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17-oxo-DHA displays additive anti-inflammatory effects with fluticasone propionate and inhibits the NLRP3 inflammasome

View Article: PubMed Central - PubMed

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function associated with increased local and systemic inflammatory markers, such as TNFα and IL-1β. Glucocorticoids are used to treat this chronic disease, however their efficacy is low and new drugs are very much required. 17-oxo-DHA is a cyclooxygenase-2-dependent, electrophilic, α,β-unsaturated keto-derivative of docosahexaenoic acid with anti-inflammatory properties. We evaluated the action of 17-oxo-DHA alone or in combination with the steroid fluticasone propionate (FP) in peripheral blood mononuclear cells (PBMCs) from COPD patients and healthy individuals exposed to lipopolysaccharide. We show that PBMCs from COPD patients released higher levels of TNFα and IL-1β compared to controls. 17-oxo-DHA displayed strong anti-inflammatory effects. The addition of 17-oxo-DHA in combination with FP showed enhanced anti-inflammatory effects through the modulation of transcriptional and post-transcriptional mechanisms. 17-oxo-DHA, but not FP, was able to suppress the release of mature IL-1β through inhibition of the NLRP3 inflammasome. Furthermore, 17-oxo-DHA inhibited inflammasome-dependent degradation of the glucocorticoid receptor (GR). Our findings suggest that 17-oxo-DHA in combination with FP or other steroids might achieve higher therapeutic efficacy than steroids alone. Combined treatment might be particularly relevant in those conditions where increased inflammasome activation may lead to GR degradation and steroid-unresponsive inflammation.

No MeSH data available.


17-oxo-DHA inhibits caspase-1 activation in CD14+ cells.PBMCs from healthy individuals were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (5 μM) for 30 min then by 10 μM nigericin for 30 min in serum-free medium. Cells were harvested, treated with a fluorescent probe recognizing active caspase-1 (FAM-YVAD-FMK), then stained for CD14. Analysis of caspase-1 activation was performed on CD14 positive cells. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for active caspase-1.
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f5: 17-oxo-DHA inhibits caspase-1 activation in CD14+ cells.PBMCs from healthy individuals were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (5 μM) for 30 min then by 10 μM nigericin for 30 min in serum-free medium. Cells were harvested, treated with a fluorescent probe recognizing active caspase-1 (FAM-YVAD-FMK), then stained for CD14. Analysis of caspase-1 activation was performed on CD14 positive cells. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for active caspase-1.

Mentions: To evaluate the effect of 17-oxo-DHA on NLRP3 inflammasome activation, LPS-primed PBMCs from healthy volunteers were stimulated with the NLRP3 inflammasome activator nigericin. The effect of 17-oxo-DHA on inflammasome activation was specifically evaluated by adding 17-oxo-DHA after LPS priming and before nigericin as previously reported2526. The effect of FP on NLRP3 inflammasome activation was also evaluated. 17-oxo-DHA effectively inhibited nigericin-induced IL-1β release while FP had no effect (Fig. 4a). The actions of 17-oxo-DHA and FP on inflammasome activation were further assessed by evaluating the formation of cleaved caspase-1 and IL-1β as well as the expression of NLRP3, pro-IL-1β and procaspase-1. 17-oxo-DHA, but not FP, completely blocked the cleavage of pro-IL-1β and procaspase-1 (Fig. 4b). In the presence of nigericin, the strong effect on the extracellular release of IL-1β was accompanied with reduced intracellular levels of pro-IL-1β (Fig. 4b). The expression of the procaspase-1 did not show significant variations among different conditions while NLRP3 was induced by LPS, which is consistent with previous reports27. The inhibitory effect of 17-oxo-DHA on caspase-1 activity was further confirmed by flow cytometry using a fluorescent probe specific for active caspase-1. Double staining with fluorescent anti-human CD14 antibody was performed on LPS-primed and nigericin-activated PBMCs to evaluate the specific contribution of CD14+ monocytes to caspase-1 activation. Caspase-1 activation was inhibited by 17-oxo-DHA, but not by FP (Fig. 5).


