Gut microbial metabolites alter IgA immunity in type 1 diabetes

Abstract

The incidence of type 1 diabetes (T1D) has been increasing among children and adolescents, in which environmental factors, including gut microbiota, play an important role. However, the underlying mechanisms are yet to be determined. Here, we show that patients with newly diagnosed T1D displayed not only a distinct profile of gut microbiota associated with decreased short-chain fatty acids (SCFAs) production, but also an altered IgA-mediated immunity compared with healthy control subjects. Using germ-free NOD mice, we demonstrate that gut microbiota from patients with T1D promoted different IgA-mediated immune responses compared with healthy control gut microbiota. Treatment with the SCFA, acetate, reduced gut bacteria-induced IgA response accompanied by decreased severity of insulitis in NOD mice. We believe our study provides new insights into the functional effects of gut microbiota on inducing IgA immune response in T1D, suggesting that SCFAs might be potential therapeutic agents in T1D prevention and/or treatment.

Keywords: Autoimmunity; Diabetes; Translation.

Figure 1. Stool SCFA production and bacteria-targeting IgA response in individuals with T1D compared with control subjects.

(A) Experimental design of the study in patients with T1D and healthy control subjects. (B–G) Stool microbiota composition was investigated by 16S rRNA sequencing (n = 19/group). Changes in α diversity were assessed by Chao richness (B) and number of observed species (C). Altered relative microbial abundances of Ruminococcaceae (D), Coprococcus (E), Roseburia (F), and Megamonas (G) between donors with T1D and controls are shown. (H and I) Stool acetate (H), and butyrate (I) concentrations from individuals with T1D and control subjects (n = 19/group). (J) Correlation between stool acetate level and serum fasting C-peptide concentration (n = 15). Data are presented as mean ± SEM and were analyzed by a 2-tailed Student’s t test (B–I). Data in (J) was analyzed using a 2-tailed Pearson correlation coefficient test and linear regression. SCFAs, short-chain fatty acids; T1D, type 1 diabetes.

Figure 2. IgA-bound bacteria and the correlations with different SCFAs in individuals with T1D and control subjects.

(A) Representative flow cytometric profiles of IgA-bound bacteria. (B) Summary of IgA-bound bacteria percentage from donors with T1D and healthy controls (n = 19/group). (C–E) Correlations between stool acetate (C), butyrate (D), or propionate (E) concentration and the level of IgA-bound bacteria. (The overall elevation or intercepts between the 2 groups was compared. The black circles show that there were more patients in the upper left areas, i.e., with a higher percentage of IgA-bound bacteria but lower stool SCFAs, n = 14–16.) (F–H) Correlations between stool acetate concentration and the relative abundances of Eubacterium (F) and Hathewayi (Firmicutes) (G), and between stool butyrate concentration and Enterococcaceae (Firmicutes) abundance (H) (n = 17/group). Data are presented as mean ± SEM and were assessed for statistical significance using a 2-tailed Student’s t test (B). Data in (F–H) were analyzed using a 2-tailed Pearson correlation coefficient test and/or linear regression. SCFAs, short-chain fatty acids; T1D, type 1 diabetes.

Figure 3. Role of gut microbiota from patients with T1D and control subjects in modulating host immune responses in GF NOD mice.

(A) Timeline for GF NOD mice gavaged with stool bacteria from patients with T1D or control subjects. (B) Unweighted principal coordinate analysis of stool microbiota from GF NOD mice received bacteria from either T1D donors or healthy control subjects (n = 22–23). (C) Gut microbiota composition at the species level (n = 22–23). (D) Gut permeability (n = 9–15). (E) Expression of Cramp (n = 16/group). (F) IgA⁺ B cell frequency in the spleen, PLN, MLN, and PP (n = 27–30). (G and H) Representative flow cytometric profiles of IgA-bound bacteria (G), and summary of IgA-bound bacteria percentage (H). Statistical analysis was performed by an analysis of similarities (B), multiple t tests with Bonferroni’s correction (C) or a 2-tailed Student’s t test(D–F and H, data combined from 2 or more independent experiments are presented as mean ± SEM). T1D, type 1 diabetes; GF, germ-free; PLN, pancreatic lymph node; MLN, mesenteric lymph node; PP, Peyer’s patch.

Figure 4. Role of acetate in modulating gut microbiota composition and IgA immune response.

