Acute vaping exacerbates microbial pneumonia due to calcium (Ca2+) dysregulation.

As electronic cigarette (E-cig) use, also known as "vaping", has rapidly increased in popularity, data regarding potential pathologic effects are recently emerging. Recent associations between vaping and lung pathology have led to an increased need to scrutinize E-cigs for adverse health impacts. Our previous work (and others) has associated vaping with Ca2+-dependent cytotoxicity in cultured human airway epithelial cells. Herein, we develop a vaped e-liquid pulmonary exposure mouse model to evaluate vaping effects in vivo. Using this model, we demonstrate lung pathology through the use of preclinical measures, that is, the lung wet: dry ratio and lung histology/H&E staining. Further, we demonstrate that acute vaping increases macrophage chemotaxis, which was ascertained using flow cytometry-based techniques, and inflammatory cytokine production, via Luminex analysis, through a Ca2+-dependent mechanism. This increase in macrophage activation appears to exacerbate pulmonary pathology resulting from microbial infection. Importantly, modulating Ca2+ signaling may present a therapeutic direction for treatment against vaping-associated pulmonary inflammation.

Introduction

Electronic cigarettes, also known as E-cigs, are electronic devices used to heat a liquid to create an aerosol that is inhaled, or “vaped”, by the user. The liquids in E-cigs, known as e-liquids, contain various compounds, which often include propylene glycol (PG), vegetable glycerin (VG), nicotine and various flavoring agents [1, 2]. With more than 400 E-cig brands available, E-cigs and e-liquids are available in a wide variety of compositions [3]. This variety of flavors and the availability of customization have made E-cigs extremely popular among middle and high school students as well as young adults [4-6]. Following the increase in E-cig use and more recently (late 2019 to early 2020) reported cases of E-cig or vaping-associated lung injury (EVALI) [7], the overall health impacts of these new products have been called into question. In vitro and in vivo studies have demonstrated the detrimental effects of these products on pulmonary health [8-11]. Furthermore, a previous assessment of JUUL e-liquids has illustrated notable signs of cytotoxicity in cultured airway epithelial cells [12]. Studies have also tied together an observable cytotoxicity after e-liquid exposure with increases in cytoplasmic calcium (Ca2+) [12-14]. These earlier studies led us to further investigate the cellular effects of e-liquids in vivo.

Ca2+ concentrations impact almost every cellular process, e.g., protein stability and programmed cell death [15]. In addition, Ca2+ is vital to cell signaling and to regulating inflammatory responses [16]. Multiple Ca2+ channels and pumps are involved in its tight regulation and include the non-store-operated calcium (SOC) transient receptor potential (TRP), the SOC Orai1 and ER-resident inositol trisphosphate (IP3) channels [15, 17]. Alterations in cellular signaling pathways such as SOC signaling and the activation of downstream SOC kinases such as PKCα lead to alterations in rates of apoptosis and cell proliferation [18].

We previously examined the responses of cultured airway epithelial cells to various e-liquids [12]. Here, we report on a series of experiments further investigating the pulmonary inflammatory response after exposure to “vaped” e-liquid in an established in vivo model. Using this exposure model, we questioned first whether acute vaping would recapitulate our previously published in vitro findings, and whether vaping might “prime” the immune inflammatory response and result in a worse prognosis after a microbial challenge, that is, vape pre-exposure would result in a worse outcome compared to no pre-exposure. For this study, we utilized our previously published Klebsiella pneumoniae mouse model [19] and evaluated for severity of disease using several metrics: gross lung morphology and weight, inflammatory cytokine panels, lung histology/H&E staining and immune cell profiles. At the same time, we also treated with well-described Ca2+ receptor antagonists to determine the effects of such compounds, if any, on inflammatory responses.

Materials and methods

Materials and cells

Klebsiella pneumoniae (BA-1705) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). K. pneumoniae cultures were routinely grown for 12–16 h in Brain Heart Infusion (BHI) broth (37°C). Bacterial numbers were calculated using serial dilution plating on BHI agar plates.

Unless otherwise noted, all reagents were purchased from either Thermo Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA) at the highest level of purity available. The utilized JUUL e-liquids/ “pods” were purchased locally from retailers in Durham, NC, USA from November 1, 2019—July 31, 2020. These products were inventoried and stored at room temperature until used. However, after being vaped using the JUUL E-cig device, the vaped e-liquid was stored at -20°C. The manufacturer’s label information stated ingredients include only vegetable glycerin (VG), propylene glycol (PG), nicotine, flavoring and benzoic acid.

