Download Commensal Bacteria Shape Intestinal Immune System

Commensal Bacteria Shape Intestinal
Immune System Development
The intestine is colonized with vast societies of microbes that promote
mucosal immune system development and contribute to host health
Heather L. Cash and Lora V. Hooper
icrobes have a knack for making
us sick. Until recently, many of
these encounters proved deadly.
In Guns, Germs, and Steel, evolutionary biologist Jared Diamond argues that such interactions were a primary force shaping human history. Therefore, it
is not surprising that human-microbe interactions often are viewed through the lens of conflict. The proliferation of antibacterial consumer
products underscores the prevailing idea that
human-bacterial contact is something to be controlled or avoided.
However, this understanding of human-microbe relationships is changing. An accumulating
body of evidence indicates that we maintain mutually beneficial relationships with the microbes that
cohabit our bodies, suggesting a profound intertwining of human and microbial biology. Wielding sophisticated new molecular tools, investigators in a number of labs are learning about the
extent to which gut microbes drive intestinal immunity. These studies are revealing that the functions of our gut immune system are only partially
encoded in our genes, and require cues from our
microbial partners for full development. As a consequence, disrupting these beneficial host-bacterial
relationships with antibiotic treatments may pave
the way for immunologic diseases.
M
Intestinal Bacteria: Partners
in Human Metabolism
The domestication of our microbial partners
begins early in life. Starting at birth, humans and
other mammals are colonized with diverse societies of bacteria that cover the surfaces of the
skin and the gastrointestinal tract, both of which
are exposed to the outer world. The vast majority of these indigenous microbes reside in the
intestine, where they are in continuous and intimate contact with host tissues, and where they
outnumber the surrounding host cells by at least
an order of magnitude.
The term “commensal” is frequently used to
describe the relationship between humans and
their intestinal bacterial cohorts. Cobbled together from Latin roots, the term means “at
table together.” This word seems especially appropriate because humans and other mammals
depend heavily on their gut bacteria to extract
maximum nutritional value from their diets.
More than 20 years ago Bernard Wostmann and
his colleagues at the University of Notre Dame,
Notre Dame, Ind., discovered that “germ-free”
rats, which are microbiologically sterile and
therefore lack intestinal microbes, require
nearly 30% more calories to maintain their
body weight than do their normally colonized
counterparts. In an environment where nutrients are in short supply, natural selection would
likely favor such host-microbe associations,
which may explain why such relationships
evolved in the first place. The vastness and diversity of the microflora ensure that this population maintains an array of metabolic talents,
allowing its members to break down a variety of
dietary compounds. The benefits associated
with these host-microbial alliances flow the
other way as well. In return for their metabolic
contributions, gut bacteria are provided with a
warm, protected, and nutrient-rich habitat in
which to multiply.
The very complexity that allows gut microflora to be valuable partners in human dietary
metabolism poses serious challenges to micro-
Heather L. Cash is
a Graduate Student
in the Molecular
Microbiology Program and Lora V.
Hooper is an Assistant Professor at
the Center for Immunology, The University of Texas
Southwestern Medical Center at Dallas.
Volume 71, Number 2, 2005 / ASM News Y 77
bial ecologists. Because intestinal bacteria are
adapted to an anaerobic environment, many
species in this population are difficult or impossible to culture outside the intestine, making it
difficult to enumerate the membership of the
gut’s microbial societies. However, new molecular techniques are allowing investigators to
make inroads on this challenge. These techniques focus primarily on 16S rRNA genes,
which are common to all bacteria but whose
precise sequences vary between species. By analyzing the 16S rRNA sequences in such populations, microbial ecologists can sidestep the need
to culture gut bacteria and thus can identify and
quantify the inhabitants of this mixed microbial
community.
Although we still have a long way to go to get
a clear picture of this society, some general
themes have been established. Mammalian
young are sterile in utero and become colonized
as they are born. The intestines of human babies
initially contain large numbers of facultative
anaerobes, including Escherichia coli and streptococci. Such species decline in number during a
critical postnatal transition: weaning from
mother’s milk onto a solid diet rich in plant
polysaccharides. During this same period, obligate anaerobes such as Bacteroides and Clostridium species gain a foothold, ultimately becoming the dominant occupiers of the adult gut
ecosystem.
The Intestinal Immune System Is a
Complex Network of Interacting Cells
The need to corral these microbial metabolic
workhorses has driven the evolution of a vast
and complex intestinal immune system. The
host cells that constitute this immune network
patrol and defend intestinal surfaces, keeping
commensal bacteria from penetrating host tissues and causing serious problems such as inflammation and sepsis. However, maintaining
these large microbial populations to serve host
nutritional needs dictates that the intestinal immune system tolerate intestinal microbial antigens. Exactly how this tolerance develops and is
maintained is a mystery.
The intestinal epithelium presents the first line
of defense against invading or attaching bacteria. In addition to presenting a physical barrier
to microbial penetration, the epithelium plays a
more active role by producing and secreting
78 Y ASM News / Volume 71, Number 2, 2005
large quantities of antimicrobial peptides.
Small-intestinal Paneth cells are key effectors of
this type of innate defense (Fig. 1). These specialized epithelial cells harbor secretory granules
that contain high concentrations of a number of
microbicidal proteins. Experiments carried out
by André Ouellette and his colleagues at the
University of California, Irvine, have shown that
Paneth cells somehow sense bacteria and react
to their presence by discharging their granule
contents into the gut lumen.
Such rapid-fire antimicrobial responses are
part of the innate immune system, which enables
the gut to deal quickly with invading microbes
and to contain commensal populations. In contrast to innate immune defenses, adaptive immune responses develop more slowly but result
in a targeted, precise response. As in other parts
of the body, adaptive immune responses in the
gut require the activation and multiplication of
B- and T-lymphocytes, which undergo key portions of their development in gut-specific lymphoid structures called Peyer’s patches (Fig. 1).
These structures punctuate the length of the
small intestine, acting as incubators for developing B- and T-lymphocytes and ensuring that
these cells mature with input from commensal
microbial populations in the lumen. Lymphocytes dispatched from Peyer’s patches are thus
primed to patrol the length of the intestine for
microbial interlopers and to respond to pathogens.
In addition to Peyer’s patch-derived lymphocytes, the gut harbors a large population of
T-cells that insinuate themselves between intestinal epithelial cells, and are thus termed intraepithelial lymphocytes (IELs). Because the intestine has a vast surface area, these cells represent
one of the largest populations of immune cells in
the body, yet their functions are still poorly
understood. Even the site of their development
remains controversial. For example, Hiromichi
Ishikawa and his colleages at the Keio University
School of Medicine in Tokyo have evidence that
these T cell populations develop entirely in the
gut, and are thus uniquely primed to deal with
local conditions. However, other investigators,
including Gerard Eberl and Dan Littman of the
New York University School of Medicine in
New York, N.Y., and Delphine Guy-Grand and
her colleagues at the Institut Pasteur in Paris,
have provided evidence indicating that these
cells develop first in the thymus and then home
FIGURE 1
The small intestinal immune system consists of a complex network of interacting cell populations. The intestinal epithelium presents a
physical barrier to microbial penetration. In addition, Paneth cells, specialized epithelial cells located at the base of small intestinal villi,
actively secrete antimicrobial proteins in response to bacterial signals. Such rapid-fire innate immune responses are bolstered by precisely
targeted adaptive immune responses that are slower to develop. The B- and T- lymphocytes that carry out such targeted responses develop
in Peyer’s patches, specialized lymphoid structures found at intervals along the length of the small intestine. Peyer’s patch dendritic cells
sample bacterial antigens and present them to the maturing lymphocytes. The B and T cells eventually exit the Peyer’s patch and guard
against microbial penetration by patrolling the subepithelial regions throughout the intestine.
to the gut, where they proliferate in response to
local signals. Despite the ongoing debate about
their origins, the positioning of IELs at the front
lines of intestinal defense suggests that they play
an important role in maintaining immune homeostasis with commensal bacterial populations.
