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Characteristics of SIM-A9 Microglia Cells: New Data

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Abstract

SIM-A9 is a line of spontaneously immortalized mouse microglial cells obtained from the brain of newborn C57BL/6 mice. The purpose of this work is to characterize the microglia of the SIM-A9 mouse line on the basis of the ratio of cells with the phenotype of resting and activated microglia in culture and to analyze the expression of markers of CD133 stem (progenitor) cells and nestin, which are receptors for growth factors CSF-1R and EGFR, as well as to analyze the karyotype of this line. Light microscopy and immunocytochemistry combined with flow cytometry and RT–PCR were used to analyze the morphology, phenotype, and expression level of pro-inflammatory cytokine genes, and the mFISH method was used to analyze the karyotype. It has been shown for the first time that SIM-A9 cells express a high level of TSPO protein and CD68, CD11b, and CD45high markers on the surface membrane of cells, which corresponds to the phenotype of activated microglia. Despite this, the cells of the line respond with additional activation in response to lipopolysaccharide stimulation, which leads to an increase in the expression of pro-inflammatory cytokine genes IL-1β, TNFα, and IL-6 and the formation of a high level of active oxygen and nitrogen metabolites. SIM-A9 cells were shown to express markers of stem and progenitor cells CD133+ and nestin, which allows us to consider them as early poorly differentiated progenitor cells, despite their phenotype corresponding to activated microglia. It was also found that SIM-A9 cells express receptors of the two growth factors CSF-1 and EGF, CSF-1R and EGFR, which indicates the possibility of stimulation of SIM-A9 cell proliferation by two alternative mechanisms under the action of corresponding factors. SIM-A9 cells have a hypotetraploid karyotype with a large number of structural and quantitative chromosome anomalies.

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REFERENCES

  1. Moskaleva, E.Yu., Rodina, A.V., Semochkina, Ju.P., and Vysotskaya, O.V., Analysis of neurons damage and level of neuroinflammation late after γ-irradiation of mice head at different doses, Radiats. Biol. Radioekol., 2022, vol. 62, no. 2, p. 171. https://doi.org/10.31857/S0869803122020059

    Article  Google Scholar 

  2. Novikov, V.E., Levchenkova, O.S., and Pozhilova, Y.V., Role of reactive oxygen species in cell physiology and pathology and their pharmacological regulation, Obzory Klin. Farmakol. Lekarstv. Ter., 2014, vol. 12, no. 4, p. 13. https://doi.org/10.17816/RCF12413-21

    Article  Google Scholar 

  3. Askew, K., Li, K., Olmos-Alonso, A., Garcia-Moreno, F., Liang, Y., Richardson, P., Tipton, T., Chapman, M.A., Riecken, K., Beccari, S., Sierra, A., Molnár, Z., Cragg, M.S., Garaschuk, O., Perry, V.H., and Gomez-Nicola, D., Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain, Cell Rep., 2017, vol. 18, p. 391. https://doi.org/10.1016/j.celrep.2016.12.041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bachiller, S., Jiménez-Ferrer, I., Paulus, A., Yang, Y., Swanberg, M., Deierborg, T., and Boza-Serrano, A., Microglia in neurological diseases: a road map to brain-disease dependent-inflammatory response, Front. Cell Neurosci., 2018, vol. 12, p. 488. https://doi.org/10.3389/fncel.2018.00488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Becher, B. and Antel, J.P., Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia, Glia, 1996, vol. 18, p. 1. https://doi.org/10.1002/(SICI)1098-1136(199609)18:1<1::AID-GLIA1>3.0.CO;2-6

    Article  CAS  PubMed  Google Scholar 

  6. Bennett, M.L., Bennett, F.C., Liddelow, S.A., Ajami, B., Zamanian, J.L., Fernhoff, N.B., Mulinyawe, S.B., Bohlen, C.J., Adil, A., Tucker, A., Weissman, I.L., Chang, E.F., Li, G., Grant, G.A., Hayden Gephart, M.G., and Barres, B.A., New tools for studying microglia in the mouse and human CNS, Proc. Natl. Acad. Sci. U. S. A., 2016, vol. 113, p. 1738. https://doi.org/10.1073/pnas.1525528113

