RIPK1 mediates TNF-induced intestinal crypt apoptosis during chronic NF-κB activation

Abstract Background and Aims TNF is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, as TNF does not kill normal IECs. Here, we investigated how chronic NF-κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice. Methods Human IBD specimens were stained for p65 and cleaved caspase-3 (cC-3). C57BL/6 mice with constitutively active IKKβ in IEC (Ikkβ(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice were injected with TNF or LPS. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole (BHA). Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation. Results NF-κB activation in human IBD correlated with appearance of cC-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkβ(EE)IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKβ facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. BHA treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo. Conclusions Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF’s destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD.