Regulation of Homeostasis and Inflammation in the Intestine

GASTROENTEROLOGY 2011;140:1768 –1775

Regulation of Homeostasis and Inflammation in the Intestine

Thomas T. MacDonald*

Ivan Monteleone‡

Massimo Claudio Fantini‡

Giovanni Monteleone‡

*Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London; and ‡Dipartimento di Medicina Interna e Centro di Eccellenza per lo Studio delle Malattie Complesse e Multifattoriali, Università Tor Vergata, Rome, Italy

The gastrointestinal tract is the largest immune interface with the environment. Exposure to large numbers of dietary and microbial antigens requires complex and highly regulated immune responses by different mucosal cell types, which result in the induction and maintenance of intestinal homeostasis. Defects in this equilibrium can disrupt the homeostatic mechanisms and lead to chronic intestinal inflammation. We review the cell populations and mechanisms involved in the control of intestinal homeostasis and inflammation, focusing on inflammatory bowel diseases. We describe some aspects of gut immunity that could alter the delicate balance between inflammatory and tolerogenic responses and result in chronic gastrointestinal tract inflammation in patients. Keywords: T Cell; Dendritic Cell; TGFbeta; IL-10; Macrophage.

I

n contrast to nearly all other tissues, the mucosa of the small and large intestine has continuous, low-grade (physiologic) inflammation, most likely because the intestine is exposed to a great antigenic load from luminal bacteria and a large variety of Toll-like receptor (TLR) ligands and potential mitogens. This is confirmed by the virtual absence of inflammatory cells in the gut of adult, germ-free mice, and the lack of inflammatory cells in the newborn human intestine.1–3 In the normal gastrointestinal (GI) tract, T cells constitute one-third of the cells in the intestinal lamina propria; the phenotypic distribution of CD4⫹ and CD8⫹ T cells is similar to that of peripheral blood lymphocytes, with a preponderance of CD4⫹ and ␣␤ T-cell receptor (TCR)–positive cells. Most CD4⫹ T-lamina propria lymphocytes (LPLs) are HLADR⫹, ␣4␤7⫹, CD62lo, CD25hi/lo, and CD45RO⫹, consistent with a population of effector-memory cells.2 Plasma cells that produce IgA are in equal abundance to T cells, and the intestine also contains many macrophages and

dendritic cells (DCs).1 The epithelial layer of the small bowel contains about 1 T cell for every 10 epithelial cells, and in the colon the ratio is 1:20. These intraepithelial lymphocytes also have markers of chronic activation.4 Figure 1 shows populations of major histocompatibility complex class II⫹ cells, macrophages and T cells in normal human colon. Healthy individuals have no pathologic features despite lamina propria infiltration with activated immune cells and separation from luminal bacteria by only a single epithelial layer and mucus. Complex regulatory pathways must therefore maintain intestinal immune homeostasis in a healthy GI tract, but also induce a protective immune response against pathogens. A fundamental question in the field of immunology is how the host distinguishes a pathogen from a member of the normal flora. Complex diseases such as inflammatory bowel diseases (IBDs) might arise partly from disruptions in these homeostatic mechanisms and recognition of the normal microbiota as pathogens.1 Many pathways have been implicated in the control of GI inflammation in animal studies, including inhibitory macrophages and DCs, T-regulatory (Treg) cells, T-cell apoptosis, and immunoregulatory cytokines. The relative importance of each individual pathway in the pathogenesis of IBDs has not been determined. However, the fact that intestinal inflammation is a common feature of many different disorders of the immune system indicates that GI immunity is finely balanced and, like nearly all complex dis-

Abbreviations used in this paper: ATP, adenosine triphosphate; DC, dendtritic cell; FoxP3, fork head box p3; GI, gastrointestinal; IL, interleukin; iTreg, inducible T regulatory; LPL, lamina propria lymphocyte; MLN, mesenteric lymph node; PP, Peyer’s patches; RA, retinoic acid; TCR, T-cell receptor; TGF, transforming growth factor; Th, T helper; TLR, Toll-like receptor; Treg, T regulatory. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.02.047

