Role of NLRP3-inflammasome in alum's adjuvanticity

描述(申请人提供)：明矾是唯一被批准在人类疫苗接种中常规使用的佐剂，尽管其佐剂的基础仍然知之甚少。NOD样受体(NLR)是一种模式识别受体，可以检测细胞质中的微生物产物和危险信号，并触发炎症体的激活，炎症体是一种多蛋白复合体，调节IL-1家族(IL-12、IL-18和IL-33)的细胞因子的分泌。我们最近证明了明矾通过激活NLR分子NLRP3来诱导IL-1家族细胞因子的分泌，更重要的是，我们证明了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，如奎拉和壳聚糖，能够在体外刺激NLRP3，提示炎症小体激活可能是颗粒佐剂的一种常见作用机制。大量研究证实了IL-1家族细胞因子的佐剂活性，但它们与明胶佐剂的关系尚不明确。在目标1中，我们将检验NLRP3-炎症体和IL-1家族细胞因子介导明矾和其他颗粒佐剂的假说。最近发表的研究结果还表明，明矾的一些作用可能是通过释放尿酸或产生活性氧物种(ROS)来调节的。明矾和尿酸都激活了NLRP3-炎症体，但它们触发的生物学效应有很大不同，这表明它们可能以不同的方式激活其他非炎症体依赖的途径。这些途径的存在可能有助于明矾的佐剂作用，NLRP3-/-小鼠对明矾疫苗的残留免疫反应支持了这些途径的存在。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，一种导致坏死的吲哚红衍生物，可以激活NLRP3-炎症体，并具有佐剂特性。已知坏死细胞会释放危险信号，如HMGB1蛋白，具有佐剂能力。在目标3中，我们想要测试的假设是，将7BIO或HMGB1加入明矾疫苗中，可以通过诱导Th1反应来改善所产生的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，并改善它们的免疫刺激光谱。 与公共卫生相关：明矾是唯一被批准在人类疫苗接种中常规使用的佐剂，尽管其免疫刺激活性的基础仍然知之甚少。这项拨款提案中描述的研究将增加我们对明矾作用机制的了解。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的疫苗的先决条件，在工业化国家和发展中国家，疫苗仍然是抗击传染病的最有前途的战略。

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.