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Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates
Department of Immunology, University of Washington, Seattle, WADepartment of Global Health, University of Washington, Seattle, WACenter for Innate Immunity and Immune Disease, University of Washington, Seattle, WA
Department of Pediatrics, University of Washington, Seattle, WADepartment of Global Health, University of Washington, Seattle, WACenter for Innate Immunity and Immune Disease, University of Washington, Seattle, WACenter for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA
Department of Obstetrics & Gynecology, University of Washington, Seattle, WADepartment of Global Health, University of Washington, Seattle, WACenter for Innate Immunity and Immune Disease, University of Washington, Seattle, WASahlgrenska Academy, Gothenburg, Sweden
Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease.
Objective
The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development.
Study Design
We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum.
Results
Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05).
Conclusion
Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.
Infection is often subclinical and thought to ascend from the lower genital tract that allows microbes to invade the placenta and amniotic fluid, which can lead to fetal bacteremia and sepsis. The fetal inflammatory response syndrome describes a condition of severe fetal inflammation that often occurs with fetal infection. The fetal inflammatory response syndrome is the counterpart to the adult condition (systemic inflammatory response syndrome) and is associated with an increased risk for multisystem fetal organ injury.
Studies have focused mainly on the relationship between the fetal inflammatory response syndrome and injury to the fetal lungs and brain, because they are often imaged and assessed postnatally. Inflammatory injury to other organs, which includes the fetal heart, has been hypothesized to occur but is more challenging to demonstrate in human neonates. Although many studies have associated prematurity, low birth weight, or fetal growth restriction with cardiovascular risk factors and heart disease later in life, the impact of perinatal infection and inflammation on fetal cardiac development is unknown.
Birth weight and systolic blood pressure in adolescence and adulthood: meta-regression analysis of sex- and age-specific results from 20 Nordic studies.
Accumulating evidence in humans and preterm sheep models implicates infection and fetal inflammation in altered fetal cardiac function. Fetal heart rate disturbances (eg, absence of variability, arrhythmias, cardiac dysfunction) have been associated with chorioamnionitis, which is an inflammation of the placental membranes often caused by infection.
In fetuses from pregnancies with preterm premature rupture of membranes, a condition often complicated by microbial invasion of the amniotic cavity, fetal echocardiography has revealed changes in diastolic ventricular function, which may increase cardiac output.
In a similar cohort, strain imaging to evaluate right ventricular function found evidence for impairment of systolic and diastolic function and, in cases with funisitis (umbilical cord inflammation), dyskinesia of the right ventricle.
These findings are consistent with observations in preterm sheep models of intraamniotic infection (Candida albicans) or inflammation (lipopolysaccharide), in which fetal inflammation was associated with a reduction in mean arterial blood pressure and oxygen saturation, depressed ventricular contractility, diastolic dysfunction, and a reduction in cardiomyocyte numbers.
The mechanism that links inflammation and fetal cardiac injury is unknown and challenging to elucidate in human neonates and sheep models for ethical reasons and the lack of genomic tools, respectively.
Our objective was to identify early biologic events in the fetal heart that occur after intrauterine infection and development of the fetal inflammatory response syndrome in a nonhuman primate. We hypothesized that development of the fetal inflammatory response syndrome is associated with fetal cardiac inflammation and changes in the gene program responsible for cardiac morphogenesis, analogous to observations that we have made on the effects of intraamniotic inflammation on fetal lung development.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council and the Weatherall report, “The use of non-human primates in research.” The protocol was approved by the Institutional Animal Care Use Committee of the University of Washington (Permit Number: 4165-01). All surgery was performed with general anesthesia, and all efforts were made to minimize discomfort.
Animals and study groups
Cases that met criteria for the fetal inflammatory response syndrome were identified retrospectively in our pregnant nonhuman primate infection program based on an elevated fetal interleukin-6 (IL-6) level (>11 pg/mL; n=5) and compared with saline controls (n=5). Fetal cardiac microarray analyses were performed on animals that were inoculated with either (1) 1–3×108 colony-forming units (CFU) of a hyperhemolytic and hypervirulent group B streptococcus (GBS) strain (GBSΔcovR; n=4) into the choriodecidual space,
(2) 5×104 CFU of E coli RS218 into the amniotic fluid (prototypic strain that causes neonatal meningitis; n=1), or (3) saline solution into the amniotic fluid and choriodecidual space (n=5)
; citations indicate publications that describe the animal experiments and pregnancy outcomes, but fetal cardiac transcriptomics and IL-1B/IL-6/IL-8 were not analyzed or reported previously. Because fetal cardiac tissue from the aforementioned saline controls was not saved to allow for protein (cytokine) analysis, an additional 4 saline controls were performed to enable the comparison of cytokines from fetal cardiac tissues of saline controls with fetal inflammatory response syndrome cases.
In our model, pregnant pigtail macaques were time mated, and fetal age was determined with the use of early ultrasound scans. Temperature in the animal quarters was maintained at 72–82°F. Animals were fed a commercial monkey diet, supplemented daily with fruits and vegetable; drinking water was always available. The animal was first conditioned to a nylon jacket/tether system for several weeks before surgery, which allows free movement within the cage but protected the catheters. On days 116–125 of pregnancy (term=172 days), catheters were implanted surgically via laparotomy into the maternal femoral artery and vein, amniotic cavity, and choriodecidual interface in the lower uterine segment (between uterine muscle and fetal membranes, external to the amniotic cavity). In the E coli case and saline controls, an additional catheter was implanted into the fetal internal jugular vein. Fetal electrocardiography electrodes and a maternal temperature probe were also implanted. Postoperative analgesia was provided by a 25-μg fentanyl patch that was applied the day before surgery, in addition to postoperative indomethacin. After 48 hours, the animals appeared to have recovered from surgery based on a return to baseline for activity, appetite, and bowel function.
