Hubert M. Tse

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.

FoxO1 Links Insulin Resistance to Proinflammatory Cytokine IL-1β Production in Macrophages

OBJECTIVE Macrophages play an important role in the pathogenesis of insulin resistance via the production of proinflammatory cytokines. Our goal is to decipher the molecular linkage between proinflammatory cytokine production and insulin resistance in macrophages. RESEARCH DESIGN AND METHODS We determined cytokine profiles in cultured macrophages and identified interleukin (IL)-1β gene as a potential target of FoxO1, a key transcription factor that mediates insulin action on gene expression. We studied the mechanism by which FoxO1 mediates insulin-dependent regulation of IL-1β expression in cultured macrophages and correlated FoxO1 activity in peritoneal macrophages with IL-1β production profiles in mice with low-grade inflammation or insulin resistance. RESULTS FoxO1 selectively promoted IL-1β production in cultured macrophages. This effect correlated with the ability of FoxO1 to bind and enhance IL-1β promoter activity. Mutations of the FoxO1 binding site within the IL-1β promoter abolished FoxO1 induction of IL-1β expression. Macrophages from insulin-resistant obese db/db mice or lipopolysaccharide-inflicted mice were associated with increased FoxO1 production, correlating with elevated levels of IL-1β mRNA in macrophages and IL-1β protein in plasma. In nonstimulated macrophages, FoxO1 remained inert with benign effects on IL-1β expression. In response to inflammatory stimuli, FoxO1 activity was augmented because of an impaired ability of insulin to phosphorylate FoxO1 and promote its nuclear exclusion. This effect along with nuclear factor-κB acted to stimulate IL-1β production in activated macrophages. CONCLUSIONS FoxO1 signaling through nuclear factor-κB plays an important role in coupling proinflammatory cytokine production to insulin resistance in obesity and diabetes.

Design of Mn porphyrins for treating oxidative stress injuries and their redox-based regulation of cellular transcriptional activities

The most efficacious Mn(III) porphyrinic (MnPs) scavengers of reactive species have positive charges close to the Mn site, whereby they afford thermodynamic and electrostatic facilitation for the reaction with negatively charged species such as O2•− and ONOO−. Those are Mn(III) meso tetrakis(N-alkylpyridinium-2-yl)porphyrins, more specifically MnTE-2-PyP5+ (AEOL10113) and MnTnHex-2-PyP5+ (where alkyls are ethyl and n-hexyl, respectively), and their imidazolium analog, MnTDE-2-ImP5+ (AEOL10150, Mn(III) meso tetrakis(N,N′-diethylimidazolium-2-yl) porphyrin). The efficacy of MnPs in vivo is determined not only by the compound antioxidant potency, but also by its bioavailability. The former is greatly affected by the lipophilicity, size, structure, and overall shape of the compound. These porphyrins have the ability to both eliminate reactive oxygen species and impact the progression of oxidative stress-dependent signaling events. This will effectively lead to the regulation of redox-dependent transcription factors and the suppression of secondary inflammatory- and oxidative stress-mediated immune responses. We have reported on the inhibition of major transcription factors HIF-1α, AP-1, SP-1, and NF-κB by Mn porphyrins. While the prevailing mechanistic view of the suppression of transcription factors activation is via antioxidative action (presumably in cytosol), the pro-oxidative action of MnPs in suppressing NF-κB activation in nucleus has been substantiated. The magnitude of the effect is dependent upon the electrostatic (porphyrin charges) and thermodynamic factors (porphyrin redox ability). The pro-oxidative action of MnPs has been suggested to contribute at least in part to the in vitro anticancer action of MnTE-2-PyP5+ in the presence of ascorbate, and in vivo when combined with chemotherapy of lymphoma. Given the remarkable therapeutic potential of metalloporphyrins, future studies are warranted to further our understanding of in vivo action/s of Mn porphyrins, particularly with respect to their subcellular distribution.

