Limits...
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

IFNγ-stimulated MBECs cross-present PbA antigen in vitro.(A) After culturing mouse brain microvessels for 10 days including puromycin selection, the resulting cells are >97% CD45-CD31+ endothelial cells. Dot plots are representative of 3 independent culture experiments. (B) MBECs were fixed and stained for the endothelial marker von Willebrand factor (green) and counterstained with DAPI (blue). (C) MBECs in quadruplicate wells of a 48-well plate were stimulated (or not) with 10 ng/ml IFNγ for 24 h, then 5 × 106 thawed PbA mature iRBCs were added (or not) for 24 h. The wells were washed and co-cultured with LR-BSL8.4a reporter cells overnight prior to X-gal staining. ****P<0.0001 compared to every other group, ANOVA with Bonferroni’s post test on log-transformed blue spot counts. (D) Cross-presentation by IFNγ-stimulated MBECs was similarly assayed after pulsing with 106 freshly isolated or freeze-thawed PbA mature stages (n = 4). ****P< 0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed blue spot counts.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4457820&req=5

ppat.1004963.g003: IFNγ-stimulated MBECs cross-present PbA antigen in vitro.(A) After culturing mouse brain microvessels for 10 days including puromycin selection, the resulting cells are >97% CD45-CD31+ endothelial cells. Dot plots are representative of 3 independent culture experiments. (B) MBECs were fixed and stained for the endothelial marker von Willebrand factor (green) and counterstained with DAPI (blue). (C) MBECs in quadruplicate wells of a 48-well plate were stimulated (or not) with 10 ng/ml IFNγ for 24 h, then 5 × 106 thawed PbA mature iRBCs were added (or not) for 24 h. The wells were washed and co-cultured with LR-BSL8.4a reporter cells overnight prior to X-gal staining. ****P<0.0001 compared to every other group, ANOVA with Bonferroni’s post test on log-transformed blue spot counts. (D) Cross-presentation by IFNγ-stimulated MBECs was similarly assayed after pulsing with 106 freshly isolated or freeze-thawed PbA mature stages (n = 4). ****P< 0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed blue spot counts.

Mentions: To further understand how brain endothelial cells cross-present during ECM, we sought to recapitulate the process in vitro using primary cultures of murine brain endothelial cells (MBECs). Brain microvessels isolated from naïve mice were cultured on collagen-coated plates using puromycin to kill non-endothelial cells for the first 2–3 days [25]. In our hands, after 10 days of culture, >97% of the cells were CD31+ endothelial cells, with essentially no CD45+ cell contamination and <0.5% NG2+ pericyte contamination (Fig 3A). The cultures also expressed the endothelial marker von Willebrand factor (Fig 3B). We always used unpassaged primary MBEC cultures to retain the original phenotype as much as possible. Unstimulated MBECs and IFNγ-stimulated MBECs were incubated with frozen-thawed PbA mature iRBCs (late trophozoites and schizonts) for 24 h, after which they were washed and co-incubated with LR-BSL8.4a reporter cells overnight. After X-gal staining of the reporter cells, only MBECs that had been exposed to both IFNγ and PbA-infected iRBCs were found to have cross-presented (Fig 3C). Both the ability of endothelial cells to cross-present PbA antigen and the necessity of IFNγ stimulation agree with our ex vivo results from infected mice. Similar results were obtained with reporter cell lines that we created to detect cross-presentation of two additional PbA epitopes (S2A and S2B Fig). Frozen mature iRBCs were used as antigen in these and many subsequent experiments for convenience; we compared these to freshly isolated PbA mature iRBCs and found no significant difference in cross-presentation efficiency (Fig 3D). We also confirmed that uninfected RBCs gave readouts indistinguishable from the background (S3A Fig).


Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

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

IFNγ-stimulated MBECs cross-present PbA antigen in vitro.(A) After culturing mouse brain microvessels for 10 days including puromycin selection, the resulting cells are >97% CD45-CD31+ endothelial cells. Dot plots are representative of 3 independent culture experiments. (B) MBECs were fixed and stained for the endothelial marker von Willebrand factor (green) and counterstained with DAPI (blue). (C) MBECs in quadruplicate wells of a 48-well plate were stimulated (or not) with 10 ng/ml IFNγ for 24 h, then 5 × 106 thawed PbA mature iRBCs were added (or not) for 24 h. The wells were washed and co-cultured with LR-BSL8.4a reporter cells overnight prior to X-gal staining. ****P<0.0001 compared to every other group, ANOVA with Bonferroni’s post test on log-transformed blue spot counts. (D) Cross-presentation by IFNγ-stimulated MBECs was similarly assayed after pulsing with 106 freshly isolated or freeze-thawed PbA mature stages (n = 4). ****P< 0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed blue spot counts.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004963.g003: IFNγ-stimulated MBECs cross-present PbA antigen in vitro.(A) After culturing mouse brain microvessels for 10 days including puromycin selection, the resulting cells are >97% CD45-CD31+ endothelial cells. Dot plots are representative of 3 independent culture experiments. (B) MBECs were fixed and stained for the endothelial marker von Willebrand factor (green) and counterstained with DAPI (blue). (C) MBECs in quadruplicate wells of a 48-well plate were stimulated (or not) with 10 ng/ml IFNγ for 24 h, then 5 × 106 thawed PbA mature iRBCs were added (or not) for 24 h. The wells were washed and co-cultured with LR-BSL8.4a reporter cells overnight prior to X-gal staining. ****P<0.0001 compared to every other group, ANOVA with Bonferroni’s post test on log-transformed blue spot counts. (D) Cross-presentation by IFNγ-stimulated MBECs was similarly assayed after pulsing with 106 freshly isolated or freeze-thawed PbA mature stages (n = 4). ****P< 0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed blue spot counts.
Mentions: To further understand how brain endothelial cells cross-present during ECM, we sought to recapitulate the process in vitro using primary cultures of murine brain endothelial cells (MBECs). Brain microvessels isolated from naïve mice were cultured on collagen-coated plates using puromycin to kill non-endothelial cells for the first 2–3 days [25]. In our hands, after 10 days of culture, >97% of the cells were CD31+ endothelial cells, with essentially no CD45+ cell contamination and <0.5% NG2+ pericyte contamination (Fig 3A). The cultures also expressed the endothelial marker von Willebrand factor (Fig 3B). We always used unpassaged primary MBEC cultures to retain the original phenotype as much as possible. Unstimulated MBECs and IFNγ-stimulated MBECs were incubated with frozen-thawed PbA mature iRBCs (late trophozoites and schizonts) for 24 h, after which they were washed and co-incubated with LR-BSL8.4a reporter cells overnight. After X-gal staining of the reporter cells, only MBECs that had been exposed to both IFNγ and PbA-infected iRBCs were found to have cross-presented (Fig 3C). Both the ability of endothelial cells to cross-present PbA antigen and the necessity of IFNγ stimulation agree with our ex vivo results from infected mice. Similar results were obtained with reporter cell lines that we created to detect cross-presentation of two additional PbA epitopes (S2A and S2B Fig). Frozen mature iRBCs were used as antigen in these and many subsequent experiments for convenience; we compared these to freshly isolated PbA mature iRBCs and found no significant difference in cross-presentation efficiency (Fig 3D). We also confirmed that uninfected RBCs gave readouts indistinguishable from the background (S3A Fig).

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