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American Journal of Physiology - Regulatory, Integrative and Comparative Physiology logoLink to American Journal of Physiology - Regulatory, Integrative and Comparative Physiology
. 2018 May 2;315(2):R336–R343. doi: 10.1152/ajpregu.00061.2018

Placental ischemia-stimulated T-helper 17 cells induce preeclampsia-associated cytolytic natural killer cells during pregnancy

Corbin A Shields 1, Maggie McCalmon 2, Tarek Ibrahim 1, Dakota L White 2, Jan M Williams 1, Babbette LaMarca 1, Denise C Cornelius 1,2,✉
PMCID: PMC6139611  PMID: 29718698

Abstract

Previous studies have demonstrated that T-helper 17 (TH17) cells and cytolytic natural killer (cNK) cells are increased in women with preeclampsia. In this study we investigated the role of placental ischemia-stimulated TH17 cells in induction of cNK cells in pregnancy. We further assessed the role of TH17 cell-mediated oxidative stress in facilitation of cNK cell activation in pregnancy by treating rats with the SOD mimetic tempol. CD4+/CD25− cells were isolated from reduced uterine perfusion pressure (RUPP) rats and differentiated into TH17 cells in vitro. On day 12 of gestation (GD12), 1 × 106 placental ischemia-stimulated TH17 cells were injected into normal pregnant (NP) rats (NP + RUPP TH17 rats), and a subset of rats were treated with tempol (30 mg·kg−1·day−1) from GD12 to GD19 (NP + RUPP TH17 + tempol rats). On GD19, cNK cells, mean arterial pressure, fetal weight, and cNK cell-associated cytokines and proteins were measured. Placental cNK cells were 2.9 ± 1, 14.9 ± 4, and 2.8 ± 1.0% gated in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Mean arterial pressure increased from 96 ± 5 mmHg in NP rats to 118 ± 2 mmHg in NP + RUPP TH17 rats and was 102 ± 3 mmHg in NP + RUPP TH17 + tempol rats. Fetal weight was 2.37 ± 0.04, 1.95 ± 0.14, and 2.3 ± 0.05 g in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Placental IFNγ increased from 1.1 ± 0.6 pg/mg in NP rats to 3.9 ± 0.6 pg/mg in NP + RUPP TH17 rats. Placental perforin increased from 0.18 ± 0.18 pg/mg in NP rats to 2.4 ± 0.6 pg/mg in NP + RUPP TH17 rats. Placental levels of granzymes A and B followed a similar pattern. Treatment with tempol did not lower placental cNK cytokines or proteins. The results of the present study identify TH17 cells as a mediator of aberrant NK cell activation that is associated with preeclampsia.

Keywords: hypertension, natural killer cell, oxidative stress, pregnancy, T-helper 17

INTRODUCTION

Preeclampsia (PE) is a multisystem disorder that affects 5–7% of pregnancies and manifests as hypertension, with or without proteinuria, and end-organ damage after week 20 of gestation (20, 34, 39). Although the primary cause of PE is not completely understood, the placenta is considered centrally responsible for inducing factors that mediate the development of maternal endothelial dysfunction and hypertension (6, 21, 22, 35). Shallow trophoblast invasion, partly due to the alteration of immune cells, such as natural killer (NK) cells and CD4+ T cell subsets, leads to inefficient remodeling of uterine spiral arteries and development of a hypoxic and undernourished placenta (20, 25, 30). The ischemic placenta, in turn, releases vasoactive factors, such as soluble fms-like tyrosine kinase 1, soluble endoglin, and inflammatory factors, including cytokines and oxidative stress molecules, which have been shown to play a role in stimulation of maternal endothelial dysfunction (36).

The chronic immune activation in PE is characterized by an increase in circulating and placental proinflammatory cytokines, such as IL-6, TNFα, and IL-17, with a concurrent decrease in anti-inflammatory cytokines, such as IL-10 and TGFβ1. Moreover, the immune cell profile is altered in women with PE. The altered immune profile is characterized by an increase in cytolytic NK (cNK) cells and an imbalance of CD 4+ T cell subsets: increased proinflammatory T-helper 17 (TH17) cells and decreased regulatory T cells compared with women with normal pregnancies (13, 16, 19, 27, 38, 42). The reduced uterine perfusion pressure (RUPP) rat model of placental ischemia mimics many of the clinical characteristics of PE, including hypertension, maternal endothelial dysfunction, intrauterine growth restriction (IUGR), and chronic immune activation. Importantly, this model has been used extensively to examine the contribution of the altered immune profile to the pathophysiology of PE.

