Effect of chronic JUUL aerosol inhalation on inflammatory states of the brain, lung, heart and colon in mice

While health effects of conventional tobacco are well defined, data on vaping devices, including the most popular e-cigarette JUUL, are less established. Prior acute e-cigarette studies demonstrated inflammatory and cardiopulmonary physiology changes while chronic studies demonstrated extra-pulmonary effects, including neurotransmitter alterations in reward pathways. In this study we investigated effects of chronic flavored JUUL aerosol inhalation on inflammatory markers in brain, lung, heart, and colon. JUUL induced upregulation of cytokine and chemokine gene expression and increased HMGB1 and RAGE in the nucleus accumbens. Inflammatory gene expression increased in colon, and cardiopulmonary inflammatory responses to acute lung injury with lipopolysaccharide were exacerbated in the heart. Flavor-dependent changes in several responses were also observed. Our findings raise concerns regarding long-term risks of e-cigarette use as neuroinflammation may contribute to behavioral changes and mood disorders, while gut inflammation has been tied to poor systemic health and cardiac inflammation to development of heart disease. One Sentence Summary Chronic, daily inhalation of pod-based e-cigarette aerosols alters the inflammatory state across multiple organ systems in mice.

Flavor-dependent changes in several responses were also observed. Our findings raise concerns regarding 44 long-term risks of e-cigarette use as neuroinflammation may contribute to behavioral changes and mood Introduction 48 Chronic inhalation of tobacco smoke is known to damage multiple cell types and cause a wide range of 49 diseases throughout the body. In particular, many pulmonary inflammatory diseases are caused and 50 affected by conventional tobacco use (1, 2). It is also known that nicotine affects brain development and 51 alters responses to addictive substances. Nicotine activates carcinogenic pathways, putting users at an 52 increased risk of cancer (3). With unproven claims to be a safer alternative to cigarette smoking, modern 53 electronic (e)-cigarette devices were introduced in 2003 as a novel nicotine delivery system (4, 5). The 54 JUUL TM , a device that gained popularity due to its sleek and concealable design, has utilized pods 55 containing e-liquids with enticing flavors such as Mango, Mint, and Crème Brulee (now discontinued)(6).

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However, the health effects of chronic inhalation of aerosols generated from pod devices remain largely 57 unknown.

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While the data on health effects of conventional tobacco are extensive, the data on e-cigarettes and vaping 60 devices are less established due to their recent entry to the market (7,8). In particular, research in this area 61 is impeded by the rapid evolution of vaping devices. The vape pens and cig-a-likes were the first e-62 cigarettes studied from 2007-2014, whereas the box Mods became highly popular and research on these 63 devices began around 2015. Pod devices, including the JUUL, were invented in 2016 and rapidly 64 dominated the market by 2017-2020, however, studies on the harmful effects of these types of vaping chronic effects of vaping are limited to e-cigarette aerosol inhalation models in animals but have 74 demonstrated more profound effects, including renal, cardiac and liver fibrosis (10), emphysema (11), 75 lung cancer (12), increased lung injury in the setting of influenza infection (13), increased arterial 76 stiffness and atherosclerosis, and activation of addiction neurocircuits in the brain (14,15).

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The health effects of vaping JUUL pods remain unknown, despite the popularity of pod-based e-devices.

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Here, we broadly assessed the effects of daily JUUL aerosol inhalation on cardiopulmonary function and 80 inflammation across organ systems, including the reward pathways in the brain. We induced acute lung 81 injury with inhaled E. coli lipopolysaccharide (LPS) to determine whether chronic JUUL use predisposes 82 to deleterious responses in the setting of common infectious challenges such as Gram-negative bacterial 83 pneumonia. We demonstrated here that daily inhalation of JUUL aerosols can lead to inflammatory 84 changes in the brain, heart, lung, and colon, as well as alterations in physiological functions.

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Chronic JUUL inhalation associated with neuroinflammation in the brain

