Fig 2: PD1n-3 DPA Rectifies Murine Resident Macrophage Phenotype and Function in ALOX15-Deficient Mice(A–C) The expression of phenotypic markers was assessed in peritoneal and splenic macrophages from ALOX15-/- mice administered PD1n-3 DPA (10 ng/mouse for 7 days) or vehicle and WT mice using flow cytometry and macrophage phenotype interrogated using PLS-DA in (A) large peritoneal macrophages, (B) small peritoneal macrophages, and (C) splenic macrophages. Results are representative of n = 8 mice per group for (A and B) and n = 3–4 mice per group for (C).(D and E) Mice were treated as in (A–C), and on day 7 administered fluorescently labeled (D) apoptotic cells (6 × 106 cells/mouse) or (E) E. coli (106 CFU/mouse) via an intraperitoneal injection. Peritoneal cells were collected after 1 hr and phagocytosis was assessed in (D) CD64+ large peritoneal macrophages (left panel) and small peritoneal macrophages (right panel), and (E) total CD64+ macrophage population. Results are mean ± SEM. n = 8 mice per group for (D) and n = 4 mice per group for (E). *p < 0.05.(F) Structures are illustrated in most likely configurations based on biosynthetic evidence. The stereochemistries for PD1n-3 DPA and 16S,17S-PDn-3 DPA are established (Aursnes et al., 2015, Aursnes et al., 2014, Dalli et al., 2013a).Related to Figures S5.

Fig 3: EPHX2 Converts 16S,17S-ePDn-3 DPA to PD2n-3 DPA in Human Monocytes and Macrophages(A) Human monocytes were isolated and differentiated using GM-CSF (20 ng/mL), IFN-? (20 ng/mL), and LPS (100 ng/mL) to produce M1 or M-CSF (20 ng/mL) and IL-4 (20 ng/mL) to obtain M2 cells and the expression of EPHX2 during the differentiation time course was evaluated using flow cytometry. Results are mean ± SEM. n = 4–6 donors per interval.(B) Human monocytes were transfected with shRNA to EPHX2 or CT shRNA (see the STAR Methods for details), cells were incubated for 10 hr at 37°C, then with E. coli for 45 min, and PDn-3 DPA concentrations evaluated using LM profiling. Results are mean ± SEM. n = 4 donors. *p < 0.05.(C) 16S,17S-ePDn-3 DPA (10 nM) was incubated with hrEPHX2 (0.2 µM; Tris buffer). Incubations were quenched using ice-cold methanol and products identified using lipid mediator profiling. Left panel: MRM chromatogram m/z 361 > 233. Center panel: MS-MS spectrum employed in the identification of PD2n-3 DPA. Right panel: PD2n-3 DPA concentrations. Results are representative of n = 4 independent experiments. *p < 0.01 versus EPHX2 incubations.(D) n-3 DPA (10 µM; Tris buffer) was incubated with hr-ALOX15 (0.2 µM), EPHX2 (0.2 µM), or a combination of the two enzymes. The incubations were quenched after 15 min and products extracted, identified, and quantified using lipid mediator profiling. Left panel: MRM chromatogram m/z 361 > 233; right panel: PD2n-3 DPA concentrations. Results are representative of n = 4 independent experiments. Results for right panels in (C) and (D) are means ± SEM. *p < 0.01 versus EPHX2 incubations; #p < 0.01 versus ALOX15 incubations.(E) EPHX2 (0.2 µM, Tris buffer) was incubated with the indicated concentrations of 16S,17S-ePDn-3 DPA. Incubations were quenched and PD2n-3 DPA concentrations were determined using lipid mediator profiling. Results are mean ± SEM. n = 3 independent experiments.