Previous studies by our group demonstrated an increased population of TH17 and cNK cells in RUPP rats compared with normal pregnant (NP) rats (15, 44). Investigation into the role of TH17 cells in PE pathophysiology revealed that adoptive transfer of TH17 cells from RUPP rats into NP rats caused a significant increase in maternal blood pressure, circulating inflammatory cytokines, renal and placental oxidative stress, and induced IUGR (10). TH17 cells can induce cellular oxidative stress, a known instigator of NK cytolytic activation (8). Furthermore, it has also been suggested that IL-17, the principal cytokine secreted by TH17 cells, may enhance cytolytic activity of human NK cells (1). We recently observed that antibody-mediated depletion of cNK cells in RUPP rats reduced maternal blood pressure and circulating and placental inflammatory cytokines and improved IUGR (15). Therefore, we hypothesized that the increased population of TH17 cells in PE may induce cNK cells as a pathophysiological mechanism. We further examined TH17 cell-mediated oxidative stress as a mechanism of cNK cell activation. Therefore, in the current study we performed adoptive transfer of placental ischemia-stimulated TH17 cells into NP rats and evaluated the circulating and placental NK cells and cytolytic factors, blood pressure, and fetal outcomes. To evaluate TH17 cell-mediated oxidative stress as a mechanism of cNK cell activation, we also treated rats with the SOD mimetic tempol. The results of the present study could identify a novel mechanism of TH17 cell-mediated pathophysiology in PE.

MATERIALS AND METHODS

Pregnant Sprague-Dawley rats were purchased from Envigo (Indianapolis, IN), where the animals were maintained on a Teklad 2018S diet. Animals were housed in a temperature-controlled (23°C) room with a 12:12-h light-dark cycle and maintained on a Teklad 8640 diet in the Center for Comparative Research at the University of Mississippi Medical Center. All experimental procedures were carried out in accordance with the National Institutes of Health guidelines for use and care of animals. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center.

Reduction in uterine perfusion pressure.

For all our in vivo experiments, ~250- to 275-g rats were used. On day 14 of gestation (GD14), isoflurane anesthesia was delivered by a vaporizer (Ohio Medical Products, Madison, WI), and RUPP surgery was performed on a subset of NP rats to induce placental ischemia. Briefly, a midline incision was made, and a constrictive (0.203-mm) silver clip was placed on the aorta superior to the iliac bifurcation, while ovarian collateral circulation to the uterus was reduced with restrictive (0.100-mm) clips to the bilateral uterine arcades at the ovarian end (2, 23, 28). Carprofen (5 mg/kg) was administered for 2 days to control postsurgical pain. Rats were excluded from the study when the clipping procedure resulted in total reabsorption of all fetuses.

TH17 cell isolation and adoptive transfer.

TH17 cells were isolated and differentiated as previously described (10). Briefly, lymphocytes were isolated from the spleen of pregnant RUPP rats at GD19 on a cushion of Ficoll-Isopaque (Lymphoprep, Accurate Chemical and Scientific, Westbury, NY) according to the manufacturer’s instruction. CD4+/CD25− T cells were isolated from the lymphocytes using FlowComp Dynabeads (Invitrogen, Oslo, Norway) according to the manufacturer’s protocol. The CD4+/CD25− population of splenocytes was incubated on anti-CD3 and anti-CD28 magnetic beads in T-helper cell medium [RPMI medium, 10% FBS, 5% penicillin-streptomycin (PenStrep), and 1% (vol/vol) 5 mM HEPES] in 96-well plates at 103 cells/well on day 0 of culture. On day 2, cells were removed from magnetic beads and cultured in T-helper cell-specific medium (T-helper cell medium, 20 ng/ml IL-6, 3 ng/ml TGFβ1, and 20 ng/ml IL-23) for 5 days. Adoptive transfer of differentiated TH17 cells was performed as previously described by our laboratory (10).

