In utero exposures to mint-flavored JUUL aerosol impair lung development and aggravate house dust mite-induced asthma in adult offspring mice

Abstract

There are few reports concerning electronic nicotine delivery system (ENDS) use during pregnancy and no studies on asthma in prenatally JUUL-exposed offspring. Here, we tested the hypothesis that in utero JUUL exposure causes unfavorable birth outcomes and lasting pulmonary health effects in adult offspring. BALB/c dams were exposed to either air or mint-flavored JUUL aerosol, 1-hr/d, 20 consecutive days during gestation. Offspring were sacrificed on post-natal day (PND) 0 or at 11-week of age, following house dust mite (HDM) challenge. Gene expression was assessed in the uterine/placental tissue of the dams and lung responses were assessed in offspring at PND0 and at 11 weeks of age. JUUL-exposed offspring exhibited decreased body weights and lengths at PND0. These birth outcomes were accompanied by dysregulation of 54 genes associated with hypoxia and oxidative stress in the uterine/placental tissues of JUUL-exposed dams, as well as 24 genes in the lungs of the offspring related to Wnt signaling, plus 9 genes related to epigenetics, and 7 genes related to inflammation. At 11 weeks of age, JUUL + HDM exposed mice exhibited pulmonary inflammation when compared to their respective air + HDM controls. Additionally, the JUUL + HDM exposure dysregulated several genes associated with allergies and asthma. Further, the JUUL + HDM females showed decreased methylation of the promoter region of the Il10ra gene. Taken together, our mouse model shows that inhalation of JUUL aerosols during pregnancy affects the intrauterine environment, impairs lung development, and heightens the effects of allergic airway responses later in life.

Introduction

From 2017–2019, JUUL a fourth-generation electronic nicotine delivery system (ENDS) device, first marketed in 2015, maintained the status of being the most purchased ENDS device in the United States (CDC, 2019). Initially this device gained popularity among teenagers and young adults due to the variety of flavored ‘pods’ offered, and to its design, which resembles a USB flash drive. JUUL became the reported “device of choice” for the 27.5% of American middle and high school students who used electronic-cigarettes (e-cigs) during that time (CDC, 2019). Additionally, recent studies put the prevalence of JUUL use between 21% and 35% for college students in the United States (Roberts et al., 2020; Ickes et al., 2020). Although JUUL has eliminated sweet flavors such as mango, crème brûlée, and mint, pod-based ENDS devices still dominate ENDS retail sales and maintain popularity among young adults. Flavored disposable ENDS, such as Vuse, Blu, Mr. Vapor, and Puff Bar, are still available in a variety of flavors, including mint. In the United States, 42.1% of women are age 24 years or younger at the birth of their first child (Mathews & Hamilton, 2016). Research published between 2007 and 2017 show that up to 15% of American women use ENDS while pregnant (Schmidt et al., 2020). In contrast to freebase nicotine found in traditional cigarettes and e-liquids, JUUL contains nicotine salts, consisting of a base nicotine plus a weak organic acid (e.g., benzoic acid), allowing users to inhale aerosols with higher concentrations of nicotine, but without a harsh “throat hit” that many users experience when inhaling freebase nicotine (National Academies of Sciences, Engineering, and Medicine, 2018; Pankow et al., 2017). Thus, JUUL devices contain highly concentrated levels of the developmental toxin nicotine, with pods containing either ~30 mg/mL or up to 59 mg/mL of nicotine salt, the latter being equivalent to the nicotine content of 1 pack of cigarettes (England et al., 2017; Talih et al., 2019). No experimental study thus far has investigated the impacts of JUUL nicotine salt-rich aerosol exposure on susceptible populations such as expectant mothers and their offspring.

Based on the developmental origin of adult diseases paradigm (Barker hypothesis), many aspects of an individual’s overall health are believed to be rooted in the environmental conditions in which fetal and early life development occurred (Jaakkola & Gissler, 2004; Infante-Rivard et al., 1999; Gilliland et al., 2001). Multiple studies have linked maternal tobacco smoking with offspring asthmatic responses later in life (Martino & Prescott, 2011; Neuman et al., 2012; Prescott & Clifton, 2009). Offspring exposed to cigarette smoke in utero have approximately double the likelihood of developing asthma in early life when compared to unexposed children (Neuman et al., 2012; Carroll et al., 2007). Newborns exposed to cigarette smoke only in utero exhibit alterations in lung function, including decreased respiratory flow, decreased respiratory compliance and modifications in tidal volume (Hoo et al., 1998; Stocks et al., 2013; McEvoy et al., 2014). As children exposed to maternal tobacco smoke mature, the rate of respiratory infections, wheezing, and childhood asthma also increase (Stoddard & Gray, 1997). In offspring who develop allergies and asthma, modified immune function is evident, even in neonatal stages, implying that these modifications result from the intrauterine environment (Martino & Prescott, 2010; Martino & Prescott, 2011; Prescott & Clifton, 2009; Prescott & Szeszko, 2019). Thus, prenatal influences, including exposures to cigarette smoke, may predispose offspring to developing allergies and asthma as children or as adults (Martino & Prescott, 2011; Prescott & Clifton, 2009).

Recently, animal studies have linked gestational use of e-cig with delayed implantation, as well as reduced weight gain, impaired lung growth, altered DNA methylation and changes in lung cytokine expression in offspring (Chen et al., 2018; Wetendorf et al., 2019; McGrath-Morrow et al., 2015; Orzabal et al., 2022). Further, we previously showed that in utero exposures of mice to cinnamon-flavored e-cig aerosol containing 36 mg/mL of nicotine lead to reduced physical growth of the newborn, impaired lung structure at birth, altered lung function at 11 days of age, delayed alveologenesis, plus dysregulation of Wnt and epigenetic signaling pathways at birth and in early neonatal life (Noël et al., 2020; Cahill et al., 2021). Thus, prenatal exposures to e-cig aerosol can have both immediate and lasting effects on lungs of mouse offspring. Another study, which investigated the effects of maternal e-cig exposure (mating, gestation, plus lactation), on subsequent asthma in offspring, concluded that perinatal e-cig exposure magnified allergy-related responses in the respiratory systems of maternally exposed BALB/c mice (Sharma et al., 2017). This was demonstrated by the heightened effects of ovalbumin-induced asthma, which e-cig exposed offspring exhibited with increases in influx of eosinophils, airway hyperresponsiveness and airway remodeling (Sharma et al., 2017). Additionally, recent evidence implies that prenatal exposure to nicotine is a major contributing factor to epigenetic changes and the impairment of pulmonary function in offspring (Rehan et al., 2007; Chhabra et al., 2014; Sekhon et al., 2001; Pierce & Nguyen, 2002; Suter et al., 2015; Orzabal and Ramadoss, 2019). Therefore, it is highly relevant to investigate whether prenatal exposure to JUUL – which contains notably high concentrations of nicotine salt – would also cause, through epigenetic mechanisms, aggravated asthmatic responses in the respiratory system of offspring.

The present study was designed to investigate the hypothesis that newborn mice exposed to JUUL aerosol in utero will exhibit impaired development, related to both physical growth and lung-specific, as well as alterations to molecular markers in the lungs – as a possible result of uterine/placental tissues insufficiency in dams. Additionally, the present study seeks to test whether exposure to JUUL in utero may have harmful pulmonary effects on offspring later in life (11 weeks old), including heightened aggravation of allergic asthma. Thus, the objectives here were 1) to determine whether a correlation exists between the effects at birth (post-natal day 0, PND0) in BALB/c mice offspring and molecular changes induced by mint-flavored JUUL aerosol exposures in the uterus/placenta of the dams; and 2) to evaluate the detrimental effects induced by in utero mint-flavored JUUL aerosol exposures of male & female mice at PND0 and after full maturity (11 weeks) by analyzing genes associated with lung organogenesis and epigenetic modifications at birth and genes associated with allergy and asthma in adulthood.

