Genetic variation near IRF8 is associated with serologic and cytokine profiles in systemic lupus erythematosus and multiple sclerosis

Abstract

Alleles of interferon (IFN) regulatory factor 8 (IRF8) are associated with susceptibility to both systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Although high-type I IFN is thought to be causal in SLE, type I IFN is used as a therapy in MS. We investigated whether IRF8 alleles were associated with type I IFN levels or serologic profiles in SLE and MS. Alleles that have been previously associated with SLE or MS were genotyped in SLE and MS patients. The MS-associated rs17445836G allele was associated with anti-double-stranded DNA (dsDNA) autoantibodies in SLE patients (meta-analysis odds ratio=1.92). The same allele was associated with decreased serum IFN activity in SLE patients with anti-dsDNA antibodies, and with decreased type I IFN-induced gene expression in peripheral blood mononuclear cell from anti-dsDNA-negative SLE patients. In secondary progressive MS patients, rs17445836G was associated with decreased serum type I IFN. Rs17445836G was associated with increased IRF8 expression in SLE patient B cells. In summary, IRF8 rs17445836G is associated with human autoimmune disease characterized by low-type I IFN levels, and this may have pharmacogenetic relevance as type I IFN is modulated in SLE and MS. The association with autoantibodies and increased IRF8 expression in B cells supports a role for rs17445836G in humoral tolerance.

Figure 1

Diagram of the IRF8 gene and LD plots. Each SNP studied is indicated by a colored line on the diagram, with the SNP rs number color-coded to the line which represents its location. The number before each rs number (1., 2., 3., etc) corresponds to the number used to designate the SNP in the linkage disequilibrium (LD) plots below. The LD plots show pairwise correlations between the SNPs studied, with increasing red shading indicating increasing correlation between the two alleles. Numbers represent the D’ statistic.

Figure 2

Serum type I IFN in SLE patients stratified by IRF8 rs17445836 genotype and presence or absence of anti-dsDNA autoantibodies. A. shows European ancestry SLE patients, and B. shows African-American SLE patients. Minor allele homozygotes were rare, and were combined with heterozygotes (AA or AG). Bars represent the median, error bars show the interquartile range, p-values calculated using the Mann-Whitney U test.

Figure 3

Serum type I IFN in MS patients stratified by clinical diagnosis. RRMS = relapsing-remitting MS, SPMS = secondary progressive MS, PPMS = primary progressive MS. Minor allele homozygotes were rare, and were combined with heterozygotes (AA or AG). Central tendency is indicated by the median, error bars show the interquartile range, p-values calculated using the Mann-Whitney U test.

Figure 4

Type I IFN-induced gene expression studies in SLE patients stratified by rs17445836 genotype. There were no minor allele homozygotes in the subjects analyzed for gene expression. Panels A., B., and C. show gene expression in SLE patient PBMC for the IFIT1, MX1, and PKR transcripts respectively. Panels D., E., and F. show expression of the same genes in WISH cells stimulated with sera from these patients taken at the same blood draw as the PBMCs. Central tendency is indicated by the median, p-values calculated using the Mann-Whitney U test.

Figure 5

Data from the ENCODE project showing likely regulatory element binding sites downstream of IRF8. Screen shots are taken from the UCSC genome browser (http://genome.ucsc.edu/). A. shows a large view including IRF8 and the downstream region, with DNAse hypersensitivity and H3K27Ac marks from the ENCODE cell lines. The location of SNP rs17445836 is marked with the arrow. Cell line GM12878 from the ENCODE project is a lymphoblastoid B cell line, and is represented in salmon-colored shading. This was the only ENCODE cell line with a significant number of H3K27Ac marks in the region. B. shows a closer view of the region surrounding SNP rs17445836, and includes a representation of the ChIP-seq data from the ENCODE project, representing transcription factor binding sites. Darker shading indicates greater evidence for binding of the transcription factor in that region.

Figure 6

IRF8 expression in SLE patient immune cell subsets, and association with rs17445836 genotype in SLE patient B cells. A. shows IRF8 expression in CD4 T cells, CD8 T cells, CD14 monocytes, and CD20 B cells from SLE patients. Cells were isolated by flow cytometric sorting for these surface markers. B. shows IRF8 expression in SLE patient B cells stratified by rs17445836 genotype. Line indicates the median, error bars show the interquartile range, p-value by Mann-Whitney U test comparing GG vs. AG or AA.