Vape distillate generation and nicotine analysis method

The e-liquid was vaped using a previously described [20] method to produce a vapor distillate. Briefly, e-liquid vapors were produced using prefilled pods and a JUUL E-cig device connected to silicon tubing and to the mouthpiece of the E-cig on one end. The other end was placed in the lower part of a 50-ml conical tube. The distillate was condensed and collected while suspended above liquid nitrogen inside of a thermal container. The JUUL device was utilized for periods of up to 5 s with at least 10 s between activations to simulate “puffs”.

The LC-MS analysis of nicotine within the vaped e-liquid samples was performed similarly as described by Pagano and coworkers [21]. Specifically, nicotine was serially diluted in LC-MS-grade methanol to the following concentrations: 1.0, 0.5, 0.1 and 0.05 mg/ml. Quinoline (45.7 μl) was diluted into 1 ml methanol. The diluted quinolone (10 μl) was added per 1 ml of each sample or standard. The vaping sample was diluted 10-fold in methanol. The samples were submitted to the Mass Spectrometry Laboratory at the Louisiana State University and Agricultural and Mechanical College (LSU) in Baton Rouge, Louisiana.

The samples were analyzed with an Agilent 5977A mass selective detector (MSD) attached to an Agilent 7890B gas chromatograph (GC). The system used a 30m DB5-MS Ultra Inert (Agilent) with a 0.25 mm inner diameter and a 0.25-μm film thickness. DB5 column contains a nonpolar phenyl arylene polymer, virtually equivalent to (5%-phenyl)-methylpolysiloxane. GC Method: Starting temp: 80°C held for 4 min, increasing at a rate of 6°/min to 300°C, and then held for 15 min. The injector was run in splitless mode at a temp of 250°C. Carrier gas was helium at a constant flow rate of 1.5 ml/min. MS Parameters: Transfer temp = 250°C, source temp = 230°C, quad temp = 150°C, and a solvent delay of 4 min. Nicotine was detected using the 84.00 m/z (83.70–84.70 m/z) fragment while the quinoline was detected with a 129 m/z (128.70–129.70 m/z) signal. The nicotine eluted at 12.6 min on the GC while the quinoline eluted at 10.3 min. The nicotine content was determined to be ~2 mg/ml, which is in contrast to an unvaped e-liquid concentration of 35 mg/ml [22]. Because each JUUL pod (0.7 ml) can produce ~200 “puffs” (juul.com), a “puff” can be calculated to contain ~125 μg of nicotine. However, for our studies, we utilized 10 μl (20 μg nicotine) doses of our vaped e-liquid (~2 mg/ml) to attempt to minimize any potential morbidity.

Mice and treatments

Mice (C57-BL/6J) were from Jackson Laboratories (Bar Harbor, MA, USA). Young adult (6- to 8-week-old male and female) mice were used for all experiments [23]. After delivery, the mice were allowed to recover from shipping stress for 1 week at the NC Central Univ. Animal Resource Complex, which is accredited by American Association for Accreditation of Laboratory Animal Care. All animal care and use were conducted in accordance with the Guide for the Care and Use of the Laboratory Animals (National Institutes of Health) and approved by the NCCU IACUC. Mice were maintained at 25°C and 15% relative humidity with alternating 12-h light /dark periods.

Once acclimated, mice were anesthetized using isoflurane and a SomnoSuite low-flow anesthesia system (Kent Scientific Corporation, Torrington, CT, USA). The e-liquid distillate, vehicle control (50:50 (vol/vol) PG/VG) or saline (10 μl) was then delivered dropwise intranasal (IN) using a micropipette, as has been previously described [24, 25], once daily for 3 days to each animal in the appropriate treatment group. On the 4th day, the mice were sedated with an i.p. injection of ketamine (100 mg/kg) and xylazine (50 mg/kg) and infected IN with a sub-lethal dose (2 × 104 CFU) of K. pneumoniae suspended in 20 μl 1× phosphate buffered saline (PBS), as previously described [19]. Body weight and health conditions was monitored daily per IACUC protocols. Humane endpoints were not used for this study as experimental time points of completion were chosen before any significant body weight change clinical signs of disease can be observed. At time points of experimental completion (24 hpi), mice were humanely euthanized using CO2 asphyxiation and cervical dislocation, as per our accepted NCCU animal protocol and NCCU Animal Resource Complex housing guidelines and conditions.

Lung wet/dry ratio

Lungs were immediately removed from euthanized mice (24 hours post infection) and weighed (wet weight). The lung tissue was then dried in an incubator (65°C) for 24 h and reweighed (dry weight). The wet/dry ratio was calculated by dividing the wet weight by the dry weight.

