We have established an in vivo model for genetic analysis of the inflammatory response by generating a transgenic zebrafish line that expresses GFP under the neutrophil-specific myeloperoxidase promoter. We show that inflammation is induced after transection of the tail of zebrafish larvae and that this inflammation subsequently resolves over a similar time course to mammalian systems. Quantitative data can be generated from this model by counting of fluorescent cells or by digital image analysis. In addition, we show that the resolution of experimentally induced inflammation can be inhibited by the addition of a pancaspase inhibitor, zVD.fmk, demonstrating that experimental manipulation of the resolution of inflammation is possible in this model. IntroductionNeutrophilic inflammation is essential for the maintenance of health and life, but failure to resolve the response in a timely manner can cause irreparable tissue damage because of the release of toxic granule contents of persisting neutrophils. 1 An understanding of the genetic basis of inflammation resolution would undoubtedly provide an important basis for the development of approaches to limiting neutrophil-mediated tissue injury. To facilitate such a genetic analysis, an animal model in which the cellular components of inflammation can be readily visualized in wild-type and genetically manipulated individuals is required.Zebrafish larvae are transparent, allowing excellent visualization of fluorescent proteins in cellular processes in vivo. Zebrafish neutrophils (heterophils) are identifiable from approximately 48 hours after fertilization, 2 and the innate immune system exists in isolation from any adaptive system, which does not arise until approximately 4 weeks after fertilization. 3 A range of tools is available for the genetic manipulation of the zebrafish, as are extensive genomics resources, including a draft sequence of the entire genome. We therefore selected this species as an ideal organism for the generation of a simplified, genetically tractable model using fluorescent neutrophils to track the inflammatory response. Here, we report the generation of a transgenic zebrafish line for use in such experiments and describe the onset and resolution of inflammation in this model. We show that inflammation proceeds with kinetics similar to those in mammalian systems and that experimental manipulation of inflammation in this system is achievable and quantifiable. Materials and methodsZebrafish were maintained according to standard protocols. 4 Reagents were from Sigma (Poole, United Kingdom) unless otherwise specified. zVD.fmk was from Bachem (Weil am Rhein, Germany). MPO::GFP lineBAC (zC91B8) was modified by the use of a red recombinase system in EL250 cells (gift of Dr Neal Copeland, National Cancer Institute, Frederick, MD). 5 EGFP with an SV40 polyadenylation site (Clontech, Palo Alto, CA) was inserted at the mpo (also called mpx) ATG start site. This BAC, linearized with PI-Sce1, was used to generate stable transgenic lines according to publis...

The oxygen-sensing transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) 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-1␣, and selective genetic manipulation of zebrafish HIF-1␣ homologs, we sought to determine the roles of HIF-1␣ in inflammation resolution. Both approaches reveal that activated Hif-1␣ 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. Sitedirected mutagenesis of Hif in vivo reveals that hydroxylation of Hif-1␣ by prolyl hydroxylases critically regulates the Hif pathway in zebrafish neutrophils. Our data demonstrate that Hif-1␣ regulates neutrophil function in complex ways during inflammation resolution in vivo. (Blood. 2011;118(3):712-722) IntroductionNeutrophilic inflammation is of fundamental importance in the innate immune response to bacterial and fungal infection in vertebrates, and it can be initiated by sterile tissue injury. Irrespective of its etiology, inflammation must resolve in a timely manner to avoid damage to surrounding tissues. 1 Persisting, noninfectious inflammation is the hallmark of inflammatory diseases, a major cause of morbidity and mortality in the developed world. The resolution phase of inflammation is critical to the restoration of normal tissue function after an inflammatory response, and thus has a central role in determining the outcome of inflammation. 2 Despite the central place of failed resolution in the pathogenesis of inflammatory disease, much remains to be known about the cellular and molecular events involved.Although neutrophil apoptosis, and subsequent uptake and removal by macrophages (efferocytosis), is well documented as a disposal route for inflammatory neutrophils, [3][4][5] there is emerging evidence that other mechanisms also may contribute to certain types of inflammation resolution. In the lung, some neutrophils are lost into the airways and expectorated, 6 and in rheumatoid arthritis, neutrophils may leave the inflammatory site while still alive and re-enter the circulation. 7 Neutrophils also can be removed by migration through tissues away from the infection site; a process termed retrograde chemotaxis, or reverse migration. [8][9][10] This process is most widely characterized in the zebrafish (Danio rerio) embryo and is less well studied in mammalian s...

Neutrophils play a pivotal role in the innate immune response. The small cytokine CXCL8 (also known as interleukin-8 or IL-8) is known to be one of the most potent chemoattractant molecules which, 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 homologue, zebrafish has two distinct CXCL8 homologues: Cxcl8-l1 and Cxcl8-l2. Cxcl8-l1 is known to be up-regulated under inflammatory conditions caused by bacterial or chemical insult but until now, the role of Cxcl8s in neutrophil recruitment has not been studied. Here, we show that both Cxcl8 genes are up-regulated 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 to control neutrophils, although the directionality was not affected. Our analysis suggests that zebrafish may possess a sub-population 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.

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.