Role of NLRP3-inflammasome in alum's adjuvanticity

描述（由申请方提供）：明矾是唯一获批用于人类疫苗接种常规用途的佐剂，但其佐剂性的基础仍知之甚少。Nod-Like受体（NLR）是模式识别受体，其检测位于细胞质中的微生物产物和“危险信号”并触发炎性体的激活，炎性体是调节属于IL-1家族的细胞因子（IL-12、IL-18和IL-33）的分泌的多蛋白复合物。我们最近已经证明，明矾通过激活NLR分子NLRP 3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NLRP 3。我们的研究结果还表明，其他颗粒佐剂，如基拉和壳聚糖，能够在体外刺激NLRP 3，这表明炎性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，但它们在明矾佐剂性中的参与尚未最终确定。在目的1中，我们将测试NLRP 3-炎性体和IL-1家族细胞因子介导明矾和其它颗粒佐剂的佐剂性的假设。最近发表的结果也表明，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。明矾和尿酸都能激活NLRP 3-炎性体，但它们触发的生物学效应有很大差异，这表明它们可能差异性地激活额外的炎性体非依赖性途径。这些途径的存在可能有助于明矾的佐剂性，这得到了NLRP 3-/-小鼠对明矾疫苗接种的残留免疫应答的支持。在目标2中，我们将测试Syk激酶、氧化应激和尿酸在明矾的佐剂性中的作用。我们最近的研究结果表明，7 BIO，一种坏死诱导靛玉红衍生物，激活NLRP 3-炎性小体，并具有佐剂特性。已知坏死细胞会释放危险信号，如具有佐剂能力的HMGB 1蛋白。在目标3中，我们想要测试以下假设：将7 BIO或HMGB 1包含到基于明矾的疫苗中可以通过诱导Th 1应答来改善所得免疫应答的质量和程度。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。本拨款申请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，这仍然是工业化国家和发展中国家防治传染病的最有希望的战略。

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.