Sample preparation from lungs

Bronchoalveolar lavage (BAL) fluid was obtained as has been previously described [19]. In brief, mice were euthanized, and a catheter attached to 1-ml syringe was inserted into the trachea. The syringe was then used to deliver 1× PBS, which was gently pipetted up and down 3× to remove fluid. BAL fluid was then directly utilized for flow studies or clarified via centrifugation for Luminex analysis. Alternatively, total lung was dissected out, then homogenized in PBS containing protease inhibitors, clarified via centrifugation and quantified using the Bradford assay.

Luminex analysis

Cytokine panels were assessed using a 10-plex mouse Luminex kit (cat. #SPRCUS1264, MILLIPLEX, EMD Millipore, Chicago, IL, USA) and either BAL fluid or clarified whole mouse lung cell lysates.

Flow cytometry

Cells isolated from BAL were stained with fluorescent-tagged antibodies as per the manufacturers’ instructions. CD170 (SiglecF)-FITC, Ly6G-PE, F4/80-AF700, CD11B-APC, CD11C-Superbright 780 and eBioscience Fixable Viability Dye eFluor 450 were purchased from Thermo Fisher Sci (eBiosciences). In brief, antibodies were chosen to sort mouse macrophages and neutrophils, similar to previously published work [26]. Data acquisition was performed using a 3-color CytoFLEX flow cytometer (Beckman Coulter, FL, USA). The CytExpert acquisition software (Beckman Coulter) was used to acquire samples while FCS Express (version 6; DeNovo Software, Pasadena, CA, USA) was then used for the analysis. For each sample, at least 30,000 events were collected.

Histopathology

At time points of experimental completion, mice were humanely euthanized using an overdose of CO2 or isoflurane. Lungs were inflated with 1 ml of 10% neutral-buffered formalin, removed and then suspended in 10% formalin for 12 h. Lungs were washed once in PBS and then immersed in 70% ethanol. Tissues were then embedded in paraffin, and three 5-μm sections (200 μm apart per lung) were stained using hematoxylin/eosin (H&E) for examination by the NCCU Histopathology Core (directed by Dr. X Chen). Histology was scored for damage associated with acute lung injury (ALI) using a rubric described by the American Thoracic Society. In short, 200X magnified H&E-stained sections were evaluated for A) neutrophils in the alveolar space, B) neutrophils in the interstitial space, C) hyaline membranes, D) proteinaceous debris filling the airspaces and E) alveolar septal thickening, with a score generated using the equation: ((20xA)+ (14xB)+ (7xC)+ (7xD)+ (2xE)/(number of fields x 100) [27].

Statistics

Statistical analyses were performed using GraphPad Prism (La Jolla, CA, USA) and Microsoft Excel analysis. Appropriate statistical tests (Student’s t-test, ANOVA) were determined after discussion with NCCU biostatistics faculty. Unless otherwise indicated, the results are shown as the mean ± the standard error of the mean (SEM). A value of P<0.05 is considered as statistically significant.

Results

“Vaped” e-liquid exposure results in cytotoxicity and a pro-inflammatory response

Our previous study investigated the consequences of exposure to resting, or “unvaped”, e-liquids using cultured airway epithelial cells [12]. Those data clearly demonstrated increased pro- inflammatory responses and cytotoxicity after the e-liquid exposures. Herein, we evaluate e-liquids after the “vaping” process. To that end, we developed a protocol utilizing commercially available E-cig devices to aerosolize the e-liquid and then condensed the vapors back into a “vaped” e-liquid. With this new reagent in hand, we recently assessed its toxicity in vitro [28]. We also performed H&E lung histology and found that the vehicle- and unvaped e-liquid-treated samples more similar to each other when compared to the vaped-e-liquid-treated tissue, which displayed more marked pathology, that is, alveolar wall thickening and the infiltration of inflammatory cells into the interstitial space ().

