Gluten-free diet in children with recent-onset type 1 diabetes: A 12-month intervention trial.

AIM: To test whether a gluten-free diet (GFD) is associated with the deceleration of the decline in beta-cell capacity in non-coeliac children with recently diagnosed type 1 diabetes. METHODS: Forty-five children (aged 10.2 ± 3.3 years) were recruited into a self-selected intervention trial: 26 started with a GFD within a median of 38 days postonset, whereas 19 remained on a standard diet. The main outcomes were the decline in C-peptide area under the curve (AUC) in mixed-meal tolerance tests (MMTTs) at 6 and 12 months relative to 1 month after diabetes onset and the difference in insulin dose, insulin dose-adjusted A1c (IDAA1c) and HbA1c assessed every 3 months. The adherence to the GFD was verified by immunoreactive gluten in the stool and by food questionnaires at every visit. Quality of life (QoL) questionnaires were administered to the participants at the end of the intervention at 12 months. The data were analysed as per protocol (in 39 subjects who duly completed the whole follow-up: 20 in the GFD group, 19 in the control group) by linear and longitudinal regression models adjusted for sex, age and baseline variables. RESULTS: At 12 months, the difference in C-peptide AUC between subjects in the GFD group and controls was 205 pmol/L (95% CI -223 to 633; P = 0.34) in a model adjusted for age, sex and body weight, and for baseline insulin dose, MMTT C-peptide AUC and HbA1c assessed at 1 month after diagnosis. In a longitudinal analysis of all three time points adjusted for age, sex and body weight, C-peptide declined more slowly in the GFD group than in controls, with the difference in trends being 409 pmol/L/year (P = 0.04). The GFD group had a marginally lower insulin dose (by 0.15 U/kg/day; P = 0.07), a lower IDAA1c (by 1.37; P = 0.01) and a lower mean HbA1c (by 0.7% [7.8 mmol/mol]; P = 0.02) than those of the controls at 12 months. There was no appreciable difference between the groups in daily carbohydrate intake (P = 0.49) or in the QoL reported by the patients (P = 0.70) and their parents/caregivers (P = 0.59). CONCLUSIONS: A GFD maintained over the first year after type 1 diabetes diagnosis was associated with better HbA1c and a prolonged partial remission period. There was a hint of slower C-peptide decline but the association was not strong enough to make definite conclusions.

Human gut microbiota transferred to germ-free NOD mice modulate the progression towards type 1 diabetes regardless of the pace of beta cell function loss in the donor.

AIMS/HYPOTHESIS: This study aimed to assess the ability of human gut microbiota to delay the onset of type 1 diabetes when transferred into germ-free NOD mice. METHODS: Two children with rapid and three children with slow beta cell function loss (as assessed by C-peptide AUC change in the mixed-meal tolerance tests performed 1 and 12 months after type 1 diabetes onset), participating in an ongoing trial with gluten-free diet, donated faeces, which were transferred into germ-free NOD mice. The mice were subsequently followed for diabetes incidence. RESULTS: The bacterial profiles of bacteriome-humanised mice had significantly (p < 10-5) lower alpha diversity than the donor material, with marked shifts in ratios between the main phyla. Diabetes onset was significantly delayed in all bacteriome-humanised colonies vs germ-free NOD mice, but the pace of beta cell loss was not transferable to the mouse model. CONCLUSIONS/INTERPRETATION: Germ-free NOD mice colonised with human gut microbiome are able to adopt a large proportion of transferred bacterial content, although the ratios of main phyla are reproduced only suboptimally. The recipient mice did not replicate the phenotype of the stool donor in relation to the pace towards type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT02867436.

Tolerogenic Dendritic Cells from Poorly Compensated Type 1 Diabetes Patients Have Decreased Ability To Induce Stable Antigen-Specific T Cell Hyporesponsiveness and Generation of Suppressive Regulatory T Cells.

