10.- ÁMBITO SINDICAL: CONOCIMIENTO Y MEDIDAS PREVENTIVAS

Interferons (IFNs) are a group of secreted cytokines which together constitute the interferon induced innate immune response. They have antiviral, anti- proliferative and immunomodulatory functions. They are classified into type I, II and III IFNs depending upon their different amino acid sequences. Type I interferon further constitutes several types of which α and β IFN genes are mainly induced in response to viruses. For this reason instead of type I IFN the term α/ β IFNs is more often used in relation to response to viruses (Randall and Goodbourn 2008). Type I Interferons are derived from intronless genes found on the short arm of human chromosome 9 (9.21p3) and they share the same receptor and have overlapping functions (Brideau- Andersen, Huang et al. 2007, Randall and Goodbourn 2008). Multiple IFNα subtypes have been found in mammalian species. IFNα subtypes have approximately 80% amino acid sequence similarity (Randall and Goodbourn 2008). Type 1 IFNs are produced by a broad range of cells in the body and induce an antiviral state in virus infected cells and surrounding tissue by setting in motion a complex signalling pathway that results in transcription of

69 hundreds of ISGs which have strong antiviral activity (reviewed in Katze, He et al. 2002,Biron 2001).

Type III IFNs were recently described and consist of IFN λ1 (IL-29), λ2 (IL- 28A) and λ3 (IL-28B) (Meager, Visvalingam et al. 2005, Zhou, Hamming et al. 2007). Their receptors have mainly an epithelial distribution (reviewed in Borden, Sen et al. 2007). These are also induced in direct response to viruses and induce a similar group of antiviral genes as IFNs α/ β (Onoguchi, Yoneyama et al. 2007).Type II IFNs are also called IFN-γ and they are not secreted in direct response to viruses but are produced by the subsets of T cells and NK cells in response to viral infection (Randall and Goodbourn 2008). IFNs α/β act on heterodimeric receptors broadly distributed in tissues to activate a signal transduction pathway which results in transcription of ISGs (Valarcher, Furze et al. 2003, reviewed in Galligan, Murooka et al. 2006). These genes are also induced directly by viral infection but the response is less effective compared to that induced by IFNs (reviewed in Randall and Goodbourn 2008). Type III IFNs are known to elicit similar response as that induced by Type I IFNs but their receptors are not widely distributed and their exact role in the antiviral response is under investigation (Meager, Visvalingam et al. 2005, Mennechet and Uze 2006, Zhou, Hamming et al. 2007). Marukian et al, has shown in a recent study that HCV infection of human foetal liver cells led to induction of type III IFN and ISGs suggesting that type III IFN may play a very important role in inducing innate immune response to HCV in liver cells (Marukian, Andrus et al. 2011).

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Figure 1.4: Different types and sub-types of Interferon

1.7.2.1. Induction of interferons (IFNs)

Type 1 interferon can be induced by Toll like receptor (TLR) dependent and TLR independent pathways. TLR dependent pathways are present primarily in cells of the myeloid lineage such as antigen presenting cells (APC) – including plasmacytoid dendritic cells and myeloid dendritic cells. This induction pathway involves TLRs which recognize and bind to the motifs present on different pathogens and leads to a wide variety of inflammatory responses as well as activation of dendritic cells and generation of specific T cell responses (Yonkers, Rodriguez et al. 2007). A number of viral motifs, including double stranded RNA (dsRNA), GU rich single stranded RNA (ssRNA) or endosomal DNA interact with TLRs (specifically TLR3, TLR7 and TLR9 respectively) which are found either extracellularly or intracellularly

71 (reviewed in Randall and Goodbourn 2008). This interaction leads to β

IFNs and other pro-inflammatory cytokines. An alternative pathway for IFN induction involves the TLR-independent pathway that is activated after cytoplasmic molecules interact with viral RNA (chiefly replicating, dsRNA). This pathway may be more ubiquitous than the TLR induction pathways and is probably important in non-myeloid cells (reviewed in Randall and Goodbourn 2008). The RNA recognition molecules are RNA helicases variously named melanoma differentiation-associated gene 5 (mda-5) or retinoid-inducible gene I (RIG-I) (Staeheli and Haller 1987, Zhou, Hassel et al. 1993). dsRNA and 5’triphosphate RNA produced by both DNA and RNA viruses are known to induce IFNs by binding to these intracellular detectors, (Mda5 and RIG-I) (reviewed in Marcus and Sekellick 1977, Marcus 1983) as shown in figure 1.5.

