Effect Of Synthetic Endotoxin Neutralizing Peptides Against Inflammatory Responses Induced By Lipopolysaccharide

Lipopolysaccharide (LPS), also named endotoxin, a major causative agent of endotoxemia and sepsis, activates host immune cells by triggering of toll like receptor 4 (TLR4) transmembrane signaling, which secrete pro-inflammatory mediators including TNF-α, and others. Excessive pro-inflammatory mediators may lead to tissues injury, multiple organ failure and even death. Thereby, inhibiting the triggering of TLR4 transmembrane signaling could act as effective therapeutic strategy for dampening LPS-induced inflammatory responses.Triggering of TLR4 transmembrane signaling is mainly mediated by four proteins: LPS-binding protein (LBP), CD14, myeloid differentiation protein-2 (MD-2) and TLR4. Among these molecules, LBP is an important carrier protein, which could markdly enhance the effect of LPS to 100-1000 times, while MD-2 is regarded as an indispensable molecule for triggering of the signaling. Hence LBP and MD-2 may be interesting and logical targets for pharmacologically intervention against triggering of TLR4 transmembrane signaling.Based on the relationship between function and structrue of LBP/MD-2, in this study, we designed and developed two kinds of novel endotoxin neutralizing peptides derived from the LPS binding site of LBP and MD-2, respectively. Further, we examined the effects of these synthetic peptides on LPS-induced inflammatory responses in vivo and in vitro, and intended to clarify their possible mechanisms.The main experimental methods and results1. In order to obtain an amphipathic peptide with high positive charges and hydrophobicity, we designed a group of mutants of endotoxin binding peptide (mEBPs) through substitution of amino acid residues of the parent peptide (LBP91-108), which named endotoxin binding peptide (EBP). Through comparing the LPS-neutralizing ability between synthetic mEBPs and EBP by a commercially chromogenic end-point tachypleus amebocyte lysate (CE-TAL) assay, mEBP9 with enhanced LPS-neutralizing ability was determined, amino acid sequence of which was WKVRASFFKLKGSFKVSV.2. In vitro, mEBP9 dose-dependently inhibited LPS-activated Limulus amebocyte lysate (LAL) and competitively inhibited rLBP binding to LPS, the effect of which was markedly better than that of the same concentration of EBP (P<0.05). In addition, pretreatment with mEBP9 attenuated LPS-induced inflammatory responses both in human THP-1 cells (induced and differentiated to macrophage-like cells by phorbol myristate acetate (PMA)) and mouse RAW264.7 cells, as indicated by reduced TNF-αand IL-6 p(?)roduction and TNF-αmRNA expression (P<0.05).3. In vivo, mEBP9 showed better protective effects than the same concentration of EBP against LPS-induced acute lung injury in mice, including reducetion of the numbers of neutrophil and macrophage, total proteins and TNF-αlevels in bronchoalveolar lavage fluid (BALF) and infiltration of inflammatory cells into the intraalveolar and interstitial of lung at 24h after LPS administration. In mouse model of severe burns combined with endotoxemia, mEBP9 also markedly decreased LPS-induced lethality within 48h, serum aminotransferases ALT/AST and TNF-αlevel.4. Based on the relationship between function and structrue of MD-2, an essential receptor of TLR4 transmembrane signaling, we developed a novel MD-2 mimetic peptide (MDMP) derived from the putative LPS-binding domain (Phe119-Lys132) and TLR4-binding domain (Cys95-Cys105) of MD-2. The amino acid sequence of MDMP is CHGHDDDYSF CFSFEGILFPKGHYR. MD-2 scrambled peptide (MDSP, sequence is RFHGCSPCDKFY HSDLDGFHGEIYF) was as negative control.5. The results of the direct binding assay and chromogenic LAL assay indicated that synthetic MDMP but not MDSP dose-dependently bound to LPS and inhibited LPS-activated LAL. Further examination showed pretreatment with MDMP dampened LPS-induced inflammatory responses in RAW264.7 cells, including down-regulation of TLR4-MD-2 complex on the cell surface, suppression of LPS binding to the cells, inhibition of mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-κB) activation, reduction of TNF-αproduction (P<0.05).6. In vivo, MDMP markedly attenuated LPS-induced acute lung injury in mice, as indicated by reduced the numbers of neutrophil and macrophage, total proteins and TNF-αlevels in BALF at 24 h after LPS administration. In addition, pretreatment with MDMP protected against LPS/D-GalN-induced liver injury either, including the notable reduction of lethality, intrahepatic inflammatory responses and hepatocellar necrosis, serum aminotransferases and TNF-αlevels .Conclusions1. Through selective mutation of the putative LPS-binding site of LBP based on bioinformatics, mEBP9 with stronger LPS-neutralizing activity than EBP was obtained. The preliminary results in vitro and in vivo indicated that mEBP9 could directly neutralize LPS and competitively inhibit LBP binding to LPS, resulting in blockage of interaction of LBP and LPS, which offer new experimental evidence for therapeutic strategy of sepsis focus on LBP.2. Synthetic MDMP could exhibit anti-inflammatory activities against LPS-induced inflammatory responses both in vitro and in vivo, which might involve in neutralization of LPS activation, down-regulation of TLR4-MD-2 expression and reduction of MAPKs and NF-κB activation. The data revealed that antagonizing MD-2 could act as one of effective strategies for inhibiting the activation of TLR4 signaling.3. The results above-mentioned indicated that endotoxin neutralizing peptides derived from both LBP and MD-2 could partly attenuate LPS-induced inflammatory responses. However, triggering of TLR4 signaling is an extremely complicated process, it may be insufficient to fully antagonize LPS bioactivity through inhibiting single signaling molecule. A further study on blockage of multiple signaling sites will be made using mEBP9 combined with MDMP in future.