BET Bromodomain Inhibition Suppresses Transcriptional Responses to Cytokine-Jak-STAT Signaling in a Gene-specific Manner in Human Monocytes

Abstract

Disruption of the interaction of bromo and extra terminal (BET) proteins with acetylated histones using small molecule inhibitors suppresses Myc-driven cancers and TLR-induced inflammation in mouse models. The predominant mechanism of BET inhibitor action is to suppress BET-mediated recruitment of positive transcription elongation factor b (pTEFb) and thus transcription elongation. We investigated the effects of BET inhibitor I-BET151 on transcriptional responses to TLR4 and TNF in primary human monocytes and also on responses to cytokines IFN-γ, IFN-γ, IL-4 and IL-10 that activate the JAK-STAT signaling pathway and are important for monocyte polarization and inflammatory diseases. I-BET151 suppressed TLR4- and TNF-induced IFN responses by diminishing both autocrine IFN-β expression and transcriptional responses to IFN-β. I-BET151 inhibited cytokine-induced transcription of STAT targets in a gene-specific manner without affecting STAT activation or recruitment. This inhibition was independent of Myc or other upstream activators. Interferon-stimulated gene transcription is regulated primarily at the level of transcription initiation. Accordingly we found that I-BET151 suppressed the recruitment of transcriptional machinery to the CXCL10 promoter and an upstream enhancer. Our findings suggest that BET inhibition reduces inflammation partially through suppressing cytokine activity and expand the understanding of the inhibitory and potentially selective immunosuppressive effects of inhibiting BET proteins.

Introduction

Human monocytes and macrophages are essential cellular components of multiple physiological activities, including innate and adaptive immunity, tissue homeostasis and systemic metabolism. Their functional diversity requires a large degree of phenotypic and functional heterogeneity, which is finely tuned by local micro-environmental factors [1, 2]. Pro- inflammatory cytokines such as TNF and interferons drive inflammatory responses (also termed M1 classical activation) in human macrophages [1, 3], while regulatory cytokines such as IL- 4/13 and IL-10 induce alternative phenotypes (also termed M2) that dampen inflammation and promote tissue repair [4-7]. Dysregulated macrophage activity has been implicated in various diseases. Macrophages contribute to chronic inflammation in human autoimmune diseases, such as rheumatoid arthritis (RA), multiple sclerosis, atherosclerosis and type I diabetes [8-13]. On the other hand, undesired alternative phenotypes facilitate tumor growth in cancer environments by dampening local immune responsiveness and producing growth factors for angiogenesis [14-18]. Thus, understanding macrophage functional regulation is pivotal for developing therapeutic approaches to suppress pathogenic macrophage functions in a multitude of human conditions. Given that to a substantial extent the complexity of macrophage phenotypes is not well reflected by animal models or immortalized cell lines, it is important to study human primary monocytes and macrophages that closely reflect cells involved in inflammatory disease pathogenesis.

Macrophage functional phenotype is determined by patterns of gene expression, which are modulated by environmental cues [2]. These environmental cues trigger core signaling pathways that activate downstream expression of genes important for classical inflammatory (M1) or alternative/resolution (M2) phenotypes. Key inducers of inflammatory responses include microbial products, TNF, and IFN-γ, which activate inflammatory genes via NF-κB, MAPK, and Jak-STAT signaling pathways and IRF family transcription factors. IL-4/13 and IL-10 induce alternative activation via, respectively, STAT6 and STAT3, and also IRF4. Recently it has become clear that the binding of signal-activated transcription factors such as NF-κB and STATs to target genes is determined by the epigenetic landscape and chromatin states at target gene loci, which determine accessibility of gene regulatory elements to transcription factor binding [1, 19]. The epigenetic landscape is set during macrophage differentiation by master transcription factors PU.1 and C/EBPα/β, which bind to and ‘open’ chromatin at gene promoters and macrophage-specific distal regulatory elements (enhancers). The enhancer repertoire of immune cells can be altered by changes in chromatin that occur during activation or priming [21-23], thereby reprogramming their responses to subsequent environmental stimuli. Acute stimulation of macrophages by inflammatory stimuli such as microbial products also requires chromatin remodeling for effective induction of a subset of inflammatory genes [24, 25].

