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. Author manuscript; available in PMC: 2017 May 15.
Published in final edited form as: J Immunol. 2016 Apr 15;196(10):4132–4142. doi: 10.4049/jimmunol.1502261

BET inhibition Attenuates H. pylori-induced Inflammatory Response by Suppressing Inflammatory Gene Transcription and Enhancer Activation

JinJing Chen *,†, Zhen Wang †, Xiangming Hu *, Ruichuan Chen ‡, Judith Romero-Gallo §, Richard M Peek Jr §, Lin-Feng Chen *,#,
PMCID: PMC4868794  NIHMSID: NIHMS771287  PMID: 27084101

Abstract

H. pylori infection causes chronic gastritis and peptic ulceration. H. pylori-initiated chronic gastritis is characterized by enhanced expression of many NF-κB-regulated inflammatory cytokines. Brd4 has emerged as an important NF-κB regulator and regulates the expression of many NF-κB-dependent inflammatory genes. In this study, we demonstrated that Brd4 was not only actively involved in H. pylori-induced inflammatory gene mRNA transcription but also H. pylori-induced inflammatory gene eRNA synthesis. Suppression of H. pylori-induced eRNA synthesis impaired H. pylori-induced mRNA synthesis. Furthermore, H. pylori stimulated NF-κB-dependent recruitment of Brd4 to the promoters and enhancers of inflammatory genes to facilitate the RNAPII-mediated eRNA and mRNA synthesis. Inhibition of Brd4 by JQ1 attenuated H. pylori-induced eRNA and mRNA synthesis for a subset of NF-κB-dependent inflammatory genes. JQ1 also inhibited H. pylori-induced interaction between Brd4 and RelA and the recruitment of Brd4 and RNAPII to the promoters and enhancers of inflammatory genes. Finally, we demonstrated that JQ1 suppressed inflammatory gene expression, inflammation, and cell proliferation in H. pylori-infected mice. These studies highlight the importance of Brd4 in H. pylori-induced inflammatory gene expression and suggest that Brd4 could be a potential therapeutic target for the treatment of H. pylori-triggered inflammatory diseases and cancer.

Keywords: Brd4, enhancer RNA, H. pylori, inflammation, JQ1, NF-κB, transcription

Introduction

Infection with H. pylori, a gram-negative bacterium that colonizes gastric epithelium and induces chronic inflammation, is the strongest risk factor for the development of gastric cancer (1, 2). The pathogenesis of H. pylori is believed to be associated with infection-initiated chronic gastritis, which is characterized by enhanced expression of many inflammatory genes. The transcription factor NF-κB plays a key role in H. pylori-initiated inflammatory response and gastritis by regulating the expression of these inflammatory genes (3–8).

NF-κB-dependent inflammatory gene expression is regulated at multiple levels, including cytoplasmic signaling events leading to the nuclear translocation of NF-κB, binding of nuclear NF-κB to various transcription factors or regulators, and the posttranslational modifications of histones and NF-κB (9, 10). Within the nucleus, NF-κB recognizes the cognate NF-κB sites on the enhancer or promoter regions of its target genes and directs the binding of co-regulators to form the transcriptional machinery for target gene expression (11). Recent studies demonstrate that posttranslational modifications of NF-κB play a key role in the recruitment of different transcription regulators to the promoters or enhancers (12, 13). For example, phosphorylation of RelA at serines 276 and 536 facilitates the recruitment of histone acetyltransferase p300/CBP, leading to the activation of NF-κB genes (14, 15). Additionally, phosphorylation-dependent acetylation of RelA at lysine-310 recruits bromodomain-containing factor Brd4 to activate the positive transcription elongation factor b (P-TEFb) complex, which phosphorylates serine 2 of RNA polymerase II for the transcription elongation of NF-κB-mediated inflammatory genes (16, 17).

In addition to its critical role in the synthesis of mRNA of inflammatory genes, emerging evidence indicates that NF-κB also contributes to the synthesis of enhancer RNA (eRNA) of inflammatory genes (18, 19). eRNAs belong to the non-coding RNAs and their transcripts are directed by enhancers. Recent studies suggest that eRNAs participate in enhancer function, creating a hierarchical cascade of transcript-mediated regulation of transcription by looping the enhancers and the promoters or by recruiting RNAPII to the promoters (20–22). Importantly, eRNAs and enhancer functions have been shown to be involved in the regulation of inflammatory transcription networks (19, 23, 24). However, it remains unclear whether eRNAs might participate in H. pylori-induced inflammatory gene expression.

Brd4 belongs to the bromodomain extra terminal (BET) family and has emerged as an important epigenetic regulator in inflammatory gene transcription and cancer development (25, 26). By binding to acetylated histones or non-histone proteins, Brd4 regulates gene transcription by recruiting different transcription components, such as Mediator and P-TEFb (25, 26). Brd4 binds to P-TEFb and activates CDK9 to phosphorylate the negative elongation factor (NELF) and C-terminal domains of RNAPII to activate NF-κB-dependent inflammatory gene expression (16, 27). Brd4 has also been shown to participate in the formation of super-enhancers and the activation of inflammatory gene expression during inflammatory responses (18, 28). Interestingly, recent studies also demonstrate that Brd4 is actively involved in the eRNA synthesis of NF-κB-dependent inflammatory genes (18, 24).

