The main findings of the present study are that persistent BDV infection of mixed neuron-glial cultures activates microglia, whereas neither direct exposure to purified virus nor persistent BDV infection of neurons alone is able to activate primary microglia in vitro. This is the first report of a casual link between microglia activation and persistent BDV infection of neurons and astrocytes in the absence of overt neurotoxicity.
Microglia activation has been shown to be triggered by several means, such as direct virus infection of microglia (e.g., HIV or visna virus), released virus particles or viral proteins (25
), double-stranded RNA (29
), neuronal cell death (43
), or by infiltrating T cells (4
). However, little was known about the mechanism of microglia activation in brains of rats following neonatal BDV infection (44
). In contrast to HIV, feline immunodeficiency virus, and visna virus that infect microglia, our study demonstrated the lack of infection of microglia by BDV in the culture. To the best of our knowledge, this is the first evidence that BDV does not infect microglia in vitro, consistent with previous reports of the absence of microglia infection by BDV in vivo (38
). We also showed that direct exposure of microglia to BDV did not lead to activation of microglia. This finding may not be completely unexpected from a physiological standpoint, given that persistent BDV infection is noncytolytic in vivo and that virus is only minimally released into extracellular compartments in culture (7
Neuronal injury is a potent physiological trigger of microglia activation as evidenced by in vivo experiments with selective neurotoxins and axotomy (43
). In neonatal BDV infection, the first signs of microglia activation (MHC-I expression and proliferation) and apoptosis of neurons are observed simultaneously, between 1 and 2 weeks p.i. (44
). Therefore, it is conceivable that BDV-associated neuropathology might act as a primary trigger of microglia activation. However, our data challenge this hypothesis because (i) we did not observe any signs of neurotoxicity in BDV-infected mixed neuron-glial cultures for up to 3 weeks p.i. and (ii) microglia activation was evident as early as 1 week p.i. Thus, our findings indicate that noncytolytic BDV infection of neurons and glia can trigger microglia activation.
The present data demonstrate that BDV infection induces microglia reaction in mixed cultures of microglia, glia, and neurons but not in cocultures of purified microglia and neurons. This finding is in agreement with several reports on inducible microglia proliferation in mixed versus pure microglia cultures (26
). On the one hand, the different outcomes of microglia activation in mixed and pure culture systems could reflect the requirement for the presence of cell-to-cell contacts between microglia and other glial cells (e.g., astrocytes) for proper maturation and reaction to infection in vitro (26
). On the other hand, the major glial cell population, astrocytes, has been shown to produce soluble microglia mitogens, such as TNF-α (14
), granulocyte-macrophage colony-stimulating factor, IL-3, and IL-5 (35
) that might activate microglia in vitro. At present, it remains unclear whether the cell-to-cell contacts or soluble factors secreted by neurons or astrocytes or both play a main role in activating microglia during BDV infection. Future studies will identify the specific mechanisms of how astrocytes contribute to the BDV-induced microglia activation.
The lack of BDV-associated increase in secretion of IL-1β, TNF-α, IL-10, or NO in mixed cultures of microglia, neurons, and astrocytes is inconsistent with previous in vivo findings of upregulation of proinflammatory cytokines in brains of newborn BDV-infected rats (22
). One of the reasons for the lack of upregulation of the proinflammatory factors could be that cell interactions normally present in the brain but not included in the in vitro study may be required. For example, effects of T cells may be of a particular interest. Transient infiltrates of low numbers of T cells in brain tissue have been reported in neonatal BDV infection at 2 to 3 weeks p.i. (38
), preceding the peak of microglial activation in the rat brain (4 weeks [38
]). T cells can be activated by BDV proteins or BDV-infected astrocytes (34
) and, in turn, can activate microglia to produce interleukins (4
). Alternatively, it is conceivable that microglia in the cultures undergo only the initial state of activation, whereas stronger stimuli would be required to attain the fully activated state with a resulting cytokine secretion. For example, proliferation of MHC-I and -II and immunoglobulin expression by microglia are early manifestations of the CNS response to infection with mouse hepatitis virus (46
) and vesicular stomatitis virus (1
). Thus, one can hypothesize that stronger activation of microglia could be conferred by infiltrating T cells or by another stimulus. It is noteworthy that an additional stimulation of microglia in the BDV-infected cultures with a strong microglia activator, LPS, resulted in a significantly greater release of TNF-α, IL-1β, and IL-10 compared to the mock-infected LPS-stimulated cell cultures.
Our results provide valuable insights into the mechanisms of BDV-mediated neuropathology. While a role of microglia in mediating BDV-associated neurodegeneration has been suggested based on the observation of microgliosis and the detection of upregulated expression of mRNAs coding for proinflammatory cytokines in the areas of a significant neuronal loss (22
), it remained unclear if activation of microglia was a secondary response to an early pathology of infected neurons or if BDV infection itself triggers microgliosis that affects neuronal survival. We believe that our results provide a missing casual link between BDV infection and microglia activation. Specifically, we hypothesize that in vivo BDV infection of neurons or glia activates microglia that trigger neuronal injury or facilitate ongoing cell damage due to direct effects of BDV. Although we did not find evidence for activation of microglia by cell death in the infected mixed cultures, it is plausible that neuronal death in the BDV-infected brain may further activate already BDV-(pre)activated microglia, leading to secretion of the proinflammatory cytokines and continuation of neuronal death. This scenario might be particularly applicable to a gradual loss of neurons in such brain regions as cerebellum (i.e., Purkinje cells) and striatum (GABA neurons) associated with chronic neuroinflammation.
In conclusion, activation of microglia in BDV-infected mixed neuron-glial cultures provides evidence of infection-driven microglia activation and represents a model for studies of neurotropic infection-mediated microglia activation in vitro.