MMP12 deficiency attenuates menthol e-cigarette plus house dust-mite effects on pulmonary iron homeostasis and oxidative stress

Pinkston, Rakeysha I.; Schexnayder, Matthew; Perveen, Zakia; Langohr, Ingeborg M.; Jelesijevic, Tomislav; Penn, Arthur L.; Noël, Alexandra

doi:10.1186/s12931-025-03213-w

Research
Open access
Published: 11 April 2025

MMP12 deficiency attenuates menthol e-cigarette plus house dust-mite effects on pulmonary iron homeostasis and oxidative stress

Rakeysha I. Pinkston^1,2^na1,
Matthew Schexnayder³^na1,
Zakia Perveen¹^na1,
Ingeborg M. Langohr^4,5^na1,
Tomislav Jelesijevic¹^na1,
Arthur L. Penn¹^na1 &
…
Alexandra Noël¹^na1

Respiratory Research volume 26, Article number: 135 (2025) Cite this article

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Abstract

Background

Little is known regarding the pulmonary effects induced by the inhalation of menthol-flavored e-cigarette aerosols on asthma exacerbation, despite the popularity of these devices and flavors among youth and young adults. In the lungs, matrix metalloproteinase 12 (MMP12) expressed and secreted by both alveolar macrophages and bronchial epithelial cells plays an essential role in airway remodeling, a key feature of severe asthma. In this study, we investigated the role of MMP12 in menthol-flavored e-cigarette aerosol exposures plus house-dust mite (HDM)-induced asthmatic responses.

Methods

We exposed wild-type (WT) and MMP12 knockout (KO) juvenile female mice to well-characterized menthol-flavored e-cigarette aerosols followed by either PBS or HDM treatment, and evaluated pulmonary outcomes in terms of iron metabolism, oxidative stress responses and pulmonary inflammation.

Results

We found high levels of iron in the menthol-flavored e-cigarette aerosol. This correlated with e-cigarette + HDM WT mice exhibiting disruption of pulmonary iron metabolism, suggesting a defense mechanism against iron-mediated toxicity. This was evidenced by altered lung protein concentrations of ferroportin, ferritin, lactoferrin, and transferrin, activation of the antioxidant response element (ARE) pathway and up-regulated expression of NQO1 in e-cigarette + HDM WT mice. Further, despite decreased neutrophilic inflammation, MUC5AC, an oxidative stress inducible mucin, was increased in the e-cigarette + HDM WT mice. In contrast, MMP12 KO mice were protected against iron-induced oxidative stress responses, highlighting a crucial role of MMP12 in this model.

Conclusion

These findings revealed in vivo evidence supporting a crucial role for iron metabolism in nicotine salt iron-rich ENDS aerosol toxicity.

Background

An electronic-cigarette (e-cig) is a handheld device that heats nicotine plus flavoring and other chemicals to generate an aerosol that can be inhaled (vaped). E-cigs are formally classified as electronic nicotine delivery systems (ENDS). ENDS aerosols are complex mixtures of: (1) carbonyls, including acetaldehyde and formaldehyde, produced during the thermal degradation of propylene glycol and glycerin, the main humectants in e-liquids; (2) metals, including chromium (Cr), copper (Cu), iron (Fe), lead (Pb) and nickel (Ni), from corrosion of ENDS device parts, e.g., alloy coil and wick; (3) particles; (4) nicotine; and (5) flavorings. Mint/menthol is the most popular ENDS flavor, accounting for 48% of the market [1]. Flavoring chemicals, including menthol, have been reported to be cytotoxic and to induce oxidative stress in lung cells in vitro [2,3,4,5,6,7,8,9]. High levels of Cr, Ni and Fe have been found in mint-flavored JUUL aerosols [10]. Mice exposed to elevated concentrations of essential metals, including Fe from ENDS aerosols (> 50 µg/kg), exhibited Fe accumulation in the central nervous system (> 26 µg/g in the striatum) [11]. These data show that the inhalation of ENDS aerosols is a source of exposure to metals, which can accumulate in the body and potentially lead to adverse effects. While cigarette smoking can exacerbate asthma symptoms and decrease lung function [12], epidemiological evidence also showing effects of ENDS aerosol exposures on asthma exacerbations is starting to emerge. Ever ENDS use is now associated with a 31% increased risk of developing asthma, even after adjusting for confounding variables, including the use of combustible tobacco products [13]. Thus, ENDS use is associated with respiratory morbidity in adolescent and adult populations [14,15,16,17,18,19,20,21]. In the United States, 4.5% of American adults [22] and 7.7% of high school students use ENDS [23]. Collectively, these data reinforce the need to study how the inhalation of ENDS aerosols, particularly menthol-flavored ENDS aerosols, affect asthma exacerbation.

Worldwide, over 300 million individuals currently suffer from asthma, a debilitating respiratory condition with more than 450,000 fatalities annually [24]. Within the United States alone, there are more than 25 million asthmatic individuals: 5 million youth plus 20 million adults [25]. These prevalence rates result in medical expenses exceeding $50 billion dollars per year [26]. Asthma exacerbations are usually triggered by common household allergens including house-dust mites (HDM), metal sensitizers, and tobacco smoke [27]. Asthma pathogenesis is characterized by airway obstruction, increased airway hyperresponsiveness, inflammation and tissue remodeling [28,29,30]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that are involved in several biological processes, including tissue remodeling. MMPs are capable of degrading extracellular matrix components during normal and pathological conditions [31]. Thus, airway remodeling can lead to structural changes in the extracellular matrix within the large and small airways, in addition to being involved in proliferation of smooth muscle within the lungs, and activation of fibroblasts. All these, if uncontrolled, can cause extensive damage to the lungs [32]. MMP9 and MMP12 (macrophage elastase) have been reported to play major roles in tissue repair and airway remodeling. MMP12 is one of the most highly expressed genes in alveolar macrophages among smokers, including those with COPD [30]. Moreover, MMP12 knockout (KO) mice have been shown to exhibit (1) decrease emphysema symptoms, marked by a reduction of pulmonary inflammation [33], as well as (2) reduction in allergen-induced exacerbations [34], highlighting key influential roles for MMP12 in emphysema and asthma pathogenesis, respectively. We previously showed that Mmp12 was dysregulated in human bronchial epithelial cell lines (BEAS-2B and H292) and murine macrophages (RAW 246.7) following in vitro air–liquid interface exposures to JUUL aerosols [35]. Collectively these findings in humans, animals, and cells strongly support the need to investigate the effects of ENDS aerosol exposures on asthmatic responses, with a particular focus on MMP12 involvement in a combined ENDS + HDM-induced asthma model. Therefore, the overall objective of this study was to investigate the role of MMP12 in the pulmonary responses caused by menthol-flavored JUUL aerosols (a fourth generation ENDS device) on HDM-induced asthmatic responses in juvenile wildtype (WT) and MMP12 KO female mice. Our study revealed that menthol-flavored JUUL aerosol has a high iron content and that the superimposition of JUUL aerosol plus HDM led to the disruption of pulmonary iron homeostasis in WT mice, while those effects were attenuated in MMP12 KO mice. Regarding the induction of asthma, while menthol-flavored JUUL aerosol suppressed immunoinflammatory responses, this exposure stimulated oxidative stress pathways and the production of airway mucus. These pulmonary outcomes were reduced in MMP12 KO mice. Overall, our data point to a mechanism involving MMP12 influence on pulmonary iron metabolism and asthmatic responses following iron-rich JUUL-menthol flavored ENDS aerosol exposures in an HDM-induced asthma model.

Methods

Animals and in vivo JUUL aerosol exposures

We used 4- to 5-week-old C57BL/6 and MMP12 KO (B6.129X-Mmp12^tm1Sds/J) female mice (Jackson Laboratories, strain # 004855). Animals were housed and handled in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All procedures and protocols were approved by the Louisiana State University (LSU) Institutional Animal Care and Use Committee (IACUC). WT and KO mice were randomly assigned to the following groups: (1) filtered air + PBS, (2) JUUL 5% menthol + PBS, (3) filtered air + house dust mite (HDM), (4) JUUL 5% menthol + HDM (n = 7–8 per group). Mice were exposed to JUUL menthol-flavored aerosols or HEPA-filtered air in 5-L whole-body exposure chambers (Scireq, Montreal, QC, Canada) for 1 h/day for 3 or 60 consecutive days (Fig. 1a). As previously described in [36], to mimic the usage of the device and activate the airflow of this closed system device, JUUL aerosols were generated by connecting a JUUL device to a peristaltic pump (MasterFlex L/S, Cole-Palmer) using 1-inch diameter tygon tubing, followed by direct connection to the 5L chamber. The aerosol was diluted with filtered air. Total particulate matter (TPM) concentration was measured gravimetrically by sampling the test atmosphere onto a 25 mm glass fiber filter with a 0.7 m pore size (catalog # AP4002500, MilliporeSigma). A MicroDustPro (Casella) instrument was also used to simultaneously record the TPM in real-time. The average TPM concentrations for JUUL exposed groups are listed in Fig. 1b.

JUUL aerosol characterization

The chemical profile of this menthol-flavored JUUL aerosol (5%) was previously reported elsewhere [38]. The chemical analysis of JUUL aerosol was performed for nicotine, propylene glycol, glycerin, a selection of carbonyls and organic acids by using gas chromatography with a flame ionization detector (GC-FID) methods (for nicotine and the humectants), using the EPA method TO-11A, based on high performance liquid chromatography (HPLC) (for carbonyls), and by ion chromatography (for the organic acids), as reported in Pinkston et al. [38]. Also, we used a scanning mobility particle sizer spectrometer (SMPS) (Model 3938L50, TSI Inc., Shoreview, MN) to determine the particle size distribution of the JUUL aerosols. The SMPS has a classifier (Model 3082), a long differential mobility analyzer (DMA) (Model 3081A), and a condensation particle counter (CPC) (Model 3750), all from TSI Inc. (Shoreview, MN). This instrument allows to determine particle size distribution with high resolution within a range of 17.2 to 982 nm. The SMPS spectrometer was operated according to the manufacturer’s recommendation. For metals, we collected 90 puffs of the JUUL aerosol on glass fiber filters and concentrations for copper (Cu), chromium (Cr), nickel (Ni), and iron (Fe) were determined by inductively coupled plasma mass spectrometry (ICP-MS). These samples were analyzed by Eurofins EAG Laboratories (Liverpool, NY).

House-dust mite (HDM) treatment

During the last 21 days of the 60-day exposure study, subsets of WT or MMP-12 KO mice were treated intranasally once a week for 3 consecutive weeks with 50 µg of HDM extract resuspended in 1X PBS to induce allergic responses or 1X PBS for the control groups, as described in [37]. As per the experimental scheme (Fig. 1a), mice were challenged with 50 µg of HDM extract (Dermatophagoides pteronyssinus, catalog # XPB70D3A2.5, Stallergenes Greer, Charlotte, NC), resuspended in 1X PBS or 1X PBS for the control groups (Fig. 1a). Approximately 10 µl of HDM extract or PBS was deposited on the outer edge of each nare using a pipette to treat the mice intranasally once a week for 3 consecutive weeks, as described in Noël et al. [37]. Mice were sacrificed 24 h after the last HDM or PBS instillation by intraperitoneal injection of Beuthanasia-D (Schering-Plough, NJ).

Whole-body plethysmography

We evaluated lung function testing in mice from all eight groups exposed for 60 days (n = 7–8 mice per group) using a non-invasive method, e.g., whole-body plethysmography. We used a plethysmograph from Buxco (Troy, NY) to record lung function parameters, namely minute volume and breathing frequency, every minute for 5 min. These measurements were then averaged to determine pulmonary function, as described previously [37].

Bronchoalveolar lavage fluid (BALF) collection

After the mice were euthanized, a lavage of the lungs was performed by inserting a 20-gauge cannula into the trachea, followed by suturing to keep the cannula in place. Next, 0.75 mL of PBS was injected through the cannula twice using a 1 mL syringe and collected. The BAL fluid was then centrifuged to obtain a pellet of the total BAL cells, and the supernatant was separated from the cells. The BAL supernatant was stored at − 80 °C until subsequent analysis. The centrifuged BAL cell pellet was used for total and differential white blood cell counts as described in Noël et al. [37]. In brief, we prepared cytology slides using a cytospin followed by staining with a Hema 3 Stat Pack (Ref 122-911, Fisher HealthCare, USA) to differentiate and determine the percentage of macrophages, neutrophils, eosinophils, and lymphocytes (in total 300 cells were evaluated per slide). The BAL slides were read by a board-certified veterinary clinical pathologist.

Serum collection, cotinine and IgE ELISAs, as well as serum iron content

Following euthanasia, blood samples were collected via cardiac puncture. Collected serum was stored at − 80 °C until analysis. Serum was used to quantify cotinine levels using a cotinine ELISA kit (cat# CO096D-100, Calbiotech, CA) according to the manufacturer’s instructions. In addition, we evaluated serum IgE concentrations via an ELISA kit (cat# EMIGHE, ThermoFisher Scientific, Waltham, MA). We performed the ELISA as per the manufacturer’s instructions. Also, the serum was used to quantify the concentration of iron content using a flame atomic absorption spectroscope (F-AAS; Agilent 240FS AA, Agilent Technologies). Serum iron content analyses were conducted at the Louisiana Animal Disease Diagnostic Laboratory (LADDL) (Baton Rouge, LA).

