Complex Immunometabolic Profiling Reveals the Activation of Cellular Immunity and Biliary Lesions in Patients with Severe COVID-19

Round 1

Reviewer 1 Report

The manuscript entitled „Complex immuno-metabolic profiling reveals activation of cellular immunity and biliary lesion in patients with severe COVID-19” consists the summary of an experimental study that summarizes the key laboratory features displayed by COVID-19 in patients with different stages of the disease. Despite the results seem to be potentially interesting and important, the comments and explanation of the presented results are poor.

Major points:

1. The Introduction should more clearly indicate the aim and rationale of the study and logical links between the experiments which were performed. Currently, it is not clear how in vitro experiments contribute to the "story".
2. The number of infected and dead people is progressively increasing and at the moment of reading the manuscript are yet an order of magnitude higher than reported. These numbers are expected to continue to increase. Please remove these numbers from the Introduction section.
3. The body of the manuscript is divided into the sectors that present the current state of knowledge about the importance of several factors in the development of COVID-19.  From this point of view, the manuscript presents the state of knowledge well known to readers who are working in the presented topic.
4. In this manuscript, the authors did not provide clear information about the physiological significance of IL-6 and IL-2 soluble receptor analysis in the analysed clinical situation. Why they chose these markers?
5. The section Patients and Methods is well and concisely written.
6. The control group eg. non-COVID-19 derived material, is missed in this study. Without additional comparison, the obtained results can be regarded as an epiphenomenon and presented conclusion is highly speculative.

Author Response

1. The main goal of our study was to provide a concise, rich dataset spanning multiple biochemical, immunological and hematological parameters in a single cohort of patients, tracking their development over time in subgroups of patients stratified based on disease severity. The parameters chosen for this study derive from previously published COVID-19 data (parameters of particular significance, e.g. neutrophils, lymphocytes, IL-6, anti-SARS-CoV-2 antibodies, liver tests) and best local clinical practice (parameters important for clinical and therapeutic management of patients). This goal is stated at the end of the Introduction on lines 73-82, which we expanded to provide more clear insight into our rationale.
2. Thank you, we agree and have removed the numbers and reworded the sentence on lines 42-43.
3. We divided the manuscript into sections based on main laboratory features of COVID-19 which readers working in the field would find immediately recognizable. Within each section we confirm previously identified characteristics within our studied cohort, and build upon them with new data, either through visualization of temporal dynamics of parameters, association between parameters (e.g. immune activation and hepatopathy), tissue immunohistochemistry and more.
4. IL-6 and soluble IL-2 receptors have both been shown to be associated with severe course of the disease, as described in the Introduction (lines 70, 71) and summarized in numerous reviews (e.g. ref 21 Cao et al). Both parameters reflect activation of proinflammatory response, with IL-6 being slightly more specific for innate immune response and sIL2R for T cell activation, as also discussed in the Discussion (lines 335, 356). Additionally, the IL-6 receptor- targeted monoclonal antibody tocilizumab has been used in treatment of severe COVID-19 patients, providing further rationale for inclusion as a predictive and meaningful marker.
5. We thank the reviewer for this praise.
6. This is an important point which is crucial to clear up and we thank the reviewer for raising it. While we acknowledge that we do not include a control cohort in our study, we suggest several reasons why this is so. First and foremost, for majority of evaluated parameters we include a healthy reference range for adults, shown in the graphs as a light gray area (which is noted in figure captions). This already allows us to put the presented findings into context and shows where values of healthy donors should lie. Further, while one may suggest that the data should be compared with patients undergoing non-COVID-19 viral infection, we would argue that this was not the purpose of our study and would be vastly more labor and time intensive, especially if these controls were to also be stratified based on disease severity. Finally, the main purpose of our study (in addition to showing pathological values exceeding healthy reference ranges) was to compare patients with different severities of disease course, e.g. mild, moderate, severe and fatal. Thus, comparisons between these groups were key and those are fully provided throughout our manuscript. We hope the reviewer understands these reasons, goals and limitations of our study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Klocperk et al compare the immune-metabolic profiles of ** patients with varying severity of presentation of Covid-19.  This is a well written, well organized study and manuscript that highlights several differences in patients that present with severe disease and those with terminal disease.  To quote, “patients with a fatal course failed to upregulate markers of inflammation, showed dyscoordination of immune response and progressed towards acute kidney failure.”

