Neutralization of Interleukin-16 Protects Nonobese Diabetic Mice From Autoimmune Type 1 Diabetes by a CCL4-Dependent Mechanism

OBJECTIVE

The progressive infiltration of pancreatic islets by lymphocytes is mandatory for development of autoimmune type 1 diabetes. This inflammatory process is mediated by several mediators that are potential therapeutic targets to arrest development of type 1 diabetes. In this study, we investigate the role of one of these mediators, interleukin-16 (IL-16), in the pathogenesis of type 1 diabetes in NOD mice.

RESEARCH DESIGN AND METHODS

At different stages of progression of type 1 diabetes, we characterized IL-16 in islets using GEArray technology and immunoblot analysis and also quantitated IL-16 activity in cell migration assays. IL-16 expression was localized in islets by immunofluorescence and confocal imaging. In vivo neutralization studies were performed to assess the role of IL-16 in the pathogenesis of type 1 diabetes.

RESULTS

The increased expression of IL-16 in islets correlated with the development of invasive insulitis. IL-16 immunoreactivity was found in islet infiltrating T-cells, B-cells, NK-cells, and dendritic cells, and within an insulitic lesion, IL-16 was derived from infiltrating cells. CD4⁺ and CD8⁺ T-cells as well as B220⁺ B-cells were identified as sources of secreted IL-16. Blockade of IL-16 in vivo protected against type 1 diabetes by interfering with recruitment of CD4⁺ T-cells to the pancreas, and this protection required the activity of the chemokine CCL4.

CONCLUSIONS

IL-16 production by leukocytes in islets augments the severity of insulitis during the onset of type 1 diabetes. IL-16 and CCL4 appear to function as counterregulatory proteins during disease development. Neutralization of IL-16 may represent a novel therapy for the prevention of type 1 diabetes.Insulitis and type 1 diabetes are mediated by the regulated homing of leukocytes to pancreatic islets by chemoattractants (1–3). Unlike chemokines that bind chemokine receptors, the interleukin-16 (IL-16) cytokine binds to CD4 and selectively recruits CD4⁺ T helper one (Th1) cells due to the functional relationship between CD4 and the CCR5 and CXCR3 chemokine receptors (4,5). Mature IL-16 is secreted after caspase-3 mediated cleavage of either neuronal IL-16 or precursor protein (pro-IL-16) (6–13). IL-16 also regulates T-cell activation, CD25 and major histocompatibility complex class II expression, IL-2 production (14), antigen-induced upregulation of CD95 expression (14), and chemokine-induced chemoattraction (15,16). Binding of IL-16 to CD4 selectively desensitizes CCL4/CCR5-induced chemotaxis, and pretreatment of T-cells with CCL4 blocks chemotaxis triggered by IL-16 binding to CD4 (15). CD4 and CCR5 may therefore share a unique relationship mediated by either IL-16 and/or CCL4 binding, and IL-16 and CCL4 may cross-regulate each other''s activity. Thus, IL-16 is an immunomodulatory and proinflammatory cytokine that influences the recruitment and activation of CD4⁺ T-cells in vivo.Given that CD4⁺ T-cells mediate the development of type 1 diabetes, we examined whether IL-16 influences CD4⁺ T-cell recruitment to pancreatic islets. We show that IL-16 potentiates insulitis and type 1 diabetes in NOD mice, and that anti-IL-16 antibody therapy protects against type 1 diabetes even when administered at the mid-late stages of disease progression. This protection results from reduced trafficking of CD4⁺ T-cells to islet lesions and requires CCL4 activity. Thus, IL-16 may represent a novel therapeutic target for the prevention of type 1 diabetes.     

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address the limited clinical efficacy of ASIT. Soluble antigen arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using either hydrolysable linkers (hSAgAs) or stable click chemistry linkers (cSAgAs). They were evaluated for the ability to block spontaneous development of disease in a nonobese diabetic mouse model of type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgAs) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers, such as CD73, interleukin-10, programmed death-1, and KLRG-1. Anaphylaxis caused by free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely nonoverlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to free peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.     

Chromogranin A Deficiency Confers Protection From Autoimmune Diabetes via Multiple Mechanisms

Recognition of β-cell antigens by autoreactive T cells is a critical step in the initiation of autoimmune type1 diabetes. A complete protection from diabetes development in NOD mice harboring a point mutation in the insulin B-chain 9–23 epitope points to a dominant role of insulin in diabetogenesis. Generation of NOD mice lacking the chromogranin A protein (NOD.ChgA^−/−) completely nullified the autoreactivity of the BDC2.5 T cell and conferred protection from diabetes onset. These results raised the issue concerning the dominant antigen that drives the autoimmune process. Here we revisited the NOD.ChgA^−/− mice and found that their lack of diabetes development may not be solely explained by the absence of chromogranin A reactivity. NOD.ChgA^−/− mice displayed reduced presentation of insulin peptides in the islets and periphery, which corresponded to impaired T-cell priming. Diabetes development in these mice was restored by antibody treatment targeting regulatory T cells or inhibiting transforming growth factor-β and programmed death-1 pathways. Therefore, the global deficiency of chromogranin A impairs recognition of the major diabetogenic antigen insulin, leading to broadly impaired autoimmune responses controlled by multiple regulatory mechanisms.     

