Immunostimulatory properties of chitin and chitosan

描述（由申请人提供）：甲壳素是一种均聚物，由2-1,4-连接的n -乙酰氨基葡萄糖胺重复单元组成，是自然界中含量第二丰富的多糖。它是真菌细胞壁、昆虫和甲壳类动物外骨骼和头足类动物喙的组成部分。壳聚糖是一种去乙酰化50%的甲壳素。由于环境中真菌的普遍存在以及食物中几丁质/壳聚糖的摄入，人类与几丁质和壳聚糖的接触非常普遍。几丁质和/或壳聚糖用于化妆品和生物材料，被认为在哮喘中起作用，并被提议作为疫苗佐剂。尽管其潜在的重要性，暴露于几丁质和壳聚糖的免疫学后果知之甚少和争议。这在很大程度上是由于葡萄糖胺和n -乙酰氨基葡萄糖胺在聚合物中的异质混合，它们的相对不溶性和现有制剂与其他免疫调节剂物质的频繁污染。该应用程序解决了几丁质和壳聚糖的免疫作用随其物理化学性质（如大小，乙酰化）而变化的中心假设。有两个相互关联的具体目标。目的1是“用于免疫检测的超纯几丁质和壳聚糖的分离和特性”。在这个目标中，几丁质和壳聚糖的超纯制剂将被制成不同的尺寸、形状和乙酰化程度。这些制剂将用于Aim 2中提出的研究，即确定“几丁质和壳聚糖差异激活炎症反应的机制基础和生物学意义”。这一目标的重点将是炎性小体（多蛋白caspase-1激活平台，控制强效促炎细胞因子IL-2和IL-18的成熟和分泌）和其他促炎细胞因子的激活需求。在初步研究中，发现壳聚糖，而不是几丁质，能有效地刺激炎性体。然而，这两种聚糖都能刺激其他促炎细胞因子，如不依赖于炎性小体组装的TNF1。使用作为Aim 1的一部分生成的试剂，在Aim 2中，我们将确定刺激炎性体和其他细胞因子反应的理化要求。甲壳素和壳聚糖与真菌细胞壁其他组分协同作用的能力将被研究。最后，将寻求体外观察的体内相关性。这些研究的完成将开始以系统的方式定义甲壳素和壳聚糖导致免疫反应激活的特性。这些结果对许多研究领域具有潜在的影响，包括：1)细胞壁几丁质和壳聚糖在对真菌的先天和适应性免疫反应中的作用，2)壳聚糖作为佐剂的使用，3)阐明几丁质在哮喘中的作用，4)含几丁质生物材料的更智能工程，以及5)炎症小体激活机制。

DESCRIPTION (provided by applicant): Chitin, a homopolymer consisting of 2-1,4-linked repeating units of N-acetylglucosamine, is the second most abundant polysaccharide in nature. It is an integral component of fungal cell walls, insect and crustacean exoskeletons and cephalopod beaks. Chitosan is chitin that has been deacetylated >50%. Human contact with chitin and chitosan is very common due to the ubiquitous presence of fungi in the environment and the ingestion of chitin/chitosan in food. Chitin and/or chitosan are used in cosmetics and biomaterials, have been postulated to play a role in asthma, and have been proposed as vaccine adjuvants. Despite its potential importance, the immunological consequences of exposure to chitin and chitosan are poorly understand and controversial. This is due, in large part, to the heterogeneous mix of glucosamine and N-acetylglucosamine in the polymers, their relative insolubility and frequent contamination of available preparations with other immune modulator substances. This application addresses the central hypothesis that the immunological effects of chitin and chitosan varies as a function of its physicochemical properties (e.g., size, acetylation). There are two interconnected specific aims. Aim 1 is "Isolation and characterization of ultrapure chitin and chitosan for use in immunological assays". In this aim, ultrapure preparations of chitin and chitosan will be made of varying sizes, shapes, and degree of acetylation. These preparations will be utilized in the studies proposed in Aim 2, which is to determine the "Mechanistic basis and biological implications for differential activation of the inflammatory response by chitin and chitosan". A focus in this aim will be on the requirements for activation of the inflammasome (multi-protein caspase-1-activating platforms that control maturation and secretion of the potent proinflammatory cytokines IL-2 and IL-18) and other proinflammatory cytokines. In preliminary studies, it was found that chitosan, but not chitin, potently stimulates the inflammasome. Yet, both glycans stimulate other proinflammatory cytokines, such as TNF1 which are not dependent upon inflammasome assembly. Using the reagents generated as part of Aim 1, in Aim 2, we will determine the physicochemical requirements for stimulation of the inflammasome and other cytokine responses. The capacity of chitin and chitosan to synergize with other components of the fungal cell wall will be studied. Finally, in vivo correlates of the in vitro observations will be sought. Completion of these studies will begin to define, in a systematic manner, the properties of chitin and chitosan that lead to activation of immune responses. The results have potential impact on many research fields, including: i) the role of cell wall chitin and chitosan in innate and adaptive immune responses to fungi, ii) the use of chitosan as an adjuvant, iii) elucidating the role of chitin in asthma, iv) smarter engineering of chitin- containing biomaterials, and v) mechanisms of inflammasome activation.