Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn’s disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn’s disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8 mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8 mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-a, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn’s disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-a-induced necroptotic cell death. Caspase-8 is a cysteineprotease critically involved in regulating cellular apoptosis. On activation of death receptors, including TNF-receptor and Fas, caspase-8 is activated by limited autoproteolysis and the processed caspase-8 subsequently triggers the caspase cascade that finally leads to apoptotic cell death. Caspase-mediated apoptosis is important for the turnover of IECs and for shaping the morphology of the gastrointestinal tract. Furthermore, recentdatahave indicated a role of caspase-mediated apoptosis of IECs in the pathogenesis of inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis. To study the function of caspase-8 in the gut, we generated mice with an IEC-specific deletion of caspase-8 (Casp8). Accordingly, mice with floxed caspase-8 alleles were bred with mice expressing the Cre recombinase under the control of the IEC-specific villin promoter. Specific deletion of caspase-8 in IECs was confirmed by polymerase chain reaction (PCR) and western blotting (Supplementary Fig. 1a, b). Casp8 mice were born at the expected Mendelian ratios and developed normally, although weighing on average slightly less than control littermates at 8 weeks of age (data not shown). Despite the paradigm that apoptosis is important for regulating epithelial cell numbers, histological and morphometrical analysis of the jejunum and colon of Casp8 mice showed no overt changes of tissue architecture or dysregulation of apoptosis (Supplementary Figs 1d–f and 2). Although this suggested that caspase-8 is not essential for the structural development of the gut, high-resolution endoscopy showed erosions in the terminal ileum—but not in the colon—of Casp8 mice (Fig. 1a and Supplementary Fig. 1c). Histological analysis demonstrated marked destruction of the architecture and signs of inflammation including bowel wall thickening, crypt loss and increased cellularity in the lamina propria (Fig. 1b) in more than 80% of all ileal specimens. This finding of spontaneous ileitis in the absence of caspase-8 in IECs was further supported by increased expression of the inflammation markers S100a9 and TNF-a (also known as Tnf) and by elevated infiltration of the lamina propria with CD4 T cells and granulocytes (Fig. 1c and Supplementary Fig. 3).