Inhibition of RIPK1 kinase does not affect diabetes development: ß-Cells survive RIPK1 activation.

OBJECTIVES: Type 1 diabetes (T1D) is caused by progressive immune-mediated loss of insulin-producing ß-cells. Inflammation is detrimental to ß-cell function and survival, moreover, both apoptosis and necrosis have been implicated as mechanisms of ß-cell loss in T1D. The receptor interacting serine/threonine protein kinase 1 (RIPK1) promotes inflammation by serving as a scaffold for NF-κB and MAPK activation, or by acting as a kinase that triggers apoptosis or necroptosis. It is unclear whether RIPK1 kinase activity is involved in T1D pathology. In the present study, we investigated if absence of RIPK1 activation would affect the susceptibility to immune-mediated diabetes or diet induced obesity (DIO). METHODS: The RIPK1 knockin mouse line carrying a mutation mimicking serine 25 phosphorylation (Ripk1S25D/S25D), which abrogates RIPK1 kinase activity, was utilized to assess the in vivo role of RIPK1 in immune-mediated diabetes or diet induced obesity (DIO). In vitro, ß-cell death and RIPK1 kinase activity was analysed in conditions known to induce RIPK1-dependent apoptosis/necroptosis. RESULTS: We demonstrate that Ripk1S25D/S25D mice presented normal glucose metabolism and ß-cell function. Furthermore, immune-mediated diabetes and DIO were not different between Ripk1S25D/S25D and Ripk1+/+ mice. Despite strong activation of RIPK1 kinase and other necroptosis effectors (RIPK3 and MLKL) by TNF+BV6+zVAD, no cell death was observed in mouse islets nor human ß-cells. CONCLUSION: Our results contrast recent literature showing that most cell types undergo necroptosis following RIPK1 kinase activation. This peculiarity may reflect an adaptation to the inability of ß-cells to proliferate and self-renewal.

NF-κB-inducing kinase (NIK) is activated in pancreatic ß-cells but does not contribute to the development of diabetes.

The transcription factor nuclear factor-κB (NF-κB) has a key role in the pathogenesis of diabetes and its complications. Although activation of the canonical NF-κB pathway in ß-cells is generally deleterious, little is known about the role of the non-canonical NF-κB signalling and its main regulator, the NF-κB-inducing kinase (NIK), on pancreatic ß-cell survival and function. Previous studies based on models of NIK overexpression in pancreatic islet cells showed that NIK induced either spontaneous ß-cell death due to islet inflammation or glucose intolerance during diet-induced obesity (DIO) in mice. Therefore, NIK has been proposed as a potential target for diabetes therapy. However, no clear studies showed whether inhibition of NIK improves diabetes development. Here we show that genetic silencing of NIK in pancreatic ß-cells neither modifies diabetes incidence nor inflammatory responses in a mouse model of immune-mediated diabetes. Moreover, NIK silencing in DIO mice did not influence body weight gain, nor glucose metabolism. In vitro studies corroborated the in vivo findings in terms of ß-cell survival, function, and downstream gene regulation. Taken together, our data suggest that NIK activation is dispensable for the development of diabetes.

Different wild type strains of zebrafish show divergent susceptibility to TNBS-induced intestinal inflammation displaying distinct immune cell profiles.

Little is known about the diversity in immune profile of the different wild type strains of zebrafish (Danio rerio), despite its growing popularity as an animal model to study human diseases and drug testing. In the case of data resulting from modeling human diseases, differences in the background Danio fishes have rarely been taken into consideration when interpreting results and this is potentially problematic, as many studies not even mention the source and strain of the animals. In this study, we hypothesized that different wild type zebrafish strains could present distinct immune traits. To address the differences in immune responses between two commonly used wild type strains of zebrafish, AB and Tübingen (TU), we used an intestinal inflammation model induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and characterized the susceptibility and immune profile in these two strains. Our data demonstrates significant differences in survival between AB and TU strains when exposed to TNBS, suggesting important physiological differences in how these strains respond to inflammatory challenges. We observed that the AB strain presented increased mortality, higher neutrophilic intestinal infiltration, decreased goblet cell numbers and decreased IL-10 expression when exposed to TNBS, compared to the TU strain. In summary, our study demonstrates strain-specific immunological responses in AB and TU animals. Finally, the significant variations in strain-related susceptibility to inflammation and the differences in the immune profile shown here, highlight that the background of each strain need to be considered when utilizing zebrafish to model diseases and for drug screening purposes, thus better immune characterization of the diverse wild type strains of zebrafish is imperative.

Short chain fatty acids (SCFAs) improves TNBS-induced colitis in zebrafish.

