64.02 Post-operative Ileus is Reduced by Pharmacologic and Genetic Inhibition of Toll-like Receptor 4

M. L. Kovler¹, C. P. Sodhi¹, M. R. Ladd¹, A. Werts¹, W. B. Fulton¹, T. Prindle¹, S. Wang¹, Y. Yamaguchi¹, D. J. Hackam^{1  1}Johns Hopkins University School Of Medicine,Baltimore, MD, USA

Introduction:

Post-operative ileus (POI) occurs frequently after abdominal surgery and leads to increased morbidity and prolonged hospitalization. Inflammation within the gut wall triggered by intestinal manipulation is recognized as an inciting event in post-operative ileus, suggesting that the innate immune receptor toll-like receptor 4 (TLR4) may be involved. We have recently identified a novel TLR4 inhibitor, C34, which is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which is safe and effective in a variety of pre-clinical studies of gut inflammation. We now hypothesize that TLR4 activation mediates POI, and that pharmacologic TLR4 inhibition with C34 will reduced this postoperative condition.

Methods:

Post-operative ileus was induced by standardized small bowel manipulation in adult wild-type (Tlr4^WT) and TLR4 knockout (Tlr4^-/-) mice, which we previously generated. Twenty-four hours after manipulation, GI transit was measured by treating mice with fluorescent dye by oral gavage. After 60 minutes, mice were sacrificed, and gastrointestinal motility was expressed as small intestinal transit (%) = the fluorescent pass distance/the total length of small intestine x 100. GI mucosal inflammation was characterized by quantitative RT-PCR measuring expression of TNFa, IL-1b, Lipocalin-2, iNOS, RORC, and FOXP3 from ileal segments. The mice in the control group did not undergo any operation. Motility and inflammatory indices were measured in mice treated with lipopolysaccharide (LPS), the main TLR4 ligand. To evaluate the effects of pharmaceutical inhibition of TLR4, the same experiments were conducted in Tlr4^WT mice after intraperitoneal injection with C34. Comparisons were by student’s t-test with p<0.05.

Results:

In Tlr4^WT and Tlr4^-/- control mice, the degree of GI motility was similar (Tlr4^WT: 100% vs. Tlr4^-/-: 95.7% p=.41), and the addition of C34 to unperturbed Tlr4^WT mice had no effect on motility (saline: 90% vs C34: 93%, NS). By contrast, Tlr4^WT mice treated with the TLR4 agonist LPS (dose 3mg/kg) showed decreased gut motility (saline: 87.5% vs. LPS 60.4%, p<0.05) and also significantly increased gut inflammation as represented by TNFa level (saline: 1.39 vs LPS 10.31, p<0.05). The induction of post-operative ileus resulted in significantly reduced transit (Tlr4^WT: 44.6%, Tlr4^-/-: 57.9% p<0.05) and increased inflammation (Tlr4^WT: 21.3, Tlr4^-/-: 9.91 p<0.05), which were milder in Tlr4^-/- mice. Strikingly, treatment of mice with the TLR4 inhibitor C34 significantly reduced inflammation and improved motility after intestinal manipulation, consistent with reversal of this post-operative complication.

Conclusion:
 

These results prove that experimental post-operative ileus is mediated through a TLR4-dependent inflammatory pathway and reveal that a novel TLR4 inhibitor can attenuate inflammation and dysmotility.