Vaping versus Smoking: A Quest for Long-term impact in a mouse model

doi:10.21203/rs.3.rs-4926091/v1

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Research Article

Vaping versus Smoking: A Quest for Long-term impact in a mouse model

https://doi.org/10.21203/rs.3.rs-4926091/v1

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Background

Most smokers consider that electronic cigarettes are safer than tobacco and are marketed as safe products. Nevertheless, recent reports show the exposure to high levels of electronic cigarette vapors (ECV) activates lung cells and triggers lung inflammation and structural alterations after chronic exposure. In order to assess the potential harmful effect of moderate exposure to ECV, we investigated in mice, its effect on lung and systemic inflammation and on lung structure and function.

Methods

To reproduce closely the situation frequently encountered in human, we exposed mice during 1h/day during 3 or 6 months with two levels of electronic cigarette power in comparison with mice exposed to cigarette smoke (CS). Lung and systemic inflammation was evaluated by measuring cell recruitment and activation as well as cytokine concentrations. Lung transcriptome, respiratory function and body weight were also measured.

Results

Our data revealed that chronic exposure to moderate levels of ECV increased specifically lung inflammation including NK cells and T lymphocyte recruitment and the production of CXCL1 and CXCL2 chemokines as well as IL-22 after 3 months, these effects being different from the profile induced by CS. Surprisingly, there is no strong overlap between the impact of the 3 types of emissions on lung transcriptome. Modulation of pro-inflammatory pathways are limited to mice exposed to e-cig set to low power. In contrast, alteration of respiratory function is observed in high-power ECV-exposed mice but not at low power, with a different profile than in CS-exposed mice.

Conclusion

Subchronic (or mid-term) exposure to ECV might alter the respiratory function independently of the inflammatory response and in a different manner than CS.

Over the past few years, new methods of delivering nicotine have emerged, and many users prefer electronic cigarettes (e-cigs) to traditional tobacco cigarettes. Invented in 2003, e-cigs have revolutionized the tobacco industry. They were introduced as a safe alternative to conventional cigarettes, and designed with the intent to provide smokers the satisfaction of conventional cigarettes without major side effects. E-cig vapors (ECV) are generated by non-combustion heating and aerosolization of an e-liquid commonly composed of glycerol, propylene glycol, nicotine and flavorings [1]. In addition to nicotine, ECV contain a variety of substances, depending on the composition of the e-liquids, the duration and volume of the puff and the power of the e-cig [2, 3]. In particular, ECV may contain low concentrations of toxic substances, some of which are also present in combustible cigarette smoke (CS) such as carbonyl compounds, polycyclic aromatic hydrocarbons and metals [4–6]. However, the concentration of these compounds is also strongly dependent of the power settings used for the e-cig.

Supporting this, recent data demonstrated that ECV induced an inflammatory reaction both in vitro, on epithelial cells [3, 7] and in vivo in mice and in smokers [1, 5]. To date, only a handful of in vivo toxicological studies have been performed on ECV.

An in vivo study conducted in a mouse model of intense exposure (5 hours per day for 3 successive days) suggested that ECV increased pro-inflammatory cytokines and diminished lung glutathione levels which are critical in maintaining cellular redox balance [8]. In another study, e-cig exposure for 2 weeks resulted in immunomodulatory effects similar to those observed after exposure to CS, with impaired pulmonary anti-microbial defenses [9]. Furthermore, the chronic inhalation of ECV (1-hour daily for 4 months) has been reported to induce airway hyper-reactivity and air space enlargement in exposed mice, some of these effects being nicotine-dependent [10]. Altogether, these data confirm that inhalation of ECV could have an impact on the inflammatory and immune response of the lungs, two stages that could constitute the background for the development of cancers or chronic bronchitis as shown with the conventional cigarette.

Nowadays, a comprehensive understanding of the long-term toxicological and immunologic consequences associated with e-cig use compared with CS are lacking. Long-term effects of ECV and the specific role of nicotine on lung parenchyma have been assessed by Roxlau ET et al [11]. Using an intense whole-body exposure protocol of 6 hours per day for 8 months, the authors showed that airway inflammation following ECV inhalation, characterized by lymphocyte recruitment and significant changes in lung structure and function, was close to mild tobacco smoke-induced alterations. However, it remains unclear if a moderate ECV exposure protocol in a more physiologic context might have similar effects. This is why we used a moderate exposure protocol (1 hour/day) to low and high power (18 W and 30 W) e-cig in nose-only exposed mice. Using this protocol, we previously reported that moderate exposure to the high-power e-cig aerosol induced oxidative DNA damage in the lung and the liver of exposed mice similarly to the effect of 3R4F cigarette smoke exposure [12]. In the present study, we compared the impact on lung function, body weight and immune response of chronic exposure (either 3 or 6 months) to ECV generated at two different powers with that of conventional cigarette (3R4F) smoke (Fig. 1).

In vivo model

Experiments were conducted on spf male BALB/c mice (Janvier Labs, Le Genest-Saint-Isle, France), 9 weeks old, 8 animals/group for each time points (3 and 6 month exposure) dispatched in 2 cages. The housing procedure respects the classical procedures with light and temperature control, free access to food and water and environmental enrichment. The number was determined according to our previous experiments with CS-exposed mice. The primary outcome measures were the alteration of the respiratory function and of the body weight. This mouse strain is described as sufficiently sensitive to oxidative stress and chemical induction of lung cancers [13].¹³ Animal procedures were in agreement with European directive 2010/63/EU for the protection of animals used for scientific purposes and obtained the Ethical Committee on Animal Experimentation (CEEA 75) approval (ref APAFIS #10363-2017062615002072v2). Animal randomisation was performed by the responsible of the animal facility at the delivery of the mice.

Animal body weights were recorded on Monday of each weak while clinical signs were monitored daily.

E-cigarettes and conventional cigarette

We chose the third generation “ModBox” model from NHOSS® (Innova, Bondues, France), used with the “Air Tank” clearomiser equipped with a 0.5 Ω kanthal coil and with a partially closed air flow. For experiments, we chose two power settings for the Modbox model: a “low” power of 18 W and a “high” power of 30 W. For the e-liquid, we chose the best-selling NHOSS® brand containing 65% propylene glycol, 35% glycerine, 16 mg/mL nicotine and the most common flavor, “blond tobacco”. Conventional 3R4F cigarettes were obtained from the University of Kentucky (Lexington, KY, USA)

To avoid chemical cross-contamination, two different pieces of equipment (dilution chamber, tubes, exposure towers and pipes) were used for e-cig and 3R4F exposures (Fig. 1). Aerosols from e-cigs and 3R4F cigarette were generated with an InExpose e-cigarette extension system on which we adapted the Modbox and a cigarette smoking robot (SCIREQ®, Emka technologies, Montreal, Quebec, Canada), respectively. Mice were exposed to aerosols by a nose-only tower (InExpose system, SCIREQ®, Emka technologies). In order to perform a comparative toxicological study of ECV and CS, we used the Health Canada Intense puff regime (55 mL puff volume, 2 s puff duration, 30 s puff period). Based on data from the literature and our preliminary study after a 4-day subacute exposure (data not shown), two exposure protocols were applied in this study for both e-cig and 3R4F emissions: a 3-month subchronic and a 6-month chronic exposure, 60 min/day and 5 days/week. For each exposure schedule, an additional group of mice was exposed to fresh conditioned air (negative control) and a period of acclimatisation of one week was performed before the exposure .

Sample Collection

Mice were euthanized 24 hours after the last exposure for both the protocols of 3 or 6 month exposures. Bronchoalveolar lavage (BAL) fluids, lungs, spleens and blood samples were collected and kept on ice until they were processed. In addition for lungs, one lobe was stored in RNA Later (Ambion,ThermoFisher Scientific, Illkirch, France) for transcriptomic analyses, posterior lobes were fixed with paraformaldehyde (PFA 4%, Labonord, Villeneuve d’Ascq, France) for histopathological analyses, and another lobe was frozen in liquid nitrogen and then stored at − 80°C until further analyses.

Broncho-alveolar lavage procedure, lung and spleen processing

BALs were performed by instilling 5 aliquots of 0,5 mL (final volume 2,5 mL) of sterile PBS. After centrifugation at 400g for 6 min at 4°C, supernatants (cell-free BAL fluid) from the first two aliquots of 0.5 ml were stored at − 80°C for cytokine analysis (ELISA), and cell pellets were used for flow-cytometry analysis.

The left lobe of the lung was mashed with a sterile blade and then digested with collagenase (Collagenase Type VI 17104–019 Gibco by Life technologies, Carlsbad, California United States) at 37°C. After 15 min of digestion, lungs were homogenized with an 18 G needle and further digested for 15 min. After centrifugation at 400g for 6 min at 4◦C, the pellets were resuspended in a 30% Percoll solution (Percoll TM GE Healthcare 17–0891-01, Chicago, IL, United States) and centrifuged at 500g for 15 min. Total spleen cells were also isolated from spleen and centrifuged at 400g for 6 min at 4°C. The lung and spleen pellets were resuspended in red blood cells (RBC) lysis buffer during 5 min at room temperature, to remove erythrocytes. The reaction of RBC lysis was stopped with PBS 2% FBS (Gibco by Life technologies, Carlsbad, California United States). After centrifugation at 400g for 6 min at 4 ◦C, pulmonary and spleen cells were resuspended in PBS 2% FBS, then enumerated and used for flow cytometry.

Flow cytometry

BAL, lung and spleen total cells were incubated with the appropriate panel of antibodies for 30 min in PBS 2% FCS. Conjugated antibodies were used against mouse CD5 (ref 130–102–574, FITC-conjugated), PBS57-loaded CD1 d Tetramer (NIH facility,PE-conjugated), NK1.1 (ref 130–103–963, PerCp-Cy5.5–conjugated), CD4 (ref 130–102– 411, PE-Cy7-conjugated), CD25 (ref 130–102–550, APC-conjugated), CD69 (ref 561–238, Alexa700-conjugated), TCRγδ (ref 130–104–016, APC-Vio770 conjugated), TCR-β (ref 130– 104–815, V450-conjugated), CD8 (ref 130–109–252, V500-conjugated), CD45 (ref BLE103140, BV605-conjugated), I-Ab (ref 130–102–168, FITC-conjugated), F4/80 (ref 130–102–422,PE conjugated), CD103 (ref 563–637, PerCP-Cy5.5-conjugated), CD11c (ref 558–079, PE Cy7-conjugated), CD86 (ref 560–581, Alexa-700 conjugated), Ly6G (ref 560–600, APC-H7conjugated), CD11b (ref 560–455, V45O conjugated), CD45 (ref 130–402–512, V500 conjugated), Ly6C (ref BLE128036, BV605-conjugated) (BD Biosciences, Franklin Lakes, United States; Biolegend, San Diego, United States and Myltenyi Biotech, Paris, France) and CCR2 (ref FAB 5538A, R&D systems, APC conjugated).

Data were acquired on a LSR Fortessa (BD Biosciences, Franklin Lakes, United States) and analyzed using FlowJo™ software v10.2 (Stanford, CA, USA). Gating strategy has been previously described [14]. Absolute cell numbers were calculated according to the total cell number and the frequency of CD45⁺ immune cells.

Cytokine Assay

Levels of IL-1β, IL-6, IL-22, IL-23, IFN-γ, CXCL1, CXCL2, CXCL5, CXCL17, TNF-α and Resistin were determined in BAL, lung and serum by enzyme-linked immunosorbent assay (ELISA) using the manufacturer’s recommendation (R&D systems, Biotechne, Minneapolis, MN, United States). In addition, concentrations of IL-17 and IL-23p19 were measured by an ELISA from Invitrogen (Waltham, MA, United States).

Lung Histology

Fixed lung lobes were paraffin-embedded and lung sections were stained with hematoxylin-eosin. Lung injury and inflammation were scored based on a scale evaluating bronchial damage, hyperplasia, inflammatory cell influx, alveolar exudates, damage, wall thickness, inflammation, and emphysema, all with a scale from 0 to 4. Moreover, hemorrhage, fibrinoid necrosis, leukocytoclasis and suppuration were noted as present or not (value of 1 and 0, respectively). This allows to generate a global histologic score from 0 to 36. The score was established in a blinded fashion by independent experts (OCV clinical research, Lille).

In order to assess the potential development of emphysema, we evaluated the mean linear intercept (MLI) on lung sections by using the Image J software (NIH).

Lung function

On the day of the sacrifice, each mouse was anaesthetized intraperitoneally with xylazine hydrochloride (15 mg/kg) / ketamine (100 mg/kg), tracheotomized, and cannulated with an 18 G metal cannula (resistance: 0.36–0.40 cmH2O.s/mL). Mice were then connected to a flexiVent FX system (SCIREQ Inc., Montreal, Qc, Canada) and operated by the flexiWare software v7.7 as previously described [15]. Immediately after connection to the ventilator, set at 150 breaths/min, two deep lung inflations were performed at least 6–12 seconds apart to recruit lung beyond any closed airway and to standardize lung volume history. This was done by inflating the lungs to 30 cm H₂O over 3 seconds and holding that pressure for another 3 seconds to allow for the lungs to equilibrate after the inflation. Mice were then submitted to a 300 breaths/min hyperventilation in order to eliminate spontaneous breathing before a 150 breaths/min return. Next, the mechanical properties of the mouse respiratory system were assessed at baseline, i.e. before the construction of a full-range pressure-volume (PV) curve. This was done using a sequence of measurements integrated by default in the flexiVent operating software (flexiWare v7.7). The Area between the PV loop inflation and deflation limbs (Hysteresis (H)) A static compliance (Cst) was calculated. Cst, the parameters A (estimate of inspiratory capacity) and K (shape constant) can be extracted from the Salazar-Knowles equation [16]. Cst reflects the intrinsic elastic properties of the respiratory system (i.e. lung + chest wall).

Transcriptomic analyses

Total RNAs were extracted from RNA later treated tissue samples using the miRNeasy mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. The RNA concentration was measured with the Biospecnano spectrophotometer (Shimadzu, Marne-la-Vallée, France). Transcriptomic experiments were carried out using 8x60k OneColor microarrays (Agilent Technologies) coupled to 60-mer oligonucleotides covering the entire mouse genome. Labeling, hybridization and lncRNA detection were carried out according to the manufacturer's instructions (Agilent Technologies). For each microarray, Cyanine 3-coupled lncRNAs were synthesized by the QuickAmp Low Input Kit from 50 ng of total RNA. Spike-in RNAs were added to each tube and used as positive controls for the labeling and amplification steps. Labeled cDNAs were purified and 600 ng of each cDNA was then hybridized to the microarrays according to the manufacturer's instructions. After washing, the microarrays were scanned and the data exported using Agilent Feature Extraction Software© (FE version 10.7.3.1). Results were then interpreted by selecting those mRNAs whose expression was at least significantly 1.5-fold higher or 1.5-fold lower than that of unexposed control mice (p < 0.05). Statistical analyses were performed with the "linear models for microarray data" (limma) package for R, using moderated t statistics with standardized data. Functional analysis of selected deregulated lung RNAs was performed using Ingenuity Pathway Analysis software (Qiagen) by selecting the top 25 of canonical pathways for each exposure condition.

Statistical analyses

The data are expressed as mean ± Standard Error of the Mean (SEM). Statistical analyses were computed using GraphPad Prism software (5.00 version, GraphPad software, San Diego, USA) in accordance with the size of samples and the nature of the experiment. Values were compared to the controls with a bilateral and non-parametric Mann-Whitney test, or with a one-sample t-test, or a two-way ANOVA test when appropriate. Statistical significance was accepted for an error risk inferior to 5% and is represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

1- Long term e-cigarette exposure impairs clinical parameters

a- Subchronic exposure (3-month treatment)

We first analyzed the impact of ECV on clinical parameters, including body weight and lung function, and compared it to that of CS. As expected [15, 17], mice exposed to CS showed slower weight gain than control mice exposed to fresh conditioned air. This effect was also observed when mice were exposed to ECV with a power of either 18W or 30W (Fig. 2A).

Interestingly, this reduction in weight gain was associated with impaired lung function in mice exposed to 30W ECV and those exposed to CS compared to the control mice (Fig. 2B). Indeed, the PV-loop curve revealed a shift towards higher volumes for mice exposed to 30W ECV but not with 18W ECV, suggesting the development of some emphysema with the Modbox 30W.We also observed the same trend with CS-exposed mice although at a lower intensity than in 30W ECV-exposed mice. Based on PV-loop curves, we calculated the static compliance (Cst) and the area under the curve (AUC). The Cst tends to be increased both in 30W ECV and CS-exposed mice (Fig. 2E) whereas the AUC was only increased in CS-exposed mice (Fig. 2G).

Afterwards, we analyzed the impact of ECV exposure on morphological lung structure. We observed an extensive lung remodeling mainly in alveolar walls and bronchial epithelium (Fig. 3). Compared to control mice, extensive thickening of respiratory epithelium due to hyperplasia and folding was observed in both ECV- and CS-exposed mice. Atypias like loss of orientation, binucleation or meganucleation were also sporadically observed in 30W ECV-exposed mice. Inflammatory cell recruitment was also detected in both peribronchial areas and alveoli. Additionally, all samples of 30W ECV and CS-exposed mice displayed periarterial edema and alveolar emphysema. MLI measurement exhibited a statistically significant (p < 0.001) difference between control mice and either 30W ECV- or CS-exposed mice but not with 18W ECV-exposed mice (Fig. 2H).

b- Chronic exposure (6-month treatment)

After 6 months of exposure, weight gain was substantially reduced in both ECV- and CS-exposed mice. Moreover, this effect tends to be greater in mice exposed to high power ECV compared to CS-exposed mice (Fig. 2C).

Lower weight gain was associated with an emphysematous-type alteration of lung function in mice which is more pronounced in 30W ECV-exposed mice (Fig. 2D). This was confirmed by a significant increase in Cst, A and AUC in the 30W ECV-exposed mice. Surprisingly, CS-exposed mice exhibited a fibrotic-like alteration of the PV loop curve and no alteration of the K, A and AUC parameters after 6 months of exposure as compared with both air and ECV-exposed mice (Fig. 2I-K, respectively). The histopathologic analysis revealed an increase in emphysema in both 30W ECV- and CS-exposed mice and a not-significant effect in 18W ECV-exposed mice as compared to air-exposed mice (Fig. 3) which were confirmed by MLI measurement (Fig. 2L). We also observed extensive lung remodeling mainly in alveolar walls and bronchial epithelium, including alveolar wall thickening, hyperplasia of bronchial epithelium and some area of cell desquamation mainly in both 30W ECV- and CS-exposed mice. This was associated with some inflammatory cell infiltrates both in the peribronchial and in alveolar area.

2- E-cigarette vapors induce pulmonary inflammation

a- Subchronic exposure (3-month treatment)

The inflammatory cell infiltrate was analysed in both lung and spleen by flow cytometry. This study revealed that mice exposed to CS during 3 months exhibited a specific immune cell signature compared to ECV-exposed mice (Fig. 4). CS-exposed mice presented reduced number of alveolar macrophages (AM) in lung tissue although these cells expressed higher levels of the activation markers CD86 and CMH class II molecules. Furthermore, we observed a statistically significant (p < 0.01) recruitment of inflammatory monocytes as well as NK and NKT cells in the BAL of CS-exposed mice (Fig. 4A) while this treatment increased the number of conventional dendritic cells type 1 and 2 (cDC1 and cDC2), CD8⁺ T Cells, CD4 + T cells, inflammatory monocytes and neutrophils in lung tissue (Fig. 4B and data not shown). This is not associated with a modulation of the expression of activation markers such as CD86 and I-Ab for monocytes and neutrophils, as well as CD25 and CD69 for lymphocytes (Fig. 4D).

In contrast, ECVs have a limited impact on innate immune cells recruitment in the BAL since they decreased the number of NK and CD8⁺ T cells whereas only 30W ECV induced a statistically higher expression (p < 0.01) of I-Ab on inflammatory monocytes (Fig. 4). In lung tissue, only 18W ECV induced the recruitment of NK cells, CD8⁺ and CD4⁺ T cells while exposure to 30W ECV increased the expression of CD25 on CD4⁺ T cells .

In order to decipher the mechanism of the inflammatory cell recruitment, we measured the levels of cytokines in the BAL and lungs of exposed mice (Fig. 5). Exposure to 30W ECV for 3 months was associated with higher levels of IL-22 and the chemokines CXCL1, CXCL2 and in BAL and a trend for CXCL17 concentrations (Fig. 5A). In mice exposed to CS, we detected higher concentrations of IL-17 and a decrease in Il-1β in the lungs compared with control mice (Fig. 5B). Meanwhile no effect was observed on Il-17 levels after exposure to ECV in either lung tissue or BAL in contrast with CS-exposed mice.

In addition, we further assessed the ability of immune cells to produce Th1 and Th17 cytokines with and without in vitro CD3 activation. Surprisingly, lung cells from CS-exposed mice produced significantly less IL-22 than those of control mice. In contrast, exposure to 30W ECV increased the levels of IL-22 by unstimulated cells but not after anti-CD3 activation while there is no change with 18W ECV.

b- Chronic exposure (6-month treatment)

After 6 months, mice exposed to ECV exhibited a significant decrease in the number of NK and CD4⁺ T cells in BAL, as well as a trend to lower number of AMs (Fig. 4A). Nevertheless, this was associated with a significant increase in activated AM and inflammatory monocytes in both ECV- and CS- exposed mice. In lung tissue, we observed a statistically significant recruitment of CD4⁺ and CD8⁺ T cells in all exposed mice (Fig. 4B). Only exposure to 30W ECV induced significant NK recruitment in lungs with a tend to recruit more NKT and AM. This was associated with higher expression of the activation marker I-Ab in AM and inflammatory monocytes in both the BAL and the lung (Fig. 4C-D). The same trend was observed in AM and inflammatory monocytes from CS-exposed mice whereas the expression of activation markers was not modulated in conventional T lymphocytes from these mice. Moreover, higher expression of CD25 in NKT cells in the lung of 30W ECV-exposed mice but not in 18W ECV-exposed mice (Fig. 4C). Interestingly, both exposure to 30W ECV and CS significantly reduce the number of cDC1 in lungs (Supplementary figure S1).

Regarding the secretion of inflammatory mediators, our data did not reveal an overproduction of cytokines in BAL and lungs from 30W ECV- and CS-exposed mice (Fig. 5A-B). In 18W ECV-exposed mice, only CXCL17 were significantly induced in the BAL and lungs as compared with controls. In contrast, we observed lower levels of IL-1β and CXCL1 in lungs for all emissions.

No significant alteration of the Th1 and Th17 cytokine secretion by lung cells was reported in mice exposed to ECV or CS.

E-cigarette vapors induce systemic inflammation.