    Article  CAS  Google Scholar 

  7. Bernal, A. and Arranz, L., Nestin-expressing progenitor cells: function, identity and therapeutic implications, Cell. Mol. Life Sci., 2018, vol. 75, p. 2177. https://doi.org/10.1007/s00018-018-2794-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., and Bistoni, F., Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus, J. Neuroimmunol., 1990, vol. 27, p. 229. https://doi.org/10.1016/0165-5728(90)90073-V

    Article  CAS  PubMed  Google Scholar 

  9. Bohnert, S., Seiffert, A., Trella, S., Bohnert, M., Distel, L., Ondruschka, B., and Monoranu, C.M., TMEM119 as a specific marker of microglia reaction in traumatic brain injury in postmortem examination, Int. J. Legal Med., 2020, vol. 134, p. 2167. https://doi.org/10.1007/s00414-020-02384-z

    Article  PubMed  PubMed Central  Google Scholar 

  10. Bonsack, F. and Sukumari-Ramesh, S., TSPO: an evolutionarily conserved protein with elusive functions, Int. J. Mol. Sci., 2018, vol. 19, p. 1694. https://doi.org/10.3390/ijms19061694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cornforth, M.N., Analyzing radiation-induced complex chromosome rearrangements by combinatorial painting, Radiat. Res., 2001, vol. 155, p. 643. https://doi.org/10.1007/978-1-4939-9432-8_15

    Article  CAS  PubMed  Google Scholar 

  12. Coniglio, S.J., Eugenin, E., Dobrenis, K., Stanley, E.R., West, B.L., Symons, M.H., and Segall, J.E., Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling, Mol. Med., 2012, vol. 18, p. 519. https://doi.org/10.2119/molmed.2011.00217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Coskun, V., Wu, H., Blanchi, B., Tsao, S., Kim, K., Zhao, J., Biancotti, J.C., Hutnick, L., Krueger, R.C., Jr., Fan, G., de Vellis, J., and Sun, Y.E., CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain, Proc. Natl. Acad. Sci. U. S. A., 2008, vol. 105, p. 1026. https://doi.org/10.1073/pnas.0710000105

    Article  PubMed  PubMed Central  Google Scholar 

  14. Douglas, M.R., Morrison, K.C., Jacques, S.J., Leadbeater, W.E., Gonzalez, A.M., Berry, M., Logan, A., and Ahmed, Z., Off-target effects of epidermal growth factor receptor antagonists mediate retinal ganglion cell disinhibited axon growth, Brain, 2009, vol. 132, p. 3102. https://doi.org/10.1093/brain/awp240

    Article  PubMed  Google Scholar 

  15. Elmore, M.R., Najafi, A.R., Koike, M.A., Dagher, N.N., Spangenberg, E.E., Rice, R.A., Kitazawa, M., Matusow, B., Nguyen, H., West, B.L., and Green, K.N., Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain, Neuron, 2014, vol. 82, p. 380. https://doi.org/10.1016/j.neuron.2014.02.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Eyo, U.B. and Dailey, M.E., Microglia: key elements in neural development, plasticity, and pathology, J. Neuroimmune Pharmacol., 2013, vol. 8, p. 494. https://doi.org/10.1007/s11481-013-9434-z

    Article  PubMed  PubMed Central  Google Scholar 

  17. Fischer, O.M., Hart, S., and Ullrich, A., Dissecting the epidermal growth factor receptor signal transactivation pathway, Methods Mol. Biol., 2006, vol. 327, p. 85. https://doi.org/10.1385/1-59745-012-X:85

    Article  CAS  PubMed  Google Scholar 

  18. Green, K.N., Crapser, J.D., and Hohsfield, L.A., To kill a microglia: a case for CSF1R inhibitors, Trends Immunol., 2020, vol. 41, p. 771. https://doi.org/10.1016/j.it.2020.07.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hagan, N., Kane, J.L., Grover, D., Woodworth, L., Madore, C., Saleh, J., Sancho, J., Liu, J., Li, Y., Proto, J., Zelic, M., Mahan, A., Kothe, M., Scholte, A.A., and Fitzgerald, M., CSF1R signaling is a regulator of pathogenesis in progressive MS, Cell Death Dis., 2020, vol. 11, p. 904. https://doi.org/10.1038/s41419-020-03084-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Han, J., Chitu, V., Stanley, E.R., Wszolek, Z.K., Karrenbauer, V.D., and Harris, R.A., Inhibition of colony stimulating factor-1 receptor (CSF-1R) as a potential therapeutic strategy for neurodegenerative diseases: opportunities and challenges, Cell. Mol. Life Sci., 2022, vol. 79, p. 219. https://doi.org/10.1007/s00018-022-04225-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Huang, Y., Xu, Z., and Xiong, S., Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion, Nat. Neurosci., 2018, vol. 21, p. 530. https://doi.org/10.1038/s41593-018-0090-8

    Article  CAS  PubMed  Google Scholar 

  22. Jenkins, S.J., Ruckerl, D., Thomas, G.D., Hewitson, J.P., Duncan, S., Brombacher, F., Maizels, R.M., Hume, D.A., and Allen, J.E., IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1, J. Exp. Med., 2013, vol. 210, p. 2477. https://doi.org/10.1084/jem.20121999

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jones, S. and Rappoport, J.Z., Interdependent epidermal growth factor receptor signalling and trafficking, Int. J. Biochem. Cell. Biol., 2014, vol. 51, p. 23. https://doi.org/10.1016/j.biocel.2014.03.014

    Article  CAS  PubMed  Google Scholar 

  24. Jurga, A.M., Paleczna, M., and Kuter, K.Z., Overview of general and discriminating markers of differential microglia phenotypes, Front. Cell Neurosci., 2020, vol. 14, p. 198. https://doi.org/10.3389/fncel.2020.00198

    Article  PubMed  PubMed Central  Google Scholar 

  25. Kalm, M., Andreasson, U., Björk-Eriksson, T., Zetterberg, H., Pekny, M., Blennow, K., Pekna, M., and Blomgren, K., C3 deficiency ameliorates the negative effects of irradiation of the young brain on hippocampal development and learning, Oncotarget, 2016, vol. 7, p. 19382. https://doi.org/10.18632/oncotarget.8400

    Article  PubMed  PubMed Central  Google Scholar 

  26. Lei, F., Cui, N., Zhou, C., Chodosh, J., Vavvas, D.G., and Paschalis, E.I., CSF1R inhibition by a small-molecule inhibitor is not microglia specific; affecting hematopoiesis and the function of macrophages, Proc. Natl. Acad. Sci. U. S. A., 2020, vol. 22, p. 23336. https://doi.org/10.1073/pnas.1922788117

    Article  CAS  Google Scholar 

  27. Lively, S. and Schlichter, L.C., Microglia responses to pro-inflammatory stimuli (LPS, IFNγ+TNFα) and reprogramming by resolving cytokines (IL-4, IL-10), Front. Cell Neurosci., 2018, vol. 12, p. 215. https://doi.org/10.3389/fncel.2018.00215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Liu, G.J., Middleton, R.J., Hatty, C.R., Kam, W.W., Chan, R., Pham, T., Harrison-Brown, M., Dodson, E., Veale, K., and Banati, R.B., The 18 kDa translocator protein, microglia and neuroinflammation, Brain Pathol., 2014, vol. 24, p. 631. https://doi.org/10.1111/bpa.12196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mansour, H.M., Fawzy, H.M., El-Khatib, A.S., and Khattab, M.M., Repurposed anti-cancer epidermal growth factor receptor inhibitors: mechanisms of neuroprotective effects in Alzheimer’s disease, Neural Regen. Res., 2022, vol. 17, p. 1913. https://doi.org/10.4103/1673-5374.332132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Michalczyk, K. and Ziman, M., Nestin structure and predicted function in cellular cytoskeletal organization, Histol. Histopathol., 2005, vol. 20, p. 665. https://doi.org/10.14670/HH-20.665

