Novel approaches to increase resistance to acute respiratory infections

Relevance . Respiratory infections are the most common in the world. In order to prevent epidemics, there is a need to improve the strategies for organizing medical care and develop new approaches in order to increase the nonspecific resistance, mobilize innate immunity. Objective . The aim of this study was to investigate the effect of glucosaminylmuramyldipeptide (GMDP) on the level of expression of markers of differentiation and activation of functionally significant subpopulations of dendritic cells in peripheral blood mononuclear cells of healthy donors,the second aim was to assess the effectiveness of GMDP in the prevention of acute respiratory infections in an unfavorable epidemiological period of the COVID-19 pandemic. Materials and Methods . An open comparative study included 309 apparently healthy participants, aged 19-22 years. At the first stage of the study, 42 participants (22 female and 20 male) took the drug Licopid 1 mg for 10 days according to the instructions, 1 tablet 3 times a day in order to prevent acute respiratory infections. Peripheral blood sampling was performed before taking the drug (day 0) and the next day after the last dose of the drug (day 12). Evaluation of the expression of markers of differentiation and activation of dendritic cell subpopulations HLA-DR, CD11c, CD123, CD80, CD83, CCR7, CD3, CD14, CD20 was assessed by flow cytometry. At the same time, mRNA was isolated from mononuclear cells of perfusion blood and, after reverse transcription, the level of gene expression was determined by RT PCR. At the next stage, the effectiveness of the prophylactic use of the drug Licopid in 267 students of the Institute of Physical Culture was assessed in order to prevent acute respiratory infections in an unfavorable epidemiological period; the observation period was 12 months. Results and Discussion . A study of the relative quantitative composition of DCs in the peripheral blood of healthy donors by flow cytometry revealed the possibility of an increase in their total number, as well as subpopulations of MDC and PDC under the influence of GMDP. There was a statistically significant increase in the receptors for the chemokine CCR7, which is responsible for the recruitment of DCs to the secondary lymphoid organs. Analysis of the levels of expression of genes XCR1, CD11b , and CD103 showed a statistically significant effect of GMDP on an increase in their expression compared to the baseline level (before GMDP intake), with the mean value being higher in participants undergoing moderate exercise. It was found that the use of the drug Licopid 1mg for the purpose of preventing and reducing the seasonal incidence of acute respiratory infections at the stage of basic training of students of the Institute of Physical Culture contributed to a decrease in the incidence of acute respiratory infections within 12 months of observation after taking the drug. The number of episodes of acute respiratory infections decreased 3.7 times, while the group with 3 or more episodes of acute respiratory infections during the year, which constituted 14.5 % of participants, completely disappeared. The maximum efficiency of GMDP was observed in the track and field command, in which the number of participants who had no episodes of acute respiratory infections during the year increased by 7 times. Conclusion . Our data complement the modern understanding of the molecular mechanism of action of GMDP and substantiate the possibility of its experimental and clinical use in order to develop new strategies for organizing medical care in order to increase the nonspecific resistance of the organism.


Introduction
Respiratory diseases are the most common in the world and occupy a leading position in the number of medical visits [1]. The variability of microorganisms, including the evolution of viruses in natural ecosystems [2], the adaptation of bacteria to existing drugs, creates a constant threat of the emergence of new infections [3]. In order to prevent epidemics, there is a need to improve strategies for organizing medical care and the development of new drugs. The development of new vaccines and antibiotics takes a long time, and therefore the task of increasing the nonspecific resistance of the organism becomes urgent, including with the help of immunomodulators, which include ligands of innate immunity receptors [4].
