ECM of the lungs

The ECM in the mammalian body is a complex and highly labile system that responds to effects of endogenous and exogenous factors by structural rearrangements of its main components. The main components of ECM include collagens, PGs, and glycoproteins.

Collagens

In turn, collagens are divided into several classes. Among the known XXIX types of collagen [39], most of which belong to non-fibrillar collagens, a smaller part constitutes fibrillar collagens [40, 41]. The latter group (type I, II, III, V, and XI) performs the formative and biomechanical function of ECM and prevents tissue compression. In the lungs, type I and type III collagens are involved in the mechanics of respiratory movements [42] and, along with surfactant, they maintain alveoli in a straightened state. Type IV collagen, having a lamellar structure with fenestrae, is part of the basement membrane of endothelial, epithelial, and other cells. Lung ECM macromolecules (collagen and elastin) are the main factors determining the mechanical properties of lung tissue. Therefore, changes in the composition of these components and their structural organization affect the physiological characteristics of the lungs [43–45]. There are relationships between tissue composition, microstructure, and macrophysiology, showing that the physiological behavior of the lungs reflects both the mechanical properties of individual tissue components and their complex structural organization [46–48], the imbalance of which can lead to impaired ventilation and perfusion, the development of tissue hypoxia and respiratory failure.

Fibrosis is characterized by excessive production of ECM components, primarily collagen, by fibroblasts and other types of cells [49, 50], such as macrophages, endothelial cells, alveolar, and bronchial epithelial cells that can produce components of ECM after lung damage [12].

ECM, present in all organs and tissues, is a complex structural and functional formation [51], changing the process of growth and development of the body [52, 53]. At the same time, the maintenance of homeostasis of the ECM ensures the implementation of its main functions, in particular, the provision of structural, trophic, signaling, intercellular interactions, etc.

In pathological conditions, the ratio of macromolecular structural components of ECM [52, 54], signaling molecules [55], and the activity of enzymes involved in anabolic and catabolic reactions [56, 57] change (Figure 1A and B).

Display full size

Figure 1. 

The main components of the lung ECM are normal, with TB and the introduction of LEDZ. (A) Normal lung; (B) pulmonary fibrosis; (C) the effect of LEDZ administration at the stabilization stage; (D) the effect of LEDZ administration at the stage of chronic inflammation. α2-MG: α2-macroglobulin. ↑: increase in the indicator; ↓: decrease in the indicator; ↓↓: a greater reduction than ↓

The appearance of type I collagen and myofibroblasts around granulomas indicated the initial stage of the forming of a fibrous membrane in this place. In addition, in lung TB, aminoterminal propeptides of procollagen types I and III were found in histological samples accumulated to a greater extent around granulomas than inside them [58], which indicates activation of collagen synthesis of types I and III more in the interstitial than inside granulomas. However, evidence exists that collagen fibers of type I are 12 times more often detected inside granulomas than outside, collagen fibers of type III and elastic fibers 5.4 times and 5.6 times, respectively [59]. It seems likely that such a difference in collagen content is determined by the age of granulomas, fibrosis phase, and duration of the disease.

The development of fibrosis in the lungs occurs at a variable rate. This is evidenced by the results of studying the content of hydroxyproline in the lungs of BCG-infected mice for 180 days (6 months) [36]. On the 3rd day after infecting, the content of hydroxyproline, a marker of collagens has already increased by almost 2 times relative to the control, on the 30th day it increased 1.5 times compared to the 3rd day, on the 60th and 90th days there was a downward trend, but on the 180th day the maximum content of hydroxyproline was twice as much as compared with the data of 3 days and 1.3 times higher compared to 30 days.

The results of the immunohistochemical study of the content of fibrous connective tissue in the lungs correspond to the data obtained for the content of hydroxyproline in different stages of BCG-induced inflammation. The finding confirms that the fibrosis development in the lungs begins as early as 3 to 30 days from the beginning of infection (the stage of acute inflammation). A slight decrease in these indicators by 60 and 90 days indicates the stabilization of fibrosis (stabilization stage), while the subsequent increase in the chronization of the process by 180 days can be considered as a stage of chronic inflammation. Thus, the dynamics of the hydroxyproline content in the lungs were characterized by stage. It increased by 30 days (the stage of acute inflammation), decreased by 60 and 90 days (the stage of stabilization), then increased again to a maximum value by 180 days (the stage of chronic inflammation).

The change in the lung content of hydroxyproline may be associated either with an increase in collagen synthesis (due to an increase in the number of fibroblasts without increasing their fibroplastic activity; an increase in their fibroplastic activity without increasing the number) or the intensity of degradation of synthesized collagen. The severity of pulmonary fibrosis in chronic granulomatous inflammation is primarily determined by the number, size of granulomas, and the degree of their “maturity”, which determines its cellular composition [60]. The dynamics of the hydroxyproline content in the interstitial of the lungs, apparently, is due to both a progressive increase in the numerical density of granulomas from 3 to 180 days, and an increase in their size [36].

PGs and GAGs

PGs and GAGs, as well as collagens, are involved in TB granulomogenesis and fibrosis. All GAGs, except hyaluronan, contain sulfate groups and are called sGAG. They constitute disaccharide chains of alternating monomers (N-acetylated hexosamine—glucosamine or galactosamine) and hexuronic acids (D-glucuronic acid or L-iduronic acid). PGs consist of a basic protein and covalently attached negatively charged sGAG.

According to aggregation PGs in the lungs are divided into three classes [61]. They constitute large chondroitin sulfate PG (versican), small leucine-rich PGs (decorin, biglycan), and heparan sulfate PGs (collagen type XVIII, perlecan, and agrin). Syndecans of types 1–4 are singled out separately [62].

It is known that in lung TB the connective tissue surrounding granulomas contains versican, large chondroitin sulfate PG, and myofibroblasts. In myofibroblasts, type I procollagen was intracellularly stained [63]. Versican (chondroitin/dermatan sulfate PG) is produced by smooth muscle cells and fibroblasts [64]. An interstitial decorin, containing one chain of chondroitin sulfate/dermatan sulfate, was localized in epithelioid cells of granuloma and some myofibroblasts [63]. As a rule, it is closely related to collagen fibrils [65, 66]. According to the authors [63], collagen synthesis is preceded by producing a preliminary matrix rich in versican, i.e., myofibroblasts at first synthesize versican, and after its content reaches sufficient value in the surrounding ECM, they begin to synthesize collagen.

PGs and GAGs are present in ECM and plasmalemma of almost all animal cells, they exist among host macromolecules primarily encountering infectious agents [67]. Hyaluronan, unsulfated GAG, is detected diffusely throughout the lung ECM and performs a structural role. Besides, it can affect many cellular functions and processes, including inflammation [68]. The involvement of hyaluronan in macrophage aggregation during the formation of the primary nucleus of TB granuloma via CD44 receptors is also discussed [49].

GAGs are the “first line” contact with the pathogen and host cells involved in the process of adhesion and invasion [69], modulating the process of inflammation [70]. There are 4 classes of GAGs in the lungs [71], in particular, hyaluronan (14%, 1st class), chondroitin sulfate and dermatan sulfate (31%, 2nd class), heparan sulfate (40–60%) and heparin (5%, 3rd class), keratan sulfate/keratan (4th class).

In the lungs of intact mice, 70–90% of sGAGs are heparan sulfate and chondroitin sulfate/dermatan sulfate [70]. Their content and qualitative composition change with inflammation (Figure 1B). In particular, an increase in sGAG and PGs was shown in patients with pulmonary TB [63]. It is noteworthy that versican, sGAG, and hyaluronan were located in the lung ECM along the edge of developing granulomas, whereas decorin is situated intracellularly in epithelioid cells.

