Both human and mouse lung epithelial cells were used. BEAS-2Bs are a non-tumorigenic, human bronchial epithelial cell line, which were obtained from the American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B cultures were grown at 37 °C with 5% CO₂ in M-199 media containing 10% fetal bovine serum (FBS). To passage the cells, media from the cell container was transferred to a waste beaker using a Pasteur pipette. The flask containing the cells was then washed with 2 mL pure Hanks’ Balanced Salt Solution (HBSS, pH 7.2), then the HBSS was transferred out of the flask and into a waste beaker. Trypsin (2–3mL) was added to the flask, and the flask was placed back in the incubator for 5–10 minutes. The flask was removed from the incubator, and 8 mL HBSS + 10% FBS (R&D Systems, Pittsburgh, PA) was added to the flask with the trypsin to inactivate the trypsin. This solution was transferred to a new 15 mL conical tube and spun down for 5–10 minutes in a centrifuge (Beckman Coulter, Brea, CA) at 193 g. The supernatant was removed, and the remaining pellet was resuspended in 5 mL of M-199 + FBS media. If applicable, cells were counted, placed into new flasks with fresh media, and placed in the incubator for cell growth and proliferation.

Wild-type C57BL/6 black mice were euthanized and the tracheas were removed and cut longitudinally to expose the inner lumen. The tracheas were incubated overnight at 4°C in 1.5 mg/mL Pronase digestion buffer (Sigma Aldrich, St. Louis, MO). Digestion was stopped with 10% FBS (R&D Systems, Pittsburgh, PA), and tracheas were removed from the media. The digestion buffer media was centrifuged at 193 g for 3 minutes to collect the cells, which were resuspended in mouse tracheal epithelial cells (MTEC) basic media (Gibco-Thermo Fisher, Waltham, MA) with 10% FBS and placed in 60 mm tissue culture dishes. After the dishes were incubated for 3–4 hours at 37 °C, the media was removed and centrifuged at 193 g for 3 minutes. The cells were resuspended in air-liquid interface (ALI) Cell Nutrient Mix consisting of MTEC basic media (Gibco-Thermo Fisher) and supplements and counted, then seeded into cell culture inserts with 100,000 cells per insert and returned to the incubator. Once cells were confluent, the media was removed. The cells were exposed to air and formed cilia after approximately 14 days. All animal procedures were approved by the University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee (IACUC Protocol #22-070-11-FC) and were in accordance with NIH guidelines for the use of rodents.

Fresh Cigarette Smoke Extract (CSE) was prepared before each experiment using reference cigarettes (85 mm, filtered, Code1R6F) obtained from the Center for Tobacco Reference Products (CTRP) located in the Kentucky Tobacco Research & Development Center (KTRDC) at the University of Kentucky (Lexington, KY). One cigarette was connected to a peristaltic pump (Model ATS-P, Bentley Laboratories, Santa Anna, CA), lit, and bubbled into 20 mL of sterile cell culture media in a 50 mL conical tube. The peristaltic pump tube was secured in the media with parafilm, and the tops and sides were wrapped to contain the smoke. Once the cigarette burned for 6 minutes, the peristaltic pump was turned off, the tube was removed from the media, and the conical was sterile filtered (0.22 μm), capped and used as 100% CSE. During this time period, the puffing regimen was 20 mL puff volume and 1.5 sec puff duration, followed by a 40 sec inter-puff interval) consistent with ISO 20778 standards ((ISO), 2018a). For cell treatments, % dilutions of an individual extract are from the 100% stock extract (Figure 3, 4). For extract comparisons between extracts, the final working concentrations of CSE and JVE were equilibrated by nicotine content for both extracts where presented as 25–100% (Figure 2, 5–8). CSE was used within 12 hours of preparation.

Fresh JUUL^™ Vapor Extract (JVE) was prepared before each experiment. A Virginia Tobacco JUUL^™ Pod (5% nicotine; 59 mg/mL) was inserted into the device and attached to one end of a peristaltic pump (Bentley Model ATS-P). Similar to the production of CSE, the other end of the tube was secured in sterile cell culture media in a 50 mL conical tube, and parafilm was wrapped around the top of the tube to prevent any vapor from escaping. During a total time period of 6 minutes, the puffing regimen was 56 mL puff volume and 7 sec puff duration, followed by a 17 sec inter-puff interval ((ISO), 2018b). The tubing was removed from the conical and capped to be used as 100% JVE. As described for CSE above, comparisons were made as either % dilution or nicotine normalized for each experiment and used within 12 hours of preparation.

Measurement of nicotine concentration was done according to a colorimetric assay previously described (Willits et al., 2002). A calibration curve of nicotine was made using a nicotine standard of 50 mg/mL serially diluted with distilled water to the following concentrations: 100 ug/mL, 50 ug/mL, 25 ug/mL, 10 ug/mL, 5 ug mL, 2.5 ug/mL, 1.25 ug/mL, 0.625 ug/mL, 0.3125 ug/mL, 0.16 ug/mL, and 0.078 ug/mL. Droplets from these serial dilutions were then read on a ThermoScientific NanoDrop One using its UV-Vis option at the wavelengths of 259 nm, 236 nm, and 282 nm. The resulting absorbance readings were then used to generate a graph of the standard nicotine calibration curve where the value of the slope at each of the three wavelengths was calculated. Next, 20 mL of 100% JVE and 100% CSE were freshly made, and one absorbance reading at each of the three wavelengths was made using the NanoDrop One’s UV-Vis option. Using the (known) nicotine standard slopes and the single absorbance readings at each of the three different wavelengths from the 100% JVE or 100% CSE tubes, the final (previously unknown) total concentration of nicotine in 20 mL of 100% JVE and 100% CSE was then calculated using Beer’s Law. For cell treatments, % dilutions of an individual extract are from the 100% stock extract (Figure 3, 4). For extract comparisons between extracts, the final working concentrations of CSE and JVE were equilibrated by nicotine content for both extracts where presented as 25–100% (Figure 2, 5–8).

Lactate dehydrogenase (LDH) release was measured in lung epithelial cells treated with 25%, 50%, 75%, and 100% cigarette smoke extract or JUUL^™ vapor extract using a Lactate Dehydrogenase Activity Assay Kit (Sigma-Aldrich, MAK066). The assay was performed with cell culture media as a negative control and lysed cells as a positive control.

BEAS-2Bs were grown in a 24-well dish to confluency. In each well, a circular “wound” was introduced to the cell monolayer using a sterile toothpick. Cell migration was monitored using a phase contrast microscope with a camera attachment (Olympus, Center Valley, PA). AmLite software (AmScope, Irvine, CA) was used to capture images of each individual wound at multiple timepoints over the span of 24 hours to track wound closure following treatments with nicotine-equivalent doses of JVE or CSE. The Wacom Intuos Pro drawing pad (Wacom, Portland, OR) and image analysis software (NIH ImageJ v1.45 for Mac OS X) were used to trace the borders of the wounds and measure the wound area in pixels at each timepoint. The percentage of the original wound area remaining after 24 hours was determined for each wound. The change in cell migration to close a wound area was measured over a 24-hour period using the concentration of CSE previously shown to produce slowing of wound closure (Allen-Gipson et al., 2013). Likewise, the nicotine equivalent concentration of JVE was diluted for this assay to 1.67% JVE.

A detailed characterization of the Sisson-Ammons Video Analysis (SAVA) system can be found in Sisson et al. (2003). SAVA uses whole-field analysis to analyze cilia beat frequency while eliminating operator bias due to selection of areas containing aberrantly fast- or slow-moving cilia. SAVA was used to detect regions of ciliated wild-type MTEC that were treated with nicotine-equivalent doses of either CSE or JVE. The change in cilia beat frequency (CBF) was measured over a 24-hour period using the concentration of CSE previously shown to produce cilia slowing (Wyatt et al., 2012). Likewise, the nicotine equivalent concentration of JVE was diluted for this assay to 1.67% JVE.

Electric cell-substrate impedance sensing (ECIS, Applied BioPhysics, Troy, NY) was used to determine barrier function and resistance in BEAS-2B cells. ECIS 8W10E+ slides were coated with collagen and BEAS-2Bs were grown to confluency. Baseline barrier function measurements were taken for 4 hours in cell culture media as a control. After 4 hours, 2 wells in each slide were treated with 10%, 25%, or 50% JVE, and at the same time, a second slide was treated with the same nicotine-equivalent doses of CSE. Resistance, impedance, and capacitance measurements were taken overnight to determine changes in barrier integrity following exposure to JVE or CSE.

Additionally, cell migration into a wounded area was also determined by ECIS. Slides were prepared in the manner described in the Barrier Function Assays. Baseline measurements were taken for 4 hours in control media, and the 10%, 25%, and 50% CSE or JVE treatments were applied. After treatments were added, the slides were left undisturbed for 6 hours in the ECIS incubator before the wound was introduced. At the 10-hour timepoint, the wells were wounded for 20 seconds at 4000 Hz. Resistance, impedance, and capacitance measurements were collected every hour until the 30-hour timepoint to determine recovery of cell monolayer barrier function following wounding.

