Toll-Like Receptors (TLRs) are a family of transmembrane proteins found on the surface or endosomal compartment of cells in the immune system. In macrophages, TLRs mediate an immune response through recognition of molecules called pathogen-associated molecular patterns (PAMPs). TLR activation triggers a signaling cascade involving diverse pathways. We have activated TLR4 by LPS stimulation and used a genome-wide siRNA screen with a human TNF reporter system to identify several intriguing prospective regulators of TLR signaling. We present here the validation and further investigation of select genes identified by the screen.
In addition to single ligand studies, we have also begun to characterize TLR signaling responses to multiple PAMPs, a scenario that more closely mimics an in vivo infection. We show that in murine macrophages, IRAK1, a kinase recruited in TLR signaling, undergoes a sol-gel transition to form clusters by activating two families of TLRs rather than just one. We explored the possibility that the supramolecular clusters are a means for spatially organizing and regulating signaling. The IRAK1 clusters are disconnected from upstream signaling members but enriched for downstream signaling proteins and might serve as sites for dampening downstream TLR signaling. This could be a means of ensuring that immune responses are tightly controlled.
Thus, through both single and dual ligand studies described above, we will be able to further characterize TLR signaling. A better understanding of the innate immune response to specific PAMPs will undoubtedly give insight into how to modulate responses to critical diseases.
This work was supported by the Intramural Research Program of NIAID, NIH.
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