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Interferon alpha bioactivity critically depends on Scavenger receptor class B type I function

ABSTRACT Scavenger receptor class B type I (SR-B1) binds pathogen-associated molecular patterns participating in the regulation of the inflammatory reaction but there is no information regarding potential interactions between SR-B1 and the interferon system. Herein, we report that SR-B1 ligands strongly regulate the transcriptional response to interferon α (IFNα) and enhance its antiviral and antitumor activity. This effect was mediated by the activation of TLR2 and TLR4 as it was annulled by the addition of anti-TLR2 or anti-TLR4 blocking antibodies. In vivo, we maximized the antitumor activity of IFNα co-expressing in the liver a SR-B1 ligand and IFNα by adeno-associated viruses. This gene therapy strategy eradicated liver metastases from colon cancer with reduced toxicity. On the other hand, genetic and pharmacological inhibition of SR-B1 blocks the clathrin-dependent interferon receptor recycling pathway with a concomitant reduction in IFNα signaling and bioactivity. This effect can be applied to enhance cancer immunotherapy with oncolytic viruses. Indeed, SR-B1 antagonists facilitate replication of oncolytic viruses amplifying their tumoricidal potential. In conclusion, SR-B1 agonists behave as IFNα enhancers while SR-B1 inhibitors dampen IFNα activity. These results demonstrate that SR-B1 is a suitable pharmacology target to enhance cancer immunotherapy based on IFNα and oncolytic viruses.


Introduction
Scavenger receptor class B type I (SR-B1) is a transmembrane glycoprotein known to bind and internalize a broad variety of ligands. 1,2 Most of the SR-B1 ligands form micellar particles containing either negatively charged phospholipids or anionic class A amphipathic a-helixes. 3 Among the best characterized SR-B1 ligands are lipoproteins, particularly high density lipoproteins (HDLs), and therefore the major protein component of HDL: apolipoprotein A-1 (Apo A-1). SR-B1 is involved in cholesterol homeostasis both at cellular and systemic levels via the selective uptake of cholesteryl esters and other lipids from HDLs and by promoting bidirectional movements of unesterified cholesterol. 4 More recently, further studies have highlighted that SR-B1 also plays a role in innate immunity by binding pathogen-associated molecular patterns or damage-associated molecular patterns and internalizing pathogens. 5 SR-B1 has been shown to modulate the macrophage response to lipopolysaccharide (LPS) response, to mediate LPS clearance by hepatocytes, to attenuate neutrophil activation and to modulate stress-induced glucocorticoid production by adrenal glands. 6-8 SR-B1 deficiency in mice is associated with enhanced production of proinflammatory cytokines and autoimmunity. 9 Apo A-I mimetic peptides such as L37pA attenuate inflammation in models of cardiac ischemia/reperfusion injury and sepsis. 10,11 However, some SR-B1 ligands such as serum amyloid A (SAA), glycated or oxidated Apo A-I or dysfunctional HDLs have been shown to promote inflammation. [12][13][14] Thus, SR-B1 plays a dual role in inflammation and may represent a novel target to design new immunotherapies against cancer.
Interferon a (IFNa) is an essential player in innate immunity 15 and links innate immune responses by acting as a signal-3 cytokine in the immunological synapse. 16 It is an essential cytokine in anti-viral and antitumor immunity but deregulation of its production may promote the development of autoimmune disorders such as lupus, thyroiditis, diabetes mellitus type I, autoimmune dermatitis, psoriasis and Sjogren's syndrome. 17 The potent immunostimulatory, antiviral and antineoplastic activities of pharmacological doses of the recombinant IFNa led to the clinical use of this cytokine for the treatment of viral infections and several type of cancers such as hairy cell leukemia, malignant melanoma, AIDS-related Kaposi's sarcoma and follicular non-Hodgkin's lymphoma. 18 . In spite of a large clinical experience, systemic side effects associated to the use of high pharmacological doses have limited the clinical utility of IFNa and is being replaced by novel targeted small molecules that are better tolerated. 19,20 Drugs that potentiate IFNa activity, but limit its toxic effects could take advantage of the huge antiviral and antitumor properties of IFNa. IFNa also plays an important role on the antitumor efficacy of gene therapy-based oncolytic viruses. On one hand, IFNa is induced upon oncolytic virus injection, limiting the replication of the vector, but on the other hand this cytokine is essential for inducing an effector antitumor immune response. Thus, transient blockade of this pathway may enhance viral replication within the tumor without affecting the induction of the antitumor immune responses. 21,22 Newcastle disease virus (NDV) is a promising oncolytic vector that can be engineered to over-express pro-inflammatory cytokines. These armed NDVs are able to achieve eradication of tumors in several preclinical models. 23 However, NDV are potent inductors of IFNa and strategies to block IFNa can benefit NDV-based antitumor therapies.
In this study, we found that SR-B1 is a critical modulator of IFNa bioactivity. SR-B1 agonists or antagonists may be used as immunomodulators with different therapeutic applications.

IFNa activity is modulated by L37pA: In vitro studies
It has been reported that the ApoA-I mimetic peptide L37pA binds SR-B1 and mediates anti-inflammatory effects. 24 We investigated whether this peptide influenced IFNa bioactivity in a mouse L929 fibroblast cell line. We found that in the presence of L37pA significantly lower doses of IFNa were required to protect fibroblasts from a lethal challenge with encephalomyocarditis virus (EMCV) (Fig. 1A). Similarly, the cytotoxic effect on mouse fibroblasts induced by higher IFNa doses was greatly amplified by L37pA (Fig. 1B). In isolation, this peptide lacked any antiviral or cytotoxic activity (data not shown). The observed synergy of the combination IFNa plus L37pA was abrogated by mutating the peptide sequence to disrupt the amphipathic a-helix structure ( Fig. 1A and B).
Microarray analysis of L929 cells treated with IFNa, with or without L37pA showed upregulation (fold change > 0.69) of 196 transcripts related to inflammation and IFNa response (data not shown). Among those with the highest fold change, eight were validated by qPCR. These included Nos2 (inducible nitric oxide synthase), Fas (a death receptor molecule mediating pro-apoptotic effects), Cxcl11 (also called interferongamma-inducible protein 9 involved in the attraction of activated T cells), Jak2 (a non-receptor tyrosine kinase involved in signaling of type II cytokine receptors including interferon receptors), Gbp2 (Interferon-induced guanylate-binding protein 2), Ifit1 (interferon-induced antiviral RNA-binding protein which inhibits the expression of viral mRNA) and Apobec3 (a cytidine deaminase with important functions in innate antiviral immunity). Fas and Cxcl11 showed the highest upregulation upon combined treatment and were selected as a gene signature of the IFNa/L37pA synergy in subsequent experiments (Fig. 1C).
The synergy between IFNa and L37pA is not exclusive to L929, as Cxcl11 and Fas were also induced by the combined treatment in other mouse cell lines, such as 3T3 fibroblasts and CT-26 murine colon cancer (Fig. S1A). More interestingly, the synergy was also observed in human cell lines such as human monocytes, hepatic HepaRG and fibroblast BJ cells (Fig. S1B).
To address whether the lipidated or delipidated status of endogenous SR-B1 ligands might determine their ability to enhance IFNa response, we analyzed the effect of HDLs, delipidated ApoA-I and SAA in L929 cells. HDLs combined with IFNa failed to upregulate Cxcl11 and Fas transcripts but delipidated ApoA-I and SAA synergized with IFNa in the induction of both transcripts ( Fig. 2A) indicating that the lipid composition of SR-B1 ligands is critical for IFNa potentiation. Finally, we evaluated the activity of Toll-like receptor (TLR) ligands in this experimental setting. Of note, TLR ligands such as Alzheimer amyloid b peptide (TLR2 and TLR4); LL37 (TLR9); phenol-soluble modulin 1 (TLR2) and LPS (TLR4 ligand) failed to upregulate Cxcl11 in combination with IFNa ( Fig. 2B) although the latter was able to enhance Fas expression (Fig. 2B).
TLR2 and TLR4 mediate the enhancement of IFNa bioactivity induced by SR-B1 agonists As members of the scavenger receptor class B family, such as CD36, have been shown to form complexes with other transmembrane proteins including TLR, we studied the role of the latter molecules in the amplification of IFNa response when cells were co-stimulated with this cytokine plus L37pA. We found that blocking antibodies to TLR-2 or TLR-4 inhibited the effect of IFNa/L37pA on their target genes (Fig. 2C). Moreover, these blocking antibodies also abrogated the activity of L37pA on IFNa-induced cytotoxicity (Fig. 2D). Both TLR can interact forming heterodimers 25,26 and indeed we detected these complexes in L929 cells used in this study (Fig. S2A). The mitogen-activated protein kinase pathway is a common signaling pathway activated by all TLRs. 27 L37pA was able to induce the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 (Fig. S2B). Our data implicate TLRs as mediators of the enhancing effects of L37pA on IFNa.

Activity of the combination of IFNa and L37pA: In vivo studies
We then assessed whether SR-B1 ligation may affect IFNa in vivo. To this end, we transduced the liver of C57BL/6 mice with an adeno-associated viral vector (AAV) encoding IFNa (AAV-IFN) plus AAV expressing luciferase (AAV-Luc) or the peptide L37pA (AAV-L37) or only with AAV-Luc (at a dose equal to the combination therapy). At week 2, after vector administration IFNa serum levels were similar in mice given AAV-IFN/AAV-Luc and in those treated with AAV-IFN/ AAV-L37 (data not shown). At week 4, however, while in the former group serum IFNa continued to rise until it reached toxic levels causing lethal pancytopenia ( Fig. 3A and B), in mice that received AAV-IFN/AAV-L37 serum IFNa dropped markedly as result of the almost complete disappearance of viral genomes from the liver (Fig. 3C). Consistently, at this time point no hematological toxicity was observed in the last group of mice which showed 100% long-term survival in sharp contrast with the 100% mortality by week 8 in all mice given AAV-IFN/AAV-Luc (Fig. 3B, C and E). Despite the elimination of AAV genomes from the liver in the AAV-IFN/AAV-L37 group, the amount of interferon-sensitive genes (ISG) transcripts were higher than both AAV-IFN/AAV-Luc and AAV-Luc groups (Fig. 3D). AAV clearance occurred in the presence of normal serum transaminases (Fig. S3) and in animals lacking T cells, B cells or NK cells ( Fig. 3E and F) supporting a noncytopathic cell autonomous mechanism for viral elimination. The high dose of AAVs used transduces more than 90% hepatocytes and a cytopathic elimination of the infected hepatocytes would have been reflected in elevated serum levels of the transaminases. In order to test whether the self-limited system formed by the combination of AAV-IFN and AAV-L37 could be used to treat liver metastasis, we inoculated MC38 colon cancer cells in the liver of mice. Four days later, the AAVs were administered and 21 d after tumor inoculation, the presence of hepatic tumors was assessed by echography (Fig. 4A). The AAV-IFN in combination with AAV-Luc or AAV-L37 displayed a potent antitumor effect eradicating tumors in more than 80% of mice (Fig. 4B). However, in the case of AAV-IFN treatment, the high levels of IFNa expressed to achieve such a potent antitumor effect were toxic and led to the death of all mice. Thus, the combined treatment was the only option that successfully eradicated tumors (Fig. 4C).

