Epstein–Barr virus-induced gene 3 negatively regulates neuroinflammation and T cell activation following coronavirus-induced encephalomyelitis Emanuele Tirotta a,1, Patrick Duncker a,1, Jean Oak a,2, Suzi Klaus a, Michelle R. Tsukamoto a, Lanny Gov a, Thomas E. Lane a,b,c,⁎ a Department of Molecular Biology and Biochemistry, University of California, Irvine 92697-3900, United States b Multiple Sclerosis Research Center, University of California, Irvine 92697-3900, United States c Sue and Bill Gross Stem Cell Center, University of California, Irvine 92697-3900, United States a b s t r a c t a r t i c l e i n f o Article history: Received 5 September 2012 Received in revised form 9 October 2012 Accepted 9 October 2012 Keywords: Virus Cytokines Neuroinflammation Demyelination Epstein–Barr virus-induced gene 3 (EBI3) associates with p28 and p35 to form the immunomodulatory cyto- kines IL-27 and IL-35, respectively. Infection of EBI3−/− mice with the neuroadapted JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality that was not associated with impaired ability to con- trol viral replication but enhanced T cell and macrophage infiltration into the CNS. IFN-γ secretion from virus-specific CD4+ and CD8+ T cells isolated from infected EBI3−/− mice was augmented while IL-10 ex- pression muted in comparison to infected WT mice. These data demonstrate a regulatory role for EBI3-associated cytokines in controlling host responses following CNS viral infection. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the CNS of susceptible strains of mice provides an excel- lent model in which to examine host response mechanisms responsi- ble for both control of viral replication within distinct cell lineages present in the brain as well as host-derived factors regulating neuroinflammation (Bergmann et al., 2006). JHMV infection results in an acute encephalomyelitis characterized by wide-spread replica- tion primarily in astrocytes and oligodendrocytes with relatively few neurons being infected (Knobler et al., 1981; Buchmeier and Lane, 1999; Parra et al., 1999; Perlman et al., 1999; Bergmann et al., 2006). Control of viral replication during acute disease relies on virus-specific CD8+ T cells, which function by two different effector mechanisms within the CNS: IFN-γ secretion is responsible for con- trolling viral replication in oligodendrocytes whereas a perforin- dependent mechanism promotes viral clearance from astrocytes (Lin et al., 1997; Parra et al., 1999). Infiltrating CD4+ T cells provide a supporting role for the maintenance and expansion of CTLs within the CNS, as well as enhancing effector responses (Stohlman et al., 1998; Phares et al., 2012). Although a robust cell-mediated immune response occurs during acute disease, sterilizing immunity is not achieved, resulting in viral persistence (Stohlman and Weiner, 1981). Histological features associated with viral persistence include the development of an immune-mediated demyelinating disease with both T cells and macrophages being important in amplifying dis- ease severity by contributing to myelin damage (Cheever et al., 1949; Perlman et al., 1999; Lane et al., 2000; Pewe and Perlman, 2002; Pewe et al., 2002). EBV-induced gene-3 (EBI3) encodes a 34 kDa protein that is sim- ilar to the p40 subunit of IL-12 (Collison and Vignali, 2008; Wojno and Hunter, 2012). EBI3 is capable of binding subunits p28 or p35 (a subunit of IL-12) to form the cytokines IL-27 and IL-35, respective- ly (Collison and Vignali, 2008; Wojno and Hunter, 2012). Both IL-27 and IL-35 have important immunoregulatory roles controlling cyto- kine secretion and inflammation (Collison and Vignali, 2008; Yoshida and Miyazaki, 2008; Wojno and Hunter, 2012). Recent stud- ies have highlighted that genetic ablation of EBI3 results in altered T cell-mediated cytokine expression (Yang et al., 2008) associated with increased inflammation as well as changes in the composition of the cellular infiltrate in experimental models of delayed-type hy- persensitivity (Tong et al., 2010), autoimmune inflammation (Igawa et al., 2009; Liu et al., 2012), as well as hepatitis (Siebler et al., 2008). The influence of EBI3 in host defense in response to infection with a neurotropic virus has not been well characterized and was the focus of the present study. Journal of Neuroimmunology 254 (2013) 110–116 ⁎ Corresponding author at: Department of Molecular Biology & Biochemistry, Multiple Sclerosis Research Center, University of California, Irvine 92697-3900, United States. Tel.: +1 949 824 5878; fax: +1 949 824 8551. E-mail address: