Securing the immune tightrope: mononuclear phagocytes in the intestinal lamina propria

The intestinal tract is the largest surface of the human body, covering an area of approximately 100 m2, and is lined by a single layer of columnar epithelial cells, which forms the barrier between the gut lumen and the host connective tissue. In addition to its constant exposure to dietary and environmental antigens, the intestine har-bours an estimated 1014 commensal bacteria, compris-ing around 1000 different species1. It is in this unique, complex and vulnerable setting that inflammatory bowel diseases (IBDs), such as Crohn’s disease and ulcerative colitis, occur in genetically predisposed individuals2. Furthermore, the intestinal tract is a major site of patho-gen entry3. To cope with these challenges, mammals have evolved robust balanced immune mechanisms that enable protection against invading pathogens, but prevent detrimental collateral damage by inflammatory responses against the commensal microbiota and food antigens.

Recent findings have indicated that intestinal mono-nuclear phagocytes, comprising dendritic cells (DCs) and macrophages, are crucial for maintaining intestinal homeostasis. These cells are strategically positioned in the intestine, residing in the gut associated lymphoid tissue (GALT) and are also dispersed throughout the subepithelial lamina propria (FIG. 1; see Supplementary information S1 (movie)). Understanding how intestinal mononuclear phagocytes interact with the microbiota, lymphocytes and the epithelium to shape intestinal immune responses will be important for identifying

therapeutic targets for Crohn’s disease and ulcerative colitis. In addition, such studies can provide valu-able insights into the orchestration of extra-intestinal immune responses. Recent reviews have comprehen-sively covered the mononuclear phagocytes of the GALT4–7; we therefore focus our attention on intesti-nal lamina propria mononuclear phagocytes, describ-ing their roles in both inflammatory and tolerogenic immune responses. We describe the recent insights into the ontogeny and function of mouse intestinal mono-nuclear phagocytes, and discuss their interaction with the microbiota. In addition, we give an overview of the current understanding of mononuclear phagocytes in the human intestinal lamina propria and discuss the contribution of these populations to the pathology of IBDs.

Mononuclear phagocytes in the mouse intestineThere have recently been large advances in our under-standing of the organization of the mononuclear phago-cyte system, in particular, with respect to the origins and functions of distinct subpopulations8. However, incon-sistency in the definition of intestinal mononuclear phagocytes between various research groups has gener-ated confusion, particularly for the outside observer of the field. In the following sections, we try to clarify this topic, by summarizing the available data on the ontog-eny, anatomical location, phenotypes and functions of intestinal mononuclear phagocytes.

*Gastroenterology and Hepatology Institute, Tel Aviv-Sourasky Medical Center, Tel Aviv, Israel.‡Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.Correspondence to S.J.e-mail: [email protected]:10.1038/nri2778

Securing the immune tightrope: mononuclear phagocytes in the intestinal lamina propriaChen Varol*, Ehud Zigmond*‡ and Steffen Jung‡

Abstract | The intestinal landscape comprises the host’s own tissue and immune cells, as well as a diverse intestinal microbiota. Intricate regulatory mechanisms have evolved to maintain peaceful coexistence at this site, the breakdown of which can result in devastating inflammatory bowel diseases (IBDs). Mononuclear phagocytes promote both innate and adaptive immune responses in the gut and, as such, are essential for the maintenance of intestinal homeostasis. Here, we review the origins and functions of the mononuclear phagocytes found in the intestinal lamina propria, highlighting the problems that have arisen from their classification. Understanding these cells in their physiological context will be important for developing new therapies for IBDs.

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Nature Reviews | Immunology

a

b

c

Peyer’s patchesGroups of lymphoid nodules present in the small intestine. 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.

Typically, studies on mononuclear phagocytes have focused on cells resident in lymphoid organs, such as the spleen or lymph nodes, rather than on populations present in peripheral tissues. mononuclear lymphocytes in the peripheral tissues have been notoriously diffi-cult to isolate and can only be retrieved after extensive enzymatic digestions. Recently, however, mono nuclear phagocytes from the lamina propria of the mouse small and large intestine have gained much attention. Classifying these subsets according to surface marker profiles has been a challenge in many of these studies. However, considerable technological progress, including advances in cell isolation protocols and in vivo imaging techniques, have enabled a better characterization of lamina propria mononuclear phagocyte subsets.

Phenotype of lamina propria mononuclear phagocytes. mononuclear phagocytes in the intestinal lamina propria have been subdivided based on the differential surface expression of the integrins CD11c (also known as αX integrin), CD11b (also known as αm integrin) and CD103 (also known as αe integrin), as well as by the expression of CX3C-chemokine receptor 1 (CX3CR1), the receptor for CX3C-chemokine ligand 1 (CX3CL1; also known as

fractalkine)9–17. Accordingly, four major populations of intestinal mononuclear phagocytes have been identi-fied (TABLE 1), although the exact classification of these different cell types as either ‘macrophages’ or ‘DCs’ has been controversial (BOX 1). In mice, CD11c has long been used as a bona fide DC marker; however, macrophages can also express CD11c. For the purposes of this Review we classify CD103+CD11c+ mononuclear phagocytes as DCs and CD11c–F4/80+ mononuclear phagocytes as macrophages. We further subdivide DCs into two subpopulations: CD11chiCD11b+CD103+CX3CR1– cells (we refer to these as CD11b+CD103+ DCs) and CD11chiCD11b–CD103+CX3CR1– cells (we refer to these as CD11b–CD103+ DCs) (TABLE 1). Finally, a population of CD11cmidCD11b+CD103–/lowCX3CR1+cells has been described with both DC and macrophage-like charac-teristics (BOX 1). Therefore, we refer to these as CX3CR1+ mononuclear phagocytes.

CX3CR1+ mononuclear phagocytes and CD11b+CD103+

DCs are the major mononuclear phagocyte popula-tions found in the intestinal lamina propria. By contrast, CD11b–CD103+ DCs resemble classic DCs present in lymphoid organs. CD11b–CD103+ DCs are generally rare in lamina propria cell preparations but prevail in the GALT, including Peyer’s patches, colonic lymphoid aggregates and small intestinal isolated lymphoid follicles. It is likely, therefore, that their detection in single cell sus-pensions derived from intestinal lamina propria tissue is due to contaminating GALT.

Intestinal lamina propria macrophages express vari-able levels of CX3CR1, and scanning electron micros-copy has confirmed the heterogeneity of these cells10. Although we classify CD11c– cells as macrophages, others have reported that only CD11cmidCD11bmidmHC class IIhi cells exhibit genuine ultrastructural macro-phage characteristics, such as phagocytic vacuoles, whereas CD11c–/lowCD11bhiCD103– cells may actually be intestinal eosinophils with uniquely shaped nuclei and eosinophilic granules10,12.

Ontogeny of lamina propria mononuclear phagocytes. Recent progress in our understanding of the origins and ontogeny of intestinal mononuclear phagocytes has helped us to appreciate the overall organization of the mononuclear phagocyte system8. Pulse–chase experiments with bromodeoxyuridine have revealed a high turnover rate for intestinal lamina propria mono-nuclear phagocytes, in particular, for both CD103+ DC subsets18. moreover, studies using small intestinal tissue transplants established that the lamina propria DC pool is maintained by circulating precursors from the blood, rather than local tissue-resident precursors18.

