Phagocytes are key effector immune cells responsible for various biological processes; from orchestrating responses against invading pathogens to maintaining tissue homeostasis and neutrophils are the most abundant population of granulocytic phagocytes present in mammalian blood (Adrover et al., 2019; Hidalgo et al., 2019; Ng et al., 2019; Yvan-Charvet and Ng, 2019; Evrard et al., 2018). Neutrophil and heterophils (a functionally analogous granulocytic phagocyte population present in non-mammals and some mammals; Montali, 1988) arise from a shared pool of haematopoietic stem cells and mitotic myeloid progenitor cells that can also differentiate into monocytes, eosinophils and basophils following exposure to the relevant growth factor (Adrover et al., 2019; Yvan-Charvet and Ng, 2019; Mehta et al., 2014). The development and life cycle of mammalian neutrophils through a continuum of multipotent progenitors to a post-mitotic mature cell has been well described and has been recently reviewed (Hidalgo et al., 2019; Yvan-Charvet and Ng, 2019).

The development and function of myeloid phagocytes is mediated through lineage-specific transcription factors and pleiotropic glycoproteins - termed colony-stimulating factors (CSFs)- acting in concert on myeloid progenitor cells. CSF1, CSF3, and their cognate receptors, are lineage-specific and responsible for the differentiation and function of monocytes/macrophages and neutrophils, respectively (for full nomenclature see Supplementary file 1; Dai et al., 2002; Yoshida et al., 1990; Umeda et al., 1996; Liu et al., 1996; Lieschke et al., 1994). There is a large body of evidence demonstrating the requirement of CSFs for cell development as multiple studies in knockout mice have shown that CSF1R/CSF1 and CSF3R/CSF3 are linked to the development of monocytes and neutrophils in vivo. The loss of CSF3 or CSF3R directly affects neutrophil populations resulting in a severe neutropenia, but not the complete loss of mature neutrophils in the models studied (Liu et al., 1996; Lieschke et al., 1994; Basu et al., 2000). The loss of CSF1 caused reduced cavity development of the bone marrow, loss of some progenitor populations, monocytopenia, and a reduced population of neutrophils in the bone marrow, although interestingly, elevated levels of neutrophils were observed in the periphery (Dai et al., 2002; Yoshida et al., 1990; Hibbs et al., 2007).

Similarly, in humans, single gene mutations have been described in both CSF3 and CSF3R resulting in severe congenital neutropenia (SCN). Furthermore, mutations in ELANE, a gene that encodes for the neutrophil granule serine protease -neutrophil elastase- and acts as a negative regulator of CSF3R signalling can also result in SCN (Piper et al., 2010; Hunter et al., 2003; Garg, 2020; Nayak et al., 2015). In contrast to some animal models, individuals present as children with early onset life-threatening infections because they lack mature neutrophils in the circulation as the neutrophil progenitors in the bone marrow do not progress beyond the myelocyte/promyelocyte stage (Lakshman and Finn, 2001). These studies demonstrate that CSF receptor/ligand pairings are essential for homeostatic neutrophil development and are intrinsically linked with neutrophil function, arguably making them ideal surrogates to study neutrophil evolution. Through multiple methods we examined the emergence of the respective CSF ligand and receptor genes and proteins across the Chordate phylum and demonstrated how CSF1R/CSF1 and CSF3R/CSF3 pairings contributed to the evolutionary adaptations of the mammalian neutrophil.

The mammalian neutrophil is a highly specialised cell that acts as a first responder to insults against a host immune system as well as acting in an equally important sentinel role (Hidalgo et al., 2019; Ng et al., 2019). They are functionally conserved across phylum Chordata and constitute the largest population of myeloid cells in the blood of birds and mammals. In humans, for example, up to one billion neutrophils per kilogram of body weight are produced in the bone marrow each day (Yvan-Charvet and Ng, 2019). The immune response has evolved in such a way as to be able to respond efficiently to a variety of threats, and it is interesting that a resource such as the bone marrow should be expended on the production and maintenance of the relatively short-lived neutrophil at the expense of the eosinophil and basophil. The timescale modelling demonstrates that prior to the advent of tetrapoda lineages, the neutrophil was in a pool of different WBC populations at the disposal of the jawed/jawless fish. However, the appearance of CSF3, altered the distribution and with each emerging animal order, a different granulocyte was favoured, presumably, for cell-specific adaptations in response to environmental challenges. Thus, suggesting that CSFR1R/CSF1 and CSF3R/CSF3 signalling conferred adaptations on the neutrophil that proved evolutionarily advantageous. The work discussed here focusses on the mammalian neutrophil within the context of blood, further work is required to define similar aspects as regards to other tissue resident populations such as macrophages or granulocytes.

The bone marrow is an essential site for granulopoiesis and myelopoiesis. As multipotent haematopoietic stem cells (HSCs) progress through different vascular niches within the bone marrow they sequentially lose their potential to form other lineages in response to environmental cues from bone marrow dwelling macrophages and stromal endothelial cells (Ng et al., 2019; Yvan-Charvet and Ng, 2019; Greenbaum and Link, 2011). The emergence of CSF1 in tetrapod lineages likely led to the appearance of bone and bone marrow. A consequence of this was the gradual re-organisation of haematopoiesis away from existing haematopoietic tissues, such as the eosinophil rich Leydig organ of sharks to tissue-specific compartments in the bone marrow. CSF1, in conjunction with another factor, receptor activator of nuclear factor-κB ligand (RANKL), co-ordinate the reabsorption of old bone through haematopoietically derived osteoclasts to allow the generation of new bone. CSF1 and RANKL (which emerged at a similar point in evolution) orchestrate bone-remodelling through their respective receptors, CSF1R and RANK (Kim and Kim, 2016). This suggests that by the time a bone structure had evolved in amphibia, the bone marrow had become the principle site of haematopoiesis, although there are some exceptions within various anurans (de Abreu Manso et al., 2009).

In contrast to peripheral blood, CSF3 is expressed on a number of cells in the bone marrow including; neutrophils, monocytes, B cells, myeloid progenitors and HSCs (Greenbaum and Link, 2011; Petit et al., 2002; Semerad et al., 2005; Lévesque et al., 2003). Although CSF3 can likely act directly on HSCs through their receptor, it is believed to indirectly mobilise HSCs through a monocytic intermediary that secretes CSF3, which leads to suppression of the CXCR4:CXCL12 axis, alteration of the bone marrow niche, and the subsequent release of HSCs (Greenbaum and Link, 2011; Petit et al., 2002; Semerad et al., 2005; Lévesque et al., 2003). In a similar process, CSF3 can suppress B cell lymphopoiesis by again targeting CXCL12 and suppressing other B cell tropic factors or stromal cells that favour the lymphoid niche (Day et al., 2015). Thus, the emergence of CSF3R/CSF3 conferred the adaptation, or advantage, of control of the biological machinery i.e. CSF3 provides a mechanism through which haematopoiesis can be shaped and deployed in favour of maximal neutrophil generation. This broadly aligns with the neutrophil starting to dominate peripheral populations in the transition from amphibia to the lizards of early squamata following the appearance of bone.

Mammalian neutrophil production occurs in the haematopoietic cords present within the venous sinuses and the daily output is approximately 1.7 × 10⁹/kg (Summers et al., 2010). As our data demonstrate high levels of neutrophil output are common across warm-blooded chordates and neutrophil/heterophils are the predominant granulocyte in birds and mammals, in what could be considered an example of convergent evolution. One of the fundamental requirements of the host innate immune response is to be able to respond rapidly to perceived threats that can be present at any site in the body, which requires the cellular arm to be constantly present and available. Theoretically this can be achieved in the steady state by having either low volumes of long-lived cells or high volumes of short-lived cells circulating in the periphery. As a consequence, there are potential biological trade-offs when considering each setting, firstly; the longer a cell survives in the periphery, the more effort is required by the host to maintain its survival in terms of providing appropriate cues and growth factors. Secondly, while a short-lived cell does not need as much host input for survival, a high turnover is required in order to ensure it doesn’t compete for growth factors with other cell types or cause damage by being retained beyond its usefulness. The latter setting fits with the observed neutrophil life cycle and could be considered an evolutionary adaptation. The CSF3R/CSF3 signalling pathway is essential for generating high neutrophil numbers without adverse effects to the host.

