Catherine A. Loynes

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.

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.

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.

Pivotal Advance: Pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish

Zebrafish are a unique model for pharmacological manipulation of physiological processes such as inflammation; they are small and permeable to many small molecular compounds, and being transparent, they permit the visualization and quantitation of the inflammatory response by observation of transgenically labeled inflammatory cell populations. Using a transgenic line specifically labeling neutrophils in vivo (mpx:GFP), we studied the effects of a range of pharmacological agents on the resolution of inflammation in vivo. These agents were selected for their ability to modulate neutrophil function and lifespan in human neutrophils in vitro. Agents delaying neutrophil apoptosis (LPS, dbcAMP, and several caspase inhibitors) all lead to a delay in resolution of neutrophilic inflammation. Reciprocally, pyocyanin and roscovitine (inducers of neutrophil apoptosis) lead to reduced neutrophil numbers. The occurrence of apoptosis was observed by time‐lapse analysis and confirmed by dual staining for neutrophil‐specific mpx activity (TSA staining) and an apoptotic marker (TUNEL). During inflammation, macrophages follow neutrophils into the inflamed site, and TUNEL/TSA dual‐positive material can be demonstrated within macrophages, consistent with their uptake of apoptotic neutrophils. This model has several advantages over mammalian models and lends itself to the study of pharmaceutical agents modulating inflammation.

Zebrafish as a model for the study of neutrophil biology

To understand inflammation and immunity, we need to understand the biology of the neutrophil. Whereas these cells can readily be extracted from peripheral blood, their short lifespan makes genetic manipulations impractical. Murine knockout models have been highly informative, and new imaging techniques are allowing neutrophils to be seen during inflammation in vivo for the first time. However, there is a place for a new model of neutrophil biology, which readily permits imaging of individual neutrophils during inflammation in vivo, combined with the ease of genetic and chemical manipulation. The zebrafish has long been the model of choice for the developmental biology community, and the availability of genomic resources and tools for gene manipulation makes this an attractive model. Zebrafish innate immunity shares many features with mammalian systems, including neutrophils with morphological, biochemical, and functional features, also shared with mammalian neutrophils. Transgenic zebrafish with neutrophils specifically labeled with fluorescent proteins have been generated, and this advance has led to the adoption of zebrafish, alongside existing models, by a number of groups around the world. The use of these models has underpinned a number of key advances in the field, including the identification of a tissue gradient of hydrogen peroxide for neutrophil recruitment following tissue injury and direct evidence for reverse migration as a regulatable mechanism of inflammation resolution. In this review, we discuss the importance of zebrafish models in neutrophil biology and describe how the understanding of neutrophil biology has been advanced by the use of these models.

MODELING INFLAMMATION IN THE ZEBRAFISH: HOW A FISH CAN HELP US UNDERSTAND LUNG DISEASE

Neutrophilic inflammation is responsible for much of the tissue damage seen in many lung diseases. For resolution of inflammation to occur, neutrophils must die by apoptosis, allowing their recognition and removal by macrophages. The molecular events controlling this important regulatory step are poorly understood, in large part due to the genetic intractability of the human neutrophil granulocyte. The authors have established a model of inflammation in the Zebrafish, which shares many features of the innate immune system with those of humans. Injury to the Zebrafish tailfin induces a reproducible and quantifiable inflammatory response, which resolves with kinetics similar to mammalian models of neutrophilic inflammation, including pulmonary inflammation. Pharmacological modulation of neutrophil apoptosis can modulate the outcome of experimentally induced inflammation. In addition, the authors have generated a construct that expresses green fluorescent protein under the myeloperoxidase promoter, allowing in vivo visualization of neutrophils during experimentally induced inflammation. The authors are also performing an unbiased forward genetic screen for mutants with defective resolution of inflammation, and to date have identified a number of putative mutants. Further study and characterization of these mutants is underway. The authors have thus established an important experimental link between apoptosis and resolution of inflammation in an in vivo system, and defined an important new model for the study of inflammation resolution. The authors hope that these tools will permit detailed study of the genetic controls of the resolution of inflammation, and provide insights with potential clinical utility.

A method for the in vivo measurement of zebrafish tissue neutrophil lifespan

Neutrophil function is thought to be regulated, in large part, by limitation of lifespan by apoptosis. A number of studies suggest that circulating neutrophils have a half-life of approximately 6 hours, although contradictory evidence exists. Measuring tissue neutrophil lifespan, however, is more problematic. It is thought that tissue neutrophils survive longer, perhaps with a half-life in the order of 3–5 days, but this has never been directly measured. Zebrafish are an emerging model organism, with several advantages for the study of vertebrate immunity. In zebrafish, neutrophils constitutively assume tissue locations allowing their direct study in vivo. Using a transgenic approach, neutrophils were labelled with a photoconvertible pigment, Kaede. Photoconversion parameters were optimised and the stability of the Kaede confirmed. Individual neutrophils were photoconverted by scanning a confocal 405 nm laser specifically over each cell and their survival monitored for 48 hours, revealing an in vivo half-life for zebrafish tissue neutrophils of around 120 hours (117.7 hrs, 95% CI 95.67–157.8). Laser energy did not extend neutrophil lifespan, and we conclude that this represents a lower bound for the lifespan of a resting tissue neutrophil in the developing zebrafish larva. This is the first direct measurement of the lifespan of an in vivo tissue neutrophil.

Measuring Inflammatory Cell Migration in the Zebrafish

A key feature of inflammatory cells is the ability to migrate to a site of injury or infection quickly and efficiently. Infectious agents can then be taken up by these inflammatory cells, preventing established infection. Inflammatory cell migration is driven by a complex interaction between inflammatory cells and their environment. In order to maintain health, inflammation needs to resolve, allowing the surrounding tissues to recover and heal. These processes are not fully understood and have been difficult to study in cell culture due to the complex interactions between cell types. We therefore use a range of techniques in near-transparent zebrafish (Danio rerio) larvae to study these migration events in a whole-organism, in vivo model. Using a transgenic zebrafish line that specifically marks neutrophils with green fluorescent protein, Tg(mpx:GFP)i114, we are able to follow neutrophil behaviour at a single cell level. Using these methods, the cellular processes involved in all phases of inflammation can be studied and better understood.

Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen

ABSTRACT Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available ‘cromones’ are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease. Summary: Zebrafish inflammation screen identifies a new series of structurally related compounds with combined anti-inflammatory and pro-resolution activity, and reveals a previously unknown mechanism of action of clinical cromones.

PGE2 production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo

Neutrophil removal from inflammatory sites is regulated by lipid mediator signals between macrophages and neutrophils. Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site—either by apoptosis and efferocytosis or by reverse migration away from the wound—for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve, due to loss of macrophage-dependent handling of eicosanoid prostaglandin E2 (PGE2) that drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous PGE synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors during injury, causing an increase in Alox12 production and switching toward anti-inflammatory eicosanoid signaling. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 (lipoxin A4) in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease.