Fig. 7: GSDMD regulates IL-18 production and caspase-4 cleavage during Shigella infection in primary human monolayers.

Human duodenal organoids were trypsinised into single cells and seeded onto culture plates pre-coated with 1:40 Matrigel:PBS. Cells were infected with S. flexneri ∆ ospc3 at an MOI of 100 for 4 h. CFUs in infected cells were enumerated (a), PI uptake was monitored using incucyte live cell imager and PI uptake was normalised per area confluency (b), images of monolayers taken 4 h post infection taken at 20x (i) scramble, uninfected; (ii) scramble, Shigella flexneri ∆ ospc3, (iii) GSDMD KD uninfected, (iv) GSDMD KD Shigella flexneri ∆ ospc3. Scale bars (indicting 200 micrometres) are depicted for each micrograph (c). Caspase-4 was measured in western blot of whole cell lysates (d), IL-18 secretion into the supernatant was measured by ELISA on cell free supernatants (e). A schematic of caspase-4 regulation (f). In (f) (i) LPS-induced caspase-4 activation (1) leads to activation of GSDMD and IL-18 (2). GSDMD pores lead to caspase-4 cleavage (3) and this stops IL-18 processing (4). In (f)(ii), during conditions of reduced GSDMD –mediated plasma pore formation, caspase-4 activation (1) leads to IL-18 processing. A lack of efficient pore formation reduces inhibitory feedback on caspase-4 (3). This leads to continued activity of caspase-4 on IL-18 and IL-18 hypersecretion (4). All experiments are representative of at least 3 experiments. Data displayed as mean ± S.E.M. *p < 0.05, ****p < 0.001. Panel (f) created with Biorender.