Targeting Ischemia/Reperfusion Stress to Inhibit Cytomegalovirus Reactivation After Lung Transplant

针对肺移植后缺血/再灌注应激抑制巨细胞病毒再激活

• 批准号: 10453246
• 负责人: Zheng J Zhang
• 金额: $ 24万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-25 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453246
• 关键词: Address; Alveolar Macrophages; Animal Model; Binding; Birds; Cells; Cellular Stress; Chemicals; Clinical; Complication; Cytomegalovirus; DNA biosynthesis; Data; Day Surgery; Disease; Donor person; Early Promoters; Enzymes; Epigenetic Process; Foundations; Future; Gene Expression; Genetic Transcription; Goals; Hour; Human; Immediate-Early Genes; Immune; Immunosuppression; In Vitro; Inflammation; Inflammatory; Infusion procedures; Inositol; Interleukin-1 Receptors; Ischemia; Kidney; Kidney Transplantation; Knockout Mice; Left; Lung; Lung Transplantation; Mediating; Molecular; Murid herpesvirus 1; Mus; Myelogenous; NF-kappa B; Opportunistic Infections; Organ; Organ Donor; Organ Preservation; Organ Transplantation; Pathway interactions; Perioperative; Peripheral; Primary Infection; Procedures; Prophylactic treatment; RNA analysis; Regimen; Reperfusion Injury; Reperfusion Therapy; Role; Salivary Glands; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Stress; System; Testing; Therapeutic; Time; Tissues; Toll-like receptors; Transcription Factor AP-1; Transcriptional Activation; Transplant Recipients; Transplantation; Transplantation Surgery; Viral; Viral Load result; Virus; Virus Replication; allograft rejection; cell type; clinical translation; clinically relevant; high risk; implantation; inhibitor; innovation; mRNA Expression; macrophage; mouse model; nanocarrier; novel; novel therapeutics; post-transplant; pre-clinical; preconditioning; prevent; reactivation from latency; response; sensor; seropositive; therapeutic evaluation; therapeutically effective; transcription factor; transcriptome; viral DNA

PROJECT SUMMARY Cytomegalovirus (CMV) is the most common opportunistic infection in lung transplant recipients, with the highest risk seen in seronegative recipients (R-) of seropositive organ donors (D+). While anti-rejection immunosuppression (IS) has been implicated as a primary cause of CMV reactivation, our studies have newly suggested otherwise. We have recently shown that CMV reactivation occurs independent of IS and seemingly independent of the allo-immune response following a D+/R- kidney transplant in mice. Treatment with a regimen of clinically relevant IS, while failing to induce CMV reactivation in latently infected mice (no transplant), does permit subsequent viral replication and dissemination following transplant, despite inhibition of the host rejection response and inflammation. These findings suggest that ischemia/reperfusion (I/R) stress inherent in a transplant procedure due to organ preservation and revascularization is necessary and sufficient to induce reactivation. However, molecular mechanisms underlying I/R stress mediated transcriptional reactivation from latency in the grafts remain elusive. The goal for this study is to identify key molecular pathways responsible for I/R stress- mediated CMV reactivation and to develop effective therapeutics to prevent both I/R injury and CMV reactivation following lung transplant. In our preliminary studies using a mouse model of syngeneic D+/R- lung transplant, we observed that expression of MCMV immediate early (IE) genes is induced within 48 hours of transplant surgery, followed by increased viral DNA replication in the lung grafts and dissemination to peripheral organs by day 14 post-transplant. We revealed that pre-transplant depletion of donor alveolar macrophages (AMs) decreased viral load in transplanted lungs and salivary glands, suggesting that AMs are primary sites of MCMV latency and reactivation, consistent with the findings from HCMV studies. We have further showed that Myeloid differentiation primary response 88 (Myd88) was critical in mediating transplant induced I/R injury, likely via activation of transcription factors (TFs) NF-kB, AP-1 and XBP-1. These TFs are shown to bind to MIEP and promote MCMV/HCMV reactivation. Thus, we hypothesize that I/R stress mediates Myd88 dependent activation of TFs in viral harboring donor lung cells (e.g. AMs); leading to reactivation of latent CMV in the transplanted lungs. Thus, tissue/cell specific inhibition of Myd88 pathways in latently infected lung donors may prevent viral reactivation. We will use a preclinical mouse model of lung transplantation to test our hypotheses. In Aim 1, we will 1) investigate how donor macrophage-derived Myd88 regulate expression of viral IE genes and viral DNA replication by utilizing genetically modified time-specific myeloid Myd88 knockout mice and 2) delineate impact of Myd88 on I/R stress mediated signaling pathways in the lung cells using single cells RNA analysis. In Aim 2, we will determine the therapeutic potentials of tissue/cell-specific targeting of Myd88 in mitigating I/R stress and MCMV reactivation and dissemination leveraging our novel nanocarrier delivery system. Data generated from this study will provide proof of concept for developing novel therapeutics for further clinical translation.

项目概要 巨细胞病毒（CMV）是肺移植受者中最常见的机会性感染， 血清反应阴性受者 (R-) 或血清反应阳性器官捐献者 (D+) 的风险最高。抗拒的同时 免疫抑制 (IS) 被认为是 CMV 重新激活的主要原因，我们的研究最新发现 否则建议。我们最近表明 CMV 重新激活的发生独立于 IS，并且似乎 独立于小鼠 D+/R- 肾移植后的同种免疫反应。治疗方案 临床相关的 IS，虽然未能在潜伏感染的小鼠中诱导 CMV 重新激活（无移植），但确实 尽管抑制了宿主排斥，但仍允许移植后随后的病毒复制和传播 反应和炎症。这些发现表明移植物固有的缺血/再灌注（I/R）应激 由于器官保存和血运重建而进行的手术对于诱导再激活是必要且充分的。 然而，I/R应激背后的分子机制介导了从潜伏期开始的转录再激活。 移植物仍然难以捉摸。本研究的目标是确定导致 I/R 应激的关键分子途径 介导的 CMV 重新激活并开发有效的治疗方法来预防 I/R 损伤和 CMV 重新激活 肺移植后。在我们使用同基因 D+/R- 肺移植小鼠模型的初步研究中， 我们观察到 MCMV 即早 (IE) 基因的表达在移植后 48 小时内被诱导 手术后，病毒 DNA 在肺移植物中复制增加，并通过以下方式传播到周围器官： 移植后第14天。我们发现移植前供体肺泡巨噬细胞 (AM) 的消耗 移植肺和唾液腺中的病毒载量减少，表明 AM 是 MCMV 的主要位点 潜伏期和再激活，与 HCMV 研究结果一致。我们进一步表明，骨髓 分化初级反应 88 (Myd88) 在介导移植诱导的 I/R 损伤中至关重要，可能通过 转录因子 (TF) NF-kB、AP-1 和 XBP-1 的激活。这些 TF 被证明与 MIEP 结合并且 促进 MCMV/HCMV 重新激活。因此，我们假设 I/R 应激介导 Myd88 依赖性激活 病毒携带的供体肺细胞中的转录因子（例如 AM）；导致移植体内潜伏的 CMV 重新激活 肺。因此，在潜伏感染的肺供体中对 Myd88 通路进行组织/细胞特异性抑制可能会预防病毒感染。 重新激活。我们将使用肺移植的临床前小鼠模型来检验我们的假设。在目标 1 中，我们 1) 研究供体巨噬细胞来源的 Myd88 如何调节病毒 IE 基因和病毒 DNA 的表达 利用基因修饰的时间特异性骨髓 Myd88 敲除小鼠进行复制和 2) 描绘影响 使用单细胞 RNA 分析，研究 Myd88 对肺细胞中 I/R 应激介导的信号通路的影响。在目标 2 中， 我们将确定 Myd88 的组织/细胞特异性靶向在减轻 I/R 应激方面的治疗潜力， 利用我们新颖的纳米载体递送系统重新激活和传播 MCMV。数据生成自 这项研究将为开发新疗法以进一步临床转化提供概念证明。