TGF-β Mediates Proinflammatory Seminal Fluid Signaling in Human Cervical Epithelial Cells

This information is current as David J. Sharkey, Anne M. Macpherson, Kelton P. of September 25, 2021. Tremellen, David G. Mottershead, Robert B. Gilchrist and Sarah A. Robertson J Immunol 2012; 189:1024-1035; Prepublished online 15 June 2012;

doi: 10.4049/jimmunol.1200005 Downloaded from http://www.jimmunol.org/content/189/2/1024

Supplementary http://www.jimmunol.org/content/suppl/2012/06/15/jimmunol.120000 Material 5.DC1 http://www.jimmunol.org/ References This article cites 73 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/189/2/1024.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TGF-b Mediates Proinflammatory Seminal Fluid Signaling in Human Cervical Epithelial Cells

David J. Sharkey,* Anne M. Macpherson,* Kelton P. Tremellen,† David G. Mottershead,* Robert B. Gilchrist,* and Sarah A. Robertson*

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-b1, TGF-b2, and TGF-b3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-b3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine , replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-b isoforms showed comparable Downloaded from ability to induce Ect1 cell expression of mRNA and for GM-CSF and IL-6, and TGF-b induced a similar IL-6 and GM- CSF response in primary cervical epithelial cells. TGF-b neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3a), or IL-1a production. Several other present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-b isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-b in the male partner’s seminal fluid may influence cervical immune function after coitus in women, and potentially be http://www.jimmunol.org/ a determinant of fertility, as well as defense from infection. The Journal of Immunology, 2012, 189: 1024–1035.

he cervix is a major inductive and effector site for immune versing the female reproductive tissues, it is now known to have responses in the female genital tract (1) that are crucial in broader actions in regulating female fertility. In animals, seminal T orchestrating the female tract response to local immune fluid interacts with epithelial cells lining the female reproductive challenges. Through secretion of chemotactic and immune-regu- tract to activate cytokine expression and elicit changes in the lating cytokines, epithelial cells at the cervical surface have a piv- abundance and behavior of infiltrating leukocyte populations, which, otal role in activating defense against bacterial and viral pathogens, in turn, promote receptivity for embryo implantation (5). In mice, by guest on September 25, 2021 and in the generation and maintenance of immune tolerance toward uterine dendritic cells recruited in response to seminal fluid are in- spermatozoa and other antigenic material present in the ejaculate. strumental in activating local responses (6, 7), including expan- The responsiveness of cervical epithelial cells to introduced stimuli sion of regulatory T (Treg) cell populations that mediate tolerance of and their subsequent influence over the cervical immune environ- the conceptus in an ensuing pregnancy (8), whereas recruited mac- ment is also implicated as a determinant of neoplasia and devel- rophages influence epithelial cell expression of glycosylated struc- opment of cervical (2–4). tures required for embryo attachment and invasion (9). Recently, we showed that, in women, the male partner’s seminal In women, we have recently reported that seminal fluid induces an fluid exerts substantial effects on immune parameters in the cervix inflammation-like response in the endocervical tissue with extensive after coitus. Although seminal plasma (SP) has conventionally infiltration of macrophages, dendritic cells, and lymphocytes into the been viewed as simply a transport medium for spermatozoa tra- epithelial and deeper stromal tissues (10), expanding upon earlier findings that semen induces a neutrophil exudate into the cervical canal (11). Leukocyte recruitment in vivo is accompanied by ele- *Robinson Institute, School of Paediatrics and Reproductive Health, University of vated expression of CSF2, IL6, IL8,andIL1A as well as several other Adelaide, Adelaide, South Australia 5005, Australia; and †Repromed, Dulwich, South Australia 5065, Australia chemokines and cytokine genes (10). The response requires contact Received for publication January 12, 2012. Accepted for publication May 11, 2012. between seminal fluid and the female cervical tissues because the This work was supported by National Health and Medical Research Council Program characteristic changes in and leukocyte recruitment Grant 453556, Project Grant 565368, and Fellowship Grant 627017 (awarded to S.A.R.). are not seen following condom-protected intercourse (10). The sequences presented in this article have been submitted to Gene Expression Seminal fluid contains a complex array of cytokines, PGs, and Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE35830. other bioactive molecules (5, 12), but, to date, the active signaling Address correspondence and reprint requests to Prof. Sarah A. Robertson, Robinson agents in human semen responsible for stimulating the female cer- Institute and School of Paediatrics and Reproductive Health, 6th Floor, Medical vical response are unidentified. There is evidence that the plasma School North, Frome Road, University of Adelaide, Adelaide, SA 5005, Australia. E-mail address: [email protected] fraction of seminal fluid is important, with in vitro experiments The online version of this article contains supplemental material. utilizing transformed or primary cervical epithelial cells showing Abbreviations used in this article: ALK, -like kinase; EpCM, epithe- that SP induces synthesis of proinflammatory cytokines and che- lial cell culture medium; KEGG, Kyoto Encyclopedia of Genes and Genomics; mokines GM-CSF, IL-6, IL-8, CCL2 (MCP-1), CCL20 (MIP-3a), KSFM, keratinocyte serum-free media; SP, seminal plasma; Treg, T regulatory; and IL-1a (13). In mice, one agent mediating at least some aspects VEGF, vascular endothelial cell growth factor. of the female response to SP is TGF-b, which is synthesized in the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 male seminal vesicles in the latent form, then activated in the female www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200005 The Journal of Immunology 1025 tract after coitus (14). TGF-b is of particular interest as a candidate Germany). Next morning, after 1-h further incubation at room temperature, signaling agent in seminal fluid because of its well-known immune- intact sheets of epithelial cells were separated from stromal tissue using deviating properties, including induction of immune tolerance me- forceps and a scalpel, and then disaggregated in DMEM containing 0.25% trypsin and 0.01% collagenase type I (Sigma-Aldrich, Castle Hill, Aus- diated by Treg cells (15–17). Because TGF-b1 and TGF-b2are tralia) at 37˚C for 30 min, with the aid of extrusion through hypodermic reported to be highly abundant in human SP (18, 19), we hypothe- needles of incrementally decreasing gauge. Epithelial cells were then sized that TGF-b may be a signaling agent mediating the actions of treated with 0.01% EDTA in DMEM at 37˚C for 10 min, and plated at 1 3 5 human SP on cervical epithelial cells. 10 cells/well in 70% DMEM/22% Hams-F12/7% FCS/1% Nutridoma-SP (Boehringer Mannheim) with 0.02 mM glutamine and 5 mM hydrocorti- The experiments described in this study aimed to define the sone (Upjohn, Rydalmere, Australia) (epithelial cell culture medium significance of SP TGF-b in eliciting production of cervical cell [EpCM]/7% FCS), in 1.5-ml culture wells (Nunc, Weisbaden, Germany). cytokines and chemokines after coitus. We used immortalized Wells were seeded 24 h prior with mitomycin C-inactivated murine 3T3 4 ectocervical epithelial (Ect1) cells, shown previously to be a faithful fibroblast cells (4 3 10 cells/well) to provide extracellular matrix com- in vitro model for cervical epithelial cell immune function and ponents required for epithelial cell attachment. Cervical epithelial cells were allowed to attach and displace the 3T3 seminal fluid responsiveness (13, 20), as well as primary ectocer- fibroblasts for 5–7 d, when unattached epithelial cells and 3T3 cells were vical epithelial cells, to evaluate the relative signaling capacity of aspirated and media was replaced with 500 ml fresh EpCM/2% FCS. This SP versus the three mammalian isoforms of TGF-b. Two strategies pretreatment supernatant was collected 12 h later and replaced with 500 ml were used, an Affymetrix microarray screen as well as detailed EpCM/2% FCS containing TGF-b1, TGF-b2, TGF-b3, or culture media alone. After 12 h the treatment was replaced with EpCM/2% FCS, which analysis of several cytokines. Our experiments show that all three in turn was collected 24 h later (posttreatment). Supernatants were mammalian isoforms of TGF-b (TGF-b1, TGF-b2, and TGF-b3) centrifuged and stored at 280˚C until ELISA. contribute to eliciting the cervical cell cytokine response to SP, Downloaded from Ect1 cervical epithelial cell culture acting via the conventional TGF-b signaling pathway to induce expression of GM-CSF and IL-6, two cytokines with key roles in Ect1 cells were propagated, as described (20, 24), in keratinocyte serum- establishing pregnancy (21, 22). Together, these results demonstrate free media (KSFM; Life Technologies, Mount Waverley, Australia) sup- that TGF-b is an important male-female signaling agent implicated plemented with 0.1 ng/ml recombinant human epidermal growth factor (Life Technologies), 0.05 mg/ml bovine pituitary extract (Life Technolo- in regulating the female immune response to seminal fluid at coitus. 5 gies), and 0.4 mM CaCl2. For experiments, 1 3 10 cells in 500 ml KSFM

were seeded in 1.5-ml culture wells (Nunc) and incubated at 37˚C/5% CO2 http://www.jimmunol.org/ Materials and Methods for 2–3 d to generate a confluent monolayer; then the medium was replaced with 500 ml fresh KSFM. This pretreatment supernatant was Cervical tissue and semen samples collected 12 h later and replaced with 500 ml KSFM containing TGF-b1, Procedures were approved by the human ethics committees of the Uni- TGF-b2, or TGF-b3 (0.5–50 ng/ml), other cytokines (all 1 ng/ml), 10% versity of Adelaide and the North Western Adelaide Health Service. Human pooled human SP (v/v), or medium alone. For TGF-b neutralization, SP was obtained from healthy, proven fertile men at Repromed (Dulwich, confluent Ect1 cells were cultured with 10% SP (v/v), or 10% SP (v/v) plus Australia). Semen was produced by masturbation and, following liquefi- neutralizing Abs specific for TGF-b1, TGF-b2, or TGF-b3 (all 15 mg/ml). cation, was centrifuged at 10,000 3 g for 10 min at room temperature For small molecule inhibition of TGF-b signaling, confluent Ect1 cells within 30 min of ejaculation to recover SP and sperm. Individual SP were pretreated for 30 min with SB431542 (0.5 or 5 mM), and then washed specimens (n = 20) or pooled SP (from n = 10 donors) was stored frozen in and cultured with inhibitor alone or 10% SP (v/v) plus inhibitor. For in- by guest on September 25, 2021 aliquots at 280˚C until use. Semen samples were excluded in the event of hibition of TGF-b signaling using the TGF-bRII-Fc receptor antagonist, abnormal sperm number, motility, and morphology parameters (according confluent Ect1 cells were cultured with TGF-bRII-Fc (2 mg/ml), 10% SP to World Health Organization criteria), high leukocytes or other symptoms (v/v), or 10% SP (v/v) preincubated for 30 min with TGF-bRII-Fc (2 mg/ of infection, or donor use of immune-modifying medications such as ml). For all experiments, the treatment was replaced with fresh KSFM at methotrexate or nonsteroidal anti-inflammatory drugs. For analysis of 12 h, which in turn was collected 24 h later (posttreatment) for cytokine TGF-b content by ELISA, whole semen and sperm were diluted in ice- analysis by ELISA or Luminex multiplex microbead assay. For Ect1 cell cold PBS containing protease inhibitor mixture (Roche) and solubilized mRNA preparation, cultures were terminated at 4 or 10 h after application using an Ultra-Turrax high-speed homogenizer (Janke and Kunkel, Stau- of treatments. 2 fen, Germany), then stored at 80˚C until assay. Affymetrix GeneChip microarray Human cervical tissue was obtained with consent from three premen- opausal women undergoing routine vaginal or total abdominal hysterectomy Quadruplicate wells of Ect1 cells were incubated in KSFM alone (con- at the Lyell McEwin Hospital (Elizabeth, South Australia) or Women’s and trol), KSFM, and 10% pooled SP, or KSFM and 5 ng/ml TGF-b3 for 10 h. Children’s Hospital (Adelaide, South Australia), for benign, noncervical Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA), pathology. according to the manufacturer’s instructions, and purified using RNeasy micro kits (Qiagen, Doncaster, Australia). RNA (10 mg per sample) from Recombinant cytokines, Abs, small molecule inhibitor, and four biological replicates, each comprising pooled material from sepa- ectodomain receptor antagonist rate sets of replicate wells, was sent to the Australian Genome Research Facility (Melbourne, Australia) for single-cycle labeling and hybridiza- Recombinant human TGF-b1, TGF-b2, and TGF-b3 were from R&D tion to Affymetrix GeneChip U133 plus 2.0 microarrays Systems (240-B, 302-B2, and 243-B3; Minneapolis, MN). Recombinant (Affymetrix, Santa Clara, CA). RNA integrity analysis, hybridization, human IL-6, IL-1a, IL-1b, IL-12, and TNF-a were purchased from and washing were performed, according to the manufacturer’s instructions. ProspecBio (CYT-213, CYT-253, CYT-208, CYT-362, and CYT-223; GCOS 1.4 software (Affymetrix) using MAS 5.0 was used to generate Rehovot, Israel). Cytokines were reconstituted to 1 mg/ml in PBS/0.1% Celestia script files for each chip. Quality control was assessed using the BSA/4 mM HCl (TGF-b isoforms), or PBS/0.1% BSA (all others). Neu- affyQC-Report package in R (v2.6.1) (25). The array data were initially tralizing Abs reactive with TGF-b1, TGF-b2, and TGF-b3 (MAB240, AF- examined using Partek Genomics Suite (Partek), after Celestia script files 302-NA, and AB-244-NA) were purchased from R&D Systems. The small were imported using robust multiarray average background correction, molecule inhibitor of TGF-b signaling SB-431542 (D8946-5MG; Sigma, guanine/cytosine content correction, and mean probe summarization, to Castle Hill, Australia) was reconstituted to 10 mM in DMSO. Recombi- generate dendrogram hierarchical clustering analysis and principal com- nant TGF-bRII-Fc receptor antagonist (341-BR; R&D Systems) was ponents analysis. Non-log2–transformed expression values for each chip reconstituted to 100 mg/ml in sterile PBS/0.1% BSA. All reagents were were submitted to IlluminaGUI (26) (free software implemented by R stored in aliquots at 280˚C. 2.6.1) after substituting Illumina probe IDs for Affymetrix IDs. Two con- Primary ectocervical epithelial cell culture trasts were analyzed, as follows: SP versus control and TGF-b3 versus control. In both cases, differentially expressed probes were identified using Ectocervical epithelial cells were prepared, as described (23). Cervical default high stringency parameters ($2.0-fold change, t test p , 0.05, tissue biopsies were washed twice in ice-cold HBSS (Invitrogen, Mount .100 difference between mean expression intensity) as well as reduced Waverley, Australia), and then incubated overnight at 4˚C in 5 ml DMEM stringency parameters ($1.4-fold change, t test p , 0.10, .50 difference (Invitrogen) containing 5 U dispase (Boehringer Mannheim, Mannheim, between mean expression intensity). The Kyoto Encyclopedia of Genes and 1026 SEMINAL FLUID TGF-b INDUCES CERVICAL CELL CYTOKINE SYNTHESIS

Table I. Relative concentrations of each of the three mammalian isoforms of TGF-b in human SP from n = 20 proven fertile men

TGF-b1 (ng/ml)a TGF-b2 (ng/ml) TGF-b3 (ng/ml) Totalb 219.3 6 13.4 5.3 6 0.7 172.2 6 32.8 Range 113.1–314.2 1.0–13.5 4.7–462.0 Bioactive (% total) 2.3 6 0.4 (1.2%) 0.25 6 0.04 (5.3%) 3.5 6 1.2 (1.8%) Range 0.3–6.9 0.01–0.80 0.16–15.9 aTGF-b1, TGF-b2, and TGF-b3 were measured by isoform-specific ELISA in SP samples from n = 20 individual healthy, proven fertile men. bTotal TGF-b1, TGF-b2, and TGF-b3 were determined by analysis after transient acid activation of SP to activate latent cytokine. Bioactive TGF-b1, TGF-b2, and TGF-b3 were determined by analysis without prior acid activation.

Genomics (KEGG) database (27) and Pathway Express and OntoExpress Luminex assays. Intra- and interassay coefficients of variation were all analysis software (28, 29) were used to assign genes to cytokine–cytokine ,10%. The TGF-b content of semen, sperm, and seminal fluid was mea- receptor interaction, TGF-b signaling, JAK/STAT signaling, or vascular sured by ELISA (TGF-b1 and TGF-b2 from R&D Systems, and TGF-b3 endothelial cell growth factor (VEGF) signaling pathways. from Promega, Madison, WI). Minimum detectable thresholds were 15.6 pg/ The microarray data discussed in this publication have been deposited ml (TGF-b1 and TGF-b2) and 31.2 pg/ml (TGF-b3). Intra- and interassay in the National Center for Biotechnology Information’s Gene Expression coefficients of variation for all TGF-b ELISAs were ,8%. All samples were Omnibus (30) and are accessible through GEO Series Accession GSE35830 assayed in duplicate, according to the manufacturers’ instructions. (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35830). Downloaded from Data analysis and statistics Quantitative RT-PCR Cytokine data were expressed as pg/105 cells/24 h. Data were excluded Ect1 cell total RNA was extracted using TRIzol solution (Invitrogen), and, from the analysis when comparable values (within 10% of control) were following treatment with RNase-free DNase I (500 IU/ml; 60 min/37˚C) not obtained for pretreatment supernatants from control and treatment (Roche, Basel, Switzerland), first-strand cDNA was reverse transcribed wells. SPSS version 15.0 (SPSS, Chicago, IL) was used to analyze com- from 5 mg RNA employing a Superscript-III Reverse Transcriptase (10 plete data sets. Student t test preceded by one-way ANOVA for multiple min/30˚C, 45 min/42˚C; Invitrogen). Primer pairs specific for cytokine and groups was used to compare differences between treatment groups. Sta- http://www.jimmunol.org/ chemokine cDNA sequences were designed and PCR conditions were tistical significance was concluded when p ,0.05. Gene symbols and optimized for each primer pair, as described (31). Primer sequences, protein names are according to nomenclature specified by HUGO Gene product sizes, and GenBank accession numbers are as published (13). Nomenclature Committee. PCR amplification employed SYBR Green PCR Master Mix kits (Ap- plied Biosystems), with 3 ml cDNA and 59 and 39 primers at concentrations of 0.1–1.0 mM, as described (31). PCR used a ABI Prism 7000 Sequence Results Detection System (Applied Biosystems) to allow amplicon quantification SP contains high concentrations of TGF-b1, TGF-b2, and using the Equation 2DCt 3 100 K21 (Applied Biosystems User Bulletin TGF-b3 #2), where Ct is the cycle number at which 50% maximum amplicon TGF-b1 and TGF-b2 are known to be present in human SP (18, synthesis occurs. Products were analyzed by dissociation curve profile and by guest on September 25, 2021 2% agarose gel electrophoresis, and were sequenced using Big Dye version 19), but the third isoform TGF-b3 has not previously been eval- 2 or 3 (Applied Biosystems) to confirm primer specificity. uated. Quantification of the total TGF-b1, TGF-b2, and TGF-b3 Cytokine ELISAs and Luminex microbead assays content of SP by isoform-specific ELISA in SP samples collected from 20 proven fertile men revealed high levels of TGF-b1, TGF- IL-8, IL-6, GM-CSF, CCL2, CCL20, and IL-1a in culture supernatants were b2, and TGF-b3 in all of 20 samples, with a total TGF-b content measured in ELISA kits (all R&D Systems) or, in some experiments as 6 6 specified, by Luminex microbead assay (Millipore). Minimum detectable of (mean SEM) 397 53 ng/ml, comprising on average 55% thresholds were 15.6 pg/ml (GM-CSF, CCL2, and CCL20), 9 pg/ml (IL-6), TGF-b1, 1% TGF-b2, and 44% TGF-b3 (Table I). All three TGF- 7.8 pg/ml (IL-1a), and 31.2 pg/ml (IL-8) for ELISA and 9.5 pg/ml or less for b isoforms were present predominantly in the latent, as opposed to

FIGURE 1. The relative abundance of TGF-b isoforms in whole semen, SP, and sperm, and the correlation between SP TGF-b content and SP ca- pacity to induce GM-CSF, IL-6, and IL-8 production in Ect1 cells. (A–C) SP was prepared by centrifu- gation, whereas diluted whole semen and sperm were disrupted by homogenization, prior to assay by ELISA specific for TGF-b1(A), TGF-b2(B), and TGF-b3(C). Data are the mean values of duplicate measurements, and are representative of two repli- cate experiments. (D–F) Ect1 cell production of GM-CSF, IL-6, and IL-8 in response to incubation with SP (10%) from 10 healthy, proven fertile men was determined in a previous study, as described (13). The total TGF-b content (ng/ml) was deter- mined as the sum of total TGF-b1, TGF-b2, and TGF-b3 (Table I). The correlation between SP TGF- b content and Ect1 cytokine production, including GM-CSF (D), IL-6 (E), and IL-8 (F), is shown. Each SP sample is plotted as an individual data point, and the Pearson’s correlation coefficient R2 and corre- sponding p values are shown for each pair. The Journal of Immunology 1027 active form (98.8% TGF-b1, 94.7% TGF-b2, and 98.2% TGF-b3) SP TGF-b content correlates with Ect1 cytokine-inducing (Table I). When the relative amount of each TGF-b isoform as- activity sociated with the plasma and sperm fractions of semen was Next, we investigated the association between SP TGF-b and quantified, the majority was present in the plasma fraction, and capacity to elicit proinflammatory cytokine production in Ect1 only 1, 15, and 1% of the total TGF-b1, TGF-b2, and TGF-b3, ectocervical epithelial cells. Previously, we quantified GM-CSF, respectively, were associated with sperm (Fig. 1A–C). IL-6, IL-8, and CCL2 production in Ect1 in response to SP

Table II. Genes of interest identified as differentially expressed using high stringency criteria in Affymetrix microarray analysis of mRNA expression in Ect1 ectocervical epithelial cells treated with SP, and their regulation by TGF-b3

