Development of Inhibitors of Supressors of Innate Immune Responses

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The discovery of Toll-like receptors (TLRs)in mammalians cells has revolutionized the understanding of innate immunity. These molecules constitute pattern recognition membrane molecules that recognize pathogenic products, including LPS, flagellin, proteoglycan, lipoteichoic acid, mycobacterium LAM, double stranded RNA, and bacterial CpG. The range of products derived from gram positive and negative bacteria, mycobacterium, fungi and viral products all activate the TLR pathways. As a consequence of TLR activation, cytokines that can activate adaptive immunity are also produced, thus they serve as a bridge between innate and adaptive immunity. Despite the pivotal role of these receptors, their involvement in defense against select agent is less well defined. In this proposal, we plan to directly examine the roles of TLRs and its downstream regulators in defense against a select agent, Yersinia pestis. We will focus on known TLRs and their downstream regulators as well as a new downstream regulator that we recently identified, the Monarch-1 protein. Monarch-1 downregulates TLR responses, including NF-.B/AP-1 activation, and TNF-. response. Thus its constitutes a novel suppressor of the TLR response that is likely to affect mmunity against r_atural infection and vaccination. Accordingly, the Aims are (1) To produce cell ines lacking known and new molecules in the TLR pathway by the use of interference RNA (RNAi :echnology). We plan to generate monocytic U937 cells that are defective in Monarch-1 as well as selected TLRs and their downstream signals including TRAF2, IRAKs, MyD88, and Md-2. (2) To determine if the removal of the genes targeted in Aim 1 causes alterations in responses to cellular products from Y. pestis. (3) To further understand how Monarch-1 inhibits inflammatory responses by determining components of the Monarch-1 protein complex.