Single-cell transcriptomics identifies altered neutrophil dynamics and accentuated T-cell cytotoxicity in tobacco-flavored e-cigarette-exposed mouse lungs

Electronic cigarettes (e-cigs) or electronic nicotine delivery systems are a relatively novel set of tobacco/nicotine and flavored products that have gained immense popularity among adolescents and young adults in many countries including the United States (US), the United Kingdom (UK), and China. Flavors are one of the key features that make these products alluring to the younger diaspora (Ma et al., 2022). Reports indicate that in 2020 about 22.5% of high school students and 9.4% of middle school students in the US were daily vapers or e-cig users with fruit (66%), mint (57.5%), and menthol (44.5%) being the most commonly used flavors (Wang et al., 2020a). However, not much is known about the flavor-specific effects of e-cig vaping on the health and immunity of an individual, especially focusing on cell types and gene expressions.

E-cig products and aerosols are known to contain harmful constituents including formaldehyde, benzaldehyde, acrolein, n-nitrosamines, volatile organic compounds, ketenes, and metal ions (Wu and O’Shea, 2020; Goniewicz et al., 2013; Lee et al., 2020). Studies have indicated that exposure to e-cigs may enhance inflammatory responses, oxidative stress, and genomic instability in exposed cells or animal systems (Wang et al., 2020b; Muthumalage et al., 2019; Lee et al., 2018b). Risk assessment (systemic) of inhaled diacetyl, a potential component of e-liquids, has estimated the non-carcinogenic hazard quotient to be greater than 1 among teens (White et al., 2021). Furthermore, clinical and in vivo studies have suggested that exposure to e-cig aerosols could impair innate immune responses in the host, thus making them more susceptible to bacterial/viral infections. The bacterial clearance, mucous production, and phagocytic responses in these individuals are shown to be affected upon use of e-cigs (Martin et al., 2016; Sussan et al., 2015; Masso-Silva et al., 2021; Madison et al., 2019; Cao et al., 2021).

However, cell-specific changes within the lung upon vaping are not fully understood, making it hard to determine the health impacts of the use of these novel products. In this respect, single-cell technology is a powerful tool to analyze gene expression changes within cell populations to study cellular heterogeneity and function (Jovic et al., 2022; Inayatullah et al., 2025; Ke et al., 2022). Such an investigation is important to deduce the health effects of acute and chronic use of e-cigs in young adults. In this study, we aim to determine the effects of acute exposure to e-cig aerosols on mouse lungs at single-cell level. To do so, we exposed C57BL/6J mice to 5-day nose-only exposure to air, propylene glycol:vegetable glycerin (PG:VG), fruit-, menthol-, and tobacco-flavored e-cig aerosols. The nose-only exposure has more translational relevance over the whole-body exposure (Kogel et al., 2021), owing to which, we chose nose-only exposure profile for this work. To limit the stress to the animals, a 1-hr exposure was chosen per day. We performed single-cell RNA sequencing (scRNA seq) on the lung digests from exposed and control animals and identified neutrophils and T cells, among others, as the major cell populations in the lung that were affected upon acute exposure. We were able to identify 29 gene targets that were commonly dysregulated among all our treatment groups upon aggregating results from the major lung cell types. These gene targets are the markers of early immune dysfunction upon e-cig aerosol exposure in vivo and could be studied in detail to understand temporal changes in their expression and function that may govern allergic responses and adverse pulmonary health outcomes upon acute and sub-acute exposures to e-cigarette aerosols.

E-cigs and associated products have constantly been under scrutiny by the US Food and Drug Administration (FDA) due to public health concerns. In February 2020, the FDA placed a regulation on all cartridge-based flavored e-cigs except for menthol and tobacco to reduce the use of e-cigs among adolescents and young adults. But it left a loophole for the sale of flavored (including menthol) disposable and open system e-cigs (Ma et al., 2022; Sindelar, 2020). Importantly, most e-cig-related bans in the US happened at the state level, thus allowing differential levels of restrictions imposed on the premarket tobacco applications and sales, which defeats the purpose of limiting their accessibility to the general public (Bhalerao et al., 2019; Azagba et al., 2023). In fact, the use of nicotine-containing e-cigs among youth and associated policy restrictions has recently been found to be linked to unintended increase in traditional cigarette use (Cheng et al., 2025). Each year, new products are introduced in the market with newer device designs and properties to lure the users (adults between the ages of 18–24 years), which makes it crucial to continue with the assessments of toxicity and health effects of e-cigs in an unbiased manner (Kramarow and Elgaddal, 2023).

