Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome

Caspase-11 WT, catalytic mutant (C254A), CDL mutant (CDL^uncl), or IDL mutant (IDL^uncl) were retrovirally expressed in Casp11^−/− BMM. Cells were primed for 12 h with Pam₃CSK₄ or 4 h with LPS, and transfected with ultrapure K12 E. coli LPS 10 μg/ml for 6 h or exposed to 5 μM nigericin for 2 h. (A) Western blot assessed expression of caspase-11 mutants in cell extracts of Pam₃CSK₄-primed, untransfected BMM. (B) Immunoblot detected mature IL-1β and the caspase-11 or caspase-1 large subunits in the culture medium (SUP) and cell extracts (XT) of Pam₃CSK₄-primed BMMs transfected with LPS for 6 h. (C) Cell death and (D) IL-1β secretion was assessed 6 h after LPS transfection or 2 h after nigericin exposure. Western blots are representative of three biological replicate experiments. Graphs are mean + SEM of four biological replicate experiments, with significance assessed using a Mann–Whitney test.

HEK293T cells were transiently transfected with pEF6-DmrB-Caspase-11 constructs depicted in (A) to mimic the p43 dimers and p32/p10 species of caspase-11 (actual predicted molecular weights of DmrB fusions are 46 kD and 35/10 kD, respectively). Transfected HEK293T cells were pre-incubated with the dimerizer drug, AP20187, for 30 min before substrate addition. (B) Cleavage of AcLEHD-afc by DmrB-Caspase-11 in digitonin-lysed HEK293T cells was monitored over 30 min. Data are mean of technical quadruplicates, and are representative of at least three biological replicate experiments. (C) HEK293T cells expressing DmrB-Caspase-11 were incubated with AP20187 for 30 min to induce dimerization. Cells were lysed with digitonin, and incubated with lysates from HEK293T expressing V5-GSDMD for 1 h at 37°C. Samples were precipitated and analysed using an immunoblot. Data are representative of three biological replicate experiments.

HEK293T cells were transfected with either DmrB alone (empty vector, EV) or DmrB-caspase-11 mutants: WT, C254A (catalytic mutant), IDL^uncl (IDL triple cleavage mutant; E266A/D277A/D285A), and single IDL mutants: E266A, D277A, and D285A. Cells were exposed to 500 nM AP20187 for 30 min and lysed with digitonin for quantification of proteolytic activity, either by (A) kinetics of AcLEHD-afc cleavage over 20 min, or (B) reaction rate (upper), and incubation with full-length GSDMD-V5 to assess the extent of cleavage to p30 after 1 h (lower). Data in (B upper) are mean + SEM of four biological replicates. Data were analysed for normality using the Shapiro–Wilk normality test, and tested for significance using parametric paired t tests (two-sided). All other data are representative of at least three biological replicates.

HEK293T cells were transiently transfected with constructs containing DmrB-Caspase-11, WT, C254A, and IDL^thr, in which a thrombin cleavage site replaces the caspase-11 IDL auto-processing site, as depicted in (A), to allow generation of unprocessed dimers (analogous to caspase-11 p43; actual predicted weight for the DmrB-caspase-11 fusion, ∼46 kD) and IDL-cleaved dimers (analogous to caspase-11 p32/p10, actual predicted weight 35/10 kD). (B) Cells were incubated with AP20187 (500 nM) for 30 min, and then AcLEHD-afc cleavage was measured with and without the addition of thrombin (20 U/ml) to the reaction. Data are mean + SEM of four biological replicates. Data were analysed for normality using the Shapiro–Wilk normality test, and tested for significance using parametric paired t tests (two-sided). (C) AcLEHD-afc kinetic trace of AP20187-treated cells expressing DmrB-Caspase-11 WT versus IDL^thr, in the presence and absence of thrombin (20 U/ml) in the reaction. (D) HEK293T expressing the DmrB-Caspase-11 constructs were exposed to AP20187 for 30 min, and then lysed and incubated for 15 min with thrombin (20 U/ml) before the addition of V5-GSDMD for 1 h. (E) Model for LPS-induced caspase-11 dimerization, auto-processing, and activation.