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Article

Placental Neutrophil Infiltration Associated with Tobacco Exposure but Not Development of Bronchopulmonary Dysplasia

by
David M. Box
1,
Abhishek Makkar
1,
Zhongxin Yu
2,
Hala Chaaban
1,
Henry H. Tran
2,
Kathryn Y. Burge
1 and
Jeffrey V. Eckert
1,*
1
Neonatal-Perinatal Medicine, Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
2
Pathology Department, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
*
Author to whom correspondence should be addressed.
Children 2022, 9(3), 381; https://doi.org/10.3390/children9030381
Submission received: 15 December 2021 / Revised: 16 February 2022 / Accepted: 3 March 2022 / Published: 9 March 2022
(This article belongs to the Section Pediatric Neonatology)
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Abstract

:
Objective: In utero inflammation is associated with bronchopulmonary dysplasia (BPD) in preterm infants. We hypothesized that maternal tobacco exposure (TE) might induce placental neutrophil infiltration, increasing the risk for BPD. Study design: We compared the composite outcome of BPD and death in a prospective pilot study of TE and no-TE mothers and their infants born <32 weeks. Placental neutrophil infiltration was approximated by neutrophil gelatinase-associated lipocalin (NGAL) ELISA, and total RNA expression was analyzed via NanoString© (Seattle, WA, USA). Result: Of 39 enrolled patients, 44% were classified as tobacco exposure. No significant difference was noted in the infant’s composite outcome of BPD or death based on maternal tobacco exposure. NGAL was higher in placentas of TE vs. non-TE mothers (p < 0.05). Placental RNA analysis identified the upregulation of key inflammatory genes associated with maternal tobacco exposure. Conclusion: Tobacco exposure during pregnancy was associated with increased placental neutrophil markers and upregulated inflammatory gene expression. These findings were not associated with BPD.

1. Introduction

Tobacco exposure (TE) during pregnancy is highly prevalent in the United States. As reported by the Center for Disease Control and Prevention (CDC) in 2016, 7.2% of mothers smoked cigarettes during pregnancy [1]. It is well recognized that maternal tobacco use during pregnancy is linked to many negative outcomes for infants, including low birthweights, preterm birth, preterm prolonged rupture of membrane (PPROM), and other birth defects [2,3,4,5].
Recently, Antonucci et al. indicated that in utero exposure to smoking is an independent risk factor for the development of bronchopulmonary dysplasia (BPD) in premature infants born weighing less than 1500 g [6]. BPD is the most prevalent sequela of preterm birth, affecting 10,000–15,000 infants annually in the United States [7]. Known postnatal risk factors for the disease include hyperoxia, mechanical ventilation, patent ductus arteriosus (PDA), and sepsis; antenatal risk factors include chorioamnionitis, preeclampsia, and hypertension [8,9,10,11,12].
Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein found predominantly in neutrophil granules. NGAL is normally expressed at low levels but is often elevated in the blood, bronchoalveolar lavage (BAL) fluid, and sputum in adults with lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD) [13]. Notably, serum levels of NGAL at birth are significantly higher in preterm infants who develop BPD than in those who do not [14], suggesting a potential role for NGAL as a biomarker for BPD.
Little is known about the mechanism by which maternal tobacco exposure is associated with the development of BPD. A previous study demonstrated a higher number of neutrophils within the placentas of mothers who smoked during pregnancy; however, the incidence of bacterial infection in that group was higher, confounding the results [15]. Recent reviews have focused on injury and its contribution to fetal lung development, identifying inflammation and tobacco exposure as major contributors [16]. In addition, a meta-analysis of tobacco smoking during pregnancy showed significant association with BPD at a postmenstrual age of 36 weeks [17]. Finally, the adverse effects of maternal tobacco exposure are supported by epidemiological and animal studies, which demonstrate disrupted pulmonary development [18,19,20]. These observations taken together establish a link between maternal tobacco exposure and BPD and raise the possibility that neutrophils play a key role in the mechanism, with elevated levels in preterm infants who develop BDP.
To further understand the effect of antenatal tobacco exposure and its association with the development of BPD, we compared placental and infant characteristics of tobacco exposure and non-tobacco exposure mothers. Our hypothesis is two-fold; (1) we hypothesized that maternal tobacco exposure would result in increased inflammatory neutrophil infiltration of the placenta of preterm infants <32 weeks gestation, and (2) we hypothesize that infants <32 weeks gestation with tobacco exposure will subsequently be at increased risk for developing BPD. Therefore, we sought to achieve two aims/objectives in our study. The first was to identify increased neutrophil infiltration in the placenta of mothers with tobacco exposure (primary outcome). The second was to follow these infants for the composite outcome of BPD or death (secondary outcome).

