The Incidence of Type-1 Diabetes in NOD Mice Is Modulated by Restricted Flora Not Germ-Free Conditions

In the NOD mouse, the incidence of type-1 diabetes is thought to be influenced by the degree of cleanliness of the mouse colony. Studies collectively demonstrate that exposure to bacterial antigen or infection in the neonatal period prevents diabetes [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], supporting the notion that immunostimulation can benefit the maturation of the postnatal immune system [11]. A widely accepted extrapolation from this data has been the notion that NOD mice maintained under germ-free conditions have an increased incidence of diabetes. However, evidence supporting this influential concept is surprisingly limited [12]. In this study, we demonstrate that the incidence of diabetes in female NOD mice remained unchanged under germ-free conditions. By contrast, a spontaneous monoculture with a gram-positive aerobic spore-forming rod delayed the onset and reduced the incidence of diabetes. These findings challenge the view that germ-free NOD mice have increased diabetes incidence and demonstrate that modulation of intestinal microbiota can prevent the development of type-1 diabetes

IL-21 limits peripheral lymphocyte numbers through T cell homeostatic mechanisms.

BACKGROUND: IL-21, a member of the common gamma-chain utilizing family of cytokines, participates in immune and inflammatory processes. In addition, the cytokine has been linked to autoimmunity in humans and rodents. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanism whereby IL-21 affects the immune system, we investigated its role in T cell homeostasis and autoimmunity in both non-autoimmune C57BL/6 and autoimmune NOD mice. Our data indicate that IL-21R knockout C57BL/6 and NOD mice show increased size of their lymphocyte population and decreased homeostatic proliferation. In addition, our experimental results demonstrate that IL-21 inhibits T cell survival. These data suggest that IL-21 acts to limit the size of the T cell pool. Furthermore, our data suggest IL-21 may contribute to the development of autoimmunity. CONCLUSIONS/SIGNIFICANCE: Taken together, our results suggest that IL-21 plays a global role in regulating T cell homeostasis, promoting the continuous adaptation of the T cell lymphoid space

Leukocyte extravasation into the pancreatic tissue in transgenic mice expressing interleukin 10 in the islets of Langerhans.

Transgenic expression of interleukin 10 (IL-10) in the islets of Langerhans leads to a pronounced pancreatic inflammation, without inflammation of the islets of Langerhans and without diabetes. A scattered infiltration of macrophages (M pi) precedes localized accumulations of CD4+ and CD8+ T lymphocytes, B lymphocytes, and M pi. This recruitment of inflammatory cells to the pancreas is somewhat surprising, since the biological activities of IL-10 in vitro indicate that IL-10 is a powerful immunosuppressive cytokine. Since endothelial cells play a major role in leukocyte extravasation, we examined if vascular changes and extralymphoid induction of peripheral and mucosal type vascular addressins contributed to IL-10-induced homing of mononuclear cells to the pancreas. The endothelium lining small vessels was highly activated in areas of inflammation, as the endothelial cells became cuboidal, and exhibited increased expression of major histocompatibility complex class II (Ia), intercellular adhesion molecule 1, and von Willebrand Factor. Furthermore, induction of vascular addressins simultaneously with accumulation of mononuclear cells around islets and vessels indicated that the endothelial cells take on the phenotype of differentiated endothelium specialized for leukocyte extravasation. In conclusion, pancreatic inflammation and vascular changes are prominent in IL-10 transgenic mice. We hypothesize that IL-10, in addition to its immuno-inhibitory properties, is a potent recruitment signal for leukocyte migration in vivo. These effects are relevant for in vivo therapeutic applications of IL-10

Identification and expansion of pancreatic stem/progenitor cells.

Pancreatic islet transplantation represents an attractive approach for the treatment of diabetes. However, the limited availability of donor islets has largely hampered this approach. In this respect, the use of alternative sources of islets such as the ex vivo expansion and differentiation of functional endocrine cells for treating diabetes has become the major focus of diabetes research. Adult pancreatic stem cells /progenitor cells have yet to be recognized because limited markers exist for their identification. While the pancreas has the capacity to regenerate under certain circumstances, questions where adult pancreatic stem/progenitor cells are localized, how they are regulated, and even if the pancreas harbors a stem cell population need to be resolved. In this article, we review the recent achievements both in the identification as well as in the expansion of pancreatic stem/progenitor cells

Production of interleukin 10 by islet cells accelerates immune-mediated destruction of beta cells in nonobese diabetic mice.

