INTRODUCTION
The last few decades have seen a dramatic increase in the global prevalence of asthma and allergies [1]. This has been particularly apparent in the western world, although similar increases have been observed in low-income countries [2]. The reasons for this increase (and reported recent decreases in some regions) have not been entirely clear. Genetic changes are unlikely to be responsible due to the short timeframe involved. The increase in allergy contrasts strongly with a reduced prevalence of infectious diseases, a result of improved public health measures, treatment, vaccination programmes and hygiene. This has also led to a corresponding decreased exposure to noninfectious microorganisms, which has been hypothesized as a possible explanation for the allergy and asthma epidemic [3]. This is commonly referred to as the ‘hygiene hypothesis’.
THE HYGIENE HYPOTHESIS
The original ‘hygiene hypothesis’ was prompted by evidence that overcrowding, unhygienic conditions and larger family size were associated with a lower prevalence of atopy, eczema, hay fever and asthma [1,4–7]. Increased infections and exposures to microorganisms and/or their components, particularly early in life, have been proposed as an explanation for these findings [8]. More recently, it has been suggested that the hygiene hypothesis may also be relevant for autoimmune conditions [9–11].
INFECTIONS
Several studies have shown associations between infections (e.g. hepatitis A, measles) or Bacillus Calmette-Guerin (BCG) immunization and a lower prevalence of atopy and allergies [12–15]. However, findings have been inconsistent, particularly for viruses (measles, mumps, rubella and chickenpox) and BCG vaccination [16–19]. It is, therefore, possible that protection is specific to certain infections, at particular times of life. For example, one study [10] found that croup [odds ratio (OR) = 0.3; 95% confidence interval (CI), 0.12–0.72] and repeated ear infections (OR = 0.58; 0.35–0.98) in the first 12 months of life were inversely associated with atopy, whereas bronchiolitis was positively associated with asthma (OR = 2.77; 1.23–6.22). Furthermore, interplay between some early-life respiratory infections and allergic sensitization may increase the risk of asthma development [9].
;)
Box 1: no caption available
A protective effect may not be limited to only microbial or viral exposure. Some helminth infections, such as Schistosoma mansonii, are associated with a strong protective effect against atopy [11]. This may be related to intensity of the infection, as parasite load was inversely associated with SPT and HDM-specific immunoglobulin E [20]. Furthermore, helminth eradication programmes are associated with an increase in atopic sensitization [11,21,22]. For example, in a large study [23] examining the effect of chronic helminth infection on allergy/atopy in school-age children, antihelminthic treatment was associated with a doubled prevalence of allergen skin test reactivity, and increased prevalence of eczema symptoms, without observed increases in asthma or rhinoconjunctivitis. Interestingly, other parasites such as Ascaris and Trichuris may not be protective [24]. It remains unclear as to what is the most important factor in protection from particular manifestations of atopy and asthma: dose, route (oral–faecal versus airborne), type of infection (viral versus bacterial versus parasitic) or location (upper versus lower airways or digestive tract).
NONINFECTIOUS MICROBES
Environmental exposures to proinflammatory microbial agents (such as bacterial endotoxin) have also been suggested to be protective [25–27]. Studies [28–30] in both rural and nonrural environments have reported a significant inverse association between indoor endotoxin levels and atopic sensitization, hay fever and atopic asthma. Recent work from International Study of Asthma and Allergies in Childhood phase II supports this, that is, combined across countries, household endotoxin levels were inversely related with asthma ever {OR = 0.53 [0.29–0.96] and current wheeze [0.77 (0.64–0.93)]}. However, the relationship with atopy was much weaker after adjustment for sex, parental allergies and exposure to cat and house dust mite allergens [31]. Overall, the available data regarding endotoxin are inconsistent. For example, one birth cohort found that early endotoxin exposure was associated with an increased risk of atopy at the age of 2 years [32], whereas two similar birth cohort studies found a protective effect on atopy in 2-year-olds [28] and asthma symptoms in 4-year-olds [30]. Endotoxin exposure has also been shown to be a risk factor for asthma as demonstrated in inner city urban environments [33,34].
Exposure to other microbial components, such as bacterial CpG, peptidoglycans [35], extracellular polysaccharides and fungal 1–3 β-d-glucans [36] may also be protective, but epidemiological evidence remains limited. There are, however, numerous studies showing a protective effect of microbial components against the development of allergic inflammation in murine models. For example, Toll-like receptor (TLR) 9 agonists inhibit the development of asthma-like inflammation [37,38], and intranasal administration of TLR2/4 ligands has been tested as a potential protective vaccine against allergic airway inflammation [39]. Protection may not be elicited through only microbial components. Short-chain fatty acids (SCFAs), produced by bacterial fermentation of dietary fibre, may play a role in gut immunoregulation and reduce allergic airway inflammation in the mouse through SCFA interaction with the G-protein-coupled receptors 41 and 43 [40].
Exposures to different types of nonpathogenic bacteria may have differential effects on atopy and asthma. One birth cohort study [41] showed that exposure to both Gram-positive and Gram-negative bacteria was inversely associated with asthma, whereas only Gram-negative bacteria were associated with allergic sensitization. More recently, studies have suggested that diversity of microbial species, rather than exposure to one particular microbial component, may be particularly protective (reviewed by Heederik and von Mutius [42]). For example, Ege et al.[43▪] showed that the diversity of environmental microorganisms to which individuals were exposed explained a substantial fraction of the inverse association between farm upbringing and asthma. Also, house dust obtained from homes in Russian Karelia (a low-allergy area) contained more Gram-positive bacteria (probably of animal origin) than neighbouring high-allergy Finnish Karelia in which samples contained more Gram-negative and proteobacteria [44]. Furthermore, daycare centres – known to be associated with a lower prevalence of allergies and asthma in attending children – have been shown to be associated with considerable bacterial diversity [45] as have households with daycare attendees or with cats and dogs [46]. Interestingly, a recent study [47] suggested that diversity of exposure may not be limited to microorganisms; in particular, it showed that atopic individuals may also have lower diversity of plant species around their homes, as well as reduced diversity of skin gamma proteobacteria, compared with nonatopics. The mechanisms through which diversity confers protection remain unclear, but one possibility may be that increased diversity simply represents exposure to higher levels of pathogen-associated molecular patterns (PAMPs).
FARMING AND ANIMAL EXPOSURE
Several studies have found consistently low prevalences of allergies and asthma in farmers’ children in both high-income and low-income countries [48–56]. These protective effects have also been observed in adult farmers [49,52,57–59] despite increased risks of other respiratory conditions such as chronic obstructive pulmonary disease [60]. The observed protective effects of farming on allergies and asthma have been particularly strong for animal contact [50,55,61,62] or unpasteurized farm milk consumption [54,55,63,64]. It remains unclear which specific factors are most important, but microbial exposures may play a role either through ingestion (lactobacilli, particularly through raw milk) or inhalation (endotoxin and other microbial components; see above). Protection conferred by farm residence may be dependent upon the type of farming conducted. For example, Ege et al.[65] found that atopic asthma was inversely associated with animal exposures and raw milk consumption, but an effect for nonatopic asthma was only observed with silage exposure and agricultural farming.
Exposure to domestic animals may also confer protection. For example, pet ownership has been shown to be inversely associated with atopy and asthma in children and adults [66–68]. This effect may be particularly strong with multiple exposures; in one birth cohort study [69], children of atopic parents who owned both a dog and a cat were less likely to be atopic at 13 years than those owning only one pet. The evidence of pets protecting against asthma is, however, mixed with some studies reporting no association, or a positive association, despite showing an inverse association with atopy [70]. In other parts of the world (Guinea-Bissau and Nepal), it has been shown that pigs and cattle in the home are associated with less atopy [15,71]. This is consistent with observations that animal contact in farmers’ children may confer protection, and as hypothesized for the farmers’ children, increased microbial exposures may play a role [72].
INTESTINAL MICROBIOTA
Ingestion of lactobacilli associated with raw milk consumption may be important as they can colonize the human gut [73] and may be involved in immunomodulation during development [74–81]; however, various microbes of the gut flora or alterations in total gut microbiota may play a role as well [82–84]. Reduced intestinal biodiversity during infancy has been associated with increased allergy at school age [85▪]. Furthermore, an assessment of gut microbiota in infants using pyrosequencing found that low diversity (reduction in members of Bacteroidetes and Proteobacteria phyla), especially in the first month of life, was associated with atopic eczema [86]. Similar findings of low-gut microbial diversity in allergic individuals have also been reported [87–89], and altering intestinal flora with antibiotics in murine models increases susceptibility to allergic airway inflammation [90]. Furthermore, it has been suggested that the composition of maternal intestinal flora may be related to wheeze in infants, possibly through indirect exposure, or maternal transfer [91▪]. In a mouse model of such prenatal exposure (albeit respiratory), maternal exposure to the cowshed bacterium Acinetobacter lwoffi F78 protected offspring from subsequent development of allergic inflammation through an epigenetic process [92] and was totally dependent upon the presence of maternal (not offspring) TLR signalling [93].
Studies in germ-free (gnotobiotic) mice have indicated that exposure to commensal microbiota is critical for appropriate immune development [94]. Germ-free mice had significantly increased allergic airway inflammation compared with specific pathogen-free (SPF) mice. Although regulatory T-cell populations and regulatory cytokine levels were unaltered, germ-free mice exhibited an increased number of basophils and decreased numbers of alveolar macrophages and plasmacytoid dendritic cells (DCs), suggesting that absence of commensals leads to alterations in allergen-presenting cell populations [95▪]. A study [96] assessing the effect of colonizing germ-free mice at different time points with different microbiota found that expression of hundreds of genes was affected, particularly those involved in type-1 interferon (IFN) induction, showing that altering the commensal microbiota leads to alterations in immune-associated gene expression.
One intriguing recent finding [97▪] suggests that reduced exposure to intestinal microbiota (i.e. in germ-free compared with SPF-specific mice) is associated with expansion of invariant natural killer T cells (iNKTs) cells in the colonic lamina propria and lung, which correlated with increased development of allergic airway inflammation and colitis. The introduction of commensal bacteria prenatally or at early age in previously germ-free mice reduced this risk, whereas inoculation of adult germ-free mice did not have a protective effect. The observed compartmental overrepresentation of iNKTs was associated with increased expression of CXCL16 (an iNKT chemotactic factor), which is hypermethylated in germ-free mice, and may be the epigenetic basis for the lack of effect observed in adult mice. This study suggested that interaction between microbiota and innate cells (such as iNKTs) may be important in protection from allergies and/or asthma, although it is currently unclear how these findings relate to allergies and asthma in humans.