2. Materials and Methods

Study design: This pilot prospective, observational study was conducted between October 2018 and December 2019 and was approved by the Institutional Review Board at the University of Oklahoma Health Sciences Center (OUHSC). Written informed consent was obtained for the mother and newborn either prior to delivery or within 24 h post-delivery. Following consent, a 9-item maternal questionnaire for self-identification of tobacco exposure during pregnancy was completed (Figure A1). Our maternal questionnaire on tobacco use was internally validated in a previous study, where cotinine levels (a nicotine metabolite) were detectable only in mothers who reported tobacco exposure [21]. Patients were stratified into two groups: TE mothers and non-TE mothers.
Study population: Participants included mothers and their preterm infants born at a gestational age of <32 weeks. Infants were excluded based on known major congenital anomalies, maternal concern for infection (e.g., clinical chorioamnionitis), maternal fever >38 °C 24 h before delivery, presence of meconium-stained fluid, maternal history of impaired immunity, or a concomitant medical condition impacting inflammatory response.
Data collection: Data were de-identified and prospectively collected and managed using a data collection sheet at OUHSC. Maternal and neonatal demographic characteristics were collected via chart review. The secondary outcome was a composite of BPD or death endpoints. BPD status was assessed at 36 weeks postmenstrual age (PMA) using the National Institutes of Health (NIH) workshop definition [22]. Mild BPD is defined as breathing room air at 36 weeks corrected or time of discharge, moderate BPD is defined as needing <30% oxygen at 36 weeks corrected/discharge, whereas severe BPD is defined as needing >30% O2 at 36 weeks corrected age/discharge. For the purpose of this study, infants were defined as having the presence or absence of BPD; absence of BPD was defined as no or mild BPD, and the presence of BPD was defined as moderate to severe BPD [22]. Additional outcomes included necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), PDA, and sepsis. A mother was considered to have received antenatal corticosteroids if she received a full or partial betamethasone or dexamethasone course. Intrauterine growth restriction (IUGR) was defined as intrauterine estimated fetal weight less than the 10th percentile. PPROM was defined as having membranes ruptured for more than 18 h. Samples from the placenta from both groups were evaluated for histological chorioamnionitis by one of two pathologists blinded to maternal tobacco exposure status. Positive tobacco exposure was defined as maternal ‘daily’ to ‘almost daily’ active smoking or ‘daily’ to ‘almost daily’ secondhand smoke exposure, as reported on the maternal tobacco exposure questionnaire (Figure A1).
To determine the contribution of tobacco exposure to the development of BPD, the groups were further subdivided into (1) TE mothers with infants developing BPD (BPD TE group); (2) non-TE mothers with infants developing BPD (BPD No TE group); (3) TE mothers with infants not developing BPD (No BPD TE group); and (4) non-TE mothers with infants not developing BPD (No BPD No TE group).
Sample collection: Fresh placenta tissue samples were collected within 24 h of delivery. Three full-thickness sections of placenta parenchyma (including fetal and maternal surfaces), one section of extraplacental membrane roll, and two sections of the umbilical cord (proximal and distal) were collected and fixed in 10% formalin for routine histopathological examination and diagnosis. One full-thickness section was split and preserved for both RNA analysis (RNAlater™, Invitrogen, Carlsbad, CA, USA) and protein analysis (snap-frozen in liquid nitrogen). All samples were stored at −80 °C until further analysis.
Immunohistochemistry (IHC): IHC was performed according to the manufacturer’s protocols using a Leica Bond-IIITM Polymer Refine Detection System (DS 9800). Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at the desired thickness (4 µm) and mounted on positively charged slides. The slides were dried overnight at room temperature and incubated at 60 °C for 45 min, followed by deparaffinization and rehydration in an automated multi-stainer (Leica ST5020). Subsequently, slides were transferred to the Leica Bond-IIITM and treated for antigen retrieval at 100 °C for 20 min in a retrieval solution, at either pH 6.0 or 9.0. Endogenous peroxidase was blocked using a peroxidase-blocking reagent, followed by 60 min of incubation with NGAL antibody (Catalog #711280, ThermoFisher Scientific, Waltham, MA, USA) diluted 1:100. Post-primary IgG-linker and/or poly-HRP IgG reagents were used as the secondary antibody. Detection was accomplished via the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB), and counterstained with hematoxylin. Completed slides were dehydrated (Leica ST5020) and mounted (Leica MM24). The antibody-specific positive control and negative control (omission of primary antibody) were parallel stained. Additionally, two pathologists blinded to smoking and BPD status semi-quantitatively scored based on anatomical location, with scores from zero to four: score ‘0′ signifying no staining; score ‘1′ for 1–10 positive cells/per high power field (HPF); score ‘2′ for 11–50 positive cells/HPF; score ‘3′ for 51–75 positive cells/HPF; and score ‘4′ for >75/HPF.
Protein analysis and enzyme-linked immunosorbent assay (ELISA): ELISA was used to quantify NGAL (Catalog #036RUO, BioPorto Diagnostics A/S, Hellerup, Denmark) following the manufacturer’s instructions. Briefly, frozen placental tissue was mechanically homogenized using a BeadBeater (Next Advance Inc., Troy, NY, USA) in a buffer containing phosphatase, protease inhibitors (Catalog #524625 and #535140, Millipore, Burlington, MA, USA) and PMSF (Sigma-Aldrich, St. Louis, MO, USA). Results were normalized to total protein concentration determined by bicinchoninic acid (BCA) assay (Catalog #23227, Pierce Biotechnology, Rockford, IL, USA).
Total RNA analysis/NanoString©: A random subset of 12 patients from the four subgroups (n = 3/group): BPD, TE group; BPD, no TE group; no BPD, TE group; and no BPD, no TE group. A BeadBeater was used to homogenize placental tissue mechanically. Total RNA was extracted per the manufacturer’s protocols using a Zymo Quick-RNA MidiPrep kit (Catalog #R1056, Zymo Research, Irvine, CA, USA). Total RNA, between 25 ng and 300 ng, was loaded onto a nCounter® Human Immunology v2 Panel (Catalog #XT-CSO-HIM2-12, NanoString, Seattle, WA, USA). This panel consisted of 594 genes of interest and 15 internal reference genes. Data were analyzed using nCounter Analysis and nCounter Advanced Analysis software. RCC output files were imported into NanoString nSolver 4.0. Default quality control (QC) settings were used to verify the quality of all data (>95% of fields of view [FOV] and binding densities between 0.2 and 0.5). The background was corrected by subtracting the mean value of 8 engineered RNA negative control sequences from the raw counts of all genes. The geometric mean was calculated for the 15 housekeeping genes, and the nine genes with the lowest coefficient of variation were used to normalize the data. Genes with mean normalized counts of less than 50 were excluded from the analysis. The control group was defined as No TE or No BPD No TE for subgroup analysis. Gene expressions are estimated to have a log2-fold change, holding all other variables constant. The 95% confidence intervals (CI) for the log2-fold change and the p values are reported. A 1.2-fold change was selected as the differential threshold.
Given the unpredictable nature of preterm deliveries, we allowed up to 24 h for placenta collection. Once collected, the placenta was immediately placed at 4 °C. The pathologist then collected full-thickness sections and stored these at −80 °C or preserved with RNAlaterTM. Although we allowed up to 24 h for placenta collection in our protocol, the majority of samples were collected within 2–12 h. This methodology allows for collection of high-quality RNA from placentas stored at 4 °C or even room temperature for up to 48 h prior to being transferred to stabilizing solution, such as RNAlaterTM [23].
Statistical methods: Our study is a pilot/preliminary study on a topic where there is little known on the association between inflammation within the placenta and development of BPD in preterm neonates. While we have directional hypotheses, we felt it would be inappropriate to quantify an effect size given the paucity of research on the topic. Descriptive statistics were computed for demographic and clinical variables. Comparisons of categorical variables between patients developing BPD or death and those who did not were evaluated with Fisher’s exact test. Continuous variables were assessed for normality, then compared between groups using a Kruskal–Wallis test or Student’s t-test, as appropriate. Frequencies and percentages were reported for categorical variables across BPD status. Count means and standard deviations are reported for continuous variables. Statistical significance is defined, in all experiments, as p < 0.05.

3. Results

In total, 95 mothers were screened, and 49 mothers were approached for study enrollment based on the inclusion and exclusion criteria. Eight mothers declined and two approached mothers aged out of this study (delivered baby >32 weeks gestation). Demographic characteristics for the remaining 39 patients were stratified by the presence and absence of tobacco exposure (Table 1), as well as by the presence or absence of the composite outcome of BPD or death (Table A1). Of enrolled mothers, 43.6% reported tobacco exposure during pregnancy (Table 1 and Table A2). Of these tobacco exposure mothers, two reported the exposure was via secondhand smoke.
No differences in birth weight, birth length, head circumference, gestational age, gender, maternal ethnicity, antenatal steroid, mode of delivery, intubation in delivery room, intubated in NICU, PDA medical or surgical treatment, IVH grade 3 or 4, ROP, IUGR <10th percentile, or death or BPD were noted with maternal tobacco exposure. There was an association with maternal age (p = 0.048), with tobacco exposure mothers being slightly older (Table 1). When comparing tobacco exposure mothers, no differences in diabetes status, maternal hypertension, prolong rupture of membranes, chorioamnionitis, antepartum hemorrhage, marijuana, or other illicit drug use were present (Table 2). No differences in the incidence of NEC, or sepsis based on maternal tobacco exposure were noted.
As expected, infants with the composite outcome of BPD or death had significantly lower (p < 0.001) birth weight, length, head circumference, and gestational age compared with the No BPD group. Additionally, more infants in the composite outcome required intubation in the delivery room (p = 0.001) or the NICU (p < 0.001), required medical management of PDA (p = 0.01), and developed threshold ROP (p = 0.017) compared to the No BPD group (Table A1). The remainder of the maternal and neonatal demographic characteristics did not differ between groups. From the maternal perspective, we found no significant association between tobacco exposure status and maternal complications, with the exception of increased incidence of antepartum hemorrhage in the composite outcome group (p = 0.003) (Table A2).
While there was no association between maternal tobacco exposure and an infant’s risk for developing BPD, IHC of placental tissues showed a higher expression of NGAL in the fetal surfaces and upper portion of the placenta parenchyma of tobacco exposure mothers (Figure 1A,C) compared to those of No TE (Figure 1B,D) mothers. The IHC for the BPD TE group (Figure 1A) showed higher expression of NGAL as compared to the BPD No TE group (Figure 1B). Regardless of BPD status, NGAL was highly expressed in the TE groups (BPD TE and No BPD TE) compared to the No TE group (BPD No TE and No BPD No TE). Additionally, NGAL intensity staining scores were higher in the chorionic plate and subchorionic space of placentas from tobacco exposure mothers, regardless of BPD status, though these differences did not reach statistical significance (Figure 1E,G; p = 0.065 and p = 0.091, respectively).
To confirm these histological findings, NGAL ELISA was performed in each of the four subgroups. As shown in Figure 2A, NGAL levels were significantly higher in the placentas of tobacco exposure compared to No TE mothers (p < 0.0001). Further subgroup analysis based on BPD outcomes showed that NGAL levels were significantly higher in infants of the BPD TE group compared to No BPD No TE infants (Figure 2B, p < 0.01). Notably, BPD No TE group also had significantly higher levels of NGAL as compared to No BPD No TE infants (Figure 2B, p < 0.001). Altogether, these data suggest that tobacco exposure during pregnancy is associated with increased neutrophil activation/infiltration in the placenta, and levels of neutrophil activation/infiltration are increased further still in the placentas of tobacco exposure infants developing BPD.
Next, the immune placental transcriptome from a subset of infants from all four subgroups was profiled using the NanoString nCounter™ Immunology Panel. Comparing BPD TE to No BPD No TE, 22 genes were significantly differentially expressed (Table 3) out of a total of 594 genes of potential interest (Table A3). Notably, transcript levels for the chemokines IL8 and CXCL10, the inflammatory molecules SA100A8/9, and the receptor CD44 were significantly upregulated in BPD TE compared to No BPD No TE infants (Table 3; p < 0.05), influencing cell signaling and inflammatory cytokine pathways (e.g., Figure A2). No other significant differences were found between the groups. We further compared the subgroups based on the neonatal outcome of BPD. Similarly, gene expression for CXCL8, CXCL10 were upregulated in the TE BPD group compared to no TE no BPD group.

4. Discussion

Bronchopulmonary dysplasia, a disease primarily affecting preterm infants, can be a challenge to manage both acutely and in the long term, as there are many persistent complications affecting patients and their families [24,25]. In this study, we sought to investigate whether tobacco exposure during pregnancy is a risk factor for developing BPD. Specifically, we questioned whether neutrophil activation/infiltration occurs in the placentas of tobacco exposure mothers and if this infiltration of neutrophils to the placenta is associated with the development of BPD or death, as a composite outcome, in preterm infants.
NGAL, neutrophil gelatinase-associated lipocalin, is a 25 kDa lipocalin originally purified from activated human neutrophils. This molecule is now known to be secreted by a variety of immune cells, hepatocytes, adipocytes, and renal tubular cells [26]. In the placenta, NGAL staining has been associated with inflammation and intra-amniotic infections [26]. NGAL levels in the plasma have also been associated with the development of BPD in preterm infants [14]. In this study, we showed for the first time that NGAL staining and NGAL protein levels are higher in the placentas of tobacco exposure mothers compared to those of No tobacco exposure mothers. Using IHC, NGAL staining was specifically high in the amniochorionic membrane and intervillous space, suggesting the presence of neutrophil activation on both the maternal and fetal surfaces. Levels of NGAL measured by ELISA in placenta homogenates were higher in BPD tabacco exposure infants compared to No BPD tobacco exposure infants. Notably, we found no difference in pathologically diagnosed chorioamnionitis or funisitis between the BPD and No BPD groups, suggesting that the observed elevated NGAL levels could be secondary to maternal tobacco exposure.
The potential physiological mechanisms associating maternal tobacco exposure with increased placental NGAL are currently unknown. However, it is reasonable to assume that tobacco exposure during pregnancy results in increased inflammation and immune cell activation, both systemically and at the placenta [27]. Immune cell activation would result in the release of inflammatory cytokines and chemotactic factors [28], potentially affecting the maturation of the fetal lungs. Previous studies have confirmed an association of elevated levels of pro-inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor-alpha [TNF-α], IL-1β, and IL-8) in amniotic fluid 5 days preceding delivery with the development of BPD, suggesting that the mechanism responsible for BPD may begin before birth [29].
To determine if tobacco exposure is associated with increased inflammation in the placenta, we profiled the placental tissues as from tobacco exposure and no tobacco exposure mothers using the nCounter® Immunology NanoString Panel, which includes over 500 immunology genes involved with activation of the inflammatory cascade, including neutrophils, natural killer cell, B cell, and T cell activation, as well as various genes responsible for complement activation. Notably, IL8 and CXCL10 mRNA were significantly upregulated in tobacco exposure compared to no tobacco exposure placenta. Both genes encode chemokines known to recruit immune cells, including neutrophils, and are associated with inflammation in the placenta [28,30]. Additionally, the SA100A8 and SA100A9 genes, upregulated in tobacco exposure placentas, encode inflammatory proteins previously shown to play a role in pregnancy loss and other complications, such as preeclampsia [31]. These expression differences further support our suggestion that maternal tobacco exposure is associated with placental inflammation, at least at the transcript level.
Surprisingly, we found no association between maternal tobacco exposure and the incidence of BPD in preterm infants born <32 weeks gestation. This lack of association could be due to the small sample size, as well as a multitude of factors known to be involved in the pathogenesis of BPD [24]. Though a previous study showed a potential association of BPD with maternal tobacco exposure, the majority of the literature indicates that maternal smoking during pregnancy is not an independent risk factor for BPD development, after controlling for additional variables [6,8,32,33]. With the exception of antepartum hemorrhage incidence, which was significantly higher in the composite outcome group compared to the No BPD group (46.7% vs. 4.2%; p = 0.003), we found no difference in known risk factors for BPD, including maternal hypertension, PPROM, and chorioamnionitis [8,9,10,11,12]. In line with other studies [7], infants with the composite outcome of BPD or death had a lower gestational age and birth weight compared to infants in the No BPD group. Composite outcome infants also required more medical interventions, such as intubation after birth, medical management of PDA, and development of threshold ROP.
Our pilot study is subject to several limitations. First, maternal tobacco exposure status was based on a self-reported questionnaire rather than biochemical measurement, such as levels of cotinine, a nicotine metabolite. We previously showed that serum cotinine levels were significantly higher in cord blood of self-reported smokers than in cord blood of non-smokers, suggesting that self-reporting smoking status could be adequate in our patient population [21]. Secondly, we did not account for the amount of tobacco exposure (e.g., number of cigarettes smoked per day, or passive versus active smoking) in our results. It is possible that active smoking has a stronger association with placental pathology than passive tobacco exposure. Third, due to the small sample size, we focused on the clinically relevant outcome of moderate to severe BPD and did not adjust for the multiple confounding variables that contribute to the development of BPD. Lastly, our focus in this study was primarily on neutrophil activation. We did not evaluate the effect of tobacco exposure on activation or placental infiltration of other leukocytes.
Our studies provide direct evidence that maternal tobacco exposure leads to neutrophil infiltration into the placenta. One possible implication of this observation is an increased inflammatory environment which could amplify other risk factors, chorioamnionitis, preeclampsia, high oxygen or mechanical ventilation, resulting in the development of BPD [16]. Additional studies need to be carried out focusing on other leukocytes present in the placenta and the cytokines the neonate is exposed to that could contribute to inflammatory injury in the developing lungs. Further, an additional larger study should be carried out to determine if an increase neutrophil infiltration into the placenta due to tobacco exposure is predictive of BPD.

5. Conclusions

In conclusion, our studies provide direct evidence that maternal tobacco exposure leads to neutrophil infiltration into the placenta. One possible implication of this observation is an increased inflammatory environment which could amplify other risk factors, chorioamnionitis, preeclampsia, high oxygen or mechanical ventilation, resulting in the development of BPD [16]. Additional studies need to be carried out focusing on other leukocytes present in the placenta and the cytokines the neonate is exposed to that could contribute to inflammatory injury in the developing lungs. Further, an additional larger study should be carried out to determine if an increase neutrophil infiltration into the placenta due to tobacco exposure is predictive of BPD.

Author Contributions

D.M.B. designed the protocol, collected data, and prepared the manuscript. Z.Y. and H.H.T. performed the standard histological analysis of placentas and IHC and NGAL. scoring. H.C. participated in the design and manuscript editing. K.Y.B. performed experiments and edited the manuscript. A.M. designed the protocol, supervised the project, analyzed the data, and edited the manuscript. J.V.E. participated in the study design, protocol design, supervised the project, performed the experiments, analyzed the data and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding; it was funded by University of Oklahoma Neonatal Section, Fellow Research Grant.

Institutional Review Board Statement

This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of The University of Oklahoma Health Sciences Center (9394 approved 23 August 2018).

Informed Consent Statement

Informed consent was obtained from all subjects involved in this study.

Data Availability Statement

Data is available on request.

Acknowledgments

We would like to thank the mothers and their infants who participated in our study. This study was funded by the Neonatal Section, at the University of Oklahoma, Fellow Research Grant. We would like to thank the Neonatal-Perinatal Medicine research team, Michael P. Anderson, PhD who assisted with statistical analysis, and Kathy Kyler who helped editing.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Children 09 00381 g0a1a 550Children 09 00381 g0a1b 550
Figure A1. Following consent, a maternal tobacco exposure questionnaire was administered within 24 h of delivery to self-identify TE during pregnancy.
Figure A1. Following consent, a maternal tobacco exposure questionnaire was administered within 24 h of delivery to self-identify TE during pregnancy.
Children 09 00381 g0a1aChildren 09 00381 g0a1b
Children 09 00381 g0a2 550
Figure A2. NanoString nSolver® pathway analysis indicating upregulation (yellow) and downregulation (blue) of genes encoding interactions between cytokines and cytokine receptors in BPD TE placentas compared to No BPD No TE.
Figure A2. NanoString nSolver® pathway analysis indicating upregulation (yellow) and downregulation (blue) of genes encoding interactions between cytokines and cytokine receptors in BPD TE placentas compared to No BPD No TE.
Children 09 00381 g0a2
Table
Table A1. Maternal and Neonatal Demographic Data by Composite Outcome.
Table A1. Maternal and Neonatal Demographic Data by Composite Outcome.
Maternal and Neonatal Demographic Data
No BPD or DeathBPD or Death
(n = 22)(n = 15)Total (n = 39)p Value
Birth weight (g)1397.7 ± 355.1711.7 ± 207.91133.8 ± 454.5<0.001
Birth length (cm)39.4 ± 2.931.7 ± 2.636.5 ± 4.7<0.001
Head circumference (cm)27.4 ± 1.822.5 ± 1.725.5 ± 3.0<0.001
Gestational age (wk)29.8 ± 1.425.5 ± 1.928.2 ± 2.6<0.001
Maternal age (yr)27.1 ± 5.628.2 ± 6.727.5 ± 6.00.643
Sex 0.083
 F14 (58.3)13 (86.7)27 (69.2)
 M10 (41.7)2 (13.3)12 (30.8)
Maternal ethnicity 0.233
 Black3 (12.5%)5 (33.3%)8 (20.5%)
 Black, Native American0 (0%)1 (6.7%)1 (2.6%)
 Hispanic7 (29.2%)1 (6.7%)8 (20.5%)
 Latino, White1 (4.2%)0 (0.0%)1 (2.6%)
 Native American3 (12.5%)1 (6.7%)4 (10.3%)
 White9 (37.5%)7 (46.7%)16(41.0%)
 White, Native American1 (4.2%)0 (0.0%)1 (2.6%)
Antenatal steroid exposure23 (95.8)15 (100)38 (97.4)1.000
Mode of delivery, C-Section14 (58.3)7 (46.7)21 (53.8)0.477
Intubated in delivery room6 (25)12 (80)18 (46.2)0.001
Intubated in NICU2 (8.3)9 (60.0)11 (28.2)<0.001
PDA medical treatment3 (12.5)8 (53.3)11 (28.2)0.010
PDA surgical treatment0 (0)1 (6.7)1 (2.6)0.385
IVH grade 3 or 41 (4.2)2 (13.3)3 (7.7)0.547
Threshold ROP, yes0 (0)4 (26.7)4 (10.3)0.017
IUGR <10th percentile, yes1 (4.2)2 (13.3)3 (7.7)0.547
All data are presented as the mean ± standard deviation or n (%). BPD—bronchopulmonary dysplasia, NICU—neonatal intensive care unit, PDA—patent ductus arteriosus, IVH—intraventricular hemorrhage, ROP—retinopathy of prematurity, and IUGR—intrauterine growth restriction.
Table
Table A2. Maternal Complications and Tobacco Exposure by Composite Outcome.
Table A2. Maternal Complications and Tobacco Exposure by Composite Outcome.
No BPD or DeathBPD or Death
(n = 24)(n = 15)Total (n = 39)p Value
Maternal diabetes4 (16.7)1 (6.7)5 (12.8)0.634
Maternal hypertension3 (12.5)2 (13.3)5 (12.8)1.000
Prolonged rupture of membranes (>18h)3 (12.5)4 (26.7)7 (17.9)0.396
Histological chorioamnionitis12 (50)8 (53.3)20 (51.3)0.839
Antepartum hemorrhage1 (4.2)7 (46.7)8 (20.5)0.003
Maternal TE11 (45.8)6 (40)17 (43.6)0.721
Maternal active smoking9 (37.5)3 (20)12 (30.8)0.305
Maternal passive smoke exposure11 (45.8)7 (46.7)18 (46.2)0.959
Illicit drugs, yes1 (4.21)1 (6.7)2 (5.1)0.765
All data are presented as n (%). BPD—bronchopulmonary dysplasia, PPROM—premature prolonged rupture of membranes, and TE—tobacco exposure.
Table
Table A3. NanoString Gene Expressions (BPD TE vs. No BPD No TE).
Table A3. NanoString Gene Expressions (BPD TE vs. No BPD No TE).
GeneLog2-Fold ChangeSELower Confidence Limit (log2)Upper Confidence Limit (log2)pTentative Function
IL84.770.8983.016.540.00034Cell Activation
S100A91.720.3391.062.380.000477Cell–Cell Signaling
S100A83.330.9121.545.120.00447Defense Response
IL1RL1−3.411.1−5.57−1.240.0115Receptor Signaling Protein Activity
CXCL103.061.090.9225.210.0187Behavior
CD441.70.6140.4932.90.02Cell–Cell Signaling
TNFRSF10C1.530.5620.4332.640.0212Integral to Membrane
PLAUR1.780.6760.4543.110.0251Behavior
IRF71.080.4150.2691.90.0261Biopolymer Metabolic Process
MALT1−0.5720.22−1−0.1410.0263Cell Development
LILRB32.040.7950.4813.60.0281Cell Surface Receptor-Linked Signal
Transduction
HLA-DRB12.741.140.5024.990.0374Antigen Presentation
HLA-DRB31.780.7460.3163.240.0384Immune Response
HFE−1.250.529−2.29−0.2160.0394Cytoplasm
TNFSF15−1.430.604−2.61−0.2440.0397Cell Development
CD991.120.4750.1862.050.0406Cytoplasm
PTPRC1.940.8470.283.60.045Integral to Membrane
PTAFR−20.881−3.73−0.2730.0466Behavior
ZBTB16−2.010.888−3.75−0.2720.0469Intracellular Organelle Part
PLA2G2A−2.250.997−4.2−0.2920.0479Cytoplasm
CXCL12−2.060.92−3.86−0.2560.0492Behavior
HRAS−1.390.621−2.6−0.1690.0497Anatomical Structure Development
SELL1.660.7570.1773.150.053Integral to Membrane
PSMB91.130.578−0.005862.260.0798Antigen Presentation
NT5E0.9170.482−0.02751.860.0862Biopolymer Metabolic Process
CCL31.660.885−0.07763.390.0907Cell Fraction
