Macpherson AJ, Harris NL.. Interactions between commensal intestinal bacteria and the immune system. Nat Rev Immunol 4: 478-485

Article (PDF Available)inNature reviews. Immunology 4(6):478-85 · July 2004with 1,490 Reads
DOI: 10.1038/nri1373 · Source: PubMed
Although we might shudder at the thought of billions of bacteria living in our lower intestine, we are colonized by these passengers shortly after birth. However, the relationship is mostly of mutual benefit, and they shape our immune system throughout life. Here, we describe our developing understanding of the far-reaching effects that the commensal flora have on mucosal and systemic immunity and their relevance to the effects of hygiene on human disease.
478 | JUNE 2004 | VOLUME 4
evolution on both sides. The bacteria benefit
from the stable habitat, which is rich in energy
sources from the food we ingest; and we sal-
vage heat energy from compounds such as cel-
lulose, which would otherwise be indigestible
because we lack the necessary enzymes. Also,
some bacterial compounds, including short-
chain fatty acids and phylloquinone (vitamin
K1), are used in host anabolic pathways.
Moreover, the commensal microflora compete
with incoming foreign microorganisms for
space and resources, thereby making it more
difficult for true pathogens to become estab-
lished. However, this seemingly ideal balance is
sometimes disturbed: the development of
(the inci-
dence of which is 1 in 500 in Western popula-
tions) and possibly some autoimmune diseases
are associated with immune responses to envi-
ronmental microorganisms or mild pathogens.
Here,we discuss the pervasive way in
which the environmental flora shapes both
the mucosal and systemic immune systems.
It is clear that immune composition and
lymphoid structures differ when this flora is
present compared with when it is absent;
however, we are only beginning to under-
stand the mechanisms and functions of the
adaptive changes that occur when environ-
mental bacteria colonize the host. One of the
consequences is to physically limit the live
bacteria to the intestinal lumen. Yet, the
functional consequences of the systemic
that the combined number of genes in these
bacteria exceeds the total number of eukaryotic
genes by a factor of at least 100.
Usually, we peacefully’ coexist with our
commensal microflora, in a good example of
MUTUALISM,which has been established through
Although we might shudder at the thought
of billions of bacteria living in our lower
intestine, we are colonized by these
passengers shortly after birth. However, the
relationship is mostly of mutual benefit, and
they shape our immune system throughout
life. Here, we describe our developing
understanding of the far-reaching effects
that the commensal flora have on mucosal
and systemic immunity and their relevance
to the effects of hygiene on human disease.
Most studies of the immune system aim to
understand the way in which it responds to
infectious pathogens. By analysing immune
mechanisms in animal models of infectious
disease, we learn how the immune system
responds to biologically important challenges.
Ye t clinically apparent microbial infections
are the exceptions in our harmonious coexis-
tence with vast numbers of non-pathogenic
microorganisms; these microorganisms enter
our body from the environment shortly after
birth and colonize the mucous membranes
and skin epithelia. In the lower intestine
alone, the density of commensal bacteria in
the lumen reaches 10
organisms per gram of
intestinal contents, with approximately 1,000
species present
(BOX 1).So when the physiol-
ogist J.B.S. Haldane once commented that
even the Archbishop of Canterbury is 65%
, he omitted to mention that the head
of the Church of England also consists of
more bacterial cells than eukaryotic cells and
Interactions between commensal
intestinal bacteria and the immune
Andrew J. Macpherson and Nicola L. Harris
Box 1 | Intestinal microorganisms
Bacteria are the main type of microorganism present in the mammalian intestine, although
other types are also found, including protozoa and fungi. The stomach and small intestine
have relatively low bacterial densities (10
organisms per gram or ml of luminal contents
in mice, consisting mainly of acid-tolerant lactobacilli and streptococci). The distal portion
of the small intestine, the ileum, is a transition zone with higher bacterial densities (10
gram) and species diversity, but the most dense colonization is in the colon (10
gram), which hosts more than 400 bacterial species. In the lower intestine, anaerobes
predominate, particularly the Bacteroides,bifidobacteria, fusobacteria and peptostreptococci
(each group present at approximately 10
per gram); by contrast, aerobes and facultative
aerobes, including enterobacteria and lactobacilli, are present at only moderate densities
per gram).
There are two main difficulties in understanding and measuring these complex flora. First,
a comparison of two techniques used to assess faecal bacterial numbers — counting colonies
of culturable bacteria and estimating numbers using smears — shows that less than 50% of
intestinal bacteria can be cultured. This is because of the precise oxygen requirements of
some species and their fastidious (and largely unknown) nutrient requirements. Second,
although most measurements have been made using faecal bacteria, the intestine is not a
homogeneous environment — groups of bacteria can also exist on the surface of the mucus
layer or deep within it.
Fortunately, there are ways of overcoming the difficulties in culturing intestinal bacteria.
The 1.5-kb gene encoding 16S ribosomal RNA is present in multiple copies in bacterial
chromosomes, and it is highly polymorphic. Therefore, the nucleotide sequence of this
gene (obtained after amplification by PCR) can be used to determine the species of each
,and the gene can serve as a target (in species-specific in situ hybridization)
for studying the spatial arrangement of each bacterial group.
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Competing interests statement
The authors declare that they have no competing financial interests.
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show that although commensal bacteria are
killed within hours by macrophages, they can
survive for several days inside dendritic cells
(DCs). However, DCs that have been loaded
with commensal bacteria in the Peyer’s
patches do not penetrate farther than the
mesenteric lymph nodes, so DCs that are
primed by live bacteria are usually restricted to
the mucosal immune system. It is important
to note that this in vivo priming effect is lost if
the bacteria are killed by heat treatment, so it is
not mediated by lipopolysaccharide and/or
other bacteria-derived immunostimulatory
molecules. (Although by internalizing live bac-
teria, DCs might concentrate these bacterial
compounds.) So, IgA production, and proba-
bly intestinal T-cell responses, can be selec-
tively induced by DCs loaded with commensal
bacteria, and this increased local secretion of
IgA limits the penetration of commensal bac-
.It has long been established that both B
and T cells are activated in the Peyer’s patches,
after which they recirculate through the
intestinal lymphatics, enter the bloodstream at
the level of the thoracic duct and home back
to the intestinal lamina propria. Because DCs
containing live commensal bacteria are con-
fined to the mucosal immune system, the
induction of T- and B-cell responses is focused
at the mucosa where it is required.
Although there is a strong locally induced
immune response, because commensal bacte-
ria do not usually penetrate beyond the
mesenteric lymph nodes, we believe that the
systemic immune system is largely ignorant of
these organisms. An example of this is that
responses that prime the production of IgG
specific for Enterobacter cloacae (the main aer-
obe in our SPF mouse colony) are not seen in
unmanipulated mice colonized with these
.This occurs as a result of IGNORANCE,
rather than tolerance, because intravenous
injection of a small dose of E. cloacae causes a
highly reproducible priming response
priming of systemic immunity to commensal
bacteria is largely unnecessary, because the
innate immune system can destroy the few
organisms that do penetrate the intestinal
barrier — mice only become unable to coexist
with their intestinal flora when there are seri-
ous deficiencies in the phagocytic biocidal
mechanisms that generate reactive oxygen
and nitric oxide radicals
So, on the whole, we believe that mice
are systemically ignorant of particulate
(live) commensal intestinal bacteria; how-
ever, soluble bacterial degradation products
that reach the systemic circulation are
probably responsible for the differences in
the organization of secondary-lymphoid
and the different concentrations
immune alterations are also important,
because there is evidence that the hygiene
status of humans influences predisposition
to allergy and/or autoimmunity. We believe
that there are important differences between
the observed improvements in human
hygiene and the current animal models that
are used to study the immunological conse-
quences of these improvements, and we con-
sider that these differences are crucial for
understanding and modelling the effects of
hygiene on systemic immunopathology.
Mucosal immune adaptation
There is no question that the host is highly
adapted to the presence of commensal
intestinal bacteria. The evidence for this comes
from comparing germ-free mice
(FIG. 1), which
have no commensal microflora, with spe-
cific-pathogen-free (SPF) animals of the
same strain, which contain a simple flora
(BOX 2).The mucosal immune system is
undeveloped in germ-free animals: they have
PEYER’S PATCHES that contain few
germinal centres, as well as greatly reduced
numbers of IgA-producing plasma cells and
T cells
(FIG. 2).The
immunological abnormalities in germ-free
animals are not confined to the mucosal
immune system: the spleen and lymph nodes
are relatively structureless, with poorly formed
B- and T-cell zones
(FIG. 2) and abnormal high
endothelial venule morphology
.The mice
also have hypogammaglobulinaemic serum,
mainly because of reduced levels of IgG
Furthermore, the gene-expression profile of
the intestinal epithelial-cell layer is altered in
the absence of commensal bacteria
.All of
these abnormalities can be reversed within
several weeks of colonizing germ-free animals
with commensal bacteria, which can be
achieved by placing an SPF mouse in a cage
that contains germ-free animals
(FIG. 1).Such
experimental colonization of germ-free ani-
mals might seem artificial, but similar colo-
nization occurs in every neonate within days
of its birth
Systemic immune ignorance
The physical barrier that separates the large
numbers of commensal intestinal bacteria
from the underlying tissues is only a simple
(single cell) epithelial layer; although it is
reinforced by a layer of mucus, the secretion of
IgA and the production of antibacterial mole-
cules (such as
DEFENSINS) by the epithelium
(FIG. 3).Considering this, it is not surprising
that in both humans and experimental ani-
mals some bacteria can penetrate this barrier
to reach deeper tissues
.Challenge studies
a b c
Figure 1 | Keeping germ-free mice in an isolator. It has been possible to keep experimental
animals in entirely germ-free conditions for the past 50 years or more, by initially delivering the pups
by sterile Caesarean section and hand rearing them aseptically. a | After this, it is less labour intensive
to keep the mice free from colonization by environmental organisms by breeding them in an isolator,
which is ventilated with sterile filtered air under positive pressure. Gnotobiotic (germ-free) animal
husbandry uses sterile food, water and bedding. It is important to note that germ-free animals have
no bacteria in the intestine or on other body surfaces, whereas specific-pathogen-free (SPF) mice
are only devoid of known mouse pathogens and do contain intestinal bacteria. b | A germ-free isolator
can be loaded with sterile supplies using a transport ring, which is small enough to fit in an animal-unit
autoclave. c | The ring is connected to a port on the isolator using a flexible plastic sleeve that
contains a glove. (The inside of the sleeve is sterilized by spraying it with peracetic acid immediately
before it is connected to the isolator and the transport ring.) The internal door on the isolator and the
external seal on the transport ring are then opened from the outside using the gloves, and sterile
materials are brought in. Samples are taken routinely from the husbandry materials and the mice,
to ensure that the colony remains germ free. Mice can either be experimentally manipulated in the
isolator, or they can be transferred into a sterile lamina-flow hood, by connecting the isolator to
a port on the side of the hood. Germ-free mice can easily be recolonized with bacteria by placing
a single mouse that has normal flora into the cage.
