Fig. 3

Azithromycin Decreases NLRP3 mRNA Stability and NF-kB Activity. a THP-1 cells were treated with azithromycin (50ug/ml), LPS (500 ng/ml), or both for the times shown prior to mRNA isolation and qPCR for relative NLRP3 mRNA measurement. n = 3. F (2,15) = 6.571, p = 0.009. b THP-1 cells were treated with azithromycin (50 ng/ml) for 6 h prior to adding actinomycin D (5ug/ml) to impair gene transcription. mRNA fold change was determined using qPCR. Percent mRNA remaining was calculated using the equation: (1 – ddCq) × 100. c THP-1 cells were treated with azithromycin (50 ng/ml) for 6 h and LPS (500 ng/ml) for 4 h prior to adding actinomycin D (5ug/ml). mRNA was quantified using qPCR. d HEK cells were lipofected with either plasmids containing an empty vector, plasmids containing a NLRP3 promoter region, or plasmids containing the 3’UTR of the NLRP3 overnight. All plasmids were linked to luciferase reporter constructs. Cells were then treated with azithromycin at concentrations shown for 1 h prior to addition of LPS (1ug/ml) for 4 h (untreated and LPS alone served as controls), and luminometry was used to measure luciferase activity. Data is representative of multiple experiments. A significant effect of azithromycin on luminescence in the cells transfected with 3’UTR was found, F (3,28) = 23.52, p < 0.0001. Dunett’s multiple comparisons test revealed a significant difference between 3’UTR luminescence in LPS-stimulated cells treated with no azithromycin and LPS-stimulated cells treated with azithromycin 100 μg/ml and 200 μg/ml (p = 0.0077 and p = 0.0001). e THP-1 Lucia cells (with NF-kB responsive luciferase construct) were treated with LPS alone at concentrations shown or LPS and azithromycin 50ug/ml for 12 h. NF-kB activity was quantitated by measuring luminescence. n = 3. A significant effect of azithromycin on luminescence was found, F (1,16) = 335.8, p < 0.0001. Bonferroni’s multiple comparison’s test also revealed significant p values for this azithromycin effect with all doses of LPS (p = 0.0002 with 0 ng/ml LPS, p < 0.0001 with 50 ng/ml LPS, p < 0.0001 with 200 ng/ml LPS, p < 0.0001 with 500 ng/ml LPS). There was also a significant effect of LPS on luminescence, F (3, 16), p = 0.0011