VitD3 调控自体DNA免疫复合物诱导的DC活化对SLE自身免疫平衡的影响及机制研究

It has been confirmed that Vitamin D deficiency was associated with susceptibility to systemic lupus erythematosus(SLE), but its mechanisms are still unclear. Dendritic cells in SLE patients are continously stimulated by plenty of autoantibody-DNA immune complexes(IC)through NFκB signal,which is the key point in SLE immune imbalance.Vitamin D can induce DC tolerance by inihibiting the gene expression of NFκB family member RelB which is a critical regulator in DC maturation and differentiation.But it is unknown whether Vitamin D could inhibit DNA-IC stimulated DC activation by blocking RelB gene expression. In this study, DNA-IC and Vitamin D are used to stimulate peripheral blood DCs and are subcutaneously injected into Skin Explant System. The maturation of DC, RelB gene expression and the acetylation level of RelB gene VDRE area are detected. At the mean time, HDAC3 gene silence is used to observe the effects of chromatin remodeling on VitD regulated DC activity. Further, the DCs pretreated by DNA-IC and VitD are cocultured with T lymphocytes in order to observe the influences and mechanisms of VitD modulating DNA-IC stimulated DC activation on SLE Treg/Th17 immune balance. Finally, the lupus mouse model is used to verify the immune regulation effects of VitD in vivo. This study is proposed to establish the theoretical basis of VitD and auto DNA-IC induced SLE immune tolerance.

研究证实VD缺乏与SLE易感性密切相关，但其机制尚不完全明确。SLE中大量自体DNA免疫复合物(IC)通过NFκB信号持续激活DC是SLE免疫失衡的中心环节。VD可抑制NFκB家族中决定DC活化的关键分子RelB的表达而诱导DC耐受，但其可否通过抑制RelB的表达从而阻断DNA-IC刺激的DC活化尚不明确。本研究拟使用VD及自体DNA-IC刺激外周血DC，并将之皮下注射体外皮肤移植系统,观察DC成熟与活化表型、RelB基因表达及启动子VDRE区组蛋白乙酰化水平，并使用HDAC3基因沉默判断染色质重塑对VD调节DC免疫活性的影响。此后进一步将上述处理的DC与T细胞共培养，观察VD调控自体DNA-IC负载的DC对 SLE Treg/Th17免疫平衡的影响及其分子机制，最后使用狼疮鼠模型在体验证VD的调节作用，为今后探索使用VD协同自体DNA-IC诱导SLE免疫耐受奠定理论基础。

研究证实VD缺乏与SLE易感性密切相关，但其机制尚不完全明确。本研究结果首次证实了自体DNA-ICs 可以刺激外周血单核细胞来源及正常皮肤中DC细胞活化，诱导Th17、抑制Treg细胞生成、促进TNF-α炎性因子分泌，从而导致SLE自身免疫反应向炎症反应方向极化；并发现DNA-ICs皮下注射正常人皮肤组织后，可刺激CD14+DC细胞吞噬DNA-ICs并明显迁移出皮肤，而VD3 可进一步促进CD14+DC 的迁移；VD3 可通过降低RelB基因启动子区组蛋白乙酰化水平，从而抑制自体DNA-ICs对DC细胞的活化作用。同时可抑制DNA-IC对外周血及皮肤中DC的炎症刺激效益，促进共培养体系中Treg产生、IL-10抗炎因子分泌，抑制Th17细胞生成及TNF-α 炎性因子分泌，使SLE中Treg/Th17 免疫失衡向免疫抑制方向发展。动物实验证实DNA-IC也可在体外刺激NZB/NZW F1狼疮鼠DC细胞活化，上调CD83,CD86表达及TNF-α分泌，VD3 可抑制DNA-IC刺激的小鼠DC 细胞活化。不同刺激物预处理DC尾静脉回输BW F1 狼疮鼠后，在体实验证实DNA-IC预处理组DC回输后可增加小鼠脾脏单个核细胞增殖，Treg/Th17细胞比率下调，同时上调狼疮鼠血清中IL-10、TGFß及anti-dsDNA浓度，促进小鼠狼疮性肾炎加重，使疾病活动度增加；VD3可降低小鼠脾脏单个核细胞增殖，上调Treg/Th17细胞比率，同时降低狼疮鼠血清中IL-10.TGF-ß及anti-dsDNA浓度，改善小鼠狼疮性肾炎，缓解疾病进程；同时VD3可缓解DNA-ICs 导致的狼疮疾病活动。通过体外及人体标本实验证实VD3调控自体DNA-IC诱导的DCs活化对 SLE自身免疫平衡的影响，并阐明了可能的分子机制，为今后探索SLE的免疫治疗奠定理论基础并开辟新的思路。

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