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Curated by eLife
Evaluation Summary:
The authors present a manuscript addressing an important unmet need, specifically focused on understanding the effects of high protein on hematopoiesis. This information can be of interest to basic biologists and clinicians who specialize in the areas of various diseases associated with elevated protein concentration (e.g. infections, inflammation, multiple myeloma, renal failure, etc). This is in part what makes for the complexity in studying this entity as the consequences of such disparate diseases are difficult to parcel out as causes of which specific disease manifestations. Furthermore, the presented work is done in an invertebrate model without additional confirmation in other model systems. Taken together, the work, which is plentiful in experiments, provides an incomplete understanding of cause and effect, leading to overinterpretation of results and overstating of derived conclusions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
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- Pharmacology and Toxicology (eLife)
Abstract
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Evaluation Summary:
The authors present a manuscript addressing an important unmet need, specifically focused on understanding the effects of high protein on hematopoiesis. This information can be of interest to basic biologists and clinicians who specialize in the areas of various diseases associated with elevated protein concentration (e.g. infections, inflammation, multiple myeloma, renal failure, etc). This is in part what makes for the complexity in studying this entity as the consequences of such disparate diseases are difficult to parcel out as causes of which specific disease manifestations. Furthermore, the presented work is done in an invertebrate model without additional confirmation in other model systems. Taken together, the work, which is plentiful in experiments, provides an incomplete understanding of cause and effect, …
Evaluation Summary:
The authors present a manuscript addressing an important unmet need, specifically focused on understanding the effects of high protein on hematopoiesis. This information can be of interest to basic biologists and clinicians who specialize in the areas of various diseases associated with elevated protein concentration (e.g. infections, inflammation, multiple myeloma, renal failure, etc). This is in part what makes for the complexity in studying this entity as the consequences of such disparate diseases are difficult to parcel out as causes of which specific disease manifestations. Furthermore, the presented work is done in an invertebrate model without additional confirmation in other model systems. Taken together, the work, which is plentiful in experiments, provides an incomplete understanding of cause and effect, leading to overinterpretation of results and overstating of derived conclusions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors present a large body of data in a silkworm model with primary high plasma protein concentration, characterizing the model with altered proportion of differentiating blood cells, possibly delayed differentiation, increased phagocytosis capacity, transiently increased autophagy and apoptosis, with evidence of increased oxidative stress. However, some of the outcomes are not specific to a type of hematopoietic cell, others are an overinterpretation of results, and no mechanistic studies are presented, making for descriptive and somewhat incomplete evaluation. Finally, the manuscript is written in a difficult to understand manner, without clearly stated reasoning for why some experiments were performed (e.g. why was an endocrine agent used to reverse effects of a high protein model?) and the …
Reviewer #1 (Public Review):
The authors present a large body of data in a silkworm model with primary high plasma protein concentration, characterizing the model with altered proportion of differentiating blood cells, possibly delayed differentiation, increased phagocytosis capacity, transiently increased autophagy and apoptosis, with evidence of increased oxidative stress. However, some of the outcomes are not specific to a type of hematopoietic cell, others are an overinterpretation of results, and no mechanistic studies are presented, making for descriptive and somewhat incomplete evaluation. Finally, the manuscript is written in a difficult to understand manner, without clearly stated reasoning for why some experiments were performed (e.g. why was an endocrine agent used to reverse effects of a high protein model?) and the conclusions are extrapolations of insufficiently rigorous assessments to warrant those conclusions. Specific comments are listed below:
- Figure 1B reveals that there are more cells early (48 and 96 hours) and fewer cells late (144 and 192 hours) in AM. It is unclear at what time point is the most relevant time point and whether fewer cells late suggests that more cells are dying or there is a differentiation block. The authors suggest in Figure 1C that there is a larger proportion of granulocytes late in AM relative to CK. What would be more helpful is to clarify which cell population is more or less prone to cell death and whether these patterns are a consequence of increased cell death in non-granulocyte lineage or increased differentiation toward granulocytes.
- Although Figure 1D-1F demonstrate some significant changes, the main interpretable result in a decrease in Gcm expression in AM. However, this data is not specific to any lineage, taking all hemocytes together and clouding the specificity of the result. Also, Figure 1G suggests that Gcm expression is equivalent in CK and AM controls, in contrast to Figure 1F results, and Gcm loss by RNAi does not appear to impact the proportion of cells differently in AM vs CK. This raises the possibility the Gcm is not an appropriate marker to measure mechanism in the current system. The authors allude to Gcm involvement in plasma cell differentiation in the Discussion section (page 30) but the relevance to increased granulocytes is missing. It may be that the authors are suggesting that decreased plasma cell differentiation leads to increased proportion of granulocytes but this is not clear from the current manuscript and the data would be indirect evidence at best.
