Abstract
BackgroundStudies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening.ResultsHere we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.ConclusionsThis approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay.See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.
Highlights
Studies on innate immunity have benefited from the introduction of zebrafish as a model system
Addition of copper sulfate to the water rapidly destroys hair cells of the lateral line system by inducing oxidative stress followed by cell death [12,13]
When we carried out in situ hybridization to detect mmp9 transcripts in control animals, we detected very low levels of expression in a few cells located within the posterior blood island (PBI) or caudal hematopoietic tissue (CHT), the areas where most myeloid leukocytes reside at this developmental stage
Summary
Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Using transgenic lines that label myeloid leukocytes (neutrophils and/or macrophages) in vivo, we observed a specific, extremely rapid and highly reproducible innate immune response to copper-induced neuromast damage. Since the wounds are localized and are elicited chemically, no invasive manipulation of fish is required and the treatment can be applied massively Exploiting this observation, we developed a quantitative measure for inflammation by counting leukocytes migrating to the lateral line neuromasts in transgenic lines or with immune cell-specific stains upon copper-induced neuromast damage. We anticipate that chemically induced inflammation assays (ChIn) will make it possible to achieve different types of highthroughput compound screens as well as genetic screens, focusing on aspects of the wound-induced inflammatory response (initiation and resolution), analyses of various types of infection-induced responses, investigation of tissue regeneration and specific cell subtype migration assays
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