T_H17- and IL-17- mediated autoantibodies and placental oxidative stress play a role in the pathophysiology of pre-eclampsia

Abstract

Pre-eclampsia is a multisystem disorder of pregnancy that affects 5–8% of pregnancies. The pathophysiologic mechanisms that lead to the development of pre-eclampsia are poorly understood. Higher than normal levels of circulating T_H17 is observed in preeclamptic women compared to women with normal pregnancy. T_H17 cells are a subset of CD4⁺ T helper cells that are characterized by their secretion of IL-17. Recent studies suggest a role for T_H17 cells and IL-17 in the pathophysiology of pre-eclampsia. In this review, we will discuss the known function of T_H17 cells and IL-17 in immunity and vascular function. We will then review the role of IL-17 and T_H17 cells in normal pregnancy and their association with pre-eclampsia, followed by a discussion of the literature to examine a potential role for IL-17 and T_H17 cells in mediating pathophysiology in pre-eclampsia.

Pre-eclampsia is a multisystem disorder of pregnancy that affects 5–8% of pregnancies. ^{1, 2} It is characterized by new-onset hypertension and proteinuria after 20 weeks gestation. The pathophysiologic mechanisms that lead to the development of preeclampsia are poorly understood, however, development of this syndrome is thought to occur in two stages. The first stage is proposed to be shallow invasion of trophoblasts into the maternal myometrium, leading to insufficient remodeling of the uterine spiral arteries, resulting in placental ischemia and a hypoxic environment. This then causes the placenta to release proinflammatory cytokines and angiogenic factors, leading to systemic inflammation and endothelial dysfunction of the maternal vasculature, and elevated blood pressure associated with a decline in renal function, the second phase of pre-eclampsia. The only effective treatment for abatement of maternal symptoms is delivery of the placenta and fetus.

In early pregnancy, the placenta is initially characterized as a hypoxic environment. This initially hypoxic placenta and an increase in oxygen and energy consumption during pregnancy account for the increase in the production of reactive oxygen species during normal pregnancy compared to the non-pregnant state. As the placenta matures, vascularization develops, changing the placenta into an oxygen-rich environment, and the enhancement of the antioxidant systems during pregnancy act as effective compensation against oxidative stress (OS).³ In contrast to normal pregnancy, women with pre-eclampsia display increased OS and decreased antioxidants, characterized by increased circulating and tissue levels of reactive oxygen species (ROS).³

Several animal models of pre-eclampsia recapitulate the oxidative stress seen in pregnant women with this disorder. Chronic placental ischemia was shown to increase blood pressure and induce the production of reactive oxygen species in the reduced uterine perfusion pressure (RUPP) rat model of pre-eclampsia. In this model, placental ischemia caused a decrease in antioxidant activity which resulted in elevated ROS, much like what is seen in pre-eclamptic women.⁴ Placental ischemia also causes release of soluble fms-like tyrosine kinase-1 (sFlt-1), an antiangiogenic factor that antagonizes vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) during pregnancy. A study was done to examine the mechanism of chronic sFlt-1 induced hypertension. Chronic infusion of sFlt-1 caused a significant increase in mean arterial pressure (MAP) which was associated with increases in both tissue and vascular ROS.⁵ Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) are suggested to be an important link between placental ischemia and the development of hypertension in pre-eclamptic women. Studies with chronic AT1-AA in pregnancy also increased blood pressure that was associated with increased ROS production.⁶ These data, taken together, implicate ROS as a mechanism by which several factors mediate hypertension and pathophysiology of pre-eclampsia.

Reactive oxygen species are natural by-products of the normal metabolism of oxygen and may arise from various sources such as mitochondrial electron transport, and oxidative enzymes.⁷ Reactive oxygen species are particularly important in innate immunity and are produced by activated neutrophils as defense mechanisms against extracellular pathogens. The recruitment and activation of neutrophils to produce reactive oxygen species can be mediated by T_H17 cells, through the release of the inflammatory cytokine, IL-17.

