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Keywords:

  • epithelial cells;
  • enterocytes;
  • mucosal immunology;
  • inflammatory bowel diseases

Abstract

  1. Top of page
  2. Abstract
  3. EPITHELIAL MONOLAYER
  4. ENTEROCYTE
  5. ENTEROCYTE–T-CELL INTERACTION
  6. CONCLUSION
  7. REFERENCES

The intestinal epithelium not only acts as a physical barrier to commensal bacteria and foreign antigens but is also actively involved in antigen processing and immune cell regulation. The inflammatory bowel diseases (IBDs) are characterized by inflammation at this mucosal surface with well-recognized defects in barrier and secretory function. In addition to this, defects in intraepithelial lymphocytes, chemokine receptors, and pattern recognition receptors promote an abnormal immune response, with increased differentiation of proinflammatory cells and a dysregulated relationship with professional antigen-presenting cells. This review focuses on recent developments in the structure of the epithelium, including a detailed account of the apical junctional complex in addition to the role of the enterocyte in antigen recognition, uptake, processing, and presentation. Recently described cytokines such as interleukin-22 and interleukin-31 are highlighted as is the dysregulation of chemokines and secretory IgA in IBD. Finally, the effect of the intestinal epithelial cell on T effector cell proliferation and differentiation are examined in the context of IBD with particular focus on T regulatory cells and the two-way interaction between the intestinal epithelial cell and certain immune cell populations. (Inflamm Bowel Dis 2011;)

The hypointestinal mucosa is constantly exposed to a milieu of both commensal bacteria and dietary antigen. The gut is therefore uniquely characterized by a state of responsiveness through carefully balanced local responses. This active process not only maintains mucosal integrity but also prevents the induction of an overwhelming immune response leading to intestinal inflammation. At the heart of this complex interface is a single monolayer of intestinal epithelium acting as a physical barrier to foreign antigen. However, in addition to this barrier function the intestinal epithelial cell (IEC) also recognizes, processes and presents antigen, produces a myriad of signaling molecules, and directly affects immune cell proliferation and differentiation.

The chronic inflammatory bowel diseases (IBD), encompassing both Crohn's disease (CD) and ulcerative colitis (UC), are characterized by inflammation at this mucosal surface. Within these complex diseases it is clear that genetic predisposition, environmental elements, and a dysregulated immune response are involved in both the pathogenesis and the chronic relapsing course that typifies these conditions.1 It is also apparent that abnormalities within the epithelial layer, such as increased permeability and abnormalities in IEC-immune cell interactions, play a key role in the disease process.2, 3

This review focuses on epithelial barrier function, epithelial cell-antigen interaction, and the role of the epithelium in orchestrating the immune response, highlighting specific dysregulated mechanisms within IBD.

EPITHELIAL MONOLAYER

  1. Top of page
  2. Abstract
  3. EPITHELIAL MONOLAYER
  4. ENTEROCYTE
  5. ENTEROCYTE–T-CELL INTERACTION
  6. CONCLUSION
  7. REFERENCES

Epithelium Structure

The proximal and distal sections of the human intestinal tract (i.e., the oral cavity, esophagus, and anus) are lined with stratified squamous epithelium and are commonly affected in CD patients. Of particular note, the oral cavity has been shown to highly express the epithelial barrier protein filaggrin (a strong genetic determinant of atopic eczema),4 with null alleles of its coding gene FLG contributing to coexistent eczema and food allergy in pediatric IBD patients.5 The epithelium lining the remainder of the gut is composed of nonciliated, columnar epithelium. This rapidly self-renewing tissue is primarily involved in the absorption of water and nutrients, but also has a vital role in acting as a barrier to luminal pathogens. The epithelial monolayer is carefully folded, producing crypts (of Lieberkühn) and villous protrusions. The crypts are an important site of IEC differentiation and it is here that the four IEC types are derived from pluripotent stem cells.6 Three of the cell types (goblet, enteroendocrine, and absorptive) migrate to the tip of the villus, where they undergo spontaneous apoptosis several days after terminal differentiation.7 On the contrary, Paneth cells remain within the crypt after differentiation and have a considerable role in antibacterial defense.8 The known genetic hierarchy of the epithelial cell lineage commitment is complex, involving two main signaling pathways: Wnt and Notch; but also includes many other transcription factors such as Hes1, Math1, and Elf3.6

The secretory lineages (Paneth, goblet, and enteroendocrine cells) are in small numbers within the epithelial layer, with the absorptive lineage (the enterocyte) accounting for over 80% of the cells.9 These abundant enterocytes are highly polarized, with an apical brush border that is in constant contact with the luminal contents and a basolateral component adjacent to the stroma.

