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. 2016 Feb;10(2):321-32.
doi: 10.1038/ismej.2015.114. Epub 2015 Aug 14.

Prolonged Antibiotic Treatment Induces a Diabetogenic Intestinal Microbiome That Accelerates Diabetes in NOD Mice

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Free PMC article

Prolonged Antibiotic Treatment Induces a Diabetogenic Intestinal Microbiome That Accelerates Diabetes in NOD Mice

Kirsty Brown et al. ISME J. .
Free PMC article

Abstract

Accumulating evidence supports that the intestinal microbiome is involved in Type 1 diabetes (T1D) pathogenesis through the gut-pancreas nexus. Our aim was to determine whether the intestinal microbiota in the non-obese diabetic (NOD) mouse model played a role in T1D through the gut. To examine the effect of the intestinal microbiota on T1D onset, we manipulated gut microbes by: (1) the fecal transplantation between non-obese diabetic (NOD) and resistant (NOR) mice and (2) the oral antibiotic and probiotic treatment of NOD mice. We monitored diabetes onset, quantified CD4+T cells in the Peyer's patches, profiled the microbiome and measured fecal short-chain fatty acids (SCFA). The gut microbiota from NOD mice harbored more pathobionts and fewer beneficial microbes in comparison with NOR mice. Fecal transplantation of NOD microbes induced insulitis in NOR hosts suggesting that the NOD microbiome is diabetogenic. Moreover, antibiotic exposure accelerated diabetes onset in NOD mice accompanied by increased T-helper type 1 (Th1) and reduced Th17 cells in the intestinal lymphoid tissues. The diabetogenic microbiome was characterized by a metagenome altered in several metabolic gene clusters. Furthermore, diabetes susceptibility correlated with reduced fecal SCFAs. In an attempt to correct the diabetogenic microbiome, we administered VLS#3 probiotics to NOD mice but found that VSL#3 colonized the intestine poorly and did not delay diabetes. We conclude that NOD mice harbor gut microbes that induce diabetes and that their diabetogenic microbiome can be amplified early in life through antibiotic exposure. Protective microbes like VSL#3 are insufficient to overcome the effects of a diabetogenic microbiome.

Figures

Figure 1
Figure 1
NOD and NOR mice harbor different gut microbiota. Microbial communities from the colon and ileum were assessed by 454 pyrosequencing. (a and b) The microbiota from NOR and NOD mice differed from each other both in the ileum (a; P=0.03) and the colon (b; P=0.009). (c) At the phyla level, NOR and NOD microbiota is similar yet the abundance of certain taxa is different in NOD versus NOR in the ileum (d) and colon (e; n=5-8; ND=none detected). *P<0.05 by FDR-corrected one way analysis of variance with Tukey's post hoc test.
Figure 2
Figure 2
Mice susceptible to diabetes harbor a microbiota that initiates insulitis in diabetes-resistant mice. (a) Schematic representation of fecal transplantation between NOR and NOD mice. Tissues were collected at 11 weeks and cross-sections of pancreas tissues were stained with H&E and scored by two blinded observers. (b) Representative pictures of islet infiltration. (c) Composite insulitis score and (d) percentage of islets in each mouse that were scored 0, 1, 2 and 3 (scale bar=100 μm; n=10–12 mice per group). *P<0.05 by Mann-Whitney U-test.
Figure 3
Figure 3
Antibiotic exposure accelerates diabetes in NOD mice and alters CD4+ T-cell populations. (a) Vancomycin and neomycin accelerated diabetes onset in NOD mice (n=17–25) *P<0.05 and **P<0.01 by log-rank survival test. (b) Serum insulin auto-antibody levels in pre-diabetic 11-week old NOD mice (n=10). (c) Percent of IFN-γ+ CD4+ T cells and (d) percent of IL17+ CD4+ T cells in the Peyer's patches of 4–6 week old NOD mice (n=12). *P<0.05, **P<0.01 and ***P<0.001 by one way analysis of variance with Tukey's post hoc test for parametric data and Kruskal-Wallis test with Dunn's post hoc test for non parametric data.
Figure 4
Figure 4
Antibiotic-induced diabetes is associated with microbial dysbiosis. Communities were profiled in the ileum and colon of control NOD mice and those treated with vancomycin and neomycin by 454 pyrosequencing. (a) Antibiotic-treated mice have less diverse intestinal microbiota. Microbial communities of antibiotic-treated mice are different compared with untreated NOD in the ileum (b; vancomycin P=0.003, neomycin P=0.003) and colon (c; vancomycin P=0.004, neomycin P=0.005). Several microbes are different in abundance in the ileum (d) and colon (e) of antibiotic-treated mice (n=4–8, ND=none detected). *P<0.05 and **P<0.01 by FDR-corrected one way analysis of variance with Tukey's post hoc test.
Figure 5
Figure 5
Metagenomic analysis of microbiota from antibiotic-treated NOD mice. PCoA plots showing clustering of predicted KEGG orthologs present in the microbiota of untreated, vancomycin-treated and neomycin-treated NOD mice in the ileum (a; neomycin P=0.59, vancomycin P=0.014) and distal colon (b; neomycin P=0.263, vancomycin P=0.001). (c) The microbiome's metabolic gene groupings in the ileum and distal colon and (d) differentially abundant gene groupings present in the colonic microbiome of NOD mice (n=4–8 per group). *P<0.05 and **P<0.01 by FDR-corrected one way analysis of variance with Tukey's post hoc test.
Figure 6
Figure 6
NOD mice given antibiotics carry a microbiota that produces fewer short-chain fatty acids. The fecal content of NOR mice contains more acetic acid than NOD mice and antibiotic treatment further reduces the acetic acid content. Vancomycin, but not neomycin, treatment reduces the amount of propionic and butyric acids found in the cecal content (n=12 mice/group). *P<0.05 and ***P<0.001 by one way analysis of variance with Tukey's post hoc test for parametric data and Kruskal-Wallis test with Dunn's post hoc test for non parametric data.
Figure 7
Figure 7
VSL#3 does not alter diabetes progression and VSL#3 bacteria do not efficiently colonize efficiently. (a) VSL#3 does not alter the onset of diabetes in NOD mice (n=17–25 mice per group). (bd) qPCR was performed on gut tissues taken from mice untreated or treated with VSL#3 in drinking water. (b) Lactobacillus and (c) Bifidobacteria spp. were not increased in either the ileum or colon. Streptococcus spp. were not increased in the ileum but were increased in the colon (n=10–12 mice per group). *P<0.05 by Mann-Whitney U-test.

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