Vape pre-exposure exacerbates K. pneumoniae-induced lung pathology

We next used our acute vape exposure model and infected animals with a published sub-lethal dose (2 × 104 CFU) of K. pneumoniae, which is a well described model for hospital acquired infections [29], known to induce early and controlled inflammatory responses post-pulmonary inoculation [30] and, therefore, ideally suited for modelling pulmonary pathology. The mice were then monitored post-infection before euthanizing the animals 24 hpi (). Because of the acute nature of infection, we did not expect to observe clinical disease behavior, though hunching and lethargy were observed in the infected animals post-vape exposure. After dissection, the lungs were weighed both wet and after drying and used to calculate the “wet/dry” ratio [31, 32], with an increase in this ratio being a well-described preclinical metric of acute lung injury and reflecting severity of lung inflammation. From these observations, it could be noted that the lungs from the vaped e-liquid pre-exposure followed by bacterial infection mice had significantly higher wet/dry ratios compared to the vaped vehicle pre-exposure followed by bacterial infection group (and bacterial infection alone) (). The lung pathology of the K. pneumoniae-infected only animals also demonstrated a mild inflammatory influx of neutrophils within the alveoli, though the majority of airways were unimpeded (). However, the lungs from animals vaped prior to K. pneumoniae infection demonstrated more significant inflammatory influx, characterized by monocytic and neutrophilic cells overwhelming within the alveoli. Acute Lung Injury (ALI) scoring of tissue sections clearly demonstrated significant disease burden within this group (). BAL fluid was then evaluated for the presence of inflammatory cytokines by Luminex analysis (). As expected, the innate immune cytokines TNFα, IL-6 and Mip-1α were upregulated in the K. pneumoniae-infected lungs, with the levels significantly increased within the lungs of the vaped animals. Interestingly, IL-17 levels were also observably activated in the vaped and K. pneumoniae-infected animals, suggesting a potential role for other inflammatory cells such as natural killer T cells.

Vaping exacerbates key clinical metrics of microbial pneumonia.

(A) Study design. (B) Lung wet/dry ratio of mice pre-exposed to either vaped vehicle or e-liquid and then infected with a sub-lethal dose of Klebsiella pneumoniae. n = 4 mice per treatment group. (C) H&E staining (200X magnification) of lung tissue isolated from vaped vehicle- or vaped e-liquid- treated mice infected with K. pneumoniae. n = 3 mice per group. Arrows indicate alveoli thickening and neutrophil influx. ALI was evaluated and scored to quantify inflammation within tissue sections using an ATS accepted rubric [27]. (D) Inflammatory cytokine analysis of BAL fluid from vaped vehicle- or vaped e-liquid- treated mice infected with K. pneumoniae (compared to the mock control) as performed via Luminex array. Symbols and bars represent the mean ± SEM compared to the mock control (* P<0.05, ** P<0.01, *** P<0.001).

Role of calcium (Ca2+) signaling in vape-primed lung pathophysiology

Based upon our previous in vitro studies investigating the role of Ca2+ in e-liquid-induced cytotoxicity and inflammation [12, 14] and our observations of enhanced acute lung pathology after vaped e-liquid pre-exposure and K. pneumoniae infection in comparison to the vaped vehicle followed by K. pneumoniae challenge (), we further queried the potential of a mechanistic role for Ca2+ signaling in this pathophysiology. To dissect out this mechanism, selective pharmacology was employed to distinguish between the major channels involved in Ca2+ influx due to tobacco/E-cig exposure, that is, store-operated calcium entry (SOCE), non-SOCE and the ER-resident inositol trisphosphate (IP3) receptor, which is the receptor regulating calcium release into the cytosol when activated [33-35]. The respective compounds used to selectively target these channels were Pyr6, Pyr10 and 2-APB [36, 37].

For this first study, we followed the treatment regime outlined in . However, along with the vape treatments on days 1–3, the mice also received either Pyr6, Pyr10, 2-APB or no drug treatment IN (10 μl of a 500 μM stock) 30 min prior to the vape treatment (). The mice were also not infected with K. pneumoniae. On day 4, the animals were euthanized, and bronchoalveolar lavage (BAL) fluid was obtained for Luminex () and flow-based assays (Figs ). From the Luminex data (), it was clear that the animals receiving either Pyr6, Pyr10 or 2-APB exhibited significantly lower levels of the pro-inflammatory cytokine IL-6 as compared to the mice receiving only the vape exposure. Additionally, IFN-γ levels were significantly lower for all treated groups, except for Pyr6 which did trend lower, compared to the vape-treated control. We complemented these data through the use of flow cytometry (), where the BAL interstitial macrophage (IM, F4/80+SiglecF-CD11b+) population of the vape-treated mice was markedly higher than the mock treatment and the 2-APB- and Pyr10-treated groups, which were more similar to the mock-treated group. In contrast, the Pyr-6 treated group was more similar to the vape-treated mice. These findings are intriguing because it is currently well-understood that the majority macrophage population within the airways, and therefore within BAL fluid, is alveolar macrophages (AM, F4/80+SiglecF+CD11b+) and not IM [38].

Based upon these observations, we next asked whether similar results would be observed if the experiments () were repeated with the addition of the K. pneumoniae challenge on day 4 followed by euthanizing the animals on day 5. As we observed prior vaping to exacerbate K. pneumoniae-dependent pathology (), we evaluated whether this exacerbation was related to Ca2+ signaling. Mice were euthanized 24 hpi, and BAL cells and fluid were harvested for analysis (). BAL fluid was assessed for inflammatory cytokines, with protein levels found to be generally increased in vaped vs mock-treated animals (). Interestingly, the three drug treatments produced differential cytokine activation profiles, with Pyr10 treatment correlating to diminished cytokine activation compared to the vaped animals. In general, however, cytokine burdens were decreased in animals administered a Ca2+ channel inhibitor, as compared to the vaped animals (). BAL cells were then identified using flow cytometry (Figs ). Previous studies have indicated that cellular chemotaxis due to K. pneumoniae infection is neutrophilic. Therefore, we investigated neutrophil populations within the collected BAL from the vaped animals. Interestingly, neutrophils (F4/80-/Lys6G+) were upregulated in K. pneumoniae-infected animals (control group) but not in the vaped animals, except for the Pyr-6 treated group, again, having a different phenotype. In contrast, the cellularity within the BAL from the vaped animals was observed to be primarily monocytes, with an increase in F4/80-/ Ly6G- cells. In fact, more IM were observed within the vaped K. pneumoniae-infected animals compared to AM, though the percentages were small in comparison to the total population collected. Interestingly, the Ca2+ inhibitor drugs appeared to increase the populations of both AM and IM within the lung compared to the vaped only animals.

Discussion

The inhalation of flavored e-liquid vapors (vaping) has significantly increased in popularity in recent years, yet potential pathologic effects are unclear. Since the introduction of E-cigs, the question of how they impact pulmonary health has been raised. With this study, we were able to further expand upon what is currently known about E-cigs and their role in Ca2+ mobilization. Using a mouse exposure model, we find that vaping alone leads to mild but observable pulmonary inflammation () [28], which is exacerbated when challenged with a microbial pulmonary infection (). Interestingly, this inflammation is clearly related to Ca2+ mobilization, as pre-treatment with various Ca2 channel antagonists differentially modulated the vaping-induced inflammatory response (Figs ). We found through this vape exposure model that vaped e-liquid potentially interacts with multiple channels, such as TRPC channels, Orai1/STIM1 and IP3R, to impact Ca2+ signaling (). Most of our utilized Ca2+ antagonists were able to somewhat reduce inflammatory cytokine production compared to the positive control. Finally, we observed a general decrease in pulmonary inflammation in the vape-treated animal groups pretreated with these same Ca2+ antagonists (prior to K. pneumoniae infection), with a more marked decrease observed with either Pyr10 or 2-APB treatment (). However, differential cytokine activation profiles were observable and clearly more work will be required to uncover the role of Ca2+ signaling within the context of our dual vape exposure and infection model.

Schematic representation of the major receptor pathways involved in Ca2+ increases due to E-cig/vape exposure.

The Ca2+ channel antagonists Pyr6, Pyr10 and 2-APB were utilized to assess their effectiveness in blocking and/or reducing vaping-associated inflammation. Pyr10, Pyr6 and 2-APB inhibit Transient Receptor Protein (TRP) channels, Orai1/STIM1 and the inositol trisphosphate receptor (IP3R), respectively. K. pneumoniae infection also elicits an inflammatory response and is also likely to alter Ca2+ homeostasis. V = Vape.

Exposure to traditional tobacco smoke is well-documented to trigger a number of inflammatory responses in the airways and often leads to ailments such as airway inflammation, acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD) [39]. ARDS, in particular, is characterized by acute respiratory failure that results from pulmonary edema and inflammation [40], and its pathophysiology is characterized by increases in circulating levels of multiple inflammatory proteins, including cytokines and chemokines [41, 42]. At the same time, both acute and chronic exposure to cigarette smoke leads to chronic elevations in intracellular Ca2+ levels, and smoke from other similar tobacco products, such as E-cigs, is expected to exert similar effects, resulting in pulmonary disease initiation and progression [34, 35, 43]. Although Ca2+ has been demonstrated to play a pivotal role in the development and disease progression of ARDS, little is known about the mechanisms of Ca2+-induced inflammatory lung diseases.

The recent literature suggests previously underappreciated levels of inflammatory effects and cytotoxicity associated with E-cig use [44-48]. At the same time, vaping renders the user’s lungs more susceptible to microbial infection/burden and, therefore, to pulmonary disease [10]. This observation is possibly due to the decreased phagocytic activity of alveolar macrophages (AMs), which has also been demonstrated with cigarette smoke exposure [49]. However, a functional gap in knowledge does still exist with regard to the effects of vaping upon airway inflammation and microbial infection, which possibly involves macrophage activity.

