Alpinone: A positive regulator molecule of immune antiviral response in Atlantic salmon kidney cells

Highlights

• Mechanisms underlying the antiviral effects of Alpinone, a flavonoid obtained from H. huascoense, are reported.

• Alpinone increased the transcriptional expression of IFNa and Mx in salmon kidney cells.

• Alpinone upregulated the RIG-I and MDA5 cytosolic receptors and genes of the downstream pathway in salmon cells.

• Mechanisms of transcriptional activation of IFNa require the kinase activity of TBK1 and IKKε.

Abstract

Alpinone is a flavonoid obtained from the resinous exudate of Heliotropium huascoense. This flavonoid shows antiviral activity against the infectious salmon anemia virus (ISAV), which causes severe disease in farmed Atlantic salmon. Here, we aim to elucidate mechanisms underlying the antiviral effects of the flavonoid. In this regard, we evaluated whether Alpinone can act upregulating the pattern-recognition receptor genes, i.e., the RIG-I-like, TLR3, and TLR9 genes, and the genes of the downstream signaling pathways. Transcriptional expression of the genes was analyzed using real-time PCR after 8, 24, and 48 h treatment of salmon kidney adherent cells with 15 μg/mL of Alpinone. First, we showed that Alpinone induced IFNa expression in the kidney adherent cells, indicating that this type of salmon cells is in part responsible for the effects previously reported in vivo. Upregulation of the IFN-induced myxovirus resistance (Mx) gene was also observed in the head kidney cells in response to the treatment. Overexpression reached a maximum level at 24 h post-treatment. Interestingly, Alpinone also induced upregulation of the cytosolic receptors of ssRNA, named Retinoic acid-inducible gene I (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5), but there were no effects on the transcriptional expression of the TLR3 and TLR9 endosomal receptors. In addition, Alpinone upregulated the expression of genes encoding the main components of the RIG-I/MDA5 signaling pathways, such as the mitochondrial antiviral-signaling protein (MAVS), TNF Receptor Associated Factor 3 (TRAF3), TANK-binding kinase 1 (TBK1), I-kappaB kinase ε (IKKε), the transcription factors IRF-3, and IRF7. The increased expression of all these genes is consistent with the upregulation of IFNa and Mx mRNAs. Because BX795 completely prevents Alpinone-dependent upregulation of IFNa and IRF3, the flavonoid targets seem to be upstream of the kinases TBK1 and IKKε. Altogether, this study contributes to elucidating the mechanisms involved in Alpinone antiviral activity in fish. Alpinone can be used to counteract virus mechanisms of evasion where the onset of interferon-mediated response is prevented or delayed.

Introduction

Viral diseases have severely harmed aquaculture worldwide, causing high fish mortality rates, economic losses, and environmental damage. Among these viruses, some can cause persistent viral infections like the Infectious Pancreatic Necrosis Virus (IPNV) (Kibenge, 2019) and some are emerging pathogens like the Salmon Infectious Anemia Virus (ISAV), Piscine Orthoreovirus (PRV) and viral hemorrhagic septicemia virus (VHSV) (Kibenge, 2019). Although there is evidence that fish have functional mechanisms of adaptive immunity (Magadan et al., 2015), and there are many vaccines against fish viruses designed to improve these types of immune responses, the rate of fish protection against infectious diseases is quite limited as they fail to avoid or resolve infection (Dahle and Jørgensen., 2019).

In the absence of tools capable of effectively stimulate the immune response and disease protection in fish, it is important to identify new bioactive compounds to induce a response able to control fish viral diseases. Indeed, nature is a source of a great variety of compounds with antiviral activity. An example of these are flavonoids, a group of compounds with different phenolic structures found in many plant roots, leaves, flowers and fruits (Panche et al., 2016). Flavonoids have a basic structure represented by a C6–C3–C6 carbon skeleton and are divided into subclasses such as flavonols, flavones, flavanones, anthocyanidins, flavanols and isoflavones (Daglia, 2012). This group of compounds has a broad spectrum of health-promoting effects like anti-viral, antioxidant, anti-allergic, anti-carcinogenic and anti-inflammatory activities (Kumar and Pandey., 2013), and are crucial components in a variety of nutraceutical, pharmaceutical, and medicinal formulations (Panche et al., 2016). Particularly, antiviral activity is useful for a variety of pathogens sharing molecular replication or infection mechanisms. For example, epigallocatechin gallate has activity against human viral infections caused by adenovirus type 2 and enterovirus 71 (Weber et al., 2003; Ho et al., 2009), fisetin exhibits antiviral activity against fish viruses like infectious hematopoietic necrosis virus (IHNV) and VHSV (Kang et al., 2012), and the flavonol isorhamnetin shows strong antiviral effects against the influenza virus (Dayem et al., 2015) while quercetin and daidzein has antiviral activity against murine norovirus (MNoV) (Seo and Choi., 2017). We also reported that flavonoid 7-O-methyleriodictyol has antiviral properties against the ISAV (Modak et al., 2012).

