Role of defensin receptor signaling in IL-1beta release

描述（由申请人提供）：IL-12的过量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不能有效地从活化的单核细胞中分泌。然而，当lps引发的单核细胞受到细胞外ATP的进一步刺激时，它们迅速释放大量成熟的IL-12和proIL-12。迄今为止，IL-12从人类单核细胞释放的机制尚不清楚。本提案的总体目标是通过研究IL-12的分子机制来确定IL-12是如何释放的？-防御素阻断人单核细胞IL-12的释放。人类吗?-防御素（HNP-1和HD-5）是一组由中性粒细胞和上皮细胞产生的抗菌肽，是人类单核细胞中唯一阻断caspase-1释放但不阻断proIL-12加工的抑制剂。这一建议并没有研究防御素在体内的作用。它仅使用防御素作为工具来识别IL-12阻断的新分子靶点。利用光亲和和共聚焦显微镜，我们发现防御素在人单核细胞中与细胞膜相关蛋白结合。我们将测试IL-12的释放和proIL-12的外化以及人类单核细胞的分泌溶酶体是由不同的信号蛋白调节的假设，这些信号蛋白可以被？-通过酶连接受体来防御素。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类单核细胞释放IL-12的受体。利用光亲和纯化和蛋白质组学方法，我们将鉴定人单核细胞中的防御素结合蛋白（DBPs）。然后，我们将通过确定sirna介导的dbp敲低对防御素阻断IL-12释放的影响来鉴定防御素受体。特定目标二：确定防御素受体介导的IL- 12释放抑制所必需的信号通路。我们将确定是否蛋白激酶C，磷脂酶C， Ca2???？依赖蛋白激酶和MAP激酶参与防御素阻断IL-12的释放，以及proIL-12和分泌溶酶体的外化。这个项目的成功完成将对基础和转化医学研究产生广泛的影响。如果我们的假设是正确的，它不仅将使IL-12生物学领域更加清晰，而且还将为许多其他重要的无前导蛋白（包括IL-18、IL-33、MIF和FGF-2）使用的ER/ golgi独立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，并可能导致新的治疗方法的发展，以预防和治疗许多危及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.