Multiple sclerosis (MS) is a human CNS autoimmune demyelinating disease. Epidemiological evidence has suggested a role for virus infection in the initiation and/or exacerbation of MS. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease serves as a relevant mouse model for MS. TMEV-infected mice develop a demyelinating disease with clinical symptoms beginning around 35 days after infection, which is associated with development of myelin-specific, PLP139–151, CD4+ T cell responses. Viruses have been suggested to initiate autoimmune disease through bystander activation of immune cells or through bystander damage to tissue during infection. We examined the effect of the innate immune response on development of autoimmune demyelinating disease by altering the innate immune response through administration of innate immune cytokines, IFN-α or IFN-β, or antiserum against the type I IFNs during the innate immune response to TMEV. Administration of IFN-β, but not IFN-α, to TMEV- infected mice led to reduced myelin-specific CD4+ T cell responses and reduced demyelinating disease, which was associated with decreased immune cell infiltration into the CNS and increased expression of IL-10 in the CNS. Conversely, administration of antiserum to IFN-β led to a more severe demyelinating disease. In addition, administration of poly(I:C), which is an innate immune agonist, to TMEV-infected mice during the innate immune response resulted in decreased myelin-specific CD4+ T cell responses and reduced demyelinating disease. These results demonstrate that activating or enhancing the innate immune response can reduce the subsequent initiation and progression of the autoimmune response and demyelinating disease.

Multiple sclerosis (MS)3 is a demyelinating disease affecting humans that is associated with an inflammatory autoimmune response in the CNS. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease serves as a mouse model of MS. TMEV infection of susceptible SJL mice results in a persistent infection of the CNS. Clinical signs of demyelinating disease first appear around 35–40 days after infection and continue as a chronic progressive disease characterized by ascending hind limb paralysis. TMEV-induced demyelinating disease is associated with development of myelin-specific CD4+ T cell responses via the process of epitope spreading. The initial autoimmune CD4+ T cell response is directed against proteolipid protein, epitope PLP139–151, which is first detected around 55 days after infection and then progresses to additional myelin Ags by epitope spreading ∼90 days after infection (1). Most significantly, the autoreactive CD4+ T cell response plays a pathologic role in chronic disease progression as shown by reduction in disease progression following induction of peripheral tolerance to PLP Ags after disease onset (2). Therefore, TMEV infection of mice results in an autoimmune-mediated demyelinating disease.

The innate immune response recognizes microbial infections and initiates the immune response to the infecting pathogen. The innate immune response recognizes pathogens by pathogen-associated molecular patterns (PAMPs), which enables the differentiation between self and non-self. Pattern recognition receptors, including the TLRs, recognize PAMPs and signal the cell to respond to the pathogen. Several agonists for TLRs have been determined including viral PAMPs such as dsRNA recognized by TLR3 and ssRNA recognized by TLR7/TLR8 (3, 4). TLRs signal through a common adaptor protein, MyD88, which leads to the downstream activation of NF-κΒ family of transcription factors (5). However, some TLRs, including TLR3, signal through a MyD88-independent pathway that leads to downstream activation of IFN response factor, IFN regulatory factor (IRF) 3, resulting in expression of IFN-inducible genes (6, 7). Recent studies have shown that intracellular innate immune receptors RIG-I and MDA-5 can also recognize viral PAMPs (8). Overall, the engagement of TLRs or other innate immune receptors leads to the transcriptional activation of cytokines, chemokines, and effector molecules as well as costimulatory molecules and MHC class I and MHC class II. Therefore, the innate immune response directly influences the development of the adaptive immune response by influencing the activation of T cells and B cells. Most recently, the innate immune response has been associated with recognition of self-tissue during tissue damage in the absence of pathogens. Damage-associated molecular patterns (DAMPs) are released from dying cells during tissue damage and include high-mobility group box 1 protein, heat shock proteins, uric acid, altered matrix proteins, and S100 proteins. DAMPs are danger signals that can mediate an inflammatory response through TLRs and the receptor for advanced glycation end products (9). A recent report has suggested a pivotal role for TLR2 and TLR4 in the immune response during spinal cord injury (10). Therefore, the innate immune response can recognize danger signals coming from pathogens during infection as well as from cell damage during injury.

Type I IFNs, IFN-α and IFN-β, are immediately induced in response to virus infections and are identified as antiviral proteins. However, several lines of evidence suggest that type I IFNs have multiple functions in the immune response to virus infection. IFN-α and IFN-β promote NK cell-mediated cytotoxicity as well as blastogenesis and proliferation (11). Type I IFNs have also been shown to enhance production of IFN-γ by T cells following virus infection (12, 13). IFN-α and IFN-β influence the adaptive immune response by driving the maturation of dendritic cells that promote CD4+ Th1-type response with high levels of IFN-γ secretion and CD4+ T cell proliferation (14, 15). Additional studies have shown that type I IFNs promote CD8+ T cell proliferation and survival (16) as well as enhance the B cell response (17). Type I IFN expression, specifically IFN-α4 and IFN-β, is induced in virus-infected cells through activation (phosphorylation) of IRF-3, which is constitutively expressed at low levels (14). IFN-α and IFN-β are then released from the infected cell and bind to the IFN-α/β receptor on the cell surface, which in turn induces expression of additional type I IFNs through activation of IRF-7 via a positive feedback mechanism (18). Most interesting, IFN-β is currently one of the primary treatments for patients with MS; however, the mechanism by which IFN-β modulates disease has not been completely determined.

The innate immune response directly influences the development of the adaptive immune response through the expression of cytokines and chemokines as well activation of APCs. The innate immune response may also be involved in activation of the autoimmune response through bystander activation of immune cells, especially autoreactive T cells, or through bystander damage of surrounding tissue. The current studies examined the effect of the innate immune response on development of demyelinating disease by altering the innate immune response during virus infection. Type I IFNs are the predominant cytokines produced following virus infection and have been shown to influence the adaptive immune response; thus, type I IFNs were altered during the innate immune response to TMEV to determine the effect on the development of demyelinating disease. Mice were administered type I IFNs to increase the amount of type I IFNs during the first 4 days of infection or mice were administered neutralizing antiserum to type I IFNs to reduce the amount of type I IFNs during the first 4 days of infection. In addition, mice were administered poly(I:C), which is a synthetic dsRNA that can activate the innate immune response to produce type I IFNs. These studies demonstrate that altering the innate immune response by administration of agents following TMEV infection can directly affect the development of demyelinating disease, suggesting that the innate immune response can influence the activation of the autoimmune response and development of demyelinating disease.

Five- to 6-wk-old female SJL mice were purchased from Harlan Laboratories. The mice were housed at the University of Wisconsin Research Animal Resource Center or at Northwestern University according to ACUC-approved protocols at each institution. Mice were infected by intracerebral injection with 2 × 106 PFU of the BeAn strain of TMEV. On the day of infection and 4 days after infection, mice were administered by i.p. injection: PBS (control), IFN-β (5000 U), anti-IFN-β (5000 NU), IFN-α4 (5000 U), anti-IFN-α (5000 NU; R&D Systems), poly(I:C) (150 μg; Sigma-Aldrich), or rabbit serum (control). Mice were followed for clinical disease signs daily and scores were assigned based on a scale of 0–5: score 1, mice show mild waddling gait; score 2, mice show more severe waddling gate; score 3, mice had a loss of righting ability associated with spastic hind limbs; score 4, mice had paralysis of hind limbs associated with dehydration; and score 5, mice were moribund.

The brain and spinal cord were removed from the infected mice at the indicated days after infection. The organs were homogenized and diluted in serum-free DMEM. BHK-21 cells were infected with the homogenized tissue dilutions. The cells were incubated at room temperature for 1 h. A 2% agar solution was diluted in DMEM supplemented with 2% serum and 200 μg/ml penicillin and 200 μg/ml streptomycin and was added to the cells. The cells were incubated at 34°C for 5 days. The cells were fixed with methanol and stained with crystal violet solution (0.12% crystal violet). The plaques were counted on each plate and multiplied by the dilution and the amount of homogenate was added to the plate to determine the PFU/ml. The weight of the tissue (mg) and homogenate volume was then used to calculate PFU/mg.

Brain, spinal cord, spleen, and lymph nodes were removed from mice at the indicated days postinfection. The RNA was isolated from the organs using TRIzol (Invitrogen). The RNA was DNase treated before converting the RNA into cDNA using oligo(dT)12–18 primers and a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). Real-time PCR were conducted with FastStart SYBR Green Master Mix (Roche Applied Science). Briefly, 0.5 μM primers, 1× SYBR Green Master Mix, and 2 μl of diluted cDNA were combined. The primers sequences were previously published (19). Real-time PCR was conducted on a Rotorgene 6000 (Corbett Research) using a hot start with cycle conditions, 40 cycles; 95°C 15 s, 60°C 10 s, and 72°C 15 s; followed by a melt from 75 to 95°C. Quantitation of the mRNA was based on standard curves derived from cDNA standards for each primer set that are run with the samples, and the samples are normalized using β-actin expression. Positive and negative cDNA controls were used for each primer set derived from known cell sources for each cytokine.

Brain, spinal cord, and spleen were removed from mice at the indicated days postinfection. The organs were homogenized and the supernatant was collected and analyzed using a LiquiChip mouse cytokine kit (Qiagen) per the manufacturer’s instructions. The beads were analyzed on an Luminex100 machine (Qiagen) and the cytokines were quantitated based on a set of standards for the assay.

Mice were perfused at the indicated days after infection and the brain, spinal cord, and spleen were removed. The brain and spinal cord were minced and digested with collagenase type IV (Invitrogen) and DNase (Invitrogen) for 1 h at 37°C. The organs were then dissociated through nylon mesh before the mononuclear cells were separated on a 70/30 Percoll gradient. The separated cells were washed with FACS buffer (saline with 5% normal goat serum) and blocked with Ab to CD16/32 (BD Biosciences) at 4°C for 30 min. The cells were then incubated for 45 min at 4°C with Abs conjugated to fluoresceinated Abs specific for CD4, CD8, CD11b, CD45, B220, and PanNK. The cells were washed and florescence was analyzed on a FACSCalibur (BD Biosciences). The CD45-positive cells were analyzed to determine the percentage of CD4 and CD8 T cells, macrophage, B cells, and NK cells infiltrating the CNS.

Spleens and lymph nodes were removed from infected mice at the indicated days following infection and dissociated through a stainless steel screen to obtain a homogenous cell suspension. The RBC were lysed with Tris-ammonium chloride. The cells were then cultured at 1 × 106 cells/well (96-well plates) in HL-1 medium (BioWhittaker) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Peptides were added to the wells at increasing concentrations from 1 to 100 μM. PLP139–151 (HSLGKWLGHPDKF), PLP178–191 (NTWTTCQSIAFPSK), VP270–86 (WTTSQEAFSHIRIPLPH), and VP3159–166 (FNFTAPFI) were purchased from CPC Scientific. The amino acid composition was verified by mass spectrometry and purity was assessed by HPLC. The plates were incubated at 37°C for 72 h and then pulsed with 1μCi of [3H]TdR for 24 h for proliferation assays. Proliferation was determined with triplicate wells for each peptide concentration and expressed as cpm ± SEM. For cytokine analysis, a duplicate set of proliferation wells was used to collect supernatants at 24, 48, and 72 h, and supernatants were analyzed using a mouse cytokine bead detection kit and read on an Luminex 100 (Qiagen) machine as described above. ELISPOT assays were performed by coating 96-well plates with Ab for IFN-γ, IL-2, IL-4, or TNF-α (BD Biosciences) before addition of cells and peptides as described for proliferation. Following a 24-h incubation, the plates were washed and stained with the corresponding Ab pair and detected by colorimetric methods. The plates were analyzed on a CTL ELISPOT reader.

A statistical comparison of the percentage of animals showing clinical disease between any two groups of mice will be performed by χ2 test using Fisher’s exact probability. Comparisons of differences in immunological assays between any two groups was determined using Student’s t test. Significance between any two groups was determined as ≥2-fold increase or decrease.

