Role of Caspases and Gasdermin A during HSV-1 Infection in Mice

Round 1

Reviewer 1 Report

The study describes the role of capspases and gasdermins during HSV-1 mouse infection models. Mice deficient in specific cell death genes (caspases-1/11, -6, -7, -8, -14 and gasdermins) were screened for their role in innate immunity against HSV-1. In their Mouse flank scarification model, they showed that caspase-6 -/- mice developed more sever lesions and showed high mortality compared to WT mice. In their footpad model caspase-6 -/- mice had significantly higher viral burden in brain at day 4 and the viral loads were not significantly different at day 7. In corneal infection model they showed that caspase-6 is dispensable for the control of HSV-1 infection. Infection with HSV-1 ?US3 strain was less infectious in Caspase -6 -/- mice than WT strain. They also showed that Casp7–/– is not required to control HSV-1 skin infection. Then they used skin inoculation route and found that RIPK3-deficient mice (Casp8+/– Ripk3–/–) developed more severe skin lesions compared with WT mice. Finally, they showed that caspase-14 and gasdermin A is not required for defense against HSV skin infection. Overall, this study showed that HSV-1 uses various pathways to evade host immune response. The study provides better insights into host innate immunity against HSV-1, however; there are few things that needs to be addressed.

Page 3, sections 2.3, 2.4 and 2.5; authors should mention the dose in PFU/mL or TCID50/mL rather than uL. They may provide the titers of the stock virus in their methods. Why have they used more dose in footpad model compared flank or corneal models?

They should also provide details in about the number of mice sacrificed at different days etc in methods or prepare a supplementary figure on their experimental design for all the models.

Page 6, section 3.2; why they have a smaller number of samples at day 7 compared to day 4? The non-significant difference here could be because of the lesser number of samples.

They have used various infection models, but they have not tested the samples for viral loads in all experiments. Is there any reason behind that?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

This work is overall uses appropriate methods and models, is very well controlled, and the conclusions justified by the data. The writeup is scholarly and clear. I commend the authors on this important work. I have only the most minor comments.

Vendors are missing for many reagents in the methods.

For western blots, samples were presumably normalized by protein concentration or some other means. Please describe.

To support the conclusion that the knockout is normal, Fig 5E could benefit from a wt mouse ear to allow the reader to visually compare the two. Otherwise, a lengthier description of what features exactly were examined, with references on their normal status, may suffice

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

This manuscript studied the effort of caspases and gasdermins contributing to innate immune defenses against HSV-1 infection. The authors utilized three different mouse models to evaluate the role of caspases-1/11, -6, -7, -8, -14, and the adaptor protein ASC involved in mediating pyroptosis. Overall, the data/analysis presented that skin lesions, tissue viral titer, and survival were not associated with caspases. But this manuscript also found that caspase-6 and caspase1/11 deficient mice are more susceptible to HSV-1 skin infection. Despite the authors did not find an impressive phenotype, the study method and analysis have been carefully presented and reasonably interpreted in this paper.

There are a few concerns listed below that need to be addressed.

1.   Figure 1: In panel B, please provide Stat information on 8 dpi and 10 dpi. In panel C, please improve the resolution of lesion pictures and also provide the lesion score next to the picture.

2.   In line 178, to minimize the effect of the microbiota, the authors infected littermate control mice for comparison viral loads in indicated tissues. But the authors should also provide the skin or gut microbiota composition data.

3.   I believe the dash-line in each dot plot represents the limit of detection. Please provide the explanation in the figure legends.

4.   Figure 5 quality is poor. Please improve the resolution of all the panels.

Author Response

Please see the attachment.

Author Response File: Author Response.docx