REVIEW

 

Innate lymphoid cells and natural killer T cells in the gastrointestinal tract immune system

Células linfoides innatas y células T natural killer en el sistema inmune del tracto gastrointestinal

 

 

Enrique Montalvillo¹, José Antonio Garrote^1,2, David Bernardo³ and Eduardo Arranz¹

¹Laboratorio de Inmunología de las Mucosas. IBGM, Universidad de Valladolid-CSIC. Valladolid, Spain.
²Department of Clinical Analysis. Hospital Universitario Río Hortega. Valladolid, Spain.
³Antigen Presentation Research Group. Imperial College London, Northwick Park & St Mark's Campus. Harrow, United Kingdom

This research was supported by Instituto de Salud Carlos III-Fondos FEDER (PI10/01647) (to EA); Junta de Castilla y León (SAN673/VA22-08) (to JAG), "BBSRC Institute Strategic Programme for Gut Health and Food Safety BB/J004529/1", United Kingdom (to DB), and Beca FPI-Junta de Castilla y León/Fondo Social Europeo (to EM).

Correspondence

 

 

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ABSTRACT

The gastrointestinal tract is equipped with a highly specialized intrinsic immune system. However, the intestine is exposed to a high antigenic burden that requires a fast, nonspecific response -so-called innate immunity- to maintain homeostasis and protect the body from incoming pathogens. In the last decade multiple studies helped to unravel the particular developmental requirements and specific functions of the cells that play a role in innate immunity. In this review we shall focus on innate lymphoid cells, a newly discovered, heterogeneous set of cells that derive from an Id2-dependent lymphoid progenitor cell population. These cells have been categorized on the basis of the pattern of cytokines that they secrete, and the transcription factors that regulate their development and functions. Innate lymphoid cells play a role in the early response to pathogens, the anatomical contention of the commensal flora, and the maintenance of epithelial integrity. Amongst the various innate lymphoid cells we shall lay emphasis on a subpopulation with several peculiarities, namely that of natural killer T cells, a subset of T lymphocytes that express both T-cell and NK-cell receptors. The most numerous fraction of the NKT population are the so-called invariant NKT or iNKT cells. These iNKT cells have an invariant TCR and recognize the glycolipidic structures presented by the CD1d molecule, a homolog of class-I MHC molecules. Following activation they rapidly acquire cytotoxic activity and secrete both Th1 and Th2 cytokines, including IL-17. While their specific role is not yet established, iNKT cells take part in a great variety of intestinal immune responses ranging from oral tolerance to involvement in a number of gastrointestinal conditions.

Key words: Intestinal innate immunity. Innate lymphoid cells. Natural killer T cells. Invariant NKT cells. CD1d. Inflammatory bowel disease.

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RESUMEN

El tracto gastrointestinal está equipado con un sistema inmune intrínseco altamente especializado. Sin embargo, el intestino soporta una gran carga antigénica que requiere de una respuesta rápida e inespecífica, denominada inmunidad innata, para mantener la homeostasis y proteger al organismo de la entrada de patógenos. En la última década, numerosos estudios han contribuido a desentrañar los requisitos particulares de desarrollo y las funciones específicas de las células que intervienen en la inmunidad innata. En esta revisión, nos centraremos en las células linfoides innatas, un nuevo y heterogéneo grupo de células derivadas de una población linfoide progenitora Id2-dependiente. Estas células han sido categorizadas en base al patrón de citocinas que producen y los factores de transcripción que regulan su desarrollo y funciones. Las células linfoides innatas intervienen en la respuesta temprana contra agentes patógenos, la contención anatómica de la flora comensal, y el mantenimiento de la integridad epitelial. Dentro de las distintas células linfoides innatas haremos especial hincapié en una subpoblación con diversas particularidades, las células T natural killer, un subtipo de linfocitos T que expresan receptores de células T y NK. La fracción más numerosa de células NKT son las denominadas NKT invariantes o iNKT. Las células iNKT, poseen un TCR invariante y reconocen estructuras glicolípidicas presentadas por la molécula CD1d, homóloga de MHC de clase I. Tras su activación, adquieren rápidamente actividad citotóxica y producen citocinas tanto Th1 como Th2, e incluso IL-17. Aunque su papel concreto no está determinado, las células iNKT intervienen en una gran variedad de respuestas inmunes intestinales, desde la tolerancia oral hasta su implicación en diversas patologías del tracto gastrointestinal.

Palabra clave: Inmunidad innata intestinal. Células linfoides innatas. Células T natural killer. Células NKT invariantes. CD1d. Enfermedades inflamatorias intestinales.

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Abbreviations list:
IEC, intestinal epithelial cell;
LP, lamina propria;
IELs, intraepithelial lymphocytes;
DC, dendritic cell;
ILC, innate lymphoid cell;
NKT, natural killer T cell;
PRR, pattern recognition receptor;
APC, antigen-presenting cell;
NK, natural killer;
LTi, lymphoid tissue inducer;
IL, interleukin;
IFN, interferon;
IBD, inflammatory bowel disease;
NCR, natural cytotoxicity receptor;
TNF, tumor necrosis factor;
CD, Crohn's disease;
MHC, major histocompatibility complex;
TCR, T-cell receptor;
iNKT, invariant NKT cell;
mNKT, mucosal NKT cell;
vNKT, variant NKT cell;
xNKT, NKT-like cell;
aGalCer, a-galactosylceramide;
iGb3, isoglobo-trihexosylceramide;
Treg, regulatory T cell;
CoD, coeliac disease;
UC, ulcerative colitis.

 

Gastrointestinal tract immune system

The gastrointestinal tract possesses the highest concentration of immune cells in the human body, and is continually exposed to a high antigenic burden made up not only of nutrients but also the saprophytic intestinal flora (1). The intestinal mucosa includes a first defensive barrier of intestinal epithelial cells (IECs), or enterocytes, that maintain epithelial integrity and are also specialized in the absorption of fluids and nutrients. In addition, the mucosa is equipped with a highly specialized intrinsic immune system (Fig. 1) that ensures optimal nutrient transportation and prevents translocation of bacteria, whether commensal or pathogenic. The intestinal mucosa-associated lymphoid tissue includes lymphoid-cell aggregates such as Peyer's patches and small-bowel mesenteric lymph nodes, as well as isolated lymphoid follicles in the large bowel, which are involved in antigen uptake, processing, and presentation. It also includes a great variety of lymphoid cells in the lamina propria (LP), and intraepithelial effector lymphocytes (IELs) interspersed in the epithelial lining (2).

 

 

Both innate and adaptive immune responses represent an integral system of host defense. Their major difference is the specificity of the adaptive immune response, which improves with successive contacts with an antigen but also requires a longer development period. However, the intestine is continually undergoing high antigenic burdens requiring a fast though nonspecific response to maintain intestinal homeostasis and protect the body against incoming pathogens. This response is dependent upon innate immunity (3).

In the past decade numerous reports have unraveled the particular developmental needs and specific functions of cells playing a role in intestinal innate immunity, including monocytes/macrophages, eosinophils, dendritic cells (DCs), and a heterogeneous, newly identified set of innate lymphoid cells (ILCs). In this review we shall focus on the various ILC types in the gastrointestinal tract, and their contribution to intestinal immunity both in health and disease, with an emphasis on a special innate lymphoid subset, namely natural killer T-cells or NKTs.

 

Innate non-lymphoid cells

While the integrity of junctions between epithelial cells and the differentiation of some of these cells into mucus-secreting goblet cells are vitally important for the defense of the intestinal mucosa, the fact that enterocytes play a much more complex role in the immune response than merely by regulating permeability is now increasingly clear (4). Similar to classic immune cells, IECs also express several pattern recognition receptors (PRRs), which allows them to identify the appropriate ligands produced both by commensal agents and intestinal pathogens. The relevance of these PRRs in IECs may be seen, for instance, in Paneth cells, a subtype of IECs with the ability to detect signals from the commensal flora and to respond by secreting various antimicrobial peptides, thus contributing to maintain intestinal homeostasis. In view of their ability to regulate the saprophytic intestinal flora, some authors have recently suggested that IECs may be considered innate cells on their own right (5).

