Interferon-stimulated genes—essential antiviral effectors implicated in resistance to Theiler’s virus-induced demyelinating disease

Experimental infection of mice with Theiler’s murine encephalomyelitis virus (TMEV) is used as an animal model of human multiple sclerosis. TMEV persists in susceptible mouse strains and causes a biphasic disease consisting of acute polioencephalomyelitis and chronic demyelinating leukomyelitis. In contrast, resistant mice eliminate the virus within 2 to 4 weeks, which seems to be based on a strong antiviral innate immune response including the activation of the type I interferon (IFN) pathway. Several interferon-stimulated genes (ISGs) such as IFN-stimulated protein of 15 kDa (ISG15), protein kinase R (PKR), and 2′5′-oligoadenylate synthetase (OAS) function as antiviral effectors and might contribute to virus elimination. Nevertheless, detailed investigations of the type I IFN pathway during TMEV-induced demyelinating disease (TMEV-IDD) are lacking. The present study evaluated microarray data of the spinal cord obtained from susceptible SJL/J mice after TMEV infection focusing on IFN-related genes. Moreover, ISG gene and protein expression was determined in mock- and TMEV-infected SJL/J mice and compared to its expression in resistant C57BL/6 mice using real- time PCR, immunohistochemistry, and immunofluorescence. Interestingly, despite of increased ISG gene expression during TMEV-IDD, ISG protein expression was impaired in SJL/J mice and mainly restricted to demyelinated lesions. In contrast, high ISG protein levels were found in spinal cord gray and white matter of C57BL/6 compared to SJL/J mice in the acute and chronic phase of TMEV-IDD. In both mouse strains, ISG15 was mainly found in astrocytes and endothelial cells, whereas PKR was predominantly expressed by microglia/macrophages, oligodendrocytes, and neurons. Only few cells were immunopositive for OAS proteins. High levels of antiviral ISG15 and PKR proteins in the spinal cord of C57BL/6 mice might block virus replication and play an important role in the resistance to TMEV-IDD.


Background
Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded ribonucleic acid (RNA) (ssRNA) Cardiovirus of the Picornaviridae family, whose large open reading frame (ORF) encodes a 2300-amino acid polyprotein that is cleaved into 12 mature proteins [1]. TO strains (BeAn, DA) of TMEV induce a biphasic disease in susceptible SJL/J mice characterized by acute mild polioencephalomyelitis and chronic demyelinating leukomyelitis with virus persistence. In contrast, resistant C57BL/6 mice eliminate the virus from the central nervous system (CNS) within 2 to 4 weeks [2]. After intracerebral inoculation, TMEV primarily infects hippocampal and cortical neurons [1]. In addition, the virus spreads to neurons within the ventral horns of the spinal cord most likely along axons and via the cerebrospinal fluid [3,4]. Approximately 2 weeks after inoculation at the peak of the specific immune response, the virus disappears from neurons but persists in white matter glial cells and macrophages [5]. Demyelination is initially induced by virusmediated axonal damage recruiting inflammatory cells to sites of Wallerian-like degeneration [6]. The subsequent release and presentation of myelin breakdown products causes a CD4 + T cell response directed against different myelin proteins such as proteolipid protein, epitope PLP 139-151 , by molecular mimicry and epitope spreading [7]. The susceptibility of mouse strains to TMEV-induced demyelination disease (TMEV-IDD) is linked to the major histocompatibility complex (MHC) class I H-2D gene [8]. Consequently, the level of TMEV-specific CD8 + cytotoxic T cells is higher in resistant compared to susceptible mice in acute lesions [9]. In addition, MHC class II-restricted CD4 + T cells play an important role in the induction of a delayed-type hypersensitivity (DTH) response initially directed against specific TMEV capsid proteins [10].
Due to similar histological and pathogenetic features, TMEV-infected susceptible mice are commonly used to model the most important demyelinating disease in humans multiple sclerosis (MS) [3,11]. The therapy of MS strongly relies on different immunomodulatory and immunosuppressive drugs including beta interferon (IFNβ), which blocks T cell activation, increases interleukin (IL)-10 expression, and maintains blood-brain barrier integrity [12,13]. Similar IFN-β-dependent mechanisms seem to reduce the demyelination in chronic TMEV-IDD [7]. Furthermore, antiviral actions of this type I interferon (IFN) have to be blocked by the TMEV leader (L) protein, a 76-amino acid protein cleaved from the N-terminal end of viral polyprotein, to establish virus persistence [14]. The L protein impedes IFN-β gene transcription by interfering with IFN regulatory factor 3 (IRF3) dimerization and nucleocytoplasmic trafficking [2,15,16]. Interestingly, IRF3 contributes not only to early control of TMEV replication but also to TMEV-induced hippocampal injury [17]. In addition, IRF3 polymorphisms strongly affect the murine innate immune response and ability of TMEV to persist in the CNS of different mouse strains [18]. Consequently, TMEV-induced demyelination might result from an insufficient infection control by the innate immune response particularly the interferon system in susceptible mice.
Type I IFN signal transduction is initiated by their cell surface receptors, IFN alpha and beta receptors (IFNAR)1 and 2, which are linked to janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2). These two tyrosine kinases activate the IFN-stimulated gene factor 3 (ISGF3) consisting of the signal transducer and activator of transcription (STAT) 1 and 2 and IRF9, which enter the nucleus and bind to IFNstimulated response elements (ISREs) of more than 300 IFN-stimulated genes (ISGs) [19,20]. The death of IFNAR knockout mice after TMEV infection supports the prominent role of ISGs in virus control [21]. IFN-stimulated protein of 15 kDa (ISG15), protein kinase R (PKR), 2′5′oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) represent classical IFN-dependent antiviral effectors detected by gene targeting studies [19]. In contrast to other ISGs, the effector functions of these proteins have been characterized in detail. In addition, studies using specific knockout mice have confirmed their antiviral activity against numerous virus families including Picornaviridae [22,23]. Consequently, mouse strainspecific differences in their expression pattern might critically influence their ability to eliminate TMEV from the CNS. ISG15 encodes an ubiquitin-like protein, which conjugates to numerous protein substrates to modulate pleiotropic cellular responses [21,24]. PKR is activated by viral double-stranded RNA (dsRNA) resulting in phosphorylation of the eukaryotic initiation factor 2α (eIF2α) and inhibition of global protein synthesis including viral protein synthesis [19,25]. OAS proteins including OAS1, OAS2, OAS3, and OASL (OAS-like) are expressed at low levels in unstimulated cells as inactive monomers, which can be activated by viral dsRNA to form tetramers [26]. These tetramers activate the latent ribonuclease L (RNase L) resulting in the degradation of viral and host cell RNAs [19,26]. Mx proteins are members of the dynamin-like large guanosine triphosphatase (GTPase) family, whose expression is exclusively controlled by type I IFNs [27]. Mx proteins bind viral nucleocapsid-like structures or other viral components to prevent the generation of new virus particles [27]. However, Mx proteins are not functional in most laboratory mouse strains [28,29] but might be biomarkers to predict the therapeutic efficacy of IFN-β treatment in MS [30].
The role of these classical ISGs in the neuropathogenesis of TMEV-IDD has already been investigated in previous studies. PKR is important for the recognition of TMEV RNA in the cytoplasm of astrocytes [31] and the ensuing expression of several pro-inflammatory cytokines such as IL-1, IL-6, IL-12p40, and TNF-α as well as chemokines including different monocyte chemoattractant proteins [32,33]. In addition, PKR is required for production of type I IFNs in response to TMEV in part by regulating IFN messenger RNA (mRNA) integrity [34]. The OAS/RNase L pathway has been described to be inhibited by the direct interaction of the ankyrin domain of RNase L with TMEV L* protein, a 156amino acid long protein encoded by an alternative TMEV ORF [35]. Surprisingly, primary mouse neurons that are exposed to type I IFNs remain highly susceptible to TMEV infection, which results from their heterogeneous response to IFN treatment revealed by a lack of Mx1 expression. In addition, they have a low level of basal ISG expression compared to fibroblasts [36]. These data underline the cell type-specific differences in their IFN response. However, all these studies have been performed in cell culture and have not investigated the expression of these antiviral effectors at the protein level. Moreover, ISG cell type-specific expression patterns in the spinal cord including white matter lesions of TMEV-infected mice remain unknown so far. Consequently, the aim of this study was to compare the expression of these four important ISGs in TMEV-IDD between SJL/J and C57BL/6 mice in order to substantiate the hypothesis that intrinsic differences in the interferon system between these mouse strains are related to their susceptibility to TMEV-induced demyelination.
Photos of ISG15, PKR, and OAS1 immunohistochemically stained sections were generated by a digital microscope (HS All-in-one Fluorescence Microscope BZ-9000 Generation II; HS All-in-one Fluorescence Microscope BZ-II Analyzer, BIOREVO, KEYENCE Deutschland GmbH, Neu-Isenburg, Germany) and evaluated using analysis software (analySIS 3.1 software package; Soft Imaging system, Münster, Germany). Light microscopy was used to count Mx-positive cells. In addition, ISG positive and negative cells present in the Virchow-Robin space of meningeal and intraparenchymal blood vessels were counted to calculate the percentage of ISG15-, PKR-, OAS1-, and Mx-positive perivascular cells. Immunohistochemical results are expressed as median with minimum and maximum.

Statistical analysis
RT-qPCR data and immunohistochemical data of SJL/J and C57BL/6 mice were analyzed using Kruskal-Wallis and non-parametric Mann-Whitney U tests (IBM SPSS, version 19.0, IBM Corporation, Armonk, NY, USA). Ratios of the geometric means and 95 % confidence intervals were calculated with independent T tests to compare ISG protein expression between C57BL/6 and SJL/J mice (IBM SPSS, version 19.0). Spearman's rank correlation coefficients were calculated to analyze the correlation between ISG protein and gene expression as well as TMEV RNA copy numbers, inflammation, and demyelination in the spinal cord of SJL/J mice (IBM SPSS, version 19.0). Data concerning TMEV, inflammation (determined on hematoxylin and eosin and immunohistochemically stained sections), and demyelination (determined on luxol fast blue stained sections) have been taken from previous publications [39,51]. Statistical significance was designated as P ≤ 0.05.

Microarray
Recently, Ulrich et al. (2010) performed a microarray analysis of mock-and TMEV-infected SJL/J mice at 14, 42, 98, and 196 dpi and detected transcriptional changes of 1001 genes in the spinal cord. In the present study, the log2-transformed normalized data were independently re-evaluated focusing on critical genes of the type I IFN pathway employing conventional statistical methods as described [41,43,52]. Most genes were significantly up-regulated by TMEV infection and peaked at 98 dpi ( Fig. 1; Table 1; Additional file 1: Table S1). Only a mild increase in Toll-like receptor (Tlr)3 gene expression (fold change <2) was found, whereas other receptors of viral RNA including Pkr, melanoma differentiationassociated gene (Mda5), Tlr7, and Tlr13 were moderately up-regulated (fold change up to 5.77). Nfkb1, lrf3, and Irf5 gene expression was barely influenced by TMEV infection. In contrast, Irf1 and Irf7 were strongly up-regulated (fold change up to 4.40 and 11.42, respectively). Interestingly, no changes were found in type I IFN gene expression, and Ifnar genes were only mildly increased in the late phase of TMEV-IDD. However, gene expression of several factors critically involved in IFN signal transduction, especially of the ISGF3 components IRF9, STAT1, and STAT2, was moderately to strongly up-regulated (fold change up to 15.39). In addition, gene expression of various IFN-dependent antiviral effectors was strongly increased in TMEV-IDD (fold change up to 31.22). Similarly, several MHC class I and II genes were strongly induced by TMEV infection (fold change up to 37.55).

RT-qPCR
RT-qPCR of selected genes was performed to validate microarray results of SJL/J mice and to compare gene expression of SJL/J and C57BL/6 mice (Additional file 2: Figure S1). In general, there was a good correlation between RT-qPCR and microarray data in SJL/J mice revealing highly similar fold changes of IRF7, ISG15, and PKR at both 14 and 98 dpi. Nevertheless, minor changes in type I IFN mRNA transcription were detected in SJL/ J mice using RT-qPCR, which were not found in the microarray analysis.
IFN-α and IFN-β transcript numbers were not significantly changed at 14 dpi in SJL/J mice (fold changes: IFN-α: 1.  Significant differences between the two mouse strains at the mRNA levels of the investigated genes were predominantly found at 98 dpi. In mock-infected mice, there were higher transcript numbers of IFN-α (P = 0.041) and IFN-β (P = 0.009) and lower mRNA levels of ISG15 (P = 0.009) in SJL/J compared to C57BL/6 mice at this time point. In TMEV-infected mice, there were higher transcript numbers of IRF7 (P = 0.008), ISG15 (P = 0.008), and PKR (P = 0.008) and lower mRNA levels of IFN-α (P = 0.016) in SJL/ J compared to C57BL/6 mice at 98 dpi. At 14 dpi, a significant difference between the two mouse strains was only detected for IFN-β, which was higher in mock-(P = 0.002) and TMEV-infected (P = 0.002) SJL/J compared to C57BL/ 6 mice.

TMEV infection induces ISG protein expression in SJL/J mice
In general, immunohistochemical analysis detected an increase in ISG15, PKR, and OAS1 protein expression in white matter lesions (Figs. 2 and 3). ISG15-positive cells particularly ependymal cells, endothelial cells, astrocytes, and microglia/ microphages were found in the spinal cord of mock-and TMEV-infected SJL/J mice at all of time points. A mild ISG15 expression was also found in some neurons (Fig. 3a, b, e). ISG15 protein levels were higher in the white matter of TMEV-compared to mock-infected mice at 14 dpi (P = 0.004) and 42 dpi (P = 0.002), whereas protein expression was only changed at 42 dpi in the gray matter (P = 0.015; Fig. 2a, b).
PKR proteins were present in microglia/macrophages, oligodendrocytes, and neurons at all investigated time points in mock-and TMEV-infected mice (Fig. 3c, d). In addition, positive gitter cells were found in the white matter of TMEV-infected SJL/J mice at 98 and 196 dpi (Fig. 3f). Similar to ISG15, PKR protein levels were increased by TMEV infection, but a significant difference was only found in the white matter at 196 dpi due to strong intralesional expression (P = 0.002; Figs. 2d and 3e, f). OAS1 was mainly expressed by neurons and microglia in mock-and TMEV-infected mice (Fig. 3g, h). In general, there was a low OAS1 protein expression at all investigated time points (Fig. 2e, f ). Nevertheless, OAS1 protein levels were significantly higher in the white matter of TMEV-compared to mock-infected mice at 42 dpi (P = 0.026) and 98 dpi (P = 0.004; Fig. 2f ). Corresponding to low IFNα and IFNβ gene expression, Mx proteins were only found in few glial cells at 14 and 98 dpi.
Higher ISG protein expression in C57BL/6 compared to SJL/J mice The ISG protein expression in the spinal cord was compared between susceptible SJL/J and resistant C57BL/6 mice at an early time point (14 dpi) and at the peak of inflammatory demyelination and ISG gene induction  ISG15 (a, b), PKR (c, d), and OAS1 (e, f) protein expression in spinal cord gray (a, c, e) and white matter (b, d, f). Shown is the percentage of immunopositive area using Box-and-Whisker plots and significant differences between groups based on Mann-Whitney U tests (*P ≤ 0.05; **P ≤ 0.01) (98 dpi). Similar to SJL/J mice, only few Mx-positive cells were detected in mock-and TMEV-infected C57BL/6 mice indicating comparable low type I IFN levels in both mouse strains. In contrast, ISG15 protein levels were significantly increased in TMEV-compared to mock-infected C57BL/6 mice in the gray (P = 0.026) and white matter (P = 0.009) at 14 dpi and in the gray matter (P = 0.017) at 98 dpi (Additional file 3: Figure S2). PKR protein levels were significantly up-regulated in TMEV-compared to mock-infected C57BL/6 mice in the gray (P = 0.030) and white matter (P = 0.004) only at 98 dpi. OAS1 protein expression was not influenced significantly by TMEV infection in C57BL/6 mice.
However, significant higher ISG15, PKR, and OAS1 protein expression was detected in the spinal cord gray matter of TMEV-infected C57BL/6 compared to SJL/J mice at 14 dpi (P = 0.007, P = 0.016, P = 0.003) and 98 dpi (P = 0.032, P = 0.001, P = 0.025; Figs. 4 and 5). h Few neurons, microglia, and perivascular immune cells express OAS1 proteins in TMEV-infected SJL/J mice at 98 dpi. Immunohistochemistry visualized by the avidin-biotin-peroxidase complex method with 3,3-diaminobenzidine as substrate and counterstaining with Mayer's hematoxylin. Bar 100 μm Significant higher ISG15, PKR, and OAS1 protein levels in the spinal cord white matter of TMEV-infected C57BL/6 compared to SJL/J mice were only found at 14 dpi (P = 0.029, P = 0.017, P = 0.004). Furthermore, there was a significant higher ISG15 expression of mock-infected C57BL/6 compared to SJL/J mice in spinal cord gray (P = 0.005) and white matter (P < 0.001) at 14 dpi (Fig. 4). PKR and OAS1 protein levels were also significantly increased in mock-infected C57BL/6 mice in the gray matter at 14 dpi (P = 0.01) and in the Fig. 4 Comparison of the ISG15, PKR, and OAS1 protein expression between mock-and TMEV-infected C57BL/6 and SJL/J mice at 14 and 98 dpi .  ISG15 (a, b), PKR (c, d), and OAS1 (e, f) protein expression in spinal cord gray (a, c, e) and white matter (b, d, f). Shown are the ratios of the geometric means and 95 % confidence intervals of immunopositive area between C57BL/6 and SJL/J mice using Box-and-Whisker plots and significant differences between groups based on independent T tests (*P ≤ 0.05; **P ≤ 0.01). Note higher protein levels in C57BL/6 compared to SJL/J mice (ratio >1) white matter at 98 dpi (P = 0.01), respectively. At 14 dpi, the difference in OAS1 expression in spinal cord white matter between the two mouse strains after mockinfection did not reach statistical significance (P = 0.052).

Correlation analysis
ISG gene and protein expression was correlated to several parameters characterizing the progression of TMEV-IDD in SJL/J using Spearman's rank correlation coefficients in order to quantify the relationship of ISGs with viral load, inflammation, and demyelination. The gene expression of ISG15, PKR, OAS1, and Mx strongly correlated with TMEV RNA, inflammatory cells including CD3 + T cells, CD45R + B cells, and MAC3 + microglia/macrophages, and demyelination (r s ≥ 0.6; P ≤ 0.05; Table 2). In addition, low but significant correlations were found between the protein expression of ISG15, PKR, and Mx and their respective gene expression, TMEV RNA, and all investigated inflammatory cells (r s ≥ 0.4; P ≤ 0.05; Table 3). ISG15 (r s = 0.3) and PKR (r s = 0.4) protein expression was also significantly correlated to the extent of demyelination in the spinal cord. OAS protein expression was only significantly correlated to the overall number of inflammatory cells (r s = 0.3) and the number of intralesional MAC3 + microglia/macrophages (r s = 0.4).

Impaired protein expression of interferon-stimulated genes in SJL/J mice
The present study investigated the expression of genes critically involved in the type I IFN pathway during TMEV-IDD. PRRs recognize pathogen-associated molecular patterns (PAMPs) such as viral and bacterial components and induce intrinsic cellular defenses. Double-stranded RNA formed during TMEV replication is typically identified by PKR, MDA5, and TLR3 [31,53,54]. PKR, MDA5, and TLR2 gene expression was moderately increased in the present study, whereas TLR3 mRNA levels were hardly affected by TMEV infection. Nevertheless, previous studies already demonstrated that TMEV induces an up-regulation of TLR2 via TLR3 signal and subsequently an activation of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) causing the expression of several pro-inflammatory cytokines and chemokines including IL-1β, IL-6, CCL2 (MCP-1), and CCL5 (RANTES) [32,33,55,56]. In contrast to IRF3 and IRF5, IRF7 was strongly up-regulated in the present study. Interestingly, the IRF7 protein must be continuously produced due to its short half-life [57]. In addition, IRF3 function can be blocked by the TMEV L protein [2,15,16]. Consequently, several PRRs might be induced by TMEV and implicated in the activation of the innate and adaptive immune response via NF-κB and IRF7. A high NF-κB expression was already described in microglia/macrophages in demyelinating TMEV lesions [55]. The activation of the type I IFN pathway by TMEV was also demonstrated by increased transcription of various ISGs including MHC class I and II genes, especially in the late phase of TMEV-IDD. Correspondingly, high correlation coefficients were found between ISG gene expression and TMEV RNA as well as inflammation and demyelination.
However, type I IFN production was blocked in the spinal cord of TMEV-infected SJL/J mice, and upregulation of ISG transcription might depend on increased expression of ISGF3 or even IRF1, which can directly induce ISG transcription and thereby exert antiviral functions in the absence of IFN [23]. The lack of type I IFN might be related to the strong OASL1 expression found in the present study, which inhibits the translation of the potent type I IFN inducer IRF7 [58]. In addition, the increase in ISG protein expression, which only weakly correlated with its gene expression, was mainly restricted to late white matter lesions. Low ISG protein levels in the early phase of TMEV-IDD in this susceptible mouse strain indicate an intrinsic and/or virus-dependent impairment of these IFN-dependent  (red, a), oligodendrocytes (red, c), and microglia/macrophages (red, e) demonstrated by an orange-yellow mixed color (arrows). No co-localization of PKR (green, arrowheads) with GFAP (red, arrows) indicates no/low PKR expression in astrocytes (red, b), whereas co-localization of PKR (green) with Olig2 (red, d) and MAC-3 (red, f) demonstrates PKR expression in oligodendrocytes and microglia (arrows). Immunofluorescence double-staining for ISG15 and PKR with GFAP, Olig2, and MAC-3. Nuclei are stained with bisbenzimide. Bar 25 μm [59] and enhance NF-κB signaling via suppression of protein phosphatase 1B (PPM1B) [60]. Downstream targets of the transcription factors IRF3 and NF-κB including IFN-β and pro-inflammatory cytokines play an important role in the innate immune response to TMEV infection. Interestingly, the main ISG15 expressing cells were astrocytes, endothelial cells, and ependymal cells, which line the ventricular system and the central canal of the spinal cord. This expression pattern puts ISG15 in the first line of defense to restrict virus spread in the CNS via the liquorogenic route [3]. PKR is required for type I IFN production in response to TMEV by acting as cytoplasmic RNA sensor and stabilizing IFN mRNA integrity [32]. Moreover, PKR is itself up-regulated by type I IFNs and exerts direct antiviral effects [25,61]. Similarly, OAS1 protein might act as antiviral effector in TMEV-infected C57BL/6 mice restraining virus spread in the spinal cord before demyelination starts. Nevertheless, due to the inhibition of the OAS/ RNase L pathway by the TMEV L* protein [35], the antiviral activity of RNase L might be similar in TMEV-infected SJL/J and C57BL/6 mice despite different OAS1 protein levels.

Conclusions
In conclusion, the type I IFN pathway is activated in TMEV-infected mice inducing the transcription of various ISGs. The translation of these genes seems to be impaired in susceptible SJL/J mice limiting their antiviral function. In contrast, a high constitutive expression of ISG15 and PKR proteins in the spinal cord white and gray matter, which was further increased by TMEV infection, might block the spread of TMEV in C57BL/6 mice and prevent demyelinating disease based on virus persistence.

Additional files
Additional file 1: Table S1. Transcriptional changes of the type I and II IFN signaling pathway in TMEV-compared to mock-infected SJL/J mice.