Deletion of Perilipin 5 Protects against Hepatic Injury in Nonalcoholic Fatty Liver Disease via Missing Inflammasome Activation

Round 1

Reviewer 1 Report

The manuscript under review by Asimakopoulou et al. describes the protective effect of Perilipin 5 deletion in a murine model of nonalcoholic fatty liver disease by attenuated NLRP3 inflammasome activation. The article is well written, easily understandable and results and methods are presented in a clear fashion. The article meets the general CELLS publication criteria.

Minor comments:

• For statistical analysis of more than two groups such as in figure 3 (4 groups) the authors should use ANOVA (or Kruskal-Wallis-testing when values are non-parametrically distributed)
• Page 2 lines 82 and 84; page 4 line 163: °C
• Figure 2 c: the H&E stainings are quiet dark and the white balance could be improved

Author Response

Dear Reviewer 1,

many thanks for your help in improving our manuscript. Please find the response to your comments in the attached pdf-file.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript by Asimakopoulou, et al., focuses on the role of PLIN5 as a regulator of hepatic homeostasis in non-alcoholic steatohepatitis development. Particularly, the authors explore the effects of PLIN5 on the regulation of NLRP3-mediated inflammatory responses in hepatocytes, using an animal model and in vitro approaches. The manuscript shows some potentially interesting findings and may be of clinical relevance considering the lack of effective therapeutic approaches for the treatment of NAFLD. However, major points should be answered in order to confirm the therapeutic significance of blocking PLIN5 expression in NAFLD/NASH patients.

 

Major comments

• In the preclinical model carried out, it seems that this high-fat diet does not achieve a significant level of NASH development, as inflammatory markers are not very high and there is no leukocyte recruitment. Consequently, it may be difficult to infer the relevance of blocking PLIN5 expression in the regulation of inflammatory signaling pathways in NASH patients. This point should be widely discussed and it would be interesting to perform key experiments in order to assess the role of PLIN5 in other models with a substantial inflammatory damage.
• Authors only present evidence obtained in a mouse model and isolated murine hepatocytes to demonstrate the importance of PLIN5 in liver homeostasis, which clearly reduces the clinical impact of the study. Authors must provide further results suggestive of similar actions in patients (e.g., obtaining liver samples from control, NAFLD and NASH patients and analyzing the expression of PLIN5, or using primary human hepatocytes and PLIN5 interference to demonstrate its role in the regulation of inflammatory responses after hepatic damage).
• In Figure 4, authors describe that PLIN5 is involved in mitochondrial structure and dynamics in liver samples, stating that Plin5-/- animals show enlarged mitochondria and increased ER-mitochondria interaction. Although they show alterations in some markers of mitochondrial dynamics, authors do not study mitochondrial function. Do alterations of Plin5 expression affect mitochondrial function in these samples? Have authors analyzed related parameters such as respiration or reactive oxygen species production?
• In this manuscript, reduction of CHOP expression in Plin5-/- mice has been related to decreased hepatic apoptosis in these animals. However, further information/experiments should be provided to support this statement, as no differences were found in other apoptosis-related markers between WT and knockout mice. For instance, TUNEL assays can be performed in liver samples to demonstrate reduced apoptosis.
• Authors should include densitometric data/quantitative analysis of the different experiments performed in all the figures that show Western blots.

 

Minor points

• In Figure 5B, a Ponceau S staining image has been shown to demonstrate equal protein loading. It should better be replaced by a loading control for proper interpretation of the blots.
• Electron microscopy images (Figure 4) are quite small and, consequently, it could be difficult to analyze the alterations described in the text.
• Name of genes should be stated properly in Table S1, in the figures and throughout the manuscript: murine gene names should be written in lowercase letters with the first capitalized, and in italics.

Author Response

Dear Reviewer 2,

many thanks for your help in improving our manuscript. Please find the response to your comments in the attached pdf-file.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I would like to thank the authors for their detailed point-by-point answer to my comments.

Although I do feel that some of the experiments suggested should have been added to the revised version of the manuscript, authors have done an remarkable effort to clarify and highlight some of the limitations of their study.

I have no further suggestions.