Neuronal P-Rex1 repression: a key factor in early-life environmental cigarette smoke exposure mediated risk of asthma

神经元 P-Rex1 抑制：生命早期环境香烟烟雾暴露介导哮喘风险的关键因素

• 批准号: 9904643
• 负责人: YAPING TU
• 金额: $ 18.19万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904643
• 关键词: Acute; Adult; Afferent Neurons; Air; Animal Model; Asthma; Attenuated; Brain-Derived Neurotrophic Factor; Bronchoalveolar Lavage Fluid; Cell model; Cells; Chemosensitization; Child; Data; Development; Ectopic Expression; Exhibits; Exposure to; Extrinsic asthma; Functional disorder; Ganglia; Goals; Health; House Dust Mite Allergens; In Vitro; Incidence; Inflammation; Inflammatory; Interleukin-6; Knockout Mice; Lead; Life; Logic; Lung; Lung diseases; Measures; Mediating; Mediator of activation protein; Mus; Neurons; Oral Administration; PRKCA gene; Pathologic; Patients; Phenotype; Phosphatidylinositols; Play; Protein Isoforms; Protein Kinase C; Protein Kinase C Inhibitor; Repression; Risk; Role; Severities; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Testing; Therapeutic Effect; Tracheostomy procedure; Vagus nerve structure; airway hyperresponsiveness; cigarette smoke; cigarette smoke-induced; cytokine; exposure to cigarette smoke; in vivo; inhibitor/antagonist; molecular modeling; nerve supply; neurite growth; neurite-inducing factor; novel; postnatal; prevent; respiratory smooth muscle; restoration; smoke-induced lung disease; therapeutic target

Early-life environmental cigarette smoke (ECS) exposure alters airway innervation and increases the incidence of asthma later in life, but the mechanisms remain undefined. We recently identified neuronal P-Rex1 as an important regulator of airway innervation and its expression was markedly down-regulated in mice exposed to early-life ECS. Objective: To define the mechanism and importance of P-Rex1 repression in early-life ECS- induced airway hyperinnervation and hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Long-term goal: to determine whether targeting neuronal P-Rex1 provides a new strategy for preventing early-life ECS-related asthma progression. Findings: 1) P-Rex1 is highly expressed in neurons but not airway cells. 2) P-Rex1 knockout (KO) mice exhibit airway smooth muscle (ASM) hyperinnervation and AHR. WT mice exposed to early-life ECS showed similar phenotypes with 60% reduction of P-Rex1 in vagal ganglia. Severing vagus nerves attenuated AHR of these mice. 3) ECS exposure enhances brain-derived neurotrophic factor (BDNF) secretion from ASM cells, serving as a target-derived signal for neurite growth of mouse vagal sensory neurons in vitro. 4) P-Rex1 over-expression blocked BDNF-stimulated neurite growth whereas loss of P-Rex1 markedly sensitized these neurons to BDNF stimulation. 5) ECS-elevated interleukin (IL)-6 down-regulates P- Rex1 and enhances BDNF-stimulated neurite growth that is blocked by a PKC inhibitor. Hypothesis: IL-6 repression of neuronal P-Rex1 plays a crucial role in early-life ECS-induced ASM hyperinnervation and AHR of asthma. We will test this hypothesis using molecular, cellular, and animal models. Aim 1: To elucidate the mechanism of early-life ECS-exposure-induced neuronal P-Rex1 repression. We hypothesize that IL-6 represses neuronal P-Rex1 via a PKC-dependent mechanism. We will first use siRNAs to silence P-Rex1 in mouse vagal sensory neurons to assess the importance of P-Rex1 in IL-6 potentiation of BDNF-induced neurite growth. We will then investigate if restoration of P-Rex1 expression attenuates IL-6 stimulatory effects. Finally, we will use inhibitors and siRNAs to identify the PKC isoforms responsible for IL-6-induced P-Rex1 repression and neurite growth. Aim 2: To investigate the pathologic importance of neuronal P-Rex1 repression in early-life ECS exposure-related asthma. We hypothesize that IL-6 repression of neuronal P-Rex1 is a critical determinant in the development and severity of early-life ECS-related asthma. WT and P-Rex1 KO mice will be exposed to ECS or air for 10 days beginning on postnatal day (PND) 2. AHR will be assessed by invasive tracheostomy 24h after a re-exposure of mice to acute insult of ECS or allergen house dust mite on PND59. Effects of early-life ECS exposure on ASM innervation and phenotype (remodeling, contractility) will be examined. Whether loss of P-Rex1 exacerbates early-life ECS-induced ASM hyper-innervation and AHR will be determined. Finally, we will determine whether oral administration of IL-6 inhibitor LMT-28 ameliorates early-life ECS-induced mouse AHR by preventing P-Rex1 repression and ASM hyperinnervation.