Materials and Methods

JUUL Aerosol Production & Chemical Characterization

We used mint-flavored JUUL pods (JUUL labs, inc., United States) that contained 5% of nicotine salt. Mouse exposures were conducted in 5-liter whole-body exposure chambers, as previously described (Noël et al., 2018; Noël et al., 2020; Cahill et al., 2021). Dams were exposed to either mint-flavored JUUL aerosol or HEPA-filtered air for 20 consecutive days during pregnancy, ceasing before the birth of offspring on day 21 or PND0. JUUL aerosol was generated by a JUUL device connected to a peristaltic pump (Masterflex^™ 77916-20, Fisher Scientific). The system was set to generate one puff every 30 seconds for a duration of 5 seconds, yielding a 55 mL puff volume. Test atmosphere was recorded both instantaneously, by a MicroDustPro (Casella), as well as gravimetrically, via a 25 mm fiber filter (AP4002500, MilliporeSigma), to determine the total particulate matter (TPM) concentrations during each exposure session. Figure 1A shows our JUUL aerosol in vivo exposure system. Based on exposure chamber TPM levels, the average concentration per puff was 0.15 mg/puff ± 0.03 (SEM). Exposure characterization data are listed in Table 1. We also performed chemical characterization of the mint-flavored JUUL aerosol for nicotine, propylene glycol, glycerin, carbonyls and organic acids, as previously described in Pinkston et al., (2020). We collected 40 puffs of the JUUL aerosol generated by the peristaltic pump, which was directly connected to a holder containing a Cambridge filter pad, which was sampled at a flow rate of 1 L/min. Nicotine was quantified using gas chromatography (GC) with a flame ionization detector (GC-FID) techniques, while carbonyls were analyzed by high performance liquid chromatography (HPLC), according to the EPA method TO-11A (Enthalpy Analytical, LLC, Durham, NC). For organic acids, the peristaltic pump generating the JUUL aerosol was directly connected to a fritted glass impinger containing 30 mL acidified 2,4-dinitrophenylhydrazine aerosol trapping solution. Subsequent analysis of the trapping solution was analyzed by ion chromatography. All samples were collected on site at the Inhalation Research Facility at Louisiana State University (LSU), School of Veterinary Medicine (SVM) and shipped overnight on dry ice to Enthalpy Analytical, LLC, which conducted all the chemical analyses. The chemical profile of the mint-flavored JUUL aerosol is showed in Figure 2.

Figure 1.

A) JUUL aerosol in vivo exposure system experimental set-up. B) Timeline of study investigating the pregnancy outcomes and pulmonary effects of murine exposure to JUUL in utero and HDM later in life.

Table 1.

Exposure characterization.

	Temperature (°C)	Relative humidity (%RH)	Total particulate matter (TPM) (mg/puff)
Air	25.8 ± 2.4	73.4 ± 7.6	--
Mint-flavored JUUL aerosol	25.3 ± 1.9	75.7 ± 6.3	0.15 ± 0.03

Values are expressed as mean ± standard error of the mean (SEM).

Figure 2. Mint-flavored JUUL aerosol chemical analysis profile.

Chemical characterization of the mint-flavored JUUL aerosol for nicotine, propylene glycol, glycerin, carbonyls and organic acids was performed. High levels of glycerin, propylene glycol and benzoic acid (> 40 μg/puff) were found in the mint-flavored JUUL aerosol.

Animal Exposure Protocols

For the experimental animal exposures, we used 12-week-old female BALB/c mice (Charles River, Wilmington, MA), which were exposed to either mint-flavored JUUL aerosol or HEPA-filtered air for 1-hour per day for 20 consecutive days during gestation. In total, 9 dams in the JUUL group and 8 dams in the air group gave birth. At birth (PND0), the numbers of live and/or stillborn neonates were documented. Individual neonates were each weighed and measured crown-to-rump. Animals were euthanized using Beuthanasia-D (Schering-Plough, NJ) via an intraperitoneal injection, followed by decapitation. Select dams (n = 4 per group), whose entire litter was necropsied at PND0 were also necropsied for tissue collection. Remaining dams (n = 5 for the JUUL group and n = 4 for the air group), were able to nurse their offspring until weaning. At 8 weeks of age, offspring were treated with intranasal instillation of either HDM to induce asthma or 1x phosphate buffered saline (PBS) for controls, once a week for 3 consecutive weeks. At 11 weeks of age, following HDM or PBS treatment, offspring were euthanized with Beuthanasia-D (Schering-Plough, NJ) via an intraperitoneal injection. Blood was collected and lungs were excised and either put on dry ice, stored in RNAlater, or formalin fixed for subsequent analysis. Experimental timeline is showed in Figure 1B. All mice were housed in an AAALAC-approved animal care facility at the Louisiana State University School of Veterinary Medicine under a 12-hour light/dark cycle (from 6:00 am to 6:00 pm). The mice had access to water and food ad libitum, except during the 1-hour exposure periods. Mice were housed and handled in accord with the NIH Guide for the Care and Use of Laboratory Animals. All procedures and protocols were approved by the Louisiana State University Institutional Animal Care and Use Committee.

House Dust Mite (HDM) Challenge (8–11 weeks)

Allergic asthma was induced in mice using HDM extract (D. pteronyssinus, catalogue number: XPB70DA2.5, Stallergenes Greer, Lenoir, NC) resuspended in 1x PBS. Mice were anesthetized, and the suspension of 25 μg of HDM was delivered intranasally with a volume of 10 μL per each nostril. The mice were challenged with HDM once weekly for three consecutive weeks. The respective control mice received intranasal instillation of 1x PBS.

Bronchoalveolar Lavage Fluid (BALF) Collection and Subsequent Cytology Analysis

Following euthanasia, BALF samples from the 11-week-old offspring were collected from the lungs via intratracheal instillation of PBS and placed immediately on ice. Lung lavages were performed with 0.5 mL PBS, administered twice, then centrifuged to obtain a cell pellet for cytological analysis, as previously described (Noël et al., 2017). Smears were stained with a modified Wright’s stain and BALF differentials were assessed on 300 cells. The remaining BALF supernatant was stored at −80°C for subsequent analyses.

Histopathology of Lungs and Morphometric Analysis of Neonatal Lung Tissue

After collections, the lungs of the PND0 and 11-week-old offspring were preserved using 10% formalin, following inflation and pressure-fixation. Tissues were sectioned and stained using hematoxylin and eosin (H&E). Morphometric analysis of PND0 lungs was performed by calculating the parenchymal airspace-to-tissue ratio, as previously described in Noël et al., (2020). Images were obtained with a Nikon Eclipse E400 microscope (Nikon, Tokyo, Japan). Lung tissue from 11-week-old mice were evaluated for intra-alveolar inflammatory cell infiltrate, tissue involvement, and alveolar septae to perivascular space ratio, using a scoring system of 0 to 4, where 0 = absent, 1 = minimal, 2 = mild, 3 = moderate and 4 = marked. Histopathological evaluations were performed by board-certified veterinary pathologists who were blinded to the treatment groups.

Immunochemistry of Placental 11-beta-hydroxysteroid dehydrogenase-2 (HSD2)

After collections, the uterine/placental tissue of the dams were preserved using 10% formalin. For tissue sectioning, all paraffin embedded sections were cut at 4 microns and air-dried at room temperature, similarly to what was previously described in Noël et al., (2020). In brief, slides were placed in a 60 °C tissue-drying oven for 45 minutes before deparaffinization and antigen retrieval. Deparaffination was done by washing the dry slides in 3 changes of xylene for 5 minute each at room temperature. Heat-induced epitope retrieval occurred at 100 °C for 20 minutes. Then slides were treated with 3% hydrogen peroxide (H₂O₂) in tris buffered saline (TBS) with tween for 10 minutes at room temperature, and then washed. The primary antibody: HSD2, [1:300] (rabbit polyclonal, catalog #LS-B2135, Lot ID: 125517, LifeSpan BioSciences, (LSBio) Inc., Seattle, WA) was incubated at 4 °C. Then, 3,3′-diaminobenzidine (DAB) [1:24], used as a chromogen, was incubated for 5 minutes at room temperature. This was followed by distilled water rinse and counterstaining with Hematoxylin.

Uterus/Placenta and Lung Extraction of RNA

At birth, uterine/placental tissue and lungs from dams, as well as lungs from offspring (at PND0 and 11 weeks of age), were collected and placed in RNA Later to preserve the tissues for RNA extraction. RNA was extracted from samples using RNAeasy Mini Kit (QIAGEN, USA) per the manufacturer’s instructions. Subsequently, quantity and purity of the uterine/placental and lung RNA obtained were verified using a NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE).

Gene Expression Analysis – qRT-PCR

We assessed the expression levels of selected inflammatory genes via quantitative PCR on cDNA samples of lung homogenates from the dams and their respective offspring at PND0. The cDNA was prepared as described in Cahill et al., (2021), using a high-Capacity cDNA kit (Applied Biosystems, P/N 4374966). The qPCR reaction mixture contained TaqMan^™ probe (Applied Biosystems) and samples were run on a QuantStudio^™ 6 (Software v1.3). Relative gene expression (ΔΔC_T) for each gene was normalized to hypoxanthine guanine phosphoribosyltransferase (Hprt1) expression and reported as fold change over control [(2^-ΔΔCT)].

