BIOLOGY OF REPRODUCTION (2016) 94(2):32, 1–13 Published online before print 16 December 2015. DOI 10.1095/biolreprod.115.134726

The Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor1

Ratana Lim,3,4 Ha Thi Tran,3,4 Stella Liong,3,4 Gillian Barker,3,4 and Martha Lappas2,3,4

3Obstetrics, Nutrition and Endocrinology Group, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia 4Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 ABSTRACT INTRODUCTION Preterm birth is the largest single cause of neonatal death Preterm birth is the leading direct cause of early neonatal and morbidity. By activating - and Toll-like death, responsible for 29% or approximately one million (TLR)-signaling pathways, infection and/or inflammation are annual neonatal deaths [1]. Spontaneous preterm birth, either strongly associated with preterm delivery. Interferon regula- due to idiopathic preterm labor or preterm prelabor rupture of tory factor-1 (IRF1) is an important regulator of the membranes (PPROM), accounts for up to 70% of all preterm inflammatory response. The aims of this study were to births [2]. For survivors, the impact of preterm birth on long- establish the effect of 1) labor on IRF1 expression in human term health includes cerebral palsy, intellectual impairment, fetal membranes and myometrium, 2) prolabor mediators on chronic lung disease, and vision and hearing loss [3]. This Downloaded from www.biolreprod.org. IRF1 expression and activity, and 3) IRF1 small interfering impacts the families, health care services, and education RNA on the expression of prolabor mediators. IRF1 expression systems [4]. The economic burden of preterm birth is large was higher in fetal membranes and myometrium after in terms of the immediate neonatal intensive care and ongoing spontaneous term labor and in preterm fetal membranes with long-term health needs [5]. infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid Infection and/or inflammation is commonly associated (poly [I:C]) significantly increased IRF1 mRNA expression and with preterm birth and is thought to contribute to PPROM transcriptional activity in human primary myometrial cells. In and also in initiating uterine contractions [6, 7]. Infection, addition, IL1B increased IRF1 activity in primary amnion cells. such as bacterial and viral, activates the maternal immune IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and response and thus the production of proinflammatory poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine , such as interleukin 1beta (IL1B), TNF, IL6, and (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 chemokines, CXCL8 (IL8), and CCL2 (MCP1). IL1B itself release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA can also induce the production of cytokines and chemokines expression and IL1B-induced prostaglandin release was also [8]. These proinflammatory cytokines and chemokines have decreased by IRF1 silencing. In conclusion, IRF1 upregulation been shown to regulate contraction-associated , in fetal membranes and myometrium after term labor uterotonic phospholipid metabolites (e.g., prostaglandins), indicates a proinflammatory role for IRF1 in human parturi- and extracellular matrix-degrading enzymes; all play a tion. IRF1 is involved in TLR- and cytokine-mediated signaling substantial role in regulating myometrial contractions, in human myometrium. These data provide new insights into cervical remodeling, and rupture of fetal membranes [7, 9– the mechanisms associated with inflammation- and infection- 12]. Thus, the biochemical pathways involved in the associated preterm birth. IRF1 inhibitors as therapeutics for formation of proinflammatory cytokines and chemokines the management of spontaneous preterm birth warrants represent potential sites for intervention that may translate to further investigation. therapeutic interventions to delay or prevent spontaneous fetal membranes, human labor, inflammation, IRF1, myometrium preterm birth. Pathogenic (viral or bacterial) stimulation of Toll-like receptors (TLRs) [13] or the interaction between pro- inflammatory cytokines and their receptors activates 1M.L. is supported by a Career Development Fellowship from the MyD88-dependent or Toll/interleukin-1 receptor (TIR)- National Health and Medical Research Council (NHMRC; grant no. containing adapter-inducing interferon-b (TRIF)-dependent 1047025). Additional funding was provided by the Medical Research signaling pathways [14–16]. These pathways activate Foundation for Women and Babies and the Mercy Research Founda- various transcription factors, including interferon regulatory tion. 2 factors (IRFs), resulting in the transcription of a number of Correspondence: Martha Lappas, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, proinflammatory . To date, nine members (IRF1–9) Level 4/163 Studley Road, Heidelberg, 3084, Victoria, Australia. have been identified, as well as virus-encoded analogs of E-mail: [email protected] cellular IRF. IRF1 was originally identified as a key regulator of type I interferons (IFNA1/IFNB1) [17]. Received: 18 August 2015. However, many studies have revealed that IRF1 also First decision: 20 September 2015. regulates the transcription of genes that play essential roles Accepted: 9 December 2015. in various physiological and pathological processes, includ- Ó 2016 by the Society for the Study of Reproduction, Inc. This article ing viral infection, tumor immune surveillance, innate and is available under a Creative Commons License 4.0 (Attribution-Non- adaptive immune responses, and inflammation [18–22]. Commercial), as described at http://creativecommons.org/licenses/by- nc/4.0/. IRF1 transcriptional activity and expression is dramatically eISSN: 1529-7268 http://www.biolreprod.org upregulated by viral infection and stimulation by pro- ISSN: 0006-3363 inflammatory cytokines [23–27]. Studies in nongestational 1 Article 32 LIM ET AL. tissues have also shown that IRF1 is involved in the contractions. All placentas collected from preterm gestations were swabbed regulation of proinflammatory cytokines induced by infec- for microbiological culture investigations and histopathological examination. tion [28–31] and inflammation [32]. Chorioamnionitis was diagnosed pathologically according to standard criteria that included histological evidence of macrophages and neutrophils There is a paucity of data on IRFs and human labor; permeating the chorionic cell layer and often infiltrating the amniotic cell. although a recent publication demonstrated that using micro- Histological chorioamnionitis is often accompanied by isolation of a array analysis, IRF1 mRNA expression was increased microbiological organism from the fetal membranes. Women with pre- following IL1B challenge in primary human amnion cells eclampsia, preexisting diabetes, asthma, multiple pregnancies, or fetuses with [33]. We hypothesized that 1) human labor and delivery chromosomal abnormalities were also excluded. Indications for preterm wouldbeassociatedwithincreasedIRF1expressioninfetal delivery (in the absence of labor) were placenta praevia, placental abruption, antepartum hemorrhage, or Rhesus isoimmunization. For the preterm labor membranes and/or myometrium, 2) proinflammatory stimuli study, fetal membranes were obtained 2 cm from the periplacental edge. The would increase IRF1 transcriptional activity, and 3) inhibition clinical details of the preterm patients are described elsewhere [35]. Of note, of IRF1 using small interfering RNA (siRNA) would be there was no difference in maternal age and body mass index, or parity of the associated with decreased expression and secretion of pro- patients recruited. Tissue samples were snap frozen in liquid nitrogen and inflammatory cytokines in the presence of infection or immediately stored at 808C for analysis by qRT-PCR and Western blot as Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 inflammation. Therefore, the aims of this study were to detailed below. Myometrium was obtained from consenting women at the time of term investigate the effect of 1) human spontaneous term labor on cesarean section (37 wk gestation). Myometrial biopsies were collected IRF1 expression in human fetal membranes and myometrium, from two groups of women: 1) pregnant women undergoing elective 2) human spontaneous preterm labor and membrane rupture cesarean section in the absence of labor (n ¼ 8 patients; mean gestational on IRF1 expression in human fetal membranes, and 3) age 39.4 6 0.3 wk) and 2) pregnant women who were delivered during bacterial and viral products and proinflammatory cytokines on active labor. Labor was defined as the presence of regular uterine IRF1 expression and transcriptional activity in primary cells contractions (every 3–4 min) resulting in cervical effacement and dilation (n ¼ 8 patients; mean gestational age 39.8 6 0.2 wk). Women were isolated from amnion and myometrium. Finally, to determine excluded from the study if they had a multiple pregnancy or evidence of an if IRF1 regulates proinflammatory and prolabor mediators, we active infection. A myometrial biopsy was obtained from the upper margin Downloaded from www.biolreprod.org. investigated the effect of IRF1 siRNA in primary myometrial of the lower uterine segment incision during the cesarean section. There was cells. In this study, we used the pro-inflammatory cytokine no difference in maternal age and body mass index, parity, or gestational IL1B, the viral double-stranded RNA (dsRNA) analog age of the patients recruited. None of these patients received any polyinosinic:polycytidylic acid (poly [I:C]), and the bacterial medications to augment or induce labor, and the average length of labor was 600 min 6 400 min. Tissue samples were fixed and paraffin embedded product fibroblast-stimulating lipopeptide (fsl-1) to mimic for immunohistochemical analysis or snap frozen in liquid nitrogen and infection and/or the cytokine environment involved in labor immediately stored at 808C for analysis by qRT-PCR and Western blot as or infection-associated labor. detailed below.

MATERIALS AND METHODS Immunohistochemistry Tissue Collection To determine the expression of IRF1 in fetal membranes and myometrium, immunohistochemistry (IHC) was performed on paraffin sections as described The Research Ethics Committee of Mercy Hospital for Women approved previously [36] using the IHC Select Horseradish Peroxidase Detection Set this study. Written, informed consent was obtained from all participating (Merck Millipore). Briefly, sections were deparaffinized followed by an antigen women. All tissues were obtained from women who delivered healthy, retrieval step (boiled in 10 mM Tris and 1 mM ethylenediaminetetraacetic acid, singleton infants. All tissues were brought to the research laboratory and pH 9.0, for 10 min followed by 20 min incubation) and then endogenous processed within 15 min of the cesarean delivery. Women with any underlying peroxidases were inactivated by adding 3% hydrogen peroxide for 10 min. medical conditions such as diabetes, asthma, polycystic ovarian syndrome, pre- After blocking (blocking reagent: normal goat serum in PBS) for 5 mins, eclampsia, or macrovascular complications were excluded. Additionally, sections were incubated with 1 lg/ml rabbit polyclonal anti-IRF1 (sc-497; women with multiple pregnancies, obese women, or fetuses with chromosomal Santa Cruz Biotechnology) in 1% (wt/vol) bovine serum albumin in PBS and abnormalities were excluded. incubated in a humidity chamber at 48C overnight. Binding sites were labeled For expression studies by Western blot analysis, fetal membranes were with biotin-conjugated rabbit anti-goat immunoglobulin G antibody followed obtained from women at 1) term no labor undergoing elective cesarean by the streptavidin-horseradish peroxidase. Negative control slides, where section (indications for cesarean section were breech presentation and/or primary antibody was replaced with rabbit immunoglobulin G, were also previous cesarean section) (n ¼ 8 patients; mean gestational age 38.8 6 0.3 performed. wk) and 2) term after spontaneous labor, spontaneous membrane rupture, and normal vaginal delivery (n ¼ 8 patients; mean gestational age 38.7 6 0.4 wk). Western Blot Analysis Fetal membranes from the nonlaboring group were obtained from the supracervical site; identification of the supracervical site was performed as Western blots were performed as previously described [37]. Twenty previously detailed [34]. In the after-labor group, fetal membranes were micrograms of total (extracted using RIPA lysis buffer) was separated obtained from the site of membrane rupture as previously described [34]. onto 10% polyacrylamide gels and transferred to nitrocellulose. Blots were None of the patients received any medications to augment or induce labor, incubated in 1 lg/ml rabbit polyclonal anti-IRF1 prepared in blocking buffer and the average length of labor was 400 min 6 100 min. Tissue samples were (5% skim milk in 50 mM Tris-Cl and 150 mM NaCl, pH 7.5, with 0.05% snap frozen in liquid nitrogen and immediately stored at 808C for analysis Tween-20) for 16 h at 48C. Membranes were viewed and analyzed using the by quantitative RT-PCR (qRT-PCR) and Western blot as detailed below. Full ChemiDoc XRS system (Bio-Rad Laboratories). Semiquantitative analysis of thickness extraplacental membranes (obtained approximately 2 cm from the the relative density of the bands in Western blots was performed using periplacental edge) were also collected, fixed, and paraffin embedded for Quantity One 4.2.1 image analysis software (Bio-Rad Laboratories). For fetal immunohistochemical analysis. membrane and primary cell studies, the levels of IRF1 were normalized to the Fetal membranes were also obtained from women at preterm birth from levels of beta-actin (ACTB) (Sigma). For myometrium studies, the level of the following groups: 1) cesarean section in the absence of labor with intact IRF1 was normalized to Ponceau S staining as described previously [38]; a membranes (artificial rupture of membranes at delivery; n ¼ 8 patients; mean section of the Ponceau S stained membrane was chosen that did not show gestational age 32.8 6 0.7 wk); 2) cesarean section in the absence of labor variation with labor status. For all Western blots, after transfer, the gel was with PROM (n ¼ 8 patients; mean gestational age 31.8 6 0.7 wk); 3) after cropped between approximately 35 and 60 kD before probing with IRF1 spontaneous labor and normal vaginal delivery without histologically antibody. confirmed chorioamnionitis (n ¼ 8 patients; mean gestational age 32.5 6 0.8 wk); and 4) after spontaneous labor and normal vaginal delivery with IRF1 Luciferase Assay histologically confirmed chorioamnionitis (n ¼ 8 patients; mean gestational age 31.6 6 4.0 wk). PROM was defined as spontaneous rupture of the A luciferase assay was utilized to determine if IL1B, poly (I:C), and fsl-1 membranes at less than 37 wk gestation at least 1 h before the onset of any regulate IRF1 transcriptional activity. The luciferase assay was performed as 2 Article 32 IRF1 AND HUMAN LABOR previously described [39], with some minor modifications. Fresh amnion 7, 16, 12, and 15 pg/ml, respectively. The limit of detection for the IL1B

(obtained 2 cm from the periplacental edge) and myometrium were obtained ELISA assay was 1.9 pg/ml. The release of PGE2 and PGF2a into the from women who delivered healthy, singleton infants at term (37–41 wk incubation medium was assayed using a commercially available competitive gestation) undergoing elective cesarean section in the absence of labor. enzyme immunoassay (EIA) kit according to the manufacturer’s specifications

Primary amnion and myometrial cells were prepared as we have previously (Kookaburra Kits; Sapphire Bioscience). The limit of detection for the PGE2 described [38, 40]. Cells at approximately 70% confluence were transfected and PGF2a EIA kits were 16 and 60 pg/ml, respectively. The interassay and with 150 ng IRF1 reporter construct (Qiagen) using FuGENE HD transfection intra-assay coefficients of variation for all ELISA and enzyme immunoassays reagent (Promega) for 48 h. The medium was then replaced with Dulbecco- were less than 10%. modified Eagle medium (DMEM)/F-12 (containing 0.5% BSA for myome- trial cells or 2% heat-inactivated fetal calf serum for amnion cells) with or Statistical Analysis without 1 ng/ml IL1B, 5 lg/ml poly (I:C), or 250 ng/ml fsl-1, and the cells incubated at 378C for an additional 24 h. The cells were harvested in lysis Statistics was performed on normalized data unless otherwise specified. buffer, and luminescence activity was measured using a Luciferase Reporter All statistical analyses were performed using GraphPad Prism (GraphPad Assay Kit (Life Research) and Renilla Luciferase Flash Assay kit (Thermo Software). For Figures 1 and 2, unpaired Student t-test was used to assess Fisher Scientific) as instructed by the manufacturers. The ratio of the firefly statistical significance between normally distributed data; otherwise, the luciferase level to the Renilla luciferase level was determined, and the results nonparametric Mann-Whitney U-test was used. For Figures 3 and 4, a one- Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 are expressed as a ratio of normalized luciferase activity under basal sample t-test, against the constant of 1, was used. For Figures 5–7, the conditions, which was set at 1. The experiments were performed from tissues homogeneity of data was assessed by the Bartlett test, and when significant, obtained from five patients. the data were logarithmically transformed before further analysis using a one-way ANOVA (using least significant difference correction to discriminate among the means). Statistical significance was ascribed to P Silencing of IRF1 with siRNA value , 0.05. Graphs are presented as mean 6 standard error of the mean Given the very low response to IRF1 in primary amnion cells, subsequent (SEM). experiments were performed in primary myometrial cells only, to investigate the effect of siRNA-mediated gene silencing of IRF1 on proinflammatory RESULTS cytokines. Fresh myometrium were obtained from women who delivered healthy, singleton infants at term (37–41 wk gestation) undergoing elective Effect of Human Term and Preterm Labor on IRF1 Downloaded from www.biolreprod.org. cesarean section in the absence of labor. Cells were isolated and cultured as we have described above. Cells at approximately 50% confluence were Expression in Fetal Membranes transfected using Lipofectamine 3000 according to the manufacturer’s To determine the effect of spontaneous labor on IRF1 guidelines (Life Technologies). IRF1 siRNA and negative control (NC) expression, fetal membranes were obtained at cesarean section siRNA was obtained from Origene. Cells were transfected with 200 nM IRF1 or 200 nM NC siRNA in DMEM/F-12 for 48 h. The medium was then in the absence of labor and after spontaneous labor and replaced with DMEM/F-12 (containing 0.5% BSA) with or without 1 ng/ml membrane rupture at term. Representative IHC images from IL1B, 5 lg/ml poly (I:C), or 