Abstract
The balance between tolerance and immunity is important for the outcome of an infection or cancer, and dendritic cells (DCs) are key regulators of this balance. DC-specific transcript (DC-SCRIPT) is a protein expressed by DCs and has been demonstrated to suppress both TLR-mediated expression of IL-10 and glucocorticoid receptor–mediated transcription of glucocorticoid-induced leucine zipper (GILZ). Because GILZ is known to promote IL-10 production, we investigated whether these two processes are linked. Dual-knockdown and inhibition experiments demonstrated that neither GILZ nor glucocorticoid receptor play a role in TLR-induced IL-10 production after DC-SCRIPT knockdown. The NF-κB pathway is another route involved in IL-10 production after DC activation. Strikingly, inhibition of NF-κB led to a decreased TLR-mediated IL-10 production in DC-SCRIPT knockdown DCs. Moreover, DC-SCRIPT knockdown DCs showed enhanced phosphorylation, acetylation, and IL10 enhancer binding of the NF-κB subunit p65. These data demonstrate that besides nuclear receptor regulation, DC-SCRIPT also modulates activation of NF-κBp65 after TLR activation in human DCs.
Introduction
Dendritic cells (DCs) are specialized immune cells uniquely capable of directing a naive T cell response toward immune activation or tolerance (1). Extracellular signals steer DC maturation either toward immunity against pathogens or tolerance to, for example, self-Ags, harmless food Ags, and commensal bacteria (1). The presence of tolerogenic DCs is crucial to maintain immune homeostasis, and they are characterized by expression of coinhibitory molecules and a high production of IL-10 (2, 3). In addition, IL-10 is also necessary to help resolve immune responses and avoid excessive immune activation leading to tissue pathology (4). Consequently, the regulation of IL-10 expression is under tight control in cells in general and in DCs in particular. Moreover, because the ability of DCs to direct the immune system is exploited in clinical vaccination settings to treat either autoimmunity (5) or cancer (6), it is of crucial importance to thoroughly understand DC activation and tolerization pathways.
Several pathways are known to regulate DC activation, including the TLR and nuclear receptor (NR) pathways. TLRs recognize structurally conserved molecules derived from diverse pathogens such as LPS and viral RNA. TLR signaling results in the maturation of the DCs, displayed by a production of proinflammatory cytokines, upregulation of costimulatory molecules, and upregulation of Ag-presenting MHC molecules (7, 8). These maturation events create DCs that are able to present Ags to and activate naive T cells. NRs are ligand-inducible transcription factors that respond to ligands such as hormones (e.g., estrogen), vitamins (e.g., vitamin D3), and metabolites (e.g., fatty acids) (9). NRs can directly bind to DNA and thereby mediate gene transcription, which involves the recruitment of several corepressor or coactivator molecules to the promoters of their target genes (9). NRs such as the glucocorticoid receptor (GR) and the vitamin D3 receptor have been found to repress proinflammatory programs induced by TLRs (10, 11).
Common for NR and TLR signaling pathways is their ability to regulate transcription by NF-κB (12, 13). TLR signaling is known to activate the NF-κB complex (13), whereas GR has been found to inhibit NF-κB–mediated transcription (10, 14–16). NF-κB resides in the cytoplasm in an inactive form by binding to the NF-κB inhibitory protein IκBα (13). Upon IκBα phosphorylation, the NF-κB–IκBα complex disintegrates, allowing NF-κB to translocate to the nucleus and activate transcription of target genes. Five different subunits of NF-κB are known, which can form either homodimers or heterodimers (17). Three of the subunits (p65, RelB, and cRel) contain a transactivation domain, making them capable of initiating transcription. The other subunits (p50 and p52) only contain a Rel-homology domain, making them capable to bind other NF-κB family members. The most frequently studied complex in the TLR-signaling pathway is the p65-p50 heterodimer, but the p52-RelB heterodimer, and the p50-p50 homodimer also have been found to play a role in DCs (13). In addition to regulation of nuclear translocation, NF-κB activation also depends on posttranslational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination, sumoylation, and nitrosylation (17). For instance, phosphorylation of S536 or S276 on p65 leads to activation of NF-κB–mediated transcription (18, 19), whereas phosphorylation of S547 or T505 on p65 renders NF-κB inactive, preventing transcription of its target genes (20, 21). Likewise, acetylation of K122 and K123 inhibits NF-κBp65 (22), whereas acetylation of K310 activates NF-κBp65–mediated transcription (23). In the past, studies on NF-κB activation focused on the induction of proinflammatory cytokines (13). Recently, however, it was shown that the NF-κBp65 subunit can also bind upstream of the IL10 gene, suggesting it can also regulate the production of the anti-inflammatory cytokine IL-10 (24). The induction of IL-10 by NF-κBp65 was recently shown to be dependent on K310 acetylation of NF-κBp65 (25).
