Background
Toll-like receptor 3 (TLR3) recognizes double stranded RNA (dsRNA), including poly IC and viral dsRNAs. TLR3 activation induces the production of a variety of cytokines, such as IL-1β, IL-6 and type I interferon (IFN) [
1‐
4]. However, the role that TLR3 activation plays in the protection from or pathogenesis of virus-induced chronic disease is still unclear. It has been reported that a dominant-negative TLR3 allele is associated with the development of herpes simplex encephalitis, suggesting that TLR3 plays a protective role in herpes simplex virus infection [
5]. In addition, TLR3 appears to play a protective role against infections with West Nile virus (WNV) [
6], Coxsackievirus B4 [
7], and mouse cytomegalovirus [
8]. However, a detrimental role of TLR3 in the induction of acute pneumonia following influenza A virus infection has also been reported [
9]. In addition, several studies have indicated that TLR3-mediated signals play either no role or a pathogenic role in viral diseases. For example, a recent study demonstrated that the absence of TLR3 did not alter viral pathogenesis after infection with single-stranded or double-stranded RNA viruses, such as lymphocytic choriomeningitis virus, vesicular stomatitis virus, and reovirus [
10]. Furthermore, TLR3-deficient mice were more resistant to lethal WNV infection, although a TLR3-mediated signal was critical for the virus to penetrate into the brain where it caused neuropathogenesis [
11].
Theiler's murine encephalomyelitis virus (TMEV) is a positive sense single-stranded RNA (ssRNA) virus of the
Picornaviridae family [
12]. TMEV establishes a persistent CNS infection in susceptible mouse strains that results in the development of demyelinating disease, which is considered a relevant viral model for human multiple sclerosis [
13‐
15]. It has previously been shown that TLR3 recognizes the dsRNAs generated as TMEV replication intermediates, and TLR3 is essential for the production of TMEV-induced inflammatory cytokines, such as type I IFNs [
16,
17]. TLR3 is constitutively expressed in a variety of cells, including antigen presenting cells (dendritic cells and macrophages) as well as glial cells, including microglia and astrocytes [
18]. In addition, the expression level of TLR3 is upregulated following TMEV infection and its expression levels are particularly high in cells from susceptible mice [
19,
20]. Furthermore, antigen presenting cells in the periphery and glial cells in the CNS are much more permissive to TMEV infection and support viral replication better than cells from resistant mice [
21,
22]. The differences appear to be, in part, due to the high intrinsic activation state of NF-κB in cells from susceptible mice [
23]. TLR3-mediated signals activate multiple NF-κB pathways and upregulate the expression of other TLRs, such as TLR2, and following TMEV infection, these secondary TLRs contribute to the production of additional proinflammatory cytokines [
17,
24]. However, dsRNAs, including synthetic dsRNA poly IC, are recognized not only by TLR3 but also by MDA5 and PKR [
16,
24]. Therefore, the relative role of TLR3-mediated signaling in the development of TMEV-induced demyelinating disease remains to be determined.
In particular, the induction of strong type I IFN production, following infection with TMEV, is mediated by TLR3 and MDA5-mediated signals [
16,
17,
24,
25]. Our previous results showed that type I IFN was critical for the prevention of rapid fatal encephalitis, by controlling the viral load and the infiltration of inflammatory cells into the CNS [
26]. However, type I IFN levels were significantly higher in susceptible SJL mice compared to resistant C57BL/6 mice [
22]. Interestingly, type I IFNs play dichotomous roles in stimulating the immune responses, i.e., up- or down-regulating T cell responses, apparently depending on IFN concentration [
21,
27]. Furthermore, the time of type I IFN presence seems to be an important factor for the function of type I IFNs against viral infection [
21]. Many recent studies utilized poly IC to activate TLR3 and/or MDA5-mediated signals in conjunction with viral infections and/or autoimmunity. For example, poly IC treatment of virus-infected mice resulted in a type I IFN-dependent reduction in viral loads and protection from virus-induced disease by enhancing the function of virus-specific T cells [
28,
29]. However, treatment with poly IC enhances the development of autoimmune diseases [
30‐
32]. Therefore, it would be important to investigate the effects of different levels of type I IFNs that are activated via TLR3 in resistant and susceptible mice to determine its impact on the development of TMEV-induced demyelinating disease, which bears both viral and autoimmunity components.
