Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death

Abstract

BackgroundWNV-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.MethodsA 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.ResultsWNV-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.ConclusionOur 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.

Encephalitic Arboviruses: Emergence, Clinical Presentation, and Neuropathogenesis

“…TNF-α impairs glutamate uptake by astrocytes, leading to neuronal death due to excitotoxicity [197]. Consistent with this, it has been shown that increased expression of TNF-α after infection with WNV and JEV is correlated with neuronal death [154,195], and that neutralizing antibodies specific for IL-1β and TNF-α protected neurons from WNV-induced cell death [195]. The release of inflammatory cytokines by neurons can trigger gliosis (i.e., microglial and astrocyte activation), which is one of the major hallmarks of arboviral neuropathogenesis [154,186,198].…”

“…Following CNS infection by arboviruses, both infected target cells and bystander cells release an array of chemokines and proinflammatory cytokines, which trigger neuroinflammation. Neurons release several inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 in response to infection by encephalitic flaviviruses, such as WNV and JEV [195]. As well as impairing BBB integrity [152], elevated levels of IL-1β can induce cell cycle arrest and apoptosis in neural precursor cells (NPCs) [196].…”

“…Activated astrocytes and microglia play an important role in inflammatory responses during natural infection with encephalitic flaviviruses, namely JEV [200], WNV [201], and TBEV [198], as well as encephalitic alphaviruses [186]. Upon activation, astrocytes and microglia release several chemokines and cytokines such as TNF-α, IL-6, C-X-C motif chemokine ligand (CXCL)10, chemokine (C-C motif) ligand 2, monocyte chemoattractant protein-1, RANTES, IFN-γ-induced protein 10, and matrix metalloproteinases [154,195,[202][203][204]. Elevated levels of matrix metalloproteinases disrupts BBB by degrading TJ proteins [203,205], allowing unrestricted entry of leukocytes into the brain, which further exacerbates neuroinflammation.…”

Differential Replication of Pathogenic and Nonpathogenic Strains of West Nile Virus within Astrocytes

“…There is circumstantial evidence that astrocytes contribute to the second wave of WNV neuroinvasion through the upregulation of MMPs, which disrupt the BBB, and proinflammatory cytokines, which recruit infected leukocytes (5,41,52,53). Indeed, propagation within astrocytes, neurons, and glial cells prior to the breakdown of the BBB is believed to be a common strategy of neuroinvasive viruses, including tick-borne encephalitis virus and HIV, to enhance dissemination within the CNS (54,55).…”

Innate immune responses regulate morphogenesis and degeneration: roles of Toll-like receptors and Sarm1 in neurons

“…Activation of TLRs in neurons produces a very low level of pro-inflammatory cytokines [11,38,39] , while microglia and astrocytes secret large amounts of cytokines [40,41] . The low level of cytokine production by neurons seems unlikely to induce a global innate immune response in the brain.…”