Preferential binding to Elk-1 by SLE-associated IL10 risk allele upregulates IL10 expression

Abstract

Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10⁻⁸, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.

Figure 1. SNPs of the IL10 gene cluster associated with SLE in European Americans.

(A) Association of 19 genotyped SNPs with SLE in EA (red), AA (yellow), AS (blue) and HS (green). Allelic P value (−log₁₀ P) of each SNP was plotted against its genomic position. (B) Association of 19 genotyped and an additional 109 imputed SNPs with SLE in EA. Genotyped and imputed SNPs were indicated as circles and triangles, respectively. Based on its pairwised LD strength with rs3024505, each SNP was highlighted as red (r²>0.9) or grey (r²<0.9). (C) Genomic structure of the IL10 gene cluster and the location of each SNP. (D) Haplotypic analysis in EA. Haplotypes were constructed using four SLE-associated SNPs shown in Figure 1B (rs3024505, rs3024495, rs3024493 and rs3122605), three previously reported SLE-associated SNPs (rs1800872, rs1800871 and rs1800896 in the promoter of IL10) and rs1518111 (the T allele associated with Bechet's disease). Risk alleles of four SLE-associated SNPs shown in Figure 1B were bolded and italicized.

Figure 2. Dose-dependent association of rs3122605 risk G-allele with elevated levels of IL10 mRNA and protein.

IL10 mRNA (A) and protein levels (B) were measured in PBMCs and plasma from EA SLE patients and healthy controls, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values.

Figure 3. Preferential binding of rs3122605-risk allele to Elk-1, which is a transcription factor activated in peripheral lymphocytes of SLE patients.

(A) Specific binding of transcription factor Elk-1 to the risk allele of rs3122605 in EMSA. Mobility-shift bands were produced by the oligodeoxynucleotide probes containing the risk allele of rs3122605 incubated with nuclear extracts of peripheral blood lymphocytes from active SLE patients, and the addition of anti-Elk-1 antibody generated a super shift band. The data are representative of two independent experiments. (B, C) Nuclear retention of activated p-Elk-1 in PBMCs from SLE patients. The presence of Elk-1 and p-Elk-1 in nuclear (B) and cytoplasmic (C) extracts of PBMCs from SLE patients and healthy controls was measured using Western blot. Lamin B (B) and β-actin (C) were used as loading controls. The data are representative of two independent experiments.

Figure 4. Co-expression of p-Elk-1 and IL-10 in PBMCs.

(A) Quantification of co-expression of p-ELK-1 and IL-10 in B cells (CD19⁺), T cells (CD3⁺) and monocytes (CD14⁺), respectively, by flow cytometry. Numbers in upper quadrants indicate the percentages of double positive (IL-10⁺p-Elk-1⁺) cells. (B) Increased proportions of IL-10⁺p-Elk-1⁺ cells in B, T cells and monocytes from SLE patients compared to controls, and increased IL-10⁺p-Elk-1⁺ cells in B cells from active compared to inactive SLE patients. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Decreased proportions of IL-10⁺p-Elk-1⁺ cells with inhibition of Elk-1 activation. Normal PBMCs were incubated with IFNα in the presence or absence of MAPK inhibitor ERK (PD 98059), JNK (SP 600125) or p38 (SB 203580), and IL-10⁺p-Elk-1⁺ cells were quantified in B, T cells and monocytes, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. P*≤0.01, P^#<0.005, P**<0.0001 (Student's t test) for the comparison of indicated groups.