- Email: [email protected]
Revisiting the hygiene hypothesis for allergy and asthma
Revisiting the hygiene hypothesis for allergy and asthma Andrew H. Liu, MD
Aurora and Denver, Colo
The hygiene hypothesis, which describes the protective influence of microbial exposures in early life on the development of allergy and asthma, has continued its swell of academic interest, investigation, and evolution. This article is focused on studies published in the past 3 years that have furthered the substance and shape of hygiene theory, primarily as it relates to allergic airways and asthma. Recent investigations have furthered an overarching ‘‘microbiome hypothesis’’ to home features, medical practices, and cleanliness behaviors that are suspects in the hygiene effect. Relatively crude markers of the protective microbial environment have been supplanted by cultureindependent microbiome science, distinguishing the characteristics of potentially protective microbiomes from pathologic features. Understanding how the microbiome is shaped and affects healthful versus harmful outcomes in the human host is relatively nascent. Good clues are emerging that give mechanistic substance to the theory and could help guide microbe-based therapeutics to fill the allergy and asthma management gap in prevention and disease modification. (J Allergy Clin Immunol 2015;136:860-5.) Key words: Asthma, hygiene hypothesis, asthma prevention
Of microbes and humans: the sequel. With the discovery of microbial pathogens in the mid- to late-1800s, a successful war against these microscopic invaders with a broad-based campaign of public and personal hygiene measures and therapeutic and preventive interventions was a victory for human health. But did this antimicrobial war, with other aspects of modern sanitized living, disrupt an immune balance between microbes and human subjects with an abruptness that did not allow for evolutionary adaptation, leading to the unintended consequence of the allergic march underlying the allergy and asthma epidemic of the past century? Since its academic re-emergence in the past few decades, the hygiene hypothesis (reviewed by Liu1) has been broadly investigated. Some have interpreted this growing body of evidence to have consistency and potential to serve the unmet need of preventive measures for allergy and asthma by mimicking nature’s ways. Others have been doubtful because a wide range of microbes (eg, respiratory viruses, bacteria, and molds) have a well-established
From the Department of Pediatrics/Section of Pulmonary Medicine, Children’s Hospital Colorado and the University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, and National Jewish Health, Denver. Disclosure of potential conflict of interest: A. H. Liu is on the Data Safety Monitoring Committee for GlaxoSmithKline and has received payment for lectures from Merck. Received for publication August 17, 2015; revised August 24, 2015; accepted for publication August 24, 2015. Corresponding author: Andrew H. Liu, MD, Children’s Hospital Colorado, The Breathing Institute, 13123 East 16th Ave, Box B395, Aurora, CO 80045. E-mail: Andrew. [email protected]. 0091-6749/$36.00 Ó 2015 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaci.2015.08.012
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Abbreviations used TLR: Toll-like receptor Treg: Regulatory T URECA: Urban Environment and Childhood Asthma Study
provocative effect on asthma symptoms. How could microbial exposures also have a paradoxical preventive effect on allergies and asthma? The seeds of this paradox were already apparent in the beginnings of allergy’s discovery, with Dr Charles Blackley’s experimental observations that the grass pollen he used for conjunctival challenges to demonstrate pollen’s causal role in ‘‘hay-fever or hay-asthma’’ was obtained from farmers who were nonallergic.2 In keeping with the current theme, let us begin by revisiting Blackley’s farmer collaborators through a recent documentary about allergy and asthma in Amish farmers in America.
THE AMISH IN AMERICA: A CASE STUDY Life on rural European farms has been a rich resource of naturally occurring evidence that microbial exposures from proximal living with domestic animals beginning in early life might protect against the development of allergies and asthma.3,4 This has provided a compelling body of evidence in support of hygiene theory. Could these farming ways indicate environments and lifestyles that are allergy and asthma protective today and were even more so in earlier times before the global increase in allergy and asthma? The Amish, a sect of Anabaptists, adhere to a lifestyle of approximately 150 years back in time, adhering to simple living, plain dress, and a traditional lifestyle eschewing modern conveniences and technology, which is fundamental to their faith. Dairy farming is a desirable lifestyle and avocation for Amish families. The Amish in America emigrated from the Swiss region of the European farm studies noted above. An allergist serving an Amish community in Indiana, Dr Mark Holbreich, in collaboration with rural European farm investigators Drs Erika von Mutius and Charlotte Braun-Fahrlander and their colleagues, determined that Amish children in Indiana have a very low prevalence of allergy, asthma, and aeroallergen sensitization when compared with both Swiss nonfarm and farm children. Results in Amish versus Swiss farm vs Swiss nonfarm children were as follows: asthma, 5.2% vs 6.8% vs 11.2%; hay fever, 0.6% vs 3.1% vs 11.6%; and atopic eczema, 1.3% vs 7.6% vs 12.1%, respectively. Also notable as an objective measure, aeroallergen sensitization was as follows: 7.2% vs 25.2% vs 44.2%, respectively.5 This Amish case study is a provocation to the notion that allergy and asthma were uncommon in a past time and influenced by environments and lifestyles still retained by European dairy farmers today. Hygiene theory implicates human subjects’ microbial environment in early life as essential
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to proper immune development, both toward protective antimicrobial and regulatory immune responses to environmental antigens and away from atopy and asthma. In addition to differences in the microbial environment, there is much to lifestyle differences from modern metropolitan living that are characteristic of Amish farming families. For example, in the study by Holbreich et al,5 the Amish cohort averaged 6 children per family, which was double the numbers seen in the Swiss farmers and nonfarmers. Could the low prevalence of allergy and asthma in these Amish be related to large family size and independent of microbial environment? This study was not large enough to discern lifestyle differences, although it is believed that these children have more physical outdoor play and are needed to participate in farm chores as soon as they are able. This hygiene anecdote is intriguing, even if it is conjecture.
CLEAN (AND NOT SO CLEAN) LIVING Recent investigations have shed light on personal hygiene behaviors suspected of contributing to the hygiene effect. In a German longitudinal birth cohort study, personal cleanliness (eg, hand washing and showering) was associated with lower levels of endotoxin (ie, a bacterial marker) and muramic acid (ie, a fungal marker) in bedding and floor dust.6 In comparison, household cleanliness (eg, cleaning floors and bathrooms, dusting, and changing towels) was associated with less dust but not with lower microbial marker levels. Endotoxin in infancy was associated with less allergic sensitization and less asthma when these children reached school age, whereas muramic acid exposure at school age, but not infancy, was associated with less school-age asthma and eczema. It might be surprising to some that neither personal nor home cleanliness activities were directly associated with atopy or asthma outcomes. This study points to the importance of early-life timing of actual microbial exposures and not cleanliness behaviors, with the influence of endotoxin exposure being in infancy. Two behaviors that could affect exposure to oral microbes were recently associated with less allergy. In a Swedish study hand dishwashing, which is presumed to be less effective in eliminating microbes when compared with machine dishwashing, was associated with less eczema and less ‘‘total allergy’’ (ie, a combination of eczema, asthma, and/or allergic rhinoconjunctivitis).7 The protective effect of hand dishwashing was stronger in children eating fermented foods, farm-bought foods, or both, raising the possibility of specific sources of food-borne microbes being transmitted through the presumably less hygienic method of hand dishwashing. The same research group reported on the parental behavior of infant pacifier ‘‘cleaning’’ by putting it in the parent’s mouth before giving it back to the infant; 48% of the cohort reported this practice in the first 6 months of life.8 Children with parents who orally ‘‘cleaned’’ their pacifiers were less likely to have allergic sensitization, eczema, and ‘‘asthma’’ by age 18 months, implicating parentto-infant oral microbe transmission as atopy protective. If or how these behaviors might significantly alter microbial exposures in an atopy-protective manner or might be behavioral markers of other protective factors is not elucidated but gives hope to the notion that allergy and asthma prevention might be as simple and benign as these frequent oral microbial exposures beginning in early life.
