Novel proresolving and tissue-regenerative resolvin and protectin sulfido-conjugated pathways

Abstract

Local mediators orchestrate the host response to both sterile and infectious challenge and resolution. Recent evidence demonstrates that maresin sulfido-conjugates actively resolve acute inflammation and promote tissue regeneration. In this report, we investigated self-limited infectious exudates for novel bioactive chemical signals in tissue regeneration and resolution. By use of spleens from Escherichia coli infected mice, self-resolving infectious exudates, human spleens, and blood from patients with sepsis, we identified 2 new families of potent molecules. Characterization of their physical properties and isotope tracking demonstrated that the bioactive structures contained a docosahexaenoate backbone and sulfido-conjugated triene or tetraene double-bond systems. Activated human phagocytes converted 17-hydro(peroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid to these bioactive molecules. Regeneration of injured planaria was accelerated with nanomolar amounts of 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Protectin sulfido-conjugates) or 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (Resolvin sulfido-conjugates). Each protectin and resolvin sulfido-conjugate dose dependently (0.1-10 nM) stimulated human macrophage bacterial phagocytosis, phagolysosomal acidification, and efferocytosis. Together, these results identify 2 novel pathways and provide evidence for structural elucidation of new resolution moduli. These resolvin and protectin conjugates identified in mice and human infected tissues control host responses promoting catabasis.

Keywords: infection; inflammation; leukocytes; resolution.

Figure 1.

In E. coli-infected mouse spleens, identification of novel sulfido-conjugates. Mice were inoculated with E. coli (1 × 10⁵ CFU/mouse) and spleens harvested. Products were extracted using C18 solid-phase columns and investigated by LC-MS-MS (see Materials and Methods). A) MRM chromatograms for the identified sulfido-conjugated products. B and C) MS-MS spectra employed for the identification of (B) 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (I) and (C) 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (II). D) Quantification of sulfido-conjugated mediators in mouse spleen during self-resolving infections (16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CY,17-OH,C₂₂H₃₀O₂ = 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid). Results for (A–C) are representative of n = 9 mice. Results for (D) are the mean ± sem (n = 3 mice per time interval).

Figure 2.

E. coli-infected mouse exudates gave novel sulfido-conjugates. Mice were inoculated with E. coli (1 × 10⁵ CFU/mouse) and exudates collected 12 or 24 h later. Products were extracted using C18 solid-phase columns and investigated by LC-MS-MS (see Materials and Methods). A) MRM chromatograms for the identified sulfido-conjugated products. B and C) MS-MS spectra employed for the identification of (B) 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and (C)16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. D) Quantification of sulfido-conjugated mediators in mouse exudates during self-resolving infections (16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 8-GS, 7,17-diOH,C₂₂H₂₉O₂ = 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; 16-CYGL,17-OH,C₂₂H₃₀O₂ = 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid). Results for (A–C) are representative of n = 18 mice. Results for (D) are the mean ± sem (n = 6 mice per interval). Lavage volume = 5 ml each mouse.

Figure 3.

Human spleen identification of novel sulfido-conjugates. Products from human spleens were extracted using C18 solid-phase columns and investigated by LC-MS-MS. A) MRM chromatograms for the identified sulfido-conjugated products. B–D) MS-MS spectra employed for the identification of (B) 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid, (C) 16-glutathionyl,17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, and (D) 16-cysteinyl,17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results are representative of n = 3 human spleens.

Figure 4.

17 lipoxygenation of DHA is precursor to novel sulfido-conjugates with human phagocytes. Human macrophages and neutrophils were incubated 30 min at 37°C with 1 μg 17-HpD plus 100 ng zymosan. A) Representative MRM chromatograms for each of the identified 17-series sulfido-conjugates in human macrophages (left panel) and PMN (right panel). B) Representative MS-MS spectra used for the identification of 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (left panel) and 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (right panel). C) Quantification of identified sulfido-conjugates. 8-GS, 7,17-diOH-C₂₂H₂₉O₂ = 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; 8-CYGL, 7,17-diOH-C₂₂H₂₉O₂ = 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; 16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CYGL,17-OH,C₂₂H₃₀O₂ = 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CY,17-OH,C₂₂H₃₀O₂ = 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results for (A) and (B) are representative of n = 3 healthy donors for each cell type. Results for (C) are the mean (n = 3 healthy donors for each cell type).

Figure 5.