Recently, we have identified and characterized the transcription regulator DC-specific transcript (DC-SCRIPT) that is expressed by all DC subsets and is involved in regulating the activation status of DCs (26–28). In the absence of DC-SCRIPT, DCs secrete enhanced amounts of IL-10 in response to TLR stimulation, and less IL-12p70, whereas TNF-α and IL-6 remain unchanged (26). In correspondence with the increased IL-10 and decreased IL-12p70 production, DC-SCRIPT knockdown also led to dampened T cell proliferation and IFN-γ production (26). Interestingly, DC-SCRIPT has also been shown to regulate NR-mediated gene transcription both in DCs and in specialized epithelial cells (29–34). For DCs, we have shown that DC-SCRIPT is involved in inhibiting GR-mediated transcription, as well as expression of its target gene glucocorticoid-induced leucine zipper (GILZ). Together with IL-10, GR and GILZ are all factors known to be involved in tolerogenic DC function (35), suggesting that these pathways may be interconnected.
In this study, we have further investigated the molecular mechanism underlying IL-10 production in DC-SCRIPT knockdown DCs, with a focus on GR, GILZ, and NF-κB. Strikingly, we found that DC-SCRIPT does not control IL-10 production via the GR and GILZ pathway, but rather affects NF-κB activation.
Materials and Methods
Generation of human monocyte-derived DCs
Human monocyte-derived DCs (moDCs) were generated from PBMCs as described previously (36). Buffy coats were obtained from healthy volunteers (Sanquin, Nijmegen, the Netherlands) after informed consent and according to institutional guidelines. Plastic-adhered monocytes were cultured for a total of 6 d in RPMI 1640 medium (Life Technologies) supplemented with 1% ultraglutamine (Cambrex), 0.5% antibiotic-antimycotic (Invitrogen), 10% (v/v) FCS (Greiner Bio-one), IL-4 (300 U/ml), and GM-CSF (450 U/ml) (both from Cellgenix). On day 3, moDCs were supplemented with new IL-4 (300 U/ ml) and GM-CSF (450 U/ml).
Small interfering RNA-mediated knockdown
On days 3–4 of DC differentiation, cells were harvested and subjected to electroporation. For DC-SCRIPT silencing, a 23-nt custom ZNF366 small interfering RNA (siRNA) termed SC38 targeting the DC-SCRIPT gene at position 2349–2369 was used (Thermo Scientific). For GILZ silencing, the SMARTpool ON-TARGETplus TSC22D3 siRNA (Thermo Scientific) was used. siRNA ON-TARGETplus Non-Targeting siRNA#1 (Thermo Scientific) was used as control. Cells were washed twice in PBS and once in OptiMEM without phenol red (Invitrogen). A total of 10 μg siRNA (5 + 5 μg for double knockdown) was transferred to a 4-mm cuvette (Bio-Rad), and 106 DCs were added in 200 ml OptiMEM and incubated for 3 min before being pulsed with an exponential decay pulse at 300 V, 150 mF, in a Genepulser Xcell (Bio-Rad), as previously described (37). Immediately after electroporation, the cells were transferred to preheated (37°C) phenol red–free RPMI 1640 culture medium supplemented with 1% ultraglutamine, 10% (v/v) FCS, IL-4 (300 U/ml), and GM-CSF(450 U/ml).
Stimulations
Immature DCs were stimulated with 4 μg/ml R848 (Axxora), 100 ng/ml LPS from E. coli 0111:B4 (Sigma-Aldrich), or 100 nM prednisolone (Centrafarm). In some experiments, DCs were pretreated 1–2 h with one of the following inhibitors: 100 nM RU-486 (Sigma-Aldrich), 5 μM BAY11-7082 (Calbiochem), 30 μM anacardic acid (AA; Calbiochem), 15 μM C646 (Calbiochem), 5 μM curcumin (Calbiochem), or 10 μM CPTH2 (Calbiochem). In some experiments, moDCs were pretreated for 1 h with 20 μg/ml DC-SIGN Ab (clone DCN46; BD Biosciences), before addition of LPS.
RNA isolation and quantitative PCR
Total RNA was isolated from cells using an RNA isolation kit (Zymo Research). RNA quantity and purity were determined on a NanoDrop spectrophotometer. RNA was treated with DNase I (amplification grade; Invitrogen) and reverse transcribed into cDNA by using random hexamers and Moloney murine leukemia virus reverse transcriptase (Invitrogen). mRNA levels for the genes of interest were determined with a CFX96 sequence detection system (Bio-Rad) with SYBR Green (Roche) as the fluorophore and gene-specific oligonucleotide primers. Primers used are as follows (forward, reverse): DC-SCRIPT (ZNF366): (5′-AAGCATGGAGTCATGGAG-3, 5′-TTCTGAGAGAGGTCAAAGG-3′), PBGD (5′-GGCAATGCGGCTGCAA-3′, 5′-GGGTACCCACGCGAATCAC-3′), GILZ (5′-AGAACCTCAATACCGACAAG-3′, 5′-CATCAGATGATTCTTCACCA-3′). Reaction mixtures and program conditions were used as recommended by the manufacturer (Bio-Rad). Quantitative PCR (qPCR) data were analyzed with the CFX Manager software (Bio-Rad) and checked for correct amplification and dissociation of the products. mRNA levels of the genes of interest were normalized to mRNA levels of the housekeeping gene PBGD and were calculated according to the cycle threshold method (38).
