IL-33 promotes IL-10 production in macrophages: a role for IL-33 in macrophage foam cell formation

Abstract

We evaluated the role of IL-10- in IL-33-mediated cholesterol reduction in macrophage-derived foam cells (MFCs) and the mechanism by which IL-33 upregulates IL-10. Serum IL-33 and IL-10 levels in coronary artery disease patients were measured. The effects of IL-33 on intra-MFC cholesterol level, IL-10, ABCA1 and CD36 expression, ERK 1/2, Sp1, STAT3 and STAT4 activation, and IL-10 promoter activity were determined. Core sequences were identified using bioinformatic analysis and site-specific mutagenesis. The serum IL-33 levels positively correlated with those of IL-10. IL-33 decreased cellular cholesterol level and upregulated IL-10 and ABCA1 but had no effect on CD36 expression. siRNA-IL-10 partially abolished cellular cholesterol reduction and ABCA1 elevation by IL-33 but did not reverse the decreased CD36 levels. IL-33 increased IL-10 mRNA production but had little effect on its stability. IL-33 induced ERK 1/2 phosphorylation and increased the luciferase expression driven by the IL-10 promoter, with the highest extent within the -2000 to -1752 bp segment of the 5'-flank of the transcription start site; these effects were counteracted by U0126. IL-33 activated Sp1, STAT3 and STAT4, but only the STAT3 binding site was predicted in the above segment. Site-directed mutagenesis of the predicted STAT3-binding sites (CTGCTTCCTGGCAGCAGAA→︀CTGCCTGGCAGCAGAA) reduced luciferase activity, and a STAT3 inhibitor blocked the regulatory effects of IL-33 on IL-10 expression. Chromatin immunoprecipitation (CHIP) confirmed the STAT3-binding sequences within the -1997 to -1700 and -1091 to -811 bp locus regions. IL-33 increased IL-10 expression in MFCs via activating ERK 1/2 and STAT3, which subsequently promoted IL-10 transcription and thus contributed to the beneficial effects of IL-33 on MFCs.

Figure 1

Serum levels of IL-33 and IL-10 and their relationship in coronary artery patients. (a) Serum levels of IL-33 and IL-10 in the included patients. (b) Serum levels of IL-10 were lower in those with low serum IL-33 concentrations. (c) Serum levels of IL-10 positively correlated with those of IL-33.

Figure 2

Validation of the presence of ST2L on MFC membrances. (a–d) Immunofluorescence staining of DAPI, CD68 and/or ST2L. (e) Flow cytometry measurements of the proportions of CD68⁺ST2L⁺ cells. (f) Western blot analysis of cell membrane proteins. upper lane, ST2L; lower lane, ATPase Na⁺/K⁺-β2. The data in a–d represent two independent experiments and the data in e, f represent three independent experiments. MFC, macrophage-derived foam cell.

Figure 3

IL-33 upregulated IL-10 expression in MFCs in a dose-dependent manner within moderate concentrations. (a) The IL-10 mRNA abundance was increased upon IL-33 stimulation. (b) IL-10 supernatant levels from MFCs were upregulated following IL-33 treatment for 24 h. *P<0.05 versus baseline (PBS treatment); ^#P<0.05 versus high IL-33 concentrations (100 ng ml⁻¹) treatment. These data represent three independent experiments. MFC, macrophage-derived foam cell.

Figure 4

Effects of IL-10 siRNA on IL-33-induced cellular cholesterol reduction and alterations of CD36 and ABCA1. (a) IL-33 reduced the cellular cholesterol content, which was abolished by IL-10 siRNA. (b, d, e) IL-33 downregulated whereas IL-10 siRNA had no effect on CD36 expression. (c, d, f) IL-33 increased IL-10 expression, which was diminished by siRNA against IL-10. *P<0.05 versus baseline (PBS treatment). These data represent three independent experiments. si-IL-10, siRNA against IL-10; si-Scr, siRNA with scramble sequences without any known target mRNA; TC, total cholesterol.

Figure 5

Effects of IL-33 on ERK 1/2 phosphorylation and its role in IL-33-induced IL-10 expression. (a) IL-33 induced ERK 1/2 phosphorylation; (b, c) an ERK 1/2 inhibitor reversed IL-10 upregulation by IL-33. These data represent three independent experiments. p-ERK 1/2, phosphorylated ERK 1/2; t-ERK 1/2, total ERK 1/2.

Figure 6

Effects of IL-33 on IL-10 mRNA production, stability and gene promoter activity. (a) A transcription factor inhibitor, ActD, abolished the up-regulatory effects of IL-10 in MFCs. (b) IL-33 had a scarcely detectable influence on IL-10 mRNA stability. (c) IL-33 activated IL-10 gene promoter, as indicated by luciferase activity. (d) Promoter deletion analysis, which identified core sequences in the IL-33-induced IL-10 production process. *P<0.05 versus PBS treatment; ^#P<0.05 versus the pGL3-Basic. These data represent three independent experiments. ActD, actinomycin D; MFC, macrophage-derived foam cell.

Figure 7

Identification of transcription and target sequences that react with IL-33. (a) Effects of IL-33 on intra-nuclear Sp1 expression as well as STAT3 and STAT4 phosphorylation. (b) Bioinformatic analysis of STAT3-binding sites within the −2000 to −1752 bp of the 5′-flank of the IL-10 transcription start site. (c) Homology analysis of the predicted binding STAT3 site among five mammalian species. (d) Effects of TTC base deletion by site-directed mutagenesis on promoter activity indicated by luciferase activity. (e, f) Effects of a STAT3 inhibitor, cryptotanshinone, on IL-33-induced IL-10 mRNA (e) and protein (f) production. (g) CHIP analysis using a STAT3 antibody and primers specific to IL-10 promoter segments following PBS or IL-33 (30 ng ml⁻¹) treatment. p-STAT3, phosphorylated STAT3; p-STAT4, phosphorylated STAT4; t-STAT3, total STAT3; t-STAT4, total STAT4; wild type, pGL3-Basic-IL10.1-Wild type; Mutant, pGL3-Basic-IL10.1-Mutant; IL-33-P1, qPCR using primer set 1; IL-33-P4, qPCR using primer set 6 (all primer sets are listed in Supplementary Materials). *P<0.05 versus PBS treatment; ^#P<0.05 versus IL-33-P1. The data in a and d–g represent three independent experiments.