Molecular Determinants of Endotoxin Interaction with Toll-like Receptor4

描述（由申请人提供）： Toll样受体将保守的和结构独特的微生物分子的分子识别与先天免疫效应系统的快速动员以及随后的适应性免疫的诱导偶联。Toll样受体4（TLR 4）对于识别甚至微量的内毒素（E）是必需的，内毒素（E）是革兰氏阴性细菌（GNB）表面上大量存在的独特糖脂，其负责宿主对许多GNB的反应。对E的有效应答需要几种细胞外和细胞表面宿主蛋白的顺序作用，包括脂多糖结合蛋白（LBP）、CD 14、MD-2，用于递送和激活TLR 4。通过TLR 4引起的内毒素依赖性应答导致MyD 88和TRIF依赖性信号通路的激活。内毒素结构的变化--如GNB中自然发生的那样--以及参与E结合和信号转导的宿主细胞蛋白水平的变化使得宿主细胞对E的反应可能极大地多样化。当局部感染不被控制时，对E的正常保护性反应可能通过加重全身炎症反应而导致严重的宿主病理，如GNB败血症。不适当的信号传导，无论是太广泛还是太温和，都可能导致各种感染性和炎性疾病，包括败血症、动脉粥样硬化、自身免疫综合征、肝病和急性肺损伤。 我们已经开发的新试剂和实验方法允许在生理相关条件下检测和定量分析特异性、高亲和力（pM）E-蛋白和TLR 4相互作用，并且因此允许确定TLR 4活化的几种天然和合成调节剂的作用机制。MD-2的稳定的水溶性单体复合物的发现和纯化，其取决于结合的E或MD-2的结构，可以在pM浓度下作为TLR 4激动剂或拮抗剂起作用，提供了独特的试剂来探测内毒素触发的TLR 4活化的剩余奥秘和MD-2和TLR 4对内毒素的模式识别的结构限制。越来越多的证据表明TLR 4的生理和病理生理作用超出了宿主对入侵GNB的反应。因此，调节TLR 4功能的分子和细胞规则的知识应该提供新的见解，什么决定的性质和强度的TLR 4依赖性反应的设置，包括但不限于GNB感染。我们工作的长期目标是更好地理解TLR 4依赖性细胞对GNB内毒素和宿主衍生的“危险分子”的反应的调节机制。更具体地，工作集中于理解1）E与MD-2的相互作用指定TLR 4的激活或拮抗的机制以及与氯化血红素激活TLR 4的结构和分子要求相反的机制; 2）TLR 4胞外结构域中天然存在的多态性变体取代D299 G和/或T399 I对TLR 4的表面表达和功能的影响;和3）E（脂质A）结构和呈递中的变量对通过活化的TLR 4诱导MyD 88相对于TRIF依赖性信号传导的影响。这两种途径与不同的细胞反应有关，因此更好地了解它们的差异调节可能对设计对TLR 4信号传导的类型和持续时间具有选择性作用的化合物（如佐剂）具有重要意义。这里提出的研究应该显着增加对TLR 4功能调节的理解，并产生适用于新型TLR 4定向免疫调节剂的设计和测试的见解。调节TLR 4功能的分子和细胞规则的知识有助于理解在包括但不限于GNB感染的环境中决定TLR 4依赖性应答的性质和强度的因素。 公共卫生相关性： 退伍军人往往有细菌感染和免疫和感染后炎症后遗症的风险。重要的炎性刺激包括有效的细菌产物如内毒素和由于组织损伤而释放的内源性宿主分子如氯化血红素。叠加侵袭性感染和组织损伤的频率强调了将我们的研究从革兰氏阴性菌的内毒素扩展到氯化血红素的重要性，以及这些研究与VA人群的相关性。随着抗生素耐药性的增加，开发非抗生素策略来扩大宿主耐药性变得越来越重要。我们的研究应该有助于更好地了解宿主识别和响应细菌入侵和组织损伤的分子和细胞基础，并提供见解，可以促进治疗药物和预防策略的基础上，引起这些主机响应的分子事件的贡献知识的发展。

DESCRIPTION (provided by applicant): ABSTRACT Toll-like receptors couple molecular recognition of conserved and structurally unique microbial molecules to rapid mobilization of innate immune effector systems and later induction of adaptive immunity. Toll-like receptor 4 (TLR4) is essential for the recognition of even minute amounts of endotoxin (E), unique glycolipids abundantly present on the surface of Gram negative bacteria (GNB), that are responsible for host responses to many GNB. Potent responses to E require the sequential action of several extracellular and cell surface host proteins, including lipopolysaccharide-binding protein (LBP), CD14, MD-2 for delivery to and activation of TLR4. Endotoxin-dependent responses elicited through TLR4 lead to activation of MyD88- and TRIF-dependent signaling pathway. Variations in endotoxin structure - as occurs naturally among GNB - and variations in levels of host cell proteins involved in E binding and signal transduction make possible great diversification of host cell responses to E. The normally protective responses to E may contribute to severe host pathology by exacerbating systemic inflammatory responses when local infection is not contained, as in GNB sepsis. Inappropriate signaling, whether too extensive or too modest, may contribute to a variety of infectious and inflammatory diseases including sepsis, atherosclerosis, autoimmune syndromes, liver disease, and acute lung injury. The novel reagents and experimental approaches we have developed allowed detection and quantitative analysis of specific, high affinity (pM) E- protein and TLR4 interactions under physiologically relevant conditions and so permitted determination of the mechanism of action of