Chronic infusion of interleukin-17 promotes hypertension, activation of cytolytic natural killer cells, and vascular dysfunction in pregnant rats

Olivia K Travis, Dakota White, W Austin Pierce, Ying Ge, Cassandra Y Stubbs, Frank T Spradley, Jan M Williams, Denise C Cornelius, Olivia K Travis, Dakota White, W Austin Pierce, Ying Ge, Cassandra Y Stubbs, Frank T Spradley, Jan M Williams, Denise C Cornelius

Abstract

Previous studies by our lab have established that placental-ischemia stimulated T-helper 17 cells (TH 17s) cause increased cytolytic natural killer (cNK) cell proliferation and activation during pregnancy; however, the exact mechanism is unknown. The objective of this study was to investigate the role of interlukin 17 (IL-17) in inducing cNK cell activation in pregnancy. We infused 150 pg/day of recombinant IL-17 into a subset of normal pregnant (NP) Sprague Dawley rats from gestation day (GD) 12-19 (NP+IL-17). On GD 19, mean arterial pressure (MAP), fetal and placental weights, cytokines, cNK cell activation, cytotoxic enzymes, and vascular reactivity were assessed. MAP significantly increased from 99 ± 3 mmHg in NP to 120 ± 1 mmHg in NP+IL-17 (P < 0.05). Fetal weight significantly decreased from 2.52 ± 0.04 g in NP to 2.32 ± 0.03 g in NP+IL-17 as did placental weight (NP: 0.65 ± 0.03 g; NP+IL-17: 0.54 ± 0.01 g, P < 0.05). Plasma levels of TNF-α increased to 281.4 ± 55.07 pg/mL in NP+IL-17 from 145.3 ± 16.03 pg/mL in NP (P < 0.05) while placental levels of VEGF decreased from 74.2 ± 6.48 pg/mg in NP to 54.2 ± 3.19 pg/mg in NP+IL-17. Total NK cells were increased in the placenta (NP: 14.3 ± 3.49%; NP+IL-17: 29.33 ± 2.76%, P < 0.05) as were cytolytic NK cells (NP: 3.31 ± 1.25%; NP+IL-17: 13.41 ± 1.81%, P < 0.05). A similar trend was observed in circulating NK cells. Plasma granzyme K increased from 3.55 ± 2.29 pg/mL in NP to 20.9 ± 7.76 pg/mL in NP+IL-17 (P < 0.05), and plasma granzyme B increased from 10.95 ± 0.64 pg/mL in NP to 14.9 ± 0.98 pg/mL in NP+IL-17(P < 0.05). In the placenta, both granzyme A (NP: 246.1 ± 16.7 pg/mg; NP+IL-17: 324.3 ± 15.07 pg/mg, P < 0.05) and granzyme B (NP: 15.18 ± 3.79 pg/mg; NP+IL-17: 27.25 ± 2.34 pg/mg, P < 0.05) increased in response to IL-17 infusion. Finally, vascular reactivity of uterine arteries was significantly impaired in response to IL-17 infusion. The results of this study suggest that IL-17 plays a significant role in the activation of cNK cells during pregnancy.

Keywords: Hypertension; IL-17; natural killer cells; pregnancy; vascular reactivity.

Conflict of interest statement

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

© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Figures

Figure 1
Figure 1
Effect of IL‐17 infusion on circulating and placental NK cell profiles in pregnant rats. Chronic IL‐17 infusion into normal pregnant (NP) rats results in increased total and cytolytic natural killer (NK) cells in the placenta and circulation. On GD 19, blood and placentas were collected and processed to obtain percentages of (A) circulating total NK cells and (B) circulating cytolytic NK cells along with (C) placental total NK cells, and (D) placental cytolytic NK cells through flow cytometry analysis. All data are expressed as mean ± SEM. Statistical analyses were performed using unpaired Student's T‐test. *P < 0.05 versus NP.
Figure 2
Figure 2
Effects of IL‐17 Infusion on placental cytokines in pregnant rats. Chronic IL‐17 infusion into normal pregnant (NP) rats results in increased inflammatory cytokines and decreased VEGF production in the placenta. On GD 19, placentas were collected and homogenized for analysis of placental cytokines. Placental levels of (A) TNF‐α, (B) IL‐12, (C) IFN‐γ, (D) MIP3α, and (E) VEGF normalized to total protein concentration are shown. All data are expressed as mean ± SEM. Statistical analyses were performed using unpaired Student's T‐test. *P < 0.05 versus NP
Figure 3
Figure 3
Effects of IL‐17 Infusion on placental cytolytic NK factors in pregnant rats: Cytolytic natural killer (NK) cell enzymes are increased in the placenta in response to chronic IL‐17 infusion in normal pregnant (NP) rats. Granzymes and perforin were measured in the placentas of animals from both groups. Placental levels of (A) granzyme A, (B) granzyme B, (C) granzyme K, and (D) perforin normalized to total protein concentration are shown. All data are expressed as mean ± SEM. Statistical analyses were performed using unpaired Student's T‐test or Mann–Whitney U test, where appropriate. *P < 0.05 versus NP.
Figure 4
Figure 4
Effects of IL‐17 Infusion on Placental Oxidative Stress and NK cytotoxicity in pregnant rats. Chronic IL‐17 infusion into normal pregnant (NP) rats causes increased oxidative stress in the placenta and results in increased cytolytic activity of placental natural killer (NK) cells. (A) Superoxide production in the placenta was analyzed using the lucigenin assay. The results are expressed as Relative Light Units (RLUs)/min/mg. (B) The cytolytic activity of isolated placental NK cells was measured using a cytotoxicity assay based on lactate dehydrogenase (LDH) release. The results are expressed as fold change in cytolytic activity. All data are expressed as mean ± SEM. Statistical analyses were performed using unpaired Student's T‐test or Mann–Whitney U test, where appropriate. *P < 0.05 versus NP.
Figure 5
Figure 5
Effects of IL‐17 Infusion on MAP, Renal Oxidative Stress, and Uterine Artery Function. Chronic Infusion of IL‐17 results in increased mean arterial pressure (MAP), increased renal oxidative stress, and decreased uterine artery vasorelaxation. (A) On GD 19, Conscious mean arterial pressure (MAP) was measured via carotid catheters. Statistical analyses were performed using unpaired Student's T‐test. *P < 0.05 versus NP (B) Renal cortex tissue was collected and homogenized for analysis of oxidative stress. Superoxide production from the kidney was analyzed using the lucigenin assay. The results are expressed as Relative Light Units (RLUs)/min/mg. Statistical analyses were performed using Mann–Whitney U test. *P < 0.05 versus NP (C) Isolated uterine arteries were constricted with 2 × 10−6 mol/L phenylephrine (Phe) and subjected to a 12‐point cumulative concentration response curve to acetylcholine (ACh: 1 × 10−10 mol/L to 3 × 10−4 mol/L). The results are expressed as %Phe constriction using the following formula: [(maximum Phe response − ACh response)/(maximum Phe response − baseline before Phe constriction)] × 100. Statistical analyses were performed using two‐way ANOVA with repeated measures followed by Sidak's multiple comparisons test. *P < 0.05 versus NP.

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