Molecular Immunology 60 (2014) 44–53

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Divergent roles of histone deacetylase 6 (HDAC6) and histone deacetylase 11 (HDAC11) on the transcriptional regulation of IL10 in antigen presenting cells Fengdong Cheng a,b,1 , Maritza Lienlaf a,b,1 , Patricio Perez-Villarroel a,b , Hong-Wei Wang a,b , Calvin Lee d,e , Karrune Woan a,b , David Woods a,b , Tessa Knox a,b , Joel Bergman f , Javier Pinilla-Ibarz a,b , Alan Kozikowski e,f , Edward Seto c , Eduardo M. Sotomayor a,b,∗ , Alejandro Villagra a,∗ a

Department of Immunology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States Department of Malignant Hematology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States Department of Molecular Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States d All Children’s Research Institute, All Children’s Hospital-Johns Hopkins Medicine, St. Petersburg, FL, United States e Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, United States f Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, IL, United States b c

a r t i c l e

i n f o

Article history: Received 26 November 2013 Received in revised form 17 February 2014 Accepted 25 February 2014 Keywords: HDAC6 HDAC11 Antigen presenting cells Tolerance IL-10

a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Antigen presenting cells (APCs) play a central role in the induction of T-cell activation as well as T-cell tolerance (Rabinovich et al., 2007). IL-10, a cytokine with immunosuppressive properties, has been shown to be critical in the generation of APCs with tolerogenic properties (Grütz, 2005; Wakkach et al., 2003) and in the prevention of self-tissue damage (Li and Flavell, 2008; Murai et al., 2009; Rubtsov et al., 2008). As such, a better understanding of the regulation of this cytokine in APCs might unveil novel molecular targets

∗ Corresponding authors at: 12902 Magnolia Drive, FOB-3 Room 5.3125, United States. Tel.: +1 813 745 1387; fax: +1 813 745 7264. E-mail addresses: Eduardo.Sotomayor@moffitt.org (E.M. Sotomayor), Alejandro.Villagra@moffitt.org (A. Villagra). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2014.02.019 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

to tip the balance of an immune response towards either tolerance or immune activation. A major regulatory mechanism for IL-10 production occurs at the transcriptional level, and it is dictated by negative and positive feedback loops involving several transcriptional regulators and signaling pathways that are cell-type specific. Important transcriptional regulators of il-10 include STAT3, Sp1, AP-1, NF␬B, C/EBP␤, and GATA3 (Saraiva and O’Garra, 2010). While some of them are required for transcriptional activation of the il-10 gene (STAT3, Sp1) others (PU.1) exert an opposite effect. The molecular mechanism(s) dictating the balance between these divergent pathways remain to be fully elucidated (Saraiva and O’Garra, 2010). Recent studies have demonstrated that in addition to genetic regulation, epigenetic modifications of specific genes influences the inflammatory status of the APC and T-cell activation versus T-cell tolerance (Medzhitov and Horng, 2009; Woan et al., 2012). Histone acetyl transferases (HATs) and histone deacetylases (HDACs)

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mediate chromatin modification by acetylation and deacetylation of histone tails respectively, a well-known mechanism of transcriptional regulation in the inflammatory response (Foster et al., 2007). Along these lines, important changes in chromatin have been observed during the activation of the il-10 gene promoter, including acetylation of specific promoter regions (Villagra et al., 2009; Zhang et al., 2006). HDACs are enzymes that are recruited by co-repressors or by multi-protein transcriptional complexes to gene promoters where they regulate gene expression through chromatin modifications (de Ruijter et al., 2003; Yang and Seto, 2008). Recently, by over-expressing or knocking down specific HDACs in murine and human APCs we found that among all the members of this family of enzymes, the primarily nuclear HDAC11 (Gao et al., 2002) is recruited to the il-10 gene promoter to negatively regulate its expression (Villagra et al., 2009). Those earlier studies also suggested that another member of this family, HDAC6, which is primarily found in the cytoplasm, might exert an opposite effect to that of HDAC11 upon il-10 gene transcriptional activity. These divergent effects led us to explore whether a “cross-talk” or interaction might exist between these two HDACs. Here we have shown that unlike HDAC11, which is a transcriptional repressor of il-10, HDAC6 is required for il-10 gene transcriptional activation in APCs. Furthermore, we have found that these two HDACs physically interact with each other in the cytoplasm and nuclei of APCs. The