ELISA
Secreted cytokines were assayed in the supernatants of 24-h–stimulated moDCs. IL-10 was measured using the human IL-10 ready-set-go kit (eBioscience), IL-6 was measured using the PeliPair human IL-6 ELISA kit (Sanquin), and TNF-α was measured using the human TNF-α ELISA set (BD Biosciences). All cytokines were measured according to the manufacturers’ instructions.
NF-κB DNA binding assay
Immature moDCs were stimulated for 1–8 h with R848, followed by two quick washes in 4°C cold PBS. Next, nuclear extracts were prepared with the NucBuster protein extraction kit (Novagen) according to the manufacturer’s protocol. Protein amount in isolated nuclei was measured using the Coomassie Plus Protein Assay (Thermo Scientific), and 7 μg protein was used as input for the TransAM NF-κB family kit (Active Motif). Binding of the NF-κB subunits to an immobilized consensus sequence for NF-κB was detected by specific mAbs for NF-κB p50, p52, p65, RelB, and cRel and secondary HRP-conjugated Abs using a colorimetric readout. The kit was used according to the manufacturer’s protocol.
Phosphorylation of NF-κBp65
Immature moDCs were plated at a cell concentration of 5 × 105/ml and allowed to rest for a minimum of 1 h before stimulation. After stimulation, cells were lysed in a concentration of 106/100 μl 4°C cold lysis buffer consisting of 62.5 mM Tris (pH 6.8; Sigma-Aldrich), 1% SDS (Invitrogen), and freshly added complete protease inhibitor mixture (Roche), 1 mM PMSF (Sigma-Aldrich), as well as the phosphatase inhibitors 1 mM Na3VO4 (Sigma-Aldrich) and 10 mM NaF (Merck). Cell lysates were mixed 1:4 with sample buffer containing 5% glycerol (Invitrogen), 6% SDS, 125 mM Tris-HCl (pH 6.8), 0.1 mg/ml bromophenol blue (Gebr. Schmid), and 10% 2-ME (Sigma-Aldrich); heated at 95°C for 5 min; and then cooled on ice. The proteins were resolved by electrophoresis on a 10% polyacrylamide gel (ratio of acrylamide to bisacrylamide, 37.5:1) and transferred to Protran nitrocellulose transfer membranes (Amersham) at 4°C. Membranes were blocked and stained in 1% skimmed milk powder (Campina) and 3% BSA (Roche) in PBS with 0.1% Tween 20 for assaying total NF-κBp65 (1:100 dilution of clone sc-8008; Santa Cruz Biotechnology), or 2% skimmed milk powder and 2% BSA in TBST for assaying S536-phosphorylated NF-κBp65 (1:1000 dilution of clone 93H1; Cell Signaling Technology). Membranes were blocked for 1 h at room temperature, stained overnight with primary Abs (including mouse [AC-40] or rabbit [20–33] anti-actin, 1:10,000 dilution; Sigma-Aldrich), and stained for 1 h at room temperature with corresponding secondary Abs (diluted 1:5000): goat anti-mouse IgG IRDye 800CW, goat anti-rabbit IgG IRDye 800CW, goat anti-mouse IRDye 680RD (all three from LI-COR Biosciences), or goat anti-rabbit IgG Alexa Fluor 640 (Invitrogen). Membranes were scanned by using an Odyssey Infrared Imaging System (LI-COR Biosciences).
Chromatin immunoprecipitation-qPCR
Chromatin immunoprecipitation (ChIP) was performed as previously described (39), with some modifications in the buffer compositions. Five million human moDCs were stimulated for 1 h with R848, followed by cross-linking with 1% formaldehyde for 15 min at room temperature. The reaction was quenched with 0.125 M glycine and washed at 4°C with three buffers: 1) PBS; 2) buffer consisting of 0.2% Triton X-100, 0.2% NP-40, 1 mM EDTA pH 8, 0.5 mM EGTA pH 8, 10 mM Tris-HCl pH 7.5, 10 mM NaCl; and 3) buffer consisting of 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES pH 7.6. Cells were then resuspended in ChIP incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, 1× protease inhibitor mixture [Roche], 0.1% BSA) and sheared using a Bioruptor Next Generation sonicator (Diagenode). Sonicated chromatin was centrifuged for 5 min at 4°C and supernatant was snap-frozen. Subsequently, the chromatin was incubated overnight with 1 μg rabbit anti-human NF-κBp65 Ab (clone D14E12; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch) and protein A/G beads (Santa Cruz). Beads were washed, eluted, and de-cross-linked for 5 h as previously described (39). De-cross-linked DNA was purified using the QIAquick PCR purification kit (Qiagen) and quantified using Qubit (Invitrogen). qPCR was performed on input and purified chromatin immunoprecipitated (ChIPed) chromatin using the primers for the IL10 enhancer: forward 5′-GAGCACAGTGTCCATCCCC-3′, reverse 5′-AAACCGGATTAAGCGCCC-3′, as previously described (40).
