BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses

Abstract

Histone acetylation regulates activation and repression of multiple inflammatory genes known to play critical roles in chronic inflammatory diseases. However, proteins responsible for translating the histone acetylation code into an orchestrated proinflammatory cytokine response remain poorly characterized. Bromodomain and extraterminal (BET) proteins are "readers" of histone acetylation marks, with demonstrated roles in gene transcription, but the ability of BET proteins to coordinate the response of inflammatory cytokine genes through translation of histone marks is unknown. We hypothesize that members of the BET family of dual bromodomain-containing transcriptional regulators directly control inflammatory genes. We examined the genetic model of brd2 lo mice, a BET protein hypomorph, to show that Brd2 is essential for proinflammatory cytokine production in macrophages. Studies that use small interfering RNA knockdown and a small-molecule inhibitor of BET protein binding, JQ1, independently demonstrate BET proteins are critical for macrophage inflammatory responses. Furthermore, we show that Brd2 and Brd4 physically associate with the promoters of inflammatory cytokine genes in macrophages. This association is absent in the presence of BET inhibition by JQ1. Finally, we demonstrate that JQ1 ablates cytokine production in vitro and blunts the "cytokine storm" in endotoxemic mice by reducing levels of IL-6 and TNF-α while rescuing mice from LPS-induced death. We propose that targeting BET proteins with small-molecule inhibitors will benefit hyperinflammatory conditions associated with high levels of cytokine production.

Figures

Figure 1. brd2 lo macrophages demonstrate a diminished inflammatory response

A. Brd2 mRNA levels in leukocytes from WT (white bar) compared to brd2 lo (black bar) mice as measured by RT-qPCR. Relative mRNA expression was normalized to GAPDH. N = 4; **, P < 0.01, as calculated by Student's t test. B. Production of pro-inflammatory cytokines by brd2 lo and WT BMDMs stimulated for 24 hours with 50 ng/ml E. coli LPS. All bars in all figures show mean values and SEM. N = 4; *, P < 0.05, as calculated by Student's t test.

Figure 2. In vitro knockdown of BET protein genes reduces the inflammatory response

A. WT BMDMs were transfected with ON-TARGET Plus siRNA pools specific for knockdown of Brd2, Brd3 or Brd4, or non-targeting control pools, then stimulated with 50 ng/ml E. coli LPS for 2 hrs before quantitation of the indicated cytokine mRNAs by RT-qPCR. Relative mRNA expression was normalized to GAPDH. N = 5; **, P < 0.01, ***, P < 0.001 as calculated by ANOVA with Dunnett’s multiple comparison. B. Cytokine protein concentrations in supernatants from LPS-stimulated (24 hours, 50 ng/ml) BMDMs after individual BET knockdown. N = 5; *. P < 0.05; **, P < 0.01; ***, P < 0.001 as calculated by ANOVA with Dunnett’s multiple comparison.

Figure 3. In vitro knockdown of Brd2 decreases NF-κB-luciferase activity

RAW264.7 cells were transfected with Brd2-specific shRNA (black bars) or empty vector (white bars) plus NF-κB luciferase reporter plasmid. Forty-eight hours after transfection, cells were stimulated with E. coli LPS for 2 hrs. NF-κB-driven luciferase activity is shown (mean and SEM). N = 3. *, P < 0.05 as calculated by Student's t test comparing empty vector and Brd2 shRNA-containing plasmids.

Figure 4. The BET family bromodomain inhibitor JQ1 reduces cytokine production in vitro

A. RAW 264.7 cells were exposed simultaneously to 50 ng/ml E. coli LPS plus 400 nM JQ1(+) or JQ1(−) for 24 hrs. Mean value and SEM for 3 determinations are shown. JQ1 enantiomers are abbreviated by (+) or (−), respectively. *, P < 0.05, ***, P < 0.001 as calculated by Student's t test comparing JQ1(+) and JQ1(−) treated samples. The chemokine KC was not detectable in RAW 264.1 macrophages under these conditions. B. BMDMs were exposed simultaneously to 50 ng/ml E. coli LPS or PBS and different concentrations of JQ1(+) or JQ1(−) for 24 hrs. Supernatant cytokines were quantified in multiplex protein assays. N = 6; ***, P < 0.001 as calculated by ANOVA with Dunnett’s multiple comparison. ND – not detected.

Figure 5. JQ1 effect on cytokine production is strain-independent and is not overcome by IFNγ priming of macrophages

A. Open bars show C57Bl/6 and BALB/c BMDMs exposed to PBS, 100 U/ml IFNγ, 50 ng/ml E. coli LPS, 50 ng/ml E. coli LPS with 400 nM JQ1(+), or primed with 100 U/ml IFNγ and stimulated with LPS 4 hours later as indicated below graph. Black bars show BMDMs primed with IFNγ or PBS and stimulated with LPS 4 hours later, with JQ1(+) being added either simultaneously with LPS (4 hours after priming with IFNγ) or simultaneously with IFNγ. Cytokine production was measured 24 hours after LPS stimulation. Supernatant cytokines were quantified in multiplex protein assays. N = 5; ***, P < 0.001; **, P < 0.01; *, P < 0.05 as calculated by ANOVA with Dunnett’s multiple comparison. #, P < 0.05 as calculated by Student's t test; ND – not detected. B. eGFP NF-κB BMDMs were stimulated were stimulated with PBS, 50 ng/ml LPS or LPS + JQ1(+) as indicated. N = 3 **, P < 0.01; *, P < 0.05 as calculated by ANOVA with Dunnett’s multiple comparison.

Figure 6. JQ1(+) displaces BET proteins Brd2 and Brd4 from inflammatory cytokine promoters

Mouse BMDMs were treated with PBS or 50 ng/ml E. coli LPS in presence of either 400 mM JQ1(−) or 400 nM JQ1(+) as indicated, then harvested for ChIP 1 hour later. A. Brd2 or Brd4 association with the IL-6 promoter. B. Brd2 or Brd4 association with the TNF-α promoter. Mean values and SEM are shown; N=3 for all datapoints except for Brd4-precipitated chromatin treated with PBS+JQ1(−), where N=2. *, P < 0.05; **, P < 0.01 as calculated by Student's t test comparing JQ1(+) and JQ1(−) results.

Figure 7. JQ1 protects mice from LPS-induced death

Mice were injected with a lethal dose of E. coli LPS (20 mg/kg, i.p.). At 2 h before and 24 h after LPS injection, mice also received JQ1 (50 mg/kg, i.p.) of one or the other enantiomer, as indicated. A. Survival curves, Kaplan-Meier analysis. N=9 per arm, P=0.001. B. Serum levels of TNF-α, IL-6, MCP-1, KC and IL-10 in LPS challenged mice. Cytokines in serum were determined 40, 60, 90, 120, and 240 min after challenge. N = 7–9. *, P < 0.05; **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 as calculated by Student's t test and comparison of JQ1(+) and JQ1(−). C. Serum SAA levels in mice challenged with lethal dose of LPS 2 hours and 4 hours after challenge. N = 6. NS indicates insignificant difference (P >0.05) as calculated by Student’s t test.