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.

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.

Early-life thermal stress mediates long-term alterations in hypothalamic microglia.

Stressful environmental events in early life have long-lasting consequences on later stress responses. We previously showed that heat conditioning of 3-day-old chicks during the critical period of heat-response development leads to heat vulnerability later in life. Here we assessed the role of early-life heat stress on the inflammatory response in the chick anterior hypothalamus (AH), focusing on hypothalamic microglia. We identified the microglial cell population in the chick AH using anti-KUL01 and anti-CD45 antibodies. Specific microglial features were also confirmed by expression of their signature genes. Under normal environmental conditions, hypothalamic microglia isolated from lipopolysaccharide (LPS)-injected chicks displayed a classical activated proinflammatory profile accompanied by a decreased homeostatic signature, demonstrating similarity of immune response with mammalian microglial cells. In accordance with our previous observations, conditioning of 3-day-old chicks under high ambient temperature decreased the number of newborn cells in the AH, among them microglial precursors. Although heat exposure did not affect microglial cell viability, it had a long-term impact on LPS-induced inflammatory response. Exposure to harsh heat led to heat vulnerability, and attenuated recruitment of peripheral monocytes and T cells into the AH following LPS challenge. Moreover, heat conditioning altered microglial reactivity, manifested as suppressed microglial activation in response to LPS. Innate immune memory developed by heat conditioning might underlie suppression of the microglial response to LPS challenge. We describe alterations in genome-wide CpG methylation profile of hypothalamic microglia, demonstrating probable epigenetic involvement in the reprogramming of microglial function, leading to heat-induced inflammatory cross-tolerance.

Cross-tolerance: embryonic heat conditioning induces inflammatory resilience by affecting different layers of epigenetic mechanisms regulating IL6 expression later in life.

A stressor can induce resilience in another, different stressor, a phenomenon known as cross-tolerance. To learn if cross-tolerance is governed by epigenetic regulation, we used embryonic heat conditioning (EHC) in chicks, during the development of the hypothalamus, to increase the immunization response. Indeed, EHC induced a lifelong systemic antibody response to immunization, in addition to reduced hypothalamic IL6 inflammatory expression following LPS challenge. Since the outcome of EHC was long-term cross-tolerance with the immune system, we studied possible epigenetic mechanisms. We first analysed the methylation and hydroxymethylation patterns of IL6. We found reduced hydroxymethylation on IL6 intron 1 in the EHC group, a segment enriched with CpGs and NFkB-binding sites. Luciferase assay in cell lines expressing NFkB showed that IL6 intron 1 is indeed an enhancer. ChiP in the same segment against NFkB in the hypothalamus presented reduced binding to IL6 intron 1 in the EHC group, before and during LPS challenge. In parallel, EHC chicks' IL6 intron 1 presented increased H3K27me3, a repressive translational modification mediated by EZH2. This histone modification occurred during embryonic conditioning and persisted later in life. Moreover, we showed reduced expression of miR-26a, which inhibits EZH2 transcription, during conditioning along with increased EZH2 expression. We demonstrate that stress cross-tolerance, which was indicated by EHC-induced inflammatory resilience and displayed by attenuated inflammatory expression of IL6, is regulated by different epigenetic layers.

Early-Life m6A RNA Demethylation by Fat Mass and Obesity-Associated Protein (FTO) Influences Resilience or Vulnerability to Heat Stress Later in Life.

Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the preoptic anterior hypothalamus (PO/AH) thermal response center and is regulated by epigenetic mechanisms. Here, we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N6-methyladenosine (m6A) RNA methylation in long-term response to heat stress. Heat conditioning of 3-d-old chicks diminished m6A RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the m6A demethylase, fat mass and obesity-associated protein (FTO). Moreover, a week later, methylation of two heat stress-related transcripts, histone 3 lysine 27 (H3K27) methyltransferase, enhancer of zeste homolog 2 (EZH2) and brain-derived neurotrophic factor (BDNF), were downregulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of m6A levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in m6A modification was negatively correlated with the expression levels of both BDNF and EZH2 Antisense "knock-down" of FTO caused an elevation of global m6A RNA methylation, reduction of EZH2 and BDNF mRNA levels, and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along BDNF coding region, and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress.