Tim-3-mediated IL-12 dysregulation in antiviral responses to HCV infection

描述（由申请人提供）：丙型肝炎病毒（HCV）在破坏人体免疫力以建立慢性感染方面表现突出;它是一个全球性健康问题，治疗选择有限，并且没有可用的疫苗。目前尚不清楚病毒通过克服宿主先天性免疫而在大部分感染肝脏中持续存在的机制， 部分原因是我们对导致这种免疫破坏的HCV-宿主相互作用的不完全理解。我们先前已经证明慢性HCV感染导致通过负性免疫调节剂的上调介导的免疫失调，所述负性免疫调节剂包括程序性死亡-1（PD-1）、细胞因子信号传导抑制剂-1（SOCS-1）以及最近的T细胞免疫球蛋白和粘蛋白结构域蛋白-3（Tim-3）。虽然Tim-3已被证明在慢性病毒感染期间的T细胞耗竭中起关键作用，但其在HCV持久性和抗病毒应答中在先天免疫细胞上的表达和功能仍然未知。我们最近发现Tim-3在Toll样受体（TLR）介导的先天性免疫应答的负调节中起关键作用，在单核细胞/巨噬细胞（M/M？）分离自慢性HCV感染者和健康的M/M？？与表达HCV的肝细胞共培养。重要的是，阻断Tim-3信号传导恢复了IL-12的表达，IL-12是一种关键的促炎细胞因子，将先天免疫应答与适应性免疫应答联系起来。这一新的观察结果表明，在慢性HCV感染的宿主中无法清除病毒可能是Tim-3介导的先天免疫损伤的功能，随后是适应性免疫应答功能障碍。因此，我们推测，HCV介导的Tim-3上调M/M？？在先天性免疫和IL-12失调中起着重要作用，因此在M/M？？可以挽救受损的抗病毒免疫反应。为了验证这一假设，我们将进行以下具体目标：1）定义的作用，蒂姆-3上调M/M？从HCV感染患者的抗病毒治疗与明确的结果，与那些自然解决的HCV感染或健康受试者相比，通过检查两个Tim-3表达和M/M？功能，特别是IL-12的产生。2)确定Tim-3在M/M？上上调的机制。使用表达HCV的肝细胞模型系统，关注特异性HCV抗原介导的Tim-3转录、翻译、生物合成和降解的调节。3)确定Tim-3信号传导在M/M？？对宿主抗病毒应答的影响，包括树突状细胞（DC）IL-12表达、病毒特异性CD 4+和CD 8 + T淋巴细胞应答、肝细胞干扰素（IFN）信号传导和HCV复制。这项建议的总体目标是采用翻译的方法，以获得一个统一的概述如何HCV介导的蒂姆-3上调M/M？改变IL-12的表达和宿主对HCV感染的先天性适应性免疫应答，从而开发有效的策略来对抗这种常见的病毒性疾病。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection; it is a global health problem with limited treatment options and no available vaccine. The mechanisms by which the virus persists in the majority of infected liver by overcoming the host innate to adaptive immunity is currently unclear, partly due to our incomplete understanding of HCV-host interactions that lead to this immune disruption. We have previously demonstrated that chronic HCV infection leads to immunodysregulation mediated through up- regulation of negative immunomodulators, including programmed death-1 (PD-1), suppressor of cytokine signaling-1 (SOCS-1), and most recently, T cell immunoglobulin and mucin domain protein-3 (Tim-3). While Tim-3 has been shown to play a critical role in T cell exhaustion during chronic viral infections, its expression and function on innate immune cells in HCV persistence and antiviral responses remain unknown. We have recently discovered that Tim-3 plays a pivotal role in negative regulation of Toll-like receptor (TLR)-mediated innate immune responses, being up-regulated on monocytes/macrophages (M/M?) isolated from chronically HCV-infected individuals and healthy M/M??co-cultured with HCV-expressing hepatocytes. Importantly, blocking Tim-3 signaling restores the expression of IL-12, a key pro-inflammatory cytokine linking innate to adaptive immune responses. This novel observation suggests that the inability to clear virus in chronically HCV-infected hosts may be a function of a Tim-3-mediated impairment of innate immunity, with subsequent dysfunction of adaptive immune responses. We thus hypothesize that HCV-mediated Tim-3 up-regulation on M/M??plays a central role in innate immune and IL-12 dysregulation, such that blockade of Tim-3 signaling in M/M??may rescue impaired antiviral immune responses. To test this hypothesis, we will carry out the following specific aims: 1) Define the role of Tim-3 up-regulation on M/M? from HCV-infected patients following antiviral therapy with defined outcomes, comparing with those naturally resolved HCV infection or healthy subjects, by examining both Tim-3 expression and M/M??functions, and in particular, IL-12 production. 2) Determine the mechanisms by which Tim-3 is up-regulated on M/M??using an HCV-expressing hepatocyte model system, focusing on specific HCV antigen-mediated regulation of Tim-3 transcription, translation, biosynthesis and degradation. 3) Determine the effects of Tim-3 signaling in M/M??on host antiviral responses, including dendritic cell (DC) IL-12 expression, virus-specific CD4+ and CD8+ T lymphocyte responses, hepatocyte interferon (IFN) signaling and HCV replication. The overall goal of this proposal is to employ a translational approach to obtain a unified overview of how HCV-mediated Tim-3 up-regulation on M/M? alters IL-12 expression and host innate to adaptive immune responses to HCV infection, so as to develop effective strategies to combat this common viral disease.