Figure 4.

Anti-bacterial host defense is enhanced in GILZ knockout macrophages. (A,B) Wildtype- (WT) and GILZ Knockout- (KO) MPI macrophages were treated with LPS (10 or 100 ng/mL) Pam₃CSK₄ (Pam, 1 μg/mL, B), or lipoteichoic acid (LTA, 5 μg/mL, B) with or without co-stimulation by IFN-γ (20 ng/mL) for 20 h. NO-production was measured by Griess assay. Data were normalized to total protein concentration and expressed as x-fold of untreated cells (n = 4, triplicates). (C–E) Phagocytic activity of WT and GILZ KO MPI cells. MPI cells were incubated with fluorescent latex particles (diameter 1.75 μm, 50 particles per cell) for 15 or 30 min and particle-associated fluorescence was quantified by flow cytometry (15 min: n = 2, 30 min: n = 6, triplicates). (C) Representative histogram for 15 min. 0 P, cells without particles; 1 P, 1 particle; 2 P, 2 particles; > 2 P, more than 2 particles. (G) Phagocytic activity as a total percentage of cells with particles. (E) The percentage of cells that engulfed 0, 1, 2 ore more than 2 particles was quantified as shown in (C). (F,G) WT and GILZ KO BMMs were left untreated (Co) or treated with LPS (1 μg/mL) and INF-γ (IFN, 20 ng/mL), IL-4 (20 ng/mL) or dexamethasone (Dex, 1 μM) for 20 h, followed by incubation with fluorescent latex particles (diameter 1.75 μm, 50 particles per cell) for 1 h. Particle uptake was quantified by flow cytometry. (F) Total percentage of cells with particle-associated fluorescence. (D) Percentage of cells with 0, 1, 2 or more than 2 particles. (H) Relative number of viable bacteria in Salmonella typhimurium-infected WT and GILZ KO BMMs. Extracellular bacteria were removed after 30 min, and the remaining bacteria were killed using gentamicin. Eight hours following infection, monolayers were lysed, and the number of intracellular bacteria was determined as Colony Forming Units (CFU) (n = 8, triplicates). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 as indicated or compared with equally treated WT cells (E,G).