-inflammatory cytokines including sCD14,sCD25,IL-2,IL-17A,IL-1?,IL-6,and IL-12 were significantly increased in GD patients(P<0.05).Additionally,the serum IL-18 level,which plays pivotal roles in intestinal inflammation,was also significantly elevated in GD patients(P<0.05).4.2 Significant changes in the composition and interrelationship of intestinal flora in GD patients.The 16S rRNA sequencing results showed that the composition of intestinal flora in GD patients altered significantly,and the richness of the flora decreased significantly in GD patients(P<0.05).At the phylum level,Firmicutes were less abundant while Bacteroidetes were more abundant(P<0.05),which were further confirmed with quantitative PCR(qPCR),thereby the ratio of Firmicutes/Bacteroidetes decreased significantly in GD patients(P<0.05).The ratio of Firmicutes and Bacteroidetes reflects the homeostasis of the intestinal flora and the decreased F/B indicated that the intestinal flora in GD patients is out of balance.The abundance of SCFAs-produced bacteria in GD patients was significantly reduced compared with healthy people(P<0.05).There is the complex interrelationship among the intestinal flora in the human body.Compared with healthy controls,the interaction of the flora in the intestine of GD patients has also changed significantly.Correlation analysis with clinical indicators showed that the SCFAs-produced bacteria are closely related to clinical indicators(T3,T4,TSH and TRAb)of GD.The result of random forest showed that a group of intestinal bacteria(Bacteroides,Alistipes,Prevo(ella)could accurately differentiate GD patients from healthy individuals with 85%accuracy.These results fully demonstrated that the intestinal flora of GD patients has changed significantly,where these changes are closely related to GD4.3 Gut metabolites including SCFAs significantly changed in GD patients.Gut bacteria,as producer and consumer of metabolites in the intestine,is closely related to the metabolites in gut.The synthesis and degradation of most metabolites in the intestine have the participation of the intestinal flora.Whether the altered intestinal flora causes changes in the metabolites.The metabolomic analysis performed by mass spectrometry showed that significant changes in metabolites in the intestine of GD patients were found.Propionic acid and butyric acid,two important immunomodulatory SCFAs,significantly decreased in GD patients compared with those in healthy individuals(P<0.05).In addition,the results of PICRUSt showed that the predicted relative abundances of butanoate and propanoate metabolism were significantly lower in GD patients(P<0.05).Through further analysis of the correlation with clinical indicators,it was found that SCFAs and clinical indicators(T3,T4,TSH and TRAb)of GD patients were strongly correlated4.4 Close correlations among clinical features of GD,SCFAs,and SCFA-producing bacteria.The results of correlation analysis between intestinal metabolites,intestinal flora and GD clinical indicators showed that propionate and butyrate were significantly positively correlated with many SCFA-producing bacteria including Roseburia,Bacteroides,and Phascolarctobacterium.In addition,propionate which significantly decreased in the gut of GD patients was strongly negatively correlated with the serum levels of TPOAb,FT3,FT4,and TRAb,while strongly positively correlated with the serum level of TSH,which suggested that propionate was closely related to the condition of patients with GD.ButA(butyryl-CoA transferase),LcdA(lactoyl-CoA dehydratase),PduP(propionaldehyde dehydrogenase),and MmdA(methylmalonyl-CoA decarboxylase)are the key enzymes to produce butyrate and propionate in gut flora.In intestinal flora of GD patients,the abundances of these key enzyme-encoding genes were all significantly decreased(P<0.05),confirming the significant reduction in their SCFA-producing ability and resulting in the lack of SCFAs in GD patients.The qPCR using the primers specific for the representative bacteria of SCFA-producing genera showed that the abundance of Bacteroides fragilis species was the most closely related to the levels of TRAb,FT3,FT4,and TSH and decreased with the most statistical significance in the intestine of GD patients(P<0.05).The above results indicate that disturbed gut flora(decreased abundance of Bacteroides fragilis)and changes of microbial metabolite profiling(decreased propionic acid)are closely related to GD5 Conclusions(1)The immune function of GD patients is disordered,including the decrease of Treg cells and the increase of Th17 cells,and this immune change is inseparable from intestinal tract.(2)The composition of the intestinal flora and the metabolite profiling characteristics of GD patients have changed significantly,especially the significantly reduced content of SCFAs-producing intestinal bacteria and SCFAs.(3)The decreased abundance of Bacteroides fragilis and reduced level of propionic acid in gut are closely related to clinical indicators of GD patients1 IntroductionGraves' disease(GD)is a typical organ-specific autoimmune disease which occurs frequently in young women.One of the main pathological features of GD is the existence of a variety of thyroid tissue-specific autoantibodies,the most important of which is thyroid stimulating hormone receptor antibody(TRAb).The higher TRAb level plays an important role in the pathogenesis of GD.In addition,the abnormality of TRAb is closely related to the immune status of the body.Many studies have shown that high levels of TRAb are often accompanied by abnormal numbers and/or functions of Treg and Th17 cells.Studies have shown that the abnormalities of Treg and Th17 cells may play an important role in the pathogenesis of GD and are closely related to the condition of GD.However,the relationship between Treg/Th17 cells and GD,and the molecular mechanism still needs to be further studiedThe human intestine harbors a complex population of microorganisms,collectively known as the gut microbiota,which co-evolved with the host by maintaining a symbiotic relationship.Gut microbiota plays a key role in the development and functional regulation of the host immune system.A number of studies have shown that gut microbiota is closely associated with the balance of Th17 and Treg.Th17 and Treg cells are two vital T cell subsets with opposing functions.Studies have shown that abnormal ratios of Th17 and Treg cells often exist in metabolic or immune-related diseases,including chronic inflammation,allergic diseases,autoimmune diseases and cancer.Understanding the relationship between specific gut microbiota organisms and the balance of Th17 cells and Tregs would therefore benefit the cure of a range of diseases caused by the imbalance of these two cell subsets.In addition,studies have shown that Yersinia enterocolitica is closely related to GD and is an important cause of GD.Taking into account the complexity of human intestinal flora and its close relationship with inflammation and autoimmunity,we believe that it is a complicated process for intestinal flora factors to affect the pathogenesis of GD.The imbalance of intestinal flora in GD patients is related to abnormal immune regulation(such as abnormal Treg and Th17 cells),but the mechanism behind this relationship is unclear.Short-chain fatty acids(SCFAs)are the main end products of indigestible carbohydrates(NDC)fermented by the intestinal flora,mainly including acetic acid,propionic acid and butyric acid.More and more studies have shown that SCFAs are closely related to the health of the body.As the preferred fuel of the epithelial cells of the colon,butyric acid is an important substrate for maintaining colonic epithelial function.In addition,SCFAs are natural ligands of free fatty acid receptor 2 and 3(FFAR2 and FFAR3),which play an important role in the body's lipid metabolism and energy homeostasis.Moreover,FFAR2 and FFAR3 were found in a variety of cell types including enteroendocrine and immune cells,suggesting that SCFAs may play a role in regulating the immune system and inflammatory response.Recently,two studies have emphasized the role of propionic acid and butyric acid in the production and function of regulatory T cells by inhibiting histone deacetylase(HDAC).In metabolic diseases(such as type 2 diabetes),a decrease in the number of propionate-producing bacteria has been observed,which is a disease characterized by low-grade inflammation Therefore,the growing evidence seems to support that SCFAs play a key role in shaping the local and peripheral immune systems,which affect host immune homeostasis through inflammatory pathwaysBacteroides is the genus with the highest content in the human intestine,accounting for about 25%of the intestinal flora and Bacteroides.fragilis is one of the most common components in the genus Bacteroides.This gram-negative obligate anaerobe is common in the human intestinal flora,and it also exists in the oral cavity,upper respiratory tract and female reproductive tract.More and more studies have shown that Bacteroides fragilis may be a probiotic in the human intestine,and it has been found that Bacteroides fragilis is closely related to many diseases,including type 2 diabetes,hypertension and obesity.In addition,recent studies have shown that Bacteroides fragilis is very important to the immune status of the body.Polysaccharide A(PSA)from Bacteroides fragilis has been identified as an immunomodulatory molecule that plays an important role in the maturation of the immune system.In addition,the intestinal flora including Bacteroides fragilis can also ferment dietary fiber to produce short-chain fatty acids(SCFAs),which