Histone deacetylase inhibition modulates indoleamine 2,3-dioxygenase–dependent DC functions and regulates experimental graft-versus-host disease in mice (original) (raw)
HDAC inhibitors regulate innate immune responses of DCs. We tested the effects of SAHA on the innate immune responses of DCs that are triggered through TLRs, cell surface molecules that recognize pathogen-associated molecular patterns (19). BM-derived DCs (BMDCs) from C57BL/6 mice (B6BMDCs) were incubated with 100 and 500 nM SAHA for 16–20 h prior to stimulation with 100 ng/ml LPS, a potent stimulus of innate immune response via TLR4. SAHA-treated DCs secreted significantly reduced amounts of TNF-α, IL-12, and IL-6 in a dose-dependent manner (Figure 1, A–C). We confirmed this effect with a second synthetic HDAC inhibitor that contains hydroxamic acid, ITF 2357. As shown in Figure 1D, pretreatment with 10 and 100 nM ITF 2357 significantly reduced the secretion of TNF-α in response to LPS. These concentrations were chosen because previous data have shown that while these agents have cytotoxic effects at micromolar concentrations, their antiinflammatory effects were observed at lower, nanomolar concentrations (8, 10, 13). Most subsequent experiments were therefore performed at the nanomolar concentrations described above.
Effect of HDAC inhibitors on LPS-induced secretion of proinflammatory cytokines by BMDCs. DCs were obtained from C57BL/6 animals as described in Methods. BMDCs were preincubated for 16–20 h with indicated concentrations of SAHA or diluent and were then stimulated overnight with LPS (100 ng/ml). Proinflammatory cytokines levels in the supernatants were measured by ELISA. (A) TNF-α levels. (B) IL-12 levels. (C) IL-6 levels. *P < 0.02 versus stimulated control DCs. (D) DCs were pretreated with the indicated concentrations of SAHA and ITF 2357 or diluent as described in A. TNF-α levels were measured in the supernatants after overnight stimulation with LPS. **P < 0.01 versus stimulated control DCs. (E) Macrophages (F4/80+) were obtained from C57BL/6 mice after peritoneal lavage and pretreated with SAHA or diluent for 14–16 h. They were then stimulated overnight with LPS, and TNF-α from the culture supernatant was measured. *P < 0.02 versus control macrophages. Data (mean ± SEM) are from 1 of 2–3 experiments with similar results. Unlabeled bar(s) at left represent unstimulated controls.
We next investigated whether HDAC inhibitors modulated LPS-mediated responses of other professional APCs, specifically macrophages. Peritoneal cells were harvested from C57BL/6 animals and normalized for F4/80 expression. The cells were incubated with diluent (sterile water), SAHA, or ITF 2357 for 16 h and then stimulated overnight with LPS. Pretreatment with SAHA significantly reduced LPS-induced secretion of TNF-α by the peritoneal macrophages (Figure 1E).
We next determined whether SAHA modulated DC responses induced by other TLR ligands. Using IL-6 production as an end point, we found that SAHA attenuated DC function after stimulation with lipoteichoic acid (LTA), a TLR4 ligand, peptidoglycan (PGN), a TLR2 ligand, the dsRNA poly(IC), a TLR3 ligand, and CpG DNA, a TLR9 ligand (Table 1) (19, 20). Similar reductions were also seen in the secretion of TNF-α by both SAHA and ITF 2357 (data not shown). Taken together, these data demonstrate that SAHA and ITF 2357 regulate DC and macrophage responses that are mediated through TLR stimulation.
Effect of SAHA on TLR ligand–mediated secretion of IL-6
HDAC inhibitors modulate allogeneic T cell–stimulatory capacity of DCs. We next determined the effects of HDAC inhibition on in vitro alloresponses by incubating BALB/c T cells with allogeneic B6BMDCs in a standard mixed leukocyte reaction (MLR) assay. As shown in Figure 2A, addition of 1 μM SAHA or more to the cultures significantly reduced T cell proliferation at 72 h by greater than 70%. IL-2 secretion in culture supernatants collected at 48 h was similarly reduced (Figure 2B).
HDAC inhibition modulates allogeneic T cell proliferation and IL-2 production in vitro. SAHA was added at the indicated concentrations to DCs isolated from C57BL/6 BM that were then immediately used as stimulators in an MLR with T cells from either BALB/c (allogeneic) or C57BL/6 (syngeneic) mice as described in Methods. (A) T cell proliferation was determined by 3H-thymidine incorporation at 72 h. Allogeneic T cell responses from control (0 μM SAHA) and SAHA-treated cultures and syngeneic T cell responses to control DCs (syn) are shown. Data are mean ± SEM of quadruplicate cultures. P = NS, control versus 0.5-μM SAHA. **P < 0.03 versus control. Results are from 1 of 3 similar experiments. (B) Supernatants of cultures with control (open symbols) or SAHA (filled symbols) were collected at 48 h, and IL-2 was measured by ELISA. Data are mean ± SEM of quadruplicate cultures. *P < 0.05 versus control. (C) B6BMDCs were pretreated with diluent or 500 nM SAHA for 16–18 h, washed, and used as stimulators with allogeneic BALB/c T cells at the indicated ratios. T cell proliferation was evaluated after 72 h of culture.
However, consistent with previous observations (9), SAHA pretreatment did not inhibit T cell responses after stimulation with anti-CD3 and anti-CD28 (Supplemental Figure 1A). Thus, we reasoned that HDAC inhibition in an MLR might primarily effect DC function. To test this directly, we pretreated BMDCs with different concentrations of SAHA for 16–20 h and then washed them thoroughly prior to use as stimulators in an MLR. As shown in Figure 2C and Figure 3, A and B, pretreatment of DCs at SAHA concentrations of 500 nM or greater significantly reduced allogeneic T cell proliferation, even with increasing the responder/stimulator ratios (Figure 2C), and also reduced IL-2 secretion by nearly 50% (Figure 3B). Similar effects were seen upon treatment with ITF 2357 (data not shown). Because cells derived from BM cultures are heterogeneous (21, 22), we purified CD11c+ DCs by fluorescence-activated cell sorting (FACS) from both C57BL/6 and BALB/c BM cultures (>98% CD11c+ DC purity) and found that SAHA had similar regulatory effects on purified DCs (62,198 ± 7,418 cpm versus 29,935 ± 1,1656 cpm, P < 0.02). In contrast, levels of TGF-β and IL-10 in MLR supernatants were unchanged (Supplemental Figure 1, B and C), and their neutralization with anti–TGF-β or anti–IL-10 mAb did not restore T cell proliferation in MLRs with SAHA-treated DCs (Figure 3, C and D). Expression of T cell activation markers, such as CD25 and CD69, was also reduced, with no increase in CD152 expression (data not shown).
HDAC inhibitors regulate in vitro and in vivo functions of DCs. B6BMDCs were pretreated with diluent or SAHA and used as stimulators in MLR cultures as described in Methods. (A) BALB/c T cell proliferation after 72 h of culture. (B) IL-2 levels in supernatants at 48 h of culture. P = NS, control versus 0.1 μM SAHA. *P < 0.02, **P < 0.01 versus control. (C and D) Addition of (C) anti–TGF-β or (D) anti–IL-10 to SAHA-treated DC cultures did not reverse SAHA-treated DC–mediated suppression. P = NS, SAHA-treated versus control DCs. *P < 0.05 versus control. (E) Naive BALB/c T cells, or those obtained after 48 h of culture with C57BL/6 (B6) DCs pretreated with 0.5 μM SAHA, were restimulated in secondary cultures with control C57BL/6 DCs either separately or together at a 1:1 ratio. T cells cultured from primary SAHA-treated DCs were restimulated with third-party C3H/HeJ BMDCs. P = NS for all between-group comparisons. Data are mean ± SEM of quadruplicate cultures. (F and G) Cd74–/– and HLA-G–/– animals were irradiated and transplanted as described in Methods. Syngeneic (n = 3–4) and some allogeneic animals (n = 4–5) received 4 × 106 to 5 × 106 control B6BMDCs, while some allogeneic recipients (n = 5) received similar numbers of SAHA-treated B6BMDCs, on days –1, 0, and 2 relative to BMT. (F) Donor CD4+ cell number was evaluated in recipients’ spleens on day 7. **P < 0.001 versus allogeneic. Data (mean ± SEM) are from 1 of 3 similar experiments. (G) Donor CD8+ cell number was evaluated in recipients’ spleens on day 21. *P < 0.05 versus allogeneic. Data (mean ± SEM) are from 1 of 2 similar experiments.
We then determined whether coculture of naive T cells with HDAC inhibitor–treated DCs altered their phenotype and/or function into suppressor T cells or Tregs. As shown in Figure 3E, BALB/c naive T cells that were incubated in primary cultures with SAHA-treated B6BMDCs proliferated normally in a secondary MLR to the same alloantigen [C57BL/6 (H2b)] and also to a third-party alloantigen, C3H/HeJ (H2k). In addition, these cells did not suppress the proliferation of freshly isolated naive BALB/c T cell responses to C57BL/6 stimulation at a 1:1 ratio (Figure 3E), and no increase in the percent or level of expression of Foxp3 in allogeneic T cells was observed after coculture with SAHA-treated DCs compared with control DCs (data not shown). To determine whether the reduction in allogeneic T cell expansion by SAHA-pretreated DCs was caused by greater apoptosis or by reduced proliferation of the allogeneic T cells, we performed serial annexin V staining and CFSE analysis. We cultured CFSE-labeled BABL/c T cells with either HDAC inhibitor–pretreated DCs or control DCs and analyzed the T cells for their proliferative responses or apoptosis by annexin V staining. As shown in Tables 2 and 3, pretreatment of DCs with SAHA reduced allogeneic T cell proliferation as determined by CFSE staining without causing significant changes in apoptosis.
Effect of HDAC inhibitor pretreatment of DCs on allogeneic T cell apoptosis, determined by annexin V staining
Effect of HDAC inhibitor pretreatment of DCs on allogeneic T cell proliferation, determined by CFSE staining
We next analyzed whether the proliferative responses were restored in the primary MLR cultures with HDAC inhibitor–treated DCs by the addition of exogenous IL-2. As shown in Supplemental Figure 3A, addition of exogenous IL-2 significantly rescued T cell proliferation, suggesting that HDAC inhibitor–treated DCs induced T cell anergy (23). Thus, pretreatment of DCs with HDAC inhibitor did not induce apoptosis of DCs, nor did it trigger regulatory function in T cells cocultured with HDAC inhibitor–treated DCs. We next examined whether SAHA-treated DCs affect the CTL functions of allogeneic T cells against allospecific targets. BALB/c T cells were stimulated in a bulk MLR with either SAHA-treated or control DCs for 5 d. T cells were harvested and tested for their CTL functions against allogeneic C57BL/6 concanavalin A blasts in a 6-h chromium release killing assay. BALB/c T cells caused equivalent lyses of the allotargets regardless of their stimulation with SAHA or control DCs (Supplemental Figure 3B) and did not cause nonspecific lysis of syngeneic BALB/c concanavalin A blasts (data not shown).
