The role of ROS-pyroptosis in PM2.5 induced air-blood barrier destruction.

Fine particulate matter (PM2.5) has attracted increasing attention due to its health-threatening effects. Although numerous studies have investigated the impact of PM2.5 on lung injuries, the specific mechanisms underlying the damage to the air-blood barrier after exposure to PM2.5 remain unclear. In this study, we established an in vitro co-culture system using lung epithelial cells and capillary endothelial cells. Our findings indicated that the tight junction (TJ) proteins were up-regulated in the co-cultured system compared to the monolayer-cultured cells, suggesting the establishment of a more closely connected in vitro system. Following exposure to PM2.5, we observed damage to the air-blood barrier in vitro. Concurrently, PM2.5 exposure induced significant oxidative stress and activated the NLRP3 inflammasome-mediated pyroptosis pathway. When oxidative stress was inhibited, we observed a decrease in pyroptosis and an increase in TJ protein levels. Additionally, disulfiram reversed the adverse effects of PM2.5, effectively suppressing pyroptosis and ameliorating air-blood barrier dysfunction. Our results indicate that the oxidative stress-pyroptosis pathway plays a critical role in the disruption of the air-blood barrier induced by PM2.5 exposure. Disulfiram may represent a promising therapeutic option for mitigating PM2.5-related lung damage.

PM2.5 induce myocardial injury in hyperlipidemic mice through ROS-pyroptosis signaling pathway.

Exposure to particulate matters with diameters below 2.5 µm (PM2.5) is considered a major risk factor for cardiovascular diseases (CVDs). The closest associations between PM2.5 and CVDs have been observed in hyperbetalipoproteinemia cases, although the detailed underpinning mechanism remains undefined. In this work, hyperlipidemic mice and H9C2 cells were used to examine the effects of PM2.5 on myocardial injury and their underlying mechanisms. The results revealed that PM2.5 exposure caused severe myocardial damage in the high-fat mouse model. Oxidative stress and pyroptosis were also observed along with myocardial injury. After inhibiting pyroptosis with disulfiram (DSF), the level of pyroptosis was effectively reduced as well as myocardial injury, suggesting that PM2.5 induced the pyroptosis pathway and further caused myocardial injury and cell death. Afterwards, by suppressing PM2.5-induced oxidative stress with N-acetyl-L-cysteine (NAC), myocardial injury was markedly ameliorated, and the upregulation of pyroptosis markers was reversed, which indicated that PM2.5-pyroptosis was also improved. Taken together, this study revealed that PM2.5 induce myocardial injury through the ROS-pyroptosis signaling pathway in hyperlipidemia mice models, providing a potential approach for clinical interventions.

Pyroptosis participates in PM2.5-induced air-blood barrier dysfunction.

Epidemiological studies have shown that particulate matters with diameter less than 2.5 µm (PM2.5) play an important role in inducing and promoting respiratory diseases, but its underlying mechanism remains to be explored. The air-blood barrier, also known as the alveolar-capillary barrier, is the key element of the lung, working as the site of oxygen and carbon dioxide exchange between pulmonary vasculatures. In this study, a mouse PM2.5 exposure model was established, which leads to an induced lung injury and air-blood barrier disruption. Oxidative stress and pyroptosis were observed in this process. After reducing the oxidative stress by N-acetyl-L-cysteine (NAC) treatment, the air-blood barrier function was improved and the effect of PM2.5 was alleviated. The level of pyroptosis and related pathway were also effectively relieved. These results indicate that acute PM2.5 exposure can cause lung injury and the alveolar-capillary barrier disruption by inducing reactive oxygen species (ROS) with the participation of pyroptosis pathway.

Oxidative stress activates Ryr2-Ca2+ and apoptosis to promote PM2.5-induced heart injury of hyperlipidemia mice.

The increased cases of hyperlipemia in China and the crucial role of PM2.5 in inducing and promoting cardiovascular diseases have attracting more and more researchers' attention. However, the effects and mechanisms of PM2.5 on cardiovascular system of hyperlipidemia people are still unclear. In this study, hyperlipidemia mice model was established by high-fat diet. Then we exposed these mice to PM2.5 or saline to explore the underling mechanism of cardiac injury in hyperlipidemia mice. The hyperlipemia mice are more susceptible to heart damage caused by PM2.5 exposure. The participation of oxidative stress, cell apoptosis and Ca2+ related mechanism could be observed in this model. After NAC (N-acetyl-L-cysteine) treatment, the oxidative stress level induced by PM2.5 exposure significantly decreased in hyperlipemia mice. NAC effectively alleviated cardiac injury, improved the imbalance of calcium and attenuated apoptosis induced by PM2.5 exposure in hyperlipemia mice. The strong oxidative stress in hyperlipemia mice could lead to calcium homeostasis imbalance and activation of apoptosis-related pathways. This mechanism of PM2.5-induced myocardial injury was also verified in vitro. In our present study, we demonstrated the contribution of the PM2.5-ROS-Ryr2-Ca2+ axis in PM2.5-induced heart injury of hyperlipidemia mice, offering a potential therapeutical target for related pathology.

Differential expression of long non-coding RNAs in the hippocampus of mice exposed to PM2.5 in Dalian, China.

Evidence is mounting that PM2.5 exposure could lead to learning disability, memory deficits, and cognitive impairment; however, the underlying mechanisms are still not well demonstrated yet. Long non-coding RNAs (LncRNAs) play a crucial role in many human diseases. Although the relationship of Alzheimer's disease (AD) and lncRNAs have been discovered, the role of lncRNA in AD-like phenotype induced by PM2.5 needs further exploration. In this study, we profiled the expression of messenger RNAs (mRNAs) and lncRNAs in hippocampus after confirming the AD-like changes in mice. Compared with the control group, a total of 478 mRNAs and 151 lncRNAs were dysregulated after PM2.5 exposure. ECM-receptor interaction, focal adhesion, complement and coagulation cascades, and AGE-RAGE signaling pathway were found dysregulated through lncRNA-co-expressed genes analysis based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Meanwhile, the genes related to microglia were significantly altered, such as CX3CR1, CD163, lncRNA Gm44750, and lncRNA Gm43509. Above evidences suggested that microglia-related lncRNAs dysregulation probably plays a crucial role in PM2.5exposure-associated learning and memory deficits.

Oxidative stress activates the TRPM2-Ca2+-NLRP3 axis to promote PM2.5-induced lung injury of mice.

PM2.5, a main particulate air pollutant, poses a serious hazard to human health. The exposure to PM2.5 increases mortality and morbidity of many respiratory diseases such as asthma, chronic obstructive pulmonary diseases and even lung cancer. The contribution of reactive oxygen species (ROS) in the PM2.5-induced acute lung injury process was confirmed in our previous research, but the molecular mechanism based for it remains unclarified. In this research, ROS-induced lung injury after exposure to PM2.5 was explored in vivo and in vitro. The in vivo study indicated that N-acetyl-L-cysteine (NAC) could attenuate the accumulation of inflammatory cells, the thickening of alveolar wall and the degree of lung injury. Furthermore, we found ROS could regulate the intracellular Ca2+ level, expression of the Transient Receptor Potential Melastatin 2 (TRPM2), NLRP3 and its downstream inflammatory factors in vivo. In vitro experiments with A549 cells and primary type II alveolar epithelium cells (SD cells) showed that ROS induced by PM2.5 exposure could mediate intracellular Ca2+ mobilization via TRPM2, with a subsequent activation of NLRP3. In our present study, we demonstrated the contribution of the ROS-TRPM2-Ca2+-NLRP3 pathway in PM2.5-induced acute lung injury and offered a potential therapeutical target valid for related pathology.

The harmful effects of acute PM2.5 exposure to the heart and a novel preventive and therapeutic function of CEOs.

Epidemiological researches have demonstrated the relationship between PM2.5 exposure and increased morbidity and mortality of cardiovascular injury. However, no effective therapeutic method was established. The purpose of this study is to investigate the effect of acute PM2.5 exposure on the mice heart tissue and explore the therapeutic effects of compound essential oils (CEOs) in this model. In this study, after mice were exposed to PM2.5 intratracheally, some obvious histopathological changes as well as some great alterations of proinflammatory cytokines were observed in the heart tissue. The imbalance of oxidative stress, the altered Ca2+ channel related proteins and the increased intracellular free Ca2+ were all involved in the heart impairment and would also be investigated in this model. The CEOs alleviated the heart impairment via its antioxidant effect rather than its anti-inflammatory function because our results revealed that oxidative stress related indicators were restored after CEOs administration. At the same time, increased concentration of intracellular free Ca2+ and ROS induced by PM2.5 were reduced after NAC (N-Acetyl-L-cysteine) administration. These data suggested that the acute PM2.5 exposure would damage heart tissue by inducing the inflammatory response, oxidative stress and intracellular free Ca2+ overload. PM2.5-induced oxidative stress probably increase intracellular free Ca2+ via RYR2 and SERCA2a. CEOs have the potential to be a novel effective and convenient therapeutic method to prevent and treat the acute heart impairment induced by PM2.5 via its antioxidant function.

The acute airway inflammation induced by PM2.5 exposure and the treatment of essential oils in Balb/c mice.

PM2.5 is the main particulate air pollutant whose aerodynamic diameter is less than 2.5 micron. The inflammation of various respiratory diseases are associated with PM2.5 inhalation. Pro-inflammatory cytokine IL-1ß generated from effected cells usually plays a crucial role in many kinds of lung inflammatory reactions. The exacerbation of Th immune responses are identified in some PM2.5 related diseases. To elucidate the underlying mechanism of PM2.5-induced acute lung inflammation, we exposed Balb/c mice to PM2.5 intratracheally and established a mice model. Acute lung inflammation and increased IL-1ß expression was observed after PM2.5 instillation. Regulatory factors of IL-1ß (TLR4/MyD88 signaling pathway and NLRP3 inflammasome) participated in this lung inflammatory response as well. Treatment with compound essential oils (CEOs) substantially attenuated PM2.5-induced acute lung inflammation. The decreased IL-1ß and Th immune responses after CEOs treatment were significant. PM2.5 may increase the secretion of IL-1ß through TLR4/MyD88 and NLRP3 pathway resulting in murine airway inflammation. CEOs could attenuate the lung inflammation by reducing IL-1ß and Th immune responses in this model. This study describes a potentially important mechanism of PM2.5-induced acute lung inflammation and that may bring about novel therapies for the inflammatory diseases associated with PM2.5 inhalation.

Th17 can regulate silica-induced lung inflammation through an IL-1ß-dependent mechanism.

Silicosis is an occupational lung disease caused by the inhalation of silica dust and characterized by lung inflammation and fibrosis. Interleukin (IL)-1ß is induced by silica and functions as the key pro-inflammatory cytokine in this process. The Th17 response, which is induced by IL-1ß, has been reported very important in chronic human lung inflammatory diseases. To elucidate the underlying mechanisms of IL-1ß and IL-17 in silicosis, we used anakinra and an anti-IL-17 monoclonal antibody (mAb) to block the receptor of IL-1ß (IL-RI) and IL-17, respectively, in a mouse model of silicosis. We observed increased IL-1ß expression and an enhanced Th17 response after silica instillation. Treatment with an IL-1 type I receptor (IL-1RI) antagonist anakinra substantially decreased silica-induced lung inflammation and the Th17 response. Lung inflammation and the accumulation of inflammatory cells were attenuated in the IL-17-neutralized silicosis group. IL-17 may promote lung inflammation by modulating the differentiation of Th1 and regulatory T cells (Tregs) and by regulating the production of IL-22 and IL-1ß during the lung inflammation of silicosis. Silica may induce IL-1ß production from alveolar macrophages and promote inflammation by initiating a Th17 response via an IL-1ß/IL-1RI-dependent mechanism. The Th17 response could induce lung inflammation during the pathogenesis of silicosis by regulating the homoeostasis of the Th immune responses and affecting the production of IL-22 and IL-1ß. This study describes a potentially important inflammatory mechanism of silicosis that may bring about novel therapies for this inflammatory and fibrotic disease.

