Epithelial cell complement production in the pathogenesis of chronic rhinosinusitis

鼻窦上皮细胞(SNEC)排列在窦腔内，在协调先天发育方面起着重要作用。 和适应性免疫反应。我们和其他人已经证明了慢性阻塞性肺疾病患者的SNEC 鼻-鼻窦炎(CRSwNP)，内源性促炎，对环境刺激高度反应，以及 促进免疫细胞的渗透和活化。最近，补体(CP)系统的多个组成部分 已被证明在CRSwNP患者的粘膜中被激活和上调，尽管包括C3 它们如何促进CRS相关的炎症尚不清楚，因此是这些拟议研究的重点。 传统上，观察到的CP因子和受体对不同类型细胞的影响被认为是中介的 仅由血清、淋巴或间质液体中产生的CP激活片段所致。然而，最近的范式 移位研究表明，细胞本身产生的CP可以在自分泌中发挥作用，而且出乎意料的是， 一种细胞内的方式，细胞内的信号对调节细胞功能是必不可少的。我们有 结果表明，SNEC细胞是C3的主要鼻腔产生者，C3是CP通路的中心蛋白， CRSwNP来源的SNEC具有可快速动员的细胞内C3/C3a的异常存储 在外源性刺激物的刺激下。在这些拟议的研究中，我们将检验以下假设 SNEC细胞内CP信号的异常在CRSwNP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特定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产生在人鼻窦上皮细胞调节中的作用 发炎。子目标1.1我们将确定细胞内C3信号与细胞外C3信号在 SNEC功能。子目标1.2将测试抑制CP介导的信号转导将减少 CRSwNP SNEC的促炎和高反应性使其功能类似于对照 主题派生的SNEC。在目标2中，确定局部C3产生在调节炎症和 特应性CRS小鼠模型的疾病严重性。次级目标2.1将确定当地生产的C3在 用我们的C3-TdT番茄报告小鼠(C3-TdT)和用小鼠过敏模型 真菌CRS(Af-CRS)检测在基线和疾病条件下产生CP的关键细胞类型。模仿，模仿 用于AIM 1、全身和局部C3的人CP抑制疗法将被Crry-Ig抑制，以及 通过使用具有新的细胞穿透性的细胞内递送CRY-Ig来抑制细胞内的C3 载体蛋白(Feldan Shuttle)。使用这些新的方法，我们将剖析上皮细胞和 系统CP活性在疾病发展中的作用(次级目标2.2)，以及抑制C3到 使用Af-CRS模型逆转已建立的疾病(次级目标2.3)。这些研究将共同提供 对升高的上皮、细胞内和局部产生的CP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.