Evolution of gut, oral and vaginal microbiota during maturation in NOD mice

To study the microbiota over time in NOD mice, we collected fecal, oral and vaginal samples from the training cohort of mice from week 3 to week 31 (details in Supplementary Fig. 1).

First we analyzed the gut microbiota of the mice in the initial training cohort at 3 to 13 weeks. It is clear that the composition of gut microbiota at 3 weeks of age was significantly different from that at 6, 8, 10 and 13 weeks of age. These differences could be seen both at the phylum and class levels. The notable decrease in the individual phyla included Actinobacteria, Bacteroidetes, Proteobacteria, TM7 and Tenericutes, whereas the phylum Firmicutes was increased after 3 weeks of age (Fig. 1A).

Figure 1 Fecal, oral and vaginal bacteria during maturation in NOD mice. *Denotes statistical significance, p < 0.05. (A) Fecal bacterial composition at phylum level, and single phyla comparison between 3w, 6w, 8w, 10w and 13w (week-old) NOD mice. (B) Selected single class comparison between NOD mice of different ages. (C) Fecal bacterial composition at class, order, family and genus level between NOD mice at different ages. (D) Oral bacterial composition at phylum level, and selected single phyla comparison between NOD mice at different ages. (E) Vaginal bacterial composition at phylum level, and selected single phyla comparison between NOD mice at different ages. Full size image

Several classes of gut bacteria also differed significantly with age. These included the increased Coriobacteriia and Bacilli classes of Actinobacteria and Firmicutes phyla, respectively, although the parental phylum Actinobacteria was decreased along with growth and maturation (Fig. 1B). The classes of Bacteroidia, Alphaproteobacteria, Epsilonproteobacteria and Mollicutes, which belong to phyla of Bacteroidetes, Proteobacteria and Tenericutes, respectively, were decreased (Fig. 1B). The differences were also observed at order, family, genus and species levels, with similar trends to their higher orders (Fig. 1C).

We also investigated the oral microbiota and did not find obvious differences between 3-week and older mice, as seen in the lower gut microbiota. Although the most abundant phylum in the oral cavity was Proteobacteria, we found a decrease in Actinobacteria and Cyanobacteria but an increase in Bacteroidetes (Fig. 1D).

Next, we analyzed the vaginal microbiota and observed a larger variation between young and adult mice and the composition changes were constant in some phyla from 3 to 13 weeks old. Actinobacteria and Deferribacteres were the highest at 3 weeks, Bacteroidetes were the highest at 6 weeks, while Cyanobacteria were the highest at 8 weeks of age. Interestingly, Firmicutes increased with maturation, whereas Proteobacteria decreased (Fig. 1E).

Differences among gut, oral and vaginal microbiota in NOD mice during maturation

Comparing the three mucosal sites, the microbiota composition was very different between gut, oral and vaginal bacteria (Fig. 1). At a young age, Proteobacteria was the dominant phylum in the oral cavity (Fig. 1D), whereas Firmicutes was the dominant phylum of gut bacteria (Fig. 1A), while the composition of vaginal microbiota was intermediate between the two, although Firmicutes appeared to be more dominant (Fig. 1E). Gut and oral microbiota became relatively stable after 6 weeks of age, while vaginal microbiota continued to evolve until 10 to 13 weeks of age, by which time they resembled gut microbiota.

Alpha-diversity of gut, oral and vaginal microbiota during maturation

We next examined alpha-diversity of the gut/oral/vaginal microbiota, and found that the greatest diversity among the three anatomical sites was in the gut microbiota. Although at 3 weeks of age, gut microbiota were less diverse compared to the same mice at a later age, there was still greater diversity compared to the oral and vaginal microbiota from the same mice at any age. The changes in alpha-diversity of oral microbiota were similar during maturation, while vaginal microbiota showed the highest diversity when the mice were 3 weeks old and then gradually decreased, similar to the oral microbiota (Fig. 2A). This was opposite to the gut microbiota of the three-week-old mice.

