Fig. 5.

Caspase-11 activation by Legionella has distinct kinetic and molecular parameters. (A) BMMs from the indicated mouse strains were pretreated with LPS and infected with flagellin-deficient Legionella (∆flaA) or E. coli for 4 h or 18 h, as indicated. Cleaved caspase-1 in the supernatant fraction (Casp1 p10) was measured by immunoblot analysis and compared with the levels of procaspase-1 and tubulin in the lysates. (B) Cell death of C57BL/6 (black bars), TRIF-deficient (Trif^Lps2/Lps2; white bars), caspase-11–deficient (Casp11^−/−; dark gray bars), or caspase-1/11–deficient (Casp1/11^−/−; light gray bars) BMMs pretreated with LPS and infected with the indicated Legionella strains or E. coli for 2 h, 4 h, or 18 h, as indicated. (C) Fluorometric plots show propidium iodide uptake (RFUs) over time to reveal the kinetics of pore formation after infection of LPS-treated BMMs derived from the indicated mouse strains and infected with Legionella strains. (D) BMMs derived from indicated mouse strains or immortalized BMMs from wild-type (iC57BL/6) or Myd88/TRIF double-knockout (iMyd88/Trif^−/−) mice were either left untreated or treated with LPS (0.1 μg/mL) for 4 h, and levels of procaspase11 and actin were measured by immunoblot analysis. (E and F) Cell death of primed BMMs derived from indicated mouse strains (E) or immortalized BMMs from wild-type (iC57BL/6) or Myd88/TRIF double-knockout (iMyd88/Trif^−/−) mice (F) infected for 2 h was measured. Cell death was determined as the percentage of LDH released compared with cells lysed with Triton X-100 (y axis). *P < 0.01 compared with the corresponding values from wild-type infections.