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.

Using in vivo zebrafish models to understand the biochemical basis of neutrophilic respiratory disease.

Neutrophilic inflammation in the lung protects against infectious disease, and usually resolves spontaneously after removal of the inflammatory stimulus. However, much lung disease is caused by a failure of resolution of neutrophilic inflammation. Our laboratory is seeking an understanding of the biochemical basis of inflammation resolution, using the zebrafish model system. Zebrafish larvae are transparent, allowing visualization of GFP (green fluorescent protein)-labelled leucocytes during inflammation in vivo, and they can be readily manipulated by a range of forward and reverse genetic techniques. This combination of advantages makes zebrafish a powerful tool for the study of in vivo inflammatory processes. Using this model, we have visualized the process of inflammation resolution in vivo, and identified a role for apoptosis in this process. In addition, we have performed a forward genetic screen for mutants with defective resolution of inflammation, and reverse genetic experiments examining the influence of candidate genes on inflammation resolution. We have established a platform for screening for compounds with anti-inflammatory activity, which has yielded a number of interesting leads. Looking forward to succeed in the future, we are working at combining mutants, transgenes and pharmacological agents to dissect the biochemical basis of inflammation resolution, and to identify compounds that might be used to treat patients with respiratory disease.