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2. Results 2.1. Genetic disruption of HDAC6 inhibits IL-10 production in macrophages and dendritic cells In previous studies we have shown that overexpression of HDAC11 in murine and human APCs resulted in decreased il-10 gene activation in response to LPS stimulation. In contrast, overexpression of HDAC6 was associated with increased il-10 gene transcriptional activity in the same cells (Villagra et al., 2009). In order to better define the role of HDAC6 in the regulation of il-10 transcriptional activity, we next examined the production of IL-10 in APCs in which HDAC6 was genetically disrupted. By using lentiviral particles carrying specific shRNA for HDAC6, we knocked down HDAC6 in the macrophage cell line RAW264.7 (HDAC6KD). As shown in Fig. 1A, wild type (WT) cells and control cells stably transfected with non-target shRNA (NT) produced equal amounts of IL-10 in response to LPS (Fig. 1A, WT or NT, LPS+). In contrast, HDAC6KD cells were unable to produce this cytokine in response

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additional demonstration that il-10 gene expression is abrogated in the absence of HDAC6, but not rescued upon additional knockdown of HDAC11, points to HDAC6 as the “driver” within this molecular complex and as a such a potential therapeutic target to disrupt the tolerogenic properties of IL-10 on the APC.

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Fig. 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 ␮g/ml) for 24 h. IL-10 production was then determined by ELISA. (B) RAW264.7 cells were transduced in vitro with lentiviral particles containing HDAC6-specific shRNA (HDAC6KD) or adenovirus carrying GFP-HDAC6 (HD6). Forty-eight hours later cells were treated with or without LPS (1 ␮g/ml) for additional 2 h. Then, total RNA was isolated and expression of IL-10 and GAPDH was measured by quantitative real-time RT-PCR. Three experiments were performed with similar results. Error bars represent standard deviation from triplicates. (C) Peritoneal elicited macrophages (PEM) and bone marrow derived DCs isolated from wild type or HDAC6KO mice were stimulated with LPS (1 ␮g/ml) in vitro for 24 h. IL-10 production was then determined by ELISA. Three experiments were performed with similar results. Error bars represent standard deviation from triplicates of these three experiments. (D) RAW264.7 cells were transduced in vitro with lentiviral particles containing HDAC11-specific shRNA (HDAC11KD) or adenovirus carrying GFP-HDAC11 (HD11). Forty-eight hours later cells were treated with or without LPS (1 ␮g/ml) for additional 2 h. Then, total RNA was isolated and expression of IL-10 and GAPDH was measured by quantitative real-time RT-PCR. Three experiments were performed with similar results. Error bars represent standard deviation from triplicates. (E) C57BL/6 and HDAC6KO mice were injected with LPS (10 ␮g/ml) given i.p. Serum from those mice were collected on 0 (no LPS), 6, and 24 h after LPS injection. IL-10 productions were determined by ELISA. Two experiments were performed with similar results.

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to LPS stimulation (Fig. 1A, HDAC6KD, LPS+). These differences in IL-10 protein production can be directly attributed to changes at the transcriptional level, since decreased expression of il-10 mRNA was observed in HDAC6KD macrophages (Fig. 1B, HDAC6KD). To further elucidate the role of HDAC6 over il-10 transcriptional activity we next over-expressed HDAC6 in macrophages. Such a manipulation resulted in, a significant increase in IL-10 production in response to LPS stimulation (Fig. 1B, HD6). The above results were confirmed in peritoneal elicited macrophages (PEM) and dendritic cells (DC) isolated from HDAC6 knock out (HDAC6 KO) mice (Zhang et al., 2008). As shown in Fig. 1C, PEM and DCs devoid of HDAC6 produced significantly less IL-10 in response to LPS as compared to APCs from WT mice. In order to confirm the antagonist role of HDAC11 over IL-10 production, we evaluated the production of this cytokine in macrophages in which HDAC11 was either overexpressed (Fig. 1D, HD11) or knocked-down (Fig. 1D, HDAC11KD) As previously reported (Villagra et al., 2009) il-10 mRNA expression was decreased when HDAC11 was overexpressed (Fig. 1D, HD11) and increased in HDAC11KD cells (Fig. 1D, HDAC11KD). Shown in Supplementary Fig. 1 are the respective controls for overexpression and knocking-down of HDAC6 and HDAC11. To assess whether HDAC6 is also required for IL-10 production in vivo, we treated HDAC6 KO mice and control mice with LPS and determined at different time points the levels of IL-10 in serum of treated mice. As shown in Fig. 1E there was significantly less IL-10 in the serum of HDAC6 KO mice as compared to their wild type counterparts, indicating the need for this HDAC in host’s cells for an effective IL-10 production in vivo. 2.2. HDAC6 physically interacts with HDAC11 in both the cytoplasmic and nuclear compartments The differential intracellular localization of HDAC11 and HDAC6, along with the prevalent view that HDAC6 lacks nuclear function (Boyault et al., 2007), made it unlikely that a physical interaction between these HDACs could explain their effects upon il-10 gene expression in APCs. However, protein extracts from murine RAW264.7 cells and the human promonocytic cell line THP.1 demonstrated that endogenous HDAC6 coimmunoprecipitated with HDAC11 (Fig. 2A). Similarly, in primary peritoneal macrophages (PEM) we identified that endogenous HDAC11 and HDAC6 are co-immunoprecipitated by using antiHDAC6 or anti-HDAC11 antibodies (Fig. 2B). To confirm these findings, we performed next confocal microscopy studies to visualize whether HDAC6 and HDAC11 co-localize within intact cells. First, a RAW264.7 cell line over expressing FLAG-HDAC11 was generated to detect the FLAG epitope using an anti-FLAG antibody and a secondary antibody conjugated to the fluorochrome Alexa Fluor 488 (green). As shown in Fig. 2C (F-HDAC11 panel), HDAC11 is localized in both the cytoplasm and nuclei. Detection of HDAC6 by using a HDAC6 antibody and a secondary antibody conjugated to the fluorochrome Alexa Fluor 647 (red), demonstrated that this HDAC is localized primarily in the cytoplasm, although some minimal expression was also observed in the nuclei (Fig. 2C, HDAC6 panel). Of note, the merged image shows that HDAC6 and HDAC11 mainly co-localize in the cytoplasm, although some co-localization was also detected in the nucleus (Fig. 2C-Merge). Finally, in order to further validate the above results, we performed co-immunoprecipitation studies in cytoplasmic (Cyt) and nuclear (Nuc) extracts prepared from FLAG-HDAC11 expressing macrophages. First, immunoblots of these extracts confirmed that HDAC11 is present in both fractions (Fig. 2D, top panel). Although HDAC6 was clearly detected in the cytoplasm, a faint band could also be detected in the nuclear fraction (Fig. 2D, ␣HDAC6). AntiHDAC1 and anti-tubulin antibodies were used as controls for nuclear and cytoplasmic localization respectively (Fig. 2D, ␣HDAC1

and ␣Tubulin). Second, by immunoprecipitating FLAG-HDAC11 in both the cytoplasmic and nuclear fractions (Fig. 2E), we found that HDAC6 interacts with HDAC11 in both cellular compartments. Finally, we determined whether the deacetylase activity of HDAC6 is required for its physical interaction with HDAC11 and IL-10 production in macrophages. We therefore over-expressed a Flag-tagged mutant version of HDAC6 lacking deacetylase activity (Flag-HDAC6 H216/611A) (Zhang et al., 2007) in RAW264.7 cells. As shown in Supplementary Fig. 2A, the deacetylase activity of HDAC6 is not necessary for its association with HDAC11. However, it seems to be required for il-10 gene expression, since minimal il-10 mRNA was observed in macrophages over-expressing FlagHDAC6 H216/611A (Supplementary Fig. 2B). 2.3. The N-terminal domain of HDAC11 and the C-terminal domain of HDAC6 are required for their physical interaction in macrophages Next, we investigated which domains of HDAC6 and HDAC11 are necessary for their putative physical interaction in vivo. We utilized constructs of FLAG-tagged HDAC6 and HDAC11 containing various deletions. For HDAC6 we used two constructs, HD6(1–840) and HD6(1–503), both lacking the C-terminal domain (Fig. 3A, top). These truncated and full-length HDAC6 constructs were overexpressed in RAW264.7 cells. Fig. 3A (middle) depicts the electrophoretic migration of these HDAC6 proteins on a Western blot (WB) probed with FLAG antibody. Immunoprecipitation (IP) with anti-FLAG antibody followed by immunoblot with anti-HDAC11 antibody demonstrates that HDAC11 coimmunoprecipitated with the full-length HDAC6 protein (Fig. 3A, bottom, 1–1215), but not with HDAC6 proteins lacking the C-terminal domain (Fig. 3A, bottom, 1–840 or 1–503), indicating that this domain is required for its physical interaction with HDAC11. Conversely, to determine which domain of HDAC11 is required for its interaction with the C-terminal domain of HDAC6, we generated three HDAC11 mutants: HD11(17–321) that lacks both the N- and C-terminal domains, HD11(66–347) that lacks only the Nterminal domain, and HD11(1–264) that lacks only the C-terminal domain (Fig. 3B, top). These mutants and full-length FLAG-tagged HDAC11(1–347) constructs were then overexpressed. The antiFLAG blot in Fig. 3B (middle) verified the expression of the expected molecular weight HDAC11 proteins. Immunoprecipitation of these HDAC11 species with anti-FLAG antibody followed by WB with HDAC6 antibody clearly indicated that HDAC6 only coimmunoprecipitated with HDAC11 proteins with an intact N-terminal domain (1–347 and 1–264 in Fig. 3B, bottom). In the absence of a N-terminal domain, no HDAC6 coimmunoprecipitated with FLAG-HDAC11 (Fig. 3B, bottom, 17–321 and 66–347 constructs), suggesting that the N-terminal domain of HDAC11 is necessary for its interaction with HDAC6. 2.4. HDAC6 and HDAC11 are simultaneously recruited to the IL-10 gene promoter The above data demonstrates that HDAC6 and HDAC11 interact with each other in macrophages. Furthermore, these two HDACs exert an opposite effect upon il-10 gene expression, with HDAC6 being required for IL-10 production and HDAC11 acting as a repressor (Fig. 1). To better understand the mechanism by which these HDACs regulate il-10 gene transcriptional activity, we examined whether endogenous HDAC6 and/or HDAC11 are recruited to the il-10 gene promoter. Previously we have reported the sequence of chromatin modifications that occur at the level of the il-10 gene promoter in macrophages stimulated with LPS (Villagra et al., 2009). Briefly, phosphorylation of Ser10 on H3 is an early event that occurs within

