Inhibitor of Apoptosis Protein-1 Regulates Tumor Necrosis Factor-Mediated Destruction of Intestinal Epithelial Cells.

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. METHODS: RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a, or Tnfrsf1b (Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. RESULTS: YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids-most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor 1. CONCLUSIONS: In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine.

A fast and simple fluorometric method to detect cell death in 3D intestinal organoids.

Organoids recapitulate the (patho)physiological processes in certain tissues and organs closer than classical cell lines. Therefore, organoid technology offers great potentials in drug development and testing, and personalized medicine. In particular, organoids can be used to study and predict drug-induced toxicity in certain tissues. However, until today few methods had been reported to analyze cell death in 3D-microtissues in a quantitative manner. Here, we describe a novel fluorometric method for the quantitative measurement of specific organoid cell death. Organoids are stained simultaneously with the cell impermeable nuclear dye propidium iodide and cell permeable Hoechst33342. While Hoechst allows in-well normalization to cell numbers, propidium iodide detects relative proportion of dead cells independent of hydrogel. Measurement and analysis time, as well as usability are drastically improved in comparison to other established methods. Parallel multiplexing of our method with established assays measuring mitochondrial activity further enhances its applicability in personalized medicine and drug discovery.