Small molecules targeting bromodomains of Brd4 display strong anti-inflammatory and anti-cancer activities (29–31). These small molecules prevent the interaction between the BET bromodomains and acetylated lysine peptides (32). For instance, one of the BET inhibitors (BETi) I-BET reduces LPS-driven expression of key inflammatory genes and suppresses LPS or bacteria-induced sepsis in mice (33). Another BETi JQ1 is able to suppress TNF-α- or IL-1β- induced inflammatory cytokine expression and abrogates super enhancer-mediated inflammatory responses and diseases by mainly targeting Brd4 (28, 34–36). These studies highlight the importance of Brd4 in regulating the expression of inflammatory genes and the therapeutic potential of Brd4 inhibitors in the treatment of inflammatory diseases. However, whether Brd4 has any role in H. pylori-mediated inflammatory gene expression and whether inhibitors of Brd4 could be therapeutics for H. pylori-induced gastric diseases remain largely undetermined.

In an effort to explore the mechanism underlying H. pylori-induced inflammatory gene expression and the role of Brd4 in the activation, we demonstrated that Brd4 was essential for the H. pylori-induced NF-κB-dependent inflammatory gene expression by activating the expression of both enhancer and messenger RNAs. Brd4 was recruited to the promoters and enhancers of inflammatory genes to facilitate the expression of eRNA and mRNA upon H. pylori infection. Inhibition of Brd4 by JQ1 down-regulated H. pylori-induced mRNA and eRNA synthesis of inflammatory genes and suppressed H. pylori-induced gastric inflammation in mice.

Materials and Methods

Cell lines, plasmids, reagents and antibodies

Human MKN28 gastric adenocarcinoma cells have been previously described (37) and cultured in 1640 supplemented with 10% fetal bovine serum (FBS). JQ1 has been previously described (36). IKK2 inhibitor IV was from EMD Millipore (Billerica, MA, USA). Antibodies against IgG, RelA and RNAPII were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-Brd2 antibody was from Cell Signaling (Danvers, MA, USA); anti-Brd3 and Brd4 antibodies were from Bethyl Laboratories (Montgomery, TX, USA); antibodies against phosphorylated Serine 2 RNAPII and anti-H3K27Ac were from Abcam (Cambridge, MA, USA); anti-H3K4Me3 and anti-H3K4Me1 antibodies were from EMD Millipore (Billerica, MA, USA); anti-actin antibody was from Sigma-Aldrich (Saint Louise, MO, USA).

H. pylori strains, H. pylori culture and infection

H. pylori G27, a cagPAI (cag pathogenicity island)-positive and virulence factor CagA-positive clinical isolate, has been previously described (38). SS1, the mouse-adapted H. pylori strain with non-functional cagPAI, was used in the mice infection experiment. H. pylori was cultured in bisulphite-free Brucella broth supplemented with 10% fetal bovine serum and 5 µg/ml vancomycin at 37°C in the presence of 10% CO2. H. pylori cultured overnight was added to MKN28 cells for infection at a multiplicity of infection of 50–100.

Immunoblotting analysis and quantitative real-time PCR analysis

Immunoblotting analysis and quantitative real-time PCR analysis were performed as previously described (16). RT-PCR array was performed according to Qiagen’s RT-PCR array for NF-κB target genes. PCR primers were purchased from Integrated DNA Technologies, Inc (Coralville, Iowa, USA).

siRNA knockdown

siRNAs targeting Brd2, Brd3, Brd4 or IL1A enhancer RNAs were purchased from Thermo Fisher (Grand Island, NY, USA) and transfected into MKN28 cells with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s protocol. Forty-eight hours post-transfection, cells were infected or harvested for further experiments.

Chromatin immunoprecipitation assays (ChIPs)

ChIP assay was performed as previously described (16). Briefly, cells were fixed in 1% formaldehyde for 10 min and sonicated using a sonicator (Cole-Parmer), and lysates were immunoprecipitated overnight with various antibodies. Protein A agarose blocked with sheared salmon sperm DNA, was used to collect antibody-chromatin complexes. DNA was extracted with DNA purification kit from Qiagen (Valencia, CA, USA). The sequence of ChIP primers will be provided upon request.

H. pylori infection in mice

C57BL/6J female mice (Harlan, Indianapolis, IN) at 6 week of age were randomly assigned to 3 groups. Group A (n=5) received Broth only as uninfected control while group B (n=4) and group C (n=4) received 108 CFU of H. pylori SS1 in broth intragastrically through oral gavage every 48 h (on days 1, 3, 5 and 7). After infection for another 11 weeks, mice in group C were intraperitoneally injected with JQ1 for 2 weeks with a dose of 50 mg/kg body weight while the other groups were administrated with the same volume of vehicle control. Stomachs were collected and rinsed with PBS to remove the gastric content. Collected stomachs consisted of the gastric mucosa beginning at the gastroesophageal junction and ending just beyond the gastroduodenal junction. The stomachs were then cut into two longitudinal sections and used for RNA extraction and histology analysis, respectively. All the animal experiments were approved by the UIUC Institutional Animal Care and Use Committee.

Hematoxylin and eosin (HE) and immunohistochemical staining

Stomach tissues were fixed in neutral buffered 10% formalin, processed by standard methods, embedded by paraffin, sectioned at 4 µm, and stained with H&E. Inflammation in the gastric corpus were each scored by a single pathologist (D.H) blinded to each group. Inflammation was graded on a 0–3 ordinal scale based on the Sydney System as follows: chronic inflammation (mononuclear cell infiltration independent of lymphoid follicles); Grade 0-no inflammation, Grade 1-mild inflammation (slight increase in mononuclear cells), Grade 2-moderate inflammation (dense but focal mononuclear inflammatory cells), Grade 3-severe inflammation (dense and diffuse mononuclear inflammatory cells). For assessment of epithelial cell proliferation, Ki-67 (BD Biosciences, San Jose, CA) labeling indices were determined. Briefly, formalin-fixed stomach samples were assessed for Ki-67 immunolabeling. The epithelial cell proliferation labeling index (LI) was semi-quantitatively scored using an online software ImmunoRatio (http://153.1.200.58:8080/immunoratio/). The percentage of positively stained nuclear cells/total cells is shown.

Statistical analysis

All data are presented as mean ± SD unless otherwise stated. Student t test, Mann-Whitney test or ANOVA with Bonferroni and Tukey correction for multiple comparisons were used to analyze the data. Statistical significance was determined using GraphPad Prism6 software (GraphPad). For all data, a p value ≤ 0.05 was considered statistically significant.

Results

JQ1 suppresses the mRNA and eRNA synthesis of a subset of H. pylori-induced NF-κB target genes

NF-κB has been shown to be essential for the H. pylori-induced inflammatory gene expression and inflammatory response (3, 4). Since JQ1 is able to inhibit the transcriptional activation of NF-κB (28, 36), we first examined the effect of JQ1 on H. pylori-induced NF-κB target gene expression. MKN28 cells with or without JQ1 treatment were infected with H. pylori G27 and the expression of 84 NF-κB-dependent genes was analyzed with quantitative real-time PCR array. Infection of MKN28 cells with H. pylori G27 for 2 h up-regulated the expression of more than half of NF-κB target genes (Figs. 1A and 1B). Specifically, 44 out of 84 genes tested displayed at least two-fold induction by H. pylori (Fig. 1B). Pre-treatment of MKN28 cells with JQ1 down-regulated about half of H. pylori up-regulated genes and 19 of 44 up-regulated genes were suppressed by JQ1 by at least 2-fold (Figs. 1A and 1B). Most of these down-regulated genes were pro-inflammatory cytokine genes, including CSF2, IL1A, IL1B and IL6, indicating that JQ1 inhibits H. pylori-induced inflammatory gene expression.

Fig. 1. JQ1 suppresses mRNA and eRNA synthesis of a subset of H. pylori-induced NF-κB-dependent proinflammatory gene.

Fig. 1

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(A) Heat map representation of expression levels of H. pylori G27- induced NF-κB target genes that were down-regulated by JQ1 (5 µM) in H. pylori-infected MKN28 cells. Log2 signal is shown and scale ranges from a signal value of 2−1.9 (green) to 27.2 (red). Fold-change values are listed. Results from two independent experiments are shown. (B) Venn diagrams display the number of H. pylori-inducible (no less than two fold, orange circle) genes that were suppressed (no less than two fold, the intersection of orange circle and green circle) or H. pylori-inducible genes that were up-regulated (no less than two fold, the intersection of orange circle and yellow circle) or H. pylori-non-inducible genes that were up-regulated (no less than two fold, the yellow circle out of the orange circle) by JQ1 treatment in MKN28 cells among 84 tested NF-κB target genes (grey circle). Table represents the distribution of down-regulated genes into functional categories. (C) MKN28 cells treated with JQ1 (5 µM) for 2 h were infected with H. pylori G27 for indicated time points and RT-PCR was performed to analyze IL1A and IL1B mRNA expression. Results are shown as mean ± SD of triplicate and are representative of three independent experiments. *p < 0.05, ***p < 0.001 versus 0 h; #p < 0.05, ###p < 0.001 versus DMSO at respective time point. (D) Mice infected with H. pylori SS1 for 12 weeks were treated with or without JQ1 for two weeks. RNA isolated from gastric tissues was analyzed for different gene expression by RT-PCR (n = 4~5 mice/group in the experiment). Results are shown as median. *p < 0.05. (E) & (F) MKN28 cells treated with JQ1 (5 µM) for 2 h were infected with H. pylori G27 for 1 h and RT-PCR was performed to analyze the synthesis of eRNA of IL1A (E) and IL1B (F). Schematic diagram of IL1A (E) and IL1B (F) genes with transcription starting site (TSS) and enhancer is shown on the top. Results are shown as mean ± SD of triplicate and are representative of at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

To further confirm the inhibitory effect of JQ1 on H. pylori-induced inflammatory gene expression, we examined the expression of IL1A and IL1B at different time points with JQ1 treatment in MKN28 cells. As expected, H. pylori activated the expression of IL1A and IL1B with a peak at 2 h (Fig. 1C). Treatment of cells with JQ1 effectively suppressed the expression of these two genes (Fig. 1C).

Furthermore, when we examined the effect of JQ1 on inflammatory gene expression in H. pylori-infected mice, we found that mice infected with the mouse adapted H. pylori SS1 for 12 weeks displayed enhanced expression of proinflammatory cytokine genes, including Il1b and Tnf, in gastric tissues (Fig. 1D). Administration of infected mice with JQ1 for two weeks dramatically reduced the expression of these genes (Fig. 1D). Collectively, these data demonstrate that JQ1 effectively inhibits the expression of a subset of H. pylori-induced NF-κB-dependent inflammatory genes.

