Michiel van der Vaart

Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system

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-ĸB 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.

Pathogen recognition and activation of the innate immune response in zebrafish.

The zebrafish has proven itself as an excellent model to study vertebrate innate immunity. It presents us with possibilities for in vivo imaging 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 with E. tarda, S. typhimurium, and M. marinum to visualize the gene expression profiles resulting from these infections. Our data illustrate that the two acute disease-causing pathogens, E. tarda and S. 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.

The DNA Damage-Regulated Autophagy Modulator DRAM1 Links Mycobacterial Recognition via TLR-MYD88 to Autophagic Defense

Autophagy is an important defense mechanism against mycobacteria, the causative agents of tuberculosis. The molecular mechanisms that link mycobacterial recognition to autophagy remain unclear. Our analysis in zebrafish and human macrophage models of mycobacterial infection reveals that the DNA damage-regulated autophagy modulator DRAM1 functions downstream of pathogen recognition by the Toll-like receptor (TLR)/interleukin-1 receptor (IL1R)-MYD88-NF-κB innate immune sensing pathway to activate selective autophagy. Mycobacterial infection of human macrophages and zebrafish embryos induced DRAM1 expression in a MYD88 and NF-κB-dependent manner. DRAM1 knockdown increased mycobacterial infection, whereas overexpression lowered infection by hyperactivating autophagy. DRAM1-mediated selective autophagic defenses require the cytosolic DNA sensor STING and the selective autophagy receptor p62/SQSTM1. Contrary to its known role in autophagy-mediated cell death and cancer, this DRAM1 function is p53 independent. We propose that DRAM1 mediates autophagic defense against a broader range of intracellular pathogens, since DRAM1 expression was also induced by the common bacterial endotoxin lipopolysaccharide.

Hypoxia Inducible Factor Signaling Modulates Susceptibility to Mycobacterial Infection via a Nitric Oxide Dependent Mechanism

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 hosts 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.

Nanoparticles as Drug Delivery System against Tuberculosis in Zebrafish Embryos: Direct Visualization and Treatment

Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.

Infectious Disease Modeling and Innate Immune Function in Zebrafish Embryos

The major cell types of the innate immune system, macrophages and neutrophils, develop during the first two days of zebrafish embryogenesis. The interaction of these immune cells with pathogenic microbes can excellently be traced in the optically transparent zebrafish embryos. Various tools and methods have recently been developed for visualizing and isolating the zebrafish embryonic innate immune cells, for establishing infections by different micro-injection techniques, and for analyzing the host innate immune response following microbial recognition. Here we provide practical guidelines for the application of these methodologies and review the current state of the art in zebrafish infectious disease research.

Naturally occurring C-terminal splice variants of nuclear receptors

Alternative mRNA splicing in the region encoding the C-terminus of nuclear receptors results in receptor variants lacking the entire ligand-binding domain (LBD), or a part of it, and instead contain a sequence of splice variant-specific C-terminal amino acids. A total of thirteen such splice variants have been shown to occur in vertebrates, and at least nine occur in humans. None of these receptor variants appear to be able to bind endogenous ligands and to induce transcription on promoters containing the response element for the respective canonical receptor variant. Interestingly, ten of these C-terminal splice variants have been shown to display dominant-negative activity on the transactivational properties of their canonical equivalent. Research on most of these splice variants has been limited, and the dominant-negative effect of these receptor variants has only been demonstrated in reporter assays in vitro, using transiently transfected receptors and reporter constructs. Therefore, the in vivo function and relevance of most C-terminal splice variants remains unclear. By reviewing the literature on the human glucocorticoid receptor β-isoform (hGRβ), we show that the dominant-negative effect of hGRβ is well established using more physiologically relevant readouts. The hGR β-isoform may alter gene transcription independent from the canonical receptor and increased hGRβ levels correlate with glucocorticoid resistance and the occurrence of several immune-related diseases. Thus, available data suggests that C-terminal splice variants of nuclear receptors act as dominant-negative inhibitors of receptor-mediated signaling in vivo, and that aberrant expression of these isoforms may be involved in the pathogenesis of a variety of diseases.

Real‐time imaging and genetic dissection of host–microbe interactions in zebrafish

Many aspects of host interactions with microbes can only be studied in the context of a whole organism. The zebrafish as a model organism has shown to be highly successful for studies of infection biology and the interactions of commensal microbiota with their hosts. Zebrafish are transparent during embryo and larval development and these early life stages are optimally suited for high‐resolution imaging of host–microbe interactions in a vertebrate organism. This is facilitated by the development of a variety of fluorescent reporter lines that mark different immune cell types or subcellular compartments where pathogens reside. The zebrafish is an excellent vertebrate model for forward genetic screening and efficient tools for gene knock‐down and targeted mutagenesis add further to the strength of this model organism. The use of zebrafish larvae for studying microbial infections has recently led to important new insights in host defence mechanisms, which are highlighted in this review focused on bacterial pathogens. Considering the highly conserved nature of the processes involved, including innate immune recognition, immunometabolism and autophagy, it is to be expected that these recent findings in zebrafish will have great translational value for biomedical applications.

Cellular Visualization of Macrophage Pyroptosis and Interleukin-1β Release in a Viral Hemorrhagic Infection in Zebrafish Larvae

ABSTRACT Hemorrhagic viral diseases are distributed worldwide with important pathogens, such as dengue virus or hantaviruses. The lack of adequate in vivo infection models has limited the research on viral pathogenesis and the current understanding of the underlying infection mechanisms. Although hemorrhages have been associated with the infection of endothelial cells, other cellular types could be the main targets for hemorrhagic viruses. Our objective was to take advantage of the use of zebrafish larvae in the study of viral hemorrhagic diseases, focusing on the interaction between viruses and host cells. Cellular processes, such as transendothelial migration of leukocytes, virus-induced pyroptosis of macrophages. and interleukin-1β (Il-1β) release, could be observed in individual cells, providing a deeper knowledge of the immune mechanisms implicated in the disease. Furthermore, the application of these techniques to other pathogens will improve the current knowledge of host-pathogen interactions and increase the potential for the discovery of new therapeutic targets. IMPORTANCE Pathogenic mechanisms of hemorrhagic viruses are diverse, and most of the research regarding interactions between viruses and host cells has been performed in cell lines that might not be major targets during natural infections. Thus, viral pathogenesis research has been limited because of the lack of adequate in vivo infection models. The understanding of the relative pathogenic roles of the viral agent and the host response to the infection is crucial. This will be facilitated by the establishment of in vivo infection models using organisms such as zebrafish, which allows the study of the diseases in the context of a complete individual. The use of this animal model with other pathogens could improve the current knowledge on host-pathogen interactions and increase the potential for the discovery of new therapeutic targets against diverse viral diseases.

Mycobacteria Counteract a TLR-Mediated Nitrosative Defense Mechanism in a Zebrafish Infection Model

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments.