PM2.5 In Aggravation Of Defective Phagocytosis Of Alveolar Macrophages And Its Effects On Immune Inflammation In Chronic Obstructive Pulmonary Disease Mice

Mechnism of aggravation of PM2.5 on defective phagocytosis in alveolar machrophages from chronic obstructive pulmonary disease mice and protective effects of Codonopsis Pilosula polysaccharidesBacground and Objective: As the main constituent of air pollutants,the increase of fine particulate matter with an aerodynamic equivalent diameter less than or equal to 2.5 ?m(PM2.5)may lead to an increase of incidence and hospital visits due to respiratory diseases.Patients with chronic obstructive pulmonary disease(COPD)was more susceptible to the increase of PM2.5 since acute exacerbations of COPD might occur.But the specific mechanism is unclear.The impaired phagocytosis of alveolar macrophages(AM)may be associated with acute exacerbation of COPD,while little was known how PM2.5 affected on the phagocytosis of AM in COPD.Codonopsis pilosula was widely used in prevention and treatment of pulmonary disease for its immune regulation,anti-oxidative and anti-inflammation effects.But due to the different content of traditional Chinese medicine,its exactly mechanism was unclear.Codonopsis pilosula polysaccharide(CPP)was supposed to improve macrophage phagocytosis and posess anti-oxidative and anti-inflammatory effects.But its function in COPD was rarely reported.This study was to investigate the aggravation effects and mechanism of PM2.5 on phagocytosis of AM,oxidative stress in lung tissue and inflammation in COPD mice.And the protective effects of CPP against PM2.5 was also determined.Methods: Fifty BALB/c male SPF mice were randomly divided into healthy control group,COPD group,COPD PM2.5 group,COPD CPP group and COPD CPP + PM2.5 group with 10 mice in each group.A COPD mouse model was established by exposure of cigarette smoking within 90 consecutive days.Meanwhile,all groups with PM2.5 intervention were exposure to PM2.5(770?g/m3)aerosol inhalation within 90 consecutive days.All groups with CPP intervention were intragastrically administered with CPP(300mg/kg)within 90 consecutive days.Mice peak inspiratory flow(PIF)and peak expiratory flow(PEF)were measured by mice noninvasive body plethysmograph.Then mice were sacrificed by cervical dislocation.Bronchoalveolar lavage fluid(BALF)was obtained by saline perfusion via tracheal.The lung tissue was obtained Sterilly.Lung histopathology and mean linear intercept(MLI)were observed.AM were isolated from lung tissue by discontinuous density gradient centrifugation.Mean fluorescence intensity(MFI)and the proportion of AM phagocytosed flurescein isothiocyanate-labeled Escherichia coli(FITC-E.coli)(AM%)were detected by flow cytometry respectively.Total antioxidative capacity(TAC)was measured by O-phenanthroline colorimetry.Malondialdehyde(MDA)was measured by thiobarbiturieacid colorimetry and glutathione peroxidase(GSH-PX)was measured by improved Hafeman colorimetry.The concentrations of interlukin(IL)-6,IL-8,and tumor necrosis factor(TNF)–? in bronchoalveolar lavage fluid(BALF)and serum were detected using Enzyme-linked immunosorbent adsorption test(ELISA)kits according to the manufacturer's instructions.Results: 1.Pulmonary function of mice: PIF and PEF in COPD group(7.63±0.94),(4.43±0.63)were significantly lower than those in healthy control group(11.85±1.28),(7.38±0.82)and COPD PM2.5 group(5.62±0.53),(3.11±0.41)were remarkably lower than in COPD group(all P<0.01).PIF and PEF in COPD CPP group(8.79±0.51),(5.98±0.34)and COPD CPP+PM2.5 group(6.45±0.45),(3.64±0.39)were respectively higher than those in COPD group and COPD PM2.5 group(all P<0.01).2.Lung histopathology: Small airway inflammation and emphysema were seen in COPD group,and those changes were much seriously in COPD PM2.5 group