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

IL-27 Mediates Neutrophils Infiltration at the Maternal and Fetal Interface in Preterm Labor With Infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IL-27 Mediates Neutrophils Infiltration at the Maternal and Fetal Interface in Preterm Labor With Infection youwen mei, Dongni Huang, Yuxin Ran, Zheng Liu, Lulu Wang, Nanlin Yin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-207641/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To reveal the role of IL-27 in neutrophils infiltration 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 infiltration 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 infiltrating neutrophils at the maternal and fetal interface. Meanwhile, LPS-induced IL-27Rα-/- mice had less neutrophil infiltration than LPS-induced WT mice. Conclusion IL-27 promotes neutrophil infiltration at the maternal and fetal interface in PTLI. Maternal & Fetal Medicine Infectious Diseases Internal Medicine IL-27 neutrophils maternal and fetal interface preterm labor with infection Figures Figure 1 Figure 2 Figure 3 Highlights 1, IL-27 signaling and neutrophils infiltration increased at the maternal and infant interface in preterm labor with infection 2, IL-27 could promote neutrophils infiltration at the maternal and fetal interface 3, Neutrophils counts in peripheral blood were higher in pregnant women from PTLI group than TL group, positively related with that at the maternal and fetal interface 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), inflammation is present throughout all gestational tissues[ 3 ]. Infiltrative leukocytes are the main resource of proinflammatory factors, of which neutrophils rank the first. In preterm labor, neutrophil abundance at the maternal and fetal interface had increased 5 to 53 fold[ 4 – 6 ] with increased survival[ 5 ]. Their gene expression profile changed from homeostatic to a proinflammatory phenotype[ 6 ], producing myeloperoxidase (MPO) or neutrophil extracellular traps et al[ 7 ]. A previous human study clearly demonstrated that intrauterine inflammation 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 significantly 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 specificity and a positive predictive value of > 90% in predicting infection, better than procalcitonin[ 11 ]. In caecal ligation puncture-induced lung inflammation mice model, elevated IL-27 levels were observed in the lung, serum, and bronchoalveolar lavage fluids, 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 proinflammatory 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 infiltration 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 Affiliated 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 defined 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 Affiliated 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 confirmed by gene identification test with mice’s tail. All mice were housed under specific 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]. Briefly, 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 sacrificed 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 Affiliated 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, Affinity, 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 quantified 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, paraffin sections were stained with hematoxylin and eosin. For IHC, following dewaxing and rehydration, microwave antigen retrieval on paraffin sections was performed. Nonspecific 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 fields at x400 magnifications. 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 significant. Results 1. 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. 1 A and Fig. 1 B). Western blot analysis confirmed that the expression of IL-27Rα in FMs was significantly higher in the PTLI group (Fig. 1 C and 1 D). Our H& E staining had showed the structure of human FMs, which consisted of epithelial cells, interstitial fibrous layer, chorion layer and decidua parietalis layer (Fig. 1 E). 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. 1 G) than TL group (Fig. 1 F). 2. Neutrophils infiltration 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. 2 A). In the immunostaining tissue sections, neutrophils (MPO + cells) could be seen in interstitial fibrous layer, chorion layer and decidua parietalis layer. The major location of initial neutrophil infiltration is choriodecidual junction (Fig. 2 B and Fig. 2 C). 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. 2 D). 3. Neutrophils infiltration at the maternal and fetal interface of mice Then, we explored neutrophils infiltration 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 significantly attenuated in IL-27Rα-/-mice compared with that in WT mice (Fig. 3 A and Fig. 3 D). In Immunohistochemistry sections, neutrophils (Ly6g + cells) could be seen in myometrium and decidua, whose number/HPF was in consistent with Ly6g mRNA expression (Fig. 3 B, 3 C and Fig. 3 E, 3 F), suggesting that IL-27 signaling could promote neutrophils infiltration in myometrium and decidua of mice model. 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 significantly higher than those in the TL group (Table 1 ). Table 1 Characteristics of pregnant women from TL and PTLI groups Characteristic Maternal age (Mean ± SD) BMI before pregnant (Mean ± SD) BMI of pregnant (Mean ± SD) Gravity times(n) Parity times(n) GA (weeks) Total leucocyte count(*10^9/L, Mean ± SD) Neutrophil count (Mean ± SD) Percentage of neutrophils (Mean ± SD) lymphocyte count (Mean ± SD) Percentage of lymphocyte (Mean ± SD) Monocyte count (Mean ± SD) Percentage of monocyte (Mean ± SD) TL(n = 36) 28.53 ± 2.24 20.17 ± 1.85 25.82 ± 2.07 1(1–2) 0(0–1) 33.09 ± 2.37 9.52 ± 1.86 7.43 ± 2.05 78.53 ± 5.13 1.46 ± 0.78 14.69 ± 4.15 0.54 ± 0.14 5.96 ± 1.34 PTLI(n = 18) 27.72 ± 4.9 20.96 ± 1.68 25.78 ± 1.63 2(1–3) 0(0–1) 39.62 ± 0.8 14.55 ± 3.31 12.25 ± 3.28 83.59 ± 5.22 1.49 ± 0.42 10.76 ± 3.85 0.72 ± 0.3 4.92 ± 1.84 P 0.408 0.137 0.951 0.101 0.384 < 0.0001 < 0.0001 < 0.0001 0.002 0.849 0.001 0.025 0.041 Discussion Neutrophils infiltration at the maternal and fetal interface is a characteristic feature of PTLI[ 18 ], accompanied by abundant proinflammatory cytokines[ 19 ]. It was observed that neutrophil depletion did reduce the levels of pro-inflammatory factors such as IL-1β at the maternal and fetal interface[ 20 ], and neutrophils in the chorionic decidua can mediate inflammation 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 inflammatory 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 infiltration 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 inflammatory process of human FMs in preterm labor[ 17 ]. Therefore,we speculated that IL-27 could contribute to the increased inflammation by affecting neutrophils infiltration at the maternal and fetal interface. The present study had the following three findings. Firstly, intrauterine infection induced neutrophils infiltration at the maternal and fetal interface in both human and mice models. This was in keeping with previous studies that massive influx 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 infiltration 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 infiltration 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 infiltration[ 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 infiltration was less abundant in our study. However, some studies conversely stated that IL-27 could downregulate neutrophil infiltration 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 firstly 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 first to study IL-27’s role on neutrophils infiltration 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 infiltration at the maternal and fetal interface in PTLI. Considering the critical role of inflammation 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. Abbreviations PTL: preterm labor, PTLI: preterm labor with infection, MPO: myeloperoxidase, IL-27:Interleukin-27, IL-27Rα:α subunit of IL-27 receptor, gp130:glycoprotein 130, TL: term labor, FMs: fetal membranes, qPCR: real-time quantitative PCR, IHC：immunohistochemistry, IL-27Rα-/-：IL-27Rα knock out, WT：wild type,CT: cycle threshold, H& E: Hematoxylin and eosin, HPF: high performance fortran Declarations Ethics approval and consent to participate Ethics approval was gained from the Ethics Committee of the First Affiliated 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 Funding This work was supported by research funding from National Natural Science Foundation of China for Youth [81801483] and the National Key R&D Program of China [No.2016YFC1000407] Author contributions Youwen Mei designed the study, performed the experiments, analyzed the data, and wrote the manuscript. Nanlin Yin and Hongbo Qi designed the study, reviewed the manuscript, and supervised the project. Dongni Huang, Yuxin Ran, Zheng Liu, and Yunqian Zhou performed the experiments, and analyzed the data. All authors approved the submitted version of the manuscript. Acknowledgements Not Applicable References Goldenberg, R. L., J. F. Culhane, J. D. Iams, and R. Romero. 2008. Epidemiology and causes of preterm birth. Lancet 371: 75–84. http://doi.org/10.1016/s0140-6736(08)60074-4 . DiGiulio, D. B., R. Romero, H. P. Amogan, J. P. 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Interleukin-27 (IL-27) Mediates Susceptibility to Visceral Leishmaniasis by Suppressing the IL-17-Neutrophil Response. Journal of immunology (Baltimore, Md.: 1950) 84 , 2289–2298. http://doi.org/10.4049/jimmunol.1501204 . Tait Wojno, E. D., C. A. Hunter, and J. S. Stumhofer. 2019. The Immunobiology of the Interleukin-12 Family: Room for Discovery. Immunity 50 , 851–870. http://doi.org/10.1016/j.immuni.2019.03.011 . Supplementary Files SupTable1.doc Supgraphicalabstract.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-207641","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":12133723,"identity":"63693c1b-aaae-4bb9-bf58-8b103dad1874","order_by":0,"name":"youwen mei","email":"","orcid":"","institution":"Chongqing University of Medical Science: Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"youwen","middleName":"","lastName":"mei","suffix":""},{"id":12133724,"identity":"9e1d6c27-07f4-459a-af12-55faef348b88","order_by":1,"name":"Dongni Huang","email":"","orcid":"","institution":"Chongqing University of Medical Science: Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dongni","middleName":"","lastName":"Huang","suffix":""},{"id":12133725,"identity":"2f8bd68e-7160-418b-b1ac-05447bc8abc5","order_by":2,"name":"Yuxin Ran","email":"","orcid":"","institution":"Chongqing University of Medical Science: Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Ran","suffix":""},{"id":12133726,"identity":"7830bf33-f7c1-46b8-b0d1-a8517f8bdee9","order_by":3,"name":"Zheng Liu","email":"","orcid":"","institution":"Chongqing University of Medical Science: Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Liu","suffix":""},{"id":12133727,"identity":"3be272ab-4b9d-422e-9da3-3dff92ab6af8","order_by":4,"name":"Lulu Wang","email":"","orcid":"","institution":"Chongqing University of Medical Science: Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lulu","middleName":"","lastName":"Wang","suffix":""},{"id":12133728,"identity":"560d0401-cf8a-4747-9ec6-827cbc3209be","order_by":5,"name":"Nanlin Yin","email":"","orcid":"","institution":"Chongqing University of Medical Science: Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nanlin","middleName":"","lastName":"Yin","suffix":""},{"id":12133729,"identity":"db847256-2685-4ab1-aaf5-853f7ba40a2a","order_by":6,"name":"Hongbo Qi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACZh4QeQDEAhESMqRoYUsAaeEhwhq4Fh4DOBcv4DvOe/Bxwa87cvwzcj6/ulFjwcPAfvjoBnxaJA/zJRvP7HtmLHEjd5t1zjGgw3jS0m7g02JwmMdMmrfncOIGidxtxjlsQC0SPGbEasl5Zpzzj1gtPD/AWpgf57YRoUXyMI+xMW8D0C9nnpkx5/ZJ8LAR8gvf+TOGj3n+AEOsPfnx55xvdXL87IeP4dUCjhHGNiAhkMAmARJgw6scpoXhDxDzH2D+QFD1KBgFo2AUjEgAABy2SQkCPohxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3776-0487","institution":"Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Qi","suffix":""}],"badges":[],"createdAt":"2021-02-04 08:08:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-207641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-207641/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6110108,"identity":"a2dd5cb2-038c-423e-bb5b-4682db5d1efa","added_by":"auto","created_at":"2021-02-18 22:10:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":716243,"visible":true,"origin":"","legend":"IL-27 and IL-27R expression in human FMs. (A) Comparison of IL-27 mRNA expression and IL-27Rα mRNA(B) in human fetal membranes of TL group（n=8） and PTLI group（n=8）. (C) Expression of IL-27Rα protein in human fetal membranes assessed by Western blotting. (D) Quantitatively analyzed band intensities of IL-27Rα, normalized against β-actin. (E) H\u0026 E staining of fetal membranes. (F) Immunohistochemical staining for IL-27Rα in human FM from TL group and (G) PTLI group. AE, amnion epithelium; CL, connective tissue layer; CT, chorionic trophoblast layer; DEC, decidua. Scale bar (E) 200 μm, (F–G) 400 μm. ∗p \u003c 0.05, ∗∗p \u003c 0.01, and ∗∗∗p \u003c 0.001","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-207641/v1/e25f4d05530f9c775c07630f.jpg"},{"id":6110218,"identity":"2ecdc4fd-f312-41e4-b48f-fe68049c1215","added_by":"auto","created_at":"2021-02-18 22:13:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":589590,"visible":true,"origin":"","legend":"Neutrophils (MPO+ cells) in human FMs. (A) Comparison of MPO mRNA expression in human FMs of TL group and PTLI group. (B) Immunohistochemical staining for MPO in human FMs of TL group and (C) PTLI group. (D) Comparison of MPO+ cells’ number/HPF in human FM of TL group(n=5) and PTLI group (n=5). Scale bar (B-C) 400 μm","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-207641/v1/28344cbb2fd36edf060d0b85.jpg"},{"id":6110104,"identity":"22704041-a43f-4539-a64f-265dad8fe257","added_by":"auto","created_at":"2021-02-18 22:10:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":333620,"visible":true,"origin":"","legend":"Leukocytes (Ly6G+ cells) in mice myometrium and decidua. (A) Comparison of Ly6G mRNA expression in mice myometrium and (D) decidua (n=8, for each group). (B) Immunohistochemical staining for Ly6G in mice myometrium and (E) decidua (n=5, for each group) (E). a: WT+PBS, b: WT+LPS, c: KO+PBS, d: KO+LPS. (C) Comparison of Ly6G+ cells number/HPF in mice myometrium and (F)decidua. WT+PBS: pregnant WT mice at gd 16.5 after PBS treatment. WT+LPS: pregnant WT mice at gd 16.5 after LPS treatment. KO+PBS: pregnant IL-27Rα-/- mice at gd 16.5 after PBS treatment. KO+LPS: pregnant IL-27Rα-/- mice at gd 16.5 after LPS treatment. Scale bar (B, E) 200μm","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-207641/v1/de4db328bfd8d077d4fd1dcc.jpg"},{"id":13667802,"identity":"613cd44a-c590-4043-a356-af2c1a4272d8","added_by":"auto","created_at":"2021-09-17 10:54:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":662783,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-207641/v1/71bb0ff6-2f73-429f-a54b-5f6dca66cf69.pdf"},{"id":6110912,"identity":"fd8675c0-c649-4bbc-9e19-708e7e15a894","added_by":"auto","created_at":"2021-02-18 22:16:24","extension":"doc","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":27136,"visible":true,"origin":"","legend":"","description":"","filename":"SupTable1.doc","url":"https://assets-eu.researchsquare.com/files/rs-207641/v1/ef6ca5af55675a8f42f864fc.doc"},{"id":6110220,"identity":"359568fd-7b39-409d-83ba-55179c05be08","added_by":"auto","created_at":"2021-02-18 