DCs are the most powerful antigen presenting cells (APCs) in the body, and serve as immune system centinels by their expression of numerous PRRs, including toll-like receptors (TLRs) (6,7). DCs may also activate in the presence of innate signals such as cytokines and/or reactive oxygen species (8). Therefore, DCs represent a nexus between innate (non-antigen-specific) immune response and the highly specialized adaptive immune response. The role of intestinal DCs has been recently reviewed in this journal (9). Under normal conditions, epithelial cells favor immature DC differentiation to tolerogenic DCs thanks to their TGFβ production and retinoic acid synthesis from vitamin A. These tolerogenic DCs controlling oral tolerance mechanisms are CD103⁺. However, other subtypes of intestinal DCs exist, including the CX3CR1⁺ population, that can send prolongations between epithelial cells and reach the intestinal lumen to directly take up antigens there. These CX3CR1⁺ DCs are capable of inducing Th17 responses in vitro (10), and of destroying certain bacteria (11-13); however, they cannot migrate to mesenteric lymph nodes (14). From all the above, CX3CR1⁺ DCs more closely resemble macrophages than classic DCs (4,15). Such observations have kindled interest in intestinal macrophages, which are the most abundant mononuclear cells in the bowel, play a role in antigen presentation within the lamina propria, and are key in maintaining intestinal homeostasis (11,16).

Until now eosinophils were considered to be IgE-dependent effector cells in inflammatory processes such as allergic hypersensitivity and parasite infestation. However, under normal conditions, eosinophils are commonly seen in the intestinal mucosa and authors such as Svenson-Frej et al. (17) advocate the notion that these cells play a crucial role in intestinal homeostasis. Other authors, as Johnsson et al. (18), note more conventional properties in these intestinal cells, and suggest their active role in various diseases, including ulcerative colitis, eosinophilic esophagitis, and respiratory allergies.

In summary, non-lymphoid cells with innate characteristics are of vital importance in gastrointestinal tract defense. However, as of today, ILCs are thought to represent the master key to innate immune responses on mucosal surfaces, and are the focus of this review as discussed below.

 

Innate lymphoid cells

Introduction

The term innate lymphoid cell refers to well-established, recently identified populations that seem to share a common origin and derive from Id2-dependent lymphoid progenitors (19-23). ILCs are defined by three major characteristics -absence of antigen-specific receptors and memory function, lack of myeloid phenotypic markers, and lymphoid morphology (24). In fact, these cells do not directly recognize pathogens but respond to cytokine pattern changes induced by pathogenic infection (21,22,25,26). A prototypal population of innate lymphoid cells is that of natural killer (NK) cells and lymphoid tissue inducer (LTi) cells. NK cells mediate in early immune responses to viruses, and are involved in the protection against cancer cells (27,28). LTi cells are key for lymph node formation during embryogenesis (27,29).

Recently, other ILC populations have been identified that, as is the case with NK and LTi cells, are also dependent on γc and ID2 for their development. These various ILC populations have different cytokine production patterns that reflect the different subpopulations of Th lymphocytes (22). For example, following activation, some ILC populations secrete Th17-related cytokines, including interleukine (IL)-17 and IL-22, whereas other ILC subsets secrete Th2 cytokines (IL-5 and IL-13) (22).

ILC populations may play effector roles in early immune responses against pathogens (30,31), in addition to their contribution to mucous tissue repair (32,33), anatomical contention of the commensal flora, and maintenance of epithelial integrity (34). On the other hand, ILC subpopulations involved in several inflammatory conditions have been found. For example, IL-17- and interferon (IFN)-γ-producing ILCs have been shown to be mediators of colitis in a murine IBD (inflammatory bowel disease) model (35), whereas Th2 cytokine-producing ILCs give rise to pulmonary inflammation in some allergic asthma models (36).

Classification of innate lymphoid cells

The recent identification of various ILC subsets has led to a new classification for them. Currently, the most widely accepted classification is that proposed by Spits et al. (24), which is based on cell phenotype and functional characteristics, and includes three groups (Fig. 2):

 

 

- Group 1 ILCs, which include classic NK cells (28,37-39). These cells rely on T-bet and IL-15 expression for optimal differentiation and function (40,41). Their stimulation may also results from IL-12 and IL-18 signaling, and NCR (natural cytotoxicity receptor) expression. Activation results in proinflammatory cytokine production, including IFNg and TNFa, or perforin and granzyme release to induce lysis in target cells, key processes for tumor suppression and immunity against certain intracellular pathogens (37-39,42). Therefore, NK cells share both homeostatic and functional similarities with CD8⁺ T-cells.

Another group 1 subpopulation has been recently identified in humans that differs from NK cells and produces IFNg but no Th17 or Th2 cytokines (43). These are known as ILC1s to differentiate them from classic NK cells. In humans they are characterized by their secretion of IFNg, lack of KIT (CD117) expression, and high levels of T-bet and low levels of RORgt expression (43). They are thought to be RORgt⁺ ILCs (included in group 3) that increase T-bet and IFNγ production, and lose RORγt when under stimulation (35,43,44).

- Group 2 ILCs require IL-7 for their development (31) and produce Th2 cytokines in response to stimulation with IL-25 (IL-17E) (45,46), IL-33 (31), and TSLP (thymic stromal lympho-poietin) (47). Only one subset has been identified in humans. These are cells characterized by their expression of ST2 (also known as IL-1RL1), which is a component of the IL-33 receptor, and of IL-17RB, which is a subunit of the IL-25 receptor (33,48). Furthermore, human ILC2s express CRTH2 (prostaglandin D2 and chemoattractant receptor-homologous molecule 2 expressed by Th2 lymphocytes) and CD161. Only human ILC2s can produce IL-4, in contrast to mouse ILC2s (48). The development of these cells is mediated by the GATA3 transcription factor (49).

- Group 3 ILCs are defined by their ability to produce IL-17A and/or IL-22, whether with NCR expression or otherwise. As with Th17 cells, the development and function of these cells are dependent upon transcription factor RORγt and IL-7 receptor. Prototypical cells in this group include LTi cells, which are crucial for the formation of secondary lymphoid organs during embryogenesis. An effector role has also been suggested for these cells within innate immunity because of their IL-17A and IL-22 secretion following stimulation (50). Surface markers on human LTi cells are identical to those in their mouse counterparts except for CD4, which is expressed by most murine but no human LTi cells (51). On the other hand, human LTi cells express NKp46 as do their murine counterparts, but also other NCRs such as NKp30 and NKp44 (30). This group 3 ILC population is heterogeneous and includes various types of cells that produce IL-17A and/or IL-22, or IL-17A, IL-22 and IFNg; these could be considered distinct stable cell subsets or, alternatively, different forms of only one cell type with plastic capabilities (24).

Functions of innate lymphoid cells

Several ILC subpopulations have been identified in human tissues, both healthy and with various conditions, including secondary lymphoid organs, peripheral blood, lungs, and gut (26,52,53). NK cells have been most thoroughly studied, and changes in their numbers and/or functions have been associated with some diseases (54). Furthermore, NK cells play a major role in various conditions such as viral infection, inflammatory disorders, pregnancy, cancer, and bone marrow transplantation (37,54). Similarly, group 2 ILCs have been recently identified that secrete IL-5 and IL-13 in the blood of healthy individuals, in the lungs and bowel of both fetal and adult donors, in the bronchoalveolar fluid of lung transplant recipients, and in the nasal polyps of patients with chronic rhinosinusitis (33,48).Group 3 ILCs, which secrete IL-17A and IL-22, are seen in the secondary lymphoid tissues and the intestinal tissue of both fetal and adult donors (30,34,51). In co-culture experiments with mesenchymal stem cells from murine models RORγt⁺ ILCs have been seen to promote the expression of adhesion molecules involved in lymphoid organogenesis (51), and the proliferation of intestinal epithelial cells (30).

ILCs play their role mainly by secreting cytokines, but they may also modify the adaptive response mediated by B cells and T cells via cell-cell interactions. An example of the above is the ability of LTi cells to interact with CD4⁺ memory cells through OX40L and CD30L (55,56). Similarly, ILCs might directly interact with the intestinal bacterial flora. While the detection of certain specific bacterial components is vital for the development of adaptive immune system cells, this requirement seems less important regarding the development of innate cells, including macrophages and NK cells, given their presence in bacteria-free mice (5). However, recent studies have shown that the bacterial flora directly regulates the role of innate cells, just as the innate immune system directly acts on the composition of the bacterial flora (42,57).

