Chronic smoke but not ENDS exposure induces lung inflammation and emphysema. We and others have shown that chronic cigarette smoke exposure activates innate and acquired immune cells in the lung and causes emphysema (26). To determine if ENDS exposure leads to similar outcomes, we first compared the lung-specific immunological effects of chronic ENDS exposure with exposure to conventional cigarette smoke. Compared with 4 months of cigarette smoke, C57BL/6J mice exposed to the same amount of nicotine delivered via ENDS (ENDS-nicotine) and those receiving ENDS without nicotine (ENDS-vehicle) showed no airway inflammation (Figure 1A). As expected, mice exposed to chronic cigarette smoke developed emphysema marked by an increase in lung volume, loss of the alveolar septa, and increased elastolytic enzymes (Figure 1, B–D). In contrast, histological assessment of the lungs in ENDS-exposed groups showed no evidence of tissue destruction when compared with the smoke-exposed group (Figure 1B); lung volume measurements with micro-computed tomography (microCT) revealed an increased lung volume in smoke-exposed mice, but not ENDS-exposed mice (Figure 1C). Consistently, matrix metalloproteinase 12 (Mmp12) expression was highly induced in smoke-exposed mice but remained unchanged in the Air-control and ENDS-exposed mice (Figure 1D).

Figure 1 Four-month exposure to ENDS does not induce inflammation in the lung. Mice were exposed to room air (Air), cigarette smoke (Smoke), ENDS-vehicle vapor, or ENDS-nicotine vapor for 4 months and the immune profiles of the lung were quantified. (A) Differential BAL cell numbers for macrophages, neutrophils, and lymphocytes in the airway (n = 5 per group). (B) Histological analysis of lung tissue following 4-month exposure. Representative micrographs of H&E staining. Scale bars: 50 μm. (C) MicroCT quantification of total lung volume (n = 5 or 6 per group). (D) BAL cell expression of RNA transcript for matrix metalloproteinase 12 by qPCR (n = 5 or 6 per group). (E) IL-17A, IL-6, and TNF-α concentrations from mouse lung homogenate measured by multiplex assay (n = 4 or 5 per group). (F) Representative and (G) cumulative flow cytometric analysis of live, CD11b+F4/80–Ly6G–CD11c+MHCII+ dendritic cells. Numbers in the upper-right corner indicate percentage positive cells for the markers (n = 5 or 6 per group). (H) Representative and (I) cumulative flow cytometric analysis of live, CD3+CD4+RORγt+IL-17A+ T lymphocytes. Numbers in the upper-right corner indicate percentage positive cells for the markers (n = 5 or 6 per group). Significance was determined by Student’s t test or 1-way ANOVA with Bonferroni’s correction for multiple comparisons. ***P < 0.001, **P < 0.01, *P < 0.05. All data shown are representative of 4 or more independent 4-month experiments with n = 5 or 6.

We and others have previously demonstrated that cigarette smoke recruits proinflammatory CD11b+CD11c+ conventional dendritic cells (cDCs) to the lung, which are critical in the differentiation of T helper 17 (Th17) cell responses in experimental emphysema (27). Therefore, we next sought to determine whether long-term ENDS exposure could induce cytokines that promote recruitment of cDCs and induction of Th17 cells. We found that chronic smoke increased IL-6, TNF-α, and IL-17A concentrations in the lung, whereas ENDS exposure (with or without nicotine) did not increase any of the same inflammatory cytokines (Figure 1E). Further, flow cytometric analysis of lung tissue homogenate revealed increased recruitment of both Th17 cells and CD11b+ cDCs in response to smoke but not in the ENDS-exposed mice (Figure 1, F–I). To better understand the immunomodulatory consequences of ENDS exposure, we next evaluated lung cytokine and chemokine profiles from mice exposed to ENDS (with and without nicotine) and Air and found similar lung cytokine profiles among the 3 groups of Air-exposed and ENDS-exposed mice (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI128531DS1). Cytokines associated with Th2, Th1, or regulatory T cells (Tregs) in the lung homogenates were not significantly different in ENDS-exposed groups when compared to Air-exposed controls (Supplemental Figure 1A). Further, no distinct induction of chemokines was observed in the airways of END-exposed mice (with or without nicotine) (Supplemental Figure 1B). Together, these data suggest that chronic ENDS exposure in C57BL/6J mice does not promote notable inflammatory responses in the lung and does not induce emphysema.

