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17-oxo-DHA displays anti-inflammatory actions and additive effects with FP in COPD patients and healthy individuals

Freshly isolated PBMCs from moderate to severe COPD patients (N = 10) and healthy individuals (N = 11, control group) were treated for 1 h with 17-oxo-DHA, FP or 17-oxo-DHA + FP, then stimulated with LPS for 18 hours. Afterwards, TNFα and IL-1β concentrations were measured in the supernatants. The induction of TNFα and IL-1β release in response to LPS stimulation was significantly higher in PBMCs from COPD patients (median (range): 17.88 ng/ml (9.003; 23.91) for TNFα and 1.528 ng/ml (1.173; 3.901) for IL-1β) compared to PBMCs from healthy individuals (median (range): 8.92 ng/ml (4.96; 11.5) for TNFα and 1.096 ng/ml (0.8440; 1.528) for IL-1β). 17-oxo-DHA and FP, when administered alone, displayed a similar effect in suppressing TNFα release, while 17-oxo-DHA was more potent than FP in inhibiting IL-1β release (). The combination of 17-oxo-DHA and FP resulted in even greater suppression of TNFα and IL-1β release, showing additive effects in both study groups. To further investigate the impact of 17-oxo-DHA on FP actions, dose-response experiments were performed and the effect of 17-oxo-DHA on half maximal inhibitory concentration (IC50) and maximum inhibition (Imax) of FP was evaluated (, ). No differences of IC50 and Imax were observed between the two study groups. When administered in combination with FP, 17-oxo-DHA significantly increased FP-Imax for both cytokines and in both study groups, without modifying FP-IC50.

17-oxo-DHA, but not FP, suppresses IL-1β release by inhibiting the NLRP3 inflammasome

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 reported. 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 (). 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 (). 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 (). 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 reports. 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 ().

17-oxo-DHA inhibits the activation of multiple inflammasomes as well as nigericin-induced pyroptosome formation and mitochondrial ROS generation

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 (). 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 (). 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 activation. Immunostaining for endogenous ASC in THP-1 cells revealed that 17-oxo-DHA, but not FP, strongly inhibited nigericin-triggered ASC pyroptosome formation (). Since the activation of the NLRP3 inflammasome is mediated by mitochondrial reactive oxygen species (ROS), we investigated the effect of 17-oxo-DHA on mitochondrial ROS generation. As reported in , 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 (). 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 , 17-oxo-DHA was able to inhibit caspase-1 activity.