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Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection.

Ovanesov MV, Ayhan Y, Wolbert C, Moldovan K, Sauder C, Pletnikov MV - J Neuroinflammation (2008)

Bottom Line: Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia.We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES.The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. ovanesov@gmail.com

ABSTRACT
Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to certain neuronal populations. Since persistent BDV infection of neurons is nonlytic in vitro, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brains remain unclear. Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia. Here, we evaluated the mechanisms whereby astrocytes can contribute to activation of microglia in neuron-glia-microglia mixed cultures. We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES. Activation of astrocytes then produces activation of microglia as evidenced by increased formation of round-shaped, MHCI-, MHCII- and IL-6-positive microglia cells. Our analysis of possible molecular mechanisms of activation of astrocytes and/or microglia in culture indicates that the mediators of activation may be soluble heat-resistant, low molecular weight factors. The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons. The data are consistent with the hypothesis that microglia activation in the absence of neuronal damage may represent initial steps in the gradual neurodegeneration observed in brains of neonatally BDV-infected rats.

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Astrocytes activated by BDV-infected neurons induce microglia activation in mixed cultures. A – Lectin staining of round microglia in mixed cultures left either uninfected and untreated (control), or BDV-infected and untreated, or uninfected but treated with conditioned media from AF5-activated astrocytes. Scale bar – 50 μm. The quantitative data for AF5-activated astrocytes are presented in C (column a). B – LPS-induced production of TNF-α and IL-1β by mixed cultures. TNF-α and IL-1β were measured by ELISA. Note a significant up-regulation of the cytokines in mixed cultures activated by conditioned media from astrocytes pre-treated with media collected from BDV-infected AF5 neuronal cells. Values are given as ratio compared to concentrations measured in mixed cultures exposed to conditioned media from astrocytes pretreated with media collected from mock infected AF5 neuronal cells, n = 3–5. C – Activation of microglia in mixed cultures following exposure to medium from astrocytes conditioned with medium from different types of infected neuronal cells. Astrocytes can be activated by persistently BDV-infected neuronal AF5 cells (a) or CA77 cells (b). Medium from persistently BDV-infected astrocytes (c) as well as LPS treatment of mixed cultures (d) were used as positive controls. Conditioned media from different pre-treated astrocyte cultures were added to mixed cultures to activate microglia. The results are numbers of round microglia cells in BDV-treatments normalized to respective mock-treated sister cultures, n = 3–6. The results of the paired t-test on two populations (mock vs. mean): *p < 0.05.
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Figure 6: Astrocytes activated by BDV-infected neurons induce microglia activation in mixed cultures. A – Lectin staining of round microglia in mixed cultures left either uninfected and untreated (control), or BDV-infected and untreated, or uninfected but treated with conditioned media from AF5-activated astrocytes. Scale bar – 50 μm. The quantitative data for AF5-activated astrocytes are presented in C (column a). B – LPS-induced production of TNF-α and IL-1β by mixed cultures. TNF-α and IL-1β were measured by ELISA. Note a significant up-regulation of the cytokines in mixed cultures activated by conditioned media from astrocytes pre-treated with media collected from BDV-infected AF5 neuronal cells. Values are given as ratio compared to concentrations measured in mixed cultures exposed to conditioned media from astrocytes pretreated with media collected from mock infected AF5 neuronal cells, n = 3–5. C – Activation of microglia in mixed cultures following exposure to medium from astrocytes conditioned with medium from different types of infected neuronal cells. Astrocytes can be activated by persistently BDV-infected neuronal AF5 cells (a) or CA77 cells (b). Medium from persistently BDV-infected astrocytes (c) as well as LPS treatment of mixed cultures (d) were used as positive controls. Conditioned media from different pre-treated astrocyte cultures were added to mixed cultures to activate microglia. The results are numbers of round microglia cells in BDV-treatments normalized to respective mock-treated sister cultures, n = 3–6. The results of the paired t-test on two populations (mock vs. mean): *p < 0.05.

Mentions: To investigate if astrocytes activated by BDV-infected neurons could in turn stimulate microglia activation, mixed cultures were treated with conditioned media from pure astrocytes that were pre-treated with conditioned media collected from mock- or BDV-infected neuronal AF5 cells (see Figure 5 for the experiment design). Compared to mixed cultures treated with media from astrocytes per-treated with media from mock-infected neurons, we found a 3.9-fold increase in the number of round microglia cells in mixed cultures treated with conditioned media collected from astrocytes pre-treated with media from BDV-infected neuronal cells (BDV-AF5-astrocyte-treated mixed cultures (Figure 6A). To confirm functional activation of round shaped microglia, we stimulated mixed cultures with LPS and observed a significantly larger release of TNF-α and IL1-β by BDV-AF5-astrocyte treated cultures vs. mock-AF5-astrocyte treated mixed cultures (Figure 6B). We also found that astrocyte activation was not limited to the AF5 neuronal cell line and was also observed after treatment of astrocytes with media collected from the BDV-infected CA77 neuronal cell line or from infected primary cortical neurons (Figure 6C). Furthermore, we used mock or BDV-infected primary cortical neurons at 10 d.p.i. in a similar experiment. Since media for primary astrocytes and primary neurons are incompatible, primary neurons were switched to serum-containing media one day before the experiment. Such conditioned media from primary BDV-infected neurons activated astrocytes as evidenced by their (i.e., astrocytes) ability to produce a 2.54 ± 0.86-fold increase in numbers of round microglia cells in mixed cultures compared to mixed cultures treated with media collected from astrocytes pre-treated with media from mock-infected primary neurons.


Astrocytes play a key role in activation of microglia by persistent Borna disease virus infection.

Ovanesov MV, Ayhan Y, Wolbert C, Moldovan K, Sauder C, Pletnikov MV - J Neuroinflammation (2008)