To investigate the role of TLR3-mediated innate immune responses on the pathogenesis of TMEV-induced demyelinating disease, we utilized TLR3-deficient mice in both the resistant C57BL/6 (B6) and susceptible SJL/J backgrounds. In addition, we administered poly IC to activate TLR3-mediated signals prior to or after TMEV infection. Our results showed that TLR3-deficient susceptible SJL mice accelerated the development of demyelinating disease, whereas TLR3-deficient resistant B6 mice remained disease free. The virus-infected TLR3-deficient SJL mice displayed increased cellular infiltration and an elevated viral load in the CNS. Therefore, TLR3-mediated signals are important in protecting susceptible mice from the development of TMEV-induced demyelinating disease, although TLR3-mediated signals appear to play a minor role in resistant mice. However, treatment with poly IC prior to viral infection exacerbated disease development in susceptible mice, while treatment after viral infection somewhat ameliorated it. This observation suggests that either a premature activation or an over-activation of TLR3 signaling during early viral infection may lead to pathogenesis, perhaps through the development of a pathogenic immune response. Therefore, our current results strongly warrant caution on the use of TLR3-mediated immune interventions against chronic viral diseases and suggest careful consideration for these treatments in conjunction with the time of viral infection.
Materials and methods
Mice
SJL/J mice were purchased from the Charles River Laboratories (Charles River, MA) through the National Cancer Institute (Frederick, MD). B6; 129S-
Tlr3
tm1Flv
/J mice (TLR3KO-B6) were purchased from Jackson Laboratories (Bar Harbor, ME). TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations to obtain TLR3KO-SJL mice. The absence/presence of TLR3 in TLR3KO-SJL and the littermate mice (NLM) were typed based on the electrophoresis patterns of TLR3 and neomycin resistant genes. PCR products from tail genomic DNA of NLM and TLR3KO-SJL mice were determined using PCR-based genotyping analysis established by the Jackson Laboratory (Additional file
1, Figure S1). Experimental procedures that were approved by the Animal Care and Use Committee of Northwestern University in accordance with NIH animal care guidelines were used in this study.
Virus
The BeAn and GDVII strains of TMEV were propagated in BHK-21 cells grown in DMEM medium supplemented with 7.5% donor calf serum. Viral titer was determined by plaque assay on BHK cell monolayers. The cells were incubated for 4-5 days in infection-medium (DMEM supplemented with 0.1% bovine serum albumin) with TMEV at 10 MOIs and the cell lysates were cleared by centrifugation. The cleared lysates yield 3-5 × 108 PFU and a pooled batch was used as a viral stock. If necessary the viral stock was diluted in DMEM before inoculation.
Assessment of clinical signs
Approximately 30 μl of TMEV was injected into the right hemisphere of 5- to 7-week-old mice anesthetized with isofluorane. Resistant B6 and TLR3KO-B6 mice were infected with 1 × 106 PFU and susceptible SJL and TLR3KO-SJL mice were infected with 2 × 105 PFU TMEV. Clinical symptoms of disease were assessed weekly on the following grading scale: grade 0 = no clinical signs; grade 1 = mild waddling gait; grade 2 = moderate waddling gait and hindlimb paresis; grade 3 = severe hind limb paralysis; grade 4 = severe hind limb paralysis and loss of righting reflex; and grade 5 = death.
Plaque assay
After cardiac perfusion with cold Hank's balanced salt solution (HBSS) (Mediatech), brain and spinal cords were removed. The tissues were homogenized in HBSS using a tissue homogenizer. A standard plaque assay was performed on BHK-21 cell monolayers [
33]. Plaques in the BHK monolayer were visualized by staining with 0.1% crystal violet solution after fixing with methanol.
Isolation of CNS-infiltrating lymphocytes
Mice were perfused through the left ventricle with 30 ml of sterile HBSS. Excised brains and spinal cords were forced through wire mesh and incubated at 37°C for 45 min in 250 μg/ml of collagenase type 4 (Worthington). CNS-infiltrating lymphocytes were then enriched at the bottom 1/3 of a continuous 100% Percoll (GE) gradient after centrifugation for 30 min at 27,000 × g.