The neutrophil has a short circulatory half-life in the steady state of approximately one day (Ng et al., 2019; Yvan-Charvet and Ng, 2019), although this does remain controversial as some estimates of the circulatory lifespan are as much as five days (Pillay et al., 2010). By contrast, a mature eosinophil has a short circulatory lifespan of approximately 18 hr before relocating to the tissue, where it will survive for a further 2–5 days (Park and Bochner, 2010). Similarly, intermediate and non-classical monocytes can survive in the periphery for four and seven days respectively (Patel et al., 2017). In human studies, CSF3 has been shown to ‘effectively’ shorten the lifespan of neutrophil myeloblasts and promyelocytes by decreasing their time spent in cell cycle and accelerating their progress to maturation (Lord et al., 1989). Although, it is unclear what the exact effect is on the lifespan of post-mitotic neutrophils, studies have shown that the addition of exogenous CSF3 can delay apoptosis in mature neutrophils (van Raam et al., 2008; Zimbelman et al., 2002). This suggests that under certain conditions; such as infection, CSF3 can extend the lifespan of a mature neutrophil. Interestingly, classical monocytes also express CSF3R and have a short circulating lifespan of approximately 24 hr (Patel et al., 2017) These studies suggest that under steady state conditions and below a certain threshold, CSF3 has an as-yet-undocumented role either directly or indirectly in maintaining a short neutrophil lifespan and thus allowing the efficient turnover required to sustain high levels of granulopoiesis.

CSF3 orchestrates the life cycle of neutrophils in the bone marrow microenvironment by marshalling neutrophil progenitors through different development stages through to maturity, which is indicative of the inductive model of differentiation (Stanley, 2009). Accordingly, in response to this, CSF3R is expressed though every life stage, although at differing levels across the neutrophil populations, with the highest levels observed on mature cells where it is expressed at between two and three-fold more than on progenitors (Demetri and Griffin, 1991). CSF3R/CSF3 signalling has a dual role in controlling the distribution of neutrophils by both retaining a population of mature neutrophils in the bone marrow as a reservoir and facilitating the egress of other neutrophils into the periphery. Thus, a major evolutionary adaptation that CSF3R/CSF3 has conferred to the neutrophil is the ability to move, both on a population-wide and individual cell level.

There is abundant production of neutrophils daily in the bone marrow; however, there are far fewer neutrophils in circulation in the blood than are produced during granulopoiesis as the total neutrophil population is effectively stored in the bone marrow or marginated in intravascular pools within the spleen and liver (Ussov et al., 1995). Neutrophils can transit between sites in response to CSF3 signalling and it is estimated that 49% of cells are present in the circulating pool and the remaining 51% are marginated in discrete vascular pools (Summers et al., 2010; Athens et al., 1961). In the event of an infection, neutrophils can be mobilised from the marginated pools and bone marrow in response to CSF3-induced production of mobilising signals such as CXCL1 (Köhler et al., 2011). The co-ordinated egress of neutrophils from the bone marrow is achieved by CSF3 interacting with the CXCR4/CXCL12 and CXCR2/CXCL2 signalling pathways. CSF3 disrupts the CXCR4-CXCL12 retention axis by reducing CXCL12 release from endothelial stromal cells and reducing CXCR4 expression on neutrophils, thus allowing the movement of mature neutrophils through the venous sinuses to the periphery. CSF3-induced expression of CXCR2 on neutrophils then causes their migration to the vasculature along a CXCL2 chemotactic gradient (Adrover et al., 2019; Eash et al., 2010; Eash et al., 2009).

As neutrophils enter the bloodstream, they need to be able to migrate easily around the circulatory system under high flow conditions. This requires the neutrophil’s physical form to have high deformability and flexibility as it encounters the different diameters of the vasculature. The neutrophil nucleus is functionally adapted to this role because of its multi-nucleated structure and the distinct protein composition of the nuclear envelope, features that are widely conserved across mammalian species (Manley et al., 2018). CSF3 in co-ordination with C/EBPε and the ETS factors; Pu.1 and GA-binding protein (GABP) are responsible for the transcriptional control of the essential neutrophil nuclear structural proteins; Lamin A, Lamin C and Lamin B receptor (LBR) (Malu et al., 2016; Cohen et al., 2008). In comparison to other cell nuclear protein compositions, neutrophils have a low proportion of Lamin A and Lamin C, which is believed to make the nucleus more flexible for easier transit (Manley et al., 2018; Malu et al., 2016; Cohen et al., 2008). In contrast, the levels of LBR are increased, which is required for nuclear lobulation and subsequent neutrophil maturation (Manley et al., 2018; Malu et al., 2016). However, recent work has also demonstrated that band cells (immature neutrophils) are capable of just as efficient migration with incomplete nuclear segmentation as mature neutrophils (van Grinsven et al., 2019). Taken together, these factors support the argument that CSF3 signalling and C/EBPε are intrinsic to the evolution of mammalian neutrophil cell motility.

The evolution of CSF3R/CSF3 has been essential to the development of the neutrophil/heterophil in chordates and its own existence as the principle neutrophil growth factor is evolutionarily advantageous. Some members of the jawed/jawless lineages are unique in that they have populations of both neutrophils and heterophils, whereas later linages favour either neutrophils or heterophils. Our analysis shows that CSF3R/CSF3 emerged before the advent of jawed/jawless fish as both are present in the Coelacanth and absent from the other lineages. Intriguingly, an IL6-like gene was present in the same syntenic location of the whale shark, suggesting the possibility that heterophils and neutrophils were independently controlled by an IL6-like protein and CSF3 in the early lineages. IL6 is an important pleiotropic pro-inflammatory cytokine that plays key roles in infection, inflammation and haematopoiesis (Tanaka et al., 2014). Although CSF3R and IIL6R, which are functional paralogues, diverged from each other many millions of years ago, there is still functional redundancy at the cell level in modern-day mammals, as neutrophils are present - though at vastly reduced numbers - in CSF3^-/- and CSF3R^-/- mice (Liu et al., 1996; Lieschke et al., 1994). IL-6 can act on neutrophil progenitors and immature neutrophils thus supporting neutrophil development in the bone marrow of CSF3-deficient mice (Basu et al., 2000). However, mature neutrophils are refractory to IL-6 signalling as the expression of the IL-6R subunit gp130 is lost during maturation (Wilkinson et al., 2018). From amphibia onwards, the protein analysis here suggests that CSF3R and CSF3 co-evolved closely together and in contrast to CSF1R/CSF1/IL34, CSF3 is likely the only ligand of CSF3R.

Using various in-silico approaches, these findings have demonstrated how essential CSF3R and CSF3 (and to a lesser extent CSF1R and CSF1) are to the predominance of mammalian neutrophils in blood. Through the course of evolution, CSF3R/CSF3 signalling has accrued many properties that are responsible for the survival of the neutrophil and its ability to function, such as cell motility and mobility. Interestingly, although CSF3 is present in jawed/jawless fish lineages, it’s not until the emergence of tetrapoda that CSF3R and CSF3 begin to dominate haematopoiesis. Our current studies have been limited to chordates in which the data is publicly available and as the datasets continue to be expanded and updated, our hypothesis may need to change to reflect that. However, we would argue that this approach is a valid method for exploring the origins of the neutrophil granulocyte and would further be supported by the physical isolation and characterisation of neutrophils, associated genes and proteins in the different animal orders within Phylum Chordata.

We also envisage exploring the phagocytic granulocyte of two model species. The amphioxus is considered the basal chordate and a macrophage-like population has been identified, although a neutrophil/heterophil has yet to be described within the rudimentary circulatory system (Han et al., 2010). The lungfish, a primitive airbreathing fish, is unique among jawed/jawless lineages as it lives in freshwater and can survive on the land for up to one year. Accordingly, the lungfish has many adaptations and is considered to be a one of the closest living relatives to tetrapods making it a good model species for further study (Takezaki and Nishihara, 2017). Given how essential motility and mobility are to neutrophil function and development, it would be useful to discern when it emerged in evolution by identifying if equivalent cells and functional gene/protein orthologues are present in either species. These comparative studies would answer fundamental questions about the origin of the neutrophilic phagocyte.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Adrover JM
   2. Del Fresno C
   3. Crainiciuc G
   4. Cuartero MI
   5. Casanova-Acebes M
   6. Weiss LA
   7. Huerga-Encabo H
   8. Silvestre-Roig C
   9. Rossaint J
   10. Cossío I
   11. Lechuga-Vieco AV
   12. García-Prieto J
   13. Gómez-Parrizas M
   14. Quintana JA
   15. Ballesteros I
   16. Martin-Salamanca S
   17. Aroca-Crevillen A
   18. Chong SZ
   19. Evrard M
   20. Balabanian K
   21. López J
   22. Bidzhekov K
   23. Bachelerie F
   24. Abad-Santos F
   25. Muñoz-Calleja C
   26. Zarbock A
   27. Soehnlein O
   28. Weber C
   29. Ng LG
   30. Lopez-Rodriguez C
   31. Sancho D
   32. Moro MA
   33. Ibáñez B
   34. Hidalgo A

   (2019) A neutrophil timer coordinates immune defense and vascular protection

   Immunity 51:966–967.

   https://doi.org/10.1016/j.immuni.2019.11.001

   • PubMed
   • Google Scholar
2. 1. Akagi T
   2. Thoennissen NH
   3. George A
   4. Crooks G
   5. Song JH
   6. Okamoto R
   7. Nowak D
   8. Gombart AF
   9. Koeffler HP