MECHANISMS UNDERLYING THE PROTECTIVE EFFECT OF MICROBIAL EXPOSURE
Microbial exposures activate pattern-recognition receptors, such as TLRs (reviewed in Medzhitov [98]) or CD14 [99], which are specific for different microbial components (or PAMPs). This process is hypothesized to lead to downstream suppression of T-helper-2 (TH2) cell expansion, and therefore TH2-mediated diseases, including allergic asthma, hay fever and eczema [100]. Originally, this was believed to function simply through altering the TH1/TH2 balance, that is, growing up in a more hygienic environment with less microbial exposure would enhance atopic (TH2) immune responses, whereas microbial pressure would drive the response of the immune system – which is known to be skewed in an atopic TH2 direction during fetal and perinatal life – into a TH1 direction and away from its tendency to develop atopic immune responses [101,102]. This model now appears to be oversimplistic, as demonstrated by parasitic infections which are associated with powerful TH2 responses but protect against atopy (see above). Furthermore, endotoxin exposure has been shown to be associated with reduced IFN-γ, tumour necrosis factor-α, interleukin (IL) 12 and IL-10 production by peripheral blood leukocytes, actually suggesting a reduced rather than increased TH1 cytokine milieu [103]. There is also evidence that the exposure dose of PAMP, for example, endotoxin, may in itself alter TH1/TH2 balance [104].
Alternatively, altered immunoregulatory mechanisms may be important. Microbial modulation (either directly or indirectly) of regulatory T cells may result in reduced expression of both TH1 and TH2 immunity. In newborns exposed to farming in utero, an increase in cord blood FoxP3+ T regulatory cells has been described [105]. Similar findings have been observed in mice in which perinatal administration of the probiotic Lactobacillus paracasei NCC2461 prevented allergic inflammation in sensitized/challenged offspring, required TLR2/4 signaling and was associated with increased Fox P3 RNA expression in the lung [106].
Furthermore, it has been reported that bacterial exposure in the airways may protect against allergic inflammation through a TLR4-dependent mechanism, without induction of either T regulatory or TH1 cells but with alterations in DC activation status [107].
Microbial exposure may also protect against nonatopic asthma
Although current data support the idea that microbial exposure is associated with reduced risk of allergies and allergic asthma, there is also evidence that it may be protective against nonallergic asthma. When assessing the effect of farm exposure upon asthma and allergy, Riedler et al.[54] found a reduced risk of asthma (1%; 3/241) in nonatopic children regularly exposed to farm stables compared with 4% (17/399) in those who were not (P = 0.034). A subsequent study [108▪] reproduced this finding, showing that farming was associated with protection against nonatopic wheeze (adjusted odds ratio 0.45, 95%CI 0.32–0.63).
Early-life bacterial exposure may also modulate immunity to more adequately respond to infectious agents. In a murine model of Streptococcus pneumoniae infection, early-life exposure to microbial components was associated with airway colonization by nonpathogenic bacteria (such as lactobacilli), increased airway expression of TLR2, 4 and 9 and decreased neutrophil recruitment [109]. Most importantly, early-life exposure was associated with prolonged survival of pneumonia (with increased expression of IFN-γ, IL-4 and monocyte chemoattractant protein-1) following infection. Thus, colonization may be responsible for increased pathogen resistance and protection against pathogen-mediated nonallergic asthma, in addition to a less inappropriate response to allergens.
Although the lower airways in humans have long been thought sterile, recent studies have shown considerable bacterial colonization, with asthma associated with a preponderance of proteobacteria compared with the Bacteriodetes found in healthy individuals [110]. There is also evidence that some asthmatics are colonized with Mycoplasma and Chlamydial species [111]. Such chronic bacterial infections may be directly responsible for at least a fraction of nonallergic asthma, although it remains to be seen whether they are cause or effect of the asthma process.
PROBLEMS WITH THE HYGIENE HYPOTHESIS
The hygiene hypothesis has gained considerable support from researchers world wide [100]. However, there is a considerable body of evidence warranting scepticizm about the hygiene hypothesis as the primary explanation for global asthma prevalence time trends in particular.
First, it has now well been established that the proportion of asthma attributable to atopy is usually less than one-half [112], with a similar proportion attributable to eosinophilic airway inflammation, the hallmark of allergic asthma [113]. The original hygiene hypothesis suggests that decreased microbial exposure would – through enhanced atopic immune responses – increase the incidence of allergies and allergic asthma. If true, then the protective effects would be most pronounced for the atopic asthma phenotype as well as other atopic conditions such as eczema. However, there is some evidence that nonatopic asthma may have increased more than atopic asthma [114]. Also, farming exposures may also be protective against nonatopic wheeze (see above), suggesting that microbial exposure may not be only affecting the prevalence of allergic conditions. Furthermore, in a repeated population survey among preschool children, an increase in asthma prevalence was not only found in children with the classic asthma pattern of wheeze but in all wheezing phenotypes including viral-induced wheezing [115]. Thus, the hygiene hypothesis may either not fully explain current time trends or the mechanisms through which the hygiene hypothesis mediates protective effects do not exclusively involve enhanced atopic immune responses.
Second, as noted above, asthma prevalence has begun to decline in both children [116,117] and adults [118] in western countries, but it appears unlikely that these countries have become less clean [100], and there is certainly no evidence that family size has increased. For example, although housing conditions are unlikely to have become more hygienic in US inner city populations, asthma prevalence has increased significantly in those populations, particularly among African–Americans living in poverty [119]. Furthermore, studies of specific infections and asthma risk have not consistently demonstrated a protective effect [120]. Also, the observed decline in asthma did not correspond with a decline in atopic eczema and food allergy, which continue to increase in prevalence [121].
A third anomaly is the high asthma prevalences in Latin America, which appear unlikely to have lower infection rates than European countries such as Spain and Portugal which have lower asthma symptom prevalence [122]. Despite this, the early epidemiological findings showing that daycare attendance and farm animal contact were protective of atopy and asthma have been replicated in Latin America (Chile), suggesting a similar effect to that originally observed [123].
Finally, although the hygiene hypothesis is generally explained as a protective effect of early exposures resulting in long-lasting health benefits, recent studies [49,124] suggest that exposures throughout life may be important (and that long-term continual exposure may be required to maintain optimal protection).
CONCLUSION
Ultimately, none of these anomalies is fatal for the hygiene hypothesis in general, but only for a very ‘narrow’ version in which early-life microbial pressure protects against atopic asthma by suppressing TH2 immune responses. A more general version of the hygiene hypothesis may, however, remain valid for asthma. In particular, it is a very useful model to explain the significant protective effects of farming on asthma observed in many studies worldwide [29]; the hygiene hypothesis is consistent with findings that pets in the home may protect against allergies and asthma [67] and many aspects of the hygiene hypothesis can be reproduced in murine models [125]. However, it is unlikely that the hygiene hypothesis can solely explain the large asthma prevalence increases observed or the decline in asthma prevalence observed more recently in western countries. As we have previously noted [100], it is important to consider the ‘forest’ of changes that occur with westernization, as well as the specific ‘trees’, and that the package of changes that come with westernization and increased hygiene may increase asthma risk but not necessarily exclusively through an imbalance of TH1/TH2 immunity. New aetiological theories of global asthma prevalence are, therefore, required that are more consistent with the epidemiological evidence and which take into account factors affecting the time trends for both allergic and nonallergic asthma.
Acknowledgements
The Centre for Public Health Research is supported by a Programme Grant from the Health Research Council (HRC) of New Zealand.
Conflicts of interest
The authors have no conflict of interest to declare.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 122).
REFERENCES
1. Asher MI, Anderson HR, Stewart AW, et al. Worldwide variations in the prevalence of asthma symptoms: International Study of Asthma and Allergies in Childhood (ISAAC). Eur Respir J 1998; 12:315–335.
2. Pearce N, Douwes J. The global epidemiology of asthma in children. Int J Tuberc Lung Dis 2006; 10:125–132.
3. Eder W, Ege MJ, von Mutius E. Current concepts: the asthma epidemic. N Engl J Med 2006; 355:2226–2235.
4. Strachan DP. Hay fever, hygiene, and household size. BMJ 1989; 299:1259–1260.
5. Strachan DP. Allergy and family size: a riddle worth solving. Clin Exp Allergy 1997; 27:235–236.
6. Ball TM, Castro-Rodriguez JA, Griffith KA, et al. Siblings, day-care attendance, and the risk of asthma and wheezing during childhood. N Engl J Med 2000; 343:538–543.
7. Kramer U, Heinrich J, Wjst M, Wichmann HE. Age of entry to day nursery and allergy in later childhood. Lancet 1999; 353:450–454.
8. Martinez FD. Role of viral infections in the inception of asthma and allergiesw during childhood: could they be protective? Thorax 1994; 49:1189–1191.
9. Sly PD, Boner AL, Bjorksten B, et al. Early identification of atopy in the prediction of persistent asthma in children. Lancet 2008; 372:1100–1106.
10. Ramsey CD, Gold DR, Litonjua AA, et al. Respiratory illnesses in early life and asthma and atopy in childhood. J Allergy Clin Immunol 2007; 119:150–156.
11. Flohr C, Tuyen LN, Lewis S, et al. Poor sanitation and helminth infection protect against skin sensitization in Vietnamese children: a cross-sectional study. J Allergy Clin Immunol 2006; 118:1305–1311.
12. Aaby P, Shaheen SO, Heyes CB, et al. Early BCG vaccination and reduction in atopy in Guinea-Bissau. Clin Exp Allergy 2000; 30:644–650.
13. Matricardi PM, Rosmini F, Ferrigno L, et al. Cross sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis A virus. BMJ 1997; 314:999–1003.
14. Shaheen SO, Aaby P, Hall AJ, et al. Cell mediated immunity after measles in Guinea-Bissau: historical cohort study. BMJ 1996; 313:969–974.
15. Shirakawa T, Enomoto T, Shimazu S, Hopkin JM. The inverse association between tuberculin responses and atopic disorder. Science 1997; 275:77–79.
16. Alm JS, Lilja G, Pershagen G, Scheynius A. Early BCG vaccination and development of atopy. Lancet 1997; 350:400–403.
17. Alm JS, Lilja G, Pershagen G, Scheynius A. BCG vaccination does not seem to prevent atopy in children with atopic heredity. Allergy 1998; 53:537.
18. Gruber C, Kulig M, Bergmann R, et al. Delayed hypersensitivity to tuberculin, total immunoglobulin E, specific sensitization, and atopic manifestation in longitudinally followed early bacille Calmette-Gue’rin-vaccinated and nonvaccinated children. Pediatrics 2001; 107:U45–U51.
