The Immuno-Inammatory Effect of IL-27 in Preterm Labor

Objective To investigate the effect of IL-27 on Th1 cells inltration in human fetal membranes (FMs) and the underlying mechanisms in preterm labor. Methods The expression of IL-27 receptor a subunit (IL-27R 𝛼 ) and Th1 cells were studied in human FMs from pregnant women with preterm labor (PL) and term labor (TL). In vitro, we investigated the effects of IL-27 in human amniotic cell lineage on the Th1 related chemokines and adhesive molecules and the underlying intracellular signaling molecules. Results The expression of IL-27R 𝛼 was higher in human fetal membranes from PL group compared with TL group. Compared with TL group, the PL group had more Th1 cells inltration in human FMs. Meanwhile, it was conrmed the expression of CXCL9, CXCL10, CXCL11 and ICAM-1 were higher in FMs from PL group to TL group. Moreover, IL-27 stimulation can up-regulate the expression of those chemokines in human amniotic cell lineage via JAK2/STAT1/STAT3 signaling pathway. The expression of IL-27R 𝛼 and number of Th1 cells inltration were higher at FMs of PL group than TL group. IL-27 could promote Th1 related chemokines and adhesive molecules in human amniotic cell lineage mainly through JAK2/STAT1/STAT3 signaling pathway. Anity Biosciences, USA)), STAT3, p-STAT3 (Tyr705,(1:1000; Anity Biosciences, USA)) after blocked in 5% nonfat milk for 2 h; Then, the membranes were incubated with an HRP-conjugated anti-IgG secondary antibody, followed by detection with an ECL chemiluminescent detection system. The blots were imaged and quantied using ImageJ software, and the results were reported as primary antibody/β-actin ratio.


Introduction
Preterm labor (PL) accounts 5 to 18% of pregnancies and is a leading cause of infant morbidity and mortality [1]. Apart from intra-amniotic in ammation, maternal-fetal immune imbalance is also an etiology of PL. A wealth of evidence showed that T cells played an important role in pregnancy [2][3][4]. As one subgroup of CD4 + T cells, Th1 cells, secreting proin ammatory cytokines such as IFN-γ, IL-2 and TNF-α, are reported to be involved in the initiation and maintenance of in ammatory process [2]. Th1-type immunity was incompatible with successful pregnancy [3], such as miscarriages [4], preeclampsia [5] or infertile polycystic ovary syndrome [6] in peripheral blood. And increased percentages of IFN-γ+/Th1 cells were found in the decidua of women who miscarried a genetically normal fetus compared with those of induced abortions women [7]. Also, the number of Th1 cells and Th1/Th2 ratio were higher in decidua of unexplained recurrent spontaneous abortion patients compared with controls [8]. As for PL, there were signi cantly higher levels of Th1 cytokines in peripheral blood from women of premature rupture of membranes [9] and PL [10]. In 2007, Curry performed a case-control study with 101,042 pregnancies, indicating that Th1 cells-inducing cytokines-IFN-γ > 90th in peripheral blood was associated with an increased risk of delivering at 30-33 weeks, while IFN-γ > 75th percentile was associated with an increased risk of delivering at 34-36 weeks [11]. At the maternal-fetal interface, higher Th1 cells-inducing cytokines were found in cervicovaginal uid [12] and placentas [13] in women with PL.
As we know, human FMs are composed of epithelial cells, interstitial brous layer, chorionic layer and parietal decidual layer. Since it is between the mother and the fetus, it could be an ideal tissue for studying the speci c immune tolerance. In addition, abundant leukocytes in ltrate at the mother and fetal interface in PL [21,22], including the participation of Th1 cells. Based on this, we speculate that IL-27 may mediate PL by promoting the in ltration of Th1 cells in FMs.

Materials And Methods
Human samples 16 pregnant women with singleton pregnancies from 28 to 36+6 weeks from the rst a liated hospital of Chongqing medical university were enrolled and divided into PL group and TL group with informed written consent. PL and TL were de ned according to the guidelines of the American College of Obstetricians and Gynecologists (ACOG). Pregnant women who suffered preterm labor with infection [23] and pregnancy complications such as pregnancy hypertension, diabetes, placental abruption or chronic diseases were excluded. The basic characteristic of these patients was presented in Table 1. Human FMs were collected within 30 minutes after delivery. These samples were stored as required for Weston blot, real-time quantitative PCR (qPCR) and Immunohistochemistry. Ethics approval was gained from the Ethics Committee of the First A liated Hospital of Chongqing Medical University (2019-137).

