Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Hygiene hypothesis wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Immune system wikipedia , lookup
Molecular mimicry wikipedia , lookup
Adaptive immune system wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
REVIEWS Immune adaptations that maintain homeostasis with the intestinal microbiota Lora V. Hooper* and Andrew J. Macpherson‡ Abstract | Humans harbour nearly 100 trillion intestinal bacteria that are essential for health. Millions of years of co-evolution have moulded this human–microorganism interaction into a symbiotic relationship in which gut bacteria make essential contributions to human nutrient metabolism and in return occupy a nutrient-rich environment. Although intestinal microorganisms carry out essential functions for their hosts, they pose a constant threat of invasion owing to their sheer numbers and the large intestinal surface area. In this Review, we discuss the unique adaptations of the intestinal immune system that maintain homeostatic interactions with a diverse resident microbiota. Microbiota The microorganisms that are harboured by normal, healthy individuals. These microorganisms live in the digestive tract and at other body sites. *The Howard Hughes Medical Institute and The Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. ‡ The Department of Clinical Research (DFK), Maurice Müller Laboratories, Universitätsklinik für Viszerale Chirurgie und Medizin (UVCM), University of Bern, 3008 Bern, Switzerland and Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Ontario L8S 4L8, Canada. e‑mails: lora.hooper@ utsouthwestern.edu; [email protected] doi:10.1038/nri2710 The mammalian intestine contains a dynamic community of trillions of microorganisms. These microorganisms establish symbiotic relationships with their hosts, making essential contributions to mammalian metabolism while occupying a protected, nutrientrich environment. Despite the symbiotic nature of this relationship, the close association of a dense bacterial community with intestinal tissues poses serious health challenges. The sheer number of intestinal bacteria presents a persistent threat of microbial breach, and the single-cell epithelial layer and huge intestinal surface area (~200 m2 in humans) further compounds this threat. Such opportunistic invasion of host tissue by resident bacteria can result in breakdown of the symbiotic host–microorganism relationship and contribute to pathologies such as bacteraemia or chronic inflammation. The intestinal immune system has an essential role in limiting tissue invasion by the resident microbiota, and is thus fundamentally important for preserving the symbiotic nature of these interactions. However, this system faces challenges unlike those faced by any other organ system, as it must continuously cope with an enormous microbial load, a high degree of microbial diversity, a vast surface area and frequent challenges from pathogenic microorganisms ingested in food and water 1. At the same time, the intestinal immune system must avoid potentially harmful overreactions that could unnecessarily damage intestinal tissues or alter the crucial metabolic functions of the microbiota. Despite these challenges, the intestinal immune system is remarkably effective at minimizing adverse health effects from the microbiota, as shown by the fact that sepsis and inflammation are rare in immunologically healthy hosts. In this Review, we discuss the unique adaptations of the intestinal immune system that allow it to limit opportunistic invasion by the resident microbiota while responding appropriately to bacterial pathogens. These include immune mechanisms that limit direct bacterial contact with epithelial cell surfaces, promote rapid detection and killing of penetrant bacteria, and minimize exposure of resident bacteria to the systemic immune system. We explore how these immune mechanisms cooperate to maintain the symbiotic nature of the host–microorganism relationship, and how these mechanisms can become dysregulated in disease. In doing so, our intention is not to provide a comprehensive catalogue of all mucosal immune mechanisms, but instead to offer a conceptual framework for understanding how the intestinal immune system manages its high bacterial load. The intestinal microbiota The human intestinal microbial community is complex and is composed of at least 1,000 distinct bacterial species (BOX 1). This diverse microbiota makes several essential contributions to human physiology and health. A primary function of intestinal bacteria is to enhance host digestive efficiency by degrading dietary polysaccharides2–4. It is this function that is thought to be the driving force behind the evolution of the mammalian nATuRe RevIeWS | Immunology volume 10 | mARcH 2010 | 159 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Sepsis A systemic response to severe infection or tissue damage, leading to a hyperactive and unbalanced network of pro-inflammatory mediators. Vascular permeability, cardiac function and metabolic balance are affected, resulting in tissue necrosis, multi-organ failure and death. Metagenome All the genetic material present in a population of microorganisms, consisting of the genomes of many individual organisms. Goblet cell A mucus-producing cell found in the epithelial cell lining of the intestine and lungs. host–microorganism relationship5. Recruitment of a complex and dynamic bacterial community has allowed mammals to acquire an adaptable ‘metagenome’ that harbours a diversity of saccharolytic enzymes that complement the limited saccharolytic diversity encoded in the host genome. commensal microorganisms such as Bacteroides thetaiotaomicron are uniquely adapted for harvesting luminal nutrients, as shown by the presence of an unusually large number of genes in these microorganisms that encode carbohydrate-degrading enzymes6. millions of years of co-evolution have led to a fundamental intertwining of mammalian and microbial biology that extends well beyond this metabolic function. For example, intestinal microorganisms provide instructive signals for several aspects of intestinal development, including epithelial cell maturation7,8, angiogenesis9 and lymphocyte development 10–12. Intestinal bacteria also have an important role in protecting their hosts against pathogenic infections. Two distinct factors contribute to this protective effect. First, many intestinal bacterial pathogens are poorly adapted to compete with commensal microorganisms for dietary nutrients, restricting their luminal colonization13. Second, symbiotic microorganisms stimulate immune responses against pathogens. For example, invasion and dissemination of Salmonella enterica subsp. enterica serovar Typhimurium are limited by immune responses induced by the stimulation of epithelial Toll-like receptors (TlRs) by symbiotic bacteria25. Intestinal commensal microorganisms also direct a protective immune response against the protozoan parasite Toxoplasma gondii by activating cytokine production by dendritic cells (Dcs)15. Box 1 | Characterizing the intestinal microbiota For decades, our understanding of the composition of intestinal microbial communities was based on the enumeration and characterization of culturable organisms. However, this approach left substantial gaps in the catalogue of intestinal bacterial species, as most gut organisms are resistant to culture by available methods. The recent development of molecular profiling methods, including high-throughput sequencing of microbial 16S ribosomal RNA genes, has revolutionized the understanding of the intestinal microbiota through culture-independent analyses of microbial community composition. These methods have allowed unprecedented insight into the make-up and diversity of intestinal microbial communities, and have even led to the identification of new bacterial species97. Molecular profiling of the human intestinal microbiota has revealed a high level of variability between individuals at the bacterial species level. This argues against the hypothesis that the human microbiota comprises a ‘core’ group of bacterial species that is common among all individuals. Nevertheless, common patterns emerge when microbial communities are compared at higher-level taxa. Firmicutes and Bacteroidetes are the predominant intestinal phyla across all vertebrates5. The intestinal Firmicutes are Gram-positive bacteria, dominated by species belonging to the Clostridia class, but also include Enterococcaceae and Lactobacillaceae families and Lactococcus spp.97. Intestinal Bacteroidetes are Gram-negative bacteria comprised of several Bacteroides species, including Bacteroides thetaiotaomicron, Bacteroides fragilis and Bacteroides ovatus97. The remaining intestinal bacteria, accounting for less than 10% of the total population, belong to the Proteobacteria, Fusobacteria, Actinobacteria, Verrucomicrobia and Spirochaetes phyla and a bacterial group that is closely related to Cyanobacteria5,97. The species variability among individuals has important implications for understanding intestinal immune system function, as it indicates that the mucosal immune system must be able to flexibly and rapidly adapt to a microbiota, the composition of which may change in unpredictable ways as a function of host diet or other interactions with the external environment. Being a member of the resident intestinal microbial community does not necessarily imply that a particular species has an entirely benign disposition towards its host. Although many gut microorganisms establish mutually beneficial relationships with their hosts, specific members of the microbiota may exist at different points on the continuum between mutualism and pathogenicity. For example, Enterococcus faecalis is a Gram-positive bacterial species that is a prominent member of the human intestinal microbiota, but it can opportunistically invade mucosal tissues to cause bacteraemia and endocarditis14. Similarly, Bacteroides fragilis is a prominent Gram-negative member of the microbiota that closely associates with mucosal surfaces and opportunistically invades intestinal tissues16. Although both E. faecalis and B. fragilis are controlled in healthy people, they pose a serious threat of invasion and disease in immunodeficient individuals. Intestinal homeostasis The relationship of the microbiota and the intestinal immune system is often described as ‘homeostatic’. In other physiological contexts, homeostasis refers to an equilibrium set point (such as blood sugar level) that is maintained by positive and negative biological feedback processes in the face of changing conditions. Similarly, intestinal host–microorganism homeostasis involves minimizing the adverse health effects of intestinal microorganisms, even during environmental perturbations such as shifts in microbial community structure, changes in host diet or overt pathogenic challenge. This involves ensuring that resident bacteria breach the barrier as rarely as possible; those that do invade are killed rapidly and do not penetrate to systemic sites. In this Review, we suggest that intestinal homeostasis is maintained by a hierarchy of three immunological barriers, each of which encompasses a distinct set of immune mechanisms. First, there are immune mediators that limit direct contact between the intestinal bacteria and the epithelial cell surface, decreasing the likelihood of tissue invasion. A second layer of immune protection involves the rapid detection and killing of bacteria that manage to penetrate intestinal tissues. Finally, a third set of immune responses minimizes exposure of resident bacteria to the systemic immune system. This occurs by distinctive anatomical adaptations that contain penetrant bacteria within the mucosal immune compartment and help to maintain systemic immune ‘ignorance’ towards the microbiota. Minimizing bacteria–epithelial cell contact A key element of the mammalian intestinal strategy for maintaining homeostasis with the microbiota is to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. This is accomplished by enhancing the physical barrier through the production of mucus, antimicrobial proteins and IgA (FIG. 1). The mucus layer. Goblet cells are specialized epithelial cells that secrete mucin glycoproteins. These mucins assemble into a viscous gel-like layer that extends up to 150 μm from the intestinal epithelial cell 160 | mARcH 2010 | volume 10 www.nature.com/reviews/immunol © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS surface, forming two structurally distinct strata17 (FIG. 1). visualization of spatial relationships between mucus, bacteria and the epithelium indicates that the inner mucus layer delineates a protected zone at the apical epithelial cell surface, whereas the outer mucus layer contains large numbers of bacteria17. Thus, the inner layer is resistant to bacterial penetration, limiting direct bacterial contact with epithelial cells. mice engineered to lack the mucin glycoprotein muc2 do not have this bacteria-free zone and suffer from spontaneous intestinal inflammation17,18, emphasizing the importance of the mucus barrier in maintaining a symbiotic relationship with the microbiota. Several pathogens have evolved specific strategies for penetrating mucus in order to gain access to the epithelial cell surface. For example, Helicobacter pylori uses urease to increase the pH in its immediate microenvironment, which in turn lowers mucus viscosity allowing the organism to propel itself through the mucus layer that coats the stomach wall19. other pathogens, such as Campylobacter jejuni and Salmonella spp., use their flagella to penetrate intestinal mucus20. However, it is important to note that flagella and mucus penetration are not sufficient for pathogenicity, as most commensal intestinal Firmicutes also have flagella, and some also penetrate the mucus layer to colonize the intestinal surface21. Defensin A class of antimicrobial peptide that has activity against Gram-positive and Gram-negative bacteria, fungi and viruses. α-defensins are produced by intestinal Paneth cells and neutrophils, and β-defensins are expressed by most epithelial cells. C-type lectin An animal receptor protein that binds to carbohydrates, frequently in a Ca2+-dependent manner. The binding activity of C-type lectins is based on the structure of the carbohydraterecognition domain, which is highly conserved among members of this family. Paneth cells A specialized epithelial cell lineage that produces most of the antimicrobial proteins in the small intestine. Antimicrobial proteins. A second immune mechanism that limits bacteria–epithelial cell contact is the secretion of antimicrobial proteins by gut epithelial cells (FIG. 1). epithelial cell-derived antimicrobial proteins are members of diverse protein families, including defensins, cathelicidins and C-type lectins. most of these proteins kill bacteria directly through enzymatic attack of the bacterial cell wall or by disrupting the bacterial inner membrane. In addition, a limited subset of antimicrobial proteins, including lipocalin 1, function by depriving bacteria of essential heavy metals such as iron22. Antimicrobial proteins are produced by virtually all intestinal epithelial cell lineages, including enterocytes, goblet cells and Paneth cells. The expression of different subsets of antimicrobial proteins is regulated by distinct mechanisms (FIG. 2). Several antimicrobial proteins, including most α-defensins, are expressed constitutively and do not require bacterial signals for their expression23. However, the expression of a key subset of antimicrobial proteins is controlled by bacterial signals through the activation of pattern recognition receptors, which recognize molecular patterns that are unique to bacteria and other microorganisms. For example, epithelial TlRs govern the expression of the antimicrobial c-type lectin regenerating islet-derived protein 3γ (ReG3γ) in the small intestine24,25. likewise, nucleotide-binding oligomerization domain-containing protein 2 (noD2) controls the expression of a distinct subset of α-defensins and defensin-related cryptdins by Paneth cells26. Biochemical measurements of antimicrobial activity indicate that antimicrobial proteins secreted by epithelial cells are retained in the mucus layer and are virtually absent from the luminal content27. This suggests that the Lumen Outer mucus layer Inner mucus layer Bacterium Mucin glycoproteins (assemble to form mucus layers) IgA Antimicrobial proteins Transcytosis Lamina propria Enterocyte Goblet cell IgA-secreting plasma cell Figure 1 | Immune mechanisms that limit bacteria– Nature Reviews | Immunology epithelial cell interactions. Several immune mechanisms work in concert to limit contact between the dense luminal microbial community and the intestinal epithelial cell surface. Goblet cells secrete mucin glycoproteins that assemble into a thick, stratified mucus layer. Bacteria are abundant in the outer mucus layer, whereas the inner layer is resistant to bacterial penetration. Epithelial cells (such as enterocytes, Paneth cells and goblet cells) secrete antimicrobial proteins that further help to eliminate bacteria that penetrate the mucus layer. Plasma cells secrete IgA that is transcytosed across the epithelial cell layer and secreted from the apical surface of epithelial cells, limiting numbers of mucosa-associated bacteria31 and preventing bacterial penetration of host tissues32,33. mucus layer protects the epithelial cell surface in at least two ways: first, by limiting the access of luminal bacteria to the epithelium, and second, by forming a diffusion barrier that concentrates antimicrobial proteins near the epithelial cell surface. This probably increases the effectiveness of antimicrobial proteins in protecting the apical surfaces of epithelial cells from microbial colonization. The suggestion that epithelial cell-derived antimicrobial proteins function primarily to protect this surface niche is supported by in vivo genetic studies of Paneth cells. Genetic ablation of these cells through Paneth cellspecific expression of a diphtheria toxin transgene 20 results in increased penetration of the epithelial cell barrier by both symbiotic and pathogenic bacteria. However, the number of luminal bacteria is not altered by the loss of Paneth cells, suggesting that Paneth cellderived antimicrobial factors do not reach bacteria that are confined to the intestinal lumen25. Several gastrointestinal pathogens have evolved specific resistance mechanisms against antimicrobial proteins that facilitate the invasion of these organisms across the epithelial cell barrier. For example, Listeria monocytogenes deacetylates its peptidoglycan, allowing it to evade enzymatic attack by lysozyme, a cellwall degrading enzyme secreted by intestinal epithelial nATuRe RevIeWS | Immunology volume 10 | mARcH 2010 | 161 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Constitutive expression TLR-dependent expression NOD2-dependent expression Bacteria Bacterial killing α-defensins Microorganismassociated molecular pattern TLR REG3γ MYD88 Muramyl dipeptide Subset of α-defensins NOD2 Paneth cell Enterocyte Figure 2 | Regulation of antimicrobial protein expression. Antimicrobial proteins are produced by virtually all intestinal epithelial cell lineages. Several antimicrobial proteins, Nature Reviews | Immunology including most α-defensins, are expressed constitutively and do not require bacterial signals for their expression23,26. The expression of the antimicrobial C-type lectin regenerating islet-derived protein 3γ (REG3γ) is controlled by microorganism-associated molecular patterns that activate Toll-like receptors (TLRs) and is dependent on the common TLR signalling adaptor molecule myeloid differentiation primary-response protein 88 (MYD88)24,25. REG3γ expression is activated in both enterocytes and Paneth cells25. The expression of a subset of α-defensins and defensin-related cryptdins is controlled by nucleotide-binding oligomerization domain-containing protein 2 (NOD2)26,90. NOD2 localizes to the cytoplasm of Paneth cells98 and recognizes muramyl dipeptide99, a constituent of bacterial peptidoglycan. Peyer’s patches Groups of lymphoid nodules present in the small intestine (usually the ileum). They occur massed together on the intestinal wall, opposite the line of attachment of the mesentery. Peyer’s patches consist of a dome area, B cell follicles and interfollicular T cell areas. High endothelial venules are present mainly in the interfollicular areas. Lamina propria Connective tissue that underlies the epithelium of the mucosa and contains various myeloid and lymphoid cells, including macrophages, dendritic cells, T cells and B cells. Plasma cell A non-dividing, terminally differentiated, immobile antibody-secreting cell of the B cell lineage. cells28. Similarly, S. Typhimurium evades the membrane disruptive activity of some antimicrobial peptides by modifying the lipid A moiety of lipopolysaccharide, which is present in the Gram-negative outer membrane29. S. Typhimurium also expresses specific factors (from the iroBCDEN gene cluster) that allow it to evade lipocalin 2 (also known as nGAl), an epithelial antimicrobial protein that functions by blocking bacterial iron acquisition30. IgA. A third mechanism for sequestering symbiotic bacteria involves IgA (FIG. 1). Secreted IgA limits bacterial association with the intestinal epithelial cell surface31 and restricts the penetration of symbiotic bacteria across the gut epithelium32. IgA specific for intestinal bacteria is produced with the aid of Dcs that sample bacteria at various mucosal sites (FIG. 3; BOX 2). Dcs located beneath the epithelial dome of Peyer’s patches sample bacteria that penetrate the overlying epithelium33. Lamina propria Dcs also actively sample the small numbers of bacteria that are present at the apical surfaces of epithelial cells, allowing them to monitor bacteria that associate with the mucosal surface34,35. The bacteria-laden Dcs induce B cells to differentiate into plasma cells that produce IgA specific for intestinal bacteria. Although these Dcs migrate from the Peyer’s patches and lamina propria to the mesenteric lymph nodes, they do not penetrate further into the body 33. IgA+ plasma cells translocate from lymphoid sites to the intestinal lamina propria and secrete IgA, which is transcytosed across the epithelial cell layer. The transcytosed IgA binds to bacteria on the luminal side of the epithelial cell barrier, limiting bacterial association with the epithelium31 and preventing bacterial penetration of host tissues32. The exact mechanisms by which IgA carries out these functions remain unclear but may involve trapping of bacteria in the mucus layer or promoting rapid phagocytic clearance of organisms that penetrate the epithelial cell barrier 36. Immune responses to penetrant bacteria Although mucus, antimicrobial proteins and secretory IgA work together to protect intestinal epithelial cell barriers from direct bacterial contact, the vast numbers of intestinal bacteria means that occasional breaches of this barrier are inevitable. A second crucial layer of intestinal immune protection relies on the rapid detection and killing of bacteria that penetrate beyond the epithelial cells. This occurs by several distinctive immune mechanisms, including bacterial uptake and phagocytosis by innate immune cells and T cell-mediated responses. Phagocytic killing. commensal microorganisms that breach the intestinal epithelial cell barrier typically succumb to rapid phagocytosis and elimination by lamina propria macrophages. macrophages are present in high numbers in the mammalian gastrointestinal tract and are frequently in close contact with the epithelium37. These cells rapidly phagocytose bacteria and kill the ingested organisms through mechanisms that include antimicrobial proteins and reactive oxygen species38. Although macrophages from many tissue sites (such as the bone marrow) secrete pro-inflammatory mediators that recruit neutrophils and activate T cells, intestinal macrophages do not mediate strong proinflammatory responses following bacterial recognition, despite retaining efficient phagocytic and bactericidal functions33,39. This probably reflects an evolutionary adaptation by these cells to the high bacterial load in the gut, preventing potentially damaging pro-inflammatory responses from being activated under normal, homeostatic conditions. Although, typically, penetrant commensal microorganisms are rapidly killed by phagocytic cells, evasion or suppression of phagocytic killing is a virulence strategy that is frequently used by intestinal pathogens, allowing them to escape the intestinal lumen where they are poorly adapted to compete with commensal microorganisms for luminal nutrients. Salmonella spp. and Shigella spp., for example, actively downregulate the microbicidal mechanisms of macrophages, allowing the pathogens to survive and replicate in host tissues 40. The susceptibility of commensal microorganisms to the antimicrobial mechanisms of macrophages is probably important for promoting mutually beneficial relationships with their hosts, as evasion of phagocytic killing could damage host tissues and perhaps compromise the microorganisms’ own intestinal niche. Susceptibility to macrophage antimicrobial mechanisms may thus reflect an evolutionary co-adaptation to the host that increases the overall evolutionary fitness of symbiotic bacteria41. 162 | mARcH 2010 | volume 10 www.nature.com/reviews/immunol © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Lumen Bacteria Epithelial cell M cell Transcytosis T cell Lamina propria DC IgA B cell IgA+ B cell Peyer’s patch Migrating DCs can induce B and T cell activation in the mesenteric lymph nodes IgA+ B cell IgA+ plasma cell Recirculation of B cells through lymph and blood Mesenteric lymph node DCs do not recirculate through lymph and blood Figure 3 | Production of IgA directed against intestinal bacteria. Dendritic cells (DCs) sample bacteria at various sites. DCs located beneath the epithelial Nature Reviews | Immunology dome of Peyer’s patches take up bacteria that penetrate the overlying epithelium33. Lamina propria DCs actively sample the small numbers of bacteria that are present at the apical surfaces of epithelial cells by extending their dendrites between the epithelial cells34,35. The bacteria-laden DCs migrate to Peyer’s patches and mesenteric lymph nodes where they induce B cells to differentiate into IgA+ plasma cells. IgA+ plasma cells home to the lamina propria and secrete dimeric IgA that is transcytosed across the epithelial cell layer and binds to intestinal bacteria, limiting bacterial association with the epithelium31 and preventing bacterial penetration of host tissue32. M cell, microfold cell. Transcytosis Process of transport of material across a cell monolayer by uptake on one side of the cell into a coated vesicle, which might then be sorted through the trans-Golgi network and transported to the opposite side of the cell. Germ-free mouse A mouse that is born and raised in isolators, without exposure to microorganisms. Intestinal macrophages have a second important role in maintaining symbiotic relationships with the intestinal microbiota by helping to restore the physical integrity of the epithelial cell barrier following injury. The presence of a dense resident microbiota means that gut epithelial cell damage can quickly lead to bacterial penetration, inflammation and sepsis. mouse models of intestinal epithelial cell damage have revealed that bacteria that penetrate damaged areas trigger the expression of a specific repair pathway in macrophages, inducing them to migrate to the damaged areas and produce growth factors42. These growth factors interact with the epithelium, triggering vigorous enterocyte proliferation that fuels the replacement of damaged epithelium with new cells. Bacteria activate this pathway by engaging TlRs expressed by macrophages42,43, although it is not yet clear whether internalization of the bacteria is required or whether the repair pathway can be activated by detection of extracellular bacteria. CD4 + T cells. Studies that compare germ-free mice and bacterially colonized mice show that the adaptive immune system, in particular the mucosal compartment, is profoundly shaped by the presence of the commensal intestinal microbiota. This includes increases in the size and number of germinal centres in Peyer’s patches44 and in the numbers of IgA-secreting plasma cells45, lamina propria cD4+ T cells and αβ T cell receptor (TcR)-expressing intraepithelial cD8αβ+ T cells46,47. In addition, the presence of commensal microorganisms in the intestine is tolerated without an acute neutrophil inflammatory infiltrate in both healthy mice and humans. cD4+ regulatory T (TReg) cells are an essential component of this mutualism. The two main subtypes of TReg cells are cD4+FoXP3+ TReg cells that are found in the colon and small intestinal lamina propria and cD4+FoXP3–Il-10+ TReg cells that are found in the small intestinal intraepithelial and lamina propria compartments48. evidence for the key role of TReg cells in intestinal immune regulation against commensal microorganisms came originally from two types of mouse model, and in both cases immune regulation is only necessary when mice are colonized with intestinal microorganisms. First, there is spontaneous intestinal inflammation in mice with specific deficiencies in regulatory cytokines (such as interleukin-10 (Il-10)49 and transforming growth factor-β (TGFβ)50) or in factors that determine TReg cell thymic selection (such as autophagy-related gene 5 (ATG5)), differentiation and/or function (such as forkhead box P3 (FoXP3) or αvβ8 integrin)51. Second, there are mouse models in which chronic intestinal inflammation is induced — for example, by the transfer of naive colitogenic cD45RBhicD4+ T cells into recombination-activating gene (Rag) deficient or severe combined immunodeficient (ScID) mice, which are lymphopenic — and is rescued by the co-transfer of TReg cell populations. The TReg cell-mediated rescue verifies the in vivo requirements for TReg cell-derived factors (such as Il-10 (ReFs 52–54) or TGFβ55) by analysing the suppressive ability of TReg cells from genetically targeted and conditioned mice in chronic intestinal inflammation. There is also evidence that TReg cell populations are induced by intestinal bacteria56 or their molecular products, such as the polysaccharide A carbohydrate expressed by B. fragilis 57 and the non-culturable clostridia-related segmented filamentous bacteria58. The expression of regulatory cytokines such as TGFβ and Il-10 is not restricted to TReg cells, but the induction of TReg cells by commensal microorganisms and the occurrence of intestinal inflammation in their absence indicate that TReg cells determine the threshold between non-inflammatory homeostasis and intestinal inflammation. The difference between these two states is important functionally: during intestinal inflammation there is a dramatic infiltrate of neutrophils and lymphocytes into the intestinal mucosa with increased epithelial cell proliferation and enhanced secretion of mucus and electrolytes. These inflammatory changes are necessary to eliminate invasive mucosal pathogens, but must usually be avoided if the intestine is to function properly in the presence of non-invasive luminal commensal microorganisms. nATuRe RevIeWS | Immunology volume 10 | mARcH 2010 | 163 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Box 2 | Intestinal dendritic cells Dendritic cells (DCs) are immune cells with characteristic projections (dendrites) acquired during development and are specialized for antigen presentation to B and T cells. For this they load peptide antigens onto MHC class II molecules that engage with the T cell receptor and provide co-stimulatory signals through, for example, B7 molecules on the DC and CD28 on the T cell. DCs can also stimulate T cells indirectly; for example, by secreting interleukin-12 (IL-12) to promote the differentiation of the CD4+ T helper 1 (TH1) cell subset. DCs also stimulate B cells through expression of tumour necrosis factor family ligands, including a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF). Conversely, when loaded with food proteins, in the absence of activating molecules (such as Toll-like receptor ligands from microorganisms), intestinal DCs promote the peripheral tolerance of B and T cells. Intestinal DCs constitutively produce retinoic acid, which promotes mucosal IgA class switch recombination (in B cells) or FOXP3– CD4+ regulatory T cell differentiation and programmes intestinal B and T cells to express CC-chemokine receptor 9 (CCR9), which in turn regulates their homing from the blood back into the mucosa. In the intestine, DCs are present in the lymphoid follicles (such as Peyer’s patches) adjacent to the epithelial cell layer and in the lamina propria beneath the epithelial cell layer. They can be separated into different classes depending on surface marker expression (classically CD11chiCD11b+CD8α–, CD11chiCD11b–CD8α+ and CD11chiCD11b–CD8α– DCs) with distinct histological distributions and functional responses. CCR6 regulates DC homing to Peyer’s patches and a large proportion of intestinal DCs express surface CD103 (also known as αE integrin). It has been shown that intestinal DCs can extend dendrites between contiguous epithelial cells to sample the external luminal milieu. Activated DCs show CCR7-dependent migration to draining lymph nodes, and, in the case of intestinal DCs, there is a constant traffic of cells from the mucosa to the mesenteric lymph nodes. Most CD103+ DCs in the mesenteric lymph nodes have probably trafficked from the lamina propria. In contrast to macrophages, DCs have poor biocidal activity, so after sampling commensal microorganisms the live bacteria are not immediately killed but persist within the DCs and can be transported to the mesenteric lymph nodes. Because DCs have a short lifespan, bacteria-laden DCs, and therefore bacteria, do not reach central systemic lymphoid structures. This is a mechanism that limits the induction of mucosal immunity by commensal microorganisms to the mucosal immune system, with the mesenteric lymph nodes functioning as a type of immune ‘firewall’. Germinal centre Located in peripheral lymphoid tissues (for example, the spleen), these structures are sites of B cell proliferation and selection for clones that produce antigen-specific antibodies of higher affinity. Recombination-activating gene (Rag). A gene expressed by developing lymphocytes. Mice that are deficient for either Rag1 or Rag2 fail to produce B or T cells owing to a developmental block in the gene rearrangement that is necessary for antigen receptor expression. Severe combined immunodeficiency (sCID). A phenotype of mice with a defect in DNA recombination. sCID mice lack B and T cells and do not reject tissue grafts from allogeneic and xenogeneic sources. A balance between the function of TReg cells and the cD4+ effector T cells in the intestinal mucosa is also crucial for homeostasis. Based on the signalling molecules and transcription factors responsible for their differentiation and their signature cytokines59, the characteristics of T helper 1 (TH1), TH2, TH17 and TReg cells are summarized in FIG. 4. The differentiation of both TH17 and FoXP3+ TReg cells requires TGFβ, although Il-6 produced by Dcs and macrophages in response to bacterial molecules, including cpG-containing oligodeoxynucleotides, drives the differentiation of the TH17 cell lineage. In the colon, FoXP3+ TReg cells can express Il-10, which reciprocally inhibits TH17 and TH1 cells. In the small intestine, another FoXP3– regulatory T cell subset, referred to as TR1 cells, is also stimulated by Il-6 to express Il-10 (FIG. 5). TReg cell-mediated regulation of effector T cells is important, as there is a substantial population of TH1 cells in the mucosa, where they are induced by the intestinal microbiota, notably by polysaccharides produced by Bacteroides spp.60,61. The differentiation of TH17 cells in the mucosa also depends on the microbiota11; however, this differentiation is driven only by a limited subset of commensal bacterial species, including mucosa-adherent segmented filamentous bacteria21,58. constitutive expression of the p40 subunit of Il-12 or Il-23, which are involved in the differentiation of TH1 and TH17 cells, respectively, by lower-small-intestinal cD8α–cD11b–cD11c+ lamina propria Dcs has also been shown to be triggered by the uptake of intestinal microorganisms62. These results are consistent with the hypothesis that T H1 and T H17 cells are needed for the elimination of the small numbers of commensal microorganisms that penetrate the surface epithelial cell layer. Indeed, mice deficient in T cells have increased persistence of translocated commensal bacteria in the mucosal tissues63. Another key function of mucosal T cells is to enhance the antimicrobial functions of phagocytic cells and epithelial cells. As discussed above, efficient microbicidal activity by phagocytic cells is essential to avoid opportunistic infections from those commensal organisms that penetrate the epithelial cell layer. The production of interferon-γ (IFnγ) by TH1 cells enhances macrophage activation. macrophages and Dcs that have been activated by microbial products secrete Il-12, which has a positive feedback effect on TH1 cell-dependent IFnγ expression. Similarly, TH17 cells secrete Il-22, which limits commensal antigen exposure by enhancing the expression of antimicrobial proteins. TH17 cells also produce Il-17A and Il-17F (which promote neutrophil recruitment and activation) (FIG. 5) and Il-21 (which inhibits TReg cell generation)64. mucosal TH2 cells are not frequently found in healthy humans and in animals that do not harbour parasites, probably owing to limited Il-4 production in the normal mucosa and antagonism from TH1 cell-derived IFnγ. A further twist in the complex interactions between TH cell subsets is shown by observations that these subsets are not necessarily terminally differentiated but that there can be conversion from TH17 to TH1 cells and TReg to TH17 cells65,66. We have discussed homeostasis between T cell subsets in the mucosa as if all commensal microorganisms are completely benign. In fact, asymptomatic carriage of potential intestinal pathogens is seen in both mice and humans; for example Clostridium difficile-associated 164 | mARcH 2010 | volume 10 www.nature.com/reviews/immunol © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Naive T cell CD4+ FOXP3– IL-12 TGFβ IL-6 IL-4 IL-6 TGFβ No IL-6 T-bet GATA3 RORγt MAF FOXP3– FOXP3+ TH1 cell TH2 cell TH17 cell TR1 cell TReg cell IFNγ IL-4 IL-5 IL-13 IL-17A IL-17F IL-6 IL-10 TGFβ Figure 4 | CD4+ T cell subset differentiation. CD4+ T cells differentiate in response to Nature Reviews | Immunology cytokine-induced signals mediated by characteristic transcription factors. Each subset of + CD4 T cells produces the characteristic cytokine profile shown. Interleukin-23 (IL-23) enhances effector function of the T helper 17 (TH17) cell subset, but it does not determine TH17 cell differentiation per se (not shown). The two classes of CD4+ regulatory T cells, conventional regulatory T (TReg) cells and TR1 cells, are characterized by the presence or absence of expression of the transcription factor forkhead box P3 (FOXP3), respectively. Although CD4+ T cell subsets have traditionally been seen as terminally differentiated cells, evidence for conversion from TH17 cells to TH1 cells and possibly from TReg cells to TH17 cells suggests plasticity between the subsets65,66. GATA3, GATA-binding protein 3; MAF, macrophage-activating factor; RORγt, retinoic acid receptor-related orphan receptor-γt; TGFβ, transforming growth factor-β. Intraepithelial CD8αα+ T cell A type of T cell that is found in the intestinal epithelium. The CD8 molecule that they express is a homodimer of CD8α, rather than the CD8αβ heterodimer that is expressed by conventional CD8+ T cells in the lymph nodes. It has been proposed that these cells are self-reactive T cells that have regulatory properties. colitis can be triggered by antibiotic usage. Therefore, it is important to briefly consider the differences between the murine models in which most of the mechanisms of mucosal immune regulation have been investigated and the ‘real life’ situation. To standardize the colonies and to eliminate pathogens, mice and rats can be maintained with a highly restricted ‘modified Schaedler’ microbiota, which is limited to eight fastidious culturable bacterial species introduced into a germ-free colony by inoculation from a pure culture. The ideal of having a fixed microbiota in this way is rarely achieved, as other species become established in most colonies through human handling. nevertheless, the simple microbiota in most immunological studies is completely different from a ‘wild’ or ‘human’ situation where a less simplistic microbiota is present that can potentially evade host immunity to establish an infection. one method to more accurately model the natural situation is the ‘infection’ of mice with Helicobacter hepaticus. Helicobacter spp. are an example of potential pathogens and are endozoonotic in wildtype mouse colonies. H. hepaticus causes an enteropathy in ScID or Rag–/– mice and this disease can be attenuated by the transfer of cD4+ TReg cells67. H. hepaticus also causes colitis in Il-10-deficient mice, protection from which can be mediated by the transfer of cD4+ TReg cells from H. hepaticus-infected (but not uninfected) wildtype mice68. Furthermore, treatment with a neutralizing Il-10-specific antibody can initiate colitis in H. hepaticusinfected wild-type mice69. The colitogenic T cells in these mice have TH1 and TH17 cell characteristics, and disease is attenuated if recipient mice lack the p19 subunit of Il-23 or are infused with TReg cells69,70. Together, these data suggest that mildly pathogenic constituents of the intestinal microbiota may be actively tolerated in the intestinal lumen, provided they do not reach a threshold of penetration into the intestinal mucosa, and that Il-10 and TReg cells have an important role in maintaining this homeostasis. Three main points emerge from the discussion of the balance between different subsets of cD4+ T cells in the intestinal mucosa. First, regulatory responses mediated by TReg cell subsets are necessary for homeostasis, especially in the presence of more potentially pathogenic microbial constituents in the microbiota. Second, the complexities of cellular and cytokinemediated regulatory mechanisms induced by constituents of the intestinal microbiota are compounded by the interconversion of individual cD4+ T cell subsets, which is now starting to be explored. It is also not clear whether this regulation of cD4+ T cell subsets occurs for all commensal microorganisms in immunocompetent animals or whether it is mainly important in the presence of microorganisms that have a predisposition to induce effector T cell subsets (such as segmented filamentous bacteria). Finally, the downstream effects of effector T cell subset activation are pleiotropic: TReg cells can stimulate IgA induction in addition to their regulatory functions, and T H17 cells also maintain epithelial cell integrity in addition to their potentially pro-inflammatory functions. CD8αα+ γδ and αβ T cell subsets. Because the numbers of innate immune leukocytes do not alter significantly when germ-free mice are colonized with a commensal microbiota, evidence for their involvement in host–commensal mutualism relies on functional data. Intraepithelial CD8αα+ T cells that express a γδ TcR produce keratinocyte growth factor (KGF) and are required for intestinal epithelial cell repair following enteropathy induced by dextran sodium sulphate (DSS)71,72. These cells recognize the stressinduced atypical mHc class I molecules mHc class I polypeptide-related sequence A (mIcA) in humans or RAe1 in mice73 through natural killer group 2, member D (nKG2D)74. In functional genomic studies of purified γδ T cells, which consist mainly of cD8αα + and double-negative (cD4 –cD8–) T cells, DSS-induced damage was shown to trigger a complex transcriptional programme, including the transcription of heat shock proteins, cytoprotective factors and antibacterial factors (including ReG3γ, although KGF was not detected)75. experiments in germ-free mice showed that this transcriptional programme was largely dependent on the presence of the intestinal microbiota. The functional consequence was that the translocation of commensal bacterial to the mesenteric lymph nodes was enhanced in γδ T celldeficient DSS-treated mice (containing a simple microbiota) compared with wild-type control mice75. There is also some evidence that the other main innate cD8αα+ T cell population, which expresses an αβ TcR, in the intraepithelial compartment may have similar protective effects for host microbial mutualism, although the evidence is indirect. When transferred into lymphopenic recipients, these cD8αα+ αβ T cells protect nATuRe RevIeWS | Immunology volume 10 | mARcH 2010 | 165 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Mucin and antimicrobial peptides NK cell IL-22 TGFβ IL-17 IL-22 Phagocytosed microorganisms Epithelium Microbial molecules and phagocytosed microorganisms Microbial molecules and phagocytosed microorganisms CD103+ IL-12 IL-6 IL-6 IL-23 DC Retinoic acid IL-6 Subepithelial macrophage TGFβ IL-10 TNF Retinoic acid IL-12 TGFβ IL-10 IL-12 IFNγ TGFβ TH17 cell FOXP3– TR1 cell FOXP3+ TReg cell IL-17A IL-17F Neutrophil chemotaxis IL-10 Negative regulation TGFβ Negative regulation TH1 cell IL-10 Figure 5 | Regulatory networks for intestinal CD4+ T cells. T helper 17 (TH17) cells are induced by transforming growth Nature Reviews | Immunology factor-β (TGFβ) and interleukin-6 (IL-6) and matured by IL-23 following the activation of intestinal dendritic cells (DCs) by phagocytosed microorganisms or stimulatory microbial molecules that have crossed the surface epithelial cell barrier and/or activated epithelial cells. The signature cytokines of TH17 cells, IL-17A and IL-17F, have pro-inflammatory effects and mediate neutrophil chemotaxis. TH17 cells also express IL-22, which contributes to epithelial homeostasis and stimulates the secretion of antimicrobial molecules. Acute inflammation is normally avoided through the induction of two classes of CD4+ regulatory T (TReg) cells that can be differentiated on the basis of their expression of the transcription factor forkhead box P3 (FOXP3). FOXP3+ TReg cells are induced by retinoic acid produced by CD103+ DCs in the presence of TGFβ. Conversely FOXP3– TR1 cells are induced by IL-6 but inhibited by retinoic acid. TReg cells secrete IL-10 and/or TGFβ, which have negative regulatory effects on effector T cells. Commensal bacteria and their associated molecules also stimulate DCs to secrete IL-12, which activates interferon-γ (IFNγ) secretion by TH1 cells, which in turn activates phagocytic activity of subepithelial macrophages. NK, natural killer; TNF, tumour necrosis factor. against colitis in the naive colitogenic cD45RBhicD4+ T cell transfer model of chronic intestinal inflammation in an Il-10-dependent manner 76. As described earlier, these colitogenic T cells require the presence of an intestinal microbiota to induce enteropathy, so cD8αα+ αβ T cells seem to be another regulatory subset relevant for mucosal immune homeostasis. Lymphoid-tissue