Objectives

Primary Sjögren’s syndrome (pSS) is a complex systemic autoimmune disease whose clinical spectrum extends from sicca syndrome to systemic involvement. T helper 17 cells (Th17) and CD4⁻CD8⁻ (double negative, DN) T cells are actively involved in the pathogenesis of pSS. Melatonin shows important immunoregulatory functions in multiple T cell-mediated autoimmune diseases. However, the effects of melatonin on the immune cells of pSS patients are unclear. Hence, this study was aimed to evaluate the effects of melatonin on the immune responses of peripheral pathogenic Th17 and DN T cells from pSS patients, and explore the underlying receptor-related mechanism.

Methods

The concentration of serum and saliva melatonin of pSS patients and healthy controls (HCs) were detected using Enzyme-linked immunosorbent assays (ELISA). Expression of arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT) were conducted in labial glands samples by immunohistochemistry. The mechanism underlying the effects of melatonin on Th17 and DN T cells responses in peripheral blood from pSS was investigated by quantitative real-time polymerase chain reaction (RT-PCR), flow cytometry, ELISA, cell viability, and proliferation assays.

Results

Serum and saliva melatonin levels were lower in pSS patients than in HCs, which were negatively correlated with disease activity. The expression levels of melatonin's biosynthetic enzymes (AANAT, HIOMT) and nuclear receptors (RORα, RORγ) were significantly increased in peripheral blood mononuclear cells (PBMCs) from pSS patients. Furthermore, in vitro melatonin administration decreased the expression of melatonin effector/receptor system in peripheral blood of pSS patients. More importantly, Melatonin inhibited pathogenic responses of peripheral Th17 and DN T cells in PBMCs from pSS, which was independent of melatonin membrane receptors. However, melatonin nuclear receptor antagonist SR1001 enhanced the inhibitory ability of melatonin on Th17 and DN T cells production, and agonist SR1078 weakened the effects of melatonin. Additionally, overexpression of the melatonin effector/receptor system in pSS patients appeared to be involved in the disease, due to that melatonin effector/receptor system expression was correlated with the frequency of Th17 or DN T cells.

Conclusion

Melatonin relieved the inflammatory responses of Th17 and DN T cells in PBMCs from pSS patients in a nuclear receptors-dependent manner, suggesting that melatonin might be beneficial to pSS.

中文翻译：

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褪黑激素通过依赖核受体的方式缓解原发性干燥综合征外周血中 Th17/CD4-CD8-T 细胞的炎症反应

目标

原发性干燥综合征 (pSS) 是一种复杂的全身性自身免疫性疾病，其临床范围从干燥综合征延伸到全身受累。T辅助17细胞（Th17）和CD4 ^{- CD8-}（双阴性，DN）T细胞积极参与pSS的发病机制。褪黑激素在多种 T 细胞介导的自身免疫性疾病中显示出重要的免疫调节功能。然而，褪黑激素对 pSS 患者免疫细胞的影响尚不清楚。因此，本研究旨在评估褪黑激素对 pSS 患者外周致病性 Th17 和 DN T 细胞免疫反应的影响，并探索潜在的受体相关机制。

方法

使用酶联免疫吸附试验 (ELISA) 检测 pSS 患者和健康对照 (HC) 的血清和唾液褪黑激素浓度。通过免疫组织化学在唇腺样品中进行芳烷基胺 N-乙酰转移酶 (AANAT) 和羟基吲哚 O-甲基转移酶 (HIOMT) 的表达。通过定量实时聚合酶链反应 (RT-PCR)、流式细胞术、ELISA、细胞活力和增殖测定研究了褪黑激素对 pSS 外周血中 Th17 和 DN T 细胞反应的影响的机制。

结果

pSS 患者的血清和唾液褪黑激素水平低于 HCs，这与疾病活动度呈负相关。pSS 患者外周血单个核细胞 (PBMC) 中褪黑激素的生物合成酶 (AANAT, HIOMT) 和核受体 (RORα, RORγ) 的表达水平显着增加。此外，体外褪黑激素给药降低了 pSS 患者外周血中褪黑激素效应/受体系统的表达。更重要的是，褪黑激素抑制来自 pSS 的 PBMC 中外周 Th17 和 DN T 细胞的致病反应，这与褪黑激素膜受体无关。然而，褪黑激素核受体拮抗剂SR1001增强了褪黑激素对Th17和DN T细胞产生的抑制能力，而激动剂SR1078减弱了褪黑激素的作用。此外，pSS 患者中褪黑激素效应/受体系统的过度表达似乎与疾病有关，因为褪黑激素效应/受体系统的表达与 Th17 或 DN T 细胞的频率相关。

结论

褪黑激素以核受体依赖性方式缓解 pSS 患者 PBMC 中 Th17 和 DN T 细胞的炎症反应， 表明褪黑激素可能对 pSS 有益。