Nrf2 regulation of alveolar macrophage immune responses in vivo to Mycobacterium tuberculosis

项目总结 结核分枝杆菌(Mtb)每年夺走近150万人的生命，是导致 世界范围内的传染病致死。由于成人肺部疾病缺乏保护性结核病疫苗， 当前抗生素治疗方案的长度和毒性，以及结核分枝杆菌耐药性的上升都很强烈 推动针对宿主的新疗法和疫苗设计策略的追求。肺泡巨噬细胞 肺中的第一批细胞在气溶胶传播后感染结核分枝杆菌，发挥着结核分枝杆菌的关键作用 呼吸道内的先天哨兵。它们必须启动宿主反应，将其他先天细胞招募到肺中。 并将细菌转移到细胞中，这些细胞可以将细菌带到引流的淋巴结处，进行有效的免疫启动。作为组织- 常驻髓系细胞，肺泡巨噬细胞也执行关键的动态平衡功能，清除碎片 在不引发肺部炎症的情况下使呼吸道通畅。目前尚不清楚肺泡巨噬细胞如何平衡其 先天感知和组织维护职责，而宿主对结核分枝杆菌启动反应的任何延迟 为细菌提供了更多的时间来不受限制地复制。我们之前的结果显示，结核分枝杆菌感染 肺泡巨噬细胞上调细胞保护信号，依赖于转录因子Nrf2。此入站 活体反应不同于先前报道的结核分枝杆菌感染的典型促炎反应 巨噬细胞的体外实验表明，从这种体内方法可以获得新的信息。我们的结果 研究表明，在没有Nrf2的情况下，感染结核杆菌的肺泡巨噬细胞更活跃，活性更低， 在感染的头10天内证明加强了对细菌的控制，但尚不清楚NRF2如何 感染早期肺泡巨噬细胞功能的调节影响疾病的后续阶段 进步。该提案的目标是开发不同的工具来调节肺泡巨噬细胞Nrf2 表达，以确定Nrf2如何影响细菌感染后免疫事件的时间和质量 呼吸道中的沉积，最终导致疾病进展或细菌遏制。首先，我们 将表征Nrf2对肺泡巨噬细胞死亡和细菌传播的调节作用 特定的Nrf2条件基因敲除菌株(目标1)。第二，我们将开发一个体外系统来研究Nrf2是如何 干扰肺泡巨噬细胞先天感知通路和shRNA慢病毒口咽递送 体内瞬时阻断肺泡巨噬细胞Nrf2表达的系统，使我们能够避免任何混淆 Nrf2在发育过程中的作用(目标2)。这些研究是由一种假设驱动的，即细胞的诱导- NRF2的保护程序阻止肺泡巨噬细胞快速有效地促炎 对结核分枝杆菌感染的反应，导致后续免疫事件的延迟，从而损害宿主的控制 感染。通过开发新的工具和试剂来研究肺泡巨噬细胞特异性Nrf2的作用 表达，我们将更好地了解结核杆菌在肺部感染的早期事件，并评估 Nrf2作为宿主导向治疗的未来靶点的潜力。

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) claims nearly 1.5 million lives each year and is one of the leading causes of death by an infectious agent worldwide. The lack of a protective TB vaccine for adult pulmonary disease, the length and toxicity of current antibiotic treatment regimens, and the rise in Mtb drug resistance all strongly motivate the pursuit of new host-directed therapies and strategies for vaccine design. Alveolar macrophages are the first cells in the lung to be infected with Mtb following aerosol transmission, performing a critical role as Mtb innate sentinels in the airway. They must initiate the host response that will recruit other innate cells into the lung and transfer bacteria to cells that can carry it to the draining lymph node for efficient immune priming. As tissue- resident myeloid cells, alveolar macrophages also perform a critical homeostatic function, clearing debris from the airway without triggering pulmonary inflammation. It is unknown how alveolar macrophages balance their innate sensing and tissue maintenance duties, while any delay in the initiation of the host response to Mtb provides the bacteria with additional time to replicate unchecked. Our previous results showed that Mtb-infected alveolar macrophages up-regulate a cell-protective signature, dependent on the transcription factor Nrf2. This in vivo response is distinct from the canonical pro-inflammatory response previously reported for Mtb-infected macrophages in vitro indicating that new information that can be gained from this in vivo approach. Our results showed that in the absence of Nrf2, Mtb-infected alveolar macrophages are more activated, less viable, and demonstrate enhanced control of bacteria within the first 10 days of infection, yet it is unknown how Nrf2 regulation of alveolar macrophage function early during infection impacts the subsequent stages of disease progression. The goal of this proposal is to develop diverse tools to modulate alveolar macrophage Nrf2 expression in order to define how Nrf2 impacts the timing and quality of the immune events following bacterial deposition in the airway, which ultimately lead to either disease progression or bacterial containment. First, we will characterize Nrf2 regulation of alveolar macrophage cell death and bacterial dissemination using myeloid- specific Nrf2 conditional knock-out strains (Aim 1). Second, we will develop an ex vivo system to study how Nrf2 interferes with alveolar macrophage innate sensing pathways and an shRNA lentiviral oropharyngeal delivery system to transiently block alveolar macrophage Nrf2 expression in vivo, allowing us to avoid any confounding effects of Nrf2 during development (Aim 2). The studies are driven by the hypothesis that the induction of a cell- protective program by Nrf2 prevents alveolar macrophages from mounting a rapid and effective pro-inflammatory response to Mtb infection, leading to delays in subsequent immune events that impair the host from controlling infection. By developing new tools and reagents to study the role of alveolar macrophage-specific Nrf2 expression, we will gain a better understanding of the early events of Mtb infection in the lung and evaluate the potential of Nrf2 as a future target for host-directed therapy.