Effects of mango and mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart, and colon in mice

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1. 

   Version published to 10.7554/elife.67621 on eLife

   Apr 12, 2022
2. 

   eLife

   Mar 31, 2022

   Author Response:

   Reviewer #1 (Public Review):

   Major Comments

   I am concerned that a lot of these studies had relatively low n numbers (n=5 in some cases) and that some of the studies may have been underpowered. Given the variability with in vivo studies, some endpoints may have been significant with more numbers. Along these lines, what is the justification for using the (parametric) ANOVA test. I'm not a statistician but I thought that the rule of thumb was that non-parametric tests should be used if n<12 since you cannot verify that the data is normally distributed. In this case, I would recommend having a statistician look at it and/or increasing some of the N's, or using the non-parametric Kruskal-Wallis test. Indeed, in some cases, the variation the variation is quite large (ie Fig 6, 7). Whilst I do not think that the low N's …

   Author Response:

   Reviewer #1 (Public Review):

   Major Comments

   I am concerned that a lot of these studies had relatively low n numbers (n=5 in some cases) and that some of the studies may have been underpowered. Given the variability with in vivo studies, some endpoints may have been significant with more numbers. Along these lines, what is the justification for using the (parametric) ANOVA test. I'm not a statistician but I thought that the rule of thumb was that non-parametric tests should be used if n<12 since you cannot verify that the data is normally distributed. In this case, I would recommend having a statistician look at it and/or increasing some of the N's, or using the non-parametric Kruskal-Wallis test. Indeed, in some cases, the variation the variation is quite large (ie Fig 6, 7). Whilst I do not think that the low N's change the ultimate conclusions, but more rigor (ie more N's) would help solidify the paper given that it will likely be of great interest and scrutinized by the scientific community.

   We conducted power analyses prior to the start of the studies to identify the number of animals per group to use, based on our past studies of inflammatory changes induced by inhalants, infections and asthma. We set the target number of mice (n) at that time, such that these studies would be powered to detect a 25% change in cytokine expression. We did go through and reviewed all of the data with our biostatisticians, we came to the conclusion that it would not be statistically appropriate to run more mice to increase the n when our primary outcome remains the same. We double-checked that the ANOVAs with corrections for multiple comparisons were correct for each particular experiment. Discussion with our statistician confirmed that ANOVA is correct as long as the data passed normality testing, which was done. An additional point, and most relevant to this specific recommendation, JUUL Mint and JUUL Mango flavors are no longer on the market, such that extensive further studies are not feasible. While these two flavors are not available anymore, they were composed of an array of chemicals commonly found in other flavors (but in different combinations), such that we believe that these data are most likely relevant to other vapes. In particular, JUUL Mint shares chemical features with JUUL Menthol, which took its place as one of the most popular JUUL flavors. The discontinuation of these flavors has been added as a limitation within the Discussion

   Fig S3. For the lung histology, please quantify the mean linear intercept per ATS guidelines and show representative BAL images.

   We have conducted the mean linear intercept (MLI) measurements on e-cigarette aerosol exposed lungs and controls per ATS guidelines and have added these data to the manuscript (new Appendix 1- Figure 4M). We paired these data with the original histology images (Appendix 1 – Figure 4A-4L). We have added appropriate methods (pages 21-22) and results (page 9) as well. Of note, the MLI data matches our original physiologic assessments of lung function (Appendix 1 – Figure 2A-2J), including elastance and compliance, which are known to change in the setting of emphysema. MLI, lung elastance and compliance were no different across inhalant groups and controls. Further, we have taken representative images of Giemsa Wright stained BAL samples, and have added these to the manuscript (new Appendix 1 Figure - 3E-3J and 3O-3T) paired with BAL cell count data.

   One of the most novel conclusions from this paper is increased inflammation in the brain which the authors speculate could lead to altered moods and or change the addiction threshold. I would tend to agree with this conclusion, but could the authors perform additional mouse psychological tests to confirm this? Also, were there observable physiological responses in the vaped mice that could be reported which may correlate this conclusion, ie changes in grooming, fur ruffling or other behavioral changes?

   We are thrilled that the Reviewer is as interested in these implications as we are, because we believe the neuroinflammation detected is quite frightening, particularly because it is likely to impact both behavior and mood. We have added further discussion regarding the potential consequences of inflammation in each of the organs (pages 13-19), with an emphasis on the effects of neuroinflammation on behavior and psychology. We have subdivided the Discussion section to highlight potential effects on each distinct organ.

   While we are not a behavioral lab, and thus running behavioral studies in mice is beyond the scope of both our lab and this manuscript, we agree that the neuroinflammation is of great interest and further studies are needed to best assess potential psychological and behavioral changes. Of note, we did not observe any overt behavioral changes - we closely observe the mice both during and after exposures and make notes regarding grouping, fur, and activity level - none of which were changed by the different vaping exposures. We have added the lack of dedicated behavioral and psychological evaluations as a limitation of this work and as an opportunity for discovery in future studies (page 19- 20).

   Minor comments Change title to state "in mouse". That this study was performed in rodents should be apparent from the outset.

   Actually, our original title does contain “in mice” at the end. Apologies if these words were cut off on your end. We do agree that the title should be apparent that the study was conducted in mice. We wanted to make the title even clearer, so replaced the brand name JUUL with the type of e-device. The title is as follow: “Effects of Mango and Mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart and colon in mice”

   No changes in collagen deposition were detected using basic histology. Have the reviewers considered performing immunohistochemistry and staining for alpha-smooth muscle actin which may be a more sensitive assay?

   We agree with the reviewer that there are more sensitive tools that can be used. We believe that, in our system, and at 3 months of exposure, JUUL Mint and Mango are not very likely to induce fibrosis, since our data of inflammatory markers and fibrosis associated genes (in homeostatic conditions, Figure 3) show that there are not significant differences, and in some markers, JUUL Mint and Mango exposed mouse lungs are even showing less inflammation than Air controls. In addition, we also showed no differences were obtain in physiological assessment (heart rate, heart rate variability or blood pressure, Appendix 1 – Figure 1). Thus, we do not expect to find significant differences even with additional assays. We are planning on challenging mice with bleomycin in the future, as it may be possible to detect differences in fibrosis in the setting of this pro-fibrotic challenge.

   "Thus long term exposure to Juul does not lead to significant changes...". I would argue that 1-3 months is not long term. Indeed, other researchers have performed 6-12 month ecigarette exposures and it takes a lifetime in humans to develop lung disease after smoking. Since you can detect pro-inflammatory changes but no altered physiology, it may be that alterations in airway physiology are only just beginning.... The authors should modify this sentence and maybe not call their studies "long term".

   We agree with the reviewer and have modified the sentence as follows for a more accurate interpretation of our results (page 9): “Thus, 1 and 3 month exposure to JUUL Mint and Mango aerosols may not cause significant changes in airway physiology, but this does not preclude the possibility that changes may occur with longer exposures, such as 6-12 months.” We have also gone through the entire the manuscript to focus on describing our exposure in terms of months instead of the descriptive terms acute / sub-acute / chronic, and we have removed the word chronic from the title.

   "Differences in LPS induced cytokine levels were no longer observed after 3 month JUUL exposure versus Air control groups". As per the major comments, this might be a power issue - there is certainly a trend for some cytokines.

   It has been seen in prior studies that chronic inhalant use (including and most notably cigarette smoke) can lead to proinflammatory changes in the first days to weeks, but opposite effects thereafter. For example, cigarette smoke inhalation leads to inflammatory changes at 4 weeks that resolve by 12 weeks. Thus, we feel that some of the cytokine findings are not unusual or surprising versus other patterns of inhalant use. However, we agree with the reviewer that IL-1b in cardiac tissue trends in the same direction at 3 months in both JUUL Mint and JUUL Mango exposed mice (Figure 8C and 8D). As per one reviewers’ comments, we combined 1 and 3 month data for merged graphs (Appendix 1 – Figure 4) and when analyzed together (data passed normality testing) further differences at 3 months were identified (see IL-1b in Appendix 1 – Figure 4 panel 4B). We have included these additional figures for each dataset in the Appendix 1 files.