In order to evaluate the effects of HDAC inhibition only on the in vivo function of BM-derived heterogeneous DCs, without the confounding effects on other nonhematopoietic tissues or expression of target antigens, we devised a model in which allogeneic CD4+ T cells would respond only to MHC class II alloantigens on exogenously administered DCs in an acute GVHD model. MHC class II–deficient (Cd74–/–) C57BL/6 mice (H2b) received 11 Gy total body irradiation and were injected with 1 × 107 BMDCs from syngeneic WT C57BL/6 (H2b) animals in 2 doses separated by 24 h. We then injected 5 × 106 T cell–depleted (TCD) BM along with 2 × 106 CD4+ T cells from either syngeneic C57BL/6 or allogeneic bm12 donors (see Methods), which differ from the recipient animals by a single MHC class II antigen. Analysis of donor T cells in the spleen at 7 d — chosen because most recipients were dead by 10 d — revealed fewer CD4+ T cells in animals that received DCs pretreated with SAHA (Figure 3F). We used a similar experimental approach to evaluate the in vivo responses of CD8+ T cells to SAHA-treated DCs in vivo. MHC class I–deficient (HLA-G–/–) recipient mice were conditioned and transplanted as described above with 3 × 106 allogeneic CD8+ T cells from bm1 donors (see Methods), which differ from the recipients by a single MHC class I antigen. SAHA pretreatment of DCs also significantly inhibited donor CD8+ T cell proliferation 21 d after BMT in this model (Figure 3G). Together, these data demonstrate that pretreatment of DCs with SAHA regulates the in vivo alloproliferation of both CD4+ and CD8+ T cells.
Ex vivo treatment of DCs with HDAC inhibitors regulates experimental GVHD. In light of these results, we evaluated whether the regulatory effects of HDAC inhibitor–treated DCs modulated clinically relevant alloresponses by using a well-established murine allogeneic BMT model [BALB/c (H2d) C57BL/6 (H2b)], in which activation of donor T cells by host APCs is critical for induction of acute GVHD (24). C57BL/6 Ly5.2 BMT recipients were injected on days –1, 0, and 2 with 4 × 106 to 5 × 106 host-type B6BMDCs treated with either diluent or 500 nM SAHA for 16–20 h. This schedule was chosen to modulate the initial donor T cell interaction with host APCs during the first 72 h after BMT (25–27). As shown in Figure 4A, injection of SAHA-treated DCs to allogeneic animals significantly improved survival compared with injection of control DCs (50% versus 10%; P < 0.01). We also observed significantly lower GVHD clinical disease scores in mice injected with SAHA-treated DCs compared with controls early after BMT (7 d, 3.7 ± 0.2 versus 5.2 ± 0.6, P = 0.037; 14 d, 2.3 ± 0.4 versus 4.4 ± 0.9, P = 0.014; 21 d, 3.1 ± 0.4 versus 4.9 ± 0.8, P < 0.05; 28 d, 4.3 ± 0.7 versus 5.1 ± 1; P = 0.21). Consistent with the reduction in clinical GVHD, injection of SAHA-treated DCs also reduced serum levels of TNF-α (Figure 4B) and attenuated the expansion of donor CD3+ lymphocytes 7 d after BMT (Figure 4C) (25). We next stained for Foxp3 on the H2d+CD4+ double-positive cells and analyzed for donor CD4+Foxp3+ Treg expansion 7 and 10 d after allogeneic BMT. There was no significant increase in the percent positivity of donor Tregs in the splenocytes of the recipient animals after infusion of SAHA-treated DCs compared with control DC infusion on days 7 and 10 (7 d, 12.6% ± 3.2% versus 8.8% ± 3.6%, P = 0.19; 10 d, 13.1% ± 2.2% versus 10.4% ± 2.9%; P = 0.23), suggesting lack of early donor Treg enhancement.
BMDCs treated ex vivo with HDAC inhibitor regulate acute GVHD. C57BL/6 animals were irradiated and transplanted with TCD BM and T cells from either allogeneic BALB/c or syngeneic C57BL/6 Ly5.2 (n = 4) animals as described in Methods. Allogeneic recipients were injected with diluent (n = 10) or with 4 × 106 to 5 × 106 control (n = 10) or SAHA-treated (n = 10) B6BMDCs on days –1, 0, and 2 relative to BMT. (A) Survival. Open circles, syngeneic; open triangles, diluent-injected allogeneic; filled circles, SAHA-treated DC–injected allogeneic; filled triangles, control DC–injected allogeneic. P < 0.05, SAHA-treated DCs versus control DCs. (B) Serum levels of TNF-α 7 d after BMT. *P < 0.05 versus allogeneic control recipients. (C) Day-7 donor T cell expansion. *P < 0.05 versus allogeneic control recipients. Data (mean ± SEM) were combined from 2 experiments and represent 2 of 3 experiments with similar results.
