Tumour necrosis factor alpha (TNFalpha) is a proinflammatory, clinically-important cytokine secreted by activated macrophages. TNFalpha has widespread roles in host defence, tumour ceil killing and homeostasis. However, excess secretion of TNFa is a major cause of tissue damage in chronic inflammatory diseases such as Crohn's disease, arthritis and in cancer. Cytokine secretion in macrophages is poorly understood at the molecular and cellular level. The goal of these studies is to generate an understanding of how TNFalpha is trafficked to the cell surface for secretion by macrophages. Recent findings have revealed a specific t-SNARE, (Syntaxin4/SNAP-23/Munc18c), a vesicle docking and fusion complex, as a key regulator of TNFa trafficking in macrophages. The proposed studies will further investigate t-SNARE expression and function in macrophages and in inflammation. The studies have three specific aims. First, to address how SNARE proteins are regulated in macrophages. LPS-responsive transcriptional and post-translational mechanisms will be studied at the level of gene and protein regulation using microarrays, then defining changes in protein modifications. Secondly, in vitro and in vivo approaches will be used to explore the function of Syntaxin4 as a rate-limiting 'rheostat' for TNFalpha secretion. Syntaxin4 levels will be titrated by expression of wild type or mutant cDNAs in cells and in transgenic mice. A mouse model will be used to investigate the roles of macrophages, TNFalpha and Syntaxin4 in inflammatory bowel disease.
The third aim uses a combination of techniques -fluorescence imaging in live cells, immunolabeling and proteomics - to characterize the TNFalpha transport vesicles and secretory pathways in macrophages. Taken together this work will generate important insights into the pathobiology of macrophages and TNFalpha. Defining the function of the Syntaxin4 t-SNARE in macrophages will be a major advance in understanding trafficking. Finally, trafficking proteins - in particular Syntaxin4 - offer potential as drug targets for novel anti-TNFalpha therapies.
