Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile.

BACKGROUND: Inhalation of biopersistent fibers like asbestos can cause strong chronic inflammatory effects, often resulting in fibrosis or even cancer. The interplay between fiber shape, fiber size and the resulting biological effects is still poorly understood due to the lack of reference materials. RESULTS: We investigated how length, diameter, aspect ratio, and shape of synthetic silica fibers influence inflammatory effects at doses up to 250 µg cm-2. Silica nanofibers were prepared with different diameter and shape. Straight (length ca. 6 to 8 µm, thickness ca. 0.25 to 0.35 µm, aspect ratio ca. 17:1 to 32:1) and curly fibers (length ca. 9 µm, thickness ca. 0.13 µm, radius of curvature ca. 0.5 µm, aspect ratio ca. 70:1) were dispersed in water with no apparent change in the fiber shape during up to 28 days. Upon immersion in aqueous saline (DPBS), the fibers released about 5 wt% silica after 7 days irrespectively of their shape. The uptake of the fibers by macrophages (human THP-1 and rat NR8383) was studied by scanning electron microscopy and confocal laser scanning microscopy. Some fibers were completely taken up whereas others were only partially internalized, leading to visual damage of the cell wall. The biological effects were assessed by determining cell toxicity, particle-induced chemotaxis, and the induction of gene expression of inflammatory mediators. CONCLUSIONS: Straight fibers were only slightly cytotoxic and caused weak cell migration, regardless of their thickness, while the curly fibers were more toxic and caused significantly stronger chemotaxis. Curly fibers also had the strongest effect on the expression of cytokines and chemokines. This may be due to the different aspect ratio or its twisted shape.

FOXF1 reverses lung fibroblasts transdifferentiation via inhibiting TGF-ß/SMAD2/3 pathway in silica-induced pulmonary fibrosis.

Silicosis is one of the most common and severe types of pneumoconiosis and is characterized by lung dysfunction, persistent lung inflammation, pulmonary nodule formation, and irreversible pulmonary fibrosis. The transdifferentiation of fibroblasts into myofibroblasts is one of the main reasons for the exacerbation of silicosis. However, the underlying mechanism of transcription factors regulating silicosis fibrosis has not been clarified. The aim of this study was to investigate the potential mechanism of transcription factor FOXF1 in fibroblast transdifferentiation in silica-induced pulmonary fibrosis. Therefore, a silicosis mouse model was established, and we found that FOXF1 expression level was significantly down-regulated in the silicosis group, and after overexpression of FOXF1 by adeno-associated virus (AAV), FOXF1 expression level was up-regulated, and silicosis fibrosis was alleviated. In order to further explore the specific regulatory mechanism of FOXF1 in silicosis, we established a fibroblasts transdifferentiation model induced by TGF-ß in vitro. In the model, the expression levels of SMAD2/3 and P-SMAD2/3 were up-regulated, but the expression levels of SMAD2/3 and P-SMAD2/3 were down-regulated, inhibiting transdifferentiation and accumulation of extracellular matrix after the overexpressed FOXF1 plasmid was constructed. However, after silencing FOXF1, the expression levels of SMAD2/3 and P-SMAD2/3 were further up-regulated, aggravating transdifferentiation and accumulation of extracellular matrix. These results indicate that the activation of FOXF1 in fibroblasts can slow down the progression of silicosis fibrosis by inhibiting TGF-ß/SMAD2/3 classical pathway, which provides a new idea for further exploration of silicosis treatment.

In vivo assessment of the toxic impact of exposure to magnetic iron oxide nanoparticles (IONPs) using Drosophila melanogaster.

Iron oxide nanoparticles (IONPs) have useful properties, such as strong magnetism and compatibility with living organisms which is preferable for medical applications such as drug delivery and imaging. However, increasing use of these materials, especially in medicine, has raised concerns regarding potential risks to human health. In this study, IONPs were coated with silicon dioxide (SiO2), citric acid (CA), and polyethylenimine (PEI) to enhance their dispersion and biocompatibility. Both coated and uncoated IONPs were assessed for genotoxic effects on Drosophila melanogaster. Results showed that uncoated IONPs induced genotoxic effects, including mutations and recombinations, while the coated IONPs demonstrated reduced or negligible genotoxicity. Additionally, bioinformatic analyses highlighted potential implications of induced recombination in various cancer types, underscoring the importance of understanding nanoparticle-induced genomic instability. This study highlights the importance of nanoparticle coatings in reducing potential genotoxic effects and emphasizes the necessity for comprehensive toxicity assessments in nanomaterial research.

Systemic inflammation indices as hematological biomarkers of inflammatory response in non-silicotic workers exposed to respirable silica dust.

This cross-sectional study was performed to assess whether systemic inflammatory indices, including systemic inflammation response index (SIRI), systemic immune­inflammation index (SII), and aggregate index of systemic inflammation (AISI), can be considered as possible inflammatory markers in silica-exposed workers with no diagnosis of silicosis. We studied 371 non-silicotic workers exposed to respirable silica dust (RSD) and 1422 reference workers. The workers' exposure to RSD were assessed and the inflammatory indices were compared between subgroups of the exposed workers based on the severity and duration of exposure. Correlations between inflammatory indices and the pulmonary function parameters were investigated. Also, the receiver operating characteristic (ROC) curve and Youden index were used to determine the cut-off values of the SII, SIRI, and AISI. Significant dose-response relationships were observed between duration of exposure and all indices except monocytes and LMR. No significant interaction was observed between duration of exposure to RSD and smoking. Borderline significant correlations were observed between AISI and SIRI with forced expiratory volume (FEV1) and FEV1 to forced vital capacity (FVC) ratio. Higher AUCs were obtained for SII and AISI, respectively. The cut-off values for these biomarkers to be considered abnormal were > 348.48 for SII, > 183.78 for AISI, and > 0.768 for SIRI. Overall, the present study showed for the first time, that SII, AISI, and SIRI might be considered as available, easy-to-obtain, and non-expensive markers of inflammation in non-silicotic workers with a long duration of exposure to RSD who are at risk of developing silicosis in subsequent years.

LncRNA MRAK052509 competitively adsorbs miR-204-3p to regulate silica dust-induced EMT process.

Silicosis is a systemic disease caused by long-term inhalation of free SiO2 and retention in the lungs. At present, it is still the most important occupational health hazard disease in the world. Existing studies have shown that non-coding RNA can also participate in complex fibrosis regulatory networks. However, its role in regulating silicotic fibrosis is still unclear. In this study, we constructed a NR8383/RLE-6TN co-culture system to simulate the pathogenesis of silicosis in vitro. Design of miR-204-3p mimics and inhibitors to overexpress or downregulate miR-204-3p in RLE-6TN cells. Design of short hairpin RNA (sh-RNA) to downregulate MRAK052509 in RLE-6TN cells. The regulatory mechanism of miR-204-3p and LncRNA MRAK052509 on EMT process was studied by Quantitative real-time PCR, Western blotting, Immunofluorescence and Cell scratch test. The results revealed that miR-204-3p affects the occurrence of silica dust-induced cellular EMT process mainly through regulating TGF-ßRΙ, a key molecule of TGF-ß signaling pathway. In contrast, Lnc MRAK052509 promotes the EMT process in epithelial cells by competitively adsorbing miR-204-3p and reducing its inhibitory effect on the target gene TGF-ßRΙ, which may influence the development of silicosis fibrosis. This study perfects the targeted regulation relationship between LncRNA MRAK052509, miR-204-3p and TGF-ßRΙ, and may provide a new strategy for the study of the pathogenesis and treatment of silicosis.

Silica Nanoparticles Decrease Glutamate Uptake in Blood-Brain Barrier Components.