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Fig. 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 antibodies. The IP fraction was then subjected to WB to evaluate for the presence of HDAC6 and HDAC11 on each condition. (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 murine macrophage RAW264.7 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. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

30 min after LPS stimulation. This is followed by increased acetylation of H3 and H4, subsequent recruitment of the transcriptional activator STAT3, and, ultimately, il-10 gene expression. Therefore, we evaluated the kinetics of HDAC6 and HDAC11 recruitment to sequential segments across the il-10 gene promoter up to 2 kb upstream in the gene promoter by chromatin immunoprecipitation (ChIP) analysis. We observed recruitment of HDAC6 and HDAC11 to only two regions of the promoter, assigned arbitrarily as proximal (−87 to −7) and distal (−1322 to −1223) regions. Analysis of the proximal region revealed that acetylation of H3 peaked at 30 min followed by a progressive decline by 2 h and a return to baseline by 7 h after LPS stimulation (Fig. 4A). Analysis of the kinetics of HDAC6

recruitment to the il-10 gene promoter has shown that its recruitment started within the first hour and achieved a first peak by 2 h. This was followed by a slight decrease at 3 h and a second (maximum) peak at 5 h post-stimulation (Fig. 4B). The recruitment of HDAC11 was also observed within 1 h, reaching a plateau by 2 h and returned to baseline levels at 5 h post-stimulation (Fig. 4C). Analysis of the distal il-10 gene promoter demonstrated that HDAC6 and HDAC11 are also recruited to this region of the gene promoter. Of note, the kinetics of recruitment seems to be similar with HDAC11 having a peak recruitment by 2 h and HDAC6 by 5 h post LPS-stimulation (Supplementary Fig. 1). We also performed a time course analysis of il-10 mRNA expression following LPS

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Fig. 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.

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Fig. 4. HDAC6 is recruited to the IL10 gene promoter. Macrophages were treated with LPS (1.0 ␮g/ml), and then harvested at baseline (time 0) or at 0.5, 1, 2, 3, 5 and 7 h 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 and −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) RAW264.7 cells were treated with LPS (1 ␮g/ml) and total RNA was isolated at the indicated times. Expression of IL-10 and GAPDH were measured by quantitative real-time RT-PCR. Three experiments were performed with similar results. Error bars represent standard deviation from triplicates. (E) 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.

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Fig. 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 ␮g/ml) for 2 h. 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 ␮g/ml) for 2 h. 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 ␮g/ml) for 24 h. The production of IL-10 protein was then determined by ELISA. Error bars represent standard deviation from triplicates of these three experiments.

stimulation. As shown in Fig. 4D, the expression of il-10 mRNA increases 2 h after LPS stimulation and remains relatively constant until 7 h post stimulation, when its expression began to decrease to reach baseline levels around 12 h post-stimulation. The findings that HDAC6 and HDAC11 were both recruited to the il-10 gene promoter led us to explore whether they might be recruited to the same DNA sequence within the promoter. We therefore performed co-chromatin immunoprecipitation (co-ChIP) for HDAC6 and HDAC11. Co-ChIP is a two-step sequential chromatin immunoprecipitation, where the first antibody is directed against HDAC11 and the second antibody against HDAC6, and viceversa (Villagra et al., 2006). As shown in Fig. 4E, both deacetylases were recruited to the il-10 gene promoter in response to LPS stimulation (black bars, ChIP␣HDAC11 and ChIP␣HDAC6, respectively). Following this first ChIP, and after proper pre-clearance, samples were subjected to a second immunoprecipitation with antibody against the other HDAC. Regardless of the order of HDAC antibodies, both co-ChIP approaches demonstrated that HDAC11 and HDAC6 are recruited to the same DNA sequence within the il-10 gene promoter (Fig. 4E, black bars, ChIP␣HDAC11, co-ChIP ␣HDAC6 and ChIP␣HDAC6, co-ChIP ␣HDAC11, respectively). This finding demonstrates a concerted recruitment of HDAC6 and HDAC11 to the same region of the il-10 gene promoter.

