Characterizing Inflammation And Downstream Effects During Chronic Viral Infection

描述（由申请人提供）：在人类和小鼠的慢性病毒感染期间，多种抑制分子创造了一个深刻的免疫抑制环境，这使得在这种情况下激发有效的免疫成为一项挑战。与健康高度相关的人类持续性病毒，包括艾滋病毒、丙型肝炎病毒和乙型肝炎病毒，仅限于人类和非人类灵长类动物，这给基于实验的研究带来了很大的限制和困难。免疫系统的复杂性不能在体外精确地重现，其研究需要使用适当的整个生物体。因此，我们选择使用慢性淋巴细胞性脉络丛脑膜炎病毒（LCMV）感染其自然宿主小鼠作为模型系统。我们发现白细胞介素6 （IL-6）在持续的LCMV感染中以一种独特的双相方式产生，晚期IL-6对病毒控制至关重要。潜在的机制涉及晚期慢性感染期间病毒特异性CD4 T细胞中IL-6转录因子BCL6的上调。这导致T滤泡辅助细胞、生殖中心和抗体反应的增加。值得注意的是，在急性LCMV感染期间没有检测到上述il - 6的作用。IL6与IL6家族细胞因子共享转导共受体gp130，其中有四种与免疫系统相关。慢性LCMV感染时，T细胞中gp130的消融比单纯缺乏il -6导致更严重的缺陷，这一点可以通过病毒特异性CD4 T细胞数量减少及其IL-21分泌（以及BCL-6表达减少）来证明。这些数据表明gp130信号细胞因子，包括但不限于IL6，在协调CD4 T细胞反应和解决体内持久性LCMV感染中发挥核心作用。我们建议研究gp130信号细胞因子在慢性LCMV感染过程中的作用及其与CD4 T细胞应答的机制联系。在Aim #1中，我们将使用抗il6r阻断抗体和具有切除或双侧IL6受体基因的小鼠来检查IL6及其直接靶细胞的时间相关性。我们还将使用过继细胞转移来辨别IL6如何驱动病毒清除。目的2将研究gp130flox/flox小鼠，以研究gp130信号在慢性LCMV感染期间不同时间和特定细胞群中的作用。我们将通过测量混合骨髓嵌合体的存活和增殖来确定gp130控制CD4 T细胞数量的机制。我们还将探索CD4 T细胞对gp130细胞因子（除IL6外）的水平和反应性，我们将使用shRNA和/或转基因小鼠来研究它们在慢性LCMV感染期间的功能。最后，在Aim #3中，我们将探索几种利用gp130信号通路来预防/减弱病毒持久性的方法。这包括单独使用pan gp130激动剂或选择的gp130细胞因子或联合阻断抑制途径。在过去的三十年中，使用LCMV小鼠感染的研究表明，小鼠和人类对持久性病毒的免疫反应具有高度保守性。因此，我相信从拟议的实验中获得的知识不仅将增强我们对基础免疫学的理解，而且还将指出在人类慢性病毒感染期间可以调节免疫反应和代表治疗靶点的重要参与者。此外，由于LCMV被认为是一种原型沙粒病毒，拟议的研究将增加我们对人类沙粒病毒发病机制的理解，沙粒病毒可引起致命的出血热。

DESCRIPTION (provided by applicant): Multiple inhibitory molecules create a profoundly immunuosuppressive environment that is conserved during chronic viral infections in humans and mice, making eliciting effective immunity in this context a challenge. Human persistent viruses highly relevant to health, including HIV, HCV and HBV are restricted to human and non-human primates, which poses great limitations and difficulties to experimental based research. The complexity of the immune system cannot be accurately recreated in vitro and its study requires the use of appropriate whole organisms. We have therefore chosen to use chronic lymphocytic choriomeningitis virus (LCMV) infection of its natural host, the mouse, as a model system. We found that interleukin 6 (IL-6) is produced in a unique biphasic manner during persistent LCMV infection, with late IL-6 being essential for viral control. The underlying mechanism involved IL-6 upregulation of the transcription factor BCL6 in virus-specific CD4 T cells during late chronic infection. This resulted in escalation of T follicular helper cell, germial center and antibody responses. Remarkably, none of the aforementioned IL6 effects could be detected during acute LCMV infection. IL6 shares the transducing co-receptor gp130 with IL6 family of cytokines of which four have been related to the immune system. Ablation of gp130 in T cells during chronic LCMV infection resulted in more profound defects than the sole absence of IL6, as indicated by reduction of virus-specific CD4 T cell numbers and their IL-21 secretion (in addition) to diminished BCL-6 expression. These data indicate that gp130 signaling cytokines, including but not limited to IL6, play a central role in orchestrating CD4 T cells responses and resolving persistent LCMV infection in vivo. We propose to investigate the role of gp130 signaling cytokines and their mechanistic link to CD4 T cell responses during chronic LCMV infection. In Aim #1 we will use anti-IL6R blocking Abs and mice with ablated or loxp-flanked IL6 receptor gene to examine the temporal relevance of IL6 and its direct target cells. We will also use adoptive cell transfer to discern how IL6 drives viral clearance. Aim #2 will study gp130flox/flox mice to investigate the role of gp130 signaling at different times and in specific cell populations during chronic LCMV infection. We will determine the mechanism underlying gp130 control of CD4 T cell numbers by measuring survival and proliferation in mixed bone marrow chimeras. We will also explore the levels and CD4 T cell responsiveness to gp130-cytokines (other than IL6) and we will use shRNA and/or genetically modified mice to investigate their function during chronic LCMV infection. Finally, in Aim #3 we will explore several approaches to harness the gp130-signaling pathway to prevent/attenuate viral persistence. This includes treatment with pan gp130-agonist or selected gp130-cytokines alone or in combination with blockade of inhibitory pathways. Studies in the past three decades using LCMV murine infection have demonstrated high conservation in the immune responses against persistent viruses in mouse and humans. Therefore I am confident that the knowledge gained from the proposed experiments not only will enhanced our understanding of basic immunology but will also point out important players that could regulate immune responses and represent therapeutic targets during chronic viral infections in humans. Furthermore, since LCMV is considered a prototypic arenavirus the proposed studies will increase our understanding of the pathogenesis of human arenaviruses, which cause fatal hemorrhagic fevers.