Nonviral-Mediated Hepatic Expression of IGF-I Increases Treg Levels and Suppresses Autoimmune Diabetes in Mice

In type 1 diabetes, loss of tolerance to β-cell antigens results in T-cell–dependent autoimmune destruction of β cells. The abrogation of autoreactive T-cell responses is a prerequisite to achieve long-lasting correction of the disease. The liver has unique immunomodulatory properties and hepatic gene transfer results in tolerance induction and suppression of autoimmune diseases, in part by regulatory T-cell (Treg) activation. Hence, the liver could be manipulated to treat or prevent diabetes onset through expression of key genes. IGF-I may be an immunomodulatory candidate because it prevents autoimmune diabetes when expressed in β cells or subcutaneously injected. Here, we demonstrate that transient, plasmid-derived IGF-I expression in mouse liver suppressed autoimmune diabetes progression. Suppression was associated with decreased islet inflammation and β-cell apoptosis, increased β-cell replication, and normalized β-cell mass. Permanent protection depended on exogenous IGF-I expression in liver nonparenchymal cells and was associated with increased percentage of intrapancreatic Tregs. Importantly, Treg depletion completely abolished IGF-I-mediated protection confirming the therapeutic potential of these cells in autoimmune diabetes. This study demonstrates that a nonviral gene therapy combining the immunological properties of the liver and IGF-I could be beneficial in the treatment of the disease.In type 1 diabetes, the immune system attacks and destroys β cells. At the clinical onset of type 1 diabetes, 15–40% of β cells are still able to produce insulin, thus blocking further autoimmune destruction even at this stage, holding great promise for arresting disease progression (1). Yet earlier intervention in individuals with documented autoimmune disease but without clinically manifest diabetes is of course the ultimate goal in any future intervention strategy to prevent diabetes. Following this rationale, a growing number of clinical intervention studies, with the common goal of blocking autoimmune disease and reestablishing long-term tolerance to β cells based on immunomodulation, have been initiated in the past decade (2).The liver has unique immunological properties that affect T-cell activation and immune regulation. Although the liver is an important site for T-cell activation, this takes place in the context of immunosuppressive cytokines and a distinctive local immune environment, so that exposure to antigens often results in tolerance rather than immunity (3). Tolerance promotion can be mediated, among other mechanisms, by the induction of regulatory T cells (Tregs) capable of inhibiting effector responses in the periphery (4). However, the key hepatic cell type responsible for initiating this phenomenon remains controversial. Whereas several reports point to hepatocytes, others indicate nonparenchymal cells (NPCs) as the cells that promote expansion of specific Treg populations able to limit autoreactive immunity (5). Engineering hepatic cells to express molecules able to induce Tregs therefore represents a potential therapeutic approach for the treatment of autoimmune disorders (5). A number of gene transfer studies have described the ectopic expression of autoantigens in the liver as a means to promote peripheral control of autoreactive lymphocytes by increasing either the number and/or the function of Tregs (6). Also, it has been shown repeatedly, in both mice and nonhuman primates, that upon in vivo liver-directed gene transfer of coagulation factor IX, transgene expression has the capacity to provide therapeutic circulating levels while inducing immune tolerance to the transgene product (7,8). Thus, in the context of an autoimmune disease such as type 1 diabetes, a liver-targeted gene therapy approach to modify peripheral control of autoreactive lymphocytes offers a unique possibility to prevent development and progression of the disease. To achieve this would require efficient and targeted expression to tolerogenic cells as well as utilization of an appropriate immunomodulatory transgene. Among the possible immunomodulatory candidate genes for type 1 diabetes is IGF-I. We have previously reported that expression of IGF-I in β cells of transgenic mice counteracts the cytotoxicity and insulitis induced by treatment with multiple low doses of streptozotocin (STZ) (9,10). Moreover, daily subcutaneous administration of human recombinant IGF-I to prediabetic NOD mice reduces the severity of insulitis and the incidence of type 1 diabetes (11–13). All these studies reveal IGF-I as a key factor able to induce protection from type 1 diabetes.In this work, we report that after delivery of a plasmid expressing IGF-I to the liver by hydrodynamic tail vein (HTV) injection, the incidence of diabetes decreases in an autoimmune mouse model of the disease. We show that the expression of IGF-I in liver NPCs leads to an increase in intrapancreatic Tregs, resulting in decreased pancreatic infiltration, reduced β-cell apoptosis, and increased β-cell replication, suggesting a blockage of the autoimmune attack against the pancreas.     

Deficiency of Phosphoinositide 3-Kinase Enhancer Protects Mice From Diet-Induced Obesity and Insulin Resistance

OBJECTIVE

Phosphoinositide 3-kinase enhancer A (PIKE-A) is a proto-oncogene that promotes tumor growth and transformation by enhancing Akt activity. However, the physiological functions of PIKE-A in peripheral tissues are unknown. Here, we describe the effect of PIKE deletion in mice and explore the role of PIKE-A in obesity development.

RESEARCH DESIGN AND METHODS

Whole-body PIKE knockout mice were generated and subjected to high-fat–diet feeding for 20 weeks. The glucose tolerance, tissue-specific insulin sensitivity, adipocyte differentiation, and lipid oxidation status were determined. The molecular mechanism of PIKE in the insulin signaling pathway was also studied.

RESULTS

We show that PIKE-A regulates obesity development by modulating AMP-activated protein kinase (AMPK) phosphorylation. PIKE-A is important for insulin to suppress AMPK phosphorylation. The expression of PIKE-A is markedly increased in adipose tissue of obese mice, whereas depletion of PIKE-A inhibits adipocyte differentiation. PIKE knockout mice exhibit a prominent phenotype of lipoatrophy and are resistant to high-fat diet–induced obesity, liver steatosis, and diabetes. PIKE knockout mice also have augmented lipid oxidation, which is accompanied by enhanced AMPK phosphorylation in both muscle and adipose tissue. Moreover, insulin sensitivity is improved in PIKE-A–deficient muscle and fat, thus protecting the animals from diet-induced diabetes.

CONCLUSIONS

Our results suggest that PIKE-A is implicated in obesity and associated diabetes development by negatively regulating AMPK activity.Obesity is a result of imbalanced energy intake and expenditure in which the accumulation of excessive fat causes disorders such as type 2 diabetes, atherosclerosis, and dyslipidemia (1). Because of its increasing prevalence in most of the world, obesity has became a major health problem (2). Although genetic linkage analysis has successfully mapped potential loci in human genome for adiposity development (3), identifying all genetic variants that contribute to differences in body weight is still one of the major goals to fully understand the mechanism of obesity progression. Recent studies using genome-wide linkage scan revealed human chromosome trait 12q14.1, where the phosphatidylinositol 3-kinase (PI 3-kinase) enhancer (PIKE) gene CENTG1 locates, has a strong correlation with serum lipid level and energy intake (4,5), suggesting PIKE may be a potential factor in regulating body weight.PIKEs are a family of GTPases that directly interact with PI 3-kinase and Akt and enhance their kinase activities (6–8). The family contains three members: PIKE-L, PIKE-S, and PIKE-A, which is generated from alternative splicing of the CENTG1 gene. Whereas PIKE-S and -L are brain specific, PIKE-A is widely expressed, such that its mRNA could be detected in brain, heart, liver, muscle, spleen, thymus, and small intestine (9,10). The mode of action of PIKE is isoform specific in different cell types. PIKE-L couples to receptors such as netrin receptor (UNC5B) and metabotropic glutamate receptors I (mGluR-I) and links the activated receptor to PI 3-kinase pathway in neurons (11,12). PIKE-S localizes in nucleus and executes the protective effects of nerve growth factor by activating the nuclear PI 3-kinase cascade (8). PIKE-A, on the other hand, substantiates the kinase activity of Akt in glioblastomas and is involved in cancer invasion activity (6,13,14). However, the role of PIKE-A in peripheral tissues remains unknown.In many cases, insulin resistance is the major associated pathologic condition of obesity. However, the molecular mechanism of this obesity-induced disorder remains ambiguous. It has been proposed that lipotoxicity is one of the candidates to explain the role of excess lipid storage in insulin resistance onset. Accumulation of excess cellular lipid changes the lipid metabolism, enhances oxidative stress, and disrupts endoplasmic reticulum homeostasis (15). Increasing cellular lipid oxidation by pharmacologic interventions in obese subjects thus represents a potential therapeutic regimen to mitigate their diabetic complications. In this regard, AMP-activated protein kinase (AMPK) is one of the targets. AMPK is the master sensor for energy status and is responsible for metabolic homeostasis (16). Activation of AMPK results in reducing hepatic gluconeogenic gene expression and glucose production, increasing fatty acid oxidation, and enhancing glucose uptake. Therefore, AMPK activators such as AICAR and metformin are effective agents in relieving the obesity-induced insulin resistance in both laboratory and clinical tests (17).To examine the role of PIKE in obesity, we developed the whole-body PIKE knockout (PIKE^−/−) mice with ablation of all PIKE isoforms. Here we report that PIKE-A is implicated in adipocyte differentiation and obesity development. PIKE knockout elicits lipoatrophy and increased insulin sensitivity by enhancing AMPK activity, leading to resistance against high-fat diet (HFD)-induced obesity and diabetes.     

Immune Depletion With Cellular Mobilization Imparts Immunoregulation and Reverses Autoimmune Diabetes in Nonobese Diabetic Mice

OBJECTIVE

The autoimmune destruction of β-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony–stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Effects of each therapy were tested in pre-diabetic and diabetic female NOD mice using measurements of glycemia, regulatory T-cell (CD4+CD25+Foxp3+) frequency, insulitis, and/or β-cell area.

RESULTS

Here, we show that combination therapy of murine ATG and GCSF was remarkably effective at reversing new-onset diabetes in NOD mice and more efficacious than either agent alone. This combination also afforded durable reversal from disease (>180 days postonset) in animals having pronounced hyperglycemia (i.e., up to 500 mg/dl). Additionally, glucose control improved over time in mice subject to remission from type 1 diabetes. Mechanistically, this combination therapy resulted in both immunological (increases in CD4-to-CD8 ratios and splenic regulatory T-cell frequencies) and physiological (increase in the pancreatic β-cell area, attenuation of pancreatic inflammation) benefits.

CONCLUSIONS

In addition to lending further credence to the notion that combination therapies can enhance efficacy in addressing autoimmune disease, these studies also support the concept for utilizing agents designed for other clinical applications as a means to expedite efforts involving therapeutic translation.Type 1 diabetes is characterized by the autoimmune destruction of β-cells, resulting in a loss of insulin production and glucose control (1,2). In both humans and the nonobese diabetic (NOD) mouse model of type 1 diabetes, the disorder''s pathogenesis appears dependent on aberrant immune regulation (3–6). A reversal of type 1 diabetes in NOD mice has been achieved, with varying levels of success, through administration of a limited number of immunosuppressive and immunomodulatory agents, some of which are controversial with respect to their translational capabilities (7–19).Antithymocyte globulin (ATG) is currently in clinical use for a variety of purposes, including the treatment of acute rejection, graft versus host disease, and conditioning for stem-cell transplantation (20–22). It has been shown to target >40 epitopes and serves to induce lymphocyte depletion, the extent of which depends upon the dose administered. Previously, we have shown that murine ATG is capable of late prevention of diabetes in NOD mice and, importantly, that this agent was capable of inducing a regulatory T-cell population (16). With this, we questioned whether the efficacy of this therapy could be improved through the use of a second immunomodulatory agent differing in its presumed mechanism of therapeutic activity. To that regard, we elected to evaluate granulocyte colony–stimulating factor (GCSF).GCSF was initially developed as a means of mobilizing neutrophils (23,24), but recent reports (25) have also indicated a GCSF-induced immunoregulatory impact. These studies indicated the ability of GCSF to induce an immunoregulatory shift from a T_H1 to a T_H2 cytokine phenotype (26), the induction of tolerogenic dendritic cells (27), and the mobilization of regulatory T-cells. In regards to type 1 diabetes, GCSF has successfully prevented the onset of disease in the NOD mouse via the induction of both tolerogenic dendritic and regulatory T-cells (28) and prevented the cyclophosphamide-mediated acceleration of diabetes (29).Hence, in this report, we examined the therapeutic efficacy of these two agents, ATG and GCSF, subject to clinical use in settings outside of type 1 diabetes, for the purpose of testing their ability to reverse disease in NOD mice as well as to monitor their ability to reinstill self tolerance. In this study, we also tested the hypothesis that combination therapy will be more effective than either monotherapy for the purposes of treating type 1 diabetes in NOD mice.     

Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance

The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad^−/−). In addition, the fDsbA-L/Ad^−/− mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders.Adiponectin is a 30-kDa adipokine with anti-inflammatory, anti–insulin resistance, antioxidant, and antiatherosclerotic properties (1–3). Adiponectin circulating in plasma exists in three major forms: trimer, hexamer, and high-molecular-weight (HMW) multimer (4–7). Serum adiponectin levels are significantly reduced in obese human subjects (8) and patients with insulin resistance (9), type 2 diabetes, and coronary artery disease (10). On the other hand, high plasma adiponectin levels are associated with increased insulin sensitivity (11), lowered incidence rate of type 2 diabetes independent of obesity (12), decreased risk of coronary artery disease (13), and extended longevity (14,15).Adiponectin is the only known adipokine whose levels are downregulated in obesity (8). Pharmacological studies have demonstrated that acutely enhancing the globular form of adiponectin in mice significantly increased fatty acid oxidation and reduced body weight (16,17). Transgenic overexpression of full-length adiponectin or the globular form of adiponectin has been shown to increase energy expenditure, insulin sensitivity, and fatty acid oxidation (16–20). Taken together, these results suggest that increasing serum adiponectin levels might be an attractive therapeutic approach for the treatment of obesity-induced metabolic diseases. However, the serum levels of adiponectin are extremely high, ranging between 1 and 20 μg/mL (21). Such a high concentration, which is at least three orders of magnitude higher than the levels of other adipokines, such as leptin and interleukin-6 (IL-6), results in technical difficulties in the development of adiponectin-based antidiabetic and antiatherogenic strategies.An important finding in the adiponectin research field is that complex distribution, rather than the total levels of adiponectin, is associated with improved insulin sensitivity in response to thiazolidinedione stimulation in mice and humans (22). Consistent with this finding, the HMW form of adiponectin has been demonstrated as having major biological functions in regulating glucose homeostasis (23–25). In contrast, impairment of adiponectin multimerization affects both secretion and function of this adipokine and is associated with diabetes and hypoadiponectinemia (4,6). These findings suggest that increasing the ratio of the HMW form rather than the total levels of adiponectin might provide an effective alternative therapeutic strategy.We have recently identified the disulfide bond A oxidoreductase-like protein (DsbA-L) as a key regulator of adiponectin multimerization in 3T3-L1 cells (26). In addition, we have found that overexpression of DsbA-L, the levels of which are significantly reduced in obese mice and human subjects (26), protected adiponectin from endoplasmic reticulum (ER) stress–induced downregulation in 3T3-L1 cells (27). However, whether overexpression of DsbA-L promotes adiponectin multimerization and improves insulin sensitivity in vivo remains unknown.In the current study, we show that adipose tissue–specific overexpression of DsbA-L increases adiponectin multimerization and stability in mice. The fat-specific DsbA-L transgenic mice (fDsbA-L) exhibited enhanced activity and energy expenditure and increased resistance to diet-induced obesity and insulin resistance. Our study also indicates that in addition to regulating adiponectin multimerization and function, DsbA-L has an additional beneficial effect on energy homeostasis. Taken together, our study suggests that increasing the expression levels of molecules such as DsbA-L could be an effective therapeutic approach for the treatment of obesity-induced insulin resistance and associated metabolic diseases.     

Vegfa Protects the Glomerular Microvasculature in Diabetes

Vascular endothelial growth factor A (VEGFA) expression is increased in glomeruli in the context of diabetes. Here, we tested the hypothesis that this upregulation of VEGFA protects the glomerular microvasculature in diabetes and that therefore inhibition of VEGFA will accelerate nephropathy. To determine the role of glomerular Vegfa in the development and progression of diabetic nephropathy, we used an inducible Cre-loxP gene-targeting system that enabled genetic deletion of Vegfa selectively from glomerular podocytes of wild-type or diabetic mice. Type 1 diabetes was induced in mice using streptozotocin (STZ). We then assessed the extent of glomerular dysfunction by measuring proteinuria, glomerular pathology, and glomerular cell apoptosis. Vegfa expression increased in podocytes in the STZ model of diabetes. After 7 weeks of diabetes, diabetic mice lacking Vegfa in podocytes exhibited significantly greater proteinuria with profound glomerular scarring and increased apoptosis compared with control mice with diabetes or Vegfa deletion without diabetes. Reduced local production of glomerular Vegfa in a mouse model of type 1 diabetes promotes endothelial injury accelerating the progression of glomerular injury. These results suggest that upregulation of VEGFA in diabetic kidneys protects the microvasculature from injury and that reduction of VEGFA in diabetes may be harmful.Diabetes is the leading cause of end-stage kidney failure in North America. A primary feature of diabetic nephropathy is dysfunction and injury of the microvasculature. To date, there has been significant scientific effort focused on understanding how hyperglycemia and other metabolic consequences of diabetes promote microvascular injury and end-organ damage. On the other hand, endogenous factors that slow or prevent development of complications are also likely to play a major role in shaping the manifestations of diabetic nephropathy. Vascular endothelial growth factor A (VEGFA) plays an important role in regulating glomerular structure and function and may also influence the outcome of diabetic kidney disease.VEGFA is a secreted glycoprotein of the platelet-derived growth factor superfamily required for glomerular endothelial cell migration, differentiation, and survival (1). Glomerular visceral epithelial cells, known as podocytes, are a major source of VEGFA in the kidney (2). Both up- and downregulation of podocyte Vegfa levels during kidney development lead to glomerular disease in mice, while a reduction of glomerular VEGFA both in patients treated with VEGFA inhibitors and in adult transgenic mice with the deletion of Vegfa causes renal thrombotic microangiopathy (TMA) (1,3–5). Thus, proper regulation of VEGFA expression is critical for the healthy glomerulus.In early stages of diabetes, Vegfa expression is increased in glomeruli. In rodents, both insulin deficiency and resistance are associated with increased production of renal Vegfa (6,7). This increase in VEGFA has been documented in renal biopsies and plasma from patients with type 1 or 2 diabetes (8,9), leading to the hypothesis that the increased level of VEGFA in diabetes is detrimental to glomerular function. In keeping with this model, overexpression of Vegfa in podocytes of transgenic mice is associated with features of diabetic nephropathy such as a thickened glomerular basement membrane and proteinuria (5,10,11). Studies of Vegfa inhibition in rodent models of diabetic nephropathy have generated mixed results, with some studies demonstrating protection from progression and others failing to show benefit (12–15). One drawback to inhibitor studies is potential lack of target specificity along with inability to determine the extent of inhibition in specific tissues.Accordingly, we took a genetic approach that allows us to extinguish Vegfa signaling in the glomerulus with a precision and degree not possible with small molecule or other pharmacologic agents. To determine the role of local Vegfa production in podocytes in the development and progression of diabetic nephropathy, we used the streptozotocin (STZ) model of type 1 diabetes in mice. Using this approach, we show that loss of Vegfa from podocytes has adverse consequences for the glomerular structure and function in diabetic mice, resulting in global sclerosis and death within a few weeks. Our results suggest that upregulation of VEGFA is not necessarily detrimental for the diabetic glomerulus and may serve a protective function.     

Dynamic Changes in Pancreatic Endocrine Cell Abundance, Distribution, and Function in Antigen-Induced and Spontaneous Autoimmune Diabetes

OBJECTIVE

Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by β-cell–specific killing by autoreactive T-cells. Less is known about β-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents.

RESEARCH DESIGN AND METHODS

We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell–mediated β-cell–destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse.

RESULTS

The proportion of pancreatic insulin-positive β-cells to glucagon-positive α-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe β-cell depletion accompanied by phenotypic β-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of α-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non–insulin-induced hypoglycemia.

CONCLUSIONS

Systematically applying multicolor flow cytometry and immunohistochemistry to track declining β-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet α-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub–islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.Although much has been learned about β-cell development and β-cell biology and function in vitro (using isolated pancreatic islets), studies designed to examine β-cell phenotype in vivo have suffered from technical limitations. For instance, currently available techniques to quantify pancreatic β-cell mass rely on laborious histomorphometric techniques (1) or on assumptions that β-cell mass correlates with β-cell function (stimulated C-peptide release) (2) or total pancreatic insulin content (3,4). Furthermore, quantifying the different endocrine islet cellular constituents by staining for the hormones produced (i.e., glucagon by α-cells, insulin by β-cells, somatostatin by δ-cells, and pancreatic polypeptide by PP-cells) has to date been challenging, relying again mostly on histomorphometry, and automated image processing setups typically allow only single-parameter analysis (5,6).Although preparative fluorescence-activated cell sorting of islet β-cells has been attempted (7–10), wider application has been limited by the lack of islet endocrine cell surface markers and insufficient resolution by autofluorescence, especially in species other than rats (11). Fluorescence reagents with islet granule affinity to identify β-cells in both mice (12) and humans (13) have limited utility, presumably because β-cells are degranulated by hyperglycemia. Other methods, such as quantitative (q)RT-PCR and analytical multicolor/multiparameter flow cytometry, capable of precise phenotypic and functional assessment, have been hampered by both the notorious difficulty to reliably prepare pancreatic RNA (14) and the fact that the pancreas is a heterogeneous organ comprised of irregularly intermixed exocrine and endocrine tissues. Even so-called “purified” isolated pancreatic islets from naïve mice (or other mammals) represent a multitude of specialized cell types (15), which is further complicated in animals with autoimmune diabetes when abundant inflammatory cells invade the islets (16).Recognizing these limitations, we adapted flow cytometry techniques for pancreatic studies and, together with qRT-PCR, confocal immunofluorescence microscopy, and quantitative immunohistochemistry, characterized the pancreatic endocrine islet cell components in naïve and recently diabetic mice. We now report that pancreatic islets isolated from mice developing T-cell–mediated β-cell–specific autoimmune diabetes demonstrate an unexpected glucagon-positive α-cell loss roughly commensurate with the expected β-cell loss.     

TM4SF5 Knockout Protects Mice From Diet-Induced Obesity Partly by Regulating Autophagy in Adipose Tissue

Transmembrane 4 L six family member 5 (TM4SF5) functions as a sensor for lysosomal arginine levels and activates the mammalian target of rapamycin complex 1 (mTORC1). While the mTORC1 signaling pathway plays a key role in adipose tissue metabolism, the regulatory function of TM4SF5 in adipocytes remains unclear. In this study we aimed to establish a TM4SF5 knockout (KO) mouse model and investigated the effects of TM4SF5 KO on mTORC1 signaling–mediated autophagy and mitochondrial metabolism in adipose tissue. TM4SF5 expression was higher in inguinal white adipose tissue (iWAT) than in brown adipose tissue and significantly upregulated by a high-fat diet (HFD). TM4SF5 KO reduced mTORC1 activation and enhanced autophagy and lipolysis in adipocytes. RNA sequencing analysis of TM4SF5 KO mouse iWAT showed that the expression of genes involved in peroxisome proliferator–activated receptor α signaling pathways and mitochondrial oxidative metabolism was upregulated. Consequently, TM4SF5 KO reduced adiposity and increased energy expenditure and mitochondrial oxidative metabolism. TM4SF5 KO prevented HFD-induced glucose intolerance and inflammation in adipose tissue. Collectively, the results of our study demonstrate that TM4SF5 regulates autophagy and lipid catabolism in adipose tissue and suggest that TM4SF5 could be therapeutically targeted for the treatment of obesity-related metabolic diseases.     

Management of Latent Autoimmune Diabetes in Adults: A Consensus Statement From an International Expert Panel

A substantial proportion of patients with adult-onset diabetes share features of both type 1 diabetes (T1D) and type 2 diabetes (T2D). These individuals, at diagnosis, clinically resemble T2D patients by not requiring insulin treatment, yet they have immunogenetic markers associated with T1D. Such a slowly evolving form of autoimmune diabetes, described as latent autoimmune diabetes of adults (LADA), accounts for 2–12% of all patients with adult-onset diabetes, though they show considerable variability according to their demographics and mode of ascertainment. While therapeutic strategies aim for metabolic control and preservation of residual insulin secretory capacity, endotype heterogeneity within LADA implies a personalized approach to treatment. Faced with a paucity of large-scale clinical trials in LADA, an expert panel reviewed data and delineated one therapeutic approach. Building on the 2020 American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD) consensus for T2D and heterogeneity within autoimmune diabetes, we propose “deviations” for LADA from those guidelines. Within LADA, C-peptide values, proxy for β-cell function, drive therapeutic decisions. Three broad categories of random C-peptide levels were introduced by the panel: 1) C-peptide levels <0.3 nmol/L: a multiple-insulin regimen recommended as for T1D; 2) C-peptide values ≥0.3 and ≤0.7 nmol/L: defined by the panel as a “gray area” in which a modified ADA/EASD algorithm for T2D is recommended; consider insulin in combination with other therapies to modulate β-cell failure and limit diabetic complications; 3) C-peptide values >0.7 nmol/L: suggests a modified ADA/EASD algorithm as for T2D but allowing for the potentially progressive nature of LADA by monitoring C-peptide to adjust treatment. The panel concluded by advising general screening for LADA in newly diagnosed non–insulin-requiring diabetes and, importantly, that large randomized clinical trials are warranted.     

Sclerostin Protects Against Vascular Calcification Development in Mice

Sclerostin is a negative regulator of the Wnt/β-catenin signaling and is, therefore, an important inhibitor of bone formation and turnover. Because ectopic vascular calcification develops in a similar way to bone formation, one might reasonably attribute a role to sclerostin in this pathological process. Ectopic calcification, especially vascular calcification, importantly contributes to mortality in elderly and patients with diabetes, osteoporosis, chronic kidney disease (CKD), and hypertension. The central players in this ectopic calcification process are the vascular smooth muscle cells that undergo dedifferentiation and thereby acquire characteristics of bonelike cells. Therefore, we hypothesize that depletion/deactivation of the Wnt/β-catenin signaling inhibitor sclerostin may promote the development of ectopic calcifications through stimulation of bone-anabolic effects at the level of the arteries. We investigated the role of sclerostin (encoded by the Sost gene) during vascular calcification by using either Sost^−/− mice or anti-sclerostin antibody. Sost^−/− and wild-type (WT) mice (C57BL/6J background) were administered an adenine-containing diet to promote the development of CKD-induced vascular calcification. Calcifications developed more extensively in the cardiac vessels of adenine-exposed Sost^−/− mice, compared to adenine-exposed WT mice. This could be concluded from the cardiac calcium content as well as from cardiac tissue sections on which calcifications were visualized histochemically. In a second experiment, DBA/2J mice were administered a warfarin-containing diet to induce vascular calcifications in the absence of CKD. Here, warfarin exposure led to significantly increased aortic and renal tissue calcium content. Calcifications, which were present in the aortic medial layer and renal vessels, were significantly more pronounced when warfarin treatment was combined with anti-sclerostin antibody treatment. This study demonstrates a protective effect of sclerostin during vascular calcification. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Costimulation and Autoimmune Diabetes in BB Rats

Costimulatory signals regulate T-cell activation. To investigate the role of costimulation in autoimmunity and transplantation, we studied the BB rat model of type 1 diabetes. Diabetes-prone BB (BBDP) rats spontaneously develop disease when 55–120 days of age. We observed that two anti-CD28 monoclonal antibodies (mAb) with different functional activities completely prevented diabetes in BBDP rats. Anti-CD154 mAb delayed diabetes, whereas treatment with CTLA4-Ig or anti-CD80 mAb accelerated disease. Anti-CD86 or anti-CD134L mAbs had no effect. Diabetes resistant BB (BBDR) rats are disease-free, but >95% of them develop diabetes after treatment with polyinosinic-polycytidylic acid and an mAb that depletes Treg cells. In the induced BBDR model, anti-CD154 mAb delayed onset of diabetes, whereas CTLA4-Ig, anti-CD134L or either of the anti-CD28 mAbs had little or no effect. In contrast, blockade of the CD134-CD134L pathway was highly effective for preventing autoimmune recurrence against syngeneic islet grafts in diabetic BBDR hosts. Blockade of the CD40-CD154 pathway was also effective, but less so. These data suggest that the effectiveness of costimulation blockade in the treatment of type 1 diabetes is dependent on both the costimulatory pathway targeted and the mechanism of induction, stage, intensity and duration of the pathogenic process.     

Redox Modulation Protects Islets From Transplant-Related Injury

OBJECTIVE

Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant.

RESEARCH DESIGN AND METHODS

The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation.

RESULTS

Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection.

CONCLUSIONS

Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.Hypoxia is the leading cause of β-cell death during islet isolation and transplantation (1), with the highest percentage of islet graft loss and dysfunction occurring just days after transplantation (2,3). Because islets are a cellular transplant, devoid of intrinsic vasculature (1,4), they are exceptionally susceptible to ischemia-reperfusion injury. Islets are also increasingly vulnerable because they have inherently decreased antioxidant capacity (5–10), making them prone to oxidative/nitrosative/free radical damage. The antigen-independent complexities of islet transplantation increase the incidence of primary graft nonfunction and β-cell death, thus requiring protection for islets at early stages of the transplant procedure (11).In addition to antigen-independent innate-mediated inflammatory injury, islet allografts are also plagued by the antigen-dependent T-cell mediated alloimmune response, which necessitates immunosuppressive drugs for allograft survival. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response, although most are harmful to islets and do not protect well from reactive oxygen species and inflammation during islet isolation and ischemia-reperfusion injury (12–14). In their review, Balamurugan et al. (13) concluded that successful islet transplantation in type 1 diabetes necessitates islet-sparing immunosuppressive agents that combat recurrent autoimmunity with low islet toxicity. Predominantly, the field of islet transplantation is devoid of cytoprotective agents that promote islet survival and function by inhibiting nonspecific innate-mediated inflammation during islet isolation and early inflammatory events in islet transplantation (11,13,15–19).The first phase of immunity involves innate immune activation and subsequent proinflammatory signals required for optimal adaptive immune function (20–22), yet the majority of immunosuppressive drugs only target adaptive immune function (17,23), the second phase of immunity. A nontoxic, cell-permeable catalytic antioxidant (CA) redox modulator, FBC-007 [manganese(II) tetrakis (N-ethylpyridium-2-yl)porphyrin], is able to depress free radical and cytokine production by antigen-presenting cells (24) and T cells in transgenic and allospecific mouse models (20,25). Additionally, redox modulation inhibits cytotoxic lymphocyte target cell lysis by reducing the production of intracellular cytolytic molecules (perforin and granzyme B) in a mixed leukocyte reaction without toxicity (25), preserves and promotes human islet function in vitro (15,16), prevents the transfer of diabetes into young NOD.scid mice (26), and inhibits innate-immune nuclear factor (NF)-κB activation (24). Thus, islet-sparing agents, which decrease the production of free radicals and, therefore, inflammatory cytokines, may have a positive impact on islet function post-transplant.Because islet transplantation can benefit from agents that inhibit early inflammatory cascades to preserve islet function (18), we hypothesize that redox modulation holds potential as a therapy in islet transplantation to decrease the incidence of β-cell primary nonfunction. To further test the effects of redox modulation using CA we treated human islets with streptozotocin (STZ) in vitro and treated mice in vivo with STZ, both in the presence or absence of CA, to mimic antigen-independent free radical damage and inflammation of post-transplant ischemia-reperfusion injury. To examine the effects of islet-directed CA treatment on innate-mediated (antigen-independent) primary islet nonfunction in vivo, we performed syngeneic (175 islets/recipient), suboptimal syngeneic (100 islets/recipient), allogeneic (300 islets/recipient), and xenogeneic (400–500 islets/recipient) islet transplants to assess islet function. Additionally, we performed (300 islets/recipient) islet transplants in diabetic recipients to assess islet function in the presence or absence of systemic redox modulation in an allogeneic transplant setting inclusive of both innate (antigen-independent) and adaptive (antigen-dependent) immune responses. Our results demonstrate that islet-directed and systemically delivered redox modulation, administered in the absence of an additional immunosuppressive regimen, preserve islet function post-transplant.     

Infection With Viruses From Several Families Triggers Autoimmune Diabetes in LEW.1WR1 Rats: Prevention of Diabetes by Maternal Immunization

OBJECTIVE

The contribution of antecedent viral infection to the development of type 1 diabetes in humans is controversial. Using a newer rat model of the disease, we sought to 1) identify viruses capable of modulating diabetes penetrance, 2) identify conditions that increase or decrease the diabetogenicity of infection, and 3) determine whether maternal immunization would prevent diabetes.

RESEARCH DESIGN AND METHODS

About 2% of LEW.1WR1 rats develop spontaneous autoimmune diabetes, but disease penetrance is much higher if weanling rats are exposed to environmental perturbants including Kilham rat virus (KRV). We compared KRV with other viruses for diabetogenic activity.

RESULTS

Both KRV and rat cytomegalovirus (RCMV) induced diabetes in up to 60% of LEW.1WR1 rats, whereas H-1, vaccinia, and Coxsackie B4 viruses did not. Simultaneous inoculation of KRV and RCMV induced diabetes in 100% of animals. Pretreatment of rats with an activator of innate immunity increased the diabetogenicity of KRV but not RCMV and was associated with a moderate rate of diabetes after Coxsackie B4 and vaccinia virus infection. Inoculation of LEW.1WR1 dams with both KRV and RCMV prior to pregnancy protected weanling progeny from virus-induced diabetes in a virus-specific manner.

CONCLUSIONS

Exposure to viruses can affect the penetrance of autoimmune diabetes in genetically susceptible animals. The diabetogenicity of infection is virus specific and is modified by immunomodulation prior to inoculation. Maternal immunization protects weanlings from virus-induced diabetes, suggesting that modification of immune responses to infection could provide a means of preventing islet autoimmunity.Type 1 diabetes results from inflammatory infiltration of pancreatic islets (insulitis), leading to destruction of insulin-producing β-cells (1). Much evidence suggests that the disease is caused by nongenetic environmental factors operating in a genetically susceptible host (2). Environmental factors thought most likely to modulate its pathogenesis include toxins, vaccination, diet, and infection.An association between type 1 diabetes and viral infection was first noted in epidemiological studies (3) and continues to attract attention (4–6). Viruses have been invoked to explain the increasing prevalence of diabetes (7), seasonal variation in onset (8), and enhanced susceptibility of transmigratory populations (9). Viruses associated with human diabetes include measles, congenital rubella, mumps, and influenza B (10–13). Coxsackie B virus RNA sequences have been found in the blood of patients early in the disease (14), and Coxsackie antigens can be recovered from children with recent diabetes onset (15). Human cytomegalovirus (HCMV) major DNA-binding protein encodes a peptide that stimulates clonal CD4⁺ T-cells that recognize the human autoantigen GAD (16). In addition, a molecular hybridization study of lymphocytes using a HCMV-specific probe found evidence of viral genome in 22% of diabetic patients but only 2.6% of control subjects (17).There are, however, no mechanistic data that link human diabetes with infection, and, on the contrary, it is plausibly argued that virus infections may prevent the disease (4,18). The “hygiene hypothesis” posits an inverse correlation between viral exposure and the prevalence of both autoimmunity and allergy (19). The role of viruses in diabetes induction has been studied in nonobese diabetic (NOD) mice, but in this model infections are associated with disease prevention. Coxsackie B virus accelerates diabetes in NOD mice but only after autoimmunity has already been initiated (20); it is also associated with exocrine pancreatitis (21), which is not a feature of human type 1 diabetes. Other studies have used transgenic mice that express viral antigen on β-cells (18), but these are inherently artificial.In rats with the high-risk major histocompatibility complex (MHC) class II RT1B/Du haplotype infection with Kilham rat parvovirus (KRV) can lead to autoimmune diabetes (22). KRV in rats has been advocated as a model for studying the problem of viral triggering (23). Our objective was to investigate rat models as platforms for evaluating the role(s) of virus infection in autoimmunity. We report that in addition to KRV, infection with rat CMV (RCMV) leads to diabetes in LEW.1WR1 rats, only 2% of which develop spontaneous disease (24). The diabetogenicity of infection is shown to be dependent on age and the preexisting state of the immune system, and innate immune activation renders Coxsackie B4 and vaccinia viruses diabetogenic. Finally, we demonstrate that inoculation of LEW.1WR1 dams with diabetogenic viruses prevents induction of diabetes in weanling progeny. Protection is both complete and virus specific.     