The innate immune response recognizes unique patterns on viruses such as dsRNA or ssRNA, which activates a signaling cascade that results in expression of innate immune cytokines. The innate immune response to virus infections is predominantly mediated by expression of type I IFNs, IFN-α and IFN-β. Most importantly, the innate immune response to infections directly contributes to the development of the adaptive immune response and possibly the autoimmune response. Thus, to determine whether the innate immune response can influence the development of autoimmune demyelinating disease after virus infection, we altered the innate immune response during TMEV infection by administration of innate immune cytokines. Mice (10 mice/group) were administered IFN-α4 or IFN-β on the day of TMEV infection and 4 days postinfection. Another group of mice (n = 10) was similarly administered poly(I:C), a synthetic dsRNA, which is recognized by innate immune receptors and induces an innate immune response that includes the expression of type I IFNs. The timing of the administration of type I IFNs and poly(I:C) was chosen to clearly differentiate the role of these molecules on early virus infection vs the effect on ongoing demyelinating disease. The TMEV-infected mice were monitored for the development and progression of demyelinating disease (Fig. 1,A). TMEV-infected mice administered IFN-α developed clinical signs of demyelinating disease similar to control-treated mice. Most interestingly, TMEV-infected mice administered IFN-β developed a less severe demyelinating disease which did not progress compared with control treated mice. Similarly, TMEV-infected mice administered poly(I:C) also developed a less severe demyelinating disease which did not progress. Coadministration of IFN-α and IFN-β to TMEV-infected mice resulted in similar clinical disease as IFN-β alone, thus there was no apparent synergistic affect between the type I IFNs (data not shown). Next, mice were administered neutralizing antiserum to type I IFNs early during TMEV infection to determine whether decreasing the innate immune cytokines altered development of subsequent demyelinating disease (Fig. 1 B). TMEV-infected mice administered antiserum to IFN-α developed demyelinating disease that was slightly, but not significantly reduced compared with control-treated mice. However, TMEV-infected mice administered antiserum to IFN-β developed a more severe demyelinating disease than that in control-treated mice. Therefore, administration of IFN-β to TMEV-infected mice during the innate immune response reduced the severity of demyelinating disease while administration of antiserum to IFN-β increased the severity of demyelinating disease. Most interestingly, administration of poly(I:C), which is an innate immune response stimulator, resulted in significantly reduced demyelinating disease.

FIGURE 1.

The innate immune response affects development of demyelinating disease. SJL mice were infected with TMEV and administered IFN-α, IFN-β, poly(I:C), or PBS (A; 10 mice/group) on the day of infection and 4 days postinfection. The mice were followed for clinical signs of demyelinating disease for 90 days following infection: PBS (▪), IFN-α (•), IFN-β (▴), and poly(I:C) (♦). The stars above the line represent a statistically significant difference in the clinical score compared with TMEV-infected mice treated with PBS. TMEV-infected mice were administered antiserum to IFN-α or IFN-β or rabbit serum (RS) (B; 10 mice/group) on the day of infection and 4 days postinfection. Mice were followed for clinical signs of demyelinating disease for 90 days following infection: RS (▪), anti-IFN-α (○), and anti-IFN-β (▵). The stars above the line represent a significant difference in clinical disease score compared with TMEV-infected mice administered rabbit serum. C, The brain and spinal cord were removed from TMEV-infected mice (three per group) on 4, 14, and 90 days postinfection and plaque assays were conducted to determine viral load. Viral load is the log of PFU per mg of tissue. The experiment was repeated five times with one representative experiment shown. D and E, The brain and spinal cord were removed from mice (three mice per group) at 7 days postinfection, and RNA was isolated from the organs and converted to cDNA. Real-time PCR was conducted on the cDNA using primers for IFN-α (D) or IFN-β (E). The concentration for each primer was based on a standard curve with cDNA of known concentration. The experiment was repeated three times with a representative of one of the experiments shown in the graph. The stars above the bars indicate a significant difference (>3-fold increase or decrease) in expression based on the expression of control-treated mice.

FIGURE 1.

The innate immune response affects development of demyelinating disease. SJL mice were infected with TMEV and administered IFN-α, IFN-β, poly(I:C), or PBS (A; 10 mice/group) on the day of infection and 4 days postinfection. The mice were followed for clinical signs of demyelinating disease for 90 days following infection: PBS (▪), IFN-α (•), IFN-β (▴), and poly(I:C) (♦). The stars above the line represent a statistically significant difference in the clinical score compared with TMEV-infected mice treated with PBS. TMEV-infected mice were administered antiserum to IFN-α or IFN-β or rabbit serum (RS) (B; 10 mice/group) on the day of infection and 4 days postinfection. Mice were followed for clinical signs of demyelinating disease for 90 days following infection: RS (▪), anti-IFN-α (○), and anti-IFN-β (▵). The stars above the line represent a significant difference in clinical disease score compared with TMEV-infected mice administered rabbit serum. C, The brain and spinal cord were removed from TMEV-infected mice (three per group) on 4, 14, and 90 days postinfection and plaque assays were conducted to determine viral load. Viral load is the log of PFU per mg of tissue. The experiment was repeated five times with one representative experiment shown. D and E, The brain and spinal cord were removed from mice (three mice per group) at 7 days postinfection, and RNA was isolated from the organs and converted to cDNA. Real-time PCR was conducted on the cDNA using primers for IFN-α (D) or IFN-β (E). The concentration for each primer was based on a standard curve with cDNA of known concentration. The experiment was repeated three times with a representative of one of the experiments shown in the graph. The stars above the bars indicate a significant difference (>3-fold increase or decrease) in expression based on the expression of control-treated mice.

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Administration of IFN-β or poly(I:C) to TMEV-infected mice during the innate immune response to TMEV reduced demyelinating disease; however, these agents were administered by i.p. injection while the infection is in the CNS. To determine whether the administration of IFN-β and poly(I:C) had an affect on the innate immune response in the CNS, the brain and spinal cord were analyzed for expression of type I IFNs at 7 days postinfection (Fig. 1, D and E). TMEV-infected mice administered IFN-α had a significant increase in the expression of IFN-α in the brain and spinal cord compared with control-treated mice. Conversely, TMEV-infected mice administered antiserum to IFN-α had a significant decrease in IFN-α expression in the CNS compared with control-treated mice. Administration of IFN-β led to a significant increase in expression of both IFN-α and IFN-β in the CNS compared with control-treated mice. Likewise, TMEV-infected mice administered poly(I:C) had a significant increase in both IFN-α and IFN-β in the CNS. TMEV-infected mice administered antiserum to IFN-β had a significant decrease in expression of IFN-β in the CNS compared with control-treated mice. Therefore, the peripheral administration of modulators of type I IFNs had a direct affect on expression of type I IFNs in the CNS of TMEV-infected mice. Furthermore, these results suggests that type I IFNs activate the innate immune response to TMEV through the IFN-α/β receptor.