Glutamate is the major excitatory amino acid in the vertebrate brain, playing an important role in most brain functions. It exerts its activity through plasma membrane receptors and transporters, expressed both in neurons and glia cells. Overstimulation of neuronal glutamate receptors is linked to cell death in a process known as excitotoxicity, that is prevented by the efficient removal of the neurotransmitter through glutamate transporters enriched in the glia plasma membrane and in the components of the blood-brain barrier (BBB). Silica nanoparticles (SiO2-NPs) have been widely used in biomedical applications and directed to enter the circulatory system; however, little is known about the potential adverse effects of SiO2-NPs exposure on the BBB transport systems that support the critical isolation function between the central nervous system (CNS) and the peripheral circulation. In this contribution, we investigated the plausible SiO2-NPs-mediated disruption of the glutamate transport system expressed by BBB cell components. First, we evaluated the cytotoxic effect of SiO2-NPs on human brain endothelial (HBEC) and Uppsala 87 Malignant glioma (U-87MG) cell lines. Transport kinetics were evaluated, and the exposure effect of SiO2-NPs on glutamate transport activity was determined in both cell lines. Exposure of the cells to different SiO2-NP concentrations (0.4, 4.8, 10, and 20 µg/ml) and time periods (3 and 6 h) did not affect cell viability. We found that the radio-labeled D-aspartate ([3H]-D-Asp) uptake is mostly sodium-dependent, and downregulated by its own substrate (glutamate). Furthermore, SiO2-NPs exposure on endothelial and astrocytes decreases [3H]-D-Asp uptake in a dose-dependent manner. Interestingly, a decrease in the transporter catalytic efficiency, probably linked to a diminution in the affinity of the transporter, was detected upon SiO2-NPs. These results favor the notion that exposure to SiO2-NPs could disrupt BBB function and by these means shed some light into our understanding of the deleterious effects of air pollution on the CNS.

High inflammatory cytokines gene expression can be detected in workers with prolonged exposure to silver and silica nanoparticles in industries.

Occupational health must be strictly considered in industries particularly in nanoparticle factories where workers were exposed to different types of chemicals. We measured the serum levels of inflammatory cytokines in workers who developed skin lesions after exposure to silver and silica nanoparticles. Using a questionnaire in this cross-sectional study, we identified 110 workers in nanoparticle industries who were exposed to silver and silica nanoparticles. We also included 40 healthy subjects as controls from the administrative department of the same factories who were not exposed to nanoparticles. Peripheral blood samples used to measure the mRNA levels of inflammatory cytokines by qRT-PCR. In comparison with the control group, the workers who developed skin lesions had significantly higher levels of interleukin IL4, IL6, IL8, and TNF-α, particularly after two or three decades of exposure to silver and silica nanoparticles. Participants who were exposed to silver had higher levels of IL6 and IL8 compared with those who were exposed to silica. Necessary measures must be considered to protect workers in nanoparticle industries against the potential toxic effects of these compounds. Our network pharmacology study suggests corresponding biochemical pathways for these disorders.

Macrophage-derived exosomal HMGB3 regulates silica-induced pulmonary inflammation by promoting M1 macrophage polarization and recruitment.

BACKGROUND: Chronic inflammation and fibrosis are characteristics of silicosis, and the inflammatory mediators involved in silicosis have not been fully elucidated. Recently, macrophage-derived exosomes have been reported to be inflammatory modulators, but their role in silicosis has not been explored. The purpose of the present study was to investigate the role of macrophage-derived exosomal high mobility group box 3 (HMGB3) in silica-induced pulmonary inflammation. METHODS: The induction of the inflammatory response and the recruitment of monocytes/macrophages were evaluated by immunofluorescence, flow cytometry and transwell assays. The expression of inflammatory cytokines was examined by RT-PCR and ELISA, and the signalling pathways involved were examined by western blot analysis. RESULTS: HMGB3 expression was increased in exosomes derived from silica-exposed macrophages. Exosomal HMGB3 significantly upregulated the expression of inflammatory cytokines, activated the STAT3/MAPK (ERK1/2 and p38)/NF-κB pathways in monocytes/macrophages, and promoted the migration of these cells by CCR2. CONCLUSIONS: Exosomal HMGB3 is a proinflammatory modulator of silica-induced inflammation that promotes the inflammatory response and recruitment of monocytes/macrophages by regulating the activation of the STAT3/MAPK/NF-κB/CCR2 pathways.

