Effect And Mechanism Of Intestinal Flora On The Development Of Collagen Induced Arthritis

Background:Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease c haracterized by joint pain,erosion of bone and cartilage,which is a crippling disease threat to human health.However,the pathogenesis of RA is not clear,genetic factors cannot fully explain its pathogenesis.Several studies have observed a significant difference in the composition of the gut microbiome in patients with RA compared to that in healthy controls.The gut microbiota and their metabolic products are considered the major environmental factors eliciting autoimmune diseases in genetically susceptible individuals [1].Molecular mimicry originating from the microbiome has also been known to trigger RA [2].And bacteria can induce arthritis symptoms in Germ free mice [3,4].Moreover,probiotic supplementation can be used to improve inflammatory status and alleviate symptoms in patients with RA [5,6].Therefore,the existing knowledge regarding the effect of gut microbiota on disease initiation or progression is controversial.In our previous study we observed an alteration in gut microbiota in patients with RA.However,how the gut microbiome changes before and after arthritis and whether these changes directly contribute to RA have not been determined.Given these large variations in the host factors and habitat-dependent environmental factors across distinct human populations [7],what is more it is difficult to collect the gut microbiota before RA happening.Autoimmune arthritis model and germ free animals hence particularly suitable for further studies the role of microbiome in arthritis pathogenesis.Collagen-induced arthritis(CIA)is an autoimmune arthritis mouse model.The incidence of arthritis in DBA1 strains of mice is approximately 80% [8].This mouse model shares both immunological and pathological features with RA,so it has been extensively studied as a model for understanding the pathogenesis of RA and testing therapeutics.Purpose:We immunized the DBA/1 mice with type II collagen(CII).A pyrosequencing of 16 S r RNA genes was performed to identify the gut bacterial composition of CIA-susceptible(develop arthritis),CIA-resistant mice(did not develop arthritis)and their changes.Key bacterial phylotypes or potential biomarkers that potentially play important roles in CIA development were characterized.Next,we colonized the faecal samples before intervention to GF mice and immunized the mice with collagen to observe the effects.The identification of pathogenic or protective effect of intestinal flora which can provide new ideas for the further understanding of the pathogenesis and treatment of RA.Previously,we demonstrated that Lactobacillus salivarius was more abundant in patients with RA than in healthy individuals.However,it is unclear whether L.salivarius exerts protective or pathogenic effect in RA.In this study,we investigated the effect of L.salivarius isolated from patients with RA on CIA in mice.Part ? Collagen induced arthritis and altered microbial diversity and compositionMethod:In this study,we made collagen induced arthritis model and performed 16 S r RNA sequencing to characterize the gut microbiota of DBA/1 mice in CIA-susceptible,CIA-resistant mice and the untreated mice.We examined microbial?diversity in the intestine,The reads,sequences,OTUs,richness estimators(ACE and Chao),and diversity indices(Shannon and Simpson)from CIA mice were calculated and compared;Secondly,the ? diversity of intestinal microflora in each group of mice was analyzed;Finally,the composition of intestinal microflora in each group was analyzed.Results: Analysis of ? diversity : In DBA/1 mice sequence reads diminished considerably after collagen induction,irrespective of arthritis onset.After the onset of arthritis CIA-susceptible mice had fewer richness estimators than untreated mice or CIA-resistant mice.With the development of arthritis happening,Shannon index increased significantly in CIA-susceptible mice group.Mice appeared to separate clearly by phylogenetics-based metrics of ?-diversity.We found that divergence in the distribution of the microbiota before arthritis onset was pronounced and statistically significant(analysis of similarity,R=0.375,P<0.01).Bacterial taxonomy comparisons differ among each group.At the family level,Desulfovibrionaceae and Lachnospiraceae numbers were lower in the Pre-CIA group than in the Pre-CIAN group,but Lactobacillaceae numbers were significantly higher.We found that the families Bacteroidaceae,Lachnospiraceae and S24-7 increased significantly after arthritis onset.Conclusion: After immunization the intestinal flora of DBA/1 mice changed,which can be divided into CIA susceptible and CIA resistant groups.The bacteria diversity and flora structure were significant differences in the intestinal flora between CIA susceptible mice and CIA resistant mice.With the development of arthritis the intestinal flora in CIA mice changed.Part ? The Role of Gut Microbiome in Modulation of Arthritis Disease ProgressionMethod:We transplanted the microbiome from CIA-susceptible mice or CIA-resistant mice to germ free mice.Following conventionalization,mice were induced with collagen II under germ-free conditions.We compared the incidence and severity of arthritis and measured serum levels of tumor necrosis factor(TNF)-?,IL-10 and IL-17.We measured the abundances of and CD3-CD11c+cells,CD3-CD19+cells,CD4+ T cells,CD8+ T cells,Th1,Th17 and Treg.Results:The characteristics of the intestinal flora of the recipient were consistent with the characteristics of the intestinal flora of the donor,indicating intestinal flora was successfully colonized.Mice conventionalized with the microbiome of CIA-susceptible mice showed an increase in arthritis incidence and more serious than that observed in the recipients of Pre-CIAN microbiome.We found that the serum concentration of IL-17 was consistently elevated in germ-free mice colonized with the microbiota from CIA-susceptible mice.Conventionalization of germ-free mice with the microbiota from CIA-susceptible mice significantly increased the abundances of total and CD8+ T cells.However,the abundances of CD3-CD11c+ cells and CD3-CD19+cells significantly decreased.Conventionalization with the microbiota from CIA-susceptible mice significantly increased the abundance of Th17 cells and reduced that of Treg cells.Conclusions: the results suggest that the gut microbiota influences susceptibility to CIA arthritis.The intestinal microbiota may impact the balance between Treg and T helper 17 and mediate arthritis in mice.Part ? Lactobacillus salivarius isolated from patients with rheumatoid arthritis suppresses collagen-induced arthritisMethods:L.salivarius UCC118 or L.plantarum WCFS1 isolated from patients with RA was administered orally for 5 weeks,starting from 2 weeks prior to the induction of arthritis in DBA/1 mice.Clinical score progression and histological changes were assessed.Bone erosion was evaluated by microcomputer tomography.Serum cytokine(TNF-?,IL-10 and IL-17)concentrations and the proportion of IL-17-producing T cells(Th17)and regulatory T cells(Tregs)in the spleen were also evaluated.Results:CIA mice treated with either L.salivarius or L.plantarum showed lower arthritis scores,milder synovial infiltration,and lesser bone erosion when compared with PBS-treated CIA mice.Both L.salivarius and L.plantarum administrations reduced Th17 cell proportion,while upregulated Tregs proportion.L.salivarius-treated CIA mice displayed significant increase in serum anti-inflammatory interleukin-10 levels.Conclusions: This study provides both L.salivarius and L.plantarum showed protective effects against chronic inflammations associated with CIA in mice.It is the first evidence that pretreatment with L.salivarius could significantly improve collagen-induced arthritis in mice.L.salivarius may be beneficial to alleviate RA in a clinical setting.