To assess the systemic impact of ECV, we examined the number and phenotype of immune cells in the spleen, as well as their ability to produce Th1 and Th17 cytokines, and cytokine concentrations within the blood.

a- Subchronic exposure (3-month treatment)

The total cell number of spleen cells are similar among all the groups (58 ± 6, 35.3 ± 4.4, 37.4 ± 3, and 49.1 ± 4.7 x10⁶ cells for Air, 18W ECV, 30W ECV and CS, respectively). Exposure to 18 and 30 W ECV was responsible for a decrease in the number of antigen-presenting cells (DC and macrophages) and lymphocytes (CD4⁺ and CD8⁺ T cells) in the spleen of ECV-exposed animals (Fig. 6A). This was associated with an increased expression in CD25 in CD8⁺ T cells in mice exposed to 30W ECV (Fig. 6B).

In contrast, the spleen of CS-exposed mice was characterized by a statistically significant recruitment of iNKT and a lower number of neutrophils, as well as a trend towards a decreased number of DC. Despite the lack of effect on their numbers, a higher expression of the activation marker I-Ab was observed in macrophages as well as CD25 in CD4⁺ and CD8⁺ T Cells after CS exposure (Fig. 6B).

Interestingly, exposure to ECV tended to increase the levels of CXCL2 and TNF-α in blood, whereas the 30W ECV-exposed mice had lower concentrations of CXCL1 (Fig. 7A). In addition, unstimulated as well as anti-CD3 stimulated spleen cells from 30W ECV- and CS-exposed mice produced significantly more IL-22 than spleen cells from controls (Fig. 7B).

b- Chronic exposure (6-month treatment)

After 6 months, ECV-exposed mice presented a statistically significant recruitment of NKT cells in the spleen, regardless of e-cig power. Moreover, only ECV of 18W Modbox increased the recruitment of CD4⁺ and CD8⁺ T cells in the spleen (Fig. 6A). No impact was noticed on cell activation.

Surprisingly ECV and CS exposures induced a drop in the neutrophil number in the spleen. This was associated with a non-significant increase in macrophage number. No effect was observed on the number and expression of activation markers by other immune cells in the spleen. Regarding the ability of spleen cells to produce cytokines (Fig. 7B), IL-22 production consecutive to anti-CD3 stimulation was enhanced in mice exposed to 18W ECV whereas IL-17 and IFN-γ production was unchanged.

Surprisingly, serum levels of IL-6, IL-22, TNF-α, CXCL1 and CXCL2 were lower in ECV- and CS-exposed mice whereas there is no change for TNF-α in CS-exposed mice (Fig. 7A). Only, CS-exposed mice exhibited increased concentrations of resistin (p < 0.0001).

We have also analyzed the cotinine levels in order to assess the level of systemic exposure of mice to CS and ECV, as well as to estimate the potential involvement of nicotine in the modulation of the inflammatory reaction. Our data revealed that the cotinine levels are higher in mice exposed to CS and ECV than in mice exposed to air (Supplementary Figure S2). However, there is a trend for higher levels of cotinine in CS-exposed mice as compared with 18W and at a lesser degree 30W ECV-exposed mice at both time courses, although the differences are not statistically significant.

In summary, exposure to ECV has an ambivalent effect on systemic inflammation, with a drop in blood cytokine concentration, and, at the opposite, some activation and enhanced capacity to produce IL-22 in splenic T lymphocytes.

4- Impact of e-cigarette on lung transcriptome

In order to determine and compare the signaling pathways deregulated by exposure to ECV or CS, lung mRNA expression profiles were characterized by pangenomic microarrays in mice exposed to emissions for 3 or 6 months.

a- Subchronic exposure (3-month treatment)

Exposure to CS for 3 months induced the deregulation of 248 genes (186 up- and 62 down-regulated). Surprisingly, the number of deregulated genes was much higher in 18W ECV-exposed mice (197 with 121 up- and 76 down-regulated) compared to those exposed to 30W ECV (72 with 50 up- and 22 down-regulated) (Fig. 8A). The fold changes and the adjusted p values were reported in the supplemental Excel table S1.

Overall, 25 genes (Table 1) were deregulated by exposure to the three types of emissions, including genes involved in cancer development (increase in Pla1a expression and down-regulation of the tumour suppressor genes Cdkn2a and Ppp2r2c) and cell metabolism (e.g. increased levels in Fmo3). Moreover, the expression of 16 genes were specifically altered by both 18W and 30W ECV (Fig. 8B), some of which are involved in the control of oxidative stress (Hmox1, Slc7a11) and cell proliferation (Fosl1).

Table 1

**Genes commonly deregulated by 3 month subchronic exposure to ECV18W, ECV30W and CS.** Fold changes are calculated in comparison with Air mice. The genes in bold are those deregulated both after 3 and 6 month exposure.
Gene Symbol	Gene Name	Fold-Change ECV18W	Fold-Change ECV30W	Fold-Change CS
Atic	5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase	-1.93	-2.02	-1.89
Calca	calcitonin/calcitonin-related polypeptide. alpha	3.04	3.16	3.38
Cckar	cholecystokinin A receptor	1.99	2.08	1.92
Cdkn2a	cyclin dependent kinase inhibitor 2A	-1.70	-1.61	-0.63
Celf3	CUGBP. Elav-like family member 3	1.82	2.30	1.82
Chac1	ChaC. cation transport regulator 1	-3.69	-3.64	-2.53
Fam167b	family with sequence similarity 167. member B	-1.68	-1.66	-1.82
Fmo3	flavin containing monooxygenase 3	2.37	2.38	1.79
Gbp11	guanylate binding protein 11	-1.59	-1.58	-1.62
Gbp4	guanylate binding protein 4	-1.55	-1.56	-1.68
Gdf15	growth differentiation factor 15	-3.53	-3.58	-2.17
Gm26641	predicted gene. 26641	1.87	2.13	1.94
Gm4610	predicted gene 4610	3.18	3.82	2.94
H1fx	H1 histone family. member X	1.57	1.76	1.57
Klhl38	kelch-like 38	2.09	1.75	1.54
Mcpt8	mast cell protease 8	-1.97	-1.61	-1.83
Nupr1	nuclear protein transcription regulator 1	-2.16	-2.34	-2.13
Pla1a	phospholipase A1 member A	1.99	2.41	2.32
Ppp2r2c	protein phosphatase 2. regulatory subunit B. gamma	-1.58	-1.86	-1.83
Scn3b	sodium channel. voltage-gated. type III. beta	-1.61	-1.56	-1.65
Selenbp2	selenium binding protein 2	1.51	1.54	2.41
Slc38a4	solute carrier family 38. member 4	2.33	2.58	1.78
Syt15	synaptotagmin XV	-1.66	-1.72	-1.72
Tcf23	transcription factor 23	2.32	1.92	1.67
Trib3	tribbles pseudokinase 3	-2.12	-2.22	-1.88