Germ-Free Mice: Unveiling Bacterial
Contributions to Intestinal Immunity
The cells of the gut immune system develop in
proximity to enormous populations of commensal bacteria in the intestinal lumen. While epithelial cells are in direct contact with commensal
bacteria, B- and T-lymphocytes are usually separated from microbial populations in the gut by
a single epithelial layer. With such a slim cellular
partition, resident bacteria are poised to influence the development of the gut’s innate and
adaptive immune systems.
To study how intestinal microflora contribute
to gut immunity, investigators often use animals
that have been raised without contact with microorganisms, taking advantage of a breeding
system that was developed roughly 50 years ago
by James Reyniers at the University of Notre
Dame. Animals are housed and bred inside sterile isolators (Fig. 2) and are manipulated using
gloves that are built into the isolator walls.
Isolator provisioning is not a trivial task, as
food, water, and bedding must be autoclaved
inside stainless steel cylinders, which are then
“docked” to the isolator before the supplies are
imported.
Animals that would otherwise be colonized
can be derived germ-free by cesarean section,
taking advantage of the fact that young animals
developing in the uterus of a healthy mother are
free of microbes. Typically, the uterus containing live pups is removed aseptically from the
mother, passed into a sterilizing solution, and
Volume 71, Number 2, 2005 / ASM News Y 79
FIGURE 2
Use of flexible film isolators for maintaining germ-free mice. Germ-free mice develop without any contact with the microbial world, and
are thus an essential tool for defining which intestinal immune system functions require interactions with commensal bacteria for full
development. The germ-free environment is created inside sterile plastic chambers (left panel) that can accommodate a number of
mouse cages. Air is supplied to each isolator by a blower attached to a filter, which allows the air to be sterilized before it enters the
isolator. Isolator interiors are sterilized by spraying with a dilute solution of Clidox (chlorine dioxide), and manipulations in the isolator are
carried out through neoprene gloves. Sterile supplies such as food, water, and bedding are autoclaved in a stainless steel cylinder (right
panel) and are transferred into the isolator through a double-door port.
then transferred into a germ-free isolator. The
pups are immediately removed and fostered to
germ-free lactating females. Although this is
now a relatively straightforward process, the
first generations of germ-free mice were derived
without benefit of germ-free foster mothers and
had to be laboriously hand-reared.
Bacterial Contributions
to Innate Immunity
Germ-free mice have yielded a number of valuable clues about how commensal bacteria shape
intestinal innate immune responses. For instance, commensal microbes alter the expression of angiogenin-4, a bactericidal protein produced in the mammalian gut, according to
Jeffrey Gordon and colleagues at Washington
University in St. Louis, Mo. Although its name
suggests a role in the formation of new blood
vessels, this protein is in fact a swift and effective
bacterial killer. Synthesized and secreted exclusively by Paneth cells (Fig. 3), angiogenin-4 specifically targets gram-positive organisms such as
Listeria monocytogenes and Enterococcus fae-
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calis, while leaving gram-negative organisms
such as Escherichia coli largely untouched. Such
specificity may assist in establishing and maintaining the predominantly gram-negative bacterial populations found in intestines of adults.
Commensal bacteria stimulate angiogenin-4
synthesis during a key developmental transition
in early postnatal life. In mice that have a normal gut flora, angiogenin-4 expression increases
dramatically when young mice switch from
mother’s milk to a regular diet and quickly
reaches adult levels. By contrast, germ-free mice
never achieve high angiogenin-4 expression levels, indicating that full expression of angiogenin-4 in Paneth cells requires interactions with
gut bacteria. However, this deficiency is reversible. By exposing germ-free mice to the mixture
of intestinal bacteria found in their colonized
counterparts, angiogenin-4 levels rapidly rise to
match those found in conventionally colonized
mice.
The early postnatal expression pattern of angiogenin-4 shows that commensal bacteria can
influence the composition of the developing
Paneth cell antimicrobial arsenal. This suggests
that bacteria and host may collaborate in shaping the composition of
the evolving gut microbial community during weaning. Moreover, by
inducing an abundance of this bactericidal protein, commensal bacteria
may help to ensure that rapid-fire
innate immune responses are primed
and ready in the event that a pathogen is encountered. However, important questions remain, including
how bacterial signals are relayed to
Paneth cells to alter antimicrobial
protein expression and whether
other Paneth cell antimicrobial proteins are regulated by interactions
with commensal flora.
