Commensal Bacteria Shape IntestinalImmune System DevelopmentThe intestine is colonized with vast societies of microbes that promotemucosal immune system development and contribute to host healthHeather L. Cash and Lora V. Hooper

Microbes have a knack for makingus sick. Until recently, many ofthese encounters proved deadly.In Guns, Germs, and Steel, evo-lutionary biologist Jared Dia-

mond argues that such interactions were a pri-mary force shaping human history. Therefore, itis not surprising that human-microbe interac-tions often are viewed through the lens of con-flict. The proliferation of antibacterial consumerproducts underscores the prevailing idea thathuman-bacterial contact is something to be con-trolled or avoided.

However, this understanding of human-mi-crobe relationships is changing. An accumulatingbody of evidence indicates that we maintain mutu-ally beneficial relationships with the microbes thatcohabit our bodies, suggesting a profound inter-twining of human and microbial biology. Wield-ing sophisticated new molecular tools, investiga-tors in a number of labs are learning about theextent to which gut microbes drive intestinal im-munity. These studies are revealing that the func-tions of our gut immune system are only partiallyencoded in our genes, and require cues from ourmicrobial partners for full development. As a con-sequence, disrupting these beneficial host-bacterialrelationships with antibiotic treatments may pavethe way for immunologic diseases.

Intestinal Bacteria: Partners

in Human Metabolism

The domestication of our microbial partnersbegins early in life. Starting at birth, humans andother mammals are colonized with diverse soci-eties of bacteria that cover the surfaces of theskin and the gastrointestinal tract, both of which

are exposed to the outer world. The vast major-ity of these indigenous microbes reside in theintestine, where they are in continuous and inti-mate contact with host tissues, and where theyoutnumber the surrounding host cells by at leastan order of magnitude.

The term “commensal” is frequently used todescribe the relationship between humans andtheir intestinal bacterial cohorts. Cobbled to-gether from Latin roots, the term means “attable together.” This word seems especially ap-propriate because humans and other mammalsdepend heavily on their gut bacteria to extractmaximum nutritional value from their diets.More than 20 years ago Bernard Wostmann andhis colleagues at the University of Notre Dame,Notre Dame, Ind., discovered that “germ-free”rats, which are microbiologically sterile andtherefore lack intestinal microbes, requirenearly 30% more calories to maintain theirbody weight than do their normally colonizedcounterparts. In an environment where nutri-ents are in short supply, natural selection wouldlikely favor such host-microbe associations,which may explain why such relationshipsevolved in the first place. The vastness and di-versity of the microflora ensure that this popu-lation maintains an array of metabolic talents,allowing its members to break down a variety ofdietary compounds. The benefits associatedwith these host-microbial alliances flow theother way as well. In return for their metaboliccontributions, gut bacteria are provided with awarm, protected, and nutrient-rich habitat inwhich to multiply.

The very complexity that allows gut micro-flora to be valuable partners in human dietarymetabolism poses serious challenges to micro-

Heather L. Cash isa Graduate Studentin the MolecularMicrobiology Pro-gram and Lora V.Hooper is an Assis-tant Professor atthe Center for Im-munology, The Uni-versity of TexasSouthwestern Med-ical Center at Dal-las.

Volume 71, Number 2, 2005 / ASM News Y 77

bial ecologists. Because intestinal bacteria areadapted to an anaerobic environment, manyspecies in this population are difficult or impos-sible to culture outside the intestine, making itdifficult to enumerate the membership of thegut’s microbial societies. However, new molec-ular techniques are allowing investigators tomake inroads on this challenge. These tech-niques focus primarily on 16S rRNA genes,which are common to all bacteria but whoseprecise sequences vary between species. By ana-lyzing the 16S rRNA sequences in such popula-tions, microbial ecologists can sidestep the needto culture gut bacteria and thus can identify andquantify the inhabitants of this mixed microbialcommunity.

Although we still have a long way to go to geta clear picture of this society, some generalthemes have been established. Mammalianyoung are sterile in utero and become colonizedas they are born. The intestines of human babiesinitially contain large numbers of facultativeanaerobes, including Escherichia coli and strep-tococci. Such species decline in number during acritical postnatal transition: weaning frommother’s milk onto a solid diet rich in plantpolysaccharides. During this same period, obli-gate anaerobes such as Bacteroides and Clos-tridium species gain a foothold, ultimately be-coming the dominant occupiers of the adult gutecosystem.

