The single-cell transcriptome of mTECs and CD4+ thymocytes under adhesion revealed heterogeneity of mTECs and a network controlled by Aire and lncRNAs.

To further understand the impact of deficiency of the autoimmune regulator (Aire) gene during the adhesion of medullary thymic epithelial cells (mTECs) to thymocytes, we sequenced single-cell libraries (scRNA-seq) obtained from Aire wild-type (WT) (Airewt/wt ) or Aire-deficient (Airewt/mut ) mTECs cocultured with WT single-positive (SP) CD4+ thymocytes. Although the libraries differed in their mRNA and long noncoding RNA (lncRNA) profiles, indicating that mTECs were heterogeneous in terms of their transcriptome, UMAP clustering revealed that both mTEC lines expressed their specific markers, i.e., Epcam, Itgb4, Itga6, and Casp3 in resting mTECs and Ccna2, Pbk, and Birc5 in proliferative mTECs. Both cocultured SP CD4+ thymocytes remained in a homogeneous cluster expressing the Il7r and Ccr7 markers. Comparisons of the two types of cocultures revealed the differential expression of mRNAs that encode transcription factors (Zfpm2, Satb1, and Lef1), cell adhesion genes (Itgb1) in mTECs, and Themis in thymocytes, which is associated with the regulation of positive and negative selection. At the single-cell sequencing resolution, we observed that Aire acts on both Aire WT and Aire-deficient mTECs as an upstream controller of mRNAs, which encode transcription factors or adhesion proteins that, in turn, are posttranscriptionally controlled by lncRNAs, for example, Neat1, Malat1, Pvt1, and Dancr among others. Under Aire deficiency, mTECs dysregulate the expression of MHC-II, CD80, and CD326 (EPCAM) protein markers as well as metabolism and cell cycle-related mRNAs, which delay the cell cycle progression. Moreover, when adhered to mTECs, WT SP CD4+ or CD8+ thymocytes modulate the expression of cell activation proteins, including CD28 and CD152/CTLA4, and the expression of cellular metabolism mRNAs. These findings indicate a complex mechanism through which an imbalance in Aire expression can affect mTECs and thymocytes during adhesion.

A novel missense mutation in the AIRE gene underlying autoimmune polyglandular syndrome type 1.

The immune regulator gene AIRE plays an essential role in the establishment of immune tolerance and the prevention of autoimmunity. This transcription factor plays a critical role in promoting self-tolerance in the thymus by regulating the expression of a large number of self-antigens that share the common feature of being tissue-restricted in their expression pattern in the periphery. Dysfunction of AIRE in humans causes a rare disease, autoimmune polyglandular syndrome type 1 (APS1), characterized by an autoimmune response against peripheral tissues, particularly endocrine tissues. Although a few dominant mutations have been described, the inactivation of AIRE is usually caused by recessive mutations. Recent data suggests that alterations in AIRE function contribute not only to APS1 but also to more common forms of autoimmune disease. Here, we present a previously unreported missense mutation (NM_000383.2:c.260 T > C) in exon 2 of the AIRE gene, predicted to cause the substitution (p.(Leu87Pro)) in the CARD domain of the AIRE protein. When inherited in conjunction with another dysfunctional AIRE allele, this mutation was associated with immune dysregulation in a pediatric patient. The presence of hypergammaglobulinemia, malabsorption syndrome, ectodermal dysplasia, mucocutaneous candidiasis, vitiligo, and hypothyroidism as well as the presence of multiple autoantibodies allowed us to confirm an APS1 diagnosis.

miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells.

BACKGROUND: The autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated. RESULTS: Here, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3'UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes. CONCLUSION: Overall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA, reducing the respective Aire protein levels; consequently, the levels of mRNAs encode tissue-restricted antigens were affected. In addition, miR-155 regulated a crucial process by which mTECs allow thymocytes' migration through chemotaxis.

The Multifaceted Roles of B Cells in the Thymus: From Immune Tolerance to Autoimmunity.

The thymus is home to a significant number of resident B cells which possess several unique characteristics regarding their origin, phenotype and function. Evidence shows that they originate both from precursors that mature intrathymically and as the entry of recirculating mature B cells. Under steady-state conditions they exhibit hallmark signatures of activated B cells, undergo immunoglobulin class-switch, and express the Aire transcription factor. These features are imprinted within the thymus and enable B cells to act as specialized antigen-presenting cells in the thymic medulla that contribute negative selection of self-reactive T cells. Though, most studies have focused on B cells located in the medulla, a second contingent of B cells is also present in non-epithelial perivascular spaces of the thymus. This latter group of B cells, which includes memory B cells and plasma cells, is not readily detected in the thymus of infants or young mice but gradually accumulates during normal aging. Remarkably, in many autoimmune diseases the thymus suffers severe structural atrophy and infiltration of B cells in the perivascular spaces, which organize into follicles similar to those typically found in secondary lymphoid organs. This review provides an overview of the pathways involved in thymic B cell origin and presents an integrated view of both thymic medullary and perivascular B cells and their respective physiological and pathological roles in central tolerance and autoimmune diseases.

Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells.

The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This "Aire complex" induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36 h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation.

Aire Gene Influences the Length of the 3' UTR of mRNAs in Medullary Thymic Epithelial Cells.

Aire is a transcriptional controller in medullary thymic epithelial cells (mTECs) modulating a set of peripheral tissue antigens (PTAs) and non-PTA mRNAs as well as miRNAs. Even miRNAs exerting posttranscriptional control of mRNAs in mTECs, the composition of miRNA-mRNA networks may differ. Under reduction in Aire expression, networks exhibited greater miRNA diversity controlling mRNAs. Variations in the number of 3'UTR binding sites of Aire-dependent mRNAs may represent a crucial factor that influence the miRNA interaction. To test this hypothesis, we analyzed through bioinformatics the length of 3'UTRs of a large set of Aire-dependent mRNAs. The data were obtained from existing RNA-seq of mTECs of wild type or Aire-knockout (KO) mice. We used computational algorithms as FASTQC, STAR and HTSEQ for sequence alignment and counting reads, DESEQ2 for the differential expression, 3USS for the alternative 3'UTRs and TAPAS for the alternative polyadenylation sites. We identified 152 differentially expressed mRNAs between these samples comprising those that encode PTAs as well as transcription regulators. In Aire KO mTECs, most of these mRNAs featured an increase in the length of their 3'UTRs originating additional miRNA binding sites and new miRNA controllers. Results from the in silico analysis were statistically significant and the predicted miRNA-mRNA interactions were thermodynamically stable. Even with no in vivo or in vitro experiments, they were adequate to show that lack of Aire in mTECs might favor the downregulation of PTA mRNAs and transcription regulators via miRNA control. This could unbalance the overall transcriptional activity in mTECs and thus the self-representation.

The AIRE Ser196Ser synonymous variant is a risk factor for systemic lupus erythematosus.

The AIRE gene influences the expression of a wide array of self-antigens in the thymus, and is essential to the negative selection of self-reactive T cells and establishment of central tolerance. Single nucleotide variants (SNVs) such as rs878081C/T (Ser196Ser) and rs2075876G/T at this locus have been associated with susceptibility to rheumatoid arthritis, mainly in Asian populations, but its role in systemic lupus erythematosus (SLE) has not been documented. We performed a case-control association study with 379 SLE patients and 460 controls from central Mexico. In addition, we replicated our finding in another group of 179 SLE patients and 97 controls from the same region of Mexico. In the first group, we identified that the AIRE Ser196Ser synonymous variant was associated with SLE (OR 1.4, p = 0.009), meanwhile, in the second group we observed the following: OR 1.7, p = 0.024. No association was found between these AIRE SNVs and lupus nephritis. Our results suggest that AIRE is a risk factor for SLE in our population. This study is the first to document an association between AIRE and SLE susceptibility.

The rs878081 polymorphism of AIRE gene increases the risk of rheumatoid arthritis in a Chinese Han population: a case-control study.