17-oxo-DHA displays additive anti-inflammatory effects with fluticasone propionate and inhibits the NLRP3 inflammasome
17-oxo-DHA inhibits caspase-1 activation in CD14+ cells.PBMCs from healthy individuals were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (5 μM) for 30 min then by 10 μM nigericin for 30 min in serum-free medium. Cells were harvested, treated with a fluorescent probe recognizing active caspase-1 (FAM-YVAD-FMK), then stained for CD14. Analysis of caspase-1 activation was performed on CD14 positive cells. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for active caspase-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121625&req=5

f5: 17-oxo-DHA inhibits caspase-1 activation in CD14+ cells.PBMCs from healthy individuals were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (5 μM) for 30 min then by 10 μM nigericin for 30 min in serum-free medium. Cells were harvested, treated with a fluorescent probe recognizing active caspase-1 (FAM-YVAD-FMK), then stained for CD14. Analysis of caspase-1 activation was performed on CD14 positive cells. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for active caspase-1.
Mentions: To evaluate the effect of 17-oxo-DHA on NLRP3 inflammasome activation, LPS-primed PBMCs from healthy volunteers were stimulated with the NLRP3 inflammasome activator nigericin. The effect of 17-oxo-DHA on inflammasome activation was specifically evaluated by adding 17-oxo-DHA after LPS priming and before nigericin as previously reported2526. The effect of FP on NLRP3 inflammasome activation was also evaluated. 17-oxo-DHA effectively inhibited nigericin-induced IL-1β release while FP had no effect (Fig. 4a). The actions of 17-oxo-DHA and FP on inflammasome activation were further assessed by evaluating the formation of cleaved caspase-1 and IL-1β as well as the expression of NLRP3, pro-IL-1β and procaspase-1. 17-oxo-DHA, but not FP, completely blocked the cleavage of pro-IL-1β and procaspase-1 (Fig. 4b). In the presence of nigericin, the strong effect on the extracellular release of IL-1β was accompanied with reduced intracellular levels of pro-IL-1β (Fig. 4b). The expression of the procaspase-1 did not show significant variations among different conditions while NLRP3 was induced by LPS, which is consistent with previous reports27. The inhibitory effect of 17-oxo-DHA on caspase-1 activity was further confirmed by flow cytometry using a fluorescent probe specific for active caspase-1. Double staining with fluorescent anti-human CD14 antibody was performed on LPS-primed and nigericin-activated PBMCs to evaluate the specific contribution of CD14+ monocytes to caspase-1 activation. Caspase-1 activation was inhibited by 17-oxo-DHA, but not by FP (Fig. 5).

View Article: PubMed Central - PubMed

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function associated with increased local and systemic inflammatory markers, such as TNFα and IL-1β. Glucocorticoids are used to treat this chronic disease, however their efficacy is low and new drugs are very much required. 17-oxo-DHA is a cyclooxygenase-2-dependent, electrophilic, α,β-unsaturated keto-derivative of docosahexaenoic acid with anti-inflammatory properties. We evaluated the action of 17-oxo-DHA alone or in combination with the steroid fluticasone propionate (FP) in peripheral blood mononuclear cells (PBMCs) from COPD patients and healthy individuals exposed to lipopolysaccharide. We show that PBMCs from COPD patients released higher levels of TNFα and IL-1β compared to controls. 17-oxo-DHA displayed strong anti-inflammatory effects. The addition of 17-oxo-DHA in combination with FP showed enhanced anti-inflammatory effects through the modulation of transcriptional and post-transcriptional mechanisms. 17-oxo-DHA, but not FP, was able to suppress the release of mature IL-1β through inhibition of the NLRP3 inflammasome. Furthermore, 17-oxo-DHA inhibited inflammasome-dependent degradation of the glucocorticoid receptor (GR). Our findings suggest that 17-oxo-DHA in combination with FP or other steroids might achieve higher therapeutic efficacy than steroids alone. Combined treatment might be particularly relevant in those conditions where increased inflammasome activation may lead to GR degradation and steroid-unresponsive inflammation.

No MeSH data available.