Administration of an SOD mimetic.

To determine a role for oxidative stress in the blood pressure response to RUPP TH17 cells, NP recipient rats of RUPP TH17 cells were treated with tempol (30 mg·kg−1·day−1), a SOD mimetic, via their drinking water, provided ad libitum, beginning on GD12 (NP + RUPP TH17 + tempol rats) (14).

Measurement of mean arterial pressure in conscious rats.

On GD18, catheters (V3 tubing) were inserted into carotid arteries for measurement of mean arterial pressure, tunneled to the back of the neck, and exteriorized under isoflurane anesthesia. On GD19, rats were placed in individual restrainers. In conscious rats, mean arterial pressure was monitored with a pressure transducer (Cobe III tranducer CDX Sema) and recorded continuously for 30 min after a 30-min stabilization period. Subsequently, blood samples were collected, placentas were harvested and weighed, and litter size and fetal weights were recorded under anesthesia.

Determination of circulating and placental TH17 and NK cell populations by flow cytometry.

Single-cell suspensions of placental leukocytes were prepared as follows. Briefly, one placenta from each rat was homogenized and filtered through a 70-μm cell strainer and resuspended in 15 ml of RPMI medium (10% FBS). Whole blood was collected in an EDTA tube and diluted with 5 ml of RPMI medium. Peripheral blood mononuclear cells and placental lymphocytes were isolated by centrifugation on a cushion of Ficoll-Isopaque (Lymphoprep) according to the manufacturer’s instructions. Single-cell suspensions (1 × 106 cells) were stained for flow cytometry after being blockedwith 10% goat and mouse serum. Antibodies used for flow cytometry were as follows: VioGreen anti-CD3 (Miltenyi Biotec, Auburn, CA), anti-ANK61 antibody (catalog no. ab36392, Abcam, Cambridge, MA), anti-mouse FITC (catalog no. ab97239, Abcam), anti-ANK44 (catalog no. ab36388, Abcam), anti-mouse Alexa Fluor 405 (catalog no. ab175663, Abcam), FITC-anti-CD4 antibody (clone 1F4, BD Biosciences, San Jose, CA), phycoerythrin-conjugated anti-CD25 (clone OX-39, BD Biosciences), and peridinin-chlorophyll-protein-conjugated anti-RORγt (clone 600380, R & D Systems, Minneapolis MN). Flow cytometry was performed on the MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed using FlowLogic software (Innovai, Sydney, Australia). Lymphocytes were gated in the forward- and side-scatter plot. After doublet exclusion, additional gates were set using fluorescence-minus-one controls. Results are expressed as percentage of cells in the gated lymphocyte population.

Determination of placental reactive oxygen species.

Superoxide production in the placenta was measured using the lucigenin technique. NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rat placentas were snap-frozen in liquid nitrogen directly after collection and stored at −80°C until further processing. Placentas were removed and homogenized using a cell lysis kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The tissue lysate was incubated with lucigenin at a final concentration of 5 μM. The samples were allowed to equilibrate for 15 min in darkness, and the luminescence [relative light units (RLUs)/min] was measured for 10 s with a plate reader (BioTek, Winooski, VT). An assay blank containing lucigenin with no homogenate was subtracted from the reading before transformation of the data. Each sample was read three times, and the average was used for data transformation. The protein concentration was measured using a protein assay with BSA standards (Pierce, Rockford, IL), and the data are expressed as RLUs·min−1·mg protein−1.

Determination of proinflammatory cytokine and cytolytic protein production.

Plasma and placental homogenate samples were assessed for circulating and placental levels of IL-17, TNFα, and IFNγ using commercial ELISA kits (Life Sciences, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Circulating and placental levels of the NK cell cytolytic proteins perforin, granzyme B, and granzyme A were measured using commercial ELISA kits (MyBioSource, San Diego, CA) according to the manufacturer’s instructions.

Statistical analysis.

Values are means ± SE. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis. P < 0.05 was considered statistically significant.

RESULTS

Adoptive transfer of RUPP TH17 cells.

Differentiated placental ischemia-stimulated TH17 cells were adoptively transferred intraperitoneally into NP rats on GD12. Flow cytometry of lymphocytes isolated from blood and placentas was used to determine circulating and placental populations of TH17 cells in rats from each group. The circulating population of TH17 cells in NP rats was 1.16 ± 0.3% gated and significantly increased to 4.24 ± 0.8% gated after adoptive transfer of placental ischemia-stimulated TH17 cells (Fig. 1A). Treatment with the SOD mimetic tempol significantly decreased circulating TH17 cells in NP + RUPP TH17 + tempol rats to 0.8 ± 0.2% gated. Placental TH17 cells were unchanged in all groups (Fig. 1B): 1.54 ± 0.6, 1.86 ± 0.6, and 0.6 ± 0.3% gated in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Adoptive transfer of placental ischemia-stimulated TH17 cells significantly increased blood pressure from 96 ± 5 mmHg in NP rats to 118 ± 2 mmHg in NP + RUPP TH17 rats and decreased blood pressure to 102 ± 3 mmHg in NP + RUPP TH17 + tempol rats. These data confirm our previous finding of a role for placental ischemia-stimulated TH17 cells in induction of hypertension during pregnancy (10).

Fig. 1.

Fig. 1.

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Adoptive transfer of placental ischemia-stimulated T-helper 17 (TH17) cells. Placental ischemia-stimulated TH17 cells were injected intraperitoneally into a subset of normal pregnant (NP) rats on day 12 of gestation (GD12) to yield NP + reduced uterine perfusion pressure (RUPP) TH17 rats. TH17 cell-mediated oxidative stress was inhibited by treatment of NP + RUPP TH17 rats with the SOD mimetic tempol from GD12 to GD19. Percentage of TH17 cells in circulation (A) and placental tissues (B) was assessed by flow cytometry on GD19. Values are means ± SE; n = 8 rats in each group. *P < 0.05 vs. NP; #P < 0.05 vs. NP + RUPP TH17 (by 1-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis).

NK cell populations in plasma and placenta after adoptive transfer of RUPP TH17 cells.

Lymphocytes isolated from blood and placental samples of animals in each group were assessed by flow cytometry to quantitate the percent gated of total NK and cNK cells in the circulation and placenta of pregnant rats from each group. Adoptive transfer of RUPP TH17 cells into NP rats caused a significant increase in the total NK cell population in the circulation from 30.9 ± 13.9% gated in NP rats to 81.3 ± 4.4% gated in NP + RUPP TH17 rats and a significant reduction to 42.8 ± 7.8% gated in NP + RUPP TH17 + tempol rats (Fig. 2A). Cytolytic activation of NK cells, indicated by the surface of expression of ANK44, was determined to be 4.0 ± 2.4% gated in NP rats, 17.1 ± 4.5% gated in NP + RUPP TH17 rats, and 2.1 ± 0.7% gated in NP + RUPP TH17 + tempol rats (Fig. 2B). A similar NK profile was also observed in the placenta after RUPP TH17 adoptive transfer and treatment with tempol. Total placental NK cells were 5.4 ± 2.0% gated in NP rats, 19.7 ± 4.6% gated in NP + RUPP TH17 rats, and 3.1 ± 1.3% gated in NP + RUPP TH17 + tempol rats (Fig. 2C). Importantly, placental cNK cells were increased from 2.9 ± 0.9% gated in NP rats to 14.9 ± 4.0% gated in NP + RUPP TH17 rats, and treatment with tempol significantly reduced cNK cells to 2.8 ± 1.0% gated in NP + RUPP TH17 + tempol rats (Fig. 2D).

Fig. 2.

Fig. 2.

