Patients with COPD have peripheral lung inflammation that may spill over into the systemic circulation, leading to skeletal muscle weakness and cachexia and increasing propensity to cardiovascular, metabolic, and bone diseases, and depression.

<p>There is an increase in circulating cytokines, including IL-1β, IL-6, IL-18, and TNFα, as well as acute-phase proteins, such as CRP and serum amyloid A. Peripheral lung inflammation may also increase the risk of developing lung cancer. There may be genetic predisposition to developing COPD in smokers, which may be shared with genetic susceptibility to comorbid diseases. Airway obstruction reduces physical activity and causes hypoxia, which may contribute to skeletal muscle weakness as well as to comorbid diseases.</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

IC₅₀ values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from BEAS-2B cells.

<p>BEAS-2B cells were pre-treated for 1h in the presence of either PF956980 or PF1367550 prior to stimulation for 20h in the absence or presence of IFNγ (10ng/ml) or IFNγ+TNFα (10ng/ml). After this time, media was harvested and the concentrations of CXCL9, CXCL10 and CXCL11 measured by ELISA. Data are presented as mean±SEM, n = 7–8.</p><p>IC₅₀ values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from BEAS-2B cells.</p

Effect of JAK Inhibitors on Release of CXCL9, CXCL10 and CXCL11 from Human Airway Epithelial Cells

<div><p>Background</p><p>CD8⁺ T-cells are located in the small airways of COPD patients and may contribute to pathophysiology. CD8⁺ cells express the chemokine receptor, CXCR3 that binds CXCL9, CXCL10 and CXCL11, which are elevated in the airways of COPD patients. These chemokines are released from airway epithelial cells via activation of receptor associated Janus kinases (JAK). This study compared the efficacy of two structurally dissimilar pan-JAK inhibitors, PF956980 and PF1367550, and the glucocorticosteroid dexamethasone, in BEAS-2B and human primary airway epithelial cells from COPD patients and control subjects.</p><p>Methods</p><p>Cells were stimulated with either IFNγ alone or with TNFα, and release of CXCL9, CXCL10 and CXCL11 measured by ELISA and expression of <i>CXCL9</i>, <i>CXCL10</i> and <i>CXCL11</i> by qPCR. Activation of JAK signalling was assessed by STAT1 phosphorylation and DNA binding.</p><p>Results</p><p>There were no differences in the levels of release of CXCL9, CXCL10 and CXCL11 from primary airway epithelial cells from any of the subjects or following stimulation with either IFNγ alone or with TNFα. Dexamethasone did not inhibit CXCR3 chemokine release from stimulated BEAS-2B or primary airway epithelial cells. However, both JAK inhibitors suppressed this response with PF1367550 being ~50-65-fold more potent than PF956980. The response of cells from COPD patients did not differ from controls with similar responses regardless of whether inhibitors were added prophylactically or concomitant with stimuli. These effects were mediated by JAK inhibition as both compounds suppressed STAT1 phosphorylation and DNA-binding of STAT1 and gene transcription.</p><p>Conclusions</p><p>These data suggest that the novel JAK inhibitor, PF1367550, is more potent than PF956980 and that JAK pathway inhibition in airway epithelium could provide an alternative anti-inflammatory approach for glucocorticosteroid-resistant diseases including COPD.</p></div

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

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

IC₅₀ values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from human primary airway epithelial cells from non-smokers, smokers and COPD patients.

<p>Human primary airway epithelial cells were pre-treated for 1h in the presence of either PF956980 or PF1367550 prior to stimulation for 20h in the absence or presence of IFNγ (10 ng/ml) or IFNγ+TNFα (10 ng/ml). After this time, media was harvested and the concentrations of CXCL9, CXCL10 and CXCL11 measured by ELISA. Data are presented as mean±SEM.</p><p>IC₅₀ values for PF956980 and PF1367550 on IFNγ and IFNγ+TNFα-stimulated release of CXCL9, CXCL10 and CXCL11 from human primary airway epithelial cells from non-smokers, smokers and COPD patients.</p