Histopathological analysis of lungs

The right lung of the mice was inflated and pressure-fixed in 10% formalin administered by intratracheal instillation, then excised and stored in 10% formalin until histologic sectioning. Tissue sections were placed on a glass slide and stained with either hematoxylin and eosin (H&E) or Periodic Acid Schiff (PAS) staining. Stained tissue sections were examined by a board-certified veterinary pathologist, who was blinded to slide identifiers. Slides were scored for the presence of leukocytes, evidence of goblet cell metaplasia, and vascular and cellular damage. To measure interstitial inflammation, peribronchial inflammation, edema, endothelialitis and pleuritis, semiquantitative scores were assigned as follows: 0: absent, 1: mild, 2: moderate, 3: marked, 4: severe. To measure the percentage of PAS-positive goblet cells lining the bronchoalveolar space, of the following scores were used: 0: < 0.5%; 1: 0.5–25%, 2: 25–50%, 3: 50–75%, 4: > 75%. The lung tissues (10 µm thick sections) were also stained with Perl’s Prussian blue (PPB) stain and counterstained with eosin to visualize iron- and hemosiderin-laden macrophages in the lungs of the mice.

Genotyping

To confirm the MMP-12 gene KO phenotype, we clipped the tails of the animals, which were subsequently sent and analyzed by Transnetyx Inc. (Cordova, TN).

Total BALF protein quantification

BALF samples were used for total protein quantification according to the manufacturer’s instructions using a Pierce™ BCA protein assay kit (Thermo Fisher, Waltham, MA, USA).

RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from the left lobe of the mouse lungs. As previously described in [36, 37], lung tissue was stabilized in RNA later and kept at − 80 °C until analyzed. Total isolation from the lung tissue and collected cells was performed using the Qiagen RNeasy Mini Kit (Cat # 74,136, Qiagen, USA). RNA quantity and quality were measured with a NanoDrop ND-1000 Spectrophotometer (260/280 nm ratio, NanoDrop 1000, Thermo Scientific). The Bio-Rad iScript cDNA synthesis kit (Cat # 1708890, Bio-Rad Laboratories Inc, Hercules, CA, USA) was used for mRNA conversion, followed by amplification using a T100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). To investigate gene expression, qRT-PCR was performed using cDNA from lung tissue with Taqman pre-developed primer–probe sets (Applied Biosystems, University Park, IL, USA). The reaction volumes used were 25 μL, with 40 reaction cycles for each gene using the Applied Biosystems 7300 Real-Time PCR System. The comparative cycle threshold (ΔΔCT) method was used to determine relative gene expression. Hypoxanthine guanine phosphoribosyltransferase (Hprt1) was used as a housekeeping gene to normalize the expression of each gene. Results are reported as fold change over control [(2^−ΔΔCT)]. A fold-change > ± 1.5 was considered significant.

RT² profiler PCR array

As previously described in [36, 37], RNA from lung tissue were analyzed for the expression of 84 genes related to the Allergy and Asthma pathway responses using a PCR array (Qiagen, catalog # PAMM-067Z) according to the manufacturer’s instructions. In Brief, 0.5 μg of total RNA was reverse transcribed with the RT² First Strand Kit (Qiagen, catalog # 330,401) according to the manufacturer’s directions. Each cDNA sample was mixed with RT² SYBR Green qPCR Master mix (Qiagen, catalog # 330503). A 25 μL reaction mixture was added to each of the PCR Array plate that contained pre-dispensed gene-specific primer sets. The plate was analyzed using Applied Biosystems model 7300 real-time cyclers. Gene expression and fold change was calculated using the ∆∆Ct method, using Qiagen’s web-based PCR Array analysis software program. ∆Ct data were calculated and normalized using the average geometric mean of the following housekeeping genes: Hsp90ab1, Gusb, Actb, and B2m (n = 4 independent samples per group).

RNA sequencing

For the WT animals only, RNA from lung tissue (n = 4 samples per group) were analyzed for global gene expression analysis. RNA sample processing and sequencing was performed by LC Sciences (Houston, TX). RNA integrity was checked upon sample receiving using an Agilent Technologies 2100 Bioanalyzer. RNA libraries were constructed, and sequencing was performed using Illumina’s TruSeq-stranded platform on Illumina’s NovaSeq 6000 sequencing system. Differentially expressed mRNAs gene transcripts were selected with log2 (fold change) > + 1.5 (up-regulation) or < − 1.5 (down-regulation). All differentially expressed genes had a p value < 0.05.

Ingenuity pathway analysis

Gene expression data from the RT² Profiler PCR array and RNA sequencing were used to investigate associated gene networks and biological pathways utilizing the Ingenuity Pathway Analysis (Qiagen, Ingenuity Systems, Redwood City, CA, USA) web-based bioinformatics application software program. Significant genes (fold-change > ± 1.5) were considered for the analysis.

In situ hybridization using RNAscope

We used the lung tissues collected for histopathology assessment (e.g., 5 µm paraffin-embedded lung section slides) to conduct RNA-fluorescence in situ hybridization for Nqo1 and Muc5ac. In situ hybridization was conducted according to the manufacturer’s instructions (ACD user manual: RNAscope™ Multiplex Fluorescent Reagent Kit v2) and using the following RNAscope mouse probes, all from ACDBio (Newark, CA), for Hprt (Mm-Hprt-C1, catalogue #312951-C1) (green), Nqo1 (Mm-Nqo1-C2, catalogue #317801-C2) (red), and Muc5ac (Mm-Muc5ac-C3, catalogue #448471-C3) (blue). Slides were visualized and images were captured using a Nikon Ni-U microscope with motorized xyz (Nikon Instruments Inc., Melville, NY).

Protein extraction

Proteins were extracted from a small piece of a mouse left inferior lung lobe. The lung tissue sample was cut into the small pieces by razor blade, and then the samples were quickly transferred to micro-centrifuge tubes containing 250 μL of RIPA lysis buffer (Santa Cruz Biotechnology, Dallas, TX, United States) and three 2.5 mm zirconia/silica beads (Biospec Products Inc.). We used a Tissue Lyser II (Qiagen, Germantown, MD, United States) set at 25 MHz for 4 min to completely lyse the tissue. Afterwards, samples were incubated on ice for 30 min. The samples were centrifuged at 10,000g for 10 min at 4 °C. We used the BCA protein assay kits (Thermo Scientific, Waltham, MA, United States) to determine the protein concentrations in the supernatants.

Assays and ELISAs for lung proteins quantification

We used the proteins extracted from the lung samples (as described above) to perform a series of ELISAs or enzyme activity assays to determine the lung concentrations or activity of specific proteins involved in iron metabolism [ferroportin-1 (LS-F7200-1), transferrin (LS-F9542-1), ferritin light chain (LS-F8829), all from LS Bio, Shirley, MA, and lactoferrin (A74883, from Antibodies, St Louis, MO)], antioxidant response (NQO1 activity, AB184867, Abcam, Waltham, MA), and airway mucus hypersecretion (MUC5AC, LS-F4842, LS Bio, Shirley, MA).

Statistical analysis

Statistical analysis for all biological outcomes were performed using GraphPad Prism 9 Software (GraphPad Software, San Diego, CA). Student t-test was performed for pairwise comparisons. One-way analysis of variance (ANOVA) followed by either Tukey's post-hoc test or, when testing 3 or more groups, by Dunnet’s post-hoc test. Results are presented as mean ± standard error of the mean (SEM). Results were considered statistically significance at p < 0.05.

Results

High levels of iron are present in JUUL menthol-flavored aerosols.

The chemical profile of this JUUL menthol-flavored aerosol (5%) was previously reported [38]. These results, summarized in Fig. 1c, show that nicotine was present at a concentration of 98.7 µg/puff and benzoic acid at a concentration of 1.38 µg/puff, while carbonyls (e.g., formaldehyde and acetaldehyde) were detected at very low concentrations < 0.0015 µg/puff (Fig. 1c). Further, the metal content analysis revealed that the menthol-flavored JUUL aerosol had elevated levels of iron (Fe) (38.9 ng/puff), while trace levels were found for chromium (Cr) (1.67 ng/puff) and copper (Cu) (1.3 ng/puff) (Fig. 1e). Regarding the physical characteristics of the JUUL aerosols, data from the SMPS showed that the JUUL device emitted particle/droplet size with a geometric mean of 255 nm and a geometric standard deviation of 1.86 (Fig. 1d). Together, these physico-chemical characteristics highlight the potential toxicity of JUUL aerosols for the lungs since they contain elevated levels of metals and have a fine particle size range that can reach the lower respiratory tract.

Mmp12 increases serum cotinine levels in HDM-treated mice exposed to JUUL menthol for 60 days

We investigated the pulmonary effects of menthol-flavored JUUL aerosol exposures in C57BL/6 WT or MMP-12 KO mice. Following 3 consecutive days of exposure, we found that short-term JUUL menthol exposure did not induce pulmonary inflammation in either WT or KO mice, compared to air controls (Additional file 1: Fig. S1). In contrast, gene expression of Mmp12 (macrophage metalloelastase) within lungs was significantly increased (1.6-fold) in WT menthol-flavored JUUL aerosol-exposed mice (Fig. 2a). These results indicate that short-term JUUL menthol aerosol exposure upregulates the expression of Mmp12, which may be a precursor of lung damage resulting from prolonged JUUL exposure. Next, we exposed mice sub-chronically for 60 days to menthol-flavored JUUL aerosols and analyzed the gene expression of Mmp12 in the lungs of WT mice. Air + HDM significantly increased the expression of Mmp12 when compared to the Air + PBS group (16.8-fold); however, JUUL + PBS had no effect on Mmp12 gene expression (Fig. 2b). JUUL + HDM also increased the expression of Mmp12 gene (7.7-fold; Fig. 2b). We also confirmed that nicotine was effectively delivered to the JUUL exposed mice in our exposure system by measuring serum cotinine concentration. We found that 60 days of menthol-flavored JUUL aerosol exposure significantly increased serum cotinine, a metabolite of nicotine, in JUUL WT exposed groups ± HDM (> 125 ng/mL; Fig. 2c) compared to respective JUUL exposed KO mice (> 55 ng/mL; Fig. 2d). Thus, while no significant changes in breathing frequency or minute volumes were noted between groups (Additional file 1: Fig. S2 ), the assessment of serum cotinine levels revealed differences among WT and KO groups, implying a role for MMP12 in nicotine salt metabolism in this particular context. In WT mice, JUUL + HDM increased serum cotinine levels compared to WT JUUL exposed animals not treated with HDM (Fig. 2c), suggesting enhanced nicotine absorption, which was accompanied in HDM treated mice by the overstimulation of the nicotinic receptor leading to its down-regulation (− 1.5-fold) [39]. A similar down-regulation was seen among JUUL + HDM MMP12 deficient mice (Fig. 2f). Overall, these data indicate that with or without MMP12, HDM treatment increased serum cotinine levels following JUUL exposures, reflecting increased nicotine absorption. The results also suggest that MMP12 deficiency reduces nicotine absorption or its metabolism. Collectively, these data highlight that MMP12 and HDM (Fig. 2) may influence the toxicokinetic of inhaled nicotine.

Exposures to menthol-flavored JUUL aerosol plus HDM disrupt pulmonary iron metabolism homeostasis, while MMP12 deficiency attenuates this effect

Since our exposure characterization data revealed high concentrations of iron in the menthol-flavored JUUL aerosols (Fig. 1e), we next evaluated serum iron concentrations as well as the concentrations of several lung proteins involved in pulmonary iron metabolism in all groups of mice. We found that the serum iron concentrations were similar between all WT groups (~ 45 µg/mL). Although these concentrations were lower than in the KO groups (~ 68 µg/mL), no statistically significant differences were observed (Additional file 1: Fig. S3 ). Thus, the high inhaled iron content from the aerosol did not translate into a systemic increase of iron levels in the blood. In contrast, when we evaluated the lung concentration of proteins involved in iron metabolism: ferroportin-1, transferrin, ferritin light chain, and lactoferrin, we saw significant differences between the mice from the different WT groups (Fig. 3). JUUL + HDM WT mice exhibited significantly increased concentrations of ferroportin and lactoferrin (Fig. 3a, c) and significantly decreased concentrations of ferritin and transferrin compared to the Air WT mice (Fig. 3e, g). No significant differences were observed in MMP12 KO mice (Fig. 3). Since these four proteins play key roles in pulmonary iron export, storage and uptake, respectively, our results indicate impaired iron metabolism in the lungs of JUUL + HDM exposed WT mice (Fig. 3). Further, although the lungs of the mice were first lavaged to collect BALF (Fig. 4), subsequent staining of the lung tissue with PPB show the presence of iron containing macrophages only in the mice from the Air WT + HDM, JUUL WT + PBS, and JUUL WT + HDM groups (Fig. 3i, arrows). Taken together, these data suggest that the high iron concentration measured in the JUUL menthol-flavored aerosol (Fig. 1e) led to a localized, as opposed to systemic (Additional file 1: Fig. S3 ), increased level of iron in the lungs of WT mice also receiving HDM, as evidence by the concentrations of iron metabolism-related lung proteins (Fig. 3a–h) and as visualized on the PPB-stained lung tissues (Fig. 3i). In contrast, pulmonary iron homeostasis was maintained in the lungs of MMP12 KO mice (Fig. 3).