Exposure to micron-grade silica particles triggers pulmonary fibrosis through cell-to-cell delivery of exosomal miR-107.

Long-term exposure to inhalable silica particles may lead to severe systemic pulmonary disease, such as silicosis. Exosomes have been demonstrated to dominate the pathogenesis of silicosis, but the underlying mechanisms remain unclear. Therefore, this study aimed to explore the roles of exosomes by transmitting miR-107, which has been linked to the toxic pulmonary effects of silica particles. We found that miR-107, miR-122-5p, miR-125a-5p, miR-126-5p, and miR-335-5p were elevated in exosomes extracted from the serum of patients with silicosis. Notably, an increase in miR-107 in serum exosomes and lung tissue was observed in the experimental silicosis mouse model, while the inhibition of miR-107 reduced pulmonary fibrosis. Moreover, exosomes helped the migration of miR-107 from macrophages to lung fibroblasts, triggering the transdifferentiation of cell phenotypes. Further experiments demonstrated that miR-107 targets CDK6 and suppresses the expression of retinoblastoma protein phosphorylation and E2F1, resulting in cell-cycle arrest. Overall, micron-grade silica particles induced lung fibrosis through exosomal miR-107 negatively regulating the cell cycle signaling pathway. These findings may open a new avenue for understanding how silicosis is regulated by exosome-mediated cell-to-cell communication and suggest the prospect of exosomes as therapeutic targets.

Cuproptosis-associated hub gene identification and immune cell infiltration patterns in silicosis.

Recent research has hinted at a potential connection between silicosis, a fibrotic lung disease caused by exposure to crystalline silica particles, and cuproptosis. The aim of the study was to explore how cuproptosis-related genes (CRGs) may influence the development of silicosis and elucidate the underlying mechanisms. An analysis of genes associated with both silicosis and cuproptosis was conducted. Key gene identification was achieved through the application of two machine learning techniques. Additionally, the correlation between these key genes and immune cell populations was explored and the critical pathways were discerned. To corroborate our findings, the expression of key genes was verified in both a publicly available silica-induced mouse model and our own silicosis mouse model. A total of 12 differentially expressed CRGs associated with silicosis were identified. Further analysis resulted in the identification of 6 CRGs, namely LOX, SPARC, MOXD1, ALB, MT-CO2, and AOC2. Elevated immune cell infiltration of CD8 T cells, regulatory T cells, M0 macrophages, and neutrophils in silicosis patients compared to healthy controls was indicated. Validation in a silica-induced pulmonary fibrosis mouse model supported SPARC and MT-CO2 as potential signature genes for the prediction of silicosis. These findings highlight a strong association between silicosis and cuproptosis. Among CRGs, LOX, SPARC, MOXD1, ALB, MT-CO2, and AOC2 emerged as pivotal players in the context of silicosis by modulating CD8 T cells, regulatory T cells, M0 macrophages, and neutrophils.

Silica-induced macrophage pyroptosis propels pulmonary fibrosis through coordinated activation of relaxin and osteoclast differentiation signaling to reprogram fibroblasts.

Silica nanoparticle (SiNP) exposure induces severe pulmonary inflammation and fibrosis, but the pathogenesis remains unclear, and effective therapies are currently lacking. To explore the mechanism underlying SiNPs-induced pulmonary fibrosis, we constructed in vivo silica exposure animal models and in vitro models of silica-induced macrophage pyroptosis and fibroblast transdifferentiation. We found that SiNP exposure elicits upregulation of pulmonary proteins associated with pyroptosis, including NLRP3, ASC, IL-1ß, and GSDMD, while the immunofluorescence staining co-localized NLRP3 and GSDMD with macrophage-specific biomarker F4/80 in silica-exposed lung tissues. However, the NLRP3 inhibitor MCC950 and classical anti-fibrosis drug pirfenidone (PFD) were found to be able to alleviate silica-induced collagen deposition in the lungs. In in vitro studies, we exposed the fibroblast to a conditioned medium from silica-induced pyroptotic macrophages and found enhanced expression of α-SMA, suggesting increased transdifferentiation of fibroblast to myofibroblast. In line with in vivo studies, the combined treatment of MCC950 and PFD was demonstrated to inhibit the expression of α-SMA and attenuate fibroblast transdifferentiation. Mechanistically, we adopted high throughput RNA sequencing on fibroblast with different treatments and found activated signaling of relaxin and osteoclast differentiation pathways, where the expression of the dysregulated genes in these two pathways was examined and found to be consistently altered both in vitro and in vivo. Collectively, our study demonstrates that SiNP exposure induces macrophage pyroptosis, which subsequently causes fibroblast transdifferentiation to myofibroblasts, in which the relaxin and osteoclast differentiation signaling pathways play crucial roles. These findings may provide valuable references for developing new therapies for pulmonary fibrosis.

Differential pulmonary toxicity and autoantibody formation in genetically distinct mouse strains following combined exposure to silica and diesel exhaust particles.

BACKGROUND: Inhalation of airborne particulate matter, such as silica and diesel exhaust particles, poses serious long-term respiratory and systemic health risks. Silica exposure can lead to silicosis and systemic autoimmune diseases, while DEP exposure is linked to asthma and cancer. Combined exposure to silica and DEP, common in mining, may have more severe effects. This study investigates the separate and combined effects of occupational-level silica and ambient-level DEP on lung injury, inflammation, and autoantibody formation in two genetically distinct mouse strains, thereby aiming at understanding the interplay between genetic susceptibility, particulate exposure, and disease outcomes. Silica and diesel exhaust particles were administered to mice via oropharyngeal aspiration. Assessments of lung injury and host response included in vivo lung micro-computed tomography, lung function tests, bronchoalveolar lavage fluid analysis including inflammatory cytokines and antinuclear antibodies, and histopathology with particle colocalization. RESULTS: The findings highlight the distinct effects of silica and diesel exhaust particles (DEP) on lung injury, inflammation, and autoantibody formation in C57BL/6J and NOD/ShiLtJ mice. Silica exposure elicited a well-established inflammatory response marked by inflammatory infiltrates, release of cytokines, and chemokines, alongside mild fibrosis, indicated by collagen deposition in the lungs of both C57BL/6J and NOD/ShilLtJ mice. Notably, these strains exhibited divergent responses in terms of respiratory function and lung volumes, as assessed through micro-computed tomography. Additionally, silica exposure induced airway hyperreactivity and elevated antinuclear antibody levels in bronchoalveolar lavage fluid, particularly prominent in NOD/ShiLtJ mice. Moreover, antinuclear antibodies correlated with extent of lung inflammation in NOD/ShiLTJ mice. Lung tissue analysis revealed DEP loaded macrophages and co-localization of silica and DEP particles. However, aside from contributing to airway hyperreactivity specifically in NOD/ShiLtJ mice, the ambient-level DEP did not significantly amplify the effects induced by silica. There was no evidence of synergistic or additive interaction between these specific doses of silica and DEP in inducing lung damage or inflammation in either of the mouse strains. CONCLUSION: Mouse strain variations exerted a substantial influence on the development of silica induced lung alterations. Furthermore, the additional impact of ambient-level DEP on these silica-induced effects was minimal.

Lung Cancer Risks Associated with Occupational Exposure to Pairs of Five Lung Carcinogens: Results from a Pooled Analysis of Case-Control Studies (SYNERGY).

BACKGROUND: While much research has been done to identify individual workplace lung carcinogens, little is known about joint effects on risk when workers are exposed to multiple agents. OBJECTIVES: We investigated the pairwise joint effects of occupational exposures to asbestos, respirable crystalline silica, metals (i.e., nickel, chromium-VI), and polycyclic aromatic hydrocarbons (PAH) on lung cancer risk, overall and by major histologic subtype, while accounting for cigarette smoking. METHODS: In the international 14-center SYNERGY project, occupational exposures were assigned to 16,901 lung cancer cases and 20,965 control subjects using a quantitative job-exposure matrix (SYN-JEM). Odds ratios (ORs) and 95% confidence intervals (CIs) were computed for ever vs. never exposure using logistic regression models stratified by sex and adjusted for study center, age, and smoking habits. Joint effects among pairs of agents were assessed on multiplicative and additive scales, the latter by calculating the relative excess risk due to interaction (RERI). RESULTS: All pairwise joint effects of lung carcinogens in men were associated with an increased risk of lung cancer. However, asbestos/metals and metals/PAH resulted in less than additive effects; while the chromium-VI/silica pair showed marginally synergistic effect in relation to adenocarcinoma (RERI: 0.24; CI: 0.02, 0.46; p = 0.05). In women, several pairwise joint effects were observed for small cell lung cancer including exposure to PAH/silica (OR = 5.12; CI: 1.77, 8.48), and to asbestos/silica (OR = 4.32; CI: 1.35, 7.29), where exposure to PAH/silica resulted in a synergistic effect (RERI: 3.45; CI: 0.10, 6.8). DISCUSSION: Small or no deviation from additive or multiplicative effects was observed, but co-exposure to the selected lung carcinogens resulted generally in higher risk than exposure to individual agents, highlighting the importance to reduce and control exposure to carcinogens in workplaces and the general environment. https://doi.org/10.1289/EHP13380.

NLRP3 inflammasome mediates abnormal epithelial regeneration and distal lung remodeling in silica­induced lung fibrosis.

NOD-like receptor protein 3 (NLRP3) inflammasome is closely related to silica particle­induced chronic lung inflammation but its role in epithelial remodeling, repair and regeneration in the distal lung during development of silicosis remains to be elucidated. The present study aimed to determine the effects of the NLRP3 inflammasome on epithelial remodeling and cellular regeneration and potential mechanisms in the distal lung of silica­treated mice at three time points. Pulmonary function assessment, inflammatory cell counting, enzyme­linked immunosorbent assay, histological and immunological analyses, hydroxyproline assay and western blotting were used in the study. Single intratracheal instillation of a silica suspension caused sustained NLRP3 inflammasome activation in the distal lung. Moreover, a time­dependent increase in airway resistance and a decrease in lung compliance accompanied progression of pulmonary fibrosis. In the terminal bronchiole, lung remodeling including pyroptosis (membrane­distributed GSDMD+), excessive proliferation (Ki67+), mucus overproduction (mucin 5 subtype AC and B) and epithelial­mesenchymal transition (decreased E­Cadherin+ and increased Vimentin+), was observed by immunofluorescence analysis. Notably, aberrant spatiotemporal expression of the embryonic lung stem/progenitor cell markers SOX2 and SOX9 and ectopic distribution of bronchioalveolar stem cells were observed in the distal lung only on the 7th day after silica instillation (the early inflammatory phase of silicosis). Western blotting revealed that the Sonic hedgehog/Glioma­associated oncogene (Shh/Gli) and Wnt/ß­catenin pathways were involved in NLRP3 inflammasome activation­mediated epithelial remodeling and dysregulated regeneration during the inflammatory and fibrotic phases. Overall, sustained NLRP3 inflammasome activation led to epithelial remodeling in the distal lung of mice. Moreover, understanding the spatiotemporal profile of dysregulated epithelial repair and regeneration may provide a novel therapeutic strategy for inhalable particle­related chronic inflammatory and fibrotic lung disease.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100 nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10 nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10 nm SiNPs significantly contributed to the production of reactive oxygen species.

Adverse effects of cigarette filter silica on lungs: Comparison with natural crystalline silica particles.

