Association Of Air Pollutants With Immunoglobulins In Children And The Mediating Role Of Mitochondrial DNA Copy Number

Air pollution is one of the most important global public health issues.Generally,air pollutant consists of particulate matter and gaseous pollutants.In recent years,the concentrations of inhalable particulate matter(PM10)and fine particulate matter(PM2.5)have been declining.However,PM2.5 is still the primary pollutant of air pollution in China,while the impact of gaseous pollutants on the health of the population is increasing,ozone has become the pollutant second only to PM2.5.Children are more vulnerable to the effects of air pollutants because of their immature immune systems.The change of immune function during children’s growth and development period may have far-reaching effects in adulthood.Immunoglobulins are important indicators of humoral immune function.Only a few epidemiological studies have suggested that exposure to air pollutants is associated with immunoglobulin levels in adults or children with asthma,but the findings are inconsistent.Immunoglobulins are immune indicators of more public health significance due to their simplicity and ease of measurement.However,the acute health effects,dose-response relationships,and individual susceptibility characteristics regarding air pollutants and immunoglobulins in healthy children are unclear.Further analysis of the relationship between the acute health effects of multiple air pollutant exposures and immunoglobulins is important based on the complex composition and real exposure of air pollution.Moreover,the potential biological mechanisms underlying the association of air pollutants with immunoglobulins have not been clarified.Previous cross-sectional studies,maternal and child studies suggest that air pollution is associated with mitochondrial DNA copy number(mtDNAcn)levels in adults and infants,and in vitro and in vivo experiments also suggest that mitochondrial DNA damage is an important trigger for immune regulation.However,it is still unknown whether mtDNAcn mediates the association between air pollutants and immunoglobulins in children.Therefore,we conducted panel study in the cities of Wuhan and Guangzhou between the ages of 4～13 among healthy children in three seasons.We aimed to explore the associations between PM and immunoglobulins,gaseous pollutant exposure and immunoglobulins,and the separate and overall relationship of exposure to PM and gaseous pollutants on immunoglobulins,as well as the potential role of mtDNAcn in the following parts.Part 1 Association of particulate matter with immunoglobulin levels in children and the mediating role of mitochondrial DNA Copy Number in peripheral bloodObjective:To investigate the association between atmospheric particulate matter and immunoglobulin levels in children,determine the lagged effect time window and exposureresponse relationship,and explore the mediating role of mitochondrial DNA copy number in relations between PM and immunoglobulin.Method:We conducted 2 longitudinal panel studies in Wuhan and Guangzhou among 279 children aged 4～13 years in autumn,winter,and summer.A microPEM real-time personal PM2.5 measurement was used to continuously monitor PM2.5 exposure 72 hours prior to the physical examination.A detailed epidemiological survey was conducted,and PM2.5,PM10,Sulfur dioxide(SO2),Nitrogen dioxide(NO2),Carbon monoxide(CO),and Ozone(O3)were collected from monitoring stations.We completed the physical examination with blood and urine samples in the morning of the fourth day.After excluding children with loss of follow-ups,missing blood drawings,and insufficient blood samples,a total of 258 children were included for further analysis.Immunoglobulin A(IgA),immunoglobulin G(IgG),and immunoglobulin M(IgM)were measured by immunoturbidimetric assay.Real-time fluorescence quantitative PCR was used to calculate mtDNAcn in peripheral blood.Linear mixed-effect models(LMEs)were used to analyze the association between PM with children’s immunoglobulin levels and mtDNAcn.Generalized additive models(GAMs)were used to fit the dose-response relationships.The analysis was stratified to explore the characteristics of children.And mediation analysis was used to explore the mediating role of mtDNAcn in the association between PM and immunoglobulins in children.The protocol was approved by the Medical Ethics Committee of Tongji Medical College,Huazhong University of Science and Technology.Result:In our study,the average age of children at baseline in the two cities was 9.0 years,57%were boys,and the mean individual PM2.5 exposure at lag 0～2 days was 39.8450.76 μg/m3.PM2.5 exposure was associated with elevated IgG and IgM levels in children,the effect of IgA was not significant,and the individual PM2.5 effect was slightly stronger than that of atmospheric PM2.5,and no significant association of PM10 with immunoglobulins was observed.After adjusting for the 4 gaseous pollutants,with each inter-quartile range(IQR)increase in PM2.5 concentration,IgG increased by 2.52%,1.35%,and 2.10%,and IgM increased by 2.57%,2.70%and 2.89%on lag 0 day,lag 1 day and cumulative lag 0 to 1 days,respectively.All above relationships showed in a dose-response manner.These associations were stronger in children aged≥7 years,boys,passive smokers,exercise duration≥1 h/day,and in Wuhan.It is noteworthy that individual PM2.5 exposure was still associated with increased IgG and IgM in children at concentrations below the Chinese National Ambient Air Quality Standards II(CNAAQS II)limits.As each 1 IQR increase in PM2.5,there was a subsequent 