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

在英国，呼吸系统疾病是一个重大的健康负担，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，呼吸系统疾病占所有死亡人数的14%。糖皮质激素（Gc）广泛用于治疗人类炎症性肺部疾病，但由于使用超生理剂量或合成Gc的效力而产生的副作用，其使用受到限制。这种生理上的要求对于克服活动性炎症所带来的部分抵抗是必要的。人们提出了多种机制来解释这种耐药性，但没有一种机制能改善治疗方法。我们已经发现了一种新的染色质重塑酶，MERM1，作为调节肺部炎症中GR功能的强有力的候选者，现在建议利用这一进展为肺部炎症疾病患者带来益处。MERM1广泛表达，但在人类中观察到的最高表达是在支气管上皮中。GR需要MERM1才能进入其在基因组中的结合位点，并调节诱导和抑制的靶基因，包括抑制促炎细胞因子。在泛素化依赖机制下，MERM1蛋白在对促炎细胞因子的反应中被降解。这伴随着GR函数的丧失。通过恢复MERM1的表达可以完全恢复GR活性。通过在一组人类肺部炎症性疾病中发现MERM1蛋白的显著缺失，证实了与人类疾病的相关性。我们现在将定义MERM1在肺部炎症中的作用。MERM1的作用机制和对GR活性的调控将在离体人类细胞中进行研究。我们将使用原代人支气管上皮细胞和原代人巨噬细胞；最大限度地发挥翻译潜力。我们将研究这些细胞在分离时对炎症激活的反应，并研究MERM1如何反应，以及MERM1如何调节糖皮质激素受体的抗炎作用。我们将把我们的观察扩展到候选GR靶点之外，以分析MERM1的全基因组作用，这些作用远远超出了增强GR的范围。这将通过针对MERM1和GR的RNA-Seq和ChIP-Seq方法来解决。我们将定义MERM1在诱导肺部炎症中的调节，MERM1在先天、过敏性和病毒诱导的肺部炎症中的功能。为了明确确定MERM1在支气管上皮中的作用，我们将产生固定的MERM1小鼠，并将它们与支气管上皮特异性cre缺失系杂交。我们已经有了缺失小鼠，并且已经证明了floxed基因切除的特异性和有效性。通过这种方式，我们可以在体内将MERM1的丢失靶向到上皮细胞。我们将研究这种损失对LPS的先天炎症挑战，室内尘螨的过敏挑战和病毒挑战的影响，使用普通感冒诱导呼吸道鼻病毒。所有三种刺激引起的肺部炎症对外源性高剂量Gc治疗有反应。因此，我们也将能够表征上皮中MERM1的丢失如何影响炎症中的Gc作用。将MERM1转化为人类炎症性肺病至关重要，这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MERM1表达来实现。此外，我们还获得了一组明确定义的严重哮喘患者。这些受试者的特点是对肌肉注射、强效类固醇治疗无反应，要么是肺功能改变，要么是痰嗜酸性粒细胞改变。我们将比较这些患者诱导痰和外周血中MERM1的表达，并与两个对照组进行比较，一个是轻度哮喘组，另一个是健康对照组。

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.