Immunostimulatory properties of chitin and chitosan

描述(申请人提供)：甲壳素是一种均聚物，由N-乙酰氨基葡萄糖的2-1，4-连接重复单元组成，是自然界第二丰富的多糖。它是真菌细胞壁、昆虫和甲壳类外骨骼以及头足类喙的组成部分。壳聚糖是脱乙酰度为50%的甲壳素。由于环境中真菌的普遍存在和食物中甲壳素/壳聚糖的摄取，人类与甲壳素和壳聚糖的接触非常普遍。甲壳素和/或壳聚糖用于化妆品和生物材料，已被认为在哮喘中发挥作用，并已被建议作为疫苗佐剂。尽管甲壳素和壳聚糖具有潜在的重要性，但人们对甲壳素和壳聚糖的免疫学后果知之甚少，也存在争议。这在很大程度上是由于聚合物中氨基葡萄糖和N-乙酰氨基葡萄糖的混合不均匀，它们的相对不溶性以及现有制剂经常与其他免疫调节剂物质污染。这一应用解决了核心假设，即甲壳素和壳聚糖的免疫效果随着其物理化学性质(例如，大小、乙酰化)的变化而变化。有两个相互关联的具体目标。目的1是用于免疫学分析的超纯甲壳素和壳聚糖的分离和鉴定。为了实现这一目标，将制备不同大小、形状和乙酰化程度的甲壳素和壳聚糖的超纯制剂。这些制剂将用于目标2中提出的研究，该研究旨在确定“甲壳素和壳聚糖不同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激活炎症体(控制强大的促炎细胞因子IL-2和IL-18的成熟和分泌的多蛋白caspase-1激活平台)和其他促炎细胞因子的要求。在初步研究中，人们发现壳聚糖，而不是甲壳素，可以有效地刺激炎症小体。然而，这两种多糖都能刺激其他致炎细胞因子，如不依赖于炎性小体组装的TNF1。使用作为目标1的一部分产生的试剂，在目标2中，我们将确定刺激炎症小体和其他细胞因子反应的物理化学要求。甲壳素和壳聚糖与真菌细胞壁的其他成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系统地确定导致免疫反应激活的甲壳素和壳聚糖的性质。这些结果对许多研究领域具有潜在的影响，包括：i)细胞壁甲壳素和壳聚糖在真菌天然和获得性免疫反应中的作用，ii)壳聚糖作为佐剂的使用，iii)阐明甲壳素在哮喘中的作用，iv)含有甲壳素的生物材料的智能工程，以及v)炎症小体激活的机制。 公共卫生相关性：人类接触甲壳素和密切相关的多糖壳聚糖是无处不在的，但这种接触的免疫学后果鲜为人知，也存在争议。在这项计划中，将制造高纯度的甲壳素和壳聚糖颗粒，然后测试它们在体外和体内刺激选定炎症反应的能力。这些研究将开始系统地确定甲壳素和壳聚糖导致免疫反应激活的特性。

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. PUBLIC HEALTH RELEVANCE: Human exposure to chitin and the closely related polysaccharide chitosan is ubiquitous but the immunological consequences of such contact are poorly understood and controversial. In this proposal, highly purified chitin and chitosan particles will be manufactured and then tested for their capacity to stimulate selected inflammatory responses in vitro and in vivo. These studies will begin to define, in a systematic manner, the properties of chitin and chitosan that lead to activation of immune responses.