Discoordination of the processes of activation and suppression of immunocompetent cells during mesotherapy with hyaluronic acid

Objective. The study aimed to assess the dynamics of the systemic immune response in patients during mesotherapy with hyaluronic acid (hA). Materials and methods. The observation group included 26 women who received the first-time mesocorrection with drugs based on hyaluronic acid (hA). Injections of low molecular weight hA were carried out according to standard methods in a course of 5 procedures with an interval of 14 days. The parameters of the innate and adaptive immune response were studied before and two weeks after the end of the course. The skin condition was assessed by dermatoscopy (Aramo Smart Lite 300, Southern Korea). Results. The clinical effect of the course of procedures was reflected in the subjective improvement in the appearance. The improvement was confirmed by hardware analysis, which recorded an increase in hydration, a smoothing of the skin relief, and a decrease in the depth of wrinkles. At the end of the course, redistribution of lymphocyte populations towards natural killer cells and B-lymphocytes was revealed with a decrease in the total number of T cells. The antibody production of immunoglobulins of classes M and G was increased, the serum content of Ig A and IgE was reduced, the number of both T-effectors and T-lymphocytes with immunosuppressive activity increased. Changes in the neutrophil system were characterized by the inhibition of the production of reactive oxygen intermediates; the dynamics of the expression of Toll-like receptors by monocytes was ambiguous. Conclusion. The results of the study confirmed the active involvement of the factors of innate and adaptive systemic response in the cosmetic effect, which manifested itself immediately after mesotherapy as systemic dysregulatory immune changes.


Introduction
P resently, esthetic medicine develops intensively. It aims to correct the appearance of a person with medical methods [1,2]. A specific peculiarity of this type of medicine is not only the diversity of methods and technologies applied but also the fact that their practical application significantly advances the theoretical justification of the mechanism of action. In this aspect, one of the leading positions (from the point of view of vast practical application) is occupied by mesotherapy with hyaluronic acid (hA) [3,4]. The technology is based on the disturbance of the skin by injections of hA that exerts expressed immune tropic effect and involves the immune system to have an effect. The ambivalence of hA effects is well-known. Its highly molecular variant exerts antiinflammatory, wound-healing, and immune-suppressive effects, while its low molecular fragments exert stimulating effects due to the impact on pattern recognizing receptors, which leads to the activation of signaling pathways of the synthesis of anti-inflammatory mediators [5]. The available modern publications on this subject are not consistent and, as a rule, they are dedicated to the characteristics of single factors of the immune response, do not contain the multicomponent analysis, and interaction of the reactions of the congenital and adaptive immunity of patients. These facts provide the rationale for the present study.
The study aimed to assess the dynamics of the systemic immune response in patients during mesotherapy with hA.

Materials and Methods
The group of observation included 26 women aged 45±10 years old) that underwent mesocorrection with hA-based drugs for the first time. The criteria of exclusion included previous mesotherapy, hormonal replacement therapy, combined oral contraceptives, acute infectious pathology, comorbid somatic pathology at the stage of subcompensation or decompensation, pregnancy, and lactation. The injection of low molecular hA was made by a standard method in a course of 5 procedures with a 14-day interval. Before the procedures and two weeks after the course, the parameters of the inborn and adaptive immunity were studied that included the evaluation of the expression of CD3, CD4, CD8, CD16, CD19, and CD25 by lymphocytes, intracellular levels of Foxp3, expression of TLR2, TLR4, and TLR9 on monocytes in the peripheral blood. The oxygen-dependent metabolic activity of neutrophils was evaluated by the nitroblue tetrazolium (NBT) test. The content of serum immunoglobulins A, M, and G was estimated by the reaction of radial immune diffusion in the gel according to the Manchini method, the level of IgEby enzyme-linked immunosorbent assay. The evaluation of the skin condition (moisture content, the depth of folds and wrinkles) was performed using an apparatus Aramo Smart Lite 300. The study was carried out at the facilities of Ekaterininskaya Clinics, LLC and Chain of European Laboratories, LLC. Statistical analysis was conducted in the software R (version 3.2, R Foundation for Statistical Computing, Vienna, Austria). The results were presented as a central tendency for the median and interquartile range (25 and 75 percentile), in the text presented as Me [LQ; UQ]. The group medians were compared using Wilcoxon's test for linked samples. The differences were statistically significant at p < 0.05.

Results
The objective reflection of the subjective perception of the course of mesotherapy with hA was the analysis of the results of dermatoscopy using Aramo Smart Lite 300. The comparison of such parameters as moisture content and skin relief, evaluated before the procedures and two weeks after the therapy, showed an increase in the parameters of moisture content from 53% to 87% and a decrease in the relief of the skin folds and wrinkles from 74% to 45%. The changes in the immune response of patients were observed at the level of leukopoiesis as a statistically significant decrease in the total number of leukocytes (before injections -6.65×10 9 /L [5.2;7.9]; after injections -6.25×10 9 /L [5.2;7.4], p = 0.003) and an increase in the relative number of lymphocytes (before injections -32.5% [27;35], after injections -36.5% [30;42], p = 0.005). The analysis of the parameters that characterize the functioning of the factors of inborn immunity showed that in comparison with the data obtained before the procedures, the changes after a

Discussion
Mesotherapy is a course intradermal injection of different drugs. In the present study, low molecular hA was used for cosmetic effect due to the improvement of the skin properties at the site of injection. The obtained results showed that all the participants reached the desirable results. This was not only a subjective perception of the appearance improvement but was also proved instrumentally by the results of dermatoscopy, which showed an increase in the moisture level, smoothening of the relief, and a decrease in the depth of wrinkles. It can be suggested that the immune system of the skin is involved in the realization of the obtained effect due to the immune trophic properties of hA. Since skin-associated lymphoid tissue is one of the of T-effectors was increased, and the differentiation of the natural killers. It should be highlighted that the data were obtained that confirmed the association of two oppositely directed reactions of immune regulation: enhancement of the processes of activation of T-lymphocytes and a shift of differentiation of T-helpers to CD4 + CD25 + Foxp3 +lymphocytes with immune suppressive activity. It should be mentioned that the parameters of the inborn immunity at the systemic level were characterized by both an increase and a decrease in the functional potency. Along with the activation of natural killers, in the system of neutrophils, a suppression of oxygen-producing activity was registered, and the changes in the monocyte cells were ambiguous: an increase in the expression of TLR 2 and TLR 9 was associated with a tendency to a decrease in the number of TLR4 + -monocytes ( Figure 1). It should be mentioned that the obtained data agree with the results obtained by other authors that indicated a consecutive involvement of pro-inflammatory [6] and anti-inflammatory [7] cytokines in response to hA, an important role of Treg [8][9][10] in the recreation and maintenance of collagen, and involvement of Toll-like receptors in the initiation of immune tropic effects of hA [11,12]. The presented publications are dedicated to the characteristics of single parameters, while the obtained data confirm the association of all the mentioned factors.

Conclusion
The results of the study reflect the dynamics of the systemic immune changes right after the end of the course of mesotherapy with hA. Further observation after the development of these patients allowed defining the period of resolution of the appeared dysregulatory changes and establishing the character and degree of the expression of these changes with the duration and quality of the clinical effect.