Sex differences in TLR4 expression in SHR do not contribute to sex differences in blood pressure or the renal T cell profile

Abstract

Hypertension is a primary risk factor for the development of cardiovascular disease. Mechanisms controlling blood pressure (BP) in men and women are still being investigated; however, there is increasing evidence supporting a role for the innate immune system. Specifically, Toll-like receptors (TLRs), and TLR4 in particular, have been implicated in the development of hypertension in male spontaneously hypertensive rats (SHR). Despite established sex differences in BP control and inflammatory markers in hypertensive males and females, little is known regarding the role of TLR4 in hypertension in females. Our hypotheses were that male SHR have greater TLR4 expression compared with females, and that sex differences in TLR4 contribute to sex differences in BP and the T cell profile. To test these hypotheses, initial studies measured renal TLR4 protein expression in 13-wk-old male and female SHR. Additional SHR were implanted with telemetry devices and randomized to treatment with either IgG or TLR4 neutralizing antibodies. Untreated control male SHR have greater TLR4 protein expression in the kidney compared with females. However, treatment with TLR4 neutralizing antibody for 2 wk did not significantly alter BP in either male or female SHR. Interestingly, neutralization of TLR4 increased renal CD3⁺ T cells in female SHR, with no alteration in CD4⁺ T cells or CD8⁺ T cells in either sex. Taken together, our data indicate that although male SHR have greater renal TLR4 expression than females, TLR4 does not contribute to the higher BP and more proinflammatory renal T cell profile in males versus females.

INTRODUCTION

Hypertension is a primary risk factor for the development of cardiovascular disease, the leading cause of death for both men and women (1). Despite increases in the incidence and prevalence of hypertension in both sexes, the mechanisms controlling blood pressure (BP) in men and women are still being investigated. Recent studies have established that hypertension is a chronic inflammatory disease characterized by increased T cell recruitment and activation (2), yet the mechanism(s) driving T cell activation in either sex are still being investigated. An increased understanding of the mechanisms inducing inflammation in hypertension will allow for the development of novel therapeutics to improve BP control rates in hypertensive males and females.

Sex differences in hypertension, with males having higher BP compared with age-matched females, have been well described in multiple experimental animal models including spontaneously hypertensive rats (SHR) (3), Dahl salt-sensitive rats (4), angiotensin II hypertension (5), and deoxycorticosterone acetate (DOCA)-salt hypertensive rats (6). Importantly, in each of these experimental models of hypertension, males also have a more proinflammatory T cell profile (7–11), implicating a role for the immune system not only in the development of hypertension but also in the development of sex differences in hypertension.

Although the specific mechanisms initiating T cell activation in hypertension are still being investigated, there is increasing evidence supporting a role for Toll-like receptors (TLRs) in hypertension (12, 13). TLRs recognize pathogen-associated molecular patterns and damage-associated molecular patterns leading to the initiation of an immune response, production of cytokines and chemokines (14), and T cell activation (15, 16). TLR4 in particular has been implicated in the development of hypertension in males (15, 17). Male SHR have greater TLR4 mRNA expression and protein abundance in mesenteric resistance arteries, vascular smooth muscle cells, and aorta compared with normotensive controls, and neutralization of TLR4 decreases BP and improves vascular function in male SHR (15, 18, 19). TLR4 knockout mice have also been shown to have an attenuated increase in BP in response to nitric oxide synthase inhibition compared with wild-type mice (20). However, not all studies in hypertensive male rodents support a decrease in BP with inhibition of TLR4 (21), and there is a lack of data regarding the role of TLR4 in experimental models of hypertension in females.

Despite established sex differences in BP control and inflammatory markers in hypertensive males and females, little is known regarding the role of TLR4 in hypertension in females. The current study was designed to test the hypothesis that male SHR have greater TLR4 expression compared with females, and that sex differences in TLR4 contribute to sex differences in BP and the T cell profile.

MATERIALS AND METHODS

Animals

Nine-week-old spontaneously hypertensive rats (SHR) were purchased from Envigo Inc. (Indianapolis, IN) and rats were studied at 13–14 wk of age. All animal procedures were approved by the Augusta University Animal Care and Use Committee (IACUC, Approval No. 2014-048) and were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were housed in humidity and temperature-controlled, light-cycled quarters, and maintained on standard chow and water ab libitum. Initial studies included control, 13-wk-old untreated male and female SHR. An additional set of rats were implanted with a radio telemetry device (Data Sciences International, St. Paul, MN) at 10 wk of age for the continuous measurement of mean arterial pressure (MAP). All rats were allowed 1-wk recovery after surgery and 1 wk of baseline BP recording before being randomized to treatment with IgG or TLR4 neutralizing antibodies. Rats received daily intraperitoneal (ip) injections of 1 µg of TLR4 neutralizing antibody in 200 µL saline (Santa Cruz Biotechnology sc-13591), or 1 µg of control IgG in 200 µL of saline. Dose and route of administration were chosen based on previous publications in male SHR (22). After 15 days of treatment, rats were anesthetized with isoflurane, a thoracotomy was performed, and a terminal blood sample was obtained by aortic puncture. The kidneys were isolated for biochemical and flow cytometric analyses.