Detection of K310 acetylation of NF-κBp65
Immature moDCs were plated at a cell concentration of 5 × 105/ml and allowed to rest for a minimum of 1 h before stimulation. After stimulation, paraformaldehyde (Merck) was added directly to the culture medium at a final concentration of 1.6%. Fixation was allowed to take place for 10 min at room temperature protected from light. Subsequently, culture medium was aspirated, and cells were resuspended in remaining medium (to avoid clumps when MeOH is added). For permeabilization, cells were vigorously resuspended in 100% ice-cold MeOH (Boom) and incubated at −20°C overnight, followed by extensive washing in PBS with 1% BSA, 0.05% NaN3 (Merck), and 2% human serum (Sanquin) (4×) to remove residual MeOH. Cells that remained adherent were scraped loose before FACS staining. Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-κBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch). Cells were stained with primary Ab/isotype control for 45 min on ice, followed by 30 min on ice with a secondary PE-Cy5.5 goat anti-rabbit IgG Ab (catalog no. L42018; Invitrogen). Data were acquired on a FACSCyan (Beckman Coulter). Isotype controls gave a staining intensity similar to unstimulated moDCs, indicating that blocking conditions were sufficient to avoid unspecific staining (data not shown). Data were analyzed using FlowJo (Tree Star).
Statistics
Statistical analysis was done using Excel (Microsoft) or Prism 5 (GraphPad Software). The specific method has been described in each figure legend.
Results
IL-10 production mediated by DC-SCRIPT knockdown is GILZ and GR independent
Previously we have shown that knockdown of the transcription regulator DC-SCRIPT leads to GILZ upregulation and IL-10 production in human moDCs, most effectively after TLR stimulation (26, 33). Because GILZ has been shown to mediate IL-10 production after GR-ligation (35), we hypothesized that GILZ is involved in IL-10 production in DC-SCRIPT knockdown moDCs (SCRIPT-KD-DCs). To investigate this, we effectively knocked down both DC-SCRIPT and GILZ in moDCs using siRNA (Fig. 1A, 1B). As previously reported (26, 33), the knockdown of DC-SCRIPT led to an increased GILZ expression (Fig. 1B) and elevated IL-10 production when compared with control nontargeting siRNA-treated moDCs (Ctrl-DCs) (Fig. 1C). Strikingly, double knockdown of DC-SCRIPT and GILZ did not restore IL-10 production to control levels (Fig. 1C), suggesting that IL-10 production is GILZ independent. Because GILZ is induced in DCs by GR, and DC-SCRIPT is known to affect GR function, we next investigated whether the IL-10 production was GR dependent. Immature SCRIPT-KD-DCs and Ctrl-DCs were treated with either the GR agonist prednisolone or the antagonist RU-486, followed by TLR7/8 triggering with R848. Neither inhibition nor stimulation of GR activity significantly altered IL-10 production in both Ctrl-DCs and SCRIPT-KD-DCs (Fig. 1D). Together, these data demonstrate that IL-10 production in SCRIPT-KD-DCs does not result from GILZ upregulation or GR signaling and that other pathways must be involved.
GILZ and GR do not affect TLR-mediated IL-10 production in human DCs. (A–C) moDCs were transfected with siRNA for DC-SCRIPT, GILZ, or in combination at day 4 of differentiation. (A and B) knockdown efficiency of the genes was assayed on day 6 by qRT-PCR. (C) Day 6 immature moDCs were stimulated with R848 for 24 h. Supernatants were collected and assayed for IL-10 by ELISA. (D) Immature DC-SCRIPT-knockdown moDCs and Ctrl-DCs were prestimulated for 1 h with RU-486 (inhibitor of GR) or prednisolone (agonist of GR), followed by a 24-h stimulation with R848. Supernatants were collected and assayed for IL-10 by ELISA. Data have been normalized to Ctrl-DCs. Statistics: ANOVA with a Bonferroni posttest (n = 3, error bars = SEM). AU, arbitrary unit. **p < 0.01, ***p < 0.01.
GILZ and GR do not affect TLR-mediated IL-10 production in human DCs. (A–C) moDCs were transfected with siRNA for DC-SCRIPT, GILZ, or in combination at day 4 of differentiation. (A and B) knockdown efficiency of the genes was assayed on day 6 by qRT-PCR. (C) Day 6 immature moDCs were stimulated with R848 for 24 h. Supernatants were collected and assayed for IL-10 by ELISA. (D) Immature DC-SCRIPT-knockdown moDCs and Ctrl-DCs were prestimulated for 1 h with RU-486 (inhibitor of GR) or prednisolone (agonist of GR), followed by a 24-h stimulation with R848. Supernatants were collected and assayed for IL-10 by ELISA. Data have been normalized to Ctrl-DCs. Statistics: ANOVA with a Bonferroni posttest (n = 3, error bars = SEM). AU, arbitrary unit. **p < 0.01, ***p < 0.01.
IL-10 production in DC-SCRIPT knockdown DCs is partially dependent on NF-κB
Because NF-κB activation is known to mediate IL-10 production in moDCs (25), we hypothesized that DC-SCRIPT may affect NF-κB signaling and thereby regulate IL-10 production. Interestingly, blocking NF-κB signaling with the NF-κB inhibitor BAY11-7082 partially inhibited IL-10 production in SCRIPT-KD-DCs (Fig. 2A).
TLR-induced IL-10 production in SCRIPT-KD-DCs depends on NF-κBp65. (A) Immature SCRIPT-KD-DCs and Ctrl-DCs were incubated with BAY11-7082 for 1 h, followed by a 24-h incubation with R848. Supernatants were harvested and assayed for the effect of NF-κB inhibition on IL-10 production. Statistics: Student two-tailed paired t test, n = 6; error bars = SEM. (B) Immature SCRIPT-KD-DCs and Ctrl-DCs were stimulated for 1 h with R848. Nuclear lysates were isolated and allowed to react with the consensus sequence for NF-κB on a precoated 96-well plate. Different subunits of NF-κB were detected using mAbs for the protein of interest, together with a colorimetric readout. Statistics: one-way ANOVA with a Bonferroni posttest; n = 4–5; error bars = SEM. *p < 0.05, **p < 0.01.
TLR-induced IL-10 production in SCRIPT-KD-DCs depends on NF-κBp65. (A) Immature SCRIPT-KD-DCs and Ctrl-DCs were incubated with BAY11-7082 for 1 h, followed by a 24-h incubation with R848. Supernatants were harvested and assayed for the effect of NF-κB inhibition on IL-10 production. Statistics: Student two-tailed paired t test, n = 6; error bars = SEM. (B) Immature SCRIPT-KD-DCs and Ctrl-DCs were stimulated for 1 h with R848. Nuclear lysates were isolated and allowed to react with the consensus sequence for NF-κB on a precoated 96-well plate. Different subunits of NF-κB were detected using mAbs for the protein of interest, together with a colorimetric readout. Statistics: one-way ANOVA with a Bonferroni posttest; n = 4–5; error bars = SEM. *p < 0.05, **p < 0.01.
Because activation of the NF-κB pathway is dependent on translocation and subsequent DNA binding of its subunits, we investigated whether the absence of DC-SCRIPT expression affects DNA binding of the different NF-κB subunits in moDCs (Fig. 2B). To this end, nuclear lysates were isolated from SCRIPT-KD-DCs and Ctrl-DCs before and 1 h after R848 treatment. As expected, and in line with previous reports from LPS-stimulated DCs (25, 41), our data show that R848 stimulation of moDCs leads to a significant increase in DNA binding of p65, p50, and cRel (p < 0.01 for Ctrl-DCs, p < 0.001 for SCRIPT-KD-DCs), whereas no significant increase was observed for p52 and RelB. Interestingly, SCRIPT-KD-DCs showed a significantly enhanced DNA binding of p65 when compared with Ctrl-DCs upon stimulation with R848. Collectively, these data link DC-SCRIPT-knockdown to NF-κB activation and enhanced nuclear DNA binding of the p65 subunit of NF-κB.
DC-SCRIPT knockdown leads to more S536 phosphorylation of NF-κBp65
NF-κBp65 has been demonstrated to regulate production of a range of different cytokines in DCs, dependent on the specific PTMs it acquires upon stimulation (13, 25). One of the most important phosphorylation sites in NF-κBp65 activation is Ser536 (17). Interestingly, the NF-κB inhibitor BAY11-7082 inhibits the IκB kinase (42), which phosphorylates NF-κBp65 exactly at this Ser536 position (18, 43). Because BAY11-7082 decreased IL-10 production in SCRIPT-KD-DCs (Fig. 2A), we determined whether NF-κBp65 is differentially phosphorylated at Ser536 in SCRIPT-KD-DCs. Intriguingly, already without stimulation (0 min) increased NF-κBp65 Ser536 phosphorylation was discerned in SCRIPT-KD-DCs compared with Ctrl-DCs (Fig. 3A, 3B). Upon stimulation with R848, both Ctrl-DCs and SCRIPT-KD-DCs increased Ser536 phosphorylation. Time-course analysis showed that levels of Ser536 phosphorylation in SCRIPT-KD-DCs remained significantly higher than Ctrl-DCs after 30-min stimulation, which leveled out at later time points. Notably, total NF-κBp65 protein expression did not change and could therefore not account for the observed effect (Fig. 3A, 3C).