The implication of chromatin remodeling in the regulation of gene expression, including in various disease settings [26, 27] has led to the targeting of chromatin regulatory proteins as a novel approach to treatment. Small molecule inhibitors of chromatin modifiers, such as histone deacetylases, DNA methyltransferases and histone 3 lysine 27 methytransferase, have been implicated in cancer treatment and bear promise for broader and more diverse applications [28-30]. Recently, inhibitors blocking the recruitment and function of the bromodomain and extra terminal domain (BET) family proteins (BrdT, Brd2, Brd3 and Brd4) have been of particular interest, as they suppress inflammation and cancer progression in disease models [31, 32]. The tandem bromodomains on BET proteins form binding pockets that recognize acetylated lysine on histones including H3 and H4 (Ac-H). This feature of BET proteins confers the ability to govern the assembly of histone acetylation-dependent chromatin complexes that results in recruitment of the active form of positive transcription elongation factor b (p-TEFb) [33]. Active p-TEFb subsequently phosphorylates RNA polymerase II on Ser2 in its C-terminal domain during promoter clearance at the start of transcription elongation [34]. In addition to its role on gene promoters, recent work has demonstrated recruitment of Brd4 to subsets of distal enhancers, including superenhancers and anti-pause enhancers, which promotes recruitment of p-TEFb and gene transcription [23], presumably via enhancer-promoter looping [35, 36]. As the enhancer repertoire is exquisitely cell-type specific [37-39] and can change with cell activation status [21, 22], this suggests that targeting of different enhancers in different cell types may contribute to cell type-specific effects of BET inhibitors.

The small molecule inhibitors I-BET151 and JQ-1 that disrupt interaction of BET proteins with acetylated histones have demonstrated efficacy in cancer models at least in part because of suppression of Myc expression [40-43], and one study has shown that I-BET suppresses induction of a subset of TLR4-induced genes in mouse macrophages and suppresses inflammation in mouse models [32]. However, the efficacy of I-BET151 in suppressing inflammatory responses in human macrophages and its effects on transcriptional responses to cytokine stimulation have not been characterized [44]. In this study, we investigated the effects of I-BET151 on transcriptional responses to TLR4 and TNF stimulation, and also to cytokines that activate the JAK-STAT signaling pathway, including IFN-β, IFN-γ, IL-4 and IL-10, in primary human macrophages. We found that I-BET151 suppressed TLR4- and TNF-induced IFN responses and interferon-stimulated gene (ISG) expression by a dual mechanism of diminishing IFN-β expression (as shown previously in mouse macrophages [32] and by suppressing transcriptional responses to IFN-β. I-BET151 inhibited cytokine-induced transcription in a gene- specific manner but did not affect activation of STATs, as assessed by tyrosine phosphorylation, nuclear translocation, and recruitment to the CXCL10 gene locus. Instead, I-BET151 suppressed the recruitment of Brd4, TBP, and RNA polymerase II to the CXCL10 promoter and an upstream enhancer. These results demonstrate that I-BET151 inhibits cytokine-induced transcriptional responses and significantly expands our understanding of the inhibitory and potentially immunomodulatory effects of inhibiting BET proteins.

Results

I-BET151 suppresses TNF- and LPS-induced CXCL10 and CXCL11 expression in human macrophages

Induction of interferon-stimulated genes (ISGs) by LPS and TNF is mediated by induction and autocrine action of IFN-β [3]; LPS and TNF induce IFNB by distinct mechanisms, mediated by, respectively, IRF3 and IRF1. Previous work showed that I-BET151 suppresses LPS-induced expression of ISGs such as Cxcl10 in mouse bone marrow-derived macrophages (BMDM) and suggested that this occurs in part by inhibition of IFN-β production [32]. We wished to test whether I-BET151 also inhibits TNF-induced ISG expression, and whether IBET151 inhibits IFN-β signaling in addition to suppressing IFN-β expression. First, we found that I-BET151 inhibits LPS-induced expression of CXCL10, CXCL11 and IFNB in a dose- and time-dependent manner in human macrophages (Fig. 1A-C), which confirms previous results in mouse BMDM [31]. I-BET151 also suppressed TNF-induced CXCL10 and CXCL11 expression in a dose-dependent manner, and effectively suppressed TNF-induced IFNB expression (Fig. 1D-F). Consistent with these results, I-BET151 suppressed LPS- and TNF-induced activation of STAT1 tyrosine phosphorylation (which is mediated by autocrine IFN-β) without changing total STAT1 levels (Fig. 1G). Cell viability was not affected by the addition of I-BET151, as assessed by an MTT assay (Fig. 1H). These results show that I-BET151 suppresses LPS- and TNF-induced expression of ISGs in human macrophages, and suggest this effect is mediated at least in part by suppression of IFN-β production.

Figure 1. I-BET151 suppresses CXCL10 and CXCL11 expression in TNFα- or LPS-stimulated human macrophages.

(A-C) Macrophages were treated with I-BET151 (0.5μM or as indicated) or control vehicle (DMSO) for 0.5 hour and then stimulated with LPS (10ng/ml) for the indicated times or for 1 hr (A). Gene expression was measured by real-time qPCR and presented as relative expression to untreated conditions. (D-F) Macrophages were treated with IBET151 (0.5μM or as indicated) or control vehicle (DMSO) for 0.5 hour and then stimulated with LPS (10ng/ml) for the indicated times or 6 hr (D). (G) Human primary macrophages were stimulated with LPS (left, 20ng/ml) or TNF (right, 10ng/ml) with or without I-BET151 treatment prior to stimulation. STAT1 Tyr701 activating phosphorylation at the indicated time points was measured by Western blot. Total STAT1 and p38 levels were used as loading controls. (H) Human primary macrophages were treated with I-BET151 alone, LPS alone, or with LPS following I-BET151 pretreatment (I+L). The MTT assay was then conducted to assess cell survival. No significant differences in cell survival were detected between these conditions by one-way ANOVA test. (B,C,E,H) Significance of expression fold changes due to I-BET151 was tested using a one-tailed t-test. p < 0.05 is considered statistically significant. (A,D) Dose responses were approximated to a line through linear regression (gene expression versus IBET151 dosage). Significance of a negative slope (β) was tested using a one-tailed t-test. Average slope: (A) CXCL10: β=-1.365 (p=0.0164); CXCL11: β=-1.612 (p=0.0288). (D) CXCL10: β=-1.408 (p=0.002); CXCL11: β=-1.6152 (p=0.0151). (N = 3 for Fig. 1A, 1B, 1C, 1E, 1F and 1H. N = 4 for Fig. 1D.)

I-BET151 suppresses IFN-β-induced transcription of CXCL10 and CXCL11

We next addressed whether, in addition to inhibiting IFN-β production, I-BET151 could also inhibit IFN-β function by suppressing cellular responses to IFN-β. Human monocytes were stimulated with IFN-β and the induction of CXCL10 and CXCL11 was compared between IBET151 treated and untreated conditions. CXCL10 and CXCL11 were significantly induced 3 hours after IFN-β treatment and their expression continued to increase at 6 hours (Fig. 2A). IBET151 suppressed their expression at both time points (Fig. 2A) and in a dose-dependent manner (Fig. 2B). However, in contrast to LPS or TNF stimulation, I-BET151 did not inhibit IFN-β-induced STAT1 tyrosine phosphorylation (Fig. 2C). Thus, I-BET151 does not inhibit proximal steps in IFN-β-induced JAK-STAT signaling. The intact activation of STAT1 suggested that I-BET151 may not inhibit all IFN-β-induced STAT target genes. Consistent with this notion, IFN-β-induced expression of the ISGs IFIT1, IFIT2, STAT1 and IRF1 was much more resistant to inhibition by I-BET151 than induction of CXCL10 and CXCL11 (Fig. 2D). In contrast, LPS- and TNF-induced expression of IFIT1, IFIT2 and IRF1 was effectively suppressed by I-BET151 (Fig. 2E and 2F), consistent with suppression of autocrine IFN-β resulting in a global reduction in ISG expression. Collectively, the results indicate that I-BET151 inhibits transcriptional responses to IFN-β in a gene-specific manner, and suggest that I-BET151 targets mechanisms downstream of STAT tyrosine phosphorylation to inhibit ISG transcription.

Figure 2. I-BET151 selectively suppresses expression of a subset of IFN-β-induced ISGs.

