Sex-specific alterations in pulmonary metabolic, xenobiotic and lipid signalling pathways after e-cigarette aerosol exposure during adolescence in mice.
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Abstract
Background
E-cigarette use is now prevalent among adolescents and young adults, raising concerns over potential adverse long-term health effects. Although it is hypothesised that e-cigarettes promote inflammation, studies have yielded conflicting evidence. Our previous work showed that JUUL, a popular e-cigarette brand, elicited minimal lung inflammation but induced significant molecular changes in adult C57BL/6 mice.Methods
Now, we have profiled immunological and proteomic changes in the lungs of adolescent male and female BALB/c and C57BL/6 mice exposed to a flavoured JUUL aerosol containing 18 mg/mL of nicotine for 14 consecutive days. We evaluated changes in the immune composition by flow cytometry, gene expression levels by reverse transcription-quantitative PCR and assessed the proteomic profile of the lungs and bronchoalveolar lavage (BAL) by tandem mass tag-labelled mass spectroscopy.Results
While there were few significant changes in the immune composition of the lungs, proteomic analysis revealed that JUUL exposure caused significant sex-dependent and strain-dependent differences in lung and BAL proteins that are implicated in metabolic pathways, including those related to lipids and atherosclerosis, as well as pathways related to immune function and response to xenobiotics. Notably, these changes were more pronounced in male mice.Conclusions
These findings raise the possibility that vaping dysregulates numerous biological responses in lungs that may affect disease risk, disproportionally impacting males and raising significant concerns for the future health of male youth who currently vape.Free full text
Sex-specific alterations in pulmonary metabolic, xenobiotic and lipid signalling pathways after e-cigarette aerosol exposure during adolescence in mice
Abstract
Background
E-cigarette use is now prevalent among adolescents and young adults, raising concerns over potential adverse long-term health effects. Although it is hypothesised that e-cigarettes promote inflammation, studies have yielded conflicting evidence. Our previous work showed that JUUL, a popular e-cigarette brand, elicited minimal lung inflammation but induced significant molecular changes in adult C57BL/6 mice.
Methods
Now, we have profiled immunological and proteomic changes in the lungs of adolescent male and female BALB/c and C57BL/6 mice exposed to a flavoured JUUL aerosol containing 18 mg/mL of nicotine for 14 consecutive days. We evaluated changes in the immune composition by flow cytometry, gene expression levels by reverse transcription-quantitative PCR and assessed the proteomic profile of the lungs and bronchoalveolar lavage (BAL) by tandem mass tag-labelled mass spectroscopy.
Results
While there were few significant changes in the immune composition of the lungs, proteomic analysis revealed that JUUL exposure caused significant sex-dependent and strain-dependent differences in lung and BAL proteins that are implicated in metabolic pathways, including those related to lipids and atherosclerosis, as well as pathways related to immune function and response to xenobiotics. Notably, these changes were more pronounced in male mice.
Conclusions
These findings raise the possibility that vaping dysregulates numerous biological responses in lungs that may affect disease risk, disproportionally impacting males and raising significant concerns for the future health of male youth who currently vape.
