Toll–like Receptor 5 Mediates ATP Release in Response to Flagellin to Activate MAPK

Abstract

Abstract: : Purpose: To determine whether or not there is functional interdependence between Toll and nucleotide receptor signaling in host defense (innate immune) responses to bacterial flagellin. Both Toll–like receptor 5 (TLR5) and nucleotide (ATP) signaling are required for flagellin–triggered activation of host defense responses, but the issue of whether these systems operate independently or cooperatively has not been addressed. Here we show that flagellin–induced autocrine nucleotide signaling requires TLR5 and that nucleotide signaling is indispensable for the activation of MAP kinase (Erk 1/2) by flagellin. Methods: We exposed mucosal epithelial cells (corneal and bronchial) to flagellin or an agonist antibody to the flagellin receptor asialoGM1. To assess host cell responsiveness, we measured NFkB reporter activity as well as phosphorylation of IkBα and Erk 1/2, all required for flagellin–induced cytokine synthesis. We also used immunofluorescence confocal microscopy to localize asialoGM1 and TLR5 as well as immunoprecipitation Western blots to assess molecular interactions between receptors. We monitored ATP release with a luciferin–luciferase bioluminescence assay and measured Ca++ fluxes in real time using Fura–2 with METAFLUOR imaging software. In some experiments, we silenced TLR5 or its downstream adaptor Myd–88 using dominant negative mutants (DNM). Nucleotide receptor transmission was blocked with Reactive Blue 2. Results: We found that flagellin–induced autocrine nucleotide signaling was dependent on TLR5. Specifically, neither ATP release nor Ca++ mobilization occurred following flagellin exposure in the presence of DNM Myd88. Conversely, we found that flagellin–induced Erk1/2 phosphorylation was ATP–dependent while NFkB activation was ATP–independent. The intersection of TLR5 and nucleotide receptor signaling pathways appears to occur at the level of flagellin–induced ATP release, which is dependent on TLR 5 in an as yet undefined manner. The pathways are not entirely co–linear, as indicated by the requirement for nucleotide signaling in the Erk1/2, but not the IkBα/NFkB response to flagellin. Conclusions: These results provide the first information linking Toll and nucleotide receptors in innate immunity.

Keywords: cornea: epithelium • signal transduction • microbial pathogenesis: experimental studies