Defining the role of mast cells during influenza A virus infection

说明(申请人提供)：甲型流感病毒(IAV)，特别是高致病性IAV毒株，如导致最近H5N1和H1N1疫情的毒株，是一个重大的公共卫生问题。这些IAV毒株导致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，可能的机制是病毒致病性增加、免疫病理学增加和合并感染增加。这项建议将研究宿主对IAV感染的早期反应元件，特别是肥大细胞和蛋白酶激活的受体在调节与IAV感染相关的发病率和死亡率方面所起的作用。肥大细胞在协调宿主对IAV呼吸道感染的反应中可能是关键的，因为它们在肺内的支气管周围被发现的频率较高。此外，我们的初步数据显示，肥大细胞缺陷小鼠发生IAV相关性发病率显著较低。因此，我们建议确定在呼吸道感染IAV期间肥大细胞是如何被激活的，以及它们在调节疾病结局中所起的作用。其次，我们将确定肥大细胞如何在IAV感染后协调有害的宿主炎症反应。具体地说，将探讨肥大细胞对肺泡上皮细胞上蛋白水解酶激活受体家族的激活，以及随后肥大细胞和蛋白酶激活受体家族对炎性趋化因子和细胞因子的调节。我们的初步数据支持肥大细胞和蛋白水解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的重要假设。因此，通过了解这些早期事件，我们希望确定新的药理靶点，可以用来限制IAV感染的发病率和死亡率。 可以看出，这项提议的重点与我之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接受了良好的培训。例如，我在利用小鼠了解包括IAV在内的许多病毒病原体的免疫反应方面获得了广泛的实验经验。此外，我还在成功完成拟议研究所需的许多技术方面接受了大量培训，包括多参数流式细胞术(我们现在经常进行12个参数的研究)、骨髓原代细胞培养、同时检测多种细胞因子/趋化因子的Luminex“微珠分析”、病毒空斑分析、过继细胞转移和ELISA法。总而言之，以前的经验提供了强大的实验基础，这将使我能够灵活地将这些知识和技术能力应用于不同的研究领域，从而发展出一个新的独立研究方向，以助理教授的身份填补病毒感染免疫调节知识的严重空白。此外，我目前和未来的研究机构为我提供了大量的技术机会来进行这些研究，包括使用10色BD LSRII细胞仪，用于制造骨髓嵌合体的g细胞辐照仪，以及用于运行Luminex平板的BioPlex200机。此外，我现在和未来大学的教职员工都提供了一个完善的知识网络，这对成功完成拟议的研究将是无价的。 每年，甲型流感病毒感染在某些人群中会导致严重的发病率和死亡率，而周期性大流行病毒株在整个人口中造成的发病率和死亡率要高得多。导致发病率和死亡率显著上升的原因仍然存在争议。这项建议将研究宿主对甲型流感病毒感染反应的早期事件，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的治疗目标。

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.