The T helper type 2 (Th2) cell product interleukin 10 (IL-10) inhibits the proliferation and function of Th1 lymphocytes and macrophages (M phi). The nonobese diabetic mouse strain (NOD/Shi) develops a M phi and T cell-dependent autoimmune diabetes that closely resembles human insulin-dependent diabetes mellitus (IDDM). The objective of the present study was to explore the consequences of localized production of IL-10 on diabetes development in NOD/Shi mice. Surprisingly, local production of IL-10 accelerated the onset and increased the prevalence of diabetes, since diabetes developed at 5-10 wk of age in 92% of IL-10 positive I-A beta g7/g7, I-E- mice in first (N2) and second (N3) generation backcrosses between IL-10 transgenic BALB/c mice and (NOD/Shi) mice. None of the IL-10 negative major histocompatibility complex-identical littermates were diabetic at this age. Furthermore, diabetes developed in 33% of I-A beta g7/d, I-E+ N3 mice in the presence of IL-10 before the mice were 10 wk old. Our findings support the notion that IL-10 should not simply be regarded as an immunoinhibitory cytokine, since it possesses powerful, immunostimulatory properties as well. Furthermore, our observations suggest that beta cell destruction in NOD mice may be a Th2-mediated event

Anti-Human Interleukin(IL)-4 Clone 8D4-8 Cross-Reacts With Myosin-9 Associated With Apoptotic Cells and Should Not Be Used for Flow Cytometry Applications Querying IL-4 Expression

Interleukin(IL)-4 is produced by T cells and other leukocytes and is a critical mediator of monocyte and B cell responses. During routine flow cytometry panel validation for the investigation of intracellular cytokines, we observed unique IL-4 expression patterns associated with the widely available monoclonal antibody 8D4-8. Namely, IL-4 (8D4-8) expression was observed in the absence of cellular activation and enhanced following staurosporine exposure. Mass spectrometry analysis of immunoprecipitates from peripheral blood lymphocytes (PBL) revealed that 8D4-8 cross-reacts with the ubiquitous cytoskeletal protein myosin-9. We confirmed these results by western blotting immunoprecipitates, using immunofluorescence among staurosporine-treated Caco-2 cells, and by surface-labeling PBL for 8D4-8 and myosin-9 and analyzing by flow cytometry. Although previously reported from several independent groups, we found no evidence to support the hypothesis that IL-4 is produced by apoptotic cells. Rather, this appears to have been myosin-9. Our data indicate clone 8D4-8 should not be used in the flow cytometric study of IL-4. Furthermore, our work calls for a reevaluation of previous flow cytometric studies that have used this clone for IL-4 analysis and highlights the importance of validation in antibody-based assays

Coordinated Induction of Antimicrobial Response Factors in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by dysregulated autoantibody production and complement activation leading to multi-organ damage. The disease is associated with increased intestinal permeability. In this study, we tested the hypothesis that SLE subjects have increased systemic exposure to bacteria. Since bacteria induce the expression of antimicrobial response factors (ARFs), we measured the levels of a series of clinically relevant ARFs in the plasma of SLE subjects. We found that levels of sCD14, lysozyme, and CXCL16 were significantly elevated in SLE subjects. A strong positive correlation was also observed between sCD14 and SELENA-SLEDAI score. Interestingly, the ratio of EndoCAb IgM:total IgM was significantly decreased in SLE and this ratio was negatively correlated with sCD14 levels. Although, there were no significant differences in the levels of lipopolysaccharide binding protein (LBP) and fatty acid binding protein 2 (FABP2), we observed significant positive correlations between lysozyme levels and sCD14, LBP, and FABP2. Moreover, galectin-3 levels also positively correlate with lysozyme, sCD14, and LBP. Since our SLE cohort comprised 43.33% males, we were able to identify gender-specific changes in the levels of ARFs. Overall, these changes in the levels and relationships between ARFs link microbial exposure and SLE. Approaches to reduce microbial exposure or to improve barrier function may provide therapeutic strategies for SLE patients

A defect in interleukin 12-induced activation and interferon gamma secretion of peripheral natural killer T cells in nonobese diabetic mice suggests new pathogenic mechanisms for insulin-dependent diabetes mellitus.

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell-mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In fact, NKT cells mainly secrete interferon (IFN)-gamma and, activated in the presence of IL-12, acquire a strong inflammatory phenotype and cytotoxic function