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SUBSTANTIATING PROTECTIVE HOME ENVIRONMENTS Recent studies have substantiated prior observations attributed to hygiene theory. David Strachan, who first demonstrated the relationship of increased sibship with less atopy, extended these associations on a global scale in the International Study of Asthma and Allergies in Childhood cohort.9 Globally, increasing numbers of siblings, especially older siblings, were negatively associated with hay fever and eczema. These associations were stronger in more affluent countries, which is consistent with the notion that less developed nations have additional significant contributors to a hygiene effect. The infant gut microbiome associated with having older siblings was recently shown in a birth cohort to have more Bifidobacterium, Lactobacillus, Escherichia, and Bacteroides genera and less Clostridia.10 This is similar to the infant gut microbiome associated with vaginal delivery versus cesarean section exhibiting greater gut microbial diversity, earlier colonization with Bacteroides, less colonization with Clostridia, and subsequently higher levels of the TH1-associated chemokines CXCL10 and CXCL11 in peripheral blood.10,11 Vaginal delivery and older siblings appear to have additive favorable effects on the gut microbiome. A recent study of ambient endotoxin exposure provides further credence for the paradoxical differences in exposure-health outcomes in adults compared with children. Dose-dependent endotoxin exposure has been associated with more asthma in adults12 and paradoxically less atopy and allergic asthma in children.3,6 In a recent nationally representative US study based on the US National Health and Nutrition Examination Survey 2005-2006, greater house dust endotoxin levels were associated with a lower risk of allergic sensitization to pets and pollens in children/adolescents; in contrast, the risk of aeroallergen sensitization was higher in adults.13 This is consistent with the German longitudinal birth cohort study noted above: endotoxin concentration in mattress dust collected at age 3 months was associated with a lower likelihood of asthma and aeroallergen sensitization at age 5 years.6 Possible explanations for this paradoxical difference include timing of exposure: preventing atopy in early life versus promoting atopy persistence in adulthood. There might also be significant differences in relevant coexposures in children compared with adults. For example, more adults might be exposed to tobacco smoke, which interacts with endotoxin to provoke asthma.14 This duality of healthful versus harmful outcomes from microbial exposures and the importance of key determinants is well exemplified by endotoxin.15,16 THE PARADOX OF US INNER CITIES Because there is a high burden of severe asthma in US inner cities, the National Institutes of Health/National Institute of Allergy and Infectious Diseases has sponsored an inner-city birth cohort study, Urban Environment and Childhood Asthma (URECA), to understand how US inner cities foster severe asthma development.17 From a hygiene hypothesis perspective, inner-city environments could be suspected of being protective on allergy and asthma development, which is a paradox. Although this study is ongoing, recent investigation of URECA participants’ first 3 years of life provides paradigmatic insights.18 First, house dust cockroach, mouse, and cat allergen exposure in the first year of life was negatively associated with recurrent
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wheezing at age 3 years. These indoor allergen exposures had a prominent additive effect, such that recurrent wheeze occurred in 51% of those with no allergen exposures versus 17% of those with all 3 allergen exposures. This protective association was not observed for allergen exposures in the second or third years of life or for allergic sensitization outcomes. This association is similar to what has been observed in other longitudinal birth cohort studies with early-life pet ownership, mostly dog ownership.19-21 However, in the URECA study dog allergen exposure was not protective. Second, a house dust microbiome that is rich and diverse in the first year of life, with an abundance of specific bacterial taxa, most notably Firmicutes and Bacteroidetes phyla, was negatively associated with allergic sensitization and atopic recurrent wheezing at age 3 years. Similarly, households with dogs have rich diverse house dust microbiomes with an abundance of Firmicutes and Bacteroidetes phyla.22 Third, combined high-level exposure to these protective allergens and microbiome features in infancy had additive and independent effects. Higher allergen levels were associated with less recurrent wheeze independent of allergic sensitization. In contrast, protective bacterial taxa were associated with less atopy. Considering these exposures together, children without atopy or recurrent wheeze at age 3 years had the highest percentage, with both high allergen levels plus protective bacterial taxa (48%). In contrast, no children with both atopy and wheeze had the combination of both protective allergens and bacterial taxa. Although it is possible that high-level exposure to cockroach, mouse, and cat allergens has a direct protective effect on recurrent wheeze, these allergens could also be environmental markers for protective microbes. In the URECA study some of the protective bacteria were associated with relevant allergen exposures, suggesting that these bacteria might come from the source of the allergens (eg, cockroaches, mice, and cats) and making it impossible to clearly disentangle the protective effect of the relevant allergens from microbes. Together, protective microbes in the gastrointestinal tract and skin might induce TH1 and tolerizing responses to allergen, as reported in animal model studies.23-25 Some allergens, such as dust mite Der p 2, have inherent innate immune stimulatory capacity26 or can be molecular carriers of microbial components; for example, cat Fel d 1 and dog Can f 1 are carriers for endotoxin.27 Taken together, cohabitation with many creatures in early life, such as pets, pests, older siblings, and domestic animals, could have an additive protective effect on allergy and asthma development, as unified by microbiome theory, working through similar and shared mechanisms.
MICROBIOME THEORY OF ALLERGY AND ASTHMA PREVENTION The microbiome has been avidly investigated as the root of the hygiene hypothesis and possibly the common thread linking the many epidemiologic observations. Microbiome investigation has taken on new depth, in part because of extraordinary advances in the scope of microbiome characterization using new cultureindependent genomic methods. The findings of new microbiome science applied to allergy and asthma has been the subject of several excellent recent reviews.16,28-30 Although the investigational methodologies for characterizing the microbiome are
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advancing and evolving rapidly, making comparisons challenging, there is some consistency of observations such that conclusive evidence is emerging.
