Azithromycin decreases NALP3 mRNA stability in monocytes to limit inflammasome-dependent inflammation

Elizabeth A. Lendermon, Tiffany A. Coon, Joseph S. Bednash, Nathaniel M. Weathington, John F. McDyer, Rama K. Mallampalli

Azithromycin, an antibiotic used for multiple infectious disorders, exhibits anti-inflammatory effects, but the molecular basis for this activity is not well characterized. Azithromycin inhibits IL-1β-mediated inflammation that is dependent, in part, on inflammasome activity. Here, we investigated the effects of azithromycin on the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) protein, which is the sensing component of the NALP3 inflammasome, in human monocytes. THP-1 cells were treated with azithromycin alone, LPS alone, or both. NALP3 and IL-1β protein levels were determined by immunoblotting. NLRP3 gene (encoding NALP3) transcript levels were determined by quantitative qPCR. In order to measure NLRP3 transcript decay, actinomycin D was used to impair gene transcription. THP-1 Lucia cells which contain an NF-κB responsive luciferase element were used to assess NF-κB activity in response to azithromycin, LPS, and azithromycin/LPS by measuring luminescence. To confirm azithromycin's effects on NLRP3 mRNA and promoter activity conclusively, HEK cells were lipofected with luciferase reporter constructs harboring either the 5' untranslated region (UTR) of the NLRP3 gene which included the promoter, the 3' UTR of the gene, or an empty plasmid prior to treatment with azithromycin and/or LPS, and luminescence was measured. Azithromycin decreased IL-1β levels and reduced NALP3 protein levels in LPS-stimulated THP-1 monocytes through a mechanism involving decreased mRNA stability of the NALP3 - coding NLRP3 gene transcript as well as by decreasing NF-κB activity. Azithromycin accelerated NLRP3 transcript decay confirmed by mRNA stability and 3'UTR luciferase reporter assays, and yet the antibiotic had no effect on NLRP3 promoter activity in cells containing a 5' UTR reporter. These studies provide a unique mechanism whereby azithromycin exerts immunomodulatory actions in monocytes by destabilizing mRNA levels for a key inflammasome component, NALP3, leading to decreased IL-1β-mediated inflammation.