Upregulation of toll-like receptor 4 (TLR4)/MD-2 complex in LPS hypersensitive livers in mice

Background: LPS sensitivity in the liver significantly worsens the outcome of gram negative sepsis. MD-2 facilitates the recognition of LPS by Toll-like receptor (TLR4). In this study we investigated the changes in TLR4 and MD-2 levels in LPS sensitive livers. Methods: C57BL/6 mice (8/group) were primed with heat-killed P. acnes (1mg, i.p., 7 day) or methionine-choline deficient (MCD) diet (3 week feeding) and stimulated with LPS (0.5mg/g b.w.). Liver histopathology (H&E), serum cytokine (ELISA), alanine aminotransferase ALT levels, MD-2 mRNA (qualitative real time-PCR) and protein levels (immunoblot) were assessed. Results: In P. acnes primed mice LPS challenge resulted resulted in extensive inflammatory cell recruitment with necrosis in the liver and significantly higher serum cytokine levels (TNFα, IFNγ, IL-12p40; p<.04) vs. non-primed mice. LPS challenge in MCD diet resulted in steatohepatitis with extended inflammatory cell invasion and significantly higher serum TNFα, IL-6 and IL-12p40 (p<.03), ALT levels (p<0.01) vs. control fed mice. In livers primed by P. acnes TLR4 protein (p<.03), MD-2 protein (p<.04) and LPS induced MD-2 mRNA expression (p<.03) rose significantly higher compared to non-primed. Similarly, MCD feeding resulted in a marked elevation in MD-2 protein (p<.05) levels and MD-2 mRNA levels (p<.04) vs. controls. In summary, in the fulminant hepatitis (P. acnes) as well as in the steatohepatitis (MCD) model increased baseline TLR4 and MD-2 protein levels and LPS induced MD-2 levels were detected. These suggest common pathophsysiological mechanism and a possible therapeutical target for LPS hypersensitivity.