Figure 1.
Plasma microparticles derived from malaria infected mice stimulate strong macrophage pro-inflammatory responses in vitro.
Microparticles were prepared from the plasma of uninfected mice (uninfected MP) and from mice infected with P. berghei ANKA (day 7: PbA MP) and were used to stimulate bone-marrow derived macrophages in vitro for 24hrs. (A) The size of plasma derived microparticles relative to 1µm beads is shown. (B) Scanning electron micrographs showing morphology of MPs. Magnification 72,000X (insert 240,000X). (C) Representative histograms showing the level of CD40 expression by bone marrow derived macrophages following stimulation with uninfected MPs, PbA MPs and LPS. (D) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. * p<0.05 between PbA MP and no-stim; ∼ p<0.05 between LPS and no stim (E) The level of TNF production by stimulated macrophages was measured in the supernatant by ELISA. * p<0.05 between PbA MP and no-stim; ∼ p<0.05 between LPS and no stim (F) The number of MPs within the uninfected and PbA derived preparations was calculated relative to a standardised number of 1µm beads. * p<0.05 between PbA MP and uninfected MP. (G) Representative histograms showing the expression level of CD40 on macrophages following stimulation with varying doses of (left plot) uninfected MPs and (right plot) PbA-derived MPs. (H) The mean fluorescence intensity of CD40 expression on macrophages stimulated with varying doses of uninfected and PbA-derived MPs. (I) TNF production by stimulated with varying doses of uninfected and PbA-derived MPs. The results are representative of 4 separate experiments.
Figure 2.
Malaria infection derived plasma microparticles promote significantly stronger macrophage activation than intact parasitized red blood cells.
Microparticles were prepared from the plasma of uninfected mice (uninfected MP) and from mice infected with P. berghei ANKA (day 7: PbA MP). Mature trophozoite and schizont stage parasitized red blood cells (pRBC) were purified in vitro from blood of PbA infected mice. (A) Representative histograms showing the level of CD40 expression by bone marrow derived macrophages following stimulation with uninfected RBCs, pRBCs and uninfected MPs and PbA MPs. (B) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. (C) The level of TNF production by stimulated macrophages was measured in the supernatant by ELISA. The results are representative of 2 separate experiments. * denotes significant difference between PbA MP and no stim.cultures.
Figure 3.
Phenotypic characterization of uninfected and P. berghei ANKA plasma derived microparticles.
Microparticles were prepared from the plasma of uninfected mice (uninfected MP) and from mice infected with P. berghei ANKA (day 7: PbA MP). The cellular sources and composition of the MPs were determined by flow cytometry. (A) Representative dot plots showing the frequency and (B) normalized numbers of Annexin V+ events within the uninfected MP and PbA MP preparation. (C) The cellular source and activation status of MPs were examined by determining the expression of TER119, CD144, CD45, F4-80, CD107a, CD41 and VCAM-1. (D) The percentage of Annexin V positive events co-expressing secondary markers. (E) The frequency and (F) numbers of flow cytometric events in uninfected and PbA MPs expressing TER119, CD144, CD45, F4-80, CD107a, CD41 and VCAM-1. The results are representative of 2 separate experiments. * p<0.05 between PbA MP and uninfected MP; ∼ P<0.05 between uninfected MP and PBS.
Figure 4.
Immunogenic malaria infection-derived MPs are produced from pRBC and contain parasite materials.
Microparticles were prepared from the plasma of uninfected mice (uninfected MP) and from mice infected with P. berghei ANKA (day 7: PbA MP). (A) The presence of parasite material in MP preparations was examined by IFAT using purified anti-P. berghei ANKA IgG antibodies followed by detection with FITC-labelled anti-mouse secondary antibodies. MPs were prepared from purified pRBC (pRBC MP) and the ability of pRBC MP to activate macrophages in vitro relative to PbA plasma derived MPs was assessed. (B) The expression of AnnexinV and TER119 on the different MP preparations. (C) Representative histograms showing the level of CD40 expression by bone marrow derived macrophages following stimulation with uninfected MPs, PbA MPs, uninfected RBC MPs (uRBC MPs) and pRBC MPs. (D) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. (E) The level of TNF production by stimulated macrophages was measured in the supernatant by ELISA. The results are representative of 2 separate experiments. * p<0.05 between PbA MP and no stim.cultures; ∼ p<0.05 between pRBC MP and no stim.cultures.
