This proposal will map expression of Toll-like receptor 7 (TLR7) in the airway and identify the mechanism of novel rapid TLR7-mediated bronchorelaxation. TLR7 is vital to human pulmonary immune responses in that TLR7 detects single-stranded RNA, common to many respiratory virus genomes, and triggers transcription of antiviral interferons, and inflammatory cytokines via NF-?B. My initial data suggest TLR7 also signals through a transcription-independent pathway, to induce nitric oxide production and cause bronchorelaxation within seconds of activation. This novel mechanism has not been described for any other TLR. Furthermore, preliminary data indicate TLR7 is expressed on airway nerves, but not smooth muscle, despite TLR7's profound relaxant effect on contracted airway tissues. The central hypothesis is that TLR7 is located on airway nerves and relaxes airway smooth muscle through production of nitric oxide by a non-classical TLR signaling pathway. The current work will define TLR7 expression in human and murine airways using immunofluorescence staining of tissue whole mount and primary cell culture preparations. TLR7 expression will be localized to specific airway nerve subtypes, and colocalized with nitric oxide synthase isozymes using high-resolution laser-scanning confocal microscopy. TLR7's rapid bronchorelaxant mechanism will be characterized using pharmacologic antagonists of potential TLR7 signal transduction pathways in wild-type and knockout murine airway tissues, and in deceased donor human airway tissues in organ bath experiments in vitro. In vitro findings will be confirmed in primary cell culture experiments using nitric oxide- and intracellula calcium-sensing fluorescent probes. This proposal's findings have significant potential to expand our understanding of TLR7's signal transduction and function in the airways, and may provide novel therapeutic targets for human airway diseases. These experiments are one component of the principle investigator's K08 career development plan that demonstrates a logical scientific progression towards research independence with the ultimate goal of obtaining R01 level funding by the end of the award period. A detailed plan for hands on scientific skill development and obtaining proficiency as a mentor are also included in this proposal. Lending additional evidence to the feasibility of this proposal is the supportive research environment at Oregon Health and Science University. The principle investigator's research progress and career development will be overseen by a select group of senior investigators, and supported by the Oregon Clinical and Translational Research Institute Scholar's Program for early career investigators.
Toll-like receptor 7 (TLR7) detects respiratory viruses in the airway and triggers immune responses. This proposal will characterize a new TLR7 mechanism that causes rapid airway relaxation in humans. This work has potential to expand our understanding of TLR7's role in lung physiology, and may provide new bronchodilatory therapeutic targets for human airway diseases.
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