Most of the hyaluronan exists in the ECM in the free state and only 1% binds with hyaladherins [40]. During infection and formation of the “primary nucleus” of granuloma, an important role is assigned to hyaluronan, which participates in the recruitment of leukocytes through its interaction with CD44 [72] and provides aggregation of macrophages in granulomas [49]. Additionally, hyaluronan is one of the main GAGs of ECM, which is regulated by TGF-β, and whose role in fibrosis has been proven [54].

As it was noted above, PGs consist of a basic protein, uronic acids, and hexosamines. It is of interest to study the structure of PGs in TB, as this will help to assess the contribution of individual PGs components in the development of fibrosis.

The study of the structure of PGs in the lungs demonstrated their maximum content on 30th days after BCG-infecting in mice [73]. The sGAG content for 30 days was higher relative to the control. After 30 days, it decreased by 1.9 times on the 60th day, by 3 times on the 90th day, and 2 times lower on the 180th day compared to 30th day. The gradual increase in sGAG by 30 days coincides with the stage of active formation of granulomas, a characteristic manifestation of the TB process. The numerical density of granulomas increased 2.2 times for 30 days [36].

The content of uronic acids, galactose, and protein in PGs in the lungs increased to maximum values on day 30 and then gradually decreased. The noted maximum sGAG content in the lungs of mice on day 30 may reflect an increase in PGs around (versican) and inside (decorin) granulomas.

The development of a pathological process in a certain target organ may affect the content of GAGs in the blood serum. The content of total GAGs in the serum of BCG-infected mice at the above specified observation stages differed from the dynamics of GAGs in the lungs [73]. The total GAG content in infected animals was higher than in the control mice. Their maximum level was observed for 90 days, the minimum for 60 days. It is known that the content of GAGs in blood serum characterizes the intensity of PGs metabolism in the body [74], and the level of hyaluronan circulating in the blood is associated with the intensity of its intake from peripheral tissues through the lymph [75].

Differences in the content of total GAG and sGAG at different infection stages suggest that the synthesis of sGAG was activated in the lungs of BCG-infected mice for 30 days, since no increase of GAGs in the serum during this stage was found. An increase of serum total GAG by 90 days against the decrease in sGAG in the lungs may be due to a shift in the GAGs composition in favor of hyaluronan. The latter is probably formed as a result of degradation of GAGs under both host and bacterial hyaluronidases activity.

It is known that heparan sulfate PG the CD44 receptor, actively participates in the catabolism of hyaluronan, creating favorable conditions for the action of hyaluronidases and the formation of low-molecular fragments of hyaluronan [40]. These fragments are involved in various signaling events, including cell proliferation, migration, differentiation, and induction of proinflammatory cytokines and chemokines [68, 72].

Changes in the metabolism of PGs/GAGs in the lungs associated with granulomogenesis are not always reflected in the blood serum. This concerns the ability of PGs and hyaluronan to facilitate the adhesion of leukocytes and their migration, to interact between PGs and MMP, while reflecting the direct regulation of MMPs activity [70]. An increase of total GAGs level in blood serum by 90 days with a simultaneous decrease in sGAG in the lungs during this stage indicates the possible involvement of low molecular weight hyaluronan in the development of inflammation and increased fibrosis of the lungs by 180 days.

During the breakdown of macromolecular aggregates consisting of hyaluronan and PGs, the released hyaluronan can be exposed to mycobacterial hyaluronidases with the formation of low molecular weight hyaluronan, which has a pro-inflammatory effect. However, another mechanism of GAGs increase in TB inflammation is not excluded, associated with increased expression of hyaluronan synthase and accumulation of hyaluronan on the surface of epithelial cells of the respiratory tract, alveoli, and around granulomas in the lungs of infected mice [76]. An interesting fact is that Mtb uses the host’s ECM hyaluronate for their growth. Degradation of PGs, including versican, the content of which is increased in lung TB [63] may be a source of increased sGAG content.