Type I IFNs have been shown to have direct antiviral activities and inhibit the replication of viruses. The development of demyelinating disease following TMEV infection has been associated with persistent virus infection of the CNS. Thus, to determine whether the reduction in demyelinating disease was associated with a reduced virus load in the CNS, plaque assays were performed with the brain and spinal cord of the TMEV-infected mice that were administered IFN-α, IFN-β, or poly(I:C) or administered antiserum to IFN-α or IFN-β (Fig. 1 C). Interestingly, the treatments did not result in significant differences in the CNS virus load in the mice at 4, 14, or 90 days postinfection. Therefore, the reduced demyelinating disease observed in TMEV-infected mice administered IFN-β or poly(I:C) was not due to a reduction in infectious virus in the CNS of the mice.

Next, we determined whether TMEV-infected mice administered IFN-β or poly(I:C) had altered expression of other cytokines in the CNS during the early antiviral immune response. TMEV-infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β and were examined at 7 days postinfection for expression of cytokines in the CNS (Fig. 2). TMEV-infected mice administered IFN-β or poly(I:C) had a significant increase in secretion of IFN-γ in the brain compared with control-treated mice (Fig. 2,A). TMEV-infected mice administered IFN-β or poly(I:C) had a significant increase in secretion of IL-6 and TNF-α in the brain and spinal cord compared with control-treated mice (Fig. 2, B and C). Most interestingly, TMEV-infected mice administered IFN-β or poly(I:C) had a significant increase in IL-10 expression in the brain and spinal cord compared with control-treated mice (Fig. 2 D). TMEV-infected mice administered IFN-α or antiserum to IFN-α had similar cytokine expression in the CNS as control-treated mice. Administration of antiserum to IFN-β resulted in similar expression of IFN-γ, IL-6, and TNF-α in the CNS as controls, but these mice had decreased expression of IL-10. Thus, TMEV-infected mice administered IFN-β or poly(I:C) had increased expression of innate immune cytokines and adaptive immune cytokines such as IFN-γ, which is associated with a proinflammatory response and IL-10, which is associated with an anti-inflammatory response.

FIGURE 2.

TMEV-infected mice administered IFN-β or poly(I:C) had increased expression of innate immune cytokines in the CNS. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-α, poly(I:C), or PBS (control) on days 0 and 4. The brain and spinal cord were removed from mice (three mice per group) at 7 days postinfection. The organs were dissociated and the supernatant was used in a cytokine bead array to determine the secretion of IFN-γ (A), IL-6 (B), or TNF-α (C). The RNA was isolated from the brain and spinal cord of mice (three mice per group) at 7 days postinfection and converted to cDNA. Real-time PCR was conducted with primers for IL-10 (D). The experiment was repeated three times with one representative experiment shown. The stars above the bars indicate a significant difference in expression levels compared with control (PBS)-treated mice.

FIGURE 2.

TMEV-infected mice administered IFN-β or poly(I:C) had increased expression of innate immune cytokines in the CNS. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-α, poly(I:C), or PBS (control) on days 0 and 4. The brain and spinal cord were removed from mice (three mice per group) at 7 days postinfection. The organs were dissociated and the supernatant was used in a cytokine bead array to determine the secretion of IFN-γ (A), IL-6 (B), or TNF-α (C). The RNA was isolated from the brain and spinal cord of mice (three mice per group) at 7 days postinfection and converted to cDNA. Real-time PCR was conducted with primers for IL-10 (D). The experiment was repeated three times with one representative experiment shown. The stars above the bars indicate a significant difference in expression levels compared with control (PBS)-treated mice.

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We next examined TMEV-infected mice to determine whether the chemokine expression may be altered in mice administered IFN-β or poly(I:C), which develop a reduced demyelinating disease. Chemokines play an important role in trafficking immune cells to the site of virus infection. TMEV-infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β at days 0 and 4 postinfection, and CNS chemokine expression was analyzed at 7 days postinfection (Fig. 3). TMEV-infected mice administered IFN-β or poly(I:C) had increased expression of MIP-1α and MCP-1 in the brain and spinal cord, whereas mice administered IFN-α had increased expression of MCP-1 in the brain (Fig. 3, A and B), suggesting that increased expression of type I IFNs leads to increased expression of chemokines in the CNS. Another group of molecules which may also be important for trafficking cells to the CNS are matrix metalloproteases (MMP), which modify matrix components. MMP9 can promote extravasation of T cells and macrophage into the CNS and has been associated with promoting breakdown of the blood-brain barrier in MS patients (20). Therefore, MMP9 expression was analyzed in TMEV-infected mice administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β (Fig. 3 C). TMEV-infected mice administered IFN-β or poly(I:C) had decreased expression of MMP9 in the brain and spinal cord compared with control-treated mice. In addition, TMEV-infected mice administered antiserum to IFN-α also had decreased expression of MMP9 in the spinal cord. Therefore, TMEV-infected mice administered IFN-β or poly(I:C) develop a less severe demyelinating disease associated with decreased expression of MMP9.

FIGURE 3.

Chemokine and MMP expression was altered in TMEV-infected mice administered IFN-β or poly(I:C). TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The brain and spinal cord was removed from mice (three mice per group) at 7 days postinfection. The RNA was isolated from the organs, converted to cDNA, and real-time PCR was conducted with primers specific for MIP-1α (A), MCP-1 (B), or MMP9 (C). The experiment was repeated three times with one representative experiment shown. The stars above the bars represent a significant difference (increase or decrease) in expression compared with control (PBS)-treated mice.

FIGURE 3.

Chemokine and MMP expression was altered in TMEV-infected mice administered IFN-β or poly(I:C). TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The brain and spinal cord was removed from mice (three mice per group) at 7 days postinfection. The RNA was isolated from the organs, converted to cDNA, and real-time PCR was conducted with primers specific for MIP-1α (A), MCP-1 (B), or MMP9 (C). The experiment was repeated three times with one representative experiment shown. The stars above the bars represent a significant difference (increase or decrease) in expression compared with control (PBS)-treated mice.

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As discussed above, TMEV-infected mice administered IFN-β or poly(I:C) have altered chemokine and MMP9 expression compared with control-treated mice, suggesting that immune cell infiltration into the CNS following administration of IFN-β or poly(I:C) may be altered. To determine whether the immune cells entering the CNS after TMEV infection were altered, TMEV-infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β and examined for infiltrating immune cells in the CNS at 7 days postinfection (Fig. 4). TMEV-infected mice administered IFN-β or poly(I:C) had reduced numbers of CD4+ and CD8+ T cells in the CNS compared with control-treated mice. Likewise, TMEV-infected mice administered IFN-β or poly(I:C) had a slight reduction in macrophages and neutrophils infiltrating into the CNS; however, the reduction was not significant compared with control-treated mice (data not shown). Thus, administration of IFN-β or poly(I:C) reduced immune cell infiltration, specifically T cells, into the CNS compared with control-treated mice.

