γδT Cell–Derived Interleukin-17A via an Interleukin-1β–Dependent Mechanism Mediates Cardiac Injury and Fibrosis in HypertensionNovelty and Significance by Yulin Li, Yina Wu, Congcong Zhang, Ping Li, Wei Cui, Jianlei Hao, Xinliang Ma, Zhinan Yin, and Jie Du Hypertension Volume 64(2):305-314 July 9, 2014 Copyright © American Heart Association, Inc. All rights reserved.

Interleukin (IL)-17A expression is elevated in T cells in angiotensin (Ang) II–induced hypertensive mouse heart. Interleukin (IL)-17A expression is elevated in T cells in angiotensin (Ang) II–induced hypertensive mouse heart. Hypertension was induced in wild-type (WT) mice by 7 days of Ang II infusion. A, Quantitative real-time polymerase chain reaction (qRT-PCR) showed that IL-17A mRNA is markedly elevated in heart at day 7 of Ang II infusion (n=5 per group). B, The induction of IL-17A mRNA in Ang II–infused heart was time dependent (n=5 per group). Statistical analysis using linear regression (R2=0.897; P<0.0001). C and D, The appearance of IL-17A+ cells in Ang II–infused heart was time dependent. Heart tissue was digested into a single-cell suspension, and analyzed for IL-17A production gated on CD45+CD3+ T cells within side scatter (SSC)lowCD45+ cells by flow cytometry (fluorescence-activated cell sorting). n=5 per group. Statistical analysis using linear regression (R2=0.913; P<0.0001). E, Signal transducer and activator of transcription 3 (STAT3) activation was quantified in heart by Western blot (n=5 per group). F, Transcription factor retinoic acid receptor–related orphan nuclear receptor γ (RoRγt) was assessed in the heart by qRT-PCR and shown to be dramatically elevated at days 3 and 7 (n=5 per group). Statistical analysis using linear regression (R2=0.718; P<0.0001). G, Localization of cardiac IL-17A expression was determined by double-immunofluorescence staining with antibodies against IL-17A (red) and CD3 (green). Bars, 50 μm. *P<0.05 vs WT+saline. Yulin Li et al. Hypertension. 2014;64:305-314 Copyright © American Heart Association, Inc. All rights reserved.

γδ T cells are a major source of interleukin (IL)-17A production in the hearts of angiotensin (Ang)-II–infused mice. γδ T cells are a major source of interleukin (IL)-17A production in the hearts of angiotensin (Ang)-II–infused mice. A, Hearts from saline or Ang II–infused wild-type (WT) mice were digested and analyzed by fluorescence-activated cell sorting (FACS). Absolute numbers of CD3+, CD4+, CD8+, and γδ+T cells were increased in Ang II–infused hearts (n=5 per group). B and C, Representative FACS analysis (B) and quantification (C) of IL-17A+ cells in different T-cell populations, showing that γδ T cell receptor (TCR)+ T cells from Ang II–infused heart had the most abundant IL-17A expression. Cells were first gated for side scatter (SSC)lowCD45+ and were then analyzed by intracellular IL-17A expression in CD45+CD3+ T cells. *P<0.05 vs WT+saline (n=5 per group). D, Representative FACS (left) and percentage (right) of CD3+γδ+ T cells from the blood of anti-γδT antibody (Ab) or isotype Ab-treated mice. *P<0.05 vs isotype Ab (n=5 per group). E and F, IL-17A mRNA and protein expression were evaluated by quantitative real-time polymerase chain reaction and immunofluorescent staining in the hearts of anti-γδT Ab-treated mice and isotype Ab-treated mice. *P<0.05 vs isotype Ab-treated mice+Ang II. Bars, 50 μm (n=5 per group). G and H, IL-17A–secreting CD3+ T cells among gated SSClowCD45+ cells were analyzed by FACS (G) in the hearts of WT, CD4–/–, and γδT–/– mice after Ang II infusion. *P<0.05 vs WT+Ang II (n=4–5 per group). Yulin Li et al. Hypertension. 2014;64:305-314 Copyright © American Heart Association, Inc. All rights reserved.

Assessment of the role of cardiac fibroblasts (CFs) in interleukin (IL)-17A production by γδ T cells. Assessment of the role of cardiac fibroblasts (CFs) in interleukin (IL)-17A production by γδ T cells. A, Cytokine production in single or combined cultures of dendritic cell (DC), activated DCs (DCact), and CF was measured by ELISA or cytometric bead array (CBA). *P<0.05 vs DC. B, DCact, CF, or DCact+CF were cocultured with CD3+ T cells (TCs) with or without anti-CD3/CD28. Cytokines production was measured by CBA. *P<0.05 vs CD3+ T cells alone. C, DCact and CF from wild-type (WT) or IL-6–/– mice were cocultured and subsequently added to CD3+ T cells. IL-6 and IL-17A production were measured by CBA. *P<0.05 vs WT DCact+WT CF. D, CD3+ T cells were cultured with supernatants of DCact. IL-17A production was measured by CBA. *P<0.05 vs CD3+ T cells alone. E and F, Purified γδ+T cells were stimulated with recombinant IL-1β, IL-23, IL-6, and transforming growth factor (TGF)-β, singly or in combination. IL-17A production was measured by CBA (E) and fluorescence-activated cell sorting (F). *P<0.05 vs γδ+T cells without cytokine treatment. G, IL-17A production was analyzed in DCact-stimulated γδ+T cells with neutralizing antibodies (Abs) against IL-1β, IL-23, IL-6, and TGF-β. *P<0.05 vs DCact-γδ+T cells without Ab treatment. Data are from 3 independent experiments. TCR indicates T cell receptor. Yulin Li et al. Hypertension. 2014;64:305-314 Copyright © American Heart Association, Inc. All rights reserved.

Monocyte-derived interleukin (IL)-1β regulates IL-17A production by γδ+T cells in hypertensive heart. Monocyte-derived interleukin (IL)-1β regulates IL-17A production by γδ+T cells in hypertensive heart. A, CD45, CD11B, and discoidin domain receptor 2 (DDR2) mRNA were assessed in CD45–, CD45+CD11B+, and CD45+CD11B– cells from heart of angiotensin (Ang II)–infused wild-type (WT) mice via quantitative real-time polymerase chain reaction (qRT-PCR) (n=5 per group). B and C, Time course of IL-1β and IL-23 mRNA expression in the CD45–, CD45+CD11B+, and CD45+CD11B– cells was assessed by qRT-PCR. *P<0.05 vs CD45– cells (n=5 per group). Statistical analysis using linear regression. IL-1β and IL-23 mRNA expression in CD45+CD11B+ has statistical significance (IL-1β, R2=0.841; P<0.0001 and IL-23, R2=0.846; P<0.0001). D, Cellular composition of CD11B+ cells, gated on CD45+ cells, was analyzed by fluorescence-activated cell sorting in Ang II–infused hearts. Numbers refer to percentage of CD11B+CD11C– and CD11B+CD11C+ cells in CD45+ cells. E, Sorted CD11B+ or CD11B- cells from the hearts of Ang II–infused mice were cultured with γδ+T cells (TCs) with anti-CD3/CD28. IL-17A pro duction was measured by cytometric bead array. *P<0.05 vs γδ+T cells alone. F, IL-17A production was analyzed in the coculture of CD11B+ and γδ+T cells with or without neutralizing antibodies (Abs) against IL-1β, IL-23, or IL-1R antagonist (IL-1Ra). *P<0.05 vs CD11B++γδ+T cells. In vitro experimental data are from 3 independent experiments. G, WT mice were treated with anti–IL-1β Ab or IL-1Ra during saline or Ang II infusion. Heart cells were stained for intracellular IL-17A and surface CD45 or CD3 (n=4 per group). *P<0.05 vs WT+Ang II. Yulin Li et al. Hypertension. 2014;64:305-314 Copyright © American Heart Association, Inc. All rights reserved.

Interleukin (IL)-17A contributes to short-term angiotensin (Ang) II–induced cardiac fibrosis. Interleukin (IL)-17A contributes to short-term angiotensin (Ang) II–induced cardiac fibrosis. Wild-type (WT) and IL-17A–/– mice were subjected to saline or Ang II infusion for 7 days. A, Masson trichrome staining of cardiac tissue sections from WT and IL-17A–/– mice. Fibrotic area (light blue) was quantified and expressed as ratio of collagen fiber:total myocardial area. Bars, 500 μm. B and C, α-SMA expression was determined by IHC (B, left), quantitative real-time polymerase chain reaction (qRT-PCR; B, right) or Western blot (C). D, Hearts from WT or IL-17A–/– mice were analyzed by fluorescence-activated cell sorting for CD45+ and CD45+F4/80+ cells. E, IL-12p40, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-10 mRNA expression in the hearts were quantified by qRT-PCR. F, transforming growth factor (TGF)-β mRNA and protein levels were determined by IHC (left) and qRT-PCR (right). Bars, 50 μm. *P<0.05 vs WT+Ang II (n=5 per group). Yulin Li et al. Hypertension. 2014;64:305-314 Copyright © American Heart Association, Inc. All rights reserved.

Interleukin (IL)-17A contributes to long-term angiotensin (Ang) II–induced cardiac fibrosis. Interleukin (IL)-17A contributes to long-term angiotensin (Ang) II–induced cardiac fibrosis. Wild-type (WT) and IL-17A–/– mice were infused with saline or Ang II for 4 weeks. A, Noninvasive blood pressure measurement is obtained via the tail-cuff method. The summarized area under the curve (AUC) of systolic blood pressure is shown (right). *P<0.05 vs WT+Ang II (n=6–8 per group). B, The quantitative analysis of fibrotic areas in saline or Ang II–infused WT and IL-17A–/– mice (n=6 per group). C, Representative Masson trichrome staining of WT and IL-17A–/– hearts with Ang II infusion (left, bars, 500 μm; middle and right, bars, 200 μm). D and E, α-SMA expression was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and histopathology (bars, 50 μm; n=6 per group). F and G, Transforming growth factor (TGF)-β expression was determined by histopathology and qRT-PCR analysis (bars, 50 μm; n=6 per group). *P<0.05 vs WT+Ang II. Yulin Li et al. Hypertension. 2014;64:305-314 Copyright © American Heart Association, Inc. All rights reserved.