(A) Human monocytes were treated with I-BET151 (0.5μM) or control vehicle (DMSO) for 0.5 hour and then stimulated with IFN-β (20ng/ml) for indicated times. Gene expression was measured by real-time qPCR and is presented as expression relative to untreated condition. (B) Dose-dependent effect of I-BET151 on IFN-β-stimulated CXCL10 and CXCL11 expression in macrophages (3 hr time point). CXCL10: β=-1.576 (p=0.0016); CXCL11: β=-1.561 (p=0.0024). (C) Human primary macrophages were stimulated with IFN-β with or without I-BET151 pretreatment. STAT1 Tyr701 phosphorylation at indicated time points was measured by Western blot. Total STAT1 and p38 were also plotted as loading controls. (D-F) The effect of I-BET151 on ISG expression induced by (D) IFN-β (E) LPS or (F) TNF stimulation. Data shown are representative of at least three experiments. Statistical analyses were performed as in Figure 1. (D) IFIT1: β=0.026 (p=0.9570); IFIT2: β=-0.087 (p=0.2045); STAT1: β=-0.787 (p=0.1454); IRF1: β=-0.394 (p=0.0603). (E) IFIT1: β=-0.7012 (p=0.3378); IFIT2: β=0.0837 (p=0.1841); IRF1: β=-0.956 (p=0.8941). (F) IFIT1: β=-0.6828 (p=0.4212); IFIT2: β=-0.393 (p=0.3553); IRF1: β=-0.705 (p=0.1303). (N = 3 for Fig. 2A-2E. N = 4 for Fig. 2F.)

Gene-specific inhibition of IFN-γ, IL-4 and IL-10 responses by I-BET151

Different JAK-STAT pathways can be activated by various cytokines. While Type I interferons are pleiotropic in inflammatory regulation, type II interferon IFN-γ strongly promotes M1 activation. Unlike Type I interferons (such as IFN-β) which function through Tyk2 and Jak1 activation and ISGF3 (STAT1/STAT2/IRF9) recruitment to IFN-stimulated response elements (ISRE) to activate transcription of IFN-inducible genes (ISGs) [45], IFN-γ signals through Jak1 and Jak2, which leads to the binding of STAT1 homodimers to IFN-γ-activated sequence (GAS) for gene activation [46]. A recent study has reported differences in the requirement of STAT1 cooperative binding between type I and type II interferon signaling [47]. Thus, we next examined whether I-BET151 affects the IFN-γ response differently from the IFN-β response. IFN-γ strongly induced expression of targets including CXCL10, CXCL11, IRF1 and STAT1 (Fig. 3A). Although IFN-γ-induced gene expression was more resistant to inhibition by I-BET151 than IFN-β-induced gene expression, we observed a similar pattern of stronger suppression of CXCL11 than of CXCL10 (Fig. 3A). Similar to IFN-β stimulation, IFN-γ-induced IRF1 and STAT1 expression was not decreased by I-BET151. The similar pattern of gene-specific inhibition of type I and type II IFN responses by I-BET151 supports the notion that repression occurs at gene loci downstream of signaling.

Figure 3. I-BET151 represses IFN-γ, IL-4 and IL-10 targets in gene-specific manner.

Human macrophages were treated with I-BET151 (0.5μM) or control vehicle (DMSO) for 0.5 hour and then stimulated with (A) IFN-γ (100 units/ml), (B) IL-10 (20ng/ml) or (C) IL-4 (40ng/ml) for the indicated times. Gene expression was measured by real-time qPCR and presented as expression relative to untreated conditions. Data shown are representative of at least three experiments. Statistical analyses were performed as in Figure 1. (N = 3 for all panels).

We wished to test more broadly the pattern of I-BET151's inhibitory effects on transcriptional responses to other cytokines and different STATs. We investigated the effect of IBET151 upon gene induction by IL-4 or IL-10, which activate, respectively, STAT6 and STAT3 and promote alternative activation and pro-resolution responses in macrophages. I-BET151 suppressed the expression of IL-10 target genes including IL-7R, ABIN3, and ENPP2 (Fig. 3B), but not SOCS3, indicating gene specificity of I-BET151 inhibition during IL-10 stimulation. IBET151 also selectively suppressed IL-4-inducible genes, demonstrated by inhibition of PPARγ, ENPP2, and MS4A4A, but lesser suppression of JMJD3, upon IL-4 stimulation (Fig. 3C). These results show that I-BET151 can inhibit transcription activation by multiple cytokines and STATs, but these inhibitory effects are gene-specific.

