Crystalline Silica Particles Research Articles

TNF-alpha mediates airway hyperreactivity in silicotic mice: Effect of thalidomide treatment.

Inhalation of crystalline silica particles causes silicosis, which is a severe inflammatory lung disease that is associated with granulomatous and fibrotic responses. We investigated whether silica-induced silicosis might promote airway hyperreactivity (AHR) and the role of TNF-α and thalidomide in this process. Mice received an intranasal instillation of silica particles (1.25, 5, and 10 mg/mouse) and given methacholine on days 2, 7, and 28 after provocation or 5-HT challenges on day 7 after provocation. AHR was assessed using invasive whole-body plethysmography. Lung-tissue samples were collected for TNF-α measurements and histological analyses. Thalidomide was given orally from days 21-27 after silica administration. We found that following aerosolised methacholine or 5-HT treatment, a state of AHR was induced with silica-particle amounts of 5 and 10 mg/mouse, but not 1.25 mg/mouse. The effect was apparent within 2 days and remained for at least 28 days. Silica-particle amounts of 5 and 10 mg/mouse also induced significant granuloma response correlating with the silica required to induce AHR. In addition, a parallel was also observed between the elevation of lung tissue levels of TNF-α and AHR. Notably, silica-induced granulomatous and AHR responses were abolished in TNFR1-/- mice compared to wild-type mice. Moreover, the blockade of ongoing TNF-α generation by thalidomide prevented both events. Our findings suggest that exposure of mice to silica particles leads to a granulomatous lung response marked by non-specific AHR induced by TNF-α. In addition, the results indicate that thalidomide can control silica-induced pathological features of the lungs by blocking TNF-α generation.

Short- and long-term pathologic responses to quartz are induced by nearly free silanols formed during crystal fracturing

BackgroundInhalation of respirable crystalline silica particles, including quartz, is associated with an increased risk of developing pathologies, including persistent lung inflammation, fibrosis, cancer, and systemic autoimmunity. We demonstrated that the nearly free silanols (NFS) generated upon quartz fracturing trigger the early molecular events determining quartz toxicity. Here, we address the involvement of NFS in driving short- and long-term pathogenic responses, including lung inflammation, fibrosis, cancer, and autoimmunity in multiple mouse models.ResultsIn vivo pulmonary responses to as-grown NFS-poor quartz (gQ) and fractured NFS-rich quartz (gQ-f) of synthetic origin were compared to two NFS-rich reference quartz dusts (Min-U-Sil 5, mQ-f). Acute and persistent inflammation, as well as fibrosis, were assessed 3 and 60 days, respectively, after administering one dose of particles (2 mg) via oropharyngeal aspiration (o.p.a.) to C57BL/6 mice. The carcinogenic potential was assessed in a co-carcinogenicity study using A/J mice, which were pre-treated with 3-methylcholanthrene (3-MC) and administered four doses of quartz particles (4 × 1 mg, o.p.a.), then sacrificed after 10 months. Autoimmunity was evaluated in autoimmune-prone 129/Sv mice 4 months after particle administration (2 × 1.25 mg, o.p.a). Mice exposed to NFS-rich quartz exhibited a strong acute lung inflammatory response, characterized by pro-inflammatory cytokine release and leukocyte accumulation, which persisted for up to 60 days. No inflammatory effect was observed in mice treated with NFS-poor gQ. Fibrosis onset (i.e., increased levels of pro-fibrotic factors, hydroxyproline, and collagen) was prominent in mice exposed to NFS-rich but not to NFS-poor quartz. Additionally, lung cancer development (tumour numbers) and autoimmune responses (elevated IgG and anti-dsDNA autoantibody levels) were only observed after exposure to NFS-rich quartz.ConclusionsCollectively, the results indicate that NFS, which occur upon fracturing of quartz particles, play a crucial role in the short- and long-term local and systemic responses to quartz. The assessment of NFS on amorphous or crystalline silica particles may help create a predictive model of silica pathogenicity.

