Multi-omic assessment shows dysregulation of pulmonary and systemic immunity to e-cigarette exposure

The use of electronic nicotine delivery systems (ENDS, also called e-cigarettes or vaping) has surged in recent years, with over 10 million adults and 3 million adolescents actively using ENDS [1]. The FDA commissioner has used the term “epidemic” levels to describe the ubiquity of vaping. However, a recent spate of hospitalizations and deaths in 2019 due to Ecig or vaping-associated acute lung injury (EVALI), as well as combined use with traditional cigarettes, suggests that ENDS may pose unanticipated risks to lung health [2]. While new products and components are being incorporated into vaping products, very little is known about specific effects of vaping on lung function, particularly impacts to immune function.

It is well known that inhaled toxicants including traditional cigarette smoking have significant impacts on immunity, including damage to respiratory epithelium [3, 4], as well as increased chronic inflammation with increased susceptibility to viral and bacterial respiratory infections [5]. Vaping has been associated with increased risk of COVID-19 [6‐8], and experimental evidence shows that vaping in animal models increases vulnerability to influenza A virus (IAV) [9], and predisposes airway epithelial cells to bacterial infection [10]. However, the precise effects of vaping and ENDS liquids on immune cells and immune function remain understudied.

ENDS products predominately use a base carrier propylene glycol (PG) plus vegetable glycerin (VG) to act as one of the most common carriers for vaping liquids [11‐14]. PG and VG are also widely used in the food and cosmetics industries, and effects of PG and/or VG on multiple biological systems including renal and respiratory function have been documented [15‐21]. PGVG is used in combination with cutting agents such as Vitamin E acetate (VEA), which has been identified as the likely causal driver of EVALI, and thus its use mostly discontinued [1, 2, 22, 23]. Alternatives to VEA include Phytol which is a diterpene alcohol that may naturally exist in cannabis products in small amounts, but it was an inexpensive diluent that many manufacturers of cannabis products were considering incorporating into products as a principal ingredient and was found in counterfeit cartridges [24]. An initial study highlighted significant pulmonary toxicity from phytol in rats and, for the most part, this finding quickly circulated to manufacturers who removed phytol from products [25]. That study used a high concentration incorporation of phytol and observed overt morbidity and mortality. In the instance of EVALI, vitamin E acetate has similarly been shown to induce severe lung inflammation in rodent models [22, 26]. While these products have been appropriately removed from commercial products, it remains unclear how these chemicals promote lung injury and, thus, what other similar chemicals may also be unsafe for general inhalation. Agents continuously change, but PGVG remains more consistent, and a better understanding of potential effects of base vaping compounds is needed.

In this study, we developed an exposure system to expose animals to PGVG vapor to study both system as well as lung specific effects of vaping exposure. Using a combination of transcriptome, proteome, metabolome, lipidome, as well as flow cytometry analyses of immune cell populations, we aim to identify novel pathways that are impacted by vaping exposure to PGVG as well as the addition of phytol. We find that these vaping exposures have both lung-specific as well as systemic effects, suggesting that vaping may have broad consequences for immune function.

As Ecig use has become more widespread, the term “epidemic” has been applied to the ubiquitous use of Ecigs. Many users switch from nicotine products to Ecigs believing vaping to be a safer alternative, but little is actually known about the effects of vaping on physiological processes. The United Kingdom has largely deemed vaping as a safer alternative to conventional smoking. However, vaping is difficult to study due to the large number of sometimes unknown additives that are contained within unregulated vaping products.

The recent outbreak of vaping-associated acute lung injury (EVALI), traced to the additive Vitamin E acetate, demonstrated the potential harm posed by vaping components. In this study, we focused on the effects of exposure to the base fluid contained in many vaping systems, PGVG, as well as a novel terpene additive, phytol found in counterfeit cartridges [24]. We find that PGVG and PGVG + 1% phytol elicits both lung-specific and systemic effects on multiple systems, including metabolites, lipids, and specific subsets of immune cells. Our results suggest that PGVG with and without phytol altered lipidomic, metabolomic, and transcriptomic profiles of multiple systems and even modified immune cell populations outside the lung. These results agree well with previous published studies showing that PGVG alone can alter lung function, lipid composition and T cell subsets [27, 28]. However, when put into the context of systemic and multi-omic pathway integration, the effects of PGVG alone are quite modest. Namely, PGVG alone did not produce a statistically significant multi-omic pathway.

