Associate with Spontaneous Preterm Birth CXCR3 Polymorphism and Expression

The Journal of Immunology

CXCR3 Polymorphism and Expression Associate with Spontaneous Preterm Birth

Minna K. Karjalainen,*,†,1 Marja Ojaniemi,*,†,1 Antti M. Haapalainen,*,†,1 Mari Mahlman,*,† Annamari Salminen,*,† Johanna M. Huusko,*,† Tomi A. Ma¨a¨tta¨,*,† Tuula Kaukola,*,† Julia Anttonen,*,† Johanna Ulvila,*,† Ritva Haataja,‡ Kari Teramo,x Stephen F. Kingsmore,{ Aarno Palotie,‖,#,**,††,‡‡,xx Louis J. Muglia,{{ Mika Ra¨met,*,†,||||,##,2 and Mikko Hallman*,†,2

Spontaneous preterm birth (SPTB) is a major factor associating with deaths and with lowered quality of life in humans. Environ- mental and genetic factors influence the susceptibility. Previously, by analyzing families with recurrent SPTB in linkage analysis, we identified a linkage peak close to the gene encoding CXCR3. Present objectives were to investigate the association of CXCR3 with SPTB in Finnish mothers (n = 443) and infants (n = 747), to analyze CXCR3 expression levels in human placenta and levels of its ligands in umbilical cord blood, and to verify the influence of Cxcr3 on SPTB-associating cytokines in mice. We detected an association between an intronic CXCR3 polymorphism, rs2280964, and SPTB in infants from families with recurrent preterm births (p = 0.009 versus term controls, odds ratio 0.52, 95% confidence interval 0.32–0.86). The minor allele was protective and undertransmitted to SPTB infants (p = 0.007). In the placenta and fetal membranes, the rs2280964 major allele homozygotes had higher expression levels than minor allele homozygotes; decidual trophoblasts showed strong CXCR3 immunoreactivity. Expres- sion was higher in SPTB placentas compared with those from elective deliveries. Concentration of a CXCR3 ligand, CXCL9, was increased in cord blood from SPTB, and the protective rs2280964 allele was associated with low CXCL9. In CXCR3-deficient mice (Mus musculus), SPTB-associating cytokines were not acutely increased in amniotic fluid after preterm birth–inducing dose of maternal LPS. Our results indicate that CXCR3 contributes to SPTB. Activation of CXCR3 signaling may disturb the maternal– fetal tolerance, and this may promote labor. The Journal of Immunology, 2015, 195: 2187–2198.

reterm birth, deﬁned as birth before 37 completed wk of involved in 30–40% of all preterm births (8); thus, infection that gestation, affects 5–18% of all deliveries and is the major causes activation of the immune system represents a major factor P cause of infant mortality worldwide (1–3). Preterm birth for SPTB. During pregnancy, the mother tolerates the fetus and represents a major global health care problem; infants born pre- extraembryonic fetal tissues as a semiallograft (9). As a conse- term are at high risk of both immediate- and long-term morbid- quence, factors affecting maternal–fetal immunotolerance may ities, with potentially lifelong consequences (4, 5). Approximately also inﬂuence the onset of preterm delivery. Although a number of 70% of all preterm deliveries occur after spontaneous onset of acquired factors (e.g., multiple pregnancy and alcohol or narcotic labor (6). However, the pathogenesis of spontaneous preterm birth addiction), diseases, and malformations contribute to the risk, the (SPTB) is poorly understood. The process leading to SPTB causes are incompletely understood, and there is no effective involves a complex interplay between signals originating both prevention for SPTB (10). from the maternal and fetal compartments (including the fetal Aggregation of preterm birth in families suggests that ge- membranes and placenta) (7). Infections are estimated to be netic factors play a role in SPTB (11–15). According to large

