Lymphoid Stress Surveillance Response Contributes to Vitiligo Pathogenesis.

Vitiligo is a chronic multifactorial depigmentation disorder characterized by the destruction and functional loss of melanocytes. Although a direct cytotoxic T cell attack is thought to be responsible for melanocyte damage, the events leading to the loss of self-tolerance toward melanocytic antigens are not understood. This research aimed to identify novel cellular and molecular factors that participate in vitiligo pathogenesis through the application of gene expression and immunofluorescence analysis of skin biopsy samples along with immunophenotyping of circulating cells. Our study provides insights into the mechanisms involved in melanocyte destruction. The upregulation of stress-ligand MICA/MICB, recognized by activating receptors on innate and innate-like T cells, imply involvement of lymphoid stress surveillance responses in vitiligo lesions. A simultaneous increase in the expression of transcription factor EOMES that is characteristic for innate-like virtual memory T cells, suggest a similar scenario. Local lymphoid stress surveillance has been previously associated with the amplification of systemic humoral responses that were mirrored in our study by increased T follicular helper cells and switched memory B cell proportions in patients with active vitiligo. In addition, microtubule-associated protein light chain 3 staining was compatible with the activation of autophagy in keratinocytes and in the remaining melanocytes of vitiligo lesional skin.

Signs of innate immune activation and premature immunosenescence in psoriasis patients.

Psoriasis is a chronic inflammatory disease that affects skin and is associated with systemic inflammation and many serious comorbidities ranging from metabolic syndrome to cancer. Important discoveries about psoriasis pathogenesis have enabled the development of effective biological treatments blocking the T helper 17 pathway. However, it has not been settled whether psoriasis is a T cell-mediated autoimmune disease or an autoinflammatory disorder that is driven by exaggerated innate immune signalling. Our comparative gene expression and hierarchical cluster analysis reveal important gene circuits involving innate receptors. Innate immune activation is indicated by increased absent in melanoma 2 (AIM2) inflammasome gene expression and active caspase 1 staining in psoriatic lesional skin. Increased eomesodermin (EOMES) expression in lesional and non-lesional skin is suggestive of innate-like virtual memory CD8+ T cell infiltration. We found that signs of systemic inflammation were present in most of the patients, correlated with the severity of the disease, and pointed to IL-6 involvement in the pathogenesis of psoriatic arthritis. Among the circulating T cell subpopulations, we identified a higher proportion of terminally differentiated or senescent CD8+ T cells, especially in patients with long disease duration, suggesting premature immunosenescence and its possible implications for psoriasis co-morbidities.

IL-6-specific autoantibodies among APECED and thymoma patients.

INTRODUCTION: Both autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and the rare thymoma patients with chronic mucocutaneous candidiasis (CMC) have neutralizing autoantibodies to Th17 cytokines and significant defects in production of IL-22 and IL-17F by their T cells. The cause of these defects is unknown. We hypothesized that they might result from autoimmunity against upstream cytokines normally responsible for generating and maintaining Th17 cells. METHODS: Luciferase immunoprecipitation (LIPS) was used to screen for autoantibodies to IL-6, IL-1ß, TGF-ß3, IL-21, and IL-23 in patients with APECED or thymoma. We used Western blotting to assess the conformation-dependence of the IL-6 autoantibodies and flow cytometric analysis of intracellular phospho-STAT3 induction to assess IL-6-neutralizing capacity in IgGs isolated from patient and control sera. We also used Luminex xMAP to measure serum cytokine levels. RESULTS: We found autoantibodies binding to conformational epitopes of IL-6 in 19.5% of 41 patients with APECED and 12.5% of 104 with thymoma-especially in those with long disease durations. The autoantibodies were predominantly of IgG1 subclass and failed to neutralize IL-6 activity. Notably, serum levels of the IL-6 and IL-17A cytokines were higher in anti-IL-6 seropositive than-negative APECED patients or healthy controls. We also detected autoantibody binding to IL-23 in 27.9% of thymoma patients, resulting from cross-recognition through the p40 subunit it shares with IL-12. CONCLUSIONS: IL-6 and IL-17A elevation in these seropositive patients suggests that antibody-binding may protect IL-6 from degradation and prolong its half-life in vivo.

