Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.
BACKGROUND
The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.


RESULTS
We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.


CONCLUSIONS
Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.
Cho
John S
JS
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA. johncho@ucla.edu
Kim
Yun C
YC
Morrison
Sherie L
SL
D000818:Animals; D016207:Cytokines / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D007249:Inflammation; D051379:Mice; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C495342:TLR2 protein, human; C495344:TLR3 protein, human; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha