Characteristics And Association Analysis Of Intestinal Flora And Serum Metabolites In Patients And Mice With Autoimmune Thyroiditis

Objective: Autoimmune thyroiditis（AIT）is the most common organ-specific Autoimmune disease,accounting for about 5% of the world population,Hashimoto’s thyroiditis（HT）is the most common AIT.Although the underlying causes of HT have not been fully elucidated,it has been pathologically established that the interaction of susceptibility genotypes and environmental triggers is the main cause of the disease.In recent years,with the deeply understanding of human intestinal flora,people have gradually discovered the important role of intestinal flora in various diseases.The intestinal tract contains 70% of the body’s immune cells,playing a powerful immune role,which is the most complex microecosystem hidden in the human body.Recently,domestic and foreign scholar have repeatedly proposed the influence of intestinal flora on autoimmune diseases,and the extensive relationship between intestinal flora and AIT has been gradually discovered.NOD.H-2^h4 mice is classic animal model of Spontaneous autoimmune thyroiditis（SAT）,in high iodine feed cases,thyroid tissue in mice immune pathological type similar to HT,lymphocyte infiltration and follicular cell damage,accompanied by thyroglobulin antibody in serum（TgAb）drop degrees higher.the effect of iodine is closely related to the development of the AIT.Recent years,articles have reported relationship between iodine and the intestinal flora.However,no one has studied whether iodine,intestinal flora and AIT are related to each other.The purpose of this study is to explore the relationship between intestinal flora and AIT in human and animal models by using microbial flora sequencing technology.In the population experiment,the characteristics of intestinal flora and serum metabolism in the AIT population were defined,and the association between intestinal flora and thyroid related indicators in the population was explored.In animal experiments,the characteristics of intestinal flora of AIT model mice were studied to explore the correlation between the degree of thyroiditis,iodine nutrition and other indicators and intestinal flora,so as to provide a basis and direction for further research on intestinal flora and thyroid diseases.Methods: 1.Experimental study on HT patients: In the first part of this study,HT patients（n=28）and healthy volunteers（n=25）were collected as controls strictly according to the inclusion and exclusion criteria of the intestinal flora study.Basic information and thyroid clinical indicators of the subjects were recorded,and stool and blood samples were collected according to the procedure for 16 S r DNA sequencing and non-targeted metabonomics determination,respectively.All the above data were integrated and bioinformatics statistical analysis was performed.2.AIT animal model test: NOD.H-2^h4 mice fed with high iodine were used to construct AIT model.NOD.H-2^h4 mice were set in the high iodine intervention group（HI）and non-intervention control group（CT）.The high iodine intervention began at 6 weeks after birth and lasted for 10 weeks.After the intervention,urine and feces of mice in each group were collected according to the standard.Tissue of spleen,thyroid and peripheral blood of mice were collected after anaesthetizing and death.The body weight and thyroid wet weight of the mice were weighed.The infiltration of lymphocytes in thyroid tissue was observed by HE staining.The titer of TgAb in serum of mice was determined by ELISA.The proportion of Th1,Th2,Th17 and Treg cell subsets in the spleen of mice was determined by flow cytometry.Iodine content in urine of mice was determined by ICP-MS.16 S rDNA sequencing of mouse feces was performed.Statistical analysis was conducted after the integration of the above information.Results: 1.Experimental study on HT patients: serum thyroid autoantibodies in HT patients were significantly higher than those in the healthy control group,and the indexes of FT3,FT4 and TSH of all subjects were within the normal reference range.Based on16 S r DNA sequencing results,we found that there were 600 same OTUs in the HT group and the control group,119 unique OTUs in the HT group and 646 unique OTUs in the control group,suggesting that there were differences in intestinal flora composition between the two groups.The α diversity of intestinal flora in HT group decreased,and the Observed_species index,Chao1 index,ACE index and PD_whole_tree index were all lower than those in healthy control group.PCo A analysis showed a tendency of separation,suggesting that there were differences in intestinal flora between the two groups.The abundance of bacteria showed that the proportion of Firmicutes/Bacteroidetes in HT group was higher than that in healthy control group.Through