JUUL exposure differentially modulates allergen-induced pulmonary inflammation in WT and MMP12 KO mice

BALF cytology results revealed that the JUUL + HDM exposure reduced neutrophilic inflammation in BALF of WT mice by 4.3-fold (p < 0.05) compared to BALF of the Air + HDM WT group (Fig. 4b, Additional file 1: Fig. S4). This suggests that JUUL menthol with nicotine salt suppresses allergen-induced lung inflammatory responses. We observed a different response for the MMP12 KO mice receiving Air + HDM compared to the WT animals receiving Air + HDM (Fig. 4b, f, Additional file 1: Fig. S4). We found that Air + HDM WT mice had increased neutrophilic inflammation in BALF compared to Air + HDM MMP12 KO mice (Fig. 4b, f, Additional file 1: Fig. S4), whereas there was an increased eosinophilic influx in Air + HDM MMP12 KO compared to Air + HDM WT mice (Fig. 4b, f, Additional file 1: Fig. S4). Having a MMP12 deficiency decreased the percentage of BALF neutrophils by 5.6-fold (p < 0.05) in the air + HDM MMP12 KO group when compared to the Air + HDM WT group, thus indicating that MMP12 promotes neutrophilic inflammation in this Air + HDM model. Additionally, we found that MMP12 deficient mice also exhibited heightened neutrophilic inflammation in JUUL + HDM-treated MMP12 KO mice by 2.3-fold (p < 0.05) versus Air + HDM MMP12 KO. Percentages of eosinophils in the BALF of JUUL + HDM KO and JUUL + HDM WT mice were comparable (30.9% and 29.0%, respectively) (Fig. 4b, f, Additional file 1:Fig. S4). This suggests that having an MMP12 deficiency, combined with exposures to nicotine salt and menthol flavoring, enhances BALF neutrophilic inflammation induced by HDM. In terms of membrane permeability, there was significant increase in total protein levels within the Air WT + HDM group, with no effect among animals exposed to JUUL with or without HDM (Fig. 4c). In the KO mice, there were slight increases in protein levels from the animals that received HDM, but not significantly (Fig. 4g). Serum IgE concentrations in WT mice demonstrated the induction of an allergic response in the Air + HDM group (Fig. 4d). Together, the results from Fig. 4a–d in the Air WT + HDM mice validate our HDM-induced asthma model, as evidenced by increased BALF total cell count, significantly increased percentage of neutrophils, significantly elevated concentration of BALF proteins, and significantly higher serum concentrations of IgE, compared to Air WT + PBS controls. In MMP12 KO mice, serum IgE levels were significantly higher in both HDM groups (Air and JUUL) as well as the JUUL + PBS group (Fig. 4h). These data suggest that when there is a deficiency in MMP12, in addition to HDM, the exposure to menthol-flavored JUUL aerosol may act as an allergen. Overall, these data indicate that JUUL menthol suppresses allergen-induced neutrophilic inflammation in juvenile WT female mice, while MMP12 deficiency has the opposite effect and promotes neutrophilic allergen-induced inflammatory responses when exposed to JUUL menthol.

These results were supported at the lung tissue level (Fig. 5). Histological assessment of lung tissue shows visible peribronchial inflammation within the Air WT animals exposed to HDM (Fig. 5a, b), along with JUUL exposure displaying a suppressive effect (Fig. 5a, b). This confirms cytology and protein analyses in BALF (Fig. 4). In addition, Air + HDM WT mice had significantly more PAS-positive mucus-producing goblet cells in the lungs compared to JUUL WT ± HDM female mice (Fig. 5d, e). Histological assessment of lung tissues of KO mice confirms the reduction of cellular inflammation as seen in Fig. 5f, g in Air MMP12 KO mice receiving HDM compared to their WT counterpart (Fig. 5a, b). Lung histopathology shows that there is increased peribronchial inflammation in JUUL + HDM MMP12 KO mice when compared to Air + PBS MMP12 KO (Fig. 5g). This further supports our findings of cellular inflammation in BALF (Fig. 4). Additionally, we found no significant difference in the number of PAS-positive goblet cells between all KO groups (Fig. 5i, j). Since there was significantly increased number of PAS-positive goblet cells only in the WT Air + HDM group, this suggests that having MMP12 deficiency may reduce the mucus production following the exposure to an allergen. Taken together, these results further confirm (1) that JUUL + menthol and nicotine salts suppress allergen-induced inflammatory responses in WT mice, and (2) that MMP12 deficiency can be beneficial to the lungs in a context of exposures to JUUL menthol aerosols alone; however, the addition of the allergen modulates this protective effect.

Molecular signatures induced by menthol-flavored JUUL aerosol exposures are reduced in the lungs of MMP12 deficient mice

We conducted RNA-sequencing on the lungs of WT and MMP12 KO mice exposed solely to menthol-flavored JUUL aerosols to determine the baseline molecular changes induced by the JUUL aerosol exposure alone (Fig. 6). We found that WT mice had a total of 50 significantly dysregulated genes (fold-change >|1.5| plus p < 0.05), with the majority of the genes being down-regulated (41 genes were down-regulated plus 9 genes were up-regulated) (Fig. 6a). This is in contrast with the lung transcriptomic alterations in MMP12 KO mice, where a total of 21 genes were significantly dysregulated, with 16 genes being down-regulated and 5 genes up-regulated (Fig. 6b). Thus, removing MMP12 resulted in > 50% reduction in the number of dysregulated lung genes. Functional annotation clustering of the dysregulated genes in the JUUL WT mice highlighted down-regulation of several genes related to immune responses and immunoglobulin production (Ccl19, Igkv3-7, Igkv4-59, Igkv8-16, Fcgbp), mucin secretion (Muc5ac and Muc5b), basal cells (Krt5 and Krt15), as well as metal-binding, particularly iron (Obscn, Tnnc1, Nr1i2, Cyp2a5, Ltf, Nrap, Zfp82, Fbxo40, Reg3g, Fer1l6, Clca1, Em2) (Additional file 1: Table S1). In the MMP12 KO mice, genes associated with hydrolase activity were down-regulated (Ctsg, Ltf, Plg) (Additional file 1: Table S1). Overall, these data show that at the lung transcriptomic level, menthol-flavored JUUL aerosol exposures can suppress immune responses and alter iron homeostasis, further supporting our lung protein results (Fig. 3; Additional file 1: Table S1). Removal of MMP12 reduces the negative molecular impact of JUUL aerosol exposures on the lungs.

MMP12 deficiency down-regulates markers of oxidative stress following menthol-flavored JUUL aerosol exposures with or without HDM

Since oxidative stress plays an important role in lung injury, we used qRT-PCR to investigate the expression of 5 genes related to xenobiotic metabolism and oxidative stress responses (Fig. 7). The aryl hydrocarbon receptor (Ahr) is a crucial transcription factor involved in the regulation of enzymes associated with xenobiotic biotransformation. Additionally, Ahr activation is essential to defend the lungs from cigarette smoke-induced damage [40]. We found that regardless of the treatment (± JUUL and ± HDM), WT mice exhibited down-regulated expression of Ahr (fold-change range from -1.9 to -3.2) (Fig. 7a). In contrast, Ahr was up-regulated by 4.1- and 2.1-fold in the Air KO + HDM and JUUL KO + HDM groups, respectively (Fig. 7b). Further, we found that key genes involved in the Keap1-Nrf2 antioxidant responsive element (ARE) signaling pathway were up-regulated in the lungs of the WT groups (± JUUL and ± HDM) (Fig. 6a). Keap1 was up-regulated by 2.3-fold in the Air WT + HDM and JUUL WT + PBS groups and Nos2 was up-regulated by more than 1.8-fold in all WT groups (Fig. 7a). In addition, Hmox1 up-regulated fold-change ranged from 3.1- to 35.1-fold and Nqo1 from 1.8- to 27.6-fold for all WT groups (Fig. 6a). These results highlight, at the transcriptome level, a strong activation of ARE pathways in response to increased oxidative stress in the lungs of JUUL ± HDM exposed WT mice. Opposite results were observed in the KO groups (Fig. 6b). All ARE associated genes either showed no change from the air control group or were down-regulated, with fold-changes ranging from -2.3- to -41.5-fold (Fig. 7b). Further, at the protein level, we evaluated the enzymatic activity of NQO1 in the lungs (Fig. 7c, d). The protein results confirmed the gene expression results since the enzymatic activity of NQO1 was significantly increased in the WT mice exposed to JUUL + HDM compared to the Air WT controls, whereas no significant changes were seen in MMP12 KO mice (Fig. 7c, d). Taken together, these results suggest that in JUUL exposed mice MMP12 plays a major role in the activation of the ARE pathway (Fig. 7a, c), whose primary function is to protect against oxidative responses; however, if this response is sustained and uncontrolled, the oxidative balance in the lungs may be impaired and lead to lung injury. Therefore, the deletion of MMP12 contributes to a reduction of oxidative stress responses in the lungs of JUUL-exposed mice (Fig. 7b, d) and may be part of the central mechanism by which MMP12 deletion protects against lung damage associated with JUUL aerosol exposures.

MMP12 deficiency reduces the number of dysregulated genes associated with allergy and asthma following menthol-flavored JUUL aerosol exposures with or without HDM

We used a PCR array containing 86 genes that play a central role in allergy and asthma mediated responses. We found that WT Air + HDM mice had similar patterns of gene expression compared to WT JUUL + HDM mice, with dysregulation of a total of 56 and 61 genes, respectively (Fig. 8a). Common upregulated genes include Il13ra2, Ccl24, Ccl17, Il10 and Il13, all of which play a role in Th2 mediated asthma responses. Common downregulated genes include Adam 33, Adrb2, Kit and Ptgdr2, all related to allergic responses or lung injury. The upregulation of Muc5ac (mucus production) by 17.2- and 12.3-fold, respectively, further confirms our results of the presence of PAS-positive mucus-producing goblet cells in both Air and JUUL WT mice that received HDM treatment (Figs. 5 and 8a). The lung protein concentration of MUC5AC was also elevated in the Air + HDM WT mice (Fig. 8c). JUUL alone dysregulated only 13 genes, of which only 2 (Ccl12, Areg) were upregulated genes. Both play roles in inflammation and immune cell recruitment (Fig. 8a). Ccl12 was the only common upregulated gene among all WT groups (Fig. 8). For MMP12 deficient animals, Air MMP12 KO + HDM dysregulated 61 total genes and JUUL MMP12 KO + HDM dysregulated 37 genes, when compared to the Air + PBS MMP12 KO group (Fig. 8b). Common upregulated genes between MMP12 KO animals with HDM treatment include Cc124, Il13ra2, and Rnase2a, all of which play a role in inflammatory cell recruitment and inflammation. Common downregulated genes in both groups include Il12a, Il18, Stat6, and Ets1. JUUL MMP12 KO without HDM treatment dysregulated only 9 genes including Rnase2a, Clca1, and Prg2. Only Ltb4r1 (leukotriene receptor) was upregulated in this group. As previously shown, when MMP12 is deficient, the HDM treatment did not lead to a significant increase in the number of PAS-positive goblet cells (Fig. 5i, j) when compared to WT Air + HDM (Fig. 5d, e). Similarly, expression of Muc5ac, a protein-coding gene responsible for mucus production, was mild with 1.5- and 2.0-fold up-regulation in the Air + HDM and JUUL + HDM MMP12 KO groups, respectively (Fig. 8b). At the lung protein levels, MUC5AC was increased in the JUUL + HDM KO mice (Fig. 8d). This contrasted with the upregulated gene expression of Muc5ac in the WT groups (17.2- and 12.3-fold, as described above) (Fig. 8a). These data highlight a marked difference between WT and MMP12 KO mice exposed to JUUL + HDM, where the gene expression of Muc5ac, a key gene involved in asthma exacerbation, is attenuated in MMP12 deficient mice. Further, MMP12 deficiency dysregulated fewer genes among MMP12 KO mice exposed to JUUL + HDM compared WT JUUL + HDM (61 WT vs 37 KO) (Additional file 1: Table S2). Also, spatial visualization of RNA transcripts for Nqo1 and Muc5ac on the lung tissues show evidence of co-localization of these two transcripts in pulmonary cells for all groups (Fig. 8e). Overall, these results suggest that MMP12 is a key modulator of HDM-induced asthmatic responses, while in MMP12 deficient mice, JUUL menthol plus nicotine salt exposures seem to have a suppressive effect.

Discussion

Fourth generation ENDS devices popularized by JUUL that contains nicotine salt are the most sold types of ENDS currently used by youth and young adults [41, 42]. Moreover, menthol is one of the top flavors used by this demographic of ENDS users [43]. Very little is known, however, regarding the pulmonary effects induced by the inhalation of menthol-flavored JUUL aerosol in the context of asthma exacerbation in both human and experimental models. In the lungs, MMP12 is expressed and secreted by both alveolar macrophages and bronchial epithelial cells, and plays a key role in airway remodeling, a feature of severe asthma [30, 31]. To our knowledge, we are the first to report the effects of menthol-flavored JUUL aerosol exposures on HDM-induced asthmatic responses in juvenile mice with MMP12 deficiency. We conducted a thorough assessment of the menthol-flavored JUUL aerosol in terms of carbonyls, organic acids, metals and particle size distribution (Fig. 1), which highlighted elevated levels of iron in the aerosol (Fig. 1e). Accordingly, we observed at both the gene and protein levels disruption of iron metabolism in the lungs of the JUUL + HDM WT mice, while iron pulmonary homeostasis was maintained in MMP12 KO mice (Additional file 1: Table S1, Fig. 3). Also, our results indicated at the gene and protein expression levels, activation of the antioxidant response element (ARE) defense system against oxidative stress responses in WT mice exposed to JUUL with or without HDM. These results suggest that having MMP12 deficiency combined with JUUL menthol + HDM exposures reduced oxidative stress responses (Fig. 7). In addition, we found that menthol-flavored JUUL aerosol suppressed immune-inflammatory responses during both, sole exposure to the JUUL aerosol and against the combined JUUL + HDM treatments in WT mice exposed for 60 days (Figs. 4, 5). It is important to bear in mind that this immunosuppressive effect may prime the lungs for exacerbation following exposures to respiratory infectious agents [44,45,46,47]. In term of key features of asthma pathogenesis, including airway mucus hypersecretion, our data further showed that gene and protein levels of MUC5AC were increased in WT mice exposed to air and JUUL with HDM, whereas MUC5AC expression levels were much lower in MMP12 KO mice (Fig. 8). This highlights that having a MMP12 deficiency may reduce the severity of asthma exacerbation by playing a role in the reduction of mucus production in this context. Overall, we showed that menthol-flavored JUUL aerosol plus house dust-mite exposures disrupt pulmonary iron homeostasis and stimulate oxidative stress responses in WT female mice, while MMP12 deficiency attenuates these effects.