In chronic BCG-induced inflammation, the expression of TNF-α by activated macrophages was noticeable from day 30, and it was 4.5 times more intense in the interstitial lung than in granulomas [36]. At 180 days of the infectious process, the expression of TNF-α was almost 2 times higher compared to that at 30 days and coincided with the maximum content of hydroxyproline in the lungs. It appears likely that the increase in lung fibrosis by the end of the experiment, as it is evident from the volume density of fibrous tissue and the content of hydroxyproline in the lungs, was induced by a high content of low-molecular-weight hyaluronan fragments with a pro-inflammatory effect in the blood serum for 90 days.

In BCG granulomas (30–90 days), the total number of cells, altered by apoptosis, increased by more than 20% [77]. It has been established that two apoptotic mechanisms are involved in this process: The receptor-mediated mechanism of apoptosis (TRAIL) is more pronounced and associated with cells expressing proinflammatory cytokine (TNF-α) than mitochondrial—mediated caspase 3. However, this does not inhibit the persistence of the pathogen in macrophages of granulomas but contributes to the formation of pronounced fibrosis in granulomas and interstitial lung.

Thus, in the mouse model of chronic BCG-induced inflammation, it was shown that the structural components of PGs in the lungs were maximally elevated for 30 days, they decreased in subsequent stages, which indicates the stage of PGs metabolism in the dynamics of fibrosis development.

The study of sGAG in the dynamics of chronic TB inflammation allowed getting an idea of their contribution to their structural diversity of developing destructive and especially fibroplastic processes, one of the main complications of TB, which are fully manifested 6 months after infecting.

It is known that bacteria, including Mtb, use disaccharide chains of PGs/GAGs as co-receptors for their adhesion and subsequent invasion into the host cells [69, 78]. It is believed that Mtb adhesion to alveolar macrophages and their phagocytosis can be facilitated by surfactants of types A and D present in the lung alveoli [79]. Mtb adhesion is a mandatory stage of interaction for the subsequent formation of granulomas. Their significance is different: For the host organism, granuloma restricts infection, for Mtb—It protects from immune attacks and maintains viability [80]. In 10% of latently infected individuals, bacilli can reactivate with a detailed clinical picture of the disease. The prognosis of the disease is largely determined by the host’s ability to eliminate Mtb.

Taking into account the “mandatory” stage of PGs/GAGs interaction with Mtb during the formation of granulomas, the expediency of developing drugs that suppress an attachment of the pathogen to the disaccharide chains of GAGs is discussed [69]. Inhibition of adhesion could prevent the invasion of Mtb into host cells, the formation of granulomas, and the long-term persistence of Mtb in them.

The quantitative assessment of PGs components in the lungs in the mice model of chronic BCG-granulomatous inflammation showed the lability of their structure at different stages of the process, reflecting the replacement of one GAGs by another. A gradual increase in sGAG, UAs, protein, and galactose during the acute stage of inflammation with a maximum on the 30th day may characterize the intensity of the response of ECM to Mtb infection with the formation of granulomas. However, the dynamics of the content of PG components differ from the dynamics of the content of hydroxyproline.

MMPs/TIMPs

Homeostasis in the ECM and the exchange of its components is regulated by a complex system of enzymes. The main enzymes that destroy ECM molecules are MMPs, a disintegrin-like and metalloproteinase domain with thrombospondin type 1 repeat (ADAMTS), and their activity is controlled by endogenous inhibitors, TIMPs [81–87]. The MMPs synthesis is regulated at the transcription level, and their proteolytic activity is controlled by the activation of proenzymes, as well as the inhibition of active enzymes by endogenous inhibitors—TIMPs and α2-MG, which play an important role in fibrosis processes [88, 89]. It is usually assumed that elevated levels of TIMPs lead to the accumulation of ECM, causing fibrosis, while their decrease leads to enhanced matrix proteolysis [90].

The MMPs family of zinc-dependent endoproteases consists of 28 representatives, whereas TIMPs are a family of four endogenous proteins [91]. Depending on the substrate specificity and domain organization, MMPs are subdivided into collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other MMPs [82, 92, 93].