Flow cytometry
CNS-infiltrating lymphocytes were isolated and Fc receptors were blocked using 100 μl of 2.4G2 hybridoma (ATCC) supernatant by incubating at 4°C for 30 minutes. The indicated antibodies were subsequently used to stain various cell types. VP3159-166-loaded H-2Ks tetramer labeled with PE was used to assess levels of virus-specific CD8+ T cells in the CNS of TMEV-infected mice. Cells were analyzed using a Becton Dickinson LSRII flow cytometer.
Intracellular cytokine staining
Freshly isolated CNS-infiltrating mononuclear cells were cultured in 96-well round bottom plates in the presence of viral or control peptides and Golgi-Plug™ (BD) for 6 h at 37°C. Cells were then incubated in 100 μl of 2.4G2 hybridoma (ATCC) supernatant for 30 minutes at 4°C to block Fc receptors. Anti-CD8 (clone 53-6.7) antibody or anti-CD4 (clone L3T4) antibody was added, and cells were incubated for an additional 30 minutes at 4°C. After two washes, intracellular IFN-γ staining was performed according to the manufacturer's instructions (BD) using PE-labeled rat monoclonal anti-IFN-γ (XMG1.2) antibody. Cells were analyzed by flow cytometry.
RT-PCR and real-time PCR
Total RNA was isolated by TRIzol reagent (Invitrogen) and reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen). The cDNAs were amplified with specific primer sets using the SYBR Green Supermix (Bio-Rad) on an iCycler (Bio-Rad). The sense and antisense primer sequences used for cytokines are as follows: TMEV (VP1), (5'-TGACTAAGCAGGACTATGCCTTCC-3' and 5'-CAACGAGCCACATATGCGGATTAC-3'); IL-1β, (5'-TCATGGGATGATAACCTGCT-3' and 5'-CCCATACTTTAGGAA-GACACGGAT-3'); IFN-α, (5'-ACCTCCTCTGACCCAGGAAG -3' and 5'-GGCTCTCCAGA-CTTCTGCTC-3'); IFN-β, (5'-CCCTATGGAGATGACGGAGA-3' and 5'-CTGTCTGCTGG-TGGAGTTGA-3'); IFN-γ, (5'-ACTGGCAAAAGGATGGTGAC-3' and 5'-TGAGCTCATT-GAATGCTT GG-3'); IL-10, (5'-GCCAAGCCTTATCGGAAATGATCC-3' and 5'-AGACA-CCTTGGTCTTGGAGCTT-3'); TNF-α, (5'-CTGTGAAGGGAATGGGTGTT-3' and 5'-GGTCACTGTCCCAGCATCTT-3'); IL-6, (5'-AGTTGCCTTCTTGGGACTGA-3' and 5'-TCCACGATTTCCCAGAGAAC-3'); IL-17, (5'-GGGGATCCATGAGTCCAGGGAGAGC-3' and 5'-CCCTCGAGTTAGGCTGCCTGGCGGA-3'); CXCL10, (5'-AAGTGCTGCCGTC-ATTTTCT-3' and 5'-GTGGCAATGATCTCAACACG-3') and GAPDH, (5'-AACTTTGG-CATTGTGGAAGGGCTC-3' and 5'-TGCCTGCTTCACCACCTTCTTGAT-3'). GAPDH expression served as an internal reference for normalization. Real-time PCR was performed in triplicate.
Statistical analyses
The statistical significance of the differences between experimental groups (two-tailed p value) was analyzed with the unpaired Student's t-test using the InStat Program (GraphPAD). Comparisons of the disease courses between 2 groups were also performed using the paired t-test. Values of P < 0.05 were considered to be significant.