inducer cell (LTi cell). A cell that is present in developing lymph nodes, Peyer’s patches and nasopharynx-associated lymphoid tissue. LTi cells are required for the development of these lymphoid organs and are characterized by expression of the transcription factor retinoic acid receptor-related orphan receptor-γt (RORγt), interleukin-7 receptor-α and lymphotoxin-α1β2. Intestinal NK cells. Intestinal nK cells have received little attention relative to other lymphocytes, but a population of intestinal nK cells has recently been described that express nKp46 (also known as ncR1), the Il-15R β-chain (also known as cD122) and the nK cell receptors nKG2D and nKG2A77. They have variable levels of nK1.1 (also known as KlRB1c) expression, and they have a phenotype that resembles immature bone marrow nK cells in that they lack expression of cD3, cD11b (also known as αm integrin), cD27, lY49c (also known as KlRA3), lY49D (also known as KlRA4), lY49H (also known as KlRA8) and lY49G2 (also known as KlRA7). The development of these intestinal nK cells was shown to depend on the transcription factor retinoic acid receptor-related orphan receptor-γt (RoRγt), suggesting that they may be derived from lymphoid-tissue inducer cells. Although they had only weak conventional nK cell effector functions, these intestinal nK cells produced Il-22, a cytokine that is known to promote epithelial cell homeostasis and the secretion of antimicrobial factors78. The picture that emerges from the role of these different immune cells and factors in the intestinal mucosa is one of tightly regulated layers of immunity that can condition epithelial cells to avoid penetration by commensal microorganisms and/or eliminate the low numbers of bacteria that penetrate without an acute inflammatory response. Presumably there is a numerical threshold beyond which regulatory mechanisms are no longer (or less) sufficient and inflammatory responses are needed for the neutralization and clearance of pathogens. Mucosal immune firewalls The mucosal immune system has the difficult task of confining commensal microorganisms to the lumen of the gut without excessive inflammation while maintaining the ability to clear intestinal infections with an 166 | mARcH 2010 | volume 10 www.nature.com/reviews/immunol © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS Specific pathogen-free (SPF) mice Mice kept in specific vivarium conditions whereby a number of pathogens are excluded or eradicated from the colony. These animals are maintained in the absence of most of the known chronic and latent persistent pathogens. Although this enables better control of experimental conditions related to immunity and infection, it also sets apart such animal models from pathogen-exposed humans or non-human primates, whose immune systems are in constant contact with potential pathogens. appropriate degree of inflammation. It is clear that there are extensive immune adaptations to intestinal commensal microorganisms and mucosal immune responses are tightly regulated. The induction of mucosal immunity depends on some sampling of live commensal microorganisms, particularly by intestinal Dcs that sample the lumen by passing dendritic processes between the tight junctions of the epithelial cell layer 34,35 or sample in the dome of intestinal lymphoid follicles or Peyer’s patches33. Such leakiness could potentially enhance the risk of mucosal infection, but both B and T cells can be activated by low numbers of penetrant bacteria, and commensal microorganisms are susceptible to phagocyte biocidal activity 33,79. nevertheless, even for microorganisms that do not contain pathogenicity islands, expressing proteins to subvert immune microbicidal mechanisms80, a system of containment, or ‘firewall’, beneath the epithelial cell layer is needed. Where is the boundary of this immune firewall? The geography of the mucosal immune system is helpful to understand this. Bacteria that enter the blood are mainly cleared by macrophages in the marginal zones of the spleen, and those that enter the mesenteric vascular system are delivered through the portal vein to the liver and cleared by Kupffer cells. By contrast, bacteria that are taken up by intestinal Dcs are contained within the mucosal tissue and can be carried by Dcs to the draining mesenteric lymph nodes, but the bacteria do not penetrate further to reach systemic secondary lymphoid structures33. As intestinal Dcs loaded with commensal bacteria can induce protective secretory IgA33, this system has the advantage that induction of mucosal immunity is confined to the mucosal tissue, but the effects of this induction can be distributed throughout the intestinal mucosa and other mucosal surfaces through the recirculation of activated B and T cells via the mucosal lymphatics and vasculature before they home back to the mucosal tissue81–83. The limited biocidal activity of Dcs84 and their limited lifespan are probably crucial for the system to function: Dcs can sample intestinal microorganisms and induce appropriate mucosal immune responses but the survival of the Dcs containing live microorganisms is limited (and, hence, so is the potential for infection). These data indicate that the mesenteric lymph nodes have a role as a firewall against bacteria that have penetrated epithelial defences or microorganisms that have been deliberately sampled by intestinal Dcs to induce mucosal immunity. In wild-type specific pathogen-free (sPF) mice, the mucosal immune system is primed by intestinal microorganisms, but the adaptive systemic immune system remains unprimed (or ignorant)32,85. It is thought that lymphoid structures are heavily influenced by the milieu of commensal bacterial molecules that penetrate host tissues, although penetration of live commensal bacteria and the induction of adaptive immune responses are absent or limited in immunocompetent hosts47,60. This adaptive systemic ignorance is lost in SPF mice in which mesenteric lymph nodes (which are a crucial part of the firewall) have been surgically removed33. under conditions in which commensal microorganisms can penetrate and persist in the systemic immune system, for example in ‘clean’ mice with severe innate immune deficiencies that affect biocidal activity, or in ‘dirty’ humans with a diverse microbiota containing some mild pathogens, systemic immune priming is consistent with eliminating the microorganisms and avoiding sepsis. The compartmentalization of mucosal and systemic priming thus requires functionality of the intestinal barriers and the innate immune system. For example, in mice deficient in IgA expression, serum IgG responses, which are indicative of a systemic immune response, are spontaneously primed against commensal bacteria32. Similarly, innate immune defects, such as a deficiency in the TlR signalling adaptor molecules myeloid differentiation primary-response protein 88 (mYD88) or TIR-domain-containing adaptor protein inducing IFnβ (TRIF; also known as TIcAm1), or the absence of the reactive oxygen burst in phagocytes also results in serum IgG priming against intestinal commensal bacteria86. The conclusion is that small numbers of commensal bacteria are probably continuously penetrating the intestinal epithelial cell layer, but they usually never reach the threshold of priming a systemic adaptive immune response because of effective phagocytosis and biocidal activity by phagocytic cells. This is supported by the occurrence of commensal microorganism-induced sepsis in mice with defects in phagocyte biocidal activity and in neutropenic and severely immunocompromised humans79. The result of the mesenteric lymph node firewall is that there is a way for the luminal microbial contents to be immunologically sampled and for relevant protective adaptive immune responses to be induced in the intestinal mucosa. The induction is confined to the mucosal immune system, but the recirculation of B and T cells through the lymph and blood to home back to mucosal tissues ensures that the local response is disseminated and averaged over the mucosal surfaces as a whole. Immunodeficiency and homeostasis Inflammatory bowel disease (IBD) comprises a broad group of disorders that are characterized by severe inflammation of the intestine. IBD can be defined as ulcerative colitis or crohn’s disease based on clinical phenotype criteria obtained from structural and histopathological analysis. Despite the prevalence of the disease in europe and north America, the exact causes of IBD remain unclear. However, increasing evidence suggests that the disease arises from dysregulated control of host–microorganism interactions. For example, patients with IBD have increased numbers of epithelial cell surface-associated bacteria87, suggesting a failure of mechanisms that normally limit direct contact between the microbiota and the epithelium. Supporting this hypothesis, several IBD risk alleles alter epithelial cell innate immune function. one of the first IBD risk alleles identified was the cytoplasmic pathogen recognition receptor noD2. Polymorphisms in the NOD2 gene are associated with ileal crohn’s disease88,89. Patients with NOD2 defects have lower α-defensin expression by Paneth cells, which coincides with severe intestinal inflammation90. one possible model is that decreased α-defensin production promotes increased association of nATuRe RevIeWS | Immunology volume 10 | mARcH 2010 | 167 © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS bacteria with the epithelial cell surface. This