Cell culture supernates were collected and measured for concentrations of released interleukins 6 and 8 using a sandwich ELISA. Flat-bottomed Immulon-II HB 96-well polystyrene ELISA plates (Thermo Electron Corporation, Milford, MA) were coated with 200 μl/well of purified (goat) anti-human IL-8 antibody diluted 1:500 (R&D Systems, Minneapolis, MN) or IL-6 antibody diluted 1:1000 in Voller’s buffer (pH 9.6) overnight at 4°C. After 3 washings in PBS-Tween 20, undiluted culture supernatants and human recombinant IL-8 or IL-6 standards (R&D Systems) were applied to the plates and incubated at room temperature for 2 hr. Plates were again washed 3 times with PBS-Tween and incubated with (rabbit) anti-human IL-8 antibody diluted 1:500 (Rockland, Gilberstville, PA) or IL-6 antibody (R & D Systems) diluted 1:1000 in PBS-Tween/BLOTTO (0.2% instant nonfat milk in PBS-Tween) for 1 h. After 3 washes, human serum-absorbed peroxidase conjugated (goat) anti-rabbit IgG (Rockland) was added at 1:2000 (IL-6) or 1:1000 (IL-8) in PBS-Tween/BLOTTO for 45 min. The plates were again washed 3 times and 200 μl/well of peroxidase substrate (10 ng/ml orthophenylenediamine (Sigma; 0.003% H2O2 in dH2O) was added. The color reaction was terminated with 27.5 μl/well of 8M sulfuric acid and plates were read at 490nm or 450nm in an automated ELISA reader (Dynex Technologies, Chantilly, VA). Values were normalized for total protein in the cell pellet for each condition using the Bradford protein assay and results expressed as pg of cytokine/mg total protein.

Precision-cut mouse lung slices were prepared using the method of Sanderson (2011). Mouse trachea were cannulated and lungs were inflated with low melting point agarose (Invitrogen, Waltham, MA) and cooled. Lungs were glued to a vibrating microtome platform (Compresstome VF-500-0Z, Precisionary Instruments, Natick, MA) and sliced to a width of 140 μm. The slices were incubated at 37 °C in M-199 media until they were treated with CSE or JVE to be used in experiments.

Precision-cut lung slices were prepared as previously described and removed from their conditioned media. The slices were flash frozen in cell lysis buffer using liquid nitrogen. Tissue were transferred into Eppendorf tubes and dissociated by sonication for 5 seconds each. The tubes were spun at 10,000 × g for 30 minutes at 4°C. The supernatant was discarded and pellets were resuspended in 250 uL cell lysis buffer. Protein kinase C (PKC) catalytic activity was determined using the method of Hannun et al. (1986). Equal volumes (20 μL) of radiolabled ATP (10 μCi/mL [γ−³²P]-ATP; Revvity Health Sciences, Waltham, MA) and PKC reaction mix (containing 24 μg/ml phorbol 12-myristate 13-acetate, 30 mM dithiotreitol, 150 μM adenosine triphosphate, 45 mM magnesium acetate, and PKC isoform-specific substrate peptide (Bachem, Torrance, CA) mixed in a 50 mM Tris-HCl buffer) were dispensed into each glass tube using a repeat pipettor. Protein sample (20 μL) was added to each tube in a 30°C water bath and each was tube incubated for exactly 10 minutes. The reaction was halted by adding 50 μL from each tube to be spotted onto phosphocellulose exchange papers (St. Vincent’s Institute of Medical Research, Fitzroy, Australia) and submerged in 85% phosphoric acid immediately.

Data are presented as the mean ± standard deviation (SD) with scatter plots depicted for each data point. Student’s t-test was used to assess statistical difference between two groups. One-way analysis of variance (ANOVA) or two-way ANOVA was used to assess statistical differences among 3 or more experimental groups with Tukey’s post hoc test for multiple comparisons between any two groups. Statistical significance accepted at a p value <0.05. All statistical analyses were performed using GraphPad Prism 10 (San Diego, CA, USA) software.

Cigarette smoke extract and JUUL^™ Vapor extract were assayed for nicotine content using a spectrophotometric assay. A nicotine standard curve was established using pure nicotine from 0 −50 μg/mL (Figure 1A). Collected CSE and JVE were assayed for nicotine content in 25 mL of media collected over a duration of 6 minutes of continual puffing. Using our collection method, JVE demonstrated an approximate 3-fold greater content of nicotine than CSE under these conditions (Figure 1B). Samples were then normalized to equal nicotine concentrations for all subsequent comparative studies.