480 | JUNE 2004 | VOLUME 4
referred to as antigen free (because they lack
full-length proteins), the animals still con-
sume milk (during lactation in the neonatal
period) and groom themselves. We argue
that the important difference between the
background immune stimulation of germ-
free animals fed a sterile (autoclaved) natural-
ingredient diet and those fed an elemental
diet probably results from the microbial
material contaminating the autoclaved food
(rather than the presence of full-length pro-
teins), because soluble proteins introduced
into the intestine are generally thought to be
tolerogenic rather than immunogenic
relative impact on immune reactivity of
reducing the levels of contaminating micro-
bial molecules or full-length proteins could
easily be tested by selective experimental
Compared with germ-free mice that are
fed autoclaved food, germ-free mice fed an
elemental diet have highly reduced levels
of serum IgG and IgA, reduced numbers of
splenic IgG-producing cells and fewer circu-
lating lymphocytes
.Despite this, serum
IgM concentrations and the diversity of the
REPERTOIRE are preserved
.When these
mice were challenged with a model antigen
together with adjuvant, they mounted a
strong specific IgG response, but the poly-
clonal response that leads to increased levels
of non-antigen-specific IgG was consider-
ably diminished. This reduction in the poly-
clonal response was also seen in germ-free
animals that were fed sterile food (in com-
parison with SPF mice), but it was more
marked in germ-free mice that were fed the
elemental diet
.In addition, the usual
increase in the frequency of somatically
hypermutated immunoglobulins (of the IgG
isotype) that occurs during ageing also
depends on exposure to environmental anti-
gens (from commensal gut microorganisms),
because this has been shown to occur to a
lesser extent in germ-free mice
than in SPF
mice. So, bacteria-driven alterations that
result from ‘bathing’ the immune system in
immunostimulatory bacterial molecules
cause a baseline level of immune activation
this increases the degree of polyclonal stimu-
lation to protein antigens administered with
adjuvants and might also improve the
immune response against invasive pathogens.
It is well known that patients with agamma-
globulinaemia are protected against infec-
tions with encapsulated bacteria by treatment
with pooled gammaglobulin preparations
from healthy donors. Furthermore, experi-
ments on rats have shown that crossreactive
antibodies can sufficiently bind the repeti-
tive capsular polysaccharides of bacteria to
are fed an elemental diet (containing hydro-
lysed amino acids and purified lipids and
carbohydrates) rather than autoclaved food,
which contains (dead) bacterial material.
Although these elemental diets are often
and diversity of serum immunoglobulins in
germ-free and SPF animals
.Indirect evi-
dence is provided by studies
that the hypogammaglobulinaemia of germ-
free animals is more pronounced when they
Germ-free mouse
Mouse colonized
with intestinal bacteria
c Intestinal IgA
(inset: Peyer's-patch IgA)
b Intestinal CD4
(inset: intestinal CD8)
a Splenic CD4
(inset: splenic CD8)
Figure 2 | The presence of intestinal bacteria has a large impact on lymphoid structures of
both the intestine and systemic tissues. Histological sections of the spleen and intestine are shown
for a germ-free wild-type C57BL/6 mouse and for a C57BL/6 mouse colonized with intestinal bacteria.
In the absence of intestinal bacteria, the spleens have relatively few germinal centres and poorly formed
T-cell (pink) and B-cell zones (a). The intestines of germ-free mice have low numbers of lamina propria
cells (brown) (b), greatly reduced numbers of IgA-producing cells (brown) (c) and hypoplastic
Peyer’s patches. The histological abnormalities of germ-free mice reverse within weeks of colonization
with intestinal bacteria, which can be achieved, for example, by placing a normal colonized mouse in
the same cage. These images are reproduced with permission from
REF. 3 (2001) Elsevier.
Box 2 | The ‘Schaedler flora’ in experimental animals
As shown in
FIG. 1, experimental animals can be bred and maintained in a sterile environment.
These germ-free or gnotobiotic animals have no microorganisms in the gut or on other body
surfaces. Starting with germ-free mice, Russell Schaedler and colleagues
at the Rockefeller
Institute for Medical Research, New York, USA, carried out experiments in the 1960s in which
they reintroduced simple mixtures of defined intestinal bacteria. They showed that inoculation
with coliform bacteria alone led to high stable levels of these bacteria, which are only a minor
component of the gut flora in a normal mouse. To attempt to introduce a simple, balanced flora
that would colonize the gut without compromising the health of the mice, a cocktail of eight
species eventually became popular: Escherichia coli var. mutabilis,Streptococcus faecalis,
Lactobacillus acidophilus, Lactobacillus salivarius,group N Streptococcus, Bacteroides distasonis,
a Clostridium species and a species of extremely oxygen-sensitive (EOS) spiral-shaped (fusiform)
bacteria. In 1987, the National Cancer Institute of the United States revised this ‘Schaedler flora
to include a Flexistipes fusiform bacterium and three further EOS fusiform species
Specific-pathogen-free (SPF) mice are defined on the basis of a negative screen for specific known
pathogens. They are usually derived from a commercial breeding colony, which might or might not
have been rederived (by Caesarean section or embryo transfer) in a germ-free environment. The
most rigorous derivation of SPF mice is to be derived germ free and then colonized with modified
Schaedler flora.
It is clear that the flora of experimental animals generated in this way is far simpler than
in humans (approximately 1,000 species) or in conventional experimental animals. Although
this defined gut population should remove much of the background variability observed in
experiments, it is often assumed that the flora remains stable while subsequent generations
of animals are bred (in SPF facilities). However, the composition of the flora (and particularly
the anaerobes) is not often checked, and it is relatively easy for new species to be introduced,
either from human handlers or from animals sourced from other SPF suppliers. Scientists are only
aware of the mouse pathogens that are routinely tested for, and the immunological community is
generally ignorant of the environmental flora of the experimental animals that they use.
to assessing whether experiments with ‘ultra-
clean mice (that is, germ-free mice or SPF
mice with a restricted flora) can help us to
understand the patterns of human disease.
