Interleukin-27 in host response to Legionella infection

项目概要：军团病是一种非典型肺炎， 在美国的发病率。尽管进行了治疗，患者的死亡率仍保持在3-30%左右。 军团菌病是由军团菌科的兼性细胞内细菌引起的， 和人工水生栖息地。吸入受污染的气溶胶可导致感染，逃避病原体 在肺泡巨噬细胞（AM）内含有空泡的军团菌中根除和持续复制。 随后的炎症促进骨髓细胞和淋巴细胞浸润到肺部。这些免疫 细胞通过细胞因子相互交流。白细胞介素-27（IL-27）是一种异源二聚体细胞因子， 亚基p28和EBI 3的非共价相互作用。IL-27由模式识别受体（PRR）诱导 单核吞噬细胞（AM、单核细胞、树突细胞）中的活化。IL-27与其独特受体的连接 淋巴细胞上的IL-27 RA启动STAT 1/STAT 3磷酸化。IL-27 RA信号传导启动多效性 这些程序可以首先加剧急性炎症，然后限制长期的T细胞激活。我们的初步 数据支持IL-27是宿主对军团菌感染应答的关键参与者的新概念。IL-27是 在患有军团病和IL-27 RA缺乏的人类患者的支气管肺泡灌洗液中升高 老鼠对感染的抵抗力更强。在这里，我们建议测试中心假设，即IL-27是由 军团菌感染单核吞噬细胞并在淋巴细胞中启动二分程序，包括 NK细胞的保护性激活和肺中不利的T细胞介导的免疫抑制。目标1）精确定位 军团菌病期间肺单核吞噬细胞亚群内IL-27的细胞来源。我们的目标 评估相关PRR免疫传感器对L.嗜肺菌样本 将研究军团病患者IL-27与PD-1/PD-L1轴和可溶性IL-27的相关性。 免疫病理学的标志物。目的2）表征组成型IL-27 RA的功能后果 在感染的IL-27 RA-/-小鼠中进行消融，并通过单细胞、多维 蛋白质转录组学（CITE-Seq/Total-Seq）。我们还将评估中和性IL-27抗体和 工程诱饵受体，以改善小鼠肺炎的严重程度。目标3）根据初步数据，我们的目标是 研究我们的NK细胞特异性缺失IL-27 RA的新型条件小鼠的改变的炎症反应。 我们将研究NK细胞成熟是否与IL-27 RA控制的转录调控开关有关。 通过参与非STAT磷蛋白信号转导程序。目的4）探讨IL-27 RA在人肝癌细胞中的作用。 常规和非常规的T细胞，其机制集中于共抑制/共刺激受体， 肺部感染L. pneumophila和L. longbeachae小鼠。总之，拟议的研究将测试 一种新的概念，即IL-27在肺宿主反应和军团菌病的结局中起关键作用， 27 RA在NK细胞相对于T细胞中启动选择性程序。

Project Summary: Legionellosis (Legionnaires’ disease) is a form of atypical pneumonia with a steep rising incidence in the United States. The mortality rates of patients stagnate around 3-30% despite treatment. Legionellosis is caused by facultative, intracellular bacteria of the Legionellaceae family, which reside in natural and engineered aquatic habitats. Inhalation of contaminated aerosols can lead to infection, evasion of pathogen eradication and continuous replication in Legionella containing vacuoles inside alveolar macrophages (AMs). The ensuing inflammation promotes an infiltration of myeloid cells and lymphocytes into lungs. These immune cells communicate with each other via cytokines. Interleukin-27 (IL-27) is a heterodimeric cytokine formed by non-covalent interactions of the subunits p28 and EBI3. IL-27 is induced by pattern recognition receptor (PRR) activation in mononuclear phagocytes (AMs, monocytes, dendritic cells). IL-27 ligation with its unique receptor chain, IL-27RA, on lymphocytes initiates STAT1/STAT3 phosphorylation. IL-27RA signaling initiates pleiotropic programs, which can first intensify acute inflammation and later limit prolonged T cell activation. Our preliminary data support the new concept that IL-27 is a critical player of the host response to Legionella infection. IL-27 is elevated in broncho-alveolar lavage fluids of human patients with Legionnaires’ disease and IL-27RA deficient mice are more resistant to infection. Here, we propose to test the central hypothesis that IL-27 is produced by Legionella infected mononuclear phagocytes and initiates dichotomous programs in lymphocytes, that include the protective activation of NK cells and adverse T cell-mediated immunosuppression in lungs. Aim 1) To pinpoint the cellular source(s) of IL-27 during Legionellosis within the subsets of lung mononuclear phagocytes. We aim to assess the contribution of relevant PRR immunosensors for IL-27 induction by L. pneumophila. Samples from Legionnaires’ disease patients will be studied for associations of IL-27 with the PD-1/PD-L1 axis and soluble markers of immunopathology. Aim 2) To characterize the functional consequences of constitutive IL-27RA ablation in infected IL-27RA-/- mice, and to study the host response programs by single-cell, multi-dimensional proteotranscriptomics (CITE-Seq/Total-Seq). We will also evaluate neutralizing IL-27 antibodies and an engineered decoy receptor to improve pneumonia severity in mice. Aim 3) Based on preliminary data, we aim to study the altered inflammatory response of our novel conditional mice with NK cell-specific deletion of IL-27RA. We will investigate whether NK cell maturation is associated with a switch of IL-27RA controlled transcriptional programs through engaging non-STAT phosphoprotein signaling. Aim 4) To investigate the roles of IL-27RA in conventional and unconventional T cells with a mechanistic focus on co-inhibitory/co-stimulatory receptors during lung infection with L. pneumophila and L. longbeachae in mice. In summary, the proposed studies will test the novel concept that IL-27 plays a critical role for the lung host response and outcome of Legionellosis and that IL- 27RA initiates selective programs in NK cells versus T cells.