The Expression of Fibrogenic Cytokines by Human Peripheral Blood Mononuclear Cells in Response to SARS-CoV-2 Spike Protein

Round 1

Reviewer 1 Report

Dear Editor;

Thank you for inquiring about my thoughts on the work titled: “The Expression of Fibrogenic Cytokines by Human Peripheral 2 Blood Mononuclear Cells in Response to SARS-CoV-2 Spike 3 Protein”.

The authors have measured the level of different cytokines expressed in peripheral blood mononuclear cells in response to SARS-Cov-2 spike protein and other antigens. The findings of the paper may be used by other investigators for further studies and experiments.

The abstract is precise and to the point. Manuscript is written beautifully, in detail and explained precisely.

There are very few grammatical errors in the manuscript that needs to be corrected.

The reviewer has a few major and minor comments listed here, after which the paper can be accepted.

Major Comments: -

1.      Results; Section 3.1. The results of this section can be presented in a tabulated form for clear and precise visualization.

2.      The authors have measured the mRNA levels of different cytokines. Out of these only two cytokines showed significant upregulation in response to VLPS. Authors did not discuss properly as to why other cytokines were not upregulated or the same cytokines are not upregulated significantly in response to SST and SNP.

3.      Lines 302-313. The figure for this data is missing in the manuscript.

Minor Comments: -

1.      Lines 236-245. This paragraph is repeated as same from the Introduction section. Kindly delete, rephrase or modify.

2.      Please provide the reason as to why the ELISA was not performed for IL-13, IL-18 and IL-22.

3.      Table 2:

I.                     All the values in the table should have same number of digits after decimal point.

II.                 What is IL-12p40? Is this an error.

III.              Please correct TNF-a to TNF-α.

4.      Lines 419-420. “Clinically….fever”. Kindly rephrase as the meaning is not clear.

5.      Line 467. ‘CACL10’ be replaced with ‘CXCL10’.

Author Response

Dear Editor,

We would like to thank reviewer 1 for taking his/her time and for his/her valuable feedback for improving our manuscript.

Various changes (shown in the correction mode) have been done according to recommendations of reviewer 1 and 2.

Addressing major comments:

1. In section 3.1., the results are now additionally illustrated by adding Table 2
2. Discussion of the difference in results when stimulating with VLP SARS-CoV-2, in comparison with spike protein trimer and nucleoprotein has been added in the Discussion section in lines 586 to 587, and 641 to 657. However, we did not add additional discussion for why certain cytokines were not up-regulated under the various stimulations, as it is difficult to tell.
3. The PCR data for all cytokines has now been added in Tables 3, 4, 5 and 6 stating the mean values of fold expression, but without additional statistical information, with the exception of indicating with * where significant differences were identified. In addition, Figure 3 has been added showing more detailed results of IL-17 mRNA expression levels.

Addressing minor comments:

1. This part of the manuscript has been adjusted according to recommendation by deleting the paragraph.
2. ELISA measurements of experimental culture supernatants were unfortunately not measured for IL-13, IL-18 and IL-22, because we were restricted with our funding and not able to purchase those kits. With frozen samples of the supernatants still available, we may do so in the future, when additional funding will be available.
3. For the Table showing the ELISA results, corresponding corrections have been done. Related to IL-12, as it is a dimer, with IL-12B (p40) the relevant protein for structure and function, the ELISA kit that was used, measured the amount of p40. However, we deleted ¨p40¨ from the Table and now put ¨IL-12¨.
4. The sentence has been adjusted to make it better understandable (now lines 439-440)
5. The typo has been corrected.

English writing has been revised for grammar, orthography and linguistics.

Several references have been added according to the requests of reviewer 2.

Reviewer 2 Report

1) SARS-CoV-2, a single stranded RNA-virus, emerged in 2019 and caused a global pandemic. Some of the infected patients develop severe complications, such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. The mechanisms leading to lung fibrosis in the context of COVID-19 are still unclear. However, there are correlations between this condition and certain inflammatory mediators that are elevated in the plasma of patients. By stimulating peripheral blood mononuclear cells (PBMC) from healthy blood donors through different formulations of SARS-CoV-2 spike protein, the expression of fibrogenic cytokines was measured. The spike protein induced significantly increased expression of IL-1 and IL-6 mRNA in PBMC. Both cytokines are important players in the COVID-19 cytokine storm, in ARDS and in the development of pulmonary fibrosis. Different receptors with affinity to the spike protein may be involved, however the exact mechanism on how the spike protein leads to a higher cytokine expression in PBMC needs further investigation. Improve the abstract and underline the novelty of the study.

