# Air pollution and IgE sensitization in 4 European birth cohorts-the MeDALL project

Erik Melén, Marie Standl, Ulrike Gehring, Hicran Altug, Josep Maria Antó, Dietrich Berdel, Anna Bergström, Jean J. Bousquet, Joachim Heinrich, Gerard H. Koppelman, et al.

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To cite this version:

Erik Melén, Marie Standl, Ulrike Gehring, Hicran Altug, Josep Maria Antó, et al. Air pollution and IgE sensitization in 4 European birth cohorts-the MeDALL project. Journal of Allergy and Clinical Immunology, 2021, 147 (2), pp.713-722. 10.1016/j.jaci.2020.08.030. hal-03141483

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# Air pollution and IgE sensitization in 4 European birth cohorts—the MeDALL project

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Erik Melén, MD, PhD,<sup>a,b</sup> Marie Standl, PhD,<sup>c</sup> Ulrike Gehring, PhD,<sup>d</sup> Hicran Altug, PhD,<sup>e</sup> Josep Maria Antó, MD, PhD,<sup>f,g,h,i</sup>

Dietrich Berdel, MD,<sup>j</sup> Anna Bergström, PhD,<sup>k</sup> Jean Bousquet, MD, PhD,<sup>l,m</sup> Joachim Heinrich, PhD,<sup>c,n,o</sup>

Gerard H. Koppelman, MD, PhD,<sup>p,q</sup> Inger Kull, PhD,<sup>a,b</sup> Christian Lupinek, MD,<sup>r</sup> Iana Markevych, PhD,<sup>c,n,s</sup>

Tamara Schikowski, PhD,<sup>e</sup> Elisabeth Thiering, PhD,<sup>c,t</sup> Rudolf Valenta, MD,<sup>r,u,v,w</sup> Marianne van Hage, MD, PhD,<sup>x</sup>

Andrea von Berg, MD,<sup>j</sup> Judith M. Vonk, PhD,<sup>q,y</sup> Magnus Wickman, MD, PhD,<sup>z</sup> Alet Wijga, PhD,<sup>aa</sup> and

Olena Gruzieva, MD, PhD<sup>k,bb</sup>

Stockholm and Eskilstuna, Sweden; Neuherberg, Düsseldorf, Wesel, and Munich, Germany;

Utrecht, Groningen, and Bilthoven, The Netherlands; Barcelona and Madrid, Spain; Montpellier, Villejuif, and Montigny le Bretonneux,

France; Melbourne, Australia; Vienna and Krems, Austria; Cracow, Poland; and Moscow, Russia

From <sup>a</sup>the Department of Clinical Sciences and Education, Karolinska Institutet, Södersjukhuset, Stockholm; <sup>b</sup>the Sachs Children's Hospital, Stockholm; <sup>c</sup>the Institute of Epidemiology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg; <sup>d</sup>the Institute for Risk Assessment Sciences, Utrecht University; <sup>e</sup>the IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf; <sup>f</sup>the ISGlobal, Barcelona Institute for Global Health, Barcelona; <sup>g</sup>the Universitat Pompeu Fabra, Barcelona; <sup>h</sup>the CIBER Epidemiología y Salud Pública, Madrid; <sup>i</sup>the Hospital de Mar Medical Research Institute, Barcelona; <sup>j</sup>the Research Institute, Department of Pediatrics, Marien-Hospital Wesel; <sup>k</sup>the Institute of Environmental Medicine, Karolinska Institutet, Stockholm; <sup>l</sup>the MACVIA-France, Contre les Maladies Chroniques pour un Vieilissement Actif en France European Innovation Partnership on Active and Healthy Ageing Reference Site, Montpellier; <sup>m</sup>the INSERM U 1168, VIMA: Ageing and Chronic Diseases Epidemiological and Public Health Approaches, Villejuif, Université Versailles St-Quentin-en-Yvelines, Montigny le Bretonneux; <sup>n</sup>the Institute and Clinic for Occupational, Social and Environmental Medicine, University Hospital, LMU Munich, Munich; <sup>o</sup>the Allergy and Lung Health Unit, Melbourne School of Population and Global Health, University of Melbourne; <sup>p</sup>the University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Department of Pediatric Pulmonology and Pediatric Allergology; <sup>q</sup>the University of Groningen, University Medical Center Groningen, Groningen Research Institute for Asthma and COPD (GRIAC); <sup>r</sup>the Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna; <sup>s</sup>the Institute of Psychology, Jagielonian University, Cracow; <sup>t</sup>the Division of Metabolic and Nutritional Medicine, Dr von Hauer Children's Hospital, University Hospital, LMU of Munich; <sup>u</sup>the Laboratory of Immunopathology, Department of Clinical Immunology and Allergy, Sechenov First Moscow State Medical University, Moscow; <sup>v</sup>the National Research Center-Institute of Immunology FMBA of Russia, Moscow; <sup>w</sup>the Karl Landsteiner University of Health Sciences, Krems; <sup>x</sup>the Division of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet and Karolinska University Hospital, Stockholm; <sup>y</sup>the Department of Epidemiology, University of Groningen, University Medical Center Groningen; <sup>z</sup>the Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna; <sup>aa</sup>the National Institute for Public Health and the Environment, Bilthoven; and <sup>bb</sup>the Centre for Occupational and Environmental Medicine, Region Stockholm, Stockholm.