Several studies have also characterized the contribution of RORγt⁺ ILCs to the pathogenesis and progression of some diseases where host-commensal flora interactions are impaired. Geremia et al. (58) observed a substantial increase in NCR-RORγt⁺, IL-17A- and IL-22-producing ILCs in the inflamed intestinal tissues of patients with Crohn's disease (CD). The potential significance of changes in the balanced production of IL-22 between NCR⁺ ILCs and NCR-RORγt ILCs in patients with IBD may be explained by a differential IL-17A expression in these two subsets (26,53,59), given the proinflammatory role of IL-17A versus the protective function of IL-22 in the bowel mucosa (60).

 

NKT cells

Natural killer T (NKT) cells are a subset of T cells that express receptors characteristic of T and NK cells (61-65). As NK cells, they are included among group 1 ILCs (24) and contain perforins and granzymes allowing them to take part in the innate immune response (61-63). In contrast to conventional T cells, which recognize peptides bound by major histocompatibility complex (MHC) class I or class II molecules, NKT cells recognize lipidic and glycolipidic structures bound by CD1d molecules (66). They may be found mainly in the liver, spleen and bone marrow, and their development is dependent upon the thymus. In the bowel, various NKT subsets have been described among IELs and in the LP (66). Intestinal NKT-cell activationcontributes to mucosal immunity against both pathogenic and commensal bacteria. Furthermore, uncontrolled or inadequate NKT-cell activation may contribute to the pathogenesis of intestinal inflammatory diseases (67).

NKT cells were initially defined by their co-expression of TCRab (T-cell receptor) and NK-cell receptors, particularly NK1.1 in selected mouse strains and CD161 in humans (64). However, conventional T cells may express NK-like receptors (61,68). Today, the most widely accepted classification is the one suggested by Wingender et al. (69), which is based on TCR composition and recognized antigen-presenting molecule, giving four distinct populations (Table I). The first two sets have a canonic or invariant TCR and interact with non-polymorphic molecules similar to MHC class I (69). The first population, designated invariant NKT or iNKT cells, includes cells that express a semi-invariant TCR composed of Vα14 - Jα18 and Vβ8.2, -7, or -2 chains in mice, or the homologous Vα24 - Jα18 and Vβ11 chains in humans (63). The second subset includes mucosal NKT or mNKT cells, characterized by their expression of an invariant TCR Va7.2 in mice, and the homologous Va19 in humans (66). A third group, called variant NKT or vNKT cells, includes NKT cells reactive to CD1d but with no fixed-composition TCR (70,71). The fourth group, that of xNKT or NKT-like cells, is most heterogeneous and includes all T cells expressing NK-like receptors. This group includes T cells not dependent on CD1d expression for their development or reactivity, and that may recognize lipids presented by CD1 molecules (CD1a, -b, -c); antigen recognition, however, is not restricted to lipids but also applies to peptides in the setting of class-I or class-II MHC molecules (69).

 

 

As discussed in this review, ILCs are of utmost importance for intestinal immunity. To gain a deeper insight into this topic, we shall now focus primarily on NKT cells within ILC1 group, particularly on the invariant NKT subset, and their relevance for the immune response in the gastrointestinal tract.

 

Invariant NKT or iNKT cells

Biology of iNKT cells

So-called invariant NKT or iNKT cells represent the most numerous and most widely studied fraction of NKT cells. As discussed above, these cells are characterized by their having a TCR made up with Vα14 - Jα18 and Vβ8.2, -7, or -2 chains in mice, or their homologous Vα24 - Jα18 and Vβ11 chain in humans (63). iNKT cells recognize glycolipidic structures presented by CD1d, a molecule homologous to class-I MHC (63). While there is a high degree of cross-reactivity between species, since mouse iNKT cells may recognize antigens presented human CD1d molecules, and vice versa, significant differences are also present between iNKT cells in both species (72).

