Stephen A. Renshaw

A model 450 million years in the making: zebrafish and vertebrate immunity

Since its first splash 30 years ago, the use of the zebrafish model has been extended from a tool for genetic dissection of early vertebrate development to the functional interrogation of organogenesis and disease processes such as infection and cancer. In particular, there is recent and growing attention in the scientific community directed at the immune systems of zebrafish. This development is based on the ability to image cell movements and organogenesis in an entire vertebrate organism, complemented by increasing recognition that zebrafish and vertebrate immunity have many aspects in common. Here, we review zebrafish immunity with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism. These unique advantages are driving forward our study of vertebrate immunity in general, with important consequences for the understanding of mammalian immune function and its role in disease pathogenesis.

Acceleration of Human Neutrophil Apoptosis by TRAIL

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-α also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-κB, suggesting TRAIL does not activate NF-κB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.

Unraveling tissue regeneration pathways using chemical genetics.

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.

Evolution of the Inflammatory Response in Vertebrates: Fish TNF-α Is a Powerful Activator of Endothelial Cells but Hardly Activates Phagocytes

TNF-α is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-α are mediated through the activation of endothelial cells. Thus, TNF-α promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-α, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-α in Ag presentation. Lastly, we found that TNF-α increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-α on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-α increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-α resulted in increased viral replication. Together, these results indicate that fish TNF-α displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.

Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish

The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186 , in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

Cxcl8 (IL-8) Mediates Neutrophil Recruitment and Behavior in the Zebrafish Inflammatory Response

Neutrophils play a pivotal role in the innate immune response. The small cytokine CXCL8 (also known as IL-8) is known to be one of the most potent chemoattractant molecules that, among several other functions, is responsible for guiding neutrophils through the tissue matrix until they reach sites of injury. Unlike mice and rats that lack a CXCL8 homolog, zebrafish has two distinct CXCL8 homologs: Cxcl8-l1 and Cxcl8-l2. Cxcl8-l1 is known to be upregulated under inflammatory conditions caused by bacterial or chemical insult but until now the role of Cxcl8s in neutrophil recruitment has not been studied. In this study we show that both Cxcl8 genes are upregulated in response to an acute inflammatory stimulus, and that both are crucial for normal neutrophil recruitment to the wound and normal resolution of inflammation. Additionally, we have analyzed neutrophil migratory behavior through tissues to the site of injury in vivo, using open-access phagocyte tracking software PhagoSight. Surprisingly, we observed that in the absence of these chemokines, the speed of the neutrophils migrating to the wound was significantly increased in comparison with control neutrophils, although the directionality was not affected. Our analysis suggests that zebrafish may possess a subpopulation of neutrophils whose recruitment to inflamed areas occurs independently of Cxcl8 chemokines. Moreover, we report that Cxcl8-l2 signaled through Cxcr2 for inducing neutrophil recruitment. Our study, therefore, confirms the zebrafish as an excellent in vivo model to shed light on the roles of CXCL8 in neutrophil biology.

A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte‐dependent resistance of zebrafish to non‐host specialized pathogens

With the emergence of multiply resistant Staphylococcus aureus, there is an urgent need to better understand the molecular determinants of S. aureus pathogenesis. A model of staphylococcal pathogenesis in zebrafish embryos has been established, in which host phagocytes are able to mount an effective immune response, preventing overwhelming infection from small inocula. Myeloid cell depletion, by pu.1 morpholino‐modified antisense injection, removes this immune protection. Macrophages and neutrophils are both implicated in this immune response, phagocytosing circulating bacteria. In addition, in vivo phagocyte/bacteria interactions can be visualized within transparent embryos. A preliminary screen for bacterial pathogenesis determinants has shown that strains bearing mutations in perR, pheP and saeR are attenuated. perR and pheP mutants are deficient in growth in vivo, and their virulence is not fully restored by myeloid cell depletion. On the other hand, saeR mutants are able to grow in vivo, and are completely restored to virulence by myeloid cell depletion. Thus specific pathogen gene function can be matched with particular facets of host response. Zebrafish are a new addition to the tools available for the study of S. aureus pathogenesis, and may provide insights into the interactions of bacterial and host genomes in determining the outcome of infection.

Activation of hypoxia-inducible factor-1α (Hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model

The oxygen-sensing transcription factor hypoxia-inducible factor-1α (HIF-1α) plays a critical role in the regulation of myeloid cell function. The mechanisms of regulation are not well understood, nor are the phenotypic consequences of HIF modulation in the context of neutrophilic inflammation. Species conservation across higher metazoans enables the use of the genetically tractable and transparent zebrafish (Danio rerio) embryo to study in vivo resolution of the inflammatory response. Using both a pharmacologic approach known to lead to stabilization of HIF-1α, and selective genetic manipulation of zebrafish HIF-1α homologs, we sought to determine the roles of HIF-1α in inflammation resolution. Both approaches reveal that activated Hif-1α delays resolution of inflammation after tail transection in zebrafish larvae. This delay can be replicated by neutrophil-specific Hif activation and is a consequence of both reduced neutrophil apoptosis and increased retention of neutrophils at the site of tissue injury. Hif-activated neutrophils continue to patrol the injury site during the resolution phase, when neutrophils would normally migrate away. Site-directed mutagenesis of Hif in vivo reveals that hydroxylation of Hif-1α by prolyl hydroxylases critically regulates the Hif pathway in zebrafish neutrophils. Our data demonstrate that Hif-1α regulates neutrophil function in complex ways during inflammation resolution in vivo.

A Zebrafish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-Inflammatory Mechanism

The proresolution therapeutic tanshinone IIA drives inflammation resolution by reverse migration. An Anti-Inflammatory Fish Story Inflammation is one way the body tries to protect itself from injury and begin the healing process. However, as with any good thing, too much inflammation can be harmful, causing bystander injuries to healthy tissue. Hence, there is an active mechanism to resolve inflammation; failed resolution contributes to diseases of chronic inflammation such as atherosclerosis and rheumatoid arthritis. Now, Robertson et al. use a zebrafish screening platform to identify new means of resolving inflammation. The authors used a transgenic zebrafish model of sterile tissue injury to screen potential factors involved in inflammation resolution. They found that tanshinone IIA, which is derived from a Chinese medicinal herb, had proresolving activity by both inducing neutrophil apoptosis and promoting reverse migration of neutrophils. What’s more, these effects were not limited to their zebrafish model but held true in human neutrophils. Although efficacy remains to be tested in actual patients, these data support “fishing” for new drug candidates for resolving inflammation. Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.

TNF-related apoptosis-inducing ligand (TRAIL) regulates inflammatory neutrophil apoptosis and enhances resolution of inflammation

Novel therapeutics targeting neutrophilic inflammation are a major unmet clinical need in acute and chronic inflammation. The timely induction of neutrophil apoptosis is critical for inflammation resolution, and it is thought that acceleration of apoptosis may facilitate resolution at inflammatory sites. We previously demonstrated that a death receptor ligand, TRAIL, accelerates neutrophil apoptosis in vitro. We examined the role of TRAIL in neutrophil‐dominant inflammation in WT and TRAIL‐deficient mice. TRAIL deficiency did not alter constitutive neutrophil apoptosis, whereas exogenous TRAIL accelerated apoptosis of murine peripheral blood neutrophils. We compared TRAIL‐deficient and WT mice in two independent models of neutrophilic inflammation: bacterial LPS‐induced acute lung injury and zymosan‐induced peritonitis. In both models, TRAIL‐deficient mice had an enhanced inflammatory response with increased neutrophil numbers and reduced neutrophil apoptosis. Correction of TRAIL deficiency and supraphysiological TRAIL signaling using exogenous protein enhanced neutrophil apoptosis and reduced neutrophil numbers in both inflammatory models with no evidence of effects on other cell types. These data indicate the potential therapeutic benefit of TRAIL in neutrophilic inflammation.