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Murine Cytomegalovirus Infection Induces Susceptibility to EAE in Resistant BALB/c Mice

In contrast to C57BL/6 mice, BALB/c mice are relatively resistant to the induction of experimental autoimmune encephalomyelitis (EAE) after challenge with MOG35–55 peptide. Here, we provide the first evidence that infection with murine cytomegalovirus (MCMV) in adulthood abrogates this resistance. Infected BALB/c mice developed clinical and histological signs similar to those seen in susceptible C57BL/6 mice. In addition to CD4+ cells, large proportion of cells in the infiltrate of diseased BALB/c mice was CD8+, similar with findings in multiple sclerosis. CD8+ cells that responded to ex vivo restimulation with MOG35–55 were not specific for viral epitopes pp89 and m164. MCMV infection favors proinflammatory type of dendritic cells (CD86+CD40+CD11c+) in the peripheral lymph organs, M1 type of microglia in central nervous system, and increases development of Th1/Th17 encephalitogenic cells. This study indicates that MCMV may enhance autoimmune neuropathology and abrogate inherent resistance to EAE in mouse strain by enhancing proinflammatory phenotype of antigen-presenting cells, Th1/Th17, and CD8 response to MOG35–55.

inTrODUcTiOn Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS) with axonal injury, characterized by varying clinical course, pathology, and inflammatory patterns (1). It develops in susceptible hosts after interaction with environmental factors which trigger the disease by promoting the activation of myelin-specific T cells that normally circulate in the peripheral lymph organs of all individuals (2). It has been suggested that some infectious agents, in particular viruses, may be potential triggers of MS (2)(3)(4). Among different infective agents, Epstein-Barr virus (EBV) has been mostly associated with increased MS risk (5). Recently, it has been shown increased CD8 + T cell response to EBV lytic antigens in active MS and also in relapses (6). Infection with murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, polarizes the adaptive immune response and heightens CNS pathology following experimental autoimmune encephalomyelitis (EAE) induction and likely, influences MS pathogenesis (7). MCMV  Experimental autoimmune encephalomyelitis is the experimental model of MS, induced in susceptible animals by active immunization with myelin antigens mixed with adjuvant (8). Immunized mice develop ascending paralysis with CD4 + T cells and macrophages in infiltrations in the white matter of the spinal cord, and with minimal brain inflammation in the majority of experimental models. However, in MS, the vast majority of myelin lesions are found within the brain parenchyma with infiltrations that contain equivalent numbers of CD8 + T and CD4 + T cells (9,10). Despite these differences, EAE is considered as a valuable tool for research of MS pathogenesis. Moreover, several therapeutics that are now being used to treat MS were developed in EAE (11). BALB/c mice are found partially or completely resistant to the induction of EAE with encephalitogenic peptide, myelin oligodendrocyte glycoprotein (MOG35-55).
Cytomegalovirus (CMV) classified within the Betaherpesvirinae subfamily establishes life-long latent infections in 70-100% of the human population (12). After a primary infection of fibroblasts, epithelial, endothelial, and smooth muscle cells (6), mostly asymptomatic in the immunocompetent host, CMV persists in myeloid precursor cells (7). During latency periodic asymptomatic reactivations occur (13). CMV contains a large number of latent and lytic genes, many of which code proteins that have the role in immunoregulation (5). When monocytes that carry CMV enter visceral parenchyma and differentiate into macrophages and myeloid dendritic cells, virus reactivates and through expression of different genes can modulate the immune response of the host (14).
Data on the role of CMV infection in etiopathogenesis of MS are controversial. CMV has been found in demyelinating plaques and the cerebrospinal fluid of MS patients (15) and causes demyelination mainly in the CNS of immunocompromised hosts (16). Further, enhancement of numbers of EBV and CMV-specific CD8 + T cells among T cells in chronic inflammatory lesions of brain of MS patients was reported (17). Several studies involving human subjects indicate correlation between CMV infection and MS development, greater rate of relapses and greater brain atrophy (18)(19)(20). Other studies indicate that CMV seropositivity is associated with a decreased MS risk and predicts a better clinical and radiological outcome in MS patient (21), suggesting a protective effect of CMV on autoimmune neuropathology (22). Furthermore, CMV encodes multiple factors that trigger immunomodulatory or evasion mechanisms, which can decrease the immune response in MS patients (23,24).
We have recently shown that deletion of an immunoregulatory pathway, IL-33/ST2 axis, may enhance susceptibility to EAE in resistant BALB/c strain (25,26). The present study was done with the aim to explore whether infection with murine CMV (MCMV) create "fertile field" (27,28) that facilitates the expansion and activation of encephalitogenic cells leading to autoimmune disease of CNS.
Here, we show that MCMV infected and MOG35-55 immunized BALB/c mice develop very pronounced neuroinflammation with extensive infiltrations in brain and spinal cord parenchyma containing large proportion of CD8 + cells in infiltrates in addition to accentuation of Th1 and Th17 immune response and skewing microglia to M1 phenotype. Our results are compatible with the notion that MCMV abrogates inherent resistance of BALB/c mice to EAE induction with MOG35-55 peptide through enhancement of inflammatory dendritic cells in the periphery, M1 type of microglia and recruitment of MOG35-55 responsive CD8 + T cells in the CNS. Thus, CMV-induced inflammatory environment may enhance autoimmunity in CNS.

MaTerials anD MeThODs
infection, induction, and scoring of eae Female 8-week-old BALB/c mice were used throughout this study. Mice were infected subcutaneously (footped) with 10 5 plaque-forming units of tissue culture MCMV, strain MW97.01 (29). EAE was induced 10 days after infection by subcutaneous administration of 200 µL suspension at two sites over the hind flanks. Depletion of CD4 + lymphocytes, where indicated, was performed with intraperitoneal injection of 100 µg of anti-CD4 mAb, 1 day prior to and 5 days after MOG35-55 immunization. The suspension consisted of 300 µg MOG35-55 peptide (Sigma-Aldrich, Germany) in 100 µL of PBS, emulsified with 100 µL complete Freund's adjuvant (Sigma-Aldrich, Germany) with 0.7 mg heat-inactivated Mycobacterium tuberculosis (strain H37 RA; Difco Laboratories, Detroit, MI, USA). Each mouse was immediately thereafter, injected intraperitoneally and 48 h later with 300 ng pertussis toxin (List Biological Laboratories, Campbell, CA, USA) in 100 µL 0.9% NaCl. Clinical signs of EAE were assessed daily by the following scoring system: grade 0, no signs; grade 1, paralyzed tail; grade 2, ataxic; grade 2.5, one hind leg paralyzed; grade 3, both hind legs paralyzed; grade 3.5, three legs paralyzed; grade 4, both hind legs and front limbs completely paralyzed; grade 5, moribund as previously described (30,31). Mice were monitored daily with fluid administration and mashed chow on the base of cages for all mice displaying a clinical score of 3. Mice were maintained in our animal facilities in a temperature-controlled environment with a 12-h light/12-h dark cycle and were administered standard laboratory food and water ad libitum. All experiments were approved by and conducted in accordance with the Guidelines of the Animal Ethics Committee of Faculty of Medical Sciences, University of Kragujevac, Serbia. Endangered animal species were not used in this study.