Menthol flavor, MMP12, HDM, and α7nAChR signaling

Flavoring chemicals found in e-liquids are important contributors to ENDS aerosols toxicity [48]. Menthol is an organic alcohol that is widely used and is generally regarded as safe to be used within foods, oral health products, and medications [49,50,51]. Additionally, menthol is used in tobacco products, including conventional cigarettes and e-liquids, due to its smooth taste and strong aroma it provides upon inhalation. Given that menthol has a high transfer rate from the e-liquid to the ENDS aerosol [52], menthol can mask the harshness of inhaled nicotine, via its cooling sensation, which allows the user to inhale more deeply [53,54,55,56]. This cooling sensation is due to the ability of menthol to activate cold sensitive transient receptor potential melastain (TRPM) channels in the lungs, as well as in nasal and oral membranes [53, 57]. Moreover, menthol has been shown to suppress nicotine metabolism [58, 59] by interacting with nicotinic acetylcholine receptors (nAChRs), including the α7nAChr, which will interfere with the binding of nicotine [53, 60, 61]. Thus, menthol, similarly to nicotine, induces an antagonizing effect on the α7nAChR [59]. It was previously reported that sub-chronic (30 days) exposure to unflavored ENDS aerosol containing propylene glycol plus 25 mg/mL of nicotine increased the expression of MMP12 at the protein level in lungs of WT male mice compared to α7nAChR KO mice, in which the MMP12 levels remained unchanged compared to the air controls [62]. This suggests that MMP12 up-regulation by ENDS aerosols containing nicotine is mediated by α7nAChR, and thus, MMP12 and α7nAChR signaling are linked. We found that Mmp12 is highly expressed among WT air animals receiving HDM (16.8-fold), and the expression of Mmp12 is significantly decreased (> 50%) when these WT mice are exposed to JUUL + HDM (7.7-fold) (Fig. 2b). This interaction suggests that nicotine salt plus menthol flavoring may play an active role in decreasing Mmp12 expression since nicotine and menthol both have an antagonizing effect on α7nAChR signaling, which is linked to Mmp12 [59, 62]. Also, we found that the concentration of cotinine in MMP12 KO mice was significantly lower compared to that in WT counterparts (Fig. 2c, d). Since we did not observe any significant difference in lung function, including in breathing frequency (Additional file 1: Fig. S2), this suggests that MMP12 influences nicotine metabolism, possibly through α7nAChR signaling [62], resulting in either increased absorption of nicotine or enhanced biotransformation of nicotine to cotinine. Further, we observed that cotinine levels were significantly lower among mice exposed solely to JUUL compared to mice exposed to JUUL + HDM in WT and MMP12 KO mice (Fig. 2c, d). This suggests that the combined exposure of JUUL (or nicotine salt) + HDM increased the absorption of nicotine. Indeed, it is well-known that when allergens, including HDM, interact with the pulmonary epithelium, the bronchial epithelial permeability can be altered, leading to enhanced delivery and absorption of inhaled toxicants [63]. Thus, our data suggest that in the presence of HDM, which can disrupt the lung epithelial barrier [63], more inhaled nicotine is absorbed in the respiratory tract of both WT and KO mice exposed to JUUL + HDM. This is reflected by increased serum cotinine levels compared to the JUUL + PBS groups (Fig. 2c, d ). Furthermore, continuous exposure to nicotine causes overstimulation of the nicotinic receptor, resulting in downregulation of active nicotinic receptors [39]. Accordingly, we found that in the case of WT mice, JUUL alone upregulated the α7nAChR (1.6-fold) and JUUL + HDM exposure led to downregulation (-1.5-fold), which suggests overstimulation of the receptor (Fig. 2e) that is reflected by increased absorption of nicotine in the presence of HDM (Fig. 2c). Collectively, our findings suggest that menthol-flavored ENDS may alter respiratory homeostasis through the interaction of menthol and nicotine salt with nicotinic receptors, an effect that is attenuated in MMP12 KO mice, and highlights the significant involvement of flavoring chemicals, particularly menthol, in ENDS aerosol toxicity.

MMP12, JUUL menthol, HDM and anti-oxidant/oxidative stress responses

Oxidative stress-facilitated signaling pathways are central to asthma pathogenesis, as production of ROS and inflammation are usually closely related and can ultimately lead to lung injury [64]. ROS and increased oxidative stress levels induce the activation of the antioxidant response element (ARE) [65]. ARE-related genes are expressed in tissue exposed to high levels of oxidative stress, including in the lungs, to provide an adequate antioxidant response [66]. The ARE system is focused on Nrf2 mediated-responses, which provides a coordinated antioxidant response, via mediators including heme oxygenase 1 (Hmox1) and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (Nqo1). NQO1 is expressed in the lungs’ epithelium [66, 67] and plays a key role in decreasing or removing ROS, free radicals, and scavenging quinones in cells [65, 68]. In addition, NQO1 has been shown to be involved in the regulation of pro-inflammatory and pro-allergic signaling pathways [65]. Overall, NQO1 is a protein-coding gene that (1) is induced by increased levels of oxidative stress and (2) is a reductase that is part of the intracellular antioxidant defense mechanism [66, 67]. In mice, we observed, in the presence of MMP12, increased NQO1 gene expression (Fig. 7a) and enzymatic activity (Fig. 7c), aiming at reducing oxidative injury (Fig. 7a); while in the absence of MMP12, NQO1 gene expression (Fig. 7b) and enzymatic activity are decreased (Fig. 7d), suggesting that there is a reduced need to combat oxidative stress (Fig. 7b). Since NQO1 is a target gene of the Nrf2 pathway, an indicator of the establishment of a robust antioxidant response to protect from inflammation and asthma exacerbation [64], the results from our model suggest that NQO1 was induced by the oxidative stress produced by menthol-flavored JUUL aerosol + HDM exposures in WT mice (Fig. 7a, c). Since KO mice showed down-regulated gene expression and decreased enzymatic activity for NQO1 (Fig. 7), these data suggest that MMP12 has a stimulating effect on oxidative stress pathways in this specific context. The oxidative stress effect of ENDS exposures has been previously reported by our laboratory along with other research groups who showed that ENDS exposures, including via JUUL, contribute to increased levels of oxidative stress in macrophages in vitro [6, 35, 69,70,71], as well as in vivo [72,73,74,75]. Moreover, human studies have shown that NQO1 polymorphism is associated with increased risk for childhood asthma in a cohort of pediatric patients from China [68]. In line with these results, another study conducted on asthmatic children from Nevada revealed an association between NQO1 polymorphism and the need for extensive pharmacological therapy to treat asthma symptoms [65]. Accordingly, NQO1 polymorphism may affect its enzymatic activity and be associated with increased risk for asthma [66]. These studies in humans strongly suggest a role for NQO1 in asthma pathogenesis. Overall, our data support a potential critical role for the ARE system, particularly NQO1, in the exacerbation of asthma following exposures to JUUL-menthol flavored aerosols in WT mice (Fig. 7a, c), an effect that is attenuated in MMP12 KO mice (Fig. 7b, d). It was previously demonstrated that MMP12 is induced by oxidative stress and that MMP12 has pro-inflammatory properties, associated with increased neutrophilia [76,77,78,79,80,81,82]. These reports support our findings of reduced oxidative stress (Fig. 7) and immune responses (Figs. 4, 5) in our mouse model with an MMP12 deficiency. Thus, reducing or eliminating the activity of MMP12 in these contexts may decrease the overwhelming oxidative stress response and correspondingly, the lungs’ antioxidant defense countermeasures caused by menthol-flavored JUUL aerosol exposures.

MMP12, JUUL menthol, HDM and pulmonary iron metabolism

In addition, as similarly observed by other research groups investigating metal levels in JUUL aerosols, including iron [10], in our study, the JUUL menthol-flavored aerosol was a significant exogenous source of inhaled iron since this aerosol contained high levels of iron (38.9 ng/puff) (Fig. 1e), equivalent to ~ 4.7 µg of daily inhaled iron intake, assuming the consumption of 120 puffs of JUUL per day. This could lead to excess of cellular and systemic iron levels. The sources of iron can be either systemic or directly from the lung microenvironment, the latter being dependent on the inhaled air. Since we did not see any significant changes in serum iron concentrations between the different groups (Additional file 1:Fig. S3), our data indicate that following JUUL exposures the contribution of iron to the pulmonary responses comes from the lung microenvironment (Fig. 3i). This is supported by the dysregulated expression of several key lung genes (Obscn, Tnnc1, Nr1i2, Cyp2a5, Ltf, Nrap, Zfp82, Fbxo40, Reg3g, Fer1l6, Clca1, Em2; Additional file 1: Table S1) and proteins (ferroportin, transferrin, ferritin, and lactoferrin) involved in pulmonary iron metabolism that was observed in the lungs of WT mice exposed to JUUL and JUUL + HDM (Fig. 3). Iron homeostasis is crucial in the functioning of a normal lung, center of oxygen exchange, as it is a vital component of some proteins, e.g., heme enzymes, and it takes part in essential cellular redox reactions [83, 84]. Thus, iron metabolism occurs in the lungs and is tightly regulated, at both the extra- and intra-cellular levels, by a high number of proteins involved in iron homeostasis, including transferrin, ferritin, and lactoferrin, which are released from leukocytes (alveolar macrophages and neutrophils), as well as bronchial epithelial and endothelial cells, and play key roles in iron import, export, storage, as well as cellular use [83, 85, 86]. It is well documented that the inhalation of cigarette smoke can cause pulmonary iron imbalance, with higher concentrations of iron and ferritin in the BAL of smokers compared to non-smokers [83, 87]. Macrophages of smokers are often described as pigment-laden cells containing organic humic-like substances that can react with metal cations, including iron [87]. This can ultimately lead to the disruption of iron metabolism within pulmonary cells [87]. Iron, Fe²⁺ or Fe³⁺, can be oxidized or reduced, and thus exhibits strong affinity for oxygen-donor ligands, and by this means, can engage in cellular processes via Haber–Weiss and Fenton reactions involving free radicals [83, 84, 88]. In fact, intracellular iron accumulation and the disruption of iron homeostasis result in augmented generation of ROS [83, 84, 88]. Excess iron and increased ROS levels result in the up-regulation of ferroportin, a membrane protein that exports iron from macrophages as well as from bronchial epithelial cells, which is also a key player in redox homeostasis [83, 85, 89, 90]. Increased ferroportin leads to reduced intracellular iron levels, reflected by reduced ferritin, a protein playing a key role in intracellular iron storage and thereby restricting extracellular iron-induced oxidative stress, a mechanism to protect against iron toxicity [83, 85, 89, 90]. Overall, this results in imbalanced iron concentrations [83, 85, 89, 90]. We observed significantly increased lung concentration of ferroportin (Fig. 3a), reflecting increased export of intracellular iron, concomitant with decreased lung concentration of ferritin (Fig. 3e), representing decreased intracellular iron storage, in WT mice exposed to JUUL + HDM, whereas non-significant differences for those iron metabolism lung proteins were seen in KO mice (Fig. 3b, f). Also, protein levels of lung transferrin that binds extracellular iron (Fe3 +), were significantly decreased in the JUUL + HDM WT group compared to the WT air controls (Fig. 3g). Since decreased levels of transferrin are an indicator of iron overload [87, 91, 92], our data demonstrate pulmonary iron overload in the JUUL + HDM WT group, whereas no significant change for that protein was seen in the KO group (Fig. 3h). Furthermore, as mentioned above, we found that when WT mice are exposed to HDM, markers of oxidative stress are increased (Fig. 7a); however, having MMP12 deficiency reduces these oxidative stress responses (Fig. 7b). Thus, our results suggest in WT mice a sequence of events where HDM + menthol-flavored JUUL aerosol, containing high concentration of iron (38.9 ng/puff) (Fig. 1e), induced local increase in pulmonary iron level (Fig. 3i) (not systemic) (Additional file 1:Fig. S3), leading to impaired pulmonary iron homeostasis (Fig. 3), and ultimately to iron-catalyzed increased ROS and oxidative stress related responses, as evidenced by the up-regulation of genes and increased enzymatic activity of modulators of the ARE antioxidant defense mechanism (Fig. 7). Since WT and MMP12 KO were exposed to the same JUUL menthol-flavored aerosol, containing iron, but only WT mice exhibited disrupted iron homeostasis (Fig. 3), which can trigger increased oxidative stress [84, 90], as we saw in the WT mice (Fig. 7), our results suggest that limiting the activity of MMP12 may reduce the pro-oxidant effects of iron, and by this means represents a protective effect associated with the removal of MMP12 in the context of menthol-flavored JUUL aerosol + HDM exposures. Further, since macrophages play a key role in pulmonary iron homeostasis, the same cell type that is mainly involved in the secretion of MMP12, there may be potential crosstalk between MMP12, iron overload conditions, and oxidative stress responses to menthol-flavored JUUL aerosol + HDM exposures. Overall, our results strongly suggest that the inhalation of iron-rich menthol-flavored JUUL aerosol (Fig. 1e) + HDM leads to iron-catalyzed oxidative stress responses (Figs. 3, 7) and show that in addition to flavoring chemicals, the presence of metals, like iron, can also significantly contribute to ENDS aerosol pulmonary toxicity.

