Silica-induced Pulmonary Fibrosis In Mice Research Articles

Myricetin Alleviates Silica-Mediated Lung Fibrosis via PPARγ-PGC-1α Loop and Suppressing Mitochondrial Senescence in Epithelial Cells.

Long-term inhalation of silica dust particles leads to lung tissue fibrosis, resulting in impaired gas exchange and increased mortality. Silica inhalation triggers the aging of epithelial cells (AECs), which is a key contributor to the development of pulmonary fibrosis. Myricetin, a flavonoid compound extracted from Myrica genus plants, possesses various biological activities, including antioxidant and immunomodulatory effects. However, the mechanisms underlying myricetin's ability to counter senescence and fibrosis need to be further studied. In vivo, the antifibrotic and anti-senescence effects of myricetin were evaluated using a silica-induced pulmonary fibrosis mouse model. To further elucidate the mechanisms by which myricetin counteracts silica-induced senescence, in vitro experiments were conducted using AECs. Our studies revealed that myricetin treatment alleviated silica-induced mortality, improved lung function, and reduced the severity of pulmonary fibrosis in mice. Immunofluorescence analysis suggests its potential in mitigating senescence of AECs. Under laboratory conditions, myricetin intervened in the cellular senescence pathway induced by silica dust by modulating mitochondrial function. It acted through the PPARγ-PGC1α axis, effectively reducing silica-induced mitochondrial oxidative stress in AECs, promoting mitophagy, and maintaining mitochondrial dynamics. However, the efficacy of myricetin was reversed under PPARγ siRNA intervention. Additionally, myricetin exhibited an enhancing effect on PPARγ and autophagy in animal models. Treatment with PPARγ and PGC-1α siRNA elucidated the role of myricetin in promoting the formation of a positive feedback loop between PPARγ and PGC-1α. Additionally, the PPARγ inhibitor GW9662 verified the in vivo effects of myricetin. Myricetin activates PPARγ, forming a PPARγ-PGC-1α loop, which promotes mitophagy and maintains mitochondrial dynamics. This alleviates epithelial cell senescence induced by silica exposure, consequently mitigating silica-induced pulmonary fibrosis in mice.

Apelin Prevents and Alleviates Crystalline Silica-induced Pulmonary Fibrosis via Inhibiting Transforming Growth Factor Beta 1-triggered Fibroblast Activation.

Silicosis is a common and ultimately fatal occupational disease, yet the limited therapeutic option remains the major clinical challenge. Apelin, an endogenous ligand of the G-protein-coupled receptor (APJ), is abundantly expressed in diverse organs. The apelin-APJ axis helps to control pathological and physiological processes in lung. The role of apelin in the pathological process and its possible therapeutic effects on silicosis have not been elucidated. In this study, we found that lung expression and circulating levels of apelin were markedly decreased in silicosis patients and silica-induced fibrotic mice and associated with the severity. Furthermore, in vivo data demonstrated that pre-treatment from day 3 and post-treatment from day 15 with apelin could both alleviate silica-induced pulmonary fibrosis in mice. Besides, apelin inhibited pulmonary fibroblast activation via transforming growth factor beta 1 (TGF-β1) signaling. Our study suggested that apelin could prevent and reverse silica-induced pulmonary fibrosis by inhibiting the fibroblast activation through TGF-β1 signaling pathway, thus providing a new potential therapeutic strategy for silicosis and other pulmonary fibrosis.

Lipid dysregulation associated with progression of silica-induced pulmonary fibrosis.

Silicosis is an irreversible, progressive, fibrotic lung disease caused by long-term exposure to dust-containing silica particles at the workplace. Despite the precautions enforced, the rising incidence of silicosis continues to occur globally, particularly in developing countries. A better understanding of the disease progression and potential metabolic reprogramming of silicosis is warranted. The low- or high-dose silica-induced pulmonary fibrosis in mice was constructed to mimic chronic or accelerated silicosis. Silica-induced mice lung fibrosis was analyzed by histology, lung function, and computed tomography scans. Non-targeted metabolomics of the lung tissues was conducted by ultra-high-performance liquid chromatography-mass spectrometry to show the temporal metabolic trajectory. The low-dose silica-induced silicosis characterized inflammation for up to 42 days, with the onset of cellular silicon nodules. Conversely, the high-dose silica-induced silicosis characterized inflammation for up to 14 days, after which the disease developed rapidly, with a large volume of collagen deposition, presenting progressive massive fibrosis. Both low- and high silica-induced fibrosis had aberrant lipid metabolism. Combined with the RNA-Seq data, this multiomics study demonstrated alterations in the enzymes involved in sphingolipid metabolism. Time-dependent metabolic reprogramming revealing abnormal glycerophospholipid metabolism was intimately associated with the process of inflammation, whereas sphingolipid metabolism was crucial during lung fibrosis. These findings suggest that lipid dysregulation, especially sphingolipid metabolism, was involved in the process of silicosis.

