Changes In Gut Microbiota In Patients With Immune-mediated Inflammatory Diseases And The Dynamic Effects Of Smoking And TNF-? Antagonist On Gut Microbiota

Immune-mediated inflammatory diseases?IMID?are a group of highly disabling diseases that chronically affect different organs and systems.It is characterized by a common inflammatory mechanism and immune disorders.Chronic inflammation is the basic manifestation of the disease.IMID is composed of more than 100 diseases,including inflammatory bowel disease?IBD?,rheumatoid arthritis?RA?,ankylosing spondylitis?AS?,multiple sclerosis,psoriasis,etc.,involving digestion,rheumatism,skin,nerves,endocrine,ophthalmology and other disciplines.The etiology is unknown.At present,it is believed that the complex interaction between genetic factors and environmental factors leads to immune disorder and disease.IMIDs have a tendency to cluster in families,and therapies bio-targeting specific sites have similar effects on these diseases.Environmental factors?smoking,drugs,dysbiosis,diet,etc.?play a key role in the pathogenesis.In this study,three environmental factors?microbiota,drugs,smoking?were studied to demonstrate whether different IMIDs have different dysbiosis? Do smoking,drugs and gut microbiota interact? Is the response of patients to TNF-? antagonists influenced by microbiota? These hypothesis are supported by the following studies.Objectives:To study the dynamic effect of tumor necrosis factor?TNF?-? antagonist on the gut microbiota of AS patients,to find out the sensitive bacteria responsed to the therapy;to study the effect of smoking on the gut microbiota and the efficacy of TNF-? antagonist in AS patients;to study the characteristics of gut microbiota of three common IMIDs?IBD,AS,RA?and healthy controls?HC?,to find out the biomarker bacteria;to explore the microbial indicators for the efficacy and disease activity monitoring.The purpose of this study is to provide experimental basis for controlling the development of disease by correcting dysbiosis in the future,andimproving the accurate clinical usage of TNF-? antagonists and investigating the pathogenesis of IMID.Methods:?1?The 16 S rRNA high-throughput sequencing was performed for 40 stool samples collected from patients with three IMIDs and healthy controls?8 IBD,10 AS,12 AS,and 10 HC?by Mi Seq method;?2?98 fecal specimens were collected from 20 patients with AS at 4 different time points of treatment with TNF-? antagonist for 6months?baseline,treatment 1,3,and 6 months were defined as AS0,AS1,AS3,and AS6 groups,respectively?and 20 health controls with gender and age matched with AS,each group was divided into two groups according to whether the subjects smoked: smoking group?s?and non-smoking group?ns?.16 S rRNA high-throughput sequencing was performed using HiSeq method;The composition,relative abundance and diversity of gut microbiota were analyzed by bioinformatics methods;The LEfSe method was used to search for microbial markers of disease;The effects of smoking on the efficacy of TNF-? antagonist and the dynamic effects on gut microbiota were analyzed.Results:Part ?: Comparison of the characteristics of gut microbiotain in three IMIDs?There are statistical differences in the following results.?1.At genus level,the number and composition?such as p_Proteobacteria?of the gut microbiota in IBD was significantly lower than that in HC and RA groups.2.Relative abundance of gut microbiota: at genus level,?1?HC>AS in 3 genera,Blautia,Coprococcus₃,etc;?2?HC > IBD in 11 genera,Coprococcus₁,Roseburia,etc.;?3?RA>HC in 3 genera,Parabacteroides,etc.;?4?RA>AS in 4genera,Barnesiella,Eggerthella,etc.;?5?RA > IBD in 14 genera,Barnesiella,Eggerthella,Prevotella₉,etc.;?6?IBD>RA Escherichia-Shigella;?7?IBD>AS Streptococcus.3.Diversity of microbiota,? and ? diversity in IBD < RA,IBD < HC.4.Biomarkers of each group: g_Coprococcus and g_Streptococcus in IBD group;g_Bifidobacterium,g_Barnesiella,etc.in RA group;g_Dialister,g_Prevotella₉,etc.in