In addition to these differentiated IECs, the epithelium is intermittently interrupted by lymphoid aggregates called Peyer's patches. These are overlaid by specialized follicle-associated epithelium containing M (microfold) cells that are structurally and positionally very different from enterocytes.10 M cells contain fewer lysosomes, more mitochondria, less mucus glycocalyx, and have direct basolateral access to underlying lymphoid cells. These cells are adept at sampling and transcytosing luminal antigen, with their differentiation recently shown to be dependent on receptor activator of NF-κB ligand (RANKL) expression in mice.11, 12 Although these cells have been shown to be vital in maintaining oral tolerance within the gut, M cells make up a small proportion of the overall intestinal surface area, and the lack of a specific marker for human M cells has severely hindered the study of this cell type in IBD.13 In view of the limited numbers of M cells within the epithelium, the role of the predominant absorptive enterocyte in the recognition, processing, and presentation of luminal antigens is likely significant.

Intraepithelial Lymphocytes

Interspersed between the single epithelial cells lining the intestine are intraepithelial lymphocytes (IELs) (Fig. 1). These cells, which express αβ T-cell receptors (TCRs) or γδTCRs, monitor for stressed or damaged IECs.14 IELs can be subdivided into two types: “type a” (expressing αβTCRs with CD4 or CD8αβ) and “type b” (expressing either αβTCRs or γδTCRs with the unique coreceptor CD8αα, and lacking markers such as cytotoxic T lymphocyte antigen-4).15 The frequency of IELs, certainly within murine models, varies between the small and large intestine with up to 10 times more residing in the colon.16 Furthermore, the abundance of naïve IELs also differs significantly within the bowel, with greater numbers in the large intestine (≈40%) compared to the small intestine (<5%).17

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Figure 1. Overview of intestinal intraepithelial lymphocytes. The interaction between IELs and IECs is complex and involves many “homing” signals which lead to multiple trafficking pathways within the lamina propria. Type “b” IELs are the predominant lymphocyte within the epithelium, with type “a” IELs also present in the blood and secondary lymphoid organs. The interaction between NKT IELs is via the MICA-NKG2D complex with other IELs utilizing the αEβ7-E-cadherin interaction at the adherens junction. In addition, RTE IELs expressing multiple ligands traffic to the intestine via α4β7-MAdCAM-1 interactions within the HEV. IEL, intraepithelial lymphocyte; IEC, intestinal epithelial cell; NKT, natural killer T cell; RTE, recent thymic emigrant; HEV, high endothelial venules. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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With regard to IBD, several pathways involving IELs are of particular interest. A proportion of IELs, so-called natural killer T (NKT) IELs, express NKG2D, which is a receptor for MHC class I polypeptide-related sequence A (MICA) and MHC class I polypeptide-related sequence B (MICB), which are nonclassical MHC-related class 1b molecules.18 MICA is preferentially expressed on γδ T cells and may function as a signal of cellular stress.19 Allez et al20 have demonstrated upregulation of MICA on the IECs of CD patients leading to the activation and subsequent increase in a subset of CD4+ effector cells (CD4+NKG2D+). This subset is increased in the lamina propria of patients with CD compared with controls and is functionally active through MICA-NKG2D interactions, producing interferon-gamma (IFN-γ) and killing targets expressing MICA. The same group20 also showed that CD4+NKG2D+ lymphocytes from patients with CD highly express interleukin-15R alpha, and that interleukin-15 increased the expression of an activating immunoreceptor complex (composed of NKG2D and DAP10) in CD4+NKG2D+ T-cell clones. Although this and other studies21 have shown the enhancement of cytotoxic effects of NKT IELs by IL-15, there is conflicting work showing the protective effects of IL-15 in renal and intestinal epithelium.22, 23 It is of further interest that IL-15R mRNA is higher in duodenal tissue specimens in those with celiac disease (which has several shared susceptibility genes with CD/IBD24), in addition to a lower gliadin-induced IL-15 response threshold that drives immune processing.25

The key chemokine receptor CCR9 (CDw199) and cellular adhesion β7 integrins are also directly involved in IEL trafficking in the gut.26 CCR9 and its ligand have been shown to play a key role in the regulation of lymphocyte homing to the small intestine,27 in particular recruitment of CD8αβ+ T cells to the epithelium.28 Clinical studies have also shown increased numbers of CCR9+ lymphocytes in the peripheral blood of patients with small bowel CD29 in addition to the increased production of inflammatory cytokines (e.g., IFN-γ and IL-17) by CCR9+ lymphocytes derived from the mesenteric lymph nodes of these patients.30 With regard to β7 integrins, the α4β7 heterodimer plays a key role in T-cell migration to the lamina propria, whereas the integrin αEβ7, expressed by IELs,31 binds E-cadherin and is involved in the retention of T effector cells in the epithelial compartment.32 The α4β7 ligand MAdCAM-1 has also been shown to be increased in sites of mucosal inflammation in IBD patients,33 along with the observation that MAdCAM-1-specific antibody alters the inflammatory response in SCID mice.34 Additionally, Staton et al35 have also recently demonstrated a specific CD8+ population of naïve IELs called recent thymic emigrants (RTEs). These nonactivated cells exclusively express α4β7 integrin, αEβ7 integrin, and CCR9 and migrate from the thymus, proliferating in response to gut-specific antigen. Both CCR9 and α4β7 have been targets of therapeutic interest in IBD with some promising results; however, their routine inclusion in the arsenal of therapeutic options in IBD is still uncertain.36, 37