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Previous studies have shown that conventional tobacco smoking increases proinflammatory cytokines in 89 the brain, specifically TNFα, IL-1β, 17). Therefore, gene expression of these inflammatory 90 cytokines were measured by qPCR in different brain regions of mice exposed to JUUL Mango and JUUL 91 Mint, as well as Air controls for 1 or 3 months. Specifically, we assessed gene expression in the nucleus 92 accumbens core and shell (NAc-core and NAc-shell), and hippocampus, regions involved in behavior 93 modification, formation of drug reward and anxious or depressive behaviors, and learning and memory, 94 respectively. We observed that TNFα gene expression was significantly increased in the NAc-core and 95 NAc-shell of mice exposed to 1 or 3 months of JUUL Mango or JUUL Mint compared to Air controls 96 ( Figure 1A-D). In contrast, TNFα levels were unchanged in the hippocampus throughout the exposures 97 ( Figure 1E-F). IL-1β gene expression was also significantly elevated in JUUL Mint-and Mango-exposed 98 mice group in both the NAc-core and NAc-shell at 1 month compared to air control group ( Figure 1G and 99 1I), but remained elevated at 3 months only in the NAc-shell ( Figure 1J) and not in NAc-core ( Figure   100 1H). The hippocampus showed unchanged levels of IL-1β gene expression at 1 and 3 months across 101 groups ( Figure 1K and 1L). In the case of IL-6, we observed a significant increase in gene expression in 102 the NAc-shell in both JUUL Mango and JUUL Mint groups at 1 and 3 months (Figure 1O and 1P), but 103 no significant differences were observed in the NAc-core and hippocampus when compared to Air 104 controls ( Figure 1M, 1N, 1Q and 1R). Overall, these data suggest that exposure to JUUL Mint and 105 JUUL Mango may induce neuroinflammation in brain regions responsible for behavior modification, drug 106 reward and formation of anxious or depressive behaviors (18).

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To further confirm the neuroinflammatory response associated with chronic JUUL exposure, we 109 measured levels of receptors for advanced glycation end products (RAGE) and its ligand high mobility 110 group box 1 (HMGB1) protein by western blot in the NAc-core, NAc-shell, and hippocampus of mice 111 exposed to JUUL Mango, JUUL Mint and Air at 1 and 3 months. RAGE and HMGB1 have been 112 implicated in inducing neuroinflammation (16), and previous studies have shown that HMBG1-1 and 113 RAGE expression are increased with exposure to cigarette smoke (19,20). No significant changes of 114 HMGB1 were observed in NAc-core at 1 or 3 months of JUUL aerosol exposure between groups ( Figure   115 2A-B). The NAc-shell, however, showed significant increase in HMGB1 at 1 and 3 months in mice 116 exposed to JUUL Mango and JUUL Mint relative to Air controls ( Figure 2C-D), and the increase was 117 more pronounced at 3 months ( Figure 2D). The hippocampus showed no changes in HMGB1 protein 118 expression at 1 month ( Figure 2E), and actually showed significant decrease in protein expression in 119 mice exposed for 3 months to either JUUL Mango or JUUL Mint as compared to Air controls ( Figure   120 2F). In the case of RAGE, the protein levels were not significantly altered in all tested brain regions 121 ( Figure 2G-I, 2K-L), except in the NAc-shell of the mice exposed to JUUL Mango or JUUL Mint for 3 122 months when compared to Air controls ( Figure 2J). Altogether, these data indicate that exposure to 123 aerosols from JUUL devices induced neuroinflammation in reward brain regions, particularly that of the 124 NAc-shell and NAc-core regions.

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Due to the altered inflammation observed in brain and cardiac tissue, we also assessed inflammation in 149 the colon, where cigarette smoking has been shown to alter inflammation and thereby promote chronic 150 digestive diseases (26,27). In terms of documenting the effects of e-cigarettes on gastrointestinal 151 inflammation, our knowledge is limited to only the study done by our research group, with a focus on 152 changes induced by aerosols generated by box Mods only (28). In order to assess JUUL induced changes 153 in the gastrointestinal tract, we examined inflammatory gene expression at 1 and 3 months of JUUL 154 exposure. JUUL Mango induced upregulation of TNFα, IL-6, and IL-8 relative to Air controls after 1 155 month exposure (Figure 4A, 4C, 4G). Interestingly, at 3 months, JUUL Mango treatment resulted in less 156 expression of TNFα, IL-6 and IL-1β than that observed in Air controls or JUUL Mint exposed mice 157 ( Figure 4B, 4D, 4F), but increased expression of CCL2 ( Figure 4J). These data suggest that exposure to 158 JUUL Mango aerosols modulates inflammation in the colon, primarily inducing key inflammatory 159 cytokines at 1 month. In JUUL Mango and Mint, there was no change in IL-1B or CCL2 at 1 month 160 ( Figure 4E and 4I) and IL-8 at 3 months ( Figure 4H).

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Daily JUUL aerosol inhalation does not alter heart rate and blood pressure.

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Chronic exposure to cigarette smoke leads to cardiovascular changes, mediated through altered autonomic 164 tone, but little is known about the chronic cardiovascular effects of e-cigarettes, especially with pod 165 devices (7). Thus, we exposed mice to JUUL aerosols and carried out assessments of cardiovascular 166 function, including blood pressure (BP), heart rate (HR), and heart rate variability (HRV). Heart rate 167 variability was determined from root-mean square differences of successive R-R intervals (RMSSD) and 168 the mean of the standard deviations for all R-R intervals (SDNN). There were no significant changes in 169 HR or HRV at 1 and 3 months of either JUUL Mint or JUUL Mango exposure relative to Air controls 170 (Supplemental Figure 1). Similarly, systolic and diastolic BP were also unchanged relative to Air 171 controls at either 1 or 3 months (Supplemental Figure 1)

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Our previous studies with mice exposed to aerosols generated from Vape pens not only found fibrosis in 198 cardiac tissue after 3-6 months of e-cigarette aerosol inhalation but also in the liver and kidneys (10).

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Cigarette smoking is also known to cause organ fibrosis (30). There were, however, no significant

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Inhaled LPS is a model of Gram-negative bacterial pneumonia and acute lung injury in mice. Mice circulation, but also in the heart (31). It is common for patients to develop myocardial inflammation and 225 even ischemia during lung infections (31, 32). Tobacco smoking is well known to increase cardiovascular 226 diseases and worsen outcomes in the setting of pneumonia (2, 33) and recently, it has been suggested that 227 dual use of e-cigarettes with conventional tobacco leads to significantly higher odds of cardiovascular 228 disease compared with cigarette smoking alone (34). Thus, we assessed the impact of acute lung injury on 229 inflammation in cardiac tissues of JUUL exposed mice.

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Enhanced inflammatory responses within tissues are known to result in fibrosis in some cases. However, 245 analysis of pro-fibrotic gene expression only revealed that Col3a1 was significantly higher after 3 month 246 JUUL Mint exposure (Figure 6T). Col1a1 and TLR4 expression were not higher in the JUUL exposed 247 groups. Indeed, periostin expression was lower in 1 month JUUL Mint and JUUL Mango compared to 248 Air ( Figure 6U) and TLR4 expression was also lower in the 3 month JUUL Mango group (Figure 6X). little is known about the effects of e-cigarette use on the brain and gastrointestinal system. In this study, 266 we found that mice exposed to flavored JUUL aerosols may induce significant neuroinflammation in the 267 brain (Figure 8). The nucleus accumbens in particular was found to have elevated levels of inflammatory 268 markers, including TNFα, IL-1β and IL-6 in both NAc-core and NAc-shell, and HMGB1-1 and RAGE in

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Exposure to drugs such as methamphetamine, cocaine and ethanol activate neuroinflammatory pathways 283 that are associated with the release of HMGB1-1 in the striatum and nucleus accumbens, potentially 284 linked to addictive behaviors and drug reward (45, 47). The overall similarity in inflammatory profiles 285 and brain regions between drugs of abuse and that observed in this model of chronic JUUL exposure is 286 certainly cause for concern as it suggests that e-cigarette use may be associated with addictive behaviors 287 (48). Further studies into the overlap of induced neuroinflammatory pathways between drugs of abuse and 288 JUUL is required to better understand these relationships. This includes studies in human subjects to 289 assess the incidence of anxiety and depression in JUUL users. A correlation had previously been observed 290 between vaping and mental health (49). Furthermore, many e-cigarette vapers, including JUUL users, are 291 also cigarette smokers. In some cases, smokers use JUULs as an attempt to help with smoking cessation.

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However, data thus far demonstrates that e-cigarettes do not increase the rates of successful smoking 293 cessation attempts (14). Indeed, neuroinflammatory effects caused by chronic, daily JUUL exposure may 294 lead to adaptations in neural circuitry that promote addictive behaviors and drug dependence, providing a 295 neurophysiological explanation for the observation that JUUL use does not help with smoking cessation.

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The basis for the variation between the two different JUUL flavored pods tested in our study is most 312 likely due to the differences in chemical flavorants. We observed significantly different inflammatory 313 gene alterations in cardiac and colonic tissue in response to chronic exposure to Mint and Mango JUUL 314 vapor (Figure 8). Ethyl maltol concentrations have been shown to be highest in Mango pods (1 mg/ml), 315 while menthol concentrations are highest in cool Mint pods (10 mg/ml)(51). The most remarkable 316 variations we observed were in response to acute lung injury through LPS challenge, where significantly 317 higher levels of cardiac inflammatory genes were seen in mice exposed to Mint relative to Mango and 318 controls. In the brain, inhalation of JUUL Mint aerosols led to higher TNFα and IL-1β in the NAc-shell 319 relative to JUUL Mango. Mint aerosols are highly similar to menthol aerosols and previous studies have 320 shown greater increases in neuronal nAChR receptors after exposure to nicotine with menthol relative to 321 nicotine exposure alone (52). As a result, we surmise that the flavoring compound menthol in "Cool 322 Mint" may be a factor leading to differences seen in the effects of Mint vs Mango. Overall, these findings 323 suggest that components other than nicotine may contribute to the observed neuroinflammatory changes.