Recent studies indicate that eRNAs play a role in gene expression and JQ1 has an inhibitory effect on the synthesis of eRNAs (18, 20, 22, 39, 40). We next determined whether H. pylori could activate the expression of eRNA of inflammatory genes and whether JQ1 had any effect on eRNA synthesis. We examined the expression of eRNAs of IL1A and IL1B, two of the most activated genes upon H. pylori infection (Fig. 1A). The enhancer of human IL1A locates around a region (from ~−8.4 kb to ~ −11.8 kb) upstream of its transcription start site (TSS) based on the data available from ENCODE Project Consortium (41, 42). The enhancer of human IL1B is located 24 kb downstream of the TSS of IL1B gene (24). H. pylori infection stimulated the expression of eRNAs of IL1A and IL1B (Figs. 1E and 1F). Importantly, treatment of cells with JQ1 dramatically suppressed H. pylori-induced IL1A and IL1B eRNA synthesis (Figs. 1E and 1F). These data suggest that JQ1 suppresses H. pylori-induced inflammatory gene eRNA synthesis.

H. pylori-induced eRNA synthesis is essential for H. pylori-mediated inflammatory gene expression

To understand the function of H. pylori-induced eRNAs, we first examined IL1A eRNA synthesis at different time points upon H. pylori infection. eRNA of IL1A was rapidly induced and reached the maximum level at 1 h after H. pylori infection (Fig. 2A), a similar pattern as the mRNA expression (Fig. 1C). We next determined the cellular localization of these inducible eRNAs. We isolated RNAs from cytoplasmic and nuclear fractions of MKN28 cells with or without H. pylori infection and measured the levels of mRNA and eRNA of IL1A. As expected, H. pylori-induced mRNA of IL1A localized in both cytosol and nucleus (Fig. 2B). Different from mRNA, the inducible eRNAs of IL1A localized predominately in the nucleus (Fig. 2C, left panel), as evidenced by the control nuclear RNA NEAT1 (nuclear-enriched abundant transcript 1) (Fig. 2C, right panel). The localization of these IL1A eRNAs in the nucleus suggests that they might have a nuclear function.

Fig. 2. H. pylori-induced eRNA synthesis is essential for H. pylori-mediated inflammatory gene expression.

Fig. 2

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(A) MKN28 cells were infected with H. pylori G27 for indicated time points and expression of IL1A eRNAs were analyzed by RT-PCR. (B&C) MKN28 cells were infected with H. pylori G27 for 1 h and RNA was isolated from cytoplasmic or nuclear fractions and levels of mRNA (B) and eRNA (C) of IL1A were analyzed by RT-PCR. NEAT1 was used as a nuclear RNA control. (D) & (E) MKN28 cells were transfected with siRNA1 against eRNA of IL1A. Forty-eight hours post-transfection, cells were infected with H. pylori G27 for 1 h and the expression of IL1A eRNA (D), IL1A, IL1B and TNF mRNA (E) was analyzed by RT-PCR. (F) & (G) MKN28 cells were transfected with siRNA2 against eRNA of IL1A. Expression of eRNA and mRNA of indicated genes was analyzed as in (D) & (E). Results are shown as mean ± SD of triplicate and are representative of at least two independent experiments (A ~ G). *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant.

One of the nuclear activities of eRNAs is to facilitate the mRNA synthesis (18, 20). We next investigated whether H. pylori-induced eRNA synthesis had any roles in H. pylori-induced inflammatory gene expression. We designed two siRNAs targeting two different sequences of the −10.9 kb enhancer region of IL1A to abolish the IL1A eRNAs and examined H. pylori-induced mRNA expression of IL1A. Inhibition of IL1A eRNA synthesis by two different siRNAs (Figs. 2D and 2F) suppressed H. pylori-induced IL1A mRNA expression (Figs. 2E and 2G, left panels). Interestingly, down-regulation of IL1A eRNAs also inhibited H. pylori-induced expression of mRNA of IL1B, which is located ~50 kb downstream of IL1A gene on the same chromosome 2 and potentially shares regulatory regions (Figs. 2E and 2G, middle panels). In contrast, down-regulation of IL1A eRNAs had no effect on the expression of TNF, which is located on chromosome 6 (Figs. 2E and 2G, right panels). These data indicate that H. pylori-induced IL1A eRNAs might be involved in the expression of more than one genes on the same chromosome.

Brd4 is essential for H. pylori-induced NF-κB-dependent inflammatory gene mRNA and eRNA synthesis

JQ1 inhibits the activity of BET family proteins, including Brd2, Brd3 and Brd4 (43). To determine which BET protein is involved in the inhibitory effect of JQ1 on H. pylori-induced inflammatory gene expression, we knocked down the expression of each BET protein with two different siRNAs and evaluated the mRNA expression by quantitative RT-PCR. Among three BET proteins, depletion of Brd4 significantly reduced H. pylori-induced IL1A and IL1B mRNA expression while depletion of Brd2 or Brd3 had little effect on the expression of IL1A but also impaired H. pylori-induced IL1B gene expression (Fig. 3A). These data suggest that BET family proteins might be differentially involved in H. pylori-induced inflammatory gene expression. Since Brd4 is involved in the expression of both IL1A and IL1B (Fig. 3A), we focused on Brd4 for its ability to regulate H. pylori-induced inflammatory gene expression. To further confirm the role of Brd4 in H. pylori-induced inflammatory gene expression, we knocked down the expression of Brd4 and examined the H. pylori-induced expression of IL1A and IL1B at different time points. Consistently, depletion of Brd4 in MKN28 cells by siRNA impaired H. pylori-induced expression of IL1A and IL1B mRNA (Fig. 3B). Similar to the reduced mRNA expression, depletion of Brd4 impaired H. pylori-induced IL1A eRNA synthesis (Fig. 3C).

