Pulmonary toxicity and global gene expression changes in response to sub-chronic inhalation exposure to crystalline silica in rats

Exposure to crystalline silica results in serious adverse health effects, most notably, silicosis. An understanding of the mechanism(s) underlying silica-induced pulmonary toxicity is critical for the intervention and/or prevention of its adverse health effects. Rats were exposed by inhalation to crystalline silica at a concentration of 15 mg/m³, 6 hr/day, 5 days/week for 3, 6 or 12 weeks. Pulmonary toxicity and global gene expression profiles were determined in lungs at the end of each exposure period. Crystalline silica was visible in lungs of rats especially in the 12-week group. Pulmonary toxicity, as evidenced by an increase in lactate dehydrogenase (LDH) activity and albumin content and accumulation of macrophages and neutrophils in the bronchoalveolar lavage (BAL), was seen in animals depending upon silica exposure duration. The most severe histological changes, noted in the 12-week exposure group, consisted of chronic active inflammation, type II pneumocyte hyperplasia, and fibrosis. Microarray analysis of lung gene expression profiles detected significant differential expression of 38, 77, and 99 genes in rats exposed to silica for 3-, 6-, or 12-weeks, respectively, compared to time-matched controls. Among the significantly differentially expressed genes (SDEG), 32 genes were common in all exposure groups. Bioinformatics analysis of the SDEG identified enrichment of functions, networks and canonical pathways related to inflammation, cancer, oxidative stress, fibrosis, and tissue remodeling in response to silica exposure. Collectively, these results provided insights into the molecular mechanisms underlying pulmonary toxicity following sub-chronic inhalation exposure to crystalline silica in rats.     

Molecular insights into the progression of crystalline silica‐induced pulmonary toxicity in rats

Identification of molecular target(s) and mechanism(s) of silica‐induced pulmonary toxicity is important for the intervention and/or prevention of diseases associated with exposure to silica. Rats were exposed to crystalline silica by inhalation (15 mg m^?3, 6 h per day, 5 days) and global gene expression profile was determined in the lungs by microarray analysis at 1, 2, 4, 8 and 16 weeks following termination of silica exposure. The number of significantly differentially expressed genes (>1.5‐fold change and <0.01 false discovery rate P‐value) detected in the lungs during the post‐exposure time intervals analyzed exhibited a steady increase in parallel with the progression of silica‐induced pulmonary toxicity noticed in the rats. Quantitative real‐time PCR analysis of a representative set of 10 genes confirmed the microarray findings. The number of biological functions, canonical pathways and molecular networks significantly affected by silica exposure, as identified by the bioinformatics analysis of the significantly differentially expressed genes detected during the post‐exposure time intervals, also exhibited a steady increase similar to the silica‐induced pulmonary toxicity. Genes involved in oxidative stress, inflammation, respiratory diseases, cancer, and tissue remodeling and fibrosis were significantly differentially expressed in the rat lungs; however, unresolved inflammation was the single most significant biological response to pulmonary exposure to silica. Excessive mucus production, as implicated by significant overexpression of the pendrin coding gene, SLC26A4, was identified as a potential novel mechanism for silica‐induced pulmonary toxicity. Collectively, the findings of our study provided insights into the molecular mechanisms underlying the progression of crystalline silica‐induced pulmonary toxicity in the rat. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Immunoglobulin responses to experimental silicosis.

Silicosis is a crippling fibrotic lung disease induced by inhalation of crystalline silica. One feature of silicosis is systemic and pulmonary immune dysfunction characterized in part by elevations in serum and bronchoalveolar lavage (BAL) immunoglobulins. A major specific aim of the current report was to demonstrate that an experimental model of silicosis previously well characterized for the development of pulmonary inflammation and fibrosis would also exhibit increased levels of serum and BAL IgG and IgM similar to those of human silicosis. We also sought to document the anatomic compartments responsible for these immunoglobulin responses. To address these specific aims, we compared levels of IgG and IgM in serum and BAL from rats with experimental silicosis induced by inhalation of silica with levels of these immunoglobulins in titanium dioxide (TiO(2))- and sham (air)-exposed controls. The ability of mononuclear cell populations from lung, lung-associated lymph node, and spleen to produce IgG and IgM ex vivo were also compared. We found that experimental silicosis was associated with elevated IgG and IgM levels in blood and BAL relative to the control groups. Our findings also suggested that draining lung-associated lymph nodes (LALN) were the most important sites for increased IgG and IgM production in experimental silicosis, with lungs contributing to a lesser degree. Increased production in the LALN appeared related to marked expansion in total numbers, but not relative proportion, of B lymphocytes.     

