Large Extracellular Vesicles Derived from Natural Killer Cells Affect the Functions of Monocytes.

Communication between natural killer cells (NK cells) and monocytes/macrophages may play an important role in immunomodulation and regulation of inflammatory processes. The aim of this research was to investigate the impact of NK cell-derived large extracellular vesicles on monocyte function because this field is understudied. We studied how NK-cell derived large extracellular vesicles impact on THP-1 cells characteristics after coculturing: phenotype, functions were observed with flow cytometry. In this study, we demonstrated the ability of large extracellular vesicles produced by NK cells to integrate into the membranes of THP-1 cells and influence the viability, phenotype, and functional characteristics of the cells. The results obtained demonstrate the ability of large extracellular vesicles to act as an additional component in the immunomodulatory activity of NK cells in relation to monocytes.

Apoptotic Receptors and CD107a Expression by NK Cells in an Interaction Model with Trophoblast Cells.

Natural killer cells (NK cells) exert cytotoxicity towards target cells in several ways, including the expression of apoptosis-mediating ligands (TRAIL, FasL). In addition, NK cells themselves may be susceptible to apoptosis due to the expression of TRAIL receptors. These receptors include TRAIL-R1 (DR4), TRAIL-R2 (DR5), capable of inducing apoptosis, and TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), the so-called "decoy receptors", which lack an intracellular domain initiating activation of caspases. Of particular interest is the interaction of uterine NK cells with cells of fetal origin, trophoblasts, which are potential targets for natural killer cells to carry out cytotoxicity. The aim of this work was to evaluate the expression of proapoptotic receptors and their ligands as well as CD107a expression by NK cells in a model of interaction with trophoblast cells. To evaluate NK cells, we used cells of the NK-92 line; cells of the JEG-3 line were used as target cells. The cytokines IL-1ß, IL-15, IL-18, TNFα, IL-10, TGFß and conditioned media (CM) of the first and third trimester chorionic villi explants were used as inducers. We established that cytokines changed the expression of apoptotic receptors by NK cells: in the presence of TNFα, the amount and intensity of Fas expression increased, while in the presence of TGFß, the amount and intensity of expression of the DR5 receptor decreased. Soluble chorionic villi factors alter the expression of TRAIL and FasL by NK-92 cells, which can reflect the suppression of the TRAIL-dependent mechanism of apoptosis in the first trimester and stimulating the Fas-dependent mechanism in the third trimester. In the presence of trophoblast cells, the expression of TRAIL and DcR1 by NK cells was reduced compared to intact cells, indicating an inhibitory effect of trophoblast cells on NK cell cytotoxicity. In the presence of chorionic villi CM and trophoblast cells, a reduced number of NK-92 cells expressing DR4 and DR5 was found. Therefore, soluble factors secreted by chorionic villi cells regulate the resistance of NK cells to death by binding TRAIL, likely maintaining their activity at a certain level in case of contact with trophoblast cells.

Deep Semantic Segmentation of Angiogenesis Images.

Angiogenesis is the development of new blood vessels from pre-existing ones. It is a complex multifaceted process that is essential for the adequate functioning of human organisms. The investigation of angiogenesis is conducted using various methods. One of the most popular and most serviceable of these methods in vitro is the short-term culture of endothelial cells on Matrigel. However, a significant disadvantage of this method is the manual analysis of a large number of microphotographs. In this regard, it is necessary to develop a technique for automating the annotation of images of capillary-like structures. Despite the increasing use of deep learning in biomedical image analysis, as far as we know, there still has not been a study on the application of this method to angiogenesis images. To the best of our knowledge, this article demonstrates the first tool based on a convolutional Unet++ encoder-decoder architecture for the semantic segmentation of in vitro angiogenesis simulation images followed by the resulting mask postprocessing for data analysis by experts. The first annotated dataset in this field, AngioCells, is also being made publicly available. To create this dataset, participants were recruited into a markup group, an annotation protocol was developed, and an interparticipant agreement study was carried out.

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFß and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFß, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFß, their cytotoxicity increased. In the case of adding TGFß to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFß in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

Effects of Microvesicles Derived from NK Cells Stimulated with IL-1ß on the Phenotype and Functional Activity of Endothelial Cells.

Microvesicles (MVs) are plasma extracellular vesicles ranging from 100 (150) to 1000 nm in diameter. These are generally produced by different cells through their vital activity and are a source of various protein and non-protein molecules. It is assumed that MVs can mediate intercellular communication and modulate cell functions. The interaction between natural killer cells (NK cells) and endothelial cells underlies multiple pathological conditions. The ability of MVs derived from NK cells to influence the functional state of endothelial cells in inflammatory conditions has yet to be studied well. In this regard, we aimed to study the effects of MVs derived from NK cells of the NK-92 cell line stimulated with IL-1ß on the phenotype, caspase activity, proliferation and migration of endothelial cells of the EA.hy926 cell line. Endothelial cells were cultured with MVs derived from cells of the NK-92 cell line after their stimulation with IL-1ß. Using flow cytometry, we evaluated changes in the expression of endothelial cell surface molecules and endothelial cell death. We evaluated the effect of MVs derived from stimulated NK cells on the proliferative and migratory activity of endothelial cells, as well as the activation of caspase-3 and caspase-9 therein. It was established that the incubation of endothelial cells with MVs derived from cells of the NK-92 cell line stimulated with IL-1ß and with MVs derived from unstimulated NK cells, leads to the decrease in the proliferative activity of endothelial cells, appearance of the pan leukocyte marker CD45 on them, caspase-3 activation and partial endothelial cell death, and reduced CD105 expression. However, compared with MVs derived from unstimulated NK cells, a more pronounced effect of MVs derived from cells of the NK-92 cell line stimulated with IL-1ß was found in relation to the decrease in the endothelial cell migratory activity and the intensity of the CD54 molecule expression on them. The functional activity of MVs is therefore mediated by the conditions they are produced under, as well as their internal contents.

NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells.

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.