Pathway and Network Analyses Identify Growth Factor Signaling and MMP9 as Potential Mediators of Mitochondrial Dysfunction in Severe COVID-19

Round 1

Reviewer 1 Report

Wang et al. described a study in which RNA samples extracted from patients infected with SARS-CoV2 (diagnosed with COVID and classified as Mild or Severe) were sequenced. The authors performed a collection of bioinformatic analyses, with a focus on mitochondrial-related genes, in order to identify key transcriptomic signatures associated with the severity of the disease. The samples collected are of high value and address an interesting and important question. However, important information and adequate controls are missing from this study. 

Major points:

1 - Additional details regarding the cohort will help interpret some of the results seen in this study. Information such as previous immunization status and what variants of SARS-CoV2 these patients were infected with should be included, if available. SARS-CoV2 RNA has been isolated before from whole blood samples (PMID: 33138836), therefore the authors should be able to deduce virus strains based on their RNA sequencing reads.

2 - A major problem with comparing transcriptomic profiles from whole blood samples is the fluctuation in cell-type diversity between "healthy controls" and infected patients. Immune cell populations will vary drastically between a healthy individual and an infected one. In particular, macrophages and neutrophils seems to be more abundant in severe cases of COVID-19. MMP9, for example, is more abundantly expressed in macrophages and neutrophils than in T or plasma cells (according to HPA, Tissue Cell enrichment analysis). The authors should mine their RNA-Seq dataset and compare representation of key cell markers (CD4, CD8, Ly6Fc, CD19, etc.) between the mild and severe groups of patients. 

3 - It's unclear why the analyses were solely focus on a small subset of genes. I understand that the authors decided to focus on mitochondrial related genes, but there's much more information in the samples that could provide better context for the interpretation of the results. The authors should consider a genome-wide analysis.

4 - It's unsettling that despite the inclusion of only mitochondrial-related genes in the analysis, no mitochondrial-specific pathways are highlighted in the gene ontology analyses (Fig3). I recommend an unbiased approach to determine if GO pathways are significantly enriched in infected patients.

5 - While the selected genes are all mitochondrial-related, the diagrams in Fig4 do not show which genes actually locate to the mitochondria. The authors should include this information.

Minor Points:

1 - What is the unit of the y-axis in Fig2A? Number of DEGs?

2 - What metric was used to calculate and plot the PCA? FPKM, normalized counts? Also, the authors perhaps would get a better segregation of the samples if all genes are included and not only mitochondrial-related genes.

3 - Fig2D-F is difficult to follow what comparisons are being made. The authors should include a title for each graph, example "MldMod vs. HC", to make it easier to follow.

4 - Fig2D-F, where are the yellow dots labelled as absLFC>0.58? Is this label necessary?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Thank you for the opportunity to review this manuscript, dealing with interesting findings entitled “Pathway and Network Analyses Identify Growth Factor Signalling and MMP9 as Potential Mediators of Immune and Metabolic Dysfunction in Severe COVID-19”. Their study aimed to understand how cellular metabolism contributes to COVID-19 disease outcomes. To prove, their hypothesis a Metacore pathway enrichment analysis was performed on RNA sequencing data from blood samples collected from healthy controls and patients with mild/moderate or severe COVID-19. When compared to the mild/moderate COVID-19 group, they find that the severe group showed overexpression of a wide range of growth factor and cell cycle signaling pathways and concurrent downregulation of interferon signaling pathways. Finally, they proposed that Five of the top 10 elevated pathways contained MMP9, suggesting that it would be a promising treatment target for COVID-19. All findings are interesting, and the article includes a balanced and critical view of the findings. However, they need to revise this manuscript with the following to publish in the "International Journal of Molecular Sciences” journal:

Though the study is relevant to the special issue requirements, the title does not match it. The Author needs to revise the title to match the title of special issues. There are several issues with the full name of the abbreviation used in the manuscript. The author needs to revise the manuscript thoroughly to write the full name of all abbreviations used the first time. Such as MMP9 etc. Why do the keywords have “DEG” while there’s no statement related to it? The authors need to write the criteria for the selection of the patients. In addition, the author needs to write exclusion and inclusion criteria in separate sections. Since there’s a significant difference between the age of the study group, does this difference and the findings are relevant to the conclusion? The author needs to explain it briefly in the discussion section. There are several reports available recently which show similar studies and findings (Int J Med Sci. 2022; 19(13): 1903–1911.; Biomolecules. 2021 Mar; 11(3): 390). Authors need to explain why their findings (except the bioinformatic approaches) are unique compared to them. Also, the author needs to cite all these recent studies in this manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

All my concerns were addressed.

Reviewer 2 Report

The revised manuscript and the author's response are satisfactory. The manuscript entitled "Pathway and Network Analyses Identify Growth Factor Signalling and MMP9 as Potential Mediators of Immune and Metabolic Dysfunction in Severe COVID-19" could be published in IJMS it's current stage.