Toll-Like Receptor 5 Modulates the Toll-Like Receptor 4-Dependent Innate Immune Response

1. Toll-Like Receptor 5Modulates the Toll-Like Receptor 4-Dependent Innate Immune Response Amy K. Dickey MD MSc, T. Eoin West MD, MPH Department of Medicine, University of Washington, and Harborview Medical Center, Seattle, WA, USA Results Background 3. TLR51174C>Tco-transfection decreases TLR4-dependent IL-8 production as compared to TLR51174C • Toll-like receptors (TLRs) are receptors that recognize conserved pathogen motifs and trigger innate immune activation. • TLR4 and TLR5 are located on the plasma membrane. Bacterial lipopolysaccharide (endotoxin) is a TLR4 agonist; bacterial flagellin is a TLR5 agonist. • TLR triggering induces transcription factor NF-κB activation and production of inflammatory cytokines such as IL-8. • A common TLR5 polymorphism, TLR51174C>T, results in a truncated TLR5 protein that does not signal in response to flagellin. • Individuals carrying TLR51174C>T are protected against death after infection with the Gram-negative, flagellated bacteria Burkholderia pseudomallei (melioidosis) • LPS stimulation of whole blood from carriers of TLR51174C>Tresults in decreased cytokine production as compared to homozygotes for TLR51174C. • Hypothesis: TLR5 modulates the TLR4-dependent innate immune response. 1. TLR5 co-transfection decreases TLR4-dependent NF-κB activation 2. TLR51174C>Tco-transfection increases TLR4-dependent NF-κB activation as compared to TLR51174C Flagellin LPS Flagellin TLR5 TLR4 TLR51174C>T ? ? NF-κB NF-κB Conclusions Inflammatory Cytokines • TLR5 modulates TLR4-dependent innate immune activation in vitro. • A hypofunctional TLR5 variant enhances TLR4-dependent NF-κB activation but suppresses TLR4-dependent IL-8 expression, suggesting a role for non-NF-κB pathways of inflammatory cytokine production. • Improved survival of melioidosis patients carrying TLR51174C>Tmay be independent of flagellin-mediated signalling. Methods HEK293 cells were transiently transfected with either control vectors, TLR4, TLR51174C (wildtype) or TLR51174C>T (variant), along with CD14, MD2, and a NF-κB reporter construct. Following transfection, cells were incubated with Salmonella typhimuriumflagellin (Flg), Escherichia coli LPS (LPS), heat-killed B. pseudomallei(BP), BP lacking flagellin(BPΔFliC), or media alone for 6 hours. NF-κB promoter activation was determined by luciferase assay and reported in relative light units (RLUs). IL-8 expression was measured in cell supernatants by ELISA. Statistical analysis was performed using the student’s t-test. *p<0.05, **p<0.005. Funding for this project was provided by R01 HL113382. Contact adickey@uw.edu for more information