After surgery, the animal was placed in the jacket and tether with the catheters/electrodes tracked through the tether system. Cefazolin and terbutaline sulfate were administered to reduce postoperative infection risk and uterine activity. Both cefazolin and terbutaline were stopped at least 72 hours before experimental start (approximately 13 half-lives for terbutaline, 40 half-lives for cefazolin, >97% of both drugs eliminated), which represented approximately a 7–10 day period of postoperative terbutaline administration. Cefazolin (1 g) was administered intravenously each day in saline controls to minimize the possibility of a catheter-related infection. Experiments began approximately 2 weeks after catheterization surgery to allow recovery (approximately 30–31 weeks human gestation).
At our center, term gestation in the noninstrumented pigtail macaque population averages 172 days.
Intraamniotic pressure was recorded, digitized, and analyzed continuously by previously described methods. The integrated area under the intrauterine pressure curve was used as a measure of uterine activity and was reported as the hourly contraction area (millimeters of mercury•seconds per hour) over 24 hours. Preterm labor was defined as >10,000 mmHg•sec/hr associated with a change in cervical effacement or dilation.
Histology
After cesarean delivery, fetal necropsy was performed in all animals; the heart was preserved in RNALater (Thermo Fisher Scientific Inc, Waltham, MA). Histopathologic examination was performed on fetal cardiac samples. A pathologist (A.B.) examined hematoxylin and eosin–stained, full-thickness paraffin sections of the fetal hearts to evaluate inflammation, necrosis, fetal vascular thrombosis, or other histopathologic findings.
Cytokine analysis
IL-6 was assayed from cardiac tissues (nonhuman primate) and umbilical cord plasma (nonhuman primate and human) with the use of Luminex multiplex cytokine kits (Millipore, Billerica, MA). Fetal cardiac tissue was not available for protein analysis from the E coli infection, which left 4 samples (GBSΔcovR) for comparison with controls (n=4).
Bacterial quantitation
Amniotic fluid and maternal blood were sampled frequently (ie, before [–24 and –0.25 hrs] and after bacterial inoculation [+0.75, +6, +12, +24 hrs] and then every 12 hrs until repeat cesarean section for fetal necropsy). At the time of cesarean section, we also collected fetal blood, fetal heart tissues, and swabs from the fetal lungs, meninges, and chorioamnion (placental membranes). For enumeration of GBS and E coli from amniotic fluid or fetal blood (100 μL), serial 10-fold dilutions were plated on trypticase soy agar (GBS) or Columbia agar with 5% sheep blood (E coli), respectively. Fetal heart tissues from GBS cases were weighed at necropsy, homogenized in sterile phosphate-buffered saline solution and 10-fold serial dilutions were plated on trypticase soy agar and incubated overnight at 37°C, 5% CO2, and enumerated the next day as described.
Other plates were incubated 24 hours at 37°C (GBS) or 72 hours at 35°C (E coli), 5% CO2, and enumerated. Swabs that were collected from tissues were aseptically removed from Port-A-Cul vials (Fisher Scientific, Pittsburgh, PA) and streaked to the same media as for blood or fluid. No bacteria were recovered from amniotic fluid, fetal blood, or fetal heart tissues from controls.
For cytokine and prostaglandin analysis, amniotic fluid and blood samples were collected in EDTA tubes. Samples were centrifuged for 5 min at 1200 rpm immediately after collection, and the supernatant was frozen and stored at –80°C. Before freezing, indomethacin (0.3 mmol/L) was added to the samples saved for cytokine and prostaglandin quantification, respectively. Fetal heart cytokine analysis was performed on tissue homogenates that were diluted 1:1 in lysis buffer (150 mmol/L NaCl, 15 mmol/L Tris, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 1% Triton X-100, supplemented with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and incubated overnight at 4°C. Lysates were then centrifuged at 4000 rpm for 5 min at 4°C, and supernatants stored at −80°C or used immediately for analysis. Approximately 100 μL of the sample was used in Luminex or enzyme-linked immunosorbent assays, as described earlier.
RNA extraction and microarray processing
To study genetic pathways in Macaca nemestrina, we used the Affymetrix Cynomolgus Array (GeneChip Cynomolgus Gene 1.0 ST; Affymetrix, Santa Clara, CA), which allows interrogation of 40,096 gene-level probe sets based on the M fascicularis genome. M nemestrina and M fascicularis are closely related macaque species and diverged within the last 5–6 million years.
RNA extraction was performed with the use of miRNeasy mini kits (Qiagen, Valencia, CA) according to the manufacturer’s established protocol for purification of total RNA from animal tissues. RNA integrity was assessed with an Agilent Bioanalyzer instrument (Agilent, Santa Clara, CA) and was judged by distinct and sharp 18s and 28s ribosomal RNA peaks that were baseline separated. RNA quantity was determined by measuring OD260 with a Thermo Scientific NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Inc). The NanoDrop instrument was also used to determine purity of RNA samples by measuring OD260/280 and OD260/230 ratios. Only samples that passed this stringent quality control were processed further. Processing of the RNA samples was carried out according to the GeneChip Whole Transcript Sense Target labeling protocol (Affymetrix). Briefly, double-stranded complementary DNA (cDNA) was synthesized with random hexamers that were tagged with a T7 promoter sequence. The double-stranded cDNA subsequently was used as a template and amplified by T7 RNA polymerase that produced many copies of antisense complementary RNA. In the second cycle of cDNA synthesis, random hexamers were used to prune reverse transcription of the complementary RNA from the first cycle to produce single-stranded DNA in the sense orientation. To reproducibly fragment the single-stranded DNA and improve the robustness of the assay, deoxyuridine triphosphate was incorporated in the DNA during the second cycle first-strand reverse transcription reaction. This single-stranded DNA sample was then treated with a combination of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 that specifically recognizes the unnatural deoxyuridine triphosphate residues and breaks the DNA strand. DNA was labeled by terminal deoxynucleotidyltransferase with the proprietary DNA Labeling Reagent (Affymetrix) that is covalently linked to biotin. The biotin-labeled DNA fragments were hybridized to GeneChip Cynomolgus Gene 1.0 ST arrays (Affymetrix), washed, and stained with fluorescent anti–streptavidin biotinylated antibody. After an additional wash step, the arrays were scanned with an GeneChip 3000 scanner (Affymetrix). Image generation and feature extraction was performed with GeneChip Command Console Software (Affymetrix).
Single gene analysis of microarray data
The microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/index.cgi; GSE98459). Analysis of the microarray data focused first on differential expression of single genes, which we refer to as the “ANOVA (analysis of variance) model.” Raw microarray data were preprocessed and analyzed with various Bioconductor packages (http://www.bioconductor.org/).
Several quality control steps were carried out to ensure data were of high quality: (1) visual inspection of the GeneChip Operating Software DAT chip images, (2) visual inspection of the chip pseudo-images generated by the Bioconductor oligo package, (3) generation and inspection histograms of raw signal intensities, and (4) generation and comparison of the Relative Log Expression and Normalized Unscaled Standard Errors using the Bioconductor oligo package. The data were normalized with the Bioconductor oligo package
We did not filter out any probe sets before analysis. From the normalized data, genes with significant evidence for differential expression were identified with the use of the limma package in Bioconductor.
Changes in expression were inferred with the use of a weighted t-test in conjunction with an empiric Bayes method to moderate the standard errors of the estimated log-fold changes. Array weights were estimated by computing the relative variability of each array and then using the inverse of the variance to weight each array.
This allows smooth adjustment for array quality without having to exclude samples. We selected genes based on an unadjusted probability value of <.05 and a 1.5-fold difference between groups.
We also fit a conventional linear model using IL-6 concentrations (log base 2) as a marker for infection severity, which we refer to as the “IL-6 model.” In this model, we excluded 2 saline controls for which we did not have an IL-6 level. We selected genes with an unadjusted probability value of <.05 and an absolute slope of >0.07. This slope can be interpreted as a 5% change in expression for every doubling of IL-6 concentration. Given that the range of observed IL-6 concentrations is >14 logs (eg, >14 doublings in concentration), this represents an approximate 1.6-fold change in expression, at minimum, over the entire range of observed IL-6 concentrations.
Gene set analysis
Next, the data were analyzed with gene set tests to investigate categories of genes, using the romer function from the Bioconductor limma package. The romer function is intended to perform a competitive test against a battery of gene sets, assessing the statistical significance of predefined gene sets/pathways as a whole rather than of single genes. This method facilitates identification of modest but concordant changes in expression of a set of genes that may be missed by single gene analysis. Gene set testing considers all the genes in the experiment and allows for the identification of gene sets that are more highly ranked, as a set, than would be expected under the null hypothesis. The probability values are based on random rotations of the residuals (9,999 random rotations), which is similar to conventional permutation tests but permits unlimited numbers of permutations to be tested. In addition, the romer function works with any linear model, not just ANOVA; we used the linear regression function against IL-6 concentration for gene set testing and Ingenuity Pathway Analysis (Qiagen, Valencia, CA). In the IL-6 model, we excluded 2 controls for which fetal plasma was not obtained and the IL-6 level was unknown (saline 1 and 3; Table 1). We used the Gene Ontology and Hallmark gene set collections from the Broad Institute (http://software.broadinstitute.org/gsea/msigdb).
Presents the mean (standard error of the mean) for amniotic fluid and fetal plasma peak cytokines; histopathologic evidence of inflammation in the placental membranes (chorioamnionitis) was diagnosed using Redline criteria89; with the exception of Escherichia coli 1, the remaining data has been published previously26-28 and are presented to provide context for the fetal cardiac transcriptomics and cytokine data
In 2 saline controls, fetal plasma was not obtained.
No
No
No growth
4
0.9
182.3
No
No
No growth
5
2.3
223.0
No
No
No growth
Mitchell et al. Disruption of fetal cardiac gene networks with fetal infection and inflammation. Am J Obstet Gynecol 2018.
a Presents the mean (standard error of the mean) for amniotic fluid and fetal plasma peak cytokines; histopathologic evidence of inflammation in the placental membranes (chorioamnionitis) was diagnosed using Redline criteria
we created a self-contained gene set using 36 of the 41 validated genes from the fetal blood transcriptome study that could be mapped to our nonhuman primate array. Using the roast function from the Bioconductor limma package, we tested whether the 36 genes in the set were, on average, expressed differentially.
