Defining the role of mast cells during influenza A virus infection

描述（由申请方提供）：甲型流感病毒（IAV），特别是高致病性IAV毒株，例如导致最近H5 N1和H1N1疫情的毒株，是一个重大的公共卫生问题。这些IAV毒株引起的发病率和死亡率明显高于季节性IAV毒株。然而，发病率和死亡率增加的确切原因仍然存在争议，病毒致病性增加，免疫病理学增加和合并感染增加被认为是可能的机制。该建议将研究IAV感染的早期宿主反应元件，特别是研究肥大细胞和蛋白酶激活受体在调节与IAV感染相关的发病率和死亡率中的作用。肥大细胞在协调宿主对IAV呼吸道感染的反应中可能至关重要，因为它们在肺内支气管周围的频率升高。此外，我们的初步数据表明，肥大细胞缺陷小鼠发展显着减少IAV相关的发病率。因此，我们建议确定肥大细胞是如何在呼吸道感染IAV期间被激活的，以及它们在调节疾病结局中起什么作用。其次，我们将确定肥大细胞是如何协调有害的主机炎症反应后，IAV感染。具体而言，肥大细胞激活肺泡上皮细胞上的蛋白酶激活受体家族，以及随后的调节炎症趋化因子和细胞因子的肥大细胞和蛋白酶激活受体家族。我们的初步数据支持这一假设，肥大细胞和蛋白酶激活受体家族在调节与呼吸道IAV感染相关的发病率方面是重要的。因此，通过了解这些早期事件，我们希望确定新的药理学靶点，可用于限制IAV感染的发病率和死亡率。 可以看出，这一建议的重点与我以前进行的研究截然不同。然而，我在成功完成拟议研究所需的实验系统和技术方面受过良好的培训。例如，我在使用小鼠了解对许多病毒病原体（包括IAV）的免疫反应方面获得了丰富的实验经验。此外，我还获得了成功完成拟定研究所需的许多技术方面的大量培训，包括多参数流式细胞术（我们目前常规进行12项参数研究）、骨髓原代细胞培养、用于同时检测多种细胞因子/趋化因子的Luminex微珠试验、病毒空斑试验、过继细胞转移和ELISA试验。总之，以前的经验提供了一个强大的实验基础，这将使我能够灵活地将这些知识和技术能力应用于一个独特的研究领域，因此开发了一个新的独立的研究方向，以解决病毒感染免疫调节知识的关键空白。此外，我目前和未来的研究机构为我提供了充足的技术机会来进行这些研究，包括使用10色BD LSRII细胞计数器，用于制造骨髓嵌合体的g细胞辐照器和用于运行Luminex板的BioPlex 200机器。此外，我现在和未来的大学的教师提供了一个良好的知识网络，这将是非常宝贵的成功完成拟议的研究。 每年，甲型流感病毒感染在某些群体中导致显著的发病率和死亡率，而周期性大流行毒株在整个人群中导致显著更高的发病率和死亡率。导致发病率和死亡率显著升高的原因仍然存在争议。该提案将研究宿主对甲型流感病毒感染反应期间的早期事件，并可能阐明限制与甲型流感病毒感染相关的发病率和死亡率的潜在新治疗靶点。

DESCRIPTION (provided by applicant): Influenza A virus (IAV) and especially the highly pathogenic IAV strains, such as those causing the recent H5N1 and H1N1 outbreaks, represent a significant public health concern. These strains of IAV cause significantly greater morbidity and mortality than seasonal IAV strains. However, the exact cause of the increased morbidity and mortality remains controversial, with increased viral pathogenicity, increased immunopathology, and increase co-infections proposed as possible mechanisms. This proposal will examine the early host response elements to IAV infection, specifically examining the role that mast cells and protease- activated receptors play in regulating the morbidity and mortality associated with IAV infection. Mast cells could be critical in orchestrating the host response to respiratory infection with IAV, because they are found at elevated frequencies around the bronchi within the lungs. Furthermore, our preliminary data show that mast cell-deficient mice develop significantly less IAV-association morbidity. Therefore, we propose to determine how mast cells are activated during respiratory infection with IAV and what role they play in regulating disease outcome. Secondly, we will determine how mast cells are orchestrating the deleterious host inflammatory response following IAV infection. Specifically, mast cell activation of the protease-activated receptor family on alveolar epithelial cells will be explored, as well as the subsequent regulation of inflammatory chemokines and cytokines by both mast cells and the protease-activated receptor family. Our preliminary data support the hypothesis that mast cells and the protease-activated receptor family are important in regulating morbidity associated with respiratory IAV infection. Thus, by understanding these early events, we hope to identify novel pharmacological targets, which can be used to limit morbidity and mortality from IAV infection. As can be seen, the focus of this proposal is quite distinct from the previous research that I have conducted. However, I have been well trained in the experimental systems and techniques needed for the successful completion of the proposed studies. For example, I have gained extensive experimental experience in the use of mice for understanding the immune response to numerous viral pathogens, including IAV. In addition, I have gained substantial training in many of the techniques required for the successful completion of the proposed studies, which include multi-parameter flow cytometry (we are routinely conducting 12 parameter studies now), primary cell culture from bone marrow, Luminex" bead assays for the detection of multiple cytokines/chemokines at the same time, viral plaque assays, adoptive cellular transfers, and ELISA assays. Together, this previous experience provides a strong experimental foundation, which will allow me the flexibility to apply this knowledge and technical ability to a distinct area of study, therefore developing a new and independent research direction that addresses critical gaps in knowledge of immune regulation of viral infection as an Assistant Professor. Furthermore, my current and future research institutes provide me ample technical opportunities to conduct these studies, including access to a 10-color BD LSRII cytometer, a g-cell irradiator for making bone marrow chimeras, and BioPlex200 machine for running Luminex plates. Additionally, faculty members at both my current and future universities provide a sound network of knowledge which will be invaluable for the successfully completion of the proposed studies. Each year, influenza A virus infection leads to significant morbidity and mortality within certain groups, while periodic pandemic strains cause significantly greater morbidity and mortality throughout the entire population. What causes the significantly elevated rates of morbidity and mortality remains controversial. This proposal will examine the early events during the host response to influenza A virus infection and could elucidate potential novel therapeutic targets for limiting morbidity and mortality associated with influenza A virus infection.