Project Summary or Abstract: Identifying the innate immune receptor and the molecular mechanism involved in rapid clearance of circulating blood-borne Lipopolysaccharide (LPS) will provide critical insights to develop therapeutic options for endotoxe- mia and several LPS-associated diseases. LPS, a major constituent of Gram-negative bacterial cell wall, is a potent microbial ligand that induces intense systemic inflammation via Toll-like receptor 4 (TLR4) in immune cells. LPS in blood circulation (endotoxemia) affects multiple organs and can cause life-threatening inflamma- tory reactions. As a proactive host defense mechanism, the liver clears LPS from blood circulation. However, the mechanisms of the immune cells and associated receptors involved in this process, and whether clearance of LPS overturns TLR4 mediated systemic inflammation, is unknown. In this proposal, we present four remarkably novel findings. First, we found that liver sinusoidal endothelial cells (LSEC) eliminate a major portion of LPS from blood circulation very rapidly within a few minutes, and that clearance of LPS is facilitated by high density lipoprotein (HDL). Secondly, LSEC clear circulating LPS-HDL via Stabilin-1 (Stab1) and Stabilin-2 (Stab2) receptor mediated endocytosis and localize to lysosomes for deg- radation. Third, the lack of both Stab1 and Stab2 in Stabilin double knock out mice results in diminished clear- ance, liver uptake and endocytosis by LSEC, but escalated systemic inflammation and early death in response to LPS. Fourth, Stab1, and to a lesser extent Stab2, participates in host defense. Fifth, Stabilin and TLR4 are functionally opposite receptors for LPS. These results lead us to hypothesize that Stabilin receptors expressed in LSEC offer innate host defense function by decreasing the plasma LPS level and subsequent systemic in- flammation by TLR4. We will test the hypothesis in three major specific aims. In Aim 1, we will determine the mechanism of LPS endocytosis by Stabilin receptors in LSEC, especially the mode of vesicular endocytic traf- ficking in the plasma membrane, the intra-cellular pathways leading to lysosomes for LPS inactivation and deg- radation, and relates the function of Stabilin receptors in LSEC and KC. In Aim 2, we will relate the expression and function of Stabilin receptors with TLR4 in LSEC to clear and endocytose LPS and regulate inflammation in both murine and human LSEC. In Aim 3, we will determine how enhancement of the endocytic function of LSEC decreases the presence of LPS in blood circulation and as a result, controls systemic inflammation. We propose that upregulation of the clearance function of LSEC can be an efficient way to control inflammation during LPS-associated diseases. This project presents a new paradigm in which LSEC and Stabilin receptors offer a host defense mechanism and protection against LPS induced inflammation during various LPS- associated diseases, and points to a novel target for future therapies that treat endotoxemia.

Project Narrative: Patients with endotoxemia and other LPS associated diseases have significantly increased risk for mortality; currently there is no specific therapy to alleviate LPS-mediated systemic inflammation and complications in ICU patients. The aim of this proposal is to investigate the novel innate immune functions of Stabilin receptors in liver sinusoidal endothelial cells which control systemic inflammation during endotoxemia. The results from this study will provide knowledge on the unknown host protective mechanism of Stabilin receptors during LPS- associated diseases, and may offer potential therapeutic targets to treat several LPS associated diseases.