CD831.640.884−0.09443.370.0935Defense Response
NFKBIA0.720.398−0.06041.50.101Apoptosis
HLA-DRA1.160.648−0.1142.430.105Cytoplasm
CLEC4A1.270.71−0.1252.660.105Cell Surface Receptor-Linked Signal
Transduction
HLA-C1.220.686−0.1242.570.106Antigen Presentation
CXCL11.660.942−0.1843.510.108Behavior
BCL31.250.717−0.1582.650.112Cytoplasm
ITGAX1.771.03−0.2433.790.115Anatomical Structure Morphogenesis
HLA-DMA0.6670.396−0.111.440.123Antigen Presentation
TRAF5−1.050.631−2.290.1830.126IκB Kinase NFκB Cascade
HLA-A0.7230.436−0.1311.580.128Antigen Presentation
GATA3−1.130.679−2.460.2040.128Anatomical Structure Morphogenesis
CD741.290.78−0.2372.820.129Biosynthetic Process
LILRB21.220.737−0.2282.660.13Cell–Cell Signaling
BST11.070.649−0.2052.340.131Humoral Immune Response
LTB4R2−0.9770.594−2.140.1870.131Behavior
RARRES3−1.230.764−2.730.2680.139Cell Proliferation
TNFSF13B0.9030.566−0.2072.010.142Cell Fraction
XBP11.340.84−0.3072.990.142DNA Binding
CD241.220.778−0.2992.750.146Cell Surface
NFKB20.8770.56−0.2211.980.148Biopolymer Metabolic Process
ITGAE−0.7750.505−1.770.2150.156Integral to Membrane
VCAM1−1.040.69−2.390.3120.162Leukocyte Adhesion
PSMD70.3420.229−0.1060.790.165Macromolecular Complex
ATG12−1.030.691−2.380.3280.168Apoptosis
CXCL21.320.896−0.4343.080.171Behavior
MAPK110.7210.497−0.2541.70.178Intracellular Signaling Cascade
IL11RA−0.8720.603−2.050.3110.179Integral to Membrane
TAL1−1.010.702−2.390.3640.18Cell Proliferation
PPBP1.060.736−0.3822.50.18Establishment of Localization
TNFRSF14−0.8540.601−2.030.3240.186Cell Surface Receptor-Linked Signal
Transduction
ITGAM0.9320.659−0.3592.220.187Integral to Membrane
C2−0.4150.294−0.9910.1610.188Defense Response
CD590.6260.446−0.2471.50.19Cell Fraction
TGFB11.150.817−0.4552.750.191DNA Metabolic Process
MIF0.6860.489−0.2721.650.191Biosynthetic Process
FCGR2A1.971.41−0.7894.720.192Phagocytosis
BATF3−0.8580.62−2.070.3580.197Biopolymer Metabolic Process
CCL40.9460.685−0.3972.290.198Anatomical Structure Morphogenesis
IFI35−0.5590.406−1.360.2370.199Nucleus
HLA-DPB11.391.01−0.5913.360.199Multi-Organism Process
FCER1G0.9530.699−0.4172.320.203Integral to Membrane
FCGR3A/B2.031.49−0.9024.950.205Immune Response
HLA-DPA1.331−0.6393.30.215Antigen Presentation
SRC−0.6550.497−1.630.320.217Cell Surface Receptor-Linked Signal
Transduction
IL1R20.8880.681−0.4462.220.221Immune Response
CXCR20.8770.675−0.4472.20.223Receptor for IL-8
ITGAL1.150.894−0.5982.910.226Leukocyte Adhesion
CFD−0.5580.433−1.410.290.226Cellular Macromolecule Metabolic
Process
SOCS30.870.681−0.4642.20.23Cell Development
IL2RG0.9730.763−0.5222.470.231Cell Surface
PECAM1−0.4910.386−1.250.2650.232Membrane
TNFRSF1B1.341.06−0.7293.410.233Receptor Activity
CASP81.271.02−0.7253.270.241Cell Development
GBP11.411.13−0.813.630.242Cell Metabolism
TLR20.7840.644−0.4782.050.251Cell Development
CDKN1A−0.6180.51−1.620.3810.253Cell Development
S1PR10.7940.662−0.5032.090.258S1P Receptor
IL180.5620.475−0.3691.490.264Anatomical Structure Morphogenesis
TFRC−0.7150.632−1.950.5230.284Cytoplasm
VTN−1.191.07−3.290.9040.291Extracellular Region
GPI1.291.16−0.9873.560.293Hemostasis
MR1−0.7630.689−2.110.5870.294Immune Response
PRKCD0.6380.578−0.4951.770.296Biopolymer Metabolic Process
BCL101.351.22−1.053.740.296Cytoplasm
MAPK141.551.4−1.214.30.297Behavior
ZEB1−0.6670.607−1.860.5210.297Biopolymer Metabolic Process
EBI30.7210.661−0.5742.020.301Biosynthetic Process
PTPN21.050.969−0.8492.950.304Biopolymer Metabolic Process
TNFRSF11A−0.4640.43−1.310.3790.306Cell–Cell Signaling
IL320.7810.733−0.6552.220.312Defense Response
C1QA1.341.27−1.153.840.317Cell–Cell Signaling
CHUK1.231.18−1.083.540.322Anatomical Structure Morphogenesis
AHR−0.6030.579−1.740.5330.323Biopolymer Metabolic Process
TGFBR2−0.330.323−0.9630.3040.331Cell Proliferation
IL13RA10.2170.214−0.2020.6370.334Cell Surface Receptor-Linked Signal
Transduction
PDCD1LG2−0.7410.732−2.180.6940.335Antigen Presentation
ETS1−0.6590.666−1.960.6470.346Hemopoiesis
FADD0.5310.538−0.5241.580.347Cell Development
HLA-B1.451.47−1.434.330.348Cell Fraction
MYD881.151.17−1.153.450.352IκB Kinase NFκB Cascade
CR10.7230.742−0.7312.180.353Integral to Membrane
TGFBI1.511.55−1.534.540.353Cell Proliferation
TRAF6−0.1320.137−0.4010.1360.358Biopolymer Metabolic Process
LTBR1.261.33−1.333.860.363IκB Kinase NFκB Cascade
TLR7−0.6040.637−1.850.6440.365Biosynthetic Process
BCAP31−0.9681.02−2.971.030.366Integral to Membrane
CD45R00.6660.707−0.7192.050.368Integral to Membrane
PSMC21.351.45−1.54.190.375Cytoplasm
CUL9−0.5470.596−1.720.6220.381Microtubule Dynamics
MAP4K40.2360.258−0.2690.7420.381Biopolymer Metabolic Process
TLR41.051.15−1.23.30.381Biosynthetic Process
STAT61.091.2−1.253.440.383DNA Binding
LTF1.281.4−1.474.020.384Endopeptidase Activity
STAT30.350.385−0.4041.10.384Biopolymer Metabolic Process
BCL60.7340.808−0.8492.320.385Intracellular Non-Membrane-Bound
Organelle
FYN0.6260.695−0.7351.990.389Behavior
IKBKAP0.8850.986−1.052.820.39Cytoplasm
PPARG0.9441.06−1.133.010.393Biopolymer Metabolic Process
IFITM11.261.42−1.524.050.395Cell Proliferation
CD40−0.6550.741−2.110.7970.398Defense Response
CASP31.031.17−1.263.330.398Cell Development
TAPBP0.3320.383−0.421.080.407Cytoplasm
IFI161.091.27−1.43.580.411Cell Development
CD45RB0.7080.825−0.912.330.411Integral to Membrane
GPR183−0.7550.882−2.480.9730.412Unknown
TAGAP−0.6710.79−2.220.8770.416Rho GTPase Activation
