Department of Internal Medicine, Division of Rheumatology, University of Nebraska Medical Center, Omaha, NE, United States.
Department of Research Services 151, Veteran Affairs Nebraska-Western Iowa Health Care System, Omaha, NE, United States.
Front Immunol. 2023 Aug 16;14:1203548. doi: 10.3389/fimmu.2023.1203548. eCollection 2023.
Post-translational protein modifications with malondialdehyde-acetaldehyde (MAA) and citrulline (CIT) are implicated in the pathogenesis of rheumatoid arthritis (RA). Although precise mechanisms have not been elucidated, macrophage-fibroblast interactions have been proposed to play a central role in the development and progression of RA. The purpose of our study was to evaluate the downstream effects of macrophage released soluble mediators, following stimulation with fibrinogen (FIB) modified antigens, on human fibroblast-like synoviocytes (HFLS).
PMA-treated U-937 monocytes (Mϕ) and macrophage-differentiated peripheral blood mononuclear cells (MP) were stimulated with FIB, FIB-MAA, FIB-CIT, or FIB-MAA-CIT. HFLS-RA cells were stimulated directly with FIB antigens or with supernatants (SN) from macrophages (Mϕ-SN or MP-SN) stimulated with FIB antigens. Genes associated with an aggressive HFLS phenotype, extracellular matrix proteins, and activated signaling pathways were evaluated.
HFLS-RA cells treated with Mϕ-SN and Mϕ-SN demonstrated significant increases in mRNA expression of genes associated with an aggressive phenotype at 24-h as compared to direct stimulation with the same antigens. Similar results were obtained using MP-SN. Cellular morphology was altered and protein expression of vimentin (p<0.0001 vs. Mϕ-SN) and type II collagen (p<0.0001) were significantly increased in HFLS-RA cells treated with any of the Mϕ-SN generated following stimulation with modified antigens. Phosphorylation of JNK, Erk1/2, and Akt were increased most substantially in HFLS-RA treated with Mϕ-SN (p<0.05 vs Mϕ-SN). These and other data suggested the presence of PDGF-BB in Mϕ-SN. Mϕ-SN contained the highest concentration of PDGF-BB (p<0.0001 vs. Mϕ-SN) followed by Mϕ-SN then Mϕ-SN. HFLS-RA cells treated with PDGF-BB showed similar cellular morphology to the Mϕ-SN generated following stimulation with modified FIB, as well as the increased expression of vimentin, type II collagen, and the phosphorylation of JNK, Erk1/2 and Akt signaling molecules.
Together, these findings support the hypothesis that in response to MAA-modified and/or citrullinated fibrinogen, macrophages release soluble factors including PDGF-BB that induce fibroblast activation and promote an aggressive fibroblast phenotype. These cellular responses were most robust following macrophage activation with dually modified fibrinogen, compared to single modification alone, providing novel insights into the combined role of multiple post-translational protein modifications in the development of RA.
与丙二醛-乙醛（MAA）和瓜氨酸（CIT）的翻译后蛋白修饰有关的类风湿关节炎（RA）发病机制。尽管确切的机制尚未阐明，但巨噬细胞-成纤维细胞相互作用被认为在 RA 的发展和进展中起核心作用。我们研究的目的是评估经过纤维蛋白原（FIB）修饰抗原刺激后，巨噬细胞释放的可溶性介质对人成纤维样滑膜细胞（HFLS）的下游影响。
用佛波醇酯（PMA）处理的 U-937 单核细胞（Mϕ）和巨噬细胞分化的外周血单核细胞（MP）用 FIB、FIB-MAA、FIB-CIT 或 FIB-MAA-CIT 刺激。直接用 FIB 抗原或用 FIB 抗原刺激的巨噬细胞（Mϕ-SN 或 MP-SN）的上清液（SN）刺激 HFLS-RA 细胞。评估与侵袭性 HFLS 表型相关的基因、细胞外基质蛋白和激活的信号通路。
与直接用相同抗原刺激相比，用 Mϕ-SN 处理的 HFLS-RA 细胞在 24 小时时与侵袭性表型相关的基因的 mRNA 表达显著增加。使用 MP-SN 也得到了类似的结果。用任何一种 Mϕ-SN 处理的 HFLS-RA 细胞的细胞形态都发生了改变，波形蛋白（p<0.0001 比 Mϕ-SN）和 II 型胶原（p<0.0001）的蛋白表达显著增加。用 Mϕ-SN 处理的 HFLS-RA 细胞中 JNK、Erk1/2 和 Akt 的磷酸化增加最多（p<0.05 比 Mϕ-SN）。这些和其他数据表明 Mϕ-SN 中存在 PDGF-BB。Mϕ-SN 中 PDGF-BB 的浓度最高（p<0.0001 比 Mϕ-SN），其次是 Mϕ-SN，然后是 Mϕ-SN。用 PDGF-BB 处理的 HFLS-RA 细胞表现出与用修饰的 FIB 刺激后产生的 Mϕ-SN 相似的细胞形态，以及波形蛋白、II 型胶原的表达增加，以及 JNK、Erk1/2 和 Akt 信号分子的磷酸化。
综上所述，这些发现支持了这样一种假设，即在 MAA 修饰和/或瓜氨酸化纤维蛋白原的作用下，巨噬细胞释放包括 PDGF-BB 在内的可溶性因子，这些因子诱导成纤维细胞激活，并促进侵袭性成纤维细胞表型。与单独单一修饰相比，用双重修饰纤维蛋白原激活巨噬细胞后，这些细胞反应最为强烈，为研究多种翻译后蛋白修饰在 RA 发展中的联合作用提供了新的见解。