Ingenuity Pathway Analysis
The Core analysis feature of the Ingenuity Pathway Analysis software (Ingenuity Systems, www.ingenuity.com [Qiagen]) was used to discover pathways and transcriptional networks in the gene expression microarray data from the IL-6 model. The functional analysis feature identified the biologic functions and/or diseases that were most significant to the data set. Given that the range of observed IL-6 concentrations is >14 logs, this represents an approximate 1.6-fold change in expression, at minimum, over the entire range of observed IL-6 concentrations. Ingenuity Pathway Analysis uses a right-tailed Fisher’s exact test to calculate a probability value to determine the probability that each biologic function and/or disease that is assigned to that data set is due to chance alone. Molecules are represented as nodes, and the biologic relationship between 2 nodes is represented as an edge (line). All edges are supported by at least 1 reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Knowledge Base (Qiagen). Human, mouse, and rat orthologs of a gene are stored as separate objects in the Ingenuity Knowledge Base but are represented as a single node in the network. The intensity of the node color indicates the degree of up- or downregulation. Nodes are displayed as various shapes that represent the functional class of the gene product. Edges are displayed with labels that describe the nature of the relationship between the nodes. Ingenuity Pathway Analysis also allows prediction of the activation or inhibition of transcription factors involved in the gene expression patterns seen in our study.
Validation of cDNA microarray by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
We used qRT-PCR to validate expression changes of genes of interest that had been identified by microarray analysis. Briefly, reverse transcription was performed according to the manufacturer’s established protocol using total RNA and the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). For gene expression measurements, 2 μL of cDNA was included in a PCR (12 μL final volume) that also consisted of the ABI TaqMan Gene Expression Assays mix and the TaqMan Gene Expression Master Mix (Applied Biosystems, ThermoFisher Scientific, Foster City, CA) according to the manufacturer’s protocol. Amplification and detection of PCR amplicons were performed with the ABI PRISM 7900 system (Applied Biosystems Inc, Foster City, CA) with the following PCR profile: 1 cycle of 95°C for 10 min, 40 cycles of 95°C for 30 sec, and 60°C for 1 min. GAPDH amplification plots derived from serial dilutions of an established reference sample were used to create a linear regression formula to calculate expression levels, and β-actin gene expression levels were used as an internal control to normalize the data.
Statistical analysis
Statistical analyses related to the transcriptomics data were detailed earlier in the respective sections. We used a t-test for the analysis of real-time PCR data. A Mann-Whitney test was also performed to test comparisons between cases and controls for cytokine quantities.
Results
Preterm labor and fetal cardiac infection in a nonhuman primate model of severe fetal inflammation
To understand biologic events induced by inflammation in the fetal heart, we used a unique, chronically catheterized, nonhuman primate model (pigtail macaque, M nemestrina) of infection-induced preterm labor. From our pregnancy infection program, we retrospectively identified 5 cases with severe fetal inflammation that was consistent with the fetal inflammatory response syndrome (GBS, 4; E coli, 1; fetal plasma IL-6, 157–29,294 pg/mL) and 3 saline controls (Table 1). In all fetal inflammatory response syndrome cases, there was acute and severe chorioamnionitis. The E coli case was delivered in preterm labor 2.5 days after inoculation. Three of the 4 GBS cases were delivered at the time of preterm labor (0.3, 1.0, and 2.0 days after inoculation). One of the 4 GBS cases was delivered 2.0 days after inoculation without preterm labor in an effort to avoid stillbirth because of a marked change in color and turbidity of the amniotic fluid that indicated heavy bacterial growth.
In four of 5 cases, the fetal inflammatory response syndrome accompanied a disseminated bacterial infection into the fetal blood and/or organs. Bacteria were cultured and quantified from fetal heart tissues in all 4 fetal inflammatory response syndrome cases that were induced by GBS inoculation (Figure 1, A ). Although fetal heart tissue was not tested specifically for bacteria from the E coli case, 107 CFUs were recovered from both fetal lungs and blood indicated a severe fetal infection. In fetal cardiac tissues, levels of IL-6 and -8 were significantly higher in fetal inflammatory response syndrome cases than in controls (Figure 1, A; P=.03). IL-1B and tumor necrosis factor-α levels were not significantly different but approached statistical significance for tumor necrosis factor-α (Figure 1, A; P=.06). Blinded evaluation of fetal cardiac tissues did not reveal significant changes in gross disease (Supplemental Table). In the case with the highest fetal plasma IL-6 levels (induced by E coli), there were areas with cytoplasmic pallor, but otherwise neutrophilic infiltrates or significant inflammation was not observed (Figure 1, B-C).
Figure 1Bacterial quantities, cytokines and gene expression in fetal heart tissues
A, Quantitation of bacterial and cytokine protein levels in the fetal heart. Quantities of group B streptococcus and fetal cardiac cytokines in cases with the fetal inflammatory response syndrome (n=4; group B streptococcus) and controls (n=4) are shown; the asterisk indicates a probability value of <.05. B and C, Hematoxylin and eosin staining of fetal cardiac tissues is shown to demonstrate cytoplasmic pallor in the Escherichia coli case (B) compared to a saline control (C), but otherwise there was an absence of neutrophilic infiltrates or significant inflammation. D, Comparison of the microarray and quantitative reverse transcriptase polymerase chain reaction analysis. The x-axis represents individual genes, and the y-axis represents fold-change in expression by either microarray (gray bars) or quantitative reverse transcriptase polymerase chain reaction (black bars). All genes shown were significant in the unadjusted microarray analysis. Nine of 10 genes selected for validation by quantitative reverse transcriptase polymerase chain reaction were significantly up- or downregulated; the asterisk indicates a probability value of <.05; 2-sided t-test). E, A heatmap is shown to depict gene expression for the “cellular morphogenesis during differentiation” gene set (P=.008; Table 3).