Product-precursor relationships for the novel sulfido-conjugates. Human macrophages (4.5 × 10⁷ cells/ml) were incubated with (A) DHA [37°C (pH 7.45)], (B and C) 17-HpD [37°C (pH 7.45)], and E. coli (1.5 × 10⁸ CFU), and product levels were assessed using LC-MS-MS (see Materials and Methods for details). D–F) Human macrophages were incubated with or without GGT inhibitor [Acivicin; 2.5 mM at 37°C (pH 7.45) for 30 min] then (D) DHA or (E and F) 17-HpD [37°C (pH 7.45)] and E. coli (1.5 × 10⁸ CFU). Incubations were quenched; precursor and product levels were assessed by LC-MS-MS. Results are the mean for n = 3 macrophage preparations.*P < 0.05 vs. vehicle macrophages.

Figure 6.

Apoptotic human PMNs produce novel sulfido-conjugates from endogenous DHA and promote E. coli clearance during infection. Apoptotic neutrophil (see Materials and Methods for details) products were obtained and profiled by LC-MS-MS metabololipidomics. A) MRM chromatograms for identified products. B) Representative MS-MS spectrum employed for the identification of 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results are representative of n = 6 apoptotic PMN preparations. C) Quantification of identified sulfido-conjugates in apoptotic PMN preparations. 8-CYGL, 7,17-diOH-C₂₂H₂₉O₂ = 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid;_;16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CYGL,17-OH,C₂₂H₃₀O₂ = 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results are the mean for n = 6 cell preparations. D and E) FVB mice were infected intraperitoneally with E. coli (1 × 10⁵ CFU/mouse). After 4 h, mice were administered saline (E. coli) or apoptotic PMN (apop PMN; 15 × 10⁶ cells per mouse; i.p.). Exudates were then collected 12 h postinfection. D) Exudate macrophage phagocytosis of E. coli was assessed by flow cytometry. E) Bacterial titers present in the peritoneum were measured. Results for (A) and (B) are representative of n = 3 neutrophil preparations. Results for (C) are the mean ± sem (n = 3 mice per group). **P ≤ 0.01 and ***P ≤ 0.001 vs. E. coli mice.

Figure 7.

Novel sulfido-conjugates accelerate tissue regeneration in planaria. A–C) Planaria were surgically injured, then kept in water containing (B) 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid plus 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (50 nM; each), (C) 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid plus 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (50 nM; each), or vehicle (water containing 0.01% EtOH), and tissue regeneration was assessed (see Materials and Methods for details). TRI₅₀, time to 50% regeneration. Results are the mean ± sem (n = 4 planaria per group). *P ≤ 0.05 and **P ≤ 0.01 vs. respective surgical injury group.

Figure 8.

17-series sulfido-conjugates display potent anti-inflammatory and proresolving actions with human macrophages. A) Human macrophages (5 × 10⁴ cells per well) were incubated with the indicated concentrations of novel sulfido-conjugated mediators [15 min at 37°C (pH 7.45)] before the addition of fluorescently labeled bacteria (2.5 × 10⁶ cells per well for 40 min at 37°C). Nonphagocytosed cells were washed, extracellular fluorescence was quenched, and phagocytosis assessed using a fluorescence plate reader. B) Human macrophages (5 × 10⁴ cells per well) were incubated with a pH-sensitive fluorophore (30 min at 37°C), then incubated with 10 nM sulfido-conjugated mediators [15 min at 37°C (pH 7.45)] prior to addition of bacteria (2.5 × 10⁶ cells per well for 60 min at 37°C), and assessment of fluorescence was performed using a BZ9000 microscope equipped with a ×20 objective. C) Human macrophages (5 × 10⁴ cells per well) were incubated with the indicated concentrations of the novel sulfido-conjugated products for 10 min at 37°C. Fluorescently labeled apoptotic PMNs (1.5 × 10⁵ cells per well) were then added (40 min at 37°C), and efferocytosis was measured using a fluorescent plate reader. Results for (B) are representative of n = 3 macrophage preparations. Results for (A) and (C) are the mean ± sem (n = 3 macrophage preparations). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 vs. vehicle incubated macrophages.

Figure 9.

Proposed biosynthesis of sulfido-protectins and sulfido-resolvins. Structures and pathways are depicted in likely stereochemistry based on biosynthetic evidence (see main text for details). Their stereochemistries as shown are tentative. PCTR1, 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; PCTR2, 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; PCTR3, 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; RCTR1, 8-glutathionyl, 7,17-hydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; RCTR2, 8-cysteinylglycinyl, 7,17-hydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; RCTR3, 8-cysteinyl, 7,17-hydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid.