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

Abstract

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.

Figure 1

WNV can infect and induce apoptosis in human neuroblastoma cell line, SK-N-SH. (A) WNV titers in culture supernatant from SK-N-SH cell line collected at 2 h and from days 1 to 4 after infection were determined by plaque assay using Vero cells. Viral titers are expressed as plaque forming units (PFU)/mL of supernatant. Data are expressed as mean ± SD for two independent experiments conducted in duplicate. (B) Cell toxicity of SK-N-SH cells from days 1 to 3 after WNV infection was assessed by cell proliferation assay and percentage cell toxicity was calculated by comparing to mock-infected cells at corresponding time points. Data are expressed as mean ± SD for three independent experiments conducted in triplicate. (C) Mock (i and ii), and WNV (MOI-1)-infected SK-N-SH cells (iii and iv) were fixed at day 2 after infection and TUNEL assay was conducted (red; i and iii). Cells were counterstained with DAPI to label nucleus. TUNEL plus DAPI images (ii and iv) indicate that number of cells in each field were similar. DNase I-treated cells were used as positive control (v). The images depict representative results of three independent experiments.

Figure 2

WNV differentially modulates the expressions of pro-inflammatory cytokines in human neuroblastoma cell line, SK-N-SH. (A) cDNA templates from mock- and WNV-infected SK-N-SH cells from days 1 to 3 after infection were used to determine the fold-change of IL-1β, -6, -8, and TNF-α by qRT-PCR. Changes in the levels of pro-inflammatory cytokines were first normalized to the GAPDH gene and the fold-change in infected cells as compared to corresponding controls was calculated. Data represents mean ± SD of five independent experiments conducted in duplicate. (B) Levels of IL-1β, -6, -8, and TNF-α in culture supernatants were determined by ELISA. WNV (MOI-1) infection significantly increased the production of pro-inflammatory cytokines. The data expressed are the mean concentration (pg/ml) ± SD of the amount of IL-1β, -6, -8 or TNF-α, secreted in the supernatant and is representative of three independent experiments. *p < 0.05. **p < 0.001.

Figure 3

Neutralization of IL-1β and TNF-α protects cell death of human neuroblastoma cell line, SK-N-SH. (A) WNV (MOI-1)- infected SK-N-SH cells were treated with neutralizing antibodies against IL-1β-, -6-, -8-, or TNF-α and cell toxicity was assessed by cell proliferation assay. Percentage cell toxicity was calculated by comparing to control cells. While neutralization of IL-8 and IL-6 did not result in significant reduction in WNV-induced cell death, it was significantly attenuated in the presence of anti-IL1β or -TNF-α at day 2 after infection. Data are expressed as mean ± SD from three independent experiments conducted in triplicates. (B) WNV (MOI-1)-infected SK-N-SH cells were treated with anti-IL-1β or -TNF-α and assessed for apoptosis at day 2 after infection. Abundant TUNEL-positive cells (red, i) co-localized with DAPI (blue; ii) were observed in WNV-infected cells. In contrast, apoptosis induced by WNV was significantly attenuated in the presence of anti-IL-1β (iii) and anti-TNF-α (v). The images depicted are representative results of three independent experiments. (C) Quantitative representation of TUNEL-positive cells in each group from three independent cover slips from three independent experiments. Quantitative analysis indicated that neutralization of IL-1β and TNF-α reduced the number of TUNEL positive cells to less than half compared to WNV-infected cells. *p < 0.05.

Figure 4

Protection of human neuroblastoma cell line, SK-N-SH, toxicity as assayed by FACS analysis. FACS analysis of TUNEL-positive cells in (A) mock and (B) WNV (MOI-1)-infected SK-N-SH cells treated with (C) anti-IL-1β or (D) anti-TNF-α at day 2 after infection. (E) DNase I-treated cells were used as positive control. The images depicted in panels A to E are representative data of three independent experiments. (F) TUNEL-positive cells reduced significantly in the presence of neutralizing antibodies against IL-1β and TNF-α. Data in panel F are expressed as mean ± SD for three independent experiments conducted in duplicate. **p < 0.001 as compared to mock, *p < 0.05 compared to corresponding infected cells.

Figure 5

UV-inactivated supernatant from WNV-infected human neuroblastoma cell line, SK-N-SH, activates astrocytes and induces expression of pro-inflammatory cytokines. (A) FACS analysis of GFAP expression in naïve HBCA cells treated with UV-inactivated supernatant derived from mock- and WNV (MOI-1)-infected SK-N-SH cells at 48 h after treatment is shown as overlapped histograms with the mean fluorescence intensity (MFI) in arbitrary units at the right. The MFI of GFAP increased significantly in HBCA cells treated with UV-inactivated supernatant from infected SK-N-SH cells (*p < 0.05). Data are representative of three independent experiments. (B) cDNA templates synthesized from RNA extracted from HBCA cells at 6, 24 and 48 h after treatment with UV-inactivated supernatant from SK-N-SH cells were used to determine the fold-change of IL-1β, -6, -8, and TNF-α by qRT-PCR. Changes in the levels of pro-inflammatory cytokines were first normalized to the GAPDH gene and then the fold-change in infected supernatant treated cells as compared to corresponding controls was calculated. Data represents mean ± SD of five independent experiments conducted in duplicate.

Figure 6

UV-inactivated supernatant from WNV-infected human neuroblastoma cell line, SK-N-SH, induce the secretion of pro-inflammatory cytokines from astrocytes. Naïve HBCA cells were either mock-treated or infected with UV-inactivated WNV or WNV at MOI-1 or treated with UV-inactivated supernatant derived from mock- and WNV (MOI-1)-infected SK-N-SH cells, and levels of IL-1β, -6, -8, and TNF-α in treated or infected HBCA culture supernatants were determined by ELISA. Supernatant derived from UV-inactivated mock- and WNV-infected SK-N-SH cells significantly increased the production of pro-inflammatory cytokines within 24 h after treatment. While WNV infection per se increased the production of these cytokines only after 48 h. Furthermore, infection of astrocytes with only UV-inactivated WNV did not induce the expression of aforementioned cytokines at any time point. The data expressed are the mean concentration (pg/ml) ± SD of the amount of IL-1β, -6, -8 or TNF-α, secreted in the supernatant and is representative of three independent experiments. *p < 0.05. **p < 0.001.