Complementarity and redundancy of IL-22-producing innate lymphoid cells.

Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.

Group 3 innate lymphoid cells continuously require the transcription factor GATA-3 after commitment.

The transcription factor GATA-3 is indispensable for the development of all innate lymphoid cells (ILCs) that express the interleukin 7 receptor α-chain (IL-7Rα). However, the function of low GATA-3 expression in committed group 3 ILCs (ILC3 cells) has not been identified. We found that GATA-3 regulated the homeostasis of ILC3 cells by controlling IL-7Rα expression. In addition, GATA-3 served a critical function in the development of the NKp46(+) ILC3 subset by regulating the balance between the transcription factors T-bet and RORγt. Among NKp46(+) ILC3 cells, although GATA-3 positively regulated genes specific to the NKp46(+) ILC3 subset, it negatively regulated genes specific to lymphoid tissue-inducer (LTi) or LTi-like ILC3 cells. Furthermore, GATA-3 was required for IL-22 production in both ILC3 subsets. Thus, despite its low expression, GATA-3 was critical for the homeostasis, development and function of ILC3 subsets.

Feeding-dependent VIP neuron-ILC3 circuit regulates the intestinal barrier.

The intestinal mucosa serves both as a conduit for the uptake of food-derived nutrients and microbiome-derived metabolites, and as a barrier that prevents tissue invasion by microorganisms and tempers inflammatory responses to the myriad contents of the lumen. How the intestine coordinates physiological and immune responses to food consumption to optimize nutrient uptake while maintaining barrier functions remains unclear. Here we show in mice how a gut neuronal signal triggered by food intake is integrated with intestinal antimicrobial and metabolic responses that are controlled by type-3 innate lymphoid cells (ILC3)1-3. Food consumption rapidly activates a population of enteric neurons that express vasoactive intestinal peptide (VIP)4. Projections of VIP-producing neurons (VIPergic neurons) in the lamina propria are in close proximity to clusters of ILC3 that selectively express VIP receptor type 2 (VIPR2; also known as VPAC2). Production of interleukin (IL)-22 by ILC3, which is upregulated by the presence of commensal microorganisms such as segmented filamentous bacteria5-7, is inhibited upon engagement of VIPR2. As a consequence, levels of antimicrobial peptide derived from epithelial cells are reduced but the expression of lipid-binding proteins and transporters is increased8. During food consumption, the activation of VIPergic neurons thus enhances the growth of segmented filamentous bacteria associated with the epithelium, and increases lipid absorption. Our results reveal a feeding- and circadian-regulated dynamic neuroimmune circuit in the intestine that promotes a trade-off between innate immune protection mediated by IL-22 and the efficiency of nutrient absorption. Modulation of this pathway may therefore be effective for enhancing resistance to enteropathogens2,3,9 and for the treatment of metabolic diseases.

Interleukin-22 protects intestinal stem cells against genotoxic stress.

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1-3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated 'sensing' of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.

Regulatory B cells: origin, phenotype, and function.

Regulatory B (Breg) cells are immunosuppressive cells that support immunological tolerance. Through the production of interleukin-10 (IL-10), IL-35, and transforming growth factor ß (TGF-ß), Breg cells suppress immunopathology by prohibiting the expansion of pathogenic T cells and other pro-inflammatory lymphocytes. Recent work has shown that different inflammatory environments induce distinct Breg cell populations. Although these findings highlight the relevance of inflammatory signals in the differentiation of Breg cells, they also raise other questions about Breg cell biology and phenotype. For example, what are the functional properties and phenotype of Breg cells? Can a Breg cell arise at every stage in B cell development? Is inflammation the primary requisite for Breg cell differentiation? Here, we use these questions to discuss the advances in understanding Breg cell biology, with a particular emphasis on their ontogeny; we propose that multiple Breg cell subsets can be induced in response to inflammation at different stages in development.

Induction of Interleukin-9-Producing Mucosal Mast Cells Promotes Susceptibility to IgE-Mediated Experimental Food Allergy.

