Figure 1.
Incubation of axenic Leishmania promastigotes with lytic human HDL reduces the infectivity of the parasites.
L. major metacyclics were observed under the microscope after co-incubation for 24 hours with lytic HDL (1.5 mg/ml) in acidic media pH 5.2 (A) or in neutral pH 7.4 media (B). BALB/c bone-marrow derived macrophages were infected with HDL-pre-treated L. major metacyclics (C,D), HDL-pre-treated L. amazonensis promastigotes (E,F) or HDL-pre-treated amastigote-like forms (G,H) at a multiplicity of infection of 6∶1, 10∶1, and 1∶1, respectively, for 24 hours in acidic media (yellow bars) or neutral media (red bars). The data represent the mean±standard deviation (SD) of duplicate cultures of one typical experiment that has been repeated twice. ** p<0.001 compared to bovine HDL, ANOVA test. L. major purified metacyclics were incubated with Alexa Fluor-488 TLF (black line) or not (blue line) for 30 min in acidic media (I) and neutral media (J), deposition of TLF was quantified by flow cytometry.
Figure 2.
TLF accumulates within the PV in L. major infected macrophages.
BALB/c bone-marrow derived macrophages were infected with L. major metacyclics for 2 hours before treatment with prelabelled-Alexa 594-TLF (10 µg/ml) for 2 hours. The grey panel is a transmission light micrograph of the imaged macrophage. The red panel depicts the uptake of labeled TLF by the macrophage. The blue panel depicts the nuclei of the macrophage (large blue area) and intracellular Leishmania parasites (small blue dots) stained with DAPI. Lysosomes were stained with anti-Lamp-1 antibodies (green). The merged panel shows a Lamp-1 delineated PV full of TLF (arrows). Samples were visualized with a Leica TCS SP2 AOBS confocal laser scanning microscope.
Figure 3.
TLF surrounds parasites in the PV but is not endocytosed by L. major.
Bone-marrow derived macrophages were infected with GFP-L. major parasites (green) for 2 hours before treatment with prelabelled-Alexa 594-TLF ((10 µg/ml) red) for 2 hours (A) and 24 hours (C). The grey panels are transmission light micrographs of the imaged macrophages. The lack of colocalisation of the two dyes (red and green) is revealed by the 2D cytoflurograms of 25 z-stacks (2 hours, B) and (24 hours, D). Samples were visualized and analyzed with a Leica TCS SP2 AOBS confocal laser scanning microscope.
Figure 4.
L. major and L. amazonensis are sensitive to lytic HDL within macrophages.
Macrophages from Swiss-Webster intra-peritoneal cavities were infected with L. major metacyclics (A). BALB/c bone-marrow macrophages were infected with L. amazonensis promastigotes (B). The multiplicity of infection is 3∶1; parasites were incubated with macrophages for 2 hours, before the addition of human or bovine HDL at 1.5 mg/ml or at the indicated concentrations. Infected macrophages were co-incubated for 2, 24, or 72 hours. The data represent the mean±standard deviation (SD) of duplicate cultures of one typical experiment that has been repeated twice. * p<0.05 compared to bovine HDL at equivalent time points, ANOVA test.
Figure 5.
L. amazonensis promastigotes but not amastigotes are sensitive to lytic HDL within macrophages.
BALB/c bone-marrow derived macrophages were infected with L. amazonensis promastigotes (A) or amastigote like forms (B) at a multiplicity of infection of 3∶1, for 2 hours before the addition of human or bovine HDL (1.5 mg/ml) for 24 hours. The data represent the mean±SD of duplicate cultures of one typical experiment that has been repeated twice. *** p<0.0001 compared to bovine HDL, ANOVA test.
Figure 6.
Lytic HDL does not generate NO nor requires the activation of macrophages to reduce the parasite burden.
BALB/c bone-marrow derived macrophages were infected with L. major metacyclics at a multiplicity of infection of 3∶1, for 2 hours before the addition of bovine or human HDL (1.5 mg/ml) or treated with IFNγ and LPS; NO production in the supernatant was measured after 24 hours (A). Bone marrow derived macrophages from iNOS−/− (B), gp91phox−/− (C), or C57BL/6 (D) mice were infected with L. major metacyclics at a multiplicity of infection of 3∶1, for 2 hours, before the addition of human or bovine HDL (1.5 mg/ml). They were co-incubated for 24 hours. The data represent the mean±standard deviation (SD) of duplicate cultures of one typical experiment that has been repeated twice. ** p<0.001, *** p<0.0001 compared to bovine HDL, ANOVA test.
Figure 7.
Protein levels of Hpr and apoL-I in HDG-TLF murine plasma compared to human plasma.
Western blot of serial dilutions of plasma from mice expressing Hpr and apoL-I from a single plasmid (apoL-I∶Hpr) 3 days post-injection, compared to normal human plasma (A). Western blot of serial dilutions of plasma from mice injected with plasmids coding for Hpr, apoL-I, both (apoL-I∶Hpr), or no protein (vector) 3 days post-injection (B). Hpr and apoL-I were detected with monoclonal antibodies.
Figure 8.
L. major is sensitive to transgenic lytic HDL (TLF) in vivo.
C57BL/6 mice (5 per group) were subjected to hydrodynamic gene delivery with a single plasmid that encodes for either apoL-I, Hpr, or both apoL-I and Hpr (apoL-I∶Hpr), or two plasmids one containing Hpr and one containing apoL-I (apoL-I+Hpr) or vector alone, before subcutaneous infection of the footpad with 1×106 (A,B) or 0.5×106 (C,D) L. major metacyclics. The size of the footpad was measured with a caliper (A,C,D). (A and B) represent the data from the same experiment, in which 19 days post-infection, mice were euthanized and footpad parasites were harvested for enumeration by serial dilution assay (B). The data shown represent the mean±SD of one typical experiment that has been repeated twice. * p<0.05, Mann Whitney test.
Figure 9.
Haptoglobin inhibits lytic HDL killing of L. major in macrophages.
BALB/c bone-marrow derived macrophages were infected with L. major promastigote metacyclics at a multiplicity of infection of 3∶1, for 2 hours before the addition of human or bovine HDL (1.5 mg/ml) in the presence or absence of Hp (1 mg/ml) for 24 hours. The data represent the mean±SD of duplicate cultures of one typical experiment that has been repeated twice. * p<0.05 compared to bovine HDL, ANOVA test.
Figure 10.
T. cruzi is resistant to transgenic lytic HDL (TLF) in vivo.
Swiss-Webster mice (3 per group) injected with a control plasmid, apoL-I or Hpr plasmid alone, or both apoL-I and Hpr were infected with 1×106 T. cruzi trypomasigotes IP three days after introduction of the genes by hydrodynamic gene delivery. The acute phase of the infection was followed by monitoring parasitemia daily. The data shown represent the mean±SD.
Figure 11.
Proposed model of action of TLF against Leishmania parasites.
The window of Leishmania susceptibility to lytic HDL is after phagocytosis of the metacyclic promastigotes during acidification of the PV and before transformation into amastigotes.