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

项目摘要 原发性移植物功能障碍（PGD）仍然是肺移植（Tx）早期发病率和死亡率的主要原因 受惠人士肺移植结果小组（LTOG）在过去的十年里一直在研究这个问题， NIH资助的LTOG相关项目（1U 01 HL 14535 -01），由Jason Christie博士（UPENN）领导，涉及长期随访- 成人肺移植受者。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HDAC 6 mRNA的前Tx水平显著升高 单核细胞我们鉴定了CD 14+单核细胞是Tx前PGD-prone中HDAC 6上调的主要来源。 在体外刺激后，观察到这些单核细胞的炎症表型，并发现HDAC 6 靶向显著降低了局部缺血/再灌注损伤的体内鼠肺模型中细胞因子的产生。 因此，我们假设在肺Tx前HDAC 6表达上调的受体血液单核细胞是 这是移植物损伤和PGD发展的重要驱动因素，单核细胞中HDAC 6表达的升高可能是 具有预后和治疗意义。我们建议通过研究来自以下人群的Tx前血液样本来验证这一假设： 2019年6月15日开始入组母U 01的患者。目的1：确定上调的HDAC 6的表型是否 在单核细胞的患者列出的肺Tx是一个危险因素，发展PGD？我们将扩大我们的初步 使用更大样本量的数据来评估1.1）单核细胞HDAC 6表达（包括mRNA和蛋白质）的前Tx水平 单核细胞活化的经典标志物的水平和共表达;和1.2）Tx后事件（PGD与等级）与单核细胞活化的关系 与Tx前单核细胞表型相关。目的2：HDAC 6改变关键单核细胞的机制是什么 特征？我们将：2.1）通过从小鼠中分离的单核细胞评估细胞因子表达（qPCR、流式细胞术、ELISA）， 2.2）研究HDAC 6表达有多高， 调节TLR/MyD 88通路活化。目的3：测试HDAC 6抑制剂（HDAC 6 i）治疗是否可以逆转或 减少单核细胞功能障碍的影响，包括离体和在PGD的鼠模型中。我们将：3.1）测试 HDAC 6 i对单核细胞活化、细胞因子产生和与其他免疫细胞相互作用的影响; 3.2）测试影响 一种或多种新的HDAC 6 i小分子（单核细胞特异性HDAC 6 i，HDAC 6锌指泛素结合结构域 抑制剂和HDAC 6 iPROTAC）;和3.3）在最近描述PGD的新鼠模型中体内应用HDAC 6 i。 重要的是，我们的研究将·描绘疾病的致病机制; ·确定影响疾病的机制或因素。 ·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.