NADPH Oxidase Deficiency Regulates Th Lineage Commitment and Modulates Autoimmunity

Reactive oxygen species are used by the immune system to eliminate infections; however, they may also serve as signaling intermediates to coordinate the efforts of the innate and adaptive immune systems. In this study, we show that by eliminating macrophage and T cell superoxide production through the NADPH oxidase (NOX), T cell polarization was altered. After stimulation with immobilized anti-CD3 and anti-CD28 or priming recall, T cells from NOX-deficient mice exhibited a skewed Th17 phenotype, whereas NOX-intact cells produced cytokines indicative of a Th1 response. These findings were corroborated in vivo by studying two different autoimmune diseases mediated by Th17 or Th1 pathogenic T cell responses. NOX-deficient NOD mice were Th17 prone with a concomitant susceptibility to experimental allergic encephalomyelitis and significant protection against type 1 diabetes. These data validate the role of superoxide in shaping Th responses and as a signaling intermediate to modulate Th17 and Th1 T cell responses.

Disruption of Innate-Mediated Proinflammatory Cytokine and Reactive Oxygen Species Third Signal Leads to Antigen-Specific Hyporesponsiveness

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-α, IL-1β, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN-γ synthesis by diminishing ROS production in the APC, which affected TNF-α levels produced by CD4+ T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4+ T cells to transition from expansion to effector function.

Preeclampsia Activates Circulating Immune Cells with Engagement of the NF‐κB Pathway

Compelling evidence implicates peripheral immune activation in the pathophysiology of preeclampsia. Polymorphonuclear neutrophils appear to be the cells most strongly affected, with changes in expression of surface markers and release of granule enzymes. Here, we investigated activation in additional leukocyte populations among women with preeclampsia.

Human proinsulin C-peptide reduces high glucose-induced proliferation and NF-κB activation in vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs) is one of the primary lesions in atherosclerosis development during diabetes. High glucose triggers VSMC proliferation and initiates activation of the transcription factor nuclear factor (NF)-kappaB. Recently, clinical studies have demonstrated that replacement therapy with C-peptide, a cleavage product of insulin, to type 1 diabetic (T1D) patients is beneficial on a variety of diabetes-associated vascular complications. However, the mechanisms underlying the beneficial activity of C-peptide on the vasculature in conditions of hyperglycemia are largely unknown. The effects of C-peptide on the proliferation of human umbilical artery smooth muscle cell (UASMC) and aortic smooth muscle cell (AoSMC) lines cultured under high glucose for 48 h were tested. To gain insights on potential intracellular signaling pathways affected by C-peptide, we analyzed NF-kappaB activation in VSMCs since this pathway represents a key mechanism for the accelerated vascular disease observed in diabetes. High glucose conditions (25 mmol/L) stimulated NF-kappaB-dependent VSMC proliferation since the addition of two NF-kappaB-specific inhibitors, BAY11-7082 and PDTC, prevented proliferation. C-peptide at the physiological concentrations of 0.5 and 1 nmol/L decreased high glucose-induced proliferation of VSMCs that was accompanied by decreased phosphorylation of IkappaB and reduced NF-kappaB nuclear translocation. These results suggest that in conditions of hyperglycemia C-peptide reduces proliferation of VSMCs and NF-kappaB nuclear translocation. In patients with T1D, physiological C-peptide levels may exert beneficial effects on the vasculature that, under high glucose conditions, is subject to progressive dysfunction.

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.

Redox modulation inhibits CD8 T cell effector function.

The evolutionary preservation of reactive oxygen species in innate immunity underscores the important roles these constituents play in immune cell activity and as signaling intermediates. In an effort to exploit these pathways to achieve control of aberrant immune activation we demonstrate that modulation of redox status suppresses cell proliferation and production of IL-2, IFN-gamma, TNF-alpha, and IL-17 in two robust CD8 T-cell-dependent in vitro mouse models: (1) response to alloantigen in an mixed leukocyte reaction and (2) CD8 T cell receptor transgenic OT-1 response to cognate peptide (SIINFEKL). To correlate these findings with cytotoxic lymphocyte (CTL) function we performed cytotoxicity assays and found that redox modulation diminishes the ability of alloantigen-specific and antigen-specific OT-1 CTLs to kill their corresponding antigen-expressing target cells. To further examine the mechanisms of redox-mediated repression of CTL target cell lysis, we analyzed the expression of the effector molecules IFN-gamma, perforin, and granzyme B and the degranulation marker CD107a (LAMP-1). In both models, redox modulation reduced the expression of these effector components by at least fivefold. These results demonstrate that redox modulation quells the CD8 T cell response to alloantigen and the T cell receptor transgenic CD8 T cell response to its cognate antigen by inhibiting proliferation, proinflammatory cytokine synthesis, and CTL effector mechanisms.

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.