Ashlee J. Tipton

Female spontaneously hypertensive rats have greater renal anti-inflammatory T lymphocyte infiltration than males

T cells contribute to hypertension in male experimental models; data in females is lacking even though women are more likely to develop immune disorders. The goal of this study was to determine whether immune cells contribute to hypertension in female spontaneously hypertensive rats (SHR) and define the T cell profile in whole blood and kidneys of male and female SHR. We hypothesized that inflammatory cells contribute to hypertension in female SHR; however, male SHR have a higher blood pressure so we hypothesize they will have a heightened inflammatory profile. The lymphocyte inhibitor mycophenolate mofetil (MMF) was administered in a dose-dependent manner to SHR. At the highest dose (50 mg·kg(-1)·day(-1)), blood pressure was significantly decreased in both sexes, yet the percent decrease in blood pressure was greater in females (female: 12 ± 1%; males: 7 ± 1%, P = 0.01). Circulating and renal T cell profiles were defined using analytical flow cytometry. Female SHR had more circulating CD3(+), CD4(+), and pro-inflammatory CD3(+)CD4(+)RORγ(+) Th17 cells, whereas males had more immune-suppressive CD3(+)CD4(+)Foxp3(+) T regulatory cells. In the kidney, females had greater numbers of CD8(+) and T regulatory cells than males, whereas males had greater CD4(+) and Th17 cell infiltration. MMF decreased circulating and renal T cells in both sexes (P < 0.0001), although the effect of MMF on T cell subtypes was sex specific with females having greater sensitivity to MMF-induced decreases in lymphocytes. In conclusion, there is a lymphocyte contribution to the maintenance of hypertension in the female SHR and sex of the animal impacts the T cell profile.

Female Spontaneously Hypertensive Rats Have a Compensatory Increase in Renal Regulatory T Cells in Response to Elevations in Blood Pressure

Female spontaneously hypertensive rats (SHR) have more regulatory T cells (Tregs) in their kidneys than males. The goal of this study was to determine the impact of blood pressure (BP) on the renal immune profile. We hypothesize that increases in BP promote a proinflammatory renal T cell and cytokine profile in SHR, although females will have greater hormone-dependent increases in Tregs and males will have greater increases in Th17 cells. Renal T cell and cytokine profiles were assessed in male and female Wistar-Kyoto rats and male and female SHR treated with vehicle or hydrochlorothiazide and reserpine (HCTZ) from 6 to 12 (6-HCTZ) or 11 to 13 weeks of age (2-HCTZ). Regardless of sex, SHR had a more proinflammatory renal immune profile than Wistar-Kyoto rats. 6-HCTZ attenuated age-related increases in BP and 2-HCTZ reversed hypertension compared with vehicle-treated SHR. Neither 6-HCTZ nor 2-HCTZ altered CD3+, CD4+, or CD8+ T cells in either sex. Both treatments decreased Tregs only in female SHR abolishing sex differences in Tregs. 6-HCTZ has no impact on Th17 cells in either sex and 2-HCTZ had a minimal impact on renal Th17 cells. To further assess mechanisms mediating sex differences in the renal immune profile, male and female SHR were gonadectomized to determine the impact of sex hormones. Gonadectomy increased proinflammatory markers in both sexes, suggesting that both male and female sex hormones are anti-inflammatory. In conclusion, BP contributes to sex differences in the renal T-cell profile of SHR; female SHR increase renal Tregs in response to increases in BP.

Female SHR have greater blood pressure sensitivity and renal T cell infiltration following chronic NOS inhibition than males

Nitric oxide is a critical regulator of blood pressure (BP) and inflammation, and female spontaneously hypertensive rats (SHR) have higher renal nitric oxide bioavailability than males. We hypothesize that female SHR will have a greater rise in BP and renal T cell infiltration in response to nitric oxide synthase (NOS) inhibition than males. Both male and female SHR displayed a dose-dependent increase in BP to the nonspecific NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME: 2, 5, and 7 mg·kg(-1)·day(-1) for 4 days each); however, females exhibited a greater increase in BP than males. Treatment of male and female SHR with 7 mg·kg(-1)·day(-1) L-NAME for 2 wk significantly increased BP in both sexes; however, prior exposure to L-NAME only increased BP sensitivity to chronic NOS inhibition in females. L-NAME-induced hypertension increased renal T cell infiltration and indices of renal injury in both sexes, yet female SHR exhibited greater increases in Th17 cells and greater decreases in regulatory T cells than males. Chronic L-NAME was also associated with larger increases in renal cortical adhesion molecule expression in female SHR. The use of triple therapy to block L-NAME-mediated increases in BP attenuated L-NAME-induced increases in renal T cell counts and normalized adhesion molecule expression in SHR, suggesting that L-NAME-induced increases in renal T cells were dependent on both increases in BP and NOS inhibition. Our data suggest that NOS is critical in the ability of SHR, females in particular, to maintain BP and limit a pro-inflammatory renal T cell profile.

Sex differences in T cells in hypertension.

PURPOSE Hypertension is a major risk factor for cardiovascular disease, stroke, and end-organ damage. There is a sex difference in blood pressure (BP) that begins in adolescence and continues into adulthood, in which men have a higher prevalence of hypertension compared with women until the sixth decade of life. Less than 50% of hypertensive adults in the United States manage to control their BP to recommended levels using current therapeutic options, and women are more likely than are men to have uncontrolled high BP. This, is despite the facts that more women compared with men are aware that they have hypertension and that women are more likely to seek treatment for the disease. Novel therapeutic targets need to be identified in both sexes to increase the percentage of hypertensive individuals with controlled BP. The purpose of this article was to review the available literature on the role of T cells in BP control in both sexes, and the potential therapeutic application/implications of targeting immune cells in hypertension. METHODS A search of PubMed was conducted to determine the impact of sex on T cell-mediated control of BP. The search terms included sex, gender, estrogen, testosterone, inflammation, T cells, T regulatory cells, Th17 cells, hypertension, and blood pressure. Additional data were included from our laboratory examinations of cytokine expression in the kidneys of male and female spontaneously hypertensive rats (SHRs) and differential gene expression in both the renal cortex and mesenteric arterial bed of male and female SHRs. FINDINGS There is a growing scientific literature base regarding the role of T cells in the pathogenesis of hypertension and BP control; however, the majority of these studies have been performed exclusively in males, despite the fact that both men and women develop hypertension. There is increasing evidence that although T cells also mediate BP in females, there are distinct differences in both the T-cell profile and the functional impact of sex differences in T cells on cardiovascular health, although more work is needed to better define the relative impact of different T-cell subtypes on BP in both sexes. IMPLICATIONS The challenge now is to fully understand the molecular mechanisms by which the immune system regulates BP and how the different components of the immune system interact so that specific mechanisms can be targeted therapeutically without compromising natural immune defenses.

Chronic Ang II Infusion Induces Sex-Specific Increases in Renal T cells in Sprague Dawley Rats

Recent studies suggest that sex of the animal and T cell impact ANG II hypertension in Rag(-/-) mice, with females being protected relative to males. This study tested the hypothesis that ANG II results in greater increases in proinflammatory T cells and cytokines in males than in females. Male and female Sprague-Dawley (SD) rats, aged 12 wk, were treated with vehicle or ANG II (200 ng·kg(-1)·min(-1)) for 2 wk. Renal CD4(+) T cells and Tregs were comparable between vehicle-treated males and females, although males expressed more Th17 and IL-17(+) T cells and fewer IL-10(+) T cells than females. ANG II resulted in greater increases in CD4(+) T cells, Th17 cells, and IL-17(+) cells in males; Tregs increased only in females. We previously showed that ANG (1-7) antagonizes ANG II-induced increases in blood pressure in females and ANG (1-7) has been suggested to be anti-inflammatory. Renal ANG (1-7) levels were greater in female SD at baseline and following ANG II infusion. Additional rats were treated with ANG II plus the ANG (1-7)-mas receptor antagonist A-779 (48 μg·kg(-1)·h(-1)) to test the hypothesis that greater ANG (1-7) in females results in more Tregs relative to males. Inhibition of ANG (1-7) did not alter renal T cells in either sex. In conclusion, ANG II induces a sex-specific effect on the renal T cell profile. Males have greater increases in proinflammatory T cells, and females have greater increases in anti-inflammatory Tregs; however, sex differences in the renal T cell profile are not mediated by ANG (1-7).

Female Spontaneously Hypertensive Rats Have a Compensatory Increase in Renal Regulatory T Cells in Response to Elevations in Blood PressureNovelty and Significance

Female spontaneously hypertensive rats (SHR) have more regulatory T cells (Tregs) in their kidneys than males. The goal of this study was to determine the impact of blood pressure (BP) on the renal immune profile. We hypothesize that increases in BP promote a proinflammatory renal T cell and cytokine profile in SHR, although females will have greater hormone-dependent increases in Tregs and males will have greater increases in Th17 cells. Renal T cell and cytokine profiles were assessed in male and female Wistar-Kyoto rats and male and female SHR treated with vehicle or hydrochlorothiazide and reserpine (HCTZ) from 6 to 12 (6-HCTZ) or 11 to 13 weeks of age (2-HCTZ). Regardless of sex, SHR had a more proinflammatory renal immune profile than Wistar-Kyoto rats. 6-HCTZ attenuated age-related increases in BP and 2-HCTZ reversed hypertension compared with vehicle-treated SHR. Neither 6-HCTZ nor 2-HCTZ altered CD3+, CD4+, or CD8+ T cells in either sex. Both treatments decreased Tregs only in female SHR abolishing sex differences in Tregs. 6-HCTZ has no impact on Th17 cells in either sex and 2-HCTZ had a minimal impact on renal Th17 cells. To further assess mechanisms mediating sex differences in the renal immune profile, male and female SHR were gonadectomized to determine the impact of sex hormones. Gonadectomy increased proinflammatory markers in both sexes, suggesting that both male and female sex hormones are anti-inflammatory. In conclusion, BP contributes to sex differences in the renal T-cell profile of SHR; female SHR increase renal Tregs in response to increases in BP.

Blood Pressure, Sex, and Female Sex Hormones Influence Renal Inner Medullary Nitric Oxide Synthase Activity and Expression in Spontaneously Hypertensive Rats

Background We previously reported that sexually mature female spontaneously hypertensive rats (SHRs) have greater nitric oxide (NO) synthase (NOS) enzymatic activity in the renal inner medulla (IM), compared to age‐matched males. However, the mechanisms responsible for this sexual dimorphism are unknown. The current study tested the hypothesis that sex differences in renal IM NOS activity and NOS1 expression in adult SHRs develop with sexual maturation and increases in blood pressure (BP) in a female sex hormone‐dependent manner. Methods and Results Renal IM were isolated from sexually immature 5‐week‐old and sexually mature 13‐week‐old male and female SHRs. Whereas NOS activity and NOS1 expression were comparable in 5‐ and 13‐week‐old male SHRs and 5‐week‐old female SHRs, 13‐week‐old females had greater NOS activity and NOS1 expression, compared to 5‐week‐old female SHRs and age‐matched males. NOS3 expression was greater in 5‐week‐old than 13‐week‐old SHRs regardless of sex. Treatment with antihypertensive therapy (hydrochlorothiazide and reserpine) from 6 to 12 weeks of age to attenuate age‐related increases in BP abolished the sex difference in NOS activity and NOS1 expression between sexually mature SHR males and females. To assess the role of female sex hormones in age‐related increases in NOS, additional females were ovariectomized (OVX), and NOS activity was studied 8 weeks post‐OVX. OVX decreased NOS activity and NOS1 expression. Conclusions The sex difference in renal IM NOS in SHR is mediated by a sex hormone‐ and BP‐dependent increase in NOS1 expression and NOS activity exclusively in females.

Sex Differences in Blood Pressure Control: Are T Lymphocytes the Missing Link?

