Regulation of MD-2 function and expression

描述(申请人提供)：宿主对许多入侵的革兰氏阴性菌(GNB)的防御依赖于对内毒素(E)的天然免疫识别，内毒素是GNB独特的、丰富的和通常保守的糖脂。依赖于TLR4的最佳反应非常敏感和稳健，但也是自我限制的。这通常是如何实现的，以及为什么TLR4激活的调节在各种病理生理条件下可能会出错，目前尚不完全清楚。对E最有效的炎症反应需要E的某些规范结构特征，以及涉及内毒素结合蛋白(LBP)、CD14、MD-2和Toll样受体(TLR)4的有序的蛋白质-E相互作用。MD-2在调节E激活TLR4的过程中发挥重要作用，它将通常由LBP和CD14启动的对E的识别连接到受体的激活或拮抗。我们对单体E.MD-2复合体作为有效的TLR4激动剂或拮抗剂的鉴定和表征，取决于E和/或MD-2的结构，清楚地表明，正是E.MD-2复合体的性质，而不是E本身，决定了TLR4的激活或受体在不激活的情况下占据。这些复合体独特的结构和功能特性为研究内毒素如何诱导TLR4激活，E结构的差异如何决定TLR4反应性的差异，以及这些新的机制见解如何转化为新型TLR4导向免疫调节剂的开发提供了新的方法。越来越多的证据表明，更多的分子可以作为TLR4的激动剂或拮抗剂，包括宿主和/或饮食衍生的脂类，这表明在许多其他情况下，MD-2/TLR4功能的调节或失调可能在宿主生理学和病理生理学中起关键作用。我们将利用新的合成、生化、结构生物学和功能方法(例如，代谢标记的天然和合成E和A类脂变异体、重组野生型和突变型人类E结合蛋白和TLR4胞外区、单抗、呼吸道感染和炎症模型)来：1)进一步定义促进与MD-2结合的配体的结构特性以及配体的作用。MD-2复合体作为TLR4激动剂或拮抗剂；2)更具体地定义E.MD-2复合体的结构特性，激活TLR4；以及3)更好地确定wt和变异体E(LipdA).MD-2在小鼠呼吸道中的体内作用。公共卫生相关性：内毒素激活依赖TLR4的细胞在宿主反应和防御许多革兰氏阴性细菌方面发挥关键作用。TLR4在周围环境中的普遍存在，以及其他微生物和宿主衍生的TLR4激动剂/拮抗剂的明显存在，表明TLR4在健康和疾病中具有更广泛的作用。更好地理解内毒素是如何诱导TLR4激活的，以及内毒素结构的差异如何决定TLR4反应的差异，可能会导致更好地理解哺乳动物宿主，包括人类，通常如何有效地应对入侵的革兰氏阴性细菌，以及某些革兰氏阴性细菌如何逃避/抵抗这些正常有效的宿主反应。新的机制洞察力也可能转化为新型TLR4导向免疫调节剂的开发，其应用可能包括但不限于，对革兰氏阴性细菌感染。

DESCRIPTION (provided by applicant): Host defense against many invading Gram-negative bacteria (GNB) depends upon innate immune recognition of endotoxin (E), unique, abundant and generally conserved glycolipids of GNB. Optimal TLR4- dependent responses are exquisitely sensitive and robust, but also self-limited. How this is normally achieved and why regulation of TLR4 activation may go awry under various patho-physiological conditions is incompletely known. The most potent inflammatory responses to E require certain canonical structural features of E and ordered protein-E and protein-protein interactions involving the LPS-binding protein (LBP), CD14, MD-2, and Toll-like receptor (TLR) 4. MD-2 plays an essential role in regulation of TLR4 activation by E, by bridging recognition of E, normally initiated by LBP and CD14, to receptor activation or antagonism. Our identification and characterization of monomeric E.MD-2 complexes as potent TLR4 agonists or antagonists, depending on E and/or MD-2 structure, have clearly demonstrated that it is the properties of the E.MD-2 complex, not E alone, that specifies TLR4 activation or receptor occupancy without activation. The unique structural and functional properties of these complexes suggest novel approaches to investigate how endotoxin induces TLR4 activation, how differences in E structure can dictate differences in TLR4 responsiveness, and how these new mechanistic insights may be translated to the development of novel TLR4-directed immunomodulators. Growing evidence of a broader array of molecules that can act as TLR4 agonists or antagonists, including host and/or diet-derived lipids, suggest many other circumstances in which the regulation or dysregulation of MD-2/TLR4 function could be pivotal in host physiology and patho-physiology. We will make use of novel synthetic, biochemical, structural biological, and functional approaches (e.g., metabolically labeled native and synthetic E and lipid A variants, recombinant wild-type and mutant human E-binding proteins and TLR4 ectodomain, monoclonal antibodies, models of airway infection and inflammation) to: 1) Further define the structural properties of ligands that promote binding to MD-2 and the action of ligand.MD-2 complexes as TLR4 agonists or antagonists; 2) Define more specifically the structural properties of E.MD-2 complexes that activate TLR4; and 3) Better define in vivo actions in the murine airway of wt and variant E(lipdA).Md-2. PUBLIC HEALTH RELEVANCE: TLR4-dependent cell activation by endotoxin plays a critical role in host response and defense against many Gram-negative bacteria. The ubiquity of endotoxin in the surrounding environment and the apparent existence of other microbe- and host-derived agonists/antagonists of TLR4 suggest an even broader role of TLR4 in health and disease. Better understanding of how endotoxin induces TLR4 activation and how differences in endotoxin structure dictate differences in TLR4 responsiveness is likely to lead to a better understanding of how mammalian hosts, including humans, normally respond effectively against invading Gram-negative bacteria and how certain Gram-negative bacterial pathogens can evade/resist these normally efficient host responses. The new mechanistic insights may also be translated to the development of novel TLR4-directed immuno-modulators whose application would likely include, but not be limited, to Gram-negative bacterial infections.