Innate immunity receptors (TLR, NLR, RLR, etc.) are widely represented in cells of all organs and tissues, but their maximum amount is found in immunocompetent cells and epithelial cells located in mucous membranes and performing barrier functions [5]. The interaction of innate immunity receptors with their ligands normally leads to the initiation of a cascade of reactions aimed at the formation of an adequate immune response and elimination of the pathogen [6][7][8]. Ligands of innate immunity receptors are widely represented in medical practice as medicines and vaccine components [9][10][11], they are also widely used in experimental models for research in the field of immunity [12,13], in particular, muramyl peptides. Muramylpeptides are part of the peptidoglycan of all known bacteria and some fungi. The sensors of muramyl peptides are NOD1 and NOD2 proteins of intracellular localization, which belong to the NLR family of innate immunity receptors. NOD1 recognize fragments of PULMONOLOGY peptidoglycan from Gram-negative bacteria, NOD2 specifically interact with fragments of peptidoglycan from Gram-negative and Gram-positive bacteria, as a result of which an anti-inflammatory response is initiated. One of the ligands of NOD2 receptor is glucosaminylmuramyl dipeptide (GMDP).
The drug based on GMDP -Licopid -has been used for more than twenty years in medicine to correct the immune status and secondary immunodeficiency states in various nosologies: in surgery -to prevent infectious complications and to correct cytopenias of various etiologies; as part of complex therapy is used in oncology.
The known mechanism of action through NOD2 receptors and its effectiveness in mobilizing an adequate immune response are the basis for the use of GMDP in various pathologies. The use of GMDP in the treatment of children with prolonged acute respiratory viral infections helped to get rid of bacterial complications and reduce relapses [14], in adults it helped to reduce the episodes of acute respiratory viral infections and normalized immunological parameters [15]. The effectiveness of GMDP in activating cellular immunity to fight viral and bacterial infections has been described in details, while the effect of GMDP on dendritic cells (DC) has not been studied. DCs play a leading role in the presentation of antigen to T cells and the formation of antigen-dependent immune responses; they play a central role in the coordination of innate and adaptive immunity, regulation and suppression of inflammatory processes. DC precursors, which are normally present in all tissues and organs, are activated under the action of PAMPs and DAMPs, and their phenotypic and functional characteristics change. The DC community is extremely diverse, several populations are distinguished based on their origin, susceptibility to various activation stimuli, phenotypic and functional characteristics. It is known that, under the action of activation stimuli, the phenotype of dendritic cell precursors changes; markers CCR7, XCR1, and CD103 are expressed on their surface, which makes it possible for them to move in tissues to form an adequate response to an activation stimulus. In addition, dendritic cells provide tolerance to harmless antigens and prevent excessive reactivity of immunocompetent cells [16]. Dendritic cells are a link between innate and adaptive immunity, coordinating the immune response to bacterial and viral infections, as well as to transformed cells [17].
The aim of this study was to study the effect of GMDP on the level of expression of markers of activation and differentiation of dendritic cells isolated from mononuclear cells of the peripheral blood of healthy donors, as well as to evaluate the effectiveness of the drug Licopid in the prevention of acute respiratory infections (ARI) in an unfavorable epidemiological period.

Materials and methods
An open comparative study involved 309 (42 and 267) participants aged 19-22 years. All study participants provided voluntary informed consent to participate in the study in accordance with the World Medical Association's Declaration of Helsinki (WMA Declaration of Helsinki -Ethical Principles for Medical Research Involving Human Subjects, 2013) and the processing of personal data.
At the first stage of the study, 42 participants (22 female and 20 male, aged 19-22) took the drug Licopid 1 mg (AO Peptek, Russia) according to the instructions for 1 tablet 3 times a day in order to prevent acute viral infections of the respiratory tract for 10 days. Peripheral blood sampling was performed before taking the drug Licopid (day 0) and the next day after the last dose of the drug (day 12). Part of the peripheral blood was used for cytometric analysis, the other part was used for isolation of mononuclear cells followed by PCR.