Tolerogenic dendritic cells (tolDCs) may offer an interesting intervention strategy to re-establish Ag-specific tolerance in autoimmune diseases, including type 1 diabetes (T1D). T1D results from selective destruction of insulin-producing ß cells leading to hyperglycemia that, in turn, specifically affects a patient's immune system. In this study, we prepared monocyte-derived tolDCs modulated by dexamethasone and vitamin D2 from 31 T1D patients with optimal glycemic control and 60 T1D patients with suboptimal glycemic control and assessed their tolerogenic properties in correlation with metabolic state of patients. tolDCs differentiated from both groups of patients acquired a regulatory phenotype and an anti-inflammatory profile. Interestingly, tolDCs from well-controlled patients expressed higher levels of inhibitory molecules IL-T3 and PD-L1. Additionally, glutamic acid decarboxylase (GAD)65-loaded tolDCs from well-controlled patients decreased significantly primary Th1/Th17 responses, induced stable GAD65-specific T cell hyporesponsiveness, and suppressed markedly control DC-induced GAD65-specific T cell activation compared with poorly controlled patients. The ability of tolDCs from poorly controlled patients to induce durable GAD65-specific T cell hyporesponsiveness was reversed once the control of glycemia improved. In both groups of patients, tolDCs were able to induce regulatory T cells from autologous naive CD4+ T cells. However, regulatory T cells from well-controlled patients had better suppressive abilities. The functionality of tolDCs was confirmed in the adoptive transfer model of NOD-SCID mice where tolDCs delayed diabetes onset. These results suggest that metabolic control of T1D affects the functional characteristics of tolDCs and subsequent effector T cell responses. Metabolic control may be relevant for refining inclusion criteria of clinical trials in the settings of T1D.

Dietary gluten and the development of type 1 diabetes.

Gluten proteins differ from other cereal proteins as they are partly resistant to enzymatic processing in the intestine, resulting in a continuous exposure of the proteins to the intestinal immune system. In addition to being a disease-initiating factor in coeliac disease (CD), gluten intake might affect type 1 diabetes development. Studies in animal models of type 1 diabetes have documented that the pathogenesis is influenced by diet. Thus, a gluten-free diet largely prevents diabetes in NOD mice while a cereal-based diet promotes diabetes development. In infants, amount, timing and mode of introduction have been shown to affect the diabetogenic potential of gluten, and some studies now suggest that a gluten-free diet may preserve beta cell function. Other studies have not found this effect. There is evidence that the intestinal immune system plays a primary role in the pathogenesis of type 1 diabetes, as diabetogenic T cells are initially primed in the gut, islet-infiltrating T cells express gut-associated homing receptors, and mesenteric lymphocytes transfer diabetes from NOD mice to NOD/severe combined immunodeficiency (SCID) mice. Thus, gluten may affect diabetes development by influencing proportional changes in immune cell populations or by modifying the cytokine/chemokine pattern towards an inflammatory profile. This supports an important role for gluten intake in the pathogenesis of type 1 diabetes and further studies should be initiated to clarify whether a gluten-free diet could prevent disease in susceptible individuals or be used with newly diagnosed patients to stop disease progression.

Dietary gluten alters the balance of pro-inflammatory and anti-inflammatory cytokines in T cells of BALB/c mice.

Several studies have documented that dietary modifications influence the development of type 1 diabetes. However, little is known about the interplay of dietary components and the penetration of diabetes incidence. In this study we tested if wheat gluten is able to induce differences in the cytokine pattern of Foxp3(+) regulatory T cells, as well as Foxp3(-) T cells, isolated from intestinal mucosal lymphoid tissue and non-mucosal lymphoid compartments in BALB/c mice. The gluten-containing standard diet markedly changed the cytokine expression within Foxp3(-) T cells, in all lymphoid organs tested, towards a higher expression of pro-inflammatory interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-2. In Foxp3(+) regulatory T cells, gluten ingestion resulted in a mucosal increase in IL-17 and IL-2 and an overall increase in IFN-γ and IL-4. The gluten-free diet induced an anti-inflammatory cytokine profile with higher proportion of transforming growth factor-ß (TGF-ß)(+) Foxp3(-) T cells in all tested lymphoid tissues and higher IL-10 expression within non-T cells in spleen, and a tendency towards a mucosal increase in TGF-ß(+) Foxp3(+) regulatory T cells. Our data shows that the gluten-containing standard diet modifies the cytokine pattern of both Foxp3(-) T cells and Foxp3(+) regulatory T cells towards a more inflammatory cytokine profile. This immune profile may contribute to the higher type 1 diabetes incidence associated with gluten intake.

