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。：慢性感染目标病原体学中肺部防御的调节：巨噬细胞（M）对于机会性酵母菌Neoformans的清除或持久性至关重要。当正确激活时，M会从L-精氨酸产生杀真菌一氧化氮，以破坏摄入的加密摄氏。在不利的临床情况下，M被激活并诱导精氨酸酶（ARG1），这是一种消耗L-精氨酸但不会诱导一氧化氮的细胞内酶。拟议的研究将重点介绍Neoformans M功能的失调，以及最近定义的毒力因子，热休克蛋白SSA1在此效率上的作用。我们的目的是检验SSA1诱导M的替代激活的假设，以这种方式促进了肺中新生虫的快速生长。我们计划确定SSA1诱导的效应是否通过SSA1结合在M上的清除剂受体A（SRA）以及随后在这些细胞中的ARG1上调来介导。研究计划：提出的研究被提出为三个相互关联的特定目的：1）确定加密局SSA1是否促进了MF的替代激活。我们提出了一系列用分离的鼠M培养物进行的实验，以分析重组SSA1蛋白的影响以及Neoformans C. Neoformans对M激活状态的SSA1表达的影响。这些体外研究将通过在小鼠和M表型分析中用SSA1产生和新生梭菌的SSA1剥落突变体的体内感染来验证。 2）确定SSA1 MF的机理是否通过SRA信号传导诱导。在此目标中，我们将确定SSA1诱导的效果以及M激活状态的变化是否需要SRA信号传导。 3）为了确定Neoformans C. Neoformans对MF功能的SSA1基因表达的影响是否可以通过ARG1抑制或具有细胞因子环境的操作来逆转。如果通过γ-干扰素和/或阻止Arginase1酶阻止M对M的替代激活，我们的最终目标将防止SSA1介导的毒力，并导致M.方法改善Neoformans的杀害。将使用抗癌球菌保护。 SSA1产生野生型梭菌的菌株：H99； SSA1敲除菌株来自H99；这些研究将使用带有恢复的SSA1基因的恢复菌株SSA1 :: SSA1诱导小鼠肺部感染或治疗原发性培养物。隐球菌重组SSA1蛋白也将用于治疗这些细胞。这些细胞产生的M，细胞表型和细胞因子的生物学将在体内和体外评估：6色流式细胞仪，QPCR和ELISA。将使用基于流动率的DAF-DT测定法评估通过M通过M生产杀真菌氧化氮。我们将使用具有破坏的SRA基因的敲除小鼠来确定SRA信号是否有助于Neoformans的M替代激活M。临床相关性：肺部传染病是HIV，器官移植受者，酗酒者，IV药物滥用者和淋巴细胞增生性恶性肿瘤患者的重要发病来源。在这些患者中，用常规抗微生物剂治疗真菌感染令人失望。我们的研究揭示了主要的机会性真菌病原体C. Neoformans如何利用M信号传导中的漏洞来建立细胞内寄生虫，并提出一种防止其发生的治疗策略。 公共卫生相关性： 与退伍军人健康的相关性：由于艾滋病，滥用药物或因恶性肿瘤，自身免疫性疾病或器官移植而导致的肺部传染病是VA患者的发病率和死亡率的主要来源。抗生素疗法通常不足；因此，对肺宿主防御的了解对于新疗法的发展至关重要，我们的研究揭示了主要的机会性真菌病原体C. Neoformans如何利用免疫系统防御中的漏洞来建立细胞内寄生虫，并提出一种治疗策略来防止其发生。表现出这些途径的重要性可能成为治疗免疫功能低下和免疫能力患者的新型治疗策略的基石。