Fig 1.
CASP8 is activated in the absence of GSDMD and CASP1/11 and it is important for restriction of L. pneumophila replication in macrophages.
(A) Bone marrow-derived macrophages from C57BL/6, Casp1/11–/–, Casp11–/–, Gsdmd–/–and Asc/Casp1/11–/–mice were left uninfected or infected with wild type L. pneumophila (WT Lp; grey bars), fliI mutants (hatched bars) or flaA mutants (flaA; black bars) at an MOI of 10 for 8 hours and the activation of CASP8 was measured using the Caspase-Glo® 8 Assay kit. (B) Macrophages were infected at an MOI of 10 for 8 hours and the cleavage of CASP8 was measured by western blot using the anti-Casp8 and anti-α-actin antibody. (C-G) Macrophages from C57BL/6, Casp1/11/Ripk3–/–, Casp8/1/11/Ripk3–/–or Nlrc4–/–mice were infected with L. pneumophila and the bacterial replication was measured for up to 4 days of infection. (C, D) Cells were infected with wild type L. pneumophila at an MOI of 0.015 (C) or 10 (D) and bacterial replication was estimated by CFU counting. (E-G) Cells were infected with wild type L. pneumophila (E), fliI mutants (F) or flaA mutants (G) expressing luciferase at an MOI of 0.015 and bacterial replication was estimated by measuring the luminescence (RLU) of each well over 4 days of infection. *, P<0.05: compared to C57BL/6. #, P<0.05: compared to Casp1/11/Ripk3–/–. Data are presented for one representative experiment of three (A- D) and four (E-G) experiments performed with similar results.
Fig 2.
CASP7 is activated downstream of CASP8 in the NAIP5/NLRC4 inflammasome.
Bone marrow-derived macrophages from Casp1/11–/–, Asc/Casp1/11–/–, Casp8/1/11/Ripk3–/–and Casp7/1/11–/–mice were left uninfected (NI, open bars) or infected with wild type L. pneumophila (WT Lp, grey bars) or flaA mutants (black bars) at an MOI of 10 for 8 hours. (A) Caspase-7 cleavage was measured by western blot using anti-Casp7 p18 and anti-α-actin antibody. (B and C) Caspase-8 cleavage was measured by western blot using anti-Casp8 p18 and anti-α-actin antibody (B) and Caspase-Glo 8 Assay kit (C). *, P<0.05. Data are presented for one representative experiment of four (A) and three (B) and two (C) experiments performed with similar results.
Fig 3.
CASP7 accounts for pore formation and pyroptosis.
Bone marrow-derived macrophages from C57BL/6, Casp1/11–/–, Casp7/1/11–/–, Casp8/1/11/Ripk3–/–and Asc/Casp1/11–/–mice were left uninfected (NI) or infected with wild type L. pneumophila (WT Lp) or flaA mutants at an MOI of 10. (A and B) Pore formation in uninfected (A) and WT Lp-infected (B) macrophages was assessed by Propidium Iodide (PI) uptake. Data are expressed as a percentage of total PI uptake (estimated using Triton-X100). (C) LDH release was assessed 7 hours after infection using CytoTox 96 Non-Radioactive Cytotoxicity Assay. (D) Lysates from C57BL/6, Casp1/11–/–, Casp7/1/11–/–and Nlrc4–/–macrophages were assessed for CASP3 cleavage using anti-Casp3 antibodies. Data are expressed as a percentage of LDH release induced by Triton-X100. Statistical significance was calculated using Student’s t test. *, P<0.05. ns: non-significant. Data are presented for one representative experiment of four (A-B) and two (C-D) experiments performed with similar results.
Fig 4.
CASP7 is important for restriction of L. pneumophila replication in vivo in the absence of CASP1/11.
Bone marrow-derived macrophages from C57BL/6, Casp7–/–, Casp1/11–/–, Casp7/1/11–/–,Casp8/1/11/Ripk3–/–and Nlrc4–/–mice were infected with wild type L. pneumophila (WT Lp, A, B) or flaA mutants (C, D). Macrophages were infected at an MOI of 0.015 and bacterial replication was assessed by measurement of luminescence (RLU) emitted by luciferase-expressing bacteria (A, C) or by estimating bacterial CFU (B, D). *, P<0.05: compared to C57BL/6. #, P<0.05: compared to Casp1/11–/–. Data are presented for one representative experiment of four (A, C) and three (B, D) experiments performed with similar results.
Fig 5.
CASP7 is important for restriction of L. pneumophila replication in vivo in the absence of CASP1/11.
(A and B) C57BL/6, Casp1/11–/–, Casp7/1/11–/–, Casp7+/–/1–/–/11–/–, Casp7+/–/1+/–/11+/–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. Data are presented for one representative experiment of four (A) and two (B) experiments performed with similar results.
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.
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.
Fig 8.
Schematic model illustrating that Gasdermin-D (GSDMD) and Caspase-7 are the key substrates of Caspase-1 and Caspase-8 downstream of the NAIP5/NLRC4 inflammasome.