Fig 1.
Endogenous MCTR from human macrophages, sepsis plasma and mouse infectious exudates match synthetic materials.
(A) MCTR biosynthetic pathway. (B-D) Endogenous MCTR were obtained from (B) human macrophages (1x107 cells/ml) incubated with E. coli (5x108 CFU/ml; 30min, 37°C, PBS+/+; n = 3 macrophage preparations). (C) Sepsis plasma (n = 3 patients) (D) mice were inoculated with 105 CFU/mouse E. coli and exudates collected after 24h (n = 3 mice). Products were extracted using C18 SPE and profiled using lipid mediator metabololipidomics. (E) Synthetic material. Results representative of 3 separate determinations. (F) MS-MS spectra for synthetic MCTR1, MCTR2 and MCTR3. Results are representative of d = 3.
Fig 2.
Synthetic MCTR dose dependently accelerate tissue regeneration in planaria.
(A-C) Planaria were surgically injured, then kept in water containing (A) MCTR1 (1 or 100nM) (B) MCTR2 (1 or 100nM) (C) MCTR3 (1 or 100nM) or vehicle (water containing 0.01% EtOH), and tissue regeneration was assessed (see Materials and Methods for details). TRI50, time to 50% regeneration. (D) Rank order potency at promoting tissue regeneration measured at Day 2 post-surgery for 100nM MCTR. Results are mean ± SEM. n = 8 planaria per group. *p<0.05, **p<0.01, #p<0.001, ##p<0.0001 vs. surgical Injury + vehicle; §p<0.05 vs. surgical Injury + MCTR1.
Fig 3.
MCTRs accelerate resolution of E. coli infections.
(A-E) Mice were administered (A) MCTR1, (B) MCTR2, (C) MCTR3 (50ng) or vehicle (saline containing 0.1% EtOH) via i.p injection; after 5 min they were inoculated with E. coli (105 CFU/mouse) and exudates collected at the indicated time intervals; peritoneal leukocyte counts and resolution indices were determined (See Materials and Methods). (D) E. coli phagocytosis by exudate leukocytes was determined as median fluorescence units (MFI) in the CD11b+E. coli+ population at 12h (E) Exudate macrophage efferocytosis was determined as MFI in the F4/80+Ly6G+ population. (F-J) Mice were inoculated with E. coli (105 CFU/mouse). After 12h, exudates were collected or (F) MCTR1, (G) MCTR2, (H) MCTR3 (100ng) or vehicle was administered via i.p. injection and peritoneal exudates collected 24h after E. coli inoculation; leukocyte counts were assessed and resolution indices determined. (I) 24h exudate leukocyte counts (see Materials and Methods). (J) E. coli phagocytosis by exudate leukocytes was determined as MFI in the CD11b+E. coli+ population. Results are mean±sem. n = 3 or 4 mice per group. *p<0.05, **p<0.01 vs. E. coli mice. #p<0.05 vs. E. coli + MCTR1.
Fig 4.
MCTRs are anti-inflammatory and pro-resolving with human phagocytes.
Human macrophages (5 × 104 cells per well) were incubated with vehicle (DPBS+/+), MCTR 1, MCTR2 or MCTR3 (at the indicated concentrations, 15 min, 37°C). Fluorescently labeled (A) E. coli (n = 8) were then added and phagocytosis assessed after 60min using a fluorescent plate reader. Results are mean ± SEM. *p<0.05, **p<0.01, #p<0.001, ##p<0.0001 vs. vehicle. (B) Human macrophages were plated onto chamber slides (6 x104 cells/well) and incubated with MCTR1, MCTR2, MCTR3 (1 nM) or vehicle control (PBS+/+) for 15 min at 37°C, followed by addition of BacLight Green-labeled E. coli. Fluorescent images were recorded every 6 min for 120 min. In each experiment, 4 fields (20X) per condition (per well) were recorded. Results are mean fluorescence of 4 fields/well. n = 3 separate macrophage preparations. (C) E. coli phagocytosis by human macrophages incubated with MCTRs individually or the indicated combinations of MCTRs added simultaneously. Results are mean ± SEM for cells from n = 4 healthy human donors, *p<0.05 vs. vehicle (macrophages with E. coli alone).