Astrocytes activated by BDV-infected neurons induce microglia activation in mixed cultures. A – Lectin staining of round microglia in mixed cultures left either uninfected and untreated (control), or BDV-infected and untreated, or uninfected but treated with conditioned media from AF5-activated astrocytes. Scale bar – 50 μm. The quantitative data for AF5-activated astrocytes are presented in C (column a). B – LPS-induced production of TNF-α and IL-1β by mixed cultures. TNF-α and IL-1β were measured by ELISA. Note a significant up-regulation of the cytokines in mixed cultures activated by conditioned media from astrocytes pre-treated with media collected from BDV-infected AF5 neuronal cells. Values are given as ratio compared to concentrations measured in mixed cultures exposed to conditioned media from astrocytes pretreated with media collected from mock infected AF5 neuronal cells, n = 3–5. C – Activation of microglia in mixed cultures following exposure to medium from astrocytes conditioned with medium from different types of infected neuronal cells. Astrocytes can be activated by persistently BDV-infected neuronal AF5 cells (a) or CA77 cells (b). Medium from persistently BDV-infected astrocytes (c) as well as LPS treatment of mixed cultures (d) were used as positive controls. Conditioned media from different pre-treated astrocyte cultures were added to mixed cultures to activate microglia. The results are numbers of round microglia cells in BDV-treatments normalized to respective mock-treated sister cultures, n = 3–6. The results of the paired t-test on two populations (mock vs. mean): *p < 0.05.
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Figure 6: Astrocytes activated by BDV-infected neurons induce microglia activation in mixed cultures. A – Lectin staining of round microglia in mixed cultures left either uninfected and untreated (control), or BDV-infected and untreated, or uninfected but treated with conditioned media from AF5-activated astrocytes. Scale bar – 50 μm. The quantitative data for AF5-activated astrocytes are presented in C (column a). B – LPS-induced production of TNF-α and IL-1β by mixed cultures. TNF-α and IL-1β were measured by ELISA. Note a significant up-regulation of the cytokines in mixed cultures activated by conditioned media from astrocytes pre-treated with media collected from BDV-infected AF5 neuronal cells. Values are given as ratio compared to concentrations measured in mixed cultures exposed to conditioned media from astrocytes pretreated with media collected from mock infected AF5 neuronal cells, n = 3–5. C – Activation of microglia in mixed cultures following exposure to medium from astrocytes conditioned with medium from different types of infected neuronal cells. Astrocytes can be activated by persistently BDV-infected neuronal AF5 cells (a) or CA77 cells (b). Medium from persistently BDV-infected astrocytes (c) as well as LPS treatment of mixed cultures (d) were used as positive controls. Conditioned media from different pre-treated astrocyte cultures were added to mixed cultures to activate microglia. The results are numbers of round microglia cells in BDV-treatments normalized to respective mock-treated sister cultures, n = 3–6. The results of the paired t-test on two populations (mock vs. mean): *p < 0.05.
Mentions: To investigate if astrocytes activated by BDV-infected neurons could in turn stimulate microglia activation, mixed cultures were treated with conditioned media from pure astrocytes that were pre-treated with conditioned media collected from mock- or BDV-infected neuronal AF5 cells (see Figure 5 for the experiment design). Compared to mixed cultures treated with media from astrocytes per-treated with media from mock-infected neurons, we found a 3.9-fold increase in the number of round microglia cells in mixed cultures treated with conditioned media collected from astrocytes pre-treated with media from BDV-infected neuronal cells (BDV-AF5-astrocyte-treated mixed cultures (Figure 6A). To confirm functional activation of round shaped microglia, we stimulated mixed cultures with LPS and observed a significantly larger release of TNF-α and IL1-β by BDV-AF5-astrocyte treated cultures vs. mock-AF5-astrocyte treated mixed cultures (Figure 6B). We also found that astrocyte activation was not limited to the AF5 neuronal cell line and was also observed after treatment of astrocytes with media collected from the BDV-infected CA77 neuronal cell line or from infected primary cortical neurons (Figure 6C). Furthermore, we used mock or BDV-infected primary cortical neurons at 10 d.p.i. in a similar experiment. Since media for primary astrocytes and primary neurons are incompatible, primary neurons were switched to serum-containing media one day before the experiment. Such conditioned media from primary BDV-infected neurons activated astrocytes as evidenced by their (i.e., astrocytes) ability to produce a 2.54 ± 0.86-fold increase in numbers of round microglia cells in mixed cultures compared to mixed cultures treated with media collected from astrocytes pre-treated with media from mock-infected primary neurons.

Bottom Line: Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia.We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES.The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. ovanesov@gmail.com

ABSTRACT
Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to certain neuronal populations. Since persistent BDV infection of neurons is nonlytic in vitro, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brains remain unclear. Our previous studies have shown that activation of microglia by BDV in culture requires the presence of astrocytes as neither the virus nor BDV-infected neurons alone activate microglia. Here, we evaluated the mechanisms whereby astrocytes can contribute to activation of microglia in neuron-glia-microglia mixed cultures. We found that persistent infection of neuronal cells leads to activation of uninfected astrocytes as measured by elevated expression of RANTES. Activation of astrocytes then produces activation of microglia as evidenced by increased formation of round-shaped, MHCI-, MHCII- and IL-6-positive microglia cells. Our analysis of possible molecular mechanisms of activation of astrocytes and/or microglia in culture indicates that the mediators of activation may be soluble heat-resistant, low molecular weight factors. The findings indicate that astrocytes may mediate activation of microglia by BDV-infected neurons. The data are consistent with the hypothesis that microglia activation in the absence of neuronal damage may represent initial steps in the gradual neurodegeneration observed in brains of neonatally BDV-infected rats.

Show MeSH
Related in: MedlinePlus