Molecular mechanisms of pulmonary response progression in crystalline silica exposed rats

An understanding of the mechanisms underlying diseases is critical for their prevention. Excessive exposure to crystalline silica is a risk factor for silicosis, a potentially fatal pulmonary disease. Male Fischer 344 rats were exposed by inhalation to crystalline silica (15?mg/m³, six hours/day, five days) and pulmonary response was determined at 44 weeks following termination of silica exposure. Additionally, global gene expression profiling in lungs and BAL cells and bioinformatic analysis of the gene expression data were done to understand the molecular mechanisms underlying the progression of pulmonary response to silica. A significant increase in lactate dehydrogenase activity and albumin content in BAL fluid (BALF) suggested silica-induced pulmonary toxicity in the rats. A significant increase in the number of alveolar macrophages and infiltrating neutrophils in the lungs and elevation in monocyte chemoattractant protein-1 (MCP-1) in BALF suggested the induction of pulmonary inflammation in the silica exposed rats. Histological changes in the lungs included granuloma formation, type II pneumocyte hyperplasia, thickening of alveolar septa and positive response to Masson’s trichrome stain. Microarray analysis of global gene expression detected 94 and 225 significantly differentially expressed genes in the lungs and BAL cells, respectively. Bioinformatic analysis of the gene expression data identified significant enrichment of several disease and biological function categories and canonical pathways related to pulmonary toxicity, especially inflammation. Taken together, these data suggested the involvement of chronic inflammation as a mechanism underlying the progression of pulmonary response to exposure of rats to crystalline silica at 44 weeks following termination of exposure.     

Lung fibrosis induced by crystalline silica particles is uncoupled from lung inflammation in NMRI mice

Previous studies in rats have suggested a causal relationship between progressive pulmonary inflammation and lung fibrosis induced by crystalline silica particles. We report here that, in NMRI mice, the lung response to silica particles is accompanied by a mild and non progressive pulmonary inflammation which is dispensable for the development of lung fibrosis. We found that glucocorticoid (dexamethasone) dramatically reduced lung injury, cellular inflammation and pro-inflammatory cytokine expression (TNF-α, IL-1β and KC) but had no significant effect on silica-induced lung fibrosis and expression of the fibrogenic and suppressive cytokines TGF-β and IL-10 in mice. Other anti-inflammatory molecules such as the COX inhibitor piroxicam or the phosphodiesterase 5 inhibitor sildenafil also reduced lung inflammation without modifying collagen, TGF-β or IL-10 lung content. Our findings indicate that the development of lung fibrosis in silica-treated NMRI mice is not driven by inflammatory lung responses and suggest that suppressive cytokines may represent critical fibrotic factors and potential therapeutic targets in silicosis.     

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure.     

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure.     

Fibrogenic and redox-related but not proinflammatory genes are upregulated in Lewis rat model of chronic silicosis

Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal (IT) silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNA samples from controls or subchronic silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of 7 genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after subchronic silica exposure and compared with 14-d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, Ccl2, and Ccl7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis models. However, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis. Data suggest that genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.     

Inhibition of gasdermin D-dependent pyroptosis attenuates the progression of silica-induced pulmonary inflammation and fibrosis

Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide, but the molecular basis underlying its development remains unclear. An accumulating body of evidence supports gasdermin D (GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases. However, there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis. In this work, we investigated the role of GSDMD-mediated pyroptosis in silicosis. Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression. Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica. Measurement of interleukin-1β release, lactic dehydrogenase activity, and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages. Additionally, we verified that both canonical (caspase-1-mediated) and non-canonical (caspase-4/5/11-mediated) signaling pathways mediated silica-induced pyroptosis activation, in vivo and in vitro. Notably, Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes, which highlighted the pivotal role of pyroptosis in this disease. Taken together, our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.     