*PEDEGO Research Center and Medical Research Center Oulu, University of Oulu, This work was supported by the Jane and Aatos Erkko Foundation (to M.H. and M.R.); 90014 Oulu, Finland; †Department of Children and Adolescents, Oulu University the Sigrid Juse´lius Foundation (to M.H.); the Competitive State Research Financing Hospital, 90029 Oulu, Finland; ‡Biocenter Oulu, University of Oulu, 90014 Oulu, of the Expert Responsibility Area of Tampere University Hospital (to M.R.); the Finland; xObstetrics and Gynecology, University of Helsinki and Helsinki University Competitive State Research Financing of the Expert Responsibility Area of Oulu Hospital, 00290 Helsinki, Finland; {Center for Pediatric Genomic Medicine, Kansas University Hospital (to M.R.); the Stiftelsen Alma och K.A. Snellman Foundation (to City, MO 64108; ‖Analytic and Translational Genetics Unit, Department of Medi- M.K.K. and M.R.); and the March of Dimes Prematurity Research Center Ohio cine, Massachusetts General Hospital, Boston, MA 02114; #Program in Medical and Collaborative (to L.J.M.). Studies with the anonymous Finnish population controls Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142; were supported by the Nordic Centre of Excellence in Disease Genetics. **Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Address correspondence and reprint requests to Dr. Minna K. Karjalainen, Depart- Cambridge, MA 02142; ††Institute for Molecular Medicine Finland, University of ment of Children and Adolescents, Institute of Clinical Medicine, University of Oulu, Helsinki, 00014 Helsinki, Finland; ‡‡Psychiatric and Neurodevelopmental Genetics 90220 Oulu, Finland. E-mail address: minna.k.karjalainen@oulu.fi Unit, Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114; xxDepartment of Neurology, Massachusetts General Hospital, Boston, MA 02114; The online version of this article contains supplemental material. {{Center for Prevention of Preterm Birth, Perinatal Institute, Cincinnati Children’s Abbreviations used in this article: CI, confidence interval; CYC1, cytochrome c-1; Hospital Medical Center, Cincinnati, OH 45229; ||||BioMediTech, University of Tam- dpc, day postcoitum; FU, fluorescence unit; GA, gestational age; HK, housekeeping; pere, 33014 Tampere, Finland; and ##Department of Pediatrics, Tampere University MAF, minor allele frequency; OR, odds ratio; PPROM, preterm premature rupture of Hospital, 33521 Tampere, Finland fetal membranes; qPCR, quantitative PCR; SNP, single-nucleotide polymorphism; 1M.K.K., M.O., and A.M.H. contributed equally to this work. SPTB, spontaneous preterm birth; TDT, transmission disequilibrium test; UPL, Uni- versal ProbeLibrary; UTR, untranslated region; WGA, whole-genome amplification; 2M.R. and M.H. contributed equally to this work. WT, wild-type. Received for publication May 22, 2015. Accepted for publication June 26, 2015. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501174 2188 CHEMOKINE RECEPTOR CXCR3 AND SPONTANEOUS PRETERM BIRTH population-based studies and genetic modeling studies, preterm premature rupture of membranes (PPROM, defined as leakage of amniotic birth and/or variation in gestational age (GA) are affected by fluid before the onset of labor) were included among the SPTB cases. maternal genetic factors (11, 13, 14, 16–19); some of these studies Inclusion criteria for SPTB cases and controls. All medically indicated suggest that fetal genes also play a role (16, 17, 19). SPTB is a preterm births without labor (intrauterine growth restriction, pre-eclampsia, and placental abruption) were excluded from the study. To include only multifactorial phenotype that is influenced by multiple interacting SPTB deliveries that involved as few as possible risk factors (other than factors, with the potential involvement of extensive gene–gene and genetic risk factors) in the study, we excluded deliveries involving the gene–environmental interactions (20). Although several small-scale following known risk factors for preterm birth: 1) multiple gestation; 2) case-control association studies of selected candidate genes for polyhydramnios; 3) acute septic infection of the mother (positive blood culture) and/or evidence of systemic inflammatory response syndrome SPTB have been published, only a few of the detected associations (WBC count .15,000/mm2 or .10% band cells and fever .38.2˚C); 4) have been replicated in subsequent studies (21). Recently, more diseases of the mother that may affect timing of the onset of delivery (those comprehensive association analyses [large-scale association studies affecting hepatorenal, cardiopulmonary, or endocrine functions and (22–24) and a family-based analysis (25)] and hypothesis-free chronic inflammatory disease); 5) alcohol or narcotic use; 6) severe acci- approaches, including linkage analyses (26–28), whole-exome se- dents; and 7) fetuses with congenital anomalies. According to our estimates, ∼20% of all preterm deliveries in Finland fulfilled our inclusion quencing (29), and a genome-wide association study (30), have criteria (corresponding to ∼1% of all deliveries). Term delivery was de- been used to study SPTB; these studies suggested significant roles fined as a delivery occurring at 37–41 wk of gestation (37 wk + 0 d to for genes involved in multiple pathways, underlining the hetero- 41 wk + 6 d); deliveries involving any pregnancy- or labor-associated geneity of the phenotype. Further studies are needed to find out the complications (intrauterine growth restriction, placental abruption, polyhydramnios, pre-eclampsia, fetuses with congenital anomalies, and re- most important factors associated with predisposition to SPTB. quirement of special care of the newborn) were further excluded from CXCR3 is a G protein–coupled receptor that plays an essential among the controls. All analyzed subjects were of Finnish origin. role in cell-mediated immunity (31, 32). There are three CXCR3 splice variants, as follows: CXCR3-A, -B, and -alt; CXCR3-A and Study populations used for genetic analyses -B are the main variants (32, 33). Five chemokines bind CXCR3, Original case-control study population from northern Finland (Population as follows: CXCL9 (also known as monokine induced by IFN-g), I). Cases included mothers with spontaneous preterm deliveries (n = 251) and their SPTB infants (n = 291; GA #36 wk). Data regarding mothers CXCL10 (IFN-g–induced protein 10), CXCL11 (IFN-inducible and infants were collected retrospectively from birth diaries of Oulu T cell a chemoattractant), CXCL4 (platelet factor 4), and University Hospital (northern Finland) during 1973–2003, and prospec- CXCL4L1 (platelet factor 4 variant) (34). CXCR3 is preferentially tively in Oulu University Hospital during 2003–2005. SPTB cases included expressed in Th1 cells, but also in other cells, including NK cells, mothers and infants from families with a single preterm delivery (n = 183) and from those with two or more preterm deliveries (n = 68). Clinical mast cells, and microvascular endothelial cells (31, 32). CXCR3 characteristics of SPTB cases are listed in Table I. Fathers of SPTB infants ligands are upregulated in proinflammatory milieus; they promote were also analyzed (n = 215; 156 fathers from families with a single migration of circulating leukocytes, mainly Th1 lymphocytes, to preterm delivery and 59 fathers from families with recurrent preterm sites of inflammation and injury (31, 34). In addition to immune births) as a part of the quality control procedure to detect potential Men- cells, CXCR3-A expression has been detected in the human pla- delian errors. Controls included mothers with exclusively at least three term deliveries (n = 192) and their singleton infants (n = 188; 102 males centa (35). During labor, immune cells infiltrate the human re- and 86 females; GA 37–41 wk) collected prospectively at Oulu University productive and gestational tissues, including the fetal membranes Hospital during 2004–2007. The selection criterion “exclusively at least and decidua at the maternal–fetal interface (36–40); several che- three term deliveries in the family” was applied for controls because women mokines are expressed by choriodecidual cells (37, 41–43) and with several uncomplicated term deliveries (and their infants) are most un- likely to carry genetic factors associated with predisposition to SPTB. upregulated during this process (39, 44). Both spontaneous term Whole-blood or buccal-cell samples were obtained from the study subjects. and preterm human labor are associated with increased expression Additional case-control study population from southern Finland of several chemokines in the reproductive and gestational tissues (Population II). The additional case-control study population included (39, 44–49). These include CXCL10 and its receptor CXCR3, SPTB (n = 98; GA ,36 wk) and term singleton infants (n = 170; 84 males whose levels are increased in choriodecidual cells during spon- and 86 females; GA 37–41 wk) collected prospectively in Helsinki Uni- taneous labor at term (39, 44). versity Central Hospital (southern Finland) during 2005–2008. Sixteen of these infants originated from families with two preterm deliveries, and 82 Previously, we used a nonbiased approach to analyze northern were from families with a single preterm delivery. The same inclusion Finnish families with SPTB and detected a linkage peak on criteria were used as for the original study population, except that the chromosome X located close to the gene encoding CXCR3 (27). In controls did not fulfill the criterion “at least three term deliveries in the the current study, we reasoned that variation of CXCR3 may affect family.” Mothers of southern Finnish origin were not available for analyses. Clinical characteristics of the SPTB cases are listed in Table I. susceptibility to SPTB. Therefore, we assessed the role of CXCR3 Whole-blood samples were obtained from the study subjects. gene in SPTB in case-control populations. We found an associa- Finnish population controls (Population III). Anonymous Finnish pop- tion in the infants between an intronic CXCR3 variant and SPTB. ulation controls from the Kuopio and Helsinki regions (50, 51) obtained We further analyzed expression of CXCR3 in placentas and fetal through the Nordic Control database (52) were used as a reference pop- membranes from preterm and term births. Next, we analyzed cord ulation (n = 732). blood expression levels of several cytokines, including two Use of Populations I–III in each step of the genetic study. The main steps of CXCR3 ligands, CXCL9 and CXCL11. Finally, we studied cy- the genetic analyses were as follows: 1) case-control association analysis of CXCR3 gene in Population I (original population of mothers and infants tokine levels in inflammation-induced preterm labor in an exper- from northern Finland); 2) case-control association analysis in Population imental model of CXCR3 deficiency. These data support our II (additional infants from southern Finland); 3) case-control association proposal that activation of CXCR3 predisposes to SPTB. analysis with infants from Populations I and II combined; 4) case-control association analysis with anonymous population controls (Population III) included in the combined population in step 