Gene Expression Analysis – RT2 PCR arrays

To prepare cDNA, extracted RNA was treated with DNAse and reverse-transcribed using the RT2 First Strand Kit (Qiagen 330401) according to manufacturer’s instructions. High quality RNase- free water was then used to dilute the resultant cDNA to a volume of 111 μl. Finally, 102 μl of the cDNA sample was combined with RT2 SYBR Green qPCR Master mix (Qiagen 330503). The mixture of diluted cDNA + SYBR Green qPCR Master mix (Qiagen 330503) was used to determine the expression of 84 Hypoxia genes (Catalog number 330231 PAMM032Z) and 84 Oxidative Stress (Catalog number 330231 PAMM-065ZA) genes in the uterine and placental tissue of the dams collected postpartum. Additionally, 84 Wnt Signaling genes (Catalog number 330231 PAMM-043ZA) and 84 Epigenetic Chromatin Modification genes (Catalog number 330231 PAMM-085ZR) were analyzed in the lungs of offspring collected at PND0, as well as 84 Asthma and Allergy related genes (Catalog number 330231 PAMM-067Z) in lung samples at 11 weeks, using RT2 profiler PCR arrays (Qiagen, USA) according to the manufacturer’s instructions. The resulting threshold cycle (Ct) values were obtained and used to calculate both gene expression and fold change, using the ΔΔCt method with the Qiagen GeneGlobe data analysis software. Housekeeping genes Actb, B2m, Gapdh, and Gusb, were used for normalization.

Gene Methylation Assay

The EpiTect^® Methyl II Signature PCR Array: Mouse Inflammatory Response and Autoimmunity (SA Bioscience, Qiagen, Hilden, Germany) was used according to the manufacturer’s instructions to identify gene promoter methylation status of 22 different inflammatory response and autoimmunity genes (Chemokines: Cx3cl1, Cxcl12, Cxcl14, Cytokines: Il7, Il11, Il13, Il18, Inha, Cytokine Receptors and Associated Proteins: Il10ra, Il13ra1, Il17ra, Il4ra, Il6ra, Il6st, Other Inflammatory Response & Autoimmunity Genes: Atf2, Fadd, Gata3, Tyk2, Ltb, Mif, Tbck, Tgfb1).

Genomic DNA was isolated using Qiagen DNeasy Blood and Tissue Kit. DNA samples were treated with RNase to remove RNA. Extracted DNA was assessed for purity and concentration by Nanodrop-One from Thermo Scientific. For reaction digestions, we prepared a reaction mix without enzymes from 1µg genomic DNA, 26µL of 5x Restriction Digestion Buffer, and RNase-DNase free water to make the final volume 120µL. Four reaction digestions were carried out: no-enzyme (Mo), methylation sensitive enzyme (Ms), methylation dependent enzyme (Md) and methylation sensitive and dependent enzymes (Msd). Each one consists of 28uL of the previous reaction mix and 2µL of RNase-DNase free water for Mo digest; 28µL of the previous reaction mix, 1µL of methylation sensitive enzyme A and 1µL of RNase-DNase free water for Ms digest; 28µL of the previous reaction mix, 1µL methylation sensitive enzyme B and 1µL of RNase-DNase free water for Md digest; 28µL of the previous reaction mix, 1µL of methylation sensitive enzyme A and 1µL of methylation sensitive enzyme B to make the final volume 30µL for Msd digest. The components were mixed gently by pipette and briefly spun down. The mixture was incubated at 37°C overnight, followed by heating for 20 min at 65°C to inactivate the enzymes.

For PCR, 300 µL of SYBR Green master mix and 300 µL of RNase-DNase free water were mixed with 30µL each of the 4 digestions (Mo, Ms, Md, and Msd). 25 µL sample volume were added to the 96 well plate. Real-time PCR was performed in an ABI 7300 (Applied Bio systems, Foster City, CA) with the cycling conditions of 95°C for 10 min for 1 cycle, 99°C for 30 s followed by 72°C for 1 min for 3 cycles, 97°C for 15 s followed by 72°C for 1 min for 40 cycles. For the dissociation curve, segment three was added to the program: 95°C for 1 min and one cycle, then, annealing at 55°C for 30 s and another denaturation at 95°C for the same period and one cycle. Data were analyzed using the EpiTect^® Methyl II PCR Array System. The system provides an integrated Excel-based template that automatically performs all ΔCt based calculations from the raw threshold cycle (Ct) values to determine gene specific DNA methylation status, using MethylScreen^™ technology. The Excel template normalizes the Ct values of both digests with the mock digestion values to calculate and report the percentage of the DNA that is methylated and un-methylated.

Gene Network and Canonical Pathway Analysis

Uterine/placenta gene expression data gathered from the Oxidative Stress and Hypoxia RT2 Profiler Arrays, along with offspring lung gene expression data gathered from the Wnt Signaling Pathway, Epigenetic Chromatin Modification and Asthma and Allergy RT2 Profiler Arrays were analyzed using QIAGEN Ingenuity Pathway Analysis (IPA) to determine most significantly enriched canonical pathways and networks of gene interactions.

Statistical Analyses

Results were analyzed using either: 1) the Student T-test for comparisons of two groups, or 2) ANOVA followed by the Tukey’s test for comparisons of multiple data sets. Outcomes are formatted as mean ± standard error of the mean (SEM) for all biological outcomes, and as median ± interquartile range (IQR) for assigned values (i.e., histopathology scores). GraphPad Prism 8 Software (GraphPad Software, San Diego, CA) was used to perform all statistical analyses. Results with a p < 0.05 were considered statistically significant. Gene expression results with fold-change > +/− 1.5 compared to respective air control group were considered significant.

Results

Mint-flavored JUUL aerosol exposures during pregnancy cause oxidant/antioxidant imbalances in the uterine/placental environment

Placental 11-β-hydroxysteroid dehydrogenase type 2 (HSD2) is a key enzyme that controls fetal development and whose reduced activity is associated with fetal growth restriction (Yang et al., 2006). Placental HSD2 protects against the fetal growth inhibiting effect of cortisol (Kajantie et al., 2003; Leslie et al., 2015). Maternal smoking has previously been linked to decreased placental HSD2 levels which correlated with low birth weight of newborns (McTernan et al., 2001; Murphy et al., 2002; Yang et al., 2006). The expression of the HSD2 enzyme, as visualized by immunohistochemistry, was slightly decreased, albeit non-significantly, in JUUL aerosol exposed dams compared to air controls (Figure 3A, B). Further, imbalances in the oxidant/antioxidant balance of the uterus and placenta have previously been linked to placental dysfunction, which can cause adverse pregnancy outcomes (Agarwal et al., 2012). We found that after gestational exposure to mint-flavored JUUL aerosol, 27 genes associated with oxidative stress and 27 genes related to hypoxia exhibited significant dysregulation in the uteri/placentas of postpartum dams (Figure 3C). Of those 54 dysregulated genes, 13 were up-regulated while 41 were down-regulated in the JUUL aerosol exposed dams compared to the air controls. Pathway analyses of these genes using IPA found that dysregulation was most significantly associated with vasoconstriction of blood vessel (9 genes), morphology of placenta (9 genes), disease of placenta (7 genes), and disorder of pregnancy (15 genes) (Figure 3D). Overall, these results demonstrate that mint-flavored JUUL aerosol exposures during pregnancy cause oxidant/antioxidant imbalances in the uterine/placental environment.

Figure 3. Mint-flavored JUUL aerosol exposures during pregnancy cause oxidant/antioxidant imbalances in the uterine/placental environment.

