In inner-city environments children with the highest exposure to specific allergens and bacteria during their first year were least likely to have recurrent wheeze and allergic sensitization. These findings suggest that concomitant exposure to high levels of certain allergens and bacteria in early life might be beneficial and suggest new preventive strategies for wheezing and allergic diseases.

Cumulative allergen exposure over the first 3 years was associated with allergic sensitization, and sensitization at age 3 years was related to recurrent wheeze. In contrast, first-year exposure to cockroach, mouse, and cat allergens was negatively associated with recurrent wheeze (odds ratio, 0.60, 0.65, and 0.75, respectively; P ≤ .01). Differences in house dust bacterial content in the first year, especially reduced exposure to specific Firmicutes and Bacteriodetes, was associated with atopy and atopic wheeze. Exposure to high levels of both allergens and this subset of bacteria in the first year of life was most common among children without atopy or wheeze.

The Urban Environment and Childhood Asthma study examined a birth cohort at high risk for asthma (n = 560) in Baltimore, Boston, New York, and St Louis. Environmental assessments included allergen exposure and, in a nested case-control study of 104 children, the bacterial content of house dust collected in the first year of life. Associations were determined among environmental factors, aeroallergen sensitization, and recurrent wheezing at age 3 years.

Wheezing illnesses affect 35% to 50% of children by the age of 3 yearsand are a leading cause for outpatient visits and hospitalizations.Wheezing in nonatopic children is often transient, but recurrent wheezing in children with early allergic sensitization or other signs of atopy during the preschool years is a risk factor for asthma.Because the prevalence and severity of asthma are high in inner cities in the United States, it is especially important to identify risk factors that contribute to the development of allergic sensitization and wheezing in this environment.

Asthma and wheezing in the first six years of life. The Group Health Medical Associates.

The indoor environment of poor urban neighborhoods can include adverse conditions that promote allergic sensitization and recurrent wheezing.Examples include stress, lack of biodiversity, and exposure to indoor pollutants and perennial allergens, such as cockroach and mouse.Conversely, farm-related microbial exposures in early life have been linked to protection against allergic diseases.Whether relationships exist between microbial exposure and allergic disease outcomes in urban settings is unknown.

Exposure to cockroach allergen in the home is associated with incident doctor-diagnosed asthma and recurrent wheezing.

Predictors of repeated wheeze in the first year of life: the relative roles of cockroach, birth weight, acute lower respiratory illness, and maternal smoking.

To examine the relationship between these conditions and development of allergic sensitization and recurrent wheezing, we studied children enrolled in an ongoing birth cohort study (Urban Environment and Childhood Asthma [URECA]). Per study design, the entire cohort was evaluated at age 3 years to test the hypothesis that high levels of exposure to sensitizing allergens, especially those associated with cockroach and mouse, is associated with the development of allergic sensitization and recurrent wheezing. In addition, a nested case-control study was designed to determine whether early-life exposure to certain microbes in house dust obtained from inner-city homes is associated with development of allergic sensitization and wheezing. The results of these 2 studies are presented in this report.

Methods used to filter and analyze microbiome data are described in the Methods section in this article's Online Repository.

The 3 allergen exposures showing a strong inverse relationship to recurrent wheeze (cockroach, mouse, and cat; see below) were combined into a single allergen exposure index based on tertiles of exposure to individual allergens (see the Methods section in this article's Online Repository). In addition, a dichotomous variable was created for exposure to each allergen (cockroach, mouse, and cat) to indicate whether the levels were greater than standard cutoffs (Bla g 1, 2 U/g; Mus m 1, 0.5 μg/g; and Fel d 1, 2 μg/g).

Demographic comparisons between recurrent wheezers and nonwheezers were tested by using Wilcoxon tests for continuous data and χtests for binary data. Univariate and multivariate analyses to determine association of exposures with sensitivity and recurrent wheeze were performed by using logistic regression. On the basis of this and previous analyses,multivariate models were adjusted for race/ethnicity (strongly correlated with site), sex, mean perceived stress of the mother in the year after birth,and number of smokers in the home.