3; 5) family-based association Materials and Methods test in Population I; and 6) analysis of the whole CXCR3 gene and selected Inclusion criteria for cases and controls in the genetic analyses fragments of the gene from a subset of samples in Population I. Definition of SPTB. SPTB was defined as preterm birth that occurred at #36 DNA sample preparation, single-nucleotide polymorphism completed wk of gestation after spontaneous onset of labor. The duration selection, genotyping, and sequencing of the pregnancy was defined on the basis of ultrasound examination performed at most 18 wk after the last menstrual period. Deliveries that occurred DNA was extracted from whole-blood and buccal-cell samples using after spontaneous onset of labor with intact membranes or after preterm standard methods, as described previously (26). DNA extracted from buccal The Journal of Immunology 2189 cells was whole-genome amplified (WGA) using the Illustra GenomiPhi were collected and protein concentrations were measured with Bio-Rad V2 DNA Amplification kit (GE Healthcare Sciences). WGA products were Protein Assay Dye Reagent (Bio-Rad Laboratories) at wavelength 595 nm. further purified on Illustra MicroSpin G-50 columns (GE Healthcare Sci- For Western blotting analysis, 20 mg total protein from each sample ences). Purified WGA samples were subjected to extensive quality control, was first separated on a 12% Bis-Tris gel (NuPAGE Novex; Life Tech- as described previously (26). nologies) and then electrotransferred onto Hybond-P polyvinylidene CXCR3 (NG_029076.1) is a small gene (2.6 kb) with only a single difluoride membrane (Amersham Biosciences), according to the manu- common (minor allele frequency [MAF] .0.01) single-nucleotide poly- facturer’s instructions. Polyvinylidene difluoride membranes were blocked morphism (SNP) (rs2280964) reported in the dbSNP database (http://www. overnight at +4˚C. CXCR3 (P49682) was detected with mouse anti-human ncbi.nlm.nih.gov/SNP) and in the 1000 Genomes sequence data (http:// CXCR3 Ab (R&D Systems; 1:500 dilution) as a primary Ab and goat anti- www.1000genomes.org) for the Finnish population. We therefore chose mouse IgG HRP (Jackson ImmunoResearch Laboratories; 1:30,000 dilu- this SNP for analysis. Genotyping of SNP rs2280964 was performed with tion) as a secondary Ab. Detection was done with the ECL Prime Western the SNaPshot Multiplex kit (Applied Biosystems). For the sake of sim- blotting Detection Reagent kit (GE Healthcare). After CXCR3 detection, plicity, rs2280964 genotypes of hemizygous males (G or A) and homo- membranes were reprobed and b-actin was used as a loading control. zygous females (GG or AA) are both referred to as GG and AA in the text. b-actin was detected by using mouse anti–b-actin (Sigma Aldrich; 1:3,000 To analyze the whole CXCR3 gene in a subset of infants and mothers from dilution) and sheep anti-mouse IgG HRP (GE Healthcare; 1:1,500 dilution) Population I, the sequence encompassing CXCR3 was further amplified by as primary and secondary Abs, respectively, together with the ECL Prime PCR with eight primer pairs described previously (53). PCR yielded eight Detection kit. After detection, intensity values of the target proteins were de- partly overlapping PCR fragments that encompassed the whole CXCR3 termined with Quantity One (4.6.9.) analysis software (Bio-Rad Laboratories). gene; the first fragment started 2 kb upstream from the first exon, and the CXCR3 immunohistochemistry and analysis of umbilical cord last ended ∼30 bp downstream from the 39 untranslated region (UTR) of the last exon. The PCR fragments were then Sanger sequenced. blood cytokines Collection of placental and umbilical cord blood samples. Placental and CXCR3 mRNA and protein analyses umbilical cord blood samples of newborn infants were collected at Oulu Collection of placental samples. Placental samples were collected at Oulu University Hospital during 1998–2002. The population has been described University Hospital during 2010–2012. There were samples from 21 pla- previously (57); it consists of very preterm infants (GA ,32 wk). Infants centas from preterm deliveries (7 from SPTB and 14 from elective de- with major congenital anomalies, metabolic diseases, chromosomal liveries), GA from 25 wk + 3 d to 35 wk + 4 d. Altogether, three were defects, or congenital TORCH (toxoplasmosis, other [syphilis, varicella- placentas from twin pregnancies. In addition, there were 27 placental zoster, parvovirus B19], rubella, CMV, and herpes) infections were ex- samples from term deliveries (14 from spontaneously initiated and 13 from cluded. Full-thickness samples of placental parenchyma were collected elective deliveries), GA 38–42 wk. Samples were collected from the basal immediately after delivery. Samples were taken from the midway point plate immediately underneath the placental surface (the maternal side of between umbilical cord insertion and placental margin and fixed in for- placenta), from the chorionic plate immediately underneath the placental malin for further analyses. Umbilical cord blood samples were collected in surface (the fetal side of placenta), and from the fetal membranes. Um- dry sterile tubes immediately after delivery. Serum was separated by bilical cord blood was collected and used for DNA extraction; the SNP centrifugation and stored at 270˚C. Blood samples were used for con- rs2280964 genotypes of corresponding infants were determined. centration measurements and DNA extraction; the SNP rs2280964 geno- Quantitative PCR. RNA was isolated from frozen placental tissue samples types of corresponding infants were determined. After exclusion of according to the manufacturer’s instructions for the High Pure RNA Tissue multiple pregnancies (23 infants) and infants without available concen- kit (Roche Diagnostics). For RNA isolation, tissue samples were first dis- tration data for the cytokines (n = 26), a total of 99 very preterm infants rupted with TissueLyser LT (Qiagen) and the stainless steel bead method. remained for analysis. Of these, 66 were born after spontaneous onset of The quality and quantity of isolated RNA were assessed by measuring ab- delivery and 33 after induced delivery or elective cesarean section without sorbance values at 230, 260, and 280 nm using NanoDrop. Altogether, 250 signs of an active labor process. ng total RNA from each sample was converted into cDNA by the standard Immunohistochemistry. Altogether, 27 and 12 placentas from SPTB and RT-PCR procedure with the Transcriptor First Strand cDNA Synthesis kit elective preterm deliveries, respectively, were analyzed using immuno- (Roche Diagnostics). Subsequently, in the corresponding quantitative PCR histochemistry. Placental samples were embedded in paraffin and cut into (qPCR) mixture, 10% of the cDNA sample was used. 4-mm slices, deparaffinized, and rehydrated. Ag retrieval was done in Tris- Relative quantifications of CXCR3 mRNA levels were done with Light- EDTA buffer. Endogenous peroxidase activity was blocked in blocking Cycler96 (Roche Diagnostics) using CXCR3 splice variant- and cytochrome solution (DAKO). Samples were incubated in a 1:2000 dilution of mouse c-1 (CYC1)–specific primers and probes; primers were designed to bind at anti-human CXCR3 mAb (MAB160 clone 49801; R&D Systems). De- the splice site. Probes were from the human Universal ProbeLibrary (UPL) tection of bound Abs was done with the Envision kit (DAKO). Two Set (Roche Diagnostics). Primers and probes were as follows: forward 59- examiners evaluated the samples independently and were blinded to ATGGTCCTTGAGGTGAGTGAC-39 and reverse 59-TCTCCATAGTCATA- clinical and genetic data. The staining intensities were visually ranked into GGAAGAGCTGA-39 for CXCR3-A (ENST00000373693) (with UPL probe three categories: faint, intermediate, and dark. 79); forward 59-CTTCCCACAGGGGTCAAGTTC-39 and reverse 59-CTCT- Analysis of cytokines. To detect the levels of selected cytokines (listed in GGAGCCCTCTCTGGTT-39 for CXCR3-alt (ENST00000373687) (with UPL Supplemental Table I) in cord blood serum, we used Ab-based protein probe 89); forward 59-CCCAGCCATGGTCCTTGAG-39 and reverse 59-AG- microarrays with fluorescent sandwich immunoassay and rolling circle GGCCGTACTTCCTCAACT-39 for CXCR3-B (ENST00000373691) (with DNA amplification (58). These cytokines were initially selected for the UPL probe 64); and forward 59-ATAAAGCGGCACAAGTGGTCA-39 and re- study based on the important role of the inflammatory pathway in the onset verse 59-GATGGCTCTTGGGCTTGAGG-39 for CYC1 (ENST00000318911) of labor (59). The fluorescent, rolling-circle amplification product was (with UPL probe 47). The qPCR amplification step consisted of two steps: detected by a microarray scanner. The amount of fluorescence reflects the 95˚C for 10 s and 60˚C for 30 s. Each measurement was done in triplicate, amount of the studied protein in the sample. Concentrations of the indi- and the mRNA levels of CXCR3 variants were normalized against CYC1 vidual cytokines are reported as fluorescence units (FU). mRNA. Relative quantifications were assessed using the DD cycle threshold method. Randomly chosen cycled qPCRs were run on agarose gels, and the Experiments in mice deficient for CXCR3 nucleotide sequences were verified. The results proved that the aforemen- Animals. Mice deficient for CXCR3 (O88410) (B6.129P2-Cxcr3tm1Dgen/ tioned primers specifically amplified the CXCR3 variants and CYC1. CYC1 J; JAX 005796) were obtained from The Jackson Laboratory and back- was chosen as a reference gene based on previous studies (54–56). For ex- crossed onto the local C57BL/6 strain over at least four generations prior to ample, Drewlo et al. (56) used nine clinical groups (healthy first trimester, experiments. All mice were genotyped, and only CXCR3-deficient mice healthy second trimester, preterm controls, vaginal and cesarian delivery were used. For the experiments, CXCR3-deficient female mice aged 3–4 mo term controls, severe early onset intrauterine growth restriction, severe pre- were mated with males of the same line. Wild-type (WT) C57BL/6 mice eclampsia, and mixed pre-eclampsia–intrauterine growth restriction) and served as controls. GA (612 h) was determined by the presence of eight commonly used housekeeping (HK) genes to determine the most stable a vaginal plug and designated as day 0 of pregnancy. HK candidates for placental research. Their data indicated that CYC1 was Mouse model of preterm birth. We used our previously established mouse one of the most stable HK genes for placental research. Additionally, in our model of LPS-induced preterm birth of live-born offspring (60) to study the study, we followed and confirmed the stability of CYC1 mRNA. role of CXCR3 in inflammation-induced preterm birth. In the current Western blotting. Protein homogenates were prepared from placental tissue study, the preterm birth–inducing dose of LPS was determined in the in lysis buffer (10 mM Tris-HCl [pH 7.5], 250 mM sucrose, and 1 mM following preliminary experiment. Timed-pregnant CXCR3-deficient and EDTA). Cells were lysed by sonication. After centrifugation, supernatants WT mice were i.p. injected at 17 d postcoitum (dpc) with different doses 2190 CHEMOKINE RECEPTOR CXCR3 AND SPONTANEOUS PRETERM BIRTH