Hygiene is even more complex to investi-
gate in the human population, because poor
hygiene usually occurs together with several
confounding factors: poor nutrition, lifestyles
with lower technology, and less access to
medical care (for registering the incidence of
disease). The importance of these variables
needs to be considered in epidemiological
Autoimmunity and allergy, which result
from inappropriate and overactive immune
responses, are disadvantages arising from
our ability to combat infectious disease. The
‘hygiene hypothesis’ states that as we
improve hygiene, there are fewer infectious
challenges, and the subsequent response of
the immune system leads to allergy
(BOX 3).
induce the fixation of complement and the
opsonization of the bacteria
. One disadvan-
tage is that the peripheral self-tolerance that
is usually generated by the presentation of
proteins on immature DCs
might be com-
promised by bystander activation of the DCs
in the presence of bacterial degradation
The presence of commensal intestinal
bacteria, therefore, has clear structural and
functional consequences for the systemic
immune system. Because even high doses of
commensal bacteria do not penetrate to sys-
temic secondary-lymphoid structures
immune changes in germ-free animals are
accentuated when they are fed a purified diet
of hydrolysed amino acids, we propose that
the baseline setting of the immune system is
a response to contamination of the immune
environment with soluble breakdown
products from microorganisms.
Interactions between the systemic and
mucosal immune systems have traditionally
been studied by examining oral tolerance.
Such studies involve the administration of a
T-CELL-DEPENDENT ANTIGEN,either by ingestion or
mucosal application, then the measurement of
suppression of the response to later systemic
immunization. Although oral tolerance can be
induced in germ-free animals
,the kinetics of
the systemic response to immunization are
,probably because of the immaturity
of systemic secondary-lymphoid organization
under germ-free conditions. This makes the
interpretation of oral-tolerance results in
germ-free animals too complicated to allow
direct comparison with SPF animals.
Improving human hygiene
We now turn from examining the effects of
experimentally manipulating the density and
diversity of environmental antigens on mice
Intestinal lumen
DC uptake of
Macrophage killing
of penetrating
Commensal bacterium
Afferent lymph
T-cell area
B cell T cell
Antibody (IgA)
IgA plasmablast
Efferent lymph
M cell
DC carrying
Blood circulation
lymph node
Induction of IgA-producing
plasma-cell formation
Figure 3 | Immune defences against commensal intestinal bacteria. Commensal bacteria are present at a high density in the intestinal lumen (up to 10
per gram of luminal contents). Most commensal bacteria reside outside the layer of mucus that covers the intestinal epithelial cells. Some bacteria can be killed by
antibacterial molecules, such as defensins, which are produced by the epithelial cells. Bacteria that penetrate the enterocyte epithelial layer are rapidly killed by the
macrophages in the lamina propria. Commensal bacteria can also penetrate the specialized follicle-associated epithelium, containing M cells, which lies over the
Peyer’s patches. These bacteria are also rapidly killed by macrophages, but small numbers can survive for several days in dendritic cells (DCs). This enables the
interaction of DCs with T and B cells in the Peyer’s patches and/or the migration of DCs to the draining mesenteric lymph nodes. (DCs that contain live bacteria induce
IgA-producing plasma cells more effectively than heat-killed bacteria.) Although DCs loaded with commensal bacteria can traffic to the mesenteric lymph nodes, the
lymph nodes function as a barrier, and the loaded DCs cannot penetrate farther to reach the systemic secondary-lymphoid tissues. The result is that the induction of
immune responses by live bacteria is confined to the mucosa itself. Following activation, B- and T-cell blasts can leave the mesenteric lymph nodes through the
efferent lymph, enter the bloodstream at the thoracic duct and home back to the intestinal mucosa.
482 | JUNE 2004 | VOLUME 4
during thymic selection can result in
.Patients with mutations in
the forkhead box P3 (FOXP3)gene (required
for the development of CD4
tory T cells
) also show systemic autoimmu-
nity, eczema and increased serum IgE levels
so a global lack of regulatory T cells does
affect immune-system homeostasis.Yet, in a
lymphocyte-replete human or experimental
animal, the ability of infections to alter regu-
latory T-cell populations is probably weak,
considering the poor antigen-specific prolif-
erative expansion of regulatory T cells
Therefore, we think that it is improbable that
functional alterations in regulatory T cells
explain the relationship between improved
hygiene and increasing incidence of allergy
and autoimmunity in the population.
Hygiene in experimental animals
Because we can manipulate the population of
environmentally derived microflora by con-
trolling the husbandry of rodents, we should
also be able to investigate the underlying
basis of the hygiene effect. It is crucial to
appreciate that whereas germ-free mice have
no microorganisms colonizing their body
surfaces, neither germ-free nor SPF animals
have been exposed to any known pathogen.
Hygienic humans have a diverse intestinal and
body-surface flora, but they take measures to
reduce their exposure to pathogens. The envi-
ronmental conditions that we manipulate for
experimental animals (when comparing
mice with a restricted commensal flora and
germ-free mice, neither of which are
exposed to pathogens) are therefore quite dif-
ferent from improvements in human hygiene
(in which early exposure to pathogens is
reduced). Humans have a much more
diverse (approximately 1,000 species) and
dense (approximately 10
bacteria per gram
of luminal contents) commensal bacterial
flora than SPF mice
(BOX 2).In fact, in con-
trast to humans, most rodent models of
autoimmunity actually show reduced inci-
dence in conditions of improved hygiene
Similarly, spontaneous and induced models
of inflammatory bowel disease are abrogated
in SPF or germ-free conditions
.There are
rare exceptions to this: notably, autoimmune
gastritis following neonatal thymectomy, the
incidence of which is unchanged in germ-
free conditions
; and diabetes in non-obese
diabetic mice, in which the incidence is
There are potentially three ways in
which microorganisms interact with the
immune system: an infection in which live
microorganisms can proliferate systemi-
cally; the penetration through the mucosa
immune deviation, thereby skewing immune
responses away from the neonatal T
2-cell bias
towards T
1-cell responses
(BOX 3).However,
it has been difficult to show this phenomenon
directly, and it does not explain three other epi-
demiological observations: first, there is a simi-
lar negative association between atopy and
infection with helminths (which are known to
induce T
2-cell cytokines)
;second, humans
with an immunodeficiency resulting from
genetic lesions that affect T
1-cell cytokine
pathways do not have an increased inci-
dence of allergic disease
; and third, the
increase in the incidence of allergy in recent
decades has been accompanied by similar
increases in autoimmune diabetes
coeliac disease
— conditions that are usu-
ally considered to be T
1-cell-biased diseases.