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Figure 1. Distribution of T cells and accessory cells in the normal human colon. CD68, the gold-standard tissue macrophage marker, identifies a population of cells predominantly below the epithelium. Major histocompatibility complex (MHC) class II, however, identifies a much larger population of cells, including subepithelial macrophages, but also populations of cells deeper in the lamina propria, which are probably DCs. T cells identified by anti-CD3 staining are sparse. Immunoperoxidase immunohistochemistry, original magnification ⫻100.

eases, IBDs arise from a combination of factors, and each conferring a relatively low risk.1

Inductive Sites of Mucosal Immune Responses and Antigen Uptake Interactions between commensal bacteria, GI antigens, and immune cells occur at distinct sites. One location is Peyer’s patches (PP) and isolated follicles, where M cells in the follicle-associated epithelium translocate bacterial antigens from the GI lumen to the dome region beneath the follicle-associated epithelium.5 Immature myeloid DC encounter and process antigen, differentiate into mature DCs, and migrate to T-cell zones in PP or to mesenteric lymph nodes (MLNs), to activate T cells. The organized gut-associated lymphoid tissue serves as the source of the activated effector cells, which

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populate the intervening mucosa; cells leave via efferent lymph, enter the blood stream, and migrate back to the lamina propria. Luminal bacterial antigens can also enter into the mucosal tissue through an alternative, M-cell–independent pathway mediated by lamina propria DCs, which extend dendrites into the lumen.6 This process requires the expression of the chemokine receptor CX3CR1.7 Numbers of dendrites that interact with bacteria are greatly reduced in mice with epithelial cells that lack the adaptor molecule MyD88, indicating that TLR signaling in epithelial cells mediates formation of the dendrites by subepithelial DCs.8 Columnar epithelial cells on the villus and colon surface can take up antigens, although less efficiently than M cells.9 This somewhat poorly described process facilitates exposure of lamina propria DCs to luminal antigens and also allows antigens to enter the lamina propria and then drain, via afferent lymphatics, to MLNs.10 GI epithelial cells also take up antigen via the neonatal Fc receptor,11 which binds IgG by a pH-sensitive mechanism that facilitates vesicular, bidirectional transport of intact IgG or IgG-antigen complexes across mucosal epithelial cells. Neonatal Fc receptor can therefore mediate the uptake of lumenal antigens and deliver them across the epithelium to underlying DCs; alternatively, the receptor can deliver IgG into the mucosal lumen and increase the host defense against epithelial cell-associated pathogens.12,13 It is important to determine whether increased uptake of antigen is a major determinant in the disruption of homeostasis that initiates chronic inflammation. Genetic links between intestinal inflammation and epithelial function have not been confirmed, but many researchers believe that patients with IBD, and their healthy relatives, have leaky GI tracts.14 Hermiston and Gordon showed that patchy disruption of the epithelial barrier in the mouse small bowel led to local focal inflammation, indicating that increased antigen uptake can precipitate mild inflammation.15

DCs and Macrophages in Maintenance of Mucosal Homeostasis DCs and macrophages are abundant in the healthy lamina propria and their interactions and roles in the development of GI inflammation are beginning to be appreciated. In mice, lamina propria macrophages might be anti-inflammatory whereas DC might be proinflammatory.16 Mice with DCs that lack the integrin ␣v␤8, which activates transforming growth factor (TGF)-␤1, develop spontaneous colitis.17 Likewise, mice with DCs that lack ␤-catenin and are therefore unresponsive to Wnt ligands change phenotype—in that the immune responses they generate switch from regulatory to proinflammatory, so these mice develop more severe colitis than normal mice when fed dextran sulfate sodium.18

