Immune surveillance and host defense against invading pathogens are mediated by neutrophils patrolling within the mammalian circulation and the tissue (1, 2). Beyond their immediate immune function, neutrophils are involved in wound healing, metastasis, autoimmunity, and chronic inflammatory disease states (3). Neutrophil recruitment to sites of inflammation crucially depends on adhesion molecules of the β₂ integrin family (CD11/CD18) that mediate adhesion, migration and extravasation as well as several host defense functions of these cells. β₂ integrins are composed of the common β subunit CD18 and different α subunits namely CD11a, CD11b, CD11c, or CD11d (4). In humans, mutations in ITGB2, the gene for CD18, result in leukocyte adhesion deficiency (LAD) type I which is characterized by neutrophilia, impaired wound healing, and recurrent infections caused by compromised neutrophil recruitment to sites of infection (5). Mice lacking CD18 show similar defects (6–8).

In humans and mice, hematopoietic stem cells in the bone marrow give rise to granulocytic progenitor cells that further differentiate into neutrophils (9). Mature neutrophils are released into the circulation upon demand, with increasing amounts during inflammatory conditions (10–12). The lifespan of circulating neutrophils which is usually limited to hours or 1-2 days is tightly regulated by controlling neutrophil apoptosis and can be prolonged by e.g. cytokines and bacterial compounds (13, 14). Neutrophil trafficking to sites of inflammation depends on a concerted cascade of strictly regulated steps to recruit free-floating neutrophils from the blood stream into the inflamed tissue. The individual steps of neutrophil recruitment have been well-described in human and murine neutrophils and include neutrophil capture, rolling, slow rolling, arrest, adhesion strengthening, intraluminal crawling, transmigration as well abluminal crawling and interstitial migration (1). Neutrophil recruitment is initiated by capture and followed by subsequent rolling of neutrophils on the inflamed endothelial cells, a step mediated by selectins expressed on the endothelial cells and the P−selectin glycoprotein ligand-1 (PSGL-1) on neutrophils (1). Slow rolling depends on selectins as well as on lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18). Subsequent neutrophil arrest is induced by different chemokines including CXC motif chemokine ligand 8 (CXCL8) in humans and CXCL1 in mice, respectively, and mediated by binding of the β₂ integrin LFA-1 to its ligands such as intercellular adhesion molecule 1 (ICAM-1) (1, 15). Outside-in signaling of β₂ integrins enables the following steps of adhesion strengthening and cell spreading (1). Intravascular crawling to extravasation sites is mediated by macrophage-1 antigen (Mac-1, CD11b/CD18), followed by extravasation into the abluminal space where neutrophils display β₂ integrin-dependent migration on activated pericytes until they eventually migrate interstitially to the site of inflammation (16, 17). In contrast to intraluminal and abluminal crawling and extravasation, subsequent interstitial migration within the tissue can occur independently of β₂ integrins as shown in a mouse model depleted of integrins (18).

The zebrafish has emerged as a novel model to study neutrophil trafficking and function (19, 20). Zebrafish neutrophils execute the same host-defense functions such as phagocytosis, degranulation, or formation of neutrophil extracellular traps (NETs) as mammalian neutrophils (21–23). Moreover, zebrafish exhibit a range of advantages that make them an attractive model to study leukocyte trafficking in vivo (23). They are easy and relatively inexpensive to keep and give rise to a large number of offspring. Many genetic tools have successfully been established to generate mutant strains including gene editing using CRISPR/Cas9 (24, 25). Zebrafish larvae are transparent, allowing neutrophil visualization in vivo in intact organisms. Most importantly, the immune system of zebrafish and mammals is highly conserved (26, 27). While the innate immune system develops within the first 2 days post fertilization (dpf), the adaptive immune system only appears after 2 weeks, enabling the exclusive analysis of innate immune cells during this period in larval zebrafish (28). Disadvantages of this model include the partial lack of tools including antibodies which are still commercially unavailable for many proteins of interest. In addition, many mammalian genes have multiple orthologous genes in the zebrafish which may complicate genetic studies accordingly.

In zebrafish, first immune cell precursors are detected approximately 12 h post fertilization (hpf) during the primitive wave of hematopoiesis (23, 29). In the initial phase, the precursor cells give rise to macrophages and a further subset differentiates into neutrophils at approximately 33 hpf (30, 31). The definitive hematopoiesis starts at approximately 24 hpf when pluripotent precursors differentiate within the posterior blood island which expands to become the caudal hematopoietic tissue (CHT), the site functionally reminiscent of the fetal liver in mammals (30, 32, 33). The CHT gives rise to macrophages and neutrophils from 2 dpf onwards. From 4 dpf onwards, the kidney marrow begins to mature and becomes the site of definitive hematopoiesis in the adult zebrafish (34).

