Th2 cell-selective enhancement of human IL13 transcription by IL13-1112C>T, a polymorphism associated with allergic inflammation

Abstract

IL-13 is a central mediator of allergic inflammation. The single nucleotide polymorphism IL13-1112C>T (rs1800925) is associated with allergic phenotypes in ethnically distinct populations, but the underlying mechanism(s) remain unknown. Using in vivo, in vitro, and in silico analysis, we show that the IL13-1112T allele enhanced IL13 promoter activity in primary human and murine CD4(+) Th2 lymphocytes. Increased expression of IL13-1112T in Th2 cells was associated with the creation of a Yin-Yang 1 binding site that overlapped a STAT motif involved in negative regulation of IL13 expression and attenuated STAT6-mediated transcriptional repression. Because IL-13 secretion was increased in IL13-1112TT homozygotes, we propose that increased expression of IL13-1112T in vivo may underlie its association with susceptibility to allergic inflammation. Interestingly, IL13-1112T had opposite transcriptional effects in nonpolarized CD4(+) T cells, paralleled by distinct patterns of DNA-protein interactions at the IL13 promoter. Our findings suggest the nuclear milieu dictates the functional outcome of genetic variation.

FIGURE 1.

IL13-1112T is the ancestral allele and resides within a putative primate-specific cis-regulatory element. Top, eShadow conservation plot (www.eshadow.dcode.org) for the region surrounding IL13-1112C>T (arrow) in humans and 12 primate species representative of distinct clades. Peaks and valleys correspond to regions of low and high variation, respectively, with 0% variation = 100% sequence identity. Areas shaded in green represent regions below the DT (15/70). Bottom, Sequence alignment for the region surrounding IL13-1112C>T (arrow) in mice, humans, and primates. Numbering is relative to the human IL13 ATG. The pairwise alignment of human (GcnBank accession No. NC_000005:132016718-132031715) and mouse (GenBank accession No. NC_000077:53392544-5337166) promoter sequences for the IL13 –1112 region was performed with AlignX, a feature of the Vector NTI suite, which is based on the Clustal W algorithm. The dashes and asterisks mark conserved positions and gaps in the sequence, respectively. The STAT6 and YY1 binding motifs revealed by the DNA-protein interaction analysis discussed later in this paper are boxed by continuous or dashed lines, respectively. OWM, Old World monkeys; NWM, New World monkeys.

FIGURE 2.

IL13-1112T enhances IL-13 promoter activity in polarized CD4⁺ Th2 cells but not in nonpolarized CD4⁺ T cells. Left, Jurkat T cells (n = 26), freshly isolated CD4⁺ T cells (n = 9), or in vitro polarized human Th2 cells (n = 5) were transiently transfected with 2.7-kb IL13 promoter reporter constructs carrying the major (C) or minor (T) −1112 allele. Cells were left in medium or stimulated with PMA (20 ng/ml) and ionomycin (1 μM) and harvested after 16–18 h. Results are expressed as fold-increase in RLA (mean ± SE) after stimulation. Right, Murine D10.G4.1 Th2 cells were nucleofected with pGL3 Basic or −1112 allelic variants of an IL13 reporter construct (n = 8). Cells were left unstimulatcd for 16 h after transfection. Results are expressed as fold increase in the activity of the IL13 reporter constructs relative to pGL3 Basic. Statistical significance of all results was determined using the Wilcoxon two-sample test.

FIGURE 3.

IL13-1112 allele-specific patterns of transcription factor binding in distinct T cell nuclear environments. EMSA analysis with IL13-1112C and T oligonucleotide probes (top) and nuclear extracts from primary polarized human Th2 cells (A), freshly isolated CD4⁺ T cells (B), and Jurkat T cells (C) cultured for 3 h in medium or PMA (P; 20 ng/ml) and ionomycin (I; 1 μM). The nuclear extracts used in lanes 1–20 of A were prepared from PMA-ionomycin-activated cells. STAT and YY1 binding sites in the probe sequence are boxed; NFAT and Oct motifs are underlined. The competitors (fold molar excess) and supershifting Abs used for each experiment are noted above the corresponding lanes in the gels. Probes are noted below the gels. FP, free probe; a (as in aSTAT), anti.

FIGURE 4.

IL13-1112T altcnuatcs STAT6-mediated repression of IL-13 transcriplion. Left, Primary polarized human Th2 cells were transienlly transfected with −1112 allelic variants of the reference 2.7-kh IL13 promoter reporter construct (−1112C/Luc or −1112T/Luc) or equivalent constructs in which the STAT site adjacent to the SNP had been disrupted (−1112C STATmut/Luc and −1112T STATmut/Luc) (n = 2). Cells were left in medium or stimulated with PMA (20 ng/ml) and ionomycin (1 μM) and harvested after 16–18 h. Results are expressed as fold increase in RLA (mean ± SE) after stimulation. Center, Murine D10.G4.1 cells were harvested 6–12 days after antigenic stimulation and nucleofected with pGL3 Basic or IL13-1112C or T promoter reporter variants. Cells were incubated with neutralizing anti (a)-murine IL-4 or control IgG1 Ab for 16 h before harvesting (n = 6). Results are expressed as fold increase in the activity of the IL13 reporter constructs relative to pGL3 Basic. Right, Jurkat T cells were cotransfected with the −1112/Luc IL13 promoter constructs and pcDNA3, STAT1 (n = 4), or a STAT6 (n = 11) expression vectors. Cells were incubated with medium or PMA (20 ng/ml) and ionomycin (1 μM). IL-4 (10 ng/ml) was added to the STAT6 cotransfections. Cells were harvested after 16–18 h. Results arc expressed as percentage of fold luciferase induction in response to stimulation for STAT-transfected cells relative to cells transfected with pcDNA3 (mean ± SE). For all panels, statistical significance was determined using the Wilcoxon two-sample test.

FIGURE 5.

STAT6 and YY1 bind at the IL13–1112 region in vivo. Naive peripheral blood CD4⁺ T cells from a healthy IL13–1112T heterozygote were differentiated in vitro for 2 wk under Th2 conditions. ChIP assays with an anti-STAT6, anti-YY1, or control Ab (mouse IgG1) were performed on cells (5 × 10⁷ per immunoprecipitation) cultured in the presence (+) or absence (−) of PMA and ionomycin (P/I) for 3 h. Top, Input DNA (5 or 10 ng) or immunoprecipitated DNA (one-twentieth and one-tenth of total) was used as template for PCR amplification of a 202-bp amplicon encompassing IL13-1112. Bottom, Input DNA (10 ng) or immunoprecipitated DNA (one-tenth of total) was used as template for PCR amplification of a 152-bp amplicon corresponding to CNS-1. Results are from one representative experiment of three independent immunoprecipitations.

FIGURE 6.

Increased IL-13 secretion in IL13-1112T homozygotes. PBMC from pregnant women enrolled in the Infant Immune Study study and genotyped for IL13-1112C>T and IL13-2044G>A were incubated with Con A (10 μg/ml) and PMA (10 ng/ml) for 18–24 h. IL-13 concentrations in culture supernatants were assessed by ELISA. Results are the mean ± SE of IL-13 concentrations measured in each IL13-1112 genotype group. Statistical significance was assessed by one-way ANOVA for the relation between IL13-1112 genotypes and IL-13 production, and a Bonferroni multiple comparison test for differences in IL-13 levels between individual genotypes.

FIGURE 7.

Model of gene-environment interactions in the nucleus at IL13-1112C>T. The results of the DNA/protein interaction analysis are summarized on the left. The corresponding IL13-1112C>T transcription data are presented on the right.