(A) Spleen cells from specific pathogen–free NOD mice were stimulated with acetate, butyrate, or propionate (all 0.1 Mm) in the presence of anti-CD40 mAb (20 μg/mL) and LPS (10 μg/mL), and secreted IgA in the culture supernatant was measured (n = 8/group). (B) Timeline of GF NOD mice gavaged with stool bacteria from patients with T1D, followed by acetate or water gavage. (C–E) Gut microbiota in fecal samples were analyzed by 16S rRNA sequencing (n = 7–8). Chao richness (C), and relative abundance of Porphyromonadaceae (D) and Staphylococcaceae (E) at family level. (F) Proportion of TCRβ⁺CD4⁺ T cells, TCRβ⁺CD8⁺ T cells, and TCRβ^–CD19⁺ B cells in PP (n = 7–8). (G) Frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells in Peyer’s patch (n = 7–8). (H) Proportion of IgA⁺ B cells in PLN (n = 6–8). (I) Proportion of IgA⁺, IgM⁺, and IgD⁺ B cells in bone marrow (n = 7–8). (J) IgA concentration from the content of small intestine, cecum and colon (n = 7–8). Data combined from 2 independent experiments are presented as mean ± SEM and were analyzed with a 1-way ANOVA, followed by a Tukey’s test with Dunn’s correction for subsequent multiple comparisons between 2 groups (A) or a 2-tailed Student’s t test (C–J). GF, germ-free; T1D, type 1 diabetes; SCFAs, short-chain fatty acids; PLN, pancreatic lymph node.

Figure 5. Long-term effect of acetate treatment on IgA response in SPF NOD mice.

(A) Timeline of SPF NOD mice gavaged with acetate or water. (B) Longitudinal proportion of IgA-bound stool bacteria before and after gavage (n = 8–9). (C) Gut permeability (n = 7–9). (D) IgA concentrations in intestinal flush (n = 6–7). (E) Percentage of IgA-bound bacteria in the intestinal flush (n = 8–9). (F) Intestinal Pigr expression post-acetate treatment (n = 14–16). (G) IgA reactive to gut bacterial products. Stool microbiota from Rag-deficient mice (without any type/form of antibodies) were used to assess IgAs reacting to gut bacterial products. IgAs reacting to the bacterial products in small intestinal flush from the mice with or without acetate treatment were determined by anti-mouse IgA by ELISA (n = 14–16). (H) Proportion of CD4⁺ or CD8⁺ T cells and CD19⁺ B cells in Peyer’s patch (n = 6–7). (I) Proportion of splenic IgA⁺ B cells (n = 6–7). Data were pooled from 2 or more independent experiments, and analyzed using either a 2-way ANOVA (B) or a 2-tailed Student’s t test (C–I, Data are presented as mean ± SEM). SPF, specific pathogen–free.

Figure 6. Long-term effect of acetate treatment on IgA response and insulitis in specific pathogen–free NOD mice.

(A) Proportion of PNA⁺GL-7⁺ germinal center B cells (n = 6–7). (B) Percentage of splenic IgA⁺ GC B cells (n = 6–7). (C) Proportion of IgA⁺, IgD⁺, and IgM⁺ B cells in bone marrow (n = 8–9). (D) Representative insulitis images of microscopic views (×200) showing differences in immune cell infiltration marked by the black arrows. (E) Summarized percentage of severity of insulitis (total of 143–164 islets were graded from 6–7 mice/group). (F) Infiltrated immune cells in the islets (n = 6–7). Data were pooled from 2 or more independent experiments, and analyzed using either a 2-tailed Student’s t test (A–C, and F, Data are presented as mean ± SEM) or a chi-square test (E). PLN, pancreatic lymph node; MLN, mesenteric lymph node; PP, Peyer’s patch.

Figure 7. Direct effect of acetate on B cells.

Ex vivo splenic B cells were purified from specific pathogen–free NOD mice gavaged with acetate or water for 10 to 12 weeks. The purified B cells were stimulated in vitro in the presence of 10 Mm acetate with 20 μg/mL anti-CD40 mAb and 10 μg/mL LPS for 5 days. (A) IgA concentration in the culture supernatant of stimulated B cells was measured by ELISA (n = 8–9). (B) Representative flow cytometric plots of intracellular IL-6 expression of B cells after acetate stimulation. (C) Summary of IL-6-expressing B cells. (D) Secreted IL-6, determined by ELISA, from the culture supernatant of B cells stimulated with acetate (n = 6–7). (E–H) Gene expression of B cells, after acetate stimulation, was assessed by qPCR: Pstα (E), Pst2β (F), Stat5b (G), and Irf4 (H). The expression levels were determined using the 2^−ΔΔCt method by normalizing the housekeeping gene Gapdh. Data combined from 2 independent experiments are presented as mean ± SEM and were analyzed using a 2-tailed Student’s t test (A and C–H).

Figure 8. Gene expression of gprs and solute carrier family 16 on B cells.

Ex vivo splenic B cells were purified from specific pathogen–free NOD mice gavaged with 200 μL of water or equivalent volume of 100 Mm acetate for 10 to 12 weeks, and were stimulated in vitro with 20 μg/mL anti-CD40 mAb and 10 μg/mL LPS in the presence of 10 Mm acetate for 5 days. Gene expression of acetate-stimulated B cells was assessed by qPCR: Gpr41 (A), Gpr43 (B), Slc16a7 (C), Slc16a1 (D), and Slc16a3 (E). All expression data were determined using the 2^−ΔΔCt method by normalization with the housekeeping gene GAPDH. Data combined from 2 independent experiments are presented as mean ± SEM and were assessed for statistical significance using a 2-tailed Student’s t test (n = 7–8).