Because the demographics of autoimmunity
are similar to those of allergy, they need to
be considered together when modelling the
effects of hygiene.
Another possible explanation for the
effects of altered hygiene on allergy and/or
autoimmunity is that there are differences in
the induction of regulatory T cells
(BOX 3).
Certain infectious agents, such as Bordetella
pertussis,have been reported to induce the
development of T regulatory 1 (TR1) cells
as a method of evading host responses
similar induction of regulatory T cells has
also been described following infection with
hepatitis C virus
, Helicobacter hepatis
.In addition, a strong bias in
and CD4
T-cell subsets
There is reasonable clinical epidemiological
support for this theory. Children from fami-
lies of lower socio-economic status or with
more siblings have decreased incidence of
atopy, presumably because of exposure to
more infectious agents
.There is also an
inverse correlation between previous infec-
tion with mycobacteria or viruses, including
hepatitis A virus (HAV), and the subsequent
development of asthma
, and other
show that children brought up on
farms are protected from the development of
asthma. However, it is usually unclear which
of these infections can actually induce a pro-
tective effect and which are surrogate markers
of poor hygiene in a complex environment.
For those exposed to HAV, protection
against asthma development is more pro-
nounced in individuals carrying a six-amino-
acid insertion at position 157 of the TIM1
(T-cell immunoglobulin domain and mucin
domain 1) gene, which encodes the cell-surface
receptor through which HAV infects human
.This receptor is also expressed by acti-
vated CD4
T helper 2 (T
2) cells
.It remains
unclear whether the polymorphism in TIM1
alters HAV infectivity or alters the immune
response against the virus. However, the results
indicate that, in this case, HAV itself is respon-
sible for protection against allergy, rather than
just being a surrogate for another protective
One suggested explanation for the observed
protection against allergy is that interaction
with ‘unhygienic’ microorganisms causes
Box 3 | The ‘hygiene hypothesis’
In 1989, Strachan
coined the term ‘hygiene hypothesis’. This hypothesis states that a leading
cause of the increased incidence of allergy in today’s population is the decrease in exposure
to common infections during early life, which occurs as a result of smaller family size and
improved hygienic conditions. Considerable epidemiological and experimental evidence
supports the hypothesis, including studies examining airborne viruses, mycobacteria, orofaecal
microorganisms and helminths
.Two popular theories that offer explanations for the
hygiene hypothesis are immune deviation and counter regulation.
It is well known that the T helper 1 (T
1)-cell cytokine interferon-γ can suppress the
differentiation of T
2 cells
and the production of IgE
,which are associated with atopy.
Because the neonatal immune system has been described as showing a T
2-cell bias
proponents of immune deviation argue that exposure to microorganisms that induce T
responses is required to prevent the development of atopic (T
2-cell) responses
The counter-regulation model states that the production of immunoregulatory factors
after exposure to microorganisms limits the development of unrelated immune-mediated
.Indeed, experimental infection of mice with Mycobacterium vaccae can elicit a
population of CD4
regulatory T cells that attenuate ovalbumin-induced airway
inflammation through the production of interleukin-10 (IL-10) and transforming growth
.IL-10-producing cells that are induced by infection with enteric helminths have
also been shown to protect mice from immunopathology associated with the subsequent
ingestion of a food allergen
We argue here that neither explanation is likely to account entirely for the long-term
consequences of altered hygiene conditions and that alterations in T- and B-cell repertoires
after pathogenic infections probably contribute more to the differences in incidence of
lymph nodes were smaller and nephritis
was reduced compared with animals on the
natural-ingredient diet, thereby demon-
strating the possible impact of microbial
products on the resulting immunopathology.
Modelling human hygiene effects
Instead of the current method of comparing
germ-free and colonized animals, in which
large differences in lymphoid structure are
apparent, we think that deliberate, defined
experimental infections will be required to
model the way in which improvements in
human hygiene during early life lead to a
greater incidence of allergy and autoimmunity.
Indeed, it is possible to show short-term
alterations in susceptibility to induced aller-
gic responses following defined experimental
infections. For example, previous infection
with Mycoplasma or Mycobacterium bovis
Bacillus Calmette-Guerin (BCG) can attenu-
ate airway inflammation that is induced
experimentally using ovalbumin, at least
when mice are challenged within 1–2 weeks
of clearing the infection
.Previous pul-
monary infection with influenza virus can
also provide protection against bronchial
hyperresponsiveness in mice
.This effect is
dependent on interferon-γ production by
lung-resident memory CD8
T cells, which
can be re-activated by nonspecific stimuli
encountered during allergen challenge
These studies provide evidence for the ability
of pathogenic infections to alter unrelated
immune responses through immune deviation
mechanisms, but they do not model the long-
term effects of early exposure to pathogenic
agents and how this prevents the development
of allergy and/or autoimmunity.
