Intestinal dendritic cells in the regulation of mucosal immunity.

Vasileios Bekiaris Emma K. Persson William W. Agace

Intestinal dendritic cells in the regulation of mucosal immunity

Authors’ addresses Vasileios Bekiaris1, Emma K. Persson1, William W. Agace1,2 1 Immunology Section, Lund University, Lund, Sweden. 2 Section of Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark.

Summary: The intestine presents a huge surface area to the outside environment, a property that is of critical importance for its key functions in nutrient digestion, absorption, and waste disposal. As such, the intestine is constantly exposed to dietary and microbial-derived foreign antigens, to which immune cells within the mucosa must suitably respond to maintain intestinal integrity, while also providing the ability to mount effective immune responses to potential pathogens. Dendritic cells (DCs) are sentinel immune cells that play a central role in the initiation and differentiation of adaptive immune responses. In the intestinal mucosa, DCs are located diffusely throughout the intestinal lamina propria, within gut-associated lymphoid tissues, including Peyer’s patches and smaller lymphoid aggregates, as well as in intestinal-draining lymph nodes, including mesenteric lymph nodes. The recognition that dietary nutrients and microbial communities in the intestine influence both mucosal and systemic immune cell development and function as well as immunemediated disease has led to an explosion of literature in mucosal immunology in recent years and a growing interest in the functionality of intestinal DCs. In the current review, we discuss recent findings from our group and others that have provided important insights regarding murine and human intestinal lamina propria DCs and highlighted marked developmental and functional heterogeneity within this compartment. A thorough understanding of the role these subsets play in the regulation of intestinal immune homeostasis and inflammation will help to define novel strategies for the treatment of intestinal pathologies and contribute to improved rational design of mucosal vaccines.

Correspondence to: William Agace Lund University – Immunology Section BMC D14 S€ olvegatan 19 Lund 22184, Sweden Tel.: +46 46 222 04 16 Fax: +46 46 222 42 18 e-mail: [email protected] V. B. and E. K. P. contributed equally to this article. Acknowledgements This work was supported by grants to W. A. from the Danish Council for Independent Research Sapere Aude Research Career program, the Swedish Medical Research € sterlund, and the IngaBritt and Arne Council, the Kocks, O Lundbergs Foundations, the Royal Physiographic Society (E. P.), and a clinical grant from the Swedish National Health Service. V. B. is supported by a scholarship from the Anna-Greta Crafoord Foundation. The authors have no conflicts of interest to declare.

Keywords: dendritic cell, mucosa, lamina propria

Introduction This article is part of a series of reviews covering Mucosal Immunity appearing in Volume 260 of Immunological Reviews.

Immunological Reviews 2014 Vol. 260: 86–101 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

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As an organ specialized in food digestion and nutrient absorption, the intestinal mucosa presents a huge surface area to the outside milieu and is continually exposed to foreign antigen derived from dietary constituents and the large numbers of microbes that reside within the intestinal lumen. Maintenance of intestinal integrity is critically dependent on the immune system’s ability to respond appropriately to such antigens and also generate protective immunity to potential pathogens that utilize the intestine as a primary site of entry and infection. Inappropriate responses to such antigens is thought to underlie several intestinal pathologies including inflammatory bowel disease (Crohn’s disease and ulcerative colitis) as well as food allergies such as Celiac’s disease (1–3). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

Bekiaris et al Intestinal dendritic cells

Dendritic cells (DCs) are key regulators of adaptive immune responses and as such are believed to play important roles in the generation and regulation of immune responses to intestinal antigens. While the definition of classical DCs (DCs) in non-lymphoid tissues has been the subject of considerable debate, the development of novel genetic tools and identification of new biomarkers has recently led to considerable progress in our understanding of mononuclear phagocyte heterogeneity, and aided in particular in the discrimination of DCs from monocyte progeny and in the identification of distinct intestinal DC subsets (Fig. 1A and B). Further, the use of such tools has started to unravel some of the in vivo functionality of DC subsets within the intestine, some of which appear lineagespecific while others appear to be common to all DCs at a particular location, and thus likely imprinted by factors within the local environment. Here, we review recent findings from our laboratory and others regarding the ontogeny of DCs in the intestinal mucosa focusing on intestinal lamina propria (LP) DC subsets and the growing body of evidence for their roles in innate and adaptive intestinal immune responses. Overview of DC subsets in the mouse and human intestinal mucosa Intestinal DCs are located within intestinal lymphoid tissues, collectively termed gut-associated lymphoid tissues (GALT), including Peyer’s patches (PPs) and solitary isolated lymphoid tissues (SILT) or diffusely throughout the intestinal LP. DC subsets in the mouse are characterized by high expression of CD11c and major histocompatibility complex class II (MHC-II) and a lack of expression of the high affinity immunoglobulin G (IgG) receptor FccR1 (CD64), which marks cells of the macrophage/monocyte lineage (4, 5). The majority of CD11c+ MHC-II+ CD64 cells in the small intestine express the integrin aE(CD103)b7, whose ligand E-cadherin is expressed on the basolateral surface of epithelial cells (6). aE(CD103)b7 serves to maintain T cells within the intestinal epithelium (7), although its function on intestinal DCs remains, as yet, unknown. Intestinal CD103+ DCs can be divided into two distinct populations, CD103+ CD11b+ and CD103+ CD11b DCs that, as discussed in detail below, differ in transcriptional factor requirements and in function. CD103+ CD11b intestinal DCs are related to lymph node (LN) resident CD8a+ DCs, and share with these cells expression of the chemokine receptor XCR1 (8, 9) (Fig. 1B). In contrast, CD103+ CD11b+ © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

intestinal DCs appear related to CD11b+ LN resident DCs sharing with these cells expression of signal regulatory protein-a (SIRPa) and DC inhibitory receptor 2 (DCIR2) (10, 11) (Fig. 1B). Finally based on lack of CD64 expression or analysis of mice expressing green fluorescence protein (GFP) under control of the DC-specific transcription factor Zbtb46, the murine intestinal LP contains a poorly defined population of CD103 CD11b+ XCR1 DCs (8, 9, 12–14). As discussed later, all three subsets can be found in intestinal-draining afferent lymph and mesenteric lymph nodes (MLNs) and are thus capable of participating in adaptive immune cell priming in draining intestinal LN. Many of the initial markers used to define DC subsets in mice and humans were not conserved between species, hampering identification of equivalent functional DC subsets. However, recent developments have lead to the identification of several markers that appear to define common functional subsets across species (10, 11, 15–17). In an early study, we demonstrated the presence of CD103+ DCs in intestinal-draining MLN that, as discussed later, appear to have similar functionality to their murine CD103+ counterparts (18), and subsequent studies by us, and others identified CD103+ DCs in the human small intestinal LP (10, 11, 19). Further phenotypic analysis of these cells allowed us to propose putative equivalents of murine intestinal CD103+ CD11b+ and CD103+ CD11b DC subsets in the human small intestine (10). Thus, we demonstrated that human intestinal CD103+ DCs could be sub-divided into two subsets: a minor subset of CD103+ SIRPa DCs that co-expressed the prototypic markers of human CD8a-like cDCs, CD141 (BDCA3), and DNGR-1 (17, 20, 21) that likely represent the human equivalent of murine CD103+ CD11b DCs and a major population of CD103+ SIRPa+ that lacked CD141 and DNGR1 expression, and likely represent the human equivalent of CD103+ CD11b+ murine cDCs (Fig. 1B). The presence of these subsets in small intestinal mucosa was recently confirmed by Watchmaker and coworkers (11), who importantly also demonstrated through comparative global gene expression and clustering analysis the inter-relationship between these cells and their murine counterparts (11). Notably these authors also identified a minor subset of small intestinal CD103 CD64 SIRPa+ DCs that expressed intermediate levels of CX3CR1 and likely represent the human counterpart of murine CD103 CD11b+ DCs. These studies, together with recent findings by others (15, 16), indicate that SIRPa and XCR1 are conserved cross-species markers that can be utilized to identify equivalent DC subsets in mice and humans (Fig. 1A