PD-1/PD-L1 inhibitor ameliorates silica-induced pulmonary fibrosis by maintaining systemic immune homeostasis

Pulmonary fibrosis induced by silica particles is defined as silicosis, which is an incurable disease. The pathogenesis of silicosis is not completely clear, but it’s certain that immune system dysfunction is closely related to it. Immune checkpoint inhibitors (ICIs) are emerging immunotherapeutic agents that mainly target adaptive immune cells, and there is abundant evidence that ICIs are of great value in cancer treatment. However, whether these attractive agents can be implemented in silicosis treatment is unclear. In this study, we explored the efficacy of small molecule inhibitors targeted PD-1/PD-L1 and CTLA-4 on silica-induced pulmonary fibrosis in mice. ICIs were injected intraperitoneally into mice that received silica instillation twice a week. The mice were sacrificed 7 and 28 days after the injection. The lungs, spleen, hilar lymph nodes, thymus, and peripheral blood of mice were collected and subjected to histological examination, flow cytometry analysis, and mRNA and protein quantification. Our results demonstrated that silica exposure caused damage to multiple immune organs in mice, leading to an imbalance in systemic immune homeostasis. Specifically, proportions and subtypes of T and B cells were significantly altered, and the expressions of PD-1, PD-L1 and CTLA-4 were abnormal on these cells. Both PD-1/PD-L1 and CTLA-4 inhibitor administration modulated silica-induced immune system disruption, however, only PD-1/PD-L1 signaling inhibition showed significant amelioration of silicosis. Our findings confirmed for the first time the potential value of ICIs for the treatment of silica-induced pulmonary fibrosis, and this may provide new ideas for the treatment of other fibrosis-related diseases.

Complement protein C1q activates lung fibroblasts and exacerbates silica-induced pulmonary fibrosis in mice

Pulmonary fibrosis is a progressive fibrotic disease with a poor prognosis and has suboptimal therapeutic options. The complement protein, C1q, which has various functions, such as promoting phagocytosis and signal transduction, has been shown to exacerbate several fibrosis-related diseases such as myofibrosis. In this study, we examined the role and cellular targets of C1q in pulmonary fibrosis. Silica-induced pulmonary fibrotic C1q-deficient mice showed improvement in fibrosis, and intratracheal administration of C1q to normal mice led to the induction of fibrotic changes. Single-cell RNA sequencing analysis revealed the early activation of fibroblasts and type 2 alveolar epithelial cells after intratracheal administration of C1q, and treatment of primary lung fibroblasts with C1q induced the expression of profibrotic genes. Thus, the inhibition of C1q may be regarded as a therapeutic target for pulmonary fibrosis.

Profibrotic properties of C1q+ interstitial macrophages in silica-induced pulmonary fibrosis in mice

Pulmonary fibrosis (PF) is a progressive fibrotic disease with poor prognosis and suboptimal therapeutic options. Although macrophages have been implicated in PF, the role of macrophage subsets, particularly interstitial macrophages (IMs), remains unknown. We performed a time-series single-cell RNA sequencing analysis of the silica-induced mouse PF model. Among the macrophage subsets in fibrotic lungs, Lyve1lo MHC IIhi IMs increased with fibrosis, and highly expressed profibrotic genes. Additionally, we identified C1q as an IM-specific marker. Experiments with C1q-diphtheria toxin receptor-GFP knock-in (C1qKI) mice revealed that IMs are distributed around fibrotic nodules. Depletion of C1q+ IMs in C1qKI mice decreased activated fibroblasts and epithelial cells; however, bodyweight loss and neutrophil infiltration were exacerbated in silica-induced PF. Collectively, these results suggest that IMs have profibrotic and anti-inflammatory properties and that the selective inhibition of the profibrotic function of IMs without compromising their anti-inflammatory effects is a potential novel therapeutic strategy for PF.

