Modulation of Pulmonary Defenses in Pathobiology of Chronic Infections

描述(由申请人提供)： Olszewski M.A.：慢性感染病理生物学中肺防御的调节目的：巨噬细胞(M)对于机会性酵母新生芽孢杆菌的清除或持续至关重要。当M被适当激活时，M会从L精氨酸中产生杀菌一氧化氮来摧毁摄入的隐球菌。在不利的临床环境中，M交替激活并诱导精氨酸酶(Arg1)，这是一种细胞内酶，消耗L精氨酸，但不诱导一氧化氮。拟议的研究将集中在新生葡萄球菌M功能的失调，以及最近定义的毒力因子，热休克蛋白Ssa1在这一过程中的作用。我们的目标是验证这样的假设，即Ssa1诱导M的交替激活，并以这种方式促进肺部新生葡萄球菌的快速生长。我们计划确定SSA1诱导的效应是否通过SSA1与M上的清道夫受体A(SRA)结合以及随后这些细胞中ARG1的上调而介导。研究计划：拟议的研究提出了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。我们用分离培养的小鼠M进行了一系列实验，以分析重组Ssa1蛋白的作用以及新生弧菌表达Ssa1对M激活状态的影响。这些体外研究将通过体内感染小鼠体内产生SSA1和缺失SSA1的新生芽孢杆菌突变株和M表型分析来验证。2)确定SSA1对Mf的机制选择性激活是否通过SRA信号诱导。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。3)研究ARG1抑制或细胞因子环境调控能否逆转新生葡萄球菌Ssa1基因表达对MF功能的影响。我们的最终目标将探索，如果阻止M的交替激活由γ-干扰素和/或阻断精氨酸酶1酶是否可以阻止Ssa1介导的毒力并导致M对新生弧菌的杀灭。方法：将使用新生弧菌感染小鼠的模型和分离的小鼠M细胞培养，这对宿主-病原体的相互作用和理解抗隐球菌保护的机制产生了重要的见解。产生SSA1的野生型菌株：H99；来源于H99的SSA1基因敲除菌株；以及恢复了SSA1基因的突变菌株SSA1：：SSA1将用于诱导小鼠肺部感染或治疗原代M培养。隐球菌重组SSA1蛋白也将用于治疗这些细胞。利用6色流式细胞术、定量聚合酶链式反应和酶联免疫吸附试验对M细胞的生物学特性、细胞表型和产生的细胞因子进行体内和体外评价。将使用基于DAF-DT的流动法来评估M产生杀菌一氧化氮的能力。我们将使用SRA基因被破坏的敲除小鼠来确定SRA信号是否有助于新生葡萄球菌对M的替代激活。临床意义：肺部感染疾病是HIV患者、器官移植受者、酗酒者、静脉药物滥用者和淋巴增生性恶性肿瘤患者的重要发病率来源。在这些患者中，使用传统抗微生物药物治疗真菌感染一直令人失望。我们的研究揭示了一种主要的机会性真菌病原体新生梭菌如何利用M信号的漏洞来建立细胞内寄生，并提出了防止其发生的治疗策略。

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.