Recent studies suggest a role for T_H17 cells and IL-17 in the pathophysiology of pre-eclampsia. Higher than normal levels of circulating T_H17 are observed in preeclamptic women compared to women with normal pregnancy. Circulating T_H17 cells have also been shown to be increased the pregnant RUPP rat. T_H17 cells are characterized by their production and secretion of IL-17, a proinflammatory cytokine most known for its recruitment and activation of neutrophils. IL-17 is also increased in preeclampsia and these findings are echoed in animal model studies of this disease. In this review, we will discuss the current knowledge on the biology of T_H17 cells and IL-17 in the immune system via stimulation of oxidative stress and their impact on normal vascular function, followed by an overview of how T_H17 cells and IL-17 may play a role in normal pregnancy and pre-eclampsia.

T_H17 CELLS AND IL-17

T_H17 and IL-17 biology and effect on immune function

T_H17 cells are a distinct lineage of CD4⁺ T cells that are distinguished by their secretion of IL-17, surface expression of the IL-23 receptor, and intracellular expression of the lineage-specific transcription factor retinoic acid receptor-related organ receptor gamma (RORγ).⁸ Like all other CD4⁺ T-helper cell subsets, T_H17 cell differentiation is determined by the milieu of cytokines present in the extracellular environment. The combination of TGFβ, IL-6, and IL-23 are required for T_H17 differentiation. TGFβ is required for the expression of RORγ expression in naïve T-cells. Expression of this transcription factor combined with activation of T cells by IL-6 and IL-23 induce the expression and production of IL-17.

T_H17 cells produce a number of effective molecules, although the cytokine they are more noted for producing is IL-17. The IL-17 family is made up of six cytokines: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. IL-17A is the prototype member, and IL-17A and IL-17F have similar functions, are genetically linked, and bind to the same heterodimeric receptor, consisting of IL-17 receptor A and C.^{8, 9} Binding of the IL-17 receptor activates the NFκB and MAPK pathways. ⁹ IL-17A and IL-17F act in a feedback loop for activation and expansion of T_H17 cells and are key cytokines responsible for proliferation, recruitment, activation, and migration of neutrophils.^{2, 10, 11} IL-17 stimulates the production of other cytokines by neutrophils for cell to cell communication, and more importantly, it stimulates these cells to release antimicrobial substances, such as reactive oxygen species. Reactive oxygen species production is a host defense mechanism used by neutrophils and macrophages against bacterial infection.¹² Thus, T_H17 cells are critical for the clearance of extracellular fungal and bacterial pathogens.⁸

T_H17s and IL-17 in vascular function and blood pressure

Both innate and adaptive immune cells populate blood vessels under normal conditions. ¹³ Low-grade inflammation is established as a mechanism involved in the progression of cardiovascular disease through increased adhesion molecules and cytokine production.¹⁴ During inflammation, leukocyte numbers in the vascular wall greatly increases due to migration and proliferation.¹³ This leads to oxidative stress and increased inflammation, causing impaired vascular function.² The inflammatory state leads to endothelial cell barrier dysfunction which results in increase vascular permeability.¹⁵ Hyperpermeability triggered by vascular and tissue inflammation or ischemia promotes edema, exacerbates disease progression, and impairs recovery.¹⁶ Development of atherosclerotic plaques is associated with immune cell invasion of vessel walls. Inflammation in this disease causes lipid uptake and foam cell formation in the vessels. Moreover, fibroid necrosis and intravascular thrombus formation are observed in small vessel vasculitis; and concentric stenosis, vascular muscular hypertrophy, fibrosis, and lipid deposition are all involved in transplant vascular disease which is largely mediated by immune cells releasing oxidative molecules that cause tissue damage.