Control Fold TGF-b Accession No. Gene Symbola Mean 6 SEMb SP Mean 6 SEMb Change p Value TGF-b Mean 6 SEMb Fold Change p Value Regulationc Cytokine– interaction (upregulated) NM_004591 CCL20 504.8 6 35.8 2344.1 6 690.1 4.6 0.008 1702.2 6 562.4 3.4 0.034 Y NM_002089 CXCL2 524.6 6 58.1 3017.9 6 868.2 5.8 0.004 734.9 6 207.9 1.4 NS N NM_002090 CXCL3 172.8 6 39.2 1233.2 6 327.2 7.1 ,0.001 369.6 6 112.1 2.1 0.098 ? NM_001945.2 HBEGF 247.6 6 39.2 1059.7 6 306.0 4.3 0.009 286.1 6 54.8 1.2 NS N NM_000874.3 IFNAR2 395.7 6 33.1 928.7 6 251.6 2.4 0.036 370.9 6 45.6 21.1 NS N NM_019618 IL1F9 69.0 6 31.7 214.0 6 58.4 3.1 0.029 174.0 6 55.7 2.5 NS N NM_004633 IL1R2 231.3 6 42.6 480.9 6 108.2 2.1 0.032 140.1 6 63.9 21.7 NS N Downloaded from NM_003856 IL1RL1 86.9 6 14.3 2057.5 6 790.3 23.7 0.013 111.0 6 4.9 1.3 0.003 N NM_000600 IL6b 218.1 6 13.2 863.8 6 95.7 4.0 ,0.001 884.1 6 276.0 4.1 0.016 Y NM_000584 IL8 230.8 6 43.7 3330.3 6 1340.3 14.4 0.020 1520.3 6 743.7 6.6 0.084 ? NM_000641 IL11 19.6 6 7.9 170.6 6 48.7 8.7 0.002 196.9 6 58.4 10.0 0.003 Y NM_001185156.1 IL24 95.2 6 29.7 408.7 6 77.7 4.3 ,0.001 124.7 6 29.8 1.3 NS N NM_002608.2 PDGFB 121.4 6 30.4 287.3 6 18.8 2.4 ,0.001 401.6 6 97.9 3.3 0.006 Y

Cytokine–cytokine receptor interaction (downregulated) http://www.jimmunol.org/ NM_001565 CXCL10 582.3 6 128.3 224.7 6 36.6 22.6 0.007 931.3 6 342.4 1.6 NS N NM_005409 CXCL11 522.4 6 87.9 172.9 6 98.1 23.0 0.008 492.0 6 12.7 21.1 NS N NM_004887 CXCL14 1732.8 6 587.3 262.5 6 159.7 26.6 ,0.001 3866.3 6 1795.8 2.2 NS N NM_000416 IFNGR1 2404.2 6 241.3 874.0 6 372.9 22.8 ,0.001 2656.8 6 565.0 1.1 NS N NM_004862.3 LITAF 3645.5 6 228.1 1775.9 6 351.5 22.1 ,0.001 3915.1 6 52.5 1.1 NS N NM_003810.3 TNFSF10 1185.5 6 124.6 597.3 6 189.0 22.0 0.009 3242.4 6 509.0 2.7 ,0.001 Yd NM_014452 TNFRSF21 2678.0 6 276.2 934.8 6 423.4 22.9 ,0.001 3718.0 6 469.4 1.4 0.056 ? TGF-b signaling (upregulated) NM_001106 ACVR2B 255.3 6 51.4 523.1 6 114.4 2.1 0.032 271.9 6 20.3 1.1 NS N NM_014482 BMP10 52.2 6 19.5 169.4 6 28.1 3.3 ,0.001 48.5 6 12.7 21.1 NS N NM_001200 BMP2 332.3 6 94.6 1122.5 6 183.5 3.4 ,0.001 475.1 6 149.9 1.4 NS N by guest on September 25, 2021 NM_ 198892.1 BMP2K 308.6 6 43.2 945.7 6 109.8 3.1 ,0.001 376.7 6 54.5 1.2 NS N NM_001718 BMP6 97.3 6 27.8 226.7 6 19.0 2.3 ,0.001 130.5 6 33.0 1.3 NS N NM_001203 BMPR1B 214.7 6 24.8 558.8 6 128.6 2.6 0.009 281.4 6 34.6 1.3 NS N NM_006350 FST 308.1 6 6.1 3089.3 6 622.4 10.0 ,0.001 689.7 6 50.0 2.2 ,0.001 Y NM_005860 FSTL3 643.9 6 47.1 2398.0 6 228.5 3.7 ,0.001 3229.3 6 809.1 5.0 ,0.001 Y NM_002192 INHBA 841.2 6 148.7 7182.6 6 1320.3 8.5 ,0.001 4900.5 6 928.8 5.8 ,0.001 Y NM_000095 THBS1 214.8 6 41.6 842.6 6 104.5 3.9 ,0.001 341.2 6 87.2 1.6 NS N NM_ 020429.2 SMURF1 138.5 6 11.1 400.9 6 71.5 2.9 ,0.001 308.8 6 74.8 2.2 0.024 Y NM_003236.3 TGF-A 2000.0 6 239.4 4305.4 6 800.9 2.2 0.006 2485.6 6 243.7 1.2 NS N NM_001042454.2 TGF-B1I1 1467.7 6 142.7 3337.2 6 666.3 2.3 0.006 2308.3 6 301.3 1.6 0.012 ? TGF-b signaling (downregulated) NM_004612.2 TGF-BR1 265.1 6 54.3 109.5 6 52.9 22.4 0.040 548.1 6 84.1 2.1 0.005 Yd NM_003243.4 TGF-BR3 239.2 6 38.9 80.4 6 11.3 23.0 ,0.001 82.2 6 29.2 22.9 ,0.001 Y JAK/STAT signaling (upregulated) NM_005188 CBL 45.5 6 5.2 210.3 6 76.0 4.6 0.031 218.8 6 13.5 4.8 ,0.001 Y NM_170662 CBLB 545.8 6 48.1 1157.4 6 202.1 2.1 0.003 561.5 6 98.3 1.0 NS N NM_015897 PIAS4 331.8 6 37.4 700.8 6 61.3 2.1 ,0.001 281.6 6 30.3 21.2 NS N NM_005026 PIK3CD 480.5 6 21.4 1310.5 6 280.7 2.7 0.003 1487.9 6 223.8 3.1 ,0.001 Y NM_003877 SOCS2 2216.1 6 142.4 6936.0 6 596.4 3.1 ,0.001 4626.6 6 707.3 2.1 ,0.001 Y JAK/STAT signaling (downregulated) NM_ 032375.3 AKT1S1 1673.0 6 299.2 704.5 6 149.2 22.4 0.004 1189.5 6 225.3 21.4 NS N NM_002645 PIK3C2A 941.0 6 104.3 391.1 6 106.3 22.4 ,0.001 1030.3 6 98.5 1.1 NS N NM_005027 PIK3R2 552.1 6 68.2 264.3 6 67.4 22.1 0.003 375.9 6 28.3 21.5 0.017 ? NM_003629.3 PIK3R3 262.4 6 35.1 45.4 6 20.7 25.8 ,0.001 100.2 6 18.0 22.6 ,0.001 Y VEGF signaling (upregulated) NM_001159920.1 FLT1 95.3 6 13.1 286.1 6 52.5 3.0 ,0.001 141.9 6 16.8 1.5 0.029 ? NM_000963 PTGS2 341.0 6 40.0 3115.0 6 729.9 9.1 ,0.001 1241.0 6 221.9 3.6 ,0.001 Y NM_001025366.2 VEGFA 1862.9 6 108.9 4531.6 6 916.3 2.4 0.004 4467.0 6 758.1 2.4 ,0.001 Y aListed genes are components of cytokine-cytokine receptor interaction, TGF-b signaling, JAK/STAT signaling, or VEGF signaling pathways, according to the KEGG database, meeting high stringency criteria for differential expression in response to treatment with SP, defined as: fold change .2; t test p , 0.05; difference between means .100. p , 0.10. Full gene titles are given in Supplemental Table I. bData are mean 6 SEM for n = 4 biological replicates from Ect1 ectocervical epithelial cells, each prepared from pools of 3 to 4 replicate wells, for three treatments: SP (10%, 10 h), TGF-b3 (5 ng/nl, 10 h), or medium alone (control). cGenes are classified as high confidence of TGF-b regulation (Y) if high stringency criteria are met for differential expression in response to 5 ng/ml TGF-b; low confidence of TGF-b regulation (?) if reduced stringency criteria (defined as: fold change .1.4; t test p , 0.10; difference between means .50) but not high stringency criteria are met, or not regulated by TGF-b (N) if reduced stringency criteria are not met. dGene is regulated in opposite direction by TGF-b compared with SP. 1028 SEMINAL FLUID TGF-b INDUCES CERVICAL CELL CYTOKINE SYNTHESIS

FIGURE 2. Effect of TGF-b on GM-CSF, IL-6, IL-8, CCL2, CCL20, and IL-1a production by Ect1 cells. Ect1 cells were incubated with TGF-b1, TGF-b2, or TGF-b3 (0.5, 5.0, or 50 ng/ml) for 12 h, and supernatants were collected 24 h later for cytokine analysis by ELISA. (A) GM-CSF, (B) IL-6, (C) IL-8, (D) CCL2, (E) CCL20, and (F) IL-1a output in pg/105 cells/24 h. Data are the mean 6 SEM of triplicate wells in each experi- mental group, and are representative of four repli- Downloaded from cate experiments. Data were analyzed by Kruskal- Wallis and Mann-Whitney U test to compare dif- ferences between the control and treatment groups. *p , 0.05 versus control. http://www.jimmunol.org/

samples from 10 individual fertile men (13). For these same 10 SP TGF-b and SP both activate cytokine signaling and related by guest on September 25, 2021 samples, there was a positive correlation between total TGF-b pathways in Ect1 cells content (sum of TGF-b1, TGF-b2, and TGF-b3) and the response To explore whether TGF-b can induce inflammatory cytokine gene of some, but not all, cytokines elicited in Ect1 cells. Total seminal transcription in Ect1 cells, and the extent to which TGF-b mimics fluid TGF-b content correlated with Ect1 GM-CSF output (Pear- changes induced by SP, a microarray experiment was undertaken. son correlation r = 0.720, p = 0.019) and IL-6 output (r = 0.829, The profile of gene expression in Ect1 cells incubated with TGF- p = 0.003) (Fig. 1D, 1E). There was a weaker correlation with b3 (5 ng/ml, 10 h) was compared with expression in cells exposed Ect1 IL-8 production (r =0.643,p = 0.045) (Fig. 1F), but no to SP (10%, 10 h). MAS 5.0 (GCOS, Gene Chip Operating correlation with CCL2 output (data not shown). Software; Affymetrix) analysis of n = 4 biological replicates for

Table III. Effect of TGF-b3 and SP on cytokine and chemokine production by Ect1 ectocervical epithelial cells