Mechanics-activated fibroblasts promote pulmonary group 2 innate lymphoid cell plasticity propelling silicosis progression

Crystalline silica (CS) particle exposure leads to silicosis which is characterized as progressive fibrosis. Fibroblasts are vital effector cells in fibrogenesis. Emerging studies have identified immune sentinel roles for fibroblasts in chronic disease, while their immune-modulatory roles in silicosis remain unclear. Herein, we show that group 2 innate lymphoid cell (ILC2) conversion to ILC1s is closely involved in silicosis progression, which is mediated by activated fibroblasts via interleukin (IL)−18. Mechanistically, Notch3 signaling in mechanics-activated fibroblasts modulates IL-18 production via caspase 1 activity. The mouse-specific Notch3 knockout in fibroblasts retards pulmonary fibrosis progression that is linked to attenuated ILC conversion. Our results indicate that activated fibroblasts in silicotic lungs are regulators of ILC2–ILC1 conversion, associated with silicosis progression via the Notch3–IL-18 signaling axis. This finding broadens our understanding of immune-modulatory mechanisms in silicosis, and indicates potential therapeutic targets for lung fibrotic diseases.

Evaluation and Comparison of Airborne Concentrations of Crystalline Silica and Total Respirable Dust in Refractory Brick Industries in Isfahan

Background and Objective: Inhalation of crystalline silica particles in the workplace can lead to occupational diseases, such as silicosis. This study aims to evaluate and compare airborne concentrations of crystalline silica and respirable dust in the refractory brick industries in Isfahan province. Materials and Methods: In this cross-sectional study, the airborne concentrations of crystalline silica and respirable dust were measured among 79 workers from large industries and 30 workers from small refractory brick industries over a three-year period. Personal sampling was conducted using individual sampling pumps and cyclones. Data analysis was performed in SPSS 24 software using the Mann-Whitney U and Kruskal-Wallis tests. Results: The results indicated that 65.1% of the samples exceeded the occupational exposure limits, while 34.9% were below the limits. There was no significant difference in the mean three-year airborne crystalline silica concentration between the two occupational groups (P=0.791). However, a significant difference was observed in the mean airborne respirable dust concentrations between the groups (P=0.002). Conclusion: The findings of this study suggest that the implementation of safety, health, and environmental management systems has effectively reduced airborne crystalline silica and respirable dust concentrations, creating a safer work environment for employees.

Benchmark dose determining airborne crystalline silica particles based on A549 lung-cell line survival in an in vitro study

Crystalline silica has emerged as a prominent occupational toxicant over extended periods, leading to the development of lung disease and cancer. The objective of this investigation is to establish a benchmark dose (BMD) for crystalline silica micro and nanoparticles based on the dehydrogenase activity of the A549 lung-cell line. The impact of exposure to crystalline silica micro-particles (C–SiO2 MPs) and crystalline silica nanoparticles (C–SiO2 NPs) on A549 epithelial lung cells was examined for durations of 24 and 72 h to evaluate cell viability using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The determination of dose-response and BMD was carried out through the BMD software v 3.2. The findings reveal a dose-dependent relationship between cell viability and both C–SiO2 MPs and -NPs. The BMDL values for 24 h treatment of C–SiO2 MPs and -NPs were determined to be 2.26 and 0.97 µg/ml, respectively, based on exponential models. Correspondingly, these values were found to be 1.17 and 0.85 µg/ml for the 72 h treatment. This investigation underscores the significance of particle size as a contributing factor in assessing occupational health risks. Moreover, the utilization of BMDL can facilitate the determination of more precise values for occupational exposures by considering various parameters associated with particle presence.

Glycolytic reprogramming governs crystalline silica-induced pyroptosis and inflammation through promoting lactylation modification

Prolonged inhalation of environmental crystalline silica (CS) can cause silicosis, characterized by persistent pulmonary inflammation and irreversible fibrosis, but the mechanism has not been elucidated. To uncover the role and underlying mechanism of glycolytic reprogramming in CS-induced pulmonary inflammation, the mouse silicosis models and glycolysis inhibition models were established in vivo. And the CS-induced macrophage activation models were utilized to further explore the underlying mechanism in vitro. The results showed that CS induced lung inflammation accompanied by glycolytic reprogramming and pyroptosis. The application of glycolysis inhibitor (2-DG) suppressed CS-induced pyroptosis and alleviated lung inflammation. In vitro, 2-DG effectively impeded CS-induced macrophage pyroptosis and inflammatory response. Mechanistically, 2-DG suppressed pyroptosis by inhibiting NLRP3 inflammasome activation both in vivo and in vitro. Furtherly, metabolite lactate facilitated NLRP3-dependent pyroptosis synergistically with CS particles, while blocking the source of lactate largely alleviated NLRP3 inflammasome activation and subsequent pyroptosis triggered by CS. More profoundly, the increment of lactate induced by CS might drive NLRP3-dependent pyroptosis by increasing histone lactylation levels. In conclusion, our findings demonstrated inhibiting glycolytic reprogramming could alleviate CS-induced inflammatory response through suppressing NLRP3 -dependent pyroptosis. Increased glycolytic metabolite lactate and protein lactylation modifications might represent significant mechanisms during CS-induced NLRP3 activation and macrophage pyroptosis.

Interleukin-11 drives fibroblast metabolic reprogramming in crystalline silica-induced lung fibrosis

Environmental exposure to crystalline silica (CS) particles is common and occurs during natural, industrial, and agricultural activities. Prolonged inhalation of CS particles can cause silicosis, a serious and incurable pulmonary fibrosis disease. However, the underlying mechanisms remain veiled. Herein, we aim to elucidate the novel mechanisms of interleukin-11 (IL-11) driving fibroblast metabolic reprogramming during the development of silicosis. We observed that CS exposure induced lung fibrosis in mice and activated fibroblasts, accompanied by increased IL-11 expression and metabolic reprogramming switched from mitochondrial respiration to glycolysis. Besides, we innovatively uncovered that elevated IL-11 promoted the glycolysis process, thereby facilitating the fibroblast-myofibroblast transition (FMT). Mechanistically, CS-stimulated IL-11 activated the extracellular signal-regulated kinase (ERK) pathway and the latter increased the expression of hypoxia inducible factor-1α (HIF-1α) via promoting the translation and delaying the degradation of the protein. HIF-1α further facilitated glycolysis, driving the FMT process and ultimately the formation of silicosis. Moreover, either silence or neutralization of IL-11 inhibited glycolysis augmentation and attenuated CS-induced lung myofibroblast generation and fibrosis. Overall, our findings elucidate the role of IL-11 in promoting fibroblast metabolic reprogramming through the ERK-HIF-1α axis during CS-induced lung fibrosis, providing novel insights into the molecular mechanisms and potential therapeutic targets of silicosis.

Activation of Sirtuin3 by honokiol ameliorates alveolar epithelial cell senescence in experimental silicosis via the cGAS-STING pathway

BackgroundSilicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair and structural integrity maintenance. Inhibiting ATII cell senescence has shown promise in silicosis treatment.However, the mechanism behind silica-induced senescence remains elusive. MethodsThe study employed male C57BL/6 N mice and A549 human alveolar epithelial cells to investigate silicosis and its potential treatment. Silicosis was induced in mice via intratracheal instillation of crystalline silica particles, with honokiol administered intraperitoneally for 14 days. Silica-induced senescence in A549 cells was confirmed, and SIRT3 knockout and overexpression cell lines were generated. Various analyses were conducted, including immunoblotting, qRT-PCR, histology, and transmission electron microscopy. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. ResultsThis study elucidates how silica induces ATII cell senescence, emphasizing mtDNA damage. Notably, honokiol (HKL) emerges as a promising anti-senescence and anti-fibrosis agent, acting through sirt3. honokiol effectively attenuated senescence in ATII cells, dependent on sirt3 expression, while mitigating mtDNA damage. Sirt3, a class III histone deacetylase, regulates senescence and mitochondrial stress. HKL activates sirt3, protecting against pulmonary fibrosis and mitochondrial damage. Additionally, HKL downregulated cGAS expression in senescent ATII cells induced by silica, suggesting sirt3's role as an upstream regulator of the cGAS/STING signaling pathway. Moreover, honokiol treatment inhibited the activation of the NF-κB signaling pathway, associated with reduced oxidative stress and mtDNA damage. Notably, HKL enhanced the activity of SOD2, crucial for mitochondrial function, through sirt3-mediated deacetylation. Additionally, HKL promoted the deacetylation activity of sirt3, further safeguarding mtDNA integrity. ConclusionsThis study uncovers a natural compound, HKL, with significant anti-fibrotic properties through activating sirt3, shedding light on silicosis pathogenesis and treatment avenues.