When neutrophils of zebrafish larvae leave the CHT, they predominantly migrate throughout the larvae in the interstitial space (33). In comparison to mammals, relatively few neutrophils are found in the circulation of zebrafish larvae whereas the majority of neutrophils is located within the tissue (35). The lifespan of neutrophils in zebrafish larvae was shown to be up to 5 days (36). In the first phase upon induction of inflammation by cutting or burning the tail fin, neutrophils mainly migrate within the tissue towards sterile injuries (37–39). Within 3-4 h post wounding, additional neutrophils are recruited from the circulation (40). The process of reverse migration of neutrophils from the inflamed tissue back into the vasculature has been discovered in zebrafish larvae and was subsequently reported in mice (41–43). Detailed information about the molecular players orchestrating neutrophil recruitment is still missing in zebrafish. P- and E-selectin expression have been described in zebrafish and it has been proposed that they have similar functions as their mammalian counterparts (44, 45). Furthermore, PSGL-1 is expressed in zebrafish and was shown to interact with human selectins (46). Similar to humans, CXCL8 binds to the CXC receptor 2 (CXCR2) in zebrafish directing neutrophils to sites of infection and mediating systemic recruitment of neutrophils from the CHT (47, 48). Previously, a zebrafish model of LAD type IV was generated by inducing a Rac2 mutation in zebrafish larvae (35). These Rac2 mutants displayed similar defects as found in human patients with the Rac2 mutation, including neutrophilia due to reduced neutrophil egress from the blood and reduced recruitment of neutrophils to sterile injury or bacterial infection. However, not all symptoms observed in human patients could be recapitulated in the zebrafish model (49).

To date, the putative role of β₂ integrins for neutrophil trafficking in zebrafish is rather elusive. In this study, we generated a model for LAD type I in zebrafish by genome editing using CRISPR/Cas9 technology to shed light on the role of CD18 for neutrophil trafficking in this model. We used sequence alignments to demonstrate that the amino acid sequences of zebrafish and human CD18 share a high similarity, supporting the putative significance of the zebrafish as a model to study integrin biology. Accordingly, CD18 knock-out (KO) zebrafish larvae recapitulated the main symptoms observed in LAD type I patients including reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation using spinning disk confocal microscopy. Thus, CD18 KO zebrafish larvae may represent an interesting model to study LAD type I in vertebrates.

Taken together, we generated CD18 KO zebrafish lines to study the role of β₂ integrins for neutrophil trafficking in the zebrafish model. CD18 deficiency in humans leads to LAD type I which is characterized by neutrophilia and recurrent infections due to a recruitment defect of neutrophils to sites of inflammation (5). To prove our hypothesis that zebrafish and human CD18 may have similar functions, we performed sequence alignments between human, murine, and zebrafish CD18 and CD11b. These alignments indicated that CD18 was highly conserved between species. In line with these findings, CD18 of human, murine and origin zebrafish share a common ancestor and is found within the same cluster of the phylogenetic tree (65). However, CD11b seems to be less conserved between zebrafish and mammals. In our alignments, both human CD11a and CD11b showed similar identities with zebrafish CD11b, suggesting that zebrafish CD11b may compensate for the absence of CD11a and thereby it may at least partially fulfill the role of CD11a in mammals. In mice, LFA-1 and Mac-1 play distinct roles for neutrophil adhesion and migration. Both LFA-1 and Mac-1 contribute to slow rolling of neutrophils on the endothelium but LFA-1 is predominantly important for neutrophil arrest (66–68). Studies with KO mice further revealed that LFA-1 deficient mice displayed leukocytosis and impaired neutrophil adhesion and extravasation, similar to CD18 deficient animals (69). However, Mac-1 also contributes to neutrophil adhesion and migration but it cannot fully compensate for the loss of LFA-1. Mammalian Mac-1 has important roles in mediating neutrophil defense functions such as phagocytosis and degranulation and promotes neutrophil apoptosis and thereby plays an important role in different inflammatory diseases such as glomerulonephritis, or thrombohemorrhagic vasculopathy (70–72). In addition, Mac-1 attenuates FcγRIIA-dependent neutrophil recruitment in response to deposited immune complexes which is key to organ damage in a model of systemic lupus erythematosus and thus prevents excessive inflammation, demonstrating that Mac-1 has diverse and partially opposing roles in mammals (73, 74). Thus, we speculate that the important roles of LFA-1 and Mac-1 in mice may be accomplished at least to some extend by only Mac-1 in zebrafish.

