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

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)在扰乱人类免疫以确定慢性感染方面是显著的；它是一个全球性的健康问题，治疗选择有限，而且没有可用的疫苗。目前尚不清楚病毒通过克服宿主固有的获得性免疫而在大多数受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丙型肝炎病毒-宿主相互作用的了解不完全。我们先前已经证明，慢性丙型肝炎病毒感染通过上调负免疫调节因子，包括程序性死亡-1(PD-1)、细胞因子信号转导抑制因子-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)而导致免疫失调。虽然TIM-3已被证明在慢性病毒感染时T细胞衰竭中起关键作用，但其在丙型肝炎病毒持续存在和抗病毒反应中对先天免疫细胞的表达和功能仍不清楚。我们最近发现TIM-3在Toll样受体(TLR)介导的天然免疫应答的负性调节中起关键作用，上调单核/巨噬细胞(M/M？)分离自慢性丙型肝炎病毒感染者和健康的M/M？与表达丙型肝炎病毒的肝细胞共培养。重要的是，阻断TIM-3信号可以恢复IL-12的表达，IL-12是一种关键的促炎细胞因子，与先天适应性免疫反应联系在一起。这一新的观察结果表明，在慢性丙型肝炎病毒感染的宿主中，无法清除病毒可能是TIM-3介导的天然免疫功能受损，继而是适应性免疫反应的功能障碍。因此，我们推测，丙型肝炎病毒介导的TIM-3上调M/M？？在先天性免疫和IL-12失调中起核心作用，阻断M/M？？的TIM-3信号可能挽救受损的抗病毒免疫反应。为了验证这一假说，我们将实现以下具体目标：1)确定TIM-3上调对M/M的作用？通过检测TIM-3的表达和M/M？？功能，特别是IL-12的产生，与那些自然缓解的丙型肝炎病毒感染患者或健康受试者进行比较。2)利用表达丙型肝炎病毒的肝细胞模型系统，研究特异的丙型肝炎病毒抗原对TIM-3转录、翻译、生物合成和降解的调节，确定TIM-3上调M/M的机制。3)确定TIM-3信号在M/M中对宿主抗病毒反应的影响，包括树突状细胞(DC)IL-12的表达、病毒特异性的CD4+和CD8+T淋巴细胞反应、肝细胞干扰素信号转导和丙型肝炎病毒复制。这项提案的总体目标是采用翻译的方法来获得关于丙型肝炎病毒介导的TIM-3如何上调M/M的统一概述？改变IL-12的表达和宿主对丙型肝炎病毒感染的天然适应性免疫反应，从而制定有效的策略来对抗这种常见的病毒疾病。

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.