2

Hygiene theory and allergy and asthma prevention

Andrew H. Liu
Division of Allergy and Clinical Immunology, Department of Pediatrics, National Jewish Medical and Research Center, University of Colorado
School of Medicine, Denver, CO, USA

Correspondence:
Andrew H. Liu, MD,
Pediatric Allergy and
Immunology, National Jewish
Medical and Research Center,
1400 Jackson Street (K1023),
Denver, CO 80206, USA.
E-mail: liua@njc.org
Summary
Liu AH. Hygiene theory and allergy and asthma prevention. Paediatric and Perinatal
Epidemiology 2007; 21(Suppl. 3): 2–7.
Epidemiological trends of allergic diseases and asthma in children reveal a global rise
in their prevalence over the past 50 years. Their rapid rise, especially in metropolitan
locales, suggests that recent changes in modern environments and/or life styles under-
lie these trends. One environmental/life style factor that may be contributing to this
trend is called the hygiene hypothesis: that naturally occurring microbial exposures in
early life may have prompted early immune maturation and prevented allergic
diseases and asthma from developing. Subsequently, children raised in modern met-
ropolitan life styles, relatively devoid of this natural microbial burden, may have
under-stimulated immune systems in infancy, thereby allowing for the ‘allergic march’
– a pattern of pro-allergic immune development and disorders that occurs in early life.
Over the past 15 years, hygiene theorising has evolved in shape and substance, in part
due to a growing and strengthening burden of evidence from epidemiological, trans-
lational and basic research. What was speculation may be key clues to allergy and
asthma prevention. The objectives of this article are to summarise the epidemiological
trends and allergic march of childhood that may be explained by hygiene theory, to
overview current hygiene theory paradigms and to exemplify the strengthening epi-
demiological evidence in support of the hygiene theory, using bacterial endotoxin
exposure as a prototypical example.

Keywords: asthma, allergy, hygiene theory, allergic rhinitis, food allergy, atopic dermatitis.

The allergic march
The ‘allergic march’ refers to a subset of allergic dis-
orders that commonly begin in early childhood:
atopic dermatitis or eczema, food allergies, allergic
rhinitis and allergy-associated asthma. Biological
markers accompanying these allergic disorders
include skin test hypersensitivity to inhaled and/or
food allergens and the presence in the blood of
specific immunoglobulin E molecules that recognise
these allergens and mediate allergic immune
responses. This biological and clinical allergic
response is called ‘atopy’. Atopy also has a hereditary
implication. In the 1920s, when this term was first
applied, atopy was observed to be a cluster of allergic
conditions that would tend to occur in individuals
and in families. Since then, we have come to realise
that environments and life styles within families may
also predispose and contribute to the basis for devel-
oping allergies and/or asthma.
Prevalence and trends
Allergic disorders are very common but vary in their
peak prevalence during childhood. In one US study of
4 to 7-year-old children, the period prevalence of
asthma was 20–30%, allergic rhinitis 30–40%, atopic
dermatitis 5–10% and food allergy 2–5%.1 By age
7 years, most children had at least one allergic condi-
tion. In addition, these allergic disorders cluster or
overlap in some children and some families.
The prevalence of asthma has increased by approxi-
mately 50% per decade in numerous countries.2 Similar
increases have also been reported in prevalence data
for other atopic disorders, including recent evidence

Paediatric and Perinatal Epidemiology, 21 (Suppl. 3), 2–7. ©2007 The Author. Journal Compilation ©2007 Blackwell Publishing Ltd
 Hygiene theory and allergy and asthma prevention
from the Centers for Disease Prevention and Control
on allergic sensitisation.3 Broad surveys of the US
population for skin test sensitisation to common aller-
gens revealed a two- to fivefold rise in prevalence in
the US, from 1976–1980 [National Health and Nutrition
Examination Survey (NHANES) II] to 1988–1994
(NHANES III) such that 54% of the US population
demonstrated allergic sensitisation in NHANES III. By
using objectively measured biomarkers of atopy such
as allergic sensitisation, these estimates in rising preva-
lence take into account the improvements in the diag-
nosis and identification of children with atopy that
have occurred over time (ascertainment bias).
A cross-sectional look at the prevalence of these
disorders across many different countries reveals con-
siderable variation in prevalence for asthma, atopic
dermatitis, and hay fever.4,5 A wide prevalence range
can exist even within single countries; for example, a
tenfold variation in asthma prevalence has been dem-
onstrated between different locales within India.4
Generally, children who grow up in rural areas of
developing countries or in farming communities are
several times to as much as 50 times less likely to have
allergic conditions or manifest allergic sensitisation or
bronchial hyper-responsiveness than children raised
in nearby metropolitan areas.6
Natural history and the allergic march
Of the atopic disorders, food allergy and atopic derma-
titis typically appear first, not at birth, but beginning in
the first 3–12 months of life, reaching their highest
prevalence during the first 2 years of life. Food allergy
and atopic dermatitis then tend to decrease in preva-
lence. The earliest appearing allergic sensitisations in a
German cohort were to common foods (chicken eggs
and cow’s milk), reaching peak prevalence at about
1 year of age and then staying at similar levels until age
6. Allergic sensitisation to wheat and soy reached peak
prevalence at 5–6 years of age.7 Allergies to nuts and
shellfish tend to appear later and persist. Some chil-
dren outgrow these atopic disorders in the preschool
years.
In contrast to food allergy and atopic dermatitis,
allergic rhinitis and asthma have a delayed onset,
steadily increasing in prevalence into the school years.
In one study of children in metropolitan San Diego
(Kaiser study), the most common atopic conditions in
the first 2 years of life were atopic dermatitis (~10%)
and food allergy (10–15%), declining in prevalence to
Paediatric and Perinatal Epidemiology, 21 (Suppl. 3), 2–7. ©2007 The Author. Journal Compilation ©2007 Blackwell Publishing Ltd
3
approximately 5% at ages 4 and 7 years.1 In compari-
son, allergic rhinitis and asthma increased steadily in
prevalence to age 7 years.1 Children who develop food
allergies and atopic dermatitis often go on to develop
airways allergic disease. The prevalence of asthma
tends to reach a plateau during school years, while the
prevalence of allergic rhinitis continues to increase
through college.
While allergic sensitisation to foods peak in preva-
lence by ages 1–2 years, allergic sensitisation to inhaled
allergens begins to appear in children by age 3 years.1
The incidence continues to increase throughout the
school years, mimicking to some extent what is seen
for allergic rhinitis and asthma. Presumably, it takes a
few seasons of exposure to inhaled allergens before the
sharp rise in prevalence is seen. In children who mani-
fest recurrent cough/wheeze in early life, inhalant
allergen sensitisation, atopic dermatitis, elevated
serum IgE levels, and/or allergic rhinitis in early child-
hood (i.e. first 3 years of life) are strong predictors of
disease persistence through later childhood.8–10
The allergic march is therefore a developmental
pattern for a cluster of allergic disorders and allergic
sensitisation that begins in early childhood, revealing
itself as a common course of atopic manifestations in
families and individual children, and subsequently as
trends in prevalence. Food allergies and atopic derma-
titis often precede and predict the development of
allergic rhinitis and asthma. Eczema and certain food
allergies (milk, soy and egg) tend to be outgrown or
improve during the preschool years. In comparison,
allergic rhinitis and other food allergies (nuts, seafood)
tend to persist into adulthood, as does allergy-
associated asthma. The severity of the atopic disorders
is predictive of their persistence and progression.
Immune developmental roots of the allergic
march and the hygiene hypothesis
Allergic sensitisation begins when the nascent immune
system develops aberrant allergic immune responses
to ubiquitous ingested, inhaled and topical exposures.
Then, chronic or recurrent exposure drives inappropri-
ate and prolonged airways injury, inflammation and
aberrant repair of injured tissues. If these aberrant pro-
cesses begin in early childhood and continue through
critical periods of postnatal lung growth and differen-
tiation, then the fully developed lung may differ from
normal. This may underlie the persistent asthma
phenotype.
4 A. H. Liu
The hygiene hypothesis specific for allergic diseases
and asthma is that nature may ‘immunise’ against the
allergic march through microbial exposures of the res-
piratory and gastrointestinal tracts (and possibly the
skin) in early life.6,11,12 The hallmark of effective anti-
microbial immune responses, the development of T
lymphocytes that produce antimicrobial interferon
(IFN)-g, so-called T-helper Type 1 cells (Th1), prevents
pro-allergic ‘Th2’ immune development and allergy,
thereby keeping ubiquitous, harmless environmental
exposures from becoming allergens. Th1 immune
responses improve host defences by inducing antiviral
mechanisms that keep respiratory viruses from prolif-
erating in respiratory epithelium and spreading down
the airways. Th1 immune responses during airways
injury and inflammation also inhibit aberrant repair
processes that underlie pathological tissue changes in
asthma. For example, IFN-g inhibits mucous gland
and smooth muscle hyperplasia, fibrotic repair pro-
cesses, and mast cell activation. However, healthy
immune responses also require the development of
anti-inflammatory responses. Microbes induce such
regulatory immune responses (e.g. IL-10-producing
regulatory T lymphocytes) to limit immune activation
and inflammation when they are no longer needed in
order to prevent further tissue damage from ‘friendly
fire’.
Contemporary paradigms in hygiene theory
Hygiene theory research suggests that a broad range of
microbial exposures and associated vectors and life
style features contribute to the microbial burden affect-
ing allergy and asthma outcomes. They can be cate-
gorised as follows:
1 infections: bacterial, parasitic, viral; pathogenic or
subclinical;
2 microbial components: such as bacterial endotoxins,
other Toll-like receptor (TLR) ligands and micro-
organism-associated molecular patterns;13
3 gastrointestinal colonisation: lactobacillus, bacteroi-
des, parasites;
4 soil microbiota: largely non-infectious, saprophytic
gram-positive bacteria (e.g. actinobacter, mycobacte-
ria, lactobacilli);14
5 forces that reduce microbial burden: antibiotics,
immunisations, public and personal hygiene
measures, reduced exposure to microbe-rich
dirt.
Paediatric and Perinatal Epidemiology, 21 (Suppl. 3), 2–7. ©2007 The Author. Journal Compilation ©2007 Blackwell Publishing Ltd
Microbes: at best, a double-edged sword
Fundamental to the hygiene hypothesis is that micro-
bial exposures can potentially cause both healthy and
harmful outcomes. This duality defies the simplistic
tendency to view the relationship as either one way or
the other. Although the bias of the hygiene hypothesis
is to understand the protective outcomes of these expo-
sures, such an unbalanced view would be akin to
studying and espousing the efficacy of a therapy
without considering the potential risks of adverse
effects when safety concerns are recognised. The
multidimensional determinants of health or disease
outcomes relative to microbial exposures include
differences in:
1 microbes or their components;
2 disease subtypes;
3 time elements such as early age, or exposures prior
to disease development vs. after the onset of atopic
pathogenesis;
4 dosage and combinations of exposures;
5 genetic contributions to the variance in response to
these exposures;
6 routes of exposure (i.e. inhaled, ingested,
cutaneous).
Although complex, skilled investigators in the basic
and clinical sciences have been teasing out these ele-
ments as well as the interactions between them.
Health – disease determinants exemplified by
bacterial endotoxin exposure
Bacterial endotoxin has received prototypical interest
in the hygiene hypothesis by those eager to investigate
the differing determinants of healthy or harmful out-
comes from microbial exposures. Endotoxin is a
lipopolysaccharide that makes up much of the outer
cell wall of all gram-negative bacteria.6 Its major
immune stimulatory component is the highly con-
served Lipid A in the cell membrane that is specifically
recognised by TLR4 and aided by several helper/
enhancer proteins, including CD14. Endotoxin has
been recognised as being carried on cotton dust and
causing byssinosis or Monday Asthma in cotton
workers.15–18 Endotoxin has since been recognised as an
asthmagenic chronic exposure in numerous other
occupational and workplace settings.19 Endotoxin is
also measured in household dust in varying levels.
Two recent epidemiological survey studies have
observed a higher prevalence of asthma with higher
 Hygiene theory and allergy and asthma prevention
levels of endotoxin in the US (National Survey of Lead
and Allergens in Housing study)20 and in South
Manchester, UK,21 implicating endotoxin as a toxic res-
piratory exposure in household settings as well.
In contrast, a number of studies, including well-
performed longitudinal birth cohort studies, have
observed that house-dust endotoxin levels are associ-
ated with less allergen sensitisation and less atopic
dermatitis in early life.22–26 Concurrently, endotoxin has
been associated with more wheezing in infancy23,26,27
but in one study, endotoxin was subsequently associ-
ated with less wheezing in later childhood.28 Although
most of these longitudinal studies have occurred in
modern metropolitan locales, these relationships were
also observed in specific subpopulations in New York
City (‘inner city’).26 In a large cross-sectional study of
school-age children living in rural European farm/
non-farm communities and where endotoxin exposure
has a much broader range, dust endotoxin levels were
associated with less allergen sensitisation, less hay-
fever and less atopy-associated wheeze in a dose-
responsive manner; at high levels of exposure,
endotoxin was associated with increased non-atopic
wheeze, suggesting the expression of a subtype of
endotoxin-hypersensitive asthma in children with
very high exposure.29
Collectively, these studies provide some consistency
of observation of the relations between endotoxin
exposure and allergy and asthma outcomes, and exem-
plify some of the determinants of healthy or harmful
outcomes outlined above: dosage, disorders of interest
(‘allergic march’ disorders – atopic dermatitis, allergen
sensitisation and hay fever vs. infant wheezing, and
occupational or endotoxin-sensitive asthma), and
timing of exposure (from birth vs. in later childhood or
adulthood; prior to disease onset vs. after asthma
onset). In addition, these parameters of dosage, timing
and disease state subtypes do not appear to completely
explain the variability in health or disease/disorder
outcomes relative to endotoxin exposure.
Since the discovery of endotoxin’s TLR4 receptor,
numerous other TLRs with other microbial ligands
have been identified. For example, microbial DNA is
recognised as distinct from non-microbial (e.g. human,
animal) DNA via TLR9, and bacterial cell wall compo-
nents other than endotoxin are recognised by TLR2.
