Figure 7.
PPAR-γ maintains cytokine-induced NK cell function and metabolism. Functional and transcriptomic analyses of splenic NK cells derived from Eµ-myc lymphoma-bearing or healthy mice. (A) Splenocytes from healthy and Eµ-myc lymphoma-bearing mice were harvested at 14 days after inoculation, and PPAR-γ protein expression was determined by flow cytometry. (B) Gene set enrichment analysis (GSEA) of Eµ-myc vs healthy NK cell transcriptomes as Figure 3 showing enrichment for KEGG pathway “PPAR signalling.” Functional significance of mediators of lipid metabolism was examined using pharmacological antagonists for PPAR-γ (PPAR-γi: 10 µM GW9662), FABP4/5 (FABP4/5i: 100 µM BMS309403), or a combination of both (FABP4/5i + PPAR-γi). (C-F) Splenic NK cells were isolated and expanded in culture in the presence of IL-15 (50 ng/mL) for 10 days, and treated with PPAR-γi, FABP4/5i, FABP4/5i + PPAR-γi, or DMSO control in the presence of IL-15 (10 ng/mL) for 24 hours, then stimulated with IL-12 (1 ng/mL) and IL-18 (1 ng/mL) for 20 hours. (C) Mitochondrial mass and membrane potential were measured by flow cytometry (n = 6). (D-E) Metabolic analysis to examine real-time changes in maximal OCR (D) and ECAR (n = 6) (E). (F) Expression of IFN-γ and GzmB was assessed by flow cytometry (n = 9). (G) Wild-type Eµ-myc lymphoma-bearing mice were administered rosiglitazone (200 µg) or vehicle (2% DMSO/H2O) daily from days 6 to 12. Spleens were harvested 1 day later, and mitochondrial mass and membrane potential were determined (n = 6-11 per group). (H) Splenocytes from mice as in panel F were stimulated with IL-12 + IL-15 + IL-18 for 20 hours. GolgiPlug was added for the last 4 hours to stop cytokine release before (left) the proportion of IFN-γ-producing NK cells and (right) the expression levels of IFN-γ were assessed by flow cytometry (n = 6-11 per group). Fold changes (FC) were calculated as δ MFI (MFI of target antibody staining or fluorescent probes for metabolism minus MFI of fluorescence mean of 0 (FMO) or isotype control staining) relative to that of DMSO controls. Data show mean ± SEM; statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001; NS, not significant) was determined by 1-way ANOVA Dunnett test (C-F) or Mann-Whitney test (G-I).

PPAR-γ maintains cytokine-induced NK cell function and metabolism. Functional and transcriptomic analyses of splenic NK cells derived from Eµ-myc lymphoma-bearing or healthy mice. (A) Splenocytes from healthy and Eµ-myc lymphoma-bearing mice were harvested at 14 days after inoculation, and PPAR-γ protein expression was determined by flow cytometry. (B) Gene set enrichment analysis (GSEA) of Eµ-myc vs healthy NK cell transcriptomes as Figure 3 showing enrichment for KEGG pathway “PPAR signalling.” Functional significance of mediators of lipid metabolism was examined using pharmacological antagonists for PPAR-γ (PPAR-γi: 10 µM GW9662), FABP4/5 (FABP4/5i: 100 µM BMS309403), or a combination of both (FABP4/5i + PPAR-γi). (C-F) Splenic NK cells were isolated and expanded in culture in the presence of IL-15 (50 ng/mL) for 10 days, and treated with PPAR-γi, FABP4/5i, FABP4/5i + PPAR-γi, or DMSO control in the presence of IL-15 (10 ng/mL) for 24 hours, then stimulated with IL-12 (1 ng/mL) and IL-18 (1 ng/mL) for 20 hours. (C) Mitochondrial mass and membrane potential were measured by flow cytometry (n = 6). (D-E) Metabolic analysis to examine real-time changes in maximal OCR (D) and ECAR (n = 6) (E). (F) Expression of IFN-γ and GzmB was assessed by flow cytometry (n = 9). (G) Wild-type Eµ-myc lymphoma-bearing mice were administered rosiglitazone (200 µg) or vehicle (2% DMSO/H2O) daily from days 6 to 12. Spleens were harvested 1 day later, and mitochondrial mass and membrane potential were determined (n = 6-11 per group). (H) Splenocytes from mice as in panel F were stimulated with IL-12 + IL-15 + IL-18 for 20 hours. GolgiPlug was added for the last 4 hours to stop cytokine release before (left) the proportion of IFN-γ-producing NK cells and (right) the expression levels of IFN-γ were assessed by flow cytometry (n = 6-11 per group). Fold changes (FC) were calculated as δ MFI (MFI of target antibody staining or fluorescent probes for metabolism minus MFI of fluorescence mean of 0 (FMO) or isotype control staining) relative to that of DMSO controls. Data show mean ± SEM; statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001; NS, not significant) was determined by 1-way ANOVA Dunnett test (C-F) or Mann-Whitney test (G-I).

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