Research ArticleIMMUNOLOGY

Fibrinogen-like protein 2 controls sepsis catabasis by interacting with resolvin Dp5

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Science Advances  13 Nov 2019:
Vol. 5, no. 11, eaax0629
DOI: 10.1126/sciadv.aax0629
  • Fig. 1 Temporal Fgl2 expression in SL versus DR inflammation.

    (A to C) Leukocyte numbers (A), plasma sFgl2 (B), and peripheral blood mononuclear cell (PBMC) mFgl2 levels (C) in the peripheral blood of healthy control, sepsis survivors, and nonsurvivors (n = 12 in each group). (D to F) Mice were induced different severity grades of sepsis by CLP as indicated in Materials and Methods (n = 10 in each group). Survival rates (D), plasma sFgl2 (E), and mFgl2 expression in PBMC (F) at indicated intervals. Inte, intermediate; Seve, severe. (G to I) Zym was injected (intraperitoneally) for acute peritonitis into male C57BL/6 mice: SL (1 mg per mouse) and DR (10 mg per mouse). Exudates were collected at indicated intervals. PMNs were enumerated (G). sFgl2 in the supernatant (H) and mFgl2 expression in the peritoneal leukocytes (I) were determined. Error bars represent mean ± SEM. For the survival rates, Mantel-Cox test was applied for the P values.

  • Fig. 2 Fgl2 expression is regulated by miR-466l and metalloproteinases.

    (A) In silico analysis of human and murine Fgl2 binding sites for miR-466l. (B) Mock or miR-466l was co-transfected with 3′UTR-luciferase reporter vector of vehicle control or Fgl2 3′UTR-mutant into mouse peritoneal macrophages (n = 4 independent experiments). Relative increased luciferase activity of respective 3′UTR reporters was normalized against control 3′UTR. (C) After mouse peritoneal MΦs were transfected with mock, miR-466l, shNC, or shmiR-466l, the expressions of miR-466l and FGL2 mRNA were determined with qPCR. Results (n = 4 independent experiments) were expressed as fold change against mock (miR-466l OE) or against shNC (miR-466l KD). (D) sFgl2 in supernatants and mFgl2 expression were determined. (E) After transfection with mock or miR-466l, human MΦs were treated with phosphate-buffered saline (PBS), GI254023X (10 μM), or TMI-1 (10 μM) for 24 hours, and the sFgl2 and mFgl2 expression was determined by enzyme-linked immunosorbent assay (ELISA). (F and G) Mice were challenged intraperitoneally with PBS (vehicle), GI254023X (40 μg/kg), or TMI-1 (40 μg/kg), plus Zym (1 mg per mouse); the peritoneal leukocytes were assessed (F); and the exudate sFgl2 and peritoneal leukocytic mFgl2 were determined with ELISA (G) at 4 and 24 hours. (H) Mice were challenged with/without moderate CLP, and the peritoneal leukocytes were collected for miR-466l determination at indicated time points. Error bars represent mean ± SEM.

  • Fig. 3 Fgl2 orchestrates inflammation resolution.

    (A to C) Survival rate (A), abscess formation (B), and histological staining for hematoxylin and eosin (H&E), CD45, and F4/80 (C) in WT and Fgl2KO mice challenged with moderate CLP. Mantel-Cox test was applied for the P values of the survival data (n = 10 each group). (D and E) WT and Fgl2KO mice were challenged with Zym (1 mg per mouse; intraperitoneal). Peritoneal PMN numbers at indicated intervals were counted, and resolution indices were calculated (D). Tmax, time point when PMN was infiltrated to maximum; Ѱmax, PMN maximum number; T50, time point when PMNs were reduced to half of Ѱmax; Ѱ50, 50% of Ѱmax; Ri, resolution interval (time interval from Tmax to T50); K50, rate of PMN reduction from Tmax to T50. Quantification of RvDp5 was obtained with a calibration curve (E). (F) Fgl2KO mice were injected intraperitoneally with Zym (1 mg) using PBS (Veh) or sFgl2 (200 ng). Peritoneal PMN numbers at indicated intervals were counted, and resolution indices were calculated as indicated in (B). (G) PMNs from WT, Fgl2KO, and FcγRIIBKO (RKO) mice induced apoptosis by UV for 2 hours with or without sFgl2 treatment (10 μg/ml). Apoptotic PMN (ANXAV+) percentages were analyzed. (H) Fgl2KO mice were challenged intraperitoneally with Zym (1 mg) ± sFgl2 (200 ng) for 12 hours, and the peritoneal apoptotic PMN (ANXAV+Ly6G+) was determined. (I) Murine peritoneal MΦs were isolated from WT, Fgl2KO (red and green), and FcγRIIBKO (RKO) mice. After treatment with or without sFgl2 (10 μg/ml) for 2 hours, MΦs were incubated with carboxyfluorescein diacetate (CFDA)–labeled apoptotic PMNs (1:3) for 1 hour, and fluorescence intensities were determined. Error bars represent mean ± SEM. ns, no significant difference. (J) Sepsis survival of WT, Fgl2KO, and RKO mice with or without sFgl2 administration (400 ng). n = 16 in WT and WT + sFgl2 group; n = 10 in other groups. Mantel-Cox test was applied for the P values. Photo credit: Yongsheng Li, Yu Zhou, and Juan Lei, Clinical Medicine Research Center, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

  • Fig. 4 sFgl2 induces RvDp5 biosynthesis.