A subset of human pancreatic beta cells express functional CD14 receptors: a signaling pathway for beta cell-related glycolipids, sulfatide and ß-galactosylceramide.

BACKGROUND: T1DM is a T-cell-mediated autoimmune disease targeting insulin-producing beta-cells. Multiple factors may contribute to the development of T1DM. Among these, the metabolic state of beta-cells and pro-inflammatory cytokines, produced by infiltrating immune cells, have been implicated in the precipitation of T1DM. METHODS AND RESULTS: In this study, confocal immunofluorescence microscopy of human pancreata revealed a distinct subset of beta-cells expressing the innate LPS co-receptor CD14. Human islets expressed fully functional CD14 as LPS stimulation led to a dose-dependent secretion of tumour necrosis factor (TNFα), interleukin (IL)-1ß and IL-8, which were substantially inhibited by a blocking anti-CD14 mAb. In addition, LPS stimulation impaired the glucose-mediated insulin secretion in rat islets. ß-GalCer and sulfatide, glycolipids that are related to insulin processing and secretion, are possibly interacting with the CD14 receptor complex. ß-GalCer had an LPS-like, serum- and CD14-dependent effect on the induction of pro-inflammatory cytokines in a human monocyte cell line. In contrast, the LPS-mediated cytokine production was inhibited by sulfatide. Human islets also responded to ß-GalCer (10 µg/mL) by secreting TNFα, IL-1ß and IL-8, whereas sulfatide partly inhibited the effect of LPS. CONCLUSIONS: A subset of human beta-cells expresses functional CD14 receptor and thus is able to recognize both exogenous bacterial (LPS) as well as endogenous ligands (e.g. glycolipids of beta-cell origin). The CD14 expression on a subset of human beta-cells may play a role in the innate surveillance of the endocrine environment but may also contribute to innate immune mechanisms in the early stages of beta-cell aggression.

Optimal Tolerogenic Dendritic Cells in Type 1 Diabetes (T1D) Therapy: What Can We Learn From Non-obese Diabetic (NOD) Mouse Models?

Tolerogenic dendritic cells (tolDCs) are explored as a promising standalone or combination therapy in type 1 diabetes (T1D). The therapeutic application of tolDCs, including in human trials, has been tested also in other autoimmune diseases, however, T1D displays some unique features. In addition, unlike in several disease-induced animal models of autoimmune diseases, the prevalent animal model for T1D, the NOD mouse, develops diabetes spontaneously. This review compares evidence of various tolDCs approaches obtained from animal (mainly NOD) models of T1D with a focus on parameters of this cell-based therapy such as protocols of tolDC preparation, antigen-specific vs. unspecific approaches, doses of tolDCs and/or autoantigens, application schemes, application routes, the migration of tolDCs as well as their preventive, early pre-onset intervention or curative effects. This review also discusses perspectives of tolDC therapy and areas of preclinical research that are in need of better clarification in animal models in a quest for effective and optimal tolDC therapies of T1D in humans.

Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice.