The detailed elucidation of the origins of intestinal mononuclear phagocytes has profited from insight into lymphoid organ mononuclear phagocytes, in particu-lar, lymphoid organ DCs. An important breakthrough came from the identification of bone marrow-resident myeloid precursors, the macrophage–dendritic cell pro-genitors (mDPs), which have lost granulocyte potential and are committed to give rise to monocytes, classical splenic DCs and macrophages19,20. Together with the

Figure 1 | Imaging intestinal CX3CR1+ lamina propria mononuclear phagocytes. a | An overhead view of the luminal surface of the colon of mice that express green fluorescent protein (GFP) under the control of the CX

3C-chemokine receptor 1 (CX

3CR1)

promoter (CX3CR1–GFP mice) showing the honeycomb-like arrangement of CX

3CR1+

lamina propria mononuclear phagocytes surrounding the crypts. Note the lymphoid aggregate (original magnification x100). b | Side-on view of villi in the small intestinal ileum of CX

3CR1–GFP mice showing CX

3CR1+ lamina propria mononuclear phagocytes (original

magnification x100). c | Intravital two-photon microscopic image of small intestinal villi in mice that have been depleted of CD11c+ cells and reconstituted with monocyte grafts yielding red and green fluorescent CX

3CR1+ lamina propria mononuclear phagocytes. The

fact that the villi exclusively host red or green cells shows that, at least in this experimental system, monocytes can reconstitute the CX

3CR1+ lamina propria mononuclear phagocyte

compartment by clonal expansion (original magnification x40). For details of the experimental procedure, see REF. 14. Image in part c is reproduced, with permission, from REF. 8 © (2010) American Association for the Advancement of Science.

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http://www.uniprot.org/uniprot/Q61549

Isolated lymphoid follicles Dynamic aggregates of lymphoid cells found in the mouse small intestinal lamina propria; similar lymphoid aggregates have also been identified in the mouse colon. Isolated lymphoid follicles have germinal centres and an overlying follicle-associated epithelium containing microfold (M) cells specialized for antigen uptake, but they lack discrete T cell zones. They are formed de novo in adult animals in response to commensal organism-derived stimuli and, although their function is not completely clear, they may be inductive sites of immunity in the intestinal lamina propria.

recent identification of a dedicated precursor of classi-cal lymphoid tissue DCs21, it is now established that the developmental pathways of monocytes and DCs diverge early in the bone marrow, downstream of mDPs8.

Using a diphtheria toxin-based conditional cell-ablation strategy and complementary precursor cell engraftment, we have shown that Ly6Chi monocytes fail to differentiate into classical splenic DCs but give rise to CX3CR1+ small intestinal lamina propria mononuclear phagocytes20. Subsequently, we and others elucidated the in vivo genealogy of small intestinal and colonic lamina propria DC subpopulations14,15 (FIG. 2). Both GALT CD11b–CD103+ DCs and CD11b+CD103+ lamina propria DCs arise from classic DC precursors, without monocytic intermediates, in a developmental path-way governed by FmS-related tyrosine kinase 3 ligand (FLT3L). Generation of CD11b+CD103+ lamina propria

DCs is further controlled by granulocyte–macrophage colony stimulating factor (Gm-CSF). Classic DC pre-cursors show negligible expression of the receptor for Gm-CSF (K. Liu, personal communication), suggest-ing the existence of an additional intermediate that is committed to this lamina propria DC subset. That CD11b–CD103+ and CD11b+CD103+ lamina propria DCs are distinct entities is further supported by the fact that the generation of CD11b–CD103+ lamina propria DCs is uniquely affected by deficiencies in inhibitor of DnA-binding 2 (ID2), interferon regulatory factor 8 (IRF8) and basic leucine zipper transcription factor, ATF-like 3 (BATF3)15,22,23. As such, CD11b–CD103+ lamina propria DCs are similar to classical lymphoid organ resident CD8α+ DCs8. However, CX3CR1+ lamina propria mononuclear phagocytes do not derive from classic DC precursors but are, as established through

Table 1 | Intestinal lamina propria mononuclear phagocyte subsets in the mouse

Subset Phenotype Precursor cell (growth factor requirements)

Functions described in the small intestine*

Functions described in the large intestine*

CX3CR1+

mononuclear phagocytes

CD11cmid CD103–/low F4/80+ CD11b+

CD14+

CD70+

CD172a+ MHC class IIhi

M-CSFR+

CD80+

CD86+

Ly6Chi monocytes

(M-CSF)

• Sense or sample lumen with transepithelial dendrites14,16,24–27

• Do not migrate to mesenteric lymph nodes17

• Poorly stimulate naive T cells17

• Induce TH1 and T

H17 cells in vitro10

• Regulate T cell-independent IgA class-switching54

• Mediate IL-23-dependent granuloma formation102

• Maintain local FOXP3+ TReg

cell pool35

• Induce colonic TH17 cells following

activation by commensal-derived ATP36

• Promote TNF-dependent colitis14

• Selectively express CD172a and are reduced in colitis-resistant CD47-deficient mice84

• Mediate IL-23-dependent granuloma formation102

CD103+CD11b+ DCs

CD11chi CD14–

CX3CR1–

CD70– MHC class IIhi M-CSFRlow

F4/80–/low CD80+

CD86+

TLR5+

CCR7+

CD172a–

Classic DC precursor

(FLT3L and GM-CSF)

• Carry antigen to mesenteric lymph nodes in a CCR7-dependent manner11,15,17,30,31

• Promote generation of functional TReg

cells 13

• Induce tolerance to orally administrated antigens30

• Imprint gut tropism (expression of CCR9 and α4β7 integrin on T cells)13,18,32

• Induce TH17 cells (TLR5-dependent)12

• Promote T cell-independent IgA class-switching (TLR5-dependent)12

• Not described

CD103+CD11b–DCs CD11chi CX3CR1– CD14– CD8+

MHC class IIhi CD80+ CD86+ F4/80– M-CSFR–


(FLT3L)

• Show phenotypic and ontogenic characteristics of lymphoid organ classic DCs14,15

• Most prevalent in Peyer’s patches and isolated lymphoid follicles15,22

• Absent in IRF8–/–, ID2–/– and BATF3–/– mice (as are CD8+ classic DCs)15,22,23

• Show phenotypic and ontogenic characteristics of lymphoid organ classic DCs14

CD11b+F4/80+ macrophages

CD11c–/low CX

3CR1– /+

MHC class II+ CD80+ CD86low CD103– PDL1+

Unknown • Secrete IL-10 spontaneously and promote the differentiation of FOXP3+ T

Reg cells 10

• Phagocytose and eradicate bacteria but are refractory to TLR stimulation47–49

• Promote colonic wound healing41

• TREM2+ macrophages promote colonic epithelial regeneration42

BATF3, basic leucine zipper transcription factor, ATF-like 3; CCR, CC-chemokine receptor; CX3CR1, CX

3C-chemokine receptor 1; DC, dendritic cell; FLT3L,

FMS-related tyrosine kinase 3 ligand; FOXP3, forkhead box P3; GM-CSF, granulocyte–macrophage colony stimulating factor; ID2, inhibitor of DNA-binding 2; IL, interleukin; IRF8, interferon regulatory factor 8; M-CSF, macrophage colony-stimulating factor; PDL1, programmed cell death ligand 1; R, receptor; T

H, T helper;

TLR, Toll-like receptor; TNF, tumour necrosis factor; TReg

cell, regulator T cell; TREM2, triggering receptor expressed on myeloid cells 2. *There is currently no evidence for distinct functions of the different mononuclear phagocyte subsets in the two tissue compartments. Classifications of the subsets have in some cases been deduced by the authors according to the provided data.