(10–25 mg/mouse) of Escherichia coli LPS (serotype 0111:B4) dissolved and term infants from Population I originating from northern in PBS. Control mice (n = 4 for both mouse lines) received an equal Finland (Tables I, II). Minor allele A was underrepresented in volume (100 ml) of PBS. The timed-pregnant mice were randomized to SPTB infants (MAF 0.148 versus 0.248, respectively, p = 0.009, receive either LPS or PBS injections. Dams were followed until the time of delivery, and the viability of the prematurely born pups was observed, as OR 0.52, 95% confidence interval [95% CI] 0.32–0.86), indicating previously described (60). The litters were called live-born when $50% of a protective effect. Alternatively, the major allele G may have the pups were born alive. a predisposing effect. A similar trend was detected between Sample collection. For cytokine analyses, mice were randomized to receive mothers with preterm deliveries and those with term deliveries; either LPS or PBS injections. Timed-pregnant CXCR3-deficient (n = 5) and this did not reach statistical significance (MAF 0.203 versus 0.255, WT (n = 4) mice were injected with a preterm birth–inducing dose of LPS (20 mg/mouse) at 17 dpc. Another set of CXCR3-deficient and WT mice respectively, p = 0.066, OR 0.74, 95% CI 0.54–1.02). Frequencies received an equal volume (100 ml) of sterile PBS (n = 4 for both mouse were similar in mothers who had had recurrent preterm deliveries lines). Sample size was calculated based on our previous studies (60), and compared with those who had experienced a single preterm de- the estimated sample size was 8–10 fetuses. Dams were anesthesized with livery. There was no difference between term infants and infants ketamine-medetomidine and killed with cervical dislocation 3 h after the from families with a single preterm delivery (Table II). Fathers of LPS or PBS injections. Amniotic fluid from the fetuses was collected and stored at 270˚C. SPTB infants had a MAF of 0.260, which is close to MAF of the Cytometric bead array. Levels of IL-6 and CCL2 in the amniotic fluid of control mothers and infants (0.255 and 0.248, respectively). CXCR3-deficient and WT mice were quantitatively measured with the To determine whether SNP rs2280964 is also associated with Cytometric Bead Array Mouse Inflammation kit (BD Biosciences), SPTB in the genetically more diverse southern Finnish population, according to the manufacturer’s instructions. In each group, amniotic fluid we analyzed SPTB and control infants originating from this region from 2–5 pups from every litter was analyzed individually. The detection limits for IL-6 and CCL2 were 5 and 53 pg/ml, respectively. (Population II). Although there was no significant difference in allele distribution between SPTB and term infants, the minor allele Statistics was underrepresented in SPTB infants in a manner similar to that in Quality control in the genetic study. Before proceeding to association the discovery population (MAF 0.181 versus 0.227, respectively, analysis, the absence of Mendelian errors in the genotype data was confirmed p = 0.28, OR 0.75, 95% CI 0.45–1.26; Table II). With infants of using Pedcheck (61). Inconsistent genotypes were recoded as missing. Populations I and II combined, a similar trend between all SPTB Statistical analysis in the genetic study. Case-control and family-based infants and term infants was evident (MAF 0.192 versus 0.238, association analyses were performed using PLINK, v. 1.07 (62). The significance of allele frequency differences was analyzed with the x2 test, respectively, p = 0.062, OR 0.76, 95% CI 0.57–1.02). The dif- and effect sizes were estimated using odds ratios (OR). To account for the ference was significant between SPTB infants from families with effect of fetal gender, fetal sex was included as a covariate in a logistic recurrent preterm deliveries and term infants when the populations regression model. Female individuals were analyzed using genotypic, were combined (MAF 0.156 versus 0.238, respectively, p = 0.016, dominant, and recessive models; analysis with these models was not ap- plicable to male infants because CXCR3 is located on the X chromosome. OR 0.59, 95% CI 0.39–0.91), further strengthening the evidence Allele frequency differences between SPTB infants from deliveries with for association of this SNP with SPTB. and without PPROM, and between very (GA ,32 wk) and moderate-to- Next, we determined the MAF of rs2280964 in a large Finnish late preterm infants (GA 32–36 wk), were also investigated. Family-based population control cohort (n = 732; population III) originating analysis was performed with the transmission disequilibrium test (TDT). from southern and eastern Finland (52) and included these con- Prediction of regulatory effects of the CXCR3 polymorphism was performed with GWAS3D (http://jjwanglab.org/gwas3d) and RegulomeDB trols in analyses. The MAF in this cohort was 0.234, that is, close (https://regulomedb.org), which use public datasets, including data from to that of our controls. With these controls included, the associ- the Encyclopedia of DNA Elements project (https://www.encodeproject. ation was significant in all SPTB infants (MAF 0.192 versus 0.235 org), as sources. in a total of 389 cases and 1090 controls, respectively, OR 0.77, Statistical analysis of mRNA, protein, and cytokine studies. RNA, protein, 95% CI 0.61–0.98, p = 0.032) and in infants from families with and cytokine analyses were performed with SPSS Statistics 20.0 (IBM Corporation). Expression level differences between genotype and pheno- recurrent SPTB (MAF 0.156, OR 0.60, 95% CI 0.41–0.89, p = type groups were assessed by the nonparametric Mann–Whitney U test or 0.011). Kruskal–Wallis test. Mann–Whitney U test was used to analyze whether Because CXCR3 is located on the X chromosome, we tested cytokine concentrations in umbilical cord blood at time of birth differen- separately for association in female and male infants in Pop- tiated between SPTB infants (infants born after spontaneous onset of labor) ulations I and II. The allele frequency difference between SPTB and preterm infants born after elective delivery (without active signs of labor). Receiver operating characteristic curves were used to specify the and term infants was detected in both female and male infants. We sensitivity and specificity of the analyzed CXCR3 ligands in predicting further analyzed the infants for SNP rs2280964 using logistic SPTB. regression, with fetal gender as a covariate. Under this model, Statistical analysis of CXCR3-deficient mice. Quantified protein concen- SNP rs2280964 was a significant factor in predicting susceptibility trations were analyzed with SPSS Statistics 20.0 (IBM Corporation). Data to SPTB in infants from families with recurrent preterm deliveries, were tested for normal distribution, and differences between experimental and control groups were analyzed using either Student t test or Mann– whereas fetal gender did not have predictive value. Analysis of Whitney U test. female infants under the dominant model revealed an association with infants of Populations I and II combined (p = 0.017); analysis Ethical approval under the genotypic, dominant, and recessive models did not re- The study had the approval of the Ethical Committees of the participating veal associations for any other group of female infants. Finally, the centers (Oulu University Hospital 79/2003 and 73/2013, Helsinki Uni- frequency distribution of SNP rs2280964 did not differ between versity Central Hospital 279/E7/2001). Informed consent was obtained from preterm infants born PPROM compared with those born after all study subjects. The animal care and experimental procedures were , conducted under a protocol approved by the National Animal Experiment deliveries without PPROM, or between very (GA 32 wk) and Board of Finland (license ESAVI/3752/04.10.03/2012). moderate-to-late preterm infants (GA 32–36 wk). A TDT in SPTB families of Population I revealed undertransmission of the minor allele A to SPTB infants in families with Results recurrent preterm deliveries (p = 0.007, Table III). In accordance CXCR3 SNP rs2280964 associated with SPTB with the results of case-control analysis, undertransmission was The frequency distribution of CXCR3 SNP rs2280964 differed not detected in families with a single preterm delivery. In the joint between SPTB infants from families with recurrent preterm births analysis of all SPTB families, this test was nearly significant The Journal of Immunology 2191

