Physiological and morphological differences between GM-CSF-derived human and bovine macrophages.
(A) hMϕ were infected with Mtb-RFP or Mbv-RFP at an MOI of 10, whereas bMϕ were infected at an MOI of 1. Intracellular bacteria were quantified based on intracellular RFP signal and expressed in bacteria area per infected cells. Each dot represents the average of 10 fields from 2 independent experiments (also with different donors). (B) Evolution of the number of bMϕ during the course of infection (2 to 72 hours post-infection). (C) Evolution of the number of hMϕ during the course of infection (2 to 120 hours post-infection). (D) Left panel: Confocal images of hMϕ infected with Mtb-RFP or Mbv-RFP after 5 days of infection. Brightfield was used to visualize the cells. Bacteria are visualized in red, cell nuclei were stained with DAPI (blue) and nuclei from dead cells in green. Scale bar: 20 μm. Right panel: Quantification of the level of cytotoxicity based on Green Live/Dead stain; uninfected. Non-infected cells (NI) were used as a control, n represents the number of cells analysed. (E) Left panel: Confocal images of hMϕ infected with Mtb-RFP or Mbv-RFP for 2 hours or 8 days. Actin (in green) was used to visualize the cells. Cell nuclei were stained with DAPI (blue) and bacteria-RFP are visualized in red. Scale bar: 20 μm. Right panel: quantification of intracellular growth expressed in bacteria area (μm2) per hMϕ. Data are representative of 2 independent experiments. (F and G) hMϕ and bMϕ after 7 days differentiation with GM-CSF. (F) Representative electron microscopy images of uninfected hMϕ and bMϕ. Scale bar: 5 μm (G) Representative confocal images of uninfected hMϕ or bMϕ fluorescently stained for the late endosomal marker Lamp-1. The regions in the white squares are highlighted on the right-hand side of the micrograph.
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