Differential regulation of lung homeostasis and silicosis by the TAM receptors MerTk and Axl.

TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 μL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-β and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.

Evaluating silicosis risk: Assessing dust constitution and influence of water as a primary prevention measure in cutting and polishing of silica agglomerates, granite and marble

An increasing number of silicosis cases have been reported related to the use of silica agglomerates. Many studies agree on the severity of this disease, which often presents with severe clinical forms in young workers and after a short latency period.Are there differences in the composition of dust generated by cutting and polishing with silica agglomerates versus granite and marble? Does the use of water injection reduce the risk associated with the use of these materials?We carried out a comparative observational-analytical study, measuring the concentration of dust generated during different machining operations on three different materials: granite, marble, and silica agglomerates. The effect of water injection on dust generation was evaluated.Personal sampling pumps were used, connected to a cyclone with polyvinyl chloride filters. The flow rate of the pumps was adjusted using a piston flowmeter. Measurements with a cascade impactor were made to assess the size distribution of respirable crystalline silica particles within the respirable fraction. In addition, environmental measurements with a spectrometer were made.10 tests were carried out on granite and silica agglomerates for each procedure. In the case of marble, with very low silica content, only 2 tests of each type were carried out. Duration of each measurement was between 6 and 25 min. Cleaning times were set for each of the operations.The amount of dust collected in the respirable fraction was 70.85, 32.50 and 35.78 mg/m3 for dry cutting; 6.50, 3.75 and 3.95 mg/m3 for wet cutting; and 21.35, 13.68 and 17.50 mg/m3 for dry polishing, for granite, marble, and silica agglomerates respectively.Dry procedures in marble, silica agglomerates and granite showed higher dust concentration of particles smaller than 0.5 μm. Silica agglomerates showed higher concentrations of respirable crystalline silica particles than granite and marble, mainly with dry procedures. The greater production of small particles in dry and wet procedures with silica agglomerates shows that water injection is an insufficient preventive measure.

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.

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.

Possible hazardous components in dental alginates: Physicochemical properties by a mineralogical and spectroscopic investigation

Alginates are products used as impression materials in dentistry and prosthetics. They consist of polymers, calcium alginates mixed with diatomite and additives. Recently, the occurrence of severe silicosis associated with exposure to respirable dust of such materials has increased the scientific interest in understanding how these materials may pose a toxicological problem to workers. The primary objective of this study is to improve the understanding of both the existence and the characteristics of the toxicant(s) contained in these materials, with the goal of better defining the risk assessment for this occupational setting.Two commercial dental alginates were subjected to a mineralogical, microchemical and spectroscopic investigation. The results indicate the presence of a significant amount of diatomite, clearly identified by micromorphology and formed mainly by cristobalite. The respirable fraction of the dust represents at least 30 % of the total number of particles, and this fraction contains a relevant amount of crystalline silica particles. Conversely, the investigated alginate materials do not exhibit the presence of radical species.The results obtained confirm that the cristobalite detected originates from the high-temperature transformation of amorphous silica during the calcination process of diatomite, prior to mixing with the other components. The same process also produces wollastonite (CaSiO3), which, like cristobalite, is a crystalline phase known for its toxicological effects. The present findings call for a rethinking of dental alginates with regard to the definition of their health risks for technical operators.

Crystalline silica-induced proinflammatory eicosanoid storm in novel alveolar macrophage model quelled by docosahexaenoic acid supplementation.

Phagocytosis of inhaled crystalline silica (cSiO2) particles by tissue-resident alveolar macrophages (AMs) initiates generation of proinflammatory eicosanoids derived from the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (ARA) that contribute to chronic inflammatory disease in the lung. While supplementation with the ω-3 PUFA docosahexaenoic acid (DHA) may influence injurious cSiO2-triggered oxylipin responses, in vitro investigation of this hypothesis in physiologically relevant AMs is challenging due to their short-lived nature and low recovery numbers from mouse lungs. To overcome these challenges, we employed fetal liver-derived alveolar-like macrophages (FLAMs), a self-renewing surrogate that is phenotypically representative of primary lung AMs, to discern how DHA influences cSiO2-induced eicosanoids. We first compared how delivery of 25 µM DHA as ethanolic suspensions or as bovine serum albumin (BSA) complexes to C57BL/6 FLAMs impacts phospholipid fatty acid content. We subsequently treated FLAMs with 25 µM ethanolic DHA or ethanol vehicle (VEH) for 24h, with or without LPS priming for 2h, and with or without cSiO2 for 1.5 or 4h and then measured oxylipin production by LC-MS lipidomics targeting for 156 oxylipins. Results were further related to concurrent proinflammatory cytokine production and cell death induction. DHA delivery as ethanolic suspensions or BSA complexes were similarly effective at increasing ω-3 PUFA content of phospholipids while decreasing the ω-6 PUFA arachidonic acid (ARA) and the ω-9 monounsaturated fatty acid oleic acid. cSiO2 time-dependently elicited myriad ARA-derived eicosanoids consisting of prostaglandins, leukotrienes, thromboxanes, and hydroxyeicosatetraenoic acids in unprimed and LPS-primed FLAMs. This cSiO2-induced eicosanoid storm was dramatically suppressed in DHA-supplemented FLAMs which instead produced potentially pro-resolving DHA-derived docosanoids. cSiO2 elicited marked IL-1α, IL-1β, and TNF-α release after 1.5 and 4h of cSiO2 exposure in LPS-primed FLAMs which was significantly inhibited by DHA. DHA did not affect cSiO2-triggered death induction in unprimed FLAMs but modestly enhanced it in LPS-primed FLAMs. FLAMs are amenable to lipidome modulation by DHA which suppresses cSiO2-triggered production of ARA-derived eicosanoids and proinflammatory cytokines. FLAMs are a potential in vitro alternative to primary AMs for investigating interventions against early toxicant-triggered inflammation in the lung.

Crystalline Silica-Induced Proinflammatory Interstitial Macrophage Recruitment through Notch3 Signaling Promotes the Pathogenesis of Silicosis.

Crystalline silica (CS) particles are ubiquitous in the environment, especially in occupational conditions, and exposure to respirable CS causes silicosis. The initial response to CS is mediated by innate immunity, where pulmonary macrophages act as central orchestrators. However, the repercussions of CS on functionally distinct macrophage subsets remain to be inconclusive. Herein, to study the effects of inhaled CS, we divided macrophages into three subsets: circulating monocytes, interstitial macrophages (IMs), and alveolar macrophages (AMs). CS-induced massive IMs increase in the lung, the phenotype and function of which differed from those of tissue-resident AMs and circulating monocytes. The augmented IMs were driven by recruitment of circulating macrophages rather than cell proliferation in situ. Moreover, the IMs predominantly exerted a classic activated (M1) phenotype and expressed proinflammatory cytokines, contributing to CS-induced lung injury. Notably, we demonstrated that IMs augmented Notch3 expression. Mechanistically, using myeloid-specific Notch3-knockout mice, we demonstrated that Notch3 signaling not only promoted IMs recruitment by regulating CCR2 expression but also manipulated the proinflammatory phenotype. Mice with conditional Notch3-knockout exhibited alleviation of CS-induced inflammation and fibrosis in lung. Overall, our study identifies IMs as critical mediators in response to CS and highlights the role of Notch3 in IMs recruitment and activation, providing new insights into CS toxicological effects in the lung.

Fibroblast-derived CXCL14 aggravates crystalline silica–induced pulmonary fibrosis by mediating polarization and recruitment of interstitial macrophages

Exposure to crystalline silica (CS) particles in worksites and dwellings can lead to silicosis due to excessive fibroblast activation. Considering their immuno-regulatory activities, the contribution of pulmonary fibroblasts in the progression of silicosis has not been thoroughly characterized. Here, we demonstrate that exposure of the lung to CS particles leads to the upregulation of fibroblast-derived C-X-C motif chemokine ligand 14 (CXCL14). By employing an in vitro co-culture system, we demonstrated activated fibroblasts recruited bone marrow–derived macrophages (BMDMs) and favored alternative macrophage polarization (M2) mediated by CXCL14. Furthermore, in vivo studies echoed that systemic CXCL14 neutralizing or fibroblast-specific Cxcl14 knockout proved CXCL14 was indispensable for the recruitment and phenotype alteration of lung macrophages, especially interstitial macrophages (IMs), under stimulation by CS particles. Mechanistically, we showed that GLI2 and p21-mediated cellular senescence were mediators of CXCL14 production following CS exposure. Accordingly, GLI2 blockage and countering cellular senescence by reviving PINK1-mediated mitophagy may be efficient strategies to reduce CXCL14 expression in activated fibroblasts during silicosis. Our findings emphasize the immuno-regulatory function of fibroblasts in silicosis via CXCL14, providing intervention targets for CS-induced pulmonary fibrosis.

Investigating erythrocyte hemolysis assay use for proinflammatory potential prediction of silica particles

Crystalline silica, which is a causative agent of silicosis (an occupational disease), is manufactured in a variety of products (particles) with different particle characteristics, such as size and surface properties. In Japan, the products are currently uniformly controlled as crystalline silica, which is a substance subject to labeling and notification requirements. However, since the toxicity of silica particles reportedly varies depending on its characteristics, businesses are encouraged to conduct appropriate risk assessments for each product to prevent silicosis. Recently, silica particles have been reported to induce lysosomal membrane damage, leading to the activation of proinflammatory factors. An indirect method to evaluate lysosomal membrane damage known as the erythrocyte hemolysis assay, in which the erythrocyte membrane is assumed to be the lysosomal membrane, was performed. This study aimed to examine the possibility of constructing a screening system for proinflammatory potential prediction of silica particles based on their erythrocyte hemolytic activity. Hemolysis assays were performed on the silica particles with different sizes, crystallinity, and surface functional groups using the erythrocytes from a healthy volunteer. Additionally, the hemolytic activity of other element particles was compared with that of the silica particles, and 27 types of commercially available crystalline silica particle products underwent screening trials. The hemolytic activity of silica particles was higher in crystalline than that in amorphous and increased with the decreasing size. The hemolytic reaction was particular to silica particles and rarely occurred in particles of other elements. Moreover, the hemolytic activity was significantly suppressed if the silica particles surface was modified with metal ions (Fe3+, Al3+). The hemolytic activities of the crystalline silica products used industrially significantly differed. This study revealed that particle properties, such as size, crystallinity, and surface functional groups, affect the hemolytic activity of silica particles. Particularly, the surface functional groups (silanol groups) that are unique to silica particles were considered to be strongly involved in hemolytic activities. Since grading the commercially available crystalline silica particle products based on the hemolytic rate was possible, hemolytic activity was suggested to be an evaluation index for predicting the proinflammatory potential of silica particles.

Crystalline silica particle functionalized by PEG for its collision-enhanced detection at ultramicroelectrode

