The prototype RORγt+ ILCs are the LTi cells, which play essential roles in the formation of secondary lymph nodes during fetal development, both in mice and humans [[25, 26]]. After birth, LTi cells are important for the formation of cryptopatches (CPs), as well as isolated lymphoid follicles (ILFs), which evolve from CPs. Within the ILFs, LTi cells are required for the production of IgA by B cells []. LTi cells are able to produce predominantly IL-17, but also some IL-22 [].
Other RORγt-dependent ILCs, which emerge after birth, have been identified [[28-35]]. These cells express the natural cytotoxicity receptor NKp46 and mostly produce IL-22, and hence they are referred to as the ILC22 subset. This subset plays several roles in the early stages of the immune response against pathogens, as exemplified by the effacing-attaching bacterium Citrobacter rodentium. This bacterium causes colitis and wasting disease, which is transient, and is cleared by T cells []. IL-22 is essential in the early response against C. rodentium as, in the absence of this cytokine, these cytokine-deficient mice succumb to the infection []. In this setting, IL-22 is mainly derived from ILCs, as deletion of the ILC22 subset in the acute phase of infection is fatal for the C. rodentium-infected mice, illustrating the importance of these cells in this type of immune response [[30, 34, 38]]. IL-22 production from ILCs is regulated by IL-23 and IL-1β [[39, 40]], and IL-22 mediates its protective effects by acting on epithelial cells, inducing proliferation and secretion of antimicrobial peptides (reviewed in []). A RORγt-dependent ILC population that produces IL-17, rather than IL-22, and is therefore called the ILC17 subset, is present in inflamed intestines in a model for inflammatory bowel disease []. Deletion of these cells ameliorates colitis suggesting that they mediate pathology in this model.
Thus far, three transcription factors have been identified that are involved in the control of development, survival, and function of Rorγt-dependent ILCs: Rorγt, Notch and AhR.
The RORC gene encodes two isoforms: RORγ (also referred to as RORγ1) and RORγt (called RORγ2). RORγ is a broadly expressed nuclear receptor []. RORγt is shorter than RORγ at the N-terminus, as the most 5’ end exons are replaced by a specific RORγt exon. ROR contains a ligand-binding domain to which different ligands can bind, such as 7 substitute oxysterols ([], and reviewed in []), but the exact nature of the agonist that binds to RORγt in different cell types is unclear. RORγt is expressed in double positive thymocytes, Th17 cells, a subset of FoxP3+ Treg cells, subsets of iNKT cells and TCRγδ cells, LTi cells and the ILC22 subset [[5, 45-47]], and genetic ablation of RORγt affects the development and functions of those cell types. RORγt-deficient mice completely lack LTi cells and, as a consequence, Rorγt−/− mice fail to develop lymph nodes, Peyer's patches and ILFs []. In Rorγt−/− mice, numbers of IL-22-producing ILCs, which express NKp46, are severely reduced as well as is their capacity to produce IL-22, whereas NK-cell numbers are unaffected [[30, 35, 41]]. The fact that RORγt is required for the development of both IL-17- and IL-22-producing Th17 cells [] and ILCs reinforces the idea that RORγt+ ILCs are the innate equivalent of Th17 cells.
AhR and Notch
AhR is a ligand-dependent transcription factor that belongs to the family of bHLH PER-ARNT-SIM transcription factors. AhR acts as a sensor of a variety of chemicals, including environmental toxins such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), and phytochemicals such as indol-3-carbinol, produced by cruciferous vegetables including cauliflower, cabbage, and broccoli []. Endogenous ligands have been identified as well, for instance the tryptophan photoproduct 6-formylindolo-(3,2-b)-carbazole (FICZ). In the cytoplasm, AhR is a component of a complex that includes chaperones like hsp90 and from which AhR is dissociated upon its activation by ligand binding. AhR associates with the AhR nuclear transporter (Arnt) prior to translocation to the nucleus to bind to promoters of a variety of genes (reviewed in []).
Only recently was a role for AhR in immunity identified. In mice, AhR controls the differentiation of Th17 cells [], and negatively affects the development of Treg cells []. Inhibition of Th17-cell differentiation by T cell-specific deletion of AhR resulted in the amelioration of collagen-induced arthritis, indicating that over-stimulation of AhR can result in pathology []. Interestingly, AhR controls the production of IL-22 by T cells, as ablation of AhR in mice completely eliminated the capacity of Th17 cells to produce IL-22 [[49, 52]]. Furthermore, AhR is involved in IL-22 production by Th22 cells in humans []. More recently, another activity of AhR emerged when it was found that AhR controls the maintenance of gut epithelium-residing CD8αα+ TCRαβ and TCRγδ cells (collectively denoted as intraepithelial lymphocytes (IELs)). Genetic ablation of AhR resulted in specific loss of IELs []. Interestingly, dietary components, in particular indol-3-carbinol, serve as ligands for AhR. Furthermore, these dietary products have been shown to be important for IEL maintenance, since mice fed with a vegetable-free diet showed reduced numbers of these cells [].
Recent work has established that AhR is not only important for the maintenance of IELs, but also for both LTi cells and the ILC22 subset that reside in the gut. Several groups reported that AhR-deficient mice had clearly reduced numbers of Rorγt+ ILCs, including LTi cells and ILC22 cells, in the gut [[54-56]]. This was also seen in Ahr−/−Rag1−/− mice, lacking T cells, as well as in sublethally irradiated wt mice receiving Ahr−/– stem cells. In addition, Rorγt+ ILC numbers were also reduced upon specific deletion of AhR in Rorγt-expressing cells (including ILCs) []. Together these data indicate that the effects of AhR-deficiency on Rorγt+ ILCs are cell intrinsic. Interestingly, the reduction of Rorγt+ ILC numbers, induced by ablation of AhR, was observed only after birth. During fetal development, and early after birth, the ILC22 numbers in AhR-deficient mice are comparable to those in wild type mice, indicating that AhR is not required for development of these cells []. However, after weaning, the numbers of Rorγt+ ILCs in AhR-deficient mice steadily decrease []. Maintenance of ILC numbers is not a consequence of AhR activation by products of colonizing microbiota, because the difference in ILC22 numbers between wt and AhR-deficient animals is not affected by treatment with a mix of antibiotics []. Also, the observation that germ-free animals do not show reductions in gut residing Rorγt+ ILC numbers [[55, 57]] is consistent with the notion that products from commensals are not required for the maintenance of these cells.
It is controversial whether dietary products are the AhR ligands responsible for the maintenance of gut-residing Rorγt+ ILCs, as observed for IELs []. In one study, it was found that mice fed with a diet free of AhR-binding phytochemicals showed decreased numbers of Rorγt+ ILCs, causing a lack of CPs and ILFs []. Addition of indole-3-carbinol, a dietary product, restored the Rorγt+ ILC numbers []. Another study, however, suggested that endogenous AhR ligands, including the tryptophane catabolite kynurenine, were potent regulators of Rorγt+ ILC maintenance as removal of dietary AhR ligands in that study did not disturb Rorγt+ ILC homeostasis and function []. The differences may be due to different types of controlled diets used by the different groups. Further experiments should aim to resolve these discrepancies.
The mechanisms by which AhR controls Rorγt+ cell numbers are not fully understood. Microarray analysis of Rorγt+ cells from wt and AhR-deficient mice suggested that Notch 1 is a downstream target of AhR []. Consistent with this, administration by gavage of the toxin TCCD (2,3,7,8-tetrachlorodibenzo-p-dioxin) resulted in the upregulation of Notch1 and Notch2 in gut Rorγt+ ILCs. Evidence for a role of Notch in AhR-mediated maintenance of Rorγt+ ILCs was provided by the observation that mice deficient for RBP-Jk, an essential partner of Notch, showed substantially reduced numbers of NKp46-expressing Rorγt+ ILCs and, although less prominently, of CD4+ Rorγt+ ILCs (LTi cells) also []. However, there were differences between the AhR- and RBP-Jk-deficient mice, in that in the latter, cryptopatches and ILFs were largely intact, whereas they were greatly reduced in AhR-deficient mice []. These data suggest that AhR mediates its effect on Rorγt+ ILCs only partly through Notch signaling; however, it should be remembered that Cre-mediated deletion of a floxed allele is rarely complete and thus there may be sufficient residual RBP-Jk present in the RBP-Jk-deficient mice for development of cryptopatches and ILFs. Notch signaling was found to be important for in vitro development of adult [] and fetal CLPs [] into RORγt+ ILCs. Interestingly, the latter study suggested a stage-specific requirement of Notch signaling in the development of RORγt+ ILCs as Notch signaling was required in an early stage of development of these cells but inhibited a subsequent step []. The relevant Notch for this role could be Notch2 [] but this has yet to be confirmed in in vivo experiments.
Rorγt+ cells in Ahr−/− mice express lower levels of the anti-apoptotic protein Bcl-2 and accordingly are more apoptotic []. Bcl-2 might be induced by the major cytokine receptors expressed on Rorγt+ ILCs, namely IL-7Rα and ckit; however, there are conflicting data with regard to the link of AhR and IL-7Rα. In one study, expression of IL-7Rα was decreased by AhR ablation [], whereas another group did not observe any change in IL-7Rα expression on Ahr−/– ILCs []. cKit, which is the receptor for stem cell growth factor, may be a direct downstream target of AhR since expression of this receptor is strongly decreased in Ahr−/− ILCs []. It is possible that the Rorγt+ ILC numbers are regulated by AhR in a cKit dependent manner. This suggestion comes from observations made in KitWv/Wv mice, which express a ckit variant with impaired kinase activity. These mice not only show diminished numbers of Rorγt+ ILCs, but also reduced numbers and sizes of CPs and ILFs. These findings strongly suggest that AhR regulates maintenance of RORγt-dependent ILCs by controlling ckit expression.
As in Th17 cells, AhR also appears to be required for optimal IL-22 production by the ILC22 population. The reduction of Rorγt+ ILC numbers in the gut, and the decreased capacity of these cells to produce IL-22, has functional consequences because AhR-deficient mice succumb to infection with C. rodentium and hydrodynamic injection of an IL-22-expressing plasmid into the tail vein reestablishes protection against C. rodentium []. In this setting, IL-23, produced by activated macrophages and DCs, controls IL-22 production by ILCs. Interestingly, AhR-deficient mice display reduced IL-23 receptor expression and IL-23 responsiveness []. It is likely that AhR directly controls IL-22 expression, as the Il22 locus contains multiple AhR-responsive elements []. Interestingly these elements are clustered with Ror-responsive elements and, in the Il22 locus, both Rorγt and AhR bind directly to their response elements. Whereas AhR recruitment to the well-known AhR target Cyp1a1 is unaffected by Rorγt, AhR binding to the Il22 locus is strongly enhanced by Rorγt [].