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Distinct subsets of DCs have been described in the mouse intestine (Table 1). These cells can be divided into 2 major classes, according to their ability to control the extent and type of T-cell activation. DC from the gut of mice are either CD11b⫹, and involved in the induction of T-helper (Th)1 and Th17 T cells, or CD103⫹, and involved in differentiation of Treg cells19 –22 (Figure 2). CD103⫹DCs also induce T and B cells to localize to the GI. For example, T cells primed with antigen-pulsed MLNs or PP DCs, but not with DCs from the spleen or peripheral lymph nodes, express CCR9, the receptor for CCL25, a chemokine that is expressed constitutively in the small intestine; the primed T cells also express high levels of ␣4␤7—an integrin that mediates localization to the GI tract and binds MadCAM-1, which is expressed on high-endothelial venules of intestinal tissues. The unique ability of gut DC to induce gut homing receptors is because they make retinoic acid (RA), a metabolite of vitamin A, which drives expression of both CCR9 and ␣4␤7.22–24 A new classification system for DCs based on lineage has been proposed. One subset, CD103⫹CX3CR1⫺ cells, arise from DC-committed precursors (pre-DC) and common monocyte and DC precursors, in response to Flt3 ligand. Another subset, CD11b⫹CD14⫹CX3CR1⫹, are derived from Ly6lo monocytes in response to granulocytemonocyte colony-stimulating factor. CX3CR1⫹ DC express higher levels of co-stimulatory molecules (eg, CD70, CD80, and CD86), produce higher amounts of the cytokine tumor necrosis factor–␣, and contain dendrites, indicating their role in activation of effector T cells.19,20 DC that are CX3CR1⫺ respond to oral antigens and migrate to local draining nodes, where they present these antigens to T cells22–24; they also promote the differentiation of Treg cells, so they might be negative regulators of effector T-cell responses and maintenance of GI homeostasis.19,20 DC that are CX3CR1⫺ express CD103, a ligand of E-cadherin, which helps them to associate with epithelial cells. Various factors released by epithelial cells in response to inflammatory cytokines or TLR stimulation (eg, thymic stromal lymphopoietin, TGF-␤1, RA,

Table 1. Dendritic Cell Subsets in the Gastrointestinal Tract DC surface markers CX3CR1 CCR6 CD11b CD8 CD11b⫺CD8⫺B220⫺ B220⫹Ly6C⫹ CD11chiCD11bCD103⫹ CD11cCX3CR1⫹CD70⫹CD11bCD103⫺ CD11chiCD11b⫹ CD11chiCD103⫹ CD11chiCD103⫺ CD8

Localization PP, subepithelial dome PP, subepithelial dome PP, subepithelial dome PP, interfollicular region PP, subepithelial dome PP, subepithelial dome Lamina propria Lamina propria, intraepithelial Lamina propria MLN MLN MLN

Figure 2. DCs can activate distinct subsets of Th cells in the gastrointestinal tract. CD103-expressing DCs can extend dendrites into the lumen and internalize antigens to promote differentiation of FoxP3-positive Treg cells. TGF-␤1 and RA contribute to differentiation of Treg cells. By contrast, CD11b⫹ DCs can produce IL-12 and IL-23 and promote the differentiation of Th1 and Th17 cells; this process is negatively regulated by Treg cells.

and interleukin [IL]-25) also regulate DC function, with the overall effect of maintaining the anti-inflammatory microenvironment of the intestine.25–27

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DC detect bacteria via a number of intracellular and membrane receptors that recognize conserved structural motifs on prokaryotic and lower eukaryotic organisms. The best known of these receptors are the TLRs, which recognize pathogen-associated molecular patterns.28 Within gut-associated lymphoid tissue, DCs can also be activated by luminal bacteria through TLR-independent mechanisms. An example of this phenomenon is represented by capsular polysaccharide A from the commensal bacterium Bacteroides fragilis. These sugar molecules are unusual in that they are taken up and processed, loaded onto major histocompatibility complex class II molecules, and presented to CD4⫹ T cells.29 Interactions between DC and polysaccharide A lead to the development of an anti-inflammatory state, mediated by IL-10 –producing regulatory cells.30 Another example of DC activation through a TLR-independent mechanism occurs in CD70hiCD11clo DCs, which are able to respond to microflora-derived adenosine triphosphate (ATP) in the absence of TLR stimulation.31 It is not known whether the ATP acts specifically, via dendrites that extend into the mucosal lumen, or more generally, on DCs in the mucosal tissues. However, the effects of ATP depend on the function of the ATP receptors P2X and P2Y and the production of IL-6 and TGF-␤ by DCs, which mediate differentiation of Th17 cells.31,32 This result is striking because certain gut bacteria, such as the segmented filamentous bacterium in mice promote production of Th17 cells, perhaps via this pathway.33 In mice, the small and large intestines contain the largest population of mature macrophages expressing the marker F4/80⫹ in the body. These cells are located in the lamina propria, in close contact with the epithelium, and also express CD11b. There are also low numbers of F4/80⫹ cells in mouse PP. Mucosal macrophages are in an ideal position to recognize any microbes or microbial products that cross the epithelial monolayer or to take up dying epithelial cells that have acquired antigens. Because macrophages avidly phagocytose particulate antigens and bacteria, it is possible that macrophages maintain intestinal homeostasis by clearing organisms that translocate across the epithelium. Intestinal macrophages express low levels of co-stimulatory molecules (CD40, CD80, and CD86) and low levels of major histocompatibility complex II. Unlike blood monocytes or macrophages present in other tissues, intestinal macrophages do not produce inflammatory cytokines or up-regulate co-stimulatory molecules in response to TLR ligands, whole bacteria, or nonmicrobial stimuli such as interferon-␥. This state of unresponsiveness has been associated with reduced expression of TLRs and other functional receptors necessary for macrophage activation (CD14; human Fc␥R1 and Fc␥RIII; complement receptors 3 and 4; and triggering receptor expressed on myeloid cells–1).34 Furthermore, intestinal macro-

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phages do not express, or down-regulate downstream signaling molecules used by TLRs to activate cells.35,36 Finally, normal GI tissue is enriched in molecules that can either desensitize macrophages (IL-10), or restrain activation (prostaglandin-E2, TGF-␤1).37,38 This state of macrophage hyporesponsiveness is not complete, because when these cells are stimulated by whole bacteria, they produce anti-inflammatory cytokines such as IL-10.39 Intestinal macrophages are therefore held in a state of partial activation that allows them to internalize and kill microbes, contribute to protective immunity, and maintain homeostasis.40 It is important to emphasize that the phenotype and function of resident intestinal macrophages differ from those of CD14⫹ macrophages derived from recently extravasated monocytes that infiltrate the mucosa in patients with IBD.41 Macrophages might also control inflammation, because mice with myeloid cell-specific deletion of signal transducer and activator of transcription factor 3, a transcription factor that mediates the inhibitor effects of IL-10, spontaneously develop colitis.42 However, signal transducer and activator of transcription factor 3’s effects might be mediated by DCs rather than macrophages. Intestinal macrophages also inhibit intestinal immune regulation. Small intestine F4/80⫹CD11b⫹CD11cdull macrophage-like cells can induce in vitro differentiation of Treg cells16; mice deficient in F4/80 have few CD8⫹ Treg cells and do not develop oral tolerance when fed protein antigens.43 Mice infected with the parasitic helminth Schistosoma mansoni are refractory to dextran sulfate sodium⫺induced colitis, and colon macrophages isolated from these mice protect against dextran sulfate sodium⫺induced colitis following transfer into recipient mice.44 Helminth infection is associated with development of alternatively activated macrophages, a subset of anti-inflammatory macrophages.45 Infection of mice with the tapeworm Hymenolepis diminuta increases markers that indicate alternatively activated macrophage differentiation, and in vitro⫺induced alternatively activated macrophages reduce colon inflammation when administered to mice.46 Further support for the anti-inflammatory role of intestinal macrophages comes from studies in which deletion of TLRs or MyD88 from bone-marrow⫺derived cells exacerbated experimental colitis.47,48 The exact mechanisms by which macrophages protected against colitis in these models are not understood, but might involve release of counter-regulatory molecules (eg, IL-10, IL-25) and cytoprotective factors.40

Control of Effector T Cells TCR signaling in lamina propria T cells is reduced compared with TCR signaling in peripheral blood T cells. For example, the former do not proliferate well when activated with anti-CD3 antibodies alone. This hyporesponsiveness has been associated with insufficient delivery of co-stimulatory signals, synthesis of low molecular

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weight, nonprotein mediators with oxidative capacities, and immunosuppressive cytokines.4 IL-22, a cytokine produced by Th17 cells and by a distinct subset of T cells (Th22 cells), might be important for GI inflammation.49 In mice, IL-22 appears to have anti-inflammatory effects, because it targets epithelial cells and induces secretion of defensins and mucus.50 Furthermore, LPL T cells undergo spontaneous apoptosis at high rates when placed in culture and in vivo.51,52 LPL T cells express the apoptotic molecule FAS, and a subpopulation express FAS ligand. They have increased levels of apoptosis compared with peripheral blood lymphocytes and respond to CD2 stimulation with increased FAS-dependent apoptosis. Patients with IBD have defects in the mechanisms that regulate T-cell apoptosis; the resistance of patients’ LPL T cells to FAS-driven apoptosis, via FLIP expression, might contribute to chronic inflammation.52,53 Response to immunosuppressive or biological therapies has consistently been associated with T-cell apoptosis.54,55

Regulatory T Cells There is enthusiasm for a model in which mucosal inflammation results from defective activity of Treg cells. In this model, effector T cells that react to the microbial flora or other GI antigens are kept in check by a population of regulatory cells; defects in these cells lead to GI inflammation. The most well-studied Treg cells include T cells that express the transcription factor fork head box p3 (FoxP3), Th3 cells, and Tr1 cells. However CD8⫹ T cells, natural killer T cells, B cells, and T cells that express the ␥␦ TCR have been also shown to suppress effector T-cell responses in various systems.56 –58 Immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome is a rare disease associated with mutations in FoxP3 that lead to defective activity of Treg cells.59,60 It is often used to demonstrate that defects in Treg cells cause colitis. Virtually all children with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome have small bowel inflammation and some have colitis. The GI manifestations, however, are associated with type I diabetes and inflammation of other endocrine organs, kidneys, liver, and skin. The patients often develop anemia or thrombocytopenia, autoantibodies, and have lymphadenopathy. It is therefore possible that children with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome have a general autoreactivity to self-antigens, and that the GI tissue is a target of autoimmune attack, rather than loss of regulation of the T-cell response to the microflora. Most data on Treg cells and gut homeostasis has been collected from studies of cells that express FoxP3. Two classes of mouse, FoxP3-expressing Treg cells have been identified. The first so-called naturally occurring Treg cells were recognized as a subpopulation of CD4⫹ T cells that develop in the thymus during the first days of postnatal life and express high levels of the IL-2 receptor

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␣ chain.60 Naturally occurring Treg cells account for 1% to 2% of peripheral CD4⫹ T cells and are believed to maintain tolerance toward self-antigens.61 In contrast, inducible Treg (iTreg) cells, which, like naturally occurring Treg cells, express FoxP3, develop from naive CD4⫹ T cells in the presence of TGF-␤1.62– 64 Although FoxP3 expression is sufficient to induce the suppressive capacity in T cells in mice, TGF-␤1–mediated induction of FoxP3 in human cells does induce regulatory functions, indicating that additional stimuli are required to induce differentiation of functional human iTreg cells.65 RA could be one of these factors because it induces FoxP3 and promotes differentiation of iTreg cells.66 The intestinal environment could be an important site for generation of iTreg cells. After antigen-specific activation, Treg cells suppress several populations of effector T cells in an antigen-independent manner.67 iTreg cells might maintain tolerance toward dietary- and microbiome-derived antigens; administration of Treg cells to lymphopenic mice prevents development of and reduces existing intestinal inflammation.68 –70 However, Treg cells have not been shown to reduce established colitis in mice with an intact immune system. The mechanism by which iTreg cells inhibit inflammation in the GI tract is not clear. Although in vitro studies indicated that cell– cell contact was required, studies in mice have shown that Treg cell–induced suppression requires soluble factors. Treg cells might express the immunosuppressive cytokine IL-10, but its involvement in Treg cell– mediated counter-regulation is not clear. In the T-cell transfer–induced model of colitis, Treg cells isolated from IL10⫺/⫺ mice suppressed colitis, similar to wild-type Treg cells.71–73 However in the same model, treatment of mice with a neutralizing antibody against IL-10 receptor reduced the ability of Treg cells to suppress colitis, indicating that IL-10 made by cells other than Treg cells help suppress mucosal inflammation.72,73 Similar results were found from other experimental models of colitis.74,75 Overall however, IL-10 production by Treg cells appears to be required for suppression of colitis. TGF-␤1 has been also implicated in the Treg cell– mediated suppression. In mice with T-cell transfer–induced colitis, administration of anti–TGF-␤ prevented Treg cell–mediated suppression of colitis.76 In the same model, CD45RBhigh cells, which express a dominant-negative form of TGF-␤ receptor type II and therefore cannot respond to TGF-␤1, are resistant to Treg-mediated suppression.77 Another molecule implicated in the function of Treg cells could be adenosine, a nucleotide with antiinflammatory properties. Administration of an adenosine agonist to mice reduces intestinal inflammation; adenosine receptor⫺deficient CD45RBhigh cells are resistant to Treg-cell suppression in animal models of colitis.78,79 Other factors involved in the Treg-mediated suppressive activity include IL-35 (a cytokine comprising EBI3 and the IL-12␣ chain)80 and IL-2.81

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Studies that evaluated the functional activity of FoxP3expressing cells from intestinal lamina propria or MLNs of IBD patients did not detect defects in the suppressive capacity of Treg cells.82,83 Moreover, there are greater numbers of FoxP3-positive cells in the inflamed GI tract of patients with IBD than healthy controls,82,84,85 yet the inflammation proceeds. It is possible that the number of Treg cells that accumulate in the GI tract of patients with IBD is insufficient to control the overwhelming influx of colitogenic lymphocytes. In vitro, effector T cells from patients with IBD are resistant to Treg-cell–mediated suppression, possibly because mucosal cells from patients with IBD express high levels of Smad7, an inhibitor of TGF-␤1 signaling. Inhibition of Smad7 with a specific antisense oligonucleotide restored the susceptibility of T cells from patients with IBD to Treg-cell–induced suppression.86 Therefore, Treg-cell therapy for IBD might not be effective, because the problem is not localization or numbers of the cells to inflamed intestine, but that in the intestine, effector cells are resistant to suppression, at least via TGF-␤1. A final problem may be plasticity, in that there is now evidence that in inflamed environments, Treg cells can become proinflammatory, Th1 or Th17 cells.87

Role of the Epithelium Although other reviews cover the topic more thoroughly (Abraham et al in this issue), the epithelium has important roles in intestinal homeostasis and inflammation. Migration of T cells to the GI tract is partially controlled by epithelial-derived chemokines. For example, the small bowel epithelium secretes CCL25 and GI-homing cells express CCR9. CCL25 is tethered to vascular endothelium in the GI tract.88 During GI inflammation, epithelial cells release chemokines such as CXCL8, which helps attract granulocytes into the tissue. In healthy individuals, GI epithelial cells produce activated TGF-␤, which can nonspecifically reduce proinflammatory T-cell responses in the lamina propria.89 In the same vein, GI epithelial cells release thymic stromal lymphopoietin, which tolerizes lamina propria DCs; interestingly, this effect is lost in patients with Crohn’s disease.90

Conclusions The GI immune system interacts with a large number of dietary and microbial antigens and promotes a state of nonpathogenic inflammation. The integrity of the intestinal epithelial barrier and the function of specialized immune cell types are required to differentiate harmful antigens and invaders from innocuous dietary components and beneficial commensal bacteria, and therefore for the maintenance of immune homeostasis and health. Defects in GI permeability and/or resident regulatory cells lead to inappropriate immune responses, even against harmless food antigens or luminal bacteria. The resulting GI leakiness

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