FIGURE 3
Bacterial Contributions
to Adaptive Immunity
Germ-free animals likewise are providing compelling evidence that
commensal bacteria serve as a driving force in the development of the
gut adaptive immune system (see table). For example, commensal bacteria play crucial roles in promoting B
cell development in Peyer’s patches,
which are underdeveloped in germBacterial contributions to small intestinal innate immunity. Commensal bacteria trigger the
free mice, according to John Cebra
expression of angiogenin-4, a bactericidal protein produced in small intestinal Paneth cells.
angiogenin-4 specifically targets gram-positive bacteria, while gram-negative organisms are
of the University of Pennsylvania in
spared. This interaction may be important for establishing and maintaining the predominantly
Philadelphia and his collaborators.
Gram negative bacterial populations found in the adult intestine. Furthermore, these findings
When commensal bacteria colonize
suggest that commensal bacteria play a central role in shaping the composition of the
Paneth cell antimicrobial arsenal. The molecular signals that commensal bacteria use to
the intestine, they initiate a series of
communicate with Paneth cells are unknown.
reactions, including those that lead
to transient expansion of germinal
center reactions between B and T
cells in the Peyer’s patches (Fig. 1) and increased
stimulate B cell IgA production. Dendritic cells,
production of immunoglobulin A (IgA) antibodwhich can wedge between gut epithelial cells,
ies by B cells. Thus, germ-free mice generate
continuously sample bacteria from the lumen
reduced amounts of IgA as compared to mice
with intestinal flora, and have decreased numbers of circulating B and T lymphocytes. MoreBacterial Contributions to Gut Adaptive
over, introducing only a single commensal bacImmunity
terial species does not restore proper development,
Formation of anatomical structures, including Peyer’s
suggesting that a diverse repertoire of bacterial
patches, which harbor developing B and T cells
species and antigens is necessary to drive full
Expansion of germinal center reactions involving B
and T cells in Peyer’s patches
development of intestinal immunity.
Increased IgA production by intestinal B cells
Recent work by Andrew MacPherson and
Generation of antibody diversity (in rabbits)
Therese Uhr, of the Institute of Experimental
Expansion of intraepithelial lymphocyte populations
Immunology in Zurich, Switzerland, has pro(␣␤TCR-bearing)
vided new insights into how commensal bacteria
Volume 71, Number 2, 2005 / ASM News Y 81
and present their products to developing gut
lymphocytes, thereby inducing B cells to synthesize IgA antibodies that specifically bind to commensal antigens. Such antibodies are ultimately
secreted into the gut lumen and are likely critically important in keeping commensal bacteria
from crossing into host tissues where they could
cause damage. In addition, stimulating IgA production may also help to keep the adaptive
immune system poised to deal rapidly with any
invading pathogens.
Katherine Knight and her collaborators at
Loyola University in Chicago, Ill., have shown
that in rabbits as in mice, commensal bacteria
help drive the formation of lymphoid structures
such as Peyer’s patches. Furthermore, although
humans and mice generate primary antibody
diversity using mechanisms that are independent of microbial colonization, her research reveals that rabbits require intestinal bacteria to
generate a diverse antibody repertoire. Thus, for
rabbits the intestinal microflora not only appear
to play a role in forming the tissues in which
intestinal lymphocytes develop but also may
influence the ability of the gut immune system to
mount a successful immune response. Confirming Cebra’s findings, Knight and her colleagues
find that a diverse consortium of bacterial species is required to fully promote immune system
development.
In addition to providing critical signals for the
development of Peyer’s patch-derived lymphocytes, commensal bacteria also influence the recruitment of IELs to the intestinal surface, where
they integrate among epithelial cells lining small
intestinal villi. Over the past decade, a number
of labs have shown that germ-free mice have
10-fold fewer IELs that bear T cell receptors
consisting of an ␣ and ␤ chain than do mice
carrying commensal bacteria. When germ-free
mice become colonized, these IEL numbers increase dramatically. In contrast, IELs that have
T cell receptors (TCR) consisting of a ␥ and ␦
chain are unaltered in germ-free mice, strongly
suggesting that this IEL population carries out
functions that are distinct from ␣␤TCR-bearing
IELs.
