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. 2021 May 27;4(3):1188–1194. doi: 10.1021/acsptsci.1c00057

14,17,18-Trihydroxy-Eicosatetraenoic Acid: A Novel Pro-Resolving Lipid Mediator from Marine Microalgae

Hans Jagusch †, Markus Werner ‡, Duco Koenis §, Jesmond Dalli §,*, Oliver Werz ‡,*, Georg Pohnert †,*
PMCID: PMC8205247  PMID: 34151208

Abstract

graphic file with name pt1c00057_0006.jpg

Specialized pro-resolving mediators (SPMs) are enzymatically oxygenated derivatives of polyunsaturated fatty acids that function as central immunoregulators in mammals. Among them are resolvins (Rvs) that stimulate the clearance of harmful stimuli and limit pro-inflammatory processes. Because of their beneficial features and their high potency, SPMs are promising molecules for anti-inflammatory therapy. Besides mammals, also marine algae form lipid mediators such as prostaglandins and leukotrienes. In particular, microalgae are attractive candidates for the production of bioactive high-value metabolites. Here, we identified the diatom Cylindrotheca closterium as a prolific producer of SPMs. The diatom forms RvE3 and novel structurally related eicosanoids, including 14S/R,17R,18R-trihydroxy-eicosatetraenoic acid, which displays inflammation-resolving and anti-inflammatory bioactivities. This pro-resolving compound might enable advancements in anti-inflammatory therapy in mammals.

Keywords: algae, inflammation, lipid mediators, natural products, oxylipins, pro-resolution

C20/22 PUFA-derived eicosanoids and docosanoids function as versatile tissue hormones in mammals.1 These potent lipid mediators are biosynthesized by oxygenating enzymes such as cyclooxygenases (COXs) and lipoxygenases (LOXs).1,2 Their effects are mainly mediated by G protein-coupled receptors (GPCRs).1 Among these molecules are pro-inflammatory leukotrienes (LTs) and prostaglandins (PGs) that contribute to acute inflammation as part of the innate host-protective immune response but are also associated with widespread disorders like asthma or arthritis.3−5 The actions of these pro-inflammatory autacoids can be counteracted by specialized pro-resolving mediators (SPMs) formed during acute inflammation to restore homeostasis.6 One class of SPMs are the resolvins (Rvs) that promote the clearance of harmful stimuli and wound healing.6 In particular, ω-3 eicosapentaenoic acid (EPA)-derived E-series Rvs limit immune cell transmigration into injured tissue counteracting pro-inflammatory LTs by antagonizing the LTB4 receptor 1 (BLT1).7,8 They also promote efferocytosis of apoptotic cells and phagocytosis of pathogens via activation of the chemerin receptor (ChemR)23.7,8 Because of their selective mode of action and bioactivities at sub-nanomolar concentrations, SPMs emerged recently as drug candidates for anti-inflammatory therapy with a potentially safe profile, given that these molecules do not suppress the immune system.9

Besides mammals, marine algae produce a variety of lipid mediators, which are involved in signaling and chemical defense.10,11 Their biosynthesis is often wound-activated by lipases releasing precursor fatty acids from membrane lipids that are rapidly oxygenated by LOXs and COXs.11,12 Red macroalgae generate arachidonic acid (AA)-derived pro-inflammatory PGs and LTs that are likely causative for food poisoning.13 In contrast, many diatoms predominantly form oxylipins derived from ω-3 fatty acids such as EPA. These microalgae are, thus, putative sources of SPMs.13 The role of oxylipin chemistry in diatom ecology is intensely investigated, but the potential of diatoms as sources for pharmacologically active oxylipins is poorly explored.14 Only one former study reported on the diatoms Coscinodiscus granii and Chaetoceros didymus producing traces of RvE2 and RvE3.15 This prompted us to systematically mine algae for mammalian-like SPMs with anti-inflammatory activity.

