Figure 2: IL-17RD deficiency enhances cell responsiveness to TLRs.

(a–c) BMDMs from wild-type (WT) and Il17rd^−/− mice were stimulated with (a) Pam2Cys (100 ng ml⁻¹), Pam3Cys (100 ng ml⁻¹), Zymosan (1 μg ml⁻¹), poly(I:C) (5 μg ml⁻¹), LPS (10 ng ml⁻¹), CL097 (1 μg ml⁻¹) and CpG (5 μg ml⁻¹), (b) poly(I:C) (5 μg ml⁻¹) and LPS (10 ng ml⁻¹) for 24 h or (c) poly(I:C) (5 μg ml⁻¹) for 24 h. Conditioned media was assayed for (a) IL-6, (b) RANTES or (c) IFN-β. (d,f) WT and Il17rd^−/− BMDMs were treated with (d) LPS (10 ng ml⁻¹) or (f) poly(I:C) (5 μg ml⁻¹) for 0–240 min and cell lysates immunoblotted with indicated antibodies. (d, inset panel) Cell lysates from WT and Il17rd^−/− BMDCs were immunoblotted for IL-17RD and β-actin expression. (e) WT and Il17rd^−/− BMDMs were stimulated with LPS (10 ng ml⁻¹) for 0–120 min and nuclear extracts assayed by EMSA for NF-κB/DNA binding. Nuclear extracts (from Il17rd^−/− cells stimulated with LPS for 60 min) were also pre-incubated for 15 min with 1 μg anti-p65 antibody (ss; supershift) or IgG control (IgG). (g) WT and Il17rd^−/− BMDCs were stimulated with poly(I:C) (5 μg ml⁻¹) for 0–120 min and nuclear extracts assayed by EMSA using an oligonucleotide containing the PRD I/III domain of the Ifnb1 promoter. Nuclear extracts (from Il17rd^−/− cells stimulated with poly(I:C) for 30 min) were also pre-incubated for 15 min with an unlabelled competitor oligonucleotide. (h–j) WT and Il17rd^−/− MEFs were infected with MSCV retroviral particles containing an empty vector (EV) plasmid or wild-type mIL-17RD-Myc (Il17rd^−/− MEFs) for 72 h. Cells were treated with (h) LPS (100 ng ml⁻¹) or (i) poly(I:C) (25 μg ml⁻¹) for 0–60 min and cell lysates immunoblotted with indicated antibodies or cells were treated with (j) LPS (100 ng ml⁻¹) or poly(I:C) (25 μg ml⁻¹) for 24 h and conditioned media assayed for levels of IL-6 and IFN-β. Data are presented (a–c,j) as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test, *P<0.05; **P<0.01 or (d–i) are representative of three independent experiments. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 8. NS, not significant.