Maternal Cigarette Smoke Exposure Does Not Impair Influenza Vaccine Responsiveness in Murine Offspring

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors evaluated the impact of maternal cigarette smoke (CS) exposure on influenza vaccine responsiveness in offspring. Their results showed that CS exposure had no significant effect on the vaccine efficacy. The experimental design is reasonable and the controls are appropriate. The results are well presented.

However, there are a few comments about the paper:

1. Under the material and method section: please provide the animal ethics approval for this study.
2. Under Section 3.1. in the text, it showed that this study used 42 female mice, with 16 in the air-exposed group and 26 in the CS-exposed group. The air-exposed group obtained 18 pups and 25 pups in CS group. However in Table 1, under the explanation for b (line 232), it states that “ bValues indicate the number of pregnant dams per group (from a total of n = 16 for the air group and n = 26 for the CS group).” The animal numbers are not consistent.
3. In Figure 2, it shows that in the air-exposed group, n = 3 animals were used as control and n = 8 animals were vaccinated, please give explanation for the rest of the animals (18-11= 7); the same for the CS-exposed group, 19 animals (control group n = 8, vaccine group n = 11) were used for the experiment, please give information about the rest 6 mice.
4. In Figure 3, the splenocytes were analyzed by cell flow cytometry, please include in the method when the spleen samples were collected.
5. In Table 1, it shows that the male mice in the CS-exposed group has a significant higher spleen-to-body weight ratio, in the discussion section, the authors did not discuss the indication of this result. If possible, please provide some explanation about this result.

Minor:

Please uniform the reference format.

Comments on the Quality of English Language

N/A

Author Response

Thank you very much for your insightful comments, which have provided valuable guidance for improving and strengthening our manuscript.

 

Comments:

1. Under the material and method section: please provide the animal ethics approval for this study.

 

Answer: The ethical approval for all animal procedures has been added to the Materials and Methods section in the revised manuscript (page 3, lines 118–122).

 

2. Under Section 3.1. in the text, it showed that this study used 42 female mice, with 16 in the air-exposed group and 26 in the CS-exposed group. The air-exposed group obtained 18 pups and 25 pups in CS group. However in Table 1, under the explanation for b (line 232), it states that “ bValues indicate the number of pregnant dams per group (from a total of n = 16 for the air group and n = 26 for the CS group).” The animal numbers are not consistent.

 

Answer: We thank the reviewer for pointing out this inconsistency. Of the 42 female mice, 16 were in the air-exposed group and 26 in the CS-exposed group. Among these, 6 air-exposed and 7 CS-exposed females became pregnant, while the remaining mice were non-pregnant (Air: 10 non-pregnant; CS: 19 non-pregnant). Table 1 has been clarified to indicate that the values correspond to pregnant dams only, and the text in Section 3.1 has been revised to reflect this (revised manuscript, page 6, lines 224-226, lines: 239-240).

 

3. In Figure 2, it shows that in the air-exposed group, n = 3 animals were used as control and n = 8 animals were vaccinated, please give explanation for the rest of the animals (18-11= 7); the same for the CS-exposed group, 19 animals (control group n = 8, vaccine group n = 11) were used for the experiment, please give information about the rest 6 mice.

 

Answer: We thank the reviewer for pointing this out. This pilot study also evaluated the effect of a dietary intervention on vaccination efficacy in the context of maternal CS exposure. Therefore, the remaining animals were allocated to dietary intervention groups (Air-vaccinated + diet: n = 7; CS-vaccinated + diet: n = 10). However, as no significant effect of maternal CS exposure on vaccine efficacy was observed, the dietary intervention groups were not relevant to the main findings reported here. To reduce confusion, the dietary intervention groups have been removed from the manuscript, and the results include only the control and vaccinated groups. The number of animals in each group is presented in the table 1. The reason for these groups exclusion is now clarified in the revised version (page 7, lines 255–260).

