FIGURE 5. LPS + Dox-induced IL-1β release is TRIF-dependent and correlated with IAP downregulation and recruitment of caspase-8/ FADD to a detergent-insoluble compartment.

(A) A model for parallel pathways of canonical NLRP3 inflammasome activation and TRIF-induced caspase-8 signaling complexes that may mediate IL-1β processing induced by LPS + doxorubicin. (B) Western blot analysis of proIL-1β and procaspase-1 in cell lysates (intra) and mature IL-1β and caspase-1 p10 subunit in extracellular supernatants (extra) from WT and Trif^−/− BMDC stimulated Pam₃CSK₄ (200 ng/ml) ± Dox (10 μM) or LPS (1 μg/ml) ± Dox for 12 h. The data are representative of 2 experiments. (C, D) IL-1β release (by ELISA) from WT or Trif^−/− BMDC treated with Pam₃CSK₄ (200 ng/ml) or LPS (1 μg/ml) for a total of 16 h and stimulated ± Dox (10 μM) (C, D) or Dox + YVAD (50 μM) (D) for the final 12 h of the LPS or Pam₃CSK₄ treatment periods). Data in (C) are representative of results from 2 experiments. Data in (D) are the mean ± SE of 3 experiments. **P < .01 or not significant (n.s) by ANOVA. (E) Western blot analysis of cIAP1, proIL-1β, procaspase-1, and actin in cell lysates from WT BMDC or Casp1/11^−/− BMDC treated ± LPS (1 μg/ml) for 4 h prior to stimulation ± Dox (10 μM) for 12 h. As a positive control, lysates from control or Dox-treated Jurkat leukemic T cells were also analysed. Results are from a single experiment. (F) WT BMDC were treated with LPS (1 μg/ml) or TNFα (50 ng/ml) for 2 h in the presence or absence of zVAD (50 μM). Alternatively, BMDC were treated with LPS for 4 h prior to stimulation with Dox (10 μM) or Dox + zVAD for another 8 h. Cell lysates were separated into detergent-soluble versus detergent-insoluble fractions for western blot analysis of caspase-8 and FADD. Results are representative of 2 experiments.