Author + information
- HaiFeng Zhang1,
- YongQing Lin1,
- WenHao Liu1,
- ShaoHua Wang1,
- ChengZhang Xu1,
- YangXin Chen1 and
- JingFeng Wang1
Both IL-33 and IL-10 benefit atherosclerosis, reduce foam cell formation and accelerate cholesterol transports. In addition, IL-33 induces Th2 response and IL-10 could be secreted by Th2 cells. We therefore evaluated the role of IL-10 on IL-33-induced cholesterol reduction in macrophages foam cell (MFC) and the mechanisms that IL-33 up-regulating IL-10.
Serum levels of IL-33 and IL-10 in coronary artery disease (CAD) patients were assessed using enzyme linked immunosorbent assay and their relationships were explored with Spearman's correlation. IL-33 was administered to THP-1 derived macrophages, which were induced into MFC by ox-LDL. The presence IL-33 trans-membrane receptor in MFC were validated by immuno-fluorescence, flow cytometry and Western blot. Cellular cholesterol were determined using commercial available Kit. Expressions of IL-10, ABCA1, CD36, p-ERK/t-ERK, p-STAT3/t-STAT3, p-STAT4/t-STAT4 and nuclear Sp1 regulated by IL-33 were confirmed by RT-qPCR and/or Western blot. The role of IL-10 in IL33-induced cellular cholesterol, CD36 and ABCA1 alterations were evaluated by siRNA against IL-10 mRNA. Transcriptional activity of -2000 bp of the 5'-flank counting from IL-10 transcription start site were determined by dual-luciferase reporter gene assay. Core sequences regulating IL-10 by IL-33 were predicted using Genomatrix MatInspector and JASPAR, which were confirmed by site-directed mutagenesis and chromatin immuno-precipitation.
In CAD patients, serum levels of IL-33 were positively correlated with those of IL-10. IL-33 decreased cellular cholesterol, up-regulated IL-10 and ABCA1 in MFC but inhibited CD36 expressions. siRNA-IL-10 partly abolished cellular cholesterol reduction and ABCA1 elevation by IL-33 but did not reverse CD36 decrease. IL-33 increased IL-10 mRNA production but had little effect on its stability. IL-33 induced ERK 1/2 phosphorylation and increased luciferase expression driven by IL-10 promoter with the highest extent within the segment containing -2000 bp to -1752 bp of the 5'-flank counting from transcription start site, which were counteracted by U0126. IL-33 induced Sp1 accumulation in nucleus, phosphorylation of STAT3 and STAT4 but only STAT3 binding sequences were predicted in the above segment. Moreover, STAT3 inhibitor dismissed the regulatory effects of IL-33 on IL-10 expression. Site-directed mutagenesis of the predicted STAT3 binding sites (CTGCTTCCTGGCAGCAGAA→CTGCCTGGCAGCAGAA) reduced luciferase activity and CHIP confirmed the STAT3 binding sequences within regions of locus of -1997 bp to -1700 bp and -1091 bp to -811 bp.
Beneficial effects of IL-33 on foam cell formation were largely mediated by up-regulation of IL-10, which was a result of the activation of ERK 1/2 and STAT3, and finally an enhancement of IL-10 genomic transcriptional activity.