Mechanisms of HDAC inhibitor–mediated regulation of DCs. We explored the cellular mechanisms critical for regulation of DCs mediated by HDAC inhibitors. Although the immunomodulatory effects were observed at low concentrations, because HDAC inhibitors can induce cellular apoptosis (28), we sought to determine whether the regulation of DC function was secondary to loss of DC viability. We found no significant increase in annexin V–positive CD11c+ DCs when pretreated with 100 and 500 nM SAHA (the dose used for most in vitro studies), but observed a significant increase at 2- and 5-μM concentrations, thus ruling out apoptosis as a significant cause of this inhibition at the nanomolar concentrations used to examine the DC immunomodulatory effects (Supplemental Table 1). We next determined whether HDAC inhibitor pretreatment increased the loss of DC viability at later time points. The numbers of DCs recovered were lower in all of the groups at 48 h after washing away SAHA, including the diluent control group (Supplemental Table 1). However, consistent with annexin V staining, no statistically significant differences were observed in the retrieval of viable DCs between the control (0 nM) and the 100- and 500-nM groups, but the viability of DCs (annexin V–negative cells) was significantly lower in the DCs treated with micromolar SAHA (Supplemental Table 1).
We also determined whether HDAC inhibitors induced lasting effects on DCs. DCs were thoroughly washed after 14–18 h of incubation with SAHA, allowed to rest for 36–40 h, and then stimulated with 100 ng/ml LPS after normalizing for the CD11c+ DCs. In contrast to immediate stimulation with LPS following pretreatment with SAHA, no significant decrease in TNF-α was observed between SAHA-treated and control DCs (329 ± 78 pg/ml versus 407 ± 103 pg/ml; P = 0.8). These data show that HDAC inhibitors induce marked effects on DC function. However, consistent with the ability of these inhibitors to reversibly inhibit HDAC enzymes, the regulatory effects on DC function may be transient.
We then evaluated the effect of HDAC inhibition on the expression of costimulatory molecules CD80, CD86, and CD40. Control B6BMDCs demonstrated high expression of CD80, CD86, and CD40, as reported previously (29, 30), and stimulated proliferation of allogeneic T cells. HDAC inhibitor treatment markedly reduced the surface expression of all these molecules (Figure 5). These data suggest that treatment of DCs with HDAC inhibitors causes active downregulation of costimulatory molecule expression.
Effect of SAHA on the phenotype of murine DCs. BM cultures enriched for DCs from C57BL/6 mice were sorted by FACS for CD11c+ DCs after being stained with FITC-conjugated anti-CD11c. Cells were then incubated with 0.5 μM SAHA or diluent for 16–18 h and stained for PE-labeled IgG (black) or anti-CD80, anti-CD86, and anti-CD40 (gray) as described in Methods. Increased expression relative to control IgG is shown by arrows and percentages. Results are representative of 3 replicate experiments.
To test the potential clinical applicability of our findings, we evaluated the effect of HDAC inhibitor pretreatment on human PBMC–derived DCs. PBMC-derived DCs from normal healthy volunteers were treated with 500 nM SAHA for 18 h and then washed prior to using them as stimulators in an allogeneic MLR. SAHA treatment of DCs reduced proliferation and IFN-γ secretion by allogeneic responder PBMCs (Supplemental Figure 2, A and B) and, consistent with a recent report (31), significantly reduced the expression of CD40 and CD80 on human PBMC–derived CD11c+ DCs (Supplemental Figure 2, C and D). Pretreatment with both SAHA and 100 nM ITF 2357 also reduced LPS-induced TNF-α expression from these cells (data not shown).
