Deciphering the Role of Lysosomal Biogenesis in Anti-Aspergillus Immune Responses

项目摘要/摘要 这份建议书描述了一项有指导的三年培训计划，将为申请者提供广泛的 真菌发病机制、细胞免疫学和细胞生物学方面的培训。赞助商的临床和实验室 建模和分析对微生物病原体的免疫反应的专业知识，专门的培训计划，以及 在免疫学、微生物发病机制和细胞生物学方面的机构优势都有助于 培训环境和成功的可能性很高。 烟曲霉是一种机会性真菌病原体，也是最常见的致病菌。 侵袭性曲霉病(IA)。尽管经常接触烟曲霉分生孢子，但免疫活性 个人很少患上IA，因为先天免疫系统的细胞被迅速招募到肺部，在那里他们 无症状地吞噬和清除分生孢子。然而，IA是免疫抑制的主要并发症。 个人，如接受实体器官或造血干细胞移植的人和患有 恶性血液病。因为这些患者群体正在扩大，目前缺乏 有效的抗真菌药物(这些风险人群的真菌可归因性死亡率超过20%) 重要的未满足的需求，以了解宿主对IA的免疫反应。 来自IA小鼠模型的初步数据表明，在肺中，中性粒细胞上调溶酶体 与真菌接触相关的基因。溶酶体生物发生的调控是由 主要转录因子TFEB和TFE3(TFEB/3)。此外，TFEB/3在巨噬细胞中被激活(MΦ) 对膨大的热致死分生孢子的刺激反应。我的培训和研究目标是将 TFEB/3和溶酶体生物发生在肺抗真菌免疫反应和免疫应答中的作用 获得这些研究所需的技术学科方面的培训。我推测烟曲霉激活了 TFEB/3通过C型凝集素受体信号在白细胞中表达，并参与杀菌免疫 在肺部，通过促进烟曲霉胞内杀灭分生孢子。 拟议研究的具体目标1将确定TFEB/3激活的分子基础 对烟曲霉菌的反应。用ΜΦ细胞系作为模型白细胞和遗传学和药理学 抑制关键信号分子的策略，我将揭示各种信号通路的相对贡献 ΜΦS TFEB/3的激活。拟议研究的具体目标2我将定义 TFEB/3具有抗曲霉免疫作用。我将使用基因工程小鼠模型来耗尽TFEB/3 髓系细胞与我们的IA模型相结合，揭示TFEB/3对小鼠存活的贡献，对 维持肺组织完整性，增强白细胞有效清除肺内分生孢子的能力。 这些研究将揭示吞噬酶体杀死的新机制，并将这些研究的意义 为IA提供新的治疗方法。

PROJECT SUMMARY/ABSTRACT This proposal describes a mentored three-year training plan that will provide the applicant with broad training in fungal pathogenesis, cellular immunology, and cell biology. The sponsor’s clinical and laboratory expertise in modeling and analyzing immune responses to microbial pathogens, the dedicated training plan, and institutional strengths in immunology, microbial pathogenesis, and cell biology all contribute to an outstanding training environment and a high likelihood of success. Aspergillus fumigatus is an opportunistic fungal pathogen and is the most common etiological agent of invasive aspergillosis (IA). Despite being constantly exposed to A. fumigatus conidia, immunocompetent individuals rarely get IA, because cells of the innate immune system are rapidly recruited to the lung where they phagocytose and clear conidia asymptomatically. However, IA is major complication for immunosuppressed individuals, such as those receiving solid organ or hematopoietic stem cell transplant and patients with hematological malignancies. Because these patient groups are expanding and there is currently a lack of effective anti-fungal drugs (fungus attributable mortality in these risk groups is upwards of 20%) there is a significant unmet need to understand the host immune response to IA. Preliminary data from a murine model of IA suggests that in the lung, neutrophils upregulate lysosome associated genes in response to fungal engagement. Regulation of lysosome biogenesis is controlled by the master transcription factors TFEB and TFE3 (TFEB/3). Additionally, TFEB/3 are activated in macrophages (MΦ) in response to stimulation with swollen heat-killed conidia. My training and research objective are to integrate the role of TFEB/3 and lysosome biogenesis in the context of the pulmonary anti-fungal immune responses and to acquire training in the technical disciplines needed for these studies. I hypothesize that A. fumigatus activates TFEB/3 in leukocytes through C-type lectin receptor signaling, and that TFEB/3 contribute to sterilizing immunity in the lung, by promoting intracellular killing of A. fumigatus conidia. Specific Aim 1 of the proposed research will determine the molecular basis for TFEB/3 activation in response to A. fumigatus. Using a ΜΦ cell line as a model leukocyte and both genetic and pharmacological strategies to inhibit key signaling molecules, I will uncover the relative contributions of various signaling pathways to the activation of TFEB/3 in ΜΦs. Specific Aim 2 of the proposed research I will define the contribution of TFEB/3 to anti-Aspergillus immunity. I will use a genetically engineered mouse model to deplete TFEB/3 from myeloid cells combined with our model of IA to uncover the contribution of TFEB/3 to murine survival, to the maintenance of lung tissue integrity, and to the ability of leukocytes to effectively clear conidia from the lung. These studies will reveal novel mechanisms of phagolysosomal killing and implications of these studies will inform novel therapeutic approaches for IA.