have beneficial functions such as providing energy for the colonic mucosa and maintaining a stable colonic environment.For example,oral supplementation of Bacteroides fragilis can significantly increase the concentration of SCFAs in the intestinal contents of rats infected with Salmonella,thereby further reducing inflammation and restoring the integrity of the intestinal barrier.Intestinal flora,especially Bacteroides fragilis,plays an important role in the body's immune inflammation,but their role in the pathogenesis of GD has not been reported yet.Our previous study found that the content of Bacteroides fragilis in the intestine of GD patients significantly decreased,and it is closely related to the clinical indicators of GD.Considering the importance of the gut microbiome in inflammation and autoimmune diseases,as well as the results of our previous research,we isolated Bacteroides fragilis from the human intestine.It has been verified at the cellular level that the decrease in propionic acid levels caused by the decrease in the abundance of Bacteroides fragilis in the intestines of GD patients is an important mechanism for the imbalance of Treg/Th17 cells.Through the further fecal microbiota transplantation(FMT)experiments,it was confirmed that the abnormal intestinal flora would promote the onset of GD,and it was proved that supplementing Bacteroides fragilis would significantly improve the immune status of GD mice.2 Objectives(1)To study the role of Bacteroides fragilis and propionic acid from the intestine in GD pathogenesis.(2)Explore the molecular mechanisms of Bacteroides fragilis and propionic acid on Treg/Th17 cells.(3)To clarify the role of intestinal flora imbalance in the pathogenesis of GD in GD mice model.(4)Explore the effect of Bacteroides fragilis on improving GD mice immune status and clinical symptoms.3 methods3.1 Collection of clinical samples:In this study,15 initially untreated GD patients and 13 healthy controls were collected.Their stool and whole blood samples were collected in the morning after fasting for 8 hours.The stool samples were used for fecal microbiota transplantation(FMT)experiments.PBMCs(Peripheral blood mononuclear cells)from whole blood samples was used to study the mechanism of Bacteroides fragilis and propionic acid on Treg and Th17 in vitro.3.2 Flow cytometric analysis of immune cells:Flow cytometry was used to detect changes in the proportion of Treg and Th17 cells in PBMCs cultured in vitro and in peripheral blood of mice3.3 ELISA test:The cytokines(IL-10,and IL-17A)and clinical indicators(T4 and TRAb)in serum or in vitro culture supernatant were detected by ELISA.3.4 Intestinal bacterial isolation and culture:Bacteroides fragilis YCH46 strain was isolated from adult healthy human feces using selective medium(M2GSC),and cultured and expanded under anaerobic environment.3.5 Real-time quantitative polymerase chain reaction(RT-PCR):The changes of Bacteroides fragilis YCH46 strain in the intestine of healthy people/GD patients,were analyzed by RT-PCR with the bacterial 16S rRNA gene as the internal control.The mRNA expression of FFAR2,HDAC6,and HDAC9 in PBMCs stimulated by Bacteroides.fragilis YCH46 from healthy people/GD patients in vitro was detected with the human ?-actin(ACTB)as the internal control.3.6 Cell experiment:The culture supernatant of Bacteroides fragilis YCH46 strain stimulated PBMCs from healthy people and GD patients in vitro.We studied the differentiation of Treg and Th17 cells and the possible mechanism3.7 Quadruple antibiotics to clear the intestinal flora of mice:Mice are administered with a quadruple antibiotic(ampicillin,neomycin,metronidazole and vancomycin)for five days,and all the four antibiotics are added to the drinking water.Antibiotics treatment lasted for two weeks to achieve the purpose of removing intestinal flora in mice3.8 Fecal microbiota transplant(FMT):Fresh feces from healthy controls and GD patients were collected,mixed with PBS and filtered to make a bacterial suspension.The suspension was transplanted to mice treated after antibiotics3.9 Sequencing of intestinal flora:The second-generation sequencing technology was used to sequence the 16S rRNA gene sequence of the intestinal flora from the donor individuals and recipient mice in the FMT experiment before and after antibiotic treatment.The composition and changes of the intestinal flora were obtained through biological analysis software such as QIIME2,USearch,and Calypso.3.10 GD mice model construction:Ad-TSHR289 was used to construct the GD mice model.The mice were first treated with antibiotics and then FMT was used to colonize the flora.Then,adenovirus injection was performed at intervals of three weeks for one injection.At the three weeks after the second adenovirus injection,TT4 and TRAb in the serum of the mice were measured to determine the success of GD mice model3.11 The effect of Bacteroides fragilis YCH46 strain on the immunity of GD mice:The GD mice were gavage with the Bacteroides fragilis YCH46 strain to observe the changes of clinical and immune indexes in the peripheral blood of mice.3.12 Statistical analysis:The statistical analysis was executed with software SPSS 25.0 and GraphPadPrism 8.0.First,we used the Shapiro-Wilk normality test to test the data for normality distribution.For normally distributed data,which were presented as mean±SD,t-test was used for comparison between two groups,while one-way ANOVA test with post hoc test(Turky or Dunnett)for multiple testing correction was used for multi-group comparison.For data not distributed normally,which were presented as median±IQR,Wilcoxon rank sum test was used for comparison between two groups,while Kruskal-Wallis test with post hoc test(Dunn or Steel-Dwass)for multiple testing correction was used for multi-group comparison.For the statistics of categorical variables,chi-square test or Fisher's exact test is used.P<0.05 or adjusted P<0.05 was considered statistically significant.4 Results4.1 Bacteroides fragilis was isolated from the intestine and cultured in vitro.According to the correlation analysis with clinical indicators,we found that Bacteroides fragilis,an intestinal bacterium which can produce SCFAs and reduced in GD patients,was most closely related to GD.The strains of Bacteroides fragilis were isolated from healthy individual feces using a selective culture medium M2GSC under anaerobic conditions and confirmed as Bacteroides fragilis YCH46 strain.RT-PCR also showed that the abundance of Bacteroides fragilis YCH46 strain in GD patients was significantly reduced compared to that in healthy controls(P<0.05),which was consistent with the trend of Bacteroides fragilis in gut.4.2 Bacteroides fragilis modulates Treg/Th17 balance through the pathway regulated by propionic acid.The supernatant of M2GSC medium culturing Bacteroides fragilis YCH46(B.f.S)could significantly increase the percentage of Treg cells and IL-10 level,while reduce the percentage of Th17 cells and IL-17A level in PBMCs from healthy individuals and GD patients in vitro(P<0.05).Our results showed that the expression of FFAR2(a propionic acid receptor)mRNA was markedly upregulated,while the expression of histone deacetylase 6 and histone deacetylase 9(HDAC6 and HDAC9)mRNA,which are the downstream molecules of FFAR2 and could suppress Treg cells,were downregulated in PBMCs from both healthy individuals and GD patients treated with B.f.S(P<0.05).These results indicated that Bacteroides fragilis YCH46 could influence the differentiation and/or proliferation of Treg and Th17 cells through the pathway regulated by propionic acid,and then modulate the Treg/Th17 balance.These results indicate that Bacteroides fragilis YCH46 exerts a role in regulating Treg and Th17 cells through propionic acid4.3 Intestinal flora contributed to the development of GD revealed by fecal microbiota transplantation in mouse model.In the process of creating GD mice model using Ad-TSHR289,we found that the GD incidence in group transplanted intestinal flora from GD patients was higher than group transplanted intestinal flora from healthy human(P<0.05).And during the process of creating GD mice model,the transplantation of intestinal flora from GD patients significantly elevated the level of IL-17A,whereas decreased the IL-10 level compared to transplanting the intestinal flora from healthy individuals in serum(P<0.05).These results indicated that the intestinal flora as an environmental factor can promote the onset of GD in mice and aggravate the immune response in mice.4.4 Adjusting the intestinal flora can improve the immune status of GD miceThe GD mice were gavage with Bacteroides fragilis YCH46,and it 'was found that in the third week after gavage,supplementing Bacteroides fragilis YCH46 could increase the proportion of Treg cells while reduce the proportion of Th17 cells in the peripheral blood of mice(P<0.05),and increase the IL-10 level while decrease the IL-17A level in the serum(P<0.05).Although supplementation of Bacteroides fragilis YCH46 to GD mice could not significantly reduce the clinical indicators(TT4 and TRAb)in serum,both of these indicators showed a downward trend5 Conclusion(1)Bacteroides fragilis YCH46 which significantly reduces in the intestine of GD patients can regulate the differentiation of Treg and Th17 cells by producing propionic acid.(2)Intestinal flora,as a predisposing factor,can significantly increase the incidence of GD in mice in the presence of Ad-TSHR289.(3)Supplementing the Bacteroides fragilis YCH46 can significantly improve the immune disorders of GD mice.(4)The intestinal flora dysbiosis is one of the important causes leading to GD.