Modulation of Inflammatory Arthritis in Mice by Gut Microbiota Through Mucosal Inflammation and Autoantibody Generation

Abstract

Objective: Observations of microbial dysbiosis in patients with rheumatoid arthritis (RA) have raised interest in studying microbial-mucosal interactions as a potential trigger of RA. Using the murine collagen-induced arthritis (CIA) model, we undertook this study to test our hypothesis that microbiota modulate immune responses leading to autoimmune arthritis.

Methods: CIA was induced by immunization of mice with type II collagen (CII) in adjuvant on days 0 and 21, with arthritis appearing on days 23 and 24. Intestinal microbiota were profiled by 16S ribosomal RNA sequencing every 7 days during the course of CIA, and intestinal mucosal changes were evaluated on days 14 and 35. Then, microbiota were depleted either early (7 days before immunization) or late (day 21 after immunization) by administration of broad-spectrum antibiotics. Disease severity, autoantibody and systemic cytokine production, and intestinal mucosal responses were monitored in the setting of microbial reduction.

Results: Significant dysbiosis and mucosal inflammation occurred early in CIA, prior to visible arthritis, and continued to evolve during the course of disease. Depletion of the microbiota prior to the induction of CIA resulted in an ~40% reduction in disease severity and in significantly reduced levels of serum inflammatory cytokines and anti-CII antibodies. In intestinal tissue, production of interleukin-17A (IL-17A) and IL-22 was delayed. Unexpectedly, microbial depletion during the late phase of CIA resulted in a >50% decrease in disease severity. Anti-CII antibodies were mildly reduced but were significantly impaired in their ability to activate complement, likely due to altered glycosylation profiles.

Conclusion: These data support a model in which intestinal dysbiosis triggers mucosal immune responses that stimulate T and B cells that are key for the development of inflammatory arthritis.

Figure 1. Microbial dysbiosis occurs early during the development of CIA

Six week old male DBA/1J mice were immunized with CII in CFA on days 0 and 21. Fecal pellets were collected from mice every 7 days from day 0 through 21 and on day 35 after the initial immunization. Fecal bacterial DNA was sequenced for 16S rRNA and analyzed. N=11 for time points 0–21 and N=6 for day 35; data are from two independent experiments. (A) The percent abundance of the top operational taxonomic units (OTU) families were compared across time points. Differences in the overall composition of microbial communities were determined by PERMANOVA. *P<0.05, **P<0.01. (B) Alpha-diversity measures for richness (Chao1), evenness, and complexity (ShannonH) across time points are shown as box-and-whisker plots. Statistical analysis was performed with ANOVA as described in Methods. *P<0.05, **P<0.01 and ^•P<0.001. (C) Statistically significant changes in family level OTU mean relative abundance ± SEM in were determined by Kruskal-Wallis with Dunn’s post-test. ***P<0.001; ****P<0.0001.

Figure 2. Mucosal inflammation parallels dysbiosis during the development of CIA

6 week old male DBA/1J mice were immunized with CII in CFA on days 0 and 21. (A) Barrier integrity was assessed by permeability of FITC-dextran orally gavaged and detected in serum 4 hours later. Data are the mean ±SEM of the serum concentration of FITC-dextran. N=10 in each group. ***, p<0.001; ****, p<0.0001 as determined by repeated-measures ANOVA. (B) Representative histology from control DBA/1J, CFA controls, and CIA mice at day 35 are shown at 40X. Bar=20 μm. (C–E) Four high-powered fields of well-oriented colon tissue per mouse were analyzed at 40X magnification for quantification of (C) inflammatory cells in the lamina propria, (D) epithelial mitotic figures, and (E) crypt length. N=3–6 mice per group. Data are the mean ± SEM; statistical analyses were performed with Mann-Whitney test *, p<0.05. (F–H) IL-17A, IL-22, and IL-23 in SI, colon, and MLNs were measured by ELISA in DBA/1J unimmunized controls, CFA controls, and in mice with CIA at days 14 and 35 after initial immunization. Cytokine concentrations were normalized to total protein and reported as pg/ml/mg protein. N=3–6 in each group. Data are the mean ±SEM and statistical analysis was performed using a Kruskal-Wallis with Dunn’s post-test. *, p<0.05; **, p<0.01.

Figure 3. Depletion of microbiota by administration of antibiotics reduces CIA severity

Six week old male DBA/1J mice were treated with broad-spectrum antibiotics in drinking water starting a week before CII immunization and stopped at day 0 (early short course) or continued until euthanasia at day 35 (early long course), or mice were treated with antibiotics from days 21 through 35 (late course). All groups were paired with control water-treated mice. All antibiotic and control-water treated groups underwent immunization with CII in CFA at days 0 and 21. (A) Diagram of experimental treatments is shown. (B) Arthritis scores were assessed every other day starting after day 21, N= 6–14 mice/group. Data are the mean arthritis score ± SEM. *, p<0.05; **, p<0.01; ****, p<0.0001 as determined by a two-way ANOVA with Bonferroni’s multiple comparisons test. (C) Serum anti-CII IgG was measured by ELISA. N=6–11 mouse/group; data are the mean U/ml of antibodies ±SEM. *, p<0.05; ****, p<0.0001 as determined by a two-way ANOVA with Bonferroni’s multiple comparisons test.

Figure 4. Microbiota are required for the development of mucosal Th17 cytokines during CIA

Six week old male DBA/1J mice were treated as in Figure 3. Intestinal tissues (SI and colon) in addition to MLN were collected either at day 14 or day 35, homogenized and tested for cytokines by multi-analyte ELISA. The final cytokine concentrations in each tissue were normalized to total protein and reported as pg/ml/mg protein. N=6–11 in each group. Data are the mean ± SEM of the tissue concentrations of cytokine. *, p<0.05, **, p<0.01 as determined by a Kruskal-Wallis test with Dunn’s post-test. (A) IL-17A, IL-22, and IL-23 in tissues from mice with CIA and CIA + early Abx at day 14. (B) IL-17A, IL-22, and IL-23 in tissues from mice with CIA and CIA + early Abx at day 35. (C) IL-17A, IL-22, and IL-23 in tissues from mice with CIA and CIA + late Abx at day 35.

Figure 5. Microbiota alter the ability of serum autoantibodies to activate complement

Activation of the classical complement pathway by day 35 serum anti-CII antibodies from mice with CIA or mice with CIA treated with antibiotics was assessed by a modified ELISA described in Methods. C3 activation is shown as the mean U/ml ± SEM. N=6–11 per group. Statistical significance was determined by a two-tailed Student’s t-test. *, p<0.05; ns, not significant. (A) CIA and CIA+ early Abx. (B) CIA and CIA+late Abx. (C) Agalactosylated, (D) galactose, and (E) sialic acid content in purified serum immunoglobulins were quantified by high-performance liquid chromatography (HPLC).

Figure 6. Proposed mechanism for microbial modulation of CIA

During the early, preclinical phase of CIA, changes in the intestinal microbiome occur, which are associated with a more permeable barrier and mucosal inflammation. Mucosal inflammation is characterized by Th17 responses, as reflected by high levels of IL-17A and IL-22 in tissue, and downstream activation of B cells and mucosal autoantibody production. Circulation of mucosa generated Th17 and B cells leads to systemic autoimmunity. Microbiota also influence autoimmune arthritis later in the disease course, by promoting complement activation by autoantibodies possibly via altered glycosylation.