Clinical significance of changes in the expression of Toll-like receptors of type 2, 4 and 9 in the formation of secondary immune dysfunction syndrome in military personnel

Introduction

The interaction among the nervous, endocrine, and immune systems plays a crucial role in maintaining body homeostasis, and to date, the cellular and molecular basis of such intersystem connections has already been described [1]. The impact of stress factors induces a change in the parameters of the functional activity of the physiological systems of the body, including various adaptive changes in the immune system. Irrespective of the nature of stress, the immune system responses are dose-dependent: acute stress has a stimulating effect, while chronic stress leads to a decrease in the immune response and the development of clinically significant disorders of the immune defense [2][3]. These reactions are also determined not only by the nature of stress factors but also by the individual characteristics of the organism, in particular, the dynamic transformation of the activity of immunocompetent cells that are responsible for the system of innate and adaptive immunity [4][5].

The study of the structural and functional potential of the system of Toll-like receptors (TLR) under conditions of various adaptive reactions is of considerable interest and is of great clinical importance as a potential target for therapeutic and prophylactic effects in both immune-mediated and various somatic disorders [6]. At present, it is known that the production of the main cytokines, regulation of immune cells, their survival and proliferation in the focus of inflammation depend on the state of signaling molecules, in particular, TLR [7]. Many experimental and clinical studies confirm the central role of TLR in the development of severe infectious diseases, and TLR2, TLR4, and TLR9 are considered potentially promising targets for the treatment of severe infectious complications [8–10]. A decrease in the TLR2 expression on monocytes reflects hyporeactivity, as well as a decrease in the immune response to bacterial damage, indicates an unfavorable course of generalized inflammation, and also negatively affects the outcome of the disease [11]. It has been proven that changes in the TLR4 expression in infectious pathology, even on the first day after infection, are a prognostic marker for the course of the pathological process: their powerful activation in the early stages after infection and the persistent elevated level for several days contribute to the containment of the pathogen in the "entrance gate" of the infection and its effective elimination in the shortest possible time [12]. At present, the role of TLR9 has been studied not only in the development of an anti-infective response to various viral agents, in particular representatives of the herpetic group, but also in the effectiveness of the therapeutic effect in this pathology [13]. The development of the immune response to the influenza virus and the introduction of the influenza vaccine also occur through the TLR9 [14].

However, at present, the transformation of TLR expressions during stress reactions remains not fully understood, the available data are contradictory and were obtained mainly in animal experiments [15][16].

This research was aimed at studying the dynamic changes in the content of monocytes expressing TLR2, TLR4, and TLR9 in military personnel under occupational stress.

Materials and methods

The research involved 37 military servicemen (the mean age was 37.3±4.8 years) who participated in special operations (military service in areas with unfavorable operational conditions during 3 months). The collection of complaints, anamnesis, assessment of the objective status, and study of the immune status parameters were performed before participation in special operations, immediately after returning from an area with unfavorable operational conditions, and also after three and six months of follow-up. The research participants were divided into two groups: Group I included military servicemen (n=27) who at the time of participation in special operations or during six months of follow-up after returning from an area with unfavorable operational conditions, had clinical manifestations of an infectious process of any etiology; while Group II included military servicemen who had no manifestations of an infectious process which was the main inclusion criterion.

The clinical research was performed in accordance with the WMA Declaration of Helsinki "Ethical Principles for Medical Research Involving Human Subjects" as amended in 2000 (2013), "Rules of Good Clinical Practice in the Russian Federation" approved by the Order of the Ministry of Health of Russia No. 266 dated June 19, 2003. Venous blood sampling was performed strictly on an empty stomach in the absence of exacerbation of infectious and somatic diseases. Phenotyping of CD14+CD282+ (TLR2), CD14+CD284+ (TLR4), CD14+CD289+ (TLR9) of peripheral blood monocytes was performed by flow cytometry (Cytomics FC 500, Beckman Coulter, USA) using kits of two- and three-color labelled monoclonal antibodies by Beckman Coulter (USA). Statistical data processing was performed using the STATISTICA 12 software package (StatSoft Inc., USA). The quantitative values ​​of the parameters were presented as the central tendency (Me) and interquartile range (25th –75th percentiles) and were designated as Me [LQ; UQ]. Analysis of changes in medians in groups was conducted using the Mann-Whitney test. Group means were compared using the paired samples Wilcoxon test. Differences were considered statistically significant at p <0.05.

Results

Dynamic monitoring of military personnel, analysis of complaints and anamnesis, and study of medical records have revealed that during participation in special operations and within six months after returning from an area with unfavorable operational conditions, 73% of the patients were diagnosed with a clinical manifestation of an infectious pathology of various etiologies and severities. During the follow-up period, clinical manifestations of acute respiratory diseases were registered in 11 military servicemen, while in one patient, the development of community-acquired pneumonia was revealed, which required a course of inpatient treatment with antibiotics; nine patients complained of recurrent herpetic eruptions of various localization; five patients were diagnosed with exacerbation of chronic infection foci; two patients noted an increase in stool with an increase in body temperature to subfebrile values, which did not require the appointment of drug therapy. The above-described changes in the state of health of military personnel were the criterion for assigning them into a separate observation group (I). Ten military servicemen showed no clinical signs of infectious pathologies of various etiologies during six months after participating in special operations, which became the criterion for their inclusion in the comparison group (II).

