Common genetic variation at the IL1RL1 locus regulates IL-33/ST2 signaling

Abstract

The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framingham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide association study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway.

Figure 1. Regional association plot of sST2 SNPs with P < 5 × 10^–8. “CEU” refers to Utah residents with northern and western European ancestry from the CEPH collection.

Recombination rates are superimposed in blue, and gene regions are in green.

Figure 2. Models of ST2 illustrate IL1RL1 missense variant locations.

Models of the (A) intracellular TIR domain (ST2-TIR) and the (B) extracellular domain (ST2-ECD) of ST2 (protein data bank codes 3O4O and 1T3G, respectively). Domains of ST2 are shown in yellow, with identified missense SNP positions represented as red spheres and labels. Note that positions 549 and 551 are near the C terminus of ST2, which is not defined in the crystal structure (protein data bank ID 1T3G, shown as dashed black line in A). Arrows point toward the transmembrane domain, which is also not observed in crystal structures.

Figure 3. IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Media from KU812 cells expressing WT and IL1RL1 missense variants were collected for ELISA analysis of (A) sST2, (B) IL-33, and (C) IL-1β levels. Horizontal bars indicate mean values, and symbols represent individual variants. *P < 0.05, **P < 0.01 vs. WT. (D) Effect of anti–IL-1β mAb on IL-33–induced sST2 expression. Dashed line indicates PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. *P < 0.05 vs. IL-33.

Figure 4. IL1RL1 missense variants modulated ST2 signaling pathways.

KU812 cells expressing WT or IL1RL1 variants were treated with PBS or IL-33. Levels of the following phosphorylated proteins were detected in cell lysates using ELISA: (A and B) phospho-NF-κB p65; (C and D) phospho-c-Jun activity; and (E and F) phospho-AKT. (A, C, and E) White bars represent basal levels, and (B, D, and F) gray bars represent relative fold increase (compared with PBS-treated group) after IL-33 treatment. *P < 0.05 vs. WT; **P < 0.01 vs. PBS-treated group. Dashed line in B, D, and F represents PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments.

Figure 5. IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

(A) sST2 mRNA expression in KU812 cells after treatment with DMSO, IL-33, or IL-33 plus signal inhibitors (wortmannin, LY294002, rapamycin, PD98059, SP60125, BAY11-7082, or SR11302). (B) ST2L mRNA and (C) sST2 mRNA expression in KU812 cells treated with PBS (white columns), rapamycin (rapa), anti-ST2 mAb, IL-33, IL-33 plus anti-ST2, IL-33 plus rapamycin, IL-33 plus rapamycin plus anti-ST2 mAb, or rapamycin plus anti-ST2. (D) IL33 mRNA expression in KU812 cells after treatment with DMSO, signal inhibitors, IL-33 plus signal inhibitors, and IL-1β plus signal inhibitors. *P < 0.05 vs. PBS-treated group; ^#P < 0.05 vs. IL-33–treated group; ^&P < 0.05 vs. IL-1b–treated group. Error bars represent mean ± SEM from 2 independent experiments. (E) A schematic model illustrating the regulation of sST2 expression by IL1RL1 missense variants through enhanced induction of IL-33 via enhanced NF-κB and AP-1 signaling and enhanced IL-33 responsiveness via increasing ST2L expression.