Guanylate-binding protein 5 licenses caspase-11 for Gasdermin-D mediated host resistance to Brucella abortus infection
Fig 7
Gasdermin-D (GSDMD) is required to induce pyroptosis and NLRP3 inflammasome activation in response to B. abortus.
BMDMs obtained from C57BL/6, Casp11-/-, Gsdmd-/- and Casp1/11-/- were left uninfected (NI) or stimulated with B. abortus. BMDMs primed with E. coli LPS (1 μg/ml) for 4h were left uninfected (A) or infected with B. abortus (B) at an MOI of 100. Pore formation was assessed fluorimetrically in real time by the uptake of propidium iodide (relative fluorescence units) into the nucleus of the permeabilized BMDMs. (C) Primed BMDMs were left uninfected (NI) or infected with B. abortus at an MOI of 100 for 8 hrs prior to the assessment of extracellular LDH. Values represent the percentage of LDH released compared with cells lysed with Triton X-100. *p < 0.05 compared with C57BL/6 cultures infected with B. abortus. (D) Immunoblot showing GSDMD p50 and p30 in lysates of primed BMDMs obtained from C57BL/6, Nlrp3-/-, Casp11-/-, Casp1/11-/- and Gsdmd-/- infected with B. abortus at an MOI of 100 for 8 hrs. (E) BMDMs were left uninfected (NI) or infected with B. abortus or mutant virB2 at an MOI of 100 for 17 hrs of infection. Supernatants were submitted to ELISA to estimate the concentration of IL-1β. (F) The same culture supernatants and lysates harvested 17 hrs postinfection were separated by SDS-PAGE, blotted and probed with a monoclonal anti-caspase-1 p20 subunit Ab. Equal loading was controlled by measuring β-actin in the corresponding cell lysates. (G) BMDMs were incubated in a medium containing 80 mM KCl 1 h before infection. Then, BMDMs were infected with B. abortus at an MOI of 100 in the same medium for 17 hrs. IL-1β in the supernatant were measured with mouse IL-1β ELISA kits. Statistically significant difference between BMDMs treated with KCl versus untreated was denoted by an asterisk for p<0.05. (H) Assessment of intracellular K+ levels, as measured by the fluorescent K+ probe APG-2. BMDMs were uninfected (NI) or infected for 6 hrs with B. abortus at an MOI of 100. Each dot represents the percentage of APG-2 fluorescence intensity in relation to the average fluorescence of control cells, and the red bars represent the mean of all analyzed cells. Statistically significant difference between infected BMDMs versus NI was denoted by * p<0.05. The graphs are representative of two independent experiments. NI: uninfected; B.a.: B. abortus; RFU: relative fluorescence unit; wt: B. abortus wild-type; virB2: mutant B. abortus. ns:statistically not significant.