Role of LPS/CD14/TLR4-mediated inflammation in necrotizing enterocolitis: pathogenesis and therapeutic implications

Abstract

Aim: To establish the roles of lipopolysaccharide (LPS)/CD14/toll-like receptor 4 (TLR4)-mediated inflammation in a rat model of human necrotizing enterocolitis (NEC).

Methods: Six pairs of intestinal samples from human NEC were collected before and after recovery for histological and molecular analysis of inflammatory cytokines and signaling components. In the rat NEC model, we isolated 10-cm jejunum segments and divided them into six groups (n = 6) for sham operation, treatment with LPS, bowel distension, combined bowel distension and LPS stimulation, and two therapeutic groups. The potential efficacy of a recombinant CD18 peptide and a monoclonal CD14 antibody was evaluated in the latter two groups. The serum and tissue levels of several inflammatory mediators were quantified by real-time polymerase chain reaction, ELISA and immunoblotting.

Results: Human acute phase NEC tissues displayed significant increases (P < 0.05) in levels of TLR4, CD14, myeloid differentiation protein (MD)-2, tumor necrosis factor (TNF)-alpha and nuclear factor-kappaB when compared to those after recovery. The histological and inflammatory picture of human NEC was reproduced in rats that were treated with combined bowel distension and LPS, but not in the sham-operated and other control rats. Serum levels of interleukin-6 and TNF-alpha were also elevated. The NEC pathology was attenuated by treating the NEC rats with a monoclonal CD14 antibody or an LPS-neutralizing peptide.

Conclusion: LPS and distension are required to produce the histological and inflammatory features of NEC. A potential treatment option is blocking LPS activation and leukocyte infiltration.

Figure 1

Pathological features of human necrotizing enterocolitis (NEC). A: Representative micrographs that show the microarchitecture of (upper panel), and the extent of macrophage infiltration (as marked by arrow) (lower panel) in jejunal tissues from NEC patients, which were collected in the acute phase and during recovery. Magnification × 200; B: Immunoblotting showed overexpression of TNF-α and upregulated nuclear content of the p65 subunit of NF-κB in jejunal tissues from NEC patients in the acute phase. Nuclear histone level was used as the loading control; C: Real-time PCR detected the mRNA transcript levels of CD14, TLR4 and MD-2 in jejunal tissues from NEC patients in the acute phase and during recovery. Statistical significance was determined by Student's t test, and all data are presented as the mean ± SD of triplicate samples from two independent experiments.

Figure 2

Histological features of rat jejunum. The severity of damage in the jejunum of rats from the different treatment groups was assessed histologically after HE staining. A: Sham-operated controls; B: LPS jejunal injection; C: Saline jejunal distension; D: Saline jejunal distension and LPS jejunal injection; E: Saline jejunal distension and LPS jejunal injection, and CD14 antibody treatment; F: Saline jejunal distension and LPS jejunal injection, and LPS-neutralizing peptide treatment. Only in (D) did the jejunal tissue show histological changes with a score of 2 for NEC (sloughing of villi and vascular congestion). Magnification × 200.

Figure 3

Macrophage infiltration into the rat jejunum. The extent of macrophage infiltration into the jejunum of rats from the different treatment groups was evaluated by immunohistochemistry using a macrophage staining reagent. Stained macrophages are marked by arrows. A: Sham-operated control; B: LPS jejunal injection; C: Saline jejunal distension; D: Saline jejunal distension and LPS jejunal injection; E: Saline jejunal distension and LPS jejunal injection, and CD14 antibody treatment; F: Saline jejunal distension and LPS jejunal injection, and LPS-neutralizing peptide treatment. Magnification × 200.

Figure 4

CD14, TLR4 and MD-2 mRNA expression in rat jejunum. Real-time PCR detected mRNA transcript levels for CD14, TLR4 and MD-2 in the jejunum of rats from the different treatment groups. Distension of jejunum with LPS-containing saline upregulated the expression of all studied transcripts, and these upregulated levels were attenuated by CD14 antibody and LPS-neutralizing peptide treatments. There were six rats in each experimental group. Data are displayed as the mean ± SD, and analyzed statistically by one-way ANOVA. ^aP = 0.00126; ^bP = 0.00498; ^cP = 0.00838; ^dP = 0.0127; ^eP = 0.0151; ^fP = 0.0187.

Figure 5

Serum TNF-α and IL-6 levels in NEC rats. Serum TNF-α and IL-6 levels in all NEC rats were measured by commercial ELISA kits. Distension of jejunum with LPS-containing increased significantly serum TNF-α and IL-6 levels, and these increases were suppressed when CD14 antibody and LPS-neutralizing peptide were added to the saline. There were six rats in each experimental group. Data are displayed as the mean ± SD, and analyzed statistically by one-way ANOVA. ^aP = 0.0237; ^bP = 0.0259; ^cP = 0.0819; ^dP = 0.0477.