Anne L. Robertson

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.

Repelled from the wound, or randomly dispersed? Reverse migration behaviour of neutrophils characterized by dynamic modelling

Following neutralization of infectious threats, neutrophils must be removed from inflammatory sites for normal tissue function to be restored. Recently, a new paradigm has emerged, in which viable neutrophils migrate away from inflammatory sites by a process best described as reverse migration. It has generally been assumed that this process is the mirror image of chemotaxis, where neutrophils are drawn into the areas of infection or tissue damage by gradients of chemotactic cues. Indeed, efforts are underway to identify cues that drive neutrophils away by the reverse process, fugetaxis. By using photoconvertible pigments expressed in neutrophils in transparent zebrafish larvae, we were able to image the position of each neutrophil during inflammation resolution in vivo. These neutrophil coordinates were analysed within a dynamic modelling framework, using different forms of the drift–diffusion equation with model selection and parameter estimation based on approximate Bayesian computation. This analysis revealed the experimental data were best fitted by a model incorporating a diffusion term but no drift term—where the presence of drift would indicate fugetaxis. This result, for the first time, provides rigorous data-driven evidence that reverse migration of neutrophils in vivo is not a form of fugetaxis, but rather a stochastic redistribution.

Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library

Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model – the Tg(mpx:GFP)i114 zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) – to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration.

Defining the phenotype of neutrophils following reverse migration in zebrafish

Stimulation of neutrophil reverse migration presents an attractive, alternative therapeutic pathway to driving inflammation resolution. However, little is known about whether the activity of wound‐experienced neutrophils is altered and whether encouraging dispersal of such neutrophils back into the body may have undesirable consequences. This study used a zebrafish tail transection inflammation model, in combination with a photoconvertible neutrophil transgenic line, to allow internally controlled, simultaneous comparison of reverse‐migrated neutrophils with naïve neutrophils in the presence and absence of secondary insult. Detailed microscopy revealed that reverse‐migrated neutrophils exhibited an activated morphology but responded normally to secondary insult and are able to mount an effective antimicrobial response to Staphylococcus aureus. These results support a model in which reverse‐migrated neutrophils exhibit no long‐term behavioral alterations and encourage the notion of enhanced reverse migration as a viable target for pharmaceutical manipulation.

Serum and Glucocorticoid-Regulated Kinase 1 Regulates Neutrophil Clearance during Inflammation Resolution

The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid–regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

Understanding the regulation of vertebrate hematopoiesis and blood disorders – big lessons from a small fish

Hematopoietic stem cells (HSCs) give rise to all differentiated blood cells. Understanding the mechanisms that regulate self‐renewal and lineage specification of HSCs is key for developing treatments for many human diseases. Zebrafish have emerged as an excellent model for studying vertebrate hematopoiesis. This review will highlight the unique strengths of zebrafish and important findings that have emerged from studies of blood development and disorders using this system. We discuss recent advances in our understanding of hematopoiesis, including the origin of HSCs, molecular control of their development, and key signaling pathways involved in their regulation. We highlight significant findings from zebrafish models of blood disorders and discuss their application for investigating stem cell dysfunction in disease and for the development of new therapeutics.

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.

Efforts to enhance blood stem cell engraftment: Recent insights from zebrafish hematopoiesis

Perlin et al. discuss recent findings in the field of zebrafish hematopoiesis, focusing on the transcriptional regulation of hematopoietic stem cell (HSC) induction and HSC–niche interactions. Manipulation of developmental signaling pathways may enhance HSC bioengineering, which would improve transplantation therapies.

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.