250 ng/ml fsl-1, and the cells were incubated at one patient for fetal membranes is shown in Figure 1A. IRF1 378C for an additional 24 h. Cells were collected and stored at 808Cuntil was present in amnion epithelium, chorionic trophoblasts, assayed for mRNA expression by qRT-PCR and protein expression by decidua, and in the fibroblasts of the connective tissue layer. Western blots as detailed below. Media were collected and stored at 808C Nonspecific staining was not present in the negative control. until assayed for cytokine and prostaglandin release as detailed below. Cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- IRF1 staining was higher in fetal membranes after labor. tetrazolium bromide proliferation assay as we have previously described [41]. Quantitative RT-PCR (Fig. 1B) and Western blot analysis (Fig. The response to IL1B, poly (I:C), and fsl-1 between patients varied greatly, as 1C) confirmed increased IRF1 expression in fetal membranes we have previously reported [14, 39]. Thus, data are presented as fold change from laboring women compared to fetal membranes from in expression relative to the expression level in the IL1B-, poly (I:C)-, or fsl- nonlaboring women at term. 1-stimulated NC siRNA-transfected cells, which was set at 1. Data could not To determine the effect of spontaneous preterm birth be normalized to NC siRNA-transfected cells alone because some of the (without histologically confirmed chorioamnionitis) on IRF1 readings were zero. Experiments were performed from myometrium obtained from five patients. expression, fetal membranes were obtained from women at preterm cesarean section with no labor (preterm no labor, no infection), and after spontaneous preterm labor and normal RNA Extraction and qRT-PCR vaginal delivery (preterm after labor, no infection). IRF1 RNA extractions and qRT-PCR was performed as previously described [39, mRNA expression (Fig. 1D) and protein expression (Fig. 1E) 42]. Total RNA was extracted using TRIsure reagent according to in fetal membranes were similar between the preterm non- manufacturer’s instructions (Bioline). RNA concentration and purity were laboring and laboring group. measured using a NanoDrop ND1000 spectrophotometer (Thermo Fisher To determine the effect of infection on IRF1 expression, Scientific). RNA concentration and purity was determined via the A260:A280 ratio. RNA was then converted to cDNA using the Tetro cDNA synthesis kit fetal membranes after spontaneous preterm labor and normal (Bioline) according to the manufacturer’s instructions. The cDNA was diluted vaginal delivery without histologically confirmed chorioamni- 50-fold, and 3 ll of this diluted cDNA was used for the qRT-PCR assay using onitis (preterm after labor, no infection) were compared to fetal SensiFAST SYBR NO-ROX Kit (Bioline) and 100 nM of predesigned and membranes after spontaneous preterm labor and normal vaginal validated QuantiTect primers (Qiagen). The qRT-PCR was performed using the delivery with histologically confirmed chorioamnionitis (pre- CFX384 Real-Time PCR detection system (Bio-Rad Laboratories). Average term after labor, infection). IRF1 mRNA expression was gene Ct values were normalized to the average ACTB Ct values from the same cDNA sample. Three housekeeping genes were assessed; however, ACTB was significantly higher in the preterm group with histologic chosen because there was no effect of either siRNA transfection or treatment on chorioamnionitis compared to the preterm group without . Fold differences were determined using the comparative Ct histologic chorioamnionitis (Fig. 1D). Western blots could method. not be performed on fetal membranes from the chorioamnio- nitis group due to protein degradation [43]. Cytokine, Chemokine, and Prostaglandin Assays We also investigated the effect of membrane rupture on IRF1 expression. There was no difference in IRF1 mRNA (Fig. Assessment of cytokine and chemokine release of TNF, IL6, CXCL8, and 1F) and protein (Fig. 1G) expression in fetal membranes CCL2 was assessed using the CytoSet sandwich enzyme-linked immunosor- bent assay (ELISA) kit according to the manufacturer’s instructions (Life obtained from women at preterm cesarean section with no labor Technologies). The concentration of IL1B in the media was determined by and intact membranes (preterm no labor, intact) compared to sandwich ELISA according to the manufacturer’s instructions (R&D Systems). those from women at preterm cesarean section with no labor The limit of detection for the TNF, IL6, CXCL8, and CCL2 ELISA assays were and PROM (preterm no labor, PROM). 3 Article 32 LIM ET AL. Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 1. IRF1 in fetal membranes. A–C) Human fetal membranes were obtained from women not in labor at term cesarean section (term no labor; n ¼ 8 patients) and women after term spontaneous labor onset and delivery (term after labor; n ¼ 8 patients). D, E) Fetal membranes were obtained from women not in labor at preterm cesarean section without histologically confirmed chorioamnionitis (preterm no labor, no infection; n ¼ 8 patients), after preterm spontaneous labor onset and delivery without histologically confirmed chorioamnionitis (preterm after labor, no infection; n ¼ 8 patients), or after preterm spontaneous labor onset and delivery with histologically confirmed chorioamnionitis (preterm after labor, infected; n ¼ 8 patients). F, G) Fetal membranes 4 Article 32 IRF1 AND HUMAN LABOR Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 2. IRF1 in myometrium. Human myometrium was obtained from nonlaboring and laboring women at term cesarean section (n ¼ 8 patients per group). A) Immunohistochemical expression of IRF1. These sections are representative of one patient sample per group. Negative control is also displayed. Original magnification 3100; inset magnification 340. Bar ¼ 100 lm. B) IRF1 gene expression was analysed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to no labor group. Data are displayed as mean 6 SEM. *P , 0.05 versus no labor (Student t-test). C) The protein expression of IRF1 was analyzed by Western blot analysis, and Ponceau S stain was used as a loading control. The fold change was calculated relative to no labor group. Data are displayed as mean 6 SEM. *P , 0.05 versus no labor (Student t-test). Representative Western blot from four patients per group is also shown.

