Txnip balances metabolic and growth signaling via PTEN disulfide reduction

Thioredoxin-interacting protein (Txnip) inhibits thioredoxin NADPH-dependent reduction of protein disulfides. Total Txnip knockout (TKO) mice adapted inappropriately to prolonged fasting by shifting fuel dependence of skeletal muscle and heart from fat and ketone bodies to glucose. TKO mice exhibited increased Akt signaling, insulin sensitivity, and glycolysis in oxidative tissues (skeletal muscle and hearts) but not in lipogenic tissues (liver and adipose tissue). The selective activation of Akt in skeletal muscle and hearts was associated with impaired mitochondrial fuel oxidation and the accumulation of oxidized (inactive) PTEN, whose activity depends on reduction of two critical cysteine residues. Whereas muscle- and heart-specific Txnip knockout mice recapitulated the metabolic phenotype exhibited by TKO mice, liver-specific Txnip knockout mice were similar to WT mice. Embryonic fibroblasts derived from knockout mice also accumulated oxidized (inactive) PTEN and had elevated Akt phosphorylation. In addition, they had faster growth rates and increased dependence on anaerobic glycolysis due to impaired mitochondrial fuel oxidation, and they were resistant to doxorubicin-facilitated respiration-dependent apoptosis. In the absence of Txnip, oxidative inactivation of PTEN and subsequent activation of Akt attenuated mitochondrial respiration, resulting in the accumulation of NADH, a competitive inhibitor of thioredoxin NADPH-reductive activation of PTEN. These findings indicate that, in nonlipogenic tissues, Txnip is required to maintain sufficient thioredoxin NADPH activity to reductively reactivate oxidized PTEN and oppose Akt downstream signaling.     

Regulatory T cells control diabetes without compromising acute anti-viral defense

While previous reports have demonstrated the efficacy of regulatory T cell therapy in the prevention of diabetes, systemic immunocompromise and Treg instability remain key safety concerns. Here we examined the influence of induced Treg (iTreg) cell therapy on anti-viral host defense and autoimmune T cell responses during acute viral infection in a murine model of autoimmune diabetes. Protective transfers of iTregs maintained IL-10 expression, expanded in vivo and controlled diabetes, despite losing FoxP3 expression. Adoptive transfer of iTregs affected neither the primary anti-viral CD8 T cell response nor viral clearance, although a significant and sustained suppression of CD4 T cell responses was observed. Following acute viral clearance, iTregs transferred early suppressed both CD4 and CD8 T cell responses, which resulted in the reversion of diabetes. These observations indicate that iTregs suppress local autoimmune processes while preserving the immunocompetent host's ability to combat acute viral infection.     

Multicomponent Plasmid Protects Mice From Spontaneous Autoimmune Diabetes

Type 1 diabetes is an autoimmune disease in which insulin-secreting β-cells are destroyed, leading to a lifelong dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immunomodulatory cytokines (transforming growth factor-β1, interleukin [IL]-10, and IL-2) capable of near-complete prevention of autoimmune diabetes in nonobese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. In preparation for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This preclinical study demonstrates that this multicomponent, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk for developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (clinical trial reg. no. {"type":"clinical-trial","attrs":{"text":"NCT04279613","term_id":"NCT04279613"}}NCT04279613, ClinicalTrials.gov).     

Combination Therapy With an Anti–IL-1β Antibody and GAD65 DNA Vaccine Can Reverse Recent-Onset Diabetes in the RIP-GP Mouse Model

