The Molecular Mechanisms Of Urban Particulate Matter-induced Inflammation And Mucus Hyperproduction In Airway Epithelium

BackgroundThe adverse health effects of exposure to outdoor particulate matter air pollution are of concern to governments and health organisations worldwide.According to 2002 World Health Report,approximately 800,000 premature deaths were attributable to air pollution worldwide annually.Air pollution is one of the great killers of our age.Exposures to ambient air pollution have been associated with increased morbidity and mortality,in particular,from respiratory diseases.With rapid industrial development and surging energy consumption,China has become the largest producer of major air pollutants worldwide,and the air pollutant levels in many cities in high-income economies exceed the upper limits stated in the WHO guidelines.Among all air pollutants,the most commonly monitored are particulate matter(PM)pollutants,sulfur dioxide(SO2),and nitrogen dioxide(NO2).Airborne pollutants,especially particulate matter(PM),pose a considerable harmful effect to human health.Particulate matter is a widespread air pollutant,which is a mixture of solid particles and liquid droplets in the air.Daily concentrations of PM2.5 in mega-cities,such as Beijing and Shanghai,may peak to 100-300 ?g/m3,which far exceeded the upper limit(10 ?g/m3)endorsed by WHO Air Quality Guideline(AQG).Epidemiological studies have shown that PM exposure is positively correlated with and increased incidence of respiratory diseases such as pneumonia,asthma and chronic obstructive pulmonary disease(COPD).Particulate matter has been found to stimulate production of pro-inflammatory cytokines,accelerate coagulation,increase the activity of endocrine systems,contribute to neurotoxicity,induce allergic sensitization,and provoke adaptive immune responses.However,the molecular mechanisms mediating the adverse effects of PM in respiratory diseases remain largely unknown.Part I Early growth response gene 1 is essential for urban particulate matter-induced pulmonary inflammation and mucus hyperproduction in airway epitheliumBackground:Particulate matter(PM)has been implicated as a risk factor for human airway disorders.However,the biological mechanisms underlying the correlation between PM exposure and adverse airway effects have not yet been fully clarified.Objective:The objective of this study was to explore the possible role of early growth response gene 1(Egr-1)in PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction in vitro and in vivo.Methods:To examine the role of Egr-1 signaling being associated with pulmonary inflammation and mucus hyperproduction,human bronchial epithelial(HBE)cells were exposed to PM in vitro.Egr-1 knockout mice were given standard PM intratracheally,and the mice were sacrificed for analyzing of the severity of inflammation and mucus hyperproduction.Results:Particulate matter exposure induced a rapid Egr-1 expression in human bronchial epithelial(HBE)cells and in mouse lungs.Genetic blockage of Egr-1 markedly reduced PM-induced inflammatory cytokines,e.g.,IL6 and IL8,and MUC5AC in HBE cells,and these effects were mechanistically mediated by the nuclear factor-KB(NF-?B)and activator protein-1(AP-1)pathways,respectively.Egr-1-knockout mice displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo.Moreover,polycyclic aromatic hydrocarbons(PAHs)contained in the PM also induced Egr-1 expression,and also played a role in the inflammatory responses and mucus production.Conclusion:Taken together,our data reveal novel Egr-1 signaling that mediates the NF-?B and AP-1 pathways to orchestrate PM-induced pulmonary inflammation and mucus hyperproduction,suggesting that Egr-1 inhibition could be an effective therapeutic approach for airway disorders or disease exacerbations induced by airborne particulate pollution.Part ? Necroptosis contributes to urban particulate matter-induced airway epithelial injuryBackground:Necroptosis,a form of programmed necrosis,is involved in the pathologic process of several kinds of pulmonary diseases.However,the role of necroptosis in particulate matter(PM)-induced pulmonary injury remains unclear.The objective of this study is to investigate the involvement of necroptosis in the pathogenesis of PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction,both in vitro and in vivo.Methods:PM was administered into human bronchial epithelial(HBE)cells or mouse airways,and the inflammatory response and mucus production were assessed.The mRNA expressions of IL6,IL8 and MUC5AC in HBE cells and Cxcl1,Cxcl2,and Gm-csf in the lung tissues were detected by quantitative real-time RT-PCR.The secreted protein levels of IL6 and IL8 in culture supernatants and Cxcl1,Cxcl2,and Gm-csf in bronchoalveolar lavage fluid(BALF)were detected by enzyme-linked immunosorbent assay(ELISA).We used Western blot to measure the protein expressions of necroptosis-related proteins(RIPK1,RIPK3,and Phospho-MLKL),NF-?B(P65 and PP65),AP-1(P-c-Jun and P-c-Fos)and MUC5AC.Cell necrosis and mitochondrial ROS were detected using flow cytometry.In addition,pathological changes and scoring of lung tissue samples were monitored using hemoxylin and eosin(H&E),periodic acid-schiff(PAS)and immunohistochemistry staining.Results:Our study showed that PM exposure induced RIP and MLKL-dependent necroptosis in HBE cells and in mouse lungs.Managing the necroptosis inhibitor Necrostatin-1(Nec-1)and GSK'872,specific molecule inhibitors of necroptosis,markedly reduced PM-induced inflammatory cytokines,e.g.,IL6 and IL8,and MUC5AC in HBE cells.Similarly,administering Nec-1 significantly reduced airway inflammation and mucus hyperproduction in PM-exposed mice.Mechanistically,we found PM-induced necroptosis was mediated by mitochondrial reactive oxygen species-dependent early growth response gene 1,which ultimately promoted inflammation and mucin expression through nuclear factor ?B and activator protein-1 pathways,respectively.Conclusions:Our results demonstrate that necroptosis is involved in the pathogenesis of PM-induced pulmonary inflammation and mucus hyperproduction,and suggests that it may be a novel target for treatment of airway disorders or disease exacerbations with airborne particulate pollution.