New role for (airway) epithelial PKD in anti-viral immunity

（气道）上皮 PKD 在抗病毒免疫中的新作用

• 批准号: 9465907
• 负责人: Janelle Marie Veazey
• 金额: $ 4.45万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9465907
• 关键词: Air; Antiviral Agents; Attenuated; Automobile Driving; Back; Biological Assay; Bone Marrow; CXCL1 gene; Cell Line; Cells; Chemotactic Factors; Chimera organism; Co-Immunoprecipitations; Data; Defect; Doctor' s Degree; Double-Stranded RNA; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Epithelial; Epithelial Cells; Flow Cytometry; Gene Expression; Gene Targeting; Genetic Transcription; Goals; Hematopoietic; Histology; Hour; Human; Immune response; Immune signaling; Immunity; Immunologist; In Vitro; Infiltration; Inflammatory; Influenza; Influenza A virus; Inhalation; Interleukin-8; Investigation; Kinetics; Lead; Learning; Leukocytes; Liquid substance; Luciferases; Lung; Lung Inflammation; Measures; Mediating; Mediator of activation protein; Messenger RNA; Methodology; Molecular; Molecular Probes; Mouse Protein; Mucosal Immune Responses; Mus; Neutrophilia; One-Step dentin bonding system; Pathology; Pattern recognition receptor; Phosphorylation; Phosphotransferases; Play; Production; Protein Isoforms; Protein Kinase; Protein Secretion; Protein-Serine-Threonine Kinases; Publishing; Receptor Signaling; Reporter; Research; Research Personnel; Research Project Grants; Respiratory Tract Infections; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Specificity; Stromal Cells; TLR3 gene; Testing; Therapeutic; Tight Junctions; Training; Universities; Viral; Viral Respiratory Tract Infection; Virus Diseases; Work; airway epithelium; antiviral immunity; career; chemokine; chromatin immunoprecipitation; combat; cytokine; design; established cell line; experimental study; in vivo; insight; knock-down; lung injury; mRNA Expression; migration; monolayer; neutrophil; new therapeutic target; novel; novel therapeutics; pathogen; protein activation; protein kinase D; receptor; recruit; respiratory; respiratory virus; response; small molecule inhibitor; transcription factor

Project Summary/Abstract Epithelial cells are a critical first line of defense against pathogens such as influenza and strategies to modulate epithelial cell signaling are therefore an appealing target for novel therapeutics. We have previously identified protein kinase D (PKD) as a key regulator of bronchial epithelial barrier integrity during viral infection and have recently identified PKD as a mediator of airway epithelial chemokine secretion. We hypothesize that stimulation with the double-stranded RNA polyI:C or Influenza A virus (IAV) infection leads to activation of PKD (specifically the PKD3 isoform) which increases epithelial chemokine release and promotes leukocyte recruitment. This project aims to determine the mechanism between PKD3 activity and epithelial human interleukin-8 (IL-8)/ murine chemokine CXCL1 secretion. To test this aim, we will assay PKD3 activity (substrate phosphorylation) following siRNA-mediated knock-down of potential receptors upstream PKD activation, and then use that information to inform identification of the adaptor molecule(s) associated with PKD (using co-immunoprecipitation). We will also assay IL-8/CXCL1 mRNA levels in human and mouse airway epithelial cell lines following knock-down of PKD isoforms to determine the isoform driving chemokine production. We will then use chromatin immunoprecipitation to identify PKD-activated transcription factors involved in IL-8/CXCL1 transcription and confirm activity with luciferase reporter assay. We further aim to investigate the therapeutic potential of PKD3 reduction/inhibition to attenuate lung injury following IAV infection. To test this aim, we will infect PKD3 deficient mice (PKD3-/-) with sub-lethal influenza A virus (IAV) and assay for leukocyte infiltration (via flow cytometry), chemokine secretion (via ELISA and multiplex array), and general pathology (histology). We will also generate bone marrow chimeric mice to assess the relative contributions of PKD3 in stromal cells vs. hematopoietic cells to lung injury and neutrophilia. We hypothesize that stimulation with the double-stranded RNA polyI:C or Influenza A virus (IAV) infection leads to activation of the PKD3 isoform which increases epithelial pro-inflammatory cytokine release and promotes leukocyte recruitment. We further hypothesize that PKD3 may play a previously unsuspected role in regulating airway epithelial anti-viral immune responses and that further study of the mechanism of PKD3-mediated cytokine release in the airway epithelium may lead to new therapeutics in combating viral respiratory infections.

项目总结/摘要 上皮细胞是抵抗病原体如流感的关键第一道防线， 因此，调节上皮细胞信号传导是新疗法的吸引人的靶点。我们先前已经 蛋白激酶D（PKD）是病毒感染期间支气管上皮屏障完整性的关键调节因子 并且最近已经将PKD鉴定为气道上皮趋化因子分泌的介质。我们假设 用双链RNA polyI：C或甲型流感病毒（IAV）感染刺激导致PKD活化 （特别是PKD 3亚型），其增加上皮趋化因子释放并促进白细胞 招聘本项目旨在研究PKD 3活性与人上皮细胞凋亡的关系， 白细胞介素-8（IL-8）/鼠趋化因子CXCL 1分泌。为了测试这一目标，我们将测定PKD 3活性， siRNA介导的PKD上游潜在受体敲低后（底物磷酸化） 激活，然后使用该信息来告知与PKD相关的衔接子分子的鉴定 （使用免疫共沉淀）。我们还将检测人和小鼠气道中IL-8/CXCL 1 mRNA的水平 在PKD同种型敲低后的上皮细胞系以确定同种型驱动趋化因子 生产然后我们将使用染色质免疫沉淀来鉴定PKD激活的转录因子 参与IL-8/CXCL 1转录，并通过荧光素酶报告基因测定证实活性。我们进一步的目标是 研究PKD 3减少/抑制减轻IAV感染后肺损伤的治疗潜力。 为了测试这一目的，我们将用亚致死性甲型流感病毒（IAV）感染PKD 3缺陷型小鼠（PKD 3-/-），并测定 用于白细胞浸润（通过流式细胞术）、趋化因子分泌（通过ELISA和多重阵列）和一般 病理学（组织学）。我们还将产生骨髓嵌合小鼠，以评估以下因素的相对贡献： 基质细胞与造血细胞中的PKD 3与肺损伤和嗜中性粒细胞相关。我们假设刺激 与双链RNA polyI：C或甲型流感病毒（IAV）感染导致PKD 3激活 增加上皮促炎细胞因子释放并促进白细胞募集的亚型。我们 进一步假设PKD 3可能在调节气道上皮抗病毒抗体中发挥先前未被怀疑的作用， 呼吸道免疫应答及PKD 3介导细胞因子释放机制的进一步研究 上皮细胞可能导致新的治疗方法，在打击病毒性呼吸道感染。