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DeltaRR vaccination protects from KA-induced seizures and neuronal loss through ICP10PK-mediated modulation of the neuronal-microglial axis.

Laing JM, Aurelian L - Genet Vaccines Ther (2008)

Bottom Line: The CM from Delta PK-infected EOC2 and EOC20 cells did not contain IL-10, but it contained TNF-alpha and RANTES.IL-10 neutralization significantly (p < 0.01) decreased, but did not abrogate, the neuroprotective activity of the CM from Delta RR-infected microglial cultures indicating that ICP10PK modulates the neuronal-microglial axis, also through induction of various microglial neuroprotective factors.Protection was associated with a significant (p < 0.001) increase in the numbers of IL-10+ microglia (CD11b+) as compared to Delta PK-treated animals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, MD 21201, USA. jlain001@umaryland.edu

ABSTRACT
Ischemic brain injury and epilepsy are common neurodegenerative diseases caused by excitotoxicity. Their pathogenesis includes microglial production of inflammatory cytokines. Our studies were designed to examine whether a growth compromised HSV-2 mutant (Delta RR) prevents excitotoxic injury through modulation of microglial responses by the anti-apoptotic HSV-2 protein ICP10PK. EOC2 and EOC20 microglial cells, which are differentially activated, were infected with Delta RR or the ICP10PK deleted virus (Delta PK) and examined for virus-induced neuroprotective activity. Both cell lines were non-permissive for virus growth, but expressed ICP10PK (Delta RR) or the PK deleted ICP10 protein p95 (Delta PK). Conditioned medium (CM) from Delta RR-, but not Delta PK-infected cells prevented N-methyl-D-aspartate (NMDA)-induced apoptosis of primary hippocampal cultures, as determined by TUNEL and caspase-3 activation (76.9 +/- 5.3% neuroprotection). Neuroprotection was associated with inhibition of TNF-alpha and RANTES and production of IL-10. The CM from Delta PK-infected EOC2 and EOC20 cells did not contain IL-10, but it contained TNF-alpha and RANTES. IL-10 neutralization significantly (p < 0.01) decreased, but did not abrogate, the neuroprotective activity of the CM from Delta RR-infected microglial cultures indicating that ICP10PK modulates the neuronal-microglial axis, also through induction of various microglial neuroprotective factors. Rats given Delta RR (but not Delta PK) by intranasal inoculation were protected from kainic acid (KA)-induced seizures and neuronal loss in the CA1 hippocampal fields. Protection was associated with a significant (p < 0.001) increase in the numbers of IL-10+ microglia (CD11b+) as compared to Delta PK-treated animals. Delta RR is a promising vaccination/therapy platform for neurodegeneration through its pro-survival functions in neurons as well as microglia modulation.

No MeSH data available.


Related in: MedlinePlus

EOC2 and EOC20 Microglia cultures are non-permissive for HSV replication. (A). EOC2 and EOC20 cells differ in morphology and the intensity of staining with CD11b antibody before, but not after virus infection. (B). EOC2 and EOC20 cells were infected with ΔRR or ΔPK or HSV-2 (1 × 106 pfu) and examined for virus growth by plaque assay as described in Materials and Methods.
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Figure 2: EOC2 and EOC20 Microglia cultures are non-permissive for HSV replication. (A). EOC2 and EOC20 cells differ in morphology and the intensity of staining with CD11b antibody before, but not after virus infection. (B). EOC2 and EOC20 cells were infected with ΔRR or ΔPK or HSV-2 (1 × 106 pfu) and examined for virus growth by plaque assay as described in Materials and Methods.

Mentions: In a first series of experiments to examine the effect of ΔRR on microglia, we asked whether: (i) microglial cells are permissive for virus growth, and (ii) permissiveness is affected by prior cell activation. We used EOC2 and EOC20 cells that differ in the levels of MHCII expression, with high levels constitutively expressed by EOC20, but not EOC2 cells [19]. Excessive activation was confirmed for EOC20 cells by their rounded morphology and high intensity staining with CD11b antibody (Fig. 2A). EOC2 cells had lower CD11b staining intensity and retained some morphologic ramification. However, high intensity staining and rounded morphology were seen after virus infection (Fig. 2A), indicative of virus-induced activation [10,28]. EOC2 and EOC20 cells were non-permissive for growth of ΔRR, ΔPK or HSV-2, as determined by plaque assay. Virus titers decreased at similar rates during the first 4 hrs p.i.. For HSV-2, the titers remained at this reduced level until 96 hrs p.i. For ΔRR and ΔPK the titers continued to decrease until 12 hrs p.i. and remained stable at this reduced level until 120 hrs p.i. During 4 – 96 hrs p.i., the titers of ΔRR and ΔPK were approximately 10-fold lower than those of HSV-2, but virus clearance after 120 hrs was similar for all viruses, with lowest titers (almost complete clearance) seen at 14 days p.i. (Fig. 2B). Infectious center assays done up to 96 hrs p.i., indicated that approximately 90% of the cells formed plaques on Vero cells. Collectively, the data indicate that: (i) microglia are non-permissive for virus growth unrelated to their activation status prior to infection, and (ii) the clearance of ΔRR and ΔPK is somewhat more efficient than that of wild type virus.