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Bromodeoxyuridine A thymidine analogue that can be incorporated into DNA during DNA replication. Treatment with bromodeoxyuridine enables the detection of cells that are dividing or have recently divided.

Ly6Chi monocytesA subset of blood monocytes that is derived from MDPs in the bone marrow and emigrates in a CCR2-dependent way to the blood. Ly6Chi monocytes can home back to the bone marrow in the steady state and differentiate into Ly6Clow monocytes. Following pathogen challenge, Ly6Chi monocytes can give rise to splenic TIP-DCs. Furthermore they are the precursors of CX3CR1+ mononuclear phagocytes in the intestinal lamina propria. In addition, Ly6Chi monocytes can contribute to tissue remodelling and healing processes.

adoptive transfer experiments, the progeny of circulat-ing Ly6Chi monocytes, with their differentiation driven by macrophage colony-stimulating factor (m-CSF; also known as CSF1)14,15.

The origin of lamina propria macrophages has not been experimentally addressed but, as is the case with other tissue macrophages, their steady-state maintenance probably involves self renewal through local proliferation and limited input from circulating precursors8.

Functions of intestinal mononuclear phagocytesThe subpopulations of mononuclear phagocytes have been found to differ in terms of their ability to take up antigen, prime intestinal T and B cell responses and induce tissue repair. It is unclear whether this functional heterogeneity results from local environmental cues or is due to distinct origin-related programs. These scenarios are not mutually exclusive, but could act in parallel.

Antigen uptake and transport. Lamina propria resi-dent mononuclear phagocytes first attracted attention when they were shown to project dendritic extensions into the gut lumen to sense and potentially sample its bacterial content24. Subsequent studies ascribed these transepithelial dendrites specifically to CX3CR1+ mono-nuclear phagocytes and reported that transepithelial dendrite formation in the terminal ileum depends on CX3CR1 expression16,25; however, this matter is still under debate26. The involvement of CX3CR1+ lamina propria mononuclear phagocytes in the uptake of bacteria from the gut lumen is supported by the fact that these cells are essential for the alternative entry of a noninvasive mutant strain of Salmonella enterica

subsp. enterica serovar Typhimurium that was shown to be independent of myeloid differentiation primary-response protein 88 (mYD88) signalling27. Consistent with a role in sampling luminal antigens, ileal CX3CR1+ mononuclear phagocytes are found adjacent to the intestinal epithelium, whereas CD11b+CD103+ DCs reside in the core of the villus17. However, the absence of transepithelial dendrites in CX3CR1-deficient animals does not impair pathogen entry or T cell priming in the mesenteric lymph nodes17, suggesting the existence of alternative pathways of antigen entry into the intesti-nal lamina propria25,28. At present it remains unclear whether these distinct antigen acquisition routes are completely redundant, or whether specific antigen entry modes affect the nature of the ensuing immune responses. Furthermore, it is unknown whether antigen acquisition through transepithelial dendrites occurs in the colon.

The initiation of mucosal T cell responses is thought to be restricted to organized lymphoid tissues and prob-ably does not occur in the lamina propria itself. As such, T cell priming towards luminal antigens acquired by lamina propria DCs requires antigen transport to the GALT. Antigens sampled in the lamina propria are pre-sented in the mesenteric lymph nodes exclusively by CD11b+CD103+ DCs. CD11b+CD103+ lamina propria DCs, but not CX3CR1+ mononuclear phagocytes, express CC-chemokine receptor 7 (CCR7), which is required for migration to the mesenteric lymph nodes11,15,17. Recently, an elegant multiphoton microscopy study revealed that only CX3CR1– lamina propria DCs migrate in affer-ent lymphatic vessels17. Analysis of the afferent lymph draining to the mesenteric lymph nodes from the intes-tinal lamina propria confirmed the absence of CX3CR1+ mononuclear phagocytes, both in the steady state and following treatment with Toll-like receptor (TLR) ago-nists that induce emigration of lamina propria DCs to the mesenteric lymph nodes29. However, CD103+ lamina propria DCs could be seen migrating in both of these set-tings17. Consistent with these findings, CD11b+CD103+ DCs are markedly reduced in the mesenteric lymph nodes of CCR7-deficient mice, and oral tolerance cannot be established in these animals11,30. In addition, CCR7-expressing DCs are required for the transport of orally administrated bacteria to the mesenteric lymph nodes31, and although orally administered Salmonella spp. is taken up by both CD11b+CD103+ and CX3CR1+ mono-nuclear phagocytes in the lamina propria, bacteria are only detected in CD11b+CD103+ DCs in the mesenteric lymph nodes15.

T cell priming and imprinting. Although mainly based on ex vivo read outs, several studies suggest that lamina propria DC subsets differ in their capacity to induce effector and regulatory T (TReg) cell differentiation. CD103+ lamina propria DCs, but not CD103– DCs, promote T cell expression of the gut homing recep-tors CCR9 and α4β7 integrin by producing retinoic acid13,18,32. CD103+ lamina propria DCs also promote the generation of inducible TReg cells through the produc-tion of retinoic acid and transforming growth factor-β

Box 1 | What’s in a name?

It has been difficult to clearly distinguish between dendritic cells (DCs) and macrophages, as these closely related cells share both phenotypical and functional characteristics. This issue becomes even more challenging during inflammation, owing to the recruitment and differentiation of further DC and macrophage subpopulations. Recent studies focusing on CX


3CR1)+ lamina propria

mononuclear phagocytes have highlighted this problem. Although these cells express classical macrophage markers, including CD11b, CD14 and F4/80, they also display high levels of MHC class II and co-stimulatory molecules, such as CD80 and CD86, which are characteristic of DCs. Electron microscopy examination of CX

3CR1+ lamina

propria cells revealed structural macrophage characteristics, such as a vacuolar cytoplasm, however these cells also exhibit morphological DC features and form extended transepithelial dendrites18.

