The BET inhibitors (JQ1 and PFI-1) reduce inflammatory mediator production.

<p>Cells were pre-treated with either JQ1 or JQ1(-) both at 500 nM for 4 hours followed by stimulation with H₂O₂ in the presence (+) or absence (-) of IL-1β (1 ng/ml) or both for 16 hours or left unstimulated. IL-6 (<b>A</b>) and CXCL8 (<b>B</b>) proteins were assayed by ELISA. <i>IL-6</i> (<b>C</b>) and <i>IL-8</i> (<b>D</b>) transcripts were quantified by RT-PCR. n = 4 independent experiments. Bar graph represents mean ± SEM *p<0.05, **p<0.01, when compared JQ1(-) with JQ1 treated cells. Under similar experimental conditions the effect of PFI-1 (1 µM) on IL-6 (<b>E</b>) and CXCL8 (<b>F</b>) proteins were assayed by ELISA. <i>IL-6</i> (<b>G</b>) and <i>IL-8</i> (<b>H</b>) mRNA levels were quantified by RT-QPCR. n = 4 independent experiments. Bar graph represent mean ± SEM *p<0.05, **p<0.01, when compared cells treated with or without PFI-1.</p

H₂O₂ induces intracellular ROS and enhances the inflammatory response.

<p>BEAS-2B cells were pre-incubated with DCFH-DA for 30 minutes in loading media followed by wash with KRH buffer. Cells were then treated with different concentrations of H₂O₂ in KRH buffer for 2 hours and intracellular ROS was measured (<b>A</b>). Cells were exposed to a range of concentrations of H₂O₂ for 2 hours and cell viability was assessed using MTT assay (<b>B</b>). Results are presented as mean ± SEM. N = 4. *p<0.05; **p<0.01; ***p<0.0001; when compared to basal level (control). BEAS-2B cells were treated with H₂O₂ for 2 hours in the absence or presence of IL-β stimulation (overnight) or left untreated as a control. IL-6 (<b>C</b>) and CXCL8 (<b>D</b>) protein levels in cell culture supernatants were quantified by ELISA. <i>IL-6</i> (<b>E</b>) and <i>IL-8</i> (<b>F</b>) transcript levels were quantified by comparative real-time PCR and were normalised by measuring <i>GNB2L1</i> transcript levels. Results are expressed as mean ± SEM of at least 4 independent experiments. * P<0.05; ** P<0.01; ***P<0.001 when compared to controls.</p

The effect of JQ1 on Brd4 and p65 binding to <i>IL-6</i> and <i>IL-8</i> promoters.

<p>Chromatin immunoprecipitation (ChIP) assay shows that IL-1β (1 ng/ml) and H₂O₂ (100 µM) induces Brd4 (<b>A</b>) and p65 (<b>B</b>) DNA binding to the <i>IL-6</i> promoter by 5-fold which is abolished in cells pre-treated with JQ1 (500 nM). Similarly, Brd4 (<b>C</b>) and p65 (<b>D</b>) DNA binding at the <i>IL-8</i> promoter is increased following H₂O₂ and IL-1β stimulation in JQ1(-) (500 nM) pre-treated cells by 10- and 4-fold. This binding is diminished in cells pre-treated with the active JQ1 (500 nM). Results are representative of at least 3 independent experiments.*p<0.05, **p<0.01, ***p<0.001 when compared with unstimulated cells.</p

NF-κB p65 acetylation and association with Brd4 protein.

<p>(<b>A</b>) BEAS-2B cells were stimulated with H₂O₂ in the presence (+) or absence (-) of IL-1β (1 ng/ml) for 2 hours, nuclear (<b>A</b>) and cytoplasmic (<b>B</b>) extracts were fractioned by Western blot and membranes were probed with anti-acetylated NF-κB p65 antibody. The blots show that acetylated-310 (Ac310) NF-κB p65 is predominantly found in the nucleus when compared with the cytoplasm. (<b>C</b>) Brd4 protein was immunoprecipitated from whole cell extracts following treatments and separated by SDS-PAGE and subsequently analysed by Western blotting using an anti-NF-κB p65 antibody. Each blot is representative of 3 independent experiments and densitometric analysis of each band is plotted as bar graph above it. TBP: TATA-binding protein; *p<0.05; **p<0.01 compared with control (unstimulated).</p

H3 acetylation, p65 and Brd4 binding to <i>IL-6</i> and <i>IL-8</i> κB promoter sites.

<p>Chromatin immunoprecipitation (ChIP) assays show that IL-1β induces p65 DNA binding to both <i>IL-6</i> (<b>A</b>) and <i>IL-8</i> (<b>B</b>) promoters. H₂O₂ by itself does not affect p65 DNA binding activity; however, when co-treated with IL-1β, the affinity is enhanced by 7-fold at <i>IL-6</i> promoter site and 20-fold at <i>IL-8</i> promoters. Brd4 is also recruited to the same κB promoter regions in the <i>IL-6</i> (<b>C</b>) and <i>IL-8</i> (<b>D</b>) promoters as p65. Histone 3 is acetylated at the <i>IL-6</i> (<b>E</b>) and <i>IL-8</i> (<b>F</b>) κB promoter sites following treatments. IgG is non-specific antibody used as a negative control. Furthermore, H3 acetylation was confirmed using confocal microscopy following IL-1β stimulation whereas H₂O₂ had no effect on AcH3 alone or in combination with IL-1β (<b>G</b>). Results are representative of at least 4 independent experiments.*p<0.05, **p<0.01 compared with control (unstimulated).</p

JQ1 reduces oxidative stress-enhanced IL-6 and CXCL8 expression in NHBE cells.

<p>NHBE cells pre-treated with JQ1 but not JQ1(-) (both at 5×10⁻⁹−10⁻⁶ M) reduced IL-1β induced release of IL-6 (A) and CXCL8 (B) in a concentration-dependent manner. Points represent mean ± SEM *p<0.05; **p<0.01;***P<0.001 when compared with IL-1β stimulation. ^#p<0.05 JQ1(-) versus JQ1. NHBE cells were treated with a range of concentrations of H₂O₂ and intracellular ROS (<b>C</b>) and cell viability (<b>D</b>) were measured using DCFH-DA and MTT assay, respectively. Results are presented as mean ± SEM. N = 4. *p<0.05; **p<0.01; ***p<0.0001; when compared to untreated cells (control). Cells were pre-treated with either JQ1 or JQ1(-) both at 5×10^-7 M for 4 hours followed by stimulation with IL-1β (1 ng/ml) in the presence (+) or absence (-) of H₂O₂ (100 µM) or both for 16 hours or left unstimulated. IL-6 (<b>E</b>) and CXCL8 (<b>F</b>) proteins were assayed by ELISA. <i>IL-6</i> (<b>G</b>) and <i>IL-8</i> (<b>H</b>) transcripts were quantified by RT-PCR. n = 5 independent experiments. Bar graph represents mean ± SEM *p<0.05; **p<0.01; ***p<0.001 when compared with controls. ^#p<0.05; ^##p<0.01; ^###p<0.001 when comparing JQ1- with JQ1(-)-treated cells.</p

Concentration dependent reduction of IL-6 and CXCL8 by BET inhibitors.

<p>Cells were pre-treated with JQ1 and JQ1 (-) enantiomers (<b>A, B</b>) or PFI-1 (<b>C, D</b>) for 4 hours followed by IL-1β (1 ng/ml) for 16 hrs. IL-6 (<b>A, C</b>) and CXCL8 (<b>B, D</b>) proteins were assayed by ELISA. The effect of JQ1 and JQ1 (-) (<b>E</b>) and PFI-1 (<b>F</b>) on cell viability was assessed by MTT assay. n = 3 independent experiments. Points represent mean ± SEM **p<0.01;***P<0.001 compared with IL-1β stimulation. ^#p<0.05 JQ1(-) versus JQ1. ^£££p<0.001when PFI-1 compared with IL-1β stimulation. ^$p<0.05; ^$$p<0.01when compared to control. (<b>G</b>) Cells were heat treated at 90°C or left untreated, mixed together and stained with LIVE/DEAD Fixable Aqua stain then analysed by flow cytometry. Cells were checked with forward scatter detector (FSC) and side scatter detector (SSC) and analysed by density graph to check cell size and granularity. Fragmented cells were excluded from the study. Histogram shows separation of live cells (left) and dead cells (right). These parameters were used to assess cell viability following treatment with JQ1 (0.5 µM) and PFI-1 (1 µM) for 16 hours. DMSO/Control (<1%) alone, PFI-1, JQ1(-) or JQ1 resulted in only 5% decrease of overall cell viability. The data is representative of 3 independent experiments.</p

Fluticasone propionate impairs GATA-3 interaction with importin-α and GATA-3 nuclear localization in vivo and ex vivo.

<p>(A and B) Co-immunoprecipitation analysis of PBMCs from steroid-naïve asthma patients treated with FP in vitro demonstrated impaired interaction between GATA-3 and importin-α measured at 60 min. Each bar represents the mean±SEM of at least three independent experiments; *** <i>p</i><0.001 compared with control as determined by ANOVA/Newman-Keuls analysis. (C and D) Co-immunoprecipitation analyses of PBMCs from steroid-naïve asthma patients treated with inhaled FP (500 µg via a spacer) in vivo demonstrated decreased association between GATA-3 and importin-α. The individual values for each treatment are presented graphically. (E) Representative Western blot showing that importin-α expression was unaffected by inhalation of FP. Blot is representative of gels from three participants.</p

Fluticasone propionate–mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.

<p>(A) Western blot analysis shows that FP (10⁻⁸ M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28–co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10⁻⁸ M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation–induced serine phosphorylation (P-Ser) of GATA-3. For (A–C), quantification of the densitometry data is also shown. Each bar represents mean±SEM of at least three independent experiments. ^###<i>p</i><0.001 compared to control, ***<i>p</i><0.001 compared to αCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner. All results are representative of at least three independent experiments and where appropriate expressed as means±SEM, *<i>p</i><0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner. Results are representative of two independent experiments. All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test.</p

Fluticasone propionate does not affect GATA-3 nuclear export.

<p>(A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10⁻⁸ M) to prevent anti-CD3/CD28–stimulated GATA-3 nuclear localization measured at 60 min. ***<i>p</i><0.001 compared to unstimulated cells, ^###<i>p</i><0.001 compared to anti-CD3/CD28–stimulated cells. (B) Western blot analysis showing that FP (10⁻⁸ M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b–j) or not (a) with anti-CD3/CD28. The effect of treating cells with FP (10⁻⁸ M, f–j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10⁻⁸ M) does not prevent anti-CD3/CD28–stimulated p65 nuclear translocation at 60 min after stimulation. **<i>p</i><0.01 compared to unstimulated cells. All results are representative of at least four independent experiments and are shown as mean±SEM. Results were analysed by ANOVA followed by Newman-Keuls test.</p