   Of note, because some JUUL flavors are no longer on the market, including JUUL Mint and JUUL Mango, we are unable to run additional studies with these flavors. We are running new studies of the impact of JUUL Tobacco and JUUL Menthol, the two remaining JUUL flavors on the market. However, these studies will take an additional 1- 2 years and thus are beyond the scope of this manuscript. We have expanded the limitation section within the discussion with regards to power, in order to clarify to the readers that some findings are limited by the number of subjects.

   Reviewer #2 (Public Review):

   Under homeostasis conditions, the authors observed sign of inflammatory responses in the brain, the heart and the colon, while no inflammation was detected in the broncho-alveolar lavage fluid of the mice following exposures to JUUL aerosols. Also, JUUL aerosol exposures mediated airway inflammatory responses in the acute lung injury model (LPS). Further, this infection affected the inflammatory responses in the cardiac tissue. Most of the biological adverse effects induced by JUUL aerosols were flavor-specific.

   Strengths include evaluating inflammation in multiple organs, as well as assessing the physiological responses in the lungs (lung function) and cardiovascular system (heart rate, blood pressure), following exposures to JUUL aerosols. Weaknesses include the fact that only female mice were used in this study. Further, the daily exposures to either air or to the JUUL aerosols lasted only 20 min per day. It is unclear how a 20-min exposure is representative of human vaping product use. Also, although daily exposures were conducted for a duration of both 1 and 3 months, time-course effects associated with JUUL aerosols are barely addressed.

   We would like to thank the reviewer for their positive comments on our manuscript. We apologize for our error; in reality we exposed mice for 20 minutes three times daily, so one hour in total per day. We have corrected this error within our Methods. We designed the exposures this way to better mimic human e-cigarette use throughout the day (instead in one intense vaping session per day, which is not the norm). We agree that there is a limitation in using only female mice in the study in case that there are sex-dependent effects, which is definitely an interesting question. We typically start with one sex of mice and then run repeat experiments with the other sex. Unfortunately, this study faced problems beyond our control that prevented us from performing further experiments. In late 2019 the FDA was moving to ban specific flavors for pod devices, which include those for Mint and Mango. In anticipation of the new regulations, JUUL ultimately decided to discontinue JUUL Mint and Mango, and soon they were out of the market. The same process occurred with the other popular JUUL flavors such as Crème Brûlée and Cucumber. We have expanded the limitation section within the Discussion, and have pointed out that because these studies were conducted in female mice alone, the results may not represent effects in males.

   Although there are a few limitations related to this study, which should be included in the manuscript, overall, the authors' claims and conclusions are based on the data that is presented through multiple figures.

   We appreciate the Reviewers comments and have added limitations about the study size, power, lack of male subjects, etc. to the discussion section.

   Reviewer #3 (Public Review):

   Weaknesses

   1. The authors observed neuroinflammation in brain regions responsible for behavior modification, drug reward and formation of anxious or depressive behaviors after exposure to JUUL. The importance of the neuroinflammation is still unclear. It would help demonstrate the pathogenic role of the neuroinflammation by testing animal behaviors. Similar issue for other organ inflammation.

   We are an immunology, inflammation, and lung physiology lab, thus, behavioral studies are beyond the scope of both our lab and this manuscript. However, we agree that the neuroinflammation is of great interest and is highly likely to impact behavior and mood. Further studies are needed to best assess potential psychological and behavioral changes. We believe this work is important to share such that dedicated behavioral science labs can undertake these important studies. We have added these important limitations to the discussion.

   1. Majority of the data are inflammatory cytokine mRNA expression. Other methods would be needed to confirm their expression.

   Of note, in the original submission, we included protein quantification data for both the brain and the lung. We have taken the reviewers comments to heart and have conducted protein-level assays on the cardiac tissues as well, yielding additional data (new Figure 4) that has been added to the methods, results, figures and discussion. Unfortunately, we do not have any additional colonic tissue for protein-level assessments, as all of the tissue was used for the gene transcription and histologic studies. But to take a step back, these studies were originally intended to examine the broad reaching impact of e-cigarette aerosols across the body. This work, and thus this manuscript, was designed to highlight changes at the gene expression level, to demonstrate that e-cigarette use is not benign and does have broad-reaching effects on gene expression. We agree that more work is needed to fully define the impact of e-cigarette use at the protein, cellular, and organ level, but the majority of that work is beyond the scope of this manuscript. To bring the focus back to gene expression, we have conducted RNAseq on the lungs of JUUL exposed mice, and have included those data herein to highlight the effects of ecigarette aerosols on gene expression in the lung, with a particular focus on differences between Mint and Mango flavors (the most popular JUUL flavors at the time of this study). These new data (new Figure 6) support the hypothesis that e-cigarette aerosol inhalation fundamentally alters the lung, which raises the specter of downstream health effects.

