慢性昼夜循环干扰诱导的白介素-6跨越式信号传导在Th17/Tr1细胞平衡调节的机制研究

Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. Modern life often involves chronic circadian disruptions, such as night shift work or jet lag, that are linked to human inflammatory diseases, such as multiple sclerosis (MS). MS is an immune-mediated disease of the CNS that is thought to result from the destruction of myelin by autoreactive T cells. CD4+ T cells characterized by the production of IFN-γ (Th1 cells) or IL-17 (Th17 cells) are considered important contributors to MS immunopathogenesis. FoxP3+ regulatory T cells (Tregs) and IL-10-secreting type 1 regulatory T cells (Tr1) regulate the activity of effector T cells, accordingly, deficits in Tregs and Tr1 cells have been described in MS. Thus, the balance between effector and regulatory T cells controls MS disease activity. . Many studies suggest that IL-6 is a putative 'sleep factor' and its circadian secretion correlates with sleep/sleepiness. Sleep also strongly enhanced IL-6 trans-signalling and concentrations of sIL-6R, exceeding wake levels by 70% at the end of sleep. Enhanced sIL-6R concentrations around the time of morning awakening agrees well with clinical observations of exacerbated symptoms in diseases during this period. Interestingly, sIL-6R shares 60% identity with the IL-12p40 sub¬unit. One clock gene NFIL3 (nuclear factor interleukin-3) is a key suppressor of IL-12 p40 gene expression in macrophages, therefore NFIL3 probably regulates the expression of sIL-6R and IL-6 trans-signalling. The circadian clock also suppresses Th17 development during nighttime through a similar NFIL3-dependent mechanism. REV-ERBα has been shown to drive Th17 cell differentiation by repression of NFIL3 transcription, which in turn will allow RORγt to induce IL17 production. An IL-27/NFIL-3 signaling axis can be a key regulator of Tr1 responses via induction of Tim-3, IL-10 and T cell dysfunction. NFIL3-/- effector T cells exhibit reduced IL-10 production, and exhibit severe defect in controlling effector function thereby resulting in more severe CNS autoimmunity. To assess whether circadian disruption by chronic light-cycle perturbations altered Th17/Tr1 cell frequencies and IL-6 trans-signalling, and whether NFIL3 has a T cell–intrinsic role in TH17/Tr1 development in vitro and in vivo, we test this general hypothesis we intend to address the following specific questions:.1. Does IL-6 trans signaling induce imbalance of Th17/Tr1 cells differentiation?.2. Does the clock gene NFIL3 regulate the expression of IL-6R on T cells and IL-6 trans signaling?.3. Does light cycle perturbation increase susceptibility to EAE via IL-6R on T cells?. The proposed study will ultimately result in identifying that the clock gene NFIL3 suppresses TH17 cell development by directly binding and repressing the IL-6R promoter. IL-6 trans signaling links imbalance of Th17/Tr1 cells differentiation to the circadian clock network through NFIL3. Light-cycle disruption elevated increased susceptibility to EAE. The results of the proposed studies will provide insights that circadian clock represents an environmental factor for affecting the development of EAE through regulating balance of Th17/Tr1 cells differentiation.

昼夜生物节律是影响免疫状态重要的环境因素，白介素-6(IL-6)是一种与生物钟节律改变及多发性硬化(MS)发病均有密切联系的炎性细胞因子，但生物钟节律紊乱与IL-6跨越式信号传导对Th17/Tr1细胞平衡调节机制的影响及其在MS发病机制中的作用尚不清楚。本研究拟通过研究慢性昼夜循环干扰以证实生物钟调节紊乱通过生物钟基因NFIL3影响IL-6跨越式信号传导进而导致Th17/Tr1细胞分化失衡而影响实验性自身免疫性脑脊髓炎(EAE)的发展，以明确生物钟调节紊乱增加小鼠EAE易感性的作用机制，揭示社会行为因素对EAE发病的影响，为MS发病机制的研究提供新的方向。

机体的免疫状态受昼夜节律等环境因素影响，免疫细胞及其分泌的因子紊乱会破坏免疫稳态进而导致多种自身免疫性疾病的发生发展，包括多发性硬化（MS）、视神经脊髓炎谱系疾病（NMOSD）等。NFIL3是调节生物昼夜节律的核心基因之一，但其在神经免疫及炎性疾病中的作用未完全清楚。本项目主要探究了昼夜节律基因NFIL3及丰富环境对实验性自身免疫性脑脊髓炎（EAE）的影响，并对MS及NMOSD等神经免疫与炎性疾病进行了系列临床研究。研究结果显示，（1）昼夜节律基因NFIL3对Th17，CD11c+DC，CD8+CD103+T细胞等免疫细胞亚群的分化发育、功能表型及IL-6、IL-10、IL-6R等的分泌、表达产生影响；敲除NFIL3能减轻MOG35-55诱导的EAE小鼠、ALD-DNA-BMDC 诱导的狼疮小鼠及K/BxN 血清诱导的关节炎小鼠的病情。NFIL3敲除后：1）EAE小鼠中枢神经系统中浸润的Th17细胞下降，外周脾脏和淋巴结中CD11c+的表面活化分子CD80、CD86等表达下降。2）在狼疮小鼠中，脾脏中B细胞减少，血清中自身抗体IgG/IgG1分泌下降。3）在关节炎小鼠脾脏中，Th1及Th17均下降。（2）丰富环境能改善EAE的病情，而这一效应可能是通过抑制PPAR-γ通路，使中枢浸润的Th1、Th17细胞减少产生的。（3）与MS患者相比，急性期NMOSD患者昼夜24小时尿量、尿钠可能更高，且NMOSD女性更容易出现高血压、高体脂。血浆高同型半胱氨酸、高抗核抗体可能影响NMOSD发生发展及疾病活动。总而言之，本研究揭示了NFIL3基因及丰富环境对EAE产生的影响，进一步表明了环境因素对机体免疫稳态起到的重要作用，同时扩大了我们对多发性硬化及视神经脊髓炎谱系疾病等神经免疫及炎性疾病临床特征的认识，为更深入研究神经免疫及炎性疾病的发病机制、药物治疗靶点等提供了新的研究方向。