Preferential binding to elk-1 by sle-associated il10 risk allele upregulates il10 expression

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

Preferential Binding to Elk-1 by SLE-Associated <i>IL10</i> Risk Allele Upregulates <i>IL10</i> Expression

<div><p>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 <i>IL10</i> 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 <i>IL10</i> gene cluster including <i>IL19</i>, <i>IL20</i> and <i>IL24</i>, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported <i>IL10</i> variant, rs3024505 located at 1 kb downstream of <i>IL10</i>, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (<i>P</i> = 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 <i>IL10</i>, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of <i>IL10</i> 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 <i>IL10</i> 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 <i>IL10</i> rs3122605-G allele upregulates <i>IL10</i> expression and confers increased risk for SLE in European Americans.</p></div

Association of rs3853839 with SLE in multiple ancestries.

*<p>AS: Previously published data in population of Eastern Asian descent (5).</p><p>Abbreviation: AS, Eastern Asian; AA, African American; EA, European American; HS, Amerindian/Hispanics; OR, odds ratio; CI, confidence interval.</p

The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.

<p>(A) Association of rs3853839 genotypes with <i>TLR7/8</i> transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter <i>in vitro</i>. <i>TLR7</i> 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in <i>TLR7</i> transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in <i>TLR7</i> transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. ^*<i>P</i><0.05. (E) Summary of the G/C allele ratio in <i>TLR7</i> transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; <i>P</i> = 0.04; paired <i>t</i> test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.</p

Allelic associations of SNPs in the <i>TLR7-TLR8</i> region with SLE.

<p>(A) The genomic structure of the <i>TLR7-TLR8</i> region and the location of all studied SNPs are indicated. (B) Association signals (−log₁₀<i>P</i>) are plotted against the position of each SNP (based on GRch37/hg19) in European Americans (EA), African Americans (AA), and Hispanics (HS). Genotyped and imputed SNPs are depicted with circles and triangles, respectively. The diamond identifies the <i>TLR7</i> 3′UTR SNP rs3853839. SNPs are highlighted using different colors according to their LD strength (r²) with rs3853839. (C) A trans-ancestral meta-analysis is conducted on 40 genotyped SNPs (circles) and 14 imputed SNPs (triangles) that are shared by the three ancestries (SNPs listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003336#pgen.1003336.s006" target="_blank">Table S1</a>) using fixed and random model, respectively. The dashed line represents the significance level of 5×10⁻⁸.</p

The SLE-risk G allele of rs3853839 displays reduced transcript modulation by miR-3148.

<p>(A) TargetScan's predicted miR-3148-binding site in <i>TLR7</i> 3′UTR. The C allele, rather than G allele of rs3853839 corresponds to the second base of this seed region. (B) Inverse correlation of miR-3148 and <i>TLR7</i> transcript levels in PBMCs from 16 patients with SLE (solid circles) and 21 controls (open diamonds). (C) HEK-293 cells were cotransfected with empty reporter vector (EV), luciferase constructs driven by <i>TLR7</i> 3′UTR segment containing either C or G allele of rs3853839 and increasing concentrations (1, 6, 12, and 48 nM) of miR-3148 or nontarget control (NC) mimics. Luciferase activity was determined 24 hours after transfection. Normalized luciferase activity is the Renilla/Firefly ratio of miR-3148-treated reporter vector compared with the same NC-treated reporter vector. Data show the mean ± SEM and are representative of cumulative data from three independent experiments. <i>P</i> = 0.0003 over all miR-3148-treated C-allele vector groups, and not significant over all miR-3148-treated G-allele or empty vector groups (ANOVA test). <i>P*</i> = 0.02, <i>P**</i><0.0001 (Student's <i>t</i> test) for the comparison of indicated groups.</p

SNPs of the <i>IL10</i> gene cluster associated with SLE in European Americans.

<p>(A) Association of 19 genotyped SNPs with SLE in EA (red), AA (yellow), AS (blue) and HS (green). Allelic <i>P</i> value (−log₁₀<i>P</i>) 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 <i>IL10</i> gene cluster and the location of each SNP. (D) Haplotypic analysis in EA. Haplotypes were constructed using four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> (rs3024505, rs3024495, rs3024493 and rs3122605), three previously reported SLE-associated SNPs (rs1800872, rs1800871 and rs1800896 in the promoter of <i>IL10</i>) and rs1518111 (the T allele associated with Bechet's disease). Risk alleles of four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> were bolded and italicized.</p

Dose-dependent association of rs3122605 risk G-allele with elevated levels of <i>IL10</i> mRNA and protein.

<p><i>IL10</i> 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.</p