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      A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis

      1 , 2 , 2 , 2 , 2 , 2 , 2 , 3 , 4 , 4 , 2 , 2 , 2 , 2 , 2 , 2 , ∗∗ , 2 , 3 ,

      Cellular and Molecular Gastroenterology and Hepatology


      Pediatrics, Premature, Organoid Model, diff, differentiated, HMGB1, high mobility group box 1, IEC, intestinal epithelial cell, IHC, immunohistochemistry, IL, interleukin, LPS, lipopolysaccharide, Mlkl, mixed lineage kinase domain-like protein, NEC, necrotizing enterocolitis, p, postnatal day, PCNA, proliferating cell nuclear antigen, pRIPK, phosphorylated receptor-interacting serine/threonine-protein kinase, qRT-PCR, real-time quantitative reverse-transcription polymerase chain reaction, Ripk, receptor-interacting serine/threonine-protein kinase, ROI, region of interest, TBST, Tris-buffered saline with 0.1% Tween-20, TLR4, Toll-like receptor 4, TNF, tumor necrosis factor, undiff, undifferentiated, 2’FL, 2'-fucosyllactose, 3NT, 3-nitrotyrosine

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          Background & Aims

          Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by Toll-like receptor 4 (TLR4)-dependent intestinal inflammation and enterocyte death. Given that necroptosis is a proinflammatory cell death process that is linked to bacterial signaling, we investigated its potential role in NEC, and the mechanisms involved.


          Human and mouse NEC intestine were analyzed for necroptosis gene expression (ie, RIPK1, RIPK3, and MLKL), and protein activation (phosphorylated RIPK3). To evaluate a potential role for necroptosis in NEC, the effects of genetic (ie, Ripk3 knockout or Mlkl knockout) or pharmacologic (ie, Nec1s) inhibition of intestinal inflammation were assessed in a mouse NEC model, and a possible upstream role of TLR4 was assessed in Tlr4-deficient mice. The NEC-protective effects of human breast milk and its constituent milk oligosaccharides on necroptosis were assessed in a NEC-in-a-dish model, in which mouse intestinal organoids were cultured as either undifferentiated or differentiated epithelium in the presence of NEC bacteria and hypoxia.


          Necroptosis was activated in the intestines of human and mouse NEC in a TLR4-dependent manner, and was up-regulated specifically in differentiated epithelium of the immature ileum. Inhibition of necroptosis genetically and pharmacologically reduced intestinal–epithelial cell death and mucosal inflammation in experimental NEC, and ex vivo in the NEC-in-a-dish system. Strikingly, the addition of human breast milk, or the human milk oligosaccharide 2 fucosyllactose in the ex vivo system, reduced necroptosis and inflammation.


          Necroptosis is activated in the intestinal epithelium upon TLR4 signaling and is required for NEC development, and explains in part the protective effects of breast milk.

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          Most cited references 46

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

          Dysfunction of the intestinal epithelium is believed to result in 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 1 . Beside the physical barrier established by the tight contact of cells, 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 2 . Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a pathogenic mechanism driving Crohn's disease (CD) in humans 3 . However, the regulation of epithelial cell death and its role in intestinal homeostasis remains poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IEC) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8 ΔIEC) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8 ΔIEC mice lacked Paneth cells and showed reduced numbers of goblet cells suggesting dysregulated anti-microbial immune cell functions of the intestinal epithelium. Casp8 ΔIEC mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumor necrosis factor (TNF) -α, was associated with increased expression of receptor-interacting protein 3 (RIP3) and could be inhibited upon blockade of necroptosis. Finally, 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. Taken together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IEC from TNF-α induced necroptotic cell death.
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            Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

            Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.
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              Kinase RIP3 Is Dispensable for Normal NF-κBs, Signaling by the B-Cell and T-Cell Receptors, Tumor Necrosis Factor Receptor 1, and Toll-Like Receptors 2 and 4

              RIP3 is a member of the RIP kinase family. It is expressed in the embryo and in multiple adult tissues, including most hemopoietic cell lineages. Several studies have implicated RIP3 in the regulation of apoptosis and NF-κB signaling, but whether RIP3 promotes or attenuates activation of the NF-κB family of transcription factors has been controversial. We have generated RIP3-deficient mice by gene targeting and find RIP3 to be dispensable for normal mouse development. RIP3-deficient cells showed normal sensitivity to a variety of apoptotic stimuli and were indistinguishable from wild-type cells in their ability to activate NF-κB signaling in response to the following: human tumor necrosis factor (TNF), which selectively engages mouse TNF receptor 1; cross-linking of the B- or T-cell antigen receptors; peptidoglycan, which activates Toll-like receptor 2; and lipopolysaccharide (LPS), which stimulates Toll-like receptor 4. Consistent with these observations, RIP3-deficient mice exhibited normal antibody production after immunization with a T-dependent antigen and normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Thus, we can exclude RIP3 as an essential modulator of NF-κB signaling downstream of several receptor systems.

                Author and article information

                Cell Mol Gastroenterol Hepatol
                Cell Mol Gastroenterol Hepatol
                Cellular and Molecular Gastroenterology and Hepatology
                19 November 2019
                : 9
                : 3
                : 403-423
                [1 ]Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland
                [2 ]Division of Pediatric Surgery, Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland
                [3 ]McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
                [4 ]Abbott Nutrition, Columbus, Ohio
                Author notes
                [] Correspondence Address correspondence to: David Hackam, MD, Department of Surgery, Johns Hopkins University, The Johns Hopkins Children’s Center, Room 7323, 1800 Orleans Street, Baltimore, Maryland 21287. fax: (410) 502-5314. Dhackam1@
                [∗∗ ]Chhinder Sodhi, PhD, Department of Surgery, Johns Hopkins University, Miller Research Building, 733 North Broadway, Room 470, Baltimore, Maryland 21205. fax: (410) 502-5314. csodhi@
                © 2020 The Authors

                This is an open access article under the CC BY-NC-ND license (

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