Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis.

BACKGROUND: The early signaling events in the development of necrotizing enterocolitis (NEC) remain undefined. We have recently shown that the endotoxin (lipopolysaccharide [LPS]) receptor toll-like receptor 4 (TLR4) on enterocytes is critical in the pathogenesis of experimental NEC. Given that the membrane receptor CD14 is known to facilitate the activation of TLR4, we now hypothesize that endotoxemia induces an early upregulation of CD14 in enterocytes and that this participates in the early intestinal inflammatory response in the development of NEC. METHODS: IEC-6 enterocytes were treated with LPS (50 microg/mL), and the subcellular localization of CD14 and TLR4 was assessed by confocal microscopy. C57/Bl6 or CD14-/- mice were treated with LPS (5 mg/kg), whereas experimental NEC was induced using a combination of gavage formula feeding and intermittent hypoxia. CD14 expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse transcriptase-polymerase chain reaction, and interleukin 6 was quantified by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. RESULTS: Exposure of IEC-6 enterocytes to LPS led to an initial, transient increase in CD14 expression. The early increase in CD14 expression was associated with internalization of CD14 to a perinuclear compartment where increased colocalization with TLR4 was noted. The in vivo significance of these findings is suggested as treatment of mice with LPS led to an early increase in CD14 expression in the intestinal mucosa, whereas the persistent endotoxemia of experimental NEC was associated with decreased CD14 expression within enterocytes. CONCLUSIONS: LPS signaling in the enterocyte is marked by an early, transient increase in expression of CD14 and redistribution of the receptor. This process may contribute to the early activation of the intestinal inflammatory response that is observed in the development of NEC.

A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and is characterized by translocation of LPS across the inflamed intestine. We hypothesized that the LPS receptor (TLR4) plays a critical role in NEC development, and we sought to determine the mechanisms involved. We now demonstrate that NEC in mice and humans is associated with increased expression of TLR4 in the intestinal mucosa and that physiological stressors associated with NEC development, namely, exposure to LPS and hypoxia, sensitize the murine intestinal epithelium to LPS through up-regulation of TLR4. In support of a critical role for TLR4 in NEC development, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates. TLR4 activation in vitro led to increased enterocyte apoptosis and reduced enterocyte migration and proliferation, suggesting a role for TLR4 in intestinal repair. In support of this possibility, increased NEC severity in C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation after mucosal injury compared with mutant mice. TLR4 signaling also led to increased serine phosphorylation of intestinal focal adhesion kinase (FAK). Remarkably, TLR4 coimmunoprecipitated with FAK, and small interfering RNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. These findings demonstrate a critical role for TLR4 in the development of NEC through effects on enterocyte injury and repair, identify a novel TLR4-FAK association in regulating enterocyte migration, and suggest TLR4/FAK as a therapeutic target in this disease.

Hypoxia causes an increase in phagocytosis by macrophages in a HIF-1alpha-dependent manner.

Phagocytosis is the process by which microbial pathogens are engulfed by macrophages and neutrophils and represents the first line of defense against bacterial infection. The importance of phagocytosis for bacterial clearance is of particular relevance to systemic inflammatory diseases, which are associated with the development of hypoxia, yet the precise effects of hypoxia on phagocytosis remain largely unexplored. We now hypothesize that hypoxia inhibits phagocytosis in macrophages and sought to determine the mechanisms involved. Despite our initial prediction, hypoxia significantly increased the phagocytosis rate of particles in vitro by RAW264.7 and primary peritoneal macrophages and increased phagocytosis of labeled bacteria in vivo by hypoxic mice compared with normoxic controls. In understanding the mechanisms involved, hypoxia caused no changes in RhoA-GTPase signaling but increased the phosphorylation of p38-MAPK significantly. Inhibition of p38 reversed the effects of hypoxia on phagocytosis, suggesting a role for p38 in the hypoxic regulation of phagocytosis. Hypoxia also significantly increased the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in macrophages, which was reversed after p38 inhibition, suggesting a link between p38 activation and HIF-1alpha expression. It is striking that small interfering RNA knockdown of HIF-1alpha reversed the effects of hypoxia on phagocytosis, and overexpression of HIF-1alpha caused a surprising increase in phagocytosis compared with nontransfected controls, demonstrating a specific role for HIF-1alpha in the regulation of phagocytosis. These data indicate that hypoxia enhances phagocytosis in macrophages in a HIF-1alpha-dependent manner and shed light on an important role for HIF-1alpha in host defense.

Toll-like receptor 4 plays a role in macrophage phagocytosis during peritoneal sepsis.

BACKGROUND: Peritoneal sepsis is a significant cause of mortality in infants with necrotizing enterocolitis, caused in part by impaired bacterial clearance. Recent studies have identified toll-like receptor-4 (TLR4) as a receptor for endotoxin (lipopolysaccharide [LPS]). We hypothesized that TLR4 regulates bacterial clearance from the peritoneal cavity and sought to investigate whether macrophage phagocytosis was involved. METHODS: Peritoneal sepsis was induced in mice expressing either functional TLR4 (TLR4-wild-type [WT]) or mutant TLR4 by intraperitoneal injection of either live Escherichia coli or LPS. Phagocytosis was assessed by measuring the uptake of opsonized red cells. To assess bacterial clearance, we irrigated peritoneal cavities of injected animals with saline and plated it on gram-negative selective media. RESULTS: LPS significantly increased the rate of phagocytosis by peritoneal macrophages from TLR4-WT mice, but not in those from TLR4-mutant mice, suggesting a role for TLR4 in phagocytosis. LPS also increased the rates of phagocytosis in cultured macrophages expressing TLR4, confirming these findings. The yield of gram-negative bacteria obtained from the peritoneal cavities of septic TLR4-WT mice was greater than that from TLR4 mutants, consistent with TLR4-dependent alterations in their septic course. CONCLUSIONS: We conclude that TLR4 plays a critical role in the response to intraperitoneal E. coli through effects on phagocytosis by macrophages, suggesting the possibility of using TLR4 as a therapeutic target in diseases of peritoneal sepsis.

Interferon-gamma inhibits intestinal restitution by preventing gap junction communication between enterocytes.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-gamma) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions. METHODS: NEC was induced in Swiss-Webster or IFN(-/-) mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and (35)S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA. RESULTS: Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN(-/-) mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution. CONCLUSIONS: IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.