Abstract：
OBJECTIVE: This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein β (C/EBPβ) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis.
METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPβ was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPβ, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPβ, IL-10, and TGF-β1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPβ.
RESULTS: TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPβ expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFβ1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPβ.
CONCLUSIONS: This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPβ expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.
METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPβ was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPβ, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPβ, IL-10, and TGF-β1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPβ.
RESULTS: TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPβ expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFβ1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPβ.
CONCLUSIONS: This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPβ expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.
摘要：
目的：本研究探讨TNF诱导蛋白3(TNFAIP3)和CCAAT/增强子结合蛋白β(C/EBPβ)在系统性硬化症相关间质性肺病(SSc-ILD)患者肺泡巨噬细胞(AMs)中的作用及其对肺纤维化的影响。
方法：用携带TNFAIP3和C/EBPβ的质粒转染HEK293T细胞和AMs，然后将AMs与肺成纤维细胞共培养。然后使用免疫印迹分析来评估TNFAIP3，C/EBPβ的表达，和胶原蛋白1型（Col1）。进行定量PCR分析以定量C/EBPβ的mRNA水平，IL-10和TGF-β1。STRING数据库分析，和免疫沉淀试验用于研究TNFAIP3和C/EBPβ之间的相互作用。
结果：TNFAIP3在SSc-ILDAMs中的表达显著降低，与成纤维细胞中Col1产生增加相关。TNFAIP3的过表达抑制了这种促纤维化活性。相反，SSc-ILDAMs中C/EBPβ表达升高，其通过TNFAIP3恢复的减少降低了促纤维化细胞因子IL-10和TGFβ1的水平。蛋白质-蛋白质相互作用研究证实了TNFAIP3和C/EBPβ之间的调节关系。
结论：本研究强调了TNFAIP3通过调节AM中C/EBPβ的表达在调节SSc-ILD肺纤维化中的重要作用。这些发现表明靶向TNFAIP3可能是治疗SSc-ILD患者的潜在治疗策略。
方法：用携带TNFAIP3和C/EBPβ的质粒转染HEK293T细胞和AMs，然后将AMs与肺成纤维细胞共培养。然后使用免疫印迹分析来评估TNFAIP3，C/EBPβ的表达，和胶原蛋白1型（Col1）。进行定量PCR分析以定量C/EBPβ的mRNA水平，IL-10和TGF-β1。STRING数据库分析，和免疫沉淀试验用于研究TNFAIP3和C/EBPβ之间的相互作用。
结果：TNFAIP3在SSc-ILDAMs中的表达显著降低，与成纤维细胞中Col1产生增加相关。TNFAIP3的过表达抑制了这种促纤维化活性。相反，SSc-ILDAMs中C/EBPβ表达升高，其通过TNFAIP3恢复的减少降低了促纤维化细胞因子IL-10和TGFβ1的水平。蛋白质-蛋白质相互作用研究证实了TNFAIP3和C/EBPβ之间的调节关系。
结论：本研究强调了TNFAIP3通过调节AM中C/EBPβ的表达在调节SSc-ILD肺纤维化中的重要作用。这些发现表明靶向TNFAIP3可能是治疗SSc-ILD患者的潜在治疗策略。
