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ATF3 is a negative regulator of inflammation in human fetal membranes
Placenta 47 (2016) 63e72
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Placenta journal homepage: www.elsevier.com/locate/placenta
ATF3 is a negative regulator of inflammation in human fetal membranes Ratana Lim a, b, Gillian Barker a, b, Stella Liong a, b, Caitlyn Nguyen-Ngo a, b, Stephen Tong b, c, Tu'uhevaha Kaitu'u-Lino b, c, Martha Lappas a, b, * a b c
Obstetrics, Nutrition and Endocrinology Group, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia Translational Obstetrics Group, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2016 Received in revised form 7 September 2016 Accepted 13 September 2016
Introduction: Infection and inflammation stimulate pro-inflammatory cytokines, prostaglandins and matrix metalloproteinase (MMP)-9, which play a central role in myometrial contractions and rupture of fetal membranes. In human and mouse immune cells, activating transcription factor 3 (ATF3) is a negative regulator of inflammation. No studies have examined the role of ATF3 in human labour. Methods: Primary amnion cells were used to determine the effect of interleukin (IL)-1b and the bacterial product fibroblast-stimulating lipopeptide (fsl-1) on ATF3 expression, and the effect of ATF3 siRNA on pro-labour mediators. ATF3 expression was assessed in fetal membranes from non-labouring and labouring women at term and preterm, and after preterm pre-labour rupture of membranes (PPROM). Results: IL-1b and fsl-1 significantly increased ATF3 expression. Silencing ATF3 significantly increased IL1b- or fsl-1-induced expression of pro-inflammatory cytokines (TNF-a, IL-1a, IL-1b, IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1 (MCP-1)); cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin PGF2a release; and MMP-9 expression. ATF3 expression was decreased in fetal membranes with term labour. There was no effect of preterm labour or PPROM on ATF3 expression. Discussion: ATF3 is a negative regulator of inflammation in human fetal membranes; in primary amnion cells, ATF3 expression is induced by IL-1b and fsl-1, and ATF3 silencing further exacerbates the inflammatory response when stimulated with these factors. Subsequently, ATF3 expression is decreased in fetal membranes after term labour and with preterm chorioamnionitis, conditions closely associated with inflammation and infection. Our data suggest that ATF3 may play a role in the terminal processes of human labour and delivery. © 2016 Elsevier Ltd. All rights reserved.
Keywords: ATF3 Human labour Inflammation Infection Fetal membranes
1. Introduction Preterm birth is the leading cause of infant morbidity and mortality [1]. For survivors, long-term risks associated with preterm birth include cerebral palsy, learning difficulties and respiratory illnesses [2]. Approximately 70% of preterm births occur spontaneously as a result of idiopathic preterm labour or preterm prelabour rupture of membranes (PPROM); PPROM is associated with higher rates of neonatal mortality and morbidity [3].
* Corresponding author. Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, 3084, Victoria, Australia. E-mail address: [email protected] (M. Lappas). http://dx.doi.org/10.1016/j.placenta.2016.09.006 0143-4004/© 2016 Elsevier Ltd. All rights reserved.
Alongside the impact preterm birth has on the child and family, data from the USA indicates the annual costs associated with preterm birth exceeds US$26 billion per year [4]. The costs imposed upon families and communities stem from the absence of effective therapeutics that can stop preterm labour. The processes involved in parturition are incompletely understood which has prevented the development of effective strategies to prevent spontaneous preterm birth. What we do know, however, is that the majority of spontaneous preterm births are due to pathological activation of the normal labour processes [3,5]. The bacterial product fibroblast-stimulating lipopeptide (fsl-1), while not a pathogenic micro-organism, is able to model the effects of such microbes [6], by stimulating the production of pro-labour mediators including pro-inflammatory cytokines, chemokines, cyclooxygenase (COX)-2 and subsequent prostaglandins, and
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2. Materials and methods
streptomycin. The media was replaced after 4 h then every 24e48 h thereafter. To determine the effect of pro-inflammatory mediators on ATF3 expression, cells at approximately 80% confluence were incubated in the absence or presence of 1 ng/ml IL-1b or 250 ng/ml fsl-1 for 24 h. Cells were collected and stored at 80 C until assayed for ATF3 protein expression by qRT-PCR or Western blotting as detailed below. Experiments were performed in amnion cells obtained from five patients. Transfection of primary amnion cells with siRNA was performed as we have previously described [29]. Briefly, cells at approximately 50% confluence were transfected using Lipofectamine 3000 according to manufacturer's guidelines (Life Technologies; Mulgrave, Victoria, Australia). SMARTpool ATF3 siRNA (siATF3) and negative control siRNA (siCONT) were obtained from Dharmacon (GE Healthcare Australia Pty. Ltd; Parramatta, NSW, Australia). Cells were transfected with 200 nM siATF3 or 200 nM siCONT in DMEM/ F-12 for 48 h. The medium was then replaced with DMEM/F-12 (containing 0.5% BSA) with or without 1 ng/ml IL-1b or 250 ng/ml fsl-1, and the cells were incubated at 37 C for an additional 24 h. Cells were collected and stored at 80 C until assayed for mRNA expression by qRT-PCR and protein expression by Western blotting as detailed below. Media was collected and stored at 80 C until assayed for cytokine, prostaglandin and MMP-9 release as detailed below. Cell viability was assessed by the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay as we have previously described [30]. The response to IL1b and fsl-1 between patients varied greatly, as we have previously reported [29]. Thus, data is presented as fold change in expression relative to the expression level in the IL-1b- or fsl-1-stimulated siCONT transfected cells, which was set at 1. Data could not be normalised to siCONT transfected cells alone as some of the readings were 0. Experiments were performed in amnion cells obtained from six patients.
2.1. Tissue collection
2.3. RNA extraction and quantitative RT-PCR (qRT-PCR)
The Research Ethics Committee of Mercy Hospital for Women approved this study. Written, informed consent was obtained from all participating women. All tissues were obtained from women who delivered healthy, singleton infants. All tissues were brought to the research laboratory and processed within 15 min of the Caesarean delivery. Tissues from women with any underlying medical conditions such as diabetes, asthma, polycystic ovarian syndrome, preeclampsia and macrovascular complications were not included. Additionally, tissues from women with multiple pregnancies, obese women, fetuses with chromosomal abnormalities were not included.
RNA extractions and qRT-PCR was performed as previously described [29]. Total RNA was extracted using TRIsure reagent according to manufacturer's instructions (Bioline; Alexandria, NSW, Australia). RNA concentration and purity were measured using a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific; Scoresby, Vic, Australia). RNA quality and integrity was determined via the A260/A280 ratio. RNA was converted to cDNA using the Tetro cDNA synthesis kit (Bioline; Alexandria, NSW, Australia) according to the manufacturer's instructions. The cDNA was diluted fifty-fold, and 4 ml of this was used to perform RT-PCR using SensiFAST™ SYBR NO-ROX Kit (Bioline; Alexandria, NSW, Australia) and 100 nM of pre-designed and validated QuantiTect primers (Qiagen; Chadstone Centre, Vic, Australia). The RT-PCR was performed using the CFX384 Real-Time PCR detection system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Average gene Ct values were normalised to the average b2-Microglobulin (B2M) and 18S ribosomal RNA (rRNA) Ct values of the same cDNA sample. Of note, there was no effect of experimental treatment on B2M or 18S rRNA gene expression. Fold differences were determined using the comparative Ct method.
metalloproteinase (MMP)-9 in fetal membranes [7,8]. These prolabour mediators participate in the uterine components of this common pathway of parturition i.e. myometrial contractility, cervical ripening and rupture of fetal membranes. Notably, and even in the absence of detected pathogens, most cases of spontaneous preterm birth have histological evidence of inflammation in the uteroplacental unit [9]. Pro-inflammatory cytokines such as IL-1b can activate the terminal processes of human labour and delivery [10e13] and induce preterm birth in mice and monkeys [14e16]. Recent studies in non-gestational tissues have demonstrated a role for activating transcription factor 3 (ATF3) as a negative regulator of inflammation [17e19]. ATF3 is a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors [20]. ATF3 functions as a transcriptional repressor [21]; while ATF3 expression is maintained at low levels in quiescent cells, ATF3 gene expression can be induced by a variety of stress signals [22], such as Toll-like receptor (TLR) agonist bacterial lipopolysaccharide (LPS). In nongestational tissues, the role of ATF3 as a negative regulator of inflammation is demonstrated whereby ATF3 is induced by TLRs, such as TLR2, 3, 4 and 9 [23], but that ATF3 silencing leads to an exacerbated inflammatory response [17e19,23e25]. While ATF2 has been considered to play a role in human myometrium [26,27], to our knowledge the expression or the role of ATF3 has not been investigated in human fetal membranes. Therefore, an aim of this study were to establish the expression of ATF3 in human fetal membranes obtained from labouring and nonlabouring women at preterm and term. Furthermore, loss-offunction studies were performed to determine whether ATF3 is involved in the genesis of pro-inflammatory and pro-labour mediators induced by inflammation (IL-1b) or infection (using the bacterial product fsl-1).