ENDS exposure alters lipids in lung macrophages and ATIIs. Mice exposed to ENDS failed to exhibit a distinct inflammatory signature; however, we found a unique morphological feature in alveolar macrophages isolated from the BAL fluid of mice exposed to ENDS when compared with the Smoke or Air control groups (Figure 2A). Notably, interrogation of multiple pathways associated with autophagy (Bcln1, Atg7, Atg14) or proapoptotic (Bax, Bad) genes failed to reveal any significant upregulation of relevant genes (Supplemental Figure 2, A and B). Further, lactate dehydrogenase (LDH) concentrations in the BAL, a marker of cytotoxicity and cell death, were not increased in the ENDS-exposed groups (Supplemental Figure 2C). We next examined whether the observed cytoplasmic inclusions in macrophages contained lipids. Oil Red O staining of ENDS-exposed macrophages showed increased lipid accumulation independent of nicotine, a feature not observed in the Air- or Smoke-exposed groups (Figure 2B). Quantification of lipid in alveolar macrophages demonstrated a significant increase among both ENDS-exposed groups when compared with Air controls (Supplemental Figure 3). To determine whether the observed lipid accumulation was derived from inhaled vaporized glycerol present in ENDS, we next quantified the VG (glycerol) content in the BAL cellular fractions and found that ENDS-exposed groups did not exhibit an increased concentration of intracellular glycerol (Supplemental Figure 4), indicating that the accumulated lipid might be arising through an endogenous, rather than exogenous, source.

Figure 2 Lipids accumulate in alveolar macrophages in chronic ENDS exposure. Mice were exposed to Air, Smoke, ENDS-vehicle, or ENDS-nicotine for 4 months. Airway immune cells were then acquired by BAL and were cytocentrifuged onto glass slides. (A) Representative H&E staining of the cytospin preparations reveals intracytoplasmic inclusions in the ENDS-vehicle and ENDS-nicotine groups (black arrows). Scale bar: 10 μm. (B) Representative Oil Red O staining of cytospin preparations reveals lipid accumulation in ENDS-vehicle and ENDS-nicotine groups. All data shown are representative of 3 or more independent 4-month experiments with n = 4 or 5 per group. Scale bar: 25 μm.

We next used transmission electron microscopy (TEM) and found numerous lipid aggregates in the cytoplasm of macrophages isolated from ENDS-exposed mice (Figure 3, A and B). Furthermore, we found an increase in the number of lysosomes in ENDS-exposed macrophages (with or without nicotine), which we confirmed with immunohistochemical staining for lysosomal-associated membrane protein 1 (LAMP-1) (Figure 3B and Supplemental Figure 5). We next examined the lamellar bodies, the specialized secretory vesicles in ATIIs that facilitate the release of the lipid constituents of pulmonary surfactant into the alveolar airspace. Similarly, the lamellar bodies in the ENDS-exposed groups also demonstrated distinct morphological changes, when compared with the Air-exposed counterparts (Figure 3, C and D). Although the absolute number of lamellar bodies was unaltered, the numbers of poorly organized, irregular lamellar bodies were increased in both ENDS-treated groups, suggesting an alteration in ATII surfactant homeostasis had occurred (Figure 3E). Collectively, these data indicate that ENDS vapor exposure disrupts lipid homeostasis in alveolar macrophages and ATIIs, warranting further systematic assessment of the airway’s lipidome.

Figure 3 TEM imaging of lipid inclusions in alveolar macrophage and alveolar type II pneumocytes. Following 4 months of exposure, lungs from Air, ENDS-vehicle, and ENDS-nicotine groups were fixed and processed for electron microscopic analysis. (A) Representative micrographs of alveolar macrophages demonstrating lipid inclusions and increased presence of lysosomal compartments in ENDS-vehicle and ENDS-nicotine groups (white arrows). Scale bars: 2000 nm. 80 kV high voltage. (B) Higher magnification of the lipid inclusions (right) and lysosomes (left) observed in ENDS-vehicle and ENDS-nicotine groups. Scale bars: 200 nm. 80 kV high voltage. (C) Representative micrographs of alveolar type II pneumocytes (ATIIs) demonstrating normal, uniform lamellar bodies in AIR-exposed mice (red arrow) and atypical lamellar body structures in ENDS-vehicle and ENDS-nicotine groups (white arrows). Scale bars: 2000 nm. 80 kV high voltage. (D) Higher magnification of the representative lamellar bodies observed in AIR, ENDS-vehicle, and ENDS-nicotine groups. Scale bars: 500 nm. 80 kV high voltage. (E) Blinded quantification of atypical lamellar bodies observed within ATIIs. The quantified results are expressed as the percentage of atypical lamellar bodies per total lamellar body count in each cell (mean ± SEM). n = 5 or 6 per group. Each data point represents a single ATII, all of which were located and imaged by scanning 3 or more independent mounted grids per experimental group. Significance was determined by Student’s t test. *P < 0.05.

ENDS-exposed mice accumulate phospholipid in BAL cells. We next used an unbiased, mass spectrometry–based lipidomic approach to assess how ENDS vapor exposure disrupts the lipid landscape in the lungs. We have found that alveolar macrophages constitute over 98% of the BAL fluid cell composition in mice exposed to 4 months of ENDS-vehicle, ENDS-nicotine, or Air control (Figure 1A and Supplemental Figure 6, A and B). Therefore, we next examined the lipidomic changes in the BAL cells in the same exposed groups of mice. Independent of nicotine exposure, we found an increase in cellular phospholipid species in the ENDS-treated groups, including the phosphatidylcholine-, phosphatidylserine-, and phosphatidylethanolamine-based lipids, with an enrichment of disaturated phospholipids and cholesterol esters (Figure 4A). These findings were specific because our comprehensive analyses of other classes of lipids (e.g., triglycerides) did not show significant alterations (Supplemental Figure 7). The observed shifts in cellular phospholipids from the lipidomic analysis were further validated by quantification of total cellular phospholipids in BAL fluid cells isolated from a separate cohort of mice exposed to chronic ENDS. Intracellular phospholipids were significantly increased in the ENDS-nicotine–treated group. (Figure 4B). To determine whether ENDS exposure altered pathways associated with lipid maintenance and clearance, we next examined gene expression patterns of lipid-associated enzymes and transport proteins. We found that expression of Abca1, a transport molecule responsible for removal of excess intracellular cholesterol and phospholipid, was reduced in ENDS-exposed groups compared with Air controls (Supplemental Figure 8A). A similar trend was also observed in the expression of Abcg1, a sterol efflux protein that works in conjunction with ABCA1 to maintain lipid balance in alveolar macrophages (Supplemental Figure 8A). Together, our findings suggest that ENDS-exposed, alveolar macrophages show defects in complex lipid processes in the airway, which may, in part, promote the accumulation of distinct lipid species.

Figure 4 ENDS exposure independent of nicotine increases phospholipids in BAL cells. (A) Heatmap demonstrating the upregulated phospholipids in ENDS-vehicle and ENDS-nicotine groups (fold change > 1.5) from the lipidomic analysis conducted on the BAL cells. Heatmap values represent averages from the 3 pooled samples per group. Changes shown are relative to the Air controls. (B) Quantification of total intracellular phospholipid content in pelleted BAL cells. n = 5–7 per group. The quantified results are expressed as means ± SEM. Significance was determined by Student’s t test and corrected for multiple comparisons. *P < 0.05. CE, cholesterol ester; TG, triglyceride; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol.

Surfactant-associated lipid species are increased in ENDS-exposed lungs. To acquire insight into the mechanisms responsible for altered intracellular lipid biosynthesis, we next examined the lipid profiles in the acellular compartment of the BAL fluid. This analysis revealed that ENDS-exposed groups have a distinct increase in phospholipid species with a concomitant decrease in neutral triglycerides (Figure 5A). The acellular fractions from ENDS-exposed mice further showed significant increases in saturated phospholipids with concurrent decreases in phospholipids bearing more double bonds (Figure 5, B and C). To ascertain whether the observed shifts in the lipid profiles of ENDS-exposed mice were related to an increase in pulmonary surfactant, we next identified and quantified lipid species that are most prominent in surfactant from the lipidomics data set: dipalmitoyl-phosphatidylcholine (DPPC), myristoyl-palmitoyl-phosphatidylcholine (MPPC), palmitoyl-stearoyl-phosphatidylcholine (PSPC), and palmitoyl-palmitoleoyl-phoshpatidylcholine (PPoPC) (Figure 5D). These phospholipids are estimated to constitute over 40% of the phospholipid compartment of the surfactant layer and primarily function to reduce alveolar surface tension (28, 29). We found that independent of nicotine, ENDS exposure significantly increased the concentration of several of these phospholipids, including MPPC and PSPC. Together, these findings show that chronic ENDS vapor exposure disturbs phospholipid homeostasis in the surfactant layer, resulting in a distinct increase in the alveolar phospholipid pool.