The Intestinal Immune System Is a

Complex Network of Interacting Cells

The need to corral these microbial metabolicworkhorses has driven the evolution of a vastand complex intestinal immune system. Thehost cells that constitute this immune networkpatrol and defend intestinal surfaces, keepingcommensal bacteria from penetrating host tis-sues and causing serious problems such as in-flammation and sepsis. However, maintainingthese large microbial populations to serve hostnutritional needs dictates that the intestinal im-mune system tolerate intestinal microbial anti-gens. Exactly how this tolerance develops and ismaintained is a mystery.

The intestinal epithelium presents the first lineof defense against invading or attaching bacte-ria. In addition to presenting a physical barrierto microbial penetration, the epithelium plays amore active role by producing and secreting

large quantities of antimicrobial peptides.Small-intestinal Paneth cells are key effectors ofthis type of innate defense (Fig. 1). These spe-cialized epithelial cells harbor secretory granulesthat contain high concentrations of a number ofmicrobicidal proteins. Experiments carried outby Andre Ouellette and his colleagues at theUniversity of California, Irvine, have shown thatPaneth cells somehow sense bacteria and reactto their presence by discharging their granulecontents into the gut lumen.

Such rapid-fire antimicrobial responses arepart of the innate immune system, which enablesthe gut to deal quickly with invading microbesand to contain commensal populations. In con-trast to innate immune defenses, adaptive im-mune responses develop more slowly but resultin a targeted, precise response. As in other partsof the body, adaptive immune responses in thegut require the activation and multiplication ofB- and T-lymphocytes, which undergo key por-tions of their development in gut-specific lym-phoid structures called Peyer’s patches (Fig. 1).These structures punctuate the length of thesmall intestine, acting as incubators for develop-ing B- and T-lymphocytes and ensuring thatthese cells mature with input from commensalmicrobial populations in the lumen. Lympho-cytes dispatched from Peyer’s patches are thusprimed to patrol the length of the intestine formicrobial interlopers and to respond to patho-gens.

In addition to Peyer’s patch-derived lympho-cytes, the gut harbors a large population ofT-cells that insinuate themselves between intes-tinal epithelial cells, and are thus termed intra-epithelial lymphocytes (IELs). Because the intes-tine has a vast surface area, these cells representone of the largest populations of immune cells inthe body, yet their functions are still poorlyunderstood. Even the site of their developmentremains controversial. For example, HiromichiIshikawa and his colleages at the Keio UniversitySchool of Medicine in Tokyo have evidence thatthese T cell populations develop entirely in thegut, and are thus uniquely primed to deal withlocal conditions. However, other investigators,including Gerard Eberl and Dan Littman of theNew York University School of Medicine inNew York, N.Y., and Delphine Guy-Grand andher colleagues at the Institut Pasteur in Paris,have provided evidence indicating that thesecells develop first in the thymus and then home

78 Y ASM News / Volume 71, Number 2, 2005

to the gut, where they proliferate in response tolocal signals. Despite the ongoing debate abouttheir origins, the positioning of IELs at the frontlines of intestinal defense suggests that they playan important role in maintaining immune ho-meostasis with commensal bacterial popula-tions.

Germ-Free Mice: Unveiling Bacterial

Contributions to Intestinal Immunity

The cells of the gut immune system develop inproximity to enormous populations of commen-sal bacteria in the intestinal lumen. While epi-thelial cells are in direct contact with commensalbacteria, B- and T-lymphocytes are usually sep-arated from microbial populations in the gut bya single epithelial layer. With such a slim cellularpartition, resident bacteria are poised to influ-ence the development of the gut’s innate andadaptive immune systems.

To study how intestinal microflora contributeto gut immunity, investigators often use animalsthat have been raised without contact with mi-croorganisms, taking advantage of a breedingsystem that was developed roughly 50 years agoby James Reyniers at the University of NotreDame. Animals are housed and bred inside ster-ile isolators (Fig. 2) and are manipulated usinggloves that are built into the isolator walls.Isolator provisioning is not a trivial task, asfood, water, and bedding must be autoclavedinside stainless steel cylinders, which are then“docked” to the isolator before the supplies areimported.

Animals that would otherwise be colonizedcan be derived germ-free by cesarean section,taking advantage of the fact that young animalsdeveloping in the uterus of a healthy mother arefree of microbes. Typically, the uterus contain-ing live pups is removed aseptically from themother, passed into a sterilizing solution, and

F I G U R E 1

The small intestinal immune system consists of a complex network of interacting cell populations. The intestinal epithelium presents aphysical 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 preciselytargeted adaptive immune responses that are slower to develop. The B- and T- lymphocytes that carry out such targeted responses developin Peyer’s patches, specialized lymphoid structures found at intervals along the length of the small intestine. Peyer’s patch dendritic cellssample bacterial antigens and present them to the maturing lymphocytes. The B and T cells eventually exit the Peyer’s patch and guardagainst microbial penetration by patrolling the subepithelial regions throughout the intestine.

Volume 71, Number 2, 2005 / ASM News Y 79

then transferred into a germ-free isolator. Thepups are immediately removed and fostered togerm-free lactating females. Although this isnow a relatively straightforward process, thefirst generations of germ-free mice were derivedwithout benefit of germ-free foster mothers andhad to be laboriously hand-reared.

Bacterial Contributions

to Innate Immunity

Germ-free mice have yielded a number of valu-able clues about how commensal bacteria shapeintestinal innate immune responses. For in-stance, commensal microbes alter the expres-sion of angiogenin-4, a bactericidal protein pro-duced in the mammalian gut, according toJeffrey Gordon and colleagues at WashingtonUniversity in St. Louis, Mo. Although its namesuggests a role in the formation of new bloodvessels, this protein is in fact a swift and effectivebacterial killer. Synthesized and secreted exclu-sively by Paneth cells (Fig. 3), angiogenin-4 spe-cifically targets gram-positive organisms such asListeria monocytogenes and Enterococcus fae-

calis, while leaving gram-negative organismssuch as Escherichia coli largely untouched. Suchspecificity may assist in establishing and main-taining the predominantly gram-negative bacte-rial populations found in intestines of adults.

Commensal bacteria stimulate angiogenin-4synthesis during a key developmental transitionin early postnatal life. In mice that have a nor-mal gut flora, angiogenin-4 expression increasesdramatically when young mice switch frommother’s milk to a regular diet and quicklyreaches adult levels. By contrast, germ-free micenever achieve high angiogenin-4 expression lev-els, indicating that full expression of angioge-nin-4 in Paneth cells requires interactions withgut bacteria. However, this deficiency is revers-ible. By exposing germ-free mice to the mixtureof intestinal bacteria found in their colonizedcounterparts, angiogenin-4 levels rapidly rise tomatch those found in conventionally colonizedmice.

The early postnatal expression pattern of an-giogenin-4 shows that commensal bacteria caninfluence the composition of the developingPaneth cell antimicrobial arsenal. This suggests

F I G U R E 2

Use of flexible film isolators for maintaining germ-free mice. Germ-free mice develop without any contact with the microbial world, andare thus an essential tool for defining which intestinal immune system functions require interactions with commensal bacteria for fulldevelopment. The germ-free environment is created inside sterile plastic chambers (left panel) that can accommodate a number ofmouse cages. Air is supplied to each isolator by a blower attached to a filter, which allows the air to be sterilized before it enters theisolator. Isolator interiors are sterilized by spraying with a dilute solution of Clidox (chlorine dioxide), and manipulations in the isolator arecarried out through neoprene gloves. Sterile supplies such as food, water, and bedding are autoclaved in a stainless steel cylinder (rightpanel) and are transferred into the isolator through a double-door port.

80 Y ASM News / Volume 71, Number 2, 2005

that bacteria and host may collabo-rate in shaping the composition ofthe evolving gut microbial commu-nity during weaning. Moreover, byinducing an abundance of this bacte-ricidal protein, commensal bacteriamay help to ensure that rapid-fireinnate immune responses are primedand ready in the event that a patho-gen is encountered. However, im-portant questions remain, includinghow bacterial signals are relayed toPaneth cells to alter antimicrobialprotein expression and whetherother Paneth cell antimicrobial pro-teins are regulated by interactionswith commensal flora.

Bacterial Contributions

to Adaptive Immunity

Germ-free animals likewise are pro-viding compelling evidence thatcommensal bacteria serve as a driv-ing force in the development of thegut adaptive immune system (see ta-ble). For example, commensal bacte-ria play crucial roles in promoting Bcell development in Peyer’s patches,which are underdeveloped in germ-free mice, according to John Cebraof the University of Pennsylvania inPhiladelphia and his collaborators.When commensal bacteria colonizethe intestine, they initiate a series ofreactions, including those that leadto transient expansion of germinalcenter reactions between B and Tcells in the Peyer’s patches (Fig. 1) and increasedproduction of immunoglobulin A (IgA) antibod-ies by B cells. Thus, germ-free mice generatereduced amounts of IgA as compared to micewith intestinal flora, and have decreased num-bers of circulating B and T lymphocytes. More-over, introducing only a single commensal bac-terial species does not restore proper development,suggesting that a diverse repertoire of bacterialspecies and antigens is necessary to drive fulldevelopment of intestinal immunity.

