The present study demonstrates that astrocytes play a key role in activating microglia by BDV infection. Our data suggest the following sequence of events that mediate activation of microglia in the course of BDV infection: BDV-infected neurons may release soluble heat-resistant molecules or virus-associated particles and/or viral proteins that can activate uninfected astrocytes as evidenced by expression of RANTES. Activated astrocytes in turn are able to stimulate microglia that acquire a round shape, express MHC I, MHC II and IL-6, and display increased secretion of TNF-α and IL1-β upon LPS priming. Activated astrocytes may secrete soluble heat-resistant low molecular weight factors that activate microglia.
Intracranial inoculation of BDV into newborn rats is associated with a gradual loss of a) granule cells of the dentate gyrus of the hippocampus, b) Purkinje cells in the cerebellum and c) GABA-ergic neurons in the cortex and striatum [1
]. However, the mechanism of BDV-associated neuronal loss is poorly understood as, unlike neurotropic lytic virus infections (e.g., cytomegalovirus, Semliki forest virus), BDV does not induce any apparent toxicity to neurons that are the primary host cells in the CNS [8
]. Given the overall strong temporal and regional association of neurodegeneration and microgliosis in BDV-infected rat brains, it is tempting to speculate that microglia activation results from BDV-induced cell death. Notably, reactive gliosis is a universal response of the innate immune system to brain pathology[37
]. The exact functions of this phenomenon are not completely understood but the available data seem to indicate that reactive gliosis may support injured neurons and/or eliminate terminally damaged cells [38
]. BDV infection, however, is non-lytic in culture, suggesting that activation of microglia by BDV infection is unlikely initiated by cell damage.
Our previous studies have demonstrated that neither exposure of microglia to the virus nor exposure of microglia to BDV-infected neurons leads to activation of microglia. It is the presence of astrocytes in mixed cultures that is required for microglia to be activated [12
]. This result is consistent with prior reports that have demonstrated the importance of astrocytes for activation of microglia. For example, astrocytes have been found to enhance LPS-induced nitric oxide production by microglial cells [39
], mediate microglia activation by trimethyltin [40
], or modulate microglia signaling pathways during neuroinflammation [41
]. Until recently, most studies interpreted the timing of activation of astrocytes to the effect that they rather play a secondary role in the initiation of events. However, there is a growing body of evidence that suggests an initiating role of astrocytes in neuroinflammation [38
]. We believe that persistent BDV infection represents another example when astrocytes play a crucial role in triggering the innate immune response in the CNS and provides a valuable model for studying the molecular mechanisms of cell-to-cell communications in the context of chronic neuroinflammation and resultant neurobehavioral abnormalities.
Our findings further indicate that direct cell contacts between neurons and astrocytes are not required for activating microglia and that soluble factors secreted by neurons and astrocytes appear responsible for microglia activation. Since uninfected astrocytes need to be activated first to mediate ensuing activation of microglia, we explored what factors could be involved in this process. Factors produced by neurons in response to infection or injury include viral proteins and various chemokines and cytokines [43
]. Based on the UV inactivation experiments as well as the enhanced astrocyte activation using increasing amount of virus stock, we speculate that certain viral BDV proteins may mediate activation of astrocytes. The data is in line with the observations that Tat or gp120 activate astrocytes by destabilizing intracellular Ca2+
, subsequently leading to release of RANTES and IL-6 [28
]. It should be pointed out that both mock and BDV virus stocks were prepared using the standard protocol [12
] and contained virus particles as well as cellular and viral protein contaminants. Thus, activation of astrocytes could be due to virus related components such as particle-associated and free viral proteins. In contrast, a contribution of cellular material unrelated to BDV appears unlikely since mock and BDV stocks were purified and used under the identical conditions throughout the study. We found that treatment of primary astrocytes with media collected from BDV-infected neurons led to up-regulation of RANTES. These results are consistent with previous studies that demonstrated an up-regulation of IP-10 and RANTES in the brains of neonatally BDV-infected rats [45
]. Interestingly, the authors have found that a likely source of chemokines in the infected brain parenchyma are astrocytes, compatible with our hypothesis that astrocytes are activated by BDV-infected neurons to initiate neuro-inflammatory cascades in situ
Activated astrocytes have been shown to produce soluble factors that are able to activate microglia [22
]. What activates microglia in the course of BDV infection remains obscure. In a search for possible candidates, we screened 22 soluble cytokines and chemokines using a mulitplex analysis. Only RANTES was found to be significantly up-regulated by BDV infection Furthermore, in our preliminary experiments, MG-CSF failed to induce round microglia formation under the conditions where BDV infection did (data not shown). Interestingly, heat inactivation of conditioned media from BDV-infected astrocytes (used as a positive control of astrocyte activation) had no effects on the ability of media to activate microglia, suggesting that putative soluble mediators are heat-resistant low-molecular weight factors. Possible candidates could include "gliotransmitters" such as ATP [47
]. These molecules secreted by neurons and astrocytes work as mediators of cell-to-cell communication in a physiological condition, and may serve as "warning molecules" in pathological conditions [22
]. We found no significant effects of BDV infection on production of ATP. In addition, the ATP concentrations in the media was very low (< 10 nM) compared to concentrations of 0.1–10 mM that have been found to activate neurons and microglia in vitro [33
]. Noteworthy, ATP in culture media is typically associated with release of intracellular ATP from lysed cells. Thus, our findings are consistent with the lack of BDV-associated cell toxicity in vitro. Future studies may help to identify if other nucleotides mediate microglia activation.
Our data provide more insights into the possible sequence of events leading to neurodegeneration in neonatally BDV-infected rats. Primary BDV infection of neurons may produce activation of astrocytes and ensuing activation of microglia. It remains unresolved whether microglia activation initiates cell demise or simply exacerbates on-going cell injury. A recent report by Solbrig et al [49
] lends an additional support for the first hypothesis. Specifically, the authors have shown that inhibition of microglia activation with a cannabinoid agonist decreases cell death in the BDV-infected rat brain [48
]. In a broader context, our report is in line with an emerging idea that chronic activation of the innate immune system in the brain contributes to neuronal injury even if a pathogen may not be directly toxic to neurons.