The zebrafish has proven itself as an excellent model to study vertebrate innate immunity. It presents us with possibilities forin vivoimaging of host-pathogen interactions which are unparalleled in mammalian model systems. In addition, its suitability for genetic approaches is providing new insights on the mechanisms underlying the innate immune response. Here, we review the pattern recognition receptors that identify invading microbes, as well as the innate immune effector mechanisms that they activate in zebrafish embryos. We compare the current knowledge about these processes in mammalian models and zebrafish and discuss recent studies using zebrafish infection models that have advanced our general understanding of the innate immune system. Furthermore, we use transcriptome analysis of zebrafish infected withE. tarda, S. typhimurium, andM. marinumto visualize the gene expression profiles resulting from these infections. Our data illustrate that the two acute disease-causing pathogens,E. tardaandS. typhimurium, elicit a highly similar proinflammatory gene induction profile, while the chronic disease-causing pathogen,M. marinum, induces a weaker and delayed innate immune response.

Summary: Toll-like receptors (TLRs) are an important class of pattern recognition receptors (PRRs) that recognize microbial and danger signals. Their downstream signaling upon ligand binding is vital for initiation of the innate immune response. In human and mammalian models, myeloid differentiation factor 88 (MYD88) is known for its central role as an adaptor molecule in interleukin 1 receptor (IL-1R) and TLR signaling. The zebrafish is increasingly used as a complementary model system for disease research and drug screening. Here, we describe a zebrafish line with a truncated version of MyD88 as the first zebrafish mutant for a TLR signaling component. We show that this immune-compromised mutant has a lower survival rate under standard rearing conditions and is more susceptible to challenge with the acute bacterial pathogens Edwardsiella tarda and Salmonella typhimurium. Microarray and quantitative PCR analysis revealed that expression of genes for transcription factors central to innate immunity (including NF-κB and AP-1) and the pro-inflammatory cytokine Il1b, is dependent on MyD88 signaling during these bacterial infections. Nevertheless, expression of immune genes independent of MyD88 in the myd88 mutant line was sufficient to limit growth of an attenuated S. typhimurium strain. In the case of infection with the chronic bacterial pathogen Mycobacterium marinum, we show that MyD88 signaling has an important protective role during early pathogenesis. During mycobacterial infection, the myd88 mutant shows accelerated formation of granuloma-like aggregates and increased bacterial burden, with associated lower induction of genes central to innate immunity. This zebrafish myd88 mutant will be a valuable tool for further study of the role of IL1R and TLR signaling in the innate immunity processes underlying infectious diseases, inflammatory disorders and cancer.

Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic intervention against tuberculosis. Tuberculosis is a mycobacterial disease that was a major cause of death until the discovery of antibiotics in the mid-twentieth century. However, TB is once again on the rise, with the emergence of strains that are multi-drug resistant. Mycobacteria are specialists in evading immune cell killing and use host immune cells as a niche in which they can proliferate and survive latently, until subsequent re-activation and spreading causing life-threatening disease. Pharmaceutical reprogramming of the immune system to kill intracellular mycobacteria would represent a therapeutic strategy, effective against currently untreatable strains and less susceptible to drug resistance. Here we use an in vivo zebrafish model of TB to show that manipulation of the host genetic pathway responsible for detecting low oxygen levels (hypoxia) causes a decrease in mycobacterial infection. This antimicrobial effect was due to a priming of immune cells with increased levels of nitric oxide, a molecule that is used by immune cells to kill bacteria. Here we show in vivo manipulation of a host-signaling pathway aids the host in combatting mycobacteria infection, identifying hypoxic signaling as a potential target for future therapeutics against TB.

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type