Reconstitution of human TLR9 yields CpG-DNA responsiveness, which is independent of MD2 and CD14. The 293 cells were transiently transfected with hTLR9 or hTLR4/hMD2 and a 6-fold NF-κB luciferase reporter plasmid (A) or with hTLR9 or hTLR4/hMD2 alone (B). After stimulation with 2 μM CpG-ODN (2006), 2 μM GpC-ODN (2006-GC), 100 ng/ml LPS or medium, NF-κB activation (A) or IL-8 production (B) was monitored. (C) The 293 cells were transiently transfected with hTLR9 or hTLR4 alone (white bars) or cotransfected with hMD2 (black bars), hCD14 (hatched bars), or hMD2 and hCD14 (gray bars). Transfected cells were stimulated with concentrations of CpG-ODN or LPS as indicated, and IL-8 production was measured by ELISA (n = 3, mean ± SD). Results are representative of at least two independent experiments.

TLR9 confers species-specific CpG motif signaling. PBMC (A) or murine splenocytes (B) were stimulated with ODN 2006 (●), ODN 2006-GC (○), ODN 1668 (▾), or ODN 1668-GC (▿) at the indicated concentrations, and IL-12 production was monitored by ELISA (n = 2, mean ± SD). The 293 cells stably transfected with a 6-fold NF-κB luciferase reporter plasmid and hTLR9 (293-hTLR9-luc) or mTLR9 (293-mTLR9-luc) (C) or with hTLR9 (293-hTLR9) or mTLR9 (293-mTLR9) alone (D) were stimulated with ODN 2006 (●), ODN 2006-GC (○), ODN 1668 (▾), or ODN 1668-GC (▿) at the indicated concentrations, and NF-κB activation or IL-8 production were measured after 12 and 48 h, respectively (n = 2, mean ± SD). Results are representative of at least two independent experiments. (E) cDNA was prepared from 293 cells (lane 1), 293-mTLR9 cells (lane 2), and 293-hTLR9 cells (lane 3), and RT-PCR for mTLR9, hTLR9, and GAPDH was performed.

Human TLR9 expression correlates with CpG-DNA responsiveness. (A) Purified human B cells, CD123⁺ dendritic cells (DC), or monocyte-derived dendritic cells (MDDC) were stimulated with 50 μg/ml E. coli DNA, 50 μg/ml DNase I-digested E. coli DNA, 2 μM CpG-ODN (2006), 2 μM GpC-ODN (2006-GC), or 100 ng/ml LPS. B cell proliferation was monitored at day 2 by [H³]thymidine uptake (n = 4, mean ± SD). IL-8 concentration was determined by ELISA (n = 2, mean ± SD; ND = not detected). (B) cDNA was prepared from MDDC (lane 1), purified CD14⁺ monocytes (lane 2), B cells (lane 3), CD123⁺ DC (lane 4), CD4⁺ T cells (lane 5), and CD8⁺ T cells (lane 6). cDNA amounts were normalized based on the GAPDH amount determined by TaqMAN-PCR. RT-PCR was performed for 30 cycles on normalized cDNA diluted 1:5 for human TLR2, TLR4, and TLR9 and diluted 1:125 for GAPDH. Results are representative of three independent experiments.

Stable Reconstitution with human TLR9 recapitulates CpG-DNA mechanisms of action. The 293 cells stably transfected with hTLR9 and a 6-fold NF-κB luciferase reporter plasmid (293-hTLR9-luc) were stimulated with 2 μM CpG-ODN (2006), 2 μM Me-CpG-ODN (methylated 2006), 2 μM GpC-ODN (2006-GC), 100 ng/ml LPS, or medium (A) or with E. coli DNA (black bars) or E.coli DNA digested with DNase I (gray bars) at concentrations indicated (B). After 12 h, NF-κB activation was measured (n = 2, mean ± SD). (C) The 293-hTLR9-luc cells were preincubated with 10 nM Bafilomycin A (gray bars) or DMSO control (black bars) for 30 min and stimulated with 0.5 μM CpG-ODN (2006), 10 ng/ml IL-1α, or 10 ng/ml TNF-α. NF-κB activation was monitored after 12 h and is presented as % yield (fold NF-κB activation with Bafilomycin A treatment/fold NF-κB activation with DMSO control × 100) (n = 2, mean ± SD). (D) 293-hTLR9-luc cells were incubated with 0.5 μM CpG-ODN (2006) (black bars) or 10 ng/ml TNF-α (gray bars) and increasing concentrations of a blocking ODN (see sequence in Materials and Methods) as indicated. NF-κB activation was monitored after 12 h and is presented as % yield (fold NF-κB activation with blocking ODN/fold NF-κB activation without blocking ODN × 100) (n = 2, mean ± SD). (E) The 293 cells stably transfected with hTLR9 (293-hTLR9) were cotransfected with a 6-fold NF-κB luciferase reporter plasmid and increasing concentrations of dominant negative human MyD88 expression vector at concentrations indicated. Cells were not stimulated (●) or stimulated with 2 μM CpG-ODN (2006) (○) or 10 ng/ml TNF-α (▾), and NF-κB activation was monitored after 12 h (n = 2, mean ± SD). Results are representative of at least two independent experiments.