   (2010) In vivo deficiency of both C/EBPβ and C/EBPε results in highly defective myeloid differentiation and lack of cytokine response

   PLOS ONE 5:e15419.

   https://doi.org/10.1371/journal.pone.0015419

   • PubMed
   • Google Scholar
3. 1. Athens JW
   2. Haab OP
   3. Raab SO
   4. Mauer AM
   5. Ashenbrucker H
   6. Cartwright GE
   7. Wintrobe MM

   (1961) Leukokinetic studies. IV. the total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects

   Journal of Clinical Investigation 40:989–995.

   https://doi.org/10.1172/JCI104338

   • PubMed
   • Google Scholar
4. 1. Basu S
   2. Hodgson G
   3. Zhang H-H
   4. Katz M
   5. Quilici C
   6. Dunn AR

   (2000) “Emergency” granulopoiesis in G-CSF–deficient mice in response to Candida albicans infection

   Blood 95:3725–3733.

   https://doi.org/10.1182/blood.V95.12.3725

   • Google Scholar
5. 1. Benson DA
   2. Karsch-Mizrachi I
   3. Lipman DJ
   4. Ostell J
   5. Sayers EW

   (2009) GenBank

   Nucleic Acids Research 37:D26–D31.

   https://doi.org/10.1093/nar/gkn723

   • PubMed
   • Google Scholar
6. 1. Borregaard N
   2. Cowland JB

   (1997) Granules of the human neutrophilic polymorphonuclear leukocyte

   Blood 89:3503–3521.

   https://doi.org/10.1182/blood.V89.10.3503

   • PubMed
   • Google Scholar
7. 1. Chih DY
   2. Chumakov AM
   3. Park DJ
   4. Silla AG
   5. Koeffler HP

   (1997) Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon)

   Blood 90:2987–2994.

   https://doi.org/10.1182/blood.V90.8.2987

   • PubMed
   • Google Scholar
8. 1. Cohen TV
   2. Klarmann KD
   3. Sakchaisri K
   4. Cooper JP
   5. Kuhns D
   6. Anver M
   7. Johnson PF
   8. Williams SC
   9. Keller JR
   10. Stewart CL

   (2008) The lamin B receptor under transcriptional control of C/EBPepsilon is required for morphological but not functional maturation of neutrophils

   Human Molecular Genetics 17:2921–2933.

   https://doi.org/10.1093/hmg/ddn191

   • PubMed
   • Google Scholar
9. 1. Dai XM
   2. Ryan GR
   3. Hapel AJ
   4. Dominguez MG
   5. Russell RG
   6. Kapp S
   7. Sylvestre V
   8. Stanley ER

   (2002) Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in Osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects

   Blood 99:111–120.

   https://doi.org/10.1182/blood.V99.1.111

   • PubMed
   • Google Scholar
10. 1. Day RB
    2. Bhattacharya D
    3. Nagasawa T
    4. Link DC

    (2015) Granulocyte colony-stimulating factor reprograms bone marrow stromal cells to actively suppress B lymphopoiesis in mice

    Blood 125:3114–3117.

    https://doi.org/10.1182/blood-2015-02-629444

    • PubMed
    • Google Scholar
11. 1. de Abreu Manso PP
    2. de Brito-Gitirana L
    3. Pelajo-Machado M

    (2009) Localization of hematopoietic cells in the bullfrog (Lithobates catesbeianus)

    Cell and Tissue Research 337:301–312.

    https://doi.org/10.1007/s00441-009-0803-0

    • PubMed
    • Google Scholar
12. 1. Demetri GD
    2. Griffin JD

    (1991) Granulocyte colony-stimulating factor and its receptor

    Blood 78:2791–2808.

    https://doi.org/10.1182/blood.V78.11.2791.bloodjournal78112791

    • PubMed
    • Google Scholar
13. 1. Eash KJ
    2. Means JM
    3. White DW
    4. Link DC

    (2009) CXCR4 is a key regulator of neutrophil release from the bone marrow under basal and stress granulopoiesis conditions

    Blood 113:4711–4719.

    https://doi.org/10.1182/blood-2008-09-177287

    • Google Scholar
14. 1. Eash KJ
    2. Greenbaum AM
    3. Gopalan PK
    4. Link DC

    (2010) CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow

    Journal of Clinical Investigation 120:2423–2431.

    https://doi.org/10.1172/JCI41649

    • PubMed
    • Google Scholar
15. 1. Engström PG
    2. Ho Sui SJ
    3. Drivenes O
    4. Becker TS
    5. Lenhard B

    (2007) Genomic regulatory blocks underlie extensive microsynteny conservation in insects

    Genome Research 17:1898–1908.

    https://doi.org/10.1101/gr.6669607

    • PubMed
    • Google Scholar
16. 1. Evrard M
    2. Kwok IWH
    3. Chong SZ
    4. Teng KWW
    5. Becht E
    6. Chen J
    7. Sieow JL
    8. Penny HL
    9. Ching GC
    10. Devi S
    11. Adrover JM
    12. Li JLY
    13. Liong KH
    14. Tan L
    15. Poon Z
    16. Foo S
    17. Chua JW
    18. Su IH
    19. Balabanian K
    20. Bachelerie F
    21. Biswas SK
    22. Larbi A
    23. Hwang WYK
    24. Madan V
    25. Koeffler HP
    26. Wong SC
    27. Newell EW
    28. Hidalgo A
    29. Ginhoux F
    30. Ng LG

    (2018) Developmental analysis of bone marrow neutrophils reveals populations specialized in expansion, trafficking, and effector functions

    Immunity 48:364–379.

    https://doi.org/10.1016/j.immuni.2018.02.002

    • PubMed
    • Google Scholar
17. 1. Forslund K
    2. Pekkari I
    3. Sonnhammer EL

    (2011) Domain architecture conservation in orthologs

    BMC Bioinformatics 12:326.

    https://doi.org/10.1186/1471-2105-12-326

    • PubMed
    • Google Scholar
18. 1. Garg B

    (2020) Inducible expression of a disease-associated

    The Journal of Biological Chemistry 295:7492–7500.

    https://doi.org/10.1074/jbc.RA120.012366

    • Google Scholar
19. 1. Genovese KJ
    2. He H
    3. Swaggerty CL
    4. Kogut MH

    (2013) The avian heterophil

    Developmental & Comparative Immunology 41:334–340.

    https://doi.org/10.1016/j.dci.2013.03.021

    • PubMed
    • Google Scholar
20. 1. Glasauer SM
    2. Neuhauss SC

    (2014) Whole-genome duplication in teleost fishes and its evolutionary consequences

    Molecular Genetics and Genomics 289:1045–1060.

    https://doi.org/10.1007/s00438-014-0889-2

    • PubMed
    • Google Scholar
21. 1. Greenbaum AM
    2. Link DC

    (2011) Mechanisms of G-CSF-mediated hematopoietic stem and progenitor mobilization

    Leukemia 25:211–217.

    https://doi.org/10.1038/leu.2010.248

    • PubMed
    • Google Scholar
22. 1. Halene S
    2. Gaines P
    3. Sun H
    4. Zibello T
    5. Lin S
    6. Khanna-Gupta A
    7. Williams SC
    8. Perkins A
    9. Krause D
    10. Berliner N

    (2010) C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via hlx

    Experimental Hematology 38:90–103.

    https://doi.org/10.1016/j.exphem.2009.11.004

    • PubMed
    • Google Scholar
23. 1. Han Y
    2. Huang G
    3. Zhang Q
    4. Yuan S
    5. Liu J
    6. Zheng T
    7. Fan L
    8. Chen S
    9. Xu A

    (2010) The primitive immune system of amphioxus provides insights into the ancestral structure of the vertebrate immune system

    Developmental & Comparative Immunology 34:791–796.

    https://doi.org/10.1016/j.dci.2010.03.009

    • PubMed
    • Google Scholar
24. 1. Hatano Y
    2. Taniuchi S
    3. Masuda M
    4. Tsuji S
    5. Ito T
    6. Hasui M
    7. Kobayashi Y
    8. Kaneko K

    (2009) Phagocytosis of heat-killed Staphylococcus aureus by eosinophils: comparison with neutrophils

    Apmis 117:115–123.

    https://doi.org/10.1111/j.1600-0463.2008.00022.x

    • PubMed
    • Google Scholar
25. 1. Hibbs ML
    2. Quilici C
    3. Kountouri N
    4. Seymour JF
    5. Armes JE
    6. Burgess AW
    7. Dunn AR

    (2007) Mice lacking three myeloid colony-stimulating factors (G-CSF, GM-CSF, and M-CSF) still produce macrophages and granulocytes and mount an inflammatory response in a sterile model of peritonitis

    The Journal of Immunology 178:6435–6443.

    https://doi.org/10.4049/jimmunol.178.10.6435

    • PubMed
    • Google Scholar
26. 1. Hidalgo A
    2. Chilvers ER
    3. Summers C
    4. Koenderman L

