Pathogenesis of tuberculosis

The development of tuberculosis inflammation depends on the reactivity of the organism and the state of its defenses, the virulence of mycobacteria tuberculosis and the duration of their persistence in the lungs. The action of various factors of the infectious process can explain the great diversity of tissue and cellular reactions of the respiratory department, where specific changes are combined with non-specific ones, in one way or another influencing the manifestation and outcome of the main process.

Each stage is a complex set of structural changes in various body systems and respiratory organs, accompanied by profound shifts in metabolic processes, intensity of metabolic reactions of the respiratory department, and is reflected in the morphofunctional state of its cellular and non-cellular elements. Of great importance is the study of the earliest mechanisms of tuberculous inflammation development, established in recent years.

[ 1 ], [ 2 ], [ 3 ], [ 4 ], [ 5 ], [ 6 ]

Microcirculation disorders and the state of the aerohematic barrier

Within 24 hours after intravenous administration of Mycobacterium tuberculosis into the lungs of mice, characteristic changes in the microcirculatory bed occur: expansion of the vascular capillary network profiles, sludge formation of erythrocytes with parietal arrangement of polymorphonuclear leukocytes can be observed. Electron microscopic analysis of the endothelial lining of pulmonary capillaries reveals activation of the luminal surface of cells, signs of intracellular edema development with disorganization of micropinocytotic vesicles and their fusion into large vacuoles. Areas of edematous, cleared cytoplasm of endotheliocytes in places form sail-shaped protrusions, differing in quantity and size in different microvessels. In some cases, local exfoliation of their cytoplasmic processes from the underlying basal layer, loosening and thickening of the latter are observed.

Regardless of the method of introduction of the tuberculosis mycobacterium, in all model experiments in the first 3-5 days an increase in the permeability of the aerohematic barrier is observed, as evidenced by the accumulation of fluid in the interstitium, the development of intracellular edema not only of endotheliocytes, but also of alveolocytes of the 1st type (A1). The changes affect their cytoplasmic processes, in which areas of clear, edematous cytoplasm appear, capable of bulging into the intraalveolar space.

In places of generalization of mycobacterium tuberculosis and development of pneumonic foci, formation of primary granulomatous accumulations of mononuclear cells and polymorphonuclear leukocytes, A1 is determined with strongly thickened, in places destroyed cytoplasmic processes, areas of exposed basement membrane. In many alveolocytes of the 2nd type (A2), swelling of apical microvilli occurs, uneven expansion of mitochondrial profiles and cytoplasmic reticulum. Hyperhydration of the alveolar epithelium is accompanied in places by the release of fluid, plasma proteins and cellular elements of inflammation into the intra-alveolar space.

Modern studies of microcirculation have established the leading role of the vascular system in the development of the initial phases of inflammation. Stimulated by cytokines, the endothelium secretes biologically active substances - adhesive molecules (selectins, integrins). various mediators (arachidonic acid metabolites) and growth factors, oxygen radicals, nitric oxide, etc., providing interaction between the endothelium and polymorphonuclear leukocytes, as well as between other cellular elements of inflammation. It has been established that L-selectin mediates the so-called "rolling neutrophil" effect, which is the initial stage of adhesion of these cells to the endothelium. Another type of selectin, P-selectin, after the effect of histamine or oxygen metabolites on endothelial cells, is translocated to their surface, facilitating the adhesion of neutrophils. E-selectin is also detected on the surface of cytokine-activated endothelial cells; It is involved in the process of interaction between the endothelium of postcapillary venules and T-lymphocytes.

Cytokines secreted by mono- and polynuclear cells cause structural rearrangement of the cytoskeleton of endothelial cells, which leads to their contraction and increased capillary permeability. In turn, the passage of polymorphonuclear leukocytes through the wall of blood vessels can be accompanied by its damage and increased permeability for fluid and plasma proteins, and a change in the composition or activity of adhesive molecules leads to increased migration of monocytes and lymphocytes, ensuring further development of the inflammatory reaction. Arising in the respiratory organs in response to the introduction of Mycobacterium tuberculosis, it affects all structures of the respiratory section.

During the formation and maturation of tuberculous granulomas, i.e. at the second stage of development of the specific process, disturbances in the structure of the interalveolar septa increase. Edema, cell proliferation and fibrillogenesis in the interstitium significantly change the morphofunctional state of the respiratory epithelium, especially near the foci of the inflammatory reaction. Disturbances in the conditions of the microenvironment and the vital activity of alveolocytes negatively affect the functional state of the aerohematic barrier and gas exchange in the lungs.

Along with the already noted changes in the interalveolar septa in the edema zone, pronounced destructive changes in the alveolar epithelium, which can be traced over a significant portion of it, attract attention. They affect both types of alveolocytes and have one direction - edematous swelling of intracellular organelles, which leads to dysfunction and then cell death. Fragments of destroyed alveolocytes, including A2, can be detected in the intraalveolar content. Macrophage elements, polymorphonuclear leukocytes, as well as a significant number of erythrocytes and eosinophils, reflecting the high permeability of the capillary network, are also located here. Fibrin threads and their conglomerates are determined among the destroyed cells.

In the alveoli that retain air, signs of edema of the tissue and cellular structures of the interalveolar septa can also be observed. In addition, on the surface of the alveolar epithelium, bubble formation processes occur, reflecting the initial stages of destruction of the aerohematic barrier and "flooding" of the alveoli. At the final stage of the development of tuberculous inflammation, a progressive increase in dystrophic and destructive changes in the structural components of the terminal sections of the lung is observed, especially in areas of the pulmonary parenchyma bordering caseous-necrotic foci or foci of tuberculous pneumonia. Microcirculatory disorders are widespread.

