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Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps.

Kober C, Rohn S, Weibel S, Geissinger U, Chen NG, Szalay AA - J Transl Med (2015)

Bottom Line: In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected.By acting as VACV traps they further reduce efficient virus infection of the tumor cells.These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter, University of Wuerzburg, Am Hubland, 97074, Würzburg, Germany. christina.kober@uni-wuerzburg.de.

ABSTRACT

Background: Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM.

Methods: VACV LIVP 1.1.1 replication in C57BL/6 and Foxn1(nu/nu) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system.

Results: We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of Iba1(+) microglia and GFAP(+) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected.

Conclusion: Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

No MeSH data available.


Related in: MedlinePlus

Impaired amount of virus progeny after stimulation of BV-2 cells with IFN-γ or IFN-γ and LPS. BV-2 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml), LPS + IFN-γ or IL-4 (10 ng/ml) for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and by detection of the percentage of MHCII+ cells by FACS analysis (c). The amount of virus progeny (pfu/ml) was analyzed by standard plaque assay in the presence of stimulating factors and in normal growth medium in cells and supernatants (d). Statistical analysis was performed related to infection medium. To analyze the recovery of viral replication, normal growth medium was applied after infection of pre-stimulated cells. Standard plaque assay was performed to determine viral progeny in cells and supernatant after 24, 48, 72 and 96 hpi (e). To analyze the relative survival of the cells in the presence of stimulating factors a MTT assay was performed (f). The cellular density of stimulated cells relative to unstimulated (w/o) cells was detected via optical density measurement after 24 and 48 h of cultivation (g). Two-sided t test with unequal variances was used for statistics *p < 0.05, **p < 0.01, ***p < 0.001. Active replication was defined as virus titer above the virus titer of the infection medium (red line). All experiments were performed in triplicate and repeated in an independent experiment.
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Fig5: Impaired amount of virus progeny after stimulation of BV-2 cells with IFN-γ or IFN-γ and LPS. BV-2 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml), LPS + IFN-γ or IL-4 (10 ng/ml) for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and by detection of the percentage of MHCII+ cells by FACS analysis (c). The amount of virus progeny (pfu/ml) was analyzed by standard plaque assay in the presence of stimulating factors and in normal growth medium in cells and supernatants (d). Statistical analysis was performed related to infection medium. To analyze the recovery of viral replication, normal growth medium was applied after infection of pre-stimulated cells. Standard plaque assay was performed to determine viral progeny in cells and supernatant after 24, 48, 72 and 96 hpi (e). To analyze the relative survival of the cells in the presence of stimulating factors a MTT assay was performed (f). The cellular density of stimulated cells relative to unstimulated (w/o) cells was detected via optical density measurement after 24 and 48 h of cultivation (g). Two-sided t test with unequal variances was used for statistics *p < 0.05, **p < 0.01, ***p < 0.001. Active replication was defined as virus titer above the virus titer of the infection medium (red line). All experiments were performed in triplicate and repeated in an independent experiment.

Mentions: Like peripheral macrophages also microglia take on either a classically activated M1 phenotype or an alternatively activated M2 phenotype [24]. We therefore set out to analyze both M1 and M2 phenotypes of BV-2 microglia for their ability to take up and support LIVP 1.1.1 virus replication. The two different phenotypes M1 and M2 are dependent on exogenous addition of certain stimuli. Such stimuli are IL-4 for induction of the M2 phenotype, and IFN-γ or LPS or a combination of both for induction of the M1 phenotype [49]. LPS is known to induce nitric oxide (NO) production in BV-2 cells, a marker for the M1 phenotype [40, 50]. As shown in Figure 5a a low NO production indicated by the measurement of nitrite via Griess assay, was induced by treating BV-2 cells with IFN-γ or LPS alone. However, when cells were treated with IFN-γ and LPS in combination, NO production was at the highest level, indirectly measured by the amount of nitrite (12.31 ± 1.61 µM). Western Blot analysis of total protein amounts extracted from treated BV-2 microglial cells indicated that the increase in NO production by IFN-γ and LPS resulted in an up-regulation of the inducible isoform of nitric oxide synthase (iNOS) (Figure 5b). In addition, treatment of BV-2 cells with IL-4 or LPS both increased the expression of arginase 1, a marker for the M2 phenotype. Treatment of BV-2 cells with IFN-γ led to a reduction of arginase 1 expression. Furthermore, treatment of BV-2 cells with IFN-γ for 24 h resulted in high-level expression of MHCII molecules, an indicator for the M1 phenotype, whereas MHCII expression was not detectable in unstimulated or IL-4 stimulated cells detected by FACS analysis (Figure 5c). Cells pre-incubated with LPS, IFN-γ, or LPS and IFN-γ and infected with LIVP 1.1.1 revealed a reduced amount of viral progeny 24 and 72 hpi (Figure 5d). In contrast, there was no difference between IL-4 or unstimulated cells. Interestingly, viral replication could be restored after medium change. We detected a continuous increase of the virus titer in cells pre-incubated with either LPS or LPS and IFN-γ when cells were cultured in normal growth medium after virus infection. The virus titer was higher than the infection medium at 72 and 96 hpi, respectively (Figure 5e). MTT assay revealed that around 50% of cells stimulated with IFN-γ or LPS and IFN-γ were alive 96 hpi, ~20% of surviving cells were detected in the LPS treated samples and less than 10% of surviving cells were detected in the IL-4 and unstimulated samples (Figure 5f). Furthermore, BV-2 cells which were stimulated with IFN-γ and/or LPS proliferated to a lesser extent than unstimulated or IL-4 stimulated cells as indicated by the cellular density at 24 and 48 h which was calculated relative to unstimulated cells. (Figure 5g). Based on virus replication and virus-mediated cell death, these results strongly indicate preferred virus infection of cells of the M2 phenotype or unstimulated form of BV-2 cells in comparison to the M1 phenotype.Figure 5


Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps.

Kober C, Rohn S, Weibel S, Geissinger U, Chen NG, Szalay AA - J Transl Med (2015)

Impaired amount of virus progeny after stimulation of BV-2 cells with IFN-γ or IFN-γ and LPS. BV-2 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml), LPS + IFN-γ or IL-4 (10 ng/ml) for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and by detection of the percentage of MHCII+ cells by FACS analysis (c). The amount of virus progeny (pfu/ml) was analyzed by standard plaque assay in the presence of stimulating factors and in normal growth medium in cells and supernatants (d). Statistical analysis was performed related to infection medium. To analyze the recovery of viral replication, normal growth medium was applied after infection of pre-stimulated cells. Standard plaque assay was performed to determine viral progeny in cells and supernatant after 24, 48, 72 and 96 hpi (e). To analyze the relative survival of the cells in the presence of stimulating factors a MTT assay was performed (f). The cellular density of stimulated cells relative to unstimulated (w/o) cells was detected via optical density measurement after 24 and 48 h of cultivation (g). Two-sided t test with unequal variances was used for statistics *p < 0.05, **p < 0.01, ***p < 0.001. Active replication was defined as virus titer above the virus titer of the infection medium (red line). All experiments were performed in triplicate and repeated in an independent experiment.
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Related In: Results  -  Collection