ITGB20.6190.732−0.8152.050.417Behavior
NFATC20.971.15−1.283.220.418Biopolymer Metabolic Process
TAP20.5750.682−0.7611.910.419Cytoplasm
TBK11.121.33−1.493.740.42IκB Kinase NFκB Cascade
NCF40.510.607−0.6811.70.421Cytoplasm
PTPN60.9841.17−1.323.290.422Biopolymer Metabolic Process
ILF31.151.38−1.553.850.423Biopolymer Metabolic Process
CASP20.9991.2−1.353.340.423Apoptosis
CD2740.3450.415−0.4681.160.425Cell Proliferation
SPP1−0.6450.779−2.170.8820.427Osteoclast Attachment
FCGR1A/B0.5070.613−0.6951.710.428Establishment of Localization
RELA0.3750.461−0.5281.280.435Cell Development
SERPING11.261.55−1.784.30.436Regulation of Complement
TNFSF101.131.41−1.643.90.441Cell Development
CSF2RB0.5790.732−0.8562.010.447Integral to Membrane
NFKBIZ0.9511.21−1.423.320.45Activation of IL-6
PTK21.171.49−1.764.10.452Cell Surface Receptor-Linked Signal
Transduction
RUNX1−0.4030.52−1.420.6170.456Biopolymer Metabolic Process
CTNNB11.441.86−2.25.080.457Cell Development
FCGR2B−0.390.507−1.380.6040.46Immune Response
TNFAIP30.5110.665−0.7921.810.46IκB Kinase NFκB Cascade
IL1R1−11.32−3.581.580.463Integral to Membrane
MAPKAPK2−0.4360.573−1.560.6870.464Biopolymer Metabolic Process
CD2090.6490.854−1.032.320.465Cell–Cell Adhesion
MX1−0.5220.689−1.870.8280.466Apoptosis
PSMB70.2420.321−0.3860.8710.467Peptide Cleavage
ATG70.8811.17−1.423.180.47Biopolymer Metabolic Process
CTSC1.311.74−2.114.720.47Cytoplasm
IL18R1−0.5350.734−1.970.9030.482Membrane
CCL5−0.4670.648−1.740.8030.487Behavior
LILRB4−0.6320.877−2.351.090.488Antigen Binding
IL2RB0.5940.83−1.032.220.491Cell Development
PSMB5−0.260.364−0.9730.4530.491Peptide Cleavage
CD461.351.9−2.375.080.492Integral to Membrane
C1QB1.21.7−2.124.530.494Extracellular Region
C14orf1661.111.58−1.984.20.499Identical Protein Binding
FCGR2A/C1.081.59−2.034.190.513Phagocyte Cell Surface Receptor
LCP20.4650.687−0.8811.810.514Cell Surface Receptor-Linked Signal
Transduction
EGR1−0.4990.746−1.960.9620.519Transcriptional Regulation
CD28−0.3930.598−1.560.7790.526Cell Development
TRAF2−0.1260.194−0.5060.2530.528Macromolecular Complex Assembly
NOD20.5240.806−1.062.10.531Cytoplasm
TLR1−0.3020.468−1.220.6160.534Biosynthetic Process
SMAD50.7621.19−1.583.10.538Cell Surface Receptor-Linked Signal
Transduction
PML0.3870.607−0.8031.580.538Cell Fraction
CXCR40.4850.764−1.011.980.54Cell Development
IRF1−0.1780.283−0.7320.3760.542Biopolymer Metabolic Process
LGALS31.021.63−2.174.20.545Carbohydrate Binding
ENTPD1−0.4830.777−2.011.040.548Hemostasis
ICOSLG−0.4150.67−1.730.8980.55Cell Activation
BCL20.4960.801−1.072.060.55Cytoplasm
STAT20.8041.3−1.743.350.55Biopolymer Metabolic Process
CD86−0.4780.773−1.991.040.55Cell Proliferation
CD2760.8441.38−1.863.540.554Biosynthetic Process
MAF0.3420.562−0.7591.440.556Biopolymer Metabolic Process
IFIT2−0.5680.944−2.421.280.56Innate Immune
CD140.8931.49−2.023.810.561Apoptosis
CD811.081.8−2.454.610.562Integral to Membrane
ITGA5−0.4590.784−21.080.571Integral to Membrane
DPP40.8841.51−2.083.840.571Immune Response
CSF1R−0.3260.564−1.430.780.577Cell Proliferation
IRF30.2270.393−0.5440.9970.577Biopolymer Metabolic Process
JAK20.7361.29−1.793.260.581Anatomical Structure Development
ICAM10.3830.673−0.9361.70.581Integral to Membrane
FKBP5−0.3960.697−1.760.9690.582Cellular Macromolecule Metabolic
Process
TNFSF120.3440.608−0.8471.540.583Cell Development
MRC10.8411.5−2.093.770.587Carbohydrate Binding
SYK0.3430.612−0.8561.540.587Anatomical Structure Morphogenesis
RELB0.3160.564−0.791.420.588DNA Binding
IRF50.3690.666−0.9351.670.591Immune Transcription Factor
IRF80.3680.681−0.9671.70.601Biopolymer Metabolic Process
SKI0.6841.27−1.83.170.602Repressor of TGF-β
C30.5170.96−1.372.40.602Cell Surface Receptor-Linked Signal
Transduction
PTGS2−0.5080.945−2.361.340.603Cytoplasm
LITAF0.3540.659−0.9381.650.603Biopolymer Metabolic Process
BLNK−0.3120.586−1.460.8360.606Anatomical Structure Development
IFNAR20.1740.326−0.4660.8130.607Cell Surface Receptor-Linked Signal
Transduction
MUC10.9351.79−2.574.440.613Integral to Membrane
NOD1−0.621.2−2.971.730.616Biosynthetic Process
PDCD20.2750.534−0.7721.320.618Apoptosis
CD82−0.3260.638−1.580.9250.62Integral to Membrane
NFIL30.5931.18−1.722.910.626Biopolymer Metabolic Process
TCF70.3630.736−1.081.810.633Biopolymer Metabolic Process
ATG10−0.5761.18−2.881.730.635Autophagocytosis
LILRB10.4050.838−1.242.050.639Integral to Membrane
FCGRT0.7571.58−2.333.850.641Immune Response
ICAM3−0.320.673−1.640.9990.645Integral to Membrane
STAT10.330.695−1.031.690.645Biopolymer Metabolic Process
IL6ST0.9051.91−2.844.650.645Cell Surface Receptor-Linked Signal
Transduction
PDGFRB−0.8631.85−4.482.760.65Phosphotransferase Activity Alcohol
Group as Acceptor
CSF10.3350.722−1.081.750.653Cell Proliferation
IL1RAP0.3580.782−1.171.890.657Cellular Component Assembly
MCL10.130.288−0.4340.6950.66Cell Development
NOTCH20.3320.744−1.131.790.665Cell Development
IFNGR10.7551.71−2.594.10.668Integral to Membrane
NFATC1−0.3460.782−1.881.190.668Biopolymer Metabolic Process
SMAD3−0.2620.594−1.430.9020.668Macromolecular Complex
STAT4−0.3580.812−1.951.230.669DNA Binding
C1S−0.6071.39−3.332.110.671Endopeptidase Activity
CX3CR10.3640.859−1.322.050.681Behavior
LTB4R0.3220.77−1.191.830.685Cell Surface Receptor-Linked Signal
Transduction
ARHGDIB0.3070.741−1.151.760.687Cytoplasm
JAK30.2390.59−0.9171.40.693Biopolymer Metabolic Process
TLR80.3010.749−1.171.770.696Biosynthetic Process
