Article Title: Infected macrophages engage alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation
Figure Lengend Snippet: GM-CSF metabolically reprograms monocytes to undergo increased aerobic glycolysis, which enhances inflammatory cytokine production (A) Isolated WT MCs were uninfected or infected with L . p . (MOI=5) and given 10 ng/mL rGM-CSF or PBS vehicle control. IL-1α, IL-1β, TNF, and IL-12 levels at 12 hpi are shown. (B) Extracellular acidification rate (ECAR) of uninfected, L . p . - infected (MOI=5), or LPS-treated (10 ng/ml) MCs incubated with rGM-CSF (10 ng/ml) or PBS vehicle control for 12 h before and after sequential treatment with glucose (10mM), oligomycin (Oligo) (1μM), and 2-DG (50 mM). (C) Heatmap depicting the fold-change in transcript levels of genes encoding glucose transporters and glycolytic enzymes in LPS- or L . p . - treated MCs incubated with or without rGM-CSF for 10 h relative to uninfected cells without rGM-CSF. Graphical model depicts an abbreviated version of the glycolytic pathway showing upregulated GLUT1, HK2 and PFKP expression following rGM-CSF treatment. (D) Immunoblot analysis of GLUT1, HK2, PFKP, pro-IL-1β, and β-actin in the lysates of LPS or L . p . - treated MCs incubated with or without rGM-CSF at 10 and 20 hpi. (E) Glut1, HK2 and Pfkp transcript levels in WT and Csf2rb -/- Ly6C hi MCs from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi. Each line represents the paired values of WT and Csf2rb -/- MCs from a given mouse. (F) Il1b, Tnf , and Il12a transcript levels in LPS- or L . p . - treated MCs treated with 10 mM 2-DG or vehicle control in the presence of rGM-CSF or PBS vehicle control at 6 hpi. (G) Immunoblot analysis of pro-IL-1β and β-actin in the lysates of WT MCs incubated in media containing glucose (10 mM) or galactose (10 mM) that were then uninfected, infected with L . p . (MOI=5), or treated with LPS (10 ng/ml) in the presence of rGM-CSF or PBS vehicle control for 10 hours.. (H) TNF and IL-12 levels in the supernatants of WT MCs incubated in media containing glucose or galactose, infected with L . p ., and treated with rGM-CSF or PBS vehicle control at 10 hpi. (I) Graphical model depicting that GM-CSF/JAK2/STAT5 signaling upregulates Glut1, HK2 and Pfkp expression and promotes aerobic glycolysis, which is critical for maximal inflammatory cytokine expression. Data shown are the pooled results of three (A, B, F and H) or two (C) independent experiments with triplicate wells per condition in each experiment. Data shown in E are the pooled results of two independent experiments with 3 mice per experiment. Data shown in D and G are representative of two independent experiments. NS, not significant; *p
Article Snippet: For intracellular cytokine staining, cell suspensions were incubated with Brefeldin A (0.1%) and monensin (0.066%) solution for 4 hours at 37°C prior to all staining, treated with BD Cytofix/Cytoperm™ buffer for 20 min at 4°C, and stained with antibodies specific for TNFα (Thermo Fisher Scientific, clone MP6-XT22), IL-12 (BioLegend, clone C15.6), GM-CSF (BioLegend, clone MP1-31G6), IL-1α (Thermo Fisher Scientific, clone ALF-161), or IL-1β (Thermo Fisher Scientific, clone NJTEN3) in perm/wash buffer for 30 min at 4°C.
Techniques: Metabolic Labelling, Isolation, Infection, Incubation, Expressing, Mouse Assay