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.

项目摘要 巨细胞病毒（CMV）是肺移植受者最常见的机会性感染，其中 在血清反应阳性器官供体（D+）的血清反应阴性受体（R-）中观察到的风险最高。虽然抗排斥 免疫抑制（IS）被认为是CMV再激活的主要原因，我们的研究最近 另有建议。我们最近的研究表明，巨细胞病毒的再激活与IS无关， 独立于小鼠中D+/R-肾移植后的同种免疫应答。治疗方案 临床相关的IS，虽然不能诱导潜伏感染小鼠（无移植）的CMV再激活， 允许随后的病毒复制和传播后移植，尽管抑制宿主排斥反应 反应和炎症。这些发现表明，移植中固有的缺血/再灌注（I/R）应激 由于器官保存和血运重建，手术是必要的，足以诱导再激活。 然而，I/R应激介导的转录再激活的潜在分子机制， 移植物仍然难以捉摸。这项研究的目标是确定负责I/R应激的关键分子途径- 介导的CMV再激活，并开发有效的治疗方法来预防I/R损伤和CMV再激活 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立即早期（IE）基因的表达在移植后48小时内被诱导， 手术后，肺移植物中的病毒DNA复制增加，并通过 移植后第14天。我们发现，移植前供体肺泡巨噬细胞（AM）的耗竭 移植肺和唾液腺中的病毒载量降低，表明AM是MCMV的主要部位 潜伏期和再激活，与HCMV研究的结果一致。我们进一步表明， 分化初级反应88（Myd 88）在介导移植诱导的I/R损伤中是关键的，可能通过 转录因子（TF）NF-κ B、AP-1和XBP-1的激活。这些TF显示与MIEP结合， 促进MCMV/HCMV再活化。因此，我们假设I/R应激介导Myd 88依赖性激活 携带病毒的供体肺细胞（例如AM）中TF的增加;导致移植肺细胞中潜伏CMV的再活化。 肺因此，在潜伏感染的肺供体中Myd 88途径的组织/细胞特异性抑制可以防止病毒感染。 重新激活我们将使用临床前小鼠肺移植模型来验证我们的假设。目标1： 将1）研究供体巨噬细胞来源的Myd 88如何调节病毒IE基因和病毒DNA的表达 通过利用遗传修饰的时间特异性骨髓Myd 88敲除小鼠进行复制，以及2）描绘影响 使用单细胞RNA分析，研究Myd 88对肺细胞中I/R应激介导的信号传导途径的影响。在目标2中， 我们将确定Myd 88的组织/细胞特异性靶向在减轻I/R应激中的治疗潜力， 利用我们的新型纳米载体递送系统的MCMV再活化和传播。生成的数据 这项研究将为开发新的治疗方法以进一步临床转化提供概念证明。