Table I. Clinical characteristics of the case study populations for SPTB

Original Population from Additional Population from Northern Finland (Population I) Southern Finland (Population II)

Characteristics Mothers Infants Infants Total numbera 251 291 98 Single preterm delivery in family 183 164 82 Two preterm deliveries in family 51 85 16 3–5 Preterm deliveries in family 17 42 0 Gestational age, wk 32.7 6 4.62 (24.1–36.0)b 33.8 6 2.08 (27.7–35.9)b Gestational age ,32 wk, frequency (n) 0.33 (97) 0.17 (17) Birthweight, g 2007 6 971 (538–3365)b 2340 6 531 (1010–3945)b PPROM, yes/no 98/193 57/41 Male/female 162/129 52/46 Maternal smoking during pregnancy, yes/no/unknown 31/159/61 31/150/110 3/95/0 aMismatch in the numbers of mothers and infants due to occasional low quality of DNA samples. bMean 6 SD (range).

(p = 0.066, Table III). Because the TDT test can only assess ered SNP rs188959001 and the novel insertion to be potentially maternal transmission of an X chromosomal SNP, transmission of interesting and therefore chose to sequence the regions encom- paternal alleles could not be estimated. passing these sites in additional subjects from Population I. Minor alleles of these polymorphisms were not detected in additional SNP rs2280964 not in linkage disequilibrium with other sequenced SPTB mothers (n = 10) and infants (n = 20). Thus, polymorphisms of CXCR3 these minor alleles are not likely to represent major SPTB- A SNP may be associated with an investigated phenotype due to predisposing factors at the population level. However, they may its direct effects on gene function. Alternatively, a phenotype- play a role in this individual family. associated SNP may be in linkage disequilibrium with the caus- Because no further common variants were revealed, we propose ative variation. To assess whether SNP rs2280964 displays linkage that the detected association between CXCR3 SNP rs2280964 and disequilibrium with any other variation within CXCR3, we se- fetal SPTB is likely a direct effect of this SNP. Indeed, based on quenced the genomic region extending from the 59 region into histone modiﬁcation marks and ChIP sequence data from the the 39 UTR in 15 mothers with preterm deliveries and their SPTB Encyclopedia of DNA Elements project, this SNP maps to a pu- infants and in 10 mothers with term deliveries and their infants tative enhancer region in several cell lines, and the intronic region from Population I. No variants correlated with the SNP analyzed encompassing the SNP interacts with two transcription factors: in the case-control association study (rs2280964) were detected. NFKB1 and PML. Furthermore, the SNP is predicted (p , 0.05) This is consistent with 1000 Genomes sequence data (http://www. to affect the binding afﬁnity of transcription factor Sp1. 1000genomes.org), which indicated that no variations within the gene are correlated with this SNP in the Finnish population. CXCR3 expression levels associated with SNP rs2280964 in In addition to SNP rs2280964, three rare polymorphic sites were placenta and fetal membranes detected within the mother–infant pairs (Supplemental Table II). CXCR3 is primarily expressed in immune cells. However, CXCR3 Two of these have been previously reported (http://www. expression has also been detected in the placenta (35). To inves- 1000genomes.org): rs56313919 and rs188959001, which results tigate this further, we analyzed samples from 48 placentas (21 in a premature stop codon in CXCR3-B. In addition, a 1-bp in- from preterm and 27 from term deliveries) using qPCR. CXCR3 sertion located in the 39 UTR was detected in a single SPTB mRNA was detected in all studied placental tissues (basal plate, mother and her male infant (Supplemental Table II). We consid- chorionic plate, and fetal membranes), and all three splice variants

Table II. Case-control association analysis of CXCR3 SNP rs2280964 in SPTB

SPTB Cases from Families SPTB Cases from All SPTB Cases with a Single Preterm Families with Recurrent Participants vs. Controls Delivery vs. Controls Preterm Deliveries vs. Controls Northern Finnish Case/control n 251/192 183/192 68/192 mothers (Population I) Case/control minor allele frequency 0.203/0.255 0.208/0.255 0.191/0.255 OR (95% CI) 0.74 (0.54–1.02) 0.76 (0.54–1.08) 0.69 (0.42–1.12) p 0.066 0.12 0.13 Northern Finnish Case/control n 291/188 164/188 127/188 infants (Population I) Case/control minor allele frequency 0.195/0.248 0.232/0.248 0.148/0.248 OR (95% CI) 0.73 (0.51–1.06) 0.92 (0.61–1.38) 0.52 (0.32–0.86) p 0.098 0.67 0.009 Southern Finnish Case/control n 98/170 82/170 16/170 infants (Population II) Case/control minor allele frequency 0.181/0.227 0.170/0.227 0.227/0.227 OR (95% CI) 0.75 (0.45–1.26) 0.71 (0.41–1.24) 1.00 (0.36–2.84) p 0.28 0.22 0.99 Infants of Populations Case/control n 389/358 247/358 143/358 I and II combined Case/control minor allele frequency 0.192/0.238 0.212/0.238 0.156/0.238 OR (95% CI) 0.76 (0.57–1.02) 0.86 (0.62–1.19) 0.59 (0.39–0.91) p 0.062 0.36 0.016 2192 CHEMOKINE RECEPTOR CXCR3 AND SPONTANEOUS PRETERM BIRTH