DC-SCRIPT knockdown increases phosphorylation of NF-κBp65. SCRIPT-KD-DCs or Ctrl-DCs were stimulated with R848 for 0–120 min. Protein lysates were assayed by Western blot for phosphorylated NF-κBp65, total NF-κBp65, and actin as loading control. (A) Representative donor displaying that DC-SCRIPT knockdown induces a higher phosphorylated NF-κBp65. (B and C) p-NF-κBp65 and total NF-κBp65 were quantified for a range of donors. Statistics: Ctrl-DCs were compared with SCRIPT-KD-DCs for each time point, using the Student two-tailed t test (n = 3–4). Error bars = SEM. *p < 0.05.
DC-SCRIPT knockdown increases phosphorylation of NF-κBp65. SCRIPT-KD-DCs or Ctrl-DCs were stimulated with R848 for 0–120 min. Protein lysates were assayed by Western blot for phosphorylated NF-κBp65, total NF-κBp65, and actin as loading control. (A) Representative donor displaying that DC-SCRIPT knockdown induces a higher phosphorylated NF-κBp65. (B and C) p-NF-κBp65 and total NF-κBp65 were quantified for a range of donors. Statistics: Ctrl-DCs were compared with SCRIPT-KD-DCs for each time point, using the Student two-tailed t test (n = 3–4). Error bars = SEM. *p < 0.05.
DC-SCRIPT knockdown leads to more K310 acetylation of NF-κBp65
In addition to phosphorylation, NF-κBp65 activity is also regulated by acetylation. Specifically, acetylation of K310 in NF-κBp65 has been linked to regulation of IL-10 production (25). Therefore, we investigated whether K310 is acetylated in SCRIPT-KD-DCs (Fig. 4). Fig. 4A and 4B shows that Ctrl-DCs do not acetylate NF-κBp65 at K310 upon R848 stimulation as expected. In contrast, SCRIPT-KD-DCs do acetylate K310 upon R848 stimulation for 30 min. Interestingly, even nonstimulated SCRIPT-KD-DCs exhibited a higher acetylation level relative to Ctrl-DCs, similar to that observed for NF-κBp65 Ser536 phosphorylation. Moreover, the baseline acetylation status of NF-κBp65 K310 in unstimulated SCRIPT-KD-DCs shows equal intensity as moDCs stimulated with the previously reported positive control compounds (Fig. 4C, Supplemental Fig. 1A) (25). Further quantification of the data confirmed the occurrence of a significant increase in NF-κBp65 K310 acetylation peaking at 30 min after stimulation (Fig. 4F). Because histone acetyltransferases (HATs) have previously been reported to acetylate NF-κB (23), we next determined whether blocking HATs with the HAT inhibitor (HATi) AA (44–46) could diminish K310 acetylation in SCRIPT-KD-DCs. As shown in Fig. 4D, 4E, and Supplemental Fig. 1B, HATi pretreatment significantly decreased NF-κBp65 K310 acetylation in SCRIPT-KD-DCs. In contrast, the level of acetylation in Ctrl-DCs was so low or absent that the HATi treatment did not mediate any change. These data indicate that DC-SCRIPT knockdown affects the HAT-dependent acetylation of NF-κBp65 at K310.
DC-SCRIPT knockdown increases acetylation of NF-κBp65. Ctrl-DCs or SCRIPT-KD-DCs (SC) were stimulated for 0–60 min with R848, followed by intracellular acetyl NF-κBp65 (K310) FACS staining. (A and B) Overlays between unstimulated and 30-min R848-stimulated moDCs. (C) Positive control: LPS + anti–DC-SIGN Ab (DCN46). (D and E) moDCs were pretreated for 1 h with the HATi AA or vehicle control before R848 stimulation to assay the effect of HATs on the observed acetylation. The line going through (A)–(E) indicates the peak of unstimulated SCRIPT-KD-DCs. (F) Summary of the relative fluorescence intensity (FI) for siRNA-treated moDCs stimulated with R848. Values are normalized to the mean FI for unstimulated Ctrl-DCs. Statistics: ANOVA with a Bonferroni posttest, n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001.
DC-SCRIPT knockdown increases acetylation of NF-κBp65. Ctrl-DCs or SCRIPT-KD-DCs (SC) were stimulated for 0–60 min with R848, followed by intracellular acetyl NF-κBp65 (K310) FACS staining. (A and B) Overlays between unstimulated and 30-min R848-stimulated moDCs. (C) Positive control: LPS + anti–DC-SIGN Ab (DCN46). (D and E) moDCs were pretreated for 1 h with the HATi AA or vehicle control before R848 stimulation to assay the effect of HATs on the observed acetylation. The line going through (A)–(E) indicates the peak of unstimulated SCRIPT-KD-DCs. (F) Summary of the relative fluorescence intensity (FI) for siRNA-treated moDCs stimulated with R848. Values are normalized to the mean FI for unstimulated Ctrl-DCs. Statistics: ANOVA with a Bonferroni posttest, n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001.