Our results regarding increased IM populations after vape exposure are intriguing alone (). However, when factoring in the effects of a simultaneous Pyr6, Pyr10 or 2-APB treatment with the vape exposure, increased Ca2+ mobilization becomes a potentially potent mechanism controlling the pro-inflammatory lung response after vape exposure. To our knowledge, there exists a current gap in knowledge surrounding Ca2+ signaling (Orai and/or TRP channel signaling ()) and macrophage activation. While the P2X family of Ca2+ channels have been investigated [50], more work must be performed to understand the regulation and function of SOC channels in macrophage phagocytosis and inflammation. Studies that have been performed have predominantly focused on immune activation in the absence of various TRP channels [51, 52] however not on the effects of perturbed Ca2+ signaling upon deleterious immune activation, as we observed in our studies. A continuation of our studies (with combined drug exposure or other variation of dosing) will undoubtedly add to this field of study and aid in the development of novel therapeutic interventions for pulmonary diseases.

Our Ca2+ channel antagonist exposure model also has potential uses for other respiratory diseases. For example, Ca2+ channel blockers are currently employed to treat hypertension and heart arrhythmias [53, 54]. Thus, these Ca2+ channel antagonists might find use in the treatment of pulmonary diseases such as COPD, cystic fibrosis or asthma. There is also the potential that Ca2+ channel antagonists might inhibit the inflammation observed in EVALI cases.

In sum, we have used an in vivo vaping model to demonstrate increased levels of macrophage chemotaxis and inflammatory cytokines after an acute exposure period. Further, we now implicate a Ca2+-dependent mechanism leading to this pro-inflammatory response and to immune cell activation. This increase in macrophage activation would likely exacerbate pneumonia symptoms due to pulmonary microbial infection. These findings indicate E-cig use has potential effects on pulmonary health and disease outcome.

An acute in vivo model of vaping indicates cytotoxicity.

Mice received either PBS, vaped PG/VG vehicle, resting e-liquid or vaped e-liquid (10 μl) once daily intranasal (IN) for 3 days and were then euthanized. n = 4 mice per treatment group. H&E staining (200X magnification) of sections of lung tissue isolated from mock-, vaped vehicle-, resting e-liquid- or vaped e-liquid- treated mice. Alveolar wall thickening was particularly observable in the lungs from the vaped e-liquid-treated mice compared to the mock control. Arrows indicate alveoli thickening and neutrophil influx.

(PPTX)

Click here for additional data file.

(A) Accompanying FACS plot data for Fig 2. (B) Multiple FACS scatter plot data frames to accompany Fig 2C. AM = SiglecF+/F4/80+ and IM = SiglecF-/F4/80+. AM = alveolar macrophage, IM = interstitial macrophage.

Fig 2

Increased intracellular Ca2+ levels mediate vape-induced inflammation.

(A) Study design. Mice were simultaneously vaped and treated with the Ca2+ channel antagonists Pyr6, Pyr10 or 2-APB for 3 days. n = 6 mice per treatment group. After being euthanized, BAL fluid was collected, and (B) a Luminex cytokine panel including IL-6 and IFN-γ was run using the clarified BAL fluid. (C) Cells collected after clarifying the BAL fluid were then analyzed using flow analysis and (D) tabulated. n = 2–3 flow analyses per group with a representative image (C) for each group shown. IM = SiglecF-/CD11b+ and AM = SiglecF+/CD11b+. Symbols and bars represent the mean ± SEM compared to mock control (* P<0.05, ** P<0.01, **** P<0.0001). AM = alveolar macrophage, IM = interstitial macrophage.

(PPTX)

Click here for additional data file.

(A) Accompanying FACS plot data for Fig 3. Includes data for all drug treatment groups (2-APB, Pyr-6 and Pyr-10) and K. pneumoniae only. (B) Multiple FACS scatter plot data frames to accompany Fig 3C. MAC = F4/80+ and NEU = LysG+. MAC = macrophage, NEU = neutrophil.

Fig 3

Ca2+ channel antagonists differentially regulate vape-induced inflammation when the treatments are supplemented with a subsequent 24-h pathogen challenge.