Infections result in an immune response
that is partly specific (T-cell clones specific for
peptides derived from pathogens and high-
affinity neutralizing antibodies specific for
surface epitopes)
and partly nonspecific
(class-switch recombination of natural anti-
body specificities, resulting mainly from
bystander help provided by specific T-cell
.We propose that infection causes
an alteration of the T-cell repertoire that
could also account for the hygiene effect,
without necessarily involving T
immune deviation or T-cell-mediated regula-
tion. The presence of a large quantity of anti-
gen can eliminate or inactivate T-cell clones;
this functional
EXHAUSTION has well-recognized
effects that abrogate antiviral
or anti-
immune responses. Exhaustion of
virus-specific T cells can also clearly occur in
wild-type immunocompetent mice that are
infected with lymphocytic choriomeningitis
.Because T cells that are specific for
of small numbers of commensal environ-
mental organisms that cannot proliferate
efficiently; and systemic penetration of solu-
ble microbial molecules that activate
on immune cells. The biggest differ-
ence between humans living under hygienic
conditions and those under ‘primitive (non-
hygienic) conditions is the decreased expo-
sure of the former to systemic infections,
whereas SPF mice are not exposed to
pathogens and even their intestinal bacteria
do not usually reach the systemic immune
system. We think that reduced exposure to
clinically obvious infections is therefore
probably crucial for the ‘hygiene effect’ in
humans, whereas the decreased nonspecific
immune activation in germ-free animals
(because of the absence of soluble microbial
products) is the key to the beneficial effect in
many autoimmune models
Evidence of the potential importance of
microbial products (and the possibility of
confusion with specific infections) is demon-
strated by the induction of autoimmune
haemolytic anaemia in mice transgenic for
erythrocyte-specific autoantibodies
pathogenic autoantibodies in this strain are
produced by B1 cells derived from the pleuro-
peritoneal lineage. In germ-free conditions,
there are few B1 cells, and no autoimmune dis-
ease develops. In SPF conditions, the animals
have B1 cells but still show no haemolytic
anaemia. The immunopathology is only seen
when the strain is maintained in conven-
tional conditions; however, defined infec-
tions of SPF animals have not yet revealed
the causative pathogen. In this case, it is clear
that a pathogen can trigger autoimmunity;
yet, because injection of lipopolysaccharide
has the same effect, the consequences of
pathogenic infection probably result from
increased exposure to microbial products.
A second example is provided by study-
ing the role of environmental antigens in the
spontaneous development of autoimmunity
in MRL/lpr mice, which have a mutation in
the Fas (CD95) gene and therefore have
defective lymphocyte apoptosis
conventional conditions, these animals
spontaneously develop an autoimmune
syndrome that is characterized by lympho-
proliferation (mainly of a CD4
subset), high serum immunoglobulin levels
(including multiple autoantibodies), vasculitis
and nephritis. When the MRL/lpr strain was
bred in a germ-free environment but fed an
autoclaved natural-ingredient diet, there was
no difference in lymphoproliferation or
autoimmune pathology compared with ani-
mals bred in conventional conditions.
However, when the germ-free MRL/lpr ani-
mals were fed a sterile elemental diet, the
A family of proteins exhibiting bactericidal properties.
They are secreted by immune cells (particularly neu-
trophils), intestinal Paneth cells and epithelial cells.
Non-responsiveness of the immune system resulting
from the deletion of specific thymocytes (central toler-
ance) and the deletion or functional inactivation of
specific T cells in the periphery (peripheral tolerance)
in the presence of large quantities of antigen.
Non-responsiveness of the immune system in the pres-
ence of a given antigen, despite the existence of specific
T and B cells capable of mounting a functional response.
Immune-mediated inflammation of the bowel. There are
two main forms: Crohn’s disease, which is a granuloma-
tous segmental inflammation affecting any part of the
intestine, and ulcerative colitis, which is a mucosal
inflammation involving the rectum and extending for a
variable distance along the colon. In developed countries,
the incidence of inflammatory bowel disease is approxi-
mately 1 in 50,000. It usually starts in early adult life and
continues afterwards with a relapsing, remitting course.
The layer of the intestine between the epithelial cells and
the most superficial smooth-muscle layer.
Resemblance between epitopes contained within
microbial and host proteins, leading to crossreactivity
of T cells in the host.
The relationship between two different species that live
in close proximity and benefit from one another.
Collections of lymphoid tissue located in the mucosa of
the small intestine, with an outer epithelial layer contain-
ing specialized epithelial cells, called M cells.
The spectrum of B or T cells. Defined according to the
specificities of the B-cell- or T-cell-receptors that are pre-
sent immediately before onset of a clinically important
To generate an antibody response to a T-cell-dependent
protein antigen requires recognition of the antigen (in
the context of MHC molecules) by helper T cells and
cooperation between those antigen-specific T cells and
B cells that recognize the same antigen.
Cell-associated pattern-recognition receptors that
recognize molecules unique to microorganisms,
resulting in immune-cell activation and production
of pro-inflammatory molecules.
484 | JUNE 2004 | VOLUME 4
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Andrew J. Macpherson and Nicola L. Harris are at
the Institute of Experimental Immunology,
Universitätsspital, Schmelzbergstrasse 12, CH8091
Zürich, Switzerland.
Correspondence to A.J.M.
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Competing interests statement
The authors declare that they have no competing financial interests.
Online links
The following terms in this article are linked online to:
Entrez Gene:
CD4 | CD8 | CD25 | CD45 | Fas | FOXP3 | IL-10 | interferon-γ |
TIM1 | transforming growth factor-β1
Access to this interactive links box is free online.
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  • ... Histology showed that the Paneth cell marker lysozyme was also strongly down-regulated in the ileum. Secretory IgA and antimicrobial factors play a key role in regu- lating contact between the epithelium and potentially harmful antigens and microbes 31,32 and may explain our observation that the microbiota were frequently seen in contact with the villus epithelium (Fig. 5). Decreased mucus production in the ileum of old mice may also be a contributing factor. ...
