Expression of TLR-2, TLR-4, NOD2 and pNF-kappaB in a neonatal rat model of necrotizing enterocolitis.

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

Scales used for the semiquantitation of the small bowel mucosal epithelial cells positive for immunohistochemical staining with an anti caspase-3 antibody.

<p>(0): with 0 to 2 cells; (1): with 2 to 5 cells; (2): with 5 to 20 cells; (3): with 20 to 50 cells; (4): more than 50 cells. Representative images are shown (original magnification 400×).</p

mRNA expression levels of TLR-2 and TLR-4 from distal jejunum were analysed by real-Time-PCR under basal and NEC conditions.

<p>(a) TLR-2 and (b) TLR-4 mRNA expression levels from distal jejunum. Data represent the means±SEM of 10 mice per group. *P<0.05 and **P<0.01, significantly different from group 1.</p

Study of morphological mucosal changes of the small bowel in NEC-induced in rats pups.

<p>(a) Histological analysis in the three pups groups (group 1: full term and were mother-fed; group 2: preterm and mother-fed; group 3: preterm, hand-gavaged with a protein rich formula after a hypoxia–reoxygenation procedure). according to the histological classification as showing in (b): intact morphology of the villi (a); sloughing of villi tips (b); mild-villous necrosis (c); loss of villi (d); complete destruction of the mucosa, transmural necrosis (e). (c) Severity of NEC in the different pups groups. Original magnifications: 120×(a,b,c,d), 60×(e).</p

Immunohistochemistry using anti anti-pNF-κB antibody in ileal samples.

<p>In group 1, there is no nuclear staining in an intestinal villous (a and e) whereas in group 2 (scored as scale (b) in H&E-stained sections), some positive cells are observed (b). These positive cells are much more numerous in the group 3 (scored as scale (c) in H&E-stained section) (c and d). (Original magnification ×400 and 800 respectively).</p

Immunohistochemistry using anti-TLR 2 antibody in ileal samples.

<p>(a) In control group, TLR-2 positive cells were located in the crypts, with weak and apical cytoplasmic staining. (b) In group 2 (scored as scale (b) in H&E-stained sections) and (c) in group 3 combining the 3 factors (scored as scale (d) in H&E-stained sections), a strong cytoplasmic TLR-2 staining was observed in the crypts and in the villi (Original magnification 200×, 400×).</p

Immunohistochemical features of epithelial cells positive for anti Ki-67 antibody staining in the small bowel mucosa.

<p>(a) normal staining is limited to the crypts; (b) abnormal staining extends beyond the crypts, and is continuous, irregular or spreads into the covering villi (<i>arrowheads</i>). (c) Histological analysis of KI-67 localisation. Representative images are shown (original magnification 400×)</p