Discussion
We have previously demonstrated that cells infected with TMEV stimulate the innate inflammatory response mainly via TLR3-mediated signaling [
16,
17]. However, the role of TMEV-induced TLR3 signaling in protection from and/or pathogenesis of demyelinating disease remains unknown. In this study, we examined the potential role of TLR3 in the progression of TMEV-induced demyelinating disease by utilizing TLR3 KO mice and administering TLR3 ligand. Our results demonstrate that TLR3-mediated signals do not play a major role in the protection of mice in the resistant C57BL/6 background against BeAn, a less virulent strain of TMEV. However, TLR3 stimulation plays a protective role in infection with GDVII, a neurovirulent TMEV strain (Figure
1). These results are consistent with previous studies demonstrating that the absence of TLR3 in B6 mice does not alter the adaptive immune response or viral pathogenesis of chronic viral infections [
10]. In contrast, it has also been reported that the presence of TLR3 provides protection from acute viral infections with West Nile virus [
6] and Coxsackievirus B4 [
7]. Therefore, it appears that TLR3 may provide some protection against acute or virulent viral infections but not against non-virulent viral infections.
In contrast to resistant C57BL/6 mice, SJL mice are susceptible to persistent chronic infection in the CNS with the less virulent BeAn strain of TMEV, and the majority of infected mice develop demyelinating disease starting from 20-35 dpi [
15]. Our current results indicate that the presence of TLR3-mediated signals provides protection from the development of TMEV-induced demyelinating disease in susceptible SJL mice, as TLR3-deficient mice with the SJL background genes showed elevated viral loads in the CNS and exacerbated disease development (Figures
2 and
3). Therefore, TLR3-mediated protection may play an important role in the susceptible host that only mounts a marginal protective response against chronic viral infections. While the early adaptive immune response to viral infections was not altered in the absence of TLR3-mediated signals (Figure
5), consistent with a previous report [
10], cellular infiltration into the CNS was markedly elevated (Figure
4A and
4B), resulting in exacerbation of TMEV-induced immune-mediated demyelinating disease (Figure
2A). The increased cellular infiltration may be due to high viral loads in the absence of TLR3 signals (Figure
2B), which leads to high levels of proinflammatory cytokine production in the CNS, thus facilitating cellular infiltration (Figure
4C). However, the elevated cytokine production in the CNS of virus-infected TLR3KO mice was unexpected, as TLR3 is essential for the production of cytokines, such as type I IFNs and IL-6, in TMEV infected glial cells [
16,
17]. Therefore, these results strongly suggest that high viral loads in the CNS led to the utilization of an alternative innate immunity pathway, such as MDA5 and/or PKR, which stimulate proinflammatory cytokine production, as previously described [
4,
24,
43,
44]. Since cells from TLR3KO mice can also produce cytokines upon stimulation with poly IC, these alternative signal-triggering molecules appear to be operational in these mice (not shown). Nevertheless, TLR3-mediated signals appear to provide a protective function, particularly in hosts susceptible to virus-induced disease.
It is interesting to note that there is a disconnect between the levels of type I IFNs and control of TMEV infection, hence TRLR3KO mice display higher levels of type I IFNs yet more susceptible to TMEV infection (Figures
2 and
4). These results are inconsistent with the previous studies with IFNIR-KO mice, which displayed fatal encephalitis upon TMEV infection [
26,
45]. Therefore, the presence of a certain level of type I IFN signaling during early TMEV infection appears to be necessary for survival of the animals. The high level of type I IFN production in TLR3KO mice is likely activated via primarily MDA5 signaling by a high viral load, because an MDA5-mediated signal is the major activator for type I IFN production in mice following infection with TMEV [
24,
46]. However, high levels of type I IFNs may not be necessarily helpful in controlling viral infection. In fact, both IFN-α and IFN-β levels were significantly higher in mice pretreated with poly IC compared to either untreated or treated at 8 dpi (Additional file
2, Figure S2). Furthermore, our previous results indicated that susceptible SJL mice produce higher levels of type I IFNs compared to resistant B6 mice and a high level of IFNs exacerbates viral infection by inhibiting induction of protective immune responses [
21]. Therefore, the exceeding levels of type I IFNs appear to play a detrimental role in the protection from virus-induced chronic demyelinating disease.
