Background: The etiology of necrotizing enterocolitis (NEC) results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC.
Methodology/principal findings: We specifically evaluated intestinal epithelial cell (IEC) expression of Toll-like receptor 2 (TLR-2), TLR-4, NOD2 and phosphorylated NF-kappaB (pNF-kappaB) after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1), neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2) or hand-gavaged with a protein rich formula after a hypoxia-reoxygenation procedure (group 3). Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-kappaB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia-reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-kappaB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged.
Conclusions/significance: These results show that this rat model of NEC induced mucosal injury, leading to a highly responsive IEC phenotype and suggesting that alterations in the innate immune system participates in the pathogenesis of NEC and are enhanced by prematurity.