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Further research is needed to better understand how specific, non-nicotine JUUL components contribute 325 to inflammatory and neuronal effects.

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Collagen expression is a hallmark of fibrosis and has previously been observed in studies involving 328 combustible cigarette smoke (30). However, compared to our prior study with Vape pens (using the nose-329 only InExpose system by SciReq) where profound increases in collagen deposition were observed across 330 cardiac, hepatic and renal issues (10), we did not find increased fibrosis in these same organs in JUUL  (26). Nicotine specifically has been previously found to decrease the expression of pro-342 inflammatory cytokines in the colon (53). Here, we saw an increase in pro-inflammatory cytokines in the 343 colon after 1 month of chronic exposure, whereas these same signals were significantly decreased after 3 344 month exposure when compared to the control group (Figure 8). Thus, over the course of the 3 months, 345 the body may adapt to these changes and downregulate these markers significantly through some yet 346 unidentified mechanism, pathway, or interaction with the specific components in the JUUL device. infarction and non-ischemic cardiac injury has been increasingly appreciated (56). For example, IL-1β 355 blockade has been shown to diminish adverse cardiac events and heart failure progression (57, 58). We 356 demonstrate here that the hearts of mice subject to chronic JUUL exposure are significantly more sensitive 357 to the effects of LPS delivered to the lung than are Air control mice, as evidenced by enhanced expression 358 of pro-inflammatory cytokines and chemokines including IL-1β. The observation that there were no 359 significant changes in vagal tone (assessed by heart rate variability) and that the pro-inflammatory 360 enhancement by JUUL exposure was largely confined to JUUL Mint, suggests that this is not due to signals 361 generated by direct nicotine action. While the mechanism by which chronic JUUL exposure predisposes to 362 LPS-induced cardiac inflammation remains to be determined, these findings suggest that chronic JUUL 363 inhalation could lead to systemic changes which sensitize maladaptive inflammatory responses that affect 364 cardiac function. 365 366 Contrary to our initial expectations, we did not find significant changes in autonomic tone or pulmonary 367 function with daily, long-term JUUL aerosol exposure. Our model is limited in that mice are primarily 368 nose breathers and we used whole-body exposure, so it is possible that the extent of e-cigarette aerosol 369 exposure at the level of the alveoli may be lower than in humans due to aerosol deposition within the 370 nasal cavity. Alternatively, our study may be underpowered to detect subtle differences induced by JUUL 371 vaping. However, it is important to mention that this is the first study assessing JUUL devices in a 372 multiorgan fashion. In addition, we found clear effects of JUUL aerosols on inflammatory responses in 373 organs other than the lungs. Thus, the effects of e-cigarette exposure may be greater on organs far 374 removed from the lungs, the first organ to come in contact with aerosols.

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The right kidney, one lobe of liver, and the base of the heart were then immediately dissected after 427 euthanasia and placed in Z-fix at 4°C. After 48 h, all organs were moved to 75% ethanol and submitted to 428 the University of California, San Diego histology core for paraffin embedding. Collagen was detected in 429 5-μm sections first by Masson's trichrome stain. All histology slides underwent quantification of fibrosis 430 by calculating the mean percent fibrotic area in 15-25 randomly acquired ~20 images using computer-431 aided morphometry performed using ImageJ. Briefly, using the color threshold with default thresholding 432 method, red threshold color and HSB color space, the total area of tissue in the slide was selected and 433 measured, later the tissue stained for Masson's trichrome blue was also selected and measured prior 434 adjustment of the "Hue" parameter (Saturation Brightness/Value Each colour shade). Then, a percentage 435 of the area stained by Masson's trichrome blue was determined relative to the total tissue area. All 436 histology slides from the same tissue group were blinded and underwent these computer analyses in an 437 identical fashion. Fibrotic area is presented relative to that of air controls.

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Isolation of RNA from the murine colonic tissue and qRT-PCR for inflammatory cytokines 440 RNA was isolated from mouse colon tissues using the Zymo miniprep kit according to the manufacturer's 441 instructions, followed by cDNA synthesis. Quantitative Real-Time PCR was conducted for target genes 442 and normalized to housekeeping gene 18S rRNA. Primer sequences are provided in Table 1