CX3CR1+ lamina propria mononuclear phagocytes originate from circulating

monocytes but not from classical DC precursors14,15. However, monocytes have also been proposed to give rise to DCs in other tissues55. Moreover, CX

3CR1+ lamina propria

mononuclear phagocytes arise from Ly6C+ monocytes and not the Ly6C– monocyte subset that seems dedicated to generate macrophages14,111. Finally, CX

3CR1+ lamina

propria cells were shown to promote T helper 17 (TH17) cell differentiation10,36 and

forkhead box P3 (FOXP3) maintenance on regulatory T cells35, suggesting that these cells can modulate adaptive immunity. However, although proficient in antigen-sampling, CX

3CR1+ cells do not express CC-chemokine receptor 7 (CCR7) and,

as a consequence, do not migrate to the mesenteric lymph nodes and only inefficiently prime naive T cells17. These cells therefore lack two major hallmarks of DCs. Taken together, these features of CX

3CR1+ cells argue against their crude classification as

bona fide DCs or macrophages, and highlight the limitations of the current terminology. For this reason, we have opted in this review for a neutral term calling them ‘mononuclear phagocytes’.

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Epithelium

CryptIsolatedlymphoid follicle

Intestinal lumen

Villus

Bacteria

Bacteria

Food antigens

Transepithelialdendrite

CD103+CD11b+ DC

CD103+CD11b– DCCD103+CD11b– DC

CD103+CD11b+ DC

CX3CR1+CD11b+

mononuclear phagocyte

CX3CR1+CD11b+


CX3CR1+CD11b+

Small intestine

Lamina propria

Mesentericlymph node

Draininglymphatic

Colon

Blood

Bone marrow

CCR7-dependentmigration

CCR7-dependentmigration tolymph node

Blood

Bone marrow

Bacteria

Lymphoidaggregate

Intestinal lumen

Coloniccrypts

CX3CR1+CD11b+


CD103+CD11b+ DC

Pre-cDC

Pre-cDC

Ly6C+ monocyte

Monocyte

Ly6C+ monocyte

Monocyte

MDP

MDP

Epithelium

Local pro- andanti-inflammatoryfunctions

CX3CR1+CD11b+


CD103+CD11b+ DCLamina propria


Initiation of tolerogenic or protective immune responses FLT3L

FLT3L and GM-CSF



FLT3L+GM-CSF M-CSFFLT3L

CD103+CD11b– DC

M-CSF

(TGFβ)13. Importantly, retinoic acid production is a specialized function of CD103+ lamina propria-derived DCs33, although stromal cells in the mesenteric lymph nodes can also produce retinoic acid34. Although TReg cell induction is considered to occur in the mesenteric lymph nodes13, CD11b+CD103+ DCs are thought to be ‘condi-tioned’ by factors in the lamina propria environment to promote TReg cell generation7.

In addition, mononuclear phagocytes residing in the lamina propria seem to promote the maintenance of functional TReg cells in the intestine. Interleukin-10 (IL-10)-secreting colonic CD11c+CD11b+F4/80+ cells were recently reported to be crucial for the in vivo main-tenance of forkhead box P3 (FoXP3) expression and the suppressive activity of TReg cells during colitis35. These

cells are probably CX3CR1+ mononuclear phagocytes, but the exact site of their interaction with the TReg cells, as well as the molecular mechanisms involved, still remain to be determined.

The population of CD11c–/lowCD11b+F4/80+ cells exhibiting macrophage characteristics are abundant in the small intestinal lamina propria and have a unique anti-inflammatory signature at this site10. In vitro, lamina propria macrophages promoted the differen-tiation of naive CD4+ T cells into FoXP3+ TReg cells10. By contrast, total CD11b+CD11c+ lamina propria DCs promoted T helper 17 (TH17) cell development10. Both reactions were dependent on the presence of TGFβ10. This study, however, did not specify whether these cells are CD11b+CX3CR1+ or CD11b+CD103+ mononuclear

Figure 2 | The emerging intestinal lamina propria dendritic cell compartment. A common precursor, the macrophage–dendritic cell (DC) precursor (MDP)19, gives rise to classical DC precursors and monocytes in the bone marrow, which enter the circulation and home to the gut. Locally, classical DC precursors give rise to CD103+ lamina propria DCs that can be subdivided into a CD11b– and a CD11b+ population — which reside in the gut-associated lymphoid tissue (GALT) or in both the GALT and the intestinal lamina propria, respectively. Differentiation of CD11b+ lamina propria DCs requires FMS-related tyrosine kinase 3 ligand (FLT3L) and granulocyte–macrophage colony stimulating factor (GM-CSF). Following antigenic challenge, CD11b+CD103+ lamina propria DCs migrate in a CC-chemokine receptor 7 (CCR7)-dependent manner to the mesenteric lymph nodes and promote antigen-specific tolerance or protective immunity11,13,15,17,18,30–32,112. Ly6C+ monocytes differentiate locally into CX


3CR1)+ lamina propria mononuclear phagocytes that can penetrate

epithelium by extending transepithelial dendrites14. CX3CR1+ lamina propria mononuclear phagocytes fail to migrate to

lymph nodes but seem to have pro and anti-inflammatory functions locally.

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Crypts of LieberkuhnTubular invaginations of the intestinal epithelium. Paneth cells are found at the base of the crypts along with continuously dividing stem cells, which are the source of all intestinal epithelial cells.

MetagenomeThe genomic content of a sample of organisms obtained from a common habitat.

phagocytes. Interestingly, in co-culture experiments lamina propria macrophages prevented the induction of TH17 cells by CD11b+ DCs suggesting that lamina propria macrophages might modify the balance between TH17 and TReg cells in the intestine10. However, it remains unclear how resident lamina propria cells can tune adap-tive immunity if they do not migrate to lymph nodes. one hypothesis is that they support the local maintenance of intestinal TH17 cells and TReg cells.

Terminal differentiation of TH17 cells has been pro-posed to occur mainly in the lamina propria, as these cells are normally rare in the mesenteric lymph nodes and Peyer’s patches36. Accordingly, small intestinal CD11b+CD11c+ lamina propria mononuclear phagocytes can induce the differentiation of antigen-specific TH17 cells through secretion of TGFβ. other studies showed that TLR5-activated CD103+CD11b+ lamina propria mononuclear phagocytes can induce the differentiation of TH17 and TH1 cell phenotypes12. Interestingly, com-mensal organism-derived ATP was shown to activate colonic CD70hiCX3CR1+ mononuclear phagocytes to promote TH17 cell differentiation36. However any dif-ferential ability of CD11b+CD103+ DCs and CX3CR1+ mononuclear phagocytes to induce TH17 cells is yet to be addressed. of note, these in vitro co-culture assays only poorly model the complexity of the intestinal lamina propria and the associated lymphoid organs. Therefore it is still unclear if the functions that have been described for intestinal mononuclear cells in vitro are reflective of their activities in vivo.

IgA class-switching. Another pivotal feature of the intesti-nal immune system is its production of immunoglobulins, in particular IgA, that can neutralize bacterial toxins, shape the microflora content and prevent breach of the epithe-lial layer by commensal bacteria and pathogens37. B cell IgA class-switching is promoted by T cell-dependent and -independent pathways38,39, and lamina propria DCs have been implicated in both types of processes. Retinoic acid synthesis by TLR5+CD11b+CD103+ lamina propria DCs is essential for class switch recombination and the upregula-tion of CCR9 on differentiated IgA+ B cells, which enables B cell retention in the lamina propria12. Importantly, how-ever, ID2-deficient mice that lack CD11b–CD103+ DCs in the GALT retain T cell-independent IgA production in the lamina propria12. Although the exact contribution of all the mononuclear phagocyte subsets to intestinal anti-body production remains to be determined, these results indicate that the lamina propria is not merely an effec-tor site of immunity, but also a site of de novo induction of immune responses. This shall be discussed in more detail below in the context of mononuclear phagocyte interactions with the intestinal microflora.

Tissue repair functions. Intestinal macrophages are highly phagocytic cells that are thought to be essential for the maintenance of tissue homeostasis owing to their functions in tissue remodelling and their ability to clear and destroy bacteria, senescent cells and other non-microbial foreign materials40. Recent work has shown the role of lamina propria macrophages in wound-healing

processes. Activated F4/80+ lamina propria macrophages were reported to move towards the colonic crypts of Lieberkuhn and to promote mYD88-dependent survival and proliferation of epithelial progenitor cells during colonic wound healing41. macrophages seem to exert this function by secreting factors that stimulate the prolifera-tion of epithelial progenitor cells, as well as through the production of metalloprotease inhibitors and extracellu-lar matrix stabilizers41. In a mouse model of acute colonic epithelial-regeneration, triggering receptor expressed on myeloid cells 2 (TRem2)-expressing lamina propria macrophages promoted wound repair by inhibiting tumour necrosis factor (TnF) and interferon-γ (IFnγ) secretion and by increasing the levels of IL-4 and IL-13 in the lamina propria42. However, the cellular components in interaction with the lamina propria macrophages and the molecular mechanisms that are involved remain to be elucidated.

Mononuclear phagocytes and the microbiotaThe mammalian intestine is densely populated with up to 1012 microorganisms per gram of intestinal contents; this represents a biomass 10 times more numerous than the body’s own cells1,43. The diverse bacterial community consists of a rapidly adaptable ‘metagenome’ that has piv-otal roles in host health by restraining pathogen coloniza-tion, complementing the digestive efficiency of the host and promoting immune homeostasis44. To enable this mutually beneficial coexistence, the microbiota and host have evolved ‘firewalls’45 and regulatory mechanisms46. In the following section we discuss recent insights into the nature of these interactions, focusing on intestinal mononuclear phagocytes and the gut microbiota.

Hyporesponsiveness of lamina propria mononu-clear phagocytes. A prerequisite for gut homeostasis is tolerance towards symbiotic bacteria. Indeed, in the steady state, colonic mononuclear phagocytes are hyporesponsive to TLR-stimulation47–49. Specifically, these cells express molecules that negatively regu-late TLR-signalling. For example, IκBnS (a molecule that is homologous to members of the IκB (inhibitor of nFκB) family) is selectively expressed by CD11b+ intestinal mononuclear phagocytes and inhibits their induction of pro-inflammatory nF-κB gene-targets47,50. mononuclear phagocytes from IκBnS-deficient mice show increased expression of the IL-12 subunit p40 and IL-6 following TLR stimulation and are highly suscepti-ble to dextran sulphate sodium (DSS)-induced colitis50. A second example of a molecular brake is TIR-domain protein 8 (TIR8, also known as SIGIRR) an IL-1 recep-tor (IL-1R) family member. TIR8 is highly expressed by immature DCs in the mouse small and large intestine and by human monocyte-derived DCs, but not by mac-rophages. TIR8 inhibits IL-1R and TLR-signalling by trapping IL-1R associated kinase 1 (IRAK1) and TnFR-associated factor 6 (TRAF6)51, and TIR8-deficient mice show increased susceptibility to DSS-induced colitis and associated cancer52,53. The exact lamina propria mononuclear phagocyte subset (or subsets) subjected to TIR8-based regulation remains unclear.

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Gnotobiotic miceMice that are born in aseptic conditions, separated from the mother by Caesarean section, and are raised in controlled environment. These animals can be monocolonized with defined microbial species in order to investigate specific relationships between the host and the microbiota.

Intestinal IgA induction. Importantly, the state of tol-erance towards commensal organisms is not due to unresponsiveness on the part of intestinal immune cells; instead, it is an active process. The microbiota promotes the generation of IgA+ plasma cells through activation of lamina propria DCs12,31,54. CD11c+ lamina propria DCs, but not macrophages, have been shown to retain ingested living commensal bacteria, enabling the transport of commensal organisms to the mesenteric lymph nodes and the induction of IgA production31. In addition, TnF and inducible nitric oxide synthase (inoS)-expressing lamina propria DCs, reminiscent of TnF and inoS producing (TIP)-DCs55, have been implicated in T cell-independent IgA class-switching through production of the TnF-superfamily members a proliferation-inducing ligand (APRIL) and B cell acti-vating factor belonging to the TnF family (BAFF). In agreement with this, transfer of wild-type CD11clow/mid

CD11b+Ly6Chi cells rescued the IgA deficiency seen in inoS-deficient mice54. Accordingly, these cells are prob-ably CX3CR1+ mononuclear phagocytes generated from Ly6Chi monocytes and producing TnF14.

Interestingly, flagellin-stimulated CD11b+CD103+ lamina propria DCs instruct IgA class-switching and promote the retention of CCR9+IgA+ B cells in the lamina propria12. However, TLR5-deficient mice har-bour normal numbers of IgA+ plasma cells and this, along with the fact that flagella are largely pathogen-associated, implies that the TLR5-signalling pathway is preferentially activated during pathogen encounters. of note, T cell-independent IgA class-switching seems to be restricted to isolated lymphoid follicles56. Accordingly, the formation of isolated lymphoid follicles in adult mice was found to depend on retinoic acid receptor-related orphan receptor-γt (RoRγt)+ lymphoid-tissue inducer cells activating stromal cells through lymphotoxin-β receptor (LTβR)56. Commensal-mediated activation of TLRs further augmented these interactions and led to recruitment of DCs and B cells, induction of activation-induced cytidine deaminase (AID), and subsequent IgA class switching. Co-culture assays have further suggested that TnF produced by CD11c+CD11b+ intestinal DCs following bacterial stimulation can induce the activation of latent TGFβ1, which facilitates IgA class switching by B cells within isolated lymphoid follicles56.

TH17 cell generation. emerging evidence indicates that commensal organisms activate specific mononuclear phagocyte subsets within the lamina propria to promote TH17 cell generation. microflora-derived ATP was shown to activate P2X and P2Y purinoceptor-expressing colonic CD70+CX3CR1+ lamina propria cells to induce TH17 cell differentiation36. of note, these CX3CR1+ mono-nuclear phagocytes are, by virtue of their trans epithelial dendrites, in direct contact with the gut lumen and the commensal organisms that reside there. moreover, germ-free mice have far fewer CX3CR1+ mononuclear phagocytes57. Interestingly, challenge with Salmonella spp. is accompanied by flagellin- and mYD88-dependent migration of Salmonella spp.-capturing CX3CR1+ mono-nuclear phagocytes into the gut lumen58. Furthermore,

flagellin-mediated TLR5-signalling by CD11b+CD103+ lamina propria DCs activates these cells to promote TH17 cell differentiation12. most intriguingly however, recent data suggest that the prevalence of TH17 cells in the intestine is directly determined by the composition of the intestinal microbiota36,59. Indeed, colonization of germ-free and gnotobiotic mice with a gram-positive clostridia-related commensal microbe, segmented fila-mentous bacterium (SFB), is sufficient to induce the development of intestinal TH17 cells60,61. In vitro, SFB induced production of serum amyloid A protein by epithelial cells in the ileum, and this conditioned intes-tinal CD11c+ cells to promote TH17 cell differentiation61. However, the specific intestinal mononuclear phago-cyte subset involved and the mechanisms of the serum amyloid A response remain elusive.

Protection against parasites. Intestinal parasites are of medical importance and are sensed by intestinal mono-nuclear phagocytes. The Toxoplasma gondii protein profilin was shown to directly activate DCs through TLR11, resulting in IL-12-dependent host responses to the parasite62,63. However, in the absence of TLR11, commen-sal organisms can function as an adjuvant during T. gon-dii infection by providing immunostimulatory signals to DCs through TLR2, TLR4, and TLR9, which leads to IL-12 production64. Interestingly, protective immu-nity following oral infection with T. gondii depends on CCR2-mediated recruitment of Ly6Chi monocytes; these monocytes give rise to F4/80+CD11b+ cells in the small intestinal lamina propria that are probably CX3CR1+ lamina propria mononuclear phagocytes65.

Mononuclear Phagocytes in the Human IntestineThe organization of the steady state intestinal mono-nuclear phagocyte compartment in humans, and whether the subsets found in the mouse have human counterparts, is poorly understood7.

Lamina propria mononuclear phagocyte subsets. The healthy human colon was reported to harbour a pop-ulation of mHC class II+CD68+ DCs, expressing the calcium-binding protein S100, that form a reticular network throughout the lamina propria. By contrast, human colonic HLA+CD68+ macrophages that express a molecule associated with late inflammatory cells (detected by the antibody 25F9) are localized in close proximity to the epithelium66. In the steady state, these cells lack expression of many innate receptors, such as CD14, and they are thought to be conditioned by TGFβ from intestinal stromal cells to have an anergic pheno-type. Despite this, these cells can still have avid bacte-riocidal activity67. However, as is the case in the mouse, classifying human intestinal mononuclear phagocytes as either DCs or macrophages has been challenging. Downregulation of CD14 is typical to human intestinal lamina propria resident macrophages expressing CD33 (REF. 67). However, several studies of human biopsies from patients with IBDs have reported the infiltration of CD14+CD33+ lamina propria mononuclear phagocytes that co-express macrophage and DC markers, such as

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Genome wide association (GWA) studiesAn approach that involves rapidly scanning single nucleotide polymorphism markers across the complete genomes of many individuals to find genetic variations associated with a particular disease.

CD68, CD205, CD209 and CX3CR1, as well as TRem1, but do not express CCR7 (REFs 68–70). Functional assays have shown that, in contrast to the CD14–CD33+ resident macrophages, these infiltrating cells produce increased amounts of pro-inflammatory cytokines, including IL-6, IL-23 and TnF68.

Mononuclear phagocytes in human GALT. Both CD11c+CD103+and CD11c+CD103– DCs can be identi-fied in human mesenteric lymph nodes. Compared with CD103– DCs, human CD103+ mesenteric lymph node DCs have a more mature phenotype, show higher levels of CCR7 expression and induce higher levels of CCR9 on CD8+ T cells by producing retinoic acid, consistent with that seen in mice18,71. In addition, CD103+ DCs in the mesenteric lymph node can also promote TReg cell development, confirming that in both mice and humans, CD103 can be a marker of tolerogenic DCs in steady state mesenteric lymph nodes72 (BOX 2). notably, a unique population of potent TnF-producing DCs, identified by the monoclonal antibody m-DC8, has been discov-ered in the subepithelial dome of the Peyer’s patches73. These cells have an immature phenotype and express CCR6 and CCR7, and are therefore similar to mouse subepithelial resident DCs (BOX 2).

Mononuclear phagocytes in IBDsIBDs are a group of idiopathic, chronic relapsing inflam-matory intestinal conditions. The two main IBDs are Crohn’s disease and ulcerative colitis, which include both overlapping and distinct clinical and pathological characteristics74. In Crohn’s disease, the terminal ileum is the most commonly affected tissue site, but lesions can occur anywhere along the entire digestive tract, from the

alimentary canal of the mouth to the anus. However, in patients with ulcerative colitis the mucosal layer of the colon is typically the only affected site. Ulcerative colitis begins in the rectum, and spreads up through the large intestine, whereas in Crohn’s disease, the inflammation is transmural and patchy.

Animal models of intestinal inflammation. Animal models have contributed considerably to our under-standing of acute and chronic small bowel inflamma-tion. Though arguably none of the mouse models truly reflects Crohn’s disease or ulcerative colitis, these models collectively have unique advantages that justify their use in research into IBDs. Specifically, animal models enable the study of the immediate events following pathogen and irritant challenge, thereby providing insights into the dynamics of disease development, the influence of emerging IBD candidate genes from genome wide asso-ciation (GWA) studies, and the efficacy of new treatments for IBDs in preclinical studies. Finally, through the study of intestinal inflammation in the mouse, we have accu-mulated a large amount of knowledge concerning the organization of the murine immune system and its vari-ous cellular players in the GALT and the lamina propria. Such information can facilitate the rational development of new therapeutic strategies for IBDs. An additional benefit of animal models is that they allow studies to be carried out in vivo.

Small animal models have revealed crucial roles for intestinal mononuclear phagocytes in the establish-ment of both intestinal inflammation and tolerance75. The direct pro-inflammatory activity of these cells is probably best highlighted in a T cell-independent coli-tis model, in which mononuclear phagocytes activated

Box 2 | Mononuclear phagocytes in gut associated lymphoid tissue

mesenteric lymph nodes Mononuclear phagocytes in the mesenteric lymph nodes comprise migrants from the lamina propria and cells that have developed locally from blood-derived precursors. Resident classical mesenteric lymph node dendritic cells (DCs) are CD11chi and can be divided into CD11b+ CD8α–, CD11b– CD8+ and CD11b– CD8– cells, the origins of which, however, remain unclear6,7. Both CD11b– and CX


3CR1)+ CD11b+ mesenteric lymph node DCs can be

reconstituted by macrophage or DC precursors, but Ly6Chi monocytes only give rise to CD11b+CX3CR1+ cells14.

CD11b+CD103+ mesenteric lymph node DCs migrate from the intestinal lamina propria in a CCR7-dependent manner, transporting antigens to activate and polarize T cells towards effector and regulatory T cell fates11,30,112. The importance of this antigen transport is highlighted by the fact that oral tolerance is impaired in CCR7-deficient mice30. For further information on mesenteric lymph node mononuclear phagocytes we refer the reader to REF. 6.