Epithelial Barrier Function

In order for the individual epithelial cells to effectively compartmentalize the underlying stroma, they must be connected by intercellular junctions that can contribute to the epithelium's overall barrier function. The three main junctions between these cells are the tight junctions (TJs), the adherens junction (AJ) (collectively referred to as the apical junctional complex [AJC]), and the desmosomes (DMs). The TJ is a highly dynamic structure that forms a continuous, circumferential barrier towards the luminal side of the intercellular junction. The three unique families of transmembrane proteins which make up the TJ are occludin, claudins, and junctional adhesion molecules (JAMs).38 These proteins help maintain cellular polarity by regulating the flux of water, electrolytes, lipids, and proteins across the epithelium. Furthermore, several of these proteins (e.g., occludin and claudins) have also been found to be directly involved in the differentiation of epithelial cells.39, 40 Immediately below the TJ lies the AJ, which is mainly composed of cadherin–catenin interactions. The cadherin superfamily plays a key role in the maintenance of cell polarity and differentiation and includes E-cadherin, which is intricately involved in cell adhesion between IECs.41 Finally, at the basal end of the IEC the DMs provide anchoring sites for keratin filaments and have intracellular proteins (plakoglobin and desmoplakin) that connect the cytoskeleton to cadherin-family adhesion proteins (Fig. 2).42

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Figure 2. The intestinal epithelial junctional complexes. Each enterocyte is connected at the tight junction (TJ), adherens junction (AJ), and desmosomes (DMs) from the luminal to lamina propria side, respectively. (A) The TJs are localized to the apical-lateral membrane junction and consist of three integral transmembrane proteins (claudins, occludin, and JAMs) with the proteins connected to the actin cytoskeleton through PDZ-domain-containing proteins via the zonulins (ZO-1,2,3) or PAR adaptor proteins. (B) E-cadherin is the main transmembrane protein at the AJ and associates with the alpha and beta catenins; this complex is further maintained through interaction with the serine/threonine phosphatase PP2A. (C) The DMs lie at the basal end of the enterocyte and are in contrast to the TJ and AJ in that they associate with the keratin filament cytoskeleton of the cell. JAMs, junctional adhesional molecules; PAR-3, partitioning defective-3; ZO, zonulin; Keratin IF, keratin intermediate filament.[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Defects in these intercellular junctions have been shown to lead to increased epithelial permeability, which has been reported in those suffering from IBD and first-degree relatives.43–46 Intestinal permeability has also been shown to predict and possibly cause relapse47, 48 and is increased in those at high risk of CD at baseline.49 Defects in E-cadherin have been of particular interest in IBD research with upregulation in areas of active inflammation50 and downregulation by pathogens implicated in the development of colitis such as invasive E. coli.51 In addition, recent work has shown that the gene encoding E-cadherin (CDH1) is associated with both CD and UC, with patients possessing the disease-associated single nucleotide polymorphisms (SNPs) having increased E-cadherin cytoplasmic accumulation.41, 52 The tight junction protein junctional adhesion molecule A (JAM-A) has also recently been studied in the context of IBD. Vetrano et al53 demonstrated a loss of epithelial JAM-A in both CD and UC patients in addition to showing that JAM-A-deficient mice were more susceptible to chemically-induced colitis. Furthermore, the gene encoding protein tyrosine phosphatase N2 (PTPN2) is a known CD susceptibility gene54 and has been shown to play a functional role in maintaining the intestinal epithelial barrier, with knockdown leading to increased intestinal permeability via IFN-γ induction55 and more severe chemically-induced colitis.56 Occludin expression,57 claudin dysregulation,58 and altered cytokine production59, 60 have also been implicated in the overall barrier disruption in IBD; however, the mechanisms involved remain unclear and often controversial.61

ENTEROCYTE

  1. Top of page
  2. Abstract
  3. EPITHELIAL MONOLAYER
  4. ENTEROCYTE
  5. ENTEROCYTE–T-CELL INTERACTION
  6. CONCLUSION
  7. REFERENCES

Enterocyte Structure

The enterocyte itself is a hyperpolarized columnar epithelial cell; the apical surface covered in a mucus coat of glycoproteins (mucin) composed of carbohydrate side chains bound to a protein skeleton.9 This viscous layer acts as a physical barrier to antigens by reducing diffusion of larger molecules. In addition, glycoproteins competitively bind to surface receptors limiting the attachment of proteins and microorganisms from the lumen.62 Directly underlying the mucin layer, a 400–500 nm thick glycocalyx layer on the negatively charged microvilli contains adsorbed enzymes responsible for digestion. This layer further limits the uptake of antigens while promoting nutrient absorption.