Additives or alternative ingredients—such as phytol added to the base liquid PGVG in vaping products—can combine for unpredictable effects, as was the case for vitamin E acetate [22, 23]. The addition of phytol modified the lung-omic profiles relative to PGVG alone, but several common outcomes were noted. Both PGVG and PGVG + 1% phytol upregulated AMP and downregulated proline, although they appear to have opposing effects on isobutyric acid, with PGVG increasing isobutyric acid levels while PGVG + phytol decreased levels. Changes in gene transcription and metabolites were quite different between PGVG alone and PGVG + phytol, and the different effects on weight gain between PGVG and PGVG + phytol are likely to result from systemic metabolic and transcriptomic effects. Connective tissue growth factor (CTGF), known for its involvement in wound healing and pathological lung remodeling [30, 31], was elevated in both exposure groups. On the other hand, 1% phytol led to a significant upregulation of MUC5B, a regulator of mucin production in the airways important for mucociliary clearance and associated with fibrosis in the lung [32, 33]. In contrast, while we found PGVG did not affect MUC5B, a previous study showed increased Muc5ac in lungs of vaping exposed animals [28], suggesting that additives and exposure conditions may affect similar lung repair pathways. PGVG did significantly change multiple lipid species including many ceramides, Phosphatidylcholine (PCs), and phosphatidylethanolamine species (PEs), while 1% Phytol + PGVG exposure evinced only modest alteration of lipids. Ceramide increases have been associated with lung injury, particularly COPD [34, 35], while PEs and PCs are components of lung surfactant, critical for alveolar expansion for gas exchange [34‐38]. Lipids have been found to be changed in the plasma of a small group of Ecig users, including similar lipid species to those identified in our studies [39]. These results point to a complex interplay and unpredictable interactive effects between additives such as phytol with base liquids. Complex addition of nicotine, cannabinoids, flavorings, etc., has the potential to affect multiple physiological systems, including metabolic and immune systems, in a manner that is currently difficult to predict.

Little is known about the novel diterpene alcohol phytol found in Ecig products [24]. An extremely high dose (100%) of phytol was shown to be lethal to rats [25], but in our studies we find that even at a very low dose, 1%, phytol can induce significant changes to lung transcription, metabolites, and even function. Using analysis combining our results showing changes in transcriptomics, lipidomics, and metabolomics, we find that phytol increases acetylcholine. Acetylcholine is a potent endogenous bronchoconstrictive agent [40‐43]. However, we find that 1% phytol possibly decreases resistance (non-significant trend) and increases lung compliance. Given the extent of lipidomic changes seen in the present study and elsewhere [9], increased acetylcholine could reflect compensatory mechanisms in vaping-exposed lungs to offset physicochemical changes in the airway surfactant. Furthermore, the upregulation of bradykinin receptor B1 could indicate a request for additional vasodilation and/or bronchoconstriction. Through the linkage to a downregulated palmitic acid, the effects observed could partially describe an inappropriate response to pathogens during sterile conditions, and an increased vulnerability to pathogen insult.

We also show phytol had significant effects on immune genes, including regulators of immune cell migration (CCL19, CCR7), inflammation (IL-1β), and immune regulation (TIMP1). Both PGVG and PGVG + phytol changed the circulating CD4 + T cell population, with decreased numbers in the blood and increased numbers in the spleen, both in total CD4s as well as CD44hi and CD25hi populations. Changes to the CD4 T cell subset were also previously observed in animals exposed to VEA in an EVALI model, suggesting that Ecig exposure may broadly affect the CD4 T cell response [44]. Interestingly, while we find no changes in overall immune subsets in the lung, one previous study did show some change in CD19 + and CD4 + cells in the lung [27]. These results suggest that changes to metabolites, lipids, and gene products in the lung may translate into systemic changes, including systemic changes to immune cell subsets. Such alterations to the systemic T cell population may only manifest under the circumstance of a lung immunological challenge (i.e., a respiratory pathogen). Epidemiological data have been reported showing individuals who vape exhibit increased susceptibility to infectious disease [6, 45]. Our results provide a potential mechanism tying exposures in the lung to systemic changes in immunity.

These results must be put into context with the operating conditions, which included a specific Ecig device, coil, and wattage. Sub-ohm vaping is not an uncommon practice among users but is more prone to high temperatures at the coil, which can generate numerous pyrolysis products [46, 47]. We endeavored to keep the puff topography (short puffs, longer intervals between puffs) to avoid overheating conditions, however, we did not explicitly measure products like metals, acrolein, or formaldehyde that could have been generated. Furthermore, additives including triacetin can be found in Ecig liquids, particularly in devices that contain THC or cannabis [48, 49], which can also lead to formation of acrolein and formaldehyde upon vaping [50]. Our addition of phytol was a simple individual study permutation that clearly altered the response phenotype. We predict that numerous other ingredients and aerosol generating conditions can also change the responses.

Changes to lipids, metabolites, and genes can lead to a multitude of downstream effects, but in the present study we have limited phenotyping showing modest alterations of lung function. Lung disease is often multifactorial, with even pathologies from long-term cigarette smoking being heavily influenced by genetics and lifestyle. The mulit-omic alterations observed in response to PGVGV and PGVG + phytol may simply amount to moderate homeostatic adjustments to an altered chemistry of the airway lining fluid. The data are concerning, however, with respect to the complex differences with a small amount of terpene added to the base, and in consideration of systemic immune cell population changes. Long-term Ecig use overlaid with a vulnerable genetic profile or in the face of a second hit (e.g., pathogens or toxicants), may lead to more pathological alterations to respiratory health. While these remain to be determined, our study demonstrates the importance of testing the effects of specific compounds in combination due to the complex interplay of toxicological exposures with biological processes.