Cytokine Synonym Controla TGF-b3 (0.5 ng/ml)a TGF-b3 (5 ng/ml)a TGF-b3 (50 ng/ml)a 10% SPa Eotaxin CCL11 3.2 6 0.1 2.9 6 0.2 2.9 6 0.1 2.9 6 0.1 6.7 6 1.5 G-CSF CSF3 3.3 6 0.8 2.4 6 0.4 4.7 6 1.7 3.2 6 1.9 232.9 6 70.7 GM-CSF CSF2 5.8 6 0.13 19.9 6 0.8 190.7 6 21.5 255.8 6 34.0 320.0 6 66.3 CXCL1 GRO 143.7 6 7.5 155.6 6 3.3 243.5 6 24.7 186.0 6 16.8 3802.8 6 919.8 IL-1a 15.2 6 0.7 7.5 6 0.2 13.1 6 2.0 14.0 6 1.5 239.08 6 24.5 IL-6 15.7 6 0.8 15.1 6 0.1 161.5 6 3.6 194.9 6 11.1 217.9 6 16.6 IL-8 146.8 6 14.8 148.2 6 12.9 203.4 6 27.5 155.0 6 19.3 1588.0 6 115.7 IL-10 BDLb BDL BDL BDL BDL IL-17 BDL BDL BDL BDL BDL IP-10 CXCL10 1090.3 6 32.7 1098.8 6 38.6 1106.4 6 97.1 606.0 6 50.4 1627.0 6 58.6 MCP-1 CCL2 4.1 6 0.0 4.1 6 0.0 4.0 6 0.0 4.1 6 0.0 83.0 6 5.8 MDC CCL22 BDL BDL BDL BDL BDL MIP-1a CCL3 BDL BDL BDL BDL BDL RANTES CCL5 8.2 6 0.5 7.5 6 0.4 8.6 6 0.5 8.1 6 0.6 28.4 6 2.6 VEGF 1286.9 6 24.0 1518.6 6 46.3 1551.0 6 104.1 1629.6 6 91.4 2161.2 6 50.4 TNFa BDL BDL BDL BDL BDL aData are mean 6 SEM of triplicate wells (pg/105 cells/24 h). bBDL, concentration of analyte is below level of detection for the assay. GRO, Growth-related oncogene; IP-10, IFN-g–inducible protein 10; MDC, macrophage-derived chemokine. The Journal of Immunology 1029 each treatment identified an average of 42.6% of probe sets as pression intensity) were applied to the array data set, another 79 present in untreated Ect1 ectocervical epithelial cells, whereas genes of interest were identified as potentially regulated by SP. Of 36.2 and 41.4% of probe sets were present in Ect1 cells incubated the total 146 genes, 51 (40.5%) met the same reduced stringency with SP or TGF-b3, respectively. Dendrograms obtained using criteria for regulation by TGF-b3 (Supplemental Table I). Among Partek software showed clustering of the array data according to these, 8 additional genes met high stringency criteria for upregu- treatment group, with greater similarity between the control and lation by TGF-b3, including cytokines CSF2, IL1A, and PDGFC; TGF-b3 treatment groups than between the SP and other treatment chemokine CXCL1, TNF family member TNFAIP8; and cytokine groups (Supplemental Fig. 1A). Principal component analysis, signaling pathway genes CBLB, SOCS1, and SOCS3 (Supplemental which displays the data after transformation and reduction to a Table I). Additional genes previously shown to be induced by SP in small number of key coordinates, also showed clustering accord- Ect1 cells (13) either showed no indication of regulation by TGF-b3 ing to treatment group. SP and (to a lesser extent) TGF-b3 arrays (CCL2), or did not meet high stringency criteria for differential both showed separation from the control arrays along the first expression (IL8). principal component, and TGF-b3 arrays showed separation from b both control and SP arrays along the second principal component TGF- stimulates cytokine production in Ect1 cells (Supplemental Fig. 1B). To further evaluate whether seminal TGF-b elicits a cytokine re- Exposure of Ect1 cells to SP resulted in differential expression of sponse in Ect1 cells, we used cytokine-specific ELISAs to analyze a total of 3955 probe sets, identified using high stringency criteria production of GM-CSF, IL-6, IL-8, CCL2, CCL20, and IL-1a by with MAS 5.0 analysis. These corresponded to 1338 genes up- Ect1 cells after incubation with each of the three TGF-b isoforms. regulated and 1343 genes downregulated by SP. TGF-b3 treatment These cytokines were chosen for detailed study on the basis of the Downloaded from of Ect1 cells resulted in differential expression of 884 probe sets, microarray experiment as well as previous in vitro experiments in corresponding to 346 upregulated genes and 229 downregulated primary cervical epithelial cells and Ect1 cells (13), together with genes. The genes differentially regulated by SP included 47 genes studies in cervical tissue biopsies that demonstrate that CSF2, IL6, of interest, relevant to the current study by virtue of their associa- IL8, and IL1A are induced in vivo after coitus (10). Ect1 cells were tion with cytokine–cytokine receptor interaction, TGF-b signaling, incubated with TGF-b1, TGF-b2, or TGF-b3 at concentrations of

JAK/STAT signaling, or VEGF signaling pathways, as specified by 0.5, 5.0, and 50 ng/ml, approximating the total TGF-b content of http://www.jimmunol.org/ the KEGG database. Of these 47 genes, 17 (36.1%) were similarly 0.1, 1, and 10% SP. All three TGF-b isoforms elicited increased regulated by both SP and TGF-b3 (Table II). Genes found to be production of GM-CSF in a generally dose-dependent manner upregulated by both SP and TGF-b3 include cytokine genes IL6 (Fig. 2A), with 50 ng/ml inducing a 4.6- to 7.1-fold increase, (4.0-fold and 4.1-fold for SP and TGF-b3, respectively), CCL20 which is 53–85% of the increase induced by 10% SP [a dilution (4.6-fold and 3.4-fold), IL11 (8.7-fold and 10.0-fold), VEGFA (both shown previously to induce maximal cytokine responses from 2.4-fold), PDGFB (2.4-fold and 3.3-fold), FST (10.0-fold and 2.2- Ect1 cells (13)]. Similarly, all three TGF-b isoforms stimulated fold), FSTL3 (3.7-fold and 5.0-fold), INHBA (8.5-fold and 5.8-fold), IL-6 production in a dose-dependent manner (Fig. 2B), with 50 ng/ and cytokine signaling pathway genes SMURF1 (2.9-fold and 2.2- ml inducing a 2.7- to 6.7-fold increase, which is 40–96% of the fold), CBL (4.6-fold and 4.8-fold, respectively), PIK3CD (2.7-fold increase induced by 10% SP. In contrast, none of the three TGF-b by guest on September 25, 2021 and 3.1-fold), SOCS2 (3.1-fold and 2.1-fold), and PTGS2 (9.1-fold isoforms significantly altered secretion of IL-8 (Fig. 2C), CCL2 and 3.6-fold, respectively). Genes downregulated by both SP and (Fig. 2D), or IL-1a (Fig. 2F), and CCL20 production was inhibited TGF-b3 include the TGF-b receptor gene TGF-BR3 (3.0-fold and by all TGF-b isoforms (Fig. 2E). 2.9-fold) and cytokine signaling pathway gene PIK3R3 (5.8-fold When the cytokine profile induced in Ect1 cells by TGF-b3 and 2.6-fold, respectively). Two genes, the TGF-b receptor TGF- was examined by Luminex microbead analysis, similar increases BR1 and TNF family member TNFSF10, were downregulated by SP in GM-CSF and IL-6, with no change in IL-8, CCL2, and IL-1a (8.7-fold and 2.0-fold, respectively) and upregulated by TGF-b3 production, were obtained (Table III). This approach revealed (2.1-fold and 2.7-fold, respectively). additional cytokines G-CSF, CXCL1 (growth-related oncogene), When reduced stringency criteria for regulation by SP ($1.4- and CCL5 (RANTES) to be induced by 10% SP in Ect1 cells, but fold change, t test p , 0.10, . 50 difference between mean ex- none of these were influenced by TGF-b3.

Table IV. Effect of GM-CSF, IL-1a, IL-1b, IL-6, IL-12, LIF, and TNF-a on cytokine and chemokine production by Ect1 ectocervical epithelial cells

Cytokine Synonym Controla GM-CSF (1 ng/ml) IL-1a (1 ng/ml) IL-1b (1 ng/ml) IL-6 (1 ng/ml) IL-12 (1 ng/ml) LIF (1 ng/ml) TNF-a (1 ng/ml) G-CSF CSF3 3.3 BDLb BDL 14.6 BDL BDL BDL 11.3 GM-CSF CSF2 BDL BDL BDL BDL BDL BDL BDL BDL CXCL1 GRO 102.2 32.4 79.3 83.7 73.7 148.6 73.2 75.6 IL-1a 16.6 ,3.2 24.5 11.3 ,3.2 ,3.2 ,3.2 16.6 IL-1ra 216.7 174.7 266.1 174.7 472.5 195.9 303.2 459.8 IL-6 47.7 70.2 54.8 49.8 50.7 19.9 42.5 25.0 IL-8 18.4 19.5 29.5 21.6 18.2 21.4 16.9 24.1 IL-10 BDL BDL BDL BDL BDL BDL BDL BDL IL-15 BDL BDL BDL BDL BDL BDL BDL BDL CXCL10 IP-10 879.9 628.7 1255.5 1839.3 603.7 1092.2 596.8 1319 CCL2 MCP-1 4.2 4.2 4.2 3.4 7.2 5.3 4.0 2.2 CCL22 MCP-3 6.9 8.5 6.9 7.1 7.1 8.0 6.9 6.6 CCL3 MIP-1b ,3.2 ,3.2 ,3.2 ,3.2 ,3.2 ,3.2 ,3.2 ,3.2 CCL5 RANTES 187.9 299.7 253.5 248.4 250.9 178.2 229.3 ,3.2 TNFa 3.1 2.5 2.4 2.5 2.3 2.4 2.6 4.3 aData are mean of duplicate wells (pg/105 cells/24 h). bBDL, concentration of analyte is below level of detection for the assay. GRO, Growth-related oncogene; IP-10, IFN-g–inducible protein 10. 1030 SEMINAL FLUID TGF-b INDUCES CERVICAL CELL CYTOKINE SYNTHESIS