Type 1 diabetes is thought to be an autoimmune condition in which self-reactive T cells attack insulin-secreting pancreatic β-cells. As a proinflammatory cytokine produced by β-cells or macrophages, interleukin-1β (IL-1β) represents a potential therapeutic target in diabetes. We reasoned IL-1β blockade could be combined with islet antigen–specific approaches involving GAD of 65 kDa (GAD65)-expressing plasmids, as previously shown in combination therapies (CTs) with anti-CD3. Thus, we investigated whether anti–IL-1β antibody alone or combined with GAD65 vaccine could reverse diabetes development in a virus-induced mouse model. Given alone, anti–IL-1β had no effect on diabetes, while GAD65 plasmid resulted in 33% disease reversal after a 5-week observation. However, CTs cured 53% of animals and prevented worsening of glycemic control in nonprotected individuals for up to 12 weeks. While the GAD65 vaccine arm of the CT was associated with increased forkhead box p3⁺ regulatory T-cell frequency in pancreatic lymph nodes, islet infiltration by CD11b^+/high cells was less frequent upon CT, and its extent correlated with treatment success or failure. Altogether, our CTs provided prolonged improvement of clinical and immunological features. Despite unsuccessful clinical trials using anti–IL-1β monotherapy, these data hold promise for treatment of type 1 diabetic patients with IL-1β blockade combined with antigen-specific vaccines.Inflammation of the pancreatic islets is observed in patients with type 1 and type 2 diabetes (1–3). Islets are sensitive to proinflammatory cytokines and the combination of interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-1β (IL-1β) can act synergistically to lead to β-cell demise (4–6). IL-1β can be produced by cells such as monocytes, macrophages, dendritic cells, or neurons (1,7). In β-cells, IL-1β secretion in conditions of high glucose concentrations can have cytotoxic effects such as altered insulin secretion and β-cell apoptosis (8–10). Therefore, IL-1β blockade as a means to prevent or reverse type 1 and type 2 diabetes onset has become a potential therapeutic target.In type 2 diabetes, neutralization of IL-1β signaling has resulted in improved glycemic control and insulin secretion in murine (11) and human studies (12,13). Microarray analyses have shown that compared with healthy control subjects, IL-1β gene expression was increased threefold in peripheral blood mononuclear cells (PBMCs) from both type 1 and type 2 recent-onset diabetic patients but significantly declined upon a 4-month course of insulin therapy and adjusted glycemic control (14). In type 1 diabetic patients, Pfleger et al. (15) found a positive association at 1 and 6 months postdiagnosis between levels of C-peptide and that of IL-1 receptor antagonist (IL-1Ra), the natural antagonist to proinflammatory cytokines IL-1α/β and the footprint of earlier IL-1 action. Moreover, short-term neutralization of IL-1β resulted in reduced chemokine receptor expression on CD11b⁺ circulating monocytes (16) in people with type 1 diabetes. More recently, two randomized, placebo-controlled phase 2a trials both enrolling 69 type 1 diabetic patients have evaluated whether IL-1 blockade could improve β-cell function in recent-onset type 1 diabetes (17). One used a human monoclonal anti–IL-1β antibody (canakinumab), whereas the other used a recombinant human IL-1Ra (anakinra). Despite no severe safety issues, neither trial met its primary or secondary end points. Thus, despite good scientific rationale, the effect of IL-1β blockade alone in overt type 1 diabetes is, if any, minor, and combination with other agents will be needed. Antigen-specific therapies, due to their lack of inducing systemic side effects, are preferable to using additional systemic immunosuppressants.Along these lines, while IL-1R–deficient NOD mice display slowed progression to diabetes but normal incidence (18), Ablamunits et al. (19) have recently shown synergistic reversal of type 1 diabetes after combined anti–IL-1β (using blocking antibody or IL-1Ra) and anti-CD3 antibody treatments. Furthermore, preclinical studies in our laboratory also demonstrated synergistic reversal of type 1 diabetes using similar anti-CD3 antibody combined with islet autoantigen-specific approaches to induce and maintain tolerance to β-cell antigens (20–22). Oral and nasal (pro)insulin were proved efficacious as part of the combination therapy (CT) in the NOD model (20,21), whereas promoter cytomegalovirus (pCMV)-encoded human glutamic acid decarboxylase of 65 kDa (pCMV-human GAD65 [hGAD65]) DNA plasmid was efficacious in the rat insulin promoter (RIP)–lymphocytic choriomeningitis virus (LCMV)–glycoprotein (GP) model (22). RIP-LCMV-GP mice express the GP from LCMV as a transgene under the RIP and turn diabetic within 2 weeks post–LCMV infection (23). Other plasmid DNA vaccines encoding mouse proinsulin II or GAD65–immunoglobulin Fc and IL-4 have also been shown to prevent and/or reverse hyperglycemia (HG) in NOD mice (24,25).In this study, we investigated whether an IL-1β–neutralizing antibody alone or combined with an islet antigen GAD65-specific vaccine could reverse recent-onset diabetes development in the RIP-GP model. For this, we chose two main time points for our studies: 1) 5 weeks posttreatment (i.e., 1 week after the last administration of anti–IL-1β antibody); and 2) 12 weeks posttreatment (i.e., up to 2 months after the treatments have been stopped).