Blackwell Publishing LtdOxford, UKCMICellular Microbiology 1462-5814© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd? 200684558564Review ArticleDendritic cells in intestinal immunityJ. H. Niess and H.-C. Reinecker

Cellular Microbiology (2006) 8(4), 558–564

doi:10.1111/j.1462-5822.2006.00694.x First published online 24 February 2006

Microreview Dendritic cells in the recognition of intestinal microbiota Jan Hendrik Niess and Hans-Christian Reinecker* Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Summary Mucosal dendritic cells (DCs) constantly survey the luminal microenvironment which contains commensal microbiota and potentially harmful organisms regulating pathogen recognition and adaptive as well as innate defense activation. Distinct mechanisms are beginning to emerge by which intestinal antigen sampling and handling is achieved ensuring specificity and contributing to redundancy in pathogen detection. Distinct DC subsets are associated with these mechanisms and regulate specific innate or adaptive immune responses to help distinguish between commensal microbiota, pathogens and self antigens. Understanding DC biology in the mucosal immune system may contribute to the unraveling of infection routes of intestinal pathogens and may aid in developing novel vaccines and therapeutic strategies for the treatment of infectious and inflammatory diseases. Introduction The gastrointestinal mucosa is in constant interaction with the luminal microenvironment which contains commensal microbiota (up to 1012 organisms per gram of intestinal content), as well as a variety of bacterial, viral and protozoan pathogens such as Shigella, Yersinia, Salmonella, Listeria, enteroinvasive, haemorrhagic and pathogenic Escherichia coli, Rotavirus, Toxoplasma gondii and Entamoeba histolytica. A single layer of intestinal epithelial cells (IECs) covers the mucosal surface of the digestive tract which is approximately the size of a tennis court (260–300 m2) preventing uncontrolled dissemination of pathogens within host tissues. In this highly antigenic environment the mucosal immune system must Received 1 December, 2005; accepted 4 January, 2006. *For correspondence. E-mail [email protected]; Tel. (+1) 617 724 2172; Fax (+1) 617 726 3673. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

maintain tolerance to commensal bacteria, food and selfantigens while initiating defensive responses to pathogens to prevent uncontrolled infection of the host. The intestine is monitored by a highly adaptable host defence system in which sentinels such as dendritic cells (DCs), macrophages and epithelial cells together monitor the microbial environment and coordinate immune responses to danger signals (Niess and Reinecker, 2005). Mucosal DCs are the critical antigen presenting cells responsible for priming of naïve T lymphocytes triggering the induction of adaptive immune responses to control tissue inflammation and to maintain immune tolerance (NaglerAnderson, 2001; Steinman et al., 2003). In recent years it has become apparent that a diversity of DC lineages with distinct characteristics contribute to innate and adaptive immune responses in the intestine. The specific role of most of these DC subsets in antigen sampling and presentation is unknown and it remains therefore unclear whether they work synergistically, as alternatives or have distinct functions in the recognition of the intestinal microbiota. The focus of effort in this field will be to understand how these diverse DC subsets cooperate in regulating the complex homeostasis and host defence in the different intestinal immune compartments. While it has become increasingly evident that several mechanisms are involved in the translocation of antigens across the intestinal barrier, it is clear that mucosal DCs play a key role in the transport and processing of intestinal antigens for presentation in Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs). However, it needs to be established whether distinct DCs subsets are associated with these pathways and if their function is linked to specific innate or adaptive immune responses to help make the distinction between commensal microbiota, pathogens and self-antigens. In this review we will discuss the key functions of mucosal DCs in the host defence mechanism in response to normal microbiota and pathogenic microorganisms. Dendritic cells in the digestive tract Dendritic cells form an extensive network in the lamina propria of the small as well as the large intestine, which is characterized by CX3CR1 (the receptor for the chemok-

Dendritic cells in intestinal immunity 559 ine fractalkine/CX3CL1) expression (Niess et al., 2005), and which may serve as a gateway for the entry of pathogenic microorganisms. The establishment of the CX3CR1+ DC subset in the lamina propria occurs partly in response to the presence of the enteric microbiota. CX3CR1+ DCs continuously sample the intestinal microbiota by extending transepithelial dendrites into the epithelium by a CX3CRl-dependent mechanism that is most developed in the terminal ileum (Rescigno et al., 2001; Niess et al., 2005). DCs transport the sampled commensals to MLNs, potentially inducing the production of IgA by B cells to limit the penetration of commensals into host tissues (Macpherson and Uhr, 2004). This DC subset could be derived from blood CX3CR1highGr1– monocytes which constitutively migrate into non-inflamed tissues and do not require inflammatory signals. In contrast, the short-lived CX3CR1lowGr1+ monocyte subset is actively recruited to inflamed tissues (Geissmann et al., 2003). CX3CR1+ DCs are distributed throughout all intestinal immune compart-

ments and it needs to be determined how they contribute to the different antigen sampling mechanisms recognized as contributing to the continuous sampling of the intestinal microbiota from the intestine. These antigen acquisition routes include IECs which themselves participate in intestinal antigen sampling by delivering antigen complexes or exosomes to lamina propria DCs (Owen, 1977; Jang et al., 2004; Yoshida et al., 2004) (Fig. 1). Aggregates of DCs are observed in cryptopatches and isolated lymph follicles of the small and large intestine, as well as in PPs (Kelsall and Leon, 2005). Most likely, the phenotype of DCs and their function is influenced by tissue-specific factors that may be produced by IECs in response to normal microbiota or pathogenic microorganisms. This is highlighted by the fact that the expression of chemokine receptors distinguishes DCs from different intestinal immune compartments. For example, CX3CR1 is expressed both by lamina propria and PP DCs, while CCR6+ DCs are restricted to PPs (J. H. Niess and H. C.

Intestinal lumen IEC

Villous M Cell dependent uptake

C×3CR1+ DC

Luminal sampling by DCs Intestinal barrier breach M Cell dependent uptake

Pathogen invasion of the FAE

Bacteria

Antigen presentation for adaptive immune defenses

C×3CR1+ DC

Innate defense activation?

CCR6+ DC B Cell

T Cell

Mesenteric lymphnode

DC

Macrophages T Cell

Local pathogen recognition

B Cell

Granulocytes T Cell

Lamina Propria

Peyer’s Patch Fig. 1. Dendritic cell subsets are responsible for antigen recognition in the intestine. CX3CR1+ dendritic cells populate the entire intestinal lamina propria, as well as dome regions of Peyer’s patches. These dendritic cells are responsible for continuous antigen acquisition from the intestinal lumen, and transport this antigen for presentation from the lamina propria to mesenteric lymph nodes. CCR6+ dendritic cells are associated with Peyer’s patches and are recruited from the interfollicular to dome region upon pathogen challenge for pathogen recognition induction of defense. It needs to be determined how lamina propria dendritic cells are integrated into innate immune responses upon intestinal epithelial barrier breaches. MLN, mesenteric lymph node, FAE, follicle associated epithelium, IEC, intestinal epithelial cell. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 558–564

560 J. H. Niess and H.-C. Reinecker Reinecker, data not shown). In addition, mucosal DCs also drive intestinal immune compartmentalization by imprinting of α4β7+ and CCR9+ T cells that home specifically to the small intestine (Mora et al., 2003). The subspecifications of DCs in intestinal immune compartments may allow the functional plasticity required for rapid adaptation to the diverse antigenic challenges in the intestine. CX3CR1+CD11c+CD11b+ cells are the predominant DC population in the terminal ileum of mice (Niess et al., 2005). However, other researchers have also observed the presence of CD11c+CD11b– DCs in the terminal ileum which constitutively express IL-23 (Becker et al., 2003). The expression of the integrin alpha chain CD103 (alpha(E)) was recently found to be characteristic of colonic lamina propria DCs (Annacker et al., 2005; Johansson-Lindbom et al., 2005) In humans the presence of immature CD11c+HLA-DR+lin– (CD3–CD14–CD16– CD19–CD34–) cells was observed in colonic and rectal biopsies (Bell et al., 2001), as well as CD83+ and DCSIGN+ lamina propria DCs (te Velde et al., 2003). In PPs, CD11chiCD11b–CD8α+CD4–, CD11chiCD11b–CD8α–CD4–, CD11chiCD11b+CD8α–CD4– DCs were found (Iwasaki and Kelsall, 1999; 2000; 2001). Upon activation, the latter produced low levels of IL-12p70 and high levels of IL-10. In contrast, the CD11chiCD11b–CD8α–CD4– DC produced low IL-10 and high IL-12 levels and were consequently able to induce Th1 immune responses. However, future work is needed to determine what physiological role the intestinal microbiota plays in driving the development of intestinal DC populations and their functional subspecifications. It has been proposed that DCs play an important role in the induction of oral tolerance, as shown by studies in which enhanced oral tolerance to intestinal antigens was observed after expansion of DCs by stimulation with Flt3-ligand (Viney et al., 1998; Horwitz et al., 2004). Several studies have suggested pathways by which DCs achieve tolerance, for example, level of maturation at the time of presenting antigens to T cells, downregulation of the costimulatory molecules CD80 and CD86, novel costimulatory molecules such as CD200 (OX-2)CD200R interaction (Gorczynski et al., 2005) and the production of immunosuppressive cytokines such as IL10, TGF-β and IFN-α (Yamagiwa et al., 2001). Understanding the crosstalk between the commensals and DC populations may aid in increasing the knowledge of how intestinal DCs contribute to the development of oral tolerance. Sensing of microbes by DCs Immature DCs are found in all peripheral tissues in an immature state where they efficiently capture foreign antigens and self-antigens (Banchereau and Steinman,

1998). When DCs are exposed to microbial products, they mature and migrate to the T cell area of secondary lymphoid organs such as MLNs, where they activate naïve T cells (Steinman, 2003). Activation and maturation of DCs occurs upon recognition of phylogenetically conserved pathogen-associated molecular patterns (PAMPs) expressed by organisms as pattern recognition receptors (PRR), such as Toll-like receptors (TLR) (Iwasaki and Medzhitov, 2004). In addition, maturation of DCs may also occur upon stimulation by pro-inflammatory mediators, such as TNF-α, IL-1 and Type I interferons, which are released after microbial or viral infection. In mice and humans DC subsets can be defined by expression of different TLRs. Isolated human plasmacytoid DCs seem to exclusively express TLR-7 and TLR-9 (Krug et al., 2001), whereas myeloid DCs express TLR-1, -2, -3, -4, -5, -6 and -8 (Liu, 2005). Intestinal lamina propria DCs show low TLR-2 and TLR-4 expression compared with blood DCs (Hart et al., 2005). In mice, only GM-CSFderived DCs from bone marrow express high TLR-4 levels, whereas most splenic DCs have low TLR-4 expression (Boonstra et al., 2003). Mouse pDCs also lack TLR3 expression but TLR-7 expression can be found on pDCs as well as CD4+DCs (Doxsee et al., 2003). It still needs to be unravelled which TLR pattern characterizes mucosal DC subsets to gain insight into their functional subspecification in response to the normal microbiota or to pathogenic organisms. In addition to the expression of TLRs, DCs express other PAMPs, such as C-type lectins and mannose receptors. Recent work has also demonstrated that DCs express the nucleotide-binding oligomerization domains NOD1 and NOD2 (Fritz et al., 2005). NOD1 recognizes muramyl-tripeptides from Gram-negative bacteria, whereas NOD2 recognizes muramyl-dipetides which are common to all peptidoglycans of all bacteria species (Girardin et al., 2003). Mutations of NOD2 are associated with an increased risk of Crohn’s disease, indicating that the dysregulated recognition of the intestinal microbiota leads to disease in genetically predisposed individuals (Hugot et al., 2001; Ogura et al., 2001). Only after oral Listeria monocytogenes administration to NOD2–/– mice show a greater dissemination of L. monocytogenes compared with wild-type mice (Kobayashi et al., 2005). Invasion of L. monocytogenes into epithelial cells by the socalled zipper mechanism requires interactions between internalin (InlA) and human E-cadherin (Cossart and Sansonetti, 2004). When proline-16 is exchanged into glutamic acid as in mouse E-cadherin (Lecuit et al., 1999), the interaction between InlA and E-cadherin cannot take place, which means that in mice L. monocytogenes most likely enter the host via M cells or a lamina propria DCdependent mechanism. Testing the hypothesis that NOD2 expressing lamina propria DCs play a key role in the

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 558–564

Dendritic cells in intestinal immunity 561 recognition of the microbiota with existing animal models will likely provide information about mechanisms that maintain immune homeostasis on mucosal surfaces. Cross communication between DCs and epithelial cells The mucous membranes lining the digestive tract are the major site for entry of pathogens (Niedergang and Kweon, 2005). A single layer of cells, the epithelium, protects the intestinal mucosa from entry of pathogens. The intestinal epithelium is highly differentiated, consisting of polarized cells that are attached to each other by tight and adherens junctions (Matter and Balda, 2003). Some bacteria, such as Salmonella and Shigella, have developed strategies that allow them to enter epithelial cells or break up tight junctions and gain access to the epithelium from the basolateral site by an active process (Zhou et al., 1999; Sakaguchi et al., 2002). While M cells have long been regarded as the main entry route for bacterial antigens for recognition by the mucosal immune system, other epithelial cells may also play an active role by sensing normal microbiota and pathogens. Epithelial cells express TLRs, which allow them to actively recognize viral and bacterial products such as lipopolysaccharide (LPS), flagellin, CpG or sRNA (Cario et al., 2000; Hornef et al., 2002). In the context of inflammation or after stimulation with IL-1β and TNF-α, epithelial cells express NOD2 which allows them to recognize intracellular muramyl-dipeptide (MDP), which leads to the activation of NF-κB (Inohara et al., 2001; Hisamatsu et al., 2003). Intestinal epithelial cells cross talk with lamina propria DCs by direct cell–cell interactions and by secretion of soluble mediators. IECs basolaterally express fractalkine/ CX3CL1 which is found in highest expression levels in the terminal ileum compared with upper parts of the small intestine (Muehlhoefer et al., 2000). Fractalkine/CX3CL1, the only member of the CX3CL1 chemokine family, is a membrane-bound chemokine which combines properties of both adhesion molecules and chemokines (Bazan et al., 1997). Expression of CX3CR1 by lamina propria DCs is required for the formation of transepithelial dendrites (Niess et al., 2005). Glycoproteins such as mucins are able to downregulate DC activation markers, such as CD40, CD86, CD83 and MHC II, and at the same time upregulate the expression of CD1a and CD206 (Rughetti et al., 2005). Recent work has demonstrated that conditioning of DCs with media derived from epithelial cell cultures drives DCs, which secrete IL-10 and IL-6 but not IL-12 and thereby promote the differentiation of Th2 polarizing T lymphocytes. Further work revealed that IECs release thymic stromal lymphopoietin (TSLP) and thereby may maintain DCs in an immature state (Rimoldi et al., 2005a).

Intestinal epithelial cells also participate in intestinal antigen uptake pathways. After transport of IgG by human neonatal Fc receptor (FcRn) across the IECs and secretion of IgG into the intestinal lumen, the secreted IgG binds cognate intestinal antigens and thus forms IgG/ antigen complexes which are recycled by FcRn and delivered to lamina propria DCs (Yoshida et al., 2004). Interaction between DCs and M cells In the intestine, M cells are predominantly found localized to specialized epithelial regions that cover lymphoid follicles, the follicle-associated epithelium (FAE). M cells have unique features that allow them to capture, traffic and transfer bacteria to DCs on their basolateral surface (Neutra et al., 2001). M cells are mainly located in the FAE of PPs, which are found in higher density in the lower small intestine compared with the upper small intestine (Mowat, 2003). Recent reports describe that M cells are scattered throughout the epithelium of small intestinal villi, which have been designated villous M cells (Jang et al., 2004). M cells do not have brush borders and their associated glycocalix, which distinguishes them from IECs, and which probably enhances their ability to transport luminal microorganisms to DCs residing in underlying tissues. Currently, the binding of lectins, especially the Ulex europeaus agglutinin 1 (UEA-1), are used as M cell markers. This, however, still lacks complete M cell specificity because UEA-1 also binds to goblet cells (Mach et al., 2005). The FAE expresses the chemokines CCL9 and CCL20 (Zhao et al., 2003), which may be required for the recruitment of DCs into the dome regions of PPs (Iwasaki and Kelsall, 2000). It has been suggested that microbial stimuli such as Salmonella typhimurium-derived flagellin also drives the expression of CCL20 by regular IECs and thereby the recruitment of DCs into the lamina propria upon Salmonella infection (Sierro et al., 2001; Rimoldi et al., 2005b). Nevertheless, M cells are ideally suited to shuttle viral particles and microbes from the intestinal lumen to DCs located in the FAE regions of PPs or to DCs in the lamina propria. M cells have fewer lysosomal compartments than IECs and may thereby favour transport of particles to intestinal DCs, which migrate from the subepithelial dome regions to B and T cell areas of PPs (Shreedhar et al., 2003). This has been shown for pathogenic microorganisms such as Shigella, Yersinia and Salmonella, as well for viruses such as mouse mammary tumour virus (MMTV), retroviruses, such as human immunodeficiency viruses (HIV), and prions. It has to be determined whether DCs extending dendrites into the intestinal lumen act synergistically with the M cell system or whether these intestinal