I-BET151 suppression is not dependent on Myc or new protein synthesis

A major mechanism of action of I-BET151 and the related inhibitor JQ-1 is to suppress the induction of Myc, and thereby suppress activation of Myc target genes [43]. Therefore we tested whether I-BET151 could inhibit cytokine-induced gene transcription indirectly through the inhibition of Myc expression and function. For that purpose, we tested whether induction of genes sensitive to I-BET151 was also sensitive to Myc inhibitor 10058-F4. Inhibition of Myc by 10058-F4 did not repress IFN-β-, IL-10- or IL-4-induced expression of genes that were sensitive to I-BET151 (Fig. 4A-C), and did not affect TNF-induced expression of CXCL10 or CXCL11 (Fig. 4D). Significant inhibition of ALOX15, a known Myc target [48], confirmed the efficacy of 10058-F4 in inhibiting Myc function (Fig. 4C). In LPS-stimulated human monocytes, 10058-F4 partially decreased CXCL10 and CXCL11 expression (Fig. 4E), but the effect was minor compared to the strong inhibitory effect of I-BET151 (Fig. 1E), suggesting that Myc may contribute to LPS-induced IFN-β production in a limited way. The addition of 10058-F4 also did not significantly affect cell viability compared to the media control or I-BET151 (Fig. 4F). The results suggest that I-BET151 inhibits expression of cytokine-induced STAT target genes analyzed in this study independently of the inhibition of Myc.

Figure 4. I-BET151 inhibitory effect on cytokine-induced gene expression is independent of cMyc inhibition.

Human macrophages were treated with the Myc inhibitor (10058-F4) or vehicle for half an hour before stimulation with (A) IFN-β, (B) IL-10, (C) Il-4, (D) TNF or (E) LPS for 3 hours. Gene expression was measured by real-time qPCR and is presented as expression relative to untreated conditions. (F) Human primary macrophages were treated with IBET151 and 10058-F4. The MTT assay was then conducted to assess cell survival. No significant differences in cell survival were detected between these conditions by one-way ANOVA. Statistical analyses were performed as in Figure 1. (N = 3 for all panels).

To test whether I-BET151 could work by suppressing cytokine-induced expression of transcription factors other than Myc, we performed experiments using cycloheximide (CHX), a protein synthesis inhibitor. Induction of the IFN-β, IL-10 and IL-4 target genes CXCL10, CXCL11, IL7R, ENPP2 and PPARG did not require de novo protein synthesis (Fig. 5A-C), consistent with direct induction of primary response STAT target genes. Interestingly, inhibition of gene induction by I-BET151 was preserved when protein synthesis was blocked (Fig. 5A-C). The addition of CHX alone or in conjunction with I-BET151 did not significantly affect total cell viability (Fig. 5D). These results showing that neither gene induction nor inhibition by I-BET151 require de novo protein synthesis exclude the possibility that I-BET151 works by blocking the induction of transcription factors that cooperate with STATs to drive gene expression. The data also suggest that the effects of I-BET151 are not mediated by de novo synthesis of transcriptional repressors. Collectively, the results argue against indirect mechanisms of IBET151 action and are consistent with the notion that I-BET151 may act directly at target gene loci to suppress transcription.

Figure 5. Gene repression by I-BET151 is independent of de novo protein synthesis.

Human macrophages were treated with I-BET151 or vehicle in the presence or absence of cycloheximide (CHX) half an hour before stimulation with (A) IFN-β, (B) IL-10 or (C) IL-4. Gene expression was measured by real-time qPCR and presented as relative expression to untreated cells. (D) Human primary macrophages were treated with I-BET15, CHX, and CHX with I-BET151 pretreatment. The MTT assay was then conducted to assess cell survival. No significant differences in cell survival were detected between these conditions by one-way ANOVA. I+C: IBET151+CHX. Statistical analysis of the effect of CHX was performed using a two-tailed t-test to compare the fold changes in the I-BET151 condition and the CHX and I-BET151 condition. The addition of CHX did not statistically significantly reverse the inhibition due to I-BET151 in any condition. (N = 3 for all panels.)

