title: Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis.
abstract: Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.
authors.affiliation: Division of Pediatric Surgery Children's Hospital of Pittsburgh and Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Neal
authors.forename: Chhinder P
authors.initials: CP
authors.lastname: Sodhi
authors.forename: Hongpeng
authors.initials: H
authors.lastname: Jia
authors.forename: Mitchell
authors.initials: M
authors.lastname: Dyer
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: Egan
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Yazji
authors.forename: Misty
authors.initials: M
authors.lastname: Good
authors.forename: Amin
authors.initials: A
authors.lastname: Afrazi
authors.forename: Ryan
authors.initials: R
authors.lastname: Marino
authors.forename: Dennis
authors.initials: D
authors.lastname: Slagle
authors.forename: Congrong
authors.initials: C
authors.lastname: Ma
authors.forename: Maria F
authors.initials: MF
authors.lastname: Branca
authors.forename: Thomas
authors.initials: T
authors.lastname: Prindle
authors.forename: Zachary
authors.initials: Z
authors.lastname: Grant
authors.forename: John
authors.initials: J
authors.lastname: Ozolek
authors.forename: David J
authors.initials: DJ
authors.lastname: Hackam
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D049109:Cell Proliferation; D020345:Enterocolitis, Necrotizing / Q000378:metabolism / Q000473:pathology; D055786:Gene Knockout Techniques; D007082:Ileum / Q000473:pathology; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D051017:Apoptosis Regulatory Proteins; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D043562:Receptors, G-Protein-Coupled; D047888:Receptors, Tumor Necrosis Factor, Type I; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D025521:Tumor Suppressor Proteins; C493597:Lgr5 protein, mouse; C507780:Myd88 protein, mouse; C488588:PUMA protein, mouse; C476286:Ticam1 protein, mouse; C493487:Tlr4 protein, mouse; C506236:Tnfrsf1a protein, mouse