Fig 1.
Up-regulated uptake of erythrocytes by macrophages infected with L. donovani in vitro.
A representative fluorescence microscopy image of RAW264.7 cells cultured with medium only (upper panel) or with L. donovani and IFN-γ (lower panel) are shown; Ld/egfp, green; RBCs, red; nuclei, blue. This experiment was conducted once. Scale bar: 20 μm.
Fig 2.
Up-regulated uptake of erythrocytes but not polystyrene beads by RAW264.7 cells infected with L. donovani.
RAW264.7 cells were cultured with live/killed L. donovani followed by incubation with RBCs or polystyrene beads. (A) A representative image of L. donovani-infected RAW264.7 cells phagocytosing RBCs (red) is shown. (B, C) The percentage of phagocytes engulfing RBCs (B) or polystyrene beads (C) were counted. The mean + SD of at least five fields are shown. These are representatives of three independent experiments with similar results. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Bonferroni's multiple comparisons test.
Fig 3.
Up-regulated uptake of erythrocytes by primary macrophages infected with L. donovani.
Peritoneal exudate cells (PECs) or bone marrow-derived macrophages (BMDMs) were cultured with L. donovani followed by incubation with RBCs. (A) Representative images of uninfected PECs and L. donovani (green)-infected PECs followed by incubation with RBCs (red) are shown. (B, C) The percentage of phagocytes engulfing RBCs were counted for PECs (B) and BMDMs (C), respectively. The mean + SD of at least four fields are shown. These are representatives of two independent experiments with similar results. *P < 0.05, **P < 0.01 by unpaired t test.
Fig 4.
Down-regulation of SIRPα in L. donovani-infected macrophages.
(A) Immunoblotting for SIRPα in naïve/L. donovani-infected RAW264.7 cells. The results of three independent experiments are shown. (B) Densitometric analysis with Image J software also showed decreased SIRPα in the infected macrophages. The mean and SD for each group are shown. *P < 0.05 by unpaired t test.
Fig 5.
Low SIRPα expression on MGCs in the spleen of L. donovani-infected mice.
Sections from the spleen of L. donovani-infected mice at 24 weeks post-infection were stained with Hematoxylin/Eosin, or immunohistochemically stained with anti-F4/80 or anti-SIRPα antibody, followed by counterstaining with hematoxylin. Arrows point to parasitized MGCs. Scale bars, 20 μm.
Fig 6.
Increased survival of L. donovani in RBC-supplemented macrophages.
(A) A representative image of uninfected macrophages (Mφ) engulfing glutaraldehyde-treated RBCs, showing highly efficient uptake of the treated RBCs. (B and C) Glutaraldehyde-treated RBCs or polystyrene beads were added to RAW264.7 cells infected with L. donovani. After 66 h, the number of infected Mφ (B) and that of amastigotes per Mφ (C) cultured in medium-only (LD only), RBC-supplemented medium (LD + RBCs) or bead-supplemented medium (LD + beads) were counted on Giemsa-stained samples. The mean + SD of triplicate are shown. These are representative of four independent experiments with similar results. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Bonferroni's multiple comparisons test.
Fig 7.
Increased Hmox1 gene expression in RBC-supplemented RAW264.7 cells.
L. donovani-infected RAW264.7 cells were cultured in medium-only (LD only), RBC-supplemented medium (LD + RBCs) or bead-supplemented medium (LD + beads). (A) Leishmania gapdh DNA were quantified by qPCR. (B) Murine Hmox1, Fpn and Fth mRNA were quantified by qPCR. The values show fold change in expression levels after normalization with murine Gapdh levels. The data are representative of two independent experiments with similar results.