Table III. TDT of CXCR3 SNP rs2280964 in families with SPTB

Study Groupa Transmitted/Untransmitted Minor Alleles (n) TDT OR (95% CI) p All families with preterm deliveries 22/36 0.61 (0.36–1.04) 0.066 Families with a single preterm delivery 17/18 0.94 (0.49–1.83) 0.87 Families with recurrent preterm deliveries 5/18 0.28 (0.10–0.75) 0.007 aTDT was performed in families of Population I originating from northern Finland.

(CXCR3-A, CXCR3-B, and CXCR3-alt) were present. These data preterm singleton infants (n = 99) (57). Umbilical cord blood indicate that CXCR3 mRNA is expressed in critical tissues related CXCL9 concentrations were higher in SPTB infants than in to SPTB, the placenta and fetal membranes. infants born after elective delivery (mean FU: 1868 versus 998, Next, we studied whether SNP rs2280964 affects CXCR3 mRNA median FU: 1101 versus 876, respectively; p = 0.006). High levels. We compared CXCR3 mRNA levels between samples from CXCL9 concentration (cutoff value 1231 FU) predicted the risk SPTB and elective preterm placentas and between SPTB and of SPTB with a sensitivity of 47.6% and specificity of 21.2% spontaneous term placentas. SNP rs2280964 was associated with (receiver operating characteristic curve shown in Supplemental expression levels of all splice variants on the basal plate and fetal Fig. 1). The SPTB-predisposing GG genotype of CXCR3 SNP membranes; the CXCR3 mRNA level was lowest in placentas rs2280964 was associated with higher CXCL9 concentrations from infants with the AA genotype (n = 6) and highest in GG (mean FU for infants with GG and AA genotypes: 1886 versus placentas (n = 33) (Fig. 1). To exclude the confounding effect of 1163, median FU: 1270 versus 761, respectively; p = 0.021). SNP rs2280964 in subsequent analyses, phenotype analyses were Umbilical cord blood CXCL11 concentrations did not differ sig- performed exclusively using placentas with the GG genotype. On nificantly between SPTB infants and infants born after elective the basal plate of GG placentas, the CXCR3 mRNA level was delivery (mean FU: 886 versus 661, median FU: 554 versus 389, higher in SPTB placentas (n = 5) compared with elective preterm respectively, p = 0.067). Moreover, SNP rs2280964 was not asso- placentas (n = 8) and in SPTB placentas compared with term ciated with CXCL11 concentration. These data indicate that elevated placentas from deliveries with a spontaneous onset (n = 10) levels of the CXCR3 ligand CXCL9 are associated with SPTB. (Fig. 2, Supplemental Table III). This was consistent for all three We further analyzed a panel of cytokines, including chemokines splice variants. Exclusion of the three twin pregnancies that were and cytokines from the hematopoietin, IL-1, and TNF families (69 included in the mRNA analyses did not notably alter the results. cytokines listed in Supplemental Table I). Several of the cytokines The splice variants were further studied at the protein level by from the very preterm infants showed increased cord blood levels Western blotting (Fig. 3). CXCR3-A and CXCR3-B were detected, in SPTB. The cytokines associated most significantly with SPTB whereas CXCR3-alt was not. When four placentas from SPTB and are shown in Table IV. Levels of these cytokines were not sig- four from spontaneous term deliveries (all with SNP rs2280964 nificantly associated with CXCR3 SNP rs2280964. IL-6 and CCL2 GG genotype) were compared, we found that expression of showed the clearest association with SPTB and were thus ana- CXCR3-A was higher compared with that of CXCR3-B in SPTB, lyzed in the subsequent mice experiment. whereas this ratio was inversed in term deliveries (p = 0.021, CXCR3 deficiency influenced SPTB-associated cytokines in Fig. 3B). This suggests that relative expression of CXCR3-A is mice higher in SPTB compared with term spontaneous delivery, which is consistent with our mRNA analyses. We hypothesized that CXCR3 may influence the trafficking of labor-inducing cytokines across the maternal–fetal barrier, and that CXCR3 immunostaining detected in specific cells of placenta this may be a conserved event in the highly species-specific labor and fetal membranes process. To investigate our hypothesis, we exposed the pregnant Because SNP rs2280964 associated with CXCR3 mRNA levels on mice to LPS and compared the specific responses between WT and the basal plate of the placenta and in the fetal membranes, we CXCR3-deficient strains. CXCR3 deficiency had no detectable further studied CXCR3 protein expression with immunohisto- influence on the duration of pregnancy or on the survival of pre- chemical analysis; only AA and GG placentas were included (n = maturely born pups. A preterm birth–inducing dose of maternal 39). Two investigators (M.O. and M.M.) blinded to the genetic LPS was injected at 17 dpc, and the acute influence of LPS on data visually ranked the staining intensities into three categories: SPTB-associating cytokines in amniotic fluid was studied. IL-6 faint, intermediate, and dark. CXCR3 staining was judged as faint and CCL2 were included due to their clear association with in 70% of SNP rs2280964 AA placental samples (n = 10), whereas SPTB in human umbilical cord blood. In WT mice, concentrations this figure was 21% in GG placental samples (n = 29). No dif- of IL-6 and CCL2 in the amniotic fluid were slightly increased 3 h ferences associated with SPTB were detected by immunohis- after LPS injection. By contrast, LPS administered to CXCR3- tochemistry. CXCR3 protein immunostaining was detected in deficient mice decreased the levels of IL-6 and CCL2 cytotrophoblasts, syncytiotrophoblasts, and extravillous tropho- (p = 0.001, Table V). blasts (Fig. 4). The decidual trophoblasts from GG placentas were strongly positive (Fig. 4G, 4H). By contrast, the capillary endo- Discussion thelia of the villi stained only faintly in all samples. Chorionic The causes of SPTB are mostly unknown. Both genetic and en- trophoblasts in fetal membranes were positive, but otherwise the vironmental factors are involved, but the actual predisposing genes fetal membranes showed only faint or no immunostaining. and underlying biochemical pathways are poorly understood (10). In this study, we provide evidence of a role for CXCR3 in preterm Umbilical cord blood levels of the CXCR3 ligand CXCL9 labor and premature birth. associated with SPTB We found an association between a fetal CXCR3 polymorphism To study the biological role of CXCR3 expression in placental (SNP rs2280964) and SPTB, with a protective role for the minor cells, we performed a post hoc analysis of two CXCR3 ligands, allele A, in a Finnish population known to be relatively genetically CXCL9 and CXCL11, in our previously collected cohort of very homogeneous (51). Further sequencing did not reveal any The Journal of Immunology 2193

