The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5’-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

BackgroundRestoration of immune tolerance to disease-relevant antigens is an appealing approach to prevent or arrest an organ-specific autoimmune disease like type 1 diabetes (T1D). Numerous studies have identified insulin as a key antigen of interest to use in such strategies, but to date, the success of these interventions in humans has been inconsistent. The efficacy of antigen-specific immunotherapy may be enhanced by optimising the dose, timing, and route of administration, and perhaps by the inclusion of adjuvants like alum. The aim of our study was to evaluate the effect of an insulin peptide vaccine formulated with alum to prevent T1D development in female non-obese diabetic (NOD) mice when administered during late-stage pre-diabetes.MethodsStarting at 10 weeks of age, female NOD mice received four weekly subcutaneous injections of an insulin B:8-24 (InsB:8-24) peptide with (Ins+alum) or without Imject® alum (Ins) as adjuvant. Diabetes incidence was assessed for up to 30 weeks of age. Insulin autoantibodies and C-peptide concentrations were measured in plasma and flow cytometric analysis was performed on pancreatic-draining lymph nodes (PLN) and pancreas using an InsB:12-20-reactive tetramer.ResultsInsB:8-24 peptide formulated in alum reduced diabetes incidence (39%), compared to mice receiving the InsB:8-24 peptide without alum (71%, P < 0.05), mice receiving alum alone (76%, P < 0.01), or mice left untreated (70%, P < 0.01). This was accompanied by reduced insulitis severity, and preservation of C-peptide. Ins+alum was associated with reduced frequencies of pathogenic effector memory CD4+ and CD8+ T cells in the pancreas and increased frequencies of insulin-reactive FoxP3+ Tregs in the PLN. Of interest, insulin-reactive Tregs were enriched amongst populations of Tregs expressing markers indicative of stable FoxP3 expression and enhanced suppressive function.ConclusionAn InsB:8-24 peptide vaccine prevented the onset of T1D in late-stage pre-diabetic NOD mice, but only when formulated in alum. These findings support the use of alum as adjuvant to optimise the efficacy of antigen-specific immunotherapy in future trials.

Immunomodulation combined with antigen therapy holds great promise to arrest autoimmune type 1 diabetes, but clinical translation is hampered by a lack of prognostic biomarkers. Lowdose anti-CD3 plus Lactococcus lactis (L. lactis) bacteria secreting proinsulin and IL-10 reversed new-onset disease in non-obese diabetic (NOD) mice, yet some mice were resistant to the therapy. Using microRNA (miRNA) profiling, six miRNAs (i.e., miR-34a-5p, miR-125a- 3p, miR-193b-3p, miR-328, miR-365-3p, and miR-671-3p) were identified as differentially expressed in plasma of responder versus non-responder mice before study entry. After validation and stratification in an independent cohort, plasma miR-193b-3p and miR-365-3p combined with age and glycemic status at study entry had the best power to predict with high sensitivity and specificity poor response to the therapy. These miRNAs were highly abundant in pancreas infiltrating neutrophils and basophils with a pro-inflammatory and activated phenotype. Here, a set of miRNAs and disease-associated parameters are presented as predictive signature for the L. lactis-based immunotherapy outcome in new-onset type 1 diabetes, hence allowing targeted recruitment of future trial participants and accelerated trial execution.

<p>Immunomodulation combined with antigen therapy holds great promise to arrest autoimmune type 1 diabetes, but clinical translation is hampered by a lack of prognostic biomarkers. Low-dose anti-CD3 plus <em>Lactococcus lactis </em>(<em>L. lactis</em>) bacteria secreting proinsulin and IL-10 reversed new-onset disease in non-obese diabetic (NOD) mice, yet some mice were resistant to the therapy. Using microRNA (miRNA) profiling, six miRNAs (i.e., miR-34a-5p, miR-125a-3p, miR-193b-3p, miR-328, miR-365-3p, and miR-671-3p) were identified as differentially expressed in plasma of responder versus non-responder mice before study entry. After validation and stratification in an independent cohort, plasma miR-193b-3p and miR-365-3p combined with age and glycemic status at study entry had the best power to predict with high sensitivity and specificity poor response to the therapy. These miRNAs were highly abundant in pancreas infiltrating neutrophils and basophils with a pro-inflammatory and activated phenotype. Here, a set of miRNAs and disease-associated parameters are presented as predictive signature for the <em>L. lactis</em>-based immunotherapy outcome in new-onset type 1 diabetes, hence allowing targeted recruitment of future trial participants and accelerated trial execution. </p>