Antibiotics in early life alter the gut microbiome and increase disease incidence in a spontaneous mouse model of autoimmune insulin-dependent diabetes

Abstract

Insulin-dependent or type 1 diabetes is a prototypic autoimmune disease whose incidence steadily increased over the past decades in industrialized countries. Recent evidence suggests the importance of the gut microbiota to explain this trend. Here, non-obese diabetic (NOD) mice that spontaneously develop autoimmune type 1 diabetes were treated with different antibiotics to explore the influence of a targeted intestinal dysbiosis in the progression of the disease. A mixture of wide spectrum antibiotics (i.e. streptomycin, colistin and ampicillin) or vancomycin alone were administered orally from the moment of conception, treating breeding pairs, and during the postnatal and adult life until the end of follow-up at 40 weeks. Diabetes incidence significantly and similarly increased in male mice following treatment with these two antibiotic regimens. In NOD females a slight yet not significant trend towards an increase in disease incidence was observed. Changes in gut microbiota composition were assessed by sequencing the V3 region of bacterial 16S rRNA genes. Administration of the antibiotic mixture resulted in near complete ablation of the gut microbiota. Vancomycin treatment led to increased Escherichia, Lactobacillus and Sutterella genera and decreased members of the Clostridiales order and Lachnospiraceae, Prevotellaceae and Rikenellaceae families, as compared to control mice. Massive elimination of IL-17-producing cells, both CD4+TCRαβ+ and TCRγδ+ T cells was observed in the lamina propria of the ileum and the colon of vancomycin-treated mice. These results show that a directed even partial ablation of the gut microbiota, as induced by vancomycin, significantly increases type 1 diabetes incidence in male NOD mice thus prompting for caution in the use of antibiotics in pregnant women and newborns.

Fig 1. Diabetes incidence in antibiotic-treated NOD mice.

The protocol used to treat NOD mice is detailed in panels (a) and (b). Antibiotics were given in drinking water. A total of 35 breeding pairs were given either vancomycin (0.2mg/ml) or an antibiotic mixture composed of streptomycin (5mg/ml), colistin (1mg/ml) and ampicillin (1mg/ml) (Strep-Col-Amp). Controls received normal drinking water. Antibiotic treatment was started at mating and administered continuously during pregnancy and pursued for the whole duration of the experiment. Incidence of T1D was significantly increased in vancomycin-treated males (c) but not in females (d). Similarly incidence of T1D was significantly increased in Strep-Col-Amp-treated males (e) but not in females (f).

Fig 2. Impact of the antibiotic treatment on Alpha and Beta-diversity of mice microbiota.

Panel (a) shows a three dimensional representation of the PCoA analysis. Panels (b) and (c) show the rarefaction curves obtained using the Chao1 and Shannon indexes, respectively.

Fig 3. Microbiota composition of control and treated mice at phylum, family and genus levels.

The bar plots show the microbiota composition at phylum, family and genus levels of the 36 mice fecal samples analyzed by 16S rRNA profiling, divided by treatment. On the right side three bar plots show the aggregate (average) bacterial composition of the three treatment conditions. Only taxa with relative abundance > 2% in at least one sample are shown.

Fig 4. Impact of the antibiotic treatment on microbiota composition of mice at genus level.

The two bar plots show the variation of bacterial taxa at genus level when mice are treated with vancomycin (a) and Strp-Col-Amp (b). Only taxa with relative abundance in at least one sample > 2% and with a variation > 0.1% are shown.

Fig 5. Immune cells in the intestinal lamina propria and spleen.

(a) Percentages of IFNγ and IL-17-producing T cells (upon 6-hr stimulation with PMA/ionomycin) among CD45+CD4+TCRαβ+ and CD45+TCRγδ+ T cells isolated from the spleen and the lamina propria of the colon and ileum of 10 week-old vancomycin-treated mice (black bars) versus controls (open bars). The data in males (upper panels) and in females (lower panels) are shown. Five animals per group were analyzed. Significant differences are indicated as follows: * p<0.05; ** p<0.01. (b) Representative dot plots of intracytoplasmic staining to detect IFNγ and IL-17-producing T cells among CD45+CD4+TCRαβ+ or CD45+TCRγδ+ T cells. (c) Percentages of Foxp3+ cells among CD4+CD45+ T cells and of CD62L+ cells and Helios+ cells among CD45+CD4+FoxP3+ lymphocytes in 10-week-old NOD males (upper panels) and females (lower panels) treated with vancomycin (black bars) or controls (open bars). Significant differences are indicated as follows: * p<0.05; ** p<0.01.

Fig 6. Analysis of gene expression in the gut.

Relative expression of Foxp3, Ifng, Il10, Il17a and Tgfb1 in the colon, ileum, Peyer’s patches and mesenteric lymph nodes of 10-week-old male (a) and female (b) mice treated with vancomycin (grey bars) and controls (open bars). Five animals per group were analyzed. Expression of Foxp3, Ifng, Il17a and Il10 was normalized to that of Cd3, while expression of Tgfb1 was normalized to Hprt expression. Results are expressed as fold of the mean expression observed in untreated mice. Only changes in expression levels outside the grey zone indicates changes in expression >2-fold increase or decrease in vancomycin-treated mice (black bars) as compared to controls (open bars). Significant differences are indicated as follows: * p<0.05; ** p<0.01.