A) Mint-flavored JUUL aerosol exposures slightly decreased the placental 11-beta-hydroxysteroid dehydrogenase-2 (HSD2) enzyme expression in the placenta. HSD2 protein expression was assessed by immunohistochemistry techniques; images of placental tissue with HSD2 antibody (20x magnification). B) Quantification of the positively stained cells for HSD2. N=4 per group. Data are expressed as mean ± SEM. C) Exposure to JUUL throughout gestation dysregulates the expression of uteri/placenta genes related to oxidative stress and hypoxia in dams. Gene expression of mice exposed to JUUL during pregnancy was normalized using gene expression results from the uterine/placental tissue of dams exposed only to HEPA-filtered air. Data expressed as fold-change compared to air control. N = 4 per group. Genes with at least a 1.5-fold up- or down-regulation were considered to have significant differences of expression compared with controls. D) The gene interaction networks altered by in utero JUUL exposure are correlated with multitude pathways that affect pregnancy outcomes. Ingenuity Pathway Analysis (IPA) of dysregulated genes in dams exposed to JUUL throughout pregnancy revealed that those dams exhibited dysregulation of genes associated with vasoconstriction of blood vessel, morphology of placenta, disease of placenta, and disorder of pregnancy, compared to the uterine/placental tissue of dams exposed only to HEPA-filtered air throughout pregnancy.

In utero mint-flavored JUUL aerosol exposure decreases body weight and length of offspring at birth

Although the difference in stillbirth numbers was not statistically significant, birth length (Figure 4A, B) and weight (Figure 4C) of neonates subjected to mint-flavored JUUL aerosol in utero were found to be significantly decreased (p < 0.05) when compared to their air-exposed counterparts – despite litter sizes being comparable. The birth weight of in utero JUUL exposed offspring was on average 10.2% lower than that of in utero air exposed offspring. This decreased body weight was sustained through 2 weeks of age (p < 0.05) (Figure 4D). These data show that exposure to mint-flavored JUUL aerosol in utero impairs the physical growth of newborns through early neonatal life.

Figure 4. Exposure to mint-flavored JUUL aerosol in utero impairs the physical growth of newborns through early neonatal life.

(A) Exposure to JUUL aerosol in utero led to a significant decrease in birth length, measured crown-to-rump, when compared to air exposed controls. Statistical analysis performed using Student’s T-Test. Data are expressed as mean ± standard error of the mean (SEM). p < 0.05 is considered statistically significant. * p < 0.05. B) Representative pictures of the body length of in utero JUUL aerosol or air exposed offspring at birth. PND0 length N = 20–29 per group. C & D) Body weight of mice exposed to either mint-flavored JUUL aerosol or filtered air in utero, recorded once weekly from birth (PND0) through 4 weeks of age (PND28). C) Significantly decreased birth weight of in utero JUUL aerosol-exposed offspring compared with the neonates exposed to only HEPA-filtered air. Statistical analysis performed using Student’s T-Test. Data are expressed as mean ± standard error of the mean (SEM). p < 0.05 is considered statistically significant. * p < 0.05. D) The significantly decreased body weight in in utero JUUL aerosol-exposed offspring was sustained at least through 2 weeks of age. PND0 N = 33–67 per group. PND7 N = 9–22 per group. PND14 N = 9–22 per group. PND21 N = 9–22 per group. PND28 N = 9–11 per group. Statistical analysis performed using a mixed ANOVA with treatment group as between factor and age of mice as within factor. Data are expressed as mean ± standard error of the mean (SEM). p < 0.05 is considered statistically significant. * p < 0.05.

Exposure to mint-flavored JUUL aerosol in utero significantly alters the lung’s tissue-to-airspace fraction of offspring at birth

In mice, at birth, the lungs are in the saccular stage, where the formation of larger saccular airspaces occurs, preceding alveologenesis (Mund et al., 2008; Warburton et al., 2010; Frank et al., 2016). As can be observed on the representative H&E-stained lung slides (Figure 5A), and as quantified by morphometric analysis (Figure 5B), the airspace fraction of the lungs of the PND0 offspring exposed in utero to JUUL aerosol is significantly decreased (p < 0.05) and the tissue fraction significantly increased (p < 0.05) when compared to in utero air-exposed offspring (Figure 5B). Overall, these data indicate that exposures to mint-flavored JUUL aerosol in utero can affect lung architecture by delaying the normal progression of lung development.

Figure 5. Exposure to mint-flavored JUUL aerosol in utero significantly alters the lung tissue-to-airspace fraction.

(A) Hematoxylin and eosin (H&E) stained segments of representative lung tissue of PND0 neonatal mice exposed to either mint-flavored JUUL aerosol or filtered air in utero. B) morphological analysis of the PND0 lung tissue showing the percentage of lung tissue-to-airspace fraction. N = 5 per group. Statistical analysis performed using Student’s T-Test. Data are expressed as mean ± standard error of the mean (SEM). A p < 0.05 is considered significantly different. * p < 0.05.

In utero exposure to mint-flavored JUUL aerosol affects maternal-fetal lung inflammatory markers at birth

We assessed lung gene expression of a selection of inflammatory markers known to be involved in lung diseases, including asthma. Exposure to JUUL aerosol throughout gestation dysregulated the expression of 7 inflammatory markers in the lungs of dams and offspring at birth (Figure 6). In dams exposed to JUUL aerosol, expression of Il-4, Il-6, Il-10, Il-13, and Hmox1 was up-regulated, while the expression of Il-5 and Stat6 was down-regulated (Figure 6B), compared to the air exposed controls. In the in utero JUUL aerosol exposed offspring, Il-5, Il-10, Il-13, Stat5a, Foxp3, and Gata3 were up-regulated, while Il-1ß was down-regulated (Figure 6B), when compared to air exposed counterparts. Il-10 and Il-13 were up-regulated in both dams (2.0− and 1.6-fold, respectively) and offspring (2.4− and 2.3-fold, respectively) exposed to mint-flavored JUUL aerosol (Figure 6B). Thus, in utero exposures to mint-flavored JUUL aerosol can promote a pro-inflammatory Th2 dominated milieu in the lungs of the offspring mice at birth.

Figure 6. Exposure to mint-flavored JUUL aerosol in utero affects maternal-fetal lung inflammatory markers at birth.

A) Exposure to JUUL aerosol throughout gestation dysregulates the expression of inflammatory markers in the lungs of dams and offspring. Gene expression of dams and offspring mice exposed to JUUL during pregnancy was normalized using the gene expression results from the respective dams and offspring exposed only to HEPA-filtered air. Data expressed as fold-change compared to respective air control group. N = 5–6 dams per group and N = 4 offspring per group. Genes with at least a 1.5-fold up- or down-regulation were considered to have significant differences of expression compared with controls. B) Venn diagram showing common dysregulated genes in JUUL aerosol in utero exposed dams and offspring mice.

In utero exposure to mint-flavored JUUL aerosol induces unique Wnt and epigenetic-related molecular signatures in the lung tissue of offspring at birth

To reveal the potential molecular mechanisms associated with impaired lung development following prenatal mint-flavored JUUL aerosol exposures, we analyzed the expression of 84 Wnt signaling genes involved in lung organogenesis, and 84 epigenetic chromatin modification genes, as early markers of epigenetic modifications, in the lungs of the PND0 offspring (Frank et al., 2016; Chen et al., 2018). At PND0, in utero mint-flavored JUUL aerosol exposures dysregulated 24 genes of the Wnt pathway and 9 genes associated with epigenetic chromatin modifications (Figure 7A), when compared to air controls. Of those 33 dysregulated genes, 14 were up-regulated, while 19 were down-regulated. Down-regulated genes included Wnt2b (−5.8-fold), Wnt3 (−1.9-fold) and Wnt6 (−1.8-fold), all protein coding genes that play essential roles in the proper morphogenesis and development of the lungs (De Langhe et al., 2008; Frank et al., 2016). Up-regulated genes included Dnmt1 (1.6-fold) and Dnmt3b (1.7-fold), DNA methyltransferases involved in epigenetic regulation of gene transcription through silencing (Lyko, 2018). Pathway analyses of the dysregulated genes from offspring prenatally exposed to mint-flavored JUUL aerosol revealed that they were most significantly associated with Wnt signaling (14 genes), regulation of epithelial-mesenchymal transition (EMT) pathway (7 genes), planar cell polarity (PCP) pathway (9 genes), methylation of DNA (3 genes), morphogenesis of embryo (10 genes), differentiation of embryonic cells (12 genes), quantity of connective tissue (8 genes), growth of epithelial tissue (10 genes), and apoptosis (15 genes) (Figure 7B). Thus, in utero exposure to mint-flavored JUUL aerosol in PND0 offspring significantly alters expression of Wnt signaling and epigenetic chromatin modification genes, both of which produce key proteins involved in lung branching and morphogenesis, as well as in the regulation of gene transcription.

Figure 7. Exposure to mint-flavored JUUL aerosol in utero significantly alters expression of Wnt signaling and epigenetic chromatin modification genes in lungs of PND0 offspring.

A) Epigenetic Chromatin Modification and Wnt Signaling Pathway-related gene dysregulation in lungs of neonates exposed to JUUL, examined at PND0. N = 4 per group. Data expressed as fold-change compared to air control. Genes with at least a 1.5-fold up- or down-regulation were considered to have significant differences of expression compared with controls. B) The gene interaction networks altered by in utero JUUL exposure are correlated with a number of vital processes in fetal and neonatal development via Ingenuity Pathway Analysis (IPA) of dysregulated genes neonates exposed to JUUL in utero. Neonates exposed to JUUL in utero exhibited dysregulation of genes associated with Wnt signaling, regulation of epithelial-mesenchymal transition pathway, PCP pathway, methylation of DNA, methylation of DNA endogenous promoter, morphogenesis of embryo, differentiation of embryonic cells, quantity of connective tissue, growth of epithelial tissue, apoptosis, and aryl hydrocarbon receptor signaling.

Exposure to mint-flavored JUUL aerosol in utero significantly aggravates HDM-induced inflammation in BALF and in lung tissue of adult 11-week-old offspring.

We also investigated whether in utero JUUL aerosol exposures could exacerbate HDM-induced asthma. Exposures to mint-flavored JUUL aerosol in utero + HDM treatment as adult induced an inflammatory response, mainly driven by a significantly increased percentage of neutrophils (p < 0.05), in the BALF of 11-week-old male offspring when compared to the air + saline controls (Figure 8A). In 11-week-old female offspring, exposure to mint-flavored JUUL in utero + HDM treatment as adult induced a significant mixed neutrophilic plus lymphocytic inflammatory response (p < 0.05) in the BALF when compared to the air + saline controls (Figure 8B). These significant results in the BALF were supported by histopathological evaluation of the lung tissue, where the total inflammation score was significantly increased (p < 0.05) in both male and female offspring exposed in utero to JUUL aerosol and challenged as adults with HDM when compared to the in utero air + saline respective controls (Figure 8C, D, E). These data reveal that in utero mint-flavored JUUL aerosol exposure exacerbates HDM-induced pulmonary inflammation at both the cellular and tissue levels later in life.

Figure 8. Exposure to mint-flavored JUUL aerosol in utero significantly aggravates HDM-induced inflammation in BALF and in lung tissue of adult 11-week-old offspring.

A & B) BALF differential cytology results of mice exposed either to mint-flavored JUUL aerosol or filtered air in utero, then challenged with HDM as adults. A) In male offspring, exposure to JUUL in utero + HDM as adults induced a mainly neutrophilic inflammatory response when compared to air + saline controls. B) In female mice, exposure to JUUL in utero + HDM as adults induced a mixed neutrophilic plus lymphocytic inflammatory response when compared to air + saline controls. N = 6 – 8 per group. Statistical analysis performed using an ANOVA followed by a Tukey post hoc test. Data are expressed as mean ± standard error of the mean (SEM). p < 0.05 is considered statistically significant. * p < 0.05. C, D & E) Histopathology total inflammation score (D) and hematoxylin and eosin (H&E) stained segments of lung tissue from (C) male and (E) female offspring exposed either to mint-flavored JUUL aerosol or filtered air in utero, then challenged with either saline or HDM as adults. N = 5 – 8 per group. Statistical analysis performed using Mann-Whitney nonparametric procedure. Outcomes are expressed as median ± interquartile range (IQR). Intra-alveolar inflammatory cell infiltrate – Score 0 = < 3 cells; 1 = 3–10 cells; 2 = 10–20 cells; and 3 = >20 cells. Tissue involvement – Score 0 = 0%; 1 = <10%; 2 = 10–50%; and 3 = >50%.

In utero mint-flavored JUUL aerosol exposure induces asthma and allergy-related molecular signatures in the lung tissue of adult 11-week-old offspring treated with HDM

To further our understanding of the underlying molecular mechanisms associated with aggravated asthmatic responses later in life following mint-flavored JUUL aerosol exposure in utero, we analyzed the expression of 84 asthma and allergy associated genes in the lungs. At 11 weeks of age, in utero JUUL aerosol exposures alone dysregulated 16 asthma and allergy related genes in the lungs of male offspring, when compared to air + saline controls (Figure 9A). Of those 16 dysregulated genes, 3 were up-regulated and 13 were down-regulated (Figure 9A). In the groups that had also been challenged with HDM, in utero JUUL aerosol exposures up-regulated 33 asthma and allergy associated genes in the male offspring, when compared to their respective in utero air + HDM treated controls (Figure 9B). Overall, in males, while the majority of the dysregulated genes were down-regulated following in utero exposures to JUUL aerosol alone, the in utero JUUL aerosol exposure in addition to the adult HDM treatment, in contrast led to a sole up-regulation of asthma-related genes (Figure 9A, B). In female offspring, at 11 weeks of age, in utero JUUL aerosol exposures alone dysregulated 19 asthma and allergy related genes, compared to air + saline controls (Figure 9A). Most of those dysregulated genes (16/19) were up-regulated, and 3 were down-regulated (Figure 9A). In the HDM-treated female offspring, in utero JUUL aerosol exposures dysregulated 53 asthma and allergy-associated genes, with 27 genes up-regulated and 26 genes down-regulated, when compared to their respective in utero air + HDM treated controls (Figure 9B). We found 12 common up-regulated genes in male and female offspring exposed to mint-flavored JUUL aerosol in utero and challenged as adults with HDM (Figure 9C). These up-regulated genes included Il-3ra (1.8− and 2.5-fold, in male and female, respectively), Il-5ra (1.6− and 2.6-fold, in male and female, respectively), Il-10 (2.0− and 5.3-fold, in male and female, respectively), and Il-25 (2.1− and 3.4-fold, in male and female, respectively) (Figure 9C). Together, these data demonstrate that exposure to mint-flavored JUUL aerosol in utero significantly alters expression of genes associated with allergy & asthma in the lungs of adult 11-week-old offspring with (Figure 9B) and without (Figure 9A) HDM challenge as adults. Further, the significantly dysregulated genes from the in utero JUUL aerosol + HDM treated groups (Figure 9B) also support, at the molecular level, the aggravated HDM-induced cellular and lung tissue inflammation observed in adult 11-week-old offspring (Figure 8).

Figure 9. Exposure to mint-flavored JUUL aerosol in utero significantly alters expression of genes associated with allergy & asthma in the lungs of adult 11-week-old offspring.

Allergy and asthma-related gene dysregulation in lungs of male and female offspring exposed to mint-flavored JUUL aerosol in utero then then challenged with intranasal installations of either saline or HDM once weekly from 8 to 11 weeks of age. A) Male and female offspring exposed to mint-flavored JUUL aerosol in utero and then treated intranasally with saline exhibited dysregulation of a number of genes associated with asthma and allergies, but to a lesser degree than those challenged with HDM intranasally. B) Both male and female mice exposed to mint-flavored JUUL aerosol in utero and then treated intranasally with HDM exhibited dysregulation of a number of genes associated with allergy and asthma. C) Venn diagram showing common dysregulated genes between male and female offspring exposed to mint-flavored JUUL aerosol in utero and then treated intranasally with HDM. Data normalized using the gene expression results from the lung tissue of offspring exposed only to HEPA-filtered air in utero + challenged with either saline or HDM as adults. N = 4 per group. Data expressed as fold-change compared to respective air control. Genes with at least a 1.5-fold up- or down-regulation were considered to have significant differences of expression compared with controls.

Exposure to mint-flavored JUUL aerosol in utero significantly affects epigenetic chromatin modification genes from birth to adulthood and alters the methylation status of Il-10ra

Since lung programming starts in utero and exposures to environmental contaminants during sensitive windows of organogenesis can affect programming resulting in altered trajectory of lung development and increased disease susceptibility throughout the lifespan (Barker, 1990; Carpinello et al., 2018), we investigated the expression of epigenetic chromatin modification enzyme-related genes in the lungs of offspring exposed in utero to mint-flavored JUUL aerosol. We recorded responses at PND0 (Figure 7A) and at 11 weeks of age following saline or HDM challenge (Figure 10A). At PND0, 8 epigenetic-related genes were up-regulated by the in utero mint-flavored JUUL aerosol exposure, while Prmt8 was the only down-regulated gene (−1.6-fold) (Figure 10A), when compared to the respective air control group. In male offspring, the in utero JUUL aerosol exposure alone dysregulated 2 epigenetic-related genes (Aurkc and Smyd1) at 11 weeks of age when compared to respective in utero air + saline controls (Figure 10A). Exposures to JUUL aerosol in utero + HDM dysregulated 4 epigenetic-related genes (Aurkc, Esco2, Smyd1, Prmt8), when compared to respective controls (Figure 10A). In 11-week-old female offspring, in utero JUUL aerosol exposure alone resulted in dysregulation of the same 2 epigenetic associated genes that were dysregulated in male offspring (Aurkc and Smyd1), compared to respective in utero air + saline controls (Figure 10A). Exposures to JUUL aerosol in utero + HDM dysregulated 7 epigenetic-related genes (Aurka, Aurkb, Aurkc, Ciita, Esco2, Smyd1, Prmt8), when compared to respective controls (Figure 10A). Noteworthy, the down-regulation of Prmt8 was conserved from birth to adulthood in both male and female offspring exposed to mint-flavored JUUL aerosol in utero (birth) and subsequently in offspring receiving HDM as adults (11 weeks of age) (Figure 10A).

Figure 10. Exposure to mint-flavored JUUL aerosol in utero significantly affects epigenetic chromatin modification genes from birth to adulthood and alters the methylation status of Il10ra.

A) Epigenetic chromatin modification enzymes related gene dysregulation in lungs of offspring exposed in utero to mint-flavored JUUL aerosol, examined at PND0 and at 11 weeks for age following saline or HDM challenge. N = 4 per group. Data expressed as fold-change compared to respective air controls. Genes with at least a 1.5-fold up- or down-regulation were considered to have significant differences of expression compared with controls. B) In utero JUUL aerosol exposure affects the methylation status of Il10ra a gene associated with asthma development. N = 4 per group. Data are expressed as mean ± standard error of the mean (SEM).

In addition, we determined the methylation status of the promoter region of 22 genes associated with immune-inflammatory responses. Only one gene of the panel was affected by the JUUL aerosol exposure. Female offspring exposed to mint-flavored JUUL aerosol in utero and treated with HDM as adults exhibited decreased methylation of the promoter region of the Il-10ra gene (Figure 10B) when compared to their in utero air + HDM treated counterparts. These data indicate lasting (up to 11 weeks) epigenetic effects of the in utero mint-flavored JUUL aerosol exposures in the lungs of the exposed mouse offspring.

Discussion

The present study using JUUL, a fourth generation ENDS device, is the first to investigate the possible link between in utero exposure to JUUL aerosols and the increased susceptibility to develop allergic asthma in mice. An individual’s overall health – both as a neonate and later in life – is shaped by the fetal environment (Almond & Currie, 2011). In this study, offspring exposed to JUUL aerosol in utero were necropsied at PND0 and at 11 weeks of age to evaluate both the immediate pulmonary effects as well as the long-term consequences of prenatal JUUL exposure on pulmonary responses following adult exposures to HDM. Overall, our data indicate that maternal JUUL use induces negative pregnancy outcomes, including decreased birth length (Figure 4A, B), decreased birth weight (Figure 4C), and decreased weight gain in offspring through the first 2 weeks of life (Figure 4D). These effects on fetal and neonatal growth correlated with disrupted uterine/placenta homeostasis, as evidenced by dysregulated expression of genes associated with hypoxia and oxidative stress in the JUUL aerosol exposed dams (Figure 3). Additionally, at birth in utero JUUL exposure altered the expression of genes associated with epigenetic chromatin modifications (9 genes), Wnt signaling (24 genes), and inflammation (7 genes), including up-regulation of Il10, in the lungs of the offspring (Figure 6 & 7A). In adulthood, in utero JUUL exposures resulted in aggravated cellular and tissue pulmonary inflammation following HDM treatment in both male and female offspring (Figure 8). Supporting these effects, several asthma and allergy related lung genes were dysregulated, including Il10, which was up-regulated in male and female offspring (Figure 9). Also, from birth to adulthood (11 weeks of age) in both male and female offspring, there was conserved down-regulation of the gene for the protein arginine methyltransferase 8 (Prmt8), which is involved in the formation of asymmetric dimethylarginine (ADMA), which has been associated with late-onset asthma (Zakrzewicz et al., 2012; Scott et al., 2014) (Figure 10A). Further, in female mice exposed in utero to JUUL aerosols and treated as adults with HDM, the methylation status of the promoter region of Il10ra was decreased when compared to the air + HDM counterparts (Figure 10B). This suggests a more active transcription of Il10ra in the in utero JUUL aerosol exposed offspring. Taken together, these data suggest that in utero JUUL exposures compromise the health of offspring at birth with lasting consequences into adulthood. More importantly, this study provides direct evidence that in utero exposures to mint-flavored JUUL aerosols can affect the lungs of the developing fetus by inducing epigenetic signatures and altering signaling pathways essential for lung organogenesis, which were accompanied by aggravated pulmonary responses to a common allergen later in life. Thus, this work emphasizes the urgent need to develop program and policy strategies to educate, prevent, and manage cessation of ENDS use by pregnant women.

Although the precise mechanisms by which JUUL aerosol induces unfavorable birth outcomes is unclear, proper development and functionality of the placenta is necessary to ensure that a fetus receives proper nutrients during intrauterine growth and maturation. One essential nutrient, oxygen, is delivered via red blood cells through the vascular system (Soares et al., 2017; Semenza, 2010). Previous studies have shown that placentation is regulated by intrauterine oxygen concentrations and that oxygen homeostasis-related mechanisms influence the formation and functionality of the placenta (Fryer & Simon, 2006; Burton, 2009; Dunwoodie, 2009). Additionally, utero-placental hypoxia has been linked with blood flow restriction to the placenta, which is typically caused by remodeling of the maternal spiral artery coupled with failed trophoblast invasion, which can cause fetal growth restriction and pregnancy complications (Benirschke et al., 2006; Wang & Zhao, 2010; Schoots et al., 2018). Besides, hypoxia leads to oxidative stress in the uterine/placental tissue due to the disrupted equilibrium of reactive oxygen species (ROS) and antioxidants (Schoots et al., 2018). In this study, we found that uterine/placental tissues of dams exposed to JUUL throughout pregnancy showed significant dysregulation of 27 genes associated with oxidative stress (fold-change ranging from −5.73 to 1.76) and 27 genes associated with hypoxia (fold-change ranging from −2.32 to 3.76) (Figure 3C). Thus, in our study, the significant dysregulation of molecular markers of oxygen imbalance in the uterine/placenta of JUUL exposed dams (Figure 3C,D), coupled with the observed fetal growth restriction of the in utero JUUL exposed offspring (Figure 4), strongly suggest that oxidative stress/hypoxia likely led to placental insufficiency, which resulted in altered maternal and fetal exchanges, and ultimately to the growth restriction of the offspring (Figure 4), independent of the placental HSD2 enzyme activity (Figure 3A,B). Previous studies have linked prenatal and early life exposure to ENDS aerosols with decreased fetal and neonatal weights and lengths (Wetendorf et al., 2019; McGrath-Morrow et al., 2015; National Academies of Sciences, Engineering, and Medicine, 2018; Noël et al., 2020; Cahill et al., 2021). However, none thus far had proposed a mechanism by which ENDS aerosol affected the physical growth of the fetuses/newborns, and therefore highlight the novelty of our study. Further, our findings are supported by previous research establishing nicotine as a major developmental toxin that alters the normal growth and weight gain of fetuses (England et al., 2017; Kramer, 1987; Lambers & Clark, 1996; Kirchengast & Hartmann, 2003). This also suggests that concentrated nicotine salt found in ENDS pods may also be a factor contributing to adverse pregnancy outcomes. Indeed, as shown in Figure 2, mint-flavored JUUL aerosols are complex mixtures containing, in addition to nicotine, high levels of propylene glycol, glycerin and benzoic acid (> 40 μg/puff), which as inhaled chemicals can also negatively impact health (Clapp and Jaspers, 2017). Therefore, we cannot delineate whether the observed effects are specific and solely due to nicotine salt exposure or if other components of the mint-flavored JUUL aerosol are also playing a role.

Alveologenesis, which is initiated at ~ PND5 in mice, is the period of maximal airway remodeling during lung development. During this stage, the saccules are sub-divided into alveoli via the thinning of the saccular/alveolar septal tissue (Bry et al., 2007; Branchfield et al., 2016; Loering et al., 2019). EMT is a key event in pulmonary remodeling, as epithelial cells transitioning to mesenchymal cells acquire the ability to produce matrix metalloproteinases (MMPs) and to deposit extracellular matrix (ECM) components, including elastin, which are essential for proper alveologenesis (Borthwick et al., 2009). Thus, early in mouse post-natal life, having the key factors necessary for normal alveologenesis, which occurs through airway remodeling processes, is crucial and involve EMT. IL-1ß is a pro-inflammatory cytokine that plays major roles in the initiation and persistence of inflammation (Bry et al., 2007). In addition to these roles, IL-1ß enhances TGF-ß1 induction of EMT in human bronchial epithelial cells (Doerner & Zuraw, 2009; Maleszewska et al., 2013). In the present study, we found that the gene expression of Il-1ß was significantly down-regulated (−1.7-fold) at birth in the lungs of the offspring exposed to JUUL aerosol in utero (Figure 6). In addition, we had previously reported that offspring exposed pre-conceptually and prenatally to cinnamon-flavored e-cig aerosols exhibited down-regulation of the Il-1ß gene in the lungs of the newborn mice (Noël et al., 2020). Moreover, in the present study, we showed through Ingenuity Pathway Analysis (Figure 7) that several genes associated with the regulation of EMT pathways, including Wnt2b, Wnt3, and Wnt6, were down-regulated by the in utero JUUL aerosol exposure. Other studies showing that Wnt signaling is involved in regulating EMT (Gao et al., 2020), combined with our results of down-regulation of Il-1ß and of several Wnt related genes (Figure 6 & 7), suggest that EMT is reduced by the in utero JUUL aerosol exposures compared to air controls, during this crucial time where the lungs need to prepare for alveologenesis. This reduction in Il-1ß, EMT and Wnt signaling was associated with increased gene expression of Il-5 and Il-13 (Figure 6), two cytokines also involved in airway remodeling (Loering et al., 2019). These molecular effects were accompanied by increased lung tissue fraction (Figure 5), representing thicker saccular septal tissue, in mice exposed in utero to JUUL aerosols when compared to the air controls (Figure 5). Thus, these results observed in the in utero JUUL exposed offspring are in contrast with what is expected during normal alveologenesis, where the saccular septal tissue becomes thinner, leading to the formation of alveoli (Bry et al., 2007). Overall, these data indicate in mice that in utero exposures to mint-flavored JUUL aerosol impair lung development by modifying lung structure and dysregulating the expression of lung genes associated with airway remodeling, EMT, and Wnt signaling. These deleterious responses are detected at PND0; thus, in utero mint-flavored JUUL aerosol exposure leads to pulmonary fragility, which could be an underlying factor for increased negative pulmonary responses following exposures to inhaled environmental pollutants, throughout the lifespan.

Asthma, a chronic respiratory disease, is prevalent among children as well as in the adult population, with more than 5 and 20 million affected individuals, respectively, in the United States (CDC data 2019). Asthma is characterized by airway hyperresponsiveness, mucus secretion and airway inflammation. These lead to increased leukocyte infiltrates, composed mainly of eosinophils, lymphocytes, and mast cells, in addition to increased production of Th2 cytokines, including IL-4 and IL-5, and increased levels of IgE (Magnan et al., 1995; Tillie-Leblond et al., 1999). The developmental origins of adult asthma include in utero exposures to cigarette smoke or secondhand smoke, which have been demonstrated in humans as well as in animal models (Penn et al., 2007; Xiao et al., 2013; McEvoy and Spindel, 2017; Noël et al., 2017; Toppila-Salmi et al., 2020; Noël et al., 2021). Further, increasing evidence points to epigenetic mechanisms regulating these latent effects (Lovinsky and Miller, 2012; Joubert et al., 2016). In the present study, we found that in utero exposures to mint-flavored JUUL aerosol significantly dysregulated the expression of 9 genes related to epigenetic chromatin modification enzymes in the lungs of exposed offspring at birth (Figure 7). This in utero exposure also aggravated HDM-induced inflammation in BALF and in lung tissue of adult 11-week-old offspring (Figure 8). While a neutrophilic inflammatory response was observed in in utero JUUL aerosol + HDM exposed male offspring compared to air controls, female offspring exposed in utero to JUUL aerosol + HDM exhibited a mixed neutrophilic plus lymphocytic inflammatory response when compared to air controls (Figure 8). Sub-phenotypes of asthmatic responses include those mainly driven by airway eosinophils or neutrophils, however, overlap between eosinophilic and neutrophilic inflammation exists (Ray and Knolls, 2018). As previously reported, neutrophilic asthma is associated with aggravation of symptoms and is usually steroid-resistant (Ray and Knolls, 2018). Further, increased levels of IFN-γ and IL-17 have been related to BALF neutrophilia in severe cases of asthma both in humans and in experimental models (Chambers et al., 2015; Raundhal et al. 2015; Ray et al. 2016). In our study, male offspring exposed in utero to JUUL aerosol and receiving HDM treatment at 11 weeks of age, had a 2.9-fold increase in the gene expression of Il-17a compared to the in utero air exposed plus HDM counterparts (Figure 9B). Whereas female offspring exposed in utero to JUUL aerosol and receiving HDM treatment at 11 weeks of age, had a 1.7-fold increase in the gene expression of Ifn-γ compared to the respective air plus HDM controls (Figure 9B). Thus, while the gene expression results suggest that JUUL-exposed offspring exhibited Th2 responses (Figure 9B), which is generally associated with eosinophilia, the increased levels of Il-17a and Ifn-γ support that the in utero JUUL aerosol exposures led to an aggravated more severe asthmatic response, which also included increased percentage of BALF neutrophils (Figures 8 & 9). Besides the cell and tissue changes observed in the lungs of JUUL-exposed offspring (Figure 8), we observed dysregulation of numerous additional genes associated with allergic and asthmatic responses in both male and female offspring (Figure 9). Multiple cytokines were found to be dysregulated in males exposed to JUUL in utero and challenged with HDM as adults, including, Il-4ra (1.5-fold), Il-9 (2.2-fold), and Il-25 (2.1-fold) (Figure 9B). Lymphokines and T cell regulatory cytokines, including Il-4, Il-9, Il-17, and Il-25, enhance asthmatic responses by increasing production of IgE, increasing number of Th2 cells, increasing number of mast cells, and indirectly increasing number of neutrophils (Barnes, 2008a & b, Bullens et al., 2006; Pene et al., 2008; Zhou et al., 2001; Fort et al., 2001; Wang et al., 2007). Thus, these molecular effects (Figure 9B) support the cellular and tissue neutrophilic inflammation observed in the in utero JUUL + HDM-exposed male mice (Figure 8). The female mice exposed to JUUL in utero and then challenged with HDM as adults exhibited dysregulation of 54 lung genes associated with allergies and asthma (Figure 9B). These dysregulated genes included Il12b (1.94-fold) and Gata3 (−1.99-fold), which were found to be associated both with eosinophilia and the development of Th2 cells (Figure 9B). Altered eosinophil and Th2 cell function was further evidenced by the significant dysregulation of Ccr3 (−2.05-fold) and Il3 (−1.69-fold), which are involved in inflammatory eosinophilic recruitment and eosinophilic differentiation and function in the lung, respectively (Figure 9B) (Asquith et al., 2008, Conroy & Williams, 2001). These data support the presence of eosinophils found in the BALF and lung tissue of in utero JUUL + HDM exposed female mice (Figure 8). Also, Foxp3, a gene of interest which was found significantly up-regulated in 11-week-old females exposed only to JUUL aerosol in utero (1.7-fold) (Figure 9A) and further exacerbated in those females subsequently challenged with HDM (3.4-fold) (Figure 9B), is associated with both airway hyperresponsiveness and inflammation of the respiratory system. Foxp3 plays a major role in the development and regulations of regulatory T cells (Tregs) (Martino & Prescott, 2011). Overall, our results reinforce those of a recent study which showed that perinatal ENDS exposure (e.g., including lactation) exacerbates the effects of ovalbumin-induced asthma in mouse offspring (Sharma et al., 2017).