19. Matricardi PM, Rosmini F, Riondino S, et al. Exposure to foodborne and orofecal microbes versus airborne viruses in relation to atopy and allergic asthma: epidemiological study. BMJ 2000; 320:412–417.
20. Rujeni N, Nausch N, Bourke CD, et al. Atopy is inversely related to schistosome infection intensity: a comparative study in Zimbabwean villages with distinct levels of schistosoma haematobium infection. Int Arch Allergy Immunol 2012; 158:288–298.
21. Lynch NR. Effect of age and helminthic infection on IgE levels in slum children. J Investig Allergol Clin Immunol 1993; 3:276–376.
22. van den Biggelaar AHJ, Rodrigues LC, van Ree R, et al. Long-term treatment of intestinal helminths increases mite skin-test reactivity in Gabonese schoolchildren. J Infect Dis 2004; 189:892–900.
23. Endara P, Vaca M, Chico ME, et al. Long-term periodic anthelmintic treatments are associated with increased allergen skin reactivity. Clin Exp Allergy 2010; 40:1669–1677.
24. Karadag B, Ege M, Bradley JE, et al. The role of parasitic infections in atopic diseases in rural schoolchildren. Allergy 2006; 61:996–1001.
25. Liu AH, Leung DYM. Modulating the early allergic response with endotoxin. Clin Exp Allergy 2000; 30:1535–1539.
26. Tulic MK, Wale JL, Holt PG, Sly PD. Modification of the inflammatory response to allergen challenge after exposure to bacterial lipopolysaccharide. Am J Respir Cell Mol Biol 2000; 22:604–612.
27. von Mutius E, Braun-Fahrlander C, Schierl R, et al. Exposure to endotoxin or other bacterial components might protect against the development of atopy. Clin Exp Allergy 2000; 30:1230–1234.
28. Bottcher MF, Bjorksten B, Gustafson S, et al. Endotoxin levels in Estonian and Swedish house dust and atopy in infancy. Clin Exp Allergy 2003; 33:295–300.
29. Braun-Fahrlander C, Lauener R. Farming and protective agents against allergy and asthma. Clin Exp Allergy 2003; 33:409–411.
30. Douwes J, van Strien R, Doekes G, et al. Does early indoor microbial exposure reduce the risk of asthma? The Prevention and Incidence of Asthma and Mite Allergy birth cohort study. J Allergy Clin Immunol 2006; 117:1067–1073.
31. Gehring U, Bolte G, Borte M, et al. Exposure to endotoxin decreases the risk of atopic eczema in infancy: a cohort study. J Allergy Clin Immunol 2001; 108:847–854.
32. Bolte G, Bischof W, Borte M, et al. Early endotoxin exposure and atopy development in infants: results of a birth cohort study. Clin Exp Allergy 2003; 33:770–776.
33. Perzanowski MS, Miller RL, Thorne PS, et al. Endotoxin in inner-city homes: associations with wheeze and eczema in early childhood. J Allergy Clin Immunol 2006; 117:1082–1089.
34. Tavernier GOG, Fletcher GD, Francis HC, et al. Endotoxin exposure in asthmatic children and matched healthy controls: results of IPEADAM study. Indoor Air 2005; 15:25–32.
35. van Strien RT, Engel R, Holst O, et al. Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid in mattress dust, and its association with respiratory health. J Allergy Clin Immunol 2004; 113:860–867.
36. Douwes J, van der Sluis B, Doekes G, et al. Fungal extracellular polysaccharides in house dust as a marker for exposure to fungi: Relations with culturable fungi, reported home dampness, and respiratory symptoms. J Allergy Clin Immunol 1999; 103:494–500.
37. Jain VV, Kitagaki K, Businga T, et al. CpG-oligodeoxynucleotides inhibit airway remodeling in a murine model of chronic asthma. J Allergy Clin Immunol 2002; 110:867–872.
38. Kline JN, Waldschmidt TJ, Businga TR, et al. Cutting edge: modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 1998; 160:2555–2559.
39. Shalaby KH, Jo T, Nakada E, et al. ICOS-expressing CD4 T cells induced via TLR4 in the nasal mucosa are capable of inhibiting experimental allergic asthma. J Immunol 2012; 189:2793–2804.
40. Maslowski KM, Vieira AT, Ng A, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009; 461:1282–2119.
41. Sordillo JE, Hoffman EB, Celedon JC, et al. Multiple microbial exposures in the home may protect against asthma or allergy in childhood. Clin Exp Allergy 2010; 40:902–910.
42. Heederik D, von Mutius E. Does diversity of environmental microbial exposure matter for the occurrence of allergy and asthma? J Allergy Clin Immunol 2012; 130:44–50.
43▪. Ege MJ, Mayer M, Normand AC, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011; 364:701–709.
This study, conducted as part of the prevention of allergy–risk factors for sensitization related to farming and anthroposophic lifestyle and GABRIELA (A multidisciplinary study to identify the genetic and environmental causes of asthma in the European community) studies in Europe, showed a strong association between bacterial diversity (as meausured in mattress dust samples using single-strand conformation polymorphism) and protection against asthma.
44. Pakarinen J, Hyvarinen A, Salkinoja-Salonen M, et al. Predominance of Gram-positive bacteria in house dust in the low-allergy risk Russian Karelia. Environ Microbiol 2008; 10:3317–3325.