2.2. Primary amnion cell culture Primary amnion cells were used to investigate the effect of proinflammatory mediators on ATF3 expression and the effect of ATF3 siRNA-mediated gene silencing on the expression of pro-labour mediators. For these studies, fresh amnion was obtained 2 cm from the peri-placental edge from women who delivered healthy, singleton infants at term (37e40 weeks gestation) undergoing elective Caesarean section in the absence of labour. Amnion cells (epithelial and mesenchymal) were prepared as previously described [28]. Briefly, amnion strips were washed in PBS and digested, twice, with 0.125% collagenase A and 0.25% trypsin in serum-free DMEM for 35 min at 37 C. The cell suspension was filtered through a cell strainer and the eluate was neutralised with 1% FCS. The cell suspensions were centrifuged at 500g for 10 min and the cells cultured in DMEM/F-12, 10% FCS and 1% penicillin-
2.4. Cytokine, chemokine and prostaglandin assays Assessment of cytokine and chemokine release of IL-6, IL-8, MCP-1 and TNF-a was performed using CytoSet™ sandwich ELISA according to the manufacturer's instructions (Life Technologies; Mulgrave, Vic, Australia). The limit of detection of the IL-6, IL-8, MCP-1 and TNF-a assays was 16, 12, 15 and 7 pg/ml, respectively.
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The concentration of IL-1a and IL-1b in the media was performed by sandwich ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The limit of detection of the IL-1a and IL-1b assays was 7.8 and 2 pg/ml, respectively. The release of PGF2a into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, NSW, Australia). The limit of detection of the PGF2a assays was 60 pg/ml. The interassay and intraassay coefficients of variation (CV) for all assays were less than 10%.
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Incubation media was also collected and assessment of enzymes of extracellular matrix (ECM) weakening and rupture (MMP-2 and MMP-9) was performed by gelatin zymography as previously described [28]. Proteolytic activity was visualized as clear zones of lysis on a blue background of undigested gelatin. Gels were scanned using a ChemiDoc XRS system (Bio-Rad Laboratories; Gladesville, NSW, Australia), images inverted to show dark bands rather than the clear zones of lysis, and densitometry performed using Quantity One image analysis software (Bio-Rad Laboratories; Gladesville, NSW, Australia).
were placenta praevia, placental abruption, antepartum haemorrhage (APH) or Rhesus isoimmunisation. PPROM was defined as spontaneous rupture of the membranes at less than 37 weeks gestation at least 1 h before the onset of any contractions. All placentas collected from preterm gestations were subject to histopathological examination and fetal membranes were swabbed for microbiological culture investigations. Chorioamnionitis was diagnosed pathologically according to standard criteria which included histological evidence of macrophages and neutrophils permeating the chorionic cell layer and often infiltrating the amniotic cell [32]. Histological chorioamnionitis is often accompanied by isolation of a microbiological organism from the fetal membranes. Women with preeclampsia, preexisting diabetes, asthma, multiple pregnancies, and fetuses with chromosomal abnormalities were not included. For the preterm labour study, the SCS could not be identified and thus, fetal membranes were obtained 2 cm from the peri-placental edge, an area approximately 5 5 cm in size. The clinical details of the preterm patients are described elsewhere [33]. Of note, there was no difference in maternal age and body mass index, or parity of the patients recruited. Tissue samples were snap frozen in liquid nitrogen and immediately stored at 80 C for analysis of ATF3 expression by qRT-PCR and Western blot as detailed below.
2.6. Tissue samples for expression studies
2.7. Western blotting
For expression studies by Western blotting, fetal membranes were obtained from women at (i) term no labour undergoing elective Caesarean section (indications for Caesarean section were breech presentation and/or previous Caesarean section) (n ¼ 8 patients; mean gestational age 38.8 ± 0.3 weeks) and (ii) term after spontaneous labour, spontaneous membrane rupture, and normal vaginal delivery (n ¼ 8 patients; mean gestational age 38.7 ± 0.4 weeks). Fetal membranes from the non-labouring group were obtained from the supracervical site (SCS). This area overlying the cervix corresponds to the site of rupture, thus providing a relative area of comparison in fetal membranes before and after labour. Identification of the SCS was performed as described previously [31]. Briefly, Bonneys blue dye was introduced through the cervix prior to Caesarean section. Upon delivery of the placenta, a blue mark was obvious on the chorion facing membrane where the dye had been applied. In the after labour group, fetal membranes were obtained from the site of membrane rupture (SOR) as previously described [6], an area approximately 5 5 cm in size. Amnion and underlying choriodecidua were collected from along the line of fetal membrane rupture. There was no difference in maternal age and body mass index, parity, or gestational age of the patients recruited. In the term after labour group none of the patients received any medications to augment or induce labour, and the average length of labour was 6 h 40 min ± 1 h 40 min. Tissue samples were snap frozen in liquid nitrogen and immediately stored at 80 C for analysis of ATF3 expression by qRT-PCR and Western blot as detailed below. Fetal membranes were also obtained from women at preterm birth from the following groups (i) Caesarean section in the absence of labour with intact membranes (artificial rupture of membranes (ARM) at delivery; n ¼ 8 patients; mean gestational age 32.8 ± 0.7 weeks); (ii) Caesarean section in the absence of labor with PPROM (n ¼ 8 patients; mean gestational age 31.8 ± 0.7 weeks); (iii) after spontaneous labour and normal vaginal delivery without histologically confirmed chorioamnionitis (n ¼ 8 patients; mean gestational age 32.5 ± 0.8 weeks); and (iv) after spontaneous labour and normal vaginal delivery with histologically confirmed chorioamnionitis (n ¼ 8 patients; mean gestational age 31.6 ± 4.0 weeks). Indications for preterm delivery (in the absence of labour)
Western blotting was performed as we have previously described [29]. Blots were incubated in 1 mg/ml rabbit polyclonal anti-ATF3 (cat # HPA001562; Sigma, St. Louis, MO, USA) prepared in blocking buffer (5% skim milk/TBS-T (0.05%)) for 16 h at 4 C. Membranes were viewed and analysed using the ChemiDoc XRS system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Semiquantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad Laboratories, Hercules, CA, USA). The levels of ATF3 were normalised to the levels of b-actin (Sigma, St. Louis, MO, USA).
2.5. Gelatin zymography
2.8. Statistical analysis Statistics was performed on the normalised data unless otherwise specified. All statistical analyses were undertaken using GraphPad Prism (GraphPad Software, La Jolla, CA). For Fig. 1, a onesample t-test, against the constant of 1, was used. For Figs. 2e5, the homogeneity of data was assessed by the Bartlett's test, and when significant, the data were logarithmically transformed before further analysis using a one-way ANOVA (using LSD correction to discriminate among the means). For Fig. 6, unpaired Student's t-test was used to assess statistical significance between normally distributed data; otherwise, the nonparametric Mann-Whitney U (unpaired) test was used. Statistical significance was ascribed to P value < 0.05. Data were expressed as mean ± standard error of the mean (SEM). 3. Results 3.1. Activation of ATF3 limits the inflammatory response Studies in non-gestational tissues have shown that ATF3 is rapidly induced by cytokines and TLR agonists [17,23,25], in order to suppress inflammation [17]. As these factors are paramount to the inflammatory response associated with preterm birth [34], we determined whether the pro-inflammatory cytokine IL-1b (i.e. inflammation) and the bacterial product fsl-1 (used as a model for intra-uterine infection) could affect ATF3 expression in fetal
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Fig. 1. Effect of pro-inflammatory mediators on ATF3 expression. Human primary amnion cells were incubated in the absence or presence of 1 ng/ml IL-1b or 250 ng/ml fsl-1 for 24 h (n ¼ 6 patients). (A) ATF3 mRNA expression was analysed by qRT-PCR. (B) ATF3 protein expression was analysed by Western blot. Representative Western blot from 1 patient is also shown. For all data, the fold change was calculated relative to basal and data displayed as mean ± SEM. *P < 0.05 vs. basal (paired sample comparison).
Fig. 2. Effect of siATF3 knockdown on IL-1b-induced pro-inflammatory cytokines and chemokines. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with 1 ng/ml IL-1b for an additional 24 h (n ¼ 5 patients). (A,B,D,F) IL-1a, IL-6, IL-8 and MCP-1 mRNA expression was analysed by qRTPCR. (C,E,G) IL-6, IL-8 and MCP-1 concentration in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL-1b-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. IL-1b-stimulated siCONT transfected cells (one-way ANOVA).
membranes. Treatment of amnion cells with IL-1b or fsl-1 significantly increased ATF3 mRNA and protein expression, as shown in Fig. 1. We next sought to determine whether ATF3 is involved in the genesis of pro-labour mediators induced by inflammation (e.g. IL-
1b) or infection (e.g. the bacterial product fsl-1). The efficacy of siATF3 knockdown in primary amnion cells is demonstrated in Supplementary Fig. 1. When compared to siCONT transfected cells, siATF3 transfection resulted in 50% decrease in ATF3 mRNA expression and 70% decrease in ATF3 protein expression. There was
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Fig. 3. Effect of siATF3 knockdown on bacterial infection-induced pro-inflammatory cytokines and chemokines. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with 250 ng/ml fsl-1 for an additional 24 h (n ¼ 5 patients). (AeC,E,G) TNF-a, IL-1b, IL-6, IL-8 and MCP-1 mRNA expression was analysed by qRT-PCR. (D,F,H) IL-6, IL-8 and MCP-1 concentration in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to fsl-1-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. fsl-1-stimulated siCONT transfected cells (one-way ANOVA).
Fig. 4. Effect of siATF3 knockdown on the prostaglandin pathway. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with 1 ng/ml IL-1b for an additional 24 h (n ¼ 5 patients). (A) COX-2 mRNA expression was analysed by qRT-PCR (B) PGF2a concentration in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL-1b-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. IL-1b-stimulated siCONT transfected cells (one-way ANOVA).
no effect of siATF3 on cell viability as determined by MTT cell viability assay. For subsequent experiments, after siRNA transfection, cells were treated with IL-1b or fsl-1 as a model of inflammation- or infection-induced preterm labour in order to define the relative importance of ATF3 in the expression of pro-
labour mediators. The effect of siATF3 on IL-1b-stimulated cytokine expression in amnion is depicted in Fig. 2. As expected, in siCONT transfected cells, IL-1b significantly increased IL-1a, IL-6, IL-8 and MCP-1 mRNA expression (Fig. 2A,B,D,F) and IL-6, IL-8 and MCP-1 secretion
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Fig. 5. Effect of siATF3 knockdown on MMP-9 expression. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with (AeD) 1 ng/ml IL-1b or (EeH) 250 ng/ml fsl-1 for an additional 24 h (n ¼ 5 patients). (A,B,E,F) MMP-2 and MMP-9 mRNA expression was analysed by qRT-PCR. (C,D,G,H) The incubation medium was assayed for MMP-2 and MMP-9 expression by gelatin zymography. Representative gelatin zymography from one patient is also shown. For all data, the fold change was calculated relative to IL-1b-or fsl-1-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. IL-1b-stimulated siCONT transfected cells (oneway ANOVA); xP < 0.05 vs. fsl-1-stimulated siCONT transfected cells (one-way ANOVA).
(Fig. 2C,E,G). In siATF3 transfected cells, IL-1b further augmented this cytokine and chemokine expression and secretion. Of note, the release of IL-1a was below the sensitivity of the assay. The effect of siATF3 on fsl-1-stimulated pro-inflammatory cytokines is demonstrated in Fig. 3. In siCONT transfected cells, fsl-1 significantly increased in TNF-a, IL-1b, IL-6, IL-8 and MCP-1 mRNA expression (Fig. 3AeC,E,G) and IL-6, IL-8 and MCP-1 secretion (Fig. 3D,F,H). This increase in the mRNA expression and release was significantly augmented in cells transfected with siATF3. Of note, the release of TNF-a and IL-1b were below the sensitivity of the assays. Fig. 4 demonstrates the effect of siATF3 knockdown on IL-1binduced COX-2 mRNA expression and release of the prostaglandin PGF2a. In cells transfected with siCONT, IL-1b induced a significant increase in COX-2 expression (Fig. 4A) and PGF2a release (Fig. 4B). In siATF3 transfected cells, IL-1b caused a further increase in COX-2 mRNA expression and PGF2a release. In siCONT transfected cells, IL-1b (Fig. 5B) and fsl-1 (Fig. 5F) induced an increase in MMP-9 mRNA expression. Likewise, by gelatin zymography, secretory pro-MMP-9 activity was also significantly higher with IL-1b (Fig. 5D) and fsl-1 (Fig. 5H) treatment. When ATF3 was silenced, there was statistically higher MMP9 mRNA and pro-MMP-9 activity. There was no effect of IL-1b, fsl-1 or siATF3 on MMP-2 mRNA expression or secretory pro-MMP-2 activity (Fig. 5A,C,E,G).
3.2. Effect of human term and preterm labour on ATF3 expression in fetal membranes Having shown that ATF3 is induced by mediators of preterm birth after 24 h and that gene silencing of ATF3 increases the inflammatory effect of IL-1b and fsl-1, we next sought to determine
the expression of ATF3 in fetal membranes obtained from term and preterm deliveries that have undergone labour and thus are likely to be associated with increased inflammation. To determine the effect of human term labour on ATF3 expression in fetal membranes, samples were obtained at term Caesarean section in the absence of labour (term, no labour) and after spontaneous labour and membrane rupture (term, after labour). ATF3 mRNA (Fig. 6A) and protein (Fig. 6B) expression was significantly lower in fetal membranes after spontaneous labour at term when compared to non-labouring tissues. To determine the effect of spontaneous preterm birth (without histologically confirmed chorioamnionitis) on ATF3 expression, fetal membranes were obtained from women at preterm Caesarean section with no labour (preterm no labour, no chorioamnionitis), and after spontaneous preterm labour and normal vaginal delivery (preterm after labour, no chorioamnionitis). ATF3 mRNA expression (Fig. 6C) and protein expression (Fig. 6D) were similar between the two groups. To determine the effect of infection on ATF3 expression, fetal membranes after spontaneous preterm labour and normal vaginal delivery without histologically confirmed chorioamnionitis (preterm after labour, no chorioamnionitis) were compared to fetal membranes after spontaneous preterm labour and normal vaginal delivery with histologically confirmed chorioamnionitis (preterm after labour, chorioamnionitis). ATF3 mRNA expression was significantly lower in the preterm group with histologic chorioamnionitis compared to the preterm group without histologic chorioamnionitis (Fig. 6C). ATF3 protein expression could not be analysed by Western blotting on fetal membranes from women with chorioamnionitis due to protein degradation in these samples [35]. To determine the effect of preterm membrane rupture on ATF3
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Fig. 6. ATF3 in fetal membranes. (A,B) Human fetal membranes were obtained from women not in labour at term Caesarean section and women after term spontaneous labour onset and delivery (n ¼ 8 patients per group). ATF3 mRNA expression was analysed by qRT-PCR and ATF3 protein expression was analysed by Western blotting. Fold change was calculated relative to the term no labour group. Data is displayed as mean ± SEM. *P < 0.05 vs. term no labour (Student's t-test). Representative Western blot from 3 patients per group is also shown. (C,D) Fetal membranes were obtained from women not in labour at preterm Caesarean section without histologically confirmed chorioamnionitis (preterm no labour, no chorioamnionitis; n ¼ 6 patients), after preterm spontaneous labour onset and delivery without histologically confirmed chorioamnionitis (preterm after labour, no chorioamnionitis; n ¼ 8 patients), or after preterm spontaneous labour onset and delivery with histologically confirmed chorioamnionitis (preterm after labour, chorioamnionitis; n ¼ 6 patients). ATF3 mRNA expression was analysed by qRT-PCR and gene expression and ATF3 protein expression was analysed by Western blotting. Fold change was calculated relative to the preterm no labour no chorioamnionitis group. Data is displayed as mean ± SEM. *P < 0.05 vs. preterm no labour no infection (Student's t-test). Representative
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expression, fetal membranes were obtained at preterm Caesarean section in the absence of labour from women with (i) intact membranes or (ii) after PPROM. There was no difference in ATF3 mRNA (Fig. 6E) or protein (Fig. 6F) expression between the two groups. 4. Discussion This study, for the first time, describes a possible role for ATF3 in the terminal processes of human labour and delivery in fetal membranes. Specifically, we found in vitro that in the presence of the pro-inflammatory mediators IL-1b and fsl-1, there was an increase in ATF3 expression in primary amnion cells; functional studies revealed a role for ATF3 in the genesis of infection- or inflammation-induced pro-labour mediators. Our data suggests that negative feedback regulation by ATF3 limits the production of pro-inflammatory mediators in response to infection and/or inflammation in human fetal membranes. Subsequently, we found that ATF3 expression in fetal membranes (combined amnion and choriodecidua) was decreased with spontaneous term labour and preterm chorioamnionitis, conditions tightly coupled to inflammation and infection. There is much evidence demonstrating that ATF3 negatively regulates inflammation; studies in non-gestational tissues have shown that ATF3 expression is induced by cytokines and TLR agonists involved in the inflammatory response [23,25]. It is therefore a molecular brake to prevent an excess inflammatory response. We first sought to determine the expression of ATF3 in response to known mediators of preterm birth; IL-1b is increased in gestational tissues at preterm labour [7] and can induce preterm labour in animal models [15], and the bacterial product and TLR2 ligand fsl-1 can activate mediators involved in the terminal effector pathways of human labour and delivery [6,28]. In primary amnion cells treated with IL-1b and fsl-1, we report an increase in ATF3 expression. This induction of ATF3 expression was also found in non-gestational tissues [23]. As a negative regulator of inflammation, ATF3 has been shown to be induced by various TLRs and cytokines [23,25] but ATF3 deficient cells promote an elevated inflammatory response. ATF3 knockdown in macrophages significantly increases interferon (IFN)-b expression [25]. A number of animal studies have shown that after LPS challenge, atf3 deficient mice have significantly increased production of inflammatory markers, such as serum IL-6 and IL-12b levels [17], chemokine CCL4 expression [36] and IL-8 and ICAM-1 expression [24]. The expression of COX-2 and prostaglandin production was found to be significantly higher in atf3 deficient mice after stimulation with TLR2/6 agonist zymosan [19]. Others have also reported increased MMP-9 expression in ATF3deficient cancer cells [37]. These studies corroborate the findings of the current study, where ATF3 knockdown in primary amnion cells is associated with increased pro-inflammatory (IL-6, IL-8 and MCP-1) and pro-labour mediators (COX-2 and prostaglandins and MMP-9) when stimulated with IL-1b and fsl-1. These findings are of significance, as these inflammatory mediators are involved in membrane rupture and cervical remodelling, essential for labour and delivery at both term and preterm. Collectively, our studies further attest that ATF3 is involved in the resolution of inflammation. Human labour is considered an inflammatory response, as an