    (2019) The neutrophil life cycle

    Trends in Immunology 40:584–597.

    https://doi.org/10.1016/j.it.2019.04.013

    • PubMed
    • Google Scholar
27. 1. Hirai H
    2. Zhang P
    3. Dayaram T
    4. Hetherington CJ
    5. Mizuno S
    6. Imanishi J
    7. Akashi K
    8. Tenen DG

    (2006) C/EBPbeta is required for 'emergency' granulopoiesis

    Nature Immunology 7:732–739.

    https://doi.org/10.1038/ni1354

    • PubMed
    • Google Scholar
28. 1. Hunter MG
    2. Druhan LJ
    3. Massullo PR
    4. Avalos BR

    (2003) Proteolytic cleavage of granulocyte colony-stimulating factor and its receptor by neutrophil elastase induces growth inhibition and decreased cell surface expression of the granulocyte colony-stimulating factor receptor

    American Journal of Hematology 74:149–155.

    https://doi.org/10.1002/ajh.10434

    • PubMed
    • Google Scholar
29. 1. Jones LC
    2. Lin ML
    3. Chen SS
    4. Krug U
    5. Hofmann WK
    6. Lee S
    7. Lee YH
    8. Koeffler HP

    (2002) Expression of C/EBPbeta from the C/ebpalpha gene locus is sufficient for normal hematopoiesis in vivo

    Blood 99:2032–2036.

    https://doi.org/10.1182/blood.V99.6.2032

    • PubMed
    • Google Scholar
30. 1. Kim JH
    2. Kim N

    (2016) Signaling pathways in osteoclast differentiation

    Chonnam Medical Journal 52:12–17.

    https://doi.org/10.4068/cmj.2016.52.1.12

    • PubMed
    • Google Scholar
31. 1. Köhler A
    2. De Filippo K
    3. Hasenberg M
    4. van den Brandt C
    5. Nye E
    6. Hosking MP
    7. Lane TE
    8. Männ L
    9. Ransohoff RM
    10. Hauser AE
    11. Winter O
    12. Schraven B
    13. Geiger H
    14. Hogg N
    15. Gunzer M

    (2011) G-CSF-mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

    Blood 117:4349–4357.

    https://doi.org/10.1182/blood-2010-09-308387

    • PubMed
    • Google Scholar
32. 1. Kovács I
    2. Horváth M
    3. Kovács T
    4. Somogyi K
    5. Tretter L
    6. Geiszt M
    7. Petheő GL

    (2014) Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes

    Free Radical Research 48:1190–1199.

    https://doi.org/10.3109/10715762.2014.938234

    • PubMed
    • Google Scholar
33. 1. Lakshman R
    2. Finn A

    (2001) Neutrophil disorders and their management

    Journal of Clinical Pathology 54:7–19.

    https://doi.org/10.1136/jcp.54.1.7

    • PubMed
    • Google Scholar
34. 1. Lekstrom-Himes J
    2. Xanthopoulos KG

    (1999) CCAAT/enhancer binding protein epsilon is critical for effective neutrophil-mediated response to inflammatory challenge

    Blood 93:3096–3105.

    https://doi.org/10.1182/blood.V93.9.3096

    • PubMed
    • Google Scholar
35. 1. Letunic I
    2. Bork P

    (2019) Interactive tree of life (iTOL) v4: recent updates and new developments

    Nucleic Acids Research 47:W256–W259.

    https://doi.org/10.1093/nar/gkz239

    • PubMed
    • Google Scholar
36. 1. Lévesque JP
    2. Hendy J
    3. Takamatsu Y
    4. Simmons PJ
    5. Bendall LJ

    (2003) Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide

    Journal of Clinical Investigation 111:187–196.

    https://doi.org/10.1172/JCI15994

    • PubMed
    • Google Scholar
37. 1. Lieschke GJ
    2. Grail D
    3. Hodgson G
    4. Metcalf D
    5. Stanley E
    6. Cheers C
    7. Fowler KJ
    8. Basu S
    9. Zhan YF
    10. Dunn AR

    (1994) Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization

    Blood 84:1737–1746.

    https://doi.org/10.1182/blood.V84.6.1737.1737

    • PubMed
    • Google Scholar
38. 1. Liu F
    2. Wu HY
    3. Wesselschmidt R
    4. Kornaga T
    5. Link DC

    (1996) Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice

    Immunity 5:491–501.

    https://doi.org/10.1016/S1074-7613(00)80504-X

    • PubMed
    • Google Scholar
39. 1. Lord BI
    2. Bronchud MH
    3. Owens S
    4. Chang J
    5. Howell A
    6. Souza L
    7. Dexter TM

    (1989) The kinetics of human granulopoiesis following treatment with granulocyte colony-stimulating factor in vivo

    PNAS 86:9499–9503.

    https://doi.org/10.1073/pnas.86.23.9499

    • PubMed
    • Google Scholar
40. 1. Malu K
    2. Garhwal R
    3. Pelletier MGH
    4. Gotur D
    5. Halene S
    6. Zwerger M
    7. Yang Z-F
    8. Rosmarin AG
    9. Gaines P

    (2016) Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

    The Journal of Immunology 197:910–922.

    https://doi.org/10.4049/jimmunol.1402285

    • Google Scholar
41. 1. Manley HR
    2. Keightley MC
    3. Lieschke GJ

    (2018) The neutrophil nucleus: an important influence on neutrophil migration and function

    Frontiers in Immunology 9:2867.

    https://doi.org/10.3389/fimmu.2018.02867

    • PubMed
    • Google Scholar
42. 1. Mehta HM
    2. Glaubach T
    3. Corey SJ

    (2014) Systems approach to phagocyte production and activation: neutrophils and monocytes

    Advances in Experimental Medicine and Biology 844:99–113.

    https://doi.org/10.1007/978-1-4939-2095-2_6

    • PubMed
    • Google Scholar
43. 1. Montali RJ

    (1988) Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals)

    Journal of Comparative Pathology 99:1–26.

    https://doi.org/10.1016/0021-9975(88)90101-6

    • PubMed
    • Google Scholar
44. 1. Morosetti R
    2. Park DJ
    3. Chumakov AM
    4. Grillier I
    5. Shiohara M
    6. Gombart AF
    7. Nakamaki T
    8. Weinberg K
    9. Koeffler HP

    (1997) A novel, myeloid transcription factor, C/EBP epsilon, is upregulated during granulocytic, but not monocytic, differentiation

    Blood 90:2591–2600.

    https://doi.org/10.1182/blood.V90.7.2591

    • PubMed
    • Google Scholar
45. 1. Nandi S
    2. Cioce M
    3. Yeung Y-G
    4. Nieves E
    5. Tesfa L
    6. Lin H
    7. Hsu AW
    8. Halenbeck R
    9. Cheng H-Y
    10. Gokhan S
    11. Mehler MF
    12. Stanley ER

    (2013) Receptor-type Protein-tyrosine Phosphatase ζ Is a Functional Receptor for Interleukin-34

    Journal of Biological Chemistry 288:21972–21986.

    https://doi.org/10.1074/jbc.M112.442731

    • Google Scholar
46. 1. Nayak RC
    2. Trump LR
    3. Aronow BJ
    4. Myers K
    5. Mehta P
    6. Kalfa T
    7. Wellendorf AM
    8. Valencia CA
    9. Paddison PJ
    10. Horwitz MS
    11. Grimes HL
    12. Lutzko C
    13. Cancelas JA

    (2015) Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

    Journal of Clinical Investigation 125:3103–3116.

    https://doi.org/10.1172/JCI80924

    • PubMed
    • Google Scholar
47. 1. Ng LG
    2. Ostuni R
    3. Hidalgo A

    (2019) Heterogeneity of neutrophils

    Nature Reviews. Immunology 19:255–265.

    https://doi.org/10.1038/s41577-019-0141-8

    • PubMed
    • Google Scholar
48. 1. Park YM
    2. Bochner BS

    (2010) Eosinophil survival and apoptosis in health and disease

    Allergy, Asthma and Immunology Research 2:87–101.

    https://doi.org/10.4168/aair.2010.2.2.87

    • Google Scholar
49. 1. Patel AA
    2. Zhang Y
    3. Fullerton JN
    4. Boelen L
    5. Rongvaux A
    6. Maini AA
    7. Bigley V
    8. Flavell RA
    9. Gilroy DW
    10. Asquith B
    11. Macallan D
    12. Yona S

    (2017) The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

    Journal of Experimental Medicine 214:1913–1923.

    https://doi.org/10.1084/jem.20170355

    • PubMed
    • Google Scholar
50. 1. Petit I
    2. Szyper-Kravitz M
    3. Nagler A
    4. Lahav M
    5. Peled A
    6. Habler L
    7. Ponomaryov T
    8. Taichman RS
    9. Arenzana-Seisdedos F
    10. Fujii N
    11. Sandbank J
    12. Zipori D
    13. Lapidot T

