BACKGROUND

WNV-associated encephalitis (WNVE) is characterized by increased production of pro-inflammatory mediators, glial cells activation and eventual loss of neurons. WNV infection of neurons is rapidly progressive and destructive whereas infection of non-neuronal brain cells is limited. However, the role of neurons and pathological consequences of pro-inflammatory cytokines released as a result of WNV infection is unclear. Therefore, the objective of this study was to examine the role of key cytokines secreted by WNV-infected neurons in mediating neuroinflammatory markers and neuronal death.

METHODS

A transformed human neuroblastoma cell line, SK-N-SH, was infected with WNV at multiplicity of infection (MOI)-1 and -5, and WNV replication kinetics and expression profile of key pro-inflammatory cytokines were analyzed by plaque assay, qRT-PCR, and ELISA. Cell death was measured in SK-N-SH cell line in the presence and absence of neutralizing antibodies against key pro-inflammatory cytokines using cell viability assay, TUNEL and flow cytometry. Further, naïve primary astrocytes were treated with UV-inactivated supernatant from mock- and WNV-infected SK-N-SH cell line and the activation of astrocytes was measured using flow cytometry and ELISA.

RESULTS

WNV-infected SK-N-SH cells induced the expression of IL-1β, -6, -8, and TNF-α in a dose- and time-dependent manner, which coincided with increase in virus-induced cell death. Treatment of cells with anti-IL-1β or -TNF-α resulted in significant reduction of the neurotoxic effects of WNV. Furthermore treatment of naïve astrocytes with UV-inactivated supernatant from WNV-infected SK-N-SH cell line increased expression of glial fibrillary acidic protein and key inflammatory cytokines.

CONCLUSION

Our results for the first time suggest that neurons are one of the potential sources of pro-inflammatory cytokines in WNV-infected brain and these neuron-derived cytokines contribute to WNV-induced neurotoxicity. Moreover, cytokines released from neurons also mediate the activation of astrocytes. Our data define specific role(s) of WNV-induced pro-inflammatory cytokines and provide a framework for the development of anti-inflammatory drugs as much-needed therapeutic interventions to limit symptoms associated with WNVE.