M1 POLARİZE MAKROFAJ FARKLILAŞMASINDA GÜNCEL TEKNİKLER: KAVRAMSAL ÇERÇEVE ENTEGRASYONU İLE KARŞILAŞTIRMALI DENEYSEL ÇALIŞMA

Öz

Amaç: Bu çalışmanın amacı, daha önce literatürde tanımlanmış ancak makrofajların fenotipik plastisite ve fonksiyonel instabilite özellikleri nedeniyle standartlaştırılmamış dört farklı protokol kullanılarak U937 hücre hattının M1 polarize makrofajlara farklılaşmasını incelemek ve elde edilen fenotipik değişiklikleri karakterize etmektir. Hücreler üzerinde farklı kimyasal indüksiyon protokolleri uygulanarak elde edilen M1 polarize makrofajların akım sitometrik profillerini ortaya koyarak, M1 polarize makrofajları içeren in vitro modellerin kullanımı hakkında yararlı veriler sağlamaktadır. Yöntem: U937 hücrelerinde yalnızca forbol-12-miristat-13-asetat (PMA) veya PMA’ya ek olarak lipopolisakkarit (LPS) ve interferon-gama (IFN-γ) gibi uyarıcıları içeren dört farklı farklılaşma stratejisi çalışıldı. Farklılaşma sonrası makrofajlara özgü CD68 (M1) ve CD206 (M2) yüzey belirteçlerinin ekspresyonu akım sitometrisi ile analiz edildi ve ilgili belirteçlerin ekspresyonuna dayalı olarak polarizasyon eğilimleri incelendi. Bulgular: Uygulanan makrofaj farklılaşma stratejileri arasında M1 makrofajına özgü hücre yüzey belirteçlerinde bir farklılık gözlemlendi. Bu durum, kullanılan protokole bağlı olarak pro-enflamatuvar (M1-benzeri) ve anti-enflamatuvar (M2-benzeri) fenotiplerin dengesinin değiştiğini ortaya koydu. Ayrıca farklılaşma stratejilerinden (2) en iyi M1 polarize makrofaj fenotipinde hücrelerin olduğu (%61) sonucunu verdi. Sonuç: Bulgular, standartlaştırılmamış farklılaşma protokollerinin, polarizasyon açısından U937 hücrelerinden farklı makrofaj benzeri popülasyonların oluşmasına neden olduğunu göstermektedir. Bu bulgular, deney tasarımlarında standardizasyonun önemini vurgulamakta ve makrofaj biyolojisi çalışmalarında uygun farklılaşma yöntemlerinin seçilmesi için bir çerçeve sunmaktadır.

Anahtar Kelimeler

• Makrofaj farklılaşması
• U937 hücreleri
• Fenotipik profilleme
• Farklılaşma protokolleri

EMERGING TECHNIQUES IN M1 POLARIZED MACROPHAGE DIFFERENTIATION: COMPARATIVE EXPERIMENTAL STUDY WITH A CONCEPTUAL FRAMEWORK INTEGRATION

Öz

Objective: This study aims to investigate the differentiation of the U937 cell line into M1-polarized macrophages using four protocols described in previous studies, but which have not been standardized on account of phenotypic plasticity and functional instability of macrophages. The resulting phenotypic alternations were characterized. By revealing the flow cytometric profiles of the M1-polarized macrophages, obtained by implementation of different chemical induction protocols on the cells, this study provides invaluable data on the use of in vitro models involving M1 polarized macrophages. Methods: Four different chemical induction strategies were implemented on U937 cells to obtain M1-polarized macrophages, each involving either phorbol-12-myristate-13-acetate (PMA) alone or combine with additional stimulants such as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). After differentiation, the expression of the macrophage-specific surface markers CD68 (M1) and CD206 (M2) was analyzed by flow cytometry, and the polarization trends were examined based on the expression of the associated markers. Results: A divergence in the M1-macrophage-specific cell surface marker was observed among the applied macrophage differentiation strategies. This finding indicates that the balance between pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes is subject to variation depending on the applied protocol. Furthermore, differentiation strategies (2) yielded optimal results in terms of cells exhibiting the M1 polarized macrophage phenotype (61%). Conclusion: The findings indicate that non-standardized differentiation protocols result in the generation of distinct macrophage-like populations from U937 cells regarding polarization. These findings emphasize the importance of standardization in experimental designs and provide a framework for selecting appropriate differentiation methods in studies of macrophage biology.

Anahtar Kelimeler

• Macrophage differentiation
• U937 cells
• Phenotypic profiling
• Differentiation protocols

Kaynakça

1. Nascimento CR, Rodrigues Fernandes NA, Gonzalez Maldonado LA, Rossa Junior C. Comparison of monocytic cell lines U937 and THP-1 as macrophage models for in vitro studies. Biochem Biophys Rep. 2022;32:101383. doi:10.1016/j.bbrep.2022.101383
2. Kulkarni P, Srivastava V, Tootsi K, et al. Synovial Fluid in Knee Osteoarthritis Extends Proinflammatory Niche for Macrophage Polarization. Cells. 2022;11(24). doi:10.3390/cells11244115
3. Haseeb A, Haqqi TM. Immunopathogenesis of osteoarthritis. Clin Immunol. 2013;146(3):185-96. doi:10.1016/j.clim.2012.12.011
4. Sreejit G, Fleetwood AJ, Murphy AJ, Nagareddy PR. Origins and diversity of macrophages in health and disease. Clin Transl Immunology. 2020;9(12):e1222. doi:10.1002/cti2.1222
5. Sundström C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer. 1976;17(5):565-577. doi:10.1002/ijc.2910170504
6. Vlahopoulos S, Boldogh I, Casola A, Brasier AR. Nuclear Factor-κB-Dependent Induction of Interleukin-8 Gene Expression by Tumor Necrosis Factor : Evidence for an Antioxidant Sensitive Activating Pathway Distinct From Nuclear Translocation. Blood. 1999;94(6):1878-1889. doi:10.1182/blood.V94.6.1878
7. Prasad A, Sedlarova M, Balukova A, et al. Reactive Oxygen Species Imaging in U937 Cells. Front Physiol. 2020;11:552569. doi:10.3389/fphys.2020.552569
8. Altuntaş C, Duruksu G, Hunç F, Yazir Y. Mitochondria transfer from mesenchymal stem cells into osteoarthritic chondrocytes ameliorate cellular functions and alleviate both inflammation and oxidative stress. Tissue and Cell. 2025;96:102990.

9. Öztürk C, Halbutoğulları ZS. Immune regulation is more effective in the U937 inflammation model with mesenchymal stem cell extracellular vesicles stimulated by pro-inflammatory cytokines. Cent Eur J Immunol. 2024;49(3):282-299. doi: 10.5114/ceji.2024.143726.
10. Abdulhadi FHS. Differentiation of U-937 monocytes to macrophage-like cells polarized into M1 or M2 phenotypes according to their specific environment: A study of morphology, cell viability, and cd markers of an in vitro model of human macrophages. 2014;
11. Kılıç KC, Duruksu G, Öztürk A, Rençber SF, Kılıç B, Yazır Y. Therapeutic potential of adult stem cells-derived mitochondria transfer combined with curcumin administration into ARPE-19 cells in age-related macular degeneration model. Tissue Cell. 2025;93:102687. doi:10.1016/j.tice.2024.102687
12. Altuntaş C, Duruksu G, Hunç F, Yazır Y. A novel isolation method for chondrocytes differentiated from synovial fluid-derived mesenchymal stem cell. Balıkesir Üniversitesi Fen Bilimleri Enstitüsü Dergisi. 2025;27(1):397-410.
13. Kiliç KC, Yazir Y, Öztürk A, et al. Investigation of impacts of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of mesenchymal stem cell through Notch/Hedgehog signaling pathways. Tissue Cell. 2023;84:102195. doi:10.1016/j.tice.2023.102195
14. Kim KW, Ivanov S, Williams JW. Monocyte Recruitment, Specification, and Function in Atherosclerosis. Cells. 2020;10(1). doi:10.3390/cells10010015
15. Kolypetri P, Weiner HL. Monocyte regulation by gut microbial signals. Trends Microbiol. 2023;31(10):1044-1057. doi:10.1016/j.tim.2023.05.006
16. Guan F, Wang R, Yi Z, et al. Tissue macrophages: origin, heterogenity, biological functions, diseases and therapeutic targets. Signal Transduct Target Ther. 2025;10(1):93. doi:10.1038/s41392-025-02124-y.
17. Chen C, Liu T, Tang Y, Luo G, Liang G, He W. Epigenetic regulation of macrophage polarization in wound healing. Burns Trauma. 2023;11:tkac057. doi:10.1093/burnst/tkac057.
18. Hasegawa T, Kikuta J, Sudo T, et al. Identification of a novel arthritis-associated osteoclast precursor macrophage regulated by FoxM1. Nat Immunol. 2019;20(12):1631-1643. doi:10.1038/s41590-019-0526-7.
19. Lin P, Ji HH, Li YJ, Guo SD. Macrophage Plasticity and Atherosclerosis Therapy. Front Mol Biosci. 2021;8:679797. doi:10.3389/fmolb.2021.679797.
20. Keren-Shaul H, Spinrad A, Weiner A, et al. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017;169(7):1276-1290.e17. doi: 10.1016/j.cell.2017.05.018.
21. Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL. IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. Mol Ther. 2022;30(6):2274-2297. doi:10.1016/j.ymthe.2022.03.008.
22. Lumeng CN, DeYoung SM, Bodzin JL, Saltiel AR. Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity. Diabetes. 2007;56(1):16-23. doi: 10.2337/db06-1076.
23. Biswas SK, Mantovani A. Orchestration of metabolism by macrophages. Cell metabolism. 2012;15(4):432-437. doi:10.1016/j.cmet.2011.11.013.
24. Shapouri-Moghaddam A. Mohammadian S. Vazini H. Taghadosi M. Esmaeili SA Mardani F. Seifi B. Mohammadi A. Afshari JT Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol. 2018;233(9):6425. doi: 10.1002/jcp.26429.
25. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol. 2000;164(12):6166-73. doi:10.4049/jimmunol.164.12.6166.
26. Holness CL, Simmons DL. Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. 1993;81(6):1607-13.
27. Scodeller P, Simón-Gracia L, Kopanchuk S, et al. Precision targeting of tumor macrophages with a CD206 binding peptide. Sci Rep. 2017;7(1):14655. doi:10.1038/s41598-017-14709-x.
28. Bosco MC. Macrophage polarization: reaching across the aisle? J Allergy Clin Immunol. 2019;143(4):1348-1350. doi: 10.1016/j.jaci.2018.12.995.
29. Kuno S, Srinoun K, Penglong T. The effects of Phorbol 12-myristate 13-acetate concentration on the expression of miR-155 and miR-125b and their macrophage function-related genes in the U937 cell line. J Toxicol Sci. 2020;45(12):751-761. doi:10.2131/jts.45.751