Caspase-1 engagement and TLR-induced c-FLIP expression suppress ASC/caspase-8-dependent apoptosis by inflammasome sensors NLRP1b and NLRC4

The caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1(-/-) macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1 beta (IL-1 beta) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation

Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells

Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient, strain-dependent, and requires expert skills. Over recent years, several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment, which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin, which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary, our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100%