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Effect of adoptive transfer of placental ischemia-stimulated T-helper 17 (TH17) cells on natural killer (NK) cell populations in pregnant rats. Placental ischemia-stimulated TH17 cells were injected intraperitoneally into a subset of normal pregnant (NP) rats on day 12 of gestation (GD12) to yield NP + reduced uterine perfusion pressure (RUPP) TH17 rats. TH17-mediated oxidative stress was inhibited by treatment of NP + RUPP TH17 rats with the SOD mimetic tempol from GD12 to GD19. Percentage of total and cytolytic NK cells in circulation (A and B) and placental tissues (C and D) was assessed by flow cytometry on GD19. Values are means ± SE; n = 8 rats in each group. *P < 0.05 vs. NP; #P < 0.05 vs. NP + RUPP TH17 (by 1-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis).

Fetal outcomes in response to RUPP TH17 cell adoptive transfer.

Fetal weights of litters were significantly lower in NP + RUPP TH17 than NP rats (1.9 ± 0.1 vs. 2.4 ± 0.04 g; Fig. 3A). Intrauterine growth restriction was attenuated after treatment with the SOD mimetic in NP + RUPP TH17 + tempol rats (2.3 ± 0.05 g; Fig. 3A). Litter size significantly decreased from 14.8 ± 0.6 fetuses/dam in NP rats to 11.8 ± 1.1 fetuses/dam in NP + RUPP TH17 rats and increased to 15.3 ± 0.4 fetuses/dam in NP + RUPP TH17 + tempol rats (Fig. 3B).

Fig. 3.

Fig. 3.

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Effect of adoptive transfer of placental ischemia-stimulated T-helper 17 (TH17) cells on fetal outcomes in pregnant rats. Placental ischemia-stimulated TH17 cells were injected intraperitoneally into a subset of normal pregnant (NP) rats on day 12 of gestation (GD12) to yield NP + reduced uterine perfusion pressure (RUPP) TH17 rats. TH17 cell-mediated oxidative stress was inhibited by treatment of NP + RUPP TH17 rats with the SOD mimetic tempol from GD12 to GD19. Fetal weight (A) and litter size (B) were assessed in all groups at time of harvest on GD19. Values are means ± SE; n = 8 rats in each group. *P < 0.05 vs. NP; #P < 0.05 vs. NP + RUPP TH17 (by 1-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis).

Placental weight and reactive oxygen species in response to adoptive transfer of RUPP TH17 cells.

Average placental weight decreased from 2.4 ± 0.04 g in NP rats to 1.9 ± 0.1 g in NP + RUPP TH17 rats and increased to 2.3 ± 0.05 g in NP + RUPP TH17 + tempol rats (Fig. 4A). Moreover, placental oxidative stress was also significantly increased after adoptive transfer of RUPP TH17 cells. Placental reactive oxygen species of 223 ± 63 RLUs·min−1·mg protein−1 in NP rats more than doubled to 574 ± 95 RLUs·min−1·mg protein−1 in NP + RUPP TH17 rats (Fig. 4B). As expected, treatment with tempol significantly decreased placental reactive oxygen species to 248 ± 72 RLUs·min−1·mg protein−1 (Fig. 4B).

Fig. 4.

Fig. 4.

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Effect of adoptive transfer of placental ischemia-stimulated T-helper 17 (TH17) cells on placentas in pregnant rats. Placental ischemia-stimulated TH17 cells were injected intraperitoneally into a subset of normal pregnant (NP) rats on day 12 of gestation (GD12) to yield NP + reduced uterine perfusion pressure (RUPP) TH17 rats. TH17 cell-mediated oxidative stress was inhibited by treatment of NP + RUPP TH17 rats with the SOD mimetic tempol from GD12 to GD19. Placental weights (A) and oxidative stress (B) were assessed in all groups at time of harvest on GD19. ROS, reactive oxygen species; RLU, relative light units. Values are means ± SE; n = 8 rats in each group. *P < 0.05 vs. NP; #P < 0.05 vs. NP + RUPP TH17 (by 1-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis).

Circulating inflammatory and cNK cell factors.

Plasma levels of the inflammatory cytokines IL-17, TNFα, and IFNγ and cytolytic proteins perforin, granzyme A, and granzyme B were measured in rats from each group. Plasma IL-17 significantly increased from 0.8 ± 0.3 pg/ml in NP rats to 5.1 ± 1.4 pg/ml in NP + RUPP TH17 rats (Fig. 5A). Treatment with tempol significantly decreased plasma IL-17 by ∼50% to 2.6 ± 0.8 pg/ml in NP + RUPP TH17 + tempol rats. Plasma TNFα was 0.8 ± 0.5, 4.6 ± 1.0, and 0.7 ± 0.5 pg/ml in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively (Fig. 5B). Plasma IFNγ significantly increased from 17.5 ± 15.8 pg/ml in NP rats to 193 ± 26.1 pg/ml in NP + RUPP TH17 rats and decreased to 44.5 ± 27.4 pg/ml in NP + RUPP TH17 + tempol rats (Fig. 5C). Plasma perforin was unchanged in all groups: 51.5 ± 21.4, 160.7 ± 28.1, and 96.5 ± 50.4 pg/ml in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively (Fig. 5D). Similarly, plasma granzyme A was unchanged in all groups: 39.3 ± 10.4, 83.7 ± 38.1, and 178.2 ± 55.8 pg/ml in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively (Fig. 5E). Importantly, plasma granzyme B significantly increased from 46.5 ± 6.7 pg/ml in NP rats to 157.6 ± 13.8 pg/ml in NP + RUPP TH17 rats and normalized to 60.5 ± 26.3 pg/ml in NP + RUPP TH17 + tempol rats (Fig. 5F).

Fig. 5.

Fig. 5.

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Effect of adoptive transfer of placental ischemia-stimulated T-helper 17 (TH17) cells on circulating proinflammatory cytokines and cytolytic proteins in pregnant rats. Placental ischemia-stimulated TH17 cells were injected intraperitoneally into a subset of normal pregnant (NP) rats on day 12 of gestation (GD12) to yield NP + reduced uterine perfusion pressure (RUPP) TH17 rats. TH17 cell-mediated oxidative stress was inhibited by treatment of NP + RUPP TH17 rats with the SOD mimetic tempol from GD12 to GD19. Plasma from rats in each group was collected on GD19 for measurement of circulating levels of IL-17 (A), TNFα (B), IFNγ (C), perforin (D), granzyme A (E), and granzyme B (F). Values are means ± SE; n = 7 NP, 8 NP + RUPP TH17, and 8 NP + RUPP TH17 + tempol. *P < 0.05 vs. NP; #P < 0.05 vs. NP + RUPP TH17 (by 1-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis).

Placental inflammatory and cNK cell factors.

Placental levels of the inflammatory cytokines IL-17, TNFα, and IFNγ and cytolytic proteins perforin, granzyme A, and granzyme B were measured in rats from each group. Placental levels of IL-17 were below the level of detection of the assay in placental homogenates from rats in all groups. Placental TNFα was unchanged in all groups: 0.14 ± 0.03, 0.23 ± 0.03, and 0.14 ± 0.02 pg/mg in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively (Fig. 6A). Placental IFNγ significantly increased from 1.1 ± 0.6 pg/ml in NP rats to 3.9 ± 0.6 pg/mg in NP + RUPP TH17 rats and was unchanged at 3.8 ± 0.3 pg/mg in NP + RUPP TH17 + tempol rats (Fig. 6B). Placental perforin significantly increased from 0.18 ± 0.18 pg/mg in NP rats to 2.4 ± 0.6 pg/mg in NP + RUPP TH17 rats and was 0.97 ± 0.6 pg/mg in NP + RUPP TH17 + tempol rats (Fig. 6C). Placental granzyme A significantly increased from 91.6 ± 18.0 pg/mg in NP rats to 180.1 ± 26.6 pg/mg in NP + RUPP TH17 rats and was 399.7 ± 54.7 pg/mg in NP + RUPP TH17 + tempol rats (Fig. 6D). Similarly, placental granzyme B significantly increased from 4.8 ± 0.7 pg/mg in NP rats to 9.3 ± 0.7 pg/mg in NP + RUPP TH17 and was unchanged at 8.3 ± 0.9 pg/mg in NP + RUPP TH17 + tempol rats (Fig. 6E).

Fig. 6.

Fig. 6.

