Figure 3.

Decrease in TLR2 signaling after TLR/NLRP3 activation or S.aureus infection is reversed by caspase/NLRP3 inhibition. PMA-primed THP-1 macrophages were stimulated with Pam3CSK4 (0.5 µg/ml) for 4 h and NLPR3 agonists for 2 h. Inhibitors were added 2 h before the inflammasome agonist (A–C). S. aureus was added 2 h after inhibitors, at the same time as NLRP3 activators and supernatants were sampled 6 h later (D). Supernatants were collected and levels of cytokines determined using ELISA( ${\left(1+x\right)}^n=1+\frac{nx}{1!}+\frac{n\left(n-1\right)x^2}{2!}+\cdots$ ). PMA-primed THP-1 macrophages were stimulated with LPS (10 ng/ml) for 4 h and NLRP3 agonists for 2 h. Cell lysates were used for MyD88 detection (E), while cell supernatants were used for active caspase-1 detection. PMA-primed THP-1 macrophages were stimulated with LPS (10 ng/ml) for 4 h and NLRP3 agonists for 2 h and then stimulated again with 10 ng/ml of LPS for 1.5 h (F-H). Supernatants were collected and levels of cytokines determined using ELISA. Data are represented as mean ± SD of at least 3 replicates (A–D, F–H). Experiments were repeated at least three times with similar results. One-tailed unpaired t-test was used for statistical analysis, **p < 0.01, ***p < 0.005.