The emerging family of RORγt+ antigen-presenting cells.

Antigen-presenting cells (APCs) are master regulators of the immune response by directly interacting with T cells to orchestrate distinct functional outcomes. Several types of professional APC exist, including conventional dendritic cells, B cells and macrophages, and numerous other cell types have non-classical roles in antigen presentation, such as thymic epithelial cells, endothelial cells and granulocytes. Accumulating evidence indicates the presence of a new family of APCs marked by the lineage-specifying transcription factor retinoic acid receptor-related orphan receptor-γt (RORγt) and demonstrates that these APCs have key roles in shaping immunity, inflammation and tolerance, particularly in the context of host-microorganism interactions. These RORγt+ APCs include subsets of group 3 innate lymphoid cells, extrathymic autoimmune regulator-expressing cells and, potentially, other emerging populations. Here, we summarize the major findings that led to the discovery of these RORγt+ APCs and their associated functions. We discuss discordance in recent reports and identify gaps in our knowledge in this burgeoning field, which has tremendous potential to advance our understanding of fundamental immune concepts.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

IL-17-driven induction of Paneth cell antimicrobial functions protects the host from microbiota dysbiosis and inflammation in the ileum.

Interleukin (IL)-17 protects epithelial barriers by inducing the secretion of antimicrobial peptides. However, the effect of IL-17 on Paneth cells (PCs), the major producers of antimicrobial peptides in the small intestine, is unclear. Here, we show that the targeted ablation of the IL-17 receptor (IL-17R) in PCs disrupts their antimicrobial functions and decreases the frequency of ileal PCs. These changes become more pronounced after colonization with IL-17 inducing segmented filamentous bacteria. Mice with PCs that lack IL-17R show an increased inflammatory transcriptional profile in the ileum along with the severity of experimentally induced ileitis. These changes are associated with a decrease in the diversity of gut microbiota that induces a severe ileum pathology upon transfer to genetically susceptible mice, which can be prevented by the systemic administration of IL-17a/f in microbiota recipients. In an exploratory analysis of a small cohort of pediatric patients with Crohn's disease, we have found that a portion of these patients exhibits a low number of lysozyme-expressing ileal PCs and a high ileitis severity score, resembling the phenotype of mice with IL-17R-deficient PCs. Our study identifies IL-17R-dependent signaling in PCs as an important mechanism that maintains ileal homeostasis through the prevention of dysbiosis.

Transcriptional regulation of the thymus master regulator Foxn1.

FOXN1 is a transcription factor critical for the development of both thymic epithelial cell (TEC) and hair follicle cell (HFC) compartments. However, mechanisms controlling its expression remain poorly understood. To address this question, we performed thorough analyses of the evolutionary conservation and chromatin status of the Foxn1 locus in different tissues and states and identified several putative cis-regulatory regions unique to TECs versus HFCs. Furthermore, experiments using genetically modified mice with specific deletions in the Foxn1 locus and additional bioinformatic analyses helped us identify key regions and transcription factors involved in either positive or negative regulation of Foxn1 in both TECs and HFCs. Specifically, we identified SIX1 and FOXN1 itself as key factors inducing Foxn1 expression in embryonic and neonatal TECs. Together, our data provide important mechanistic insights into the transcriptional regulation of the Foxn1 gene in TEC versus HFC and highlight the role of FOXN1 in its autoregulation.

Extrathymic expression of Aire controls the induction of effective TH17 cell-mediated immune response to Candida albicans.

Patients with loss of function in the gene encoding the master regulator of central tolerance AIRE suffer from a devastating disorder called autoimmune polyendocrine syndrome type 1 (APS-1), characterized by a spectrum of autoimmune diseases and severe mucocutaneous candidiasis. Although the key mechanisms underlying the development of autoimmunity in patients with APS-1 are well established, the underlying cause of the increased susceptibility to Candida albicans infection remains less understood. Here, we show that Aire+MHCII+ type 3 innate lymphoid cells (ILC3s) could sense, internalize and present C. albicans and had a critical role in the induction of Candida-specific T helper 17 (TH17) cell clones. Extrathymic Rorc-Cre-mediated deletion of Aire resulted in impaired generation of Candida-specific TH17 cells and subsequent overgrowth of C. albicans in the mucosal tissues. Collectively, our observations identify a previously unrecognized regulatory mechanism for effective defense responses against fungal infections.

A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer.

Medullary thymic epithelial cells (mTECs), which produce and present self-antigens, are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with a specific subset of DCs. Using several relevant Cre-based mouse models that control for the expression of fluorescent proteins, we have found that, in regards to CAT, each subset of thymic DCs preferentially targets a distinct mTEC subset(s). Importantly, XCR1+-activated DC subset represented the most potent subset in CAT. Interestingly, thymic DCs can also acquire antigens from more than one mTEC, and of these, monocyte-derived dendritic cells (moDCs) were determined to be the most efficient. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

Mechanistic dissection of dominant AIRE mutations in mouse models reveals AIRE autoregulation.