Figure 5 ENDS exposure independent of nicotine increases disaturated phospholipid pools in BAL fluid. (A) Heatmap depicting the upregulated phospholipid species in the ENDS-vehicle and ENDS-nicotine groups from the lipidomic analysis conducted on the BAL fluid. n = 3 pooled samples per group. (B and C) Quantification of BAL fluid phospholipids based on saturation of the lipid acyl groups. n = 3 per group. The quantified results are expressed as percentage of total lipid signal (mean ± SEM). DB, double bond. (D) Quantification of known surfactant-associated species in BAL fluid. n = 3 per group. The quantified results are expressed as percentage of total lipid signal (mean ± SEM). Significance was determined by 1-way ANOVA with Bonferroni’s correction for multiple comparisons. *P < 0.05. CE, cholesterol ester; TG, triglyceride; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol; SM, sphingomyelin; MG, monoacylglycerol; MGDG, mono/diacylglycerol.

To further characterize the effect of ENDS vapor on the biosynthetic function of ATIIs, we assessed the expression of proteins that are critical for the assembly and transport of pulmonary surfactant. We found that Abca3, a type II–specific lipid transport protein that aids in the building of lamellar bodies, was upregulated in response to ENDS-vehicle, with ENDS-nicotine demonstrating an additive effect (Supplemental Figure 8B). We also found that expression of Pcyt1a and Lpcat1, genes for the phosphatidylcholine-synthesizing enzymes CTP:phosphacholine cytidylyltransferase and lysophosphatidylcholine acyltransferase 1, were upregulated in ENDS-nicotine–exposed groups (Supplemental Figure 8B).

The cytokine granulocyte macrophage colony–stimulating factor (GM-CSF) plays an important role in macrophage physiology and lipid metabolism (30). Therefore, we next sought to determine if expression of GM-CSF is altered in mice with abnormal lipid homeostasis in response to ENDS exposure. Interestingly, we found no significant alteration in GM-CSF protein levels from whole lung homogenate, GM-CSF receptor (Csf2ra), or PU.1 expression, a downstream transcription factor that regulates expression of proteins necessary for surfactant maintenance and catabolism in BAL fluid cells (Supplemental Figure 9, A–C). Together with the disruption in lipid homeostasis, these data suggest that chronic ENDS exposure alters the surfactant-related lipid output in the terminal airway and disrupts key metabolic pathways by which alveolar macrophages process and catabolize the lipids at the alveoli’s air-liquid interface in a manner that is independent of GM-CSF.

ENDS exposure alters innate immune functions of ATIIs and lung macrophages. Although ENDS exposure resulted in increased abundance of lipid species associated with the surfactant proteins, mice receiving ENDS showed significantly reduced SP-D concentrations in the BAL fluid (Figure 6A). Furthermore, gene expression analysis revealed that mRNA transcripts for 2 opsonins, Sfptd and Sfpta, were significantly reduced in the lung homogenates from ENDS-exposed mice when compared with Air controls (Figure 6B). Notably, these findings were specific because no significant changes were found in Sfptb and Sfptc (Figure 6C). Together, this demonstrates that ENDS vapor exposure impairs production of an essential class of opsonins in the distal airway.

Figure 6 Four-month exposure to ENDS vapor reduces the expression of surfactant proteins. (A) Quantification of SP-D by ELISA from BAL fluid. n = 4 or 5 per group. (B and C) Relative gene expression of surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D) from whole lung homogenate. n = 4 or 5 per group. The quantified results are expressed as means ± SEM. Significance was determined by 1-way ANOVA with Bonferroni’s correction for multiple comparisons. **P < 0.01. NS, not significant.

Using the same exposure protocol, we found that lipids are deposited in lung macrophages as early as 2 weeks (Supplemental Figure 10). Therefore, to examine the early immunological effects of ENDS vapor exposure on lung macrophages, we isolated F4/80+ macrophages from the whole lung tissue of mice after 1 month of exposure. We found similar cell viability and expression of M2 polarization markers (e.g., Arg1) in ENDS-exposed F4/80+ lung macrophages when compared with Air-exposed controls (Figure 7, A and B). In contrast, lung macrophages showed significantly reduced M1-associated markers, including Nos2 (Figure 7C), proinflammatory cytokines (Il1b and Tnfa) (Figure 7D), B7 costimulatory molecules (Cd80 and Cd86) (Figure 7E), and Tlr7, a pattern-recognition receptor that recognizes single-stranded RNA elements from viral species (Figure 7F).