I-BET151 suppresses recruitment of general transcription factors to the CXCL10 locus

We then analyzed how I-BET151 affects IFN-β-mediated transcription factor recruitment and histone modifications at the CXCL10 locus. We found that I-BET151 did not affect IFN-β-induced recruitment of STAT2 to the CXCL10 promoter (Fig. 6A). Accordingly, IBET151 did not affect IFN-β-induced increases in histone acetylation (Fig. 6B), which are thought to be mediated by STATs. However, as expected, I-BET151 suppressed recruitment of BRD4 to the CXCL10 promoter (Fig. 6C) and also to a putative enhancer located 4 kb upstream of the CXCL10 TSS (Fig. 6C), which is consistent with its major proposed mechanism of action. In addition, I-BET151 suppressed the recruitment of general transcription factors RNA polymerase II and TBP to the CXCL10 promoter (Fig. 6D and 6E) and enhancer (Fig. 6F and 6G). Consistent with the observation that I-BET151 did not significantly inhibit IFN-β-induced IRF1 or STAT1 (Fig. 2D), Pol II recruitment to the regulatory regions of these two genes were not significantly affected by I-BET151 (Fig. 6D). These results are distinct from the model that I-BET151 predominantly suppresses transcription elongation in most cell types [32, 49, 50], but are consistent with a previous report that I-BET151 suppressed LPS-induced RNA pol II occupancy near the TSS in mouse macrophages [32].

Figure 6. I-BET151 inhibits RNA polymerase II complex assembly at the CXCL10 promoter and enhancer.

Human primary macrophages were stimulated with IFN-β (100ng/ml) for 3 hours with or without I-BET151 pre-treatment for half an hour. ChIP assays were used to measure recruitment of STAT2 (A), levels of histone 4 acetylation (B), BRD4 recruitment (C) and pol II (D) and TBP recruitment (E) to the CXCL10 promoter. ChIP was used to measure BRD4 (C), pol II (F), and TBP (G) recruitment to an enhancer located 5 kb upstream of the CXCL10 transcription start site. Occupancy of the hemoglobin (HBB) promoter was used as a negative control. Data shown are representative of two to three experiments. Statistical analyses were performed as in Figure 1. (N=3 for Fig. 6C and 6D, and N=2 for the remaining panels.)

Materials and Methods

Reagents

I-BET151 was from GlaxoSmithKline. Recombinant human TNF-α and recombinant IFN-β were from R&D Systems. Escherichia coli LPS used for cell stimulation was from Chemicon International. Recombinant human IL-4 and recombinant human IL-10 were from Peprotech. Cycloheximide (cat #: C4859), Myc inhibitor, 10058-F4 (cat #: F3680), and MTT reagent, Thiazolyl Blue Tetrazolium Bromide (cat #: M5655), were from Sigma Aldrich.

Cell culture

Peripheral blood mononuclear cells were obtained from blood leukocyte preparations (purchased from the New York Blood Center) by density-gradient centrifugation with lymphoprep (Accurate Chemical, cat #: AN1001969) following a protocol approved by the Hospital for Special Surgery Intuitional Review Board. CD14+ monocytes were purified from fresh peripheral blood mononuclear cells with anti-CD14 magnetic beads (Miltenyi Biotec, cat #: 130-050-201) as recommended by the manufacturer. Macrophages were derived by culture of monocytes for 24 hours in MEM-α medium (Invitrogen, cat #: 12561-072) supplemented with 10% (vol/vol) FBS (Hyclone, cat #: SH30070.03), 10% (vol/vol) L-Glutamine (Invitrogen, cat #: 25030-081), 10% (vol/vol) Penicillin/Streptomycin (Invitrogen, cat #: 15070-063), and human macrophage colony-stimulating factor (M-CSF; 10ng/ml; Peprotech, cat#: 300-25). DMSO or IBET151 (500nM) was added thirty minutes prior to the addition of the indicated stimulus. The stimuli were recombinant human TNF-α (10ng/ml), LPS (10ng/ml for mRNA analysis and 50ng/mL for ChIP), recombinant IFN-β (20units/ml for mRNA analysis and 200 units/ml for ChIP), recombinant human IFN-γ (100 units/ml), recombinant human IL-4 (100ng/ml), or recombinant human IL-10 (50ng/ml). The samples were harvested at the indicated time points.

MTT assay

Human primary monocytes (2.5x10⁵ cells/well) were cultured in a 96-well plate at 37 °C, and were treated with inhibitors and stimuli for indicated hours. Then 10% thiazoyl blue tetrazolium bromide (5mg/mL in PBS) was added. Cells were incubated for 3.5 hours at 37 °C. MTT solvent (4mM HCl, 0.1% NP-40 in isopropanol) equal to culture volume was added to dissolve the formazan crystals. Absorbancies were read at 590nm with a reference filter of 620nm [51].