HDAC inhibitors cause immunodominant regulation of DCs. We explored whether HDAC inhibition would dominantly regulate the function of DCs. SAHA-treated DCs were cocultured at increasing ratios with control DCs, whose numbers were kept constant. As shown in Figure 6A, SAHA-treated DCs significantly inhibited the ability of normal DCs to stimulate allogeneic T cell proliferation when they were cocultured only at a high, 1:1 ratio. We next evaluated whether regulation at this high ratio was contact dependent. As shown in Figure 6B, when SAHA-treated DCs were separated from control B6BMDCs and BALB/c allogeneic T cells by transwell, allogeneic T cells proliferated briskly. These data show that HDAC inhibition dominantly regulates the allostimulation of control DCs in a contact-dependent manner.
HDAC inhibitor–treated DCs regulate T cell proliferation induced by normal DCs. BMDCs were obtained and treated with SAHA as described in Methods. Control and SAHA-treated DCs were cocultured with allogeneic T cells at the indicated ratios as described in Methods. (A) Coculture at a 1:1 ratio reduced T cell proliferation at 72 h. *P < 0.05 versus 1:0 ratio. (B) Separation of the control DCs and SAHA DCs at a 1:1 ratio in a transwell caused robust T cell proliferation. P = NS, 1:1 versus 1:0. *P < 0.05 versus 1:0 ratio. (C) Coculture of control DCs from WT C57BL/6 and SAHA-treated DCs from Cd74–/– animals at a 1:1 ratio caused BALB/c CD4+ T cell proliferation. P = NS, control DCs versus 1:1. **P < 0.01 versus control DCs. Data are mean ± SEM.
Next, to address whether the contact-dependent regulation of HDAC inhibitor–treated DCs requires contact with T cells or control DCs, we devised a 3-cell experiment in which SAHA-treated DCs lacked the capacity to present antigens to T cells. We reasoned that if regulation depended on contact with control DCs alone, rather than T cells, it would be preserved even if the SAHA-treated DCs were deficient in MHC molecules and thus incapable of direct interaction with responder T cells. Cd74–/– (H2b) mouse B6BMDCs were treated with SAHA as described above and then cocultured with WT (H2b) B6BMDCs together with purified BALB/c (H2d) responder CD4+ T cells in an MLR. Contrary to our hypothesis, allogeneic CD4+ T cells proliferated briskly in this 3-cell experiment (Figure 6C), demonstrating that the critical points of contact in the regulation of T cell proliferation lie between HDAC inhibitor–treated DCs and T cells. These data also provide an explanation for the reduction of GVHD by ex vivo–treated DCs we observed and thus collectively show that HDAC inhibition can dominantly regulate the function of DCs both in vitro and in vivo.
HDAC inhibitors induce the expression of IDO. We next sought to determine the potential molecular mechanisms underpinning the regulation of DCs by HDAC inhibitors. IDO is an enzyme that degrades tryptophan, which suppresses DC function and induces T cell anergy (32, 33). Therefore, to elucidate the molecular mechanism of the effect of HDAC inhibition on DC responses, we tested the hypothesis that treatment of DCs with HDAC inhibitors enhances the expression of IDO. B6BMDCs were harvested and treated overnight with increasing concentration of SAHA or ITF 2357; the controls were treated with diluent alone. Cells were harvested and analyzed for the induction of IDO mRNA by RT-PCR, as described in Methods. As shown in Figure 7A, both SAHA and ITF 2357 increased the expression of IDO mRNA, which suggests that HDAC inhibition increases the transcription of IDO. We confirmed the presence of IDO protein by Western blot analysis using these same cellular preparations (data not shown), which suggests that the regulatory effects of HDAC inhibitors on DCs correlates with increased expression of the immunoregulatory enzyme IDO. We next evaluated whether HDAC inhibitors regulate the transcription of IDO by performing a chromatin immunoprecipitation assay (see Methods). As shown in Supplemental Figure 4A, acetylated H4 was bound to the IDO promoter, demonstrating a direct role for acetylation of histones in promoting transcription of IDO by SAHA.
