Gut Microbiota Composition And Fecal Metabolic Phenotype In Patients With Acute Anterior Uveitis

BackgroundUveitis,an inflammatory disease,is one of the primary causes of visual impairment and blindness in the world.As the most common uveitis entity,acute anterior uveitis(AAU)damages the normal function and structure of the anterior segment of the eye.It is strongly associated with the human leucocyte antigen B27(HLA-B27)and is a common extra-articular manifestation of other inflammatory diseases such as spondyloarthropathies(SpA),ankylosing spondylitis(AS)and inflammatory bowel disease(IBD).Several immunological factors play an important role in the pathogenesis of the disease,such as the imbalance of Th17/Treg cells as well as an increased expression of IL-17.The human gut microbiota and its effects on the metabolic phenotype in the host have been shown to play a critical role in maintaining immune homeostasis.Microbial components have been thought to be involved in the regulation of the immune response such as activating IL-17-producing T cells in the gut environment.The presence of Collinsella,for instance,showed a strong correlation with alpha-aminoadipic acid and asparagine and was able to promote the production of the proinflammatory cytokine IL-17A.Previous studies furthermore showed that gut bacteria,such as Yersinia Enterocolitica and Salmonella typhimurium,are involved in the pathogenesis of SpA.Significantly decreased genera such as Roseburia and butyrate production have been observed in Behcet's patients as compared with healthy controls.Commensal bacteria are thought to be a potential trigger and a factor which can influence the development of disease in experimental autoimmune uveitis(EAU).Antibiotics,such as metronidazole or vancomycin,can increase the abundance of some bacteria including Dorea and Clostridium and reduce the mean uveitis score in EAU.Although microbiota and metabolites are thought to play a role in the development of immune diseases,whether these two factors play a role in the pathogenesis of AAU is still unknown.Based on the above background information,this study focused on the following two parts:(1)Whether the composition of gut microbiota and metabolites is different in AAU as compared to controls?(2)If these two factors contribute to the disease,what is the exact mechanism?Through the study of the above two issues,it may be possible to further clarify the pathogenesis of AAU and find out new targets in the treatment of AAU.PART I GUT MICROBIOTA COMPOSITION AND FECAL METABOLIC PHENOTYPE IN PATIENTS WITH ACUTE ANTERIOR UVEITISObjective:Gut microbiota and fecal metabolic play important roles in the development of immune disease.The purpose of this part was to investigate gut microbiota composition and the fecal metabolic phenotype in patients with AAU.Methods:1.Fecal DNA was extracted from 78 fecal samples(38 AAU patients and 40 family members of patients or sex-and age-matched healthy controls)and then sequenced by high-throughput 16S rDNA analysis.2.Gas chromatographic mass spectrometry(GC-MS)based metabolomics was performed on 60 fecal samples(30 AAU patients and 30healthy controls).3.Spearman correlation between the level of fecal metabolites and the relative abundance of genera was performed by the cor.test function from the stats R package.Results:A significant difference was observed in beta diversity between AAU patients and healthy controls.Eight genera including Roseburia were reduced in AAU patients,and Veillonella was increased in AAU patients as compared with healthy controls.Significance was however lost after false discovery rate(FDR)correction.The expression of seven fecal metabolites including 6-deoxy-D-glucose 1,linoleic acid,N-Acetyl-beta-D-mannosamine 3,shikimic acid,azelaic acid,isomaltose 1 and palmitoleic acid was increased in AAU patients.Linoleic acid showed a significant correlation with Roseburia and Veillonella according to Spearman correlation analysis.Conclusions:Our results did not reveal a difference in gut microbiota composition,but did show that the fecal metabolic phenotype in AAU patients was significantly different from healthy controls.Part II Analysis of the role of palmitoleic acid in acute anterior uveitisObjective:PA has recently been shown to have beneficial effects on the function of the immune system.In the previous part,it has been revealed that a significant difference in the fecal PA concentration was identified in AAU patients as compared with healthy individuals.Whether PA has a direct effect on the pathogenesis of AAU is unknown and was therefore the subject of this part presented here.Methods:1.PA levels in feces from AAU patients were measured by GC-MS and compared with samples obtained from healthy individuals.2.Enzyme linked immunosorbent assay(ELISA)and flow cytometry(FCM)were used to assess the effect of PA on dendritic cells(DCs)and CD4~+T cells obtained from mice,AAU patients and healthy individuals.3.C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis(EAU)was induced with a human retinal IRBP651-670 peptide.Disease severity of EAU was evaluated by clinical manifestation and histology.Differentiation of splenic Type 1 helper T cells(Th1)and Th17 cells was evaluated by FCM.4.Tandem mass tag(TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA.5.FCM and western blot(WB)were used to investigate the effect of PA on DCs apoptosis.Results:The fecal concentration of PA was increased in AAU patients as compared with healthy individuals.In vitro,PA promoted apoptosis of DCs and inhibited the secretion of TNF-?from mouse bone-marrow-derived dendritic cells(BMDCs)as well as in DCs from AAU patients and healthy individuals.It only decreased DC surface marker expression and IL-12p70secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs.PA also inhibited the differentiation of Th cells and secretion of IFN-?and IL-17 in CD4+T cells from mice,AAU patients and healthy individuals.In vivo,PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group.PA feeding inhibited differentiation of splenic Th17 cells,whereas Th1 cells were not affected.Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls.Conclusions:PA was shown to inhibit the function of DCs and CD4~+T cells and was able to inhibit disease severity in an animal model of uveitis in mice.We also showed that PA promotes apoptosis and regulates protein expression in DCs.