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

描述(由申请人提供)：在实体器官和骨髓移植的受者中，经常观察到潜伏的巨细胞病毒(CMV)的重新激活，这也是新生儿和免疫低下宿主发病率和死亡率的重要原因。目前可用的针对病毒复制的抗病毒疗法与治疗范围狭窄有关，从而导致显著的毒性，包括骨髓抑制，特别是在肾功能障碍的患者中。此外，对这些药物的耐药率越来越高，需要使用有效但毒性更大的药物。人们普遍认为，在潜伏感染中，即刻早期(IE)基因在转录上是沉默的，重新激活IE基因的表达是病毒重新激活的关键第一步。用阻止IE基因表达重新激活的药物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基因表达重新激活的机制还不是很清楚。由于缺乏研究体内感染巨细胞病毒的动物模型，我们等人利用巨细胞病毒作为模型来研究巨细胞病毒的潜伏期和再激活。我们之前已经利用小鼠肾移植模型在体内诱导了MCMV的转录重新激活。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上调。我们推测，移植后MCMV即刻早期(IE)基因表达的转录重新激活是由先天免疫反应通过Toll样受体与缺血损伤受损的细胞配体相互作用而启动的，这种反应通过识别MHC不相容的细胞而进一步放大。我们假设这会导致炎性细胞因子如肿瘤坏死因子的表达增加，而这些细胞因子本身可以激活增强子，而这些因子激活的信号通路会导致染色质重塑和IE基因表达的重新激活。我们的初步数据表明，TLR2在激活同种异体移植的细胞免疫反应中发挥了重要作用，从而重新激活了MCMV。通过利用TLR2基因缺陷小鼠和利用siRNA技术阻断体内表达，我们建议在该模型中测试对TLR2以及信号转接子MyD88和Trif重新激活MCMV IE基因表达的需求。在移植前用小干扰核糖核酸等针对启动重新激活所需步骤的药物治疗捐献器官将是一种新的方法，它将大大减少对受者的毒性问题。因此，这些研究可能导致开发新的策略来预防CMV的重新激活及其后遗症。公共卫生相关性：潜伏的巨细胞病毒的重新激活可导致免疫功能低下的移植患者的显著发病率或死亡率。目前可用的针对病毒复制的抗病毒疗法与严重毒性和不断增加的耐药率有关。这项建议的目标是了解触发潜伏病毒重新激活的分子途径，并开发防止潜伏病毒重新激活的新策略。

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.