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

描述（由申请方提供）：在实体器官和骨髓移植受者中经常观察到潜伏性巨细胞病毒（CMV）的再活化，这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疗法与狭窄的治疗范围相关，导致显著的毒性，包括骨髓抑制，特别是在肾功能不全患者中。此外，对这些药物的耐药性增加，需要使用有效但毒性更大的药物。人们普遍认为，立即早期（IE）基因在潜伏感染中是转录沉默的，IE基因表达的再激活是病毒再激活的关键第一步。用防止IE基因表达再活化的试剂治疗将是一种新的方法，其将避免与试图对抗病毒复制相关的问题。IE基因表达再激活的机制尚不清楚。由于缺乏研究体内HCMV感染的动物模型，我们和其他人使用MCMV作为模型来研究CMV潜伏期和再激活。我们之前已经利用小鼠肾移植模型来诱导体内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同种异体肾移植中上调。我们假设，MCMV立即早期（IE）基因表达的转录再激活响应移植是由先天性免疫反应通过Toll样受体与缺血性损伤损伤的细胞配体的相互作用启动的，这是进一步放大的识别MHC不相容的细胞。我们假设这导致炎性细胞因子如TNF的表达增加，这些细胞因子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基因表达的重新激活。我们的初步数据表明，TLR2在激活对同种异体移植物的细胞免疫应答中起重要作用，因此，对MCMV的再激活起重要作用。通过使用TLR2缺陷小鼠和使用siRNA技术阻断体内表达，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基因表达的再激活中的需求。在移植前用靶向启动再活化所需步骤的试剂如siRNA处理供体器官将是一种新的方法，其将显著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，以防止再激活CMV及其后遗症的发展。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。目前可用的靶向病毒复制的抗病毒疗法与严重毒性和耐药率增加相关。该提案的目标是了解触发潜伏病毒再激活的分子途径，并开发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.