(A) Study design. Mice were simultaneously vaped and treated with the Ca2+ channel antagonists Pyr6, Pyr10 or 2-APB, then challenged with a sub-lethal dose of K. pneumoniae and euthanized for BAL fluid and lung tissue collection. n = 6 mice per treatment group. (B) Clarified BAL was run using the Luminex cytokine panel. (C) Collected BAL fluid cells were then analyzed via flow and (D) tabulated. IM = SiglecF-/CD11b+ and AM = SiglecF+/CD11b+. AM = alveolar macrophage, IM = interstitial macrophage, neutrophils = F4/80-/Lys6G+. Symbols and bars represent the mean ± SEM compared to the mock control (* P<0.05).

(PPTX)

Click here for additional data file.

23 Jun 2021

PONE-D-21-13261

Acute Vaping Exacerbates Microbial Pneumonia due to Calcium (Ca2+) Dysregulation

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a very well-written and surprisingly useful mouse study. The information is current, useful and important for interested pulmonary health providers. I have just a few thoughts.

Traditionally the materials and methods are placed before the results, correct?

Vaping resulting in acute cellular lung damage which increases the pathology process in mice infected with K.pneumoniae is important basic information. But why K.pneumoniae? Is it the mouse equivalent of pneumococcal in humans?

Reviewer #2: Zhang et al developed a mouse model of vaped e-liquid pulmonary exposure and studied the effects of calcium ion channel inhibitors in cohorts of mice that received either vaping or vaping+Klebsiella pneumonia. While some of the results are encouraging, the authors overstated their findings – the tested inhibitors barely showed therapeutic effects in vaping+bacterial pneumonia model.

Please proof-read and correct typographical/grammar issues.

Abstract:

The authors wrote – “Using this model, we demonstrate through the use of clinical measures, that is, the lung wet:dry ratio and lung histology/H&E staining, lung pathology.” Although these parameters are clinically used but only in EVALI-related dead patients or post-mortem. These parameters are widely used in pre-clinical models. Lung histology and lung pathology can be synonymously used, therefore one of these two words can be deleted.

The authors wrote – “This increase in macrophage activation appears to exacerbate pneumonia when due to pulmonary microbial infection.” Not clear, please paraphrase.

Results:

The authors wrote “To that end, we developed a protocol utilizing commercially available E-cig devices to aerosolize the e-liquid and then condensed the vapors back into a “vaped” e-liquid. With this new reagent in hand, we first assessed its toxicity in vitro (data not shown, manuscript under review).” In my opinion, the manuscript under review should be removed.

Page 6 – “lung pathology of the K. pneumoniae-infected only animals also demonstrated a mild inflammatory influx of neutrophils within the alveoli, though the majority of airways were unimpeded (Fig. 1B).” It should be Fig. 1C.

In figure 1B, on the y-axis, lung weight (mg) is written. Ratios have no units.

In figure 1C, instead of pixilation, please use scoring methods such as ATS scoring for ALI An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals - PubMed (nih.gov) (PMID: 21531958).

In Figures 2A and 2B, please use consistent labeling for axes. Present vape+control bar after sham (control) group for easy interpretation here and subsequent bar graphs. If possible, please present bar graphs with scatter plots to see the variation of the data.

For ease of interpretation, please present the study paradigm (as in Figure 1A) in a panel for figures 2 and 3. By looking at legends for figures 2 and 3, it is difficult to differentiate the study paradigm.

**********

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Reviewer #1: No

Reviewer #2: No

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13 Jul 2021

2. We ask that you please remove citations for unavailable and unpublished work, including manuscripts that have been submitted but not yet accepted (e.g., “unpublished work,” “data not shown”).

We have removed all “data not shown” and replaced with a citation for our recent publication. Lines 209 and 323.

Please amend your current ethics statement to confirm that your named ethics committee specifically approved this study.

All animal care and use were conducted in accordance with the Guide for the Care and Use of the Laboratory Animals (National Institutes of Health). The study was carried out in strict accordance with the NCCU IACUC (protocol number VS-10-09-2019). Mice were maintained at 25°C and 15% relative humidity with alternating 12-h light /dark periods. All exposures and surgeries were performed under isofluorane anesthesia, and all efforts were made to minimize suffering.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was supported in part by a grant from the National Cancer Institute (NCI) NIH 5-U54-

CA156733-10, National Minority Health Disparities Institute (NIMHD) NIH-3-U54MD012392 and

by funds from NCCU BRITE."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The author(s) received no specific funding for this work."

We apologize for this mistake. This has been updated in the cover page. Please include the following text for our funding statement:

“This work was supported in part by a grant from the National Cancer Institute (NCI) NIH 5-U54-CA156733-10, National Minority Health Disparities Institute (NIMHD) NIH-3-U54MD012392 and by funds from NCCU BRITE.”

