Fig 2: LPS activated non-canonical inflammasomes cleave IL-1ß and IL-18.(A) HEK 293T cells stably expressing IL-18-V5 and either CASP4 or CASP5 were transfected with LPS (25 µg/mL) for 24 h before samples were collected and analyzed by immunoblotting. (B,C) HEK 293T cells stably expressing 2xFLAG-CASP11 were transiently transfected with the indicated constructs. After 24 h, samples were transfected with LPS (25 µg/mL) for 24 h then analyzed for LDH release (B) and immunoblotting (C). Data are means ± SEM of three biological replicates. ***P < 0.001, **P < 0.01 and *P < 0.05 by two-sided Student’s t-test compared with control. Data are representative of two or more independent experiments. *Represents non-specific bands in immunoblot. The small m or h represents mouse or human versions of the proteins respectively.

Fig 3: Caspase-4 and caspase activity are required for IFN-γ-primed inflammasome responses to L. pneumophila. (A–C) Primary hMDMs were unprimed or primed with IFN-γ (100 U/mL) overnight and treated with YVAD, ZVAD, or DMSO vehicle control for 1 h followed by infection with T4SS+ Lp for 4 h. (D, E) Primary hMDMs were transfected with 30 nM of scrambled control siRNA (siControl), or siRNAs targeting CASP4 (siCASP4), CASP5 (siCASP5), or both (siCASP4/5), unprimed or primed with IFN-γ (100 U/mL) overnight, and infected with T4SS+ Lp or PBS (Mock) for 4 h. (A, D) Cell death was measured using the LDH assay and normalized to mock-infected cells. (B, C, E) IL-1β and IL-18 levels in the supernatant were measured by ELISA. (A, B, C) Shown are the pooled results of four to six independent experiments using hMDMs from different healthy human donors. Each data point represents the mean of triplicate infected wells from an individual donor. *P < 0.05, **P < 0.01, and ***P < 0.001 by paired t-test. (D, E) Shown are the pooled results of three independent experiments using hMDMs from three different healthy human donors. Each data point represents the mean of triplicate infected wells from an individual donor. *P < 0.05 by paired t-test.

Fig 4: Elevated inflammatory response in CASP5 knockdown fibroblasts. a qRT-PCR of CASP5 in cells with 3 different doxycycline-inducible shCASP5 (Sh1, Sh2, and Sh3), normalized to GAPDH, are shown relative to non-induced culture (Ctrl). * indicates statistical significance. b Cytokine induction in Sh3 culture in response to 24-h exposure of 1 μg/ml lipopolysaccharide (LPS), normalized with GAPDH mRNA. c, d Cytokine induction in Sh3 culture in response to 24-h exposure to TNF-α, 10 ng/ml or 20 ng/ml, normalized with GAPH mRNA. Sh1 and Sh2 cultures showed similar trends (data not shown)

Fig 5: CASP5 variant and expression in a progeroid pedigree. a Proband II:3 at age 54 and the affected brother II:4 at age 51. Patients provided written consent to use these photographs. b Family pedigree with the proband ( ), affected (•■), and carriers ( ). c CASP5 sequencing showing the familial variant. d Western analysis of caspase 5 in individuals with 3 genotypes. The nonspecific band is shown as a reference. e qRT-PCR analysis of CASP5 mRNA in the same individuals as in c