mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-EOM-2276mimmun-2416Research ArticleМАТЕРИАЛЫ ФОРУМА "ДНИ ИММУНОЛОГИИ В СПБ" 2021ВЛИЯНИЕ РАСТВОРИМЫХ ФАКТОРОВ МАКРОФАГОВ М2-ФЕНОТИПА НА ДИФФЕРЕНЦИРОВКУ КЛЕТОК ЛИНИИ SH-SY5YEFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLSРащупкинИ. М.RashchupkinI. M.

аспирант лаборатории клеточной иммунотерапии,

630099, г. Новосибирск, ул. Ядринцевская, 14

Postgraduate Student, Laboratory of Cellular Immunotherapy,

630099, Novosibirsk, Yadrintsevskaya str., 14

ШевелаЕ. Я.ShevelaE. Ya.

д.м.н., ведущий научный сотрудник лаборатории клеточной иммунотерапии,

630099, г. Новосибирск, ул. Ядринцевская, 14

PhD, MD (Medicine), Leading Research Associate, Laboratory of Cellular Immunotherapy,

630099, Novosibirsk, Yadrintsevskaya str., 14

ЧерныхЕ. Р.ChernykhE. R.

д.м.н., профессор, член-корр. РАН, заведующая лабораторией клеточной иммунотерапии,

630099, г. Новосибирск, ул. Ядринцевская, 14

PhD, MD (Medicine), Professor, Corresponding Member, Russian Academy of Sciences, Head, Laboratory of Cellular Immunotherapy,

630099, Novosibirsk, Yadrintsevskaya str., 14

ФГБНУ «Научно-исследовательский институт фундаментальной и клинической иммунологии»РоссияResearch Institute of Fundamental and Clinical ImmunologyRussian Federation202117102021234677684Copyright © Ращупкин И.М., Шевела Е.Я., Черных Е.Р., 20212021Ращупкин И.М., Шевела Е.Я., Черных Е.Р.Rashchupkin I.M., Shevela E.Y., Chernykh E.R.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/2416

Известно, что макрофаги играют важную роль в запуске и регуляции процессов нейрорегенерации. Макрофаги характеризуются выраженной пластичностью, что проявляется в их способности изменять свой фенотип в зависимости от сигналов микроокружения. В условиях патологии одним из индукторов формирования противовоспалительного М2-фенотипа макрофагов является взаимодействие с апоптотическими клетками (эффероцитоз). Ранее нами был разработан оригинальный протокол генерации М2-подобных макрофагов, поляризованных в результате эффероцитоза в условиях депривации сыворотки. Целью настоящего исследования явилась оценка нейрорегенеративного потенциала таких макрофагов (M2 (LS), Low Serum). Исследовали влияние кондиционных сред М2 (LS) на процесс дифференцировки клеток линии SH-SY5Y в сравнении c ретиноевой кислотой (RA). В качестве морфологических критериев дифференцировки оценивали относительное содержание дифференцированных клеток, т.е. клеток с длиной нейритов, превышающей длину тела клетки, а также среднюю длину нейритов на 3-и, 7-е и 13-е сутки. Наряду с этим оценивали соотношение нейроноподобных (N-тип) и эпителиально-подобных (S-тип) клеток в культурах. Клетки SHSY5Y характеризовались низким уровнем спонтанной дифференцировки как в стандартных условиях (10% FBS), так и при депривации сыворотки (1% FBS). При обработке ретиноевой кислотой клетки SH-SY5Y прекращали делиться и подвергались нейрональной дифференцировке. Культивирование клеток SH-SY5Y в присутствии кондиционной среды (КС)-M2 (LS) (30% v/v) также приводило к значимому возрастанию относительного содержания дифференцированных клеток, средней длины нейритоподобных отростков, а также изменению баланса клеток S- и N-типа в сторону выраженного преобладания последних. При этом выраженность морфологических признаков дифференцировки на ранних этапах дифференцировки (3-и сут.) была достоверно снижена по сравнению с уровнем RA-индуцированных изменений и достигала уровня позитивного контроля только на более поздних стадиях (к 13-м сут.) (p < 0,05). В отличие от ретиноевой кислоты, КС-М2 (LS) индуцировали нейрональную дифференцировку клеток SH-SY5Y без подавления их пролиферативной активности. Полученные данные могут свидетельствовать о нейрорегенеративном потенциале M2 (LS) макрофагов in vitro, который опосредуется через растворимые факторы и проявляется в усилении дифференцировки клеток SH-SY5Y. 

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. 

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The authors declare that there are no conflicts of interest present.