45. Lee L, Tin S, Kelley ST. Culture-independent analysis of bacterial diversity in a child-care facility. BMC Microbiol 2007; 7:27.
46. Maier RM, Palmer MW, Andersen GL, et al. Environmental determinants of and impact on childhood asthma by the bacterial community in household dust. Appl Environ Microbiol 2010; 76:2663–2667.
47. Hanski I, von Hertzen L, Fyhrquist N, et al. Environmental biodiversity, human microbiota, and allergy are interrelated. Proc Natl Acad Sci USA 2012; 109:8334–8339.
48. Braun-Fahrlander C, Gassner M, Grize L, et al. Prevalence of hay fever and allergic sensitization in farmer's children and their peers living in the same rural community. Clin Exp Allergy 1999; 29:28–34.
49. Douwes J, Travier N, Huang K, et al. Lifelong farm exposure may strongly reduce the risk of asthma in adults. Allergy 2007; 62:1158–1165.
50. Downs SH, Marks GB, Mitakakis TZ, et al. Having lived on a farm and protection against allergic diseases in Australia. Clin Exp Allergy 2001; 31:570–575.
51. Ernst P, Cormier Y. Relative scarcity of asthma and atopy among rural adolescents raised on a farm. Am J Respir Crit Care Med 2000; 161:1563–1566.
52. Kilpelainen M, Terho EO, Helenius H, Koskenvuo M. Farm environment in childhood prevents the development of allergies. Clin Exp Allergy 2000; 30:201–208.
53. Portengen L, Sigsgaard T, Omland O, et al. Low prevalence of atopy in young Danish farmers and farming students born and raised on a farm. Clin Exp Allergy 2002; 32:247–253.
54. Riedler J, Braun-Fahrlander C, Eder W, et al. Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey. Lancet 2001; 358:1129–1133.
55. Riedler J, Eder W, Oberfeld G, Schreuer M. Austrian children living on a farm have less hay fever, asthma and allergic sensitization. Clin Exp Allergy 2000; 30:194–200.
56. Von Ehrenstein OS, Von Mutius E, Illi S, et al. Reduced risk of hay fever and asthma among children of farmers. Clin Exp Allergy 2000; 30:187–193.
57. Braback L, Hjern A, Rasmussen F. Trends in asthma, allergic rhinitis and eczema among Swedish conscripts from farming and nonfarming environments: a nationwide study over three decades. Clin Exp Allergy 2004; 34:38–43.
58. Eduard W, Douwes J, Omenaas E, Heederik D. Do farming exposures cause or prevent asthma? Results from a study of adult Norwegian farmers. Thorax 2004; 59:381–386.
59. Smit LAM, Zuurbier M, Doekes G, et al. Hay fever and asthma symptoms in conventional and organic farmers in the Netherlands. Occup Environ Med 2007; 64:101–107.
60. Schenker MB, Christiani D, Cormier Y, et al. Respiratory health hazards in agriculture. Am J Respir Crit Care Med 1998; 158:S1–S76.
61. Douwes J, Pearce N. Commentary: the end of the hygiene hypothesis? Int J Epidemiol 2008; 37:570–572.
62. Remes ST, Iivanainen K, Koskela H, Pekkanen J. Which factors explain the lower prevalence of atopy amongst farmers’ children? Clin Exp Allergy 2003; 33:427–434.
63. Waser M, Michels KB, Bieli C, et al. Inverse association of farm milk consumption with asthma and allergy in rural and suburban populations across Europe. Clin Exp Allergy 2007; 37:661–670.
64. Wickens K, Lane JM, Fitzharris P, et al. Farm residence and exposures and the risk of allergic diseases in New Zealand children. Allergy 2002; 57:1171–1179.
65. Ege MJ, Frei R, Bieli C, et al. Not all farming environments protect against the development of asthma and wheeze in children. J Allergy and Clin Immunol 2007; 119:1140–1147.
66. de Meer G, Toelle BG, Ng K, et al. Presence and timing of cat ownership by age 18 and the effect on atopy and asthma at age 28. J Allergy Clin Immunol 2004; 113:433–438.
67. Hesselmar B, Aberg N, Aberg B, et al. Does early exposure to cat or dog protect against later allergy development? Clin Exp Allergy 1999; 29:611–617.
68. Oryszczyn MP, Van Ree R, Maccario J, et al. Cat sensitization according to cat window of exposure in adult asthmatics. Clin Exp Allergy 2009; 39:1515–1521.
69. Mandhane PJ, Sears MR, Poulton R, et al. Cats and dogs and the risk of atopy in childhood and adulthood. J Allergy Clin Immunol 2009; 124:745–750.
70. Kerkhof M, Wijga AH, Brunekreef B, et al. Effects of pets on asthma development up to 8 years of age: the PIAMA study. Allergy 2009; 64:1202–1208.
71. Melsom T, Brinch L, Hessen JO, et al. Asthma and indoor environment in Nepal. Thorax 2001; 56:477–481.
72. Douwes J, Pearce N, Heederik D. Does environmental endotoxin exposure prevent asthma?[comment]. Thorax 2002; 57:86–90.
73. Johansson ML, Molin G, Jeppsson B, et al. Administration of different lactobacillus strains in fermented oatmeal soup: in vivo colonisation of human intestinal mucosa and effect on the indigenous flora. Appl Environ Microbiol 1993; 59:15–20.