Transcapillary passage of plasma proteins of blood promotes entry of circulating immune complexes (CIC) into the interstitium of the lung, promoting the development of both immunological and secondary immunopathological reactions in it. The role of the latter in the pathogenesis of tuberculosis has been proven, and it is caused by intrapulmonary deposition of CIC, a defect in the phagocyte system, and an imbalance in the production of cytokines, regulating intercellular interactions.

The area of the air pulmonary parenchyma is reduced to 30% of the section area, its areas alternate with areas of pronounced intraalveolar edema, distelectasis and atelectasis, emphysematous expansion of the alveoli. Despite the progressive nature of the development of untreated tuberculous inflammation, compensatory and restorative processes take place in the pulmonary parenchyma free of foci. As our studies have shown, in the perifocal zone of inflammation, the functional activity of A2 is aimed mainly at maintaining the integrity of the alveolar epithelium, restoring the A1 population, which is most sensitive to the action of tuberculous process factors. The fact of A2 participation in regeneration processes as a cellular source of respiratory epithelium is generally recognized today. A marked increase in the proliferative activity of A2 in these zones is indicated by the detection of 6-10 young alveolocytes located nearby - "growth buds" with a uniform well-developed nuclear structure, a significant content of mitochondria and polyribosomes in the cytoplasm, a small number of secretory granules. Sometimes mitotic figures can be seen in these cells. At the same time, intermediate-type alveolocytes, reflecting the transformation of A2 into A1, are extremely rare. The gas exchange function of the organ is maintained due to alveolar hypertrophy, the formation of growth points and the transformation of A2 into A1 in remote areas of the pulmonary parenchyma. Ultrastructural signs of the active secretory function of A2 are also observed here.

These data correlate with the results of electron microscopic examination of alveolar epithelium in surgical material. In patients with healing of tuberculosis infection foci, adenomatous structures are formed that resemble alveolar ducts. The cells lining them have an A2 ultrastructure, preserving single secretory granules. It is characteristic that the transformation of A2 into A1 does not occur (intermediate-type alveolocytes are not detected), which does not allow these structures to be classified as newly formed alveoli, as noted by some authors.

The processes of restoration of the respiratory epithelium, the formation of transitional alveolocytes are observed only in the more distant pulmonary parenchyma, where nodular growths of alveolocytes corresponding to "growth buds" are determined. The main gas exchange function of the lungs is also carried out here, the cells of the aerohematic barrier have a well-developed ultrastructure with a large number of micropinocytic vesicles.

The study of various models of tuberculous inflammation showed that the development of specific inflammation in the lungs is associated not only with certain destructive changes in the respiratory section directly in the foci of infection, but affects the entire pulmonary parenchyma, where signs of impaired microcirculation are observed. increased permeability of the vessels of the interalveolar septa. With the progression of the inflammatory process, the phenomena of edema increase, which affects the state of the alveolocytes, especially A1. The lumens of many alveoli are partially or completely filled with fluid and cellular elements of inflammation. Hypoxia and fibrous changes in the interalveolar septa affect the gas exchange function of the aerohematic barrier, lead to the development of respiratory failure and death of experimental animals.

[ 7 ], [ 8 ], [ 9 ], [ 10 ]

The role of lung macrophages

Lung macrophages are a component of the mononuclear phagocyte system, which is common to the entire body and originates from the pluripotent stem cell of the bone marrow. During stem cell division, monocyte precursors are produced - monoblasts and promonocytes. Monocytes circulate in the blood and partially enter the interstitial tissue of the lungs, where they can remain inactive for some time. In the presence of differentiation inducers, they are activated, move to the surface of the respiratory and bronchial epithelium, where they undergo several stages of maturation, turning into alveolar and bronchial macrophages, respectively. The main function of these cells - absorptive - is associated with their ability to phagocytose foreign material. Being one of the factors of the body's natural resistance, they protect those regions of the lungs that are the first to come into contact with microbes and abiogenic agents, i.e. they maintain the sterility of the epithelial lining of the lungs throughout its entire length. Most of the foreign material, as well as fragments of destroyed cellular elements, are almost completely digested after conjugation of the phagosomal vacuole of the macrophage (necrophage, hemosiderophage) with lysosomes containing proteolytic enzymes. Lung macrophages are characterized by a high content of acid phosphatase, nonspecific esterase, cathepsins, phospholipase A2, and enzymes of the Krebs cycle, especially succinate dehydrogenase. At the same time, it is known that pathogens of a number of infectious diseases, and above all M. tuberculosis, can persist for a long time in the cytoplasm of alveolar macrophages, since they have highly resistant cell walls that resist the action of lysosomal enzymes. In model experiments in untreated animals, despite the pronounced activation of acid phosphatase and other hydrolases, a certain proliferative activity of Mycobacterium tuberculosis and the formation of small colony-like clusters by the pathogen can be observed in the cytoplasm of alveolar macrophages.

Low microbicidal activity of lung macrophages is associated with organ-specific features of phagocytes, as they function in an environment with a high oxygen content. Energy processes in their cytoplasm are supported mainly by oxidative phosphorylation of lipoproteins, with the catabolism of which one of the main functions of these cells, which are part of the pulmonary surfactant system, is associated. Energy extraction, localization of oxidative processes affect the mitochondrial system, the development of which correlates with the functional state of the phagocyte. Superoxide dismutase is also localized here - an antioxidant protection enzyme that catalyzes the dismutation of singlet oxygen formed during the passage of electrons along the respiratory chain. This fundamentally distinguishes lung macrophages from polymorphonuclear leukocytes, which receive oxygen and bioenergy mainly due to glycolysis. In the latter case, the cleavage of the substrate occurs directly in the cytosol, and activated oxygen and hydrogen peroxide formed with the help of myeloperoxidase constitute the main bactericidal potential for action on bacteria.

