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. 2022 Apr 12:11:e67621.
doi: 10.7554/eLife.67621.

Effects of mango and mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart, and colon in mice

Alex Moshensky #  1   2 , Cameron S Brand #  3 , Hasan Alhaddad #  4 , John Shin  1   2 , Jorge A Masso-Silva  1   2 , Ira Advani  1   2 , Deepti Gunge  1   2 , Aditi Sharma  5 , Sagar Mehta  1   2 , Arya Jahan  1   2 , Sedtavut Nilaad  1   2 , Jarod Olay  1   2 , Wanjun Gu  2 , Tatum Simonson  2 , Daniyah Almarghalani  4 , Josephine Pham  1   2 , Samantha Perera  1   2 , Kenneth Park  1   2 , Rita Al-Kolla  1   2 , Hoyoung Moon  3 , Soumita Das  5 , Min Kwang Byun  2   6 , Zahoor Shah  7 , Youssef Sari  4 , Joan Heller Brown  3 , Laura E Crotty Alexander  1   2
Affiliations

Effects of mango and mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart, and colon in mice

Alex Moshensky et al. Elife. .

Abstract

While health effects of conventional tobacco are well defined, data on vaping devices, including one of the most popular e-cigarettes which have high nicotine levels, are less established. Prior acute e-cigarette studies have demonstrated inflammatory and cardiopulmonary physiology changes while chronic studies have demonstrated extra-pulmonary effects, including neurotransmitter alterations in reward pathways. In this study we investigated the impact of inhalation of aerosols produced from pod-based, flavored e-cigarettes (JUUL) aerosols three times daily for 3 months on inflammatory markers in the brain, lung, heart, and colon. JUUL aerosol exposure induced upregulation of cytokine and chemokine gene expression and increased HMGB1 and RAGE in the nucleus accumbens in the central nervous system. Inflammatory gene expression increased in the colon, while gene expression was more broadly altered by e-cigarette aerosol inhalation in the lung. Cardiopulmonary inflammatory responses to acute lung injury with lipopolysaccharide were exacerbated in the heart. Flavor-specific findings were detected across these studies. Our findings suggest that daily e-cigarette use may cause neuroinflammation, which may contribute to behavioral changes and mood disorders. In addition, e-cigarette use may cause gut inflammation, which has been tied to poor systemic health, and cardiac inflammation, which leads to cardiovascular disease.

Keywords: JUUL; e-cigarette; immunology; inflammation; medicine; mouse; neuroinflammation; vaping.

Plain language summary

The use of e-cigarettes or ‘vaping’ has become widespread, particularly among young people and smokers trying to quit. One of the most popular e-cigarette brands is JUUL, which offers appealing flavors and a discrete design. Many e-cigarette users believe these products are healthier than traditional tobacco products. And while the harms of conventional tobacco products have been extensively researched, the short- and long-term health effects of e-cigarettes have not been well studied. There is even less information about the health impacts of newer products like JUUL. E-cigarettes made by JUUL are different relative to prior generations of e-cigarettes. The JUUL device uses disposable pods filled with nicotinic salts instead of nicotine. One JUUL pod contains as much nicotine as an entire pack of cigarettes (41.3 mg). These differences make studying the health effects of this product particularly important. Moshensky, Brand, Alhaddad et al. show that daily exposure to JUUL aerosols increases the expression of genes encoding inflammatory molecules in the brain, lung, heart and colon of mice. In the experiments, mice were exposed to JUUL mint and JUUL mango flavored aerosols for 20 minutes, 3 times a day, and for 4 and 12 weeks. The changes in inflammatory gene expression varied depending on the flavor. This suggests that the flavorings themselves contribute to the observed changes. The findings suggest that daily use of pod-based e-cigarettes or e-cigarettes containing high levels of nicotinic salts over months to years, may cause inflammation in various organs, increasing the risk of disease and poor health. This information may help individuals, clinicians and policymakers make more informed decisions about e-cigarettes. Further studies assessing the impact of these changes on long-term physical and mental health in humans are desperately needed. These should assess health effects across different e-cigarette types, flavors and duration of use.

