A Metagenomic Study Of The Gut Microbiome In Behcet's Disease

Background:Behcet's disease(BD)is a chronic,multisystemic,inflammatory autoimmune disorder characterized by recurrent attacks of oral ulcers,genital ulcers,skin lesions and uveitis.As the disease occurs in young and mid-aged individuals and is one of the most common causes of blindness,it has important public health concerns.The exact cause of BD is unknown.Studies have shown that deregulated autoimmunity in the context of genetic and infectious factors may contribute to the disease.An abnormal balance of T helper 1(Th1),T helper 17(Th17)and regulatory T(Treg)cells were observed in BD patients.Recent studies showed that the gut microbiome may play a crucial role in modulating Th1,Th17 and Treg cells.Dysbiosis of the gut microbiome has been observed in many inflammatory autoimmune diseases,including BD.Although some researchers have already studied the microbial diversity based on the method of 16 S rRNA sequencing in BD,they did not study their functions.In contrast of the limitations of 16 S rRNA sequencing,the method of metagenomic sequencing provides us more information.Based on thesebackgrounds,the project was performed as followes:(1)A pre-study was designed using method of 16 S rRNA sequencing to explore whether immunosuppressive treatment has an effect on the gut microbiome in patients with BD.(2)Investigate the composition and function of the gut microbiome in BD patients by method of metagenomic sequencing.(3)A supplementary 16 S rRNA study on salivary samples of BD was performed to understand the profile of the oral microbiome and its association with the gut microbiome in BD patients.PART ONE: EFFECTS OF IMMUNOSUPPRESSIVE AGENTS ON GUT MICROBIOME IN BD PATIENTSPurpose:To explore whether immunosuppressive treatment has an effect on the gut microbiome in active BD patients.Method : The feces of patients with untreated active BD patients,immunosuppressive treated active BD patients and normal controls were collected,PCR were then amplified.The fecal microbial community was sequenced by Illumina MiSeq sequencing system and analyzed by bioinformatics analysis.Result:Comparing to the differences between treated-BD patients and healthy controls,there were more significantly differences between untreated-BD patients and healthy controls.Conclusion:The immunosuppressant drugs had an effect on the gut mocriobome in active BD patients.These treatments could partly restore the unbalanced gut microbiome in BD patients.In view of these findings,we strictly selected untreated active BD patients as study objects in our further investigations.PART TWO: METAGENOMIC STUDY IN BD PATIENTSPurpose : To investigate the composition and function of the gut microbiome in BD patients by method of metagenomic sequencing.To analyse the potential mechanism of gut microbiome affecting BD.Method:The feces of patients with untreated active BD patients and normal controls were collected.The fecal microbial community was sequenced by Illumina TruSeq sequencing system and analyzed by bioinformatics analysis.Results : 1.The abundance of 248 genera and 933 species was significantly different between BD patients and healthy controls.Of those,lactate-producing bacteria,sulphate-reducing bacteria and a low abundance of opportunistic pathogens were enriched in BD patients.On the contrary,butyrate-producing bacteria and methanogens were enriched in healthy controls.2.The MGS analysis showed that there were 13 MGS identified according to the abundance profiles.Using the 13 MGSs as microbiome markers to discriminate the BD group from the control group,the area under the receiver operating characteristic curve(AUC)was 82.69% and the 95 % CI was 71.24%-94.14%.3.To analyze the potential functional roles of the gut microbiome in BD patients,we identified 449 KEGG(Kyoto Encyclopedia of Genes and Genomes database)orthologues(KO)and 1,859 eggNOG(evolutionary genealogy of genes: Non-supervised Orthologous Group database)orthologues(OG)to be significantly different between BD patients and healthy controls.The BD patients were enriched in orthologue markers of membrane transport,amino acid transport and metabolism,coenzyme transport and metabolism,energy production and conversion,inorganic ion transport and metabolism.We further found that two OG markers related to HSP90 were enriched in BD patients and one KO marker related to HSP20 was enriched in healthy controls.At the module or pathway level,the BD patients were enriched in peptidoglycan biosynthesis,sulfur and lipid metabolism,capsular polysaccharides transport system,Type ? and Type? secretion systems and some others in line with the BD-enriched KO/OG markers mentioned above.Conclusion:The compositions and functions of gut microbiome were significant differences between BD patients and healthy controls.The enriched lactate-producing bacteria,sulphate-reducing bacteria and low abundance of opportunistic pathogens in association with depleted butyrate-producing bacteria and methanogens were correlated with BD.These alterations could take part in the occurrence of BD according to synthesis of peptidoglycan synthesis,sulfur and lipid metabolism,capsularpolysaccharide transport system,bacterial type ? and type ? secretion system and HSP90.A model explaining the association of the gut microbiome with BD pathogenesis is proposed.PART THREE: THE STUDY OF ORAL MICROBIAL IN BD PATIENTSPurpose : To explore the profile of the oral microbiome and its association with the gut microbiome in BD patients.Method : The saliva samples of patients with untreated active BD patients,immunosuppressive treated active BD patients and normal controls were collected,PCR were then amplified.The oral microbial community was sequenced by Illumina MiSeq sequencing system and analyzed by bioinformatics analysis.Result : Lactate-producing bacteria including Bifidobacteriaceae,Coriobacteriaceae Lactobacillaceae and sulphate-reducing bacteria including Desulfovibrio were enriched in BD patients,while Neisseriales was enriched in normal controls.Conclusion: The alterations of oral microbiome were consistent with the abnormalities of gut microbiome in these BD patients.The study revealed oral microbiome could be related with gut microbiome in BD patients,and they both involved in the development of BD.