Cutting edge: Negative regulation of dendritic cells through acetylation of the nonhistone protein STAT-3

Abstract

Histone deacetylase (HDAC) inhibition modulates dendritic cell (DC) functions and regulates experimental graft-vs-host disease and other immune-mediated diseases. The mechanisms by which HDAC inhibition modulates immune responses remain largely unknown. STAT-3 is a transcription factor shown to negatively regulate DC functions. In this study we report that HDAC inhibition acetylates and activates STAT-3, which regulates DCs by promoting the transcription of IDO. These findings demonstrate a novel functional role for posttranslational modification of STAT-3 through acetylation and provide mechanistic insights into HDAC inhibition-mediated immunoregulation by induction of IDO.

Figures

Figure 1:

(A) HDAC inhibition acetylates STAT-3: BM DCs were harvested and treated with HDAC inhibitors SAHA and ITF 2357. Whole cell lysates were prepared and immunoprecipitated with anti-Stat3 and then analyzed with antibodies to acetylated lysine. Data shown are from one of 3 similar experiments. (B) HDAC inhibition induces IDO protein: BM DCs were harvested and treated with HDAC inhibitors SAHA and ITF 2357. Cell lysates were analyzed for IDO by Western Blot. Data are from one of two similar experiments. (C) Blockade of STAT-3 signaling with JSI-124 reverses HDAC inhibitor mediated suppression TNFα secretion from BM DCs: BM DCs were pretreated with 5 μM of JSI-124 or diluent for 30 min and then treated with 500 nM of SAHA or 200 nM ITF2357 or H20 for another 12–16 hours. They were then stimulated with 1μg/ml of LPS for 8 hours after which supernatants were harvested and measured for TNFα by ELISA. Results are representative of 3 experiments. (D) JSI-124 reverses the suppression of allogeneic T cell proliferation by the HDAC inhibitor treated BM DCs: BM DCs were pretreated with 5 μM of JSI-124 or diluent and then treated with 500 nM of SAHA or 200 nM ITF2357 or H20 for another 12–16 hours. They were then washed and used as stimulators in allogeneic MLR. T cell proliferation was determined after 72 h of culture. Results are representative of 2 experiments. (E) HDAC inhibitors acetylate but do not enhance phosphorylation of STAT-3. BM DCs were treated with SAHA, ITF 2357, LPS or the diluent control. Whole cell lysates were processed for immunoprecipitation with STAT-3 antibody and then blotted with phosphorylated 705-tyrosine STAT-3 antibody. The data are representative of 2 similar experiments. (F): Quantification of normalized pSTAT-3 levels combined from the above experiments is shown.

Figure 2:

(A) Inhibition of HDAC inhibitor mediated induction of IDO by JSI-124: BM DCs were pretreated with 5 μM of JSI-124 or diluent for 30 min and then treated with 500 nM of SAHA or 200 nM ITF2357 or H20 for another 14–16 hours. Expression of IDO was evaluated with RT-PCR for IDO mRNA. Results combined from three similar representative similar experiments are shown in bar graph. P < 0.05, solid bars vs. open bars (B) IDO promoter analysis: Nucleotide sequence of the 5’ region of the mouse IDO gene (−1500 bp upstream of IDO gene start codon) was obtained from GenBank database and analyzed with TESS promoter analysis software (http://www.cbil.uppen.edu/tess/). STAT binding sites (GAS) and NF-κB binding site (κB site) are shown. (C) STAT-3 binds to IDO promoter: BM DCs were treated with SAHA or diluent for 14–18 hours and assessed for the occupancy of STAT-3 to the IDO GAS regions. DCs were harvested and ChIP assay was performed as described in Materials and Methods. Chromatin complexes were immunoprecipitated with antibodies to STAT-3 or with control rabbit IgG. One-tenth of the total lysates were used as for total genomic DNA as input DNA control.

Figure 3.

(A): STAT-3 binding and IDO promoter driven luciferase: BM DCs were transfected with different IDO promoter driven luciferase constructs as in Materials and Methods. DCs were treated with JSI-124 and then with SAHA as above. The expression of luciferase was analyzed as in Materials and Methods. The error bars and statistics are representative of the technical replicates from one of 3 similar experiments. (B): STAT-3 acetylation is required for induction of IDO: PC3 cell lines expressing pcDNA3 empty vector (STAT-3 null), wild type STAT-3 (WT) and STAT-3 mutant K685R (K685R) were treated with either SAHA, ITF 2357 or diluent for 12–14 hours. They were then harvested and analyzed for IDO by RT-PCR. Data are from 1 of 2 experiments with similar results. (C): Quantification of normalized IDO levels from the above experiments is shown.solid bars vs. striped bars, P < 0.05.