Fig 2: 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 3: 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 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-dependent JAK2-STAT5 signaling is required to enhance inflammatory cytokine expression during Legionella infection. (A) THP-1 human monocytes were pretreated with PBS or rGM-CSF for 1 h. Cells were harvested at 6 h after infection to perform immunoblot analysis for phospho-STAT5, total STAT5, or β-actin as loading control. Lanes from one membrane have been cropped to depict the appropriate conditions, as indicated by the dashed lines. No changes were made to the original image during the editing. (B to C) THP-1 monocytes were pretreated with vehicle control, (B) the JAK2 inhibitor NVP-BSK805, or (C) the STAT5 inhibitor SH-4-54 for 1 h. Cells were then treated with PBS or rGM-CSF for 30–60 min and left uninfected or infected with L.p. Cells were harvested at 6 h post-infection (hpi) to measure IL1A, IL1B, and IL6 transcript levels by qPCR. Data represent the mean ± SEM of triplicate wells from at least two (C) or three (B) 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.