Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes.

AIM: Much focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells. METHODS: We induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry. RESULTS: We found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35's protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells. CONCLUSION: In conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

Interleukin-35 Prevents Development of Autoimmune Diabetes Possibly by Maintaining the Phenotype of Regulatory B Cells.

The anti-inflammatory role of regulatory B cells (Breg cells) has been associated with IL-35 based on studies of experimental autoimmune uveitis and encephalitis. The role of Breg cells and IL-35+ Breg cells for type 1 diabetes (T1D) remains to be investigated. We studied PBMCs from T1D subjects and healthy controls (HC) and found lowered proportions of Breg cells and IL-35+ Breg cells in T1D. To elucidate the role of Breg cells, the lymphoid organs of two mouse models of T1D were examined. Lower proportions of Breg cells and IL-35+ Breg cells were found in the animal models of T1D compared with control mice. In addition, the systemic administration of recombinant mouse IL-35 prevented hyperglycemia after multiple low dose streptozotocin (MLDSTZ) injections and increased the proportions of Breg cells and IL-35+ Breg cells. A higher proportion of IFN-γ+ cells among Breg cells were found in the PBMCs of the T1D subjects. In the MLDSTZ mice, IL-35 administration decreased the proportions of IFN-γ+ cells among the Breg cells. Our data illustrate that Breg cells may play an important role in the development of T1D and that IL-35 treatment prevents the development of hyperglycemia by maintaining the phenotype of the Breg cells under an experimental T1D condition.

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Kinetics of immune cell responses in the multiple low-dose streptozotocin mouse model of type 1 diabetes.

In type 1 diabetes (T1D), the insulin-producing ß cells are destructed by immune mechanisms. It has been hypothesized that the very first immune response in T1D onset comes from innate immune cells, which further activates the adaptive immune cells to attack the islets. Despite intensive research on characterization of islet-infiltrating immune cells, the kinetics of different immune cells in multiple low-dose streptozotocin (MLDSTZ)-induced T1D mouse model is still much unclear. Therefore, we investigated the proportions of innate immune cells such as neutrophils, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), macrophages, natural killer (NK) cells, and adaptive immune cells (T and B lymphocytes) in thymi, pancreatic-draining lymph nodes, and spleens of MLDSTZ mice on days 3, 7, 10, and 21 after the first injection of STZ by flow cytometry. The proportions of DCs and B cells were increased from day 3, while the proportions of B-1a lymphocytes and interferon-γ+ cells among NK cells were increased, but NK cells were decreased on day 10 in MLDSTZ-treated mice, illustrating that the initial immune response is induced by DCs and B cells. Later, the proportions of T helper 1 and cytotoxic T cells were increased from day 7, suggesting that the innate immune cells precede adaptive immune cell response in MLDSTZ mice. Altogether, our data demonstrate a possible sequence of events regarding the involvement of DCs, pDCs, NK cells, B-1a lymphocytes, B, and T cells at the early stage of T1D development.

Pancreatic islets of bank vole show signs of dysfunction after prolonged exposure to high glucose concentrations in vitro.

Bank voles develop glucose intolerance/diabetes mellitus when kept in captivity. We have characterized beta-cell function of glucose intolerant/diabetic animals, and found that this animal model has features of both human type 1 and type 2 diabetes. The aim of this study was to study the functional alterations of islets isolated from glucose tolerant bank voles after a prolonged exposure to various glucose concentrations in vitro. For this purpose, pancreatic islets from normal (glucose tolerant) male and female bank voles were cultured at different glucose concentrations (5.6, 11.1 (control), or 28 mM) whereupon islet functions were examined. Overall, islet insulin output was lowered at 5.6 mM glucose, and similar to control, or enhanced after culture in 28 mM glucose. High glucose culture led to decreased insulin contents, but there was no change in islet DNA content and in morphological assessments of cell death, with the latter findings suggesting that the so-called glucotoxicity had not evolved. A slight gender difference was observed in that islets isolated from females exhibited a glucose-regulated (pro)insulin biosynthesis rate and insulin gene expression. In conclusion, we have found that islets isolated from female and male bank voles are affected by glucose concentrations in vitro in that some signs of dysfunction were observed upon high glucose exposure. A minor gender difference was observed suggesting that the islets of the females may more readily adapt to the elevated glucose concentration than islets of the male bank voles. It could be that these in vitro gender differences observed may represent a mechanism underlying the gender difference in diabetes development observed among bank voles.

Antiviral treatments reduce severity of diabetes in Ljungan virus-infected CD-1 mice and delay onset in diabetes-prone BB rats.

The effects of LV in two different species, CD-1 mice, without a genetic disposition for diabetes, and BB rats prone to T1D were examined. Male CD-1 mice that had been exposed to LV in utero developed a type 2-like diabetes with increased blood glucose, insulin levels and epididymal fat at the age of 10-15 weeks. Combination therapy including LV-antiserum and an antiviral drug, Pleconaril, significantly reduced the levels of blood glucose and insulin and the amount of abdominal fat. In BB rats, LV has been found in both prediabetic- and diabetic diabetes-prone rats, as well as in diabetes-resistant rats. To evaluate whether the presence of LV has any influence on the onset of T1D, prediabetic BB rats were treated with an antiserum against LV or a combination of the antiviral drugs Pleconaril and Ribavirin. In the group treated with antiviral drugs, the onset was significantly delayed. These results indicate that the presence of LV can be involved in the pathogenesis of diabetes in these animal models.

Suppression of bank vole pancreatic islet function by proinflammatory cytokines.

Bank voles kept in captivity may develop diabetes. We recently characterized beta-cell function of pancreatic islets from normal and glucose intolerant/diabetic bank voles. These animals had features of both human type 1 and type 2 diabetes. Cytokines may impair beta-cell function in both types of diabetes. Presently, we studied how pancreatic islets isolated from normal, i.e. glucose tolerant bank voles are affected by proinflammatory cytokines in vitro. Islets were exposed to hIL-1beta (25U/ml) alone or in combination with hTNF-alpha (1000U/ml)+mIFN-gamma (1000U/ml) for 48h, whereupon islet functions were assessed. Cytokines markedly reduced insulin gene expression and the (pro)insulin biosynthesis rate, which was accompanied by a profound depletion of the islet insulin content. The cytokines did not affect the culture medium insulin accumulation and the glucose oxidation rate, but caused a modest increase in medium nitrite, an indicator of nitric oxide (NO) generation. Cytokine-induced decrease in islet insulin content was not prevented by the preferential inducible NO synthase inhibitor aminoguanidine. These findings suggest that the reduction in islet insulin content is not attributed to enhanced exocytosis or related to altered glucose metabolism, but is rather due to a decline in insulin production. The suppressive effects of islet functions elicited by cytokines seem to be mediated by an NO-independent mechanism. In relation to previous studies on cytokine effects on islets from various species, the bank vole islets show a pattern which more resembles human islets than rat or murine islets.

Characterization of beta-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus.

Bank voles (Clethrionomys glareolus) kept in captivity develop diabetes mellitus to a significant extent. Also in wild bank voles, elevated blood glucose has been observed. A newly isolated picornavirus named Ljungan virus (LV) has been found in the pancreas of these bank voles. Moreover, LV infection in combination with environmental factors may cause glucose intolerance/diabetes (GINT/D) in normal mice. The aim of the present study was to investigate the functional characteristics of pancreatic islets, isolated from bank voles, bred in the laboratory but considered LV infected. About 20% of all males and females were classified as GINT/D following a glucose tolerance test. Of these animals the majority had become diabetic by 20 weeks of age, with a tendency towards an earlier onset in the males. GINT/D animals had increased serum insulin levels. Islets were tested on the day of isolation (day 0) and after 1 week of culture for their insulin content and their capacity to synthesize (pro)insulin, secrete insulin and metabolize glucose. Functional differences could be observed between normal and GINT/D animals as well as between genders. An elevated basal insulin secretion was observed on day 0 indicating beta-cell dysfunction among islets isolated from diabetic males. In vitro culture could reverse some functional changes. The increased serum insulin level and the increased basal islet insulin secretion may suggest that the animals had developed a type 2 diabetes-like condition. It is likely that the putative stress imposed in the laboratory, maybe in combination with LV infection, can lead to an increased functional demand on the beta-cells.