The short-chain fatty acids (SCFAs) are metabolites originated from the fermentation of dietary fibers and amino acids produced by the bacteria of the intestinal microbiota. The most abundant SCFAs, acetate, propionate, and butyrate, have been proposed as a treatment for inflammatory bowel diseases (IBDs) due to their anti-inflammatory properties. This work aimed to analyze the effects of the treatment of three combined SCFAs in TNBS-induced intestinal inflammation in zebrafish larvae. Here, we demonstrated that SCFAs significantly increased the survival of TNBS-exposed larvae, preserved the intestinal endocytic function, reduced the expression of inflammatory cytokines and the intestinal recruitment of neutrophils caused by TNBS. However, SCFAs treatment did not appear to avoid TNBS-induced tissue damage in the intestinal wall and did not restore the number of mucus-producing goblet cells. Finally, exposure to TNBS induced dysbiosis of the microbiota with an increase in Betaproteobacteria and Actinobacteria, while the treatment with SCFAs maintained these population levels similar to control. Thus, we demonstrate that the treatment of three combined SCFAs presented anti-inflammatory properties previously seen in mammals, opening an opportunity to use zebrafish to explore the potential benefit of these and other metabolites to treat inflammation.

Short chain fatty acids (SCFAs) improves TNBS-induced colitis in zebrafish

The short-chain fatty acids (SCFAs) are metabolites originated from the fermentation of dietary fibers and amino acids produced by the bacteria of the intestinal microbiota. The most abundant SCFAs, acetate, propionate, and butyrate, have been proposed as a treatment for inflammatory bowel diseases (IBDs) due to their anti-inflammatory properties. This work aimed to analyze the effects of the treatment of three combined SCFAs in TNBS-induced intestinal inflammation in zebrafish larvae. Here, we demonstrated that SCFAs significantly increased the survival of TNBS-exposed larvae, preserved the intestinal endocytic function, reduced the expression of inflammatory cytokines and the intestinal recruitment of neutrophils caused by TNBS. However, SCFAs treatment did not appear to avoid TNBS-induced tissue damage in the intestinal wall and did not restore the number of mucus-producing goblet cells. Finally, exposure to TNBS induced dysbiosis of the microbiota with an increase in Betaproteobacteria and Actinobacteria, while the treatment with SCFAs maintained these population levels similar to control. Thus, we demonstrate that the treatment of three combined SCFAs presented anti-inflammatory properties previously seen in mammals, opening an opportunity to use zebrafish to explore the potential benefit of these and other metabolites to treat inflammation.

Immunometabolism: A target for the comprehension of immune response toward transplantation.

Organ transplantation is a life-saving procedure, however predicting graft survival is still challenging. Understanding immune-cell pathobiology is critical to the development of effective therapies to prevent rejection. Over the recent years it has become progressively evident that the complex nature of immune cell behavioral dynamics is strongly dependent on cellular metabolism, which in turn, relies on competition for nutrients, oxygen and metabolites with other immune cells and microbiota. Furthermore, the influence of the inflammatory state can lead to substantial changes in conditions within the tissue micro-environment. Considering the context of immunity, alterations in metabolic pathways (glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, the fatty acid oxidation and synthesis, and the amino acid metabolic pathways) will influence the production of different sets of cytokines and affect transplantation outcome. It is now known that naïve, resting and effector cells acquire different metabolic profiles and studies have shown that specifically targeting some of these metabolic routes can prevent differentiation of effector T cells in favor of Tregs. Ultimately, to develop effective therapies that will prevent graft loss and understanding how cell metabolism impacts the fate and function of immune cells is now a critical point of discussion. The distinct metabolic features and requirements observed in effector and suppressive cell subsets offer promising opportunities for selective regulation of the immune responses in transplantation and will be discussed in this review.

Intestinal barrier and gut microbiota: Shaping our immune responses throughout life.

The gastrointestinal (GI) tract is considered the largest immunological organ in the body having a central role in regulating immune homeostasis. Contrary to earlier belief, the intestinal epithelial barrier is not a static physical barrier but rather strongly interacts with the gut microbiome and cells of the immune system. This intense communication between epithelial cells, immune cells and microbiome will shape specific immune responses to antigens, balancing tolerance and effector immune functions. Recent studies indicate that composition of the gut microbiome affects immune system development and modulates immune mediators, which in turn affect the intestinal barrier. Moreover, dysbiosis may favor intestinal barrier disruption and could be related to increased susceptibility to certain diseases. This review will be focused on the development of the intestinal barrier and its function in host immune defense and how gut microbiome composition throughout life can affect this role.

Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti-CD3 Depends on the Induction of Foxp3-Positive T Cells.