Ferulic acid ameliorates the progression of pulmonary fibrosis via inhibition of TGF-β/smad signalling

Natural products are one of the best sources for the discovery of novel drugs and compounds for multiple diseases. Pulmonary fibrosis (PF) is a chronic, progressive, irreversible, and fatal fibrotic disorder of lungs with unknown etiology and finite therapeutic choices. The use of naturally occurring phytomedicines has emerged to counteract many fibrotic disorders involving oxidative stress and inflammation. In the present study, we evaluated the protective effects of ferulic acid (FA), in an animal model of silica-induced PF. Pulmonary function of mice was evaluated by performing radiological analysis, bronchoalveolar lavage fluid (BALF), inflammatory cytokines, histology and protein expression studies. Our findings revealed that mice challenged with silica displayed characteristic features of pulmonary injury and fibrosis. However, treatment with FA significantly restored the accumulation of inflammatory cells in BALF. FA led to a partial reversal of silica-induced fibrotic changes in the pulmonary tissue. Subsequently, FA halts the progression of PF in a dose-dependent manner by ameliorating the expression of fibrotic proteins including collagen-I, TGF-β, p-smad2/3 and prevented epithelial-mesenchymal transition (EMT). Collectively, the present study suggests that the inhibition of oxidative stress, inflammatory and TGF-β/smad signalling might be involved in the observed anti-fibrotic benefits of FA against silica-induced PF in mice.

MiR-503 suppresses fibroblast activation and myofibroblast differentiation by targeting VEGFA and FGFR1 in silica-induced pulmonary fibrosis.

Inhalation and deposition of crystalline silica particles in the lung can cause pulmonary fibrosis, then leading to silicosis. Given the paucity of effective drugs for silicosis, new insights for understanding the mechanisms of silicosis, including lung fibroblast activation and myofibroblast differentiation, are essential to explore therapeutic strategies. Our previous research showed that the up‐regulation of miR‐503 alleviated silica‐induced pulmonary fibrosis in mice. In this study, we investigated whether miR‐503 can regulate the TGF‐β1‐induced effects in lung fibroblasts. Mimic‐based strategies aiming at up‐regulating miR‐503 were used to discuss the function of miR‐503 in vivo and in vitro. We found that the expression level of miR‐503 was decreased in fibroblasts stimulated by TGF‐β1, and the up‐regulation of miR‐503 reduced the release of fibrotic factors and inhibited the migration and invasion abilities of fibroblasts. Combined with the up‐regulation of miR‐503 in a mouse model of silica‐induced pulmonary fibrosis, we revealed that miR‐503 mitigated the TGF‐β1‐induced effects in fibroblasts by regulating VEGFA and FGFR1 and then affecting the MAPK/ERK signalling pathway. In conclusion, miR‐503 exerted protective roles in silica‐induced pulmonary fibrosis and may represent a novel and potent candidate for therapeutic strategies in silicosis.

Polyphenol-rich blue honeysuckle extract alleviates silica-induced lung fibrosis by modulating Th immune response and NRF2/HO-1 MAPK signaling

Blue honeysuckles are rich in polyphenol compounds that exert many beneficial effects. Previously, we demonstrated that polyphenol-rich blue honeysuckle extract (BHE) could alleviate silica-induced lung inflammation; however, its role in anti-fibrosis effects remains unknown. Here, we applied polyphenol-rich BHE to silica-injured mice to investigate its anti-fibrosis effect and the underlying mechanism. The results showed that BHE alleviated silica-induced pulmonary fibrosis in mice by altering regulatory T cell activity and mitigating T helper (Th)1/Th17 responses, resulting in reduced Th2 cytokine and transcription-factor mRNA levels. Additionally, cell-signaling detection revealed that p38 and c-Jun N-terminal kinase phosphorylation were effectively blocked by BHE treatment, which also downregulated the expression of inducible nitric oxide synthase and upregulated two antioxidant mediators [nuclear factor (erythroid-derived 2)-like 2 and heme oxygenase-1] in the lung. These findings support the use of BHE as functional food to modulate immune responses and alleviate lung fibrosis.