5. We note that you have included the phrase “data not shown” in your manuscript.

We have removed all “data not shown” and replaced with a citation for our recent publication. Lines 209 and 323.

Reviewers' comments:

Traditionally the materials and methods are placed before the results, correct? ---------Now corrected.

Vaping resulting in acute cellular lung damage which increases the pathology process in mice infected with K.pneumoniae is important basic information. But why K.pneumoniae? Is it the mouse equivalent of pneumococcal in humans? ----------Excellent point by the reviewer. We now include text for our rationale of using K. pneumoniae. Lines 215-217.

Reviewer #2: Zhang et al developed a mouse model of vaped e-liquid pulmonary exposure and studied the effects of calcium ion channel inhibitors in cohorts of mice that received either vaping or vaping+Klebsiella pneumonia. While some of the results are encouraging, the authors overstated their findings – the tested inhibitors barely showed therapeutic effects in vaping+bacterial pneumonia model.

-------------We have now amended the text to indicate the obtained data are promising but not complete and that further study will be required to understand this mechanism (Line 329-335).

Abstract:

The authors wrote – “Using this model, we demonstrate through the use of clinical measures, that is, the lung wet:dry ratio and lung histology/H&E staining, lung pathology.” Although these parameters are clinically used but only in EVALI-related dead patients or post-mortem. These parameters are widely used in pre-clinical models. Lung histology and lung pathology can be synonymously used, therefore one of these two words can be deleted.----------Edited and now done. Lines 50-52.

The authors wrote – “This increase in macrophage activation appears to exacerbate pneumonia when due to pulmonary microbial infection.” Not clear, please paraphrase. ---------Edited. Lines 54-56.

Results:

The authors wrote “To that end, we developed a protocol utilizing commercially available E-cig devices to aerosolize the e-liquid and then condensed the vapors back into a “vaped” e-liquid. With this new reagent in hand, we first assessed its toxicity in vitro (data not shown, manuscript under review).” In my opinion, the manuscript under review should be removed. -----------Removed and replaced. We now reference our recent publication (Sivaraman et al., Front. Physiol., 2021) for these data, which are now freely available. Lines 208-209.

Page 6 – “lung pathology of the K. pneumoniae-infected only animals also demonstrated a mild inflammatory influx of neutrophils within the alveoli, though the majority of airways were unimpeded (Fig. 1B).” It should be Fig. 1C.--------- Done. Line 228.

In figure 1B, on the y-axis, lung weight (mg) is written. Ratios have no units. --------Done.

In figure 1C, instead of pixilation, please use scoring methods such as ATS scoring for ALI An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals - PubMed (nih.gov) (PMID: 21531958).---------- ATS scoring for ALI has now been performed and is now included within Figure 1C. Please see also lines 187-193 and 243-244.

In Figures 2A and 2B, please use consistent labeling for axes. Present vape+control bar after sham (control) group for easy interpretation here and subsequent bar graphs. ---------Done.

If possible, please present bar graphs with scatter plots to see the variation of the data. ---------This is an excellent point. These data sets are, however, very large and were collected and analyzed by the NC Central University Flow Core. We did ask for and receive additional raw data scatter plots from the Core and now include these plots to allow for readers to observe the variation of the data (Supplementary Figures 2B and 3B). The quantitative data are currently within Figures 2 and 3.

For ease of interpretation, please present the study paradigm (as in Figure 1A) in a panel for figures 2 and 3. By looking at legends for figures 2 and 3, it is difficult to differentiate the study paradigm.----------- Study paradigms are now included for Figures 2 and 3, i.e., Figures 2A and 3A.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Aug 2021

Acute Vaping Exacerbates Microbial Pneumonia due to Calcium (Ca2+) Dysregulation

PONE-D-21-13261R1

Dear Dr. Sivaraman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Shama Ahmad, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: N/A

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Well-done, interesting and educational paper. The author addressed and answered all of the reviewers' observations and questions adequately and completely. The selection of the specific microorganism was explained in a complete and referenced sentence. This is overall worth reading for primary care and specialty care providers and, in my opinion, should be accepted and published. While I am thinking about this, I have decided that I don't like to work with a 'minimum character count' and since I am volunteering my effort, I will ask that you remove my name from your list of reviewers.

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

4 Aug 2021

PONE-D-21-13261R1

Acute Vaping Exacerbates Microbial Pneumonia due to Calcium (Ca2+) Dysregulation

Dear Dr. Sivaraman:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Shama Ahmad

Academic Editor

PLOS ONE