Ferroptosis can be defined as a form of controlled cell death induced by lipid peroxidation resulting from iron-mediated release of free radicals or oxidative stress [93, 94]. Ferroptosis is associated with labile iron or intracellular non-protein-bound iron that is redox active [93, 94]. Markers for ferroptosis include CD71, also known as transferrin receptor 1, as well as transferrin [94]. In our study, we evaluated multiple proteins in the lung tissue associated with iron metabolism, including ferroportin, lactoferritin, ferritin, and transferrin (Fig. 3). We did not evaluate the transferrin receptor 1 (or CD71) since we evaluated transferrin, the specific ligand for this receptor, which is also a marker of ferroptosis [94]. As per Fig. 3g, h, we found that the levels of lung transferrin were significantly decreased in the lungs of the JUUL WT + HDM compared to air controls, while no significant change was seen in the MMP12 KO mice. Yoshida et al. [93] showed in vitro and in vivo that cigarette smoke disrupted pulmonary iron homeostasis, which led to ferritinophagy prior to the induction of ferroptosis in COPD models. Thus, these data suggest that ferroptosis occurs following alterations in iron pulmonary homeostasis, and that it can be a progressive effect requiring specific prior stages or damage such as ferritinophagy. Taken together, it is plausible that our results show iron-mediated toxicity following JUUL aerosol exposure as an early event potentially leading to ferroptosis. Since we did not measure cell death whether by apoptosis, necrosis or ferroptosis, we cannot conclude whether ferroptosis played a role in our model. However, based on the transferrin results (reduction; Fig. 3g), it is unlikely that ferroptosis, at this timepoint, was a main cell death mechanism in our model. Future studies should investigate the effects of vaping on this specific mechanism of cell death.

MMP12, JUUL menthol, HDM, and pulmonary inflammation

Nicotine, in its free base chemical form, has both anti-inflammatory as well as stimulating properties [95, 96]. It was previously shown in humans that nicotine salt suppressed immune-inflammatory responses in sputum samples [97]. Since nicotinic receptors are ubiquitously expressed within macrophages and lung epithelial cells, they play a key role in regulating pulmonary inflammatory and anti-inflammatory responses [98, 99]. Menthol, with its cooling properties, may also suppress immune-inflammatory responses [100]. Thus, when nicotine salt is coupled with menthol, they may act in concert to suppress immune-inflammatory responses leading to reduced pulmonary inflammation. This is in line with our findings comparing the BALF results of Air WT + HDM and JUUL WT + HDM groups (Fig. 4b). We found that in Air WT mice, HDM treatment increases membrane permeability and causes mixed eosinophilic/neutrophilic inflammatory response with significant influx of neutrophils. This response is suppressed when WT mice treated with HDM are exposed to JUUL menthol (Fig. 4a–c). These results were supported by histopathological evaluations showing that although peribronchal inflammation and number of PAS-positive goblet cells are significantly increased in Air WT + HDM mice, inflammation is suppressed among WT + HDM mice following exposure to JUUL, suggesting that JUUL menthol suppresses allergen-induced neutrophilic inflammation (Fig. 5a–e). A similar interaction between ENDS aerosol exposures and HDM was seen in another study [101], which reported that exposure to flavored ENDS aerosol (12 mg/mL nicotine) suppressed HDM induced inflammation in Balb/c mice, an effect that was suggested to be due to nicotine [101]. This is not, however, necessarily a beneficial outcome, as a suppressed immune pulmonary defense system may prolong subsequent respiratory infections. This has been shown in several other in vivo studies employing secondary bacterial or viral exposures [44,45,46,47]. Furthermore, in vitro, macrophage polarization between M1 and M2 phenotypes is influenced by iron levels, with iron overload conditions leading to a M2 anti-inflammatory phenotype and suppression of the M1 pro-inflammatory phenotype [102]. Thus, it is plausible that iron accumulation in the lungs of WT mice, as evidenced by our genes and proteins iron metabolism associated dysregulation (Fig. 3; Additional file 1: Table S1), led to a M2 anti-inflammatory phenotype, which suppressed the activation of pro-inflammatory M1 macrophages following the HDM exposures, resulting in the dampen pulmonary inflammatory responses we observed in the JUUL WT + HDM compared to the Air WT + HDM groups (Fig. 4b). When KO mice with HDM were exposed to JUUL, there was significant influx of neutrophils (Fig. 4f). This suggests that MMP12 is a modifier of neutrophilic inflammation, in which MMP12 suppresses neutrophil trafficking due to HDM induced exacerbations when combined with JUUL menthol exposure. In our model, BALF cytology results show a neutrophilic HDM-induced pulmonary inflammation (Fig. 4b, f), which typically correlates with an increased severity in asthma pathogenesis [103]. As for the baseline groups, the WT mice exposed to JUUL + PBS showed no signs of inflammation after 3 or 60 days of JUUL aerosol exposures (Additional file 1: Fig. S1 , Fig. 4a–c). This is in contrast with results from a study by Been et al. [72], where C57BL/6 J mice were exposed to mint-flavored JUUL aerosols for 3 days. They found that the BALF of exposed mice were mostly composed of macrophages, with significant neutrophilic influx in both male and female mice compared to their respective air controls [72]. Differences between these results and ours may be due to the duration of the JUUL aerosol exposures, since we performed a 1 h/day continuous exposure at 2 puffs per minute compared to three vaping sessions per day for 20 min each, at 4 puffs per minute in Been et al. [72]. There were 120 daily puffs in our study in comparison to 240 puffs in [72]. This suggests that the total exposure level used in Been et al. [72] was almost twice that of our exposure level. In addition, another major difference was the JUUL flavor used, menthol vs. mint, despite the fact that mint flavoring contains menthol [7, 104]. Our results from this study, for JUUL + PBS, are in line with those of other groups that saw no visible signs of cellular inflammation or lung tissue inflammation after exposure to JUUL mint flavored aerosol for 15 days [105] or for 1–3 months [106] in C57BL/6 WT mice. Overall, MMP12 in our model plays a role in modulating neutrophilic inflammatory responses following exposure to menthol-flavored JUUL aerosol during asthma induced inflammation.

MMP12, JUUL menthol, HDM, and mucin hypersecretion

MUC5AC, a mucin, is a protein predominantly present in the airway epithelium of the central conducting airways [107, 108]. Mucin can be stained with PAS to identify airway goblet cell hyperplasia/metaplasia, with mucus hypersecretion being a pathological feature of severe asthma and its exacerbation [67, 107]. We found that in WT mice the Air + HDM exposure resulted in significantly increased percentage of BALF neutrophils compared to MMP12 KO mice exposed Air + HDM (Fig. 4). Accordingly, we found significantly increased number of PAS-positive mucus-producing goblet cells only in the WT Air + HDM group (Fig. 5d, e), which was accompanied by the up-regulation of Muc5ac by 17.2-fold, whereas this gene was up-regulated by 12.3-fold in the JUUL + HDM WT mice (Fig. 8a). In the MMP12 deficient mice, the HDM treatment did not lead to significant increase in the number of PAS-positive goblet cells (Fig. 5i, j). Accordingly, the expression of Muc5ac was mild with 1.5- and 2.0-fold up-regulation in the Air MMP12 KO + HDM and JUUL MMP12 KO + HDM groups, respectively (Fig. 8b). These data highlight a marked difference between WT and MMP12 KO mice exposed to HDM with or without JUUL, where the expression of Muc5ac, a key gene involved in asthma exacerbation, is attenuated in MMP12 deficient mice. Muc5ac can be induced by increased levels of Il-13 and Il-33 [107, 109]. In our model, however, there were minimal differences in gene expression for IL-13 as well as for IL-33 in the respective WT and MMP12 KO groups (IL-13: 10.4- and 4.7-fold in WT Air + HDM and JUUL + HDM, respectively, and 13.1- and 4.6-fold in MMP12 KO Air + HDM and JUUL + HDM, respectively) (IL-33: 2.6- and 2.1-fold in WT Air + HDM and JUUL + HDM, respectively, and 1.6- and 1.6-fold in MMP12 KO Air + HDM and JUUL + HDM, respectively) (Fig. 8). These results strongly suggest that another differential mechanism for the increased mucus secretion observed in the WT mice may be at play. It was previously demonstrated in human bronchial epithelial cells that oxidative stress leads to increased gene expression of Muc5ac [110]. Other studies in human nasal and bronchial epithelial cells similarly showed that increased oxidative stress levels driven by H₂O₂ led to increased intracellular ROS and enhanced Muc5ac production via EGFR-MAPK signaling [111, 112]. In addition, mice deficient in the Nrf2 gene, a key regulator of the antioxidant response, exhibit, among others, increased goblet cell metaplasia, putting in evidence the critical role of oxidative stress in mucus hypersecretion [113]. Furthermore, NQO1 and MUC5AC gene expression positively correlate in patients with asthma [67]; while in a mouse model, PM_2.5-induced MUC5AC expression was driven by NQO1 oxidative stress responses [67]. Thus, induction of Muc5ac gene and protein expression by increased level of oxidative stress in both bronchial cells and in mice have been demonstrated [67, 110,111,112,113,114]. Further, in our model, we show with 2-D spatial transcriptomics for Nqo1 and Muc5ac that these two RNA transcripts are co-localized in airway cells (Fig. 8e). This strongly suggests that, in our model, since MMP12 stimulates oxidative stress responses induced by JUUL + HDM exposure (Fig. 7), there may be an intermediary role of MMP12 in the up-regulation of Muc5ac gene and protein expression in WT mice (Fig. 8). Taken together, our data suggest that in WT mice menthol-flavored JUUL + HDM exposure-induced oxidative stress led to increased gene expression of Muc5ac, an effect that was diminished in MMP12 KO mice (Fig. 8), indicating a key role for MMP12 in the production of ROS in the lungs and subsequently, Muc5ac gene expression after exposure to menthol-flavored JUUL aerosol + HDM. Thus, these results suggest that MMP12 is involved in mucin production induced by oxidative stress in the airways.

JUUL menthol, iron-mediated pulmonary toxicity, oxidative stress, mucus hypersecretion, and asthma

Asthma is a complex disease in which inflammation and oxidative stress play key roles. This is in addition to iron homeostasis, which is also a multifaceted essential phenomenon involving several proteins achieving different functions related to iron metabolism. There are both clinical [115,116,117] and experimental [116, 118,119,120] data supporting an association between disrupted pulmonary iron homeostasis, leading to accumulation of iron in cells or airway tissue, and asthma. Our animal model is complex and contained multiple variables, since we exposed WT and MMP12 KO juvenile female mice to menthol-flavored JUUL, a nicotine salt and iron-rich aerosol, followed by HDM treatment. MMP12 is involved in extracellular matrix remodeling and structural alterations to the small and large airways are phenotypical changes found in severe asthma [121]. Macrophages secrete MMP12 and are also key players involved in regulating immune homeostasis through the production of oxidative stress mediators, including ROS [122, 123]. Increased in cellular oxidative stress in bronchial epithelial cells and macrophages can be caused by iron, an event that can also initiate mucus hypersecretion in chronic lung diseases, including asthma [124, 125]. Together, our data point to and correlate with iron-rich menthol-flavored JUUL aerosol plus HDM treatment disrupting pulmonary iron homeostasis, suggesting a potential defense mechanism against iron-mediated toxicity. This is evidenced by the iron metabolism lung protein results (Fig. 3), which likely led to iron-catalyzed generation of ROS and increased oxidative stress levels that activated the ARE signaling pathway and up-regulated the expression of NQO1 (Fig. 7), and subsequently the expression of MUC5AC (Fig. 8) in WT mice. On the other hand, MMP12 KO mice seemed to be protected against iron-induced oxidative stress responses (Figs. 3, 7), highlighting a crucial role of MMP12 in this model (Fig. 9). Our data suggest a crucial role for iron regulation in nicotine salt iron-rich ENDS aerosol toxicity. We believe we are the first to propose a mechanism with in vivo evidence supporting a crosstalk between menthol-flavored ENDS aerosol metals content, more specifically, iron, disruption of pulmonary iron metabolism, ROS production, and mucus hypersecretion, as an underlying mechanism of menthol-flavored ENDS aerosol pulmonary toxicity in an asthma model (Fig. 9). This may represent an underlying path ultimately leading to lung damage induced by the inhalation of iron-rich ENDS aerosols.

Limitations for our in vivo study are first, that we only used female mice. We used female mice since it is well-known that adolescent and adult females have higher prevalence of asthma exacerbations compared to males [126]. We also used MMP12 whole-body KO instead of tissue specific MMP12 KO mice. Additionally, we only investigated lung function using non-invasive whole-body plethymography (Additional file 1: Fig. S2) in opposition to the determination of more precise lung function measurements. Further, the timing of the HDM treatment, in this case, at the end and concurrent with the JUUL aerosol exposure, may affect the BALF inflammatory responses. We observed decreased percentages of leukocytes in the BALF of the JUUL WT + HDM mice compared to the Air WT + HDM mice (Fig. 4b). Different timing of allergen exposure, for instance before or after the ENDS aerosol exposure, may lead to different outcomes. This phenomenon was previously reported for other allergens and inhaled pollutants [127,128,129]. Also, more research is needed to better understand the understudied interrelationships between MMP12, menthol flavor, cotinine, and α7nAChR signaling, which may influence biological outcomes. Subsequent work could investigate the effects of treating menthol-flavored ENDS exposed mice with an intracellular iron chelator (e.g., deferiprone, citric acid) or using an equivalent approach to target the accumulation of iron in the lungs. Future studies should also target iron-related pathways, including ferroptosis, to uncover potential mechanisms of toxicity associated with metal-rich ENDS aerosol exposures.

