The innate immune system that defends mammals from many Gram-negative bacteria is triggered when host cells sense the lipid A moiety of the bacterial cell wall lipopolysaccharide (LPS). The resulting inflammatory response then usually kills the bacteria. LPS-induced inflammation can also be harmful, however, and animals have numerous ways to limit its magnitude and duration. One important control mechanism is a highly conserved lipase, acyloxyacyl hydrolase (AOAH) that selectively removes from LPS the fatty acyl chains that are required for sensing by MD-2--TLR4, the mammalian LPS receptor. Mice that have a disabling mutation in the AOAH gene respond to low doses of LPS by producing large quantities of antibodies and experiencing prolonged immunosuppression. If the LPS are given intravenously, they also develop striking enlargement of the liver. LPS that is not deacylated by AOAH remains stimulatory for weeks in vivo. We now request continuing support to test the hypothesis that providing AOAH will prevent prolonged reactions to LPS and Gram-negative bacteria in vivo, thereby benefiting animals with serious Gram-negative bacterial infections. In Specific Aim 1, we will find out how LPS deacylation by phagocytes in a subcutaneous tissue bed regulates its ability to traffic to lymph nodes and interact with a second LPS-responsive target cell, the B lymphocyte. These studies should provide the first quantitative information about how LPS processing by phagocytes prevents activation of other cells in extravascular tissue spaces. In Specific Aim 2 we will define AOAH's role in the liver, the organ that normally detoxifies most bloodborne endotoxin, and determine the cellular basis for the persistent hepatomegaly that LPS induces in Aoah-/- mice. These experiments should indicate how AOAH prevents prolonged LPS- induced reactions in the intravascular (systemic) compartment. In Specific Aim 3, we will use three complementary strategies to find out whether increasing LPS deacylation in vivo can protect animals with serious Gram-negative bacterial infections from dying and/or prevent prolonged post-infection immunosuppression. In addition to defining the enzyme's potential for controlling responses to LPS in vivo, the proposed studies should break new ground in understanding the "recovery" phase of Gram- negative bacterial infections, an important yet understudied aspect of bacterial pathogenesis and host defense. Project narrative - relevance for public health: Gram-negative bacteria cause a large fraction of the serious infections that occur in the U.S. today. In patients who are infected with these bacteria, a bacterial molecule called "endotoxin" is the most important trigger for the lethal condition known as septic shock. Over 100,000 Americans die each year from septic shock due to these bacteria. The subject of our research is a human enzyme that can detoxify bacterial endotoxin. Having discovered the enzyme, acyloxyacyl hydrolase (AOAH), and studied it intensively, we want now to find out if it can prevent or treat this lethal reaction to Gram-negative bacterial infection. Since the currently available drugs for septic shock are not very effective, finding that AOAH can prevent or reverse the septic reaction could have an enormous impact on public health. No other laboratory has worked as intensively on AOAH or has the ability to do these tests.