NADPH oxidase deficiency regulates Th lineage commitment and modulates autoimmunity - PubMed (original) (raw)
NADPH oxidase deficiency regulates Th lineage commitment and modulates autoimmunity
Hubert M Tse et al. J Immunol. 2010.
Abstract
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.
Figures
FIGURE 1
Ncf1m1J mutation decreases the respiratory burst capacity of bone marrow macrophages, neutrophils, and splenic T cells without decreasing the percentage of cells. The percentage of bone marrow neutrophils (A) and macrophages (C) from C57BL/6.H2g7, C57BL/6.H2g7.Ncf1m1J, C57BL/6, ALR, NOD, and NOD.Ncf1m1J mice was assessed by flow cytometry with gating for macrophage (Gr1− CD11b+)- or neutrophil (Gr1+ CD11b+)-specific populations. The respiratory burst capacity of bone marrow neutrophils (B) and macrophages (D) was assessed by loading 5 × 106 cells/ml with 0.99 M dihydrorhodamine and stimulated with 98 nM PMA for 40 min. The oxidation of dihydrorhodamine to rhodamine was assessed by flow cytometry with gating for macrophage (Gr1− CD11b+)- or neutrophil (Gr1+ CD11b+)-specific populations. Whole-cell lysates of cell-sorted macrophages were used in an immunoblot analysis for p47_phox_ and actin expression (E). T cell ROS production was measured by labeling purified CD4+ T cells with DCFDA. Cells were activated by adding hamster anti-mouse CD3 and then cross-linked with rabbit anti-hamster IgG. Cells were analyzed at 0, 30, and 60 min. ROS generation was determined by the increase in DCFDA fluorescence upon anti-CD3 stimulation over unstimulated control (G). The percentage of splenic CD4+ T cells was assessed by flow cytometry by gating for CD4+ T cell-specific populations (CD3+CD4+CD8−) (F). Results are representative of three independent experiments.
FIGURE 2
NOD.Ncf1m1J mice resist spontaneous diabetes. Kaplan-Meier survival curve of NOD (n = 20) and NOD.Ncf1m1J (n = 20) age-matched female mice for spontaneous diabetes incidence (A). Insulitis score of 7- and 22-wk-old female NOD and NOD.Ncf1m1J mice (B). H&E staining of pancreatic sections from 7- and 22-wk-old female NOD (C, E) and NOD.Ncf1m1J (D, F) mice. Mice were diabetic after consecutive blood glucose readings of >300 mg/dl.
FIGURE 3
Anti-CD3 and anti-CD28 stimulation of NOD.Ncf1m1J T cells exhibits a decrease in Th1 cytokine profile concomitant with an increase in Th17 cytokine synthesis. Immunoblot analysis of p47_phox_ expression in negatively selected and purified NOD and NOD.Ncf1m1J T cells (A). Proliferation and cytokine synthesis of purified NOD and NOD.Ncf1m1J T cells (2.5 × 105 cells) after stimulation with polyclonal Abs for 72 h, as tested by cytokine-specific ELISAs and Luminex Bio-plex assay for cytokine synthesis (B_–_K). Proliferation was assessed by 3H-TdR incorporation. Results are representative of three independent experiments done in triplicate. n.m., Not measurable.
FIGURE 4
NOD.Ncf1m1J naive CD4+CD62L+ T cells exhibit a Th17 cytokine profile after polyclonal stimulation. The percentages of splenic naive (CD62L+CD69−CD44−), effector (CD62L−CD69+CD44+), and memory (CD62L−CD69−CD44+) CD4+ T cells were assessed by flow cytometry (A). Intracellular cytokine staining of CD4+/IFN-γ+ (B) and CD4+/IL-17A+ (C) after 72-h stimulation of purified naive CD4+CD62L+ T cells from the spleen of NOD and NOD.Ncf1m1J mice.
FIGURE 5
NOD.Ncf1m1J T cells do not express Th1 lineage-specific transcription factors upon polyclonal stimulation. Immunoblot analysis of whole-cell lysates (20 μg protein) of negatively selected and purified NOD and NOD.Ncf1m1J T cells after anti-CD3 and anti-CD28 stimulation for 72 h for T-bet, STAT4, and STAT1α. Actin was also probed to confirm equal protein loading on the gels (A). STAT3 activation was assessed by using phospho-specific STAT3 Abs (Y705 and S727) in an immunoblot analysis of anti-CD3– and anti-CD28–stimulated NOD and NOD.Ncf1m1J T cells for 48 and 72 h. Actin was also probed to confirm equal protein loading on the gels (C). Cumulative data from three independent experiments employing Image J software (NIH) were used to determine the area under the curve values for T-bet, STAT4, STAT1α, P-STAT3 (Y705), P-STAT3 (S727), and actin controls. The data are expressed both as the ratio of T-bet, STAT4, or STAT1α to actin (B) or P-STAT3 (Y705) and P-STAT3 (S727) to actin (D, E) and as the change after NOD T cell samples were normalized to a value of 1. **p < 0.01 versus NOD T cells.
FIGURE 6
NOD.Ncf1m1J macrophages exhibit a skewed Th1 to Th17 proinflammatory cytokine response after LPS stimulation. NOD and NOD.Ncf1m1J bone marrow-derived macrophages were stimulated with 1 mg/ml LPS for 4–24 h. Supernatants were harvested, and the levels of cytokines and chemokines were measured with cytokine-specific ELISAs and with a Luminex Bio-plex cytokine panel (A–H). Nitrite levels were measured using a Greiss assay. Results are representative of three independent experiments done in triplicate.
FIGURE 7
NOD.Ncf1m1J mice exhibit both Th17 and Th1 T cell responses after HEL immunization. Ag-recall crisscross assay with negatively selected and purified T cells and APCs from the LN of HEL-immunized NOD and NOD.Ncf1m1J mice. T cells (2 × 105) and APCs (2 × 105) were combined in a crisscross fashion with 25 μg HEL and stimulated for 72 h. Supernatants were collected and cytokine synthesis was measured with cytokine-specific ELISAs and a Luminex Bio-plex cytokine panel (A_–_F). Results are representative of three independent experiments.
FIGURE 8
NOD mice with Ncf1m1J mutation do not have increased numbers of Treg cells. The total splenocytes and Treg (CD4+CD25+ FoxP3+) cell number (A) and percentage of Treg (B) from NOD and NOD. Ncf1m1J mice were assessed by flow cytometry. Total splenocytes were counted on Beckman Coulter (Fullerton, CA) counter. Splenocytes were gated on CD4+CD8− T cells, and CD25+FoxP3+ population was selected to determine percentage of Treg cells.
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