MICA: Epigenetic regulation of GR function in pulmonary inflammation: the role of MERM1

呼吸系统疾病是英国的一个重大健康负担，估计有370万人患有慢性阻塞性肺疾病（COPD），520万人正在接受哮喘治疗。2010年，呼吸道疾病占所有死亡人数的14%。糖皮质激素（Gc）被广泛用于治疗人类炎症性肺病，但其使用受到由使用超生理剂量或合成Gc的效力导致的副作用特征的限制。这种超生理要求是克服活动性炎症所产生的部分抵抗所必需的。已经提出了各种机制来解释这种耐药性，但没有一个导致了改进的治疗。我们发现了一种新的染色质重塑酶，MERM 1，作为一个强有力的候选人调节GR功能在肺部炎症，现在建议利用这一进展的好处与肺部炎症性疾病的患者。MERM 1是广泛表达，但在人类中观察到的最高表达是在支气管上皮。MERM 1是GR进入其在基因组中的结合位点所必需的，并调节诱导和抑制的靶基因，包括抑制促炎细胞因子。MERM 1蛋白在响应促炎细胞因子时以泛素化依赖性机制降解。这是伴随着GR功能的丧失。GR活性可以通过恢复MERM 1表达而完全拯救。在一组人类肺部炎症性疾病中发现MERM 1蛋白的显著缺失证实了MERM 1与人类疾病的相关性。我们现在将确定MERM 1在肺部炎症中的作用。MERM 1的作用机制以及GR活性的调节将在体外人类细胞中进行。我们将使用原代人支气管上皮细胞和原代人巨噬细胞，以最大限度地提高翻译潜力。我们将研究这些细胞对炎症激活的反应，并研究MERM 1如何反应，以及MERM 1如何调节糖皮质激素受体的抗炎作用。我们将扩展我们的观察超出候选GR目标，以分析MERM 1的全基因组作用，其远远超出增强GR。这将通过靶向MERM 1和GR的RNA-Seq和ChIP-Seq方法来解决。我们将定义MERM 1在诱导的肺部炎症中的调节，MERM 1在先天性，过敏性和病毒诱导的肺部炎症中的功能。为了特异性确定MERM 1在支气管上皮中的作用，我们将产生floxed Merm 1小鼠，并将它们与支气管上皮特异性cre deletor系杂交。我们有删除小鼠，并且已经显示了floxed基因切除的特异性和效率。以这种方式，我们可以在体内将MERM 1损失靶向上皮。我们将研究这种损失对LPS先天性炎症激发、屋尘螨过敏激发和普通感冒诱导呼吸道鼻病毒激发反应的后果。由所有三种刺激诱导的肺部炎症对外源性高剂量Gc治疗有反应。因此，我们也将能够研究上皮中MERM 1的缺失如何影响炎症中Gc的作用。翻译为人类炎症性肺病是必不可少的，将通过分析肺组织病理样本和肺源性细胞（主要是气道巨噬细胞）中的MERM 1表达来实现。此外，我们还获得了一组明确定义的重度哮喘患者。这些受试者的特征为对肌内、强效类固醇治疗无应答，伴肺功能变化或痰液嗜酸性粒细胞增多变化。我们将比较MERM 1在诱导痰和外周血细胞中的表达，并将这些患者与两个对照组（一个轻度哮喘组和一个健康对照组）进行比较。

Respiratory diseases are a significant health burden in the UK, with an estimated 3.7 million people with chronic obstructive pulmonary disease (COPD) and 5.2 million people being treated for asthma. In 2010, respiratory diseases accounted for 14% of all deaths. Glucocorticoids (Gc) are widely used to treat human inflammatory lung diseases, but their use is limited by side-effect profile resulting from the use of supraphysiological doses, or potencies of synthetic Gc. This supraphysiological requirement is necessary to overcome the partial resistance conferred by active inflammation. Various mechanisms to explain this resistance have been proposed, but none has led to improved therapy.We have discovered a novel chromatin remodelling enzyme, MERM1, as a strong candidate for regulating GR function in pulmonary inflammation, and now propose to exploit this advance for the benefit of patients with pulmonary inflammatory disease.MERM1 is widely expressed, but the highest expression observed in humans was in bronchial epithelium. MERM1 is required for GR to access its binding sites in the genome, and to regulate both induced and repressed target genes, including repression of pro-inflammatory cytokines. MERM1 protein is degraded in response to pro-inflammatory cytokines, in a ubiquitinylation-dependent mechanism. This is accompanied by loss of GR function. GR activity can be completely rescued by restoring MERM1 expression. Relevance to human disease was confirmed by finding marked loss of MERM1 protein in a panel of human lung inflammatory diseases.We will now define the role of MERM1 in pulmonary inflammation.The mechanisms of MERM1 action, and regulation of GR activity will be pursued in human cells ex-vivo. We will use primary human bronchial epithelial cells, and primary human macrophages; to maximise translation potential. We will investigate the responses of these cells in isolation to inflammatory activation, and investigate how MERM1 responds, and how MERM1 regulates the anti-inflammatory actions of the glucocorticoid receptor. We will extend our observations beyond candidate GR targets to analyse the genome-wide actions of MERM1, which extend well beyond potentiating the GR. This will be addressed by RNA-Seq and ChIP-Seq approaches targeting MERM1, and GR.We will define the regulation of MERM1 in induced pulmonary inflammation, the function of MERM1 in innate, allergic, and viral induced lung inflammation. To specifically determine the role of MERM1 in the bronchial epithelium we will generate floxed Merm1 mice, and cross them to a bronchial epithelial-specific cre deletor line. We have the deletor mice, and have already shown the specificity and efficiency of floxed gene excision. In this way we can target MERM1 loss to the epithelium in-vivo. We will investigate the consequences of this loss for response to innate inflammatory challenge with LPS, allergic challenge using house dust mite, and viral challenge, using the common cold inducing respiratory rhinovirus. The pulmonary inflammation induced by all three stimuli responds to exogenous, high-dose Gc therapy. Therefore we will also be able to characterise how MERM1 loss in the epithelium affects Gc action in inflammation. Translation to human inflammatory lung disease is essential, and will be achieved by analysing MERM1 expression in pathological samples of lung tissue, and lung-derived cells (mainly airway macrophages). In addition, we have access to a cohort of well-defined severe asthma patients. These subjects have been characterised as not responding to intramuscular, potent steroid therapy, either with a change in lung function, or a change in sputum eosinophilia. We will compare MERM1 expression in induced-sputum, and peripheral blood cells from these patients compared to two control groups, one with mild asthma, and one of healthy controls.