A highly conserved NF-κB-responsive enhancer is critical for thymic expression of Aire in mice.

Autoimmune regulator (Aire) has a unique expression pattern in thymic medullary epithelial cells (mTECs), in which it plays a critical role in the activation of tissue-specific antigens. The expression of Aire in mTECs is activated by receptor activator of nuclear factor κB (RANK) signaling; however, the molecular mechanism behind this activation is unknown. Here, we characterize a conserved noncoding sequence 1 (CNS1) containing two NF-κB binding sites upstream of the Aire coding region. We show that CNS1-deficient mice lack thymic expression of Aire and share several features of Aire-knockout mice, including downregulation of Aire-dependent genes, impaired terminal differentiation of the mTEC population, and reduced production of thymic Treg cells. In addition, we show that CNS1 is indispensable for RANK-induced Aire expression and that CNS1 is activated by NF-κB pathway complexes containing RelA. Together, our results indicate that CNS1 is a critical link between RANK signaling, NF-κB activation, and thymic expression of Aire.

Mechanisms of IFN-γ-induced apoptosis of human skin keratinocytes in patients with atopic dermatitis.

BACKGROUND: Enhanced apoptosis of keratinocytes is the main cause of eczema and spongiosis in patients with the common inflammatory skin disease atopic dermatitis (AD). OBJECTIVE: The aim of the study was to investigate molecular mechanisms of AD-related apoptosis of keratinocytes. METHODS: Primary keratinocytes isolated from patients with AD and healthy donors were used to study apoptosis by using annexin V/7-aminoactinomycin D staining. Illumina mRNA Expression BeadChips, quantitative RT-PCR, and immunofluorescence were used to study gene expression. In silico analysis of candidate genes was performed on genome-wide single nucleotide polymorphism data. RESULTS: We demonstrate that keratinocytes of patients with AD exhibit increased IFN-γ-induced apoptosis compared with keratinocytes from healthy subjects. Further mRNA expression analyses revealed differential expression of apoptosis-related genes in AD keratinocytes and skin and the upregulation of immune system-related genes in skin biopsy specimens of chronic AD lesions. Three apoptosis-related genes (NOD2, DUSP1, and ADM) and 8 genes overexpressed in AD skin lesions (CCDC109B, CCL5, CCL8, IFI35, LYN, RAB31, IFITM1, and IFITM2) were induced by IFN-γ in primary keratinocytes. The protein expression of IFITM1, CCL5, and CCL8 was verified in AD skin. In line with the functional studies and AD-related mRNA expression changes, in silico analysis of genome-wide single nucleotide polymorphism data revealed evidence of an association between AD and genetic markers close to or within the IFITM cluster or RAB31, DUSP1, and ADM genes. CONCLUSION: Our results demonstrate increased IFN-γ responses in skin of patients with AD and suggest involvement of multiple new apoptosis- and inflammation-related factors in the development of AD.

DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues.

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.

MicroRNA expression profiles of human blood monocyte-derived dendritic cells and macrophages reveal miR-511 as putative positive regulator of Toll-like receptor 4.

Dendritic cells (DCs) and macrophages (MFs) are important multifunctional immune cells. Like other cell types, they express hundreds of different microRNAs (miRNAs) that are recently discovered post-transcriptional regulators of gene expression. Here we present updated miRNA expression profiles of monocytes, DCs and MFs. Compared with monocytes, ∼50 miRNAs were found to be differentially expressed in immature and mature DCs or MFs, with major expression changes occurring during the differentiation. Knockdown of DICER1, a protein needed for miRNA biosynthesis, led to lower DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and enhanced CD14 protein levels, confirming the importance of miRNAs in DC differentiation in general. Inhibition of the two most highly up-regulated miRNAs, miR-511 and miR-99b, also resulted in reduced DC-SIGN level. Prediction of miRNA-511 targets revealed a number of genes with known immune functions, of which TLR4 and CD80 were validated using inhibition of miR-511 in DCs and luciferase assays in HEK293 cells. Interestingly, under the cell cycle arrest conditions, miR-511 seems to function as a positive regulator of TLR4. In conclusion, we have identified miR-511 as a novel potent modulator of human immune response. In addition, our data highlight that miRNA influence on gene expression is dependent on the cellular environment.