Table 1. Number of male and female offspring in the six study groups.

 

Maternal Exposure	Total Offspring	Sex (M/F)	Control (M/F)	Vaccine (M/F)	Diet Intervention (M/F)
Air	18	7/11	1/2	3/5	3/4
CS	29	9/20	2/6	4/7	3/7
Total	47	16/31	3/8	7/12	6/11

 

4. In Figure 3, the splenocytes were analyzed by cell flow cytometry, please include in the method when the spleen samples were collected.

 

Answer: The time of spleen sample collection has been added to the Materials and Methods section in the revised manuscript (page 5, lines 179-180).

 

5. In Table 1, it shows that the male mice in the CS-exposed group has a significant higher spleen-to-body weight ratio, in the discussion section, the authors did not discuss the indication of this result. If possible, please provide some explanation about this result.

 

Answer: We have added further details about spleen-to-body weight ratio in the discussion section in the revised manuscript (page 9: lines 312-327).

Minor:

6. Please uniform the reference format.

Answer: The references have been reformatted and organized according to the journal’s instructions for authors in the revised version.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The paper is well written.

However, my biggest problem with the paper is what is relevant about your paper? You either have results that you weren't expecting that fit what has already been done or results that show that exposure doesn't harm the vaccines. 

Yes, negative results can be important, but I believe that the literature already has shown much of what countered your hypotheses.

So without something new what is the importance of your paper? I saw nothing in your results, discussion, or conclusion indicating anything new or of interest.

Without this there is nothing of particular value in your paper.

Perhaps there is and you can rewrite the paper in such a way that it emphasizes the importance of your results, but right now you have failed to accomplish this.

I am sorry to have to write such a negative review as the topic of indoor air pollution is important and your paper is otherwise good.

Author Response

Vaccines-3775023

 

Title: Maternal Cigarette Smoke Exposure Does Not Impair Influenza Vaccine Responsiveness in Murine Offspring

 

Thank you very much for your insightful comments, which have provided valuable guidance for improving and strengthening our manuscript.

 

Reviewer 2

Comment:

However, my biggest problem with the paper is what is relevant about your paper? You either have results that you weren't expecting that fit what has already been done or results that show that exposure doesn't harm the vaccines. 

Yes, negative results can be important, but I believe that the literature already has shown much of what countered your hypotheses.

So without something new what is the importance of your paper? I saw nothing in your results, discussion, or conclusion indicating anything new or of interest.

Without this there is nothing of particular value in your paper.

Perhaps there is and you can rewrite the paper in such a way that it emphasizes the importance of your results, but right now you have failed to accomplish this.

I am sorry to have to write such a negative review as the topic of indoor air pollution is important and your paper is otherwise good.

Answer: Thank you for your thoughtful feedback. We acknowledge the concern regarding the novelty and relevance of our findings. While maternal CS exposure did not significantly impair vaccine efficacy under the conditions tested, our study provides important and novel insights for several reasons.

Although some immune parameters were altered by maternal CS exposure, notably an increase in activated Th2 cells, our results demonstrate that influenza vaccine efficacy remains intact in the offspring. This suggests a remarkable resilience of vaccine-induced protection despite environmental immunomodulation, a finding that contrasts with concerns that pollutants may compromise vaccine responses. These results provide novel insight into the robustness of vaccine performance in adverse environmental contexts, indicating that current vaccination strategies may still be effective even in populations exposed to air pollution. We also emphasize that these results should be interpreted cautiously, as variations between studies are likely influenced by differences in exposure routes, pollutant composition, exposure duration, and study protocols. Nonetheless, our findings contribute to a better understanding of early-life immune development in the context of maternal pollutant exposure and lay a foundation for future studies examining broader aspects of immune function and long-term health outcomes. This point, originally discussed in the Discussion section, has now also been included in the Conclusion section of the revised manuscript (page 12, lines 444–448).