Figure 2 Comparison of gut, oral and vaginal microbiota during maturation in NOD mice. (A) Alpha-diversity of gut, oral and vaginal microbiota of NOD mice at different ages. (B) Beta-diversity of gut, oral and vaginal microbiota of NOD mice at different ages. Full size image

Beta-diversity of gut, oral and vaginal microbiota during maturation

Beta-diversity analysis showed that 3-week-old mice have different gut microbiota compared with the same mice at later time points. It is interesting that we did not find noticeable differences in beta-diversity of microbiota between the oral and vaginal sites regardless of the age (from 3w to 13w; Fig. 2B). This indicated that comparing the three mucosal sites, gut microbiota are the most dynamic during mouse maturation. The gut microbiota in the NOD female mice undergo significant changes from weaning (3 weeks) to maturation (8 weeks) and become stable thereafter. Although oral and vaginal microbiota also underwent some changes during this period, the differences were not significant.

Diabetes incidence of NOD mice

While investigating the microbiota in the three anatomical sites, we also observed the experimental training cohort of mice for diabetes development. Of the 63 female NOD mice, 39 mice developed diabetes from 12 to 30 weeks of age. The overall incidence of diabetes was 61.9%, which was comparable to the diabetes incidence in our NOD mouse colony (a total of 144 females; Supplementary Fig. 2).

Gut, oral and vaginal microbiota in non-diabetic and pre-diabetic NOD mice

Next, we compared the gut microbiota in fecal samples, at different taxonomic levels and different ages, from the mice that were non-diabetic at 31 weeks (unlikely to develop diabetes beyond this age based on extensive experience of studying our NOD mouse colony) and those that were pre-diabetic (mice that developed diabetes before 31 weeks but had not developed diabetes at the time of gut microbiota sampling). Although the overall composition was similar between 3 and 13 weeks of age (Fig. 3A), several taxa showed significant differences after the Sidak-Bonferroni correction and there were more differences at the family and genus levels (Fig. 3B). Importantly, most of the differences were observed at 8 and 10 weeks of age, indicating that the age of around 2 months is important for the establishment of gut microbiota and this also may be the critical time for predicting whether a NOD female mouse would later develop diabetes.

Figure 3 Comparison of gut microbiota between non-diab (mice that had not developed diabetes at the end of the observation period) and pre-diab (mice that later developed diabetes) NOD mice at different ages. (A) Gut bacterial composition at the order level is shown. (B) Selected single phyla, class, family and genus that show significantly different abundance between non-diab and pre-diab mice. Each figure title indicates the taxon and time point. *P < 0.05, **P < 0.01, ***P < 0.001 by multiple t test with Sidak-Bonferroni correction. (C) Alpha-diversity of gut, oral and vaginal microbiota between non-diab and pre-diab NOD mice at different ages. At week 8, non-diab and pre-diab NOD mice showed significantly different alpha-diversity in gut microbiota. **P < 0.01 by two-way ANOVA. Full size image

We found that the composition of oral microbiota between non-diabetic and pre-diabetic mice was similar; however, the composition of vaginal microbiota was very different between non-diabetic and pre-diabetic mice (Supplementary Fig. 3). Since we did not observe a significant correlation between the vaginal bacterial profile and diabetes onset, as the variation between individual mice was very large, we did not pursue this further.

Alpha-diversity between non-diabetic and pre-diabetic NOD at different ages

We also compared the alpha-diversity between mice that were ultimately non-diabetic and pre-diabetic mice (these mice later developed diabetes) (Fig. 3C). At week 8, we found significantly greater alpha-diversity in the gut microbiota of the non-diabetic group compared with that of the pre-diabetic group. It is possible the changes at this age might have some impact on the host later in life, since this is the time when islet infiltration has begun but diabetes has not yet developed. In the oral and vaginal microbiota, there were no significant differences between the non-diabetic and the pre-diabetic groups, except at week 13. However, the overall diversity in the oral and vaginal microbiota was much lower than that in the gut microbiota.

