Modulation of Immune Defenses in Pathobiology of Chronic Infections

描述(由申请人提供)： 新生隐球菌是一种常见的、毁灭性的机会性真菌病原体，在世界范围内可导致大量死亡。目前对感染新生葡萄球菌或相关真菌病原体的退伍军人的治疗方法漫长，往往有毒，而且往往无效。需要在较长的时间内产生和维持这种1型或“经典激活”的免疫反应，以实现对新生葡萄球菌的完全清除，并防止非常常见的持续性感染和/或复发。越来越多的处方抗炎肿瘤坏死因子�中和型单抗治疗的副作用之一是对真菌感染(包括新生葡萄球菌)的易感性。我们的初步数据有力地表明，肿瘤坏死因子�S是编程1型树突状细胞(Dc1)和随后发展稳定的、保护性的Th1/Th17反应所必需的中心分子。本研究旨在确定肿瘤坏死因子�如何有助于髓系树突状细胞的编程，以在清除新生隐球菌所需的延长时间内维持保护性的Th1/Th17反应。假设：我们的中心假设是，有效清除新生隐球菌需要肿瘤坏死因子�编辑的髓系树突状细胞的Dc1编程，以在清除新生隐球菌所需的延长时间段内维持保护性的Th1/Th17反应。我们进一步假设，DC和/或其髓系前体中的肿瘤坏死因子�信号有助于执行表观遗传修饰，以支持Dc1表型的稳定性，并阻止Dc2表型的发展。为了严格检验这一假说，我们构建了以下目标。目的1：确定在对隐球菌感染的保护性反应中，肿瘤坏死因子�S是否需要稳定的Dc1编程。目的：研究肿瘤坏死因子�是否参与了新生隐球菌感染肺中髓系前体细胞和/或DC的表观遗传过程。目的3：确定肿瘤坏死因子�诱导的Dc1编程对于产生保护性免疫应答是否必要和充分。研究计划和方法：我们的建议利用中等毒力的新生葡萄球菌24067株感染Cba/J小鼠的高度翻译模型和一系列的体外研究，以确定在感染时使用一剂单抗瞬时消除肿瘤坏死因子�的效果。这些研究将使我们能够确定肿瘤坏死因子�是否以及在多大程度上提高了Dc1计划的稳定性。我们将确定在DC发育的哪个阶段(骨髓前体、DC分化或成熟)，肿瘤坏死因子�将DC表化。我们将确定Dc1表型稳定性是否可以归因于负责表观遗传染色质修饰的酶的变化以及对Dc1程序执行至关重要的基因启动子区域组蛋白甲基化特征的变化。随后，将在一系列过继转移实验中测试DC及其髓系前体细胞对肺部执行稳定保护性免疫反应的贡献。这些研究的完成将证明肿瘤坏死因子�稳定对新生葡萄球菌的保护性免疫反应的新作用。这项翻译研究将提供表观遗传染色质修饰和体内肿瘤坏死因子�生物效应之间的联系，并提供对使用抗肿瘤坏死因子�抗体治疗的患者真菌感染风险增加的潜在机制的洞察，我们实验室的小鼠模型非常有效地模拟了这种机制。

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a common and devastating opportunistic fungal pathogen that causes substantial mortality worldwide. Current therapies for veterans infected with C. neoformans or related fungal pathogens are lengthy, frequently toxic, and often ineffective. Generation and maintenance of this type 1 or "classically-activated" immune response over extended time is required to achieve complete clearance of C. neoformans and to prevent the all too common development of persistent infection and/or relapses. One of the side effects of the increasingly prescribed anti-inflammatory TNF�eutralizing monoclonal antibody therapy is susceptibility to fungal infections (including C. neoformans). Our preliminary data strongly suggest that TNF�s a central molecule required for programing of type 1 dendritic cell (DC1) and the subsequent development of a stable, protective Th1/Th17 response. The present studies are designed to determine how TNF�ontributes to programming of myeloid DC to sustain a protective Th1/Th17 response throughout the extended time period required for clearance of C. neoformans. Hypothesis: Our central hypothesis is that effective clearance of C. neoformans requires the TNF�ediated DC1 programming of myeloid DC to sustain a protective Th1/Th17 response throughout the extended time period required for clearance of C. neoformans. We further hypothesize that TNF�ignaling in DC and/or their myeloid precursors contributes to execution of epigenetic modifications that support stability of the DC1 phenotype and prevents the development of a DC2 phenotype. The following aims have been constructed to rigorously test this hypothesis. Aim 1: To determine whether TNF�s required for stable DC1-programing during the protective response to cryptococcal infection. Aim 2: To determine if TNF�ediates epigenetic programing of the DC1 phenotype in myeloid precursors and/or DC in C. neoformans infected lungs. Aim 3: To determine whether TNF�nduced DC1 programming is necessary and sufficient for generating protective immune responses to C. neoformans. Research Plan and Methods: Our proposal utilizes highly translational model of infection of CBA/J mice with the moderately virulent C. neoformans strain 24067 and series of in vitro studies that will define the effects of transient TNF�epletion using one dose of monoclonal antibodies at the time of infection. These studies will allow us to define whether, and to what degree, TNF�nduces stability of the DC1 program. We will define at which stage of DC development (bone marrow precursor, DC differentiation or maturation) TNF�tabilizes DC. We will determine whether the DC1 phenotype stability can be attributed to changes in enzymes responsible for epigenetic chromatin modification and resultant changes in histone methylation signatures at gene promoter regions crucial for execution of DC1 program. The contribution of DC and their myeloid precursors to the execution of stable protective immune responses in the lungs will be then tested in a series of adoptive transfer experiments. Completion of these studies will demonstrate a novel role of TNF�n stabilization of a protective immune response to C. neoformans. This translational study will provide a link between epigenetic chromatin modification and biological effects of TNF�n vivo and provide an insight into a potential mechanism of increased risk of fungal infections in patients treated with anti-TNF�ntibodies, which our lab's mouse model very effectively mimics.