Peyer’s patches Although anatomically similar to lymph nodes, Peyer’s patches lack afferent lymphatics and instead receive antigen through specialized epithelial microfold (M) cells113. Peyer’s patch DCs comprise CD11b+ DCs (in the subepithelial dome), CD8α+ DCs (in the T cell-rich interfollicular regions) and CD11b–/lowCD8α– DCs (found in the sub-epithelial dome, the interfollicular regions and the follicular-associated-epithelial area)114. CD11b+ Peyer’s patch DCs promote T helper 2 (T

H2)

cell generation, but other DCs in the Peyer’s patches induce TH1-type polarization114. Oral administration of Salmonella

spp. induces the migration of CC-chemokine receptor 6 (CCR6)+ Peyer’s patch DCs from the subepithelial dome to the follicle-associated epithelium115. In addition, viral antigens derived from infected apoptotic cells of the follicle-associated epithelium can be ingested by CD11blowCD8– DCs in the sup-epithelial dome region and presented to CD4+ T cells. Similarly to CD11b+ lamina propria DCs, CD11b+ Peyer’s patch DCs uniquely secrete interleukin-6 (IL-6) and promote the class-switch recombination of co-cultured naive B cells into IgA-producing cells. Furthermore, Peyer’s patch DCs imprint α4β7 integrin expression on T cells and promote gut homing. The origins of Peyer’s patch DCs remain unclear, but grafted Ly6Chi monocytes can differentiate into functional subepithelial dome resident CX

3CR1+ CD11b+ DCs16. Interestingly, in

this study, Ly6Chi monocytes gave rise to a morphologically distinct Peyer’s patch mononuclear phagocyte population16. For a more detailed description of Peyer’s patches we refer the reader to REF. 116, an excellent recent review.

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SLAN (6-sulfo LacNAc). A carbohydrate modification of the P-selectin glycoprotein 1 (PsGL1). sLAN is expressed by a subset of DCs found in human blood and is recognized by the monoclonal antibody MDC8.

by CD40 engagement produce IL-23 and drive intes-tinal inflammation76. In addition, specifically deleting key immunoregulatory genes in intestinal mononuclear phagocytes can result in gut inflammation. An interest-ing example is the T-bet–/–Rag–/– ulcerative colitis (TRUC) model77, in which a restricted haematopoietic deficiency of the T-box transcription factor T-bet in the absence of adaptive immunity results in a spontaneous and com-municable colitis resembling human ulcerative colitis. notably, selectively restoring T-bet expression in CD11c+ cells was sufficient to decrease colonic inflammation and prevent colonic neoplasia78. In this model, T-bet nega-tively regulates TnF production by CD11c+ intestinal mononuclear phagocytes, thereby moderating host–commensal relationships; however the specific intestinal mononuclear phagocyte subset involved has not been identified77. Additional evidence for the importance of DCs for gut homeostasis has come from the study of ani-mals deficient in αvβ8 integrin. This integrin is required for the activation of the anti-inflammatory cytokine TGFβ; mice with intestinal mononuclear phagocytes that lack either the αv integrin or β8 integrin subunit develop spontaneous colitis79,80, which is associated with a failure to induce TReg cells.

Recent data from our group has suggested that intes-tinal homeostasis depends on the maintenance of a del-icate equilibrium between CD103+ DCs and CX3CR1+ lamina propria mononuclear phagocytes14. Using a combination of CD11c+ cell ablation and complemen-tary adoptive transfer of precursor cells, we showed that mice persistently or transiently depleted of lamina propria mononuclear phagocytes did not develop spon-taneous intestinal inflammation and were not overtly susceptible to colitis development16. By contrast, ani-mals that predominantly harboured monocyte-derived CX3CR1+ cells in their lamina propria developed severe signs of colitis in response to DSS challenge14. moreover the resulting disease was specifically driven by TnF secreted by graft-derived CX3CR1+ lamina propria mono nuclear phagocytes14. These findings highlight the crucial importance of a balanced lamina propria mono-nuclear phagocyte compartment for the maintenance of intestinal homeostasis.

Ablation of CD11c+ cells has previously been shown to attenuate DSS colitis81; however, in the case of prior TLR9 activation of the host, ablation of CD11c+ cells exacer-bates disease82. Interestingly, TLR9 is expressed at much higher levels on CD103–CX3CR1+ cells than CD103+ small intestinal lamina propria DCs57,83. CD103– lamina propria DCs selectively express CD172a (also known as SHPS1), the receptor for CD47, which is a membrane protein that prevents ingestion of cells by phagocytes. CD47-deficient mice are protected from TH17-associated trinitrobenzene sulphonic acid (TnBS)-induced colitis and have fewer CD103– CD172a + DCs in the lamina propria; however, CD103–CD172a+ DCs accumulate in colitic wild-type mice84 Therefore, this TnBS-induced colitis model suggests that CD47 is necessary for the accumulation of TH17-inducing CD103–CD172a+ DCs (that are most probably CX3CR1+ cells) in the lamina propria and for the development of inflammation.

mice deficient for the immunoregulatory cytokine IL-10 spontaneously develop enterocolitis85. A pro-inflammatory role for mononuclear phagocytes in this model is suggested by the finding that depleting intes-tinal mononuclear phagocytes by rectal administration of cytotoxic microspheres prevents colitis in IL-10-deficient mice86. However, intestinal mononuclear phagocytes also have crucial immunoregulatory roles, as intestinal CD11c+CD11b+F4/80+ cells (which are prob-ably CX3CR1+ lamina propria mononuclear phagocytes) are an important source of IL-10 in the gut35. IL-10 pro-duced by these cells in the intestinal lamina propria and mesenteric lymph nodes maintains FoXP3 expression by TReg cells and is sufficient to prevent colitis induced by the adoptive transfer of CD4+CD45RBhi T cells35.

Characteristics of mononuclear phagocytes in human IBDs. The immunopathology of human IBDs remains poorly understood, but several studies have suggested that mononuclear phagocyte dysfunction has pivotal roles in its aetiology. mononuclear phagocyte numbers increase in the inflamed intestinal mucosa of patients with active IBDs, and a significant positive correlation has been noted between the number of infiltrating intes-tinal mononuclear phagocytes and the degree of crypt inflammation in ulcerative colitis87,88. Aberrant activa-tion of resident intestinal mononuclear phagocytes or the recruitment of inflammatory mononuclear phago-cytes from the blood (which have not been conditioned by the local tolerogenic microenvironment) is proposed to contribute to the development and progression of IBDs69,70,89.

Phenotypical characterization of colonic DCs from patients with Crohn’s disease revealed two distinct DC populations: CD209+IL-12+IL-18+ cells scattered throughout the mucosa and CD83+IL-12–IL-18– cells present in aggregated lymphoid nodules and as single cells in the lamina propria90. The subepithelial dome of the Peyer’s patches of patients with Crohn’s disease har-bours increased numbers of these CD209+ and CD83+ DC subsets, possibly owing to decreased CCR7 expres-sion and impaired migration of these cells. The CD209+ and CD83+ DCs exhibit increased co-localization with bacteria, display higher levels of TLR4 and produce more TnF91. Furthermore, the inflamed ileal mucosa of patients with active Crohn’s disease also comprises a prominent population of monocyte-derived sLAN+ (6-Sulfo LacnAc) DCs: these cells secrete large amounts of TnF in response to lipopolysaccharide, and may be a fundamental TnF source in these patients73. Increased numbers of mature mucosal CD83+ DC producing macro phage migration-inhibitory factor (mIF) might also contribute to neutrophil recruitment in patients with ulcerative colitis92.

