Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells
Hung Le, Paul Spearman, Stephen N. Waggoner, Karnail Singh
Hung Le, Paul Spearman, Stephen N. Waggoner, Karnail Singh
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Research Article Immunology Infectious disease

Ebola virus protein VP40 stimulates IL-12– and IL-18–dependent activation of human natural killer cells

Abstract

Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles (VLPs) comprising glycoprotein and matrix protein VP40 provides rapid, NK cell–mediated protection against Ebola challenge. We used Ebola VLPs as the viral surrogates to elucidate the molecular mechanism by which Ebola virus triggers heightened NK cell activity. Incubation of human peripheral blood mononuclear cells with Ebola VLPs or VP40 protein led to increased expression of IFN-γ, TNF-α, granzyme B, and perforin by CD3–CD56+ NK cells, along with increases in degranulation and cytotoxic activity of these cells. Optimal activation required accessory cells like CD14+ myeloid and CD14– cells and triggered increased secretion of numerous inflammatory cytokines. VP40-induced IFN-γ and TNF-α secretion by NK cells was dependent on IL-12 and IL-18 and suppressed by IL-10. In contrast, their increased degranulation was dependent on IL-12 with little influence of IL-18 or IL-10. These results demonstrate that Ebola VP40 stimulates NK cell functions in an IL-12– and IL-18–dependent manner that involves CD14+ and CD14– accessory cells. These potentially novel findings may help in designing improved intervention strategies required to control viral transmission during Ebola outbreaks.

Authors

Hung Le, Paul Spearman, Stephen N. Waggoner, Karnail Singh

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Figure 1

Generation of EBOV GP-VP40 293F stable cell line and characterization of EBOV GP-VP40 VLPs (Ebola VLPs).

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Generation of EBOV GP-VP40 293F stable cell line and characterization of...
(A) EBOV GP-VP40 293F cells were cultured with or without doxycycline (Dox) for 24 hours and cell lysates probed for EBOV GP, EBOV VP40, or actin by Western blotting using specific antibodies. A representative Western blot is shown. (B) Cell lysates and VLPs collected from EBOV GP-VP40 293F cell cultures induced with doxycycline for 40 hours were probed by Western blotting using anti-EBOV GP and anti-EBOV VP40 antibodies. The majority of the EBOV GP and EBOV VP40 were secreted out of the cells as part of the VLPs. (C) Negative stain electron microscopy analysis of EBOV GP-VP40 VLPs showed filamentous particles of different shapes and sizes, covered abundantly with EBOV GP spikes. Scale bars are indicated with black lines (from left to right, 500 nm, 200 nm, and 100 nm). (D) Buoyancy density analysis of EBOV GP-VP40 VLPs. Ebola VLPs fractionated on 20% to 60% sucrose density gradient were analyzed by Western blotting for EBOV GP and EBOV VP40. Buoyance density of each fraction was calculated by measuring its refractive index. Buoyancy densities of the fractions enriched for Ebola VLPs (identified by the relative EBOV GP and VP40 contents) are shown with vertical arrows above those fractions. Numbers at left of blots indicate kilodaltons.