Fig 1: 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; ?max, PMN maximum number; T50, time point when PMNs were reduced to half of ?max; ?50, 50% of ?max; 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 Fc?RIIBKO (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 MFs were isolated from WT, Fgl2KO (red and green), and Fc?RIIBKO (RKO) mice. After treatment with or without sFgl2 (10 µg/ml) for 2 hours, MFs 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 2: FGL2 promoted accumulation of hepatic neutrophils via enhanced expression of ICAM-1, Cxcl1/2, and Cxcr2 in MHV-3 infection. Liver neutrophil infiltration was detected by Ly6G staining using (A) immunohistochemistry and (B) flow cytometry gated by CD45+CD11b+LY6G+. Scale bars: 50 µm. (C) Protein levels of hepatic ICAM-1, E-selectin, and L-selectin were measured by Western blot and analyzed by Image lab. (D) Relative mRNA levels of Cxcl1, Cxcl2, Cxcl5, and Cxcr2 in liver were measured by quantitative reverse-transcription polymerase chain reaction and expressed as a ratio to ß-actin. For bar graphs, n = 5 in each group. *P < .05, **P < .01, ***P < .001, and ****P < .0001, determined by 1-way analysis of variance followed by the Tukey follow-up test. CXCL, Chemokine (C-X-C Motif) Ligand; CXCR, CXC chemokine receptor.
Fig 3: 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 MFs 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 MFs 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 MFs 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 Fc?RIIB, sFgl2 promotes the apoptosis of PMN, which recruit monocytes and MFs for efferocytosis. The exudate sFgl2 enhances the expression of 12/15-LOX in MFs 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.
Fig 4: Neutrophil-specific FGL2 enhanced NETs formation and aggravated liver injury in MHV-3–infected mice. (A–D) Fgl2-/- and WT mice were challenged with 100 plaque forming unit (PFU) of MHV-3. (A) H&E staining of liver tissue was shown at 24, 48, and 72 hours after MHV-3 injection (n = 5 in each group). Arrows indicate necrotic areas. (B) Representative images of liver tissues were shown at 72 hours after MHV-3 infection. (C) Serum ALT, AST, (D) TNF-a, IL1ß, and IL6 levels were measured at the indicated time points after MHV-3 infection (n = 5 in each group). (E) Fgl2-/- or WT BM neutrophils were adoptively transferred intravenously into fgl2-/- mice as the time shaft indicated (n = 5 in each group). (F) Hepatic neutrophil infiltration after the adoptive transfer experiment were detected using immunochemistry. (G) Histologic liver tissues at 48 hours after MHV-3 injection were observed. (H) Serum ALT, AST, (I) TNF-a, IL1ß, IL6, and (J) MPO-DNA levels (n = 5 in each group) were measured at 48 hours after MHV-3 infection. (K) Hepatic NETs formation was detected by immunofluorescence staining as previously described. (L) Fibrin deposition of liver tissues at 48 hours after MHV-3 injection was measured by immunohistochemistry. Scale bars: 50 µm. For immunochemical analysis, 10 microscopic fields per liver section from 3 mice in each group were counted using ImageJ. *P < .05, **P < .01, ***P < .001, and ****P < .0001, determined by 1-way analysis of variance.
Fig 5: 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.
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