Enterocyte–Antigen Interaction

There is increasing evidence that IECs not only provide a physical barrier to antigens but can also act independently as antigen-presenting cells (APCs) to more tightly control the immune response.63 In order to act as an APC the enterocyte must therefore possess the apparatus capable of internalizing, processing, and efficiently presenting foreign antigen.

Antigen Recognition

As alluded to in the introduction, the intestinal epithelium is constantly exposed to an enormous dietary antigen load and a multitude of bacterial phylotypes.64 Individuals may harbor up to 160 different species of bacteria, with considerable interperson variation, in addition to variations between those with UC and CD.65 Commensal bacteria have become of particular interest recently with metagenomic sequencing now able to better characterize the gut flora in addition to highlighting differences between health and disease.65 The lack of a massive mucosal inflammatory response within this environment is not due to passive unresponsiveness but rather an active process involving the gut-associated lymphoid tissue, lamina propria cells, and the IEC. In order to actively suppress any inflammatory response the gut employs numerous mechanisms such as limiting the initial innate immune response, recruiting immune regulatory cells, and secreting antiinflammatory cytokines.

With regard to the recognition and response to enteric bacteria, the strongest link to dysregulation in IBD came from the identification of nucleotide oligomerization domain-2 (NOD2/CARD15) mutations predisposing to CD.66 This pathogen recognition molecule recognizes muramyl dipeptide (MDP), a specific motif of bacterial peptidoglycan, with CD-associated mutations altering the recognition of MDP at the NOD2/CARD15 leucine-rich repeat region.67 In addition, it has been recently shown that NOD2/CARD15-deficient mice harbor an increased number of commensal bacteria with a diminished ability to prevent colonization of pathogenic bacteria.68 Another group of receptors that has gained considerable interest with regard to innate pattern recognition in IBD are the Toll-like receptors (TLRs). A variant allele (Asp299Gly) in the gene encoding TLR4, which recognizes bacterial lipopolysaccharide and viral glycoproteins,69, 70 with other genes in the TLR4 pathway are also recently implicated.71 TLR4 is associated with the activation of NF-κB leading to the release of proinflammatory cytokines,72, 73 in addition to other mechanisms such as intestinal restitution (resealing of superficial erosions by IEC migration) and apoptosis (controlled cell-death).74 Other TLRs such as TLR9, which recognizes CpG motifs, have also been shown to be important in IBD. Török et al. demonstrated that the −1237C allele at the TLR9 locus was more common in IBD patients compared to controls, with this finding replicated in a more recent meta-analysis of CD patients.75, 76 Sanchez et al77 have also recently shown that TLR9 mRNA is significantly upregulated in rectal biopsies of UC patients. One further molecule involved in innate immune recognition which has been shown to be dysregulated in IBD is CARD9. SNPs in the region of CARD9 were found to confer susceptibility to CD,78 with CARD9 also been shown to be important in NOD2/CARD15 signaling,79 dendritic cell maturation,80 and most recently the antiviral response through retinoic acid inducible gene-I (RIG)-like receptors (RLRs).81

Recently a renewed interest in the role of mucosal dendritic cells (DCs) in IBD pathogenesis has occurred.82, 83 This has mainly been as a result of work demonstrating that DCs are primed locally to undertake tissue-specific roles within the intestine.84 Some of these local functions include driving the differentiation of certain T-cell populations,85, 86 the development of IgA-producing B cells,87 and the selective imprinting of gut-homing T cells.88 There is considerable crosstalk between the IEC and lamina propria DCs, with IECs presenting antigen to DCs,89 in addition to DCs playing an important role in IEC homeostasis.90 The observation that DCs could express tight junction proteins and sample luminal antigens by intercalating their dendrites between IECs91 has led to further work demonstrating the ability of the IEC to promote the differentiation of tolerogenic CD103+ DCs, a capacity lost in CD.92 It has also been recently demonstrated that monocyte-derived inflammatory DCs expressing E-cadherin, a receptor for CD103 (or alpha E), are associated with the pathogenesis of intestinal inflammation.93 In addition, the therapeutic potential of DCs in IBD has also been recognized, with TGF-β1 gene-modified and regulatory probiotic-induced DCs suppressing murine models of intestinal inflammation.94, 95 Work on thymic stromal lymphopoietin (TSLP), a hematopoietic cytokine secreted by the IEC, has shown that dysregulated IEC-intrinsic TSLP expression directly affects the production of DC-derived proinflammatory cytokines.96 It has also been shown that TSLP and as-yet unidentified IEC-derived factors induce TSLP receptor expression on mucosal DCs, which may skew the normal Th1/Th2 balance, with altered expression in CD patients also described.97 The more general role of mucosal DCs in IBD is discussed in detail elsewhere.82, 98