Analysis of the pathways affected by CS treatments (Fig. 9C) revealed that most of the deregulated pathways are involved in the metabolism of xenobiotics (in particular the biotransformation of PAHs with increased expression of Cyp1a1 and Cyp1b1) and oxidative stress (increased levels of Nqo1, Gsta3, Gsto1). Pathways deregulated by 18W ECV are mainly related to inflammation linked to IL-17 signaling (decreased expression of Ccl2 and Il6) (Fig. 9A), oxidative stress (deregulation of Abcc1, Chac1, Gclc, Hmox1 and Myc) and lipid metabolism (Pla1a, Pmvk, Plcb1, Plcb4).

b- Chronic exposure (6-month treatment)

While we found an increase in the number of deregulated genes after 6 months of exposure to 18W ECV (431 with 388 up- and 43 down-regulated), the opposite was observed with ECV 30W, which altered the expression of only 40 genes (12 up- and 28 down-regulated) (Fig. 8C). The fold changes and the adjusted p values were reported in the supplementary Excel table S2. Thirty-four of them were commonly deregulated by the vapors of the 2 wattages.

CS exposures changed the expression of 615 genes (336 up- and 279 down-regulated), including 398 in a specific manner. Furthermore, 216 genes were deregulated in common with 18W ECV.

Overall, 23 genes (Table 2) were deregulated by exposure to the three types of emissions (Fig. 8D), including genes involved in inflammatory response (decreased expression of Il6 and Tnip3) and in cancer development (down-regulation of Fosl1 and Sfn and up-regulation of Plk5).

Table 2

**Genes commonly deregulated by 6 month chronic exposure to ECV18W, ECV30W and CS.** Fold changes are calculated in comparison with Air mice. The genes in bold are those deregulated both after 3 and 6 month exposure.
Gene Symbol	Gene Name	Fold-Change ECV18W	Fold-Change ECV30W	Fold-Change CS
Adm2	adrenomedullin 2	-2.15	-1.92	-1.92
Atic	5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase	-2.04	-2.31	-2.86
Chac1	ChaC. cation transport regulator 1	-2.79	-2.27	-2.45
Diras2	DIRAS family. GTP-binding RAS-like 2	1.71	1.53	1.84
Dpp6	dipeptidylpeptidase 6	2.43	1.72	1.56
Fam161a	family with sequence similarity 161. member A	2.17	1.71	2.06
Fmo3	flavin containing monooxygenase 3	2.61	2.45	3.11
Fosl1	fos-like antigen 1	-4.03	-4.10	-3.04
Galnt5	polypeptide N-acetylgalactosaminyltransferase 5	1.76	1.67	1.54
Gdf15	growth differentiation factor 15	-3.45	-2.55	-2.64
Gjb2	gap junction protein. beta 2	-2.18	-2.19	-1.55
Il6	interleukin 6	-2.18	-2.80	-1.73
Itprid1	ITPR interacting domain containing 1	3.45	2.23	1.57
Lrrc26	leucine rich repeat containing 26	3.54	2.11	1.84
Mab21l3	mab-21-like 3 (C. elegans)	-2.20	-2.27	-2.34
Mgat3	mannoside acetylglucosaminyltransferase 3	3.08	2.21	2.21
Nupr1	nuclear protein transcription regulator 1	-2.39	-2.59	-4.13
Phgdh	3-phosphoglycerate dehydrogenase	-1.57	-1.50	-1.87
Plk5	polo like kinase 5	3.13	1.80	2.38
Rptoros	regulatory associated protein of MTOR. complex 1. opposite strand	2.32	1.67	1.79
Sfn	stratifin	-3.67	-3.88	-2.57
Tnip3	TNFAIP3 interacting protein 3	-1.55	-1.67	-1.70
Trib3	tribbles pseudokinase 3	-1.84	-1.87	-2.17

After 6 months of exposure, the main pathways affected by CS were related to the inflammatory response (e.g. decreased levels of Il6, Il1b and Mmp9) and xenobiotic metabolism (e.g. modulation of Ahrr, Cyp1a1, Cyp1b1 and Gsta3) (Fig. 10C). The 18W ECV mostly affected nucleotide biosynthesis, metabolism of xenobiotics (Aldh18a1, Aldh1l2 et Aldh3a1) and inflammation (Ccl20, Cxcl6, Il6, Muc5ac) (Fig. 10A).

Altogether, the transcriptomic data showed that mRNA profiles are very different between 3 and 6 months of exposure, for both 18W ECV and CS. Moreover, the impact of 30W ECV on the lung transcriptome appeared to be much lower than that of 18W ECV. We failed to clearly identify signalling pathways altered by 30W ECV due to the low number of modulated genes (Figs. 9B and 10B).