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Conflict of interest statement

AM, CB, HA, JS, JM, IA, DG, AS, SM, AJ, SN, JO, WG, TS, DA, JP, SP, KP, RA, HM, SD, MB, ZS, YS, JH, LC No competing interests declared

Figures

Figure 1.
Figure 1.. Three months of JUUL aerosol inhalation leads to an increase of pro-inflammatory cytokines in different regions of the brain.
Brains were harvested at the end point and the regions for NAc-core, NAc-shell and Hippocampus were harvested and frozen. RNA was extracted and qPCR was performed to quantify the expression of Tnfa, Il1b, Il6. Tnfa expression is shown from NAc-core at (A) 1 month and (B) 3 months, from NAc-shell at (C) 1 month and (D) 3 months, and from Hippocampus at (E) 1 month and (F) 3 months. Il1b expression is shown from NAc-core at (G) 1 month and (H) 3 months, from NAc-shell at (I) 1 month and (J) 3 months, and from Hippocampus at (K) 1 month and (L) 3 months. Il6 expression is shown from NAc-core at (M) 1 month and (N) 3 months, from NAc-shell at (O) 1 month and (P) 3 months, and from Hippocampus at (Q) 1 month and (R) 3 months. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons for each brain region and timepoint. Data are presented as individual data points ± SEM with n = 5–6 mice per group. *p < 0.05, **p < 0.01, *** p < 0.001 and **** p < 0.0001.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Inflammatory gene expression changes in the central nervous system in mice exposed to JUUL Mango and JUUL Mint.
These data are graphed with each exposure type (air – blue, mango – orange, and mint – green) grouped for ease of comparison of gene expression at 1 month (white columns) and 3 months (solid columns) of exposure. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each brain region and gene of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group. *p < 0.05, **p < 0.01, *** p < 0.001 and **** p < 0.0001.
Figure 2.
Figure 2.. Three months of JUUL aerosol inhalation leads to an increase of inflammatory mediators HMGB1 and RAGE.
Brains were harvested at the end point and the regions for NAc-core, NAc-shell and Hippocampus were sectioned. Later, protein was extracted and Western Blot was performed to quantify the expression of HMGB1-1 and RAGE. HMGB1-1 relative protein level are shown from NAc-core at (A) 1 month and (B) 3 months, from NAc-shell at (C) 1 month and (D) 3 months, and from Hippocampus at (E) 1 month and (F) 3 months. RAGE protein levels are shown from NAc-core at (G) 1 month and (H) 3 months, from NAc-shell at (I) 1 month and (J) 3 months, and from Hippocampus at (K) 1 month and (L) 3 months. Changes in proteins levels are relative to Air controls. Data are presented as individual data points ± SEM with n = 5–6 mice per group. *p < 0.05, **p < 0.01 and *** p < 0.001.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Inflammatory protein level changes in the central nervous system in mice exposed to JUUL Mango and JUUL Mint.
These data are graphed with each exposure type (air – blue, mango – orange, and mint – green) grouped for ease of comparison of protein levels at 1 month (white columns) and 3 months (solid columns) of exposure. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each brain region and protein of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group. *p < 0.05, **p < 0.01, and *** p < 0.001.
Figure 3.
Figure 3.. Three months of inhalation of JUUL aerosols alters inflammatory and fibrosis associated gene expression in cardiac tissue.