As occurs with conventional T-cells, NKT cells develop from thymic precursors. Immature CD4⁺CD8⁺ T cells derived from these precursors give rise to the NKT cell lineage that depends on signals via CD1d molecules expressed by cortical thymocytes, which may present self-glycolipids (73). As iNKT cells differentiate in the thymus, they express a surface marker pattern (CD69⁺, CD44^high, CD11a^high, CD62^low, CD122⁺) that is typically associated with memory or activated T cells (69). Following activation, iNKT cells rapidly develop cytotoxic activity, and produce both Th1 (IFNγ and TNFα) and Th2 (IL-4, IL-10, and IL-13) cytokines (74); also, some iNKTs have been recently shown to secrete IL-17 (74,75). iNKT cells may be involved in the early stages of a great variety of immune responses, ranging from oral tolerance to autoimmunity development, including responses to both pathogenic and tumor agents (64,68).

Following their development in the thymus, a major fraction of iNKT cells stays there and the rest migrate to peripheral sites, where they make up a relevant T-cell subset in the bone marrow, spleen, blood and liver, being less common in lymph nodes (67). Some of these cells reach the intestinal and pulmonary mucosa. Interestingly, iNKT cells are less common in most human versus murine organs, and their prevalence varies greatly between subjects because of yet unknown reasons (67). Recent studies show that ageing results in a rapid and significant decrease in peripheral blood iNKT cells associated with an increased proportion of CD4⁺ iNKTs and fewer double-negative iNKT cells. Furthermore, iNKT cells from older subjects secrete more IL-4 when compared to younger individuals (76). These results may also explain age-related Th1/Th2 cytokine profile changes and shed light on the mechanisms of immunosenescence. Given the significance of iNKT cells for immune response initiation and regulation, these results may also help in understanding the increased incidence of both infections and malignancies, as well as the increased severity of autoimmune conditions, in the elderly (76,77).

Several lipidic or glycolipidic antigens have been identified, which may be presented by CD1d and activate iNKT cells. KRN7000, an α-galactosylceramide (α-GalCer) that was originally discovered in a sea sponge and possesses anti-metastatic properties in mice, is prototypical (78). α-GalCer likely derives from the Sphingomonas bacteria that colonize this sponge (79-81). The current hypothesis establishes that iNKT cells can recognize natural glycolipidic structures in several bacterial pathogens, including Borrelia burgdorferi, Ehrlicha bacteria, Streptococcus pneumoniae (79) and Bacteroide fragilis (82).

The effector phenotype of iNKT cells and their constitutive expression of IL-4 and IFNγ mRNAs suggests that these cells undergo strong antigenic stimulation during differentiation (64). In this regard, it is suggested that mature iNKT cells may, under certain circumstances, recognize CD1d-bound endogenous glycolipids. As of today, the best such candidate is a glycosphingolipid that is present in the lysosomes of some cells -isoglobotrihexosylceramide (iGb3) (83). However, recent data raise doubts regarding the role of iGb3 as single endogenous antigen in iNKT-cell development, as mice deficient in iGb3- synthase have been found with a normal count of functional iNKT cells (84). A third class of iNKT ligands has been identified from dietary lipids used as emulsifiers and thickeners. These compounds are similar to glycolipids found in the walls of some bacteria, including the genus Mycobacteria (85), and may activate iNKT cells. The affinity of iNKT TCR for CD1d-bound antigens is not always enough to predict cytokine response types (Th1 or Th2). Available data suggest that response polarization in iNKT cells is determined by antigen-CD1d binding strength, cell surface complex longevity, and APC type (86).

iNKT cells may also be directly activated by proinflammatory cytokines such as IFNγ, IL-12, and IL-18, either alone or combined, which are produced by macrophagesand DCs early on after bacterial or viral infection (87). While, in some instances, iNKT cells have been shown to require the recognition of CD1d-presented self-endogenous ligands for activation in this setting, direct activation most often ensues without TCR-related antigen recognition. This type of direct activation prompts iNKT cells to produce IFNγ but not IL-4 (87).