IFNa activity is abolished by disrupting SR-B1
To determine whether IFNa bioactivity was dependent on functional SR-B1, we knocked-down this molecule using specific siRNA (Fig. 5A). We found that IFNa-mediated upregulation of the ISGs Isg15, Usp18 and 2 0 5 0 OAS was significantly impaired in SR-B1 deficient cells (Fig. 5B). Further supporting the role of SR-B1 on IFNa bioactivity, we found that mouse embryonic fibroblasts (MEFs) from SR-B1 knockout mice required higher doses of IFNa to achieve the same protection against EMCV than wild type cells to a lethal challenge with this virus (Fig. S4A) and that the induction of ISGs such as Pbms8, Tap2, Irf1 and Isg15 was dampened in SR-B1 deficient cells (Fig. S4B).We then tested if known SR-B1 antagonists also affected cell response to IFNa. We used three different SR-B1 chemical inhibitors: block lipid transport 1 (BLT-1) 28 , ITX-5061 (ITX) 29 and glyburide 30 and we found that upregulation of Isg15, Usp18 and 2 0 5 0 OAS by IFNa was blunted in the cells treated with all these SR-B1 antagonists (Fig. 5C). A doseresponse curve with BTL-1 showed that concentrations in the order of 1 mM were able to dampen IFNa activity (Fig. S5A). The impact of SR-B1 inhibitors on IFNa response was similar in L929 mouse fibroblasts and in other murine cell lines such as MC38 colon cancer cells and RAW macrophages (Fig. S5B). Moreover, BLT-1 inhibited ISGs induction by IFNa in HepaRG human cells and human monocytes (Fig. S5C).
Notably, we observed that the impaired response to IFNa in BLT-1 treated cells was associated with a decreased display of IFNAR1 on the cell membrane (Fig. 5D) and with reduced activation of STAT-1 upon cell stimulation with IFNa (Fig. 5E). Accordingly, BLT-1-treated cells required higher amounts of IFNa to survive a lethal inoculum of EMCV and were less sensitive to the cytotoxic effects of high doses of IFNa ( Fig. 5F and G). Finally, we analyzed whether other interferons were blocked by BLT-1. The activity of other type I IFN such as IFNb was affected by BLT-1 but this compound did not modify the activity of IFNg, a type II IFN ( Fig. S5D and E).

SR-B1 inhibitors block clathrin-dependent endocytosis
It has been shown that that IFNa signaling requires clathrin-dependent IFNAR endocytosis and recycling. [31][32][33] We confirmed that chlorpromazine (CPZ), a clathrin inhibitor, similarly to BTL-1, reduced ISGs upregulation in IFNatreated L929 cells (Fig. 6A) and decreased the expression of IFNAR1 on the cell membrane at 37 C. At 4 C, neither CPZ nor BLT-1 affected the IFNAR1 levels on the cell membrane (Fig. 6B). To investigate whether BLT-1 could block clathrin-endocytosis, we analyzed its effect on the internalization of fluorescent-labeled bovine serum albumin (BSA). We found that both BLT-1 and CPZ reduced the internalization of this molecule by clathrin at 37 C (Fig. 6C). Moreover, both compounds attenuated the internalization of the SR-B1 ligand L37pA at 37 C (Fig. 6D). At 4 C, neither BSA nor L37pA were internalized. In addition, downregulation of SR-B1 by siRNA also decreased internalization of L37pA (Fig. S6A). Taken together, these data indicate that SR-B1 is essential for IFNAR internalization and recycling.

Blockade of SR-B1 function promotes Newcastle disease virus infection
Next, we analyzed whether SR-B1 function was required for endogenous IFNa activity. To this end, we infected L929 mouse fibroblasts with NDV encoding green fluorescent protein (NDV-GFP) and transgene expression was evaluated by flow cytometry. We found that BLT-1 increased the percentage of GFP-expressing cells and the intensity of fluorescence in the infected cells (Fig. 7A). In line with these results, NDV infection induced the transcription of several ISGs but blockade of SR-B1 by BLT-1 profoundly altered this process. With this treatment, 6 h after viral infection, we detected reduced transcription of ISGs. 24 h after NDV infection, cells treated or not with BLT-1 expressed high levels of the ISGs (Fig. 7B). Thus, the effect of BLT-1 on IFNa signaling is transient. To confirm that SR-B1 was involved in this process, we infected murine L929 fibroblasts with NDV-GFP after silencing SR-B1 with specific siRNA. We noted increased fluorescence intensity and an elevated proportion of GFP-positive cells (Fig. S6B) in association with blunted ISGs expression in SR-B1 deficient cells (Fig. S6C).
We further studied whether SR-B1 inhibitors might foster intratumoral replication of oncolytic viruses. To this purpose, we inoculated CT-26 or MC-38 colon cancer cells subcutaneously and when mean tumor diameter was superior to 5 mm, we performed intratumoral injection of NDV encoding GFP (NDV-GFP) to CT-26 tumor-bearing mice or NDV encoding interleukin 2 (NDV-IL2) to MC-38 tumor-bearing mice with or without BLT-1. These treatments were repeated 72 h later. We found that BLT-1CNDV-GFP or NDV-IL2 achieved a stronger antitumor effect than the oncolytic virus alone (Fig. S7).

