Fig. 1: IAV infection results in cleavage of GSDMD in lung epithelial cells.
From: Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection
A, B Wildtype and Gsdmd−/− mice were infected with 104 PFU of HKx31 IAV. A Immunoblot of GSDMD and tubulin protein in lung tissue lysates from uninfected and IAV-infected mice on day 3 and 5. Please note lanes 4 and 5 were inversely loaded unintentionally. Arrows indicate full length (FL) and active N-terminal p30 GSDMD subunit. Individual wildtype (horizontal solid black lines) and Gsdmd−/− (dotted blue lines) mice are shown. * indicates non-specific band. Data are representative of one of two independent experiments, each consisting of 3 or 4 mice per group. B Expression of cleaved GSDMD (green) in E-Cadherin+ (red) epithelial cells in lung tissue sections measured by confocal microscopy on day 3 and 5 post-infection. Hoechst nuclear stain is shown in blue. Images were analyzed with ImageJ software. Bronchiole (BR), alveolus (AL) and a blood vessel (BV) are labeled. Representative images at 40× magnification (scale bar = 20 μm). C Quantification of cleaved GSDMD staining determined using ImageJ software. Data presented as the mean % positive cells per field of view (FOV) ± SD, with each data point representing an individual animal. n = 3 or 4 per group. *p < 0.05, **p < 0.01, one-way ANOVA. D–G Human normal bronchial epithelial HBEC3-KT cells were infected with human IAV: HKx31 (HK; H3N2), Brazil/78 (BR; H1N1), Solomon Islands/04 (SI; H1N1), Tasmania/09 (Tas; pandemic H1N1), and Perth/09 (Perth; H3N2). Data are representative of one of two independent experiments. D Immunoblot of GSDMD in cell supernatants (SNF; top) and cell lysates (bottom) at 24 h post-infection. Uninfected (Un) cells are shown. Arrows indicate full length (FL), and active p43 and p30 subunits of GSDMD. * indicates non-specific bands. Immunoblot of pro-caspase 1 (p45), caspase-1 subunit p20 and tubulin protein in cell lysates is also shown. E Levels of LDH and F IL-1β and G IL-18 in cell supernatants at 24 h post-infection, determined by colorimetric assay (OD; optical density) and ELISA, respectively. Data shown as experimental triplicates ± SD. **p < 0.01, ****p < 0.0001, One-way ANOVA.