iNKT cells in the human bowel

The percentage of iNKT cells present in the human bowel is controversial. To date, these cells may only be unequivocally detected through mRNA quantification for invariant TCR chains or by flow cytometry CD1d-αGalCer tetramers (69). Via flow cytometry analysis, the proportions of CD161-expressing T cells to total lymphocytes are: 50-70 % of IELs in the small bowel (88,89), 40-45 % of IELs in the large bowel (88,89), and 9 % of lymphocytes in the large bowel LP (90). However, only 1.6 -1.7 % of intestinal CD3ε/CD161 cells express Va24 (88,89), and immunohistochemical data suggest that most of these cells reside in the LP (91). By using CD1d-αGalCer tetramers for flow cytometry the proportion of iNKT cells in the human bowel is estimated to be 0.4 % of all T cells, with the LP being their primary site (90). However, despite their low numbers these cells can produce huge amounts of cytokines following activation with α-GalCer (89).

CD1d expression is prerequisite for antigen-specific iNKT-cell activation (86). This molecule resembles those in the class-I MHC, consisting of a light chain (β2-microglobulin) covalently bonded with a heavy chain (92), and is structurally related to HLA-A, HLA-B and HLA-C molecules in IECs (62). CD1d is expressed by professional APCs, including dendritic cells, macrophages, and B cells, and non-professional APCs, including liver cells and IECs (93-95).

A controversial aspect of human IECs is CD1d expression, as most of these cells express a CD1d form not associated with β2-microglobulin, mainly intracellular, and with surface expression restricted to the apical pole (93,96-98). While its function is not clear, and no evidence exists on the recognition of this CD1d form by iNKTs, it has been suggested that some T cells, probably vNKT cells, do recognize it (93,99,100). IECs weakly express native CD1d, preferably on their basal pole (98). However, these human IECs may activate iNKT cells in vitro via CD1d (101,102). A CD1d-dependent feedback pathway has also been found to play a role in IL-10 signaling and production in the intestinal mucosa via IEC activation (86). The abundance of CD1d in the intestine and the power of CD1d-dependent iNKT activation suggest their involvement in intestinal homeostasis, bacterial regulation in the bowel, and protection against pathogens such as Salmonella typhimurium and Toxoplasma gondii (103-105).

The immune system's ability to discriminate between pathogenic and non-pathogenic antigens is the basis of immune tolerance. A relevant component of oral tolerance against dietary and saprophytic flora antigens is represented by regulatory intestinal cells, including IL-10-secreting regulatory T (Treg) cells, tolerogenic DCs, and iNKT cells (106). Treg lymphocytes may inhibit iNKT activity by cell-to-cell contact (107), whereas iNKT cells increase regulatory intestinal cell activity by producing cytokines such as IL-2 and TGFβ (108). Treg lymphocytes can suppress Th1 and Th2 responses (109), whereas iNKTs suppress CD8⁺ T-cell activation, henceTh1 responses, and either increase or suppress Th2 responses (110,111). Furthermore, iNKT cells can induce immature DC maturation to tolerogenic DCs (106). When tolerance mechanisms fail, an inappropriate immune response against dietary and saprophytic flora antigens ensues, giving rise to inflammatory bowel conditions such as coeliac disease (CoD) or IBD, respectively.

The role of iNKT cells in intestinal diseases

Few studies exist on iNKT-cell activation in patients with IBD; however, studies in murine models and our current understanding of iNKT-cell biology suggest a relevant role. Furthermore, an increase in CD1d expression has been seen in the terminal ileal epithelium of patients with CD, and in the involved cecal area of patients with ulcerative colitis (UC), which might increase the recruitment of CD1d-dependent proinflammatory cells and bring about the destruction of the intestinal mucosa in IBD (112). However, a more recent study suggests that, in contrast to IECs in healthy individuals, IECs in patients with IBD do not express CD1d, which results in abnormal NKT-cell regulation (101).

Clinical studies have shown a significant reduction in peripheral blood iNKT cells in patients with CD, assessed as Vα24/Vβ11⁺ cells or cells reactive against tetramer CD1d-αGalCer (113,114), as well as a reduced expression of Vα24 and a decrease in iNKT-cell numbers in the bowel (113). However, aberrant iNKT-cell activation is likely given that these cells may secrete huge amounts of IFNg through IL-12 mediation (89). Interestingly, IECs in the terminal ileum, the primary site for CD, show numerous lipid-containing lysosomes that may act as potent iNKT-cell activators (115,116). IL-13-producing NKT cells have been identified in UC patients. In contrast with the murine oxazolone-induced colitis model, these cells do not express the invariant TCR Vα24 chain, hence they are no iNKTs despite their CD1d-dependent activation (117).

