Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling

Proc Natl Acad Sci U S A. 2013 Jun 4;110(23):9451-6. doi: 10.1073/pnas.1219997110. Epub 2013 May 6.


Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS(-/-) mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate--a precursor for enteral generation of nitrite and nitric oxide--and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.

Keywords: infant formula; neonatal inflammation; neonatal nutrition; prematurity; sepsis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Animals
  • Animals, Newborn
  • Enterocolitis, Necrotizing / drug therapy
  • Enterocolitis, Necrotizing / etiology*
  • Enterocolitis, Necrotizing / metabolism
  • Infant Formula / chemistry
  • Infant Formula / pharmacology
  • Intestinal Mucosa / blood supply*
  • Mice
  • Mice, Knockout
  • Microcirculation / drug effects
  • Microcirculation / physiology*
  • Microscopy, Confocal
  • Milk, Human / chemistry
  • Nitrates / analysis
  • Nitrates / metabolism
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type III / genetics
  • Nitric Oxide Synthase Type III / metabolism
  • Nitrites / metabolism
  • Piperazines / pharmacology
  • Piperazines / therapeutic use
  • Purines / pharmacology
  • Purines / therapeutic use
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Sildenafil Citrate
  • Sulfones / pharmacology
  • Sulfones / therapeutic use
  • Toll-Like Receptor 4 / deficiency
  • Toll-Like Receptor 4 / metabolism*


  • Nitrates
  • Nitrites
  • Piperazines
  • Purines
  • Sulfones
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • Nitric Oxide
  • sodium nitrate
  • Sildenafil Citrate
  • Nitric Oxide Synthase Type III
  • Nos3 protein, mouse