Pharmacological effects of RvD₂ on ALX / FRP2 protein expression in TNF-α-pretreated human bronchi.

<p><b>a.</b> Human bronchial homogenates derived from control (untreated) bronchi or pre-treated with 10 ng/ml TNF-α, TNF-α + 300 nM RvD₂ or TNF-α + RvD₂ + 1 μM WRW4 were stained using specific antibodies against ALX / FRP2 and β-actin. <b>b.</b> Quantitative analyses of ALX / FRP2 density ratio. Staining densities of ALX / FRP2 are expressed as a function of β-actin staining level (n = 5, * <i>P</i> < 0.05).</p

Pro-Resolving Effects of Resolvin D₂ in LTD₄ and TNF-α Pre-Treated Human Bronchi

<div><p>Inflammation is a major burden in respiratory diseases, resulting in airway hyperresponsiveness. Our hypothesis is that resolution of inflammation may represent a long-term solution in preventing human bronchial dysfunctions. The aim of the present study was to assess the anti-inflammatory effects of RvD₂, a member of the D-series resolving family, with concomitant effects on ASM mechanical reactivity. The role and mode of action of RvD₂ were assessed in an <i>in vitro</i> model of human bronchi under pro-inflammatory conditions, induced either by 1 μM LTD₄ or 10 ng/ml TNF-α pre-treatment for 48h. TNF-α and LTD₄ both induced hyperreactivity in response to pharmacological stimuli. Enhanced 5-Lipoxygenase (5-LOX) and cysteinyl leukotriene receptor 1 (<a href="http://www.ejog.org/action/doSearch?searchType=quick&occurrences=all&ltrlSrch=true&searchScope=series&searchText=CysLTR1&seriesISSN=0301-2115" target="_blank">CysLTR1</a>) detection was documented in LTD₄ or TNF-α pre-treated human bronchi when compared to control (untreated) human bronchi. In contrast, RvD₂ treatments reversed 5-LOX/β-actin and CysLTR1/β-actin ratios and decreased the phosphorylation levels of AP-1 subunits (c-Fos, c-Jun) and p38-MAP kinase, while increasing the detection of the ALX/FPR2 receptor. Moreover, various pharmacological agents revealed the blunting effects of RvD₂ on LTD₄ or TNF-α induced hyper-responsiveness. Combined treatment with 300 nM RvD₂ and 1 μM WRW4 (an ALX/FPR2 receptor inhibitor) blunted the pro-resolving and broncho-modulatory effects of RvD₂. The present data provide new evidence regarding the role of RvD₂ in a human model of airway inflammation and hyperrresponsiveness.</p></div

Effect of RvD₂ on LTD₄–pretreated human bronchi in response to pharmacologically-induced tone and 5-LOX/CysLTR1 expression.

<p><b>a.</b> Corresponding bar graph showing mean contractile amplitudes induced by 1 μM methacholine (MCh), 1 μM histamine or 30 nM U-46619, either under control (untreated) conditions or after 1 μM LTD₄ pre-treatment in the absence or presence of 300 nM RvD₂ (n = 7–22, *<i>P</i> < 0.05). <b>b.</b> Western blot analyses of the 5-LOX / β-actin ratio assessing the putative effects of 1 μM LTD₄ and 1 μM LTD₄ + 300 nM RvD₂ on human bronchial homogenates compared with control conditions (<i>n</i> = 6, *<i>P</i> < 0.05). <b>c.</b> Western blot analyses using <a href="http://www.ejog.org/action/doSearch?searchType=quick&occurrences=all&ltrlSrch=true&searchScope=series&searchText=CysLTR1&seriesISSN=0301-2115" target="_blank">CysLTR1</a> and β-actin antibodies. Densities of the immunoreactive bands are expressed as a function of the β-actin-immunoreactive band (<i>n</i> = 6, * <i>P</i> < 0.05).</p

Effect of RvD₂ on TNF-α-induced reactivity and inflammation in human bronchi.

<p><b>a.</b> Bar graph of the contractile activity induced by bronchoactive agents (MCh, His and U-46619) on 48-h cultured human bronchi in control (untreated, n = 7–12) conditions, 10 ng/ml TNF-α (n = 14–19), TNF-α + 300 nM RvD₂ (n = 15–26), TNF-α + RvD₂ + 300 nM WRW4 (n = 11–14) or TNF-α + RvD₂ + 1 μM WRW4 (n = 10–11), *<i>P</i> < 0.05. <b>b.</b> Western blot analyses using specific antibodies against P-p38-MAPK and total p38-MAPK. Staining densities of P-p38-MAPK are expressed as a function of p38-MAPK levels (n = 5, * <i>P</i> < 0.05). <b>c.</b> Western blot analyses were performed using antibodies against p-c-Fos and total c-Fos. The relative density ratio of p-c-Fos to total c-Fos was used to quantify the comparative effects of RvD₂ on TNF-α-pretreated human bronchi (n = 5, * <i>P</i> < 0.05). <b>d.</b> Western blot analyses were performed using antibodies against p-c-Jun and total c-Jun. Staining densities of p-c-Jun are expressed as a function of c-Jun levels (n = 5, * <i>P</i> < 0.05).</p

Effect of RvD₂ and WRW4 treatments on 5-LOX and CysLTR1 expression in TNF-α-pre-treated human bronchi.

<p><b>a.</b> Western blot analyses of bronchial homogenates derived from control, TNF-α, TNF-α + 300 nM RvD₂, TNF-α + 300 nM RvD₂ + 300 nM WRW4 and TNF-α + RvD₂ + 1 μM WRW4-pretreated bronchial rings for 48h, using specific antibodies against 5-LOX, <a href="http://www.ejog.org/action/doSearch?searchType=quick&occurrences=all&ltrlSrch=true&searchScope=series&searchText=CysLTR1&seriesISSN=0301-2115" target="_blank">CysLTR1</a> and β-actin, respectively. <b>b.</b> Quantitative analysis of various 5-LOX / β-actin density ratios (n = 5, * <i>P</i> < 0.05). <b>c.</b> Bar graph of mean <a href="http://www.ejog.org/action/doSearch?searchType=quick&occurrences=all&ltrlSrch=true&searchScope=series&searchText=CysLTR1&seriesISSN=0301-2115" target="_blank">CysLTR1</a> / β-actin density ratios in human bronchial homogenates (n <i>=</i> 5, * <i>P</i> < 0.05).</p