Naturally transmitted segmented filamentous bacteria segregate with diabetes protection in nonobese diabetic mice

Abstract

Vertebrates typically harbor a rich gastrointestinal microbiota, which has coevolved with the host over millennia and is essential for several host physiological functions, in particular maturation of the immune system. Recent studies have highlighted the importance of a single bacterial species, segmented filamentous bacteria (SFB), in inducing a robust T-helper cell type 17 (Th17) population in the small-intestinal lamina propria (SI-LP) of the mouse gut. Consequently, SFB can promote IL-17-dependent immune and autoimmune responses, gut-associated as well as systemic, including inflammatory arthritis and experimental autoimmune encephalomyelitis. Here, we exploit the incomplete penetrance of SFB colonization of NOD mice in our animal facility to explore its impact on the incidence and course of type 1 diabetes in this prototypical, spontaneous model. There was a strong cosegregation of SFB positivity and diabetes protection in females, but not in males, which remained relatively disease-free regardless of the SFB status. In contrast, insulitis did not depend on SFB colonization. SFB-positive, but not SFB-negative, females had a substantial population of Th17 cells in the SI-LP, which was the only significant, repeatable difference in the examined T-cell compartments of the gut, pancreas, or systemic lymphoid tissues. Th17-signature transcripts dominated the very limited SFB-induced molecular changes detected in SI-LP CD4(+) T cells. Thus, a single bacterium, and the gut immune system alterations associated with it, can either promote or protect from autoimmunity in predisposed mouse models, probably reflecting their variable dependence on different Th subsets.

Fig. 1.

SFB status of mice housed in different animal facilities. (A) Variable SFB colonization of different strains at the different sites. PCR-determined relative SFB levels in mice housed at TAC, JAX, or the NRB facility. Each bar represents the average level +/− SEM for six mice (three males and three females). Fecal pellets taken from mice that were shipped to the NRB facility from TAC or JAX were collected and processed or were frozen within 24 h of arrival. Mice were 5–6 wk of age at the time of feces collection. SFB negativity in the NOD strain was confirmed 10 months later with additional sets of mice at 6–8 wk of age. (B) SFB colonization through four generations of NOD mice housed at the NRB facility. Squares represent males; circles indicate females. Black symbols indicate SFB-positive mice; white symbols indicate SFB-negative mice; mixed black/white symbols indicate mice of unknown SFB status. Each litter is delineated by the brackets below, with the date of birth (month/day) within 2010 indicated. SFB status of pups was assessed by PCR of fecal DNA at 4–6 wk of age except for breeding cages A–J. Parental feces were collected and examined for the presence of SFB within 2–3 wk of the litter encompassing the line of descent. The parental age at the time of feces collection varied between 2.5 and 6 mo.

Fig. 2.

Diabetes incidence and insulitis scores in SFB-negative and SFB-positive NOD mice at the NRB facility. (A and B) Cumulative incidence curves for female (A) and male (B) NOD mice identified as SFB positive or SFB negative at 4–6 wk of age. Diabetes development was monitored weekly starting at 10 wk of age. The difference between the two groups was statistically significant in females (P < 0.0001, log-rank test) but not in males. (C) Insulitis scores from SFB-colonized female NOD mice at 10 wk of age and age-matched SFB-negative controls. Pancreata were prepared and stained with H&E, and islet cell infiltration was scored as described in SI Materials and Methods. Each bar represents an individual mouse.

Fig. 3.

Induction of Th17 cells in the SI-LP of SFB-positive NOD females. (A) Lymphocytes were isolated from the indicated lymphoid tissues of SFB-positive or SFB-negative female NOD mice at 6–8 wk of age. Intracellular cytokine expression was enhanced by a 4-h culture in the presence of phorbol ester and ionomycin. Cells were gated by side-scatter and as CD45⁺, CD19⁻, CD8⁻, TCRβ⁺, and CD4⁺. Shown is a representative plot for IL-17 and IFN-γ staining from six mice analyzed. (B) Frequencies of IL-17–producing CD4⁺TCRβ⁺ cells in various lymphoid organs of SFB-positive and SFB-negative NOD mice. Gating was done as per A. Statistical analysis was performed using the nonparametric Mann–Whitney test. (C) Correlation of relative SFB levels in fecal pellets (determined as per Fig. 1) and percentage of IL-17–expressing CD4⁺TCRβ⁺ cells (as per A and B). Statistical analysis was performed using the Pearson correlation. (D) Frequencies of IFN-γ–producing CD4⁺TCRβ⁺ cells in the same lymphoid organs of the NOD mice depicted in B. Gating was done as per A. (E and F) Representative flow cytometry plots of three mice analyzed and frequencies of Foxp3-expressing CD4⁺TCRβ⁺ cells for various lymphoid tissues of age-matched SFB-positive and SFB-negative NOD mice. Cells were gated by side-scatter and as CD45⁺, CD19⁻, CD8⁻, TCRβ⁺, and CD4⁺. Ns, not significant.

Fig. 4.

Comparison of Th17 cell numbers in the SI-LP of female versus male NOD mice classed by SFB status. Frequencies of IL-17–producing CD4⁺TCRβ⁺ cells in various lymphoid organs of SFB-positive and SFB-negative female and male NOD mice. Lymphocytes were isolated and stained and were gated as per Fig. 3A. IL-17 expression levels from four SFB-positive and four SFB-negative female NOD mice from Fig. 3 were paired with SFB-positive and SFB-negative male mice processed on the same day. Statistical analysis was performed using the paired t test. No statistically significant differences between SFB-positive and SFB-negative NOD mice were found in any compartment except the SI-LP. The P values for SI-LP IL-17 levels from SFB-positive versus SFB-negative NOD mice are 0.0097 for females and 0.0459 for males.

Fig. 5.

Specific induction of Th17 transcripts in SI-LP CD4⁺ T cells of SFB-positive NOD mice. (A) Affymetrix microarray analysis of SI-LP CD4⁺ T cells from SFB-negative (x axis) versus SFB-positive (y axis) NOD mice 6–10 wk of age. Highlighted are several genes characteristic of Th17 cells (red type), other CD4⁺ T-cell subsets (blue type), or genes located within the idd3 diabetes susceptibility interval (orange type). Values in the upper left and lower right corners refer to the numbers of loci up- or down-regulated, respectively, by more than twofold. (B) As in A, except that values for colonic lamina propria transcripts are plotted. (C) An expression heatmap for transcription factors and cytokines diagnostic of Th17 cells and other CD4⁺ T-cell subsets. Data are from SI-LP or splenic CD4⁺ T cells from NOD mice that were positive or negative for SFB. All mice were 6–10 wk of age. Data are row-normalized.