Effects of early‐life exposure to dust mite allergen and endotoxin on the development of asthma and wheezing: The Japan Environment and Children's Study

Abstract Background The effects of early‐life exposure to house dust mite allergen and endotoxin on the development of asthma are unclear in the literature. We investigated the association of early‐life exposure (0–36 months old) to house dust mite allergen and endotoxin with asthma incidence. Methods In this novel, large‐scale, nationwide birth cohort study, 5017 participants were randomly selected from those who met the eligibility criteria. House dust was vacuum‐sampled from the children's mattresses within homes and assayed for the presence of dust mite allergen (Der 1) and endotoxin. The participants were classified into four quartiles (Q1–Q4) according to exposure levels. We defined the incidence of asthma and wheezing using questionnaires at 12, 24, and 36 months old. Odds ratios (ORs) of the incidence of asthma and wheezing by age in Der 1 and endotoxin exposure level were estimated using logistic regression. Results The cumulative incidence rates of asthma and wheezing during 0–36 months were 10.4% and 38.1%, respectively. Significant ORs were observed in asthma onset during 12–24 months old, asthma onset during 24–36 months old, and wheezing onset during 0–12 months old in the Q4 Der 1 group. In the Q4 endotoxin group, significant positive associations between endotoxin exposure and asthma (OR 2.00, 95% confidence interval [CI]: 1.03–3.85) and wheezing (OR 1.78, 95% CI: 1.01–3.12) onset during 24–36 months old were found. Conclusions Our results indicated that high levels of early‐life exposure to Der 1 and endotoxin in mattresses may be involved in the development of asthma.


Conclusions:
Our results indicated that high levels of early-life exposure to Der 1 and endotoxin in mattresses may be involved in the development of asthma.

K E Y W O R D S
asthma, dust mite, early life, endotoxin, wheeze

| INTRODUCTION
The prevalence of paediatric allergic diseases has recently increased in developed countries. Adoption of Western lifestyle was noted as a contributing factor, but it has not been sufficiently verified. 1,2 Western lifestyle is characterised by an increase in the time spent indoors, highly insulated airtight houses, and a more hygienic environment; in addition, it may also be characterised by changes in the pattern of exposure to house dust mite allergen and microorganisms that influence the prevalence of paediatric allergic diseases. 2 There is sufficient scientific evidence for dust mite allergen as a factor aggravating asthma symptoms, but there is no evidence that it is a risk factor for asthma incidence. 3,4 A European large-scale birth cohort study showed that exposure to a high concentration of allergens immediately after birth influences the development of allergic sensitisation. 5,6 Moreover, exposure to dust mite allergens is associated with dust mite sensitisation. 7 However, intervention studies reported no associations between dust mite exposure in infancy and asthma onset at 3 8 or 8 years of age. 9 Previous studies suggest no consistency in the association between endotoxin, a cell wall component of gram-negative rods and microorganism exposure marker, and asthma development.
Braun-Fahrländer et al. reported that the endotoxin concentrations in mattress dust were higher in children from farming household than in those from non-farming household, and significantly fewer children of farmers had hay fever, asthma, and atopic sensitisation. 10 Mendy et al. reported a positive correlation between the endotoxin concentration in dust (endotoxin concentration per gram of dust) and wheezing in infants and toddlers (0-3 years old) but an inverse correlation with asthma after they reached school age. 11 Furthermore, recent studies in adult Americans revealed that the influence on allergic disease differs due to climate and lifestyle because of differences in exposure to endotoxin and indoor allergens such as mites and moulds. 12,13 Therefore, it is necessary to clarify the association of exposure to dust mite allergen and endotoxin with asthma development in Asian regions under different climatic regions and lifestyles in comparison with that in Western countries. Although large-scale birth cohort studies have been conducted in Western countries, there are few reports from Asia, including Japan.
We conducted a large-scale birth cohort study on the effects of early-life exposure to dust mite allergen and endotoxin in mattresses on asthma and wheezing development by 0-36 months old in Japan.