The research leading to these results has received funding from the European Community's Seventh Framework Program under grant agreement numbers: 211250 (European Study of Cohorts for Air Pollution Effects [ESCAPE]), and 261357 (Mechanisms of the Development of ALLergy [MeDALL]). Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) was supported by The Swedish Research Council, The Swedish Heart-Lung Foundation, Region Stockholm (ALF project, and database maintenance), the Strategic Research Programme in Epidemiology at Karolinska Institutet, the Swedish Research Council Formas and the Swedish Environment Protection Agency, the Swedish Asthma and Allergy Research Foundation, the Cancer and Allergy Foundation. E.M. is supported by a grant from the European Research Council (grant agreement 757919, TRIBAL). O.G. is supported by the Swedish Research Council for Health, Working Life and Welfare (FORTE 2017-01146). R.V. is supported by the Austrian Science Fund (grant F4605) and is a recipient of a Megagrant of the Government of the Russian Federation (grant 14.W03.31.0024). The Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study was supported by project grants from The Netherlands Organization for Health Research and Development; The Netherlands Organization for Scientific Research; The Netherlands

Asthma Fund; The Netherlands Ministry of Spatial Planning, Housing, and the Environment; and The Netherlands Ministry of Health, Welfare, and Sport. U.G. was supported by a Grant of The Netherlands Organization for Scientific Research. The German Infant Study on the Influence of Nutrition Intervention PLUS Environmental and Genetic Influences on Allergy Development (GINIplus) study was mainly supported for the first 3 years of the Federal Ministry for Education, Science, Research and Technology (interventional arm) and Helmholtz Zentrum Munich (formerly GSF) (observational arm). The 4-year, 6-year, 10-year, and 15-year follow-up examinations of the GINIplus study were covered from the respective budgets of the 5 study centers (Helmholtz Zentrum Munich [formerly GSF], Research Institute at Marien-Hospital Wesel, LMU Munich, TU Munich), and from year 6 onward it was also supported with funding from IUF-Leibniz Research Institute for Environmental Medicine at the University of Düsseldorf and by a grant from the Federal Ministry for Environment (IUF Düsseldorf [grant FKZ 20462296]). Furthermore, the 15-year follow-up examination of the GINIplus study was supported by the Commission of the European Communities, the Seventh Framework Program MeDALL project, and by the companies Mead Johnson and Nestlé. The Influences of Lifestyle-Related Factors on the Human Immune System and Development of Allergies in Childhood (LISA) study was mainly supported by grants from the Federal Ministry for Education, Science, Research and Technology and also from Helmholtz Zentrum Munich (formerly GSF), the Helmholtz Centre for Environmental Research-UFZ, Leipzig, the Research Institute at Marien-Hospital Wesel, Pediatric Practice, and Bad Honnef for the first 2 years. The 4-year, 6-year, 10-year, and 15-year follow-up examinations of the LISA study were covered from the respective budgets of the involved partners (Helmholtz Zentrum Munich [formerly GSF], the Helmholtz Centre for Environmental Research-UFZ, Leipzig, the Research Institute at Marien-Hospital Wesel, Pediatric Practice, Bad Honnef, and IUF-Leibniz-Research Institute for Environmental Medicine at the University of Düsseldorf) and also by a grant from the Federal Ministry for Environment (IUF Düsseldorf [grant FKZ 20462296]). Furthermore, the 15-year follow-up examination of the LISA study was supported by the Commission of the European Communities, the Seventh Framework Program MeDALL project. I.M. is supported by a grant from the NeuroSmog: Determining the Impact of Air Pollution on the Developing Brain (grant POIR.04.04.00-1763/18-00) which is implemented as part of the TEAM-NET programme of the Foundation for Polish Science and cofinanced with funding from European Union resources obtained from the European Regional Development Fund under the Smart Growth Operational Programme.

Disclosure of potential conflict of interest: R. V. reports that he has received research funding from Viravaxx, Vienna, Austria, and serves as a consultant for the company. M. v.H. serves as consultant for Biomay, Vienna, Austria.

Received for publication April 30, 2020; revised August 13, 2020; accepted for publication August 21, 2020.

Available online September 11, 2020.

Corresponding author: Olena Gruzieva, MD, PhD, Karolinska Institutet, Institute of Environmental Medicine, Nobels väg 13, Box 210, SE-17177, Stockholm, Sweden. E-mail: olena.gruzieva@ki.se.

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© 2020 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/)).

[https://doi.org/10.1016/j.jaci.2020.08.030](https://doi.org/10.1016/j.jaci.2020.08.030)
**Background:** Whether long-term exposure air to pollution has effects on allergic sensitization is controversial.

**Objective:** Our aim was to investigate associations of air pollution exposure at birth and at the time of later biosampling with IgE sensitization against common food and inhalant allergens, or specific allergen molecules, in children aged up to 16 years.

**Methods:** A total of 6163 children from 4 European birth cohorts participating in the Mechanisms of the Development of ALLergy [MeDALL] consortium were included in this meta-analysis of the following studies: Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) (Sweden), Influences of Lifestyle-Related Factors on the Human Immune System and Development of Allergies in Childhood (LISA)/German Infant Study on the Influence of Nutrition Intervention PLUS Environmental and Genetic Influences on Allergy Development (GINIplus) (Germany), and Prevention and Incidence of Asthma and Mite Allergy (PIAMA) (The Netherlands). The following indicators were modeled by land use regression: individual residential outdoor levels of particulate matter with aerodynamic diameters less than 2.5 µm, less than 10 µm, and between 2.5 and 10 µm; PM<sub>2.5</sub> absorbance (a measurement of the blackness of PM<sub>2.5</sub> filters); and nitrogen oxides levels. Blood samples drawn at ages 4 to 6 (n = 5989), 8 to 10 (n = 6603), and 15 to 16 (n = 5825) years were analyzed for IgE sensitization to allergen extracts by ImmunoCAP. Additionally, IgE against 132 allergen molecules was measured by using the MedALL microarray chip (n = 1021).

**Results:** Air pollution was not consistently associated with IgE sensitization to any common allergen extract up to age 16 years. However, allergen-specific analyses suggested increased risks of sensitization to birch (odds ratio [OR] = 1.12 [95% CI = 1.01-1.25] per 10-µg/m<sup>3</sup> increase in NO<sub>2</sub> exposure). In a subpopulation with microarray data, IgE to the major timothy grass allergen *Phleum pratense* 1 (Phl p 1) and the cat allergen *Felis domesticus* 1 (Fel d 1) greater than 3.5 Immuno Solid-phase Allergen Chip standardized units for detection of IgE antibodies were related to PM<sub>2.5</sub> exposure at birth (OR = 3.33 [95% CI = 1.40-7.94] and OR = 4.98 [95% CI = 1.59-15.60], respectively, per 5-µg/m<sup>3</sup> increase in exposure).

**Conclusion:** Air pollution exposure does not seem to increase the overall risk of allergic sensitization; however, sensitization to birch as well as grass pollen Phl p 1 and cat Fel d 1 allergen molecules may be related to specific pollutants. (J Allergy Clin Immunol 2021;147:713-22.)

**Key words:** Allergy, allergen, air pollution, children, cohort, IgE, sensitization, meta-analysis

Associations between exposure to outdoor air pollution and several adverse health conditions in children and adolescents, including lower lung function and higher respiratory morbidity, have been widely demonstrated<sup>1-4</sup>; however, the evidence for associations of air pollution with the risk of allergic diseases remains conflicting because of the small number of studies available in the literature and their inconsistent results. Experimental studies provide a biologic basis for air pollutants being risk factors for allergic sensitization by demonstrating enhanced IgE production following exposure to particulates.<sup>5-7</sup> To date, there have been only a few prospective cohort studies following children from birth up to school age, with objective assessments

Abbreviations used

BAMSE: Children, Allergy, Milieu, Stockholm, Epidemiology

ESCAPE: European Study of Cohorts for Air Pollution Effects

Fel d 1: *Felis domesticus* 1

GINIplus: German Infant Study on the Influence of Nutrition Intervention PLUS Environmental and Genetic Influences on Allergy Development

ISAC: Immuno Solid-phase Allergen Chip

ISU-E: Immuno Solid-phase Allergen Chip standardized units for detection of IgE antibodies

LISA: Influences of Lifestyle-Related Factors on the Human Immune System and Development of Allergies in Childhood

LUR: Land use regression

MeDALL: Mechanisms of the Development of ALLergy

NO<sub>2</sub>: Nitrogen dioxide

NO<sub>x</sub>: Nitrogen oxides

OR: Odds ratio

Phl p 1: *Phleum pratense* 1

PIAMA: Prevention and Incidence of Asthma and Mite Allergy

PM<sub>2.5</sub>: Mass concentration of particles less than 2.5 µm in size

PM<sub>10</sub>: Mass concentration of particles less than 10 µm in size

PM<sub>2.5</sub> absorbance: Measurement of the blackness of PM<sub>2.5</sub> filters

PM<sub>coarse</sub>: Mass concentration of particles between 2.5 and 10 µm in size

of specific sensitization to allergen extracts as well as assessments of exposure to air pollution at an individual level.<sup>8-13</sup> In an earlier multicohort analysis with harmonized exposure and health data from 5 European cohorts (including the 4 cohorts participating in this analysis), we found no apparent association between exposure to air pollution and allergic sensitization in children at either 4 years or 8 years of age.<sup>14</sup> Recent narrative and systematic reviews evaluating the body of evidence on air pollution exposure and allergy outcomes have provided inconclusive results, acknowledging a high degree of heterogeneity between existing studies.<sup>15-17</sup> Importantly, most previous studies have been limited to cross-sectional analyses of prevalence of specific IgE sensitization measured only up to school age. In a longitudinal analysis of 2 German birth cohorts that were followed for 10 years, no consistent evidence that exposure to air pollution increases the risk of aeroallergen sensitization in later childhood was found.<sup>13</sup> To our knowledge, as yet there has been no combined longitudinal analysis of the relationship between air pollution exposure and prevalence of IgE sensitization against common allergens or defined allergen molecules in individuals up to 16 years of age based on prospective birth cohort studies.

Therefore, we conducted the present study within the framework of the European collaborative Mechanisms of the Development of ALLergy (MeDALL)<sup>18</sup> project with the aim of evaluating the association of exposure to air pollution with prevalence of IgE sensitization in children during the first 16 years of life. We implemented a meta-analysis based on already collected and harmonized health data from 4 European birth cohorts and applied uniform exposure assessment methodology developed
<table><caption>TABLE I. Overview of the tested allergen sources in the 4 birth cohorts</caption><thead><tr><th>Cohort</th><th>Test system</th><th>Tested allergens</th></tr></thead><tbody><tr><td>BAMSE</td><td>ImmunoCAP System, Thermo Fisher/Phadia AB, Uppsala, Sweden (Phadiatop/fx5)</td><td>Inhalant allergen sources:<ul><li>Outdoor: birch, timothy grass, mugwort</li><li>Indoor: cat, dog, mold (Cladosporium herbarum), house dust mite (Der p)</li></ul>Food allergen sources: cow's milk, egg white, soy bean, peanut, cod fish and wheat</td></tr><tr><td>PIAMA</td><td>Radioallergosorbent test-like method used at the Sanquin Laboratories (Amsterdam, The Netherlands)</td><td>Inhalant allergen sources:<ul><li>Outdoor: birch, dactylis glomerata</li><li>Indoor: cat, dog, Alternaria alternata, house dust mite (Der p)</li></ul>Food allergen sources: egg, milk</td></tr><tr><td>LISA/GINI South, LISA/GINI North</td><td>CAP-RAST FEIA (Pharmacia Diagnostics, Freiburg, Germany): SX1/FX1</td><td>Inhalant allergen sources:<ul><li>Outdoor: birch, timothy grass, mugwort</li><li>Indoor: cat, dog, mold (Cladosporium herbarum), house dust mite (Der p)</li></ul>Food allergen sources: cow's milk, egg white, soy bean, peanut, cod fish, rye, and wheat</td></tr></tbody></table>

Der p, Dermatophagoides pteronyssinus.

within the European Study of Cohorts for Air Pollution Effects (ESCAPE) project (http://www.escapeproject.eu).¹⁹ Furthermore, the availability of MeDALL microarray data on IgE against 132 allergen molecules²⁰ in 2 of the cohorts (Children, Allergy, Milieu, Stockholm, Epidemiology [BAMSE] and German Infant Study on the Influence of Nutrition Intervention PLUS Environmental and Genetic Influences on Allergy Development [GINIplus]) provided us for the first time with a unique opportunity to investigate associations of ambient air pollution with sensitization to defined inhalant outdoor and indoor allergen molecules, as well as to food and venom allergen molecules. Finally, we investigated the importance of timing of long-term exposure to air pollution by utilizing information on exposures early in life and at the time of later biosampling.

## METHODS

The Methods section in this article's Online Repository (available at jacionline.org) provides additional details on the study populations, assessment of exposure to air pollution, IgE microarray measurement, and statistical analyses used in this study.

