Membrane and Cell BiologyMembrane and Cell BiologyБиологические мембраны0233-47553034-5219The Russian Academy of Sciences66746110.31857/S0233475524020058xttsenArticlesСТАТЬИResearch ArticleA New Approach to Analyze the State of the Complement System in Patients with COVID-19. Pilot StudyНовый подход к анализу состояния системы комплемента у больных COVID-19. Пилотные исследованияAvdoninP. P.АвдонинП. П.
Russian Federation
ppavdonin@gmail.comKomlevaL. А.КомлеваЛ. А.
Russian Federation
ppavdonin@gmail.comBlinovaM. S.БлиноваМ. С.
Russian Federation
ppavdonin@gmail.comIvanovaЕ. S.ИвановаЕ. С.
Russian Federation
ppavdonin@gmail.comKotenkoO. N.КотенкоО. Н.
Russian Federation
ppavdonin@gmail.comFrolovaN. F.ФроловаН. Ф.
Russian Federation
ppavdonin@gmail.comStolyarevichE. S.СтоляревичЕ. С.
Russian Federation
ppavdonin@gmail.comRybakovaE. Y.РыбаковаЕ. Ю.
Russian Federation
ppavdonin@gmail.comAvdoninP. V.АвдонинП. В.
Russian Federation
ppavdonin@gmail.comKoltzov Institute of Developmental Biology, Russian Academy of SciencesИнститут биологии развития им. Н. К. Кольцова РАНMoscow City Clinical Hospital 52ГБУЗ Городская клиническая больница № 52 Департамента здравоохранения города Москвы1406202441213914826022025Copyright ©; 2024, The Russian Academy of SciencesCopyright ©; 2024, Российская академия наук2024The Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0233-4755/article/view/667461

One of the risk factors for infection caused by SARS-CoV-2 is hyperactivation of the complement system, which can lead to activation and damage to the endothelium, thrombosis and, in some severe cases, the development of multiple organ failure. Using quantitative immunohistochemistry, we studied the opsonization of human umbilical vein endothelial cells (HUVEC) by complement factors C3/C3b when exposed to blood plasma from patients with a confirmed diagnosis of COVID-19 in order to evaluate the possibility of using this approach to study the state of the complement system in COVID-19 ex vivo. For these purposes, FITC-labeled antibodies specific to the C3c domain of factors C3/C3b were used. The integral intensity of the recorded fluorescence and the number of C3/C3b-immunopositive structures were chosen as parameters for assessing the binding of C3/C3b to cells. We have shown that when HUVEC are incubated with plasma from patients, the total number of C3/C3b-immunopositive structures recorded on the membrane of these cells is 5.8 ± 2.8 times (mean ± SD, p < 0.001) higher than when exposed to plasma from healthy people. In this case, the integrated fluorescence intensity increased by 6.3 ± 3.2 times (mean ± SD, p ≤ 0.0001) compared to the control. The area of immunopositive structures recorded after exposure to plasma cells from healthy donors and patients with COVID-19 and selected for analysis ranged from 2.2 to 70 μm². Immunopositive particles with an area of 2.2–10.9 μm² after incubation with plasma of COVID-19 patients had a more elongated shape compared to controls. The average number of particles per cell was 0.49 ± 0.06 (mean ± SD, n = 6) in the control, and 2.4 ± 0.4 (mean ± SD, n = 13, p < 0.001) during incubation with patient plasma. Analysis of particle area distribution showed that the most pronounced differences in the number of C3/C3b-immunopositive structures compared to the control were observed among large particles. Thus, we showed an increase in the level of opsonization of endothelial cells by complement factors C3/C3b in the presence of plasma from COVID-19 patients in comparison with control plasma. The increase is due to an increase in the number of C3/C3b-immunopositive structures, mostly large ones. We assume that the proposed approach will allow us to study the role of the complement system in damage to vascular endothelial cells in patients with COVID-19 using an ex vivo model, as well as to evaluate the level of complement activation in the plasma of patients and the effectiveness of their treatment.

Одним из факторов риска при инфекции, вызванной SARS-CoV-2, является гиперактивация системы комплемента, которая может приводить к активации и повреждению эндотелия, тромбозу и, в ряде тяжелых случаев, развитию полиорганной недостаточности. Методом количественной иммуноцитохимии мы исследовали опсонизацию факторами комплемента С3/С3b эндотелиальных клеток пупочной вены человека (HUVEC) при воздействии на них плазмы крови пациентов с подтвержденным диагнозом COVID-19, чтобы оценить возможность использования данного подхода к изучению состояния системы комплемента при COVID-19 ex vivo. Для этих целей были использованы FITC-меченые антитела, специфичные к С3с домену факторов С3/С3b. В качестве параметров для оценки связывания С3/С3b с клетками были выбраны интегральная интенсивность регистрируемой флуоресценции и число С3/С3b- иммунопозитивных структур. Мы показали, что при инкубации HUVEC с плазмой больных общее число C3/C3b-иммунопозитивных структур, регистрируемых на мембране этих клеток, в 5.8±2.8 раз (mean ± SD, p < 0.001) выше, чем при воздействии плазмы здоровых людей. При этом интегральная интенсивность флуоресценции возрастала в 6.3±3.2 раз (mean ± SD, p ≤ 0.0001) по сравнению с контролем. Площадь иммунопозитивных структур, регистрируемых после воздействия на клетки плазмы здоровых доноров и больных COVID-19 и выбранных для анализа, составляла от 2.2 до 70 мкм2. Иммунопозитивные частицы площадью 2.2–10.9 мкм2 после инкубации с плазмой больных COVID-19 имели более вытянутую форму в сравнении с контролем. Среднее число частиц на клетку составляло в контроле 0.49 ± 0.06 (mean±SD, n = 6), при инкубации с плазмой больных 2.4 ± 0.4 (mean ± SD, n = 13 р < 0.001). Анализ распределения частиц по площади показал, что наиболее выраженные отличия в количестве C3/C3b-иммунопозитивных структур в сравнении с контролем наблюдались среди крупных частиц. Таким образом, мы показали рост уровня опсонизации эндотелиальных клеток факторами комплемента С3/С3b в присутствии плазмы больных COVID-19 в сравнении с контрольной плазмой. Прирост обусловлен увеличением числа C3/C3b-иммунопозитивных структур, в большей степени крупных. Мы предполагаем, что разработанный нами подход позволит исследовать роль системы комплемента в повреждении клеток эндотелия сосудов у больных COVID-19 на модели ex vivo, а также оценивать уровень активации комплемента в плазме больных и эффективность их лечения.

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