Figure 5.

Plasma and extracted plasma RNA from subjects with ARDS activates TLR8 in vitro. (A) Plasma from subjects with ARDS activates TLR8. TLR8-HEK-Blue cells were stimulated with 40 μl of plasma from mechanically ventilated subjects with no lung injury or with ARDS, and TLR8-dependent NF-κB SEAP activity was measured by a plate reader at 650 nm after 16 hours. *P = 0.014 by Mann-Whitney U test compared with subjects with no lung injury. (B) TLR8-HEK-Blue cells were stimulated with extracted plasma RNA from subjects with ARDS, and TLR8-dependent NF-κB SEAP activity was measured by plate reader at 650 nm after 16 hours. ***P = 0.002 by Mann-Whitney U test compared with empty vector. (C) Plasma from subjects with ARDS (n = 11) was aliquoted into two 20-μl samples. One 20-μl aliquot was treated with RNase I (100 μg/ml) for 30 minutes, and both aliquots were added to TLR8-HEK-Blue cells. OD values at 650 nm were recorded after 16 hours. (D) TLR8-HEK-Blue cells were transfected with R848 or miRNA mimics for miR-16, miR-92, miR-181, or miR-223, and TLR8-dependent NF-κB SEAP activity was measured by plate reader at 650 nm after 16 hours. ***Adjusted P < 0.005 by Dunnett multiple comparisons test, one-way ANOVA.