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Fig. 1. Dendritic cell subsets in the mouse and human intestinal lamina propria. (A) Lamina propria (LP) dendritic cell (DC) subsets in the mouse and human can be identified by the differential expression of CD103 and the marker CD11b in the mouse or SIRPa in human and mouse. The indicated transcription factors have been implicated in regulating the development of each of the subsets. (B) Surface markers that can be used to identify human and mouse DC subsets in the intestinal LP. Highlighted are common markers expressed by both murine and human DC subsets. (C) Pie charts indicating the relative distribution of CD103+ CD11b , CD103+ CD11b+, and CD103 CD11b+ DCs in the murine small intestine and colon (data are a mean ratio from six mice).

and B), and their usage as phenotypic markers in future studies should help alleviate some of the historical problems in comparing DC subset functionality between species. It is important to note that CD103 is not a marker of a particular DC lineage, but is, we believe, induced on CD11b+ and CD11b DCs during their residence within the intestine. In this regard, the relationship between intestinal CD103 CD11b+ and CD103+ CD11b+ DCs warrants further study. Intestinal DC progenitors Tissue DCs are continuously replenished with bone marrow (BM)-derived lineage-restricted progenitors. Early studies indicated that the earliest BM progenitor committed to the mononuclear phagocyte lineage is the macrophage and dendritic cell progenitor (MDP), which can generate myeloid and DC but no other hematopoietic lineages (22, 23). These cells then transition to common dendritic cell progenitors (CDP) that have lost their myeloid potential but maintain

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the ability to develop into plasmacytoid DCs (pDCs) and DCs (24, 25). Subsequently, CDPs were found to generate a pre-cDC intermediate, which gave rise to DCs but had lost pDC potential (23). However while MDPs and CDPs are found only in the BM, pre-cDCs are also found in blood and tissues where they can differentiate into all mature tissue DC subsets (23). More recent clonal analysis studies have suggested pre-existing commitment to either the DC or the pDC lineage within CDPs (24) and Zbtb46 expression in both CDP and pre-cDC appears to identify subsets of cells that have lost pDC potential (14). Further, genetic tracing of DNGR-1 expression history was recently suggested to define precursors restricted to the DC lineage. Thus, DNGR-1 expression marked a majority of CDPs and a minor proportion of MDPs, and adoptive transfer of lineage DNGR-1+ cells specifically gave rise to DCs but not pDCs, indicating that DNGR-1 expression is a useful marker of DC restricted progenitors (26). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014


Regarding intestinal DCs, adoptive transfer of DC progenitors into diphtheria toxin (DT)-treated CD11c-DTR (DT Receptor) or wildtype mice has demonstrated that pre-cDCs, but not Ly6Chi monocytes, can give rise to CD103+ CD11b and CD103+ CD11b+ DC in the intestinal mucosa, whereas MDPs give rise to all DC and macrophage subsets (27, 28). The ability of MDPs or pre-cDCs to give rise to intestinal CD103 DCs was not addressed in these studies as CD64 or other markers that would help distinguish CD103 CD11b+ DCs from CD11b+ macrophages were not used. It is of note, however, that in the study by Varol et al. (28), adoptive transfer of pre-cDCs gave rise not only to CD11c+ CX3CR1 cells but also a population of CD11c+ CX3CR1int cells, which could represent CD103 DCs. Moreover, two reports (4, 5) have demonstrated that Ly6Chi monocytes, when adoptively transferred, give rise exclusively to F4/80+ CD64+ cells suggesting that intestinal CD103 DCs do not derive from monocytes. A recent study examining the origin of mononuclear phagocyte subsets in the intestinal LP surprisingly found that in DT-treated Zbtb46-DTR mice, CD103+ CD11b+ DCs in the small intestinal LP were only decreased by approximately 50%, whereas CD103+ CD11b DCs were almost completely abolished (29). Further, in mice where monocytes and monocyte-derived cells were specifically ablated (DT-treated LysmCre 9 Csf1rLsL-DTR mice), CD103+ CD11b+ DCs were partially depleted, suggesting that CD103+ CD11b+ DCs are derived partly from DC progenitors and partly from monocytes (29). The reason for the discrepancies between these findings and those described above currently remains unclear. In addition to pre-cDCs, Zeng et al. (30) recently identified a subset of lineage CD11c+ a4b7+ B220+ CCR9 progenitors that, upon adoptive transfer, preferentially gave rise to CD103+ (mostly CD11b ) DCs in the intestinal LP. While these findings suggest the presence of a putative gut-specific DC progenitor, which the authors termed pre-l (mucosal) DCs, these cells also gave rise to splenic and lung DCs (also preferentially CD11b ) as well as pDCs (30). Finally a pDC progenitor expressing PDCA1, B220, and Siglec-H, but not the chemokine receptor (CCR)9 was identified as having DC potential (31). When this subset was cultured with intestinal epithelial cell-conditioned media it developed into a CD11b+ DC population that could efficiently express DCassociated transcription factors, high levels of MHC-II, and prime T cells (31). A follow-up study by the same group demonstrated that CCR9 progenitors derive from CDPs that in vivo give rise to intestinal and other tissue DCs, at least partially through imprinting within the local tissue microenvironment (32). Collectively these data suggest that © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

multiple DC progenitors can potentially serve as precursors to intestinal cDCs. The relationship between these DC progenitors and their relative contribution to intestinal DC subsets homeostasis remain to be established. Transcriptional networks in the generation and functionality of intestinal LP DCs A considerable portion of a cell’s developmental fate and functional capacity is predetermined by the collective action of its transcriptional network—that is the sum of the constitutively expressed transcription factors (TFs). Although many TFs are shared among different immune cells, there is considerable evidence for functional specificity of TFs that most often define a lineage or provide the cell with unique properties. Below, we discuss recent developments regarding the role of TFs in DC subset development focusing on those TFs that have helped enhance our understanding of the relationship between intestinal CD103+ DCs subsets and their LN resident counterparts (Fig. 1A). Transcription factors implicated in the development and maintenance of intestinal CD103+ CD11b DCs BATF3 BATF, BATF2, and BATF3 constitute a sub-family of basic leucine zipper (bZIP) TFs that form heterodimers with JUN proteins (33). JUN is known to interact with the bZIP protein FOS and form the heterodimeric TF activator protein-1 (AP1), which recognizes the consensus DNA site TGA(G/C)TCA (AP-1 binding site) (33). The association of the BATF–JUN composite factor on AP-1 binding sites has been linked with gene silencing via competition with FOS–JUN complexes that are positive regulators of gene transcription (34). Although BATF2 is broadly expressed, BATF and BATF3 are almost exclusive to the immune system. BATF3 is highly expressed in CD8a+ LN resident DCs (35) and genetic ablation of BATF3 results in a loss of CD8a+ DCs (35) strongly suggesting the in vivo relevance of BATF3-JUN dimers as positive regulators of normal DC development. More recent analyses showed that BATF3 also plays a critical role in the development of intestinal CD103+ CD11b DCs (36, 37), indicating that these cells are developmentally related to LN resident CD8a+ DCs. Mechanistically, BATF3 appears not to be required for pre-cDCs to commit to the DC lineage but is important to sustain the survival of committed DCs (38, 39). In this regard, AP-1 sites are found in the promoters of numerous genes that have been associated with cell growth, metabolism, differentiation and apoptosis (34) and in T cells, BATF induces ATP production