AS group;f_Lachnospiraceae in HC group.Part ?: Dynamic effect of TNF-? antagonist on gut microbiota in patients with ankylosing spondylitis1.Dynamic changes in the relative abundance of gut microbiota in AS patients treated with TNF-? antagonists for 6 months: the relative abundance fluctuates above the normal level in 6 bacteria,and fluctuate below the normal level in 4 bacteria;After1 month of treatment,the abundance of g_Bifidobacterium rose to normal levels,and g_Klebsiella dropped below baseline;According to above characteristics,the flora is divided into: Sensitive bacteria?the abundance reaches normal level after therapy?,such as g_Escherichia-Shigella ? g_Klebsiella,etc.;Insensitivity bacteria?no significant change in abundance after therapy?,such as g_Dialister?f_Prevotellace,etc.;Resistant bacteria?abundance away from normal level after therapy?,as g_Collinsella?f_Actinomycetaceae,etc.2.Bacterial diversity: ? diversity was higher in AS0 group than in HC,there was no difference after therapy;? diversity was lower in AS than in HC group,and normal after one month of treatment;that was higher in ASns than in ASs,higher in HCns than in HCs,and lower diversity in smokers.3.During treated with TNF-? antagonist in AS patients,disease activity indexes showed dynamic changes,which correlated with the relative abundance of gut microbiota.The thoracic expansion intensity began to increase in the second week,and the other eight indexes began to decrease in the second week.Among them,MASES fell fastest.The correlation analysis showed that 30 bacteria were associated with 10 disease activity indices.4.Functional gene prediction: The functional genes of AS were significantly enriched in metabolic pathways in membrane transport,carbohydrate and amino acid metabolism,drug resistance,etc.;that of HC group were enriched in cell motility,signal transduction,lipid and amino acid metabolism,cell cycle control,cell division,gene replication,recombination and repair,etc.Part ?: Effect of smoking on gut microbiota and TNF-? antagonists in AS patients1.The Smokers shared g_Ruminiclostridium₉ and g_RuminocaceaeUCG-003 as common bacteria.ASs shared g_colinsella and g_dorea.2.The dynamic effect of smoking and TNF-? antagonist on microbiotaabundance in AS: The increase or decrease of microbiota abundance in AS patients is affected by smoking or not.The abundance of ASns tends to increase,and the ASs tends to decrease.3.The improvement rate of ASDAS in ASns at 6 months was higher than that in ASs?2.297 vs 1.736?.There are 35 correlations between the relative abundance of 30 bacteria at genus level and the improvement rate of ASDAS in AS.76.29% of related bacteria belong to p_Firmicutes,65.38% were in ASns group.Conclusions:This study confirmed for the first time: 1.Smoking can reduce the diversity of gut microbiota,affect the composition and abundance ofmicrobiota,reduce the efficacy of TNF-? antagonist and the sensitivity of flora to TNF-? antagonist.2.The response of gut microbiota to the treatment of TNF-? antagonists is heterogeneous.According to this,the flora can be divided into sensitive bacteria,insensitive bacteria and resistant bacteria.3.The common bacteria of smoking in AS patients were g_Collinsella and g_Dorea.4.The relative abundance of gut microbiota in AS patients changed dynamically during the treatment with TNF-? antagonist.The diversity of gut microbiota in AS patients decreased,and TNF-? antagonist treatment can make them return to normal.This study also found that: 5.The three IMIDs?IBD,AS,RA?showed dysbiosis,and that were similar in IBD and AS patients.The biomarker bacteria of each disease were different.6.The microbiota highly related to AS disease activity indices can be used as microbial indicators for disease activity monitoring.The findings of this study have theoretical significance for further study of the pathogenesis of IMID,and have potential practical value for improving the clinical application of TNF-? antagonists.