Interestingly, premature activation of TLR3 via administration of poly IC prior to viral infection promoted disease progression. In contrast, additional TLR3 signals by poly IC after viral infection yielded a clinical improvement and less pathogenic immune responses in the CNS (Figure
6). These results suggest that TLR3 signaling provides differential protection against viral infection, depending on the time of the signals with respect to viral infection. It was previously shown that the presence of external poly IC mainly stimulates TLR3-mediated signals for the production of various proinflammatory cytokines in many different cell types, including macrophages, microglia, and astrocytes [
3,
16,
19]. Poly IC, a TLR3 ligand, has previously been used to protect the host from acute viral infections. Administration of poly IC between < 72 hours prior to infection and < 24 post infection with foot and mouth disease virus protected mice from death [
47]. Similarly, poly IC treatment at 1 day prior to infection through 4 hours post virus challenge effectively prolonged the survival of mice from herpes simplex virus 2 challenges [
28]. Therefore, the efficacy of TLR3-mediated protection from acute viral infection appears to be limited to a narrow time window. Furthermore, such poly IC treatment prior to viral infection may exacerbate the development of chronic virus-infection induced immune-mediated diseases, such as TMEV-induced demyelinating disease (Figure
6). Interestingly, it has recently been shown that poly IC treatment enhances autoimmune disease in a retinal autoimmunity model [
32]. Therefore, it is conceivable that the exacerbation of virus-induced disease by pretreatment with poly IC may not be limited to the development of chronic viral infection-induced immune mediated disease.
In contrast to the treatment with poly IC prior to viral infection, poly IC administration at 8 days after TMEV infection ameliorated disease development (Figure
6A). Recently, it has been shown that poly IC treatment of mice at 4 and 8 days after infection with Friend retrovirus reduces viral loads and promotes protection from the development of chronic viral infection-induced leukemia over a period of several weeks [
29]. Therefore, TLR3-mediated signaling during chronic viral infection, particularly infections leading to immune-mediated diseases, appears to be protective, whereas premature activation of the signals prior to and/or at the time of viral infection may exacerbate the pathogenesis.
Our further analyses of the immune response in poly IC treated mice showed marked reductions in protective, virus-specific IFN-γ-producing CD4
+ and CD8
+ T cell responses in poly IC pretreated mice, in contrast to increases in poly IC post-treated mice (Figure
6E and
6F). Furthermore, poly IC-pretreated mice displayed elevated expression of a T cell inhibitor, PDL-1, and an increased generation of regulatory FoxP3
+ CD4
+ T cells in the CNS, while poly IC-post-treated mice expressed reduced levels of PDL-1 and FoxP3
+ CD4
+ T cells (Figure
7). The engagement of PD-1/2 or CD80 with PDL-1 exerts a powerful inhibitory function for CD4
+ as well as CD8
+ T cells in many virus systems (13). In addition, poly IC treatment is also known to upregulate PDL-1 expression [
36,
37]. Furthermore, it is interesting to note that poly IC-pretreated mice uniquely showed an increased level of FoxP3
+ regulatory CD4
+ T cells in the CNS of virus-infected mice. Although the underlying mechanisms for the increase are unknown, elevated levels of cytokines in the CNS of mice with high viral loads favoring the generation of FoxP3
+ CD4
+ T cells may contribute to the increase of the regulatory T cells. Nevertheless, FoxP3
+ CD4
+ T cells generated in virus-infected hosts, including TMEV-infected mice, inhibit virus-specific CD4
+ as well as CD8
+ T cell function [
42,
48]. Therefore, these results strongly suggest that the activation of TLR3 signaling prior to virus infection may induce premature stimulation of regulatory immune mechanisms, hindering anti-viral immune cell function and leading to viral persistence. On the other hand, further activation of TLR3 signaling after viral infection appears to enhance anti-viral T cell function by reducing the expression of inhibitory PDL-1 and preventing the generation of regulatory T cells. These observations are particularly important, as the results imply that TLR-mediated stimulation of innate immunity as an intervention strategy for the treatment of viral infections could exacerbate the development of chronic immune-mediated disease. Therefore, the timing of innate immunity stimulation should be carefully considered.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YHJ and MHK investigated disease development. YHJ performed immunological studies and contributed to writing the manuscript. HSK generated peptide-loaded tetramers. TK and CSK performed histological studies and contributed to analysis. CSK and BSK analyzed the data and wrote the manuscript. All authors have seen and approved the final version of the manuscript.