Hearts were harvested, and RNA was extracted from the left ventricle and qPCR was performed to quantify the gene expression of different cytokines, chemokines and fibrosis-associated genes. Cytokines include Tnfa at (A) 1 month and (B) 3 months, Il1b at (C) 1 month and (D) 3 months, Il6 at (E) 1 month and (F) 3 months, and Il18 at (G) 1 month and (H) 3 months. Chemokines include Ccl2 at (I) 1 month and (J) 3 months, Ccl3 at (K) 1 month and (L) 3 months, Cxcl1 at (M) 1 month and (N) 3 months, and Cxcl2 at (O) 1 month and (P) 3 months. Fibrosis-associated genes include Col1a1 at (Q) 1 month and (R) 3 months, Col3a1 at (S) 1 month and (T) 3 months, Postn at (U) 1 month and (V) 3 months, and Tlr4 at (W) 1 month and (X) 3 months. Changes in expression levels are relative to Air controls. Data are presented as individual data points ± SEM with n = 5–11 mice per group. *p < 0.05 and **p < 0.01.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Inflammatory gene expression changes in the hearts of mice exposed to JUUL Mango and JUUL Mint.
Mice exposed to JUUL Mint aerosols three times daily (60 min total) for 1 month had diminished expression of Tnfa, Il6, Col1a1, and Col3a1 relative to air controls. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each gene of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group. *p < 0.05, **p < 0.01, and *** p < 0.001.
Figure 4.
Figure 4.. Chronic inhalation of JUUL aerosols alters Ccl2 and Il13 levels in cardiac tissue.
Cardiac apex tissue was lysed, total protein isolated, and inflammatory proteins quantified by Bio-Plex Pro Mouse Cytokine 23-plex Assay. Both Il13 and Ccl2 levels were diminished in cardiac tissue from mice exposed for 3 months to JUUL Mint (green) and JUUL Mango (orange) aerosols, relative to Air controls (blue). Data was analyzed by two-way ANOVA with Dunnett’s corrections for multiple comparisons, and are presented as individual data points ± SEM, with n = 4–6 mice per group. *p = 0.017, **p < 0.01, and ****p < 0.0001.
Figure 5.
Figure 5.. Three months of JUUL aerosol inhalation alters pro-inflammatory markers in colon.
Inflammation was assessed in the colon at 1 and 3 months. Panels show inflammation markers in the colon in Tnfa (A) 1 month and (B) 3 months, Il6 at (C) 1 month and (D) 3 months, Il1b at (E) 1 month and (F) 3 months,Il8 (G) and (H), and Ccl2I 1 month and (J) 3 months. Data for inflammation markers is presented as individual data points ± SEM. * p < 0.01 and ** p < 0.001.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Inflammatory gene expression changes in the colon of mice exposed to JUUL Mango and JUUL Mint.
These data are graphed with each exposure type (air – blue, mango – orange, and mint – green) grouped for ease of comparison of gene expression at 1 month (white columns) and 3 months (solid columns) of exposure. Notably, inhalation of JUUL Mango aerosols three times daily for 1 month led to increased expression of Tnfa, Il6, and Il8 in the colon. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each gene of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group. *p < 0.05, **p < 0.01, *** p < 0.001 and **** p < 0.0001.
Figure 6.
Figure 6.. Unique RNAseq signatures in the lungs exposed to different flavors of JUUL aerosols.
(A) Venn diagram of gene expression unique to JUUL Mango (155) and JUUL Mint (Alhaddad et al., 2014b). Gene expression changes common to both aerosols (99) suggest that they are due to chemicals found in both of the flavored e-liquids (nicotinic salts, propylene glycol, glycerin, benzoic acid, etc). (B) Greatest gene expression changes associated with nicotine and flavorant chemicals within aerosols. (C) Volcano plots demonstrating gene expression changes specific for each flavor after sub-acute exposure (1 month; top row) and 3 months of exposure (bottom row). (D) Gene expression changes associated with inhalation of JUUL aerosols with different flavors in the setting of inflammatory challenge with inhaled LPS.(E) Heat map of the pathways most notably impacted by 1- and 3-month daily exposures to JUUL Mint and Mango, as well as in the setting of LPS challenge at 3 months. IFNg: interferon gamma; CCR: C-C motif chemokine related; IFN: interferon; BP: binding protein; TGFb: transforming growth factor beta; TNF: tumor necrosis factor; SOCS: suppressor of cytokine signaling; CCL: C-C motif ligand.
Figure 7.
Figure 7.. JUUL exposure alters airway inflammatory responses in the setting of inhaled LPS challenge.
