Determiningmacrophageversusneutrophilcontributionstoinnate immunity … · innate immunity, and the...

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REVIEW Determining macrophage versus neutrophil contributions to innate immunity using larval zebrafish Emily E. Rosowski ABSTRACT The specific roles of the two major innate immune cell types neutrophils and macrophages in response to infection and sterile inflammation are areas of great interest. The larval zebrafish model of innate immunity, and the imaging capabilities it provides, is a source of new research and discoveries in this field. Multiple methods have been developed in larval zebrafish to specifically deplete functional macrophages or neutrophils. Each of these has pros and cons, as well as caveats, that often make it difficult to directly compare results from different studies. The purpose of this Review is to (1) explore the pros, cons and caveats of each of these immune cell-depleted models; (2) highlight and place into a broader context recent key findings on the specific functions of innate immune cells using these models; and (3) explore future directions in which immune cell depletion methods are being expanded. KEY WORDS: Innate immunity, Larval zebrafish, Macrophages, Neutrophils Introduction The immune system is best known as a defense against pathogens, but it is also involved in other aspects of human health and disease: wound healing, allergy, auto-immunity, cancer, metabolism, aging and neurological diseases. At a cellular level, this system is composed of two main arms adaptive immunity (e.g. T cells and B cells) and innate immunity (e.g. macrophages and neutrophils) as well as components that bridge these arms (e.g. dendritic cells). In order to improve human disease outcomes, it is important to understand the specific functions of these cell types in different inflammatory contexts. Much recent research has focused on the role of adaptive immune cells in human health [most clearly illustrated by the explosion of research on the role of T cells in cancer therapy (Leach et al., 1996; Iwai et al., 2005)], but innate immune cells, including macrophages and neutrophils (Box 1), also play key roles in human health (Nielsen and Schmid, 2017; Coffelt et al., 2016; Kolaczkowska and Kubes, 2013). The immune response involves a complex crosstalk between many cells. The clearest way to experimentally define the function of a cell is to deplete that specific cell type in a whole-animal in vivo model. Such depletion experiments in mice have contributed major advances on the roles of both macrophages (Hua et al., 2018) and neutrophils (Daley et al., 2008). However, many questions remain unanswered, and murine models have limitations. The larval zebrafish model has emerged as an attractive supplementary model in which to interrogate these questions. The immune system of zebrafish is largely conserved with humans, and, during the larval stage, the adaptive immune system is not yet developed, allowing for the study of innate responses in isolation (Yoder et al., 2002) (Box 2). Excellent recent zebrafish innate immunity reviews have focused on findings related to the specific functions of macrophages (Yoshida et al., 2017; Torraca et al., 2014) or neutrophils (Henry et al., 2013; Harvie and Huttenlocher, 2015), or immunity in specific contexts such as infection (Gomes and Mostowy, 2019; Rosowski et al., 2018b; Masud et al., 2017). The purpose of this Review is to provide a broader view of the role of these cell types in diverse biological situations, and to compare and contrast different depletion methods to perhaps explain disparate results and interpretations in the literature. I first briefly discuss mouse models used to study macrophage and neutrophil function (Table 1) and highlight some of the first studies to utilize these models in order to provide historical context. Then, I dive deeper into the larval zebrafish model, first discussing how existing cell depletion methods work, highlighting the most recent findings that were made possible because of these immune cell-depleted models, and exploring their future prospects. Innate immune cell depletion in mice Macrophages and monocytes In terms of macrophage function (Box 1), the use of clodronate (see Glossary, Box 3) liposome-mediated depletion has historically identified important roles for macrophages in mice, especially in murine cancer and infection models (Van Rooijen and Sanders, 1994; Moreno, 2018). Macrophages phagocytose these liposomes, releasing clodronate inside the cell, leading to cell death (Frith et al., 1997; Lehenkari et al., 2002). In cancer models, clodronate liposome administration led to decreased tumor growth in multiple studies, demonstrating a role of macrophages in supporting tumor development (Banciu et al., 2008; Zeisberger et al., 2006; Fritz et al., 2014). However, it has become clear that the role and phenotypes of tumor- associated macrophages can vary widely depending on the specific tissue context (Yang et al., 2018; Hobson-Gutierrez and Carmona- Fontaine, 2018). In wound healing, clodronate liposome administration can decrease scarring, suggesting a role of macrophages in fibrosis at a wound (Zhu et al., 2016; Lu et al., 2014). In the context of infection, clodronate depletion experiments in mice revealed that macrophages are required for a successful immune response against multiple pathogens. For example, during infection with viruses [e.g. Herpes simplex virus (Pinto et al., 1991)], bacteria [e.g. Pseudomonas aeruginosa (Kooguchi et al., 1998; Manicone et al., 2009), Listeria monocytogenes (Pinto et al., 1991), Klebsiella pneumoniae (Cheung et al., 2000)] and fungi [e.g. Candida albicans (Qian et al., 1994), Aspergillus fumigatus (Bhatia et al., 2011)], macrophage depletion can lead to decreased mouse survival and/or increased infectious burden. Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA. *Author for correspondence ([email protected]) E.E.R., 0000-0002-6761-1098 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 © 2020. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2020) 13, dmm041889. doi:10.1242/dmm.041889 Disease Models & Mechanisms

Transcript of Determiningmacrophageversusneutrophilcontributionstoinnate immunity … · innate immunity, and the...

Page 1: Determiningmacrophageversusneutrophilcontributionstoinnate immunity … · innate immunity, and the imaging capabilities it provides, is a source of new research and discoveries in

REVIEW

Determining macrophage versus neutrophil contributions to innateimmunity using larval zebrafishEmily E. Rosowski

ABSTRACTThe specific roles of the two major innate immune cell types –

neutrophils and macrophages – in response to infection and sterileinflammation are areas of great interest. The larval zebrafish model ofinnate immunity, and the imaging capabilities it provides, is a sourceof new research and discoveries in this field. Multiple methods havebeen developed in larval zebrafish to specifically deplete functionalmacrophages or neutrophils. Each of these has pros and cons, aswell as caveats, that often make it difficult to directly compare resultsfrom different studies. The purpose of this Review is to (1) explore thepros, cons and caveats of each of these immune cell-depletedmodels; (2) highlight and place into a broader context recent keyfindings on the specific functions of innate immune cells using thesemodels; and (3) explore future directions in which immune celldepletion methods are being expanded.

KEY WORDS: Innate immunity, Larval zebrafish, Macrophages,Neutrophils

IntroductionThe immune system is best known as a defense against pathogens,but it is also involved in other aspects of human health and disease:wound healing, allergy, auto-immunity, cancer, metabolism, agingand neurological diseases. At a cellular level, this system iscomposed of two main arms – adaptive immunity (e.g. T cells and Bcells) and innate immunity (e.g. macrophages and neutrophils) – aswell as components that bridge these arms (e.g. dendritic cells). Inorder to improve human disease outcomes, it is important tounderstand the specific functions of these cell types in differentinflammatory contexts. Much recent research has focused on therole of adaptive immune cells in human health [most clearlyillustrated by the explosion of research on the role of T cells incancer therapy (Leach et al., 1996; Iwai et al., 2005)], but innateimmune cells, including macrophages and neutrophils (Box 1), alsoplay key roles in human health (Nielsen and Schmid, 2017; Coffeltet al., 2016; Kolaczkowska and Kubes, 2013).The immune response involves a complex crosstalk between

many cells. The clearest way to experimentally define the functionof a cell is to deplete that specific cell type in a whole-animal in vivomodel. Such depletion experiments in mice have contributed majoradvances on the roles of both macrophages (Hua et al., 2018) andneutrophils (Daley et al., 2008). However, many questions remainunanswered, and murine models have limitations. The larval

zebrafish model has emerged as an attractive supplementarymodel in which to interrogate these questions. The immunesystem of zebrafish is largely conserved with humans, and, duringthe larval stage, the adaptive immune system is not yet developed,allowing for the study of innate responses in isolation (Yoder et al.,2002) (Box 2).

Excellent recent zebrafish innate immunity reviews have focusedon findings related to the specific functions of macrophages (Yoshidaet al., 2017; Torraca et al., 2014) or neutrophils (Henry et al., 2013;Harvie and Huttenlocher, 2015), or immunity in specific contextssuch as infection (Gomes and Mostowy, 2019; Rosowski et al.,2018b; Masud et al., 2017). The purpose of this Review is to providea broader view of the role of these cell types in diverse biologicalsituations, and to compare and contrast different depletion methods toperhaps explain disparate results and interpretations in the literature.I first briefly discuss mouse models used to study macrophage andneutrophil function (Table 1) and highlight some of the first studies toutilize thesemodels in order to provide historical context. Then, I divedeeper into the larval zebrafish model, first discussing how existingcell depletion methods work, highlighting the most recent findingsthat were made possible because of these immune cell-depletedmodels, and exploring their future prospects.

Innate immune cell depletion in miceMacrophages and monocytesIn terms of macrophage function (Box 1), the use of clodronate (seeGlossary, Box 3) liposome-mediated depletion has historicallyidentified important roles for macrophages in mice, especially inmurine cancer and infection models (Van Rooijen and Sanders,1994; Moreno, 2018). Macrophages phagocytose these liposomes,releasing clodronate inside the cell, leading to cell death (Frith et al.,1997; Lehenkari et al., 2002). In cancer models, clodronate liposomeadministration led to decreased tumor growth in multiple studies,demonstrating a role of macrophages in supporting tumor development(Banciu et al., 2008; Zeisberger et al., 2006; Fritz et al., 2014).However, it has become clear that the role and phenotypes of tumor-associated macrophages can vary widely depending on the specifictissue context (Yang et al., 2018; Hobson-Gutierrez and Carmona-Fontaine, 2018). In wound healing, clodronate liposome administrationcan decrease scarring, suggesting a role of macrophages in fibrosis at awound (Zhu et al., 2016; Lu et al., 2014).

In the context of infection, clodronate depletion experiments inmice revealed that macrophages are required for a successfulimmune response against multiple pathogens. For example, duringinfection with viruses [e.g. Herpes simplex virus (Pinto et al.,1991)], bacteria [e.g. Pseudomonas aeruginosa (Kooguchiet al., 1998; Manicone et al., 2009), Listeria monocytogenes(Pinto et al., 1991), Klebsiella pneumoniae (Cheung et al., 2000)]and fungi [e.g. Candida albicans (Qian et al., 1994), Aspergillusfumigatus (Bhatia et al., 2011)], macrophage depletion can lead todecreased mouse survival and/or increased infectious burden.

Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA.

*Author for correspondence ([email protected])

E.E.R., 0000-0002-6761-1098

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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However, other conflicting studies report that macrophages are notrequired for immunity to some of these same pathogens, includingboth bacteria [e.g. P. aeruginosa (Koh et al., 2009; Cheung et al.,2000)] and fungi [e.g. A. fumigatus (Mircescu et al., 2009)]. Onepossible variable in these studies is the method of liposomeadministration. Clodronate liposomes can be injected intravenouslyto systemically deplete macrophages. However, they can also beadministered locally to deplete cells in just one organ such as thelungs (Cheung et al., 2000; Kooguchi et al., 1998). Independent ofthe administration route and target, macrophage depletion is difficultto monitor in multiple tissues in an intact, live mouse.Genetic models for macrophage depletion in mice have recently

been reviewed elsewhere (Hua et al., 2018), and herewe just providea brief overview of the most commonly used of these methods.

Specific cells can be depleted with a diphtheria toxin receptor(DTR; Box 3) system (Saito et al., 2001). DTR expression iscontrolled by a specific promoter and, upon diphtheria toxinadministration, it leads to the death of DTR+ cells. This system hasbeen used with both LysM andCD11b (also known as Itgam; Box 3)promoters. Although LysM and CD11b are expressed on multiplemyeloid lineages (Gordon et al., 1974; Keshav et al., 1991; Faustet al., 2000; Dziennis et al., 1995), DTR-mediated ablation of cellsexpressing these markers only seems to target macrophages, notneutrophils (Duffield et al., 2005; Goren et al., 2009). Both LysM:DTR and CD11b:DTR mice were used to study macrophagefunction in response to wounding, finding that macrophagespromote wound healing, primarily at the early stages of woundresponse (Mirza et al., 2009; Lucas et al., 2010).

Ccr2+ monocytes (Box 3) can also be specifically depleted withthe DTR system (Hohl et al., 2009). Ccr2 is required for monocyteinfiltration to an infection site, and Ccr2−/− mice are also used tointerrogate the function of these cells at sites of inflammation(Serbina and Pamer, 2006). Using DTR-mediated depletion,inflammatory monocytes were found to be important for clearanceof both bacterial [e.g. L. monocytogenes (Kurihara et al., 1997),Mycobacteria tuberculosis (Peters et al., 2001; Scott and Flynn,2002)] and fungal [e.g. A. fumigatus (Hohl et al., 2009), C. albicans(Ngo et al., 2014)] infections. Ultimately, the most completeinformation can be gleaned from using multiple depletion models inconjunction, as illustrated by a recent studyon the role of macrophagesin the response to Vaccinia virus, which used a variety of methods,including systemic and local clodronate administration, LysM:DTRand Ccr2−/−, to conclude that local and systemic macrophagepopulations have different functions in the control of viral replicationand dissemination (Davies et al., 2017).

NeutrophilsThe primary method for depleting neutrophils in mice isadministration of an antibody targeting Ly6G (also known as Gr-1;Box 3), first done primarily with the RB6-8C5 monoclonal antibody(Tepper et al., 1992; Conlan andNorth, 1994). Themechanism of thisdepletion is not fully understood, but seems to depend on the presenceof macrophages (Bruhn et al., 2016). The RB6-8C5 antibody clonewas used extensively to determine the function of neutrophils inmultiple inflammatory contexts, including infection, wounding andcancer, as detailed below.

Box 1. Macrophages and neutrophils, the basicsMacrophages and neutrophils are two major cell types of the innateimmune system, the primary function of which is to combat infection.These cells are the primary phagocytic cells, able to take up and destroyboth pathogens and cellular debris. However, these cells have amultitudeof functions, including secreting cytokines, growth factors, and lipidsignaling molecules to orchestrate the behavior of other immune cells.Macrophages can also efferocytose apoptotic cells and promote tissueremodeling, while neutrophils can form neutrophil extracellular traps(NETs) to combat pathogens too large to ingest. Tissue-residentmacrophages reside in almost every tissue, ready to respond to anylocal inflammatory signals. Monocytes can also be recruited from thecirculation into inflamed tissue to differentiate into macrophages. Likemonocytes, neutrophils primarily reside in circulating blood and aregenerally the first cells recruited to a source of infection or tissue damage.Both zebrafish macrophages (Torraca et al., 2014) and neutrophils (Henryet al., 2013) are remarkably similar to their mammalian counterparts.Zebrafish macrophages are capable of phagocytosis (Herbomel et al.,1999), pro-inflammatory gene expression and polarization (Nguyen-Chiet al., 2015), and granuloma formation (Davis et al., 2002). Neutrophilshave conserved motility mechanisms (Rosowski et al., 2016), and arecapable of phagocytosis (Le Guyader et al., 2008) and generating NETs(Palic et al., 2007).

Myeloid cell precursors develop by 12 h postfertilization in zebrafishand functional macrophages and neutrophils are present by 30 hpostfertilization (Herbomel et al., 1999; Le Guyader et al., 2008;Lieschke et al., 2002). Both of these cell types derive from a commonmyeloid progenitor, and their development is dependent on Pu.1 (alsoknown as Spi1b) (Rhodes et al., 2005; Li et al., 2011). While neutrophilsonly require minimal Pu.1 activity, macrophage development requiresearly and continual Pu.1, in conjunction with another transcription factor,Irf8 (Tenor et al., 2015; Li et al., 2011; Shiau et al., 2015). Neutrophildevelopment and function is also controlled by colony stimulating factor 3(Csf3; also known as Gcsf) and its receptor Csf3r (also known as Gcsfr)(Panopoulos and Watowich, 2008).

In zebrafish, these cells are marked by well-established reporters thatuse cell-specific promoters that are generally different from those used inmice. Macrophages are typically marked by the mpeg1.1 gene (Ellettet al., 2011); however, other genes are also specific for this cell type,including csf1ra (Gray et al., 2011) and mfap4 (Walton et al., 2015). Onecaveat is that these markers do not distinguish between tissue-residentmacrophages and inflammatory monocytes that can be recruited to sitesof inflammation. Neutrophils are typically marked by themyeloperoxidase(mpx) promoter (Mathias et al., 2006; Renshaw et al., 2006), an enzymemost highly expressed by neutrophils. Other promoters include lysozyme(lyz) (Kitaguchi et al., 2009), and although this genemay be expressed bymacrophages early in development, by 2 days postfertilization, it is specificfor neutrophils (Meijer et al., 2008). This finding does highlight anothercaveat for thesemarkers; while their expression is well studied in the larvalstage of zebrafish, it is unknown if they maintain their specificity throughthe juvenile and adult stages of the animal.

Box 2 . The advantages of larval zebrafish

As an intermediate model, larval zebrafish have many advantages overhigher vertebrates. The most highly touted aspect of larvae is that theyare relatively small (∼5 mm) and optically transparent, allowing for high-resolution imaging of immune cells throughout an entire live, intactorganism. Simple genetic methods utilizing both targeted gene mutation(e.g. CRISPR/Cas9) and exogenous transgene insertion (e.g. Tol2system) allow experimenters to test the role of specific genes in theseresponses, even within specific cell populations (Ablain et al., 2015;Zhou et al., 2018) and at specific times (Gerety et al., 2013). More than100 larvae can be obtained every week from one adult female, allowingfor experiments with high statistical power. Larval zebrafish are also idealfor drug screens as small molecules are well absorbed through their skinand inhibitors can be utilized by simply adding them to the larval water(Zon and Peterson, 2005). Adaptive immunity does not becomefunctionally mature until 4-6 weeks postfertilization (Lam et al., 2004),also allowing innate immunity to be studied in isolation in theseorganisms.

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Table 1. Key methods for macrophage and neutrophil depletion in mice and zebrafish

Method Type Pros Cons Reference

Mice Macrophages Clodronateliposomes

Toxin based High specificity of depletionProvides temporal control

Resulting cell carcasses mayinduce inflammation

Difficult to monitor efficiencyand breadth of depletion inreal time

(Van Rooijen andSanders, 1994)

Ccr2−/− Genetic Specifically inhibits migration ofinflammatory monocytes

Not a true ablation method,monocytes are present butdeficient in motility

Lack of temporal control

(Serbina and Pamer,2006)

Ccr2:DTR Toxin based Provides temporal control Resulting cell carcasses mayinduce inflammation

Difficult to monitor efficiencyand breadth of depletion inreal time

Requires treatment with DTRwhich may have off-targeteffects

(Hohl et al., 2009)

Neutrophils α-Ly6G Antibodymediated

Provides temporal control Resulting cell carcasses mayinduce inflammation

Antibody clones can havevarying specificity

Difficult to monitor efficiencyand breadth of depletion inreal time

(Daley et al., 2008)

Zebrafish Macrophages pu.1 (spi1b) MO Genetic High specificity of depletion Lack of temporal controlOnly a short-term knockdown

(Rhodes et al., 2005)

irf8 mutant/MO Genetic High specificity of depletion Lack of temporal controlOnly a short-term knockdownAlso causes increasedneutrophil numbers

(Li et al., 2011; Shiauet al., 2015)

Clodronateliposomes

Toxin based High specificity of depletionProvides temporal control

Resulting cell carcasses mayinduce inflammation

(Bernut et al., 2014)

NTR system Toxin based Provides temporal controlHighly penetrant stabletransgenic lines

Resulting cell carcasses mayinduce inflammation

Requires treatment with MTZwhich may have off-targeteffects

(Gray et al., 2011; Petrieet al., 2014)

Microglia and tissue-residentmacrophages

csf1ra−/− Genetic Modulates peripheralmacrophage establishmentspecifically

Effects on global macrophagepopulation still unclear

Lack of temporal control

(Herbomel et al., 2001)

il34 crispant/MO Genetic May prevent colonization ofmicroglia more specifically thancsf1ra knockdown

Effects on global macrophagepopulation still unclear

Lack of temporal control

(Wu et al., 2018; Kuilet al., 2019)

Neutrophils csf3r MO Genetic Can disrupt basal and/oremergency granulopoeisis

Can affect macrophagepopulations

Lack of temporal control

(Liongue et al., 2009)

Tg(mpx:cxcr4bWHIM)

Genetic Highly penetrant stabletransgenic line

Not a true ablation method,neutrophils are present butdeficient in motility

Lack of temporal control

(Walters et al., 2010)

Tg(mpx:rac2D57N)

Genetic Highly penetrant stabletransgenic line

Not a true ablation method,neutrophils are present butdeficient in motility

Lack of temporal control

(Deng et al., 2011)

NTR system Toxin based Provides temporal controlHighly penetrant stabletransgenic line

Resulting cell carcasses mayinduce inflammation

Requires treatment with MTZwhich may have off-targeteffects

(Prajsnar et al., 2012)

Ccr2, chemokine (C-C motif) receptor 2; csf1ra, colony-stimulating factor 1 receptor a; csf3r, colony-stimulating factor 3 receptor; cxcr4b, CXC chemokinereceptor 4b; DTR, diphtheria toxin receptor; il34, interleukin 34; irf8, interferon regulatory factor 8; MO, morpholino;mpx,myeloperoxidase; MTZ, metronidazole;NTR, nitroreductase; WHIM, warts, hypogammaglobulinemia, immunodeficiency and myelokathexis.

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In response to infection, such studies found that neutrophilsprotect the host against a large range of pathogens, includingbacteria [e.g. Listeria monocytogenes, Salmonella typhimurium,Yersinia enterocolitica (Conlan, 1997), P. aeruginosa (Koh et al.,2009), Staphylococcus aureus (Mölne et al., 2000)] and fungi[e.g. C. albicans (Romani et al., 1997), A. fumigatus (Stephens-Romero et al., 2005)]. However, excess neutrophil function wasalso found to harm the host, most likely due to excess tissuedamage (Gresham et al., 2000; Romani et al., 1997). In the contextof wounding, the role of neutrophils depended on the age of themice, with neutrophils promoting wound repair in older mice buthaving little effect in young mice (Nishio et al., 2008). The role ofneutrophils in cancer also varied in studies using the RB6-8C5antibody. Neutrophils were found to promote growth (Pekarek et al.,

1995; Seung et al., 1995) and metastasis (Tazawa et al., 2003; Spiceret al., 2012) of injected cancer cells. However, in an experiment inwhich the injected cancer cells were engineered to express colony-stimulating factor 3 (Csf3; also known as GCSF; Box 3), neutrophilscould promote tumor regression (Stoppacciaro et al., 1993).