influx of leukocytes into the fetal membranes produces proinflammatory cytokines, such as IL-6, IL-8 and IL-1b [38,39]. In turn, these pro-inflammatory cytokines can increase MMP-9 expression and activity in the fetal membranes, as well as COX-2 expression and subsequent prostaglandin production. Of note, human gestational tissues can all produce these inflammatory mediators as cultured cells, regardless of leukocyte infiltration. The current study demonstrates that ATF3 expression is decreased in fetal membranes after term labour, compared to non-labouring fetal membranes. While much of the literature reports increased ATF3 expression during a disease state, ATF3 is down-regulated in human prostate cancer [40,41] and esophageal squamous cell carcinomas [42]. In contrast to term studies, there was no change in ATF3 expression in fetal membranes after preterm labour. That the SCS could not be identified in the preterm samples is a limitation to our study, and could possibly be responsible for why we saw no change in ATF3 expression. Additionally, in the absence of labour, there was no change in ATF3 expression associated with PPROM, compared to membranes that were artificially ruptured at delivery. A possible reason why there was no change in ATF3 expression in these groups is that there is an overall increase in inflammation associated with preterm deliveries [43] that blanketed any further effect of labour or rupture of membranes. An important contributor to preterm birth is intrauterine inflammation, which commonly presents as chorioamnionitis. Intrauterine infection and/or inflammation are the pathological processes that provide a firm causal link to preterm birth [9,43]. Pregnancies affected by infection characteristically display elevated levels of pro-inflammatory cytokines in maternal serum, amniotic fluid and gestational tissues [9]. Expression of ATF3 was found to be decreased in fetal membranes from preterm deliveries with chorioamnionitis. Collectively, our studies correspond to the established role of ATF3 as a negative regulator of inflammation; IL-1b and fsl-1 treatments (for 24 h in vitro) induces ATF3 expression, but ATF3 knockdown augments the inflammatory response induced by said mediators. That we also saw a decrease in ATF3 expression in fetal membranes after term labour and with preterm chorioamnionitis further attests this role for ATF3; these conditions can be considered endpoints that are already in a chronically inflamed state, as the processes of labour can last over 24 h. The chronic stimulation of the inflammatory process could decrease ATF3, leading to the inflammatory processes that dominate labour and delivery. This study demonstrates that ATF3 expression is decreased in fetal membranes after term labour and with preterm chorioamnionitis. Up-regulation by pro-labour mediators IL-1b and fsl1 and the exacerbation of the inflammatory response in ATF3deficient amnion cells indicate that ATF3 is a negative regulator of the inflammatory response in human gestational tissues. This is of significance given the driving role of inflammation and bacterial infection in the processes of preterm birth [44]. Improvement of our understanding of the mechanisms associated with spontaneous preterm birth will lead to more effective interventions. Funding ML, KT and ST are supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC #1047025, #1050765, #1062418). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer
Western blot from 3 patients per group is also shown. (E,F) Fetal membranes were obtained from women at preterm Caesarean section not in labour with intact membranes (n ¼ 6 patients) and from women at preterm Caesarean section not in labour with PROM (n ¼ 6 patients). ATF3 mRNA expression was analysed by qRT-PCR and ATF3 protein expression was analysed by Western blotting. Fold change was calculated relative to the intact membranes group. Data is displayed as mean ± SEM. *P < 0.05 vs. intact membranes (Student's ttest). Representative Western blot from 3 patients per group is also shown.
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Medical Research Foundation and the Mercy Research Foundation. Disclosure summary The authors have nothing to declare. Conflict of interest The authors have nothing to declare. Acknowledgements The following are gratefully acknowledged: Clinical Research Midwives Genevieve Christophers, Gabrielle Pell, and Rachel Murdoch for sample collection; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2016.09.006. References [1] L. Liu, H.L. Johnson, S. Cousens, J. Perin, S. Scott, J.E. Lawn, I. Rudan, H. Campbell, R. Cibulskis, M. Li, C. Mathers, R.E. Black, Child Health Epidemiology Reference Group of WHO and Unicef. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000, Lancet 379 (9832) (2012) 2151e2161. [2] K. Flood, F.D. Malone, Prevention of preterm birth, Semin. Fetal Neonatal Med. 17 (1) (2012) 58e63. [3] R. Menon, S.J. Fortunato, Infection and the role of inflammation in preterm premature rupture of the membranes, Best Pract. Res. Clin. Obstet Gynaecol. 21 (3) (2007) 467e478. [4] Preterm birth: causes, consequences, and prevention, in: R.E. Behrman, A.S. Butler (Eds.), National Acadamies Press, Washington (DC), 2007. [5] R.S. Gibbs, J.D. Blanco, Premature rupture of the membranes, Obstet. Gynecol. 60 (6) (1982) 671e679. [6] R. Lim, G. Barker, M. Lappas, The TLR2 ligand FSL-1 and the TLR5 ligand Flagellin mediate pro-inflammatory and pro-labour response via MyD88/ TRAF6/NF-kappaB-dependent signalling, Am. J. Reprod. Immunol. 71 (5) (2014) 401e417 (New York, NY : 1989). [7] J.M. Bowen, L. Chamley, J.A. Keelan, M.D. Mitchell, Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition, Placenta 23 (4) (2002) 257e273. [8] I. Christiaens, D.B. Zaragoza, L. Guilbert, S.A. Robertson, B.F. Mitchell, D.M. Olson, Inflammatory processes in preterm and term parturition, J. Reprod. Immunol. 79 (1) (2008) 50e57. [9] R.L. Goldenberg, J.C. Hauth, W.W. Andrews, Intrauterine infection and preterm delivery, N. Engl. J. Med. 342 (20) (2000) 1500e1507. [10] J.A. Keelan, M. Blumenstein, R.J. Helliwell, T.A. Sato, K.W. Marvin, M.D. Mitchell, Cytokines, prostaglandins and parturitionea review, Placenta 24 (Suppl A) (2003) S33eS46. [11] J.A. Keelan, K.W. Marvin, T.A. Sato, M. Coleman, L.M. McCowan, M.D. Mitchell, Cytokine abundance in placental tissues: evidence of inflammatory activation in gestational membranes with term and preterm parturition, Am. J. Obstet. Gynecol. 181 (6) (1999) 1530e1536. [12] R. Romero, S. Durum, C.A. Dinarello, E. Oyarzun, J.C. Hobbins, M.D. Mitchell, Interleukin-1 stimulates prostaglandin biosynthesis by human amnion, Prostaglandins 37 (1) (1989) 13e22. [13] R. Romero, Y.K. Wu, D.T. Brody, E. Oyarzun, G.W. Duff, S.K. Durum, Human decidua: a source of interleukin-1, Obstet. Gynecol. 73 (1) (1989) 31e34. [14] R. Romero, M. Mazor, B. Tartakovsky, Systemic administration of interleukin-1 induces preterm parturition in mice, Am. J. Obstet. Gynecol. 165 (4 Pt 1) (1991) 969e971. [15] D.W. Sadowsky, K.M. Adams, M.G. Gravett, S.S. Witkin, M.J. Novy, Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model, Am. J. Obstet. Gynecol. 195 (6) (2006) 1578e1589. [16] R. Romero, B. Tartakovsky, The natural interleukin-1 receptor antagonist prevents interleukin-1-induced preterm delivery in mice, Am. J. Obstet. Gynecol. 167 (4 Pt 1) (1992) 1041e1045. [17] M. Gilchrist, V. Thorsson, B. Li, A.G. Rust, M. Korb, K. Kennedy, T. Hai, H. Bolouri, A. Aderem, Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4, Nature 441 (7090) (2006) 173e178.
