Lung Macrophages Accumulate Novel Cytosolic Inclusions With Vaping: A Case Report From an Asymptomatic Habitual Vaper

DA Armstrong (  David.A.Armstrong@hitchcock.org ) Dartmouth-Hitchcock Medical Center https://orcid.org/0000-0003-1748-2520 Radu V Stan Dartmouth College Geisel School of Medicine John Dessaint Dartmouth-Hitchcock Medical Center Daniel S Aridgides Dartmouth-Hitchcock Medical Center Diane L Mellinger Dartmouth-Hitchcock Medical Center Haley F Hazlett Dartmouth College Geisel School of Medicine Xiaoying Liu Dartmouth-Hitchcock Medical Center Alix Ashare Dartmouth-Hitchcock Medical Center


Background
The electronic nicotine delivery system (ENDS), entered the US market in 2007 and rapidly gained popularity among both tobacco smokers and non-smokers (1)(2)(3)(4). E-juice, the liquid medium used in ENDS devices, contains one or more of the following chemicals: nicotine, THC, propylene glycol (PG) and vegetable glycerin (VG). In addition, polyethylene glycol, vitamin E acetate, or triglycerides are often added to e-juice to impact its viscosity (5). Vaping has short-term detrimental health effects on the lungs in certain individuals, a condition known as e-cigarette/vaping product use-associated lung injury (EVALI), and will likely have chronic effects that will not fully be realized for decades. Numerous EVALI case reports highlight alterations in lung macrophages (LMs) and suggest that the innate immune system may be affected in vape users (6)(7)(8). However, there are presently no studies in habitual vapers to examine/characterize pulmonary innate immune cells. We present a novel macrophage phenotype observed in BAL-derived LMs isolated from an asymptomatic habitual vaper.

Case Presentation
We present a case of an asymptomatic 25-year-old female frequent vape user with no underlying respiratory disease. The subject reported that she has been vaping for approximately 3.5 years with a daily consumption of 2 milliliters of e-juice including use of both nicotine-based and THC-containing products. The device she currently uses is SMOK brand, but the subject has also used JUUL brand. This subject is a previous cigarette smoker (3 years duration).

Methods
This study was approved by the Dartmouth-Hitchcock Institutional Review Board (#22781). Subject underwent exible bronchoscopy as previously described (9). Bronchoalveolar lavage (BAL) uid was obtained from tertiary airways via instillation of 20 ml of sterile saline followed by 10 ml of air and repeated for a total of 5 times per airway. LMs were isolated as previously described (9,10). Cells were stained with Oil Red O (ORO) to assess lipid accumulation (11). Cellular ultrastructure was examined by transmission electron microscopy (TEM). Cells prepared for TEM from a healthy non-smoking 27-year-old male were used for ultrastructural comparison.

Results
A heterogeneous distribution of ORO stain was seen across the LM cell population ( Figure 1A Variable density inclusions were not observed in LMs of the non-smoking subject.

Discussion
The long-term effects of habitual vaping on pulmonary immune cells is unknown. Macrophages are among the rst cells in the lung to respond to inhaled particles or pathogens. To explore possible phenotype alterations to LMs with vaping, we examine the cytology and ultrastructure of LMs from BAL uid of a habitual vaping device user. We have identi ed a novel cytosolic accumulation of heterogeneous electron density which we refer to as a VIB, a vaping-associated inclusion body.
Historically, cytology-based studies utilizing Oil Red O staining have been used to demonstrate the presence of lipid-laden macrophages in the lung (7,11,14,15) There are limited ultrastructural studies showing lipid-like cytosolic inclusions in lung macrophages from BAL uid. M. tuberculosis infection studies (16,17), mineral oil-exposure related (18,19) or petroleum aspiration(20) reports come closest to describing similar inclusions, nonetheless these structures are clearly not the heterogeneous structures we have observed here in the vaping subject.

Conclusion
This is the rst report describing lung macrophage ultrastructure associated with habitual vape product use. Most notably, we observed a novel pattern of cytosolic accumulation within membrane bound compartments with heterogenous electron density. This pattern does not resemble any pattern published in the prior literature, including vaping and aspiration pneumonia. One hypothesis of the likely composition of VIBs is that they are the vaporized derivatives of PG/VG in the e-juice. Future lipidomics analysis as well as organelle marker studies of LMs from habitual vapers may shed light on this question.