Reviewer 3 Report

Comments and Suggestions for Authors

Overall Impression

This manuscript describes an experiment in which female mice were exposed to air or cigarette smoke (CS) during pregnancy and lactation. The offspring were vaccinated as young adults and some immune parameters in the offspring were measured. The key idea examined was whether or not CS exposure early in life would skew immune responses of mice in the Th2 direction relative to unexposed mice. The immune parameters selected were designed to help differentiate relative differences in Th1 (cell mediated) and Th2 (humoral) type immune responses in the peripheral blood (IgG1 and IgG2a antibodies), spleen (Th1, Th2, Treg and Th17 cell markers), and skin (delayed type hypersensitivity responses).

The manuscript is well-written. The methods are adequately described and are based on procedures regularly employed by these researchers. The experiment is well-designed and includes controls measures. The parameters measured are relevant for the hypothesis examined. Appropriate references are provided. The Discussion is a bit long, given the essentially negative results observed (relative to the hypothesis examined).

The manuscript suffers from unremarkable results relative to the framework described in the Introduction and the hypothesis examined. In other words, cigarette smoke did little in this study. The main factor that influenced the dependent variables examined was vaccination.

 

Comments for Authors

1. The evidence that CS did anything to the young mice is limited. The statistical results provided basically tell us CS decreased the body weight of pups and increased the spleen/body weight ration of male pups. The most internally consistent aspect of the results confirm that the vaccinations seemed to influence most, but not all, measures of the immune system examined irrespective of CS or air exposure.
2. The statistical tools used to evaluate the data are provided in the Materials and Methods. The general description is adequate. However, details of the specific factors used in the analyses, especially the ANOVA, are not reported in detail. This is important as the results in Table 1 suggest a statistically significant increase in spleen/body weight ratio in male offspring but not female offspring. This suggests an examination of sex in analyses of cells from the spleen (splenocytes) and perhaps other markers should include sex as a factor. Perhaps sex influenced the antibody or DTH responses as well as spleen/body wright ratios? Such results are not provided; thus, potentially interesting interactions of treatment x sex may be lurking in either/both of the antibody, DTH, or splenocyte data.
3. CS exposure was given to female mice during pregnancy and to mothers with their pups during lactation. This means any effects of CS in the pups might be the result of CS exposure in utero, or during lactation, or a combination of the two. The authors briefly acknowledge some timing limitations of CS exposure; differentiating in utero from post-natal CS exposure might be important to understanding what is going on. This is certainly a place for future research.
4. The selection of BALB/c mice for this experiment is important. These mice are predominantly a Th2-biased mouse. Might the results be different in a Th1-biased mouse, such as those on the C57BL/6 background? Perhaps a test of a Th2 effect of CS would emerge from testing mice with a lesser propensity to generate Th2 responses? This is something the authors might want to consider.
5. The results obtained may point to the possibility that the immune parameters (e.g., DTH and antibodies) and selected or the immune compartment examined (spleen and peripheral blood) to test the CS hypothesis of Th1-Th2 immune modulation might not be the best markers for what the authors want to learn. The examination of Treg and Th17 cell markers may be an attempt to get at this issue; it is unfortunate these markers did not result in effects of CS/air or vaccination status.
6. The first sentence of the Abstract can be removed since the data from the experiment conducted are not consistent with any clinical outcomes and no clinical outcomes were measured in the mice used in this experiment.
7. The statement in the Discussion (page 10, line 377) that states ‘offspring were not exposed to CS postnatally’ seems inconsistent with the Methods described (page 4, line 142) stating ‘exposure continued daily for 6 weeks from gestational day 4 to the end of lactation’. This apparent conflict should be resolved.

 

Author Response

Vaccines-3775023

 

Title: Maternal Cigarette Smoke Exposure Does Not Impair Influenza Vaccine Responsiveness in Murine Offspring

 

Thank you very much for your insightful comments, which have provided valuable guidance for improving and strengthening our manuscript.

 

Reviewer 3

Comments:

This manuscript describes an experiment in which female mice were exposed to air or cigarette smoke (CS) during pregnancy and lactation. The offspring were vaccinated as young adults and some immune parameters in the offspring were measured. The key idea examined was whether or not CS exposure early in life would skew immune responses of mice in the Th2 direction relative to unexposed mice. The immune parameters selected were designed to help differentiate relative differences in Th1 (cell mediated) and Th2 (humoral) type immune responses in the peripheral blood (IgG1 and IgG2a antibodies), spleen (Th1, Th2, Treg and Th17 cell markers), and skin (delayed type hypersensitivity responses).

The manuscript is well-written. The methods are adequately described and are based on procedures regularly employed by these researchers. The experiment is well-designed and includes controls measures. The parameters measured are relevant for the hypothesis examined. Appropriate references are provided. The Discussion is a bit long, given the essentially negative results observed (relative to the hypothesis examined).

The manuscript suffers from unremarkable results relative to the framework described in the Introduction and the hypothesis examined. In other words, cigarette smoke did little in this study. The main factor that influenced the dependent variables examined was vaccination.

Answer: we appreciate your comment. We agree that CS exposure did not produce strong effects on vaccine outcomes in our study. However, we believe this finding is still important. Negative or modest results help refine hypotheses and avoid publication bias toward only strong or positive effects. Our data show that, although maternal CS exposure enhanced activated Th2 cells in offspring, vaccine efficacy, as measured by DTH responses and antibody levels, remained intact. This resilience of vaccine-induced immunity under CS exposure conditions (dose, duration, route, and timing) provides novel insight, as few studies have directly addressed vaccine responses in this context. We also acknowledge the limitations of our model and emphasize the need for further studies under different exposure scenarios, as discussed in the Discussion (page 10, lines 356–358) and highlighted in the Conclusion (page 12, lines 444–448) sections.

Comments for Authors

1. The evidence that CS did anything to the young mice is limited. The statistical results provided basically tell us CS decreased the body weight of pups and increased the spleen/body weight ration of male pups. The most internally consistent aspect of the results confirm that the vaccinations seemed to influence most, but not all, measures of the immune system examined irrespective of CS or air exposure.

 

Answer: We agree that the most consistent and statistically robust findings in our study relate to the effects of vaccination on immune outcomes, while the effects of maternal CS exposure were limited to reduced body weight in pups and increased spleen/body weight ratio in male offspring. Nevertheless, we believe the subtle effects of CS observed are informative, as they suggest that under the current exposure conditions, vaccine-induced immunity appears resilient, despite measurable changes in growth and spleen to body weight ratio. The substantial impact of vaccination on the immune system, alongside the limited effects of maternal CS exposure, has been highlighted in the revised version of the Conclusion (page 12, lines 433-434).

 

2. The statistical tools used to evaluate the data are provided in the Materials and Methods. The general description is adequate. However, details of the specific factors used in the analyses, especially the ANOVA, are not reported in detail. This is important as the results in Table 1 suggest a statistically significant increase in spleen/body weight ratio in male offspring but not female offspring. This suggests an examination of sex in analyses of cells from the spleen (splenocytes) and perhaps other markers should include sex as a factor. Perhaps sex influenced the antibody or DTH responses as well as spleen/body wright ratios? Such results are not provided; thus, potentially interesting interactions of treatment x sex may be lurking in either/both of the antibody, DTH, or splenocyte data.

 

Answer: We thank the reviewer for this important comment. We have revised the Materials and Methods section to provide additional details on the statistical analyses, including the specific factors used in the two-way ANOVA. Treatment (CS vs. air) and vaccination (PBS vs. influenza vaccine) were included as factors.