Reduction of IL-17A might suppress the Th1 response and promote the Th2 response by boosting the function of Treg cells during silica-induced inflammatory response in vitro.

Silica inhalation can induce chronic lung inflammation and fibrosis. Upon silica stimulation, activated macrophages trigger the T-lymphocyte which can differentiate into many different types of Th cells, including the recently discovered Th17 cells. IL-17A, the typical Th17 cytokine, is reported in some inflammatory diseases. However, the role of IL-17A in silica-induced inflammatory response is still not clear. The regulatory mechanism of silica-induced Th17 response also needs to be investigated. So we established a mice primary cell coculture system (macrophage and lymphocyte) to investigate the role of IL-17A in silica-induced inflammatory response in vitro, by using anti-IL-17A mAb and IL-1Ra. Both anti-IL-17A mAb and IL-1Ra decreased the level of IL-17A and increased the function of Treg cells. The Th1 response was suppressed and the Th2 response was promoted by the addition of anti-IL-17A mAb or IL-1Ra. IL-1Ra treatment decreased the level of IL-6, whereas the levels of IL-23 and ROR- γ t were increased. Our study demonstrated that IL-17A reduction altered the pattern of silica-induced Th responses by boosting the function of Treg cells in vitro. Blocking the function of IL-1 signal pathway could suppress the level of IL-17A, which played the major role in modulating silica-induced Th responses in vitro.

Neutralization of interleukin-17A delays progression of silica-induced lung inflammation and fibrosis in C57BL/6 mice.

Silica exposure can cause lung inflammation and fibrosis, known as silicosis. Interleukin-17A (IL-17A) and Th17 cells play a pivotal role in controlling inflammatory diseases. However, the roles of IL-17A and Th17 cells in the progress of silica-induced inflammation and fibrosis are poorly understood. This study explored the effects of IL-17A on silica-induced inflammation and fibrosis. We used an anti-mouse IL-17A antibody to establish an IL-17A-neutralized mice model, and mice were exposed to silica to establish an experimental silicosis model. We showed that IL-17A neutralization delayed neutrophil accumulation and progression of silica-induced lung inflammation and fibrosis. IL-17A neutralization reduced the percentage of Th17 in CD4+ T cells, decreased IL-6 and IL-1ß expression, and increased Tregs at an early phase of silica-induced inflammation. Neutralization of IL-17A delayed silica-induced Th1/Th2 immune and autoimmune responses. These results suggest that IL-17A neutralization alleviates early stage silica-induced lung inflammation and delays progression of silica-induced lung inflammation and fibrosis. Neutralization of IL-17A suppressed Th17 cell development by decreasing IL-6 and/or IL-1ß and increased Tregs at an early phase of silica-induced inflammation. Neutralization of IL-17A also delayed the Th1/Th2 immune response during silica-induced lung inflammation and fibrosis. IL-17A may play a pivotal role in the early phase of silica-induced inflammation and may mediate the Th immune response to influence silica-induced lung inflammation and fibrosis in mice.

1,3-ß-glucan affects the balance of Th1/Th2 cytokines by promoting secretion of anti-inflammatory cytokines in vitro.

1,3-ß-glucan is considered a fungal biomarker and exposure to this agent induces lung inflammation. Previous studies have shown that 1,3-ß-glucan affects Th1 and Th2 immune responses. Interleukin (IL)-10 and transforming growth factor (TGF)-ß, as typical anti-inflammatory cytokines, suppress the Th1 immune response. To investigate the effects of 1,3-ß-glucan on the secretion of cytokines in co-cultured mouse macrophages and lymphocytes in vitro, mice were exposed to 1,3-ß-glucan or phosphate-buffered saline (PBS) by intratracheal instillation. Following extraction and co-culture of macrophages and lymphocytes, which were treated with or without 1,3-ß-glucan in vitro, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of cytokines and real-time reverse transcription (RT)-polymerase chain reaction (PCR) was used to investigate the mRNA expression of forkhead box p3 (Foxp3) in the cells. We showed that 1,3-ß-glucan exposure in vitro decreased the secretion of Th1 cytokines and increased the secretion of Th2 cytokines in the culture media. Furthermore, 1,3-ß-glucan exposure in vitro increased the secretion of IL-10 and TGF-ß in the culture media. According to these results, 1,3-ß-glucan exposure in vitro is suggested to promote the secretion of anti-inflammatory cytokines, which may lead to a decrease in the levels of Th1 cytokines and an increase in the levels of Th2 cytokines. 1,3-ß-glucan is suggested to induce regulatory lymphocytes, which partly contributes to an increased secretion of anti-inflammatory cytokines in co-cultured mouse macrophages and lymphocytes in vitro.

T(reg) cells may regulate interlukin-17 production by modulating TH1 responses in 1,3-ß-glucan-induced lung inflammation in mice.

1,3-ß-glucan is considered a fungal biomarker and exposure to this agent can induce lung inflammation. Complement activation plays an important role in early immune responses to ß-glucan. Previous studies showed that T-regulatory cells (Tregs) regulated 1,3-ß-glucan-induced lung inflammation by modulating the maintenance of immune homeostasis in the lung. Both interleukin (IL)-17 and TH17 cells play pivotal roles in inflammation associated with lung disease and share reciprocal developmental pathways with Tregs. However, the effect of Tregs on IL-17 and TH17 responses in 1,3-ß-glucan-induced lung inflammation remains unclear. In this study, mice were exposed to 1,3-ß-glucan by intratracheal instillation. To investigate the effects of Tregs on IL-17 and TH17 cells in the induced lung inflammation, a Treg-depleted mice model was generated by administration of anti-CD25 mAb. The results indicated that Treg-depleted mice showed more severe pathological inflammatory changes in lung tissues. Tregs depletion reduced IL-17 expression in these tissues, and increased those of TH1 cytokines. The expression of IL-17 increased at the early phase of the inflammation response. There were no significant effects of the Tregs on expression of RORγt and IL-6 or the amount of CD4(+)IL-17(+) cells in the lungs. When taken together, the late phase of the 1,3-ß-glucan-induced inflammatory response in the mice was primarily mediated by TH1 cytokines rather than IL-17. In contrast, the early phase of the inflammatory response might be mediated in part by IL-17 along with activated complement. Tregs might be required for IL-17 expression during the late phase inflammatory response in mice. The increased IL-17 mRNA observed during the 1,3-ß-glucan induced inflammatory response were attributed to cells other than TH17 cells.

Tregs promote the differentiation of Th17 cells in silica-induced lung fibrosis in mice.

BACKGROUND: Silicosis is an occupational lung disease caused by inhalation of silica dust and characterized by lung inflammation and fibrosis. Previous study showed that Tregs regulate the process of silicosis by modulating the maintenance of immune homeostasis in the lung. Th17 cells share reciprocal developmental pathway with Tregs and play a pivotal role in the immunopathogenesis of many lung diseases by recruiting and activating neutrophils, but the regulatory function of Tregs on Th17 response in silica induced lung fibrosis remains to be explored. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the role of Th17 and IL-17 in the development of silicosis and their interaction with Tregs, Treg-depleted mice model was generated and exposed to silica to establish experimental model of silica-induced lung fibrosis. Here we showed that silica increased Th17 response in lung fibrosis. Tregs depletion enhanced the neutrophils accumulation and attenuated Th17 response in silica induced lung fibrosis. Both mRNA and protein results showed that Tregs exerted its modulatory function on Th17 cells and IL-17 by regulating TGF-ß1 and IL-1ß. CONCLUSION/SIGNIFICANCE: Our study suggested that Tregs could promote Th17 cells differentiation by regulating TGF-ß1 and IL-1ß in silica induced lung fibrosis of mice, which further the understanding of the progress of silicosis and provide a new insight in the regulatory mechanism of Th17 by Tregs in lung inflammation.

