Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna , 1090 Vienna, Austria.
Anal Chem. 2017 Feb 7;89(3):1945-1954. doi: 10.1021/acs.analchem.6b04433. Epub 2017 Jan 20.
During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.
在炎症过程中，蛋白质和脂质协同作用，需要进行联合分析。成纤维细胞是慢性炎症的强大介质。然而，人们对人成纤维细胞中花生四烯酸形成的了解甚少。本研究旨在分析人成纤维细胞在炎症刺激后及随后用强力抗炎药物地塞米松处理时，包括蛋白质和脂质在内的最相关炎症介质的形成。无标记定量用于蛋白质组谱分析，而基于高分辨率质谱的内部建立的数据依赖性分析方法用于分析花生四烯酸代谢物。此外，通过靶向分析还确定了 188 种代谢物。发现 40 种蛋白质（包括细胞因子、蛋白酶和其他炎症激动剂）和 14 种促炎和 9 种抗炎花生四烯酸显著诱导分泌，而几种酰基辅酶 A 和神经鞘磷脂则显著下调。地塞米松处理显著下调了大多数细胞因子和蛋白酶，阻断了促炎和抗炎花生四烯酸的形成，并恢复了酰基辅酶 A 的正常水平，但神经鞘磷脂没有。此外，已知有助于慢性炎症的趋化因子 CXCL1、CXCL5、CXCL6 和补体 C3 也未被地塞米松调节。人间充质干细胞也得到了类似的发现，并且通过多重反应监测的靶向分析证实了结果。关于其他细胞的比较蛋白质组谱分析表明，除其他外，形成花生四烯酸的酶以及相关转录因子的合成具有细胞类型特异性，使我们能够更好地理解炎症介质的细胞类型特异性调节，并为成纤维细胞在慢性炎症中的作用提供新的见解。
