Solitary chemosensory cell development and function

项目摘要 气道上皮研究的新方法拓宽了我们对气道上皮细胞多样性的理解， 化妆，发育调节，对损伤的反应，以及参与局部组织功能的细胞相互作用。 我们小组和其他人最近的工作已经证明了气道孤立化学感受细胞（SCC）的作用。 在检测气道表面液体内的刺激物时，通过典型苦味的信号传导 信号效应器和启动多方面的免疫反应。有趣的是， 在小鼠和人类肺部研究中基于RNAseq的方法表明SCC之间可能存在关系 以及其他罕见的气道细胞类型，包括离子细胞和神经内分泌细胞。 我们的中心假设是，人类SCC包含一个异质性群体， 过敏性炎症环境，并独特表达细胞特异性化学感受受体，以检测人类 疾病相关化合物。这一假设是根据出版物提出的， K23指导研究期间申请人实验室生成的初步数据，以及Co-I Vladar对气道上皮修复的长期兴趣。为了测试这一点，并生成工具， 机制研究-我们将利用逆转录病毒基因转移的平台来遗传修饰人类SCC 细胞培养我们将追求两个目标：（1）使用hTRPM5p-GFP表征SCC分化 正常和变应性人原代鼻窦上皮细胞培养物中慢病毒报告基因;和（2）测试人 使用实时TRPM5-mCherry/GCaMP6对疾病相关空气刺激物的SCC功能反应 原代HSNEC培养物中的Ca 2+报告基因。 我们的长期目标是确定SCC和化学感受细胞类型在气道中的功能作用， 上皮内稳态，并将这些发现转化为对气道粘膜 免疫力对人类健康和疾病的影响。值得注意的是，用于标记和分离这种蛋白的可用方法的局限性， 来自人类组织的罕见细胞类型导致了将发现从小鼠转化为人类的挑战。 这一创新提案在概念上与现状有很大不同， 新的方法来测试特定的假设，并在一个鲜为人知的领域进行普遍发现。的 拟议的研究是重要的，因为它有望促进对SCC如何发挥作用的理解， 调节上皮细胞内环境稳定的环境感觉器。最终，这些知识有可能成为 开发成为治疗炎症性气道疾病的有效疗法，鉴于这一重大需求， 这些疾病的发病率和负担。

PROJECT SUMMARY Novel approaches to airway epithelial study have broadened our understanding of its diverse cellular makeup, developmental regulators, response to injury, and cell interactions involved in local tissue function. Recent work by our group and others has demonstrated a role for airway solitary chemosensory cells (SCCs) in detection of irritants within the airway surface liquid, signal transduction through canonical bitter taste signaling effectors, and initiation of a multifaceted immune response. Interestingly, lineage implications from RNAseq based approached in mouse and human lung studies suggest a possible relationship between SCCs and other rare airway cell types including the ionocyte and the neuroendocrine cell. Our central hypothesis is that human SCCs encompass a heterogeneous population that is modulated in the allergic inflammatory setting, and uniquely express cell-specific chemosensory receptors to detect human disease-associated compounds. This hypothesis has been formulated on the basis of publications and preliminary data produced in the applicant's laboratory during the K23 mentored research period, and Co-I Vladar's long-standing interest in airway epithelial repair. To test this—and generate tools for further mechanistic study—we will utilize a platform of retroviral gene transfer to genetically modify SCCs in human cell culture. We will pursue two aims: (1) characterization of SCC differentiation using the hTRPM5p-GFP lentiviral reporter in normal and allergic human primary sinonasal epithelial cell culture; and (2) test human SCC functional responses to disease-associated airborne irritants using a real-time TRPM5-mCherry/GCaMP6 Ca2+ reporter in primary HSNEC culture. Our long-term goal is to determine the functional role(s) of SCCs and chemosensory cell types in airway epithelial homeostasis, and to translate these findings into a cohesive understanding of airway mucosal immunity in human health and disease. Notably, limitations of available methods for labeling and isolating this rare cell type from human tissues have resulted in challenges in translating findings from mouse to human. This innovative proposal represents a significant conceptual departure from the status quo, and will utilize novel methodologies to test specific hypotheses and for general discovery in an area where little is known. The proposed research is significant because it is expected to advance understanding of how SCCs function as environmental sensory that regulate epithelial homeostasis. Ultimately, such knowledge has the potential to be developed into effective therapies for inflammatory airway disorders, a pressing need given the significant incidence and burden of these diseases.