22:13:25","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":374460,"visible":true,"origin":"","legend":"","description":"","filename":"Supgraphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-207641/v1/256361df7264e5495f10af4d.tif"}],"financialInterests":"","formattedTitle":"\u003cp\u003eIL-27 Mediates Neutrophils Infiltration at the Maternal and Fetal Interface in Preterm Labor With Infection\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1, IL-27 signaling and neutrophils infiltration increased at the maternal and infant interface in preterm labor with infection\u003c/p\u003e\n\u003cp\u003e2, IL-27 could promote neutrophils infiltration at the maternal and fetal interface\u003c/p\u003e\n\u003cp\u003e3, Neutrophils counts in peripheral blood were higher in pregnant women from PTLI group than TL group, positively related with that at the maternal and fetal interface\u003c/p\u003e"},{"header":"Introduction","content":" \u003cp\u003eInfection is associated with about 40% preterm labor (PTL), which accounts for 75% perinatal mortality and over 50% long-term morbidity[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In preterm labor with infection (PTLI), inflammation is present throughout all gestational tissues[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Infiltrative leukocytes are the main resource of proinflammatory factors, of which neutrophils rank the first. In preterm labor, neutrophil abundance at the maternal and fetal interface had increased 5 to 53 fold[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] with increased survival[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Their gene expression profile changed from homeostatic to a proinflammatory phenotype[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], producing myeloperoxidase (MPO) or neutrophil extracellular traps et al[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A previous human study clearly demonstrated that intrauterine inflammation increased the risk for PTL[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], thus neutrophils at the maternal and fetal interface was speculated to increase the risk of PTL.\u003c/p\u003e \u003cp\u003eInterleukin-27 (IL-27), a member of IL-6/IL-12 family, is secreted mainly by antigen-presenting cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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 significantly enhance TNF-α and IL-6 secretion from THP-1 cells, promoting sepsis progression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. IL-27 was also a novel candidate diagnostic biomarker for bacterial infection in critically ill children. At a cut-point value of \u0026ge;\u0026thinsp;5 ng/ml, serum IL-27 had a specificity and a positive predictive value of \u0026gt;\u0026thinsp;90% in predicting infection, better than procalcitonin[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In caecal ligation puncture-induced lung inflammation mice model, elevated IL-27 levels were observed in the lung, serum, and bronchoalveolar lavage fluids, and IL-27 neutralizing antibody could reduce lung injury and improve survival[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our previous team work also demonstrated that IL-27 could induce a proinflammatory response in human fetal membrane, mediating in preterm labor[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In regards of the above evidence, we hypothesized that IL-27 could participate in PTLI by regulating neutrophils infiltration at the maternal and fetal interface.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eHuman samples \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePregnant women who had suffered PTLI from September,2018 to September,2020 in the First Affiliated 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 defined according to the guidelines of the American College of Obstetricians and Gynecologists. The criteria of infection was based on the following items: temperature \u0026gt;37.6 centigrade, white cell count \u0026gt;15*10^9/L, C-reactive protein \u0026gt;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\u0026rsquo; informed consent was obtained, and ethics approval was gained from the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (2019-137).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-27R\u0026alpha; knock out (IL-27R\u0026alpha;-/-) 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-27R\u0026alpha; gene was confirmed by gene identification test with mice\u0026rsquo;s tail. All mice were housed under specific 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]. Briefly, at gestational day of 16.5, the pregnant mice were intraperitoneally administered with LPS (25\u0026mu;g in 200\u0026mu;l PBS) or PBS 200\u0026mu;l. Then, these mice were sacrificed 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 Affiliated Hospital of Chongqing Medical University (2019-137).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of total RNA and qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 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 \u0026beta;-actin gene were calculated with comparative threshold cycle (CT) method, and primers for each target gene were presented in Sup Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 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 \u0026mu;g) was electrophoresed on 10% SDS-polyacrylamide gels (Invitrogen) and blotted onto PVDF membranes. The membranes were incubated overnight with IL-27R\u0026alpha; (1:1000, Affinity, 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 quantified using ImageJ software, and the results were reported as IL-27R\u0026alpha;/\u0026beta;-actin ratio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and eosin (H\u0026amp; E) staining and immunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor H\u0026amp; E staining, paraffin sections were stained with hematoxylin and eosin. For IHC, following dewaxing and rehydration, microwave antigen retrieval on paraffin sections was performed. Nonspecific 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-27R\u0026alpha; 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 fields at x400 magnifications. All measurements were carried out by two independent researchers without knowing the experimental protocols in advance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed by Prism software. Student\u0026rsquo;s t test or Mann\u0026ndash;Whitney U test were used to assess continuous variables according to its distribution. Chi-square test was used to assess categorical variables. P value\u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003e1. Expression of IL-27/IL-27R\u0026alpha; at the maternal and fetal interface from PTLI and TL groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous team work had proved that serum IL-27 level was higher in the PTL group than women in the TL group[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the present study, qPCR analyses showed increased IL-27 and IL-27R\u0026alpha; mRNA expression in human FMs from PTLI group compared to TL group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Western blot analysis confirmed that the expression of IL-27R\u0026alpha; in FMs was significantly higher in the PTLI group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Our H\u0026amp; E staining had showed the structure of human FMs, which consisted of epithelial cells, interstitial fibrous layer, chorion layer and decidua parietalis layer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). In the immunostaining tissue sections, IL-27R\u0026alpha; was expressed in amnion cells, chorion cells and decidua cells. It could be observed that the number of IL-27R\u0026alpha; positive cells and color intensity were enhanced in PTLI group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG) than TL group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Neutrophils infiltration at the maternal and fetal interface of human\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeutrophils\u0026rsquo; 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the immunostaining tissue sections, neutrophils (MPO\u0026thinsp;+\u0026thinsp;cells) could be seen in interstitial fibrous layer, chorion layer and decidua parietalis layer. The major location of initial neutrophil infiltration is choriodecidual junction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In parallel with qPCR analyses, the number of MPO\u0026thinsp;+\u0026thinsp;cells / high performance fortran(HPF)was higher in PTLI group than that in TL group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Neutrophils infiltration at the maternal and fetal interface of mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThen, we explored neutrophils infiltration in myometrium and decidua in pregnant WT and IL-27R\u0026alpha; -/- 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 significantly attenuated in IL-27R\u0026alpha;-/-mice compared with that in WT mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). In Immunohistochemistry sections, neutrophils (Ly6g\u0026thinsp;+\u0026thinsp;cells) could be seen in myometrium and decidua, whose number/HPF was in consistent with Ly6g mRNA expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB,\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE,\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF), suggesting that IL-27 signaling could promote neutrophils infiltration in myometrium and decidua of mice model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Neutrophils in peripheral blood in pregnant women of PL group and TL group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs neutrophils at the maternal and fetal interface were entirely maternal origin[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], we compared the neutrophils number in peripheral blood according to the routine blood test on admission between PTLI group(n\u0026thinsp;=\u0026thinsp;18) and TL group(n\u0026thinsp;=\u0026thinsp;36). As a result, neutrophils number and percentage of neutrophils in the PTLI group were significantly higher than those in the TL group (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCharacteristics of pregnant women from TL and PTLI groups\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMaternal age\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBMI before pregnant\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBMI of pregnant\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGravity times(n)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eParity times(n)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGA (weeks)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal leucocyte\u003c/p\u003e\n\u003cp\u003ecount(*10^9/L,\u003c/p\u003e\n\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNeutrophil count\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePercentage of neutrophils\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003elymphocyte count\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePercentage of lymphocyte\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMonocyte count\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePercentage of monocyte\u003c/p\u003e\n\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTL(n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1(1\u0026ndash;2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0(0\u0026ndash;1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.53\u0026thinsp;\u0026plusmn;\u0026thinsp;5.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePTLI(n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2(1\u0026ndash;3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0(0\u0026ndash;1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.59\u0026thinsp;\u0026plusmn;\u0026thinsp;5.22\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.408\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.137\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.951\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.101\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.384\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.849\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.025\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.041\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eNeutrophils infiltration at the maternal and fetal interface is a characteristic feature of PTLI[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], accompanied by abundant proinflammatory cytokines[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It was observed that neutrophil depletion did reduce the levels of pro-inflammatory factors such as IL-1β at the maternal and fetal interface[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and neutrophils in the chorionic decidua can mediate inflammation and immune imbalance[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 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 inflammatory chemokine/cytokine production during infection[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. 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[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and anti-IL-6 receptor monoclonal antibody abrogated neutrophil recruitment[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In mice model of streptococcus pneumoniae infection, IL-12 can promote pulmonary neutrophil recruitment[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, it may be rational to speculate that IL-27 may also promote neutrophil infiltration under certain context. Indeed, IL-27R signaling contributed to Ly6G\u0026thinsp;+\u0026thinsp;neutrophils accumulation in diseased aorta of mice model with aortic aneurysm[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our previous work had suggested that IL-27 can promote the inflammatory process of human FMs in preterm labor[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore,we speculated that IL-27 could contribute to the increased inflammation by affecting neutrophils infiltration at the maternal and fetal interface.\u003c/p\u003e \u003cp\u003eThe present study had the following three findings. Firstly, intrauterine infection induced neutrophils infiltration at the maternal and fetal interface in both human and mice models. This was in keeping with previous studies that massive influx of neutrophils was detected at decidua in mice model of LPS induced PTL[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Secondly, it demonstrated that IL-27 was positively related with neutrophils infiltration 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[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeutrophils infiltration is mainly regulated by chemokines[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and adhesive factors, such as L-selectin and intercellular cell adhesion molecule-1[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A previous study demonstrated that IL-27 could augment CXCL8 expression in cord blood dendritic cells[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. CXCL8 was an important chemokine for neutrophils infiltration[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and genes encoding CXCL8 were associated with labor onset in humans[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. IL-27 also augmented the secretion of intercellular cell adhesion molecule-1[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The above conclusions helped to explain that in LPS-treated IL-27Rα-/- mice, neutrophil infiltration was less abundant in our study. However, some studies conversely stated that IL-27 could downregulate neutrophil infiltration in zymosan-induced peritonitis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and in mice model with C. parapsilosis infection[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the mother-fetal interface is intricate and affected by many factors. Based on this, our study firstly 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 first to study IL-27\u0026rsquo;s role on neutrophils infiltration 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.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn all, IL-27 promote neutrophils infiltration at the maternal and fetal interface in PTLI. Considering the critical role of inflammation 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.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003ePTL: preterm labor, PTLI: preterm labor with infection, MPO: myeloperoxidase, IL-27:Interleukin-27, IL-27R\u0026alpha;:\u0026alpha; subunit of IL-27 receptor, gp130:glycoprotein 130, TL: term labor, FMs: fetal membranes, qPCR: real-time quantitative PCR, IHC：immunohistochemistry, IL-27R\u0026alpha;-/-：IL-27R\u0026alpha; knock out, WT：wild type,CT: cycle threshold, H\u0026amp; E: Hematoxylin and eosin, HPF: high performance fortran\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval was gained from the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (2019-137), and Written informed consent for participation was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for participation was obtained from the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData and materials would be provided if requested\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were no competing interests to declare\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by research funding from National Natural Science Foundation of China for Youth [81801483] and the National Key R\u0026amp;D Program of China [No.2016YFC1000407]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYouwen Mei designed the study, performed the experiments, analyzed the data, and wrote the manuscript. Nanlin Yin and Hongbo Qi designed the study, reviewed the manuscript, and supervised the project. Dongni Huang, Yuxin Ran, Zheng Liu, and Yunqian Zhou performed the experiments, and analyzed the data. All authors approved the submitted version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoldenberg, R. L., J. F. Culhane, J. D. Iams, and R. Romero. 2008. Epidemiology and causes of preterm birth. \u003cem\u003eLancet\u003c/em\u003e 371: 75\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1016/s0140-6736(08)60074-4\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDiGiulio, D. B., R. Romero, H. P. Amogan, J. P. Kusanovic, E. M. Bik, F. Gotsch, C. J. Kim, O. Erez, S. Edwin, and D. A. Relman. 2008. 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The Immunobiology of the Interleukin-12 Family: Room for Discovery. \u003cem\u003eImmunity 50\u003c/em\u003e, 851\u0026ndash;870.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1016/j.immuni.2019.03.011\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"IL-27, neutrophils, maternal and fetal interface, preterm labor with infection","lastPublishedDoi":"10.21203/rs.3.rs-207641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-207641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo reveal the role of IL-27 in neutrophils infiltration at the maternal and fetal interface in preterm labor with infection (PTLI).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe expression of IL-27 receptor and the number of neutrophils (MPO\u0026thinsp;+\u0026thinsp;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 infiltration at the maternal and fetal interface was investigated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe 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 infiltrating neutrophils at the maternal and fetal interface. Meanwhile, LPS-induced IL-27Rα-/- mice had less neutrophil infiltration than LPS-induced WT mice.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIL-27 promotes neutrophil infiltration at the maternal and fetal interface in PTLI.\u003c/p\u003e","manuscriptTitle":"IL-27 Mediates Neutrophils Infiltration at the Maternal and Fetal Interface in Preterm Labor With Infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-18 22:10:22","doi":"10.21203/rs.3.rs-207641/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa564e40-01cc-47a7-bb7a-95c984521022","owner":[],"postedDate":"February 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2489874,"name":"Maternal \u0026 Fetal Medicine"},{"id":2489875,"name":"Infectious Diseases"},{"id":2489876,"name":"Internal Medicine"}],"tags":[],"updatedAt":"2021-03-04T19:07:29+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-18 22:10:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-207641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-207641","identity":"rs-207641","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}