Commensal Bacteria and the Emergence
of Inflammatory Bowel Diseases
Modern Western societies place a strong emphasis on cleanliness and hygiene. The wide avail-
82 Y ASM News / Volume 71, Number 2, 2005
ability of antibiotics and antibacterial products
promotes a nearly antiseptic existence, cleansed
of unwanted interactions with the microbial
world. However, Western countries are also increasingly afflicted with several immune disorders, such as allergies, that are virtually nonexistent in nonindustrialized nations where access
to antibiotics is far more limited.
Agnes Wold of Göteborg University in Sweden has proposed that excessive hygiene and
antibiotic use, and their accompanying effects
on the normal gut flora, might help to explain
the rise of immune disorders such as allergy in
heavily industrialized regions such as North
America and Europe. Originally proposed in
1989 by epidemiologist David Strachan of the
London School of Hygiene and Tropical Medicine, this idea is known as the “hygiene hypothesis.”
Inflammatory bowel diseases are a group of
immune disorders in which the gut is chronically
inflamed. Those who suffer from IBDs experience a range of symptoms, including severe diarrhea, abdominal cramps, fever, and rectal
bleeding. Most people experience IBD as a painful recurring condition with alternating periods
of remission and exacerbation. Unfortunately,
there is currently no cure for IBD. Although the
causes of IBDs are poorly understood, these
disorders are thought to stem from an overly
harsh, gut-damaging immune response to the
commensal microflora. IBDs and allergies are
thus similar in that both are characterized by
inappropriate immune responses to otherwise
harmless environmental antigens.
Epidemiologists have found that IBDs, like
allergies, are found mainly in North America
and Europe. IBDs currently affect more than 1
in 1,000 individuals in the United States. Moreover, the incidence of IBDs has risen dramatically in North America and Europe over the past
50 years. Although genetics plays a role in IBDs,
many investigators have invoked the hygiene
hypothesis to explain the increasing prevalence
of these diseases. The idea is that exposure to
key microbes, particularly during childhood,
may be critical for directing the maturing gut
immune system to develop tolerance to commensal flora. Disrupting the interactions between commensal bacteria and intestinal immune cells by exposure to broad-spectrum
antibiotics or excessively clean environments
may compromise the development of normal,
measured immune responses to commensal gut
bacteria.
Outlook
We are beginning to appreciate more fully the
extent to which commensal microbes contribute
to human biology. In our nutrient-rich society,
we may no longer rely on the metabolic contributions of our prokaryotic cohorts for survival.
However, the rise of immunologic diseases such
as IBDs underscores the fact that we have intricate, co-evolved relationships with our microbial populations, and that these interactions are
likely essential for the normal, healthy development of our immune systems.
Building a truly comprehensive understanding of human biology will thus entail developing
a detailed molecular picture of our interactions
with our commensal microbial populations.
Such a picture must include a better grasp of the
composition of these populations, how these
microbial societies communicate with host cells,
and precisely how microbial signals shape the
intestinal immune system. This challenge calls
for a multidisciplinary line of attack, blending
expertise and experimental approaches from
fields such as microbial ecology, developmental
biology, and immunology. Meeting this challenge will undoubtedly also yield new insights
about the consequences of broad-spectrum antibiotic use and its impact on intestinal immunity. By fully elucidating microbial contributions to intestinal immunity, we will be better
equipped to harness the power of our bacterial
allies in maintaining our health.
ACKNOWLEDGMENTS
We are grateful to our colleagues Cassie Behrendt, Anisa Ismail, and Cecilia Whitham for many helpful discussions. Work in
the Hooper lab is supported by grants from the Crohn’s and Colitis Foundation of America and from the Burroughs-Wellcome
Foundation (Career Award in the Biomedical Sciences to L.V.H.).
SUGGESTED READING
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