Effects of early dexamethasone treatment on several markers of inflammation and fibrosis in an animal model of lung silicosis in rats – A pilot study

Lung silicosis, a serious fibrotizing disease, is primarily caused by massive or long-term inhalation of particles of silicon oxide (or silica) (1). Background of the pathological changes in silica-injured lungs has not been completely understood; however, they presumably result from direct cytotoxic effect of silica on macrophages, activation of macrophage surface receptors, lysosomal rupture, generation of reactive oxygen species (ROS), activation of inflammasome, overproduction of cytokines and chemokines, cell apoptosis/pyroptosis, and ongoing lung fibrosis (2-5). Nevertheless, despite a huge progress in understanding the complex interactions among the proinflammatory, prooxidant and profibrotic mechanisms in the pathogenesis of lung silicosis in the last years, there is still a lack of effective therapy. Various anti-inflammatory, antioxidant, and anti-fibrotic treatments including herbal compounds have been tested in experimental models of lung silicosis as well as in patients suffering from lung silicosis; however, effects of the given treatments were often contradictory (6-8).

With respect to a wide therapeutic action of a long-acting corticosteroid dexamethasone, the purpose of this pilot study was to evaluate early effects of dexamethasone on several markers of inflammation and lung fibrosis in a rat model of silicosis. We have presumed that early administration of dexamethasone may positively influence a development of the inflammation and thereby could mitigate the inflammation-associated lung fibrosis.

This study was approved by the National Veterinary Board of Slovakia (Ro-1068/19-221/3) and the local Ethical Committee of Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava (EK 6/2019). Adult male Wistar rats of the mean body weight of 270-320 g were supplied by a certified animal breeding station (VELAZ, Czech Republic). Animals were kept in a certified faculty animal house where they underwent a 7-day quarantine and acclimation, with available food and water ad libitum.

Then, the animals (n=48 in total) were divided into three groups. In Sil group of animals, a model of lung silicosis was induced by a single transoral intratracheal instillation of silica (50 mg/ml/animal) in the inhalation anesthesia (4% isoflurane), while the control animals (Sal group) received an equivalent volume of sterile saline. The instillation of silica/saline was performed on an angled board by hooking the front teeth while positioning of the tongue laterally enables the instillation of silica suspension/saline by micropippetor into the trachea through opened vocal cords during inspiration (modified according to 9, 10). In a half of the silica-instilled animals, the treatment with intraperitoneal dexamethasone (Dexamed, Medochemie, Cyprus) initiated the next day after the silica instillation and was given twice a week at a dose of 1 mg/kg (Dex group). The animals were euthanized 14 days (Sil14, Sal14, and Dex14 subgroups, each of n=8) or 28 days (Sil28, Sal28, and Dex28 subgroups, each of n=8) after the treatment onset by an overdosing of anesthetics (Zoletil, Virbac, France). The analysis after 14 days enables an observation of inflammatory changes, while the analysis after 28 days reveals an observation of early fibrotic changes (11, 12).

Sample of blood was taken by a direct puncture of the heart. The total count of leukocytes was determined by a veterinary hematologic analyzer (Sysmex XT-2000iV, Japan). The differential count of leukocytes was estimated microscopically after staining by May-Grünwald/Giemsa-Romanowski and expressed in percents (%) and in absolute values (x 109/L).

The lung was excised. The left lung was lavaged with saline (0.9% NaCl, 2x10 ml/kg b.w.). The total count of cells was measured by a cell analyzer Countess (Thermo Fisher Scientific, USA). Then, the BAL fluid was centrifuged at 1500 rpm for 15 min. The differential count of cells in the BAL fluid sediment was evaluated microscopically after staining by May-Grünwald/Giemsa-Romanowski and expressed in percents (%) and in absolute values (x 103/mL).

The right lung was washed by saline and stored in 4% formaldehyde. The presence of collagen (by Sirius red staining) and smooth muscle mass (detection of smooth muscle actin, SMA) in the walls of bronchioles and vessels of the right lung was verified immunohistochemically by a qualified histologist. The lung tissue was dehydrated through the series of graded ethanol baths, infiltrated with paraffin and cut into 4μm thick sections.

Detection of collagen by Sirius red: after deparaffinization the slides were firstly stained with Weigert's hematoxylin for visualization of the cell nuclei. After washing in running tap water (10 min), the slides were stained in picro-sirius red solution (Millipore Sigma, USA) for 1 hour. After washing twice in acidic distilled water, and dehydration in 100% ethanol, the slides were mounted in Entellan (Millipore Sigma, USA). The result of staining was red collagen fibers on pale yellow background. The slides were viewed with an Olympus BX43 microscope (Olympus, Japan). The image capture and airway wall thickness measurement was performed with the Quick Photo Micro software, version 3.2 (Olympus, Japan).