Depletion of CD4+CD25+Foxp3+ regulatory T cells with anti-CD25 antibody may exacerbate the 1,3-ß-glucan-induced lung inflammatory response in mice.

1,3-ß-Glucan was a major cell wall component of fungus. The existing studies showed that 1,3-ß-glucan exposure could induce lung inflammation that involved both Th1 and Th2 cytokines. Regulatory T cells (Treg cells) played a critical role in regulating immune homeostasis by adjusting the Th1/Th2 balance. The role of Treg cells and regulatory mechanism in 1,3-ß-glucan-induced lung inflammation is still unclear. In our study, mice were exposed to 1,3-ß-glucan by intratracheal instillation. To investigate the role of Treg cells in response to 1,3-ß-glucan, we generated Treg-depleted mice by intraperitoneal administration of anti-CD25 mAb. The Treg-depleted mice showed more inflammatory cells and severer pathological inflammatory change in lung tissue. Depletion of Treg cells led to increased Th1 cytokines and decreased Th2 cytokines. Treg-depleted mice showed a decreased expression of anti-inflammation cytokine and lower-level expression of CTLA-4. In all, our study indicated that Treg cells participated in regulating the 1,3-ß-glucan-induced lung inflammation. Depletion of Treg cells aggravated the 1,3-ß-glucan-induced lung inflammation, regulated the Th1/Th2 balance by enhancing Th1 response. Treg cells exerted their modulation function depending on both direct and indirect mechanism during the 1,3-ß-glucan-induced lung inflammation.

CD4+CD25+Foxp3+ regulatory T cells depletion may attenuate the development of silica-induced lung fibrosis in mice.

BACKGROUND: Silicosis is an occupational lung disease caused by inhalation of silica dust characterized by lung inflammation and fibrosis. Previous study showed that Th1 and Th2 cytokines are involved in silicosis, but Th1/Th2 polarization during the development of silicosis is still a matter of debate. Regulatory T cells (Treg cells) represent a crucial role in modulation of immune homeostasis by regulating Th1/Th2 polarization, but their possible implication in silicosis remains to be explored. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the implication of Treg cells in the development of silicosis, we generated the Treg-depleted mice model by administration of anti-CD25 mAbs and mice were exposed to silica by intratracheal instillation to establish experimental model of silica-induced lung fibrosis. The pathologic examinations show that the Treg-depleted mice are susceptive to severer inflammation in the early stage, with enhanced infiltration of inflammatory cells. Also, depletion of Treg cells causes a delay of the progress of silica-induced lung fibrosis in mice model. Further study of mRNA expression of cytokines reveals that depletion of Tregs leads to the increased production of Th1-cytokines and decreased production of Th2-cytokine. The Flow Cytometry and realtime PCR study show that Treg cells exert the modulation function both directly by expressing CTLA-4 at the inflammatory stage, and indirectly by secreting increasing amount of IL-10 and TGF-ß during the fibrotic stage in silica-induced lung fibrosis. CONCLUSION/SIGNIFICANCE: Our study suggests that depletion of Tregs may attenuate the progress of silica-induced lung fibrosis and enhance Th1 response and decelerate Th1/Th2 balance toward a Th2 phenotype in silica-induced lung fibrosis. The regulatory function of Treg cells may depend on direct mechanism and indirect mechanism during the inflammatory stage of silicosis.