Results and Discussion

A screening of various microalgae species revealed that the diatom Cylindrotheca closterium produces substantial amounts of C20 oxylipins after a mechanical wounding of the cells (see Table S1 in the Supporting Information).10,16 Using commercially available standards, we identified 15-hydroxy-eicosapentaenoic acid (15-HEPE 10), 18-HEPE (9), and the pro-resolving mediator RvE3 (8) from this alga (Figure 1). Upon optical rotation measurements, 8 was assigned as 17R,18R-configured [[α]D20+32° (c = 0.025 in methanol)] in accordance to the literature [17R,18R-RvE3 [α]D+34° (c = 0.16 in methanol)].17 Considering the 17R,18R-configuration of 8, we indirectly deduced the suggested absolute configuration of 9 as 18R, which is in line with the proposed biosynthetic pathway (see the section below).

Figure 1.

Figure 1

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UHPLC-MS profile of an extract from wounded C. closterium. The novel triol oxylipins 1–4 eluted at 6.1–7.4 min. Methoxylated analogues 5 and 6 eluted at 7.9–8.7 min. The epoxy-alcohol 7 eluted at 8.8 min next to RvE3 (8) at 8.9 min, 18R-HEPE (9) at 10.3 min, and 15-HEPE (10) at 10.5 min. The oxylipin profile was recorded on a QTRAP-mass spectrometer with gradient 2 (see the Supporting Information) in negative ionization mode, and the TIC in full MS is plotted.

Further, six novel eicosanoids with similar mass spectra (1–6) and a dominant, unknown metabolite with a mass that would correspond to a C20-epoxy-alcohol 7 were detected (Figure 1). The novel oxylipins have a mass of m/z 351.2166 [M-H]− for compounds 1–4 indicative for triols derived from EPA, and m/z 365.2324 [M-H]− for compounds 5 and 6 consistent with methoxylated analogues (Figures 1 and 2).

Figure 2.

Figure 2

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Structures of 1–6 with characteristic NMR correlations given as bold lines for 1H,1H COSY and arrows for characteristic HMBC signals.

The mass spectrometry (MS2) spectra of 1 and 2, 3 and 4, and 5 and 6 are identical, suggesting that these metabolites are each pairs of diastereomers (Figures S5, S8, and S11). This was confirmed by nuclear magnetic resonance (NMR) data (Tables S2–S4). The double-bond position and conformation as well as the positions of the OH groups were deduced from characteristic 1H–1H couplings and heteronuclear multiple bond correlation (HMBC) signals (Tables S2–S4). The methoxy groups in 5 and 6 were located by HMBC signals (see Table S4). Thus, the novel oxylipins were elucidated as 14,17,18-trihydroxy-eicosatetraenoic acid (HETE) (1, 2) consistent with its source of origin and bioactivity described below, 16,17,18-triHETE (3, 4), and 17,18-dihydroxy-14-methoxy-ETE (5, 6) (Figure 2). The oxylipins were produced in quantities of 30–370 fg cell–1 (Figure S4).

To assign the stereochemistry of the algal metabolites, optical rotation measurements, circular dichroism (CD) spectra, as well as biosynthetic rationales were used. All molecules are nonracemic with 1 and 2, 3 and 4, and 5 and 6 likely being pairs of epimers that differ from each other at one stereogenic center (see Figure S7, S10, and S13). In addition, we performed nuclear Overhauser effect (NOE) NMR experiments for 1–6 to deduce the relative configuration. On the basis of the proposed biosynthesis we assume that the absolute configuration at C18 is 18R, identical to that of 8. This leads to the assignments of 17R,18R for 1 and 2 as well as for 5 and 6 and 17S,18R for 3 and 4. The configuration at C14 is 14S for 1, 14R for 2, 14R for 5, and 14S for 6 as well as at C16 with 16R for 3 and 16S for 4. According to its MS2 fragmentation pattern (see Figure S14) in conjunction with biosynthetic considerations (see the section below), we tentatively assigned 7 as 16,17-epoxy-18-HETE.

