Table_1_Vitamin D3 Attenuates Viral-Induced Inflammation and Fibrotic Responses in Bronchial Smooth Muscle Cells.pdf

Toll-like receptor 3 (TLR3) activation by viral infections plays a key role in promoting inflammatory immune responses that contribute to pulmonary fibrosis in chronic inflammatory respiratory diseases. Vitamin D3 has been shown to be beneficial to patients with asthma and chronic obstructive pulmonary disease (COPD) through its anti-inflammatory and anti-fibrotic properties. Smooth muscle cells are one of the major contributors to airway remodeling in asthma and COPD. We therefore aimed to investigate the effect of vitamin D3 treatment on viral-induced TLR3 responses in Bronchial Smooth Muscle Cells (BSMCs) as a mechanism contributing to pulmonary fibrosis in asthma and COPD. Primary BSMCs from patients with asthma (n=4), COPD (n=4), and healthy control subjects (n=6) were treated with polyinosinic: polycytidylic acid (polyI:C), TLR3 agonist in the presence or absence of vitamin D3 (1,25D3). Here we report the mRNA expression and protein levels of pro-inflammatory and pro-fibrotic markers (IL-6, IFN-β1, CCL2/MCP-1, fibronectin 1 and type I collagen) among BSMCs groups: asthma, COPD, and healthy controls. We show that at the baseline, prior to polyI:C stimulation, asthma and COPD BSMCs presented increased pro-inflammatory and pro-fibrotic state compared to healthy control subjects, as measured by quantitative PCR and immunoassays (ELISA/Flow Cytometry. Ligation of TLR3 by polyI:C in BSMCs was associated with increased TLR3 mRNA expression, and 1,25D3 treatment significantly reduced its expression. In addition, 1,25D3 decreased the expression of IL-6, IFN-β1, CCL2, FN1 and COL1A1 induced by polyI:C in BSMCs. The regulatory effect of 1,25D3 treatment on polyI:C-stimulated BSMCs was further confirmed at protein levels. Our findings suggest that vitamin D3 attenuates TLR3 agonist-induced inflammatory and fibrotic responses in BSMCs and support the clinical relevance of vitamin D3 supplementation in patients with viral infections having chronic respiratory diseases, such as asthma and COPD.

病毒感染激活的Toll样受体3（Toll-like receptor 3，TLR3）在促炎免疫应答中发挥关键作用，此类应答可加剧慢性炎症性呼吸系统疾病中的肺纤维化进程。既往研究证实，维生素D3可通过抗炎与抗纤维化特性，使哮喘与慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）患者获益。平滑肌细胞是哮喘与COPD患者气道重塑的主要贡献者之一，因此本研究旨在探讨维生素D3干预对病毒诱导的支气管平滑肌细胞（Bronchial Smooth Muscle Cells，BSMCs）中TLR3应答的调控效应，以此阐明哮喘与COPD患者肺纤维化发生的潜在机制。本研究招募哮喘患者（n=4）、COPD患者（n=4）及健康对照受试者（n=6），分离其原代BSMCs并分组干预：在存在或不存在1,25D3的条件下，使用聚肌苷酸-聚胞苷酸（polyinosinic:polycytidylic acid，polyI:C，TLR3激动剂）对细胞进行刺激处理。本研究检测了哮喘组、COPD组及健康对照组BSMCs中促炎与促纤维化标志物的mRNA表达水平与蛋白含量，涉及的标志物包括白细胞介素6（interleukin 6，IL-6）、干扰素β1（interferon β1，IFN-β1）、趋化因子配体2/单核细胞趋化蛋白1（chemokine ligand 2/monocyte chemoattractant protein 1，CCL2/MCP-1）、纤连蛋白1（fibronectin 1，FN1）及I型胶原（type I collagen）。结果显示，在聚肌苷酸-聚胞苷酸刺激前的基线状态下，相较于健康对照组，哮喘组与COPD组的BSMCs已呈现出显著升高的促炎与促纤维化表型，该结论通过定量聚合酶链反应（quantitative PCR）与免疫检测方法（酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）/流式细胞术（Flow Cytometry））得以验证。BSMCs经聚肌苷酸-聚胞苷酸结合TLR3后，可上调TLR3的mRNA表达水平，而1,25D3干预可显著降低该表达水平。此外，1,25D3可抑制聚肌苷酸-聚胞苷酸诱导的BSMCs中IL-6、IFN-β1、CCL2、FN1及I型胶原α1链（collagen type I alpha 1 chain，COL1A1）的表达。1,25D3对聚肌苷酸-聚胞苷酸刺激的BSMCs的调控效应进一步在蛋白水平得到证实。本研究结果表明，维生素D3可减弱BSMCs中TLR3激动剂诱导的炎症与纤维化应答，支持对合并病毒感染的慢性呼吸系统疾病（如哮喘与COPD）患者补充维生素D3的临床应用价值。