IL-17 has been linked to oxidative stress and has been shown to induce expression of cytokines in non-immune cells, including endothelial cells.^{11, 17, 18} Studies in mice show that Angiotensin II (Ang-II) induced-hypertension is dependent on IL-17 signaling. ^{1, 19} IL-17 deficient mice were resistant to Ang II-induced hypertension. Moreover, IL-17 inactivation was also associated with a decrease in vessel infiltration by T cells, and a decrease in oxidative stress.^{1, 18} This suggests a role for IL-17 in recruitment of leukocytes during hypertensive vascular remodeling. Further studies are needed to determine how IL-17 production results in leukocyte migration into blood vessels. Inhibition of angiotensin converting enzyme (ACE) decreased T_H17 cells and disease severity in a model of experimental autoimmune encephalitis, further establishing a role for angiotensin in IL-17 production.^{13, 18}

Angiotensin may mediate IL-17 production through aldosterone, a downstream effector of the Ang II pathway.^{13, 19} Antigen presenting cells, such as dendritic cells, have a role in mediating the differentiation of T effector cells. Dendritic cells treated with aldosterone cause an increase in the production of IL-17 by CD4⁺ T cells.^{13, 19} Numerous other studies have established a pathogenic role for IL-17 in vascular conditions such as atherosclerosis, primary vasculitis, systemic lupus erythematosus (SLE), and thrombotic events.¹³

T_H17 cells and IL-17 in normal pregnancy and pre-eclampsia

T_H17 cells are found in the peripheral blood and decidua during normal pregnancy. ^{20, 21} The population of IL-17 expressing lymphocytes is higher in the decidua compared to peripheral blood.²⁰ IL-17 mRNA expression has been observed in trophoblasts and macrophages in human placentas, ²² and in stromal cells at the fetomaternal interface in pregnant mice.²³ IL-17 has also been reported to induce secretion of progesterone and increase invasiveness of placental cells.^{24, 25} Moreover, IL-17 receptor is expressed on extravillous trophoblasts.²⁴ These data, taken together, suggest a role for IL-17 in the establishment (implantation and placentation) and maintenance of pregnancy.²¹ Nakashima et al. reported that peripheral blood levels of T_H17 cells are constant throughout pregnancy,²⁰ while Santner-Nanan et al. report an increase in T_H17 cells late in pregnancy.²⁶ Other studies that report increased IL-17 levels in the third trimester of healthy pregnant women with term labor supports the observations of Santner-Nanan et al. and suggests that IL-17 and T_H17 cells might induce inflammation and labor.^{21, 27} Since IL-17 expression has been observed in both T-cells and other decidual cells, it is plausible that IL-17 expression in pregnancy is not solely due to the T_H17 population.

It has been reported that the population of T_H17 cells are significantly increased in pre-eclampsia compared to normal pregnancy. ^{21, 28} Santner-Nanan et al. report a decrease in the percentage of T_H17 cells in normal pregnancy that was not seen in pre-eclampsia.²⁶ Other studies have also reported an increased percentage of T_H17 cells in the peripheral blood of pre-eclamptic patients compared to normal pregnant women.^{29, 30} A more recent study that examined maternal plasma levels of a number of cytokines in pregnancy reported a decrease in plasma IL-17 in pre-eclamptic women.³¹ Therefore, more studies may be required to confirm how IL-17 levels differ between normal pregnancy and pre-eclampsia. Other evidence supports an increase in IL-17 levels in pre-eclampsia. For example, mRNA expression of the T_H17 specific transcription factor, RORγ, is also increased in pre-eclampsia.³² Inhibition of TGFβ signaling is one possible mechanism of increasing T_H17 cells in pre-eclampsia.²¹ Soluble endoglin, an inhibitor of the TFGβ receptor, is increased in pre-eclampsia.^{21, 33–35} Therefore, inhibition of TGFβ signaling by the increased soluble endoglin in pre-eclampsia could explain the expansion of the T_H17 cell populations observed in pre-eclamptic patients. However, further studies need to be performed to determine this cascade during pregnancy. Additionally, the increased production of inflammatory cytokines IL-6 and IL-1β, two cytokines that are involved in T_H17 cells, have been reported to be increased in pre-eclampsia, and indicates another proposed mechanism of increased T_H17 cells in pre-eclampsia.²¹