As a common additive in cigarette filters, nanosilica has been implemented to reduce the release of harmful substances in cigarette smoke. However, the potential risk of occupational exposure for cigarette factory workers is unknown. We collected physical examination data from 710 cigarette factory workers to evaluate the adverse effects of cigarette filter silica exposure. We also established mouse models induced by cigarette filter silica and crystalline silica separately to compare the lung inflammation, pulmonary function, apoptosis, and fibrosis of the two models. Workers in the rolling and packing workshop exposed to cigarette filter silica had a higher rate of abnormal lung function (17.75%) than those in the cutting workshop (0.87%). Animal experiments showed that compared with the same dose of crystalline silica, cigarette filter silica resulted in higher levels of inflammatory factors in the bronchoalveolar lavage fluid (BALF) of mice at day 7, and lower levels of total lung capacity (TLC), inspiratory capacity (IC), vital capacity (VC), and forced vital capacity (FVC) in mice at day 28. Additionally, both exposed groups of mice showed increased levels of caspase 3, collagen I (Col-Ⅰ), α-smooth muscle actin (α-SMA) and hydroxyproline (HYP) in the lungs, as well as collagen accumulation and fibrous nodules at day 28, with no significant difference between the two groups. The results suggested that cigarette filter silica caused more severe early lung inflammation and late ventilation impairment than the same dose of crystalline silica. In the future, we need to pay more attention to nanosilica protection in cigarette factories to prevent pulmonary dysfunction in workers.

Reduced lysosomal activity and increased amyloid beta accumulation in silica-coated magnetic nanoparticles-treated microglia.

Nanoparticles have been used in neurological research in recent years because of their blood-brain barrier penetration activity. However, their potential neuronanotoxicity remains a concern. In particular, microglia, which are resident phagocytic cells, are mainly exposed to nanoparticles in the brain. We investigated the changes in lysosomal function in silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]-treated BV2 murine microglial cells. In addition, we analyzed amyloid beta (Aß) accumulation and molecular changes through the integration of transcriptomics, proteomics, and metabolomics (triple-omics) analyses. Aß accumulation significantly increased in the 0.1 µg/µl MNPs@SiO2(RITC)-treated BV2 cells compared to the untreated control and 0.01 µg/µl MNPs@SiO2(RITC)-treated BV2 cells. Moreover, the MNPs@SiO2(RITC)-treated BV2 cells showed lysosomal swelling, a dose-dependent reduction in proteolytic activity, and an increase in lysosomal swelling- and autophagy-related protein levels. Moreover, proteasome activity decreased in the MNPs@SiO2(RITC)-treated BV2 cells, followed by a concomitant reduction in intracellular adenosine triphosphate (ATP). By employing triple-omics and a machine learning algorithm, we generated an integrated single molecular network including reactive oxygen species (ROS), autophagy, lysosomal storage disease, and amyloidosis. In silico analysis of the single triple omics network predicted an increase in ROS, suppression of autophagy, and aggravation of lysosomal storage disease and amyloidosis in the MNPs@SiO2(RITC)-treated BV2 cells. Aß accumulation and lysosomal swelling in the cells were alleviated by co-treatment with glutathione (GSH) and citrate. These findings suggest that MNPs@SiO2(RITC)-induced reduction in lysosomal activity and proteasomes can be recovered by GSH and citrate treatment. These results also highlight the relationship between nanotoxicity and Aß accumulation.

IRF4 Participates in Pulmonary Fibrosis Induced by Silica Particles through Regulating Macrophage Polarization and Fibroblast Activation.

Long-term exposure to silica dust can cause silicosis, which is characterized by chronic progressive inflammatory injury, fibroblast activation, and the deposition of extracellular matrix. IRF4 is involved in immune response. However, the potential regulation of IRF4 in silicosis and pulmonary fibrosis remains largely unexplored. In this study, RNA-seq analysis identified the upregulated expression of IRF4 in fibrotic lung tissues of mice exposed to silica particles. And we verified the increased expression of IRF4 in SiO2-treated macrophages and TGF-ß1-treated fibroblasts. We further found that the down-regulation of IRF4 impeded the macrophage polarization and the release of pro-fibrotic factors. Moreover, the down-regulation of IRF4 alleviated the migration, invasion, and the expression of fibrotic molecules in fibroblasts. Using ChIP-qPCR assay, we confirmed that IRF4 regulated the transcriptional activity of the IL-17A promoter, thus stimulated fibroblast activation, migration and invasion. In vivo experiment, the AAV-siIRF4 was designed to interfere with the expression of IRF4 in lung tissues of mice exposed to silica particles. Whole blood, bronchoalveolar lavage fluid and lung tissues were obtained from mice at 7, 14, 28 and 56 days after silica exposure. The results showed that the leukocyte content and inflammatory factors reached a peak at day 14 and remained peak for a long time after IRF4 knockdown. Furthermore, the fibrotic responses of mouse lung tissues were alleviated after IRF4 knockdown. Our study explored the important roles of IRF4 in inflammatory and fibrotic responses, which provided a new target for the treatment of silicosis and pulmonary fibrosis.