Fetal cardiac single gene analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation
To identify changes in the fetal cardiac gene program, we performed a single gene analysis of microarray results by 2 methods: (1) ANOVA (case-control comparison) and (2) linear regression that correlated fetal plasma IL-6 to gene expression, regardless of experimental group (IL-6 model). In the ANOVA analysis, 609 probe sets differentially were expressed >1.5-fold (P<.05) between fetal inflammatory response syndrome cases and controls. According to the IL-6 model, 1162 probe sets were significantly differentially expressed (P<.05). Genes significantly downregulated in both types of analyses included SFRP4, NPPA, PLA2G7, GRIA3, MTHFR, EPHA4, and ANGPTL7 (ANOVA: log2 fold changes ranged from –2.7 to –1.3 [P<.05]; IL-6 model: P<.02; Table 2, Supplemental Figure). In both analyses, genes that were upregulated significantly included S100A8, FAM69C, PLA2G2, CFTR, STEAP4, and IRX4 (case-control: log2 fold changes ranged from 1.2–2.6 [P<.05]; IL-6 model: P<.02; Table 2). Of the 609 significant genes from the ANOVA analysis, 452 were also significant in the IL-6 model (with consistent direction of change). The probability that this would occur by chance, under a hypergeometric null distribution, corresponds to a probability value of <1×10–16. To validate our microarray data, we performed qRT-PCR on 10 genes of interest. Nine of 10 genes were significantly differentially expressed between fetal inflammatory response syndrome cases and controls: SFRP4, NPPA, MAL2, ANGPTL7, FAM69C, ACE2, CFTR, STEAP4 and IRX4 (P<.05; Figure 1, D).
Table 2Select differentially expressed probe sets in the fetal heart of animals that develop the fetal inflammatory response syndrome vs saline controls
To explore relationships among differentially expressed genes, we identified gene sets and pathways with concordant changes in expression by performing gene set analysis for data obtained with the IL-6 model. Multiple gene sets related to morphogenesis that were significantly downregulated with increasing IL-6 level included cellular morphogenesis during differentiation (P=.008; Figure 1, E), neuron differentiation (P=.008), axonogenesis (P=.008), and regulation of DNA replication (P=.03; Table 3). To place the findings of disruptions in gene networks for cellular morphogenesis within a visual construct, the Ingenuity Pathway Core Analysis feature was used to map functional networks of relevant genes. We also used the data obtained with the IL-6 model as input for the Ingenuity Pathway Analysis. Multiple pathways that were regulated differentially within the Ingenuity Pathway Analysis “Diseases and Biological Functions” category were related to cardiac morphogenesis and vasculogenesis, which included morphologic evaluation of the cardiovascular system (Figure 2), cellular movement and migration of cells, growth of smooth muscle, morphologic evaluation of vessel, growth of muscle tissue, and vasculogenesis and migration of endothelial cells (Table 3; probability value range: 4×10–8 to 7×10–21). Ingenuity Pathway Analysis also has the capability to predict activation states of transcriptional regulators based on the activation or suppression of downstream genes. The top transcription factors that predicted to be associated with changes in gene expression were tumor necrosis factor, transforming growth factor beta 1, and IL-1B (probability value range: 2×10–10 to 2×10–14). These data suggest that multiple pathways related to fetal cardiac morphogenesis may be impacted by the development of the fetal inflammatory response syndrome and/or fetal bacteremia.
Table 3Select differentially expressed gene sets and Ingenuity Pathway Analysis (Qiagen, Valencia, CA) pathways that are downregulated in the fetal heart of animals with the fetal inflammatory response syndrome
The Ingenuity Pathway Functional Analysis of a Network identified biologic functions and/or diseases most significant to the molecules in the network with the use of a right-tailed Fisher’s exact test
Transcription factor analysis is based on previous knowledge of expected effects between transcription factors and their target genes stored in the Ingenuity Pathway Analysis library; the overlap probability value measures whether there is a statistically significant overlap between the dataset genes and the genes that are regulated by a transcription factor with the use of the Fisher’s Exact Test.
Predicted activation state
Activation Z-score
P value of overlap
Top transcription factors
Tumor necrosis factor
Activated
4.2
2×10–14
Interleukin-1β
Activated
3.7
1×10–11
Transforming growth factor β1
Inhibited
–2.0
2×10–10
Mitchell et al. Disruption of fetal cardiac gene networks with fetal infection and inflammation. Am J Obstet Gynecol 2018.
a Gene set and Ingenuity Pathway Analyses were based on the interleukin-6 model and select results are reported in the Table
b The Ingenuity Pathway Functional Analysis of a Network identified biologic functions and/or diseases most significant to the molecules in the network with the use of a right-tailed Fisher’s exact test
c Transcription factor analysis is based on previous knowledge of expected effects between transcription factors and their target genes stored in the Ingenuity Pathway Analysis library; the overlap probability value measures whether there is a statistically significant overlap between the dataset genes and the genes that are regulated by a transcription factor with the use of the Fisher’s Exact Test.
A, Heatmap of gene expression for the Ingenuity Pathway Analysis (Qiagen, Valencia, CA) gene set, “morphology of cardiovascular system”, is shown across all samples. The rows represent genes, and the columns represent either fetal inflammatory response syndrome cases (red) or controls (blue). B, A visual construct of the “morphology of cardiovascular system” gene set depicts the regulatory network of genes that were identified in the nonhuman primate fetal cardiac tissues. “Morphology of cardiovascular system” was a significantly downregulated gene set that was identified through Ingenuity Pathway Analysis in the interleukin-6 model (P=4×10–11; right-tailed Fisher’s exact test). Each node represents a gene with different colored lines that show types of connectivity and functional relationships. Green and red nodes represent significant gene down- and upregulation, respectfully. Protein-protein interactions from the Ingenuity Knowledge Base (Qiagen, Valencia, CA) were used to connect genes. Network data were then exported into Cytoscape (version 3.2.1) to make custom network figures.