Antigen Uptake

Following the identification of pathogens, the IEC must then begin the process of internalization in order to fully process and present the relevant antigens. There are two main pathways that are utilized by the IEC in order to initiate antigen uptake, namely, fluid phase and receptor-mediated endocytosis.99

Although IECs lack true phagocytic activity, they have been shown to ingest antigen by fluid phase pinocytosis (or “cell drinking”).42 Within the IEC this process likely occurs via three different mechanisms: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis.9 These complex pathways are possibly maturity-dependent, with increased endocytosis demonstrated within fetal enterocytes.100 During pinocytosis the solubility of the antigen, regardless of size, has been found to be most important, with kinetic studies showing that the IEC has significantly slower uptake than professional APCs.101

IECs are also able to take up antigen by receptor-mediated endocytosis, delivering molecules to endolysomal compartments, or for transport of intact macromolecules across the cell.99 There are numerous receptors involved in this process but many of the mechanisms involved are still unclear. A receptor that has gained considerable interest is the neonatal Fc receptor (FcRn), a β2-microglobulin-associated, type 1 transmembrane protein with similarities in structure to classical MHC class I molecules.89, 99 The major ligands for this receptor are immunoglobulins (mainly IgG) and albumin.102 In mouse models the FcRn has been found to deliver IgG across the IEC to the lumen, and to then reverse the process by transporting the IgG-antigen complex to the basal surface to facilitate presentation to interstitial DCs.89 Kobayashi et al103 have recently shown that antibacterial IgG antibodies are involved in the pathogenesis of colitis, with the FcRn found to be required for its presentation. In addition, a similar low-affinity receptor for IgE, FcεRII(CD23), is expressed on the IEC104 and has been found to be upregulated on biopsy specimens from IBD patients.105 Although the gene encoding FcεRII (FCER2) lies at the IBD6 susceptibility locus on chromosome 16p, SNPs in the gene itself have not been found to confer susceptibility to IBD,106 unlike a similar low-affinity IgG receptor whose gene (FCGR2A) has recently been shown to confer susceptibility to UC.107

Antigen Processing

Initial work by Hershberg et al108 demonstrated that IECs possess some of the major proteases (e.g., cathepsins) involved in antigen processing through endosomal compartments. Further details of the IECs ability to process antigen has been evidenced by work that suggests that undegraded antigens are internalized and enter apical or basal early endosomes, leading to either the recycling of antigen to the appropriate surface or proceeding through the endocytic pathway.9, 109 Furthermore, recent work has shown that one group of major histocompatibility complex-II enriched compartments (MIICs), the multivesicular body (MVB), seems to be principally responsible for class II-associated antigen processing in IECs, irrespective of mucosal inflammation, and constitutes the origin of MHC II-loaded exosomes.110 Due to the fact that no differences were identified in this process during mucosal inflammation suggests that processing and editing within the MVBs may account for MHC-II restricted antigen presentation in health and disease.

There is currently some evidence that there is increased uptake of luminal antigens into endosomes in the ileum of CD patients, possibly mediated by TNF-α.111 In addition, a recently described distinct enterocyte population termed rapid antigen uptake into the cytosol enterocytes (RACE) cells have been shown to be increased in IBD with numbers correlating with the degree of mucosal inflammation.112 These cells show increased antigen transport to late endosomes and the trans-Golgi network in addition to a disassembled cytoskeleton.112

The process of autophagy (i.e., the delivery of portions of the cytoplasm to the lysosome for degradation) has received increasing attention, with three loci containing autophagy-related genes (ATG16L1, IRGM, and LRRK2) reaching genome-wide significance in the CD GWAS meta-analysis54 and two (ATG16L1 and IRGM) in UC.52 Recent work in this area has shown that in the ileal epithelium ATG16L1 is crucial for Paneth cell biology,113 with CD patients homozygous for the risk allele displaying Paneth cell granule abnormalities.114 In addition, Kuballa et al115 showed that the CD-associated ATG16L1 variant impaired Salmonella handling in human IECs. IRGM, a member of the GTPase family, has similarly been shown to have key roles in the protection against adherent-invasive E. coli,116 the protection of effector CD4+ cells against IFN-γ-induced autophagic cell death117 and macrophage motility,118 with the role of LRRK2 in the selective autophagic response in CD currently under intense investigation. It should also be noted that a link between innate immune recognition molecules (NOD1 and NOD2) and autophagy has now been established,119, 120 with alterations in the ubiquitin-proteasome system (which accounts for the remaining 80% of intracellular component degradation) also demonstrated in IBD.121 It is not yet clear how dysregulated pathways in antigen processing affect IBD etiopathogenesis; however, it is likely that a more detailed insight into these complex mechanisms will expand our knowledge of potential abnormalities in intracellular processing.