Detecting individual particulate matter is highly significant in many areas, such as mine safety, environment, and human health. The analytical method based on single entity electrochemistry (SEE) has shown great potential in detecting, counting, and measuring individual particles, especially conductive metals or carbon particles, based on their unique charge transfer reactions at an ultramicroelectrode (UME). In this study, we report an innovative SEE method for improving the sensitivity of the detection of electrochemical inert crystalline silica particles by functionalizing silica particles with polyethylene glycol (PEG) molecules. The PEG surface functionalization of the silica was characterized by Fourier-transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) techniques. The morphology of silica particles was characterized by a scanning electron microscope (SEM), and a transmission electron microscope (TEM) was employed to calibrate size distribution and determine the elemental composition of silica particles. The surface charges of silica particles were measured by dynamic light scattering techniques. The collision behaviors of crystalline silica particles with UME were investigated by cyclic voltammetric experiments, which are rarely reported in the literature. The crystalline silica particles were detected based on electrochemically blocking the flux of the redox mediator at the surface of UME, which showed significant signal amplification in the proposed method. Our method was demonstrated for detecting crystalline silica functionalized with or without PEG, acquiring the limit of quantification (LOQ) values of 0.391 μM (23.45 μg/L) and 0.824 μM (49.45 μg/L), respectively, which confirmed that a more than two times improvement in LOQ could be achieved over the PEG functionalized silica particles. We further presented a theoretical model using finite element simulations with COMSOL Multiphysics. We deduced a quantitative relation between the distribution of the current step size and the size distribution of silica particles. Therefore, the reported method here provides a paradigm for SEE-based detection of electrochemically inert crystalline silica particles, which extends the previous report substantially concerning particle detection.

Gasdermin D membrane pores orchestrate IL-1α secretion from necrotic macrophages after NFS-rich silica exposure

IL-1α is an intracellular danger signal (DAMP) released by macrophages contributing to the development of silica-induced lung inflammation. The exact molecular mechanism orchestrating IL-1α extracellular release from particle-exposed macrophages is still unclear. To delineate this process, murine J774 and bone-marrow derived macrophages were exposed to increasing concentrations (1–40 cm2/ml) of a set of amorphous and crystalline silica particles with different surface chemical features. In particular, these characteristics include the content of nearly free silanols (NFS), a silanol population responsible for silica cytotoxicity recently identified. We first observed de novo stocks of IL-1α in macrophages after silica internalization regardless of particle physico-chemical characteristics and cell stress. IL-1α intracellular production and accumulation were observed by exposing macrophages to biologically-inert or cytotoxic crystalline and amorphous silicas. In contrast, only NFS-rich reactive silica particles triggered IL-1α release into the extracellular milieu from necrotic macrophages. We demonstrate that IL-1α is actively secreted through the formation of gasdermin D (GSDMD) pores in the plasma membrane and not passively released after macrophage plasma membrane lysis. Our findings indicate that the GSDMD pore-dependent secretion of IL-1α stock from macrophages solely depends on cytotoxicity induced by NFS-rich silica. This new regulated process represents a key first event in the mechanism of silica toxicity, suitable to refine the existing adverse outcome pathway (AOP) for predicting the inflammatory activity of silicas.

ATX-LPA-Dependent Nuclear Translocation of Endonuclease G in Respiratory Epithelial Cells: A New Mode Action for DNA Damage Induced by Crystalline Silica Particles.

Crystalline silica particles (CSi) are an established human carcinogen, but it is not clear how these particles cause necessary mutations. A well-established scenario includes inflammation caused by retained particles in the bronchioles, activated macrophages, and reactive oxygen species (ROS) that cause DNA damage. In previous studies, we showed that CSi in contact with the plasma membrane of human bronchial epithelium induced double strand breaks within minutes. A signaling pathway implicating the ATX-LPA axis, Rac1, NLRP3, and mitochondrial depolarization upstream of DSB formation was delineated. In this paper, we provide in vitro and in vivo evidence that this signaling pathway triggers endonuclease G (EndoG) translocation from the mitochondria to the nucleus. The DNA damage is documented as γH2AX and p53BP1 nuclear foci, strand breaks in the Comet assay, and as micronuclei. In addition, the DNA damage is induced by low doses of CSi that do not induce apoptosis. By inhibiting the ATX-LPA axis or by EndoG knockdown, we prevent EndoG translocation and DSB formation. Our data indicate that CSi in low doses induces DSBs by sub-apoptotic activation of EndoG, adding CSi to a list of carcinogens that may induce mutations via sub-apoptotic and "minority MOMP" effects. This is the first report linking the ATX-LPA axis to this type of carcinogenic effect.