The article presents the results of a study of the effect of anthocyanins on cellular immunity in rats on a model of alimentary obesity. The aim of the study was to study the effect of an anthocyanin- enriched diet on cellular immunity in diet induced obesity in rats. The study was carried out on male Wistar rats with an initial body weight of 108±2 g. The animals were randomized by body weight into 3 groups (8 pcs. in group). For 12 weeks, rats of the 1st (control) group received a complete modified diet of AIN93M; rats of the 2nd group consumed a high-calorie choline-deficient diet (HCChDD), the fat content of which was 45%, fructose – 20% of the energy value of the diet; rats of the 3rd group received HCChDD with the addition of standardized blueberry and blackcurrant extract (30% anthocyanins) at an average daily dose of 11 mg anthocyanins/kg body weight. The expression of differentiation markers of peripheral blood lymphocytes was carried out by flow cytofluorimetry. As a result of the study, it was found that in rats of the 2nd group with alimentary obesity, the relative content in the peripheral blood of T helpers (CD3+CD4+) was increased (p < 0.05) (75.75±1.11% versus 70.07±0 49% – group 1, 72.14±0.91% – group 3) and reduced (p < 0.05) content of T cytotoxic lymphocytes (CD3+CD8+) (22.54±1.14% versus 28.09±0.72% – 1st group, 26.07±0.87% – 3rd group). The CD3/CD4 ratio in rats of the 2nd group exceeded (p < 0.05) this index in rats of the 1st and 3rd groups (3.44±0.25 versus 2.47±0.09 – 1st group, 2.79±0.13 – 3rd group). Enrichment of the HCChDD with the blueberry and blackcurrant extract led to the normalization of these parameters of cellular immunity. The number of B lymphocytes (CD45R+), Т lymphocytes (CD3+) and NK cells (CD161+) in the rat peripheral blood of all experimental groups had no statistically significant differences. The results of the study of cellular immunity in rats with alimentary obesity indicate the presence of metainflammation. The received data indicate the prospect of using biologically active substances.
Macrophages are involved in the regulation of fibrogenesis and turnover of the extracellular matrix. One way to perform this function is through the production of profibrotic and fibrolytic factors including fibronectin, laminin, collagen, and extracellular matrix proteases. The production of most of them has been well studied in experimental models; however, much remains unclear regarding human macrophages. Therefore, the aim of this study was to study the content of extracellular matrix proteases (MMP-2 and MMP-9, cathepsin L), their inhibitors (TIMP-1), and collagen (type I) in supernatants of differently activated human macrophages. We compared macrophages differentiated by M-CSF or GM-CSF and further polarized in M1 with lipopolysaccharide, in M2a with IL-4, and in M2c with dexamethasone. Macrophages was obtained from peripheral blood monocytes. The content of MMPs, TIMP, cathepsin, and collagen was determined using appropriate ELISA kits. The results obtained demonstrate that differentiation factors are more important for the production of the above factors compared to polarizing stimuli (lipopolysaccharide, IL-4, dexamethasone). Moreover, macrophages differentiated by M-CSF showed predominantly antifibrotic activity because of pronounced MMPs production, while GM-CSF-induced cultures, on the contrary, were characterized by profibrotic properties due to the high level of TIMP-1 and type I collagen. M1, M2a, and M2c, induced by M-CSF, differed only in MMP-2 production, and M2a produced this metalloproteinase more than other subtypes. In the case of GM-CSF-differentiated cells, a higher level of production of TIMP-1 and, to a lesser extent, type I collagen was characteristic of M1, whereas M2c have minimal concentration of them among GM-CSF-induced macrophage subtypes. Concerning the level of cathepsin L production was relatively constant and did not depend on the generation conditions (differentiation and polarizing signals). Thus, the data obtained help to identify macrophage subtypes with anti- or profibrotic potential and may be useful for the development of cell therapy for diseases associated with fibrogenesis dysregulation.
Heat shock proteins 70 kDa (HSP70) protect intracellular proteins from the damaging effects of stress factors of various natures. Moreover, HSP70 play an important role in the vital activity of cells under normal physiological conditions, performing chaperone functions. These functions are realized in the intracellular space; however, in some cases, these proteins are also found on the cell surface and in the extracellular environment. The causes and mechanisms of HSP70 translocation to the cell surface and secretion into the extracellular space have not yet been well understood, but such an unusual localization of HSP70 activates the immune system. The surface HSP70 and their extracellular pool stimulate the cytotoxic activity of NK cells. However, direct experimental evidence for the internalization of HSP70 molecules by NK cells has not yet been demonstrated. This paper presents the results of the interaction of the extracellular HSP70 pool with NK cells from the peripheral blood. The results demonstrated the confirmation of the internalization of exogenous HSP70 molecules by NK cells. To this end, fluorescently labeled recombinant stress-inducible human HSP70 were obtained. The electrophoretic data indicated the absence of protein degradation during the labeling process, the purity and stability of the modified protein. To assess the interaction of HSP70 with NK cells, the fluorescently labeled HSP70 was added to an in vitro culture of NK cells isolated by magnetic separation from the peripheral blood mononuclear fraction and analyzed by confocal microscopy. This analysis indicated that living NK cells internalize extracellular HSP70 with localization both in lysosomes and in phagosomes. Our experiments illustrated for the first time the process of penetration of the extracellular form of HSP70 into these cells. The results suggest that the activation of NK cells under the action of exogenous HSP70 could be associated with the internalization of these protein molecules.