FIGURE 4.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced immune cell infiltration into the CNS early during infection. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The brain and spinal cord were removed from the mice (three mice per group) at 7 days postinfection, and the mononuclear cells were isolated. The mononuclear cells were stained with fluoresceinated Abs specific for CD4 (A) or CD8 (B). The number of cells staining positive for each Ab was determined based on the total number of CD45-positive cells in the CNS of each mouse. The experiment was repeated four times with one representative experiment shown. The stars above the bars represent a significant difference compared with control (PBS)- treated mice.

FIGURE 4.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced immune cell infiltration into the CNS early during infection. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The brain and spinal cord were removed from the mice (three mice per group) at 7 days postinfection, and the mononuclear cells were isolated. The mononuclear cells were stained with fluoresceinated Abs specific for CD4 (A) or CD8 (B). The number of cells staining positive for each Ab was determined based on the total number of CD45-positive cells in the CNS of each mouse. The experiment was repeated four times with one representative experiment shown. The stars above the bars represent a significant difference compared with control (PBS)- treated mice.

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The CD4+ and CD8+ T cell response to TMEV is activated by 7 days postinfection, which correlates with peak virus loads in the CNS. We next examined whether TMEV-infected mice administered IFN-β or poly(I:C) have an altered virus-specific CD4+ and CD8+ T cell responses. TMEV-infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β at days 0 and 4 after infection, and the T cell responses in the spleen and lymph nodes were determined at 7 days after infection to TMEV-specific CD4+ T cell epitope VP270–86 or TMEV-specific CD8+ T cell epitope VP3159–166 (Fig. 5). Both the VP270–86-specific CD4+ T cell response and the VP3159–166-specific CD8+ T cell response in the TMEV- infected mice administered IFN-β or poly(I:C) were similar to the response in the control-treated mice. Therefore, administration of IFN-β or poly(I:C) to TMEV-infected mice does not alter the peripheral T cell response to virus. Thus, TMEV-infected mice administered IFN-β or poly(I:C) have similar virus loads in the CNS and similar virus-specific T cell responses as control-treated mice, suggesting that IFN-β and poly(I:C) do not affect virus clearance by altering the virus-specific T cell response.

FIGURE 5.

The CD4+ and CD8+ T cell responses against TMEV were not affected by altering the innate immune response. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The spleens were removed from the mice (three mice per group) at 7 days postinfection, and a single-cell suspension was obtained followed by lysis of the RBC. IFN-γ-specific ELISPOT assays were conducted by adding the splenocytes and CD8+ T cell-specific peptide VP3159–166 (A) or CD4+ T cell-specific peptide VP270–86 (B). The number of IFN-γ-expressing cells per 5 × 105 splenocytes was determined. The experiment was repeated five times with one representative experiment shown.

FIGURE 5.

The CD4+ and CD8+ T cell responses against TMEV were not affected by altering the innate immune response. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The spleens were removed from the mice (three mice per group) at 7 days postinfection, and a single-cell suspension was obtained followed by lysis of the RBC. IFN-γ-specific ELISPOT assays were conducted by adding the splenocytes and CD8+ T cell-specific peptide VP3159–166 (A) or CD4+ T cell-specific peptide VP270–86 (B). The number of IFN-γ-expressing cells per 5 × 105 splenocytes was determined. The experiment was repeated five times with one representative experiment shown.

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TMEV-infected mice develop an autoimmune CD4+ T cell response to myelin Ags, which can first be detected ∼55 days postinfection, and is directed to the immunodominant PLP epitope PLP139–151 (1). As disease progresses, epitope spreading of the CD4+ T cell response results in reactivity to additional myelin Ags, such as PLP178–191, at ∼90 days after infection. Previous studies have shown that tolerizing TMEV-infected mice to myelin-specific CD4+ T cells reduced disease progression, which directly demonstrated that the myelin-specific CD4+ T cell response is involved in chronic TMEV-induced demyelinating disease (2). Since TMEV-infected mice treated with IFN-β or poly(I:C) develop a reduced demyelinating disease, these mice were examined for development of myelin-specific CD4+ T cell responses. TMEV- infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β at days 0 and 4 postinfection. At 63 days after infection, mice were analyzed for the CD4+ T cell response to virus and myelin Ags in the spleen and lymph nodes by performing a proliferation assay (Fig. 6,A). TMEV-infected mice have a viral-specific VP270–86 CD4+ T cell response throughout TMEV-induced demyelinating disease. At 63 days postinfection, TMEV-infected mice have a myelin, PLP139–151-specific CD4+ T cell response but have not yet developed a PLP178–191-specific CD4+ T cell response. TMEV- infected mice administered IFN-β or poly(I:C) did not develop a significant virus, VP270–86-specific CD4+ T cell response. Most interestingly, TMEV-infected mice administered IFN-β or poly(I:C) did not develop myelin-specific CD4+ T cell responses to the immunodominant myelin epitope PLP139–151 or to the spread epitope PLP178–191. Next, the supernatants from the proliferation assays were examined to determine the activation of CD4+ T cells and to determine whether the CD4+ T cell responses were Th1- or Th2-type responses (Fig. 6, B–D). TMEV-infected mice had virus- and myelin-specific CD4+ T cells that secrete IL-2 and IFN-γ but not IL-4, demonstrating activation of the CD4+ T cells to a Th1-type phenotype. TMEV-infected mice administered IFN-β or poly(I:C) did not develop virus- or myelin-specific CD4+ T cell responses that secrete IL-2, IFN-γ, or IL-4 during demyelinating disease. Therefore, TMEV-infected mice administered IFN-β or poly(I:C) during the innate immune response do not develop myelin-specific CD4+ T cell responses, corresponding with the reduced demyelinating disease in these mice.