Fig. 3. Brd4 is essential for H. pylori-induced NF-κB-dependent inflammatory gene mRNA and eRNA synthesis.

Fig. 3

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(A) MKN28 cells transfected with control or siRNA against Brd2, Brd3 and Brd4 as indicated were infected with H. pylori G27 for 2 h and RT-PCR was performed to analyze IL1A and IL1B gene expression. Levels of Brd2, Brd3, Brd4 and actin in siRNA-transfected cells are shown in the right panels. (B) MKN28 cells transfected with control or siRNA against Brd4 were infected with H. pylori G27 for indicated time points and RT-PCR was performed to analyze IL1A and IL1B gene expression. *p < 0.05, ***p < 0.001 versus control siRNA at respective time point. (C) MKN28 cells transfected with control or siRNA against Brd4 were infected with H. pylori G27 for 1 h and RT-PCR was performed to analyze IL1A eRNA synthesis. (D ~ E) MKN28 cells treated with IKK2 inhibitor IV (5 µM) for 1 h were infected with H. pylori G27 for 1 h and RT-PCR was performed to analyze IL1A eRNA synthesis (D) and mRNA expression (E) of IL1A and IL1B. Results are shown as mean ± SD of triplicate and are representative of at least two independent experiments (A ~ E). *p < 0.05, **p < 0.01, ***p < 0.001.

Since NF-κB has been shown to be critical for the expression of eRNAs of inflammatory genes (18), we next assessed whether NF-κB was involved in H. pylori-induced inflammatory gene eRNA synthesis. We inhibited NF-κB activation using IKK2 inhibitor IV, which inhibits IKK2 activation and NF-κB nuclear translocation. Treatment of the cells with IKK2 inhibitor IV down-regulated H. pylori-induced IL1A and IL1B eRNA synthesis (Fig. 3D and data not shown), suggesting an indispensable role of NF-κB in H. pylori-induced eRNA synthesis. Consistently, inhibition of NF-κB by IKK2 inhibitor IV suppressed H. pylori-induced mRNA expression of IL1A and IL1B (Fig. 3E). All together, these results demonstrate that Brd4 is critically involved in NF-κB-dependent mRNA and eRNA synthesis of a subset of inflammatory genes upon H. pylori infection.

H. pylori stimulates the recruitment of RelA and Brd4 to the enhancers and promoters of inflammatory genes

Having identified Brd4 as a key regulator for the H. pylori-induced NF-κB-dependent mRNA and eRNA synthesis, we next investigated whether Brd4 was recruited to the promoter and enhancer of NF-κB target genes, an essential step for Brd4-mediated mRNA and eRNA synthesis (40, 44). Promoters and enhancers are often associated with unique histone modification marks with histone H3K4Me3 found to be associated with promoters and H3K27Ac and H3K4Me1 found to be associated with active enhancers (42). Consistent with this notion, we found that enhancer of IL1A was associated with higher levels of H3K27Ac and H3K4Me1 marks, while promoter of IL1A was associated with higher levels of H3K4Me3 mark (Figs. 4B, 4C and 4D). These results further confirm the active enhancer and promoter elements of IL1A. Interestingly, while the H3K27Ac signal was relatively low at the promoter region, it was enhanced upon H. pylori infection on both promoter and enhancer (Fig. 4B). However, H3K4Me1 or H3K4Me3 signal was barely changed upon H. pylori infection (Figs. 4C and 4D).

Fig. 4. H. pylori stimulates the recruitment of RelA and Brd4 to the enhancer and promoter of IL1A.

Fig. 4

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(A ~ G) MKN28 cells were infected with H. pylori G27 for 1 h. ChIP assays were performed using antibodies against IgG (A), H3K27Ac (B), H3K4Me1 (C), H3K4Me3 (D), RelA (E), Brd4 (F) and RNAPII (G) and probed for the promoter and enhancer of IL1A. (H ~ K) MKN28 cells treated with IKK2 inhibitor IV (5 µM) for 1 h were infected with H. pylori G27 for 1 h. ChIP assays were performed as in (A ~ G) for the recruitment of IgG (H), RelA (I), Brd4 (J) and RNAPII (K) to the promoter and enhancer of IL1A. Results are shown as mean ± SD of triplicate and are representative of at least two independent experiments (A ~ K). *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant.

We next examined the recruitment of RelA and Brd4 to the promoter and enhancer of IL1A in response to H. pylori infection since Brd4 is essential for the NF-κB-dependent IL1A mRNA and eRNA synthesis (Fig. 3). Without infection, binding of RelA to the promoter or enhancer of IL1A was barely detectable (Fig. 4E). Upon H. pylori infection, RelA was recruited to both the promoter and enhancer of IL1A (Fig. 4E). Similar to RelA, H. pylori also stimulated the recruitment of Brd4 to the promoter and enhancer of IL1A (Fig. 4F). We then examined the recruitment of RNAPII to the promoter and enhancer of IL1A, an important step for the transcription of RNAPII-dependent gene. H. pylori stimulated the recruitment of RNAPII to the promoter and enhancer of IL1A (Fig. 4G), indicating that H. pylori activates RNAPII to stimulate inflammatory gene transcription.