2.5. HDAC6, but not HDAC11, is required for transcriptional activation of the IL-10 gene promoter in APCs To better define the contribution of these HDACs in regulating il-10 gene transcriptional activity we next evaluated the chromatin changes in the il-10 gene promoter when HDAC6, HDAC11, or both are depleted. First, we examined the changes in RAW264.7 cells in which HDAC6 was knocked down. As expected, minimal HDAC6 recruitment to the il-10 gene promoter in response to LPS was found in HDAC6KD cells as compared to non-target shRNA control cells (Fig. 5A, gray bar versus open bar). In the absence of HDAC6, there was a slight increase in HDAC11 recruitment to the il-10 gene promoter, but this was not significantly different than the levels observed in non-target shRNA control cells (Fig. 5B). Notably, in HDAC6KD cells there was a significant decrease in the acetylation of H3 at the level of the il-10 gene promoter (Fig. 5C). A similar decrease in H3 acetylation and HDAC6 recruitment to the il-10 gene promoter was observed in PEM treated with Tubastatin A, an isotype-selective HDAC6 inhibitor (Kozikowski et al., 2008) (Supplementary Fig. 4A). Reminiscent of our findings in HDAC6KD or

HDAC6KO APCs, a decrease in IL-10 protein production was also observed in PEM from wild-type mice treated with Tubastatin A in vitro (Supplementary Fig. 4B). Previously, we have shown the chromatin modifications that occur at the level of the il-10 gene promoter in macrophages in which HDAC11 was knocked down (Villagra et al., 2009). In those studies, we did not determine whether HDAC11 manipulation influences the recruitment of HDAC6 to the il-10 gene promoter. As shown in Fig. 5D, primary macrophages isolated from HDAC11KO mice have increased H3 acetylation. However, HDAC6 was still recruited to the il-10 gene promoter in cells lacking HDAC11. The above results suggest that in the absence of the other interacting partner, HDAC11 or HDAC6 can still be recruited to the il-10 gene promoter. However, in HDAC6KD or KO cells in which HDAC11 was over-expressed, no IL-10 production was observed in response to LPS (Fig. 1), whereas in HDAC11 KD or KO cells in which HDAC6 is present, an enhanced IL-10 production was observed (Villagra et al., 2009). We therefore hypothesized that if HDAC6 is the “driver” for IL-10 production within this molecular complex, knocking down HDAC6 should abrogate the enhancement in IL-10 production in macrophages already lacking HDAC11. Fig. 5E shows the LPS-induction of IL-10 production by primary macrophages and those lacking each or both HDACs. Unlike wild-type cells (white bar), HDAC6KD (gray bar) do not produce IL-10 in response to LPS. In sharp contrast, macrophages lacking HDAC11 display an enhanced production of IL-10 in response to LPS (black bar). However, this enhancement was no longer present in macrophages lacking both HDAC11 and HDAC6, indicating that the transcriptional activation of the il-10 promoter that occurs in the absence of HDAC11 is dependent upon the presence of HDAC6.

3. Discussion In this study we have clearly established that HDAC6 associates with HDAC11 to control IL-10 production by APCs. Prior studies have shown that HDAC11 and HDAC6 can interact at the same time with the Vitamin D3 Receptor (VDR) to regulate the expression of MYC (Toropainen et al., 2010). Furthermore, by over-expressing FLAG-tagged versions of HDAC11 in 293 cells, Gao et al. have shown that HDAC6 can be co-immunoprecipitated with FLAG-HDAC11 (Gao et al., 2002). However, our study is the first to demonstrate that endogenous HDAC6 and HDAC11 physically interact with each other in the cytoplasm and nuclei of APCs and are recruited to the il-10 gene promoter to regulate its transcriptional activity.

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Fig. 6. Representative scheme of the sequential recruitment of HDAC6 and HDAC11 to the IL-10 promoter gene.