The next stage of the investigation was to assess the effectiveness of glucosaminylmuramyl dipeptide in the prevention of acute viral infections of the respiratory tract during an unfavorable epidemiological period. The investigation involved 267 students (130 female and 137 male) studying at the Kyrgyz State Academy of Physical Training and Sports (Bishkek, Kyrgyzstan) at the stage of basic training with moderate physical activity (2-3 hours a day). The participants were divided into two groups: participants in the control group of 124 people (66 female and 58 male) took GMDP 3 mg sublingually per day for 10 days. The drug Licopid was used according to the indications of the instructions for the prevention of acute respiratory infections. The participants in the comparison group of 143 people (71 female and 72 male) did not use GMDP. The distribution of participants by sport is shown in Table 1. The distribution of participants into groups was carried out based on the number of ARI episodes in the previous period (1 year) in order to evenly distribute participants in each group who had 1-2 (28 %), 3 or more (14 %) ARI episodes per year. Time of the study: April 2020 -March 2021. During the observation period, the number of ARI episodes of participants was recorded, data was collected on the number of cases with a confirmed diagnosis of COVID-19 among study participants and their families.
Inclusion criteria were relative health, age 19-20 years, and willingness to participate in the study.
The exclusion criteria for all surveyed groups were smoking, chronic inflammatory and autoimmune diseases.
Statistical data processing was performed using the Mann -Whitney and Wilcoxon tests using the SPSS and Microsoft Excel software. Data are presented as mean and interquartile range. Values at p <0.05 were considered statistically significant.

Results and discussion
The study of the relative amount and phenotypic characteristics of DCs in the peripheral blood of healthy participants by cytofluorimetry showed the DC content of 1.41 % (0.22-1.85 %). After a 10-day course of GMDP application, the amount of the total DC pool increased to 2.72 % (0.93-2.95 %). Along with this, significant changes in functionally significant populations of DC phenotypes were revealed in relation to the initial values before the intake of GMDP, the level of activated myeloid (MDC) and plasmacytoid (PDC) increased 1.9 times, while the ratio between these populations remained at the same level of -1.8 ( Fig. 1). It is known that the ratio of MDC and PDC in healthy donors is 1.5-2 [19]. The level of deviation of the ratio of MDC and PDC from the level of healthy individuals indicates chronic inflammation, autoimmune processes, oncology and may be the cause of miscarriage [20][21][22]. In particular, in rheumatoid arthritis, the amount of MDC exceeds the amount of PDC by 10.2 times. This change in subtypes was caused by a 6-fold decrease in PDC compared to PDC healthy donors [23].
It is known that DCs are professional antigenpresenting cells with a complex and heterogeneous phenotype and functional plasticity [24]. DC precursors are formed in the bone marrow and through the blood enter the secondary lymphoid organs, the mucous epithelium of the barrier tissues (lungs, intestinal mucosa, etc.), and the skin. After interacting with the antigen, DCs differentiate and change their phenotype. The differentiation markers that appeared on their surface -chemotaxis receptors -determine the possibility of DC migration into secondary lymphoid organs for the presentation of antigen to T cells. The interaction of DCs with T cells is determined by surface receptors for DC activation, and the type of T-cell response is regulated by cross-presentation. Depending on the antigen, microenvironment and cross-presenting markers, different T-cell populations can be activated: Th1, Th2, Th17, Treg [17,20]. In our study, maintaining the ratio of PDC and MDC at the same level under the influence of GMDP indicates the absence of chronic and autoimmune diseases in the study participants. MDC populations were determined by HLA-DR+ CD3-CD14 CD20 CD11c+CD123-, PDC was determined by markers HLA-DR+D3-CD14-CD20-CD11c+CD123+ (Fig. 2). The number of DCs and optimal ratio of their subpopulations is a necessary condition for maintaining immune homeostasis. The degree of their maturity is also of great importance. In various pathologies, defects were revealed at the stage of DC differentiation. For example, in rheumatoid arthritis and psoriatic arthritis, the absence of mature DC has been shown [19]. The main markers that determine DC maturity are CD80, CD83, and CCR7, which ensure DC migration to lymph nodes and interaction with T cells. The study showed that GMDP increases the expression of differentiation markers CD80, CD83 and CCR7 (Fig. 3-5). The ability to influence the degree of DC differentiation can serve as a justification for the previously discovered clinical efficacy of GMDP in psoriasis [11].  A significant increase in MDC and PDC with the CCR7 receptor was found compared to the baseline value, when comparing the percentage of dendritic cells expressing the CCR7 marker (CD197) before and after taking GMDP (Fig. 5). CCR7 is a receptor for β-chemokines CCL19 and CCL21, it ensures the migration of DCs to afferent lymphatic vessels, homing of T cells to secondary lymphoid organs, and also coordinates the movement of B cells in lymph node follicles to the border of B and T cell regions for interaction with Th. In the external endothelial venules, CCL21 is expressed, interacting with which CCR7 directs immune cells to the secondary lymphoid organs. Thus, CCR7 is important for the formation of an antigen-specific immune response, ensuring the transport of DCs to secondary lymphoid organs [25][26][27]. In addition, constitutively expressed CCR7 provides self-tolerance [28] as well as tolerance to harmless antigens, including food and inhalation antigens [26,27].
