Klas Katharina, Ondracek Anna S, Hofbauer Thomas M, Mangold Andreas, Pfisterer Karin, Laggner Maria, Copic Dragan, Direder Martin, Bormann Daniel, Ankersmit Hendrik Jan, Mildner Michael
Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Department of Thoracic Surgery, Medical University of Vienna, 1090 Vienna, Austria.
Aposcience AG, 1200 Vienna, Austria.
Antioxidants (Basel). 2022 Aug 11;11(8):1559. doi: 10.3390/antiox11081559.
Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.
中性粒细胞胞外诱捕网（NET）形成是快速清除感染的一种重要防御机制。然而，已有研究表明，过度的NET形成会对损伤后的组织再生产生负面影响。由于我们之前的研究揭示了应激外周血单核细胞分泌组（PBMCsec）具有强大的组织保护和再生特性，因此我们在此研究了PBMCsec对NET形成的影响。使用流式细胞术、图像流式分析和释放的细胞外DNA定量，在体外评估PBMCsec对来自健康供体的离子霉素刺激的中性粒细胞NET形成的影响。分析了PBMCsec对NET形成所涉及的分子机制的影响，包括钙通量、蛋白激酶C活性、活性氧产生和蛋白精氨酸脱亚氨酶4活性。我们的结果表明，PBMCsec显著抑制NET形成。对PBMCsec中发现的不同生物物质类别的研究表明，NET形成仅部分减少，提示存在协同效应。从机制上讲，PBMCsec处理不干扰钙信号传导和PKC激活，但具有抗氧化活性，这在PBMCsec处理的中性粒细胞中表现为活性氧水平降低以及血红素加氧酶1和缺氧诱导因子1上调。此外，PBMCsec强烈抑制蛋白精氨酸脱亚氨酶4（PAD4）的激活，最终导致NET形成受到抑制。由于目前尚无拮抗过度NET形成的治疗方法，我们的研究为因NET形成过度导致的多种病症提供了一种有前景的新治疗选择。