Antigen Presentation

In order to efficiently present the internally processed antigen IECs must express major histocompatibility complex (MHC) class molecules. It has now been clearly shown that IECs not only constitutively express MHC class-II122 but also nonclassical MHC class I molecules (e.g., MICA/B, HLA-E, CD1d, MR1, and UL16-binding protein).123 This latter group has been of interest in recent years, with Mayer's group demonstrating altered expression of several of these molecules in IECs from IBD patients.124 In addition, a recent Chinese study has demonstrated UC-related risk alleles in the gene encoding MICA.125 Although potential ligands for molecules such as MR1126 and a possible role of CD1d expression on IECs in the regulation of intestinal bacterial colonization have been suggested,127 the overall role of this diverse group of molecules remains elusive.

In addition, IECs have been found to secrete exosomes both in basal and inflammatory conditions.128 Again, enterocyte polarity is crucial, with exosomes released from the apical and basal surfaces containing different molecules.129 In noninflammatory conditions exosomes express MHC class I and class II molecules with upregulation during inflammation, with more recent evidence suggesting the exosomes released by IECs interact preferentially with gut DCs, further increasing the IECs role in immune regulation.128

Enterocyte Secretory Function

The epithelium produces a large repertoire of antimicrobial proteins in order to prevent microbial invasion of the intestinal tissue. These proteins are grouped into major classes such as the enzymatically active proteins (e.g., lysozymes and secretory phospholipid A2), defensins, cathelicidins, RNases, and C-type lectins. The defensins are a group of antimicrobial peptides that are secreted by a variety of cell types.130 They exert their bactericidal activity by forming microspores which disrupt the phospholipid bilayer of the bacterial membrane.131 Several defects have been shown to be present in this group of peptides including the reduction of human defensin 5 (HD5) and human defensin 6 (HD6) in both adult and treatment-naïve pediatric CD.132, 133 In addition, the cathelicidin LL-37 has been found to be upregulated in UC,134 with the C-type lectin MGL1/CD301a having been shown to play an antiinflammatory role in murine experimental colitis through the upregulation of IL-10.135 Furthermore, recent genetic analysis has demonstrated that an SNP in the region of CLEC16A (which encodes a protein containing a C-type lectin domain) was associated with CD patients specifically lacking the three known CD-associated NOD2/CARD15 mutations.136

Secretory IgA (sIgA)

Another element of the IECs barrier function is the transport of immunoglobulins to the intestinal lumen. Immunoglobulin A (IgA) is the most abundant antibody isotype produced in the body and provides mucosal protection via interaction with the polymeric Ig receptor (pIgR).137 IgA dimers are transcytosed to the apical surface following binding to pIgR on the basolateral aspect of the IEC and sIgA is generated at the surface.138 Secretory IgA plays several protective roles at the mucosal surface including the reduction of intestinal proinflammatory signaling and bacterial epitope expression,139 facilitation of antigen-sampling by M cells,140 and clearance of microorganisms via FcαRI (CD89).141 pIgR-deficient mice show greater intestinal disease severity compared with IgA-deficient mice when challenged with dextran-sulfate sodium (DSS), suggesting that pIgR and/or the secretory component are important in maintaining epithelial integrity.142 Furthermore, FcαRI has recently been shown to interact with dimeric IgA during the recruitment of neutrophils with excessive IgA-antigen complexes sustaining the inflammatory process in UC.143

Enterocyte–Cytokine Interaction

In the last decade the arrival of monoclonal antibodies (such as anti-TNF-α) in clinical practice has led to dramatic changes in IBD treatment.144 Although the indications and treatment regimens for these drugs are still under debate,145, 146 several of these drug therapies have proven efficacy, with newer biologicals showing promising results in early trials.147, 148 Even the most effective of these biologicals (anti-TNF-α) only induces remission in around 50% of adult CD patients,149 although higher rates are reported in pediatric studies.150, 151 There are therefore renewed efforts to identify other cytokine pathways involved in IBD etiopathogenesis and although a myriad of cytokines are likely to be involved in IBD, only the most recently implicated are discussed below.

