S. A. Morrison1, S. Hamarneh1, T. Tantillo1, T. Phupitakphol1, M. Gharedaghi1, K. Economopoulos1, S. Hyoju1, S. S. Gul1, M. N. Kohnehshahri1, R. A. Hodin1 1Massachusetts General Hospital,Surgery,Boston, MA, USA
Introduction: Despite the increasing prevalence and morbidity of inflammatory bowel disease (IBD), the mechanistic pathogenesis remains elusive. Established clinical practice in the management of Crohn’s disease rest on the premise that exposure to the fecal stream is intimately correlated to disease expression. This tenet is supported by numerous studies in the literature; however, the mechanism underlying the toxicity of the luminal stream has never been thoroughly studied. This study was based upon the hypothesis that constituents within the fecal stream serve to aberrantly activate TLR-mediated pathways and that the brush border enzyme, intestinal alkaline phosphatase (IAP), known for its anti-inflammatory properties, may play an antagonistic role in attenuating this dysregulated immune response.
Methods: 8 week old female C57BL/6 wild type mice were used in this study. Study groups consisted of a control and experimental group. Colitis was induced in the experimental group by the addition of 3% Dextran Sodium Sulfate (DSS) to the drinking water for 7 days, at which time all animals were sacrificed, and luminal fluid samples were collected. These samples were then applied to HEK-293 cell lines, engineered to express a specific Toll-Like Receptor (TLR); 2, 4, 5, or 9. Prior to application, samples were incubated +/- IAP. Resulting IL-8 production was then assayed.
Results: Specific TLR cell lines were first tested in vitro to demonstrate an appropriate response to known target ligands, as well as antigens that served as negative controls. These cell lines demonstrated significant IL-8 production as anticipated in response to known receptor ligands, and this response was counteracted by incubation with IAP. In studying the activation of TLR pathways in response to luminal fluid, TLR-4 and TLR-9 pathways appeared relatively quiescent when incubated with luminal contents from the control group; however incubation with luminal samples from the colitis group demonstrated a hyperactivation of these pathways (TLR 4: 640.5pg/ml v. 1675.9pg/ml, p=0.009, TLR9: 138.4pg/ml v. 1063.5pg/ml, p=0.005; Control v. DSS), suggesting a role in the pathogenesis of disease expression. This response was significantly attenuated by incubation with IAP (TL4: 1675.9pg/ml v. 1083.5pg/ml, p=0.03, TLR 9:1063.5pg/ml v. 438.4pg/ml, p=0.01; DSS v. +IAP). The TLR-2 pathway was noted to be activated by luminal contents from both groups, implying some basal activation of this pathway (TLR2: 1670.8pg/ml v. 2216.6 pg/ml, p=0.68; Control v. DSS). The TLR-5 pathway was relatively inactive in response to luminal contents from both control and colitis groups (TLR5: 29.1pg/ml v. 20.9pg/ml, p=0.17; Control v. DSS).
Conclusion: Following the initiation of DSS colitis, the luminal contents of mice contribute to the generation of inflammation, at least in part through the aberrant over-stimulation of TLR-4 and TLR-9 pathways. This response was attenuated by incubation with IAP.