A novel immunomodulatory mechanism of ribavirin in suppressing natural killer cell function
Graphical abstract
Introduction
Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide), a synthetic nucleoside analog, is a broad-spectrum inhibitor of RNA and DNA viruses that is currently licensed for the treatment of severe respiratory syncytial virus (RSV) disease [1] and chronic hepatitis C virus (HCV) infection [2], [3]. Anti-viral properties of ribavirin were ascribed to inhibition of inosine monophosphate dehydrogenase, interference with viral RNA capping reactions, inhibition of viral polymerase and induction of error catastrophe resulting from the accumulation of lethal mutations in viral genome [4].
Apart from direct anti-viral effects, ribavirin was reported to have immunomodulatory effects on different constituents of the immune system. Ribavirin induced a switch in T-helper (Th) cell phenotype from type 2 to type 1 [5]. In patients with chronic HCV infection, ribavirin was shown to exert anti-inflammatory effect by reducing the blood levels of interferon (IFN)-γ and the expression of IFN-γ by activated T cells [6], thus thereby helping to suppress IFN-γ-driven T cell activation and liver damage [6]. In macrophages, ribavirin inhibited the induction of pro-inflammatory cytokines like interleukin (IL)-1β and tumor necrosis factor (TNF)-α[7]. In dendritic cells, ribavirin suppressed the production of TNF-α, IL-10, and IL-12(p70) [8]. In murine natural killer (NK) cells, ribavirin inhibited IFN-γ production in a dose-dependent manner [9].
NK cells provide the first line of defense and substantially contribute to the elimination of virus-infected cells as well as antitumor immune response [10]. Moreover, IL-15 is known to play a pivotal role in NK cell development in vivo as well as in NK cell activation and proliferation and is secreted in response to infectious pathogens to mediate NK cytotoxicity [11]. IL-15 and IL-2 receptor gamma (IL-2Rγ) genes, both involved in lymphocyte activation and signaling, were reported to be down-regulated by ribavirin in RSV-infected human A549 pulmonary type II epithelial cells [12]. In NK cells, the IL-15 receptor includes IL-15Rα, IL-2/15Rβ and γ chain subunits (β and γc subunits are shared with IL-2) [11]. As IL-2 and IL-15 share common signaling components, most evidence suggests that the interaction of IL-2 or IL-15 with their respective receptor complex in various cell types leads to a series of similar if not identical signaling events [13]. IL-15Rβ is associated with Janus kinase (Jak)-1 and the γc is associated with Jak-3, resulting in signal transducer and activator of transcription (STAT)-3 and STAT-5 phosphorylation respectively, following ligation with IL-15 [13]. IL-15 also activates the phosphorylation of STAT-1 [14], [15]. In addition to signaling via IL-15/IL-15R, NK cell activation is also tightly regulated by a delicate balance between signaling through inhibitory [killer immunoglobulin-like receptors (KIR), CD94-NK group 2, member A (NKG2A)] and activating receptors [natural cytotoxic receptors (NCRs-NKp30, NKp44 and NKp46), NK group 2, member D (NKG2D) and DNAX accessory molecule-1 (DNAM-1)] [10]. The expressions of these receptors are up-regulated upon treatment with IL-15 [16], [17]. Inhibitory receptors consist of a single polypeptide containing cytoplasmic immunoreceptor tyrosine-based inhibitory motifs that recruit tyrosine phosphatases and abort signaling. On the other hand, activating receptors typically couple to signaling adaptors that contain either an immunoreceptor tyrosine-based activating motif (ITAM, such as DAP12, FcRγ or CD3ζ) or ‘YXNM’ motif, where ‘X’ is any amino acid (such as the signaling adaptor DAP10) through interactions specified by their transmembrane regions [18]. Interestingly, signaling through DAP10, an adaptor that transmits important signals for NKG2D [19], was shown to be coupled to IL-15R signaling pathway [20]. Here, DAP10 was shown to specifically bind β- and γ-chains of IL15R [20].
In this report, we have studied the direct effect of ribavirin on effector functions (including cytolytic activity and IFN-γ production) of IL-15-activated human NK cells as well as molecular mechanisms by which ribavirin influences NK cell functions. We observed that ribavirin exerts immunomodulatory activities on NK cells by down-regulating the expression of IL-15Rβ and γ and thus inhibiting downstream events involved in NK cell signaling (IL-15-activation of Jak/STAT pathway) and degranulation [extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) signaling pathways].
Section snippets
Reagents and monoclonal antibodies
Ribavirin (Virazole®) was obtained from Valeant Pharmaceuticals Germany GmbH (Eschborn, Germany), DETA NONO-ate and tetrahydrobiopterin were purchased from Sigma–Aldrich (Taufkirchen, Germany), recombinant human IL-15 was from Cell Concepts (Umkirch, Germany). The following phycoerythrin (PE)-conjugated anti-human monoclonal antibodies (mAbs) were used: NKp30, NKp44, NKp46, NKG2D, DNAM-1, KIR- KIR2DL2/DL3 and KIR3DL1, all from Miltenyi Biotec (Bergisch Gladbach, Germany), NKG2A, IL-2Rβ and
Effect of RBV on viability of NK cells
We previously demonstrated that ribavirin neither induced a block in the cell cycle progression nor exerted cytotoxic effects in primary HUVEC at concentrations up to 20 μg/ml and 100 μg/ml respectively [25]. We therefore studied effects of ribavirin at concentrations ranging from 5 μg/ml to 80 μg/ml on viability of IL-15-activated NK cells. For this purpose, IL-15-activated NK cells were treated for 5 days with ribavirin. Dead cells were identified with fractional DNA content (“sub-G1 fraction”).
Discussion
The data presented in this study show for the first time that ribavirin inhibits human NK cell function. Moreover, evidence is presented about the molecular mechanisms that underlie the immunomodulatory activities of ribavirin in NK cells. NK cells mediate host cell defenses by direct lysis of target cells and/or by production of IFN-γ[30]. Suppression of NK-cell cytotoxicity may be associated with the inhibition of IFN-γ production [26], [27]. Our results show that while ribavirin had no
Acknowledgement
The authors gratefully acknowledge the support by the organization “Hilfe für krebskranke Kinder, Frankfurt/Main e.V.”, by the foundation “Frankfurter Stiftung für krebskranke Kinder.”, and by the European Commission-funded Co-operative Research and Specific Targeted Research Projects; COOP-CT-2004, Contract Nr. 512864 and LSHB-CT-2004, Contract Nr. 512054 respectively.
The authors wish to thank Rosy Schmidt for her excellent technical assistance.
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