The vicinal diol 8 is likely formed biotically by a sequential 18-LOX/peroxidase/15-LOX/peroxidase catalysis (Figure 3) following a mechanism proposed by Isobe et al. in 2012.18,19 In contrast to mammals, 18-LOX have been found in algae allowing alternative transformations of oxylipins.20,21 Aside to 8, we also found the acid-labile epoxy-alcohol 7 (Figure 1). On the basis of experiments in acidified solvent–water mixtures, it is the precursor for 1–4 (Figure 3). They might be formed in a mixed reaction with an abiotic acid catalysis and enzymatic contribution, as nonacidified samples also contained the oxylipins 1–4 (see Figure S2). A water-assisted epoxide opening can occur via an attack at C14 or C16, accounting for the entire product family. On the basis of the stereochemistry of the transformation products, we deduced a 16S,17S-epoxy-18R-hydroxy moiety for 7. This structure is likely generated in a sequential reaction by an 18-LOX/hydroperoxide isomerase (Figure 3, left).22,23 To prove the hydrolytic epoxide opening suggested in Figure 3, we incubated the epoxy-alcohol 7 in H218O (pH 1.5) and found 18O incorporation at position C14 or C16 resulting in singly 18O-labeled 1–4 (see Figures S3 and S22–S25). This rules out an alternative pathway via a 17,18-epoxide with a carbocation at C16 or a spontaneous process from the hydroperoxide.24 The dihydroxy-methoxy-ETEs 5 and 6 were most likely generated during the methanolic workup and can be considered as non-natural product artifacts (see Figure S3). This is confirmed by the observation that 5 and 6 are methoxylated at C14 and were not formed upon sample preparation with acetonitrile (see Figure S3). 15-HEPE 10 is likely formed independently by a 15-LOX and subsequent reduction of the intermediate hydroperoxide.

Figure 3.

Figure 3

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Proposed pathway for formation of RvE3 (8) and structurally related triHETEs (1–4) in C. closterium. When wounded, EPA is released from lipids and oxygenated by an 18-LOX forming the intermediary 18R-hydroperoxy-eicosapentaenoic acid. This molecule is subsequently either processed by a hydroperoxide isomerase (left branch) or by a peroxidase (right branch). Following the left route, the catalytic enzyme generates in consecutive reaction steps the precursor 16S,17S-epoxy-18R-HETE (7), which is hydrolyzed to 14S/R,17R,18R-triHETE (1, 2) or 16R/S,17S,18R-triHETE (3, 4). This enzymatic and/or acid-catalyzed reaction occurs at C14 (black) or C16 (gray). According to the right route, the hydroperoxy intermediate is reduced by a peroxidase to the corresponding alcohol 18R-HEPE (9) that is again oxygenated by a 15-LOX to 17R-hydroperoxy-18R-HETE and reduced by a peroxidase to RvE3 (8).18−21

Because of the structural similarity of 1–6 to RvE3 (8), we reasoned that algal oxylipins might also evoke anti-inflammatory and pro-resolving activities in mammals. This assumption was substantiated by previous observations demonstrating anti-inflammatory effects of raw extracts of C. closterium.25,26 We therefore investigated compounds 1–6 for typical SPM bioactivities against human innate immune cells, focusing on their ability to regulate phagocytosis, efferocytosis, and chemotaxis in these cells.2,5−8,27,28 Among the novel algal oxylipins 1–6, 1 and 2 mediated inflammation-resolving and anti-inflammatory features at concentrations comparable to RvE1 (Figures 4, 5, and S56).

Figure 4.

