Local tissue damage, such as that caused by Metchnikoff's thorn, induces the release of endogenous damage-associated molecular patterns (DAMPs), a pro-inflammatory cocktail of proteins and small-molecule mediators. In vertebrates, these signals result in the recruitment of phagocytes - neutrophils and macrophages - to the site of the lesion. Nowadays, models of the innate immune response often use sterile tissue injury as the inciting stimulus; the earliest investigation into neutrophil recruitment in the zebrafish, performed in the laboratory of Graham Lieschke, used a simple tailfin transection as the inflammatory stimulus [3
]. The subsequent development of transgenic lines of zebrafish, in which innate immune cell populations are specifically labeled with fluorescent proteins and readily imaged in the transparent larva, has allowed unprecedented visualization of immune cell recruitment and inflammation resolution in vivo
] and references cited therein, and [5
]). This assay has provided the basis for genetic and pharmacological screens for novel modulators of inflammation [4
]. But although robust and reproducible, the tailfin injury assay is time consuming and requires considerable technical skill, limiting its applicability to high-throughput screens. In the absence of efficient means of automating the process, there was a pressing need for alternative methods of initiating inflammation in the fish. d'Alençon et al.
] now describe one such alternative; they show that a robust inflammatory response can be induced by localized cell death of sensory hair cells in the zebrafish lateral-line system, a process that they had previously found can be induced simply by immersion of zebrafish larvae in copper sulfate solution [8
]. The response of neutrophils to copper exposure is extremely rapid: cells can be seen to start migrating towards the damaged neuromasts - discrete clusters of nerve cells regularly distributed along the length of the body - within 15 minutes of immersion of the larvae in the CuSO4
solution, and large accumulations of active neutrophils are apparent after 2 hours (Figure ). The authors show that this response can be suppressed both by known anti-inflammatory drugs and by a mutation in the gene encoding the Wiskott-Aldrich syndrome protein (WASP) [9
]. Moreover, by combining automated imaging with a custom software script that maps the fluorescent expression domains in the larvae, they have established a platform for high-throughput screening.
Figure 1 ChIn analysis. Compound transgenic zebrafish larvae expressing green fluorescent protein in neuromasts (arrowed in top panel) and dsRed in neutrophils. Upper panel shows recruitment of red fluorescent neutrophils to injured neuromasts in green. In the (more ...)
This innovation - which the authors call the chemically induced inflammation assay, or 'ChIn' assay for short - frees the neutrophil-recruitment model from the constraints imposed by manual intervention, thereby opening the door to the automation of both pharmacological and genetic screens. The zebrafish has been used for forward genetic analysis for more than 20 years, but more recently its suitability for pharmacological or 'chemical genetic' screens has become equally apparent [10
]. Such whole-organism screens provide a powerful approach to the discovery of biologically active compounds that can have utility either as reagents for the dissection of biological processes or as leads for the development of therapeutics. Indeed, the first compound identified in this type of zebrafish screen is currently in phase I clinical trials to improve engraftment of transplanted cord blood stem cells, and our own experience suggests that many more will follow. The ChIn protocol will now bring screens for modulators of the innate immune response within reach of many more labs than those with established expertise in the tailfin transection assay, allowing investigators studying the molecular basis of leukocyte recruitment and inflammation resolution to add the zebrafish model to their experimental armory.
While hypothesis-driven experiments are a crucial driver of scientific knowledge, the complementary benefits of unbiased screens are clear to see. Key advances in the field of innate immunity have been made by phenotype-driven approaches and the technological innovation of d'Alençon and colleagues should accelerate both the identification of novel genes underlying the innate immune response as well as novel therapeutic approaches to its manipulation. It is worth remembering that aspirin, one of the best selling anti-inflammatory drugs of all time, and the founding member of entire classes of medicines, was similarly identified by a phenotype: the ability to reduce fever.