TLR4 Western Blot

Kidneys were harvested from male and female SHR and 30 mg of whole kidney was homogenized. Whole homogenate was used for Western blot analysis as previously described (23). Briefly, 30 µg of kidney homogenate was resolved on 4%–20% Tris-glycine-SDS gel (Bio-Rad, Hercules, CA) and proteins were transferred to PVDF membranes (Millipore, Sigma, Burlington, MA). Protein expression was determined using two-color immunoblots using a primary antibody to TLR4 (Cat. No 48–2300, 1:300 dilution; Thermo Fisher Scientific). The antibody used has previously been shown to be specific for TLR4 in rats (24). Specific protein bands were detected using an Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE) with Alexa Fluor 680 conjugated secondary antibody (Molecular Probes, Eugene, OR; 1:10,000 dilution). Protein concentrations were determined by standard BCA reagent (Thermo Scientific) using BSA as the standard. Protein loading was normalized to β-actin (Cat. No. A1978, Sigma Aldrich, St. Louis, MO; 1:10,000 dilution).

Quantitative-Polymerase Chain Reaction

Total RNA was extracted from kidneys (n = 4 or 5/group) using RNA extraction miRNeasy mini-kit according to the manufacturer’s instructions (Qiagen; Cat No. 217004). One microgram of total RNA was reverse transcribed to prepare cDNA using iScript Reverse Transcription supermix according to the manufacturer’s instructions (Bio-Rad; Cat. No.1708844). qPCR was performed using iTaq Universal SYBR Green supermix according to the manufacturer’s instructions (Bio-Rad; Cat. No. 1725121). The following gene-specific predesigned primers were used from Qiagen: Rat IL-1β (Cat. No. QT00181657) and rat IL6 (Cat. No. 330001). Rat TNFα from IDT (5′-AAATGGGCTCCCTCTCATCAGTTC-3′; 5′-TCTGCTTGGTGGTTTGCTACGAC-3′). The amount of DNA was normalized to GAPDH (Qiagen; Cat. No. QT00199633). Data were normalized to control and expressed as fold change from control male SHR. All amplifications were conducted in duplicate.

Analytical Flow Cytometry

T cells and cytokines were measured in kidney samples as previously described (25, 26). Briefly, phenotypic and intracellular analyses were performed by incubating cells with antibodies for T cell surface markers CD3 (1:100, Invitrogen, Cat. No. 46–003082), CD4 (1:100, BD Biosciences, Cat. No. 554837), and CD8 (1:100, BD Biosciences, Cat. No. 554857). After a wash, cells were fixed and permeabilized using fix/perm concentrate (eBioscience, San Diego, CA) before incubation with antibodies for intracellular staining of Foxp3 (1:100, Invitrogen, Cat. No. 17–577382) to identify regulatory T cells (Tregs; expressed as %CD3⁺ CD4⁺ cells), related orphan receptor (RoR)-γt (1:100, R&D Systems, Cat. No. IC60006p) to identify T helper (Th)17 cells (expressed as %CD3⁺ CD4 cells), as well as IL-10 cytokines (1:100, NovusBio, Cat. No. 151262; expressed as %CD3⁺ CD4 cells). Cells were then washed and run through a four-color flow cytometer (FACSCalibur, BD Biosciences), and data were collected using CellQuest software as previously described (25, 26). Proper compensation was set to ensure that the median fluorescence intensities of negative and positive cells were identical and then was used to gate the population. Gating excluded dead cells and debris using forward and side scatter plots. Data were analyzed using either CellQuest Pro (BD Biosciences) or FlowJo 10.5.3 software (Becton, Dickinson & Company, Franklin Lakes, NJ).

Statistical Analysis

Western blot data of TLR4 expression in male and female kidneys were compared via Student’s t test. Telemetry data were analyzed using repeated-measures analysis of variance (ANOVA). Effect of sex and treatment on renal T cell profile, TLR4 expression, and renal cytokine mRNA expression were analyzed using a two-way ANOVA. Tukey’s post hoc analyses were performed for multiple comparison. Statistical analyses were performed using GraphPad Prism version 9.01 software (GraphPad Software, La Jolla, CA). All data were presented as means ± SE. P < 0.05 was considered significant.