DeltaRR vaccination protects from KA-induced seizures and neuronal loss through ICP10PK-mediated modulation of the neuronal-microglial axis.

Laing JM, Aurelian L - Genet Vaccines Ther (2008)

EOC2 and EOC20 Microglia cultures are non-permissive for HSV replication. (A). EOC2 and EOC20 cells differ in morphology and the intensity of staining with CD11b antibody before, but not after virus infection. (B). EOC2 and EOC20 cells were infected with ΔRR or ΔPK or HSV-2 (1 × 106 pfu) and examined for virus growth by plaque assay as described in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268933&req=5

Figure 2: EOC2 and EOC20 Microglia cultures are non-permissive for HSV replication. (A). EOC2 and EOC20 cells differ in morphology and the intensity of staining with CD11b antibody before, but not after virus infection. (B). EOC2 and EOC20 cells were infected with ΔRR or ΔPK or HSV-2 (1 × 106 pfu) and examined for virus growth by plaque assay as described in Materials and Methods.
Mentions: In a first series of experiments to examine the effect of ΔRR on microglia, we asked whether: (i) microglial cells are permissive for virus growth, and (ii) permissiveness is affected by prior cell activation. We used EOC2 and EOC20 cells that differ in the levels of MHCII expression, with high levels constitutively expressed by EOC20, but not EOC2 cells [19]. Excessive activation was confirmed for EOC20 cells by their rounded morphology and high intensity staining with CD11b antibody (Fig. 2A). EOC2 cells had lower CD11b staining intensity and retained some morphologic ramification. However, high intensity staining and rounded morphology were seen after virus infection (Fig. 2A), indicative of virus-induced activation [10,28]. EOC2 and EOC20 cells were non-permissive for growth of ΔRR, ΔPK or HSV-2, as determined by plaque assay. Virus titers decreased at similar rates during the first 4 hrs p.i.. For HSV-2, the titers remained at this reduced level until 96 hrs p.i. For ΔRR and ΔPK the titers continued to decrease until 12 hrs p.i. and remained stable at this reduced level until 120 hrs p.i. During 4 – 96 hrs p.i., the titers of ΔRR and ΔPK were approximately 10-fold lower than those of HSV-2, but virus clearance after 120 hrs was similar for all viruses, with lowest titers (almost complete clearance) seen at 14 days p.i. (Fig. 2B). Infectious center assays done up to 96 hrs p.i., indicated that approximately 90% of the cells formed plaques on Vero cells. Collectively, the data indicate that: (i) microglia are non-permissive for virus growth unrelated to their activation status prior to infection, and (ii) the clearance of ΔRR and ΔPK is somewhat more efficient than that of wild type virus.

Bottom Line: The CM from Delta PK-infected EOC2 and EOC20 cells did not contain IL-10, but it contained TNF-alpha and RANTES.IL-10 neutralization significantly (p < 0.01) decreased, but did not abrogate, the neuroprotective activity of the CM from Delta RR-infected microglial cultures indicating that ICP10PK modulates the neuronal-microglial axis, also through induction of various microglial neuroprotective factors.Protection was associated with a significant (p < 0.001) increase in the numbers of IL-10+ microglia (CD11b+) as compared to Delta PK-treated animals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, MD 21201, USA. jlain001@umaryland.edu

ABSTRACT
Ischemic brain injury and epilepsy are common neurodegenerative diseases caused by excitotoxicity. Their pathogenesis includes microglial production of inflammatory cytokines. Our studies were designed to examine whether a growth compromised HSV-2 mutant (Delta RR) prevents excitotoxic injury through modulation of microglial responses by the anti-apoptotic HSV-2 protein ICP10PK. EOC2 and EOC20 microglial cells, which are differentially activated, were infected with Delta RR or the ICP10PK deleted virus (Delta PK) and examined for virus-induced neuroprotective activity. Both cell lines were non-permissive for virus growth, but expressed ICP10PK (Delta RR) or the PK deleted ICP10 protein p95 (Delta PK). Conditioned medium (CM) from Delta RR-, but not Delta PK-infected cells prevented N-methyl-D-aspartate (NMDA)-induced apoptosis of primary hippocampal cultures, as determined by TUNEL and caspase-3 activation (76.9 +/- 5.3% neuroprotection). Neuroprotection was associated with inhibition of TNF-alpha and RANTES and production of IL-10. The CM from Delta PK-infected EOC2 and EOC20 cells did not contain IL-10, but it contained TNF-alpha and RANTES. IL-10 neutralization significantly (p < 0.01) decreased, but did not abrogate, the neuroprotective activity of the CM from Delta RR-infected microglial cultures indicating that ICP10PK modulates the neuronal-microglial axis, also through induction of various microglial neuroprotective factors. Rats given Delta RR (but not Delta PK) by intranasal inoculation were protected from kainic acid (KA)-induced seizures and neuronal loss in the CA1 hippocampal fields. Protection was associated with a significant (p < 0.001) increase in the numbers of IL-10+ microglia (CD11b+) as compared to Delta PK-treated animals. Delta RR is a promising vaccination/therapy platform for neurodegeneration through its pro-survival functions in neurons as well as microglia modulation.

No MeSH data available.


Related in: MedlinePlus