FIGURE 6.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced autoimmune CD4+ T cell responses. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The spleen was removed from mice (three mice per group) at 60 days postinfection and then dissociated into a single-cell suspension. The splenocytes were used in a proliferation assay with CD4+ T cell peptides specific for virus Ag VP270–86 or myelin Ags PLP139–151 and PLP178–191 (A). Proliferation was determined by pulsing the cells with 1 μCi of [3H]TdR for the last 24 h of culture. The cpm were determined for each peptide group. The supernatants from the proliferation cultures were removed and analyzed for IFN-γ (B)-, IL-2 (C)-, or IL-4 (D)-specific responses using ELISA kits. The stars above the bars indicate a significant increase compared with naive mice. The experiment was repeated five times with one representative experiment shown.

FIGURE 6.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced autoimmune CD4+ T cell responses. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The spleen was removed from mice (three mice per group) at 60 days postinfection and then dissociated into a single-cell suspension. The splenocytes were used in a proliferation assay with CD4+ T cell peptides specific for virus Ag VP270–86 or myelin Ags PLP139–151 and PLP178–191 (A). Proliferation was determined by pulsing the cells with 1 μCi of [3H]TdR for the last 24 h of culture. The cpm were determined for each peptide group. The supernatants from the proliferation cultures were removed and analyzed for IFN-γ (B)-, IL-2 (C)-, or IL-4 (D)-specific responses using ELISA kits. The stars above the bars indicate a significant increase compared with naive mice. The experiment was repeated five times with one representative experiment shown.

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As shown above, TMEV-infected mice administered IFN-β or poly(I:C) develop a reduced demyelinating disease that is associated with reduced autoreactive CD4+ T cell responses. TMEV-induced demyelinating disease is associated with infiltration of activated T cells and macrophages into the spinal cord, which mediate myelin destruction. Therefore, TMEV-infected mice administered type I IFNs or poly(I:C) were next examined for infiltration of immune cells into the CNS during demyelinating disease. TMEV-infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β at days 0 and 4 postinfection. At 63 days postinfection, the mice were examined for infiltration of immune cells into the CNS by flow cytometric analysis (Fig. 7). TMEV-infected mice had significant numbers of CD4+ T cells, CD8+ T cells, and macrophages (CD11b+CD11cCD45high) in the spinal cord with fewer numbers of B cells and dendritic cells (CD11c+) (Fig. 7). Administration of IFN-β or poly(I:C) significantly reduced the numbers of CD4+ T cells, CD8+ T cells, macrophages, B cells, and dendritic cells in the CNS compared with control-treated mice during demyelinating disease, which correlates with reduced demyelination (data not shown) and reduced clinical disease in these mice.

FIGURE 7.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced immune cell infiltration into the CNS during demyelinating disease. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The spinal cord was removed from mice (three mice per group) at 60 days postinfection. The mononuclear cells were isolated from the organs and stained with fluoresceinated Abs specific for CD45, CD4, CD8, CD11b, B220, and CD11c. Flow cytometric analysis was conducted to determine the number of CD45high cells in the CNS per mouse that were (A) CD4+ T cells, (B) CD8+ T cells, (C) macrophages (CD45highCD11b+CD11c), (D) B cells (B220+), and (E) dendritic cells (CD11c+ and CD11b+CD11c+). The stars above the bars represent a significant difference in the number of cells in the CNS compared with control (PBS)-treated mice. The experiment was repeated five times with one representative experiment shown.

FIGURE 7.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced immune cell infiltration into the CNS during demyelinating disease. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and 4 postinfection. The spinal cord was removed from mice (three mice per group) at 60 days postinfection. The mononuclear cells were isolated from the organs and stained with fluoresceinated Abs specific for CD45, CD4, CD8, CD11b, B220, and CD11c. Flow cytometric analysis was conducted to determine the number of CD45high cells in the CNS per mouse that were (A) CD4+ T cells, (B) CD8+ T cells, (C) macrophages (CD45highCD11b+CD11c), (D) B cells (B220+), and (E) dendritic cells (CD11c+ and CD11b+CD11c+). The stars above the bars represent a significant difference in the number of cells in the CNS compared with control (PBS)-treated mice. The experiment was repeated five times with one representative experiment shown.

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Demyelinating disease is associated with a Th1-type, proinflammatory immune response in the CNS, and reduction in demyelinating disease has been associated with a Th2-type response. Therefore, to determine whether the TMEV-infected mice administered IFN-β or poly(I:C) have altered cytokine expression in the CNS, mice were analyzed for expression of cytokines which promote either a Th1- or Th2-type immune response. TMEV-infected mice were administered IFN-α, IFN-β, poly(I:C), or antiserum to IFN-α or IFN-β during the innate immune response. At 63 days postinfection, the brain and spinal cord were removed from the mice and analyzed for expression of IFN-γ, TNF-α, IL-12, or IL-10 (Fig. 8). TMEV-infected mice expressed significant amounts of proinflammatory cytokines, IFN-γ, TNF-α, and IL-12 in the CNS during demyelinating disease. Administration of IFN-β or poly(I:C) significantly reduced expression of IFN-γ, TNF-α, and IL-12 in the CNS compared with control-treated mice. Most interestingly, TMEV-infected mice administered IFN-β or poly(I:C) had significantly increased expression of IL-10 in the CNS compared with control-treated mice. The increased expression of IL-10 was mediated by both resident microglia cells as well as infiltrating cells which included macrophages and CD4+ and CD8+ T cells (supplemental Fig. 14). TMEV-infected mice administered antiserum to IFN-α had a significantly reduced expression of IL-12 in the CNS as well as a slightly reduced expression of TNF-α. Therefore, administration of IFN-β or poly(I:C) during the innate immune response resulted in a long-lasting alteration of cytokine expression in the CNS during demyelinating, with decreased expression of proinflammatory cytokines and increased expression of the anti-inflammatory cytokine IL-10.

FIGURE 8.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced expression of proinflammatory cytokines in the CNS during demyelinating disease. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and day 4 postinfection. The brain and spinal cord were removed from mice (three mice per group) at 60 days postinfection. The RNA was isolated from the organs, converted to cDNA, and real-time PCR was conducted with primers specific for IFN-γ (A), IL-10 (B), TNF-α (C), or IL-12 (D). The concentration for each primer was determined by a standard curve with cDNA of a known concentration. The stars above the graph represent a significant difference in expression compared with control (PBS)-treated mice. The experiment was repeated three times with one representative experiment shown.

FIGURE 8.

TMEV-infected mice administered IFN-β or poly(I:C) had reduced expression of proinflammatory cytokines in the CNS during demyelinating disease. TMEV-infected mice were administered IFN-α, anti-IFN-α, IFN-β, anti-IFN-β, poly(I:C), or PBS (control) on days 0 and day 4 postinfection. The brain and spinal cord were removed from mice (three mice per group) at 60 days postinfection. The RNA was isolated from the organs, converted to cDNA, and real-time PCR was conducted with primers specific for IFN-γ (A), IL-10 (B), TNF-α (C), or IL-12 (D). The concentration for each primer was determined by a standard curve with cDNA of a known concentration. The stars above the graph represent a significant difference in expression compared with control (PBS)-treated mice. The experiment was repeated three times with one representative experiment shown.

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TMEV infection of SJL mice results in a persistent virus infection of the CNS which leads to the development of a chronic progressive demyelinating disease that has immunological and pathological similarities to MS. MS has been associated with an inflammatory immune response in the CNS, and myelin-specific CD4+ T cells have been isolated from MS patients. The causative agent of MS is unknown; however, epidemiological evidence suggests that an infectious agent may be involved in initiation of disease. Viruses have been proposed to trigger the development of autoimmune diseases through bystander activation of immune cells or bystander damage of local tissue during infection. The innate immune response is the immediate immune response to a virus infection, and it distinguishes self from non-self. The innate immune response induces the secretion of cytokines which initiate the antiviral immune response and influence the development of the adaptive immune response. The innate immune response also induces the secretion of chemokines which attract immune cells to the site of infection. Finally, the innate immune response activates NK cells and neutrophils which control the spread of the virus and activates APCs which mediate the Ag-specific adaptive immune response. Thus, the innate immune response controls the spread of infection and damage to local tissue as well as activates the adaptive immune response that will ultimately clear the virus from the host. Therefore, the innate immune response may influence the development of an autoimmune response following virus infection through bystander activation of autoreactive T cells at the site of infection or through bystander damage to tissue at the site of infection. We examined the role of the innate immune response during early TMEV infection on the development and progression of autoimmune demyelinating disease. Since the innate immune response to virus infection is predominantly mediated by type I IFNs, IFN-α and IFN-β, our studies focused on determining the effect of altering the amount of type I IFN present during the innate immune response to TMEV on development of the anti-myelin-specific immune responses and on development of demyelinating disease.

Administration of either type I IFNs, IFN-α and IFN-β, or poly(I:C) to TMEV-infected mice during the innate immune response resulted in increased expression of the respective cytokines in the CNS. The most abundant source of type I IFN expression in the CNS following TMEV infection are microglial cells and, to a lesser extent, astrocytes (21, 22). Conversely, administration of antiserum to IFN-α or IFN-β during the innate immune response in TMEV-infected mice led to decreased CNS expression of IFN-α or IFN-β. Manipulations of the levels of IFN-β in TMEV-infected mice led to significant changes in subsequent disease in that administration of IFN-β resulted in less severe demyelinating disease, while administration of antiserum to IFN-β resulted in a more severe demyelinating disease. These results indicate that IFN-β expressed during the innate immune response mediates protection from development of demyelinating disease. In contrast, manipulations of the levels of IFN-α in TMEV-infected mice had no significant effects on disease development. IFN-α and IFN-β share a common receptor complex composed of two major subunits, IFNAR-1 and IFNAR-2. Although IFN-α and IFN-β bind to the same receptor complex and can elicit similar responses, IFN-α and IFN-β can also have different effects on the immune response. Previous studies have suggested that IFN-β may form a more stable interaction with the receptor complex than IFN-α, leading to conformational differences that influence the response elicited by receptor engagement (23). Thus, even though IFN-α and IFN-β bind to the same receptor, our results suggest that they have different effects on the innate immune response and development of demyelinating disease. Most interestingly, administration of poly(I:C) in TMEV-infected mice during the innate immune response resulted in less severe demyelinating disease. These results show that altering the innate immune response during early virus infection directly affects the development of demyelinating disease, indicating that the innate immune response to virus infection directly affects development of autoimmune disease.

The development of demyelinating disease following TMEV infection is dependent on the establishment of persistent virus infection of microglia and macrophages in the CNS (22). Type I IFNs are antiviral proteins that can directly inhibit virus replication. Therefore, one possible explanation for the less severe demyelinating disease may be reduced levels of infectious virus in the CNS of the mice. However, the TMEV-infected mice administered type I IFNs during the innate immune response had similar amounts of infectious virus in the CNS compared with controls, suggesting that the reduction in demyelinating disease may be due to an effect on the immune response during virus infection.

The innate immune response is mediated by the expression of innate immune cytokines that coordinate the innate immune response as well as influence the development of the adaptive immune response. TMEV is associated with the development of a Th1-type, proinflammatory immune response in the CNS. TMEV-infected mice administered IFN-β or poly(I:C) had increased expression of proinflammatory cytokines (IFN-γ, IL-6, and TNF-α) during the innate immune response compared with control-treated mice. Most interestingly, TMEV-infected mice administered IFN-β or poly(I:C) also had increased the expression of IL-10, which is normally associated with an anti-inflammatory immune response. These results suggest that the innate immune response was enhanced in the mice administered IFN-β or poly(I:C) and that the increased expression of IL-10 may play a role in reducing the development of the proinflammatory immune response associated with demyelinating disease. Furthermore, the innate immune response induces the expression of chemokines and trafficking molecules that aid in the attraction of immune cells to the site of infection. TMEV-infected mice administered IFN-β or poly(I:C) had increased expression of chemokines early postinfection; however, these mice had decreased infiltration of T cells and macrophages into the CNS. Interestingly, TMEV-infected mice administered IFN-β or poly(I:C) had decreased expression of MMP9, which is a molecule that facilitates cell migration across the blood-brain barrier (24). Therefore, IFN-β and poly(I:C) may be mediating the trafficking of cells within the CNS but may be reducing the number of immune cells infiltrating from the periphery by maintaining the integrity of the blood-brain barrier.