RESULTS

Male SHR Have Greater Renal TLR4 Protein Abundance Compared with Females

Initial studies measured TLR4 protein abundance in whole kidney homogenates from control, untreated 13-wk-old male and female SHR via Western blot analysis. TLR4 protein was detected in the kidney of both male and female SHR. However, male SHR had greater TLR4 protein abundance compared with females (Fig. 1; P = 0.049).

Figure 1.

Male SHR have greater renal TLR4 protein level vs. females. TLR4 protein abundance was measured via Western blot analysis in whole kidney tissue homogenate in 13-wk-old male (M) and female (F) spontaneously hypertensive rats (SHR; n = 12 rats/sex). A: representative blot and B: average densitometric analysis. TLR4 protein abundance is presented normalized to β-actin. Data are expressed as means ± SE. Males are represented by squares and females by circles. Data were compared via Student’s t test. SHR, spontaneously hypertensive rats; TLR, Toll-like receptor.

Anti-TLR4 Treatment Decreases Renal Inflammation in Both Sexes

TLR4 activation leads to the activation and release of proinflammatory mediators, including TNF-α and IL-1β (15, 21, 27). Neutralization of TLR4 in male SHR has previously been shown to decrease the release of TNF-α and IL-1β and decrease TLR4 expression (22, 28, 29). To confirm that anti-TLR4 treatment was successful, renal TNF-α and IL-1β renal mRNA expression were measured by RT-qPCR and TLR4 abundance was measured by Western blot in vehicle and anti-TLR4-treated male and female SHR (Fig. 2). Males had greater renal mRNA expression of both TNF-α (P_sex = 0.05) and IL-1β (P_sex = 0.04) than females. Fifteen days of anti-TLR4 treatment significantly decreased renal TNF-α (P_treatment = 0.0001; P_interaction = 0.83) and IL-1β (P_treatment = 0.0001; P_interaction = 0.59) mRNA expression to a comparable degree in both sexes. TLR4 protein abundance was not altered in male or female SHR treated with anti-TLR4 compared with IgG-treated control groups (P_sex= 0.26 P_treatment = 0. 35; P_interaction = 0.067).

Figure 2.

Anti-TLR4 treatment decreases renal inflammation in both sexes. Renal TNF-α (A) and IL-12 (B) mRNA expression were measured via RT-PCR and TLR4 abundance (C) was measured via Western blot in male and female SHR randomized to control anti-IgG (n = 4–5 rats/sex) or anti-TLR4 SHR (n = 4 or 5). C: representative blot with average densitometric analysis. Filled symbols indicate control IgG-treated animals, unfilled symbols indicate anti-TLR4 antibody-treated animals. Males are represented by squares and females by circles. Data are expressed as means ± SE. Data were analyzed using two-way ANOVA with Tukey’s post hoc analyses for multiple comparisons. SHR, spontaneously hypertensive rats; TLR, Toll-like receptor.

Treatment with TLR4-Neutralizing Antibody Did Not Alter BP in SHR

TLR4 has been implicated in the development of hypertension in male SHR (15, 21, 22). One goal of this study was to determine the relative contribution of TLR4 to BP control in male and female SHR. Twenty-four-hour mean arterial BP was measured via telemetry in male and female SHR treated with control or TLR4-neutralizing antibody from 12 to 14 wk of age (Fig. 3). Before treatment was initiated, males had a higher BP than females (150 ± 1 vs. 133 ± 1; P < 0.0001). BP was comparable between control and anti-TLR4-treated males (151 ± 2 vs. 149 ± 2 mmHg, respectively; P > 0.99) and females before treatment (131 ± 1 vs. 134 ± 2 mmHg, respectively; P > 0.99). Treatment with anti-TLR4 did not significantly alter BP in either male or female SHR. Terminal BP’s were not significantly different between control and anti-TLR4-treated male (153 ± 2 vs. 148 ± 2 mmHg; P = 0.98) or female SHR (134 ± 2 vs. 140 ± 5; P = 0.46). However, males maintained a higher BP than females throughout the study (P < 0.0001).

Figure 3.

