Activation of macrophages decreases phagosomal copper availability and impairs proliferation of Ctr3-deficient yeasts.

Phagosome copper dynamics following cytokine activation of macrophages (A, B, and D) or in vivo administration of IFN-γ (C). (A) Representative differential interference contrast (DIC) and fluorescence (GFP) images of intracellular H. capsulatum yeasts with the CTR3 promoter-gfp fusion in non-activated BMDMs (top panel) or BMDMs activated with 1000 U/mL IFN-γ (bottom panel). Scale bar represents 10 μm. (B) BMDMs were treated with IFN-γ, TNF-α, or GM-CSF and infected with H. capsulatum yeast (MOI 1:2) with TEF1 (PTEF1) or CTR3 (PCTR3) promoter-gfp fusions. After 48 hours CTR3 promoter activity of intramacrophage yeasts was determined by lysis of macrophages, recovery of yeasts, and measurement of GFP-fluorescence by microscopy (n > 100 yeasts for each sample) and normalized to the average TEF1 promoter activity. (C) Activation of the CTR3 promoter in vivo following IFN-γ treatment was determined by infecting mice intranasally with wild-type H. capsulatum yeasts with the PTEF1 or PCTR3 promoter gfp fusions. 2000 U of IFN-γ was delivered to the lungs at day 4 and 5 post-infection, and at 6 days post-infection, lungs were harvested, lung cells lysed to release intracellular yeasts, and the GFP fluorescence quantified by microscopy (n > 100 yeasts). (B-C) Data represent the CTR3 promoter activity among biological replicates (n = 3) after normalization to TEF1 promoter activity. Box plot shows upper and lower quartiles with the median value (horizontal bar). Vertical bars indicate 10% to 90% of the data distribution. Asterisks indicate significant differences compared to non-activated macrophages (* P P P (D) BMDMs were activated with IFN-γ, TNF-α, or both cytokines and infected with Ctr3-expressing (CTR3, black), Ctr3-deficient (ctr3, red) and complemented (ctr3/CTR3, blue) H. capsulatum yeasts (MOI 1:2). After 48 hours, intracellular yeasts were recovered and enumerated by plating for CFU. Data represent the average fold change ± standard deviation compared to yeasts at the start of the assay among biological replicate infections (n = 3). Asterisks indicate significant differences compared to the intracellular proliferation of CTR3 yeasts (* P < 0.05).