Airway Inflammation due to the Mutated CFTR and Modulation with Macrolide Antibiotics

弥漫性泛细支气管炎（DPB）是一种主要影响日本等亚洲国家成人的慢性炎症性气道疾病，其特征性肺部表现与囊性纤维化（CF）相似。由于大环内酯类抗生素（MA）如红霉素和克拉霉素治疗DPB患者已经成功并显著改善了疾病的预后，并且我们之前的研究表明CFTR基因突变在DPB患者中非常普遍，我们假设CFTR可能至少在部分发病机制中起作用，CFTR的表达也可能被MA调节。首先，我们分析了CFTR mRNA表达的可能调控以及CFTR外显子9与MA的剪接。有趣的是，MA并没有改变CFTR mRNA的水平，但外显子9-CFTR mRNA的数量以剂量依赖性的方式增加，这表明可能对功能性CFTR mRNA进行了负调节。在CFTR通道特性方面，利用稳定转化的LLCPK和LLCPKΔF508细胞系进行体外氯化物外排实验，这些细胞系具有正常的CFTR cDNA和突变的CFTRΔF508。MA可能上调CFTR通道活性的功能。接下来，我们继续评估CF， DPB和先天性双侧输精管缺失的日本患者的CFTR基因型。通过这项研究，我们发现了多个罕见或独特的突变，这些突变没有被存入全球CF突变数据库。最后，通过构建劳斯肉瘤病毒启动子驱动的表达质粒载体，并将其稳定转染到不产生cftr的细胞中，研究了在日本个体中检测到的突变125C和Q1352H的功能特性。实验和分析目前正在进行中。

Diffuse panbronchiolitis(DPB), a chronic inflammatory airway disease affecting mainly adults in Asian countries such as Japan, presents characteristic pulmonary manifestations similar to those of cystic fibrosis(CF). Since the introduction of macrolide antibiotics(MA) such as erythromycin and clarithromycin to treat patients with DPB has successfully and dramatically improved the prognosis of the disease, and our previous studies demonstrated a high prevalence of CFTR gene mutations in those patients with DPB, we have hypothesized that CFTR might be responsible, at least in part, for the pathogenesis of DPB, and the expression of CFTR could be modulated with MA as well. First, we have analyzed possible modulation of CFTR mRNA expression and splicing of CFTR exon 9 with MA. Interestingly, MA did not alter the levels of CFTR mRNA, but the amounts of exon 9-CFTR mRNA increased in a dose-dependent manner, suggesting the likely negative modulation of functional CFTR mRNA. In terms of CFTR channel property, LLCPK and LLCPKΔF508, which were stably transformed cell lines with normal CFTR cDNA and mutated CFTRΔF508, were utilized for in vitro chloride efflux assay. It seemed that MA could upregulate the function of CFTR channel activity. Next, we have continued to evaluate the CFTR genotypes in Japanese patients with CF, DPB and congenital bilateral absence of vas deferens. Through the study, we have found multiple rare or unique mutations which have not been deposited to the world-wide CF Mutation Database. Finally, the functional properties of the mutations detected in Japanese individuals such as 125C and Q1352H have been conducted by constructing expression plasmid vectors driven by Rous sarcoma virus promoter and stably transfecting those plasmids into CFTR-non-producing cells. The experiments and analyses are currently underway.