FIGURE 1. Association of SNP rs2280964 with CXCR3 mRNA levels in placenta and fetal membranes. Relative levels of CXCR3-A, CXCR3-B, and CXCR3-alt mRNA were determined and normalized to expression levels of the HK gene CYC1 using qPCR. Fetal G/GG and A/AA genotypes were compared from the following compartments: basal plate of the placenta (A–C), chorionic plate of the placenta (D–F), and fetal membranes (G–I). G and A refer to genotypes of male fetuses (hemizygotes), and GG and AA to genotypes of female fetuses (homozygotes). Differences between study groups were analyzed by nonparametric Mann–Whitney U test. Median of each group is shown as a horizontal line. Significant change (p , 0.05) indicated by an asterisk. NS, no significant change. polymorphisms that displayed linkage disequilibrium with T cells. Therefore, we suggest that CXCR3 rs2280964 may con- rs2280964. Because there were no linked variations for rs2280964 tribute to SPTB through transcriptional effects in the placenta. either in our sequence data or in that of the 1000 Genomes Finnish In the placenta, only expression of CXCR3-A mRNA has been population, we conclude that the detected association is most previously reported (35). To our knowledge, this is the first study likely a direct effect of this SNP that was previously shown to be to report expression of all three CXCR3 splice variants in this a functional polymorphism (expression SNP), associated with tissue. Our protein analyses suggest that the ratio of the two main expression levels of CXCR3 in human T cells and changes in splice variants, CXCR3-A/CXCR3-B, is higher in SPTB com- immune cell responses to chemokine–cytokine signals (63). pared with spontaneous term delivery. These preliminary data Therefore, we assessed whether this SNP was associated with suggest that increased expression of CXCR3-A, in particular, is expression levels of CXCR3 in the placenta and fetal membranes. associated with SPTB, whereas CXCR3-B is expressed at a rela- Our results demonstrate that on the basal plate of the placenta and tively higher level in the placenta during normal term parturition. in the reflected fetal membranes, minor allele A was associated This is an interesting finding, because CXCR3-A has been shown with decreased CXCR3 mRNA expression. This decreased mRNA to promote chemotaxis and growth, whereas CXCR3-B mediates expression is consistent with the results of experiments performed growth-inhibitory signals (35, 64). in human T cells by Choi et al. (63). We also detected higher In several abnormal conditions, CXCR3 levels increase. For expression levels in SPTB placentas compared with placentas instance, in renal carcinoma (65) and ovarian cancer (66), the from elective preterm or spontaneous term deliveries. It is likely expression level of CXCR3 is correlated with the disease patho- that the associated SNP is located within a regulatory region and genesis. Additionally, expression of CXCR3 in CD4+ T cells was the G-to-A change modulates binding of regulatory proteins, reported to be increased in the peripheral blood of women with which leads to alterations in transcription initiation. Indeed, Choi multiple miscarriages (67). Our finding of higher expression of et al. (63) were able to demonstrate this phenomenon in cultured CXCR3 in SPTB placentas suggests a role for this molecule in the 2194 CHEMOKINE RECEPTOR CXCR3 AND SPONTANEOUS PRETERM BIRTH

FIGURE 2. Increased CXCR3 mRNA levels in SPTB placentas. The following phenotype groups were compared: SPTB versus elective preterm birth (A–C) and SPTB versus spontaneous term birth (D–F). All samples were from the basal plate of the placenta. Only placentas with the fetal G/GG genotype were included; G and GG refer to genotypes of male (hemizygotes) and female (heterozygotes) fetuses, respectively. As in Fig. 1, relative levels of CXCR3-A, CXCR3-B, and CXCR3-alt mRNA were determined and normalized to expression levels of the HK gene CYC1. Differences were analyzed with nonparametric Mann–Whitney U test. Horizontal line denotes the median of each group. Significant change (p , 0.05) indicated by an asterisk. NS, no significant change. preterm labor process. This proposed role of CXCR3 in labor is immunohistochemical staining. This is in concordance with the further supported by a study by Gomez-Lopez et al. (39) in which labor-associated upregulation of several chemokines, including CXCR3 expression in choriodecidual leukocytes was shown to be CXCL10, in choriodecidual membranes (39, 44). According to the increased in spontaneous term labor. The placental cells that po- current proposal, the choriodecidua transmits signals required for tentially contribute to the success or failure of a pregnancy may be the activation of labor-producing uterine contractions. Likewise, located in the decidua (maternal side of the placenta), in which fetal membranes overlying the internal orifice of the cervix could fetal and maternal cells are in direct contact (68). We determined potentially transmit signals for cervical ripening (70). that CXCR3 was localized in both decidual and villous cyto- In the decidua, NK cells produce CXCL10, which promotes the trophoblasts, as well as in syncytiotrophoblasts at the feto– migration and invasion of fetal trophoblasts during formation of the maternal interface. Trophoblasts are involved in several key placenta (71). The decidua also contains macrophages and T cells. functions of the placenta, including hormone synthesis, molecular Nancy et al. (72), however, showed that the decidua did not ac- transport, metabolic regulation, and immune defense (69). All cumulate T cells, and that the T cells could not be recruited from trophoblasts are of fetal origin, which is intriguing considering blood in early pregnancy in mice. This was due to low expression that the association of SNP rs2280964 with SPTB was detected in of the chemokines CXCL9 and CXCL10, which results from the re- the fetus. Moreover, the chorionic trophoblasts showed significant pressive methylation histone mark that appears upon decidualization. The Journal of Immunology 2195

FIGURE 3. Immunoblot analyses of CXCR3 expression levels on the basal plate of the placenta. (A) Two phenotype groups were analyzed: SPTB (samples 1–4) and spontaneous term birth (samples 5–8). All analyzed cases were of the fetal CXCR3 SNP rs2280964 G/GG genotype; G and GG refer to genotypes of male (hemizygotes) and female (heterozygotes) fetuses, respectively. The two forms of CXCR3 receptor, CXCR3-A (40.7 kDa) and CXCR3-B (45.5 kDa), and the loading control (b-actin) are indicated by arrows. For more speciﬁc experimental details, see Materials and Methods.(B) Protein expression level ratios of CXCR3-A and CXCR3-B. Intensities of CXCR3-A and CXCR3-B in (A) were determined. Sub- sequently, the CXCR3-A/CXCR3-B (A/B) ratio was calculated for each of the eight samples in (A). SDs are shown. In term placentas, the A/B ratio was ,1, indicating that CXCR3-B was the predominant splice variant. The A/B ratio of preterm placentas was .1. The difference in the A/B ratio between preterm and term placentas was signiﬁcant (*p = 0.021).

