Promotion of hypertension and end-organ injury by Interleukin 17A mediated by Aldosterone-MR and ENaC

Hypertension is one of the most important risk factors for cardiovascular diseases and mortality worldwide. Despite available lifestyle modifications and pharmacological antihypertensive medication high blood pressure remains undiagnosed in more than 50% of hypertensive patients, and poorly controlled in 12-15% of patients. Furthermore, patients with type 2 diabetes have more prevalent resistant hypertension and rarely reach treatment goals.


Evidence from preclinical studies suggest that the adaptive and innate immune system, involving both T-cells, macrophages and dendritic cells play significant roles in the pathogenesis of hypertension. The major focus of this PhD project is to investigate the role of the pro-inflammatory interleukin 17A (IL-17A) produced by T-helper (Th)-17 lymphocytes in hypertension. Mineralocorticoid receptor (MR) is expressed on several adaptive and innate immune cells including T-lymphocytes, macrophages, and dendritic cells. Differentiation of naïve CD4+ T-cells into pro-inflammatory Th17 cells is dependent on the MR in MRdependent-hypertension in rats, and aldosterone-MR activation in vitro. Thus, a mechanistic link between aldosterone-MR and Th17-mediated inflammation contributing to hypertension can be suggested. This could also include the epithelial sodium channel ENaC that, besides being expressed in the distal nephron, also is expressed on lymphocytes and dendritic cells, and shown to be involved in promoting hypertension in vivo. We sought to translate these findings that IL-17A is a major culprit in immune-mediated hypertension in animals by two approaches: 1) In retrospective explorative studies we examined whether well-known antihypertensive drugs (spironolactone and amiloride) target release of IL-17A and related upstream and downstream cytokines in patients in vivo. 2) By direct intervention in mice, IL17A was administered and effects on blood pressure and heart rate were measured. The following overall hypotheses were tested: 1) IL-17A is directly pro-hypertensive by vascular and renal actions in a concentration-dependent manner; 2) IL-17A contributes non-redundantly to immune mediated hypertension through aldosterone-MR interaction; and 3) Dendritic or macrophage and/or Th17 lymphocyte-ENaC channels contribute to IL-17A production in hypertensive patients and accounts for salt-sensitivity. Hypotheses were addressed directly by IL-17 infusion into mice and indirectly, by testing the impact of MR inhibitors and ENaC blocker in vitro in inflammation-stimulated macrophages treated with MR and ENaC blockers, and in vivo in patient plasma samples from intervention studies, by measuring the cytokine IL17A, and other cytokines that are either produced downstream of IL-17A production (IL-6), involved in IL-17A production (IL-6, IL-1β), and produced by macrophage activation (TNF, IL-6, and IL-10) or Th-1 cell activation (IFN-γ). The obtained results and conclusions of this overall study resulted in 4 independent manuscripts as follows.


Study I: The mineralocorticoid receptor blocker spironolactone lowers plasma
interferon-γ and interleukin-6 in patients with type 2 diabetes and treatment resistant
hypertension


We hypothesized that MR antagonism by spironolactone selectively suppresses Th-17 derived IL-17A and other macrophage- and Th-1 cell derived cytokines, and that this relates to reduction in blood pressure, albuminuria, and a plasma potassium increase. Plasma samples from patients with type 2 diabetes mellitus (T2DM) and treatment resistant hypertension, included in a double-blind, placebo-controlled interventional trial, were assessed for plasma cytokine levels before and after intervention with spironolactone (25-50 mg/daily) for 16 weeks. It was previously shown that spironolactone intervention mediated blood pressure and urine albumin/creatinine ratio (UACR) decreases, and plasma potassium increase. In the present study it was shown that plasma IL-17A was unchanged upon spironolactone intervention, but plasma levels of IFN-γ and IL-6 were decreased with no changes observed in placebo treated patients. At baseline serum aldosterone concentrations and night-time diastolic blood pressure (DBP) correlated positively. There were no relations between blood pressure and plasma cytokine levels at baseline; between serum aldosterone and plasma cytokine levels at baseline; and between plasma cytokine and blood pressure changes after spironolactone treatment, except for IFN-γ, which was inversely related to blood pressure changes. Plasma potassium increases related to blood pressure decrease, but not to plasma cytokine decrease after spironolactone intervention. Furthermore, baseline plasma IL-6 related positively to baseline UACR. Finally, in vitro macrophage cell studies revealed a reduction in macrophage derived cytokines upon spironolactone intervention in LPS stimulated cells. We conclude that blood pressure lowering by MR blockade with spironolactone in T2DM patients with treatment resistant hypertension was not mediated by Th-17-derived IL-17A reduction. Instead, spironolactone exerts anti-inflammatory actions in vivo through suppression of Th-1 and macrophage-derived cytokines (IFN-γ and IL-6)

Study II: Amiloride decreases TNF and IL-6 but not IL-17A in patients with
hypertension and type 2 diabetes


