Nicotine in E-Cigarettes Dysregulates Pulmonary Inflammation and MMP-12 Expression without Effecting Respiratory Syncytial Virus Virulence
and Patrick Geraghty 2,*
Round 1
Reviewer 1 Report
Manuscript by Goldenberg et al offers interesting findings on the impact of nicotine while using in vitro and A/J mice exposed to E-cig vapors. Study also highlights the importance of inflammation in RSV infection. There are several concerns and comments when addressed would help in improving the quality of the manuscript. In general, single doses for e-cig vapor, diacetyl and nicotine would be difficult to arrive at meaningful conclusions.
Specific Comments
Figure 1A. The JUUL e-cig had a flavor of Kiwiberries Ice pod, but same e-cig was not used for animal or in vitro experiments. Authors decided not to use flavored e-cig for in vitro and in vivo experiments yet they included diacetyl as one of the components which is a chemical used to flavor e-cigs. The inclusion of diacetyl in the study should be justified.
Figure 1B. The trace showing methanol, ethanol and diacetyl could be moved to right so it is away from the propylene glycol peak. The picture quality of traces could be improved.
Figure 2A. When vapor inhalation from e-cig, 3 ‘puffs’ were passed through the artificial lung structure how long was the puff? What was the inter-puff interval? What was the composition of PG:VG in the e-liquid?
Figure 3. How were the cells exposed to the e-cig vapors? Were nicotine and diacetyl added directly to the growth medium? If the goal is to explore impact of nicotine in e-cig then nicotine containing e-cig vapor would be ideal. How did the authors decide on the doses for 1% e-cig vapor (puff profile not provided), nicotine and diacetyl? Perhaps growth curves could be provided as supplemental data for readers to understand the dose-range studied. The experimental design or the legend do not explain these points. In addition, labels for the genes need to be consistent and ‘n>4 subjects per group’ should be corrected as these are cell lines.
Figure 4. What was the e-cig profile for exposing animals (was 0.4 ml of e-liquid vaped using JUUL device? Was it done for per animal or per cage? Was PBS alone exposure done in presence of PG:VG?).
Figure 4 B and D. The PV loops can vary just based on the weight of animals, was this put into consideration for calculating AUC? The PV loops and AUC need to include the Mock+PBS to resolve this issue, especially when in 4E the same group is included for comparison.
Figure 5A. There is no change in overall inflammation score in the lungs yet there are differences in inflammatory cytokines in the BALF: this should be discussed further, a supporting evidence could be provided by number of immune populations involved. Were the lungs from mice undergoing lung function analysis taken for the histological analysis as well? Data in this figure is depicting protein levels for MMP-12 and TNF-alpha in BALF as well as IFN-beta gene expression yet the legend is misleading into stating gene expression for all the three genes, please clarify. It would certainly be helpful to support the data with changes in protein levels in the BALF but methodology and discussion on these need to be included in the body of the manuscript. Also, discussion in lines 251-253 is not supported by data. Could the authors also comment on possible influence on alveolar injury by nicotine, since they already have the histology sections.
Minor points:
Line 203: states cigarette vapor with nicotine increased MMP12 expression; should be e-cigarette vapor
Line 212: each measurement was performed with n= or> 4 subjects per group but clearly there are 6-7 data points for each gene presented; please verify.
Line 255: II-6 should be written as IL-6
Author Response
There are several concerns and comments when addressed would help in improving the quality of the manuscript. In general, single doses for e-cig vapor, diacetyl and nicotine would be difficult to arrive at meaningful conclusions.
Response: We appreciate the thoughtful input from the reviewer. Regarding a single dose, we do think it is important to examine changes due to acute exposure (i.e., a single dose) versus chronic exposure (animal experiment where disease parameters are altered at the sampling stage). Are these changes due to the vaping directly or a subsequent change due to disease establishment? In this case, it appears that short exposure to recondensed vaping liquid influence these changes. For this reason, we have left the single-dose data in the manuscript but have included the following sentence into the manuscript on lines 246 and of the manuscript copy with track changes:
… a short exposure to recondensed vaping liquid modulated these changes and remained altered in our chronic exposure model in mice.
And
… we utilized a single dose in our vitro model. This was undertaken to determine whether an acute exposure to nicotine and e-cigarette vapor could alter MMP-12, TNFα and IFNβ responses or whether tissue remodeling was required to change these parameters.
Figure 1A. The JUUL e-cig had a flavor of Kiwiberries Ice pod, but same e-cig was not used for animal or in vitro experiments. Authors decided not to use flavored e-cig for in vitro and in vivo experiments yet they included diacetyl as one of the components which is a chemical used to flavor e-cigs. The inclusion of diacetyl in the study should be justified.
Response: We have now removed the diacetyl data from the manuscript and included the following sentence on line 251 of the manuscript copy with track changes:
“Due to the recent ban on flavors in e-cigarette that target young users this year in the USA [25], we have primarily focused on the other components of the e-cigarette liquid in our in vitro cell and in vivo mouse studies. However, we focus on non-flavorants, we do want to stress the importance of detecting Inhaled diacetyl vapors were detected in our GC analysis using flavored e-cigarette liquids and diacetyl is associated with flavorings-related lung disease, such as bronchiolitis obliterans [26].”
Figure 1B. The trace showing methanol, ethanol and diacetyl could be moved to right so it is away from the propylene glycol peak. The picture quality of traces could be improved.
Response: We have moved the trace as suggested. We have included high-resolution images as EPS for each figure not embedded into the word file. Embedding the images into word as recommended by the journal does impact the quality of the image
Figure 2A. When vapor inhalation from e-cig, 3 ‘puffs’ were passed through the artificial lung structure how long was the puff? What was the inter-puff interval? What was the composition of PG:VG in the e-liquid?
Response:
For figure 1 and 3 the following was added on lines 76-81: “The syringe was used to draw in 40 mL of vapor in 4 seconds from one puff generated from the e-cigarettes. Although this method is manual, a 4-second puff was utilized as suggested based on previously e-cigarette topography [17]. In order to mimic a smoking regime, three 4-second puffs separated by 10-second intervals between puffs were manually drawn into the syringe. Subsequent recondensed vapor was utilized for gas chromatography analysis and cell experiments. There was a 50:50 ratio of PG:VG in all e-cigarette liquids.”
Regarding the artificial lung, we performed a similar approach to above and have added additional information in the methodology on lines 95-96 of the manuscript copy with track changes:
“Samples were collected from three 4-second puffs separated by 10-second intervals at the end of the artificial bronchi and were analyzed via GC-FID.”
Figure 3. How were the cells exposed to the e-cig vapors? Were nicotine and diacetyl added directly to the growth medium? If the goal is to explore impact of nicotine in e-cig then nicotine containing e-cig vapor would be ideal. How did the authors decide on the doses for 1% e-cig vapor (puff profile not provided), nicotine and diacetyl? Perhaps growth curves could be provided as supplemental data for readers to understand the dose-range studied. The experimental design or the legend do not explain these points. In addition, labels for the genes need to be consistent and ‘n>4 subjects per group’ should be corrected as these are cell lines.
Response: E-cigarette liquid was vaporized and collected manually with a syringe as outlined above. The subsequent recondensed vapor was added to the media. The e-cigarette vapor did contain nicotine. We have now added another group to figure 3, e-cigarette vapor without nicotine, and removed the diacetyl group. The concentrations of vapor and nicotine were determined based on cell viability assays. We chose concentrations that resulted in no loss of cell viability. We have now included cell viability data for several concentrations and removed the diacetyl data in Figure 3. We have added more detail to the methods for the puff profile. This information is present in the methodology on lines 76-81 and 102-107 of the manuscript copy with track changes.
We have also adjusted labels throughout the manuscript and corrected the text to reflect cell line and not subjects. This information is present in the methodology on lines 198-199 of the manuscript copy with track changes:
“Data are represented as mean ± S.E.M., where each measurement was performed on several independent days”
Figure 4. What was the e-cig profile for exposing animals (was 0.4 ml of e-liquid vaped using JUUL device? Was it done for per animal or per cage? Was PBS alone exposure done in presence of PG:VG?).
Response: Yes, 0.4 ml of e-cigarette liquid was vaporized using the JUUL device into the mouse aerosol pie cage. This amount of vapor was for the entire cage and not per animal. PBS was added alone. E-cigarette vapor without nicotine was the PG:VG control. This information is present in the methodology on lines 123-126 of the manuscript copy with track changes:
“During exposures, up to 12 animals were housed in a mouse aerosol pie cage (Braintree Scientific, Braintree, MA) and vapor was generated from 0.4 ml of PBS alone or e-cigarette (PG:VG with and without nicotine) and delivered to all twelve animals. Puffs were separated by 60-second intervals.”
Figure 4 B and D. The PV loops can vary just based on the weight of animals, was this put into consideration for calculating AUC? The PV loops and AUC need to include the Mock+PBS to resolve this issue, especially when in 4E the same group is included for comparison.
