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4. Caspase 8 maintains epithelial cell adhesion and intestinal homeostasis in vivo through regulation of clathrin-dependent endocytosis and autophagy

G. Pineton de Chambrun1, C. Manthey2, C. McAllister2, M. Spehlmann2, A. Till3, M. Kagnoff2, P. Desreumaux4, D. Stupack5, J. Wang5, L. Eckmann2, 1Lille University Hospital, Gastroenterology, Lille, France, 2UCSD, Medicine, La Jolla, United States, 3UCSD, Molecular biology, La jolla, United States, 4North of France University, Inserm U995, Lille, France, 5UCSD, Moores Cancer center, La Jolla, United States

Background

Caspase 8 is a key mediator of ligand-activated apoptosis, but has other functions, including regulation of endocytosis in epithelial cells. The physiological importance of these different activities is poorly understood. The aim of this study was to study the physiological functions of caspase 8 in intestinal epithelial cells (IECs).

Methods

We generated mice specifically lacking Casp8 in IECs (Casp8ΔIEC) by crossing Casp8fl/fl mice to Tg(Vil-cre) mice, and examined them for cell death and endocytosis under constitutive and challenge conditions. The role of TNF-dependent signaling and autophagy was tested by additional ablation of Tnfr1 (Casp8ΔIEC/Tnfr1−/−) or Atg7 in IECs (Casp8ΔIEC/Atg7ΔIEC).

Results

At 6 weeks Casp8ΔIEC mice did not present any macroscopic or microscopic intestinal abnormalities compared with Casp8fl/fl mice. However, IEC from Casp8ΔIEC mice lacked the normal constitutive degradation of proteins important for clathrin-dependent endocytosis. To examine the consequences of this disturbance in endocytosis, we infected mice with the murine attaching/effacing pathogen, C. rodentium. Casp8ΔIEC mice displayed significantly more weight loss and mortality than Casp8fl/fl mice after infection. Microscopically, Casp8ΔIEC mice presented a complete destruction of small intestinal villi and ileitis. Furthermore, stimulation of Casp8ΔIEC mice with LPS for 4 hours caused marked detachment of small intestine IECs and led to villus destruction and mucosal inflammation. Immunofluorescence analysis revealed disappearance of basal β1-integrin in IECs of Casp8ΔIEC mice after LPS stimulation, suggesting active endocytosis of adhesion molecules. Consistent with this, LPS-induced IEC detachment was prevented in Casp8ΔIEC mice treated with the endocytosis inhibitor chlorpromazine. LPS-induced IEC detachment was prevented in Casp8ΔIEC/Tnfr1−/− mice, demonstrating a role of TNFα in these events. Furthermore, Casp8ΔIEC mice treated with LPS showed abnormal autophagic activation in IECs compared to Casp8fl/fl mice, and IEC detachment was absent in Casp8ΔIEC/Atg7ΔIEC mice. Casp8ΔIEC/Atg7ΔIEC mice were also protected from intestinal inflammation induced by C. rodentium.

Conclusion

Caspase 8 controls IECs adhesion and maintains intestinal barrier integrity in response to infectious stimuli by regulating clathrin-dependent endocytosis and autophagy. This work demonstrates a major physiologic role of caspase 8 in maintaining intestinal homeostasis and controlling intestinal inflammation.