The relevance of our discovery lies on several levels. First, unlike HDAC11 which is a transcriptional repressor of IL-10, HDAC6 is required for the transcriptional activation of il-10 gene expression in macrophages and dendritic cells (DCs). Second, these two HDACs with opposite effects upon IL-10 expression physically interact with each other in the APC. Third, our demonstration that HDAC6 and HDAC11 elicit opposite effects upon il-10 gene expression is the first evidence that two different HDACs can interact with the same target to dictate divergent transcriptional responses. Finally, the additional demonstration that il-10 gene expression is abrogated in the absence of HDAC6, but not rescued upon additional knockdown of HDAC11, points to HDAC6 as the “driver” within this regulatory complex. HDAC6 therefore represents a potential novel target to disrupt the anti-inflammatory effects of IL-10 upon the APC supporting our findings, we have also shown that treatment of macrophages with the HDAC6 selective inhibitor Tubastatin A decreases IL-10 mRNA and protein production by these cells. Furthermore, we have recently found that Tubastatin A-treated APCs are better activators of antigen-specific T-cells in both in vitro and in vivo studies (data not shown). HDAC6 is a 131 kDa protein predominantly found in the cytoplasm where it regulates the acetylation of multiple proteins, including tubulin, Hsp90, and cortactin (Valenzuela-Fernández et al., 2008). HDAC6 has been primarily recognized as a key regulator of cytoskeleton, cell migration and cell–cell interactions (Aldana-Masangkay and Sakamoto, 2010). However, emerging evidence also implicates this protein in the regulation of immune responses, in particular at the level of the APC/T-cell immune synapse (Serrador et al., 2004) and in the suppressive function of regulatory T-cells (de Zoeten et al., 2011). Although these functions of HDAC6 may not be exclusively due to the deacetylation of histones, it has been named according to its homology to other HDACs and its weak ability to deacetylase histones in in vitro assays (Todd et al., 2010). Here, we have expanded upon the previously described properties of HDAC6 showing that it forms a complex with HDAC11 that is detected in both the cytoplasm and nuclear compartments. This interaction is mediated by the carboxyterminus of HDAC6 and the aminoterminus of HDAC11. In response to LPS stimulation, HDAC6 and HDAC11 are both recruited to the same DNA region within the IL-10 gene promoter, an effect that occurs within the first 2 h of LPS stimulation. However, 5 h after LPS stimulation, a divergent outcome is observed with HDAC6 being retained at the promoter region and HDAC11 disappears from the surrounding chromatin. Show in Fig. 6 is a model summarizing these findings. Notably, individual knockdowns of HDAC6 or HDAC11 demonstrated that each deacetylase can still be recruited to the il-10

gene promoter without its interacting partner, suggesting that their nuclear translocation might be independently controlled, perhaps by transcription factors involved in il-10 gene regulation. HDACs are widely known as potent negative regulators of transcription. Therefore, we are left to explain the unexpected role of HDAC6 as a transcriptional activator of IL-10. Since HDAC6 has minimal effects on histone deacetylation, it is plausible that the observed outcome might be due to the effects of HDAC6 over nonchromatin transcriptional regulators. For instance, HDAC6 might be an active member of the local chromatin composition of the IL-10 gene promoter via the deacetylation of transcriptional regulators of IL-10. We are currently exploring whether the several known transcriptional regulators of il-10 could be putative targets of HDAC6. Indeed, in recent preliminary experiments we have found that STAT3 and PU1 interact with HDAC6 (data not shown). Of note, gene array analysis of macrophages in which HDAC6 was knocked down showed that several STAT3-target genes such as Bclxl, Cmyc, Socs3, Vegf and il-6 are among the most down regulated. Needless to say, we are currently evaluating whether the two identified transcription factors also interact with HDAC11 and if knocking down this HDAC might also affect the expression of STAT3 target genes. It is plausible therefore that this dual HDAC6/HDAC11 complex might participate in the regulation of other immune-related genes beyond their regulatory role upon Il-10 gene expression. In summary, we have unveiled a previously unknown mechanism for the transcriptional regulation of IL-10 that involves HDAC6 and HDAC11. Their dynamic interaction and the dynamic changes in the expression of IL-10 might explain the intrinsic plasticity of the APC in influencing T-cell activation versus T-cell tolerance.

4. Methods 4.1. Mice Male BALB/c or C57BL/6 mice (6- to 8-weeks old) were obtained from the National Institutes of Health (Frederick, MD). HDAC6 KO mice (H-2b ) were kindly provided by Dr. P. Matthias (Zhang et al., 2008) (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland). HDAC11KO mice were provided by Dr. Edward Seto (H. Lee Moffitt, Tampa, FL). Experiments involving the use of mice were performed in accordance with protocols approved by the Animal Care and Use Committee of the University Of South Florida College Of Medicine.

F. Cheng et al. / Molecular Immunology 60 (2014) 44–53

4.2. Cell lines

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The macrophage cell line RAW264.7 and THP1 cells have been described previously (Vicente-Suarez et al., 2007; Villagra et al., 2009). Cells were cultured in vitro in RPMI 1640 media, supplemented with 10% FCS, penicillin/streptomycin (50 U/ml), lglutamine (2 mM), and ␤-mercaptoethanol (50 ␮M), and grown at 37 ◦ C and 5% CO2 .

hours later, cells expressing each protein variant were lysed and the respective recombinant proteins purified by anti-FLAG affinity columns. Cell extracts isolated from PEM or BMDM were incubated with the FLAG-tagged proteins previously purified. Protein complexes were immunoprecipitated using anti-FLAG-agarose beads (Sigma A-2220). Immune complexes were eluted using 200 ␮g/ml FLAG peptide (Sigma F-3290) in whole-cell extract buffer and analyzed by immunoblot.

