Figure 5. Inhibition of Endosomal Receptor Recycling Prevents TFAM + CpGA DNA-induced Splenocyte TNFα Release.

(A) The presented data was derived from at least 5 independent experiments. Pre-treatment (30 minutes) of Flt3L-expanded splenocytes (1 × 10⁶ cells/ml) with inhibitors of endothelin converting enzyme-1 (ECE-1) (SM-19712, 10 µM) or phosphoramidon (PPRMD, 100 µM), which impede endosomal receptor recycling, totally prohibited TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced TNFα release 24 hours post-treatment (*p < 0.01, relative to no treatment; † p < 0.01, compared to the TFAM + CpGA treatment group). (B) Representative photomicrograph of a Western blot showing degradation of TLR9 (50 µM) over time by in vitro incubation with ECE-1 (0.3 µg/ml) under optimal (6.0) and normal (7.4) pH conditions. (C) The presented data is representative of at least 3 independent experiments. Relative band expression of TLR9 was dramatically reduced at all time-points and under both assay pH conditions following co-incubation with ECE-1. As expected, evident degradation appeared more marked under optimal conditions (*p < 0.05, relative to matching TLR9 Alone treatment group; † p < 0.05, compared to matching TLR9 Alone and TLR9 + ECE-1 @ 5 min treatment groups).