isolation of Mononuclear cells from cns and lymph nodes
At day 15 post-EAE induction (mean clinical score of 3 for MCMV EAE mice), mice were perfused with PBS, and brain and spinal cord were carefully removed. The mononuclear cells from CNS were isolated as described previously (25). Briefly, the brains and spinal cords were homogenized in RPMI 1640 (Sigma-Aldrich) with 10% FBS and 1 mg/mL collagenase type I (Sigma-Aldrich) and incubated at 37°C for 60 min. After digestion, the tissue was passed through a 40-µm mesh, pelleted, resuspended in 10 mL 30% Percoll (Sigma-Aldrich), overlaid onto 5 mL 70% Percoll, and centrifuged at 390 g for 20 min. The myelin layer was removed, and the mononuclear cells accumulated in the intermediate phase were collected, washed twice in PBS, and resuspended in RPMI 1640 containing 10% FBS. Total cell numbers were determined by counting on a hemocytometer, and viability was assessed by trypan blue exclusion. Lymph nodes were minced in RPMI 1640 (Sigma-Aldrich) and forced gently through 40-µm cell-strainer nylon mesh (Falcon) using a sterile syringe plunger and centrifuged at 400 g for 5 min. Pellet from lymph nodes was resuspended in RPMI 1640 containing 10% FBS.

Tetramer staining
Immune cells were isolated as described above. Cells isolated from CNS were incubated with H-2L(d)/IE-1/pp89 (168-176 YPHFMPTNL) and H-2D(d)/m164 (257-265 AGPPRYSRI) tetramers provided by NIH tetramer core facility. Cells stained with tetramer and anti-CD8 and anti-CD3 antibodies were incubated for 30 min at room temperature and then washed. Data were acquired using a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star).

immunohistochemistry and evaluation of Brain and spinal cord Pathology
Brains and spinal cords were fixed in 4% buffered formalin fixative overnight. Paraffin wax embedded sections (5 µm) were stained with hematoxylin and eosin and CD3 (ab699; Abcam) immunohistochemical staining. The slides were analyzed on light microscope (BX51; Olympus), and digital images were acquired by digital camera. The level of infiltration was graded using the following score: 0, no inflammatory cells; 1, a few scattered inflammatory cells; 2, organization of inflammatory infiltrates into perivascular cuffs; 3, extensive perivascular cuffing with extension into adjacent subarachnoid space and CNS parenchyma, and 4, extensive perivascular cuffing with increasing subarachnoid and parenchymal inflammation (32). Slides were analyzed on Olympus BX51 microscope, and digital images were acquired by Olympus digital camera (DP71).

interferon-γ assay
Mononuclear cells isolated from CNS, 10 5 in 100 µL complete media were put on 96-well plate in duplicates, and 100 µL of media, or MOG35-55 (1 μg/well) were added. After incubation for 1 h on 37°C, 0.2 µL of Brefeldin A in 10 µL of medium was added to each well and incubated for 4 h on 37°C. Cells were washed and then incubated with anti-CD8 and anti-CD4 antibodies on +4°C for 15 min. After washing, cells were resuspended in Cytofix/ Cytoperm buffer for 30 min on ice, washed twice, and incubated with anti-IFNγ antibodies diluted in Perm wash buffer (30 min, on ice) and resuspended in FACS media.

statistical analysis
All statistics were carried out using SPSS 18.0 for Windows software. Results were analyzed using the Student's t-test or Mann-Whitney test and ANOVA or Kruskal-Wallis. Data in this study were expressed as the mean + SEM. Values of P < 0.05 were considered significant.

McMV infection in adult life abrogates resistance to eae in BalB/c Mice
BALB/c mice immunized with MOG35-55 did not develop clinical signs of EAE, while BALB/c mice infected with MCMV 8 weeks after birth and 10 days later challenged with MOG35-55 in CFA and pertussis toxin (MCMV + MOG35-55) developed clinical signs that correspond to EAE manifestations seen in C57BL/6 mice ( Figure 1A). Based on evaluation of clinical course ( Figure 1A) and mean maximal clinical score (Figure 1B), MCMV-infected BALB/c mice developed disease indistinguishable from disease in susceptible C57BL/6 mice. Infiltration in CNS of MCMV + MOG35-55, expressed by mean histological score ( Figure 1D) and total cell number ( Figure 1C), was significantly higher compared with BALB/c mice immunized with MOG35-55 only mice. BALB/c mice infected with MCMV and 10 days later immunized with MOG35-55 developed subarachnoid and perivascular infiltrations in the brain cortex, perivascular infiltrations in brainstem and cerebellum with spreading to parenchyma (Figure 1E), and white matter spinal cord infiltrations ( Figure 1F). Single-cell infiltrates were detected in brains and spinal cords of MOG35-55-immunized mice only, and mild perivascular infiltrations were detected in brainstem of MCMV-infected mice (Figures 1E,F). Immunostaining of spinal cord sections showed presence of CD3 + cells in the infiltrates ( Figure 1G). cns infiltrates of McMV + MOg 35-55 Mice contain higher amounts of T1/T17 cD4 + and cD8 + T cells Further analysis showed significantly higher number of CD4 + and CD8 + T cells in the infiltrates of MCMV + MOG35-55 mice compared with MOG35-55 mice (Figure 2A). In the CNS of MCMV + MOG35-55 mice, there was higher number of CD4 + T cells then CD8 + T cells, similar to typical EAE in C57BL/6 mice.

McMV infection induces inflammatory Phenotype of antigen-Presenting cells in Periphery and in cns
It is known that viral infection induces antiviral immune response mediated by NK cells, CD8 + and CD4 + lymphocytes (33). Such inflammatory microenvironment in peripheral lymph organs can affect activation of antigen-presenting cells and thus indirectly contribute to development of inflammatory lymphocytes. Therefore, we explored possible influence of viral infection on changes of phenotype of antigen-presenting cells. To this end, mononuclear cells were isolated from inguinal lymph node 12 days after MCMV was administered in foot pad and compared with cells isolated from mice immunized with MOG35-55 only. Lymph nodes of MCMV-infected and MCMV + MOG35-55 mice had significantly higher percentage of CD11c + dendritic cells and CD11c + PDCA1 + plasmocitoid dendritic cells in inguinal lymph nodes compared to MOG35-55-immunized mice ( Figure 5A). Also higher percentage of dendritic cell-expressing CCR2 chemokine receptor was found in both groups of MCMVinfected mice (Figure 5B). More importantly, lymph nodes of MCMV-infected and MCMV + MOG35-55 mice contained higher percentage of activated CD86 + and CD40 + (Figure 5C) and inflammatory IL-12 + dendritic cells ( Figure 5E). Higher  expression of markers of activation, CD86 + and CD40 + was noticed in MCMV-infected mice compared with MOG35-55immunized mice ( Figure 5D). These data suggest that inflammatory phenotype of dendritic cells is achieved in BALB/c mice with viral infection but not with encephalitogenic challenge only, as it was seen in C57BL/6 mice.
It is known that systemic inflammation in mice causes activation of microglia that persists for months (34,35). Since MCMV infection in BALB/c mice causes systemic inflammation, we wanted to see effect of MCMV infection on phenotype of antigen-presenting cells in CNS. We analyzed expression of markers of classical (CD11c) and alternative activation  (CD206) in the population of microglia (CD45 int CD11b + ) in healthy mice, MOG35-55-immunized mice, MCMV-infected, and MCMV + MOG35-55 mice. As shown in Figure 5F, microglia of mice with viral infection in adult life, with and without EAE, had proinflammatory, M1 phenotype. Significantly higher percentage of CD11c-expressing cells in microglia population was found in both groups of infected mice compared to healthy and MOG35-55immunized mice. On the other hand, the highest percentage of (type 2) CD206-expressing microglia was found in MOG35-55 BALB/c mice. Higher percentage of M2 microglia was also found in MCMV + MOG35-55-immunized mice compared to healthy and MCMV-infected mice but lower compared to MOG35-55immunized mice. Thus, high percentage of M2 microglia in mice at the peak of EAE could be also the compensatory mechanism that precedes disease attenuation.

