Pre-Transplant Monocyte HDAC6 Expression and Risk of Primary Graft Dysfunction in Clinical Lung Transplant Recipients

项目摘要 原发移植物功能障碍(PGD)仍然是肺移植(TX)早期发病率和死亡率的主要原因 收件人。肺移植结果小组(LTOG)在过去的十年里一直在研究这个问题，并推出了一项新的 NIH资助的LTOG相关项目(1U01HL14535-01)，由宾夕法尼亚大学Jason Christie博士领导，涉及长期跟踪- 成人肺移植受者的数量。在与LTOG合作进行的初步研究中，我们发现患者 随后发生PGD的患者外周血中HDAC6mRNA水平显著升高 单个核细胞。我们发现CD14+单核细胞是Pre-Tx PGD倾向患者HDAC6上调的主要来源 患者，观察这些单核细胞在体外刺激后的炎症表型，发现HDAC6 靶向显著减少在体小鼠肺缺血/再灌注损伤模型中细胞因子的产生。 因此，我们假设受者单核细胞在肺前Tx上调了HDAC6的表达。 移植物损伤和PGD发生的重要驱动因素，单核细胞中HDAC6表达的增加可能是 对预后和治疗有重要意义。我们建议通过研究TX前的血液样本来检验这一假设 患者参加了2019年6月15日开始的家长U01。目的1：确定上调的HDAC6的表型 在被列为肺部疾病的患者的单核细胞中，TX是发生PGD的危险因素吗？我们将在我们的初步工作基础上展开 使用更大样本量的数据来评估1.1)TX前单核细胞HDAC6表达水平，包括mRNA和蛋白质 单核细胞活化的经典标志物的水平和共表达；1.2)TX后事件(PGD与分级)的关系 到TX前单核细胞表型。目的2：HDAC6改变关键单核细胞的机制是什么 特征是什么？我们将：2.1)评估细胞因子的表达(qPCR、流式细胞仪、酶联免疫吸附试验) TX前受者和健康供者与他们的HDAC6水平的关系；以及2.2)研究HDAC6的高表达程度 调节TLR/MyD88通路的激活。目的3：检测HDAC6抑制剂(HDAC6i)治疗能否逆转或 减少单核细胞功能障碍的影响，包括体外和PGD小鼠模型。我们将：3.1)测试 HDAC6i对单核细胞活化、细胞因子产生及与其他免疫细胞相互作用的影响；3.2)测试效果 一种或多种新型HDAC6i小分子(单核细胞特异性HDAC6i、HDAC6锌指泛素结合域 抑制剂和HDAC6i PROTAC)；以及3.3)在最近描述的一种新的PGD小鼠模型中应用HDAC6i。 重要的是，我们的研究将·描述疾病的致病机制；·确定影响 和/或预测治疗反应；以及·发现或验证疾病发展和/或进展的生物标志物。

Project Summary Primary Graft Dysfunction (PGD) remains a leading cause of early morbidity and mortality in lung transplant (Tx) recipients. The Lung Transplant Outcomes Group (LTOG) has been studying this problem for the past decade and a newly NIH-funded LTOG-related project (1U01HL14535-01), headed by Dr. Jason Christie (UPENN), involves long-term follow- up of adult lung transplant recipients. In preliminary studies, undertaken in collaboration with LTOG, we found that patients who subsequently developed PGD had significantly elevated pre-Tx levels of HDAC6 mRNA in their peripheral blood mononuclear cells. We identified CD14+ monocytes as the main source of upregulated HDAC6 in pre-Tx PGD-prone patients, observed an inflammatory phenotype of those monocytes after stimulation in vitro, and found that HDAC6 targeting significantly decreased cytokine production in an in vivo murine lung model of ischemia/reperfusion injury. Accordingly, we hypothesize that recipient blood monocytes with upregulated HDAC6 expression pre-lung Tx are important drivers of graft injury and development of PGD, and that such elevated HDAC6 expression in monocytes may be of prognostic and therapeutic significance. We propose to test this hypothesis by studying pre-Tx blood samples from patients enrolled in the parent U01 that began June 15, 2019. Aim 1: Determine if the phenotype of upregulated HDAC6 in monocytes of patients listed for lung Tx is a risk factor for developing PGD? We will expand upon our preliminary data using a larger sample size to evaluate 1.1) pre-Tx levels of monocyte HDAC6 expression, including mRNA and protein levels, and co-expression of classical markers of monocyte activation; and 1.2) post-Tx events (PGD with grade) in relation to the pre-Tx monocyte phenotype. Aim 2: What are the mechanisms by which HDAC6 alters key monocyte characteristics? We will: 2.1) evaluate cytokine expression (qPCR, flow cytometry, ELISA) by monocytes isolated from pre-Tx recipients and healthy donors, in relation to their HDAC6 levels; and 2.2) study how high HDAC6 expression regulates TLR/MyD88 pathway activation. Aim 3: Test whether HDAC6 inhibitor (HDAC6i) therapy can reverse or diminish the effects of monocyte dysfunction, including ex vivo and in a murine model of PGD. We will: 3.1) test effects of HDAC6i on monocyte activation, cytokine production and interaction with other immune cells; 3.2) test effects of one or more novel HDAC6i small molecules (monocyte specific HDAC6i, HDAC6 zinc-finger ubiquitin binding domain inhibitor and HDAC6i PROTAC); and 3.3) apply HDAC6i in vivo in a recently described new murine model of PGD. Importantly, our studies will • delineate pathogenic mechanisms of disease; • identify mechanisms or factors that influence and/or predict response to treatment; and • discover or validate biomarkers of disease development and/or progression.