The Role of Innate IL-17 Responses to Aspergillus fumigatus

 描述（由申请人提供）：呼吸道粘膜利用先天性和适应性免疫系统保护正常呼吸功能免受入侵生物的侵害。然而，当这些防御之一存在缺陷时，宿主变得容易受到烟曲霉（Af）的攻击。这种无处不在的真菌作为孢子或休眠分生孢子进入气道，但通常被完整的呼吸道防御系统清除。然而，当个体免疫功能低下时，Af分生孢子更容易萌发并形成称为菌丝的丝状结构。作为这种形态型，Af可以成为侵袭性的，特别是在中性粒细胞计数低的人中。据估计，全世界每年有20万人被诊断患有侵袭性曲霉病，高达90%的人死于感染。AF还可以定植于患有哮喘或囊性纤维化的人的气道，引起过敏性支气管肺曲霉病（ABPA），其每年影响全世界超过400万人。 本文提出的研究旨在进一步了解Af分生孢子的完整先天免疫反应，特别是白细胞介素-23（IL-23）和白细胞介素-17（IL-17）的参与。更好地了解这种反应可能会发现与易感染Af的宿主缺陷相关的细微差别，并可能为合理的疫苗设计和针对Af的免疫治疗提供信息。Af分生孢子在感染的前24小时内从宿主气道中诱导IL-23和IL-17的产生。已知这些细胞因子对于适应性TH 17应答是重要的。然而，它们在针对Af的先天免疫中的作用在很大程度上是未知的。已显示IL-23增加IL-17的产生，并且IL-17又促进中性粒细胞募集。IL-23和IL-17之间的关系被称为IL-23/IL-17轴，该提案旨在系统地表征该轴在针对Af分生孢子的先天应答中的每个部分，并测试该应答是否是针对该真菌病的保护所需的。 为了表征IL-23的时间产生模式，我们将通过ELISA在感染的前72小时内定期测量该细胞因子的水平。通过初步筛选，我们发现了可能参与IL-23产生的候选细胞类型。我们的目的是通过体内和体外细胞内细胞因子染色来确认这些来源。为了测试IL-23产生是否对Af感染中的死亡率具有保护作用，将野生型（WT）小鼠的存活率与功能性IL-23敲除菌株（IL-23 pl 9-/-）进行比较。最后，我们建议创建混合骨髓嵌合体，以测试是否需要任何特定的细胞来源的IL-23来保护免受Af（特异性目的1）。 IL-17产生的时间模式及其来源也将使用上述方法在Af感染的前72小时内表征。为了测试IL-23是否响应于Af而先天地增加IL-17产生，将在IL-23 pl 9-/-小鼠和WT小鼠中评估IL-17水平。此外，我们有证据表明，IL-23和IL-17 A是由一种先天性细胞类型在响应Af时共同产生的，我们建议测试和剖析这种细胞类型中任何潜在的自分泌机制。最后，还将通过监测IL-17 RA-/-小鼠和WT小鼠的存活来测试IL-17在保护免受感染中的作用（具体目标2）。 由于许多IA的风险是移植患者谁是医源性免疫抑制，知识的因素，导致对曲霉菌病的保护也可以告知发展有针对性的免疫抑制剂，保持防御机会性感染的完整。

 DESCRIPTION (provided by applicant): The respiratory mucosa employs the innate and adaptive immune system to protect normal respiratory function from invading organisms. However, when there is a defect in one of these defenses the host becomes vulnerable to Aspergillus fumigatus (Af). This ubiquitous fungus enters the airways as a spore, or resting conidium but is generally cleared by intact respiratory defenses. However, when individuals are immunocompromised, Af conidia are more likely to germinate and form filamentous structures called hyphae. As this morphotype, Af can become invasive particularly in persons with low neutrophil counts. An estimated 200,000 people are diagnosed with invasive aspergillosis annually worldwide, and up to 90% die from the infection. Af can also colonize the airways of those who suffer from asthma or cystic fibrosis causing allergic bronchopulmonary aspergillosis (ABPA), which affects over 4 million people annually worldwide. Studies proposed here seek to further understand the intact innate immune response to Af conidia, particularly the involvement of interleukin-23 (IL-23) and interleukin-17 (IL-17). A better understanding of this response may uncover nuances associated with the host defects that predispose to infection with Af, as well as potentially inform rational vaccine design and immunotherapies against Af. Af conidia elicit IL-23 and IL-17 production from the host airways within the first 24 hours of infection. These cytokines are known to be important for the adaptive TH17 response. However their role in innate immunity against Af is largely unknown. IL-23 has been shown to augment IL-17 production, and in turn IL-17 elicits neutrophil recruitment. The relationship between IL-23 and IL-17 is referred to as the IL-23/IL-17 axis and this proposal aims to systematically characterize each portion of this axis in the innate response against Af conidia, and test whether this response is required for protection against this mycosis. In order to characterize the temporal production pattern of IL-23, we will measure levels of this cytokine at regular intervals in the fist 72 hours of infection by ELISA. From a preliminary screen, we have uncovered candidate cell types that may be involved in the production of IL-23. We aim to confirm these sources by in vivo and ex vivo intracellular cytokine staining. To test whether IL-23 production is protective against mortality in Af infection, the survival rates of wild-type (WT) mice will be compared to a functional IL-23 knock-out strain (IL-23p19-/-). Finally, we propose to create mixed bone marrow chimeras to test whether any specific cellular source of IL-23 is required for protection against Af (Specific Aim 1). The temporal pattern of production for IL-17 and its source will also be characterized in the first 72 hours of infection with Af using methods described above. To test whether IL-23 augments IL-17 production innately in response to Af, IL-17 levels will be assessed in IL-23p19-/- mice and WT mice. In addition, we have evidence that IL-23 and IL-17A are co-produced by one innate cell type in response Af, we propose to test and dissect any potential autocrine mechanisms in this cell type. Finally, the role of IL-17 in protection against f infection will also be tested by monitoring the survival of IL-17RA-/- mice and WT mice (Specific Aim 2). Because many at risk for IA are transplant patients who are iatrogenically immunosuppressed, knowledge of the factors leading to protection against aspergillosis could also inform development of targeted immunosuppressive agents that keep defenses against opportunistic infections intact.