Gasdermin-D and Caspase-7 are the key Caspase-1/8 substrates downstream of the NAIP5/NLRC4 inflammasome required for restriction of Legionella pneumophila
Fig 7
CASP7 and GSDMD are important for NAIP5/NLRC4/CASP1-dependent pore formation and restriction of L. pneumophila replication in vivo.
(A-C) Bone marrow-derived macrophages from C57BL/6, Casp7–/–, Gsdmd–/–, Gsdmd/Casp7–/–, Casp7/1/11–/–and Nlrc4–/–mice were left uninfected (NI) or infected with wild type L. pneumophila (WT Lp) or flaA mutants (flaA) at an MOI of 10. Pore formation was assessed by Propidium Iodide (PI) uptake. Data are expressed as a percentage of PI uptake induced by Triton-X100. (D and E) Bone marrow-derived macrophages from C57BL/6, Casp1–/–, Casp7–/–, Gsdmd–/–and Gsdmd/Casp7–/–mice were stimulated with 4μg/ml Pa and 2μg/ml LFn-Fla (FlaTox). (D) The percentage of propidium iodide uptake was estimated by assessing the fluorescence (RFU). Data is shown as percentage of the total PI uptake (estimated using Triton-X100). (E) LDH release was assessed 4 hours after FlaTox treatment using CytoTox 96 Non-Radioactive Cytotoxicity Assay. (F and G) Bone marrow-derived macrophages from C57BL/6, Casp7–/–, Gsdmd–/–, Gsdmd/Casp7–/–, Casp7/1/11–/–and Nlrc4–/–mice were infected with WT Lp (F) or flaA (G) expressing luciferase at an MOI of 0.015 and bacterial replication was estimated by measuring the luminescence (RLU) of each well. *, P<0.05: compared to C57BL/6. Data are presented for one representative experiment of two (A-C), one (D-E) and two (F-G) experiments performed with similar results. (H) C57BL/6, Casp7–/–, Gsdmd–/–, Gsdmd/Casp7–/–and Nlrc4–/–mice were infected intranasally with 105 wild type L. pneumophila, lungs were harvested at the indicated time points and homogenates were plated for CFU determination. Each dot represents a single animal, and the horizontal lines represent the averages. *, P<0.05. ns, P>0.05. Data presented in (H) are a pool of two independent experiments performed.