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Gut microbiota (GM) and diet both appear to be important in the pathogenesis of type 1 diabetes. Fermentable fibres (FFs), of which there is an ample supply in natural, diabetes-promoting diets, are used by GM as a source of energy. Our aim was to determine whether FFs modify GM and diabetes incidence in the NOD mouse.Female NOD mice were weaned to a semisynthetic diet and the effects of FF supplementation on diabetes incidence and insulitis were evaluated. Real-time quantitative PCR was employed to determine the effects imposed to gene transcripts in the colon and lymph nodes. Changes to GM were analysed by next-generation sequencing.NOD mice fed semisynthetic diets free from FFs were largely protected from diabetes while semisynthetic diets supplemented with the FFs pectin and xylan (PX) resulted in higher diabetes incidence. Semisynthetic diet free from FFs altered GM composition significantly; addition of PX changed the composition of the GM towards that found in natural-diet-fed mice and increased production of FF-derived short-chain fatty acid metabolites in the colon. The highly diabetogenic natural diet was associated with expression of proinflammatory and stress-related genes in the colon, while the semisynthetic diet free from FFs promoted Il4, Il22, Tgfβ and Foxp3 transcripts in the colon and/or pancreatic lymph node. PX in the same diet counteracted these effects and promoted stress-related IL-18 activation in gut epithelial cells. 16S RNA sequencing revealed each diet to give rise to its particular GM composition, with different Firmicutes to Bacteroidetes ratios, and enrichment of mucin-degrading Ruminococcaceae following diabetes-protective FF-free diet.FFs condition microbiota, affect colon homeostasis and are important components of natural, diabetes-promoting diets in NOD mice.

Dietary and microbial factors and the gut immune system are important in autoimmune diabetes. We evaluated inflammatory activity in the whole gut in prediabetic NOD mice using ex vivo imaging of reactive oxygen and nitrogen species (RONS), and correlated this with the above-mentioned factors.NOD mice were fed a normal diet or an anti-diabetogenic casein hydrolysate (CH) diet. RONS activity was detected by chemiluminescence imaging of the whole gut. Proinflammatory and T cell cytokines were studied in the gut and islets, and dietary effects on gut microbiota and short-chain fatty acids were determined.Prediabetic NOD mice displayed high RONS activity in the epithelial cells of the distal small intestine, in conjunction with a proinflammatory cytokine profile. RONS production was effectively reduced by the CH diet, which also controlled (1) the expression of proinflammatory cytokines and colonisation-dependent RegIIIγ (also known as Reg3g) in ileum; (2) intestinal T cell activation; and (3) islet cytokines. The CH diet diminished microbial colonisation, increased the Bacteroidetes:Firmicutes ratio, and reduced lactic acid and butyric acid production in the gut.Epithelial RONS production and proinflammatory T cell activation appears in the ileum of NOD mice after weaning to normal laboratory chow, but not after weaning to an anti-diabetogenic CH diet. Our data suggest a link between dietary factors, microbial colonisation and mucosal immune activation in NOD mice.

NOD.Igµ null mice lacking mature B cells are highly resistant to diabetes and display poor CD4 T cell responses to autoantigens. Nevertheless, the degree to which different B cell subsets contribute to diabetes in NOD mice remains unresolved. Due to their role in the recognition of microbial and autoantigens, peritoneal B cell characteristics were examined in NOD mice to see if they differ developmentally, phenotypically or functionally in aspects relevant to diabetogenesis. The population dynamics, activation state, migratory behaviour and antigen presentation function were investigated in NOD peritoneal B cells. NOD peritoneal B cells were found to express abnormally high levels of co-stimulatory molecules (CD40, CD86 and CD69). In contrast, the expression of l-selectin and integrin α4β1 was markedly reduced in NOD mice compared with BALB/c and C57BL/6 mice. The number of B cells in the peritoneum was lower in NOD than in control mice throughout development; migration of B cells from the peritoneum to the pancreatic lymph nodes in NOD mice was enhanced tenfold. NOD B cells showed no chemotactic response to sphingosine-1-phosphate, which normally acts to retain B cells in the peritoneum. Peritoneal B cells of NOD mice also presented insulin autoantigen to CD4 T cells, inducing T cell proliferation. NOD peritoneal B cells are hyperactivated, migrate to the pancreatic lymph nodes and are capable of driving insulin-specific CD4 T cell activation. These characteristics could make them important for inducing or amplifying T cell responses against islet-antigens.