   1. The author seemed to assume the difference between JUUL Mango and JUUL Mint is flavor and then came up with the conclusion regarding flavor-dependent changes in several inflammatory responses. Evidence is needed to approve the assumption.

   Although the formulation of JUUL e-liquids is proprietary, their website claims simplicity (https://www.juul.com/learn/pods) in that they use pharmaceutical grade propylene glycol and glycerol (which makes up the majority of their e-liquids), in order to form an aerosol which carries pharmaceutical grade nicotine and benzoic acid (when combined, create a nicotine salt), and flavors (which can be a mixture of natural and artificial ingredients). Thus, according to their website the only difference among the different JUUL pods would be the flavoring components. Hence, we concluded that differences observed in our study between Mint vs Mango should be most likely due to flavor-dependent effects, since base components should be the same. To support this flavor-dependent effect, a study from Omaiye et al in 2019 (PMID: 30896936) showed the variety of different flavoring chemical in all JUUL flavors and how the different JUUL vapors induce different level of cytotoxicity in BEAS-2B cells in vitro based their flavor. We have added relevant discussion to the manuscript.

   1. In most cases, the change of inflammatory cytokines is mild ~2 fold. The author should demonstrate how these marginal changes could affect pathophysiology.

   We agree with the reviewer that the majority of changes in cytokines were relatively small. However, the fact that multiple cytokines are changing in concert indicates a significant shift in immunophenotyping across organs. We are most concerned about how these shifts in the inflammatory state will alter an e-cigarette vapers response to common clinical challenges. In Dr. Kheradmand’s recent work, mice exposed to e-cigarette aerosols with and without nicotine were much more susceptible to acute lung injury in the setting of viral pneumonia. In our work, we utilized the LPS model of acute lung injury to take a first look at the potential impact of JUUL inhalation in particular on susceptibility to lung inflammation. Further work is needed to truly define how the subtle, broad shifts in the cytokine milieu across organs will impact the health of e-cigarette vapers. We have added relevant discussion to the manuscript.

   1. To fully evaluate the health impact of evolving cigarette, it would be informative to included other tobacco or vaping device as control.

   We agree that such comparisons are likely to provide insight into the differences between devices and formulations and versus cigarette smoke, and thus will be incredibly important for the field. However, these comparisons were beyond the scope of this study, whose main goal was to assess the inflammatory and physiological aspects of JUUL in particular. We believe this to be important because JUUL e-cigarettes are the most popular of all e-cigarette devices, and many young users do not use other e-devices or conventional tobacco. Thus, our primary objective of this work was to specifically assess the safety or risk of this device in particular (versus not using any inhalant at all). However, because we have run parallel studies in the past with vape pens, box mods, and conventional tobacco, we are hopeful to start combining data to look for trends and differences across inhalant exposures. For example, we recently published our work on differences in metabolites in the circulation of mice exposed to a wide variety of ecigarette based inhalants (Moshensky et al. Vaping induced metabolomic signatures in the circulation of mice are driven by device type, eliquid, exposure duration and sex. ERJ Open. July 2021 PMID: 34262972). This study is one of the few studies that have employed animal models to test JUUL devices and the only one assessing their effects in different organs, and although we agree that comparisons with other devices is important, it was not the goal of this study.

   1. The longest exposure in the study is 3 months. It is not convicting to come up with conclusions regarding chronic exposure. Some organ showing no difference may be due to the timing.

   We have altered the wording throughout the manuscript to clarify that the 3-month duration is equivalent to 10 to 20 years of inhalant use versus 40 to 50 years for a 6 to 12 month model. We have also removed many instances of the descriptive terms acute, sub-acute and chronic across the manuscript, as focused on using the absolute duration of exposure instead, to avoid accidental extrapolation to longer exposures. Because we utilized cellular and molecular based assays, we were not relying on identifying organ level pathology such as fibrosis, emphysema, and organ dysfunction, all of which would require longer exposures.

   Read the original source
3. 

   eLife

   Mar 29, 2022

   Evaluation Summary:

   This is a potentially important paper that aimed to investigate the effects of vaping on multiple organs. The authors show multi-organ inflammatory responses of JUUL exposure in mice. While the rationale of the current study if of high interest and timely, the manuscript in its current form remains largely descriptive and some of the conclusions are not clearly supported by the data. A major limitation is the lack of investigation of (causal) pathophysiological consequences/general organ outcomes that might be driven by the reposted inflammatory response.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
4. 

   eLife

   Mar 29, 2022

   Reviewer #1 (Public Review):

   This is a very interesting and potentially very important paper that shows multi-organ effects of JUUL exposure in mice. I have some major and minor comments which are listed below.

   Major Comments:

   I am concerned that a lot of these studies had relatively low n numbers (n=5 in some cases) and that some of the studies may have been underpowered. Given the variability with in vivo studies, some endpoints may have been significant with more numbers. Along these lines, what is the justification for using the (parametric) ANOVA test. I'm not a statistician but I thought that the rule of thumb was that non-parametric tests should be used if n<12 since you cannot verify that the data is normally distributed. In this case, I would recommend having a statistician look at it and/or increasing some of the N's, or using …

   Reviewer #1 (Public Review):

   This is a very interesting and potentially very important paper that shows multi-organ effects of JUUL exposure in mice. I have some major and minor comments which are listed below.

   Major Comments:

   I am concerned that a lot of these studies had relatively low n numbers (n=5 in some cases) and that some of the studies may have been underpowered. Given the variability with in vivo studies, some endpoints may have been significant with more numbers. Along these lines, what is the justification for using the (parametric) ANOVA test. I'm not a statistician but I thought that the rule of thumb was that non-parametric tests should be used if n<12 since you cannot verify that the data is normally distributed. In this case, I would recommend having a statistician look at it and/or increasing some of the N's, or using the non-parametric Kruskal-Wallis test. Indeed, in some cases, the variation the variation is quite large (i.e. Fig 6, 7). Whilst I do not think that the low N's change the ultimate conclusions, but more rigor (i.e. more N's) would help solidify the paper given that it will likely be of great interest and scrutinized by the scientific community.

   Fig S3. For the lung histology, please quantify the mean linear intercept per ATS guidelines and show representative BAL images.

   One of the must novel conclusions from this paper is increased inflammation in the brain which the authors speculate could lead to altered moods and or change the addiction threshold. I would tend to agree with this conclusion, but could the authors perform additional mouse psychological tests to confirm this? Also, were there observable physiological responses in the vaped mice that could be reported which may correlate this conclusion, i.e. changes in grooming, fur ruffling or other behavioural changes?

   Minor comments:

   Change title to state "in mouse". That this study was performed in rodents should be apparent from the outset.

   No changes in collagen deposition were detected using basic histology. Have the reviewers considered performing immunohistochemistry and staining for alpha-smooth muscle actin which may be a more sensitive assay?

   "Thus long term exposure to Juul does not lead to significant changes...". I would argue that 1-3 months is not long term. Indeed, other researchers have performed 6-12 month e-cigarette exposures and it takes a lifetime in humans to develop lung disease after smoking. Since you can detect pro-inflammatory changes but no altered physiology, it may be that alterations in airway physiology are only just beginning.... The authors should modify this sentence and maybe not call their studies "long term".

   "Differences in LPS induced cytokine levels were no longer observed after 3 month JUUL exposure versus Air control groups". As per the major comments, this might be a power issue - there is certainly a trend for some cytokines.