Types of microbes The explosion of microbiome interest has been largely about the bacterial component in taxa-level characterization and commensal bacterial mechanisms of action. Methodological advancements, including metagenomic shotgun sequencing approaches applied to investigating the microbiome’s fungal and viral components, are well underway and likely to provide insights into differences in health and disease similar to discoveries with bacteria.29 Microbiome location The microbiome differs dramatically in different locations on or in the body and surrounding environment.31 The microbiomes of the healthy airways are surprisingly homogeneous from top to bottom32; in contrast, microbiomes of the airways, oropharynx, gastrointestinal tract, vaginal tracts of peripartum mothers, skin, surrounding indoor and outdoor environments, and ingested foods differ in the context of health versus allergic/asthmatic disease.29 Investigators are meeting the challenge of extending these observations beyond associations to determine whether specifics of these differing microbial compositions might have causal roles, protective roles, or both. Importance of early life The microbiome in these different locations can evolve rapidly in early life. For example, the microbiome of the infant gastrointestinal tract seems to evolve almost daily during life’s beginning.33 Yet, as noted based on recent investigations, the transitional microbiome in the first year of life but not subsequently is relevant to the development of allergy and asthma.18,34 This is supported by mouse model studies in which early-life but not adult gut colonization with commensal Clostridia induced regulatory T (Treg) lymphocyte development and prevented the allergic asthma phenotype.35 Can maternal preterm exposures significantly influence infant immunity? In a Finnish study of newborn children living in relatively impoverished and ‘‘primitive’’ Karelia, Russia, where allergies and asthma are less common than in modern Finnish cities, cord blood transcriptomes of Karelian neonates were skewed toward innate Toll-like receptor (TLR) responses when compared with those of Finnish neonates, suggesting immune poising for antimicrobial responses at birth.36 Helminths have been inversely associated with allergy and asthma.37,38 The possibility that this protective effect of helminths begins prenatally is suggested by a large randomized controlled trial in which antihelminth treatment with albendazole during pregnancy was associated with an increased likelihood of eczema and recurrent wheeze in the offspring of treated mothers.39 Recent mouse model studies have demonstrated that prenatal helminth (ie, schistosome) infection can prevent the induction of the allergic asthma phenotype only if pregnancy occurred during the TH1 phases of the infection (eg, chronic) and in a maternal IFN-g–dependent manner, but not if pregnancy occurred during the acute TH2 phase of infection.40
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Microbiome pathogens and protectors Clearly, some bacteria are pathogens, such as the common respiratory pathogens Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae in patients with pneumonia, sinusitis, and otitis media and Staphylococcus aureus in patients with atopic dermatitis. These same organisms can be pathogenic microbiome colonizers and indicators of subsequent and severe disease without causing infectious illness or pathology. For example, with asthma, nasopharyngeal carriage of these same respiratory pathogens without manifestations of infectious illness both in infancy and in children with asthma in later childhood was associated with persistent asthma, severe disease, asthma exacerbations, and sputum neutrophilia.34,41,42 This implicates these respiratory pathogens as microbiome indicators and possibly determinants of asthma development, persistence, severity, and risk of exacerbations. In contrast, some commensal bacteria are thought to confer disease protection. Recently, to fulfill one of Koch’s 4 postulates to establish a causal relationship between a microbe and disease (or prevention), microbiome pathogens versus protectors have been identified in human subjects and tested in mouse models of disease. Chronic rhinosinusitis pathogens and protectors. In a study using modern profiling techniques to determine the sinus microbiome in chronic rhinosinusitis, an abundance of a single fastidious species, Corynebacterium tuberculostearicum, and depletion of Lactobacillus sakei was identified and compared with healthy control subjects.43 C tuberculostearicum was then demonstrated in a mouse model to be a sinus pathogen after pretreatment with a common antibiotic used for respiratory pathogens (amoxicillin–clavulanic acid). Importantly, protection against this organism was conferred by pretreatment with L sakei. Microbial pathogens affecting corticosteroid responsiveness in asthmatic patients. The microbiomes from bronchoalveolar lavage samples obtained from corticosteroidresistant and corticosteroid-sensitive asthmatic patients were compared.44 Although their microbiomes did not differ in richness, evenness, diversity, or community composition at the phylum level, some patients with corticosteroid-resistant asthma had a microbiome expansion of Haemophilus parainfluenzae. Preincubation of asthmatic airway macrophages with this organism inhibited in vitro corticosteroid responses through activation of inhibitory pathways. This was not observed when preincubation of airway macrophages was performed with the commensal bacterium Prevotella melaninogenica. Asthma-protective microbiomes of home environments with dogs. Dog ownership in early life can have a protective influence on allergy and asthma.19,20,45 Households with dogs have rich diverse house dust microbiomes when compared with households without pets, with an abundance of Firmicutes and Bacteroidetes phyla.22 In a mouse model of allergen-induced asthma, oral exposure to extracts of house dust from homes with dogs prevented the development of allergyassociated asthma while altering the cecal microbiome, including enrichment of Lactobacillus johnsonii.46 Oral supplementation with L johnsonii alone prevented development of allergic asthma and respiratory syncytial virus–induced bronchiolitis in this mouse model. In the respiratory syncytial virus model heat-killed L johnsonii supplementation did not confer a protective effect. L johnsonii has also been shown in rodent
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models to mitigate the development of type 1 diabetes and atopic dermatitis.47,48
Mechanisms of action How does a single species of bacterium in the wider microbiome sea of a tolerant gut make a difference in allergy and asthma outcomes? Community leadership. Some bacterial species are community leaders and shape the microbiome around them. As noted above, Bacteroides are abundant in microbiomes associated with less allergy and asthma.10,11,18,22 Bacteroides fragilis and Bacteroides stercoris appear to be keystone bacteria that have a disproportionate influence on intestinal microbiome composition, even though they are only found in moderate abundance.49 Intestinal colonization with B fragilis is dependent on glycan synthesis for the synthesis of essential capsular polysaccharides.50 Mouse studies of B fragilis colonization have determined that bacterial polysaccharide pathways are upregulated and important for B fragilis to penetrate colonic mucus to reach and reside deep in colonic crypt channels, which seem to be the reservoir necessary for long-term colonization of the gut.51 This microbial polysaccharide pathway is necessary for stable B fragilis colonization after microbiome disruption by antibiotic treatment or Citrobacter rodentium infection. The investigation by Fujimura et al46 discussed above also provided insights into how oral supplementation with a single microbe, L johnsonii, might exert its protective effect by altering microbiome composition and function. L johnsonii oral supplementation caused a significant change in cecal microbiome phylogeny by enriching for Rikenellaceae and reducing Clostridium and Bacteroides genera. Predicted cecal microbiome functions shared by L johnsonii–supplemented animals included N-glycan biosynthesis, an important pathway for B fragilis colonization that is downregulated in patients with severe asthma.52 Cellular and molecular mechanisms through which a single microbial species within a vast microbiome can exert their influence on the immune system have been revealed. In some mice, colonization of the small intestine with commensal segmented filamentous bacterium induced lamina propria dendritic cells to induce TH17-type lymphocyte development and enhance resistance to the rodent intestinal pathogen C rodentium.53 B fragilis, which has been associated with allergy and asthma protection, as described above, makes polysaccharide A, which induces forkhead box protein 3–positive Treg lymphocytes directly through TLR2 expressed on these cells, thereby inhibiting pathogenic inflammation, and also symbiotically promotes microbial colonization.54-56 Another bacterium, Acinetobacter lwoffii, has been associated with rural farm cowsheds (believed to be a major source of protective microbial exposures)57 and recently, with greater abundance in the skin of nonatopic versus atopic children.58 A lwoffii induced TH1-type polarization in human monocyte-derived dendritic cells through this bacterium’s LPS (ie, endotoxin).59 Mouse models have further demonstrated A lwoffii’s prenatally induced protective effect on the allergic asthma phenotype in mice through maternal TLR signaling and TH1 induction, which is potentially mediated by epigenetic histone modifications affecting IFN-g promoter accessibility and expression.57,58,60,61
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Helminths are of compelling interest because they induce robust TH2 immune responses while paradoxically being inversely associated with allergy and asthma, which is believed to result from a dominant multifaceted regulatory immune induction.37,38 Some of the molecular products through which helminths induce regulatory immune responses and development include schistosome-specific lysophosphatidylserine, which induces dendritic cells to induce IL-10–producing Treg lymphocytes.62 The intestinal helminth Heligmosomoides polygyrus secretes immunomodulatory molecules that induce forkhead box protein 3–positive Treg lymphocytes, inhibit type 2 innate lymphoid cells, and inhibit allergic airways inflammation in mouse models of asthma.63,64 Thematic to these examples are the broad array of small molecules (eg, proteins, polysaccharides, and lipids) generated by microbes to inhibit allergic immune responses and disease through innate-adaptive regulatory pathways.
CONCLUSIONS Translating hygiene/microbiome theory to microbe-based therapeutics has been considered often.65,66 Effective translation to help address the unmet need of allergy and asthma prevention in human health has been elusive. Compelling scientific leads have been followed in randomized clinical trials without significant benefit for patients and doctors in airways allergy and asthma clinics. For example, regarding probiotic supplementation, a recent systematic review and meta-analysis of 17 randomized controlled trials of probiotic supplementation during pregnancy and/or early life, and a recent long-term study have demonstrated a lasting and statistically significant, although relatively slight, effect on eczema without improvement in airways allergies or asthma.67,68 Also, based on microbial DNA’s immune recognition by TLR9 and inhibition of allergic immune responses, microbebased immunostimulatory DNA was conjugated to the major ragweed allergen Amb a 1 and used in allergen-specific immunotherapy, with improved efficacy and safety in a 6-week regimen69; however, this microbe-based allergy therapeutic is not available for patient care. Clinical trials with helminths have thus far fallen short of the clinics.70 There are potentially effective and safe options on the horizon,16 microbiome research is exploding, and inspiration can be gleaned from recent clinical trials demonstrating effective and safe immunomodulatory prevention of peanut allergy with intervention in infancy71 and an asthma prevention trial with prenatal maternal vitamin D supplementation that is nearing conclusion.72 Controversial and paradoxical, hygiene theory doubters are unlikely to become believers until microbe-based preventive interventions are developed and demonstrated to be effective and safe, which is the end goal for current believers. REFERENCES 1. Liu AH. Hygiene hypothesis for allergy and asthma. In: Martin RJ, Sutherland ER, editors. Asthma and infections. Lung biology in health and disease. New York: Informa Healthcare; 2010. pp. 32-59. 2. Blackley CH. Experimental researches on the causes and nature of catarrhus aestivus (hay-fever or hay-asthma). London: Balliere, Tindall & Cox; 1873. pp. 1-202. 3. Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002;347:869-77. 4. Ege MJ, Mayer M, Normand AC, Genuneit J, Cookson WO, Braun-Fahrlander C, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011;364:701-9.
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5. Holbreich M, Genuneit J, Weber J, Braun-Fahrlander C, Waser M, von Mutius E. Amish children living in northern Indiana have a very low prevalence of allergic sensitization. J Allergy Clin Immunol 2012;129:1671-3. 6. Weber J, Illi S, Nowak D, Schierl R, Holst O, von Mutius E, et al. Asthma and the hygiene hypothesis. Does cleanliness matter? Am J Respir Crit Care Med 2015; 191:522-9. 7. Hesselmar B, Hicke-Roberts A, Wennergren G. Allergy in children in hand versus machine dishwashing. Pediatrics 2015;135:e590-7. 8. Hesselmar B, Sjoberg F, Saalman R, Aberg N, Adlerberth I, Wold AE. Pacifier cleaning practices and risk of allergy development. Pediatrics 2013;131: e1829-37. 9. Strachan DP, Ait-Khaled N, Foliaki S, Mallol J, Odhiambo J, Pearce N, et al. Siblings, asthma, rhinoconjunctivitis and eczema: a worldwide perspective from the International Study of Asthma and Allergies in Childhood. Clin Exp Allergy 2015;45:126-36. 10. Penders J, Gerhold K, Stobberingh EE, Thijs C, Zimmermann K, Lau S, et al. Establishment of the intestinal microbiota and its role for atopic dermatitis in early childhood. J Allergy Clin Immunol 2013;132:601-7.e8. 11. Jakobsson HE, Abrahamsson TR, Jenmalm MC, Harris K, Quince C, Jernberg C, et al. Decreased gut microbiota diversity, delayed Bacteroidetes colonisation and reduced Th1 responses in infants delivered by caesarean section. Gut 2014;63: 559-66. 12. Thorne PS, Kulhankova K, Yin M, Cohn R, Arbes SJ Jr, Zeldin DC. Endotoxin exposure is a risk factor for asthma: the national survey of endotoxin in United States housing. Am J Respir Crit Care Med 2005;172:1371-7. 13. Min KB, Min JY. Exposure to household endotoxin and total and allergen-specific IgE in the US population. Environ Pollut 2015;199:148-54. 14. Matsui EC, Hansel NN, Aloe C, Schiltz AM, Peng RD, Rabinovitch N, et al. Indoor pollutant exposures modify the effect of airborne endotoxin on asthma in urban children. Am J Respir Crit Care Med 2013;188:1210-5. 15. Liu AH. Endotoxin exposure in allergy and asthma: reconciling a paradox. J Allergy Clin Immunol 2002;109:379-92. 16. Martinez FD. The human microbiome. Early life determinant of health outcomes. Ann Am Thorac Soc 2014;11(suppl 1):S7-12. 17. Gern JE, Visness CM, Gergen PJ, Wood RA, Bloomberg GR, O’Connor GT, et al. The Urban Environment and Childhood Asthma (URECA) birth cohort study: design, methods, and study population. BMC Pulm Med 2009;9:17. 18. Lynch SV, Wood RA, Boushey H, Bacharier LB, Bloomberg GR, Kattan M, et al. Effects of early-life exposure to allergens and bacteria on recurrent wheeze and atopy in urban children. J Allergy Clin Immunol 2014;134:593-601.e12. 19. Ownby DR, Johnson CC, Peterson EL. Exposure to dogs and cats in the first year of life and risk of allergic sensitization at 6 to 7 years of age. JAMA 2002;288: 963-72. 20. Remes ST, Castro-Rodriguez JA, Holberg CJ, Martinez FD, Wright AL. Dog exposure in infancy decreases the subsequent risk of frequent wheeze but not of atopy. J Allergy Clin Immunol 2001;108:509-15. 21. Bufford JD, Reardon CL, Li Z, Roberg KA, DaSilva D, Eggleston PA, et al. Effects of dog ownership in early childhood on immune development and atopic diseases. Clin Exp Allergy 2008;38:1635-43. 22. Fujimura KE, Johnson CC, Ownby DR, Cox MJ, Brodie EL, Havstad SL, et al. Man’s best friend? The effect of pet ownership on house dust microbial communities. J Allergy Clin Immunol 2010;126:410-2, e1-3. 23. Haapakoski R, Karisola P, Fyhrquist N, Savinko T, Lehtimaki S, Wolff H, et al. Toll-like receptor activation during cutaneous allergen sensitization blocks development of asthma through IFN-gamma-dependent mechanisms. J Invest Dermatol 2013;133:964-72. 24. Noval Rivas M, Burton OT, Wise P, Zhang YQ, Hobson SA, Garcia Lloret M, et al. A microbiota signature associated with experimental food allergy promotes allergic sensitization and anaphylaxis. J Allergy Clin Immunol 2013; 131:201-12. 25. Oertli M, Noben M, Engler DB, Semper RP, Reuter S, Maxeiner J, et al. Helicobacter pylori gamma-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance. Proc Natl Acad Sci U S A 2013;110:3047-52. 26. Trompette A, Divanovic S, Visintin A, Blanchard C, Hegde RS, Madan R, et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature 2009;457:585-8. 27. Herre J, Gronlund H, Brooks H, Hopkins L, Waggoner L, Murton B, et al. Allergens as immunomodulatory proteins: the cat dander protein Fel d 1 enhances TLR activation by lipid ligands. J Immunol 2013;191:1529-35. 28. Huang YJ, Boushey HA. The microbiome in asthma. J Allergy Clin Immunol 2015;135:25-30. 29. Fujimura KE, Lynch SV. Microbiota in allergy and asthma and the emerging relationship with the gut microbiome. Cell Host Microbe 2015;17:592-602.
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30. Penders J, Gerhold K, Thijs C, Zimmermann K, Wahn U, Lau S, et al. New insights into the hygiene hypothesis in allergic diseases: mediation of sibling and birth mode effects by the gut microbiota. Gut Microbes 2014;5:239-44. 31. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207-14. 32. Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, Frank I, Yadav A, et al. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 2011;184:957-63. 33. Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, et al. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci U S A 2011;108(suppl 1):4578-85. 34. Bisgaard H, Hermansen MN, Buchvald F, Loland L, Halkjaer LB, Bonnelykke K, et al. Childhood asthma after bacterial colonization of the airway in neonates. N Engl J Med 2007;357:1487-95. 35. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 2011;331:337-41. 36. Kallionpaa H, Laajala E, Oling V, Harkonen T, Tillmann V, Dorshakova NV, et al. Standard of hygiene and immune adaptation in newborn infants. Clin Immunol 2014;155:136-47. 37. Smits HH, Akdis CA. In utero priming by worms protects against respiratory allergies. J Allergy Clin Immunol 2014;134:1280-1. 38. Yazdanbakhsh M, Kremsner PG, van Ree R. Allergy, parasites, and the hygiene hypothesis. Science 2002;296:490-4. 39. Mpairwe H, Webb EL, Muhangi L, Ndibazza J, Akishule D, Nampijja M, et al. Anthelminthic treatment during pregnancy is associated with increased risk of infantile eczema: randomised-controlled trial results. Pediatr Allergy Immunol 2011;22:305-12. 40. Straubinger K, Paul S, Prazeres da Costa O, Ritter M, Buch T, Busch DH, et al. Maternal immune response to helminth infection during pregnancy determines offspring susceptibility to allergic airway inflammation. J Allergy Clin Immunol 2014;134:1271-9.e10. 41. Kloepfer KM, Lee WM, Pappas TE, Kang TJ, Vrtis RF, Evans MD, et al. Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations. J Allergy Clin Immunol 2014;133:1301-7, e1-3. 42. Green BJ, Wiriyachaiporn S, Grainge C, Rogers GB, Kehagia V, Lau L, et al. Potentially pathogenic airway bacteria and neutrophilic inflammation in treatment resistant severe asthma. PLoS One 2014;9:e100645. 43. Abreu NA, Nagalingam NA, Song Y, Roediger FC, Pletcher SD, Goldberg AN, et al. Sinus microbiome diversity depletion and Corynebacterium tuberculostearicum enrichment mediates rhinosinusitis. Sci Transl Med 2012;4:151ra24. 44. Goleva E, Jackson LP, Harris JK, Robertson CE, Sutherland ER, Hall CF, et al. The effects of airway microbiome on corticosteroid responsiveness in asthma. Am J Respir Crit Care Med 2013;188:1193-201. 45. Lodge CJ, Allen KJ, Lowe AJ, Hill DJ, Hosking CS, Abramson MJ, et al. Perinatal cat and dog exposure and the risk of asthma and allergy in the urban environment: a systematic review of longitudinal studies. Clin Dev Immunol 2012;2012:176484. 46. Fujimura KE, Demoor T, Rauch M, Faruqi AA, Jang S, Johnson CC, et al. House dust exposure mediates gut microbiome Lactobacillus enrichment and airway immune defense against allergens and virus infection. Proc Natl Acad Sci U S A 2014;111:805-10. 47. Valladares R, Sankar D, Li N, Williams E, Lai KK, Abdelgeliel AS, et al. Lactobacillus johnsonii N6.2 mitigates the development of type 1 diabetes in BB-DP rats. PLoS One 2010;5:e10507. 48. Inoue R, Nishio A, Fukushima Y, Ushida K. Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga. Br J Dermatol 2007;156:499-509. 49. Fisher CK, Mehta P. Identifying keystone species in the human gut microbiome from metagenomic timeseries using sparse linear regression. PLoS One 2014;9: e102451. 50. Coyne MJ, Chatzidaki-Livanis M, Paoletti LC, Comstock LE. Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis. Proc Natl Acad Sci U S A 2008;105:13099-104. 51. Lee SM, Donaldson GP, Mikulski Z, Boyajian S, Ley K, Mazmanian SK. Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 2013;501:426-9.
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52. Orsmark-Pietras C, James A, Konradsen JR, Nordlund B, Soderhall C, Pulkkinen V, et al. Transcriptome analysis reveals upregulation of bitter taste receptors in severe asthmatics. Eur Respir J 2013;42:65-78. 53. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 2009;139:485-98. 54. Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005;122:107-18. 55. Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, et al. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science 2011;332:974-7. 56. Round JL, Mazmanian SK. Inducible Foxp31 regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci U S A 2010;107:12204-9. 57. Debarry J, Garn H, Hanuszkiewicz A, Dickgreber N, Blumer N, von Mutius E, et al. Acinetobacter lwoffii and Lactococcus lactis strains isolated from farm cowsheds possess strong allergy-protective properties. J Allergy Clin Immunol 2007; 119:1514-21. 58. Fyhrquist N, Ruokolainen L, Suomalainen A, Lehtimaki S, Veckman V, Vendelin J, et al. Acinetobacter species in the skin microbiota protect against allergic sensitization and inflammation. J Allergy Clin Immunol 2014;134:1301-9.e11. 59. Debarry J, Hanuszkiewicz A, Stein K, Holst O, Heine H. The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharide. Allergy 2010;65:690-7. 60. Conrad ML, Ferstl R, Teich R, Brand S, Blumer N, Yildirim AO, et al. Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78. J Exp Med 2009;206:2869-77. 61. Brand S, Teich R, Dicke T, Harb H, Yildirim AO, Tost J, et al. Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes. J Allergy Clin Immunol 2011;128:618-25, e1-7. 62. van der Kleij D, Latz E, Brouwers JF, Kruize YC, Schmitz M, Kurt-Jones EA, et al. A novel host-parasite lipid cross-talk. Schistosomal lysophosphatidylserine activates toll-like receptor 2 and affects immune polarization. J Biol Chem 2002;277:48122-9. 63. Grainger JR, Smith KA, Hewitson JP, McSorley HJ, Harcus Y, Filbey KJ, et al. Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway. J Exp Med 2010;207:2331-41. 64. McSorley HJ, Blair NF, Robertson E, Maizels RM. Suppression of OVA-alum induced allergy by Heligmosomoides polygyrus products is MyD88-, TRIF-, regulatory T- and B cell-independent, but is associated with reduced innate lymphoid cell activation. Exp Parasitol 2015 [Epub ahead of print]. 65. Versini M, Jeandel PY, Bashi T, Bizzaro G, Blank M, Shoenfeld Y. Unraveling the hygiene hypothesis of helminthes and autoimmunity: origins, pathophysiology, and clinical applications. BMC Med 2015;13:81. 66. Stiemsma LT, Reynolds LA, Turvey SE, Finlay BB. The hygiene hypothesis: current perspectives and future therapies. ImmunoTargets Ther 2015;4:143-57. 67. Zuccotti G, Meneghin F, Aceti A, Barone G, Callegari ML, Di Mauro A, et al. Probiotics for prevention of atopic diseases in infants: systematic review and meta-analysis. Allergy 2015 [Epub ahead of print]. 68. Simpson MR, Dotterud CK, Storro O, Johnsen R, Oien T. Perinatal probiotic supplementation in the prevention of allergy related disease: 6 year follow up of a randomised controlled trial. BMC Dermatol 2015;15:13. 69. Creticos PS, Schroeder JT, Hamilton RG, Balcer-Whaley SL, Khattignavong AP, Lindblad R, et al. Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis. N Engl J Med 2006;355:1445-55. 70. Evans H, Mitre E. Worms as therapeutic agents for allergy and asthma: understanding why benefits in animal studies have not translated into clinical success. J Allergy Clin Immunol 2015;135:343-53. 71. Du Toit G, Roberts G, Sayre PH, Bahnson HT, Radulovic S, Santos AF, et al. Randomized trial of peanut consumption in infants at risk for peanut allergy. N Engl J Med 2015;372:803-13. 72. Litonjua AA, Lange NE, Carey VJ, Brown S, Laranjo N, Harshfield BJ, et al. The Vitamin D Antenatal Asthma Reduction Trial (VDAART): rationale, design, and methods of a randomized, controlled trial of vitamin D supplementation in pregnancy for the primary prevention of asthma and allergies in children. Contemp Clin Trials 2014;38:37-50.
