Transcriptomic Analysis Suggests the M1 Polarization and Launch of Diverse Programmed Cell Death Pathways in Japanese Encephalitis Virus-Infected Macrophages
Round 1
Reviewer 1 Report
This is a descriptive study of transcriptome analysis of JEV-infected macrophages. Although the study topic is interesting, there are several issues that need to be addressed before this manuscript is acceptable for publication.
It is important to show the purity of peritoneal macrophages isolated from the mice. What is the justification for using peritoneal macrophages?
It is also important to know if these peritoneal macrophages even get infected with JEV. No virus data is shown. What is the virus titers in the serum and isolated macrophages of these mice?
What is the cut-off fold-change used for differentially expressed genes (1.5 fold? 2-fold?). Looks like no cut-off was used in this study. Therefore so many up and down-regulated genes which may or may not be physiological relevant.
Authors must verify selected genes by qRT-PCR and protein levels by Western or ELISA.
Authors should also do some knock-out studies to show the function of selected significantly modulated genes in JEV infection.
There have been several previously published studies with macrophages and JEV infection and ROS (see papers from Basu group). Authors should discuss their data in relation to the published studies.
Title is inappropriate. Not enough data to conclude M1 polarization.
Discussion looks more like repeat of the. results.
This manuscript needs extensive english editing.
Author Response
Thank you for your time and kind consideration of our manuscript for possible publication in Viruses. We feel very grateful to you for your kind comments and constructive advice on our manuscript refinement. The following is the point-by-point answers to your comments and suggestions.
Reviewer1:
Q1: It is important to show the purity of peritoneal macrophages isolated from the mice. What is the justification for using peritoneal macrophages?
A1: Yes, we acknowledge the importance of the purity of isolated macrophages, which we have previously examined by using immunofluorescent (IF) staining of F4/80, a specific marker of macrophages. The purity of peritoneal macrophages isolated from mock-treated and JEV-infected mice was 91% and 94%, respectively, indicating most of the peritoneal cells isolated were macrophages. This data has been added to the Results 3.1 (Line 169-171) and Figure 1A and 1B now.
The reason why we use peritoneal macrophages is that macrophages have been proved as one of the major target cells for early flavivirus infection, such as ZIKV, DENV, and JEV (Wan SW et al., J Biomed Sci, 2018; Quicke KM et al., Cell Host Microbe, 2016). Intraperitoneal injection is a widely used and accepted method to inoculate JEV (Justin TH et al., Nat Commun, 2019; Wang et al., J Virol, 2017). Previously we have discovered that the peritoneal macrophages are highly susceptible to JEV infection, and play very important roles in early viral replication and pathogenesis (unpublished data). Accordingly, we have chosen peritoneal macrophages as the target cells for JEV infection in this study
Q2: It is also important to know if these peritoneal macrophages even get infected with JEV. No virus data is shown. What is the virus titers in the serum and isolated macrophages of these mice?
A2: Yes, the well infection of peritoneal macrophages is very important for this study. Actually, we have previously examined the JEV infection rate in peritoneal macrophages, the JEV RNA load in peritoneal lavages and mouse sera at 24 hours post infection (hpi), which we have added to Results 3.1 (Line 172-174) and Figure 1C-1F now. Briefly, the JEV infection rate of peritoneal macrophages was 50% at 24 hpi. The JEV RNA load in peritoneal lavages (each mouse was washed with 4 mL of PBS) and mouse sera was 10^3.7 copies/mL and 10^3.9 copies/mL, respectively.
Q3: What is the cut-off fold-change used for differentially expressed genes (1.5-fold? 2-fold?). Looks like no cut-off was used in this study. Therefore, so many up and down-regulated genes which may or may not be physiological relevant.
A3: We understand your concern, and acknowledge that the appropriate setting of cut-off value is of great importance for both data analysis and interpretation. Currently, for samples with no biological replicates, the cut-off value of fold change is usually set as log2|Fold Change| > 1 & padj < 0.05 [edgeR (Robinson et al., Bioinformatics, 2010)], while for samples with biological replicates, the cut-off value is usually set as log2|Fold Change| > 0 & padj < 0.05 [DESeq2 (Love MI et al., Genome Biol, 2014)]. Notwithstanding, the setting of cut-off value is flexible within a certain range, which depends on researchers’ aims and further GO and KEGG enrichment analysis. Because we have five biological replicates for each treatment, and we want to find out as many changed genes as possible to facilitate further GO and KEGG enrichment analysis, we thus use Love MI’ criteria, namely log2| Fold Change| > 0 & padj < 0.05, which we have now emphasized in Materials and Methods 2.9 and Line 144.
Q4: Authors must verify selected genes by qRT-PCR and protein levels by Western or ELISA.
A4: Thank you for your kind advice, and we know that the qRT-PCR validation of selected genes is usually necessary. Actually, we have directly examined the protein expression of as many selected genes as possible by using IF staining (Figure 5I and Figure 7). We are very willing to validate the expression of the other selected genes by qRT-PCR and WB to refine this manuscript. However, as you may have known, our country is being confronted with a big public health threat of 2019-nCoV, and all the labs have been temporarily closed. We will perform this work immediately once we can begin our experimental work in the near future.
Q5: Authors should also do some knock-out studies to show the function of selected significantly modulated genes in JEV infection.
A5: Thank you for your kind advice. We are planning to do such work in the future researches.
Q6: There have been several previously published studies with macrophages and JEV infection and ROS (see papers from Basu group). Authors should discuss their data in relation to the published studies.
A6: Many thanks. Actually, we have cited two published papers from Dr. Basu’s team (please see Reference 24 and 30), and as well as other online papers (please see Reference 29) regarding JEV infection and ROS. According to your suggestion, we have now cited another relevant paper from Basu’s team to discuss the relationship between JEV infection and ROS (please see Reference 34) now.
Q7: Title is inappropriate. Not enough data to conclude M1 polarization.
A7: M1 polarization is immune killing and pro-inflammatory, and has some specific markers. In our study, transcriptomic data showed extensive activation of various immune and inflammatory pathways upon JEV infection. Furthermore, several M1 polarization markers including iNOS, CD68, CD80 and CD86 showed significant upregulation, while CD163 and CD200R3, two M2 polarization markers, showed significant downregulation. The upregulation of iNOS and downregulation of CD163 were also validated by IF staining. Based on above data, we tend to believe that JEV-infected macrophages might undergo M1 polarization, at least at transcriptomic level. According to your comment, we’d like to replace “revealed’’ with “suggests” in the tile in view that “suggests” may be less confirmative than “revealed”. The new title is “Transcriptomic analysis suggests M1 polarization and launch of diverse programmed cell death pathways in JEV-infected macrophages” now.
Q8: Discussion looks more like repeat of the results.
A8: Many thanks. Now we have revised the discussion.
Q9: This manuscript needs extensive English editing.
A9: We have carefully re-edited the language now.
Reviewer 2 Report
In this study the authors perform transcriptomic analysis on JEV-infected or mock infected mouse peritoneal macrophages and report the data following standard pathway analyses. Overall the approach is inherently sound and the study should provide some foundational data for the field. However, it is not clear how many biological replicates were sequenced (to ensure rigor and reproducibility) nor have most of the key inferences derived from the sequencing study been independently validated. These and other points to improve the study are outlined below:
Major Points:
- The Results section starts by discussing QC of sequencing data without outlining a priori in detail what was sequenced and why. Thus, the experiment needs to be set up more effectively for the reader to fully digest the work and its implications.
- I apologize if I missed it, but its not clear how many biological replicates were sequenced to obtain the data in the study. I would strongly recommend that greater than a single biological sample per condition be required to be sequenced and analyzed to ensure the rigor and reproducibility of the study.
- 5: What do the error bars represent in the graphs? They should represent independent biological replicates and not technical replicates that simply report assay error rather than biological relevance.
- To ensure rigor throughout the study, it is necessary to validate key findings from the RNAseq data independently via independent samples and RT-PCR.
Minor Points:
- Line 73: italicize Aedes albopictus
- There are several minor improvements to the use of the English language that can be made throughout the study
Author Response
Thank you for your time and kind consideration of our manuscript for possible publication in Viruses. We feel very grateful to you for your kind comments and constructive advice on our manuscript refinement. The following is the point-by-point answers to your comments and suggestions.
Major Points:
Q1: The Results section starts by discussing QC of sequencing data without outlining a priori in detail what was sequenced and why. Thus, the experiment needs to be set up more effectively for the reader to fully digest the work and its implications.
A1: We have re-organized the demonstration of results, and made a schematic graph in Figure 1G and added details about the research design in Results 3.1.
Q2: I apologize if I missed it, but it’s not clear how many biological replicates were sequenced to obtain the data in the study. I would strongly recommend that greater than a single biological sample per condition be required to be sequenced and analyzed to ensure the rigor and reproducibility of and the study.
A2: We feel sorry for not having described clearly the number of biological replicates used, though we have mentioned that there were five biological replicates for each treatment in Materials and Methods. To facilitate understanding, we have now clearly described the number of biological replicates used in Results 3.1 (Line 176-177) and in Figure 1G.
Q3: What do the error bars represent in the graphs? They should represent independent biological replicates and not technical replicates that simply report assay error rather than biological relevance.
A3: The error bars represent the standard errors of five independent biological replicates. We have now emphasized this information in the legend of Figure 6.
Q4: To ensure rigor throughout the study, it is necessary to validate key findings from the RNAseq data independently via independent samples and RT-PCR.
A4: Thank you for your kind advice, and we know that the RT-PCR validation of key genes is usually necessary. Actually, we have directly examined the protein levels of as many selected genes as possible by using IF staining (Figure 5I and Figure 7). We are very willing to validate the expression of the other selected genes by RT-PCR to refine this manuscript. However, as you may have known, our country is being confronted with a big public health threat of 2019-nCoV, and all the labs have been temporarily closed. We will perform this work immediately once we can begin our experimental work in the near future.
Minor Points:
Q1: Line 73, italicize Aedes albopictus.
A1: Aedes albopictus has now been italicized.
Q2: There are several minor improvements to the use of the English language that can be made throughout the study.
A2: The language has been re-edited in this version.
Round 2
Reviewer 1 Report
Manuscript is acceptable now.
Author Response
Thank you very much for your constructive advice on the refinement and kind consideration for the publication of this manuscript . According to your suggestion, we have now re-edited our language by using MDPI’s English pre-edit services. The attachment is the re-edited manuscript, please see it.
Best regards.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors have effectively responded to most of the points raised in the original round of critiques. I find the study to be significantly improved and much more convincing. I also understand the limitations on additional experimentation now due to lab closures. I would recommend that the manuscript be carefully reviewed for English language usage as a number of issues still remain in this regard.
Author Response
Thank you very much for your constructive advice on the refinement and kind consideration for the publication of this manuscript . According to your suggestion, we have now re-edited our language by using MDPI’s English pre-edit services. The attachment is the re-edited manuscript, please see it.
Best regards.
Author Response File: Author Response.pdf