It was found that PDCs expressing CCR7 had a more significant increase when exposed to GMDP than MDCs (3,3 and 1.9 times, respectively). It should be noted that MDCs represent a heterogeneous population of DCs, which are divided into subpopulations DC1 and DC2, which differ phenotypically and functionally. Despite the fact that both subpopulations are able to capture antigen, migrate to secondary lymphoid organs PULMONOLOGY and present antigen to T cells, DC1 and DC2 differ in their ability to respond to different components of the microenvironment and activate T cells. The DC1 subpopulation expresses XCR1 and CD103 is capable of activating cytotoxic T cells and shifting the T helper balance towards Th1. DC2 express CD11b and shift the balance towards Th2, while both subpopulations are able to activate Th17 [29].
It is known that in normal peripheral blood DC1 and DC2 are present in insignificant amounts: 0.3-0.8 % DC1 and 0.02-0.06 % DC2, in contrast to other types of cells, such as MPC and Langerhans cells [29]. In this regard, changes in the expression of genes of activation markers under the influence of GMDP for these subpopulations were studied by PCR. For these purposes, the XCR1, CD11b and CD103 genes were selected. The choice of these genes is due to the fact that infections of bacterial and viral etiology induce the appearance on the membrane of chemokine receptors (CCR7, XCR1), a receptor for the complement component (CD11b), and a receptor for E-cadherin -Integrin Subunit Alpha E (CD103). Expression of XCR1, CD11b and CD103 receptors indicates the activation of cells, promotes their migration into the follicles of the lymph nodes with subsequent movement into the zone of inflammation of the barrier tissues and indicates the implementation of the immune response.
XCR1 is a chemokine receptor that takes part in the cooperative interaction of T cells and DCs in response to an antigen in barrier tissues. In this study, the level of transcription of the XCR1 gene increased after the application of GMDP by 1.5 times (Fig. 6).
It is known that dendritic cells expressing the XCR1 receptor play a major role in maintaining immune homeostasis of the intestinal mucosa [30] and the mucous membranes of the upper respiratory tract [31]. The presence of XCR1 on DC ensures the formation of memory T cells and, as a consequence, a rapid immune response during secondary infection with viral and parasitic infections [32]. In addition, expression of XCR1 on DCs is also required for the maturation of regulatory T cells, for maintaining selftolerance and suppressing inflammation [33]. One of the mechanisms for controlling excessive inflammation in the mucous membrane by dendritic cells with XCR1 is the production of transforming growth factor-β1 and retinoic acid from vitamin A, which causes the induction of peripheral T-regulatory cells and the synthesis of anti-inflammatory cytokines. [34]. The discovered ability of GMDP to increase the expression of XCR1 demonstrates the capacity of GMDP to control excessive inflammation in mucosal tissues.

CD103 -ITGAE (alpha E integrin subunit) is an E-cadherin receptor and mediates adhesion to epithelial cells. E-cadherin is one of the main molecules of Ca 2+ -dependent cell-cell adhesion in epithelial tissues. CD103 is present on T cells and DCs for migration
to the site of inflammation [35][36][37]. CD103+ DCs located in the lungs, intestines and skin, through crosspresentation, present a cytotoxic T cell-processed mechanism, providing protection against infection. CD103+ DCs influence the formation of regulatory T cells, limiting inflammatory responses and maintaining homeostasis [38]. The effect of GMDP on the expression of the CD103 gene is shown in (Fig. 6).
