Immunostimulatory properties of chitin and chitosan

描述（由申请人提供）：甲壳素，一种由2- 1，4-连接的N-乙酰葡糖胺重复单元组成的均聚物，是自然界中第二丰富的多糖。它是真菌细胞壁、昆虫和甲壳类动物外骨骼以及头足类动物喙的组成部分。壳聚糖是脱乙酰化> 50%的甲壳质。由于环境中真菌的普遍存在以及食物中甲壳素/壳聚糖的摄入，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，已被假定在哮喘中起作用，并已被提议作为疫苗佐剂。尽管其潜在的重要性，暴露于甲壳素和壳聚糖的免疫后果是知之甚少和有争议的。这在很大程度上是由于葡糖胺和N-乙酰葡糖胺在聚合物中的异质混合物、它们的相对不溶性以及可用制剂与其他免疫调节剂物质的频繁污染。本申请解决了中心假设，即几丁质和壳聚糖的免疫学作用作为其物理化学性质的函数而变化（例如，尺寸、乙酰化）。有两个相互关联的具体目标。目的1是“用于免疫学测定的超纯甲壳素和壳聚糖的分离和表征”。在这个目标下，超纯的甲壳素和壳聚糖制剂将被制成不同的尺寸、形状和乙酰化程度。这些制剂将用于目标2中提出的研究，即确定“几丁质和壳聚糖对炎症反应的差异活化的机制基础和生物学意义”。这一目标的重点将是激活炎性体（控制强效促炎细胞因子IL-2和IL-18的成熟和分泌的多蛋白胱天蛋白酶-1激活平台）和其他促炎细胞因子的要求。在初步研究中，发现壳聚糖而不是几丁质有效地刺激炎性小体。然而，这两种聚糖都刺激其他促炎细胞因子，如不依赖于炎性小体组装的TNF 1。使用作为目标1的一部分产生的试剂，在目标2中，我们将确定刺激炎性小体和其他细胞因子应答的理化要求。将研究几丁质和壳聚糖与真菌细胞壁的其他组分协同作用的能力。最后，将寻求体外观察结果的体内相关性。这些研究的完成将开始以系统的方式定义导致免疫应答激活的几丁质和壳聚糖的性质。这些结果对许多研究领域具有潜在的影响，包括：i）细胞壁几丁质和壳聚糖在对真菌的先天性和适应性免疫应答中的作用，ii）壳聚糖作为佐剂的用途，iii）阐明几丁质在哮喘中的作用，iv）含几丁质生物材料的智能工程，以及v）炎性小体活化的机制。 公共卫生相关性：人类暴露于甲壳素和密切相关的多糖壳聚糖是无处不在的，但这种接触的免疫后果知之甚少，有争议的。在这个提议中，将制造高度纯化的几丁质和壳聚糖颗粒，然后测试它们在体外和体内刺激选定炎症反应的能力。这些研究将开始以系统的方式定义导致免疫应答激活的几丁质和壳聚糖的性质。

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.