The Roles of Bacteria and TLR4 in Rat and Murine Models of Necrotizing Enterocolitis

Abstract

Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes.

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).…”

“…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

“…The reason for this hypersensitivity is only partly understood but recent findings have shed some light on the underlying mechanisms. Unlike in the mature, healthy intestinal epithelial cell, where TLR4 is poorly expressed, in the neonate that has experienced asphyxia and formula feeding, TLR4 is up-regulated on the luminal side of the intestinal epithelium, thereby allowing for gram negative bacteria or their cell wall components to activate TLR4 signaling [73]. Another line of evidence suggests that IĸB expression in epithelial cells is lower in fetal and premature neonatal intestine and activation of TLR signaling results in higher levels of IĸB phosphorylation in these premature cells than in more mature enterocytes [72].…”

“…These findings together suggest that a combination of higher receptor expression, diminished inhibitory capacity by IĸB and more active signaling contribute to the exaggerated NFĸB activation and inflammatory mediator production in the premature gut. The pathological significance of TLR4-dependent and NFĸB-mediated proinflammatory signaling in NEC is underscored by the observation in two independent studies that TLR4 mutant mice are protected from experimental NEC and additional findings that NFĸB inhibitors reduce the risk for experimental NEC [71,73].…”