The downstream events that follow the activation of interferon inducers are less well established but activation of NFkβ and IRF-3 have a role in induction of IFN-β (Honda and Taniguchi 2006). Upon receipt of appropriate signals IRF-3 and NFkβ are translocated to the nucleus and bind to the IFN-β promoter leading to induction of IFN-β (Murray 2007). The mechanism of induction of IFN-α is less well defined (Meurs, Chong et al. 1990). It is thought that production of primary IFN i.e. IFN-β may lead to the induction of other transcription factors i.e. IRF-1, IRF-7 and IRF-9 which play a role in the induction of secondary IFN genes i.e. IFN-α genes (Morin, Braganca et al. 2002, Cebulla, Miller et al. 1999). In the context of HCV infection in

72 hepatocytes it is thought that TLR-independent pathway is induced after an HCV specific PAMP is recognised by the host receptor, specifically RIG-I, which leads to the production of IRF-3 which in turn mediates IFN and interferon stimulated genes (ISGs) induction and induces an innate antiviral defence response within days of HCV exposure limiting host cell permissiveness. RIG-I recognises blunted end dsRNA or ssRNA rich in polyuridine runs or bearing 5' triphosphates (Kato, Takahasi et al. 2011). Although RIG-I in Huh-7 cells recognises PAMP and responds to HCV RNA, Huh-7.5 cell line, which is a derivative of Huh-7 cell line, has defective RIG-I signalling which leads to blunted innate immune response, part of the reason why this cell line is so permissive to HCV infection in culture. In one study it was found that restoration of RIG-I signalling rendered Huh-7.5 cells non- permissive to HCV infection (Sumpter, Loo et al. 2005).

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Figure 1.5: Mda5 and RIG-I dependent pathway for IFN-β induction (TLR-independent pathway): Viral dsRNA activates both mda5 and RIG-I and their specific domains recruit Cardif/VISA/MAVS/IPS-1, which in turn recruits signalling molecules which feed into NFкβ and IRF-3 pathway and thus lead to IFN-β production.

1.7.2.2. Interferon induced signalling

Pioneering studies using mutagenised cells that were selected for the loss of the cellular response to interferon have confirmed the pivotal role of the JAK- STAT signal transduction pathway in the cellular response to type I IFN. The JAK-STAT pathway is comprised of Janus kinases (JAK, a tyrosine kinase which is recruited to many receptor types after binding of ligands) and signal transducer and activators of transcription (STATs) (Stark, Kerr et al. 1998, Platanias 2005, Stark 2007). STATs are phosphorylated by JAKs and bind to

74 DNA (Murray 2007). IFNs α/ β binds to their heterodimeric cognate receptor composed of IFNAR1 and IFNAR2. IFNAR1 is linked to tyrosine kinase 2 (Tyk2) while IFNAR2 is linked to JAK1. Following binding of IFN to the receptor the JAKs are phosphorylated and then bind both STAT1 and STAT2 which in turn are phosphorylated by JAK1 and Tyk2 respectively. On phosphorylation STAT1 and STAT2 dimerise and this complex then binds to IRF-9 before translocating into the nucleus where it binds with Interferon stimulated response elements (ISRE) which are present in the promoters of all ISGs. The above signal transduction leads to the activation of ISGs which create the antiviral state in the cell (figure 1.6). Similar signalling is observed in response to IFN II in which IFN-γ binds to its cognate receptors leading to phosphorylation of STAT1 and its homodimerization to form gamma- activated factor (GAF) complex which activates the transcription of IFN-γ responsive genes (reviewed in Randall and Goodbourn 2008, Platanias 2005), Murray 2007, Gunji, Kato et al. 1994).

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Figure 1.6: Signalling pathway activated by IFNs α/ β leading to the production of ISGs: phosphorylated JAKs phosphorylate/dimerise STAT1 and STAT2 into a complex which binds with IRF-9 and translocates into the nucleus to produce ISGs.