Epithelial cell complement production in the pathogenesis of chronic rhinosinusitis

慢性鼻窦炎发病机制中上皮细胞补体的产生

• 批准号: 9886648
• 负责人: Carl Atkinson
• 金额: $ 42.6万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9886648
• 关键词: Allergic; Anatomy; Carrier Proteins; Cell Culture System; Cell Line; Cell physiology; Cells; Chronic; Chronic Obstructive Airway Disease; Clinical Research; Clinical Trials; Complement; Complement 3a; Complement Activation; Complement Inactivators; Development; Disease; Epithelial; Epithelial Cells; Epithelium; Functional disorder; Generations; Goals; Heart failure; Human; Immune; Immune System Diseases; In Vitro; Infiltration; Inflammation; Inflammatory; Intercellular Fluid; Irritants; Laboratories; Location; Lymph; Mediating; Modeling; Mucositis; Mucous Membrane; Mus; Nasal Polyps; Nature; Nose; Outcome Study; Pathogenesis; Patients; Play; Production; Proteins; Quality of life; Recording of previous events; Regulation; Reporter; Reporting; Role; Serum; Severity of illness; Shapes; Signal Transduction; Sinus; Sinusitis; Source; Stimulus; Structure of respiratory epithelium; Systemic Therapy; T-Lymphocyte; Tamoxifen; Testing; Therapeutic; Therapeutic Agents; Therapy Clinical Trials; Tissues; Tomatoes; Topical application; Translations; adaptive immune response; airway epithelium; autocrine; cell type; chronic rhinosinusitis; clinically relevant; complement C3 precursor; complement pathway; complement system; conditional knockout; design; early phase clinical trial; extracellular; innovation; insight; knockout animal; mouse Cre recombinase; mouse model; novel; novel strategies; novel therapeutics; pre-clinical; receptor; success; tool; tumor-immune system interactions

Sinonasal epithelial cells (SNEC) line the sinus cavity and play important roles in orchestrating innate and adaptive immune responses. We, and others, have demonstrated that SNEC from patients with chronic rhinosinusitis (CRSwNP), are endogenously pro-inflammatory, hyper-responsive to environmental stimuli, and promote immune cell infiltration and activation. Recently, multiple components of the complement (Cp) system have been shown to be activated and up regulated in the mucosa of CRSwNP patients, including C3, though how they contribute to CRS-related inflammation is unknown and thus the focus of these proposed studies. Traditionally, the observed effect of Cp factors and receptors on various cell types was thought to be mediated solely by Cp activation fragments generated in the serum, the lymph, or interstitial fluids. Yet, recent paradigm shifting studies have shown that Cp generated by the cell itself can function in an autocrine, and unexpectedly, an intracellular fashion, and that intracellular signaling is essential for regulating the cells functions. We have shown that SNEC cells are the primary sinonasal cavity producers of C3, the central protein in the Cp pathway, and that SNECs from CRSwNP have dysregulated intracellular stores of C3/C3a that can be rapidly mobilized upon stimulation with exogenous irritates. In these proposed studies we will test the hypothesis that dysregulated intracellular Cp signaling in SNEC plays a role in the epithelial cell dysfunction seen in CRSwNP. We will test our hypothesis by executing two inter-related but independent specific aims. In Aim 1 we will determine the role of local and intracellular Cp production in the regulation of human sinonasal epithelial cell inflammation. Sub-Aim 1.1 we will to determine the role of intracellular versus extracellular C3 signaling on SNEC functions. Sub-Aim 1.2 will test the hypothesis that inhibition of Cp-mediated signaling will reduce the pro-inflammatory and hyper-responsive nature of CRSwNP SNEC such that they will function similarly to control subject-derived SNEC. In Aim 2, determine the role local C3 production in the regulation of inflammation and disease severity in a murine model of atopic CRS. Sub-Aim 2.1 will determine the role of locally produced C3 in the sinonasal cavity using our C3-TdT tomato reporter mouse (C3-TdT), and by using a murine model of allergic fungal CRS (Af-CRS) test the key cell types that generate Cp at baseline and under disease conditions. To mimic the human Cp inhibitory therapies utilized in Aim 1, systemic and local C3 will be inhibited with Crry-Ig, and intracellular C3 will be inhibited through the use of intracellular delivery of Crry-Ig with a novel cell penetrating carrier protein (Feldan Shuttle). Using these novel approaches, we will dissect the impact of epithelial and systemic Cp activity on disease development (sub-Aim 2.2), and the therapeutic potential of inhibiting C3 to reverse established disease (sub-Aim 2.3) using the Af-CRS model. Together these studies will provide mechanistic insights into how elevated epithelial intracellular and locally produced Cp can shape the local immune microenvironment.