A potential involvement of iNKT cells as regulatory or protective agents in IBD has also been posited. In the murine DSS (dextran sodium sulfate)-induced colitis model the use of the iNKT-cell activator αGalCer (118,119), or its analogue OCH (120), has been seen to result in great improvement. This is due to iNKT-cell polarization towards a Th2 profile with an increase in IL-4 and IL-10 production, and a decrease in IFNγ (120).

The innate and adaptive characteristics of iNKT cells, and their ability to produce huge amounts of IL-4 and IFNγ, also suggest their involvement in CoD (91,114,121). The various studies performed to quantify circulating or intestinal iNKT cells in celiac patients have yielded controversial findings (91,114). In some cases a decreased number of iNKT cells have been found in the duodenum of these patients (91,122). However, prior findings by our team show an increase of these cells only in the epithelial compartment during active disease. These findings, together with the correlation between Vα24 mRNA expression and total intraepithelial iNKT-cell count, suggest that iNKT cells may be a source of IFNγ in CoD (manuscript awaiting publication). Today, natural ligands of iNKT cells are thought to include glycolipids in the cytoplasm of enterocytes, which are released into the extracellular matrix after apoptosis or necrosis (123) in the presence of IFNγ and IL-15, as occurs in CoD (124), while IL-15 plays a central role in the activation and biologic functions of these cells (125). Finally, a decrease in total circulating iNKT-cell numbers has been seen in refractory CoD; however, whether this is a cause or a result of disease malignant progression remains unknown (126).

A predominance of the CD4⁺ regulatory phenotype versus the proinflammatory phenotype of intestinal iNKT cells has been recently described in healthy individuals. However, HIV (human immunodeficiency virus) infection results in a decrease in this intestinal iNKT population that directly correlates with poorer immune response, the hallmark of disease progression (127).

Multiple studies have shown the high potential of iNKT cells to initiate an effective anti-tumor response. The activation of these cells as a result of αGalCer stimulation has demonstrated anti-tumor effects in several experimental models and in spontaneous skin, liver, lung and intestine metastatic models, including colon-26 adenocarcinoma, EL-4 T-cell lymphoma, sarcoma, melanoma, and carcinoma (128-130). αGalCer rapidly activates iNKT cells and apoptosis ensues, which suggests that iNKT cells initiate the primary antitumor response by direct cytotoxicity, and then activate more persistent immune mechanisms aimed at the destruction of tumor cells (121).

 

Conclusion

The innate immune response is key for the maintenance of epithelial integrity, homeostasis, and early response to pathogens in the intestinal mucosa. Innate lymphoid cells seem to be key components of such response. Group 1 ILCs include NKT cells, a subset with various peculiarities that play an active role in this response. The most important fraction in this subpopulation is iNKT cells. Due to their effector phenotype and great capability to produce huge amounts of cytokines upon activation, their taking part in several immune processes has been suggested, including intestinal homeostasis, defense against tumors and several pathogens, and an active role in the development of a number of inflammatory conditions. Their activation results from the recognition of glycolipids presented by CD1d. Although the source of these immunogenic glycolipids that trigger active, iNKT-mediated responses remains unknown in humans, a number of both endogenous and exogenous origins may be posited. Among these, intracellular lipids from apoptotic enterocytes released during inflammation, dietary glycolipids modified by non-physiological enzymes, and glycolipids from bacteria within the intestinal lumen stand out. Identifying the specific role of these cells in each process, as well as their specific ligands, is key for the development of promising therapies for the treatment of intestinal inflammatory diseases.

 

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Correspondence:
David Bernardo.
Antigen Presentation Research Group.
Imperial College London.
Northwick Park &
St Mark's Campus, Level 7W,
St. Mark's Hospital, Watford Road,
Harrow, HA1 3UJ, United Kingdom
e-mail: d.bernardo-ordiz@imperial.ac.uk

Received: 17-02-2014
Accepted:10-03-2014