Since NF-κB was required for Brd4-mediated mRNA and eRNA synthesis (Fig. 3), we also assessed whether NF-κB was required for the recruitment of Brd4 to the promoter and enhancer. As expected, inhibition of NF-κB by IKK2 inhibitor IV blocked H. pylori-induced recruitment of RelA to the promoter and enhancer of IL1A (Fig. 4I). NF-κB inhibition also impaired the recruitment of Brd4 and RNAPII to the promoter and enhancer of IL1A (Figs. 4J and 4K). Together, these data suggest that H. pylori infection facilitates the NF-κB-dependent recruitment of Brd4 to the promoter and enhancer to activate RNAPII for the synthesis of eRNA and mRNA of IL1A gene.

JQ1 inhibits the recruitment of Brd4 and RNAPII to the enhancers and promoters of inflammatory genes

Brd4 is recruited to the promoters or enhancers via its binding to acetylated histone and non-histone proteins to activate target gene expression (45). Since H. pylori infection stimulates RelA-dependent recruitment of Brd4 to the promoter and enhancer of IL1A (Fig. 4), we next determined the effect of JQ1 on the recruitment of Brd4 to the promoter and enhancer of IL1A. Treatment of cells with JQ1 had no effect on the H. pylori-induced recruitment of RelA to the promoter or enhancer of IL1A (Fig. 5B). However, H. pylori-induced recruitment of Brd4 to the promoter and enhancer was significantly inhibited by JQ1 (Fig. 5C). JQ1 also inhibited H. pylori-induced recruitment of RNAPII to the promoter and enhancer of IL1A (Fig. 5D) and H. pylori-induced serine 2 phosphorylation of RNAPII (Fig. 5E). The reduced Brd4 recruitment after JQ1 treatment is likely due to the reduced interaction between RelA and Brd4 (Fig. 5F) and might account for the impaired RNAPII activation and RNAPII-mediated eRNA and mRNA synthesis.

Fig. 5. JQ1 inhibits the recruitment of Brd4 to the enhancer and promoter of IL1A.

Fig. 5

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(A~ D) MKN28 cells were pretreated with JQ1 (5 µM) for 2 h followed by H. pylori G27 infection for 1 h. ChIP assays were performed using antibodies against IgG (A), RelA (B), Brd4 (C) and RNAPII (D) and probed for the promoter and enhancer of IL1A. Results are shown as mean ± SD of triplicate and are representative of at least two independent experiments. *p < 0.05, **p < 0.01, ns = non-significant. (E) MKN28 cells were pretreated with JQ1 (5 µM) for 2 h followed by H. pylori G27 infection for 1 h. Levels of phosphorylated RNAPII and total RNAPII were detected by anti-phosphorylated RNAPII and anti-RNAPII antibodies, respectively. (F) MKN28 cells were pretreated with JQ1 (5 µM) for 2 h followed by H. pylori G27 infection for another 1 h. Brd4 was immunoprecipitated and the associated RelA were detected by anti-RelA antibodies.

JQ1 suppresses H. pylori-induced inflammation in mice

JQ1 and other BET inhibitors have been shown to possess anti-inflammatory activities and could be potential therapeutics for the treatment of inflammatory diseases (28, 33, 45). Since JQ1 inhibited H. pylori-induced inflammatory gene expression (Fig. 1A), we next determined the potential therapeutic activity of JQ1 against H. pylori SS1-induced inflammation in mice. We infected the mice with SS1 for 12 weeks and administrated solvent control or JQ1 to the infected mice daily for 2 weeks (Fig. 6A), followed by the isolation of the gastric tissues from non-infected, JQ1-treated and solvent-treated SS1-infected mice. We first evaluated the effect of JQ1 on the H. pylori SS1-induced gastric inflammation by hematoxylin and eosin (H&E) staining of the mouse gastric tissues. As expected, H. pylori SS1-infected mice developed pathological changes with increased infiltration of immune cells, including lymphocyte and plasma cells, and increased statistical inflammation scores (Figs. 6B and 6C). Treatment of the infected mice with JQ1 for 2 weeks significantly attenuated the inflammation with reduced number of infiltrated immune cells and decreased inflammation scores (Figs. 6B and 6C). These data are consistent with the changes of inflammatory cytokine expression in H. pylori SS1-infected mice with or without JQ1 treatment (Fig. 1D), suggesting an anti-inflammatory activity of JQ1 in H. pylori SS1-induced gastric inflammation.

Fig. 6. JQ1 suppresses H. pylori-induced inflammatory response and cell proliferation in mice.

Fig. 6

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(A) C57BL/6J female mice at 6 week of age were randomly assigned to 3 groups. The infection groups received 108 CFU of H. pylori SS1 in broth intragastrically through oral gavage every 48 h (on days 1, 3, 5 and 7). After infection for another 11 weeks, mice in infection plus JQ1 treatment group were intraperitoneally injected with JQ1 for 2 weeks with a dose of 50 mg/kg body weight while the other groups were administrated with the same volume of vehicle control. (B) Representative images of H&E staining of gastric tissues from mice without infection (left), with H. pylori infection for 12 weeks without (middle) or with JQ1 treatment for two weeks (right) are presented. H. pylori infection induced gastric inflammation associated with infiltrated immune cells (plasma cells and lymphocytes) which are marked with arrows. (C) The quantitative inflammation score was assessed by a pathologist and the results are shown as median (n = 4~5 mice/group). *p < 0.05. (D) Immunohistochemical staining of Ki-67 in gastric mucosa of mice without infection (left), with H. pylori infection for 12 weeks without (middle) or with JQ1 treatment for two weeks (right). (E) Ki-67 LI was measured in corpus mucosa as described in the “Materials and Methods”. Data are shown as median (n = 4 mice/group). *p < 0.05. (F) Schematic model for the function of binding of Brd4 to the enhancers and promoters of NF-κB-dependent genes for their eRNA and mRNA synthesis upon H. pylori infection. H. pylori stimulates the NF-κB-dependent recruitment of Brd4 to the promoter and enhancer, facilitating RNAPII-mediated eRNA and mRNA synthesis. eRNAs also regulates the mRNA synthesis. JQ1 blocks the interaction between RelA and Brd4 to suppress the eRNA and mRNA synthesis and H. pylori-induced inflammatory response.