However, although the RB6-8C5 antibody clone was chosen totarget Ly6G, it was later found to cross-react with Ly6C (Box 3), amarker that is also expressed on macrophages and monocytes, andthus this antibody can also deplete subsets of these cells (Daley et al.,2008; Lee et al., 2013). A different monoclonal antibody (1A8),which was originally less commonly used, is more specific forneutrophil depletion, targeting Ly6G but not Ly6C (Daley et al.,2008), but this issue highlights the difficulty in developing trulyspecific immune cell depletion methods.

Box 3. GlossaryBlastema: a collection of cells competent for growth and regeneration oftissues.Ccr2+ monocytes: Chemokine (C-C motif ) receptor 2 (Ccr2) is a receptorcontrolling the recruitment of monocytes out of the blood circulation and intoinflamed tissues. Ccr2+ monocytes found in tissues are also calledinflammatory monocytes.CD11b: subunit of the cell-surface exposed integrin Mac-1. Although foundon multiple cell types, including neutrophils, macrophages, and dendriticcells, high expression is often used as a marker for macrophage lineages.Chemokines: a subset of cytokines that specifically modulate migration ofcells, especially by attracting immune cells to a source of inflammation.Clodronate (also known as dichloromethylene diphosphonate): ananalog of pyrophosphate that is metabolized by cells to create a non-hydrolyzable form of ATP, blocking mitochondrial respiration and leading tocell death. When packaged in liposomes and injected into animals, it isspecifically phagocytosed by, and leads to depletion of, macrophages.Complement system: part of innate immunity, this system comprisessmall, soluble proteins that can be cleaved and activated in a cascade toeither directly target pathogen membranes or activate phagocytes.Colony-stimulating factor 1 receptor (Csf1r; also known as c-fms):receptor for multiple cytokines [including Colony-stimulating factor-1 (Csf1)and Interleukin 34 (Il34)] that has effects on the production, differentiation,migration and activity of macrophages. Zebrafish have two copies of thegene: csf1ra and csf1rb.Colony-stimulating factor 3 [Csf3; also known as Granulocyte colony-stimulating factor (GCSF)]: a cytokine that stimulates new neutrophilproduction from the hematopoietic tissue and their release into the blood.Crispant: an F0 embryo or larvae that was injected with Cas9 protein andguide RNAs (gRNAs) for CRISPR-based genome editing during the veryearliest developmental stages (1-4 cells). Depending on the efficiency of theCas9/gRNA, crispants can have stable mutations at the target locus in themajority of cells, but are genetically mosaic at this locus.Cryptococcus neoformans: an opportunistic fungal pathogen primarily ofimmunosuppressed individuals, especially HIV/AIDS patients. Infection inhealthy individuals is rare. It grows as a haploid yeast form, but can alsoundergo mating and meiosis to produce spores that germinate into yeast.CXCchemokine receptor-1/2 (Cxcr1/2): the primary chemokine receptorscontrolling neutrophil migration to sites of inflammation. In humans andzebrafish, the primary ligand for Cxcr1/2 is Chemokine (C-X-C motif ) ligand8 (also known as Interleukin-8). Mice lack Cxcl8, and Cxcl1/2 can bind tothis receptor instead and control neutrophil migration.Cytokines: small, usually secreted proteins that affect the activation andbehavior of cells, especially immune cells.Diphtheria toxin receptor (DTR): this receptor can be used to target andkill specific cell populations in a time-controlled manner. The receptor isexogenously expressed under a cell-type specific promoter, and, whendiphtheria toxin is administered, only DTR-expressing cells are affected andkilled.

Emergency granulopoiesis: a response to infection or inflammatorystimuli that results in increased production of neutrophils from thehematopoietic tissue.Gr-1: a marker/antigen originally thought to be specific for neutrophils butthat includes epitopes from both Ly6G and Ly6C, and is therefore found onboth neutrophils and monocytes.Interleukin 1 beta (Il1b): a major pro-inflammatory cytokine. Requiresprocessing by caspase enzymes to cleave off an inhibitory pro domainbefore it can be secreted and active. Binds to the Interleukin 1 receptor (Il1r).Lipopolysaccharide (LPS): molecules that are a fundamental componentof the outer membrane of the cell wall of Gram-negative bacteria.Recognized by Toll-like receptor 4 (TLR4) in humans and mice.LysozymeM (LysM): antimicrobial enzyme that can degrade peptidoglycan,a major component of Gram-positive bacterial cell walls. Can be expressedby multiple myeloid lineages, but is often used as a marker for neutrophils.Ly6C: cell surface protein, expressed on both neutrophils and monocyte/macrophage lineages. A previously used antibody clone used to depleteneutrophils in mice (RB6-8C5) was found to cross-react with this target,leading to unintended depletion of monocytes and macrophages.Ly6G: cell surface protein, expressed primarily on neutrophils. Targeted bythe antibody clone 1A8 to specifically deplete neutrophils.Mechanosensory hair cell: sensory receptor cells that contain membranechannels that open in response to mechanical stimulation. In humans andmice, these are found in the auditory system responding to sound vibration;in fish, they can be found in the lateral line to detect movement in thesurrounding water.Morpholino: antisense oligonucleotides made from synthetic, stabilizednucleic acids. Used to inhibit protein expression either by directly blockingtranslational initiation or by blocking mRNA splice sites, leading to mis-splicing, and inclusion of introns or exclusion of exons.Myeloid cells: cells that arise from a common myeloid progenitor in thehematopoietic tissue, including neutrophils, basophils, eosinophils, mastcells, monocytes, macrophages and some dendritic cells.Parabiosis: the joining of two separate individual animals such that theyshare a circulatory system and can exchange cells through the blood flow.Synteny: conservation of the physical architecture of the genome. Forexample, the existence of similar blocks of genes in similar positions inmultiple organisms.Tumor necrosis factor alpha (Tnfa): a major pro-inflammatory cytokine.Signals through the TNF receptors Tnfr1 and Tnfr2, leading to activation ofthe NF-κB transcription factor and MAP kinase pathways.GAL4-UAS system:GAL4 is a yeast-derived protein that binds to upstreamactivation sequence (UAS) enhancers and activates transcription of genesdownstream. This allows for construction of genetic lines with promoter-specific expression of gene targets where these two pieces (promoter:GAL4; UAS: gene target) are separable and interchangeable.Vascular endothelial growth factor A (Vegfa): a growth factor that targetsendothelial cells, promoting vascular permeability, angiogenesis and cellmigration.

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Immune cell depletion in larval zebrafish: how they work,pros, cons and caveatsOverall, only a few systems for specific and reproducible depletionof innate immune cell types are available in mice, and experimentsare complicated by the difficulty of monitoring cell depletionglobally in the animal. As immune cells can be easily monitored inthe entire living organism through microscopy, the larval zebrafishemerged as a popular model for the study of innate immune cellsin vivo (Levraud et al., 2008) (Box 2).Zebrafish have both innate and adaptive immune systems that are

similar to those of mammals, including the key cell types (e.g. T andB cells, macrophages, neutrophils, eosinophils and natural killercells), cytokines (Box 3; e.g. TNF, IFNs, IL-10, IL-12 and TGFβ),receptors (e.g. TLRs) and soluble factors [e.g. complement systemand antibodies (van der Sar et al., 2004; Renshaw and Trede, 2012)].The genome of zebrafish is also relatively conserved with humans:70% of human genes have an ortholog in zebrafish (Howe et al.,2013), and this conservation is often also accompanied bysignificant synteny (Box 3) (Barbazuk et al., 2000). Whilegenome duplication in teleost fishes contributed to the existenceof multiple copies of some human gene orthologs (Glasauer andNeuhauss, 2014), zebrafish carry orthologs for 84% of humandisease-associated genes (Howe et al., 2013).The function of innate immune cells and pathways can be studied

in isolation in larval-stage zebrafish. Adaptive immunity does notfunctionally mature until 4-6 weeks postfertilization (Lam et al.,2004), while myeloid cell (Box 3) precursors develop by 12 hpostfertilization (Lieschke et al., 2002). Functional macrophagesand neutrophils are present by 30 h postfertilization (Herbomelet al., 1999; Le Guyader et al., 2008). Their small size, opticaltransparency and the existence of reliable markers for bothneutrophils and macrophages (Box 1) also allow for monitoringdepletion of both the cell type of interest and the possible off-targetcells in the entire larvae (Ellett et al., 2011; Mathias et al., 2006;Renshaw et al., 2006; Walton et al., 2015). The simplicity of geneticmanipulation in zebrafish has promoted the rapid increase inavailable models in which these cell types are depleted, and findingscan now be validated with multiple depletion strategies.The rest of this Review will focus on innate immune cell

depletion techniques in the larval zebrafish model system (Table 1,Fig. 1) and highlight some of the most recent advances from thissystem in the understanding of cell type-specific contributions toinnate immunity in the context of infection, cancer and tissue repair(Fig. 2).

MacrophagesMacrophages are the first immune cell to develop in zebrafishlarvae, with primitive macrophages developing as early as 22 hpostfertilization (Masud et al., 2017; Herbomel et al., 1999, 2001).Methods that target macrophage developmental pathways preventthe generation of these cells. Moderate knockdown of pu.1 with alow-dose morpholino (Box 3) (Tenor et al., 2015; Rhodes et al.,2005), and morpholinos against or mutations in irf8 (Li et al.,2011; Shiau et al., 2015), inhibit macrophage development.However, with irf8 knockdown, the cells that would have becomemacrophages are diverted towards the neutrophil lineage, effectivelychanging the ratio of neutrophils to macrophages instead of simplydepleting macrophages (Li et al., 2011; Shiau et al., 2015). Althoughthis can complicate the analysis of the macrophages’ contribution to aphenotype, in theory, it can demonstrate that even increasedneutrophil numbers cannot compensate for lack of a macrophage-mediated function.

While research shows that morpholino-induced phenotypes can bethe result of off-target effects (Robu et al., 2007; Gentsch et al., 2018;Kok et al., 2015), fully established and validated morpholinos aregenerally accepted (Stainier et al., 2017). They offer the ability toknock down gene expression in any zebrafish line without themaintenance of a transgenic or mutant background, but they do notoffer much temporal control or long-term knockdown. To temporallymodulate the number of macrophages in zebrafish larvae, twodifferent methods are used: clodronate liposome injection or thenitroreductase (NTR) system. As the NTR system can be applied to avariety of cell types, it is discussed separately below.

Systemic intravenous or local clodronate liposome injection wasfirst established as a method for macrophage depletion in mice,discussed above (Van Rooijen and Sanders, 1994), but is alsoapplied to larval zebrafish (Bernut et al., 2014). An intravenousinjection depletes macrophages throughout the body, includingmicroglia and peripheral macrophages. Depletion of macrophages isobserved as early as 6 h postinjection, although most studiesperform this injection ∼24 h prior to further experimentation.Macrophage depletion can last for at least 72 h, with no effect onneutrophil numbers (Bojarczuk et al., 2016). A control injection ofphosphate-buffered saline-containing liposomes accounts for theeffect of the injection, which itself is an injury. A major advantageof this method is that depletion can be timed during specificperiods of interest. However, one concern is that this depletionstrategy is based on killing already existing cells, leaving behinddead cells that may activate immune signaling pathways (Zitvogelet al., 2010).

MicrogliaMicroglia are specialized macrophages that reside in braintissue, where they play a major role in maintaining brainhomeostasis and are arguably more sensitive to changes in theirenvironment than other tissue-resident macrophages (Gehrmannet al., 1995). However, because of their similarities to macrophages,macrophage depletion strategies also deplete microglia. Larvalmicroglia originate not from the caudal hematopoietic tissue (CHT)and definitive hematopoeisis, but instead from early-arising cellsaround the yolk, in the rostral blood island (RBI) (Herbomel et al.,1999; Xu et al., 2015). One of the first innate immune-deficientzebrafish strains, panther, is a csf1ra (Box 3) mutant, and in theselarvae, primitive macrophages largely fail to migrate from the RBIto colonize tissues, including the brain, resulting in larvae that aremicroglia deficient (Herbomel et al., 2001). These early cells in theRBI express Csf1ra and their colonization is directed by a source ofthe Csf1ra ligand Il34 in the brain (Wu et al., 2018; Kuil et al.,2019). il34 mutants therefore also have fewer microglia (Wu et al.,2018; Kuil et al., 2019). Some tissue-resident macrophages alsocome from these early-arising cells, and csf1ra−/− or il34 crispant(Box 3) larvae have fewer peripheral macrophages with fewerprotrusions that respond poorly to tail fin injury (Kuil et al., 2019;Wu et al., 2018; Pagán et al., 2015; Morales and Allende, 2019).

NeutrophilsAs with macrophages, targeting the development pathways ofneutrophils can be an effective depletion strategy. Morpholinosagainst csf3r, which is the central controller of neutrophildevelopment, inhibit the generation of neutrophils, but macrophagepopulations can also be affected (Liongue et al., 2009). There is alsointerest in the role of Csf3r in emergency granulopoiesis (Box 3), andwhile this receptor can be depleted to study emergency granulopoiesis(Willis et al., 2018; Hall et al., 2012), this pathway has roles in both

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basal and induced modes of neutrophil production (Liongue et al.,2009).Other effective models to study neutrophil-dependent functions

do not necessarily decrease neutrophil numbers, but instead preventneutrophil migration from hematopoietic tissues to sites of damageand inflammation, and therefore can still be used as models ofneutrophil deficiency in response to inflammatory stimuli. In somecases, blocking the main signals that govern neutrophil recruitment,Cxcl8 and Cxcr1/2 (Box 3), either through genetic manipulation ordrug inhibition, is sufficient to prevent neutrophil recruitment (deOliveira et al., 2013; Powell et al., 2018). Two transgenic lines thatexemplify the strategy ofmodulating neutrophil motility are a zebrafish

model of the rare congenital warts, hypogammaglobulinemia,immunodeficiency and myelokathexis (WHIM) syndrome [Tg(mpx:cxcr4bWHIM)] (Walters et al., 2010) and a zebrafish strain expressing adominant-negative mutant of Rac2 [Tg(mpx:rac2D57N)] (Deng et al.,2011). In normal neutrophil development, these cells are held in theCHT by Cxcl12 (also known as SDF-1 or Cxcl12a), which signalsthrough Cxcr4. As neutrophils mature, Cxcr4 is internalized, allowingtheir release from the hematopoietic tissue. Cxcr4WHIM is atruncated mutant form that cannot be internalized, resulting in apersistent retention signal. The Rac2D57N dominant-negativemutation directly targets neutrophil motility. Rac2 is a smallGTPase that coordinates many cellular functions, including

Myeloid cellpu.1+

pu.1irf8

csf3r

Cell death

B Clodronate liposome injection

Tg(mpx:rac2D57N)

No directional migration

Tg(mpx:cxcr4bWHIM)

Retention inhematopoietic tissue

D57N

Rac

Cxcl12

Cxcr4bWHIM

Retention

C Cell-specific NTR expression and MTZ treatment

MTZ

DNA damage,cell death

Tg(mpx:NTR)Tg(mpeg1:NTR)

