www.nature.com/scientificreports OPEN Histological Chorioamnionitis Induces Diferential Gene Expression in Human Cord Blood Received: 9 October 2018 Accepted: 21 March 2019 Mononuclear Leukocytes from Term Published: xx xx xxxx Neonates Suhita Gayen nee’ Betal1, Swati Murthy1, Michael Favara1, Gina Fong1, Joanna S. Y. Chan2, Sankar Addya3, Thomas H. Shafer1, Jay Greenspan1, Vineet Bhandari 4, Irfan Rahman 5 & Zubair H. Aghai1 Histological chorioamnionitis (HCA) is an infection of fetal membranes and complicates 5.2% to 28.5% of all live births. HCA is associated with increased mortality and morbidity in both premature and term neonates. Exposure to HCA may have long-term consequences, including an increased risk for allergic disorders and asthma later in childhood, the mechanism(s) of which are still not yet well understood. The objective of this study was to determine the mRNA transcriptome of cord blood mononuclear leukocytes from term neonates to identify key genes and pathways involved in HCA. We found 366 diferentially expressed probe IDs with exposure to HCA (198 upregulated, 168 downregulated). These transcriptomes included novel genes and pathways associated with exposure to HCA. The diferential gene expression included key genes regulating infammatory, immune, respiratory and neurological pathways, which may contribute to disorders in those pathways in neonates exposed to HCA. Our data may lead to understanding of the role of key genes and pathways identifed on the long-term sequelae related to exposure to HCA, as well as to identifying potential markers and therapies to prevent HCA- associated complications. Chorioamnionitis (CA) is an infection of the fetal membranes and placenta that complicates 5.2% of all live births1. Te prevalence of histological CA (HCA) in term neonates with spontaneous labor is 23.6–28.7%2,3. Approximately 500,000 to one million infants in the United States are born each year to mothers diagnosed with HCA. Exposure to HCA may have long-term consequences, including an increased risk for allergic disorders and asthma later in childhood4–6. Additionally, studies have demonstrated that exposure to HCA can also lead to developmental delay and cerebral palsy in preterm and term infants7–9. Asthma and other allergic chronic diseases during childhood afect greater than 7 million children in the United States10. Although HCA is associ- ated with the development of asthma, allergic disorders, and neurodevelopmental impairment, the exact mecha- nism for these complications is unknown. Exposure to infection during late fetal life is likely to incite epigenetic changes, which may modulate the immune and neurological systems and increase the risk for development of neurological and allergic disorders later in life. Gene expression studies of cells and tissues have become a major tool for discovery in the pathogenesis of var- ious diseases. Global gene expression by transcriptomic analysis can uncover gene signatures and help delineate molecular pathways involved in the development of asthma, allergy, and neurodevelopmental impairment in neo- nates born to mothers with HCA. Previous studies have reported diferential gene expression patterns associated 1Neonatology, Thomas Jeferson University/Nemours, Philadelphia, PA, USA. 2Department of Pathology, Thomas Jefferson University, Philadelphia, PA, USA. 3Cancer Genomics and Bioinformatics Laboratory, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA. 4Section of Neonatology, Department of Pediatrics, St. Christopher’s Hospital for Children, Drexel University College of Medicine, Philadelphia, PA, USA. 5Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY, United States. Correspondence and requests for materials should be addressed to Z.H.A. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:5862 | https://doi.org/10.1038/s41598-019-42205-x 1 www.nature.com/scientificreports/ www.nature.com/scientificreports HCA Group Control Group Fold ProbeID Gene Symbol Average Expression Average Expression Change Up/Down P-value TC17001604.hg.1 LRRC37A4P 1389.16 344.89 4.01 0.0245 TC04002938.hg.1 LOC285505 364.56 136.24 2.68 0.012 TC07000288.hg.1 LINC01061 1652 643.59 2.58 0.0117 TC03000255.hg.1 CCR2 765.36 304.44 2.51 0.0417 TC04001682.hg.1 FAM198B 643.59 256 2.5 0.0167 Up TC17000604.hg.1 LRRC37A2 13216.02 5556.65 2.37 0.0312 TC07001299.hg.1 TRGV4 140.07 60.13 2.32 0.0002 TC07001300.hg.1 TRGV3 179.77 79.89 2.24 4.50xE-05 TC04002939.hg.1 TMEM144 240.52 112.99 2.12 0.0173 TC17001603.hg.1 LRRC37A4P; LRRC37A2 20594.91 9946.68 2.08 0.0478 TC01003638.hg.1 PTGS2 252.48 9089.59 −35.91 0.0205 TC04002953.hg.1 AREG 313 1884.54 −6.03 0.001 TC01001749.hg.1 G0S2 2352.53 13034.07 −5.54 0.008 TC05001854.hg.1 HBEGF 385.34 2005.85 −5.18 0.0022 TC19001593.hg.1 PLAUR 1448.15 7082.29 −4.89 0.0138 Down TC21001069.hg.1 SAMSN1 3956.48 17438.64 −4.42 