Structural changes in the thymus under the pathogenic factors action

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Structural changes in the thymus under the pathogenic factors action

Prykhodko O.O.

Sumy State University (Sumy, Ukraine)

The thymus is the primary lymphoid (immune) organ in which antigen-independent proliferation and differentiation of T-lymphocytes occur and protects the body against various pathogenic factors. A comprehensive study of the morphological features of the thymus is relevant and one of the primary tasks of theoretical and practical medicine. The goal is to analyze and generalize data from the modern scientific literature on structural changes in the thymus under exposure to various exo- and endoantigens. It is possible to predict its further changes and develop methods for their prevention and correction, having followed the patterns of structural reorganization of the thymus under the influence of specific pathogens. The article analyzes data from the literature on the effects of cyclophosphamide, immunofan, salt extract of Hirudo verbana, tobacco smoke, formaldehyde, staphylococcal toxoid, polyoxypropylenetriol triglycidyl ester aqueous solution, adjuvant arthritis, light stress, reticuloendotheliosis virus, ablation therapy on the structural components of the thymus. Data are presented that emphasize the severity of the age-related clinical picture of COVID-19 and SARS-CoV-2, which is explained by insufficient antiviral immune function and an excessive self-destructive immune reaction, which includes T-cell immunity and is associated with already existing chronic inflammation in the human body of elderly. In addition, there is current information on the structure of the thymus and patterns of changes in its structural organization in age-related involution and atrophy. In most cases, due to the short-term exposure of the research animals to various exo- and endoantigens, changes in the thymus of the type of compensatory-adaptive reaction develop; that is, they are not specific.

Key words: thymus, experiment, cortical substance, brain substance, lymphocytes.

Connection of the publication with planned research works.

This work is a fragment of the research topic "Morphological aspects of experimental pathology of internal organs and musculoskeletal system", state registration number 0123U101135.

Introduction

The thymus belongs to the primary lymphoid (immune) organs [1]. Since antigen-independent proliferation and differentiation of T-lymphocytes takes place in the thymus, it is of great interest to study the possibilities of controlling thymus function for therapeutic purposes in the conditions of autoimmune diseases, immunodeficiency, immunotherapy and immunocor- rection [2].

In general, the structure of the rat thymus is typical and close to the structure of this gland in humans. Because of what this experimental model is justified and probable, the obtained data can be extrapolated to a person. Each lobe of the thymus is formed by lobules in which the cortex and medulla are visualized [1]. Small lymphocytes are characterized by a densely stained nucleus, surrounded by a narrow cytoplasmic rim filled with ribosomes and containing up to ten mitochondria, a small zone of the Golgi apparatus and endoplasmic reticulum. Large lymphocytes are structurally interme-diate between small lymphocytes and monocytes, but they lack monocytic granules [3].

The thymus is necessary for the development and maturation of T cells. It is susceptible to atrophy, in which thymus cellularity and disruption of its architecture are lost. It can lead to a decrease in the output of naive T cells. Atrophy of the thymus is often associated with ageing. The thymic function is critical for reducing morbidity and mortality associated with various clinical conditions, including infections and transplantation. Therefore, therapeutic interventions that increase thy- mopoietic potential and reduce thymus atrophy are relevant. These treatments increase thymus production, which is vital in producing favourable outcomes in the clinical setting. Such methods include the administration of IL7, zinc, keratinocyte growth factor, and ablation of sex steroids during thymic atrophy treated with leptin [4]. T-lymphocytes are essential mediators of immunity produced by the thymus in proportion to its size [5].

The study of the structural reorganization of the thymus parenchyma is an urgent task for both morphologists and practising physicians.

The aim of the study

thymus pathogenic factors action

Analysis and generalization of data from modern scientific literature regarding structural changes in the thymus under exposure to various exo- and endoantigens. Following the patterns of structural reorganization of the thymus under the influence of certain pathogens, it is possible to predict its further changes and develop methods of prevention and correction.

Main part

The thymus specific density significantly depends on the number of degranulated mast cells and the absolute number of microcirculatory vessels, especially lymphatic vessels. The specific density index and the lymphocyte-epithelial index change in a wave-like manner during the first week of life every 12 hours. These changes are accompanied by changes in the structure of the extracellular matrix, the absolute number of small lymphocytes in the cortical substance of the thymus lobes, and the adhesive properties of lymphocytes. Grigorieva O.A. and Apt O.A. studied the dynamics of changes in the period from 0 to 2, from 12 to 16 and from 108 to 120 hours after birth. At this time, there was a discrete migration of lymphocytes from the thymus through the paravasal lymphatic vessels. After the temporary local oedema reduction, the lymphatic vessels emptied, and their number decreased. Therefore, the emigration of lymphocytes from the thymus occurs along the lymphatic vessels at the top of periodic shortterm local oedema [6].

Yerokhina V.V. and Avilova A.V. describe a study in which male rats were given a single dose of cyclophosphamide at 200 mg/kg. Immunocorrection was performed with immunofan at a dose of 50 ^g/kg of rat body weight. As it turned out, on the 3rd day after the correction of induced immunosuppression with immu- nophane, signs of accidental involution are characteristic of the structural organization of the thymus. Chromatin in the nucleus of lymphocytes is condensed in lumps and is located on the periphery; the thin rim of the cytoplasm contains ribosomes, polysomes, and single mitochondria. On the 30th day, after the correction, an increase in the proportion of plasma cells is described in the cortical substance of the lobules of the thymus; the proportion of cells with signs of mitosis increases, so there is a process of restoration of the population of lymphoid cells, as well as macrophages [7].

Aminov R.F., with co-authors, analyzed the morphological parameters of rats' thymus under the influence of Hirudo verbana salt extract. Female rats were injected with a standardized saline extract from medical leech bodies twice before and twice after mating. The animals were withdrawn from the experiment on the 60th day after the last administration of the substance. The material was collected on the 15th, 30th, 45th and 60th day of post-embryonic ontogenesis. As a result, it was found in the offspring that the number of cells per unit area (400 ^m2) increases in both the cortical and medulla of the thymus lobes, the relative area of the cortical matter increases, the relative area of the medulla decreases, and the cortical-cerebral index increases. All the results obtained by the authors indicate that the salt extract of Hirudo verbana has a stimulating effect on immunogen- esis in the primary organs of the lymphoid system [8].

Tobacco smoke harms the normal functioning of the thymus, not only in animals that were directly exposed to negative influence but also in offspring. V. Tkachenko conducted an experiment in which adult rats were exposed to cigarette smoke in a closed chamber for 15 minutes. The level of cotinine was studied in the blood of the offspring. Rats were mechanically injured on the outer surface of the right hind limb and removed from the experiment 24 and 48 hours later. As a result, there was a significant increase in the level of cotinine in the blood, which confirms the tobacco intoxication of the rats. The most pronounced changes in the thymus were found two days after thymus injury, where only male rats were exposed to tobacco, and after 24 and 48 hours in the group of progeny, where both male and female rats were exposed to smoking. An increase in the mass of the thymus and the area of the nuclei of epitheliocytes was observed. The wounding process in the offspring had signs of purulent inflammation with histolysis and the development of phlegmon and necrotic changes. Therefore, the influence of tobacco smoke on the progeny has been proven as a result of the intoxication of the parents of rat pups, which ultimately leads to a violation of wound healing due to a decrease in the reactivity of the entire organism [9].

N.A. Voloshyn, with co-authors, conducted research on white rats on 1, 2, 3, 5, 9, 14, 21, and 30 days after birth. An experimental group of animals was intrauteri- neally injected with 0.05 ml of staphylococcal toxoid (1:10) on the 18th day of pregnancy. Within 30 days after birth, the group of rats injected with staphylococcal antigen showed increased expression of SC5 receptors with a peak in the first three days and on the 14th day after birth. The area occupied by CK5+ - epitheliore- ticulocytes increased significantly, namely by 1.5 times in rats of the experimental group from the 1st to the 9th day after birth with a maximum on the 1st day, then decreased and almost reached the value of the intact and control groups rats until the 30th day of observation. All the changes described above by the authors indicate an increase in the functional activity of epithelioreticulo- cytes as cells of the microenvironment [10].

Vash I.Yu. investigated formaldehyde's effect on the thymus's structural organisation. Inhalations of formaldehyde at a concentration of 2.766 mg/m3 were carried out once a day for 60 minutes for 20, 30, 60 and 90 days. The number of cells on an area of 2500 ^m2 of the subcapsular zone of the thymus in animals under the influence of formaldehyde for 10, 20 and 30 days did not change reliably. After 40 and 60 days, this indicator was lower than the control by 8.90% and 9.63%. Inhalation of formaldehyde led to a decrease in the relative area of the cortical substance of the thymus lobes, and the phenomenon of inversion of the cortical substance and the brain substance was observed. Under the influence of formaldehyde, a picture of the so-called "starry sky" was observed in the cortical substance of the thymus lobes. A decrease in the severity of changes in the thymus of rats in the rehabilitation group, which after 60 days of exposure to formaldehyde stayed for 30 days in standard vivarium conditions, compared to animals that received 90 exposures, indicates the reversibility of the above-described changes [11].

