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Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Bretscher P, Egger J, Shamshiev A, Trötzmüller M, Köfeler H, Carreira EM, Kopf M, Freigang S - EMBO Mol Med (2015)

Bottom Line: While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown.Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2).Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.

No MeSH data available.


Related in: MedlinePlus

Oxidized phospholipids are potent inhibitors of the pro-inflammatory response of myeloid cellsA, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown. (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.G–I Splenic dendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4 T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenic dendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4 T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenic dendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
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fig01: Oxidized phospholipids are potent inhibitors of the pro-inflammatory response of myeloid cellsA, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown. (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.G–I Splenic dendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4 T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenic dendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4 T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenic dendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Mentions: To examine the effects of OxPL signaling on innate immune responses, synthetic PAPC was either oxidized by metal-catalyzed oxidation or autoxidized by exposure to ambient air, and the bioactivity of the resulting oxidized PAPC (OxPAPC) mixtures was evaluated in vitro. Exposure of bone marrow-derived dendritic cells (BMDC) to OxPAPC strongly inhibited their subsequent ability to produce the pro-inflammatory cytokines interleukin (IL)-6 and IL-12 in response to stimulation of the Toll-like receptor (TLR) 7 with imiquimod (Fig1A). This potent anti-inflammatory bioactivity of OxPAPC could be directly attributed to the oxidative modification of PAPC, since treatment with 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DPPC), a phospholipid with no unsaturated acyl chains that is therefore inert to oxidation, did not show such effect (Fig1A). OxPAPC-treated cells produced reduced levels of IL-6 and IL-12 messenger RNA upon TLR stimulation, indicating that OxPL signaling regulated these cytokine responses at the transcriptional level (Fig1B). While copper-catalyzed PAPC oxidation appeared to be the most rapid and potent method to generate anti-inflammatory OxPL species, also iron-oxidized and autoxidized OxPAPC preparations comparably inhibited the inflammatory response of myeloid cells (Fig1C). We concluded from this result that modification of PAPC by reactive oxygen species generates anti-inflammatory OxPL irrespective of the method used, albeit with different efficacy and kinetics. Weak anti-inflammatory bioactivity was already detectable after 2 h of copper-catalyzed PAPC oxidation (Fig1D). The bioactivity of the OxPAPC preparation progressively increased to reach its maximum at 24 h of oxidation and remained constant at this level during further oxidation for at least 5 days (Fig1D). This observation suggested that generation of the anti-inflammatory OxPL species required a certain degree of oxidation to occur. Still, once formed, the bioactive OxPL species appeared to be relatively stable, at least under the conditions used for PAPC oxidation in vitro.


Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Bretscher P, Egger J, Shamshiev A, Trötzmüller M, Köfeler H, Carreira EM, Kopf M, Freigang S - EMBO Mol Med (2015)

Oxidized phospholipids are potent inhibitors of the pro-inflammatory response of myeloid cellsA, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown. (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.G–I Splenic dendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4 T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenic dendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4 T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenic dendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
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fig01: Oxidized phospholipids are potent inhibitors of the pro-inflammatory response of myeloid cellsA, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown. (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.G–I Splenic dendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4 T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenic dendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4 T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenic dendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
Mentions: To examine the effects of OxPL signaling on innate immune responses, synthetic PAPC was either oxidized by metal-catalyzed oxidation or autoxidized by exposure to ambient air, and the bioactivity of the resulting oxidized PAPC (OxPAPC) mixtures was evaluated in vitro. Exposure of bone marrow-derived dendritic cells (BMDC) to OxPAPC strongly inhibited their subsequent ability to produce the pro-inflammatory cytokines interleukin (IL)-6 and IL-12 in response to stimulation of the Toll-like receptor (TLR) 7 with imiquimod (Fig1A). This potent anti-inflammatory bioactivity of OxPAPC could be directly attributed to the oxidative modification of PAPC, since treatment with 1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DPPC), a phospholipid with no unsaturated acyl chains that is therefore inert to oxidation, did not show such effect (Fig1A). OxPAPC-treated cells produced reduced levels of IL-6 and IL-12 messenger RNA upon TLR stimulation, indicating that OxPL signaling regulated these cytokine responses at the transcriptional level (Fig1B). While copper-catalyzed PAPC oxidation appeared to be the most rapid and potent method to generate anti-inflammatory OxPL species, also iron-oxidized and autoxidized OxPAPC preparations comparably inhibited the inflammatory response of myeloid cells (Fig1C). We concluded from this result that modification of PAPC by reactive oxygen species generates anti-inflammatory OxPL irrespective of the method used, albeit with different efficacy and kinetics. Weak anti-inflammatory bioactivity was already detectable after 2 h of copper-catalyzed PAPC oxidation (Fig1D). The bioactivity of the OxPAPC preparation progressively increased to reach its maximum at 24 h of oxidation and remained constant at this level during further oxidation for at least 5 days (Fig1D). This observation suggested that generation of the anti-inflammatory OxPL species required a certain degree of oxidation to occur. Still, once formed, the bioactive OxPL species appeared to be relatively stable, at least under the conditions used for PAPC oxidation in vitro.

Bottom Line: While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown.Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2).Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.

No MeSH data available.


Related in: MedlinePlus