Bioactive natural compounds obtained from species of the Heliotropium genus, section Cochranea were isolated and characterized in our laboratory (Torres et al., 1996). These plants grow in arid zones and produce a resinous exudate covering leaves and stems as a defense mechanism (Torres et al., 1996). The resin is formed mainly by flavonoids and aromatic geranyl derivatives in minor quantities (Luebert, 2013). One of the compounds obtained from the resin is Alpinone (Fig. 1), a flavanonol with hydroxyl substituents at positions 3 and 5, and methoxylated at position 7 (Urzúa et al., 2000). There is little information about the potential biological properties of Alpinone, and the only known ethnomedicinal use of the leaves of this species is for vaginal washes reported in Pichasca, Limarí Province, Chile (Luebert, 2013). However, in a recent study, Espinoza reported that Alpinone increases the proliferation of a murine macrophage-like cell line (RAW 264.7 cells) and induces the formation of intracellular reactive oxygen species (ROS), which suggests the activation of the macrophage cell line (Espinoza et al., 2017). Other studies reported that Alpinone increases the transcriptional expression of the antiviral cytokine IFNa, the proinflammatory cytokines TNF-α, IL-1 and IFN-γ, and the anti-inflammatory cytokine TGF-β1 in the SHK-1 cell line and Atlantic salmon (Valenzuela et al., 2018). In addition, Alpinone induced the secretion of antiviral compounds in SHK-1 cells, which effectively inhibit the replication of the Infectious Salmon Anemia Virus (strain ISAV 752_09) (Valenzuela et al., 2018).

In fish, as in mammals, an important part of the antiviral immunity relies on the type I interferon (Dahle and Jørgensen., 2019). also (Secombes and Wang., 2012). PPRs and components of the activation pathways are conserved throughout evolution in vertebrates and have been found in salmonid fish (Collet, 2014). The most studied and characterized PRRs are toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), some of them capable to detect viral-type RNA. TLRs are a family of transmembrane receptors with a N-terminal ligand recognition domain, a single transmembrane helix, and a C-terminal cytoplasmic signaling domain (Botos et al., 2011). In Atlantic salmon, the head kidney and spleen express TLRs, and CpG DNA, Poly I:C and imiquimod (agonists of TRL9, TLR3 and TLR7, respectively) strongly upregulated inflammatory cytokines involved in antiviral immunity (Arnemo et al., 2014). The RLRs are located in the cytoplasm and activate intracellular signaling cascades during viral infection. In mammals, RLRs are a family of receptors that include retinoic acid-inducible gene I (RIG-I), laboratory of genetics and physiology 2 (LGP2), and melanoma differentiation-associated gene 5 (MDA5). In response to viral infection, RIG-I and MDA5 bind viral ssRNA and trigger the production of proinflammatory cytokines and type I interferon (IFN–I) through a common adapter called mitochondrial antiviral signaling protein (MAVS), also known as IPS-1. This signals two activation pathways; one activates the IKK complex, resulting in a pro-inflammatory response, while the other activates the TANK/IKKγ/ε/TBK1 complex through TRAF3. This facilitates the phosphorylation of interferon regulatory factor (IRF)-3 and IRF7, which are key transcription factors for IFN production (Liu et al., 2017). The role of TBK1 and IKKε in the production of IFN-I in macrophages stimulated with polyinosinic:polycytidylic acid (poly I:C) has been pharmacologically demonstrated using BX795, a potent and relatively specific inhibitor of these two kinases. BX795 suppressed pTBK1 signaling by inhibiting its ability to phosphorylate IRF3 (Clark et al., 2009). The secreted IFN-I binds to their receptors and initiates the downstream cascade of signaling, activating the expression of interferon-stimulated genes (ISGs). Myxovirus resistance (Mx) genes are the most extensively studied ISGs involved in viral inhibition (Raftery and Stevenson., 2017). As in mammals, Mx genes in fish are inducible by the double-stranded RNA (dsRNA) such as poly I:C (Strandskog et al., 2011).

The orthologous genes RIG-I, MDA5 and LGP2 have been found in several teleost fish species (Chen et al., 2017). In rainbow trout, MDA5 and LGP2 are upregulated with poly(I:C) in response to infection by VHSV and salmon alphavirus (SAV) (Chang et al., 2011). RIG-I and MDA5 from Atlantic salmon have also been cloned (Biacchesi et al., 2009; Lauksund et al., 2009; Nerbøvik et al., 2017), and the expression of these genes have been up-regulated with SAV subtype 3 in salmon TO cells (Chang et al., 2011; Xu et al., 2016). Interestingly, synthetic dsRNA poly(I:C) has been shown to bind recombinant MDA5 and LGP2 proteins in vitro in rainbow trout (Xu et al., 2016). RIG-I and MDA5 recruit MAVS in the signaling pathway activated after recognition of viral PAMPs. Orthologous genes to MAVS have been reported in teleost fish and have been cloned from several species, including Atlantic salmon (Lauksund et al., 2009). MAVS exhibits a conserved function in the RIG-I-like receptor-mediated signaling pathway, containing protein domains like mammals. MAVS activates the transcription factor IRF3 through signaling molecules such as TRAF3, which recruits two kinases: TBK1 and IKKϵ. These kinases activate the IRF pathway through direct phosphorylation of IRF3 and IRF7 (Hemmi et al., 2004). Once it has been phosphorylated, IRF3 is translocated to the nucleus for induction of type I IFN and the transcription of some interferon-stimulated genes (ISGs) (Chen et al., 2017).

Here, we aim to elucidate the mechanisms underlying the antiviral effects of Alpinone observed in fish. Since Alpinone increases the expression of IFNa in Atlantic salmon and induces protection against two RNA viruses, i.e., ISAV (Valenzuela et al., 2018) and IPNV (not published), we hypothesized that Alpinone could also regulate the expression of upstream like those encoding the pattern recognition receptors and the signal cascade trigger by viral RNA stimulation. In this regard, we first seek to verify its ability to induce transcription of Mx, usually observed in conjunction with IFNa, in head kidney cells of Atlantic salmon. In addition, we assess the Alpinone effects on the transcriptional expression of the pattern-recognition receptors, i.e., the RIG-I-like, TLR3, and TLR9 receptors and the genes of the associated signaling pathways.