Exposure to bacterial DNA and TLR2 ligands also
appears to vary, in levels that correlate yet are distinct
from endotoxin.30,31 These ligands also interact and
alter the cellular and immune responses to endotoxin.
Paediatric and Perinatal Epidemiology, 21 (Suppl. 3), 2–7. ©2007 The Author. Journal Compilation ©2007 Blackwell Publishing Ltd
5
For example, microbial DNA is not potent relative to
endotoxin in activating and stimulating cells; however,
in co-stimulation with endotoxin, microbial DNA in
dust samples can augment anti-inflammatory, immune
regulatory and Th1 cytokine release, thereby shifting
the nature of the immune response away from allergy
and inflammation.30
The interaction of microbial exposures with func-
tional polymorphisms that affect response to these
exposures has provided compelling evidence for a role
for specific microbial exposures in influencing disease
outcomes. Again, endotoxin exposure can serve as an
instructive example. In dose–response challenge
studies of healthy persons with inhaled endotoxin, a
small proportion were hypersensitive to endotoxin,
consistent with the observations noted above of
endotoxin-sensitive asthma in occupational and rural
farm settings.32 There were also a small proportion of
individuals who demonstrated essentially no response
to inhaled endotoxin; most of these individuals had
dominant loss-of-function mutations in their TLR4
gene33 and may therefore be protected from disease
mediated by high levels of endotoxin exposure in
workplace or home settings.
Another instructive example can be found in CD14,
a membrane-bound and soluble protein that aids
endotoxin detection by TLR4. A promoter region poly-
morphism at position -260 in the CD14 gene alters the
expression of CD14, such that ‘T’ vs. ‘C’ alleles at this
locus are associated with increased CD14 expression,
reduced Th2-type allergic immune responses, and
fewer atopic markers.34,35 In a longitudinal birth cohort
study in Manchester, UK, the relationship of early
endotoxin exposure with less allergen sensitisation
and less atopic dermatitis was affected by CD14 geno-
type: only the subgroup of children with the low-
responder ‘CC’ CD14 allele (28% of their cohort) had a
significantly decreased risk of allergen sensitisation
and atopic dermatitis, and increased risk of non-atopic
wheeze associated with increased endotoxin levels in
infancy.36 In a cross-sectional study of asthma in adults
living in Barbados, those with the high-responder ‘TT’
CD14 genotype (9% of their cohort) had a significantly
decreased risk of asthma; lower risk in this CD14 sub-
group occurred at lower house-dust endotoxin levels,
while increased risk occurred at high endotoxin
levels.37 These epidemiological studies of gene–
environment interactions strengthen the evidence for
the role of microbial exposures in two ways: they
provide supportive ‘forensic DNA’ evidence that
6 A. H. Liu
further implicate specific microbial exposures in the
pathogenesis of allergic disorders and asthma; they
also exemplify the potential for gene polymorphisms
to contribute to the variability in outcomes due to
microbial exposures.
Conclusions
The quantity and quality of this growing burden of
evidence suggests that the hygiene hypothesis for
allergic disorders and asthma is evolving beyond
theory. Included in this evidence is the maturation of
clinical epidemiological studies, from small cross-
sectional to larger and longitudinal studies designed to
test causal relationships. The relationships between
microbial exposures and disease outcomes are not
simple or unimodal. Indeed, the evidence reveals multi-
dimensional relationships; the evidence developing
around endotoxin exposure is a prototypical example.
A few caveats are in order: the current evidence is
strengthening but is far from being beyond doubt. For
example, the veracity of today’s epidemiological
studies is limited by the current confines of our
research tools to measure and quantify specific micro-
bial exposures. Most of the epidemiological research in
this area has been in children, with longitudinal
studies largely in early life. This is logical as the pro-
tective influence of microbial exposures is believed to
occur on the allergic march and on asthma that most
often begins in early life; however, this implies that
what we understand about hygiene theory as it relates
to later childhood and adulthood is commensurately
limited.
Even if the hygiene hypothesis is true, it is already
apparent that it is only part of the story of causation in
allergic disorders and asthma. The hygiene hypothesis
is also susceptible to extrapolations that would indi-
cate that antimicrobial pathogen practices may be
harmful, when the benefits of such practices have
already been well established. That said, evidence sup-
porting this theory provides hope for the future that
preventive environmental and life style interventions
can improve allergic disease and asthma outcomes.
Conflicts of interest
The author has declared no conflicts of interest.
Acknowledgements
Source of support: NIH#K23-HL-04272 (Liu).
Paediatric and Perinatal Epidemiology, 21 (Suppl. 3), 2–7. ©2007 The Author. Journal Compilation ©2007 Blackwell Publishing Ltd
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