Aurora and Denver, Colo
The hygiene hypothesis, which describes the protective influence of microbial exposures in early life on the development of allergy and asthma, has continued its swell of academic interest, investigation, and evolution. This article is focused on studies published in the past 3 years that have furthered the substance and shape of hygiene theory, primarily as it relates to allergic airways and asthma. Recent investigations have furthered an overarching ‘‘microbiome hypothesis’’ to home features, medical practices, and cleanliness behaviors that are suspects in the hygiene effect. Relatively crude markers of the protective microbial environment have been supplanted by cultureindependent microbiome science, distinguishing the characteristics of potentially protective microbiomes from pathologic features. Understanding how the microbiome is shaped and affects healthful versus harmful outcomes in the human host is relatively nascent. Good clues are emerging that give mechanistic substance to the theory and could help guide microbe-based therapeutics to fill the allergy and asthma management gap in prevention and disease modification. (J Allergy Clin Immunol 2015;136:860-5.) Key words: Asthma, hygiene hypothesis, asthma prevention
Of microbes and humans: the sequel. With the discovery of microbial pathogens in the mid- to late-1800s, a successful war against these microscopic invaders with a broad-based campaign of public and personal hygiene measures and therapeutic and preventive interventions was a victory for human health. But did this antimicrobial war, with other aspects of modern sanitized living, disrupt an immune balance between microbes and human subjects with an abruptness that did not allow for evolutionary adaptation, leading to the unintended consequence of the allergic march underlying the allergy and asthma epidemic of the past century? Since its academic re-emergence in the past few decades, the hygiene hypothesis (reviewed by Liu1) has been broadly investigated. Some have interpreted this growing body of evidence to have consistency and potential to serve the unmet need of preventive measures for allergy and asthma by mimicking nature’s ways. Others have been doubtful because a wide range of microbes (eg, respiratory viruses, bacteria, and molds) have a well-established
From the Department of Pediatrics/Section of Pulmonary Medicine, Children’s Hospital Colorado and the University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, and National Jewish Health, Denver. Disclosure of potential conflict of interest: A. H. Liu is on the Data Safety Monitoring Committee for GlaxoSmithKline and has received payment for lectures from Merck. Received for publication August 17, 2015; revised August 24, 2015; accepted for publication August 24, 2015. Corresponding author: Andrew H. Liu, MD, Children’s Hospital Colorado, The Breathing Institute, 13123 East 16th Ave, Box B395, Aurora, CO 80045. E-mail: Andrew. [email protected]. 0091-6749/$36.00 Ó 2015 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaci.2015.08.012
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Abbreviations used TLR: Toll-like receptor Treg: Regulatory T URECA: Urban Environment and Childhood Asthma Study
provocative effect on asthma symptoms. How could microbial exposures also have a paradoxical preventive effect on allergies and asthma? The seeds of this paradox were already apparent in the beginnings of allergy’s discovery, with Dr Charles Blackley’s experimental observations that the grass pollen he used for conjunctival challenges to demonstrate pollen’s causal role in ‘‘hay-fever or hay-asthma’’ was obtained from farmers who were nonallergic.2 In keeping with the current theme, let us begin by revisiting Blackley’s farmer collaborators through a recent documentary about allergy and asthma in Amish farmers in America.
THE AMISH IN AMERICA: A CASE STUDY Life on rural European farms has been a rich resource of naturally occurring evidence that microbial exposures from proximal living with domestic animals beginning in early life might protect against the development of allergies and asthma.3,4 This has provided a compelling body of evidence in support of hygiene theory. Could these farming ways indicate environments and lifestyles that are allergy and asthma protective today and were even more so in earlier times before the global increase in allergy and asthma? The Amish, a sect of Anabaptists, adhere to a lifestyle of approximately 150 years back in time, adhering to simple living, plain dress, and a traditional lifestyle eschewing modern conveniences and technology, which is fundamental to their faith. Dairy farming is a desirable lifestyle and avocation for Amish families. The Amish in America emigrated from the Swiss region of the European farm studies noted above. An allergist serving an Amish community in Indiana, Dr Mark Holbreich, in collaboration with rural European farm investigators Drs Erika von Mutius and Charlotte Braun-Fahrlander and their colleagues, determined that Amish children in Indiana have a very low prevalence of allergy, asthma, and aeroallergen sensitization when compared with both Swiss nonfarm and farm children. Results in Amish versus Swiss farm vs Swiss nonfarm children were as follows: asthma, 5.2% vs 6.8% vs 11.2%; hay fever, 0.6% vs 3.1% vs 11.6%; and atopic eczema, 1.3% vs 7.6% vs 12.1%, respectively. Also notable as an objective measure, aeroallergen sensitization was as follows: 7.2% vs 25.2% vs 44.2%, respectively.5 This Amish case study is a provocation to the notion that allergy and asthma were uncommon in a past time and influenced by environments and lifestyles still retained by European dairy farmers today. Hygiene theory implicates human subjects’ microbial environment in early life as essential
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to proper immune development, both toward protective antimicrobial and regulatory immune responses to environmental antigens and away from atopy and asthma. In addition to differences in the microbial environment, there is much to lifestyle differences from modern metropolitan living that are characteristic of Amish farming families. For example, in the study by Holbreich et al,5 the Amish cohort averaged 6 children per family, which was double the numbers seen in the Swiss farmers and nonfarmers. Could the low prevalence of allergy and asthma in these Amish be related to large family size and independent of microbial environment? This study was not large enough to discern lifestyle differences, although it is believed that these children have more physical outdoor play and are needed to participate in farm chores as soon as they are able. This hygiene anecdote is intriguing, even if it is conjecture.
CLEAN (AND NOT SO CLEAN) LIVING Recent investigations have shed light on personal hygiene behaviors suspected of contributing to the hygiene effect. In a German longitudinal birth cohort study, personal cleanliness (eg, hand washing and showering) was associated with lower levels of endotoxin (ie, a bacterial marker) and muramic acid (ie, a fungal marker) in bedding and floor dust.6 In comparison, household cleanliness (eg, cleaning floors and bathrooms, dusting, and changing towels) was associated with less dust but not with lower microbial marker levels. Endotoxin in infancy was associated with less allergic sensitization and less asthma when these children reached school age, whereas muramic acid exposure at school age, but not infancy, was associated with less school-age asthma and eczema. It might be surprising to some that neither personal nor home cleanliness activities were directly associated with atopy or asthma outcomes. This study points to the importance of early-life timing of actual microbial exposures and not cleanliness behaviors, with the influence of endotoxin exposure being in infancy. Two behaviors that could affect exposure to oral microbes were recently associated with less allergy. In a Swedish study hand dishwashing, which is presumed to be less effective in eliminating microbes when compared with machine dishwashing, was associated with less eczema and less ‘‘total allergy’’ (ie, a combination of eczema, asthma, and/or allergic rhinoconjunctivitis).7 The protective effect of hand dishwashing was stronger in children eating fermented foods, farm-bought foods, or both, raising the possibility of specific sources of food-borne microbes being transmitted through the presumably less hygienic method of hand dishwashing. The same research group reported on the parental behavior of infant pacifier ‘‘cleaning’’ by putting it in the parent’s mouth before giving it back to the infant; 48% of the cohort reported this practice in the first 6 months of life.8 Children with parents who orally ‘‘cleaned’’ their pacifiers were less likely to have allergic sensitization, eczema, and ‘‘asthma’’ by age 18 months, implicating parentto-infant oral microbe transmission as atopy protective. If or how these behaviors might significantly alter microbial exposures in an atopy-protective manner or might be behavioral markers of other protective factors is not elucidated but gives hope to the notion that allergy and asthma prevention might be as simple and benign as these frequent oral microbial exposures beginning in early life.