A prosed pathophysiological role for T_H17 cells and IL-17 in pre-eclampsia

Effect of IL-17 infusion on hypertension, oxidative stress, and autoantibody production in pregnancy

Clinical studies show an association between pre-eclampsia and increased T_H17 cells and IL-17. However, a role for T_H17 and IL-17 in mediating the pathophysiology of pre-eclampsia has not been established. To determine if increased IL-17 is pathophysiologically relevant in PE, Dhillon et al. examined the effect of IL-17 infusion on blood pressure via mediation by oxidative stress and production of the angiotensin II type I receptor autoantibody (AT1-AA).³⁶ In this study, IL-17 infusion into normal pregnant rats resulted in a significant increase in mean arterial pressure (MAP), increased oxidative stress as measured by urinary isoprostane and NADPH-stimulated placental reactive oxygen species (ROS), an increase in the population of circulating T_H17 cells, and production of AT1-AA. Blockade of the AT1 receptor by administration of losartan attenuated the blood pressure response and decreased placental production of ROS in response to chronic IL-17. Additionally, depletion of B cells, with Rituximab, blunted the blood pressure response and decreased circulating T_H17 cells in IL-17 infused rats. Finally, administration of the superoxide dismutase mimetic, tempol, attenuated the increase in blood pressure, decreased placental production of ROS, and significantly decreased circulating AT1-AAs produced in response to IL-17 infusion.³⁶ The effect of tempol on IL-17 induced pathophysiology suggests that the ROS stimulated by T_H17 cells, via IL-17, may be an important signaling molecule for the activation of B cells to produce AT1-AAs. This data suggests that IL-17 causes pathophysiology associated with pre-eclampsia, including hypertension, via placental oxidative stress and activation of the AT1 receptor.

The endothelin pathway was also investigated as a mechanism by which IL-17 mediates hypertension in response to placental ischemia. Although administration of IL-17 at various doses caused an increase in blood pressure, mRNA levels of preproendothelin were decreased in a dose dependent manner in both the placenta and renal cortex. To determine if this was an indirect compensatory response during pregnancy, we investigated the direct effect of IL-17 to induce endothelin-1 (ET-1) secretion from human umbilical vein endothelial cells (HUVECs). In vitro experiments in which HUVECs were cultured in serum-free media supplemented with IL-17 did not alter ET-1 secretion.³⁷ In fact, increasing IL-17 directly reduced secretion of ET-1 by HUVECs. HUVEC ET-1 secretion decreased from that seen in serum free media, 42.7±7.7 pg/mL, to 36.2±5.9 pg/mL at 10 pg IL-17 and 31.3±5.1 pg/mL at 10 μg IL-17. These studies suggest a direct effect of IL-17 to suppress ET-1 pathway, however, further studies are still needed to elucidate the mechanism by which the IL-17 signaling pathway mediates its effect on vascular function to lead to the development of hypertension.

Effect of reduced uterine perfusion pressure to increase T_H17 and IL-17

The reduced uterine perfusion pressure (RUPP) rat model is a commonly used whole animal model system for the study of mechanisms and pathways involved in the pathophysiology of pre-eclampsia.³⁸ In the RUPP model, a 0.203 mm ID silver clip is placed around the aorta above the iliac bifurcation on day 14 of gestation. To control for compensatory ovarian blood flow to the placenta, 0.100 mm ID silver clips are also placed on the right and left uterine artery arcades. This results in reduced uterine flow in the gravid rat by ~40%.³⁹ Many of the symptoms characteristic of pre-eclampsia are duplicated in the RUPP rat model, including increased IL-17 and T_H17 cells. Additionally, chronic inflammation, another hallmark characteristic of pre-eclampsia has been demonstrated numerous times in the RUPP rat model with increased plasma IL-6 and TNF-α.^{40, 41} Wallace et al. demonstrated that RUPP induced an increase in CD4⁺ T cells in the pregnant rat characterized by increased T_H17 and decreased T_Regs, similar to what is seen in pre-eclampsia patients. Specifically, the percentage of T_H17 cells in peripheral blood increased significantly from 7% in normal pregnant rats to 22% in RUPP rats. This same study recapitulated the increased IL-6 and TNF-α previously seen in the RUPP model and also demonstrated a significant increase in plasma IL-17.⁴² This data further confirms the appropriateness of using the RUPP model to study pre-eclampsia, by duplicating characteristics of the clinical disease in an animal model.

