Chronic Lung Allograft Dysfunction: Role for Tumor Suppressor LKB1 in Exosomes

慢性肺同种异体移植功能障碍：肿瘤抑制因子 LKB1 在外泌体中的作用

• 批准号: 10644007
• 负责人: THALACHALLOUR MOHANAKUMAR
• 金额: $ 41.05万
• 依托单位: ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644007
• 关键词: 5' -AMP-activated protein kinase; Acute; Aldehydes; Allografting; Automobile Driving; Binding; Biological Markers; Biological Models; Biopsy; Bronchiolitis Obliterans; CD80 gene; CD86 gene; CDC37 gene; Cell Culture Techniques; Cell Line; Cells; Chronic; Clinical; Cyclic AMP-Dependent Protein Kinases; Data; Development; Diagnosis; Down-Regulation; Early identification; Epithelial Cells; Epithelium; Family; Fibrosis; Flow Cytometry; Functional disorder; Genetic Transcription; Goals; Heat-Shock Proteins 90; Human; Immunologics; In Vitro; Intervention; Liver; Lung; Lung Transplantation; Lung diseases; MHC Class II Genes; Mediating; Mesenchymal; Metabolism; Methods; Modeling; Molecular; Morphology; Pathogenesis; Pathology; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Plasma; Platelet-Derived Growth Factor Receptor; Play; Property; Prostaglandin D2; Receptor Signaling; Receptor Up-Regulation; Regulation; Reporting; Risk; Risk Factors; Role; STK11 gene; Sampling; Seasons; Signal Pathway; Signal Transduction; Syndrome; TNFRSF5 gene; Testing; Therapeutic; Transforming Growth Factor beta; Transplant Recipients; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Up-Regulation; Viral Respiratory Tract Infection; Western Blotting; airway epithelium; cell growth; clinical diagnosis; donor-specific antibody; epithelial to mesenchymal transition; exosome; graft dysfunction; high risk; in vitro Model; knock-down; liver function; lung allograft; multicatalytic endopeptidase complex; nano-string; novel; prevent; primary endpoint; receptor expression; receptor upregulation; secondary endpoint; statistics; therapeutically effective; transcription factor

Project Summary/Abstract Lung transplantation (LTx) is a therapeutic option for patients with advanced lung diseases. Long‐term survival after LTx is, however, limited by chronic lung allograft dysfunction (CLAD). CLAD most commonly manifests itself as bronchiolitis obliterans syndrome (BOS) and about 50% of recipients (LTxRs) will develop BOS within 5 years post-LTx. Epithelial-mesenchymal-transition (EMT) and fibrosis have been implicated in the pathogenesis of BOS. We demonstrated that liver kinase 1 (LKB1), a tumor suppressor gene, is downregulated in BOS but not in stable biopsies using both western blotting and aldehyde bead conjugated exosomes by flow cytometry. We also demonstrated that LKB1 knockdown induces exosome release from airway epithelial cell line, BEAS-2B, and another human airway epithelial cell line, HPBEC. Exosomes released from LTxRs with BOS also induces EMT which was regulated by LKB1 in BEAS-2B and HPBEC. NanoString analyses identified LKB1 knockdown induced PDGFRβ expression in human airway epithelial cells. We also demonstrated that biopsies from BOS LTxRs had reduced LKB1 and increased PDGFβR with inverse correlation. These novel findings indicate an important role for the tumor suppressor gene LKB1 in the regulation of PDGFβR and, therefore, fibrosis development. Studies proposed using both clinical samples and in vitro cell culture model, we will define the mechanism by which exosomes with downregulated LKB1 released from transplanted lungs mediate EMT leading to CLAD. Aim 1 of the proposal is to determine serially whether inactivation of LKB1 in exosomes isolated from plasma from LTxRs with known risk factors (primary graft dysfunction [PGD]), acute rejection [AR] and respiratory viral infections [RVI]) can be useful as a non- invasive biomarker for LTxRs at risk for CLAD. Our hypothesis is that persistent downregulation of LKB1 in exosomes will be a biomarker for LTxRs at risk for developing CLAD. The second goal is to determine and quantitate exosomes with LKB1/AMPK1 using serial retrospectively stored plasma from LTxRs with known clinical diagnosis will be a more sensitive marker for CLAD and to determine its potential to differentiate restrictive allograft syndrome and BOS by defining their immunological and molecular properties. Our third goal is to define the mechanisms by which loss of LKB1 results in EMT and upregulation of PDGFRβ and promotes the pathogenesis of CLAD. Towards this; a) we will define the regulatory mechanisms suppressing LKB1 in LTxRs with PGD, AR and RVI, risk factors for CLAD, and b) we will determine the mechanisms by which LKB1 downregulation leads to upregulation of PDGFRβ and its signaling pathways which contributes towards development of fibrosis. Results from these studies will provide novel information for the role of LKB1, in EMT and fibrosis related pathologies including CLAD following LTx.

项目总结/摘要 肺移植（LTx）是晚期肺部疾病患者的治疗选择。长期生存率 然而，LTx后的肺移植受到慢性肺移植物功能障碍（CLAD）的限制。CLAD最常见的表现是 本身为闭塞性细支气管炎综合征（BOS），约50%的接受者（LTxR）将在 LTx后5年。上皮-间质转化（EMT）和纤维化已被牵连在 BOS的发病机制。我们证明了肝激酶1（LKB 1），一种肿瘤抑制基因， 在BOS中下调，但在稳定的活检中未下调，使用蛋白质印迹和醛珠缀合 通过流式细胞术检测外泌体。我们还证明了LKB 1敲低诱导外泌体从细胞中释放， 气道上皮细胞系BEAS-2B和另一种人气道上皮细胞系HPBEC。外泌体释放 在BEAS-2B和HPBEC中，BOS诱导LTxRs的EMT，并受LKB 1的调节。NanoString 分析鉴定了在人气道上皮细胞中LKB 1敲低诱导的PDGFRβ表达。我们也 证明BOS LTxR的活检减少了LKB 1并增加了PDGFβR，并且呈相反趋势 相关性这些新的发现表明肿瘤抑制基因LKB 1在肿瘤发生中的重要作用。 PDGFβR的调节，因此，纤维化的发展。使用临床样本和 在体外细胞培养模型中，我们将定义具有下调的LKB 1的外泌体 从移植的肺中释放介导EMT导致CLAD。该提案的目标1是连续确定 从具有已知风险因素的LTxR的血浆中分离的外泌体中的LKB 1的失活（主要 移植物功能障碍[PGD]）、急性排斥反应[AR]和呼吸道病毒感染[RVI]）可以用作非- LTxR的侵入性生物标志物具有CLAD风险。我们的假设是，LKB 1的持续下调可能是一个潜在的原因。 外泌体将是处于发展CLAD风险中的LTxR的生物标志物。第二个目标是确定和 使用来自LTxR的连续回顾性储存的血浆定量具有LKB 1/AMPK 1的外泌体， 临床诊断将是CLAD更敏感的标志物，并确定其鉴别诊断的潜力。 限制性同种异体移植物综合征和BOS通过定义其免疫学和分子特性。我们的第三个目标 目的是确定LKB 1缺失导致EMT和PDGFRβ上调的机制， CLAD的发病机制。为此; a）我们将定义抑制LKB 1的调节机制， LTxRs与PGD、AR和RVI、CLAD的风险因素，以及B）我们将确定LKB 1 下调导致PDGFRβ及其信号通路上调， 纤维化的发展。这些研究结果将为LKB 1在EMT中的作用提供新的信息 和纤维化相关的病理，包括LTx后的CLAD。

项目成果

Downregulation of a tumor suppressor gene LKB1 in lung transplantation as a biomarker for chronic murine lung allograft rejection.

• DOI: 10.1016/j.cellimm.2023.104690
• 发表时间: 2023-02
• 期刊: Cellular immunology
• 影响因子: 4.3
• 作者: Mohammad Rahman;R. Ravichandran;N. Sankpal;S. Bansal;A. Sureshbabu;T. Fleming;S. Perincheri;A. Bharat