Beta-diversity between non-diabetic and pre-diabetic NOD at different ages

By multivariate testing (ANOSIM), adding diabetic/non-diabetic and age as separation variants, we found at age of 8 weeks, the non-diabetic and pre-diabetic NOD mice showed significant differences in OTU composition based on Bray-Curtis distance (P = 0.019, Fig. 4A). PCoA analysis and Pearson’s correlation also showed some differences between these two groups at week 8 (Fig. 4B,C). However, there were no differences at weeks 3, 6, 10 and 13 (Supplementary Fig. 4), although there were some differences at week 3, but only by PCoA analysis (data not shown). From these results, we further concluded that the age of around two months is important for establishment of homeostasis of gut microbiota, and it may be possible to predict later diabetes development by gut microbiota at week 8.

Figure 4 Multivariate testing using pre-diabetic (D) or non-diabetic (ND) and age as well as OTUs as variants analyzed by Calypso. (A) ANOSIM based on Bray-Curtis distance. (B) Principal Coordinate Analysis (PCoA) based on Bray-Curtis OTU. (C) Pearson’s correlation by OTU cluster. Full size image

Gut microbiota evolution after mice become diabetic

We also studied the fecal samples collected from the mice after diabetes diagnosis (within 5 days) and found there was reduced alpha-diversity in the diabetic mice (Supplementary Fig. 5). It is interesting that the level of alpha-diversity in diabetic mice was similar to that seen in immature 3-week old mice. Moreover, the lower alpha-diversity was also observed in pre-diabetic mice at week 8 compared to the non-diabetic mice (Fig. 3C). However, the reduction in alpha-diversity was more evident after developing diabetes. Taxonomic changes were also different in non-diabetic and diabetic mice at the age of 16 to 27 weeks, comparing to the differences in non-diabetic and pre-diabetic mice at same ages, especially at the Genus level (Supplementary Fig. 6). Some of these alterations included Coprobacillus, Staphylococcus, and Escherichia, which are often reported to be pathogenic15,16, and were significantly increased (Supplementary Fig. 6).

Altered Gram-positive (G+)/gram-negative (G−) ratio during maturation and with diabetes development

Among the eight major phyla, Actinobacteria, Firmicutes, TM7 and Tenericutes are G+, while Bacteroides, Cyanobacteria, Deferribacteres and Proteobacteria are G−. Our results in the training cohort revealed that the ratio of G+to G− bacteria was significantly increased when the mice became diabetic. Importantly, the increased G+/G− ratio in diabetic mice was not associated with age of diabetes onset (Supplementary Fig. 7A). From week 3 to week 13, we also found an overall higher trend of G+/G− ratio in pre-diabetic NOD mice, while the most significant difference was seen at week 6 and 8 (Supplementary Fig. 7B). Thus, the G+/G− ratio could be used as a marker to predict whether an individual young mouse may eventually develop diabetes.

Overall taxonomic changes of gut microbiota between non-diabetic and pre-diabetic NOD mice in the training cohort

Analyzing the overall taxonomic changes between non-diabetic and pre-diabetic mice at week 8 in the training cohort, we found decreased phyla Bacteroidetes, Cyanobacteria, Proteobacteria, TM7 and Tenericutes, classes of Coriobacteriia, Bacteroidia, 4C0d-2, Erysipelotrichi, Proteobacteria (alpha/beta/delta/gamma), TM7–3, Mollicutes but increased classes of Actinobacteria, Bacilli, Clostridia and epsilon Proteobacteria (Supplementary Fig. 8). There were clear changes between non-diabetic and pre-diabetic mice in most of the families from the phyla Bacteroidetes and Firmicutes, especially the class of Erysipelotrichi. These changes are presented as a heatmap shown in Supplementary Fig. 9. There is a clear profile showing that the abundance of Bacteroidetes was decreased in the pre-diabetic mice. However, although the overall abundance of Firmicutes was increased in the pre-diabetic mice, the family of Erysipelotrichaceae showed the opposite profile, i.e., reduced in pre-diabetic mice (Supplementary Fig. 9). Since the phylum Bacteroidetes and family Erysipelotrichaceae were the most significantly different between non-diabetic and pre-diabetic groups, we chose these two taxa as indicators to predict whether our experimental test mice would eventually develop diabetes in the later studies.

