Role of defensin receptor signaling in IL-1beta release

描述(申请人提供)：IL-12的过度产生与自身免疫性疾病和微生物感染有关。新合成的无铅ProIL-12不能有效地从激活的单核细胞中分泌出来。然而，当内毒素诱导的单核细胞进一步用细胞外ATP刺激时，它们会迅速释放大量成熟的IL-12和ProIL-12。到目前为止，人类单核细胞释放IL-12的机制尚不清楚。该提案的总体目标是通过研究β-防御素阻止人类单核细胞释放IL-12的分子机制来确定IL-12是如何释放的。人β-防御素(HNP-1和HD-5)是由中性粒细胞和上皮细胞产生的一组抗菌肽，是目前唯一能阻断caspase-1释放但不能抑制caspase-1加工的ProIL-12的抑制剂。这项建议没有研究防御素在体内的作用。它只使用防御素作为工具来识别IL-12阻断的新分子靶点。利用光亲和共聚焦显微镜，我们发现防御素与人单核细胞的细胞膜相关蛋白结合。我们将验证这一假设，即IL-12的释放以及人单核细胞中前IL-12和分泌性溶酶体的外化受到不同信号蛋白的调节，这些信号蛋白可以通过酶连接的受体被β-防御素抑制。为了实现这一目标，我们确定了两个特定目标：特定目标一：确定负责防御素介导的抑制人单核细胞释放IL-12的受体。利用光亲和纯化和蛋白质组学方法，我们将在人类单核细胞中鉴定防御素结合蛋白(DBPs)。然后，我们将通过确定siRNA介导的这些DBPs的敲除对防御素阻断IL-12释放的影响来鉴定防御素受体。具体目的二：明确防御素受体介导的抑制IL-12释放所必需的信号通路。我们将确定蛋白激酶C、磷脂酶C、钙依赖的蛋白激酶和MAP激酶是否参与防御素阻断IL-12的释放以及人单核细胞分泌IL-12和分泌型溶酶体的外化。该项目的成功完成将对基础医学研究和转化医学研究产生广泛影响。如果我们的假设是正确的，它不仅将为IL-12生物学领域带来更多的澄清，而且还将为许多其他重要的无领导蛋白，包括IL-18，IL-33，MIF和FGF-2所使用的ER/高尔基非依赖性分泌途径提供新的见解。拟议的研究还将导致发现IL-12阻断的新分子靶点，并可能导致开发新的治疗方法来预防和治疗许多威胁生命的微生物感染和炎症性疾病。 公共卫生相关性：防御素是由白细胞和上皮细胞产生的多肽。使用防御素作为工具，我们将确定促炎症细胞因子IL-12是如何从人类单核细胞释放出来的。拟议的研究将导致发现IL-12阻断的新分子靶点，并可能导致开发新的治疗方法来预防和治疗许多威胁生命的微生物感染和炎症性疾病。

DESCRIPTION (provided by applicant): Overproduction of IL-12 is associated with autoimmune diseases and microbial infections. Newly synthesized leaderless proIL-12 cannot be efficiently secreted from activated monocytes. However, when LPS-primed monocytes are further stimulated with extracellular ATP, they rapidly release large amounts of mature IL-12 and proIL-12. Thus far, the mechanism by which IL-12 is released from human monocytes is not understood. The overall goal of this proposal is to determine how IL-12 is released by investigating the molecular mechanisms by which ?-defensins block the release of IL-12 from human monocytes. Human ?-defensins (HNP-1 and HD-5), a group of antimicrobial peptides produced by neutrophils and epithelial cells, are the only inhibitors that block the release but not the processing of proIL-12 by caspase-1 in human monocytes. This proposal does not investigate the in vivo role of defensins. It only uses defensins as a tool to identify novel molecular targets for IL-12 blockade. Using photo-affinity and confocal microscopy, we have found that defensins bind to cell-membrane-associated proteins in human monocytes. We will test the hypothesis that IL-12 release and the externalization of proIL-12 and secretory lysosomes from human monocytes are regulated by distinct signaling proteins which can be inhibited by ?-defensins via an enzyme- linked receptor. To accomplish that, we have identified two specific aims: Specific Aim One: Identify the receptor that is responsible for defensin-mediated inhibition of IL-12 release from human monocytes. Using photo-affinity purification and proteomic approaches, we will identify defensin-binding proteins (DBPs) in human monocytes. We will then identify the defensin receptor by determining the effect of siRNA-mediated knockdown of these DBPs on defensin blockade of IL-12 release. Specific Aim Two: Define the signaling pathway essential for defensin receptor-mediated inhibition of IL- 12 release. We will determine whether protein kinase C, phospholipase C, Ca2????dependent protein kinase, and MAP kinases are involved in defensin blockade of IL-12 release and the externalization of proIL-12 and secretory lysosomes from human monocytes. Successful completion of this project will have broad impacts on both basic and translational medical research. If our hypotheses are correct, it will not only bring much clarity to the field of IL-12 biology, but also provide novel insights to the ER/Golgi-independent secretory pathway used by many other important leaderless proteins, including IL-18, IL-33, MIF, and FGF-2. The proposed studies will also lead to the discovery of novel molecular targets for IL-12 blockade and may lead to the development of new therapeutic approaches to prevent and treat many life-threatening microbial infections and inflammatory diseases. PUBLIC HEALTH RELEVANCE: Defensins are peptides produced by white blood cells and epithelial cells. Using defensins as a tool, we will determine how proinflammatory cytokine IL-12 is released from human monocytes. The proposed studies will lead to the discovery of novel molecular targets for IL-12 blockade and may lead to the development of new therapeutic approaches to prevent and treat many life-threatening microbial infections and inflammatory diseases.