Death Receptor-Mediated Apoptotic Signaling Is Activated in the Brain following Infection with West Nile Virus in the Absence of a Peripheral Immune Response

Abstract

Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS).N euronal apoptosis is an important mechanism of virus-induced pathogenesis within the central nervous system (CNS) (1). During West Nile virus (WNV) encephalitis, the proapoptotic executioner caspase, caspase 3, is activated, and mice lacking caspase 3 have reduced neuronal death and tissue injury following West Nile virus infection (2). Despite the importance of apoptosis in WNV pathogenesis, the exact pathways involved in triggering apoptotic cell death in the CNS have not yet been defined.Activation of initiator caspases 8 (3, 4) and 9 (3) occurs in cultured neuronal cells infected with WNV, and inhibition of these initiator caspases leads to reduced cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase (PARP) (3, 4). These studies indicate that both extrinsic and intrinsic apoptotic signaling pathways are activated in vitro following WNV infection and are consistent with in vitro studies demonstrating that mitochondrial apoptotic signaling protein Bax is upregulated in neuronal cells following WNV infection (5) and that cytochrome c is released from the mitochondria (3). However, it remains to be seen whether the same apoptotic pathways are also activated in the intact brain during WNV encephalitis.Innate and adaptive immune responses also influence WNV pathogenesis within the CNS. Toll-like receptors 3 and 7 (TLR3 and TLR7) and the cytoplasmic proteins encoded by retinoic acidinducible gene I (RIG-I) and melanoma-differentiation-associated gene 5 (MDA5) are important for detection of WNV within the CNS, and defects in these, or additional, components of the interferon (IFN) response show enhanced viral burden and increased lethality (6). Although IFN restricts infection, pathogenic WNV strains attenuate IFN function at several steps of the induction and signaling cascade, allowing the virus to establish infection (6). Studies performed in mice (7-11) and humans (12-14) have also highlighted the role of CD8 ϩ T cells and the associated importance of pathways involving Fas ligand (10) and tumor necrosis factor (TNF)-related apoptosis-inducing factor ligand (TRAIL) (15) effector mechanisms in containing WNV CNS infection.In this report, we demonstrate that genes involved in death receptor (DR) apoptotic signaling are upregulated in the mouse brain following WNV infection. We also show for the first time that the activity of the DR-associated initiator caspase, caspase 8, is increased in the brain following WNV infection. WNV-induced activation of caspase 8 in the CNS is associated with cleavage of the proapoptotic Bcl-2 family protein Bid and with activation of caspase 9, suggesting that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of WNV-infected ex vivo brain slice cultures (BSC), a novel model of WNV encephalitis, revealed that inhibition of caspase 8 decreased virus-induced activation of caspase 3 an...

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