During late human pregnancy, on the contrary, the maternal observation that Cxcr3 knockout mice show delayed rejection. circulating T cells have been shown to infiltrate into decidua, as Hancock et al. (74) also suggested that this phenomenon was due reviewed by Gomez-Lopez et al. (73). Another study suggested to infiltration of activated T cells into the site of rejection. Fur- that CXCR3 ligands play important roles in transplant rejection; thermore, in cases of villitis of unknown etiology, the placental Hancock et al. (74) showed that acute cardiac transplant rejection inflammatory lesion affecting the villous tissues (rather than de- was associated with much higher expression in mice not only of cidual tissue in SPTB) has been proposed to be analogous to graft CXCR3, but also of its ligands. This was further supported by the rejection. Indeed, concentrations of CXCR3 ligands in the maternal

FIGURE 4. Expression of CXCR3 in preterm placentas from infants with SNP rs2280964 A/AA and G/GG genotypes. A simpliﬁed illustration of the placenta is shown with sampling sites indicated by black squares. Representative placental tissues were immunostained with CXCR3 Ab. Black arrows show the immunostaining in the cytotrophoblastic cells, and black arrowheads in endothelial cells. Syncytiotrophoblasts are shown in big open arrows, and small open arrows show decidual trophoblasts. Original magniﬁcation 320 for all photomicrographs. (A and B) Samples from SPTB placentas without histological chorioamnionitis. (C and D) Samples from SPTB placentas with histological chorioamnionitis. (E–H) Samples from elective preterm deliveries without histological chorioamnionitis and with cesarean section due to maternal symptoms. G and A refer to genotypes of male fetuses (hemizygotes), and GG and AA to genotypes of female fetuses (homozygotes). Samples from villous (A–F) and decidual (G and H) sections of the placenta. Scale bar, 100 mm. 2196 CHEMOKINE RECEPTOR CXCR3 AND SPONTANEOUS PRETERM BIRTH

Table IV. Cytokines with increased umbilical cord blood induced by i.p. injection of LPS into either CXCR3-deficient or concentrations in SPTB WT pregnant mice. We were able to demonstrate that levels of the two cytokines associated most significantly with human SPTB, Relative Median IL-6 and CCL2, tended to be acutely increased in the amniotic fluid Concentration Cytokinea in SPTB Infantsb p of WT mice, whereas in CXCR3-deficient mice, these cytokines were significantly decreased after administration of preterm birth– CCL2 2.25 1 3 1025 CCL4 1.82 1 3 1024 inducing LPS. This observation raises several important questions CCL23 1.75 8 3 1025 to be studied, including the possibility that in human pregnancy CXCL6 1.59 1 3 1024 CXCR3 regulates the trafficking of cytokine signals across the 2 IL-6 3.56 1 3 10 6 maternal–fetal barrier. IL-1b 1.50 3 3 1024 2 The strengths of our investigation include the study populations TNFR superfamily 1.90 2 3 10 5 member 1A (TNFRSF1A) that were rather homogenous with carefully defined phenotypes TNFRSF11A 1.45 9 3 1025 and thorough analysis of the whole CXCR3 gene by sequencing. 2 TNFRSF14 1.45 4 3 10 4 Results of the mRNA and protein analyses were consistent and aCytokines significant after Bonferroni correction for multiple testing of 69 cyto- demonstrated by several techniques. The major limitation of the kines (p , 7 3 1024) are shown. genetic, mRNA, and protein studies was the relatively small bCytokine levels were initially reported as fluorescence units and are expressed relative to the median of levels in infants born before 32 wk of pregnancy after sample size. Thus, our findings require verification in additional elective birth without labor. larger sample sets. In mice, we chose to study the two cytokines that had the strongest association with SPTB in human cord blood. Further analysis of additional cytokines, such as IL-1b, would and fetal plasma have been reported to be elevated in villitis of bring more thorough information about the overall effect of unknown etiology (75). It is tempting to speculate that a process CXCR3 deficiency in cytokine levels in the mouse model of LPS- analogous to allograft protection/rejection is evident in the pla- induced preterm birth. SPTB is a heterogeneous phenotype, which centa. However, the idea that a phenomenon related to allograft consists of several phenotypic subgroups, such as PPROM, and rejection requiring high CXCR3 expression is occurring during most likely involves the activation of different biological path- SPTB will have to be investigated in future studies. ways, the most important of which are inflammation/infection, To provide further evidence for the role of CXCR3 in the preterm activation of hypothalamic-pituitary-adrenal axis, decidual labor process, we evaluated the umbilical cord blood concen- hemorrhage/thrombosis, and uterine overdistension (59, 78). Ac- trations of two CXCR3 ligands, CXCL9 and CXCL11, in a cohort cordingly, in recent large-scale association studies, genes from of 99 very preterm infants. Previously, serum concentrations of several pathways, for example, the follicle-stimulating hormone CXCL10, one of the CXCR3 ligands, were reported to be higher in pathway (22), the cell communication and extracellular matrix mothers with preterm delivery than in those with term delivery (76), receptor interaction pathway (25), insulin-like growth factor sig- and increased expression of CXCL9, CXCL10, and CXC11 in naling (26), and complement and coagulation cascade (29), have fetal membranes was reported to associate with chronic cho- been suggested to associate with preterm birth or gestational age. rioamnionitis, one of the phenotypical subgroups of preterm birth However, in a recent genome-wide association study of early (77). We detected increased levels of CXCL9 in the umbilical cord spontaneous preterm delivery, none of the detected suggestive blood of SPTB infants. Furthermore, the SPTB-predisposing ge- associations could be replicated in an additional studied pop- notype of the CXCR3 polymorphism was associated with high ulation (30). The search for SPTB-predisposing genetic factors is CXCL9 levels. This finding and the previous findings of increased complicated by its heterogeneous nature, and thus, studies fo- CXCR3 ligand levels associated with both spontaneous term and cusing on the phenotypic subgroups and utilizing larger sample preterm labor (39, 44, 76, 77) implicate that changes in the ex- sizes are needed. Our findings of association of CXCR3 poly- pression of components of the CXCR3 pathway most likely pre- morphism and expression with SPTB are in line with the evidence cede the onset of labor. However, the detected sensitivity and of the importance of the inflammatory pathway in the onset of specificity values of CXCL9 were relatively low for predicting the preterm and term labor (59, 73). Our hypothesis of the involve- risk of SPTB, and CXCL9 is only one of the several cytokines ment of CXCR3 as a receptor augmenting the SPTB-predisposing whose concentrations associate with SPTB. Based on these find- cytokine signaling requires further mechanistic studies, including ings, we speculate that high CXCR3 expression and increased transgenic techniques that, however, are complicated by high cytokine response may predispose to SPTB. Because SPTB- species specificity of the labor process (10). predisposing adverse events (e.g., from infections and inflamma- In conclusion, we found an association between a fetal CXCR3 tory diseases) often generate a cytokine response and this response polymorphism and SPTB and provide evidence of an association is transmitted to the fetal compartment, the processing and am- between this polymorphism and both the expression levels of plification of the signal at the interface between fetal extraem- CXCR3 and the levels of its ligand CXCL9. Furthermore, we bryonic tissue and the maternal decidua may be critical. We tested provide preliminary experimental evidence about the role of this hypothesis by using an experimental model of preterm birth CXCR3 in enhancing the expression of labor-associated cytokines.

Table V. Amniotic ﬂuid cytokine levels of WT and CXCR3-deﬁcient fetuses after maternal LPS or PBS injections

WT Mice CXCR3-Deﬁcient Mice

Cytokine LPS PBS LPS/PBS p LPS PBS LPS/PBS p CCL2 (pg/ml) 4611 6 1531a 3114 6 3774a 1.5 0.338 2564 6 3187a 6042 6 3564a 0.42 0.001 IL-6 (pg/ml) 232 6 120a 197 6 371a 1.2 0.139 127 6 126a 451 6 376a 0.28 0.001 Levels of CCL2 and IL-6 in amniotic ﬂuid of WT and CXCR3-deﬁcient fetuses after maternal LPS (20 mg/mouse) or PBS injections at 17 dpc are shown. aMean 6 SD. The Journal of Immunology 2197

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