Analysis of mRNA

For quantitative real-time PCR, DNA-free RNA was extracted with an RNeasy Mini Kit (Qiagen, cat #: 74104) with DNase (Qiagen, cat #: 79254) treatment. 0.2mg of total RNA was reverse-transcribed with a First Strand cDNA Synthesis kit (Fermentas, cat #: K1622). Real-time PCR was done in triplicate with the SYBR Green Master Mix (Applied Biosystems, cat #: 4385618), oligonucleotide primers (Life Technology), and the 7500 Fast Real-Time PCR System (Applied Biosystems). Gene expression is normalized to the expression of glyceraldehyde phosphate dehydrogenase (GAPDH) and is presented relative to the expression of untreated cells (set as 1). Primer sequences are as following: GAPDH forward: 5’-ATCAAGAAGGTGGTGAAGCA-3’; reverse: 5’- GTCGCTGTTGAAGTCAGAGGA-3’. CXCL10 forward: 5’-ATTTGCTGCCTTATCTTTCTG-3’; reverse: 5’- TCTCACCCTTCTTTTTCATTGTAG-3’). CXCL11 forward: 5’- GAAGGATGAAAGGTGGGTGA-3’; reverse: 5’-AAGCACTTTGTAAACTCCGATG-3’. IFNB forward: 5’- AGCAGTTCCAGAAGGAGGAC-3’; reverse: 5’- TGATAGACATTAGCCAGGAGGTT-3’.

Immunoblot

Whole-cell extracts and nuclear extracts were obtained and protein amounts were quantified by the Bradford assay (Biorad) as described [52]. For immunoblot analysis, 5μg cell lysates were separated by 7.5% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, cat #: IPVH00010). The membranes were incubated with specific antibodies overnight and chemiluminescence (Perkin Elmer, cat #: NEL105) was used for detection. Rabbit polyclonal rabbit antibody to STAT1 (cat #: sc-345), STAT2 (cat #: sc-476), and p38α (cat #: sc-535) were from Santa Cruz. Rabbit polyclonal antibody to phosphorylated STAT1 at Tyr701 (cat #: 9171S) and Akt (cat #: 9272S) was from Cell Signaling Technology. Rabbit polyclonal antibody to Lamin B1 (cat #: ab16048) was from Abcam.

Chromatin Immunoprecipitation (ChIP)

Macrophages plated in tissue culture dishes were fixed with formaldehyde and the fixing reaction stopped with glycine. Fixed cells were lysed and sonicated at the high power output for 20 cycles with the Bioruptor (Diagenode). The samples were then incubated with antibody conjugated to Dynabead Protein A (Invitrogen, cat#: 100-02D) at 4°C overnight. The DNA fragments were purified with the Qiaquick PCR purification kit (Qiagen, cat#: 28106) and analyzed by quantitative real-time PCR. Their enrichment was normalized relative to input DNA amount. Primer sequences are as following:

CXCL10 promoter forward: 5’-GTGCTGAGACTGGAGGTTCC-3’; reverse: 5’-GGGAGGGAAAATGGCTTTGC-3’. CXCL10 enhancer forward: 5’-CCGTTTCAGTCGCTATTGATTT-3’; reverse: 5’-CTGATGTCCTCCTGCTCACTTT-3’. HBB promoter forward: 5’-GAGGGCTGAGGGTTTGAAGT-3’; reverse: 5’-TGCTCCTGGGAGTAGATTGG-3’.

Statistical Analysis

Statistical analyses were performed using one-tailed t-test or ANOVA as indicated in figure legends. p-values less than 0.05 were considered significant.

Discussion

Recent years have witnessed an increasing interest in epigenetic targets in small molecule drug development, which provide additional possibilities for addressing drug resistance and combinational therapy. The BET protein inhibitor I-BET151 and its analogs have been tested for their inhibitory effects on inflammation and tumor growth in mouse models [31, 32, 40-43]. However, I-BET151's efficacy in human cells is less well documented. Considering the important role of monocytes and macrophages in various physiological conditions and the broad application of their modulation in clinical settings, we characterized the inhibitory effects of IBET151 in human primary monocytes after stimulation with factors that promote either classical or alternative polarization. We found that I-BET151 inhibited transcriptional responses to multiple cytokines that activate distinct STATs in a gene-specific manner, but did not suppress IFN-induced activation of STATs or their recruitment to target gene loci. I-BET151 suppressed ISG expression independently of its effects on Myc or other upstream activators. These results suggest that a subset of ISGs are direct targets for inhibition by I-BET151, and that inhibition of cytokine-induced transcriptional responses may contribute to the efficacy of I-BET151 in suppressing inflammatory responses.

