IL-27 Activates Human Trophoblasts to Express IP-10 and IL-6: Implications in the Immunopathophysiology of Preeclampsia

Purpose. To investigate the effects of IL-27 on human trophoblasts and the underlying regulatory signaling mechanisms in preeclampsia. Methods. The expression of IL-27 and IL-27 receptor (WSX-1) was studied in the placenta or sera from patients with preeclampsia. In vitro, we investigated the effects of IL-27 alone or in combination with inflammatory cytokine tumor necrosis factor (TNF-α) on the proinflammatory activation of human trophoblast cells (HTR-8/SVneo) and the underlying intracellular signaling molecules. Results. The expression of IL-27 and IL-27 receptor α (WSX-1) was significantly elevated in the trophoblastic cells from the placenta of patients with preeclampsia compared with control specimens. In vitro, IL-27 could induce the expression of inflammatory factors IFN-γ-inducible protein 10 (CXCL10/IP-10) and IL-6 in trophoblasts, and a synergistic effect was observed in the combined treatment of IL-27 and TNF-α on the release of IP-10 and IL-6. Furthermore, the production of IP-10 and IL-6 stimulated by IL-27 was differentially regulated by intracellular activation of phosphatidylinositol 3-OH kinase-AKT, p38MAPK, and JAK/STAT pathways. Conclusions. These results provide a new insight into the IL-27-activated immunopathological effects mediated by distinct intracellular signal transduction molecules in preeclampsia.


Introduction
Preeclampsia is a complex pregnancy-specific hypertensive syndrome, and it is a leading cause of maternal and neonatal death worldwide. As a systemic inflammation is common to all pregnancies [1], it is proposed that an excessive maternal inflammatory response to pregnancy may cause preeclampsia [2]. Besides, an angiogenic imbalance also plays an important role in the pathogenesis of preeclampsia which was associated with blood pressure, renal and endothelial dysfunction, and trophoblast deportation, as well as with a shorter duration of pregnancy, fetal growth restriction, and the severity and preterm onset of the disease in preeclampsia [3].
Recently, more studies have focused on the role of trophoblast cells which could mediate inflammation through a wide range and complex mechanisms in the development of preeclampsia. Cytokines and chemokines are the most important inflammatory mediators contributing to inflammation. In PE, trophoblast cells express inflammatory cytokines including interleukins (ILs) 1 , 2, 4, 6, 8, 10, 12, and 18, transforming growth factor (TGF)-beta1, IFN-inducible protein 10/IP-10, tumor necrosis factor (TNF)-, interferon (IFN)-, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 [4,5]. Among them, IL-6 is a classic multifunctional proinflammatory cytokine which is produced by the activated vascular endothelial cell and placenta [6]. The increased maternal serum levels of IL-6 are associated with the severity and onset of preeclampsia [7]. IP-10 is a cytokine of the CXC chemokine family which binds CXCR3 receptor to induce chemotaxis, apoptosis, cell growth, and angiostasis [8]. Preeclampsia was found to be associated with a higher median maternal serum concentration of IP-10 than normal pregnancy [9]. IP-10 2 Mediators of Inflammation has proinflammatory and antiangiogenic properties, and this chemokine has been proposed to be a potential link between inflammation and antiangiogenesis in preeclampsia [10]. The identification of factors and the understanding of the mechanisms regulating the production of cytokines are considered fundamental in the comprehension of the genesis in the inflammatory process of PE. IL-27 is a heterodimeric cytokine composed of p28, an IL-12p35-related subunit, and EBI3 (Epstein-Barr virusinduced gene 3), an IL-12p40-related subunit, which belongs to IL-6/IL-12 family. IL-27 receptor is composed of the IL-27Ra (WSX-1/TCCR) subunit, unique for binding of IL-27, and the gp130 subunit which is shared with the IL-6R [11,12]. IL-27R has been found to be expressed on monocytes, dendritic cells, T and B lymphocytes, natural killer (NK) cells, mast cells, and endothelial cells, whereas IL-27 is mainly produced by antigen-presenting cells (APC) [13,14]. It could activate a variety of cellular targets, resulting in the production of various inflammatory mediators, including TNF-, IL-1 , IL-6, IL-18, MIP-1 , MIP-1 , and -defensin-2, thereby enhancing inflammatory reactions that can occur during some human diseases [15]. However, the role of IL-27 in the pathogenesis of PE has not been elucidated. It was therefore hypothesized that there might be an aberrant production of IL-27 in patients with PE. The aim of this study was to investigate how IL-27 activates human trophoblast cells in PE and its underlying signaling pathways.

Subjects.
Preeclampsia ( = 20) and normal pregnant woman ( = 28) were recruited for this study, and we divided them into two groups. The matched conditions included age (±3 years), parity (0, 1-3, and 4+), and gestational age (±14 days). All cases and controls had singleton pregnancies with no known fetal abnormality. Case characteristics are detailed in Table 1.
Preeclampsia diagnosis was based on ACOG guidelines.  Freshly obtained placentas were snap frozen immediately for processing and fixed with 10% formalin for immunohistochemistry studies. Blood samples were taken from an antecubital vein into EDTA anticoagulation tubes and then centrifuged at 4 ∘ C with a relative centrifugal force of 3000 g for 10 minutes. The serum was stored at −80 ∘ C until the analysis was performed.