Discussion
In this study, we describe a novel role of SR-B1 agonist and antagonist modulating IFNa biology. Tissue fibroblasts and stromal cells are important sentinels to initiate tissue-adapted immune responses. Upon direct cell damage or recognition of pathogen-associated molecular patterns or damage-associated molecular patterns, these tissue-resident cells release antimicrobial peptides, chemokines or cytokines that modulate the adaptive immune response initiated by tissue-resident dendritic cells. 34 Thus, the identification of the mechanism by nonimmune cells is of great interest to understand the activation of the immune system and the mechanisms that trigger immunemediated disorders.
SR-B1 is a promiscuous receptor that binds and internalizes amphipathic molecules. TLR4 and TLR9 ligands are able to bind to SR-B1. In these cases, SR-B1 acts as a scavenger reducing the bioavailability of the TLR ligands and thus limiting the immune response. 35,36 Here, we show that SR-B1 can present certain ligands to TLR2 and TLR4. Heterodimers of these molecules have been reported to be activated by ethanol 25 and hemoglobin. 26 In our study, we used the Apo A-1 mimetic peptide L37pA as a model of the SR-B1 ligand. This peptide is a tandem repetition of a model amphipatic a-helix of 18 aminoacids linked by a proline. 37 SR-B1 is able to bind and internalize L37pA 24 and we confirmed the role of SR-B1 in L37pA internalization using siRNA in L929. It was likely that endogenous ligands were able to trigger the inflammatory response initiated by SR-B1. Indeed, the two most important endogenous ligands of SR-B1, SAA and ApoA-I, were able to trigger this effect. Of note, only delipidated ApoA-I was active indicating an inhibitory effect of the lipid bound to the SR-B1 ligands.
Moreover, the SR-B1-mediated activity was sensitive to the activity of chloroquine (data not shown), a molecule that can inhibit TLR2 activities that require internalization of the TLR. 38,39 Finally, L37pA induces the phosphorylation of ERK. ERK is implicated in the signaling of all TLRs and many cytokines including interferon a. 27,40 Indeed, in the L929 fibroblasts, IFNa alone phosphorylated this protein and although the combination did not increase the phosphorylation, it is likely that the modification of the kinetics may exert an impact on cytokine-induced transcriptional activity.
The potent effect against AAV exerted when combined with IFNa indicated the great potential of these ligands. AAV infection generates stable episomal viral genomes that persist over time, and this stabilization of the AAV genome is not abrogated even upon treatment with potent immunostimulatory cytokines such as IFNa and interleukin 12. Here, we show that the AAV genome can be eliminated when IFNa is combined with L37pA. Interestingly, the AAV genome elimination was not dependent on T, B, NK cells and was probably due to a phenomenon of intrinsic immunity. 41 In addition to the effect of SR-B1 agonist on IFNa biology, we also show the effect of SR-B1 inhibitors on IFNa signaling. Selective uptake of lipids from HDLs by SR-B1 has been shown to be independent of endocytosis 42 and only SR-B1 is implicated as this transport can be reproduced in artificial membranes containing only purified SR-B1. 43 Moreover, it has been shown that BLT-1 does not affect clathrin-mediated endocytosis analyzing transferrin and epidermal growth factor uptake in Hela cells 28 or transferrin in ldlA[mSR-BI], a Chinese hamster ovary (CHO) cell line that overexpresses SR-B1. 44 The interference of BLT-1 with caveolin-mediated endocytosis was evaluated analyzing the entry of cholera toxin into BSC-1 cells and no effect was observed. 28 However, at least three isoforms of SR-B1 are produced by alternatively splicing of the mRNA. The isoform SR-B1.2 mediates a clathrin-mediated endocytosis of ligands that accumulate within the transferrin-positive endosomal recycling compartment. 45,46 This isoform only differs from SR-B1.1 in the carboxyl-terminal cytoplasmatic tail. SR-B1.1 represents 85-90% of SR-B1 protein present in the liver but in other tissues, SR-B1.2 is the dominant isoform. 47,48 In our mouse cell lines, we were able to detect the mRNA of both isoforms (data not shown) and both isoforms were inhibited by the siRNA and by the pharmacologic inhibitors. Our experiments show that SR-B1 not only mediated the internalization of an ApoA-1 mimetic ligand, such as L37pA, but also that interference with SR-B1 activity by genetic or pharmacological targeting reduced clathrin-dependent endocytosis of albumin. This finding may have broad implications. Among these, we focused our research on the effect on interferon a signaling. The type I interferon receptor is composed of IFNAR1 and IFNAR2 subunits. 49 Upon ligand binding, the ternary complex is internalized through a clathrin-dependent endocytosis. The endocytosis is required for the activation of STAT1 and for the biological responses induced by IFNa. 33 We corroborated this model using CPZ as a clathrin inhibitor. In fact, the activity of this inhibitor was similar to BLT-1 on SR-B1-dependent internalization, clathrin-dependent internalization, abrogation of ISGs induction and decrease of IFNAR1 levels on the cell membrane. Thus, it is likely that SR-B1 activity is required to maintain clathrin-enriched microdomains and drugs that block SR-B1 can affect cell processes mediated by clathrin. In addition, IFNb activity was inhibited by BLT-1 but it does not affect IFNg activity. These results are in line with the clathrin-dependent type I IFN activity and clathrin-independent IFNg activity. 33 Interferon a is an important cytokine due to its antiviral, antiproliferative and immunomodulatory activity. However, unbalanced production or unbalanced response may produce deleterious effects leading to autoimmune responses such systemic lupus erythematous and Sjogren's syndrome. 17 Moreover, chronic interferon induction may be a strategy used by a diverse range of pathogens, including some viruses to increase host susceptibility. 50 Thus, blockade of IFNa signaling is an appealing therapeutic target and we present an alternative strategy to the conventional targets present in the IFN signaling pathway.
In this study, we have focused on other applications of IFNa blockade: the enhancement of the efficacy of oncolytic viruses. IFNa plays a dual role in the biology of oncolytic viruses. While the induction of IFNa may be essential for triggering an antitumor immune response, 51,52 the antiviral activity of the released IFNa may limit the spreading of the oncolytic virus. 21,22 Thus, a transient inhibition during the initial phase of virus infection may allow optimal virus replication and induction of a strong immune response. That is what we achieved using BLT-1. This SR-B1 inhibitor allowed NDV to infect more cells and express a higher amount of the transgene. In vivo, this strategy clearly enhanced the antitumor efficacy of an NDV expressing GFP or IL-2. Thus, BLT-1 or another inhibitor could be included as an adjuvant when oncolytic viruses are administered.
In conclusion, we demonstrate in this study that SR-B1 controls IFNa activity through the modulation of clathrin-mediated endocytosis and through the presentation of certain ligands to TLR2 and TLR4. These findings have broad implications for the design of novel treatments for IFNa-mediated autoimmune diseases, to improve oncolytic virotherapy, or to improve IFNa-based therapies.