Aeroallergen sensitization was defined by a wheal 3 mm or more larger than that elicited by the saline control on skin prick testing or a specific IgE level of 0.35 kU/L or greater. Recurrent wheeze was defined as parental report of at least 2 wheezing episodes, with at least 1 episode occurring in the third year. Eczema was defined as a score of 1.0 or greater on the Eczema Area and Severity Indexat age 3 years. Children at higher risk for asthma were identified by using the modified Asthma Predictive Index.

Household dust samples from the living room (chair or sofa and floor) and child's bedroom (mattress and floor) were collected, as described in the Methods section in this article's Online Repository at www.jacionline.org , and assayed for allergenic proteins, including Bla g 1 (cockroach), Can f 1 (dog), Fel d 1 (cat), Der f 1 and Der p 1 (house dust mites), and Mus m 1 (mouse), by using ELISA (Indoor Biotechnologies, Charlottesville, Va). A subsample (n = 104) of living room dust specimens collected at 3 months of age underwent culture-independent microbiome profiling with a 16S rRNA-based phylogenetic microarray (G3 PhyloChip; Second Genome, San Bruno, Calif; see the Methods section in this article's Online Repository for details) to generate a high-resolution profile of both dominant and rare microbiota members in each sample for comparative and correlative analyses. An approximately equal number of dust samples was randomly selected from each of 4 categories defined by clinical outcomes at age 3 years: (1) recurrent wheeze and aeroallergen sensitivity, (2) recurrent wheeze alone, (3) aeroallergen sensitivity alone, and (4) neither outcome (see Table E1 in this article's Online Repository at www.jacionline.org ). This substudy population did not differ from the remainder of the cohort with respect to demographic characteristics or environmental exposures in the first year (see Table E2 in this article's Online Repository at www.jacionline.org ).

Allergen-specific IgE (ImmunoCAP; Phadia, Uppsala, Sweden) levels were measured annually for milk, egg, peanut, and German cockroach. At 2 and 3 years of age, specific IgE levels for dust mites, dog, cat, mouse, and Alternaria species were also measured. Skin prick testing was performed at age 33 months for 14 common indoor and outdoor allergens.

URECA is a longitudinal birth cohort study in 4 urban areas: Baltimore, Boston, New York City, and St Louis.Selection criteria included residence in an area with more than 20% of residents below the poverty level; mother or father with allergic rhinitis, eczema, and/or asthma; and birth at 34 weeks' gestation or later. Maternal questionnaires were administered prenatally, and participant questionnaires were administered every 3 months thereafter. Clinic visits occurred at 12, 24, 33, and 36 months, and homes were visited annually beginning at age 3 months for an environmental survey and house dust collection. Between February 2005 and March 2007, 1850 families were screened; 889 met the eligibility criteria, and 560 were enrolled. Informed consent was obtained from the parent or legal guardian of the infant.

Given the above associations, we thought it important to examine how combined early-life exposures to the selected allergens (cockroach, mouse, and cat) and bacteria (richness of either the total community or the 82 taxa of interest) affected clinical outcomes at 3 years. Our analysis of the nested case-control study population showed that the combined exposure patterns differed significantly across the 4 outcome groups, and this was true whether the richness of the total bacterial community ( Fig 3 , A) or of the 82 taxa of interest ( Fig 3 , B) was considered in the analysis. Of the children in the Both group, the largest proportion (42%) were exposed to low levels of allergens and low bacterial community richness, whereas the smallest proportion (8%) of this group had been exposed to high levels of selected allergens and bacterial richness. Conversely, the children in the Neither group were more likely to have been exposed to high levels of allergens and high bacterial richness (41%) and were least likely (14%) to have had low-level allergen and bacterial exposure ( Fig 3 ). These comparisons reached higher levels of significance when only the 82 taxa of interest were used in the analysis ( Fig 3 , B). For example, the largest proportion (42%) of children in the Both group had low-level exposure to both the specified allergens and the 82 bacteria of interest, whereas none of the children in this group were exposed to high levels of allergen and of these specific bacteria.

