Limits...
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

Stimulation of IMA 2.1 cells had no effect on relative survival after viral infection. IMA 2.1 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml) LPS + IFN-γ, IL-4 (10 ng/ml), FGF (5 ng/ml) or FGF + LPS for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and FACS analysis by detection of MHCII+ cells (c). d MTT assay was performed to detect the relative percentage of VACV-mediated cell death in the presence of stimulating factors. e Viral replication was analyzed by standard plaque assay in the presence of stimulating factors. All experiments were performed in triplicate.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4492094&req=5

Fig6: Stimulation of IMA 2.1 cells had no effect on relative survival after viral infection. IMA 2.1 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml) LPS + IFN-γ, IL-4 (10 ng/ml), FGF (5 ng/ml) or FGF + LPS for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and FACS analysis by detection of MHCII+ cells (c). d MTT assay was performed to detect the relative percentage of VACV-mediated cell death in the presence of stimulating factors. e Viral replication was analyzed by standard plaque assay in the presence of stimulating factors. All experiments were performed in triplicate.

Mentions: Polarization towards the M1 or M2 phenotypes is also known to occur in activated astrocytes [29]. The cell line IMA2.1 is known to respond similarly to stimuli like primary astrocytes [37]. We tested the ability of different pro-inflammatory cytokines (IL-4 and IFN-γ) and LPS to induce NO production in IMA2.1 cells. We also tested the effect of bFGF on IMA2.1 cells since bFGF like IL-4 is known to enhance the proliferation of astrocytes [51]. We have found that there was no increase in NO levels after treatment with different stimulating factors shown as measurement of nitrite in Figure 6a. In addition, iNOS expression levels were not detectable via Western blot. Only arginase 1 was detectable after IL-4 treatment (Figure 6b). Surprisingly, FACS analyses revealed increased MHCII expression in IMA2.1 cell cultures after stimulation with IFN-γ for 24 h as marker for the M1 phenotype (Figure 6c).Figure 6


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)

Stimulation of IMA 2.1 cells had no effect on relative survival after viral infection. IMA 2.1 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml) LPS + IFN-γ, IL-4 (10 ng/ml), FGF (5 ng/ml) or FGF + LPS for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and FACS analysis by detection of MHCII+ cells (c). d MTT assay was performed to detect the relative percentage of VACV-mediated cell death in the presence of stimulating factors. e Viral replication was analyzed by standard plaque assay in the presence of stimulating factors. All experiments were performed in triplicate.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492094&req=5

Fig6: Stimulation of IMA 2.1 cells had no effect on relative survival after viral infection. IMA 2.1 cells were stimulated with LPS (10 µg/ml), IFN-γ, (10 ng/ml) LPS + IFN-γ, IL-4 (10 ng/ml), FGF (5 ng/ml) or FGF + LPS for 24 h in medium with 2% FBS. Polarization of cells was analyzed by Griess assay (a), Western blot (b) and FACS analysis by detection of MHCII+ cells (c). d MTT assay was performed to detect the relative percentage of VACV-mediated cell death in the presence of stimulating factors. e Viral replication was analyzed by standard plaque assay in the presence of stimulating factors. All experiments were performed in triplicate.
Mentions: Polarization towards the M1 or M2 phenotypes is also known to occur in activated astrocytes [29]. The cell line IMA2.1 is known to respond similarly to stimuli like primary astrocytes [37]. We tested the ability of different pro-inflammatory cytokines (IL-4 and IFN-γ) and LPS to induce NO production in IMA2.1 cells. We also tested the effect of bFGF on IMA2.1 cells since bFGF like IL-4 is known to enhance the proliferation of astrocytes [51]. We have found that there was no increase in NO levels after treatment with different stimulating factors shown as measurement of nitrite in Figure 6a. In addition, iNOS expression levels were not detectable via Western blot. Only arginase 1 was detectable after IL-4 treatment (Figure 6b). Surprisingly, FACS analyses revealed increased MHCII expression in IMA2.1 cell cultures after stimulation with IFN-γ for 24 h as marker for the M1 phenotype (Figure 6c).Figure 6

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