Figure 4

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Clearance of bacteria (phagocytosis) and apoptotic HL-60 cells (efferocytosis) by human immune cells. Phagocytosis of pHrodo Green-labeled S. aureus by mononuclear phagocytes (left) or macrophages (middle) labeled with Hoechst 33342, or efferocytosis of pHrodo Red SE-labeled apoptotic HL60 cells by macrophages (right) labeled with CellBrite Green. The immune cells were preincubated with vehicle (medium), 14S,17R,18R-triHETE (1) (top), 14R,17R,18R-triHETE (2) (middle), or 5S,12R,18R-triHEPE (RvE1, bottom) for 15 min prior to the fluorescence measurements over 2 h. For phagocytosis, the ratio between the fluorescence intensity of S. aureus and the cell count of the respective immune cell type is depicted as Δ% of the vehicle (medium). For efferocytosis, the ratio between the cell count of apoptotic HL60 cells and the cell count of macrophages is depicted as Δ% of the vehicle (medium). All values are shown as means ± standard error of measurement. Statistical evaluation: one-way analysis of variance with Tukey Posthoc test against the vehicle, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Figure 5.

Figure 5

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Induced transmigration of neutrophils in a Boyden chamber assay. (A) The immune cells were preincubated with vehicle (medium), 14S,17R,18R-triHETE (1) (left), 14R,17R,18R-triHETE (2) (middle), or RvE1 (right) for 30 min prior to stimulation with 10 nM LTB4. Transmigration was conducted over 1 h. Cells were subsequently stained with CellTiter-Glo for 10 min prior to the cellular luminescence measurement. A standard curve was applied to convert the values into a cell number. Depicted is the number of migrated cells in percentage to vehicle (medium). (B) Competition with LTB4 for 100 nM 1 (left) or 100 nM RvE1 (right). Depicted is the number of migrated cells in percentage to vehicle (medium). All values are shown as means ± standard error of measurement. Statistical evaluation: one-way analysis of variance with Dunnett’s Posthoc test against the vehicle (medium), * P ≤ 0.05; ** P ≤ 0.01.

We observed a stereospecific activity of the C14 epimers. 14S,17R,18R-triHETE (1) effectively suppressed LTB4-induced chemotaxis of neutrophils (Figure 5A), while its phagocytic and efferocytic efficiency was less pronounced (Figure 4). The migration-inhibitory activity of 1 was only weakly affected by LTB4 (Figure 5B). In contrast, 14R,17R,18R-triHETE (2) markedly stimulated the clearance of pathogens and apoptotic cells (Figure 4) but was less potent at diminishing LTB4-induced chemotaxis (Figure 5).

We thus introduce 14S/R,17R,18R-triHETE here as a novel SPM. Since the algal oxylipins 16R/S,17S,18R-triHETE (3, 4) and 17R,18R-dihydroxy-14R/S-methoxy-ETE (5, 6) were ineffective (see Figures S32–S47 and S52–S55), it follows that, in particular, the vicinal diol moiety at C17/18, unsubstituted C16, and hydroxylated C14 are crucial for the pro-resolving bioactivity. Since migration-inhibitory effects of 1 were weakly affected by LTB4, a competitive action via BLT1 is possibly less pronounced. 14S/R,17R,18R-triHETE (1, 2) might be an agonist of ChemR23 and/or another receptor as discussed for the structurally related 8.29 We conclude that the diatom C. closterium that can be reared in an aquaculture might be a supplier of pharmaceutically promising SPMs, such as 14S/R,17R,18R-triHETE (1, 2), that promote inflammation-resolving and anti-inflammatory pharmacotherapy in mammals.

In conclusion, our systematic screening of microalgae revealed the diatom C. closterium as a source for mammalian-like lipid mediators. We have identified novel SPMs, namely, 14S/R,17R,18R-triHETE (1, 2), that show inflammation-resolving and anti-inflammatory bioactivities against human cells including the promotion of pathogen and apoptotic cell clearance as well as suppression of neutrophil chemotaxis with similar potency as RvE1. On the basis of structure–activity relationship studies, it arises that a vicinal diol at C17/18 and unsubstituted C16 are crucial for the RvE-related bioactivity of 14S/R,17R,18R-triHETE (1, 2), whereas substitution at C14 with a methoxy group is detrimental in this respect. The bioactivity is stereospecific, with the C14 epimers 1 and 2 being differently potent. These discoveries might allow novel applications in health and disease.