Additionally, although DCs in the healthy intes-tine express little TLR2 or TLR4, expression of both of these TLRs is found on DCs during IBDs93. DCs from inflamed intestinal tissue of patients with Crohn’s disease also express substantially higher levels of the co-stimulatory molecule CD40 and secrete the pro-inflammatory cytokines IL-6 and IL-12 (REF. 93). In

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AutophagyAny process involving degradative delivery of a portion of the cytoplasm to the lysosome that does not involve direct transport through the endocytic or vacuolar protein sorting pathways.

Paneth cellsPaneth cells are found at the base of intestinal crypts and produce antimicrobial proteins and peptides, including phospholipase A2 and defensins.

addition, when stimulated with heat shock protein 70, CD40L or lipopolysaccharide, DCs from patients with Crohn’s disease produce greater levels of TnF and IL-12 than DCs obtained from non-inflamed mucosa94.

The engagement of TRem1 might also promote pro-inflammatory functions of intestinal mononuclear phagocytes. TRem1 is expressed on monocytes and neu-trophils and its activation leads to increased secretion of inflammatory cytokines and chemokines95. myeloid cells of the healthy intestine generally lack TRem1 expression. However, in patients with IBDs (and in mouse models of IBDs) intestinal TRem1 expression is upregulated spe-cifically on CD14+CD89+ intestinal mononuclear phago-cytes70. TRem1 expression by intestinal mononuclear phagocytes correlates with disease activity and enhanced secretion of pro-inflammatory mediators70. Patients with Crohn’s disease, but not patients with ulcerative colitis or healthy subjects, have increased numbers of CD14+CD33+TRem1+ intestinal mononuclear phago-cytes, and these cells are a major source of IL-23 and TnF. In the presence of commensal organisms, these CD14+ cells are thought to promote TH17 polarization, which can further contribute to inflammatory intestinal responses89.

Mononuclear phagocyte dysfunction in human IBDs. mesenteric lymph node DCs have been implicated in the generation of TH1 and TH17 cells. T cells isolated from mesenteric lymph nodes of patients with Crohn’s disease, but not from patients with ulcerative colitis or normal controls, produce higher levels of IFnγ and IL-17. This is possibly due to higher production of IL-23 and lower production of IL-10 by CD11chimHC class IIhi mesenteric lymph node DCs96. These data suggest that the cytokine milieu produced by mesenteric lymph node DCs affects the TH1–TH17 balance in the intestine, an imbalance of which is associated with Crohn’s disease 97,98.

IL-23 has also been shown to be a key contributor to pathology in mouse models of IBDs99–101. Interestingly, a unique intestinal mononuclear phagocyte subset pro-ducing large amounts of IL-23 and characterized by the expression of CD11c, CD11b and F4/80 can be identified in experimental models of intestinal inflammation102. These cells may be related to CX3CR1+ mononuclear phagocytes and directly induce the development of granulomas, a distinctive characteristic of Crohn’s dis-ease, by secreting IL-23 in response to the enteric flora102. Importantly, IL-23 receptor polymorphisms were associated with IBDs in a GWA study103.

mutations in nucleotide-binding oligomerization domain 2 (NOD2) have also been described as pre-disposing to Crohn’s disease104. This intracellular innate immune receptor is triggered by muramyl di peptide, a product of microbial peptidoglycan catabolism. Human blood DCs incubated with muramyl dipeptide promoted TH17 cell induction by secreting IL-1 and IL-23, but DCs derived from patients with Crohn’s disease who have noD2 mutations failed to do so105. Interestingly, prior exposure of human DCs to muramyl dipeptide inhibited their subsequent production of inflammatory cytokines following TLR ligation. These

findings suggest an anti-inflammatory regulatory role of the muramyl dipeptide–noD2 pathway, the absence of which leads to increased susceptibility to Crohn’s disease87. However, the link between noD2 and TH17 induction requires further investigation in order to determine its role in the breakdown of tolerance that occurs in Crohn’s disease.

GWA studies have linked the autophagy-related gene products ATG16L1 (autophagy-related 16-like 1) and IRGm (immunity-related GTPase family m) with the pathogenesis of Crohn’s disease106,107. However, whereas animal studies have suggested the involvement of Paneth cells in disease development108, a firm link between dysregulated intestinal mononuclear phago-cyte autophagy and gut inflammation has not been established109. Interestingly, noD2-mediated activation of human monocyte-derived DCs was recently shown to trigger autophagy, and this pathway was impaired in DCs from patients with Crohn’s disease who had NOD2 mutations110.

Collectively, the emerging evidence105,110 suggests an intriguing link between three Crohn’s disease suscepti-bility genes (IL23R, NOD2 and ATG16L) and human DC functions, as noD2 activation promotes IL-1 and IL-23-mediated TH17 cell induction and autophagy responses that are required to maintain host hyporesponsiveness to the commensal flora. However, it will be important to explore these pathways in intestinal mononuclear phagocytes in vivo using animal models of intestinal inflammation.

Conclusions and future perspectivesIntestinal mononuclear phagocytes have central roles in the initiation, perpetuation and resolution of innate and adaptive immune reactions in the intestinal mucosa. Improved experimental approaches have revealed the complexity of the intestinal mononuclear phagocyte compartment. Importantly, recent studies have estab-lished that there are several subsets of intestinal mono-nuclear phagocytes with distinct developmental origins and growth factor requirements. A number of studies have described different functions for these subsets in vitro, which might be relevant either locally in the lamina propria or following migration to gut associated lymphoid tissues.

The challenge ahead is to investigate the functions of these intestinal mononuclear populations in an in vivo context, exploring their interaction with the microbiota, epithelium and other cells in the tissue. The availability of increasingly sophisticated conditional gene-targeting approaches and adoptive cell transfer strategies should enable major breakthroughs in this area. equally impor-tant, are the efforts to align the intestinal mononuclear phagocyte subsets identified in mouse with their equiva-lents in the human mucosa: it is possible that such stud-ies will reveal notable species-specific differences. If research into human IBDs can effectively capitalize on the advances we have made in understanding intestinal mononuclear phagocytes in the mouse, this should spur the development of new strategies for treating acute and chronic intestinal inflammation.

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AcknowledgementsThis work was supported by the Israel Science Foundation, The Thyssen Foundation and a research grant from the estate of Edith F. Goldensohn Center (S.J.).

Competing interests statementThe authors declare no competing financial interests.

DATABASESUniProtKB: http://www.uniprot.orgBATF3 | CCR7 | CD11b | CD11c | CD47 | CD103 | CD172a | CX

3CR1 | F4/80 | FLT3L | GM-CSF | ID2 | IRF8 | Ly6C | M-CSF |

MIF | MYD88 | profilin | SIGIRR | TLR2 | TLR4 | TLR5 | TLR9 | TLR11 | TREM1 | TREM2

FURTHER INFORMATIONSteffen Jung’s homepage: http://www.weizmann.ac.il/immunology/jung/

SUPPLEMENTARY INFORMATIONSee online article: S1 (movie)

All lInkS ARe ACTIve In The onlIne PdF

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