could contribute to uncontrolled inflammation, perhaps in conjunction with other genetic defects. Similarly, autophagy 16 relatedlike 1 (ATG16l1), encoded by a crohn’s disease risk allele, contributes to intestinal inflammation by impairing exocytosis of Paneth cell secretory granules, thereby inhibiting antimicrobial protein release91. The mechanism by which ATG16L1 contributes to abnormal Paneth cell exocytosis remains unclear. However, as in the case of NOD2 risk alleles, defects in ATG16L1 could impair the ability of Paneth cells to limit bacterial association with the mucosal surface, thereby increasing the likelihood of bacterial penetration and mucosal inflammation. Finally, loss of the transcription factor X-box-binding protein 1 (XBP1), which is required for normal development of Paneth and goblet cells, triggers spontaneous intestinal inflammation in mice. Hypomorphic variants of XBP1 are also linked to IBD in humans92 indicating a requirement for normal Paneth and goblet cell development in intestinal homeostasis. Together, these studies suggest that defects leading to reduced antimicrobial protein and/or mucus production may increase the likelihood of bacterial invasion of the epithelial cell barrier with consequent inflammation. However, it is important to note that epithelial cell defects, such as genetic Paneth cell ablation, are insufficient to produce inflammation in mice25,93. This suggests that the manifestation of inflammatory disease in humans may require additional genetic defects that Mathers, C. D., Boerma, T. & Ma Fat, D. Global and regional causes of death. Br. Med. Bull. 92, 7–32 (2009). 2. Xu, J. et al. A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 299, 2074–2076 (2003). 3. Sonnenburg, J. L. et al. Glycan foraging in vivo by an intestine-adapted bacterial symbiont. Science 307, 1955–1959 (2005). 4. Martens, E. C., Chiang, H. C. & Gordon, J. I. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell Host Microbe 4, 447–457 (2008). 5. Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R. & Gordon, J. I. Worlds within worlds: evolution of the vertebrate gut microbiota. Nature Rev. Microbiol. 6, 776–788 (2008). 6. Xu, J. & Gordon, J. I. Inaugural article: honor thy symbionts. Proc. Natl Acad. Sci. USA 100, 10452–10459 (2003). 7. Hooper, L. V. et al. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291, 881–884 (2001). This paper shows that commensal bacteria manipulate host cell functions and widely influence host biology, revealing the essential nature of the interactions between resident microorganisms and their mammalian hosts. 8. Hooper, L. V., Stappenbeck, T. S., Hong, C. V. & Gordon, J. I. Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nature Immunol. 4, 269–273 (2003). 9. Stappenbeck, T. S., Hooper, L. V. & Gordon, J. I. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl Acad. Sci. USA 99, 15451–15455 (2002). 10. He, B. et al. Intestinal bacteria trigger T cellindependent immunoglobulin A2 class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 26, 812–826 (2007). 11. Ivanov, I. I. et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 4, 337–349 (2008). 1. affect, for example, the ability of phagocytic cells to remove bacteria that breach the epithelial cell barrier. Thus, multiple genetic lesions that target different levels of immune control of the microbiota may be required before inflammatory disease is manifested. In addition to classical IBD, there are many patients with chronic nonspecific (or ‘indeterminate’) intestinal inflammation where an infectious cause cannot be found. occasionally, patients with classical immunodeficiencies — such as common variable immunodeficiency 94, other B cell disorders or chronic granulomatous disease — can present with intestinal inflammation95. Although human immunodeficiency has been classically regarded as a set of conditions starting in childhood with severe recurrent opportunistic infections, it is increasingly recognized that immunodeficiencies with a mild phenotype can be seen in adults with no previous family history, and the immunodeficiency is sometimes triggered by a narrow range of pathogens or opportunistic microorganisms96. The disease phenotypes may be determined by patientspecific microbiota, although we are only beginning to have the tools to address this. Paradoxically, this level of uncertainty offers some improved future perspectives that we can do more for the many cases where empirical application of steroids, antibiotics, immunosuppressants or tumour necrosis factor-specific biological agents offers inadequate control of the debilitating symptoms of intestinal inflammation and provides a hope that the natural history of the diseases can be altered in the future. 12. Hall, J. A. et al. Commensal DNA limits regulatory T cell conversion and is a natural adjuvant of intestinal immune responses. Immunity 29, 637–649 (2008). 13. Stecher, B. et al. Comparison of Salmonella enterica serovar Typhimurium colitis in germfree mice and mice pretreated with streptomycin. Infect. Immun. 73, 3228–3241 (2005). 14. Klare, I., Werner, G. & Witte, W. Enterococci. Habitats, infections, virulence factors, resistances to antibiotics, transfer of resistance determinants. Contrib. Microbiol. 8, 108–122 (2001). 15. Benson, A., Pifer, R., Behrendt, C. L., Hooper, L. V. & Yarovinsky, F. Gut commensal bacteria direct a protective immune response against Toxoplasma gondii. Cell Host Microbe 6, 187–196 (2009). 16. Kuwahara, T. et al. Genomic analysis of Bacteroides fragilis reveals extensive DNA inversions regulating cell surface adaptation. Proc. Natl Acad. Sci. USA 101, 14919–14924 (2004). 17. Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105, 15064– 15069 (2008). This paper clearly visualizes the spatial relationships between the microbiota and the intestinal epithelial cell surface, and it shows that mucus glycoproteins are essential for limiting direct contact between luminal bacteria and epithelial cells. 18. Van der Sluis, M. et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology 131, 117–129 (2006). 19. Celli, J. P. et al. Helicobacter pylori moves through mucus by reducing mucin viscoelasticity. Proc. Natl Acad. Sci. USA 106, 14321–14326 (2009). 20. Guerry, P. Campylobacter flagella: not just for motility. Trends Microbiol. 15, 456–461 (2007). 21. Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009). 22. Flo, T. H. et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432, 917–921 (2004). 168 | mARcH 2010 | volume 10 23. Putsep, K. et al. Germ-free and colonized mice generate the same products from enteric prodefensins. J. Biol. Chem. 275, 40478–40482 (2000). 24. Brandl, K., Plitas, G., Schnabl, B., Dematteo, R. P. & Pamer, E. G. MyD88-mediated signals induce the bactericidal lectin RegIIIγ and protect mice against intestinal Listeria monocytogenes infection. J. Exp. Med. 204, 1891–1900 (2007). 25. Vaishnava, S., Behrendt, C. L., Ismail, A. S., Eckmann, L. & Hooper, L. V. Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface. Proc. Natl Acad. Sci. USA 105, 20858–20863 (2008). 26. Kobayashi, K. S. et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307, 731–734 (2005). 27. Meyer-Hoffert, U. et al. Secreted enteric antimicrobial activity localizes to the mucus surface layer. Gut 57, 764–771 (2008). 28. Boneca, I. G. et al. A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system. Proc. Natl Acad. Sci. USA 104, 997–1002 (2007). 29. Guo, L. et al. Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell 95, 189–198 (1998). 30. Raffatellu, M. et al. Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype Typhimurium for growth and survival in the inflamed intestine. Cell Host Microbe 5, 476–486 (2009). 31. Suzuki, K. et al. Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut. Proc. Natl Acad. Sci. USA 101, 1981–1986 (2004). 32. Macpherson, A. J. et al. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 288, 2222–2226 (2000). 33. Macpherson, A. J. & Uhr, T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303, 1662–1665 (2004). This study shows that DCs harbouring live commensal bacteria are restricted to the mucosal immune compartment by mesenteric lymph nodes, which thus function as an immune firewall that limits systemic penetration of commensal bacteria. www.nature.com/reviews/immunol © 2010 Macmillan Publishers Limited. All rights reserved REVIEWS 34. Rescigno, M. et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunol. 2, 361–367 (2001). 35. Niess, J. H. et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 307, 254–258 (2005). 36. Fagarasan, S. & Honjo, T. Intestinal IgA synthesis: regulation of front-line body defences. Nature Rev. Immunol. 3, 63–72 (2003). 37. Lee, S. H., Starkey, P. M. & Gordon, S. Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80. J. Exp. Med. 161, 475–489 (1985). 38. Kelsall, B. Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages. Mucosal Immunol. 1, 460–469 (2008). 39. Smythies, L. E. et al. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. J. Clin. Invest. 115, 66–75 (2005). 40. Sansonetti, P. J. War and peace at mucosal surfaces. Nature Rev. Immunol. 4, 953–964 (2004). 41. Macpherson, A. J., Geuking, M. B. & McCoy, K. D. Immune responses that adapt the intestinal mucosa to commensal intestinal bacteria. Immunology 115, 153–162 (2005). 42. Pull, S. L., Doherty, J. M., Mills, J. C., Gordon, J. I. & Stappenbeck, T. S. Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury. Proc. Natl Acad. Sci. USA 102, 99–104 (2005). 43. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004). 44. Shroff, K. E., Meslin, K. & Cebra, J. J. Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. Infect. Immun. 63, 3904–3913 (1995). 45. Benveniste, J., Lespinats, G. & Salomon, J. Serum and secretory IgA in axenic and holoxenic mice. J. Immunol. 107, 1656–1662 (1971). 46. Guy-Grand, D. et al. Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J. Exp. Med. 173, 471–481 (1991). 47. Macpherson, A. J. & Harris, N. L. Interactions between commensal intestinal bacteria and the immune system. Nature Rev. Immunol. 4, 478–485 (2004). 48. Barnes, M. J. & Powrie, F. Regulatory T cells reinforce intestinal homeostasis. Immunity 31, 401–411 (2009). 49. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K. & Muller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263–274 (1993). 50. Shull, M. M. et al. Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease. Nature 359, 693–699 (1992). 51. Nedjic, J., Aichinger, M., Emmerich, J., Mizushima, N. & Klein, L. Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance. Nature 455, 396–400 (2008). 52. Powrie, F. et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1, 553–562 (1994). 53. Asseman, C., Mauze, S., Leach, M. W., Coffman, R. L. & Powrie, F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190, 995–1004 (1999). 54. Asseman, C., Read, S. & Powrie, F. Colitogenic Th1 cells are present in the antigen-experienced T cell pool in normal mice: control by CD4+ regulatory T cells and IL-10. J. Immunol. 171, 971–978 (2003). 55. Li, M. O., Wan, Y. Y. & Flavell, R. A. T cell-produced transforming growth factor-β1 controls T cell tolerance and regulates Th1- and Th17-cell differentiation. Immunity 26, 579–591 (2007). 56. Cong, Y., Weaver, C. T., Lazenby, A. & Elson, C. O. Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora. J. Immunol. 169, 6112–6119 (2002). 57. Mazmanian, S. K., Round, J. L. & Kasper, D. L. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620–625 (2008). 58. Gaboriau-Routhiau, V. et al. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Immunity 31, 677–689 (2009). 59. Neurath, M. F. et al. The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn’s disease. J. Exp. Med. 195, 1129–1143 (2002). 60. Mazmanian, S. K., Liu, C. H., Tzianabos, A. O. & Kasper, D. L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–118 (2005). 61. Mazmanian, S. K. & Kasper, D. L. The love–hate relationship between bacterial polysaccharides and the host immune system. Nature Rev. Immunol. 6, 849–858 (2006). 62. Becker, C. et al. Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells. J. Clin. Invest. 112, 693–706 (2003). 63. Gautreaux, M. D., Gelder, F. B., Deitch, E. A. & Berg, R. D. Adoptive transfer of T lymphocytes to T-cell-depleted mice inhibits Escherichia coli translocation from the gastrointestinal tract. Infect. Immun. 63, 3827–3834 (1995). 64. Weaver, C. T., Harrington, L. E., Mangan, P. R., Gavrieli, M. & Murphy, K. M. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 24, 677–688 (2006). 65. Lee, Y. K., Mukasa, R., Hatton, R. D. & Weaver, C. T. Developmental plasticity of Th17 and Treg cells. Curr. Opin. Immunol. 21, 274–280 (2009). 66. Lee, Y. K. et al. Late developmental plasticity in the T helper 17 lineage. Immunity 30, 92–107 (2009). 67. Maloy, K. J. et al. CD4+CD25+ TR cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197, 111–119 (2003). 68. Kullberg, M. C. et al. Bacteria-triggered CD4+ T regulatory cells suppress Helicobacter hepaticusinduced colitis. J. Exp. Med. 196, 505–515 (2002). 69. Kullberg, M. C. et al. IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis. J. Exp. Med. 203, 2485–2494 (2006). 70. Hue, S. et al. Interleukin-23 drives innate and T cellmediated intestinal inflammation. J. Exp. Med. 203, 2473–2483 (2006). 71. Boismenu, R. & Havran, W. L. Modulation of epithelial cell growth by intraepithelial γδ T cells. Science 266, 1253–1255 (1994). 72. Chen, Y., Chou, K., Fuchs, E., Havran, W. L. & Boismenu, R. Protection of the intestinal mucosa by intraepithelial γδ T cells. Proc. Natl Acad. Sci. USA 99, 14338–14343 (2002). 73. Groh, V., Steinle, A., Bauer, S. & Spies, T. Recognition of stress-induced MHC molecules by intestinal epithelial γδ T cells. Science 279, 1737–1740 (1998). 74. Suemizu, H. et al. A basolateral sorting motif in the MICA cytoplasmic tail. Proc. Natl Acad. Sci. USA 99, 2971–2976 (2002). 75. Ismail, A. S., Behrendt, C. L. & Hooper, L. V. Reciprocal interactions between commensal bacteria and γδ intraepithelial lymphocytes during mucosal injury. J. Immunol. 182, 3047–3054 (2009). 76. Poussier, P., Ning, T., Banerjee, D. & Julius, M. A unique subset of self-specific intraintestinal T cells maintains gut integrity. J. Exp. Med. 195, 1491–1497 (2002). 77. Sanos, S. L. et al. RORγt and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells. Nature Immunol. 10, 83–91 (2009). 78. Zheng, Y. et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nature Med. 14, 282–289 (2008). 79. Shiloh, M. U. et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38 (1999). This study shows the essential role of microbicidal mechanisms in containing the commensal intestinal microbiota. 80. Sansonetti, P. Phagocytosis of bacterial pathogens: implications in the host response. Semin. Immunol. 13, 381–390 (2001). 81. Gowans, J. L. & Knight, E. J. The route of re-circulation of lymphocytes in the rat. Proc. R. Soc. Lond. B Biol. Sci. 159, 257–282 (1964). 82. Husband, A. J. & Gowans, J. L. The origin and antigendependent distribution of IgA-containing cells in the intestine. J. Exp. Med. 148, 1146–1160 (1978). This is a landmark study of the immune geography of the mucosal immune system, showing the dissemination of IgA+ plasma cells that have been induced in the intestinal mucosa through the lymph and blood. 83. Pierce, N. F. & Gowans, J. L. Cellular kinetics of the intestinal immune response to cholera toxoid in rats. J. Exp. Med. 142, 1550–1563 (1975). nATuRe RevIeWS | Immunology 84. Nagl, M. et al. Phagocytosis and killing of bacteria by professional phagocytes and dendritic cells. Clin. Diagn. Lab. Immunol. 9, 1165–1168 (2002). 85. Konrad, A., Cong, Y., Duck, W., Borlaza, R. & Elson, C. O. Tight mucosal compartmentation of the murine immune response to antigens of the enteric microbiota. Gastroenterology 130, 2050–2059 (2006). 86. Slack, E. et al. Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science 325, 617–620 (2009). 87. Swidsinski, A., Weber, J., Loening-Baucke, V., Hale, L. P. & Lochs, H. Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. J. Clin. Microbiol. 43, 3380–3389 (2005). 88. Hugot, J. P. et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411, 599–603 (2001). 89. Ogura, Y. et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411, 603–606 (2001). References 88 and 89 show that NOD2 polymorphisms account for a proportion of the genetic risk of Crohn’s disease. This was the first of a large number of linked genetic loci to be identified and the suspected dysregulation of host–microorganism mutualism as a cause of IBD was reinforced by the finding that NOD2 senses a bacterial cell wall component. 90. Wehkamp, J. et al. Reduced Paneth cell α-defensins in ileal Crohn’s disease. Proc. Natl Acad. Sci. USA 102, 18129–18134 (2005). 91. Cadwell, K. et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 456, 259–263 (2008). 92. Kaser, A. et al. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134, 743–756 (2008). 93. Garabedian, E. M., Roberts, L. J., McNevin, M. S. & Gordon, J. I. Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice. J. Biol. Chem. 272, 23729–23740 (1997). 94. Chapel, H. et al. Common variable immunodeficiency disorders: division into distinct clinical phenotypes. Blood 112, 277–286 (2008). 95. Notarangelo, L. et al. Primary immunodeficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005. J. Allergy Clin. Immunol. 117, 883–896 (2006). 96. Casanova, J. L. & Abel, L. Primary immunodeficiencies: a field in its infancy. Science 317, 617–619 (2007). 97. Eckburg, P. B. et al. Diversity of the human intestinal microbial flora. Science 308, 1635–1638 (2005). This landmark study uses molecular profiling to reveal the diversity of the human microflora and establish the dominant bacterial phylotypes that inhabit the human gastrointestinal tract. 98. Ogura, Y. et al. Expression of NOD2 in Paneth cells: a possible link to Crohn’s ileitis. Gut 52, 1591–1597 (2003). 99. Inohara, N. et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J. Biol. Chem. 278, 5509–5512 (2003). Acknowledgements L.V.H. thanks the students and colleagues from her laboratory for the many discussions that contributed to the ideas in this manuscript. Work in L.V.H.’s laboratory is supported by the Howard Hughes Medical Institute, the US National Institutes of Health (DK070855), the Burroughs Wellcome Foundation and the Crohn’s and Colitis Foundation. A.M. acknowledges K. McCoy, E. Slack, S. Hapfelmeier, M. Stoehl and M. Geuking. Competing interests statement The authors declare no competing financial interests. DATABASES UniProtKB: www.uniprot.org ATG16L1 | IL-22 | KGF | lipocalin 1 | lipocalin 2 | MICA | MUC2 | NOD2 | REG3γ | XBP1 FURTHER INFORMATION Lora V. Hooper’s homepage : http://www.hhmi.org/ research/investigators/hooper_bio.html Andrew J. Macpherson’s homepage: http://www-fhs. mcmaster.ca/medicine/gastro/faculty-member_ macpherson.htm All lInks ARe ACTIve In The onlIne PDF volume 10 | mARcH 2010 | 169 © 2010 Macmillan Publishers Limited. All rights reserved