Our zebrafish model presented with an increased number of neutrophils detectable in the circulation by spinning disk confocal microscopy which is in line with Rac2 deficient zebrafish larvae as a model for LAD type IV which also show increased neutrophil numbers in the circulation (35). CD18 deficient humans and mice display increased neutrophil numbers in the bone marrow and in the circulation (5, 8, 75). The neutrophilia in CD18 KO mice is not mainly caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. Here, the genetic absence of CD18 causes the disruption of a feedback loop involving G-CSF and IL-17 and drives granulopoiesis as neutrophils cannot migrate into peripheral tissues (76). In addition to an increased release of neutrophils from the bone marrow into the circulation, reduced extravasation of neutrophils at sites of inflammation and delayed apoptosis of neutrophils may contribute to neutrophilia upon CD18 deficiency in mammals to some extend (77). In zebrafish, evidence has been reported that a CD18 knock-down may induce the expansion of hematopoietic pluripotent stem cells in the CHT which opens the possibility that CD18 may influence granulopoiesis in this model as well (78).

Analysis of random interstitial migration of neutrophils in the head region demonstrated that CD18 was widely dispensable for migration in this model. This is in line with findings in mice where integrins are not required for interstitial leukocyte migration (18). In sharp contrast, we did observe a strong and significantly reduced neutrophil recruitment to sites of sterile injury at 6 h post wounding in the CD18 KO zebrafish larvae. As neutrophil migration was largely unaffected by CD18 deficiency, a cell-intrinsic impairment of the migratory capacity can be excluded as relevant mechanism for defective neutrophil recruitment to the site of the tail fin injury 6 h after the onset of inflammation. At this time point, neutrophils are mainly recruited from the circulation (40). Thus, the impaired neutrophil accumulation in the inflamed tissue despite elevated neutrophil numbers in the circulation suggests that neutrophils may not be able to egress from the blood stream in the genetic absence of CD18. However, the exact molecular mechanisms underlying this extravasation defect in zebrafish larvae requires further investigation. This is especially true for the endothelial counterreceptors of the β₂ integrins which are largely unknown in this model (79). In accordance with our findings, small molecules specifically interfering with Mac-1 have been reported to reduce the recruitment of neutrophils in wounded tail fins in zebrafish larvae within 4 h after the onset of the experiment further supporting of role for Mac-1 in neutrophil extravasation (80).

Our zebrafish CD18 KO model presents a global KO of the itgb2 gene similar to the patients’ situation and therefore effects of the deletion of CD18 in other tissue than neutrophils cannot be ruled out. However, itgb2 is expressed in zebrafish larvae only in leukocytes as previously published by Xue et al. (78). Here, itgb2 expression is restricted to the region of the CHT and hematopoietic cells at 2, 3, and 4 dpf as shown with whole-mount in situ hybridization (WISH). Furthermore, expression of itgb2 is restricted to neutrophils, macrophages, and thymus according to the UCSC cell browser database (81). Since we analyzed zebrafish larvae at 3 dpf and 5 dpf no cells of the adaptive immune system are present which start to develop two weeks after fertilization.

Of note, zebrafish larvae represent an embryonic model and the data obtained in the present study are therefore not directly transferable to the situation in human patients and murine models. Hence, future studies characterizing the CD18 KO zebrafish lines at the adult stage can shed additional light on the role of CD18 on neutrophil trafficking in zebrafish. Only recently, the existence of a substantial pool of neutrophils present in the tissue under steady state conditions has been reported in mammals which modulate organ function but can also influence pathological states including e.g. tumor cell infiltration (2). Beyond the immediate role of neutrophils in innate immunity, our knowledge of the homeostatic function of tissue neutrophils only starts to emerge and zebrafish larvae may represent an extremely valuable tool to decipher the versatile roles of neutrophils under steady state conditions by live imaging in the intact organism.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Regierung von Oberbayern Maximilianstraße 39 80538 München.

AB carried out research, analyzed data and wrote the manuscript. JG and TR carried out research and analyzed data. BS participated in generation of the zebrafish knock-out. BW analyzed data and wrote the manuscript. DM-B designed the study, carried out research, analyzed data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the Deutsche Forschungsgemeinschaft [SFB 914/A02 (DM-B and BW) and Z03 (BW)].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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