IL-10 production from SCRIPT-KD-DCs depends on p300/CBP
To link the increase in acetylation in SCRIPT-KD-DCs to the increase in IL-10 production, we stimulated SCRIPT-KD-DCs and Ctrl-DCs in the presence or absence of HATi. In addition to the broad specificity HATi AA (44–46), we also included the p300/CBP-specific HATi’s curcumin (47, 48) and C646 (49) because p300/CBP has previously been implicated in NF-κB acetylation (23). As shown in Fig. 5, all three HATis significantly decreased IL-10 production in SCRIPT-KD-DCs to a similar extent. These data imply that p300/CBP is involved in IL-10 production in SCRIPT-KD-DCs.
Induction of IL-10 in SCRIPT-KD-DCs is dependent on p300/CBP. Immature Ctrl-DCs or SCRIPT-KD-DCs were preincubated with the HATis AA (broad specificity), curcumin (specific for p300/CBP), or C646 (specific for p300/CBP) for 1 h. Subsequently, the moDCs were stimulated for 24 h with R848, and the supernatants were assayed for IL-10. Cells treated with the inhibitor were compared with vehicle control, and statistical significance was calculated by a Student two-tailed paired t test (n = 4–6, error bars = SEM). *p < 0.05, **p < 0.01.
Induction of IL-10 in SCRIPT-KD-DCs is dependent on p300/CBP. Immature Ctrl-DCs or SCRIPT-KD-DCs were preincubated with the HATis AA (broad specificity), curcumin (specific for p300/CBP), or C646 (specific for p300/CBP) for 1 h. Subsequently, the moDCs were stimulated for 24 h with R848, and the supernatants were assayed for IL-10. Cells treated with the inhibitor were compared with vehicle control, and statistical significance was calculated by a Student two-tailed paired t test (n = 4–6, error bars = SEM). *p < 0.05, **p < 0.01.
DC-SCRIPT knockdown increases NF-κBp65 binding to the IL10 enhancer
Until recently, NF-κBp65 was mainly considered to induce inflammatory cytokine expression (13), but recently it was found that specific signaling through Raf-1 leads to both p300/CBP-mediated acetylation of NF-κBp65 and increased binding of NF-κBp65 to a IL10 cis-regulatory enhancer element (24, 25, 40). To determine whether DC-SCRIPT would also affect the binding of NF-κBp65 to this specific IL10 enhancer, we stimulated SCRIPT-KD-DCs and Ctrl-DCs for 1 h with R848 and ChIPed with a NF-κBp65 Ab or isotype Ab as control. Subsequent qPCR for the IL10 enhancer showed that SCRIPT-KD-DCs had a significantly higher binding of NF-κBp65 to the IL10 enhancer compared with Ctrl-DCs and isotype control (Fig. 6). These data suggest that DC-SCRIPT decreases NF-κBp65 binding to the IL10 enhancer, and thereby limits IL-10 production in human DCs.
DC-SCRIPT knockdown increases NF-κBp65 binding to the IL10 enhancer. SCRIPT-KD-DCs and Ctrl-DCs were stimulated for 60 min with R848 and ChIPed with an NF-κBp65 Ab or isotype control (IgG). The ChIPed product and input chromatin for the ChIP was assayed for IL10 enhancer binding by qPCR. The recovery was calculated by relating the ChIPed product to the input. Statistics: ANOVA with a Bonferroni posttest; n = 4; error bars = SEM. *p < 0.05, **p < 0.01.
DC-SCRIPT knockdown increases NF-κBp65 binding to the IL10 enhancer. SCRIPT-KD-DCs and Ctrl-DCs were stimulated for 60 min with R848 and ChIPed with an NF-κBp65 Ab or isotype control (IgG). The ChIPed product and input chromatin for the ChIP was assayed for IL10 enhancer binding by qPCR. The recovery was calculated by relating the ChIPed product to the input. Statistics: ANOVA with a Bonferroni posttest; n = 4; error bars = SEM. *p < 0.05, **p < 0.01.
Discussion
In this study, we sought to increase our understanding of the molecular pathways the NR coregulator DC-SCRIPT uses to regulate TLR-mediated IL-10 production by DCs. We report that the IL-10 production induced in SCRIPT-KD-DCs was neither dependent on GILZ nor on GR. Instead, IL-10 production in the absence of DC-SCRIPT is at least in part dependent on NF-κB signaling and can be blocked by HATis. Moreover, the NF-κB subunit p65 exhibits increased PTMs and an enhanced IL10 enhancer-binding capacity in SCRIPT-KD-DCs.