Animal handling
Female BALB/c or C57BL/6 mice were purchased from Harlan laboratories (Barcelona, Spain). The experimental design was approved by the Ethical Committee for Animal Testing of the University of Navarra. Experiments were performed with mice between 5-7 weeks old. For subcutaneous tumors, C57BL/6 or BALB/c mice were subcutaneously inoculated with MC-38 cells or CT-26 cells, respectively (5 £ 10 5 ) in the right posterior flank in a total volume of 100 mL. Two intratumoral injections of 6.5 £ 10 6 NDV, PBS and/or 360 ng of BLT-1 or DMSO as vehicle were administrated at day 7 and 9 after cell inoculations in a total volume of 50 mL. Hepatic tumors were established by direct implantation of 5 £ 10 5 MC38 cells in the left liver lobe of C57BL/6 mice following medial laparotomy in isofluoraneanaesthetized animals. Recombinant AAVs were inoculated via retro-orbital injection with 5 £ 10 11 viral genomes in a total volume of 200 mL in female C57BL/6 mice. Rag1 knockout mice, Rag2gc knockout mice, SR-B1 knockout mice all in a C57BL/6 background were breed at the University of Navarra.

AAV production
Recombinant AAV vectors were constructed to express the IFNa, L37pA or the reporter gene luciferase under transcriptional control of elongation factor 1a promoter (EF). The signal peptide of the mouse gene for IFNa1 was used to secrete the L37pA. AAV vectors were produced by co-transfection of the plasmids pDP8.ape and pAAV containing IFNa1, L37pA or luciferase into 293T cells. Two days later, AAV was purified from the cell lysates by ultracentrifugation in Optiprep Density Gradient Medium-Iodixanol (Sigma-Aldrich, St Louis MO). To titer the AAV productions, viral DNA was isolated using "The High Pure Viral Nucleic Acid" kit (Roche Applied Science. Mannheim, Germany). The concentration of viral particles was subsequently determined by real-time quantitative PCR using primers specific to the EF promoter (Table S1).

NDV vectors production
To generate recombinant NDV expressing GFP or mIL2, A549 cells and Hep2 cells were plated in a 6-well plate and were infected with MVA-T7 at a multiplicity of infection of 1 and then transfected with the NDV full-length clone together with the following expression plasmids: pTM1-NP (nucleoprotein), pTM1-P (phosphoprotein), and pTM1-L (RNA-dependent RNA polymerase). After overnight incubation, transfected cells were cocultured for an additional 2 d. Cells and supernatants were collected and injected into the allantoic cavities of 9-or 10 d old chicken embryonated eggs. Three days later, allantoic fluids were harvested and virus growth was confirmed by hemagglutination and immunofluorescent assays. Original virus stock was frozen at ¡80 C. Viruses were amplified by injecting 1000 infection units of the virus stock into 10 d old embryonated eggs. Allantoid fluid was collected after 2 d alliquoted and quick frozen in dry ice. Stocks were kept at ¡80 C before titration by immunofluoresce in Hep2 cells.

Antiviral assay
Antiviral assay was performed by using a cytopathic-effect reduction assay with EMCV. Cells were plated in 96-well plates (3 £ 10 4 cells/well) and stimulated for 24 h with 1:2 serial dilutions of 125 U/mL murine IFNa. Then, the virus was added for 24 h. When maximum cytopathic effect (CPE) was achieved in control cells, viable IFN-protected cells were measured by luminometry with the ViaLight Plus Kit (Lonza, Rockland, ME).

IFNa cytotoxicity
Mouse L929 cells were plated in 96-well plates (3 £ 10 3 cells per well) and incubated with IFNa (1500 U/mL) in the presence or absence of BLT-1 (15 mM). After 72 h, cell death was determined by the release of adenylate kinase with the Toxi-Light BioAssay Kit (Lonza).

Quantitative RT-PCR analysis
Cells were seeded in 24-well plates using DMEM complete medium for 24 h to achieve 80% confluence. Then, the cells were washed twice with PBS and stimulated with mouse (200 U/mL) or human (100 U/mL) IFNa, L37pA or IFNa plus L37pA for 3 h. For experiments with blockers of lipid transport, the cells were pre-incubated for 1 h with 15 mM of BLT-1, 30 mM of ITX-506, 500 mM of glyburide or 10 mM of CPZ in DMEM prior stimulation with mouse (200 U/mL) or human (100 U/mL) IFNa. Total RNA from liver tissue and cell lines was extracted using the automated Maxwell system from Promega (Madison, WI). RNA was treated with DNase I and reverseretrotranscribed to cDNA with MMLV RT in the presence of RNase OUT (all reagents from Invitrogen) according to the manufacturers instructions. Primer sequences are described in Table S1. Real time PCR reaction was performed using SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). As RPLP0 levels remained constant across different experimental conditions, they were used to standardize gene expression. The amount of each transcript was expressed by the formula 2 DCt (2 ct(RPLP0)ct(gene) ), ct being the point at which the fluorescence rises significantly above background fluorescence.