than in COPD group.Comparing to COPD group and COPD PM2.5 group,small airway inflammation and emphysema were alleviated respectively in COPD CPP group and COPD CPP+PM2.5 group.MLI in COPD group(55.51±2.17)was significantly larger than that in healthy control group(32.92±1.30)and COPD PM2.5 group(61.69±1.84)was remarkably larger than in COPD group(all P<0.01).PIF and PEF in COPD CPP group and COPD CPP+PM2.5 group(43.25±1.50?57.78±1.69)were respectively lower than those in COPD group and COPD PM2.5 group(P<0.01).3.Phagocytosis of AM: MFI and AM% in COPD group(4939±924),(30.90±5.19)% were significantly decreased than those in healthy control group(10222±1567),(69.11±5.59)%(all P<0.01);MFI and AM% in COPD PM2.5 group(3293±543),(20.91±3.51)% were remarkably decreased than those in COPD group(all P<0.01);Comparing to COPD group and COPD PM2.5 group respectively,MFI and AM% in COPD CPP group(5903±484),(38.53±2.84)% and COPD CPP+PM2.5 group(4064±418),(26.88±1.84)% were increased(all P<0.01).4.Oxidative stress: The level of TAC and GSH-PX in lung homogenate from COPD group(6.83±0.36),(46.49±2.59)were lower than those from healthy control group(17.99±0.09),(84.32±5.65)and those from COPD PM2.5 group(3.61±0.29),(32.37±3.78)were lower than those from COPD group(all P<0.01).Comparing to COPD group and COPD PM2.5 group,TAC and GSH-PX in COPD CPP group(8.80±0.26),(53.40±4.02)and COPD CPP+PM2.5 group(5.43±0.30),(42.42±3.95)were increased respectively(all P<0.01).The content of MDA in lung homogenate in COPD group(2.73±0.22)were higher than that in healthy control group(1.74±0.37)(P<0.01).MDA in COPD PM2.5 group(3.55±0.33)were higher than that in COPD group(P<0.01).MDA from COPD CPP group(2.22±0.28)and COPD CPP+PM2.5 group(2.72±0.44)were respectively decreased than that from COPD group and COPD PM2.5 group(all P<0.01).5.The level of IL-6,IL-8 and TNF-? in BALF and serum from COPD group(1136.17±79.12,456.74±38.58),(336.80±36.57,248.77±31.92),(201.72±37.65,29.17±9.77)were higher than those from healthy control group(350.42±71.94,224.23±32.84),(50.25±7.79,62.65±11.90),(44.58±20.35,0.60±0.35)and those from COPD PM2.5 group(1351.67±41.64,753.64±44.39),(562.38±39.37,535.94±51.09),(401.49±35.98,46.76±11.17)were higher than those from COPD group(all P<0.01).Comparing to COPD group and COPD PM2.5 group,IL-6,IL-8 and TNF-? in BALF and serum from COPD CPP group(762.33±32.79,365.63±39.21),(289.02±48.57,143.14±33.66),(134.35±35.34,14.63±4.93)and COPD CPP+PM2.5 group(778.17±20.07,558.73±38.07),(427.27±36.35,331.53±43.59),(258.09±36.74,34.72±11.94)were decreased respectively(all P<0.01).6.Correlation analysis: Positive correlations were existed between MFI,AM% and TAC,GSH-PX,while negative correlations were existed between MFI,AM% and MDA in helthy control group,COPD group,PM2.5 COPD group,CPP COPD group,CPP + PM2.5 COPD group.Conclusions: 1.Amouse model of COPD can be established by cigarette smoke exposure.2.Phagocytosis of E.coli is defective in AM from COPD mice.Oxidative stress damage and a high level of inflammation response of lung and circulation are existed in COPD mice.3.PM2.5 impairs phagocytosis of AM and exacerbates oxidative stress and inflammation in COPD mice.4.CPP can improve the phagocytosis of AM and alleviate oxidative stress and inflammation in COPD mice and PM2.5 damage COPD mice.5.The protection of CPP may be closely related to relieving imbalance of oxidative/antioxidative and resolution of inflammation.Effects of PM2.5 on Th17/Treg imbalance and immune inflammation in COPD miceBackgroud and Objective: Abnormal secretion of inflammatory cytokines due to imbalance of T cell subpopulation and the accompanying immune inflammation is one of the important pathogenesis of COPD.An excess of Th17 cells with defective inhibition by Treg cells can cause chronic inflammation.This is due to