Mechanisms of regulation in the interferon factor 3 (IRF- 3) pathway

Interferon regulatory factor 3 (IRF-3) plays a critical role in the host cell response to both bacterial and viral infection. IRF-3 is activated by Toll-like receptors (TLRs) and cytoplasmic nucleic acid sensors, and serves to upregulate interferon beta and interferon stimulated genes (ISGs), thereby providing a quick and effective response to infection. In this work, two novel mechanisms of regulation in the IRF-3 pathway are revealed. The first part of this thesis work shows that upon binding to lipopolysaccharide, TLR-4 signaling activates phospholipase C gamma 2 (PLCg2), which cleaves phosphatidylinositol (4,5)-bisphosphate (PIP₂) into Inositol 1,4,5-triphosphate (IP₃) and regulates IP₃R activity. Inositol 1,4,5-triphosphate receptors (IP₃Rs) mediate the release of calcium from the endoplasmic reticulum into the cytosol. The hypothesis presented here is that, upon calcium release, the calcium-dependent phosphatase calcineurin becomes activated and activates dynamin-dependent endocytosis of the LPS-TLR4 complex. Signaling occurs from the early endosomes via the TRAM- TRIF pathway, and induces the IRF3-dependent gene response. Our results suggest a novel means by which the IRF3 pathway is regulated. The second part of this thesis work describes the Schlafen (Slfn) gene family and its complex role in the immune response. The family is comprised of nine murine members, which are divided into short (Slfn 1 and 2), medium (Slfn 3 and 4) and long (Slfn 5, 8, 9, 10,14) subtypes. The short subtype member, Slfn2, is a cytoplasmic protein that binds to foreign nucleic acids and inhibits protein translation, putatively as a host defense strategy. However, short and medium subtypes, Slfn2 and Slfn3, appear to inhibit Interferon stimulated gene (ISG) production via the IRF3 pathway, thus indicating a role in attenuating the immune response. Moreover, Slfn8, a long subtype member, does not appear to have any effect on ISG induction, indicating divergent roles of different individual Slfn family members. We propose that the Slfn family is differentially regulated during a response to infection, thus initially mediating cellular defense and, later, downregulation of response through the varying participation of individual Slfn proteins within a comple