Dysregulated Inflammatory Cytokine Levels May Be Useful Markers in a Better Up-Dated Management of COVID-19

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

Dear Author,

 

I did like to read the present paper submitted to current issues in molecular biology:  

Inflammatory cytokines dysregulation levels may be useful markers in a better up-dated management of COVID-19

 

 

It is an interesting study; the authors investigated different inflammatory cytokines in Anti-SARS-CoV-2 infections in the acute and post-acute stage in order to identify markers predictable for the disease. The paper is interesting; however, I have some points, which could potentially improve the quality of the manuscript:

 

1. I would recommend to spell out abbreviations first; even in the abstract. Some readers are not familiar with them.  

 2. I also would describe in the abstract shortly parameters which were investigated.  

3. In the introduction I would recommend to elucidate more the rationale behind the study. The introduction contains lot of general information.

4. The results are presented well.

5. I would always begin the section discussion with a short summary of the main results of the presented study and based on that starting the discussion.   

6. I would structure the discussion and discuss point by point.

7. Are there some limitations, are there some strengthens?

8. I would temper conclusions related to long COVID. It is speculative.

9. Some discussion on clinical impact would be beneficial. Could be implemented some of the finding in clinical SOPs?  

Author Response

1. I would recommend to spell out abbreviation first; even in abstract. Some readers are not familiar with them.

• Abbreviations have been inserted throughout the text when used first and also into the abstract, as recommended by the reviewer.

2. I also would describe in the abstract shortly parameters which were investigated.

• We described shortly which parameters have been investigated. To fit into the abstract length, as requested by the journal’s instructions, we modified some part of the abstract as following: “Coronavirus disease 2019 (COVID-19) is an infection characterized by the dysregulation of systemic cytokine levels. The measurement of serum levels of inflammatory cyto-/chemokines has been suggested as a tool in the management of COVID-19. The aim of this study is to highlight the significance of measured levels of inflammatory cyto-/chemo-kine interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-12(p70), IL-27, interferon (IFN)γ, interferon gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α in serum samples from infected and recovered subjects, possibly predictive of severity and/or duration of the disease. Serum samples from healthy (HD), positive at hospital admittance (T0), and recovered subjects (T1, 31-60 or 70-200 days post negativization), were collected and tested for 11 inflammatory cyto-/chemo-kines through a bead-based cytometric assay and confirmed through ELISA. IL-10 levels were increased in T0 group compared to both HD and T1. IL-27 significantly decreased in 31-60 group. IL-1β significantly increased in 70-200 days group. TNF-α significantly decreased in T0 compared to HD and in 31-60 days group versus HD. IP-10 significantly increased in T0 compared to HD. Collectively, these results suggest that IP-10 increase in severe COVID-19 patients sera could represent an early marker of clinical worsening, whereas IL-10 levels at the follow-up in patients recovered from pneumonia, might be indicative of the possible settlement of post-COVID-19 long syndrome.”

3. In the introduction I would recommend to elucidate more the rationale behind the study. The introduction contains lot of general information.

• To accomplish the request of the reviewer we have decided to modify the introduction by adding this paragraph at line 65: “The rapid evolution of COVID-19 is challenging to identify and manage such disease. The study of classical inflammatory mediators of the host could represent a valid system to individuate a constant trace alongside the virus evolution. Moreover, it is reasonable to think that only a specific combination of cyto-/chemo-kines alteration could effectively be predictive for the disease evolution.”

4. The results are presented well.
5. I would always begin the section discussion with a short summary of the main results of the presented study and based on that stating the discussion.

• A summary of the main significance and the main results accomplished on this study has been added at the beginning of the discussion section (line 293); specifically: “The measurement of serum levels of inflammatory cyto-/chemokines has been suggested as a tool in the management of COVID-19. In this study it has been analyzed the significance of 11 cyto-chemokines (i.e., IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12(p70), IL-27, IFNγ, IP-10, MCP-1, TNF-α) in serum samples from infected and recovered subjects, possibly predictive of severity and/or duration of the disease. The serial use of wide range analysis with a cytometric assay and with ELISA assay allowed to find that: IL-10 levels were increased in T0 group compared to both HD and T1; IL-27 significantly decreased in 31-60 group; IL-1β significantly increased in 70-200 days group; TNF-α significantly decreased in T0 compared to HD and in 31-60 days group versus HD; and IP-10 significantly increased in T0 compared to HD.”

6. I would structure the discussion and discuss point by point.

• The discussion section has been formatted in a point by point like structure. Specifically, every paragraph of the discussion explains every result obtained. Here the list of the paragraphs and which result explain.
  • Line 293-304 “resume of the results”
  • Line 305-323 “resume of the results obtained by using flow cytometry assay”
  • Line 324-327 “discussion of IL-27 result”
  • Line 328-333 “discussion of IL-8 result”
  • Line 334-337 “discussion of IP-10 result”
  • Line 338-346 “discussion of IL-1b result”
  • Line 347-357 “resume of the results obtained by using ELISA assay”
  • Line 358-363 “some other consideration on IL-27 result”
  • Line 364-374 “some other consideration on IP-10 result”
  • Line 375-389 “discussion of IL-10 result”

7. Are there some limitations, are there some strengthens?

• Some limitations in our study should be underlined: i) the small number of subjects in our cohorts; ii) the recruitment of subjects of the same ethnic group (Caucasian) and located in the province of Latina, Italy; iii) the short period of recruitment from March 2020 to the beginning of 2022 that excludes from the analysis the last variants of SARS-CoV-2. By contrary, some strengths can be also highlighted: flow cytometry assay has the ability to determinate a plethora of mediators at the same time; the subsequent ELISA analysis reinforces the previous type of analysis therefore providing more complete results.

We added this part at the end of the Conclusions paragraph.

8. I would temper conclusions related to long COVID. It is speculative.

• We understand the comment of the reviewer but this argument is still in progress. Even if our group at the state of the art can only speculate on the role of the cyto-/chemokines in the development of long COVID-19 because of the short duration of this study, our work is also in progress about it, since various authors have begun to describe this association:
  • Queiroz MAF et al. Cytokine Profiles Associated With Acute COVID-19 and Long COVID-19 Syndrome. Front Cell Infect Microbiol. 2022 Jun 30;12:922422. doi: 10.3389/fcimb.2022.922422. PMID: 35846757; PMCID: PMC9279918.
  • Williams ESCP et al. Plasma cytokine levels reveal deficiencies in IL-8 and gamma interferon in Long-COVID. medRxiv [Preprint]. 2022 Oct 5:2022.10.03.22280661. doi: 10.1101/2022.10.03.22280661. PMID: 36238724; PMCID: PMC9558442.
  • Gomes SMR et al. High levels of pro-inflammatory SARS-CoV-2-specific biomarkers revealed by in vitro whole blood cytokine release assay (CRA) in recovered and long-COVID-19 patients. PLoS One. 2023 Apr 5;18(4):e0283983. doi: 10.1371/journal.pone.0283983. PMID: 37018291; PMCID: PMC10075475.

9. Some discussion on clinical impact would be beneficial. Could be implemented some of the finding in clinical SOPs?

Our opinion starts from the predictive role of the cyto-/chemokines. In this scenario, definition of a specific pattern of cyto-/chemokines plasma concentration can be implemented in clinical SOPs to more rapidly define the correct procedure to manage the patient and improve the prognosis.

Reviewer 2 Report

Comments and Suggestions for Authors

In the present study, the authors found that IL-1β, IL-8, IL-10, IL-27, IP-10, and TNF-α are deregulated in serum from patients with COVID-19 compared to healthy donors. Specifically, they tested increases of both IL-10 and IP-10 in the PZ T0 group compared to HD controls. In addition, the levels of IP-10 and TNF-α dysregulations are associated to COVID severity. This manuscript is well written, and the data is well presented.

1.     Method: What are the including and excluding criteria? Did the recovered patients get other infections after recovery from COVID-19?

2.     Results: All the tables should be presented in three-line form.

3.     When was this study conducted? Please include the study period.

4.     Line 166, 199, delete “()”.

 

 

Author Response

1. Method: What are the including and excluding criteria? Did the recovered patients get other infections after recovery from COVID-19?

• We decided to not introduce very strong excluding criteria in order to find out which combination of cyto-/chemokines could be significative in COVID-19 disease evolution in the majority of the cases. We decided to exclude all of the subjects that had documented chronic inflammatory diseases, immunodepression and immunodistraction. Since a great number of the subjects showed a body mass index around 30 we decided to not use this parameter as an exclusion criteria. All the recovered patients within 60 days from the end of the infection did not manifest other infections. For the recovered patients recruited after longer time we only had the information given us by the subject declaring they had not significative event during the period from the end of the infection and the blood donation.

2. Results: all the tables should be presented in three-line form.

• We understand the request of the reviewer, but the tables are formatted by the statistical analysis software and a reduction or a modification of the style can alter the data presentation and a loss of the correct data interpretation. Unfortunately, for these reasons we prefer to leave the tables in the original format.

3. When was this study conducted? Please include the study period

• The study was conducted from March 2020 to the beginning of 2022. This sentence has been inserted on line 81 of the manuscript.

4. Line 166, 199 delete ()

• Typos have been substituted with:
  • Line 166 (in the new version of the manuscript line 174) “(Figure 1)”;
  • Line 199 (in the new version of the manuscript line 199) “(Figure 2)”.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

the first sentence in the discussion should be deleted. the sentence is not necessary. Otherwise the paper is ready.

Author Response

The first sentence in the discussion should be deleted. The sentence is not necessary. Otherwise, the paper is ready.

• The sentence “The measurement of serum levels of inflammatory cyto-/chemokines has been suggested as a tool in the management of COVID-19.” at the beginning of the Discussion has been deleted according to the indication of the reviewer.