Disease-related defects in dendritic cell processing of bacterial antigens

描述(申请人提供)：细菌感染在很大程度上是通过特殊吞噬细胞类型的吞噬作用清除的。被吞噬的细菌在越来越恶劣的条件下被杀死，因为它们被隔离在其中的吞噬小体通过一系列有序的膜融合和裂变事件成熟，导致酸化，随之而来的水解酶激活，以及活性氧和氮物种的合成。在传统的树突状细胞(DC)中，吞噬细菌(如单核细胞增生性李斯特菌)的抗原被主要组织相容性复合体(MHC)I类(MHC-I)和MHC-II(MHC-II)分子处理和提呈，分别激活NAVE病原体特异性CD8+和CD4+T细胞。这些功能需要修改正常的吞噬小体成熟程序，在该程序中，抗原被迅速摧毁。因此，由于细菌逃避策略或宿主遗传缺陷引起的DC吞噬小体成熟缺陷，可以通过限制抗原的处理和提呈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，吞噬小体成熟和随之而来的MHC-II抗原递呈发生了变化。HPS是一组多系统疾病，以眼皮肤白化、出血过多和其他症状为特征，这些症状是由于未能正确形成组织特异性细胞内间隔而导致的，统称为溶酶体相关细胞器(LRO)。缺乏内体蛋白分选复合体AP-3的HPS2患者还会患有免疫缺陷和中性粒细胞减少症。HPS7和HPS8患者缺乏一种不同的复合体，BLOC-1，它在其他类型的LRO产生细胞中调节不同的运输步骤；这些患者不是免疫缺陷的。这表明AP-3是免疫细胞中LRO功能所特需的。值得注意的是，树突状细胞中的LRO参与了几种功能，包括与吞噬小体融合，中和吞噬小体的pH，从而促进抗原存活，从而允许MHC-I逃逸和呈递。随后的酸化是促进MHC II呈递的多肽产生所必需的。根据我们的初步数据，我们假设AP-3而不是BLOC-1调节DC中的吞噬小体成熟步骤，这是MHC-II最佳呈递吞噬细胞抗原所必需的。我们将在以下特定目标中验证这一假说：1.确定BLOC-1或AP-3是否需要(A)吞噬或其他形式的体外吞噬后抗原的最佳DC提呈，以及(B)在体内受到细菌病原体(单增李斯特菌)攻击时的T细胞免疫反应。2.检测在缺乏BLOC-1或AP-3的HPS小鼠模型中，骨髓来源的DC(BMDCs)的LRO生物发生和/或吞噬小体成熟是否发生改变。 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，患者出现一系列症状，包括大量出血、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，这与一种免疫细胞--细胞毒性T淋巴细胞的缺陷有关。在这项建议中，我们将使用HPS 2型小鼠模型来测试另一种不同类型的免疫细胞-树突状细胞是否也存在缺陷，以及这种故障是否导致对细菌病原体(如李斯特菌)的免疫缺陷。

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.