Nox2 in regulatory T cells promotes angiotensin II–induced cardiovascular remodeling
Amber Emmerson, Silvia Cellone Trevelin, Heloise Mongue-Din, Pablo D. Becker, Carla Ortiz, Lesley A. Smyth, Qi Peng, Raul Elgueta, Greta Sawyer, Aleksandar Ivetic, Robert I. Lechler, Giovanna Lombardi, Ajay M. Shah
Amber Emmerson, Silvia Cellone Trevelin, Heloise Mongue-Din, Pablo D. Becker, Carla Ortiz, Lesley A. Smyth, Qi Peng, Raul Elgueta, Greta Sawyer, Aleksandar Ivetic, Robert I. Lechler, Giovanna Lombardi, Ajay M. Shah
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Research Article Cardiology Immunology

Nox2 in regulatory T cells promotes angiotensin II–induced cardiovascular remodeling

Abstract

The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II–induced (Ang II–induced) pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline, whereas Ang II–induced effector T cell (Teff) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of Ang II–induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 antibody depletion of Tregs. Mechanistically, Nox2–/y Tregs showed higher in vitro suppression of Teff proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on Ang II–induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling.

Authors

Amber Emmerson, Silvia Cellone Trevelin, Heloise Mongue-Din, Pablo D. Becker, Carla Ortiz, Lesley A. Smyth, Qi Peng, Raul Elgueta, Greta Sawyer, Aleksandar Ivetic, Robert I. Lechler, Giovanna Lombardi, Ajay M. Shah

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Figure 7

Deficiency of Nox2 in Tregs increases nuclear levels of FoxP3 and NF-κB activation.

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Deficiency of Nox2 in Tregs increases nuclear levels of FoxP3 and NF-κB ...
(A) Representative ImageStream pictures (left) and mean similarity scores (right) for the colocalization of FoxP3 and DAPI in CD4+FoxP3+ cells (Tregs) under basal conditions. (B) Representative ImageStream pictures (left) and mean similarity scores (right) for the colocalization of FoxP3 and p65 in Tregs after anti-CD3 plus anti-CD28 stimulation. (C) Nuclear localization of p65 in Tregs after anti-CD3 plus anti-CD28 stimulation. CS, coefficient of similarity. Representative overlay histogram of similarity of p65/DAPI in WT Tregs and Nox2–/y Tregs is shown to the right. (D) Colocalization of FoxP3 and p65 in the nucleus of Tregs by confocal microscopy. Scale bars: 7.5 μm. (E) NF-κB transcriptional activity assessed by a luciferase promoter assay in Jurkat cells preincubated with a specific Nox2 peptide inhibitor, gp91ds-tat (gp91ds, 30 μM), or a scrambled peptide control, scrambled-tat (sc-tat; 30 μM). Cells transfected with minimal promoter and thymidine kinase Renilla were used as controls. RLU, relative lumen units. (F) mRNA levels of CD25 in purified Tregs (CD4+CD25+). (G) Level of STAT5 phosphorylation (p-STAT5, Y694) assessed in purified Tregs (CD4+CD25+FoxP3+) by flow cytometry after 30 minutes of IL-2 (100 IU/ml) stimulation. Representative histogram shown to the right. MFI, mean fluorescence intensity. *P < 0.05 for highlighted comparisons by unpaired t test (A, B, and F), 2-way ANOVA (G), or 1-way ANOVA followed by Tukey’s post-test (C and E); n = 3 independent experiments except where shown otherwise.