Introduction
Since their commercial debut in 2007, e-cigarette use has surged to 86 million users.1 E-cigarettes heat a liquid into an aerosol for inhalation as a means of delivering nicotine to the brain. While e-cigarettes were initially marketed as safer alternatives to cigarette smoking due to the presence of fewer combustion products,2 the risk of their long-term use is still unknown, particularly for tobacco-naïve users. The increased prevalence of e-cigarette use among youth has raised concerns that these products may be associated with adverse health effects over time.3 In 2022, >2.5 million US middle and high school students regularly used e-cigarettes.4 The availability of popular flavours, combined with their small compact and sleek design, has contributed to the dramatic increase in e-cigarette use among adolescents.5,12 This increase in e-cigarette popularity coincided with the emergence of JUUL in 2015.13 JUUL quickly became the top selling e-cigarette brand and one of the most frequently used among youth e-cigarette users.14,16
E-cigarettes contain a battery, the atomiser which contains the wick and a metal filament that aerosolises the e-liquid when heated.17 E-liquids contain varying amounts of the solvents propylene glycol (PG) and vegetable glycerin (VG) as well as flavouring agents, water and nicotine.18 Although some of the ingredients in e-liquids are considered to be ‘Generally Recognised as Safe’ for ingestion, there is little information on the safety of these chemicals when inhaled.19 The newness of e-cigarettes on the market means that population-level chronic health data in humans are not available. This makes the use of preclinical models an invaluable tool to detect potential disease induction by vaping and explore the underlying mechanisms. Evidence from preclinical studies suggests that many flavoured e-liquids decrease cell viability and induce oxidative stress and inflammation.20,23 There are some immunological changes that occur in the lungs from JUUL exposure.24 25 However, recruitment of early response immune cells, such as neutrophils, to the lungs from e-cigarette exposure is variable and inconsistent between studies.26,28 Such differences in outcomes between studies may be related to the e-cigarette brand/liquid, exposure regime, age and/or mouse strain. Moreover, studies comparing the inflammatory response with that of cigarette smoke skews interpretation of emerging data towards e-cigarettes being less harmful or even safe.29
Other challenges in assessing e-cigarette toxicology include the wide variety of e-cigarette products available to consumers and the lack of a standardised preclinical animal model. Current preclinical studies differ in fundamental ways, including the strain of inbred mouse used. Two of the common strains used in respiratory health research are BALB/c and C57BL/6, which display inherent differences in the innate and adaptive immune systems30 and susceptibility to respiratory diseases from inhaled toxicants.31,33 We recently reported that there are important sex-specific differences in biological responses to e-cigarettes in C57BL/6 mice, including significant changes in the proteins found within extracellular vesicles (EVs) obtained from the bronchoalveolar lavage (BAL) fluid.24 However, there remain considerable gaps in the literature on the pulmonary consequences of e-cigarette exposure. To identify potential mechanisms of lung damage caused by e-cigarette exposure, we used our established preclinical vaping model to evaluate the pulmonary proteomic and immunophenotypic landscape of male and female C57BL/6 and BALB/c mice exposed to JUUL aerosols that begins during the critical adolescent phase of development. Here, we report that there are widespread changes to proteins within the lungs from JUUL exposure that are associated with key biological pathways including metabolism and response to xenobiotics. These proteomic changes largely affected male mice but occurred without changes in immune cell infiltration to the lungs. These results continue to support the notion that e-cigarette aerosols are not inert and elicit changes within the lungs that may predispose to disease aetiology if used chronically.
Methods
E-cigarette exposures
Male and female BALB/c and C57BL/6 mice were bred in-house and used for experimentation when 6–7 weeks of age; this age range represents adolescence in mice.34 Mice were housed in regular cages (3–5 mice per cage) with ad libitum access to food and water, under a regular diurnal light cycle. Mice were monitored every day for signs of stress. No experimental animals we excluded from the study. In a non-blinded fashion, mice were randomly allocated to control or treatment groups at the time of weaning. At weaning, the mice were removed from the breeding cage and placed randomly in either the sex-specific air-only, PG/VG or JUUL (each cage containing only mice of one sex). Mice comprising the air group were placed in the exposure system but were exposed to room air only. Mice in the PG/VG exposure group were exposed to an aerosol derived from a liquid composed of a 30:70 ratio of PG and VG purchased from Fusion Flavours (fusionflavors.ca). Finally, mice in the JUUL exposure group were exposed to an aerosol derived from a mint-flavoured JUUL pod containing 18 mg/mL nicotine purchased at a local retailer. Two daily exposures were performed 5 hours apart for 14 consecutive days and all exposures were done using a SCIREQ inExpose inhalation system and an e-cigarette extension compatible with JUUL devices as described.24 25 Briefly, aerosols were delivered according to a pre-established e-cigarette puffing profile which was programmed with the flexiWare software, consisting of a 70 mL puff volume and 3.3s puff duration.35 A 2 L/min bias flow provided the mice uninterrupted air in between puffs which were delivered at a rate of 2 puffs/min for 30
min per session, two sessions per day, for 14 consecutive days. The two daily exposure sessions were 5 hours apart. To minimise the effect of residual aerosols between treatments, the air-only group was exposed first, followed by the PG/VG group and the JUUL group last. In between exposures, the entire exposure system was cleaned to ensure no residual aerosol build-up inside the system. Male and female mice do not exhibit differences in serum cotinine levels.25
Tissue harvest and BAL fluid collection
Mice were anaesthetised with Avertin (2,2,2-tribromoethanol, 250 mg/kg intraperitoneal; Sigma-Aldrich, St. Louis, Missouri, USA) 12 hours after the last exposure. Verification that the mice had been completely anaesthetised was done using the toe-pinch technique and euthanasia performed via cardiac puncture followed by laceration of the pneumothorax. The lungs were lavaged twice using 0.5 mL of cold phosphate-buffered saline (PBS). The right lobe was used to prepare single cell suspensions, and the left lobe was frozen in liquid nitrogen and stored at −80°C. The BAL fluid was centrifuged for 5min at 5000 rpm at 4°C. The cell-free supernatant of each sample was transferred into a microcentrifuge tube and stored at −80°C. The remaining cell pellets were resuspended with 50 μL of ACK lysis buffer (Thermo Fisher Scientific, Grand Island, New York, USA) and incubated for 2
min. Then, BAL cells were resuspended in cold FACS buffer containing 5 g of bovine serum albumin (BSA) (GE Healthcare Life Sciences, Logan, Utah, USA) and 4 mL of 0.5 M EDTA (Thermo Fisher Scientific, Vilnius, Lithuania) dissolved in a final volume of 1 L of PBS.
Flow cytometry
Lungs were manually sheared and enzymatically digested with Collagenase IV (Sigma-Aldrich) for 1 hour at 37°C. Tissue samples were passed through 70 μm cell strainers (CellTreat, Pepperell, Massachusetts, USA) to generate single-cell suspensions. After centrifugation at 1500 rpm for 10min at 4°C, cell pellets were resuspended and incubated for 10
min in 2 mL of ACK lysis buffer (Thermo Fisher Scientific) to lyse red blood cells. Cells were counted using the AcT differential cell counter (Beckman Coulter) and resuspended in FACS buffer. Lung tissue and BAL cells were transferred to 96-well round bottom plates at 1 million cells per well. Cells were stained with the viability dye eFluor 506 (Thermo Fisher Scientific, Carlsbad, California, USA) for 30
min at 4°C, washed twice with FACS buffer and subsequently incubated with a blocking buffer prepared by diluting 0.5 µL of anti-CD16/32 antibody (BioLegend, San Diego, California, USA) in 70 µL FACS buffer per well for 15
min at 4°C. Afterwards, lung tissue and BAL fluid cells were stained with a mix of fluorochrome-conjugated antibodies (online supplemental table 1). Innate immune cells were identified as shown in online supplemental figure 1A. In a separate panel, adaptive immune cells were identified in the lung tissue as shown in online supplemental figure 1B. The complete gating strategy is shown in online supplemental figure 2. Fluorescence compensation for each fluorochrome was set with single-stained UltraComp eBeads compensation beads (Invitrogen, Eugene, Oregon, USA). Cells were acquired on the FACSCanto II or LSR Fortessa X-20 cytometers (BD Biosciences), and data were analysed on FlowJo.
Mass spectrometry
BAL fluid and lung tissue from air-exposed and JUUL-exposed mice were analysed using tandem mass tag (TMT)-labelled mass spectrometry. Samples were treated with TMT-16plex reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Labelled peptides were fractionated using Pierce High pH Reversed-Phase Peptide Fractionation Kit into eight fractions. Each fraction was re-solubilised in 0.1% aqueous formic acid and 2 μg of each was loaded onto a Thermo Scientific Acclaim PepMap (75 μM internal diameter×2 cm C18 3 μM beads) precolumn. Then, fractions were loaded onto an Acclaim PepMap Easyspray (Thermo Scientific, 75 μM×15 cm with 2 μM C18 beads) analytical column for separation using a Dionex Ultimate 3000 UHPLC at 250 nL/min with a gradient of 2%–35%organic (0.1% formic acid in acetonitrile) over 3
hours, running at the default settings for MS3-level SPS TMT quantitation.36 Fractions were run on an Orbitrap Fusion instrument (Thermo Fisher Scientific) operated in DDA-MS3 mode. MS1 scans were collected at 120
000 resolution, scanning from 375 to 1500
m/z, collecting ions for 50 ms or until the AGC target of 4e5 was reached. Precursors with a charge state of 2–5 were included for MS2 analysis, which were isolated with an isolation window of 0.7 m/z. Ions were collected for up to 50 ms or until an AGC target value of 1e4 was reached and fragmented using CID at 35% energy; these were then read out on the linear ion trap in rapid mode. Subsequently, the top 10 (height) sequential precursor notches were selected from MS2 spectra for MS3 quantitative TMT reporter ion analysis, isolated with an m/z window of 2 m/z, and fragmented with HCD at 65% energy. Resulting fragments were read out in the Orbitrap at 60
000 resolution, with a maximum injection time of 105 ms or until the AGC target value of 1e5 was reached. To translate .raw files into protein identifications (SeQuest) and TMT reporter ion intensities, Proteome Discoverer 2.3 (Thermo Fisher Scientific) was used with the built-in TMT reporter ion quantification workflows. Default settings were applied, with trypsin as enzyme specificity. Spectra were matched against the mouse protein FASTA database obtained from Uniprot (2023). Dynamic modifications were set as oxidation (M) and acetylation on protein N-termini. Cysteine carbamidomethyl was set as a static modification, together with the TMT tag on both peptide N-termini and K residues.
Proteomics analysis
To obtain a list of quantified proteins with relative abundances, all proteins identified by mass spectrometry were filtered to a 1% Protein FDR Confidence. No imputation of missing values was performed. Relative protein abundances were calculated for the following comparisons: JUUL versus Air (in male mice), JUUL versus Air (in female mice), male versus female (in air-exposed mice) and male versus female (in JUUL-exposed mice). To identify differentially expressed proteins (DEPs), −log10(p)>1.3 (ie, p<0.05) was used as the threshold for both upregulated and downregulated proteins. Proteomics data were analysed for differences related to strain (BALB/c or C57BL/6), exposure (JUUL or Air) or the biological sex of the mice (male or female). Venn diagrams of all DEPs were generated using an online Venn diagram generator and enrichment analysis of DEPs was performed on Metascape.37 To visualise protein-protein interaction (PPI) networks, the Search Tool for Retrieval of Interacting Genes (STRING) (https://string-db.org) database was employed. Active interaction sources for constructing the network include text mining, experiments, databases and co-expression. The species was limited to ‘Mus musculus’ and a minimum required interaction score was set to 0.4 and k-means clustering was employed to group proteins.
Statistics
All statistical analyses were performed using GraphPad Prism V.9 (GraphPad Software, San Diego, California, USA). Statistically significant changes were identified with two-way analysis of variance with Tukey’s multiple comparisons tests. A p value <0.05 was considered statistically significant.
Patient and public involvement
Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Results
E-cigarette aerosols have minimal effect on the lung inflammatory profile in BALB/c and C57BL/6 mice. To date, there has been no comprehensive immunophenotypic analysis of the pulmonary system in JUUL-exposed inbred strains of mice. Therefore, we first characterised innate and adaptive immune cell populations in the respiratory system of male and female BALB/c and C57BL/6 mice. JUUL exposure had minimal impact on innate immune cells within the lung (figure 1). There were some changes in the frequency of adaptive immune cell between strains of mice and in response to JUUL (figure 2). In the BAL fluid, there were few changes in innate immune cells from JUUL exposure (online supplemental figure 3). We also measured the messenger RNA (mRNA) expression of select genes in lung tissue. JUUL exposure significantly upregulated Tnfα, Col3a1 and Muc5b mRNA only in the lungs of BALB/c mice (online supplemental figure 4). Overall, there are minimal changes in the immune composition of the lungs in response to JUUL.
JUUL aerosol exposure significantly alters the proteomic landscape in the lungs of inbred strains of mice. Our results suggest that e-cigarettes do not exert significant lung inflammation, consistent with observations by others.38 This would imply that e-cigarettes are unlikely to damage the lungs, and thus could be interpreted as being ‘safe’ to use. This interpretation of data assumes that the nature of the inflammatory response will be similar/identical to that of other inhaled toxicants, particularly tobacco smoke. It is possible, however, that changes are occurring within the lungs from e-cigarette aerosols that are fundamentally distinct from tobacco smoke and are not being captured by traditional methodology (eg, flow cytometry). Therefore, we next performed quantitative proteomics on the cell-free BAL fluid and lung tissue using isobaric chemical labelling with TMT. TMT are isobaric chemical tags, which are identical in mass but dissociate to yield reporter ions of distinct mass.39 The main advantage of TMT quantitative proteomics is that it allows for the accurate determination of the relative protein abundance of many different samples at the same time through multiplexed protein quantification.39 To our knowledge, this technique has not been used to profile changes in the lungs exposed to e-cigarette aerosol. We first compared the total proteins in the lungs versus BAL fluid of C57BL6 mice and BALB/c mice. We selected these two lung compartments because BAL samples the airway lumen, while the lung tissue analysis provides information on changes within the tissue. The complete list of proteins quantified in each strain is available in online supplemental table 2 (BAL) and (lungs) available at https://doi.org/10.6084/m9.figshare.26351740.v1. First, these data, which include proteins from both air-exposed and JUUL-exposed mice, show that in the lungs of C57BL/6 mice, there were 1226 proteins quantified, and in BAL, there were 958 proteins (figure 3A). There were 550 proteins common between both lung compartments in C57BL/6 mice, including numerous serpins (serine protease inhibitors). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed similarities in pathways related to the phagosome (mmu04145), cholesterol metabolism (mmu04979), COVID-19 (mmu05171) and glycolysis/gluconeogenesis (mmu00010) (figure 3B; online supplemental table 3A). Overall, these data show overlap in pathways between lung and BAL proteins and associated pathways in C57BL/6 mice.
We next assessed which proteins were significantly changed in response to JUUL in the lungs and BAL of C57BL/6 mice. The list of DEPs between JUUL versus air for male and female mice are listed in online supplemental table 3B. First, we compared DEPs in BAL versus lung which revealed that the majority of proteins significantly changed by JUUL exposure were different between the lungs and BAL (figure 3C). KEGG pathway analysis revealed distinct pathways for DEPs between the lungs and BAL in response to JUUL aerosols (figure 3D; online supplemental table 3C). There were numerous pathways in the lungs affected by JUUL exposure related to metabolism. We also evaluated PPIs using k-means clustering in STRING to visualise associations between DEPs in each compartment (figure 3E). In the lungs, there was significant PPI (p<1.0e-16; online supplemental table 3D). Proteins in cluster 1 (red) were related to numerous biosynthetic pathways similar to those identified by KEGG analysis in figure 3D. Proteins in cluster 2 (green) were related to fatty acid oxidation, steroid metabolism and fat digestion pathways, with significant enrichment in the respiratory system (FDR=0.00014). Group 3 (blue) proteins are largely involved in xenobiotic and glutathione metabolism. PPI analysis of BAL proteins also revealed significant interaction (PPI enrichment p=1.53e-06; online supplemental table 3E). Cluster 1 (red) contained proteins related to immune function and/or control of infection. Cluster 2 (green) also contained proteins related to immune and oxidative stress responses. Finally, proteins in cluster 3 (blue) were related to cellular redox homeostasis and anti-oxidant activity. Thus, these data highlight that in C57BL/6 mice, JUUL aerosol exposure significantly affects proteins within the lungs and BAL associated with lipid metabolism, immune regulation and cellular redox states.
We performed identical analysis on the proteomics data from BALB/c mice (figure 4). There were a total of 1316 proteins quantified in the lung and 464 in BAL (figure 4A). Pathway analysis revealed variations in top enriched KEGG pathways between the lungs and BAL (figure 4B; online supplemental table 4A) with enrichment in pathways related to platelet activation (mmu04611) only in the lungs. When we compared how JUUL exposure affects the proteomics profile of BALB/c mice, we found that there were 67 DEPs in the lungs and 23 in BAL (figure 4C; online supplemental table 4B). KEGG analysis similarly showed little overlap in enriched pathways (figure 4D; online supplemental table 4C). In BAL, there was only enrichment for the complement and coagulation cascades (mmu04610) and COVID-19 (mmu05171). In the lungs, enriched pathways included those that were related to lipid and atherosclerosis (mmu05417) and chemical carcinogenesis-reactive oxygen species (mmu05208). STRING analysis also revealed significant PPI (PPI enrichment p=1.2e-12) in the lungs (figure 4E; online supplemental table 4D). Proteins in cluster 1 (red) were related to protein folding and intracellular transport, whereas those in cluster 2 (green) were associated with cellular detoxification and response to xenobiotic stimuli. Tissue expression was linked to club cells and the respiratory epithelium, and included proteins such as Cyp2f2, which we also identified in a previous study.24 Finally, cluster 3 (blue) were proteins involved in translation. Lastly, we evaluated interactions for proteins in BAL (figure 4E; online supplemental table 4E). Cluster 1 (red) contained proteins associated with the immune response (including several complement proteins), whereas Cluster 2 (green) contained the serpins. There was no significant enrichment for the proteins grouped in cluster 3 (blue).
We also performed analyses to better visualise these strain-dependent differences (online supplemental table 5). While there was a striking difference in the total number of proteins quantified in the BAL fluid between BALB/c and C57BL/6 mice (figure 5A), there was considerable overlap in pathways in BAL proteins, particularly those related to metabolism (figure 5B; online supplemental table 5A). Similarly, there was considerable overlap in proteins (figure 5C) and pathways (figure 5D) in the lungs (online supplemental table 5B). DEPs between murine strains were distinct, and only one pathway was enriched in BAL of BALB/c mice related to the complement and coagulation cascade (mmu04610) (figure 5E; data not shown). In the lungs, there was also minimal overlap in proteins (figure 5F) or KEGG pathways (figure 5G; online supplemental table 5C) between strains. However, there were two pathways in common between JUUL-exposed BALB/c and C57BL/6 mice, one of which was lipid and atherosclerosis (mmu05417) (figure 5G). When considered together, these results highlight that: (1) there is little overlap in the proteome of the lungs and BAL and (2) JUUL exposure changes protein expression in the respiratory system.
Sex-dependent differences in the proteomic profile caused by JUUL aerosol exposure. Our analysis revealed there are sex-dependent differences in the proteome of the respiratory system, including differences in metabolic pathways in BAL and lungs in naïve male versus female mice (online supplemental figure 5). In response to JUUL exposure, these results further show that there is little overlap in DEPs in BAL (figure 6A) or lungs (figure 6B) between male and female C57BL/6 mice. In BAL, there was no enrichment of KEGG pathways between males and females (online supplemental table 6A). STRING analysis reveals that protein clustering in male mice centred on prostaglandin synthesis and regulation (cluster 1: red; FDR=0.0429), immune system physiology (FDR=0.0103) and oxidative stress responses in BAL (cluster 2: green; FDR=8.42E-05) (figure 6C; online supplemental table 6B). Furthermore, DEPs in cluster 3 (blue) of male mice were related to the respiratory system (FDR=0.0241). In BAL of female mice, proteins clustered in processes related to acetylation (FDR=0.00011) and lipocalin (0.0111) (online supplemental table 6C). Pathway enrichment of proteins in the lungs showed distinct differences between male and female mice, with proteins in JUUL-exposed male mice enriching for numerous pathways including PPAR signalling (mmu03320) as well as lipid and atherosclerosis (mmu05417) (figure 6D; online supplemental table 6D).
Finally, we analysed for differences between male and female BALB/c mice exposed to JUUL (figure 7A,B). There was no enrichment of pathways in BAL of male mice, whereas KEGG analysis of DEPs in BAL of female mice revealed pathways related to complement and coagulation cascades (mmu04610) and COVID-19 disease (mmu05171) (online supplemental table 7A). In the lungs of male mice, enriched pathways included lipids and atherosclerosis (mmu05417). In female mice, enriched pathways were fewer and included pathways related to chemical carcinogenesis (mmu05208) and infection, the latter of which overlapped with male mice (figure 7C; online online supplemental table 7B). Collectively, these data highlight that e-cigarette exposure affects numerous biological pathways suggestive of alterations in lipid metabolism, infection and oxidative stress responses, with distinct differences between male and female mice. Importantly, changes in the proteome of the respiratory system are not accompanied by significant alterations in the lung’s immunological profile. Thus, e-cigarette aerosols are causing significant sex-specific changes in the lungs that may be a prelude to the development of chronic disease.
Discussion
There is an urgent need to understand the health consequences of inhaling e-cigarette aerosols. E-cigarettes are often perceived as harmless due to comparisons with tobacco smoke and savvy marketing. Compared with smoking, e-cigarette aerosols have been shown to contain less particulate matter40 and, if they promote inflammation, do so at considerably lower levels.24 25 38 However, the health effects of e-cigarette use in never-smokers is unknown, which is of significant concern given the meteoric rise in youth vaping rates. Although there are now hundreds of e-cigarette brands on the market, JUUL became the most popular brand among youth. Although there have been efforts from regulatory agencies to ban JUUL in response to the youth vaping epidemic, JUUL recently launched a new device in the UK and Canada, along with novel flavoured pods that contain more e-liquid than the original devices.41 Due to the relative novelty of JUUL, and the rapidly evolving nature of these devices and e-liquid composition, there remains limited public health data. Because information on disease causation does not yet exist, preclinical models are an invaluable tool to explore underlying mechanisms of damage to the lungs and other organs. Using our optimised in vivo e-cigarette exposure paradigm, which mimics the patterns of human e-cigarette users,25 we now demonstrate that inhalation of JUUL aerosols during the adolescence phase of development causes significant sex-specific dysregulation in proteins that are involved in important metabolic, oxidative stress and immunological pathways.
Inflammation is an important driver of many chronic diseases, but our results have shown that e-cigarette aerosols evoke minimal immune cell recruitment and no change in lung cytokine levels.24 25 Absence of a lung inflammatory response to e-cigarette aerosols has also been reported by others.38 In our previous studies, we used C57BL/6 mice and have thus considered that the lack of inflammatory response may be due to strain-dependent differences in immunobiology. The most common inbred strains in respiratory health research are the BALB/c and C57BL/6, which exhibit strain-dependent differences in the susceptibility to respiratory diseases such as cigarette smoke-induced emphysema in C57BL/6 mice and allergic asthma in BALB/c mice.31 32 42 Our results show that there are few immunological changes in the lungs and airways in response to JUUL irrespective of mouse strain. While it is possible that the lack of inflammatory response could be due to the fact that we sacrificed the mice 12 hours after the last exposure, and thus the inflammatory cells retuned to baseline. However, we do not think that this is the case for several reasons. First, inhalation exposure to toxicants typically evokes a lung inflammatory response that remains elevated for at least 24 hours after cessation.43 Second, our previous work also showed that there is minimal inflammation in the respiratory system, even when the mice are sacrificed within hours of the last JUUL exposure.24 25 Thus, we conclude that exposure to JUUL aerosols evokes a minimal inflammatory response to the lungs and airways.
However, there were significant differences in the proteome caused by JUUL in both strains. This includes alterations in a number of metabolic pathways, which may lead to impairment of innate defence mechanisms. Innate defence mechanisms in the lungs include the secretion of host-defence molecules such as lysozyme and surfactant proteins (SP)-A and SP-D by lung epithelial cells and the phagocytic capacity of alveolar macrophages. Proper cellular metabolism is a critical feature of tissue response to injury, being necessary for lung repair44 as well as macrophage function. For example, metabolic reprogramming is associated with defective alveolar macrophage function caused by smoking.45 46 This includes defects in lipid metabolism that impairs phagocytosis, reduces surfactant catabolism and results in ineffective efferocytosis.47 Several lines of evidence suggest that e-cigarette aerosols and/or e-liquid constituents negatively impact important aspect of immune cell function, including neutrophil and macrophage phagocytosis and the oxidative burst,48 49 leading to reduced antimicrobial activity and increased infection burden.50 51 Together, these data support that perturbations in cell and tissue metabolism may be mechanistically linked to decreased functions of innate immune cells in the absence of an inflammatory response.
We have also uncovered biological pathways that are significantly different between male and female mice, underscoring the importance of sex in disease susceptibility. One of these centres on our findings that common pathways involve lipid homeostasis. Adequate lipid metabolism is essential for lung function. Important in this regard is surfactant, a lipoprotein complex composed of phospholipids that prevents alveolar collapse. Surfactant is produced by type II alveolar cells and degraded/recycled by alveolar macrophages.52 Our results show that JUUL exposure significantly affects protein involved in numerous pathways related to lipid homoeostasis including ‘lipid and atherosclerosis’, ‘peroxisome’ and ‘peroxisome proliferator-activated receptor (PPAR) signalling pathway’ in male mice. Among the proteins that were significantly increased by JUUL exposure included CD36. CD36 is a scavenger receptor expressed on alveolar macrophages, where it plays an important role in the internalisation of apoptotic cells and pathogens.53 CD36 also interacts with oxidised phospholipids and contributes to the formation of atherosclerotic lesions.54 CD36 expression is controlled at the transcriptional level by PPARs. There are three PPAR family members: PPARα, PPARβ/δ and PPARγ,55 with PPARγ being particularly important in regulating lipid homeostasis in the lungs and other organs by regulating cholesterol metabolism in part through the transcriptional upregulation of CD36. PPARγ is activated by oxidised fatty acid derivatives and other products of oxidative stress, suggesting that inhaled e-cigarette aerosols may be activating PPARs to transcriptionally increase key proteins important in lipid metabolism as a compensatory mechanism. These data are in line with our recent publication showing that JUUL exposure causes early pro-atherogenic changes in an inducible model of atherosclerosis,56 suggesting that dysregulation of lipid metabolism may be an important component in potential disease risk associated with vaping, particularly among males.
Our data also show that proteins involved in controlling cellular redox status are affected by JUUL exposure including catalase and peroxiredoxin 1; proteins related to glutathione metabolism, including glutamate-cysteine ligase catalytic subunit, NAD(P)H quinone dehydrogenase 1 and thioredoxin reductase 1 were also affected. In general, these are proteins important in protecting against oxidative stress, suggesting that their downregulation may render the lungs vulnerable to further oxidative insult. Mechanistically, many of these proteins are regulated by Nrf2, a transcription factor that when activated binds to the antioxidant response element to induce the transcriptional upregulation of genes that protect the lungs from oxidative insult. Although we did not see changes in the transcriptional level of the Nrf2 gene, this might not be surprising given that Nrf2 expression is generally regulated through stabilisation of the protein on dissociation from Keap1.57 It is interesting to note that proteins enriched in pathways related to detoxification and responses to oxidative stress and xenobiotic stimulus were significantly affected in both mouse strains, although there were notable differences between the strains, particularly cytochrome P450 isoforms. In the lungs of C57BL/6 mice, JUUL exposure significantly increased Cyp4b1, a predominantly extrahepatic Cyp isoform which has its highest expression in the lungs.58 In addition to bioactivation of several xenobiotics including 4-ipomeanol, a toxin that causes lung damage, Cyp4b1 also plays an important role in fatty acid metabolism. Moreover, the expression of Cyp4b1 is controlled by androgens and thus has higher expression in males than females, consistent with our proteomics data. Cyp4b1 can also be regulated by PPAR,59 further suggesting that the sex-specific changes observed in our study may be due to PPAR activation by JUUL aerosol constituents and/or resultant lipid oxidation, leading to downstream alterations in key metabolic pathways.
In this study, we demonstrated that there are strain-dependent alterations in biological pathways in BALB/c and C57BL/6 mice caused by a subchronic exposure to JUUL aerosol. Critically, even in the absence of overt changes in pulmonary immune cell composition, exposure to e-cigarette aerosols induced significant changes in pathways related to lipid metabolism and response to xenobiotics. This study is not without its limitations, including the fact that we report the percentage of inflammatory cells (rather than absolute numbers) and the relatively short 2-week exposure time. Regardless, our findings contribute vital information to a growing body of work that aims to understand the impact of e-cigarette use on pulmonary health and raises the possibility that vaping may induce important cellular metabolic changes in susceptible users, the outcomes of which may contribute to disease development if used long term. Carefully designed experimental studies with appropriate animal models are thus needed to generate evidence to support a comprehensive understanding of the consequences of vaping on pulmonary health.
supplementary material
online supplemental file 1
online supplemental table 1
online supplemental table 2
online supplemental table 3
online supplemental table 4
online supplemental table 5
online supplemental table 6
Acknowledgements
This work is based on the thesis found at: https://www.proquest.com/docview/3061277733.
Footnotes
Funding: This work was supported by the Quebec Respiratory Health Research Network (QRHN) and the Canadian Institutes for Health Research (CIHR; PJT-168836 to CB and a Vaping Catalyst Grant HEV-172889 to KKM and CB). CB was supported by the Fonds de Recherche du Québec-Santé (FRQS).
Provenance and peer review: Not commissioned; externally peer reviewed.
Patient consent for publication: Not applicable.
Ethics approval: All procedures were approved by the McGill University Animal Care Committee and performed in accordance with the Canadian Council on Animal Care Committee (protocol number 2013-7421).
Data availability free text: Proteomics data supporting the conclusions of this paper can be found at DOI: 10.6084/m9.figshare.26351740.
Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Data availability statement
Data are available in a public, open access repository. Data are available on reasonable request.
References
Articles from BMJ Open Respiratory Research are provided here courtesy of BMJ Publishing Group
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Funding
Funders who supported this work.
Canadian Institutes of Health Research (2)
Grant ID: HEV-172889
Grant ID: PJT-168836