Low biocidality of lung macrophages can be considered as a kind of price for adaptation to aerobic conditions of functioning. Apparently, therefore, they fight tuberculosis mycobacteria together with polymorphonuclear leukocytes and exudate monocytes (they are also called inflammatory macrophages). Pathogenetically important is that not all lung macrophages that have captured tuberculosis mycobacteria are removed from the lungs with the drift of surfactant and bronchial secretion - some of them develop in the interstitium, which is the trigger for the formation of characteristic cell clusters - granulomas.

Getting into the interstitium, rich in blood vessels, lung macrophages with incomplete phagocytosis begin to produce inflammatory cytokines, activating the adjacent endothelium. On the membranes of the latter, the expression of immunoglobulins increases, with the help of which selective adhesion of monocytes is carried out. Having left the vascular bed, these cells are transformed into exudate macrophages, producing inflammatory mediators, attracting not only mono-, but also polynuclears to the focus.

At the same time, the signal for the development of a granulomatous reaction comes from sensitized T-lymphocytes - effectors of delayed-type hypersensitivity. Among the lymphokines that these cells begin to produce, the factor inhibiting the migration of monocytes and IL-2 are of great importance for granulomatogenesis. They accelerate the influx and fix monocytes in the infection site, regulate their transformation into phagocytic, secreting and antigen-presenting macrophages.

It should be emphasized that, being a mechanism of cellular protection of the respiratory organs from penetration of the pathogen, the granulomatous reaction of the lungs in tuberculous inflammation ultimately reflects the failure of mononuclear phagocytes to fight tuberculosis mycobacteria. Therefore, macrophages are forced to constantly proliferate (increase the number of populations) and differentiate into larger phagocytes (increase the quality of proteolysis). which are giant cells of the foreign body type. In the phagosomes of the latter, under an electron microscope, one can see not only tuberculosis mycobacteria, but also large apoptotic cells, fragments of destroyed polymorphonuclear leukocytes. At the same time, the ultrastructural signs of proteolytic activity (the degree of development of the lysosomal apparatus) in such phagocytes per unit area of the cytoplasm do not differ significantly from mononuclear ones. In this regard, lung macrophages constantly attract polymorphonuclear leukocytes, which have greater biocidal properties, to the lesion. Activation of the latter is accompanied by the release of a significant amount of hydrolases and oxidants into the extracellular environment, which leads to tissue breakdown and the formation of caseous masses in the center of the lesion.

The most pronounced metabolic disorders are observed in patients with acutely progressive forms of pulmonary tuberculosis, occurring with a predominance of exudative and alterative inflammatory reaction, and the course of progressive forms of pulmonary tuberculosis is characterized, as a rule, by pronounced T-cell immunodepression. Suppression of T-cell immunity, pronounced lymphopenia lead to disruption of intercellular interactions, inhibition of the granulomatous reaction.

Deficiency of activated monocytes and lymphocytes, combined with their morpho-functional insufficiency, may be a consequence of increased apoptosis. The cytokine imbalance that occurs in such cases may serve as a marker of a defect in the immune system. The process of apoptosis has characteristic morphological features: chromatin condensation at the nuclear membrane, nucleolus disintegration, formation of cellular fragments (apoptotic bodies) and their phagocytosis by macrophages.

The peculiarities of the functioning of lung macrophages are associated with their ability not only to phagocytosis, but also to produce a large number of cytokines necessary for the activation and regulation of many extracellular reactions and processes occurring in the focus of tuberculosis inflammation. With their help, self-regulation of the renewal and differentiation of mononuclear cells is carried out, intercellular interactions are built under the conditions of a specific process and regeneration.

The universal mediator of intercellular interactions is IL-1, the target of which are lymphocytes, polymorphonuclear leukocytes, fibroblasts, endotheliocytes and other cellular elements. At the same time, the secretory function of lung macrophages is based on the principles of self-regulation, when the same cell secretes not only regulators of extracellular processes, but also inhibitors that block their action. Secretory macrophages differ significantly from phagocytic ones in their ultrastructural organization. They rarely contain phagosomal vacuoles and secondary lysosomes, but have a developed vesicular apparatus and other ultrastructural signs of secretion. They are especially well expressed in epithelioid cells, which are hyperactive secretory macrophages.

Certain stages of differentiation of lung macrophages can be clearly traced under a light and especially an electron microscope in the bronchoalveolar lavage material. Depending on the structural organization of the nucleus and cytoplasm, young non-activated and biosynthetic mononuclears, as well as mature phagocytic and secreting macrophages are determined among them. Young non-activated cells (15-18 μm in diameter) usually make up about 1/5 of all macrophage elements. They have a round nucleus with smooth contours: the cytoplasm is weakly basophilic, does not contain any inclusions. Under an electron microscope, rare profiles of the cytoplasmic reticulum and mitochondria, several small lysosome-like granules, and free ribosomes are visible in these cells.

Activated, biosynthetic macrophages are larger in size (18-25 μm in diameter), the nucleus is distinguished by wavy contours and a distinct nucleolus. They have basophilic cytoplasm, which contains developed long canals of the granular cytoplasmic network and numerous polysomes. Elements of the lamellar complex are detected simultaneously in two or three zones, where primary lysosomes accumulate. Secondary lysosomes are represented by single inclusions; phagosomes are rarely detected, which reflects the readiness of the cell for phagocytic function.

