Toll-like receptors (TLRs) are principal actors in innate immunity. Their detection of exogenous danger signals from pathogens as well as endogenous injury signals from host tissues initiates homeostatic inflammatory responses involving multiple cell types and the concomitant expression of many soluble mediators. Although these TLR mediated responses have obviously been positively selected by evolution, at times they have undesirable and even lethal side effects. Septic shock has long been recognized as a potentially lethal hyper response to infection. More recently, sterile injury such as renal ischemia reperfusion injury, systemic autoimmunity, experimental autoimmune encephalomyelitis, and cardiovascular disease have all been shown to have significant TLR involvement in that their severity is lessened by deficiency of one or more TLR. Intracellular signaling by most TLRs requires their association with MyD88. We have developed an assay which detects the association of TLRs with MyD88. The assay is based on the ability of ?-lactamase (Bla) to be split into two inactive fragments. When these two fragments are brought into juxtaposition they will complement with each other to reform the active enzyme. This reassociation is readily detected through the use of a fluorescent Bla substrate. Thus in the assay we have already developed, TLR4 and MyD88 are expressed in HeLa cells as chimeras with fragments of Bla. When the TLR4 and MyD88 interact productively Bla activity is readily observed. A stable cell line expressing these chimeras of TLR4 and MyD88 was useful in high throughput screening of a 16,000 compound library in that it identified 5 compounds that were found to inhibit TLR4 - MyD88 association. Through this proposal we hope to expand our approach to identify inhibitors of signaling initiated by additional TLRs. Ultimately we hope that this work will lead to the development of drugs useful for therapeutic application as well as research. There should be no doubt that such drugs are needed. We propose three specific aims for this work: 1) To develop stable cell lines in which a MyD88 chimera associates with a chimera of TLR2 or TLR9 and leads to active Bla. 2) To demonstrate that the stable cell lines are useful for high throughput screening by screening a 16,000 compound subset of the NIH MLSCN library. 3) To characterize the compounds so identified as to their method of action, to define their efficacy in vitro, and to begin to characterize their efficacy in vivo.
Inflammation, whether it is in response to infection or in response to injury, can be deleterious and in some cases even life threatening even though it is an essential part of the healing response. The goal of this application is to develop techniques for finding new inhibitors of inflammation. ? ? ?
