Superoxide production by macrophages and T cells is critical for the induction of autoreactivity and type 1 diabetes

Abstract

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 β-cell-directed autoimmunity by T cells.

FIG. 1.

Congenic introduction Ncf1^m1J mutation ablates bone marrow cell superoxide production and oxygen consumption in response to PMA. Bone marrow from NOD (black bars) and NOD-Ncf1^m1J (white bars) mice were (A) stimulated with PMA (98 nmol/L) and superoxide production was evaluated by measuring rate reduction of cytochrome C. Purified superoxide dismutase 1 (SOD1; 0.5 units/mL) was added to confirm production of superoxide. B: Oxygen consumption was measured with an oxygen electrode for 30 min. Results are from five independent experiments.

FIG. 2.

NOD-Ncf1^m1J mice are protected from spontaneous autoimmune diabetes. Age-matched female NOD (black square), NOD-Ncf1^+/+ littermate controls (black diamond), and NOD-Ncf1^m1J (open triangle) mice were monitored by glucosuria for the onset of spontaneous type 1 diabetes (T1D).

FIG. 3.

Cultured islets from NOD.Rag (black bars) and NOD-Ncf1^m1J (white bars) mice were treated with cytokines IFN-γ, IL-1β, and TNF-α. Islets were (A) susceptible to proinflammatory cytokine-mediated damage and (B) produced NO after treatment. Data representative of three independent experiments performed in triplicate. ND, none detected.

FIG. 4.

AI4 transgenic T cells, but not BDC-2.5 cells, are able to induce diabetes in NOX-deficient mice. A: Splenocytes (2 × 10⁷ cells) from NOD-AI4 donors were transferred to sublethally (7.5 Gy) irradiated NOD (black square) and NOD-Ncf1^m1J (open triangle) recipients. B: BDC-2.5 CD4⁺ T cells were harvested, activated, and transferred to age-matched NOD and NOD-Ncf1^m1J recipients. Mice were monitored by glucosuria for the onset of type 1 diabetes (T1D). Mice were considered diabetic after two consecutive readings of blood glucose above 250 mg/dL. AT, adoptive transfer.

FIG. 5.

Macrophages, but not neutrophils, are critical for type 1 diabetes (T1D) induction. A: Age-matched NOD female mice were treated with 500 μg 2 days a week and 750 μg 1 day a week anti-Gr-1 RB6–8C5 (black diamond), 1 mg 3 days a week anti-Gr-1 1A8 (open triangle), or PBS vehicle control (black square) starting at 4 weeks of age. Mice were monitored by glucosuria for the onset of diabetes and considered diabetic after two consecutive readings of blood glucose above 250 mg/dL. Peripheral blood was used to confirm depletion of (B) neutrophils (CD11b⁺Ly6G⁺) and (C) macrophages (CD11b⁺F4/80⁺) by flow cytometry. D: Blood samples were gated on CD11b⁺ cells and analyzed for Ly6G and F4/80 expression (representative plot of treated mice at 16 weeks of age). Statistical differences noted above signify *P < 0.0001 or #P < 0.002, respectively.

FIG. 6.

NOX-deficient NOD-Ncf1^m1J immune system has a reduced capacity to transfer type 1 diabetes (T1D) to immune-deficient hosts. Splenocytes (2 × 10⁷ cells) from (A) 20-week-old diabetic or (B) 8-week-old prediabetic donor NOD (black square) and NOD-Ncf1^m1J (open triangle) female mice were transferred intraperitoneally to NOD-Scid hosts. C: A total of 1 × 10⁷ purified CD4⁺ and CD8⁺ T cells at a ratio of 3:1 were transferred intraperitoneally to NOD-Scid recipients. Transfers were divided into four groups: group 1) NOD CD4⁺ + NOD CD8⁺, group 2) NOD-Ncf1^m1J CD4⁺ + NOD-Ncf1^m1J CD8⁺, group 3) NOD-Ncf1^m1J CD4⁺ + NOD CD8⁺, or group 4) NOD CD4⁺ + NOD-Ncf1^m1J CD8⁺. *Letters denote significance. Groups/lines with different letters were statistically significant (P < 0.05), whereas those with the same letter were not statistically different. Comparisons of the groups led to the following P values: group 1 vs. group 2, P = 0.047; group 1 vs. group 3, P = 0.67; group 1 vs. group 4, P = 0.049; group 2 vs. group 3, P = 0.043; group 2 vs. group 4, P = 0.64; and group 3 vs. group 4, P = 0.021. In all three adoptive transfer (AT) experiments, mice were monitored by glucosuria for the onset of diabetes and confirmed by blood glucose measurement. Mice were considered diabetic after two consecutive readings of blood glucose above 250 mg/dL.