several natural and synthetic regulators of TLR4 activation. The discovery and purification of stable, water-soluble monomeric complexes of E.MD-2 that, depending on the structure of bound E or MD-2, can act at pM concentrations as TLR4 agonists or antagonists, have provided unique reagents to probe the remaining mysteries of endotoxin-triggered TLR4 activation and the structural limits of pattern recognition of endotoxin by MD-2 and TLR4. Increasing evidence suggests physiologic and patho-physiologic roles of TLR4 extending beyond host responses to invading GNB. Thus, knowledge of the molecular and cellular rules regulating TLR4 function should provide new insights into what determines the nature and the strength of TLR4-dependent responses in settings including but not limited to GNB infection. The long-term goals of our work are to better understand the mechanism(s) by which TLR4-dependent cellular responses to GNB endotoxin and host-derived "danger molecules" are regulated. More specifically, the work is focused on understanding 1) the mechanism(s) by which E interactions with MD-2 specify activation or antagonism of TLR4 and the contrasting structural and molecular requirements for hemin activation of TLR4; 2) the effects of the naturally occurring polymorphic variant substitutions D299G and/or T399I in the TLR4 ectodomain on surface expression and function of TLR4; and 3) the effects of variables in E (lipid A) structure and presentation on induction of MyD88- vs. TRIF-dependent signaling by activated TLR4. The two pathways are linked to different cellular responses so a better understanding of their differential regulation could have important implications for the design of compounds such as adjuvants with selective effects on the type and duration of TLR4 signaling. The studies proposed here should significantly increase understanding of the regulation of TLR4 function and produce insights applicable to the design and testing of novel TLR4-directed immune-modulators. Knowledge of the molecular and cellular rules regulating TLR4 function contributes to the understanding of what determines the nature and the strength of TLR4-dependent responses in settings including, but not limited to, GNB infection. PUBLIC HEALTH RELEVANCE: Veterans are often at risk to bacterial infections and immunologic and post-infectious inflammatory sequelae. Important inflammatory stimuli include potent bacterial products such as endotoxin and endogenous host molecules released as a result of tissue injury such as hemin. The frequency of superimposed invasive infection and tissue injury underscores the importance of extending our studies from endotoxin of Gram negative bacteria to hemin and the relevance of these studies to the VA population. As antimicrobial resistance increases, the development of non-antibiotic strategies to amplify host resistance becomes increasingly important. Our studies should contribute toward a better understanding of the molecular and cellular bases of host recognition and response to bacterial invasion and tissue injury and provide insight that could facilitate the development of therapeutic agents and prophylactic strategies based on knowledge of the contribution of molecular events that evoke these host responses.