Numerous studies indicate increased oxidative stress, DNA damage, and loss of neutrophil function due to exposure to e-cig aerosols in vitro and in vivo (Muthumalage et al., 2019; Lamb et al., 2023; Lamb et al., 2020; Corriden et al., 2020; Jasper et al., 2024; Ren et al., 2022). However, we do not have much knowledge about the cell populations and biological signaling mechanisms that are most affected upon exposure to differently flavored e-cig aerosols at a single-cell level. To bridge this gap in knowledge, we studied the transcriptional changes in the inflammatory responses due to acute (1 hr nose-only exposure including 120 puffs for 5 consecutive days) exposure to fruit-, menthol-, and tobacco-flavored e-cig aerosols using single-cell technology. Short-term (2 hr/day for 3 or 5 consecutive days) e-cigarette exposure studies conducted by others and our group have shown an increase in the pro-inflammatory cytokines and oxidative stress markers in both pulmonary and vascular tissues (Wang et al., 2019; Kuntic et al., 2020; Wang et al., 2020c). Since nose-only exposures are more direct than whole-body, we reduced the daily exposures of mice to 1 hr for 5 consecutive days. This dose and duration were found to show sex-dependent changes in MMP-2 and -9 activity and expression (Lamb et al., 2023). Prior literature formed the basis of the current study to assess the effect of acute exposure to differently flavored e-cig aerosols on the lung cell population using a nose-only exposure system.

Reports indicate that the release of metal ions due to the burning of metal coil is a major source of variation during e-cig exposures (Zhao et al., 2020; Zhao et al., 2019; Alcantara et al., 2023). A 2021 study reported the presence of 21 elements in the pod atomizers from different manufacturers identifying a high abundance of 11 elements including nickel, iron, zinc, and sodium among others (Omaiye et al., 2021). Previous studies have also shown the presence of similar elements in the e-cig aerosols which could have a possible adverse health effect on the vapers (Olmedo et al., 2018; Rastian et al., 2022). More importantly, our study points toward a very important issue, which pertains to the product design of the e-cig vapes. We believe that the aerosol composition varies based on the type of atomizer, coil resistance, coil composition, and chemical reactivity of the e-liquid being used. While much work is done on the chemical composition of the flavors and e-liquids, the other aspects of device design remain understudied and must be an area of research in the future. In fact, a recent publication by our group emphasizes this aspect of product design by studying the exposure profile from low- and high-resistance coil in an e-cig (Effah et al., 2025). Another factor that may limit the interpretation of our results pertains to the correlation between the leached metal and the observed transcriptional changes. Since our study provides proof of day-to-day variation in the leaching of metal ions from the same liquid using the same atomizer in the emitted aerosols, it could be possible to develop a statistical model correlating the differential metal exposure to the gene expression changes to get a good assessment of the dose of each toxicant deposited on the lung surface, which has not been done in this study. This raises a pertinent question about what parameters should be controlled to design a comparative study between different flavors of e-cigs in the market per the standards of particle distribution and characteristics. Flavor-dependent variations in daily leaching of metals from an e-cig coil may result in variable deposition of toxicants onto the epithelial lining of both mouth and lungs in frequent vapers. Thus, future studies need to consider conditions like flavor, wattage, coil resistance, coil composition, and atomizer life when designing in vitro and in vivo studies to deduce the acute and chronic toxicities of vaping in humans.

E-cig vaping has been known to affect the innate and adaptive immune responses among vapers (Jasper et al., 2024; Kalininskiy et al., 2021; Rebuli et al., 2021; Reidel et al., 2018), but flavor-specific effects on immune function are not fully explored. While we expected to see flavor-dependent changes in our experiment, we did not anticipate observing interesting sex-dependent variation in the lung tissues using flow cytometry. In this respect, recent studies show concurring evidence suggesting sex-specific changes in lung inflammation, mitochondrial damage, gene expression, and even DNA methylation in mice exposed to e-cig aerosols (Song et al., 2023; Chirumamilla et al., 2024).

It is important to highlight here that while the changes in macrophage and neutrophil frequencies ascertained by scRNA seq corroborate with that observed using flow cytometry, similar correlation was not observed for the CD4⁺ and CD8⁺ T-cell data. The possible explanation for such a discrepancy could be high gene dropout rates in scRNA seq (Qiu, 2020), different analytical resolution for the two techniques (Palit et al., 2019), and pooling of samples in our single-cell workflow. Also, while we were able to identify some interesting changes in the eosinophil population within the lungs upon exposure to e-cigs using our flow cytometry data, we could not identify a cluster for eosinophils in our scRNA seq dataset. This could be due to the loss of this cell type during the sample preparation during scRNA seq capture or filtering out as empty droplets during data analyses. Theoretically, eosinophils are known to have lower RNA content and express fewer numbers of genes, which may result in them being identified as empty droplets and removed when running Cell Ranger (Goss et al., 2025). Despite the lack of data from scRNA seq, our findings are of relevance as an increased neutrophilic, but a decreased eosinophilic response is characteristic of severe inflammation, infection, and asthma (Jukema et al., 2022; Flinkman et al., 2023; Lourda et al., 2021). Further probing into the role of these two cell types in shaping the immune landscape within the lung upon e-cig aerosol exposure is paramount in understanding the specific toxicities and impact of each e-cig flavor on human health and well-being.

One of the most interesting discoveries from our single-cell analyses was the identification of a cluster of immature neutrophils without Ly6G surface marker. While we observed an increase in the cell percentages of these cells in our treatment groups, little to no change in the gene expression was noted (data not shown), which could be indicative of impaired function of mature neutrophil population, a fate being reported by various previous studies pertaining to e-cig exposures (Madison et al., 2019; Kalininskiy et al., 2021). In fact, our study identified (a) a moderate increase in the neutrophil count in menthol- and tobacco-flavored e-cig aerosol treated mouse lungs through scRNA seq analyses, (b) corroboration of the findings from scRNA seq using flow cytometry with more pronounced increase in Ly6G⁺ neutrophils for male menthol-flavored e-cig aerosol exposed mice, and (c) identification of decreased co-localization for Ly6G and S100A8 in the lungs of tobacco-flavored e-cig aerosols as compared to air control through co-immunostaining. Ly6G is an important marker of neutrophil recruitment and maturation in mammalian cells and has been reported in relation to various bacterial and parasitic infections in previous studies (Deniset et al., 2017; Kleinholz et al., 2021). Of note, this cluster did not express SiglecF and had high expression of other neutrophil markers including S100A8/9, Lcn2, and Il1b, thus negating the possibility of eosinophils being misrepresented as ‘Ly6G⁻ neutrophils’ in our analyses.

