W. Li1,2, M. Deng1, B. Lu3, Y. Tang3, M. Scott1, Q. Wang1, T. R. Billiar1 1University Of Pittsburgh,Surgery,Pittsburgh, PA, USA 2The 3rd Xiangya Hospital Of Central South University,Surgery,Changsha, HUNAN, China 3The 3rd Xiangya Hospital Of Central South University,Department Of Hematology And Research Institute Of Immunology,Changsha, HUNAN, China
Introduction: Caspase-11, a recently described intracellular receptor for endotoxin (LPS) initiates the noncanonical inflammasome and induces pyroptotic cell death in immune cells during endotoxemia. It is unknown how LPS is delivered to cytosolic caspase-11 in endotoxemia or sepsis. We hypothesis that HMGB1, a known LPS-binding protein that contributes to lethality in sepsis, would deliver LPS to the cytosol of macrophages.
Methods: Experiments were carried out in cultured murine macrophages exposed to LPS in vitro and in a CLP sepsis model in vivo. We generated global caspase-11-/- , TLR4-/- and RAGE-/- mice as well as mice with selective deletion of caspase-11, HMGB1 or TLR4 from myeloid cells or hepatocytes. All in vivo experiments included 6-20 mice per group.
Results: In vitro experiments established that HMGB1 efficiently delivered LPS to the cytosol of cultured macrophages through RAGE-dependent uptake of HMGB1-LPS complexes. This in turn, led to LPS delivery to caspase-11 in the cytosol and pyroptosis.
In vivo, hepatocyte specific deletion of TLR4, caspase-11 or HMGB1 suppressed circulating HMGB1 levels, improved survival and bacterial clearance, and prevented immune cell death in the spleen during sepsis. In contrast, myeloid cell specific deletion of TLR4 or HMGB1 had no effect on circulating HMGB1 levels or immune cell death in sepsis, while caspase-11 deletion prevented immune cell death.
Conclusion: These findings indicate that hepatocytes are the dominant source of HMGB1 in sepsis. Surprisingly, the release of HMGB1 from the liver requires both TLR4 and caspase-11 in hepatocytes. The systemic release of HMGB1 drives caspase-11 dependent pyroptosis in immune cells. These studies identify a novel circuit that involves the LPS-sensing pathway in the liver for the regulation of systemic release of HMGB1 in sepsis. This liver-derived HMGB1 is required for caspase-11 dependent immune cell pyroptosis and lethality.