To the Editor: Environmental factors play a role in the development of type 1 diabetes. There is growing enthusiasm towards assessing the role of gut microbiota as an environmental modifier of autoimmunity in type 1 diabetes. This is largely based on the increasing awareness of the role of microbiota in regulating gut homeostasis and immune responses in the gut [1]. Microbiota composition varies between individuals and is modified by diet [2]. An emerging number of studies suggest that the abundance of Bacteroidetes is associated with autoimmunity in type 1 diabetes [3]. Thus, a diet promoting an abundance of Bacteroidetes could be an environmental risk modifier in type 1 diabetes. The notion of Bacteroides as ‘diabetogenic’ species is now further supported by a recent study in which Bacteroides dorei was found to be abundant in children who subsequently developed diabetes-related autoantibodies in a cohort of 76 children participating in the Diabetes Intervention and Prevention Project (DIPP) study in Southwest Finland [4]. In NODmice, diabetes incidence is effectively modified by diet [5]. We have studied the effect of two common nutritional fibres, pectin and xylan, on microbial colonisation, gut homeostasis and diabetes development in NOD mice [6]. Pectin and xylan dramatically increased colonisation of the intestine by Bacteroidetes, including B. dorei (Fig. 1 and data in [6]), increased the expression of stress-relatedmolecular transcripts and inflammatory cytokines in the gut, and altered the balance of T cell cytokines in pancreatic lymph nodes. In terms of disease-promoting mechanisms, our findings alluded to a pathophysiological model [6] well aligned with the model recently discussed by Davis-Richardson and Triplett in Diabetologia [3]. The core idea is that, by displacing beneficial butyrate-producing taxa, Bacteroidetes have adverse effects on epithelial cells [3, 6]. Our findings provided experimental evidence in support of this and indicated that at least some Bacteroides species play a role in diabetes-related autoimmunity. In addition, our results identified dietary fibres as novel candidates for environmental modifiers of the pathogenesis of diabetes. Interestingly, induction of low-grade inflammation in the gut and microbiota encroachment was recently reported in mice following administration of two other fibre compounds used as dietary emulsifiers [7]. According to Finnish healthcare recommendations, fermentable fibres including pectin and xylan (hemicellulose, a constituent of all plant cell walls) are introduced into the diet of infants in the form of berry cocktails. In Finland, parents are recommended to start giving berry cocktails to their infants at 4 months age, an age preceding the peak in the observed abundance of B. dorei [3]. * Arno L. M. Hanninen arno.hanninen@utu.fi

Microbial factors influence the development of diabetes in NOD mice. Studies in germ-free animals have revealed important roles of microbiota in the regulation of Th17 and forkhead box P3 (FOXP3)(+) T regulatory (Treg) activation in the intestine. However, the effects of intestinal microbiota in immune regulation and diabetes development in NOD mice are still poorly understood.A colony of germ-free NOD mice was established to evaluate the effects of intestinal microbiota on regulatory immunity in the gut, and on the development of insulitis and diabetes in NOD mice.Diabetes developed in roughly equal numbers in germ-free and specific pathogen-free NOD mice. Insulitis was accentuated in germ-free NOD mice; yet insulin preservation was unaltered. Germ-free NOD mice showed increased levels of Il17 (also known as Il17a) mRNA in the colon, and of Th17 and Th1 cells in the mesenteric and pancreatic lymph nodes, while Foxp3 mRNA and FOXP3(+) Tregs were reduced. In the islet infiltrates, FOXP3(+)CD4(+) T cells were slightly increased in germ-free mice. B cells appeared less activated in the peritoneum and were less abundant in islet infiltrates.These results indicate that lack of intestinal microbiota promotes an imbalance between Th1, Th17 and Treg differentiation in the intestine. This imbalance is associated with accelerated insulitis, but intact recruitment of FOXP3(+) Tregs into islets, suggesting: (1) a microbial dependence of local induction of Treg in the gut and draining lymph nodes; but (2) a potentially compensatory function of naturally occurring Tregs in the islets, which may help control diabetogenic T cells.