Sex-specific differences were not analyzed in this study, as the primary objective was to evaluate the effects of maternal CS exposure on vaccine efficacy in offspring. The power calculation was based on offspring randomization without sex stratification, and the relatively small numbers in some subgroups limited the feasibility of sex-specific analyses. We acknowledge this as a limitation and have added it to the Material and Methods (page 6, lines 216–217) and Discussion sections (pages 11-12, lines 424-431) of the revised manuscript. We agree that future studies with larger sample sizes should explore potential sex-specific variations in immune responses.

 

3. CS exposure was given to female mice during pregnancy and to mothers with their pups during lactation. This means any effects of CS in the pups might be the result of CS exposure in utero, or during lactation, or a combination of the two. The authors briefly acknowledge some timing limitations of CS exposure; differentiating in utero from post-natal CS exposure might be important to understanding what is going on. This is certainly a place for future research.

Answer: We acknowledge this as a limitation and have added it to the Discussion section of the revised manuscript (page 11, lines 411-416).

4. The selection of BALB/c mice for this experiment is important. These mice are predominantly a Th2-biased mouse. Might the results be different in a Th1-biased mouse, such as those on the C57BL/6 background? Perhaps a test of a Th2 effect of CS would emerge from testing mice with a lesser propensity to generate Th2 responses? This is something the authors might want to consider.

Answer: We acknowledge this as a limitation and have added it to the Discussion section of the revised manuscript (page 10, lines 364-367).

 

5. The results obtained may point to the possibility that the immune parameters (e.g., DTH and antibodies) and selected or the immune compartment examined (spleen and peripheral blood) to test the CS hypothesis of Th1-Th2 immune modulation might not be the best markers for what the authors want to learn. The examination of Treg and Th17 cell markers may be an attempt to get at this issue; it is unfortunate these markers did not result in effects of CS/air or vaccination status.

 

Answer: We appreciate your thoughtful point regarding the selection of immune markers. Our rationale for focusing on DTH, antibody responses, and cells from spleen and peripheral blood was that these are well-established and frequently used readouts to assess Th1–Th2 modulation following vaccination (DOI: 10.3389/fimmu.2021.797152, DOI: 10.3389/fimmu.2018.00452, DOI: 10.3390/vaccines9030254).

We agree that Treg and Th17 cells represent important immune compartments that could further elucidate the role of cigarette smoke in shaping immune modulation. For this reason, we included analyses of these subsets. Although no significant effects were observed in our study, this negative finding is still informative, suggesting that the impact of CS exposure on vaccine-induced responses might not primarily involve Treg/Th17 pathways, at least under the conditions tested.

We acknowledge that other immune compartments or functional assays (e.g., lung-resident cells or cytokine profiling) may provide additional insight into the mechanisms underlying CS-induced modulation of vaccine responses. We acknowledge this as a limitation and have added it to the Discussion section of the revised manuscript (page 11, lines 421-423).This is an important avenue for future research.

 

6. The first sentence of the Abstract can be removed since the data from the experiment conducted are not consistent with any clinical outcomes and no clinical outcomes were measured in the mice used in this experiment.

Answer: We thank the reviewer for this suggestion. The first sentence of the Abstract has been removed. The revised Abstract now focuses solely on the experimental findings.

 

7. The statement in the Discussion (page 10, line 377) that states ‘offspring were not exposed to CS postnatally’ seems inconsistent with the Methods described (page 4, line 142) stating ‘exposure continued daily for 6 weeks from gestational day 4 to the end of lactation’. This apparent conflict should be resolved.

Answer: We thank the reviewer for pointing out this inconsistency. The conflicting text has been corrected in the revised manuscript to ensure consistency between the Methods and Discussion.

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript is well written and describes a relevant subject, the impact of air pollutants on offspring development through maternal functions. 

Author Response

Comment: 

This manuscript is well written and describes a relevant subject, the impact of air pollutants on offspring development through maternal functions. 

Answer: We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the relevance of the topic.