The diameter of mature lung macrophages varies widely (30-55 μm), depending on the activity and functional orientation of the cells. The largest sizes are characteristic of macrophages with structural signs of pronounced phagocytosis. The surface of such cells forms numerous microgrowths and long pseudopodia. The oval or round nucleus is often located acentrically, has wavy contours. A significant amount of condensed chromatin lies near the nuclear membrane, the nucleolus is small (1-1.2 μm). Inclusions, short canals of the granular cytoplasmic reticulum, cisterns and vacuoles of the lamellar complex, and free ribosomes are determined in the cytoplasm. The cells contain a significant number of mitochondria, primary (0.5-1 μm) and secondary (1.2-2 μm) lysosomes, as well as phagosomal vacuoles that vary in size and number. The latter contain fragments of destroyed cellular elements and tuberculosis mycobacteria (“necrophages”, “hemosiderophages”), lamellar inclusions of phospholipid nature (“phospholipophages”) and/or granules of neutral fat (“lipophages”), particles of dust, tobacco resin, kaolin (“coniophages”, “smoker’s macrophages”).

In the presence of a constant object of phagocytosis, multinuclear macrophages (more than 70 μm in diameter) with five or more nuclei appear. Typical foreign body cells - the final stage of differentiation of a macrophage with phagocytic function - are determined in the granulomas and granulation tissue of tuberculous foci. Lung macrophages with pronounced secretory activity (25-40 μm in diameter) usually do not have typical pseudopodia. The nature of the surface can be compared to a thin lace indentation formed by numerous, relatively short micro-outgrowths. The round or oval nucleus contains a small amount of condensed chromatin, a clear large nucleolus (1.5-2 μm). The transparent cytoplasm practically does not contain large inclusions. Short canals of the granular cytoplasmic network are represented by single profiles, while well-developed elements of the lamellar complex are numerous vacuoles and vesicles with electron-transparent or osmiophilic contents. The same structures are detected in the ectoplasm, where they merge directly with the plasmalemma. Even in long-term smokers, in whom all phagocytic cells contain characteristic inclusions of tobacco tar, secreting macrophages have a small number of secondary lysosomes and single phagosome-like formations, i.e. they practically do not absorb foreign material. Macrophages with ultrastructural signs of secretory activity under normal conditions make up no more than 4-8% of bronchoalveolar lavage. Since the function of these cells is associated with metabolism, synthesis and release of many biologically active substances into the extracellular environment, any disturbances in the mechanisms of specific and non-specific protection lead to an increase in their number, the formation of macrophages with an increased secretory potential - epithelioid cells. They form symplasts or, as a result of incomplete mitotic division, turn into characteristic multinuclear Pirogov-Langhans cells - the final differentiation of a macrophage with secretory activity.

Depending on the body's resistance, the nature of the action, and the conditions of the microenvironment, the processes of transformation of the build-up of phagocytic, secretory, or antigen-presenting activity have their own characteristics. It has been shown that calculating the relative percentage of morphofunctional types of macrophages in bronchoalveolar lavage (determining the macrophage formula) helps in the differential diagnosis of tuberculosis and other pulmonary granulomatosis, and allows one to evaluate the effectiveness of the etiotropic treatment.

The ratio of the number of actively phagocytic and synthesizing lung macrophages not only reflects the nature of the tissue reaction in the area of tuberculosis inflammation, but can serve as an indicator of the activity of the pathological process. The problem of the completion of phagocytosis in tuberculosis also remains relevant. The results of our studies of experimental and clinical material show that the outcome of the interaction between phagocytosis and the pathogen depends on the functional state of the macrophage and the biological properties of the microorganism.

Surfactant system status

Achievements of the experimental and theoretical direction in the study of pulmonary surfactants have made it possible to formulate a modern concept of surfactant as a multicomponent system of cellular and non-cellular elements, the structural and functional unity of which ensures normal biomechanics of respiration.

By now, a certain amount of factual material has been accumulated, which testifies not only to the significant adaptive capabilities of the surfactant system in conditions of profound restructuring of pulmonary ventilation and hemodynamics, but also to the pronounced sensitivity of its components to many unfavorable factors of the tuberculosis process, the specific nature of which is determined by the duration of the pathogen persistence, the wave-like course of the process, and profound disturbances of the microcirculatory bed. The changes observed in this case affect not only the zones of formation of foci of infection, but also remote, actively functioning areas of the pulmonary parenchyma. In this regard, it is extremely important to evaluate the morpho-functional usefulness of various components of the surfactant system, to highlight those changes that can be used to diagnose surfactant-dependent disorders of the respiratory function and their timely correction.

The earliest signs of pulmonary surfactant destruction can be observed in model experiments using special lung fixation methods. At the initial stage of tuberculous inflammation development, they are local in nature and are expressed mainly in the zones of intra-alveolar edema. Under an electron microscope, various stages of peeling and destruction of the outer film - the surfactant membrane by edematous fluid can be observed. These changes are fully manifested in the foci of tuberculous inflammation, where the material of destroyed surfactant is determined everywhere in the composition of the intra-alveolar contents.

The noted changes in the extracellular lining of the alveoli occur in foci of various bacterial pneumonias. In this case, part of A2, primarily in the perifocal alveoli, carries out compensatory production of surfactants. A different picture is observed in the respiratory organs during the development of tuberculous inflammation, since the pathogen has an adverse effect on the processes of intracellular surfactant synthesis. Direct introduction of tuberculosis mycobacteria into the lung of dogs (chest puncture) showed that disorganization of the cytoplasmic reticulum and mitochondria profiles is observed in A2 already in the first 15-30 minutes; after several hours, alveolocytes are completely destroyed at the site of infection. Rapid development of surfactant deficiency leads to collapse of the alveoli and rapid spread of the inflammatory process into the surrounding parenchyma. In the alveoli adjacent to the foci, small young A2 with single small secretory granules or large cells with signs of vacuolization of intracellular structures, sometimes with completely destroyed cytoplasm, predominate. In those alveolocytes where there are developed elements of the cytoplasmic network and lamellar complex, giant osmiophilic lamellar bodies (GLB) are detected, which indicates a delay (inhibition) in the release of intracellular surfactant to the surface of the alveoli.