- Figure 2 presents data measuring DNA replication. Although Figure 2B suggests that there is a delay in DNA replication in AM vs CK, the statistics are not included and there is no evidence whether if maintained longer, whether DNA replication returns to baseline levels. An alternative interpretative hypothesis would be that DNA replication is increased in later time points. Because there is no specific lineage of hemocytes and they are all increasing and decreasing at different time points, this finding is difficult to interpret. Furthermore, the Edu differences appear to possibly be driven by changes in granulocytes (Figure 2C) but this is not clear from the data presentation and no specific cell lineage effect is visible in Figure 2A. Figure 2D demonstrates evaluation of cell cycle from the 96-hour time point but it is unclear why this was selected and whether the results would be comparable if a different time point was selected. Finally Figure 2G-2H is presented without a clear explanation what this adds to the manuscript.
- Figure 3 aims to evaluate signaling via the JAK/STAT pathway. However, it is unclear which STAT is relevant in the silkworm and whether changes in phosphorylation, which is typically how signaling occurs was evaluated. STAT mRNA expression and total STAT protein concentration are not sufficient to demonstrate signaling changes along this pathway.
- Figure 4 presents data demonstrating increased phagocytosis in AM. However, as already noted in Figure 1, AM results in more granulocytes which have a strong phagocytic function among blood cells. Are the authors evaluating the specific function of granulocytes or all hemocytes together? Are there more particles per cell or more cells with particles? Without focusing the analysis on granulocytes specifically, the conclusion that AM has a higher phagocytic function is an overstatement.
- Figure 6 demonstrates increased apoptosis in blood cells, especially granulocytes, but Figure 1 demonstrates more granulocytes. How do the authors reconcile this conundrum?
- Figure 8 demonstrates use of 20E but the purpose of this approach is not made clear. Furthermore, the treatment is applied at 24 hours after modeling and presented in a way that cannot be directly compared with data from treatment applied at time 0. In addition, no CM treated with 20E or JAK inhibitor A490 is presented for comparison. It is altogether unclear the mechanism by which several of the presented parameters are restored. Again, signaling via JAK/STAT cannot be assessed on the basis of mRNA expression of STAT. Finally, JAK inhibitor A490 does not appear to have a cell specific effect in Figure 8I, confounding the results in Figure 8H.
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Reviewer #2 (Public Review):
In their manuscript entitled "Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model", Wang and colleagues set out to study the role of hyperproteinemia in circulating blood cell homeostasis by using an invertebrate model.
I have several major concerns about this paper.
In the introduction the authors mentioned that no model of hyperproteinemia exist, however it is unclear why animal models other than Bombyx mori could not be developed. This is particularly important as it seems evident that substantial differences exist between this model and mammals, not last the fact that hematopoiesis occurs in the bone marrow in humans, but it occurs in the peripheral blood in this model. It is unclear how data derived via this model could apply to human diseases.
The animal model is …
Reviewer #2 (Public Review):
In their manuscript entitled "Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model", Wang and colleagues set out to study the role of hyperproteinemia in circulating blood cell homeostasis by using an invertebrate model.
I have several major concerns about this paper.
In the introduction the authors mentioned that no model of hyperproteinemia exist, however it is unclear why animal models other than Bombyx mori could not be developed. This is particularly important as it seems evident that substantial differences exist between this model and mammals, not last the fact that hematopoiesis occurs in the bone marrow in humans, but it occurs in the peripheral blood in this model. It is unclear how data derived via this model could apply to human diseases.
The animal model is not even briefly discussed in the introduction and its limitations are not addressed in the discussion.
The authors bundle together the concept of HPPC whether this is driven by a malignant process (such as multiple myeloma) or a severe inflammatory state (such as sepsis). This appears rather risky as the nature of the proteins contributing to HPPC is substantially different and it could certainly play a role in its effect on hematopoiesis.
The manuscript is difficult to read and lacks of sustained focus and scientific scrutiny. Rather a number of circumstantial, descriptive data without strong cause-effect relationship are presented. A number of signaling pathways are examined without a clear underlying hypothesis or preliminary data to suggest relevance. Conclusions are not well supported by data. For instance, based on the presented data, the increase in STAT can not be completely explained by transcription only. However, stabilization of the protein was not assessed.
The authors state that the animal model is lethal, however no information is provided regarding the time of death in relationship to the induction of the model. It is also unclear if a cause underlying the death of animals could be established. This is an important point as most of the differences occur late after induction of the model and could be potentially due to impending demise.
There is no attempt to evaluate margination and transmigration of blood cells as a potential cause for changes in circulating blood cells over time.
Finally, several of the information provided in the discussion and introduction is incorrect such as that the effect of multiple myeloma on hematopoiesis is secondary to HPPC, while most likely than not it is due to bone marrow myelophtisis as it is present even in patients without significant elevation in plasma protein concentration. Or that multiple myeloma is secondary to hyperproteinemia or the implication that JAK/STAT inhibition is therapeutic in myeloma due to its effect on HPPC.
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