Targeting T2 inflammation-evoked mechanical endotypes of ASM shortening in asthma

项目概要： 哮喘的特点是慢性炎症和支气管阻塞，由于人体气道畅通 肌肉（HASM）缩短。然而，增强缩短或过度收缩的潜在基础 HASM在哮喘中状态尚不清楚。此外，我们对2型（T2）炎症的不完全理解- HASM缩短中受调节的兴奋-收缩（E-C）偶联阻碍了新的 具有60多年新作用机制的HASM支气管扩张剂。本申请旨在获得 关于哮喘中HASM缩短的机械内型的基础知识（炎症依赖性 和非依赖性），并确定改善的支气管扩张剂， 受哮喘免疫炎症反应的影响，关注以前未被认识的机制 由HASM上表达的苦味受体（TAS 2 Rs）诱发。我们的初步数据，在临床前模型中， 支持这样一个前提，即与持续性免疫相关的免疫和/或致病机制 HASM缩短的机械强化，以及β2-肾上腺素能受体（β2AR）介导的 支气管扩张，涉及多梳组（PcG）蛋白EZH 2的转录抑制功能 （zeste同源物2的增强子）。此外，我们的初步研究发现microRNA-214的机制作用， （miR-214）在TAS 2 R诱发的EZH 2翻译抑制中的作用。基于这些结果，我们假设， HASM上的TAS 2 Rs抑制HASM缩短中T2甘氨酸调节的E-C偶联和HASM的生理损失。 β2AR以EZH 2和miR-214依赖性方式发挥功能。我们的目标是，首先，描述T2和非T2- 介导的分子动力学和HASM缩短中的E-C偶联机制，第二，确定miR- 表观遗传联系（即，非遗传机制），TAS 2 R活化通过其促进 β 2 AR和抑制哮喘中HASM缩短的机械内源性。为此，我们将 利用我们独特的单分子和单细胞微机械方法的技术创新 以及临床相关的人类精确切割肺切片的综合遗传学和基因组学方法 （hPCLS）和来源于患有和不患有严重哮喘的患者的供体肺的原代HASM细胞。当 成功，从这些实验和计算研究中获得的知识将：1）揭示新的光 HASM缩短中E-C偶联的炎症依赖性和非炎症依赖性调节; 2）揭示 先前未鉴定的TAS 2 R范例，以减轻β2AR功能的生理损失;和3）建立 新的药物靶点和药物治疗β2-激动剂不敏感的大型队列患者难以 控制和严重哮喘。这一系列的研究在哮喘中被低估了，代表了哮喘的明显转变。 哮喘治疗范例。

PROJECT SUMMARY: Asthma is characterized by chronic inflammation and bronchial obstruction due to human airway smooth muscle (HASM) shortening. However, the underlying basis for an enhanced shortening or the hyper-contractile state of HASM in asthma is not known. Further, our incomplete understanding of type 2 (T2) inflammation- regulated excitation-contraction (E-C) coupling in HASM shortening has hindered the development of new HASM bronchodilators with a novel mechanism of action for over 60 years. This application seeks to gain a foundational knowledge on the mechanical endotypes of HASM shortening in asthma (inflammation-dependent and -independent) and identify improved bronchodilators that are less susceptible to tolerance and less affected by immune inflammatory responses in asthma, focusing on previously unrecognized mechanisms evoked by bitter taste receptors (TAS2Rs) expressed on HASM. Our preliminary data, in pre-clinical models, support a premise that the immunologic and/or pathogenic mechanisms associated with a sustained mechanical reinforcement of HASM shortening, and the loss of β2-adrenoceptor (β2AR)-mediated bronchodilation, involve a transcriptional repressor function of the polycomb group (PcG) protein EZH2 (enhancer of zeste homolog 2). Further, our preliminary studies find a mechanistic role for microRNA-214 (miR-214) in TAS2R-evoked translational inhibition of EZH2. Based on these results, we hypothesize that TAS2Rs on HASM inhibit T2 cytokine-regulated E-C coupling in HASM shortening and the physiological loss of β2AR function in EZH2- and miR-214-dependent manners. Our goals are, first, to characterize T2- and non-T2- mediated molecular kinetics and mechanics of E-C coupling in HASM shortening and, second, determine miR- epigenetic nexus (i.e., non-genetic mechanisms) by which TAS2R activation promotes the functional efficacy of β2ARs and inhibits the mechanical endotypes of HASM shortening in asthma. Toward this end, we will leverage our unique technological innovations of single-molecule and single-cell micromechanical methods and integrative genetics and genomics approaches in clinically relevant human precision cut lung slices (hPCLS) and primary HASM cells derived from donor lungs of patients with and without severe asthma. When successful, the knowledge gained from these experimental and computational studies will: 1) shed new light on inflammation-dependent and -independent regulation of E-C coupling in HASM shortening; 2) uncover previously unidentified TAS2R paradigms to mitigate the physiological loss of β2AR function; and 3) establish new druggable targets and agents to treat β2-agonist-insenstivity in a large cohort of patients with difficult-to- control and severe asthma. This line of research is underappreciated in asthma and represents a clear shift in the asthma treatment paradigm.