Abstract
BackgroundProduction of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia.MethodsOxidation of 2', 7'-dichlorodihydrofluorescin diacetate (H2DCFDA) was used to measure production of intracellular ROS in microglial cell cultures following viral infection. Virus-induced cytokine and chemokine mRNA and protein levels were assessed using real-time RT-PCR and ELISA, respectively. Virus-induced phosphorylation of microglial p38 and p44/42 (ERK1/2) MAPKs was visualized using Western Blot, and levels of phospho-p38 were quantified using Fast Activated Cell-based ELISA (FACE assay). Diphenyleneiodonium (DPI) and apocynin (APO), inhibitors of NADPH oxidases, were used to investigate the role of virus-induced ROS in MAPK activation and cytokine, as well as chemokine, production.ResultsLevels of intracellular ROS were found to be highly elevated in primary murine microglial cells following infection with HSV and the majority of this virus-induced ROS was blocked following DPI and APO treatment. Correspondingly, inhibition of NADPH oxidase also decreased virus-induced proinflammatory cytokine and chemokine production. In addition, microglial p38 and p44/42 MAPKs were found to be phosphorylated in response to viral infection and this activation was also blocked by inhibitors of NADPH oxidase. Finally, inhibition of either of these ROS-induced signaling pathways suppressed cytokine (TNF-α and IL-1β) production, while chemokine (CCL2 and CXCL10) induction pathways were sensitive to inhibition of p38, but not ERK1/2 MAPK.ConclusionsData presented herein demonstrate that HSV infection induces proinflammatory responses in microglia through NADPH oxidase-dependent ROS and the activation of MAPKs.
Highlights
Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage
Viral infection induces intracellular ROS generation by murine microglia To determine the role of redox responses in virusinduced cytokine and chemokine production, we first examined ROS production by herpes simplex virus (HSV)-stimulated microglia
We examined whether HSV infection activated p38 and p44/42 mitogen-activated protein kinases (MAPKs) in our primary murine microglia
Summary
Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. Like other phagocytic cells, generate reactive oxygen species (ROS) as a mechanism to eliminate invading pathogens. The imbalance between the generation of ROS and the cell’s ability to detoxify these same mediators produces a state known as oxidative stress [1]. It is well-established that oxidative stress is an important contributing factor to many pathologic and neurodegenerative processes in the central nervous system (CNS) including HIV-associated neurocognitive disease (HAND), Alzheimer’s disease, Parkinson’s disease, and Amyotrophic lateral sclerosis [2,3].
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