RIP3 AND pMLKL promote necroptosis-induced inflammation and alter membrane permeability in intestinal epithelial cells

tBackground: Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL).Aims: The aim of this study is to examine in depth in vitro and ex vivo the contribution of necroptosis tointestinal inflammation.Methods: In vitro: we used an intestinal cell line, HCT116RIP3, produced in our laboratory and overex-pressing RIP3. Ex vivo: intestinal mucosal biopsies were taken from patients with inflammatory boweldisease (IBD) (20 with Crohn’s disease; 20 with ulcerative colitis) and from 20 controls.Results: RIP3-induced necroptosis triggers MLKL activation, increases cytokine/alarmin expression (IL-8,IL-1, IL-33, HMGB1), NF-kBp65 translocation and NALP3 inflammasome assembly. It also affects mem-brane permeability by altering cell–cell junctional proteins (E-cadherin, Occludin, Zonulin-1). Targetingnecroptosis through Necrostatin-1 significantly reduces intestinal inflammation in vitro and in culturedintestinal explants from IBD.Conclusion: We show for the first time in vitro and ex vivo that RIP3-driven necroptosis seriously affectsintestinal inflammation by increasing pMLKL, activating different cytokines and alarmins, and alteringepithelial permeability. The inhibition of necroptosis causes a significant decrease of all these effects.These data strongly support the view that targeting necroptosis may represent a promising new optionfor the treatment of inflammatory enteropathies