CMV Infection as Immunomodulator in an MS Model Figure 2. MRI results. Ventricular volumetry at 8 months post TMEV infection. Lower numbers represent less atrophy. The observed close to 30% lower atrophy in the MCMEV/TMEV group only showed a trend (p = 0.19) due to low statistical power, which was the consequence of the relatively high standard deviation observed in these groups. Of note, ventricular volumetry of SJL/J mice undergoing normal aging does not demonstrate the development of significant atrophy as demonstrated earlier [15]. doi:10.1371/journal.pone.0032767.g002 Infection of mice with MCMV reflects the infection of humans with HCMV in many respects. MCMV infects numerous tissues and cell types during the acute phase of infection and establishes a lifelong persistent or latent infection similar to HCMV [13]. Similar to HCMV, MCMV encodes viral genes which function to evade or alter the host immune response [9,10]. Some of these appear to contribute to the observed lifelong viral persistence, while others exploit immune cells that contribute to antiviral immunity [24]. Both HCMV and MCMV inhibit MHC class I expression on infected cells [9,24,26], and MCMV has been shown to impair IFN-gamma induced MHC class II-dependent antigen presentation by macrophages [27,28]. HCMV was also demonstrated to inhibit the induction of HLA class II antigens by IFN-beta dependent and independent molecules including ICAM- 1 and VCAM-1, which are thought to be involved in MS pathogenesis [29]. In addition, CMV-infected endothelial cells have the capability to induce IFN-beta production [30]. Interferon beta represents the most commonly used disease modifying agent for relapsing forms of MS [1]. Another possible mechanism to explain the effects of prior MCMV infection on TMEV-mediated MS development could result from modulation of the immune response to TMEV infection itself. In mouse infection studies with either MCMV or other viruses, prior infection of mice with one virus influences the immune response to other heterologous infections [31,32,33]. Thus, prior MCMV infection in the TMEV model of MS could lead to attenuation of the MS-like disease as shown in our studies by modulation of the immune response to TMEV infection. In addition, cytokines released as part of an antiviral immune response can activate the hypothalamo-hypo- physeal axis, resulting adrenal glucocorticoid release, which in turn provides strong negative feedback on the further synthesis and release of cytokines, and exerts an overall protective effect from the detrimental consequences of an overactive immune response [34,35]. Lastly, as another potential, but at this stage purely speculative explanation to our observations of increased propor- tion of Mac-1+ cells, these cells may contribute to a more efficient elimination of TMEV infected CNS cells, and as such led to a better overall outcome. In addition, macrophages may exert immunosuppressive effects on T-cells, as commonly demonstrated in cancer models.[36,37] Given that our study was designed as a pilot project paving the way to future larger scale proposals, there are clear limitations to our data. These include the relatively low number of mice per group, which resulted in being underpowered from the standpoint of demonstrating significant MRI-based differences. In addition, due to the same limitation, only one time point was studied with FACS, and we only studied brain and not spinal cord samples - ideally both should be assessed given the prevalence of TMEV in the spinal cord at late time points. We utilized the most commonly studied immune cell markers in the FACS based studies; however, additional immune subset markers, cytokine assay, microarray studies would enable us to study this phenomenon in more details, and all the above are planned in future extensions of this study. The 2-week lag between MCMV and TMEV infections was PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32767