See related article, pp 384–390 There is an ever expanding literature base implicating T lymphocytes in the development and progression of numerous cardiovascular diseases, including hypertension. T lymphocytes contribute to the development of hypertension in genetic, angiotensin II (Ang-II), and salt-sensitive male experimental animals.1 Among the most definitive studies implicating T lymphocytes in hypertension are studies conducted in Rag-1−/− mice, which lack B and T lymphocytes. Guzik et al2 were the first to demonstrate that these mice have a blunted hypertensive response to Ang-II infusion. Adoptive transfer of T lymphocytes into male Rag−/− mice restored the hypertensive response to Ang-II; adoptive transfer of B lymphocytes did not alter the blood pressure (BP) response. Although low-grade inflammation, and T lymphocytes in particular, are now a recognized hallmark of hypertension, the majority of basic science literature in this field has been conducted exclusively in males, despite the fact that females account for ≈50% of all hypertensive cases in the United States. Therefore, it was with great interest that we read the study by Pollow et al3 in the current issue of Hypertension , which was designed to determine (1) whether there are sex differences in the ability of T lymphocytes to induce Ang-II–dependent hypertension and (2) whether sex affects central or renal T lymphocytes infiltration after Ang-II hypertension. Of particular interest, they found that male mice exhibited a significant increase in BP and renal damage to Ang-II after the adoptive transfer of CD3+ T lymphocytes from wild-type male mice. In contrast, BP responses and renal injury to Ang-II were not significantly altered in female Rag−/− mice after adoptive transfer of T lymphocytes from males. Male Rag−/− mice also had greater renal CD3+, CD4+, CD8+, and T-regulatory cells (Tregs) …

Greater transforming growth factor-β in adult female SHR is dependent on blood pressure, but does not account for sex differences in renal T-regulatory cells

Female spontaneously hypertensive rats (SHR) have more renal regulatory T cells (Tregs) than males, and greater levels of Tregs in female SHR are dependent on blood pressure (BP). However, the molecular mechanism responsible for greater Tregs in female SHR is unknown. Transforming growth factor (TGF)-β is a pleiotropic cytokine critical in the differentiation of naïve T cells into Tregs, and female SHR have higher TGF-β excretion than male SHR. The goals of the current study were to test the hypotheses that 1) female SHR have greater renal TGF-β expression than male SHR, which is dependent on BP and 2) neutralizing TGF-β will decrease renal Tregs in female SHR. Renal cortices were isolated from 5- and 13-wk-old male and female SHR, and TGF-β levels were measured via Western blot and ELISA. Adult female SHR have more free, active TGF-β1 than 5-wk-old female SHR (46% more) or male SHR (44% more than 5-wk-old males and 56% more than 13-wk-old male SHR). We confirmed greater TGF-β1 in adult female SHR was due to increases in BP and not sexual maturation by measuring TGF-β1 levels following treatment with BP-lowering drugs or ovariectomy. Separate female SHR were treated with an antibody to TGF-β1,2,3; BP was measured, and T cells were assessed in whole blood and the kidney. Neutralizing TGF-β had no effect on BP, although circulating Tregs decreased by 32%, while Th17 cells increased by 64%. Renal Tregs were not altered by antibody treatment, although Th17 cells were decreased by 61%. In conclusion, although TGF-β promotes circulating Tregs in female SHR, it does not account for the sex difference in renal Tregs in SHR.

Female SHR Have a Compensatory Increase in Renal Regulatory T Cells in Response to Elevations in Blood Pressure

Female spontaneously hypertensive rats (SHR) have more regulatory T cells (Tregs) in their kidneys than males. The goal of this study was to determine the impact of blood pressure (BP) on the renal immune profile. We hypothesize that increases in BP promote a proinflammatory renal T cell and cytokine profile in SHR, although females will have greater hormone-dependent increases in Tregs and males will have greater increases in Th17 cells. Renal T cell and cytokine profiles were assessed in male and female Wistar-Kyoto rats and male and female SHR treated with vehicle or hydrochlorothiazide and reserpine (HCTZ) from 6 to 12 (6-HCTZ) or 11 to 13 weeks of age (2-HCTZ). Regardless of sex, SHR had a more proinflammatory renal immune profile than Wistar-Kyoto rats. 6-HCTZ attenuated age-related increases in BP and 2-HCTZ reversed hypertension compared with vehicle-treated SHR. Neither 6-HCTZ nor 2-HCTZ altered CD3+, CD4+, or CD8+ T cells in either sex. Both treatments decreased Tregs only in female SHR abolishing sex differences in Tregs. 6-HCTZ has no impact on Th17 cells in either sex and 2-HCTZ had a minimal impact on renal Th17 cells. To further assess mechanisms mediating sex differences in the renal immune profile, male and female SHR were gonadectomized to determine the impact of sex hormones. Gonadectomy increased proinflammatory markers in both sexes, suggesting that both male and female sex hormones are anti-inflammatory. In conclusion, BP contributes to sex differences in the renal T-cell profile of SHR; female SHR increase renal Tregs in response to increases in BP.