A comparative analysis of the content of monocytes expressing TLR2, TLR4, and TLR9 in military personnel with clinical manifestation of an infectious syndrome allowed revealing statistically significant changes in the follow-up dynamics compared to baseline data: during the first month after returning from an area with unfavorable operational conditions, a decrease in the expression of TLR4 on monocytes (CD14+CD284+) was noted, which persisted throughout the entire follow-up period (six months) and reached the lowest value in the third month of the follow-up period. Statistically significant differences in the values ​​of the indicators concerned both the relative and absolute number of peripheral blood flow monocytes expressing TLR4 on their surface. A downward trend in the content of monocytes expressing TLR2 (CD14+CD282+) was noted during the entire observation period in military personnel with clinical manifestations of the infectious syndrome after returning from an area with unfavorable operational conditions. During the sixth month of follow-up, these differences reached significant values ​​compared to the initial values of absolute parameters. The number of monocytes expressing TLR9 (CD14+CD289+) remained unchanged throughout the whole follow-up period. The results of the dynamic observation are presented in Table 1.

In the group of military personnel participating in special operations who showed no clinical manifestations of the infectious syndrome (II) during six months after returning from an area with unfavorable operational conditions, the content of monocytes expressing TLR2 and TLR9 of peripheral blood did not differ. A statistically significant decrease in the content of monocytes expressing TLR4, both in absolute and relative values, was noted from the third month of the follow-up period (p <0.05). The results of the dynamic follow-up period are presented in Table 2.

Discussion

Interest in the study of the innate immunity system in various clinical situations is due to the described property of memory formation for these structures, which is called "trained immunity" [17]. The mechanism of memory formation is probably induced by changes in the epigenome of innate immunity cells and the transformation of their functional potential [18]. Dysregulation of the body's anti-infective potential manifests itself in the chronicization of inflammatory processes, the transition of inflammation from a normergic to a hypo- or hyperergic form, and the formation of persistent immune dysfunction [19]. Therefore, the processes of regulation of inflammation, the causes, mechanisms, and consequences of dysregulation continue to be the subject of study.

In this research, the expression of TRL on peripheral blood monocytes was studied for the first time in practically healthy military servicemen who have experienced extreme stress conditions. It is known that the central mechanism of the inflammatory response and response to stress is the activity of the monocyte-macrophage system, and the persistence of inflammatory stimuli over time is a biological background that favors the development of chronic inflammation, a higher incidence of infections and chronic diseases [20].

The immune response efficiency depends on TLR-mediated pathogen recognition and further TLR-mediated activation of intracellular signaling pathways. It has been demonstrated that impaired functioning of TLRs and their signaling pathways can increase the risk of developing not only infectious but also autoimmune, oncological, and cardiovascular diseases [21]. It has also been revealed that hyperactivation of TLRs under the action of endogenous ligands can induce the development of an excessive inflammatory response [22].

During the dynamic follow-up period, a decrease in the expression of TLR4 was detected in all military personnel participating in special operations, including those patients who had no clinical manifestations of infectious pathology. Among the military servicemen of the main observation group who had infectious diseases of various etiologies during the observation period, a violation of the initial stages of recognition of nonspecific pathogen-associated molecular patterns was recorded due to a decrease in the expression of not only TLR4 but also of TLR2. It has been revealed that these types of receptors recognize the largest number of PAMPs of gram-positive and gram-negative bacteria and, accordingly, play an essential role in the systemic response to bacterial damage [9]. Some studies have also revealed a decrease in the expression of TLR4 on monocytes of peripheral blood flow in first-year military medical students during adaptation to training at a military training center, which the authors of the studies considered as an alarming factor in a possible disruption of the adaptive reserves of the immune response system [23]. Acute and chronic stresses affect the immune system through the secretion of hormones, which in turn affects the ability to form an effective immune response [24]. It is possible that it is the functional state of the innate immunity system under conditions of adaptation that becomes the basis for initiating the development of further immune-mediated pathology.

Conclusion

The complex influence of occupational stress factors on military personnel, even in the absence of clinical manifestations, leads to a disruption in the functional activity of the cellular structures of innate immunity and inhibits antigenic recognition through the TLR system. The most sensitive to stress-induced adaptive transformations were TLR4 whose expression disturbance on monocytes was recorded in all participants of the research. An additional decrease in the content of monocytes expressing TLR2 may significantly exacerbate immune surveillance disorders and induce the development of the clinical picture of various infectious pathologies. The above changes were recorded in military personnel within six months after participating in special operations, which allows classifying these individuals as a risk group for the development of persistent immune dysfunction.