Recruited Lung Dendritic Cells and the Orchestration of Local Immune Responses

目的：新生隐球菌是导致艾滋病患者致死性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，未能根除 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性感染。 损坏。目前的治疗方法冗长，疗效有限，而且往往是有毒的。宿主受损或异常 免疫反应是病原体清除失败的先决条件，是导致肺损伤的原因之一。 与持续性感染有关。然而，人们对这些答复知之甚少。目前的研究是 旨在确定免疫调节性单核细胞来源的肺树突状细胞(i-moDC)如何协调 细胞和分子网络损害cneo清除并促进持续性肺部感染和 慢性肺部炎症。[对这些异常免疫反应的更好理解可能会产生新的 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易感性和持久性]结果来自 交替激活基因IL-10参与的I-moDC信号通路的异常发展 产品和PD-1/PD-L1。为了严格检验这一假说，我们构建了以下目标。 目的1：确定自分泌的单核细胞来源的DC产生IL-10是否是必要和充分的 隐球菌抗原上调交替激活基因和PD-L1/PD-L2的表达 (体外)或活动性真菌肺部感染(体内)。 目的2：确定肺I-moDC是否促进T调节细胞的极化和扩增 [在易患持续性肺部感染的小鼠中。] 目的3：确定阻断IL-10或PD-1/PDL-1信号通路是否[改善真菌 清除和]预防持续的隐球菌肺部感染。 研究计划和方法：我们的方案利用已建立的隐球菌肺小鼠模型 以免疫调节性树突状细胞聚集为特征的易感C57BL/6小鼠感染， T调节细胞和Th2细胞的扩张以及与免疫介导的肺相关的持续感染 损坏。肺树突状细胞在真菌清除失败和持续感染中的作用 表型将使用以下实验技术来确定：1)比较流式细胞术 分析和细胞分类，2)对分离的肺白细胞进行定量聚合酶链式反应，3)真菌CFU分析，4)组织学， 5)体外细胞培养和共培养，6)体内过继细胞转移。我们的调查将得到加强 通过我们选择的使用：a)IL-10缺陷小鼠，b)IL-10报告小鼠(Vert-X小鼠)，c)F1代小鼠 (从培育C57BL/6小鼠和BALB/c小鼠获得)，d)含有白喉毒素的转基因小鼠 白喉毒素注射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IL-10基因，以及f)注射抗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 许可。]

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.]