Naoko Satoh-Takayama

Microbial Flora Drives Interleukin 22 Production in Intestinal NKp46+ Cells that Provide Innate Mucosal Immune Defense

Natural killer (NK) cells are innate lymphocytes with spontaneous antitumor activity, and they produce interferon-gamma (IFN-gamma) that primes immune responses. Whereas T helper cell subsets differentiate from naive T cells via specific transcription factors, evidence for NK cell diversification is limited. In this report, we characterized intestinal lymphocytes expressing the NK cell natural cytotoxicity receptor NKp46. Gut NKp46+ cells were distinguished from classical NK cells by limited IFN-gamma production and absence of perforin, whereas several subsets expressed the nuclear hormone receptor retinoic acid receptor-related orphan receptor t (RORgammat) and interleukin-22 (IL-22). Intestinal NKp46+IL-22+ cells were generated via a local process that was conditioned by commensal bacteria and required RORgammat. Mice lacking IL-22-producing NKp46+ cells showed heightened susceptibility to the pathogen Citrobacter rodentium, consistent with a role for intestinal NKp46+ cells in immune protection. RORgammat-driven diversification of intestinal NKp46+ cells thereby specifies an innate cellular defense mechanism that operates at mucosal surfaces.

Lineage Relationship Analysis of RORγt+ Innate Lymphoid Cells

Innate Innit? Innate lymphocytes (ILCs) are a recently described population of immune cells that produce cytokines like those associated with T helper cells, but lack the recombined antigen receptors characteristic of T cells. Again, like some T helper cell lineages, a proportion of ILCs express the transcription factor RORγt. These include lymphoid tissue inducer (LTi) cells required for fetal lymphoid tissue organogenesis and a population of natural killer (NK)–like cells that function in gut immune responses. Sawa et al. (p. 665; see the Perspective by Veldhoen and Withers) wondered whether the RORγt-expressing ILCs all develop from the same progenitor population. Indeed, they found a fetal liver progenitor that gave rise to several phenotypically distinct populations. However, the LTi cells were not progenitors for the NK-like cells. It seems the trajectory of different ILC populations is developmentally regulated, and postnatally ILCs are favored that play a role in intestinal defense before the gut is fully colonized by intestinal microbiota. Immune cells develop to preempt intestinal colonization by microbial symbionts. Lymphoid tissue–inducer (LTi) cells initiate the development of lymphoid tissues through the activation of local stromal cells in a process similar to inflammation. LTi cells express the nuclear hormone receptor RORγt, which also directs the expression of the proinflammatory cytokine interleukin-17 in T cells. We show here that LTi cells are part of a larger family of proinflammatory RORγt+ innate lymphoid cells (ILCs) that differentiate from distinct fetal liver RORγt+ precursors. The fate of RORγt+ ILCs is determined by mouse age, and after birth, favors the generation of cells involved in intestinal homeostasis and defense. Contrary to RORγt+ T cells, however, RORγt+ ILCs develop in the absence of microbiota. Our study indicates that RORγt+ ILCs evolve to preempt intestinal colonization by microbial symbionts.

IL-7 and IL-15 independently program the differentiation of intestinal CD3−NKp46+ cell subsets from Id2-dependent precursors

The natural cytotoxicity receptor NKp46 (encoded by Ncr1) was recently shown to identify a subset of noncytotoxic, Rag-independent gut lymphocytes that express the transcription factor Rorc, produce interleukin (IL)-22, and provide innate immune protection at the intestinal mucosa. Intestinal CD3−NKp46+ cells are phenotypically heterogeneous, comprising a minority subset that resembles classical mature splenic natural killer (NK) cells (NK1.1+, Ly49+) but also a large CD127+NK1.1− subset of lymphoid tissue inducer (LTi)–like Rorc+ cells that has been proposed to include NK cell precursors. We investigated the developmental relationships between these intestinal CD3−NKp46+ subsets. Gut CD3−NKp46+ cells were related to LTi and NK cells in requiring the transcriptional inhibitor Id2 for normal development. Overexpression of IL-15 in intestinal epithelial cells expanded NK1.1+ cells within the gut but had no effect on absolute numbers of the CD127+NK1.1−Rorc+ subset of CD3−NKp46+ cells. In contrast, IL-7 deficiency strongly reduced the overall numbers of CD3−NKp46+NK1.1− cells that express Rorc and produce IL-22 but failed to restrict homeostasis of classical intestinal NK1.1+ cells. Finally, in vivo fate-mapping experiments demonstrated that intestinal NK1.1+CD127− cells are not the progeny of Rorc-expressing progenitors, indicating that CD127+NK1.1−Rorc+ cells are not canonical NK cell precursors. These studies highlight the independent cytokine regulation of functionally diverse intestinal NKp46+ cell subsets.

Gata3 drives development of RORγt+ group 3 innate lymphoid cells

The transcription factor Gata3 is required for the generation of group 3 innate lymphoid cells (ILC3) that protect mucosal surfaces.

The Natural Cytotoxicity Receptor NKp46 Is Dispensable for IL-22-Mediated Innate Intestinal Immune Defense against Citrobacter rodentium

Natural cytotoxicity receptors (including NKp30, NKp44, and NKp46 in humans and NKp46 in mice) are type I transmembrane proteins that signal NK cell activation via ITAM-containing adapter proteins in response to stress- and pathogen-induced ligands. Although murine NKp46 expression (encoded by Ncr1) was thought to be predominantly restricted to NK cells, the identification of distinct intestinal NKp46+ cell subsets that express the transcription factor Rorc and produce IL-22 suggests a broader function for NKp46 that could involve intestinal homeostasis and immune defense. Using mice carrying a GFP-modified Ncr1 allele, we found normal numbers of gut CD3−GFP+ cells with a similar cell surface phenotype and subset distribution in the absence of Ncr1. Splenic and intestinal CD3−NKp46+ cell subsets showed distinct patterns of cytokine secretion (IFN-γ, IL-22) following activation via NK1.1, NKp46, IL-12 plus IL-18, or IL-23. However, IL-22 production was sharply restricted to intestinal CD3−GFP+ cells with the CD127+NK1.1− phenotype and could be induced in an Ncr1-independent fashion. Because NKp46 ligands can trigger immune activation in the context of infectious pathogens, we assessed the response of wild-type and Ncr-1-deficient Rag2−/− mice to the enteric pathogen Citrobacter rodentium. No differences in the survival or clinical score were observed in C. rodentium-infected Rag2−/− mice lacking Ncr1, indicating that NKp46 plays a redundant role in the differentiation of intestinal IL-22+ cells that mediate innate defense against this pathogen. Our results provide further evidence for functional heterogeneity in intestinal NKp46+ cells that contrast with splenic NK cells.

Lymphotoxin‐β receptor‐independent development of intestinal IL‐22‐producing NKp46+ innate lymphoid cells

The natural cytotoxicity receptor NKp46 is an activating receptor expressed by several distinct innate lymphoid cell (ILC) subsets, including NK cells, some γδ T cells and intestinal RORγt+IL‐22+ cells (NCR22 cells, IL‐22‐producing NKp46+ cell). NCR22 cells may play a role in mucosal barrier function through IL‐22‐mediated production of anti‐bacterial peptides from intestinal epithelial cells. Previous studies identified a predominant proportion of NCR22 cells in gut cryptopatches (CP), lymphoid structures that are strategically positioned to collect and integrate signals from luminal microbes; however, whether CP or other lymphoid structures condition NCR22 cell differentiation is not known. Programmed and inducible lymphoid tissue development requires cell‐surface‐expressed lymphotoxin (LT)α1β2 heterotrimers (provided by lymphoid tissue inducer (LTi) cells) to signal lymphotoxin‐β receptor (LTR)+ stromal cells. Here, we analyzed NCR22 cells in LTβR‐deficient Ncr1GFP/+ mice that lack organized secondary lymphoid tissues. We found that NCR22 cells develop in the absence of LTβR, become functionally competent and localize to the lamina propria under steady‐state conditions. Following infection of LTβR−/− mice with the Gram‐negative pathogen Citrobacter rodentium, IL‐22 production from NCR22 cells was not affected. These results indicate that organized lymphoid tissue structures are not critical for the generation of an intact and fully functional intestinal NCR22 cell compartment.

IL-22 is produced by γC-independent CD25+ CCR6+ innate murine spleen cells upon inflammatory stimuli and contributes to LPS-induced lethality.

IL‐22 is a Th17 cytokine that plays a key role in immune responses against extracellular bacteria. In mucosal lymphoid tissues, IL‐22 production is mainly due to an IL‐23‐responsive NK‐like cell subset that shares some markers with lymphoid tissue inducer (LTi) cells. Here, we identified a new spleen cell population responsible for IL‐22 production upon either in vitro stimulation by anti‐CD3 antibodies or in vivo stimulation by lipopolysaccharide (LPS) via IL‐2‐ and an IL‐23‐dependent mechanisms, respectively. These cells represent 1% of spleen cells from recombination activating gene (Rag2)‐deficient mice, and correspond to a discrete innate lymphoid cell population expressing CD25, CCR6 and IL‐7R. This population comprises 60–70% CD4+ cells, which produce IL‐22, and are still present in common γ chain‐deficient mice; the CD4− subset coexpresses IL‐22 and IL‐17, and is common γ chain‐dependent. The importance of IL‐22 production for the LPS‐triggered response is highlighted by the fact that IL‐22‐deficient mice are more resistant to LPS‐induced mortality.

Phenotypic and Functional Plasticity of Murine Intestinal NKp46+ Group 3 Innate Lymphoid Cells

Group 3 innate lymphoid cells (ILC3) actively participate in mucosal defense and homeostasis through prompt secretion of IL-17A, IL-22, and IFN-γ. Reports identify two ILC3 lineages: a CCR6+T-bet− subset that appears early in embryonic development and promotes lymphoid organogenesis and a CCR6−T-bet+ subset that emerges after microbial colonization and harbors NKp46+ ILC3. We demonstrate that NKp46 expression in the ILC3 subset is highly unstable. Cell fate mapping using Ncr1CreGFP × Rosa26RFP mice revealed the existence of an intestinal RFP+ ILC3 subset (Ncr1FM) lacking NKp46 expression at the transcript and protein levels. Ncr1FM ILC3 produced more IL-22 and were distinguishable from NKp46+ ILC3 by differential CD117, CD49a, DNAX accessory molecule-1, and, surprisingly, CCR6 expression. Ncr1FM ILC3 emerged after birth and persisted in adult mice following broad-spectrum antibiotic treatment. These results identify an unexpected phenotypic instability within NKp46+ ILC3 that suggests a major role for environmental signals in tuning ILC3 functional plasticity.

Conditional ablation of NKp46+ cells using a novel Ncr1greenCre mouse strain: NK cells are essential for protection against pulmonary B16 metastases

To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1greenCre mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre‐expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell‐specific expression of RFP in Ncr1greenCreRosa‐dtRFP reporter mice. We generated Ncr1greenCreIl2rgfl/fl mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc), which is an essential receptor subunit for cytokines (IL‐2, ‐4, ‐7, ‐9, ‐15, and ‐21) that stimulate lymphocyte development and function. In Ncr1greenCreIl2rgfl/fl mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK‐cell‐deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1greenCreIl2rgfl/fl mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.

Dissecting Human NK Cell Development and Differentiation

Our understanding of human NK cell biology lags behind that of the mouse NK cell biology; this is in a large part because of the ethical and logistical restrictions to the access of healthy human lymphoid tissue and the experimental manipulation in vivo. Nevertheless, in-depth analyses in genetically modified mice have provided us with models for NK cell development, differentiation, and function that guide our thinking about the role of NK cells in immune defense. Collectively, mouse and human studies have unveiled a number of conserved transcription factors, cytokines, cell surface receptors, and associated signaling proteins that are essential for normal NK cell development. Still, human and mouse NK cells differ with regard to expression of several key cell surface receptors, kinetics of development, and frequency in adult lymphoid organs (Huntington ND, Vosshenrich CA, Di Santo JP. Nat Rev Immunol 7:703–714, 2007). Accordingly, the specific biological roles for NK cells in human immune responses remain poorly described. New preclinical animal models that allow the analysis of human immune system development in function may provide a means to further our understanding of the biology of human NK cells in vivo.