We next evaluated the effect of JQ1 on H. pylori SS1-mediated proliferation of gastric epithelial cells since H. pylori-induced inflammation stimulates the proliferation of gastric epithelial cells and the levels of mucosal proliferation are directly related to the intensity of the gastric inflammation (46). Immunohistochemical staining of proliferation marker Ki-67 demonstrated that a small number of proliferative cells were present in the isthmus regions of corpus mucosa in uninfected mice (Fig. 6D). The Ki-67 labeling index (LI) was remarkably increased upon H. pylori SS1 infection with an increased number of Ki-67 positive cells in expanded areas (Figs. 6D and 6E). Treatment of the mice with JQ1 for two weeks reduced Ki-67 LI and the proliferative cells were restricted back to the isthmus region of the corpus mucosa (Figs. 6D and 6E) while JQ1 had little effect on the Ki-67 staining in the uninfected mice (data not shown). These data suggest that JQ1 effectively suppresses H. pylori SS1 infection-induced cell proliferation in mice.

Discussion

H. pylori infection is the major risk factor for the development of gastric diseases, including gastritis and gastric cancer, and infection-associated inflammation plays a key role in the pathogenesis of H. pylori (47, 48). Understanding the molecular mechanism by which H. pylori induces inflammation would provide new insights into the prevention and treatment of H. pylori infection-associated gastric diseases. H. pylori-mediated inflammation is associated with its ability to activate NF-κB and NF-κB-dependent inflammatory gene expression. In this study, we found that H. pylori not only activated NF-κB-dependent inflammatory gene mRNA synthesis but also activated NF-κB-dependent eRNA synthesis (Fig. 6F). The pathogenicity island of H. pylori and its encoded virulence factor CagA are known to be critical for H. pylori-induced inflammatory gene expression by activating NF-κB (3). Since NF-κB is essential for H. pylori-induced eRNA synthesis (Fig. 3D), it is likely that pathogenicity island or CagA might also be involved in the inducible eRNA synthesis. Supporting this, we found that cagA-deficient G27 failed to activate the IL1A eRNA synthesis (data not shown).

In this study, we also identified Brd4 as a novel regulator of H. pylori-induced inflammatory response. Brd4 facilitates H. pylori-induced NF-κB-dependent inflammatory gene transcription and enhancer activation (Fig. 6F). It is well established that Brd4 regulates transcription by recruiting P-TEFb to proximal promoter regions to stimulate RNAPII-dependent mRNA synthesis (25). Brd4 utilizes a similar mechanism to facilitate NF-κB-dependent inflammatory gene expression (16). In line with this, we found that H. pylori stimulated NF-κB-dependent recruitment of Brd4 and RNAPII to the promoters of inflammatory genes, including IL1A and IL1B (Fig. 4 and data not shown). Recruitment of Brd4 and RNAPII to the promoters is critical for H. pylori-induced inflammatory gene expression since inhibiting the recruitment of Brd4 and RNAPII to the promoters by JQ1 was associated with the down-regulated inflammatory gene expression (Figs. 1 and 5).

In additional to its role in mRNA synthesis, Brd4 also regulates H. pylori-induced eRNA synthesis. Upon H. pylori infection, Brd4 was recruited to the active IL1A enhancer which is associated with H3K27Ac and H3K4Me1 marks (Figs. 4B and 4C). More importantly, Brd4 co-occupied eRNA-producing IL1A enhancer with RelA and RNAPII (Fig. 4) and was essential for H. pylori-induced IL1A eRNA synthesis (Fig. 3C). The recruitment of Brd4 and RNAPII to IL1A enhancer relied on the recruitment of RelA since inactivation of NF-κB impaired such recruitments and IL1A eRNA synthesis (Figs. 3D, 4J and 4K). These data suggest that similar to mRNA synthesis, eRNA synthesis also relies on a NF-κB-directed coordinated recruitment of Brd4 and RNAPII to the enhancers of inflammatory genes (Fig. 6F).

