Supplemental Data
Figure Legends
Supplemental Figure 1.
Fecal transplantation alters the microbiota in the ileum and colon of the chimeric mice . The abundance of several microbes in the ileum (A) and colon (B) was examined before and after fecal transplantation by qPCR using specific primer sets (n = 6-8 mice/group).
Supplemental Figure 2. qPCR confirms the 454 pyrosequencing data . The abundance of several microbes found to be altered through 454 sequencing analysis was confirmed through qPCR analysis using specific primers to bacterial groups identified. Bacterial groups were assessed in NOR and NOD mice (A, ileum. B, colon) and in the antibiotics treated mice (C, ileum. D, colon). (n = 5-8 mice/group).
Supplemental Figure 3. Diabetogenic microbes induced by antibiotics do not induce barrier dysfunction.
Levels of FITC-dextran were measured in the sera 4-hours post oral-gavage to measure intestinal permeability. Fecal transplanted NOD mice carrying NOR microbiota was found to improve barrier function B. Both neomycin and vancomycin treatment improved barrier function (n=4-5).
Table S1 : Bacterial primers used for qPCR
Materials and Methods
DNA extraction and Real-Time PCR Analysis: DNA was extracted from tissue segments of the colon and ileum from mice that were 11-20 weeks old and qPCR was performed as described
(Gibson et al 2015).
Primers are listed in table S1.
FITC-dextran barrier function assay : The FITC-dextran (FD4) assay for barrier function was performed
as previously described (Lee et al 2010). 11 week old mice received 150 µl of 80 mg/mL FITC-dextran
by oral gavage. 4 hours later, mice were anaesthetized and blood was collected by cardiac puncture and fluorescence was measured in plasma.
Statistical analysis: Graphing and statistical analysis was performed using Graphpad Prism. For comparisons between > 2 groups, *<0.05, **<0.01 and ***<0.0001 by one way ANOVA with
Tukey’s post-hoc test for parametric data and Kruskal-Wallis test with Dunn’s post-hoc test for non-parametric data.
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Results
Fecal transplanted microbial analysis
To examine the microbiota in fecal transplanted mice, we examined particular microbes before and after transplantation by qPCR (supplemental Figure 1). Several microbes were transferred to the NOD mice that received NOR transplants including Akkermansia muciniphila , Desulfovibrio spp., Enterobacteriaceae in the ileum and A. muciniphila and Enterobacteriaceae in the colon. In addition, microbes were lost in the NOR mice that received NOD transplants including
Segmented Filamentous bacteria in the ileum and Bacteroides acidifaciens in the colon. Since we saw differences in insulitis between the NOD and NOR transplanted mice, these results suggest this is due to particular microbes or microbial products that had been transplanted from the stool gavages.
Microbial Analysis
Due to limitations inherent in broad sequence analysis and to support our conclusions regarding the microbiome comparison based on our 454 pyrosequencing data, we confirmed our data using qPCR. Our qPCR data is consistent with % abundance data observed using high throughput sequencing (Supplemental Figure 2).
Barrier dysfunction is not required for the accelerated diabetes
Our group has previously shown that NOD mice exhibit dysfunctional intestinal barriers prior to diabetes onset compared to other mouse strains that are resistant to diabetes (Lee et al 2010).
Moreover intestinal barrier dysfunction is also seen humans with T1D (Bosi et al 2006, Sapone et al 2006). To determine if barrier function was related to the microbiota in the NOD and NOR mice, we examined intestinal permeability to FITC-dextran in the fecal transplanted chimeric mice (supplemental Figure 3A). The NOR
H
+ NOD
M
had more FITC-dextran translocating to their blood compared to NOR
H
+ NOR
M
indicating the NOD microbiota increased intestinal barrier dysfunction. Likewise, the NOD
H
+ NOR
M
displayed less FITC-dextran in their blood compared to NOD
H
+ NOD
M
indicating the NOR microbiota could protect against intestinal barrier dysfunction. These results support the hypothesis that NOD mice harbor pathogenic microbes capable of disrupting the normal intestinal barrier, potentially driving inflammation of the pancreas. However, when we assessed the function of the intestinal barrier in the antibiotic-
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treated NOD mice, we found that both vancomycin and neomycin treated mice had significantly improved barrier dysfunction compared to untreated NOD mice, despite their accelerated diabetes (supplemental Figure 3B). We conclude from our study that although barrier dysfunction often correlates with insulitis/diabetes development, it is not a necessary factor, since although antibiotic treatment reduced barrier dysfunction, it led to accelerated diabetes development.
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