Disease-related defects in dendritic cell processing of bacterial antigens

描述（由申请人提供）：细菌感染主要通过专门的吞噬细胞类型内的吞噬作用清除。吞噬的细菌受到越来越苛刻的条件，并最终杀死吞噬体，其中它们被隔离成熟，通过一系列有序的膜融合和裂变事件，导致酸化，随后激活水解酶，并合成活性氧和氮物种。在传统的树突状细胞（DC）中，来自吞噬细菌（如单核细胞增生李斯特菌）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，以分别激活新生病原体特异性CD 8+和CD 4 + T细胞。这些功能需要修改正常的吞噬体成熟程序，其中抗原被迅速破坏。因此，由细菌逃避策略或宿主遗传缺陷引起的DC吞噬体成熟缺陷可以通过限制抗原加工和呈递来限制适应性免疫应答。我们的初步研究结果表明，吞噬体成熟和随之而来的MHC-II抗原呈递改变了从遗传性疾病，Hermansky-Pudlak综合征（HPS）2型（HPS 2）的小鼠模型的DC。HPS是一组多系统疾病，其特征在于眼皮肤白化病、过度出血和由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（LRO））导致的其他症状。缺乏内体蛋白分选复合物AP-3的HPS 2患者还患有免疫缺陷和中性粒细胞减少症。HPS 7和HPS 8患者缺乏不同的复合物BLOC-1，其介导其他LRO产生细胞类型中的不同转运步骤;这些患者不是免疫缺陷的。这表明AP-3是免疫细胞中LRO功能所特别需要的。值得注意的是，DC中的LRO涉及几种功能，包括与吞噬体融合以中和吞噬体pH，从而促进抗原存活以允许逃逸和由MHC-I呈递。随后的酸化是必要的，以促进肽的产生，由MHC II呈递。基于我们的初步数据，我们假设AP-3，而不是BLOC-1，调节吞噬体成熟的步骤，所需的最佳呈递吞噬的细菌抗原的MHC-II的DC。我们将在以下具体目标中检验这一假设：1。为了确定BLOC-1或AP-3是否是（a）在体外吞噬作用或其它形式的内吞作用后抗原的最佳DC呈递和（B）在用细菌病原体（L.单核细胞增多症）。2.为了测试在缺乏BLOC-1或AP-3的HPS小鼠模型中骨髓来源的DC（BMDC）中的LRO生物发生和/或吞噬体成熟是否改变。 公共卫生相关性：Hermansky-Pudlak综合征（HPS）是一组遗传性疾病，在波多黎各异常流行，患者患有多种症状，包括出血过多，眼皮肤白化病和肺功能障碍。在一类疾病（HPS 2型）中，患者还患有轻度免疫缺陷，这与一种类型的免疫细胞（细胞毒性T淋巴细胞）的缺陷有关。在这项提议中，我们将使用HPS 2型小鼠模型来测试不同类型的免疫细胞（树突状细胞）是否也存在缺陷，以及这种功能障碍是否是导致免疫缺陷的原因。

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.