    (A) Biosynthesis of RvDp5. (B) mRNA expression of key enzymes in PUFA metabolism. (C) Murine peritoneal MΦs were isolated from WT and Fgl2KO mice. Fgl2KO MΦs were treated with PBS or sFgl2 (10 μg/ml) for 4 hours. Quantification of RvDp5, LXA4, and LTB4 was obtained with UPLC-MS/MS. Results are presented as mean ± SEM (n = 4 to 6). (D) Murine peritoneal MΦs were treated with PBS, baicalein (10 μM), and/or sFgl2 (10 μg/ml) for 4 hours, and the levels of RvDp5 and LXA4 were obtained with UPLC-MS/MS. Error bars represent mean ± SEM. Two-tailed Student’s t test was applied for the P values.

  • Fig. 5 RvDp5 activates ALX/FPR2 to display dual anti-inflammatory and proresolving actions.

    (A) Mice were injected intraperitoneally with Zym (1 mg) using PBS or RvDp5 (200 ng). Infiltrated PMNs were enumerated (upper panel), and resolution indices were calculated as indicated in Fig. 3B (bottom panel). (B) Three-dimensional (3D) chart of RvDp5 binding with ALX/FPR2. (C) Binding mode of RvDp5 structure ZINC35876755 in the binding pocket of ALX/FPR2. Important amino acid residues were shown, and the red lines indicated that the hydrogen bonds formed in the corresponding residues. (D) Diagrammatic principle of β-arrestin system. (E) Ligand (RvDp5 or LXA4)–receptor interaction was monitored in Chinese hamster ovary (CHO) cells using a β-arrestin system overexpressing ALX/FPR2. RLU, relative luminescence unit. (F) Mouse was treated intraperitoneally with Zym (1 mg/mouse; vehicle) with or without 200 ng of RvD2, LXA4, or RvDp5 for 4 hours, and the exudate levels of IL-1β, IL-10, MCP-1, and TNF were determined with ELISA. Error bars represent mean ± SEM. Two-tailed Student’s t test was applied for the P values.

  • Fig. 6 RvDp5 and sFgl2 synergistically accelerate sepsis catabasis.

    (A) WT C57BL/6 mice were injected intraperitoneally with Zym (1 mg) using PBS or RvDp5 (200 ng), and exudate sFgl2 and peritoneal leukocytic mFgl2 were assessed. (B) Murine MΦs were treated with Zym (100 ng/ml), RvD1 (10 nM), RvDp5 (10 nM), and WRW4 (1 μM) as indicated. The supernatant expression of sFgl2 was detected with ELISA. Error bars represent mean ± SEM. Numbers upon the groups were P values as compared with vehicle. P1 represents the comparison between RvD1 and RvD1 + WRW4, while P2 represents the comparison between RvDp5 and RvDp5 + WRW4. (C) After human MΦs were treated with PBS (Veh), RvDp5 (10 nM) with or without GI254023X (10 μM), TMI-1 (10 μM), GW280264X (10 μM), or H89 (10 μM) for 24 hours, sFgl2 in the supernatant was assessed with ELISA. (D) WT C57BL/6 murine MΦs were treated with RvDp5 (0 to 10 nM) with or without sFgl2 (10 μg/ml) for 1 hour and then coincubated with CFDA-labeled apoptotic PMNs (1:3) for another 1 hour. The percent increases of efferocytosis are shown. (E) Fgl2KO mice were injected intraperitoneally with Zym (1 mg) using PBS, RvDp5 (200 ng), and/or sFgl2 (200 ng). The exudate PMNs were enumerated. Error bars represent mean ± SEM. P1 represents value compared with vehicle; P2 represents value compared with RvDp5 alone. Error bars represent mean ± SEM. (F) Schematic mechanism of Fgl2 and RvDp5 programming inflammation resolution. Inflammatory stimuli trigger the infiltration of PMN and ADAM10/17-mediated Fgl2 shedding. By binding to FcγRIIB, sFgl2 promotes the apoptosis of PMN, which recruit monocytes and MΦs for efferocytosis. The exudate sFgl2 enhances the expression of 12/15-LOX in MΦs and the production of SPM, including RvDp5 and LXA4. RvDp5 and LXA4 activate ALX/FPR2 to promote ADAM17-mediated sFgl2 shedding, thereby synergistically facilitating inflammation resolution.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax0629/DC1

    Table S1. Characteristics of patients.

    Table S2. The predicted binding free energy components for the ALX/FPR2-RvDp5 and ALX/FPR2-LXA4 complexes using the MM/PBSA method.

    Fig. S1. Inflammation profiles in SL versus DR inflammation.

    Fig. S2. Fgl2 expression is regulated by miR-466l and metalloproteinases.

    Fig. S3. Deficiency of Fgl2 promotes inflammation initiation and delays resolution.

    Fig. S4. Fgl2 deficiency reduces RvDp5 production.

    Fig. S5. Fgl2 regulates PMN apoptosis and phagocytes of MΦs.

    Fig. S6. An overview of binding modes for the four isomers of RvDp5 and LXA4 in the binding pocket of ALX/FPR2.

    Fig. S7. RvDp5 modulates Fgl2 and improves sepsis survival.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. Characteristics of patients.
    • Table S2. The predicted binding free energy components for the ALX/FPR2-RvDp5 and ALX/FPR2-LXA4 complexes using the MM/PBSA method.
    • Fig. S1. Inflammation profiles in SL versus DR inflammation.
    • Fig. S2. Fgl2 expression is regulated by miR-466l and metalloproteinases.
    • Fig. S3. Deficiency of Fgl2 promotes inflammation initiation and delays resolution.
    • Fig. S4. Fgl2 deficiency reduces RvDp5 production.
    • Fig. S5. Fgl2 regulates PMN apoptosis and phagocytes of MΦs.
    • Fig. S6. An overview of binding modes for the four isomers of RvDp5 and LXA4 in the binding pocket of ALX/FPR2.
    • Fig. S7. RvDp5 modulates Fgl2 and improves sepsis survival.

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