Conclusion

Overall, our in vivo results suggest that following exposures to menthol-flavored ENDS aerosols MMP12 limits neutrophilic inflammation while increasing iron-catalyzed oxidative stress responses in the context of allergen-induced inflammation. This study further adds to the current body of knowledge highlighting that 4th generation ENDS are not safe and users with respiratory conditions, including asthma, should take caution. Recent meta-analysis and cross-sectional studies reported that use of ENDS is highly associated with symptoms of asthma among former or current ENDS users [13, 130, 131]. More research is needed to determine whether the disruption of pulmonary iron metabolism is a central mechanism of pulmonary toxicity induced by 4th generation ENDS aerosols, rich in nicotine salt and in elemental metals.

Availability of data and materials

No datasets were generated or analysed during the current study.

References

Gaiha SM, Lempert LK, McKelvey K, Halpern-Felsher B. E-cigarette devices, brands, and flavors attract youth: Informing FDA’s policies and priorities to close critical gaps. Addict Behav. 2022;126: 107179.
Article PubMed Google Scholar
Alexander LEC, Vyas A, Schraufnagel DE, Malhotra A. Electronic cigarettes: the new face of nicotine delivery and addiction. J Thorac Dis. 2015;7(8):E248.
Google Scholar
Bengalli R, Ferri E, Labra M, Mantecca P. Lung toxicity of condensed aerosol from E-CIG liquids: influence of the flavor and the in vitro model used. Int J Environ Res Public Health. 2017;14(10):1254.
Article PubMed PubMed Central Google Scholar
Go YY, Mun JY, Chae S-W, Chang J, Song J-J. Comparison between in vitro toxicities of tobacco-and menthol-flavored electronic cigarette liquids on human middle ear epithelial cells. Sci Rep. 2020;10(1):1–9.
Article Google Scholar
Leigh NJ, Lawton RI, Hershberger PA, Goniewicz ML. Flavourings significantly affect inhalation toxicity of aerosol generated from electronic nicotine delivery systems (ENDS). Tobacco Control. 2016;25(Suppl 2):ii81–7.
Muthumalage T, Prinz M, Ansah KO, Gerloff J, Sundar IK, Rahman I. Inflammatory and oxidative responses induced by exposure to commonly used e-cigarette flavoring chemicals and flavored e-liquids without nicotine. Front Physiol. 2018;8:1130.
Article PubMed PubMed Central Google Scholar
Omaiye EE, McWhirter KJ, Luo W, Tierney PA, Pankow JF, Talbot P. High concentrations of flavor chemicals are present in electronic cigarette refill fluids. Sci Rep. 2019;9(1):2468.
Sassano MF, Davis ES, Keating JE, Zorn BT, Kochar TK, Wolfgang MC, Glish GL, Tarran R. Evaluation of e-liquid toxicity using an open-source high- throughput screening assay. PLoS Biol. 2018;16(3): e2003904.
Article PubMed PubMed Central Google Scholar
Singh J, Luquet E, Smith DP, Potgieter HJ, Ragazzon P. Toxicological and analytical assessment of e-cigarette refill components on airway epithelia. Sci Prog. 2016;99(4):351–98.
Article CAS PubMed PubMed Central Google Scholar
Pappas RS, Gray N, Halstead M, Valentin-Blasini L, Watson C. Toxic metal-containing particles in aerosols from pod-type electronic cigarettes. J Anal Toxicol. 2021;45(4):337–47.
Article CAS PubMed Google Scholar
Re DB, Hilpert M, Saglimbeni B, Strait M, Ilievski V, Coady M, Talayero M, Wilmsen K, Chesnais H, Balac O, Glabonjat RA, Slavkovich V, Yan B, Graziano J, Navas-Acien A, Kleiman NJ. Exposure to e-cigarette aerosol over two months induces accumulation of neurotoxic metals and alteration of essential metals in mouse brain. Environ Res. 2021;202: 111557.
Article CAS PubMed PubMed Central Google Scholar
Woodruff PG, Koth LL, Yang YH, Rodriguez MW, Favoreto S, Dolganov GM, Paquet AC, Erle DJ. A distinctive alveolar macrophage activation state induced by cigarette smoking. Am J Respir Crit Care Med. 2005;172(11):1383–92.
Article PubMed PubMed Central Google Scholar
Xie W, Kathuria H, Galiatsatos P, Blaha MJ, Hamburg NM, Robertson RM, Bhatnagar A, Benjamin EJ, Stokes AC. Association of electronic cigarette use with incident respiratory conditions among US adults from 2013 to 2018. JAMA Netw Open. 2020;3(11): e2020816.
Article PubMed PubMed Central Google Scholar
Choi K, Bernat D. E-cigarette use among Florida youth with and without asthma. Am J Prev Med. 2016;51(4):446–53.
Article PubMed PubMed Central Google Scholar
Fedele DA, Barnett TE, Dekevich D, Gibson-Young LM, Martinasek M, Jagger MA. Prevalence of and beliefs about electronic cigarettes and hookah among high school students with asthma. Ann Epidemiol. 2016;26(12):865–9.
Article PubMed Google Scholar
Kim SY, Sim S, Choi HG. Active, passive, and electronic cigarette smoking is associated with asthma in adolescents. Sci Rep. 2017;7(1):1–8.
Article Google Scholar
Larsen K, Faulkner GE, Boak A, Hamilton HA, Mann RE, Irving HM, To T, Network CRR. Looking beyond cigarettes: are Ontario adolescents with asthma less likely to smoke e-cigarettes, marijuana, waterpipes or tobacco cigarettes? Respir Med. 2016;120:10–5.
Article PubMed Google Scholar
McConnell R, Barrington-Trimis JL, Wang K, Urman R, Hong H, Unger J, Samet J, Leventhal A, Berhane K. Electronic cigarette use and respiratory symptoms in adolescents. Am J Respir Crit Care Med. 2017;195(8):1043–9.
Article PubMed PubMed Central Google Scholar
Turner E, Fedele DA, Thompson L, Salloum RG. Patterns of electronic cigarette use in youth with asthma: results from a nationally representative sample. Ann Allergy Asthma Immunol. 2018;120(2):220–2.
Article PubMed Google Scholar
Martinasek MP, White RM, Wheldon CW, Gibson-Young L. Perceptions of non-traditional tobacco products between asthmatic and non-asthmatic college students. J Asthma. 2019;56(5):498–504.
Article PubMed Google Scholar
Xie W, Tackett AP, Berlowitz JB, Harlow AF, Kathuria H, Galiatsatos P, Fetterman JL, Cho J, Blaha MJ, Hamburg NM, Robertson RM. Association of electronic cigarette use with respiratory symptom development among US young adults. Am J Respir Crit Care Med. 2022;205(11):1320–9.
Kramarow EA, Elgaddal N. Current electronic cigarette use among adults aged 18 and over: United States, 2021. NCHS Data Brief, no 475. Hyattsville, MD: National Center for Health Statistics. 2023. https://doiorg.publicaciones.saludcastillayleon.es/10.15620/cdc:129966.
US FDA. Results from the Annual National Youth Tobacco Survey. https://www.fda.gov/tobacco-products/youth-and-tobacco/results-annual-national-youth-tobacco-survey.
World Health Organization. (2022, May 11). Asthma. Retrieved October 12 from https://www.who.int/news-room/fact-sheets/detail/asthma.
Centers for Disease Control and Prevention. More than 2.5 Million Youth Reported E- Cigarette Use in 2022. Retrieved October 12, 2022 from https://www.cdc.gov/media/releases/2022/p1007-e-cigarette-use.html
Loftus PA, Wise SK. Epidemiology and economic burden of asthma. Int Forum Allergy Rhinol. 2015;5(S1):S7–10.
World Health Organization. What triggers an asthma attack? Retrieved April 19, 2019 from https://www.who.int/features/qa/46/en/.
Demedts IK, Brusselle GG, Bracke KR, Vermaelen KY, Pauwels RA. Matrix metalloproteinases in asthma and COPD. Curr Opin Pharmacol. 2005;5(3):257–63.
Erle DJ, Sheppard D. The cell biology of asthma. J Cell Biol. 2014;205(5):621–31.
Article CAS PubMed PubMed Central Google Scholar
Houghton AM. Matrix metalloproteinases in destructive lung disease. Matrix Biol. 2015;44:167–74.
Article PubMed Google Scholar
Lavigne MC, Thakker P, Gunn J, Wong A, Miyashiro JS, Wasserman AM, Wei SQ, Pelker JW, Kobayashi M, Eppihimer MJ. Human bronchial epithelial cells express and secrete MMP-12. Biochem Biophys Res Commun. 2004;324(2):534–46.
Hough KP, Curtiss ML, Blain TJ, Liu R-M, Trevor J, Deshane JS, Thannickal VJ. Airway remodeling in asthma. Front Med. 2020;7:191.
Article Google Scholar
Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science. 1997;277(5334):2002–4.
Article CAS PubMed Google Scholar
Mukhopadhyay S, Sypek J, Tavendale R, Gartner U, Winter J, Li W, Page K, Fleming M, Brady J, O'Toole M, Macgregor DF. Matrix metalloproteinase-12 is a therapeutic target for asthma in children and young adults. J Allergy Clin Immunol. 2010;126(1):70–6.
Pinkston R, Zaman H, Hossain E, Penn AL, Noel A. Cell-specific toxicity of short-term JUUL aerosol exposure to human bronchial epithelial cells and murine macrophages exposed at the air-liquid interface. Respir Res. 2020;21(1):269.
Article CAS PubMed PubMed Central Google Scholar
Cahill KM, Johnson TK, Perveen Z, Schexnayder M, Xiao R, Heffernan LM, Langohr IM, Paulsen DB, Penn AL, Noël A. In utero exposures to mint-flavored JUUL aerosol impair lung development and aggravate house dust mite-induced asthma in adult offspring mice. Toxicology. 2022;477: 153272.
Article CAS PubMed Google Scholar
Noël A, Perveen Z, Xiao R, Hammond H, Le Donne V, Legendre K, Gartia MR, Sahu S, Paulsen DB, Penn AL. Mmp12 Is upregulated by in utero second-hand smoke exposures and is a key factor contributing to aggravated lung responses in adult emphysema, asthma, and lung cancer mouse models. Front Physiol. 2021;12: 704401.
Article PubMed PubMed Central Google Scholar
Pinkston R, Penn AL, Noël A. Increased oxidative stress responses in murine macrophages exposed at the air-liquid interface to third- and fourth-generation electronic nicotine delivery system (ENDS) aerosols. Toxicol Rep. 2023;11:40–57.
Article CAS PubMed PubMed Central Google Scholar
Herman M, Tarran R. E-cigarettes, nicotine, the lung and the brain: multi-level cascading pathophysiology. J Physiol. 2020;598(22):5063–71.
Article CAS PubMed Google Scholar
Shivanna B, Chu C, Moorthy B. The aryl hydrocarbon receptor (AHR): a novel therapeutic target for pulmonary diseases? Int J Mol Sci. 2022;23(3):1516.
Article CAS PubMed PubMed Central Google Scholar
Barrington-Trimis JL, Gibson LA, Halpern-Felsher B, Harrell MB, Kong G, Krishnan- Sarin S, Leventhal AM, Loukas A, McConnell R, Weaver SR. Type of e-cigarette device used among adolescents and young adults: findings from a pooled analysis of eight studies of 2166 vapers. Nicotine Tob Res. 2018;20(2):271–4.
Article PubMed Google Scholar
Fadus MC, Smith TT, Squeglia LM. The rise of e-cigarettes, pod mod devices, and JUUL among youth: Factors influencing use, health implications, and downstream effects. Drug Alcohol Depend. 2019;201:85–93.
Do EK, O’Connor K, Diaz MC, Schillo BA, Kreslake JM, Hair EC. Relative increases in direct-to-consumer menthol ads following 2020 FDA guidance on flavoured e-cigarettes. Tobacco Control. 2023;32(6):779–81.
Hwang JH, Lyes M, Sladewski K, Enany S, McEachern E, Mathew DP, Das S, Moshensky A, Bapat S, Pride DT. Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria. J Mol Med. 2016;94(6):667–79.
Article CAS PubMed Google Scholar
Madison MC, Landers CT, Gu BH, Chang CY, Tung HY, You R, Hong MJ, Baghaei N, Song LZ, Porter P, Putluri N, Salas R, Gilbert BE, Levental I, Campen MJ, Corry DB, Kheradmand F. Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine. J Clin Invest. 2019;129(10):4290–304.
Article PubMed PubMed Central Google Scholar
Miyashita L, Suri R, Dearing E, Mudway I, Dove RE, Neill DR, Van Zyl-Smit R, Kadioglu A, Grigg J. E-cigarette vapour enhances pneumococcal adherence to airway epithelial cells. Eur Respir J. 2018;51(2).
Sussan TE, Gajghate S, Thimmulappa RK, Ma J, Kim J-H, Sudini K, Consolini N, Cormier SA, Lomnicki S, Hasan F. Exposure to electronic cigarettes impairs pulmonary anti-bacterial and anti-viral defenses in a mouse model. PLoS ONE. 2015;10(2): e0116861.
Article PubMed PubMed Central Google Scholar
Noël A, Ghosh A. Carbonyl profiles of electronic nicotine delivery system (ENDS) aerosols reflect both the chemical composition and the numbers of e-liquid ingredients-focus on the in vitro toxicity of strawberry and vanilla flavors. Int J Environ Res Public Health. 2022;19(24):16774.
Article PubMed PubMed Central Google Scholar
US Food and Drug Administration. 2022. Food Additive Status List. Retrieved October 12 from https://www.fda.gov/food/food-additives-petitions/food-additive-status-list
Eccles R. Menthol: effects on nasal sensation of airflow and the drive to breathe. Curr Allergy Asthma Rep. 2003;3(3):210–4.
Article PubMed Google Scholar
Prasad N, Vijay S, Reddy AY, Nonitha S. Effects of menthol-flavored substances at the cellular level on oral mucosal sites. Dent Res J. 2019;16(1):7.
Article Google Scholar
Erythropel HC, Anastas PT, Krishnan-Sarin S, O'Malley SS, Jordt SE, Zimmerman JB. Differences in flavourant levels and synthetic coolant use between USA, EU and Canadian Juul products. Tobacco Control. 2021;30(4):453–5.
Kabbani N. Not so Cool? Menthol’s discovered actions on the nicotinic receptor and its implications for nicotine addiction. Front Pharmacol. 2013;4:95.
Article PubMed PubMed Central Google Scholar
Lee YO, Glantz SA. Menthol: putting the pieces together. Tobacco Control. 2011;20(Suppl 2):ii1–7.
Rosbrook K, Green BG. Sensory effects of menthol and nicotine in an e-cigarette. Nicotine Tobacco Res. 2016;18(7):1588–95.
US Food and Drug Administration. (2009, March 13). Menthol and other flavors in tobacco products. Retrieved April 20, 2019 from menthol and other flavors in tobacco products.
Fan L, Balakrishna S, Jabba SV, Bonner PE, Taylor SR, Picciotto MR, Jordt SE. Menthol decreases oral nicotine aversion in C57BL/6 mice through a TRPM8-dependent mechanism. Tobacco Control. 2016;25(Suppl 2):ii50–4.
Benowitz NL. Clinical pharmacology of nicotine: implications for understanding, preventing, and treating tobacco addiction. Clin Pharmacol Ther. 2008;83(4):531–41.
Article CAS PubMed Google Scholar
Benowitz NL, Herrera B, Jacob P. Mentholated cigarette smoking inhibits nicotine metabolism. J Pharmacol Exp Ther. 2004;310(3):1208–15.
Article CAS PubMed Google Scholar
Ashoor A, Nordman JC, Veltri D, Yang K-HS, Al Kury L, Shuba Y, Mahgoub M, Howarth FC, Sadek B, Shehu A. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors. PLoS ONE. 2013;8(7): e67674.
Article CAS PubMed PubMed Central Google Scholar
Hans M, Wilhelm M, Swandulla D. Menthol suppresses nicotinic acetylcholine receptor functioning in sensory neurons via allosteric modulation. Chem Senses. 2012;37(5):463–9.
Article CAS PubMed PubMed Central Google Scholar
Wang Q, Sundar IK, Li D, Lucas JH, Muthumalage T, McDonough SR, Rahman I. E-cigarette-induced pulmonary inflammation and dysregulated repair are mediated by nAChR α7 receptor: role of nAChR α7 in SARS-CoV-2 Covid-19 ACE2 receptor regulation. Respir Res. 2020;21(1):1–17.
Article Google Scholar
De Alba J, Raemdonck K, Dekkak A, Collins M, Wong S, Nials AT, Knowles RG, Belvisi MG, Birrell MA. House dust mite induces direct airway inflammation in vivo: implications for future disease therapy? Eur Respir J. 2010;35(6):1377–87.
Article PubMed Google Scholar
Noel S, Saams J, Ong MJ, Breysse P, Diette G, Biswal S, Matsui EC. Opposing effects of nasal epithelial NAD(P)H dehydrogenase quinine 1 and heme oxygenase 1 expression on upper and lower airway symptoms in adolescents with asthma. J Allergy Clin Immunol. 2011;128(2):422-4.e3.
Article CAS PubMed PubMed Central Google Scholar
Goodrich GG, Goodman PH, Budhecha SK, Pritsos CA. Functional polymorphism of detoxification gene NQO1 predicts intensity of empirical treatment of childhood asthma. Mutat Res. 2009;674(1–2):55–61.
Article CAS PubMed Google Scholar