Cell culture
Human fetal membranes could secret chemokines recruiting T cells [24]. Therefore, human amniotic cell lineage was used as the targeting cells in the present study. Human amniotic cell lineage-WISH (Wistar Institute Susan Hay ick) cell line was purchased from the commercialized company. They were cultivated in MEM (Gibco, USA) with 10% fetal bovine serum (PAN, Germany), 1% nonessential Amino Acid Solution, 100µg/ml streptomycin and 100 U/ml penicillin (Beyotime Biotechnology, Shanghai, China). Cells were kept in a wetted 5% CO 2 atmosphere at 37 o C and the medium was changed every two days.

Extraction of total RNA and qPCR test
Total RNA was extracted by RNAiso Plus (Takara Bio Inc., Tokyo, Japan) and quanti ed by measuring the ratio of A 260nm/A 280nm. Total RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara Bio Inc., Tokyo, Japan). Thereafter, generated cDNA was subjected to qPCR analysis using SYBR Premix Ex Taq II kit (MCE, Shanghai, China) with speci c primers ( Table 2). Ampli cation and detection of mRNA was performed using Thermal Cycler Dice Real Time System (TP800; Takara Bio, Shiga, Japan). Relative quantity of target gene expression to β-actin gene was measured by comparative Ct method as described previously.

Hematoxylin and eosin (H& E) staining and immunohistochemistry (IHC)
Fetal membrane tissues were formalin xed, and para n-embedded sections were stained with hematoxylin and eosin. IHC was used to localize IL-27Rα+ and T-bet+ cells within the fetal membranes. Para n sections of human FMs were dewaxed and rehydrated, followed by microwave antigen retrieval. Then, these sections were blocked with 3% H2O2 followed by 10% normal goat serum. Following that, these tissue sections were incubated with primary antibodies overnight at 4 centigrade (IL-27Rα, (1:200, santa, China); T-bet, (1:300, protein tech, China)). After thorough washes, they were incubated with biotinylated goat anti-mouse/Rabbit IgG. Positive antibody binding was detected with diaminobenzidine, followed by staining with hemotoxylin (Sigma).

ELISA Assay
Supernatant of cells culture were harvested and stored at -80°C until analyzed by Elisa Assay. Frozen human FMs were thawed on ice and solubilized in icecold PBS containing protease inhibitors PMSF. Samples were centrifuged at 5,000 g for 10 min at 4°C, and supernatant was stored at -80°C until analyzed by Elisa Assay. The total protein of human FMs homogenates was determined using Bradford's reagent (Bio-Rad Protein Assay; Bio-Rad) according to the manufacturer's instructions. Both the supernatant of cells culture and FMs tissue homogenate were measured by a human CCL2, CXCL9, CXCL10, and CXCL11 Elisa kit (Jiubang, China). All the experiments were conducted according to the manufacturer's instructions. The absorbance of the supernatant was assessed at 450nm in a Multi-skan GO plate reader (Thermo Fisher Scienti c, Waltham, MA, USA).

Statistical Analysis
Statistical analysis was performed by prism soft-ware. Student's t test or Mann-Whitney U test were used to assess continuous variables according to its distribution. Chi-square test were used to assess categorical variables. A p value< 0.05 was considered statistically signi cant.

Expression of IL-27Rα in Human FMs in PTLI and TL Group
Our previous team work had proved that serum IL-27 level was higher in the PL group than women in the TL group [25]. In the present study, qPCR test (Fig. 1a) and western blot analysis ( Fig. 1b and 1c) con rmed that the expression of IL-27Rα in FMs was signi cantly higher in PL group than TL group. Our H& E staining had showed the structure of human FMs, which mainly consisted of amnion layer, interstitial brous layer, chorion layer and decidua parietalis layer (Fig. 1d). In the immunostaining tissue sections, IL-27Rα was expressed in amnion cells, chorion cells and decidua cells (Fig. 1e and Fig. 1f).

Th1 (T-bet+) Cells In ltration at the Maternal-fetal Interface of Human
The expression of four main CD4 + T cells subgroups' transcription factors (T-bet-Th1, GATA3-Th2, RORγt-Th17, FOXP3-Treg) were analyzed by qPCR in human FMs. As a result, T-bet mRNA expression was mostly elevated in PL group compared with that in TL group (Fig. 2a). Then, T-bet expression was analyzed by Western blot analysis and IHC in human FMs. As a result, T-bet protein expression was higher in FMs from PL group than those in TL group ( Fig. 2b and Fig. 2c). In the immunostaining tissue sections, the major location of Th1 cells was decidua parietalis layer and chorion layer (Fig. 2d and   Fig. 2e).

The Expression of In ammatory Mediators were Higher at FMs from PL Group than TL Group
The expression of CXCL9, CXCL10, CXCL11 and ICAM-1 were compared in human FMs between PL and TL groups. The mRNA expression of CXCL9, CXCL10, CXCL11, and ICAM-1 were higher at human FMs of PL group than TL group. Furthermore, the protein levels of CXCL9, CXCL10, CXCL11 in the human FMs homogenate in PL group were also higher compared with those in TL group. In addition, Weston blot results showed that the protein expression of ICAM-1 was higher in PL group than TL group.

IL-27 Could Upregulate the Expression of Th1 cells Related Chemokines and Adhesive Molecules in WISH cells
As T cells were mainly attracted to the maternal-fetal interface by chemokines and adhesive molecules, we analyzed Th1 cells related chemokines-CXCL9, CXCL10, CXCL11 and ICAM1 in WISH cells stimulated by IL-27 or not. The mRNA expression of CXCL9, CXCL10, CXCL11 and ICAM1 were analyzed after IL-27 treatment for 6,12 or 24 h at different dose (10 ng/mL and 50 ng/ mL). As a result, IL-27 could signi cantly upregulate the mRNA expression of CXCL9, CXCL10, CXCL11, mainly peaking at 12 hours ( Fig. 4a and Fig. 4b). And this upregulation was dose dependent, peaking at 50 ng/mL IL-27 (Fig. 4c). Therefore, we treated WISH cells with 50 ng/ mL IL-27 for further analysis. The Elisa assay con rmed that upregulation of CXCL9, CXCL10, CXCL11 protein levels in the supernatant of WISH cells (Fig. 4d,e,f), which were consistent with qPCR analysis. Weston blot analysis also showed that IL-27 could upregulate ICAM-1 expression in WISH cells (Fig. 5e).
5. The Role of IL-27 in the Activation of the JAK2/STAT1/STAT3 Signaling Pathways at Fetal Membranes As JAK/STAT signaling pathway played a critical role in Th1 related reactions, we investigate if IL-27 could upregulate its expression in WISH cells. IL-27 was added at 10, 20, 30, 60, 120, 240 min in the culture medium of WISH cells, and total protein and phosphate protein of JAK2, STAT1, and STAT3 were investigated by Weston blot analysis (Fig. 5a). As a result, phosphorylated JAK2 was peaked at 10 minutes and then weakened gradually. As time accumulated, phosphorylated STAT1 / STAT3 increased sequentially. To further investigate the role of JAK/STAT signaling pathway, JAK2 inhibitor-AG490 (10uM) was added in WISH cells one hour before IL-27 was added. The data of qPCR (Fig. 5b),Elisa (Fig. 5c) and Weston blot (Fig. 5d and 5e) revealed that AG490 could effectively inhibit the expression of CCL2, CXCL9, CXCL10, CXCL11, however, except for ICAM-1.

Discussion
The immune tolerance of human FM plays an important role in maintaining pregnancy, and excessive in ammation is one of the mechanism leading to PL [26]. A wealth of evidence had demonstrated that Th1 cells were associated with PL by secreting proin ammatory cytokines such as IFN-γ and TNF- [2].
However, there are few literatures to study the underlying mechanism of Th1 cells in ltration in FMs of PL. According to our previous data, we hypothesized that IL-27 can mediate Th1 cells in ltration at human FM involving in the pathological mechanism of PL.
As a well-known speci c transcription factor of Th1 cells, T-bet regulates the differentiation and development of Th1 cells. Thus, we measured the expression of T-bet to represent Th1 cells, as previous literature reported [26,27].The results showed that Th1 cells expression at FMs from PL group were more than those from TL group, suggesting that Th1 cells may related with PL, which was consistent with previous studies [28]. And the immunohistochemistry analysis revealed that Th1 cells were mainly located in partial decidua and chorion layer, similar with the ndings of other literature [29]. Studies have shown that the leukocytes residing at FM could be maternal origin [30], and the blood vessels in the chorionic and decidual layers may be the main source of these in ltrating cells.
Next, we are thinking about what factors can regulate the changes of Th1 cells at the maternal-fetal interface? It is well known that the migration of Th1 cells is related to CXCL9, CXCL10 and CXCL11, which belong to the CXC chemokine family. In addition, the chemotactic effect of leukocytes often depends on ICAM-1, which is recognized to regulate rolling and stagnation [31]. Previous studies have shown that IL-27 can signi cantly up-regulate the expression of CXCL9 and CXCL10 in human oral epithelial cells [32] and rheumatic arthritis broblast-like synovial cells in vitro [33], and enhance the expression of CXCL9, CXCL10 and CXCL11 in mouse models of psoriasis-like skin diseases [33] and Con A-induced hepatitis [34] in vivo. At the same time, the up-regulation of ICAM-1 was also found in human coronary artery endothelial cells [35] and broblast-like synovial cells [19] stimulated by IL-27. Therefore, we speculate whether the changes of Th1 cells at the maternal-fetal interface are related to the regulation of IL-27? Indeed, our results found that IL-27 can up-regulate the expression of CXCL9, CXCL10, CXCL11 and ICAM-1 in WISH cells. A previous study had con rmed that the expression of CXCL9, CXCL10 and CXCL11 mRNA in chorion-amniotic membrane of chronic chorioamnionitis was higher than the control group [36]. Meanwhile, it was also con rmed that IL-27 can up-regulate the expression of ICAM-1 in WISH cells. This was in consistent with a study of 622 pregnant women with threatened preterm birth, ICAM-1 was found to be signi cantly associated with PL [37]. Therefore, these results help us better understand the reason why Th1 cells accumulated at the maternal-fetal interface in PL. It also should be noted that in ltrative CD4 + T cells could also promote the local production of CXCL9 and CXCL10 [38]. Thus, there was a positive feedback loop in this process.
Then, what is the speci c molecular mechanism of IL-27 regulating these chemotactic mediators? In our study, IL-27 could up-regulate the expression of CXCL9, CXCL10, CXCL11 in WISH cells by activating the JAK/STAT signaling pathway. This was similar with the conclusions in the previous studies. IL-27 could up-regulate the expression of CXCL9, CXCL10 and CXCL11 in human hepatocytes through STAT1 [34] and enhance the expression of CXCL10 in human keratinocytes by STAT1 and STAT3 [39], Furthermore, AG490(JAK2 inhibitor)could also inhibit the expression of SOCS1 in activated CD4 + T cells from cord blood mononuclear cells [40] and ABCA1 expression in macrophages [41] stimulated by IL-27.
In this experiment, we rst tried to explore the regulatory relationship between IL-27 and Th1 cells at the maternal-fetal interface, in order to partly explain the reason for Th1 cell in ltration in PL. However, it still has certain limitations. For example, these T-bet positive cells cannot be completely interpreted as Th1 cells, and their biological functions and mechanisms need further studies. Secondly, it is worth noting that the difference in Th1 cells in the maternal-fetal interface cannot be completely explained by the difference in IL-27 expression. It may be affected by many factors such as gestational age. Meanwhile, it should be noted that the number of Th1 cells in chorion layer was relatively small, further studies are needed to determine their contribution to PL.
In conclusion, our research shows that IL-27 may play a role in the pathogenesis of PL by regulating the in ltration of Th1 cells in FM. This effect is mainly achieved by activating the JAK2/STAT1/STAT3 signaling pathway to up-regulate the expression of CCL2, CXCL9, CXCL10 and CXCL11. Based on the maternal-fetal interface, this research provides new insights into the regulation of Th1 cell in ltration by IL-27 and further enriches the pathogenesis theory of PL.

Declarations
Ethics approval and consent to participate Ethics approval was gained from the Ethics Committee of the First A liated Hospital of Chongqing Medical University (2019-137), and Written informed consent for participation was obtained from all participants.

Consent for publication
Written informed consent for participation was obtained from the participants.

Availability of data and materials
Data and materials would be provided if requested

Competing interests
There were no competing interests to declare