Zebrafish genetic approaches to understanding the regulation of inflammatory cell apoptosis.

In many inflammatory diseases, white blood cells called neutrophils cause damage to tissues. Examples in the lung include common conditions such as COPD, asthma and interstitial lung diseases. Current treatments are poorly effective and have many side effects. Neutrophils normally fight infection, but can also harm tissues if inappropriately activated and in health this is prevented by programmed cell death (apoptosis), which allows their removal. This process is blocked in inflammatory disease. I am looking for ways to circumvent this block and thus suppress the inflammatory response. Apoptosis is regulated by activation of a group of specialised enzymes called caspases in various types of caspase activating platform (CAP) - complexes of proteins that regulate activation of caspases. I propose apoptosis is regulated in a unique way in neutrophils, by activation of specific caspases in a new type of CAP. I will explore this idea using a model of the inflammatory response I have developed in the zebrafish. This organism allows us to observe neutrophils in living animals and to screen for genetic mutations or chemicals that modify neutrophil behaviour. In this way I aim to discover the genes that control neutrophil apoptosis and develop new drugs to treat inflammatory disease.

My aim is to build on the successes of my CSF, using the power of zebrafish models to understand the molecular controls of neutrophilic inflammation. Tissue damage caused by neutrophil granule contents is the cause of pathology across all organ systems. Neutrophil inflammatory potential is limited by apoptotic cell death, which is uniquely rapid in neutrophils, but suppressed at sites of inflammation. Neither the mechanisms for neutrophil apoptosis, nor their suppression are understood. I hypothesise that neutrophil apoptosis is driven by a neutrophil-specific caspase activating platform (CAP) analogous to the inflammasome, and that this CAP is directly regulated by Mcl-1, a critical regulator of neutrophil survival, induced by survival signals such as GMCSF. I further hypothesise that levels of Mcl-1 are in part determined by the action of microRNAs to suppress Mcl-1 translation. Experimental Plan: 1a) I will use biotinylated, irreversible caspase inhibitors to bind and block apical caspases from human neutrophils, then use these to pull down the CAP. I will knockdown expression of the CAP components using neutrophil-specific siRNA in transgenic zebrafish, and then investigate CAP assembly in vivo using transgenically-expressed tagged CAP components and FRET in zebrafish. 1b) Mutants identified in forward genetic screens performed during my CSF will be characterised and mapped to identify the drivers for neutrophil apoptosis in an unbiased way. 2a) I will assess binding to and regulation of the CAP by Mcl-1, both in pull down experiments from human neutrophils (+/- GMCSF) and in vivo in transgenic zebrafish using FRET. I will also generate a transgenic zebrafish line expressing a constitutively-active GMCSF receptor beta subunit (CA-GMCSF-R) specifically in neutrophils, and use this to examine the effects of GMCSF signalling on CAP assembly. 2b) I will investigate the role of microRNAs in regulating GMCSF-stimulated neutrophil survival, using microarrays and Taqman quantitative PCR, clone new microRNAs from ultrapure human neutrophils and search for targets of microRNAs in transgenic zebrafish overexpressing those microRNAs. 2c) Finally I will use the CA-GMCSF-R line in a suppressor screen for mutants correcting the transgenic phenotype of delayed inflammation resolution. The interaction of Mcl-1 and the putative CAP is a important target for rational drug design, and the suppressor screen described here is ideal for use in a screen for compounds correcting pathologically-suppressed neutrophil apoptosis, and as such might identify compounds with therapeutic potential in treating inflammatory disease.