The Diverse Roles of Phagocytes During Bacterial and Fungal Infections and Sterile Inflammation: Lessons From Zebrafish

Abstract

The immediate and natural reaction to both infectious challenges and sterile insults (wounds, tissue trauma or crystal deposition) is an acute inflammatory response. This inflammatory response is mediated by activation of the innate immune system largely comprising professional phagocytes (neutrophils and macrophages). Zebrafish (danio rerio) larvae possess many advantages as a model organism, including their genetic tractability and highly conserved innate immune system. Exploiting these attributes and the live imaging potential of optically transparent zebrafish larvae has greatly contributed to our understanding of how neutrophils and macrophages orchestrate the initiation and resolution phases of inflammatory responses. Numerous bacterial and fungal infection models have been successfully established using zebrafish as an animal model and studies investigating neutrophil and macrophage behavior to sterile insults have also provided unique insights. In this review we highlight how examining the larval zebrafish response to specific bacterial and fungal pathogens has uncovered cellular and molecular mechanisms behind a variety of phagocyte responses, from those that protect the host to those that are detrimental. We also describe how modeling sterile inflammation in larval zebrafish has provided an opportunity to dissect signaling pathways that control the recruitment, and fate, of phagocytes at inflammatory sites. Finally, we briefly discuss some current limitations, and opportunities to improve, the zebrafish model system for studying phagocyte biology.

Keywords: infection; innate immunity; macrophages; neutrophils; phagocytes; sterile inflammation; zebrafish.

Figure 1

Schematic illustration of the different delivery routes in larval zebrafish for pathogens. (A, left side) and to model sterile inflammation (B, right side). In the sterile injury section, the stimulus is specified by blue writing. All delivery routes show an exemplary reference, with the ones covered in this review highlighted in bold.

Figure 2

Schematic illustration of the phagocyte responses to the bacterial pathogens M. marinum (A), M. leprae (B), B. cenocepacia (C), and S. aureus (D). (A) Macrophages phagocytose M. marinum (1) and release ESAT-6 (2). ESAT-6-driven Mmp9 production by epithelial cells leads to macrophage recruitment (3) and granuloma formation (4). Newly-arriving macrophages become infected by engulfing dying infected macrophages (5). Infected macrophages can establish secondary granulomas (6). Low TNF levels promote intracellular bacterial growth and macrophage necrosis (7). High TNF levels promote mROS production within infected macrophages that, although initially bactericidal, also leads to necrosis (8). Necrosis results in the release of bacteria into the extracellular milieu (9). Neutrophils can phagocytose infected macrophage debris (10) and kill M. marinum by NADPH oxidase-mediated ROS production and Hif-1α-dependent reactive nitrogen species production (11). (B) M. leprae-infected macrophages migrate along nerve axons (1), where PGL-1 (2) stimulates iNOS-driven nitric oxide production in macrophages (3) that damages mitochondria in adjacent axons (4). (C) Following i.v. delivery, macrophages phagocytose B. cenocepacia (1) providing a replication niche (2). Infected macrophages produce Il1b (3) that attracts neutrophils and macrophages (4), leading to tissue damage resulting from degranulating neutrophils (5). The inflammatory response also leads to myeloid cell ablation that favors the survival of infected macrophages (6). B. cenocepacia can disseminate through non-lytic escape from infected macrophages (7). Following s.c. infection, neutrophils phagocytose B. cenocepacia (8) but are inefficient in killing the bacteria and instead release the bacteria into the extracellular milieu (9). (D) Following phagocytosis of S. aureus by neutrophils (1), NADPH oxidase activity (2) contributes to the formation of non-acidic Lc3-positive phagosomes (3) that provide a replication niche. Phagosome membrane damage results in the release of bacteria into the cytosol (4), neutrophil death and bacterial dissemination (5).

Figure 3

Schematic illustration of phagocyte responses to the fungal pathogens A. fumigatus (A), T. marneffei (B) and C. neoformans (C) in larval zebrafish. (A) Macrophages phagocytose A. fumigatus conidia and form tight clusters around the fungus, which inhibits fungal germination (1). Fungal germination can occur in the late phagosomes of infected macrophages (2) causing macrophage necroptosis (3). Transfer of A. fumigatus conidia can occur from infected and dying macrophages to recipient macrophages (4). Neutrophils kill A. fumigatus hyphae with their effector functions, including phagocytosis and NETosis (5). Infected neutrophils can transfer A. fumigatus to recipient macrophages through shuttling (6). (B) T. marneffei exists as filamentous conidia at 30°C and as pathogenic yeast form at 37°C (1). T. marneffei spores can transition to the yeast form within macrophages (2). Neutrophils can also phagocytose T. marneffei conidia (3) and transfer them to recipient macrophages through shuttling (4). (C) Macrophages phagocytose C. neoformans (1) where it can be killed (2) or persist and proliferate (3). WASP-Arp2/3 regulates vomocytosis and fungal dissemination (4).

Figure 4

Schematic illustration of the signaling pathways and mechanisms that help control phagocyte migration and abundance during larval zebrafish acute tail fin injury. (A) A gradient of H₂O₂, generated at the wound margin, is sensed by neutrophils through oxidation of Lyn, leading to directed neutrophil migration (1). Neutrophil-delivered Mpx consumes H₂O₂ producing hypochlorous acid (HOCl) (2). (B) Macrophage arrival at the wound site is promoted by NADPH oxidase activity and Yrk, in addition to Cxcr3.2/Cxcl11(1). In this context, Cxcr3.3 acts as a scavenger receptor to negatively regulate Cxcr3.2 function. Macrophage-delivered PGE2 promotes neutrophil retrograde chemotaxis (2) together with NADPH/Yrk-dependent contact-mediated guidance from macrophages (3). Cxcr2 signaling also contributes to initiate neutrophil retrograde chemotaxis (4). (C) Cxcr4/Cxcl12 signaling contributes to the retention of neutrophils at the wound site (1) along with Hif-1 activation (2). Macrophages at the wound site also remove apoptotic neutrophil debris (3).