2.81%increase in mtDNAcn on lag 2 day with an exposure-response relationship.Association of PM2.5 with mtDNAcn was still observed after adjusting for the 4 gaseous pollutants.And the association was more significant in children who were passive smokers,with an interaction effect(Pinteraction=0.003).However,IgG decreased with increase in mtDNAcn,more significantly in children aged≥7 years,boys,without passive smoking,and with exercise duration≥1 h/day.No significant mediating role of mtDNAcn in the association of individual PM2.5 with IgG was found.Conclusion:PM2.5 exposure was associated with elevated IgG and IgM and mtDNAcn with a significant linear dose-response relationship,and remained associated with elevated IgG and IgM in children when PM2.5 was below CNAAQS Ⅱ.However,a significant mediating role of mtDNAcn in the association of PM with immunoglobulins was not found.Part 2 Association of gaseous pollutants with immunoglobulin levels in children andthe mediating role of mitochondrial DNA Copy Number in peripheral bloodObjective:To clarify the dose-response relationships between multiple gaseous pollutants and immunoglobulins in children,and examine the mediating role of mtDNAcn in relations between gaseous pollutants and immunoglobulins.Method:The study design and subjects were same as above.LMEs were used to analyze the association between four gaseous pollutants,SO2,NO2,CO,and O3,and children’s immunoglobulin and mtDNAcn levels.Moreover,GAMs were used to fit the dose-response relationship,stratified analysis of individual characteristics,and further Bayesian kernel machine regression models were used to analyze the association between gaseous pollutant mixtures with immunoglobulins and mtDNAcn levels.mediation analysis and Bayesian kernel machine regression-causal mediation analysis were used to analysis the mediating role of mtDNAcn in the association between individual and mixture exposure of gaseous pollutant and immunoglobulin,respectively.Result:SO2 was positively associated with elevated immunoglobulin IgG,IgM,and IgA,and NO2 was positively associated with elevated IgM,whereas O3 was associated with decreased IgG and IgM,all with exposure-response relationships,while no significant association of CO with immunoglobulins was observed.And the association above was more evident in children aged≥7 years,boys,passive smokers,and those exercising duration≥1 h/day,all with an interaction effect.In addition,gaseous pollutants mixture was related to elevated IgG and IgM at lag 2 day,with SO2 and O3 may play an important role.With an IQR increase of NO2,the increment of 5.08%in mtDNAcn was related at lag 1 day.And each IQR increase of O3 was related to the increment in mtDNAcn at lag 0 day to lag 2 day,respectively.Such relations in O3 were the strongest at lag 0 day and declined over time.Above associations showed in a dose-response manner.Children who had a history of passive smoking had a more significant increase in mtDNAcn.The overall effect of gaseous pollutants mixture related to the increment of mtDNAcn,with O3 may play an important role.Moreover,mtDNAcn mediated the proportion of 5.56%in the relation of O3 and IgG decreased at lag 2 day.However,mtDNAcn not mediated the association of other gaseous pollutants and multiple gaseous pollutants mixture with immunoglobulins.Conclusion:Both individual and mixture exposure of gaseous pollutants were associated with immunoglobulin and mtDNAcn changed in a linear dose-response manner.Also,mtDNAcn might be partly involved in the association between O3 with IgG.Part 3 Association of air pollutant mixture exposure with immunoglobulin levels in children and the mediating role of mtDNAcn in peripheral bloodObjective:To investigate the association between air pollutant mixture exposure and immunoglobulins in children,and explore the interaction between PM2.5 and gaseous pollutants on immunoglobulins.Method:The study design and subjects were same as above.And after adjusting PM2.5,LMEs were used to analyze the relation of SO2,NO2,and O3,on children’s immunoglobulin and mtDNAcn levels.The dose-response relationship was fitted by GAMs,and bayesian kernel machine regression models were used to analyze the relation of air pollutant mixtures on immunoglobulins and mtDNAcn.Moreover,the role of mtDNAcn in the association above was explored using bayesian kernel machine regression-causal mediation analysis.Result:It was still observed that SO2 was associated with elevated IgG and IgM,NO2 with elevated IgM,and O3 with decreased IgG and IgM,and all had dose-response manners after adjusting PM2.5 in the model.The relation of SO2,NO2,and O3 exposure on IgG and IgM were more significant at high levels of PM2.5,and there was an interaction effect.In addition,exposure to air pollutants mixture was also related to elevated IgG and IgM at lag 2 day,where SO2,O3,and PM2.5,may play an important role.Moreover,the increment of mtDNAcn was related with increase of NO2 at lag 1 day,and with increase of O3 at lag 3 days after adjusting PM2.5.Above associations showed in a dose-response manner.The association between O3 and increased mtDNAcn was more significant at high PM2.5 levels with an interaction effect.The overall effect of air pollutants mixture related to the increment of mtDNAcn,with O3 may play an important role.However,mtDNAcn not mediated the association of air pollutants mixture with immunoglobulins.Conclusion:Air pollutant mixture exposure was associated with elevated IgG,IgM,and mtDNAcn,with more significant associations of gaseous pollutants with immunoglobulin and mtDNAcn at high levels of PM2.5.