The introduction of ß-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4+Foxp3+ T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-ß partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3+ Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4+Foxp3+ T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.

Combining MK626, a novel DPP-4 inhibitor, and low-dose monoclonal CD3 antibody for stable remission of new-onset diabetes in mice.

Combining immune intervention with therapies that directly influence the functional state of the ß-cells is an interesting strategy in type 1 diabetes cure. Dipeptidyl peptidase-4 (DPP-4) inhibitors elevate circulating levels of active incretins, which have been reported to enhance insulin secretion and synthesis, can support ß-cell survival and possibly stimulate ß-cell proliferation and neogenesis. In the current study, we demonstrate that the DPP-4 inhibitor MK626, which has appropriate pharmacokinetics in mice, preceded by a short-course of low-dose anti-CD3 generated durable diabetes remission in new-onset diabetic non-obese diabetic (NOD) mice. Induction of remission involved recovery of ß-cell secretory function with resolution of destructive insulitis and preservation of ß-cell volume/mass, along with repair of the islet angioarchitecture via SDF-1- and VEGF-dependent actions. Combination therapy temporarily reduced the CD4-to-CD8 distribution in spleen although not in pancreatic draining lymph nodes (PLN) and increased the proportion of effector/memory T cells as did anti-CD3 alone. In contrast, only combination therapy amplified Foxp3+ regulatory T cells in PLN and locally in pancreas. These findings open new opportunities for the treatment of new-onset type 1 diabetes by introducing DPP-4 inhibitors in human CD3-directed clinical trials.

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.

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.

Effects of vitamin D on antigen-specific and non-antigen-specific immune modulation: relevance for type 1 diabetes.

Vitamin D is a fat-soluble precursor of the circulating 25-hydroxyvitamin D3 (25(OH)D3)which can be converted by the 1α-hydroxylase (1α(OH)ase) enzyme into the bioactive hormonal metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), generally known to promote bone mineralization through its ability to enhance calcium absorption from the gut. Importantly, in humans, vitamin D is mainly derived from endogenous production of vitamin D3 from ultraviolet (UV) radiation exposure to the skin while a small part (<10%) is obtained via dietary intake of dairy products and fatty fish (1). Taking these factors into account, geographic distribution and seasonality, skin pigmentation, age, and lifestyle may predispose certain populations to be at a higher risk of developing vitamin D insufficiency or deficiency (2). The first valid reports correlating the importance of an adequate vitamin D status to optimal human health originate from the early part of the 20th century, when vitamin D was described to prevent and treat the bone disease rickets. Since then, the findings that vitamin D receptors (VDR) are present in many body tissues and that vitamin D metabolizing enzymes can be found in various cells outside the kidney, including the intestine, prostate, immune cells, and within the skin itself (reviewed in reference 3), have revolutionized the vitamin D business. In this review, we will mainly focus on vitamin D as a component of immune regulation and on the role of vitamin D in antigen-specific and non-specific therapies with potential relevance for type 1 diabetes (T1D).

1,25-Dihydroxyvitamin D3 curtails the inflammatory and T cell stimulatory capacity of macrophages through an IL-10-dependent mechanism.

The vitamin D receptor (VDR) is a hormone nuclear receptor regulating bone and calcium homeostasis. Studies revealing the expression of VDR on immune cells point toward a role for VDR-dependent signaling pathways in immunity. Here we verified the ability of the natural VDR ligand, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to interfere in inflammatory and T cell stimulatory capacity of macrophages, in particular within a chronic inflammatory disease features of experimental type 1 diabetes (T1D). We demonstrated that VDR is constitutively expressed in macrophages and both the levels of VDR and its downstream targets, are clearly induced by 1,25(OH)(2)D(3). In control mice, macrophage programming with 1,25(OH)(2)D(3) partially abrogated the activation-provoked expression of IL-12p40, TNFα and iNOS as well as the effector T cell-recruiting chemokines, CXCL9, CXCL10 and CXCL11. Targeting VDR signaling in macrophages counteracted their T-cell stimulatory ability despite essentially unaltered expression of antigen-presenting and costimulatory molecules. Furthermore, even in non-obese diabetic (NOD) mice, where macrophages/monocytes featured a heightened responsiveness toward danger signals and a superior T cell stimulatory capacity, 1,25(OH)(2)D(3) successfully curtailed these basic macrophage-mediated functions. Interestingly, the inhibitory action of the active compound was associated with an IL-10-dependent mechanism since 1,25(OH)(2)D(3)-treatment of IL-10-deficient macrophages failed to reproduce the characteristic repression on inflammatory mediators or T cell proliferation. Combined, these results highlight the possible therapeutic applicability of this natural immunomodulator, due to its ability to counteract macrophage inflammatory and T cell-activating pathways.

Vitamin D and diabetes.

There is no doubt that vitamin D deficiency is the cause of several metabolic bone diseases, but vitamin D status is also linked to many major human diseases including immune disorders. Mounting data strengthen the link between vitamin D and diabetes, in particular T1D and T2D. Despite some inconsistencies between studies that associate serum 25(OH)D levels with the risk of developing T1D or T2D, there seems to be an overall trend for an inverse correlation between levels of 25(OH)D and both disorders. There is also compelling evidence that 1,25(OH)2D regulates b-cell function by different mechanisms, such as influencing insulin secretion by regulating intracellular levels of Ca2+, increasing ß-cell resistance to apoptosis, and perhaps also increasing ß-cell replication. The capacity of vitamin D, more specifically 1,25(OH)2D, to modulate immune responses is of particular interest for both the therapy and prevention of diabetes. In the case of T1D, vitamin D supplementation in prediabetic individuals could help prevent or reduce the initiation of autoimmune processes possibly by regulating thymic selection of the T-cell repertoire, decreasing the numbers of autoreactive T cells, and inducing Treg cells. Although immune modulation is generally discussed for the treatment of T1D, it is also relevant for T2D. Indeed, recent studies have shown that T2D patients have increased systemic inflammation and that this state can induce ß-cell dysfunction and death. Supplementation trials with regular vitamin D for the protection against the development of T1D and T2D have generated some contradictory data, but many weaknesses can be identified in these trials as most were underpowered or open-labeled. However, the overwhelming strength of preclinical data and of the observational studies make vitamin D or its analogues strong candidates for the prevention or treatment of diabetes or its complications. However, proof of causality needs well-designed clinical trials and if positive, adequate dosing, regimen, and compound studies are needed to define the contribution of vitamin D status and therapy in the global diabetes problem. There are many confounding factors that need to be taken into consideration when translating successful vitamin D therapies in animal models into humans, for example, gender, age, lifestyle, and genetic background. To come to solid conclusions on the potential of vitamin D or its analogues in the prevention of or therapy for all forms of diabetes, it is clear that large prospective trials with carefully selected populations and end points will be needed, but should also receive high priority.

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.

Early IL-10 production is essential for syngeneic graft acceptance.

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival.

Vitamin D and diabetes.

Type 1 (T1D) and type 2 (T2D) diabetes are considered multifactorial diseases in which both genetic predisposition and environmental factors participate in their development. Many cellular, preclinical, and observational studies support a role for vitamin D in the pathogenesis of both types of diabetes including: (1) T1D and T2D patients have a higher incidence of hypovitaminosis D; (2) pancreatic tissue (more specifically the insulin-producing beta-cells) as well as numerous cell types of the immune system express the vitamin D receptor (VDR) and vitamin D-binding protein (DBP); and (3) some allelic variations in genes involved in vitamin D metabolism and VDR are associated with glucose (in)tolerance, insulin secretion, and sensitivity, as well as inflammation. Moreover, pharmacologic doses of 1,25-dihydroxyvitamin D (1,25(OH)(2)D), the active form of vitamin D, prevent insulitis and T1D in nonobese diabetic (NOD) mice and other models of T1D, possibly by immune modulation as well as by direct effects on beta-cell function. In T2D, vitamin D supplementation can increase insulin sensitivity and decrease inflammation. This article reviews the role of vitamin D in the pathogenesis of T1D and T2D, focusing on the therapeutic potential for vitamin D in the prevention/intervention of T1D and T2D as well as its complications.

Vitamin D: modulator of the immune system.

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, is known to regulate calcium and phosphorus metabolism, thus being a key-player in bone-formation. However 1,25(OH)(2)D(3) also has a physiological role beyond its well-known role in skeletal homeostasis. Here, we describe 1,25(OH)(2)D(3) as an immunomodulator targeting various immune cells, including monocytes, macrophages, dendritic cells (DCs), as well as T-lymphocytes and B-lymphocytes, hence modulating both innate and adaptive immune responses. Besides being targets, immune cells express vitamin D-activating enzymes, allowing local conversion of inactive vitamin D into 1,25(OH)(2)D(3) within the immune system. Taken together, these data indicate that 1,25(OH)(2)D(3) plays a role in maintenance of immune homeostasis. Several epidemiological studies have linked inadequate vitamin D levels to a higher susceptibility of immune-mediated disorders, including chronic infections and autoimmune diseases. This review will discuss the complex immune-regulatory effects of 1,25(OH)(2)D(3) on immune cells as well as its role in infectious and autoimmune diseases, more in particular in tuberculosis and type 1 diabetes (T1D).