Gluten-free diet reduces autoimmune diabetes mellitus in mice across multiple generations in a microbiota-independent manner.

Experimental and clinical data suggest that a gluten-free diet attenuates the development of type 1 diabetes. A gluten-free diet changes the gut microbiota composition, and such microbial changes are expected to reduce the autoimmune responses. However, in experiments with laboratory mice, a gluten-free diet changes the gut microbiota differently under varying experimental settings, questioning the specific role of the gut microbes. Here we show that a maternal gluten-free diet until weaning of their pups, delayed type 1 diabetes in both dams (parent generation) and offspring (F1 generation) of untreated non-obese diabetic (NOD) mice and in mice treated with a full cocktail of antibiotics that eradicates most of the existing microbiota. Breeding a second (F2) generation of NOD mice, never exposed to the gluten-free diet or the associated microbial changes, also demonstrated a preventative effect on type 1 diabetes even though their parents (the F1 generation) had only been on a gluten-free diet very early in life. Collectively, the experimental data, thus, points towards microbiota-independent dietary protection. Furthermore, both the perinatal gluten-free diet and antibiotic treatment reduced inflammation in the salivary glands and improved glucose challenged beta cell function in the F1 offspring. However, in contrast to the autoimmune response in the pancreas, those changes appeared to be microbiota dependent, as they were missing in the antibiotic treated mice, and do, therefore, not seem to be related to the preventative effect on type 1 diabetes. Interestingly, adoptive transfer of splenocytes from gluten-free fed mice protected NOD.SCID mice from developing diabetes, demonstrating that the anti-diabetic effect of a gluten-free diet was based on early life changes in the evolving immune system. In particular, genes involved in regulation of lymphocyte activation, proliferation, and cell adhesion were highly expressed in the spleen in gluten-free fed mice at weaning compared to control fed mice of the F1 generation, which suggested that gluten promotes autoimmunity by inhibiting immune regulation, though the involvement of the specific genes needs further investigation. In conclusion, gluten-free diet reduces autoimmune inflammation in salivary glands and pancreas in NOD mice in a microbiota-dependent and -independent manner respectively, and has preventative effect on type 1 diabetes by modulating the systemic immune system.

Targeting gut microbiota and barrier function with prebiotics to alleviate autoimmune manifestations in NOD mice.

AIMS/HYPOTHESIS: Adopting a diet containing indigestible fibre compounds such as prebiotics to fuel advantageous bacteria has proven beneficial for alleviating inflammation. The effect of the microbial changes on autoimmunity, however, remains unknown. We studied the effects of prebiotic xylooligosaccharides (XOS) on pancreatic islet and salivary gland inflammation in NOD mice and tested whether these were mediated by the gut microbiota. METHODS: Mother and offspring mice were fed an XOS-supplemented diet until diabetes onset or weaning and were compared with a control-fed group. Diabetes incidence was monitored, insulitis and sialadenitis were scored in histological sections from adult mice, and several metabolic and immune variables were analysed in mice before the development of diabetes. Gut barrier function was assessed using an in vivo FITC-dextran permeability test. The importance of XOS-mediated gut microbial changes were evaluated in antibiotic-treated mice fed either XOS or control diet or given a faecal microbiota transplant from test animals. RESULTS: Diabetes onset was delayed in the XOS-fed mice, which also had fewer cellular infiltrations in their pancreatic islets and salivary glands. Interestingly, insulitis was most reduced in the XOS-fed groups when the mice were also treated with an antibiotic cocktail. There was no difference in sialadenitis between the dietary groups treated with antibiotics; the mice were protected by microbiota depletion regardless of diet. Faecal microbiota transplantation was not able to transfer protection. No major differences in glucose-insulin regulation, glucagon-like peptide-1, or short-chain fatty acid production were related to the XOS diet. The XOS diet did, however, reduce gut permeability markers in the small and large intestine. This was accompanied by a more anti-inflammatory environment locally and systemically, dominated by a shift from M1 to M2 macrophages, a higher abundance of activated regulatory T cells, and lower levels of induction of natural killer T cells and cytotoxic T cells. CONCLUSIONS/INTERPRETATION: Prebiotic XOS have microbiota-dependent effects on salivary gland inflammation and microbiota-independent effects on pancreatic islet pathology that are accompanied by an improved gut barrier that seems able to heighten control of intestinal diabetogenic antigens that have the potential to penetrate the mucosa to activate autoreactive immune responses.

Dietary LPS traces influences disease expression of the diet-induced obese mouse.

Lipopolysaccharides (LPS) from Gram negative bacteria are generally present in laboratory animal chow diets in unknown amounts, which has been correlated to significant immunological differences between animals receiving diets with either low or high "naturally" occurring LPS content. LPS in the blood stream has been linked to glucose intolerance through Toll-like receptor mediated release of pro-inflammatory cytokines, metabolic endotoxemia, adipose tissue inflammation. LPS uptake increases when co-administered with fat, therefore uncontrolled LPS levels in a high-fat diet may increase variation in development of disease when high-fat diets are used to induce obesity and type 2 diabetes. Three experiments were conducted, in which C57BL/6NTac mice received high-fat (60%) or low fat (10%) diets with or without LPS for 8 or 20 weeks investigating the short and long term effects. Three different doses of LPS were used to investigate dosage effect, and ampicillin to isolate the effect of dietary LPS. Dietary LPS increased LPS levels in the blood stream, and affected the level of glycated haemoglobin (HbA1c), a key parameter in this model, in a dose dependant manner (p < 0.05). There was a strong tendency toward slower glucose uptake in the LPS supplemented groups once obesity was established, but the differences disappeared after 20 weeks. A high-fat diet slightly increased serum LPS and altered ileal expression of il10 and tnfa (p < 0.05). In conclusion, LPS seems to affect the glucose metabolism in a time-dose dependant manner, and uncontrolled variation in LPS levels of a diet may therefore increase inter-study variation.

Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice.

Transferring gut microbiota from one individual to another may enable researchers to "humanize" the gut of animal models and transfer phenotypes between species. To date, most studies of gut microbiota transfer are performed in germ-free mice. In the studies presented, it was tested whether an antibiotic treatment approach could be used instead. C57BL/6 mice were treated with ampicillin prior to inoculation at weaning or eight weeks of age with gut microbiota from lean or obese donors. The gut microbiota and clinical parameters of the recipients was characterized one and six weeks after inoculation. The results demonstrate, that the donor gut microbiota was introduced, established, and changed the gut microbiota of the recipients. Six weeks after inoculation, the differences persisted, however alteration of the gut microbiota occurred with time within the groups. The clinical parameters of the donor phenotype were partly transmissible from obese to lean mice, in particularly ß cell hyperactivity in the obese recipients. Thus, a successful inoculation of gut microbiota was not age dependent in order for the microbes to colonize, and transferring different microbial compositions to conventional antibiotic-treated mice was possible at least for a time period during which the microbiota may permanently modulate important host functions.