Experimental IgE-mediated food allergy depends on intestinal anaphylaxis driven by interleukin-9 (IL-9). However, the primary cellular source of IL-9 and the mechanisms underlying the susceptibility to food-induced intestinal anaphylaxis remain unclear. Herein, we have reported the identification of multifunctional IL-9-producing mucosal mast cells (MMC9s) that can secrete prodigious amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induced MMC9 development that required T cells, IL-4, and STAT6 transcription factor, but not IL-9 signals. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, which resulted in decreased food allergy symptoms that could be restored by adoptively transferred MMC9s. Finally, atopic patients that developed food allergy displayed increased intestinal expression of Il9- and MC-specific transcripts. Thus, the induction of MMC9s is a pivotal step to acquire the susceptibility to IgE-mediated food allergy.

Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells.

T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1-3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.

Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism.

The mammalian gut is colonized by numerous microorganisms collectively termed the microbiota, which have a mutually beneficial relationship with their host. Normally, the gut microbiota matures during ontogeny to a state of balanced commensalism marked by the absence of adverse inflammation. Subsets of innate lymphoid cells (ILCs) and conventional T cells are considered to have redundant functions in containment and clearance of microbial pathogens, but how these two major lymphoid-cell populations each contribute to shaping the mature commensal microbiome and help to maintain tissue homeostasis has not been determined. Here we identify, using advanced multiplex quantitative imaging methods, an extensive and persistent phosphorylated-STAT3 signature in group 3 ILCs and intestinal epithelial cells that is induced by interleukin (IL)-23 and IL-22 in mice that lack CD4+ T cells. By contrast, in immune-competent mice, phosphorylated-STAT3 activation is induced only transiently by microbial colonization at weaning. This early signature is extinguished as CD4+ T cell immunity develops in response to the expanding commensal burden. Physiologically, the persistent IL-22 production from group 3 ILCs that occurs in the absence of adaptive CD4+ T-cell activity results in impaired host lipid metabolism by decreasing lipid transporter expression in the small bowel. These findings provide new insights into how innate and adaptive lymphocytes operate sequentially and in distinct ways during normal development to establish steady-state commensalism and tissue metabolic homeostasis.

Transcription factor c-Maf mediates the TGF-ß-dependent suppression of IL-22 production in T(H)17 cells.

Interleukin 22 (IL-22), which is produced by cells of the T(H)17 subset of helper T cells and other leukocytes, not only enhances proinflammatory innate defense mechanisms in epithelial cells but also provides crucial protection to tissues from damage caused by inflammation and infection. In T(H)17 cells, transforming growth factor-ß (TGF-ß) regulates IL-22 and IL-17 differently. IL-6 alone induces T cells to produce only IL-22, whereas the combination of IL-6 and high concentrations of TGF-ß results in the production of IL-17 but not IL-22 by T cells. Here we identify the transcription factor c-Maf, which is induced by TGF-ß, as a downstream repressor of Il22. We found that c-Maf bound to the Il22 promoter and was both necessary and sufficient for the TGF-ß-dependent suppression of IL-22 production in T(H)17 cells.

The chemokine receptor CXCR6 controls the functional topography of interleukin-22 producing intestinal innate lymphoid cells.

Interleukin-22 (IL-22) plays a critical role in mucosal defense, although the molecular mechanisms that ensure IL-22 tissue distribution remain poorly understood. We show that the CXCL16-CXCR6 chemokine-chemokine receptor axis regulated group 3 innate lymphoid cell (ILC3) diversity and function. CXCL16 was constitutively expressed by CX3CR1(+) intestinal dendritic cells (DCs) and coexpressed with IL-23 after Citrobacter rodentium infection. Intestinal ILC3s expressed CXCR6 and its ablation generated a selective loss of the NKp46(+) ILC3 subset, a depletion of intestinal IL-22, and the inability to control C. rodentium infection. CD4(+) ILC3s were unaffected by CXCR6 deficiency and remained clustered within lymphoid follicles. In contrast, the lamina propria of Cxcr6(-/-) mice was devoid of ILC3s. The loss of ILC3-dependent IL-22 epithelial stimulation reduced antimicrobial peptide expression that explained the sensitivity of Cxcr6(-/-) mice to C. rodentium. Our results delineate a critical CXCL16-CXCR6 crosstalk that coordinates the intestinal topography of IL-22 secretion required for mucosal defense.

The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair.

Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3(-/-) bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and by impaired caspase 1-mediated processing of interleukin-1ß (IL-1ß). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3(-/-) mice exhibited an impaired axis of injury-induced IL-1ß, IL-23, and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild-type DCs, but not Ripk3(-/-) DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory-cytokine expression, and injury-induced tissue repair.

Human pregnancy levels of estrogen and progesterone contribute to humoral immunity by activating TFH /B cell axis.

Circulating TFH (cTFH ) cells express CXCR5, PD-1, and, when activated, ICOS, and release IL-21. According to the production of IFN-γ, IL-4, and IL-17 and expression of FoxP3, these cells are also classified as cTFH 1, cTFH 2, cTFH 17, and cTFR cells, respectively. This CD4+ T-cell subset is pivotal to efficient humoral immunity, and pregnancy appears to favor IgG production. Here, not only pregnancy amplified the in vivo production of anti-HBsAg IgG in HBV immunized women, but the frequency of cTFH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-ß-estradiol (E2) directly increased the percentage of different cTFH subsets. While E2 and progesterone (P4) increased the proportion of differentiated TFH cells derived from naïve CD4+ T-cells, only E2 amplified the release of IL-21 in those cell cultures. In addition, E2 and P4 increased the proportion of memory B cells and plasma cells, respectively. In SEB-activated B/TFH cell co-cultures, E2, in the presence of P4, increased the production of total IgG. Finally, among the hormones, P4 was stronger in upregulating the percentage of IL-10+ TFR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cTFH and B cell subsets.

Dysregulation in B-cell responses and T follicular helper cell function in ADA2 deficiency patients.

Adenosine deaminase 2 deficiency (DADA2) is an autoinflammatory disease characterized by inflammatory vasculopathy, early strokes associated often with hypogammaglobulinemia. Pure red cell aplasia, thrombocytopenia, and neutropenia have been reported. The defect is due to biallelic loss of function of ADA2 gene, coding for a protein known to regulate the catabolism of extracellular adenosine. We therefore investigated immune phenotype and B- and T-cell responses in 14 DADA2 patients to address if ADA2 mutation affects B- and T-cell function. Here, we show a significant decrease in memory B cells, in particular class switch memory, and an expansion of CD21low B cells in DADA2 patients. In vitro stimulated B lymphocytes were able to secrete nonfunctional ADA2 protein, suggesting a cell intrinsic defect resulting in an impairment of B-cell proliferation and differentiation. Moreover, CD4+ and CD8+ T cells were diminished; however, the frequency of circulating T follicular helper cells was significantly increased but they had an impairment in IL-21 production possibly contributing to an impaired B cell help. Our findings suggest that ADA2 mutation could lead to a B-cell intrinsic defect but also to a defective Tfh cell function, which could contribute to the immunodeficient phenotype reported in DADA2 patients.

The molecular basis for IL-31 production and IL-31-mediated itch transmission: from biology to drug development.

Atopic dermatitis (AD) is one of the most prevalent chronic inflammatory skin diseases in the world. It is characterized by recurrent eczematous lesions and intense itch, and many cytokines are involved in the pathogenesis of AD. Among them, much attention has been paid to interleukin 31 (IL-31) as an AD-associated itch mediator. IL-31 is mainly produced by CD4+ helper T cells and transmits the signals via a heterodimeric receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR), both of which are expressed in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms of how IL-31 is produced in helper T cells upon stimulation and transmits the itch sensation to the brain were largely unknown. Recently, by using original mouse models of AD, we have identified endothelial PAS domain 1 (EPAS1) and neurokinin B (NKB) as key molecules critical for IL-31 production and IL-31-mediated itch transmission, respectively. These molecules could be novel drug targets for AD-associated itch. This review highlights our recent findings, which show the functional significance of these molecules in the IL-31-induced itch sensation, referring to their application to drug development.