    Article  CAS  PubMed  Google Scholar 

  31. Muñoz-Garcia, J., Cochonneau, D., Télétchéa, S., Moranton, E., Lanoe, D., Brion, R., Lézot, F., Heymann, M.F., and Heymann, D., The twin cytokines interleukin-34 and CSF-1: masterful conductors of macrophage homeostasis, Theranostics, 2021, vol. 11, p. 1568. https://doi.org/10.7150/thno.50683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Nagamoto-Combs, K., Kulas, J., and Combs, C.K., A novel cell line from spontaneously immortalized murine microglia, J. Neurosci. Methods, 2014, vol. 15, p. 187. https://doi.org/10.1016/j.jneumeth.2014.05.021

    Article  CAS  Google Scholar 

  33. Onyango, I.G., Jauregui, G.V., Čarná, M., Bennett, J.P., Jr., and Stokin, G.B., Neuroinflammation in Alzheimer’s disease, Biomedicines, 2021, vol. 9, p. 524. https://doi.org/10.3390/biomedicines9050524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pannell, M., Economopoulos, V., Wilson, T.C., Kersemans, V., Isenegger, P.G., Larkin, J.R., Smart, S., Gilchrist, S., Gouverneur, V., and Sibson, N.R., Imaging of translocator protein upregulation is selective for pro-inflammatory polarized astrocytes and microglia, Glia, 2020, vol. 68, p. 280. https://doi.org/10.1002/glia.23716

    Article  PubMed  Google Scholar 

  35. Prater, K.E., Aloi, M.S., Pathan, J.L., Winston, C.N., Chernoff, R.A., Davidson, S., Sadgrove, M., McDonough, A., Zierath, D., Su, W., Weinstein, J.R., and Garden, G.A., A subpopulation of microglia generated in the adult mouse brain originates from prominin-1-expressing progenitors, J. Neurosci., 2021, vol. 41, p. 7942. https://doi.org/10.1523/JNEUROSCI.1893-20.2021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Qu, W.S., Tian, D.S., Guo, Z.B., Fang, J., Zhang, Q., Yu, Z.Y., Xie, M.J., Zhang, H.Q., Lü, J.G., and Wang, W., Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury, J. Neuroinflammation, 2012, vol. 9, p. 178. https://doi.org/10.1186/1742-2094-9-178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Qu, W.S., Liu, J.L., Li, C.Y., Li, X., Xie, M.J., Wang, W., and Tian, D.S., Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia, Neurochem. Int., 2015, vol. 90, p. 85. https://doi.org/10.1016/j.neuint.2015.07.007

    Article  CAS  PubMed  Google Scholar 

  38. Ramprasad, M.P., Terpstra, V., Kondratenko, N., Quehenberger, O., and Steinberg, D., Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein, Proc. Natl. Acad. Sci. U. S. A., 1996, vol. 93, p. 14833. https://doi.org/10.1073/pnas.93.25.14833

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sasaki, Y., Ohsawa, K., Kanazawa, H., Kohsaka, S., and Imai, Y., Iba1 is an actin-cross-linking protein in macrophages/microglia, Biochem. Biophys. Res. Commun., 2001, vol. 286, p. 292. https://doi.org/10.1006/bbrc.2001.5388

    Article  CAS  PubMed  Google Scholar 

  40. Stanley, E.R. and Chitu, V., CSF-1 receptor signaling in myeloid cells, Cold Spring Harb. Perspect. Biol., 2014, vol. 6, p. a021857. https://doi.org/10.1101/cshperspect.a021857

