Identification of 14-series sulfido-conjugated mediators that promote resolution of infection and organ protection - PubMed (original) (raw)

Identification of 14-series sulfido-conjugated mediators that promote resolution of infection and organ protection

Jesmond Dalli et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Upon infection and inflammation, tissue repair and regeneration are essential in reestablishing function. Here we identified potent molecules present in self-limited infectious murine exudates, regenerating planaria, and human milk as well as macrophages that stimulate tissue regeneration in planaria and are proresolving. Characterization of their physical properties and isotope tracking indicated that the bioactive structures contained docosahexaenoic acid and sulfido-conjugate (SC) of triene double bonds that proved to be 13-glutathionyl, 14-hydroxy-docosahexaenoic acid (SCI) and 13-cysteinylglycinyl, 14-hydroxy-docosahexaenoic acid (SCII). These molecules rescued Escherichia coli infection-mediated delay in tissue regeneration in planaria, improving regeneration intervals from ∼ 4.2 to ∼ 3.7 d. Administration of SCs protected mice from second-organ reflow injury, promoting repair via limiting neutrophil infiltration, up-regulating Ki67, and Roof plate-specific spondin 3. At nanomolar potencies these conjugates also resolved E. coli infections by limiting neutrophil infiltration and stimulating bacterial phagocytosis and clearance as well as efferocytosis of apoptotic cells. Together, these findings identify previously undescribed conserved chemical signals and pathways in planaria, mouse, and human tissues that enhance host responses to contain infections, stimulate resolution of inflammation, and promote the restoration of function.

Keywords: eicosanoids; inflammation; leukocytes; omega 3; regeneration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

New pathway promotes tissue regeneration. (A) Leukocyte recruitment following E. coli (105 CFUs per mouse, i.p.) inoculation (Materials and Methods). Results are mean ± SEM for n = 4 mice per time point. (B) Surgically injured planaria were incubated with SPE-C isolate fraction 2 from E. coli resolving infectious exudates (REs), human milk, maresin 1 (100 nM), or vehicle (surgical injury; water containing 0.01% EtOH). Tissue regeneration indices were determined. _T_50, time interval corresponding to 50% of maximal tissue regeneration (_TRI_50). (C) Gene expression in regenerating blastemas (Bottom). Results are mean ± SEM (n = 3 per group pooled from blastemas of 9 animals). (D) Injured planaria were incubated with SPE-C isolate fraction 2 from human macrophages transfected with shRNA for 12-lipoxygenase, control scrambled sequence (CS shRNA), or vehicle. (B and D) Results are mean ± SEM for n = 9 planaria per incubation. *P < 0.05, **P < 0.0001 vs. surgical injury group. #P < 0.01 vs. 12-LOX shRNA at day 4.

Fig. 2.

Fig. 2.

Identification of previously undescribed signals in SPE-C isolate fraction 2. (A and B) Infectious exudates were obtained at 24 h after inoculation with E. coli (105 CFUs per mouse). Exudates were then incubated with DHA (1 µg/mL, 37 °C, 45 min), products were extracted and signals investigated by LC-MS-MS. (A) MS-MS spectrum for signals under peaks III and IV. (B) MS-MS spectrum for signals under peaks I and II. Results are representative of n = 4 mice. (C and D) Planaria were surgically injured; after 3 d, products were extracted and signals were investigated by LC-MS-MS. Results are representative of n = 20 planaria. (E_–_G) Human macrophages were transfected with shRNA for 12-LOX or CS sequence; products were then extracted and signals were investigated by LC-MS-MS. (G) MRM quantification for products under peaks III and IV (Left) and peaks I and II (Right). Results for E and F are representative of n = 3 macrophage preparations. (G) Mean ± SEM expressed as peak area ion counts. n = 3 macrophage preparations. *P < 0.05 vs. CS shRNA transfected macrophages.

Fig. 3.

Fig. 3.

New sulfido conjugates promote regeneration. Human macrophages (1 × 107 cells per mL) were incubated with 14-HpDHA (1 µM, PBS+/+) and E. coli (1 × 108 CFUs per mL, 30 min, 37 °C), and products were isolated by RP-UV-HPLC and assessed by LC-MS-MS. Representative MS-MS spectra used in the identification of (A) SCI from peaks III and IV and (B) SCII from peaks I and II. Results represent n = 10 macrophage preparations. (C) After surgical injury, planaria were incubated with SCI plus SCII (10 nM) or vehicle (surgical injury; water containing 0.01% EtOH) and regeneration indices determined. (D) After surgical injury, planaria were incubated with lipoxygenase inhibitor (L.I.; 100 μM) or vehicle (surgical injury; water containing 0.01% EtOH). SCI (Left) and SCII (Right) were quantified by LC-MS-MS. Results are mean ± SEM. n = 3 representative of 40 planaria per group. (E) Surgically injured planaria, were incubated with lipoxygenase inhibitor (L.I.; 100 μM), L.I. plus SCI plus SCII (SC; 100 nM) or vehicle. (F) After surgical injury, planaria were incubated with E. coli (108 CFUs), E. coli plus SCI and SCII (100 nM), or vehicle (surgical injury; water containing 0.01% EtOH), and regeneration indices were determined. Results are mean ± SEM for n = 9 planaria per group. *P < 0.05, **P < 0.01 vs. the respective control group. #P < 0.01, ##P < 0.001 vs. the respective L.I. group.

Fig. 4.

Fig. 4.

SCI and SCII promote tissue regeneration and regulate key signaling pathways in planaria. After surgical injury, planaria were incubated with (A) SCI (100 nM), (B) SCII (100 nM), or vehicle (surgical injury; water containing 0.01% EtOH), and regeneration indices were determined. Results represent two independent experiments and are mean ± SEM for n = 9 planaria per group. (C) After surgical injury, planaria were incubated with SCI (100 nM), SCII (100 nM), or vehicle, and gene expression was assessed 2 d postinjury in regenerating head blastemas. Results are mean ± SEM for n = 3 per incubation pooled from blastemas of 9 animals. *P < 0.05, **P < 0.01, ***P < 0.001 vs. surgical injury group.

Fig. 5.

Fig. 5.

Regulation of tissue regeneration and SC production by planaria GST. (A) DjGst expression by WISH in uninjured animals (Left; representative of n = 9) and time course for DjGst expression in regenerating blastemas (Right; mean ± SEM for n = 4 per interval pooled from blastemas of 12 animals). (B_–_D) Planaria were fed homogenized beef liver containing DjGst dsRNA or beef liver (WT). (B and C) After 8 d, DjGst expression was assessed by WISH (B) and tissue regeneration kinetics were determined (C). (D) SC levels 3 d postsurgery. Results are mean ± SEM for n = 13 planaria per group. *P < 0.05, **P < 0.01 vs. surgical injury group. MCTR, maresin conjugates in tissue regeneration.

Fig. 6.

Fig. 6.

Endogenous SCI is converted to SCII with human macrophages and planaria. (A) Human macrophages (3 × 107 cells) were incubated with DHA (37 °C, pH 7.45) and E. coli (1.5 × 108 CFUs), and product levels were assessed using LC-MS-MS. Results are mean for n = 3 separate incubations. (B) Human macrophages were incubated with or without γ-glutamyl transferase inhibitor (GTI, Acivicin; 2.5 mM, 37 °C, pH 7.45, 30 min) and then DHA (37 °C, pH 7.45) and E. coli (1.5 × 108 CFUs), and precursor and product levels were assessed by LC-MS-MS. Results are mean ± SEM for n = 3 distinct incubations. (C) Planaria were surgically injured and then incubated with or without GTI (2.5 mM). SCI and SCII were assessed 3 d after injury using LC-MS-MS. Results are mean ± SEM for n = 20 planaria per group. *P < 0.05 vs. surgical injury group, **P < 0.01 vs. macrophages plus E. coli.

Fig. 7.

Fig. 7.

SCs resolve infection and stimulate efferocytosis. (A and B) Mice were inoculated with E. coli (105 CFUs per mouse, i.p.) followed by either SCI plus SCII (50 ng per mouse each, i.p.) or vehicle (saline containing 0.1% EtOH) 12 h later (A) Peritoneal leukocyte counts and resolution indices were determined (Materials and Methods). (B) In vivo E. coli phagocytosis. Results are mean ± SEM for n = 4 mice per interval. *P < 0.05, **P < 0.01 vs. E. coli. (C) Human macrophages (5 × 104 cells per well) were incubated with SCI (Left), SCII (Right), or MaR1, and fluorescently labeled apoptotic PMN and efferocytosis were assessed. Results are mean ± SEM for n = 4 macrophage preparations. *P < 0.05, **P < 0.01 vs. vehicle group. MFI, mean fluorescence intensity.

Fig. 8.

Fig. 8.

SCs are organ-protective. Mice were subjected to hind-limb ischemia (1 h) followed by reperfusion (3 h). Ten minutes before reperfusion, vehicle (saline containing 0.1% EtOH; R.I., reflow injury), SCI plus SCII (50 ng each) were administered intravenously. Lungs were then collected. (A) Tissue H&E staining. (Scale bars, 100 µm.) Black arrows indicate leukocyte-mediated tissue damage; blue arrows indicate intact alveolar regions. (B) Immunofluorescence staining. Nuclear material (DAPI, blue), Ki67 (Alexa 488, green), RSPO3 (Alexa 594, red). (Scale bars, 100 µm.) Results are representative of n = 4 mice per group.

Fig. 9.

Fig. 9.

Proposed SC biosynthetic scheme. Structures are depicted in likely conformations based on biosynthetic evidence (see main text and Table S1 for further details). The stereochemistry of Maresin 1 and the maresin-epoxide intermediate are established (21).

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