Bukreieva T.V. Peculiarities of the effect of umbilical cord-derived multipotent mesenchymal stromal cells on the immune system of patients with pneumonia caused by the SARS-CoV-2 coronavirus – qualifying scientific work оn the rіght оf а mаnuscrірt. The thesіs fоr а scіentіfіc degree оf Dоctоr оf Рhіlоsорhy by sрecіаlіty 091 – Bіоlоgy (09 – Bіоlоgy) – Іnstіtute оf Mоleculаr Bіоlоgy аnd Genetіcs NАS оf Ukrаіne, Kyіv, 2024. The dissertation is devoted to the characterization of dynamic changes in the subpopulations of immune cells in the peripheral blood of patients with acute respiratory distress syndrome (ARDS) caused by severe COVID-19 pneumonia, the characterization of lung damage degree changes, levels of cytokines, chemokines and miRNAs after transplantation of cryopreserved allogeneic umbilical cord-derived MSCs, and the search of MSC action targets. Replication of SARS-CoV-2 caused coronavirus disease COVID-19 and leads to direct damage of lung tissue and the development of ARDS. Infected cells recruit T-lymphocytes, monocytes, and neutrophils, which release excessive amounts of cytokines. Changes in miRNAs associated with inflammation were also observed. Thus, the pathogenesis of ARDS during COVID-19 is the result of an overreaction of the host's immune system. The study of changes in the patients’ immune system functions is important for understanding the pathophysiology of COVID-19 and the treatment strategy, and normalizing the imbalance of the immune system and reducing the consequences of ARDS is a promising task for effective treatment. MSCs are safe when used in cell therapy and can modulate both innate and adaptive immunity through direct intercellular interactions with immune cells or by paracrine effect via the secretion of chemokines, growth factors, and cytokines. In this work, human UC-MSCs are characterized in context of their application for ARDS therapy dirung COVID-19. Repeated transfusions of UC-MSC have been shown to be safe and have no significant side effects. It has been proven that MSC therapy has a long-term (up to one year) improvement in lungs fibrosis in ARDS patients according to CT scans. It is shown that the presence of myelocytosis, leukopenia, and lymphopenia in early disease stages, shifting to leukocytosis during treatment. For the first time, we showed an increased content of CD4+ and CD8+ regulatory, activated, exhausted, and senescent memory cells from the day 14 after hospitalization; and an increase in the content of double-positive T cells, CD127- and CD25-expressing T cells, CD3+PD-1low and CD3-PD-1low T cells, monocytes and dendritic cells during 28 days. This research highlights the initial predominance of monocytes and low levels of dendritic cells, followed by a shift indicating adaptive immunity development. For the first time, significantly higher relative expression levels of miR-221-3p, miR-27a-3p, miR-133a-3p, and miR-126-3p were detected at the beginning of hospitalization of patients with ARDS caused by SARS-CoV-2. When comparing clinical and molecular indicators in patients with ARDS with traditional therapy and with three consecutive infusions of UC-MSC, the latter showed accelerated recovery of lymphocytes and the reduction of C-reactive protein level, erythrocytes sedimentation rate and banded neutrophils count, and a significant increase in the content of the anti-inflammatory cytokine IL-10 and pro-inflammatory chemokines IP-10, MIP- 1α and G-CSF. MSC therapy did not significantly affect the relative expression levels of inflammation-associated miRNAs in blood plasma. In vitro experiments revealed that UC-MSCs promote the expression of genes involved in the development of the immune response, the activation of neutrophils, leukocyte migration and phagocytosis in the PBMC of patients with ARDS, while reducing the expression of genes related to apoptosis, and increase the expression of early-activated CD69 marker in T cells and intermediate-stage CD25 marker among CD3+ subpopulations with a decrease in percentages of effector, senescent effector CD8 and CD4 T cells, and memory CD8 T cells. Overall, this dissertation established that triple intravenous transplantation of MSCs with an interval of 3 days at a dose of 1 million of cells per kilo of body weight in the early/middle stage of ARDS caused by SARS-CoV-2 is safe, does not cause significant side effects, and has an immunomodulatory effect, leading to a significant reduction in lung fibrosis after 1 year of observation.