Tolerogenic DCs (tolDCs) are being researched as a promising intervention strategy also in autoimmune diseases including type 1 diabetes (T1D). T1D is a T-cell-mediated, organ-specific disease with several well-defined and rather specific autoantigens, i.e., proinsulin, insulin, glutamic acid decarboxylase 65 (GAD65), that have been used in animal as well as human intervention trials in attempts to achieve a more efficient, specific immunotherapy. In this study, we have tested tolerogenic DCs for their effectiveness to prevent adoptive transfer of diabetes by diabetogenic splenocytes into non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) recipients. While i.p. application of tolDCs prepared from bone marrow of prediabetic NOD mice by vitamin D2 and dexamethasone significantly reduced diabetes transfer into the NOD-SCID females, this effect was completely abolished when tolDCs were loaded with the mouse recombinant GAD65, but also with a control protein-ovalbumin (OVA). The effect was not dependent on the presence of serum in the tolDC culture. Similar results were observed in NOD mice. Removal of possible bystander antigen-presenting cells within the diabetogenic splenocytes by negative magnetic sorting of T cells did not alter this surprising effect. Tolerogenic DCs loaded with an immunodominant mouse GAD65 peptide also displayed diminished diabetes-preventive effect. Tolerogenic DCs were characterized by surface maturation markers (CD40, CD80, CD86, MHC II) and the lipopolysaccharide stability test. Data from alloreactive T cell proliferation and cytokine induction assays (IFN-γ) did not reveal the differences observed in the diabetes incidence. Migration of tolDCs, tolDCs-GAD65 and tolDCs-OVA to spleen, mesenteric- and pancreatic lymph nodes displayed similar, mucosal pattern with highest accumulation in pancreatic lymph nodes present up to 9 days after the i.p. APPLICATION: These data document that mechanisms by which tolDCs operate in vivo require much better understanding for improving efficacy of this promising cell therapy, especially in the presence of an antigen, e.g., GAD65.

Cell-based Tolerogenic Therapy, Experience from Animal Models of Multiple Sclerosis, Type 1 Diabetes and Rheumatoid Arthritis.

Cell-based tolerogenic therapy is a promising approach for the treatment of autoimmune diseases and transplant rejection. Regulatory T cells and tolerogenic dendritic cells have been particularly explored in the treatment of various autoimmune disorders in experimental models of disease. Although some of these cells have already been tested in a limited number of clinical trials, there is still a need for preclinical research on tolerogenic cells in animal models of autoimmunity. This review will focus on the relevance of data obtained from studies in experimental animal models for the use of tolerogenic cell-based therapy in humans. Also, perspectives for further improvement of tolerogenic cell preparation towards enhanced suppressive activity and stability of the cells will be discussed.

Gluten-Free Diet Only during Pregnancy Efficiently Prevents Diabetes in NOD Mouse Offspring.

Studies have documented that the pathogenesis of autoimmune diabetes is influenced by the intake of gluten. Aims. To investigate the importance of gluten exposure during pregnancy and the subsequent development of autoimmune diabetes in offspring. Methods. Nonobese diabetic mice were divided into 7 groups to receive combinations of gluten-free and standard diet before, during, or after pregnancy. Diabetes incidence in offspring was followed in each group (n = 16-27) for 310 days. Insulitis score and intestinal expression of T-cell transcription factors (RT-QPCR) were evaluated in animals from the different diet groups. Results. If mothers were fed a gluten-free diet only during pregnancy, the development of autoimmune diabetes in offspring was almost completely prevented with an incidence reduction from 62.5% in gluten-consuming mice to 8.3% (p < 0.0001) in the gluten-free group. The islets of Langerhans were less infiltrated (p < 0.001) and the intestinal expression of RORγt (Th17) (p < 0.0001) reduced in mice whose mothers were Gluten-free during pregnancy. Conclusion. A gluten-free diet exclusively during pregnancy efficiently prevents autoimmune diabetes development in offspring and reduces insulitis and intestinal expression of RORγt (Th17).

Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases.