    (2002) G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4

    Nature Immunology 3:687–694.

    https://doi.org/10.1038/ni813

    • PubMed
    • Google Scholar
51. 1. Pillay J
    2. den Braber I
    3. Vrisekoop N
    4. Kwast LM
    5. de Boer RJ
    6. Borghans JA
    7. Tesselaar K
    8. Koenderman L

    (2010) In vivo labeling with 2h2o reveals a human neutrophil lifespan of 5.4 days

    Blood 116:625–627.

    https://doi.org/10.1182/blood-2010-01-259028

    • PubMed
    • Google Scholar
52. 1. Piper MG
    2. Massullo PR
    3. Loveland M
    4. Druhan LJ
    5. Kindwall-Keller TL
    6. Ai J
    7. Copelan A
    8. Avalos BR

    (2010) Neutrophil elastase downmodulates native G-CSFR expression and granulocyte-macrophage colony formation

    Journal of Inflammation 7:5.

    https://doi.org/10.1186/1476-9255-7-5

    • Google Scholar
53. 1. Pruitt KD
    2. Tatusova T
    3. Maglott DR

    (2007) NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins

    Nucleic Acids Research 35:D61–D65.

    https://doi.org/10.1093/nar/gkl842

    • PubMed
    • Google Scholar
54. 1. Sayers EW
    2. Barrett T
    3. Benson DA
    4. Bryant SH
    5. Canese K
    6. Chetvernin V
    7. Church DM
    8. DiCuccio M
    9. Edgar R
    10. Federhen S
    11. Feolo M
    12. Geer LY
    13. Helmberg W
    14. Kapustin Y
    15. Landsman D
    16. Lipman DJ
    17. Madden TL
    18. Maglott DR
    19. Miller V
    20. Mizrachi I
    21. Ostell J
    22. Pruitt KD
    23. Schuler GD
    24. Sequeira E
    25. Sherry ST
    26. Shumway M
    27. Sirotkin K
    28. Souvorov A
    29. Starchenko G
    30. Tatusova TA
    31. Wagner L
    32. Yaschenko E
    33. Ye J

    (2009) Database resources of the national center for biotechnology information

    Nucleic Acids Research 37:D5–D15.

    https://doi.org/10.1093/nar/gkn741

    • PubMed
    • Google Scholar
55. 1. Scott LM
    2. Civin CI
    3. Rorth P
    4. Friedman AD

    (1992) A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells

    Blood 80:1725–1735.

    https://doi.org/10.1182/blood.V80.7.1725.1725

    • Google Scholar
56. 1. Screpanti I
    2. Romani L
    3. Musiani P
    4. Modesti A
    5. Fattori E
    6. Lazzaro D
    7. Sellitto C
    8. Scarpa S
    9. Bellavia D
    10. Lattanzio G

    (1995) Lymphoproliferative disorder and imbalanced T-helper response in C/EBP beta-deficient mice

    The EMBO Journal 14:1932–1941.

    https://doi.org/10.1002/j.1460-2075.1995.tb07185.x

    • PubMed
    • Google Scholar
57. 1. Segaliny AI
    2. Brion R
    3. Mortier E
    4. Maillasson M
    5. Cherel M
    6. Jacques Y
    7. Le Goff B
    8. Heymann D

    (2015) Syndecan-1 regulates the biological activities of interleukin-34

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1853:1010–1021.

    https://doi.org/10.1016/j.bbamcr.2015.01.023

    • Google Scholar
58. 1. Semerad CL
    2. Christopher MJ
    3. Liu F
    4. Short B
    5. Simmons PJ
    6. Winkler I
    7. Levesque JP
    8. Chappel J
    9. Ross FP
    10. Link DC

    (2005) G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow

    Blood 106:3020–3027.

    https://doi.org/10.1182/blood-2004-01-0272

    • PubMed
    • Google Scholar
59. 1. Shamri R
    2. Xenakis JJ
    3. Spencer LA

    (2011) Eosinophils in innate immunity: an evolving story

    Cell and Tissue Research 343:57–83.

    https://doi.org/10.1007/s00441-010-1049-6

    • PubMed
    • Google Scholar
60. 1. Smith LT
    2. Hohaus S
    3. Gonzalez DA
    4. Dziennis SE
    5. Tenen DG

    (1996) PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells

    Blood 88:1234–1247.

    https://doi.org/10.1182/blood.V88.4.1234.bloodjournal8841234

    • PubMed
    • Google Scholar
61. 1. Stanley ER

    (2009) Lineage commitment: cytokines instruct, at last!

    Cell Stem Cell 5:234–236.

    https://doi.org/10.1016/j.stem.2009.08.015

    • PubMed
    • Google Scholar
62. 1. Styrt B

    (1989) Species variation in neutrophil biochemistry and function

    Journal of Leukocyte Biology 46:63–74.

    https://doi.org/10.1002/jlb.46.1.63

    • PubMed
    • Google Scholar
63. 1. Summers C
    2. Rankin SM
    3. Condliffe AM
    4. Singh N
    5. Peters AM
    6. Chilvers ER

    (2010) Neutrophil kinetics in health and disease

    Trends in Immunology 31:318–324.

    https://doi.org/10.1016/j.it.2010.05.006

    • PubMed
    • Google Scholar
64. 1. Takezaki N
    2. Nishihara H

    (2017) Support for lungfish as the closest relative of tetrapods by using slowly evolving Ray-finned fish as the outgroup

    Genome Biology and Evolution 9:evw288.

    https://doi.org/10.1093/gbe/evw288

    • Google Scholar
65. 1. Tanaka T
    2. Akira S
    3. Yoshida K
    4. Umemoto M
    5. Yoneda Y
    6. Shirafuji N
    7. Fujiwara H
    8. Suematsu S
    9. Yoshida N
    10. Kishimoto T

    (1995) Targeted disruption of the NF-IL6 gene discloses its essential role in Bacteria killing and tumor cytotoxicity by macrophages

    Cell 80:353–361.

    https://doi.org/10.1016/0092-8674(95)90418-2

    • PubMed
    • Google Scholar
66. 1. Tanaka T
    2. Narazaki M
    3. Kishimoto T

    (2014) IL-6 in inflammation, immunity, and disease

    Cold Spring Harbor Perspectives in Biology 6:a016295.

    https://doi.org/10.1101/cshperspect.a016295

    • PubMed
    • Google Scholar
67. 1. Umeda S
    2. Takahashi K
    3. Shultz LD
    4. Naito M
    5. Takagi K

    (1996)

    Effects of macrophage colony-stimulating factor on macrophages and their related cell populations in the osteopetrosis mouse defective in production of functional macrophage colony-stimulating factor protein

    The American Journal of Pathology 149:559–574.

    • PubMed
    • Google Scholar
68. 1. Ussov WY
    2. Aktolun C
    3. Myers MJ
    4. Jamar F
    5. Peters AM

    (1995) Granulocyte margination in bone marrow: comparison with margination in the spleen and liver

    Scandinavian Journal of Clinical and Laboratory Investigation 55:87–96.

    https://doi.org/10.3109/00365519509075382

    • PubMed
    • Google Scholar
69. 1. van Grinsven E
    2. Textor J
    3. Hustin LSP
    4. Wolf K
    5. Koenderman L
    6. Vrisekoop N

    (2019) Immature neutrophils released in acute inflammation exhibit efficient migration despite incomplete segmentation of the nucleus

    The Journal of Immunology 202:207–217.

    https://doi.org/10.4049/jimmunol.1801255

    • PubMed
    • Google Scholar
70. 1. van Raam BJ
    2. Drewniak A
    3. Groenewold V
    4. van den Berg TK
    5. Kuijpers TW

    (2008) Granulocyte colony-stimulating factor delays neutrophil apoptosis by inhibition of calpains upstream of caspase-3

    Blood 112:2046–2054.

    https://doi.org/10.1182/blood-2008-04-149575

    • PubMed
    • Google Scholar
71. 1. Wilkinson AN
    2. Gartlan KH
    3. Kelly G
    4. Samson LD
    5. Olver SD
    6. Avery J
    7. Zomerdijk N
    8. Tey SK
    9. Lee JS
    10. Vuckovic S
    11. Hill GR

    (2018) Granulocytes are unresponsive to IL-6 due to an absence of gp130

    The Journal of Immunology 200:3547–3555.

    https://doi.org/10.4049/jimmunol.1701191

    • PubMed
    • Google Scholar
72. 1. Yamanaka R
    2. Barlow C
    3. Lekstrom-Himes J
    4. Castilla LH
    5. Liu PP
    6. Eckhaus M
    7. Decker T
    8. Wynshaw-Boris A
    9. Xanthopoulos KG

    (1997) Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein -deficient mice

    PNAS 94:13187–13192.

