Sodium Pyruvate Ameliorates Influenza A Virus Infection In Vivo

Round 1

Reviewer 1 Report

Very interested study, well designed, conclusions justified by the results.

I think that the data in Tab. 2 aren't necessary for this paper, short descriptions with references would be enough.

Author Response

We thank the editor and reviewers for their time and careful consideration of our manuscript titled, “Sodium Pyruvate Ameliorates Influenza A Virus Infection In Vivo.” We greatly appreciate the suggestions and that the reviewers found the study “interesting, well designed, and relevant.” Below, we have provided a point-by-point response.

Reviewer 1

Very interested study, well designed, conclusions justified by the results.

1. I think that the data in Tab. 2 aren't necessary for this paper, short descriptions with references would be enough.

As two reviewers have recommended the removal of table 2, we have removed it and summarized it in the manuscript.

Reviewer 2 Report

In the current manuscript, the author expand on their previous report demonstrating that pyruvate treatment affected the inflammatory responses of macrophages following in vitro influenza A virus (IAV) infection. The rationale for this study, to evaluate the impact of sodium pyruvate administration on de novo IAV infection, is sound. And the experimental approaches the authors utilize to test their hypothesis are relevant, well-characterized in the field of IAV immunobiology, and adequately described in the manuscript. While their results are clearly presented, my initial enthusiasm for the study was dampened by its incomplete nature. My comments:

 

1. While Table 2 provides useful information concerning previous clinical findings with N115 and human respiratory infections, it's placement in the beginning of the text is a bit odd. The study's focus is a murine model of IAV infection and the impact of N115 treatment, rather than human respiratory infections directly. The Table also discusses age differences and co-morbidities- variables not addressed in the authors' in vivo studies. As such, the Table would fit better as Supplemental Data.
2. The authors test for potential toxic effects with their "in-house"  nebulized NaPyr treatment but not with N115. Presumably the authors noted no toxic or adverse effects with N115 treatment.
3.  How was the 20mM N115 dose established? Was this the only dose the authors were able to test? Any way to compare this dose with the 10mM NaPyr the authors used in Figure 2?
4. Peak IAV titers in mice are typically detected between 2-4 days post-infection. While the data for day 7 is statistically significant, I suspect that the authors would have detected a greater difference in viral titers if they looked earlier.
5. Concerning Figure 4, since the data are combined from 2 independent experiments I would recommend using a scatter plot with means or medians indicated. This is key to showing whether the data is skewed to the point where non-parametric tests should be used rather than the Student's T-test.
6. For cytokine data, how was the assay normalized? Interpretation of the ELISA data is critically dependent on ensuring that the total protein in the homogenates is similar. At the very least, the tissue weight/mL should be similar.
7. The study just seems to end, leaving many questions unanswered. It would have been useful to examine lung pathology to establish whether N115 treatment reduced lung damage. The finding that IL-1b levels are reduced begs the question- does N115 treatment impact NLRP3 inflammasome activity in vivo? No Western blot data nor RT-qPCR data. I realize that it may be difficult/impossible to collect such data, but these approaches should at least be mentioned in the Discussion as future directions.
8. The Conclusion is severely lacking in development. The authors restate their observation by fail to address questions that readers in the fields of IAV biology and immunobiology would be asking themselves (see #7). The authors fail to mention next steps in their studies. Does N115 represent a modulator of inflammasome activity? Does N115 treatment protect as viral challenge increases? What potential impact would N115 have on the development of IAV immunity? Would this work in other animal models of IAV infection (ferrets, NHP).  

Author Response

We thank the editor and reviewers for their time and careful consideration of our manuscript titled, “Sodium Pyruvate Ameliorates Influenza A Virus Infection In Vivo.” We greatly appreciate the suggestions and that the reviewers found the study “interesting, well designed, and relevant.” Below, we have provided a point-by-point response.

-Reviewer 2

• In the current manuscript, the author expand on their previous report demonstrating that pyruvate treatment affected the inflammatory responses of macrophages following in vitro influenza A virus (IAV) infection. The rationale for this study, to evaluate the impact of sodium pyruvate administration on de novo IAV infection, is sound. And the experimental approaches the authors utilize to test their hypothesis are relevant, well-characterized in the field of IAV immunobiology, and adequately described in the manuscript. While their results are clearly presented, my initial enthusiasm for the study was dampened by its incomplete nature. My comments:

 

1. While Table 2 provides useful information concerning previous clinical findings with N115 and human respiratory infections, it's placement in the beginning of the text is a bit odd. The study's focus is a murine model of IAV infection and the impact of N115 treatment, rather than human respiratory infections directly. The Table also discusses age differences and co-morbidities- variables not addressed in the authors' in vivo studies. As such, the Table would fit better as Supplemental Data.

We thank the reviewer for the recommendation. In agreement with reviewer 1, we have discussed the results in table 2 in the manuscript but removed the table.

2. The authors test for potential toxic effects with their "in-house” nebulized NaPyr treatment but not with N115. Presumably the authors noted no toxic or adverse effects with N115 treatment.

We have not observed any toxic effects of N115. We have performed toxicity experiments and now include these data in Figure 3A.

3. How was the 20mM N115 dose established? Was this the only dose the authors were able to test? Any way to compare this dose with the 10mM NaPyr the authors used in Figure 2?

The 20mM N115 dose was established by EmphyCorp and their research on chronic obstructive pulmonary disease and other respiratory diseases. This is the only concentration of N115 available to us. We have used the clinical grade N115 provided by Emphycorp and did not have other doses available.

4. Peak IAV titers in mice are typically detected between 2-4 days post-infection. While the data for day 7 is statistically significant, I suspect that the authors would have detected a greater difference in viral titers if they looked earlier.

These data have been included into Figure 3D. Although viral titers are highest on day 3, we did not observe a difference between N115 treated and control mice in viral titers. This is in agreement with weight loss in Figure 3B, where we only saw significant improvements after day 7. This may suggest an immunological role in decreasing virus titers rather than a direct effect on virus replication.

5. Concerning Figure 4, since the data are combined from 2 independent experiments I would recommend using a scatter plot with means or medians indicated. This is key to showing whether the data is skewed to the point where non-parametric tests should be used rather than the Student's T-test.

We appreciate the reviewer’s thoughts on this, and we agree. We have made these changes.

6. For cytokine data, how was the assay normalized? Interpretation of the ELISA data is critically dependent on ensuring that the total protein in the homogenates is similar. At the very least, the tissue weight/mL should be similar.

ELISA samples were normalized using individual lung weights/ml. This has been added to the method section.

7. The study just seems to end, leaving many questions unanswered. It would have been useful to examine lung pathology to establish whether N115 treatment reduced lung damage. The finding that IL-1b levels are reduced begs the question- does N115 treatment impact NLRP3 inflammasome activity in vivo? No Western blot data nor RT-qPCR data. I realize that it may be difficult/impossible to collect such data, but these approaches should at least be mentioned in the Discussion as future directions.

We have included the future directions to further investigate the NLRP3 immunomodulation in vivo. In vitro we established the mechanism in BMDMs, but we do agree that in future directions, we should look to ensure that NaPyr is immunomodulating the NLRP3 inflammasome in vivo or if it could be a combination of mechanisms. These changes are in lines 305-333.

8. The Conclusion is severely lacking in development. The authors restate their observation by fail to address questions that readers in the fields of IAV biology and immunobiology would be asking themselves (see #7). The authors fail to mention next steps in their studies. Does N115 represent a modulator of inflammasome activity? Does N115 treatment protect as viral challenge increases? What potential impact would N115 have on the development of IAV immunity? Would this work in other animal models of IAV infection (ferrets, NHP).

We have added some ideas for future directions including other animal models. This new information is included in lines 305-333.

Reviewer 3 Report

The manuscript contain lots of important information, including the references and addressing a results of the previous studies. However I would recommend to include into a Introduction, more information about therapeutic properties of Sodium Pyruvate discovered for the potential treatment of other pathological conditions, including heart failure, hyperkeratotic disorder, diabetes, liver problems, hair loss, etc. In the Discussion I would suggest to focus on more detailed description of the mechanism of sodium pyruvate caused molecular changes in host metabolic pathways leading to the decrease of morbidity and weight loss, as well as improvement of pro-inflammatory cytokine production and lower virus titers. 

Author Response

We thank the editor and reviewers for their time and careful consideration of our manuscript titled, “Sodium Pyruvate Ameliorates Influenza A Virus Infection In Vivo.” We greatly appreciate the suggestions and that the reviewers found the study “interesting, well designed, and relevant.” Below, we have provided a point-by-point response.

-Reviewer 3

• The manuscript contain lots of important information, including the references and addressing a results of the previous studies.

1. However I would recommend to include into a Introduction, more information about therapeutic properties of Sodium Pyruvate discovered for the potential treatment of other pathological conditions, including heart failure, hyperkeratotic disorder, diabetes, liver problems, hair loss, etc.

We appreciate the reviewer’s suggestions for the introduction. We have included more references and uses of Pyr/NaPyr in lines 55-78.

2. In the Discussion I would suggest to focus on more detailed description of the mechanism of sodium pyruvate caused molecular changes in host metabolic pathways leading to the decrease of morbidity and weight loss, as well as improvement of pro-inflammatory cytokine production and lower virus titers.

We have included in the discussion the future directions to further investigate the NLRP3 immunomodulation in vivo. In vitro we established the mechanism in BMDMs, but we do agree that in future directions, we should look to ensure that NaPyr is modulating the NLRP3 inflammasome in vivo or if it could be a combination of mechanisms. These changes are in lines 305-333.

Round 2

Reviewer 2 Report

The authors have thoroughly and sufficiently addressed each of the concerns I raised during my review of their initial submission. The revised manuscript is much improved. No additional revisions are needed.