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. 2024 Sep 18;11(1):e002423.
doi: 10.1136/bmjresp-2024-002423.

Sex-specific alterations in pulmonary metabolic, xenobiotic and lipid signalling pathways after e-cigarette aerosol exposure during adolescence in mice

Sofia Paoli  1   2 , David H Eidelman  1   3 , Koren K Mann #  2   4 , Carolyn Baglole #  5   2   3
Affiliations

Sex-specific alterations in pulmonary metabolic, xenobiotic and lipid signalling pathways after e-cigarette aerosol exposure during adolescence in mice

Sofia Paoli et al. BMJ Open Respir Res. .

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.

Keywords: inflammation.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1. The percentage of innate immune cells in the lung tissue of BALB/c and C57BL/6 mice are not affected by JUUL exposure. The frequency of total CD45+ immune cells (A), neutrophils (B), eosinophils (C), macrophages (D), alveolar macrophages (E), dendritic cells (F) and monocytes (G) are shown. Data represent pooled samples from two independent experiments. Data are expressed as mean±SEM. Differences were analysed by two-way analysis of variance (*p≤0.05, **p≤0.01). PG, propylene glycol; VG, vegetable glycerin.
Figure 2
Figure 2. Differential effects on adaptive immune cell populations in the lung tissue of BALB/c and C57BL/6 mice after JUUL exposure. The frequency of total CD45+ immune cells (A), CD19+ B cells (B), CD4+ T cells (C) and CD8+ T cells (D) are shown. CD4+ T cells were higher among all BALB/c mice compared with C57BL/6 mice. CD8+ T cells were decreased by JUUL exposure only in C57BL/6 mice. Data represent pooled samples from two independent experiments and are expressed as mean±SEM. Differences were analysed by two-way analysis of variance (*p≤0.05, ***p<0.001, ****p≤0.0001). PG, propylene glycol; VG, vegetable glycerin.
Figure 3
Figure 3. Proteins quantified in the lungs and BAL fluid of C57BL/6 mice are significantly affected by JUUL exposure. (A) Total proteins—C57BL6: in the BAL fluid of C57BL/6 mice, 958 proteins were quantified and 1226 were quantified in lung tissue. There were 550 proteins in common between the two lung compartments. (B) KEGG pathways—C57BL/6: there were numerous enriched pathways in BAL and lungs with considerable overlap between the lung compartments. (C) JUUL versus Air—DEPs: there were significant changes in proteins in the lungs and BAL of C57BL/6 mice in response to JUUL with minimal overlap. (D) JUUL versus Air—KEGG: pathway enrichment analysis revealed that in the lungs, the proteins affected by JUUL are related to many metabolic pathways and immunological functions. (E) JUUL versus Air—STRING: STRING analysis showed significant PPI interaction in the lungs and BAL in pathways related to immune and oxidative stress responses as well as lipid metabolism. DEP, differentially expressed protein; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction; STRING, Search Tool for Retrieval of Interacting Genes.
Figure 4
Figure 4. Proteins quantified in the lung and BAL fluid of BALB/c mice are significantly affected by JUUL exposure. (A) Total proteins—BALB/c: there were 1316 proteins quantified in the lung tissue and 464 in BAL; the proteins found in both lung compartments are shown. (B) KEGG pathways—BALB/c: there were more enriched pathways in the lungs compared with BAL with considerable overlap between them. (C) JUUL versus Air—DEPs: there was a significant change in proteins from JUUL exposure, but none were common between the lungs and BAL. (D) JUUL versus Air—KEGG: pathway enrichment analysis revealed pathways related to infection and chemical carcinogenesis, with some pathways showing distinct patterns between lung compartments. (E) JUUL versus Air—STRING: there was enrichment in the lungs for pathways related to protein folding (red), detoxification (green) and translation (blue) whereas in BAL only two pathways were enriched and were related to immune function (red) and serpins (green). DEP, differentially expressed protein; KEGG, Kyoto Encyclopedia of Genes and Genomes; STRING, Search Tool for Retrieval of Interacting Genes.
Figure 5
Figure 5. Comparative proteomic analysis of the lungs and BAL between C57BL/6 and BALB/c mice. (A) Total proteins—BAL: there was overlap in the majority of proteins quantified in BAL between inbred strains of mice. (B) KEGG pathways—BAL total proteins: pathways were similar between C57BL/6 and BALB/c mice. (C) Total proteins—lungs: most of the proteins quantified in the lungs were similar between mouse strains. (D) KEGG pathways—lungs total proteins: there were minimal differences in pathways from lung proteins between strains of mice. (E) DEPs BAL JUUL versus Air: only two DEPs were found in BAL of both strains of mice exposed to JUUL. Most proteins that were significantly affected by JUUL were distinct. (F) DEPs—lungs JUUL versus Air: three proteins were common in the lungs of JUUL-exposed mice. Most of the proteins significantly affected by JUUL exposure were distinct between C57BL/6 and BALB/c mice. (G) KEGG—lung JUUL versus Air DEPs: there were few common pathways enriched in the lungs of JUUL-exposed C57BL/6 and BALB/c mice. DEP, differentially expressed protein; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 6
Figure 6. Sex-specific changes in the expression of proteins caused by JUUL exposures in C57BL/6 mice. (A) C57BL/6 BAL DEPs: there was minimal overlap in DEPs in BAL between male and female mice. (B) C57BL/6 lungs DEPs: there were more DEPs in the lungs of male mice after JUUL exposure with little overlap with proteins changed by JUUL in female mice. (C). STRING—BAL: there was significant pathway enrichment in BAL fluid of BALB/c mice, including pathways involved in prostaglandin synthesis (red), oxidative stress (green) and acetylation (blue). (D) KEGG pathways—lungs: there was no overlap in enriched pathways in DEPs between males and female mice. DEP, differentially expressed protein; KEGG, Kyoto Encyclopedia of Genes and Genomes; STRING, Search Tool for Retrieval of Interacting Genes.
Figure 7
Figure 7. Sex-specific changes in the expression of proteins caused by JUUL exposures in BALB/c mice. (A) BALB/c BAL DEPs: there was no overlap in DEPs between male and female mice, with more proteins being affected by JUUL in female BABL/c mice. (B) BALB/c lungs DEPs: there were more DEPs in the lungs of JUUL-exposed male mice, with little overlap with DEPs in the lungs of female mice. (C) KEGG pathways—lungs: only one pathway was in common between male and female mice. DEP, differentially expressed protein; KEGG, Kyoto Encyclopedia of Genes and Genomes.
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