4.3. Isolation of macrophages and dendritic cells

4.7. Confocal studies

C57BL/6 (H-2b ), HDAC11KO (H-2b ) or HDAC6 KO (H-2b ) mice were injected intraperitoneally (i.p.) with 1 ml of thioglycollate (DIFCO Laboratories, Detroit, MI). Four days later, peritoneal elicited macrophages (PEM) were isolated by peritoneal lavage as previously described (Sotomayor et al., 1991). Dendritic cells (DCs) were generated from murine bone marrow using RPMI 1640 medium supplemented with 10% FCS, 20 ng/ml murine recombinant GMCSF, and 10 ng/ml IL-4 (both from RDI, Flanders, NJ). The cultures were maintained at 37 ◦ C in 5% CO2 humidified atmosphere. On day 3 of culture, floating cells were gently removed and fresh medium with cytokines was replaced. On day 5, cells were collected and DCs were enriched by centrifugation on metrizamide gradient (Accurate Chemicals, Westbury, NY).

RAW264.7 cells were subjected to immunofluorescent staining using primary antibodies against HDAC6 (1:200) and FLAG (1:25), secondary antibodies Alexa Fluor 488 donkey anti-mouse IgG (1:200) and Alexa Fluor 594 donkey anti-rabbit IgG (1:200), respectively. Samples were viewed with a Leica DMI6000 inverted microscope, TCS SP5 confocal scanner, and a 63X/1.40NA Plan Apochromat oil immersion objective (Leica Microsystems GmbH, Wetzlar, Germany). 405 Diode, Argon, and HeNe 594 laser lines were applied to excite the samples and a tunable AOBS was adjusted to minimize crosstalk between fluorochrome emissions. Gain, offset, and pinhole settings were identical for all samples within the treatment group. Images were captured with photomultiplier detectors and prepared with the LAS AF software version 2.5.0 (Leica Microsystems GmbH, Wetzlar, Germany).

4.4. Reagents 4.8. Quantitative real time (qRT)-PCR analysis LPS (Escherichia coli 055:B5, catalog number L-2880) and shRNA lentiviral particles were purchased from Sigma–Aldrich (St. Louis, MO). Lipofectamine 2000 and Lipofectamine LTX were from Invitrogen (Carlsbad, CA). HDAC6 mutants were kindly provided by Dr. Zhang’s lab at University of South Florida (Zhang et al., 2007) and HDAC11 mutants were generated in Dr. E. Seto’s lab (Glozak and Seto, 2009). 4.5. Antibodies and immunoblotting Anti-HDAC6 (C0226) antibody was purchased from Assay Biotech. Anti-FLAG (F1804) antibody was from Sigma. Anti-GAPDH (sc-25778), anti-Tubulin (sc-32293), anti-HDAC1 (sc-7872) and anti-acetylated tubulin (sc-23950) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-acetylated H3 (06599) antibody was purchased from Millipore (Billerica, MA). Three different antibodies against HDAC11 were used; ab47036 from ABCAM (Cambridge, MA), ab3611 from Biovision (Milpitas, CA) for immunoblot and abH4539 from Sigma for ChIP analysis. 4.6. Overexpression and knocking-down experiments Already validated shRNA lentiviral transduction particles for murine HDAC11 (NM144919, TRCN0000039224), murine HDAC6 (NM010413, TRCN0000008415), and non-target shRNA (SHC002V) were all from Sigma. Sequences for their respective targets are available from the company (Sigma–Aldrich, St. Louis, MO). For transduction of lentiviral particles for shRNA HDAC6 and HDAC11, we followed the protocol provided by the manufacturer using a final MOI of 75. Lentiviral particles for the non-target control were a single random sequence not present in the human or mouse genome. We generated at least three different stable cell lines for HDAC6 and HDAC11 following the instructions provided by the manufacturer (Sigma), and Western blots were performed to evaluate protein expression. The over-expression of full length mutants for HDAC6 (reported previously by Dr. Seto, 2007) and HDAC11 were performed in macrophages RAW264.7 cells transfected with plasmids encoding for FLAG-tagged version of the respective proteins. Forty-eight

