Alterations Of Gut Microecological Characteristics And Oral Microecological Characteristics In Patients With Autoimmune Hepatitis

BackgroundAutoimmune hepatitis（AIH）is a chronic,immune-mediated nonspecific liver inflammation.Epidemiological statistics show that the incidence of AIH in China is up to 20/100000,and the average annual incidence rate of white people in northern Europe is 1.07-1.9/100000[1].Its etiology is correlated with several genetic and environmental factors but has not yet been fully elucidated.AIH is globally distributed and occurs in children and adults.It is characterized by etiological and clinical heterogeneity and is distinct from other hypothetical autoimmune liver diseases such as primary bile cirrhosis（PBC）and primary sclerosing cholangitis（PSC）.Long-term untreated AIH is associated with increased risk of cirrhosis and/or hepatocellular carcinoma（HCC）with extremely high mortality and,therefore,rapid and timely diagnosis is important[2].Although the clinical diagnostic criteria for AIH have been established and widely validated,due to the variability of autoantibodies,there are still some atypical autoimmune hepatitis such as autoantibody-negative AIH,drug-induced AIH,similarities among AIH,PBC,and PSC,overlap syndrome（OS）,and post-transplant AIH,which are difficult to rely on existing AIH diagnostic criteria for diagnosis or observational efficacy[3].Recently,the vigorous development of the microbial field has continuously improved the status of microorganisms in the pathogenesis,diagnosis or treatment of many diseases.At present,the microbial community in the human body has been considered as a very vulnerable part of the surrounding environment,and thousands of microorganisms are gradually forming a unique system that imperceptibly affects the immune function and metabolism of the host[4].The alterations in the intestinal flora characterized by perturbations in microbial composition and function were associated with autoimmune diseases such as rheumatoid arthritis（RA）[5],autoimmune liver disease（AILD）[6,7],multiple sclerosis（MS）[8],and type 1 diabetes（T1D）[9].Over time,the position of human microbiota in the field of prevention and treatment of a variety of diseases is constantly increasing rapidly,and more and more experts are setting their sights on the relevant research of human microbial communities.Considering the close association between the human microecosystem and some chronic liver diseases found in numerous studies in recent years,we have the following question:Are the composition and distribution of gut and oral microbial communities in autoimmune hepatitis（AIH）patients different from those in the healthy individuals?If the differences exist,is it possible to use gut or oral microbes as noninvasive biomarkers to differentiate autoimmune hepatitis patients from healthy individuals?First of all,relevant experiments based on mouse models have laid the foundation for gut microbiota to participate in the process of AIH occurrence and development[10].In addition,a recent study sequenced the 16S rRNA gene in 91 AIH patients and 98 healthy controls（HCs）,and found that the intestinal microbial alpha diversity in AIH patients was low and the microbial composition was significantly different from that in HCs[6].Previous experiments and studies have supported the importance of gut microbiota for AIH diagnosis.Secondly,as for oral microecology,dysbiosis of oral microbial flora not only contributes to oral diseases such as dental caries,periodontitis,oral mucosal diseases,but also concerns with systemic diseases including gastrointestinal system diseases like inflammatory bowel disease（IBD）（Said HS,2014）[11],liver cirrhosis（LC）（Bajaj JS,2015）[12],pancreatic cancer（Ren Z,2017）[13],nervous System diseases like Alzheimer’s disease（AD）（Aguayo S,2018）[14],endocrine system diseases like diabetes（Xiao E,2017）[15],adverse pregnancy outcomes（Corwin EJ,2017）[16],obesity and polycystic ovary syndrome（Akcali A,2014）[17],immune system diseases like rheumatoid arthritis（RA）（Zhang X,2015）[18]and cardiovascular system diseases like atneroselerosis（AS）（Koren O,2011）[19].A large number of published literatures have shown that oral microbes play an important role in the development of many diseases,but so far,the oral microorganisms of patients with AIH have not been scientifically studied,and there is a lack of adequate systematic randomized controlled trials in this