In pro-inflammatory responses, the CXCR3-binding family of chemokines CXCL10 and CXCL11 are induced by various stimuli in human macrophages with significant relevance in disease pathologies. CXCL9/10/11 function as potent Th1-recruiting chemokines in the pathophysiology of multiple diseases including asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, ulcerative colitis (UC), and Crohn's disease. In particular, studies of various anti-CXCL10 human monoclonal antibodies are in phase II clinical trials for the treatment of RA, UC, and Crohn's disease [53, 54]. I-BET151 potently suppresses the induction of CXCL10 and CXCL11 by multiple stimuli including LPS, TNF, IFN-β and IFN-γ. In the case of TNF or LPS, I-BET151 suppresses CXCL10 and CXCL11 induction by dual and complementary mechanisms of suppressing autocrine IFN-β production and suppressing IFN-β-induced transcriptional responses.

In IFN-β-stimulated cells, expression of a subset of ISGs including CXCL10 and CXCL11 was decreased by I-BET151 despite unaffected JAK-STAT signaling and recruitment of STATs to the promoter. This suggests that BET proteins play an essential role downstream of STAT recruitment in the expression of certain genes, including CXCL10 in IFN-β-stimulated human monocytes, implying greater selectivity compared to JAK inhibitors in IFN responses. Notably, I-BET151 exhibited differential inhibitory effects on IFN-β target gene expression. The expression of genes such as IFIT1 and STAT1 was minimally reduced by I-BET151 compared to that of CXCL10 and CXCL11. As BRD4 recruitment to the promoters of all four of the aforementioned genes upon IFN-β stimulation has been observed [55], the most likely explanation for differential sensitivity to I-BET151 is recruitment of BET proteins to some genes by mechanisms independent of interaction with acetylated histones [55].

The major mechanism of I-BET151 action in disrupting BET-mediated recruitment of p-TEFb and thereby suppressing transcription elongation has been defined primarily in transformed cell lines and proliferating cells such as embryonic stem cells [31, 32, 56, 57]. IBET151 sensitive cells are dependent on Myc for proliferation and survival, and thus a major component of I-BET151 action is mediated by suppression of Myc expression. In contrast, in terminally differentiated nonproliferating primary human macrophages I-BET151 inhibited STAT target gene expression independently of Myc inhibition. In addition, ISG transcription is regulated primarily at the level of transcription initiation [58-60], although a recent report suggests that recruitment of elongation suppressors NELF and DSIF occurs after IFN stimulation [55]. Consistent with the important role of transcription initiation in ISG expression, we found that I-BET151 suppressed recruitment of pol II, and also of TBP, which is associated only with transcription initiation complexes, to the CXCL10 locus. A function for I-BET151 in suppressing transcription initiation in non-proliferating macrophages is also supported by a previous report showing that I-BET151 suppressed pol II recruitment at the TSS in LPS-stimulated mouse macrophages [32]. The mechanism by which I-BET151 can suppress pol II and TBP recruitment will need to be clarified in future work but is likely related to the interaction of BET protein with various distinct transcription regulators [35, 61].

In conclusion, this study explored the inhibitory effect and mechanism of action of IBET151 on gene expression in response to cytokines that activate the Jak-STAT pathway and regulate the phenotype of human primary monocytes. Inhibition of transcription of ISGs that are regulated at the level of transcription initiation extends the mechanisms of action of BET inhibitors. Gene-specific inhibition of various STAT target genes suggests a broader consideration of the potential therapeutic utility of BET inhibitors. Selectiveness of gene repression by I-BET151 could be further advanced by genome-wide profiling of gene expression, which would allow more comprehensive investigation of the characteristics of genes in each category. This would yield further insight into the nature of the inhibitory mechanism by I-BET151 and provide guidance for the therapeutic application of this inhibitor. In addition, ChIP-seq could also be utilized to evaluate the extent I-BET151's effect on gene expression through either transcriptional initiation or transcriptional elongation on a genome-wide scale.

Abbreviations

BET

bromo and extra-terminal

C/EBP

CCAAT/enhancer-binding protein alpha

ISGF

interferon-stimulated gene factor

ISRE

interferon-sensitive response element