Figure 5.
LPS-inflammation induced MPs do not activate macrophages.
Microparticles were prepared from the plasma of uninfected mice (uninfected MP), from mice injected with 20µg LPS (Day 3: LPS MP) and from mice infected with P. berghei ANKA (day 7: PbA MP). (A) Representative dot plots showing the expression of Annexin V in the MP preparations. (B) The frequency and (C) normalized numbers of Annexin V+ events within the MP preparations. (D) The frequency of TER119+ and CD41+ events within the Annexin V+ populations. (E) Representative histograms showing the level of CD40 expression by bone marrow derived macrophages following stimulation with MPs: shaded histogram no stimulation; dark line LPS MP; light line PbA MP. (F) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. (G) The level of TNF production by stimulated macrophages was measured in the supernatant by ELISA. The results are representative of 2 separate experiments. * p<0.05 between PbA MP and no stim.; ∼ p<0.05 between LPS MP and no stim.; + p<0.05 between PbA MP and LPS MP.
Figure 6.
The generation of TER119+ immunogenic MPs depends on the stage of malaria infection.
Macrophages were stimulated for 24hrs with microparticles prepared from the plasma of mice infected with P. berghei ANKA. (A) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. (B) The level of TNF production by stimulated macrophages was measured in the supernatant by ELISA. (C) The number of MPs within the PbA derived preparations was calculated relative to a standardized number of 1µm beads. (D) The expression of TER119 on the different MP preparations. (E) The percentage parasitaemia on days 3, 5 and 7 post-infection. The results are representative of 2 separate experiments. * p<0.05 between day 5 PbA MP and day 7 PbA MP; + p<0.05 between day 3 PbA MP and day 5 PbA MP; ∼ p<0.05 between day 3 PbA MP and day 7 PbA MP; # p<0.05 between day 7 PbA MP and all groups.
Figure 7.
Microparticles generated during malaria infection independently from inflammation promote macrophage activation.
Microparticles were prepared from the plasma of uninfected mice (uninfected MP) and from WT, TNF−/−, IL-12p40−/−, IFN-γ−/− and RAG-1−/− mice infected with P. berghei ANKA (day 7: PbA MP). (A) The presence of parasite material in the separate MP preparations was examined by IFAT. (B) Representative histograms showing the level of CD40 expression by bone marrow derived macrophages following stimulation with MPs. (C) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. (D) The level of TNF production by stimulated macrophages was measured in the supernatant by ELISA. The results are representative of 2 separate experiments. * denotes significant difference between no stim and all infection derived MP preparations.
Figure 8.
Microparticle driven macrophage activation is MyD88 and TLR-4 dependent.
Microparticles were prepared from the plasma of mice infected with P. berghei ANKA (day 7: PbA MP). Bone marrow derived macrophages were generated from WT (B6), MyD88−/−, TLR-2−/−, TLR-4−/−, TLR-2/4−/− and TLR-9−/− mice. The ability of PbA MPs to activate (A–C) MyD88−/− macrophages and (D–F) TLR-2−/−, TLR-4−/−, TLR 2/4−/− and TLR-9−/− macrophages was examined. (A, D) Representative histograms showing the level of CD40 expression by macrophages following stimulation with PbA MPs. (B) Mean fluorescence intensity of CD40 expression by macrophages following stimulation. (E) normalized CD40 expression by macrophages relative to non-stimulated controls. (C, F) The level of TNF production by stimulated macrophages was measured by ELISA. The results are representative of 2 separate experiments. (B, C) * p<0.05 between WT non-stimulated and PbA MP stimulated; ∼ p<0.05 between WT PbA MP stimulated and MyD88−/− PbA MP stimulated. (E, F) * p<0.05 between WT PbA MP stimulated and TLR-4−/− PbA MP stimulated; ∼ p<0.05 between WT PbA MP stimulated and TLR-2/4−/− PbA MP stimulated; + p<0.05 between WT PbA MP stimulated and TLR-2−/− PbA MP stimulated; # p<0.05 between WT PbA MP stimulated and TLR-9−/− PbA MP stimulated.