### Study populations

The current study included data from the following 4 European birth cohorts participating in the MeDALL project: BAMSE (Sweden), Prevention and Incidence of Asthma and Mite Allergy (PIAMA [The Netherlands]), Influences of Lifestyle-Related Factors on the Human Immune System and Development of Allergies in Childhood (LISA [Germany]), and GINIplus (Germany). Participants were recruited from 1994 to 1999. Detailed descriptions of study design, enrollment, and procedures for data collection in each cohort have been published elsewhere.²¹⁻²³

### Air pollution exposure assessment

Annual mean concentrations of ambient particulate matter with an aerodynamic diameter less than 2.5 µm (PM<sub>2.5</sub>) or 10 µm (PM<sub>10</sub>), coarse particulate matter (PM<sub>2.5-10</sub>), PM<sub>2.5</sub> absorbance (an indicator for black carbon particulate matter content), nitrogen dioxide (NO<sub>2</sub>), and nitrogen oxides (NO<sub>x</sub>) at the residential addresses of the study participants were estimated through land use regression (LUR) models developed for each study area within the framework of the ESCAPE project; they have been extensively described elsewhere,²⁴,²⁵ as well as in the Online Repository.

## Measurement of IgE sensitization

At the age of 4 years (6 years for the LISA/GINI cohorts), 8 years (10 years for the LISA/GINI cohorts), and 16 years (15 years for the LISA/GINI cohorts) children were invited for clinical examinations including biosampling. Blood samples were drawn for the analysis of IgE sensitization by measuring specific serum IgE levels against panels of common inhalant and food allergen sources (natural allergen extracts) by using the ImmunoCAP System (Thermo Fisher/Phadia AB, Uppsala, Sweden) or equivalent test systems (ie, a radioallergosorbent test-like method used at the Sanquin Laboratories [Amsterdam, The Netherlands] and the CAP-RAST FEIA [Pharmacia Diagnostics, Freiburg, Germany]). The range of tested inhalant allergen sources was comparable across the studies and included cat, dog, mold, house dust mite, birch pollen, and grass pollen for all cohorts, as well as mugwort for all cohorts except PIAMA (Table I). The panel of food allergen sources includes cow's milk and egg for all cohorts and soy bean, peanut, cod fish, and wheat, in all cohorts except PIAMA (milk and egg only). Details regarding the set of specific allergens tested in each cohort are given in Table I. Sensitization was defined as an IgE antibody level of at least 0.35 kU<sub>A</sub>/L to any of the tested allergen extracts.

In addition, IgE sensitization to 132 allergen molecules corresponding to 51 allergen sources was analyzed in BAMSE at the age of 16 years (n = 743), as well as in GINIplus at the age of 15 years (n = 278); the analysis was performed with an allergen chip based on Immuno Solid-phase Allergen Chip (ISAC [Thermo Fisher]) standardized units for detection of IgE antibodies technology (ISU-E), which was developed within the MeDALL project.²⁶ Serum aliquots of 30 µL were incubated on the microarray for 2 hours at room temperature, and the slides were washed and then incubated with fluorescence-labeled anti-IgE antibodies (Thermo Fisher) for 30 minutes. The chips were then washed, dried, and analyzed with a Laser Scan Confocal microarray reader (LuxScan 10K/A [Capital-Bio, Beijing, China]). A complete list of included molecules and the prevalence of IgE sensitization against those in the 2 cohorts are presented in Table E1 (available in the Online Repository at www.jacionline.org). The cutoff for IgE detection was set at 0.3 ISU-E.

### Statistical analyses

Associations of exposure to outdoor air pollution with prevalence of sensitization until 16 years of age were assessed in each cohort separately by using generalized estimating equation models with an unstructured correlation structure to account for correlations between repeated observations within the same subject. Separate analyses were conducted with exposure at the birth address and at the address at which the participant lived at the time of biosampling. The models incorporated interaction terms between time
<figure><img src="image_5.png" /><figcaption>FIG 1. Air pollution exposure at birth address in 4 European birth cohorts. Each box contains the middle 50% of the data, with the right edge (hinge) of the box indicating the 75th percentile and the left edge indicating the 25th percentile (interquartile range). The line in the box represents the median. The ends of the horizontal lines ("whiskers") indicate 1.5 × IQR.</figcaption></figure>

indicator variable (age) and exposure to evaluate age-specific effects of exposure.

Adjusted cohort-specific odds ratios (ORs) and 95% CIs were then meta-analyzed by using a random effects model,²⁷ taking into account possible nonrandom variability within and between cohorts. Statistical heterogeneity among studies was evaluated by using I² statistics.²⁸ A P value less than .05 was considered significant. The results of microarray-wide analyses were adjusted for multiple testing by using Bonferroni correction applied to 132 tests.²⁹ All analyses were performed with STATA software, release 13.1 (StataCorp, College Station, Tex).

## RESULTS

The population of the present study comprises individuals with available data on repeated IgE measurements throughout childhood and adolescence and air pollution exposure at birth (n = 6163) or at the time of biosampling (n = 5771). A short description of selected characteristics related to the enrolment and follow-ups of the included cohorts is provided in Table E2 (available in the Online Repository at www.jacionline.org). The distribution of potential risk factors at the ages of 4 to 6, 8 to 10, and 15 to 16 years in each cohort is presented in Tables E3 to E5, respectively (available in the Online Repository at www.jacionline.org). Notably, in the subset of children with IgE measurements in the BAMSE cohort, the proportion of

children with allergic heredity was lower than in the other cohorts. This is partly explained by differences in study designs between the cohorts. Fig 1 presents the distribution of air pollution exposure concentrations at the participants' birth addresses. For most pollutants, the levels were lower for the Stockholm County area, which is where the BAMSE cohort is based. Median air pollution levels ranged from 12.4 µg/m³ (BAMSE) to 23.2 µg/m³ (LISA/GINI North) for NO₂ and from 8.1 µg/m³ (BAMSE) to 17.2 µg/m³ (LISA/GINI North) for PM₂.₅, whereas the concentrations of PMcoarse were largely comparable between the cohorts. The levels of air pollution at home addresses at the time of respective biosampling are summarized in Table E6 (available in the Online Repository at www.jacionline.org). There were no major changes in estimated air pollution levels at the addresses at different ages. Although more than half of the study subjects moved at least once during follow-up, exposure levels at the birth address and at the follow-up addresses were moderately to highly correlated (see Fig E1 in the Online Repository at www.jacionline.org). The strongest correlations were observed for the LISA/GINI North cohort (r = 0.59-0.77 for the 15-year addresses) and lowest for the BAMSE cohort (r = 0.35-0.45 for the 16-year addresses).

