Using the germline mutagen N-ethyl-N-nitrosourea (ENU), we induced and then positionally cloned a mutation called Lps2. This mutation revealed that the mammalian LPS sensing pathway has two branches, one of which also forms the basis of an important viral sensing pathway. In brief, the dsRNA receptor (TLR3) and the LPS receptor (TLR4) utilize a common TIR adapter molecule, disrupted by the Lps2 mutation. Also known as Trif, or as Ticam-1, this adapter is essential for all MyD88-independentsignaling from both receptors, but does not transduce signals from the other TLRs. In vivo, the Trif Lps2/Lps2 genotype dramatically diminishes sensitivity to LPS, and increases susceptibility to infection by mCMV (and most probably many other viruses) by ablating the type I interferon response. We have shown that Trif, rather than MyD88, is of key importance in dendritic cell maturation, and by implication, in activation of the adaptive immune response as it occurs in the context of microbial infection. Moreover, we have ascertained the existence of Trif-dependent and Trif-independent macrophages, as well as poly I:C-responsive and poly I:C-unresponsive macrophages, and suggest that based on these phenotypic characteristics, macrophages must display afferent functional specificity. Our decipherment of TLR4 signaling pathways has thus raised many new questions to replace those that it has answered. We now propose to complete our phenotypic characterization of Lps2, to examine the functional specificity of macrophages with respect to signal transduction through the Trif axis, and also, to implement broadened phenotypic screens that will detect still other genes that influence responses to LPS and mCMV. The new mutations that are found--and those that have already been found in preliminary efforts--will be isolated by positional cloning.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060031-09
Application #
7210572
Study Section
Special Emphasis Panel (ZRG1-SSS-F (01))
Program Officer
Dunsmore, Sarah
Project Start
2000-03-01
Project End
2009-03-31
Budget Start
2007-04-01
Budget End
2009-03-31
Support Year
9
Fiscal Year
2007
Total Cost
$659,277
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Hoebe, Kasper; Du, Xin; Goode, Jason et al. (2003) Lps2: a new locus required for responses to lipopolysaccharide, revealed by germline mutagenesis and phenotypic screening. J Endotoxin Res 9:250-5

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