Interleukin-21 (IL-21) is a recently described T-cell derived cytokine152 which signals through the common γ chain of the IL-2 receptor and its own receptor IL-21R.153 Binding of IL-21 to its receptor enhances the proliferation of prestimulated T cells154 and also regulates B and NK cell proliferation.155 IL-21R expression is upregulated on IECs from IBD patients compared with controls and stimulation of IECs with IL-21 leads to increased MIP-3α synthesis (a T-cell chemoattractant).154 IL-21-deficient mice have furthermore been found to be largely protected against DSS- and TNBS-induced colitis, with an inability to upregulate Th17-associated molecules during inflammation.156 There is also evidence that IL-21 plays an important role in the balance between effector and regulatory T cells in the gut.157

Interleukin-22 (IL-22) binds at the cell surface to two chains, namely, IL-22R1 and IL-10R2, and has been implicated in the inflammatory processes of hepatic,158 pancreatic,159 and skin disease.160 In response to IL-22 stimulation, colonic epithelial cells have been shown to overexpress acute phase proteins including α-antichymotrypsin and serum amyloid A,161 both of which have been shown to be markers of disease activity in IBD.162, 163 In addition, Brand et al164 have recently shown that IL-22 increases IEC proliferation and β-defensin production and is significantly upregulated in inflamed CD lesions as well as in DSS-induced murine colitis.

Interleukin-31 (IL-31) is a helical cytokine belonging to the gp130/IL-6 family, which also includes IL-27.165 In vitro the biological activity of IL-31 on overall cell proliferation is cell type-, cell density-, and cytokine concentration-dependent.166 This has specifically been shown in the IEC line HCT116, in which suppression of cell proliferation occurs at low cell density but the effect is lost, and in some cases reversed, in conditions of high cell density.167 IL-31 has been found to mediate certain transcription factors which in turn enhance IL-8 expression in the IEC167 and may also have roles in IEC proliferation and migration.168

Interleukin-32 (IL-32) is a proinflammatory cytokine expressed in various tissues including the spleen, thymus, small intestine, and colon.169 Its proinflammatory properties consist of stimulating the secretion of IL-1α, TNF-α, IL-6, and IL-8 via the activation of NF-κB.170 With regard to its role in the pathogenesis of IBD, studies have shown that IL-32α is overexpressed in the inflamed mucosa of IBD patients.171 This upregulation was in contrast to weak expression in normal colonic mucosa and the intestinal mucosa of patients with ischemic colitis. Furthermore, work by Netea et al170 has shown that IL-32 synergizes with the NOD2 pathway by augmenting the production of IL-1β and IL-6 in response to MDP.

Chemokines

The chemokines are a large group of chemotactic cytokines with roles in leukocyte migration in addition to hematopoietic progenitor cell trafficking.172 Many chemokines are produced by the enterocyte itself, with their functions and structures varying widely. Chemokines are classified into four families based on the arrangement of highly conserved cysteine residues, namely, CXC, CC, XCL1/lymphotactin, and CX3CL1/fractalkine.172 These families can be further classified into constitutive/homeostatic and inducible/inflammatory forms. Chemokines exert their actions through the binding and activation of specific 7 transmembrane G-protein-coupled receptors173 leading to additional roles in angiogenesis,174 immune tissue development175 and epithelial healing.176 An array of inducible/inflammatory chemokines such as CXCL 8177 and CCL20178 are upregulated in IBD, with early attempts to regulate chemokine levels in IBD patients in vivo showing promising results.179 In addition to this, constitutive/homeostatic chemokines such as CXCL12 and CCL14 have also been shown to be dysregulated in IBD,180, 181 with others such as CCL25 and CCL28 involved in the trafficking of specific T-cell subsets in the small and large intestine respectively.28, 182

ENTEROCYTE–T-CELL INTERACTION

  1. Top of page
  2. Abstract
  3. EPITHELIAL MONOLAYER
  4. ENTEROCYTE
  5. ENTEROCYTE–T-CELL INTERACTION
  6. CONCLUSION
  7. REFERENCES

T-cell Proliferation

In both murine and human models the ability of the IEC to alter T-cell proliferation has been demonstrated, although the mechanisms involved remain unclear. An early study by Christ et al183 studied the effect of IEC supernatant on CD3-mediated T-cell proliferation. Using a series of epithelial cell lines and both peripheral and intraepithelial T cells they showed that IEC supernatant contains factors that inhibit T-cell proliferation. In a similar experiment Cruickshank et al184 used a coculture system of murine IECs with splenic CD4+ T cells. Here it was demonstrated that CD4+ cells remained in the G1 phase of the cell cycle when cultured with IECs, and that IECs prevented the activation of these CD4+ cells by professional APCs. However, they found that this suppression of T-cell activation was cell-contact-dependent and independent of TGF-β. Several studies have shown that T-cell proliferation is dependent on the expression of MHC class-II by the IEC,185, 186 with other studies showing different inhibitory and stimulatory responses to peripheral blood and lamina propria T cells.187, 188 In addition, many of these early studies used irradiated epithelial cells, almost certainly altering the vital epithelial cell–T cell crosstalk. With regard to the mechanisms involved in IEC-T cell proliferation, IECs do not express classical costimulatory molecules (such as CD80, CD86), which are needed to interact with local T cells; however, they do express other costimulatory molecule ligands such as ICOSLG.189 Recent genome-wide association studies have delineated a possible germline mutation of ICOSLG as a possible risk locus for CD and UC in both the adult and pediatric populations, suggesting that costimulation may also be involved in IBD pathogenesis.190, 191