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Il34

RBI: primitive hematopoiesis

CHT: definitive hematopoiesis

Key

Neutrophil

E Neutrophil-specific dominant-negative transgenes keep cells in the CHT

D Targeting developmental pathways

Macrophage

A Disrupting microglial and tissue-resident macrophage colonization signaling

NTR Toxic DNA cross-linking agent

Fig. 1. Immune cell depletion methods in larval zebrafish. Multiple techniques can be used in larval zebrafish to modulate numbers of macrophages andneutrophils. (A) Primitivemacrophages that develop in theRBI seed peripheral tissues, especially the brain to formmicroglia, via expression of the Csf1ra receptorand recognition of Il34 produced in tissues. Genetically targeting either of the genes encoding these proteins can abolish this seeding and prevent thedevelopment of microglia. (B) Injected clodronate liposomes are taken up specifically by macrophages, leading to macrophage cell death and ablation.(C) NTR can be transgenically expressed in a specific cell type and, upon treatment with MTZ, the drug is converted to a toxic compound, leading to cell death,specifically in NTR-expressing cells. (D) Genes that are required for differentiation from progenitor cells (pu.1 or irf8 in macrophages, csf3r in neutrophils)can be targeted genetically to prevent the development of these cells. (E) Dominant-negative forms of proteins with roles in neutrophil motility and release fromCHT are expressed under a neutrophil-specific promoter, preventing these cells from migrating to sites of inflammation. CHT, caudal hematopoietic tissue;Csf1ra, colony-stimulating factor 1 receptor a; csf3r, colony-stimulating factor 3 receptor; Cxcl12, CXC motif chemokine ligand 12; Cxcr4b, CXC chemokinereceptor 4b; Il34, interleukin 34; irf8, interferon regulatory factor 8; mpx, myeloperoxidase; MTZ, metronidazole; NTR, nitroreductase; RBI, rostral bloodisland; WHIM, warts, hypogammaglobulinemia, immunodeficiency and myelokathexis.

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actin polymerization required for directed cell migration.Rac2D57N cannot bind guanosine triphosphate (GTP) andmonopolizes guanine nucleotide exchange factors required forthe Rac GTPase cycle (Williams et al., 2000). Experiments with

both of these lines have demonstrated that they are deficient inneutrophil activity, validating their use as models tointerrogate neutrophil function (Yang et al., 2012; Gratacapet al., 2017).

Vegfa

Earlyresponse

Lateresponse

Il1bTnfa

Il1bTnfa

Kras+

Repair of nerve tissue

Transformed cellproliferation

Control of invasive infection

Pathogen survivaland growth

Mechanical forces

Angiogenesisand vascular repair

Resistance to pathogens

Woundinflammatoryenvironment

Epithelial breach

Key

Neutrophil

Macrophage

Nervous tissue

Transformed cell

LC3

Fig. 2. Recent key advances on cell-specific innate immune functions in larval zebrafish. Recent studies have highlighted many different roles formacrophages and neutrophils in a range of immune contexts. In response to pathogens, macrophages can target and kill these microbes; for example, inresponse to the bacterial pathogen Salmonella, macrophages use LC3-associated phagocytosis to control this pathogen (Masud et al., 2019). But, in otherinfections, such as the bacterial pathogen B. cenocepacia (Mesureur et al., 2017) or the fungal pathogens A. fumigatus (Rosowski et al., 2018a), T. marneffei(Ellett et al., 2018) or the C. neoformans spore form (Davis et al., 2016), macrophages actually provide a protective niche for pathogen survival and growth. Insterile wounding conditions, macrophages can modulate the inflammatory microenvironment (Tsarouchas et al., 2018; Hasegawa et al., 2017; Nguyen-Chi et al.,2017), use Vegfa activation and mechanical forces to promote angiogenesis and vascular repair (Liu et al., 2016; Gurevich et al., 2018), and promote the repairand regrowth of damaged nerve tissue (Carrillo et al., 2016; Tsarouchas et al., 2018). In response to infection, neutrophils often have roles in controlling pathogeninvasive growth at later stages of infection (Gratacap et al., 2017). Neutrophils also have a role in the microenvironment of transformed cells, includingglioblastoma cells, promoting their proliferation (Powell et al., 2018). Il1b, interleukin 1 beta; LC3, microtubule-associated protein light chain 3; Tnfa, tumornecrosis factor alpha; Vegfa: vascular endothelial growth factor A.

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NTR systemPerhaps the most popular technique to deplete specific cell typesin zebrafish is the NTR system (Curado et al., 2008). First, abacterial NTR transgene is expressed in a cell population ofchoice using a cell type-specific promoter. The non-toxicpro-drug metronidazole (MTZ) is then given to and taken up bylarvae through bath immersion. NTR reduces MTZ into a DNAcross-linking agent, leading to DNA damage and cell deathspecifically in the cells in which NTR is expressed. In zebrafishlarvae, this system was first applied to heart, pancreas andliver cells (Curado et al., 2007; Pisharath et al., 2007), but hassince been expanded to other cell types, including macrophages(Gray et al., 2011; Petrie et al., 2014) and neutrophils (Prajsnaret al., 2012). It was also combined with the GAL4-UAS system(Box 3) for easy interchange of other cell-specific promoters, suchas mpeg1.1 and mpx for macrophages and neutrophils,respectively (Box 1) (Davison et al., 2007; Ellett et al., 2011;Mathias et al., 2006).This system has similar issues and advantages as clodronate

liposomes, discussed above. MTZ treatment of these cells alsocauses cell death, possibly leading to immune activation. Temporalcontrol of depletion is one of the main advantages of the NTRsystem. MTZ can be added at any time during experimentation totest the effect of depletion at different stages of the immuneresponse. The duration of treatment required for full target cellpopulation ablation varies depending on the experimental system,with most studies initiating treatment at least a day before analysis. Itshould also be noted that MTZ may have NTR-independent effectson some phenotypes of interest (Oehlers et al., 2015) and MTZtreatment of non-NTR larvae should always be included as a controlcondition.

Recent findings made possible by these modelsThe innate immune depletion models in larval zebrafish discussedabove can be used to investigate the role of macrophages andneutrophils in a variety of immune contexts including infection,wounding and cancer. Here, we highlight a few recent studies (Fig. 2).

Macrophages as controllers of infectionThe role of macrophages in the innate immune system’s response toinfection is different for each infection context. Macrophages arerequired for full control of the bacterial pathogen Salmonella(Masud et al., 2019). Using the live-imaging capabilities of larvalzebrafish, Masud et al. (2019) found that LC3-associatedphagocytosis by macrophages promotes control of this pathogen.NTR-mediated macrophage depletion resulted in 100% larval deathby 24 h postinfection and increased bacterial replication, results thatwere confirmed with an anti-irf8 morpholino. Neutrophil depletionwith the NTR system also somewhat increased bacterial burden, butit was clear that macrophages play a larger role in the early control ofthis infection.Two other recent papers also reported a role for macrophages in

controlling the growth of a pathogen, in this case the fungalpathogen Cryptococcus neoformans (Box 3) (Tenor et al., 2015;Bojarczuk et al., 2016). Both studies injected zebrafish larvae withthe yeast form of a highly virulent strain of C. neoformans, H99.Although the yeast could in some cases replicate inside ofmacrophages, removal of macrophages with either low-dose anti-pu.1 morpholino (Tenor et al., 2015) or clodronate liposomes(Bojarczuk et al., 2016) decreased the survival of larvae andincreased fungal growth. Bojarczuk et al. (2016) also tookadvantage of the temporal control offered by clodronate liposome

injection, demonstrating that removal of macrophages after aninfection is established still leads to an increased fungal burden.

Macrophages as a protective niche for pathogensDespite their importance in fighting infection, macrophages do notcontrol growth of all pathogens and can, for some infections, serveas a protective or proliferative niche. By residing in macrophages,pathogens are protected from recognition and destruction by bothsoluble factors such as the complement system and by other immunecells. Macrophages can be a protective niche for bacteria as well,and Mesureur et al. (2017) recently investigated this question in thecontext of Burkholderia cenocepacia infection. Using either low-dose anti-pu.1morpholino or the NTR system, this study found thatlarvae without macrophages had increased survival after infection.Although bacteria could replicate in the absence of macrophages,the presence of macrophages increased bacterial growth. Similarneutrophil depletion experiments with the NTR system or a csf3rmorpholino had no effect on larval survival, again demonstrating aspecific role for macrophages as a proliferative niche (Mesureuret al., 2017).

A different study of infection with the fungal pathogen C.neoformans found that when zebrafish larvae are infected with a lessvirulent strain and with spores instead of the yeast form of thepathogen, macrophages can play a pathogen-protective role (Daviset al., 2016). These fungal spores are phagocytosed by macrophagesbut later escape back into the vasculature. Removal of this earlyintracellular niche in an irf8 mutant actually resulted in a lowerfungal burden, although the increased neutrophil numbers in thisgenetic background may also contribute to fungal clearance (Daviset al., 2016).

Two other studies came to similar conclusions with fungalpathogens recently, using multiple models of macrophage depletion(Rosowski et al., 2018a; Ellett et al., 2018). Rosowski et al. (2018a)reported that macrophage deficiency, in either an irf8 mutant orupon clodronate liposome injection, led to increased clearance of afast-growing strain of the fungus A. fumigatus, but not of a slower-growing strain. The ability of macrophages to inhibit fungalgermination and growth actually inhibited neutrophil-mediatedkilling of the faster-growing strain. In a Taloromyces marneffeifungal infection, the infectious burden also decreased whenmacrophages were removed, either by anti-irf8 morpholino or bythe NTR system (Ellett et al., 2018). Conversely, neutrophildepletion, either through anti-csf3r morpholino or the NTRsystem, had little effect on fungal burden. In fact, depletion ofboth cell types with a combination of anti-pu.1 and anti-csf3rmorpholinos also decreased early fungal burden, underlining theimportance of the macrophage intracellular niche for fungal growth(Ellett et al., 2018).

Macrophages promote vascular repairThe role of macrophages in the repair of blood vessels has becomean area of active research in larval zebrafish, finding thatmacrophages can mediate vascular repair (Liu et al., 2016;Gurevich et al., 2018; Gerri et al., 2017). Mechanical or laser-mediated damage to blood vessels in either the brain or the tailrecruits macrophages to the injury site, where they wrap around oradhere to the wounded vessels (Liu et al., 2016; Gurevich et al.,2018). Knockdown of macrophages with anti-irf8morpholino resulted in deficient blood vessel repair in the brain(Liu et al., 2016), while depletion with either the NTR system orclodronate liposomes resulted in a failure of tail fin vessel repair(Gurevich et al., 2018). Csrf1ra−/− larvae also have

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impaired healing of the tail vasculature, suggesting that peripheralmacrophages in particular promote this repair (Gurevich et al.,2018). By temporally controlling macrophage depletion withadministration of MTZ or clodronate liposomes after initial vesselrepair already occurred, Gurevich et al. (2018) also implicatedmacrophages in vessel pruning at later stages of regeneration.How do macrophages mediate this repair and regrowth? In the

case of targeted brain blood vessel damage with a multi-photonlaser, a single macrophage is often recruited to the injury site andmediates repair through mechanical forces, adhering to theruptured vessel ends and pulling them together (Liu et al.,2016). Interestingly, if the first macrophage to arrive to the injurysite is also laser-ablated, this loss impairs repair; althoughanother macrophage is recruited to phagocytose the dead cell,this second macrophage does not engage with the injured vessel(Liu et al., 2016). Several studies also implicate Vegfa (alsoknown as Vegfaa; Box 3) in macrophage-mediated angiogenesisand repair (Gurevich et al., 2018; Oehlers et al., 2015; Britto et al.,2018).

Macrophages increase repair of damaged nerve tissueLarval zebrafish can effectively repair and regenerate nerve cells,and macrophages have been implicated in this process. Two recentstudies demonstrated that, after nerve damage, both neutrophils andmacrophages are recruited to the injury site, with neutrophilnumbers peaking early and then resolving away, whereasmacrophages are more persistent at the wound (Carrillo et al.,2016; Tsarouchas et al., 2018). In a model of mechanosensory haircell (Box 3) damage caused by exposure of lateral line hair cells tocopper, macrophage depletion by either low-dose anti-pu.1morpholino or local clodronate liposome injection delaysregeneration of these hair cells (Carrillo et al., 2016). Larvalzebrafish can repair spinal cord injuries, including complete spinalcord transection. In a transection model, Tsarouchas et al. (2018)found that altering the level of overall inflammation by treatmentwith a glucocorticoid drug or lipopolysaccharide (LPS; Box 3)modulates regeneration, with increased immune cell recruitmentassociated with better outcome. Using an irf8 mutant, they foundthat macrophages are not required for the initial repair steps but arerequired for complete recovery of spinal cord function, as measuredby larval swimming. Csf1ra−/− larvae do not have defects in thisrepair, suggesting that this regeneration function of macrophages isdue to recruited macrophages, not tissue-resident cells or microglia(Tsarouchas et al., 2018).

Macrophages regulate the inflammatory environment at sterilewoundsMultiple recent papers have focused on the role of macrophages inmodulating the immune environment at an injury site, especiallythrough regulation of the pro-inflammatory cytokines il1b and tnfa(Box 3) (Tsarouchas et al., 2018; Morales and Allende, 2019;Nguyen-Chi et al., 2017; Hasegawa et al., 2017). These pro-inflammatory mediators are turned on early in the wound responseand downregulated in later stages of repair (Tsarouchas et al., 2018;Hasegawa et al., 2017). In contrast, in macrophage-deficientzebrafish, this early expression is impaired but increases later.Morpholino-mediated knockdown or drug inhibition of pro-inflammatory cytokine expression impairs regeneration in wild-type larvae, but improves repair in macrophage-deficient ones(Hasegawa et al., 2017; Nguyen-Chi et al., 2017). Overall, thesestudies suggest that although early expression of these pro-inflammatory genes is required for efficient repair, their

expression must be downregulated to promote late healing andimplicate macrophages in both early and late healing phases(Tsarouchas et al., 2018; Hasegawa et al., 2017; Nguyen-Chi et al.,2017). Work in a csf1ra mutant identified peripheral macrophagesas the cells responsible for downregulating il1b expression (Moralesand Allende, 2019). One function of tnfa signaling may be tosupport the proliferation of cells in the blastema (Box 3) (Nguyen-Chi et al., 2017). Using a tnfa expression reporter, Nguyen-Chi et al.(2015) identified macrophages as sources of tnfa at the wound site,with early macrophages expressing tnfawhen first responding to theinjury and then converting to a tnfa-negative phenotype later inrepair. This group then used parabiosis (Box 3) experiments toconfirm that macrophage-produced Tnfa can signal to stromal cellsto support the proliferation of blastemal cells (Nguyen-Chi et al.,2017).

However, there are twomajor confounding factors that could alterthe interpretation of these macrophage-depletion experiments andthe assignment of specific roles to macrophages in wound repair.First, the presence of neutrophils at a wound may be increased inmacrophage-deficient larvae, and, as neutrophils can cause tissuedamage, an increased presence of neutrophils may be the factor thatdelays repair and regeneration. This is especially true forexperiments with irf8 mutants or morphants that have increasedtotal numbers of neutrophils, but may also occur with othermacrophage depletion methods, as macrophages can promoteneutrophil resolution from wounds (Tauzin et al., 2014; Loyneset al., 2018). In a blood vessel repair model, more neutrophils wereobserved at the injury site after macrophage depletion (Gurevichet al., 2018). Additionally, in a spinal cord injury model, neutrophilswere identified as a major producer of il1b, and depletion ofneutrophils with anti-pu.1 and anti-csf3rmorpholinos in irf8mutantzebrafish larvae improved repair when compared to irf8 mutationalone (Tsarouchas et al., 2018).

A second issue is that wounding leads to a significant level ofcell death, and a major role of macrophages is to phagocytose deadcells and debris (Tabas, 2010). Several studies documented anincrease in the number of dead cells at the wound and in theblastema after tail fin amputation in macrophage-deficient larvae(Hasegawa et al., 2015, 2017; Loynes et al., 2018; Morales andAllende, 2019). However, the interpretation of this observation hasdiffered, with studies concluding either that macrophages producea signal to promote the survival of these cells (Hasegawa et al.,2015, 2017) or that these are cells that would normally be clearedby phagocytic macrophages (Loynes et al., 2018). It is also unclearwhat the effect is of these dead cells on tnfa, il1b and other pro-inflammatory cytokine levels, as it is possible that their veryappearance or failure to be removed activates inflammatorypathways (Zitvogel et al., 2010). The role of phagocytosis inthese phenotypes was addressed in the context of spinal cordinjury, where chemical inhibition of phagocytosis alone did notimpair regeneration (Tsarouchas et al., 2018), but remains unclearin other models.

Neutrophils control invasive infectionNeutrophils are often the first responders to infection or tissue damage,and some infections are characterized by early neutrophil recruitmentand neutrophil-mediated immunity (Rosowski et al., 2016; Williset al., 2018). However, in other cases, such as mycobacterialinfection (Yang et al., 2012) or infections with the fungal pathogensA. fumigatus (Rosowski et al., 2018a) or C. neoformans (Davis et al.,2016), neutrophils arrive later and control late-stage infection,combating invasive growth of the pathogen.

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A recent study by Gratacap et al. (2017) illustrates this late role ofneutrophils in controlling invasive growth in a model of mucosalcandidiasis in zebrafish. Modulating neutrophil activity at theinfection site with two different methods, Tg(mpx:rac2D57N) andchemical inhibition of Cxcr2, resulted in increased larval host death.Rac2D57N neutrophil-defective larvae have increased fungalfilamentation, leading to increased damage to the epithelial barriersurrounding the infection site, suggesting that neutrophils controlthis later, invasive growth stage of infection (Gratacap et al., 2017).

Neutrophils promote transformed cell proliferationThe innate immune system also plays a role in the response totransformed cells and cancer, with neutrophils having both pro- andanti-tumor functions (Giese et al., 2019). Neutrophils promote earlystages of cancer progression in multiple transformed cell models inlarval zebrafish (Freisinger and Huttenlocher, 2014; Feng et al.,2012). In fact, increasing neutrophil recruitment to a developingclone of transformed cells by creating a nearby tissue woundincreases the proliferation of that clone (Antonio et al., 2015).Powell et al. (2018) recently expanded these studies to a model ofglioblastoma with Kras-transformed astrocytes. In Rac2D57N

neutrophil-defective larvae, Kras+ cells are less proliferative.Additionally, cxcr1−/− larvae or larvae treated with a Cxcr1/2inhibitor had decreased neutrophil recruitment to and proliferationof Kras+ cells, identifying a major signaling axis for neutrophilstimulation of transformed cell proliferation (Powell et al., 2018).

Future directionsInnate immune cell subsetsThe models discussed above were developed to deplete entire cellpopulations, either neutrophils, macrophages or tissue-residentmacrophages such as microglia, but these cell types exist in a varietyof activation states (Murray et al., 2014; Silvestre-Roig et al., 2019),and one future direction will be to determine the roles of theindividual cell subsets through subset-specific depletion strategies.One categorization of macrophage subtypes relies on anatomicalsource – tissue-resident versus recruited (Box 1). Mutation of csf1rais one method to modulate peripheral macrophages specifically,including microglia as discussed above, but it is still unclear whatpercentage of non-microglial peripheral macrophages depend on theCsf1ra pathway for their localization (Xu et al., 2015; Herbomelet al., 2001). The Ramakrishnan laboratory has also delineatedseparate functions of tissue-resident and infiltrating macrophages inthe response to mycobacterial infection (Cambier et al., 2014,2017). They find that Mycobacterium marinum escape killing bytissue-resident macrophages, and instead recruit fewer microbicidalCcr2+ monocytes. The recruitment of these permissive monocytesand their function at the infection site can be inhibited bymorpholinos against either ccr2 or its ligand, ccl2 (Cambier et al.,2014), but the role of the Ccl2–Ccr2 signaling axis in otherinflammatory responses is unknown. The main marker ofmacrophage transcriptional polarization used in zebrafish hasbeen tnfa expression (Nguyen-Chi et al., 2015). Either tnfa ortnfr1 morpholinos can block this signaling, but it remains unclearhow this knockdown affects overall macrophage polarization or thebehavior of other immune cells (Nguyen-Chi et al., 2017).

Disease-specific deficiency modelsLarval zebrafish have emerged as models of a variety of humangenetic diseases. In fact, two of the neutrophil-defective modelsdiscussed here, the WHIM and Rac2D57N models, were developedas models for human disease mutations (Walters et al., 2010; Deng

et al., 2011). Although most work on innate immune cell function inzebrafish has focused on the role of macrophages and neutrophils inwild-type larvae, the zebrafish presents a highly useful modelsystem in which to understand the requirement for (and defectivephenotypes of) these cells in the context of other immunedeficiencies. Two such genetic diseases that already havezebrafish models are cystic fibrosis (Bernut et al., 2019) andphagocyte oxidase deficiency (Tauzin et al., 2014), the cause ofchronic granulomatous disease. Another disease factor in humans,high-fat diet, was recently modeled in larval zebrafish in the contextof liver cancer (de Oliveira et al., 2019). Here, macrophagespromoted hepatocellular carcinoma progression specifically inanimals that were fed a high-fat diet, highlighting the importanceof combining these immune deficiency models with other diseasefactors to fully understand disease mechanisms and identifytherapeutic opportunities (de Oliveira et al., 2019).

Innate immune cell reconstitution with mammalian cellsImmune depletion models in fish will also allow researchers toreconstitute the immune system with cells derived from humans ormammalian models, in order to directly visualize the behavior ofthese cells in complex tissues, similar to ‘humanized’mouse models(Ito et al., 2018). Mouse neutrophils co-injected with C. albicanswere at least partially functional as they could somewhat decreasefungal burden in Rac2D57N neutrophil-defective larvae (Gratacapet al., 2017). Transplantation of murine bone marrow cells(Parada-Kusz et al., 2018), human hematopoietic stem cells(Hamilton et al., 2018) and human macrophages (Paul et al.,2019) into zebrafish is possible but has not yet been applied tostudies of the behavior of these cells in response to infection orinjury.

ConclusionsDetermining the specific requirements and functions of differentinnate immune cells in response to insults such as infection,wounding and cancer is key for future development andimplementation of patient treatments. Knowing how different celltypes’ activities improve or worsen disease progression isparamount for deciding whether to use treatments that seek tomodulate the numbers of a given immune cell type, such as Csf3administration to increase neutrophil production, and for identifyingnew molecular targets that modulate innate immune cell activity. Asdiscussed in this Review, the function of each of these cells can varygreatly in each disease context. While much continues to be learnedfrom the mouse model, the expansion of cell depletion models andlive imaging methods in larval zebrafish make this animal model afruitful system for research on innate immune function.

Competing interestsThe author declares no competing or financial interests.

FundingE.E.R. was supported by the National Institute of Allergy and Infectious Diseases ofthe National Institutes of Health under award number K22AI134677. The content issolely the responsibility of the author and does not necessarily represent the officialviews of the National Institutes of Health.

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