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during cellular activation, which is a necessary step for differentiation (40), suggesting that a similar function may be in place for BATF3-dependent migratory DCs in the gut. Interestingly, production of IL-12 during bacterial infection restores the lack of LN resident CD8a+ DCs in Batf3-deficient mice via induction of BATF and BATF2 revealing functional co-operation between BATF proteins for DC development and homeostasis (41). Whether intestinal CD103+ CD11b DC development is also restored in Batf3 / mice during mucosal infection or inflammation remains to be determined. IRF8 The IRF family of TFs regulate type I IFN responses and their expression is often turned on or sustained by inducible or tonic IFNa/b signaling (42). However, IRF4 and IRF8 display constitutive, IFNa/b-independent and restricted expression in certain immune cell types (42). Early studies using genedeficient animals demonstrated that IRF8 regulates the development of CD8a+ CD11b DCs and pDCs (43, 44). Thus, Irf8-deficient mice display a near complete loss of LN resident CD8a+ DCs (43, 44). Irf8 deficiency also results in a loss of CD103+ CD11b DCs in the intestine, as well as other peripheral tissues, mirroring the defects found in Batf3 / mice, and further emphasizing the developmental relationship between these cells and LN resident CD8a+ DCs (36, 45). Recently, L-Myc, which is a homolog of c-Myc and is encoded by Mycl1, was identified as an IRF8-regulated gene in DCs that shows high expression in CD103+ CD11b DCs (46). Genetic ablation of Mycl1 resulted in a significant reduction in intestinal CD103+ CD11b DCs (46). Id2 Inhibitor of DNA-2 (Id2) is a member of the basic helix-loophelix (bHLH) TF family and, as its name suggests, inhibits other bHLH TFs from binding to DNA. Id2 is strongly associated with lymphoid development as it is necessary for the differentiation of natural killer and innate lymphoid cells (ILCs) (47). Id2 is highly expressed in DC progenitors (38) and is critical for the generation of intestinal CD103+ CD11b but not CD103+ CD11b+ DCs (45), linking Id2 with the IRF8/ BATF3-regulated differentiation program. Transcription factors implicated in the development and maintenance of intestinal CD103+ CD11b+ DCs IRF4 Irf4-deficient animals fail to generate CD11b+ DCs in vitro and have significantly reduced numbers of splenic CD11b+

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DCs (48). Similarly, we recently demonstrated that mice whose DCs are deficient in IRF4 have reduced numbers of CD11b+ but normal numbers of CD8a+ LN resident DCs (10). These mice also display an approximate 50% reduction in intestinal CD103+ CD11b+ DCs and almost complete loss of such cells in draining MLN (10, 13), linking their development with LN resident CD11b+ DCs. One of the proposed mechanisms by which IRF4 regulates intestinal CD103+ CD11b+ DC numbers is by sustaining their survival (10). Thus, we observed that Irf4-deficient CD103+ CD11b+ DCs are more prone to enter apoptosis than their wildtype counterparts (10). These findings are in agreement with chromatin immune precipitation-sequencing (ChIP-seq) data showing that IRF4 regulates expression of the anti-apoptotic molecule Bcl-2, at least in T cells (49, 50) and with data showing that Irf4-deficient CD11b+ DCs in the lung are also highly susceptible to apoptotic death (13). IRF4 binds the CCR7 promoter in in vitro generated DCs (51), and has been suggested to regulate the migration of dermal CD11b+ DCs to draining LN (52), however we found that Irf4-deficient CD103+ CD11b+ DCs expressed similar levels of CCR7 as their wildtype counterparts following culture in vitro (10), suggesting that the marked reduction in these cells in MLN compared with the small intestinal LP is not a result of reduced CCR7-dependent migration. Interestingly, a recent study identified IRF4 as a key regulator of the metabolic state of activated CD8+ T cells (53). More specifically IRF4 was important for lymphocytes to switch to aerobic glycolysis, which is necessary for high-energy functions such as proliferation, cytokine production, or general cellular activation (53). Given the presence of continuous activation signals in the gut (e.g. bacterial and food products), it seems plausible that IRF4 shapes the metabolic status of CD103+ CD11b+ DCs to withstand the demand for highenergy functions in the gut and to sustain the continued need for activation and migration. In this regard, TLR stimulation was recently shown to switch on aerobic glycolysis in DCs (54) raising the possibility of a cross-talk between IRF4 and the sensing of bacteria. Finally IRF4 was recently shown to regulate the expression of genes associated with the MHC-II processing and presentation machinery in DCs (51), indicating that this TF also plays a subsequent role in regulating the functional activity of DCs. Notch2 The Notch signaling pathway is highly conserved among species and within the hematopoietic system regulates the © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014


commitment of lymphoid progenitors to different lineages (55). Notch receptors (e.g. Notch2) interact with ligands of the Delta-like or Jagged family and upon cleavage translocate to the nucleus where they interact with activating TFs such as RBP-J and induce the expression of generic as well cellspecific genes (56). Notch2 is critical for the development of splenic CD11b+ DCs (57), particularly for a subset that expresses high levels of the surface marker ESAM (58). In the LP and MLN, loss of Notch2 or RBP-J results in an almost complete loss of CD103+CD11b+ DCs but has no effect on the CD103+CD11b DC subset (59) suggesting a functional relationship between IRF4 and Notch2. Notch2 deficiency does not impact on the generation of pre-cDCs but it is currently believed to be required for the proper maturation or survival of CD103+CD11b+ DCs (59). Notably expression of Notch2 target genes is unaffected in Irf4-deficient intestinal DCs (authors unpublished observations) indicating that Notch2 acts upstream of IRF4. Notch2 levels have been shown to regulate the switch from oxidative phosphorylation to glycolysis in tumor cells (60), suggesting that it may also regulate DC metabolism. Potential role of additional transcription factors in intestinal DC development The TFs Bcl-6 and Blimp1 are transcriptional repressors to one another and are tightly associated with the differentiation of both B and T cells (61, 62). A recent study, however, demonstrated that lack of Bcl-6 expression led to a loss of intestinal CD103+ CD11b DCs, whereas Blimp1 deficiency correlated with a significant reduction in CD103+ CD11b+ LP DCs (11). Bcl-6 and Blimp1 additionally showed differential expression in these two intestinal DC subsets (11), perhaps reflecting their reciprocal repressive activities. Signal transducer of activation and transcription (STAT) proteins are a family of TFs integrating signals downstream of many cell surface receptors and display pleiotropic functions in the immune system. STAT3 and STAT5 have both been implicated in the differentiation of DCs (63). Although neither have been decisively linked to specific DC subsets, based on in vitro Flt3L cultures, STAT3 is likely to affect IRF8/BATF3dependent DCs (64). A recent study showed a decrease in the numbers of CD103-expressing DCs in the MLN of STAT5deficient mice (65); however, no further phenotyping of these cells was performed. It seems possible that the physical interactions of IRF4 and STAT3 in CD4+ T cells (66) may represent a conserved mechanism to integrate diverse IRF4dependent (or IRF8) gene transcription in DCs. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