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Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
ATF3 is a negative regulator of inflammation in human fetal membranes Ratana Lim a, b, Gillian Barker a, b, Stella Liong a, b, Caitlyn Nguyen-Ngo a, b, Stephen Tong b, c, Tu'uhevaha Kaitu'u-Lino b, c, Martha Lappas a, b, * a b c
Obstetrics, Nutrition and Endocrinology Group, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia Translational Obstetrics Group, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2016 Received in revised form 7 September 2016 Accepted 13 September 2016
Introduction: Infection and inflammation stimulate pro-inflammatory cytokines, prostaglandins and matrix metalloproteinase (MMP)-9, which play a central role in myometrial contractions and rupture of fetal membranes. In human and mouse immune cells, activating transcription factor 3 (ATF3) is a negative regulator of inflammation. No studies have examined the role of ATF3 in human labour. Methods: Primary amnion cells were used to determine the effect of interleukin (IL)-1b and the bacterial product fibroblast-stimulating lipopeptide (fsl-1) on ATF3 expression, and the effect of ATF3 siRNA on pro-labour mediators. ATF3 expression was assessed in fetal membranes from non-labouring and labouring women at term and preterm, and after preterm pre-labour rupture of membranes (PPROM). Results: IL-1b and fsl-1 significantly increased ATF3 expression. Silencing ATF3 significantly increased IL1b- or fsl-1-induced expression of pro-inflammatory cytokines (TNF-a, IL-1a, IL-1b, IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1 (MCP-1)); cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin PGF2a release; and MMP-9 expression. ATF3 expression was decreased in fetal membranes with term labour. There was no effect of preterm labour or PPROM on ATF3 expression. Discussion: ATF3 is a negative regulator of inflammation in human fetal membranes; in primary amnion cells, ATF3 expression is induced by IL-1b and fsl-1, and ATF3 silencing further exacerbates the inflammatory response when stimulated with these factors. Subsequently, ATF3 expression is decreased in fetal membranes after term labour and with preterm chorioamnionitis, conditions closely associated with inflammation and infection. Our data suggest that ATF3 may play a role in the terminal processes of human labour and delivery. © 2016 Elsevier Ltd. All rights reserved.
Keywords: ATF3 Human labour Inflammation Infection Fetal membranes
1. Introduction Preterm birth is the leading cause of infant morbidity and mortality [1]. For survivors, long-term risks associated with preterm birth include cerebral palsy, learning difficulties and respiratory illnesses [2]. Approximately 70% of preterm births occur spontaneously as a result of idiopathic preterm labour or preterm prelabour rupture of membranes (PPROM); PPROM is associated with higher rates of neonatal mortality and morbidity [3].
* Corresponding author. Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, 3084, Victoria, Australia. E-mail address: [email protected] (M. Lappas). http://dx.doi.org/10.1016/j.placenta.2016.09.006 0143-4004/© 2016 Elsevier Ltd. All rights reserved.
Alongside the impact preterm birth has on the child and family, data from the USA indicates the annual costs associated with preterm birth exceeds US$26 billion per year [4]. The costs imposed upon families and communities stem from the absence of effective therapeutics that can stop preterm labour. The processes involved in parturition are incompletely understood which has prevented the development of effective strategies to prevent spontaneous preterm birth. What we do know, however, is that the majority of spontaneous preterm births are due to pathological activation of the normal labour processes [3,5]. The bacterial product fibroblast-stimulating lipopeptide (fsl-1), while not a pathogenic micro-organism, is able to model the effects of such microbes [6], by stimulating the production of pro-labour mediators including pro-inflammatory cytokines, chemokines, cyclooxygenase (COX)-2 and subsequent prostaglandins, and
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2. Materials and methods
streptomycin. The media was replaced after 4 h then every 24e48 h thereafter. To determine the effect of pro-inflammatory mediators on ATF3 expression, cells at approximately 80% confluence were incubated in the absence or presence of 1 ng/ml IL-1b or 250 ng/ml fsl-1 for 24 h. Cells were collected and stored at 80 C until assayed for ATF3 protein expression by qRT-PCR or Western blotting as detailed below. Experiments were performed in amnion cells obtained from five patients. Transfection of primary amnion cells with siRNA was performed as we have previously described [29]. Briefly, cells at approximately 50% confluence were transfected using Lipofectamine 3000 according to manufacturer's guidelines (Life Technologies; Mulgrave, Victoria, Australia). SMARTpool ATF3 siRNA (siATF3) and negative control siRNA (siCONT) were obtained from Dharmacon (GE Healthcare Australia Pty. Ltd; Parramatta, NSW, Australia). Cells were transfected with 200 nM siATF3 or 200 nM siCONT in DMEM/ F-12 for 48 h. The medium was then replaced with DMEM/F-12 (containing 0.5% BSA) with or without 1 ng/ml IL-1b or 250 ng/ml fsl-1, and the cells were incubated at 37 C for an additional 24 h. Cells were collected and stored at 80 C until assayed for mRNA expression by qRT-PCR and protein expression by Western blotting as detailed below. Media was collected and stored at 80 C until assayed for cytokine, prostaglandin and MMP-9 release as detailed below. Cell viability was assessed by the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay as we have previously described [30]. The response to IL1b and fsl-1 between patients varied greatly, as we have previously reported [29]. Thus, data is presented as fold change in expression relative to the expression level in the IL-1b- or fsl-1-stimulated siCONT transfected cells, which was set at 1. Data could not be normalised to siCONT transfected cells alone as some of the readings were 0. Experiments were performed in amnion cells obtained from six patients.
2.1. Tissue collection
2.3. RNA extraction and quantitative RT-PCR (qRT-PCR)
The Research Ethics Committee of Mercy Hospital for Women approved this study. Written, informed consent was obtained from all participating women. All tissues were obtained from women who delivered healthy, singleton infants. All tissues were brought to the research laboratory and processed within 15 min of the Caesarean delivery. Tissues from women with any underlying medical conditions such as diabetes, asthma, polycystic ovarian syndrome, preeclampsia and macrovascular complications were not included. Additionally, tissues from women with multiple pregnancies, obese women, fetuses with chromosomal abnormalities were not included.
RNA extractions and qRT-PCR was performed as previously described [29]. Total RNA was extracted using TRIsure reagent according to manufacturer's instructions (Bioline; Alexandria, NSW, Australia). RNA concentration and purity were measured using a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific; Scoresby, Vic, Australia). RNA quality and integrity was determined via the A260/A280 ratio. RNA was converted to cDNA using the Tetro cDNA synthesis kit (Bioline; Alexandria, NSW, Australia) according to the manufacturer's instructions. The cDNA was diluted fifty-fold, and 4 ml of this was used to perform RT-PCR using SensiFAST™ SYBR NO-ROX Kit (Bioline; Alexandria, NSW, Australia) and 100 nM of pre-designed and validated QuantiTect primers (Qiagen; Chadstone Centre, Vic, Australia). The RT-PCR was performed using the CFX384 Real-Time PCR detection system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Average gene Ct values were normalised to the average b2-Microglobulin (B2M) and 18S ribosomal RNA (rRNA) Ct values of the same cDNA sample. Of note, there was no effect of experimental treatment on B2M or 18S rRNA gene expression. Fold differences were determined using the comparative Ct method.
metalloproteinase (MMP)-9 in fetal membranes [7,8]. These prolabour mediators participate in the uterine components of this common pathway of parturition i.e. myometrial contractility, cervical ripening and rupture of fetal membranes. Notably, and even in the absence of detected pathogens, most cases of spontaneous preterm birth have histological evidence of inflammation in the uteroplacental unit [9]. Pro-inflammatory cytokines such as IL-1b can activate the terminal processes of human labour and delivery [10e13] and induce preterm birth in mice and monkeys [14e16]. Recent studies in non-gestational tissues have demonstrated a role for activating transcription factor 3 (ATF3) as a negative regulator of inflammation [17e19]. ATF3 is a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors [20]. ATF3 functions as a transcriptional repressor [21]; while ATF3 expression is maintained at low levels in quiescent cells, ATF3 gene expression can be induced by a variety of stress signals [22], such as Toll-like receptor (TLR) agonist bacterial lipopolysaccharide (LPS). In nongestational tissues, the role of ATF3 as a negative regulator of inflammation is demonstrated whereby ATF3 is induced by TLRs, such as TLR2, 3, 4 and 9 [23], but that ATF3 silencing leads to an exacerbated inflammatory response [17e19,23e25]. While ATF2 has been considered to play a role in human myometrium [26,27], to our knowledge the expression or the role of ATF3 has not been investigated in human fetal membranes. Therefore, an aim of this study were to establish the expression of ATF3 in human fetal membranes obtained from labouring and nonlabouring women at preterm and term. Furthermore, loss-offunction studies were performed to determine whether ATF3 is involved in the genesis of pro-inflammatory and pro-labour mediators induced by inflammation (IL-1b) or infection (using the bacterial product fsl-1).