excessive secretion of IL-17 with a reduction of IL-10,which leads to a defective resolution of inflammation and plays a vital role in development of COPD.But effects of PM2.5 on Th17 and Treg proportion and secretion of IL-17 and IL-10 in COPD is not clear.Delta1/4-Notch1 signaling pathway is involved in the differentiation of naive T cells into different subgroups.Whereas the effect of this pathway on Th17 and Treg differentiation and the effect of PM2.5 on Delta1/4-Notch1 signaling pathway in COPD is rarely known.IL-6 is also involved in the regulation of Th17 differentiation.But it is also unclear whether IL-6 is involved in regulation of Th17 differentiation and effects of PM2.5 in COPD.Cytokines such as IL-6 can be secreted by AM and leads to inflammation,but it is not known that whether this can regulate imbalance of Th17/Treg is not known.This study is to investigate the effects of PM2.5 on imbalance of Th17/Treg proportion and release of IL-17 and IL-10 in COPD mice,and its correlationship with expression of Delta1/4 on AM,Notch1 on T cells and IL-6 levels in serum.Methods: Fifty BALB/c male SPF mice were randomly divided into healthy control group,COPD group,COPD PM2.5 group,COPD ?-secretase inhibitor(GSI)group and COPD GSI + PM2.5 group with 10 mice in each group.A COPD mouse model was established by exposure of cigarette smoking within 90 consecutive days.Meanwhile,all groups with PM2.5 intervention were exposure to PM2.5(770?g/m3)aerosol inhalation within 90 consecutive days.All groups with GSI intervention were injected intraperitoneally with Notch signaling inhibitor GSI every other day from day 61 to day 90.PIF and PEF were measured by mice noninvasive body plethysmograph.Then mice were sacrificed by cervical dislocation.Lung tissue was obtained Sterilly.Lung histopathology and MLI were observed.AM were isolated from lung tissue by discontinuous density gradient centrifugation.Spleen T cells were isolated from lung tissue by continuous density gradient centrifugation and purified by nylon cyber column.Flow cytometry were utilized to detect the Th17 and Treg proportion in CD4+ T cells and ratio of Th17/Treg was calculated.Real-time quantitative polymerase chain reaction(RT-PCR)and Western blot were used to detect the expression of Delta 1/4 and Notch 1 m RNA and protein respectively.The concentrations of IL-6,IL-17 and IL-10 in serum were detected using ELISA kits according to the manufacturer's instructions.Results: 1.Pulmonary function and lung histopathology: PIF and PEF in COPD group(7.63±1.12),(4.44±0.70)were significantly lower than those in healthy control group(12.03±1.35),(7.17±0.63)(P<0.01).Small airway inflammation and emphysema were seen in COPD group,and while there were no such changes in healthy control group.MLI in COPD group(56.31±3.27)was significantly larger than that in healthy control group(33.32±2.28)(P<0.01).2.Proportion of Th17:Propotion of Th17 in COPD group(17.69±5.08)were significantly increased than those in healthy control group(5.59±1.49)(all P<0.01);Propotion of Th17 in COPD PM2.5 group(32.16±6.06)were remarkably increased than those in COPD group(all P<0.01);Comparing to COPD group and COPD PM2.5 group respectively,Propotion of Th17 in COPD GSI group(12.11±1.83)and COPD GSI+PM2.5 group(27.57±7.38)were decreased(P<0.05 or P<0.01).Proportion of Treg: Propotion of Treg in COPD group(0.84±0.29)were significantly decreased than those in healthy control group(1.18±0.60)(P<0.05);Propotion of Treg in COPD PM2.5 group(0.45±0.16)was remarkably decreased than that in COPD group(P<0.05);Comparing to COPD group and COPD PM2.5 group respectively,Propotion of Th17 and ratio of Th17/Treg in COPD GSI group(1.15±0.25)and COPD GSI+PM2.5 group(0.77±0.23)were decreased(P<0.05 or P<0.01).Ratio of Th17/Treg: Ratio of Th17/Treg in COPD group(22.50±7.72)were