Systemic multi-organ injury is an important component associated with sepsis. Multi-organ dysfunction during sepsis can be caused by a myriad of factors, including endotoxin and systemic production of proinflammatory cytokines, such as TNFalpha and IL-1beta. The liver is a major source of cytokine expression in response to endotoxin and hence plays a critical role in mediating systemic organ injury during sepsis. Hepatic responses to LPS that control proinflammatory cytokine production include a number of receptor-mediated signal transduction pathways, of which NF-kappaB is one of the most important. In this context, NF-kappaB activation in the liver directly controls the induction of TNFalpha in response to LPS and mediates signals that are important to hepatic cell survival. We have determined that IL-1beta and LPS share interesting similarities and differences in their mechanisms of NF-kappaB activation. Both involve redox-sensitive pathways that appear to be controlled by NADPH oxidase activation and converge at the level of the IKB kinase (IKK) complex. LPS and IL-1beta activation of NF-kappaB also utilize two related receptors (TLR4 and IL-1R) that share similar effector complexes. Despite the similar dependence of both these pathways on the intracellular production of reactive oxygen species (ROS), LPS and IL-1beta activate NF-kappaB through distinct subunits of the IKK complex. The manner in which ROS uniquely control NF-kappaB activation by LPS and IL-1beta remains unclear. This project proposes to dissect the redox-mediated events that control these activation pathways using both in vitro and in vivo mouse models. In vitro studies will evaluate LPS and IL-1beta responses in hepatocytes and Kupffer cell line models in order to identify the redox-regulated components of the TLR4 and IL-1R receptor complexes that mediate activation of specific IKK kinases. An important aspect of these mechanisms involves the formation of redox-active endosomes (termed redoxosomes) that appear to cluster ligand-activated receptors into NADPH oxidase active endosomes. We hypothesize that redoxosomes help partition ROS to redox-dependent TLR4 and IL-1R effector domains. Through this process, endosomal compartmentalization of ROS allows only ligand-activated receptor/effector complexes to be influenced by NADPH oxidase. In vivo studies will utilize mouse models of endotoxemia to study redoxosomal functions in vivo and their contribution to hepatic NF-kappaB activation pathways. Findings from these studies may lead to alternative therapeutic approaches targeting the liver by which to abrogate the detrimental effects of systemic cytokine production during sepsis.
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