Atherosclerosis Impairs Naive CD4 T-Cell Responses via Disruption of Glycolysis.

Objective: CD4 T cells are important regulators of atherosclerotic progression. The metabolic profile of CD4 T cells controls their signaling and function, but how atherosclerosis affects T-cell metabolism is unknown. Here, we sought to determine the impact of atherosclerosis on CD4 T-cell metabolism and the contribution of such metabolic alterations to atheroprogression. Approach and Results: Using PCR arrays, we profiled the expression of metabolism genes in CD4 T cells from atherosclerotic apolipoprotein-E knockout mice fed a Western diet. These cells exhibited dysregulated expression of genes critically involved in glycolysis and fatty acid degradation, compared with those from animals fed a standard laboratory diet. We examined how T-cell metabolism was changed in either Western diet­fed apolipoprotein-E knockout mice or samples from patients with cardiovascular disease by measuring glucose uptake, activation, and proliferation in CD4 T cells. We found that naive CD4 T cells from Western diet­fed apolipoprotein-E knockout mice failed to uptake glucose and displayed impaired proliferation and activation, compared with CD4 T cells from standard laboratory diet­fed animals. Similarly, we observed that naive CD4 T-cell frequencies were reduced in the circulation of human subjects with high cardiovascular disease compared with low cardiovascular disease. Naive T cells from high cardiovascular disease subjects also showed reduced proliferative capacity. Conclusions: These results highlight the dysfunction that occurs in CD4 T-cell metabolism and immune responses during atherosclerosis. Targeting metabolic pathways within naive CD4 T cells could thus yield novel therapeutic approaches for improving CD4 T-cell responses against atheroprogression.

Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

OBJECTIVE: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4+ T cells have been extensively studied in CVD, the importance of CD8+ T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8+ T (TN) cell population expressing CD95 (termed CD95+CD8+ stem cell memory T [CD8 TSCM] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95lo cells within the TN compartment. We found that CD8 TSCM cells positively correlated with CVD risk in humans, while CD8+ TN cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice also displayed respective 7- and 2-fold increases in CD8+ TSCM frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8+ TSCM cells were 1.7-fold increased in aortas from western diet fed ApoE-/- mice compared with normal laboratory diet-fed ApoE-/- mice. Importantly, transfer of TSCM cells into immune-deficient Rag.Ldlr recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis. CONCLUSIONS: CD8+ TSCM cells are increased in humans with high CVD. As these TSCM cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.

Neuropilin-1 Expression on CD4 T Cells Is Atherogenic and Facilitates T Cell Migration to the Aorta in Atherosclerosis.

Neuropilin 1 (Nrp1) is a type I transmembrane protein that plays important roles in axonal guidance, neuronal development, and angiogenesis. Nrp1 also helps migrate thymus-derived regulatory T cells to vascular endothelial growth factor (VEGF)-producing tumors. However, little is known about the role of Nrp1 on CD4 T cells in atherosclerosis. In ApoE-/- mice fed a Western diet for 15 wk, we found a 2-fold increase in Nrp1+Foxp3- CD4 T cells in their spleens, periaortic lymph nodes, and aortas, compared with chow-fed mice. Nrp1+Foxp3- CD4 T cells had higher proliferation potential, expressed higher levels of the memory marker CD44, and produced more IFN-γ when compared with Nrp1- CD4 T cells. Treatment of CD4 T cells with oxLDL increased Nrp1 expression. Furthermore, atherosclerosis-susceptible mice selectively deficient for Nrp1 expression on T cells developed less atherosclerosis than their Nrp1-sufficient counterparts. Mechanistically, we found that CD4 T cells that express Nrp1 have an increased capacity to migrate to the aorta and periaortic lymph nodes compared to Nrp1- T cells, suggesting that the expression of Nrp1 facilitates the recruitment of CD4 T cells into the aorta where they can be pathogenic. Thus, we have identified a novel role of Nrp1 on CD4 T cells in atherosclerosis. These results suggest that manipulation of Nrp1 expression on T cells can affect the outcome of atherosclerosis and lower disease incidence.

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 ß cell destruction in T1D by influencing proinflammatory M1 macrophage responses, and mechanistically linking oxidative stress, inflammation, and diabetogenic virus infections.