We hypothesized that ENaC inhibition with amiloride reduces plasma IL-17A in patients with T2DM and treatment resistant hypertension, and that this reduction in plasma IL-17A relates to amiloride-mediated reduction in blood pressure, albuminuria, and plasma potassium increase. In a follow-up, open label study of the previously described patient cohort (study I), T2DM patients with treatment resistant hypertension received amiloride for 8 weeks (5-10 mg/day). Previously, it was shown that amiloride lowered blood pressure and UACR and increased plasma potassium. In the present study, we show that amiloride decreased plasma TNF and IL-6 but had no effect on plasma IL-17A. At baseline, plasma IL-1β correlated positively to night-time mean arterial pressure (MAP) and DBP. Baseline plasma IL-17A correlated positively to systolic blood pressure (SBP). No correlations were observed between TNF, IL-6, and blood pressure decreases after amiloride intervention. Baseline UACR correlated positively with baseline SBP, and with baseline plasma IL-17A, TNF, IL-6, and IL1β concentrations. Plasma potassium increase did not relate to blood pressure decrease or plasma TNF and IL-6 changes after amiloride intervention. In vitro studies show that LPSstimulated macrophages increase release of cytokines whilst amiloride co-incubation caused a significant reduction in IL-6, IL-1, and IL-10 but had no effect on TNF. Co-incubation with benzamil, a more potent ENaC inhibitor, reduced all 4 cytokines at nmol/L concentrations. In conclusion, amiloride-mediated blood pressure reduction in the hypertensive patients was not mediated by IL-17A reduction. By contrast, amiloride lowered TNF and IL-6 both in vitro and in vivo. Thus, ENaC contributes to macrophage stimulation and is a relevant non-renal target to suppress inflammation and potentially blood pressure in patients with type 2 diabetes and hypertension.


Study III: Mineralocorticoid receptor blockade with spironolactone has no direct effect
on plasma IL-17A but lowers injury markers in urine from kidney transplant patients


We hypothesized that MR antagonism by spironolactone reduces IL-17A production in plasma and relates to spironolactone-mediated blood pressure changes and renal epithelial protection in kidney transplant patients. Plasma and urine samples were obtained from kidney transplant patients from a multicenter, double-blind, placebo-controlled intervention study including kidney transplant patients, that received either spironolactone (25-50 mg/daily) or placebo for 3 years. Plasma and urine samples from study inclusion and after 1 year intervention were analyzed for cytokine or kidney injury markers, respectively. Plasma IL-17A was unchanged in response to spironolactone treatment but increased in the placebo group. No changes were observed in other T-cell and macrophage-derived cytokines. Plasma aldosterone did not relate to 24hr ambulatory MAP or plasma IL-17A at baseline. Plasma IL-17A did also not relate to 24h ambulatory MAP measurements at baseline. The kidney injury markers calbindin and trefoil factor 3 (TFF3) were decreased after 1-year spironolactone treatment with no changes in the placebo group. Baseline urine calbindin/creatinine and TFF3/creatinine ratios did not relate to baseline plasma aldosterone, but TFF3/creatinine ratios related positively to blood pressure at baseline. When assessing differences in cytokine response of patients that received angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARBs), and patients that did not receive these drugs, no difference in plasma cytokine production was observed. We conclude that MR blockade may stabilize IL-17A production and suppress epithelial injury markers associated with the distal nephron.


Study IV: Interleukin 17A lowers blood pressure at baseline and after ANGIIhypertension in conscious unrestrained male WT mice


We hypothesized that 1) chronic i.v. infusion of IL-17A in conscious mice would increase blood pressure; that 2) ANGII-induced blood pressure elevation would be accentuated by IL-17A by renal mechanisms; and that 3) acute bolus i.v. infusion of IL-17A would cause acute blood pressure increase by vascular effects. When mice received chronic i.v. IL-17A infusion for 8 days in a step-up model, hypertensive effects were not observed, rather significant hypotensive effects were detected. IL-17A also did not accentuate ANGII-mediated hypertension in mice, but rather lowered ANGII-mediated blood pressure elevation compared to blood pressure in ANGII-vehicle co-infused mice. Acute bolus infusion did not show any acute vascular effects either. Plasma IL-17A levels were measured to be up to 3500 times above baseline levels and activation of the downstream inflammatory IL-6 cytokine was observed by increased plasma IL-6 in mice and in vitro in IL-17A stimulated fibroblasts. We concluded that IL-17A by itself, does not have any direct hypertensive effects, but instead lowers blood pressure.


Altogether the 3 clinical studies including hypertensive patients treated with a MR inhibitor, spironolactone, or ENaC blocker, amiloride, revealed no effect on IL-17A production in plasma of these patients. Instead, our data indicated a minor, but non-redundant antiinflammatory effect by detection of reduced levels of other T-cell and macrophage-derived cytokines including IFN-γ, IL-6, and TNF. Chronic and acute i.v. infusion of IL-17A in mice showed no direct hypertensive effect, but rather a blood pressure lowering effect at nonphysiologically high concentrations. Thus, it is concluded that IL-17A by itself is not a hypertensive protein but may in concert with other cytokines or unknown factors, contribute to inflammation-mediated hypertension.