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Disconnection of man and the soil: Reason for the asthma and atopy epidemic?

      Intense search has been going on to find factors responsible for the asthma and atopy epidemic in Western societies. Attention has increasingly been devoted to environmental saprophytes, which, in addition to gut commensals, might be the major players in the development and fine tuning of immunologic homeostasis. This review outlines current evidence for the role of environmental saprophytes in the development of atopic disease and considers the consequences of urbanization in reducing contacts with soil microorganisms. The major microbial components that have been shown to possess immunomodulatory capacity and their respective Toll-like receptors are also discussed, as are the possible mechanisms underlying the ability of saprophytes to confer protection against atopic disease.

      Key words

      Abbreviations used:

      TLR (Toll-like receptor), Treg cell (Regulatory T cell)
      The current asthma and atopy epidemic in Western societies has raised a common concern and questions of factors involved. Although in some countries prevalence rates in atopic diseases appear to have leveled off,
      • Braun-Fahrländer C.
      • Gassner M.
      • Grize L.
      • Takken-Sahli K.
      • Neu U.
      • Sticker T.
      • et al.
      No further increase in asthma, hay fever and atopic sensitisaton in adolescents living in Switzerland.
      • Ronchetti R.
      • Villa M.P.
      • Barreto M.
      • Rota R.
      • Pagani J.
      • Martella S.
      • et al.
      Is the increase in childhood asthma coming to an end? Findings from three surveys of schoolchildren in Rome, Italy.
      • Verlato G.
      • Corsico A.
      • Villani S.
      • Cerveri I.
      • Migliore E.
      • Accordini S.
      • et al.
      Is the prevalence of adult asthma and allergic rhinitis still increasing? Results of an Italian study.
      • Yura A.
      • Shimizu T.
      Trends in the prevalence of atopic dermatitis in school children: longitudinal study in Osaka Prefecture, Japan, from 1985 to 1997.
      trends are still on the increase in many other countries.
      • Bråbäck L.
      • Hjern A.
      • Rasmussen F.
      Trends in asthma, allergic rhinitis and eczema among Swedish conscripts from farming and non-farming environments. A nationwide study over three decades.
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.
      Numerous studies have consistently shown that high asthma and atopy rates are associated with urbanization and Western lifestyle.
      • von Mutius E.
      The environmental predictors of allergic disease.
      • von Hertzen L.
      • Haahtela T.
      Asthma and atopy—the price of affluence?.
      Accumulating data suggest that something that is necessary for the normal maturation of the immune system might be lacking in our affluence.
      • Ring J.
      • Krämer U.
      • Schäfer T.
      • Behrendt H.
      Why are allergies increasing?.
      Conversely, farm environment and a more traditional lifestyle in nonaffluent countries appear to confer protection against atopic disease.
      • von Hertzen L.
      • Haahtela T.
      Asthma and atopy—the price of affluence?.
      • von Mutius E.
      Influences in allergy: epidemiology and the environment.
      • Björksten B.
      • Dumitrascu D.
      • Foucard T.
      • Khetsuriani N.
      • Khaitov R.
      • Leja M.
      • et al.
      Prevalence of childhood asthma, rhinitis and eczema in Scandinavia and Eastern Europe.
      Although the ultimate factors responsible for the asthma and atopy epidemic have remained unidentified, a common denominator for both living on a farm and in a nonaffluent environment is the heavy exposure to microorganisms in soil and vegetation.
      Most of the microorganisms we encounter do not cause any overt infection but are still recognized by the innate immune system. Microbes in this respect need not be alive because even nonviable microbial components interact with the innate immune system. Persistent and moderate environmental exposure to microbial components might play a decisive role in the normal maturation of the immune system in childhood.
      • Kabesch M.
      • Lauener R.P.
      Why Old McDonald had a farm but no allergies: genes, environment, and the hygiene hypothesis.
      It has been proposed that certain microorganisms that have been present throughout the mammalian evolutionary history are recognized by the innate immune system as “no danger” signals and thus do not trigger inflammatory responses but instead have the ability to induce tolerance through rapid regulatory T (Treg) cell responses.
      • Rook G.A.W.
      • Adams V.
      • Hunt J.
      • Palmer R.
      • Martinelli R.
      • Brunet L.R.
      Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders.
      These organisms include saprophytic mycobacteria, lactobacilli, and some intestinal parasites that are able to elicit Treg cell responses in vivo
      • Zuany-Amorim C.
      • Manlius C.
      • Trifilieff A.
      • Brunet L.R.
      • Rook G.
      • Bowen G.
      • et al.
      Long-term protective and antigen-specific effect of heat-killed Mycobacterium vaccae in a murine model of allergic pulmonary inflammation.
      • Adams V.C.
      • Hunt J.R.
      • Martinelli R.
      • Palmer R.
      • Rook G.A.
      • Brunet L.R.
      Mycobacterium vaccae induces a population of pulmonary CD11c+ cells with regulatory potential in allergic mice.
      • van den Biggelaar A.H.
      • van Ree R.
      • Rodrigues L.C.
      • Lell B.
      • Deelder A.M.
      • Kremsner P.G.
      • et al.
      Decreased atopy in children infected with Schistosoma haematobium: a role for parasite-induced interleukin-10.
      • Yazdanbakhsh M.
      • Kremsner P.
      • van Ree R.
      Allergy, parasites and the hygiene hypothesis.
      and in vitro.
      • Smits H.
      • Engering A.
      • van der Kleij D.
      • de Jong E.
      • Schipper K.
      • van Capel T.
      • et al.
      Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin.
      The list of such microbes will certainly grow in the next few years.
      The focus of the research in the context of the hygiene hypothesis has largely shifted from overt infections and the TH1/TH2 paradigm to noninfectious organisms, Treg cells, and Toll-like receptors (TLRs), as new data have been accumulated and the paradigm was found to be unable to unambiguously explain some important epidemiologic findings.
      • Smit J.J.
      • Folkerts G.
      • Nijkamp F.P.
      Mycobacteria, genes and the ‘hygiene hypothesis’.
      • Horner A.A.
      • Redecke V.
      • Raz E.
      Toll-like receptor ligands: hygiene, atopy and therapeutic implications.
      Indeed, diseases of immune dysregulation, including atopic diseases, are now considered to develop, more or less, as a result of failure in Treg cell function.
      • Rook G.A.W.
      • Adams V.
      • Hunt J.
      • Palmer R.
      • Martinelli R.
      • Brunet L.R.
      Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders.
      Immune defense mechanisms that evolved during the long history of humankind in a hostile environment appear now to be less appropriate when living in a clean environment.
      • LeSouëf P.
      • Goldblatt J.
      • Lynch N.
      Evolutionary adaptation of inflammatory immune responses in human beings.

      Hypothesis

      In this review we propose a hypothesis that one major factor in the current asthma and atopy epidemic might be the disconnection of man and the soil.

      Evidence to support the hypothesis

      There is abundant literature on adverse respiratory health effects attributable to exposure to environmental bioparticles (eg, bacteria, molds, and fugal spores).
      • Hauswirth D.W.
      • Sundy J.S.
      Bioaerosols and innate immune responses in airway diseases.
      Paradoxically, data are now accumulating to suggest that exposure to microbes in soil and vegetation might be beneficial, even necessary, for the normal maturation of the immune system.
      Several lines of evidence indicate that settings associated with high-level exposure to microorganisms in soil are associated with reduced risk for asthma and atopy. Such settings include farm environments, environments in nonaffluent Eastern countries, and rural areas, particularly in developing countries.

       Farm environment and atopic disease

      More than 30 studies from the last 6 years have consistently shown that children who have lived or are living on a farm are less likely to have atopic disease than their counterparts not living on a farm. The issue of farming and atopic disease has been thoroughly reviewed elsewhere and is not reiterated here.
      • von Hertzen L.
      • Haahtela T.
      Asthma and atopy—the price of affluence?.
      • von Mutius E.
      Influences in allergy: epidemiology and the environment.
      • Braun-Fahrländer C.
      Environmental exposure to endotoxin and other microbial products and the decreased risk of childhood atopy: evaluating developments since April 2002.
      In many of these studies, the effect of parental farming on the development of atopic disease in the child has been found to be dose dependent,
      • Braun-Fahrländer C.
      • Gassner M.
      • Grize L.
      • Neu U.
      • Sennhauser F.H.
      • Varonier H.S.
      • et al.
      Prevalence of hay fever and allergic sensitization in farmers' children and their peers living in the same rural community.
      • von Ehrenstein O.
      • von Mutius E.
      • Illi S.
      • Bauman L.
      • Böhm O.
      • von Kries R.
      Reduced risk of hay fever and asthma among children of farmers.
      • Riedler J.
      • Braun-Fahrländer C.
      • Eder W.
      • Schreuer M.
      • Waser M.
      • Maisch S.
      • et al.
      Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey.
      and many of these studies have also revealed “frequent contacts with farm animals” as one of the major factors responsible for this effect.
      • von Ehrenstein O.
      • von Mutius E.
      • Illi S.
      • Bauman L.
      • Böhm O.
      • von Kries R.
      Reduced risk of hay fever and asthma among children of farmers.
      • Waser M.
      • von Mutius E.
      • Riedler J.
      • Nowak D.
      • Maisch S.
      • Carr D.
      • et al.
      Exposure to pets, and the association with hay fever, asthma, and atopic sensitization in rural children.
      • Remes S.
      • Iivanainen K.
      • Koskela H.
      • Pekkanen J.
      Which factors explain the lower prevalence of atopy amongst farmers' children?.
      However, frequent contacts with farm animals can also be a surrogate marker for exposure to microorganisms in soil and vegetation because farm animals (and pets) are likely to serve as a secondary source of exposure to such microorganisms. In addition, frequent contacts with farm animals could also reflect general activity of the child on the farm. The effect of farming on conferring protection against asthma and atopy might not be restricted to early life only because current parental farming has been found to be an even stronger protective factor than that in early life.
      • Remes S.
      • Iivanainen K.
      • Koskela H.
      • Pekkanen J.
      Which factors explain the lower prevalence of atopy amongst farmers' children?.

       Environments in nonaffluent societies: The effect of traditional lifestyle

      Frequent compost and waste handling, wood handling, and animal excreta and manure handling are examples of high-level microbial exposure
      • Hauswirth D.W.
      • Sundy J.S.
      Bioaerosols and innate immune responses in airway diseases.
      associated with a traditional lifestyle. Unchlorinated surface water from lakes and rivers might be used as domestic water, untreated waste water might be used for irrigation, and animal excreta might be used as manure. Traditional lifestyle might also be associated with a microbe-rich diet (eg, frequent use of fermented vegetables).
      • Horner A.A.
      • Redecke V.
      • Raz E.
      Toll-like receptor ligands: hygiene, atopy and therapeutic implications.
      We found recently that occurrences of atopy (determined by means of skin prick tests) and atopic diseases were substantially lower among schoolchildren and their mothers in Russian Karelia compared with that seen in their counterparts in North Karelia, Finland, irrespective of the geographic proximity of the areas and similar geoclimatic and vegetative conditions (see Fig E1 in the Online Repository at www.jacionline.org). Analysis of generational differences revealed that in Finland children had higher atopy rates than their mothers, whereas in Russia the opposite trend, children having lower atopy rates than their mothers, emerged. No signs of westernization, with atopy prevalence as a proxy, were yet discernible in Russian Karelia, which was part of the Soviet Union until 1991.
      • von Hertzen L.
      • Mäkelä M.J.
      • Petäys T.
      • Jousilahti P.
      • Kosunen T.U.
      • Laatikainen T.
      • et al.
      Growing disparities in atopy between the Finns and the Russians—a comparison of two generations.
      The results are in line with those reported earlier from other Eastern countries in transition crisis.
      • Björksten B.
      • Dumitrascu D.
      • Foucard T.
      • Khetsuriani N.
      • Khaitov R.
      • Leja M.
      • et al.
      Prevalence of childhood asthma, rhinitis and eczema in Scandinavia and Eastern Europe.
      The East-West gradient in light of the occurrence of atopic diseases has been thoroughly reviewed.
      • von Hertzen L.
      • Haahtela T.
      Asthma and atopy—the price of affluence?.

       Rural areas and atopic disease: Evidence from relocation studies

      Data both from Western and particularly from developing countries, in which great differences in lifestyle still exist between urban and rural areas, have shown that living in rural areas might confer protection against atopic disease, even in a dose-dependent manner.
      • Viinanen A.
      • Munhbayarlah S.
      • Zevgee T.
      • Narantsetseg L.
      • Naidansuren T.
      • Koskenvuo M.
      • et al.
      Prevalence of asthma, allergic rhinoconjunctivitis and allergic sensitisation in Mongolia.
      • Yemaneberahn H.
      • Bekele Z.
      • Venn A.
      • Lewis S.
      • Parry E.
      • Britton J.
      Prevalence of wheeze and asthma and relation to atopy in urban and rural Ethiopia.
      • Perzanowski M.
      • Ng'ang'a L.
      • Carter M.
      • Odhiambo J.
      • Ngari P.
      • Vaughan J.
      • et al.
      Atopy, asthma and antibodies to Ascaris among rural and urban children in Kenya.
      • Braun-Fahrländer 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.
      • Nilsson L.
      • Castor O.
      • Löfman O.
      • Magnusson A.
      • Kjellman N.I.
      Allergic disease in teenagers in relation to urban or rural residence at various stages of childhood.
      A recent study in Mongolia that compared the occurrence of atopy and allergic disorders in 3 different environments of various degree of urbanization—a city area, rural towns, and villages—found significant increasing trends in the prevalence of allergic rhinitis and atopy, as determined by using skin prick tests, with increasing degree of urbanization.
      • Viinanen A.
      • Munhbayarlah S.
      • Zevgee T.
      • Narantsetseg L.
      • Naidansuren T.
      • Koskenvuo M.
      • et al.
      Prevalence of asthma, allergic rhinoconjunctivitis and allergic sensitisation in Mongolia.
      Analysis of the effect of relocation revealed that continuous living in a village since birth was most protective against atopy and allergic rhinitis, whereas those who relocated from villages to towns in adolescence or adulthood acquired allergic conditions at rates approaching those found in subjects who had always lived in towns.

      Viinanen A. Occurrence and risk factors of asthma, allergic rhinoconjunctivitis and allergic sensitisation in rural and urban Mongolia [thesis]. Turku (Finland): University of Turku; 2004. Annales Universitatis Turkuensis D/604.

      The results are in line with those of other migrant studies showing that sensitizaton rates and profiles among immigrants shift along with time, resembling finally those in natives,
      • Kalyoncu A.F.
      • Stålenheim G.
      Serum IgE levels and allergic spectra in immigrants to Sweden.
      • Grüber C.
      • Illi S.
      • Plieth A.
      • Sommerfeld C.
      • Wahn U.
      Cultural adaptation is associated with atopy and wheezing among children of Turkish origin living in Germany.
      thus supporting the view that there might not be any strictly limited window period in early life during which the individual is susceptible to immunomodulatory effects of the environment; rather, susceptibility to immunomodulation probably continues to adolescence, even to adulthood.
      • Matricardi P.M.
      • Yazdanbakhsh M.
      Mycobacteria and atopy, 6 years later: a fascinating, still unfinished, business.
      • Kemp A.
      • Björksten B.
      Immune deviation and the hygiene hypothesis: a review of the epidemiological evidence.
      • Linneberg A.
      Hypothesis: urbanization and the allergy epidemic—a reverse case of immuno-therapy.
      However, it must be borne in mind that disparities between asthma and atopic conditions in this respect might exist.
      • von Mutius E.
      Influences in allergy: epidemiology and the environment.

       Indicators of urbanization and atopic disease

      Before urbanization, humans have lived in close contact with soil, either directly or indirectly through food, water, and air,
      • Santamaria J.
      • Toranzos G.A.
      Enteric pathogens and soil: a short review.
      and heavy exposure to environmental microorganisms has occurred through inhalation, ingestion, and skin contact.
      • Horner A.A.
      • Redecke V.
      • Raz E.
      Toll-like receptor ligands: hygiene, atopy and therapeutic implications.
      Inhalation of bioaerosols (composed of microbes and their components, such as products of plants and fecal material from animals) has been considered to represent the major route of exposure.
      • Hauswirth D.W.
      • Sundy J.S.
      Bioaerosols and innate immune responses in airway diseases.
      This natural exposure to microbes, particularly in soil, has been dramatically reduced along with urbanization characterized by living in environments covered with asphalt and concrete.
      There are no unambiguous and commonly accepted criteria for urbanization. Many of the suggested criteria are based on population density and are not relevant for sparsely populated countries, such as Finland.
      We performed time-series analyses of occurrence of atopic diseases and urbanization using the asphalt index (use of asphalt, tons per inhabitant per year, years 1960-1990; The Road Administration, the Ministry of Traffic and Communication, and The Finnish Asphalt Association. Census statistics; Statistics Finland; http://statfin.stat.fi) and the decreasing proportion of farmers among the population (years 1966-2000, Statistics Finland; http://statfin.stat.fi) as indicators of urbanization here because both are closely related to reduced contacts with soil.
      Prevalences of asthma and allergic rhinitis were based on our recent data on occurrence of atopic disease among military conscripts.
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.
      The database here covered the years 1966 through 2000 and comprised more than 1 million military conscripts aged 18 to 19 years. The men had been examined to establish their fitness for service at the call up. Similar diagnostic codes for asthma and allergic rhinitis have been used throughout the study period on the basis of ICD-8 and ICD-9 in 1966 through 1996 and ICD-10 in 1997 through 2000.
      We found that the use of asphalt, which in Finland started at the end of the 1950s and was very modest still in the early 1960s (The Road Administration and The Finnish Asphalt Association, unpublished data), increased 10-fold in 3 decades. A nearly similar increase was also found in asthma prevalence among military conscripts, from 0.3% in 1966 to 2.6% in 1995 during a 30-year period, and the trend was upward for the whole study period (Fig 1, A).
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.
      Figure thumbnail gr1
      Fig 1A, Use of asphalt (tons per inhabitant per year) from 1960 through 1990 and occurrence of diagnosed asthma among military conscripts from 1966 through 2000 in Finland.
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.
      B, Proportion of farmers among the whole population from 1970 through 2000 and occurrence of diagnosed allergic rhinitis among military conscripts from 1966 through 2000 in Finland.
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.
      Supplemental information is available in the Online Repository at www.jacionline.org.
      Along with urbanization, heavy structural changes have occurred in agriculture and forestry. In Finland, the proportion of farmers among the population has decreased from 17.3% in 1970 to 4.9% in 2000. During the same time, the occurrence of allergic rhinitis, as assessed among young Finnish men, increased almost exponentially, from 0.1% in 1966 to 8.9% in 2000 (Fig 1, B).
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.
      The proportion of population that is continuously in natural connection with soil has thus diminished since the 1960s and will evidently still diminish, whereas the opposite has occurred for the prevalence of allergic rhinitis.
      Urbanization can also be characterized by living in apartment houses, which is, similarly to the use of asphalt and decrease in farming occupation, likely to reduce contact with soil. Dwelling type has indeed been shown to affect the magnitude of exposure to microorganisms in the environment. A study among 81 randomly selected teachers showed that both personal exposure to microorganisms (assessed with transportable inhalable aerosol samplers) and microbial concentrations in their homes were higher among persons living in family (single) houses compared with those in apartment houses, and this was considered partly to be due to increased outdoor activities among those living in family houses.

      Toivola M. Personal exposure to microbial aerosols [thesis]. Kuopio (Finland): University of Kuopio; 2004. Publications of the National Public Health Institute A13/2004.

      A sedentary lifestyle with little outdoor activity might not only be involved in the association between asthma and obesity
      • Brisbon N.
      • Plumb J.
      • Brawer R.
      • Paxman D.
      The asthma and obesity epidemics: the role played by the built environment—a public health perspective.
      but can also increase the risk of atopy through reduced exposure to saprophytes in the environment.
      Although exposure to pathogens has been found to be inversely associated with atopic diseases
      • Bach J.F.
      The effect of infections on susceptibility to autoimmune and allergic diseases.
      and undoubtedly is able to exert immunomodulatory effects in early life, infectious agents might represent only a minimal part of our total exposure to microorganisms. The largely neglected group of saprophytes in the environment might play a decisive role, in addition to gut microbiota,
      • Rakoff-Nahoum S.
      • Paglino J.
      • Eslami-Varzaneh F.
      • Edberg S.
      • Medzhitov R.
      Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis.
      in the development and maintenance of immunologic homeostasis.

       Source of drinking water and atopic disease

      An important issue closely related to soil is the runoff of soil microorganisms into natural waters
      • Lindström E.S.
      • Bergström A.K.
      Community composition of bacterioplankton and cell transport in lakes in two different drainage areas.
      and the use of such waters as drinking water. We found that in Russian Karelia, where atopy and atopic diseases are uncommon,
      • von Hertzen L.
      • Mäkelä M.J.
      • Petäys T.
      • Jousilahti P.
      • Kosunen T.U.
      • Laatikainen T.
      • et al.
      Growing disparities in atopy between the Finns and the Russians—a comparison of two generations.
      surface water bodies, lakes and rivers, are used as domestic water, frequently without any chemical or other treatment. Previous data have shown that consumption of unpasteurized milk in early life is associated with reduced risk of asthma and atopy in later life independently from other determinants.
      • Riedler J.
      • Braun-Fahrländer C.
      • Eder W.
      • Schreuer M.
      • Waser M.
      • Maisch S.
      • et al.
      Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey.
      It is reasonable to assume that consumption of untreated surface water could have similar effects and could be involved in the low atopy prevalence in Russian Karelia. Indeed, this view is supported by recent data from Ethiopia showing that consumption of river water in rural areas, as contrasted with consumption of pipe water in urban areas, conferred protection against atopic eczema.
      • Haileamlak A.
      • Dagoye D.
      • Williams H.
      • Venn A.J.
      • Hubbard R.
      • Britton J.
      Early life risk factors for atopic dermatitis in Ethiopian children.
      In another study among schoolchildren in a rural area of Latin America, consumption of river water was found to be weakly protective against atopy.
      • Cooper P.J.
      • Chico M.E.
      • Rodriguest L.C.
      • Strachan D.P.
      • Anderson H.R.
      • Rodrigues E.A.
      • et al.
      Risk factors for atopy among school children in a rural area of Latin America.

      Soil microbiota

      Soil is considered the most complicated biomaterial and at the same time the most diverse and important ecosystem on the planet.
      • Young I.M.
      • Crawford J.W.
      Interactions and self-organization in the soil-microbe complex.
      The definition of the microbial composition of a typical soil has proved to be problematic because of this diversity of soil types and the complexity and variability of the physicochemical circumstances. Nonetheless, the majority of soil bacteria are considered to belong to the lineage of gram-positive bacteria,
      • Liesack W.
      • Janssen P.H.
      • Rainey F.
      • Ward-Rainey N.
      • Stackebrandt E.
      Microbial diversity in soil: the need for a combined approach using molecular and cultivation techniques.
      and members of the phylum Actinobacter have been found to predominate in the soil.
      • Gisi U.
      • Schkendel R.
      • Schulin R.
      • Standelmann F.
      • Sticker H.
      Bodenökologie.
      This phylum includes genera such as Mycobacterium species, Streptomyces species, Actinomyces species, Corynebacterium species, and Bifidobacterium species.
      Fungi are often dominant in soils in terms of their biomass, particularly fungi dominate in acid temperate or polar soils that are oligotrophic,
      • Thorn G.
      The fungi in soil.
      • Bakken L.R.
      Culturable and nonculturable bacteria in soil.
      whereas bacteria predominate in near-neutral or moderately alkaline soils.
      • Bakken L.R.
      Culturable and nonculturable bacteria in soil.
      Some estimates of the density of microorganisms in a normal near-neutral organic soil obtained by means of cultivation and microscopy have been reported: Actinobacter, for example, might occur at the concentrations of 10
      • Ring J.
      • Krämer U.
      • Schäfer T.
      • Behrendt H.
      Why are allergies increasing?.
      • von Mutius E.
      Influences in allergy: epidemiology and the environment.
      • Björksten B.
      • Dumitrascu D.
      • Foucard T.
      • Khetsuriani N.
      • Khaitov R.
      • Leja M.
      • et al.
      Prevalence of childhood asthma, rhinitis and eczema in Scandinavia and Eastern Europe.
      • Kabesch M.
      • Lauener R.P.
      Why Old McDonald had a farm but no allergies: genes, environment, and the hygiene hypothesis.
      • Rook G.A.W.
      • Adams V.
      • Hunt J.
      • Palmer R.
      • Martinelli R.
      • Brunet L.R.
      Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders.
      bacteria/dm3 soil and other bacteria at the level of 108/dm3 soil.
      • Gisi U.
      • Schkendel R.
      • Schulin R.
      • Standelmann F.
      • Sticker H.
      Bodenökologie.
      Cultivation, which has traditionally been used to measure bacterial densities in soil samples, probably greatly underestimates the true values. The more modern methods, such as PCR tests and fatty acid analyses, have revealed that a considerable proportion of all bacteria in soil is in a dormant (metabolically inactive) stage.
      Mycobacteria, one of the major bacterial groups in soil and natural waters, including more than 80 saprophytic species,
      • Rook G.A.W.
      • Adams V.
      • Hunt J.
      • Palmer R.
      • Martinelli R.
      • Brunet L.R.
      Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders.
      has received considerable attention during the last decade as a potential immunomodulatory agent in alleviating symptoms of atopic disease
      • Arkwright P.D.
      • David T.J.
      Intradermal administration of a killed Mycobacterium vaccae suspension (SRL 172) is associated with improvement in atopic dermatitis in children with moderated-to-severe disease.
      and even conferring protection against them.
      • Zuany-Amorim C.
      • Manlius C.
      • Trifilieff A.
      • Brunet L.R.
      • Rook G.
      • Bowen G.
      • et al.
      Long-term protective and antigen-specific effect of heat-killed Mycobacterium vaccae in a murine model of allergic pulmonary inflammation.
      • Adams V.C.
      • Hunt J.R.
      • Martinelli R.
      • Palmer R.
      • Rook G.A.
      • Brunet L.R.
      Mycobacterium vaccae induces a population of pulmonary CD11c+ cells with regulatory potential in allergic mice.
      In addition, much of the current research of atopic diseases has been focused on lactobacilli, which are considered one of the potential groups of immunomodulatory agents with prophylactic and therapeutic potential.
      • Isolauri E.
      Dietary modification of atopic disease: use of probiotics in the prevention of atopic dermatitis.
      • Kalliomäki M.
      • Isolauri E.
      Role of intestinal flora in the development of allergy.
      • Ogden N.S.
      • Bielory L.
      Probiotics: a complementary approach in the treatment and prevention of pediatric atopic disease.
      Notably, lactobacilli are originally common inhabitants of plants and grow at the expense of the nutrients liberated from decomposing plant material.
      • Stanier R.Y.
      • Doudoroff M.
      • Adelberg E.A.
      General microbiology.
      Results from our laboratory indicate that, indeed, gram-positive bacteria represent the great majority (90%) of all bacteria in settled barn dust measured by means of exact fatty acid analyses (Saris et al, unpublished data). However, the proportions of gram-negative and gram-positive bacteria might vary to some extent according to the season,
      • Andersson A.M.
      • Weiss N.
      • Rainey F.
      • Salkinoja-Salonen M.S.
      Dust-borne bacteria in animal sheds, schools and children's day care centres.
      particularly in northern boreal latitudes, where the soil is frozen and covered with snow for months.

      Major components of microorganisms with immunomodulatory potential and their interaction with the innate immunity

      The idea that microbial products have immunomodulatory potential and could be used as immunotherapeutic agents in asthma and allergies dates back to the 1950s.
      • Frankland A.W.
      • Hughes W.H.
      • Gorrild R.H.
      Autogenous bacterial vaccines in the treatment of asthma.
      Bacterial extracts obtained mainly from species associated with upper respiratory tract and urinary tract infections administered subcutaneously were earlier used for such purposes but are not in use today because several double-blind studies showed no efficacy in asthma, possibly because of overly low concentrations of bacterial material in these extracts, nonoptimal route of administration, and overly long intervals between the doses (1 week or longer). No studies on the preventive effect of oral bacterial extracts are available.
      • Matricardi P.M.
      • Björksten B.
      • Bonini S.
      • Bousquet J.
      • Djukanovic R.
      • Dreborg S.
      • for the EAACI Task Force 7
      • et al.
      Microbial products in allergy prevention and therapy. Position paper.
      Because both viable and nonviable bacterial components have been found to be immunobiologically active, a renewed interest in bacterial cell-wall components has been raised, although the literature on immunologic effects of cell-wall components other than LPS (endotoxin) is relatively scarce. However, in addition to LPS, 2 other ubiquitous bacterial components, lipoteichoic acids and peptidoglycans, might be of importance in this respect, and the cell-wall components of fungi (eg, β-glucans) have also been found to have immunomodulatory potential. In addition, a potent immunomodulator appears to be bacterial DNA, the unmethylated CpG oligonucleotide.

       TLRs

      TLRs, the receptors that recognize conserved microbial structures, represent an ancient system of host defense but were not discovered until 1989.
      • Janeway Jr., C.A.
      Approaching the asymptote? Evolution and revolution in immunology.
      • Heine H.
      • Lien E.
      Toll-like receptors and their function in innate and adaptive immunity.
      This discovery brought the formerly underappreciated innate immunity into the focus of research. TLRs have been extensively reviewed during the last few years.
      • Heine H.
      • Lien E.
      Toll-like receptors and their function in innate and adaptive immunity.
      • Takeda K.
      • Kaisho T.
      • Akira S.
      Toll-like receptors.
      • Netea M.G.
      • van der Graaf C.
      • van der Meer J.W.M.
      • Kullberg B.J.
      Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system.
      • Akira S.
      • Sato S.
      Toll-like receptors and their signalling mechanisms.
      To date, at least 10 different TLRs have been identified in mammals, and they have been found to play a decisive role in recognizing microbes and bridging the innate and acquired immune responses.
      • Netea M.G.
      • van der Graaf C.
      • van der Meer J.W.M.
      • Kullberg B.J.
      Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system.
      Although the role of TLRs has been mostly shown in infectious systems, there are good reasons to believe that TLRs are equally important in repeated exposure to saprophytic bacteria in the environment and commensals in the gut and are involved in the induction of tolerance. TLR2, TLR4, and TLR9 are briefly considered here in the light of exposure to environmental saprophytes. TLR2 is the principal receptor and signaling molecule for gram-positive bacteria, lipoteichoic acid, mycobacteria, mycobacterial lipoarabinomannan, bacterial lipoproteins, and fungal β-glucan. TLR4 is the primary receptor for LPS from gram-negative bacteria, and TLR9 recognizes bacterial unmethylated CpG oligonucleotides.
      • Heine H.
      • Lien E.
      Toll-like receptors and their function in innate and adaptive immunity.

       Major cell-wall components of bacteria and fungi

       LPS (endotoxin)

      The chemical composition of LPS, the major cell-wall component of gram-negative bacteria, has been known for more than 50 years, and its physicochemical properties, stability and heat resistance, are also well established.
      • Liu A.H.
      Endotoxin exposure in allergy and asthma: reconciling a paradox.
      Abundant literature exists concerning the biologic effects of this macromolecule. In several studies the relative lack of exposure to endotoxin has been suggested to be one major reason for the asthma and atopy epidemic.
      • Braun-Fahrländer 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.
      • Gereda J.E.
      • Leung D.Y.
      • Thatayatikom A.
      • Streib J.E.
      • Price M.R.
      • Klinnert M.D.
      • et al.
      Relation between house-dust endotoxin exposure, type 1 T-cell development, and allergen sensitisation in infants at high risk of asthma.
      • Gehring U.
      • Bolte G.
      • Borte M.
      • Bischof W.
      • Fahlbusch B.
      • Wichmann H.E.
      • et al.
      Exposure to endotoxin decreases the risk of atopic eczema in infancy: a cohort study.
      The relationship between LPS and the occurrence of asthma and atopy has also been thoroughly reviewed elsewhere.
      • Liu A.H.
      Endotoxin exposure in allergy and asthma: reconciling a paradox.
      • Liu A.H.
      • Murphy J.R.
      Hygiene hypothesis: fact or fiction?.
      • Eder W.
      • von Mutius E.
      Hygiene hypothesis and endotoxin: what is the evidence?.
      It has also been known since the 1950s that the biologic activity of LPS resides in the lipid A moiety (the theory of the “endotoxic principle”
      • Westphal O.
      • Lüderitz O.
      Chemische Erforschung von Lipopolysacchariden Gram-negativer Bakterien.
      ). Moreover, it is now well established that the immune response to LPS is dependent on the chemical structure and molecular conformation of the lipid A moiety. Lipid A consists of a phosphorylated glucosamine disaccharide (the backbone), to which fatty acids are attached. It has been found that biologic effects of LPS from different gram-negative bacteria are not similar.
      • Netea M.G.
      • van Deuren M.
      • Kullberg B.J.
      • Cavaillon J.M.
      • van der Meer J.W.M.
      Does the shape of lipid A determine the interaction of LPS with Toll-like receptors?.
      The critical determinants here are the length and number of acyl (fatty acid) chains, the asymmetry of these chains, and the number and distribution of negative charges.
      • Netea M.G.
      • van Deuren M.
      • Kullberg B.J.
      • Cavaillon J.M.
      • van der Meer J.W.M.
      Does the shape of lipid A determine the interaction of LPS with Toll-like receptors?.
      • Seydel U.
      • Schromm A.B.
      • Blunck R.
      • Brandenburg K.
      Chemical structure, molecular conformation and bioactivity of endotoxins.
      Interestingly, the widely used test to detect LPS in various samples, the Limulus amebocyte lysate test, has been found to be specific for the LPS glucosamine backbone and is thus not a measure of biologic activity. Fatty acid analyses might better correlate with biologic activity because the conformation and number of acyl (fatty acid) chains in lipid A appear to be central to the capacity of LPS to interact with TLRs and to induce cytokine production.
      • Seydel U.
      • Hawkins L.
      • Schromm A.B.
      • Heine H.
      • Scheel O.
      • Koch M.H.
      • et al.
      The generalized endotoxic principle.

       Peptidoglycan and teichoic acid

      With the exception of a few groups of some minor bacteria, such as Mycoplasma and Chlamydia species, all members in the domain Bacteria have one common denominator, the presence of peptidoglycan as the main strengthening and shape-determining constituent of the cell wall. In gram-positive bacteria, peptidoglycan accounts for at least 50%, and often more, of the total dry weight of the wall, but in gram-negative bacteria, it might comprise less than 10% of the dry weight of the wall.
      • Stanier R.Y.
      • Doudoroff M.
      • Adelberg E.A.
      General microbiology.
      In gram-negative bacteria peptidoglycan is localized in the innermost layer of the wall and, although extremely thin, is still capable of retaining the shape of the cell.
      • Stanier R.Y.
      • Doudoroff M.
      • Adelberg E.A.
      General microbiology.
      Peptidoglycan is composed of 2 amino sugars, N-acetylglucosamine and N-acetylmuramic acid, and a side chain of 4 amino acids that can vary from species to species.
      • Jawetz E.
      • Melnick J.L.
      • Adelberg E.A.
      Review of medical microbiology.
      The other of these amino sugars, N-acetylmuramic acid, is a molecule specific to the domain Bacteria and can thus be used for laboratory diagnostic purposes. In a recent study, van Strien et al
      • van Strien R.T.
      • Engel R.
      • Holst O.
      • Bufe A.
      • Eder W.
      • Waser M.
      • et al.
      Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid in mattress dust, and is association with respiratory health.
      showed that muramic acid can be found in dust from children's mattresses and in higher concentrations in those of farmer's children. Wheezing and asthma were inversely associated with the muramic acid concentration, independently from LPS, whereas no association was found for atopic sensitization. Increased muramic acid concentrations were found in homes heated with wood or coal, independent of whether it was a farm home, which suggests that settings associated with traditional lifestyle and increased exposure to microorganisms, rather than farming per se, is responsible for this effect.
      The other characteristic cell-wall components of most gram-positive bacteria are teichoic acids (up to 50% of the dry weight of the wall). Teichoic acids are water-soluble polymers containing ribitol or glycerol residues joined through phosphodiester linkages.
      • Jawetz E.
      • Melnick J.L.
      • Adelberg E.A.
      Review of medical microbiology.
      In lipoteichoic acids there is a single lipid side chain anchored to the ribitol or glycerol backbone.
      • Cleveland M.G.
      • Gorham J.D.
      • Murphy T.L.
      • Tuomanen E.
      • Murphy K.M.
      Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway.
      Teichoic and lipoteichoic acids have been found to exert potent inflammatory responses.
      • Keller R.
      • Fischer W.
      • Keist R.
      • Bassetti S.
      Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids.
      • Keller R.
      • Keist R.
      • Joller P.W.
      Macrophage response to bacteria and bacterial products: modulation of Fc gamma receptors and secretory and cellular activities.
      • Suda Y.
      • Tochio H.
      • Kawano K.
      • Takada H.
      • Yoshida Y.
      • Kotani S.
      • et al.
      Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790.
      As stated above, teichoic acids are recognized by TLR2. Peptidoglycan has similarly been considered to be recognized by TLR2,
      • Heine H.
      • Lien E.
      Toll-like receptors and their function in innate and adaptive immunity.
      • Akira S.
      • Sato S.
      Toll-like receptors and their signalling mechanisms.
      but this view has recently been challenged by the identification of intracellular proteins, the nucleotide-binding oligomerization domain 1 and 2, as the principal receptors for peptidoglycan fragments.
      • Boneca I.G.
      The role of peptidoglycan in pathogenesis.
      • Travassos L.H.
      • Girardin S.E.
      • Philpott D.J.
      • Blanot D.
      • Nahori M.A.
      • Werts C.
      • et al.
      Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition.

       Fungal cell-wall components

      Fungal cell walls differ from those of bacteria by lacking peptidoglycan, teichoic acids, and LPS. In their place are the external and antigenic peptidomannans embedded in matrices of α- and β-glucans, and structural rigidity is provided by chitin.
      • Drutz D.J.
      Fungal diseases.
      The principal sterol in fungal cell membranes is ergosterol (corresponding cholesterol in mammalian cells), which has been used, in addition to β-glucans, in laboratory diagnostics of environmental samples. The immunobiology of β-glucans has been reviewed earlier.
      • Williams D.L.
      Overview of (1, 3)-beta-D-glucan immunobiology.
      Most of the recent studies on fungal recognition by TLRs have been centered on a few potentially pathogenic fungi, such as Candida albicans and Aspergillus fumigatus. It has been found that both TLR2 and TLR4 can be important for their recognition
      • Netea M.G.
      • van der Graaf C.
      • van der Meer J.W.M.
      • Kullberg B.J.
      Recognition of fungal pathogens by Toll-like receptors.
      and that TLR2 might be involved in maintaining prolonged candidasis by mediating anti-inflammatory signals leading to IL-10 production and generation of regulatory T cells.
      • Netea M.G.
      • Sutmuller R.
      • Hermann C.
      • van der Graaf C.A.
      • van der Meer J.W.
      • Adema G.
      • et al.
      Toll-like receptor 2 inhibits cellular responses against Candida albicans through pathways mediated by IL-10 and CD4+CD25+ regulatory T cells.
      In addition, a new coreceptor for β-glucan, dectin-1, expressed on macrophages, dendritic cells, and monocytes, has been found to be involved in mediating proinflammatory responses to fungi together with TLR2.
      • Brown G.D.
      • Herre J.
      • Williams D.L.
      • Willment J.A.
      • Marshall A.S.
      • Gordon S.
      Dectin-1 mediates the biological effects of beta-glucans.
      • Gantner B.N.
      • Simmons R.M.
      • Canavera S.J.
      • Akirta S.
      • Underhill D.M.
      Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.
      The recognition of fungi by the innate immune system appears to be more complex than that of bacteria because fungi can exist in 2 forms, as hyphae or conidiae.

       Bacterial CpG oligonucleotides

      Since the late 1980s, bacterial DNA has been known to possess immunostimulatory properties
      • Tokunaga T.
      • Yamamoto S.
      • Shimada S.
      • Abe H.
      • Fukuda T.
      • Fujisawa Y.
      • et al.
      Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Isolation, physicochemical characterization, and antitumor activity.
      that are not found in vertebrate DNA.
      • Yamamoto S.
      • Yamamoto T.
      • Shimada S.
      • Kuramoto E.
      • Yano O.
      • Kataoka T.
      • et al.
      DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth.
      The activating element was identified as an unmethylated CpG oligonucleotide that was found to be 20-fold more abundant in bacterial than in vertebrate DNA, and when present in vertebrate DNA, about 70% of it was found to be methylated.
      • Bird A.P.
      DNA methylation and the frequency of CpG in animal DNA.
      Unmethylated CpG oligonucleotides have the ability to elicit a multifaceted innate immune response characterized by the production of IL-12, IL-18, and IFN-γ and the upregulation of costimulatory molecules by antigen-presenting cells, B cells, and natural killer cells.
      • Horner A.A.
      • Raz E.
      Immunostimulatory sequence oligodeoxynucleotide-based vaccination and immunomodulation: two unique but complementary strategies for the treatment of allergic diseases.
      They have both direct and indirect effects on the commitment of CD4+ cells to a TH1 phenotype and are thus able to downregulate or reverse TH2 responses.
      • Horner A.A.
      • Raz E.
      Immunostimulatory sequence oligodeoxynucleotide-based vaccination and immunomodulation: two unique but complementary strategies for the treatment of allergic diseases.
      • Hussain I.
      • Kline J.N.
      DNA, the immune system, and atopic disease.
      • Wild J.S.
      • Sur S.
      CpG oligonucleotide modulation of allergic inflammation.
      Mammalian DNA or methylated bacterial DNA, in contrast, does not induce these responses.
      • van Uden J.
      • Raz E.
      Immunostimulatory DNA and applications to allergic disease.
      In addition, CpG oligonucleotides have been found to strongly induce IL-10 release, which inhibits both TH1 and TH2 responses in a dose-dependent manner.
      • Kitagi K.
      • Jain V.V.
      • Businga T.R.
      • Hussain I.
      • Kline J.N.
      Immunomodulatory effects of CpG oligonucleotides on established Th2 responses.
      This IL-10 is the key cytokine in the development of adaptive regulatory T (Treg) cells, which in turn are able to downregulate antigen-specific IgE responses and promote tolerance to allergens.
      • Cottrez F.
      • Hurst S.D.
      • Coffman R.L.
      • Groux H.
      T regulatory cells 1 inhibit a Th2-specific response in vivo.
      • Curotto de Lafaille M.A.
      • Muriglan S.
      • Sunshine M.J.
      • Lei Y.
      • Kutchukhidze N.
      • Furtado G.C.
      • et al.
      Hyper immunoglobulin E response in mice with monoclonal populations of B and T lymphocytes.
      Data on synthetic CpG oligonucleotides in murine models of atopic diseases and as vaccine adjuvants and therapeutic agents in human subjects with allergic disorders are promising.
      • Horner A.A.
      • Raz E.
      Immunostimulatory sequence oligodeoxynucleotide-based vaccination and immunomodulation: two unique but complementary strategies for the treatment of allergic diseases.
      • Hussain I.
      • Kline J.N.
      DNA, the immune system, and atopic disease.
      • Wild J.S.
      • Sur S.
      CpG oligonucleotide modulation of allergic inflammation.
      • Bohle B.
      CpG motifs as possible adjuvants for the treatment of allergic diseases.
      In a study by Roy et al,
      • Roy S.R.
      • Schiltz A.M.
      • Marotta A.
      • Shen Y.
      • Liu A.H.
      Bacterial DNA in house and farm barn dust.
      bacterial DNA and LPS contents in dust from urban, rural, and farm homes and from farm barns were quantified (by means of PCR specific for bacterial ribosomal DNA and the Limulus test, respectively) to determine whether there are differences in the immune stimulatory capacity between different dust samples. The highest bacterial DNA levels were found in farm barns, followed by rural homes, farm homes, and urban homes. Farm barn DNA significantly potentiated LPS-induced IL-10 and IL-12p40 release from PBMCs, whereas DNA from urban homes did not show this effect, probably because of the low content of bacterial DNA in urban home dust; only approximately 3% of the total DNA content in urban samples was bacterial in origin. Increased IL-10 and IL-12 release shown after stimulation of PBMCs with barn dust DNA and LPS might be crucial in the context of environments conferring protection against atopic diseases.
      • Roy S.R.
      • Schiltz A.M.
      • Marotta A.
      • Shen Y.
      • Liu A.H.
      Bacterial DNA in house and farm barn dust.
      Furthermore, we found a 3.5-fold higher bacterial DNA content (measured by means of bacterial ribosome-specific PCR) in barn dust compared with urban-suburban home dust (5127 vs 1479.5 ng bacterial DNA/g dust; Saris et al, unpublished data). It is known that vertebrate DNA does not possess immunostimulatory capacity but might neutralize or even inhibit the immunostimulatory effects of bacterial CpG motifs.
      • Chen Y.
      • Lenert P.
      • Weerantna R.
      • McCluskie M.
      • Wu T.
      • Davis H.L.
      • et al.
      Identification of methylated CpG motifs as inhibitors of the immune stimulatory CpG motifs.
      • Pisetsky D.S.
      • Reich C.F.
      Inhibition of murine macrophage IL-12 production by natural and synthetic DNA.
      The only known TLR for unmethylated CpG oligonucleotides is TLR9.
      The innate immunity recognizes saprophytic bacteria, which results in release of proinflammatory cytokines. We have shown in vitro that robust responses are elicited in murine macrophages when they are stimulated by common soil microorganisms isolated from barn dust, such as Streptomyces species, Sphingomonas species, and Macrococcus species (Pylkkänen et al, unpublished data), and a dose-dependent and rather similar response was found in the production of, for example, TNF-α for all 3 organisms, contrary to Bacillus species, which showed minimal response in this in vitro setting (Pylkkänen et al, unpublished data). None of these genera represents true gram-negative bacteria, because Sphingomonas species, although categorized as gram-negative, do not possess LPS but have sphingolipids instead. Interestingly, we found that the dominant (>85%) bacterial genus in dust from urban-suburban homes was Bacillus species (Saris and Andersson, unpublished data), lending further support to the view that urban home dust might have minimal, if any, immunomodulatory capacity. Nonetheless, the ability of the common soil saprophytes Streptomyces species, Sphingomonas species, and Macrococcus species to elicit robust proinflammatory cytokine responses in vitro raises the question of tolerance, which must have evolved during the long history of coexistence of these saprophytes and man.
      • Rook G.A.W.
      • Adams V.
      • Hunt J.
      • Palmer R.
      • Martinelli R.
      • Brunet L.R.
      Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders.

      Tolerance and Treg cells

      Repeated or persistent exposure appears to be one fundamental factor in the induction of tolerance. Repeated intranasal antigen exposure leads to decreased bronchial reactivity and tolerance in TH2-sensitized mice.
      • Takabayashi K.
      • Vibet L.
      • Chisholm D.
      • Zubeldia J.
      • Horner A.A.
      Intranasal immunotherapy is more effective than intradermal immunotherapy for the induction of airway allergen tolerance in Th2-sensitized mice.
      • Hall G.
      • Houghton C.G.
      • Rahbek J.U.
      • Lamb J.R.
      • Jarman E.R.
      Suppression of allergen reactive Th2 mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production.
      In addition, allergen desensitization therapy (injection of a specific allergen extract at increasing doses) has for years been successfully used, particularly in patients with hay fever and insect venom allergy.
      • Hawrylowicz C.M.
      • O'Garra A.O.
      Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.
      Tolerance is mediated by several mechanisms, including anergy and deletion of effector T-cell clones, and particularly by the induction and function of Treg cells.
      • Akbari O.
      • Stock P.
      • DeKruyff R.H.
      • Umetsu D.T.
      Mucosal tolerance and immunity: regulating the development of allergic disease and asthma.
      These Treg cells are defined as cells that actively control the function of other cells, mostly in an inhibitory way.
      • Umetsu D.T.
      • Akbari O.
      • DeKruyff R.H.
      Regulatory T cells control the development of allergic disease and asthma.
      Two major lineages of Treg cells have been identified: (1) naturally occurring, thymus-derived Treg cells expressing the transcription factor Foxp3, which are associated primarily with the control of autoantigens, and (2) induced (adaptive), antigen-specific Treg cells, which require IL-10, TGF-β, or both for their differentiation and function.
      • Hawrylowicz C.M.
      • O'Garra A.O.
      Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.
      • Bluestone J.A.
      • Abbas A.K.
      Natural versus adaptive regulatory T cells.
      These cells ameliorate inflammation through the release of IL-10, TGF-β, or both in repeated or persistent exposure to prevent immune pathology (a form of tolerance) and maintain the persistence of low numbers of antigens in the body, which is necessary in certain cases to provide long-lasting immunity against reinfections.
      • Mills K.H.G.
      • McGuirk P.
      Antigen-specific regulatory T cells—their induction and role in infection.
      Induction of antigen-specific Treg cells has been performed by administration of heat-killed Mycobacterium vaccae and allergen (ovalbumin) into mice. These specific Treg cells were found to release IL-10 and TGF-β and suppress eosinophilia and bronchial hyperresponsiveness.
      • Zuany-Amorim C.
      • Sawicka E.
      • Manlius C.
      • Le Moine A.
      • Brunet L.R.
      • Kemeny D.M.
      • et al.
      Suppression of airway eosinophilia by killed Mycobacterium vaccae-induced allergen-specific regulatory T-cells.
      Desensitization therapy has also been found to operate through IL-10.
      • Hawrylowicz C.M.
      • O'Garra A.O.
      Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.
      In general, Treg cells are able to prevent the development of highly polarized TH cells,
      • Curotto de Lafaille M.A.
      • Lafaille J.J.
      The role of regulatory T cells in allergy.
      and one of the mechanisms involved in the development of asthma and atopy has been suggested to be a failure in Treg cell function.
      • Hawrylowicz C.M.
      • O'Garra A.O.
      Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.
      • Umetsu D.T.
      • Akbari O.
      • DeKruyff R.H.
      Regulatory T cells control the development of allergic disease and asthma.
      • Curotto de Lafaille M.A.
      • Lafaille J.J.
      The role of regulatory T cells in allergy.
      Several excellent review articles of Treg cells are available for further reading.
      • Hawrylowicz C.M.
      • O'Garra A.O.
      Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.
      • Umetsu D.T.
      • Akbari O.
      • DeKruyff R.H.
      Regulatory T cells control the development of allergic disease and asthma.
      • Bluestone J.A.
      • Abbas A.K.
      Natural versus adaptive regulatory T cells.
      • Curotto de Lafaille M.A.
      • Lafaille J.J.
      The role of regulatory T cells in allergy.
      Treg cells can be preferentially induced at mucosal surfaces, particularly in the gut and respiratory tract.
      • Mills K.H.G.
      • McGuirk P.
      Antigen-specific regulatory T cells—their induction and role in infection.
      In urbanized Western societies the natural environment might no longer have the ability to maintain the respiratory and gut mucosal system in a state that favors the development of Treg cells and mucosal tolerance to harmless bioparticles.
      • Umetsu D.T.
      • Akbari O.
      • DeKruyff R.H.
      Regulatory T cells control the development of allergic disease and asthma.
      Persistent exposure to saprophytic bacteria in soil and vegetation, in addition to commensals in the gut and respiratory tract, might be needed to stimulate the production of IL-10 and TGF-β through the innate immune system, which in turn are required for the development of inducible Treg cells.

      TLRs and gene-environment interaction

      Innate immunity is now recognized as a central element also in the gene-environment interaction. The significance of particularly the TLR2 gene in this respect has been demonstrated in several studies. In European children TLR2 gene expression has been found to be higher in blood cells obtained from farmers' children compared with that seen in children not growing up on a farm. A similar difference was not found for the TLR4 gene.
      • Lauener R.P.
      • Birchier T.
      • Adamski J.
      • Braun-Fahrländer C.
      • Bufe A.
      • Herz U.
      • et al.
      Expression of CD14 and Toll-like receptor 2 in farmers' and non-farmers' children.
      Furthermore, a genetic variation in the TLR2 gene was shown to be a major determinant of reduced susceptibility to asthma and atopy in farmers' children but not in nonfarmers' children.
      • Eder W.
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      Toll-like receptor 2 as a major gene for asthma in children of European farmers.
      No clear association could be found between variations in the TLR4 gene and asthma or hay fever either in farmers' or nonfarmers children in most,
      • Eder W.
      • Klimecki W.
      • Yu L.
      • von Mutius E.
      • Riedler J.
      • Braun-Fahrländer C.
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      Toll-like receptor 2 as a major gene for asthma in children of European farmers.
      • Raby B.A.
      • Klimecki W.T.
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      Polymorphisms in toll-like receptor 4 are not associated with asthma or atopy-related phenotypes.
      albeit not all,
      • Fageras Bottcher M.
      • Hmani-Aifa M.
      • Lindström A.
      • Jenmalm M.C.
      • Mai X.M.
      • Nilsson L.
      • et al.
      A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-linked interleukin-12(p70) responses in Swedish children.
      studies.
      These studies provide convincing evidence for gene-environment interactions: a certain polymorphism is expressed only in a certain environment. They also underscore the significance of TLR2 in environments associated with high exposure to soil microorganisms. Because TLR2 is the main receptor for gram-positive bacteria and their structural molecules, lipoteichoic acid and lipoproteins,
      • Heine H.
      • Lien E.
      Toll-like receptors and their function in innate and adaptive immunity.
      • Akira S.
      • Sato S.
      Toll-like receptors and their signalling mechanisms.
      these studies might also point to the significance of particularly gram-positive bacteria in this context. Interaction of TLR2 with its ligands has been found to lead to rapid release of IL-10, which can block the induction of IL-12p35 and IFN-γ by TLR3 and TLR4.
      • Re F.
      • Strominger J.L.
      IL-10 released by concomitant TLR2 stimulation blocks the induction of a subset of Th1 cytokines that are specifically induced by TLR4 or TLR3 in human dendritic cells.
      It remains to be clarified whether TLR2 is the crucial Toll receptor in mediating IL-10 release for the development of inducible Treg cells.

      Concluding remarks

      Several lines of evidence support the view that the environment in modern industrialized societies is unable to provide the stimulation for the developing immune system that might be beneficial or even necessary: disruption of the ancient connection of humankind and the soil might have had unexpected consequences.
      The immunomodulatory role of saprophytic bacteria in soil and vegetation is now increasingly recognized. The innate immune system recognizes such saprophytes or their nonviable components encountered at respiratory and gut mucosal surfaces; however, robust inflammatory responses are not normally elicited but kept in tight control through mechanisms that involve the function of Treg cells that in turn might control the development of atopic diseases. A number of microorganisms and their components have been found to induce the production of IL-10, the differentiation factor of inducible Treg cells, by innate cells.
      • Mills K.H.G.
      • McGuirk P.
      Antigen-specific regulatory T cells—their induction and role in infection.
      • McGuirk P.
      • Mills K.H.G.
      Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases.
      Here we proposed a hypothesis that the disconnection of man and the soil might be one major factor in the current asthma and atopy epidemic, as shown in Fig 2. The hypothesis could be tested in animal models and in further comparative and more clearly defined epidemiologic settings, and if correct, a strategy that involves enhancement of the development and activity of Treg cells
      • Fageras Bottcher M.
      • Hmani-Aifa M.
      • Lindström A.
      • Jenmalm M.C.
      • Mai X.M.
      • Nilsson L.
      • et al.
      A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-linked interleukin-12(p70) responses in Swedish children.
      without concomitant induction of inflammation by bacterial products is evidently the goal to pursue. Central to the outcome of this strategy is probably timing (with respect to the primary sensitization), duration, dose, and route of exposure. The nature of the microbe might also play a role, although it is likely that there is no single agent or agent group behind the protective effect; rather, a mixture, including members of the phylum Actinobacter, might be involved. In addition, the product might not be based on bacterial cell-wall components only because bacterial DNA might have beneficial effects in this respect.
      Figure thumbnail gr2
      Fig 2A model of the asphalt theory in the development of atopic diseases. Supplemental information is available in the Online Repository at www.jacionline.org. Because of the implementation of new technologies, including recycling of asphalt material, the figures for the use of asphalt from 1991 onward are not comparable with the earlier ones and have not been included in the analysis. Figures for the years 1960 through 1974 are based on unofficial data from the Road Administration, Ministry of Traffic and Communication, because no official statistics are available for that period. Data on the prevalence of diagnosed asthma and allergic rhinitis are based on Latvala et al.
      • Latvala J.
      • von Hertzen L.
      • Lindholm H.
      • Haahtela T.
      Trends in prevalence of asthma and allergy in Finnish young men: a nationwide study from 1966 to 2003.

      Appendix. Supplementary data

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