Mathematical modeling of the secretory function of A2 in foci-free pulmonary parenchyma with increased functional load showed that despite the increase in the volume and numerical density of mature secretory granules, the reserve potential of the population did not change significantly. It was found that under conditions of increased vascular permeability, development of hypoxia and fibrous changes in the interalveolar septa, the balance of the processes of OPT formation and maturation is disrupted towards the predominance of the latter. Accelerated maturation of OPT often leads to an increase in the electron-transparent substance of the matrix in the composition of secretory granules, whereas the content of osmiophilic surfactant material in them may be insignificant; the lamellar material of surface-active substances is loosely packed, occupying only 1/3-1/5 of the volume of the secretory granule. The appearance of a significant number of A2 with vacuolated OPT can be explained by the disruption of the initial stages of secretion formation. Such cells usually have ultrastructural signs of destruction (clearing of the cytoplasmic matrix, edematous swelling of the mitochondria, tubules of the cytoplasmic reticulum and lamellar complex), which indicates a decrease in the processes of intracellular surfactant production.

It is characteristic that the decrease in the synthesis of surface-active phospholipids is accompanied by the appearance of neutral lipid granules in the cytoplasm of A2. An adequate reflection of lipid metabolism disorders in the lung affected by tuberculosis of experimental animals and humans is the accumulation of macrophages-lipophages (foam cells) of varying degrees of maturity in the alveoli and bronchoalveolar lavage material. In parallel, a reliable increase in the content of neutral lipids and a decrease in the proportion of total phospholipids are observed in the lavage fluid.

One of the early signs of surfactant destruction in the experiment and clinical picture of tuberculosis of the respiratory organs is the loss of the ability of its membranes to form structures of reserve material. Instead, on the surface of the alveoli, in the phagosomes of alveolar macrophages and directly in the material of bronchoalveolar lavage, one can see membranes twisted into balls ("giant layered balls") without the characteristic three-dimensional organization. The depth of destructive changes in the surfactant system is also evidenced by the frequency of detection of discharged A2 in the washout. These data correlate with the results of biochemical and physicochemical studies of pulmonary surfactants.

Taking into account all the identified features, three degrees of its disorders are currently distinguished to characterize the state of the surfactant system: minor, severe, widespread. The latter reflects an increased risk of developing surfactant-dependent respiratory failure in patients with widespread destructive forms of the disease.

The results of the studies show that the basis of the disturbances that occur in the surfactant system of the lungs during tuberculosis are processes associated with an increase in the permeability of the air-blood barrier:

• damage to the surfactant on the alveolar surface;
• metabolic changes and damage to A2;
• disruption of the mechanisms for removing waste surfactant from the alveoli.

At the same time, studies have established that the main cytological mechanism supporting the functional potential of the surfactant system in the lung altered by tuberculous inflammation is an increase in the number of hypertrophied A2, mainly in the lung parenchyma distant from the specific focus.

[ 11 ], [ 12 ], [ 13 ], [ 14 ], [ 15 ]

Genetic aspects of susceptibility to tuberculosis

Before we begin our analysis of the current state of research in the field of mechanisms of anti-tuberculosis immunity and tuberculosis immunogenetics, we consider it necessary to dwell on some general positions.

• First, mycobacteria are known to multiply and be destroyed primarily in macrophages. Very little data (and they are contradictory) indicate that there are any factors that can destroy mycobacteria extracellularly.
• Second, there is no compelling evidence that the neutrophil phagocyte system plays a significant role in defense against tuberculosis infection.
• Third, there is no compelling evidence that anti-TB antibodies can either destroy mycobacteria extracellularly or promote their intracellular destruction in macrophages or any other cell types.
• Fourth, there is a large number of facts supporting the position that the central link in anti-tuberculosis immunity is T-lymphocytes and that they exert their regulatory influence through the phagocyte system.
• Fifth, there is a body of evidence that hereditary factors play a significant role in tuberculosis infection.

The data indicating the important role of genetic factors in susceptibility to tuberculosis in humans are quite convincing. First of all, this is indicated by the fact that with an extremely high infection rate of M. tuberculosis (approximately one third of the adult population of the planet), the disease develops in only a small proportion of people. This is also indicated by the different levels of susceptibility to infection in different ethnic groups and the nature of the inheritance of susceptibility and resistance to tuberculosis in families with multiple cases of the disease. Finally, evidence of this position is the significantly increased concordance of the occurrence of clinically expressed tuberculosis in monozygotic (identical) twins compared to dizygotic twins.

Traditional genetic testing for tuberculosis

The role of the major histocompatibility complex and NRAMP*

Identification of genes and their alleles, the expression of which determines sensitivity or resistance to tuberculosis, would not only allow deep insight into the fundamental mechanisms of immunity and the development of the pathological process in tuberculosis, but would also bring closer to reality the use of genetic typing methods to identify individuals among healthy people with a genetically increased risk of contracting tuberculosis, requiring priority preventive measures, in particular, a special approach to vaccination.

* - Natural resistance-associated macrophage protein - macrophage protein associated with natural resistance.

There are a significant number of experimental studies that show the role of a number of genetic systems and individual genes (H2, BCG1, Tbc1, xid, etc.) in resistance (sensitivity) to tuberculosis in mice. In humans, the most studied genes include the major histocompatibility complex (MHC) class II genes, among which the allele complex of the HLA-DR2 family (human) reveals a fairly high degree of association with increased morbidity in several ethnically distant populations, and the alleles of the HLA-DQ locus affect the clinical picture of tuberculosis. Recently, the first successes have been achieved in analyzing the connection of the NRAMP1 gene with tuberculosis in humans. These data are particularly noteworthy because this gene has a high degree of homology with the NRAMP1 gene (formerly called BCG 1, since it controls susceptibility to M. bovisBCG), which is selectively expressed in mouse macrophages and which undoubtedly influences susceptibility to intracellular pathogens (including mycobacteria).

