A Novel Approach to Target Neutrophilic Airway Inflammation and Airway Hyperresponsiveness in Therapy-Resistant (Refractory) Asthma.

呼吸道高反应性(AHR)和呼吸道炎症是哮喘的特征。难治性哮喘 尽管使用了大剂量口服皮质类固醇和长效β2-激动剂，仍有持续症状的表现 支气管扩张剂，并构成一个重大的医疗挑战。对机制的理解和发展 克服治疗耐药性的策略构成了一个重大的未得到满足的需求。呼吸道平滑肌(ASM) 过度收缩是AHR的一个关键因素，而T辅助17(Th17)细胞促进类固醇不敏感的中性粒细胞 呼吸道炎症(NAI)。我们发现RhoA激活剂p63Rhogef在难治性哮喘中起着至关重要的作用。 我们还开发了p63Rhogef的小分子抑制剂CXN-8。目的：确定 以及p63Rhogef调节哮喘素质的机制以及CXN-8是否抑制p63Rhogef 改善AHR和NAI。长期目标：开发治疗难治性哮喘的新疗法。调查结果：1)P63- Rhogef在哮喘患者中选择性上调，并在控制ASM的RhoA激活中发挥关键作用 过度收缩与Th17细胞分化。2)CXN-8抑制ASM收缩，诱导ASM松弛 气道口。3)CXN-8可改善哮喘模型小鼠的AHR和NAI。4)CXN-8与p63Rhogef相互作用 阻止RhoA激活。假设：靶向p63Rhogef刺激的ASM过度收缩和Th17细胞 与CXN-8的差异可改善AHR和NAI，CXN-8疗法代表着一种新的治疗策略 难治性哮喘。我们将在体外和体内检验这一假说。目标1.定义潜在的机制 P63Rhogef和CXN-8对AHR的调节。我们将使用RNAi和抑制剂来确定关键作用和 P63Rhogef对人哮喘ASM细胞收缩功能增强的作用机制我们将分析CXN- 8调节β-2激动剂敏感/不敏感的HASM的RhoA活性、Ca~(2+)信号、收缩/松弛 细胞。我们将结合RNAi、突变和表面等离子共振来阐明其特异性和 CXN-8调控p63Rhogef的机制。P63基因缺失是否能改善AHR并降低 CXN-8的作用将在屋尘螨(HDM)驱动的哮喘小鼠模型中进行检测。目标2.目标 阐明p63Rhogef和CXN-8调控NAI的机制。我们将研究以下方面的影响 CXN-8对Th17细胞分化的影响我们将使RhoA沉默或表达活跃的RhoA突变体以建立其 CXN-8抑制Th17细胞分化的重要性。我们还将确定p63Rhogef的丢失是否 减轻RhoA活化和Th17分化及HDM诱导的小鼠NAI，并降低CXN-8 体外和体内的抑制作用。目的3.研究CXN-8对小鼠肾功能不全的治疗作用。 难治性哮喘。我们将确定吸入CXN-8是否是一种急性有效的小鼠支气管扩张剂 β-2激动剂不敏感的AHR模型。我们将测试肺部靶向、长效CXN-8微粒以缓解 AHR/NAI在皮质激素不敏感哮喘小鼠模型中的有限全身副作用。其影响 在这些研究中还将检测p63Rhogef缺失对AHR/NAI的影响以及CXN-8的治疗效果。

Airway hyperresponsiveness (AHR) and airway inflammation are hallmarks of asthma. Refractory asthma manifests with persistent symptoms despite use of high-dose oral corticosteroids and long-acting β2-agonist bronchodilators and poses a major healthcare challenge. Understanding the mechanisms and developing strategies to overcome therapeutic resistance poses a significant unmet need. Airway smooth muscle (ASM) hypercontraction is a key factor of AHR, and T-helper 17 (Th17) cells promote steroid-insensitive neutrophilic airway inflammation (NAI). We found p63RhoGEF, a RhoA activator, plays crucial roles in refractory asthma. We also developed CXN-8, a small molecule inhibitor of p63RhoGEF. Objective: To determine the importance and mechanisms by which p63RhoGEF modulates the asthma diathesis and if CXN-8 inhibits p63RhoGEF to ameliorate AHR and NAI. Long-term goal: To develop new therapies for refractory asthma. Findings: 1) p63- RhoGEF is selectively upregulated in asthmatics and plays a critical role in RhoA activation that controls ASM hypercontraction and Th17 cell differentiation. 2) CXN-8 inhibits ASM contraction and induces relaxation of airway. 3) CXN-8 ameliorates AHR and NAI in murine asthma models. 4) CXN-8 interacts with p63RhoGEF to block RhoA activation. Hypothesis: Targeting p63RhoGEF-stimulated ASM hypercontractility and Th17 cell differentiation with CXN-8 ameliorates AHR and NAI and that CXN-8 therapy represents a novel strategy for refractory asthma. We will test this hypothesis in vitro and in vivo. Aim 1. To define the mechanism underlying p63RhoGEF and CXN-8 regulation of AHR. We will use RNAi and inhibitors to determine the pivotal role and mechanism of p63RhoGEF in hypercontractility of asthmatic human ASM (HASM) cells. We will analyze CXN- 8 regulation of RhoA activity, Ca2+ signaling, contraction/relaxation of β2-agonist-sensitive/insensitive HASM cells. We will combine RNAi, mutagenesis, and surface plasmon resonance to elucidate the specificity and mechanism for CXN-8 regulation of p63RhoGEF. Whether p63RhoGEF loss ameliorates AHR and reduces the effects of CXN-8 will be examined in a house dust mite (HDM)-driven murine model of asthma. Aim 2. To elucidate the mechanism underlying p63RhoGEF and CXN-8 regulation of NAI. We will examine the effects of CXN-8 on Th17 cell differentiation. We will silence RhoA or express an active RhoA mutant to establish its importance in CXN-8 inhibition of Th17 cell differentiation. We will also determine if loss of p63RhoGEF attenuates RhoA activation and Th17 differentiation in cells and HDM-induced NAI in mice and reduces CXN-8 inhibitory effects in vitro and in vivo. Aim 3. To examine the therapeutic potential of CXN-8 in murine models of refractory asthma. We will determine if inhaled CXN-8 is an acute and effective bronchodilator in a murine model of β2-agonist insensitive AHR. We will test lung targeted, long-acting CXN-8 microparticles to alleviate AHR/NAI with limited systemic side-effects in a murine model of corticosteroid-insensitive asthma. The impact of p63RhoGEF loss on AHR/NAI and therapeutic effects of CXN-8 will also be examined in these studies.