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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Azithromycin Decreases NALP3 Protein in THP-1 Monocytes. a THP-1 cells were treated with azithromycin at concentrations shown for 12 h overnight prior to immunoblot analysis for indicated proteins. b THP-1 cells were plated and rested overnight. Cells were treated with LPS at concentrations shown for 4 h prior to immunoblot analysis. c THP-1 cells were treated with azithromycin at concentrations shown for 12 h overnight and then treated with LPS for 4 h prior to immunoblot analysis. The data representative of 2–3 separate experiments. d Peripheral blood mononuclear cells were treated with azithromycin or clarithromycin at concentrations shown for 12 h overnight. Plate-adherent monocytes were then treated with LPS 500 ng/ml for 4 h prior to lysis and immunoblot analysis for indicated proteins. Data shown is representative of multiple experiments. e NALP3:actin densitometry of immunoblot shown in Figure D

Fig. 2

Kinetics of Azithromycin Effects on NALP3 Protein in THP-1 Monocytes. a THP-1 cells were treated with azithromycin 50ug/ml for the times shown prior to immunoblot analysis for indicated proteins. b THP-1 cells were treated with LPS 500 ng/ml for times shown prior to analysis. c THP-1 cells were treated with azithromycin (50ug/ml) and LPS (500 ng/ml) for times shown prior to immunoblot analysis. The data representative of 2–3 separate experiments

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