Dysregulated immune responses to lipopolysaccharide (LPS, endotoxin) can lead to many inflammatory diseases. The innate immune system detects LPS through a series of pattern recognition receptors. One of the key receptors, LPS-binding protein (LBP), recognizes LPS, disassembles LPS aggregates in extracellular fluids, and then delivers LPS to the receptors on immune cell surfaces. It is documented that the appropriate concentration of LBP can lead to over 1,000-fold enhancement of LPS-induced immune response. The recent clinical study of endotoxemia suggested targeting LPS is a better strategy than antimicrobial therapy to reduce mortality in multiple-trauma patients with septic shock. Therefore, the neutralization of LBP function leading to the reduction of LPS-induced immune response becomes an attractive therapeutic approach. Prior in vivo and in vitro studies show that inhibition of LBP could attenuate LPS-induced inflammation. However, lack of fundamental knowledge of LBP is the main obstacle to the development of LBP-neutralization therapy. Prior studies reported that delivery of LPS to immune cell surface is activated by the adsorption/intercalation of LBP to cell membrane. The PIs propose to discover a set of cell membrane molecules involved in LBP adsorption; thus, the assembly of these molecules can serve as a high LBP affinity reagent for endotoxin shock intervention. LBP adsorption is determined by cooperative actions between multiple membrane molecules rather than a single membrane molecule. In order to observe the synergistic effects of different membrane molecules, a new Membrane Perturbation Method developed by the PIs will be applied. In brief, macrophage membranes will be reconstituted onto the sensor surface and then perturbed by inserting the membrane molecules purified from native macrophages. Because the reconstituted membrane preserves the molecules in native cell membranes, the complex interplay between membrane molecules can be identified. To complete this complex analysis, large data sets are required to survey many different experimental conditions. Thus, the PIs will use their unique nanocube-based membrane array, which enables large-scale quantitative analysis of protein binding to cell membrane surface. The high-throughput and easy-to-use features of this unique tool address these critically needed aspects. After identifying the major molecules contributing to LBP-membrane adsorption, the PIs will fabricate synthetic liposomes containing these high affinity molecules to attenuate LPS-induced inflammation in RAW 264.7 murine macrophages. Pro- (TNF-?, IL-1, IL-6) and anti-inflammatory cytokines (IL-10, IL-4) will be measured to determine the LBP-neutralization efficiency. The efficacy of LPS detoxification will be quantified by the rate of fluorescence conjugated LPS transport to synthetic liposomes. This preliminary study will provide a new strategy for the intervention of LPS related inflammatory diseases.
Lipopolysaccharide-binding protein (LBP) is an essential immunological receptor which responds to lipopolysaccharide. LBP neutralization is an attractive intervention for many inflammatory diseases; however, lack of fundamental knowledge of LBP is the main obstacle to the development of LBP-neutralization therapy. We propose to use our unique detection tools to dissect the LBP-immune cell interactions in order to design efficient reagents for LBP neutralization.