Effect of Human Term Labor on IRF1 Expression in the term cesarean section in the absence of labor (term, no Myometrium labor) and during spontaneous labor onset (term, in labor). IHC revealed that IRF1 staining was present in the To determine the effect of spontaneous labor on IRF1 longitudinal and transverse muscle fibers (Fig. 2A). expression in the myometrium, samples were obtained at Nonspecific staining was not present in the negative

3 were obtained from women at preterm cesarean section not in labor with intact membranes (preterm no labor, intact; n ¼ 8 patients) and from women at preterm cesarean section not in labor with PROM (preterm no labor, PROM; n ¼ 8 patients per group). A) Immunohistochemical expression of IRF1. These sections are representative of one patient sample per group. Negative control is also displayed; ae, amniotic epithelium; cl, connective tissue layer; ct, cytotrophoblast layer; dec, decidua. Original magnification 3100; inset magnification 340. Bar ¼ 100 lm. B, D, F) IRF1 gene expression was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and data are displayed as mean 6 SEM. *P , 0.05 versus term no labor (Student t- test); **P , 0.05 versus preterm after labor, no infection (Student t-test). C, E, G) The protein expression of IRF1 was analyzed by Western blots, normalized to b-actin protein expression, and data are displayed as mean 6 SEM. *P , 0.05 versus term no labor (Student t-test). Representative Western blot from four patients per group is shown. 5 Article 32 LIM ET AL. Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021

FIG. 3. Effect of proinflammatory mediators on IRF1 expression and transcriptional activity. A, B) Human primary amnion (A) or myometrial (B) cells were cotransfected with 150 ng IRF1-luc reporter construct for 48 h, then treated for an additional 24 h with 1 ng/ml IL1B, 5 lg/ml poly (I:C), or 250 ng/ml fsl-1 (n ¼ 5 patients per treatment). activity (normalized with Renilla expression) is expressed as a ratio of luciferase activity of IRF1 reporter under basal conditions. Each bar represents the mean 6 SEM. *P , 0.05 versus basal (one-sample t-test). C) Human primary myometrial cells were treated with or without 1 ng/ml IL1B, 5 lg/ml poly (I:C), or 250 ng/ml fsl-1 (n ¼ 5 patients per treatment). IRF1 gene expression was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to basal. Data are displayed as mean 6 SEM. *P , 0.05 versus basal. Downloaded from www.biolreprod.org. control. IHC (Fig. 2A), qRT-PCR (Fig. 2B), and Western Effect of IRF1 siRNA Knockdown on Proinflammatory blots (Fig. 2C) showed an increase in IRF1 expression after Cytokines and Chemokines in Primary Myometrial Cells term labor compared to the no labor group. A section of Ponceau S stained gel that did not vary with labor status We next sought to determine the effect of IRF1 siRNA was chosen for normalization of IRF1 protein expression. on proinflammatory cytokines and chemokines. For these Housekeeping proteins such as actin and tubulin labor studies, we used primary cells isolated from fresh could not be used because their expression in the laboring myometrium. As depicted in Figure 4A, IRF1 mRNA myometrium samples varied greatly. These proteins are expression was decreased in IRF1 siRNA-transfected cells by approximately 70%, and protein expression was reduced cytoskeletal proteins; there is now increasing evidence of by 50% (Fig. 4B). As presented in Figure 4, there was no the importance of the smooth muscle cytoskeleton in the effect of IRF1 siRNA knockdown on the mRNA expression regulation of uterine contractility, suggesting that labor and release of the proinflammatory cytokine IL6 (Fig. 4, C may indeed alter the expression of cytoskeletal proteins and D). Similar results were obtained for the mRNA [44]. Notably, the use of general protein stains for expression of the proinflammatory cytokines TNF and IL1B normalization of data is becoming increasingly used and as well as the chemokines CXCL8 and CCL2 (data not accepted; it was recently shown to be the most stable for shown). IL6, CXCL8, and CCL2 release was also not the placenta [45]. different between NC siRNA-transfected cells when com- pared to IRF1 siRNA-transfected cells (data not shown). Effect of Infection and Inflammation on IRF1 The release of TNF and IL1B were below the sensitivity of Transcriptional Activity in Human Amnion and Myometrial the assays. There was also no effect of IRF1 siRNA on cell viability, compared to NC siRNA-transfected cells, as Cells determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- A range of proinflammatory mediators, known to induce 2H-tetrazolium bromide cell viability assay (absorbance at the terminal effector pathways involved in preterm birth, 570 nM for NC siRNA 0.65 6 0.12 vs. IRF1 siRNA 0.71 were used to determine which could affect IRF1 transcrip- 6 0.12). tional activity in primary cells isolated from fresh amnion For subsequent experiments, after IRF1 siRNA transfec- and myometrium. We tested the proinflammatory cytokine tion, cells were treated with IL1B, poly (I:C), or fsl-1. We IL1B, the viral dsRNA analog poly (I:C), and the bacterial used IL1B, poly (I:C), or fsl-1 as models of inflammation product fsl-1. For primary amnion cells, IL1B significantly and/or infection associated with preterm labor in order to increased IRF1 transcriptional activity with a 3.5-fold define the relative importance of IRF1 in the expression of proinflammatory cytokines and chemokines. For all