DiscUssiOn
Here, we provide the first evidence that MCMV infection results in breaking resistance of BALB/c mice to EAE induction with MOG35-55 peptide, as indicated by typical clinical manifestations and massive inflammatory infiltration in the CNS (Figure 1). The role of MCMV infection in EAE is not studied, while its significance in MS is controversial. There are several prospective clinical studies that indicate protective effect of CMV infection on MS risk (21,22,36), while recent well-powered meta-analysis found no significant difference in the rate of CMV seropositivity between MS patients and healthy controls based on pooled samples from all studies to date (37). On the other hand, CMV has been found in demyelinating plaques and the liquor of MS patients (15), and several clinical studies support the role of CMV in MS pathogenesis (18)(19)(20). Additionally, CMV infection induces expansion of inflammation-seeking/proinflammatory effector-memory CD4 + CD28 null T cells that are attracted to MS lesions via a CX3CL1 gradient (38,39) and are mostly found in MS patients (40). Stimulation of these cells with myelin autoantigens results in their proliferation and release of cytotoxic granules, and thus may contribute to MS pathology (41). Some experimental animal studies support the role of CMV in EAE development. Cross-reactivity between CMV981-1,003 and MOG35-55 peptides was found in Lewis rats immunized with MOG35-55 (42), while crossreactivity between CMV981-1,003 and MOG34-56 was found in rhesus monkeys (43). Immunization of rhesus monkeys with human CMV981-1,003 peptide induced expansion of MOG34-56-specific T cells (43). Female SJL/J mice primed with vaccinia virus that contain PLP gene and later challenged with MCMV-developed lesions in white matter regions in the brains (28). Other studies on primates also support the role of CMV in MS pathogenesis (44,45 Figure 1A) seen in susceptible C57BL/6 mice and massive brain and spinal cord infiltrations (Figures 1E,F). It should be noted that brain infiltrates are more significant in BALB/c mice treated with encephalitogen then in "classical" EAE in C57BL/6 mice. It appears that the disease after infection + MOG35-55 challenge in otherwise resistant mice is more similar to MS than classical EAE (50). While in C57BL/6 mice, CD4 + cells dominate in CNS infiltrates in the experiments presented here, there was similar number of CD4 + and CD8 + cells in the infiltrates (Figure 1).
Encephalitogenity of CD4 + T cells in the infiltrates in BALB/c mice is further documented by their expression of chemokine receptors CXCR3 (Figure 2E), whose blockade during EAE induction attenuated the disease (51), and CCR6 receptor known to have the key role in development of initial autoimmune infiltration in the CNS (52,53). While γHV-68 infection in C57BL/6 mice with EAE leads to almost exclusive infiltration with Th1 cells (7), in the CNS of MCMV-infected BALB/c mouse with EAE there is almost equal participation of Th1 and Th17 cellexpressing IFN-γ and T-bet (Th1) and IL-17 and RORγt (Th17) (Figures 2C,D). Moreover, inflammatory infiltrates in MCMVpretreated BALB/c mice immunized with MOG35-55 contained CD8 + cell-expressing T1 and T17 transcriptional factors and corresponding cytokines TNF-α and IFN-γ (Tc1) and IL-17 (Tc17 cells) (Figures 2C,D). Interestingly, it was suggested that Tc17 cells are required for Th17 accumulation and development of MS (54). Patients with early-stage MS harbor a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood that contribute to the initiation of CNS autoimmunity (54). Since in the CNS of BALB/c mice with neonatal MCMV infection but without immunization with MOG35-55 dominates Tc1 cells (IFN-γ and T-bet + ) (55), it appears that the newly developing autoimmune process attracts a different population of CD8 + cells (IL-17 producing) that contribute to autoimmune process (54).
Significant accumulation of CD8 + cells in the infiltrates and their role in autoimmune pathogenesis is documented by clinical signs and typical pathology complemented by specificity of infiltrating cells. Ex vivo restimulation with MOG35-55 leads to significant increase of CD8 + cell-producing IFN-γ in MCMV + MOG35-55 BALB/c mice ( Figure 3C). This finding is at variance with our finding in C57BL/6 mice. CD8 + cells from EAE C57BL/6 mice did not responded to restimulation with MOG35-55 ( Figure 3C). Further, MOG35-55 responsive CD8 + cells in MCMV + MOG35-55 BALB/c mice were not specific for viral epitopes pp89 and m164 ( Figure 3D) implicating that CD8 + cell contributes to autoimmune process in this model of EAE. This finding is in line with previous report that initiation of autoimmune process in CNS with CD4 + T cells is followed with spreading to myelin-specific CD8 + T cells that are capable of direct recognition of oligodendrocytes and contribute to tissue damage (56). Similarly, heighten EAE in C57BL/6 mice infected with γHV-68 is accompanied with infiltration of brain parenchyma with CD8 + IFN-γ + granzyme + cells (7). However, specificity for autoantigen of these inflammatory and cytolytic CD8 + cells in γHV-68-infected C57BL/6 mice was not studied (7). Finding that mice with depletion of CD4 + cells after MCMV infection and before MOG35-55 immunization did not develop the disease ( Figure 3A) contributes to the conclusion that MCMVinfected BALB/c mice developed autoimmune neuropathology. Persistence of CNS infiltrations and MOG35-55-specific CD4 + and CD8 + cells in CNS of BALB/c mice 2 months after MOG35-55 immunization that were infected 3 months previously (Figure 4) also proving autoimmune nature of the disease. Thus although CD4 + cells are required, it appears that CD8 + cells are the main effector cells.
MCMV infection significantly increases proportion of dendritic cells (CD11c + ), plasmocitoid dendritic cells (CD11c + PDCA1 + ) in peripheral lymph nodes ( Figure 5A) compared to immunization with MOG35-55. Further higher percentage of dendritic cells in lymph nodes in MCMV-infected mice is accompanied with higher percentage of CCR2 + dendritic cells (Figure 5B). It is known that MCMV encodes proinflammatory factor (MCK-2), analog of chemokine CCL2 (57) that enhances monocyte recruitment and viral dissemination (58). Then, higher percentage of dendritic cells in inguinal lymph nodes of MCMV-infected mice could be the consequence of CCL2 analog production, since CCL2 goes to lymph nodes where is presented on the surface of high endothelial venules for recruitment of monocytes (59). Although it was previously shown that MCMV attracts monocytes that have the immunosuppressive role (60), here, we found higher percentage of dendritic cell-expressing markers of activation CD86 and CD40 ( Figure 5C) and containing Th1 promoting cytokine, IL-12 (Figures 5D,E). Our results indicate that MCMV infection of BALB/c mice induces increase of inflammatory dendritic cells in peripheral lymph nodes and thus enables development of encephalitogenic T cells. This finding is in correlation with previous report that MCMV-infected mice are resistant to bacterial infection due to prolonged production of the antiviral cytokine IFN-γ and systemic activation of macrophages (61). Significantly, there was an increase of classically activated microglia (CD45 med CD11b + CD11c + ) in the CNS of BALB/c mice 25 days after MCMV infection compared to MOG35-55-immunized mice without previous infection that had mostly alternatively activated microglia (CD45 med CD11b + CD206 + ) ( Figure 5F). Lower percentage of alternatively activated microglia in MCMV + MOG35-55 mice observed on day 15 after immunization and day 25 after infection in compared to MOG35-55 treated mice, and prevalence of M1 microglia in virus-infected mice may contribute to chronic disease in MCMV-infected MOG35-55-immunized BALB/c mice. Previously, it was shown that systemic MCMV infection elicited a significant increase in the number of microglia with morphological signs of activation and M1 phenotype (62). Thus, it appears that at least one level of resistance of BALB/c mice to EAE is the inability to convert microglia into M1 phenotype.
In summing up, we report here that MCMV infection may promote autoimmune neuropathology and convert resistant mice into susceptible to EAE induction. This was achieved by activation of antigen-presenting cells and promoting M1 phenotype of microglia as well as participation of CD8 + encephalitogen-specific T cells in the autoimmune pathogenesis.