CFTR and Toll-Like Receptor Signaling

 描述（由申请人提供）：在囊性纤维化（CF）中，肺部疾病是由于多层功能障碍（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吞噬细胞（包括巨噬细胞）的损伤都有助于CF相关的免疫功能障碍。关于介导囊性纤维化跨膜调节因子（CFTR）蛋白（其在突变时负责CF）与参与细胞免疫调节的受体之间的串扰的机制知之甚少。我们已经确定了一个途径，在CF巨噬细胞中被破坏，并干扰了严格调节的先天免疫反应的炎症触发。我们已经发现CF巨噬细胞不能诱导支架蛋白小窝蛋白1（CAV 1）响应于LPS和铜绿假单胞菌的表达，这最终导致Toll样受体4（TLR 4）信号传导的负调控受损。最近，我们发现CAV 1表达的降低是由于高水平的microRNA-199 a-5 p，其在TLR 4信号传导期间持续存在于CF巨噬细胞中，并靶向CAV 1。此外，CF MΦ中高水平的miR-199 a-5 p是由于响应于炎症触发物的钝化的磷脂酰肌醇3-激酶（PI 3 K）/蛋白激酶B-1（Akt 1）信号传导。现在，我们有初步的数据表明，这一途径也是功能失调的CF上皮。因此，我们已经确定了一种新的失调的细胞途径CF影响免疫调节。 我们假设CFTR介导质膜上调节平台的组装，这是诱导保护性PI 3 K/Akt 1/miR 199 - 5 p/CAV 1通路响应TLR活化所必需的。此外，响应于炎症和感染的这种保护性途径的低效下游诱导降低了CF细胞响应炎性应激源的能力，导致持续的过度炎症，降低的抗氧化反应，并且最终导致细胞不能重建细胞稳态和存活。 在本提案中，我们将：表征CF细胞中miR-199 a-5 p水平失调的机制（目标1）;研究功能性CFTR的缺乏改变TLR 4/MyD 88信号传导过程中Akt 1/miR-199 a-5 p轴诱导的机制（目标2）;测试调节AKT/miR-199 a/CAV 1通路的药物（目标3）。 我们的研究结果有可能确定CF细胞治疗干预的新分子靶点，这可能会阻止慢性致命性气道过度炎症和感染的进展，并延迟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.