Shyian D. and co-authors studied the xenobiotic's effect on the thymus's structural components. In the study, sexually mature male rats were injected with aqueous solutions of triglycidyl ester of polyoxypropyl- enetriol (TEPPT) at a dose of 1/10 LD50 of 5.75 g/kg for 7, 15, 30, and 45 days. The study indicates that exposure to polyoxypropylenetriol triglycidyl ester caused marked organometric changes in the thymus of rats. A 100% effect on all morphometric parameters of the thymus under the influence of TEEPT was noted. However, more pronounced changes were observed on the 7th and 30th days. The study revealed that the greatest limits of parameter fluctuations and their significant variability were the indicators of the length and width of the thymus. All obtained morphological data indicate an active reaction of the thymus to the induced xenobiotic [12].

Thymic epithelial cells (TECs) are believed to play an important role in T cell development and have been identified primarily in mice by lectin binding and antikeratin antibodies. Sawanobori Y. et al. performed a study using putative marker antibodies and novel monoclonal antibodies (i.e., ED 18/19/21 and anti- CD205 antibodies) that revealed that rat TECs are phenotypically divided into three subsets: ED18 +ED19+/- keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21-K5-K8+Ulex europaeus lectin 1 (UEA-1)+CD205-medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1-CD205- medullary TECs (mTEC2s). Thymic cells were identified in smears as ED18+ED19+/-K5+K8+ subset of cTEC. mTEC1 preferentially expresses MHCII, claudin-3, claudin-4, and an autoimmune regulator. Using the ED18 and ED21 antibodies also identified three TEC subsets in mice. The authors found two clear zones, free of TEC, in the sub- capsular area and the medulla [13].

Zimecki M. and co-authors demonstrated the therapeutic utility of calf thymus extract to alleviate the symptoms of autoimmune encephalomyelitis in rats by clinical, immunological, histological and ultrastructural parameters [14].

With age, the thymus rapidly atrophies, which leads to a progressive decrease in the formation of new T cells. This reduced output is compensated by the duplication of existing T cells, but this leads to a gradual dominance of memory T cells and a reduced ability to respond to new pathogens or vaccines. Griffith A.V., with coauthors, emphasize that accelerated and irreversible atrophy of the thymus results from a stromal deficiency of the reducing enzyme catalase, which leads to increased damage by hydrogen peroxide produced as a result of aerobic metabolism. Genetic supplementation of catalase in stromal cells would reduce atrophy, as would chemical antioxidants, thus providing a mechanistic link between antioxidants, metabolism, and normal immune function [5].

It is known that the thymus atrophies during infections. Majumdar S. and Nandi D. conducted a systematic study of changes in thymocyte subpopulations, highlighting the main points. In particular, there is a block in the path of development of CD4-CD8- double negative (DN) thymocytes; CD4+CD8+ double positive (DP) thymocytes, mainly in DP1 (CD5loCD3lo) and DP2 (CD5hiCD3int), but not DP3 (CD5intCD3hi); single positive (SP) thymocytes are more resistant to exhaustion, but their maturation is delayed, resulting in the accumulation of CD24hiCD3hi SPs. A study in Ifny-/- mice demonstrated that endogenous Ifny produced during infection enhances the depletion of DN2-DN4 subsets, promotes DP3 accumulation and delays thymocyte SP maturation [15].

Thymic epithelial cells (TEC) form a three-dimensional mesh that supports the development and maturation of thymocytes. In their study, Sun L. and co-authors emphasize that CD45(-)FSP1(+) thymus non-hematopoietic cells represent a unique subset of fibroblast-derived specific protein 1 (FSP1)(-) cells. Deletion of these cells in FSP1-TK transgenic mice caused thymic atrophy through loss of TECs, especially mature medullary TECs (MHCII (high), CD80 (+) and Aire (+)). In a model of thymic injury and regeneration induced by cyclophosphamide, the absence of the non-hematopoietic sub-population of CD45(-)FSP1(+) fibroblasts significantly delayed thymic regeneration. In fact, FSP1(+) thymic fibroblasts released more IL-6, FGF7, and FSP1 into the culture medium than their FSP1(-) counterparts. Their subsequent experiments showed that the FSP1 protein could directly enhance the proliferation and maturation of TECs in in vitro culture systems. FSPl-deficient mice had significantly smaller thymus size and smaller TECs than controls [16].

Juvenile rheumatoid arthritis (JRA) is a chronic autoimmune systemic disease of the connective tissue that develops before age 16, with damage to the joints and internal organs. The pathogenesis of this disease lies in activating cellular and humoral links of immunity. The thymus is a unique complex organ of the neuroendocrine and immune system, capable of producing various biologically active substances that play a significant role in immunological and many other physiological processes. Adjuvant arthritis is accompanied by a typical autoimmune reaction, the main link of which is T- cell immunity. The most commonly developed model of rheumatoid polyarthritis in rats is close to human rheumatoid arthritis in terms of clinical course, pathological and histological data. Therefore, it is reliable in reproduction and allows for studying the links of rheumatoid arthritis pathogenesis and revealing the preventive and therapeutic effects of drugs. Kaladze N.N. and co-authors in reconstructed adjuvant arthritis with the help of an electron microscopic study showed the development of changes in the cellular elements of the thymus according to the type of hydropic dystrophy. Based on the obtained results, the authors believe that the changes in the mentioned pathology negatively affect the body's immune status [17].

Sorokina I.V. and Bocharova T.V. studied the effect of light stress on rabbits' thymus and spleen under constant illumination. In the thymus, changes appeared after two months of the experiment; in particular, hyperplasia of the cortical substance, an increase in the density of the location of cells, and an acceleration of the proliferation and differentiation of lymphocytes were observed. After six months, a decrease in the mass of the thymus, a decline in the number of lymphoid nodules, signs of involution, and cells in a state of apoptosis were found [18].

Reticuloendotheliosis virus or avian retrovirus can infect various birds and cause immunosuppression. Therefore, Fu L. and co-authors conducted a study to establish the relationship between thymic lymphocyte apoptosis and the proliferation of T-cell subtypes with immunosuppression. The results indicate that REV infection causes thymic lymphocyte apoptosis, suppresses T-lymphocyte proliferation, changes T-cell subsets, and enhances the immunosuppressive effect. In addition, several new causes of reticuloendotheliosis virus- induced immunosuppression in the thymus of infected chickens have also been identified [19].

The external structure and topography of the thymus of children change as the normal process of its involution takes place. Wee T. and co-authors emphasize that thymus tissue can be localized orthotopically within the anterior mediastinum or ectopically, related to the course of its embryonic development. This phenomenon in children during imaging studies can lead to incorrect interpretation of a normal thymus as a pathology [20].

Epithelial cells of the thymus play an essential role in the differentiation of T cells. It is known that several transcription factors are necessary for the development and functioning of thymus epithelial cells. Recent advances in understanding thymic epithelial cell heterogeneity have provided some new insights into the transcriptional requirements of epithelial cell subtypes. However, it is unknown whether epithelial cell progenitors exist in the adult thymus; thus, the factors linking putative progenitors to differentiated cell types are poorly understood. Martinez-Ruiz G.U. et al. believe that new single-cell transcriptomic and epigenomic technologies should provide rapid progress in this field [2].

The thymus undergoes a decline in functional capacity during ageing, leading to a progressive decline in naive T-cell output. Atrophy of the organ is evidenced by the deterioration of the thymus microenvironment, including limited epithelial-mesenchymal transitions, fibrosis and adipogenesis. The study of cellular changes in the thymus at different stages of life, including mouse models with single-cell RNA sequencing, reveals an expansion in the number of different cell types that influence thymic function [21].

In the process of proliferation and differentiation of T-lymphocytes in the thymus, they interact with many different types of cells in the microenvironment, for example, stromal cells, which include epithelial, mesenchymal, and other non-T-lineage immune cells. "Scavenger cells" are also necessary for cleaning the thymus parenchyma from apoptotic thymocytes, such as macrophages [22].

Age-related involution is accompanied by a decrease in the number of epithelial cells and a reduction in the proliferation of T cells. Yang B. and co-authors investigated the therapeutic effect of metformin on thymus degeneration in rats with accelerated ageing of mice, which was achieved by intraperitoneal injection of D- galactose (120 mg/kg/day) for eight weeks. Metformin was administered intragastrically at a dose of 100 or 300 mg/kg body weight per day, respectively, for six weeks. A histological study showed that using metformin can alleviate thymus atrophy, which is caused by D-galactose. In addition, metformin therapy increased mitochondrial membrane potential, decreased mitochondrial reactive oxygen species, malondialdehyde and superoxide dismutase levels, and restored mitochondrial balance through increased expression of dynamin-related protein 1. Thus, metformin demonstrated a positive effect on thymus atrophy [23].

