Disease-related defects in dendritic cell processing of bacterial antigens

描述（由申请人提供）：细菌感染主要通过特定吞噬细胞类型的吞噬作用清除。被吞噬的细菌所处的环境越来越恶劣，随着它们被隔离的吞噬体通过一系列有序的膜融合和裂变事件成熟，导致酸化，随后水解酶的激活，以及活性氧和活性氮的合成，最终被杀死。在传统的树突状细胞（dc）中，来自被吞噬细菌（如单核增生李斯特菌）的抗原被主要组织相容性复合体（MHC） I类（MHC-I）和II类（MHC-II）分子加工和呈递，分别激活新病原体特异性CD8+和CD4+ T细胞。这些功能需要对正常的吞噬体成熟程序进行修改，在该程序中抗原被迅速破坏。因此，DC吞噬体成熟的缺陷，无论是由细菌逃避策略引起的，还是由宿主遗传缺陷引起的，都可以通过限制抗原的加工和呈递来限制适应性免疫反应。我们的初步结果表明，在遗传性疾病Hermansky-Pudlak综合征（HPS） 2型（HPS2）小鼠模型的dc中，吞噬体成熟和随之而来的MHC-II抗原呈递发生了改变。HPS是一组多系统疾病，其特征是皮肤白化病、过度出血和其他症状，这些症状是由于不能正确形成组织特异性细胞内区室（统称为溶酶体相关细胞器（LROs））而引起的。HPS2患者缺乏内体蛋白分选复合物AP-3，还会出现免疫缺陷和中性粒细胞减少症。HPS7和HPS8患者缺乏不同的复合物block -1，该复合物在其他LRO生成细胞类型中介导不同的运输步骤；这些患者没有免疫缺陷。这表明免疫细胞中LRO功能特异性需要AP-3。值得注意的是，dc中的LROs涉及多种功能，包括与吞噬体融合以中和吞噬体pH，从而促进抗原存活，从而允许mhc - 1逃逸和呈递。随后的酸化是必要的，以促进肽的产生，以供MHC II呈递。根据我们的初步数据，我们假设AP-3，而不是block -1，调节dc中吞噬体的成熟步骤，这是MHC-II最佳呈现被吞噬细菌抗原所必需的。我们将在以下具体目标中检验这一假设：为了确定block -1或AP-3是否需要(a)体外吞噬或其他形式的内吞作用后抗原的最佳DC呈递和(b)体内细菌病原体（单核增生乳杆菌）攻击时T细胞的免疫反应。2. 在缺乏block -1或AP-3的HPS小鼠模型中，检测骨髓源性dc （bmdc）的LRO生物发生和/或吞噬体成熟是否发生改变。

DESCRIPTION (provided by applicant): Bacterial infections are largely cleared by phagocytosis within specialized phagocytic cell types. Engulfed bacteria are subjected to increasingly harsh conditions and eventually killed as the phagosome in which they are sequestered matures through an ordered series of membrane fusion and fission events, leading to acidification, consequent activation of hydrolases, and synthesis of reactive oxygen and nitrogen species. In conventional dendritic cells (DCs), antigens from phagocytosed bacteria - such as Listeria monocytogenes - are processed and presented by major histocompatibility complex (MHC) class I (MHC-I) and class II (MHC-II) molecules to activate naove pathogen-specific CD8+ and CD4+ T cells, respectively. These functions require modifications to the normal phagosome maturation program in which antigens are rapidly destroyed. As such, defects in DC phagosome maturation, arising either from bacterial evasion strategies or from host genetic deficiencies, can restrict the adaptive immune response by limiting antigen processing and presentation. Our preliminary results suggest that phagosome maturation and consequent MHC-II antigen presentation are altered in DCs from a mouse model of the genetic disease, Hermansky-Pudlak syndrome (HPS) type 2 (HPS2). HPS is a group of multi-system disorders characterized by oculocutaneous albinism, excessive bleeding, and other symptoms resulting form the failure to properly form tissue-specific intracellular compartments known collectively as lysosome-related organelles (LROs). HPS2 patients, who lack the endosomal protein sorting complex AP-3, additionally suffer from immunodeficiency and neutropenia. HPS7 and HPS8 patients lack a different complex, BLOC-1, that mediates different transport steps in other LRO- producing cell types; these patients are not immunodeficient. This suggests that AP-3 is specifically required for LRO function in immune cells. Notably, LROs in DCs have been implicated in several functions, including fusion with phagosomes to neutralize phagosomal pH and thereby promote antigen survival to allow for escape and presentation by MHC-I. Subsequent acidification is necessary to promote peptide generation for presentation by MHC II. Based on our preliminary data, we hypothesize that AP-3, but not BLOC-1, regulates phagosome maturation steps in DCs that are required for optimal presentation of phagocytosed bacterial antigens by MHC-II. We will test this hypothesis in the following Specific Aims: 1. To determine whether BLOC-1 or AP-3 are required for (a) optimal DC presentation of antigens following phagocytosis or other forms of endocytosis in vitro and (b) T cell immune responses upon challenge with a bacterial pathogen (L. monocytogenes) in vivo. 2. To test whether LRO biogenesis and/or phagosome maturation in bone marrow-derived DCs (BMDCs) is altered in HPS mouse models lacking BLOC-1 or AP-3. PUBLIC HEALTH RELEVANCE: Hermansky-Pudlak syndrome (HPS) is a group of genetic diseases that is unusually prevalent in Puerto Rico and in which patients suffer from a number of symptoms, including excessive bleeding, oculocutaneous albinism, and lung dysfunction. In one class of the disease (HPS type 2), patients also suffer from a mild immunodeficiency that has been linked to defects in one type of immune cell, the cytotoxic T lymphocyte. In this proposal, we will use a mouse model of HPS type 2 to test whether there are also defects in a different type of immune cell, the dendritic cell, and whether this malfunction is responsible for immunodeficiency against bacterial pathogens such as Listeria monocytogenes.