The NFjB TF member RelB has been tightly associated with the development and function of DCs in vitro and in vivo (67). Early studies indicated that RelB binds and trans-activates the promoter of the Irf4 gene inducing its expression (68). RelB-dependent transcriptional regulation of IRF4 may be of physiological relevance as mice deficient in RelB (67) or the lymphotoxin-b receptor (LTbR) (69), which signals almost exclusively to induce RelB activation (70), display reduced numbers of IRF4 expressing CD8a CD11b+ DCs in the spleen. While the relevance of RelB and LTbR in intestinal DC homeostasis remains to be determined, in competitive BM reconstitution experiments, wildtypederived CD103+ CD11b+ DCs outcompeted their Ltbr-deficient counterparts both in the LP and MLN (59). These results are indicative that the LTbR-RelB axis may be important for intestinal DC homeostasis. In contrast to intestinal CD103+ DC subsets, the TFs required for the development of recently identified CD103 CD11b+ intestinal LP DCs remain to be fully assessed, primarily because most studies assessing mice with a DC deficiency in a given TF have not distinguished these cells from CD103 CD11b+ CD64+ monocyte/macrophages. This DC subset is not however reduced in the small intestine of mice with a DC deficiency in IRF4 (13), indicating that CD103 CD11b+ DCs are not related to CD103+ CD11b+ DCs or that IRF4 is only required subsequent to upregulation of CD103 on these cells. DC localization within the intestine The use of flow cytometry analysis to assess LP DC subset composition along the length of the intestine is complicated as tissues digests inevitably contain DC-rich microscopic SILT whose numbers vary with mouse strain and within different intestinal segments. With this caveat in mind, the proportion of each of the DC subsets described above appears to change dramatically along the length of the mouse intestine with CD103+ CD11b+ DCs representing the major DC subset in the murine small intestine LP, while in the colon, these cells are dramatically reduced, resulting in higher proportions of CD103+ CD11b and CD103 CD11b+ DCs (Fig. 1C) (71). The reason for these differences remains to be assessed but could reflect differential accumulation of distinct DC precursors between the two sites, tissue specific cues that induce the development of distinct DC subsets from the same DC precursor or to some extent reflect differences in the level of CD103 induction on SIRPa+ CD11b+ DCs. Whether similar variation in DC subset composition occurs along the length of the human intestinal tract remains to be determined.

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Detailed analysis of DC subset localization within the intestine is largely lacking, in part because of the multiple parameters required to unambiguously define these populations. CD103+ CD11c+ MHC-II+ DCs are present within villous LP of both the human and murine small intestine (10, 11, 18, 19, 72) (Fig. 2) and within SILT (18, 73). A variable number of LN resident CD11b , but not CD11b+, DCs express CD103 (74), and consistent with this, most CD103+ DCs in PP are CD103+ CD11b DCs. Thus, inclusion of PP in small intestinal digests results in enhanced proportions of CD103+ CD11b DCs (27, authors’ unpublished observations). Nevertheless, CD103+ CD11b DCs are present in small intestinal preparations of RORct-deficient mice, which lack PP and SILT, demonstrating that GALT are dispensable for their final differentiation (75). Further CD103+ DNGR-1+ DCs, that represent the equivalent of murine CD103+ CD11b DCs are readily identified diffusely dispersed throughout the human small intestinal LP (21). Whether CD103+ CD11b+ and CD103+ CD11b DCs share the same or distinct niches with the intestinal LP remains to be determined, as does the location of CD103 DCs. Intestinal lamina propria DC subsets in antigen uptake and T-cell priming Antigen uptake by intestinal DCs The intestinal immune system constantly samples and reacts to luminal contents of the intestine, forming the basis for

Fig. 2. CD103+ dendritic cells in the human intestinal mucosa. Immunofluorescence staining of the human small intestine, depicting CD103+ DCs (yellow) within the intestinal mucosa. The intestine is stained with antibodies to MHC-II (green) and CD103 (red) and the cell nuclei are counterstained with DAPI (white). Image courtesy of Dr Heli Uronen-Hansson.

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immune homeostasis in the gut. Initial findings from our group showed that, following intra-luminal injection of fluorescently labeled chicken ovalbumin (OVA) into exteriorized intestinal loops, antigen is primarily taken up by CX3CR1hi macrophages, while only a minor proportion of CD103+ DCs acquire antigen (72). OVA uptake by intestinal macrophages was independent of CX3CR1 as intestinal macrophages in CX3CR1GFP/GFP mice, that lack CX3CR1 expression, took up OVA as efficiently as their wildtype counterparts (72). Subsequent studies, using fluorescent microscopy and flow cytometric analysis, have confirmed that intestinal CX3CR1hi macrophages more efficiently acquire soluble luminal antigen when compared with CD103+ DCs (76, 77). In contrast to our findings, Mazzini et al. observed that uptake of soluble antigen by CX3CR1hi macrophages after intra-gastric antigen administration was dependent on CX3CR1 expression (77). The reason for these discrepant findings currently remains unclear, however T-cell priming in the MLN in response to orally administered soluble antigen is unaffected in CX3CR1-deficient mice (72), indicating that CX3CR1-dependent uptake of antigen is not required for the induction of T-cell responses to luminal soluble antigen. The mechanism by which intestinal DCs acquire soluble luminal antigen remains incompletely understood but likely involves several pathways. Farache et al. (76) demonstrated that CD103+ DCs can move across the intestinal basement membrane underlying the epithelium and may directly acquire soluble antigen from the lumen. Further, McDole et al. (78) identified sites of soluble antigen accumulation in the small intestinal epithelium, termed goblet cell-associated passages (GAPs) and demonstrated sampling of antigen from GAPs by underlying CD11c+ cells, which they identified as CD103+ DCs. Finally Mazzini et al. (77) recently suggested that CD103+ CD11b+ DCs may acquire antigenic material from CX3CR1hi macrophages via gap junctions formed by connexin proteins, in particular connexin 43, expressed on both cell subsets. CD4+ T-cell priming in MLN in response to luminal soluble antigen was however unaffected in mice lacking connexin 43 in CD11c+ cells, although iTreg conversion appeared marginally compromised. CD4+ T-cell priming in the MLN of these mice was proposed to be due to orally administered antigen directly reaching MLN in free form, however we and others have demonstrated that T-cell priming in MLN in response to similar concentrations of soluble luminal antigen does not occur in Ccr7-deficient mice indicating that such responses requires DC-mediated transport of antigen from the LP (79, 80). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014