| Participants
This is a sub-cohort study of the Japan Environment and Children's Study (JECS). The study design and recruitment methods have been described in detail previously. 14,15 In brief, JECS is a large-scale birth cohort study involving 100,000 pairs of parents and children in 15 nationwide regions in Japan, wherein participants were observed from the foetal period to the age of 13 years old. Participants were recruited between January 2011 and March 2014; 103,060 pregnancies were registered, of which 100,304 were live births. For the sub-cohort study, 5017 participants were randomly selected from those who met the eligibility criteria and consented to participate in the sub-cohort study. 16 Data for this study were used from the dataset released in October 2019 (dataset jecs-ta-20190930). A schematic summarising the sample selection methodology is shown in

| Questionnaire
Questionnaires were sent to the participants by post at the age of 12, 18, 24 and 36 months old, and the parents filled them out and sent them back. Those questionnaires were a modified version of ISAAC for children aged 6-7 years, with translation validation in Japanese. [17][18][19] Okabe et al. investigated the validity of asthma symptoms based on ISAAC questionnaires involving 0-60-monthold infants by using asthma diagnosis made by allergists as criteria, 20 wherein the sensitivity and specificity of asthma symptoms were 0.91 and 0.64 in 0-24-month-old children and 0.88 and 0.68 in 36-60-month-old children, respectively. In our study, asthma and wheezing were defined according to the definition by Okabe et al. 20,21 The incidence rates of wheezing and asthma by age (0-12, 12-24, and 24-36 months old) were defined as a ratio of participants initially judged as having wheezing/asthma at each age compared to the that of all participants.
Information on lower respiratory tract inflammation and respiratory syncytial virus (RSV) infection was obtained from the questionnaires at the age of 12, 18, and 24 months. Information concerning the presence of indoor pets and passive smoke after birth was obtained from the questionnaires at 18 months old.

| Blood and urinal examination of mothers
Blood samples were obtained from mothers during the first trimester of pregnancy. Total serum IgE titres, assayed using ImmunoCAP (Thermo Fisher Scientific, Inc.), were analysed by an independent clinical laboratory. Mothers with high levels of total serum IgE were defined as those with a total IgE level of 214 IU/ml or above. 22 Maternal spot urine was collected at mid-pregnancy, and the urinary cotinine level was measured. Sample collection and measuring methods were described in detail previously. 23 The cut-off values for active smoking and passive smoking during pregnancy were set at 36.8 and 0.31 μg/g creatinine, respectively. 23

| Blood examination of children
The mothers and children visited the test site when their children were 24 months old; blood samples were obtained from children. Total serum IgE titres, assayed using ImmunoCAP (Thermo Fisher Scientific, Inc.,), were analysed by an independent clinical laboratory. As the cutoff value of high levels of total serum IgE in 24-month-old children is unclear, it was set at the upper 10th percentile of the total IgE level.

| Exposure assessment (mite allergen/ endotoxin)
Trained investigators visited homes when the children were 18 months old and collected house dust from mattresses on which the children slept. A 50-cm � 100-cm frame exclusive for sampling was placed on the mattress, and a vacuum cleaner (Model DC61, Dyson, Japan) was run over the area in the frame for 2 min ( Figure S1). The collected dust was frozen until analysis.
Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1) in the dust were measured using an enzyme-linked immunosorbent assay kit (Indoor Biotechnologies Ltd). Endotoxin levels in the dust were measured by kinetic chromogenic limulus amoebocyte lysate assays (Kinetic-QCL), obtained from Lonza Japan.
There is high structural similarity in the corresponding allergen between Der p 1 and Der f 1. Once sensitised, there is strong crossreactivity even when sensitised with either type. 24 Thus, we used the total exposure to both mite species Der 1 (Der p 1 + Der f 1) in the analysis of the association with health outcomes.

F I G U R E 1 Flowchart of the selection of study participants
HASUNUMA ET AL.

| Statistical analysis
Dust mite allergen and endotoxin concentrations were expressed as 'amount per m 2 of dust sampling area' and 'amount per unit mass of dust', 25 and Spearman's rank correlation coefficient was calculated for these exposure indices. The correlation coefficient for the 'amount per gram of dust' and 'amount per m 2 dust sampling area' of Der 1 was 0.86, and that in endotoxin between the 'amount per mg dust' and 'amount per m 2 dust sampling area' was 0.79, demonstrating a high correlation between these exposure indices. Thus, the 'amount per m 2 dust sampling area' was analysed in the main analysis thereafter. Analysis of the 'amount per unit mass of dust' was performed as a sensitivity analysis. If the data were lower than those of the method detection limit, then a value of one-half was assigned. In the analysis of the association with health outcomes, the participants were classified into the first-to-fourth quartile (Q1-Q4) groups.
Regarding the association between the dust mite allergen or endotoxin exposure level and health outcome, the odds ratios (ORs) of the exposure level in the Q2-Q4 groups in comparison with the Q1 group were estimated using a logistic regression model.

| Participant characteristics
The characteristics of the 3089 participants included in the analysis sets are shown in Table 1. The cumulative incidence rates of asthma and wheezing were 10.4% and 38.1%, respectively. The incidence rates of asthma by age were 2.7%, 4.8%, and 3.0% at 0-12, 12-24, and 24-36 months old, respectively, and were highest at 12-24 months old. The incidence rates of wheezing by age were 20.0%, 13.6%, and 4.6%, respectively, and were highest at 0-12 months old.
The total serum IgE level was measured at 24 months old in 2900 children. The upper 10th percentile of the total serum IgE level in 24month-old children was 180 IU/ml, and the serum IgE level was high

| Mite allergen and endotoxin
The levels of dust mite allergen and endotoxin in dust are shown in

| Association of mite allergen with the incidence of asthma or wheezing
The adjusted ORs of dust mite allergen exposure level Der 1 in the Q2-Q4 groups in comparison with the Q1 group are shown in Figure 2. The ORs of the cumulative incidence rates of asthma and wheezing were significantly higher in the Q4 group during

| Association of endotoxin with the incidence of asthma or wheezing
The adjusted ORs of the endotoxin exposure level are shown in Figure 3. The adjusted OR of the cumulative incidence rate of asthma during 0-36 months old was 1.59 (95% CI:

| Association of mite allergen or endotoxin with high levels of total serum IgE
The adjusted ORs for high levels of total serum IgE at 24 months old in the Q2-Q4 groups of the dust mite allergen exposure level and endotoxin exposure level in comparison with the Q1 group are shown in Figure 4. A significant OR (95% CI) was noted in both dust mite allergen and endotoxin exposure levels in the Q4 group in compari-

| DISCUSSION
In this study, the risks of the cumulative incidence rates of asthma and wheezing increased during 0-36 months old in children with a high level of dust mite allergen exposure in bed dust. Regarding the endotoxin exposure level, a significant positive OR was occasionally noted in the onset of asthma and wheezing in the high exposure group. In the simultaneous exposure to allergen, microorganisms, fungi, and air pollutants, these were found to interact with each other and promote or prevent allergic disease. 26 As dust mite allergen and endotoxin were simultaneously investigated in only limited epidemiological studies, these are individually discussed below.