Inhibition of macrophage pyroptosis ameliorates silica-induced pulmonary fibrosis.

Macrophage pyroptosis has recently been involved in some inflammatory and fibrosis diseases, however, the role of macrophage pyroptosis in silica-induced pulmonary fibrosis has not been fully elucidated. In this study, we explored the role of macrophage pyroptosis in silicosis in vivo and in vitro. A mouse model of silicosis was established and mice were sacrificed at 7, 14, and 28 days after exposure of silica. The results revealed that the expression of GSDMD and other pyroptosis-related indicators was up-regulated obviously at 14 days after silica exposure, indicating that silica induced pyroptosis in vivo. In vitro, human monocytic leukemia cells (THP-1) and human lung fibroblasts (MRC-5) were used to detect the relationship between macrophage pyroptosis and lung fibroblasts. It showed that silica increased the levels of GSDMD and other pyroptosis-related indicators remarkably in macrophages and the supernatant of macrophage stimulated by silica could promote the upregulation of fibrosis markers in fibroblasts. However, GSDMD knockdown suppressed silica-induced macrophage pyroptosis and alleviated the upregulation of fibrosis markers in fibroblasts, suggesting the important role of macrophage pyroptosis in the activation of myofibroblasts during the progression of silicosis. Taken together, it showed that silica could induce macrophage pyroptosis and inhibiting macrophage pyroptosis could be a feasible clinical strategy to alleviate silicosis.

C5a/C5aR1 axis as a key driver promotes epithelial-to-mesenchymal transition in airway epithelial cells in silica nanoparticles-induced pulmonary fibrosis.

Previous studies have shown that silica nanoparticles (SiNPs) exposure can affect the respiratory, cardiovascular, reproductive and other systems, with the lung being the primary target organ for the direct effect, causing damage with a central feature of pulmonary inflammation and fibrosis. However, the underlying mechanisms of pulmonary fibrosis due to SiNPs are not fully understood. The aim of the study was to investigate the role of complement anaphylatoxin C5a in SiNPs-induced pulmonary fibrosis. A mouse model of SiNPs-induced pulmonary fibrosis was established, and pulmonary fibrosis-related indicators, epithelial-to-mesenchymal transition (EMT), C5a/C5aR1 and high mobility group protein B1 (HMGB1) proteins were measured. An in vitro study using the human lung epithelial cell line BEAS-2B investigated whether C5a leads to epithelial-to-mesenchymal trans-differentiation. In vivo studies revealed that SiNPs-induced pulmonary fibrosis mainly manifested as EMT trans-differentiation in airway epithelial cells, which subsequently led to excessive deposition of extracellular matrix (ECM). Furthermore, we found that C5a and C5aR1 proteins were also increased in SiNPs-induced pulmonary fibrosis tissue. In vitro studies also showed that C5a directly activated HMGB1/RAGE signaling and induced EMT in BEAS-2B cells. Finally, treatment of SiNPs-exposed mice with the C5aR1 inhibitor PMX205 effectively reduced C5aR1 levels and inhibited the activation of HMGB1/RAGE signaling and the expression of EMT-related proteins, culminating in a significant alleviation of pulmonary fibrosis. Taken together, our results suggest that C5a/C5aR1 is the main signaling pathway for SiNPs-induced pulmonary fibrosis, which induces EMT in airway epithelial cells via the HMGB1/RAGE axis.