Modulation of Pulmonary Defenses in Pathobiology of Chronic Infections

慢性感染病理学中肺防御的调节

• 批准号: 8259074
• 负责人: Michal A Olszewski
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259074
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Alveolar; Antibiotic Therapy; Arginine; Binding; Biological Assay; Biology; Cell Culture Techniques; Cells; Chronic; Clinical; Code; Color; Communicable Diseases; Cryptococcus; Cryptococcus neoformans; Cryptococcus neoformans infection; Cytokine Network Pathway; Data; Development; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Exhibits; Flow Cytometry; Gene Activation; Gene Deletion; Gene Expression; Genes; Grant; Growth; HIV; Health; Heat shock proteins; Heat-Shock Proteins 70; Homologous Gene; Host Defense; Hour; Immune System Diseases; Immune response; Immune system; Immunocompetent; Immunocompromised Host; In Vitro; Industrial fungicide; Infection; Interferon Type II; Interleukin-4; Knock-out; Knockout Mice; Knowledge; Lead; Life; Lung; M cell; Malignant Neoplasms; Mediating; Methods; Modeling; Morbidity - disease rate; Mouse Strains; Mus; Mycoses; NOS2A gene; Nitric Oxide; Nitric Oxide Synthase; Organ Transplantation; Organism; Outcome; Pathway interactions; Patients; Pattern; Phase; Phenotype; Process; Production; Proteins; Recombinants; Research; Role; Series; Signal Transduction; Source; Substance abuse problem; Testing; Transplant Recipients; Up-Regulation; Veterans; Virulence; Virulence Factors; Yeasts; antimicrobial drug; arginase; base; clinically relevant; cytokine; improved; in vivo; insight; intracellular parasitism; killings; macrophage; mortality; mutant; novel therapeutics; pathogen; prevent; problem drinker; rapid growth; receptor; research study; scavenger receptor; stem; substance abuser; treatment strategy

DESCRIPTION (provided by applicant): Olszewski M.A.: Modulation of Pulmonary Defenses in Pathobiology of Chronic Infections Objectives: Macrophages (M) are crucial for either clearance or persistence of the opportunistic yeast C. neoformans. When properly activated, M generate fungicidal nitric oxide from L-arginine to destroy ingested cryptococci. In unfavorable clinical circumstances, M become alternatively activated and induce Arginase (ARG1), an intracellular enzyme that consumes L-arginine but does not induce nitric oxide. The proposed studies will focus on dysregulation of M function by C. neoformans, and the role of recently defined virulence factor, heat shock protein Ssa1 in this porcess. Our objective is to test the hypothesis that Ssa1, induces the alternative activation of M and in this fashion promotes rapid growth of C. neoformans in the lungs. We plan to determine, if Ssa1-induced effects are mediated through Ssa1-binding to the scavenger receptor A (SRA) on M and through subsequent upregulation of ARG1 in these cells. Research Plan: The proposed research is presented as three interrelated specific aims: 1) To determine if cryptococcal Ssa1 promotes alternative activation of MF. We propose a series of experiments with cultures of isolated murine M to analyze the effects of recombinants Ssa1 protein and the effects of Ssa1 expression by C. neoformans on M activation status. These in vitro studies will be validated by in vivo infections with Ssa1 producing and Ssa1-deleted mutants of C. neoformans in mice and M phenotype analysis. 2) To determine if the mechanism alternative activation of MF by Ssa1 is induced via the SRA signaling. In this aim, we will determine if SRA signaling is required for Ssa1-induced effects and for the changes in M activation status. 3) To determine if the effects of Ssa1 gene expression by C. neoformans on MF function can be reversed by ARG1 inhibition or manipulations with cytokine environment. Our final aim will explore, if preventing the alternative activation of M by gamma- interferon and/or blocking Arginase1 enzyme would prevent the Ssa1-mediated virulence and lead to improved killing of C. neoformans by M. Methods: Models of murine C. neoformans infections and the isolated mouse M cell cultures, which yielded significant insights into the host-pathogen interaction and understanding mechanisms of anticryptococcal protection, will be used. Ssa1 producing wild type strain of C. neoformans: H99; Ssa1 knockout strain derived from H99; and the revertant strain ssa1::SSA1 with restored SSA1 gene will be used in these studies to induce pulmonary infections in mice or to treat the primary M cultures. Cryptococcal recombinant Ssa1 protein will be also used to treat these cells. Biology of M, cell phenotypes and cytokines produced by these cells will be evaluated in vivo and in vitro, utilizing: 6-color flow cytometry, qPCR and ELISA. Production of fungicidal nitric oxide by M will be evaluated using flow-ctometry based DAF-DT assay. We will use the knockout mice with disrupted SRA gene to determine if SRA signaling contributes to alternative activation of M by C. neoformans. Clinical Relevance: Pulmonary infectious diseases are significant source of morbidity in patients with HIV, organ transplant recipients, alcoholics, IV substance abusers and patients with lymphoproliferative malignancies. Treatment of fungal infections with conventional anti-microbial agents in these patients has been disappointing. Our studies reveal how a major opportunistic fungal pathogen C. neoformans may exploit the loopholes in M signaling to establish intracellular parasitism and propose a treatment strategy to prevent its occurrence. PUBLIC HEALTH RELEVANCE: Relevance to the Veterans Health: Pulmonary infectious diseases are a major source of morbidity and mortality in VA patients with compromised immune systems as a result of AIDS, substance abuse, or stemming from treatment of malignancy, auto-immune disease, or organ transplantation. Antibiotic therapy is often inadequate; thus knowledge of pulmonary host defense is of central importance to the development of new therapies Our studies reveal how a major opportunistic fungal pathogen C. neoformans may exploit the loopholes in the immune system defenses to establish intracellular parasitism and propose a treatment strategy to prevent its occurrence. Demonstrating importance of these pathways may become a cornerstone for novel therapeutic strategies for treatment of cryptococcosis in both immunocompromised and immunocompetent patients.