鼻窦上皮细胞（SNEC）排列在鼻窦腔内，在协调先天性方面发挥着重要作用 和适应性免疫反应。我们和其他人已经证明，慢性病患者的 SNEC 鼻窦炎 (CRSwNP) 具有内源性促炎性，对环境刺激过度反应，并且 促进免疫细胞浸润和活化。最近，补体（Cp）系统的多个组成部分 已被证明在 CRSwNP 患者的粘膜（包括 C3）中被激活并上调，但 它们如何导致 CRS 相关炎症尚不清楚，因此是这些拟议研究的重点。 传统上，观察到的 Cp 因子和受体对各种细胞类型的影响被认为是介导的 仅由血清、淋巴液或间质液中产生的 Cp 激活片段引起。然而，最近的范式 转移研究表明，细胞本身产生的 Cp 可以在自分泌中发挥作用，并且出乎意料的是， 细胞内的方式，细胞内信号传导对于调节细胞功能至关重要。我们有 表明 SNEC 细胞是 C3 的主要鼻腔生产者，C3 是 Cp 途径的核心蛋白， 来自 CRSwNP 的 SNEC 细胞内 C3/C3a 储存失调，可以快速动员 受到外源性刺激物的刺激后。在这些拟议的研究中，我们将检验以下假设： SNEC 中细胞内 Cp 信号传导失调在 CRSwNP 中观察到的上皮细胞功能障碍中发挥着一定作用。 我们将通过执行两个相互关联但独立的具体目标来检验我们的假设。在目标 1 中，我们将 确定局部和细胞内 Cp 产生在人鼻腔上皮细胞调节中的作用 炎。子目标 1.1 我们将确定细胞内与细胞外 C3 信号传导对 SNEC 功能。子目标 1.2 将检验以下假设：抑制 Cp 介导的信号传导将减少 CRSwNP SNEC 的促炎性和高反应性，因此它们的功能与对照相似 主题衍生的 SNEC。在目标 2 中，确定局部 C3 生成在炎症调节中的作用和 特应性 CRS 小鼠模型中疾病的严重程度。分目标 2.1 将确定本地生产的 C3 在 使用我们的 C3-TdT 番茄报告小鼠 (C3-TdT) 以及过敏性小鼠模型 真菌 CRS (Af-CRS) 测试在基线和疾病条件下产生 Cp 的关键细胞类型。模仿 目标 1 中使用的人类 Cp 抑制疗法，全身和局部 C3 将被 Crry-Ig 抑制，并且 通过使用具有新型细胞穿透性的 Crry-Ig 细胞内递送，可以抑制细胞内 C3 载体蛋白（Feldan Shuttle）。使用这些新方法，我们将剖析上皮细胞和 全身 Cp 活性对疾病发展的影响（子目标 2.2），以及抑制 C3 的治疗潜力 使用 Af-CRS 模型逆转既定疾病（子目标 2.3）。这些研究将共同​​提供 关于上皮细胞内和局部产生的 Cp 升高如何塑造局部细胞的机制见解 免疫微环境。