field.Therefore,our study of oral microorganisms in the field of AIH is well-founded and necessary.Moreover,regional differences are important influen cing factors for gut microbiota differences[20].Herein,We aimed to determine the difference between gut and oral microbiota between AIH patients and healthy individuals（HCs）in central China and to verify the effectiveness of gut and oral microbiota as a diagnostic tool for AIH.ObjectiveWe aimed to determine the difference between gut and oral microbiota between AIH patients and HCs in central China and to verify the effectiveness of gut and oral microbiota as a diagnostic tool for AIH.MethodsWe prospectively collected 115 fecal samples and 138 saliva samples in the infectious diseases department and physical examination department of the first affiliated hospital of zhengzhou university,including stool samples from patients with aih（n=37）,stool samples from matched healthy controls（n=78）,saliva samples from patients with AIH（n=46）and saliva samples from matched HCs（n=92）.16S rRNA gene sequencing was performed on all fecal and saliva samples.Subsequently,to explore microbial composition characteristics of feces and saliva,and identify microbial markers of AIH,we performed a series of microecological data analyses,including species composition analysis,alpha diversity analysis,and beta diversity analysis.The specific steps were as follows:（1）screening and clinical characteristics of participants;（2）samples collection and DNA extraction;（3）PCR amplification,MISEQ sequencing;（4）sequence data processing;（5）bacterial diversity and taxonomic analysis;（6）gene function prediction;（7）determination of OTU biomarkers and disease probability;and（8）statistical analysis.Results1.Fecal microbial diversity showed a downward trend in AIH compared with HCs.Fecal microbial communities significantly differed between both groups.At the phylum level,Verrucomicrobia abundance was significantly increased,while Lentisphaerae and Synergistetes were significantly decreased in the AIH patients versus the HCs group.Compared to the HCs group,fifteen genera,including Veillonella,Faecalibacterium and Akkermansia,were enriched,while nineteen genera,such as Pseudobutyrivibrio,Lachnospira and Ruminococcaceae,were decreased in the AIH patients.Five microbial biomarkers including Lachnospiraceae,Veillonella,Bacteroides,Roseburia and Ruminococcaceae were deemed optimal diagnostic tools for AIH through a fivefold cross-validation on a random forest model.The probability of disease（POD）was significantly increased in AIH group versus HCs,achieving 83.25%value of area under the curve.2.Oral microbiome diversity was significantly higher in AIH patients than in HCs.The oral microbial communities significantly differed between these groups.7 genera including Fusobacterium,Actinomyces,Capnocytophaga,Peptostreptococcus,Incertae_Sedis,Solobacterium and Sphingomonas were dominant in HCs group,while 51 genera including Streptococcus,Veillonella,Leptotrichia,Gemella,Aggregatibacter,Selenomonas and Rothia were enriched in AIH group compared to the HCs,which were considered as microbial dominant genera.Notably,we found 41 gene functions,including 6_phosphofructokinase₁,L_lactate dehydrogenase,pyruvate dehydrogenase,L_serine dehydratase,dominant in AIH and 21 gene functions prevailing in HCs.The optimal 5 microbial markers including OTU412（Selenomonas）;OTU277（Corynebacterium）;OTU315（Leptotrichia）;OTU42（Rothia）and OTU484（Selenomonas）were identified through a fivefold cross-validation on a random forest model and achieved an area under the curve of 99.88%between 46 AIH and 92 HCs samples.Conclusion1.We present the gut and oral microbiome characteristics of AIH patients in Central China for the first time.For one thing,five gut microbial biomarkers,including Lachnospiraceae,Veillonella,Bacteroides,Roseburia and Ruminococcaceae,achieved a high potential distinguishing AIH patients from healthy individuals.For another,the oral microbial combination of Leptotrichia,Selenomonas,Corynebacterium and Rothia were determined to be highly efficacious at distinguishing AIH patients from healthy individuals.2.This work highlights diagnosis potential of gut and oral microbiome for AIH,providing a novel diagnostic strategy.Notably,this is the first analytical study that combines gut with oral microbes in AIH patients as well as healthy individuals,providing a basis for future AIH microbial research fields.