Examining the Role of ECSIT in Innate Immune Responses

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) was identified a number of years ago as a TNF receptor associated factor 6 (TRAF6) interacting partner and was suggested to participate in the NF-kappaB signaling pathway. Subsequent generation of knockout animals revealed that ECSIT deficiency results in early embryonic lethality, thus complicating the characterization of ECSIT function in vivo. In this thesis, I present data demonstrating that ECSIT localizes predominately to mitochondrial membranes, and functions to regulate reactive oxygen species (ROS) production from the mitochondrial respiratory chain. Although antimicrobial ROS production by the phagosomal NADPH oxidase is well established, the contribution of mitochondrial-derived ROS (mROS) in the innate immune response and their interplay with phagosomal ROS remains unclear. Here I show that engagement of a subset of Toll-like receptors (TLRs 1, 2 and 4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of TRAF6 to mitochondria where it engages and ubiquitinates ECSIT, a component of mitochondrial Complex I. Interaction with TRAF6 leads to re-localization of ECSIT to the mitochondrial periphery and results in an increase in both mitochondrial and cellular ROS. Consequently, ECSIT and TRAF6 depleted macrophages exhibit decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Furthermore, mitochondrial-targeted catalase expression results in significantly reduced ROS production and defective bacterial killing, thereby supporting the role of mROS in bactericidal activity.;In addition to demonstrating a role for mROS in antibacterial immunity, I also show that ECSIT-regulated mROS contributes to immune signaling and the stabilization of hypoxia inducible factor-1 alpha (HIF-1alpha) upon TLR ligation. Macrophages and tissues from ECSIT deficient mice display reduced HIF-1alpha levels after TLR1/2/4 ligation, and consequently, ECSIT heterozygous mice are less susceptible to LPS-induced septic shock, a phenotype resembling that of mice lacking HIF-1alpha in macrophages and neutrophils. 'Thus, TRAF6-ECSIT signaling represents a novel route to ROS production utilized by the innate immune system for robust antibacterial responses and ROS-dependent inflammatory signaling.