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Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.

Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in preterm infants. It results from an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin into the circulation. Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In this article, we develop a mathematical model of migration of enterocytes during experimental necrotizing enterocolitis. The model is based on a novel assumption of elastic deformation of the cell layer and incorporates the following effects: i), mobility promoting force due to lamellipod formation, ii), mobility impeding adhesion to the cell matrix, and iii), enterocyte proliferation. Our model successfully reproduces the behavior observed for enterocyte migration on glass coverslips, namely the dependence of migration speed on the distance from the wound edge, and the finite propagation distance in the absence of proliferation that results in an occasional failure to close the wound. It also qualitatively reproduces the dependence of migration speed on integrin concentration. The model is applicable to the closure of a wound with a linear edge and, after calibration with experimental data, could be used to predict the effect of chemical agents on mobility, adhesion, and proliferation of enterocytes.

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.

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.

Background & Aims: Experimental necrotizing enterocolitis (NEC) is characterized by circulating endotoxin (lipopolysaccharide [LPS]) and impaired enterocyte migration. We hypothesized that LPS increases integrin function and cell-matrix adhesion, leading to impaired enterocyte migration in the pathogenesis of NEC., Methods: NEC-like intestinal injury was induced in newborn rats by hypoxia/gavage feedings, and restitution was determined by assessing bromodeoxyuridine-labeled enterocytes along the crypt-villus axis. Newborn mice were injected with 5 mg/kg LPS. IEC-6 cells were treated with LPS +/- LY294002 or wortmannin, and beta 1- and alpha 3-integrins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescence. Beta 1-integrin function was determined by adherence of fibronectin beads to IEC-6 monolayers. Migration of IEC-6 cells into a scraped wound was measured by time-lapse microscopy., Results: Newborn intestinal injury was associated with decreased intestinal restitution and increased alpha 3- and beta 1-integrin expression in the ileal mucosa, which also was observed after LPS injection. In IEC-6 cells, LPS caused an increase in the expression of alpha 3- and beta 1-integrins, a shift of beta 1-integrins from the cytoplasm to the plasma membrane and an increase in fibronectin bead adhesion during which beta 1-integrins accumulated underneath attached beads. These effects could be reversed with LY294002 or wortmannin, suggesting phosphatidylinositol-3-phosphate kinase (PI3K) dependence. The increased integrin-matrix adhesion by LPS led to an inhibition of enterocyte migration, which could be reversed by anti-beta 1-antibodies., Conclusions: Enterocyte migration is inhibited by LPS through increased expression and function of alpha 3- and beta 1-integrins. Modulation of enterocyte migration via integrins may provide novel insights into the pathogenesis of NEC, in which intestinal restitution is impaired.