Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction

Abstract

We have shown previously that T cells are required for the full development of angiotensin II-induced hypertension. However, the specific subsets of T cells that are important in this process are unknown. T helper 17 cells represent a novel subset that produces the proinflammatory cytokine interleukin 17 (IL-17). We found that angiotensin II infusion increased IL-17 production from T cells and IL-17 protein in the aortic media. To determine the effect of IL-17 on blood pressure and vascular function, we studied IL-17(-/-) mice. The initial hypertensive response to angiotensin II infusion was similar in IL-17(-/-) and C57BL/6J mice. However, hypertension was not sustained in IL-17(-/-) mice, reaching levels 30-mm Hg lower than in wild-type mice by 4 weeks of angiotensin II infusion. Vessels from IL-17(-/-) mice displayed preserved vascular function, decreased superoxide production, and reduced T-cell infiltration in response to angiotensin II. Gene array analysis of cultured human aortic smooth muscle cells revealed that IL-17, in conjunction with tumor necrosis factor-alpha, modulated expression of >30 genes, including a number of inflammatory cytokines/chemokines. Examination of IL-17 in diabetic humans showed that serum levels of this cytokine were significantly increased in those with hypertension compared with normotensive subjects. We conclude that IL-17 is critical for the maintenance of angiotensin II-induced hypertension and vascular dysfunction and might be a therapeutic target for this widespread disease.

Figure 1. Effect of angiotensin II on IL17 production from T cells

C57BL/6J mice were treated for 14 d with vehicle (sham) or angiotensin II. T lymphocytes were then isolated, cultured, and stimulated with anti-CD3 antibodies or PMA-ionomycin. (A) ELISA for IL17 released into the media (n=6–8 per group). (B) Intracellular FACS staining for IL17 expressed as percentage of CD4+ cells (n=5–6 per group). Data are expressed as mean±SEM. *p<0.05 vs sham, **p<0.01 vs sham.

Figure 2. Effect of IL17 on angiotensin II-induced hypertension

(A) Non-invasive blood pressure measurements obtained via the tail cuff method (n=8–9 per group). (B) Sample traces of telemetry recordings from freely moving mice after 4 weeks of angiotensin II infusion obtained via indwelling carotid artery catheters. Systolic and diastolic blood pressure (C), mean arterial pressure (MAP) and heart rate (D) from telemetry recordings (n=4–6 per group). Data are expressed as mean±SEM. *p<0.05 vs IL17^−/− angiotensin II, **p<0.01 vs IL17^−/− angiotensin II, n.s. = not significant.

Figure 3. Effect of angiotensin II on IL17 protein in vessels

Immunoflourescence for IL17 in sections from the thoracic aorta of C57BL/6J or IL17^−/− mice treated with vehicle or angiotensin II for 4 weeks. (A) Representative confocal images taken at 60x/oil. (B) Integrated density quantification made from 20x Axiocam images (n=3–4 per group). Data are expressed as mean±SEM. p-values are shown on graph.

Figure 4. Effect of IL17 on vascular function and superoxide production

Aortic rings were isolated from C57BL/6J and IL17^−/− mice exposed to 4 weeks of vehicle or angiotensin II. (A) Endothelium-dependent relaxation to increasing doses of acetylcholine (n=7–13 per group). (B) Endothelium-independent relaxation to increasing doses of sodium nitroprusside (n=7–13 per group). (C) Contraction to increasing doses of phenylephrine (n=5–9 per group). (D) Aortic superoxide production as measured by dihydroethidium/HPLC analysis (n=6–7 per group). (E) Superoxide production in mesenteric vessels as measured by dihydroethidium/HPLC analysis (n=4–5 per group). Data are expressed as mean±SEM. **p<0.001 vs remaining 3 groups. *p<0.05 vs C57BL/6J Sham, ^†p<0.01 vs IL17^−/− Sham and IL17^−/− angiotensin II.

Figure 5. Effect of IL17 on aortic T cell infiltration

(A) Examples of flow cytometric analysis of collagenase-digested aortic single cell suspensions from C57BL/6J and IL17^−/− mice exposed to 4 weeks of vehicle or angiotensin II. Fluorescent staining was performed to detect CD45 (total leukocytes) and CD3 (T cells). Absolute numbers of total leukocytes (CD45+ cells, panel B) and total T cells (CD3+ cells, panel C) in whole aortas from vehicle-infused/sham treatment (open bars) and angiotensin II-infused (solid bars) mice (n=7–9 per group). p-values are shown on graph.