The transcription regulator DC-SCRIPT is not only an important NR coregulator important in DC and cancer biology (30–34, 50), it also affects TLR-mediated cytokine production by DCs (26). Our current findings now show that the pleiotropic protein DC-SCRIPT modulates NF-κBp65 activity in DCs. DC-SCRIPT has previously been shown to bind the transcription corepressors receptor-interacting protein 140 (RIP140) and C-terminal binding protein 1, as well as the histone deacetylase 1 (HDAC1), HDAC3, and HDAC6 (27, 29). We now show that the IL-10 production observed after DC-SCRIPT knockdown is dependent on NF-κB and p300/CBP. Interestingly, both HDAC3 and RIP140 have been demonstrated to interact with NF-κBp65 (51–53) and HDAC3 has been found to mediate deacetylation of NF-κBp65 (53). Our finding that DC-SCRIPT knockdown leads to more K310 acetylation and subsequent IL-10 production is in line with a previous report that shows that concomitant triggering of DC-SIGN and TLR4 leads to HAT p300/CBP-dependent K310 acetylation of NF-κBp65 with subsequent IL-10 induction (25). In addition to enhanced acetylation of K310, we also show that DC-SCRIPT knockdown leads to enhanced phosphorylation of Ser536 NF-κBp65. Interestingly, it has been shown that enhanced phosphorylation of this specific site is linked to enhanced interaction between NF-κBp65 and p300/CBP (54). Therefore, it is tempting to speculate that DC-SCRIPT modulates p300/CBP-dependent acetylation of NF-κBp65 K310 by reducing the level of Ser536 NF-κBp65 phosphorylation. Subsequent recruitment of the corepressors such as HDAC3 and RIP140 would then result in decreased DNA binding of NF-κBp65, thereby controlling IL-10 production in DCs. However, further studies are required to define the exact molecular mechanism.
NF-κBp65 also regulates other cytokines besides from IL-10, such as IL-6 and TNF-α. However, previously we have demonstrated that neither IL-6 nor TNF-α is affected by DC-SCRIPT knockdown (26). It is therefore likely that the combination of the specific S536 phosphorylation and K310 acetylation on NF-κBp65 is specific for only a subset of NF-κBp65–regulated genes like the observed increase in IL-10 production. Interestingly, under basal conditions, SCRIPT-KD-DCs readily displayed enhanced phosphorylation of S536 and acetylation of K310 NF-κBp65 compared with Ctrl-DCs. This suggests that already in immature DCs, DC-SCRIPT controls activation of NF-κBp65 and subsequent IL-10 production. After TLR7/8 triggering, the effect of DC-SCRIPT on IL-10 production was even more pronounced, as can be expected in mature DCs where NF-κB activity is strongly increased (25). Possibly, DC-SCRIPT increases the threshold for IL-10 induction in immature professional Ag-presenting DCs to prevent a premature tolerogenic phenotype and allows for the full immune potentiation function of mature DCs, by limiting IL-10–mediated feedback control. In the absence of DC-SCRIPT, NF-κBp65 thus appears to be more preactivated. Intriguingly, NF-κBp65 has been found to be constitutively active in prostate carcinoma (55), a cancer found to correlate with low DC-SCRIPT expression (30). This may suggest that, both in the immune system and in cancer, DC-SCRIPT is involved in controlling signaling cascades that lead to enhanced activation of NF-κBp65.
Inhibiting the NF-κB pathway or blocking p300/CBP showed only a partial reduction in IL-10 secretion after TLR ligation of SCRIPT-KD-DCs. This suggests that DC-SCRIPT not only dampens the NF-κB pathway but may also regulate other pathways that control IL-10 production in TLR-stimulated DCs. Therefore, it will be very interesting in future experiments to perform genome-wide analyses in moDCs to further investigate the pathways that are affected by DC-SCRIPT.
In conclusion, our current data provide novel insight into how DCs regulate expression of the important immune-dampening cytokine IL-10, and indicate that DC-SCRIPT may play a key role in the immunogenic potential of DCs. Understanding the immunogenic potential of DCs is crucial for exploiting these cells in immunotherapy to the benefit of patients with cancer, autoimmunity, or infectious diseases.
Acknowledgements
We thank Prof. Dr. Teunis B.H. Geijtenbeek (Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands) for helpful advice in assays concerning DNA binding and acetylation of NF-κB.
Footnotes
This work was supported by Dutch Cancer Society Grants KUN2011-5229 and KUN2009-4402.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- AA
anacardic acid
- ChIP
chromatin immunoprecipitation
- ChIPed
chromatin immunoprecipitated
- Ctrl-DC
control nontargeting siRNA-treated moDC
- DC
dendritic cell
- DC-SCRIPT
DC-specific transcript
- GILZ
glucocorticoid-induced leucine zipper
- GR
glucocorticoid receptor
- HAT
histone acetyltransferase
- HATi
HAT inhibitor
- HDAC
histone deacetylase
- moDC
monocyte-derived DC
- NR
nuclear receptor
- PTM
posttranslational modification
- qPCR
quantitative PCR
- RIP140
receptor-interacting protein 140
- SCRIPT-KD-DC
DC-SCRIPT knockdown moDC
- siRNA
small interfering RNA.
References
Disclosures
The authors have no financial conflicts of interest.