Detection of smooth muscle mass by SMA: after deparaffinization, revitalisation and rehydratation, the tissue slides were treated with 3% H₂O₂ solution for 10 min for blocking endogenous peroxidases. Washing with Tris buffer was used after each handling step. The sections were incubated with the primary rabbit polyclonal smooth muscle actin (SMA; 1:300, Cell Signaling Technology, USA) for 30 min at room temperature. The slides were then incubated by sequential 10 min incubation with LSAB2 System-HRP for use on rat specimens (Dako, Denmark), which detects primary mouse and rabbit antibodies. The sections were then counterstained with Mayer´s hematoxylin (Himedia Laboratories, India) and mounted with an Entellan (Merck, USA). The sections were viewed with an Olympus BX43 microscope (Olympus, Japan) equipped with a photo camera Canon E0S 2000D. The Quick Photo Micro program, version 3.2 (Olympus, Japan) was used to image capture the sections and measure the thickness of the smooth muscle layer in the bronchial wall and the tunica media in the blood vessel wall (expressed by dark brown cytoplasm of SMA-positive cells).

For analysis of the data, the statistical package SYSTAT for Windows (Systat Software Inc., USA) was used. Differences among the groups were analyzed by one-way ANOVA with posthoc Fisher’s LSD test. A value of p<0.05 was considered statistically significant. The data are expressed as means ± SD.

Inhaled silica particles may interact with pulmonary epithelial cells and resident macrophages, but also with dendritic cells and other immune cells contributing to both innate and adaptive immune responses. Our pilot study demonstrated that significant mobilization of leukocytes and first fibrotic changes in the walls of bronchioles and pulmonary vessels can be found already 14 days after a silica instillation into the rat lung. Early administration of dexamethasone mitigated the above mentioned changes suggesting its potential use in the treatment of silicosis.

Inhaled silica immediately reacts with immune cells, which causes their activation and subsequent overproduction of ROS and massive generation of proinflammatory cytokines via an increased expression of transcription factors including nuclear factor (NF)-κB (3, 13, 14). Our results demonstrated that the inflammatory changes detected after the silica instillation were associated with an increased total count of leukocytes in the blood and elevated absolute numbers of circulating lymphocytes and slightly increased counts of neutrophilsand eosinophils. Higher absolute numbers of lymphocytes, neutrophils, and eosinophils were also found in the BAL fluid of the silica-instilled compared to the saline-instilled animals after both 14 and 28 days of the experiment. The decreased percentage of circulating monocytes in the blood of the silica-instilled animals compared to the saline-instilled controls could indicate that these cells migrated from the circulation into the injured lung. However, the percentage of macrophages in the BAL fluid even decreased compared to the saline-instilled group. This may indicate that a part of macrophages could be destroyed by a direct cytotoxic action of silica. The macrophages which survived their contact with extremely toxic silica can move to the lung interstitium where they can transform to activated interstitial macrophages and participate in the progression of lung fibrosis (15), which may partially explain their shortage in the alveolar compartment and lumen of the airways from which the BAL fluid is extracted.

On the other hand, significant increases in neutrophils, eosinophils, and lymphocytes in the BAL fluid were also observed by other authors (16-19). For instance, a significant increase in total cell counts, but particularly in neutrophil counts, in the BAL fluid together with an increased count of neutrophils in the blood was found already in the first days of silica exposure in the silica-aerosol induced model of silicosis (13).

A persistence of inflammation because of an ineffective clearance of phagocyted material and a release of macrophage-derived products is associated with activation of profibrotic mechanisms, which finally results into irreversible fibrotization of the lung tissue (2, 18, 19) and pathological changes in the airways (17). The comparison of the silica-instilled and the saline-instilled animals in our study showed a significant accumulation of collagen and smooth muscle mass in the walls of bronchioles and pulmonary vessels in the silica-injured animals already after 14 days of silica exposure and the extent of fibrotization enlarged during the following 14 days of the experiment. This finding is in agreement with the study of Porter et al. (13) who found a progressive worsening of the lung inflammation and fibrosis with increasing time after the silica exposure. Surprisingly, slight fibrotic changes were also detected in the saline-instilled animals after 14 days when compared to 28 days of the experiment. We can hypothesize that the orotracheal instillation of sterile saline in the control group could initiate some local inflammatory response resulting into a slight increase in collagen formation which was detected only by Sirius red staining, but not by antibody against SMA. Our results are in agreement with other authors who found a significant but transient increase in annexin A5, a marker of apoptosis (20), or sligh but non-sig nificant increases in lactate dehydrogenase, a marker of cell injury, and protein content in BAL fluid (21), or elevations in hydroxyproline, a component of collagen, and malo nyldialdehyde, a marker of lipid peroxidation in the lung tissue (22) of the saline-instilled animals.

