Alantolactone prevents double-stranded DNA induced inflammasome activation in human immune cells

Abstract Introduction The AIM2 inflammasome, a cytosolic sensor of double-stranded DNA (dsDNA), plays a crucial role in innate immunity and is implicated in cardiovascular diseases such as atherosclerosis. Identifying inhibitors specific to the inflammasome may offer novel therapeutic strategies. Methods Over 6,000 drugs and drug-like compounds from the Spectrum and Selleckchem Bioactive compound libraries were screened for inflammasome modulation by measuring changes in propidium iodide fluorescence in response to inflammasome activation. The screen identified alantolactone, a naturally occurring sesquiterpene lactone. For validation, THP-1 cells were primed overnight with 100 ng/ml lipopolysaccharide (LPS) and treated with 100 nM alantolactone or the equivalent volume of solvent (DMSO). The following day, cells were transfected with 1 µg/ml polydA:dT using Lipofectamine 2000 and incubated for 4 hours to activate the inflammasome. Effects on cell proliferation, viability, and chemosensitivity were measured by a WST-1 assay. IL-1β concentrations were quantified in cell supernatants by ELISA. NFκB-activity was assessed by immunoblotting to determine the ratio of p-IκBα to IκBα. Binding to AIM2 was tested using a DARTS assay and Co-Immunoprecipitation of AIM2 and ASC. Results Alantolactone (100 nM) reduced IL-1β release in response to nigericin in THP-1 cells (276.9 vs. 48.7 pg/ml, p0.05). IL-1β release in response to double-stranded DNA (polydA:dT) was also reduced (69.8 vs. 16.5 pg/ml). IL-1β release induced by Bacillus anthracis lethal factor (NeedleTox) remained unchanged. The inhibitory effect of alantolactone on polydA:dT-induced IL-1β release reached a maximum at 1 μM compared to solvent control (58.2 vs. 4.8pg/ml, p0.05, Fig. A). At this concentration, alantolactone did not impair cell viability measured by WST-1 assay (Fig. B). Phosphorylation of IκBα quantitated by Western Blot remained unchanged after treatment with 100 nM alantolactone. Mechanistically, alantolactone does not bind directly to AIM2 which was observed using DARTS assay and Co-Immunoprecipitation of AIM2 and ASC. Ex vivo, interleukin release in peripheral blood mononuclear cells (PBMCs) was significantly reduced without relevant toxicity at the respective concentrations (-76.1 %, p0.05, Fig. C). Conclusion Alantolactone inhibits inflammasome activity in response to double-stranded DNA in THP-1- and peripheral blood mononuclear cells in an NF-κB-independent manner. Figure: A: Concentration-response curve of LPS and polydA:dT transfected PBMCs depicting IL-1β release. B: Cell viability analyzed via WST-1 assay. C: IL-1β concentration in the supernatant of LPS-primed and polydA:dT-transfected PBMCs. Cells were treated as indicated with 100 nM Alantolactone, 10 µM MCC950, or solvent control prior to LPS-priming. Data are presented as mean + SD. Data were analyzed using Student’s t-test. *p0.05.