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SUBSTANTIATING PROTECTIVE HOME ENVIRONMENTS Recent studies have substantiated prior observations attributed to hygiene theory. David Strachan, who first demonstrated the relationship of increased sibship with less atopy, extended these associations on a global scale in the International Study of Asthma and Allergies in Childhood cohort.9 Globally, increasing numbers of siblings, especially older siblings, were negatively associated with hay fever and eczema. These associations were stronger in more affluent countries, which is consistent with the notion that less developed nations have additional significant contributors to a hygiene effect. The infant gut microbiome associated with having older siblings was recently shown in a birth cohort to have more Bifidobacterium, Lactobacillus, Escherichia, and Bacteroides genera and less Clostridia.10 This is similar to the infant gut microbiome associated with vaginal delivery versus cesarean section exhibiting greater gut microbial diversity, earlier colonization with Bacteroides, less colonization with Clostridia, and subsequently higher levels of the TH1-associated chemokines CXCL10 and CXCL11 in peripheral blood.10,11 Vaginal delivery and older siblings appear to have additive favorable effects on the gut microbiome. A recent study of ambient endotoxin exposure provides further credence for the paradoxical differences in exposure-health outcomes in adults compared with children. Dose-dependent endotoxin exposure has been associated with more asthma in adults12 and paradoxically less atopy and allergic asthma in children.3,6 In a recent nationally representative US study based on the US National Health and Nutrition Examination Survey 2005-2006, greater house dust endotoxin levels were associated with a lower risk of allergic sensitization to pets and pollens in children/adolescents; in contrast, the risk of aeroallergen sensitization was higher in adults.13 This is consistent with the German longitudinal birth cohort study noted above: endotoxin concentration in mattress dust collected at age 3 months was associated with a lower likelihood of asthma and aeroallergen sensitization at age 5 years.6 Possible explanations for this paradoxical difference include timing of exposure: preventing atopy in early life versus promoting atopy persistence in adulthood. There might also be significant differences in relevant coexposures in children compared with adults. For example, more adults might be exposed to tobacco smoke, which interacts with endotoxin to provoke asthma.14 This duality of healthful versus harmful outcomes from microbial exposures and the importance of key determinants is well exemplified by endotoxin.15,16 THE PARADOX OF US INNER CITIES Because there is a high burden of severe asthma in US inner cities, the National Institutes of Health/National Institute of Allergy and Infectious Diseases has sponsored an inner-city birth cohort study, Urban Environment and Childhood Asthma (URECA), to understand how US inner cities foster severe asthma development.17 From a hygiene hypothesis perspective, inner-city environments could be suspected of being protective on allergy and asthma development, which is a paradox. Although this study is ongoing, recent investigation of URECA participants’ first 3 years of life provides paradigmatic insights.18 First, house dust cockroach, mouse, and cat allergen exposure in the first year of life was negatively associated with recurrent
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wheezing at age 3 years. These indoor allergen exposures had a prominent additive effect, such that recurrent wheeze occurred in 51% of those with no allergen exposures versus 17% of those with all 3 allergen exposures. This protective association was not observed for allergen exposures in the second or third years of life or for allergic sensitization outcomes. This association is similar to what has been observed in other longitudinal birth cohort studies with early-life pet ownership, mostly dog ownership.19-21 However, in the URECA study dog allergen exposure was not protective. Second, a house dust microbiome that is rich and diverse in the first year of life, with an abundance of specific bacterial taxa, most notably Firmicutes and Bacteroidetes phyla, was negatively associated with allergic sensitization and atopic recurrent wheezing at age 3 years. Similarly, households with dogs have rich diverse house dust microbiomes with an abundance of Firmicutes and Bacteroidetes phyla.22 Third, combined high-level exposure to these protective allergens and microbiome features in infancy had additive and independent effects. Higher allergen levels were associated with less recurrent wheeze independent of allergic sensitization. In contrast, protective bacterial taxa were associated with less atopy. Considering these exposures together, children without atopy or recurrent wheeze at age 3 years had the highest percentage, with both high allergen levels plus protective bacterial taxa (48%). In contrast, no children with both atopy and wheeze had the combination of both protective allergens and bacterial taxa. Although it is possible that high-level exposure to cockroach, mouse, and cat allergens has a direct protective effect on recurrent wheeze, these allergens could also be environmental markers for protective microbes. In the URECA study some of the protective bacteria were associated with relevant allergen exposures, suggesting that these bacteria might come from the source of the allergens (eg, cockroaches, mice, and cats) and making it impossible to clearly disentangle the protective effect of the relevant allergens from microbes. Together, protective microbes in the gastrointestinal tract and skin might induce TH1 and tolerizing responses to allergen, as reported in animal model studies.23-25 Some allergens, such as dust mite Der p 2, have inherent innate immune stimulatory capacity26 or can be molecular carriers of microbial components; for example, cat Fel d 1 and dog Can f 1 are carriers for endotoxin.27 Taken together, cohabitation with many creatures in early life, such as pets, pests, older siblings, and domestic animals, could have an additive protective effect on allergy and asthma development, as unified by microbiome theory, working through similar and shared mechanisms.
MICROBIOME THEORY OF ALLERGY AND ASTHMA PREVENTION The microbiome has been avidly investigated as the root of the hygiene hypothesis and possibly the common thread linking the many epidemiologic observations. Microbiome investigation has taken on new depth, in part because of extraordinary advances in the scope of microbiome characterization using new cultureindependent genomic methods. The findings of new microbiome science applied to allergy and asthma has been the subject of several excellent recent reviews.16,28-30 Although the investigational methodologies for characterizing the microbiome are
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advancing and evolving rapidly, making comparisons challenging, there is some consistency of observations such that conclusive evidence is emerging.
Types of microbes The explosion of microbiome interest has been largely about the bacterial component in taxa-level characterization and commensal bacterial mechanisms of action. Methodological advancements, including metagenomic shotgun sequencing approaches applied to investigating the microbiome’s fungal and viral components, are well underway and likely to provide insights into differences in health and disease similar to discoveries with bacteria.29 Microbiome location The microbiome differs dramatically in different locations on or in the body and surrounding environment.31 The microbiomes of the healthy airways are surprisingly homogeneous from top to bottom32; in contrast, microbiomes of the airways, oropharynx, gastrointestinal tract, vaginal tracts of peripartum mothers, skin, surrounding indoor and outdoor environments, and ingested foods differ in the context of health versus allergic/asthmatic disease.29 Investigators are meeting the challenge of extending these observations beyond associations to determine whether specifics of these differing microbial compositions might have causal roles, protective roles, or both. Importance of early life The microbiome in these different locations can evolve rapidly in early life. For example, the microbiome of the infant gastrointestinal tract seems to evolve almost daily during life’s beginning.33 Yet, as noted based on recent investigations, the transitional microbiome in the first year of life but not subsequently is relevant to the development of allergy and asthma.18,34 This is supported by mouse model studies in which early-life but not adult gut colonization with commensal Clostridia induced regulatory T (Treg) lymphocyte development and prevented the allergic asthma phenotype.35 Can maternal preterm exposures significantly influence infant immunity? In a Finnish study of newborn children living in relatively impoverished and ‘‘primitive’’ Karelia, Russia, where allergies and asthma are less common than in modern Finnish cities, cord blood transcriptomes of Karelian neonates were skewed toward innate Toll-like receptor (TLR) responses when compared with those of Finnish neonates, suggesting immune poising for antimicrobial responses at birth.36 Helminths have been inversely associated with allergy and asthma.37,38 The possibility that this protective effect of helminths begins prenatally is suggested by a large randomized controlled trial in which antihelminth treatment with albendazole during pregnancy was associated with an increased likelihood of eczema and recurrent wheeze in the offspring of treated mothers.39 Recent mouse model studies have demonstrated that prenatal helminth (ie, schistosome) infection can prevent the induction of the allergic asthma phenotype only if pregnancy occurred during the TH1 phases of the infection (eg, chronic) and in a maternal IFN-g–dependent manner, but not if pregnancy occurred during the acute TH2 phase of infection.40