Some minor points should be revised prior to publication as listed below by line:

35. The manuscript starts with a figure which needs a legend and citation within the text.

48. typo: damage IS not yet known

109. grammar: add comma: immunohistochemistry, 3…………

131. Should not start sentence with number: rephrase as: Patients suffering from a severe course of the disease (n=7, 19%)…….

133. see line 131

139. Comment should be made regarding any differences in the characteristics that would be normal for an elderly individual as apposed to one with Covid-19, if any.

162. The Y axis scale is so large that differences in values are hard to see on the graphs. Possibly switch to a log scale. This is not a requirement.

186. It was not clear to this reader how long patients with severe disease but not fatal disease were followed to ensure that it was not fatal.

322. Add to this list for fatal course, activated T cells (as indicated by HLA-DR and CD38) and that sIL2R is also a marker for inflammation. Comment can be made that the presence of sIL2R favors Tregs rather than Th1 (and Th17) which would be important in an antiviral response.

338. rephrase as: and particularly CD8 T cell depression, BUT AN UNUSUAL PRESENCE OF ACTIVATED CD38, HLA DR EXPRESSING CD8 T CELLS.

343. Just a note to the authors: activated T cells are more permissive for virus replication and hence susceptible.

348. replace ‘scarcely’ with ‘rarely’

356. This is a very good qualifying statement.

This is a very good qualifying statement.

Author Response

1. This figure is a graphical abstract, rather than a main figure of the manuscript. As such, it is not referred to in the main text and is not supposed to have its own figure caption.
2. Thank you, fixed this typo.
3. Thank you, fixed this typo.
4. Thank you, we re-worded the sentence to avoid starting with a numeral.
5. Thank you, we re-worded the sentence to avoid starting with a numeral.
6. This is a helpful suggestion, unfortunately the line numbers were lost from the text of the review (perhaps the word processing software interpreted them as a numbered list?) and we are therefore unsure which line and which particular parameters does this comment refer to. However, we would like to point out that for the vast majority of parameters we also provide healthy adult reference ranges visualized in the graphs as light grey background areas, which allow the reader to readily see which patients had sub/supranormal values.
7. We thank the reviewer for the suggestion. We believe the major differences (e.g. “severe” × the rest) were easy to discern even in linear axes, but we agree that the differences between the remaining cohorts were hard to evaluate especially for procalcitonin, IL-6 and immature granulocyte numbers, and to a lesser extent for bilirubin and ALP values. Thus, we re-created the relevant graphs with log(10) basis and included them in the revised version of the figures 1, 2 and 3.
8. To the best of our knowledge, all the patients from the severe cohort are alive at time of resubmission 2-3 months after initial data analysis. We have added a qualifying statement about their status at time of inclusion in the study into section 2.1 Patient cohort and study design, lines 91-93.
9. Thank you for this suggestion, we have added a note regarding the unique sIL2R and activated CD8 T cell trend to lines 305-309. The relationship between sIL2R, Tregs, Th1 and Th17 cells seems to not be clear, with reports suggesting negative correlation between sIL2R and Tregs in Kawasaki disease in vivo (http://www.ijcem.com/files/ijcem0027287.pdf), positive correlation in a follicular lymphoma study in vitro (https://ashpublications.org/blood/article/118/10/2809/28683/Soluble-IL-2R-facilitates-IL-2-mediated-immune), positive correlation between sIL2R and Th1/17 in murine models (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047748 and more. While certainly a fascinating topic, we feel that this particular facet of our study would require discussion that would exceed the scope of our manuscript. If the reviewer suggests a particular study that would be helpful in this context, we would be very appreciative.
10. Thank you, we re-worded the sentence.
11. Thank you for this helpful note which helps put our findings into context. We added a sentence and a reference to the Discussion reflecting this information.
12. Thank you, replaced.
13. We are glad the reviewer appreciates our endeavor to provide a realistic interpretation of our data.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has been slightly improved and focus on the topic of discussion. Despite the methodological errors (eg. missed controls) have been not improved, the authors clearly explained the purpose of the presented study. However, I recommend considering these suggestions in the planned experiments in future. I recommend this manuscript for publication after some English editing to gain clarity and precision.

Author Response

We thank the reviewer for his/her positive assessment of the changes we made and justification of approach taken.

Language editing will be arranged through inhouse services provided by MDPI.

Author Response File: Author Response.pdf