The recent discovery of non-NK ILCs complicated the interpretation of older literature on NK cells in tissues because these studies sometimes fail to distinguish NK cells from non-NK ILCs. Nevertheless, NKp46+ NKp44− RORγt− lymphocytes with a transcriptional profile similar to splenic NK cells can be found throughout the human small and large intestine (Fig. 2).[82, 83] These gut NK cells display a phenotype similar to peripheral blood CD56bright NK cells with high expression of NKG2A, intermediate levels of intracellular effector molecules, and low to absent levels of CD16 and KIRs. Interestingly, in humans, a lamina propria Lin− c-kit+ precursor population, expressing Id2 and PU.1, could upon culture up-regulate CD56, start to express effector molecules, and gain the capacity to perform cytotoxicity, suggesting that the human gut contains NK cell precursors. The role of NK cells in gut pathology has been examined. In mice, Listeria monocytogenes infection induced gut NK cell IFN-γ and this contributed to control bacterial dissemination. Furthermore, patients with inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, exhibit phenotypic alterations in their gut NK cell compartment.[83-85] The significance of these correlative results is further corroborated by population genetic studies indicating that certain NK cell receptors are linked to IBD susceptibility. Whereas less is known with respect to the interplay between gut NK cells and the intestinal microbiota, non-mucosal NK cells are dysfunctional in germ-free mice. While the broader implications of this finding still remain unclear, the relationship between gut NK cell function, inflammation and the intestinal microbiota merit further investigation.
Figure 2. Tissue-specific functions of innate lymphoid cells (ILCs). In the mouse gut, ILC2s provide defence against parasite infections whereas natural cytotoxicity receptor-positive (NCR+) ILC3s, through their production of interleukin-22 (IL-22), mediate direct tissue protective effects. In gut inflammation, natural killer (NK) cells, ILC1s, and NCR− ILC3s have all been implicated in contributing to inflammation via production of interferon-γ (IFN-γ) and/or IL-17. As in the gut, IL-22-producing NCR+ ILC3s seem to exhibit a tissue protective role in the airways. Interestingly, lung ILC2s seem to mediate both tissue-protective effects and type 2-mediated inflammation, the latter caused by the type 2 cytokines produced by these cells. NK cells are important for pulmonary virus infections but also seem to contribute to tissue damage and inflammation, including asthma. Relatively little is known about the role for ILCs in the liver. However, as in the lungs, NK cell action is both protective, providing anti-viral defence, and tissue damaging through production of pro-inflammatory molecules such as TRAIL. IL-22 production from NCR+ ILC3s might help to limit such collateral liver damage, although this remains to be determined. In the placenta, CD56superbright CD16− NK cells are important for trophoblast invasion and spiral artery remodelling, a process that is critical for the establishment of the placenta during pregnancy. In addition, NCR+ ILCs producing tissue-protective IL-22 have been found in second-trimester decidual tissue but the exact function of these cells in reproduction remains to be elucidated.
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ILC1s and ILC3s – yin and yang of the gut?
In mice, NKp46+ RORγt-dependent ILC3s were shown to reside in the gut lamina propria and in cryptopatches, but less in the intraepithelial layer.[5, 63, 88, 89] In the gut, these cells are important sources of IL-22, which is indispensable for homeostatic maintenance of epithelial barrier function in mice. This is exemplified by the notion that mice lacking NKp46+ RORγt-dependent ILC3s (RAG2−/− IL-2Rγc−/−) or IL-22 (IL-22−/−) rapidly succumb to infection with Citrobacter rodentium, which typically triggers IL-23-dependent IL-22 production from ILC3s.[5, 65, 66] The mechanism behind the gut epithelial barrier protective role of IL-22-producing ILC3s has been extensively studied in several mouse models of colitis and recently also in a model of graft-versus-host disease.