In summary, our results show that alpha-diversity, G+/G− ratio and microbiota composition change (especially the phylum Bacteroidetes and Family Erysipelotrichaceae) may be used for prediction of future diabetes development in NOD mice at the age of 8 weeks. We next used each of the three indicators, as well as the three markers combined, to predict diabetes development in individual mice in the subsequent test experiments.

Using alpha-diversity for prediction of diabetes development

We carried out two independent test experiments, with 12 and 17 female NOD mice in each test cohort and predicted diabetes development using the sequencing data of their gut microbiota at the age of 8 weeks. We compared the gut bacterial alpha-diversity of the mice from the two independent experiments to the average gut bacterial alpha-diversity from the training cohort. As there was a significantly higher gut bacterial alpha-diversity in the non-diabetic mice when they were 8 weeks old in the training cohort, we plotted the gut bacterial alpha-diversity of each individual mouse in the test cohort, and compared to the average gut bacterial alpha-diversity of pre-diabetic and non-diabetes group in the training cohort (Fig. 5A). In both experimental groups, some mice showed the gut bacterial alpha-diversity close to or lower than the pre-diabetic mice in the training cohort (red line) whereas others showed gut bacterial alpha-diversity close to or higher than the average gut bacterial alpha-diversity of non-diabetic mice in the training cohort (blue line) (Fig. 5A). We predicted that the mice having gut bacterial alpha-diversity below the red line would develop diabetes, whereas the mice that had gut bacterial alpha-diversity above it would not develop diabetes (predicted result and the actual observation result are listed in Table 1). The accuracy of our prediction was 69% (20 out of 29 mice had the correct diabetes or non-diabetes prediction) if gut bacterial alpha-diversity was used as the sole prediction marker.

Figure 5 Prediction of diabetes development in NOD mice at 8 weeks of age. The prediction experiment was repeated using two test groups, comprising 12 and 17 mice, respectively. (A) Use of gut bacterial alpha-diversity for prediction of diabetes development. The individual mouse numbers in the figure are listed in the order of the mouse that had the highest diversity of gut bacteria to the lowest diversity. Non-diab (filled blue circles) and pre-diab (filled red squares) represent the average diversity of the bacteria from non-diab mice and pre-diab mice, respectively, from the training cohorts used as the reference points for comparison with the test experiment. Mice with gut bacterial alpha diversity higher than pre-diab were predicted not to develop diabetes by the end of observation (i.e., mice 9, 10, 7 and 12 in experiment 1 and 19, 23, 25, 26, 28, 22, 15 and 17 in experiment 2). (B) Use of gut bacterial G+/G− ratio for diabetes prediction. The cut-off line was drawn at 15.3 to separate the non-diabetic and pre-diabetic groups according to ROC analysis. Non-diab and pre-diab were the gut bacterial G+/G− ratio of non-diab mice and pre-diab mice, respectively, from the training cohorts. G+/G− ratio of single experimental mice were plotted and the mouse was predicted to be non-diab if the ratio was below the cut-off line or pre-diab if the ratio was above the cut-off line. (C) Use of gut taxonomic profile for prediction. The abundance of Phylum Bacteroidetes and Family Erysipelotrichaceae of the 29 experimental mice were plotted on this 2D scatter diagram. #30 (the red solid circle) represents the average abundance of Bacteroidetes and Erysipelotrichaceae of all non-diabetic NOD mice in the training cohort. Full size image

Table 1 Diabetes prediction of NOD mice at week 8 of age according to gut microbiota. Full size table

Using G+/G− ratio to predict diabetes development

To test whether G+/G− ratio could also be used as a marker to predict diabetes development, we calculated gut bacterial G+/G− ratio of all the mice used in the training cohort at 8 weeks of age (P = 0.001 by unpaired t-test comparing non-diabetic and pre-diabetic mice) and used the calculated value to predict whether the mice in the two test cohorts would develop diabetes. The optimal cut-off value at 15.3 achieved 100% sensitivity and 57% specificity to discriminate the pre-diabetic mice from non-diabetic ones according to ROC analysis. The gut bacterial G+/G− ratios of the individual mice in the test groups are shown in Fig. 5B, with the prediction and actual diabetes observation result are listed in Table 1. The accuracy of our prediction was 72% if using the gut bacterial G+/G− as the sole marker.

Using microbiota composition change to predict diabetes development

We plotted the percentage of Bacteroidetes and Erysipelotrichaceae in a 2D scatter graph. As the abundance of these two taxa was significantly altered in pre-diabetic mice, we proposed that the mice having a lower abundance of Bacteroidetes and Erysipelotrichaceae may later develop diabetes (lower left quadrant, Fig. 5C), compared to the average abundance of these two taxa in the non-diabetic mice from the training cohort. Table 1 lists the results from our prediction and the results from the actual observation. The accuracy of our prediction was 69% if using the percentage of Bacteroidetes and Erysipelotrichaceae as the only marker.

Prediction of diabetes development using the combined indicators

Finally, we used all three sets of data together to predict the diabetes development of the 29 experimental mice in the two independent experimental test cohorts, and monitored glycosuria for diabetes development weekly to 31 weeks (Table 1). If 2 or 3 of the three parameters indicated diabetic (or non-diabetic), we predicted the mouse would have (or not) developed diabetes. Thus, we predicted 16 mice would have developed diabetes (14 mice did develop diabetes later and 2 did not) and 13 mice would have not developed diabetes (9 mice did not develop diabetes by the time the experiments ended but 4 developed diabetes during the observation period). Thus, using the combined parameters, we were able to achieve ~80% predictive accuracy (23 out of 29 mice had the correct prediction) (Table 1). We also performed ROC analysis to test the sensitivity and specificity of the parameters used for diabetes prediction (Fig 6 and Table 2), with area under the curve (AUC) of 0.776. Gut bacterial G+/G− ratios or taxa alone gave the highest specificity or sensitivity, respectively. However, prediction utilizing all three parameters gave overall higher sensitivity, specificity, positive and negative predictive values (Table 2).

Figure 6 ROC analysis of sensitivity and specificity of the three-indicator-model for prediction of diabetes development in NOD mice at 8 weeks of age. Full size image

Table 2 ROC analysis of diabetes prediction using different indicators. Full size table

Immune cell characterization

To identify whether there were changes in immune cells correlating with the subsequent development of diabetes, as shown for the gut microbiota, we characterized the immune cells in the peripheral blood of the mice when 8 weeks old, by flow cytometry. All the mice tested showed similar frequencies of T cells (using anti-mouse CD4+ and CD8+ antibodies), B cells (using anti-mouse B220/CD19 antibodies), macrophages (using anti-mouse CD11b antibody), dendritic cells (using anti-mouse CD11c antibody) and the cytokine profiles of the immune cells (using anti-mouse IL-10/IL-17/IFN-γ antibodies) were also similar in all the mice tested (Supplementary Fig. 10). Furthermore, the co-stimulatory marker expression (using anti-mouse CD80 antibody), Treg (defined by gated CD4+CD25+FoxP3+ cells), Breg (defined by gated CD1dhighCD5+B220+CD19+ cells) and transitional B cells (defined by gated CD21+CD24+B220+CD19+ cells) were also similar between non-diabetic and diabetic groups (Supplementary Fig. 10). Our results showed that at the early time point of 8 weeks of age in NOD mice, there were no distinct immune cell features in the peripheral blood that correlated with marked differences in microbiota, comparing those mice that were ultimately non-diabetic with those that were pre-diabetic mice. Our data also suggested that the alteration of gut microbiota is likely to be an early biomarker for prediction of T1D development.