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 558–564

562 J. H. Niess and H.-C. Reinecker antigen uptake systems encompass two independent systems with specific roles in regulating host responses. Dendritic cells and pathogenic microorganisms Lamina propria DCs either directly sample pathogenic microorganism such as Salmonella by extending transepithelial dendrites into the intestinal lumen in a CX3CR1 dependent manner, or phagocytose bacteria delivered by M cells or IECs. The degradation of internalized antigens by DCs is delayed compared with macrophages, which favours efficient accumulation, processing, dissemination and presentation of antigens to T cells by DCs (Delamarre et al., 2005). Pathogens have developed different strategies by which they interact with macrophages and DCs to overcome the host’s immune system, which is illustrated for Salmonella, Shigella and Yersinia. After translocation of the SipB effector spi1 via the type III secretion system (TTSS) into the cytoplasm of macrophages, caspase-1 is activated, which induces apoptosis in mammalian cells and secretion of pro-inflammatory cytokines (Monack et al., 2000). Then the effectors of the sip2 genes are expressed, which inhibit apoptosis and allow remodelling of phagosomes by engineering the Salmonella-containing vacuole. Salmonella spp. survive in the intracellular compartments and are further disseminatted resulting in septicaemia (Garcia-del Portillo and Finlay, 1995; VazquezTorres et al., 2000). In contrast, Shigella induces apoptosis of macrophages and monocytes through translocation of IpaB effectors via the TTSS resulting in the activation of caspase-1 and the release of pro-inflammatory cytokines such as IL-1β and IL-18, which disrupt the integrity of the epithelial barrier (Zychlinsky et al., 1992). Yersinia has developed a strategy which inhibits phagocytosis by inactivation of the cytoskeleton through YopE, YopH and YopT injection (Juris et al., 2000). When Yersinia is phagocytosed, this pathogen then induces apoptosis of the phagocytes (Thirumalai et al., 1997). Although these mechanisms have mainly been shown for interactions with macrophages and monocytes, one can conclude that the same mechanisms may also take place in interactions with DCs. Conclusion Dendritic cells play a major role in regulating the complex interactions between pathogens, the gut microbiota and the innate and adaptive immune systems leading to tolerance or immunity. However, the work of unravelling the complexity of emerging DC interactions with commensals and pathogens in health and disease has just begun. An important question that remains to be answered is which microbial signals result in tolerance or immunity induction by DCs. Nevertheless, the investigation of DC biology in

the intestine has yielded important contributions to the understanding of host–pathogen interaction. Genetic mouse model systems, as well as genetically engineered mutant pathogens, which allow the in vivo analysis of host–pathogen interactions are becoming increasingly available and contribute to the understanding of intestinal DC subsets in inducing mucosal immunity. Further insights will be gained about pathways that pathogens use to enter the host and to overcome the host’s immune system, which may aid in developing novel therapeutic approaches for intestinal infections and immunization strategies.

Acknowledgements This work was supported by Grants DK068181 and DK33506 to H.C.R. J.H.N. is supported by a Research Fellowship Award from the Crohn’s and Colitis Foundation of America (CCFA).

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