Figure 5.

Multiple lysosomal proteases are required for TLR9 cleavage. (a) BMDCs from wild-type mice or mice lacking cathepsin L (Cat L-KO), cathepsin S (Cat S-KO) or cathepsin K (Cat K-KO) were stimulated with poly I:C (100 µg/ml), LPS (1 µg/ml), Imiquimod (10 µg/ml) or CpG DNA (1 µM) for 4 h in the presence of brefeldin A at day 6 of BMDC culture. Cells were fixed and stained with anti-TNF, and intracellular TNF was measured by flow cytometry. (b) Top, RAW macrophages expressing TLR9-Myc were pretreated for 12 h with DMSO, z-FA-FMK (10 µM) or the selective cathepsin L inhibitors Clik195 and Clik148 (Cat Li, 10 µM), the cathepsin S inhibitor LHVS (Cat Si, 10 nM), the cathepsin K inhibitor II (Cat Ki, 1 µM), or combinations thereof. Cells were metabolically labeled for 1.5 h followed by a 5 h chase period. TLR9 was immunoprecipitated and re-immunoprecipitated from lysates with anti-Myc, and was digested with EndoF. Bottom, RAW macrophages were treated for 12 h with the inhibitors listed and stimulated for 2 h with the indicated TLR agonists. Secreted TNF was analysed by ELISA. (c) BMDCs from Cat L-KO mice were retrovirally transduced with vectors encoding GFP-tagged wild-type TLR9 or GFP-tagged C-terminal TLR9 fragment (471–1032) or were left untransduced (−) at day 1 of BMDC culture. At day 6, cells were stimulated with CpG (1µM) for 4 h in the presence of brefeldin A, fixed and stained with anti-TNF. TNF was measured by flow cytometry in GFP+ (transduced) cells. Data are representative of two (a,c) or three (b) independent experiments (a-c; average, s.d.).