In the 4 cohorts, the prevalence of sensitization to a combination of common inhalant and food allergen extracts
A

NO₂

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C

PM₂.₅

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E

PM₁₀

<figure><img src="image_8.png" /></figure>

B

NOₓ

<figure><img src="image_9.png" /></figure>

D

PM₂.₅bs

<figure><img src="image_10.png" /></figure>

F

PMcoarse

<figure><img src="image_11.png" /></figure>

FIG 2. Cohort-specific and combined associations of air pollution exposure at birth with IgE sensitization to any common inhalant and/or food allergen extract up to 16 years of age in 4 European birth cohorts (n = 6163): NO₂ (A), NOₓ (B), PM₂.₅ (C), PM₂.₅ absorbance (D), PM₁₀ (E), and PMcoarse (F). Adjusted for sex, maternal smoking during pregnancy, anyone smoking at the child's home, breast-feeding, atopic parents, parental education, mold at home, furred pets at home, older siblings, gas cooking, study arm (GINIplus cohort), and region (for the BAMSE cohort only). Combined ORs and 95% CIs have been derived by random effects methods. Effects are presented for increments of 10 µg/m³ (NO₂, PM₁₀), 1×10⁻⁵/m (PM₂.₅ absorbance), 5 µg/m³ (PM₂.₅, PMcoarse), and 20 µg/m³ (NOₓ).
<table><caption>TABLE II. Meta-analyses of the associations between air pollution exposure at birth or at the time of biosampling and IgE sensitization to selected pollen allergen extracts up to age 16 years in 4 European birth cohorts</caption><thead><tr><th rowspan="2">Air pollution indicator</th><th colspan="3">Birch pollen extract</th><th colspan="3">Timothy grass pollen extract</th></tr><tr><th>OR</th><th colspan="2">95% CI</th><th>OR</th><th colspan="2">95% CI</th></tr></thead><tbody><tr><td>At birth</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cases (no./total no.)</td><td colspan="3">1494 of 6163</td><td colspan="3">1965 of 6163</td></tr><tr><td>NO<sub>2</sub></td><td>0.99</td><td>0.83</td><td>1.18</td><td>0.99</td><td>0.86</td><td>1.15</td></tr><tr><td>NO<sub>x</sub></td><td>0.98</td><td>0.84</td><td>1.15</td><td>1.00</td><td>0.87</td><td>1.15</td></tr><tr><td>PM<sub>2.5</sub></td><td>1.25</td><td>0.79</td><td>1.97</td><td>1.20</td><td>0.88</td><td>1.63</td></tr><tr><td>PM<sub>2.5</sub> absorbance</td><td>0.97</td><td>0.66</td><td>1.42</td><td>0.94</td><td>0.71</td><td>1.25</td></tr><tr><td>PM<sub>10</sub></td><td>0.97</td><td>0.77</td><td>1.22</td><td>1.03</td><td>0.83</td><td>1.27</td></tr><tr><td>PM coarse</td><td>0.89</td><td>0.62</td><td>1.30</td><td>0.94</td><td>0.73</td><td>1.20</td></tr><tr><td>At the time of biosampling</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cases (no./total no.)</td><td colspan="3">1409 of 5771</td><td colspan="3">1842 of 5771</td></tr><tr><td>NO<sub>2</sub></td><td><strong>1.12</strong></td><td><strong>1.01</strong></td><td><strong>1.25</strong></td><td>0.99</td><td>0.88</td><td>1.10</td></tr><tr><td>NO<sub>x</sub></td><td>1.09</td><td>0.99</td><td>1.20</td><td>0.99</td><td>0.89</td><td>1.11</td></tr><tr><td>PM<sub>2.5</sub></td><td>1.21</td><td>0.98</td><td>1.49</td><td>1.25</td><td>0.87</td><td>1.82</td></tr><tr><td>PM<sub>2.5</sub> absorbance</td><td>1.07</td><td>0.83</td><td>1.38</td><td>1.03</td><td>0.78</td><td>1.36</td></tr><tr><td>PM<sub>10</sub></td><td><strong>1.24</strong></td><td><strong>1.03</strong></td><td><strong>1.50</strong></td><td>1.11</td><td>0.85</td><td>1.44</td></tr><tr><td>PM coarse</td><td><strong>1.23</strong></td><td><strong>1.07</strong></td><td><strong>1.40</strong></td><td>1.05</td><td>0.94</td><td>1.18</td></tr></tbody></table>

Effects are presented for increments of 10 µg/m³ (NO₂, PM₁₀), 1×10⁻⁵/m (PM₂.₅ absorbance), 5 µg/m³ (PM₂.₅, PMcoarse), and 20 µg/m³ (NOₓ). Adjusted for sex, maternal smoking during pregnancy, anyone smoking at the child's home, breast-feeding, atopic parents, parental education, mold at home, furred pet at home, older siblings, gas cooking, study arm (for GINIplus cohort), and region (for the BAMSE cohort only). Boldface indicates statistical significance.

ranged from 24.1% to 40.4% at the age of 4 to 6 years (the lowest rates were in the BAMSE cohort and the highest in the PIAMA cohort), from 34.8% to 47.9% at the age of 8 to 10 years (in the BAMSE and LISA/GINI South cohorts, respectively), and from 41.8% to 51.2% at the age of 15 to 16 years (in the LISA/GINI North and LISA/GINI South cohorts, respectively), as shown in Table E7 (available in the Online Repository at www.jacionline.org).

