Nitroxolin is a novel inhibitor of NLRP3-dependent pyroptosis

Introduction: Pyroptosis is an inflammatory cell death resulting in the release of inflammatory cytokines like Interleukin-1β (IL-1β). Pyroptosis in immune cells contributes to cardiovascular diseases such as atherosclerosis, ischemia-reperfusion injury and ventricular remodeling. We aimed to identify novel inhibitors of pyroptosis in mononuclear cells. 

Materials and Methods: Approximately 6,280 drugs or drug-like compounds were screened in the “Spectrum Collection” (Microsource) and the “Bioactive Compound Library” (Selleckchem). NLRP3-mediated pyroptosis was induced in THP1 ASC–GFP monocytes by lipopolysaccharide (LPS) and nigericin treatment and quantified by propidium iodide staining. Effects on cell proliferation, viability, and chemosensitivity were measured by a Celltiter-Glo assay. IL-1β concentrations were quantified in cell supernatants using an 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 NLRP3 was confirmed using a Drug Affinity Responsive Target Stability (DARTs) assay. ASC-specks representing inflammasome formation were imaged via immunocytochemistry and quantified using flow cytometry. Ex vivo experiments were performed in isolated peripheral blood mononuclear cells (PBMCs).

Results:  The screening revealed 22 potentially anti-pyroptotic substances. One of the most promising candidates was Nitroxolin that is used as an antibiotic in the first-line treatment of urinary tract infections. Cell culture studies showed that Nitroxolin at the concentration of 80 µM inhibited monocyte pyroptosis by 38 % and had no toxic effect on the cells. IL-1β release was significantly inhibited (198 pg/ml vs. 11 pg/ml, p<0.05). The ratio between p-IκBα to IκBα (0.73 vs 0.74) and translocation of p65 to the nucleus was not significantly different from solvent control, indicating no regulation of NFκB-activity. Treatment with Nitroxolin reduced inflammasome formation (47.0 vs 17.4% of ASC-speck positive cells, p<0.05, Figure 1A, B). Mechanistically, Nitroxolin protected NLRP3 from protease degradation in the DARTs assay, implying a direct interaction of NLRP3 and Nitroxolin (Figure 1C). The addition of Nitroxolin significantly reduced the release of IL-1β by PBMCs after LPS and ATP treatment (4978 pg/ml vs. 2 pg/ml, p<0.05). 

Conclusion:  The study identifies Nitroxolin as a novel NFκB-independent inhibitor of NLRP3-mediated pyroptosis. Nitroxolin reduces inflammasome formation by binding to NLRP3, thereby reducing IL-1β secretion in THP-1 cells and in human PBMCs. Therefore, Nitroxolin is a promising candidate for further characterization in the context of NLRP3-mediated inflammatory conditions such as atherosclerosis.

Figure 1: (A) THP-1 ASC-GFP cells were treated with LPS and nigericin. DMSO or 80 µM Nitroxolin were added as indicated. Left-tilted arrows point out ASC-GFP specks indicating inflammasome formation, right-tilted arrows point out cells remaining in primed state. (B) Flow cytometry analysis of ASC-speck formation in THP-1 ASC-GFP cells treated with LPS and nigericin. DMSO or Nitroxolin was added as indicated. (C) Western Blot of NLRP3 protein with and without protein degradation by Pronase and with and without Nitroxolin incubation. *p < 0.05