CASP8 C362A KI MLKL KO e16.5 intestine RNAseq

Caspase-8 is a protease with both pro-death and pro-survival functions: it is required for apoptosis induced by death receptors such as TNFR1 (tumor necrosis factor receptor 1) 1, and it has a critical role in suppressing necroptosis mediated by the kinase RIPK3 (receptor interacting protein kinase 3) and the pseudokinase MLKL (mixed lineage kinase-like) 2-4. Mice lacking caspase-8 display MLKL-dependent embryonic lethality 4, as do mice expressing catalytically inactive caspase-8 mutant C362A. However, Casp8C362A/C362A Mlkl-/- mice die in the perinatal period, whereas Casp8-/- Mlkl-/- mice are viable 4, indicating that inactive caspase-8 also has a pro-death scaffolding function. Here we show that inactive caspase-8 activates pyroptosis in MLKL-deficient intestinal epithelial cells around embryonic day 18, triggering the formation of ASC specks. Accordingly, intestinal atrophy and perinatal lethality in Casp8C362A/C362A Mlkl-/- mice was prevented by loss of caspase-1. In transfection studies, inactive caspase-8 mutants C362A or C362S were found in both the triton X-100 insoluble and soluble fractions, whereas wild-type caspase-8 existed only in the soluble fraction. Moreover, inactive caspase-8 shifted co-transfected ASC into the insoluble fraction, whereas wild-type caspase-8 did not. Thus, a defense mechanism is revealed that would allow intestinal epithelial cell death in the face of pathogens expressing virulence factors to inhibit caspase-8-dependent apoptosis and necroptosis. RNA was extracted from e16.5 mouse intestine for the following genotypes - Casp8 wildtype MLKL knockout (Casp8 WT MLKL KO), Casp8 MLKL double knockout (Casp8 KO MLKL KO), and Casp8 C362A knockin MLKL knockout (Casp8 KI MLKL KO).半胱天冬酶-8（Caspase-8）是一种兼具促死亡与促存活功能的蛋白酶（protease）：它参与肿瘤坏死因子受体1（tumor necrosis factor receptor 1, TNFR1）等死亡受体介导的细胞凋亡（apoptosis）过程1，同时对抑制由激酶RIPK3（受体相互作用蛋白激酶3，receptor interacting protein kinase 3）与假激酶MLKL（混合谱系激酶样蛋白，mixed lineage kinase-like）介导的细胞坏死性凋亡（necroptosis）发挥关键作用2-4。缺失半胱天冬酶-8的小鼠会出现MLKL依赖的胚胎致死表型4，表达催化失活型半胱天冬酶-8突变体C362A的小鼠亦表现出相同表型。然而，Casp8C362A/C362A Mlkl-/- 小鼠会在围产期死亡，而Casp8-/- Mlkl-/- 小鼠则可存活4，这表明失活型半胱天冬酶-8同时具备促死亡的支架功能。本研究证实，在胚胎第18天左右的MLKL缺陷型肠上皮细胞中，失活型半胱天冬酶-8可激活细胞焦亡（pyroptosis），并触发ASC斑点（ASC specks）的形成。据此，Casp8C362A/C362A Mlkl-/- 小鼠的肠道萎缩与围产期致死表型可因Caspase-1缺失而得到挽救。在转染实验中，研究人员发现失活型半胱天冬酶-8突变体C362A或C362S同时存在于Triton X-100不溶性组分与可溶性组分中，而野生型半胱天冬酶-8仅存在于可溶性组分中。此外，失活型半胱天冬酶-8可将共转染的ASC迁移至不溶性组分，而野生型半胱天冬酶-8则无此效应。综上，本研究揭示了一种防御机制：当病原体表达毒力因子以抑制半胱天冬酶-8依赖的细胞凋亡与坏死性凋亡时，该机制可介导肠上皮细胞死亡。本研究从胚胎期16.5天（e16.5）的小鼠肠道中提取RNA，用于以下三种基因型样本的分析：Casp8野生型MLKL敲除（Casp8 WT MLKL KO）、Casp8与MLKL双敲除（Casp8 KO MLKL KO）以及Casp8 C362A敲入型MLKL敲除（Casp8 KI MLKL KO）。