Lindsey E. Padgett

The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease characterized by the destruction of insulin‐secreting pancreatic β cells. In humans with T1D and in nonobese diabetic (NOD) mice (a murine model for human T1D), autoreactive T cells cause β‐cell destruction, as transfer or deletion of these cells induces or prevents disease, respectively. CD4+ and CD8+ T cells use distinct effector mechanisms and act at different stages throughout T1D to fuel pancreatic β‐cell destruction and disease pathogenesis. While these adaptive immune cells employ distinct mechanisms for β‐cell destruction, one central means for enhancing their autoreactivity is by the secretion of proinflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1. In addition to their production by diabetogenic T cells, proinflammatory cytokines are induced by reactive oxygen species (ROS) via redox‐dependent signaling pathways. Highly reactive molecules, proinflammatory cytokines are produced upon lymphocyte infiltration into pancreatic islets and induce disease pathogenicity by directly killing β cells, which characteristically possess low levels of antioxidant defense enzymes. In addition to β‐cell destruction, proinflammatory cytokines are necessary for efficient adaptive immune maturation, and in the context of T1D they exacerbate autoimmunity by intensifying adaptive immune responses. The first half of this review discusses the mechanisms by which autoreactive T cells induce T1D pathogenesis and the importance of ROS for efficient adaptive immune activation, which, in the context of T1D, exacerbates autoimmunity. The second half provides a comprehensive and detailed analysis of (1) the mechanisms by which cytokines such as IL‐1 and IFN‐γ influence islet insulin secretion and apoptosis and (2) the key free radicals and transcription factors that control these processes.

Superoxide Production by Macrophages and T Cells Is Critical for the Induction of Autoreactivity and Type 1 Diabetes

OBJECTIVE The role of reactive oxygen species (ROS) and their dissipation in type 1 diabetes pathogenesis have garnered considerable controversy. Our recent work has demonstrated the importance of NADPH oxidase (NOX) activity for type 1 diabetes development and modulating T-cell autoreactivity. We previously linked decreased monocyte ROS with diabetes resistance in the alloxan-resistant mouse, and NOD-Ncf1m1J mice with a genetic ablation of NOX activity had reduced and delayed type 1 diabetes compared with NOD mice. RESEARCH DESIGN AND METHODS To determine the required cellular sources of ROS that are necessary for type 1 diabetes initiation, we used antibody depletion and adoptive transfer experiments into NOD and NOD-Scid females, respectively. After receiving treatment, female mice were monitored for hyperglycemia and overt diabetes. RESULTS Depletion of macrophages and neutrophils fully protected NOD mice from type 1 diabetes. However, elimination of neutrophils alone showed no significant reduction or delay. Type 1 diabetes induction in NOD-Scid mice by adoptive transfer with NOD-Ncf1m1J splenocytes was significantly delayed compared with NOD splenocytes, suggesting macrophage ROS and modulation of effector responses are critical for diabetes. The adaptive immune response was also altered by the absence of NOX activity, as purified T cells from NOD-Ncf1m1J mice exhibited delayed transfer kinetics. Cotransfer experiments demonstrated the defect was intrinsic to NOX-deficient CD8+ T cells. After stimulation, cytotoxic T cells exhibited decreased effector function in the absence of superoxide production. CONCLUSIONS These data demonstrate that the impaired autoreactive response of NOX-deficient NOD-Ncf1m1J immune system results from an alteration in the antigen-presenting cell–T-cell axis rather than failure of neutrophils to act as effector cells and that ROS signaling is important for the initiation of β-cell–directed autoimmunity by T cells.

Loss of NADPH Oxidase-Derived Superoxide Skews Macrophage Phenotypes to Delay Type 1 Diabetes

Macrophages are early islet-infiltrating cells seen in type 1 diabetes (T1D). While proinflammatory M1 macrophages induce T1D, M2 macrophages have been shown to delay this autoimmune disease in nonobese diabetic (NOD) mice, but the environmental cues that govern macrophage polarization and differentiation remain unresolved. We previously demonstrated the importance of reactive oxygen species (ROS) in T1D, as NOD mice deficient in NADPH oxidase (NOX)-derived superoxide (Ncf1m1J) were protected against T1D partly because of blunted Toll-like receptor–dependent macrophage responses. We provide evidence that NOX-derived ROS contribute to macrophage differentiation in T1D. During spontaneous diabetes progression, T1D-resistant NOD.Ncf1m1J islet-resident macrophages displayed a dampened M1 and increased M2 phenotype. The transfer of diabetogenic T cells into NOX-deficient NOD.Rag.Ncf1m1J recipients resulted in decreased TNF-α+ and IL-1β+ islet-infiltrating M1 macrophages and a concomitant enhancement in arginase-1+ M2 macrophages. Mechanistic analysis of superoxide-deficient bone marrow–derived macrophages revealed a marked diminution in a proinflammatory M1 phenotype due to decreased P-STAT1 (Y701) and interferon regulatory factor 5 compared with NOD mice. We have therefore defined a novel mechanistic link between NOX-derived ROS and macrophage phenotypes, and implicated superoxide as an important factor in macrophage differentiation. Thus, targeting macrophage redox status may represent a promising therapy in halting human T1D.

Hydrogen-Bonded Multilayers of Tannic Acid as Mediators of T-Cell Immunity

Type 1 diabetes is an autoimmune-mediated disease resulting in the destruction of insulin-secreting pancreatic β-cells. Transplantation of insulin-producing islets is a viable treatment to restore euglycemia in Type 1 diabetics; however, the clinical application remains limited due to the use of toxic immunosuppressive therapies to prevent immune-mediated rejection. A nanothin polymer material with dual antioxidant and immunosuppressive properties capable of modulating both innate and adaptive immune responses crucial for transplantation outcome is presented. Through the use of hollow microparticles (capsules) composed of hydrogen-bonded multilayers of natural polyphenol (tannic acid) with poly(N-vinylpyrrolidone) (TA/PVPON) and with poly(N-vinylcaprolactam) (TA/PVCL), proinflammatory reactive oxygen and nitrogen species are efficiently dissipated and the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α proinflammatory cytokines is attenuated by cognate antigen-stimulated autoreactive CD4+ T cells. These results provide evidence that TA-containing capsules are efficacious in immunomodulation and may provide physical transplant protection and prevent diabetogenic autoreactive T-cell responses. Future studies will determine if xeno- and allotransplantation with (TA/PVPON)- or (TA/PVCL)-coated pancreatic islets will decrease the risk of graft rejection due to attenuation of oxidative stress and IFN-γ, and restore euglycemia in Type 1 diabetics.

NADPH Oxidase–Derived Superoxide Provides a Third Signal for CD4 T Cell Effector Responses

Originally recognized for their direct induced toxicity as a component of the innate immune response, reactive oxygen species (ROS) can profoundly modulate T cell adaptive immune responses. Efficient T cell activation requires: signal 1, consisting of an antigenic peptide–MHC complex binding with the TCR; signal 2, the interaction of costimulatory molecules on T cells and APCs; and signal 3, the generation of innate immune-derived ROS and proinflammatory cytokines. This third signal, in particular, has proven essential in generating productive and long-lasting immune responses. Our laboratory previously demonstrated profound Ag-specific hyporesponsiveness in the absence of NADPH oxidase–derived superoxide. To further examine the consequences of ROS deficiency on Ag-specific T cell responses, our laboratory generated the OT-II.Ncf1m1J mouse, possessing superoxide-deficient T cells recognizing the nominal Ag OVA323–339. In this study, we demonstrate that OT-II.Ncf1m1J CD4 T cells displayed a severe reduction in Th1 T cell responses, in addition to blunted IL-12R expression and severely attenuated proinflammatory chemokine ligands. Conversely, IFN-γ synthesis and IL-12R synthesis were rescued by the addition of exogenous superoxide via the paramagnetic superoxide donor potassium dioxide or superoxide-sufficient dendritic cells. Ultimately, these data highlight the importance of NADPH oxidase–derived ROS in providing a third signal for adaptive immune maturation by modulating the IL-12/IL-12R pathway and the novelty of the OT-II.Ncf1m1J mouse model to determine the role of redox-dependent signaling on effector responses. Thus, targeting ROS represents a promising therapeutic strategy in dampening Ag-specific T cell responses and T cell–mediated autoimmune diseases, such as type 1 diabetes.

Loss of NOX-derived superoxide exacerbates diabetogenic CD4 T cell effector responses in Type 1 Diabetes

Reactive oxygen species (ROS) play prominent roles in numerous biological systems. While classically expressed by neutrophils and macrophages, CD4 T cells also express NADPH oxidase (NOX), the superoxide-generating multisubunit enzyme. Our laboratory recently demonstrated that superoxide-deficient nonobese diabetic (NOD.Ncf1m1J) mice exhibited a delay in type 1 diabetes (T1D) partially due to blunted IFN-γ synthesis by CD4 T cells. For further investigation of the roles of superoxide on CD4 T-cell diabetogenicity, the NOD.BDC-2.5.Ncf1m1J (BDC-2.5.Ncf1m1J) mouse strain was generated, possessing autoreactive CD4 T cells deficient in NOX-derived superoxide. Unlike NOD.Ncf1m1J, stimulated BDC-2.5.Ncf1m1J CD4 T cells and splenocytes displayed elevated synthesis of Th1 cytokines and chemokines. Superoxide-deficient BDC-2.5 mice developed spontaneous T1D, and CD4 T cells were more diabetogenic upon adoptive transfer into NOD.Rag recipients due to a skewing toward impaired Treg suppression. Exogenous superoxide blunted exacerbated Th1 cytokines and proinflammatory chemokines to approximately wild-type levels, concomitant with reduced IL-12Rβ2 signaling and P-STAT4 (Y693) activation. These results highlight the importance of NOX-derived superoxide in curbing autoreactivity due, in part, to control of Treg function and as a redox-dependent checkpoint of effector T-cell responses. Ultimately, our studies reveal the complexities of free radicals in CD4 T-cell responses.

Superoxide Production by NADPH Oxidase Intensifies Macrophage Antiviral Responses during Diabetogenic Coxsackievirus Infection

Coxsackievirus B infections are suspected environmental triggers of type 1 diabetes (T1D) and macrophage antiviral responses may provide a link to virus-induced T1D. We previously demonstrated an important role for NADPH oxidase (NOX)–derived superoxide production during T1D pathogenesis, as NOX-deficient NOD mice (NOD.Ncf1m1J) were protected against T1D due, in part, to impaired proinflammatory TLR signaling in NOD.Ncf1m1J macrophages. Therefore, we hypothesized that loss of NOX-derived superoxide would dampen diabetogenic antiviral macrophage responses and protect from virus-induced diabetes. Upon infection with a suspected diabetogenic virus, Coxsackievirus B3 (CB3), NOD.Ncf1m1J mice remained resistant to virus-induced autoimmune diabetes. A concomitant decrease in circulating inflammatory chemokines, blunted antiviral gene signature within the pancreas, and reduced proinflammatory M1 macrophage responses were observed. Importantly, exogenous superoxide addition to CB3-infected NOD.Ncf1m1J bone marrow–derived macrophages rescued the inflammatory antiviral M1 macrophage response, revealing reduction-oxidation–dependent mechanisms of signal transducer and activator of transcription 1 signaling and dsRNA viral sensors in macrophages. We report that superoxide production following CB3 infection may exacerbate pancreatic β cell destruction in T1D by influencing proinflammatory M1 macrophage responses, and mechanistically linking oxidative stress, inflammation, and diabetogenic virus infections.

Antiretroviral therapy potentiates high-fat diet induced obesity and glucose intolerance

Objective Breakthroughs in HIV treatment, especially combination antiretroviral therapy (ART), have massively reduced AIDS-associated mortality. However, ART administration amplifies the risk of non-AIDS defining illnesses including obesity, diabetes, and cardiovascular disease, collectively known as metabolic syndrome. Initial reports suggest that ART-associated risk of metabolic syndrome correlates with socioeconomic status, a multifaceted finding that encompasses income, race, education, and diet. Therefore, determination of causal relationships is extremely challenging due to the complex interplay between viral infection, ART, and the many environmental factors. Methods In the current study, we employed a mouse model to specifically examine interactions between ART and diet that impacts energy balance and glucose metabolism. Previous studies have shown that high-fat feeding induces persistent low-grade systemic and adipose tissue inflammation contributing to insulin resistance and metabolic dysregulation via adipose-infiltrating macrophages. Studies herein test the hypothesis that ART potentiates the inflammatory effects of a high-fat diet (HFD). C57Bl/6J mice on a HFD or standard chow containing ART or vehicle, were subjected to functional metabolic testing, RNA-sequencing of epididymal white adipose tissue (eWAT), and array-based kinomic analysis of eWAT-infiltrating macrophages. Results ART-treated mice on a HFD displayed increased fat mass accumulation, impaired glucose tolerance, and potentiated insulin resistance. Gene set enrichment and kinomic array analyses revealed a pro-inflammatory transcriptional signature depicting granulocyte migration and activation. Conclusion The current study reveals a HFD-ART interaction that increases inflammatory transcriptional pathways and impairs glucose metabolism, energy balance, and metabolic dysfunction.