Cohort-specific and combined overall associations of air pollution exposure at birth and at the time of biosampling and IgE sensitization to mixes of common inhalant and/or food allergen extracts up to 15 to 16 years are shown in Fig 2 and Fig E2 (available in the Online Repository at www.jacionline.org), respectively. The heterogeneity between cohort-specific effect estimates was generally moderate ($I^2$ = 0-72%). In general, no consistent evidence was found for either exposure at birth or at the time of biosampling to be associated with sensitization to any common allergen up to 15 or 16 year of age. In the cohort-specific analyses, we observed statistically significant positive associations with several air pollutants in the PIAMA cohort, but not in the other included cohorts. The combined adjusted meta-analysis ORs for air pollution exposure at birth ranged from an OR of 0.99 (95% CI = 0.83-1.17) for a 10-µg/m³ increase in PM₁₀ exposure to an OR of 1.32 (95% CI = 0.95-1.84) for a 5-µg/m³ increase in PM₂.₅ exposure. Similar results were obtained in relation to air pollution exposure at the time of biosampling (see Fig E2). The results of the fully adjusted analyses were largely comparable to those based on the basic model (see Table E8 in the Online Repository at www.jacionline.org). Further, increasing cutoffs for IgE positivity to any of the tested inhalant and/or food allergen extracts (ie, ≥0.70 kUₐ/L and ≥3.5 kUₐ/L) had no major impact on the overall results (see Table E9 in the Online Repository at www.jacionline.org). On the basis of analyses with exposure-age interaction terms, we did not find any indication of differences in age-specific associations with either exposure at birth or exposure at the time of biosampling (see Table E10 in the Online Repository at www.jacionline.org).

When we distinguished between sensitization against mixes of food and inhalant allergen extracts, there was a general trend of higher risk estimates for food allergens in relation to all studied pollutants, although the corresponding CIs largely overlapped (see Tables E11 and E12 in the Online Repository at www.jacionline.org). However, by looking separately at associations with sensitization against single-allergen extracts we observed statistically significantly higher odds of sensitization against birch pollen extract for exposure at the time of biosampling to several markers of air pollution (adjusted meta-analysis OR = 1.12 [95% CI = 1.01-1.25] for a 10-µg/m³ increase in NO₂ exposure; adjusted meta-analysis OR = 1.24 [95% CI = 1.03-1.50] for a 10-µg/m³ increase in PM₁₀ exposure; and adjusted meta-analysis OR = 1.23 [95% CI = 1.07-1.40] for a 5-µg/m³ increase in PMcoarse exposure [Table II and see Table E13 in the Online Repository at jacionline.org]). No consistent association was found with sensitization against timothy grass pollen extract (see Table E14 in the Online Repository at www.jacionline.org).

Further, we evaluated the association of air pollution exposure with IgE sensitization to microarrayed allergen molecules at the age of 15 to 16 years as determined with the MeDALL chip. In the initial discovery microarray-wide association meta-analysis based on the BAMSE and GINIplus cohorts, no allergen molecule appeared to be significantly associated with air pollution exposure after multiple testing corrections (data not shown). In the candidate analysis, which was limited to 4 “risk molecules” previously linked to the risk of respiratory allergy,²⁰ we observed positive associations of sensitization against these molecules with PM₂.₅ exposure at birth address that did not reach the level of statistical significance (see Table E15 in the Online Repository at www.jacionline.org). However, when we defined IgE sensitization to allergen molecules based on a higher cutoff of more than 3.5 ISU-E, statistically significant positive associations of air pollution exposure at the birth address with sensitization against Phleum pratense 1 (Phl p 1) and Felis domesticus 1 (Fel d 1) were seen (adjusted meta-analysis OR = 3.33 [95% CI: 1.40-7.94] and adjusted meta-analysis OR = 4.98 [1.59-15.60],
<table><caption>TABLE III. Meta-analyses of the associations of air pollution exposure at birth or at the time of biosampling with levels of identified risk molecules (>3.5 ISU-E) measured at age 15 to 16 years in the BAMSE and GINIplus cohorts</caption><thead><tr><th rowspan="2">Air pollution indicator</th><th colspan="2">Bet v 1 (birch)</th><th colspan="2">Phl p 1 (grass)</th><th colspan="2">Fel d 1 (cat)</th></tr><tr><th>OR</th><th>95% CI</th><th>OR</th><th>95% CI</th><th>OR</th><th>95% CI</th></tr></thead><tbody><tr><td colspan="7"><strong>At birth</strong></td></tr><tr><td>Cases (no./total no.)</td><td colspan="2">176 of 1021</td><td colspan="2">203 of 1021</td><td colspan="2">95 of 1021</td></tr><tr><td>NO<sub>2</sub></td><td>1.14</td><td>0.22</td><td>5.83</td><td>1.20</td><td>0.58</td><td>2.49</td><td>1.19</td><td>0.46</td><td>3.07</td></tr><tr><td>NO<sub>x</sub></td><td>0.84</td><td>0.24</td><td>2.96</td><td>1.10</td><td>0.67</td><td>1.80</td><td>1.00</td><td>0.57</td><td>1.76</td></tr><tr><td>PM<sub>2.5</sub></td><td>1.96</td><td>0.37</td><td>10.36</td><td><strong>3.33</strong></td><td><strong>1.40</strong></td><td><strong>7.94</strong></td><td><strong>4.98</strong></td><td><strong>1.59</strong></td><td><strong>15.60</strong></td></tr><tr><td>PM<sub>2.5</sub> absorbance</td><td>0.80</td><td>0.003</td><td>234.25</td><td>2.05</td><td>0.17</td><td>24.08</td><td>3.01</td><td>0.12</td><td>73.18</td></tr><tr><td>PM<sub>10</sub></td><td>0.86</td><td>0.30</td><td>2.43</td><td>1.27</td><td>0.73</td><td>2.20</td><td>1.36</td><td>0.64</td><td>2.87</td></tr><tr><td>PM coarse</td><td>0.37</td><td>0.03</td><td>4.22</td><td>0.82</td><td>0.34</td><td>1.94</td><td>0.84</td><td>0.32</td><td>2.23</td></tr><tr><td colspan="7"><strong>At time of biosampling</strong></td></tr><tr><td>Cases (no./total no.)</td><td colspan="2">165 of 933</td><td colspan="2">190 of 933</td><td colspan="2">90 of 933</td></tr><tr><td>NO<sub>2</sub></td><td>0.64</td><td>0.24</td><td>1.72</td><td>0.84</td><td>0.59</td><td>1.18</td><td>0.81</td><td>0.51</td><td>1.30</td></tr><tr><td>NO<sub>x</sub></td><td>0.56</td><td>0.16</td><td>2.03</td><td>0.91</td><td>0.67</td><td>1.23</td><td>0.85</td><td>0.56</td><td>1.30</td></tr><tr><td>PM<sub>2.5</sub></td><td>0.90</td><td>0.35</td><td>2.26</td><td>1.15</td><td>0.52</td><td>2.55</td><td>1.43</td><td>0.30</td><td>6.87</td></tr><tr><td>PM<sub>2.5</sub> absorbance</td><td>0.51</td><td>0.08</td><td>3.28</td><td>0.90</td><td>0.29</td><td>2.81</td><td>1.04</td><td>0.28</td><td>3.93</td></tr><tr><td>PM<sub>10</sub></td><td>0.55</td><td>0.11</td><td>2.66</td><td>0.87</td><td>0.53</td><td>1.42</td><td>1.05</td><td>0.56</td><td>1.95</td></tr><tr><td>PM coarse</td><td>0.44</td><td>0.06</td><td>3.35</td><td>0.93</td><td>0.65</td><td>1.31</td><td>0.84</td><td>0.34</td><td>2.11</td></tr></tbody></table>