Li YF, Tseng PJ, Lin CC, Hung CL, Lin SC, Su WC, Huang YL, Sung FC, Tai CK. NAD(P)H: Quinone oxidoreductase 1, glutathione S-transferase M1, environmental tobacco smoke exposure, and childhood asthma. Mutat Res. 2009;678(1):53–8.
Article CAS PubMed Google Scholar
He X, Zhang L, Xiong A, Ran Q, Wang J, Wu D, Niu B, Liu S, Li G. PM2. 5 aggravates NQO1-induced mucus hyper-secretion through release of neutrophil extracellular traps in an asthma model. Ecotoxicol Environ Saf. 2021;218:112272.
Guo SL, Liu F, Ren CJ, Xing CH, Wang YJ. Correlations of LTα and NQO1 gene polymorphisms with childhood asthma. Eur Rev Med Pharmacol Sci. 2019;23(17):7557–62.
PubMed Google Scholar
Ghosh A, Beyazcicek O, Davis ES, Onyenwoke RU, Tarran R. Cellular effects of nicotine salt-containing e-liquids. J Appl Toxicol. 2021;41(3):493–505.
Article CAS PubMed Google Scholar
Scott A, Lugg ST, Aldridge K, Lewis KE, Bowden A, Mahida RY, Grudzinska FS, Dosanjh D, Parekh D, Foronjy R, Sapey E, Naidu B, Thickett DR. Pro- inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages. Thorax. 2018;73(12):1161–9.
Article PubMed Google Scholar
Zhao J, Zhang Y, Sisler JD, Shaffer J, Leonard SS, Morris AM, Qian Y, Bello D, Demokritou P. Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches. J Hazard Mater. 2018;344:549–57.
Article CAS PubMed Google Scholar
Been T, Traboulsi H, Paoli S, Alakhtar B, Mann KK, Eidelman DH, Baglole CJ. Differential impact of JUUL flavors on pulmonary immune modulation and oxidative stress responses in male and female mice. Arch Toxicol. 2022;96(6):1783–98.
Article CAS PubMed Google Scholar
Cirillo S, Vivarelli F, Turrini E, Fimognari C, Burattini S, Falcieri E, Rocchi MBL, Cardenia V, Rodriguez-Estrada MT, Paolini M. The customizable e- cigarette resistance influences toxicological outcomes: Lung degeneration, inflammation, and oxidative stress-induced in a rat model. Toxicol Sci. 2019;172(1):132–45.
Article CAS PubMed Google Scholar
Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, McIntosh S, Robinson R, Rahman I. Vapors produced by electronic cigarettes and e-juices with flavorings induce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung. PLoS ONE. 2015;10(2): e0116732.
Article PubMed PubMed Central Google Scholar
Schweitzer RJ, Wills TA, Tam E, Pagano I, Choi K. E-cigarette use and asthma in a multiethnic sample of adolescents. Prev Med. 2017;105:226–31.
Article PubMed PubMed Central Google Scholar
Kameda K, Matsunaga T, Abe N, Hanada H, Ishizaka H, Ono H, Saitoh M, Fukui K, Fukuda I, Osanai T, Okumura K. Correlation of oxidative stress with activity of matrix metalloproteinase in patients with coronary artery disease: possible role for left ventricular remodelling. Eur Heart J. 2003;24(24):2180–5.
Nénan S, Boichot E, Lagente V, Bertrand CP. Macrophage elastase (MMP-12): a pro-inflammatory mediator? Mem Inst Oswaldo Cruz. 2005;100(Suppl 1):167–72.
Article PubMed Google Scholar
Gencer S, Cebeci A, Irmak-Yazicioglu MB. Matrix metalloproteinase gene expressions might be oxidative stress targets in gastric cancer cell lines. Chin J Cancer Res. 2013;25(3):322–33.
Kim MJ, Nepal S, Lee ES, Jeong TC, Kim SH, Park PH. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages. Toxicol Appl Pharmacol. 2013;273(1):77–89.
Article CAS PubMed Google Scholar
Razavian M, Bordenave T, Georgiadis D, Beau F, Zhang J, Golestani R, Toczek J, Jung JJ, Ye Y, Kim HY, Han J, Dive V, Devel L, Sadeghi MM. Optical imaging of MMP-12 active form in inflammation and aneurysm. Sci Rep. 2016;6:38345.
Article CAS PubMed PubMed Central Google Scholar
Nighot M, Ganapathy AS, Saha K, Suchanec E, Castillo EF, Gregory A, Shapiro S, Ma T, Nighot P. Matrix metalloproteinase MMP-12 promotes macrophage transmigration across intestinal epithelial tight junctions and increases severity of experimental colitis. J Crohns Colitis. 2021;15(10):1751–65.
Article PubMed PubMed Central Google Scholar
Amor M, Bianco V, Buerger M, Lechleitner M, Vujić N, Dobrijević A, Akhmetshina A, Pirchheim A, Schwarz B, Pessentheiner AR, Baumgartner F. Genetic deletion of MMP12 ameliorates cardiometabolic disease by improving insulin sensitivity, systemic inflammation, and atherosclerotic features in mice. Cardiovasc Diabetol. 2023;22(1):327.
Zhang WZ, Butler JJ, Cloonan SM. Smoking-induced iron dysregulation in the lung. Free Radical Biol Med. 2019;133:238–47.
Article CAS Google Scholar
Ying JF, Lu ZB, Fu LQ, Tong Y, Wang Z, Li WF, Mou XZ. The role of iron homeostasis and iron-mediated ROS in cancer. Am J Cancer Res. 2021;11(5):1895–912.
CAS PubMed PubMed Central Google Scholar
Ghio AJ. Asthma as a disruption in iron homeostasis. Biometals. 2016;29(5):751–79.
Article CAS PubMed Google Scholar
Kim J, Wessling-Resnick M. The role of iron metabolism in lung inflammation and injury. J Allergy Ther. 2012;3(Suppl 4):004.
PubMed PubMed Central Google Scholar
Ghio AJ, Pavlisko EN, Roggli VL, Todd NW, Sangani RG. Cigarette smoke particle-induced lung injury and iron homeostasis. Int J Chron Obstruct Pulmon Dis. 2022;17:117–40.
Article CAS PubMed PubMed Central Google Scholar
Galli TT, de Campos EC. Effects of environmental exposure to iron powder on healthy and elastase-exposed mice. Sci Rep. 2024;14(1):9134.
Article CAS PubMed PubMed Central Google Scholar
Kaisar MA, Sivandzade F, Bhalerao A, Cucullo L. Conventional and electronic cigarettes dysregulate the expression of iron transporters and detoxifying enzymes at the brain vascular endothelium: In vivo evidence of a gender-specific cellular response to chronic cigarette smoke exposure. Neurosci Lett. 2018;682:1–9.
Article CAS PubMed PubMed Central Google Scholar
Tangudu NK, Alan B, Vinchi F, Wörle K, Lai D, Vettorazzi S, Leopold K, Vujić Spasić M. Scavenging reactive oxygen species production normalizes ferroportin expression and ameliorates cellular and systemic iron disbalances in hemolytic mouse model. Antioxid Redox Signal. 2018;29(5):484–99.
Article CAS PubMed PubMed Central Google Scholar
Gkouvatsos K, Papanikolaou G, Pantopoulos K. Regulation of iron transport and the role of transferrin. Biochem Biophys Acta. 2012;1820(3):188–202.
Article CAS PubMed Google Scholar
Wafriy CI, Nor-Ashikin MNK, Kamsani YS, Muid SA, Sarbandi MS. Iron-related genes and proteins involved in iron homeostasis in animal models of allergic asthma: a systematic review. Biol Trace Elem Res. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12011-024-04183-8.
Article PubMed Google Scholar
Yoshida M, Minagawa S, Araya J, Sakamoto T, Hara H, Tsubouchi K, Hosaka Y, Ichikawa A, Saito N, Kadota T, Sato N. Involvement of cigarette smoke-induced epithelial cell ferroptosis in COPD pathogenesis. Nat Commun. 2019;10(1):3145.
Gao M, Monian P, Quadri N, Ramasamy R, Jiang X. Glutaminolysis and transferrin regulate ferroptosis. Mol Cell. 2015;59(2):298–308.
Article CAS PubMed PubMed Central Google Scholar
Mabley J, Gordon S, Pacher P. Nicotine exerts an anti-inflammatory effect in a murine model of acute lung injury. Inflammation. 2011;34(4):231–7.
Article CAS PubMed Google Scholar
Zhang W, Lin H, Zou M, Yuan Q, Huang Z, Pan X, Zhang W. Nicotine in inflammatory diseases: anti-inflammatory and pro-inflammatory effects. Front Immunol. 2022;13:826889.
Hickman E, Payton A, Duffney P, Wells H, Ceppe AS, Brocke S, Bailey A, Rebuli ME, Robinette C, Ring B, Rager JE. Biomarkers of airway immune homeostasis differ significantly with generation of e-cigarettes. Am J Respir Crit Care Med. 2022;206(10):1248–58.
Báez-Pagán CA, Delgado-Vélez M, Lasalde-Dominicci JA. Activation of the macrophage α7 nicotinic acetylcholine receptor and control of inflammation. J Neuroimmune Pharmacol. 2015;10(3):468–76.
Article PubMed PubMed Central Google Scholar
Zia S, Ndoye A, Nguyen V, Grando S. Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells. Res Commun Mol Pathol Pharmacol. 1997;97(3):243–62.
CAS PubMed Google Scholar
Hammons G. What is the biological fate of mentholated cigarette smoking? Addiction. 2010;105:8–10.
Article Google Scholar
Chapman DG, Casey DT, Ather JL, Aliyeva M, Daphtary N, Lahue KG, van der Velden JL, Janssen-Heininger YMW, Irvin CG. The effect of flavored e-cigarettes on murine allergic airways disease. Sci Rep. 2019;9(1):13671.
Article PubMed PubMed Central Google Scholar
Agoro R, Taleb M, Quesniaux VFJ, Mura C. Cell iron status influences macrophage polarization. PLoS ONE. 2018;13(5): e0196921. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0196921.
Article CAS PubMed PubMed Central Google Scholar
Choy DF, Hart KM, Borthwick LA, Shikotra A, Nagarkar DR, Siddiqui S, Jia G, Ohri CM, Doran E, Vannella KM, Butler CA. TH2 and TH17 inflammatory pathways are reciprocally regulated in asthma. Sci Transl Med. 2015;7(301):301ra129.
Omaiye EE, McWhirter KJ, Luo W, Pankow JF, Talbot P. High-nicotine electronic cigarette products: toxicity of JUUL fluids and aerosols correlates strongly with nicotine and some flavor chemical concentrations. Chem Res Toxicol. 2019;32(6):1058–69.
Article CAS PubMed PubMed Central Google Scholar
Matsumoto S, Fang X, Traber MG, Jones KD, Langelier C, Hayakawa Serpa P, Calfee CS, Matthay MA, Gotts JE. Dose-dependent pulmonary toxicity of aerosolized vitamin E acetate. Am J Respir Cell Mol Biol. 2020;63(6):748–57.
Article CAS PubMed PubMed Central Google Scholar
Moshensky A, Brand CS, Alhaddad H, Shin J, Masso-Silva JA, Advani I, Gunge D, Sharma A, Mehta S, Jahan A. Effects of mango and mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart, and colon in mice. Elife. 2022;11: e67621.
Article CAS PubMed PubMed Central Google Scholar
Silva MA, Bercik P. Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells. Lab Investig. 2012;92(6):937–48.
Janssen WJ, Stefanski AL, Bochner BS, Evans CM. Control of lung defence by mucins and macrophages: ancient defence mechanisms with modern functions. Eur Respir J. 2016;48(4):1201–14.
Article CAS PubMed PubMed Central Google Scholar
Morris CR, Habibovic A, Dustin CM, Schiffers C, Lin MC, Ather JL, Janssen-Heininger YMW, Poynter ME, Utermohlen O, Krönke M, van der Vliet A. Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease. Mucosal Immunol. 2022;15(5):977–89.
Article CAS PubMed PubMed Central Google Scholar
Nishida Y, Yagi H, Ota M, Tanaka A, Sato K, Inoue T, Yamada S, Arakawa N, Ishige T, Kobayashi Y, Arakawa H, Takizawa T. Oxidative stress induces MUC5AC expression through mitochondrial damage-dependent STING signaling in human bronchial epithelial cells. FASEB bioAdvances. 2023;5(4):171–81.
Article CAS PubMed PubMed Central Google Scholar
Takeyama K, Dabbagh K, Jeong Shim J, Dao-Pick T, Ueki IF, Nadel JA. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils. J Immunol. 2000;164(3):1546–52.
Kim HJ, Park YD, Moon UY, Kim JH, Jeon JH, Lee JG, Bae YS, Yoon JH. The role of Nox4 in oxidative stress-induced MUC5AC overexpression in human airway epithelial cells. Am J Respir Cell Mol Biol. 2008;39(5):598–609.
Article CAS PubMed Google Scholar
Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, Yamamoto M, Kensler TW, Tuder RM, Georas SN, Biswal S. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med. 2005;202(1):47–59.
Article CAS PubMed PubMed Central Google Scholar
Zheng S, Byrd AS, Fischer BM, Grover AR, Ghio AJ, Voynow JA. Regulation of MUC5AC expression by NAD(P)H:quinone oxidoreductase 1. Free Radic Biol Med. 2007;42(9):1398–408. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.freeradbiomed.2007.01.040
Kocyigit A, Armutcu F, Gurel A, Ermis B. Alterations in plasma essential trace elements selenium, manganese, zinc, copper, and iron concentrations and the possible role of these elements on oxidative status in patients with childhood asthma. Biol Trace Elem Res. 2004;97(1):31–41.
Article CAS PubMed Google Scholar
Ali MK, Kim RY, Brown AC, Mayall JR, Karim R, Pinkerton JW, Liu G, Martin KL, Starkey MR, Pillar AL, Donovan C. Crucial role for lung iron level and regulation in the pathogenesis and severity of asthma. Eur Respir J. 2020;55(4):1901340.
Wang Z, He Y, Cun Y, Li Q, Zhao Y, Luo Z. Transcriptomic analysis identified SLC40A1 as a key iron metabolism-related gene in airway macrophages in childhood allergic asthma. Front Cell Dev Biol. 2023;11:1164544.
Article PubMed PubMed Central Google Scholar
Bibi H, Vinokur V, Waisman D, Elenberg Y, Landesberg A, Faingersh A, Yadid M, Brod V, Pesin J, Berenshtein E, Eliashar R, Chevion M. Zn/Ga-DFO iron-chelating complex attenuates the inflammatory process in a mouse model of asthma. Redox Biol. 2014;2:814–9.
Article CAS PubMed PubMed Central Google Scholar
Yang N, Shang Y. Ferrostatin-1 and 3-methyladenine ameliorate ferroptosis in OVA-induced asthma model and in IL-13-challenged BEAS-2B cells. Oxid Med Cell Longev. 2022;2022:9657933.
PubMed PubMed Central Google Scholar
Zeng Z, Huang H, Zhang J, Liu Y, Zhong W, Chen W, Lu Y, Qiao Y, Zhao H, Meng X, Zou F, Cai S, Dong H. HDM induce airway epithelial cell ferroptosis and promote inflammation by activating ferritinophagy in asthma. FASEB J. 2022;36(6): e22359.
Article CAS PubMed Google Scholar
Araujo BB, Dolhnikoff M, Silva LF, Elliot J, Lindeman JH, Ferreira DS, Mulder A, Gomes HA, Fernezlian SM, James A, Mauad T. Extracellular matrix components and regulators in the airway smooth muscle in asthma. Eur Respir J. 2008;32(1):61–9.
Article CAS PubMed Google Scholar
Chandra M, Panchatcharam M, Miriyala S. Biomarkers in ROS and role of isoprostanes in oxidative stress. In: Free Radicals and Diseases; 2016. pp. 131–48.
Di Meo S, Reed TT, Venditti P, Victor VM. Role of ROS and RNS sources in physiological and pathological conditions. Oxidat Med Cell Long. 2016;2016(1):1245049.
Na HG, Kim YD, Choi YS, Bae CH, Song SY. Allethrin and prallethrin stimulates MUC5AC expression through oxidative stress in human airway epithelial cells. Biochem Biophys Res Commun. 2018;503(1):316–22.
Article CAS PubMed Google Scholar
Galaris D, Barbouti A, Pantopoulos K. Iron homeostasis and oxidative stress: An intimate relationship. Biochimica et Biophysica Acta (BBA)-Mol Cell Res. 2019;1866(12):118535.
McHugh MK, Symanski E, Pompeii LA, Delclos GL. Prevalence of asthma among adult females and males in the United States: results from the National Health and Nutrition Examination Survey (NHANES), 2001–2004. J Asthma. 2009;46(8):759–66.
PubMed PubMed Central Google Scholar
Steerenberg PA, Withagen CE, Dormans JA, van Dalen WJ, van Loveren H, Casee FR. Adjuvant activity of various diesel exhaust and ambient particles in two allergic models. J Toxicol Environ Health A. 2003;66(15):1421–39.
Article CAS PubMed Google Scholar
Dong CC, Yin XJ, Ma JY, Millecchia L, Barger MW, Roberts JR, Zhang XD, Antonini JM, Ma JK. Exposure of brown Norway rats to diesel exhaust particles prior to ovalbumin (OVA) sensitization elicits IgE adjuvant activity but attenuates OVA-induced airway inflammation. ToxicolSci. 2005;88(1):150–60.
CAS Google Scholar
van Zijverden M, de Haar C, van Beelen A, van Loveren H, Penninks A, Pieters R. Coadministration of antigen and particles optimally stimulates the immune response in an intranasal administration model in mice. Toxicol Appl Pharmacol. 2001;177(3):174–8.
Article PubMed Google Scholar
Li X, Zhang Y, Zhang R, Chen F, Shao L, Zhang L. Association between e-cigarettes and asthma in adolescents: a systematic review and meta-analysis. Am J Prevent Med. 2022;62(6):953–60.
Xian S, Chen Y. E-cigarette users are associated with asthma disease: a meta-analysis. Clin Respir J. 2021;15(5):457–66.
Article PubMed Google Scholar

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Acknowledgements

The authors thank Ms. Blaire Holliday and Mrs. Sultan Yilmaz of the Louisiana State University School of Veterinary Medicine for their excellent technical assistance.

Funding

Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health and the FDA Center for Tobacco Products (CTP) under Award Number K01HL149053 (AN). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Food and Drug Administration (FDA). This research was also supported by a grant to AP from the Louisiana Governor’s Biotechnology Initiative GBI-BOR#013.

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Rakeysha I. Pinkston and Alexandra Noël have equal contributions.

Authors and Affiliations

Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USA
Rakeysha I. Pinkston, Zakia Perveen, Tomislav Jelesijevic, Arthur L. Penn & Alexandra Noël
Department of Environmental Toxicology, Southern University and A & M College, Baton Rouge, LA, USA
Rakeysha I. Pinkston
IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, ME, 04092, USA
Matthew Schexnayder
Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USA
Ingeborg M. Langohr
Global Discovery Pathology and Multimodal Imaging, Sanofi, Cambridge, MA, 02141, USA
Ingeborg M. Langohr

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Rakeysha I. Pinkston
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Matthew Schexnayder
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Zakia Perveen
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Ingeborg M. Langohr
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Tomislav Jelesijevic
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Arthur L. Penn
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Alexandra Noël
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Contributions

RP, AP and AN designed the study. RP, MS, ZP, IL, and TJ carried out the assays and AN assisted with and interpreted data analysis. The manuscript was drafted by RP and revised by AN. All authors read and approved the final manuscript.

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Correspondence to Alexandra Noël.

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Animals were housed and handled in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All procedures and protocols were approved by the Louisiana State University (LSU) Institutional Animal Care and Use Committee (IACUC).

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Additional file 1.

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Pinkston, R.I., Schexnayder, M., Perveen, Z. et al. MMP12 deficiency attenuates menthol e-cigarette plus house dust-mite effects on pulmonary iron homeostasis and oxidative stress. Respir Res 26, 135 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-025-03213-w

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Received: 02 December 2024
Accepted: 31 March 2025
Published: 11 April 2025
DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-025-03213-w

MMP12 deficiency attenuates menthol e-cigarette plus house dust-mite effects on pulmonary iron homeostasis and oxidative stress

Abstract

Background

Methods

Results

Conclusion

Background

Methods

Animals and in vivo JUUL aerosol exposures

JUUL aerosol characterization

House-dust mite (HDM) treatment

Whole-body plethysmography

Bronchoalveolar lavage fluid (BALF) collection

Serum collection, cotinine and IgE ELISAs, as well as serum iron content

Histopathological analysis of lungs

Genotyping

Total BALF protein quantification

RNA isolation and quantitative real-time PCR (qRT-PCR)

RT2 profiler PCR array

RNA sequencing

Ingenuity pathway analysis

In situ hybridization using RNAscope

Protein extraction

Assays and ELISAs for lung proteins quantification

Statistical analysis

Results

High levels of iron are present in JUUL menthol-flavored aerosols.

Mmp12 increases serum cotinine levels in HDM-treated mice exposed to JUUL menthol for 60 days

Exposures to menthol-flavored JUUL aerosol plus HDM disrupt pulmonary iron metabolism homeostasis, while MMP12 deficiency attenuates this effect

JUUL exposure differentially modulates allergen-induced pulmonary inflammation in WT and MMP12 KO mice

Molecular signatures induced by menthol-flavored JUUL aerosol exposures are reduced in the lungs of MMP12 deficient mice

MMP12 deficiency down-regulates markers of oxidative stress following menthol-flavored JUUL aerosol exposures with or without HDM

MMP12 deficiency reduces the number of dysregulated genes associated with allergy and asthma following menthol-flavored JUUL aerosol exposures with or without HDM

Discussion

Menthol flavor, MMP12, HDM, and α7nAChR signaling

MMP12, JUUL menthol, HDM and anti-oxidant/oxidative stress responses

MMP12, JUUL menthol, HDM and pulmonary iron metabolism

MMP12, JUUL menthol, HDM, and pulmonary inflammation

MMP12, JUUL menthol, HDM, and mucin hypersecretion

JUUL menthol, iron-mediated pulmonary toxicity, oxidative stress, mucus hypersecretion, and asthma

Conclusion

Availability of data and materials

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval and consent to participate

Clinical trial number

Consent for publication

Competing interests

Additional information

Publisher's Note

Supplementary Information

Additional file 1.

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About this article

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Keywords

Respiratory Research

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