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Effect of adoptive transfer of placental ischemia-stimulated T-helper 17 (TH17) cells on placental proinflammatory cytokines and cytolytic proteins in pregnant rats. Placental ischemia-stimulated TH17 cells were injected intraperitoneally into a subset of normal pregnant (NP) rats on day 12 of gestation (GD12) to yield NP + reduced uterine perfusion pressure (RUPP) TH17 rats. TH17 cell-mediated oxidative stress was inhibited by treatment of NP + RUPP TH17 rats with the SOD mimetic tempol from GD12 to GD19. Placentas from rats in each group were collected on GD19 and homogenized for measurement of tissue levels of TNFα (A), IFNγ (B), perforin (C), granzyme A (D), and granzyme B (E). Values are means ± SE; n = 7 NP, 8 NP + RUPP TH17, and 8 NP + RUPP TH17 + tempol. *P < 0.05 vs. NP (by 1-way ANOVA with Tukey’s multiple-comparisons test as post hoc analysis).

DISCUSSION

NK cells are immune factors of the innate immune system that target virally infected or neoplastic cells for cytotoxic destruction. Cytotoxicity of NK cells is regulated by a balance between activating and inhibitory signals (31). IL-2 signaling activates NK cell cytotoxicity and production of IFNγ and TNFα (4, 29, 40). IL-2 levels are usually decreased during normal pregnancy (32). Thus, peripheral NK cell populations are decreased in pregnancy, while a distinct uterine NK cell population characterized by release of anti-inflammatory cytokines and angiogenic growth factors is increased (31). This unique population of NK cells has integral roles in implantation, uterine spiral artery remodeling, and maintenance of maternal immune tolerance toward the fetus, during early pregnancy (17, 33, 43). Alternatively, in PE the population of peripheral and decidual NK cells undergoes a type 1 shift and is less able to regulate uterine spiral artery remodeling, leading to improper placental vascularization and development of placental ischemia (7). Indeed, NK cells in women with PE are more cytotoxic than NK cells in normal pregnant women and secrete significant amounts of the type 1 cytokines IFNγ and TNFα (18). We previously demonstrated a similar shift in the peripheral and placental populations of NK cells in the RUPP rat model of placental ischemia (15). In the current study we tested the hypothesis that placental ischemia-stimulated TH17 cells could induce cNK cells and their associated cytokines and proteins during pregnancy. We further investigated if inhibition of TH17 cell-mediated oxidative stress would attenuate the activation of cNK cells and improve PE pathophysiology. We observed that placental ischemia-induced TH17 cells induce cytotoxic activation of NK cells in the circulation and placenta during pregnancy and are associated with increased arterial pressure, circulating and placental inflammatory cytokines, placental oxidative stress, and fetal demise. Furthermore, treatment with the SOD mimetic tempol reduced arterial pressure, inflammatory cytokines, and oxidative stress and improved IUGR but did not decrease the expression of cNK cell proteins and inflammatory cytokines in the placenta.

In addition to increased cNK cells, women with PE and RUPP rats also have an increased population of circulating TH17 cells (5, 13, 15, 16, 19, 25, 44). TH17 cells are a proinflammatory subset of CD4+ T cells that primarily secrete IL-17 and are implicated in autoimmune disorders and hypertension (9, 24, 26, 41). Elegant studies by Al Omar et al. demonstrated that in vitro stimulation with IL-17 induced proliferation and increased perforin expression in human NK cells (1). Additionally, IL-17 has recently been shown to be essential for NK cell-mediated antifungal immunity (3) and enhances NK cell activation and antitumor activity (37). Thus the increased population of TH17 cells and IL-17 could mediate the increase in cNK cells in PE. Our study shows that adoptive transfer of placental ischemia-stimulated TH17 cells causes a significant increase in circulating and placental populations of total NK cells. Importantly, expression of the NK cell activation marker ANK44 was significantly increased in response to RUPP TH17 cells, suggesting an increase in activation of NK cells. Activated NK cells secrete IFNγ, TNFα, perforin, and granzymes to mediate their cytotoxic activity against target cells. Adoptive transfer of placental ischemia-stimulated TH17 cells into NP rats resulted in a significant increase in circulating IFNγ, TNFα, and granzyme B. Furthermore, placental IFNγ, perforin, granzyme A, and granzyme B were significantly elevated in the placentas of NP + RUPP TH17 rats compared with NP rats. These data indicate that TH17 cells stimulate cytotoxic activation of NK cells in the placenta during pregnancy.

Recently, we observed that TH17 cell/IL-17-mediated oxidative stress is a mediator of hypertension, IUGR, and increased plasma levels of IL-6 and TNFα in placental ischemia (10–12, 14). In the current study we examined whether TH17 cell-induced oxidative stress would activate cNK cells and induce hypertension and IUGR. We inhibited TH17 cell-mediated oxidative stress with the SOD mimetic tempol. Administration of tempol attenuated hypertension, IUGR, and placental oxidative stress. Additionally, expression of the NK cell activation surface marker ANK44 and circulating inflammatory cytokines was decreased. However, placental IFNγ, TNFα, and NK cell cytolytic proteins perforin and granzymes A and B remained elevated in NP + RUPP TH17 cells after treatment with tempol. This suggests that TH17 cells induce placental cNK cells independent of oxidative stress. Previous studies demonstrate a role for IL-17 in mediating cNK cell polarization in vitro and in vivo (1, 37). In this study, placental levels of IL-17 were undetectable; therefore, we cannot definitely identify TH17 cell-mediated IL-17 signaling as a mechanism of cNK cell activation. Simultaneous in vitro stimulation of NK cells with IL-12, IL-18, and IL-15 induces increased expression of the IL-2 receptor and, thus, enhances IL-2-mediated activation of NK cells (29). It is possible that TH17 cell signaling increases NK cell sensitivity to IL-2-mediated activation. However, investigation into this mechanism is beyond the scope of the current study.

Adoptive transfer of placental ischemia-stimulated TH17 cells had a profound effect on proliferation and activation of cNK cell proteins and cytokines in the placenta of NP rats. However, limitations of this study require further investigation. The effects of placental ischemia-stimulated TH17 cell adoptive transfer on placental NK cell activation were shown to be independent of oxidative stress and placental IL-17 levels; thus the specific mechanism by which TH17 cells activate cNK cells remains unclear. Furthermore, as the focus of this study was on activation of placental NK cell activation, the impact of TH17 cell adoptive transfer on NK cell activation or infiltration in the kidney remains to be investigated because of the established role of the kidney in long-term blood pressure regulation. Finally, TH17 cell adoptive transfer had a differential effect on circulating and placental population of NK cells in pregnancy, which suggests that different mechanisms may be involved in cNK cell activation in the periphery and placenta. Investigations to identify these differential mechanisms may help identify specific targets that could inhibit placental cNK cells in late pregnancy while preserving cNK cells in the periphery for immune protection.

In conclusion, the current study demonstrates, for the first time, that placental ischemia-stimulated TH17 cells polarize NK cells toward a cytotoxic phenotype in the circulation and placenta during pregnancy. Importantly, this polarization occurs independent of oxidative stress in the placenta, as treatment with the SOD mimetic tempol did not attenuate cNK cell activation in the placenta of NP recipients of RUPP TH17 cells. We previously demonstrated a pathophysiological role of cNK cells in PE (15). However, because of their important roles in establishment and maintenance of pregnancy and immune defense, depletion of total NK cells is not a practical option during pregnancy. This study suggests that targeting TH17 cells may be a therapeutic option to inhibit the aberrant polarization of NK cells that occurs in PE and, possibly, improve maternal and fetal outcomes.

Perspectives and Significance

This study demonstrates an important role for placental ischemia-induced TH17 cells in mediating cNK cell polarization and activation during pregnancy. The data identify TH17 cell-mediated oxidative stress as a mechanism that facilitates PE pathophysiology, but not cNK cell polarization and activation. Downregulation of the increased TH17 cells in PE could attenuate the aberrant NK cell activation, hypertension, and IUGR associated with PE. Improvement in these outcomes could lower the morbidity and mortality of PE.

GRANTS

This work was supported by National Institutes of Health Grants HL-130456, HD-067541, GM-104357, and DK-109133.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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

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