Response: All animal weights were inputted into the Scireq software prior to PV loops and the software takes these weights into consideration when generating the PV loops. Therefore, the current PV loops and AUC were corrected for weight. We have now included the Mock+PBS data into the PV loops and AUC graphs/curves. Please see figure 4D and lines 138-139 for these changes.
Figure 5A. There is no change in overall inflammation score in the lungs yet there are differences in inflammatory cytokines in the BALF: this should be discussed further, a supporting evidence could be provided by number of immune populations involved. Were the lungs from mice undergoing lung function analysis taken for the histological analysis as well? Data in this figure is depicting protein levels for MMP-12 and TNF-alpha in BALF as well as IFN-beta gene expression yet the legend is misleading into stating gene expression for all the three genes, please clarify. It would certainly be helpful to support the data with changes in protein levels in the BALF but methodology and discussion on these need to be included in the body of the manuscript. Also, discussion in lines 251-253 is not supported by data. Could the authors also comment on possible influence on alveolar injury by nicotine, since they already have the histology sections.
Response: Unfortunately, immune cells were not collected from the BALF to quantify immune cell populations. Separate groups of animals were used for histology/BALF and pulmonary function. We do observe that occasionally small traces of blood can get into the lungs during the pulmonary function testing (just an observation when processing samples). Therefore, we quantified BALF and tissues from mice without pulmonary function processing. We have discussed the inflammation changes observed here in greater depth on lines 276-285, 300-304, 307-313, and 319-321 of the manuscript with track changes.
We have changed the text in the figure legends not to confuse the reader for protein and gene expression changes. We unsuccessfully tried to quantify protein levels of IFN-beta in BALF. Therefore, we utilized gene expression only for this target. We have listed this and other limitations in the discussion on lines 323-333 of the manuscript with track changes.
We have modified the wording on lines 251-253 to better reflect the data.
We have made additional comments on alveolar injury on lines 276-285.
Line 203: states cigarette vapor with nicotine increased MMP12 expression; should be e-cigarette vapor
Response: We have made this change.
Line 212: each measurement was performed with n= or> 4 subjects per group but clearly there are 6-7 data points for each gene presented; please verify.
Response: The reviewer is correct. We have made this change to be n= or > 6.
Line 255: II-6 should be written as IL-6
Response: We have made this change.
Reviewer 2 Report
The article “Nicotine in e-cigarettes dysregulates pulmonary inflammation and MMP-12 expression without effecting respiratory syncytial virus virulence” is an interesting study aiming to clarify the possible effect of e-cigarettes vapor on the immune response to infections. The interest in the e-cigarette vapor effect is a topical theme, given the growing amount of people using these devices.
The article is well-written, and the exposure of the content is clear and effective.
Although, some revisions are requested, mainly concerning the English style and some misprints.
Abstract. The authors should consider the possibility to stress the relevance of the results obtained.
Introduction. The sentences starting at lines 40 and 45 are a bit cumbersome and could be re-phrased in order to make their comprehension more immediate.
Results. The title of the y-axis of the second panel of figure 5B reported: “IFNγ” instead of “IFNβ” as described in the figure caption and in the main text.
Discussion. The correct use of “conventual” at line 217 should be checked (maybe in this case the correct word could be “conventional”). The authors may also consider the possibility to give some additional information on the function of the biomarkers and molecules studied, in order to better contextualize the meaning of the results, stress the relevance of these results for human beings, and thus make the whole article more readable, even for readers who come from other fields.
Author Response
Although, some revisions are requested, mainly concerning the English style and some misprints.
Response: We appreciate the thoughtful input from the reviewer.
Abstract. The authors should consider the possibility to stress the relevance of the results obtained.
Response: We have modified the abstract as requested.
Introduction. The sentences starting at lines 40 and 45 are a bit cumbersome and could be re-phrased in order to make their comprehension more immediate.
Response: We have modified these sentences as requested.
Results. The title of the y-axis of the second panel of figure 5B reported: “IFNγ” instead of “IFNβ” as described in the figure caption and in the main text.
Response: We have corrected this typo in Figure 5B.
Discussion. The correct use of “conventual” at line 217 should be checked (maybe in this case the correct word could be “conventional”). The authors may also consider the possibility to give some additional information on the function of the biomarkers and molecules studied, in order to better contextualize the meaning of the results, stress the relevance of these results for human beings, and thus make the whole article more readable, even for readers who come from other fields.
Response: We have added additional information on lines 300-304, 307-313, and 319-321 of the manuscript with track changes. We do think that it should remain pulmonary focused as this is a pulmonary journal. We have corrected the typo on line 217.
Round 2
Reviewer 1 Report
The authors have adequately responded to the reviewer's comments and made the necessary changes.
