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IL-27 Mediates Neutrophils Inltration at the Maternal and Fetal Interface in Preterm Labor With Infection

Objective To reveal the role of IL-27 in neutrophils inltration at the maternal and fetal interface in preterm labor with infection (PTLI). Methods The expression of IL-27 receptor and the number of neutrophils (MPO + cells) at the maternal and fetal interface of pregnant women were compared between PTLI group and term labor (TL) group. Using LPS-induced preterm labor IL-27Rα-/- mice, the role of IL-27 in neutrophils inltration at the maternal and fetal interface was investigated. The expression of IL-27Rα and neutrophils number at the maternal and fetal interface in the PTLI group were higher than those in the TL group in pregnant women. Compared with PBS-treated mice, LPS-treated mice had increased inltrating neutrophils at the maternal and fetal interface. Meanwhile, LPS-induced IL-27Rα-/- mice had less neutrophil inltration than LPS-induced WT mice. and blotted onto PVDF membranes. The membranes were incubated overnight with IL-27Rα (1:1000, Anity, Jiangsu, China) antibody after blocked in 5% nonfat milk for 2 hours. Then, the PVDF membranes were incubated with an HRP-conjugated anti-IgG secondary antibody, followed by band detection with an ECL chemiluminescent detection system. The blots were imaged and quantied using ImageJ software, and the results were reported as IL-27Rα/β-actin ratio. Hematoxylin and eosin (H& E) staining and immunohistochemistry (IHC) For H& E staining, paran sections were stained with hematoxylin and eosin. For IHC, following dewaxing and rehydration, microwave antigen retrieval on paran sections was performed. Nonspecic staining was blocked with 3% H2O2, followed by nonimmune block with 10% normal goat serum. Then, tissue sections were incubated with primary antibodies overnight at 4 centigrade (IL-27Rα 1(cid:0)200,Santa Crue, China; MPO 1:300, protein-tech, China; Ly6g:1:200, protein-tech, China). After thorough washes, the sections were incubated with biotinylated goat anti-mouse/Rabbit IgG. Positive antibody binding was detected with diaminobenzidine, followed by hematoxylin staining. The cells positive for each biomarker in gestational tissues were enumerated on three elds at x400 magnications. All measurements were carried out by two independent researchers without knowing the experimental protocols in advance.


Objective
To reveal the role of IL-27 in neutrophils in ltration at the maternal and fetal interface in preterm labor with infection (PTLI).

Methods
The expression of IL-27 receptor and the number of neutrophils (MPO + cells) at the maternal and fetal interface of pregnant women were compared between PTLI group and term labor (TL) group. Using LPS-induced preterm labor IL-27Rα-/-mice, the role of IL-27 in neutrophils in ltration at the maternal and fetal interface was investigated.

Results
The expression of IL-27Rα and neutrophils number at the maternal and fetal interface in the PTLI group were higher than those in the TL group in pregnant women. Compared with PBS-treated mice, LPS-treated mice had increased in ltrating neutrophils at the maternal and fetal interface. Meanwhile, LPS-induced IL-27Rα-/-mice had less neutrophil in ltration than LPS-induced WT mice.
Conclusion IL-27 promotes neutrophil in ltration at the maternal and fetal interface in PTLI.

Introduction
Infection is associated with about 40% preterm labor (PTL), which accounts for 75% perinatal mortality and over 50% long-term morbidity [1,2]. In preterm labor with infection (PTLI), in ammation is present throughout all gestational tissues [3]. In ltrative leukocytes are the main resource of proin ammatory factors, of which neutrophils rank the rst. In preterm labor, neutrophil abundance at the maternal and fetal interface had increased 5 to 53 fold [4][5][6] with increased survival [5]. Their gene expression pro le changed from homeostatic to a proin ammatory phenotype[6], producing myeloperoxidase (MPO) or neutrophil extracellular traps et al [7]. A previous human study clearly demonstrated that intrauterine in ammation increased the risk for PTL [8], thus neutrophils at the maternal and fetal interface was speculated to increase the risk of PTL.
Interleukin-27 (IL-27), a member of IL-6/IL-12 family, is secreted mainly by antigen-presenting cells [9]. IL-27 receptor (IL-27R) is a heterodimer composed of IL-27Rαlpha (IL-27Rα) and glycoprotein 130 subunits. IL-27Rα is unique to IL-27R, while gp130 is also a subunit of IL-6 receptor and IL-35 receptor. IL-27 could signi cantly enhance TNF-α and IL-6 secretion from THP-1 cells, promoting sepsis progression [10]. IL-27 was also a novel candidate diagnostic biomarker for bacterial infection in critically ill children. At a cut-point value of ≥ 5 ng/ml, serum IL-27 had a speci city and a positive predictive value of > 90% in predicting infection, better than procalcitonin [11]. In caecal ligation puncture-induced lung in ammation mice model, elevated IL-27 levels were observed in the lung, serum, and bronchoalveolar lavage uids, and IL-27 neutralizing antibody could reduce lung injury and improve survival [12,13]. Our previous team work also demonstrated that IL-27 could induce a proin ammatory response in human fetal membrane, mediating in preterm labor [14]. In regards of the above evidence, we hypothesized that IL-27 could participate in PTLI by regulating neutrophils in ltration at the maternal and fetal interface.

Materials And Methods
Human samples Pregnant women who had suffered PTLI from September,2018 to September,2020 in the First A liated Hospital of Chongqing Medical University were enrolled as study group-PTLI group, while those who had term labor at the parallel period were randomly selected as control group-TL group. Preterm labor and term labor were de ned according to the guidelines of the American College of Obstetricians and Gynecologists. The criteria of infection was based on the following items: temperature >37.6 centigrade, white cell count >15*10^9/L, C-reactive protein >10 mg/L, or histological signs of chorioamnionitis [15]. Those patients with pregnancy complications such as pregnancy hypertension, intrahepatic cholestasis of pregnancy, placenta abruption and chronic diseases were excluded. Human FMs were collected within 30 minutes after delivery. These samples were stored as required for westen blot, real-time quantitative PCR (qPCR) and immunohistochemistry (IHC). The patients' informed consent was obtained, and ethics approval was gained from the Ethics Committee of the First A liated Hospital of Chongqing Medical University (2019-137).

Mouse Models
IL-27Rα knock out (IL-27Rα-/-) mice on C57BL/6 background were purchased from Jackson Laboratory in the USA, and C57BL/6 mice purchased from experimental animal center of Chongqing medical university were designated as wild type (WT) mice. The absence of IL-27Rα gene was con rmed by gene identi cation test with mice's tail. All mice were housed under speci c pathogen free conditions during the whole course of the study. Two female mice (8-12 weeks) were mated with one male of the same genotype at dawn. The vaginal plugs were checked the next morning, whose presence indicated gestational day of 0.5. The mice model of PTLI was established as previously reported [16,17]. Brie y, at gestational day of 16.5, the pregnant mice were intraperitoneally administered with LPS (25μg in 200μl PBS) or PBS 200μl. Then, these mice were sacri ced and gestational tissues including fetal membrane, uterus myometrium, and decidua were harvested 6 hours after LPS/PBS injection. All human and animal experiments were approved by the Clinical Research Ethics Committee of the First A liated Hospital of Chongqing Medical University (2019-137).
Extraction of total RNA and qPCR Total RNA was extracted by RNAiso Plus (Takara Bio Inc., Tokyo, Japan), followed by reverse transcription using a PrimeScript RT Reagent Kit (Takara Bio Inc., Tokyo, Japan). Thereafter, generated cDNA from 1 ug RNA was subjected to real-time PCR analysis with SYBR Premix Ex Taq II kit (MCE, Shanghai, China), using thermal cycler dice real time system. Relative quantity of target gene expression to β-actin gene were calculated with comparative threshold cycle (CT) method, and primers for each target gene were presented in Sup Table 1.

Western Blot
Total protein was harvested from human FMs by RIPA lysis buffer (ZSGB-BIO, Beijing, China) containing PMSF (ZSGB-BIO, Beijing, China). Equal amount of protein (40 μg) was electrophoresed on 10% SDS-polyacrylamide gels (Invitrogen) and blotted onto PVDF membranes. The membranes were incubated overnight with IL-27Rα (1:1000, A nity, Jiangsu, China) antibody after blocked in 5% nonfat milk for 2 hours. Then, the PVDF membranes were incubated with an HRP-conjugated anti-IgG secondary antibody, followed by band detection with an ECL chemiluminescent detection system. The blots were imaged and quanti ed using ImageJ software, and the results were reported as IL-27Rα/β-actin ratio.

Hematoxylin and eosin (H& E) staining and immunohistochemistry (IHC)
For H& E staining, para n sections were stained with hematoxylin and eosin. For IHC, following dewaxing and rehydration, microwave antigen retrieval on para n sections was performed. Nonspeci c staining was blocked with 3% H2O2, followed by nonimmune block with 10% normal goat serum. Then, tissue sections were incubated with primary antibodies overnight at 4 centigrade (IL-27Rα 1 200,Santa Crue, China; MPO 1:300, protein-tech, China; Ly6g:1:200, protein-tech, China). After thorough washes, the sections were incubated with biotinylated goat anti-mouse/Rabbit IgG. Positive antibody binding was detected with diaminobenzidine, followed by hematoxylin staining. The cells positive for each biomarker in gestational tissues were enumerated on three elds at x400 magni cations. All measurements were carried out by two independent researchers without knowing the experimental protocols in advance.

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

Expression of IL-27/IL-27Rα at the maternal and fetal interface from PTLI and TL groups
Our previous team work had proved that serum IL-27 level was higher in the PTL group than women in the TL group [14]. In the present study, qPCR analyses showed increased IL-27 and IL-27Rα mRNA expression in human FMs from PTLI group compared to TL group ( Fig. 1A and Fig. 1B). Western blot analysis con rmed that the expression of IL-27Rα in FMs was signi cantly higher in the PTLI group ( Fig. 1C and 1D). Our H& E staining had showed the structure of human FMs, which consisted of epithelial cells, interstitial brous layer, chorion layer and decidua parietalis layer (Fig. 1E). In the immunostaining tissue sections, IL-27Rα was expressed in amnion cells, chorion cells and decidua cells. It could be observed that the number of IL-27Rα positive cells and color intensity were enhanced in PTLI group (Fig. 1G) than TL group (Fig. 1F).

Neutrophils in ltration at the maternal and fetal interface of human
Neutrophils' marker MPO was analyzed by qPCR in human FMs. As a result, MPO mRNA expression was higher in PTLI group than that in TL group ( Fig. 2A).
In the immunostaining tissue sections, neutrophils (MPO + cells) could be seen in interstitial brous layer, chorion layer and decidua parietalis layer. The major location of initial neutrophil in ltration is choriodecidual junction (Fig. 2B and Fig. 2C). In parallel with qPCR analyses, the number of MPO + cells / high performance fortran(HPF)was higher in PTLI group than that in TL group (Fig. 2D).

Neutrophils in ltration at the maternal and fetal interface of mice
Then, we explored neutrophils in ltration in myometrium and decidua in pregnant WT and IL-27Rα -/-mice. qPCR analyses demonstrated that Ly6g mRNA expression was enhanced in myometrium and decidua in LPS-treated mice than their corresponding PBS-treated mice. Furthermore, the enhancement was signi cantly attenuated in IL-27Rα-/-mice compared with that in WT mice ( Fig. 3A and Fig. 3D). In Immunohistochemistry sections, neutrophils (Ly6g + cells) could be seen in myometrium and decidua, whose number/HPF was in consistent with Ly6g mRNA expression (Fig. 3B,3C and Fig. 3E,3F), suggesting that IL-Page 4/9 4. Neutrophils in peripheral blood in pregnant women of PL group and TL group As neutrophils at the maternal and fetal interface were entirely maternal origin[6], we compared the neutrophils number in peripheral blood according to the routine blood test on admission between PTLI group(n = 18) and TL group(n = 36). As a result, neutrophils number and percentage of neutrophils in the PTLI group were signi cantly higher than those in the TL group (Table 1).  [19]. It was observed that neutrophil depletion did reduce the levels of pro-in ammatory factors such as IL-1β at the maternal and fetal interface [20], and neutrophils in the chorionic decidua can mediate in ammation and immune imbalance [6]. Furthermore, neutrophil recruitment at the maternal and fetal interface may contribute to tissue injury by vital neutrophil extracellular traps formation, prolonged neutrophil viability, and neutrophil degranulation, reactive oxygen species production and in ammatory chemokine/cytokine production during infection [7]. IL-27 is a member of IL-6/IL-12 family. Previous studies reported that functional inhibition of IL-6 led to reduced systemic and pulmonary neutrophilia [21], and anti-IL-6 receptor monoclonal antibody abrogated neutrophil recruitment [22]. In mice model of streptococcus pneumoniae infection, IL-12 can promote pulmonary neutrophil recruitment [23]. Therefore, it may be rational to speculate that IL-27 may also promote neutrophil in ltration under certain context. Indeed, IL-27R signaling contributed to Ly6G + neutrophils accumulation in diseased aorta of mice model with aortic aneurysm [24]. Our previous work had suggested that IL-27 can promote the in ammatory process of human FMs in preterm labor [17]. Therefore,we speculated that IL-27 could contribute to the increased in ammation by affecting neutrophils in ltration at the maternal and fetal interface.
The present study had the following three ndings. Firstly, intrauterine infection induced neutrophils in ltration at the maternal and fetal interface in both human and mice models. This was in keeping with previous studies that massive in ux of neutrophils was detected at decidua in mice model of LPS induced PTL [4,6]. Secondly, it demonstrated that IL-27 was positively related with neutrophils in ltration upon LPS exposure. Thirdly, counts and percentage of neutrophils in peripheral blood were higher in the PTLI group than those in the TL group, positively related with neutrophils at the maternal and fetal interface. It suggested that neutrophils extravasated from peripheral blood quickly to the maternal and fetal interface [25].
Neutrophils in ltration is mainly regulated by chemokines[26] and adhesive factors, such as L-selectin and intercellular cell adhesion molecule-1 [27]. A previous study demonstrated that IL-27 could augment CXCL8 expression in cord blood dendritic cells [28]. CXCL8 was an important chemokine for neutrophils in ltration [29] and genes encoding CXCL8 were associated with labor onset in humans [30]. IL-27 also augmented the secretion of intercellular cell adhesion molecule-1 [31,32]. The above conclusions helped to explain that in LPS-treated IL-27Rα-/-mice, neutrophil in ltration was less abundant in our study. However, some studies conversely stated that IL-27 could downregulate neutrophil in ltration in zymosan-induced peritonitis [33] and in mice model with C.
parapsilosis infection [34]. This discrepancy may be a presentation of the dual role of IL-27 in different context, affected by multiple factors such as disease phase, animal models and interventional methods[35].
Since the mother-fetal interface is intricate and affected by many factors. Based on this, our study rstly explores the relationship between IL-27 and neutrophils in preterm birth, which helps to further clarify the dual mechanism of IL-27 and lay the foundation for further research. To the best of our knowledge, the present study was one of the rst to study IL-27's role on neutrophils in ltration at the maternal and fetal interface. Furthermore, neutrophils at the maternal and fetal interface and in peripheral blood were linked up and studied. There was also certain limitation in our manuscript, such as the sampling time point was relatively simple, thus these results should be cautious to interpret.

Conclusion
In all, IL-27 promote neutrophils in ltration at the maternal and fetal interface in PTLI. Considering the critical role of in ammation in the pathogenesis of preterm birth which may contributed by neutrophils, therefore, IL-27 and neutrophils might be important intervention targets in the pathogenesis of PTLI.