4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice.

Silicosis is caused by exposure to crystalline silica (CS). We have previously shown that blocking 4-1BB signaling attenuated CS-induced inflammation and pulmonary fibrosis. However, the cells that express 4-1BB, which plays a vital role in promoting fibrosis, are still unknown. In this study, we demonstrated that the expression of 4-1BB is elevated in alveolar macrophages (AMs) in the lungs of CS-injured mice. CS exposure also markedly enhanced the expression of 4-1BB in macrophage-like, MH-S cells. In these cells, activation of the 4-1BB signaling with an agonist antibody led to upregulated secretion of pro-fibrotic mediators. Consistently, blocking 4-1BB downstream signaling or genetic deletion of 4-1BB alleviated pro-fibrotic responses in vitro, while treatment with a 4-1BB fusion protein promoted pro-fibrotic responses. In vivo experiments showed that blocking 4-1BB signaling decreased the expressions of pro-fibrotic mediators and fibrosis. These data suggest that 4-1BB signaling plays an important role in promoting AMs-mediated pro-fibrotic responses and pulmonary fibrosis. Our findings may provide a potential molecular target to reduce CS-induced fibrotic responses in occupational lung disease.

Retraction: Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice.

Retraction: Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice.

Secreted Phosphoprotein 1 and Sex-Specific Differences in Silica-Induced Pulmonary Fibrosis in Mice.

Background:Fibrotic lung diseases occur predominantly in males, and reports describe better survival in affected females. Male mice are more sensitive to silica-induced lung fibrosis than silica-treated female mice. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) increases in pulmonary fibrosis, and Spp1 transcription may be regulated by estrogen or estrogen receptor–related receptors.Objective:We determined whether differences in silica-induced SPP1 levels contribute to sex differences in lung fibrosis.Methods:Male and female mice were treated with 0.2 g/kg intratracheal silica, and lung injury was assessed 1, 3, or 14 days post-exposure. Gene-targeted (Spp1–/–) mice, control Spp1+/+ (C57BL/6J) mice, ovariectomized (OVX) female mice, and estrogen-treated male mice were treated with silica, and lung injury was assessed.Results:Silica-induced SPP1 in lung tissue, bronchoalveolar lavage, and serum increased more in male than in female mice. Following silica treatment, bronchoalveolar lavage cell infiltrates decreased in female Spp1–/– mice compared with female Spp1+/+ mice, and lung hydroxyproline decreased in male Spp1–/– mice compared with male Spp1+/+ mice. OVX female mice had increased lung SPP1 expression in response to silica compared with silica-treated sham female mice. Silica-induced lung collagen and hydroxyproline (markers of fibrosis), and SPP1 levels decreased in estrogen-treated males compared with untreated males.Conclusion:These findings suggest that sex-specific differences in SPP1 levels contribute to the differential sensitivity of male and female mice to the development of silica-induced fibrosis.Citation:Latoche JD, Ufelle AC, Fazzi F, Ganguly K, Leikauf GD, Fattman CL. 2016. Secreted phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice. Environ Health Perspect 124:1199–1207; http://dx.doi.org/10.1289/ehp.1510335

Reduced Supply of Monocyte-Derived Macrophages Leads to a Transition from Nodular to Diffuse Lesions and Tissue Cell Activation in Silica-Induced Pulmonary Fibrosis in Mice

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses invivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuselesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, andFgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.

Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.

Pranlukast, a Cysteinyl Leukotriene Type 1 Receptor Antagonist, Attenuates the Progression but Not the Onset of Silica-Induced Pulmonary Fibrosis in Mice