The autoimmune regulator (AIRE), a transcriptional regulator expressed in medullary thymic epithelial cells, plays an important role in thymocyte education and negative selection. Several citations studying the association between the rs878081 exon polymorphism of the AIRE gene and the risk of rheumatoid arthritis (RA) in different populations have yielded conflicting findings. Thus, this case-control study involving 300 RA cases and 300 controls was aimed to identify whether such association existed in a Chinese Han population from East China. The rs878081 polymorphism of the AIRE gene was genotyped. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using the chi-squared test, genetic model analysis, and stratification analysis. Genetic model analysis showed significant correlations between the TT genotype and the risk of RA (OR: 1.89, 95%CI: 1.03-3.47 in TT vs CC; OR: 1.84, 95%CI: 1.02-3.31 in TT vs CC+TC). Stratification analyses of sex, age, smoking, and alcoholism suggested that the rs878081 polymorphism of the AIRE gene increased RA risk among non-smokers. In conclusion, rs878081 polymorphism of AIRE gene increases the risk of RA in a Chinese Han population.

The heterogeneity of autoimmune polyendocrine syndrome type 1: Clinical features, new mutations and cytokine autoantibodies in a Brazilian cohort from tertiary care centers.

Autoimmune polyendocrine syndrome type 1 (APS1) is characterized by multiorgan autoimmunity. We aim at characterizing a multi-center Brazilian cohort of APS1 patients by clinical evaluation, searching mutation in the AIRE gene, measuring serum autoantibodies, and investigating correlations between findings. We recruited patients based on the clinical criteria and tested them for AIRE mutations, antibodies against interferon type I and interleukins 17A, 17F and 22. We identified 12 unrelated families (13 patients) with typical signs of APS1 in the proband, and the screening of relatives recognized an asymptomatic child. Candidiasis was present in all cases, and 19 other manifestations were observed. All patients carried one of 10 different mutations in AIRE, being 3 new ones, and were positive for anti-interferon type I serum antibody. Anti-interleukin-17A levels inversely correlated with the number of manifestations in each patient. This negative correlation may suggest a protective effect of anti-interleukin-17A with a potential therapeutic application.

Minipuberty and Sexual Dimorphism in the Infant Human Thymus.

AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women more susceptible to autoimmune disorders. Here we investigated the effects of minipuberty on male and female infant human thymic tissue in order to verify if this initial transient increase in sex hormones - along the first six months of life - could affect thymic transcriptional network regulation and AIRE expression. Gene co-expression network analysis for differentially expressed genes and miRNA-target analysis revealed sex differences in thymic tissue during minipuberty, but such differences were not detected in the thymic tissue of infants aged 7-18 months, i.e. the non-puberty group. AIRE expression was essentially the same in both sexes in minipuberty and in non-puberty groups, as assessed by genomic and immunohistochemical assays. However, AIRE-interactors networks showed several differences in all groups regarding gene-gene expression correlation. Therefore, minipuberty and genomic mechanisms interact in shaping thymic sexual dimorphism along the first six months of life.

Aire Disruption Influences the Medullary Thymic Epithelial Cell Transcriptome and Interaction With Thymocytes.

The function of medullary thymic epithelial cells (mTECs) is associated with thymocyte adhesion, which is crucial for the negative selection of autoreactive thymocytes in the thymus. This process represents the root of central tolerance of self-components and prevents the onset of autoimmune diseases. Since thymic epithelia correspond to an important target of donor T cells during the onset of chronic graft-vs-host-disease, mTEC-thymocyte adhesion may have implications for alloimmunity. The Aire and Fezf2 genes function as transcriptome controllers in mTECs. The central question of this study is whether there is a mutual relationship between mTEC-thymocyte adhesion and the control of the mTEC transcriptome and whether Aire is involved in this process. Here, we show that in vitro mTEC-thymocyte adhesion causes transcriptome changes in mTECs and upregulates the transcriptional expression of Aire and Fezf2, as well as cell adhesion-related genes such as Cd80 or Tcf7, among others. Crispr-Cas9-mediated Aire gene disruption demonstrated that this gene plays a role in the process of mTEC-thymocyte adhesion. Consistent with the nuclear localization signal (NLS) encoded by Aire exon 3, which was targeted, we demonstrate that Aire KO-/- mTECs impair AIRE protein localization in the nucleus. Consequently, the loss of function of Aire reduced the ability of these cells to adhere to thymocytes. Their transcriptomes differed from their wild-type Aire+/+ counterparts, even during thymocyte adhesion. A set of mRNA isoforms that encode proteins involved in cell adhesion were also modulated during this process. This demonstrates that both thymocyte interactions and Aire influence transcriptome profiling of mTEC cells.

The Role of AIRE in the Immunity Against Candida Albicans in a Model of Human Macrophages.

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1ß, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.

Update on Aire and thymic negative selection.

Twenty years ago, the autoimmune regulator (Aire) gene was associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, and was cloned and sequenced. Its importance goes beyond its abstract link with human autoimmune disease. Aire identification opened new perspectives to better understand the molecular basis of central tolerance and self-non-self distinction, the main properties of the immune system. Since 1997, a growing number of immunologists and molecular geneticists have made important discoveries about the function of Aire, which is essentially a pleiotropic gene. Aire is one of the functional markers in medullary thymic epithelial cells (mTECs), controlling their differentiation and expression of peripheral tissue antigens (PTAs), mTEC-thymocyte adhesion and the expression of microRNAs, among other functions. With Aire, the immunological tolerance became even more apparent from the molecular genetics point of view. Currently, mTECs represent the most unusual cells because they express almost the entire functional genome but still maintain their identity. Due to the enormous diversity of PTAs, this uncommon gene expression pattern was termed promiscuous gene expression, the interpretation of which is essentially immunological - i.e. it is related to self-representation in the thymus. Therefore, this knowledge is strongly linked to the negative selection of autoreactive thymocytes. In this update, we focus on the most relevant results of Aire as a transcriptional and post-transcriptional controller of PTAs in mTECs, its mechanism of action, and its influence on the negative selection of autoreactive thymocytes as the bases of the induction of central tolerance and prevention of autoimmune diseases.

Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.

Aire-dependent peripheral tissue antigen mRNAs in mTEC cells feature networking refractoriness to microRNA interaction.

The downregulation of PTA genes in mTECs is associated with the loss of self-tolerance, and the role of miRNAs in this process is not fully understood. Therefore, we studied the expression of mRNAs and miRNAs in mTECs from autoimmune NOD mice during the period when loss of self-tolerance occurs in parallel with non-autoimmune BALB/c mice. Although the expression of the transcriptional regulator Aire was unchanged, we observed downregulation of a set of PTA mRNAs. A set of miRNAs was also differentially expressed in these mice. The reconstruction of miRNA-mRNA interaction networks identified the controller miRNAs and predicted the PTA mRNA targets. Interestingly, the known Aire-dependent PTAs exhibited pronounced refractoriness in the networking interaction with miRNAs. This study reveals the existence of a new mechanism in mTECs, and this mechanism may have importance in the control of self-tolerance.

Expression profile of peripheral tissue antigen genes in medullary thymic epithelial cells (mTECs) is dependent on mRNA levels of autoimmune regulator (Aire).

In the thymus of non-obese diabetic (NOD) mice, the expression of the autoimmune regulator (Aire) gene varies with age, and its down-regulation in young mice precedes the later emergence of type 1 diabetes mellitus (T1D). In addition, the insulin (Ins2) peripheral tissue antigen (PTA) gene, which is Aire-dependent, is also deregulated in these mice. Based in these findings, we hypothesized that the imbalance in PTA gene expression in the thymus can be associated with slight variations in Aire transcript levels. To test this, we used siRNA to knockdown Aire by in vivo electro-transfection of the thymus of BALB/c mice. The efficiency of the electro-transfection was monitored by assessing the presence of irrelevant Cy3-labeled siRNA in the thymic stroma. Importantly, Aire-siRNA reached medullary thymic epithelial cells (mTECs) down-regulating Aire. As expected, the in vivo Aire knockdown was partial and transient; the maximum 59% inhibition occurred in 48 h. The Aire knockdown was sufficient to down-regulate PTA genes; however, surprisingly, several others, including Ins2, were up-regulated. The modulation of these genes after in vivo Aire knockdown was comparable to that observed in NOD mice before the emergence of T1D. The in vitro transfections of 3.10 mTEC cells with Aire siRNA resulted in samples featuring partial (69%) and complete (100%) Aire knockdown. In these Aire siRNA-transfected 3.10 mTECs, the expression of PTA genes, including Ins2, was down-regulated. This suggests that the expression profile of PTA genes in mTECs is affected by fine changes in the transcription level of Aire.