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Fig5: Impaired amount of virus progeny after stimulation of BV-2 cells with IFN-γ or IFN-γ and LPS. BV-2 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml), LPS + IFN-γ or IL-4 (10 ng/ml) for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and by detection of the percentage of MHCII+ cells by FACS analysis (c). The amount of virus progeny (pfu/ml) was analyzed by standard plaque assay in the presence of stimulating factors and in normal growth medium in cells and supernatants (d). Statistical analysis was performed related to infection medium. To analyze the recovery of viral replication, normal growth medium was applied after infection of pre-stimulated cells. Standard plaque assay was performed to determine viral progeny in cells and supernatant after 24, 48, 72 and 96 hpi (e). To analyze the relative survival of the cells in the presence of stimulating factors a MTT assay was performed (f). The cellular density of stimulated cells relative to unstimulated (w/o) cells was detected via optical density measurement after 24 and 48 h of cultivation (g). Two-sided t test with unequal variances was used for statistics *p < 0.05, **p < 0.01, ***p < 0.001. Active replication was defined as virus titer above the virus titer of the infection medium (red line). All experiments were performed in triplicate and repeated in an independent experiment.
Mentions: Like peripheral macrophages also microglia take on either a classically activated M1 phenotype or an alternatively activated M2 phenotype [24]. We therefore set out to analyze both M1 and M2 phenotypes of BV-2 microglia for their ability to take up and support LIVP 1.1.1 virus replication. The two different phenotypes M1 and M2 are dependent on exogenous addition of certain stimuli. Such stimuli are IL-4 for induction of the M2 phenotype, and IFN-γ or LPS or a combination of both for induction of the M1 phenotype [49]. LPS is known to induce nitric oxide (NO) production in BV-2 cells, a marker for the M1 phenotype [40, 50]. As shown in Figure 5a a low NO production indicated by the measurement of nitrite via Griess assay, was induced by treating BV-2 cells with IFN-γ or LPS alone. However, when cells were treated with IFN-γ and LPS in combination, NO production was at the highest level, indirectly measured by the amount of nitrite (12.31 ± 1.61 µM). Western Blot analysis of total protein amounts extracted from treated BV-2 microglial cells indicated that the increase in NO production by IFN-γ and LPS resulted in an up-regulation of the inducible isoform of nitric oxide synthase (iNOS) (Figure 5b). In addition, treatment of BV-2 cells with IL-4 or LPS both increased the expression of arginase 1, a marker for the M2 phenotype. Treatment of BV-2 cells with IFN-γ led to a reduction of arginase 1 expression. Furthermore, treatment of BV-2 cells with IFN-γ for 24 h resulted in high-level expression of MHCII molecules, an indicator for the M1 phenotype, whereas MHCII expression was not detectable in unstimulated or IL-4 stimulated cells detected by FACS analysis (Figure 5c). Cells pre-incubated with LPS, IFN-γ, or LPS and IFN-γ and infected with LIVP 1.1.1 revealed a reduced amount of viral progeny 24 and 72 hpi (Figure 5d). In contrast, there was no difference between IL-4 or unstimulated cells. Interestingly, viral replication could be restored after medium change. We detected a continuous increase of the virus titer in cells pre-incubated with either LPS or LPS and IFN-γ when cells were cultured in normal growth medium after virus infection. The virus titer was higher than the infection medium at 72 and 96 hpi, respectively (Figure 5e). MTT assay revealed that around 50% of cells stimulated with IFN-γ or LPS and IFN-γ were alive 96 hpi, ~20% of surviving cells were detected in the LPS treated samples and less than 10% of surviving cells were detected in the IL-4 and unstimulated samples (Figure 5f). Furthermore, BV-2 cells which were stimulated with IFN-γ and/or LPS proliferated to a lesser extent than unstimulated or IL-4 stimulated cells as indicated by the cellular density at 24 and 48 h which was calculated relative to unstimulated cells. (Figure 5g). Based on virus replication and virus-mediated cell death, these results strongly indicate preferred virus infection of cells of the M2 phenotype or unstimulated form of BV-2 cells in comparison to the M1 phenotype.Figure 5

Bottom Line: In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected.By acting as VACV traps they further reduce efficient virus infection of the tumor cells.These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter, University of Wuerzburg, Am Hubland, 97074, Würzburg, Germany. christina.kober@uni-wuerzburg.de.

ABSTRACT

Background: Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM.

Methods: VACV LIVP 1.1.1 replication in C57BL/6 and Foxn1(nu/nu) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system.

Results: We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of Iba1(+) microglia and GFAP(+) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected.

Conclusion: Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

No MeSH data available.


Related in: MedlinePlus