significantly increased than those in healthy control group(5.81±3.03)(all P<0.01);Ratio of Th17/Treg in COPD PM2.5 group(77.32±22.14)were remarkably increased than those in COPD group(all P<0.01);Comparing to COPD group and COPD PM2.5 group respectively,Ratio of Th17/Treg in COPD GSI group(11.07±3.13)and COPD GSI+PM2.5 group(38.72±15.04)were decreased(all P<0.01).3.Exprssion of m RNA and protein of Delta1,Delta4 and Notch1: Exprssion of m RNA and protein of Delta1,Delta4 and Notch1 in COPD group(6.34±0.88,0.78±0.05),(10.03±1.32,0.70±0.06),(11.56±1.39,0.51±0.07)were higher than those from healthy control group(1.20±0.72,0.54±0.48),(1.06±0.38,0.51±0.07),(1.03±0.28,0.29±0.05)and those from COPD PM2.5 group(9.97±3.38,1.05±0.07),(19.08±3.40,0.88±0.05),(23.11±5.98,0.77±0.06)were higher than those from COPD group(P<0.05 or P<0.01).Comparing to COPD group and COPD PM2.5 group,Exprssion of m RNA and protein of Delta1,Delta4 and Notch1 in COPD GSI group(2.23±0.56,0.69±0.05),(7.27±1.27,0.63±0.05),(7.74±1.01,0.41±0.05)and COPD GSI+PM2.5 group(4.00±3.54,0.90±0.10),(12.66±2.07,0.77±0.06),(17.05±2.40,0.63±0.06)were decreased respectively(P<0.05 or P<0.01).4.Level of IL-6,IL-17 and IL-10: The level of IL-6,IL-17 and IL-10 in serum from COPD group(492.64±61.05,60.70±2.57,2.60±0.47)were higher than those from healthy control group(224.13±33.67,44.14±3.86,4.15±1.41)and those from COPD PM2.5 group(744.70±42.78,71.15±3.69,0.57±0.19)were higher than those from COPD group(all P<0.01).Comparing to COPD group and COPD PM2.5 group,IL-6,IL-8 and TNF-? in BALF and serum from COPD GSI group(430.42±43.21,54.30±3.00,3.32±0.14)and COPD GSI+PM2.5 group(643.03±25.04,65.19±2.64,1.51±0.12)were decreased respectively(all P<0.01).5.Correlation analysis: Positive correlations were existed between protein of Delta1/4 and IL-6 in serum in helthy control group,COPD group,PM2.5 COPD group,GSI COPD group,GSI + PM2.5 COPD group.Positive correlations were existed between IL-6 in serum and protein of Notch1 in all groups.Positive correlations were existed between protein of Notch1 and proportion of Th17 and ratio of Th17/Treg in all groups,while negative correlations were existed between protein of Notch1 and proportion of Th17 in all groups.Positive correlations were existed between IL-17 in serum and proportion of Th17 in all groups,while negative correlations were existed between IL-17 in serum and ratio of Th17/Treg in all groups.Positive correlations were existed between IL-10 in serum and proportion of Treg in all groups,while negative correlations were existed between IL-10 in serum and ratio of Th17/Treg in all groups.Conclusions: 1.Th17 predominant imbalance of Th17/Treg exists in COPD mice and PM2.5 can aggravate the imbalance.IL-17 is increased and IL-10 and the inflammation is decreased due to imbalance of Th17/Treg.PM2.5 can aggravate the inflammation.2.Expression of Delta1/4 in AM and Notch1 in T cells are all upregulated in COPD mice and PM2.5 further enhance the upregulated expression.3.Imbalance of Th17/Treg characterized by abnormal elevation of Th17 and inflammation with elevated levels of IL-17 were significantly associated with the up-regulation of Delta1/4 and Notch1 expression before and after PM2.5 exposure in COPD mice.GSI could partially reverse the immune inflammation response before and after PM2.5 exposure,wich is related with inhibition of Delta1/4-Notch1 activation.4.The levels of IL-6 is increased in serum of COPD mice and PM2.5 enhance the excessive release of IL-6,which is involved in the regulation of abnormal elevation of Th17.The up-regulation of Delta1/4 in AM was related to the increase of IL-6,suggesting that AM may be involved in IL-6 abnormal release,leading to abnormal increase of Th17.These are involved in immune inflammation in COPD before and after PM2.5 exposure.