Seminal fluid cytokines IL-6, IL-1a, IL-1b, IL-12, GM-CSF, and TNF-a do not stimulate cytokine production in Ect1 cells SP contains several cytokines in addition to TGF-b. Some of these, including IL-6, IL-1a, IL-1b, IL-12, GM-CSF, and TNF-a (32– 34), were evaluated for their capacity to induce inflammatory cytokines in Ect1 cells. Luminex microbead analysis showed that none of these induced any substantial production of GM-CSF, IL-6, IL-8, CCL2, or IL-1a in Ect1 cells (Table IV). TGF-b stimulates cytokine production in primary ectocervical epithelial cells Next, the cytokine response of primary ectocervical epithelial cells to TGF-b was examined by incubation with 5.0 ng/ml TGF-b1, TGF-b2, or TGF-b3. Each TGF-b isoform elicited increased GM-CSF and IL-6 release in cells recovered from each of three women, which was similar in scale to changes seen in Ect1 cells (Fig. 3A, 3B) and comparable to the response to 10% SP in our previous experiments (13). Similar to the response in Ect1 cells, each TGF-b isoform induced moderate suppression of CCL20 Downloaded from production in primary ectocervical cells (Fig. 3E). However, the response of primary ectocervical cell preparations to TGF-b was different to that seen in Ect1 cells for three cytokines, with rela- tively small but significant increases in CCL2 and IL-1a (Fig. 3D, 3F) and moderate inhibition of IL-8 (Fig. 3C). http://www.jimmunol.org/ TGF-b neutralizing Abs, signaling inhibitors, and TGF-BRII receptor antagonist inhibit SP-induced cytokine production in Ect1 cells TGF-b neutralizing Abs were added to Ect1 cells incubated with SP to determine the proinflammatory activity attributable to each TGF-b isoform. Concentrations of each Ab sufficient to com- pletely neutralize the quantities of TGF-b1, TGF-b2, and TGF-b3 present in SP were determined in preliminary experiments (data not shown). Neutralizing Abs reactive with TGF-b1, TGF-b2, or by guest on September 25, 2021 TGF-b3 inhibited SP-induced Ect1 cell synthesis of GM-CSF by 37, 36, and 56%, respectively. Neutralization of all three isoforms FIGURE 3. Effect of TGF-b on GM-CSF, IL-6, IL-8, CCL2, CCL20, reduced GM-CSF production by 43% (Fig. 4A). and IL-1a production by primary ectocervical epithelial cells. Primary SP-induced increases in IL-6 were decreased by 13, 20, and 52% cervical epithelial cells were incubated with TGF-b1, TGF-b2, or TGF-b3 after neutralization of TGF-b1, TGF-b2, and TGF-b3, respec- (5.0 ng/ml) for 12 h, and supernatants were collected 24 h later for cyto- tively, and neutralization of all three isoforms of TGF-b reduced kine analysis by ELISA. (A) GM-CSF, (B) IL-6, (C) IL-8, (D) CCL2, (E) IL-6 production by 77% (Fig. 4D). In contrast, neutralization of CCL20, and (F) IL-1a output in pg/105 cells/24 h. Data are the mean 6 TGF-b1, TGF-b2, TGF-b3, or all three TGF-b isoforms had no SEM of triplicate wells in each experimental group, and are representative effect on SP-induced production of IL-8 (Fig. 4G), or CCL2, of three replicate experiments with cells from three patients. Data were analyzed by Kruskal-Wallis and Mann-Whitney U test to compare differ- CCL20, or IL-1a (data not shown). Irrelevant mouse and goat Ab ences between the control and treatment groups. *p , 0.05 versus control. did not change GM-CSF or IL-6 production (data not shown). When Ect1 cells were preincubated with SB-431542, a specific b inhibitor of the activin receptor-like kinase (ALK) receptor ALK5 TGF- activates CSF2 and IL6 cytokine mRNA expression in (TGF-BR1 receptor) and related ALK4 and ALK7 receptors re- Ect1 cells quired for TGF-b and activin signaling (35), SP-induced increases To confirm microarray data indicating that TGF-b regulates cy- in both GM-CSF and IL-6 were significantly impaired. The in- tokine expression at the transcriptional level, additional prepara- crease in GM-CSF induced by 10% SP was reduced by 67 and tions of Ect1 cells were incubated with 10% SP or 5 ng/ml TGF- 91% when cells were pretreated with 0.5 or 5 mM SB-431542, b1, TGF-b2, or TGF-b3 for 4 or 10 h, and cytokine mRNA ex- respectively (Fig. 4B). Likewise, the SP-induced increase in IL-6 pression was quantified by RT-PCR. SP induced substantial ex- production was reduced by 51 and 67%, respectively, by SB- pression of CSF2, IL6, and IL8 with evidence of a later peak in IL6 431542 inhibitor (Fig. 4E). SB-431542 did not alter SP-induced compared with CSF2 and IL8 (Fig. 5). All three isoforms of TGF- production of IL-8 (Fig. 4H), CCL2, CCL20, or IL-1a (data not b induced increased CSF2 expression at both time points, showing shown). that elevated GM-CSF production is regulated at the mRNA level The effect of rTGF-bRII-Fc receptor ectodomain antagonist on (Fig. 5A). TGF-b2 and TGF-b3, but not TGF-b1, also stimulated SP-induced Ect1 cell production of cytokines was also investi- increased IL6 mRNA expression, although this was evident only gated. Preincubation of SP with rTGF-bRII-Fc prior to addition to at 10 h, not 4 h (Fig. 5B). Conversely, none of the three TGF-b Ect1 cells reduced GM-CSF and IL-6 production by 73 and 77%, isoforms induced expression of IL8 mRNA (Fig. 5C). Similarly, respectively (Fig. 4C, 4F). TGF-bRII-Fc receptor ectodomain whereas SP clearly induced CCL2, CCL20, and IL1A expression antagonist did not affect production of IL-8 (Fig. 4I), CCL2, in Ect1 cells consistent with the microarray data, this effect was CCL20, or IL-1a (data not shown). not replicated by any of the TGF-b isoforms (data not shown). The Journal of Immunology 1031 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Effect of TGF-b neutralization or inhibition of TGF-b signaling with SB431542 or TGF-bRII ectodomain receptor antagonist on SP-induced production of GM-CSF (A–C), IL-6 (D–F) or IL-8 (G–I) by Ect1 cells. Ect1 cells were incubated with 10% SP (v/v) in the presence or absence of neutralizing Abs specific for TGF-b1, TGF-b2, or TGF-b3 (all 15 mg/ml) or a combination of all three Abs (A, D, G); the ALK4/ALK5/ALK7 inhibitor SB431542 (0.5 or 5 mM) (B, E, H), or TGF-bRII-Fc receptor antagonist (2 mg/ml) (C, F, I) for 12 h, and supernatants were collected 24 h later for cytokine analysis by ELISA or Luminex multiplex microbead assay. Data are the mean 6 SEM of triplicate wells in each experimental group, and are representative of three replicate experiments. Data were analyzed by Kruskal-Wallis and Mann-Whitney U test to compare differences between the 10% SP and treatment groups. *p , 0.05 versus control.

Discussion cytokines. Because GM-CSF and IL-6 are identified as centrally Seminal fluid elicits an inflammation-like response in the cervix involved in the physiological response to seminal fluid after coitus after coitus by inducing a surge in proinflammatory cytokine and in women (10), these results strongly implicate seminal fluid TGF- chemokine synthesis (10). The experiments described in this study b as responsible, at least in part, for triggering the postcoital in- identify TGF-b as a key factor in human SP that regulates in- flammatory cascade in the human cervix. flammatory cytokine production in cervical epithelial cells. With Our findings extend earlier reports of TGF-b1 and TGF-b2in isoform-specific assays, we showed that all three mammalian iso- human seminal fluid (18, 36) to show that TGF-b3 is present at forms, TGF-b1, TGF-b2, and TGF-b3, are present in high concen- levels comparable to TGF-b1 and substantially greater than TGF- trations in human SP. Using microarray analysis, we demonstrated b2. The TGF-b content of semen is extraordinarily high, ∼5-fold that TGF-b3 induces a pattern of gene expression in Ect1 cells that of serum, and similar to colostrum, which is the most potent sharing features with the Ect1 cell response to SP, including several biological source of TGF-b known (17). TGF-b is abundant in the gene pathways linked with progression of the cervical inflamma- seminal fluid of other animals, including pigs (37) and mice (14), tory response to seminal fluid in vivo (10). We then focused on six indicating the likely conservation of this signaling pathway across cytokines identified previously as central to the cervical cell re- mammalian species. As in the mouse and pig, where TGF-b is sponse (10, 13) and found that TGF-b1, TGF-b2, and TGF-b3 each synthesized in male accessory glands in the latent form, the ma- promote synthesis of GM-CSF and IL-6 in both transformed Ect1 jority of TGF-b present in human SP is latent and would require cells and primary ectocervical epithelial cells. This is consistent activation to bind to receptors on cervical cells. Together with with the strong correlation between the differential capacity of SP proteolytic enzymes endogenous to seminal fluid (38, 39), activa- samples to induce GM-CSF and IL-6 and their TGF-b content, and tion after coitus would be facilitated by the acid pH of the vaginal the finding that neutralizing any or all of the TGF-b isoforms in SP, environment, proteases such as and metalloproteinases pro- or inhibition of TGF-b signaling, impairs SP induction of these duced in the female reproductive tissues (40, 41), and the expression 1032 SEMINAL FLUID TGF-b INDUCES CERVICAL CELL CYTOKINE SYNTHESIS

bRI is less abundant (46, 47). TGF-b receptor expression in the endometrium is regulated by ovarian steroid hormones (47), but whether similar regulation occurs in cervical tissue is not known. Consistent with the self-limited TGF-b response in other cell lineages (48), the microarray data indicate that Ect1 cells down- regulate expression of all three TGF-b receptors, TGF-BR1, TGF- BR2, and TGF-BR3, after exposure to SP, and, at least for TGF- BR3, TGF-b contributes to this response (Supplemental Table I). Downregulation of the TGF-b response would be further facili- tated by induction of SMURF1, which encodes Smad ubiq- uitination regulatory factor-1, a homologous to E6-AP carboxy terminus-type ubiquitin ligase that regulates quantity of TGF-b receptors and various Smads via the ubiquitin–proteasome path- way (49). In the physiological setting, the interaction between seminal fluid and female tract epithelial cells is important in inducing tolerance of paternal Ags required for sperm survival and adap- tation for pregnancy (50). The cytokine environment prevailing

when these Ags are first encountered is pivotal in controlling APC Downloaded from recruitment, which in turn determines the strength and quality of the ensuing T cell response, and thus capacity to tolerate sperm and the implanting embryo (51). Because sexually transmitted infectious agents are introduced in the context of seminal fluid, the cytokine environment in the tract after coitus will also impact the

course of infection and immune defense to these organisms. http://www.jimmunol.org/ The cytokines induced by TGF-b in human cervical cells are consistent with contributing to reproductive function and fit with observations in the mouse, where seminal fluid TGF-b induces GM-CSF production after mating (14). GM-CSF is a key factor regulating fertility that targets both reproductive tract dendritic FIGURE 5. Effect of TGF-b on CSF2, IL6,andIL8 mRNA expression cells and the developing embryo (21). CSF2 null mutant mice in Ect1 cells. Ect1 cells were incubated with TGF-b1, TGF-b2, or TGF- have reduced expression of uterine dendritic cell MHCII, scav- b3 (5.0 ng/ml) for 4 or 10 h, when RNA was recovered for quantitative enger receptor, and costimulatory molecules, causing stunted

