Neutrophil chemotaxis to sites of inflammation is a critical process during normal immune responses to tissue injury and infection and pathological immune responses leading to chronic inflammation. Although progress has been made in understanding the mechanisms that promote neutrophil recruitment to inflamed tissue, the mechanisms that regulate the resolution phase of the inflammatory response have remained relatively elusive. To define the mechanisms that regulate neutrophil-mediated inflammation in vivo, we have developed a novel transgenic zebrafish in which the neutrophils express GFP under control of the myeloperoxidase promoter (zMPO:GFP). Tissue injury induces a robust, inflammatory response, which is characterized by the rapid chemotaxis of neutrophils to the wound site. In vivo time-lapse imaging shows that neutrophils subsequently display directed retrograde chemotaxis back toward the vasculature. These findings implicate retrograde chemotaxis as a novel mechanism that regulates the resolution phase of the inflammatory response. The zMPO:GFP zebrafish provides unique insight into the mechanisms of neutrophil-mediated inflammation and thereby offers opportunities to identify new regulators of the inflammatory response in vivo.

SummaryPseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

The hallmark of chronic inflammation is the infiltration and persistence of leukocytes within inflamed tissue. Here, we describe the first zebrafish chronic inflammation mutant identified in an insertional mutagenesis screen for mutants that exhibit abnormal tissue distribution of neutrophils. We identified a mutant line with an insertion in the Hepatocyte growth factor activator inhibitor 1 gene (hai1; also known as Spint1) that showed accumulation of neutrophils in the fin. The mutant embryos exhibited inflammation in areas of epidermal hyperproliferation that was rescued by knock-down of the type II transmembrane serine protease Matriptase 1 (also known as St14), suggesting a novel role for Hai1-Matriptase 1 pathway in regulating inflammation. Using time-lapse microscopy of mutant embryos that express GFP from a neutrophil-specific promoter, we found that individual neutrophils in inflamed tissue displayed random motility characterized by periods of pausing alternating with periods of motility. During periods of persistent movement the cells were highly polarized, while the pausing modes were characterized by a loss of cell polarity. In contrast to responses to acute injury, neutrophils did not exhibit clear retrograde chemotaxis or resolution of inflammation in the mutant. These findings illustrate the utility of zebrafish as a new model system to study chronic inflammation and to visualize immune responses with high resolution in vivo.

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

Zebrafish have emerged as a powerful model system to study leukocyte recruitment and inflammation. Here we characterize the morphology and function of inflammatory macrophages in zebrafish larvae. These macrophages can be distinguished from neutrophils by immunolabeling of L-Plastin without MPO co-expression and by an elongated morphology. Live imaging of transgenic zMPO:GFP larvae demonstrate that GFPlo macrophages migrate to wounds by extension of thin pseudopods and carry out phagocytosis of tissue debris, and FACS analysis of leukocyte markers indicates expression of CSF1R in these macrophages. These findings identify distinct functional and morphological characteristics of inflammatory macrophages in zebrafish larvae.