Fig 4: Inhibiting 15-Lipoxygenase Activity Reduces PDn-3 DPA Production Dysregulating Macrophage Phenotype and Function(A) Human monocytes were incubated with M-CSF (20 ng/mL) and either a ALOX15 inhibitor or vehicle (37°C, 5% CO2). On day 7 incubations were quenched, lipid mediators were extracted, identified, and quantified using lipid mediator profiling (see the STAR Methods for details). Results are mean ± SEM. n = 6 donors. *p < 0.05.(B) Human monocytes were isolated and incubated with GM-CSF (20 ng/mL), IFN-? (20 ng/mL), and LPS (100 ng/mL) to produce M1 or M-CSF (20 ng/mL) and IL-4 (20 ng/mL) to obtain M2 cells, and the expression of ALOX15 and ALOX15B was evaluated during the differentiation time course using flow cytometry. Results are mean ± SEM n = 4–6 donors per interval.(C and D) Human monocytes were incubated with vehicle or ALOX15 inhibitor and then with M-CSF (20 ng/mL) for 7 days, and (C) expression of lineage markers was determined using fluorescently labeled antibodies and flow cytometry on day 7, and interrogated using OPLS-DA. n = 6 donors. (D) Phagocytosis of fluorescently labeled apoptotic cells investigated. Results for are mean ± SEM. n = 6 donors. *p < 0.05.(E) Peritoneal macrophages were harvested from wild-type (WT) and ALOX15-/- mice, and the expression of lineage markers on CD64+ cells was determined using flow cytometry. Results were interrogated using OPLS-DA and are representative of n = 7 mice.(F) Fluorescently labeled apoptotic cells were administered to WT and ALOX15-/- mice via intraperitoneal injection. After 1 hr peritoneal cells were harvested, and phagocytosis of apoptotic cells by CD64+ cells was evaluated using flow cytometry. Results are mean ± SEM. n = 7 mice per group. *p < 0.05.Related to Figures S1 and S2 and Tables S1–S3.

Fig 5: Increased ALOX15-derived lipid mediators by Treg precursors leads to FOXP3 upregulation.(A–D) CD4+ T-cells were isolated from healthy volunteers and incubated for 30 min with p38 inhibitor (SB202190) or vehicle (0.01% DMSO), then with TGF-ß+CD3/CD28 for (A, B) 12 h for the expression of (A) p-smad2/3, (B) p-STAT1 and p-STAT3, (C, D) or for 24 h and the expression of (C) ALOX15 and (D) FOXP3 assessed using flow cytometry. Results are mean ± s.e.m. and expressed as percent change from cells incubated with TGF-ß+CD3/CD28 alone; n = 4–6 healthy volunteers; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, One sample t test or Unpaired t test with Welch’s correction. (E) CD4+ T-cells were incubated with a FOXP3 inhibitor peptide (P60 peptide; 100 µM) or vehicle (0.01% DMSO) and cells differentiated to Tregs (see methods for details) for 7 days. ALOX15 expression was determined using flow cytometry. (F) HEK 293 cells were co- transfected with a vector containing the promoter region of ALOX15 tagged with luciferase and either a vector containing a FOXP3 or a control vector and luminescence was determined after 24 h. Results are mean ± s.e.m. n = 7 cell preparations from three independent experiments. For E ***p < 0.0001, One sample t test, For F **p < 0.01, Unpaired t test with Welch’s correction. (G) CD4+ T-cells were isolated from healthy volunteers and incubated with or without the Treg differentiation cocktail for 1 h (37 °C). Incubations were quenched and lipid mediator profiles determined using lipid mediator profiling. Pathway analysis for the differential expression of mediators from the (left panel) DHA and n-3 DPA, and (right panel) EPA and AA bioactive metabolomes in cells incubated with the Treg differentiation cocktail when compared to Th0 cells incubated in media alone. Statistical differences between the normalized concentrations (expressed as the fold change) of the lipid mediators from the cells incubated with Treg cocktail and Th0 cells were determined using a Mann–Whitney test followed by a multiple comparison correction using Benjamini–Hochberg procedure. Results are representative of n = 6 donors from three distinct experiments. (H) Human Tregs were obtained as detailed above, in the presence or absence of ALOX15 inhibitor (5 µM) and with RvD3, RvD5n-3 DPA (1nM) each or vehicle (0.1% EtOH; 37 °C; 7 days). Abundance of FOXP3 positive cells (left panel) and expression of FOXP3 per cell (right panel). Results are mean ± s.e.m. and expressed as percent change from Tregs + ALOX15 inhibitor, n = 4 donors from two independent experiments. *p < 0.05, One sample t test or Unpaired t test with Welch’s correction. (I) Scheme summarizing the proposed ALOX15-initiated Treg differentiation pathway.