Role of NLRP3-inflammasome in alum's adjuvanticity

描述（由申请人提供）：明矾是唯一被批准用于人类疫苗接种常规使用的佐剂，尽管其佐剂作用的基础仍然知之甚少。 Nod 样受体 (NLR) 是一种模式识别受体，可检测位于细胞质中的微生物产物和“危险信号”，并触发炎症小体的激活，炎症小体是一种调节 IL-1 家族（IL-12、IL-18 和 IL-33）细胞因子分泌的多蛋白复合物。我们最近证明明矾通过激活 NLR 分子 NLRP3 诱导 IL-1 家族细胞因子的分泌，更重要的是，我们表明明矾的佐剂作用在很大程度上依赖于 NLRP3。我们的结果还表明其他颗粒佐剂，例如QuilA和壳聚糖，能够在体外刺激NLRP3，这表明炎症小体激活可能是颗粒佐剂的常见作用机制。许多研究记录了 IL-1 家族细胞因子的佐剂活性，但它们与明矾佐剂作用的关系尚未确定。在目标 1 中，我们将检验 NLRP3 炎症小体和 IL-1 家族细胞因子介导明矾和其他颗粒佐剂的佐剂作用的假设。最近发表的结果还表明，明矾的一些作用可能是通过释放尿酸或产生活性氧（ROS）来介导的。明矾和尿酸都会激活 NLRP3 炎症小体，但它们引发的生物效应有很大不同，这表明它们可能会不同地激活其他不依赖炎症小体的途径。这些途径的存在可能有助于明矾的佐剂作用，并得到 NLRP3-/- 小鼠对明矾疫苗接种的残余免疫反应的支持。在目标 2 中，我们将测试 Syk 激酶、氧化应激和尿酸在明矾佐剂中的作用。我们最新的结果表明，7BIO（一种诱导坏死的靛玉红衍生物）可激活 NLRP3 炎症小体并具有佐剂特性。众所周知，坏死细胞会释放危险信号，例如具有佐剂能力的 HMGB1 蛋白。在目标 3 中，我们想要测试以下假设：将 7BIO 或 HMGB1 纳入明矾疫苗中可以通过诱导 Th1 反应来提高免疫反应的质量和程度。我们研究的长期目标是在分子水平上了解明矾和其他颗粒佐剂的作用机制，并改善其免疫刺激谱。 公共卫生相关性：明矾是唯一被批准用于人类疫苗接种常规使用的佐剂，尽管其免疫刺激活性的基础仍知之甚少。本拨款提案中描述的研究将增加我们对明矾作用机制的理解。更深入地了解决定佐剂免疫刺激特性的机制是合理设计更复杂疫苗的先决条件，疫苗仍然是工业化国家和发展中国家对抗传染病的最有前途的策略。

DESCRIPTION (provided by applicant): Alum is the only adjuvant approved for routine use in human vaccination although the basis for its adjuvanticity remains poorly understood. Nod-Like receptors (NLR) are pattern recognition receptors that detect microbial products and "danger signals" situated in the cytoplasm and trigger activation of the inflammasome, a multiprotein complex that regulates secretion of cytokines belonging to the IL-1 family (IL-12, IL-18, and IL-33). We have recently demonstrated that alum induces secretion of IL-1 family cytokines by activating the NLR molecule NLRP3 and, more importantly, we showed that the adjuvant effect of alum is in good part dependent on NLRP3. Our results also show that other particulate adjuvants, such as QuilA and chitosan, are able to stimulate NLRP3 in vitro, suggesting that inflammasome activation may be a common mechanism of action of particulate adjuvants. Numerous studies documented the adjuvant activities of the IL-1 family cytokines, yet their involvement in alum adjuvanticity has not been established conclusively. In aim 1 we will test the hypothesis that the NLRP3-inflammasome and the IL-1 family cytokines mediate the adjuvanticity of alum and other particulate adjuvants. Recently published results also suggest that some of alum's effects may be mediated by release of uric acid or generation of reactive oxygen species (ROS). Both alum and uric acid activate the NLRP3-inflammasome, yet they differ substantially in the biological effects they trigger, suggesting they may differentially activate additional inflammasome-independent pathways. The existence of these pathways, which may contribute to alum's adjuvanticity, is supported by the residual immune response of NLRP3-/- mice to alum vaccination. In aim 2 we will test the role of Syk kinase, oxidative stress, and uric acid in alum's adjuvanticity. Our most recent results show that 7BIO, a necrosis-inducing indirubin derivative, activate the NLRP3-inflammasome and possess adjuvant properties. Necrotic cells are known to release danger signals, such as the HMGB1 protein, that possess adjuvant ability. In aim 3 we want to test the hypothesis that inclusion of 7BIO or HMGB1 into alum-based vaccines can improve the quality and extent of the resulting immune response by inducing Th1 responses. The long-term goal of our studies is to understand at the molecular level the mechanism of action of alum and other particulate adjuvants and to improve their immunostimulatory spectrum. PUBLIC HEALTH RELEVANCE: Alum is the only adjuvant approved for routine use in human vaccination although the basis for its immunostimulatory activity remains poorly understood. The studies described in this grant proposal will increase our understanding of the mechanism of action of alum. A deeper understanding of the mechanisms that determine the immunostimulatory properties of adjuvants is a prerequisite for the rational design of more sophisticated vaccines, which remain the most promising strategy to fight infectious diseases in both industrialized and developing countries.