Recent work by Andrew MacPherson andTherese Uhr, of the Institute of ExperimentalImmunology in Zurich, Switzerland, has pro-vided new insights into how commensal bacteria

stimulate B cell IgA production. Dendritic cells,which can wedge between gut epithelial cells,continuously sample bacteria from the lumen

F I G U R E 3

Bacterial contributions to small intestinal innate immunity. Commensal bacteria trigger theexpression of angiogenin-4, a bactericidal protein produced in small intestinal Paneth cells.angiogenin-4 specifically targets gram-positive bacteria, while gram-negative organisms arespared. This interaction may be important for establishing and maintaining the predominantlyGram negative bacterial populations found in the adult intestine. Furthermore, these findingssuggest that commensal bacteria play a central role in shaping the composition of thePaneth cell antimicrobial arsenal. The molecular signals that commensal bacteria use tocommunicate with Paneth cells are unknown.

Bacterial Contributions to Gut AdaptiveImmunity

Formation of anatomical structures, including Peyer’spatches, which harbor developing B and T cells

Expansion of germinal center reactions involving Band T cells in Peyer’s patches

Increased IgA production by intestinal B cellsGeneration of antibody diversity (in rabbits)Expansion of intraepithelial lymphocyte populations

(��TCR-bearing)

Volume 71, Number 2, 2005 / ASM News Y 81

and present their products to developing gutlymphocytes, thereby inducing B cells to synthe-size IgA antibodies that specifically bind to com-mensal antigens. Such antibodies are ultimatelysecreted into the gut lumen and are likely criti-cally important in keeping commensal bacteriafrom crossing into host tissues where they couldcause damage. In addition, stimulating IgA pro-duction may also help to keep the adaptiveimmune system poised to deal rapidly with anyinvading pathogens.

Katherine Knight and her collaborators atLoyola University in Chicago, Ill., have shownthat in rabbits as in mice, commensal bacteriahelp drive the formation of lymphoid structuressuch as Peyer’s patches. Furthermore, althoughhumans and mice generate primary antibodydiversity using mechanisms that are indepen-dent of microbial colonization, her research re-veals that rabbits require intestinal bacteria togenerate a diverse antibody repertoire. Thus, forrabbits the intestinal microflora not only appearto play a role in forming the tissues in whichintestinal lymphocytes develop but also mayinfluence the ability of the gut immune system tomount a successful immune response. Confirm-ing Cebra’s findings, Knight and her colleaguesfind that a diverse consortium of bacterial spe-cies is required to fully promote immune systemdevelopment.

In addition to providing critical signals for thedevelopment of Peyer’s patch-derived lympho-cytes, commensal bacteria also influence the re-cruitment of IELs to the intestinal surface, wherethey integrate among epithelial cells lining smallintestinal villi. Over the past decade, a numberof labs have shown that germ-free mice have10-fold fewer IELs that bear T cell receptorsconsisting of an � and � chain than do micecarrying commensal bacteria. When germ-freemice become colonized, these IEL numbers in-crease dramatically. In contrast, IELs that haveT cell receptors (TCR) consisting of a � and �chain are unaltered in germ-free mice, stronglysuggesting that this IEL population carries outfunctions that are distinct from ��TCR-bearingIELs.

Commensal Bacteria and the Emergence

of Inflammatory Bowel Diseases

Modern Western societies place a strong empha-sis on cleanliness and hygiene. The wide avail-

ability of antibiotics and antibacterial productspromotes a nearly antiseptic existence, cleansedof unwanted interactions with the microbialworld. However, Western countries are also in-creasingly afflicted with several immune disor-ders, such as allergies, that are virtually nonex-istent in nonindustrialized nations where accessto antibiotics is far more limited.

Agnes Wold of Goteborg University in Swe-den has proposed that excessive hygiene andantibiotic use, and their accompanying effectson the normal gut flora, might help to explainthe rise of immune disorders such as allergy inheavily industrialized regions such as NorthAmerica and Europe. Originally proposed in1989 by epidemiologist David Strachan of theLondon School of Hygiene and Tropical Medi-cine, this idea is known as the “hygiene hypoth-esis.”

