Protective major histocompatibility complex allele prevents type 1 diabetes by shaping the intestinal microbiota early in ontogeny

Abstract

Certain MHC-II or HLA-D alleles dominantly protect from particular autoimmune diseases. For example, expression of the MHC-II Eα:Eβ complex potently protects nonobese diabetic (NOD) mice, which normally lack this isotype, from spontaneous development of type 1 diabetes. However, the underlying mechanisms remain debated. We investigated MHC-II-mediated protection from type 1 diabetes using a previously reported NOD mouse line expressing an Eα transgene and, thereby, the Eα:Eβ complex. Eα16/NOD females vertically protected their NOD offspring from diabetes and insulitis, an effect that was dependent on the intestinal microbiota; moreover, they developed autoimmunity when treated with certain antibiotics or raised in a germ-free environment. Genomic and proteomic analyses revealed NOD and Eα16/NOD mice to host mild but significant differences in the intestinal microbiotas during a critical early window of ontogeny, and transfer of cecal contents from the latter to the former suppressed insulitis. Thus, protection from autoimmunity afforded by particular MHC/HLA alleles can operate via intestinal microbes, highlighting potentially important societal implications of treating infants, or even just their pregnant mothers, with antibiotics.

Keywords: NOD mice; autoimmune disease; microbiome; neonatal; type 1 diabetes.

Fig. 1.

Eα16/NOD dams transmitted protection from insulitis and T1D vertically to their NOD progeny. (A) Diabetes incidence in a cohort of NOD mice born to NOD dams (n = 34), NOD mice born to Eα16/NOD dams (n = 24), and Eα16/NOD mice (n = 20). ***P = 0.0004 (Gehan–Breslow–Wilcoxon test). (B) Proportion of islets with insulitis or peri-insulitis at 10 wk of age. NOD mice born to NOD dams mated with NOD sires, NOD mice born to NOD dams mated with Eα16/NOD sires, NOD mice born to Eα16/NOD dams mated to NOD sires, and Eα16/NOD mice. (C) Composite insulitis score for each mouse in B. ***P = 0.001, **P = 0.005 (Mann–Whitney test). (D) Composite insulitis scores for vancomycin-treated Eα16/NOD dams that received oral vancomycin during the last 7–10 d of pregnancy. ***P = 0.0005, *P = 0.02 (Mann–Whitney test).

Fig. 2.

Antibiotic treatment induced insulitis and altered the intestinal microbiome in Eα16/NOD mice. (A) Insulitis composite score in Eα16/NOD mice treated with oral antibiotics provided in their drinking water from 3 to 6 wk of age. (B) Pancreas histology of 10-wk-old NOD mice (Left), Eα16/NOD mice (Center) or Eα16/NOD mice treated with vancomycin from 3 to 6 wk of age (Right). (C) Composite insulitis score of Eα16/NOD mice treated with vancomycin over different time periods, which include the last 7–10 d of pregnancy through weaning of the pups at 3 wk of age, 3–6 wk of age, or 6–10 wk of age. **P = 0.002, *P = 0.03 (Mann–Whitney test). (D) Phylum-level representation of 16S rRNA fecal microbiome from control or vancomycin-treated parents and their progeny at 3 and 10 wk of age. P, parents; 3 wk, 3-wk-old progeny; 10 wk, 10-wk-old progeny. (E) PCoA of unweighted UniFrac distances calculated from 16S rRNA gene sequencing of these fecal samples. (F) Insulitis scores from NOD, Eα16/NOD, and GF Eα16/NOD and NOD mice at 16–20 wk of age. *P = 0.02 (unpaired t test). (G) Pancreas histology from Eα16/NOD and GF Eα16/NOD mice.

Fig. 3.

NOD and Eα16/NOD mice host distinctive intestinal microbiomes. (A) Boxplots of cecal microbiome α-diversity (PD whole tree) normalized as the ratio of each sample’s α-diversity to its cage mean. n = 25 Eα16/NOD and n = 25 NOD mice (**P = 0.004). (B) Boxplots of cecal microbiome β-diversity (weighted UniFrac distance) (**P = 0.0098). Unpaired t test with 10,000 Monte-Carlo simulations. (C) Boxplots of the relative abundance of the order Clostridiales in cecal contents and tissue. **P < 0.01, ***P < 0.001. (D) Cecal contents from NOD or Eα16/NOD donors were gavaged to NOD pups twice weekly from 2 to 5 wk of age. Insulitis was assessed at 10 wk of age. *P = 0.04 (unpaired t test).

Fig. S1.

Boxplots of the relative abundance of the order Clostridiales and the genus Blautia in cecal contents and tissue of 18-d-old NOD and Eα16/NOD mice showing distribution by individual mice (A) and individual cages (B). n = 25 Eα16/NOD and n = 25 NOD mice for cecal contents, and n = 21 Eα16/NOD and n = 21 NOD mice for cecal tissue. **P < 0.01, ***P < 0.001.

Fig. S2.

(A) Boxplots of NOD mouse fecal metaproteomic α-diversity (Shannon index) normalized as the ratio of each sample’s α-diversity to its cage mean. n = 4 and 8 Eα16/NOD mice, and n = 4 and 8 NOD mice at day 18 and day 70, respectively (*P = 0.026 and ***P = 0.0002). (B) PCoA plot of NOD mice fecal metaproteomic β-diversity at day 18 and day 70.

Fig. 4.

IgA-bound repertoire of cecal bacteria is similar in NOD and Eα16/NOD mice. (A and B) Volcano plot comparing the ICI of the cecal microbiotas from NOD and Eα16/NOD mice at 18 and 35 d of age. (C and D) Comparison of the representation of specific IgA-coated taxa between the cecal microbiomes of 18- and 35-d-old NOD and Eα16/NOD mice.

Fig. S3.

(A and B) Frequency and mean fluorescence intensity of IgA-coated cecal bacteria in 18- and 35-d-old NOD and Eα16/NOD mice. (C and D) Heatmaps depicting specific OTUs whose IgA-coated microbes may differ in 18- and 35-d-old NOD and Eα16/NOD mice.

Fig. 5.

Eα16/NOD mice had an enlarged representation of cecal T_reg. (A) Cells were isolated from littermates at day 18 and analyzed by flow cytometry. Representative plot of Foxp3⁺CD4⁺ cell population. Cells were gated as TCRβ⁺CD19⁻CD45⁺. (B) Percentage of cecal lamina propria Foxp3⁺CD4⁺ T-cell from three independent experiments. (C) Percentage of Foxp3⁺CD4⁺ T cells. *P < 0.05.

Fig. S4.

Flow cytometric comparison of the ILC3 cell population cytokine production in littermate NOD and Eα16/NOD mice. (A) ILC3 gating strategy and representative plot. (B) Cytokine production by colonic ILC3 cell populations in NOD and Eα16/NOD mice.