Chloroquine Suppresses the Development of Hypertension in Spontaneously Hypertensive Rats

Abstract

Background: Innate immune system responses to damage-associated molecular patterns (DAMPs) are involved in hypertension. However, the mechanisms of this contribution are not well understood. Circulating mitochondrial DNA is a DAMP that activates Toll-like receptor (TLR) 9 and is elevated in spontaneously hypertensive rats (SHR). Therefore, we hypothesized that lysosomotropic agent chloroquine (CQ) would impair TLR9 signaling, as well as prevent the development of hypertension and immune cell recruitment to the vasculature, in SHR.

Methods: Initially, adult SHR and Wistar-Kyoto (WKY) rats (12 weeks old), as well as a group of young SHR (5 weeks old), were treated with CQ (40mg/kg/day) or vehicle (saline) via intraperitoneal injections for 21 days and then TLR9-myeloid differentiation primary response protein (MyD88) signaling proteins were assessed in mesenteric resistance arteries (MRA) via western blot. Subsequently, young SHR and WKY were treated from 5-8 weeks of age and then were allowed to mature without further treatment. Blood pressure was measured pretreatment, posttreatment, and after maturation, and immune cell recruitment to the vasculature was measured via flow cytometry after maturation.

Results: In MRA from adult SHR, CQ increased the expression of MyD88-dependent proteins, whereas young SHR MRA exhibited a decrease. This inhibition was subsequently associated with suppression of blood pressure, as well as decreased counts of circulating T cells and vascular infiltrating leukocytes in SHR, when CQ was administered during the prehypertensive phase.

Conclusions: These data bring into question the participation of TLRs during the maintenance phase of hypertension and promote the exploration of innate immune system therapy during the critical developmental phase.

Keywords: Toll-like receptors.; blood pressure; hypertension; immune system.

Figure 1.

CQ inhibited MyD88-dependent signaling proteins in young, but not adult, SHR MRA. MRA were isolated from SHR treated with CQ or Veh for 21 days starting at 12 weeks of age (adult) or 5 weeks of age (young). Protein expression analysis for (a) TLR3, (b) TLR7, (c) TLR8, (d) TLR9, (e) MyD88, (f) IRAK, (g) TRAF6, and (h) TRIF, all normalized for β actin, in young and adult SHR MRA. Above, representative images of immunoblots; below, densitometric analysis. Sample sizes for statistical analyses are presented within figure. Student’s t-test: *P < 0.05 vs. Veh. Abbreviations: CQ, chloroquine; IRAK, interleukin-1-receptor-activated kinase; MRA, mesenteric resistance arteries; MyD88, myeloid differentiation primary response protein; SHR, spontaneously hypertensive rats; TRAF6, tumor-necrosis factor receptor-associated factor 6; TRIF, Toll-interleukin-1 receptor-domain-containing adaptor inducing interferon-β; Veh, vehicle.

Figure 2.

CQ inhibited noncanonical NF-κB signaling in young, but not adult, SHR MRA. MRA were isolated from SHR treated with CQ or Veh for 21 days starting at 12 weeks of age (adult) or 5 weeks of age (young). Protein expression analysis for (a) phospho-IκB normalized for total IκB and (b) phospho-p65 and total p65 normalized for β actin, as well as phospho-p65 normalized for total p65, in young and adult SHR MRA. Above, representative images of immunoblots; below, densitometric analysis. Sample sizes for statistical analyses are presented within figure. Student’s t-test: *P < 0.05 vs. Veh. Abbreviations: CQ, chloroquine; IκB, inhibitor of κB; MRA, mesenteric resistance arteries; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SHR, spontaneously hypertensive rats; Veh, vehicle.

Figure 3.

Prophylactic treatment with CQ during the prehypertensive phase of SHR prevented the development of hypertension and immune cell recruitment to the vasculature upon maturation to adulthood. (a) The values of SBP before treatment (5 weeks old), concluding treatment (8 weeks old), and following maturation to adulthood (12 weeks old) were measured in Veh- or CQ-treated WKY and SHR. Immune cell recruitment analysis included: (b) aortic accumulation of leukocytes (CD45⁺), (c) CD44⁺ expression on T cells (CD3⁺) from aorta, (d) circulating T cell (CD3⁺) counts (left, quantification; right, dot plots representing live gating for T cells from whole blood cells), and (e) CD44⁺ expression on circulating T cells (CD3⁺) from whole blood cells (left, quantification; right, representative histogram based on live gating for T cells—Figure 3E dot plots). Sample sizes for statistical analyses are presented within figure. Two-way (treatment × age) ANOVA and 1-way ANOVA: *P < 0.05 vs. WKY-Veh; ^† P < 0.05 vs. SHR-Veh. Abbreviations: ANOVA, analysis of variance; CQ, chloroquine; SBP, systolic blood pressure; SHR, spontaneously hypertensive rats; Veh, vehicle; WKY, Wistar–Kyoto.

Figure 4.

Hypothesized schematic of how the innate and adaptive immune systems synergize during the development and maintenance of hypertension. After an inappropriate spike in blood pressure due to genetics or a prohypertensive factor such as angiotensin II, high salt, or chronic stress, the innate immune system initially responds. With the continued presence of prohypertensive factors and increasing blood pressure, the contribution of the innate immune system begins to wane and the adaptive immune system becomes increasingly engaged. In a feed-forward manner, chronic pressure- and ischemic-induced cell death and remodeling in hypertension fosters the escalating presence of DAMPs and the continued participation of the adaptive immune system during the maintenance phase of hypertension. Therefore, we propose a critical therapeutic window during the development of hypertension if the innate immune system is to going be targeted. Abbreviation: DAMPs, damage-associated molecular patterns.