Wahn et al. showed a dose-response relationship between in-
door allergen exposure and sensitisation during the first 3 years of life. 27 In an integrated analysis of five geographically different European birth cohort studies, an association was noted between dust mite allergen exposure (2-6 months old) and sensitisation to house dust mite (≤6 years old), but no association was observed with an increase in the risk of asthma (≤6 years old). 6  but a trend was observed between dust mite allergen exposure and the cumulative incidence rates of asthma and wheezing in the present study. This may have been due to differences in the dust mite allergen sampling point and the difference in the unit of Der 1 (μg/m 2 , μg/g).
Regarding the sampling point, dust on the floor of the children's bedroom was collected in the European cohort studies, whereas dust mite allergen in bed dust was evaluated in our study. The dust mite allergen concentration is higher in bed dust than that in dust on the bedroom floor and the dust mite allergen exposure level in children was also higher. 25 Although it is necessary to pay attention to a simple comparison of the Der 1 concentration, the geometric mean was within a range of 0.06-0.97 μg/g in the European cohort studies, but the Der 1 concentration was higher at 3.89 μg/g in our study performed in Japan. Regarding the second point, the difference in the unit of Der 1 level, μg/g was used in the European cohort studies, whereas it was measured as μg/m 2 in our study. The risk of mite sensitisation markedly increased when the Der 1 level exceeded 2 μg/g. 28 In the present study, based on analysis of the amount per gram dust (unit: μg/g), no significant increase in total F I G U R E 2 Associations among dust mite allergen, Der 1 (μg/m 2 ), and incidence of asthma or wheezing using a logistic regression model adjusted for high levels of maternal total serum IgE, active smoking/passive smoking based on the urinary cotinine level in mid-pregnancy, passive smoke after birth, children's sex, lower respiratory inflammation, respiratory syncytial virus infection, presence of indoor pets and climatic region F I G U R E 3 Associations between endotoxin (EU/m 2 ) and the incidence of asthma or wheezing using a logistic regression model adjusted for high levels of maternal total serum IgE, active smoking/passive smoking based on the urinary cotinine level in mid-pregnancy, passive smoke after birth, children's sex, lower respiratory inflammation, respiratory syncytial virus infection, presence of indoor pets, and climatic region IgE levels was noted in the Q3 (3.17-8.37 μg/g) or Q4 (8.37 μg/g or higher) groups ( Figure S5)  33 whereas that in dust in the present study was 16.0 EU/mg, which is within a range similar to that seen in the United States.
The main strength of this study was that this is the first largescale birth cohort study wherein an environmental measurement for both dust mite allergen and endotoxin and health influence were evaluated using standardised methods involving approximately 3000 infants. The cumulative incidence rate of wheezing and asthma in early life could be evaluated by conducting annual questionnaire surveys. However, this study has the following limitations. First, dust mite allergen and endotoxin exposure were evaluated only at 18 months old. Exposure to high concentrations of allergens immediately after birth strongly influences the advancement of paediatric allergic disease. 5,6 Samples were not collected immediately after birth in our study, but dust was collected at the age of 18 months old. The association with allergic disease over school age after growth will be investigated in a follow-up survey. Second, asthma symptoms were investigated using self-administered questionnaires filled out by mothers.

F I G U R E 4
Associations between dust mite allergen, Der 1 (μg/m 2 ), or endotoxin (EU/m 2 ) and high serum IgE levels in children aged 24 months old using a logistic regression model adjusted for high levels of maternal total serum IgE, active smoking/passive smoking based on the urinary cotinine level in mid-pregnancy, passive smoke after birth, children's sex, lower respiratory inflammation, respiratory syncytial virus infection, presence of indoor pets, and climatic region However, a validation study of questionnaires that followed the diagnostic criteria of asthma used by allergists reported that the sensitivity and specificity of asthma symptoms were 0.91 and 0.64, respectively, in 0-24-month-old children. 20 Third, total IgE levels at 24 months old alone were used as the cut-off for outcome evaluation of high serum IgE levels, and the specific IgE level could not be used. Soto-Quiros et al. reported that higher levels of specific IgE to dust mite allergen were correlated significantly with total IgE among school children. 34 The associations with specific IgE and with changes accompanying growth remain to be investigated.
As the evaluation was performed for up to 3 years in our study, the risk (or preventive effect) of dust mite or endotoxin on the development of asthma at school age will be investigated through a follow-up survey in the future.

| CONCLUSION
Early-life exposure to dust mite allergen was associated with the onset of wheezing during the age of 0-12 months old and asthma during the age of 12-36 months old. Early-life exposure to endotoxin was also associated with the onset of wheezing and asthma during the age of 24-36 months old. Our results indicated that high levels of early-life exposure to dust mite allergen and endotoxin may be involved in the development of asthma.