    https://doi.org/10.1073/pnas.94.24.13187

    • Google Scholar
73. 1. Yáñez A
    2. Coetzee SG
    3. Olsson A
    4. Muench DE
    5. Berman BP
    6. Hazelett DJ
    7. Salomonis N
    8. Grimes HL
    9. Goodridge HS

    (2017) Granulocyte-Monocyte Progenitors and Monocyte-Dendritic Cell Progenitors Independently Produce Functionally Distinct Monocytes

    Immunity 47:890–902.

    https://doi.org/10.1016/j.immuni.2017.10.021

    • Google Scholar
74. 1. Yates AD
    2. Achuthan P
    3. Akanni W
    4. Allen J
    5. Allen J
    6. Alvarez-Jarreta J
    7. Amode MR
    8. Armean IM
    9. Azov AG
    10. Bennett R
    11. Bhai J
    12. Billis K
    13. Boddu S
    14. Marugán JC
    15. Cummins C
    16. Davidson C
    17. Dodiya K
    18. Fatima R
    19. Gall A
    20. Giron CG
    21. Gil L
    22. Grego T
    23. Haggerty L
    24. Haskell E
    25. Hourlier T
    26. Izuogu OG
    27. Janacek SH
    28. Juettemann T
    29. Kay M
    30. Lavidas I
    31. Le T
    32. Lemos D
    33. Martinez JG
    34. Maurel T
    35. McDowall M
    36. McMahon A
    37. Mohanan S
    38. Moore B
    39. Nuhn M
    40. Oheh DN
    41. Parker A
    42. Parton A
    43. Patricio M
    44. Sakthivel MP
    45. Abdul Salam AI
    46. Schmitt BM
    47. Schuilenburg H
    48. Sheppard D
    49. Sycheva M
    50. Szuba M
    51. Taylor K
    52. Thormann A
    53. Threadgold G
    54. Vullo A
    55. Walts B
    56. Winterbottom A
    57. Zadissa A
    58. Chakiachvili M
    59. Flint B
    60. Frankish A
    61. Hunt SE
    62. IIsley G
    63. Kostadima M
    64. Langridge N
    65. Loveland JE
    66. Martin FJ
    67. Morales J
    68. Mudge JM
    69. Muffato M
    70. Perry E
    71. Ruffier M
    72. Trevanion SJ
    73. Cunningham F
    74. Howe KL
    75. Zerbino DR
    76. Flicek P

    (2020) Ensembl 2020

    Nucleic Acids Research 48:D682–D688.

    https://doi.org/10.1093/nar/gkz966

    • PubMed
    • Google Scholar
75. 1. Yoshida H
    2. Hayashi S
    3. Kunisada T
    4. Ogawa M
    5. Nishikawa S
    6. Okamura H
    7. Sudo T
    8. Shultz LD
    9. Nishikawa S

    (1990) The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene

    Nature 345:442–444.

    https://doi.org/10.1038/345442a0

    • PubMed
    • Google Scholar
76. 1. Yvan-Charvet L
    2. Ng LG

    (2019) Granulopoiesis and neutrophil homeostasis: a metabolic, daily balancing act

    Trends in Immunology 40:598–612.

    https://doi.org/10.1016/j.it.2019.05.004

    • PubMed
    • Google Scholar
77. 1. Zhang D-E
    2. Zhang P
    3. Wang N-d
    4. Hetherington CJ
    5. Darlington GJ
    6. Tenen DG

    (1997) Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein -deficient mice

    PNAS 94:569–574.

    https://doi.org/10.1073/pnas.94.2.569

    • Google Scholar
78. 1. Zhang P
    2. Nelson E
    3. Radomska HS
    4. Iwasaki-Arai J
    5. Akashi K
    6. Friedman AD
    7. Tenen DG

    (2002) Induction of granulocytic differentiation by 2 pathways

    Blood 99:4406–4412.

    https://doi.org/10.1182/blood.V99.12.4406

    • PubMed
    • Google Scholar
79. 1. Zhang P
    2. Iwasaki-Arai J
    3. Iwasaki H
    4. Fenyus ML
    5. Dayaram T
    6. Owens BM
    7. Shigematsu H
    8. Levantini E
    9. Huettner CS
    10. Lekstrom-Himes JA
    11. Akashi K
    12. Tenen DG

    (2004) Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP alpha

    Immunity 21:853–863.

    https://doi.org/10.1016/j.immuni.2004.11.006

    • PubMed
    • Google Scholar
80. 1. Zimbelman J
    2. Thurman G
    3. Leavey PJ
    4. Ellison MC
    5. Ambruso DR

    (2002) In vivo treatment with granulocyte colony-stimulating factor does not delay apoptosis in human neutrophils by increasing the expression of the vacuolar proton ATPase

    Journal of Investigative Medicine 50:33–37.

    https://doi.org/10.2310/6650.2002.33515

    • PubMed
    • Google Scholar

Acceptance summary:

In this study, Pinheiro and colleagues examine how neutrophils and other myeloid cells evolved. Using a wide range of relational taxonomic data, the authors show how pairings of genes in the granulocyte colony-stimulating factor receptor family (CSF3R/CSF3) and in the macrophage colony-stimulating factor receptor family (CSF1R/IL34/CSF1) evolved and contributed to granulocyte adaptations. This work sheds light into the evolution of mammalian neutrophils.

Decision letter after peer review:

Thank you for submitting your article "Analysis of receptor-ligand pairings and distribution of myeloid subpopulations across the animal kingdom reveals neutrophil evolution was facilitated by colony-stimulating factors" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Patricia Wittkopp as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Leo Koenderman (Reviewer #1); Clare Pridans (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

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.

Essential revisions:

1) First of all Figure 4, is missing. This is an important figure as the text (as it is now) is difficult to understand.

2) At several locations in the article the authors imply that G-CSF is inducing differentiation fitting with an inductive model (e.g. Introduction). 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.

3) 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.

4) 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.

5) The part described in the subsection “The emergence of CSF3R/CSF3 and onset of endothermy likely influenced the distribution of neutrophils in Chordates during evolution”/Supplementary Figure 1 is not adding much to the article. It is only human with no evolutionary perspective. Consider removing.

6) 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.

7) Discussion. 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.

8) Discussion. 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. van Grinsven et al., 2019 ).

9) Introduction. 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?

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

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

12) Regarding the CBC data – the authors should mention in the text if all the samples were obtained from adults. While we appreciate that n values 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.

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

14) The authors may want to mention that there are other receptors for IL-34 which may explain its expression (in fish, Figure 2A) in the absence of Csf1r.

15) Please provide a supplementary file containing all the NCBI protein accession numbers used for Figure 3A.

16) Please include isotype controls on histogram in Supplementary Figure 1A, C and D.

17) Please include full gating strategy for Supplementary Figure 1A.

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

19) Supplementary Figure 1C – please include the antibodies in the Lin cocktail for flow cytometry in the figure legend.

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

21) 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?

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

23) It would be helpful to check the sequence conservation of the receptors across these taxonomic 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?

Essential revisions:

1) First of all Figure 4, is missing. This is an important figure as the text (as it is now) is difficult to understand.

We thank the reviewer for bringing this to our attention. This was a problem with transfer from BioXriv and eLife (pre-print copy had Figure 4 included). We have now resolved the issue with editorial team and Figure 4 is present in its entirety.

2) At several locations in the article the authors imply that G-CSF is inducing differentiation fitting with an inductive model (e.g. Introduction). 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.

We thank the reviewers for raising these interesting points. As highlighted, functional redundancy is a prominent feature of myelopoiesis, as in the knock-out models identified, IL-6 and CSF2 can both fully or partially function as surrogates for either CSF3, IL5 or both [1, 2] (all references at end of this document). Although this is indicative that redundancy favours the stochastic model, we would argue in homeostatic conditions CSF3 favours an inductive model, as it is exclusively associated with neutrophil development and function. Competitive repopulation assays in CSF3R null mice have shown that CSF3 is responsible for almost all basal granulopoiesis including the regulation, production and maintenance of committed myeloid progenitors [3]. Similarly, in vitro CSF3 treatment of colony forming units -granulocyte, monocyte (CFU-GM), which are capable of producing monocyte or neutrophil populations, resulted in the uniform production of neutrophils, which is supportive of the inductive model [4, 5]. These studies also support a similar model where CSF3 is considered an instructive cytokine as opposed to permissive [6]. The bioavailability of CSF3R could be considered another indicator of the inductive model, as although it is also expressed on haematopoietic stem cells (HSCs), higher levels of expression are restricted to specific neutrophil progenitors increasing through to maturity [7, 8]. Furthermore, in basal conditions, CSF3 is present at higher concentrations in blood serum than CSF2, therefore the CSF3/CSF3R axis “effectively” outcompetes the CSF2/CSF2Ra/CSF2rb axis [8]. Within this context, it could be argued that the elevated presence of CSF3/CSF3R as the default or principle mechanism for neutrophil generation, in mammals at least, strongly supports an inductive model of development. We have now added a new sentence to reflect this hypothesis (Discussion).