74. Bjorksten B, Naaber P, Sepp E, Mikelsaar M. The intestinal microflora in allergic Estonian and Swedish 2-year-old children. Clin Exp Allergy 1999; 29:342–346.
75. Hessle C, Hanson LA, Wold AE. Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production. Clin Exp Immunol 1999; 116:276–282.
76. Kalliomaki M, Salminen S, Arvilommi H, et al. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001; 357:1076–1079.
77. Murosaki S, Yamamoto Y, Ito K, et al. Heat-killed Lactobacillus plantarum L-137 suppresses naturally fed antigen-specific IgE production by stimulation of IL-12 production in mice. J Allergy Clin Immunol 1998; 102:57–64.
78. Pessi T, Sutas Y, Hurme H, Isolauri E. Interleukin-10 generation in atopic children following oral Lactobacillus rhamnosus GG. Clin Exp Allergy 2000; 30:1804–1808.
79. Sepp E, Julge K, Vasar M, et al. Intestinal microflora of Estonian and Swedish infants. Acta Paediatr 1997; 86:956–961.
80. Shida K, Makino K, Morishita A, et al. Lactobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte cultures. Int Arch Allergy Immunol 1998; 115:278–287.
81. Shida K, Takahashi R, Iwadate E, et al. Lactobacillus casei strain Shirota suppresses serum immunoglobulin E and immunoglobulin G1 responses and systemic anaphylaxis in a food allergy model. Clin Exp Allergy 2002; 32:563–570.
82. Bjorksten B, Sepp E, Julge K, et al. Allergy development and the intestinal microflora during the first year of life. J Allergy Clin Immunol 2001; 108:516–520.
83. Bottcher MF, Nordin EK, Sandin A, et al. Microflora-associated characteristics in faeces from allergic and nonallergic infants. Clin Exp Allergy 2000; 30:1590–1596.
84. Kalliomaki M, Kirjavainen P, Eerola E, et al. Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin Immunol 2001; 107:129–134.
85▪. Bisgaard H, Li N, Bonnelykke K, et al. Reduced diversity of the intestinal microbiota during infancy is associated with increased risk of allergic disease at school age. J Allergy Clin Immunol 2011; 128:646–652.
This study assessed bacterial flora (using 16 s RNA-based typing) at 1 or 2 months, and found that diversity of microbiota was inversely associated with allergic sensitization but not subsequent development of asthma or atopic dermatitis.
86. Abrahamsson TR, Jakobsson HE, Andersson AF, et al. Low diversity of the gut microbiota in infants with atopic eczema. J Allergy Clin Immunol 2012; 129:434–440.
87. Sjogren YM, Jenmalm MC, Bottcher MF, et al. Altered early infant gut microbiota in children developing allergy up to 5 years of age. Clin Exp Allergy 2009; 39:518–526.
88. Stsepetove J, Sepp E, Julge K, et al. Molecularly assessed shifts of Bifidobacterium ssp and less diverse microbial communities are characteristic of 5-year-old allergic children. FEMS Immunol Med Microbiol 2007; 51:260–269.
89. Wang M, Karlsson C, Olsson C, et al. Reduced diversity in the early fecal microbiota of infants with atopic eczema. J Allergy Clin Immunol 2008; 121:129–134.
90. Russell SL, Gold MJ, Hartmann M, et al. Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma. EMBO Rep 2012; 13:440–447.
91▪. Lange NE, Celedon JC, Forno E, et al. Maternal intestinal flora and wheeze in early childhood. Clin Exp Allergy 2012; 42:901–908.
This study of 60 pregnant women observed that higher counts of total aerobes and enterococci in maternal stool samples were associated with increased risk of infant wheeze (OR 2.32 for 1 log increase in colony-forming unit/g stool), but no microorganisms were associated with either eczema or atopic wheeze.
92. Brand S, Teich R, Dicke T, et al. Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes. J Allergy Clin Immunol 2011; 128:618–625.
93. Conrad ML, Ferstl R, Teich R, et al. Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78. J Exp Med 2009; 206:2869–2877.
94. Tlaskalova-Hogenova H, Stepankova R, Kozakova H, et al. The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases. Cell Mol Immunol 2011; 8:110–120.
95▪. Herbst T, Sichelstiel A, Schaer C, et al. Dysregulation of allergic airway inflammation in the absence of microbial colonization. Am J Respir Crit Care Med 2011; 184:198–205.
In a mouse model of allergic airway inflammation, numbers of infiltrating lymphocytes and eosinophils were elevated in the airways of allergic germ-free mice compared with control mice. This could be reversed by recolonization of germ-free mice with the complex commensal flora of SPF mice.
96. Yamamoto M, Yamaguchi R, Munakata K, et al. A microarray analysis of gnotobiotic mice indicating that microbial exposure during the neonatal period plays an essential role in immune system development. BMC genomics 2012; 13:335.
97▪. Olszak T, An D, Zeissig S, et al. Microbial exposure during early life has persistent effects on natural killer T cell function. Science 2012; 336:489–493.
In germ-free mice, reduced exposure to intestinal microbiota was associated with expansion of iNKT cells in the intestine and lung, correlating with development of allergic airway inflammation and colitis. The introduction of commensal bacteria prenatally or at early age in previously germ-free mice reduced this risk, whereas inoculation of adult germ-free mice did not have a protective effect.
98. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001; 1:135–145.
99. Ege MJ, Bieli C, Frei R, et al. Prenatal farm exposure is related to the expression of receptors of the innate immunity and to atopic sensitization in school-age children. J Allergy Clin Immunol 2006; 117:817–823.
100. Douwes J, Pearce N. Asthma and the westernization ’package’. Int J Epidemiol 2002; 31:1098–1102.
101. Holt PG, Sly PD, Bjorksten B. Atopic versus infectious diseases in childhood: a question of balance? Pediatr Allergy Immunol 1997; 8:53–58.
102. Martinez FD, Holt PG. Role of microbial burden in aetiology of allergy and asthma. Lancet 1999; 354:S12–S15.
103. Braun-Fahrlander C, Riedler J, Herz U, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002; 347:869–877.
104. Eisenbarth SC, Piggott DA, Huleatt JW, et al. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002; 196:1645–1651.
105. Schaub B, Liu J, Hoppler S, et al. Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells. J Allergy Clin Immunol 2009; 123:774–782.e5.
106. Schabussova I, Hufnagl K, Tang MLK,
et al. Perinatal maternal administration of Lactobacillus paracasei NCC 2461 prevents allergic inflammation in a mouse model of birch pollen allergy. Plos One 2012; 7:e40271.
107. Nembrini C, Sichelstiel A, Kisielow J, et al. Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism. Thorax 2011; 66:755–763.
108▪. Fuchs O, Genuneit J, Latzin P,
et al. Farming environments and childhood atopy, wheeze, lung function, and exhaled nitric oxide. J Allergy Clin Immunol 2012; 130:382.
In the GABRIELA study (including 8023 children aged 6–12 years), exposure to farming environments affected the prevalence and degree of atopy, the prevalence of transient wheeze (adjusted odds ratio, 0.78; 95% CI, 0.64–0.96) and the prevalence of current wheeze among nonatopic patients (adjusted odds ratio, 0.45; 95% CI, 0.32–0.63). No farm effect on lung function and exhaled nitric oxide levels was observed.
109. Yasuda Y, Matsumura Y, Kasahara K, et al. Microbial exposure early in life regulates airway inflammation in mice after infection with Streptococcus pneumoniae with enhancement of local resistance. Am J Physiol Lung Cell Mol Physiol 2010; 298:L67–L78.
110. Hilty M, Burke C, Pedro H,
et al. Disordered microbial communities in asthmatic airways. Plos One 2010; 5:e8578.
111. Lemanske RF. Is asthma an infectious disease? Thomas A. Neff lecture. Chest 2003; 123:385S–390S.
112. Pearce N, Pekkanen J, Beasley R. How much asthma is really attributable to atopy? Thorax 1999; 54:268–272.
113. Douwes J, Gibson P, Pekkanen J, Pearce N. Noneosinophilic asthma: importance and possible mechanisms [comment]. Thorax 2002; 57:643–648.
114. Thomsen SF, Ulrik CS, Larsen K, Backer V. Change in prevalence of asthma in Danish children and adolescents. Ann Allergy Asthma Immunol 2004; 92:506–511.
115. Kuehni CE, Davis A, Brooke AM, Silverman M. Are all wheezing disorders in very young: (preschool) children increasing in prevalence? Lancet 2001; 357:1821–1825.
116. Asher MI, Montefort S, Bjorksten B, et al. Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC phases one and three repeat multicountry cross-sectional surveys. Lancet 2006; 368:733–743.
117. Pearce N, Ait-Khaled N, Beasley R, et al. Worldwide trends in the prevalence of asthma symptoms: phase III of the International Study of Asthma and Allergies in Childhood (ISAAC). Thorax 2007; 62:758–766.
118. Chinn S, Jarvis D, Burney P, et al. Increase in diagnosed asthma but not in symptoms in the European Community Respiratory Health Survey. Thorax 2004; 59:646–651.
119. Crater DD, Heise S, Perzanowski M. Asthma hospitalization trends in Charleston, South Carolina, 1956 to 1997: twenty-fold increase among black children during a 30-year period. Paediatrics 2001; 108:E97.
120. Pearce N, Douwes J, Beasley R. Oxford textbook of public health. 4th ed. In: Tanaka H, editor. Asthma. Oxford: Oxford University Press; 2002. pp. 1255–1277.
121. Ponsonby A-L, Glasgow N, Pezic A,
et al. A temporal decline in asthma but not eczema prevalence from 2000-2005 at school entry in the Australian Capital Territory with further consideration of country of birth. Int J Epidemiol 2008; 37:559–569.
122. Pearce N, Douwes JT. The Latin American exception: why is childhood asthma so prevalent in Brazil? J Paediatr 2006; 82:319–321.
123. Boneberger A, Haider D, Baer J, et al. Environmental Risk factors in the first year of life and childhood asthma in the central south of Chile. J Asthma 2011; 48:464–469.
124. Douwes J, Le Gros G, Gibson P, Pearce N. Can bacterial endotoxin exposure reverse atopy and atopic disease? J Allergy Clin Immunol 2004; 114:1051–1054.
125. Lundy SK, Berlin AA, Lukacs NW. Interleukin-12-independent down-modulation of cockroach antigen-induced asthma in mice by intranasal exposure to bacterial lipopolysaccharide. Am J Pathol 2003; 163:1961–1968.