These gut ILC3s are in constant interaction with the microbiota and the net signalling from dietary and epithelium-derived factors as well as IL-23 probably determine the outcome of ILC3 effector functions. In mice, it was recently demonstrated that the source of IL-23 following systemic flagellin administration was a subset of gut CD103+ dendritic cells, which, in turn, promoted IL-22-dependent RegIIIγ production from gut epithelial cells. However, during homeostatic conditions, the levels of IL-23 are likely to be low. Here, dietary compounds, such as AHR ligands, might instead trigger IL-22 production from gut-residing ILC3s.[69-71] Recently, AHR was also shown to be important for IL-23 responsiveness as AHR−/− mice failed to respond to IL-23 production induced by Citrobacter rodentium and rapidly succumbed to infection. In addition to induction of anti-microbial peptides, such as RegIIIa and RegIIIb, IL-22 also has an important role in maintaining barrier function in the gut via its interaction with colonic epithelial cells where signalling through IL-22 receptors, via signal transducer and activator of transcription 3, promotes epithelial proliferation and gut wound healing.[65, 88, 91]
In addition to maintaining gut mucosal barrier function via IL-22 production, ILC3s have been implicated in colitis as demonstrated by work performed in several mouse models. For instance, it was suggested that IL-17 could drive Helicobacter hepaticus-induced colitis where induction of colitis elicited a population of ILCs producing IL-17 and IFN-γ. Interestingly, neutralization of IL-17 had little effect on colitis severity, whereas ablation of IFN-γ restored the phenotype, suggesting that IFN-γ is important in this particular model. In line with these data, colitis could be induced via anti-CD40 injection into RAG2−/− mice promoting a population of IFN-γ-producing NKp46+ ILCs that were distinct from NK cells. Adoptive transfer of such cells was enough to induce colitis, whereas depletion ameliorated the gut inflammation, again suggesting that IFN-γ-producing ILC1s can drive colitis. These findings are further corroborated by results from the human setting where ILC1s with a Th1 phenotype accumulate in the inflamed ileum of patients with Crohn's disease. These ILC1s were distinct from NK cells because they lacked expression of prototypic NK cell markers, including cytotoxic molecules, and did not develop into NK cells in vitro. Intriguingly, although RORγt− precursors cannot be ruled out, these ILC1s could be generated from RORγt+ IL-22-producing ILC3s under the influence of IL-12/18. Interleukin-12/18 stimulation led to down-regulation of RORγt, up-regulation of T-bet, and the capacity to produce IFN-γ. Furthermore, in the absence of T-bet, using Tbx21−/− RAG2−/− (TRUC) mice, colitis can be driven by ILC1s producing excessive amounts of IL-23-triggered and tumour necrosis factor-triggered IL-17A. Hence, there seems to be a redundancy in the system, with IL-17 and IFN-γ both being able to drive gut inflammation. Given that blocking of these two cytokines in human IBD have largely generated disappointing results,[94, 95] more information is needed regarding the role of these cytokines and of ILCs in the pathogenesis of IBD.
In summary, ILC3s seem to be important for the maintenance of gut barrier function and homeostasis (Fig. 2). However, similar to T helper cells, these cells are plastic and may be dysregulated to participate in inflammation through reprogramming by the surrounding pro-inflammatory microenvironment.
The ILC2s, dedicated to production of type 2 cytokines, were the third population of ILCs to be described in the mouse. Several groups established that these cells were critical for gut parasite expulsion (Fig. 2),[14-16] although this still remains to be verified in the human setting. In RAG−/− mice, a population of Lin− IL-7Ra+ ckit+ Sca1+ T1/ST2+ ILC2s, associated with the mesenteric fat, mediated helminthic parasite expulsion from the gut. This population produced type 2 cytokines upon parasite infection or after treatment with IL-33. The ILC2s could induce all the classical features of type 2-mediated inflammation in the gut, including goblet cell hyperplasia and support of B1-cell-mediated IgA production. Absence of ILC2s, as in RAG−/− IL-2Rγc−/− mice, was associated with failure to handle parasitic infection, whereas adoptive transfer of ILC2s to these mice rescued them from A. brasiliensis-associated gut pathology. Corroborating these observations, additional reports, using either IL-4 or IL-13 reporter mice, pinpointed the source of IL-13 in the gut. A population of IL-13-producing Lin− lymphocytes, termed nuocytes by the authors, was induced by IL-25, or IL-33, and was largely absent in IL-25R-deficient animals (IL-17BR−/−). As a consequence, IL-17BR−/− mice failed to clear gut parasitic infection, whereas this function was restored upon adoptive transfer of IL-13-producing ILC2s. Similarly, with IL-13 reporter mice, the presence of a Lin− cell population elicited by IL-25 administration or Nippostrongylus brasiliensis infection was demonstrated. Interestingly, in this model, IL25−/− mice developed severe IFN-γ- and IL-17-mediated infection-induced gut inflammation. This suggests that ILC2s in gut might not only be mediators of type 2 immunity, but might also have immune regulatory functions limiting gut inflammation.
In the human setting, ILC2s have been reported in fetal gut, as well as in the adult gut of IBD and non-IBD patients. However, the frequencies are low, and so far no definite physiological or inflammatory role has been ascribed to these cells. Future studies, aimed at unravelling the role of these cells in human gut parasite infections, which is a major health burden in many low-income countries, are warranted.