CMV Infection as Immunomodulator in an MS Model Figure 3. Flow cytometric analysis of brain infiltrating immune cells in 8 months TMEV infected mice that were either sham/PBS injected, pre- or post-infected with MCMV. Shown are CD45+ cells that are: A.) CD3+, B.) CD4+, and C.) Mac1+, respectively. The observed reduction in CD3+ cells compared to PBS controls was significant (p = 0.026), and so was the increase in MAC1+ cells (p = 0.003). The decrease in CD4+ T-cells demonstrated a trend (p = 0.17). doi:10.1371/journal.pone.0032767.g003 chosen because MCMV virus titers peak in the salivary gland at 2 weeks and all other tissues are infected and some are even starting to be cleared from the tissue, indicating virus-specific immune response. However, it is possible that a different lag time may have resulted in enhanced immunomodulatory effects. We also did not demonstrate the effects of MCMV alone on the observed outcome measures, including there was no control group where SJL/J mice would be infected with MCMV alone. Histology time course analysis was also not done, but given that we clearly documented significant differences in motor performance, and motor perfor- mance in this model is determined by the extent of both demyelination and axonal loss, we anticipate that quantitative measures of the aforementioned would also have demonstrated significant differences. In conclusion, in the studied chronic-progressive model of MS, MCMV infection prior to demyelinating disease induction resulted in an attenuated clinical phenotype. Overall, the mechanisms by which human or mouse CMV effectively results in reduced disease activity in MS or in the studied MS model remain unclear. However, this study recapitulates in a rodent model the clinical observation that underlying HCMV infection is protective in human MS. We plan to study the observed phenomenon in additional details in future extensions to this study, which will address all the limitations of the current study, and include additional time points and outcome measures. In our view, the presented new model system will enable us to gain insights into the beneficial immunomodulatory mechanism(s) associated with this common viral infection, and may pave the way to novel therapeutic strategies in MS. Materials and Methods Mice and Experimental Infection The study was approved by the institutional committee for animal care and use at the University of Cincinnati (approval number 06-10-09-01). 4 week old female SJL/J mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). Four groups of 8 mice were studied: MCMV inoculation either preceded the infection with TMEV by 2 weeks, or was established 2 weeks after TMEV. We also used 2 control groups where PBS was used as control for the MCMV inoculation, either 2 weeks before or 2 weeks after infection. To induce the MS-like demyelinating disease, all groups received TMEV infection by intracranial (i.c.) injection of 105 PFU of TMEV from the DAV strain as published earlier [38,39]. Mice that received MCMV were inoculated by intraperitoneal (i.p.) injection of 16105 PFU PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32767