CFTR and Toll-Like Receptor Signaling

 描述(申请人提供)：在囊性纤维化(CF)中，肺部疾病是由多层功能障碍(感染、炎症和粘液脱水)引起的。在气道表面液体调节中起主要作用的支气管上皮细胞和在炎症反应和细菌清除中起主要作用的巨噬细胞等吞噬细胞的损伤都是CF相关免疫功能障碍的原因。目前对囊性纤维化跨膜调节蛋白(CFTR)和参与细胞免疫调节的受体之间的相互作用机制知之甚少。我们已经确定了一条在CF巨噬细胞中被破坏的途径，并干扰了严密调控的对炎症刺激的先天免疫反应。我们发现巨噬细胞在内毒素和铜绿假单胞菌的刺激下不能诱导支架蛋白小窝蛋白1(CAV1)的表达，最终导致Toll样受体4(TLR4)信号的负调控受损。最近，我们发现CAV1表达的降低是由于高水平的microRNA-199a-5p，它在TLR4信号转导过程中持续存在于CF巨噬细胞中，并以CAV1为靶标。此外，在CFMΦS中，miR-199a-5p的高水平是由于炎症刺激下磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B-1(Akt1)信号的钝化所致。现在我们有初步的数据表明，这一途径在CF上皮细胞中也是功能失调的。因此，我们已经确定了一种新的影响免疫调节的调节失调的细胞通路。我们假设CFTR介导了质膜上调控平台的组装，这是诱导保护性PI3K/Akt1/miR199-5p/CAV1通路响应TLRs激活所必需的。此外，下游对炎症和感染反应的保护途径的低效诱导降低了CF细胞对炎症应激源的反应能力，导致持续的炎症反应，降低抗氧化反应，最终导致细胞无法重新建立细胞内环境平衡和生存。在这项建议中，我们将：表征CFR-199a-5p在CF细胞中水平失调的机制/S(目标1)；研究功能性CFTR缺失改变TLR4/MyD88信号转导过程中Akt1/miR-199a-5p轴诱导的机制/S(目标2)；测试调节AKT/miR-199a/CAV1通路的药物(目标3)。我们的发现有可能为CF细胞的治疗干预找到新的分子靶点，可以阻止慢性致死性呼吸道超炎症和感染的进展，并延缓CF患者的肺恶化。

 DESCRIPTION (provided by applicant): In Cystic Fibrosis (CF), lung disease arises due to multiple layers of dysfunction (infection, inflammation, and mucus dehydration). Impairment of bronchial epithelial cells, which play a major role in airway surface liquid modulation, and of phagocytes, including macrophages, which play a major role in the inflammatory response and bacterial clearance, both contribute to CF-related immune dysfunctions. Little is known about the mechanisms mediating the crosstalk between the cystic fibrosis transmembrane regulator (CFTR) protein, which is responsible for CF when mutated, and receptors involved in the immune regulation of the cells. We have identified a pathway that is disrupted in CF macrophages and interferes with the tightly regulated innate immune response to inflammatory triggers. We have discovered that CF macrophages fail to induce expression of the scaffold protein caveolin 1 (CAV1) in response to LPS and Pseudomonas aeruginosa, which, ultimately, leads to impaired negative regulation of Toll like receptor 4 (TLR4) signaling. More recently, we found that decreased CAV1 expression is due to high levels of microRNA-199a-5p, which persists in CF macrophages during TLR4 signaling, and targets CAV1. Furthermore, high levels of miR-199a-5p in CF MΦs are due to blunted phosphatidylinositol 3-kinase (PI3K)/protein kinase B-1 (Akt1) signaling in response to inflammatory triggers. Now we have preliminary data showing that this pathway is also dysfunctional in CF epithelium. Thus, we have identified a novel dysregulated cellular pathway in CF that affects immune regulation. We hypothesize that CFTR mediates the assembly of a regulatory platform at the plasma membrane, which is necessary for the induction of the protective PI3K/Akt1/miR199-5p/CAV1 pathway in response to activation of TLRs. Moreover, inefficient downstream induction of this protective pathway in response to inflammation and infection reduces the ability of CF cells to respond to inflammatory stressors, leading to persistent hyper-inflammation, reduced anti-oxidative response, and, ultimately, the inability of the cells to re-establish cellular homeostasis and to survive. In this proposal we will: characterize the mechanism/s by which miR-199a-5p levels are dysregulated in CF cells (Aim 1); investigate the mechanism/s by which lack of functional CFTR alters Akt1/miR-199a-5p axis induction during TLR4/MyD88 signaling (Aim 2); test drugs that modulate the AKT/miR-199a/CAV1 pathway (Aim 3). Our findings have the potential to identify new molecular targets for therapeutic intervention in CF cells, which could halt the progression of chronic fatal airway hyper-inflammation and infection and delay lung deterioration in CF patients.