2) 1. Introduction L28-34. SARS-CoV-2, a single stranded positive RNA-virus, emerged in 2019. The infection  is caused via binding of the angiotensin-converting enzyme 2 (ACE2) receptors expressed  on lung epithelial cells in the upper respiratory tract. Infected patients can develop severe  complications, such as atypical pneumonia or acute respiratory distress syndrome  (ARDS), which can lead to death (1,2). A certain proportion of the ARDS patients develop  pulmonary fibrosis, which can create a dry cough and breathlessness, and may need pul-  monary rehabilitation and oxygen therapy (3). Please improve the introduction and discuss some important recent references:

a-Severe COVID-19 ARDS Treated by Bronchoalveolar Lavage with Diluted Exogenous Pulmonary Surfactant as Salvage Therapy: In Pursuit of the Holy Grail?. J Clin Med. 2022;11(13):3577. Published 2022 Jun 21. doi:10.3390/jcm11133577

b-Long COVID: major findings, mechanisms and recommendations [published correction appears in Nat Rev Microbiol. 2023 Apr 17;:]. Nat Rev Microbiol. 2023;21(3):133-146. doi:10.1038/s41579-022-00846-2

c-  Risk and Protective Factors for COVID-19 Morbidity, Severity, and Mortality. Clin Rev Allergy Immunol. 2023;64(1):90-107. doi:10.1007/s12016-022-08921-5

3) L83- 86 The aim of this in-vitro human study was to identify fibrogenic cytokines expressed  by human peripheral blood mononuclear cells (PBMC) in response to the SARS-CoV-2  spike protein. For that purpose, PBMC were stimulated with various formulations of  spike protein. Expression of profibrotic cytokines were measured by using quantitative  reverse-transcription PCR (RT-qPCR) and ELISA. Please improve the description of study aim. 

4) 3. Results 211 3.1. Proliferation assay 212 The unknown immune status towards SARS-CoV-2 was assessed through T cell pro- 213 liferation assays. PBMC were accordingly cultured in the presence (PHA for maximum 214 positive stimulation, various formulations of SARS-Cov-2 or dengue virus antigens) or 215 absence (negative control) of stimulation. Please clarify this paragraph.

5) 4. Discussion 403 The aim of this in-vitro human study was to identify fibrogenic cytokines expressed 404 by PBMC in response to SARS-CoV-2 spike protein. In the RT-qPCR, two statistically sig- 405 nificant results were obtained under stimulation with the spike protein. This was the case 406 for IL-1 and IL-6 mRNA expression under the VLP-SARS condition. Furthermore, 407 CXCL10 mRNA expression was increased by the SARS-CoV-2 nucleoprotein stimulation. Please improve the summary of study aim.

6) 5. Conclusions 518 In conclusion, through this in-vitro human study, we could show that the expression 519 of fibrogenic cytokines by PBMC in response to SARS-CoV-2 spike protein could be in- 520 duced. For IL-1 and IL-6 an increase in their mRNA expression under a spike protein 521 condition was observed. The CXCL10 mRNA expression was increased under the nucle- 522 oprotein condition. These cytokines all play an important role in the cytokine storm, in 523 the development of ARDS and the complication into pulmonary fibrosis. Additionally, 524 these inflammatory mediators represent possible targets for COVID-19 therapy. To study 525 the influence of immune status on the cytokine expression, more data would be needed. 526 We assume, based on the experimental results, that the spike protein has a direct effect on 527 PBMC cytokine expression, however the underlying mechanism needs to be studied more 528 in detail. Underline the novelty of the study and the possible clinical implications.

Minor editing of English language is required

Author Response

Dear Editor,

We would like to thank reviewer 2 for taking his/her time and for his/her valuable feedback for improving our manuscript.

Various changes (shown in the correction mode) have been done according to recommendations of reviewer 1 and 2.

Addressing the 6 points:

1. Improving the abstract and underline the novelty of the study: Lines 16 to 18 has been added to the abstract for addressing this recommendation.
2. Improving the introduction by adding 3 recommended journal articles: The 3 recommended articles have been included (Reference 4, 7 and 13). We added also reference 14, with a corresponding statement (line 54 to 59) for addressing this recommendation.
3. Improving the description of the study aim: This recommendation has been addressed in line 93 to 102
4. Results section 3.1.: Making this paragraph better understandable:We added Table 2 and some additional information to paragraph 3.1. for addressing the recommendation.
5. Discussion section: Improving the summary of the study aim: Tis recommendation has been addressed in line 517 to 525.
6. Conclusion section: Underlining the novelty of the study and the possible clinical implications: Line 672 to 676 has been added to address this recommendation.

In addition, several tables have been added as requested by reviewer 1. English writing has been revised for grammar, orthography and linguistics.

Round 2

Reviewer 1 Report

Authors should mention the limitations they faced to conduct some studies because of non-availability of the resources in the manuscript.

Author Response

Dear Editor, dear Reviewer 1

Thank you for asking for the following addition: Authors should mention the limitations they faced to conduct some studies because of non-availability of the resources in the manuscript.

We included in section 2. Materials and Methods line 209-210 the following sentence: Unfortunately, secretion of IL-13, IL-18 and IL22 could not be measured, due to insufficient funding.