Role of Toll-like Receptors in Transplant-Induced Reactivation of Cytomegalovirus

描述（由申请人提供）：潜伏巨细胞病毒（CMV）的再激活在实体器官和骨髓移植接受者中经常观察到，也是新生儿和免疫功能低下宿主发病和死亡的重要原因。目前可用的靶向病毒复制的抗病毒治疗与治疗范围狭窄相关，导致显著的毒性，包括骨髓抑制，特别是在肾功能不全的患者中。此外，由于对这些药物的耐药性越来越高，必须使用有效但毒性更大的药物。人们普遍认为，即时早期（IE）基因在潜伏感染中转录沉默，IE基因表达的重新激活是病毒重新激活的关键第一步。使用防止IE基因表达再激活的药物进行治疗将是一种新的方法，可以避免与试图对抗病毒复制相关的问题。IE基因表达的再激活机制尚不清楚。由于缺乏动物模型来研究HCMV在体内的感染，我们和其他人使用MCMV作为模型来研究CMV的潜伏期和再激活。我们之前利用小鼠肾移植模型在体内诱导MCMV的转录再激活。我们的初步研究表明Toll样受体诱导的基因在异基因肾移植中表达上调。我们假设MCMV即时早期（IE）基因表达在移植反应中的转录再激活是由先天免疫反应启动的，通过toll样受体与缺血性损伤的细胞配体相互作用，并通过mhc不相容细胞的识别进一步放大。我们假设这会导致炎症细胞因子（如TNF）的表达增加，TNF本身可以激活增强子，并且这些因子激活的信号通路导致染色质重塑和IE基因表达的再激活。我们的初步数据表明，TLR2在激活同种异体移植的细胞免疫应答中起着重要作用，因此，在MCMV的再激活中起着重要作用。通过使用TLR2缺陷小鼠，并使用siRNA技术在体内阻断表达，我们提出在该模型中测试TLR2以及信号适配器MyD88和Trif在MCMV IE基因表达再激活中的需求。在移植前用siRNA等靶向启动再激活所需步骤的药物治疗供体器官将是一种新方法，可以显着减少对受体的毒性问题。因此，这些研究可能会导致新的策略的发展，以防止巨细胞病毒的再激活及其后遗症。公共卫生相关性：在免疫功能低下的移植患者中，潜伏巨细胞病毒的再激活可导致显著的发病率或死亡率。目前可用的针对病毒复制的抗病毒治疗与严重的毒性和不断增加的耐药性有关。本提案的目标是了解触发潜伏病毒再激活的分子途径，并制定新的策略来防止潜伏病毒的再激活。

DESCRIPTION (provided by applicant): Reactivation of latent cytomegalovirus (CMV) is frequently observed in recipients of solid organs and bone marrow transplants and is also a significant cause of morbidity and mortality in newborns and in immunocompromised hosts. Currently available anti-viral therapies which target viral replication are associated with narrow therapeutic margins resulting in significant toxicities, including bone marrow suppression, especially in patients with renal dysfunction. Also, the increasing rates of resistance to these agents necessitates the use of efficacious but more toxic agents. It is generally accepted that immediate early (IE) genes are transcriptionally silent in latent infection, and that reactivation of IE gene expression is a critical first step in reactivation of the virus. Treatment with agents which prevent reactivation of IE gene expression would be a novel approach which would avoid problems associated with attempting to combat viral replication. The mechanisms by which reactivation of IE gene expression occurs are not well understood. Because of the lack of animal models to study HCMV infection in vivo, we and others have used MCMV as a model to study CMV latency and reactivation. We have previously utilized a mouse kidney transplant model to induce transcriptional reactivation of MCMV in vivo. Our preliminary studies indicate that genes induced by Toll like receptors are up-regulated in allogeneic kidney transplants. We hypothesize that transcriptional reactivation of MCMV immediate early (IE) gene expression in response to transplantation is initiated by an innate immune response through interaction of Toll-like receptors with cellular ligands damaged by ischemic injury, and this is further amplified by recognition of MHC-incompatible cells. We hypothesize that this leads to increased expression of inflammatory cytokines such as TNF which can themselves activate the enhancer, and that signaling pathways activated by these factors leads to chromatin remodeling and reactivation of IE gene expression. Our preliminary data suggests a major role for TLR2 in activation of the cellular immune response to allogeneic transplants, and thus, for reactivation of MCMV. Through the use of TLR2 deficient mice and the use of siRNA technology to block expression in vivo, we propose to test the requirement for TLR2 as well as signaling adapters MyD88 and Trif in reactivation of MCMV IE gene expression in this model. Treatment of the donor organ prior to transplantation with agents such as siRNA that target steps required for initiation of reactivation would be a novel approach which would significantly reduce the problem of toxicity to the recipient. These studies may therefore lead to the development of new strategies to prevent reactivation of CMV and its sequelae. PUBLIC HEALTH RELEVANCE: Reactivation of latent cytomegalovirus can cause significant morbidity or mortality in immunocompromised transplant patients. Currently available anti-viral therapies, which target viral replication, are associated with severe toxicities and increasing rates of resistance. The goal of this proposal is to understand the molecular pathways that trigger reactivation of latent virus and to develop new strategies to prevent reactivation of latent virus.