Viral infections and diabetes.

Type 1 diabetes mellitus (T1DM) is a multi-factorial autoimmune disease determined by the interaction of genetic, environmental and immunologic factors. One of the environmental risk factors identified by a series of independent studies is represented by viral infection, with strong evidence showing that viruses can indeed infect pancreatic beta cells with consequent effects ranging from functional damage to cell death. In this chapter we review the data obtained both in man and in experimental animal models in support of the potential participation of viral infections to Type 1 diabetes pathogenesis, with a particular emphasis on virus-triggered islet inflammation, beta-cell dysfunction and autoimmunity.

Increased expression of microRNA miR-326 in type 1 diabetic patients with ongoing islet autoimmunity.

BACKGROUND: The current paradigm that microRNAs represent a new layer of gene regulation has generated much interest in this field. MicroRNAs have emerged as important regulatory factors involved in the developmental processes and in the regulation of insulin secretion and signalling. Furthermore, recent studies revealed an altered microRNA profiling in lymphocytes of patients with autoimmune diseases like multiple sclerosis, in which a hyperexpression of miR-326 was reported. Here, we analysed the expression levels of miR-326 in peripheral blood lymphocytes from type 1 diabetic (T1D) patients in relationship with ongoing islet autoimmunity. METHODS: Peripheral blood lymphocytes were obtained from 19 T1D patients; 4/19 patients were positive for both glutamic acid decarboxylase (GAD) and islet cell antigen 512 autoantibodies; 10/19 were single GAD or IA-2 Ab positive and 5/19 were GAD antibodies and IA-2 antibodies (IA-2A) negative. Quantitative analysis of miR-326 was performed using specific stem-loop primers followed by real-time polymerase chain reaction. All values were normalized to endogenous control U6. RESULTS: miR-326 resulted increased in Ab-positive versus Ab-negative T1D subjects. Its expression levels were 2.05±0.38-fold increased in peripheral blood lymphocytes from patients expressing both GADA and IA-2A and 2.93±0.46-fold increased in single Ab-positive versus Ab-negative individuals (p<0.05). CONCLUSION: In conclusion, we have shown that miR-326 is expressed at higher levels in T1D subjects with ongoing islet autoimmunity, similar to what has been observed in multiple sclerosis, in which levels of this microRNA were highly correlated with disease severity. Interestingly, an online search of miR-326 predicted targets revealed vitamin D receptor and Erythroblastosis virus E26 oncogene homologue 1, two molecules highly involved in immune regulation.

Dietary supplementation with high doses of regular vitamin D3 safely reduces diabetes incidence in NOD mice when given early and long term.

High doses of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], prevent diabetes in the NOD mouse but also elicit unwanted calcemic side effects. Because immune cells themselves can convert vitamin D3 into 1,25(OH)2D3 locally, we hypothesized that dietary vitamin D3 can also prevent disease. Thus, we evaluated whether dietary administration of high doses of regular vitamin D3 (800 IU/day) during different periods of life (pregnancy and lactation, early life [3-14 weeks of age], or lifelong [3-35 weeks of age]) safely prevents diabetes in NOD mice. We found that only lifelong treatment raised serum 25-hydroxyvitamin D3 from 173 nmol/L in controls to 290 nmol/L, without inducing signs of calcemic or bone toxicity, and significantly reduced diabetes development in both male and female NOD mice. This diabetes protection by vitamin D3 correlated with preserved pancreatic insulin content and improved insulitis scores. Moreover, vitamin D3 treatment decreased interferon-γ-positive CD8(+) T cells and increased CD4(+)(CD25(+))FoxP3(+) T cells in pancreatic draining lymph nodes. In conclusion, this study shows for the first time that high doses of regular dietary vitamin D3 can safely prevent diabetes in NOD mice when administered lifelong, although caution is warranted with regards to administering equivalently high doses in humans.

Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice.

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370-575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional ß-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4(+)Foxp3(+)CD25(+) regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.

The case for virus-induced type 1 diabetes.

PURPOSE OF REVIEW: Type 1 diabetes (T1D) results from the immune-mediated destruction of pancreatic insulin-producing cells because of the interaction among genetic susceptibility, the immune system and environmental factor(s). A possible role of viral infections in T1D pathogenesis has been hypothesized for some time; however, only in the most recent years, studies performed at the molecular and cellular level are starting to shed light on this issue. RECENT FINDINGS: Studies in animal models and in man have shown that viruses can indeed infect pancreatic beta-cells, inducing islet inflammation and functional damage. In addition, recent in-situ investigations performed on pancreatic tissue samples have provided evidence that in addition to adaptive immune response, innate immunity is involved in T1D pathogenesis and the whole pancreas (not only its endocrine portion) is infiltrated by immune-mediated phenomena. SUMMARY: The established role of inflammation in the insulitic process and the increasing evidence in support of the contribution of viral infections to a proinflammatory islet scenario are strongly suggestive that viruses may indeed contribute to beta-cell damage and dysfunction, thus setting the stage for the design of antiviral strategies (e.g. vaccines and antiviral drugs) aimed at protecting the beta-cells.

Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice.

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.

Innate immunity and the pathogenesis of type 1 diabetes.

Type 1 diabetes mellitus is an autoimmune disease caused by the immune-mediated destruction of insulin-producing pancreatic beta cells occurring in genetically predisposed individuals, with consequent hyperglycemia and serious chronic complications. Studies in man and in experimental animal models have shown that both innate and adaptive immune responses participate to disease pathogenesis, possibly reflecting the multifactorial pathogenetic nature of this autoimmune disorder, with the likely involvement of environmental factors occurring at least in a subset of individuals. As a consequence, components of both innate and adaptive immune response should be considered as potential targets of therapeutic strategies for disease prevention and cure. Here we review the contribution of innate immune response to type 1 diabetes, with a particular emphasis to Toll-like receptors (TLR) and NK cells.