   Read the original source
5. 

   eLife

   Mar 29, 2022

   Reviewer #2 (Public Review):

   Under homeostasis conditions, the authors observed sign of inflammatory responses in the brain, the heart and the colon, while no inflammation was detected in the broncho-alveolar lavage fluid of the mice following exposures to JUUL aerosols. Also, JUUL aerosol exposures mediated airway inflammatory responses in the acute lung injury model (LPS). Further, this infection affected the inflammatory responses in the cardiac tissue. Most of the biological adverse effects induced by JUUL aerosols were flavor-specific.

   Strengths include evaluating inflammation in multiple organs, as well as assessing the physiological responses in the lungs (lung function) and cardiovascular system (heart rate, blood pressure), following exposures to JUUL aerosols. Weaknesses include the fact that only female mice were used in this …

   Reviewer #2 (Public Review):

   Under homeostasis conditions, the authors observed sign of inflammatory responses in the brain, the heart and the colon, while no inflammation was detected in the broncho-alveolar lavage fluid of the mice following exposures to JUUL aerosols. Also, JUUL aerosol exposures mediated airway inflammatory responses in the acute lung injury model (LPS). Further, this infection affected the inflammatory responses in the cardiac tissue. Most of the biological adverse effects induced by JUUL aerosols were flavor-specific.

   Strengths include evaluating inflammation in multiple organs, as well as assessing the physiological responses in the lungs (lung function) and cardiovascular system (heart rate, blood pressure), following exposures to JUUL aerosols. Weaknesses include the fact that only female mice were used in this study. Further, the daily exposures to either air or to the JUUL aerosols lasted only 20 min per day. It is unclear how a 20-min exposure is representative of human vaping product use. Also, although daily exposures were conducted for a duration of both 1 and 3 months, time-course effects associated with JUUL aerosols are barely addressed.

   Although there are a few limitations related to this study, which should be included in the manuscript, overall, the authors' claims and conclusions are based on the data that is presented through multiple figures.

   Read the original source
6. 

   eLife

   Mar 29, 2022

   Reviewer #3 (Public Review):

   In this study, Alex Moshensky et al. investigated effects of chronic aerosol inhalation of flavored JUUL on inflammatory markers in several organs, including brain, lung, heart, and colon in a mouse model. They found that JUUL inhalation upregulated a number 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 LPS were exacerbated in the heart. They also found flavor-dependent changes in several responses.
   Overall, it is a descriptive study and the conclusions was not clearly supported by the data.

   Strengths:

   Due to the rapid evolution of vaping devices, the data on health effects of Pod devices are scarce. This study provides useful information on …

   Reviewer #3 (Public Review):

   In this study, Alex Moshensky et al. investigated effects of chronic aerosol inhalation of flavored JUUL on inflammatory markers in several organs, including brain, lung, heart, and colon in a mouse model. They found that JUUL inhalation upregulated a number 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 LPS were exacerbated in the heart. They also found flavor-dependent changes in several responses.
   Overall, it is a descriptive study and the conclusions was not clearly supported by the data.

   Strengths:

   Due to the rapid evolution of vaping devices, the data on health effects of Pod devices are scarce. This study provides useful information on the inflammatory change caused by chronic JUUL aerosol inhalation.

   Weaknesses:

   1. The authors observed neuroinflammation in brain regions responsible for behavior modification, drug reward and formation of anxious or depressive behaviors after exposure to JUUL. The importance of the neuroinflammation is still unclear. It would help demonstrate the pathogenic role of the neuroinflammation by testing animal behaviors. Similar issue for other organ inflammation.

   2. Majority of the data are inflammatory cytokine mRNA expression. Other methods would be needed to confirm their expression.

   3. The author seemed to assume the difference between JUUL Mango and JUUL Mint is flavor and then came up with the conclusion regarding flavor-dependent changes in several inflammatory responses. Evidence is needed to approve the assumption.

   4. In most cases, the change of inflammatory cytokines is mild ~2 fold. The author should demonstrate how these marginal changes could affect pathophysiology.

   5. To fully evaluate the health impact of evolving cigarette, it would be informative to included other tobacco or vaping device as control.

   6. The longest exposure in the study is 3 months. It is not convicting to come up with conclusions regarding chronic exposure. Some organ showing no difference may be due to the timing.

   Read the original source
7. 

   Version published to 10.1101/2021.03.09.434442v1 on bioRxiv

   Mar 10, 2021