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    Aging significantly increases the vulnerability to gastrointestinal (GI) disorders but there are few studies investigating the key factors in aging that affect the GI tract. To address this knowledge gap, we used 10-week- and 19-month-old litter-mate mice to investigate microbiota and host gene expression changes in association with ageing. In aged mice the thickness of the colonic mucus layer was reduced about 6-fold relative to young mice, and more easily penetrable by luminal bacteria. This was linked to increased apoptosis of goblet cells in the upper part of the crypts. The barrier function of the small intestinal mucus was also compromised and the microbiota were frequently observed in contact with the villus epithelium. Antimicrobial Paneth cell factors Ang4 and lysozyme were expressed in significantly reduced amounts. These barrier defects were accompanied by major changes in the faecal microbiota and significantly decreased abundance of Akkermansia muciniphila which is strongly and negatively affected by old age in humans. Transcriptomics revealed age-associated decreases in the expression of immunity and other genes in intestinal mucosal tissue, including decreased T cell-specific transcripts and T cell signalling pathways. The physiological and immunological changes we observed in the intestine in old age, could have major consequences beyond the gut.
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    Diverse commensal populations are now regarded as key to physiological homeostasis and protection against disease. Although bacteria are the most abundant component of microbiomes, and the most intensively studied, the microbiome also consists of viral, fungal, archael, and protozoan communities, about which comparatively little is known. Host-defense peptides (HDPs), originally described as antimicrobial, now have renewed significance as curators of the pervasive microbial loads required to maintain homeostasis and manage microbiome diversity. Harnessing HDP biology to transition away from non-selective, antibiotic-mediated treatments for clearance of microbes is a new paradigm, particularly in veterinary medicine. One family of evolutionarily conserved HDPs, β-defensins which are produced in diverse combinations by epithelial and immune cell populations, are multifunctional cationic peptides which manage the cross-talk between host and microbes and maintain a healthy yet dynamic equilibrium across mucosal systems. They are therefore key gatekeepers to the oral, respiratory, reproductive and enteric tissues, preventing pathogen-associated inflammation and disease and maintaining physiological normality. Expansions in the number of genes encoding these natural antibiotics have been described in the genomes of some species, the functional significance of which has only recently being appreciated. β-defensin expression has been documented pre-birth and disruptions in their regulation may play a role in maladaptive neonatal immune programming, thereby contributing to subsequent disease susceptibility. Here we review recent evidence supporting a critical role for β-defensins as farmers of the pervasive and complex prokaryotic ecosystems that occupy all body surfaces and cavities. We also share some new perspectives on the role of β-defensins as sensors of homeostasis and the immune vanguard particularly at sites of immunological privilege where inflammation is attenuated.
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    The importance of the microbiome to human health is increasingly recognized and has become a major focus of recent research. However, much of the work has focused on a few aspects, particularly the bacterial component of the microbiome, most frequently in the gastrointestinal tract. Yet humans and other animals can be colonized by a wide array of organisms spanning all domains of life, including bacteria and archaea, unicellular eukaryotes such as fungi, multicellular eukaryotes such as helminths, and viruses. As they share the same host niches, they can compete with, synergize with, and antagonize each other, with potential impacts on their host. Here, we discuss these major groups making up the human microbiome, with a focus on how they interact with each other and their multicellular host.
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    Background Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Chronic intestinal inflammation develops against resident intestinal bacteria in genetically susceptible hosts. We hypothesized that host intestinal immunoglobulin (Ig) G can be used to identify bacteria involved in IBD pathogenesis. Results IgG-bound and -unbound microorganisms were collected from 32 pediatric terminal ileum aspirate washes during colonoscopy [non-IBD (n = 10), Crohn disease (n = 15), and ulcerative colitis (n = 7)], and composition was assessed using the Illumina MiSeq platform. In vitro analysis of invasive capacity was evaluated by fluorescence in situ hybridization and gentamicin invasion assay; immune activation was measured by qPCR. Despite considerable inter-individual variations, IgG binding favored specific and unique mucosa-associated species in pediatric IBD patients. Burkholderia cepacia, Flavonifractor plautii, and Rumminococcus sp. demonstrated increased IgG binding, while Pseudomonas ST29 demonstrated reduced IgG binding, in IBD. In vitro validation confirmed that B. cepacia, F. plautii, and Rumminococcus display invasive potential while Pseudomonas protogens did not. Conclusion Using IgG as a marker of pathobionts in larger patient cohorts to identify microbes and elucidate their role in IBD pathogenesis will potentially underpin new strategies to facilitate development of novel, targeted diagnostic, and therapeutic approaches. Interestingly, this method can be used beyond the scope of this manuscript to evaluate altered gut pathobionts in a number of diseases associated with altered microbiota including arthritis, obesity, diabetes mellitus, alcoholic liver disease, cirrhosis, metabolic syndrome, and carcinomas. Electronic supplementary material The online version of this article (10.1186/s40168-018-0604-3) contains supplementary material, which is available to authorized users.
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    In anti-red blood cell autoantibody transgenic (autoAb Tg) mice almost all B cells are deleted except for B-1 cells in the peritoneal cavity and the gut. About one-half of the auto Ab Tg mice suffer from autoimmune hemolytic anemia (AIHA) in the conventional condition. Oral administration of lipopolysaccharides activates B-1 cells and induces autoimmune symptoms in the Tg mice, suggesting that the autoimmune disease in anti-RBC autoAb Tg mice is triggered by infections. To examine the association of bacterial infections with the generation of B-1 cells and the occurrence of the autoimmune disease, we analyzed anti-RBC autoAb Tg mice bred in germ-free and specific pathogen-free conditions. In germ-free conditions, few peritoneal B-1 cells were detected, while a significant number of peritoneal B-1 cells existed in specific pathogen-free conditions. In both conditions, no mice suffered from AIHA. However, when these Tg mice were transferred to the conventional condition or injected with lipopolysaccharide, peritoneal B-1 cells expanded and some of these mice suffered from AIHA. These results clearly showed that bacterial infections are responsible for both the expansion of B-1 cells and the onset of the autoimmune disease in these Tg mice.