2.2. Primary amnion cell culture Primary amnion cells were used to investigate the effect of proinflammatory mediators on ATF3 expression and the effect of ATF3 siRNA-mediated gene silencing on the expression of pro-labour mediators. For these studies, fresh amnion was obtained 2 cm from the peri-placental edge from women who delivered healthy, singleton infants at term (37e40 weeks gestation) undergoing elective Caesarean section in the absence of labour. Amnion cells (epithelial and mesenchymal) were prepared as previously described [28]. Briefly, amnion strips were washed in PBS and digested, twice, with 0.125% collagenase A and 0.25% trypsin in serum-free DMEM for 35 min at 37 C. The cell suspension was filtered through a cell strainer and the eluate was neutralised with 1% FCS. The cell suspensions were centrifuged at 500g for 10 min and the cells cultured in DMEM/F-12, 10% FCS and 1% penicillin-
2.4. Cytokine, chemokine and prostaglandin assays Assessment of cytokine and chemokine release of IL-6, IL-8, MCP-1 and TNF-a was performed using CytoSet™ sandwich ELISA according to the manufacturer's instructions (Life Technologies; Mulgrave, Vic, Australia). The limit of detection of the IL-6, IL-8, MCP-1 and TNF-a assays was 16, 12, 15 and 7 pg/ml, respectively.
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The concentration of IL-1a and IL-1b in the media was performed by sandwich ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The limit of detection of the IL-1a and IL-1b assays was 7.8 and 2 pg/ml, respectively. The release of PGF2a into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Kookaburra Kits from Sapphire Bioscience, NSW, Australia). The limit of detection of the PGF2a assays was 60 pg/ml. The interassay and intraassay coefficients of variation (CV) for all assays were less than 10%.
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Incubation media was also collected and assessment of enzymes of extracellular matrix (ECM) weakening and rupture (MMP-2 and MMP-9) was performed by gelatin zymography as previously described [28]. Proteolytic activity was visualized as clear zones of lysis on a blue background of undigested gelatin. Gels were scanned using a ChemiDoc XRS system (Bio-Rad Laboratories; Gladesville, NSW, Australia), images inverted to show dark bands rather than the clear zones of lysis, and densitometry performed using Quantity One image analysis software (Bio-Rad Laboratories; Gladesville, NSW, Australia).
were placenta praevia, placental abruption, antepartum haemorrhage (APH) or Rhesus isoimmunisation. PPROM was defined as spontaneous rupture of the membranes at less than 37 weeks gestation at least 1 h before the onset of any contractions. All placentas collected from preterm gestations were subject to histopathological examination and fetal membranes were swabbed for microbiological culture investigations. Chorioamnionitis was diagnosed pathologically according to standard criteria which included histological evidence of macrophages and neutrophils permeating the chorionic cell layer and often infiltrating the amniotic cell [32]. Histological chorioamnionitis is often accompanied by isolation of a microbiological organism from the fetal membranes. Women with preeclampsia, preexisting diabetes, asthma, multiple pregnancies, and fetuses with chromosomal abnormalities were not included. For the preterm labour study, the SCS could not be identified and thus, fetal membranes were obtained 2 cm from the peri-placental edge, an area approximately 5 5 cm in size. The clinical details of the preterm patients are described elsewhere [33]. Of note, there was no difference in maternal age and body mass index, or parity of the patients recruited. Tissue samples were snap frozen in liquid nitrogen and immediately stored at 80 C for analysis of ATF3 expression by qRT-PCR and Western blot as detailed below.
2.6. Tissue samples for expression studies
2.7. Western blotting
For expression studies by Western blotting, fetal membranes were obtained from women at (i) term no labour undergoing elective Caesarean section (indications for Caesarean section were breech presentation and/or previous Caesarean section) (n ¼ 8 patients; mean gestational age 38.8 ± 0.3 weeks) and (ii) term after spontaneous labour, spontaneous membrane rupture, and normal vaginal delivery (n ¼ 8 patients; mean gestational age 38.7 ± 0.4 weeks). Fetal membranes from the non-labouring group were obtained from the supracervical site (SCS). This area overlying the cervix corresponds to the site of rupture, thus providing a relative area of comparison in fetal membranes before and after labour. Identification of the SCS was performed as described previously [31]. Briefly, Bonneys blue dye was introduced through the cervix prior to Caesarean section. Upon delivery of the placenta, a blue mark was obvious on the chorion facing membrane where the dye had been applied. In the after labour group, fetal membranes were obtained from the site of membrane rupture (SOR) as previously described [6], an area approximately 5 5 cm in size. Amnion and underlying choriodecidua were collected from along the line of fetal membrane rupture. There was no difference in maternal age and body mass index, parity, or gestational age of the patients recruited. In the term after labour group none of the patients received any medications to augment or induce labour, and the average length of labour was 6 h 40 min ± 1 h 40 min. Tissue samples were snap frozen in liquid nitrogen and immediately stored at 80 C for analysis of ATF3 expression by qRT-PCR and Western blot as detailed below. Fetal membranes were also obtained from women at preterm birth from the following groups (i) Caesarean section in the absence of labour with intact membranes (artificial rupture of membranes (ARM) at delivery; n ¼ 8 patients; mean gestational age 32.8 ± 0.7 weeks); (ii) Caesarean section in the absence of labor with PPROM (n ¼ 8 patients; mean gestational age 31.8 ± 0.7 weeks); (iii) after spontaneous labour and normal vaginal delivery without histologically confirmed chorioamnionitis (n ¼ 8 patients; mean gestational age 32.5 ± 0.8 weeks); and (iv) after spontaneous labour and normal vaginal delivery with histologically confirmed chorioamnionitis (n ¼ 8 patients; mean gestational age 31.6 ± 4.0 weeks). Indications for preterm delivery (in the absence of labour)
Western blotting was performed as we have previously described [29]. Blots were incubated in 1 mg/ml rabbit polyclonal anti-ATF3 (cat # HPA001562; Sigma, St. Louis, MO, USA) prepared in blocking buffer (5% skim milk/TBS-T (0.05%)) for 16 h at 4 C. Membranes were viewed and analysed using the ChemiDoc XRS system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Semiquantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad Laboratories, Hercules, CA, USA). The levels of ATF3 were normalised to the levels of b-actin (Sigma, St. Louis, MO, USA).
2.5. Gelatin zymography
2.8. Statistical analysis Statistics was performed on the normalised data unless otherwise specified. All statistical analyses were undertaken using GraphPad Prism (GraphPad Software, La Jolla, CA). For Fig. 1, a onesample t-test, against the constant of 1, was used. For Figs. 2e5, the homogeneity of data was assessed by the Bartlett's test, and when significant, the data were logarithmically transformed before further analysis using a one-way ANOVA (using LSD correction to discriminate among the means). For Fig. 6, unpaired Student's t-test was used to assess statistical significance between normally distributed data; otherwise, the nonparametric Mann-Whitney U (unpaired) test was used. Statistical significance was ascribed to P value < 0.05. Data were expressed as mean ± standard error of the mean (SEM). 3. Results 3.1. Activation of ATF3 limits the inflammatory response Studies in non-gestational tissues have shown that ATF3 is rapidly induced by cytokines and TLR agonists [17,23,25], in order to suppress inflammation [17]. As these factors are paramount to the inflammatory response associated with preterm birth [34], we determined whether the pro-inflammatory cytokine IL-1b (i.e. inflammation) and the bacterial product fsl-1 (used as a model for intra-uterine infection) could affect ATF3 expression in fetal
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Fig. 1. Effect of pro-inflammatory mediators on ATF3 expression. Human primary amnion cells were incubated in the absence or presence of 1 ng/ml IL-1b or 250 ng/ml fsl-1 for 24 h (n ¼ 6 patients). (A) ATF3 mRNA expression was analysed by qRT-PCR. (B) ATF3 protein expression was analysed by Western blot. Representative Western blot from 1 patient is also shown. For all data, the fold change was calculated relative to basal and data displayed as mean ± SEM. *P < 0.05 vs. basal (paired sample comparison).
Fig. 2. Effect of siATF3 knockdown on IL-1b-induced pro-inflammatory cytokines and chemokines. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with 1 ng/ml IL-1b for an additional 24 h (n ¼ 5 patients). (A,B,D,F) IL-1a, IL-6, IL-8 and MCP-1 mRNA expression was analysed by qRTPCR. (C,E,G) IL-6, IL-8 and MCP-1 concentration in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL-1b-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. IL-1b-stimulated siCONT transfected cells (one-way ANOVA).
membranes. Treatment of amnion cells with IL-1b or fsl-1 significantly increased ATF3 mRNA and protein expression, as shown in Fig. 1. We next sought to determine whether ATF3 is involved in the genesis of pro-labour mediators induced by inflammation (e.g. IL-
1b) or infection (e.g. the bacterial product fsl-1). The efficacy of siATF3 knockdown in primary amnion cells is demonstrated in Supplementary Fig. 1. When compared to siCONT transfected cells, siATF3 transfection resulted in 50% decrease in ATF3 mRNA expression and 70% decrease in ATF3 protein expression. There was
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Fig. 3. Effect of siATF3 knockdown on bacterial infection-induced pro-inflammatory cytokines and chemokines. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with 250 ng/ml fsl-1 for an additional 24 h (n ¼ 5 patients). (AeC,E,G) TNF-a, IL-1b, IL-6, IL-8 and MCP-1 mRNA expression was analysed by qRT-PCR. (D,F,H) IL-6, IL-8 and MCP-1 concentration in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to fsl-1-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. fsl-1-stimulated siCONT transfected cells (one-way ANOVA).