Methods

More information on methods can be found in the Supporting Information

Algae Extraction

The diatom C. closterium was obtained from the BCCM/DCG Diatom Collection and cultivated on-site in 14 h light/10 h darkness cycles at 70–80 μmol m–2 s–1 and 10–12 °C before harvest. Microalgal cultures were centrifuged at 11 000g for 10 min (Sorvall Evolution RC), and pelleted cells were sonicated with a Sonopuls HD 2070 MS73 for 4 min on ice with cycle 5 × 10 s at 100% power. Samples of wounded algae were incubated for 20 min at 22 °C. Ice-cold methanol was added in a ratio of 1:2 v/v. The oxylipins were extracted by a solid-phase extraction (SPE) according to a published protocol.30

Oxylipin Profiling

For an untargeted analysis, the algal extracts were analyzed via ultrahigh-performance liquid chromatography mass spectrometry (UHPLC-MS) (Q-Exactive Plus) using gradient 1 (see the Supporting Information). For targeted experiments, the extracts were analyzed via UHPLC-MS (QTRAP) using gradient 2 (see the Supporting Information). The targeted approach analyzed the algal oxylipins and precursor fatty acids in comparison to a defined set of lipid mediator standards (see Table S5).31 Unidentified compounds were then assigned in accordance to their high-resolution mass, sum formula, and MS2 data compared to the literature.32

Isolation of Human Immune Cells

Human neutrophils were isolated from the freshly withdrawn peripheral blood of healthy adult donors, obtained from the Institute of Transfusion Medicine at the University Hospital Jena as described.33,34 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. Leukocyte concentrates were obtained from heparinized blood preparations by centrifugation (4000 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. Contaminating erythrocytes in the pelleted neutrophil fraction were removed by hypotonic lysis. Neutrophils were suspended in an ice-cold phosphate-buffered saline (PBS) at pH 7.4 containing 0.1% glucose (PG-buffer) and counted on a Vi-CELL XR (Beckman Coulter). Human peripheral blood mononuclear cells (PBMC) were procured from the NHS Blood and Transplant Bank. PBMC were isolated using the Histopaque 1077 (MilliporeSigma) density centrifugation as previously described.35

Cultivation and Differentiation

For the differentiation of mononuclear phagocytes, PBMC were plated on 96-well plates at 6 × 104 cells per well in X-VIVO 15 medium containing 10% human serum, 2 mM l-glutamine, and 1% P/S as previously described.35,36 The cells were incubated for 24 h (37 °C, 5% CO2) prior to the functional assays. For the differentiation of human monocyte-derived macrophages, a previously published protocol was used.30 Briefly, PBMC were diluted in PBS with 1 mM CaCl2/MgCl2 (PBS+/+) to 30 × 106 cells mL–1, seeded in nontreated 10 cm culture dishes, and incubated for 60 min (37 °C, 5% CO2). Subsequently, the cells were washed with PBS without Ca2+ and Mg2+ (PBS–/–), and adherent cells were incubated in RPMI 1640 medium containing 10% human serum, 1% P/S, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng mL–1) for 6 d (37 °C, 5% CO2). Medium and differentiation agents were replaced after 3–4 d. After 6 d, the medium was removed, and the remaining cells were washed with PBS–/–. Adherent macrophages were detached using PBS with 2 mM ethylenediaminetetraacetic acid (EDTA) and incubated for 15 min (37 °C, 5% CO2). The cells were scraped into a 50 mL Falcon tube and spun down at 400g for 5 min. The macrophages were then seeded onto 96-well plates as indicated in RPMI-1640.