Loss-of-function mutations

Several genes were identified, changes in which, leading to a complete loss of the ability to code a functionally active product (gene knockout), particularly affected the ability of mice to develop a protective immune response to mycobacteria infection. These are the genes encoding IFN-γ, IL-12, TNFα, as well as the receptors of immune system cells to the listed cytokines. On the other hand, with a knockout of the genes encoding IL-4 and IL-10, the course of tuberculosis infection was practically no different from that in wild-type (initial) mice. These data confirmed at the genetic level the primary protective role in tuberculosis of the ability of the immune system (primarily T1 lymphocytes) to respond to infection by producing type 1 cytokines, but not type 2.

The applicability of these data to mycobacterial infections in humans has been demonstrated. In very rare families in which children suffered from recurrent mycobacterial infections and salmonellosis from an early age, the extremely high susceptibility is due to homozygous non-conservative mutations in the genes encoding cell receptors for IFN-γ and IL-12, inherited from parents heterozygous for these mutations; as expected, with such inheritance of rare mutations, the marriages turned out to be closely related. However, such gross violations lead to such a high susceptibility to infections that they practically do not allow the child to survive more than a few years, and then only in almost sterile conditions.

These same considerations give rise to a somewhat skeptical assessment of the approach of modeling infections in animals with knockout mutations in genes that play a primary role in protecting against these infections. Such mutations lead to the expression of phenotypes that have no chance of survival under normal conditions and would be quickly eliminated by selection. Thus, mice that do not express MHC class II products and, as a result, do not have a normal pool of CD4 lymphocytes die from disseminated infection in a short time after infection with M. tuberculosis. A very similar course of tuberculosis in humans is observed with a pronounced drop in the number of CD4 cells in the late stages of AIDS. When solving the issues of genetic determination of risk groups and, in general, for understanding the genetic causes of increased susceptibility within the normal population distribution, the researcher deals with individuals that are, although not optimal (according to this feature), but quite viable. This aspect of the problem speaks in favor of using more traditional experimental models for genetic analysis, for example, interlinear differences in the course of tuberculosis in mice.

Genome screening and previously unknown tuberculosis susceptibility genes

As early as the 1950s and 1960s, it was shown that the inheritance of traits of susceptibility and resistance to tuberculosis in laboratory animals is complex and polygenic. In this situation, firstly, it is necessary to select clearly expressed, "extremely different" phenotypes between susceptible and resistant animals or individuals, i.e. characteristics of the disease, and then study the nature of their inheritance. Secondly, it is necessary to take into account that a priori we have no idea how many genes are involved in disease control and how they are located in the genome. Therefore, it is necessary either to reduce the genetic diversity in the population under study in advance, segregating according to the trait under study, using genetic techniques (which is only possible in animal experiments), or to screen the entire genome using statistical methods of quantitative genetics rather than Mendelian genetics, or to combine these techniques. After the development of genome scanning methods using PCR for microsatellite DNA regions and statistical processing and interpretation of the results, genetic analysis of susceptibility to tuberculosis began at a new level.

The approaches mentioned above have recently been successfully applied in genetic experiments on linear mice by two groups of researchers. A group of authors from the Central Research Institute of Tuberculosis, Russian Academy of Medical Sciences, together with colleagues from the Centre for the Study of Host Resistance at McGill University (Montreal, Canada) and the Royal Stockholm Institute were the first to conduct a genomic screen for the inheritance of the severity of the disease caused by intravenous administration of a high dose of M. tuberculosis strain H37Rv in mice. The A/Sn (resistant) and I/St (sensitive) lines were used as parental lines with opposite susceptibility to tuberculosis. A reliable linkage of susceptibility in females was found to at least three different loci located on chromosomes 3, 9 and 17. More recently, linkage to loci in the proximal part of chromosome 9 and the central part of chromosome 17 was also shown for males. The strongest linkage to susceptibility was found for the locus on chromosome 9. Another group of researchers in the United States screened the mouse genome to determine the inheritance pattern of the susceptibility trait in M. tuberculosa strain Erdman. In a combination of the C57BL/6J (resistant in their model) and C3HeB/FeJ (sensitive) mouse lines, a locus in the central part of chromosome 1 controlling the severity of the disease was mapped in the analysis of F2 hybrids and then BC1 offspring. After the initial mapping, a more precise localization of the locus was achieved using recombination analysis, and its effect on such an important phenotypic trait as the severity of granulomatous lung tissue damage was established in backcrossed mice (generation BC3), i.e. after the genetic diversity among the animals under study was significantly reduced using genetic techniques. It is important to note that the mapped locus. designated sst1 (susceptibility to tuberculosis 1), although located on chromosome 1, is clearly not identical to the NRAMP1 locus. This is evidenced by both its localization on the chromosome and the fact that C57BL/6 mice carry the allele of sensitivity to BCG for the NRAMP1 gene, but the allele of resistance to M tuberculosis for the sst1 locus.

The data published in recent years on the presence in the mouse genome of loci that fundamentally influence the nature of the tuberculosis process allow us to hope for significant progress in this area and in the analysis of genetic susceptibility in humans. The fantastically rapid progress in genomic analysis will most likely make it possible to make the transition from the genetics of mouse tuberculosis to the genetics of human tuberculosis very fast, since the complete sequence of the genome of both humans and mice has been practically deciphered.

Macrophage-mycobacterium interaction

Macrophages play an extremely important role in the defense against tuberculosis infection both at the phase of antigen recognition and elimination of mycobacteria.