Fig. 4. Effect of siATF3 knockdown on the prostaglandin pathway. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with 1 ng/ml IL-1b for an additional 24 h (n ¼ 5 patients). (A) COX-2 mRNA expression was analysed by qRT-PCR (B) PGF2a concentration in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL-1b-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. IL-1b-stimulated siCONT transfected cells (one-way ANOVA).
no effect of siATF3 on cell viability as determined by MTT cell viability assay. For subsequent experiments, after siRNA transfection, cells were treated with IL-1b or fsl-1 as a model of inflammation- or infection-induced preterm labour in order to define the relative importance of ATF3 in the expression of pro-
labour mediators. The effect of siATF3 on IL-1b-stimulated cytokine expression in amnion is depicted in Fig. 2. As expected, in siCONT transfected cells, IL-1b significantly increased IL-1a, IL-6, IL-8 and MCP-1 mRNA expression (Fig. 2A,B,D,F) and IL-6, IL-8 and MCP-1 secretion
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Fig. 5. Effect of siATF3 knockdown on MMP-9 expression. Human primary amnion cells were transfected with or without 200 nM siATF3 or siCONT for 48 h and then treated with (AeD) 1 ng/ml IL-1b or (EeH) 250 ng/ml fsl-1 for an additional 24 h (n ¼ 5 patients). (A,B,E,F) MMP-2 and MMP-9 mRNA expression was analysed by qRT-PCR. (C,D,G,H) The incubation medium was assayed for MMP-2 and MMP-9 expression by gelatin zymography. Representative gelatin zymography from one patient is also shown. For all data, the fold change was calculated relative to IL-1b-or fsl-1-stimulated siCONT transfected cells and data displayed as mean ± SEM. *P < 0.05 vs. IL-1b-stimulated siCONT transfected cells (oneway ANOVA); xP < 0.05 vs. fsl-1-stimulated siCONT transfected cells (one-way ANOVA).
(Fig. 2C,E,G). In siATF3 transfected cells, IL-1b further augmented this cytokine and chemokine expression and secretion. Of note, the release of IL-1a was below the sensitivity of the assay. The effect of siATF3 on fsl-1-stimulated pro-inflammatory cytokines is demonstrated in Fig. 3. In siCONT transfected cells, fsl-1 significantly increased in TNF-a, IL-1b, IL-6, IL-8 and MCP-1 mRNA expression (Fig. 3AeC,E,G) and IL-6, IL-8 and MCP-1 secretion (Fig. 3D,F,H). This increase in the mRNA expression and release was significantly augmented in cells transfected with siATF3. Of note, the release of TNF-a and IL-1b were below the sensitivity of the assays. Fig. 4 demonstrates the effect of siATF3 knockdown on IL-1binduced COX-2 mRNA expression and release of the prostaglandin PGF2a. In cells transfected with siCONT, IL-1b induced a significant increase in COX-2 expression (Fig. 4A) and PGF2a release (Fig. 4B). In siATF3 transfected cells, IL-1b caused a further increase in COX-2 mRNA expression and PGF2a release. In siCONT transfected cells, IL-1b (Fig. 5B) and fsl-1 (Fig. 5F) induced an increase in MMP-9 mRNA expression. Likewise, by gelatin zymography, secretory pro-MMP-9 activity was also significantly higher with IL-1b (Fig. 5D) and fsl-1 (Fig. 5H) treatment. When ATF3 was silenced, there was statistically higher MMP9 mRNA and pro-MMP-9 activity. There was no effect of IL-1b, fsl-1 or siATF3 on MMP-2 mRNA expression or secretory pro-MMP-2 activity (Fig. 5A,C,E,G).
3.2. Effect of human term and preterm labour on ATF3 expression in fetal membranes Having shown that ATF3 is induced by mediators of preterm birth after 24 h and that gene silencing of ATF3 increases the inflammatory effect of IL-1b and fsl-1, we next sought to determine
the expression of ATF3 in fetal membranes obtained from term and preterm deliveries that have undergone labour and thus are likely to be associated with increased inflammation. To determine the effect of human term labour on ATF3 expression in fetal membranes, samples were obtained at term Caesarean section in the absence of labour (term, no labour) and after spontaneous labour and membrane rupture (term, after labour). ATF3 mRNA (Fig. 6A) and protein (Fig. 6B) expression was significantly lower in fetal membranes after spontaneous labour at term when compared to non-labouring tissues. To determine the effect of spontaneous preterm birth (without histologically confirmed chorioamnionitis) on ATF3 expression, fetal membranes were obtained from women at preterm Caesarean section with no labour (preterm no labour, no chorioamnionitis), and after spontaneous preterm labour and normal vaginal delivery (preterm after labour, no chorioamnionitis). ATF3 mRNA expression (Fig. 6C) and protein expression (Fig. 6D) were similar between the two groups. To determine the effect of infection on ATF3 expression, fetal membranes after spontaneous preterm labour and normal vaginal delivery without histologically confirmed chorioamnionitis (preterm after labour, no chorioamnionitis) were compared to fetal membranes after spontaneous preterm labour and normal vaginal delivery with histologically confirmed chorioamnionitis (preterm after labour, chorioamnionitis). ATF3 mRNA expression was significantly lower in the preterm group with histologic chorioamnionitis compared to the preterm group without histologic chorioamnionitis (Fig. 6C). ATF3 protein expression could not be analysed by Western blotting on fetal membranes from women with chorioamnionitis due to protein degradation in these samples [35]. To determine the effect of preterm membrane rupture on ATF3
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Fig. 6. ATF3 in fetal membranes. (A,B) Human fetal membranes were obtained from women not in labour at term Caesarean section and women after term spontaneous labour onset and delivery (n ¼ 8 patients per group). ATF3 mRNA expression was analysed by qRT-PCR and ATF3 protein expression was analysed by Western blotting. Fold change was calculated relative to the term no labour group. Data is displayed as mean ± SEM. *P < 0.05 vs. term no labour (Student's t-test). Representative Western blot from 3 patients per group is also shown. (C,D) Fetal membranes were obtained from women not in labour at preterm Caesarean section without histologically confirmed chorioamnionitis (preterm no labour, no chorioamnionitis; n ¼ 6 patients), after preterm spontaneous labour onset and delivery without histologically confirmed chorioamnionitis (preterm after labour, no chorioamnionitis; n ¼ 8 patients), or after preterm spontaneous labour onset and delivery with histologically confirmed chorioamnionitis (preterm after labour, chorioamnionitis; n ¼ 6 patients). ATF3 mRNA expression was analysed by qRT-PCR and gene expression and ATF3 protein expression was analysed by Western blotting. Fold change was calculated relative to the preterm no labour no chorioamnionitis group. Data is displayed as mean ± SEM. *P < 0.05 vs. preterm no labour no infection (Student's t-test). Representative
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expression, fetal membranes were obtained at preterm Caesarean section in the absence of labour from women with (i) intact membranes or (ii) after PPROM. There was no difference in ATF3 mRNA (Fig. 6E) or protein (Fig. 6F) expression between the two groups. 4. Discussion This study, for the first time, describes a possible role for ATF3 in the terminal processes of human labour and delivery in fetal membranes. Specifically, we found in vitro that in the presence of the pro-inflammatory mediators IL-1b and fsl-1, there was an increase in ATF3 expression in primary amnion cells; functional studies revealed a role for ATF3 in the genesis of infection- or inflammation-induced pro-labour mediators. Our data suggests that negative feedback regulation by ATF3 limits the production of pro-inflammatory mediators in response to infection and/or inflammation in human fetal membranes. Subsequently, we found that ATF3 expression in fetal membranes (combined amnion and choriodecidua) was decreased with spontaneous term labour and preterm chorioamnionitis, conditions tightly coupled to inflammation and infection. There is much evidence demonstrating that ATF3 negatively regulates inflammation; studies in non-gestational tissues have shown that ATF3 expression is induced by cytokines and TLR agonists involved in the inflammatory response [23,25]. It is therefore a molecular brake to prevent an excess inflammatory response. We first sought to determine the expression of ATF3 in response to known mediators of preterm birth; IL-1b is increased in gestational tissues at preterm labour [7] and can induce preterm labour in animal models [15], and the bacterial product and TLR2 ligand fsl-1 can activate mediators involved in the terminal effector pathways of human labour and delivery [6,28]. In primary amnion cells treated with IL-1b and fsl-1, we report an increase in ATF3 expression. This induction of ATF3 expression was also found in non-gestational tissues [23]. As a negative regulator of inflammation, ATF3 has been shown to be induced by various TLRs and cytokines [23,25] but ATF3 deficient cells promote an elevated inflammatory response. ATF3 knockdown in macrophages significantly increases interferon (IFN)-b expression [25]. A number of animal studies have shown that after LPS challenge, atf3 deficient mice have significantly increased production of inflammatory markers, such as serum IL-6 and IL-12b levels [17], chemokine CCL4 expression [36] and IL-8 and ICAM-1 expression [24]. The expression of COX-2 and prostaglandin production was found to be significantly higher in atf3 deficient mice after stimulation with TLR2/6 agonist zymosan [19]. Others have also reported increased MMP-9 expression in ATF3deficient cancer cells [37]. These studies corroborate the findings of the current study, where ATF3 knockdown in primary amnion cells is associated with increased pro-inflammatory (IL-6, IL-8 and MCP-1) and pro-labour mediators (COX-2 and prostaglandins and MMP-9) when stimulated with IL-1b and fsl-1. These findings are of significance, as these inflammatory mediators are involved in membrane rupture and cervical remodelling, essential for labour and delivery at both term and preterm. Collectively, our studies further attest that ATF3 is involved in the resolution of inflammation. Human labour is considered an inflammatory response, as an
