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.免疫调节细胞因子白细胞介素-10（IL-10）在系统性红斑狼疮（SLE）患者的血清中水平升高，且与疾病活动度呈正相关。鉴于IL10与SLE及其他自身免疫性疾病的既定关联，我们旨在精细定位潜在致病变异并探究其潜在分子机制。我们针对涵盖IL10基因簇（包含IL19、IL20与IL24）的19个标签单核苷酸多态性（tag SNPs），在来自4个遗传血统群体的15533名病例及对照受试者中，评估其与SLE的关联强度。此前报道的位于IL10下游1kb处的IL10变异rs3024505，展现出最强的关联信号，并在欧裔美国人（EA）队列中得到验证（P=2.7×10⁻⁸，比值比OR=1.30），但在非欧裔血统群体中未观测到该关联。对欧裔美国人数据集开展单核苷酸多态性插补（SNP imputation）分析后，我们发现了另外3个由rs3024505标记的SLE相关SNPs：rs3122605、rs3024493与rs3024495，分别位于IL10上游9.2kb区域、IL10的内含子3与内含子4区域。上述SNPs的SLE风险等位基因，与SLE患者及对照者外周血单个核细胞（PBMCs，peripheral blood mononuclear cells）中IL10 mRNA水平升高、循环IL-10蛋白水平升高呈剂量依赖性相关。我们采用SLE患者外周血细胞的核提取物开展电泳迁移率变动分析（electrophoretic mobility shift assays），发现转录因子Elk-1可特异性结合携带rs3122605风险等位基因（G）的寡脱氧核苷酸序列，提示rs3122605是调控IL10表达的最可能致病变异。已知Elk-1可通过磷酸化修饰及核定位被激活以诱导靶基因转录。有趣的是，仅在SLE患者外周血单个核细胞核提取物中检测到的磷酸化Elk-1（p-Elk-1）水平，似乎随疾病活动度升高而上升。p-Elk-1与IL-10的共表达水平在SLE患者的T细胞、B细胞与单核细胞中均显著升高，且在SLE患者B细胞中该共表达水平与疾病活动度增加呈正相关；二者的表达可被ERK抑制剂最有效地下调。综上，我们的研究结果表明，激活状态的Elk-1优先结合IL10基因rs3122605位点的G型风险等位基因，从而上调IL10的表达，并使欧裔美国人罹患SLE的风险升高。