Concanavalin A-induced hepatitis: Gut-derived LPS, intestinal permeability, and TLR4
Beth L. Mallard1,4, Ayako Mabuchi1,4, Sachiko Akashi-Takamura2, Jacguie Harper3, Leo R. Quinlan1, Antony M. Wheatley1,4
1Physiology, NUI Galway, Galway, Ireland; 2Institute of Medical Sciences, University of Tokyo, Tokyo, Japan; 3Malaghan Institute of Medical Research, Wellington, New Zealand; 4Physiology, University of Otago, Dunedin, New Zealand
Background & Aims The plant lectin Concanavalin A (ConA) induces T cell-mediated acute hepatitis in mice that is manifested by histological changes and elevated plasma ALT up to 24 hrs after injection. TLR4 has been implicated in a wide range of liver patologies including ischaemia-reperfusion injury, hepatitis B and C and primary and secondary biliary cirrhosis. Here we tested the hypothesis that the LPS/TLR4 pathway played a role in ConA-induced hepatitis. Materials & Methods The effect of iv administration of ConA was tested in TLR4-/and wildtype C57/BL6, in C3H/HeN (LPS-sensitive) and C3H/HeJ mice (LPS-insensitive) and in C57/BL6 mice pretreated with the anti-TLR4 mAb, MTS510. Plasma levels of cytokine INF-γ, TNF-α and IL-6 were measured using the BioPlex suspension array system. The effect of ConA on TLR4 expression by T cells and Kupffer cells was FACS analysis. To test if gut-derived bacteria were involved, conA was given to mice treated for 5 days with antibiotics (Polymyxin B/Neomycin). Gut permeability was tested both in vivo (FITCalbumin by oral gavage) and in vitro (Ussing chamber experiment using native colon segments). Blood flow in the hepatic microcirculation was measured by laser Doppler flowmetry (LDF) and by intravital microscopy (IVM). Results ConA caused elevation of plasma ALT and histological sings of injury in C57/BL6 and C3H/HeN mice but not in TLR-/-, C3H/HeJ and MTS510-treated C57/BL6 mice. In mice depleted of Gram-negative bacteria, ConA did not induce liver injury. Plasma TNF-α (peak, 2 hr), INF-γ and IL-6 (peak, both 6 hr) were significantly elevated after ConA in wildtype but not in TLR4-/- or MTS510 treated mice. FACS analysis revealed that ConA administered in vivo induced increase increases in the percentage of TLR4positive CD3+ (T cells) and F4/80+ (Kupffer cells). ConA caused a 15-fold increased the permeability of the gut to FITCdextran, that was absent in TLR4-/- mice. In native colon, basolaterally applied ConA rapidly (<10min) reduced transepithelial electrical resistance (increased permeability) compared to control (∼20%). The lack of TLR4 was without impact on the ConA induced reduction in hepatic perfusion (LDF), but IVM revealed fewer rolling/adherent leukocytes in TLR4-/- mice than in control mice. Conclusions Our result indicate that ConA-induced hepatitis reguires (1)the presence of Gram-negative bacteria in the gut (2) ConA-induced increase in intestinal permeability and (3) the presence of a functionalTLR4. Thus Gram-negative bacteria or their products (eg, LPS) would appear to play a central role on ConA-induced acute hepatitis.
The following people have nothing to disclose: Beth L. Mallard, Ayako Mabuchi, Sachiko Akashi-Takamura, Jacguie Harper, Leo R. Quinlan, Antony M. Wheatley