Genetic Deficiency of Itgb2 or ItgaL Prevents Autoimmune Diabetes Through Distinctly Different Mechanisms in NOD/LtJ Mice

OBJECTIVE

Insulitis is an important pathological feature of autoimmune diabetes; however, mechanisms governing the recruitment of diabetogenic T-cells into pancreatic islets are poorly understood. Here, we determined the importance of leukocyte integrins β₂(Itgb2) and αL (ItgaL) in developing insulitis and frank diabetes.

RESEARCH DESIGN AND METHODS

Gene-targeted mutations of either Itgb2 or ItgaL were established on the NOD/LtJ mouse strain. Experiments were performed to measure insulitis and diabetes development. Studies were also performed measuring mutant T-cell adhesion to islet microvascular endothelial cells under hydrodynamic flow conditions. T-cell adhesion molecule profiles and adoptive transfer studies were also performed.

RESULTS

Genetic deficiency of either Itgb2 or ItgaL completely prevented the development of hyperglycemia and frank diabetes in NOD mice. Loss of Itgb2 or ItgaL prevented insulitis with Itgb2 deficiency conferring complete protection. In vitro hydrodynamic flow adhesion studies also showed that loss of Itgb2 completely abrogated T-cell adhesion. However, ItgaL deficiency did not alter NOD T-cell adhesion to or transmigration across islet endothelial cells. Adoptive transfer of ItgaL-deficient splenocytes into NOD/Rag-1 mice did not result in development of diabetes, suggesting a role for ItgaL in NOD/LtJ T-cell activation.

CONCLUSIONS

Together, these data demonstrate that genetic deficiency of Itgb2 or ItgaL confers protection against autoimmune diabetes through distinctly different mechanisms.Autoimmune diabetes results from insulitis, the infiltration of T-cells into the pancreatic islets, leading to significant β-cell death. This instigates dysregulation of blood glucose levels, which has both genetic and environmental causes. The impetus for T-cell accumulation within pancreatic islets has been debated; however, activation of autoreactive CD4⁺Th1 (type 1 T-helper) cells (1,2), defective regulatory T-cell activity (3), local production of chemokines (4,5), and increased adhesion molecule expression are prime culprits (6–9). Although many different types of leukocytes are involved in insulitis, i.e. CD4⁺and CD8⁺, T-cells are essential in the pathogenesis of diabetes and islet damage. Moreover, the specific molecules and pathways regulating T-cell recruitment into pancreatic islets remain largely unknown.Regulation of T-cell recruitment is controlled by a host of cellular events within the microcirculation involving rolling, cellular activation, firm adhesion, and subsequent transmigration across endothelial cell monolayers. Several different adhesion molecules expressed by both T-cells and endothelial cells regulate these interactions with selectin proteins primarily governing rolling and integrin proteins regulating firm adhesion and transmigration (10,11). The multiple overlapping nature of these molecules helps to enable immune responses; however, certain adhesion molecules may play a more dominant role in the process, thereby controlling immune cell recruitment. Two classes of integrins, β₁(Itgb1) and β₂(Itgb2), have been identified in regulating T-cell adhesion and homing to various organs and may be important for homing of diabetogenic T-cells (12–15). Importantly, both clinical and animal studies demonstrate that several members of these integrin protein families are upregulated or differentially engaged during autoimmune diabetes (16–19). However, no specific information exists regarding which of the integrin proteins are critically necessary for development of autoimmune diabetes or the mechanism by which this occurs.The Itgb2 protein family is exclusively expressed on leukocytes and forms heterodimeric molecules consisting of a common Itgb2 chain paired with a specific α chain of either αL (ItgaL), αM (ItgaM), αX (ItgaX), or αD (ItgaD). We previously reported that the Itgb2 chain is important for the development of murine autoimmune lupus and streptozotocin-induced diabetes (12,13). However, the importance of various heterodimer α chains and the molecular disease mechanisms involving Itgb2 proteins during the development of autoimmune diabetes is not known. Therefore, we generated NOD/LtJ mice containing gene-targeted null deletions of Itgb2 or ItgaL to determine whether these molecules play a role in the development of spontaneous autoimmune diabetes. Here, we report that genetic deficiency of either Itgb2 or ItgaL confers protection against the development of frank diabetes and insulitis in NOD mice. However, the protective mechanisms resulting from the loss of these proteins are distinctly different from one another. These findings also demonstrate that leukocyte Itgb2 predominate over other adhesion molecules during the development of diabetes, and they identify key molecular targets for possible therapeutic intervention.