Autoimmune regulator (Aire) controls the expression of microRNAs in medullary thymic epithelial cells.

The autoimmune regulator (Aire) is a transcription factor that controls the ectopic expression of a large set of peripheral tissue antigen (PTA) genes in medullary thymic epithelial cells (mTECs). Recent evidence has demonstrated that Aire releases stalled RNA polymerase II (RNA Pol II) from blockage at the promoter region of its target genes. Given that, in addition to messenger RNAs (mRNA), RNA Pol II also transcribes microRNAs (miRNAs), we raised the hypothesis that Aire might play a role as an upstream controller of miRNA transcription. To test this, we initially analyzed the expression profiles of 662 miRNAs in control and Aire-silenced (siRNA) murine mTEC 3.10 cells using microarrays. The bioinformatics programs SAM and Cluster-TreeView were then used to identify the differentially expressed miRNAs and their profiles, respectively. Thirty Aire-dependent miRNAs were identified in the Aire-silenced mTECs, of which 18 were up- and 12 were down-regulated. The down-regulated miR-376 family was the focus of this study because its members (miR-376a, miR-376b and miR-376c) are located in the genome within the Gm2922 open-reading frame (ORF) gene segment on the chromosome 12F1. The T-boxes (TTATTA) and G-boxes (GATTGG), which represent putative RNA Pol II promoter motifs, were located in a portion spanning 10 kb upstream of the ATG codon of Gm2922. Moreover, we found that Gm2922 encodes an mRNA, which was also down-regulated in Aire-silenced mTECs. These results represent the first evidence that Aire can play a role as a controller of transcription of miRNAs located within genomic regions encompassing ORF and/or mRNA genes.

Autoimmune polyendocrine syndrome type 1: case report and review of literature.

Autoimmune polyendocrine syndrome type 1 (APECED) is a rare autosomal recessive disorder characterized by autoimmune multiorgan attack. The disease is caused by mutations in the autoimmune regulator gene (AIRE), resulting in defective AIRE protein, which is essential for selftolerance. Clinical manifestations are widely variable. Although the classic triad is composed by mucocutaneous candidiasis, hypoparathyroidism and adrenal failure, many other components may develop. Treatment is based on supplementation of the various deficiencies, and patients require regular follow-up throughout their lifespan. This article describes the case of a patient with the disease, and reviews literature data on the epidemiology, clinical course, immunogenetic aspects, diagnosis and treatment of the syndrome.

Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-α production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1.

BACKGROUND: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. OBJECTIVE: To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of ß-glucan through the Dectin-1 pathway, which is required for defense against Candida species. METHODS: Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. RESULTS: PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. CONCLUSION: AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome.

Decreased AIRE expression and global thymic hypofunction in Down syndrome.

The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 ± 49.59 AIRE-positive cells/mm(2) in DS versus 154.70 ± 61.16 AIRE-positive cells/mm(2) in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm(2) was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-related genes included those involved in 1) Ag processing and presentation (HLA-DQB1, HLA-DRB3, CD1A, CD1B, CD1C, ERAP) and 2) thymic T cell differentiation (IL2RG, RAG2, CD3D, CD3E, PRDX2, CDK6) and selection (SH2D1A, CD74). It is noteworthy that relevant AIRE-partner genes, such as TOP2A, LAMNB1, and NUP93, were found hypoexpressed in DNA microarrays and quantitative real-time PCR analyses. These findings on global thymic hypofunction in DS revealed molecular mechanisms underlying DS immune phenotype and strongly suggest that DS immune abnormalities are present since early development, rather than being a consequence of precocious aging, as widely hypothesized. Thus, DS should be considered as a non-monogenic primary immunodeficiency.