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.]

目的：新型隐球菌（Cneo）是艾滋病患者致死性真菌感染的主要原因 也是移植患者中第二常见的真菌感染在存活的患者中， Cneo或类似的肺部病原体通常会导致与慢性肺部相关的持续感染 损害目前的治疗方法耗时长，疗效有限，而且往往有毒。受损或异常宿主 免疫应答是病原体清除失败的先决条件，并导致肺损伤 与持续感染有关。然而，这些反应却知之甚少。目前的研究是 旨在确定免疫调节性单核细胞衍生的肺树突状细胞（i-moDC）如何协调 损害Cneo清除并促进持续性肺部感染的细胞和分子网络， 慢性肺部炎症[对这些异常免疫反应的进一步理解可能会产生新的 用于治疗由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]是由 通过涉及IL-10、替代激活基因的途径的i-moDC信号传导的异常发展 产品和PD-1/PD-L1。下面的目标是为了严格检验这一假设。 目的1：确定自分泌单核细胞来源的DC IL-10的产生是否是必要的和足够的， 响应隐球菌抗原上调交替激活基因和PD-L1/PD-L2的表达 (in体外）或活动性真菌肺部感染（体内）。 目的2：探讨肺内i-moDC是否促进调节性T细胞的极化和扩增 [in易受感染的小鼠，发展为持续性肺部感染。 目的3：确定阻断IL-10或PD-1/PDL-1信号通路是否[改善真菌感染]。 清除和]防止持续性隐球菌肺部感染。 研究计划和方法：我们的建议利用了一个完善的隐球菌肺小鼠模型 易感C57 BL/6小鼠感染，其特征为免疫调节树突状细胞的积累， 调节性T细胞和Th 2细胞的扩增，以及与免疫介导的肺部感染相关的持续感染， 损害肺树突状细胞对[真菌清除失败和]持续感染的贡献 表型将使用以下实验技术确定：1）比较流式细胞术 分析和细胞分选，2）对分离的肺白细胞进行qPCR，3）真菌CFU分析，4）组织学， 5)体外细胞培养和共培养，和6）体内过继细胞转移。我们的调查会得到加强 通过我们选择使用：a）IL-10缺陷小鼠，B）IL-10报告小鼠（Vert-X小鼠），c）F1代小鼠 d）含有白喉毒素的转基因小鼠 在白喉毒素施用后允许细胞特异性耗竭的受体，e）转基因小鼠 含有允许细胞特异性IL-10缺乏的floxed IL-10基因，和f）施用抗IL-10和 抗PD-L1受体阻断抗体，以破坏免疫调节信号传导网络[并改善真菌感染] 清除。]