The use of electronic nicotine delivery systems, commonly known as e-cig, has gained significant in popularity as an alternative to conventional cigarette or even as a trend among adolescents. While the well-established health risks associated with CS exposure are widely acknowledged, the potential long-term effects of e-cig usage have only recently begun to receive attention. In this study, we aim to investigate the impact of moderate (1h/day) and chronic exposure to e-cig set at 2 different wattages (i.e. 18W or 30W) in comparison to CS on a range of major physiological parameters. Interestingly, exposure to ECV induced an alteration of lung function but this effect was not related to a chronic lung inflammation as revealed by the data obtained with 30W ECV. Moreover, the impact of ECV on transcriptome, inflammation and lesions within the lung are mostly specific compared to the effect of CS.

Exposure to ECV has some major impact on physiological mechanisms including host metabolism and respiratory function although this latter is restricted to 30W ECV. The impact on host metabolism was evidenced by a lower weight gain in mice exposed to ECV. This effect is similar with both low and high power e-cig and does not seem to be related to blood nicotine levels (Supplementary figure S2). Furthermore, the impact of CS on weight gain was similar to that of ECV, although blood cotinine concentrations were higher in these mice. We also observed a strong effect of 30W ECV on lung function, with a shift in the PV-loop curve towards an emphysematous phenotype associated with a major impact on static compliance and the area of the curve. The presence of emphysema was confirmed by a significant increase in MLI in 30W ECV-exposed mice, close to the effect observed in CS-exposed mice. However, our protocol of CS exposure has a very different effect on the PV-loop curve with no significant effect on compliance and area under the curve. A previous study has reported a similar impact of ECV on lung function with emphysema and alteration of static compliance, an effect that has been shown to be nicotine-dependent [11]. In parallel, we also reported on the same mice that the ECV exposure induced important disturbances of proliferation, inflammation, and permeability in both ileum and colon, which reveals that ECV-induced lesions are not restricted to respiratory mucosa [18].

To determine whether a chronic inflammatory response could be responsible for the altered lung function observed in mice exposed to ECV, we assessed pulmonary and systemic inflammation in these mice by measuring leukocyte recruitment and activation, as well as cytokine concentrations. In contrast to a previous study using an intense ECV exposure (5-6h/day) protocol [11], we did not detect any effect on neutrophil recruitment within the lung after ECV exposure; the number of these granulocytes was even reduced within the spleen. Nevertheless, we observed a transient increase of neutrophil-active chemokine secretion which suggests that neutrophil recruitment requires persistent chemokine production not detectable after chronic exposure to moderate doses of ECV, such as those used in the present study. However, our protocol of exposure using Modbox 30W was associated with higher activation of antigen-presenting cells such as AM and inflammatory monocytes in the BAL and lung tissue although their numbers were similar. This stimulation was associated with the mobilization of NK cells and T lymphocytes within lung tissue even if activation of these cells could not be observed. An activation of CD4⁺ T lymphocytes was only detected in 30W ECV- and CS-exposed mice. Altogether, these data showed that exposure to e-cig induced a specific profile of cell recruitment and activation as compared to CS. Moreover, no clear impact was detected on neutrophil count and phenotype in contrast to CS.

Regarding cytokine production, we observed some upregulation of neutrophil-chemotactic cytokines such as CXCL1, CXCL2 and CXCL17 in the BAL of mice exposed to 30W ECV for 3 months. This transient upregulation was not associated with neutrophil recruitment, probably because chemokine concentrations are too low, or because of a defect in structural cell activation required for leucocyte diapedesis. While IL-17 levels are unaffected by ECV exposure, IL-22 concentrations are higher in the BAL and in the supernatant of unstimulated lung cells from 30W ECV-exposed mice. Although IL-17 has been identified as a key player in the development of CS-induced COPD, IL-22 production seems to be preferentially targeted by ECV. In the present study, the role of IL-22 in the lung injury remains to be determined although some reports show that IL-22 can induce airway remodeling and can also participate in COPD development [19]. Interestingly, spleen cells from ECV-exposed mice produced higher levels of IL-22 after anti-CD3 stimulation. Exposure to ECV seems to induce a mobilization of both antigen presenting cells and T lymphocyte in the spleen at 3 months, followed by a replenishment of NKT cells and T cells at 6 months. Some T cell activation was also detected at 3 months of exposure, as evidenced by modulation of CD25 expression and enhanced capacity to produce IL-22. This suggests that the effect of ECV is not restricted to the lung and could induce some recirculation of immune cells towards the lung and/or intestinal mucosa although we did not observe a cytokine response in the blood.

In parallel with the modulation of the immune response, ECV exposure affects clinical parameters including body weight and lung function with some similarities to the impact of CS. Previous data confirm this impact of ECV in mice exposed in adulthood [20, 21] but also in mice exposed in utero, where both lung dysfunction and structural impairments persist into adulthood. Although the effects observed with our protocol using moderate doses of ECV appear to be close to those following exposure to high concentrations of ECV, the mechanisms underlying these effects are probably different. At high concentrations, ECV induced the recruitment of inflammatory cells such as neutrophils, and the upregulation of metalloproteinases such as MMP9 and MMP12 [11, 21]. At lower concentrations, we reported that chronic exposure to ECV did not promote persistent inflammation and MMP expression whereas this treatment enhances the expression of IL-22, an essential cytokine for the maintenance of the epithelium and remodeling of mucosa [22].