Cell-intrinsic IL-27 and gp130 cytokine receptor signaling regulates virus-specific CD4⁺ T cell responses and viral control during chronic infection.

The outcome of chronic viral infections, which affect millions of people worldwide, is greatly dependent on CD4⁺ T cells. Here we showed that T cell-specific ablation of the common interleukin-6 (IL-6) family receptor, gp130, profoundly compromised virus-specific CD4⁺ T cell survival, T follicular helper responses, and IL-21 production at late stages of chronic lymphocytic choriomeningitis virus (LCMV) infection. These effects were cell intrinsic for CD4⁺ T cells and were accompanied by a reduction of CD8⁺ T cells, antibodies, and a severe failure in viral control. We identified IL-27 as a gp130 cytokine that promoted antiviral CD4⁺ T cell accumulation in vivo and that rapidly induced IL-21 ex vivo. Furthermore, IL-27R was critical for control of persistent LCMV in vivo. These results reveal that gp130 cytokines (particularly IL-27) are key regulators of CD4⁺ T cell responses during an established chronic viral infection, empowering both humoral and cytotoxic immunity.

Group 3 innate lymphoid cells inhibit T-cell-mediated intestinal inflammation through aryl hydrocarbon receptor signaling and regulation of microflora.

Aryl hydrocarbon receptor (Ahr) is crucial for the maintenance and function of group 3 innate lymphoid cells (ILCs), which are important in gut immunity. Because Ahr promotes T helper 17 (Th17) cell differentiation in vitro, it is reasonable to expect that Ahr would enhance Th17 cells in vivo. Instead, we show that Ahr deficiency caused increased intestinal Th17 cells, raising the possibility that group 3 ILCs could negatively regulate Th17 cells. Reduced innate interleukin-22 (IL-22) in Ahr-deficient mice allowed expansion of commensal segmented filamentous bacteria (SFB), known to promote Th17 cells. Compared to Rorc(+/+)Ahr(-/-) mice, Rorc(gfp/+)Ahr(-/-) mice had further reduced group 3 ILCs and were prone to spontaneous colitis with increased SFB and Th17 cells. Innate expression of Ahr played a protective role in T-cell-mediated experimental colitis by suppressing pathogenic Th17 cells. Our data reveal an intricate balance between ILCs and Th17 cells regulated by Ahr and commensal flora.

Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence.

Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed, but how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glial­ILC3­epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably, ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit, revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals.

IL-30 ameliorates imiquimod and K14-VEGF induced psoriasis-like disease by inhibiting both innate and adaptive immunity disorders.

Psoriasis is a severe skin disease with significant physical and psychological health consequences. As a typical type of immune disease, both innate and adaptive immunity disorders play key roles in the development of psoriasis. Interleukin (IL)-30 was thought as a natural antagonist of gp130-mediated signaling that affects T helper type 1 and 17 cell polarization by inhibiting IL-6 and IL-27 signaling pathways. Here, we found that, in vitro, IL-30 reduced cytokine levels of HaCaT keratinocytes and dendritic cells (DCs), weakened the maturationS of DCs, inhibited DC-mediated T cell proliferation, and blocked the activation of nuclear factor-κB. In vivo, IL-30 inhibited the development of skin disease in two animal models: Krt14-Vegfa and imiquimod (IMQ)-induced psoriasis-like skin disease. Thus, IL-30 may be useful as a therapeutic agent for controlling psoriasis.

Spotlight on IL-22-producing NK cell receptor-expressing mucosal lymphocytes.

New findings show that a subpopulation of mucosal ROR gammat+ cells expresses natural killer cell receptors and produces interleukin 22. These innate immune cells may be pivotal in maintaining mucosal homeostasis.