After mycobacteria enter the lungs, the situation can develop according to four main patterns:

• the primary host response may be sufficient to completely eliminate all mycobacteria, thereby eliminating the possibility of tuberculosis;
• In the case of rapid growth and reproduction of microorganisms, a disease known as primary tuberculosis develops;
• in latent infection, the disease does not develop, but mycobacteria persist in the body in a so-called dormant state, and their presence is manifested only in the form of a positive skin reaction to tuberculin;
• In some cases, mycobacteria are able to transition from a dormant state to a growth phase, and latent infection is replaced by reactivation of tuberculosis.

The first line of defense against infection, after mycobacteria have reached the lower respiratory tract, are alveolar macrophages. These cells are capable of directly suppressing the growth of bacteria by phagocytizing them. They also participate in a wide range of cellular anti-tuberculosis immunity reactions - through antigen presentation, stimulation of T-lymphocyte accumulation at the site of inflammation, etc. It is important to note that the specific mechanisms of binding of virulent and relatively avirulent strains of mycobacteria to phagocytes may differ.

There is sufficient evidence to indicate that the process of vacuole or phagosome formation during the interaction of M. tuberculosis with a mononuclear phagocyte is mediated by the attachment of the microorganism to complement receptors (CR1, CR3, CR4), mannose receptors, or other cell surface receptors. The interaction between the mannose receptors of phagocytic cells and mycobacteria is mediated, apparently, by the glycoprotein of the mycobacterial cell wall - lipoarabinomannan.

Cytokines of T-helpers type 2 - prostaglandin E2 and IL-4 - stimulate the expression of CR and MR, and IFN-γ, on the contrary, suppresses the expression and function of these receptors, which leads to a decrease in the adhesion of mycobacteria to macrophages. Data on the participation of receptors for surfactant proteins in the attachment of bacteria to cells also continue to accumulate.

The role of the CD14 molecule (phagocyte marker) was demonstrated using a model of interaction between mycobacteria and microglia, resident phagocytes of brain tissue. It was found that antibodies to CD14 prevented infection of microglial cells with the virulent laboratory strain H37Rv. Since the CD14 molecule does not penetrate the cell membrane through and thus has no direct contact with the cytoplasm, it is unable to transmit the lipoprotein-induced signal independently, but requires a coreceptor to activate intracellular signal transmission pathways. The most likely candidates for such coreceptors are representatives of the Toll-like receptor family. Microbial lipoproteins, through activation of these receptors, can, on the one hand, potentiate the host organism's defense mechanisms, and, on the other hand, cause tissue damage through the induction of apoptosis. At the same time, apoptosis is able to inhibit the immune response by eliminating cells involved in immune reactions, thereby reducing the damage caused to tissues.

In addition to the above, it seems quite likely that a significant role in the process of attachment of mycobacteria to phagocytic cells is played by the so-called “scavenger” receptors, which are located on the surface of macrophages and have affinity for a number of ligands.

The fate of M. tuberculosis after phagocytosis is suppression of its growth by macrophages. After entering the phagosome, pathogenic bacteria are exposed to a number of factors aimed at their destruction. Such factors include fusion of the phagosome with lysosomes, synthesis of reactive oxygen radicals and synthesis of reactive nitrogen radicals, especially nitric oxide. The death of mycobacteria inside the macrophage can occur by several mechanisms as a result of complex cytokine-mediated interactions between lymphocytes and phagocytes. It is possible that the ability of mycobacteria to avoid the toxic effects of reactive oxygen and nitrogen radicals is a key step in the transition to the latent stage of infection. The ability of the macrophage to suppress the growth of M. tuberculosis significantly depends on the stage of cell activation (at least partially) and on the balance of cytokines (primarily, probably, platelet-derived growth factor alpha (TGF-α) and IFN-γ).

An important component of the mechanism of antimycobacterial activity of macrophages is apparently apoptosis (programmed cell death). In the model of culturing M.bovis BCG in monocytes, it was shown that apoptosis (but not necrosis) of macrophages is accompanied by a decrease in the viability of phagocytosed mycobacteria.

The role of T-lymphocytes in anti-tuberculosis immunity

T-lymphocytes are known to be the main component of acquired immunity in tuberculosis infection. Immunization of experimental animals with mycobacterial antigens, as well as the course of tuberculosis infection, are accompanied by the generation of antigen-specific lymphocytes CD4 ⁺ and CD8 ⁺.

Deficiency of CD4 and, to a lesser extent, CD8 lymphocytes observed in CD4, CD8, MHCII, MHCI gene knockout mice, as well as upon administration of antibodies specific to CD4 or CD8 antigens, leads to a significant decrease in the resistance of mice to M. tuberculosis infection. It is known that AIDS patients, who are characterized by a deficiency of CD4 ⁺ lymphocytes, have an extremely high sensitivity to tuberculosis. The relative contribution of CD4 ⁺ and CD8 ^{+ lymphocytes to the protective immune response can change at different stages of infection. Thus, in the lung granulomas of mice infected with M. bovis BCG, CD4+} T lymphocytes predominate at the early stages of infection (2-3 weeks), while the content of CD8 ⁺ lymphocytes increases at later stages. During adoptive transfer, CD8 ^{+ lymphocytes, especially their CD44hl} subpopulation, have high protective activity. In addition to CD4 ⁺ and CD8 ⁺ lymphocytes, other lymphocyte subpopulations, in particular γδ and CD4 ⁺ CD8 ^{+ lymphocytes,}, restricted by non-polymorphic molecules of the MHC class CD1. also, apparently, contribute to the protective immunity against tuberculosis infection. The mechanisms of the effector action of T-lymphocytes are reduced mainly to either the production of soluble factors (cytokines, chemokines) or cytotoxicity. In mycobacterial infections, predominant formation of T1 occurs, the characteristic features of which are the production of cytokines IFN-γ and TNF-α. Both cytokines are capable of stimulating the antimycobacterial activity of macrophages, which is primarily responsible for the protective effect of CD4 lymphocytes. In addition, IFN-γ is capable of suppressing the severity of inflammatory reactions in the lungs and thereby reducing the severity of tuberculosis infection. TNF-α is necessary for granuloma formation, full cooperation of macrophages and lymphocytes, and tissue protection from necrotic changes. In addition to its protective effect, TNF-α also has a "pathological" effect. Its production can lead to fever, weight loss, and tissue damage - symptoms characteristic of tuberculosis infection. T lymphocytes are not the only source of TNF-α. Its main producers are macrophages. The effect of TNF-α is largely determined by the level of production of other cytokines of types 1 and 2 in the inflammation focus. In conditions of predominant production of cytokines of type 1 and the absence of production of cytokines of type 2, TNF-α has a protective effect, and with the simultaneous production of cytokines of types 1 and 2, it has a destructive effect. Since, as noted above, mycobacteria stimulate mainly T1 lymphocytes, the course of mycobacterial infections is usually not accompanied by an increase in the production of IL-4 and IL-5. At the same time, in severe forms of infection, as well as in its late stages, there may be a local and systemic increase in the production of IL-4 and IL-5. Whether increased production of type 2 cytokines is a cause of more severe tuberculosis infection or a consequence of it is unclear.

Cytotoxicity towards infected target cells is exhibited by CD8 ⁺ cells as well as "non-classical" CD8 ⁺ lymphocytes restricted by CDlb molecules, CD4 ⁺ CD8 ⁺ lymphocytes, and CD4 ⁺ lymphocytes. The importance of cytotoxicity in protection against tuberculosis is indicated by a decrease in the cytotoxic activity of CD8 ⁺ lymphocytes and the content of perforin in tuberculosis patients compared to healthy donors. It is essential to answer the question of how lysis of infected target cells can influence the course of the infectious process: does it lead to a decrease in the intensity of reproduction of mycobacteria, which are intracellular parasites, or, on the contrary, does it promote the release of mycobacteria from infected macrophages and the infection of new cells. The data of S. Stronger (1997) seem to be able to contribute to understanding this problem. The authors showed. that cytotoxic lymphocytes contain granulysin molecules, which have a bactericidal effect on mycobacteria. For granulysin to penetrate into infected cells, lymphocytes must secrete proteins that form pores in the membrane of target cells. Thus, for the first time, data were obtained on the direct destruction of mycobacteria (in macrophages) by T-lymphocytes, thereby demonstrating the possibility of direct participation of T-lymphocytes in protection against mycobacterial infections.

Regulation of T-cell immune response

The response of T lymphocytes and their production of effector cytokines are regulated by cytokines produced by antigen-presenting cells, including infected macrophages. IL-12 shifts the differentiation of T lymphocytes towards the formation of Th1 cells and stimulates the production of IFN-γ. Infection of mice with IL-12 ^% M.bovis BCG leads to progressive development of the infection, increased dissemination of mycobacteria and is accompanied by the absence of granuloma formation in the lungs. In mice with IL-12p40 ^% infected with M. tuberculosis, uncontrolled growth of mycobacteria is noted, associated with a violation of both natural resistance and acquired immunity and caused by a significant decrease in the production of proinflammatory cytokines IFN-γ and TNF-β. Conversely, administration of recombinant IL-12 to mice followed by infection with M. tuberculosis Erdmann leads to an increase in their resistance to infection.

IL-10 is a regulatory cytokine that stimulates the development of humoral immunity reactions and suppresses many reactions of cellular immunity. It is believed that the effect of IL-10 on the T-cell response may be mediated by its action on macrophages: IL-10 inhibits the presentation of antigens by macrophages and suppresses the synthesis of proinflammatory cytokines TNF-α, IL-1, IL-6, IL-8 and IL-12, GM-CSF, G-CSF by macrophages. IL-10 also has an anti-apoptotic effect. Such a spectrum of action, it would seem, should determine the significant effect of IL-10 on the intensity of anti-tuberculosis immunity, however, the data on the dependence of protective immunity on IL-10 production are extremely contradictory.

TGF-β is a unique factor in suppressing cellular immunity. Its production level correlates with the severity of tuberculosis, and administration of anti-TGF-β antibodies or natural TGF-β inhibitors to mice infected with M. tuberculosis corrects the reduced T-cell response.

It should be noted that the effector role of T-lymphocytes is not limited to the production of cytokines and cellular cytotoxicity. Other processes occurring during the establishment of direct contact between T-lymphocytes and macrophages, as well as the production of chemokines by T-lymphocytes, can make a significant contribution to the development of local inflammatory reactions. The latter, in turn, are caused not only by the response of macrophages and T-lymphocytes. Neutrophils, eosinophils, fibroblasts, epithelial and other cells can be active participants in the processes occurring in the lungs during tuberculosis infection.

Morphological studies of the process of granuloma formation, as well as the results of determining the dynamics of the formation of a specific T-cell response, allow, in our opinion, to distinguish several stages of the interaction of mycobacteria with the macroorganism. The first is characterized by progressive proliferation of mycobacteria in the absence of a specific response of T-lymphocytes and lasts about 2-3 weeks. The second occurs after the formation of mature T-lymphocytes and is characterized by stabilization of mycobacterial growth. As a rule, this is followed by the decompensation stage, which coincides in time with the destruction of lymphoid formations and the appearance of necrotic changes in the lungs. The vaccine effect may be due to a reduction in the first phase of the response.