The therapeutic action of corticosteroids is mediated by complex genomically- and non-genomically-mediated mechanisms (23, 24). These mechanisms result into effective suppression of inflammation via modulation of inflammatory cascades on various levels and influencing various cells including respiratory epithelium, endothelium, macrophages, eosinophils, neutrophils, and lymphocytes, as well as into the reduction of oxidative and fibrotic changes (25-27). However, the effects of corticosteroids may be largely non-homogenous depending on the type, dose, delivery route, and timing of the given therapy, as we previously published elsewhere (28). In this study, early treatment with dexamethasone decreased the total count of circulating leukocytes, showed a clear trend to reduce the mobilization of inflammatory cells into the BAL fluid and prevented fibrotic changes in the lung as expressed by a decrease in production of collagen and lower accumulation of smooth muscle mass in the walls of bronchioles and vessels in comparison to the non-treated Sil group. In agreement with our results, partial alleviation of inflammatory infiltrate, granulomatous res ponse, and generation of collagen after dexamethasone treatment was demonstrated in other two studies in silica-instilled mice (19, 29). However, in the study by Rabolli et al. (19) the reduction in inflammatory response after dexamethasone was not associated with antifibrotic effect. Variability in the antifibrotic response to given dexamethasone may be related to inter-species differences of laboratory animals which were used for preparing the model of silicosis. Thus, dexamethasone may reduce the pulmonary fibrotic response in rats (30), but it may omit such effect in mice (31). The decreased markers of inflammation and oxidative stress, mitigated expression of NF-κB, and lower total counts and neutrophil counts in the BAL fluid after dexamethasone treatment was demonstrated by other authors in the silica-instilled animals (32, 33) but also by us in the models of acute lung injury (34, 35). Dexamethasone-induced inhibition of chemotactic factors may result in a reduction of neutrophil margination and influx into the lungs, with their simultaneous increase in the blood (36, 37) as also demonstrated in our previous studies (34, 35). On the other hand, the potential of dexamethasone to reduce the numbers of eosinophils, neutrophils and lymphocytes in the blood and BAL fluid shown in our study is in agreement with the action of corticosteroids in the treatment of other respiratory diseases including bronchial asthma (38-40), chronic obstructive pulmonary disease (41-43), community-aquired pneumonia (44), or various types of acute lung injury (45).

Of course, we are aware of several limitations of our experimental study. In this phase of the project, only male animals were used. Female animals are more sensitive to induction of various forms of lung fibrosis including silicosis, which may be associated with more frequent complications and higher mortality (46, 47), therefore, a majority of animal studies with modeling of lung fibrosis or silicosis use male rats or mice. Moreover, dynamics of progression of pulmonary silicosis in patients inhaling silica particles in higher or smaller concentrations is gradual and develops within the years. In this context, the distribution of the silica particles after an artificial instillation in the laboratory animals is different. However, as demonstrated in many previous studies (2, 13, 14, 19, 31), silica induces rather homogenic local response in the immune cells and the silica-induced inflammatory and fibrotic changes in the lung of laboratory animals are fully comparable with the changes observed in patients suffering from lung silicosis. The other objection may arise from the use of corticosteroids in the treatment of silicosis regarding their wide adverse effects in long-term use. In this study, the long-acting corticosteroid dexamethasone was used for only 28 days to evaluate its potential in administration in an early phase of the development of silicosis. Our results showed that dexamethasone given early may prevent later development of massive inflammatory and fibrotic changes in the lung. These findings may subsequently provoke the following research regarding the early or even preventive use of any anti-inflammatory agents (either synthetically produced or natural compounds) which may prevent the development and progression of irreversible fibrotic changes after silica inhalation.

Our pilot study has shown that early administration of dexamethasone exerted anti-inflammatory, anti-fibrotic and anti-remodeling effects in the rat model of lung silicosis suggesting a future perspective for the early use of anti-inflammatory drugs including corticosteroids in the treatment of lung fibrosis.