Data regarding: Seasonal variations in macrophages/microglia underlie changes in the mouse model of multiple sclerosis severity

Multiple sclerosis (MS) patients show seasonal variations in relapses, lesion numbers and activity and in several cytokines important for the disease. These variations have been associated with environmental factors, as solar exposure, either during adult life or during perinatal period. Data regarding seasonal variations in the MS animal model, experimental autoimmune encephalomyelitis (EAE), are scarce, but suggest that these variations may be endogenous, since they were detected in animals maintained under a controlled environment. We aim to address the effect of both the season of birth and of EAE induction on EAE severity and on immune pathogenic (Th1, Th17 cells, macrophages/microglia) and protective (T regulatory cells) responses in the central nervous system (CNS). A total of 51 independent experiments in which EAE was induced in C57BL/6 mice maintained under a 12:12 light:dark cycle and controlled conditions of temperature and humidity were retrospectively analyzed. EAE induction protocol and EAE score assessment, and protocols for isolating 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. 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. CNS (brain + spinal cord) isolated at the peak of disease from EAE mice perfused with cold PBS were digested with 1.25 mg/ml of collagenase IV and 0.5 mg/ml of DNase I for 35 minutes at 37°C, and mononuclear cells were isolated using a 30%:70% discontinuous Percoll gradient. CNS mononuclear cells were cultured at final density of 3x106 cells/ml (RPMI 1640 supplemented with 5% fetal bovine serum, 2 mM L-glutamine and 50 U/ml of penicillin/streptomycin). T effector cells (Th1 and Th17 responses) were studied by flow cytometry on CNS-infiltrating mononuclear cells cultured overnight with MOG 10 μM and incubated with brefeldin A for the last 5 hours of culture. Treg cells and macrophages/microglia were studied in ex-vivo CNS-infiltrating mononuclear cells. Our data show that summer-born or summer-immunized animals developed a milder disease, which coincided with variations in numbers of T effector/Treg subsets, and significantly low count of macrophages/microglia. Moreover, both the season of birth and the season in which EAE was induced were independently associated with EAE susceptibility. These data suggest that endogenous rhythms in immune responses might cause seasonal variations in EAE severity and that they might be related to macrophages/microglia.多发性硬化（Multiple sclerosis, MS）患者的复发、病灶数量与活性，以及多种与疾病相关的重要细胞因子均存在季节变异。此类变异与环境因素相关，例如成年期或围产期的日光暴露。目前关于多发性硬化动物模型——实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE）的季节变异数据较为匮乏，但已有研究提示此类变异可能为内源性的：在可控环境下饲养的动物中也检测到了该现象。 本研究旨在探讨出生季节与EAE诱导季节分别对EAE严重程度，以及中枢神经系统（Central Nervous System, CNS）内免疫致病应答（Th1、Th17细胞、巨噬细胞/小胶质细胞）与保护性应答（调节性T细胞，T regulatory cells, Treg）的影响。我们回顾性分析了共计51项独立实验，这些实验均在光照:黑暗为12:12的循环条件、温度与湿度可控的环境下饲养的C57BL/6小鼠中诱导EAE。EAE诱导方案、EAE评分评估方法，以及分离中枢神经系统单核细胞与检测免疫细胞亚群的实验流程，详见Álvarez-Sánchez等人发表于《Brain, Behavior, and Immunity》2015年的研究。简言之，EAE诱导方案为：皮下注射100 μg溶于完全弗氏佐剂（Complete Freund's Adjuvant, CFA）的MOG₃₅₋₅₅肽段，该佐剂中含有50 μg热灭活结核分枝杆菌（Mycobacterium tuberculosis, H37Ra），并于第0天与第2天腹腔注射两次400 ng百日咳毒素。EAE临床症状评分标准如下：0分：无临床症状；1分：尾巴松弛无力；2分：翻正反射受损；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。 于疾病峰值期处死EAE模型小鼠，经冷磷酸盐缓冲液（PBS）灌流后分离中枢神经系统（脑+脊髓），将组织于37℃下用1.25 mg/ml胶原酶IV与0.5 mg/ml DNase I消化35分钟，随后采用30%:70%不连续Percoll梯度离心法分离单核细胞。中枢神经系统单核细胞以终密度3×10⁶ cells/ml进行培养（培养基为添加5%胎牛血清、2 mM L-谷氨酰胺与50 U/ml青霉素/链霉素的RPMI 1640培养基）。对于T效应细胞（Th1与Th17应答），采用流式细胞术检测经10 μM MOG肽段体外过夜培养、并于培养最后5小时加入布雷菲尔德菌素A处理的中枢神经系统浸润单核细胞。对于调节性T细胞与巨噬细胞/小胶质细胞，则直接采用离体的中枢神经系统浸润单核细胞进行检测。 本研究数据显示，夏季出生或夏季诱导EAE的小鼠疾病症状更轻微，该现象与T效应细胞/调节性T细胞亚群数量的变化以及巨噬细胞/小胶质细胞数量显著降低相吻合。此外，出生季节与EAE诱导季节均分别与EAE易感性相关。上述数据提示，免疫应答的内源性节律可能导致EAE严重程度的季节变异，且该变异可能与巨噬细胞/小胶质细胞相关。