Atopic dermatitis (AD) is one of the most common skin disorders among children. Disease etiology involves genetic and environmental factors, with 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated the potential epigenetic mechanisms responsible for the genetic susceptibility of CD4+ T cells. To understand the differences in gene regulatory activity in peripheral blood T cells in AD, we measured chromatin accessibility (an assay based on transposase-accessible chromatin sequencing, ATAC-seq), nuclear factor kappa B subunit 1 (NFKB1) binding (chromatin immunoprecipitation with sequencing, ChIP-seq), and gene expression levels (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD, as well as in age-matched and non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of the AD risk loci overlapping AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF-κB DNA-binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD- or control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF-κB DNA-binding motifs. Surprisingly, control-specific NFKB1 ChIP-seq peaks were not enriched for NFKB1 motifs, but instead contained motifs for other classes of human transcription factors, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of altered genotype-dependent chromatin accessibility at 36 AD risk variant loci (30% of AD risk loci) that might lead to genotype-dependent gene expression. Based on these findings, we propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease- and genotype-dependent chromatin accessibility alterations involving NFKB1 binding.
Atopic dermatitis (AD) is one of the most common skin disorders in children. Disease etiology involves genetic and environmental factors, with the 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated epigenetic mechanisms that may account for genetic susceptibility in CD4+ T cells. To understand gene regulatory activity differences in peripheral blood T cells in AD, we measured chromatin accessibility (ATAC-seq), NFKB1 binding (ChIP-seq), and gene expression (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD and age-matched, non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of AD risk loci overlapping with AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NFκB DNA binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD-specific or Control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NFκB DNA binding motifs. Surprisingly, Control-specific NKFB1 ChIP-seq peaks were not enriched for NFKB1 motifs, instead containing motifs for other classes of human TFs, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of genotype-dependent chromatin accessibility at 36 AD risk variants (30% of AD risk loci) that could lead to genotype-dependent expression at these loci. We propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease and genotype-dependent chromatin accessibility involving NFKB binding.AUTHOR SUMMARYStimulated CD4+ T cells from patients with atopic dermatitis have disease-dependent regulation of how gene expression is regulated. This regulation is disease dependent and the way the DNA is accessible and the transcription factor NFKB1 binds is enriched for genetic risk variants. Clinically, the CD4+ T cells in the peripheral blood of patients with AD respond to stimulation in a disease and genotype-dependent manner.
OBJECTIVES/GOALS: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatologic disease childhood and a cause of pain and potential disability. JIA has a strong genetic component and no known cure. The goal of this study is to evaluate allele-dependent effects of a novel JIA risk variant at 1q24.3. METHODS/STUDY POPULATION: JIA patients meeting criteria for the two most common disease subtypes (oligoarticular and RF neg polyarthritis) were genotyped using the Immunochip, an Illumina array with dense coverage of the HLA region and 186 other loci previously reported in autoimmune diseases. Phase I association findings (Hinks, 2013) and Phase II analysis (unpublished) of an expanded cohort (4,271 JIA and 14,390 controls) identified new risk loci, including rs78037977 at 1q24.3. We prioritized rs78037977 and predicted possible impacted mechanisms based on Bayesian predictions of attributable risk, the surrounding chromatin landscape, and transcription factor binding data. A luciferase reporter assay was used to assess allele-dependent enhancer activity. RESULTS/ANTICIPATED RESULTS: rs78037977 is located between FASLG and TNFSF18 at chromosome 1q24.3 is associated with JIA (p = 6.3x10−09), and explains 94% of the posterior probability at this locus; no other SNPs in linkage disequilibrium (r2>0.6). The chromatin landscape around rs78037977 contains H3K4Me1 and H3K27Ac marks, which are indicative of enhancer activity. Further, >160 transcription factors have chromatin immunoprecipitation followed by sequencing (ChIP-seq) peaks overlapping rs78037977 in various cellular contexts. In luciferase reporter assays, the region around rs78037977 containing the reference A allele had ~2-fold increased enhancer activity compared to the non-reference allele. DISCUSSION/SIGNIFICANCE OF IMPACT: This work provides in vitro evidence to support allele-dependent enhancer activity of a novel JIA-risk variant at 1q24.3. Our ongoing work investigates the effect of the DNA-containing region of rs78037977 on gene expression and differential transcription factor binding at rs78037977.
RATIONALE: Epithelial cell-derived cytokines are critical regulators in the pathogenesis of atopic dermatitis (AD). We examined mast cell expression of IL-33, ST2 and TSLPR in skin biopsies from patients with AD after intradermal allergen challenge. METHODS: Intradermal challenges with allergen and saline control were conducted in patients with moderate-to-severe AD. Punch biopsies were collected from the site of challenge 24 hours later, and stained with immunofluorescent antibodies to tryptase, IL-33, ST2, and TSLPR. Images were obtained and analysed by selecting regions of interest, and cells positive for the selected markers were expressed as cells per mm2 of the area examined. RESULTS: Compared to saline challenge, there was a significant increase in the number of tryptase-positive cells in the dermis (2-fold) and in the epidermis (3-fold) at 24 hours post-allergen (p<0.05). There was also an increase in the number of IL-33-positive cells in the dermis (2-fold) and epidermis (8-fold) after allergen compared to saline (p<0.05). Postallergen, most cells expressing IL-33 were tryptase-positive. Furthermore, there was a significant increase in the number of cells in the dermis and epidermis co-localizing tryptase and IL-33 after allergen compared to saline (p<0.05). In contrast, few tryptase-positive cells expressed ST2 and TSLPR, and expression of these receptors on tryptase-positive cells was not different between allergen and saline. CONCLUSIONS: Most cells expressing IL-33 post-allergen are tryptasepositive, and these cells that co-localize tryptase and IL-33 increase significantly after allergen challenge. These data suggest that mast cells are a major source of IL-33 in the skin following exposure to allergen. Abstracts AB65 SATURDAY
Systemic Lupus Erythematosus (SLE) is an incurable, debilitating autoimmune disease characterized by widespread inflammation and rampant production of autoantibodies. The most prominent and highly replicated set of genes up-regulated in the immune cells of patients with SLE are the type I interferons (IFN-I) and IFN-responsive genes. IFN-I are predominantly made by plasmacytoid dendritic cells (pDCs), and their expression is directly regulated by the transcription factor interferon regulatory factor 7 (IRF7). While IRF7 is an established SLE risk locus, the variants responsible for disease pathology remain unknown. We hypothesize that an amino-acid changing SLE risk variant in IRF7 (rs1131665) alters expression of disease-relevant IFN-I in clinically-relevant cells to increase SLE risk. The functional genomic consequences of the SLE-associated variant were assessed in human cell lines and in genome-edited mice with an introduced SLE-risk variant at Irf7. Our data demonstrate greater than 2-fold genotype-dependence in IFN-stimulated response element-driven luciferase activity and inflammatory cytokine secretion detected in supernatant after toll-like receptor-7 stimulation. Gene expression differences in cells with IRF7/Irf7 risk variants are consistent with those dysregulated in SLE patients. In the present study, we demonstrate the functional consequences of an amino acid substitution in a critical type I interferon regulator. Understanding these mechanisms will enhance development of more effective clinical practices for autoimmune patients expressing the risk variant for IRF7.
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