T-cell Differentiation

The area of T-cell differentiation and its role in inflammatory diseases is expanding rapidly, with multiple T-cell lineages now recognized.192 The aberrant differentiation of this immune cell population has been postulated to be involved in the pathogenesis of several chronic inflammatory conditions including IBD, most notably the recently described Th17 subset.193 In contrast to the above findings with regard to T-cell proliferation, Mayer and colleagues have demonstrated that T-cell coculture of IECs from IBD patients and normal controls produce significantly different T-cell populations.3, 189, 194 During an experiment looking at the role of costimulatory molecules in IEC-T cell interaction, they found that normal IECs predominantly induced CD8+ T cells directly, whereas those from IBD patients mainly stimulated CD4+ cells.189 This work was reproduced and expanded in later studies that showed that there was also increased IFN-γ secretion by IBD IECs and no significant difference in IL-2 secretion between disease and control groups.3

Regulatory T Cells

T regulatory cells (Tregs) are T cells that have the ability to suppress effector T cells and are identified by the expression of the main transcription factor forkhead box P3 (Foxp3).195 These cells produce minimal IL-2 and mediate their inhibitory effect by expressing molecules such as CTLA-4 and the production of antiinflammatory cytokines (e.g., IL-10, TGF-β).196 It has been recognized that Treg cells play a key role in oral tolerance,197 and that a defect in Treg response may be important in IBD pathogenesis.198 Although the role of Tregs in human IBD is not fully understood, patients do show a reduced number of Tregs in the blood and colon with the remaining cells functional in vitro.199 Westendorf et al200 have recently shown that in a TCR-HA mouse model (where mice express the MHC class II-restricted epitope of the hemagglutin protein), antigen presentation by primary IECs is sufficient to expand antigen-specific CD4+Foxp3+ Tregs efficiently. Other regulatory cells are currently under investigation, most notably the CD28-CD101+CD103+ subset of CD8+ T cells, having been shown to expand following interaction with healthy IECs and reduced in IBD.201

Effect of IL-10 and Immune Cells on the Epithelium

The epithelium is being constantly renewed with proliferation, migration, and differentiation in the crypts and apoptosis at the villus tips. It is now becoming clear that certain immune-mediators play key roles in this epithelial homeostasis and that the interaction between IECs and immune-regulators is bidirectional.

Animal models using IL-10-deficient mice have shown that this antiinflammatory cytokine is involved in IEC homeostasis202 and that deficiency leads to colitis in the presence of a normal mucosal microflora.203 IL-10 is likely to play an important part in the balance between IEC differentiation and proliferation and recent work has also shown its production by regulatory T cells,198 and that the frameshift mutation in the NOD2 gene (3020insC) leads to inhibition of IL-10 transcription.204 Lymphocytes and macrophages have also been shown to directly affect IECs. Lymphocytes within Peyer's patches have the ability to differentiate human enterocytes (CaCo-2 cells) into specialized M cells205 with activated γδ IELs producing keratinocyte growth factor leading to increased IEC proliferation and preservation of mucosal integrity.206 Barrier integrity has also shown to be influenced by immune cell populations, with human mucosa-derived lymphocytes (MDL) likely to attenuate barrier function at high MDL:IEC ratios207 and CD4+ T cells increasing permeability in Peyer's patches.208 In addition, macrophages have been found to be intricately involved in the colonic epithelial progenitor niche, which is responsible for mounting an appropriate response to tissue injury at the epithelial crypts.209

CONCLUSION

  1. Top of page
  2. Abstract
  3. EPITHELIAL MONOLAYER
  4. ENTEROCYTE
  5. ENTEROCYTE–T-CELL INTERACTION
  6. CONCLUSION
  7. REFERENCES

It can be seen that the intestinal epithelium is not merely a passive barrier but an active tissue involved in antigen recognition, processing, and immune cell regulation. The emergence of newly described T-cell lineages and immune pathways provides an opportunity to delineate the role of this complex tissue. The two-way interaction between the IEC and immune cells is also of great interest, with new work highlighting possible new therapeutic targets for those with IBD. In addition, the ever-growing body of genetic studies in IBD and the acceleration in mucosal immunology will further define the dysregulated pathways already implicated in IBD pathogenesis.

REFERENCES

  1. Top of page
  2. Abstract
  3. EPITHELIAL MONOLAYER
  4. ENTEROCYTE
  5. ENTEROCYTE–T-CELL INTERACTION
  6. CONCLUSION
  7. REFERENCES