Journal Article

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

Egan, Charlotte E., Billiar, Timothy R., Branca, Maria F., Lin, Joyce, Yazji, Ibrahim, Lee, Elizabeth K., Good, Misty, Sodhi, Chhinder P., Jia, Hongpeng, Richardson, Ward M., Binion, David G., Neal, Matthew D., Prindle, Thomas, Gladwin, Mark T., Afrazi, Amin, Hackam, David J., Ozolek, John, Ma, Congrong

2013

Overview

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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Analysis of Variance / Animals / Animals, Newborn / Babies / Biological Sciences / Blood circulation / Breast milk / Endothelial cells / endothelial nitric oxide synthase / Endothelium / enterocolitis / Enterocolitis, Necrotizing - drug therapy / Enterocolitis, Necrotizing - etiology / Enterocolitis, Necrotizing - metabolism / genes / humans / Infant Formula - chemistry / Infant Formula - pharmacology / Intestinal Mucosa - blood supply / Intestines / ischemia / lipopolysaccharides / Mice / Mice, Knockout / Microcirculation - drug effects / Microcirculation - physiology / Microscopy, Confocal / Milk / Milk, Human - chemistry / necrosis / Necrotizing enterocolitis / Nitrates / Nitrates - analysis / Nitrates - metabolism / Nitric oxide / Nitric Oxide - metabolism / Nitric Oxide Synthase Type III - genetics / Nitric Oxide Synthase Type III - metabolism / Nitrites / Nitrites - metabolism / Perfusion / Piperazines - pharmacology / Piperazines - therapeutic use / premature birth / Purines - pharmacology / Purines - therapeutic use / repletion / Rodents / Signal transduction / Signal Transduction - drug effects / Signal Transduction - physiology / Sildenafil Citrate / small intestine / Sodium / sodium nitrate / Sodium nitrates / Sulfones - pharmacology / Sulfones - therapeutic use / T cell receptors / Toll-like receptor 4 / Toll-Like Receptor 4 - deficiency / Toll-Like Receptor 4 - metabolism