Impact of Airway Epithelium on Innate and Adaptive Immunity in the Lung

项目概要 研究项目 2 呼吸道病毒，例如呼吸道合胞病毒（RSV），是哮喘恶化的主要诱因。 儿童和成人。该项目的总体假设是气道上皮细胞（AEC）协调 对呼吸道病毒感染和空气过敏原的反应。此外，AEC 之间的内在差异 哮喘儿童和健康儿童在感染和接触病毒期间会改变先天反应和适应性反应 过敏原。我们假设患有 RSV 或 HRV 的哮喘儿童感染 AEC， 与过敏原暴露相结合，诱导固有先天细胞，包括树突状细胞 (DC) 和 先天淋巴细胞 (ILC)，触发 2 型反应。我们将使用隔离的 AEC 来测试这个想法 在空气/液体界面 (ALI) 中生长并感染呼吸道病毒的哮喘儿童和健康儿童 过敏原挑战的背景。我们将检查与这些 AEC 共培养的 DC 和 ILC 的反应，以及 它们通过以下目标在促进 2 型炎症中发挥作用： 目标 1. 检验 AEC 控制局部 DC 成熟和功能的假设。由于 AEC 在 哮喘患者可能因屏障缺陷而“渗漏”，我们首先确定是否有更多的抗原通过 使用改良的 RSV 菌株将哮喘患者的 AEC 转化为 DC 的效果优于健康的 AEC。然后，我们将测试 AEC 是否来自哮喘 儿童在促进 DC 分化和功能方面存在差异，以及病毒感染和过敏原暴露如何影响 这种互动。最后，我们将评估暴露于哮喘儿童 AEC 的 DC 驱动 CD4 的能力 共培养测定中的 T 细胞分化和增殖。 目标 2. 检验以下假设：在接触过敏原和病毒感染后，哮喘患者和健康患者 AEC 通过脂质介质和血管活性肠肽对肺 ILC2 产生不同的影响。我们会 评估 AEC 在调节 ILC2 中的作用，ILC2 是肺部最丰富的 ILC。我们期望找到 来自哮喘儿童的 AEC 在病毒感染和过敏原挑战期间增强了 ILC2 功能。在我们的 根据初步数据，我们发现人肺 ILC2 的两个不同子集，它们差异表达受体 氧甾醇、视黄酸和血管活性肠肽。因此，我们建议定义这些脂质的影响 和神经肽介质对不同 ILC2 群体激活的影响。 目标 3. 检验 ICOS ILC 调节肺部免疫反应的假设。我们创作了小说 观察发现肺部含有一个不寻常的 ICOS ILC 子集。这些细胞不适合 ILC1、ILC2、 ILC3 范式，表达调节性淋巴细胞标记物，并富含免疫抑制物质 环境相关。因此，我们假设这些细胞可能具有调节功能。为了测试这一点，我们 建议分离肺ICOS ILC并定义其离体转录组谱及其功能 体外能力，包括细胞因子和趋化因子的分泌。此外，我们建议测试他们的 对来自暴露于 HDM 和/或病毒的 ALI 培养物的刺激的反应性。

PROJECT SUMMARY RESEARCH PROJECT 2 Respiratory viruses, such as respiratory syncytial virus (RSV), are major triggers of asthma exacerbation in children and adults. The overarching hypothesis of this project is that airway epithelial cells (AECs) coordinate responses to respiratory virus infection and aeroallergens. Moreover, intrinsic differences between AECs of asthmatic and healthy children change both innate and adaptive responses during infection and exposure to allergens. We hypothesize that infection of AECs from asthmatic children with RSV or HRV, in combination with allergen exposure, induces resident innate cells, including dendritic cells (DCs) and innate lymphoid cells (ILCs), to trigger type-2 responses. We will test this idea using AECs isolated from asthmatic and healthy children, grown at air/liquid interface (ALI) and infected with respiratory viruses in the context of allergen challenge. We will examine the response of DCs and ILCs co-cultured with these AECs, and their role in promoting type-2 inflammation through the following aims: Aim 1. Test hypothesis that AECs control local DC maturation and function. Since AECs in asthmatics may be “leaky” due to barrier defects, we will first determine whether more antigen is transferred by asthmatic than healthy AECs to DCs using modified RSV strains. Then, we will test whether AECs from asthmatic children differ in promoting DC differentiation and function, and how virus infection and allergen exposure impact this interaction. Finally, we will assess the ability of DCs exposed to AECs from asthmatic children to drive CD4 T cell differentiation and proliferation in co-culture assays. Aim 2. Test hypothesis that after allergen exposure and viral infections, asthmatic and healthy AECs differentially impact lung ILC2 through lipid mediators and vasoactive intestinal peptide. We will assess the role of AECs in regulating ILC2s, which are the most abundant ILCs in the lung. We expect to find that AECs from asthmatic children enhance ILC2 function during viral infection and allergen challenge. In our preliminary data, we found two distinct subsets of human lung ILC2s that differentially express receptors for oxysterols, retinoic acid, and vasoactive intestinal peptide. Thus, we propose to define the impact of these lipidic and neuropeptide mediators on the activation of different ILC2 populations. Aim 3. Test hypothesis that ICOS+ ILCs regulate lung immune responses. We made the novel observation that the lung contains an unusual subset of ICOS+ ILCs. These cells do not fit into the ILC1, ILC2, ILC3 paradigm, express a marker of regulatory lymphocytes and are enriched in an immunosuppressive environment associated. Thus, we hypothesize that these cells may have regulatory functions. To test this, we propose to isolate lung ICOS+ ILCs and define their transcriptome profile ex vivo as well as their functional capabilities in vitro, including cytokine and chemokine secretion. Moreover, we propose to test their responsiveness to stimuli derived from ALI cultures exposed to HDM and/or viruses.