The expression of MMPs and TIMPs by cells is regulated by many cytokines [in particular, interleukin-1 (IL-1)], growth factors, and hormones, some of which are specific to the cell type, while others are ubiquitous (for example, TGF-β) [94]. High levels of TGF-β1 were detected in tissue fibrosis [4, 95, 96] and fibrous granulomas [6].

The profibrotic properties of TGF-β1 are associated with its participation in stimulating the proliferation, differentiation of fibroblasts, and their transformation into myofibroblasts [97, 98]. It is known that TGF-β induces connective tissue growth factor (CTGF) in fibroblasts through various signaling pathways [99, 100]. Only when interacting with TGF-β, CTGF over expression and pronounced fibrosis were observed in vivo [101, 102]. The effect of another cytokine IL-10 consisted in reducing fibrosis and inhibiting the synthesis of TGF-β1 during pulmonary fibrosis [103–106].

MMPs and TIMPs are considered as potential regulators of TB infection [107]. It was shown that the levels of MMP-1, MMP-8, MMP-9, and MMP-12 in blood plasma were significantly increased in patients with pulmonary TB compared to individuals with extra-pulmonary TB, latent TB, and healthy control. On the contrary, the level of MMP-7 was significantly reduced in patients with pulmonary TB compared to those with extra-pulmonary TB. Similarly, the levels of MMP-1, MMP-7, and MMP-13 were significantly increased in patients with extra-pulmonary TB compared to those with latent TB and healthy control. However, the level of TIMP-2 was significantly reduced, and TIMP-3 was significantly increased in people with extra-pulmonary TB compared to latent TB and healthy control.

When infected with Mtb, MMPs regulate cell migration, reconstruct lung tissue, and initiate granuloma formation [108]. A violation of the regulation of MMPs activity leads to the progression of various pathologies, which can be grouped into: 1) tissue destruction, 2) fibrosis, and 3) matrix weakening [86]. Both “classical immune cells” of myeloid and lymphoid origin and “non-classical immune cells” are involved in all these processes [35]. Type II alveolar pneumocytes infected with Mtb secrete MMPs, which destroy collagens, and contribute to the formation of cavities and the spread of bacilli. The destruction of lung tissue with the formation of caseous necrosis and caverns [109] is also facilitated by the activation of collagenase—MMP-1 associated with the accumulation of hypoxia-inducible factor-1α [83].

Numerous ex vivo and in vitro studies demonstrate that Mtb induces the secretion of MMP-1 by host cells in an inactive form—pro-MMP-1, requiring proteolytic activation [110]. In addition, Mtb secretes serine proteases [111, 112], which indicates a potential proteolytic cascade in which Mtb both directly induces and activates MMP-1 in its microenvironment, contributing to the destruction of the matrix.

The participation of MMPs in the pathogenesis of TB is evidenced by the results of studies of other MMPs. There was a significant increase in the level of MMP-1 by 15 times, MMP-3 by 3.6 times, but a decrease in TIMP-1, TIMP-2 in sputum and bronchoalveolar lavage fluid in patients with pulmonary TB relative to control [113]. However, MMP-8 was not detected, and the content of MMP-2, MMP-7, and MMP-9 did not differ from the control. At the same time, it was shown that the level of circulating MMP-9 was increased in patients with TB relative to the healthy individuals and correlated with the severity of TB [114]. The content of MMP-2 did not differ from the level in the control. In addition, in transgenic mice that express human MMP-1 in the lungs, Mtb infection increased the expression of MMP-1, which led to alveolar destruction in lung granulomas and significantly greater collagen breakdown compared to wild mice.

In patients with infiltrative pulmonary TB with pronounced fibrous changes (70% of patients with multidrug resistance) after the intensive phase of chemotherapy, a more than 8 times decrease in the content of MMP-9 was noted [115]. However, no changes in other enzymes, pro-MMP-1, MMP-8, TIMP-1, and α2-MG were detected. With positive clinical dynamics and complete closure of destruction, the content of MMP-9 decreased by 4.5 times, while in maintaining destruction and decay cavities it fell only by 1.2 times.

Another study showed that in patients at the end of TB treatment, the concentration of MMPs remained elevated due to the influence of pro-inflammatory chemokines [116]. The persistent increase in TIMP-1 and TIMP-2, noted after 6 months of treatment, is associated with ongoing tissue remodeling and possible fibrosis.

An imbalance in the MMPs/TIMPs system leads to degradation of the connective tissue ECM and pathological remodeling, forming a morphological basis for violations of external respiration. This is evidenced by the correlations found in pulmonary TB patients between the concentrations of MMP-9 and MMP-8 in the blood with the extent of lesion of the pulmonary parenchyma when comparing tuberculomas and fibrous-cavernous TB, MMP-9, and TIMP-1—with changes in pulmonary volumes, MMP-8—with functional disorders of gas exchange. However, changes in MMP-1, MMP-3, and α2-MG did not correlate with the volume of lesion of the pulmonary parenchyma and with a decrease in lung function [47].

When BCG was administered to CBA mice 1.5 months after being infected, an increase in the total activity of MMP-2 and MMP-7 in the blood serum was observed [117]. According to the authors [117], such an increase, on the one hand, can ensure the migration of macrophages to the focus of inflammation and lead to the formation of granulomas, and on the other hand, indicate the development of destructive processes in the ECM.

The immunohistochemically measured MMP-9 and the number of myofibroblasts are detected 9 and 12 times more often, respectively, inside granulomas than outside them [59].

There are observations that over expression of TIMP-2 increased the activity of autophagy and decreased the levels of IL-6 and TNF-α in Mtb-infected A549 cells [118]. An increase in the expression of MMP-1 and a decrease in TIMP-3 in New Zealand rabbits is associated with the progression of TB by the formation of cavities in the lungs with a high bacterial load [119].

Incubation of peritoneal mouse macrophages with viable BCG and studies in vivo with infection of BALB/c BCG mice showed increased secretion of MMP-9 and activity of the enzyme [120]. Exogenous addition of TNF-α or IL-18 induced expression of macrophages by MMPs, whereas immunoregulatory cytokines, interferon-γ, IL-4, and IL-10 all suppressed BCG-induced MMP production.

Intravenous infecting of C57BL/6 mice with virulent Mtb Erdman strain leads to differential expression of MMPs in the lungs [121]. The activity of MMP-9 increased by 1 week after infection, while the activity of MMP-2 after 2 weeks. Reverse transcription polymerase chain reaction (RT-PCR) analysis for the expression of gelatinase genes and their corresponding inhibitors showed a slight increase in MMP-9 by 1 week, no changes were observed in TIMP-1 and MMP-2, as well as a significant decrease in TIMP-2 by 4 weeks. According to the authors [121], glycolipid lipoarabinomannan and other Mtb components induce the expression of MMP-9. In the mouse model of BCG-induced inflammation, 6 months after infection, the total activity of MMPs increased by 9 times, hyaluronidase by 4 times, α2-MG by 10 times, TIMP-1 by 11 times, and TIMP-2 by 13 times in the lungs [122, 123], that indicates high both protease and antiprotease activity of enzymes.

The participation of the MMPs/TIMPs system in the mechanisms of fibrosis regulation is multifaceted. It is known that collagenases (MMP-1, MMP-8, and MMP-13) reduce fibrosis by degradation of collagens, MMP-2—by reducing the expression of type I collagen, MMP-12 indirectly stimulates fibrosis by suppressing MMP-13 and activating fibroblasts, MMP-7 indirectly mediates fibrosis by increasing the influx and activation of neutrophils that stimulate tissue destruction, epithelial damage and increase fibrosis [57].

An evaluation of the activity of the MMPs/TIMPs system in the lungs in an animal model, apparently, characterizes the direct responses of enzymes to infection and treatment whereas clinical data obtained by measuring biochemical parameters in serum, plasma, sputum, and bronchoalveolar lavage fluid provide oblique results. However, in both cases, the results can vary depending on the stage of the inflammatory process, on scheme and time of initiation of treatment, comorbidity, and the development of complications.

