15‐Hydroperoxy‐PGE2: Intermediate in Mammalian and Algal Prostaglandin Biosynthesis

Abstract Arachidonic‐acid‐derived prostaglandins (PGs), specifically PGE2, play a central role in inflammation and numerous immunological reactions. The enzymes of PGE2 biosynthesis are important pharmacological targets for anti‐inflammatory drugs. Besides mammals, certain edible marine algae possess a comprehensive repertoire of bioactive arachidonic‐acid‐derived oxylipins including PGs that may account for food poisoning. Described here is the analysis of PGE2 biosynthesis in the red macroalga Gracilaria vermiculophylla that led to the identification of 15‐hydroperoxy‐PGE2, a novel precursor of PGE2 and 15‐keto‐PGE2. Interestingly, this novel precursor is also produced in human macrophages where it represents a key metabolite in an alternative biosynthetic PGE2 pathway in addition to the well‐established arachidonic acid‐PGG2‐PGH2‐PGE2 route. This alternative pathway of mammalian PGE2 biosynthesis may open novel opportunities to intervene with inflammation‐related diseases.


Instrumentation
NMR All 1D ( 1 H, 13 C) and 2D (HSQC, 1 H-1 H-COSY, HMBC) NMR spectra were recorded in deuterated solvents on an AVANCE III 600 MHz (equipped with an inverse coil) instrument (Bruker, Germany) at room temperature (22 °C). The chemical shifts are reported in parts per million (δ) relative to the resonance of the residual solvent signal (δ (CD3)2SO = 2.50 for 1 H spectra and 39.51 for 13 C spectra). Coupling constants (J) are reported in Hertz (Hz). NMR spectra were evaluated using the TopSpin® software.
Drying and freeze-drying Samples were dried on a TurboVap® (Biotage, Sweden) and an OIL MIST FILTER EMF3 high vacuum pump (Edwards High Vacuum Int., Great Britain). For freeze-drying an Alpha 1-2 LD Laborgefriertrockner (Martin Christ Gefriertrocknungsanlagen, Germany) was used.

Statistical and image processing software
For statistical evaluation and quantification the Excel and GraphPad Prism software was used. Chemical formulae were created with the ChemDraw software. As graphic processing programs GraphPad Prism and OriginPro were used.

Algae extraction
Algae collection and extraction was conducted according to according to Jagusch et al. 2019 and was slightly modified. [1] Briefly, field samples of the red alga G. vermiculophylla (Baltic Sea near Kieler Förde, Germany) were collected by hand in the intertidal region and frozen immediately in liquid nitrogen. Algae were characterized according to morphological features by the taxonomic expert Florian Weinberger (IFM Geomar, Kiel). Samples were stored at -80 °C until utilization. For extraction, the frozen tissue was taken out of the freezer (-80 °C) and ground in a mortar under liquid nitrogen. For analysis of intact tissue the cold sample was extracted. For samples of wounded algae, the homogenate was incubated for 20 min at 22 °C before transfer to 50 mL Falcon tubes. Ice-cold methanol was added in a ratio of 1:2 w/v. The oxylipins were extracted by SPE according to a published protocol which was slightly modified. [3] The following steps were performed on ice. A sample Falcon tube was centrifuged on a Heraeus Multifuge® X3R (Thermo Fisher Scientific) for 5 min at 3,488 g. Subsequently, the supernatant was decanted into a fresh Falcon tube and diluted 1:4 v/v with water acidified with PBS-HCl (see SI- Table 8) and mixed thoroughly (final pH 3.5). The extract was loaded on a 10 g silica-bond C18 Vac cartridge (Sep-Pak®, 35 cc, 55-105 µm, 125 Å, WAT 043345, Waters) that was previously equilibrated with 20 mL methanol and 20 mL water. Next, the cartridge was washed with 20 mL water and 20 mL hexane. The oxylipins were eluted with 12 mL methyl formate into test tubes. The organic solvents were evaporated under a nitrogen stream. The residue was taken up in methanolwater (50:50 v/v for UHPLC-MS or 72:28 v/v for semi-preparative HPLC, maximum solubility: extract of 37 g alga mL -1 ) and centrifuged at 3,488 g for 5 min (Heraeus Multifuge® X3R). The solution was transferred into a 2 mL Eppendorf tube and subsequently centrifuged twice at 21,130 g for 5 min in a Centrifuge 5424 R (Eppendorf, Germany). The purified extract was decanted into a new tube and stored in a freezer (-20 °C).

Thermal degradation and reduction
Two different treatments with 3 were conducted: 1) The sample (stock in methanol, 69 mM) was diluted 1:1000 v/v in methanolwater (50:50 v/v). Four aliquots were prepared accordingly which were either incubated at -20 °C, 22 °C, 37 °C or 60 °C for 24 h. 2) 78.7 µL of the stock solution (in methanol, 69 mM) were diluted with 21.32 µL methanol v/v (final concentration 54.3 mM) and equimolar NaBH4 was added thereafter. The sample was stirred at 0 °C for 5 min. The reaction was quenched with some droplets of water and the mixture was then diluted 1:500 v/v in methanol-water (50:50 v/v). All samples were analyzed by UHPLC-MS as indicated using gradient 1 (Q-Exactive Plus) with 3 µL injection volume.

