Data regarding: Melatonin impairs T cell priming, migration and reactivation in the CNS, and induces neuroprotection in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE), the pre-clinical animal model of human multiple sclerosis, is triggered by myelin-specific T effector cells that are primed in peripheral lymph nodes and migrate to the central nervous system (CNS) where they are reactivated and induce demyelination. While melatonin ameliorates EAE disease symptoms, modulates encephalitogenic and suppressive responses, and reduces demyelination, its mechanism of action is not clear. Our objective was to study the effects of melatonin on key processes in EAE: the peripheral priming of T cells, their migration to the CNS and their reactivation therein. In this way, the actions of melatonin on co-stimulatory/co-inhibitory and adhesion molecules, on chemokine ligands/receptors expressed by different immune and CNS cell populations, on the blood-brain barrier (BBB) and on brain-derived neurotrophic factor (BDNF) levels in the CNS were studied in EAE. EAE induction protocol and EAE score assessment, and protocols for isolating draining lymph node (dLN) and CNS mononuclear cells and assessing immune subsets cells can be found in Álvarez-Sánchez et al. Brain, Behavior, and Immunity. 2015. Briefly, EAE was induced by s.c. administration of 100 μg of MOG35-55 emulsified in CFA containing 50 μg of heat-killed Mycobacterium tuberculosis (H37Ra), followed by two i.p. doses of 400 ng of pertussis toxin on days 0 and 2. Melatonin (80 mg/kg in ethanol/saline 8%) or vehicle was administered i.p. daily from day 0 onwards. Clinical signs of EAE were assessed as follows: 0: no clinical signs; 1: limp tail; 2: impaired righting reflex; 3: partial hind limb paralysis; 4: complete hind limb paralysis; 5: complete hind limb paralysis + frontal limb paralysis; 6: dead or moribund. Mononuclear cells or RNA were isolated from dLNs and CNS at days 5, 10 and 15 post-induction (p.i.) and analyzed by flow cytometry (co-stimulatory/co-inhibitory molecules, adhesion molecules and chemokyne receptors on APCs and T cells) or real-time quantitative PCR (expression of chemokyne ligands and receptors, adhesion molecules, BDNF). The integrity of the BBB was assessed in spinal cords by the immunohistochemical detection of albumin in the capillaries and by Evans Blue extravasation. Melatonin regulated many of the key pathological steps in EAE by regulating the immune synapse as well as chemoattractant and adhesion molecules and suppressive andneuroprotective responses, both in the periphery and in the CNS.

实验性自身免疫性脑脊髓炎（EAE）是人类多发性硬化的临床前动物模型，其由髓鞘特异性效应T细胞触发——此类细胞在外周淋巴结中被致敏，并迁移至中枢神经系统（CNS），在该处再次被激活并诱导脱髓鞘病变。虽然褪黑素可改善EAE的疾病症状、调节致脑炎应答与抑制性应答并减轻脱髓鞘，但目前其确切作用机制仍不明确。本研究旨在探究褪黑素对EAE关键进程的影响：包括T细胞的外周致敏、向中枢神经系统的迁移以及在中枢神经系统内的再次激活。据此，本研究针对EAE模型中褪黑素对共刺激/共抑制分子、黏附分子的作用，对不同免疫细胞与中枢神经系统细胞群体所表达的趋化因子配体/受体的影响，对血脑屏障（BBB）的作用，以及对中枢神经系统内脑源性神经营养因子（BDNF）水平的影响展开了分析。

EAE造模方案、EAE评分评估方法，以及分离引流淋巴结（dLN）与中枢神经系统单个核细胞、鉴定免疫细胞亚群的实验方案可参考Álvarez-Sánchez等人发表于《Brain, Behavior, and Immunity》2015年的研究。简要而言，EAE造模采用皮下注射100 μg溶于完全弗氏佐剂（CFA）的髓鞘少突胶质细胞糖蛋白35-55肽（MOG₃₅₋₅₅），其中佐剂含50 μg热灭活结核分枝杆菌H37Ra菌株，随后于第0天和第2天两次腹腔注射400 ng百日咳毒素。自造模第0天起，每日腹腔注射褪黑素（80 mg/kg，溶于8%乙醇生理盐水溶液）或相应溶媒作为对照。

EAE临床症状评分标准如下：0分：无临床症状；1分：尾巴无力；2分：翻正反射障碍；3分：单侧后肢不完全瘫痪；4分：双侧后肢完全瘫痪；5分：双侧后肢瘫痪伴前肢瘫痪；6分：死亡或濒死状态。分别于造模后第5、10、15天，从引流淋巴结与中枢神经系统中分离单个核细胞或总RNA，通过流式细胞术检测抗原呈递细胞与T细胞表面的共刺激/共抑制分子、黏附分子及趋化因子受体，或通过实时定量PCR检测趋化因子配体/受体、黏附分子及BDNF的表达水平。通过免疫组化检测脊髓毛细血管内白蛋白表达，以及伊文思蓝渗出实验，评估血脑屏障的完整性。

本研究结果显示，褪黑素可通过调节免疫突触、趋化因子与黏附分子，以及外周与中枢神经系统内的抑制性应答和神经保护性应答，调控EAE的诸多关键病理进程。