A B C by guest on September 25, 2021 RT-PCR analysis. ( ) CSF2,( ) IL6, and ( ) IL8 expression, normalized CD4+ and CD8+ T cell responses to seminal fluid Ags (7), linked to the housekeeping gene ACTB, expressed in arbitrary mRNA units as with impaired capacity to clear bacteria from the reproductive a percentage of the mean value of the control group, calibrated so that the tract after coitus, depressed fertility, and elevated fetal loss (52). It mean value of the control group = 100. Data are the mean 6 SEM of triplicate wells in each experimental group, and are representative of three is not known whether GM-CSF exerts similar actions in women, replicate experiments. Data were analyzed by Kruskal-Wallis and Mann- but reduced GM-CSF production in early pregnancy is linked with Whitney U test to compare differences between the control and treatment recurrent miscarriage (53). The significance of seminal fluid in- groups. *p , 0.05 versus control. duction of IL-6 in cervical cells is less clear, but a function in controlling progression of the inflammatory response through by cervical epithelial cells of factors that bind TGF-b and induce regulation of chemokine-mediated monocyte recruitment seems its conformational rearrangement, including -1 (42) likely (54). Several studies show reduced IL6 expression in the and avb6 integrin (43). reproductive tract tissues of women experiencing miscarriage (31, The three isoforms of TGF-b share similar target cell lineages, 55), and Il6-deficient mice are fertile, but show elevated rates of and each signals through heterotetrameric complexes of type I and fetal loss in midgestation (56). type II receptor serine-threonine kinases (TGF-bRI and TGF- Seminal fluid induction of other cytokines in Ect1 cells, IL-8, bRII), resulting in signal transduction to the nucleus through the CCL2, CCL20, and IL-1a, occurred independently of TGF-b. action of specific members of the SMAD family of transcriptional This implies that other seminal fluid agents contribute to the cer- regulators (44). The different isoforms display differential affini- vical cell cytokine response and that the array of cytokines and ties for type II receptors, with TGF-b2 requiring colocalization of chemokines produced is differentially regulated. A factor likely additional transmembrane-docking molecules to facilitate receptor to interact with TGF-b is PGE, which is present in high concen- binding. In the current experiments, all three TGF-b isoforms trations in SP and mediates immune regulatory functions (12, 57). exerted qualitatively similar GM-CSF and IL-6 responses in Ect1 PGE can induce IL-8 in cervical explants (58) as well as VEGF in and primary ectocervical cells, but TGF-b3 was consistently cervical carcinoma cells (3). TLR-mediated signaling is likely to found to be moderately more potent than TGF-b1 and TGF-b2, be an important pathway whereby seminal fluid microorganisms and correlations between this isoform and the GM-CSF and IL-6 influence the cervical cytokine response (59). Another possibility response in Ect1 cells were stronger than the other isoforms (data is a sequential cascade of induction, whereby endogenous cyto- not shown). This may be due to differences in the relative ex- kines amplify the cascade. The later onset of IL6 expression pression of TGF-b receptors by ectocervical epithelial cells, be- compared with CSF2 suggests the possibility of indirect induction, cause TGF-b1 and TGF-b3 have high affinity for TGF-bRII, and, in turn, IL-6 trans-signaling is a candidate for regulating che- whereas TGF-b2 binds with low affinity and requires coexpression mokine synthesis (54, 60). Evidence supporting this includes the of TGF-bRI for successful signal transduction (45). TGF-bRII is array data showing elevated IL6ST (GP130) expression in response highly expressed in the cervix and endometrium, whereas TGF- to seminal fluid (Supplemental Table I), increased CCL2 produc- The Journal of Immunology 1033 tion in Ect1 cells induced by IL-6 (Table IV), and delayed onset of The immune-regulatory properties of TGF-b would also con- CCL2 expression compared with other factors (13). tribute to the known actions of seminal fluid in promoting maternal A limitation in this study is the use of Ect1 cervical cells, immune tolerance for pregnancy. Our studies in mouse models in- generated by transformation with human papilloma virus, as a dicate that seminal fluid exposure is essential for boosting uterine model for primary ectocervical cells. Ect1 cells have a comparable Treg cells prior to embryo implantation (8, 71), and experiments morphology and gene expression profile to their primary parent administering exogenous TGF-b to mice after coitus show effects cells (24) and are more responsive to SP than endocervical or on Treg cells that support a central role for this cytokine (72). Ag vaginal epithelial cell lines (13). The similarity between Ect1 delivery in the presence of TGF-b favors development of Treg cells cells and primary cells in GM-CSF and IL-6 induction by TGF-b (73, 74), and naive CD4+CD252 T cells express FOXP3 when supports the validity of conclusions concerning those factors, and TGF-b is present (75). TGF-b is also implicated in proliferation of their relevance to the cervical response in vivo. However, there mature Treg cells through modifying the function and signaling are important differences in the cytokine response of Ect1 cells capabilities of dendritic cells (76). It remains to be determined and primary cells that caution against complete extrapolation. In whether TGF-b in seminal fluid acts on cervical lymphocytes and primary cells, but not Ect1 cells, TGF-b moderately inhibited IL-8 dendritic cells to induce tolerogenic phenotypes in women, and and induced modest increases in IL-1a and CCL2. Additionally, whether this has consequences for immune tolerance in pregnancy. primary cells show considerably higher IL-8 and CCL2 output, In summary, this study advances understanding of the mecha- consistent with reports that immortalizing cervical keratinocytes nisms by which seminal fluid interacts with cervical cells in women. with human papilloma virus DNAs can suppress cytokine secre- TGF-b is identified as a key signaling agent for inducing cytokines tion (61). In particular, reduced CCL2 expression in Ect1 cells has that control cervical tissue immune responses to seminal fluid Ags, Downloaded from been attributed to active suppression by viral oncogenes E6 and E7 and influence the quality of the ensuing effector phase of the re- (62). Thus, in the in vivo setting, cervical epithelial cells may sponse to skew the balance toward tolerance as opposed to immunity. respond to seminal fluid TGF-b stimulation with a wider range of Depending on whether the female tract has evolved mechanisms to cytokine response than observed in this study in Ect1 cells. discriminate seminal Ags from opportunistic pathogens, there may In addition to cytokine production, the microarray experiment be a detrimental cost of seminal TGF-b in inhibiting protective

revealed that TGF-b accounts for other aspects of the Ect1 cell immunity to agents of sexually transmitted disease, including HIV. http://www.jimmunol.org/ response to seminal fluid, which warrant further detailed evalua- Whereas SP TGF-b content appears unaltered when sperm pa- tion. These include effects on the VEGF pathway involved in an- rameters are abnormal (19), whether TGF-b abundance is different giogenesis and neovascularization, with compelling evidence for in the SP of individuals with unexplained infertility warrants fur- upregulation of genes VEGFA and PTGS2 (COX-2). This extends ther investigation. earlier findings that SP induces these genes (3, 4), and indicates that TGF-b might contribute to possible effects of seminal fluid Disclosures exposure on neoplastic transformation and tumor progression in The authors have no financial conflicts of interest. the cervix of sexually active women. 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Supplemental Table I. Genes of interest identified as differentially expressed using reduced stringency criteria in Affymetrix microarray analysis of

mRNA expression in Ect1 ectocervical epithelial cells treated with seminal plasma, and their regulation by TGF3.

Accession # Gene Symbola Gene Title Control SP Fold- P TGF Fold- P TGF mean ± SEMb mean ± SEMb change value mean ± SEMb change value regulationc

Cytokine – Cytokine Receptor Interaction (up-regulated): NM_002982 CCL2 CCL2, MCP-1 9.0 ± 1.5 27.3 ± 10.7 3.0 0.089 8.5 ± 0.6 -1.1 NS N NM_002984 CCL4 CCL4, MIP-1 16.3 ± 7.4 60.7 ± 23.4 3.7 0.070 15.0 ± 4.4 -1.1 NS N NM_002986 CCL11 CCL11, Eotaxin 46.7 ± 9.4 141.8 ± 31.8 3.0 0.004 47.0 ± 16.8 1.0 NS N NM_004590 CCL16 CCL16, LEC 28.3 ± 9.6 82.6 ± 11.8 2.9 <0.001 43.9 ± 5.2 1.6 NS N NM_002988 CCL18 CCL18, MIP-4 18.2 ± 4.3 39.6 ± 12.0 2.2 0.093 18.9 ± 2.8 1.0 NS N b NM_004591 CCL20 CCL20, MIP-3 504.8 ± 35.8 2344.1 ± 690.1 4.6 0.008 1702.2 ± 562.4 3.4 0.034 Y NM_000758 CSF2 CSF2, GM-CSF 39.7 ± 23.8 90.12 ± 31.4 2.3 NS 180.4 ± 67.3 4.5 0.048 Y NM_001511 CXCL1 CXCL1; melanoma growth 195.3 ± 17.8 1029.8 ± 464.4 5.3 0.072 448.9 ± 112.4 2.3 0.025 Y stimulating activity b NM_002089 CXCL2 CXCL2, MIP-2 524.6 ± 58.1 3017.9 ± 868.2 5.8 0.004 734.9 ± 207.9 1.4 NS N b NM_002090 CXCL3 CXCL3, MIP-2 172.8 ± 39.2 1233.2 ± 327.2 7.1 0.001 369.6 ± 112.1 2.1 0.098 ? NM_002994 CXCL5 CXCL5, ENA-78 3.1 ± 1.2 63.2 ± 29.0 20.1 0.030 13.9 ± 8.9 4.4 NS N NM_001005609 EDA Ectodysplasin A, ED1 48.2 ± 19.5 145.3 ± 44.7 3.0 0.046 66.4 ± 25.9 1.4 NS N NM_ 005711.3 EDIL3 EGF-like repeats and discoidin I- 37.3 ± 14.2 123.0 ± 41.2 3.3 0.049 36.9 ± 6.6 -1.0 NS N like domains 3 NM_001945.2 HBEGF -binding EDF-like growth 247.6 ± 39.2 1059.7 ± 306.0 4.3 0.009 286.1 ± 54.8 1.2 NS N factor NM_02175.2 IFNA21 Interferon, alpha 21 10.1 ± 2.5 70.0 ± 34.2 7.0 0.080 14.1 ± 6.3 1.4 NS N NM_000629.2 IFNAR1 Interferon (alpha, beta and omega) 329.1 ± 26.3 522.3 ± 59.6 1.6 0.003 304.6 ± 19.4 -1.1 NS N receptor 1 NM_000874.3 IFNAR2 Interferon (alpha, beta and omega) 395.7 ± 33.1 928.7 ± 251.6 2.4 0.036 370.9 ± 45.6 -1.1 NS N receptor 2 NM_020124.2 IFNK Interferon, kappa 13.8 ± 6.7 83.2 ± 19.6 6.0 0.001 32.1 ± 14.8 2.3 NS N NM_000575 IL1A IL-1 1319.2 ± 185.6 2660.7 ± 841.1 2.0 NS 4201.2 ± 868.3 3.2 <0.001 Y NM_000576 IL1B IL-1 997.8 ± 85.5 1833.5 ± 172.4 1.8 <0.001 1497.2 ± 454.7 1.5 NS N 1

NM_019618 IL1F9 IL-1, Interleukin-1 family member 69.0 ± 31.7 214.0 ± 58.4 3.1 0.029 174.0 ± 55.7 2.5 NS N 9 NM_000877 IL1R1 IL-1R1, IL-1 receptor type 1 512.8 ± 26.8 996.1 ± 170.2 1.9 0.008 364.6 ± 41.9 -1.4 0.102 ? b NM_004633 IL1R2 IL-1RII, IL-1 receptor type 2 231.3 ± 42.6 480.9 ± 108.2 2.1 0.032 140.1 ± 63.9 -1.7 NS N b NM_003856 IL1RL1 IL-RL1, ST2 86.9 ± 14.3 2057.5 ± 790.3 23.7 0.013 111.0 ± 4.9 1.3 0.003 ? NM_000564 IL5RA IL5-RA, IL-5 receptor alpha 17.7 ± 2.3 78.2 ± 29.7 4.4 0.042 42.3 ± 14.1 2.4 0.084 ? b NM_000600 IL6 IL-6 218.1 ± 13.2 863.8 ± 95.7 4.0 <0.001 884.1 ± 276.0 4.1 0.016 Y NM_002184 IL6ST IL-6RB, GP130 215.5 ± 14.6 604.8 ± 203.1 2.8 0.056 306.6 ± 21.6 1.4 <0.001 ? b NM_000584 IL8 IL-8, CXCL8 230.8 ± 43.7 3330.3 ± 1340.3 14.4 0.020 1520.3 ± 743.7 6.6 0.084 ? b NM_000641 IL11 IL-11 19.6 ± 7.9 170.6 ± 48.7 8.7 0.002 196.9 ± 58.4 10.0 0.003 Y NM_138284 IL17D IL-17D, IL-27 43.1 ± 14.1 105.2 ± 20.6 2.4 0.013 62.2 ± 9.3 1.4 NS N NM_052872 IL17F IL-17F, IL-24 28.6 ± 7.2 79.0 ± 11.1 2.8 <0.001 33.5 ± 20.7 1.2 NS N NM_020505 IL22 IL-22 13.6 ± 7.0 47.5 ± 17.5 3.5 0.07 13.4 ± 9.8 -1.0 NS N NM_001185156.1 IL24 IL-24 95.2 ± 29.7 408.7 ± 77.7 4.3 <0.001 124.7 ± 29.8 1.3 NS N NM_022789 IL25 IL-25, IL-17E 24.1 ± 3.6 79.1 ± 14.7 3.3 <0.001 62.8 ± 29.2 2.6 NS N NM_172138 IL28A IL-28A 70.1 ± 8.8 164.0 ± 44.1 2.3 0.037 118.3 ± 14.4 1.7 0.004 ? NM_008721.1 LIF Leukaemia Inhibitory Factor 59.8 ± 14.3 123.3 ± 12.6 2.1 0.001 77.9 ± 7.4 1.3 NS N NM_ 005928.2 MFGE8 Milk fat globule-EGF factor 8 24.4 ± 8.3 97.0 ± 24.9 4.0 0.006 13.1 ± 1.8 -1.9 NS N protein NM_002608.2 PDGFB Platelet-derived growth factor beta 121.4 ± 30.4 287.3 ± 18.8 2.4 <0.001 401.6 ± 97.9 3.3 0.006 Y polypeptide NM_001080437.1 SNED1 Sushi, nidogen and EGF-like 49.8 ± 17.8 151.1 ± 51.7 3.0 0.064 69.5 ± 13.8 1.4 NS N domains 1 NM_003692.4 TMEFF1 Transmembrane protein with EGF- 1204.4 ± 71.0 2025.9 ± 180.7 1.7 <0.001 2272.5 ± 121.9 1.9 <0.001 ? like and two follistatin-like domains 1 NM_021137.4 TNFAIP1 , alpha 901.3 ± 64.7 1438.9 ± 193.7 1.6 0.009 1554.0 ± 56.0 1.7 <0.001 ? induced protein 1 (endothelial) NM_001077654.1 TNFAIP8 Tumor necrosis factor, alpha- 1839.7 ± 110.3 2919.4 ± 364.9 1.6 0.005 4210.2 ± 689.2 2.3 <0.001 Y induced protein 8 NM_024575.4 TNFAIP8L2 Tumor necrosis factor alpha-like 40.3 ±11.3 104.7 ± 29.8 2.6 0.043 88.8 ± 28.7 2.2 NS N protein 8 like 2 NM_003839.2 TNFRSF11A Tumor necrosis factor superfamily , 67.2 ± 6.9 127.7 ± 28.1 1.9 0.037 62.5 ± 8.9 -1.1 NS N member 11a, NFKB activator 2

NM_001204344.1 TNFSF15 Tumor necrosis factor (ligand) 41.7 ± 4.5 93.7 ± 24.5 2.2 0.037 61.7 ± 15.7 1.5 NS N superfamily, member 15 NM_005092.3 TNFSF18 Tumor necrosis factor (ligand) 68.2 ± 11.0 135.8 ± 35.0 2.0 0.065 87.2 ± 5.8 1.3 NS N superfamily, member 18 NM_001252385.1 TNIP1 TNFAIP3 interacting protein 1 2281.7 ± 41.9 4618.9 ± 542.0 2.0 <0.001 4479.1 ± 2.0 0.065 ? 1189.6 Cytokine – Cytokine Receptor Interaction (down-regulated):

NM_133459 CCBE1 Collagen and calcium-binding EGF 383.1 ± 58.5 209.9 ± 7.9 -1.8 0.003 273.4 ± 29.9 -1.4 0.095 ? domain-containing protein 1 NM_001250.4 CD40 CD40 molecule, Tumor necrosis 801.8 ± 98.6 462.4 ± 92.0 -1.7 0.012 654.6 ± 52.7 -1.2 NS N factor receptor superfamily member 5 NM_005202 CSFR1 CSFR1, colony stimulating factor 1 333.8 ± 38.0 193.7 ± 32.2 -1.7 0.005 562.6 ± 38.8 1.7 <0.001 ? receptor b NM_001565 CXCL10 CXCL10, IP-10 582.3 ± 128.3 224.7 ± 36.6 -2.6 0.007 931.3 ± 342.4 1.6 NS N b NM_005409 CXCL11 CXCL11, IP-9 522.4 ± 87.9 172.9 ± 98.1 -3.0 0.008 492.0 ± 12.7 -1.1 NS N b NM_004887 CXCL14 CXCL14, BRAK 1732.8 ± 587.3 262.5 ± 159.7 -6.6 <0.001 3866.3 ± 2.2 NS N 1795.8 NM_022059 CXCL16 CXCL16, C-X-C motif chemokine 1239.4 ± 44.0 702.7 ± 198.9 -1.8 0.010 1042.5 ± 90.1 -1.2 0.073 N 16 NM_001039349 EFEMP1 EGF-containing fibulin-like 12611.4 ±743.1 7814.0 ±1752.2 -1.6 0.012 14064.7 ± 1.1 NS N extracellular matrix protein 1 937.7 NM_005228 EGFR epidermal growth factor (EGF) 3604.2 ± 216.8 1901.9 ± 387.9 -1.9 <0.001 4860.7 ± 838.8 1.4 NS N receptor NM_ 000043.4 FAS Fas (TNF receptor superfamily, 1032.1 ± 37.8 529.1 ± 142.4 -2.0 0.001 984.7 ± 97.4 -1.1 NS N member 6) NM_000416 IFNGR1 IFNGR, receptor 2404.2 ± 241.3 874.0 ± 372.9 -2.8 0.001 2656.8 ± 565.0 1.1 NS N 1 NM_000565 IL6R IL-6R, IL-6 receptor 86.2 ± 16.3 17.7 ± 4.6 -4.9 <0.001 30.4 ± 6.8 -2.9 0.002 ? NM_004512 IL11RA IL-11RA, IL-11 receptor alpha 185.7 ± 17.2 131.7 ± 10.5 -1.4 0.007 138.6 ± 3.5 -1.3 0.007 N NM_001559 IL12RB2 IL-12RB2, IL-12 receptor beta 2 1084.3 ± 127.2 652.8 ± 132.5 -1.7 0.018 1333.2 ± 72.4 1.2 0.089 N NM_000640 IL13RA2 IL-13RA2, IL-13 receptor alpha 2 86.2 ± 16.3 17.8 ± 4.6 -4.9 <0.001 30.4 ± 6.8 -2.8 0.002 ? NM_018725 IL17RB IL-17RB, IL-17 receptor B 159.4 ± 56.1 72.93 ± 34.5 -2.2 0.180 75.0 ± 43.0 -2.1 NS N NM_ 001203263 IL17RC IL-17RC, IL-17 receptor C 85.4 ± 22.3 32.9 ± 8.3 -2.6 <0.028 64.0 ± 13.8 -1.3 NS N 3

NM_014432 IL20RA IL-20RA, IL-20 receptor alpha 131.7 ± 19.9 40.7 ± 17.6 -3.2 <0.001 39.5 ± 16.3 -3.3 <0.001 ? NM_144717 IL20RB IL-20RB, IL-20 receptor beta 7254.9 ± 293.7 3686.4 ± 413.8 -2.0 <0.001 5058.7 ± 200.2 -1.4 <0.001 ? NM_145659 IL27 IL-27, IL-27 alpha 91.3 ± 22.2 21.3 ± 4.1 -4.3 0.002 133.7 ± 36.4 1.5 NS N NM_139017 IL31RA IL-31RA, IL-31 receptor alpha 173.3 ± 15.0 88.92 ± 40.0 -2.0 0.048 156.9 ± 18.5 -1.1 NS N NM_004862.3 LITAF Lipopolysaccharide-induced tumor 3645.5 ± 228.1 1775.9 ± 351.5 -2.1 <0.001 3915.1 ± 52.5 1.1 NS N necrosis factor-alpha factor NM_ 206943.2 LTBP1 LTBP-1, Latent-transforming growth 83.2 ± 13.9 27.8 ± 5.6 -3.0 <0.001 28.1 ± 3.2 -3.0 <0.001 ? factor beta-binding protein 1

NM_001080497 MEGF9 Multiple EGF-like domains protein 152.6 ± 14.9 70.6 ± 38.9 -2.2 0.049 139.0 ± 25.9 -1.1 NS N 9, EGFL5 NM_ MET met proto-oncogene (hepatocyte 800.9 ± 42.7 356.4 ± 113.0 -2.3 <0.001 1223.6 ± 225.0 1.5 0.065 ? 001127500.1 growth factor receptor), HEGFR NM_016205 PDGFC Platelet derived growth factor C, 521.1 ± 85.8 288.4 ± 37.6 -1.8 0.013 1050.9 ± 132.7 2.0 <0.001 Y VEGF-E NM_006291 TNFAIP2 Tumor necrosis factor alpha- 1094.1 ± 272.3 527.3 ± 162.5 -2.1 0.074 2799.3 ± 2.6 NS N induced protein 2 1286.4 NM_ 003842.4 TNFRSF10B Tumor necrosis factor receptor 499.9 ± 28.9 309.4 ± 27.5 -1.6 <0.001 600.3 ± 48.1 1.2 0.073 N superfamily member 10B, TRAILR2 NM_014452 TNFRSF21 Tumor necrosis factor receptor 2678.0 ± 276.2 934.8 ± 423.4 -2.9 0.001 3718.0 ± 469.4 1.4 0.056 ? superfamily member 21 NM_003810.3 TNFSF10 Tumor necrosis factor ligand 1185.5 ± 124.6 597.3 ± 189.0 -2.0 0.009 3242.4 ± 509.0 2.7 <0.001 Y superfamily member 10, TRAIL NM_003811 TNFSF9 Tumor necrosis factor ligand 554.1 ± 51.0 285.9 ± 32.4 -1.9 <0.001 432.6 ± 45.8 -1.3 0.076 N superfamily member 9 NM_003789 TRADD Tumor necrosis factor receptor type 879.2 ± 52.2 613.5 ± 67.5 -1.4 0.002 1058.0 ± 95.0 1.2 0.099 N 1-associated DEATH domain protein NM_ 015650.3 TRAF3IP1 Tumor necrosis factor receptor- 516.4 ± 14.7 340.6 ± 74.7 -1.5 0.021 412.5 ± 26.8 -1.3 <0.001 N associated factor 3 interacting protein 1 NM_032271 TRAF7 Tumor necrosis factor receptor- 1169.8 ± 67.1 735.8 ± 181.4 -1.6 0.025 1203.4 ± 180.0 1.0 NS N associated factor 7

4

TGF Signalling (up-regulated):

b NM_001106 ACVR2B Activin receptor type IIB 255.3 ± 51.4 523.1 ± 114.4 2.1 0.032 271.9 ± 20.3 1.1 NS N NM_000479 AMH Anti-Muellerian hormone 84.0 ± 12.5 151.7 ± 23.3 1.8 0.011 91.4 ±22.3 1.1 NS N NM_001199.3 BMP1 BMP-1, Bone morphogenetic 33.9 ± 13.4 86.6 ± 28.9 2.6 0.098 27.7 ±4.3 -1.2 NS N protein 1 NM_014482 BMP10 BMP-10, Bone morphogenetic 52.2 ± 19.5 169.4 ± 28.1 3.3 <0.001 48.5 ±12.7 -1.1 NS N protein 10 NM_001200 BMP2 BMP-2, Bone morphogenetic 332.3 ± 94.6 1122.5 ±183.5 3.4 <0.001 475.1 ±149.9 1.4 NS N protein 2 NM_ 198892.1 BMP2K BMP-2 inducible kinase 308.6 ± 43.2 945.7 ± 109.8 3.1 <0.001 376.7 ±54.5 1.2 NS N NM_021073 BMP5 BMP-5, Bone morphogenetic 78.8 ± 22.0 145.4 ± 21.2 1.9 0.029 96.1 ±14.0 1.2 NS N protein 5 NM_001718 BMP6 BMP-6, Bone morphogenetic 97.3 ± 27.8 226.7 ± 19.0 2.3 <0.001 130.5 ±33.0 1.3 NS N protein 6 NM_001203 BMPR1B Bone morphogenetic protein 214.7 ± 24.8 558.8 ± 128.6 2.6 0.009 281.4 ±34.6 1.3 NS N receptor, type IB, ALK6 b NM_006350 FST Follistatin 308.1 ± 6.1 3089.3 ± 622.4 10.0 <0.001 689.7 ± 50.0 2.2 <0.001 Y b NM_005860 FSTL3 follistatin-like 3 (secreted 643.9 ± 47.1 2398.0 ± 228.5 3.7 <0.001 3229.3 ± 809.1 5.0 <0.001 Y glycoprotein) b NM_002192 INHBA inhibin, beta A (activin A) 841.2 ± 148.7 7182.6 ± 1320.3 8.5 <0.001 4900.5 ± 928.8 5.8 <0.001 Y b NM_000095 THBS1 thrombospondin 1 214.8 ± 41.6 842.6 ± 104.5 3.9 <0.001 341.2 ± 87.2 1.6 NS N b NM_005092 SMAD3 SMAD, mothers against DPP 692.2 ± 50.3 1058.55 ± 67.0 1.5 <0.001 1245.1 ± 278.7 1.8 0.051 ? homolog 3 (drosophila) NM_ 020429.2 SMURF1 SMAD specific E3 ubiquitin protein 138.5 ± 11.1 400.9 ± 71.5 2.9 <0.001 308.8 ± 74.8 2.2 0.024 Y ligase 1 NM_003236.3 TGFA Transforming growth factor, alpha 2000.0 ± 239.4 4305.4 ± 800.9 2.2 0.006 2485.6 ± 243.7 1.2 NS N NM_001042454.2 TGFB1I1 Transforming growth factor beta 1 1467.7 ± 142.7 3337.2 ± 666.3 2.3 0.006 2308.3 ± 301.3 1.6 0.012 ? induced transcript 1 TGF Signalling (down-regulated):

NM_000020 ACVRL1 Activin A receptor type II-like 1, 59.0 ± 17.4 22.7 ± 2.0 -2.6 0.038 85.6 ± 22.7 1.5 NS N ALK-1

5

NM_004612.2 TGFBR1 Transforming growth factor, beta 265.1 ± 54.3 109.5 ± 52.9 -2.4 0.040 548.1 ± 84.1 2.1 0.005 Yd receptor I NM_003242.5 TGFBR2 Transforming growth factor, beta 199.6 ± 38.0 91.1 ± 41.1 -2.2 0.053 215.4 ± 27.9 1.1 NS N receptor II NM_003243.4 TGFBR3 Transforming growth factor, beta 239.2 ± 38.9 80.4 ± 11.3 -3.0 <0.001 82.2 ± 29.2 -2.9 0.001 Y receptor III, betaglycan NM_ TGIF2 TGFB-induced factor homeobox 2 771.1 ± 95.0 438.9 ± 123.4 -1.8 0.033 528.5 ± 117.4 -1.5 NS N 001199514.1

JAK / STAT signalling (up-regulated):

NM_005465.4 AKT3 v-akt murine thymoma viral 66.7 ± 11.2 160.5 ± 45.9 2.4 0.047 106.5 ± 5.6 1.6 0.002 ? oncogene homolog 3 (protein CBL Cas-Br-Mki B (murine) ) ecotropic 45.5 ± 5.2 210.3 ± 76.0 4.6 0.031 218.8 ± 13.5 4.8 <0.001 Y NM_005188 retroviral transforming sequence CBLB Cas-Br-M (murine) ecotropic 530.8 ± 74.3 823.4 ± 124.3 1.6 0.043 1412.8 ± 362.8 2.7 0.017 Y NM_170662 retroviral transforming sequence b CBLC NM_012116 Cas-Br-M (murine) ecotropic 545.8 ± 48.1 1157.4 ± 202.1 2.1 0.003 561.5 ± 98.3 1.0 NS N retroviral transforming sequence c NM_015897 PIAS4 Protein inhibitor of activated STAT, 331.8 ± 37.4 700.8 ± 61.3 2.1 <0.001 281.6 ± 30.3 -1.2 NS N NM_005026 PIK3CD Phosphoinositide-3-kinase, 480.5 ± 21.4 1310.5 ±280.7 2.7 0.003 1487.9 ± 223.8 3.1 <0.001 Y catalytic, delta polypeptide NM_003745 SOCS1 Suppressor of cytokine signalling 1 219.1 ± 6.7 387.1 ± 71.5 1.8 0.019 443.0 ± 32.4 2.0 <0.001 Y (SOCS1) b NM_003877 SOCS2 Suppressor of cytokine signalling 2 2216.1 ± 142.4 6936.0 ± 596.4 3.1 <0.001 4626.6 ± 707.3 2.1 <0.001 Y (SOCS2) NM_003955 SOCS3 Suppressor of cytokine signalling 1 370.2 ± 103.4 172.1 ± 30.6 2.2 0.066 413.2 ± 90.4 2.4 0.012 Y (SOCS3)

JAK / STAT signalling (down-regulated):

NM_ 005163.2 AKT1 v-akt murine thymoma viral 1735.0 ± 179.7 887.8 ± 280.4 -2.0 0.011 1478.8 ± 189.3 -1.2 NS N oncogene homolog 1 NM_ 032375.3 AKT1S1 AKT1 substrate 1 (proline-rich) 1673.0 ± 299.2 704.5 ± 149.2 -2.4 0.004 1189.5 ± 225.3 -1.4 NS N 6

NM_001626 AKT2 v-akt murine thymoma viral 312.1 ± 24.2 168.3 ± 31.5 -1.9 <0.001 186.9 ± 27.1 -1.7 <0.001 ? oncogene homolog 2 NM_ 001012398 AKTIP AKT interacting protein 744.7 ± 86.6 436.4 ± 121.0 -1.7 0.038 870.9 ± 65.1 1.2 NS N

NM_016166 PIAS1 Protein inhibitor of activated STAT, 700.4 ± 137.3 362.4 ± 33.2 -1.9 0.017 499.4 ± 45.5 -1.4 NS N 1 NM_173206 PIAS2 Protein inhibitor of activated STAT, 636.0 ± 69.7 407.5 ± 55.2 -1.6 0.010 692.6 ± 58.0 1.1 NS N 2 NM_006099 PIAS3 Protein inhibitor of activated STAT, 900.7 ± 54.2 492.9 ± 62.6 -1.8 <0.001 865.7 ± 94.6 -1.0 NS N 3 NM_002645 PIK3C2A Phosphoinositide-3-kinase, class 2, 941.0 ± 104.3 391.1 ± 106.3 -2.4 <0.001 1030.3 ± 98.5 1.1 NS N alpha polypeptide NM_005027 PIK3R2 Phosphoinositide-3-kinase, 552.1 ± 68.2 264.3 ± 67.4 -2.1 0.003 375.9 ± 28.3 -1.5 0.017 ? regulatory subunit 2 (beta) NM_003629.3 PIK3R3 Phosphoinositide-3-kinase, 262.4 ± 35.1 45.4 ± 20.7 -5.8 <0.001 100.2 ± 18.0 -2.6 <0.001 Y regulatory subunit 3 (gamma)

VEGF signalling (up-regulated): NM_001159920.1 FLT1 fms-related tyrosine kinase 1, 95.3 ± 13.1 286.1 ± 52.5 3.0 <0.001 141.9 ± 16.8 1.5 0.029 ? VEGFR1 b NM_000963 PTGS2 prostaglandin-endoperoxidase 341.0 ± 40.0 3115.0 ± 729.9 9.1 <0.001 1241.0 ± 221.9 3.6 <0.001 Y synthase 2; cyclooxygenase-2 (COX-2) NM_001025366.2 VEGFA Vascular endothelial growth factor 1862.9 ± 108.9 4531.6 ± 916.3 2.4 0.004 4467.0 ± 758.1 2.4 <0.001 Y A NM_005429.2 VEGFC Vascular endothelial growth factor 2158.2 ± 57.9 3884.4 ± 729.8 1.8 0.018 3734.0 ± 227.9 1.7 <0.001 ? C

a Listed genes are components of cytokine-cytokine receptor interaction, TGF signalling, JAK/STAT signalling or VEGF signalling pathways according to the KEGG database, meeting reduced stringency criteria (defined as: fold change>1.4; t-test P<0.10; difference between means>50) for differential

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expression in response to seminal plasma. Listed genes also include genes of interest CSF2 and IL1A (P=0.200 and P=0.119 for 10% SP, respectively). SP = seminal plasma. NS = not significant, P<0.10. b Data are mean ± SEM for n=4 biological replicates from Ect1 ectocervical epithelial cells, each prepared from pools of 3-4 replicate wells, for three treatments; seminal plasma (10%, 10 h), TGF3 (5 ng/nl, 10 h) or medium alone (control). c Genes are classified as (1) high confidence of TGF-regulation (‘Y’) if high stringency criteria (defined as: fold change>2.0; t-test P<0.05; difference between means>100) are met for differential expression in response to 5 ng/ml TGFβ; low confidence of TGF-regulation (‘?’) if reduced stringency criteria but not high stringency criteria are met, or not regulated by TGF (‘N’) if reduced stringency criteria are not met. d Gene is regulated in opposite direction by TGFβ compared with seminal plasma.

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