Phagocytosis

Phagocytosis assays were performed as described previously.35 Briefly, human monocyte-derived macrophages were seeded onto 96-well plates at 3 × 104 cells per well and incubated at least for 3 h before use (37 °C, 5% CO2). Macrophages, or mononuclear phagocytes plated 24 h in advance at 6 × 104 cells per well, were stained with Hoechst 33342 for 1 h (37 °C, 5% CO2) and washed with PBS+/+. Cells were subsequently incubated in RPMI 1640 containing the respective test compound for 15 min (37 °C, 5% CO2). pHrodo Green-labeled S. aureus bioparticle conjugates were opsonized by incubation in PBS containing 20% human serum (v/v) for 30 min (37 °C, 5% CO2). Opsonized pHrodo Green-labeled S. aureus bioparticles were added to the cells at a final concentration of 5 μg per well. The increase of the pHrodo Green signal was quantified over 2 h with 10 min measurement points using a ZEISS Celldiscoverer 7 high-content imaging system (Carl Zeiss).

Efferocytosis

Efferocytosis assays were performed in accordance to a previously published protocol.35 Briefly, human monocyte-derived macrophages were seeded onto 96-well plates at 3 × 104 cells per well and incubated at least for 3 h before use (37 °C, 5% CO2). Apoptotic cells for efferocytosis were generated by seeding human promyelocytic HL-60 cells at 1 × 106 cells mL–1 onto 30 mm dishes prior to irradiation with UV-C light (254 nm) for 15 min and incubation for 2 h (37 °C, 5% CO2). Apoptotic HL-60 cells were subsequently washed with PBS+/+ and stained with 1 μM pHrodo Red SE for 30 min at 22 °C. Macrophages were stained with CellBrite Green for 1 h (37 °C, 5% CO2) and washed with PBS+/+. Cells were subsequently incubated in RPMI-1640 containing the respective test compound for 15 min (37 °C, 5% CO2). Apoptotic pHrodo Red-labeled HL-60 cells were added directly to the macrophages at 9 × 104 cells per well. The increase of the pHrodo Red signal was quantified over 2 h with 10 min measurement points using a ZEISS Celldiscoverer 7 high-content imaging system (Carl Zeiss).

Chemotaxis

A neutrophil migration was conducted via a Boyden chamber assay (ChemoTX System 101-5; Neuro Probe Inc.) using bioluminescence measurements (CellTiter-Glo) in accordance with previously published protocols.37−39 Neutrophils were resuspended in RPMI medium (containing 0.5% FCS and 1% P/S) to a density of 1.6 × 106 cells mL–1. Subsequently two different setups were applied: the inhibition of immune cell transmigration or a competition with LTB4. The respective test compounds were added to the cells and preincubated for 30 min (37 °C, 5% CO2). Then, 30 μL of LTB4 or vehicle (n = 3) was added in the lower chamber of the ChemoTX System. For inhibition studies, 10 nM LTB4 was used, whereas for competition studies 0.1–100 nM LTB4 was applied. Next, the migration membrane (5 μm pores, 4000 pores mm–2, 8 mm2) was put on top of the lower chamber, and 25 μL of the preincubated cell suspensions (4 × 104 neutrophils) was added. The ChemoTX System was closed and incubated for 1 h (37 °C; 5% CO2). Finally, 25 μL of the medium of the lower chamber was transferred in a new 384-well plate containing 25 μL of CellTiter-Glo and then incubated for another 10 min (37 °C, 5% CO2). The bioluminescence was measured with a NOVOstar microplate reader (BMG Labtechnologies) at 240–740 nm.

Acknowledgments

S. Bartram (Max Planck Institute for Chemical Ecology Jena), D. Weiß, and T. Baumeister (both Friedrich Schiller University Jena) are thanked for help on CD and optical rotation measurements as well as on graphic design. The authors furthermore thank M. Walker (Queen Mary University of London) and P. Bellstedt (Friedrich Schiller University Jena) for the support with biological assays and NMR measurements. We are grateful for a Kekulé-Stipend (H.J.) of the German chemical industry association Verband der Chemischen Industrie (VCI). This work was supported by the German Research foundation within the framework of the collaborative research centre SFB 1127/2 ChemBioSys-239748522. J.D. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant No. 107613/Z/15/Z).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00057.

  • Experimental details and supporting data (PDF)

The authors declare no competing financial interest.

Supplementary Material

pt1c00057_si_001.pdf (3.5MB, pdf)

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