TMEV infection is associated with a virus-specific CD4+ and CD8+ T cell response which can be detected at 7 days after infection, which correlates with peak virus load in the periphery and CNS, and the virus-specific CD4+ T cell response can be detected throughout the persistent infection of the CNS. The demyelinating phase of the disease is associated with development of a myelin-specific CD4+ Th1-type T cell response directed against the immunodominant myelin epitope PLP139–151, which arises via epitope spreading. Interestingly, TMEV-infected mice administered type I IFNs or poly(I:C) have similar virus-specific CD4+ and CD8+ T cell responses as the controls, suggesting that the virus-specific immune response was not significantly altered by the innate immune response. In contrast, TMEV-infected mice administered IFN-β or poly(I:C) did not develop autoreactive CD4+ T cell responses to either the immunodominant myelin Ag PLP139–151 or to other identified myelin Ags. Furthermore, administration of IFN-β or poly(I:C) reduced the expression of Th1-type cytokines in the CNS during chronic demyelinating disease and increased the expression of IL-10, which has been associated with a Th2-type response. Therefore, altering the innate immune response to TMEV significantly affected the development of the autoreactive CD4+ T cell response.

IFN-β was approved for MS treatment more than 10 years ago and remains a predominate treatment for MS. IFN-β administration to patients with relapsing-remitting disease reduces the relapse rate by 30–50% and reduces progression to disability by 30% (25). Patients also show a reduction in active lesions in the CNS by magnetic resonance imaging (26). The mechanism by which IFN-β reduces MS progression is not completely understood; however, studies suggest that the main action of IFN-β is through modulation of the inflammatory response. IFN-β has been shown to reduce the proliferation of T cells and to restore suppressor functions to T cells (27). Treatment with IFN-β has also been associated with a reduction in the proinflammatory Th1-type cytokines and an increase in Th2-type cytokines such as IL-10 resulting in a decrease in the inflammatory response. Furthermore, treatment with IFN-β in MS patients has been shown to decrease the expression of adhesion molecules and MMP9, which prevent autoreactive T cells from entering the CNS (28). The results from our studies demonstrate that administration of IFN-β to TMEV-infected mice in only two doses during the innate immune response resulted in the alteration of cytokine expression from a Th1- to a Th2-type response, the reduction of T cell infiltration associated with decreased MMP9 expression, and the decreased activation/proliferation of autoreactive myelin-specific T cells. Thus, IFN-β expression may be affecting several mechanisms of the immune response that alter the progression of demyelinating disease. In addition, our studies show that when these effects are induced during the innate immune response, IFN-β can inhibit the development of autoimmune disease. Thus, if MS is exacerbated by virus infections or sustained by a persistent/latent infection, IFN-β may be reducing the effects of the virus infection on CNS disease progression.

Poly(I:C) is a synthetic dsRNA that mimics viral RNA and has been shown to be a potent activator of the innate immune response. Our previous studies have shown that microglia isolated from the CNS of mice are activated following stimulation with poly(I:C) to express innate and adaptive immune cytokines and to become effective APCs (19). The current studies have shown that administration of poly(I:C) to mice during the innate immune response to TMEV alters the development of autoimmune demyelinating disease. The reduction in demyelinating disease following poly(I:C) administration was associated with altered cytokine expression from a Th1- to a Th2-type response, reduced infiltration of T cells and macrophages into the CNS, and reduced activation of autoreactive T cells. Thus, poly(I:C) had a similar effect on the immune response as IFN-β and is likely exerting its effect by inducing the expression of IFN-β. Poly(I:C) also reduced experimental autoimmune encephalomyelitis disease when administered before disease onset, and the action of poly(I:C) was shown to be mediated through IFN-β production and the induction of MCP-1 (29). However, poly(I:C) is an agonist for TLR3 as well as Mda5, both of which are innate immune receptors that upon recognition of poly(I:C) can activate several components of the adaptive immune response. These include activation of APCs, specifically dendritic cells, induction of CD8+ T cell expansion, and the production of Abs (30). Poly(I:C) does not induce T cell proliferation and does not induce disease when used as an adjuvant for experimental autoimmune encephalomyelitis induction (31). Thus, poly(I:C) may also be exerting its effects on the development of autoimmune disease through mechanisms other than expression of type I IFNs. Further studies will be required to differentiate the mechanism of action by which poly(I:C) alters the immune response during TMEV-induced demyelinating disease.

The current studies demonstrate that altering the innate immune response during TMEV infection of the CNS resulted in an alteration in the development and progression of chronic autoimmune-mediated demyelinating disease. The results suggest that the innate immune response to CNS virus infections may be involved in the development of autoimmune demyelinating disease through bystander activation of immune cells, including autoreactive T cells. Administration of IFN-β or poly(I:C) reduced the infiltration of immune cells into the CNS, specifically T cells, which could be activated to become autoreactive cells. This is consistent with our recent finding that naive PLP139–151-specific CD4+ T cells get activated in the inflamed CNS in TMEV-infected mice. Administration of IFN-β or poly(I:C) also altered the cytokine expression to reduce the inflammatory response and to switch the response toward a Th2 response which does not promote activation of autoreactive T cells. Thus, these results suggest that a more robust innate immune response may inhibit bystander activation of immune cells in the CNS. Likewise, the innate immune response to virus infection may also affect the development of autoimmune demyelinating disease through bystander damage. Virus- infected cells are killed directly by the virus as well as by components of the innate and adaptive response in an effort to reduce the spread of infection. The damaged tissue may form DAMPs that are recognized by innate immune receptors which may promote a proinflammatory immune response. The increased expression of IFN-β may protect the CNS cells from the cytolytic actions of the virus and reduce cell death of neurons during the innate immune response (32, 33). Therefore, the innate immune response to virus infection of the CNS may be involved in the development of autoimmune demyelinating disease by bystander activation or bystander damage. However, depending on the particular innate immune response initiated, the virus can either promote or inhibit the development of autoimmune disease. Interestingly, several viruses, including encephalitis viruses that infect the CNS, encode a protein that inhibits the expression of IFN-β which may promote initiation of autoimmune disease (34). Furthermore, MS patients have been shown to express lower levels of IFN-β than control patients, thus, the innate immune response to a particular virus may vary between individuals, which could result in differential development of autoimmune disease in individuals infected with the same virus (35).

We thank Jenna Bowen and Erin Johnson at the University of Wisconsin for technical assistance on the cytokine expression assays.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Multiple Sclerosis Society Grants RG3625 and RG3629 and National Institutes of Health Grant NS023349.

3

Abbreviations used in this paper: MS, multiple sclerosis; PLP, proteolipid protein; TMEV, Theiler’s murine encephalomyelitis virus; PAMP, pathogen-associated molecular pattern; IRF, IFN regulatory factor; DAMP, damage-associated molecular pattern; MMP, matrix metalloproteinase.

4

The online version of this article contains supplemental material.

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