Mitchell et al. Disruption of fetal cardiac gene networks with fetal infection and inflammation. Am J Obstet Gynecol 2018.
and provides an opportunity to compare gene expression directly in different fetal compartments between human and nonhuman primate fetuses. Thirty-six of the 41 validated genes reported by Madsen-Bouterse et al could be mapped to our nonhuman primate array. A self-contained gene set test was then used to determine whether any of the 36 genes that were identified in the human fetal blood transcriptome were expressed differentially in the nonhuman primate fetal cardiac tissues. We observed that 33% of these genes were upregulated in our ANOVA model (P=.04) and that 44% were upregulated in the IL-6 model (P=.008). Overall, there was significant overlap between the transcriptomes that are derived from human blood and nonhuman primate cardiac tissues in the setting of severe fetal inflammation.
Comment
Principal findings of the study
Our study is the first to demonstrate that fetal infection and inflammation were associated with changes in the cardiac developmental gene program that involved a disruption in gene networks for morphogenesis and vasculogenesis in a nonhuman primate model of infection-associated preterm labor (conceptual model; Figure 3). Principal findings of the study are (1) proinflammatory cytokines (IL-6, IL-8) are elevated in the fetal myocardium without significant evidence of histopathologic inflammation during an early stage of the infection and inflammatory response, (2) a consistent set of genes is regulated differentially with fetal infection and fetal inflammatory response syndrome by either case-control analysis or correlation with IL-6 levels (eg, NPPA), and (3) genes differentially expressed in the nonhuman primate fetal heart are involved in cardiac morphogenesis and vasculogenesis.
Figure 3Conceptual model of events leading to fetal cardiac injury in utero
First, bacteria from the vagina traffic into the choriodecidual space, which was modeled in our experiments by inoculation of bacteria into this space with the use of an implanted catheter in a nonhuman primate. Inflammatory cytokines are then produced by the decidua and chorioamniotic membranes that diffuse into the amniotic fluid and fetal lung leading to a placental and fetal inflammatory response. When fetal inflammation is severe, which is typical in cases of early preterm birth complicated by infection, disruptions in the expression of gene networks for morphogenesis and vasculogenesis may occur in the fetal heart.
Mitchell et al. Disruption of fetal cardiac gene networks with fetal infection and inflammation. Am J Obstet Gynecol 2018.
Birth weight and systolic blood pressure in adolescence and adulthood: meta-regression analysis of sex- and age-specific results from 20 Nordic studies.
Our results of perturbations in the fetal cardiac developmental gene program in cases with preterm labor, infection, and the fetal inflammatory response syndrome provide a link between preterm birth and adult cardiovascular disease. During pregnancy, diastolic dysfunction has been reported in human fetuses after preterm premature rupture of membranes, which is a condition typically complicated by an intraamniotic infection.
In preterm sheep models, amniotic fluid inoculation of either lipopolysaccharide or C albicans was associated with fetal cardiac dysfunction (diastolic dysfunction, reductions in ventricular contractility and cardiomyocyte density, hypertrophy of mature cardiomyocytes).
Our work extends these observations in pregnancy by associating intraamniotic infection with the production of proinflammatory cytokine mediators in the fetal heart, severe fetal inflammation, and perturbations in the preterm cardiac developmental gene program.
The long-term effects of infectious and/or inflammatory injury on the developing fetal heart are unknown, but the idea that adverse events in fetal life might predispose towards adult disease is consistent with a body of literature often referred to as the “Barker hypothesis.”
David Barker, a British epidemiologist, proposed a causal association between intrauterine growth retardation, low birthweight, and preterm birth with the development of cardiovascular disease, hypertension, and diabetes mellitus in middle age based on a cohort in the United Kingdom.
Gestational age and growth rate of fetal mass are inversely associated with systolic blood pressure in young adults: an epidemiologic study of 165,136 Swedish men aged 18 years.
Clinical cardiovascular risk during young adulthood in offspring of hypertensive pregnancies: insights from a 20-year prospective follow-up birth cohort.
which is a major risk factor for coronary heart disease and stroke. A recent large epidemiologic study with a Swedish birth cohort found a 17-fold increased risk of heart failure in the first year of life after preterm birth (95% confidence interval, 7.96–36.3), which provides even stronger evidence for an ominous effect of preterm birth on cardiac development.
A series of physiologic and imaging studies in 102 adults who were born preterm revealed significant changes in their myocardial structure and function compared with controls.
Experiments in animal models and observations from human neonates who were born preterm also demonstrate altered cardiac structure (cardiomyocyte and cardiac hypertrophy) after preterm birth
Activation of the cardiac renin-angiotensin system in high oxygen-exposed newborn rats: angiotensin receptor blockade prevents the developmental programming of cardiac dysfunction.
Our data suggest that preterm birth complicated by severe fetal inflammation is more likely to be associated with a long-term risk of heart disease for surviving neonates.
Proinflammatory response in the fetal heart without evidence of histopathologic inflammation in the acute stages of fetal infection and the fetal inflammatory response syndrome
Neutrophils can injure cardiomyocytes through respiratory burst and oxidative injury,
but neutrophilic infiltration was minimal or absent in the heart tissues in our study, which suggests that either bacteria or cytokines played a greater role in perturbing fetal cardiac developmental gene networks. Although it is well established that bacterial cell wall components (eg, lipopolysaccharide) and inflammatory cytokines can induce direct cardiodepressive effects on adults with sepsis, evidence is only beginning to accumulate that the fetal heart is similarly vulnerable to infectious and inflammatory injury.
