The Impact Of PM On Acute Exacerbation Of Allergic Airway Inflammation And Its Molecular Mechanism

BackgroundAsthma is a common chronic respiratory disease affecting 300 million patients globally.There were 30 million asthma patients in China and the morbidity increase annually.An epidemiological finding showed that the morbidity of asthma over the year of 14 was 1.24%.Among them,59.5% of the population has not met the goals of asthma management recommended in Global Initiative for Asthma(GINA)causing social and economic burden on patients.As we all known,the long-term goals of asthma management are to achieve good symptom control,and to minimize future risk of asthma-related mortality,exacerbations,persistent airflow limitation and side-effects of treatment.Acute exacerbation of asthma increased the risk of asthma-related disability to death.Recognizing risk factors and triggering factors of asthma exacerbations is helpful for the prevention and long-term management.Quite a lot of epidemiology researches showed that air pollution is a major environmental issue affecting health globally,which has become a concern of governments and World Health Organization(WHO).Air pollution could increase cardiovascular and respiratory related emergency visits,hospital admission,mortality and acute exacerbations of chronic respiratory disease with decreased lung function.Particulate matters(PM),sulfur dioxide(SO2),nitrogen dioxide(NO2)and O3 are most common air pollutants.PM is a ubiquitous atmospheric aerosol composes with Solid particles and droplets.In Beijing and Shanghai,the concentrations of PM could reach 100 to 300 ?g/m3 which exceeded WHO's criteria of 10?g/m 3.Epidemiology researches showed that PM exposure is related with new-onset respiratory diseases such as asthma,chronic obstructive pulmonary disease(COPD)and pneumonia.Researches have found that PM could stimulate the production of pro-inflammatory cytokines,adjust coagulation and endocrine system,result neurotoxicity and allergy and affect adjust immune reaction.However,the role of PM in respiratory disease,especially acute exacerbation of pre-existing asthma and underlying molecular mechanism need urgent exploration.Part I Impact of PM on acute exacerbation of allergic airway inflammation Background: Particulate matters(PMs)have been implicated as one of the risk factors for acute exacerbation of asthma and allergic airway diseases.However,the impact of PMs on acute exacerbation of allergic airway inflammation and the underlying molecular mechanism were still unknown.Objective: To investigate the impact of PM exposure on acute exacerbation of allergic airway inflammation in vivo and possible molecular mechanism.Methods: Six-to eight-week-old female C57BL/6 mice were separated into vehicle group,ovalbumin(OVA)group,and OVA /PM group.Allergic airway disease murine model was sensitized and challenged with OVA.PM exposure was achieved by the oropharyngeal instillation.Bronchoalveolar lavage fluid(BALF)was obtained for total leukocytes and differential cell counting with Wright-Giemsa stain.Murine lung tissues were stained with hematoxylin and eosin(H&E)and periodic acid-Schiff(PAS)to explore the pathological changes.The expression of Toll like receptor(TLR2)and NOD-like receptor pyridine containing 3(NLRP3)inflammasome in murine lung was examined by Western Blot analysis.The supernatants of BALF were used for cytokine analysis with ELISA.Results: PM exposure led to increased airway inflammation and expression of TLR2.Compared with the vehicle group,airway wall thickened,some small airway collapsed accompanied with increased inflammatory cell recruitment,goblet cell hyperplasia,mucus secretion and accumulation of inflammatory cells and inflammatory cytokines interlukin-6(IL-6),cytokines interlukin-18(IL-18)in BALF and immunoglobulin E(Ig E)in serum in OVA-allergic WT mice significantly.Furthermore,we observed a marked increase in inflammatory cells recruiting to the peri-bronchial regions,goblet cell hyperplasia and mucus secretion surrounding the airway in the OVA/PMs-induced mice compared with that in the OVA-induced mice as well as marked accumulations of total inflammatory cells and neutrophils in the BALF.There was a significant increase of protein level of TLR2 and NLRP3 inflammasome expression in OVA-challenged mice than the vehicle group.Post-OVA/PMs challenge could further enhanced the expression of TLR2 and NLRP3 inflammasome.Conclusion: PMs exacerbate the allergic airway inflammation mediated by the TLR2/ NLRP3 signaling pathway.Part II Molecular mechanism involved in PM induced acute exacerbation of allergic airway inflammationBackground: Exposure to particulate matters(PMs)can lead to an acute exacerbation of allergic airway diseases,increasing the severity of symptoms and mortality.However,little is known about the underlying molecular mechanism.Objective: To investigate the role of TLR2 and possible downstream signaling pathway in PM induced acute exacerbation of allergic airway inflammation in vivo and in vitro and seek potential therapeutic targets for allergic airway inflammation.Methods: In vitro,methyl thiazolyl tetrazolium(MTT)was used to test the toxic effect of PM on Raw264.7 cell.Tlr2 was knocked down by small-interfering RNA or the NF-?B inhibitor JSH-23 was used,the expression of TLR2,NF-?B and NLRP3 inflammasome was examined by Western Blot analysis;immunofluorescence was used to test expression of P-p65 in P–induced Raw264.7 cell.In vivo,six-to eight-week-old C57BL/6 mice and Tlr2-/-mice in the C57BL/6 back-ground formed four groups: the wild-type(WT)OVA group,WT OVA/PM group,Tlr2-/-OVA group,and Tlr2-/-OVA/PM group.BALF was obtained for total leukocytes and differential cell counting with Wright-Giemsa stain.Murine lung tissues were stained with hematoxylin and eosin and periodic acid-Schiff to explore the pathological changes.The expression of TLR2 and NLRP3 inflammasome in murine lung was examined by Western Blot analysis.The supernatants of BALF were used for cytokine analysis with ELISA.Results: PM exposure led to increased expression of TLR2 and NLRP3 inflammasome in Raw 264.7 cells and murine lung.The P-induced NLRP3 activation in the RAW 264.7 cell line was diminished by the knockdown of Tlr2 or JSH-23 treatment in vitro indicating the mediating role of TLR2/ NF-?B/ NLRP3 signaling pathway in vitro.In vivo,OVA/PMs-induced mice further confirmed that Tlr2 deficiency effectively inhibited the airway inflammation and NLRP3 inflammasome.Conclusion: PMs exacerbate the allergic airway inflammation possibly mediated by the TLR2/ NF-?B/NLRP3 signaling pathway.Targeting TLR2 and inhibition of NF-?B seems to be a possible treatment.