(A) L. pneumophila activates TLR5 through FliC. CHO cells were stably transfected with an NF-κB luciferase promoter and either a control vector (pEF6, shaded bars) or TLR5^RNF (white bars). Cells were stimulated with S. typhimurium FliC at 10 ng/ml⁻¹, S. minnesota LPS at 100 ng/ml⁻¹, or heat-killed L. pneumophila (multiplicity of infection = 250:1). Strains of L. pneumophila are as follows: LpWT, wild-type Corby strain; LpFlaA−, Corby strain FliC mutant; Boven, Bovenkarspel; Phil, Philadelphia; Knox, Knoxville. RLU, relative luciferase unit. (B and C) L. pneumophila stimulates macrophages through MyD88, but not TLR4. Macrophages were derived from mouse bone marrow and stimulated with PBS (white bars), S. minnesota LPS at 10 ng ml⁻¹ (shaded bars), or LpWT (multiplicity of infection = 100:1; black bar). After 16 h of stimulation, supernatants were assayed by ELISA for TNF-α (B) or IL-6 (C). WT, wild type. T4, TLR4^−/−. M88, MyD88^−/− mouse strains. (A–C) Assays performed in triplicate with standard deviations indicated. (A) Transfected cells were stimulated for 4 h before determining luminescence levels.

TLR5 polymorphisms and FliC signaling. (A) Sequence chromatogram indicating a polymorphism at base pair 1174. Amino acids are numbered for the respective protein depictions. The diagonal stripes at amino acid 643–660 indicate the transmembrane domain. (B) DdeI restriction digest to verify C1174T SNP. Lanes 1 and 2, genotype 1174 CC; lanes 3 and 4, genotype 1174 CT. Lanes 1 and 3, no DdeI; lanes 2 and 4, with DdeI. (C) Western immunoblot. CHO cells were transfected with control vector (lane 1, pEF6 without insert), TLR5^RNF (lane 2), TLR5^RNL (lane 3), TLR5^RSF (lane 4), or TLR5^392STOP (lane 5). Immunoblot probed with an anti-V5 antibody. (d and e) Luciferase assay in CHO (D) and HEK293 (E) cells. Cells were transfected with pEF6 control (⋄), TLR5^RNF (○), or TLR5–392^STOP (•), an NF-κB luciferase reporter, and pRL-TK as a transfection control. (F) Luciferase assay in HEK293 cells transfected with: pEF6 control (⋄), TLR5^RNF (○), TLR5^RNL (•), or TLR5^RSF (×). RLU, relative luciferase unit. Assays performed in triplicate with standard deviations indicated. (D–F) Transfected cells were stimulated for 4 h with S. typhimurium FliC at the indicated concentration before determining luminescence levels.

FliC stimulation of cytokine production. (A–E) Cells were stimulated with LPS (•), purified S. typhimurium FliC (○), heat-killed wild-type L. pneumophila (⋄), or FlaA− (FliC mutant) L. pneumophila (×). (A and B) A549 cells; (C and D) Calu-3 cells; and (E) whole blood. (F–G) PBMCs were harvested from individuals with homozygous wild-type TLR5 (392RR; n = 6) or stop codon TLR5 heterozygotes (392R*; n = 4). Cells were stimulated with FliC at 30 ng/ml⁻¹ (F) or LPS at 10 ng/ml⁻¹ (G). (A–G) Cells were stimulated for 18 h and supernatants were assayed for cytokine production by ELISA. Data are representative of experiments performed at least twice in triplicate with standard deviations indicated. (F and G) The mean level and standard deviation of IL-6 were derived from averaging the responses of different individual's cells stimulated in triplicate.