EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation. 

1. Agholme L., Lindstrom T., Kagedal K., Marcusson J., Hallbeck L. An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J. Alzheimers Dis., 2010, Vol. 20, no. 4, pp. 1069-1082.

2. Butovsky O., Ziv Y., Schwartz A., Landa G., Talpalar A.E., Pluchino S., Martino G., Schwartz M. Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/ progenitor cells. Mol. Cell. Neurosci., 2006, Vol. 31, no. 1, pp. 149-160.

3. Chernykh E.R., Shevela E.Ya., Sakhno L.V., Tikhonova M.A., Petrovsky Ya.L., Ostanin A.A. The generation and properties of human M2-like macrophages: potential candidates for CNS repair? Cell Ther. Transplant., 2010, Vol. 2, no. 6. doi: 10.3205/ctt-2010-en-000080.01.

4. Chernykh E.R., Shevela E.Ya., Starostina N.M., Morozov S.A., Davydova M.N., Menyaeva E.V., Ostanin A.A. Safety and therapeutic potential of M2-macrophages in stroke treatment. Cell Transplant., 2016, Vol. 25, no. 8, pp. 1461-1471.

5. Encinas M., Iglesias M., Liu Y., Wang H., Muhaisen A., Cena V., Gallego C., Comella J.X. Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J. Neurochem., 2000, Vol. 75, no. 3, pp. 991-1003.

6. Fanti Z., Martinez-Perez M.E., De-Miguel F.F. NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images. Dev. Neurobiol., 2013, Vol. 71, no. 10, pp. 870-881.

7. Huang D., Shen S., Cai M., Jin L., Lu J., Xu K., Zhang J., Feng G., Hu Y., Zheng K., Feng X. Role of mTOR complex in IGF-1 induced neural differentiation of DPSCs. J. Mol. Histol., 2019, Vol. 50, no. 3, pp. 273-283.

8. Jacobi А., Bareyre F.M. Regulation of axonal remodeling following spinal cord injury. Neural Regen. Res., 2015, Vol. 10, no. 10, pp. 1555-1557.

9. Jin K., Zhu Y., Sun Y., Mao X.O., Xie L., Greenberg D.A. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in svivo. Proc. Natl Acad. Sci. USA, 2002, Vol. 99, no. 18, pp. 11946-11950.

10. Lange C., Storkebaum E., De Almodóvar C.R., Dewerchin M., Carmeliet P. Vascular endothelial growth factor: a neurovascular target in neurological diseases. Nat. Rev. Neurol., 2016, Vol. 12, no. 8, pp. 439-454.

11. Lavenius E., Parrow V., Nanberg E., Pahlman S. Basic FGF and IGF-I promote differentiation of human SHSY5Y neuroblastoma cells in culture. Growth Factors, 1994, Vol. 10, no. 1, pp. 29-39.

12. Louissaint (Jr) A., Rao S., Leventhal C., Goldman S.A. Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain. Neuron, 2002, Vol. 34, no. 6, pp. 945-960.

13. Mattsson M.E., Enberg G., Ruusala A.I., Hall K., Pahlman S. Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation. J. Cell Biol., 1986, Vol. 102, no. 5., pp. 1949-1954.

14. Miron V.E., Boyd A., Zhao J.W., Yuen T.J., Ruckh J.M., Shadrach J.L., van Wijngaarden P., Wagers A.J., Williams A., Franklin R.J. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat. Neurosci., 2013, Vol. 16, no. 9, pp. 1211-1218.

15. Nicoletti J.N., Shah S.K., McCloskey D.P., Goodman J.H., Elkady A., Atassi H., Hylton D., Rudge J.S., Scharfman H.E., Croll S.D. Vascular endothelial growth factor is up-regulated after status epilepticus and protects against seizure-induced neuronal loss in hippocampus. Neuroscience, 2008, Vol. 151, no. 1, pp. 232-241.

16. Peng B., Kong G., Yang C., Ming Y. Erythropoietin and its derivatives: from tissue protection to immune regulation. Cell Death Dis., 2020, Vol. 11, no. 2, 79. doi: 10.1038/s41419-020-2276-8.

17. Popova D., Karlsson J., Jacobsson S.O.P. Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity. BMC Pharmacol Toxicol., 2017, Vol. 18, no.1, p. 42.

18. Pregi N., Vittori D., Pérez G., Pérez Leirós C., Nesse A. Effect of erythropoietin on staurosporine-induced apoptosis and differentiation of SH-SY5Y neuroblastoma cells. Biochim. Biophys. Acta, 2006, Vol. 1763, no. 2, pp. 238-246.

19. Rashchupkin I.M., Maksimova A.A., Sakhno L.V., Ostanin A.A., Shevela E.Ya., Chernykh E.R. The effect of M2 macrophage-derived soluble factors on proliferation and apoptosis of SH-SY5Y cells. Bulletin of Experimental Biology and Medicine, 2021, Vol. 171, no. 1, pp. 59-63. (In Russ.)

20. Rawji K.S., Mishra M.K., Yong V.W. Regenerative capacity of macrophages for remyelination. Front Cell Dev. Biol., 2016, Vol. 4, 47. doi:10.3389/fcell.2016.00047.

21. Ross R.A., Spengler B.A., Biedler J.L. Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J. Natl Cancer Inst., 1983, Vol. 71, no. 4, pp. 741-747.

22. Sakhno L.V., Shevela E.Y., Tikhonova M.A., Ostanin A.A., Chernykh E.R. The phenotypic and functional features of human M2 macrophages generated under low serum conditions. Scand. J. Immunol., 2016, Vol. 83, no. 2, pp. 151-159.

23. Shevela E., Davydova M., Starostina N., Yankovskaya A., Ostanin A., Chernykh Е. Intranasal delivery of M2 macrophage-derived soluble products reduces neuropsychological deficit in patients with cerebrovascular disease: a Pilot Study. J. Neurorestoratol., 2019, Vol. 7, no. 2, pp. 89-100.

24. Ulrich H., Resende R. Mechanisms of neural differentiation and integration. Semin. Cell Dev. Biol., 2019, Vol. 93, pp. 1-3.

25. Xiang X., Li Y., Fu M., Xin H-B. Polarizing macrophages in vitro. Methods Mol. Biol., 2018, Vol. 1784, pp. 119-126.

26. Xiong X.Y., Liu L., Yang Q.W. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke. Prog. Neurobiol., 2016, Vol. 142, pp. 23-44.

27. Yuste V., Sánchez-López I., Solé C., Encinas M., Bayascas J., Boix J., Comella J. The prevention of the staurosporine-induced apoptosis by Bcl-xL, but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells. J. Neurochem., 2002, Vol. 80, no. 1, pp. 126-139.

28. Zhu Y., Jin K., Mao X.O., Greenberg D.A. Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression. FASEB J., 2003, Vol. 17, no. 2, pp. 186-193.