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    Human immune responses are heterogeneous and may involve antagonism between T helper (TH) lymphocyte subsets and their cytokines. Atopy is characterized by immediate immunoglobulin E (IgE)-mediated hypersensitivity to agents such as dust mites and pollen, and it underlies the increasingly prevalent disorder asthma. Among Japanese schoolchildren, there was a strong inverse association between delayed hypersensitivity to Mycobacterium tuberculosis and atopy. Positive tuberculin responses predicted a lower incidence of asthma, lower serum IgE levels, and cytokine profiles biased toward TH1 type. Exposure and response to M. tuberculosis may, by modification of immune profiles, inhibit atopic disorder.
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    Germfree mice were given food contaminated with pure cultures of various bacterial species isolated from ordinary healthy mice. The cultures were given singly, or in association, or consecutively at weekly intervals. Whatever the technique of administration, the lactobacilli and anaerobic streptococci immediately established themselves throughout the gastrointestinal tract, and became closely associated with the walls of the organs. In contrast, the organisms of the bacteroides group were found in large numbers only in the large intestine. Within a week after exposure, the populations of these three bacterial species reached levels similar to those found in ordinary mice. They remained at these characteristic levels throughout the period of observation (several months). Their presence resulted in a progressive decrease in the size of the cecum which eventually became normal in gross appearance. Coliform bacilli multiplied extensively and persisted at high levels in all parts of the gastrointestinal tract of germfree mice, even after these had become colonized with lactobacilli, anaerobic streptococci and bacteroides. However, the coliform population fell precipitously within a few days after the animals were fed the intestinal contents of healthy pathogen-free mice.
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    The study of the IgA content of serum, spleen, cecum and salivary glands of axenic and gnotoxenic mice leads us to these conclusions: only the intestinal IgA system works in axenic mice to secrete an immunoglobulin which is immunologically comparable to the 11S IgA described in man; this system is independent of the serum IgA system which does not function until several weeks after the establishment of a normal intestinal flora. Perhaps only after flora has triggered intense proliferation and kinetic activation of the intestinal lympho-epithelial system can the gut epithelium perform its “instructive function” and influence the maturation of the serum and the other exocrine IgA systems.
  • Chapter
    The indigenous gastrointestinal microflora consists of microorganisms that normally inhabit the gastrointestinal tract. The indigenous microflora primes the host immune response so that it can respond more effectively to exogenous pathogens. Keywords: GI microflora; indigenous microflora; indigenous bacteria; gastrointestinal bacteria; natural antibodies; immunostimulation by indigenous bacteria; bacterial translocation; opportunistic infections
  • Article
    We previously investigated the primary and secondary responses and hyperimmunization to the T cell-dependent antigen 2,4-dinitrophenyl keyhole limpet hemocyanin (DNP-KLH) in antigen-free (AF), germ-free (GF) and conventional (CV) mice. Both the absolute and relative numbers of DNP-specific IgG-secreting cells in the spleen of AF mice were considerably higher compared to GF and CV mice, especially after hyperimmunization. In the present study we measured the total and DNP-specific IgG concentration in the sera of these hyperimmunized mice using a sensitive sandwich enzyme-linked immunosorbent assay. With respect to the total IgG concentration before and after hyperimmunization, the AF mice showed an almost 13-fold increase after boosting with the antigen; the GF mice showed an approximately 8-fold increase. A slight but non-significant increase was observed in the CV mice. The total as well as the DNP-specific IgG levels in the AF-immunized mice were 2-fold and 5-fold higher compared to GF and CV mice, respectively. With the use of Surface Plasmon Resonance instrumentation (BIAcoreTM, Pharmacia, Uppsala, Sweden) we obtained mean binding affinities (KA) of the polyclonal samples of the three groups of hyperimmunized mice. IgA and IgM samples displayed low affinity for DNP-lysine. The AF mice displayed the highest KA value among IgG antibodies, followed by GF mice, while CV mice showed a 3-fold lower KA compared to AF mice. These differences were mainly determined by the dissociation rate constant (kdiss), since no significant changes were observed in the association rate constant (kass). Furthermore, the sera of the CV mice have a lower percentage of high-affinity antibodies compared to GF and AF mice. These results suggest that besides a higher overall binding affinity seen in AF mice, and to a lesser extent in GF mice, the relative contribution of high-affinity IgG is greater in AF mice compared to CV mice.
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    IgA is the most abundant immunoglobulin produced in mammals; most is secreted as a dimer across mucous membranes. This review discusses the different mechanisms of induction of IgA, and its role in protecting mucosal surfaces against pathogenic and non-pathogenic microorganisms.
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    Gut translocation of bacteria has been shown in both animal and human studies. Evidence from animal studies that links bacteria translocation to the development of postoperative sepsis and multiple organ failure has yet to be confirmed in humans. To examine the spectrum of bacteria involved in translocation in surgical patients undergoing laparotomy and to determine the relation between nodal migration of bacteria and the development of postoperative septic complications. Mesenteric lymph nodes (MLN), serosal scrapings, and peripheral blood from 448 surgical patients undergoing laparotomy were analysed using standard microbiological techniques. Bacterial translocation was identified in 69 patients (15.4%). The most common organism identified was Escherichia coli (54%). Both enteric bacteria, typical of indigenous intestinal flora, and non-enteric bacteria were isolated. Postoperative septic complications developed in 104 patients (23%). Enteric organisms were responsible in 74% of patients. Forty one per cent of patients who had evidence of bacterial translocation developed sepsis compared with 14% in whom no organisms were cultured (p < 0.001). Septic morbidity was more frequent when a greater diversity of bacteria resided within the MLN, but this was not statistically significant. Bacterial translocation is associated with a significant increase in the development of postoperative sepsis in surgical patients. The organisms responsible for septic morbidity are similar in spectrum to those observed in the mesenteric lymph nodes. These data strongly support the gut origin hypothesis of sepsis in humans.