早期生活环境香烟烟雾（ECS）暴露会改变气道神经并增加发病率 生命后期的哮喘，但机制仍然不确定。我们最近将神经元P-rex1确定为 在暴露于 早期生活EC。目的：定义p-rex1抑制在早期生活ECS中的机制和重要性 诱导的气道高感染和反应性高反应性（AHR），哮喘的病理学标志。 长期目标：确定针对神经元P-REX1是否提供了一种防止的新策略 早期生活与EC相关的哮喘进展。调查结果：1）p-rex1在神经元中高度表达，而不是气道 细胞。 2）P-REX1敲除（KO）小鼠表现出气道平滑肌（ASM）高吸毒和AHR。 WT小鼠 暴露于早期寿命的EC显示出类似的表型，而迷走神经节中P-Rex1的降低了60％。切断 迷走神经减弱了这些小鼠的AHR。 3）EC暴露增强了脑衍生的神经营养因子 （BDNF）从ASM细胞分泌，用作靶向的小鼠迷走性感觉神经突生长的信号 体外神经元。 4）p-rex1过表达阻塞BDNF刺激的神经突生长，而p-rex1的丧失 这些神经元显着敏感到BDNF刺激。 5）EC升高的白介素（IL）-6下调p- REX1并增强了PKC抑制剂阻断的BDNF刺激的神经突生长。假设：IL-6 神经元P-REX1的抑制在早期寿命ECS诱导的ASM高吸毒和AHR中起着至关重要的作用 哮喘。我们将使用分子，细胞和动物模型检验这一假设。目标1：阐明 早期生活ECS暴露诱导的神经元P-REX1抑制的机制。我们假设IL-6 通过PKC依赖性机制抑制神经元P-REX1。我们将首先使用sirnas沉默p-rex1 小鼠迷走神经感觉神经元评估p-rex1在BDNF诱导的IL-6增强中的重要性 神经突的生长。然后，我们将调查P-REX1表达的恢复是否会减弱IL-6刺激作用。 最后，我们将使用抑制剂和siRNA来识别负责IL-6诱导的P-REX1的PKC同工型 抑制和神经突的生长。目的2：研究神经元P-REX1的病理意义 早期EC暴露有关的哮喘的抑制作用。我们假设IL-6抑制神经元P-REX1 是与ECS相关哮喘的发展和严重程度的关键决定因素。 WT和P-REX1 KO 从产后日开始（PND）2，小鼠将暴露于ECS或空气中10天。AHR将由AHR评估 侵入性气管造口术后24小时后，小鼠急性侮辱ECS或过敏原房屋尘螨 PND59。早期ECS暴露对ASM神经和表型的影响（重塑，收缩力）将 被检查。 P-REX1的损失是否加剧早期寿命ECS诱导的ASM高温和AHR 将确定。最后，我们将确定口服IL-6抑制剂LMT-28是否可以改善 通过防止P-REX1抑制和ASM过度连接，早期ECS诱导的小鼠AHR。