Divergent roles of histone deacetylase 6 (HDAC6) and histone deacetylase 11 (HDAC11) on the transcriptional regulation of IL10 in antigen presenting cells

Abstract

The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.

Keywords: Antigen presenting cells; HDAC11; HDAC6; IL-10; Tolerance.

Figure 1. HDAC6 is required for IL-10 production by APCs

(A) RAW264.7 cells were stably transduced with lentiviral particles containing HDAC6-specific shRNA (HDAC6KD) or with non-targeting shRNA control following manufacture's protocol (NT). These cells, as well as wild-type (WT) RAW264.7 were treated with or without LPS (1 μg/ml) for 24 hours. IL-10 production was then determined by ELISA. (B) Peritoneal elicited macrophages (PEM) and bone marrow derived DCs isolated from wild type or HDAC6KO mice were stimulated with LPS (1 μg/ml) in vitro for 24 hours. IL-10 production was then determined by ELISA. Three experiments were performed with similar results. Error bars represent standard deviation from triplicates.

Figure 2. HDAC6 interacts with HDAC11 in the cytoplasmic and nuclear compartments of APCs

Cell extracts from RAW264.7 or THP.1 cells (A) or from primary peritoneal macrophages (B) were immunoprecipitated (IP) using an anti-HDAC11 or anti-HDAC6 antibody, respectively. The IP fraction was then subjected to WB to evaluate for the presence of HDAC6 or HDAC11 respectively. (C) RAW264.7 cells were stably transfected with a plasmid carrying FLAG-HDAC11 (F-HDAC11). Cells were then analyzed by confocal microscopy with an anti-FLAG antibody and a secondary antibody conjugated to the fluorochrome Alexa Fluor 488 (green). To detect endogenous HDAC6 we used an anti-HDAC6 antibody and a secondary antibody conjugated to the fluorochrome Alexa Fluor 647 (red). Shown is a representative set of pictures from three experiments with similar results. (D) Nuclear and cytoplasmic fractions from macrophages over-expressing FLAG-HDAC11 were subjected to immunoblotting to evaluate the intracellular distribution of HDAC11 and endogenous HDAC6. Immunoblotting of HDAC1 and tubulin were used as controls for nuclear and cytoplasmic fractions respectively. (E) Nuclear and cytoplasmic fractions from macrophages over-expressing FLAG-HDAC11 were immunoprecipitated with an anti-FLAG antibody and the presence of HDAC6 was then evaluated by western blot. Data is from a representative experiment of three independent experiments with similar results.

Figure 3. HDAC6/HDAC11 interaction requires the C-terminus domain of HDAC6 and the N-terminus domain of HDAC11

(A) Constructs of HDAC6 coding for truncated forms of the protein carrying the FLAG epitope (top) were expressed in RAW264.7 cells (middle). Whole cell lysates were then subjected to immunoprecipitation and the presence of HDAC11 was evaluated by western blotting (bottom). (B) Constructs of HDAC11 coding for truncated forms of the protein carrying the FLAG epitope (top) were expressed in RAW264.7 cells (middle). Whole cell lysates were subjected to immunoprecipitation and the presence of HDAC6 was evaluated by western blotting (bottom). Data is from a representative experiment of two independent experiments with similar results.

Figure 4. HDAC6 is recruited to the IL10 gene promoter

Macrophages were treated with LPS (1.0 μg/mL), and then harvested at baseline (time 0) or at 0.5, 1, 2, 3, 5 and 7 hours after treatment. Cells were then subjected to ChIP analysis using antibodies against acetylated H3 (A), HDAC11 (B), or HDAC6 (C). Quantitative real time PCR analysis was performed in the region between −87 to −7 of the IL-10 gene promoter. Error bars represent standard deviation from triplicates. Shown is a representative experiment of two independent experiments with similar results. (D) Sequential chromatin immunoprecipitation (co-ChIP) of HDAC6 and HDAC11 on the IL-10 promoter. Data is from a representative experiment of two independent experiments with similar results.

Figure 5. Transcriptional activation of the IL-10 gene requires HDAC6

(A-C) HDAC6KD RAW264.7 cells or non-target shRNA control cells were treated with LPS (1.0 μg/mL) for two hours. Cells were then subjected to ChIP analysis using antibodies against HDAC6, HDAC11, and acetylated H3. Data is from a representative experiment of two independent experiments with similar results. (D) PEM isolated from HDAC11KO or wild-type mice were treated with LPS (1.0 μg/mL) for 2 hours. Then, cells were subjected to ChIP analysis using antibodies against acetylated H3, and HDAC6. (E) PEM from WT or HDAC11KO mice were transiently infected with a lentivirus carrying shRNA specific for HDAC6 and were stimulated with LPS (1.0 μg/ml) for 24 hours. The production of IL-10 protein was then determined by ELISA. Data is from a representative experiment of three independent experiments with similar results.

Figure 6

Representative scheme of the sequential recruitment of HDAC6 and HDAC11 to the IL-10 promoter gene. (A) A Initial recruitment of HDAC6 and HDAC11 to the IL-10 promoter upon stimulation. Transcriptional activity of the promoter is minimal. (B)Maximal transcriptional activity of the IL-10 promoter characterized by a decrease recruitment of HDAC11. Other transcription factors uncharacterized are noted as question marks.