Role of the Gut Microbiome in Modulating Arthritis Progression in Mice

Abstract

Genetics alone cannot explain most cases of rheumatoid arthritis (RA). Thus, investigating environmental factors such as the gut microbiota may provide new insights into the initiation and progression of RA. In this study, we performed 16S rRNA sequencing to characterise the gut microbiota of DBA1 mice that did or did not develop arthritis after induction with collagen. We found that divergence in the distribution of microbiota after induction was pronounced and significant. Mice susceptible to collagen-induced arthritis (CIA) showed enriched operational taxonomic units (OTUs) affiliated with the genus Lactobacillus as the dominant genus prior to arthritis onset. With disease development, the abundance of OTUs affiliated with the families Bacteroidaceae, Lachnospiraceae, and S24-7 increased significantly in CIA-susceptible mice. Notably, germ-free mice conventionalized with the microbiota from CIA-susceptible mice showed a higher frequency of arthritis induction than those conventionalized with the microbiota from CIA-resistant mice. Consistently, the concentration of the cytokine interleukin-17 in serum and the proportions of CD8+T cells and Th17 lymphocytes in the spleen were significantly higher in the former group, whereas the abundances of dendritic cells, B cells, and Treg cells in the spleen were significantly lower. Our results suggest that the gut microbiome influences arthritis susceptibility.

Figure 1. Analysis of microbial diversity.

Mice were induced with collagen II on days 0 and 21. On day 35, mice that developed arthritis were classified as CIA-susceptible, and those that did not were classified as CIA-resistant. Faecal samples were collected before (on day 28) and after (on day 35) clinical onset of arthritis. (A) Number of reads for operational taxonomic units (OTUs) and (B) the ACE and Chao richness estimators per group were determined on day 35 (cross-sectional study). (C) Changes in the number of reads and (D) Shannon diversity index scores per group before and after collagen-mediated induction (cohort study). Each symbol represents an individual mouse. Error bars indicate SEM. Data in (A,B) were compared by one-way analysis of variance, while those in (C,D) were compared by paired t-test. n = 5; *p < 0.05; **p < 0.01. CIA, mice with collagen-induced arthritis; CIAN, mice without arthritis after collagen-mediated induction; NC, untreated control.

Figure 2. Differences in microbiome composition between CIA-susceptible and -resistant mice.

(A) The distribution of bacterial families with relative abundance >1% in at least onesample was compared by Student’s t-test in mice prior to arthritis onset (left panel) and by Student’s paired t-test in CIA-susceptible mice (right panel). n = 5 per group; *p < 0.05; **p < 0.01. (B) Heat map of the relative abundances of genera in each group. The vertical columns represent one group (n = 5 mice per group), and the horizontal rows depict genera. The colour coding ranges from blue (not detected), through green (low abundance) and yellow (medium abundance), to red (high abundance). The genera shown with red and blue blocks were more abundant in CIA-susceptible mice prior to arthritis onset and CIA-resistant mice, respectively. Yellow and green blocks indicate genera with greater abundance in CIA and untreated control (NC) mice, respectively.

Figure 3. Separation of mice according to their susceptibility to CIA.

(A) Clustering of unweighted UniFrac distances between samples colour-coded by breeding pair, as determined by the UniFrac-based unweighted pair group method with arithmetic analyses. (B) The top two principal components of unweighted UniFrac distances between untreated and collagen-induced mice prior to arthritis onset. Symbols represent data from individual mice. Red circles indicate CIA-susceptible mice. Blue and green circles indicate CIA-resistant and untreated mice, respectively.

Figure 4. Analyses of faecal microbiomes of germ-free mice conventionalized with CIA-susceptible or CIA-resistant mice.

(A) NMDS ordination for all 12 mice following conventionalization with the microbiome of CIA-susceptible or CIA-resistant mice. (B) LEfSe-identified LDA bar graphs of taxa/clades with differential abundance following conventionalization with the microbiome of the CIA-susceptible (green) and the CIA-resistant (red) mice. Taxa in this graph were statistically significant (p < 0.05) and had an LDA Score > ± 2.0, which was considered a significant effect size.

Figure 5. Higher incidence and severity of arthritis in CIA-susceptible mice.

Germ-free mice were conventionalized with microbiota obtained from CIA-susceptible or CIA-resistant mice after induction with collagen. Four weeks after conventionalization, recipient mice were induced with collagen II under germ-free conditions. (A) Clinical scores progression was assessed, and (B) the incidence of arthritis was determined. Data indicate means ± SEM from six animals per group. *p < 0.05. (C) Representative sections of ankle joint tissue stained with hematoxylin and eosin. Magnification, 100×. Histopathological evaluation revealed severe inflammation in the joint sections of CIA mice (arrowhead). (D) Serum tumour necrosis factor-α, interleukin-17, and interleukin-10 as estimated by enzyme-linked immunosorbent assay. Data indicate mean ± SEM (n = 6). *p < 0.05 by Student’s t-test.

Figure 6. Phenotypic analysis of splenocytes from germ-free mice conventionalized with the microbiota from CIA-susceptible or -resistant mice.

Lymphocytes were harvested from the spleen after 9 weeks of immunization with collagen. Cells were stained with CD3, CD19, CD11c, CD4, and CD8 antibodies and analysed by flow cytometry. Representative contour plots showing CD3 vs. CD19 (A, left panel) and CD11c (B, left panel), and CD4 vs. CD8 (C, left panel) expression. Data indicate mean ± SEM (n = 6). *p < 0.05; **p < 0.001 by Student’s t-test. CD3-CD19+ B cells (A, right panel) and CD3-CD11c+ dendritic cells (B, right panel) were more abundant in germ-free mice conventionalized with the microbiota from CIA-resistant mice than in those conventionalized with the microbiota from CIA-susceptible mice. CD4-CD8+ T cells (C, right panel) were more abundant in recipients of CIA-susceptible microbiota. CD4+ T cells were stained with intracellular anti-IFN-γ antibody, anti-Foxp3 antibody, and anti-IL-17A antibody. Numbers in each quadrant indicate the percentages of CD4+ T cells positive for each cytokine (D, right panel). Mean ± SEM of the IL17+Foxp3-, IL17-Foxp3+, and IL17-IFN-γ+ T cell subsets (D, left panel). Data indicate mean ± SEM (n = 6). *p < 0.05; **p < 0.001 by Student’s t-test.