BAL was harvested at the endpoints, and cytokines and chemokines were quantified by ELISA. Ccl2 at (A) 1 month, and (B) 3 months, Cxcl1 at (C) 1 month and (D) 3 months, Cxcl2 at (E) 1 month and (F) 3 month, RANTES at (G) 1 month and (H) 3 months, Tnfa at (I) 1 month and (J) 3 months, Il1b at (K) 1 month and (L) 3 months, Il6 at (M) 1 month and (N) 3 months. Data are presented as individual data points ± SEM with n = 5–11 mice per group. **p < 0.01 and **** p < 0.0001.
Figure 7—figure supplement 1.
Figure 7—figure supplement 1.. Inflammatory cytokine levels in the BAL of mice exposed to JUUL Mango and JUUL Mint prior to inhaled LPS challenge.
These data are graphed with each exposure type (air – blue, mango – orange, and mint – green) grouped for ease of comparison of protein levels at 1 month (white columns) and 3 months (solid columns) of exposure. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each brain region and gene of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group.
Figure 8.
Figure 8.. Cardiac inflammation induced by inhaled LPS challenge is increased in the setting of 3 months of JUUL aerosol inhalation.
Hearts were harvested, and RNA was extracted from the left ventricle and qPCR was performed to quantify the gene expression of different cytokines, chemokines and fibrosis-associated genes. Cytokines include Tnfa at (A) 1 month and (B) 3 months, Il1b at (C) 1 month and (D) 3 months, Il6 at (E) 1 month and (F) 3 months, and Il18 at (G) 1 month and (H) 3 months. Chemokines include Ccl2 at (I) 1 month and (J) 3 months, Ccl3 at (K) 1 month and (L) 3 months, Cxcl1 at (M) 1 month and (N) 3 months, and Cxcl2 at (O) 1 month and (P) 3 months. Fibrosis-associated genes include Col1a1 at (Q) 1 month and (R) 3 months, Col3a1 at (S) 1 month and (T) 3 months, Postn at (U) 1 month and (V) 3 months, and Tlr4 at (W) 1 month and (X) 3 months. Changes in expression levels are relative to Air controls. Data are presented as individual data points ± SEM with n = 5–11 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 8—figure supplement 1.
Figure 8—figure supplement 1.. Inflammatory gene expression changes in the hearts of mice exposed to JUUL Mango and JUUL Mint and challenged with inhaled LPS.
Mice exposed to JUUL Mint aerosols three times daily (60 min total) for 1 month prior to LPS challenge had increased expression of Tnfa, Il1b, Ccl2, Ccl3, Cxcl1, and Cxcl2, and decreased Postn, relative to air controls, while mice exposed to JUUL Mango aerosols for 1 month had increased Ccl2 and decreased Postn. After 3 months of JUUL Mint exposure, cardiac tissue had increased Il1b, Ccl2, Il1b, Ccl2, and Col3a1 in the setting of LPS challenge, while JUUL Mango had increased Il1b. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each gene of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group. *p < 0.05, **p < 0.01, *** p < 0.001, and ****p < 0.0001.
Figure 9.
Figure 9.. Three months of JUUL aerosol inhalation does not alter inflammatory markers in the setting of by inhaled LPS challengein the gastrointestinal tract.
Inflammation was assessed in the colon at 1 and 3 months. Panels show inflammation markers in the colon in Tnf (A) 1 month and (B) 3 months, Il6 at (C) 1 month and (D) 3 months, Il1b at (E) 1 month and (F) 3 months. Data for inflammation markers are presented as individual data points ± SEM.
Figure 9—figure supplement 1.
Figure 9—figure supplement 1.. Inflammatory gene expression in the colon of mice exposed to JUUL Mango and JUUL Mint and challenged with inhaled LPS.
These data are graphed with each exposure type (air – blue, mango – orange, and mint – green) grouped for ease of comparison of gene expression at 1 month (white columns) and 3 months (solid columns) of exposure. Data were analyzed with one-way ANOVA with Holm-Sidak multiple comparisons for each gene of interest. Data are presented as individual data points ± SD with n = 5–6 mice per group.
Figure 10.
Figure 10.. Overview of JUUL aerosol induced inflammatory changes across organs.
Appendix 1—figure 1.
Appendix 1—figure 1.. JUUL aerosol inhalation does not alter heart rate, heart rate variability or blood pressure.
Before assessment of lung function, mice underwent heart rate and blood pressure measurements using Emka non-invasive ECG Tunnels and the CODA non-invasive blood pressure system at 1 and 3 months. Heart rate (A–B), heart rate variability as measured by RMMSD and SDNN (C–F), systolic blood pressure (G–H) and diastolic blood pressure (I–J) were unaltered by chronic e-cigarette aerosol inhalation. Data are presented as individual data points ± SEM with n = 5–11 mice per group.
Appendix 1—figure 2.
Appendix 1—figure 2.. Chronic exposure of JUUL does not increase airways resistance or induce airways hyperreactivity.
At end points prior to harvest, mice underwent tracheostomy and attached to the FlexiVent mouse ventilator (SciReq). Airways resistance, lung elastance and pressure-volume (PV) loops were measured by mechanics scans using the FlexiVent mouse ventilator (SciReq) (A–F), followed by the assessment of responses to methacholine (MCH) challenge at 0, 6, 12 and 24 mg/mL (G–J). These parameters were assessed after 1 and 3 months of exposure of JUUL. Panels show airways resistance (Rrs) at (A) 1 month and (B) 3 months, Elastance at (C) 1 month and (D) 3 months, and PV loops at (E) 1 month and (F) 3 months. There were no differences in responses to methacholine challenge by Rrs at (G) 1 month and (H) 3 months, or Ers at (I) 1 month and (J) 3 months. Data for airways resistance and lung elastance are presented as individual data points ± SEM with n = 5–11 mice per group, and PV loops as means with n = 6 mice per group.
Appendix 1—figure 3.
Appendix 1—figure 3.. Chronic JUUL exposure does not affect leukocyte levels in the airways or influx into the airways and parenchyma in the setting of inhaled LPS challenge.
BAL was obtained, leukocyte counts were performed. BAL total cell counts in air versus JUUL exposed mice were no different at (A) 1 month and (B) 3 months, nor were neutrophils counts at (C) 1 month and (D) 3 months. Representative pictures from Giemsa Wright stained BAL cells are shown in (E,F,G) for 1 month and (H,I,J) for 3 months. In the setting of acute lung injury induced by inhaled LPS challenge, cell counts in the BAL were no different across groups (K–N), with representative images from BAL cells demonstrating the same (O–T). Data for cell counts are presented as individual data points ± SEM with n = 5–11 mice per group.
Appendix 1—figure 4.
Appendix 1—figure 4.. Chronic JUUL exposure does not affect lung parenchyma at baseline or in the setting of inhaled LPS challenge.
The left lung lobe was fixed with formalin at 25 cm3 water pressure and stained with H&E. Representative pictures from H&E staining of lung tissue are shown in A,B,C for 1 month and (D,E,F) for 3 months. In the setting of acute lung injury induced by inhaled LPS challenge, lung inflammation was no different by histologic evaluation (G–L). Mean linear intercept (MLI) was calculated for all lungs and were no different across groups at 1 and 3 months (M). n = 5–11 mice per group.
Appendix 1—figure 5.
Appendix 1—figure 5.. Chronic exposure of JUUL for 3 months does not induce fibrosis in the liver, heart, and kidney.
Collagen deposition was quantified by image analysis (using ImageJ) of lung histological slides stained with Masson’s trichrome. Representative pictures are shown for liver tissue in (A) Air control, (B) JUUL Mango, (C) JUUL Mint and (D) Quantification of collagen percentage in liver tissue. Representative pictures for heart tissue are shown in (E) Air control, (F) JUUL Mango, (G) JUUL Mint and (H) Quantification of collagen percentage in heart tissue. Representative pictures for heart tissue are shown in (I) Air control, (J) JUUL Mango, (K) JUUL Mint and (L) Quantification of collagen percentage in kidney tissue. Data for quantification is presented as individual data points ± SEM with n = 4–6 mice per group.
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