Fig 1.
NMII grows robustly in C57BL6/J BMDM differentiated with rmM-CSF.
(A) BMDM in 24-well plates were infected with NMII at an MOI of 0.1 or 10. Bacterial growth was assessed by quantifying genomic equivalents (GE). Results are expressed as the means of three biological replicates from one experiment and are representative of three independent experiments. Error bars indicate the standard deviations of the means. (B) BMDM on coverslips in 24-well plates were infected with NMII at an MOI of 10. Cells were fixed immediately after infection (Day 0), and at 1 and 3 days post-infection. Cells were stained for NMII (green) and DNA (blue), and fluorescent images overlaid with the corresponding phase contrast image. Bar, 10 μm.
Fig 2.
The NMII-occupied vacuole of C57BL/6J BMDM has lysosomal characteristics.
(A) BMDM infected for 3 days and stained for NMII (red), LAMP1 (green), and DNA (blue). (B) BMDM infected for 3 days and incubated with DQ Green BSA to detect lysosomal proteolytic activity (green). Arrows denote Coxiella-containing vacuoles. An arrowhead demarks an uninfected cell. In both panels, nuclei are stained with Hoescht (blue) and fluorescent images are overlaid with the corresponding phase contrast image. Bars, 5 μm.
Fig 3.
Permissive C57BL/6J BMDM display an M0 phenotype.
BMDM were mock infected or infected with NMII at an MOI of 10. At 56 h post-infection, cells were treated with medium (no stimulation), 100 ng/ml LPS, or 20 ng/ml IL4. Six h prior to treatment with LPS, cells were also treated with 20 ng/ml IFNγ. Flow cytometry was conducted at 72 h post-infection. (A) Cells were gated on live CD11b+ F4/80+ cells with additional gating on infected cells based on NMII signal. (B) Expression of CD38 and early growth response protein 2 (Egr2), markers of classically activated (M1) and alternatively activated (M2) macrophages, respectively. Results are representative of three independent experiments.
Fig 4.
NMII-infected C57BL/6J BMDM produce moderate amounts of TNF and nitric oxide.
BMDM were infected with NMII at an MOI of 50. At 8 h post-infection, cells were mock treated or treated with 100 ng/ml LPS (TLR4 ligand), 10 μg/ml zymosan (TLR2 ligand), or 10 ng/ml TNF (nitrite assay only). Following an 18 h treatment, culture supernatants were assayed for TNF by using an ELISA kit (A) and nitric oxide by determining nitrite concentration with the Griess reagent (B). Results are expressed as the means of three biological replicates from one experiment and are representative of two independent experiments. Error bars indicate the standard deviations of the means.
Fig 5.
Wild type and Nramp1-positive ER-Hoxb8 macrophages are permissive for NMII growth.
(A) Bone marrow-derived myeloid progenitors from wild type and C57BL/6J Nramp1+ mice were immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. Macrophages were derived from immortalized progenitors using M-CSF and infected with NMII at an MOI of 10. Approximately 2 logs of NMII growth, based on genome equivalents (GE), was observed in both C57BL/6J and C57BL/6J ER-Hoxb8 Nramp1+ macrophages over a 6 day time course. Results are expressed as the means of three biological replicates from one experiment and are representative of two independent experiments. Error bars indicate the standard deviations of the means. (B) Confocal fluorescence micrographs of ER-Hoxb8 micrographs showing large vacuoles harboring NMII (red) in both cell types at 3 days post-infection. DNA is stained with DAPI (blue). Bar, 10 μm. (C) Primary BMDM from wild type and C57BL/6J Nramp1+ mice differentiated with rmM-CSF were infected with NMII at an MOI of 0.1. Approximately 2 logs of NMII growth, based on genome equivalents (GE), was achieved at early stationary phase (3 days post-infection) in both cell types. (D) Primary BMDM from wild type and C57BL/6J Nramp1+ mice differentiated with rmM-CSF were infected with Salmonella Typhimurium at an MOI of 5. Colony forming units (CFUs), representing intracellular bacteria, were enumerated at 1, 2, and 10 h post-infection by plating macrophage lysates on LB-agar plates. Significantly less CFU were recovered from Nramp1+ macrophages then from wild type macrophages at 10 h post-infection (asterisks, p <0.01). Results are expressed as the means of three biological replicates from one experiment and are representative of two independent experiments. Error bars indicate the standard deviations of the means.