In-silico analysis of myeloid cells across the animal kingdom reveals neutrophil evolution by colony-stimulating factors

  1. Damilola Pinheiro
  2. Marie-Anne Mawhin
  3. Maria Prendecki
  4. Kevin J Woollard

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Abstract

Neutrophils constitute the largest population of phagocytic granulocytes in the blood of mammals. The development and function of neutrophils and monocytes is primarily governed by the granulocyte colony-stimulating factor receptor family (CSF3R/CSF3) and macrophage colony-stimulating factor receptor family (CSF1R/IL34/CSF1) respectively. Using various techniques this study considered how the emergence of receptor:ligand pairings shaped the distribution of blood myeloid cell populations. Comparative gene analysis supported the ancestral pairings of CSF1R/IL34 and CSF3R/CSF3, and the emergence of CSF1 later in lineages after the advent of Jawed/Jawless fish. Further analysis suggested that the emergence of CSF3 lead to reorganisation of granulocyte distribution between amphibian and early reptiles. However, the advent of endothermy likely contributed to the dominance of the neutrophil/heterophil in modern-day mammals and birds. In summary, we show that the emergence of CSF3R/CSF3 was a key factor in the subsequent evolution of the modern-day mammalian neutrophil.

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  1. Version published to 10.7554/elife.60214 on eLife
  2. eLife

    ###Reviewer #3:

    PREreview of "Analysis of receptor-ligand pairings and distribution of myeloid subpopulations across the animal kingdom reveals neutrophil evolution was facilitated by colony-stimulating factors" Authored by Damilola Pinheiro et al. and posted on bioRxiv DOI: 10.1101/2020.06.19.161059

    Review authors in alphabetical order: Monica Granados and Katrina Murphy

    This review is the result of a virtual, live-streamed preprint journal club organized and hosted by PREreview and eLife. The discussion was joined by 8 people in total, including researchers from several regions of the world, a preprint author, and the event organizing team.

    Overview and take-home message: Pinheiro et al. have made advances in understanding neutrophil evolution and receptor-ligand participation by using a wide range of relational taxonomic data to show how CSF1/CSF1R and CSF3/CSF3R pairings evolved and contribute to granulocyte adaptations. Neutrophils are the most prolific granulocytes of the mammalian myeloid cells involved in the immune response. The research team bridged the gap in our knowledge on how the receptor-ligand pairing signals of CSF1R/CSF1 helped with bone marrow development, where these short-lived cells are generated, and CSF3R/CSF3 signaled the maximum production volume of the neutrophils and their movement as both a cell population and a single cell for distribution. Although this work is of significant importance in the field, below we outlined some concerns that could be addressed in the next version of this manuscript.

    Positive feedback:

    -(The findings were) Super novel! I love the breadth of taxa that are covered.

    -The intersection of cell biology and evolution is quite interesting!

    -This preprint could be a great model for future research/analysis.

    -The bolded subtitles for the different results sections were clear and helpful!

    -Increased understanding in neutrophils is important because children with immature neutrophils end up with recurrent early-onset life-threatening infections, e.g. severe congenital neutropenia. The more we can learn about neutrophils the more we can take steps to fight this type of infection.

    -I believe there is sufficient information in the materials and methods section to allow for the reproduction of the experiment.

    -The format made sense and the flow could be followed.

    -Cells have a tendency to call out domestic and evolutionary elements which are beneficial, so learning how receptor and ligand interactions evolve in different taxa is relevant.

    -It's interesting that gene complexes are associated with specific morphological aspects (e.g. exotherms and endotherms); the gene expression is obvious.

    -Figure 1 was cool to see. An expansion of Figure 1 might be of interest, where the phylogenetic tree changes over time to show the loss and gain of specific granulocytes.

    -Gene sequencing data was pulled from NCBI Gene and Ensembl databases to create Figure 2a. This is a great example of having a very specific question/hypothesis that can be answered with existing data.

    Can other types of physiology be tracked similarly in future research, e.g. scales, breathing - anything that could be mapped?

    Are there other groups that could relate to metabolism e.g. brain studies?

    It would be interesting to see the level of degradation, e.g. for fish - mapping physiology to a specific gene or brain size (the brain is more developed in different taxa).

    -The preprint can be relevant for myeloid phagocyte development and across species geometric morphology/computational anatomy particularly as it can relate to brain structure and sizes. More genetic data across species and homologous brain areas is helpful.

    -Overall there was a connection between the results and the research questions, yes, I would say the conclusions were supported by the data.

    -Even though we don't have this specific field expertise, as a group, we recommend this manuscript to both others and further peer review.

    Major concerns:

    -Since this is a large selection of taxa groups, can specification (of a subset) be divided into more detail?

    -Please note, taxonomy is not a field I am familiar with. It would be helpful to check the sequence conservation of the receptors across these taxa families and see whether there are any minor evolution instances where they mutated. If the receptors have mutated, do they have a particular residue that mutated?

