PONE-D-24-19541Acute blood loss in mice forces terminal differentiation of the CD45-negative erythroid cells and up-regulation of the pathogen-sensing gene Clec5a in bone marrow erythroid cellsPLOS ONE

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Reviewer #1: Partly

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Reviewer #1: Yes

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Reviewer #1: No

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Reviewer #1: Yes

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Reviewer #1: In this manuscript, Nazarov et al. aimed at investigating the immune function of the erythrocytes induced by acute blood loss (ABL). To this end, the authors collected the bone marrow and spleens of mice three days after blood drawn, and performed flow cytometry analysis of the different stages of erythrocytes, Nanostring gene expression profiling of immune response genes, and BioPlex analysis of multiple cytokines in the media conditioned with cultured erythrocytes. While the potential immune function of the erythrocytes upon ABL is interesting, this manuscript, at least in its current form, seems to be too preliminary and does not achieve its aim very well, because it lacks sufficient information for describing the background, rationale for experimental design, and inter-validation of the different parts of the results. Specific concerns include:

1. The notion that erythrocyte differentiation can be stimulated by ABL has been well documented (e.g., reviewed by Valent et al. Haematologica. 2018, 103(10):1593-1603; Srole and Ganz. J Cell Physiol. 2021, 236(7):4888-4901). Although the current study might provide more details, the authors should cite the previous studies and make necessary comparisons. Any new finding or discrepancy (e.g., the decrease of reticulocytes upon ABL, as shown in Fig. 3) should be described and explained in detail.

2. In Figs. 1 and 3, besides the statistical analysis results, representative results of flow cytometry analysis of the bone marrow and spleen samples upon ABL should also be provided for readers’ better understanding and for objective evaluation of the quality of the data.

3. In the “Acute blood loss leads to the up-regulation of the pathogen-sensing Clec5a gene and the down-regulation of the early erythroid cell genes Itga4 and Itgb1 in Ter-119+ erythroid cells” section, too many gene names were simply listed in the main text. It would be better to put them in a table, together with the values of their expression levels in different samples.

4. Clec5a seems to be just one of the many differentially expressed genes. Without more technical validation, determination of its protein level, or functional experiments to explain its role in the erythrocyte differentiation or immune response, it is not reasonable to emphasize Clec5a too much over other genes, let alone highlighting it in the title of the manuscript.

5. There are several problems regarding the BioPlex analysis of cytokine levels. First, as this assay was performed using the media of cultured erythrocytes, the results may not reflect the in vivo cytokine secretion functions of the ABL-stimulated cells. Second, as most of the cytokines seemed to be undetectable in this assay, it is questionable whether the viability and functions of the cells are comparable with those in vivo or fresh isolated. Third, there is no inter-validation between the gene expression profiling and the BioPlex analysis results. These problems should be explained or at least discussed by providing more supporting information.

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Reviewer #1: Yes: Xiao-Jian Sun

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Acute blood loss in mice forces differentiation of both CD45-positive and CD45-negative erythroid cells and leads to a desreased CCL3 chemokine production by bone marrow erythroid cell

PONE-D-24-19541R1

Dear Dr. Sennikov,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Gary S. Stein

Academic Editor

PLOS ONE

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