2.4.
Immunohistochemistry. Formalin-fixed paraffinembedded human placental sections were deparaffinized in xylene and then rehydrated in a series of graded alcohol. The sections were rinsed twice with PBS for 10 min and then blocked with 5% (wt/vol) nonfat milk/PBS for one hour to reduce nonspecific bindings after quenching the activity of endogenous peroxidase with 3% (vol/vol) H 2 O 2 in PBS for 30 min. Sections were incubated with anti-IL-27 mAb (Abcam115671, HK) and anti-WSX-1 mAb (RD AF1479), diluted in 5% (wt/vol) nonfat milk for 16 h at 4 ∘ C. Negative controls were performed with the same progress. Super Sensitive Link-Label IHC detection System (BioGenex, San Ramon, CA) was used after rinsing twice with PBS and the specific immunostaining was visualized with 3,3diaminobenzidine liquid substrate system (Sigma, St. Louis, MO). All sections were counter-stained with hematoxylin for 40 seconds and mounted with UltraKit (J. T. Baker, Deventer, The Netherlands). Five fields for each placental group were chosen at random and three placentae from each group were used.

Cell
Culture. The HTR-8/SVneo cell line was kindly provided by Doctor CH Graham of Queen's University, Kingston, ON, Canada. Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin (Invitrogen Paisley, Scotland, UK) at 37 ∘ C in a 5% CO 2 atmosphere. Cells were treated with trypsin, removed from culture flasks, and then seeded at a density of 1 × 10 6 cells/mL. After 24-48 h culture, semiconfluent monolayers were exposed to treatments.
2.6. PCR. RNA was extracted from cells by Trizol reagent (Invitrogen, Carlsbad, CA), which was then followed by DNAse I digestion and reverse transcription with TaqMan Reverse Transcription Reagents (Applied Biosystems Inc., Foster City, CA, USA). The sequences of PCR primers were described in Table 2. Briefly, the reaction of quantitative realtime PCR was performed in a 25 uL volume with 2 uL cDNA, 400 nm each of sense and antisense primers, and 12.5 uL Brilliant SYBR Green QPCR Master Mix (Takara Bio Inc., Tokyo, Japan) on ABI PRISM 7000 (Applied Biosystems, Foster City, CA). The reaction performed for 40 cycles, with denaturation at 95 ∘ C for 30 seconds, annealing at 53 ∘ C for 30 seconds and extension at 72 ∘ C for 10 seconds. The gene of GAPDH was amplified as an endogenous reference. The comparative threshold cycle (CT) method was applied for relative quantification in data analysis.

2.7.
ELISA. IL-27 was measured in serum using a human IL-27 ELISA kit with precoated plates (BioLegend, San Diego, USA). According to the manufacturer's instructions, IP-10 and IL-6 levels in culture supernatant with equal cell numbers were assayed by ELISA kit with precoated plates (R&D Systems, MN, USA). For each assay both standards and samples were tested in triplicate.  2.8. Western Blot. Cells and tissues were washed and lysed and an equal amount of proteins to ensure equal protein loading was subjected to SDS-PAGE and then blotted onto PVDF membrane (GE Healthcare Corp., Piscataway, NJ, USA). The membrane was blocked with 5% bovine serum albumin and probed with specific primary antibody at 4 ∘ C overnight. After washing, the membrane was incubated with secondary antibody coupled to horseradish peroxidase (GE Healthcare) for 45 minutes at 37 ∘ C. Antibody-antigen complexes were then detected using an ECL chemiluminescent detection system (GE Healthcare).
2.9. Statistical Analysis. All data were expressed as mean ± SD from three independent experiments. Differences between groups were analyzed by Kruskal-Wallis test, Mann-Whitney U test, Student's t-test, or one-way ANOVA analysis. To test correlations between the two parameters, the nonparametric Spearman rank correlation coefficient was used. < 0.05 was considered significantly different.

Analysis of IL-27 and IL-27 Receptor Expression in
Placental Tissues from PE Patients. IL-27 serum levels were measured in women with preeclampsia and normal pregnant women ( Figure 1) and there was no significant difference between the two groups. The expression of IL-27 receptors in placental tissues of PE and control subjects was also assessed. Western blot analysis confirmed that the expression of WSX-1 in placenta tissues was significantly higher in the PE group than that in control group (Figure 2(a)). In the immunostaining of formalin-fixed paraffin-embedded serial sections, IL-27 (Figure 2(b)) and WSX-1 (Figure 2 also upregulated in the trophoblastic cells from the placenta of preeclampsia compared with control specimens.

IL-27 Could Upregulate IP-10 and IL-6 Expression in HTR-8/SVneo Cells.
Since there was an elevated expression of IL-27 and WSX-1 in the placental tissues from PE patients, we then determined the effects of IL-27 on human trophoblast cells (HTR-8/SVneo). First, it was demonstrated that the IL-27 receptor complex including WSX-1 and gp130 was constitutively expressed in human trophoblast cells on both mRNA and proteinlevels (Figures 3(a) and 3(b)). Then quantitative real-time PCR was applied to screen the inflammatory mediators expressed in trophoblast cells activated by IL-27 (50 ng/mL; 0-4 h) (Figure 3(c)). The expression of IP-10 and IL-6 was increased, while IL-1 , IL-1 , IL-10, and MMP-9 were not induced by IL-27 (Figures 3(d) and 3(e)). In addition, the protein levels of IP-10 and IL-6 in the supernatants were significantly increased (Figures 3(f) and  3(g)).

Discussion
In normal pregnancy, inflammation is necessary during several stages of fetal development, but it should be tightly regulated to prevent tissue injury at the fetomaternal interface. During this study, it was firstly demonstrated a dysregulated expression of IL-27 and IL-27R (WSX-1) in women with PE and that IL-27 had a proinflammatory activation on human trophoblasts, which provides a possible link between hypertensive disorders of pregnancy and inflammation.
The serum levels of IL-27 in PE patients and normal controls were determined. However, there was no significant difference between the two groups. Intriguingly, the expression of WSX-1 in placenta tissues identified by Western blot demonstrated a significant difference between PE patients and normal controls. Further immunohistochemical staining assay demonstrated that the expression of IL-27 and IL-27R in the trophoblast cells from placenta tissues was significantly enhanced when compared with normal controls, suggesting that there was a dysregulated expression of IL-27 and IL-27R , which may play an important role in the pathogenesis of PE.

Mediators of Inflammation
Trophoblast cells are central participants in the pathogenesis of PE. We therefore took HTR-8/SVneo cells as a cell model to study the potential role of IL-27 in the induction of inflammatory mediators from trophoblast cells in vitro. It was found that IL-27 could induce a significantly higher amount of IP-10 and IL-6 in HTR-8/SVneo. In previous studies, it has been confirmed that patients with preeclampsia have significantly higher serum concentrations of IP-10 and IL-6 than normal pregnant women [7,9]. IL-6 is involved in trophoblast invasion, proliferation, and oxidative stress, which play an important role in the pathogenesis of PE [17,18]. IP-10 has potent antiangiogenic and promotes adhesion, migration, and invasion of trophoblast cell properties which are associated with the pathogenesis of PE [9,[19][20][21][22][23][24]. Furthermore, IL-6 and IP-10 have been shown to be highly expressed in the maternal serum of preeclampsia compared with control specimens. Here, we found that IL-27 was a novel inducer of IP-10 and IL-6 in trophoblast cells, suggesting that the IL-27-IP-10/IL-6 axis may be particularly important in the development of PE.
There are many other inflammatory mediators involved in the pathogenesis of PE [16,25]. Therefore, we further investigated whether IL-27 could augment the production of IP-10 or IL-6 from trophoblast cells separately stimulated with proinflammatory cytokine (TNF-alpha), IL-6/IL-12 family cytokine (IL-12 and IL-23), Th1 cytokine (IFN-), or IL-17C. It was demonstrated that IL-27 could augment IP-10 and IL-6 production from trophoblast cells in combination with TNF-. TNF-overproduction has been observed in patients with preeclampsia [26,27] and in animal models of preeclampsia [28], which suggests that it plays an important role in maternal physiological response observed in preeclampsia [29].
To elucidate the molecular signaling mechanisms regulating the induction of IP-10 and IL-6 from trophoblast cells by IL-27, the inhibition assay and Western bolt analysis were used to determine the signaling pathways in trophoblast cells. Using the selective specific signaling molecule inhibitors, we showed that inhibition of JAK/STAT, p38MAPK, and PI3K-Akt could partially suppress the production of IP-10 and IL-6 by IL-27, suggesting that activation of JAK/STAT, p38MAPK, and PI3K-Akt signaling pathways by IL-27 in trophoblast cells may contribute to the development of PE.
IL-27 is a proinflammatory factor which is involved in the pathogenesis of many human inflammatory diseases, such as psoriasis, arthritis, and asthma [30][31][32]. Therefore, IL-27 represents a potential candidate for a therapeutic approach to manage some diseases. In view of the application of antibodies for the blockade of TNF-, IL-1 , and IL-6 receptor as treatment for RA, it was hypothesized that IL-27 may offer an alternative target for therapeutic intervention for PE [33][34][35]. Although many human studies are informative in describing how the immunobiology of IL-27 may be translated, it should be considered how individual polymorphisms might impact the expression patterns of IL-27. The present study provides new clues for the development of a novel treatment for IL-27-mediated PE inflammation, and further studies should investigate whether IL-27 may be a direct target for therapeutic intervention of PE.
Taken together, our results provide evidence that IL-27 could play an important role in PE. IL-27 was found to induce the expression of IP-10 and IL-6 in trophoblast cells via the activation of the JAK/STAT, p38MAPK, and PI3K-Akt signaling pathways. Elucidating the interactions between IL-27 and IP-10/IL-6 would be helpful in understanding and treating PE.