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Microbiome pathogens and protectors Clearly, some bacteria are pathogens, such as the common respiratory pathogens Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae in patients with pneumonia, sinusitis, and otitis media and Staphylococcus aureus in patients with atopic dermatitis. These same organisms can be pathogenic microbiome colonizers and indicators of subsequent and severe disease without causing infectious illness or pathology. For example, with asthma, nasopharyngeal carriage of these same respiratory pathogens without manifestations of infectious illness both in infancy and in children with asthma in later childhood was associated with persistent asthma, severe disease, asthma exacerbations, and sputum neutrophilia.34,41,42 This implicates these respiratory pathogens as microbiome indicators and possibly determinants of asthma development, persistence, severity, and risk of exacerbations. In contrast, some commensal bacteria are thought to confer disease protection. Recently, to fulfill one of Koch’s 4 postulates to establish a causal relationship between a microbe and disease (or prevention), microbiome pathogens versus protectors have been identified in human subjects and tested in mouse models of disease. Chronic rhinosinusitis pathogens and protectors. In a study using modern profiling techniques to determine the sinus microbiome in chronic rhinosinusitis, an abundance of a single fastidious species, Corynebacterium tuberculostearicum, and depletion of Lactobacillus sakei was identified and compared with healthy control subjects.43 C tuberculostearicum was then demonstrated in a mouse model to be a sinus pathogen after pretreatment with a common antibiotic used for respiratory pathogens (amoxicillin–clavulanic acid). Importantly, protection against this organism was conferred by pretreatment with L sakei. Microbial pathogens affecting corticosteroid responsiveness in asthmatic patients. The microbiomes from bronchoalveolar lavage samples obtained from corticosteroidresistant and corticosteroid-sensitive asthmatic patients were compared.44 Although their microbiomes did not differ in richness, evenness, diversity, or community composition at the phylum level, some patients with corticosteroid-resistant asthma had a microbiome expansion of Haemophilus parainfluenzae. Preincubation of asthmatic airway macrophages with this organism inhibited in vitro corticosteroid responses through activation of inhibitory pathways. This was not observed when preincubation of airway macrophages was performed with the commensal bacterium Prevotella melaninogenica. Asthma-protective microbiomes of home environments with dogs. Dog ownership in early life can have a protective influence on allergy and asthma.19,20,45 Households with dogs have rich diverse house dust microbiomes when compared with households without pets, with an abundance of Firmicutes and Bacteroidetes phyla.22 In a mouse model of allergen-induced asthma, oral exposure to extracts of house dust from homes with dogs prevented the development of allergyassociated asthma while altering the cecal microbiome, including enrichment of Lactobacillus johnsonii.46 Oral supplementation with L johnsonii alone prevented development of allergic asthma and respiratory syncytial virus–induced bronchiolitis in this mouse model. In the respiratory syncytial virus model heat-killed L johnsonii supplementation did not confer a protective effect. L johnsonii has also been shown in rodent
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models to mitigate the development of type 1 diabetes and atopic dermatitis.47,48
Mechanisms of action How does a single species of bacterium in the wider microbiome sea of a tolerant gut make a difference in allergy and asthma outcomes? Community leadership. Some bacterial species are community leaders and shape the microbiome around them. As noted above, Bacteroides are abundant in microbiomes associated with less allergy and asthma.10,11,18,22 Bacteroides fragilis and Bacteroides stercoris appear to be keystone bacteria that have a disproportionate influence on intestinal microbiome composition, even though they are only found in moderate abundance.49 Intestinal colonization with B fragilis is dependent on glycan synthesis for the synthesis of essential capsular polysaccharides.50 Mouse studies of B fragilis colonization have determined that bacterial polysaccharide pathways are upregulated and important for B fragilis to penetrate colonic mucus to reach and reside deep in colonic crypt channels, which seem to be the reservoir necessary for long-term colonization of the gut.51 This microbial polysaccharide pathway is necessary for stable B fragilis colonization after microbiome disruption by antibiotic treatment or Citrobacter rodentium infection. The investigation by Fujimura et al46 discussed above also provided insights into how oral supplementation with a single microbe, L johnsonii, might exert its protective effect by altering microbiome composition and function. L johnsonii oral supplementation caused a significant change in cecal microbiome phylogeny by enriching for Rikenellaceae and reducing Clostridium and Bacteroides genera. Predicted cecal microbiome functions shared by L johnsonii–supplemented animals included N-glycan biosynthesis, an important pathway for B fragilis colonization that is downregulated in patients with severe asthma.52 Cellular and molecular mechanisms through which a single microbial species within a vast microbiome can exert their influence on the immune system have been revealed. In some mice, colonization of the small intestine with commensal segmented filamentous bacterium induced lamina propria dendritic cells to induce TH17-type lymphocyte development and enhance resistance to the rodent intestinal pathogen C rodentium.53 B fragilis, which has been associated with allergy and asthma protection, as described above, makes polysaccharide A, which induces forkhead box protein 3–positive Treg lymphocytes directly through TLR2 expressed on these cells, thereby inhibiting pathogenic inflammation, and also symbiotically promotes microbial colonization.54-56 Another bacterium, Acinetobacter lwoffii, has been associated with rural farm cowsheds (believed to be a major source of protective microbial exposures)57 and recently, with greater abundance in the skin of nonatopic versus atopic children.58 A lwoffii induced TH1-type polarization in human monocyte-derived dendritic cells through this bacterium’s LPS (ie, endotoxin).59 Mouse models have further demonstrated A lwoffii’s prenatally induced protective effect on the allergic asthma phenotype in mice through maternal TLR signaling and TH1 induction, which is potentially mediated by epigenetic histone modifications affecting IFN-g promoter accessibility and expression.57,58,60,61
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Helminths are of compelling interest because they induce robust TH2 immune responses while paradoxically being inversely associated with allergy and asthma, which is believed to result from a dominant multifaceted regulatory immune induction.37,38 Some of the molecular products through which helminths induce regulatory immune responses and development include schistosome-specific lysophosphatidylserine, which induces dendritic cells to induce IL-10–producing Treg lymphocytes.62 The intestinal helminth Heligmosomoides polygyrus secretes immunomodulatory molecules that induce forkhead box protein 3–positive Treg lymphocytes, inhibit type 2 innate lymphoid cells, and inhibit allergic airways inflammation in mouse models of asthma.63,64 Thematic to these examples are the broad array of small molecules (eg, proteins, polysaccharides, and lipids) generated by microbes to inhibit allergic immune responses and disease through innate-adaptive regulatory pathways.
CONCLUSIONS Translating hygiene/microbiome theory to microbe-based therapeutics has been considered often.65,66 Effective translation to help address the unmet need of allergy and asthma prevention in human health has been elusive. Compelling scientific leads have been followed in randomized clinical trials without significant benefit for patients and doctors in airways allergy and asthma clinics. For example, regarding probiotic supplementation, a recent systematic review and meta-analysis of 17 randomized controlled trials of probiotic supplementation during pregnancy and/or early life, and a recent long-term study have demonstrated a lasting and statistically significant, although relatively slight, effect on eczema without improvement in airways allergies or asthma.67,68 Also, based on microbial DNA’s immune recognition by TLR9 and inhibition of allergic immune responses, microbebased immunostimulatory DNA was conjugated to the major ragweed allergen Amb a 1 and used in allergen-specific immunotherapy, with improved efficacy and safety in a 6-week regimen69; however, this microbe-based allergy therapeutic is not available for patient care. Clinical trials with helminths have thus far fallen short of the clinics.70 There are potentially effective and safe options on the horizon,16 microbiome research is exploding, and inspiration can be gleaned from recent clinical trials demonstrating effective and safe immunomodulatory prevention of peanut allergy with intervention in infancy71 and an asthma prevention trial with prenatal maternal vitamin D supplementation that is nearing conclusion.72 Controversial and paradoxical, hygiene theory doubters are unlikely to become believers until microbe-based preventive interventions are developed and demonstrated to be effective and safe, which is the end goal for current believers. REFERENCES 1. Liu AH. Hygiene hypothesis for allergy and asthma. In: Martin RJ, Sutherland ER, editors. Asthma and infections. Lung biology in health and disease. New York: Informa Healthcare; 2010. pp. 32-59. 2. Blackley CH. Experimental researches on the causes and nature of catarrhus aestivus (hay-fever or hay-asthma). London: Balliere, Tindall & Cox; 1873. pp. 1-202. 3. Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002;347:869-77. 4. Ege MJ, Mayer M, Normand AC, Genuneit J, Cookson WO, Braun-Fahrlander C, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011;364:701-9.
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