3) 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.

We thank the reviewers for highlighting this important point. We agree the majority of myeloid cells are expected to reside within tissue. However, the focus of our work was to examine the blood microenvironment and evolution of bona fide blood myeloid cells. Correlating the evolution of CSFs with heterogeneity of myeloid subsets. We provocatively highlight that adaption of granulocyte like behaviour (migration between tight junctions) may correlate with arrival of lungs, blood vessels and acceleration of microbial infection diversity. More work will now be needed to define evolution of other cells outside the vasculature, such as tissue resident macrophages and bone fide subpopulations of granulocytes, such as neutrophils. We have now added text to reflect this in the Discussion.

4) 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.

We thank the reviewer for their comments and agree the section needs further clarity. Three members of the C/EBP gene family; C/EBPε, C/EBPβ and C/EBPε have been shown to be required for neutrophil development. C/EBPβ, is the earliest expressed member during neutrophil development and is present on HSCs, common myeloid progenitors (CMPs) and granulocyte monocyte progenitors (GMPs) [9]. C/EBPβ is necessary for the transition from the CMP to GMP and when absent, granulopoiesis is impeded and does not progress beyond the CMP stage [9-13]. C/EBPβ is the next member to be expressed and is present on GMPs through to maturation and is required for emergency and/or cytokine induced granulopoiesis. C/EBPβ-deficient mice failed to mobilise granulocytes in response to systemic fungal infection or cytokine stimuli [9, 14-17]. Interestingly, although C/EBPβ is not required for steady-state haematopoiesis, it has been shown in models to functionally compensate for the loss of C/EBPβ [18]. Finally, C/EBPε is required for the terminal differentiation of granulocytes and is the last of the three to be expressed and is only present on neutrophil-committed promyelocytes until maturation [19-24]. We have now expanded this section of work by including a table (new Supplementary file 2) that further details the C/EBP-related knock-out studies to help the reader understand the relevance of these transcription factors to neutrophil development. We agree that system redundancy is not necessarily an appropriate measure and have re-written the section to reflect that the C/EBP gene family is very strongly evolutionarily conserved across Phylum Chordata, indicative of their importance to neutrophil development (subsection “Interrogation of CSF1/CSF1R and CSF3/CSF3R and C/EBP gene family reveals their loss in the early lineages”).

5) The part described in the subsection “The emergence of CSF3R/CSF3 and onset of endothermy likely influenced the distribution of neutrophils in Chordates during evolution”/Supplementary Figure 1 is not adding much to the article. It is only human with no evolutionary perspective. Consider removing.

We thank the reviewers for their comments and agree that as the work currently stands it does not add much to the in silico work presented. We have decided to remove this work, as more lab work is required to complete the preliminary studies discussed and is out of scope of current in silico studies. We are now seeking grant funding to complete this work.

6) 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.

We thank the reviewers for their comments. We have considered how eosinophils and neutrophils are specialized towards intra- and extracellular killing respectively Firstly, neutrophils have an arsenal of primary and secondary granules such as; defensins, cathelicidins and lysozymes, which have known antimicrobial properties [25]. In contrast, although eosinophils have a similar set up of primary and secondary granules, eosinophil cationic protein (ECP) and major basic protein (MBP) are both key for extracellular killing, as they are toxic to large targets such as helminth parasites and hemoflagellates as well bacteria [26]. While both eosinophils and neutrophils use respiratory bursts as a killing function, the mechanisms differ. Eosinophils are known to have more O₂^- species than neutrophils, and this is believed to be required for the extracellular targeting of the respiratory burst, a feature unique to eosinophilic granulocytes and implicated in host-pathogen defences against parasites [27-29]. In contrast, neutrophils favour intracellular respiratory bursts for the destruction of internalised pathogens [27-29]. Interestingly, although eosinophils can phagocytose extracellular S. aureus and E. coli, they are unable to kill them as efficiently as neutrophils, who are by far the more efficient cell type [30].

Our analysis suggests that as early as the emergence of amphibians, neutrophils and eosinophils had likely acquired their respective functional properties, however, the population proportions are relatively similar. Therefore, it could be posited that the re-organisation of blood granulocyte populations is linked to the environment and likely corresponds with the type and prevalence of pathogens, i.e. pathogen exposure shapes the host myeloid cell pool and selects for the most appropriate cell type. In some ectothermic lineages, where presumably parasites and viral or bacterial pathogens are more prevalent, there is a near equal distribution of eosinophils and neutrophils. However, it could be hypothesised that following the transition from ectothermy to endothermy, there would have been changes to existing pathogens such as loss of pathogenicity or function as they failed to cross over and infect the newly emerging endothermic species, as evidenced by the inability of some viruses and fungi to infect modern day mammals [31-33]. Ultimately, this “thermic switch” would lead to the emergence of novel pathogenic strains that could survive in endothermic hosts (or both). As neutrophils and heterophils dominate the blood of land-dwelling mammals and birds respectively, this again supports an argument that in response to external selection pressures, the host species are redistributing their myeloid blood cell pool and selecting for neutrophil predominance. We have commented further on this within the manuscript (subsection “The emergence of CSF3R/CSF3 and onset of endothermy likely influenced the distribution of neutrophils in Chordates during evolution”).

7) Discussion. 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.

We thank the reviewer for their comment. We have adjusted our statement in the manuscript to reflect that it applied to the blood microenvironment only (Discussion), where neutrophils are the largest population of myeloid cells. Also see point 3 above.

8) Discussion. 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. van Grinsven et al., 2019 ).

We thank the reviewers for bringing this important and highly relevant article to our attention. It is now evident that immature neutrophils (band cells) can efficiently migrate with incomplete segmentation, suggesting at least, that it is not always a requirement for movement and trans-cellular migration in all neutrophil populations. We have amended the text of the manuscript to reflect this (Discussion).

9) Introduction. 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?

We thank the reviewers for their interesting comments, and we have further considered the role of ELANE in the model. The ELANE gene encodes for the neutrophil granule serine protease- neutrophil elastase- and is a negative regulator of CSF3R [34-37]. in vitro studies have shown the ELANE can downregulate CSF3R expression on the surface of mature neutrophils and can abrogate proliferative signals provided by CSF3 for expansion of the relevant committed myeloid progenitors [34, 35]. Similarly, it has also been shown that ELANE can antagonise the in vitro activity of CSF3 [34, 35]. Therefore, ELANE is heavily implicated in the regulation of neutrophil development via CSF3R/CSF3 signalling. During SCN the arrest of neutrophil development at the promyelocyte stage and concomitant apoptosis of those precursors results in acute neutropenia. There are several theories as to how aberrant ELANE expression could be implicated in this. Some studies have shown that improper ELANE expression results in endoplasmic reticulum stress (ER stress) which can lead to subcellular mislocalization and subsequent cell death of progenitors [36, 37]. Alternatively, other groups posit that disrupted or truncated CSF3R/CSF3 signalling as mediated by the aberrant expression of mutated ELANE could be a possible mechanism for the subsequent loss of neutrophils [34, 35]. A recent study that used an inducible expression system in mice, showed that a specific ELANE mutation diminished enzymatic activity which resulted in impaired granulocytic differentiation and reduced expression of key neutrophil associated genes such as GFi1, C/EBPδ, C/EBPε and CSF3R [37]. Taken together, these studies suggest there could be multiple mechanisms through which ELANE can dysregulate granulopoiesis and is likely mutation specific. ELANE fits into our model as the disruption caused be improperly functioning ELANE to granulopoiesis is significant as it directly impacts on CSF3R/CSF3 signalling and through this has downstream consequences for the development of neutrophils from committed progenitors. We have added a line to reflect that mutations in the ELANE gene are also involved in SCN pathogenesis (Introduction).

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

We thank the reviewer for their comments. Heterophils are described as a phagocytic leukocyte that is functionally analogous to the mammalian neutrophil. Heterophils can be distinguished from neutrophils using Romanowsky staining techniques as they do not stain neutral because of the presence of red -orange eosinophilic cytoplasmic granules [38, 39] A definition has been added to manuscript (Introduction).

11) 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.

We thank the reviewer for their comments. A supplementary file (Supplementary file 3) has been added that contains all the references included in the meta-analysis.

12) Regarding the CBC data – the authors should mention in the text if all the samples were obtained from adults. While we appreciate that n values 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.

We thank the reviewer for their comments and have clarified within the Materials and methods section, that datasets used for the metanalysis used sub-adult and adult data and were pooled with a minimum of male and female set where the data was available. It was not possible to take this approach for every species considered and within the context of this current study it is not possible to comment definitively on if there are gender-specific differences observed.

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

We thank the reviewer for their comments. A supplementary file (Supplementary file 4) has been added that contains all the ID numbers for each gene, in each species examined.

14) The authors may want to mention that there are other receptors for IL-34 which may explain its expression (in fish, Figure 2A) in the absence of Csf1r.

We thank for the reviewer for bringing these receptors to our attention. We have now included a line referencing the IL-34 receptors, Protein tyrosine phosphatase zeta and CD138 (Sydecan), but have cited this in the general discussion regarding the emergence and co-evolution of CSF1R and IL34 (subsection “Analysis of Chordate orthologous protein homology further supports the ancestral pairing of CSF1R/IL34 and CSF3R/CSF3 in early lineages”).

15) Please provide a supplementary file containing all the NCBI protein accession numbers used for Figure 3A.

We thank the reviewer for their comments. A supplementary file (Supplementary file 5) has been added that contains all the ID numbers for each protein, in each species examined.

16) Please include isotype controls on histogram in Supplementary Figure 1A, C and D.

We thank for the reviewer for their comments, this section of work has now been removed.

17) Please include full gating strategy for Supplementary Figure 1A.

We thank for the reviewer for their comments, this section of work has now been removed.

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

We thank for the reviewer for their comments, this section of work has now been removed.

19) Supplementary Figure 1C – please include the antibodies in the Lin cocktail for flow cytometry in the figure legend.

We thank for the reviewer for their comments, this section of work has now been removed.

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

We thank for the reviewer for their comments, this section of work has now been removed.

21) 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?

We thank for the reviewer for their comments, this section of work has now been removed.

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

We thank the reviewers for their comments. Where applicable larger taxonomic groups have been subdivided into smaller groups such as reptilia being divided in squamata, crocodilia and testudines. Similarly, mammalia has also been subdivided into the respective groups of monotremata, marsupalia and placentalia. For the purpose of these studies, the cell population of interest does not further subset as determined by CSF3R.

23) It would be helpful to check the sequence conservation of the receptors across these taxonomic 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?

We thank the reviewers for this good suggestion. However, we feel that this is beyond the scope of the original focus of the study. The work here addresses changes on a species or sub-species wide scale and how this might have contributed overall to the functional properties of mammalian neutrophils. Although the suggested idea is very interesting, we feel that focussing on changes at the residue level would not really fit in with the wider macro level view and might confuse the overall message.

References

1) Walker, F., et al., IL6/sIL6R complex contributes to emergency granulopoietic responses in G-CSF– and GM-CSF–deficient mice. Blood, 2008. 111(8): p. 3978-3985.2)Kopf, M., et al., IL-5-deficient mice have a developmental defect in CD5⁺ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses. Immunity, 1996. 4(1): p. 15-24.3) Richards, M.K., et al., Pivotal role of granulocyte colony-stimulating factor in the development of progenitors in the common myeloid pathway. Blood, 2003. 102(10): p. 3562-3568.4) Sonoda, Y., et al., Analysis in serum-free culture of the targets of recombinant human hemopoietic growth factors: interleukin 3 and granulocyte/macrophage-colony-stimulating factor are specific for early developmental stages. Proceedings of the National Academy of Sciences, 1988. 85(12): p. 4360.5) Avalos, B.R., Molecular analysis of the granulocyte colony-stimulating factor receptor. Blood, 1996. 88(3): p. 761-777.6) Stanley, E.R., Lineage Commitment: Cytokines Instruct, At Last! Cell Stem Cell, 2009. 5(3): p. 234-236.7) Demetri, G.D. and J.D. Griffin, Granulocyte colony-stimulating factor and its receptor. Blood, 1991. 78(11): p. 2791-808.8) Lee, K.Y., et al., Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes. Experimental and Molecular Medicine, 2000. 32(4): p. 210-215.9) Scott, L.M., et al., A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood, 1992. 80(7): p. 1725-35.10) Smith, L.T., et al., PU.1 (Spi-1) and C/EBP α regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood, 1996. 88(4): p. 1234-47.11) Ma, O., et al., Granulopoiesis requires increased C/EBPα compared to monopoiesis, correlated with elevated Cebpa in immature G-CSF receptor versus M-CSF receptor expressing cells. PLoS One, 2014. 9(4): p. e95784.12) Zhang, P., et al., Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBP α. Immunity, 2004. 21(6): p. 853-63.13) Ford, A.M., et al., Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP α, -β, and -δ during granulocyte-lineage specification. Proc Natl Acad Sci U S A, 1996. 93(20): p. 10838-43.14) Zhang, P., et al., Induction of granulocytic differentiation by 2 pathways. Blood, 2002. 99(12): p. 4406-12.15) Hirai, H., et al., C/EBPbeta is required for 'emergency' granulopoiesis. Nat Immunol, 2006. 7(7): p. 732-9.16) Screpanti, I., et al., Lymphoproliferative disorder and imbalanced T-helper response in C/EBP β-deficient mice. EMBO J, 1995. 14(9): p. 1932-41.17) Tanaka, T., et al., Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell, 1995. 80(2): p. 353-61.18) Jones, L.C., et al., Expression of C/EBPbeta from the C/ebpalpha gene locus is sufficient for normal hematopoiesis in vivo. Blood, 2002. 99(6): p. 2032-6.19) Yamanaka, R., et al., Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice. Proc Natl Acad Sci U S A, 1997. 94(24): p. 13187-92.20) Morosetti, R., et al., A novel, myeloid transcription factor, C/EBP epsilon, is upregulated during granulocytic, but not monocytic, differentiation. Blood, 1997. 90(7): p. 2591-600.21) Verbeek, W., et al., C/EBPepsilon -/- mice: increased rate of myeloid proliferation and apoptosis. Leukemia, 2001. 15(1): p. 103-11.22) Chih, D.Y., et al., Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon). Blood, 1997. 90(8): p. 2987-94.23) Lekstrom-Himes, J. and K.G. Xanthopoulos, CCAAT/enhancer binding protein epsilon is critical for effective neutrophil-mediated response to inflammatory challenge. Blood, 1999. 93(9): p. 3096-105.24) Gombart, A.F., et al., Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency. J Leukoc Biol, 2005. 78(5): p. 1153-65.25) Borregaard, N. and J.B. Cowland, Granules of the Human Neutrophilic Polymorphonuclear Leukocyte. Blood, 1997. 89(10): p. 3503-3521.26) Shamri, R., J.J. Xenakis, and L.A. Spencer, Eosinophils in innate immunity: an evolving story. Cell and tissue research, 2011. 343(1): p. 57-83.27) Bolscher, B.G.J.M., et al., NADPH:O2 oxidoreductase of human eosinophils in the cell-free system. FEBS Letters, 1990. 268(1): p. 269-273.28) Lacy, P., et al., Divergence of Mechanisms Regulating Respiratory Burst in Blood and Sputum Eosinophils and Neutrophils from Atopic Subjects. The Journal of Immunology, 2003. 170(5): p. 2670.29) Kovács, I., et al., Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes. Free Radical Research, 2014. 48(10): p. 1190-1199.30) Hatano, Y., et al., Phagocytosis of heat-killed Staphylococcus aureus by eosinophils: comparison with neutrophils. APMIS, 2009. 117(2): p. 115-23.31) Robert, V.A. and A. Casadevall, Vertebrate Endothermy Restricts Most Fungi as Potential Pathogens. The Journal of Infectious Diseases, 2009. 200(10): p. 1623-1626.32) Chinchar, V.G., Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Archives of Virology, 2002. 147(3): p. 447-470.33) Wirth, W., et al., Ranaviruses and reptiles. PeerJ, 2018. 6: p. e6083-e6083.34) Hunter, M.G., et al., Proteolytic cleavage of granulocyte colony-stimulating factor and its receptor by neutrophil elastase induces growth inhibition and decreased cell surface expression of the granulocyte colony-stimulating factor receptor. Am J Hematol, 2003. 74(3): p. 149-55.35) Piper, M.G., et al., Neutrophil elastase downmodulates native G-CSFR expression and granulocyte-macrophage colony formation. J Inflamm (Lond), 2010. 7(1): p. 5.36) Nayak, R.C., et al., Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells. J Clin Invest, 2015. 125(8): p. 3103-16.37) Garg, B., et al., Inducible expression of a disease-associated. J Biol Chem, 2020. 295(21): p. 7492-7500.38) Montali, R.J., Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals). Journal of Comparative Pathology, 1988. 99(1): p. 1-26.39) Horobin, R.W., How Romanowsky stains work and why they remain valuable — including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme. Biotechnic and Histochemistry, 2011. 86(1): p. 36-51.

Author details

1. Damilola Pinheiro

   Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft

   For correspondence

   d.pinheiro@imperial.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0294-9423

2. Marie-Anne Mawhin

   Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

3. Maria Prendecki

   Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7048-7457

4. Kevin J Woollard

   Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Supervision, Methodology, Writing - review and editing

   For correspondence

   k.woollard@imperial.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9839-5463

• 1,382

  Page views

• 139

  Downloads

• 5

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.