描述（由申请人提供）： Olszewski M.A.：慢性感染病理学中肺防御的调节目标：巨噬细胞 (M) 对于机会性酵母菌新生隐球菌的清除或持续存在至关重要。当正确激活时，M 从 L-精氨酸中产生杀菌一氧化氮，以消灭摄入的隐球菌。在不利的临床情况下，M 会交替激活并诱导精氨酸酶 (ARG1)，这是一种消耗 L-精氨酸但不诱导一氧化氮的细胞内酶。拟议的研究将重点关注新型隐球菌对 M 功能的失调，以及最近确定的毒力因子热休克蛋白 Ssa1 在此过程中的作用。我们的目的是检验以下假设：Ssa1 诱导 M 的替代激活，并以这种方式促进肺部新型隐球菌的快速生长。我们计划确定 Ssa1 诱导的效应是否是通过 Ssa1 与 M 上的清道夫受体 A (SRA) 结合以及随后这些细胞中 ARG1 的上调来介导的。研究计划：拟议的研究分为三个相互关联的具体目标：1）确定隐球菌 Ssa1 是否促进 MF 的替代激活。我们提出了一系列用分离的小鼠 M 培养物进行的实验，以分析重组 Ssa1 蛋白的影响以及新型隐球菌 Ssa1 表达对 M 激活状态的影响。这些体外研究将通过小鼠体内产生 Ssa1 和 Ssa1 删除的新型隐球菌突变体的体内感染和 M 表型分析来验证。 2) 确定Ssa1对MF的替代激活机制是否是通过SRA信号传导诱导的。为此，我们将确定 Ssa1 诱导的效应和 M 激活状态的变化是否需要 SRA 信号传导。 3) 确定新型隐球菌 Ssa1 基因表达对 MF 功能的影响是否可以通过 ARG1 抑制或细胞因子环境操作来逆转。我们的最终目标是探索，通过γ-干扰素和/或阻断精氨酸酶1酶来防止M的替代激活将阻止Ssa1介导的毒力并导致M对新型隐球菌的杀伤力增强。方法：小鼠新型隐球菌感染模型和分离的小鼠M细胞培养物，这对宿主-病原体相互作用和理解新型隐球菌的机制产生了重要的见解。 抗隐球菌保护，将被使用。产生Ssa1的新型隐球菌野生型菌株：H99； Ssa1 敲除菌株源自 H99；具有恢复的SSA1基因的回复菌株ssa1::SSA1将在这些研究中用于诱导小鼠肺部感染或治疗原代M培养物。隐球菌重组 Ssa1 蛋白也将用于治疗这些细胞。 M 的生物学、细胞表型和这些细胞产生的细胞因子将在体内和体外进行评估，利用：6 色流式细胞术、qPCR 和 ELISA。 M 产生的杀菌一氧化氮将使用基于流式细胞仪的 DAF-DT 测定进行评估。我们将使用 SRA 基因被破坏的敲除小鼠来确定 SRA 信号传导是否有助于新型隐球菌对 M 的替代激活。临床相关性：肺部感染性疾病是艾滋病毒患者、器官移植受者、酗酒者、静脉注射药物滥用者和淋巴增殖性恶性肿瘤患者发病的重要来源。使用传统抗微生物药物治疗这些患者的真菌感染效果令人失望。我们的研究揭示了一种主要的机会性真菌病原体新型隐球菌如何利用 M 信号传导的漏洞建立细胞内寄生，并提出了一种预防其发生的治疗策略。 公共卫生相关性： 与退伍军人健康的相关性：肺部传染病是因艾滋病、药物滥用或恶性肿瘤、自身免疫性疾病或器官移植治疗而导致免疫系统受损的退伍军人患者发病和死亡的主要原因。抗生素治疗常常是不够的；因此，了解肺部宿主防御对于新疗法的开发至关重要。我们的研究揭示了主要的机会性真菌病原体新型隐球菌如何利用免疫系统防御中的漏洞建立细胞内寄生，并提出了一种预防其发生的治疗策略。证明这些途径的重要性可能成为治疗免疫功能低下和免疫功能正常患者隐球菌病的新治疗策略的基石。