It should be noted that CD103 plays a key role in lung clearance from influenza virus [39] with crosspresentation protecting dendritic cells from moreover, ПУЛЬМОНОЛОГИЯ cross-presentation protects dendritic cells from infection with influenza virus [40]. The presence of CD103 + DC and CD103 + T cells is a predictor of a favorable outcome in oral cancer therapy [41]. The ability to enhance the expression of CD103, discovered in this study explains the positive clinical effects of GMDP in pulmonology [10,14,15].
Integrin alpha-M (CD11b) is a receptor for the C3b component of complement, recognizes fibrinogen and cell adhesion molecules present on the membranes of endothelial cells and leukocytes. The effect of GMDP on the increase in the expression of the CD11b gene is shown in (Fig. 6). It is known that CD11b participates in site-specific localization of DCs together with [34], constitutively supporting the maturation of Th17 [42]. CD11b+ DCs express proteins that provide contacts with epithelial cells in order to capture by dendrites, representatives of microorganisms of the intestinal lumen [43].
Dendritic cells represent a heterogeneous population of cells with a high level of plasticity. The appearance of various markers on their surface upon activation is finely regulated by inflammatory stimuli, and, which is important, triggers feedback mechanisms that stop and complete the process of inflammation. The study of the effect of GMDP on the expression of phenotypic markers of DC can supplement our understanding of the mechanisms of the implementation of the immune response upon activation of innate immunity receptors, manifested not only in the initiation of inflammatory stimuli, but also in their arrest, preventing excessive inflammation observed in experiment and clinical practice [44,45].
Аn increase in the expression levels of differentiation markers via GMDP indicates its ability to regulate DC recruitment and the ability to influence the direction of the immune response.
It should be noted that when analyzing the results of gene expression under the influence of GMDP, the expression results were grouped according to the survey data, which reflected the number of ARIs per year, the level of physical activity, food preferences, and diet in order to identify significant factors. It turned out that gender, the number of ARIs per year and food preferences in this group were not significant, and the participants grouped according to these characteristics had the same values as the entire group. At the same time, the study participants (N = 9), experiencing moderate physical activity (2-3 hours per day), had median values in relation to the rest of the participants were higher (000 versus 000), which confirms the beneficial effect of moderate physical activity on stabilization and maintenance of the reactive ability of immunocompetent cells.
Thus, a statistically significant effect of GMDP on the expression of genes of DC activation markers XCR1, CD11b, and CD103 was observed in comparison with the initial state (before taking the drug Licopid) of healthy donors 19-20 years old, and the mean value was higher in participants experiencing moderate physical exercise.
The data obtained explain the clinical results with the use of GMDP for the prevention of acute respiratory diseases in 267 students of the Institute of Physical Culture, experiencing moderate physical activity (2-3 hours per day).
The use of the drug Licopid 1mg in order to prevent and reduce the seasonal incidence of acute respiratory infections at the stage of basic training of students of the Institute of Physical Culture contributed to a decrease in the incidence of acute respiratory infections within 12 months of observation after taking the drug. If, before taking the drug Licopid, 42.7 % of participants had a history of acute respiratory infections in the previous 12 months of observation, then after using the drug, this value dropped to 11.4 % ( Table 2).