HDAC inhibitors induce IDO and regulate DC function. (A) B6BMDCs were treated with diluent control or with SAHA or ITF 2357 at the indicated concentrations for 16 h, and expression of IDO was evaluated with RT-PCR for IDO mRNA. GAPDH was used to control for RNA loading. Data are from 1 of 2 similar experiments. (B) Blockade of IDO with siRNA. BMDCs were transfected with IDO-specific siRNA or control scrambled (sc) siRNA and treated with diluent control, 500 nM SAHA, or 10 ng/ml IFN-γ. Knockdown of IDO mRNA was evaluated with RT-PCR as described in A. SOD2 expression by IFN-γ was used as control for the specificity of IDO knockdown, and GAPDH was used to control for RNA loading. (C) BMDCs were transfected with IDO-specific or control siRNA and treated with either diluent or SAHA as described above. LPS was added for the last 18 h of culture, and TNF-α was measured in the supernatants. Data (mean ± SEM) are from 1 of 3 similar experiments. (D) BMDCs were obtained from either WT C57BL/6 or Indo–/– C57BL/6 mice. Cells were treated with diluent, SAHA, or ITF 2357 and stimulated overnight with control media or LPS, after which TNF-α was measured in the supernatants. Data (mean ± SEM) are from 1 of 2 similar experiments. *P < 0.05; **P < 0.03.
IDO is critical for HDAC inhibitor–mediated regulation of DCs. To address whether IDO is critical for HDAC inhibitor–mediated regulation of DCs, we used 3 distinct but complementary approaches with siRNA, with pharmacologic inhibition by 1-MT, and with DCs from Indo–/– mice (34, 35). B6BMDCs were treated with 500 nM SAHA, 10 ng/ml IFN-γ, or diluent and were transfected with either IDO-specific or control siRNA as described in Methods. At 48 h, IDO-specific siRNA efficiently silenced the mRNA expression of IDO both in the SAHA group and in IFN-γ–treated controls (Figure 7B). The lack of effect of siRNA on the increased expression of SOD2 in IFN-γ–treated cells further confirmed the specificity of the silencing of Indo gene expression.
We directly examined the functional role of IDO in mediating the effects of SAHA by measuring the response of DC production of TNF-α to LPS stimulation. B6BMDCs were treated with SAHA or diluent, transfected with either scrambled siRNA control or IDO-specific siRNA, and then stimulated with LPS for the last 12 h of a 48-h culture (see Methods). TNF-α levels were measured in the supernatants to determine DC function as described above. As we observed in Figure 1A, SAHA treatment suppressed TNF-α production by more than 70% (Figure 7C). Blockade of IDO by siRNA significantly, but not completely, reversed this suppression of TNF-α production by SAHA-treated DCs (Figure 7C). Marked reversal of suppression of the allogeneic T cell proliferation was also observed when IDO was knocked down (data not shown).
We further confirmed the critical role of IDO in HDAC inhibitor–mediated regulation by stimulating BMDCs from Indo–/– mice and B6BMDCs with LPS. We observed significantly less suppression of TNF-α secretion by SAHA-treated Indo–/– B6BMDCs (Figure 7D), confirming a substantial role for IDO in HDAC inhibitor–mediated suppression of DCs. The role for functional IDO expression by HDAC inhibitor treatment of DCs in the suppression of allogeneic T cell proliferation was further confirmed by cotreatment with 1-MT (data not shown). Thus, IDO directly mediates, at least in part, the regulation of DCs by HDAC inhibitors.
We next determined whether the effects of HDAC inhibitor–treated DCs modulated in vivo GVHD responses in an IDO-dependent manner. We once again used the same allogeneic BMT model (BALB/c into C57BL/6), in which activation of donor T cells by host APCs are critical for induction of acute GVHD. C57BL/6 Ly5.2 recipients were injected on days –1, 0, and 2 relative to BMT with 4 × 106 to 5 × 106 host-type B6BMDCs derived from Indo–/– animals that were treated with either diluent or 500 nM SAHA for 18 h. As shown in Figure 8, A and B, injection of SAHA-treated Indo–/– DCs to allogeneic animals did not alter the survival or the clinical severity of GVHD compared with control-treated Indo–/– DCs (32.1% versus 12.9%; P = 0.72), demonstrating that induction of IDO in DCs by HDAC inhibition is critical for regulating DC function.
