We previously reported low‐level intestinal inflammation in NOD compared to disease‐protected mice. 6 It is unknown the extent to which genetically driven gut inflammation is a barrier to therapies designed to restore a healthy microbiota in T1D. In this study, we have investigated whether the genetic effect on the NOD microbiota composition could be overcome by the introduction of a highly dissimilar microbiota from diabetes‐protected mice and whether this could reduce intestinal inflammation and disease progression.

A number of studies have described alterations in the gut microbiota associated with type 1 diabetes (T1D) which is thought to be linked to disease pathogenesis. 1 - 5 Recently, we have shown that genetic susceptibility to T1D contributes to the structure of the intestinal microbiota in both T1D susceptible nonobese diabetic (NOD) mice and humans. 6 In NOD mice, manipulations that lead to major disturbances in the microbiota can both protect from and accelerate disease. 7 - 9 This suggests that a balance between host genetics and environment contributes to dysbiosis associated with T1D risk.

Immune regulation was not restored by cohousing NOD and B6 mice. NOD mice cohoused long‐term from birth (LCoH), not‐cohoused NOD (NCoH) and B6 mice culled at 12 weeks of age and lymph node and spleen tissue analyzed by FACS. (a) Foxp3 + CD4 + Treg frequency within CD4 + T cells. (b) IGRP‐specific and (c) insulin‐specific tetramer+ cell proportion within CD8 + T cells. Dotted line: mean frequency of control LLO‐specific tetramer+ cells. Mean and s.e.m. are shown. * P <0.05, *** P <0.001, **** P <0.0001. LCOH: long‐cohoused. The experiment was performed once.

Regulatory immune T cells (Tregs) play an important role in suppressing autoimmunity and defects in Tregs activity, contribute to the development of T1D. 12 - 14 Specific members of the gut microbiota have been shown to impact peripheral induction of Tregs. 15 We examined Treg frequencies in the mesenteric, pancreatic and inguinal lymph nodes and spleen. B6 mice had significantly higher proportions of CD4 + Foxp3 + Tregs in their lymph nodes and relatively less in spleen compared with both NOD and LCoH NOD mice as previously described (Figure 4 a). 16 No difference was seen between the proportions of Tregs in LCoH and noncohoused NODs. Islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP) is a major autoantigen in the NOD mouse. 17 Several species of the Bacteroides genera express an epitope that mimics IGRP and can promote recruitment of diabetogenic CD8 + T cells to the gut. 18 , 19 We did not observe any differences in the frequency of IGRP‐specific CD8 + T cells in lymph nodes of NOD and LCoH NOD mice using IGRP‐specific tetramers (Figure 4 b). Strikingly, insulin‐specific CD8 + T cells were significantly decreased in the pancreatic and mesenteric lymph nodes of LCoH NOD mice (Figure 4 c). We concluded that while cohousing NOD mice with B6 mice did not have a significant effect on peripheral Tregs, autoreactive CD8 + T cells were differentially impacted depending on their specificity.

Intestinal architecture, inflammation, goblet cell mucous production, and lysozyme production were not altered by cohousing NOD mice. Histological sections from NOD mice cohoused long term from birth (LCoH), not‐cohoused NOD (NCoH) and B6 mice culled at 12 weeks of age were scored (maximum possible score 29) (a) Examples of H&E stained colonic tissue and ileum Paneth structures with summary of gut scoring from n = 6 to 8 mice per group. (b) Goblet PAS staining of goblet cells from colonic tissue and quantification of goblet cell area from n = 6 to 8 mice per group. (c) IHC staining of lysozyme P production by Paneth cells from the ileum and quantification of staining intensity in n = 6 or 7 mice per group. *** P <0.001, **** P <0.0001. The experiment was performed once.

We next questioned whether cohousing had any effect on the subclinical intestinal inflammation seen in NOD mice. 6 As expected, control NOD had significantly higher inflammation scores compared to B6 mice (Figure 3 a). B6 mice had an increased goblet cell area (Figure 3 b) and Paneth cell staining of lysozyme P in the ileum to NOD mice (Figure 3 c). However, there were no statistically significant differences between LCoH and control NOD mice in inflammatory scoring, goblet cell area or lysozyme P staining. We concluded that the altered abundance of the taxa shown in Figure 1 d did not impact intestinal inflammation present in NOD mice.

The effect of cohousing on diabetes frequency and insulitis. Diabetes frequency in (a) NOD mice not cohoused (NCoH, n = 23) or cohoused from weaning (CoH@W NOD n = 22) and (b) in control NOD mice (NCoH, n = 20), NOD mice cohoused from birth short‐term (SCoH n = 20) or long‐term cohoused (LCoH n = 34). P ‐values from a log‐rank test are shown. Insulitis scoring from individual mice (c) and mean insulitis scores (d) from mice cohoused continually from birth at 12 weeks of age. ** P <0.01. Mean and s.e.m. are shown. Each experiment was performed once.

We next investigated whether introduction of B6‐derived taxa by cohousing impacted the onset of diabetes. Cohousing from weaning showed a trend to delaying the progression of diabetes (Figure 2 a, P = 0.056). LCoH NOD mice showed a trend to a reduced frequency of diabetes; however, this was not significant (Figure 2 b, P = 0.244). SCoH NOD mice had a similar disease onset to control NOD mice (Figure 2 b, P = 0.24). There was also no difference in insulitis scores between LCoH and control NOD mice (Figure 2 c, d). Thus, the changes in the NOD microbiota brought about by cohousing with B6 mice were not sufficient to significantly prevent or slow progression to diabetes.

We also compared the microbiota of the cohoused mice at weaning when the LCoH and SCoH groups were separated from each other. As expected, while the microbiota profiles of cohoused and noncohoused NOD mice were different at weaning ( P = 0.02), the LCoH and SCoH groups were not significantly different at the time of separation ( Supplementary figure 3a ). Likewise, the Shannon diversity index was increased at 3 weeks of age in both LCoH and SCoH groups compared with the control NOD mice ( Supplementary figure 3b ). These data indicate that cohousing during the neonatal period is essential for effective transfer of bacterial species into the NOD gut, although maintaining cohousing past weaning was required to preserve these changes to a maximal effect.

By 10–12 weeks of age, sPLS‐DA multivariate analysis showed that cohousing from weaning modestly altered the NOD microbiota (Figure 1 b, NOD control vs CoH@W P = 0.05). Noncohoused NOD and B6 mice had distinct gut microbial communities ( P < 0.001, Supplementary figure 1a ). Only two genera ( Clostridiaceae and Clostridium ) had altered abundances in NOD CoH@W compared to control NOD mice ( Supplementary figure 1a ), but not in the direction of the B6 mice. Consistent with this, the Shannon diversity index was not increased by cohousing from weaning ( Supplementary figure 2a ). In contrast, both LCoH and SCoH NOD mice had a significantly altered microbiota to control NOD mice (Figure 1 c, P = 0.018 and P = 0.003, respectively). Eight genera were significantly altered in LCoH compared with the control NOD mice (Figure 1 d), though many other genera were not altered ( Supplementary figure 1b ). Some taxa that were significantly increased in abundance by long‐term cohousing were not altered or had a partial increase following short‐term cohousing (e.g. Parabacteroides , unclassified YS2 and Clostridium ). Consistent with this, the Shannon diversity index was not increased in SCoH NOD mice but was in the LCoH NOD mice ( Supplementary figure 2b ). Together, these data suggest that the majority of changes to the microbiota were introduced prior to weaning, but the NOD intestinal environment may cause the microbiota to revert back to a steady state over time.

Long‐term cohousing of NOD and B6 mice from birth results in an altered gut microbiota. (a) Schematic of cohousing design. (b) sPLS‐DA multivariate analysis of mice cohoused from weaning and control noncohoused B6 and NOD mice ( n = 6 or 7 per group). (c) sPLS‐DA analysis of mice cohoused from birth ( n = 10 or 11 per group). Ellipses show 95% confidence intervals. (d) Genera with differential abundance in cohoused NOD versus control NOD mice from (c) (adj P < 0.05). CoH@W NOD: Cohoused at weaning. NCoH NOD Not cohoused (control). SCoH NOD: short‐term cohoused. LCoH NOD: long‐term cohoused. Mean and s.d. are shown. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was performed once.

We aimed to test whether cohousing T1D susceptible NOD and disease‐protected C57BL/6 (B6) mice would result in the NOD microbiota becoming more B6‐like and protect from disease. The study was split into two experiments (Figure 1 a). In the first experiment, NOD pups were cohoused with age‐matched B6 pups from weaning (CoH@W). In the second experiment, NOD mice were cohoused with B6 mice from birth either continually (long‐cohoused: LCoH) or until weaning (short cohoused: SCoH). Fecal samples were collected at weaning and 10–12 weeks of age and used to profile the microbial community composition.

Discussion

We hypothesized that cohousing NOD and B6 mice would change the gut microbiota of the NOD mice to become more B6‐like. However, the effects of cohousing were modest and terminating cohousing at weaning reduced the effect. Exposure during the preweaning period was essential for optimal establishment of a new microbiota. The changes induced in the gut bacterial communities of the NOD mice were not sufficient to alter either the gut inflammatory environment or to prevent diabetes development. Notably, insulin but not IGRP‐specific CD8+ T cells were modulated by cohousing suggesting that the microbiota influence autoreactive T cells in an epitope‐specific manner.

In our colony, the B6 mice had a microbiota dominated by Allobaculum while the NOD mice had a predominance of Lactobacillus. Surprisingly, neither cohousing from before birth or from weaning changed the abundance of these genera. The taxa that were altered were all obligate anaerobes, which might infer that cohousing leads to a less oxidative intestinal environment. An aerobic intestinal environment has been associated with dysbiosis and disease.20 However, intestinal inflammation observed histologically in NOD mice was not reduced by cohousing.

Introduction of a new microbiota into NOD mice may be dependent on the specific strain and sex of the donor. For example, cross‐fostering NOD pups to nonobese resistant mothers permanently altered the microbiota of the offspring.21 However, this only resulted in a significant reduction in diabetes in the male offspring. An adult male NOD microbiota transferred to females by oral gavage was able to protect from diabetes and alter the recipient's microbiota.22 Others have reported that B6 foster parents did not transfer diabetes protection to NOD mice, whereas ICR foster parents did.23 It is therefore likely that the microbiota composition of the donor strain is critical for determining protection.

The small effects on the NOD microbiota may be because of passive microbiota transfer through cohousing. While we introduced the B6 and their microbiota, we did not cross‐foster the NOD pups onto B6 dams. This would have allowed closer interaction between the NOD pups and the B6 females as well as exposing them to maternal antibodies from the B6 mothers. Maternal antibodies contribute to shaping the microbiota of the offspring.24

The small changes in the microbiota we observed may have been due to an inherent incompatibility for one another's microbiota because of genetic pressure. Constraints to colonization may occur that allow only an inflammation tolerant microbiome in the NOD strain. An enterotype associated with low‐grade inflammation has been described in NOD mice,25 suggesting that subclinical inflammation contributes to shaping the gut microbiota.

The implication of our findings is that a therapeutic strategy aimed at inducing permanent changes in the gut microbiota of individuals at risk of T1D would be more effective if initiated in early life and continued long‐term. A T1D susceptible host genetic background imposes a barrier to fundamentally changing the microbiota composition. Major community remodeling may require therapy to reverse underlying inflammation to induce long‐lasting effects.