Less is known about the mechanisms by which intestinal DCs gain access to particular antigen present in the gut lumen. In this regard, Hapfelmeier et al. (81) have suggested that invasion-deficient Salmonella co-localize predominantly with cells expressing CD11c and CX3CR1, markers associated with intestinal macrophages, in the intestinal LP. In contrast, Farache et al. (76) recently visualized a population of CD103+ DCs within the epithelial layer, able to sample bacteria directly from the intestinal lumen via dendrites extended between epithelial cells. The number of intra epithelial CD103+ DCs increased in response to luminal administration of Salmonella and CD103+ DCs within the epithelium were more likely to take up Salmonella compared with those in the LP. Moreover, they were able to take up invasive and noninvasive strains of Salmonella with equal efficiency (76). Collectively, these results suggest that intestinal DCs can acquire bacteria directly from the intestinal lumen and possibly also via macrophages. Further studies are required to clarify possibly redundancies between these mechanisms of particulate antigen uptake by intestinal DCs as well as potential differences between intestinal DC subsets in this process. DC migration from the intestine to draining MLNs Intestinal DCs are intimately linked with the initiation of adaptive immune responses to luminal antigen, which in turn is tightly associated with their ability to migrate to draining LNs where they present these antigens to na€ıve T cells. The observation that CD103+ DCs were selectively reduced in the MLNs but not LP of Ccr7-deficient mice provided the first indication that these cells represent a major LP-derived migratory DC population (79, 80). Consistent with this, we subsequently demonstrated in BrdU pulsechase experiments that CD103+ DCs accumulate with delayed kinetics in the MLN compared to the LP and as compared to CD103 DCs in the MLNs (18). Using confocal imaging to study afferent lymph vessels ex vivo, as well as flow cytometric analysis of intestinal lymph, we subsequently provided direct evidence that the majority of CD11c+ cells in intestinaldraining lymph are CD103+ (72). More recently, Cerovic et al. (75), assessing cannulated thoracic duct lymph from mice which had undergone mesenteric lymphadenectomy, showed that CD103+ CD11b and CD103+ CD11b+ DCs as well as a minor population of CD103 DCs constitutively traffic in intestinal lymph from the intestinal LP. Of DCs in collected lymph, 75–85% were CD103+ with CD103+ CD11b+ and CD103+ CD11b DCs present at an approximate ratio of 1.5:1. Notably CD103 DCs in intestinal © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

lymph could be further split into CD11b+ and CD11b populations, and the latter were missing in the lymph of RORctdeficient mice indicating that they may derive from GALT (75). Of note, we and others failed to detect monocyte-derived CX3CR1hi macrophages in intestinal-draining steady-state lymph, or in draining MLNs (72, 75), although these cells make up a major population of MHC-IIhi cells in the intestinal LP (see companion review by Mowat and coworkers). In contrast, Diehl et al. (82) recently reported the presence of CD103 CX3CR1hi macrophages in steady-state lymph. While the reason for these discrepant findings currently remains unclear it seems possible that the CX3CR1hi cells described by Diehl et al. in fact represent the CD103 CD11b+ DCs identified by Cerovic et al., which are CX3CR1int (75, 82). DC migration from the intestinal LP to draining MLNs can be dramatically enhanced by oral or intra-peritoneal administration of adjuvants including TLR agonists (10, 83, 84). Given the differential expression of pattern recognition receptors by DC subsets (85, 86) as well as noted indirect mechanisms by which TLRs can induce DC migration (83), it seems possible that mucosal exposure to different microbial products will result in the preferential migration of distinct LP-derived DC subsets to draining intestinal LNs that may have important consequences on downstream adaptive immune responses. Antigen presentation by intestinal DC subsets in MLNs MLN DCs can be divided into migratory DCs, arriving into the LNs from afferent lymph, and LN-resident DCs, that derive from circulating DC precursors and enter the LNs from the circulation. In the steady state, MLN-resident DCs can be distinguished from migratory subsets based on lower levels of MHC-II and higher levels of CD11c expression (10). These DCs, similar to resident DCs in other LNs, consist of two major DC subsets, CD11b+ and CD8a+ DCs (10). In contrast, migratory DCs in MLNs are characterized by higher expression of MHC-II. MHC-IIhi MLN DCs consist of CD103+ DCs, with roughly equal numbers of CD103+ CD11b+ and CD103+ CD11b DCs, and a minor population of CD103 DCs, most of which are CD11b+ (10). The subset composition of MHC-IIhi MLN DCs thus closely resembles that in the intestinal-draining lymph and LP (10, 13, 75). Thus, all three major DC subsets present in the intestinal LP migrate in lymph and enter the MLN in the steady state. As described above, intestinal-derived migratory DC subsets are thought to play crucial roles in the initiation of

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adaptive immune responses to luminal antigen in MLNs. Thus, Ccr7-deficient mice fail to mount T-cell responses to orally administered soluble antigen (79, 80), and we, and others, have demonstrated that bulk CD103+, but not CD103 DCs isolated from the MLNs of OVA-fed mice induce proliferation of OVA-specific CD4+ and CD8+ T cells ex vivo (18, 87). There is also evidence to suggest that migratory intestinal DCs carry particulate luminal antigen to the MLN. Initial studies demonstrated that after intra-gastric administration of commensal bacteria Enterobacter cloacae or Salmonella, live bacteria were found within CD11c+ CD11b+ cells in the MLN (88). Subsequently, following oral administration of Salmonella, bacteria were detected primarily within CD11c+ cells in the MLN (89) or CD103+ CD11b+ DCs (27). Voedisch et al. (89) further demonstrated that oral administration of the TLR7/8 agonist R848, or treatment with exogenous Flt3 ligand, which leads to increased numbers of DCs in MLNs, correlated with significantly increased numbers of Salmonella recovered from the MLNs, while Ccr7deficient mice had reduced numbers of Salmonella in MLNs compared to wildtype controls. Together these studies suggest key roles for LP-derived migratory DCs in the transport and presentation of luminal-derived antigen to adaptive immune cells in MLNs. As yet it remains unclear whether the different subsets of intestinal-derived migratory DCs are capable of processing and presenting the same or distinct luminal antigens to adaptive immune cells in MLNs and whether these subsets participate in priming distinct arms of the adaptive immune system in response to luminal antigen. Functionality of intestinal DC subsets Intestinal DCs in the generation of gut-homing T cells During their priming in LNs, naive T cells not only receive signals to proliferate and differentiate but also alter their expression of homing receptors, imparting on these cells the ability to migrate into extra-lymphoid tissues (90, 91). In early studies we, and others, demonstrated that the guthoming receptors chemokine receptor 9 (CCR9) and a4b7 integrin are more efficiently induced on T cells primed in intestinal-draining MLNs compared with T cells primed in the spleen or skin-draining LNs (92, 93). Further, PP or MLN DCs displayed an enhanced ability to induce CCR9 and a4b7 on responding T cells in vitro compared to DCs isolated from other sites (94–96). The ability to generate gut-homing T cells was not common to all MLN DCs but subsequently shown to be restricted to small intestinal LP-derived migratory CD103+ DCs (18, 80, 97). In these early studies,

