https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Leipzig Klinik und Poliklinik für Kardiologie Leipzig, Deutschland; 2Universität Leipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie Leipzig, Deutschland
Introduction: The inflammasome family plays a pivotal role in the innate immune system by detecting molecules of viral or microbial origin. Pathogen-associated molecular patterns (PAMPs) and host-derived danger-associated molecular patterns (DAMPs) target different members of the inflammasome superfamily. The AIM2 inflammasome differs from other inflammasomes by its location in the cytosol, where it acts as a double-stranded DNA (dsDNA) sensor. DNA damage response contributes to vascular senescence and atherosclerosis
Materials and Methods: To identify novel inflammasome modulators over 6000 drugs or drug-like compounds were evaluated by measuring the change in propidium iodide fluorescence in response to treatment with lipopolysaccharides and nigericin. In general, cells were primed overnight with 100 ng ml-1 lipopolysaccharides (LPS) and 100 nM alantolactone or the equivalent volume of the solvent control (DMSO). 1 µg ml-1 polydA:dT was transfected using Lipofectamine 2000 the following day and incubated for 4 h to induce AIM2 inflammasome activation. Effects on cell proliferation, viability, and chemosensitivity were measured by a Celltiter-Glo 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 and p65 immunostaining, revealing translocation of p65 to the nucleus. Binding to Aim2 was confirmed using a DARTS assay and Co-Immunoprecipitation of NLRP3 and ASC.
Results: Treatment with 100 nM alantolactone reduced IL-1β release in response to potassium efflux induced by nigericin (276.9 vs. 48.7 pg ml-1, p<0.05). IL-1β release in response to double-stranded DNA (polydA:dT) was also reduced (69.8 vs. 16.5 pg ml-1). Interestingly, IL-1β release induced by intracellular pathogens like the Bacillus anthracis lethal factor (NeedleTox) remained unchanged. The reduction of Il-1β – release in response to activation with polydA:dT reached its maximum at a concentration of 1 μM compared to solvent control (58.2 vs. 4.8 pg ml-1, p<0.05). At this concentration, alantolactone had no toxic effect on cell metabolism measured by CellTiterGlo.
Phosphorylation of IκBα measured by Western Blot remained unchanged after treatment with 100 nM alantolactone. Additionally, translocation of p65 in PMA-differentiated THP1-macrophages to the cell nucleus measured by microscopy was not significantly altered after alantolactone treatment.
Conclusion: In summary alantolactone inhibits inflammasome activity in response to double-stranded DNA mediated by the AIM-2 inflammasome in THP-1 cells. Alantolactone inhibits pyroptosis, gasdermin D cleavage, and interleukin release in an NF-κB-independent manner.
Figure 1: THP-1 cells were primed with 1 µg ml-1 lipopolysaccharide (LPS) for 4h and either 1h nigericin or 20 ng ml-1 Anthrax Protective Antigen with 100 ng ml-1 LFnNeedle (NeedleTox) for NLRP3- and NLRC4 inflammasome activation. AIM2-inflammasome activation was induced by priming with 100 ng ml-1 LPS overnight and transfection of 1 µg ml-1 polydAdT for 4h. 100 nM Alantolactone (ALA) were added prior to LPS-priming.