Polymicrobial infections are common in cases of early preterm birth, and there is evidence that cell wall fragments from gram-positive and gram-negative bacteria (eg, peptidoglycan and lipopolysaccharide) can also synergize to induce release of cytokines and injure organs.
A cell wall component from pathogenic and non-pathogenic gram-positive bacteria (peptidoglycan) synergises with endotoxin to cause the release of tumour necrosis factor-alpha, nitric oxide production, shock, and multiple organ injury/dysfunction in the rat.
The latency between bacterial inoculation and delivery was fairly short in our study (maximum, 2.5 days); therefore, fetuses exposed to infection for a longer time in utero may have a greater degree of leukocytic infiltration in their cardiac tissues, more complex mechanisms of cardiac injury, and changes in the expression of their developmental gene networks.
Differential gene expression in the fetal heart with infection and the fetal inflammatory response syndrome
A consistent set of genes was regulated differentially in association with the diagnosis of the fetal inflammatory response syndrome or rising IL-6 levels and included SFRP4, NPPA, MAL2, ANGPTL7, FAM69C, ACE2, CFTR, STEAP4, and IRX4. MYH6 was also significantly downregulated when correlated with IL-6 levels. Of these genes, NPPA and MYH6 have known roles in cardiac morphogenesis, and gene mutations have been linked to the development of arrhythmias and cardiomyopathy in the adult.
NPPA encodes atrial natriuretic peptide, which is an early and specific marker for functional myocardium of the embryonic heart; expression of NPPA is essential for formation and expansion of the cardiac chambers.
During fetal life, there is a dynamic and patterned expression of NPPA throughout the heart, and atrial natriuretic peptide suppresses proliferation of cardiomyocytes near term in response to growth-promoting stimuli.
A premature downregulation of NPPA may restrict growth potential of the fetal heart. ACE2 expression is thought to have a protective role for the heart in pathologic settings, and downregulation of ACE2 may also be harmful for development.
Several single genes with altered expression in our study have known functions in cardiac development or are implicated in heart disease or cardiac injury repair.
Downregulation of cardiac morphogenesis and vasculogenesis gene networks
Multiple gene sets, biologic pathways, and transcription factors related to morphogenesis and vasculogenesis (cellular movement, TGF-β signaling, epithelial-mesenchymal transition, cellular morphogenesis during differentiation, morphology of the cardiovascular system, and morphology of blood vessel) were disrupted in the context of fetal infection and inflammation. By gene set analysis and Ingenuity Pathway Analysis, multiple pathways related to cardiac morphogenesis and vasculogenesis were regulated differentially in cases with fetal infection and the fetal inflammatory response syndrome that suggested multiple mechanisms and pathways for injury. Although the atria and ventricles have formed by week 10 of gestation, the fetal heart continues to develop and grow until birth;
disruptions in cardiomyocyte growth patterns imposed by an infection in the late second or third trimester may restrict growth potential and lead to compensatory changes such as cardiomyocyte hypertrophy. Downregulation of vasculogenesis during fetal life is particularly important because coronary vessels continue to develop de novo in postnatal life, which is necessary to augment growth of the coronary vasculature.
There was also significant overlap between our data in nonhuman primate cardiac tissues and the human blood transcriptome from fetuses with the fetal inflammatory response syndrome;
we conclude that an overlap in gene expression between our studies reflects a shared biologic response across preterm cardiomyocytes and leukocytes to systemic infection and inflammation, particularly as leukocytes were minimal or absent in fetal cardiac tissues. Notably, a similar transcriptomic profile of downregulation in morphogenesis pathways was observed in our previous studies of gene expression in the nonhuman primate fetal lung in response to intraamniotic inflammation with a lesser degree of fetal inflammation.
The question of whether infection and fetal inflammation disrupts fetal heart development is of critical importance, because an intraamniotic infection occurs in approximately one-half of pregnancies with preterm premature rupture of membranes.
Further, an elevated plasma IL-6 level, which is diagnostic of the fetal inflammatory response syndrome, is associated with neonatal hypotension, which can be the result of low cardiac output and altered myocardial contractility.
Our study provides critical evidence that infection and severe inflammation in utero disrupts the developmental genetic program of the fetal heart before birth. The extent to which an interrupted genetic program for organ development can be restored after birth is unknown, but a number of interventions in the neonatal intensive care unit are also associated with an inflammatory stress (eg, mechanical ventilation, supplemental oxygen).
Overall, this study suggests that, in addition to risks of neonatal death and neurologic and pulmonary morbidity, some preterm infants have altered cardiac development that begins with inflammatory injury in utero.
Research implications
A nonhuman primate model of infection-associated preterm labor affords a rare opportunity to investigate the fetal origins of abnormal cardiac development that can lead to adult heart disease. Many important questions remain as to how this risk may be amplified by common neonatal interventions and whether antiinflammatory therapeutics that are administered in utero or postnatally might lessen this risk. In our study, fetuses were exposed to intraamniotic infection and had severe systemic inflammation, but whether a disruption of cardiac development occurs in preterm labor cases with a lesser degree of fetal inflammation is unknown. Our experiments were not designed originally to investigate heart function, and additional work is needed to correlate changes in fetal cardiac transcriptomics with heart function and anatomic changes in the myocardium and vascularity. Whether therapeutic inhibition of inflammation in preterm infants may reduce the long-term risks of heart disease is unknown but is a critical question for prevention of life-course morbidity. Therapeutics that target inflammation in preclinical studies of preterm labor should be studied for their effect on the transcriptomic profile of fetal organs to determine whether disruptions in developmental gene networks can be ameliorated in the fetal heart.
Strengths and weaknesses
The strength of our study is in the novel finding of a disruption in morphogenesis gene networks in the fetal heart after intraamniotic infection and development of the fetal inflammatory response syndrome. Furthermore, the nonhuman primate shares many key features with human pregnancy (eg, placentation, hormonal onset of labor) that differ in other animal models of preterm birth (eg, murine, sheep). The main study limitation is the modest sample size, which is necessary for ethical reasons, conservation of nonhuman primates, and expense of the studies. Notably, our nonhuman primate model is consistent with the effects of a disseminated bacterial infection and severe systemic fetal inflammation; whether our results are applicable to inflammation in the absence of bacteremia or a lesser magnitude of the fetal inflammatory response syndrome is unknown.
Another important feature of our model is the short time course from bacterial inoculation to delivery and fetal necropsy (typically 2 days); histopathologic inflammation or overt signs of cardiac injury in the fetal heart may not have had sufficient time to develop. Our results are best applied to a severe infectious/inflammatory insult to the fetus in the late second or early third trimester (approximately 28 weeks) because the influence of infection on fetal development is likely gestational-age dependent.
The combination of cases with the use of 2 different microbes (GBS and E coli) as inciting agents of the fetal inflammatory response syndrome was necessary to establish sufficient power for the analysis; however, this mix of microbes is also typical of human case series of preterm labor and the fetal inflammatory response syndrome. Finally, we acknowledge that the effect of systemic fetal inflammation and preterm birth on gene expression in the fetal heart may involve multiple factors beyond simply the infectious/inflammatory component that may also function to increase the risk of cardiovascular disease in adulthood such as a reduction in fetal systolic blood pressure because of sepsis,
Our study offers new insight into the effect of fetal infection and inflammation on the dynamic regulation of genetic pathways that program fetal cardiac morphogenesis and vasculogenesis. These data provide a framework by which to understand how early biologic changes impart a risk for cardiovascular disease later in life. Rapid changes in the fetal cardiac genetic program after bacterial inoculation suggest that inflammatory injury to the fetal heart occurs quickly after a virulent bacterial strain invades the amniotic cavity. An important question is whether an intervention that combines antibiotic and antiinflammatory therapeutics can ameliorate fetal organ inflammatory injury and restore a normal developmental genetic program in the fetal heart.
Acknowledgments
We thank Dr Chuck Murry for his assistance with interpretation of the histopathology evidence, Mrs Jan Hamanishi for technical assistance with graphic design, Ms Geena Gallardo for administrative assistance, and Dr Marie-Terese Little for technical editing.
Data availability
The microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/index.cgi; GSE98459) and is publicly available.
Appendix
Supplemental FigureLinear fit between log interleukin-6 concentration and log myosin heavy chain 6, phospholipase A2, group VII, and secreted frizzled-related protein 4 expression
The plots depict how the log expression of each gene decreases with increasing log interleukin-6 concentration. A, For myosin heavy chain 6, the slope of the line is –0.48, which indicates that, for every unit change in log interleukin-6 (log scale), the log expression of myosin heavy chain 66 is reduced by 0.48. On a linear scale, this means that a doubling in interleukin-6 concentration results in an approximate reduction of 28% in myosin heavy chain 6 gene expression. Over the observed range of interleukin-6 concentrations, myosin heavy chain 6 expression decreases 76-fold. For B, phospholipase A2, group VII and C, secreted frizzled-related protein 4, there is an approximate reduction of 15% and 18% in gene expression for every doubling of interleukin-6 concentration, respectively. Across the range of interleukin-6 concentrations in our samples, there was a 13-fold decrease in phospholipase A2, group VII and a 19-fold decrease in secreted frizzled-related protein 4 expression.
IL, interleukin; MYH6, myosin heavy chain 6; PLA2G7, phospholipase A2, group VII; SFTP4, secreted frizzled-related protein 4.
Mitchell et al. Disruption of fetal cardiac gene networks with fetal infection and inflammation. Am J Obstet Gynecol 2018.
Birth weight and systolic blood pressure in adolescence and adulthood: meta-regression analysis of sex- and age-specific results from 20 Nordic studies.
Gestational age and growth rate of fetal mass are inversely associated with systolic blood pressure in young adults: an epidemiologic study of 165,136 Swedish men aged 18 years.
Clinical cardiovascular risk during young adulthood in offspring of hypertensive pregnancies: insights from a 20-year prospective follow-up birth cohort.
Activation of the cardiac renin-angiotensin system in high oxygen-exposed newborn rats: angiotensin receptor blockade prevents the developmental programming of cardiac dysfunction.
A cell wall component from pathogenic and non-pathogenic gram-positive bacteria (peptidoglycan) synergises with endotoxin to cause the release of tumour necrosis factor-alpha, nitric oxide production, shock, and multiple organ injury/dysfunction in the rat.
Supported by the March of Dimes ( 21-FY08-562 ), National Institute of Allergy and Infectious Diseases and National Center for Research Resources of the National Institute of Health under award numbers [R01AI100989, R01AI33976, R01AI112619, R21AI09222, K08AI067910, P30HD002274], the University of Washington Department of Obstetrics & Gynecology; the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health ( P51OD010425 ) through the Washington National Primate Research Center, and the UW Intellectual and Developmental Disabilities Research Center ( 5U54HD083091-04 ) funded by the Eunice Kennedy Shriver National Institute of Child & Human Development.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funders. The sponsors had no role in the study design, collection, analysis and interpretation of the data, writing of the report or the decision to submit the article for publication.
The authors report no conflict of interest.
Cite this article as: Mitchell T, MacDonald JW, Srinouanpranchanh S, et al. Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates. Am J Obstet Gynecol 2018;218:438.e1-16.
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