Adam D. Werts 1 , William B. Fulton 2 , Mitchell R. Ladd 2 , Ali Saad-Eldin 2 , Yue X. Chen 2 , Mark L. Kovler 2 , Hongpeng Jia 2 , Emilyn C. Banfield 3 , Rachael H. Buck 4 , Karen Goehring 4 , Thomas Prindle Jr. 2 , Sanxia Wang 2 , Qinjie Zhou 2 , Peng Lu 2 , Yukihiro Yamaguchi 2 , Chhinder P. Sodhi 2 , ∗∗ , David J. Hackam 2 , 3 , ∗
19 November 2019
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
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.