Research Article: Murine Gammaherpesvirus M2 Protein Induction of IRF4 via the NFAT Pathway Leads to IL-10 Expression in B Cells

Date Published: January 2, 2014

Publisher: Public Library of Science

Author(s): Udaya S. Rangaswamy, Samuel H. Speck, Vera L. Tarakanova.


Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner – leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4 – a key player in plasma cell differentiation – which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.

Partial Text

Gammaherpesviruses are lymphotropic viruses that are associated with the development of lymphoproliferative diseases and lymphomas (reviewed in [1]). The two human γ-herpesviruses, Epstein Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), exhibit very narrow host tropism, making in vivo studies challenging and accentuating the need for relevant small animal models. One of the best-characterized animal models of gammaherpesvirus pathogenesis is murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice [2], [3]. Intranasal inoculation of mice with MHV68 results in an acute lytic phase in the lung that is largely cleared by days 10–12 post infection. Latency is established in the spleen and, similar to EBV, memory B cells are the primary long-term latency reservoir. Periodic reactivation results in productive infectious virus capable of reseeding new latency reservoirs. Efficient trafficking of MHV68 to the spleen requires B cells, as evidenced by the delayed kinetics of establishment of splenic latency in B cell-deficient mice [4]–[7].

Plasma cell differentiation-driven virus reactivation is a common theme in gammaherpesviruses biology [15], [69]–[71]. In contrast to MHV68 where we have previously shown that the viral M2 antigen can promote plasma cell differentiation [15], for both EBV and KSHV there is no information regarding how plasma cell differentiation of latently infected B cells is regulated. Although there are no obvious homologs of the MHV68 M2 gene in the human gammaherpesviruses, it seems reasonable to speculate that EBV and KSHV encode viral gene products that are capable of promoting plasma differentiation – ensuring that virus reactivation occurs at both the appropriate time and anatomical site(s) (e.g., mucosal surfaces). Here, we describe the signaling pathway that ensues upon expression of M2. Expression of M2 in primary murine B cells results in secretion of IL-10, a cytokine associated with plasma cell differentiation in humans [36] and in mice [72]. We therefore hypothesized that identifying the mechanism underlying M2-driven IL-10 production will provide clues about pathways required for plasma cell differentiation driven virus reactivation. As a first step, we created an inducible cell line expressing M2 in a tightly regulated temporal manner. As described in (Figure 1C), this cell line makes IL-10 only when M2 expression is induced by doxycycline treatment, thereby providing us a means to study M2 mediated IL-10 production in a systematic fashion.




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