IDO is critical for regulation of GVHD by HDAC inhibitors. (A and B) WT C57BL/6 mice were irradiated and transplanted with TCD BM and T cells from either allogeneic BALB/c or syngeneic C57BL/6 Ly5.2 (black line; n = 8) animals. Allogeneic recipients were injected with 4 × 106 to 5 × 106 of either diluent-treated control (gray line; n = 14) or SAHA-treated (dotted line; n = 14) B6BMDCs from Indo–/– mice on days –1, 0 and 2. All mice were evaluated for survival (A) and clinical GVHD severity (B). P = NS, SAHA-treated versus control allogeneic recipients. Data (mean ± SEM) were combined from 2 experiments with similar results. (C and D) [C57BL/6→C57BL/6] and [C57BL/6→_Indo–/–_C57BL/6] chimeras were generated as described in Methods and used as recipients in allogeneic BMT. Chimeras were reirradiated and transplanted with TCD BM and T cells from either allogeneic BALB/c or syngeneic C57BL/6 Ly5.2 (gray line; n = 4) animals. Allogeneic [C57BL/6→C57BL/6] recipients were injected with either diluent (thin line; n = 6) or ITF 2357 (thick line; n = 6) as described in Methods. [C57BL/6→_Indo–/–_C57BL/6] recipients were also injected with diluent (dotted line; n = 5) or ITF 2357 (dashed line; n = 6). All mice were evaluated for (C) survival and (D) clinical severity of GVHD. *P < 0.05 versus ITF 2357. Data (mean ± SEM) are from 1 of 2 similar experiments.
Next, to directly demonstrate the in vivo relevance of IDO induction by HDAC inhibitors, we first confirmed in naive C57BL/6 mice the dose and schedule of ITF 2357 sufficient to reach the required drug levels in vivo for induction of IDO. Mouse splenocytes were harvested after administration of either 5 mg/kg ITF 2357 or the diluent control for 4 d and analyzed for IDO expression by PCR. As shown in Supplemental Figure 4B, administration of ITF 2357 induced IDO in vivo, which suggests that sufficient tissue and plasma levels of the drug were obtained to evaluate for the role of IDO induction.
We then generated C57BL/6 BM chimeras using TCD BM from _Indo–/–_C57BL/6 mice transplanted into recipient C57BL/6 mice ([_Indo–/–_C57BL/6→C57BL/6] animals) so that BM-derived APCs would be incapable of IDO expression. Chimeras were reirradiated on day –1 relative to BMT, then injected on day 0 with TCD BM and 3 × 106 purified CD3+ T cells from either allogeneic BALB/c or syngeneic C57BL/6 donors. The allogeneic recipients were injected with either diluent or 5 mg/kg ITF 2357 on days –1, 0, 1, and 2 relative to BMT in order to modulate the host hematopoietic-derived APCs that initiate graft-versus-host reaction (27). As expected, all allogeneic BALB/c→[C57BL/6→C57BL/6] mice treated with diluent died of GVHD (Figure 8C), while 50% of allogeneic animals that received ITF 2357 died, and survivors demonstrated mild clinical GVHD (Figure 8, C and D). Conversely, all allogeneic [_Indo–/–_C57BL/6→C57BL/6] recipients died of GVHD, regardless of treatment with ITF 2357 (Figure 8, C and D). However, no significant survival difference was noted between the control-treated [C57BL/6→C57BL/6] and [_Indo–/–_C57BL/6→C57BL/6] animals. GVHD was also confirmed by target organ histopathology (i.e., gastrointestinal tract and liver; data not shown). These data collectively demonstrate that expression of IDO by host APCs alone was not critical for the modulation of GVHD in control-treated animals but was critical for HDAC inhibitor–mediated regulation of GVHD.