Commensal microflora (normal microflora, indigenous microbiota) consists of those micro-organisms, which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). The number of bacteria colonising mucosal and skin surfaces exceeds the number of cells forming human body. Commensal bacteria co-evolved with their hosts, however, under specific conditions they are able to overcome protective host responses and exert pathologic effects. Resident bacteria form complex ecosystems, whose diversity is enormous. The most abundant microflora is present in the distal parts of the gut; the majority of the intestinal bacteria are Gram-negative anaerobes. More than 50% of intestinal bacteria cannot be cultured by conventional microbiological techniques. Molecular biological methods help in analysing the structural and functional complexity of the microflora and in identifying its components. Resident microflora contains a number of components able to activate innate and adaptive immunity. Unlimited immune activation in response to signals from commensal bacteria could pose the risk of inflammation; immune responses to mucosal microbiota therefore require a precise regulatory control. The mucosal immune system has developed specialised regulatory, anti-inflammatory mechanisms for eliminating or tolerating non-dangerous, food and airborne antigens and commensal micro-organisms (oral, mucosal tolerance). However, at the same time the mucosal immune system must provide local defense mechanisms against environmental threats (e.g. invading pathogens). This important requirement is fulfilled by several mechanisms of mucosal immunity: strongly developed innate defense mechanisms ensuring appropriate function of the mucosal barrier, existence of unique types of lymphocytes and their products, transport of polymeric immunoglobulins through epithelial cells into secretions (sIgA) and migration and homing of cells originating from the mucosal organised tissues in mucosae and exocrine glands. The important role of commensal bacteria in development of optimally functioning mucosal immune system was demonstrated in germ-free animals (using gnotobiological techniques). Involvement of commensal microflora and its components with strong immunoactivating properties (e.g. LPS, peptidoglycans, superantigens, bacterial DNA, Hsp) in etiopathogenetic mechanism of various complex, multifactorial and multigenic diseases, including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, multiorgan failure, colon cancer has been recently suggested. Animal models of human diseases reared in defined gnotobiotic conditions are helping to elucidate the aetiology of these frequent disorders. An improved understanding of commensal bacteria-host interactions employing germ-free animal models with selective colonisation strategies combined with modern molecular techniques could bring new insights into the mechanisms of mucosal immunity and also into pathogenetic mechanisms of several infectious, inflammatory, autoimmune and neoplastic diseases. Regulation of microflora composition (e.g. by probiotics and prebiotics) offers the possibility to influence the development of mucosal and systemic immunity but it can play a role also in prevention and treatment of some diseases.

Prevention or early cure of type 1 diabetes by intranasal administration of gliadin in NOD mice.

Induction of long-term tolerance to ß-cell autoantigens has been investigated both in animal models and in human type 1 diabetes (T1D) in order to prevent the disease. As regards external compounds, the dietary plant protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevent T1D in animal models. Herewith we investigated whether intranasal (i.n.) administration of gliadin or gluten may arrest the diabetogenic process. I.n. administration of gliadin to 4-week-old NOD mice significantly reduced the diabetes incidence. Similarly, the insulitis was lowered. Intranasal gliadin also rescued a fraction of prediabetic 13-week-old NOD mice from progressing to clinical onset of diabetes compared to OVA-treated controls. Vaccination with i.n. gliadin led to an induction of CD4(+)Foxp3(+) T cells and even more significant induction of γδ T cells in mucosal, but not in non-mucosal lymphoid compartments. This prevention strategy was characterized by an increased proportion of IL-10 and a decreased proportion of IL-2, IL-4 and IFN-γ-positive CD4(+)Foxp3(+) T cells, and IFN-γ-positive γδ T cells, preferentially in mucosal lymphoid organs. In conclusion, i.n. vaccination with gliadin, an environmental antigen with possible etiological influence in T1D, may represent a novel, safer strategy for prevention or even early cure of T1D.

Pepsin digest of wheat gliadin fraction increases production of IL-1ß via TLR4/MyD88/TRIF/MAPK/NF-κB signaling pathway and an NLRP3 inflammasome activation.

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1ß and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1ß and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1ß production in celiac PBMC show that PDWGF-induced de novo pro-IL-1ß synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1ß. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1ß release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3(-/-) and ASC(-/-) knockout mice. Moreover, treatment of human PBMC as well as MyD88(-/-) and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-ß (TRIF)(-/-) BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1ß in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD.

Impact of dietary gluten on regulatory T cells and Th17 cells in BALB/c mice.

Dietary gluten influences the development of type 1 diabetes (T1D) and a gluten-free (GF) diet has a protective effect on the development of T1D. Gluten may influence T1D due to its direct effect on intestinal immunity; however, these mechanisms have not been adequately studied. We studied the effect of a GF diet compared to a gluten-containing standard (STD) diet on selected T cell subsets, associated with regulatory functions as well as proinflammatory Th17 cells, in BALB/c mice. Furthermore, we assessed diet-induced changes in the expression of various T cell markers, and determined if changes were confined to intestinal or non-intestinal lymphoid compartments. The gluten-containing STD diet led to a significantly decreased proportion of γδ T cells in all lymphoid compartments studied, although an increase was detected in some γδ T cell subsets (CD8(+), CD103(+)). Further, it decreased the proportion of CD4(+)CD62L(+) T cells in Peyer's patches. Interestingly, no diet-induced changes were found among CD4(+)Foxp3(+) T cells or CD3(+)CD49b(+)cells (NKT cells) and CD3(-)CD49b(+) (NK) cells. Mice fed the STD diet showed increased proportions of CD4(+)CD45RB(high+) and CD103(+) T cells and a lower proportion of CD4(+)CD45RB(low+) T cells in both mucosal and non-mucosal compartments. The Th17 cell population, associated with the development of autoimmunity, was substantially increased in pancreatic lymph nodes of mice fed the STD diet. Collectively, our data indicate that dietary gluten influences multiple regulatory T cell subsets as well as Th17 cells in mucosal lymphoid tissue while fewer differences were observed in non-mucosal lymphoid compartments.

The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases.

Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.

Gluten-free but also gluten-enriched (gluten+) diet prevent diabetes in NOD mice; the gluten enigma in type 1 diabetes.

BACKGROUND: Environmental factors such as nutrition or exposure to infections play a substantial role in the pathogenesis of type 1 diabetes (T1D). We have previously shown that gluten-free, non-purified diet largely prevented diabetes in non-obese diabetic (NOD) mice. In this study we tested hypothesis that early introduction of gluten-enriched (gluten+) diet may increase diabetes incidence in NOD mice. METHODS: Standard, gluten-free, gluten+ modified Altromin diets and hydrolysed-casein-based Pregestimil diet were fed to NOD females and diabetes incidence was followed for 310 days. Insulitis score and numbers of gut mucosal lymphocytes were determined in non-diabetic animals. RESULTS: A significantly lower diabetes incidence (p < 0.0001) was observed in NOD mice fed gluten-free diet (5.9%, n = 34) and Pregestimil diet (10%, n = 30) compared to mice on the standard Altromin diet (60.6%, n = 33). Surprisingly, gluten+ diet also prevented diabetes incidence, even at the level found with the gluten-free diet (p < 0.0001, 5.9%, n = 34). The minority of mice, which developed diabetes on all the three diabetes-protective (gluten+, gluten-free, Pregestimil) diets, did that slightly later compared to those on the standard diet. Lower insulitis score compared to control mice was found in non-diabetic NOD mice on the gluten-free, and to a lesser extent also gluten+ and Pregestimil diets. No substantial differences in the number of CD3(+), TCR-gammadelta(+), and IgA(+) cells in the small intestine were documented. CONCLUSIONS: Gluten+ diet prevents diabetes in NOD mice at the level found with the non-purified gluten-free diet. Possible mechanisms of the enigmatic, dual effect of dietary gluten on the development of T1D are discussed.

Diabetes preventive gluten-free diet decreases the number of caecal bacteria in non-obese diabetic mice.

BACKGROUND: A gluten-free diet reduces the incidence of diabetes mellitus in non-obese diabetic (NOD) mice, but the mechanism is not known. The aim of this study was to examine the possible influence of the diet on the caecal bacterial flora, which may affect the intestinal physiology and mediate disease prevention. METHODS: Two groups of NOD mice from the age of 3 weeks were fed either a gluten-free diet or a standard diet. Each diabetic mouse, when diagnosed, along with a non-diabetic mouse from the same diet group and two non-diabetic mice from the alternate diet group were euthanized and sampled for classical bacteriological examination. RESULTS: Nine out of 19 (47%) standard-fed mice and 1 out of 19 (5%) gluten-free-fed mice developed diabetes (p < 0.01). Mice on the gluten-free diet had significantly fewer aerobically (p < 0.01) and microaerophilically (p < 0.001) cultivated bacteria in their intestines than standard-fed mice. Non-diabetic mice also had significantly fewer microa erophilic and anaerobic bacteria than diabetic mice (p < 0.05). These differences were primarily due to a difference in the Gram-positive flora. CONCLUSIONS: The gluten-free diet compared to the standard diet both qualitatively and quantitatively substantially altered the composition of the caecal bacterial flora in NOD mice. Although Gram-positive bacteria might influence the beta cells through certain digestive products, it is more likely to assume that any effect on diabetes incidence is immunological.

TCR gamma delta intraepithelial lymphocytes are required for self-tolerance.

Neonatal thymectomy (NTX) impairs T cell regulation and leads to organ-specific autoimmune disease in susceptible mouse strains. In the NOD mouse model of spontaneous type 1 diabetes, we observed that NTX dramatically accelerated autoimmune pancreatic beta cell destruction and diabetes. NTX had only a minor effect in NOD mice protected from diabetes by transgenic expression of the beta cell autoantigen proinsulin in APCs, inferring that accelerated diabetes after NTX is largely due to failure to regulate proinsulin-specific T cells. NTX markedly impaired the development of intraepithelial lymphocytes (IEL), the number of which was already reduced in euthymic NOD mice compared with control strains. IEL purified from euthymic NOD mice, specifically CD8alphaalpha TCRgammadelta IEL, when transferred into NTX-NOD mice, trafficked to the small intestinal epithelium and prevented diabetes. Transfer of prototypic CD4+CD25+ regulatory T cells also prevented diabetes in NTX-NOD mice; however, the induction of these cells by oral insulin in euthymic mice depended on the integrity of TCRgammadelta IEL. We conclude that TCRgammadelta IEL at the mucosal interface between self and nonself play a key role in maintaining peripheral tolerance both physiologically and during oral tolerance induction.

TCR-mediated activation promotes GITR upregulation in T cells and resistance to glucocorticoid-induced death.

T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR-/-). We also compared the action of GC on GITR+ and GITR- T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR-/- mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR-/- cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10(-8) to 10(-6) M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.

Mucosal immunity: its role in defense and allergy.

The interface between the organism and the outside world, which is the site of exchange of nutrients, export of products and waste components, must be selectively permeable and at the same time, it must constitute a barrier equipped with local defense mechanisms against environmental threats (e.g. invading pathogens). The boundaries with the environment (mucosal and skin surfaces) are therefore covered with special epithelial layers which support this barrier function. The immune system, associated with mucosal surfaces covering the largest area of the body (200-300 m(2)), evolved mechanisms discriminating between harmless antigens and commensal microorganisms and dangerous pathogens. The innate mucosal immune system, represented by epithelial and other mucosal cells and their products, is able to recognize the conserved pathogenic patterns on microbes by pattern recognition receptors such as Toll-like receptors, CD14 and others. As documented in experimental gnotobiotic models, highly protective colonization of mucosal surfaces by commensals has an important stimulatory effect on postnatal development of immune responses, metabolic processes (e.g. nutrition) and other host activities; these local and systemic immune responses are later replaced by inhibition, i.e. by induction of mucosal (oral) tolerance. Characteristic features of mucosal immunity distinguishing it from systemic immunity are: strongly developed mechanisms of innate defense, the existence of characteristic populations of unique types of lymphocytes, colonization of the mucosal and exocrine glands by cells originating from the mucosal organized tissues ('common mucosal system') and preferential induction of inhibition of the responses to nondangerous antigens (mucosal tolerance). Many chronic diseases, including allergy, may occur as a result of genetically based or environmentally induced changes in mechanisms regulating mucosal immunity and tolerance; this leads to impaired mucosal barrier function, disturbed exclusion and increased penetration of microbial, food or airborne antigens into the circulation and consequently to exaggerated and generalized immune responses to mucosally occurring antigens, allergens, superantigens and mitogens.