It should be noted that the use of GMDP completely disappeared the group with 3 or more episodes of acute respiratory infections during the year, which accounted for 14.5 % of participants. At the same time, in the comparison group, the number of ARIs during the observed period did not change significantly. Obviously, the number of ARI episodes after taking GMDP has decreased to a much greater extent, since the group that had 3 or more ARI episodes per year has completely disappeared. Analysis of the results of the GMDP effect by kind of sport showed that the greatest efficiency was observed in the student group track and field, in which the number of participants who had no episodes of acute respiratory infections during the year increased by 7 times. In sports using general equipment (volleyball and basketball), the number of students of the Institute of Physical Culture, who have never been sick has increased by 50 % or more. In contact sports -boxing and martial arts -the use of GMDP also significantly contributed to a decrease in ARI episodes. At the same time, there was no statistically significant change in fencing, but the most favorable situation was observed in this kind of sport -85 % of students specialized in this kind of sport, did not have acute respiratory infections during the year. It is known that, with the exception of injuries, upper respiratory tract diseases are the most frequent manifestation of the disease in [46][47][48]. Although the frequency of ARI in athletes is comparable to the frequency in the general population, the timing differs from typical seasonal fluctuations, episodes appear more often in the pre-competition period and during the competition [49]. The unfavorable state of the upper respiratory tract has a negative impact on aerobic processes, muscle contraction and information processing ability [50][51]. The maximum number of episodes of acute respiratory infections in the group of athletes in comparison with athletes of other sports indicates the tension of mucosal immunity and a decrease in the adaptive capabilities of the organism. A 7-fold reduction in this group of ARI episodes when taking GMDP confirms its effectiveness in increasing the body's resistance to acute viral infections of the respiratory tract, previously established for children and adults [52].
The next stage of the study was to analyze the effect of GMDP on the body's resistance to acute viral infections of the respiratory tract in a difficult epidemiological situation against the background of an increase in the incidence of COVID-19.
During the entire observation period, cases of COVID-19 diseases were recorded in students of the Institute of Physical Culture and their family members of the main group (Table 3) and the comparison group (Table 4). All COVID-19 episodes in both groups were mild, moderate and severe, with no deaths. In the group taking GMDP, 48.4 % of participants were in contact with a COVID-19 patient, while 98.4 % of participants did not have COVID-19.

ПУЛЬМОНОЛОГИЯ
Comparison of the episodes of the disease among the participants revealed that in the group taking GMDP, 2 people had a history of confirmed COVID-19, which is 1.6 %. In the comparison group, who did not take GMDP, this figure is 6 times higher -9.8 % (14 people) (Fig. 7).
It should be noted that 2 study participants who took GMDP and had a history of confirmed COVID-19 had a mild coronavirus infection, unlike those in the group who did not receive GVDP: 4 out of 14 required medical attention.
Adverse reactions after the use of GMDP corresponded to those set out in the instructions, were mild and did not require discontinuation of the drug, 75.4 % of the participants did not experience adverse reactions.
Thus, the use of the drug Licopid contributed to a 3.7-fold decrease in the number of students of the Institute of Physical Culture who had ARI during the year, and the number of ARI episodes decreased to a much greater extent, since the group that had 3 or more ARI episodes per year completely disappeared. The use of GMDP contributed to an increase in nonspecific resistance and protection of 98.4 % of participants from COVID-19 during an unfavorable epidemiological period (April 2020 -March 2021). For the first time, the study covers a large group of participants with moderate physical activity, who used Licopid for prophylaxis. This clinical observation is consistent with the previously obtained results of the positive effect of GMDP in the prevention of acute respiratory infections in children and adults [14, 15,52].

PULMONOLOGY
The limitation of this study includes the narrow age range of the participants.
Our data reveal the molecular mechanism of GMDP action on functionally significant DC subpopulations and substantiate the clinical efficacy of GMDP use in the prevention of acute respiratory infections in 267 apparently healthy donors with moderate physical activity.

1.
A new way of managing the recruitment of immune competent cells was discovered.
2. The possibility of a directed change in the phenotype of functionally significant subpopulations of dendritic cells under the action of an agonist of NOD2 receptors of innate immunity was established.
3. Glucosaminylmuramyldipeptide (GMDP) promotes an increase in the amount of DCs, and significantly increases the expression of markers of differentiation and activation of DC.
4. Thus, new molecular mechanisms of GMDP action on DCs have been revealed and its clinical efficacy in the prevention of acute respiratory infections has been substantiated.
5. The experimental data explain the molecular mechanism of action of GMDP in the prevention of acute respiratory infections in people leading an active lifestyle, with moderate physical activity.