    Article  PubMed  PubMed Central  Google Scholar 

  41. Stansley, B., Post, J., and Hensley, K., A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s disease, J. Neuroinflammation, 2012, vol. 9, p. 115. https://doi.org/10.1186/1742-2094-9-115

    Article  PubMed  PubMed Central  Google Scholar 

  42. Takamori, Y., Mori, T., Wakabayashi, T., Nagasaka, Y., Matsuzaki, T., and Yamada, H., Nestin-positive microglia in adult rat cerebral cortex, Brain Res., 2009, vol. 1270, p. 10. https://doi.org/10.1016/j.brainres.2009.03.014

    Article  CAS  PubMed  Google Scholar 

  43. Waller, R., Baxter, L., Fillingham, D.J., Coelho, S., Pozo, J.M., Mozumder, M., Frangi, A.F., Ince, P.G., Simpson, J.E., and Highley, J.R., Iba-1-/CD68+ microglia are a prominent feature of age-associated deep subcortical white matter lesions, PLoS One, 2019, vol. 25, p. e0210888. https://doi.org/10.1371/journal.pone.0210888

    Article  CAS  Google Scholar 

  44. Wohl, S.G., Schmeer, C.W., Friese, T., Witte, O.W., and Isenmann, S., In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo, PLoS One, 2011, vol. 6, p. e22408. https://doi.org/10.1371/journal.pone.0022408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wong, A.M., Patel, N.V., Patel, N.K., Wei, M., Morgan, T.E., de Beer, M.C., de Villiers, W.J., and Finch, C.E., Macrosialin increases during normal brain aging are attenuated by caloric restriction, Neurosci. Lett., 2005, vol. 390, p. 76. https://doi.org/10.1016/j.neulet.2005.07.058

    Article  CAS  PubMed  Google Scholar 

  46. Yan, P., Wu, X., Liu, X., Cai, Y., Shao, C., and Zhu, G., A causal relationship in spinal cord injury rat model between microglia activation and EGFR/MAPK detected by overexpression of microRNA-325-3p, J. Mol. Neurosci., 2019, vol. 68, p. 181. https://doi.org/10.1007/s12031-019-01297-w

    Article  CAS  PubMed  Google Scholar 

  47. Yang, Y., Sun, Y., Hu, R., Yan, J., Wang, Z., Li, W., and Jiang, H., Morphine promotes microglial activation by upregulating the EGFR/ERK signaling pathway, PLoS One, 2021, vol. 16 P. e0256870. https://doi.org/10.1371/journal.pone.0256870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by National Research Center “Kurchatov Institute” and the Joint Institute for Nuclear Research.

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Authors and Affiliations

  1. National Research Center “Kurchatov Institute”, 123182, Moscow, Russia

    D. A. Shaposhnikova, E. Yu. Moskaleva, Yu. P. Syomochkina & O. V. Vysotskaya

  2. Joint Institute for Nuclear Research, 141980, Dubna, Russia

    O. V. Komova, E. A. Nasonova & I. V. Koshlan

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  1. D. A. Shaposhnikova
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  2. E. Yu. Moskaleva
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Correspondence to D. A. Shaposhnikova.

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Abbreviations: AFN—active form of nitrogen; ROS—reactive oxygen species; AChr—chromosomal aberration; LPS—lipopolysaccharide; SC—stem cells, EDTA—ethylenediaminetetraacetic acid; EGTA—ethylene glycol-di-(2-aminoethyl)-tetraacetic acid; CNS—central nervous system; CSF-1—colony stimulating factor-1; CSF-1R—CSF-1 receptor; EGF—epidermal growth factor; EGFR—EGF receptor; mFISH—multicolor fluorescent hybridization in situ; PBS—phosphate-buffered saline solution.

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Shaposhnikova, D.A., Moskaleva, E.Y., Syomochkina, Y.P. et al. Characteristics of SIM-A9 Microglia Cells: New Data. Cell Tiss. Biol. 17, 503–516 (2023). https://doi.org/10.1134/S1990519X23050127

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