A genome-wide siRNA screen reveals NF-{kappa}B independent regulators of NOD2-induced IL-8 secretion [Signal Transduction]

August 28th, 2014 by Warner, N., Burberry, A., Pliakas, M., McDonald, C., Nunez, G.

NOD2 encodes an intracellular multi-domain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohns disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates pro-inflammatory and anti-microbial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen we identify numerous genes that regulate secretion of the pro-inflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing sub-networks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, protein ubiquitination and trafficking. A TNF╬▒ counter-screen to induce IL-8 secretion in a NOD2 independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the Ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2 induced cytokine secretion. Interestingly, several genes that affect NOD2 induced IL-8 secretion are present in loci associated with CD risk by GWAS, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8.