A novel mucosal RORγtNKp46 cell subset is a source of interleukin-22.

Lymphoid tissue-inducer cells are hematopoietic cells essential for the organogenesis of several lymphoid structures during both fetal and adult life, whereas natural killer cells are key effector lymphocytes of the innate immune system. A series of recent reports has identified RORγt(+)NKp46(+) interleukin-22-producing cells in gut and tonsils that share features with both lymphoid tissue-inducer cells and natural killer cells and that may be involved in mucosal immunity and homeostasis.

Introduction and context

The internal surfaces of the body are covered by distinct types of mucosal epithelia. These tissues constitute the first physical and immunological defense against various environmental insults and enclose local immune systems characterized by tissue-specific organization and complexity [1]. Gut-associated lymphoid tissues (GALTs) include lymphoid structures harboring different degrees of organization, such as mesenteric lymph nodes, Peyer's patches, cryptopatches, and isolated lymphoid follicles (ILFs), as well as of a network of immune cells scattered in both epithelium and underlying lamina propria connective tissue [2]. Tonsils belong to the nasopharynx-associated lymphoid tissue (NALT), which shares functional similarities with GALT but differs in its ontogenetic program [3].

Lymphoid tissue-inducer (LTi) cells are hematopoietic cells required for lymphoid organogenesis during both fetal and adult life [4,5]. LTi cell interaction with stromal organizer cells via lymphotoxin beta (LTβ) and tumor necrosis factor signals induces chemokine secretion and adhesion molecule expression upregulation, both crucial events for LTi cell and mature lymphocyte recruitment and subsequent lymphoid tissue organization [4]. In mouse embryo, LTi cell development and function are dependent on the expression of transcription factors such as retinoic acid-related orphan receptor gamma t (RORγt) and Id2 [6,7] as well as of interleukin (IL)-7Rα receptor and LTα1β2 cytokine [4]. In the adult mouse, the LTi cells are found mainly within cryptopatch and ILF gut lymphoid structures, in close contact with dendritic cells [8], as well as in the spleen [9-11].

Natural killer (NK) cells are lymphocytes belonging to the innate immune system that are able to exert two major effector functions, cytotoxicity and cytokine secretion [12]. Therefore, NK cells can limit the spreading of microbial infections as well as of cells undergoing several kinds of cellular stress, and participate in the orchestration of adaptive immune responses. NK cells are ubiquitously expressed in both lymphoid and non-lymphoid organs, where they harbor distinct phenotypic and functional properties, depending on tissue locations [13-15]. An NK cell subset resident in mucosal tissues, such as human tonsils, has been documented [16]. However, the presence and the function of NK cells in GALT have been poorly investigated [17-19].

Major recent advances

By using the NK cell marker NKp46 [20], which is the most selective NK cell marker described so far in humans and mice [21], several groups have recently described a new population of RORγt+NKp46+ IL-22-producing cells in the adult mouse gut [22-25]. Within the mouse gut, RORγt+NKp46+ cells have been identified in at least three different locations. First, RORγt+NKp46+ cells are detectable in variable proportions among RORγt+ cells constituting cryptopatches (Figure 1, right panel) [22,23]. Second, a significant fraction of NKp46+ cells present in the lamina propria also express RORγt, thus revealing the existence of two distinct RORγt+NKp46+ and RORγt-NKp46+ cell subpopulations (Figure 1, left panel) [22-24]. As described below, these two subsets harbor distinct developmental programs as well as phenotypic and functional properties. Finally, RORγt+NKp46+ and RORγt-NKp46+ cells are also found within Peyer's patches, in particular in subepithelial dome and interfollicular areas [22,25].

Figure 1.

Immunofluorescence histology of mouse gut intestine

Immunofluorescence histology of frozen sections of mouse gut intestine stained with anti-NKp46 (green), anti-RORγt (red), and anti-CD3 (blue). Nuclei were counterstained with Sytox (grey). The left panel shows small intestini villi; the right panel shows cryptopatches. Magnification ×40. RORγt, retinoic acid-related orphan receptor gamma t.

Interestingly, RORγt+NKp46+ and RORγt-NKp46+ cell subsets have distinct ontogenetic programs. While RORγt+NKp46+ cell development requires both RORγt expression and exposure to commensal flora [22-24], RORγt-NKp46+ cell subset generation is dependent upon IL-2/IL-15 signaling [23,24]. RORγt+NKp46+ cells thus share some developmental requisites with LTi cells, whereas RORγt-NKp46+ cell subset ontogeny appears to be related to that of conventional NK cells. RORγt-NKp46+ cells express the mouse NK cell marker NK1.1 and contain perforin and interferon-gamma (IFN-γ), though at lower levels than in lymphoid NK cells. In contrast, RORγt+NKp46+ cells express low amounts of the NK cell marker NK1.1, are devoid of messages encoding perforin and IFN-γ, but are equipped to secrete IL-22 [22-25]. IL-22, an IL-10 family member, induces epithelial secretion of various antimicrobial products, such as defensins and S100 calcium-binding proteins, and of various molecules involved in epithelial tissue proliferation and homeostasis, thus contributing to greater epithelial resistance to injury after microbial infection of the lung and the gut [26]. Consistent with this protective function of IL-22, mucosal RORγt+NKp46+ cells appear to play a critical role in the defense against the extracellular enteric pathogen Citrobacter rodentium [24,25]. Furthermore, IL-22-expressing NK-like cells have been recently shown to contribute to protection from experimental inflammatory bowel disease [27].

A subset of CD56+NKp44+ cells represents the human functional equivalent of the mouse gut RORγt+NKp46+ population. Indeed, these cells reside within human tonsils and intestines, contain mRNA encoding human RORγt, and constitutively produce IL-22, but not IL-17A [25]. Like mouse RORγt+NKp46+ cells, these human IL-22-producing cells express some NK cell-associated markers, including NKp46 (H Spits, personal communication), yet lack perforin and granzyme and are unable to produce IFN-γ. The human mucosal RORγt+CD56+NKp44+ cell subset is possibly the progeny of newly discovered human LTi cells [28]. Indeed, the phenotype of RORγt+CD56+ cells generated in vitro from human fetal LTi cells [28] resembles the mucosal RORγt+ cells found in human tonsil and intestines [25]. However, unlike adult cell populations found postnatally, fetal human LTi cells and RORγt+NKp46+ cells produce only very low amounts of IL-22 message yet constitutively express IL-17A [28]. A switch from IL-17 to IL-22 expression has also been reported in mouse LTi cells from birth to adult life [29].

Future directions

Several questions regarding this novel mucosal RORγt+NKp46+ cell subset remain unanswered. First, what are the stimuli that promote the expression of NK cell receptors on these cells? In particular, what is the potential role of commensal flora exposure in inducing the expression of NK cell receptors and their respective ligands? Second, the features shared by both LTi and NK cells in mouse RORγt+NKp46+ and human RORγt+CD56+NKp44+ cells call for a closer look into their developmental relationships. Finally, the ability to secrete IL-22 highlights the potential contribution of this new cell type to mucosal tissue protection and homeostasis (for example, epithelium repair) upon diverse environmental insults and paves the way for the development of novel strategies to target these functions.