influx of leukocytes into the fetal membranes produces proinflammatory cytokines, such as IL-6, IL-8 and IL-1b [38,39]. In turn, these pro-inflammatory cytokines can increase MMP-9 expression and activity in the fetal membranes, as well as COX-2 expression and subsequent prostaglandin production. Of note, human gestational tissues can all produce these inflammatory mediators as cultured cells, regardless of leukocyte infiltration. The current study demonstrates that ATF3 expression is decreased in fetal membranes after term labour, compared to non-labouring fetal membranes. While much of the literature reports increased ATF3 expression during a disease state, ATF3 is down-regulated in human prostate cancer [40,41] and esophageal squamous cell carcinomas [42]. In contrast to term studies, there was no change in ATF3 expression in fetal membranes after preterm labour. That the SCS could not be identified in the preterm samples is a limitation to our study, and could possibly be responsible for why we saw no change in ATF3 expression. Additionally, in the absence of labour, there was no change in ATF3 expression associated with PPROM, compared to membranes that were artificially ruptured at delivery. A possible reason why there was no change in ATF3 expression in these groups is that there is an overall increase in inflammation associated with preterm deliveries [43] that blanketed any further effect of labour or rupture of membranes. An important contributor to preterm birth is intrauterine inflammation, which commonly presents as chorioamnionitis. Intrauterine infection and/or inflammation are the pathological processes that provide a firm causal link to preterm birth [9,43]. Pregnancies affected by infection characteristically display elevated levels of pro-inflammatory cytokines in maternal serum, amniotic fluid and gestational tissues [9]. Expression of ATF3 was found to be decreased in fetal membranes from preterm deliveries with chorioamnionitis. Collectively, our studies correspond to the established role of ATF3 as a negative regulator of inflammation; IL-1b and fsl-1 treatments (for 24 h in vitro) induces ATF3 expression, but ATF3 knockdown augments the inflammatory response induced by said mediators. That we also saw a decrease in ATF3 expression in fetal membranes after term labour and with preterm chorioamnionitis further attests this role for ATF3; these conditions can be considered endpoints that are already in a chronically inflamed state, as the processes of labour can last over 24 h. The chronic stimulation of the inflammatory process could decrease ATF3, leading to the inflammatory processes that dominate labour and delivery. This study demonstrates that ATF3 expression is decreased in fetal membranes after term labour and with preterm chorioamnionitis. Up-regulation by pro-labour mediators IL-1b and fsl1 and the exacerbation of the inflammatory response in ATF3deficient amnion cells indicate that ATF3 is a negative regulator of the inflammatory response in human gestational tissues. This is of significance given the driving role of inflammation and bacterial infection in the processes of preterm birth [44]. Improvement of our understanding of the mechanisms associated with spontaneous preterm birth will lead to more effective interventions. Funding ML, KT and ST are supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC #1047025, #1050765, #1062418). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer
Western blot from 3 patients per group is also shown. (E,F) Fetal membranes were obtained from women at preterm Caesarean section not in labour with intact membranes (n ¼ 6 patients) and from women at preterm Caesarean section not in labour with PROM (n ¼ 6 patients). ATF3 mRNA expression was analysed by qRT-PCR and ATF3 protein expression was analysed by Western blotting. Fold change was calculated relative to the intact membranes group. Data is displayed as mean ± SEM. *P < 0.05 vs. intact membranes (Student's ttest). Representative Western blot from 3 patients per group is also shown.
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Medical Research Foundation and the Mercy Research Foundation. Disclosure summary The authors have nothing to declare. Conflict of interest The authors have nothing to declare. Acknowledgements The following are gratefully acknowledged: Clinical Research Midwives Genevieve Christophers, Gabrielle Pell, and Rachel Murdoch for sample collection; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2016.09.006. References [1] L. Liu, H.L. Johnson, S. Cousens, J. Perin, S. Scott, J.E. Lawn, I. Rudan, H. Campbell, R. Cibulskis, M. Li, C. Mathers, R.E. Black, Child Health Epidemiology Reference Group of WHO and Unicef. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000, Lancet 379 (9832) (2012) 2151e2161. [2] K. Flood, F.D. Malone, Prevention of preterm birth, Semin. Fetal Neonatal Med. 17 (1) (2012) 58e63. [3] R. Menon, S.J. Fortunato, Infection and the role of inflammation in preterm premature rupture of the membranes, Best Pract. Res. Clin. Obstet Gynaecol. 21 (3) (2007) 467e478. [4] Preterm birth: causes, consequences, and prevention, in: R.E. Behrman, A.S. Butler (Eds.), National Acadamies Press, Washington (DC), 2007. [5] R.S. Gibbs, J.D. Blanco, Premature rupture of the membranes, Obstet. Gynecol. 60 (6) (1982) 671e679. [6] R. Lim, G. Barker, M. Lappas, The TLR2 ligand FSL-1 and the TLR5 ligand Flagellin mediate pro-inflammatory and pro-labour response via MyD88/ TRAF6/NF-kappaB-dependent signalling, Am. J. Reprod. Immunol. 71 (5) (2014) 401e417 (New York, NY : 1989). [7] J.M. Bowen, L. Chamley, J.A. Keelan, M.D. Mitchell, Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition, Placenta 23 (4) (2002) 257e273. [8] I. Christiaens, D.B. Zaragoza, L. Guilbert, S.A. Robertson, B.F. Mitchell, D.M. Olson, Inflammatory processes in preterm and term parturition, J. Reprod. Immunol. 79 (1) (2008) 50e57. [9] R.L. Goldenberg, J.C. Hauth, W.W. Andrews, Intrauterine infection and preterm delivery, N. Engl. J. Med. 342 (20) (2000) 1500e1507. [10] J.A. Keelan, M. Blumenstein, R.J. Helliwell, T.A. Sato, K.W. Marvin, M.D. Mitchell, Cytokines, prostaglandins and parturitionea review, Placenta 24 (Suppl A) (2003) S33eS46. [11] J.A. Keelan, K.W. Marvin, T.A. Sato, M. Coleman, L.M. McCowan, M.D. Mitchell, Cytokine abundance in placental tissues: evidence of inflammatory activation in gestational membranes with term and preterm parturition, Am. J. Obstet. Gynecol. 181 (6) (1999) 1530e1536. [12] R. Romero, S. Durum, C.A. Dinarello, E. Oyarzun, J.C. Hobbins, M.D. Mitchell, Interleukin-1 stimulates prostaglandin biosynthesis by human amnion, Prostaglandins 37 (1) (1989) 13e22. [13] R. Romero, Y.K. Wu, D.T. Brody, E. Oyarzun, G.W. Duff, S.K. Durum, Human decidua: a source of interleukin-1, Obstet. Gynecol. 73 (1) (1989) 31e34. [14] R. Romero, M. Mazor, B. Tartakovsky, Systemic administration of interleukin-1 induces preterm parturition in mice, Am. J. Obstet. Gynecol. 165 (4 Pt 1) (1991) 969e971. [15] D.W. Sadowsky, K.M. Adams, M.G. Gravett, S.S. Witkin, M.J. Novy, Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model, Am. J. Obstet. Gynecol. 195 (6) (2006) 1578e1589. [16] R. Romero, B. Tartakovsky, The natural interleukin-1 receptor antagonist prevents interleukin-1-induced preterm delivery in mice, Am. J. Obstet. Gynecol. 167 (4 Pt 1) (1992) 1041e1045. [17] M. Gilchrist, V. Thorsson, B. Li, A.G. Rust, M. Korb, K. Kennedy, T. Hai, H. Bolouri, A. Aderem, Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4, Nature 441 (7090) (2006) 173e178.
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