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Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.

Couper KN, Barnes T, Hafalla JC, Combes V, Ryffel B, Secher T, Grau GE, Riley EM, de Souza JB - PLoS Pathog. (2010)

Bottom Line: In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells.Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells.MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling.

View Article: PubMed Central - PubMed

Affiliation: Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.

ABSTRACT
There is considerable debate as to the nature of the primary parasite-derived moieties that activate innate pro-inflammatory responses during malaria infection. Microparticles (MPs), which are produced by numerous cell types following vesiculation of the cellular membrane as a consequence of cell death or immune-activation, exert strong pro-inflammatory activity in other disease states. Here we demonstrate that MPs, derived from the plasma of malaria infected mice, but not naive mice, induce potent activation of macrophages in vitro as measured by CD40 up-regulation and TNF production. In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells. Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells. MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling. Similar levels of immunogenic MPs were produced in WT and in TNF(-/-), IFN-gamma(-/-), IL-12(-/-) and RAG-1(-/-) malaria-infected mice, but were not produced in mice injected with LPS, showing that inflammation is not required for the production of MPs during malaria infection. This study therefore establishes parasitized red blood cell-derived MPs as a major inducer of systemic inflammation during malaria infection, raising important questions about their role in severe disease and in the generation of adaptive immune responses.

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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.
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ppat-1000744-g004: 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.

Mentions: The phenotypic characterisation of PbA infection-induced MPs and uninfected MPs demonstrated clear differences in the expression level of AnnexinV, TER119 and CD45, suggesting that the predominant cellular sources of PbA infection induced MPs and uninfected MPs varied, potentially explaining the inflammatory nature of the malaria infection induced MPs. As the frequency and numbers of RBC-derived particles was increased in the PbA infection induced MP preparation, we hypothesised that infection-induced MPs may either be formed by vesiculation of the pRBC membrane during intra-erythrocytic parasite maturation and/or pRBC rupture at schizogeny, in which case MPs would be expected to contain malaria parasite-derived components, or that they may be derived from uninfected RBCs, which are known to undergo bystander lysis during acute malaria infection, contributing to the rapid onset of anaemia [3]. To distinguish between these two possibilities, we analysed PbA infection-induced MPs for the presence of PbA-specific antigens by immunofluorescence using purified anti-PbA IgG. As expected, no parasite-derived material was detected in uninfected MP preparations (Fig. 4A). In contrast a large proportion of MPs from PbA-infected mice bound the anti-PbA IgG (Fig. 4A), indicating the presence of significant quantities of parasite-derived material in the infection-derived MPs. Although a number of parasite moieties are likely to be incorporated within the malaria infection induced MP preparation, we failed to detect hemozoin in the plasma derived preparation by beta-hematin formation assay (results not shown).


Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.

Couper KN, Barnes T, Hafalla JC, Combes V, Ryffel B, Secher T, Grau GE, Riley EM, de Souza JB - PLoS Pathog. (2010)

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.
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Related In: Results  -  Collection

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ppat-1000744-g004: 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.
Mentions: The phenotypic characterisation of PbA infection-induced MPs and uninfected MPs demonstrated clear differences in the expression level of AnnexinV, TER119 and CD45, suggesting that the predominant cellular sources of PbA infection induced MPs and uninfected MPs varied, potentially explaining the inflammatory nature of the malaria infection induced MPs. As the frequency and numbers of RBC-derived particles was increased in the PbA infection induced MP preparation, we hypothesised that infection-induced MPs may either be formed by vesiculation of the pRBC membrane during intra-erythrocytic parasite maturation and/or pRBC rupture at schizogeny, in which case MPs would be expected to contain malaria parasite-derived components, or that they may be derived from uninfected RBCs, which are known to undergo bystander lysis during acute malaria infection, contributing to the rapid onset of anaemia [3]. To distinguish between these two possibilities, we analysed PbA infection-induced MPs for the presence of PbA-specific antigens by immunofluorescence using purified anti-PbA IgG. As expected, no parasite-derived material was detected in uninfected MP preparations (Fig. 4A). In contrast a large proportion of MPs from PbA-infected mice bound the anti-PbA IgG (Fig. 4A), indicating the presence of significant quantities of parasite-derived material in the infection-derived MPs. Although a number of parasite moieties are likely to be incorporated within the malaria infection induced MP preparation, we failed to detect hemozoin in the plasma derived preparation by beta-hematin formation assay (results not shown).

Bottom Line: In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells.Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells.MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling.

View Article: PubMed Central - PubMed

Affiliation: Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.

ABSTRACT
There is considerable debate as to the nature of the primary parasite-derived moieties that activate innate pro-inflammatory responses during malaria infection. Microparticles (MPs), which are produced by numerous cell types following vesiculation of the cellular membrane as a consequence of cell death or immune-activation, exert strong pro-inflammatory activity in other disease states. Here we demonstrate that MPs, derived from the plasma of malaria infected mice, but not naive mice, induce potent activation of macrophages in vitro as measured by CD40 up-regulation and TNF production. In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells. Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells. MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling. Similar levels of immunogenic MPs were produced in WT and in TNF(-/-), IFN-gamma(-/-), IL-12(-/-) and RAG-1(-/-) malaria-infected mice, but were not produced in mice injected with LPS, showing that inflammation is not required for the production of MPs during malaria infection. This study therefore establishes parasitized red blood cell-derived MPs as a major inducer of systemic inflammation during malaria infection, raising important questions about their role in severe disease and in the generation of adaptive immune responses.

Show MeSH
Related in: MedlinePlus