Inflammatory bowel diseases are a group ofimmune disorders in which the gut is chronicallyinflamed. Those who suffer from IBDs experi-ence a range of symptoms, including severe di-arrhea, abdominal cramps, fever, and rectalbleeding. Most people experience IBD as a pain-ful recurring condition with alternating periodsof remission and exacerbation. Unfortunately,there is currently no cure for IBD. Although thecauses of IBDs are poorly understood, thesedisorders are thought to stem from an overlyharsh, gut-damaging immune response to thecommensal microflora. IBDs and allergies arethus similar in that both are characterized byinappropriate immune responses to otherwiseharmless environmental antigens.

Epidemiologists have found that IBDs, likeallergies, are found mainly in North Americaand Europe. IBDs currently affect more than 1in 1,000 individuals in the United States. More-over, the incidence of IBDs has risen dramati-cally in North America and Europe over the past50 years. Although genetics plays a role in IBDs,many investigators have invoked the hygienehypothesis to explain the increasing prevalenceof these diseases. The idea is that exposure tokey microbes, particularly during childhood,may be critical for directing the maturing gutimmune system to develop tolerance to com-mensal flora. Disrupting the interactions be-tween commensal bacteria and intestinal im-mune cells by exposure to broad-spectrumantibiotics or excessively clean environmentsmay compromise the development of normal,

82 Y ASM News / Volume 71, Number 2, 2005

measured immune responses to commensal gutbacteria.

Outlook

We are beginning to appreciate more fully theextent to which commensal microbes contributeto human biology. In our nutrient-rich society,we may no longer rely on the metabolic contri-butions of our prokaryotic cohorts for survival.However, the rise of immunologic diseases suchas IBDs underscores the fact that we have intri-cate, co-evolved relationships with our micro-bial populations, and that these interactions arelikely essential for the normal, healthy develop-ment of our immune systems.

Building a truly comprehensive understand-ing of human biology will thus entail developing

a detailed molecular picture of our interactionswith our commensal microbial populations.Such a picture must include a better grasp of thecomposition of these populations, how thesemicrobial societies communicate with host cells,and precisely how microbial signals shape theintestinal immune system. This challenge callsfor a multidisciplinary line of attack, blendingexpertise and experimental approaches fromfields such as microbial ecology, developmentalbiology, and immunology. Meeting this chal-lenge will undoubtedly also yield new insightsabout the consequences of broad-spectrum an-tibiotic use and its impact on intestinal immu-nity. By fully elucidating microbial contribu-tions to intestinal immunity, we will be betterequipped to harness the power of our bacterialallies in maintaining our health.

ACKNOWLEDGMENTS

We are grateful to our colleagues Cassie Behrendt, Anisa Ismail, and Cecilia Whitham for many helpful discussions. Work inthe Hooper lab is supported by grants from the Crohn’s and Colitis Foundation of America and from the Burroughs-WellcomeFoundation (Career Award in the Biomedical Sciences to L.V.H.).

SUGGESTED READING

Bandeira, A., T. Mota-Santos, S. Itohara, S. Degermann, C. Heusser, S. Tonegawa, and A. Coutinho. 1990. Localization of�� T cells to the intestinal epithelium is independent of normal microbial colonization. J. Exp. Med. 172:239–244.Hooper, L.V. 2004. Bacterial contributions to mammalian gut development. Trends Microbiol. 12:129–134.Jiang, H. Q., M. C. Thurnheer, A. W. Zuercher, N. V. Boiko, N. A. Bos, and J. J. Cebra. 2003. Interactions of commensal gutmicrobes with subsets of B- and T-cells in the murine host. Vaccine 22:805–811.Loftus, E. V., Jr. 2004. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmentalinfluences. Gastroenterology 126:1504–1517.Macpherson, A. J., and N. L. Harris. 2004. Interactions between commensal intestinal bacteria and the immune system.Nature Rev. Immunol. 4:478–485.Mowat, A. M. 2003. Anatomical basis of tolerance and immunity to intestinal antigens. Nature Rev. Immunol. 3:331–341.Rhee, K., P. Sethupathi, A. Driks, D. K. Lanning, and K. L. Knight. 2004. Role of commensal bacteria in development ofgut-associated lymphoid tissues and preimmune antibody repertoire. J. Immunol. 172:1118–1124.Suau, A., R. Bonnet, M. Sutren, J. J. Godon, G. R. Gibson, M. D. Collins, and J. Dore. 1999. Direct analysis of genes encoding16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl. Environ. Microbiol.65:4799–4807.Wostmann, B. S., C. Larkin, A. Moriarty, and E. Bruckner-Kardoss. 1983. Dietary intake, energy metabolism, and excretorylosses of adult male germfree Wistar rats. Lab. Anim. Sci. 33:46–50.

Volume 71, Number 2, 2005 / ASM News Y 83