Distribution of allergen and bacterial exposure among children with atopy, recurrent wheeze, atopy with wheeze, and neither outcome. Exposure to allergen is classified as high or low with respect to the median of the allergen exposure index and richness of exposure to all microbes (A) or taxa of interest (B) . The distribution of exposure in each outcome group is compared with the distribution in the Neither group.

Fig 3 Distribution of allergen and bacterial exposure among children with atopy, recurrent wheeze, atopy with wheeze, and neither outcome. Exposure to allergen is classified as high or low with respect to the median of the allergen exposure index and richness of exposure to all microbes (A) or taxa of interest (B) . The distribution of exposure in each outcome group is compared with the distribution in the Neither group.

We next asked whether bacteria that were inversely related to atopy and atopy with wheeze were also related to allergen exposure. Of the 82 taxa of interest significantly enriched in the Neither group, 14 and 10 were also significantly correlated with levels of Mus m 1 and Bla g 1 allergens, respectively, and these taxa were primarily members of the Bacteroidetes (eg, Prevotellaceae and Rikenellaceae; see Table E7 in this article's Online Repository at www.jacionline.org ). Blattabacterium species, known endosymbionts of cockroaches,were highly positively correlated with Bla g 1 levels, indicating that some of these bacterial constituents of house dust might originate from the enteric contents of cockroaches. None of the 82 taxa of interest were associated with Fel d 1 allergen levels.

To identify the specific taxa within the house dust microbiome that discriminated among these groups, we next compared taxon relative abundance between the Neither group and each of the other groups. The greatest number of taxa exhibiting significant difference in relative abundance was identified in the Both versus Neither comparison (82 taxa; Fig 2 , E). All of these “taxa of interest” were significantly increased in the first-year dust samples of the Neither group, raising the possibility that exposure to certain bacteria in house dust during the first year of life might play a role in protection against atopic wheeze. These putatively protective bacteria belonged to several phyla but were primarily members of the Bacteroidetes and Firmicutes, particularly the Prevotellaceae, Lachnospiraceae, and Ruminococcaceae families (see Table E6 in this article's Online Repository at www.jacionline.org ). Fewer taxa showed significant differences in relative abundance in the Neither versus Atopy group comparison ( Fig 2 , D), but again, of those that did, all were relatively more abundant in the Neither group and were thus associated with a reduction in the risk of atopy. These taxa largely belonged to the same families as those identified from the Neither versus Both group comparison (see Table E6 ). No taxa were found to differ significantly in relative abundance between the Neither and Wheeze groups ( Fig 2 , F).

Relative bacterial richness, a measure of the number of bacterial taxa detected in each sample, was significantly different among the 4 groups (P < .02) and was considerably lower in first-year dust samples from the Atopy or Both groups relative to the Neither group (see Fig E3 in this article's Online Repository at www.jacionline.org ), indicating reduced bacterial exposure in these environments. Multivariate analysis of house dust bacterial community composition further demonstrated that the Neither group house dust microbiome was compositionally distinct from that of the Atopy (P = .002; Fig 2 , A) and Both (P = .005; Fig 2 , B) groups but not from the Wheeze group ( Fig 2 , C).

House dust microbiome composition is associated with clinical outcomes. A-C, Similarity or dissimilarity of house dust microbiota composition is indicated by the distance between samples (colored dots). Samples plotted close together are compositionally similar, and greater intersample distance indicates compositionally distinct bacterial communities. Ellipses represent 95% CIs for each group. D-F, Mean taxon relative abundance across house dust samples from the Neither group was compared with those of the Atopy (Fig 2, D), Both (Fig 2, E), and Wheeze (Fig 2, F) groups to identify specific bacterial taxa associated with clinical outcomes. Taxa in the left lower quadrant are underrepresented in the Atopy (Fig 2, D) and Both (Fig 2, E) groups compared with the Neither group. Colors indicate phylum-level classification of individual taxa, and the horizontal lines indicate P values of less than .05 after correction for false discovery rates.

Fig 2 House dust microbiome composition is associated with clinical outcomes. A-C, Similarity or dissimilarity of house dust microbiota composition is indicated by the distance between samples (colored dots). Samples plotted close together are compositionally similar, and greater intersample distance indicates compositionally distinct bacterial communities. Ellipses represent 95% CIs for each group. D-F, Mean taxon relative abundance across house dust samples from the Neither group was compared with those of the Atopy (Fig 2, D), Both (Fig 2, E), and Wheeze (Fig 2, F) groups to identify specific bacterial taxa associated with clinical outcomes. Taxa in the left lower quadrant are underrepresented in the Atopy (Fig 2, D) and Both (Fig 2, E) groups compared with the Neither group. Colors indicate phylum-level classification of individual taxa, and the horizontal lines indicate P values of less than .05 after correction for false discovery rates.

To test the hypothesis that early-life exposure to certain microbes in house dust is associated with protection against allergic sensitization and wheezing, we conducted a nested case-control study of the microbes present in house dust of 104 URECA households. The number of samples were relatively evenly distributed across 4 distinct groups: children with recurrent wheeze alone (“Wheeze,” n = 26), atopy alone (“Atopy,” n = 25), both recurrent wheeze and atopy (“Both,” n = 24), or neither recurrent wheeze nor atopy (“Neither,” n = 29). The inverse association between first-year allergen exposure and recurrent wheeze at age 3 years in the nested case-control study population was similar to that observed in the whole population (see Table E5 in this article's Online Repository at www.jacionline.org ).

However, in contrast to our expectations, significant inverse relationships were found between first-year exposure to cockroach, mouse, and cat, but not house dust mite or dog, allergens and recurrent wheeze at age 3 years (odds ratio, 0.60, 0.65, 0.75, 0.97, and 1.01, respectively; Table III ). An additive reduction in wheeze was observed with exposure to more than 1 of these 3 allergens ( Fig 1 ). When categorized as exposed or not exposed by using standard cutoffs, recurrent wheeze decreased from 51% in those with no exposures (n = 96) to 17% in those exposed to all 3 allergens (n = 18; Fig 1 , A). By using an index that reflects exposure to all 3 of these allergens in the first year of life, the prevalence of recurrent wheeze was inversely related to the index over the entire range of exposures ( Fig 1 , B). The negative relationship between first-year exposures and wheeze persisted when stratified by aeroallergen sensitization and when adjusted for covariates ( Fig 1 , C, and Table III ). Our data revealed that the timing of allergen exposure was important; the inverse relationship with recurrent wheeze at age 3 years was significant only for exposures in the first year of life and not for those encountered in the second or third years ( Fig 1 , D and E).

Relationships between specific allergen exposures and recurrent wheezing. A, Probability of recurrent wheeze (95% CIs) according to the number of allergen exposures (cockroach, mouse, and/or cat). B-E, Probability of recurrent wheeze (95% CI) determined by using logistic regression. Fig 1, C is shown stratified by aeroallergen sensitivity (pink = sensitive, blue = not sensitive). Fig 1, D and E, show the probability of recurrent wheeze for allergen exposures during years 2 and 3.

Fig 1 Relationships between specific allergen exposures and recurrent wheezing. A, Probability of recurrent wheeze (95% CIs) according to the number of allergen exposures (cockroach, mouse, and/or cat). B-E, Probability of recurrent wheeze (95% CI) determined by using logistic regression. Fig 1, C is shown stratified by aeroallergen sensitivity (pink = sensitive, blue = not sensitive). Fig 1, D and E, show the probability of recurrent wheeze for allergen exposures during years 2 and 3.

Individual exposure models are adjusted for the respective sensitization. The exposure index is adjusted for overall aeroallergen sensitization.

∗ Individual exposure models are adjusted for the respective sensitization. The exposure index is adjusted for overall aeroallergen sensitization.