eRNAs have emerged as new regulatory non-coding RNAs that have been shown to regulate cis localized mRNA expression, including mRNA of inflammatory genes, in various types of cells (20, 22, 39). Consistent with eRNA’s role in gene transcription, knockdown of IL1A eRNAs by siRNA reduced H. pylori-induced IL1A mRNA synthesis (Fig. 2). Interestingly, knockdown of IL1A eRNAs also down-regulated H. pylori-induced IL1B but not TNF mRNA synthesis (Fig. 2). Signal-dependent eRNA synthesis has been shown to be highly correlated with corresponding signal-dependent transcriptional changes in promoters of nearby genes (20). Both IL1A and IL1B genes localize on chromosome 2 whereas TNF gene localizes on chromosome 6. Therefore, H. pylori-induced IL1A eRNAs might regulate the transcription of IL1B on the same chromosome, consistent with an in cis action of eRNA in gene transcription (20). The non-coding regulatory regions between IL1A and IL1B genes, which include enhancers of IL1A and IL1B, contain multiple high levels of RelA binding signals upon TNF stimulation (42). The intense NF-κB binding signals at multiple sites within a regulatory domain have been shown to be a characteristic of inducible super-enhancer of inflammatory genes (18). It is possible that the regulatory region between IL1A and IL1B is part of a super enhancer that coordinates the transcription of multiple genes, including IL1A and IL1B. Supporting this possibility, we found that the regulatory region between Il1a and Il1b is part of a super enhancer for Il1a and Il1b gene expression in mice (18). A search in the super enhancer database dbSUPER (49) also reveals that the TSS-10.9k regulatory region of IL1A is part of the super enhancer region of human IL1A and IL1B genes.

NF-κB-dependent eRNA synthesis has been shown to be an essential element in NF-κB-dependent inflammatory gene expression (18, 28). For example, NF-κB-dependent IL1B eRNA expression regulates LPS-induced expression of IL1B mRNA (24). H. pylori-infection stimulates the expression of many NF-κB-dependent inflammatory genes (Fig. 1). It is likely that enhancer activation and eRNA synthesis might be involved in other H. pylori-induced inflammatory gene expression. eRNAs regulate gene transcription via enhancing the interaction between enhancers and promoters or facilitating the RNAPII recruitment and activation on the promoters of target genes (21, 50–52). It remains to be determined whether IL1A eRNAs might utilize similar mechanisms to regulate the transcription of IL1A and IL1B.

Recent studies reveal that inhibitors of BET family proteins display therapeutic activity in a variety of pathologies, including cancer and inflammation (45). Many hematological cancers and solid tumors are sensitive to BET protein inhibition (45, 53). BETi also exhibit potent anti-inflammatory effects in several inflammatory disease models, including sepsis, atherosclerosis, rheumatoid arthritis, and some autoimmune diseases (28, 33, 34, 54, 55). Clinical trails with different BETi, including JQ1, against tumors and inflammation have been initiated (56). The anti-inflammatory activity of BETi is largely due to their abilities to inhibit the transcriptional activation of NF-κB by interfering NF-κB interaction with Brd4, leading to down-regulated inflammatory gene expression (28, 33, 36). In this study, we demonstrated that JQ1 also possessed strong anti-inflammatory effects in a mouse model of H. pylori-mediated inflammatory disease. JQ1 suppressed inflammation and cell proliferation in H. pylori-infected mice (Fig. 6). JQ1 had no effect on the colonization of H. pylori on the mice stomachs (data not shown). As such, the reduced inflammatory response in mice was attributed to the down-regulated expression of inflammatory genes (Fig. 1), likely due to the reduced NF-κB-dependent recruitment of Brd4 and RNAPII to the promoters and enhancers of inflammatory genes (Fig. 5). The acetylation of RelA at lysine-310 appears be critical for Brd4 recruitment since mutation of lysine-310 to arginine or deletion of bromodomains of Brd4 abolished H. pylori-induced RelA and Brd4 interaction (data not shown). Consistently, JQ1, which blocks the interaction between acetylated lysine-310 and Brd4 (36), blocked H. pylori-induced Brd4 and RelA interaction and the recruitment of Brd4 to the promoter and enhancer (Fig. 5).

JQ1 is an inhibitor targeting all BET family proteins, which include Brd2, Brd3, Brd4, and a testis specific Brdt (57). It is possible that other BET proteins might also be involved in H. pylori-induced inflammatory gene expression since H. pylori-induced expression of IL1B was down-regulated in Brd2 or Brd3 knockdown cells (Fig. 3A). In line with our finding, the potential role of other BET proteins in inflammatory gene expression is also found in LPS-treated macrophages (34). Whether other BET proteins use a similar mechanism like Brd4 to regulate inflammatory gene expression remains unclear and merits further investigation. Nevertheless, the overall inhibitory effect of JQ1 in H. pylori-induced inflammation in mice might reflect a combined inhibitory effect on different BET family proteins.

In conclusion, we have identified a novel function of Brd4 in H. pylori-induced inflammatory response. In addition to its ability to regulate mRNA synthesis, we found that Brd4 was critically involved in the synthesis of eRNA of H. pylori-induced inflammatory genes. By blocking the interaction between Brd4 and NF-κB, JQ1 inhibited H. pylori-induced mRNA and eRNA synthesis and suppressed inflammation and cell proliferation in H. pylori-infected mice. Since H. pylori-associated chronic inflammation is a pre-requisite for H. pylori-triggered gastric cancer, it would be interesting and necessary to investigate whether JQ1 could also be an effective agent for the treatment of H. pylori-induced gastric cancer. Identification of Brd4 as a novel regulator of H. pylori-induced inflammatory gene expression not only provides new insights into the regulation of H. pylori-induced inflammation, but also provides potential therapeutic approaches for H. pylori-associated gastric diseases, including gastritis and gastric cancer.

Acknowledgments

We thank K. F. D for H&E and immunohistochemical staining; D.H. for pathological analysis; members in the Chen lab for discussion.

Funding information

This work is supported in part by fund provided by UIUC (to L.F.C.), NIH-DK-085158 (to L.F.C.), NIH-CA179511 (to L.F.C.), NSFC-81361120386 (to R.C) and NSFC-81328024 (to W.Z. and L.F.C.).

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