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Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

Howland SW, Poh CM, Rénia L - PLoS Pathog. (2015)

Bottom Line: The main source of antigen appears to be free merozoites, which were avidly phagocytosed.A human brain endothelial cell line also phagocytosed P. falciparum merozoites.Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore.

ABSTRACT
In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

No MeSH data available.


Related in: MedlinePlus

Endothelial cells and leukocytes are the cross-presenting cells in the brain during PbA infection.(A–B) Brains from naïve (n = 5) and PbA-infected mice exhibiting signs of ECM (n = 11) were subjected to automated dissociation using papain. After myelin removal, the single cell suspensions were stained and sorted into four populations: CD45+ microglia and leukocytes, CD45-CD31+ endothelial cells, CD45-CD140b+ cells including pericytes, and cells negative for all three markers. Each sorted population was tested for cross-presentation by incubating with LR-BSL8.4a cells and staining with X-gal. Data were pooled from 3 independent experiments. (A) Raw spot counts. **P< 0.01, separate Mann-Whitney U tests comparing naïve vs infected for each population. (B) Only data from infected mice are shown, after background subtraction (the mean of all naïve wells) and normalization by the number of sorted cells added. Negative data are plotted as zeroes. (C) Brains from naïve (n = 5) and PbA-infected mice (n = 10) were manually dissociated and digested with collagenase. After myelin removal, the single cell suspensions were stained and sorted for microglia (CD45intCD11b+) and peripheral leukocytes together with PVM (CD45hi), which were assayed for cross-presentation as above. Data were pooled from 2 independent experiments. **P< 0.01, Mann-Whitney U test. See S1 Fig for representative FACS plots.
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ppat.1004963.g002: Endothelial cells and leukocytes are the cross-presenting cells in the brain during PbA infection.(A–B) Brains from naïve (n = 5) and PbA-infected mice exhibiting signs of ECM (n = 11) were subjected to automated dissociation using papain. After myelin removal, the single cell suspensions were stained and sorted into four populations: CD45+ microglia and leukocytes, CD45-CD31+ endothelial cells, CD45-CD140b+ cells including pericytes, and cells negative for all three markers. Each sorted population was tested for cross-presentation by incubating with LR-BSL8.4a cells and staining with X-gal. Data were pooled from 3 independent experiments. (A) Raw spot counts. **P< 0.01, separate Mann-Whitney U tests comparing naïve vs infected for each population. (B) Only data from infected mice are shown, after background subtraction (the mean of all naïve wells) and normalization by the number of sorted cells added. Negative data are plotted as zeroes. (C) Brains from naïve (n = 5) and PbA-infected mice (n = 10) were manually dissociated and digested with collagenase. After myelin removal, the single cell suspensions were stained and sorted for microglia (CD45intCD11b+) and peripheral leukocytes together with PVM (CD45hi), which were assayed for cross-presentation as above. Data were pooled from 2 independent experiments. **P< 0.01, Mann-Whitney U test. See S1 Fig for representative FACS plots.

Mentions: Ultimately, we succeeded using a papain-based Neural Tissue Dissociation Kit (Miltenyi) with automated mechanical dissociation of the entire brain. Single cell suspensions from naïve and PbA-infected mouse brains were separated into four populations after antibody labeling. First, all CD45+ cells (microglia and leukocytes) were gated, then the CD45- cells were further subdivided into CD31+ endothelial cells, a CD140b+ population including pericytes, and triple negative cells such as astrocytes and neurons (S1A Fig). After overnight incubation with LR-BSL8.4a reporter cells and X-gal staining, only the CD45+ and CD31+ populations gave rise to elevated numbers of blue spots comparing infected mice to naïve mice (Fig 2A). The numbers of blue spots induced by sorted endothelial cells were much smaller than those typically arising from brain microvessel fragments (tens compared to hundreds). One reason could be the low yield of endothelial cells with the Neural Tissue Dissociation Kit, averaging 1.7 × 103 cells sorted per brain compared to 1.6 × 104 cells from Liberase digestion of isolated brain microvessels. Further, while papain was selected because it largely preserves CD31 staining, its ability to cleave the heavy chain of MHC class I molecules is well-known. Despite reducing the papain concentration, it is possible that we were only able to detect “new” Pb1-H-2Db complexes that were generated in vitro from intracellular antigen stores. Because endothelial cells were isolated with much lower efficiency than leukocytes, we have also plotted the numbers of blue spots normalized by the input cell number, making it evident that endothelial cells were by far the most efficient cross-presenters on a per-cell basis (Fig 2B).


Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

Howland SW, Poh CM, Rénia L - PLoS Pathog. (2015)

Endothelial cells and leukocytes are the cross-presenting cells in the brain during PbA infection.(A–B) Brains from naïve (n = 5) and PbA-infected mice exhibiting signs of ECM (n = 11) were subjected to automated dissociation using papain. After myelin removal, the single cell suspensions were stained and sorted into four populations: CD45+ microglia and leukocytes, CD45-CD31+ endothelial cells, CD45-CD140b+ cells including pericytes, and cells negative for all three markers. Each sorted population was tested for cross-presentation by incubating with LR-BSL8.4a cells and staining with X-gal. Data were pooled from 3 independent experiments. (A) Raw spot counts. **P< 0.01, separate Mann-Whitney U tests comparing naïve vs infected for each population. (B) Only data from infected mice are shown, after background subtraction (the mean of all naïve wells) and normalization by the number of sorted cells added. Negative data are plotted as zeroes. (C) Brains from naïve (n = 5) and PbA-infected mice (n = 10) were manually dissociated and digested with collagenase. After myelin removal, the single cell suspensions were stained and sorted for microglia (CD45intCD11b+) and peripheral leukocytes together with PVM (CD45hi), which were assayed for cross-presentation as above. Data were pooled from 2 independent experiments. **P< 0.01, Mann-Whitney U test. See S1 Fig for representative FACS plots.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4457820&req=5

ppat.1004963.g002: Endothelial cells and leukocytes are the cross-presenting cells in the brain during PbA infection.(A–B) Brains from naïve (n = 5) and PbA-infected mice exhibiting signs of ECM (n = 11) were subjected to automated dissociation using papain. After myelin removal, the single cell suspensions were stained and sorted into four populations: CD45+ microglia and leukocytes, CD45-CD31+ endothelial cells, CD45-CD140b+ cells including pericytes, and cells negative for all three markers. Each sorted population was tested for cross-presentation by incubating with LR-BSL8.4a cells and staining with X-gal. Data were pooled from 3 independent experiments. (A) Raw spot counts. **P< 0.01, separate Mann-Whitney U tests comparing naïve vs infected for each population. (B) Only data from infected mice are shown, after background subtraction (the mean of all naïve wells) and normalization by the number of sorted cells added. Negative data are plotted as zeroes. (C) Brains from naïve (n = 5) and PbA-infected mice (n = 10) were manually dissociated and digested with collagenase. After myelin removal, the single cell suspensions were stained and sorted for microglia (CD45intCD11b+) and peripheral leukocytes together with PVM (CD45hi), which were assayed for cross-presentation as above. Data were pooled from 2 independent experiments. **P< 0.01, Mann-Whitney U test. See S1 Fig for representative FACS plots.
Mentions: Ultimately, we succeeded using a papain-based Neural Tissue Dissociation Kit (Miltenyi) with automated mechanical dissociation of the entire brain. Single cell suspensions from naïve and PbA-infected mouse brains were separated into four populations after antibody labeling. First, all CD45+ cells (microglia and leukocytes) were gated, then the CD45- cells were further subdivided into CD31+ endothelial cells, a CD140b+ population including pericytes, and triple negative cells such as astrocytes and neurons (S1A Fig). After overnight incubation with LR-BSL8.4a reporter cells and X-gal staining, only the CD45+ and CD31+ populations gave rise to elevated numbers of blue spots comparing infected mice to naïve mice (Fig 2A). The numbers of blue spots induced by sorted endothelial cells were much smaller than those typically arising from brain microvessel fragments (tens compared to hundreds). One reason could be the low yield of endothelial cells with the Neural Tissue Dissociation Kit, averaging 1.7 × 103 cells sorted per brain compared to 1.6 × 104 cells from Liberase digestion of isolated brain microvessels. Further, while papain was selected because it largely preserves CD31 staining, its ability to cleave the heavy chain of MHC class I molecules is well-known. Despite reducing the papain concentration, it is possible that we were only able to detect “new” Pb1-H-2Db complexes that were generated in vitro from intracellular antigen stores. Because endothelial cells were isolated with much lower efficiency than leukocytes, we have also plotted the numbers of blue spots normalized by the input cell number, making it evident that endothelial cells were by far the most efficient cross-presenters on a per-cell basis (Fig 2B).

Bottom Line: The main source of antigen appears to be free merozoites, which were avidly phagocytosed.A human brain endothelial cell line also phagocytosed P. falciparum merozoites.Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore.

ABSTRACT
In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

No MeSH data available.


Related in: MedlinePlus