Inhibition of TLR4 does not alter blood pressure (BP) in male or female SHR. Twenty-four-hour mean arterial pressure (MAP) was measured via telemetry in male (M) and female (F) SHR randomized to control anti-IgG (n = 5 rats/sex) or anti-TLR4 treatment (n = 5 rats/sex). Filled symbols indicate control IgG-treated animals, unfilled symbols indicate anti-TLR4 antibody-treated animals. Males are represented by squares and females by circles. Data are expressed as means ± SE. Data within each sex were analyzed using repeated-measures ANOVA with Tukey’s multiple-comparisons test and between group comparisons were made using Student’s t test. SHR, spontaneously hypertensive rats; TLR, Toll-like receptor.

TLR4 Does Not Contribute to Sex Differences in the Renal T Cell Profile

TLR4 activation is proinflammatory and can lead to T cell activation (17, 30, 31), which is known to contribute to the development of hypertension. Renal T cells were measured via flow cytometric analysis (Fig. 4). Interestingly, female SHR had more CD3⁺ T cells (P_sex = 0.008), Tregs (P_sex < 0.0001), and IL-10⁺ kidney cells (P_sex = 0.002) than males whereas males had more CD4⁺ T cells (P_sex = 0.003) and RORgγ T cells (P_sex: P = 0.03). Renal CD8⁺ T cells were comparable between the sexes (P_sex = 0.18). Following 15 days of anti-TLR4 treatment, there was a significant increase in CD3⁺ T cells only in females (P_treatment = 0.023; P_interaction = 0.0009). Anti-TLR4 did not alter CD4⁺ T cells (P_treatment = 0.99; P_interaction = 0.81), CD8⁺ T cells (P_treatment = 0.48; P_interaction = 0.14), Tregs (P_treatment = 0.57; P_interaction = 0.63), RORgγ T cells (P_treatment = 0.37; P_interaction = 0.23), or IL-10⁺ kidney cells in either sex (P_treatment = 0.42; P_interaction = 0.58).

Figure 4.

Renal T cell profiles were largely unaltered by anti-TLR4 treatment in male or female SHR. Renal T cell profiles were measured via flow cytometric analysis in male and female SHR randomized to control anti-IgG (n = 5) and anti-TLR4 SHR (n = 5 rats/sex). Total CD3⁺ T cells are expressed as a percentage of total renal cells (A). CD4⁺ T cells (B) and CD8⁺ T cells (C) are expressed as a percentage of total CD3⁺ T cells. Foxp3⁺ T regulatory cells (Tregs; D) and related orphan receptor (ROR)γt⁺ T helper (Th)17 cells (E) are expressed as a percentage of CD3⁺CD4⁺ T cells. IL-10⁺ T cells are expressed as a percentage of total CD3⁺CD4⁺ T cells (F). Filled symbols indicate control IgG-treated animals, unfilled symbols indicate anti-TLR4 antibody-treated animals. Males are represented by squares and females by circles. Data are expressed as means ± SE. Data were analyzed using two-way ANOVA with Tukey’s post hoc analyses for multiple comparisons. SHR, spontaneously hypertensive rats; TLR, Toll-like receptor.

DISCUSSION

There are well-established sex differences in both the development of hypertension and the inflammatory response to hypertension, with males having greater increases in BP and inflammation than females (7, 8, 11). Previous studies have implicated TLR4 in the development and maintenance of hypertension in male experimental animals (14–16, 21, 22). However, there is a lack of information regarding the role of TLR4 in BP control in hypertensive females. The major finding of the current study is that despite male SHR having greater renal TLR4 protein abundance versus females, TLR4 does not contribute to sex differences in either BP or the renal T cell profile.

Male SHR have greater TLR4 expression than male Wistar rats in mesenteric arteries, aorta, and vascular smooth muscle cells and TLR4 expression increases with age and the development of hypertension in male SHR (15). Interestingly, treatment of male SHR with the AT₁ receptor blocker losartan decreases TLR4 expression in male SHR (18), further suggesting that TLR4 expression and BP are related. Previous studies focused on males. However, consistent with our recent findings (32), male SHR have greater renal TLR4 protein abundance versus females. Established sex differences in both BP and inflammatory markers in SHR (7, 33), coupled with the fact that TLR4 activation is proinflammatory lead to the further investigation of the relative contribution of TLR4 to BP control and the renal T cells profile in male and female SHR.

