Increased Production of Interleukin-10 and Tumor Necrosis Factor-Alpha in Stimulated Peripheral Blood Mononuclear Cells after Inhibition of S100A12

Round 1

Reviewer 1 Report

Circulating S100A12 is elevated in sepsis patients and is associated with a higher risk of death.

The reference given for treatment of patients (Intensive 364 Care Med 2017, 43, 304-377.) cites a wide range of options. A brief summary of the treatment(s) used in this study should be given, including whether the more seriously affected patients were treated differently.

Of the 4 markers elevated in patents’ sera compared with controls (Table 2), sRAGE and S100A12 had statistical significance at day 1. A link was made between sRAGE and lung damage. At day 7 only S100A12 distinguished non-survivors. This may have some value for clinical management although it would have been useful to assay relevant cytokines such as IL10, TNF and IL6.

Patients’ PBMCs were then challenged in vitro with LPS to compare production of TNF-a, IL-10 and IL-12. These cytokines are known to be increased in sepsis  (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378830/). IL10A and TNF were further increased slightly in the presence of S100A12 inhibition.

The proposal in Fig. 3 is plausible but activation of some of the proposed intermediates such as TLR4, IL10 and RAGE should be measured to support it. This should be feasible for the in vitro assay, with a few control donors. Its not clear why RAGE, HMGB1 and AGE weren’t measured for PBMCs, to compare with Table 2 results.

Author Response

Circulating S100A12 is elevated in sepsis patients and is associated with a higher risk of death.

The reference given for treatment of patients (Intensive 364 Care Med 2017, 43, 304-377.) cites a wide range of options. A brief summary of the treatment(s) used in this study should be given, including whether the more seriously affected patients were treated differently.

Ans:  Brief summary for the management of sepsis was added (Page 3, Line 108-111). The treatment may be different since the severity is different. For example, patients with septic shock were prescribed with vasopressor and patients with renal failure received renal replacement therapy.

Of the 4 markers elevated in patents’ sera compared with controls (Table 2), sRAGE and S100A12 had statistical significance at day 1. A link was made between sRAGE and lung damage. At day 7 only S100A12 distinguished non-survivors. This may have some value for clinical management although it would have been useful to assay relevant cytokines such as IL10, TNF and IL6.

Ans:  Yes. We agree your opinions. The possible meaning in the difference of Day 7 S100A12 levels between survivors and non-survivors was discussed in the Discussion section (Line 245-256).

Patients’ PBMCs were then challenged in vitro with LPS to compare production of TNF-a, IL-10 and IL-12. These cytokines are known to be increased in sepsis (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378830/). IL10A and TNF were further increased slightly in the presence of S100A12 inhibition.

Ans:  Yes. We also added this article (In Vivo. Nov-Dec 2013;27(6):669-84.) in references to explain why these cytokines were measured (Page 4, Line 147-148).

The proposal in Fig. 3 is plausible but activation of some of the proposed intermediates such as TLR4, IL10 and RAGE should be measured to support it. This should be feasible for the in vitro assay, with a few control donors. Its not clear why RAGE, HMGB1 and AGE weren’t measured for PBMCs, to compare with Table 2 results.

Ans:        We agree your opinions. The proposed schema (Figure 3) was speculative because RAGE, TLR4, NF-κB, STAT3, and Ras were not measured. Well-controlled animal model or cell line studies are necessary to demonstrate our proposed hypothesis. This was mentioned in limitations. The causes why RAGE, HMGB1 and AGE weren’t measured for the production from stimulated PBMCs are the followings: 1) sRAGE lacks the transmembrane and the signaling domain and is hypothesized to counteract the detrimental action of the full-length RAGE; 2) HMGB1 is an intracellular DNA-binding protein which are released by necrotic cells passively, and by active secretion from macrophages, natural killer cells, and dendritic cells; 3) AGE are proteins or lipids that become glycated as a result of exposure to sugars. Briefly, most of RAGE and AGE are not produced by PBMCs and circulatory HMGB1 is not mainly released by PBMCs.

Reviewer 2 Report

The publication titled "Increased production of interleukin-10 and tumor necrosis ..." by Huang-Pin Wu et al. Is quite a good job but in my opinion, it needs a lot of corrections before it can be published.
Below are my comments.
1. I don't know if the title should contain abbreviations? Let the authors decide for themselves whether it can be written otherwise, if not, it can stay that way.
2. The first part of the abstract definitely needs to be rewritten.
3. First of all, the introduction is probably two times too short. And secondly, the last paragraph in which the purpose of the research should be described is rather carelessly written. It must be corrected.
4. Figure 1 - illegible. And the graphics are so careless - please improve these figers!
5. Fig 2 - the same - terribly unreadable graphics quality.
6. Conclusions - definitely underdeveloped. Nothing comes out of it. Please think it over and rewrite it.
In summary, the work is not bad, but it is terribly underdeveloped, I do not know where it comes from. Before publication, the authors must definitely improve this work.

Author Response

The publication titled "Increased production of interleukin-10 and tumor necrosis ..." by Huang-Pin Wu et al. Is quite a good job but in my opinion, it needs a lot of corrections before it can be published.

Below are my comments.

1. I don't know if the title should contain abbreviations? Let the authors decide for themselves whether it can be written otherwise, if not, it can stay that way.

Ans:  Thanks for your comment. Generally, title did not contain abbreviations. We suggest to keep the written title.

2. The first part of the abstract definitely needs to be rewritten.

Ans:  The first part of the abstract was revised.

3. First of all, the introduction is probably two times too short. And secondly, the last paragraph in which the purpose of the research should be described is rather carelessly written. It must be corrected.

Ans:  The introduction section, especially the purpose of the research was revised (Page 2, Line 74-84).

4. Figure 1 - illegible. And the graphics are so careless - please improve these figers!

Ans:  The titles of mediators may be too small in sizes. We have revised this and let it readable easily.

5. Fig 2 - the same - terribly unreadable graphics quality.

Ans:  As the same to the above comment, the titles may be too small in sizes. We have revised figure and legend, and let it readable easily.

6. Conclusions - definitely underdeveloped. Nothing comes out of it. Please think it over and rewrite it.

Ans:  The conclusion has been rewritten (Page 10, Conclusion section).

In summary, the work is not bad, but it is terribly underdeveloped, I do not know where it comes from. Before publication, the authors must definitely improve this work.

Ans:        Thanks for your all comments. We have revised this manuscript as possible as we can. If there is something needs to be revised, please inform us directly.

Round 2

Reviewer 1 Report

No further comments

Reviewer 2 Report

The authors improved really well this manuscript. I think that we can publish this manuscript in this journal. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.