Therapeutic measures used to treat cancer provoke damage to the thymus and accordingly limit the restoration of protective immunity. Therefore, Cosway E.J. and colleagues conducted a study demonstrating that eosinophils are essential to the type 2 intrathymic immune network that ensures thymic recovery after ablation therapy. Eosinophil regulation of thymic regeneration occurs through the concerted action of natural killer cells, which trigger CCL11 production via IL4 receptor signalling in the thymic stroma and ILC2, which are the intrathymic source of IL5. This cytokine therapeutically accelerates thymic regeneration after injury. The above findings identify an intrathymic network consisting of many innate immune cells that restore thymic function during the recovery of the adaptive immune system [24].

Non-T-lineage thymic stromal cells, namely thymic epithelial cells, endothelial cells, mesenchymal/fibroblast cells, dendritic cells, and B cells, provide signals and functions necessary for thymocyte development as well as homeostasis of the thymic stroma itself. In addition, Thymus stromal cells in the early stages of T-cell development play the homing precursors of the thymus, contribute to the induction of differentiation of T-lineage cells and support the proliferation of thymocytes [25].

Dendritic cells (DCs) in the thymus are involved in the formation of central tolerance, but they also perform other functions, such as pathogen recognition. Li Y. and his co-authors conducted a study in which all common DC subgroups were found in the thymus of people of different ages. It was noted that most DC accumulates in the epithelial space of the human thymus. It was found that the cortical substance of the human thymus lobules atrophies relatively faster than the medulla, which leads to a gradual increase in the ratio of the area of the medulla to the cortical substance with increasing age. The density of DC subsets in the human thymus showed various changes with increasing age, which contributed to a change in the composition of DC subsets. The density of plasmacytoid DCs in the human thymus gradually increases with ageing, suggesting that these cells play another, as yet unexplored, role in addition to providing central tolerance [26].

Understanding the pathogenesis of the effect of various viral agents on the body is essential for developing new treatment methods. With the beginning of the coronavirus pandemic (SARS-CoV-2) at the beginning of 2020, the direction of studying the effect of this virus on immune organs became relevant. Lins M.P. and Smaniotto S. claim that the new virus causes lymphopenia and other probable pathological changes in the thymus parenchyma [27].

The severe course of COVID-19 disease is most often observed in the elderly and the presence of inflammatory diseases. Dysfunction of the thymus can be a predisposing factor and worsening of its course. Any disorders of the thymus from childhood can lead to its abnormal function and explain the severe course of COVID-19 disease among younger people. Kellogg C. and Equils O. treated patients with thymic dysfunction prophylacti- cally with convalescent serum or recombinant antibodies and may respond better to high-dose or adjuvant therapy with a COVID-19 vaccine. It has been concluded that therapeutic measures that stimulate thymus regeneration can improve patients' general health and can be included in the treatment protocol for COVID-19 [28].

The severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) and the global pandemic coronavirus disease 2019 (COVID-19) has particularly severe symptoms and mortality in the elderly. A growing body of evidence shows that the features of the severity of the age-related clinical picture of COVID-19 are explained by impaired antiviral immune function and an excessive self-destructive immune response, which includes T-cell immunity and is associated with already existing chronic inflammation in the body of the elderly. Age-related changes, i.e., T-cell immunodeficiency, are not only characterized by a limited diversity of T-cell receptors, the accumulation of exhausted and/or senescent memory T cells, as well as increased self-reactivity of T cells and cells of innate immunity induced by chronic inflammation. Many of these changes can be traced to age-related involution/degeneration of the thymus [29].

"Immunoaging" includes a decline in innate and acquired immunity and increased production of inflammatory cytokines. This scenario of immunological dysfunction and its association with disease development in the elderly has been widely studied, particularly in infections that can be fatal, such as influenza and COVID-19 [30].

The limited number of tissue-specific stem epithelial cells of the thymus in vitro seriously hinders the implementation of thymus regenerative therapy. Current solutions are mainly based on growth factors that can stimulate the differentiation of pluripotent stem cells into tissue-specific ones. Target-specific small chemical compounds represent an alternative solution that can induce and maintain clonal expansion of tissue-specific thymic stem epithelial cells and reversibly block their differentiation into mature cells. Furthermore, these compounds can be used as part of culture media intended to reproduce these cells in vitro and develop preparations for the regeneration of the thymus in vivo. It should help to achieve the ultimate goal - autologous regeneration of thymus tissue in children who have had it removed during cardiac surgery [31].

Conclusions.

Under the short-term influence of any factor on the body of experimental animals, all changes in the thymus manifest a compensatory-adaptive reaction and are not specific. Under the conditions of long-term exposure, a decrease in cell density, apoptosis of lymphocytes, and loss of functional capabilities of the organ is observed.

Prospects for further research. They consist in the study of changes in the structural organization of the thymus parenchyma under conditions of general, cellular and extracellular dehydration.

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