Fig 1: 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 2: 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 3: The effects of recombinant cytokines on cell growth of human mesothelial cells. MeT-5A cells were cultured with (A) G-CSF, GM-CSF, IL-1α, IL-1β, IL-3, IL-5, IL-13 or IL-17A, and cell growth was evaluated using WST-1 assays. Some groups of cells were cultured with a mixture of all cytokines or (B) with various combinations of cytokines. Concentrations were selected according to the concentrations of cytokines in peripheral blood mononuclear cell cultures determined by the Luminex system. Values are expressed as the ratio relative to the control and as the mean ± standard deviation. Statistical significance is expressed as *P<0.01 vs. controls. G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; OD450 nm, optical density at 450 nm.
Fig 4: Cytokine analysis of murine FMC63 and humanized VH4vκ1 CAR T cells after macrophage activation. (A) Schema showing macrophage activation experiment. NSG mice were engrafted with Raji tumor followed by murine and humanized CAR treatment. Serum was collected 3 days after CAR treatment and added to GM-CSF–activated donor-matched macrophages. The supernatant was collected 48 hours later and Luminex cytokine analysis was carried out. (B) Serum collected from mice before adding to the macrophages was analyzed for baseline cytokine levels. (C) Macrophage-derived cytokines. Data are shown as mean ± SEM; n = 4 mice per group and respective macrophage conditions; P > .05.
Fig 5: GM-CSF enhances inflammatory responses in human monocytes during Legionella infection. (A and B) THP-1 human monocytes or (C to E) primary human monocytes were pretreated with PBS or rGM-CSF for 30–60 min. Cells were then left uninfected (UI), infected with L.p. or treated with the TLR2 agonist Pam3CSK4. (A and C) Cells were harvested at 6 h post-infection (hpi) to measure IL1A, IL1B, and IL6 transcript levels by qPCR, or (B and D) cells and supernatants harvested at 24 h after infection to measure IL-1α, IL-1β, and IL-6 release by ELISA or (E) intracellular IL-1α and IL-1β protein levels by immunoblot with β-actin as loading control. Data represent the mean ± SEM of triplicate wells from at least three independent experiments. Data were analyzed by two-way ANOVA with Šidák’s multiple comparisons test; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.
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