Figure 7 One-month exposure to ENDS vapor attenuates lung-resident macrophage function. Lung-resident F4/80+ macrophages were isolated from whole lung tissue using magnetic beads following 1 month of exposure. Cells were cultured for 24 hours following isolation and supernatants and cells were harvested for the analyses. Individual data points represent technical replicates from pooled lungs of 4 mice per treatment group. (A) Absorbance values following the colorimetric, lactate dehydrogenase (LDH) cytotoxicity assay from 24-hour cultures of Air-, ENDS-vehicle–, and ENDS-nicotine–exposed groups. (B and C) Relative gene expression for Arg1 and Nos2 derived from RNA samples acquired from the cells after 24-hour culture. (D–F) Relative gene expression for (D) cytokines Tnfa and Il1b, (E) costimulatory molecules Cd86 and Cd80, and (F) viral recognition receptor Tlr7, derived from RNA samples in each treatment group acquired from the cells after 24-hour culture. (G) Relative gene expression for the transcription factor Irf7, a critical factor for type I IFN production, derived from RNA samples acquired from the cells in each treatment group after 24-hour culture. Cells were treated with either polyinosinic:polycytidylic acid (poly I:C) at a concentration of 10 μg/mL or PBS vehicle. All quantified results are expressed as means ± SEM. n = 4 or 5 per group. Significance was determined by 1-way ANOVA with Bonferroni’s correction for multiple comparisons. ****P < 0.0001, **P < 0.01, *P < 0.05. All data shown are representative of 3 or more independent 1-month experiments with n = 4 or 5 per group. NS, not significant; ND, none detected.

To determine the functional significance of reduced M1-associated cytokines and molecules, we next used polyinosinic:polycytidylic acid (poly I:C), a synthetic analog of viral dsRNA, to stimulate macrophages isolated from the lungs of ENDS-exposed mice. Consistently, in response to poly I:C, the macrophages from ENDS-exposed groups showed reduced expression of interferon (IFN) response factor 7 (Irf7) (Figure 7G). IRF7 is a master transcription factor that governs type 1 IFN induction in response to viral stimuli, which is critical for rapid antiviral immunity (31). This reduction, along with the decrease in M1-associated markers, strongly indicates that one potentially important physiological consequence of ENDS vapor exposure is an impaired response to inhaled viruses.

ENDS-exposed mice exhibit delayed immune responses to influenza. To further examine the translational relevance of reduction of M1-associated markers, impaired macrophage responses to poly I:C, and surfactant dysregulation in mice exposed to ENDS vapor, we next assessed defenses against acute viral infection. We have previously shown that mice exposed to chronic cigarette smoke, before sublethal infection with influenza A, fail to mount the appropriate antiviral responses and exhibit increased morbidity (32). In a similar manner, mice were exposed to Air (control), chronic ENDS-vehicle (PG/VG), or ENDS-nicotine for 3 months and were subsequently challenged with the influenza A virus (45 TCID 50 /mouse). Mice receiving ENDS responded poorly by day 10 following infection, and the ENDS-vehicle group demonstrated a significant increase in mortality when compared with Air controls (Figure 8A). To better understand the effects of ENDS on the recovery from acute infection, we next exposed mice to ENDS vapor for 3 months and infected with a sublethal dose of influenza A (20 TCID 50 /mouse). Compared with the Air group, ENDS-nicotine–exposed mice showed significantly impaired responses to influenza A, as detected by augmented weight loss within 8 days following infection (Figure 8B). As expected, ENDS-exposed macrophages in the BAL showed the same lipid-filled morphology (Figure 8C). The Air-exposed control group showed resolution of inflammation and had regained some of the normal lung architecture at day 14; however, histological evaluation of the lung parenchyma in ENDS-exposed mice showed increased lung inflammatory cells and edema with significant distortion of the lung tissue and increased hemorrhage in ENDS-exposed mice when compared with the Air–exposed mice that were infected with flu (Figure 8, D and E). Consistent with the increase in inflammatory infiltrates in the lungs, there was persistent IFN-γ expression in the lungs of ENDS-nicotine–treated groups, and TNF-α in the ENDS-vehicle–exposed mice (Figure 8F). We further found increases in total and HA-specific IgG titers in the lung homogenate of ENDS-exposed mice 14 days following viral infection (Figure 8, G and H). Together, our data demonstrate that exposure to ENDS vapor reduces innate immune responsiveness, and long-term exposure impairs the ability of mice to control pulmonary infection with influenza.