Recruited Lung Dendritic Cells and the Orchestration of Local Immune Responses

招募肺树突状细胞和局部免疫反应的协调

• 批准号: 8633079
• 负责人: JOHN OSTERHOLZER
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8633079
• 关键词: Acquired Immunodeficiency Syndrome; Adoptive Cell Transfers; Antibiotic Therapy; Antigens; Apoptosis; Asthma; Bacteriophages; Blocking Antibodies; Breeding; C57BL/6 Mouse; Cell Culture Techniques; Cell Death; Cell Separation; Cells; Cessation of life; Chronic; Chronic Obstructive Airway Disease; Chronic lung disease; Coculture Techniques; Cryptococcus; Cryptococcus neoformans; Data; Dendritic Cells; Development; Diphtheria Toxin; Failure; Generations; Genes; Histology; Host Defense; Human; IL10 gene; Immune; Immune System Diseases; Immune response; Immune system; Immunity; Immunocompetent; Immunocompromised Host; In Vitro; Inbred BALB C Mice; Infection; Inflammatory; Interferons; Interleukin-1; Interleukin-10; Investigation; Ions; Knowledge; Length; Leukocytes; Lung; Lung Inflammation; Malignant Neoplasms; Mediating; Methods; Microbe; Modeling; Molecular; Morbidity - disease rate; Mus; Mycoses; Myeloid Cell Activation; Organ Transplantation; Pathway interactions; Patients; Phenotype; Play; Predisposition; Production; Proteins; Pulmonary Fibrosis; Recruitment Activity; Regulatory T-Lymphocyte; Reporter; Research; Research Design; Resistance; Role; Signal Pathway; Signal Transduction; Site; Source; Stimulus; Substance abuse problem; System; T-Lymphocyte; Techniques; Testing; Th2 Cells; Transgenic Mice; Transplant Recipients; Transplantation; Vaccines; Veterans; arginase; autocrine; comparative; diphtheria toxin receptor; immunopathology; improved; in vivo; insight; macrophage; monocyte; mortality; mouse model; novel; novel therapeutic intervention; pathogen; patient population; prevent; programs; public health relevance; receptor; research study; response; stem

Objectives: [Cryptococcus neoformans (Cneo) is the leading cause of fatal fungal infection in AIDS patients and the second most common fungal infection in transplant patients. In surviving patients, failure to eradicate Cneo, or similar pulmonary pathogens, often results in persistent infection associated with chronic lung damage. Current therapies are lengthy, have limited efficacy, and are often toxic. Impaired or aberrant host immune responses are a prerequisite for failed pathogen clearance and contribute to the lung damage associated with persistent infections. Yet these responses are poorly understood.] The present studies are designed to determine how immunomodulatory monocyte-derived lung dendritic cells (i-moDC) orchestrate the cellular and molecular networks which impair Cneo clearance and promote persistent pulmonary infections and chronic lung inflammation. [Improved understanding of these aberrant immune responses may yield novel therapies for the treatment of pulmonary infections caused by Cneo or related pathogens.] Hypothesis: [Susceptibility and persistence in response to cryptococcal lung infection] results from the aberrant development of i-moDC signaling through pathways involving IL-10, alternative activation gene products, and PD-1/PD-L1. The following aims have been constructed to rigorously test this hypothesis. Aim 1: To determine whether autocrine monocyte-derived DC IL-10 production is necessary and sufficient to up-regulate expression of alternatively-activated genes and PD-L1/PD-L2 in response to cryptococcal antigens (in vitro) or active fungal lung infection (in vivo). Aim 2: To determine whether pulmonary i-moDC promote the polarization and expansion of T regulatory cells [in susceptible mice that develop persistent lung infection.] Aim 3: To determine whether blockade of IL-10 or the PD-1/PDL-1 signaling pathways [improves fungal clearance and] prevents persistent cryptococcal lung infection. Research Plan and Methods: Our proposal utilizes a well-established murine model of cryptococcal lung infection in susceptible C57BL/6 mice characterized by an accumulation of immunomodulatory dendritic cells, an expansion in T regulatory and Th2 cells, and persistent infection associated with immune-mediated lung damage. The contribution of lung dendritic cells to [failed fungal clearance and] the persistently-infected phenotype will be determined using the following experimental techniques: 1) comparative flow cytometric analysis and cell sorting, 2) qPCR performed on isolated lung leukocytes, 3) fungal CFU analysis, 4) histology, 5) in vitro cell culture and co-culture, and 6) in vivo adoptive cell transfers. Our investigations will be enhanced by our selected use of: a) IL-10 deficient mice, b) IL-10 reporter mice (Vert-X mice), c) F1 generation mice (obtained from breeding C57BL/6 mice and BALB/c mice), d) transgenic mice containing the diphtheria toxin receptor permitting cell-specific depletion following diphtheria toxin administration, e) transgenic mice containing a floxed IL-10 gene permitting cell specific IL-10 deficiency, and f) administration of anti-IL-10 and anti-PD-L1 receptor blocking antibodies to disrupt immunomodulatory signaling networks [and improve fungal clearance.]

目标：[Cryptococcus Neoformans（CNEO）是艾滋病患者致命真菌感染的主要原因 以及移植患者中第二常见的真菌感染。在幸存的患者中，未能消除 CNEO或类似的肺病原体，通常会导致与慢性肺有关的持续感染 损害。当前的疗法很长，功效有限，并且通常是有毒的。受损或异常宿主 免疫反应是病原体清除失败的先决条件，并导致肺部损伤 与持续感染有关。然而，这些回答知之甚少。]当前的研究是 旨在确定免疫调节的单核细胞衍生的肺树突状细胞（I-MODC）如何编排 细胞和分子网络会损害CNEO清除并促进持续的肺部感染和 慢性肺部炎症。 [提高对这些异常免疫反应的理解可能会产生新颖 治疗由CNEO或相关病原体引起的肺部感染的疗法。] 假设：[对隐球菌肺部感染的敏感性和持久性] I-MODC信号通过涉及IL-10，替代激活基因的途径的异常发展 产品，PD-1/PD-L1。已经构建了以下目标来严格检验该假设。 目的1：确定自分泌单核细胞衍生的DC IL-10是否需要并且足以足以 替代激活的基因和PD-L1/PD-L2的上调表达对隐球菌抗原响应 （体外）或活性真菌肺部感染（体内）。 目标2：确定肺I-MODC是否促进了调节细胞的极化和膨胀 [在发展持续性肺部感染的敏感小鼠中。] AIM 3：确定IL-10或PD-1/PDL-1信号传导途径的封锁[改善真菌 清除和]防止持续的加密肺癌感染。 研究计划和方法：我们的提案采用了一个公认的加密肺癌的鼠模型 易感C57BL/6小鼠的感染，其特征是免疫调节的树突状细胞积累， T调节和Th2细胞的扩展，以及与免疫介导的肺有关的持续感染 损害。肺树突细胞对[真菌清除失败的贡献，]持续感染 表型将使用以下实验技术确定：1）比较流式细胞仪 分析和细胞分选，2）在分离的肺白细胞上进行的qPCR，3）真菌CFU分析，4）组织学， 5）体外细胞培养和共培养，6）体内产卵细胞转移。我们的调查将得到增强 通过我们选定的：a）IL-10缺陷小鼠，b）IL-10记者小鼠（vert-X小鼠），c）F1 f1 oferation小鼠 （从育种C57BL/6小鼠和BALB/C小鼠中获得）D）含有白喉毒素的转基因小鼠 毒素毒素给药后允许细胞特异性耗竭的受体，e）转基因小鼠 包含允许细胞特异性IL-10缺乏的floxed IL-10基因，f）给药抗IL-10和 抗PD-L1受体阻断抗体破坏免疫调节信号网络[并改善真菌 清除。]