Macrophage cell line, primary murine macrophages or DCs were plated at 2 × 106 cells per 35 mm well and cultured under conditions detailed under each experiment. Total RNA was extracted using TriZol reagent (Invitrogen, NY) and cDNA obtained with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Target mRNA was quantified using MyIQ single color real time PCR detection system (Bio-Rad) and iQ SYBR green Supermix (Bio-Rad, Hercules, CA). Mouse IL-10 (left, 5 -CAGGGATCTTAGCTAACGGAAA3 ; right, 5 -GCTCAGTGAATAAATAGAATGGGAAC-3 ) were used for PCR amplification (cycling parameters 3 min 95 ◦ C, 15 s 95 ◦ C, 30 s 60 ◦ C 40 reps, 1 min 95 ◦ C). Single product amplification was confirmed by melting curve analysis and primer efficiency was near 100% in all the experiments performed. Quantification is expressed in arbitrary units and target mRNA levels were normalized to GAPDH expression using the method of Pfaffl (2001). 4.9. Chromatin immunoprecipitation (ChIP) assays ChIP studies were performed as previously described (Villagra et al., 2009). All steps were carried out at 4 ◦ C. RAW264.7 or PEM were incubated for 10 min with 1% formaldehyde under gentle agitation. Cross-linking was stopped by the addition of 0.125 M glycine for 5 min. Then, cells were washed with 10 ml of PBS, scraped off in the same volume of PBS, and collected by centrifugation at 1000 × g for 5 min. The cell pellet was re-suspended in lysis buffer (50 mM HEPES [pH 7.8], 20 mM KCl, 3 mM MgCl2 , 0.1% NP40, and a cocktail of protease inhibitors) and incubated for 10 min on ice. The cell extract was collected by centrifugation at 1000 × g for 5 min, resuspended in sonication buffer (50 mM HEPES [pH 7.9], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, and a cocktail of protease inhibitors), and incubated for 10 min on ice. To reduce the length of the chromatin fragments to approximately 500 bp (confirmed by electrophoretic analysis and PCR), the extract was sonicated using a BioruptorTM (Diagenode). Eight pulses of 30 s with 30 s rest time were repeated 6 times at a frequency of 20 kHz. After centrifugation at 16,000 × g, the supernatant was collected, frozen in liquid nitrogen, and kept at −80 ◦ C. An aliquot was used for A260 measurements. Cross-linked extracts (6 U A260) were

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resuspended in a sonication buffer to a final volume of 500 ␮l. The samples were pre-cleared by incubation with normal mouse IgG plus protein A/G agarose (Santa Cruz Biotechnology) for 2 h at 4 ◦ C with agitation. After centrifugation at 1000 × g for 5 min, the supernatant was collected and immunoprecipitated with specific antibodies as detailed for each experiment. The immunocomplexes (except from Flag/agarose conjugates) were recovered with the addition of 30 ␮l of protein-A agarose beads and subsequent incubation for 2 h at 4 ◦ C with agitation. The complexes were washed twice with sonication buffer plus 500 mM NaCl, twice with LiCl buffer (20 mM Tris–HCl [pH 8.0], 250 mM LiCl, 1 mM EDTA, and 0.5% Triton X-100), and twice with TE buffer (2 mM EDTA and 50 mM Tris–HCl [pH 8.0]). The solution was incubated for 5 min at 4 ◦ C. The protein–DNA complexes were eluted by incubation with 100 ␮l of elution buffer (50 mM NaHCO3 and 1% SDS) for 15 min at 65 ◦ C. After centrifugation at 1000 × g for 5 min, the supernatant was collected and incubated with 10 ␮g of RNase A per ml for 1 h at 42 ◦ C. NaCl was added to the mixture to a final concentration of 200 mM and incubated at 65 ◦ C to reverse the cross-linking. The proteins were digested with 200 ␮g/ml of proteinase K for 2 h at 50 ◦ C. The DNA was recovered using Qiagen QiaquickTM columns. Two different set of primers were used for IL-10: IL-10 (proximal region) sense 5 -GGAGGAGGAGCCTGAATAAC-3 and antisense 5 -CTGTTCTTGGTCCCCCTTTT-3 , and IL-10 (distal region) sense 5 -AACTCAGCCTGGAACTGACC-3 and antisense 5 -GCCTCTCCTCCTGACACTCTT-3 . All samples and inputs were quantified using MyIQ single color real time PCR detection system (Bio-Rad) and iQ SYBR green Supermix (Bio-Rad). Single product amplification was confirmed by melting curve analysis and primer efficiency was near or close to 100% in all experiments performed. Quantification is expressed in arbitrary units, and target sequence levels were normalized to the input signal using the method of Pfaffl (2001). All ChIP experiments were repeated twice starting from the crosslinking, and final quantitative real time PCR was done in triplicates. The co-chromatin immunoprecipitation (co-ChIP) for HDAC6 and HDAC11 were performed using the same experimental procedure described above adding an extra clearance step between the sequential immunoprecipitations. Briefly, the eluted fraction after the first immunoprecipitation was pre-cleared by incubation with protein A/G agarose (Santa Cruz Biotechnology) for 2 h at 4 ◦ C with agitation. After centrifugation at 1000 × g for 5 min, the supernatant was collected and immunoprecipitated using specific antibodies as detailed under each experiment.

4.10. In vivo treatment with LPS HDAC6KO as well as control mice were injected with 10 ␮g/ml of LPS given i.p. Serum were collected from those mice on 0 h (no LPS injection), 6, and 24 h after LPS injection. IL-10 production from serum were determined by ELISA.

4.11. Statistical analysis All experiments were repeated at least three times unless indicated otherwise. Unpaired t-tests were performed using Microsoft Excel Software (Microsoft, Redmond, WA) with significance at p < 0.05.

Conflict of interest The authors have no financial conflict of interest.

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