Lefse analysis and T test,we found a total of 8 different bacteria genera between groups,among which the different bacteria were mainly concentrated in Lachnospiraceae.After Spearman correlation analysis between the intestinal flora of high abundance and clinical indicators,we found that a total of and 14 bacteria genera were significantly correlated with thyroid autoantibodies,among which Collinsella,Adlercreutzia,and downstream bacteria genera of Trichelicaceae,including Lachnoclostridium,Dorea,and Romboutsia,were significantly positively correlated with thyroid autoantibodies.In terms of untargeted metabolomics,the partial least squares discriminant analysis（PLS-DA）showed that there was a distinct tendency of separation between HT group and healthy control group,suggesting a significant difference between the two groups.Combined with the PLS-DA model,the variables that contributed the most to the classification（VIP>1）A total of 119 differential metabolites were screened out as important variables（all P>0.05）.Spearman correlation analysis was performed between serum differential metabolites detected by metabonomics and all clinical indicators.Eighty metabolites were positively correlated with at least one HT autoantibody,and 33 metabolites were negatively correlated with at least one HT autoantibody.Annotation of metabolites in The HMDB（The Human Metabolome Database）Database,these differential metabolites are mainly lipid metabolism molecules,benzene ring compounds and amino acid compounds.Compared with the healthy control group,the levels of different kinds of fatty acids in serum were increased in HT group,and there was a significant positive correlation with thyroid autoantibodies.Metabolic pathway analysis of differential metabolites showed that there were abnormalities in cortisol synthesis and metabolism and metabolic pathway of Cushing’s syndrome in the HT group.2.AIT animal model test: After 10 weeks of high iodine feeding,there was no statistical difference in the body weight of NOD.H-2^h4 mice between the two groups;The wet weight and relative weight of thyroid gland in HI group were higher than those in CT group（P<0.05）;The degree of inflammatory infiltration of thyroid cells,the incidence of AIT and the histological score of thyroid inflammation in HI group were significantly higher than those in CT group（P<0.05）;The serum TgAb titer and urinary iodine concentration in HI group were significantly higher than those in CT group（P<0.05）;The proportion of CD4⁺CD25⁺Foxp3⁺Treg cells in HI group was significantly lower than that in CT group.The proportion of CD4⁺IL-17A⁺Th17 cells in HI group was significantly higher than that in CT group.The proportion of CD4⁺IFNγ⁺Th1 cells in HI group was significantly higher than that in CT group（All P<0.05）.Based on 16 S rDNA sequencing results,we found that there were 607 OTUs in the HI group and the control group,243 unique OTUs in the HI group and 281 unique OTUs in the control group,suggesting differences in intestinal flora composition between the two groups.The αdiversity of intestinal flora in HI group was lower than that in CT group,but the difference was not statistically significant.PCo A analysis showed a tendency of separation,suggesting that there were differences in intestinal flora between the two groups.Through Lefse analysis and T test,we found a total of 6 bacteria genera with differences between the groups.After Spearman correlation analysis of the intestinal flora with high abundance and clinical indicators,we found that 8 bacterial genera were significantly correlated with histological score;and 13 bacterial genera were significantly correlated with TgAb;17 bacterial genera were significantly correlated with urinary iodine concentration.Conclusions: The intestinal flora of patients with AIT has its own characteristics in terms of its outcome and function,which is significantly different from that of normal population.Our study found that the changes in intestinal flora of HT patients were mainly concentrated in Lachnospiraceae and its downstream bacteria,Collinsella and other bacteria genera.The increase in the abundance of these bacteria genera has also been widely reported in other diseases,which may be one of the important links causing HT.We found that serum lipid metabolism was disturbed in HT patients,and serum fatty acids were significantly enriched,which were positively correlated with HT antibodies.In the animal model,iodine intervention had a significant impact on intestinal flora,and we found that the downstream bacteria of the family Lachnospiraceae were also significantly positively correlated with urinary iodine,TgAb,histological score and other indicators,which was similar to the human experiment.This may be the key point for iodine to play a mitigating role in the pathogenesis of AIT.