Profiling by LC-MS
Nontargeted metabolomics experiments were performed with extracts (in methanol-water 50:50 v/v; 5 g algal extract mL -1 ) from wounded or intact (data not shown) G. vermiculophylla that were analyzed via UHPLC-MS (Q-Exactive Plus) using gradient 1 with 1 µL injection volume. SI- Figure 2. UHPLC-MS profiles for 3 (black, red at 7.36 min) and products 1 and putative PGF2α/β (red at 7 min and 7.2 min) formed upon treatment with NaBH4. All profiles were measured in negative ionization mode and the total ion count in full MS is plotted. Figure 3. UHPLC-MS profiles for 3 (black, red at 7.36 min) and products 2 (black at 6.5 min), 1 (black, red at 7 min), and putative PGF2α/β (red at 7.2 min) formed under thermally controlled conditions (black: 22 °C, 24 hours) or upon treatment with NaBH4 (red) in comparison to 1 isolated from alga (green) at 7 min and prostaglandin standards: 1 (blue) at 7 min; 2 (purple) at 6.5 min. All profiles were measured in negative ionization mode and the total ion count in full MS is plotted.

Analytical data for 15-hydroperoxy-PGE2
The structure of 3 was assigned with MS 2 (Q-Exactive Plus, as indicated) and NMR ( 1 H, 13 C, HSQC, 1 H, 1 H-COSY, HMBC) (see SI- Table 1). MS 2 fragmentation was conducted with 10 eV (see SI- Figure 4). The chromophore of 3 in comparison to 1 and 2 was confirmed by the UV spectrum showing a maximum absorption at 215 nm (see SI- Figure 5). The absolute configuration was deduced by LC and CD. Thermal degradation products of 3 co-eluted with the commercially available standards 1 and 2 (Q-Exactive Plus, as indicated). CD spectra of 3 and isolated 1 were in accordance with the commercially available standard 1 also excluding racemic mixtures (see SI- Figure 6).

Isolation of human immune cells
Human peripheral blood mononuclear cells (PBMC) were collected from freshly withdrawn peripheral blood of healthy adult human donors, obtained from the Institute of Transfusion Medicine at the University Hospital Jena (Germany). The experimental protocol was approved by the local ethical committee at the University Hospital Jena. All methods were conducted in accordance with the relevant guidelines and regulations. Informed consent was given from all donors. PBMC were isolated as described. [2] Briefly, leukocyte concentrates were obtained from heparinized blood preparations by centrifugation (4,000 g, 20 min, 20 °C) on a Heraeus Multifuge® X3R (Thermo Fisher Scientific). Subsequently, leukocyte concentrates were subjected to dextran sedimentation and centrifuged (872 g, 4 °C, 10 min) on LymphoPrep. The PBMC fractions were combined and suspended in ice-cold PBS containing 0.1 % glucose (PG buffer) and counted on a Vi-CELL® XR (Beckman Coulter, United States).

Differentiation and polarization
For differentiation and polarization toward human M1 or M2 macrophages, published criteria were used. [3] Briefly, isolated PBMC were diluted and resuspended in PBS plus 1 mM CaCl2 / MgCl2 to 180x10 6 cells 15 mL -1 and incubated for 1 h (37 °C, 5 % CO2) in a 175 cm flask. The medium was removed and 15 mL RPMI 1640 medium supplemented with 10 % FBS, penicillin-streptomycin (RPMIFP), and 5 mM L-glutamine were added to remove nonadherent lymphocytes. Monocytes were subsequently differentiated either to M0 macrophages with GM-CSF or M-CSF (20 ng mL -1 , respectively) for six days. Medium and differentiation agents were replaced after three days. After six days, the medium was removed and remaining cells were washed with 10 mL PBS. Adherent macrophages were detached using 10 mL PBS plus 5 mM EDTA and incubated for 30 min (37 °C, 5 % CO2). The flask was shaken and cells were scraped into a 50 mL Falcon tube and spun down at 1200 g for 5 min at 4 °C. The pellet was taken up in 3 mL RPMIFP and macrophages were resuspended to obtain 2x10 6 cells 3 mL -1 per well in 6-well plates which were then incubated for 2 h (37 °C, 5 % CO2). Polarization toward M1 or M2 macrophages was achieved applying 20 ng mL -1 INF-γ plus 100 ng mL -1 LPS or 20 ng mL -1 IL-4 followed by incubation for 48 h (37 °C, 5 % CO2).

Stimulation of macrophages for lipid mediator production
Macrophages were stimulated to initiate lipid mediator production. Therefore, a well-established protocol was used and slightly modified. [2] Experiments were implemented with simplicates of a maximum of six different human donors. Briefly, RPMIFP was removed and 1 mL PBS plus 1 mM CaCl2 was added per well and incubated for 3 min. Two approaches were applied for M1 and M2 macrophages: 1) general analysis of lipid mediator profiles (stimulated n=4, nonstimulated n=3), and 2) modification of the lipid mediator spectrum (n=6) using 3 (final concentration 100 nM) or methanol (0.5 %, vehicle) by preincubation for 10 min. The cells were then stimulated with ionophore A23187 (final concentration 2.5 µM) or methanol (0.5 %) for another 10 min. 3 was resolved in 1 mL PBS plus 1 mM CaCl2 in the absence of cells (control) and incubated for 20 min (n=6). Lipid mediator biosynthesis was stopped with 2 mL ice cold methanol containing 10 µL deuterated standard mix (see SI- Table 6) for UHPLC-MS quantification.

Extraction
Solid phase extraction (SPE) of macrophage samples was conducted as published, [3] with slight modifications. The 3 mL incubation mixture (see above) was brought to -20 °C for 30 min to precipitate proteins. The sample was then centrifuged on a Heraeus Multifuge® X3R (Thermo Fisher Scientific) for 10 min at 1,200 g. Subsequently, the supernatant was decanted into a fresh Falcon tube and diluted 1:4 v/v with water acidified with PBS-HCl (see SI- Table 8) and mixed thoroughly (final pH 3.5). The extract was loaded on a 500 mg silica-bond C18 Vac cartridge (Sep-Pak®, 6 cc, 55-105 µm, 125 Å, WAT 043395, Waters) that was equilibrated with 6 mL methanol and 2 mL water. Next, the cartridge was washed with 6 mL water and 6 mL hexane. The lipid mediators were eluted with 6 mL methyl formate. The organic solvents were evaporated under a nitrogen stream. The residue was taken up in 200 µL methanol-water (50:50 v/v for UHPLC-MS) and centrifuged at 3,488 g for 5 min (Heraeus Multifuge® X3R). The solution was transferred into a 2 mL Eppendorf tube and subsequently centrifuged twice at 21,130 g for 5 min in a Centrifuge 5424 R (Eppendorf). The purified extract was decanted into a new tube and stored in a freezer (-20 °C).

LC-MS analysis and quantification
The lipid mediators produced by M1 or M2 macrophages were analyzed via UHPLC-MS (QTRAP) according to the literature. [2,4] A selected set of external standards (see SI- Table 7) was used. Standard curves for 3 and 2 were recorded separately (see SI- Figures 7 and 8). Six-point calibrations (0 nM, 5 nM, 25 nM, 50 nM, 100 nM, and 200 nM, n=3, respectively) with isolated 3 and commercially available 2 in methanol-water (50:50 v/v) were applied. Calculated values were normalized to the deuterated internal standard mix (see SI- Table 6). All samples were investigated using gradient 2 with 10 µL injection volume. Change in PG amounts upon treatment of cells with 3 were calculated as follows: the total amount of 1 or 2 in the respective cell type was subtracted by the amounts formed endogenously and by the amounts generated in the equally handled compound control. For statistical evaluation, amounts of PGs produced by differently treated M1 or M2 macrophages are shown as means ± SEM and a one way ANOVA with Tukey Post-hoc test was performed, * P≤0.05; ** P≤0.01, *** P≤0.001.  SI- Figure 9. Amounts of 2 produced in 2x10 6 stimulated (n=4) or nonstimulated (n=3) human M1 or M2 macrophages shown as means ± SEM. Statistical evaluation: one way ANOVA with Tukey Post-hoc test, * /+ P≤0.05; ** /++ P≤0.01; *** /+++ P≤0.001. Cells were suspended in 1 mL PBS plus 1 mM CaCl2 and incubated for 10 min at 37 °C with 2.5 µM A23187 or vehicle (0.5 % methanol). Figure 10. Production of 2 in 2x10 6 stimulated or nonstimulated A) M1 or B) M2 macrophages, either treated with 100 nM 3 (stimulated, nonstimulated n=6, respectively) or vehicle (0.5 % methanol) (nonstimulated n=3; stimulated n=4). Cells were suspended in 1 mL PBS plus 1 mM CaCl2 and preincubated with 3 or vehicle for 10 min at 37 °C, and subsequently stimulated with 2.5 µM A23187 or vehicle (0.5 % methanol) and incubated for another 10 min at 37 °C. 3 was dissolved in 1 mL PBS plus 1 mM CaCl2 and incubated in absence of cells for 20 min at 37 °C as control (n=6) to determine formation of 2 due to degradation. All values are shown as means ± SEM. Statistical evaluation: one way ANOVA with Tukey Post-hoc test, * /+ P≤0.05; ** /++ P≤0.01; *** /+++ P≤0.001. S12 Na2HPO4·2H2O, 5.28 g L -1 NaCl, 90 g L -1