S100A8 acts as damage-associated molecular patterns and is responsible for neutrophil activation and neutrophil extracellular trap (NET) formation (Sprenkeler et al., 2022; Guo et al., 2021). While scRNA seq results in our study identified expression of S100A8/A9 genes in both neutrophil clusters – Ly6G⁺ and Ly6G⁻, the expression of Ly6G was totally absent for clusters identified as Ly6G⁻. Co-immunoprecipitation results also showed expression of both S100A8 and Ly6G markers within the lungs of treated and control lungs, but the co-localization of the two markers was more prominent in the control lungs, thus pointing toward a possible shift in the neutrophil function and activity upon exposure to e-cig aerosols. We are not the first to report the Ly6G deficiency among neutrophils. Previous work by Deniset et al., 2017 and Kleinholz et al., 2021 has highlighted the importance of Ly6G deficiency in relation to infection (Deniset et al., 2017; Kleinholz et al., 2021). Deniset et al., 2017 study reported the presence of two populations of neutrophils in the splenic tissue of Streptococcus pneumoniae infected mice, based on the Ly6G expression – Ly6G^high and Ly6G^intermediate. They found that while the former corresponds to mature neutrophils with ability of tissue migration and bacterial clearance, the latter constitutes the immature, immobile pool of neutrophils responsible for proliferation and replenishment of the mature pool of neutrophils (Deniset et al., 2017). The later study by Kleinholz et al., 2021 demonstrated decreased uptake of Leishmania major in Ly6G-deficient mice, thus leading to delayed recruitment to and pathogen capture by neutrophils at the site of infection. Taken together, these studies provide evidence for a protective/compensatory role of the loss of Ly6G in the neutrophil population (Kleinholz et al., 2021). This, in addition to the recent findings by Jasper et al., 2024 where neutrophils from healthy volunteers demonstrated a reduction in neutrophil chemotaxis, phagocytic function, and NET formation upon exposure to e-cig aerosols (Jasper et al., 2024), points toward a probable shift in the neutrophil dynamics upon exposure to e-cig aerosols. It is important to mention here that though our results from co-immunostaining using Ly6G and S100A8 pointed toward a shift in innate immune responses upon exposure to e-cig aerosols, it must not be confused with them being only expressed by neutrophil population. S100A8 is expressed in myeloid population including neutrophils, monocytes, and macrophages (Averill et al., 2012; Wang et al., 2018), and a subgroup of eosinophils is also known to express Ly6G (Mair et al., 2021). Thus, an in-depth characterization of these identified populations is important to understand the cellular and molecular responses toward e-cig aerosol exposure in vivo.

The myeloid and lymphoid limbs of immunity are interconnected (Shanker and Marincola, 2011; Carroll and Prodeus, 1998). In our study, we find an incidental shift in the neutrophil dynamics in menthol- and tobacco-flavored e-cig exposed mouse lungs. In contrast, we report an increase in the T-cell responses in the form of increased CD8⁺ T cells from both scRNA seq and flow cytometric analyses in male mice. In fact, increased expression of genes including Malt1, Serpinb9b, and Sema4c is indicative of enhanced T-cell-mediated immune response in the lungs of mice exposed to fruit (mango) flavored e-cig aerosols (Wang et al., 2020d; Bird et al., 2014; Beland et al., 2014). Contrary to this, exposure to menthol-flavored e-cig aerosols had a much milder effect on the lymphoid population within the lungs of C57BL/6J mice. We found evidence for increased lymphoid cell proliferation due to activation of cyclin-dependent protein kinase signaling mediated via expression of genes including Cdk8 and Camk1d in these cells (Jin et al., 2022; Szilagyi and Gustafsson, 2013). Exposure to tobacco-flavored e-cig aerosol provided evidence for decreased chaperone-mediated protein folding, due to the downregulation of Chaperonin Containing TCP-1 (CCT) family of proteins. Chaperonin Containing TCP-1 proteins are important to regulate the production of native actin, tubulin, and other proteins crucial for cell cycle progression and cytoskeletal organization (Brackley and Grantham, 2009). This is in conjunction with the upregulation of Klra4 and Klra8 that is indicative of increased protein misfolding and cytotoxic responses in the lymphoid cells of tobacco-exposed e-cig aerosols (Berry et al., 2013; Bolanos and Tripathy, 2011).

Overall, we provide evidence of altered innate immune responses due to variable neutrophilic–eosinophilic function and increased T-cell proliferation and cytotoxicity in a flavor-dependent manner upon exposure to e-cig aerosols in this study. An increase in the levels of CCL17, CCL20, CCL22, IL2, and Eotaxin in the lung digests from tobacco-exposed mouse lungs further supports this deduction as these cytokines/chemokines are associated with T-cell-mediated immune responses (Israr et al., 2022; Li et al., 2020; Wang et al., 2024; Ross and Cantrell, 2018; Rapp et al., 2019; Jinquan et al., 1999; Chia et al., 2023; Böttcher et al., 2015). Importantly, CXCL16 attracts T cells and natural killer cells to activate cell death. It is involved in LPS-mediated acute lung injury, an outcome which has also been linked with e-cig exposures in humans (Tu et al., 2019; Christiani, 2020).

Further, we compiled a list of commonly dysregulated genes in a flavor-independent manner and identified 29 gene targets. Signal-transducing adaptor protein-2 (Stap1), which is commonly upregulated upon exposure to e-cig aerosols, is known to regulate T-cell activation and airway inflammation, which is in agreement with the overall outcome of our findings (Kagohashi et al., 2024). Another gene that was found to be consistently dysregulated in many cell types was cold-inducible RNA binding protein (Cirbp). Cirbp is a stress response protein linked with stressors like hypoxia. Its upregulation upon e-cig exposure supports that vaping induces oxidative stress and can have adverse implications on the exposed cell types (Zhu et al., 2024). Importantly, this gene is involved in DNA repair mechanisms, thus making it crucial for cell survival pathways (Firsanov et al., 2025). 5-Hydroxytryptamine receptor 2C (Htr2c) and Klra8 are other genes in this category of commonly dysregulated genes that are associated with enhancing inflammation and cell death (Berry et al., 2013; Bolanos and Tripathy, 2011; Mikulski et al., 2010). Overall, we provide a cell-specific resource of immune responses upon exposure to differently flavored e-cig aerosols.

Considering that scRNA technology has not been commonly used for e-cig research, ours is one of the first studies employing this technique to identify possible changes in the cellular composition and gene expressions. Importantly, we use the nose-only exposure system for our experiment to avoid exposure through other routes. However, despite the novel approach and state-of-the-art exposure system, we had a few limitations. First, we used a small sample size to identify the changes in the mouse lungs upon exposure to e-cig aerosols at a single-cell level. Due to the expensive nature of single-cell sequencing technology and limited information in the literature, we chose to design this experiment with small sizes of experimental and validation cohorts. But, based on the encouraging findings from this study, future studies could be designed with a larger sample size, longer durations of exposure, and more targeted approach to identify the acute and chronic effects of vaping in vivo. Second, we could not expand upon the sex-dependent changes observed through our work upon exposure. This was because such an effect was not anticipated when we conceived the idea of a short-term exposure in mice. However, considering the evidence from the current study, future experimental designs in our lab are considering sex as a crucial confounder for studying the effects of e-cig exposure in translational contexts. Third, the inclusion of PG:VG + Nic group was streamlined in this study, but in future work, the inclusion of this group for scRNA seq analyses to delineate the effects of nicotine alone on gene transcription is necessary. Fourth, we did not anticipate changes in the metal release on consecutive days of exposure at the start of our study. Later, our data pointed toward the importance of device design in e-cig exposures. Future studies need to identify the factors that may affect the daily composition of e-cig aerosols and devise a method of better monitoring these possible confounders. However, in this regard, our experiment does mimic the real-life scenario, as such variations due to prolonged storage of e-liquid and differences arising due to vape design must be common among human vapers.

In conclusion, this study identified cell-specific changes in the gene expressions characterized by altered neutrophil dynamics and accentuated T-cell cytotoxicity upon exposure to tobacco-flavored e-cig aerosols using single-cell technology. Furthermore, a set of top 29 dysregulated genes was identified that could be studied as markers of toxicity/immune dysfunction in e-cig research. Future work with larger sample sizes and sex distribution is warranted to understand the health impacts of long-term use of these novel products in humans.

The datasets generated are deposited on NCBI Gene Expression Omnibus under accession code GSE263903' The data will be publicly available upon publication or on February 1, 2026, whichever is earlier. All other relevant data supporting the key findings of this study are available within the article and its supplementary files. Source data are provided with this paper.

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Author details

1. Gagandeep Kaur

   Department of Environmental Medicine, University of Rochester Medical Center, Rochester, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2674-9947

2. Thomas Lamb

   Department of Environmental Medicine, University of Rochester Medical Center, Rochester, United States

   Contribution

   Data curation, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Ariel Tjitropranoto

   Department of Environmental Medicine, University of Rochester Medical Center, Rochester, United States

   Contribution

   Data curation, Software, Formal analysis, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

4. Irfan Rahman

   Department of Environmental Medicine, University of Rochester Medical Center, Rochester, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   Irfan_Rahman@urmc.rochester.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2274-2454

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