Silica dust exposure induces autophagy in alveolar macrophages through switching Beclin1 affinity from Bcl‐2 to PIK3C3

Increased deposition of silica dust in pulmonary interstitial tissues leads to silicosis, in which autophagy plays a defensive role in silica dust‐associated stress response and cell death. Our previous studies revealed that silica dust exposure contributed to autophagy in pulmonary macrophages in vivo, while the specific regulatory mechanism is still unclear. This study aimed to figure out the regulatory mechanism as well as the role of autophagy in the pathogenesis of experimental silicosis. We used 3‐methyladenine (3‐MA) and ABT‐737 to suppress the expression of phosphatidylinositol 3‐kinase catalytic subunit type 3 (PIK3C3) and B cell leukemia/lymphoma 2 (Bcl‐2), two critical initiators of autophagy, and detected and evaluated the autophagy in NR8383 cells with or without silica dust exposure. We found that exposure of silica dust increased autophagy in NR8383 cells and elevated the expression of Beclin1 and PIK3C3, but it reduced the expression of Bcl‐2. The relationship among Beclin1, PIK3C3, and Bcl‐2 were then investigated using immunoprecipitation analysis, and we found that suppression of PIK3C3 and/or Bcl‐2 using 3‐MA and/or ABT‐737 could alter the autophagy induced by silica dust in NR8383 cells, and the complexes of Beclin1/PIK3C3 and Beclin1/Bcl‐2 were both downregulated, which may be that inhibition of PIK3C3 and Bcl‐2 altered the affinity of Beclin1 with PIK3C3 and Bcl‐2 and lead to the silence of PIK3C3 signaling. These findings indicate that silica dust exposure induces autophagy via changing the connectivity of Beclin1 from Bcl‐2 to PIK3C3.     

Pulmonary glass particles may persist in the lung suppressing function of immune cells

The health effects of silica may depend on the inherent properties of crystalline silica or on external factors affecting the biological activity or distribution of its polymorphs. Inhaled crystalline silica is classified as a Group I carcinogen, however, information on the health effects of amorphous silica is still insufficient. Considering that alveolar macrophages play a key role in both innate and adaptive immune responses for removal of foreign bodies that enter via the respiratory system, we treated sheet‐like glass particles (SGPs), a type of noncrystalline amorphous silica, to MH‐S cells, an alveolar macrophage cell line. SGPs reduced the generation of ROS and NO and induced cell death via multiple pathways. Although the expression of CD80, CD86, and CD40, increased by exposure to SGPs, the expression of MHC class II molecules had not notably changed. Additionally, expression of ICAM‐1 tended to decrease. In mice, SGPs were distributed in the interstitial region of the lung without notable pathological lesion on day 14 after a single intratracheal instillation. Pulmonary total cell number increased significantly with the highest dose, but the levels of all measured inflammatory cytokines and chemokines, except IL‐1, were lower in BAL fluid from SGP‐treated mice compared to control. More interestingly, the expression of antigen presentation‐related proteins was enhanced in the lungs of SGP‐exposed mice concomitant with an increase in the number of mature dendritic cells, whereas the expression of ICAM‐1, an important adhesion molecule for helper T cell recruitment, was suppressed. Taken together, we suggest that SGPs may induce adverse health effects by down‐regulating function of immune cells in the lungs. Furthermore, ICAM‐1 may play a key role in immune response to remove pulmonary SGPs.     

ZC3H4 mediates silica-induced EndoMT via ER stress and autophagy

BackgroundInflammatory reactions induced by alveolar macrophages and excessive fibroblast activation lead to pulmonary fibrosis in silicosis. The endothelial-mesenchymal transition (EndoMT) is a key source of myofibroblasts. ZC3H4 is a member of the CCCH zinc finger protein family that participates in macrophage activation and epithelial mesenchymal transition (EMT). However, whether ZC3H4 is involved in EndoMT in silicosis has not yet been elucidated. Therefore, we conducted further studies into the role of ZC3H4 in silica-induced EndoMT in pulmonary vessels.MethodsWestern blotting and immunofluorescence staining were used to detect the regulatory influences of SiO₂ on pulmonary fibrosis and EndoMT. ZC3H4 was specifically downregulated using CRISPR/Cas9 to explore whether ZC3H4 regulated EndoMT during silicosis. C57BL/6 J mice were administered with SiO₂ via the trachea to establish a silicosis animal model.Results1) SiO₂ exposure increased ZC3H4 expression in pulmonary vessels. 2) ZC3H4 was involved in EndoMT induced by silica. 3) ZC3H4 mediated EndoMT via endoplasmic reticulum stress (ER stress) and autophagy.ConclusionsZC3H4 greatly affects the progression of SiO₂-induced EndoMT via ER stress and autophagy, which provides the possibility that ZC3H4 may become a novel target in pulmonary fibrosis treatment.     

Neutralization of interleukin-1 beta attenuates silica-induced lung inflammation and fibrosis in C57BL/6 mice

The inflammation and fibrosis induced by silica dust are considered to be substantial responses in silicosis progression. Interleukin-1 beta (IL-1β) plays an important role in silica-induced lung inflammation, but the mechanisms that underlie the influence of IL-1β on the progression of silicosis remain unclear. In this study, the role of IL-1β in silica-induced inflammation and fibrosis was evaluated by administering a suspension of 2.5-mg silica dust, either with or without 40 μg anti-mouse IL-1β monoclonal antibody (mAb), to the lungs of male C57BL/6 mice. Silica + anti-IL-1β mAb-treated mice showed the depletion of IL-1β as well as the attenuation of inflammation, as evaluated in the bronchoalveolar lavage fluid (BALF) and histological sections from 1 to 84 days after silica exposure. Further study of the BALF indicated that inhibition of IL-1β could reduce the contents of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. The real-time PCR and pathology results showed that the neutralization of IL-1β attenuated silica-induced fibrosis by inhibiting the gene expression of transforming growth factor-beta 1, collagen I and fibronectin. The examination of Th1-cytokine and Th2-cytokine suggested that depletion of IL-1β decelerated the Th1/Th2 balance toward a Th2-dominant response. In conclusion, the present study suggests that the neutralization of IL-1β attenuates silica-induced inflammation and fibrosis by inhibiting other inflammatory and fibrogenic mediators and modulating the Th1/Th2 balance.     

Dynamic changes of mononuclear phagocytes in circulating,pulmonary alveolar and interstitial compartments in a mouse model of experimental silicosis

Context: Silicosis is a devastating, irreversible lung fibrosis condition exposed to crystalline silica. The mononuclear phagocyte system plays an important role in the pathogenesis of silicosis.

Objective: The present study was aimed to explore the dynamic changes of mononuclear phagocytes in circulating, pulmonary alveolar and interstitial compartments in experimental silicosis model.

Materials and methods: A mouse model of lung fibrosis was developed with crystalline silica particles (2?mg/40?μL via oropharyngeal instillation) using male C57BL/6 mice, and were killed on days 1, 3, 7, 14, and 28. The lung inflammation and fibrosis was investigated using hematoxylin–eosin staining and bronchoalveolar lavage fluid (BALF) analysis, Masson’s trichrome staining, and immunofluorescence. Circulating monocyte subsets (Ly6C^hi and Ly6C^lo), polarization state of BALF-derived alveolar macrophages (AM?) and lung interstitial macrophages (IM?, derived from enzymatically digested lung tissue) were analyzed by flow cytometry.

Results: The percentage of Ly6C^hi monocytes significantly increased on day 1 after silica exposure, which reached the peak level from day 7 till day 28. Moreover, M2 (alternative activation) AM? (PI???CD64?+?CD206+) was dramatically and progressively increased from day 1 to day 28. A parallel increase in IM? with M2 polarization (PI-CD64?+?CD11b?+?CD206+) was also observed from day 1 to day 28.

Conclusion: Our data demonstrate a dynamic view of mononuclear phagocyte change in three compartments after silica challenge, which highlights the remodeling of mononuclear phagocyte system as a potential therapeutic target for silicosis.     

基于GEO结直肠癌芯片数据的生物信息学分析

目的 利用生物信息学分析方法,挖掘结直肠癌(CRC)的关键基因并探索其发病机制.方法 在公共基因芯片数据库(GEO)中下载结直肠癌表达谱芯片数据,在GCBI实验室中筛选出结直肠癌显著差异的基因,分别对显著差异基因作GO富集分析、KEGG通路分析、蛋白质相互作用(PPI)网络分析.进一步利用cytoscape将PPI结果建立互作模块.结果 通过差异分析得出在正常结直肠组织、结直肠腺瘤、结直肠癌中表达量逐步明显下调的基因有492个,逐步明显上调的有248个,共有740个显著差异基因.GO富集分析主要体现在各种代谢过程、细胞增殖、信号调节、RNA聚合酶Ⅱ转录因子活性等.KEGG信号通路主要富集在癌症转录失调、细胞周期及p53信号通路等.并利用互作模型筛选出CDK1、MCM2、CDC6、CCNA1、CCNB2、CDKN1B、ORC1、E2F1、CHEK1、PCNA等45个与结直肠癌发生发展关系密切的关键基因.关键基因主要富集在细胞周期、病毒致癌机理、癌症相关、p53及PI3K-Akt等信号通路.结论 通过生物信息学对基因芯片数据的分析,能获取结直肠癌的关键基因及其相关通路,为后续研究提供依据.