treat- change (Fig. 3A). There was, however, no effect of poly ments, IRF1 mRNA expression was decreased in IRF1 (I:C) and fsl-1 on IRF1 transcriptional activity in primary siRNA-transfected cells by approximately 70% (data not amnion cells (data not shown). On the other hand, as shown shown). Figure 5 demonstrates the effect of IRF1 siRNA on in Figure 3B, treatment of primary myometrial cells with myometrial cells stimulated with the proinflammatory IL1B, poly (I:C), or fsl-1 significantly increased IRF1 cytokine IL1B. As expected, IL1B significantly increased transcriptional activity, with fold changes of 8.1, 2.7, and TNF, IL6, CXCL8, and CCL2 mRNA expression and release 3.5, respectively. We also assessed the effect of IL1B, poly of IL6, CXCL8, and CCL2 in NC siRNA-transfected cells (I:C), or fsl-1 on IRF1 mRNA expression in primary (Fig. 5). In cells transfected with IRF1, there was a myometrial cells. As shown in Figure 3C, treatment of significant decrease in IL1B-stimulated IL6, CXCL8, and myometrial cells with IL1B, poly (I:C), or fsl-1 significantly CCL2 mRNA expression and release, and mRNA expres- increased IRF1 mRNA expression. sion of TNF. Similarly, in NC siRNA-transfected cells 6 Article 32 IRF1 AND HUMAN LABOR Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 4. Effect of IRF1 siRNA on basal expression of proinflammatory cytokines. Human primary myometrial cells were transfected with or without 200 nM IRF1 or NC siRNA for 48 h (n ¼ 3 patients). A, C) IRF1 and IL6 gene expression was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to the NC siRNA-transfected cells. Data are displayed as mean 6 SEM. *P , 0.05 versus NC siRNA (one-sample t-test). B) IRF1 protein expression was analyzed by Western blot. Protein expression was normalized to ACTB, and the fold change was calculated to the NC siRNA-transfected cells. Data are displayed as mean 6 SEM. *P , 0.05 versus NC siRNA-transfected cells (Student t-test). D)The incubation medium was assayed for concentration of IL6 by ELISA. The fold change was calculated relative to NC siRNA-transfected cells. Data are displayed as mean 6 SEM. treated with the viral analog poly (I:C) (Fig. 6) or the significant decrease in IL1B-induced PTGS2 mRNA expres- bacterial product fsl-1 (Fig. 7), there was a significant sion and prostaglandin release (Fig. 8A–C). Additionally, increase in TNF,IL1B,IL6,CXCL8,and CCL2 mRNA IRF1 siRNA was associated with a significant decrease in expression and release of IL6, CXCL8, and CCL2. This poly (I:C)- or fsl-1-induced PTGS2 mRNA expression (Fig. 8, increase was abrogated in cells transfected with IRF1 D and E). Of note, there was no effect of IL1B, poly (I:C), fsl- siRNA. 1, or IRF1 silencing on COX-1 mRNA expression (data not shown). Effect of IRF1 siRNA Knockdown on the PTGS2- Prostaglandin Pathway in Primary Myometrial Cells DISCUSSION The final aim of this study was to determine the effect of To our knowledge, this is the first study to show that IRF1 silencing on the PTGS2 prostaglandin pathway in the IRF1 plays a role in the effector pathways involved in the presence of IL1B, poly (I:C), or fsl-1. As expected, in NC terminal processes of human preterm labor and delivery. siRNA-transfected myometrial cells, treatment with IL1B IRF1 expression was increased in laboring fetal membranes (Fig. 8A), poly (I:C) (Fig. 8D), or fsl-1 (Fig. 8E) induced a and myometrium at term. IRF1 expression was also significant increase in PTGS2 mRNA expression. Likewise, increased in fetal membranes obtained from women with histologically confirmed chorioamnionitis at preterm birth. the release of PGE2 and PGF2a was significantly augmented by IL1B in NC siRNA-transfected cells (Fig. 8, B and C). The In keeping with this, stimulation with the bacterial product concentration of prostaglandins was below the sensitivity of fsl-1, the viral product poly (I:C), and pro-inflammatory the assay for poly (I:C)- or fsl-1-stimulated primary human cytokine IL1B, which were used to mimic infection and the myometrial cells. The effect of IRF1 silencing resulted in a inflammatory environment that is associated with preterm 7 Article 32 LIM ET AL. Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 5. Effect of IRF1 siRNA on IL1B-induced proinflammatory cytokines and chemokines. Human primary myometrial cells were transfected with or without 200 nM IRF1 or NC siRNA for 48 h and then treated with 1 ng/ml IL1B for an additional 24 h (n ¼ 5 patients). A–D) Gene expression for TNF, IL6, CXCL8, and CCL2 was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to IL1B-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus IL1B-stimulated NC siRNA-transfected cells (one-way ANOVA). E–G) The incubation medium was assayed for concentration of IL6, CXCL8, and CCL2 by ELISA. The fold change was calculated relative to IL1B-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus IL1B-stimulated NC siRNA-transfected cells (one-way ANOVA). labor, significantly increased IRF1 expression and transcrip- due to reasons such as placental abruption and antepartum tional activity in primary cells isolated from human amnion hemorrhage, which are closely associated with inflamma- and myometrium. Furthermore, we found that IRF1 is tion [48]. Thus, a possible reasoning for no change in IRF1 involved in the genesis of proinflammatory cytokines, expression due to preterm labor may be that IRF1 chemokines, and prostaglandins mediated by infection and expression was already higher due to the increased