Activation of the innate immune system and TLRs is an attractive mechanism to explain increases in BP and inflammation in hypertension (14). Activation of TLR4 is of particular interest since TLR4 activation results in the increase of its own expression leading to further increases in inflammation (34). Indeed, TLR4 expression on peripheral monocytes is higher in patients with noncontrolled hypertension versus those with well-controlled hypertension, and there is a positive correlation between hypertension duration and TLR4 expression (35). Therefore, inhibiting TLR4 has been explored as a novel mechanism to decrease BP in hypertension (21, 36). In contrast to our findings, previous studies showed that treatment of 15-wk-old male SHR with daily intraperitoneal injection of 1 µg of anti-TLR4 antibody for 15 days significantly decreased terminal BP when measured via a pressure transducer in the right carotid artery (15, 18) or tail-cuff (18, 22). Similarly, angiotensin (ANG) II infusion in male C57BL6 mice increases aortic TLR4 expression and anti-TLR4 treatment attenuates ANG II-induced increases in systolic BP (19). In contrast, and consistent with our findings, anti-TLR4 treatment had no effect on systolic or diastolic BP when measured via carotid artery cannulation in 21-wk-old male SHR (37). Moreover, ANG II hypertension is not altered in TLR4 knockout mice (27, 38) and anti-TLR4 treatment did not reduce terminal BP in male C57BL6 mice following 28 days of ANG II infusion (39). It is important to note that the current study is the only one to continuously measure BP via telemetry during systemic delivery of anti-TLR4 antibodies. Moreover, all rats in the current study were allowed 2 wk to recover from telemetry implantation before initiating anti-TLR4 treatment. In contrast, studies implanting a carotid catheter to measure terminal BP would induce an inflammatory response, which could be mitigated by anti-TLR4. Alternatively, SHR are a model of hyperactivity (40), therefore it is plausible that anti-TLR4 treatment modulates an acute stress response associated with handling to limit increases in BP. Moreover, the rats in the current study were younger than those used in previous work in the literature, therefore it is possible that older SHR would have a different response. Further studies are needed to understand the relative contribution of age-dependent activation of TLR4 on BP regulation in SHR.

Previous studies in male SHR have further shown that anti-TLR4 treatment decreases contractility to noradrenaline (15, 22) and phenylephrine and increases dilation to acetylcholine (18). Anti-TLR4 treatment also attenuates cardiac remodeling in 21-wk-old male SHR (37). Therefore, even in the absence of a significant BP effect, inhibition of TLR4 has consistently been shown to have cardiovascular protective effects in males. This is likely related to the finding that anti-TLR4 treatment decreases inflammation (34, 41–44), a finding that is confirmed in the current study. TLR4 activation results in the activation of NF-κB, which mediates the transcription of COX-2, proinflammatory cytokines, and increases in oxidative stress (45). Therefore, it is not surprising that treatment of male SHR with anti-TLR4 antibody decreases IL-6 in serum and the vasculature, NF-κB activation, and COX-2 (15, 22, 37). The current study extends these findings to hypertensive females treated with anti-TLR4. We previously published that vascular function is impaired in male SHR versus both female SHR and male WKY while vascular function in female SHR is comparable with that in female WKY (46). As a result, sex differences in TLR4 may also contribute to sex differences in vascular function.

Despite the known proinflammatory effects of TLR-4 activation, less is known regarding the role of TLR4 in T cell activation in hypertension. Because of the central role of the kidney on long-term BP control and previous studies displaying significant sex differences within the renal immune cell profiles (7, 8, 11), studies assessed the renal T cell profile in male and female SHR following control and anti-TLR4 treatment. Our findings suggest that sex differences in TLR4 do not contribute to the T cell profile in either sex or to sex differences in renal T cells. However, TLR4 has been shown to have renal (45), central (29), and vascular effects (19, 22); future studies will examine the relative impact of treatment on different organ systems and if the dose and route of administration affects all pathways effectively and/or equally.

Perspectives and Significance

There remains significant interest in identifying the mechanisms controlling immune cell activation in hypertension. Despite evidence implicating TLR4 in hypertension, neutralization of TLR4 had no effect on BP control or the T cell profile in male or female SHR. However, BP is not the only cardiovascular complication associated with the activation of the innate immune system. Additional studies are needed to fully assess the cardiovascular implications of differential TLR4 expression in hypertensive males versus females.

GRANTS

This work was supported by the National Institutes of Health Grant P01 HL134604-05 (to J.C.S.) and the American Heart Association Grants EIA33410565 (to J.C.S.) and 20CDA35310362 (to R.M.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.C.W. and J.C.S. conceived and designed research; K.M.B. and R.M. performed experiments; K.M.B., R.M., and J.C.S. analyzed data; K.M.B. and J.C.S. interpreted results of experiments; K.M.B., R.M., and J.C.S. prepared figures; K.M.B. and J.C.S. drafted manuscript; K.M.B., R.M., R.C.W., and J.C.S. edited and revised manuscript; K.M.B., R.M., R.C.W., and J.C.S. approved final version of manuscript.