Japanese encephalitis virus stimulates nitric oxide production by glial cells

• Main Authors: Jian-Hong Chen, 陳建宏
• Other Authors: Chun-Jung Chen, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/44742177237922219678

碩士 === 國立中興大學 === 生物醫學研究所 === 92 === The activation of inducible nitric oxide synthase (iNOS) and consequent production of nitric oxide (NO) in neural cells play important roles in the regulation of brain inflammation. NO exerts two opposite biological functions in the central nervous system (CNS) including neuroprotective and neurotoxic effects. Clinically, inflammatory responses such as the infiltration of macrophages, small numbers of T cells and B cells as well as production of pro-inflammatory cytokines are detected in the brain, plasma or cerebrospinal fluids of Japanese encephalitis virus (JEV)-infected patients. The immunohistochemical studies reveal that JEV antigen was mainly localized in neurons. However, the role of glial cells in the replication or pathogenesis of JEV is limited. In animal study, the infection with JEV in mice is characterized at the pathologic level by significant infiltration of inflammatory cells and the production of pro-inflammatory factors. Among the measurable factors, NO exerts antiviral effect by inhibiting the growth of JEV and increasing the survival rate of infected mice. In the beginning of our study, we found that JEV-infected neuron/glia cells, consisting of neuron, astrocyte, and microglia, produced NO. The generation of NO in in vitro neural cells after JEV infection is mimicked the in vivo animal study activating nitric oxide synthase activity in brain tissues after JEV inoculation. Generally, large amounts of NO are produced by activated glial cells. Therefore, we assessed whether glial cells played roles in the induction of NO synthesis after JEV infection and elucidated the underlying molecular mechanisms. In addition to neuron/glia, mixed glia and microglia were able to produce NO after JEV infection. However, neurons and astrocytes were unable to release NO. Next, we examined whether JEV infection stimulated glial activation by the following assays including morphological change, cytokine expression, and cell proliferation. Obvious change in morphology was only observed in microglia after JEV infection. JEV-infected mixed glia, astrocyte, or microglia all released various cytokines with variable efficiency. JEV infection stimulated cell proliferation in mixed glia. Whereas, JEV-induced cell proliferation could not be found in both astrocyte and microglia cultures. In short conclusion, JEV infection activated glial cells, especially microglia, and the activated microglia could produce NO. The mechanisms of JEV-induced NO production were further characterized. Both UV-inactivated and heat-inactivated JEV lost their ability to stimulate glial cells release NO. PKR inhibitor 2-AP attenuated JEV-induced NO production in glial cells. These findings imply that viral amplification event is critical to NO production in JEV-infected glial cells. Regarding the intracellular signaling pathways, we found that treatment of glial cells with ERK/MEK inhibitor U0126 reduced the production of NO, expression of iNOS protein and mRNA level. Based on the western blot analysis, JEV infection markedly increased the phosphorylation level of ERK in glial cells and the increase was blocked by the treatment with U0126, indicating the importance of ERK signaling pathway in the induction of iNOS activity after JEV infection in glial cells. The activation of iNOS gene expression is mostly through the transcriptional control, especially via transcription factor NF-kB. JEV infection increased the DNA binding ability of NF-kB in glial cells and the increase was attenuated by the treatment with U0126. These results revealed that transcription factor NF-kB played an important role in the induction of iNOS in JEV-infected glial cells. Finally, we tried to elucidate the biological functions of released NO after JEV infection in glial cells. We hypothesized that NO might exert antiviral effect and the elevation of NO production after JEV infection could suppress JEV amplification and reduced JEV-induced cellular alterations. In this study, increased NO production was achieved by treatment of cells with organic NO donors such as SNP or SNAP. In contrast, the suppression of JEV-induced NO production was done by treating cells with NOS inhibitor L-NAME or NO scavenger PTIO. Treatment of JEV-infected glial cells with SNP and SNAP reduced viral amplification but, L-NAME and PTIO increased that. These four agents modulated JEV-induced cytokine production with distinct characteristics. Taken together, we found that microglia responded to JEV infection by release of NO. The event of viral amplification, ERK signaling pathways, and transcription factor NF-kB were critical for iNOS gene expression. Finally, viral infection induced NO production in glial cells and the consequent NO production could suppress JEV replication and modulate JEV-induced cytokine expression.