Association between bedroom dust allergen exposure in the first year of life with recurrent wheeze in year 3

Table III Association between bedroom dust allergen exposure in the first year of life with recurrent wheeze in year 3

Allergen levels from the first-year dust samples varied by site (see Fig E2 in this article's Online Repository at www.jacionline.org ), and there was also moderate within-subject variation over the 3 years (intraclass correlation, 0.32-0.56). Cumulative exposure (summed over the 3 years) to cockroach, mouse, and dust mite (Dermatophagoides farinae) was positively associated with sensitization to those allergens at age 3 years (odds ratio, 1.27-1.68; see Table E4 in this article's Online Repository at www.jacionline.org ), and as expected, allergic sensitization was positively associated with recurrent wheeze ( Table II ). In contrast, exposure to allergens in the first year had little or no association with sensitization at age 3 years (see Table E4 ).

Of the 560 children in the URECA cohort, 478 (86%) remained in the study at age 3 years; 467 (83%) had sufficient data to assess recurrent wheeze, and 383 (68%) had serum IgE data available at age 3 years. Children included in the primary analysis (with complete follow-up data on wheeze, sensitization, and home allergen exposure data) differed from those not included in terms of study site and race/ethnicity but not allergen exposure (see Table E3 in this article's Online Repository at www.jacionline.org ). Of these, 44% were sensitized to at least 1 aeroallergen, 36% had recurrent wheeze, and 9% had eczema (see Fig E1 in this article's Online Repository at www.jacionline.org ). Furthermore, 12% of the cohort met the criteria for the modified Asthma Predictive Index, indicating a high risk for subsequent asthma. Factors related to recurrent wheeze were an annual family income of less than $15,000, lower birth weight and gestational age, and the number of smokers in the household ( Table I ). Children with recurrent wheeze had a median of 6 wheezing episodes (range, 2-26 wheezing episodes) by age 3 years, and 77% had been prescribed albuterol, 27% had been prescribed an inhaled corticosteroid, and 33% had been prescribed at least 1 course of oral corticosteroid for wheezing.

Discussion

Using a birth cohort and a nested case-control study, we assessed the relationships between exposure to allergens and bacteria over the first 3 years of life and the development of recurrent wheeze and atopy. As hypothesized, cumulative allergen exposure over the first 3 years of life was associated with allergic sensitization, and allergic sensitization was associated with recurrent wheeze. However, the major finding from the birth cohort study was that high levels of cockroach, mouse, and, to a lesser extent, cat allergen in the first-year inner-city house dust samples had a strong inverse relationship with recurrent wheeze at age 3 years. This association was strongest for allergen levels in the first-year dust samples, suggesting that the first few months of life is a critical time period in childhood allergic disease development. In addition, changes in the bacterial house dust environment, characterized by reduced exposure to bacterial richness, as well as specific bacteria, were significantly associated with the development of atopy with wheezing or atopy alone. Some of the bacterial taxa related to favorable clinical outcomes were positively associated with mouse and cockroach allergen levels, raising the possibility that household pests might be the source of some of the beneficial bacteria in the inner-city environment. Finally, combined analysis of exposure to both allergens and bacteria revealed that the group of children with neither wheeze nor atopy had the highest first-year exposure to allergens and the bacterial species identified in this study as potentially protective against atopy and recurrent wheeze. To our knowledge, this is the first scientific report of exposure to high levels of allergens combined with an environment rich in specific bacterial families as having a protective effect against atopy and atopy with wheezing in early childhood.

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et al. Relationship of indoor allergen exposure to skin test sensitivity in inner-city children with asthma. A prevalent conceptual model is that exposure to perennial indoor allergens contributes to the development of allergic sensitization with subsequent development of wheezing.Earlier cross-sectional studies of inner-city populations supported this concept by showing a dose-response relationship between Bla g 1 levels in house dust and allergic sensitization to cockroach.Our findings are in some ways consistent with this model in showing that cumulative exposure to cockroach and mouse allergens in the dust of inner-city houses over the first 3 years of life is associated with allergic sensitization and that allergic sensitization is associated with recurrent wheeze.

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et al. Effects of dog ownership and genotype on immune development and atopy in infancy. Where our findings importantly differ from this conceptual model is in showing an inverse relationship between the levels of cockroach, mouse, and cat allergen in dust samples collected in the first months of life and recurrent wheeze at age 3 years. The temporal nature of the relationship is similar to that previously observed between early-life exposure to pets and reduced rates of allergic sensitization and asthmaand supports a mounting body of evidence that exposures in the first few months of life are important in shaping allergic and respiratory outcomes. This counterintuitive effect of early-life exposure to certain allergens might help explain the failure of several previous studies to find straightforward relationships between allergen exposure and the development of atopy and asthmaor to demonstrate that allergen avoidance protects against development of these conditions.Notably, in contrast to other reports,dog exposure was not protective for atopy or wheeze in this study. We speculate that the reason for the lack of protection in urban homes might be that dogs can be kept for security and thus interact less with young children and might also be less likely to bring soil into the home from outdoor sources, which are sparse in urban neighborhoods.

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et al. Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers. Our findings point to particular families of bacteria as being protective against the development of atopy and atopic wheeze. These findings are in line with experimental data in animals and observational studies in Western Europe showing that microbial exposures in early life are critical contributors to respiratory health.Certainly, the URECA findings have a very different context; overall rates of wheezing and allergic sensitization in low-income, urban US populations are high, likely influenced by adverse factors related to the urban environment (eg, indoor and outdoor pollutants) and increased rates of low birth weight, prematurity, household tobacco smoking, and maternal stress and depression. Among families exposed to these adverse conditions, it is remarkable that the general tenets of the hygiene hypothesis are applicable, in that some of the residences in this environment contain bacteria that reduce the risk of allergic sensitization with or without recurrent wheezing through exposure. Whether these bacteria are similar in phylogeny, quantity, or function to those found in farmhouses remains to be determined.

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Hegde R.S.

Madan R.

et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. There are several possible explanations for the associations between early microbial and allergen exposures and 3-year outcomes. The infant microbiome exhibits a temporal program of community assembly over the first year of life, and patterns of early-life gastrointestinal colonization have previously been linked to immune development,allergic disease,and the response to viral respiratory tract infection.Most of the “protective” taxa identified in our study belong to bacterial families (eg, Prevotellaceae, Lachnospiraceae, and Ruminococcaceae), which are human colonizers and important producers of immunomodulatory metabolites, such as short-chain fatty acids.This suggests that early-life exposure to house dust containing these bacteria might inoculate the developing gastrointestinal microbiome with species that produce metabolites that protect against development of atopy and atopic wheeze. It is also plausible that these microbial exposures could exert a similar effect at airway mucosal surfaces. Accordingly, the respiratory microbiome is altered in asthmatic patients,and experiments in mice demonstrate that environmental microbes in early life can modify lung mucosal immunity to reduce inflammatory responses to allergens.How allergen exposure modifies the risk of recurrent wheeze, which frequently is caused by viral respiratory infections, is unknown, but the mechanisms could be related to direct biological effects of allergens (eg, activation of innate immune receptors) or allergen-associated biologic agents (eg, proteases, chitin, and DNA).Whether exposure to these immunologically active substances in early life affects the development of mucosal immune and antiviral responses remains to be determined.

The strengths of our investigation include its examination of an inner-city population at high risk for respiratory morbidity, high rate of study participant retention, and prospective assessments of environmental exposures. Previous clinical studies of the microbiome have used lower-resolution culture- or molecular-based approaches to distinguish a limited selection of microbes. The URECA study used a phylogenetic microarray to profile thousands of organisms in parallel and provided data at the phylum, family, and species level. This is a multicenter study with site differences in exposure, race/ethnicity, and the prevalence of recurrent wheeze, but the main study findings were unaffected by adjustment for these factors. One limitation of the study is that the microbial analysis was performed in a subset of homes, and therefore relationships between specific bacterial taxa and outcomes, although false discovery rate corrected, await confirmation in other populations. Furthermore, the array-based method in this study provides neither information about absolute quantity of bacteria nor functional analysis of metabolic activity that could mediate effects on immune development. Finally, it is important to consider that effects of environmental exposures could change with age and also in relationship to age-dependent phenotypes (eg, recurrent wheezing vs persistent asthma).