0.0257 TC12001216.hg.1 OLR1 178.53 765.36 −4.27 0.0365 TC03000276.hg.1 CAMP 657.11 2683.69 −4.09 0.0011 TC09000508.hg.1 NR4A3 143.01 580.04 −4.07 0.0128 TC03001357.hg.1 LTF 2683.69 9345.14 −3.49 0.0208 Table 1. Top 10 diferentially up- or down-regulated genes afer exposure to HCA. with preterm labor and lipopolysaccharide (LPS) stimulation of cord blood leukocytes11,12. More recently, difer- ential gene expression in whole blood from preterm neonates exposed to HCA has been published13. However, fetal infammatory and immune response to microbial infection difer between preterm (born before 37 weeks gestation) and term infants14–16. Te efects of HCA on gene expression profle on cord blood mononuclear leuko- cytes in human term neonates have not been characterized. Our objective was to determine the mRNA transcrip- tome of cord blood mononuclear leukocytes from term neonates and identify key genes and pathways involved in HCA. Results Ten term infants were enrolled in the study. Five infants had histological chorioamnionitis (HCA group), and fve infants with no evidence of histological chorioamnionitis on placental histopathology served as the control group. Diferential Gene Expression. Comparison of the HCA group array data with the control group using transcriptome array console software revealed that 366 probe IDs were differentially expressed with a fold change ≥ 1.5 (p < 0.05). Of these, 198 probe IDs were signifcantly up-regulated (Supplementary Table 1), 60 with annotated genes and 138 non-annotated genes. One hundred and sixty-eight probe IDs were signifcantly down-regulated (Supplementary Table 2), of which 105 had annotation with gene symbols and 63 were not anno- tated. Te top 10 up- and down-regulated genes based on the fold change are reported in Table 1. Te top 10 up-regulated genes included chemokine (C-C motif) receptor-2 (CCR2), a pro-infammatory chemokine impor- tant in infammatory diseases including asthma17,18. Te top down-regulated genes included two important anti- microbial proteins, lactoferrin (LTF) and cathelicidin antimicrobial peptide (CAMP). Differential Gene Expressions Related to Immune Response. Thirty genes related to immune response and important for regulation of infammatory pathways were identifed (Table 2). Twelve genes were up-regulated and 18 genes were down-regulated. Te up-regulated genes have been previously associated with asthma; hyper-reactivity and allergy (CCR2, HRH2, PTEN); lung development and apoptosis (CHRNA7, RTKN2); and immune function (GIMAP4, GIMAP7, KLRC3 and CD99) (Table 3). Similarly, the down-regulated genes have roles in asthma and airway modeling (PTGS2, AREG, HBEGF, PLAUR, CAMP, LTF, FCAR, PDE4B, ZNF331 and IRAK2) and immune function (NR4A3, NFIL3, ADGRE3, TARM1 and ZC3H12A). Te down-regulated gene CXCL1 has a role in lung development and BPD (Table 4). Ingenuity Pathway Analysis. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) sofware (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) by loading 366 probe sets that were diferentially expressed with HCA19. Seventy-three functions were modifed by diferen- tial gene expression with exposure to HCA. Important functions modifed with the exposure to HCA are listed in Table 5. Te modifcation of functions with HCA included genes related to immune system, infammatory response, connective tissue disorders, neurodevelopmental disorders, hematological development and disorders, and respiratory development and disorders. Ingenuity Canonical Pathway Analysis identifed 207 pathways that SCIENTIFIC REPORTS | (2019) 9:5862 | https://doi.org/10.1038/s41598-019-42205-x 2 www.nature.com/scientificreports/ www.nature.com/scientificreports HCA Group Control Group Gene Symbol Up/Down Fold Change P-value Average Expression Average Expression CCR2 2.51 0.0417 765.36 304.44 TRGV4 2.32 0.0002 140.07 60.13 GIMAP4 2.29 0.0118 52498.92 23010.42 TRGV3 2.24 0.000045 179.77 79.89 KLRC3 1.8 0.0433 1458.23 809.00 HRH2 1.74 0.0233 1734.13 1002.93 CHRNA7 1.61 0.0201 33.36 20.68 Up-regulated Genes GIMAP7 1.61 0.0086 4482.23 2778.33 RTKN2 1.59 0.0294 153.28 96.34 CD99 1.56 0.0121 2646.74 1698.45 PTEN 1.55 0.0275 694.58 448.82 GIMAP1- GIMAP5; 1.53 0.0155 989.12 648.07 GIMAP5; GIMAP1 PTGS2 −35.91 0.0205 252.48 9089.59 AREG −6.03 0.001 313.00 1884.54 HBEGF −5.18 0.0022 385.34 2005.85 PLAUR −4.89 0.0138 1448.15 7082.29 CAMP −4.09 0.0011 657.11 2683.69 NR4A3 −4.07 0.0128 143.01 580.04 LTF −3.49 0.0208 2683.69 9345.14 NFIL3 −2.77 0.0262 1192.69 3304.00 FCAR −2.35 0.0023 2256.70 5330.30 Down-regulated Genes PDE4B −2.33 0.0125 280.14 652.58 ADGRE3 −2.3 0.042 680.29 1573.76 CXCL1 −2.05 0.0404 112.21 229.13 TARM1 −2.02 0.0059 116.16 235.57 SNAI1 −1.93 0.0083 47.18 91.14 ZNF331 −1.77 0.0308 288.01 512.00 ZC3H12A −1.76 0.0422 256.00 451.94 IRAK2 −1.74 0.048 164.28 284.05 NFKBIE −1.59 0.0351 413.00 657.11 Table 2.