P006 Intestinal alkaline phosphatase ameliorates experimental colitis via TLR4-dependent pathway
S.W. Hwang*, J.W. Lee, J. Chun, H. Yoon, C. Lee, J.P. Im, J.S. Kim
Seoul National University College of Medicine, Department of Internal Medicine and Liver Research Institute, Seoul, Korea, Republic of
Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme, and the expression of epithelial IAP was reduced in patients with inflammatory bowel disease (IBD). IAP has a protective effect on colonic inflammation possibly due to dephosphorylation of lipopolysaccharide (LPS), but the precise mechanism on colitis has not been clarified. The aim of the present study was to evaluate whether the effect of IAP was mediated via LPS/Toll-like receptor 4 (TLR4)/NF-κB pathway.
Peritoneal macrophages from wild-type (WT) and TLR4-/- mice were pretreated with IAP, and stimulated with LPS. The secretion of pro-inflammatory cytokines was measured by ELISA. The effect of IAP on NF-κB signaling was evaluated by Western blot and Electrophoretic mobility shift assay (EMSA). Immunofluorescence was performed to confirm the effect of IAP in WT and TLR4-/- macrophages. For in vivo study, dextran sulfate sodium (DSS) was given for 7 days in WT and TLR4-/- mice, and IAP was administered by oral gavage as preventive or therapeutic models. The effect of IAP on colitis was evaluated by disease activity score and histology, and NF-κB activity in colitis tissue was assessed by phosphorylated IκB-α and p65 immunohistochemistry.
The secretion of TNF-α and IL-6 was significantly inhibited by IAP in the LPS-stimulated WT macrophages, whereas the effect of IAP was much decreased in the LPS-stimulated TLR4-/- macrophages. In the WT macrophages, the IκB-α phosphorylation and NF-κB DNA binding activity were inhibited by IAP. However, the inhibition of NF-κB signaling by IAP was attenuated in the TLR4-/- macrophages. The immunofluorescence staining confirmed that the inhibitory effect of IAP on p65 nuclear translocation in the LPS-stimulated WT macrophages, but not in the TLR4-/- macrophages. In the preventive and therapeutic models of WT mice, oral administration of IAP significantly reduced loss of body weight, disease activity score and histologic grade of colitis. However, the protective effect of IAP was attenuated in the both models of TLR4-/- mice. The inhibitory effect of IAP on p65 and phosphorylated IκB-α expressions was also found in the WT colitis tissue, but the effect was decreased in the TLR4-/- colitis.
These Results revealed that IAP has a protective effect on experimental colitis mainly via TLR4-dependent pathway. The consideration of TLR4 expression or polymorphisms could be useful when administrating exogenous IAP in patients with IBD.