Mtb and Mbv differentially replicate and localise within human or bovine macrophages.
(A) Confocal images of monocyte-derived GM-CSF differentiated-human or bovine Mϕ (hMϕ or bMϕ) infected with RFP-expressing M. tuberculosis H37Rv (Mtb-RFP) or M. bovis (Mbv-RFP) for 2, 24 and 72 h. Brightfield was used to visualize the cells. Cell nuclei were stained with DAPI (blue) and bacteria-RFP are visualized in red. Scale bar: 20 μm. (B and C) Quantification of intracellular growth expressed in bacteria area (μm2) per infected cell of Mtb-RFP and Mbv-RFP within hMϕ (B) and bMϕ (C). (D and E) Electron microscopy images of hMϕ (D) and bMϕ (E) infected with Mtb or Mbv for 24 h. Asterisks mark the intracellular bacteria. Images were selected to illustrate free cytosolic bacteria (left-hand panels), phagosomal bacteria (middle panels) and bacteria surrounded by multiple membranes (right-hand panels). Scale bar, 200 nm. (F) Quantification by stereology of the proportion of bacteria contained in each compartment. Black represents the proportion of cytosolic bacteria; green, the proportion of single membrane bound bacteria, and grey, the bacteria surrounded by multiple membranes. “n” represents the number of cells analysed. The total number of bacteria analysed corresponds to the number in brackets. (G) Quantification by RT-qPCR of the relative fold change mRNA expression of interferon-β (IFNB1) in hMϕ (left graph) and bMϕ (right graph) infected with Mtb or Mbv for 24 h. Data are normalized to Mtb. (Housekeeping gene used: GAPDH). Non-infected cells (NI) were use as control. For all the figures, p-value is considered significant when < 0.05 and indicate as follow: *p<0.05; ** p<0.01; *** p<0.001; ns: not significant.