RAF10.5331.33−2.073.140.697Biopolymer Metabolic Process
CSF3R0.220.556−0.871.310.701Defense Response
SIGIRR0.2720.693−1.091.630.703Membrane
ATG16L1−0.4361.13−2.661.790.709Autophagy
SOCS1−0.5531.46−3.412.30.712Biopolymer Metabolic Process
POU2F20.260.694−1.11.620.715Biopolymer Metabolic Process
HLA-DMB−0.3110.839−1.961.330.719Antigen Presentation
MAP4K2−0.521.41−3.282.240.72Biopolymer Metabolic Process
IFIH10.1860.509−0.8121.180.722B-Cell Differentiation
TGFBR10.51.38−2.213.210.726Biopolymer Metabolic Process
B2M−0.2120.59−1.370.9440.726Antimicrobial Protein
STAT5A−0.2860.804−1.861.290.729DNA Binding
IGF2R0.4361.27−2.052.920.738Cytoplasm
CD34−0.1750.516−1.190.8360.741Carbohydrate Binding
ITGB1−0.1910.563−1.290.9120.741Cell–Cell Adhesion
TLR3−0.3290.974−2.241.580.742Biosynthetic Process
CCL13−0.3110.926−2.131.50.744Behavior
LAMP30.2480.739−1.21.70.744Cell Proliferation
CCBP2−0.1740.522−1.20.850.746Behavior
IDO10.2610.787−1.281.80.747Tryptophan Catabolism
MME0.2440.739−1.21.690.748Cell–Cell Signaling
MSR10.4871.48−2.43.380.748Establishment of Localization
C7−0.2920.908−2.071.490.754Integral to Membrane
CD360.6031.88−3.094.30.755Cell Fraction
IL16−0.1470.462−1.050.7590.757Extracellular Region
CISH−0.2320.733−1.671.210.759Suppressor of Cytokine Signaling
CCL20.2560.827−1.371.880.763Biopolymer Metabolic Process
CD163−0.2650.886−21.470.771Integral to Membrane
STAT5B0.5021.69−2.83.810.772DNA Binding
SLC2A1−0.1230.419−0.9440.6980.776Cell Fraction
IRAK4−0.3721.29−2.912.160.779Activates NFκB
DUSP40.2030.708−1.181.590.78Biopolymer Metabolic Process
CEBPB−0.1580.553−1.240.9250.78Biopolymer Metabolic Process
ITGA40.2130.748−1.251.680.782Identical Protein Binding
CTSS0.220.819−1.391.830.794Cellular Macromolecule Metabolic
Process
IKZF2−0.3621.35−3.012.280.794Lymphocyte Development
LY960.1890.711−1.21.580.796Cell Surface Receptor-Linked Signal
Transduction
CLEC7A0.2140.812−1.381.810.797Cell Activation
HAVCR2−0.2070.787−1.751.340.798Th1 Surface Protein
ICAM2−0.3881.48−3.292.510.798Integral to Membrane
PSMB80.3541.35−2.330.799Antigen Presentation
C1R−0.2430.945−2.091.610.802Endopeptidase Activity
ABL1−0.3751.48−3.272.520.805Biopolymer Metabolic Process
TLR5−0.1170.466−1.030.7960.807Innate Immunity
BST2−0.4061.64−3.632.810.81IκB Kinase NFκB Cascade
IL6R0.1590.653−1.121.440.813Cell Surface Receptor-Linked Signal
Transduction
TMEM1730.1390.642−1.121.40.833Innate Immunity
IL10RA−0.180.835−1.821.460.833Interleukin Binding
CDH5−0.4011.9−4.133.330.837Cell–Cell Adhesion
PSMB100.2731.31−2.32.850.839Humoral Immune Response
NFATC30.2571.24−2.172.680.84Biopolymer Metabolic Process
BCL2L110.1080.551−0.9721.190.849Apoptosis
EDNRB−0.3081.62−3.492.870.853Integral to Membrane
TICAM10.1190.632−1.121.360.854IκB Kinase NFκB Cascade
JAK1−0.2861.56−3.352.770.858Interferon Signal Transduction
MAP4K10.1150.632−1.121.350.859Biopolymer Metabolic Process
C1QBP−0.07830.452−0.9640.8070.866Immune Response
CD970.3021.76−3.153.750.867Cell–Cell Signaling
CMKLR1−0.2711.6−3.412.860.869Behavior
ABCB1−0.1090.655−1.391.170.871Cell Fraction
ITGA6−0.05690.384−0.810.6960.885Cellular Component Assembly
CFI−0.1230.832−1.751.510.885Endopeptidase Activity
TOLLIP−0.2271.55−3.272.810.886Cell–Cell Signaling
CFH−0.1441.02−2.141.850.89Extracellular Region
NFKB1−0.1771.26−2.652.30.891Biopolymer Metabolic Process
FN1−0.09870.729−1.531.330.895Cytoplasm
IRAK30.1140.846−1.541.770.896Biopolymer Metabolic Process
CXCR60.09950.75−1.371.570.897Cell Surface Receptor-Linked Signal
Transduction
TCF40.08360.707−1.31.470.908Biopolymer Metabolic Process
LILRB50.08310.747−1.381.550.914Cell Surface Receptor-Linked Signal
Transduction
IKBKB0.05950.55−1.021.140.916Activates NFκB
IKBKE0.05590.578−1.081.190.925Biopolymer Metabolic Process
CD190.1261.36−2.542.790.928Cell Surface Receptor-Linked Signal
Transduction
IRAK1−0.1191.39−2.852.610.934Cellular Component Assembly
UBE2L3−0.1121.49−3.032.80.942Biopolymer Metabolic Process
CD530.1261.73−3.263.520.943Membrane
TRAF4−0.11.41−2.872.670.945DNA Binding
THY1−0.06370.979−1.981.860.949Cell Surface
ATG5−0.1081.75−3.533.310.952Cytoplasm
CEACAM10.04890.824−1.571.660.954Cell Fraction
CCND30.08441.55−2.963.130.958Biopolymer Metabolic Process
MAPK1−0.09321.94−3.893.70.963Behavior
CD164−0.09061.98−3.963.780.964Cell–Cell Adhesion
NOTCH10.030.657−1.261.320.965Cell Development
CRADD−0.02720.617−1.241.180.966Apoptosis
TP53−0.06821.76−3.523.380.97Cell Fraction
BAX0.06661.81−3.473.610.971Cytoplasm
CASP10.05241.43−2.752.860.971Cellular Protein Metabolic Process
IKBKG−0.04161.19−2.372.280.973Cell Development
TYK20.04671.37−2.632.730.973Biopolymer Metabolic Process
CLEC4E−0.03511.08−2.162.090.975Carbohydrate Binding
CFB0.02830.901−1.741.790.976Complement Activation
LAIR1−0.01910.739−1.471.430.98Inhibitory Receptor
CD58−0.03541.4−2.792.720.98T Cell Activation
KCNJ20.007990.384−0.7440.760.984Establishment of Localization
PLAU−0.0160.788−1.561.530.984Behavior
TRAF30.01120.57−1.111.130.985Apoptosis
CCR10.02711.38−2.682.740.985Cell–Cell Signaling
CD9−0.01380.765−1.511.480.986Anatomical Structure Morphogenesis
APP−0.006680.389−0.7690.7560.987Cell Surface
ARG20.01060.761−1.481.50.989Cytoplasm
IL4R0.01591.64−3.213.240.992Immune Response
CD40.01141.48−2.882.910.994Cell Activation
CYBB0.002690.554−1.081.090.996Defense Response
KIT−4.48 × 10 −160.576−1.131.131Phosphotransferase Activity Alcohol
Group as Acceptor

References

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Children 09 00381 g001 550
Figure 1. Representative diaminobenzidine (brown) and hematoxylin (blue) staining (A–D, 400X magnification), and staining quantification (E–H) for placental NGAL. (A) BPD, TE group (n = 5), with strong (3 to 4+) NGAL-positive staining in a chorionic plate and subchorionic space. (B) BPD, no TE group (n = 9), with mild (1–2+) NGAL-positive staining only in subchorionic space. (C) No BPD, TE group (n = 10), with strong (3–4+) NGAL-positive staining in the chorionic plate and subchorionic space. (D) No BPD, no TE group (n = 12), with rare (0–1+) NGAL-positive staining only in subchorionic space. (E,F) Quantification of chorionic plate staining stratified by maternal smoking status and subgroup analysis. (G,H) Quantification of subchorionic space staining stratified by maternal smoking status and subgroup analysis. NGAL—neutrophil gelatinase-associated lipocalin, BPD—bronchopulmonary dysplasia, and TE—tobacco exposure.
Figure 1. Representative diaminobenzidine (brown) and hematoxylin (blue) staining (A–D, 400X magnification), and staining quantification (E–H) for placental NGAL. (A) BPD, TE group (n = 5), with strong (3 to 4+) NGAL-positive staining in a chorionic plate and subchorionic space. (B) BPD, no TE group (n = 9), with mild (1–2+) NGAL-positive staining only in subchorionic space. (C) No BPD, TE group (n = 10), with strong (3–4+) NGAL-positive staining in the chorionic plate and subchorionic space. (D) No BPD, no TE group (n = 12), with rare (0–1+) NGAL-positive staining only in subchorionic space. (E,F) Quantification of chorionic plate staining stratified by maternal smoking status and subgroup analysis. (G,H) Quantification of subchorionic space staining stratified by maternal smoking status and subgroup analysis. NGAL—neutrophil gelatinase-associated lipocalin, BPD—bronchopulmonary dysplasia, and TE—tobacco exposure.
Children 09 00381 g001
Children 09 00381 g002 550
Figure 2. ELISA for NGAL within placental tissue comparing (A) TE status and further comparing (B) TE and BPD status. (A) Compares TE group (n = 17) and No TE group (n = 22)—the NGAL is significantly higher in the TE group compared to No TE group (**** p < 0.0001). (B) Further subgroup analysis based on BPD status had significantly higher NGAL in the BPD TE group compared to No BPD No TE group (** p < 0.01). BPD No TE group also had significantly higher levels of NGAL as compared to No BPD No TE infants (*** p < 0.001). ELISA—enzyme-linked immunosorbent assay, NGAL—neutrophil gelatinase-associated lipocalin, BPD—bronchopulmonary dysplasia, and TE—tobacco exposure.
Figure 2. ELISA for NGAL within placental tissue comparing (A) TE status and further comparing (B) TE and BPD status. (A) Compares TE group (n = 17) and No TE group (n = 22)—the NGAL is significantly higher in the TE group compared to No TE group (**** p < 0.0001). (B) Further subgroup analysis based on BPD status had significantly higher NGAL in the BPD TE group compared to No BPD No TE group (** p < 0.01). BPD No TE group also had significantly higher levels of NGAL as compared to No BPD No TE infants (*** p < 0.001). ELISA—enzyme-linked immunosorbent assay, NGAL—neutrophil gelatinase-associated lipocalin, BPD—bronchopulmonary dysplasia, and TE—tobacco exposure.
Children 09 00381 g002
Table
Table 1. Maternal and Neonatal Demographic Data by Composite Outcome.
Table 1. Maternal and Neonatal Demographic Data by Composite Outcome.
Tobacco Exposure
NoYes
(n = 22)(n = 17)Total (n = 39)p Value
Birth weight, g (SD)1.141 (458)1.125 (464)1.134 (454)>0.9
Birth length, cm (SD)36.3 (4.9)36.6 (4.6)36.5 (4.7)0.9
Head circumference, cm (SD)25.56 (2.87)25.48 (3.25)25.53 (3.00)>0.9
Gestational age, wk (SD)28.76 (2.64)28.40 (2.68)28.60 (2.63)0.5
Maternal age, yr (SD)25.7 (4.9)29.8 (6.6)27.5 (6.0)0.048
Sex 0.4
 F17 (77%)10 (59%)27 (69%)
 M5(23%)7(41%)12(31%)
Maternal ethnicity 0.4
 Black5 (23%)3 (18%)8 (21%)
 Black, Native American0 (0%)1 (5.9%)1 (2.6%)
 Hispanic6 (27%)2 (12%)8 (21%)
 Latino, White1 (4.5%)0 (0.0%)1 (2.6%)
 Native American1 (4.5%)3 (18%)4 (10%)
 White9 (41%)7 (41%)16 (41%)
 White, Native American0 (0%)1 (5.9%)1 (2.6%)
Antenatal steroid, yes9 (41%)9 (53%)18 (46%)0.4
Mode of delivery, C-Section11 (50%)10 (59%)21 (54%)0.8
Intubated in delivery room, yes9 (41%)9 (53%)18 (46%)0.7
Intubated in NICU, yes7 (32%)4 (24%)11 (28%)0.7
PDA medical treatment, yes9 (41%)2 (12%)11 (28%)0.073
PDA surgical treatment, yes1 (4.5%)0 (0%)1 (2.6%)>0.9
IVH grade 3 or 4, yes2 (9.1%)1 (5.9%)3 (7.7%)>0.9
Threshold ROP, yes3 (14%)1 (5.9%)4 (10%)0.6
IUGR <10th percentile, yes3 (14%)0 (0%)3 (7.7%)0.2
Death or BPD, yes15 (38%)9 (41%)6 (35%)>0.9
All data are presented as the mean (standard deviation) or n (%). Statistical tests performed: Wilcoxon rank-sum test; chi-square test of independence; Fisher’s exact test. BPD—bronchopulmonary dysplasia, NICU—neonatal intensive care unit, PDA—patent ductus arteriosus, IVH—intraventricular hemorrhage, ROP—retinopathy of prematurity, and IUGR—intrauterine growth restriction.
Table
Table 2. Maternal Complications and Tobacco Exposure by Composite Outcome.
Table 2. Maternal Complications and Tobacco Exposure by Composite Outcome.
Tobacco Exposure
NoYes
(n = 22)(n = 17)Total (n = 39)p Value
Maternal diabetes, yes3 (14%)2 (12%)5 (13%)>0.9
Maternal hypertension, yes3 (14%)2 (12%)5 (13%)>0.9
Prolonged rupture of membranes (>18h), yes4 (18%)3 (18%)7 (18%)>0.9
Chorioamnionitis, yes10 (45%)10 (59%)20 (51%)0.6
Antepartum hemorrhage, yes4 (18%)4 (24%)8 (21%)0.7
Marijuana use, yes1 (4.5%)1 (5.9%)2 (5.1%)0.4
Illicit drugs, yes0 (0%)2 (12%)2 (5.1%)0.4
All data are presented as n (%). Statistical tests performed: Fisher’s exact test; chi-square test of independence.
Table
Table 3. Significantly Differential Gene Expressions (TE vs. No TE).
Table 3. Significantly Differential Gene Expressions (TE vs. No TE).
GeneAnnotationLog2-Fold ChangeSEpTentative Function
IL8Interleukin 84.770.8980.00034Neutrophil Chemotaxis
S100A9S100 Calcium-Binding Protein A91.720.3390.000477Leukocyte Activation
S100A8S100 Calcium-Binding Protein A83.330.9120.00447Leukocyte Activation
IL1RL1Interleukin 1 Receptor Like 1−3.411.10.0115IL-33 Receptor/Inflammatory Signaling
CXCL10C-X-C Motif Chemokine Ligand 103.061.090.0187Peripheral Immune Cell Activation
CD44CD44 Molecule1.70.6140.02Cell–Cell Signaling
TNFRSF10CTNF Receptor Superfamily Member 10c1.530.5620.0212Anti-Apoptosis
PLAURPlasminogen Activator, Urokinase Receptor1.780.6760.0251Plasminogen Activation/Extracellular Matrix Degradation
IRF7Interferon Regulatory Factor 71.080.4150.0261Anti-viral Immune Response
MALT1MALT1 Paracaspase−0.5720.220.0263NF-κB Activation
LILRB3Leukocyte Immunoglobulin-Like Receptor B32.040.7950.0281Anti-B Cell Activation
HLA-DRB1Major Histocompatibility Complex, Class II, DR Beta 12.741.140.0374Antigen Presentation
HLA-DRB3Major Histocompatibility Complex, Class II, DR Beta 31.780.7460.0384Antigen Presentation
HFEHomeostatic Iron Regulator−1.250.5290.0394Regulates Iron Absorption
TNFSF15TNF Superfamily Member 15−1.430.6040.0397Endothelial Inflammatory Signaling
CD99CD99 Molecule1.120.4750.0406Leukocyte Migration
PTPRCProtein Tyrosine Phosphatase Receptor Type C1.940.8470.045T Cell Activation
PTAFRPlatelet-Activating Factor Receptor−20.8810.0466Receptor for Inflammatory PAF
ZBTB16Zinc Finger- and BTB Domain-Containing 16−2.010.8880.0469Transcription Repression/Myeloid Maturation
PLA2G2APhospholipase A2 Group IIA−2.250.9970.0479Phospholipid Metabolism
BPD—bronchopulmonary dysplasia, TE—tobacco exposure, and SE—standard error.
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Box, D.M.; Makkar, A.; Yu, Z.; Chaaban, H.; Tran, H.H.; Burge, K.Y.; Eckert, J.V. Placental Neutrophil Infiltration Associated with Tobacco Exposure but Not Development of Bronchopulmonary Dysplasia. Children 2022, 9, 381. https://doi.org/10.3390/children9030381

AMA Style

Box DM, Makkar A, Yu Z, Chaaban H, Tran HH, Burge KY, Eckert JV. Placental Neutrophil Infiltration Associated with Tobacco Exposure but Not Development of Bronchopulmonary Dysplasia. Children. 2022; 9(3):381. https://doi.org/10.3390/children9030381

Chicago/Turabian Style

Box, David M., Abhishek Makkar, Zhongxin Yu, Hala Chaaban, Henry H. Tran, Kathryn Y. Burge, and Jeffrey V. Eckert. 2022. "Placental Neutrophil Infiltration Associated with Tobacco Exposure but Not Development of Bronchopulmonary Dysplasia" Children 9, no. 3: 381. https://doi.org/10.3390/children9030381

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