Caspase-8 self-cleavage is necessary for apoptosis but not cytokine responses.

<p>(A) Schematic of strategy used to generate <i>Casp8</i>^<i>DA/DA</i> mice using CRISPR/Cas9. GuideRNA is in blue. (B) <i>Casp8</i>^<i>+/+</i>, <i>Casp8</i>^<i>DA/+</i>, <i>Casp8</i>^<i>DA/DA</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were infected with YopJ-deficient (ΔYopJ) and wild type <i>Yersinia</i> and caspase-8 processing was measured by western analysis. (C) BMDMs were pretreated with GSK’ 872 or vehicle control 1 hr prior to infection with <i>Yersinia</i>. Cytotoxicity was measured by LDH release 4 hrs post-infection. (D) Cleaved caspase-3 was measured by flow cytometry in BMDMs 2 hrs post-indicated treatments. (E-G) <i>Casp8</i>^<i>+/+</i>, <i>Casp8</i>^<i>DA/+</i>, <i>Casp8</i>^<i>DA/DA</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were treated with PAMPs and cytokine production was measured after 6 hrs by flow cytometry. Representative flow plots of IL-12p40 production in response to LPS (100 ng/mL) (E), quantification of percentage of IL-12p40⁺ in response to LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) (F), IL-1β⁺ and IL-6⁺ in response to LPS (100 ng/mL) (G) (H) BMDMs were treated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) for 6 hrs and TNF production was measured by ELISA. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. Student’s unpaired two tailed <i>t</i>-test. Representative of 3–5 independent experiments.