Nlrp3 inflammasome activation and Gasdermin D-driven pyroptosis are immunopathogenic upon gastrointestinal norovirus infection
Fig 3
Nlrp3-dependent GSDMD cleavage mediates pyroptosis in MNV-infected macrophages.
(A-C) Unprimed WT bone-marrow-derived macrophages (BMDMs) were infected with either UV-inactivated or live MNV at a MOI 5. (A) Sytox Green (SG) uptake and Caspase-3/-7 (C3/7) activity were assessed for 24 hours by IncuCyte real-time monitoring every 30 minutes; (B) confocal microscopic picture of Propidium Iodide uptake and cell morphology at 13 hours post-infection. White arrows indicate cells displaying swollen necrotic morphology; (C) cell lysates were prepared at indicated time points post-infection and were immunoblotted for GSDMD and caspase-3 processing. (D-I) BMDMs from WT and indicated KO mice were (D) left untreated (unprimed) or were (E-I) TLR2-primed, after which they were infected with either UV-inactivated or live MNV at a MOI 5. (D, E) Sytox Green (SG) uptake was assessed for 24 hours by IncuCyte real-time monitoring every 30 minutes. (F-H) Cell lysates were prepared at 24 hours post-infection and were immunoblotted for (F-G) GSDMD processing along with (H) IL-1β and caspase-1 maturation. (I) Cell culture supernatants collected at indicated time points post-infection were analyzed for secreted IL-1β. Data shown in (A, D-E) are the means of duplicate wells from a representative experiment out of two independent experiments; data shown in (B-C, F-H) are representative for at least two independent experiments; data shown in (I) are the means ± SD of triplicate wells from a representative experiment out of two independent experiments.