Transcriptomic analyses were carried out to identify the signalling pathways affected by exposure to ECV and attempt to characterize the mechanisms involved in the observed effects of these emissions. The pathways deregulated by CS are deregulated more significantly than by ECV. Unsurprisingly, following exposure to CS, the biological functions most affected are the metabolism of xenobiotics and the inflammatory response. In mice exposed to CS, deregulated genes contribute mainly to the AhR pathway, which is characteristic of a response to exposure to PHAs such as those found in CS [3]. The overexpressed genes code in particular for the cytochromes CYP1A1 and CYP1B1, enzymes that may be involved in the generation of highly reactive intermediate metabolites capable of inducing DNA damage. In contrast, exposure to ECV did not induce gene expression of these two cytochromes, but rather modulated gene expression of aldehyde dehydrogenases (Aldh18a1, Aldh1l2 et Aldh3a1) possibly involved in the metabolism of carbonyl compounds, and most likely in the biotransformation of propylene glycol, one of the main compounds in e-liquids. Chronic exposure to ECV also altered the expression of gene involved in oxidative stress, a mechanism which can explained the oxidative DNA damage in the lung that we previously reported in this experimental model [12]. Notably, the absence of activation of the AhR pathway by ECV is consistent with the very low levels of PAHs measured in these emissions [4]. The involvement of major genes of LXR/RXR pathway, which are also deregulated following exposure to CS and ECV, has already been shown in the exacerbation of macrophage-mediated lung inflammation in cases of CS exposure [23]. In addition, our data show that the number of deregulated genes was greater after exposure to 18W ECV than 30W ECV. These results are surprising given the lower quantity of toxic compounds measured in 18W ECV compared to 30W ECV. Indeed, we had previously shown that the 18W ECV generated levels of carbonyl compounds that are around 2 times lower than for the ECV 30W [4, 24]. This difference in the transcriptomic impact of ECV could be explained by an adaptive mechanism at the higher e-cig power and by the kinetics of the effects, which could differ according to the power of the e-cig. After a short-term exposure (one week), we observed a dose-dependent effect of ECV on transcriptome with more deregulations at the highest e-cig power (data not shown).

In ECV-exposed mice, transcriptomic data revealed deregulation of genes involved in lipid metabolism (Pla1a, Pmvk, Plcb1, Plcb4). This effect could explain the altered metabolism and weight gain in exposed mice, and could also contribute to the observed impairment of lung function in these mice. These genes have already been described as being affected in regular e-cig users, which could reflect the excessive lipid load provided by the glycerol contained in e-liquids. This excessive load could then disrupt surfactant secretion in the lungs and impact lung function [25].

Altogether, our data showed that long term exposure to moderate levels of ECV (1 h / day) can induce some alteration of lung function and host metabolism whereas only limited effect on the inflammatory reaction can be exhibited. Il-22 upregulation is associated with these changes suggesting a physiopathologic role for this cytokine. Our transcriptomic analyses highlighted certain signalling pathways common to exposure to CS and ECV (although the number of genes involved is much lower with ECV), in particular the Th17 pathway involved in the development of chronic inflammation and the progression of COPD. Exposure of C57BL/6 mice to CS for chronic exposure had already demonstrated this increase in Th17 signaling pathway [15, 19, 26]. However, the alteration of IL-22 production had never been demonstrated after exposure to ECV. Overall, the impact on the transcriptome of ECV revealed some alteration in the expression of genes involved in inflammation, oxidative stress, and metabolism of lipids and xenobiotics. These data about ECV associated with its genotoxic effect the lesions in the intestine [12, 18] confirm the urgent need of information in the general population about regular use of e-cigarette and the necessity to complete by studying the consequences of e-cigarette smoking in the general population even at moderate levels.

Availability of data and materials:

The dataset(s) supporting the conclusions of this article are available within the manuscript or supplementary information files.

Acknowledgements

We acknowledge Dr Martin Figeac from the Functional and Structural Platform (CHU Lille, France) for transcriptomic analyses and Dr OLivier Molendi-Coste and Hélène Bauderlique from the Bicell platform (Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, US 41—UAR 2014—PLBS, 59000 Lille, France) for the flow cytometry analysis. We also thanks the NIH facility for providing murine iNKT tetramer.

Conflicts of Interest:

The authors declare they have no conflicts of interest related to this work to disclose.

Funding

This work benefited grant from the IRESP (Institut de recherche en Santé Publique) and the French National Cancer Institute (INCa): Contract No. INCa_11505.

Ethics Approval and Consent to Participate

This study does not involve human participants.

Animal procedures were in agreement with European directive 2010/63/EU for the protection of animals used for scientific purposes and obtained the Ethical Committee on Animal Experimentation of the Hauts de France (CEEA 75) approval (ref APAFIS 10363-2017062615002072v2).

Author information

Author notes : Nothing to declare

Authors and Affiliations

¹Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 9017, Center for Infection and Immunity of Lille, Lille, France.

²Univ. Lille, CHU Lille, Institut Pasteur de Lille, ULR 4483, IMPECS - IMPact de l’Environnement Chimique sur la Santé, F-59000, Lille, France

³Univ. Lille, CNRS, Inserm, CHU Lille, UMR9020 - UMR1277 - Canther - Cancer Heterogeneity, Plasticity and Resistance to Therapies, 59000 Lille, France

Contributions

Conceptualization, PG, JMLG, SA.; methodology, LM, AO, JMLG, SA, JK, AP, MP and PG.; formal analysis, LM, AO, RD, JMLG, SA, PG.; investigation, LM, AO, RD, GK, JMLG, SA, JK, AP, MP and PG.; writing-original draft, LM, AO, RD, JMLG, SA, PG.; writing-review and editing, all authors.; supervision, PG and SA.; funding acquisition, JMLG, SA, JK, AP, MP and PG. All authors have read and agreed to the published version of the manuscript.

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Vaping versus Smoking: A Quest for Long-term impact in a mouse model

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

E-cigarettes and conventional cigarette

Sample Collection

Broncho-alveolar lavage procedure, lung and spleen processing

Flow cytometry

Cytokine Assay

Lung Histology

Lung function

Transcriptomic analyses

Statistical analyses

Results

1- Long term e-cigarette exposure impairs clinical parameters

a- Subchronic exposure (3-month treatment)

b- Chronic exposure (6-month treatment)

2- E-cigarette vapors induce pulmonary inflammation

a- Subchronic exposure (3-month treatment)

b- Chronic exposure (6-month treatment)

a- Subchronic exposure (3-month treatment)

b- Chronic exposure (6-month treatment)

4- Impact of e-cigarette on lung transcriptome

a- Subchronic exposure (3-month treatment)

b- Chronic exposure (6-month treatment)

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1