We have recently reported that HSV-induced ROS production by microglial cells is responsible for lipid peroxidation, oxidative damage, and toxicity to neurons in culture, and that viral recognition is mediated, at least in part, through Toll-like receptor (TLR)-2 . In several other systems, engagement of TLRs has been demonstrated to induce NADPH oxidase activation, with corresponding ROS generation, which subsequently activates NF-κB to induce proinflammatory cytokine production [19–21]. Following up on our previous work, the present study examined the effect of HSV-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) and driving cytokine, as well as chemokine, expression in primary murine microglia. Data obtained during these studies clearly demonstrate that intracellular ROS are generated following viral infection of murine microglia and are associated with a marked increase in the expression of NADPH oxidase mRNA. Viral infection was found to induce microglial cell-produced ROS as early as 3 h in individual cells, however, additional time was required to reach statistical significance when the entire culture was assessed.
ROS are important second messengers in redox signaling. Viral brain infection initiates robust inflammatory responses pivoting on the production of cytokines and chemokines by microglial cells . We have previously reported that microglial cells undergo an abortive, non-productive infection with HSV-1 in which immediate early gene (e.g., ICP4) expression occurs, but late gene expression (e.g., such as glycoprotein D, gD) and viral replication are blocked . These cells respond to HSV infection by inducing a burst of cytokine and chemokine production, followed by apoptotic death. It has previously been reported that microglial ROS, produced largely through the action of NADPH oxidases, precedes cytokine and chemokine production in response to HIV Tat or M. tuberculosis 30-kDa Ag [12, 22]. In the present study, inhibition of NADPH oxidase with either DPI or APO was also found to decrease subsequent HSV-induced cytokine and chemokine production. These data demonstrate that NADPH-derived ROS drive cytokine and chemokine expression by microglia in response to viral infection.
Phosphorylation of p38 and p44/p42 ERK1/2 MAPK is commonly associated with TLR signaling and has been implicated in TLR-associated ROS production [11, 19, 23, 24]. Because these MAPKs play an important role in regulating the expression of immune mediators following stimulation with viruses, viral proteins, and other inflammatory factors [9, 14, 17, 25–27], we next investigated the role of p38 and p44/p42 ERK1/2 activation in HSV-infected microglia. In these studies, we first found that viral infection induced the phosphorylation of both MAPKs. We then went on to perform experiments using the inhibitors DPI and APO to determine whether NADPH oxidase-derived ROS drive viral activation of p38 and p44/p42 ERK1/2 MAPKs. In these studies, treatment of microglial cells with the NADPH oxidase inhibitors was found to blunt HSV-induced MAPK phosphorylation by Western Blot (p38 and p44/p42 ERK1/2) and FACE (p38) assay.
In our last set of experiments we investigated the effect of blocking specific MAPK pathways on HSV-induced cytokine and chemokine production. Using human microglia, we have previously reported that while an inhibitor of p38 MAPK (SB202190) blocked both HSV-induced cytokine and chemokine production, treatment with the ERK1/2 inhibitor (U0126) inhibited the induction of cytokines (i.e., TNF-α, IL-1β), but not chemokines (i.e., CCL5 and CXCL10), . In the present study, very similar differential cytokine and chemokine results are found using HSV-infected murine microglia. HSV-induced TNF-α and IL-1β production was found to be susceptible to inhibition by both the p38 MAPK inhibitor SB203580 and the p44/p42 ERK1/2 inhibitor U0126, while virus-induced CXCL10 and CCL2 was suppressed by SB203580, but the p44/p42 ERK1/2 inhibitor had no inhibitory effect at any concentration tested. Taken together, it is likely that insufficient activation of these MAPK pathways following the inhibition of NADPH oxidase, and decreased ROS generation, is responsible for the attenuated cytokine production.
A number of studies have shown that beneficial neuroimmune responses, for example those needed to purge infectious virus from the brain, can develop into chronic pathological inflammation with progressive neurodegeneration . Restoration of redox balance may be an important determinant in returning activated microglia back to a resting state following viral infection and neuroinflammation. The findings presented herein support the idea that ROS-driven microglial cell activation, and its associated neurotoxicity, may be a target for therapeutic modulation through the stimulation of opposing anti-oxidative responses.