Adjusted for sex, maternal smoking during pregnancy, anyone smoking at the child's home, breast-feeding, atopic parents, parental education, mold at home, furred pets at home, older siblings, gas cooking, study arm (GINIplus cohort), and region (for the BAMSE cohort only). Combined ORs and 95% CIs derived by random effects methods. Effects are presented for increments of 10 µg/m³ (NO₂, PM₁₀), 1×10⁻⁵/m (PM₂.₅ absorbance), 5 µg/m³ (PM₂.₅, PMcoarse), and 20 µg/m³ (NOₓ). Boldface indicates statistical significance.

respectively, for a-5 µg/m³ increase in PM₂.₅ exposure [Table III and see Table E16 in the Online Repository at jacionline.org]). No similar associations with exposure at the time of biosampling were detected. We were not able to run corresponding analysis with sensitization to the fourth risk molecule, the major peanut allergen molecule *Arachis hypogea* 1, because of its low prevalence in the present study sample.

## DISCUSSION

The present study constitutes a major extension of our previous collaborative project to examine the impact of outdoor air pollution exposure on the prevalence of allergic sensitization to common inhalant and/or food allergens in children and adolescents who were followed from birth until 16 years of age. The combined results from the 4 birth cohort studies indicated that air pollution exposure was generally not associated with IgE sensitization. However, analyses based on specific IgE to allergen extracts suggest increased risks of sensitization to birch in relation to several air pollution indicators. Further, higher air pollution exposure at birth address appeared to be associated with elevated levels of the grass allergen molecule Phl p 1, as well as with the cat allergen molecule Fel d 1 in a subset of the study population with available data on IgE sensitization against allergen molecules.

We observed no overall association between exposure to the studied air pollution components and any allergic sensitization up to adolescent age, which is in line with our previous meta-analysis based on the same cohorts exploiting data from 4- and 8-year follow-ups.¹⁴ Similarly, recent longitudinal analysis of LISA/GINI cohorts followed for 10 years did not find consistent evidence that air pollution exposure is associated with a higher risk of sensitization in later childhood.¹³ In the analyses based on specific IgE to allergen extracts, however, we found higher risks of sensitization to birch pollen in relation to exposure at the time of biosampling. Even though the observed associations appeared to be sensitive to multiple testing adjustment, we would like to acknowledge a consistent direction of positive association

of sensitization against birch pollen extracts with estimated exposures at the time of biosampling to all considered pollutants, supporting the biologic plausibility of such associations. Notably, the strength of the observed association was moderate (eg, for sensitization to birch allergen extract, OR = 1.12 [95% CI = 1.01-1.25] per 10-µg/m³ increase in NO₂ exposure); however, because air pollution exposures affect large parts of the population, even a moderately increased risk estimate may lead to a high number of extra cases resulting from this particular exposure. Some of the earlier published cohort-specific results indicated that air pollution exposure was related to pollen sensitization at age 4 to 6 years in the BAMSE cohort (OR = 1.83 [95% CI = 1.02-3.28] per 46.7-µg/m³ increase in NOₓ exposure during infancy)⁸ and LISA/GINI cohorts (OR = 1.45 [95% CI = 1.21-1.74] per 1.5-µg/m³ increase in PM₂.₅ exposure),³⁰ whereas in the PIAMA cohort associations between air pollution exposure to PM₂.₅, soot, and NO₂ at birth addresses and specific sensitization were limited to food allergens.¹¹ In contrast, no association between long-term air pollution exposure and sensitization to any allergen was seen in the PIAMA cohort at the age of 8 years,¹² or in 9- to 10-year-old children in Oslo, Norway.¹⁰ Another study by Janssen et al also found a positive association between NO₂ and sensitization to inhalant allergens (OR = 1.70 [95% CI = 1.03-2.81] per 17.6-µg/m³ increase in NO₂) among Dutch children 7 to 12 years of age.³¹

There is limited evidence on the association between air pollution exposure and allergic sensitization in children beyond primary school age. Recent longitudinal analysis of LISA/GINI cohorts followed for 10 years did not find consistent evidence that air pollution exposure is associated with a higher risk of sensitization in later childhood.¹³ One study from the United States demonstrated that prenatal air pollution exposure was associated higher total IgE measured at early adolescent age (median age of 12 years).³² However, this study considered only total IgE data without investigating specific IgE to allergen extracts.
We found a statistically significant positive association of air pollution exposure at birth address with sensitization at the age of 16 years against the major grass allergen Phl p 1, as well as against the major cat allergen molecule Fel d 1, which have been recognized as the allergen molecules capable of predicting development of respiratory allergies at later ages.²⁰,³³ As no previous study has examined IgE sensitization using allergen molecules in relation to air pollution exposure, our results represent a novel finding. It should also be noted that these analyses were limited to a subsample of individuals from 2 participating cohorts and that this observation needs to be confirmed in future larger studies.

