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 6
CASP7 is activated and contributes to restriction of L. pneumophila replication in the absence of CASP8.
(A–D) Bone marrow-derived macrophages were left uninfected (NI) or were infected with wild type (WT Lp) or flaA mutants (flaA) L. pneumophila for the indicated time points to assay CASP7 and CASP8 activation. (A) Lysates from C57BL/6 and Casp1/11–/–macrophages infected with WT Lp for 1 to 8 h were assessed by Western blot using anti-Casp7 p18 antibodies and anti-α-actin. (B) C57BL/6 and Casp1/11–/–macrophages were infected with WT Lp for 5, 6, 7 and 8 h and Caspase-8 cleavage was measured by western blot anti-Casp8 p18 antibody and anti-α-actin. (C) C57BL/6 and Casp1/11–/–macrophages were infected for 8 h and CASP8 activation was measured using the Caspase-Glo 8 Assay kit. (D) Lysates from C57BL/6, Casp1/11–/–, Casp8/Ripk3–/–and Nlrc4–/–macrophages were assessed for CASP7 cleavage using anti-Casp7 p18 antibodies and anti-α-actin after 6 h infection. (E–H) Macrophages from C57BL/6, Ripk3–/–, Casp1/Ripk3–/–, Casp8/Ripk3–/–, Casp8/1/Ripk3–/–, Casp8/1/11/Ripk3–/–and Nlrc4–/–mice (E-F) or from C57BL/6, Gsdmd/Ripk3–/–, Gsdmd/Casp8/Ripk3–/–and Nlrc4–/–mice (G-H) were infected with wild type L. pneumophila (WT Lp) or flaA mutants (flaA) expressing luciferase at an MOI of 0.015 and bacterial replication was estimated by measurement of luminescence (RLU). Student’s t test. *, P<0.05 compared to C57BL/6. #, P<0.05 compared to Casp1/Ripk3–/–. Data are presented for one representative experiment of three (C) and two (A, B, E-H) experiments performed with similar results.