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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, but not FP, reduces nigericin-induced mitochondrial ROS generation in THP-1 cells.THP-1 cells were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (10 μM) for 30 min, then by 10 μM nigericin for 30 min in serum-free medium. Mitochondrial superoxide generation was measured by flow cytometric analysis after staining the cells with the fluorescent probe MitoSOX red. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for MitoSox staining.
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f8: 17-oxo-DHA, but not FP, reduces nigericin-induced mitochondrial ROS generation in THP-1 cells.THP-1 cells were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (10 μM) for 30 min, then by 10 μM nigericin for 30 min in serum-free medium. Mitochondrial superoxide generation was measured by flow cytometric analysis after staining the cells with the fluorescent probe MitoSOX red. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for MitoSox staining.

Mentions: To further investigate the molecular mechanisms of inflammasome inhibition by 17-oxo-DHA and consistent with the activation of the inflammasome in peripheral blood monocytes, our analysis was expanded using the human monocyte-like cell line THP-1. The strong inhibitory effect of 17-oxo-DHA, but not FP, on nigericin-induced release of IL-1β, and cleavage of procaspase-1 and pro-IL-1β was confirmed (Fig. 6). Similar to PBMCs, the expression of procaspase-1 did not vary among conditions while pro-IL-1β was induced by the LPS treatment. The action of 17-oxo-DHA on NLRP3 inflammasome activation was confirmed also in response to ATP and MSU crystals (Supplementary Figure S4). Stimulation of LPS-primed THP-1 cells with nigericin induces the formation of the pyroptosome, a large supramolecular assembly of ASC that is believed to mediate caspase-1 activation28. Immunostaining for endogenous ASC in THP-1 cells revealed that 17-oxo-DHA, but not FP, strongly inhibited nigericin-triggered ASC pyroptosome formation (Fig. 7). Since the activation of the NLRP3 inflammasome is mediated by mitochondrial reactive oxygen species (ROS)29, we investigated the effect of 17-oxo-DHA on mitochondrial ROS generation. As reported in Fig. 8, 17-oxo-DHA, unlike FP, inhibited nigericin-induced mitochondrial ROS generation. We also tested the effects of 17-oxo-DHA on other inflammasome complexes. We found that 17-oxo-DHA significantly inhibited the activation of AIM2 and NAIP-NLRC4 inflammasomes induced by dA-dT and flagellin from S. typhimurium, respectively (Supplementary Figure S5). In addition, we performed a colorimetric assay using human recombinant caspase-1 to test whether the effects of 17-oxo-DHA were mediated by inhibition of caspase-1. As reported in Supplementary Figure S5, 17-oxo-DHA was able to inhibit caspase-1 activity.


17-oxo-DHA displays additive anti-inflammatory effects with fluticasone propionate and inhibits the NLRP3 inflammasome
17-oxo-DHA, but not FP, reduces nigericin-induced mitochondrial ROS generation in THP-1 cells.THP-1 cells were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (10 μM) for 30 min, then by 10 μM nigericin for 30 min in serum-free medium. Mitochondrial superoxide generation was measured by flow cytometric analysis after staining the cells with the fluorescent probe MitoSOX red. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for MitoSox staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121625&req=5

f8: 17-oxo-DHA, but not FP, reduces nigericin-induced mitochondrial ROS generation in THP-1 cells.THP-1 cells were treated with 1 μg/ml LPS for 3.5 h, followed by FP (10 nM) or 17-oxo-DHA (10 μM) for 30 min, then by 10 μM nigericin for 30 min in serum-free medium. Mitochondrial superoxide generation was measured by flow cytometric analysis after staining the cells with the fluorescent probe MitoSOX red. Representative histograms of three independent experiments are shown. Numbers indicated in the graphs represent the percentage of cells positive for MitoSox staining.
Mentions: To further investigate the molecular mechanisms of inflammasome inhibition by 17-oxo-DHA and consistent with the activation of the inflammasome in peripheral blood monocytes, our analysis was expanded using the human monocyte-like cell line THP-1. The strong inhibitory effect of 17-oxo-DHA, but not FP, on nigericin-induced release of IL-1β, and cleavage of procaspase-1 and pro-IL-1β was confirmed (Fig. 6). Similar to PBMCs, the expression of procaspase-1 did not vary among conditions while pro-IL-1β was induced by the LPS treatment. The action of 17-oxo-DHA on NLRP3 inflammasome activation was confirmed also in response to ATP and MSU crystals (Supplementary Figure S4). Stimulation of LPS-primed THP-1 cells with nigericin induces the formation of the pyroptosome, a large supramolecular assembly of ASC that is believed to mediate caspase-1 activation28. Immunostaining for endogenous ASC in THP-1 cells revealed that 17-oxo-DHA, but not FP, strongly inhibited nigericin-triggered ASC pyroptosome formation (Fig. 7). Since the activation of the NLRP3 inflammasome is mediated by mitochondrial reactive oxygen species (ROS)29, we investigated the effect of 17-oxo-DHA on mitochondrial ROS generation. As reported in Fig. 8, 17-oxo-DHA, unlike FP, inhibited nigericin-induced mitochondrial ROS generation. We also tested the effects of 17-oxo-DHA on other inflammasome complexes. We found that 17-oxo-DHA significantly inhibited the activation of AIM2 and NAIP-NLRC4 inflammasomes induced by dA-dT and flagellin from S. typhimurium, respectively (Supplementary Figure S5). In addition, we performed a colorimetric assay using human recombinant caspase-1 to test whether the effects of 17-oxo-DHA were mediated by inhibition of caspase-1. As reported in Supplementary Figure S5, 17-oxo-DHA was able to inhibit caspase-1 activity.

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.