Limanaqi Fiona, Zecchini Silvia, Ogno Pasquale, Artusa Valentina, Fenizia Claudio, Saulle Irma, Vanetti Claudia, Garziano Micaela, Strizzi Sergio, Trabattoni Daria, Clerici Mario, Biasin Mara
Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy.
Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.
Front Cell Dev Biol. 2024 Jul 5;12:1421360. doi: 10.3389/fcell.2024.1421360. eCollection 2024.
Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson's disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear. Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1 μM) for 4 h and 24 h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C-C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages. Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4 h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24 h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4 h αS-exposed THP-1 cells showed increased spontaneous migration, while 24 h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia. In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.
α-突触核蛋白（αS）异常扩散是帕金森病的一个标志，已知其会促进外周炎症，部分原因是单核细胞/巨噬细胞的功能改变。然而，潜在的细胞内机制仍不清楚。在此，我们研究了过量αS在人THP-1单核细胞系、THP-1衍生的巨噬细胞以及至少初步在原代单核细胞衍生的巨噬细胞（MDM）中的亚细胞、分子和功能效应。在用/不用重组αS（1 μM）培养4小时和24小时的细胞中，通过共聚焦显微镜、蛋白质免疫印迹、逆转录定量聚合酶链反应、酶联免疫吸附测定和流式细胞术，我们评估了：i）αS内化；ii）细胞因子/趋化因子表达/分泌以及C-C基序趋化因子受体2（CCR2）水平；iii）自噬（LC3II/I、溶酶体相关膜蛋白1/LysoTracker、p62、磷酸化核糖体蛋白S6/总S6）；以及iv）脂滴（LDs）积累和胆固醇途径基因表达。采用Transwell迁移试验测量THP-1细胞迁移/趋化性，而用异硫氰酸荧光素标记的免疫球蛋白珠子试验分析吞噬能力以及THP-1衍生巨噬细胞中吞噬货物的命运。细胞外αS被THP-1细胞、THP-1衍生的巨噬细胞和MDM内化。在THP1细胞中，αS诱导了一种普遍的促炎表型，并且来自暴露于αS的THP-1细胞的条件培养基能有效吸引未刺激的细胞。然而，CCL2分泌在αS处理4小时时达到峰值，与其受体CCR2的早期内化一致，而在αS暴露24小时时这种情况减弱，此时CCR2循环回到质膜。同样，暴露于αS 4小时的THP-1细胞显示出自发迁移增加，而暴露于αS 24小时的细胞显示趋化性降低。这发生在没有细胞毒性的情况下，并且与自噬/溶酶体标志物的上调相关，表明存在针对应激相关炎症的促存活/耐受机制。相反，在THP-1衍生的巨噬细胞中，αS随时间增强细胞内积累以及促炎介质的释放。这伴随着轻度毒性、自噬-溶酶体标志物减少、脂滴形成缺陷以及吞噬作用受损，并且出现被吞噬货物吞噬的停滞溶酶体，表明巨噬细胞耗竭状态类似于摄食减少。总之，尽管单核细胞和巨噬细胞具有明显相似的促炎表型，但它们在细胞存活、代谢和功能方面对细胞内αS积累的反应不同。我们的结果表明，在外周，αS发挥细胞和环境特异性的生物学效应，连接自噬、脂质动态和炎症途径的改变。