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.

IL18 signaling promotes homing of mature Tregs into the thymus.

Foxp3+ regulatory T cells (Tregs) are potent suppressor cells, essential for the maintenance of immune homeostasis. Most Tregs develop in the thymus and are then released into the immune periphery. However, some Tregs populate the thymus and constitute a major subset of yet poorly understood cells. Here we describe a subset of thymus recirculating IL18R+ Tregs with molecular characteristics highly reminiscent of tissue-resident effector Tregs. Moreover, we show that IL18R+ Tregs are endowed with higher capacity to populate the thymus than their IL18R- or IL18R-/- counterparts, highlighting the key role of IL18R in this process. Finally, we demonstrate that IL18 signaling is critical for the induction of the key thymus-homing chemokine receptor - CCR6 on Tregs. Collectively, this study provides a detailed characterization of the mature Treg subsets in the mouse thymus and identifies a key role of IL18 signaling in controlling the CCR6-CCL20-dependent migration of Tregs into the thymus.

Toll-like receptor signaling in thymic epithelium controls monocyte-derived dendritic cell recruitment and Treg generation.

The development of thymic regulatory T cells (Treg) is mediated by Aire-regulated self-antigen presentation on medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), but the cooperation between these cells is still poorly understood. Here we show that signaling through Toll-like receptors (TLR) expressed on mTECs regulates the production of specific chemokines and other genes associated with post-Aire mTEC development. Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirpα+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines. Consistently, the cellularity of CD14+moDC is diminished in mice with MyD88-deficient TECs, in which the frequency and functionality of thymic CD25+Foxp3+ Tregs are decreased, leading to aggravated mouse experimental colitis. Thus, our findings describe a TLR-dependent function of mTECs for the recruitment of CD14+moDC, the generation of Tregs, and thereby the establishment of central tolerance.

Aire-expressing ILC3-like cells in the lymph node display potent APC features.

The autoimmune regulator (Aire) serves an essential function for T cell tolerance by promoting the "promiscuous" expression of tissue antigens in thymic epithelial cells. Aire is also detected in rare cells in peripheral lymphoid organs, but the identity of these cells is poorly understood. Here, we report that Aire protein-expressing cells in lymph nodes exhibit typical group 3 innate lymphoid cell (ILC3) characteristics such as lymphoid morphology, absence of "classical" hematopoietic lineage markers, and dependence on RORγt. Aire+ cells are more frequent among lineage-negative RORγt+ cells of peripheral lymph nodes as compared with mucosa-draining lymph nodes, display a unique Aire-dependent transcriptional signature, express high surface levels of MHCII and costimulatory molecules, and efficiently present an endogenously expressed model antigen to CD4+ T cells. These findings define a novel type of ILC3-like cells with potent APC features, suggesting that these cells serve a function in the control of T cell responses.

Enteric α-defensins on the verge of intestinal immune tolerance and inflammation.

The gut is the biggest immune organ in the body that encloses commensal microbiota which aids in food digestion. Paneth cells, positioned at the frontline of host-microbiota interphase, can modulate the composition of microbiota. Paneth cells achieve this via the delivery of microbicidal substances, among which enteric α-defensins play the primary role. If microbiota is dysregulated, it can impact the function of the local mucosal immune system. Importantly, this system is also exposed to an enormous number of antigens which are derived from the gut-resident microbiota and processed food, and may potentially trigger undesirable local inflammatory responses. To understand the intricate regulations and liaisons between Paneth cells, microbiota and the immune system in this intestinal-specific setting, one must consider their mode of interaction in a wider context of regulatory processes which impose immune tolerance not only to self, but also to microbiota and food-derived antigens. These include, but are not limited to, tolerogenic mechanisms of central tolerance in the thymus and peripheral tolerance in the secondary lymphoid organs, and the intestine itself. Defects in these processes can compromise homeostasis in the intestinal mucosal immunity. In this review, which is focused on tolerance to intestinal antigens and its relevance for the pathogenesis of gut immune diseases, we provide an outline of such multilayered immune control mechanisms and highlight functional links that underpin their cooperative nature.

A novel conditional Aire allele enables cell-specific ablation of the immune tolerance regulator Aire.

Medullary thymic epithelial cell (mTEC)-restricted expression of autoimmune regulator (Aire) is essential for establishment of immune tolerance. Recently, Aire was also shown to be expressed in cells of hematopietic and reproductive lineages. Thus, the generation of Airefl/fl mouse strain enables the investigation of the cell-specific function of Aire.

Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse.

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.

Gastrointestinal Autoimmunity Associated With Loss of Central Tolerance to Enteric α-Defensins.

BACKGROUND & AIMS: Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is an autoimmune disorder characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, but patients also develop intestinal disorders. APECED is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE, which regulates immune tolerance) that allow self-reactive T cells to enter the periphery. Enteric α-defensins are antimicrobial peptides secreted by Paneth cells. Patients with APECED frequently have gastrointestinal symptoms and seroreactivity against secretory granules of Paneth cells. We investigated whether enteric α-defensins are autoantigens in humans and mice with AIRE deficiency. METHODS: We analyzed clinical data, along with serum and stool samples and available duodenal biopsies from 50 patients with APECED collected from multiple centers in Europe. Samples were assessed for expression of defensins and other molecules by quantitative reverse transcription polymerase chain reaction and flow cytometry; levels of antibodies and other proteins were measured by immunohistochemical and immunoblot analyses. Histologic analyses were performed on biopsy samples. We used Aire(-/-) mice as a model of APECED, and studied the effects of transferring immune cells from these mice to athymic mice. RESULTS: Enteric defensins were detected in extraintestinal tissues of patients with APECED, especially in medullary thymic epithelial cells. Some patients with APECED lacked Paneth cells and were seropositive for defensin-specific autoantibodies; the presence of autoantibodies correlated with frequent diarrhea. Aire(-/-) mice developed defensin-specific T cells. Adoptive transfer of these T cells to athymic mice resulted in T-cell infiltration of the gut, loss of Paneth cells, microbial dysbiosis, and the induction of T-helper 17 cell-mediated autoimmune responses resembling those observed in patients with APECED. CONCLUSIONS: In patients with APECED, loss of AIRE appears to cause an autoimmune response against enteric defensins and loss of Paneth cells. Aire(-/-) mice developed defensin-specific T cells that cause intestinal defects similar to those observed in patients with APECED. These findings provide a mechanism by which loss of AIRE-mediated immune tolerance leads to intestinal disorders in patients with APECED.

HIC1 Tumor Suppressor Loss Potentiates TLR2/NF-κB Signaling and Promotes Tissue Damage-Associated Tumorigenesis.

UNLABELLED: Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses "danger" signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells. IMPLICATIONS: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells.

NKD1 marks intestinal and liver tumors linked to aberrant Wnt signaling.

The activity of the Wnt pathway undergoes complex regulation to ensure proper functioning of this principal signaling mechanism during development of adult tissues. The regulation may occur at several levels and includes both positive and negative feedback loops. In the present study we employed one of such negative feedback regulators, naked cuticle homolog 1 (Nkd1), to follow the Wnt pathway activity in the intestine and liver and in neoplasia originated in these organs. Using lineage tracing in transgenic mice we localized Nkd1 mRNA to the bottom parts of the small intestinal crypts and hepatocytes surrounding the central vein of the hepatic lobule. Furthermore, in two mouse models of intestinal tumorigenesis, Nkd1 expression levels were elevated in tumors when compared to healthy tissue. We utilized a collection of human intestinal polyps and carcinomas to confirm that NKD1 represents a robust marker of neoplastic growth. In addition, expression analysis of NKD1 in liver cancer showed that high expression levels of the gene distinguish a subclass of hepatocellular carcinomas related to aberrant Wnt signaling. Finally, our results were confirmed by bioinformatic analysis of large publicly available datasets that included gene expression profiling and high-throughput sequencing data of human colon and liver cancer specimens.

Eosinophils from patients with type 1 diabetes mellitus express high level of myeloid alpha-defensins and myeloperoxidase.

Type 1 diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of pancreatic beta cells. Recently, small cationic α-defensin molecules have been implicated in the pathogenesis of certain inflammatory and autoimmune diseases. The purpose of this study was to assess the α-defensin expression in patients with T1D and elucidate the cellular source of their production. Our results show that 30% of patients exhibit increased levels of α-defensin mRNAs in their capillary blood. Quantitative RT-PCR performed on FACS-sorted granulocytes identified CD15(dull)/CD14(weak) population as the cellular source of α-defensins. Surprisingly, this granulocyte subpopulation displayed augmentation of α-defensin expression in all T1D patients tested. The determination of cell surface markers, expression of cell-specific genes and confocal microscopy identified CD15(dull)/CD14(weak) cells as eosinophils. The presence of transcriptionally active eosinophils in diabetic patients suggests that eosinophils could be a part of an intricate innate immune cellular network involved in the development of diabetes.

Regularity-induced separation of intrinsic and collective dynamics.

We propose that the adiabatic separation of collective and intrinsic motions in many-body systems is related to increased regularity of the intrinsic dynamics. The surmise is verified on the separation of rotations from intrinsic vibrations in the interacting boson model of nuclear structure.