inflammation. inflammatory environment that is associated with indicated The current study found that IRF1 expression is preterm births [49], and as such, no further increase in increasedinbothfetalmembranesandmyometriumafter IRF1 expression would be observable when compared to spontaneous onset of labor at term compared to no labor. It fetal membranes obtained from women with spontaneous is noted that in term fetal membranes after labor, there is preterm labor. Although we did not assess temporal increased thickness of the cytotrophoblast layer; this may expression of IRF1, there is a possibility that there is an contributetotheincreaseinIRF1expressionseenin increase in IRF1 expression in preterm samples compared laboring fetal membranes, as determined by qRT-PCR and to term. Studies have shown increased expression of pro- Western blot. This increase in IRF1 expression is in inflammatory cytokines (IL6, IL1B) and chemokines accordance with studies in nongestational tissues, where (CXCL8) in gestational tissues and amniotic fluid from IRF1 expression is increased during inflammatory states preterm deliveries when compared to term deliveries [50, [46, 47]. In preterm deliveries in the absence of labor, there 51]. Moreover, this study has shown that IL1B increases wasnodifferenceinIRF1expressioninfetalmembranes IRF1 expression and activity. Given that the preterm from women with PROM compared with intact membranes nonlaboring samples were obtained from indicated preterm that were artificially ruptured at delivery. Furthermore, and deliveries, it is feasible that an increase in IRF1 expression in contrast to term studies, in preterm fetal membranes in is due to this heightened inflammatory state when the absence of infection, there was no effect of spontaneous compared to term nonlaboring samples obtained from preterm labor on IRF1 expression when compared to nonindicated term cesarean deliveries. It is also worth membranes obtained from nonlaboring women. In this noting that other studies have also shown that preterm and study, indicated preterm birth in the absence of labor was term labor differ in their pathophysiology. For example, 8 Article 32 IRF1 AND HUMAN LABOR Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 6. Effect of IRF1 siRNA on viral-induced proinflammatory cytokines and chemokines. Human primary myometrial cells were transfected with or without 200 nM IRF1 or NC siRNA for 48 h and then treated with 5 lg/ml poly (I:C) for an additional 24 h (n ¼ 5 patients). A–E) Gene expression for TNF, IL1B, IL6, CXCL8, and CCL2 was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to poly (I:C)-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus poly (I:C)-stimulated NC siRNA-transfected cells (one-way ANOVA). F–H) The incubation medium was assayed for concentration of IL6, CXCL8, and CCL2 by ELISA. The fold change was calculated relative to poly (I:C)-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus poly (I:C)-stimulated NC siRNA-transfected cells (one-way ANOVA).

Tattersalls and colleagues [52] have shown that prolabor The significance of our finding that IRF1 expression is gene expression is different in term and preterm laboring both cytoplasmic and nuclear is unknown at this time. IRF1 myometrial samples. is translocated to the nucleus after translation, and once Infection and inflammation are considered to be the within the nucleus, it is active. However, IRF1 has also biggest etiological factor for the majority of early preterm been shown to be active in the cytoplasm where it can births [2]. Studies in nongestational tissues have demon- interact with other proteins. Of interest to our study, IRF1 strated increased IRF1 expression is associated with has been shown to interact with MyD88 [59], a cytoplasmic endotoxemia [53] and inflammation [54, 55]. Thus, we adaptor protein critical for TLR signaling and human labor sought to also elucidate the expression of IRF1 in preterm and delivery [14]. Further studies may be conducted to pregnancies complicated by chorioamnionitis and in explore this possible interaction. response to mediators known to induce preterm birth. We Functional studies were then performed to determine if found that IRF1 was increased in fetal membranes from IRF1 plays a mechanistic role in the terminal effector women with preterm histologically confirmed chorioamni- pathways involved in preterm labor induced by infection onitis. Moreover, IL1B, which is increased in human (fsl-1 and poly [I:C]) and inflammation (IL1B). We showed gestational tissues at preterm labor [9] and induces preterm that human primary myometrial cells transfected with IRF1 labor in animal models [56], significantly induced IRF1 siRNA decreased the production of proinflammatory transcriptionalactivityinbothhumanprimaryamnionand cytokines IL6 and TNF and chemokines CXCL8 and myometrial cells. Similarly, the bacterial product and TLR2 CCL2 when stimulated with IL1B, fsl-1, and poly (I:C). ligandfsl-1andtheviraldsRNAanalogandTLR3ligand These findings are of significance as proinflammatory poly (I:C) significantly increased IRF1 transcriptional cytokines and chemokines play an essential role in the key activity. This is of significance given that fsl-1 has been terminal effector pathways of preterm labor and delivery, shown to activate mediators involved in the terminal that is, rupture of fetal membranes, cervical ripening, and effector pathways of preterm labor and delivery in human myometrial contractions. Increased infiltration of leuko- gestational tissues [14, 57] and that poly (I:C) has been cytes into the myometrium during spontaneous preterm shown to induce preterm delivery in pregnant mice [58]. labor is associated with increased production of pro- 9 Article 32 LIM ET AL. Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 7. Effect of IRF1 siRNA on bacterial infection-induced proinflammatory cytokines and chemokines. Human primary myometrial cells were transfected with or without 200 nM IRF1 or NC siRNA for 48 h and then treated with 250 ng/ml fsl-1 for an additional 24 h (n ¼ 5 patients). A–E) Gene expression for TNF, IL1B, IL6, CXCL8, and CCL2 was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to fsl-1-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus fsl-1-stimulated NC siRNA-transfected cells (one-way ANOVA). F–H) The incubation medium was assayed for concentration of IL6, CXCL8, and CCL2 by ELISA. The fold change was calculated relative to fsl-1-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus fsl-1-stimulated NC siRNA-transfected cells (one-way ANOVA). inflammatory cytokines (including TNF, IL1B, and IL6) expression is increased in gestational tissues during and chemokines (such as CXCL8 and CCL2) [60–62]. parturition [68]. Studies in nongestational tissues have IL1B stimulates the production of contraction-associated demonstrated IRF1 regulates PTGS2 expression by binding proteins [63] and induces preterm labor in animal models to the PTGS2 promoter region [69, 70]. For example, LPS- [56]. Increased expression of IL6, CXCL8, and CCL2 is induced PTGS2 expression was attenuated in IRF1- associated with cervical ripening [64] because these deficient mice [70]. In agreement, this study found cytokines can increase production of metalloproteinases IL1B-, poly (I:C)-, and fsl-1-induced PTGS2 expression, [9]. We therefore hypothesize that during preterm labor and IL1B-induced prostaglandin secretion was significant- (triggered by inflammation or infection), IRF1 is activated, ly attenuated in IRF1-deficient primary human myometrial leading to increased expression of proinflammatory cyto- cells. kines and chemokines. The upregulation of these pro- This study demonstrates that IRF1 expression is increased inflammatory cytokines and chemokines leads to fetal in fetal membranes and myometrium after term labor. There membrane rupture, cervical ripening, and myometrial was no change in IRF1 expression in preterm fetal contractions, which then eventually leads to labor and membranes either after spontaneous labor or with PROM. delivery. IRF1 may thus be a possible point of intervention IRF1 transcriptional activity was induced in primary for preterm birth; the inhibition of IRF1 could effectively myometrial cells with inflammation and bacterial and viral suppress labor mediators that lead to preterm labor and stimuli, and with IL1B in primary amnion cells. Further- delivery. more,transfectionwithIRF1siRNAwasassociatedwitha Prostaglandins play a key role in labor and delivery by decrease in inflammation- and infection-induced expression potentiating uterine contractility and cervical ripening [65]. of proinflammatory cytokines and chemokines. This is of Proinflammatory cytokines IL6, IL1B, and TNF have been significance given the importance of the inflammatory shown to stimulate prostaglandin synthesis in intrauterine response via inflammation and bacterial and viral infection tissues leading to spontaneous preterm labor and delivery in the processes of preterm birth [13, 60]. Figure 9 depicts [66, 67]. PTGS2 is a proinflammatory enzyme that is our working model of how IRF1 regulates spontaneous responsible for the formation of prostaglandins, and its preterm birth. Infection and/or inflammation increase IRF1 10 Article 32 IRF1 AND HUMAN LABOR Downloaded from https://academic.oup.com/biolreprod/article/94/2/32, 1-13/2434373 by guest on 01 October 2021 Downloaded from www.biolreprod.org.

FIG. 8. Effect of IRF1 siRNA on PTGS2-prostaglandin pathway. Human primary myometrial cells were transfected with or without 200 nM IRF1 or NC siRNA 48 h and then treated with 1 ng/ml IL-1b (A–C), 5 lg/ml poly (I:C) (D), or 250 ng/ml fsl-1 (E) for an additional 24 h (n ¼ 5 patients). A, D, E) Gene expression for PTGS2 was analyzed by qRT-PCR. Gene expression was normalized to ACTB mRNA expression, and the fold change was calculated relative to IL1B-, poly (I:C)-, or fsl-1-stimulated NC siRNA-transfected cells. Data displayed as mean 6 SEM. *P , 0.05 versus IL1B-stimulated NC siRNA- transfected cells (one-way ANOVA); §P , 0.05 versus poly (I:C)-stimulated NC siRNA-transfected cells (one-way ANOVA); #P , 0.05 versus fsl-1- stimulated NC siRNA-transfected cells (one-way ANOVA). B, C) The incubation medium was assayed for concentration of PGE2 and PGF2a release by EIA. Data displayed as mean 6 SEM. *P , 0.05 versus IL1B-stimulated NC siRNA-transfected cells (one-way ANOVA). expression, leading to increased production of proinflamma- models, however, will be required to establish whether IRF1 tory cytokines, chemokines, and prostaglandins; pro- inhibitors can prevent infection-induced preterm labor in inflammatory cytokines can also further induce IRF1 vivo. expression. The upregulation of these prolabor mediators can lead to fetal membrane rupture, and thus PROM, or can lead to increased uterine contractility and preterm labor, which then culminates in spontaneous preterm birth. Animal 11 Article 32 LIM ET AL.

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