BEAS-2Bs were treated with CSE at concentrations of 25%, 50%, 75%, and 100% and assayed for LDH release. CSE caused significant decreases in cell viability at concentrations of 75% and 100% compared to 0 – 50% CSE (Figure 2A). No change in LDH release was observed over baseline detection media levels with 25% and 50% CSE. An equal number of lysed cells was used as the positive control for maximum LDH release in the assay. The LDH assay was also performed with BEAS-2Bs treated with JVE at concentrations of 25%, 50%, 75%, and 100% for 24 hours (Figure 2B). No significant changes in cell viability were observed in epithelial cells treated with any concentration of JVE.

Measurement of cilia beat frequency (CBF) in MTEC with SAVA shows that treatment with CSE (Figure 3A) causes decreased cilia beating at 24 hours, but treatment with nicotine-equivalent JVE (Figure 3B) does not. In MTEC treated with CSE, there was an increase in CBF from baseline at 1 hour due to the known cilia response to particles in CSE (Navarrette et al., 2012). No stimulation of CBF was observed with JVE in either 0.22 μm filtered or unfiltered JVE. At the 3 hour timepoint, CBF returned to baseline levels, but after 24 hours, cilia slowed to frequencies below baseline. No significant increase or decrease in baseline CBF was seen in MTEC treated with JVE over 24 hours. This suggests that while CSE causes changes in CBF, gradually slowing cilia over time, JVE has no effect on CBF under similar conditions.

Confluent monolayers of BEAS-2B were subjected to circular scratch wounding, and cell migration, which corresponds with wound recovery, was monitored. Under baseline culture conditions, complete wound closure was observed in wounds by 20–24 hours (Figure 4A). Cells treated with 5% CSE resulted in only 50% wound closure after 24 hours, confirming that CSE causes a decrease in cell migration into the wounded area (Amatngalim et al., 2016). In contrast, the nicotine-equivalent dose (1:3 dilution vs CSE) of JVE did not impede epithelial cell migration or wound recovery, with no significant difference compared to media control after 24 hours. Furthermore, no significant difference in wound closure was observed at any concentration of JVE (Figure 4B). Similarly, in electrically-induced wounding experiments using ECIS, treatment of BEAS-2Bs with CSE showed a decrease in cell migration into the wound (Figure 5A). CSE also impaired monolayer recovery back to baseline resistance levels following introduction of an electrical wound. In comparison, monolayers treated with JVE demonstrated an identical pattern of recovery similar to the media control (Figure 5B). Monolayers also recovered to pre-wound baseline resistance levels within 8 hours. These results indicate that CSE causes decreased cell migration following manual wounding and electrical wounding, whereas JVE has no adverse impact on epithelial cell migration and wound recovery.

Epithelial cell barrier function was continuously monitored over 24 hours following exposure to 10%, 25%, and 50% CSE and JVE. All concentrations of CSE caused a dose-dependent decrease in BEAS-2B epithelial cell barrier function over 24 hours compared to the media control (Figure 6A). JVE treatment had no impact on barrier integrity at any concentration out to 24 hours of exposure (Figure 6B). Collectively, this reveals that CSE compromises epithelial cell barrier function in a concentration-dependent manner, whereas JVE has no impact on barrier function.

Epithelial cell release into culture media of interleukin-6 (IL-6) and interleukin-8 (IL-8) was collected 24 hours following exposure to 10%, 25%, and 50% CSE and JVE and compared to control media under serum-free conditions. CSE treatment revealed a dose-dependent increase in IL-6 and IL-8 in both 16HBE cells (Figure 7A) and BEAS-2B cells (Figure 7B) compared to the media control. JVE treatment resulted in no significant release of these cytokines compared to media controls at the same time point. These data indicate a significant difference between CSE and JVE with regard to stimulated proinflammatory cytokine release of IL-6 and IL-8.

Cigarette smoke is a known activator of epithelial cell and mouse lung PKC (Allen-Gipson et al., 2005) (Simet et al., 2010). Precision-cut mouse lung slices were treated with 0 – 50% concentrations of both CSE and JVE. Following treatment for one hour, 10%, 25%, and 50% CSE induced activation of protein kinase C (Figure 8A) and also increased enzyme activity in the particulate fraction which demonstrates post-activation translocation (Figure 8B). Lung slices treated with nicotine-equivalent concentrations of JVE had no effect on PKC activity or kinase translocation.