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CD103+ MLN DCs were directly compared with CD103 MLN DCs, the majority of which represent migratory or LN resident DCs, respectively. However, a recent article by Cerovic et al. (75) provided evidence that the minor population of migratory CD103 DCs in intestinal lymph also have the ability to induce gut-homing receptors on responding T cells, suggesting this is a property of all small intestinalderived DCs. Indeed, T cells primed in the MLN of Ccr7deficient mice, which lack LP-derived migratory DCs, fail to express CCR9 following intra-peritoneal antigen administration (80). The ability of small intestinal DCs to efficiently generate gut-tropic T cells is tightly linked to their ability to generate the Vitamin A metabolite, retinoic acid (RA). RA binds the RA receptor (RAR) that together with the retinoid X receptor (RXR) forms a heterodimeric TF that recognizes RA response elements in target genes (98). RA induces expression of CCR9 and a4b7 on anti-CD3 activated T cells and addition of a pan-retinoic acid receptor inhibitor blocks the ability of intestinal DCs to induce gut-homing receptors on responding T cells in vitro (99, 100). Importantly, mice deficient in Vitamin A display reduced numbers of small intestinal T cells (100), and T cells primed in the MLN of such mice fail to express CCR9 and a4b7 (101), demonstrating a key role for RA in the generation of gut-tropic T cells in vivo. Studies to dissect the molecular mechanism by which RA induces CCR9 and a4b7 have shown that expression of both molecules is regulated at the transcriptional level. Thus, RARa can bind the promoter of the gene encoding the integrin-a4 subunit, Itga4 and induce its transcription (102). Moreover, the RARa/RXRa complex associates with NFATc2 forming a trimer that binds the promoter region of the Ccr9 gene at both NFAT sites and retinoic acid responsive elements (103). A more recent report showed that BATF-deficient mice had significantly reduced T cells in the gut and this correlated with low levels of CCR9 and a4b7 expression (104). Mechanistically, BATF appears important for the binding of RARa to the promoters of Ccr9 and Itga4, partly by preventing histone deacetylase-mediated inhibition of RARa (104). The generation of RA occurs through a two-step enzymatic oxidation of retinol. Retinol is converted to retinal by alcohol dehydrogenases and the subsequent oxidation of retinal to RA is mediated by retinal dehydrogenases, of which RALDH2, encoded by the aldh1a2 gene is highly expressed in small intestinal DCs. There has been considerable focus in trying to identify the factors in the small intestine that imprint local DCs with their enhanced capacity to metabolize Vitamin A © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014


(reviewed in 105). Several groups, including our own, recently demonstrated that small intestinal and MLN DCs in Vitamin A-deficient mice fail to express aldh1a2 and have lost their capacity to metabolize RA (101, 106–108) indicating a key role for RA in small intestinal DC imprinting. Using retinoic acid responsive reporter (DR5) mice, we found that DCs constitutively receive enhanced RA signals in the small intestine compared with the colon and further demonstrated that retinol levels are considerably higher in the murine small intestine and MLNs compared with other sites outside the liver (the main storage site for Vitamin A), potentially explaining why small intestinal DCs are preferentially imprinted with Vitamin A metabolizing activity (101). We further demonstrated that small intestinal DCs remain imprinted with Vitamin A metabolizing activity when Vitamin A is removed from the diet indicating that continual intake of dietary Vitamin A is not required for small intestinal DC imprinting. In this regard, we observed that bile, which drains directly into the duodenum, not only contains high levels of Vitamin A, but can also induce Vitamin A metabolizing activity in DCs in an RA-dependent manner (101). Collectively these findings suggest that Vitamin A derived from the diet and/or bile plays a central role in imprinting small intestinal DCs with Vitamin A metabolizing activity. Deficiency in the ubiquitin-modifying enzyme A20, which has been tightly linked with negative regulation of the NFjB pathway, results in overproduction of RA by BMderived DCs and a subsequent increased ability to induce CCR9 and a4b7 expression in T cells (109). The inhibitory properties of A20 are linked with its ability to function as both a de-ubiquitylation enzyme degrading K63 ubiquitin chains but also as a ubiquitin ligase catalyzing K48 ubiquitination (110). Using model cell lines, it has been shown that the expression levels of RARa and RXR and their cognate interaction can be partially regulated by ubiquitylation (111, 112), whereas in the liver alcohol dehydrogenase turnover is also linked to ubiquitin-mediated proteasomal degradation (113). Thus, A20 or similar modifying enzymes may regulate the function of the RARa/RXR complex or turnover of alcohol or retinal dehydrogenases. Alternatively, A20 may interfere with RA synthesis indirectly via regulation of the NFjB pathway. Despite these findings, direct evidence that migratory DCderived RA is critical for the generation of gut-tropic T cells in vivo is currently lacking. For example, we recently found that the generation of gut-tropic CD4+ T cells in MLNs is unaffected in mice lacking IRF4-dependent CD103+ CD11b+ DCs (10). Furthermore, at steady state, intestinal T cells in © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

Batf3-deficient mice, which lack CD103+ CD11b DCs, expressed normal levels of CCR9 and a4b7 on T cells (36). These results suggest that different migratory DC subsets may play redundant roles in providing RA to responding T cells or that additional cells within the MLN environment contribute as additional sources of RA. Regarding the former, mice lacking both intestinal CD103+ subsets were recently shown to display a marked reduction in CCR9 expression on FoxP3+ Tregs, although other T-cell populations were not assessed (37). Regarding the latter, LN transplantation experiments have suggested that MLN stromal cells may contribute to CCR9 and a4b7 induction on Ag-specific T cells (108, 114). Thus, Hammerchmidt et al. (114) observed that when peripheral LNs were transplanted to the gut mesenteries they failed to support the generation of gut-homing T cells, despite entry of small intestinal LP-derived DCs, while Molenaar et al. (108) demonstrated that when MLNs were transplanted into the popliteal fossa, and thus lacking intestinal-derived DCs, they could support a4b7 but not CCR9 induction on responding T cells. Together these results indicate a complex interplay between intestinal-derived migratory DCs and MLN stromal components in the generation of gut-tropic T cells in vivo. Intestinal DCs in iTreg generation and the development of oral tolerance The intestinal immune system is continually exposed to foreign antigens derived from our diet and the huge numbers of microbes that reside within the intestinal lumen. Maintenance of intestinal homeostasis critically depends upon the immune system’s ability to tolerate exposure to such antigens and prevent responses that could result in a state of overt inflammation. One of the characterized responses of the intestinal immune system to food antigen is the phenomenon of oral tolerance, by which oral administration of innocuous antigen leads to a state of local and systemic immunological unresponsiveness to subsequent administration of the same antigen (115, 116). Early studies in mice lacking PPs, MLNs, or both, as a result of in utero manipulation of the lymphotoxin pathway, suggested that MLNs are necessary and sufficient for the generation of oral tolerance (117). Subsequently, Worbs et al. (79) elegantly demonstrated that surgical removal of MLNs in wildtype mice prevents establishment of oral tolerance, and also found an absence of oral tolerance induction in Ccr7deficient mice. The latter was associated with a complete lack of T-cell activation in the MLN of Ccr7-deficient mice following oral but not intravenous antigen administration. Collectively these studies suggest a key role for DC mediated antigen

