Epithelial cell complement production in the pathogenesis of chronic rhinosinusitis

鼻窦上皮细胞（SNEC）排列在鼻窦腔中，在协调先天性 和适应性免疫反应。我们和其他人已经证明，慢性胰腺炎患者的SNEC 鼻窦炎（CRSwNP）是内源性促炎性的，对环境刺激反应过度， 促进免疫细胞浸润和活化。最近，补体（Cp）系统的多个组分 已经显示在CRSwNP患者的粘膜中被激活和上调，包括C3， 它们如何导致CRS相关的炎症是未知的，因此是这些拟议研究的焦点。 传统上，观察到的Cp因子和受体对各种细胞类型的作用被认为是介导的。 仅通过血清、淋巴液或间质液中产生的Cp激活片段。然而，最近的范例 转移研究表明，细胞自身产生的Cp可以自分泌方式发挥作用，并且出乎意料地， 细胞内的方式，细胞内的信号是必不可少的调节细胞功能。我们有 显示SNEC细胞是C3的主要鼻窦腔生产者，C3是Cp途径中的中心蛋白， 并且来自CRSwNP的SNEC具有失调的C3/C3 a细胞内储存，其可以被快速动员， 受到外源性刺激物的刺激。在这些拟议的研究中，我们将测试假设， SNEC中失调的细胞内Cp信号传导在CRSwNP中观察到的上皮细胞功能障碍中起作用。 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， 确定局部和细胞内Cp产生在人鼻窦上皮细胞调节中的作用， 炎症子目标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将检验抑制Cp介导的信号传导将降低 CRSwNP SNEC的促炎性和高反应性性质，使得它们将与对照类似地起作用 受试者衍生的SNEC。在目标2中，确定局部C3产生在炎症调节中的作用， 在特应性CRS的鼠模型中的疾病严重程度。次级目标2.1将确定当地生产的C3在以下方面的作用： 使用我们的C3-TdT番茄报告小鼠（C3-TdT），并通过使用小鼠过敏模型， 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Cp抑制疗法，全身和局部C3将被Crry-Ig抑制，并且 细胞内C3将通过使用Crry-Ig的细胞内递送与新的细胞穿透剂来抑制， 载体蛋白（Feldan Shuttle）。使用这些新的方法，我们将剖析上皮细胞的影响， 系统Cp活性对疾病发展的影响（子目标2.2），以及抑制C3至 使用Af-CRS模型逆转既定疾病（子目标2.3）。这些研究将提供 关于上皮细胞内和局部产生的Cp升高如何塑造局部 免疫微环境

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.