Background: Although cysteinyl leukotrienes (CysLTs) have been implicated in the etiology of acute inflammatory diseases, recent studies have suggested that they also directly stimulate fibroblasts. However, their precise role in the pathogenesis of pulmonary fibrosis is unclear. Methods: In this study, we evaluated the effect of both short- and long-term treatment with pranlukast, a CysLT type 1 (CysLT₁) receptor antagonist, on silica-induced pulmonary fibrosis in mice, which is characterized by persistent progression of fibrosis in the chronic phase. Pranlukast (30 mg/kg/day) was administered orally to mice for 2 or 10 weeks after intratracheal silica instillation. Results: Pranlukast treatment for 10 weeks significantly attenuated the progression of pulmonary fibrosis, and decreased the content of CysLTs and LTB₄, which were markedly increased in the bronchoalveolar lavage fluid (BALF) and lung tissues of silica-instilled mice in the chronic phase. However, pranlukast treatment for 2 weeks neither affected the acute inflammatory response induced by silica instillation nor inhibited the onset of fibrosis. The expression of TGF-β1 and TNF-α was not affected by pranlukast treatment for either 2 or 10 weeks. Conclusions: Pranlukast attenuates the progression of pulmonary fibrosis in the chronic phase but has no effect on the acute inflammatory response or on the onset of pulmonary fibrosis. The antifibrotic effect of pranlukast may be exhibited by antagonizing the direct profibrotic effect of CysLTs, without affecting the expression of other profibrotic cytokines such as TGF-β1 and TNF-α, and also by decreasing the production of CysLTs and LTB₄.

The specific chymase inhibitor TY-51469 suppresses the accumulation of neutrophils in the lung and reduces silica-induced pulmonary fibrosis in mice

ABSTRACTChymase is a chymotrypsin-like serine protease that is present in mast cells. Its activities include various effects associated with inflammatory responses. But little is known about the effects of chymase in pulmonary fibrosis. The mouse silicosis model was induced by intratracheal injection of 10 mg silica. The Ashcroft pathological score and the hydroxyproline content of lungs were measured to evaluate the effect of a chymase inhibitor, 2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl] thiazole-4-carboxylic acid (TY-51469). The cellular composition and cytokine levels in bronchoalveolar lavage fluid (BALF) were also examined. Following TY-51469 treatment, the lung fibrosis score and hydroxyproline level were significantly reduced, and the number of neutrophils and the levels of macrophage inflammatory protein-2, monocyte chemoattractant protein-1, and transforming growth factor-β1 in BALF were reduced on day 21. The administration of TY-51469 at an early stage showed a greater reduction of fibrosis compared to administration at a later stage. The neutrophil number in BALF in mice treated with TY-51469 both at an early stage and late stage was significantly reduced. The level of mouse mast cell proteinase-4 mRNA increased with time in silica-induced fibrosing lung tissue. These results show that the chymase inhibitor TY51469 suppresses the migration of neutrophils, which results in the suppression of lung fibrosis.

Involvement of leukotrienes in the pathogenesis of silica-induced pulmonary fibrosis in mice

ABSTRACTThe authors investigated the role of leukotrienes (LTs) in the pathogenesis of silica-induced pulmonary fibrosis in mice during the progression from acute to chronic phases. Intratracheal instillation of silica particles induced progressive pulmonary fibrosis. The tissue content of cysteinyl (Cys) LTs and LTB4 was markedly increased in the acute phase after silica instillation, concurrently with the up-regulation of LTB4 receptor, transforming growth factor (TGF)-β1, and tumor necrosis factor (TNF)-α, along with down-regulation of the CysLT type 2 receptor. Importantly, the tissue content of CysLTs and mRNA levels of TGF-β1 and TNF-α were increased in the fibrotic lung in the chronic phase. Furthermore, strong immunohistochemical staining for the CysLT type 1 receptor, TNF-α, and TGF-β1, but not for the CysLT type 2 receptor, was codetected in the pathological lesions during both acute and chronic phases. These findings suggest that an increase in LT production in the lung and modulation of homeostatic balance among LT receptors may contribute to the progression of pulmonary fibrosis.

Requirement of tumour necrosis factor for development of silica-induced pulmonary fibrosis

The deposition of silica particles in the lung of man or experimental animals leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. It has long been suspected that the phagocytosis of silica by pulmonary macrophages induces the secretion of fibrogenic factors. Several potentially fibrogenic cytokines released by macrophages have been identified, including interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF), platelet-derived growth factor, basic fibroblast growth factor and transforming growth factor-beta (TGF-beta). Here we show that TNF plays an important part in silica-induced pulmonary fibrosis in mice in that (1) a single instillation of silica leads to a marked increase in the level of lung TNF messenger RNA which lasts for greater than 70 days, while there are no obvious changes in the amounts of IL-1 alpha or TGF-beta mRNAs; and (2) silica-induced collagen deposition is almost completely prevented by anti-TNF antibody, but is significantly increased by continuous infusion of mouse recombinant TNF.