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transport from the intestinal LP to the MLNs in the establishment of oral tolerance. The MLN is a site of enhanced FoxP3+ iTreg generation (87, 118–120) and recent findings have suggested a key role for FoxP3+ iTreg generation in the establishment of oral tolerance (121, 122). In this regard, CD103+ DCs from the small intestinal LP and MLN appear particularly potent in converting naive T cells into FoxP3+ iTregs in vitro (87, 118– 120), a property linked initially to their enhanced capacity to generate RA that acts as a co-factor for TGFb to enhance iTreg conversion (87, 118–120). Nevertheless, mice kept on a Vitamin A-deficient diet or RARa-deficient mice display normal numbers of small intestinal FoxP3+ Tregs (123, 124). More recently, CD103+ DCs were shown to express high levels of the integrin avb8, an activator of latent TGFb, and intestinal CD103+ DCs from mice whose DCs are deficient in either of these integrin subunits displayed a reduced capacity to generate iTregs in vitro (125). Further, these mice fail to generate iTregs in MLNs in response to orally administered antigen (125, 126). Molecular insights into the ability of DCs to induce iTreg are largely missing. However, a recent study showed that deletion of the mitogen activated protein kinase p38a in DCs leads to impaired oral tolerance, which was associated with decreased iTreg generation and gut-homing receptor induction in vitro and in vivo. Mechanistically, p38a was required for optimal expression of aldh1a2 by CD103+ MLN DCs. Further, p38a-deficient CD103+ MLN DCs expressed significantly lower levels of Tgfb2 and Itgb8 and in vitro conversion of iTregs by CD103+ MLN DCs was found to be dependent on TGF-b2 (127). Moreover, expression of B7 family members of co-stimulatory molecules programmed death ligand-1 (PDL1) and PDL2 by MLN DCs has also been implicated in the induction of oral tolerance via regulation of the Foxp3+ iTreg compartment (128). Complementary to their importance in oral tolerance to soluble antigens, intestinal DCs have also been implicated in the generation of tolerance to the microbiota at least partially via a TNF receptor associated factor-6 (TRAF6)-dependent mechanism that regulates IL-2-mediated generation of iTreg cells, as shown in mice lacking TRAF6 expression in their CD11c compartment (129). While the studies described above implicate an important role for LP-derived migratory DCs in iTreg generation in MLNs, as well as in the establishment of oral tolerance, it remains unclear whether this is a property of a particular LPderived migratory DC subset or of all intestinal-derived DCs. Our preliminary findings suggest that intestinal CD103+ CD11b DCs but not CD103+ CD11b+ DCs express avb8

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integrin (author’s unpublished observation). Nevertheless, studies assessing the ability of intestinal- or MLN-derived CD103+ CD11b and CD103+ CD11b+ DCs to drive iTreg conversion in vitro have produced conflicting results (71, 127). Further, Batf3-deficient mice, which lack intestinal CD103+ CD11b DCs, have normal numbers of intestinal FoxP3+ Tregs (36) as do mice with a DC deficiency in IRF4, which display a 90% reduction in CD103+ CD11b+ MLN DCs (10), or mice lacking intestinal and MLN CD103+ CD11b+ DCs (37). However, it was recently demonstrated that mice lacking both intestinal CD103+ DC subsets have reduced numbers of intestinal FoxP3+ Tregs indicating potential redundancy between these subsets in FoxP3+ Treg generation and/or homing (37). While oral tolerance induction in these mice has yet to be assessed, Mazzini et al. recently found that mice whose DCs lack expression of the gap junction protein connexin 43 are compromised in their ability to generate oral tolerance and proposed this to be due to a reduced capacity of intestinal CD103+ CD11b+ DCs to acquire antigen from intestinal macrophages (77). This hypothesis however does not fit with our preliminary findings that mice with a DC deficiency in IRF4 retain their ability to develop oral tolerance (Persson et al., manuscript in preparation). Induction of CD4+ T-cell immunity The finding that small intestinal CD103+ DCs could efficiently promote iTreg differentiation in vitro lead to initial proposals that these cells represent tolerogenic DCs, and unfortunately CD103 expression alone has occasionally been used as a marker to define DCs with tolerogenic activity. It is now clear that intestinal migratory DCs however can also promote the generation of effector Th cells, and there is emerging evidence that LP-derived migratory DC subsets display some specialization in the generation of distinct Th cell subsets. Th17 cell differentiation Recent findings by us, and others, have suggested a key in vivo role for intestinal CD103+ CD11b+ DCs in intestinal Th17 homeostasis (10, 13, 37, 59). Thus, mice with a selective reduction or absence of intestinal-derived CD103+ CD11b+ DCs, as observed in mice with a DC-specific deletion in IRF4 or Notch2, or human Langerin-DTA mice, display significantly reduced numbers of Th17 cells in the small intestine, colon, and MLN (10, 13, 37, 59). Notably however, mice specifically lacking MHC-II on CD103+ CD11b+ DCs have normal numbers of intestinal Th17 cells © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014


(37), indicating that cognate interactions between these cells and CD4+ T cells are not required for maintaining intestinal Th17 cell homeostasis. Further, a role for IRF4-dependent DCs in Th17 induction appears not to be restricted to the intestine as mice lacking IRF4 in DCs also have significantly reduced numbers of Th17 cells in the lungs and in lungdraining LNs in the steady state or after infection with the fungal pathogen Aspergillus fumigatus (13). Using an OVA-specific TCR transgenic (OT-II) adoptive transfer model, we further demonstrated that intestinal migratory + + CD103 CD11b DCs are critical for driving Th17 cell differentiation in draining MLNs following intra-peritoneal administration of OVA in combination with aCD40 and LPS (10), suggesting that deficient Th17 cell generation may, at least in part, underlie reduced intestinal Th17 cell numbers in these mice. Mechanistically, the capacity of activated intestinal CD103+ CD11b+ DCs to induce Th17 differentiation in MLNs appears to be linked, at least in part, to their production of IL-6 (10, 71). Thus, in vitro or in vivo activated intestinal- and MLN-derived CD103+ CD11b+ DCs produce high levels of IL-6 compared with CD103+ CD11b DCs and display an enhanced capacity to induce IL-6-dependent Th17 differentiation in vitro (10, 71). Further, we found that

the differentiation of na€ıve OT-II cells to Th17 cells in MLNs following intra-peritoneal administration of OVA in combination within aCD40 and LPS was IL-6 dependent (10), and an absence of CD103+ CD11b+ DCs in MLNs lead to significantly reduced levels of DC-derived IL-6 (10) (Fig. 3). In vitro, ligands for several TLRs including TLR2, TLR5, and TLR9 induce IL-6 production by CD103+ CD11b+ DCs and increase their capacity to promote Th17 differentiation (10, 75, 86), indicating that inflammatory signals are required for their ability to drive Th17 cell generation. Consistent with this, CD103+ CD11b+ MLN DCs from LPS and aCD40 treated, but not from untreated mice, induce Th17 differentiation in vitro (10). The range of in vivo signals that imprint a Th17 polarizing capacity in CD103+ CD11b+ DCs however currently remains unclear, but is unlikely to be restricted to Myd88-dependent TLR signaling. For example, serum amyloid A, which is induced after segmented filamentous bacteria (SFB) colonization, induces IL-6 production in intestinal DCs and imprints these cells with Th17 differentiation capacity (130). Further, Myd88-deficient mice have been reported to display normal numbers of intestinal Th17 cells (131, 132), although conflicting data have also been presented (133).

Intestinal lamina propria

Intestinal lumen

Activating signals

CD103 + CD11b+ IL-23 ?