Human and animal studies can appear inconsistent regarding whether levels of IL-10, a Th2-derived pleiotropic cytokine, are increased or decreased in allergies and asthma responses (Borish et al., 1996; Tournoy et al., 2000; Robinson et al., 1996; Wong et al., 2001). Indeed, several studies have shown that levels of IL-10 are decreased in asthma patients (Borish et al., 1996; Grunig et al., 1997; John et al., 1998; Wilson et al., 2007; Tournoy et al., 2000), while other studies reported that levels of IL-10 were increased in individuals with allergies and asthma (Magnan et al., 1995; Robinson et al., 1996; KleinJan et al., 1998; Tillie-Leblond et al., 1999; Wong et al., 2001). These conflicting results may be due to the fact that while IL-10 is most widely known for its anti-inflammatory and immunosuppressive properties, it also has pro-inflammatory properties (Borish, 1998). IL-10 anti-inflammatory properties are related to the inhibition of Th1 cells cytokines (e.g., TNF-a, Il-1b, IL-6, IL-12, IFN-y) and suppression of T-cells proliferation (Robinson et al., 1996; Borish, 1998). Its pro-inflammatory properties include activation of humoral and of cytotoxic immune responses, up-regulation of B cell production and differentiation, increased proliferation of mast cells, and increased IgE synthesis (Borish, 1998; Koulis and Robinson, 2000; Hyun et al., 2013). Moreover, Il-10 is released after T-cell activation and thus appears later in the allergen-induced inflammatory response (Robinson et al., 1996; Borish, 1998). In fact, maximal levels of Il-10 mRNA are seen 12 to 24 hours after exposure to an allergen, and thus correlate with the timing at which levels of pro-inflammatory mediators from the early response phase are decreasing (Robinson et al., 1996; Borish, 1998). This supports the autoregulatory immunosuppressive properties of IL-10, involving a negative feedback mechanism where IL-10 production is induced by increasing levels of pro-inflammatory cytokines, with IL-10 appearing later in the course of the response, to dampen, control and resolve the early phase inflammatory reaction (Robinson et al., 1996; Borish, 1998; Koulis and Robinson, 2000; Coomes et al., 2015). Overall, both the level of expression as well as the timing of IL-10 appearance are crucial in determining whether IL-10 exhibits pro- or anti-inflammatory properties, as well as for optimal resolution of 1) the early pro-inflammatory phase response and 2) potential infections (Couper et al., 2008). Thus, any disruption of this control mechanism, whether abnormal activation or suppression, could worsen pulmonary responses. We evaluated the effects of in utero JUUL aerosol exposures on gene-environment interactions in adult asthma and found that expression of Il-10 was up-regulated in the lungs of the in utero mint-flavored JUUL aerosol exposed offspring at PND0 (2.4-fold) (Figure 6) and in both male and female offspring exposed in utero to JUUL aerosol and treated as adult with HDM (2.0− and 5.3-fold, respectively) (Figure 9), thus, revealing that in utero JUUL aerosol exposure leads to an abnormal immune activation of IL-10. It was previously reported that overexpression or untimely presence of Il-10 can suppress the pro-inflammatory reaction, including to specific pathogens (e.g., Leishmania major), which could then evade immune control and lead to persistent infection (Couper et al., 2008). Thus, by inhibiting inflammatory responses, Il-10 also dampens pathogen control, which could lead to increased risk of infection. Further, using an ovalbumin-induced asthma model in IL-10 knockout mice, Mäkela et al., (2000) showed that IL-10 plays a crucial role in airway hyperresponsiveness after the development of the inflammatory reaction. This supports the up-regulated presence of Il-10 in the late phase response and demonstrates, in allergic mice, that IL-10 is involved in modulating lung function; namely, increasing airway resistance and decreasing dynamic compliance following methacholine challenge (Mäkela et al., 2000). It was suggested that Il-10 may act directly on smooth muscle in the presence of leukocyte infiltrates. These data, along with our findings, imply that the in utero JUUL aerosol-induced up-regulation of Il-10 may increase the risk for respiratory infections and aggravate airway hyperresponsiveness following exposures to pathogens or allergens later in life. These data are supported by the higher prevalence of respiratory infections observed in infant prenatally exposed to tobacco products (Weitzman et al., 1990; Wu et al., 2012; Fuseini et al., 2017; McEvoy et al., 2017). Furthermore, we found decreased methylation of the promoter region of Il-10ra, the receptor for IL-10 (Figure 10B). Decreased methylation or reduced silencing of gene transcription implies active gene transcription, compared to baseline (Lyko, 2018). Thus, decreased methylation of the promoter region of Il-10ra would result in increased gene expression and consequently, increased presence of this receptor. Although the gene expression of Il-10ra in the lungs of the offspring at birth and at 11 weeks of age was not directly evaluated, given that the IL-10 signaling axis is cell-specific, with all cells producing IL-10 also expressing IL-10ra (Avdiudhko et al., 2001; Lim et al., 2003), it is likely that the increased gene expression of Il-10 in the lungs of the in utero mint-flavored JUUL aerosol exposed mice at birth and at 11 weeks of age (Figures 7 and 9) correlated with a possible increase in the Il-10ra gene. Taken together, our data show that exposure to mint-flavored JUUL aerosol in utero decreases the methylation status of Il-10ra, which correlates with the up-regulation of the Il-10 gene at birth and following HDM treatment as adults. These results suggest that the aggravated HDM-induced asthmatic responses observed in the in utero JUUL aerosol exposed mice may have a fetal epigenetic lung programming origin.

Overall, the data collected during the present in vivo study suggest that gestational exposure to JUUL aerosol can be harmful to the reproductive health of the mother, and subsequently affect the overall health of the offspring. This is based on the observed hypoxia- and oxidative stress-related transcriptomic dysregulation of maternal uterine/placental tissue (Figure 3) accompanied by decreased birth length and weight (Figure 4A–C) as well as prolonged decrease in weight gain (Figure 4D). We believe the potential mechanisms associated with these effects are a consequence of the nicotine salt-rich JUUL aerosol-induced vasoconstriction, resulting in hypoxia and oxidative stress in the uterus/placenta, leading to placental dysfunction, and subsequently causing intrauterine growth restriction of the offspring. Further, our in utero JUUL aerosol exposure data are consistent with sex-based differences with regards to the susceptibility for asthma development in adolescent and adult females (increased number of allergy and asthma related genes for females compared to males, Figure 9). Indeed, there is a higher prevalence of asthma in girls/women during adolescence and adulthood when compared to boys/men (Fuseini et al., 2017). Finally, the present study indicates that in utero exposure to aerosols produced by a 4^th generation ENDS device causes both short- and long-term pulmonary effects in mouse offspring. We showed that ENDS aerosol exposure alone disrupts pulmonary development (Figure 5–7) and causes lasting effects in the respiratory systems of offspring (Figure 9A), most probably through induction of epigenetic modifications, and alterations of immune cells in the respiratory system of the offspring. This can affect lung structure and function, as well as increase airway inflammation later in life when offspring are challenged with HDM (Figure 8–9).

Although this study has several strengths, including the investigation of how exposures to mint-flavored JUUL aerosols during pregnancy affect 1) the uterine/placental milieu in dams; 2) the fetal growth of the offspring; and 3) the pulmonary health of the offspring both at birth and later in life, thus covering the critical period of maternal-fetal-neonatal interactions, it also has limitations associated with the nature of the exposures. Indeed, it is well-known that numerous factors can affect ENDS aerosol toxicity, including the type of ENDS device used (either from the first, second, third or fourth generation), operational ENDS device settings (power), vaping topography, and e-liquid composition (propylene glycol/glycerin ratios, flavoring chemicals and nicotine concentration) (Stefaniak et al., 2022). Thus, the resulting pulmonary toxicity may be specific to each distinct ENDS aerosols generated. Similarly, regarding the responses observed in our study, with exposures to mint-flavored JUUL aerosols containing 5% of nicotine salt, the findings we report here may be flavor, nicotine form and concentration specific. Therefore, drawing inference on the generalizability of these research findings performed once should be made with caution. Nonetheless, it is also important to emphasize that accumulating experimental evidence are showing adverse pulmonary effects in prenatally ENDS aerosol-exposed offspring (Chen et al., 2018; Wetendorf et al., 2019; McGrath-Morrow et al., 2015; Noël et al., 2020; Wang et al., 2020; Cahill et al., 2021; Orzabal et al., 2022).

Conclusion

The data presented here in mouse models demonstrate that inhalation of ENDS aerosols during pregnancy is not harmless: it affects the intrauterine environment, as well as lung morphology at birth and gene expression in both neonates and adult offspring. Further research is necessary to unravel the precise mechanisms by which ENDS aerosol exposures disrupt lung development, as well as to establish epigenetic markers which are precursors of allergic airway disease development.

Our data support the conclusions 1) that ENDS use should be discontinued during pregnancy; 2) that expectant mothers should not view ENDS devices as harmless alternatives to traditional smoking; and 3) that health care providers should inform pregnant patients about potential risks involved with prenatal ENDS usage.