Differential regulation of antiviral and proinflammatory cytokines and suppression of Fas-mediated apoptosis by NS1 of H9N2 avian influenza virus in chicken macrophages

Abstract

The NS1 protein is known to suppress immune responses in influenza virus-infected hosts. However, the role of NS1 in apoptosis in infected cells is disputed. In this study, through the use of a mutant A/pheasant/California/2373/1998 (H9N2) avian influenza virus (AIV) with a truncated NS1, we have demonstrated that a functional NS1 protein suppresses the induction of interferons in chicken macrophages. However, NS1 appeared to be irrelevant to the regulation of cytokines interleukin (IL)-1β and IL-6, indicating that distinct mechanisms may be employed in the regulation of antiviral and proinflammatory cytokines in chicken immune cells. Our study also showed that this H9N2 AIV induced apoptosis extrinsically through the Fas/Fas ligand (FasL)-mediated pathway. We found that NS1 suppressed the apoptotic process through suppression of the induction of FasL, but not tumour necrosis factor-α or TNF-related apoptosis-inducing ligand. Furthermore, our data indicated that the disruption of a potential binding site for the p85β subunit of phosphoinositide 3-kinase in the carboxyl terminus of NS1, while having no effect on the regulation of IFN induction, may contribute to the suppression of Fas/FasL-mediated apoptosis. Therefore, suppression of Fas/FasL-mediated apoptosis by NS1 is one of the critical mechanisms necessary to increase infectivity in AIV-infected chicken macrophages.

Roles of the ERK MAPK in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with H9N2 avian influenza virus

“…It was shown previously that some of the TNF family members are upregulated in H9N2-infected HTC cells, triggering the extrinsic apoptotic pathway (Xing et al, 2009). In H9N2-infected cells, 15-and 25-fold increases in FasL transcripts were detected at 8 and 16 h p.i.…”

“…We have observed that the HTC cells underwent aggressive apoptosis in response to H9N2 infection (Xing et al, 2008b(Xing et al, , 2009). However, in HTC cells treated with U0126 (10 mM), we found that apoptosis was more pronounced than in untreated controls at various time points p.i.…”

“…(b) Regulation of proinflammatory cytokines IL-1b, IL-6, IL-8 and IFN-b transcripts in infected or non-infected cells 8 and 16 h p.i. After total RNA was extracted from the cells and reverse transcribed, specific primers were used for a SYBR Green-based real-time RT-PCR as described previously (Xing et al, 2009). The data were normalized to GAPDH reference (*P,0.05; **P.0.05; t test; mean+1.96¾SEM).…”

“…In AIV-induced apoptosis, the Fas/ Fas ligand (FasL)-mediated extrinsic pathway has been identified in H9N2-infected chicken macrophages. Despite this, it is unclear why among three major tumour necrosis factor (TNF) ligands, only FasL is upregulated while TNF-a and the TNF-related apoptosis inducing ligand (TRAIL) are either unchanged or even downregulated in response to AIV infections (Xing et al, 2008b(Xing et al, , 2009). Cytokine and apoptotic responses are presumably key elements in the pathogenesis of AIV infections.…”

Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

“…The full biological consequences of NS1-mediated PI3K activation are far from clear, although an association with regulating lung epithelium ion channel activity has been proposed (11). Early studies also suggested that NS1-activated PI3K contributes to the suppression of cellular apoptosis during virus infection (12)(13)(14)(15), but very recent data now indicate that this may not be the case (16). Thus, the precise role of this important signaling pathway with respect to the influenza A virus replication cycle has yet to be firmly established.…”