Th17

ILC3

CD103 + CD11b +

Mesenteric Lymph Node

Naive T cell

Th17

IL-6

Fig. 3. Role of CD103+ CD11b+ lamina propria dendritic cells in the regulation of intestinal immunity. CD103+ CD11b+ dendritic cells (DCs) in the lamina propria (LP) have been linked with regulating both adaptive and innate immunity. Exogenous signals from TLRs, cytokines, and the microbiota have been shown to activate production of IL-23 and IL-6 by CD103+ CD11b+ DCs. Although somewhat controversial, several studies have indicated that CD103+ CD11b+ DCs are an important source of IL-23 in the intestine, that can induce IL-22 production in group 3 ILCs (ILC3) and may potentially support intestinal Th17 cell homeostasis and functionality. Following their activation in the LP, CD103+ CD11b+ DCs migrate via afferent lymph to draining mesenteric lymph nodes, where they provide an important source of IL-6 to drive naive T cell differentiation into Th17 cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014

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Th1 and Th2 cell differentiation In contrast to Th17 cell differentiation, it currently remains unclear whether intestinal migratory DC subsets have specialized roles in the generation of Th1- and Th2-type mucosal responses. In vitro studies indicate that both CD103+ CD11b and CD103+ CD11b+ SI DCs can drive Th1 polarization (86), and recent findings by Cerovic et al. (75) assessing the activity of DCs isolated from intestinal afferent lymph suggested that migratory CD103 CD11b+ DCs are superior to both CD103+ CD11b and CD103+ CD11b+ migratory DCs in inducing IFN-c production by T cells. The role of intestinal CD103+ CD11b and CD103 CD11b+ DCs in driving Th1 responses in vivo has yet to be assessed; however, mice lacking IRF4-dependent DCs have normal or slightly increased numbers of intestinal Th1 cells in the SI and colonic LP (10, 13, 37), and these DCs are dispensable for the induction of antigen-specific IFN-c-producing T cells in MLNs (10). Regarding Th2 differentiation, in early reports it was demonstrated that CD11b+ PP-derived DCs were more efficient than their CD8a+ counterparts at inducing IL-4 production by T cells in vitro, although this also correlated with induction of enhanced CD4+ T-cell proliferation by this subset (134). While the role of LP-derived migratory DC subsets in driving intestinal Th2 responses in vivo has yet to be addressed, recent studies have demonstrated a key role for IRF4-dependent DCs in driving Th2 responses in the skin (135, 136) as well as in house dust mite-induced allergic airway inflammation (137). These findings suggest that assessment of the role of IRF4dependent intestinal DCs in immune responses to intestinal Th2 polarizing pathogens merits investigation. Potential roles for intestinal DC subsets within the intestinal LP In addition to their role in antigen transport, T-cell priming and T-cell differentiation in intestinal-draining LNs, it remains likely that intestinal DC subsets have immune-modulatory functions during their residence within the intestinal LP. Although controversial, one of the major cytokines suggested to be provided by intestinal DCs and in particular intestinal CD103+ CD11b+ DCs is IL-23. Thus, Satpathy et al. (59) demonstrated that mice with a DC-specific deletion in Notch2, which as discussed above, lack most intestinal CD103+ CD11b+ DCs, succumb to infection with the attaching-and-effacing bacteria, Citrobacter rodentium, which they linked to a reduction in IL-23 production and a subsequent lack of IL-22 production by group 3 innate lymphoid cells (ILCs). In addition, intravenous administration of

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recombinant bacterial flagellin induces rapid production of IL-22 in the intestinal mucosa that is dependent on TLR5/ MyD88 expression by DCs (138). Utilizing a combination of in vivo depletion experiments, the authors showed that DCs in general are necessary for the induction of IL-22 in the LP, a finding that was later confirmed (37). Based on higher expression of IL-12p40 and IL-23p19, it was suggested IL-23 production by CD103+ CD11b+ DCs might be driving the IL-22 response (138), the major source of which was ILCs (139), again suggesting a potential link between CD103+ CD11b+ DCs and the functionality of ILCs (Fig. 3). However, a more recent study in human LangerinDTA mice, which selectively lack CD103+ CD11b+ DCs, demonstrated that the response to flagellin was independent of this population (37), suggesting a potential redundancy between DC subsets. The same study also concluded that CD103+ CD11b+ DCs were not important for clearing Citrobacter rodentium (37) in marked contrast to mice that lack Notch2 in the CD11c compartment (59). While the reason for these discrepant findings currently remains unclear, in the latter study, the investigators only measured weight-loss after Citrobacter rodentium and did not correlate their results with bacterial burden, ILC activation, or IL-22 production. Nevertheless, it remains plausible that Notch2 deficiency may affect the functionality of additional DC subsets (e.g. the CD103 CD11b+ subset in the colon) or unrelated CD11cexpressing cells that may in turn regulate the ILC response to Citrobacter rodentium. Despite these somewhat conflicting findings, collectively these data suggest that IL-23 produced by LP DCs contribute to driving IL-22 responses in the mucosa. Moreover, given the role of IL-23 in promoting the expansion and functionality of Th17-committed cells (140, 141), LP DC-derived IL-23 may also contribute to intestinal Th17 cell homeostasis and/or function (Fig. 3). Summary Intestinal DCs play important and diverse roles in the regulation of T-cell responses. Thus, DCs promote effector T-cell differentiation, induce oral tolerance, and direct the migration of activated T cells. Recent evidence has demonstrated the presence of distinct intestinal DC sub-populations with different developmental requirements and there is currently considerable effort in trying to understand the functional diversity of each subset. The generation of in vivo models that lack a specific intestinal DC subset, now allows us to dissect the individual and combined contribution of intestinal DC populations to immunity and tolerance and to © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 260/2014


correlate their relevance with their human counterparts, particularly in disease settings such as chronic intestinal inflammation or food allergy. The complete characterization of the

genes and environmental factors that regulate DC subset development and functionality will likely provide us with novel therapeutic targets in these diseases.

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Immunity and Tolerance Induced by Intestinal Mucosal Dendritic Cells.

Mucosal dendritic cells shape mucosal immunity.

Regulation of intestinal immune system by dendritic cells.

Contrasuppressor cells in mucosal immunity.

A role for intestinal T lymphocytes in bronchus mucosal immunity.

Intestinal dendritic cells.

MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium.

Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.

Wnt signaling in dendritic cells: its role in regulation of immunity and tolerance.

Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

Gut barrier structure, mucosal immunity and intestinal microbiota in the pathogenesis and treatment of HIV infection.

The dual role of nod-like receptors in mucosal innate immunity and chronic intestinal inflammation.

Altered inactivation of commensal LPS due to acyloxyacyl hydrolase deficiency in colonic dendritic cells impairs mucosal Th17 immunity.

Development of intestinal mucosal immunity in fetal life and the first postnatal months.

CD8αα⁺ innate-type lymphocytes in the intestinal epithelium mediate mucosal immunity.

Dietary Apostichopus japonicus enhances the respiratory and intestinal mucosal immunity in immunosuppressive mice.

Probiotic Bacillus pumilus SE5 shapes the intestinal microbiota and mucosal immunity in grouper Epinephelus coioides.

Mucosal immunity and the microbiome.

Metabolic reprogramming in macrophages and dendritic cells in innate immunity.

Activation-Induced TIM-4 Expression Identifies Differential Responsiveness of Intestinal CD103+ CD11b+ Dendritic Cells to a Mucosal Adjuvant.

HIV and mucosal immunity.

Dendritic Cells in Tolerance and Immunity against Pathogens.

Mucosal immunity and vaccination.

Human cytomegalovirus tropism for mucosal myeloid dendritic cells.