The pandemic of the new COVID-19 coronavirus infection has created a public health emergency in the Russian Federation in 2020-2022. COVID-19 causes various consequences, often manifested by the endocrine system dysfunction. The rationale for our study is insufficient data on T and NK cell immunity in patients with hyperglycemia after COVID-19. The study was aimed at the features of T cell immunity in individuals with post-COVID syndrome and disorders of carbohydrate metabolism, depending on the NK cells count. Materials and methods: Sixty-four post-COVID patients with carbohydrate metabolism disorders were divided into three groups: with reduced, normal, or elevated NK cell counts. Carbohydrate metabolism disorders included impaired glucose tolerance (n = 36) and type 2 diabetes mellitus (n = 28). The comparison group comprised 60 post-COVID persons with no history of carbohydrate metabolism disorders. The assessment of the lymphocytic link of immunity included the definition of: CD45 + CD3 + (T lymphocytes), CD45 + CD3 + CD4 + (T helpers), CD45 + CD3 + CD8 + (T cytotoxic), CD45 + CD3 + CD16 + CD56 + (T-NK cells), CD45 + CD3 - CD16 + CD56 + (natural killers), CD45 + CD3 + CD4 + CD25 + (T lymphocytes – early activation), CD45 + CD3 + HLA-DR + (T lymphocytes – late activation). Results and Discussion. As the study showed, a decrease in the number of natural killers was accompanied by a higher level of T helpers in the group with carbohydrate metabolism disorders, the latter is probably associated with a compensatory increase in T lymphocytes and dysregulation of the T cell link of the immune system. It can also be concluded that in patients with long-COVID and CMD, with normal NK cell counts, an altered subpopulation composition remains, namely, a significant increase in total T lymphocytes. Many authors associate the obtained data on a significant decrease in T-NK lymphocytes with a decrease in the antiviral activity of the immune system, which can lead to a poor response to new viral agents or contribute to the activation of chronic viral infections. Dysregulation of the T cell link in individuals with long-COVID and disorders of carbohydrate metabolism requires a more detailed study, including an assessment of the cytokine profile in this category of patients.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that primarily suppress T lymphocytes in healthy pregnancies and pathologies. MDSCs are one of the key regulators of immune responses. Finding ways to control them is important for the treatment of cancer, autoimmune diseases, miscarriage, and post-transplant complications. The mechanisms of immune suppression by MDSC are: expression of CD73, ADAM17, PD -L1, production of Arg 1, iNOS, IDO, IL -10 and TGF-b1. Pregnancy-specific b1-glycoprotein (PSG) has modulatory effects on dendritic cells and macrophages that mediate the shift of T cell phenotypes toward Th2 and Treg. We have previously shown that native PSG suppresses Th17 differentiation and cytokine production, stimulates the production of IDO by monocytes and the differentiation of Tregs. Considering the immunomodulatory properties of PSG and the key role of MDSCs in pathologies, the aim of our work was to investigate the effect of native and recombinant PSG on the differentiation of MDSCs in vitro . MDSCs were differentiated from CD11b + peripheral blood cells. Cells were cultured for 7 days and received stepwise GM-CSF, IL-1b, and LPS. Native (n) (1; 10 and 100 mg/mL) and recombinant (r) (1 and 10 mg/mL) PSG were introduced into the cultures three days before the end of incubation. Flow cytometry was used to determine the percentage of MDSC among the cells in culture and the percentage of M-, PMN-, and e-MDSC among the total number of MDSCs. It was found that rPSG (1 mg/mL) increased the percentage of MDSCs in culture. Both nPSG (1 and 10 mg/mL) and rPSG (10 mg/mL) increased the proportion of M-MDSC, whereas rPSG (10 mg/mL) decreased the number of PMN-MDSC. Thus, the cytokine background in CD11b + cell cultures favored the differentiation of predominantly M-MDSC, similar to the tumor microenvironment, whereas native and recombinant PSG enhanced this effect. Thus, nPSG and rPSG are able to modulate the differentiation of MDSCs by increasing their number, mainly due to the monocytic subpopulation. This fact opens perspectives for new research on targeted manipulation of MDSCs.
