Pro-Inflammatory Stem Cell Variants in Cystic Fibrosis

炎症在囊性纤维化（CF）的进行性病理学中起主要作用，并且通常被认为是 是对CF肺部微生物定植增加的反应。然而，最近的研究涉及正常和 在广谱抗生素下饲养的CFTR突变雪貂在CF肺中显示出强烈的炎症， 没有细菌病原体。此外，虽然CFTR调节剂的革命性类别改善了肺 但是，由于它们不能发挥功能并减少恶化，因此它们在减轻CF肺的炎症方面不太成功。这些 这些发现提出了这样一种可能性，即炎症以及CF的其他致病特征是由以下因素维持的： 这些因素出现在疾病中，但随后独立于CFTR活性驱动进展。类似的 在慢性阻塞性肺疾病（COPD）中，炎症和疾病 尽管戒烟，病情仍在继续。在COPD中，最近的研究表明， （p<10-16）在促炎性小气道上皮细胞的出现和疾病本身之间。 这些致病性变异体也存在于无COPD的对照患者和胎儿中，尽管水平较低。 肺。对CF肺的类似分析显示，它们被致病性干细胞变异高度淹没。 与COPD中观察到的相关，沿着两种以前未在COPD中发现的新的高度炎症变异， 慢性阻塞性肺病。我们假设这些CF干细胞变异体在CF的进展中起关键作用， 代表由CFTR基因型触发但独立于CFTR基因型的这种疾病的致病因素。到 检验这一假设，并扩展我们对这些变异在CF疾病中的潜在意义的理解 过程中，我们将在三个具体目标，1）确定关键的炎症驱动因素， 使用CRISPR-Cas9定向突变和异种移植模型的人CF变体，2）测试以下的依赖性： 在CF患者中发现的这三种变体的促炎表型对CFTR活性的影响使用基因 互补和CFTR调节药物如依伐卡托、依拉卡托和替扎卡托，以及3）利用我们的 最近开发的克隆雪貂气道干细胞的方法，以确定致病性 CF进展的雪貂条件模型中的变体。我们预计这些研究将提供背景 并深入了解占CF肺主导地位的变异干细胞的贡献，评估新的 CF疗法对这些细胞的致病特征，以及确定节点基因在炎症 这些变体的特征，其抑制可能对这些患者具有治疗益处。

Inflammation plays a major role in the progressive pathology of cystic fibrosis (CF), and is generally thought to be a response to increased microbial colonization of CF lungs. However, recent studies involving normal and CFTR-mutant ferrets raised under broad-spectrum antibiotics show robust inflammation in the CF lung despite the absence of bacterial pathogens. Moreover, while the revolutionary class of CFTR modulators improve lung function and reduce exacerbations, they are less successful in mitigating inflammation of the CF lung. These findings raise the possibility that inflammation, and perhaps other pathogenic features of CF, are maintained by elements that emerge in the disease but then drive progression independent of CFTR activity. An analogous scenario may be operating in chronic obstructive pulmonary disease (COPD), where inflammation and disease progression continues despite smoking cessation. In COPD, recent studies have shown a strong correlation (p<10-16) between the emergence of pro-inflammatory, small airway epithelial cells and the disease itself. These pathogenic variants are also present, albeit at low levels, in control patients without COPD and in fetal lung. A similar analysis of CF lungs has revealed them to be inundated by pathogenic stem cell variants highly related to those seen in COPD, along with two novel, hyperinflammatory variants not previously identified in COPD lungs. We hypothesize that these CF stem cell variants play key roles in the progression of CF, and represent pathogenic elements of this disease triggered by, and yet independent of, the CFTR genotype. To test this hypothesis and extend our understanding of the potential significance of these variants in CF disease processes, we will, in three specific aims, 1) identify key inflammatory drivers in the three, hyperinflammatory human CF variants using CRISPR-Cas9-directed mutations and xenograft models, 2) test the dependence of the pro-inflammatory phenotype of these three variants found in CF patients on CFTR activity using gene complementation and CFTR-modulating drugs such as ivacaftor, elexacaftor, and tezacaftor, and 3) exploit our recently developed methods for cloning ferret airway stem cells to determine the dynamics of the pathogenic variants in a ferret conditional model of CF progression. We anticipate that these studies will provide context and insight into the contributions of variant stem cells that dominate CF lungs, assess the impact of the new CF therapeutics on the pathogenic features of these cells, as well as identify nodal genes in the inflammatory signatures of these variants whose suppression could be of therapeutic benefit to these patients.