Western blotting
L929 and MEF cells were seeded in 6-well plates using DMEM at a cell density of 3 £ 10 5 per well and incubated for 24 h. BLT-1 (15 mM) treatment was performed for 1 h. IFNa, L37pA or IFNa plus L37pA stimulations were performed for 30 min and then Western blotting was accomplished using the appropriate antibodies.

Co-immunoprecipitation
Lysates from L929 cells treated or not with L37pA for 30 min were immunoprecipitated with anti-TLR4 beads. Cell samples were lysed for 2 h at 4 C in radioimmune precipitation assay buffer with protease inhibitors. Separately, protein G magnetic beads (Dynabeads Protein G, Novex, San Diego, CA) were washed and resuspended according to the manufacturer's instructions and incubated overnight at 4 C with 2 mg of anti-TLR4 antibody. The antibody-bound beads were washed three times, and samples containing 50 mg of cell lysate were added and incubated for 2 h at 4 C in a volume of 500 mL. After washing, the immunoprecipitated proteins were eluted by heating at 60 C for 15 min in Laemmli sample buffer. Samples of equal total protein content were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA in TBS/Tween and incubated overnight at 4 C with anti-TLR2 (1:1000) antibody. After washing in TBS, horseradish peroxidase-conjugated secondary antibody was applied, and immunoreactive bands were detected by chemiluminescence.

Determination of IFNa by ELISA analysis
Serum IFNa levels were measured using a VeriKine TM Mouse Interferon Alpha ELISA Kit (PBL Biomedical Laboratories, Piscataway, NJ) following the manufacturer's recommendations.

Hemogram
After 2 or 4 weeks of AAV injection, blood samples were collected in Microvette EDTA-coated tubes (Sarstedt Inc., Newton, NC). Hemograms were analyzed using the Drew Scientific HemaVet Hematology Analyzer according to the manufacturer's recommendations.
Flow cytometry analysis 8 £ 10 4 L929 cells were seeded in 24-well plates using complete DMEM and incubated for 24 h to achieve 80% confluence. Then, cells were washed twice with PBS and incubated with BLT-1 (15 mM) or CPZ (10 mM) in DMEM. For IFNAR1 analyses, cells were incubated for 3 h with BLT-1 or CPZ, detached using citrate buffer and labeled with PE anti-mouse IFNAR1 antibody. For uptake studies, cells were incubated for 1 h with BLT-1 and CPZ. Then, 10 mg/mL of L37pA-or BSA-FITC were added for 3 h, detached using 0.05% trypsin-EDTA, washed twice with PBS and resuspended in 200 mL of PBS containing 0.2 mg/mL of trypan blue. Analyses were performed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and data were analyzed using FlowJo software (Tree Star Inc., San Carlos, CA).

Statistical analysis
Prism software (GraphPad Software, Inc.) was employed for statistical analysis. We used the log-rank test to determine the significance of differences in survival curves. Mean differences were compared with t-tests for 2 group comparisons or oneway ANOVA followed by a multiple comparison test for three or more group comparisons. The CPE reduction assay was fitted to a Hill equation and compared with the Extra sum-ofsquares F test. p values < 0.05 were considered to be statistically significant.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.

Funding
This work was supported by the grant PI13/00207 from Instituto de Salud Carlos III, financed by the FEDER program of the European Union, by a grant from the FAECC and by the EC 's H2020 PROCROP project, under grant agreement 635122. Pedro Berraondo was supported by a Miguel Servet and Miguel Servet II (CPII15/00004) contract from Instituto de Salud Carlos III.