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.Ncf1(m1J) mouse, possessing superoxide-deficient T cells recognizing the nominal Ag OVA323-339 In this study, we demonstrate that OT-II.Ncf1(m1J) 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.Ncf1(m1J) 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.

Nonclassical monocytes potentiate anti-tumoral CD8+ T cell responses in the lungs.

CD8+ T cells drive anti-cancer immunity in response to antigen-presenting cells such as dendritic cells and subpopulations of monocytes and macrophages. While CD14+ classical monocytes modulate CD8+ T cell responses, the contributions of CD16+ nonclassical monocytes to this process remain unclear. Herein we explored the role of nonclassical monocytes in CD8+ T cell activation by utilizing E2-deficient (E2-/-) mice that lack nonclassical monocytes. During early metastatic seeding, modeled by B16F10-OVA cancer cells injected into E2-/- mice, we noted lower CD8+ effector memory and effector T cell frequencies within the lungs as well as in lung-draining mediastinal lymph nodes in the E2-/- mice. Analysis of the myeloid compartment revealed that these changes were associated with depletion of MHC-IIloLy6Clo nonclassical monocytes within these tissues, with little change in other monocyte or macrophage populations. Additionally, nonclassical monocytes preferentially trafficked to primary tumor sites in the lungs, rather than to the lung-draining lymph nodes, and did not cross-present antigen to CD8+ T cells. Examination of the lung microenvironment in E2-/- mice revealed reduced CCL21 expression in endothelial cells, which is chemokine involved in T cell trafficking. Our results highlight the previously unappreciated importance of nonclassical monocytes in shaping the tumor microenvironment via CCL21 production and CD8+ T cell recruitment.

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression.

Functional crosstalk between T cells and monocytes in cancer and atherosclerosis.

Monocytes and monocyte-derived cells, including Mϕs and dendritic cells, exhibit a diverse array of phenotypic states that are dictated by their surrounding microenvironment. These cells direct T cell activation and function via cues that range from being immunosuppressive to immunostimulatory. Solid tumors and atherosclerotic plaques represent two pathological niches with distinct immune microenvironments. While monocytes and their progeny possess a phenotypic spectrum found within both disease contexts, most within tumors are pro-tumoral and support evasion of host immune responses by tumor cells. In contrast, monocyte-derived cells within atherosclerotic plaques are usually pro-atherogenic, pro-inflammatory, and predominantly directed against self-antigens. Consequently, cancer immunotherapies strive to enhance the immune response against tumor antigens, whereas atherosclerosis treatments seek to dampen the immune response against lipid antigens. Insights into monocyte-T cell interactions within these niches could thus inform therapeutic strategies for two immunologically distinct diseases. Here, we review monocyte diversity, interactions between monocytes and T cells within tumor and plaque microenvironments, how certain therapies have leveraged these interactions, and novel strategies to assay such associations.

Dampening Antigen-Specific T Cell Responses with Antigens Encapsulated in Polyphenolic Microcapsules.

Efficient T cell activation and effector responses require an antigenic peptide presented on the MHC complex to the TCR (signal 1), costimulatory molecule interactions between T cells and APCs (signal 2), and the synthesis of innate immune-derived proinflammatory cytokines and reactive oxygen species (signal 3). We previously demonstrated that the third signal dissipation impairs autoreactive T cell activation. In this study, we tested the hypothesis that encapsulation of Ag with an antioxidant-containing biomaterial would induce Ag-specific hyporesponsiveness. We cocultured bone marrow-derived dendritic cells with microcapsules composed of multilayer-assembled poly(N-vinylpyrrolidone) (PVPON) and the antioxidant tannic acid (TA). LPS-activated dendritic cells cocultured with (PVPON/TA) microcapsules displayed a decrease in TNF-α, IL-12p70, and CXCL10 synthesis. To study Ag-specific T cell responses, we incorporated chicken OVA into the (PVPON/TA) multilayers and stimulated OT-II splenocytes in a primary recall assay. Flow cytometric analysis demonstrated a significant inhibition of CD4 T cell activation markers, upregulation of CTLA-4 and PD-1, and blunted secretion of IL-2, IFN-γ, TNF-α, and CXCL10 by ELISA. To test microcapsule efficacy in vivo, we immunized OT-II mice with (PVPON/TA)-OVA microcapsules and performed an OVA recall assay. Immunization of OT-II mice with (PVPON/TA)-OVA microcapsules elicited a decrease in CD4 T cell differentiation and effector responses including IFN-γ, TNF-α, CCL3, and CCL5 by ELISA compared with OVA immunization alone. These data show that microcapsules composed of antioxidant and encapsulated Ags can effectively blunt innate immune-derived proinflammatory third signal synthesis necessary for Ag-specific effector T cell responses and present a prospective strategy for T cell-mediated autoimmunity.

Apolipoprotein AI prevents regulatory to follicular helper T cell switching during atherosclerosis.

Regulatory T (Treg) cells contribute to the anti-inflammatory response during atherogenesis. Here we show that during atherogenesis Treg cells lose Foxp3 expression and their immunosuppressive function, leading to the conversion of a fraction of these cells into T follicular helper (Tfh) cells. We show that Tfh cells are pro-atherogenic and that their depletion reduces atherosclerosis. Mechanistically, the conversion of Treg cells to Tfh cells correlates with reduced expression of IL-2Rα and pSTAT5 levels and increased expression of IL-6Rα. In vitro, incubation of naive T cells with oxLDL prevents their differentiation into Treg cells. Furthermore, injection of lipid-free Apolipoprotein AI (ApoAI) into ApoE-/- mice reduces intracellular cholesterol levels in Treg cells and prevents their conversion into Tfh cells. Together our results suggest that ApoAI, the main protein in high-density lipoprotein particles, modulates the cellular fate of Treg cells and thus influences the immune response during atherosclerosis.

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.

Islet encapsulation with polyphenol coatings decreases pro-inflammatory chemokine synthesis and T cell trafficking.

Type 1 Diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of islet-infiltrating leukocytes involved in pancreatic ß-cell lysis. One promising treatment for T1D is islet transplantation; however, clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to preserve islet function and prevent immune-mediated rejection after transplantation. We previously demonstrated that a novel cytoprotective nanothin multilayer coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), was efficacious in dampening in vitro immune responses involved in transplant rejection and preserving in vitro islet function. However, the ability of (PVPON/TA) to maintain islet function in vivo and reverse diabetes has not been tested. Recent evidence has demonstrated that modulation of redox status can affect pro-inflammatory immune responses. Therefore, we hypothesized that transplanted (PVPON/TA)-encapsulated islets can restore euglycemia to diabetic mice and provide an immunoprotective barrier. Our results demonstrate that (PVPON/TA) nanothin coatings can significantly decrease in vitro chemokine synthesis and diabetogenic T cell migration. Importantly, (PVPON/TA)-encapsulated islets restored euglycemia after transplantation into diabetic mice. Our results demonstrate that (PVPON/TA)-encapsulated islets may suppress immune responses and enhance islet allograft acceptance in patients with T1D.

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 (Ncf1(m1J)) 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.Ncf1(m1J) islet-resident macrophages displayed a dampened M1 and increased M2 phenotype. The transfer of diabetogenic T cells into NOX-deficient NOD.Rag.Ncf1(m1J) recipients resulted in decreased TNF-α(+) and IL-1ß(+) 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 ß-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.

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.Ncf1(m1J)) 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.Ncf1(m1J) (BDC-2.5.Ncf1(m1J)) mouse strain was generated, possessing autoreactive CD4 T cells deficient in NOX-derived superoxide. Unlike NOD.Ncf1(m1J), stimulated BDC-2.5.Ncf1(m1J) 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-12Rß2 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.

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 ß cells. In humans with T1D and in nonobese diabetic (NOD) mice (a murine model for human T1D), autoreactive T cells cause ß-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 ß-cell destruction and disease pathogenesis. While these adaptive immune cells employ distinct mechanisms for ß-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 ß cells, which characteristically possess low levels of antioxidant defense enzymes. In addition to ß-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-Ncf1(m1J) 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-Ncf1(m1J) 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-Ncf1(m1J) 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-Ncf1(m1J) 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 ß-cell-directed autoimmunity by T cells.