It has been suggested that air pollution may increase sensitivity of the airway epithelium to inhalant allergens. Our findings of elevated risk for sensitization against pollen allergens are also consistent with experimental data demonstrating that inhalation of traffic-related air pollutants enhances the immune responses to airborne allergens, such as pollens.⁶,³⁴ It has been shown that certain pollen allergens such as the major birch pollen allergen *Betula verrucosa* 1 and the major grass pollen allergen *Lolium perenne* 1 can bind to respirable small particles, which may enhance the induction of respiratory allergy.³⁵,³⁶ However, pollutants may also contribute to increased allergic sensitization by other mechanisms. For example, it has been demonstrated that air pollution exposure strongly upregulates the expression of grass pollen allergens and may thus increase allergen loads during the pollen season.³⁷ The finding that nitration of the major birch pollen allergen *Betula verrucosa* 1 enhances its immunogenicity and allergenic potential provides yet another possible mechanism by which pollution may increase allergic sensitization.³⁸ This possibility is further supported by the altered morphology of pollen in polluted areas.³⁹

Our study has several strengths. To our knowledge, it is one of the first to assess effects of air pollution exposure on the development of allergic sensitization throughout childhood up to adolescent age based on combined analyses of prospective birth cohort studies with a total sample size of more than 6000 individuals and use of a meta-analytic approach. The 4 birth cohorts included in this study were specifically designed to examine the development of allergic diseases during childhood. Participation in the European collaborative projects ESCAPE and MeDALL provided a unique opportunity to utilize standardized individual-level exposure data generated from dedicated monitoring campaigns and area-specific LUR modeling, along with repeatedly measured outcome and confounder data harmonized across all the included cohorts. In addition, high-throughput MeDALL microarray measurements in 2 of the participating cohorts enabled investigation of IgE sensitization against allergen molecules in relation to air pollution exposure.

We assigned the exposure levels to the birth addresses and the addresses at which children resided at the time of biosampling to investigate potential critical time periods of susceptibility to air pollution. One limitation related to exposure assessment is that we used exposure models based on air pollution measurement campaigns from 2008-2010 to assess exposure to air pollution for the entire duration of follow-up. Although the measurement campaigns concurred with the most recent follow-ups of the cohorts, it may not be optimal for estimation of historical exposures. However, we have extensively investigated this issue in our previous publications based on the same data by performing sensitivity analyses using modeled concentrations of

considered pollutants that were extrapolated back in time, and this did not have any influence on the results.³,¹⁴,⁴⁰ Furthermore, several validation studies have investigated the rationale of using LUR models developed from current data to estimate exposure back in time by comparing modeled and measured data. For instance, a Dutch study demonstrated that a LUR model developed from 2007 data could explain almost 80% of the variability in NO₂ concentrations measured in 1999-2000.⁴¹ Similar observations were made in other geographic areas (ie, Vancouver, Rome, and Oslo),⁴²⁻⁴⁴ thus justifying the use of LUR models developed from 2008-2010 measurement data as valid tools to assess the spatial variation of air pollution levels at earlier time points. By using spatial LUR we did not take into account long-term trends in air pollution concentrations. Previous studies have shown decreasing trends for several air pollutants, including NO₂ and PM₂.₅ in the Stockholm area (BAMSE cohort) during the period from 1999 to 2009, but not in the other study areas.⁴⁵ This change might have biased associations with recent air pollution exposures, but it is less of a concern for analyses with early life exposures. Further potential limitation of the exposure assessment is that LUR models were applied only to the residential addresses, which may lead to exposure misclassification, as children spend part of their time at day care and school. However, previous studies have demonstrated high correlation between estimated air pollution exposure levels based on residential addresses only and those accounting for multiple locations, possibly on account of the fact that day care centers and primary schools are often located within the same neighborhood.⁸,⁴⁶ Further, the modeled individual concentrations account only for outdoor air pollution and, therefore, may not fully reflect personal exposure. A recent study in The Netherlands, Finland, and Spain compared pollutant estimates from the ESCAPE LUR models against measurements from personal monitors, showing good agreement between modeled and measured concentrations of PM₂.₅ absorbance (coefficient of determination r² = 0.83), NO₂ (r² = 0.79), and NOₓ (r² = 0.54).⁴⁷ Although some misclassification of true individual exposure may affect our results, the assessments of both exposure and disease were done independently from one another, thus making potential misclassification likely to be nondifferential. Further, we have adjusted for a wide set of potential confounders; however, as in most epidemiologic studies, the possibility of residual confounding cannot be ruled out. Unfortunately, we do not have empiric data on pollen counts to check potential differences in pollen exposure levels between the specific study areas.

In conclusion, the results of this study based on the data from 4 birth cohorts did not provide consistent evidence of an association between air pollution exposure and overall allergic sensitization in children up to 16 years of age. However, analyses of specific IgE to allergen extracts suggest higher risks of sensitization to birch pollen in relation to several air pollution indicators, as well as to the grass allergen molecule Phl p 1 and the cat allergen molecule Fel d 1, defined by a higher threshold of more than 3.5 ISU-E, in relation to PM₂.₅ exposure at birth.

We would like to thank the families who participated in the included studies and the staff for their hard work and effort. We additionally acknowledge Alexandra Ek, Sandra Ekström, Niklas Andersson, André Lauber (the BAMSE study) and Marjan Tewis (the PIAMA study) for excellent data management.
## Key messages

* The associations of residential exposure to air pollution with IgE sensitization to common inhalant and food allergens were studied in combined analyses of 4 European birth cohorts with a standardized exposure assessment following a common protocol.

* Overall, the analysis results did not provide clear evidence of an association between air pollution exposure and development of IgE sensitization to common inhalant or food allergens in children up to 16 years of age.

* However, analyses based on specific IgE to allergens extracts suggested increased odds of sensitization to birch, as well as to the grass allergen component molecule Phl p 1 and the cat allergen Fel d 1 (defined by a higher threshold of more than 3.5 ISU-E) in relation to air pollution.

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