Introduction of LEDZ at the stage of stabilization of BCG-induced granulomatosis

Intraperitoneal administration of LEDZ at the stage of stabilization of BCG granulomatosis in mice (3 months after infection with BCG vaccine, LEDZ administration for 2 months), when fibrotic processes in the lungs are slowed down [36, 73], was accompanied by significant changes in the structure of pulmonary PGs, a decrease in the content of sGAG, uronic acids, and an increase in galactose (Figure 1C) [126]. This, apparently, indicates in favor of an increase in the content of keratan sulfate PG, which in the lungs of intact mice is no more than 10% [70]. The introduction of LEDZ at this stage did not affect the content of the hydroxyproline and its fractions. At the same time, the activity of MMPs increased, but not the content of TIMP-1 and TIMP-2. This ratio may be associated with the activation of MMP performs and increased expression by MMP-1 and MMP-9 macrophages, since earlier in an in vitro experiment such activation of enzymes was observed when LEDZ was introduced into a culture medium [127].

LEDZ inhalations performed during the stabilization stage of BCG granulomatosis proved to be more effective in terms of reducing the severity of fibrosis in the lungs [128]. The antifibrotic effect during this stage was reproduced by an increase in degradation (i.e., through the increase in free hydroxyproline) and a decrease in collagen synthesis (via a decrease in the content of total hydroxyproline, peptide-bound hydroxyproline, and protein-bound hydroxyproline) (Figure 1C). The decrease in hydroxyproline fractions during inhalation was due to the direct LEDZ entry into the respiratory tract and alveoli followed by its capture by macrophages.

In another mouse model, intraperitoneal administration of LEDZ (4 months after infection with BCG vaccine, LEDZ administration for 2 months) suppressed the expression of MMP-9 mRNA and type III collagen α1 chain in the lungs, which indicates inhibition of fibroblast differentiation into myofibroblasts [129]. Inhalations of LEDZ in this experiment had no effect on the studied indicators.

Introduction of LEDZ at the stage of chronic BCG-induced granulomatosis

Under the intraperitoneal LEDZ administration in the stage of chronic BCG granulomatosis (6 months after infection with BCG vaccine, LEDZ administration for 3 months), the lung contents of hyaluronan, perlecan, peptide-bound hydroxyproline, and protein-bound hydroxyproline decreased, but the content of free hydroxyproline increased (Figure 1D), indicating increased degradation of collagens and suppression of their synthesis [123, 130]. The introduction of LEDZ at this stage of BCG granulomatosis led to a decrease in the activity of MMPs and specific and non-specific inhibitors (TIMP-1, TIMP-2, and α2-MG), which also implies a decrease in the activity of destructive-inflammatory potential [122]. The combined decrease in MMPs and α2-MG activity, level in TIMP-1 and TIMP-2 indicates the suppression of protease, antiprotease activity, and inflammatory response due to the influence of LEDZ.

After inhalation of LEDZ, the content of hyaluronan, perlecan, protein-bound hydroxyproline, peptide-bound hydroxyproline, as well as total hydroxyproline and an increase in free hydroxyproline in the lungs were also reduced (Figure 1D) [123, 131]. However, the activity of MMPs did not differ from the BCG group, while their inhibitors decreased, TIMP-1, TIMP-2, and α2-MG, but remained elevated compared with intraperitoneal administration of LEDZ.

Thus, this section shows the effects of LEDZ on the components of ECM and enzymes that control the exchange of PGs and collagen. The effects of LEDZ depend on the stage of infection and the route of administration. It should be noted that when LEDZ is administered, fibrolysis is activated in both routes of administration. This antifibrotic effect is more pronounced when the composition was administered in the stage of chronic BCG granulomatosis than at the stage of stabilization.

It can be assumed that the components of ECM and their local regulators involved in TB granulogenesis can serve as a target for the development of new approaches to chemotherapy that will improve the prognosis of the disease and reduce the risk of complications.