School of Respiratory Therapy, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei, 11031, Taiwan.
Respiratory Therapy, Division of Pulmonary Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.
Lung. 2024 Jun;202(3):343-356. doi: 10.1007/s00408-024-00701-6. Epub 2024 Apr 28.
Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-β. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-β-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs).
CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-β-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay.
An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-β-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-β-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-β-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-β-treated NHLFs. TP213 alleviated TGF-β-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-β-induced p65 and α-SMA expression in NHLFs.
These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.
重度哮喘的特征是炎症和气道重塑，涉及成纤维细胞分化为表达α-SMA 的肌成纤维细胞。这个过程导致纤维连接蛋白和结缔组织生长因子 (CTGF) 的产生，由转化生长因子 (TGF)-β 等因素驱动。此外，肌成纤维细胞的持续存在与抗细胞凋亡和线粒体功能障碍有关。趋化因子 (C-X3-C 基序) 配体 1 (CX3CL1) 在组织纤维化中发挥作用。然而，目前尚不清楚中和 CX3CL1 是否会减少正常人类肺成纤维细胞 (NHLFs) 中 TGF-β 诱导的成纤维细胞分化和线粒体功能障碍。
通过免疫荧光 (IF) 或卵清蛋白挑战小鼠的免疫组织化学 (IHC) 染色分析 CX3CL1/C-X3-C 基序趋化因子受体 1 (CX3CR1)。通过 ELISA 检测 CX3CL1 释放。通过 Western blot 或 IF 染色检测中和 CX3CL1（TP213）处理后 NHLFs 中 TGF-β 诱导的 CTGF、纤维连接蛋白和 α-SMA 表达。通过 JC-1 测定和 Seahorse 测定检测线粒体功能。通过末端尿嘧啶核苷尼克末端标记 (TUNEL) 测定观察细胞凋亡。
IF 染色显示卵清蛋白诱导的哮喘小鼠肺组织中 CX3CL1 表达增加。IHC 染色显示 CX3CR1 在气道的上皮下层增加。此外，CX3CR1 小干扰 (si)RNA 下调 NHLFs 中 TGF-β 诱导的 CTGF 和纤维连接蛋白表达。CX3CL1 诱导 NHLFs 中 CTGF 和纤维连接蛋白的表达。TGF-β 诱导 NHLFs 中 CX3CL1 的分泌。此外，TP213 降低了 NHLFs 中 TGF-β 诱导的 CTGF、纤维连接蛋白和 α-SMA 表达。在 TGF-β 处理的 NHLFs 中中和 CX3CL1 后，检查了与线粒体相关的差异表达基因 (DEG)。TP213 减轻了 NHLFs 中 TGF-β 诱导的线粒体功能障碍和抗细胞凋亡。CX3CL1 以时间依赖性方式诱导 p65、IκBα 和 IKKα 磷酸化。此外，p65 siRNA 降低了 CX3CL1 诱导的纤维连接蛋白表达和 JC-1 单体。TP213 降低了 NHLFs 中 TGF-β 诱导的 p65 和 α-SMA 表达。
这些发现表明中和 CX3CL1 可减轻肺成纤维细胞的激活和线粒体功能障碍。了解 CX3CL1 中和对成纤维细胞线粒体功能的影响可能有助于开发管理重度哮喘气道重塑的治疗策略。
