MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo

Abstract

Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an "adjuvant" during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity.

Keywords: MHC transcription; antigen presentation; autoimmunity; inflammation; vitiligo.

Fig. 1.

Vitiligo association in the MHC class II region of human chromosome 6p. Nucleotide positions, HLA-DRB1 and HLA-DQA1 genes, transcriptional orientation, and the three SNPs that define the vitiligo high-risk haplotype are shown. Layered H3K27Ac, H3K4Me1, and H3K4Me3 marks, hidden Markov model chromatin state segmentation (ChromHMM), DNase I hypersensitive site cluster (DNase I Clusters), transcription factor chromatin immunoprecipitation sequencing (Txn Factor ChIP-seq), and CTCF ChIP-seq data are from ENCODE (9). For layered H3K27Ac, H3K4Me1, H3K4Me3 marks, data are shown for the seven cell lines studied by ENCODE; red indicates data from GM12878 lymphoblastoid cells. For ChromHMM, data shown are for GM12878; orange indicates strong enhancers, yellow indicates weak/poised enhancers, and green indicates weakly transcribed regions. For DNase clusters, darkness indicates relative signal strength in 125 cell types from ENCODE (V3). For Txn factor ChIP-seq, darkness indicates relative signal strength of aggregate binding of 161 transcription factors and green bars indicate ENCODE Factorbook (42) canonical motifs for specific transcription factors. For CTCF ChIP-seq, data are from GM12878.

Fig. 2.

Expression of HLA-DR and HLA-DQ on blood monocytes of low-risk and high-risk subjects. (A) Representative dot plots showing the gating strategy and down-regulation of CD14 of unstimulated compared with C. albicans-stimulated monocytes. The frequency of CD11b⁺CD14^+/low monocytes is reported in each dot plot. (B) Representative histogram plots of HLA-DQ and HLA-DR expression on unstimulated monocytes from the blood of low risk (LR) and high risk (HR) subjects. Fluorescence minus one (FMO) is shown as a negative staining control. (C) Summary of the MFI of unstimulated monocytes from five low-risk and five high-risk subjects. (D) Summary of the MFI of C. albicans-stimulated monocytes from low-risk and high-risk subjects. (E) Summary of the MFI of S. epidermidis-stimulated monocytes from low-risk and high-risk subjects. Statistical significance of differences between groups was determined with the unpaired t test.

Fig. 3.

Production of IFN-γ, IL-10, IL-2, IL-18, IL-1β, IL-1Ra, IL-6, and TNF-α by PBMCs of low-risk and high-risk subjects. Mean ± SEM production of IFN-γ (A), IL-10 (B), IL-2 (C), IL-18 (D), IL-1β (E), IL-1Ra (F), IL-6 (G), TNF-α (H). Data are expressed as fold-change of baseline cytokine production, which was as follows: IFN-γ: LR median 28 pg/mL, range 15–50; HR median 39; range 15–45; IL-10: LR median 7 pg/mL, range 3–62; HR median 5, range 3–42; IL-2: LR median 10 pg/mL, range 2–17; HR median 8, range 2–20; IL-18: median 21 pg/mL, range 12–97; HR median 31, range 16–184; IL-1β: LR median 23 pg/mL, range 2–56; HR median 16; range 2–77; IL-1Ra: LR median 180 pg/mL, range 14–1016; HR median 232, range 36–829; IL-6: LR median 35 pg/mL, range 9–199; HR median 23, range 14–92; TNF-α: LR median 15 pg/mL, range 7–114; HR median 28, range 13–114. For each determination, data were normalized to the number of lymphocytes or monocytes. Statistical significance of differences between groups was determined with the unpaired t test. LR (n = 12), HR (n = 6); ns, nonsignificant; *P < 0.05; **P < 0.01.