A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair

Abstract

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.

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

“…After prolonged exposure to LPS, as was observed in a model of chronic murine endotoxemia in experimental NEC, the expression of CD14 within the intestinal mucosa is decreased. Given recent findings from our laboratory and the Caplan laboratory demonstrating the importance of LPS-mediated signaling in the pathogenesis of NEC (9,10), we now propose a novel role for CD14 in the regulation of LPS signaling, potentially contributing to the initiating events in the development of NEC.…”

“…Our group and the group of Caplan and colleagues have recently shown that TLR4 plays a key role in the development of experimental NEC in vivo, as animals with mutations in TLR4 were found to be protected from the development of this disease as compared to wild-type counterparts (9,10). We have shown that the role of TLR4 in the pathogenesis of NEC involves an exacerbation of enterocyte injury and an inhibition of mucosal repair mechanisms (9). Expression of TLR4 was also found to be increased in the early stages of NEC (9,10).…”

“…We have shown that the role of TLR4 in the pathogenesis of NEC involves an exacerbation of enterocyte injury and an inhibition of mucosal repair mechanisms (9). Expression of TLR4 was also found to be increased in the early stages of NEC (9,10). LPS-dependent TLR4 activation is known to involve a family of co-receptors including MD2 and CD14 (11).…”

Intestinal Epithelial Cell Apoptosis, Immunoregulatory Molecules, and Necrotizing Enterocolitisb

“…An evidence for such defective repair mechanisms was found when the role of TLR4 was evaluated in a neonatal murine model of NEC. It was found that in experimental NEC, in addition to increased apoptosis as was shown earlier, there was a reduced epithelial cell proliferation and defective migration along the crypt villus axis; these defects required intact TLR4 signaling and were accompanied with an increased phosphorylation of focal adhesion kinase (FAK) on S722, i.e., a phosphorylation state of FAK that inhibits epithelial cell migration [82]. In vitro, TLR4 activation resulted in FAK phosphorylation and inhibited enterocyte migration.…”

“…Additionally, we have shown that PAF does not only directly regulate apoptosis, but that signaling via the PAFR induces TLR4 expression in enterocytes (unpublished data). TLR4, the receptor for LPS, has been shown to take part in NEC pathogenesis [73,82], causes enterocyte apoptosis [101] and defective enterocyte migration [82]. Furthermore, TLR4 activation leads to induction of inflammatory molecules such as TNFα [102] and nitric oxide (NO) [103]; both of these molecules have been shown to be involved in NEC pathogenesis [15,81,104] and have been shown to be pro-apoptotic for enterocytes [81,105].…”