Conservative estimates hold that in the United States alone, more than 20,000 people die each year as a result of septic shock brought on by Gram-negative infection. The lethality is linked to the biological effects of the bacterial cell wall component lipopolysaccharide (LPS), which prompts the production and release of proinflammatory cytokines such as TNF-alpha, and IL-1. LPS binds to the Toll-like receptors (TLRs), a receptor family which also includes the IL-l receptor and is part of the evolutionary conserved innate immune system. We have identified a novel signaling pathway that is initiated by LPS or IL-1, and leads through the activation of Interferon Regulatory Factor 3 (IRF-3) to the induction of Interferon Stimulated Genes (ISGs). We had previously found this pathway to be activated upon viral infection, indicating that IRF-3 plays a crucial role in the host defense against either viral or bacterial pathogens. Furthermore, IRF-3 activation by IL-1 suggests that this signaling cascade might also contribute to inflammatory processes such as rheumatoid arthritis. The three aims of Part I are focused on identifying the activation mechanism of IRF-3.
In Aim 1 we will identify the phosphorylation sites in IRF-3 after LPS or IL-I stimulation, In Aim 2 we will generate phosphospecific antisera against IRF-3.
Aim 3 proposes experiments to isolate and identify the kinase(s), which phosphorylates IRF-3 in response to LPS or IL-1. Part II of the proposal contains two aims that investigate the biological role of IRF-3.
In Aim 4 we will examine the crosstalk between IL-1- and LPS-induced activation of IRF-3 and ISGs. We will further explore whether IL-l and/or LPS treatment interfere with viral infection.
In Aim 5 we propose to generate transgenic mice that express a dominant-negative mutant of IRF-3 either ubiquitously or in a myeloid restricted manner. We will investigate the susceptibility of these animals to septic shock and collagen-induced arthritis as well as viral and bacterial infection. Results from these proposed studies will not only facilitate our understanding of the mechanism of IRF-3 activation, but will also shed light on the role of IRF-3 in physiological and pathological processes.