Adoptive transfer of CD4+ T cells from RUPPS into NP rats induced hypertension and increases in TNFα and sFlt-1, similar to that seen in RUPP rats.⁴² Other studies demonstrate that adoptive transfer of RUPP CD4⁺ T cells into NP rats induced AT1-AA production and activation of the endothelin- 1 system. T_H17 cells are a predominate cell type among the RUPP CD4⁺ T cells, suggesting that T_H17s may have an important role in mediating chronic inflammation and hypertension in pregnancy. Moreover, the pathophysiology that results from infusion of IL-17 duplicates much of the pathophysiology associated with placental ischemia, such as hypertension, placental ROS, and AT1-AA production, suggesting that IL-17 may have a role to cause placental oxidative stress to increase blood pressure during pregnancy. However more conclusive studies linking elevated T_H17s and increased IL-17 in response to placental ischemia need to be performed.

The effect of IL-17 blockade on MAP, intrauterine growth restriction, oxidative stress, and AT1-AA production in RUPP

Many studies suggest a role for IL-17 and T_H17 cells in mediating pathophysiology in pre-eclampsia via oxidative stress. To support a proposed role for IL-17 in oxidative stress mediated pathophysiology in pregnancy, we performed studies to examine the effect of IL-17 blockade on various pathways activated in the RUPP rat model of pre-eclampsia. A soluble form of the IL-17 receptor C (IL-17RC) was infused from gestational day 14–19 in RUPP rats to block the IL-17 signaling cascade. This resulted in significantly decreased circulating T_H17 cells, thereby suggesting a feed forward response between the cytokine and T_H17 cells. Importantly, this decrease in T_H17 cells resulted in significantly lower blood pressure, improved intrauterine growth restriction (IUGR), and attenuated oxidative stress and AT1-AA in RUPP rats. To examine the mechanism of IUGR inhibition, placental weight and uterine artery resistance index were measured. Infusion of IL-17RC caused an increase in pup weight which was accompanied by increased placental weight and decreased uterine artery resistance index.¹² Thus inhibition of the IL-17 signaling pathway could normalize placental inflammation and attenuate fetal demise which could be a result of decreased placental oxidative stress. These studies show that IL-17 signaling is a modulator of placental oxidative stress which leads to development of pre-eclamptic symptoms and inhibition of specific immune pathways stimulating ROS leads to a fetal protective response. Therefore, innovative therapies of immunosuppression that would be safe to administer during pregnancy may have an important role to preserve the health in both mother and fetus.

Conclusions

Clinical studies have shown an association between pre-eclampsia and increased T_H17 cells and IL-17. However, a pathophysiological role for the IL-17 signaling pathway had not been examined in pre-eclampsia. Several studies have been performed to establish a mechanism whereby T_H17 cells may mediate pathophysiology in pre-eclampsia.^{12, 36, 42} Data presented in these studies show that T_H17 cells, likely via their secretion of IL-17 induce placental and renal oxidative stress which leads to production of AT1-AA and placental vascular dysfunction resulting in the development of hypertension. Further studies are needed to confirm a role for T_H17 cells as the source of increased IL-17 and their capability to cause pathology in the RUPP placental ischemia model and in preeclamptic women. Moreover, further studies are needed to elucidate the mechanism of action by which IL-17 causes pathology. Nevertheless, animal model studies identify IL-17 and T_H17 cells as potential targets for the effective treatment of pre-eclampsia. Increased understanding of the mechanistic pathways used by these immune components could lead to the identification of other targets and the development of treatment options that could prolong pregnancy and lead to better outcomes for both mother and fetus.