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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Azithromycin decreases NALP3 mRNA stability in monocytes to limit inflammasome-dependent inflammation
Elizabeth A Lendermon1, Tiffany A Coon1, Joseph S Bednash1,2
1Pulmonary, Allergy, & Critical Care Medicine, Department of Medicine, University of Pittsburgh, UPMC Montefiore, NW 628, Pittsburgh, PA, 15213, USA.
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.
Insights
Azithromycin reduces inflammation by decreasing levels of NALP3 inflammasome components in monocytes. This antibiotic works by destabilizing NLRP3 mRNA, thereby lowering IL-1β production and mediating its anti-inflammatory effects.
Area of Science:
- Immunology
- Pharmacology
Background:
- Azithromycin, an antibiotic, has known anti-inflammatory effects, but the underlying molecular mechanisms are not fully understood.
- Inflammasome activity, particularly IL-1β-mediated inflammation, is a key target of azithromycin's anti-inflammatory action.
- This study focuses on the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome in human monocytes.
Purpose of the Study:
- To investigate the precise molecular mechanisms by which azithromycin modulates NALP3 inflammasome activity in human monocytes.
- To elucidate how azithromycin affects NALP3 protein and NLRP3 gene expression.
- To determine the impact of azithromycin on NF-κB signaling pathway.
Main Methods:
- THP-1 monocytes were treated with azithromycin and LPS to assess NALP3 and IL-1β protein levels via immunoblotting.
- Quantitative PCR (qPCR) and mRNA stability assays (using actinomycin D) were employed to analyze NLRP3 gene transcript levels and decay.
- Luciferase reporter assays in THP-1 Lucia and HEK cells were used to evaluate NF-κB activity and the effect of azithromycin on NLRP3 promoter and UTR activity.
Main Results:
- Azithromycin significantly reduced IL-1β and NALP3 protein levels in LPS-stimulated monocytes.
- The antibiotic was found to decrease NLRP3 mRNA stability, accelerating transcript decay, and also reduced NF-κB activity.
- Reporter assays confirmed that azithromycin affects 3' UTR activity but not the NLRP3 promoter activity.
Conclusions:
- Azithromycin exerts immunomodulatory effects in monocytes by destabilizing NLRP3 mRNA, a key inflammasome component.
- This mRNA destabilization leads to reduced NALP3 protein levels and subsequently decreased IL-1β-mediated inflammation.
- The findings reveal a novel mechanism for azithromycin's anti-inflammatory action involving inflammasome regulation at the mRNA level.
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