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Reciprocal Expression and Signaling of TLR4 and TLR9 in the Pathogenesis and Treatment of Necrotizing Enterocolitis

We show that the the reciprocal nature of TLR4 (the receptor for bacterial endotoxin, LPS) and TLR9 (the receptor for bacterial DNA, CpG-DNA) signaling within the neonatal intestine plays a role in the development of NEC and provides novel therapeutic approaches to this disease. Using a novel ultrasound-guided in utero injection system, we administered LPS directly into the stomachs of early and late gestation fetuses to induce TLR4 signaling and demonstrated that TLR4-mediated signaling within the developing intestine follows its expression pattern. Murine and human NEC were associated with increased intestinal TLR4 and decreased TLR9 expression, suggesting that reciprocal TLR4 and TLR9 signaling may occur in the pathogenesis of NEC. Mice treated enternally with an adenovirus expressing a mutant form of TLR4 increased NEC severity and increased intestinal TLR9 expression. Treatment of mice with CpG-DNA significantly reduced NEC severity, whereas TLR9-deficient mice exhibited increased NEC severity.

 

As shown above, TLR4 and TLR9 are reciprocally expressed in the developing intestinal mucosa and in experimental and human NEC. A, Quantitative RT-PCR showing the expression relative to _-actin of TLR4 (blue) and TLR9 (red) in the intestine of Swiss Webster mice during gestation and in the postnatal period (P0) at the indicated times. Shown are mean and SEM from three separate experiments with 3 animals per group; _, p _ 0.05 vs expression of TLR4 in E14 animals by ANOVA; †, p _ 0.05 vs expression of TLR9 in E14 animals by ANOVA. B–E, Embryonic stomach was injected with fluorescein-labeled LPS (5 _l of 1 mg/ml solution) at E14 and E18 as described in Materials and Methods using ultrasound-guided backscatter microscopy. The ultrasound micrographs made during injection at E14 (B) and E18 (C) are shown in which the locations of the needle and stomach can be seen. Fluorescent emission of the harvested intestine at E14 (D) and E18 (E). F, Quantitative RT-PCR showing the expression of the proinflammatory cytokines IL-6 (blue) and inducible NO synthase (iNOS; red) in the intestine of mice that were injected with either LPS or saline (control) at E14 or E18 as shown. G, SDS-PAGE showing the expression of TLR4 and TLR9 in small intestinal mucosa obtained from healthy controls and patients or mice with NEC. Results shown are representative of four separate experiments.

 
Authors
Gribar SC, Sodhi CP, Richardson WM, Anand RJ, Gittes GK, Branca MF, Jakub A, Shi XH, Shah S, Ozolek JA, Hackam DJ
J Immunol
2009 Jan 1
 
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Last Update
January 22, 2011
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Last Update
January 22, 2011
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