    Acknowledgments:

    We thank all participants for attending the live-streamed preprint journal club. We are especially grateful for both the first author's contributions to the discussion and for those that engaged in providing constructive feedback.

  3. eLife

    ###Reviewer #2:

    The article is well written.

    1. Please provide a supplementary file containing all the references used for Figure 1b (complete blood count data; CBC). This would be a useful source of data for researchers interested in other blood cell types.

    2. Regarding the CBC data - the authors should mention in the text if all the samples were obtained from adults. Whilst I appreciate that n are low for some species, do you obtain the same result if you analyse males and females separately? This may be worth mentioning given that neutrophil numbers have been reported to be higher in women.

    3. Please provide a supplementary file containing all the NCBI gene and Ensembl accession numbers for each gene, in each species (Figure 2a).

    4. The authors may want to mention that there are other receptors for IL-34 which may explain its expression (in fish, Fig2a) in the absence of Csf1r.

    5. Please provide a supplementary file containing all the NCBI protein accession numbers used for Fig3a.

    6. Please include isotype controls on histogram in Supplementary figure 1a, 1c and 1d.

    7. Please include the full gating strategy for Supplementary figure 1a.

    8. Why was 72h chosen for the mobility assays (Supplementary Fig 1b)? At this point, monocytes cultured in CSF1 would begin differentiating into macrophages, and this may affect their mobility.

    9. Supplementary Fig 1c - please include the antibodies in the Lin cocktail for flow cytometry in the figure legend.

    10. Please mention in text and figure legend that human blood was used (there is no mention of it within text).

    11. Was a dead cell exclusion dye used for flow cytometry of human blood and neutrophils? And did you look at FSC-A v FSC-H to exclude doublets? If not, how can you exclude the possibility that the Cxcr4 hi neutrophils are not dying or doublets?

  4. eLife

    ###Reviewer #1:

    Pinheiro and colleagues have described a fascinating view on the evolution of neutrophils and other myeloid cells. This is a very original and potentially important piece of work. To follow neutrophil evolution in the evolutionary tree through co-analysis of the expression of G-CSF/G-CSFR and M-CSF/M-CSFR in the same tree is smart and interesting. The article is not easy to read and some issues need some more clarification(s). So the article would benefit when (random order):

    1. At several locations in the article the authors imply that G-CSF is inducing differentiation fitting with an inductive model (eg. introduction lines 41-51). At the same time the authors rightly mention the presence of mature neutrophils in G-CSF-/- mice (as well as mature eosinophils in IL5R-/- mice) more pointing at a stochastic model. This latter model assumes that expression of CSF-R's is more random, and only committed progenitors expressing these receptors will proliferate rather than differentiate in response to these CSF. Please provide sufficient arguments for the inductive model or change part of the interpretations when a stochastic model is more likely.

    2. In the whole article data are provided on numbers in peripheral blood. Only a minority of myeloid cells reside in the blood, the majority is in the tissues. The situation with neutrophils is uncertain. Please discuss.

    3. The part on C-EBP transcription factors is difficult to follow. Please help the reader understand why they are so important (based on KO strategies) while there is no clear picture in evolution as the genes are sometimes present, sometimes not. Some species have many, some only one. Simply stating redundancy in the system does not really fit the knock-out studies.

    4. The part described in lines 372-409/Supplemental figure 1 is not adding much to the article. It is only human with no evolutionary perspective. Consider removing.

    5. Please provide some more insight into the issue of eosinophils versus neutrophils. Now it is implied that the co-evolution with endothermia is relevant. Many articles suggest that eosinophils are more specialized in killing large targets (extracellular killing/e.g. parasites) vs neutrophils small targets (intracellular killing/e,g, bacteria). Can the authors provide their ideas about the functional difference of the cells in the evolutionary perspective.

    6. line 466: it is stated that neutrophils comprise the largest population of myeloid cells in mammals. This needs supportive evidence, as macrophages are thought to be the largest population at least in the tissues.

    7. lines 582 - 585. Although the issue of the lamins is well taken formal proof that the segmented nuclear morphology of neutrophils is important for movement and trans-cellular migration is yet to be determined (e.g. J Immunol January 1, 2019, 202 (1) 207-217; DOI: https://doi.org/10.4049/jimmunol.1801255 ).

    8. Lines 61-64 young children with SCN often have mutations in the ELANE gene rather than the GSF-R gene. Can the authors discuss how ELANE fits with the model they are presenting?

    9. Please provide the definitions of neutrophils and heterophils as they can be present as different cells in the same species.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    Pinheiro and colleagues have described a fascinating view on the evolution of neutrophils and other myeloid cells. The authors used a wide range of relational taxonomic data to show how CSF1/CSF1R and CSF3/CSF3R pairings evolved and contributed to granulocyte adaptations. This is a very original and potentially important piece of work that sheds light into the evolution of mammalian neutrophils.

  6. Version published to 10.1101/2020.06.19.161059 on bioRxiv