Research ArticleInflammation

Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses

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Science Translational Medicine  24 Aug 2016:
Vol. 8, Issue 353, pp. 353ra111
DOI: 10.1126/scitranslmed.aaf7483
  • Fig. 1. Proresolving lipid mediators dose-dependently reduce TNF-α from CD8+ and CD4+ T cells.

    (A) Chemical structures of the SPMs RvD1, RvD2, and MaR1. (B) Peripheral blood mononuclear cells (PBMCs) (1 × 106 cells per well) were left untreated (Veh) or treated with different concentrations (1 to 100 nM) of SPMs (RvD1, RvD2, and MaR1) for 30 min. Cells were then stimulated with PMA/ionomycin for 6 hours, stained at the cell surface and intracellularly, and analyzed by flow cytometry. (C) Representative cytofluorimetric plot of the gating strategy for TNF-α evaluation from CD8+ and CD4+ T cells. PBMCs were appropriately gated according to physical parameters. (D) Percentages of intracellular cytokine production in both CD8+ and CD4+ T cells. Data are means ± SEM of four independent experiments. *P < 0.05 [one-way analysis of variance (ANOVA)].

  • Fig. 2. Proresolving lipid mediators reduce CD8+ and CD4+ T cell responses.

    (A to D) PBMCs (1 × 106 cells per well) were left untreated or treated with RvD1, RvD2, and MaR1 (10 nM) for 30 min. Cells were then stimulated with PMA/ionomycin for 6 hours [(A) and (B)] or with anti-CD3/CD28 beads [(C) and (D)], stained at the cell surface and intracellularly, and analyzed by flow cytometry, as detailed in Materials and Methods. Percentages of intracellular production of TNF-α and IFN-γ from CD8+ and of TNF-α, IFN-γ, and IL-17 from CD4+ T cells are shown as means ± SEM of eight independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

  • Fig. 3. Proresolving lipid mediators inhibit IL-2 production from TCR-activated CD8+ and CD4+ T cells without affecting their viability.

    PBMCs (1 × 106 cells per well) were left untreated or treated with RvD1, RvD2, and MaR1 (10 nM) for 30 min. Cells were then stimulated with anti-CD3/CD28 for 8 hours, stained at the cell surface and intracellularly, and analyzed by flow cytometry. (A) Percentages of intracellular production of IL-2 from CD8+ and CD4+ T cells are shown as means ± SEM of six independent experiments. *P < 0.05 (one-way ANOVA). (B) Cell death of CD8+ and CD4+ T cells after stimulation with anti-CD3/CD28 beads through staining for annexin V and PI flow cytometry analysis. The percentage of annexin V–positive/PI-negative cells (early apoptotic cells) and annexin V–positive cells (total apoptotic cells) is reported in the cumulative graph. Data are means ± SEM of four independent experiments. (C) Cell proliferation of CD3+ T cells after stimulation with anti-CD3/CD28 beads (day 4) shown by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution. (D) Surface expression of FasL, PD-1, and CTLA-4 in CD3+ T cells after stimulation with anti-CD3/CD28. A representative experiment (of four independent experiments) of receptor expression is shown (the isotype is shown in gray).

  • Fig. 4. Proresolving lipid mediators affect TH cell polarization.

    (A) Schematic representation of TH1, TH17, and TH2 generation. ELISA, enzyme-linked immunosorbent assay. (B) Percentages of intracellular cytokine production from polarized TH1, TH17, and TH2 cells in the presence or absence of RvD1, RvD2, or MaR1 (10 nM), assessed after 6 hours of restimulation with anti-CD3/CD28. Data are means ± SEM of six independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA). (C) ELISA of IFN-γ, IL-17, and IL-4 in supernatants of TH1, TH17, and TH2 cells polarized in the presence or absence of RvD1, RvD2, or MaR1 (10 nM), measured after 24 hours of restimulation with anti-CD3/CD28. Data are means ± SEM of six independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA). (D) Real-time quantitative polymerase chain reaction (qRT-PCR) analysis of the expression of T-bet, RORc, and GATA-3 in TH1, TH17, and TH2 cells. Cycling threshold values are normalized to those of mRNA encoding ribosomal protein L34, and data are normalized to the maximum value obtained for each donor. Data are expressed as arbitrary units (A.U.) and are means ± SEM of four independent experiments. (E) Percentages of intracellular production of IFN-γ and IL-17 from CD4+ T cells of splenocytes obtained from wild-type (WT), Elovl2 knockout (Elovl2−/−), and Elovl2−/− + DHA mice. Data are means ± SEM of four different mice per experimental group and performed in duplicate. *P < 0.05 versus WT, #P < 0.05 versus Elovl2−/− (one-way ANOVA). (F) Percentages of intracellular production of IFN-γ and IL-17 from peripheral blood CD4+ T cells obtained from WT and Elovl2 knockout (Elovl2−/−) mice injected intraperitoneally with 100 ng of RvD1 for 15 min and then with 50 μg of anti-CD3 for 3 hours. Data are means ± SEM of four different mice. *P < 0.05 versus WT or Elovl2−/− (t test).

  • Fig. 5. Proresolving lipid mediators promote de novo generation of Foxp3-expressing Treg cells.

    (A) Schematic representation of iTreg generation. (B) Flow cytometry analysis of iTreg cells gated on CD4+CD25high and CD127 cells and expressing Foxp3, CTLA-4, and granzyme B, and ELISA of IL-10 in supernatants of iTreg cells. Data are means ± SEM of four independent experiments. *P < 0.05 versus iTreg, **P < 0.01 versus iTreg (one-way ANOVA). (C) Percentages of intracellular expression of Foxp3 in CD4+CD25high T cells of splenocytes obtained from WT, Elovl2 knockout (Elovl2−/−), and Elovl2−/− + DHA mice. Data are means ± SEM of five different mice. *P < 0.05 versus WT, #P < 0.05 versus Elovl2−/− (one-way ANOVA).

  • Fig. 6. The effects of proresolving lipid mediators on T cells are mediated by GPR32 and ALX/FPR2 receptors.

    (A) qRT-PCR analysis and immunoblotting of GPR32 and ALX/FPR2 in polarized TH0, TH1, TH2, TH17, and iTreg, as detailed in Materials and Methods. Data are means ± SEM of four independent experiments. *P < 0.05 versus TH0 (one-way ANOVA for TH1, TH2, and TH17, and t test for iTreg). A flow cytometry representative analysis of GPR32 and ALX/FPR2 is shown in resting and anti-CD3/CD28–activated total peripheral CD3+ T cells. (B and C) Intracellular cytokine production from CD8+ and CD4+ T cells, as well as from polarized TH1, TH17, and iTreg, pretreated with neutralizing antibodies against GPR32 or ALX/FPR2 (alone or in combination; 2 μg/ml) in the presence of RvD1 and after anti-CD3/CD28 stimulation for 8 hours. Data are means ± SEM of four independent experiments. *P < 0.05 versus anti-CD3/CD28–activated cells, #P < 0.05 versus RvD1-treated cells (one-way ANOVA).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/353/353ra111/DC1

    Fig. S1. AT-RvD3 dose-dependently reduces TNF-α, and RvD1, RvD2, and MaR1 do not affect long-term cell death in both CD8+ and CD4+ T cells.

    Fig. S2. RvD1, RvD2, and MaR1 do not affect TH0 cells.

    Fig. S3. Gating strategy for Treg identification.

    Fig. S4. Schematic representation of SPM effects on T cell subsets.

    Table S1. Antibodies used for T cell surface staining and for analysis of intracellular cytokine production.

  • Supplementary Material for:

    Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses

    Valerio Chiurchiù,* Alessandro Leuti, Jesmond Dalli, Anders Jacobsson, Luca Battistini, Mauro Maccarrone, Charles N. Serhan*

    *Corresponding author. Email: v.chiurchiu{at}hsantalucia.it (V.C.); cnserhan{at}zeus.bwh.harvard.edu (C.N.S.)

    Published 24 August 2016, Sci. Transl. Med. 8, 353ra111 (2016)
    DOI: 10.1126/scitranslmed.aaf7483

    This PDF file includes:

    • Fig. S1. AT-RvD3 dose-dependently reduces TNF-α, and RvD1, RvD2, and MaR1 do not affect long-term cell death in both CD8+ and CD4+ T cells.
    • Fig. S2. RvD1, RvD2, and MaR1 do not affect TH0 cells.
    • Fig. S3. Gating strategy for Treg identification.
    • Fig. S4. Schematic representation of SPM effects on T cell subsets.
    • Table S1. Antibodies used for T cell surface staining and for analysis of intracellular cytokine production.

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