Pituitary Adenylate Cyclase Activating Peptide Deficient Mice Exhibit Impaired Thymic and Extrathymic Regulatory T Cell Proliferation during EAE

We have shown that mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) manifest enhanced sensitivity to experimental autoimmune encephalomyelitis (EAE), supporting the anti-inflammatory actions described for this neuropeptide. In addition to an increased proinflammatory cytokine response in these mice, a reduction in regulatory T cell (Treg) abundance in the lymph nodes (LN) was observed, suggesting altered Treg kinetics. In the present study, we compared in PACAP deficient (KO) vs. wild type mice the abundances and rates of proliferation FoxP3+ Tregs in three sites, the LN, central nervous system (CNS) and thymus and the relative proportions of Th1, Th2, and Th17 effector subsets in the LN and CNS. Flow cytometry analyses revealed a decrease in Treg proliferation and an increased T effector/Tregs ratio in the LN and CNS of PACAP KO mice. In the thymus, the primary site of do novo natural Treg production, the total numbers and proliferative rates of FoxP3+ Tregs were significantly reduced. Moreover, the expression of IL-7, a cytokine implicated in thymic Treg expansion during EAE, failed to increase at the peak of the disease in the thymus and LN of PACAP KO mice. In addition to these Treg alterations, a specific reduction of Th2 cells (about 4-fold) was observed in the lymph nodes in PACAP KO mice, with no effects on Th1 and Th17 subsets, whereas in the CNS, Th1 and Th17 cells were increased and Th2 decreased. Our results suggest that endogenous production of the neuropeptide PACAP protects against EAE by modulating Treg expansion and Th subsets at multiple sites.本研究团队既往研究证实，垂体腺苷酸环化酶激活肽（pituitary adenylate cyclase-activating polypeptide, PACAP，基因名ADCYAP1）缺陷小鼠对实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）的易感性显著增强，这一结果支持了该神经肽的抗炎生物学功能。相较于野生型小鼠，PACAP缺陷小鼠体内促炎细胞因子应答增强，同时其淋巴结（lymph nodes, LN）中调节性T细胞（regulatory T cell, Treg）丰度降低，提示Treg的增殖或存活动力学发生了改变。 本研究中，我们对比了PACAP缺陷型（KO）与野生型小鼠在三处免疫相关位点——淋巴结、中枢神经系统（central nervous system, CNS）及胸腺——中FoxP3+ Treg的丰度与增殖速率，并分析了淋巴结与中枢神经系统内Th1、Th2、Th17效应性T细胞亚群的相对比例。流式细胞术（flow cytometry）分析结果显示，PACAP缺陷型小鼠的淋巴结与中枢神经系统内，Treg的增殖能力显著下降，且效应性T细胞与Treg的比值升高。 在天然Treg生成的核心场所胸腺中，FoxP3+ Treg的总数量与增殖速率均出现显著降低。此外，在EAE疾病峰值期，PACAP缺陷型小鼠的胸腺与淋巴结内，IL-7（一种在EAE进程中参与胸腺Treg扩增的细胞因子）的表达未出现预期的上调。 除上述Treg相关的异常改变外，PACAP缺陷型小鼠的淋巴结中还观察到Th2细胞出现特异性减少（降幅约4倍），而Th1与Th17细胞亚群未受明显影响；但在中枢神经系统内，Th1与Th17细胞的数量显著增加，Th2细胞则出现减少。 本研究结果表明，内源性神经肽PACAP可通过调控多部位的Treg扩增与Th细胞亚群分化，从而对实验性自身免疫性脑脊髓炎发挥保护作用。