Model of M2 mediated IL-10.

<p>Upon BCR engagement, multiple protein kinases including Src family kinases (Lyn,Fyn,Src,Blk,Yes), Syk, Btk get activated leading to the activation of PLCγ2.Activated PLCγ2 catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate [(PtdIns(4,5)P2] to DAG and IP3. Binding of IP3 to its receptor on the ER membrane leads to release of ER Ca²⁺ stores. However, this increase in Ca²⁺ is only transient and following depletion of ER Ca²⁺ stores, an influx of extracellular Ca²⁺ occurs (termed as SOCE-Store Operated Calcium Entry). This prolonged and sustained increase in Ca²⁺ leads to activation of the Calcineurin-NFAT pathway. In the case of M2, <i>Miranda et al</i> have shown that M2 can interact with Fyn and PLCγ2. Our model suggests that interaction of M2 with proximal membrane signaling molecules such as Src kinase and/or PLCγ2 potentially mimics events that occur upon BCR engagement resulting in the Ca²⁺ mediated activation of the NFAT pathway. Induction of IRF4 by M2 is dependent, at least partially by the activation of NFAT pathway. IRF4 can activate regulatory elements in the IL-10 promoter locus (termed CNS-Conserved Noncoding Sequences) in T cells and in our case, in B cells as well. These series of events leads to eventual increase in the levels of IL-10 observed upon induction of M2.</p

IRF4 expression in B cells leads to IL-10 secretion and M2 synergizes with IRF4 to enhance IL-10 levels.

<p>(A) 3×10⁶ DS10 cells were transfected with 10 µg of either an empty vector (pMSCV-IRES-GFP, designated pMIG-EV) or an IRF4 expression vector (pMSCV-IRF4-IRES-GFP, designated pMIG-IRF4) and the reaction was divided into two wells-one well received doxycycline treatment for 48 hours and the other well was left uninduced. Each reaction was performed in triplicates. Supernatants were collected at 48 hours post induction with doxycycline and IL-10 levels were measured by ELISA (B) 1×10⁶ M12 cells per well were nucleofected with an expression vector containing M2 or M2stop, together with either pMIG-EV or pMIG-IRF4.Supernatants were harvested 48 hours after nucleofection and analyzed for IL-10 levels by ELISA. (C) DS10 cells were nucleofected as in (A) above with either pMIG-EV or pMIG-IRF4 along with 2.5 µg of IL-10pCNS9-luc reporter construct. Each reaction was divided into two reactions as above and luciferase activity was measured 48 hours post doxycycline induction. (D and E) The indicated cell types (A20, BCL1-3B3, P3xAg and M12) were plated at a density of 2.5×10⁵ cells in each well of a 6-well plate. Supernatants and whole cell lysates were harvested at 48 hours and analyzed for IL-10 levels by ELISA (D) and IRF4 expression by immunoblotting (E).</p

M2stop infected mice exhibit a decreased frequency of infected B cells expressing IRF4.

<p>C57BL6/J mice were infected intraperitoneally with 1000 PFU of the indicated viruses and splenocytes were analyzed on day 14 post infection. (A) Representative flow plots illustrating the gating strategy. Splenocytes were gated to remove doublets following gating on the live cell population using a live/dead stain (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#s4" target="_blank">Materials and Methods</a>). (B) Frequency of live, singlet YFP+ cells that were IRF4 positive between M2stop.HY and M2MR.HY viruses. Data shown is from individual mice with five mice per virus group.</p

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

<div><p>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 <i>in vivo</i> 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.</p></div

M2 induction of IRF4 by M2 is partially dependent on NFAT pathway.

<p>(A) DS10 and C30p cells were either left uninduced or induced with or without CsA for 48 hours in the culture media. Whole cell lysates were harvested at 48 h post induction and 5×10⁵ cell equivalents were analyzed for levels of IRF4 protein by immunoblotting. PMA and Ionomycin treatment for 6 hours served as a positive control for IRF4 induction. P3X63Ag8 cell lysates served as a stimuli independent positive control. (B) Duplicate gel run using whole cell lysates from (A) to verify expression of M2 using antibody for AU1 tag. (C) Supernatants from cells in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#ppat-1003858-g004" target="_blank">Figure 4A</a> were analyzed for IL-10 levels by ELISA.</p

IRF4 is required for establishment of MHV68 latency as well as for reactivation from latency.

<p>IRF4^fl/fl mice were infected with 1000 PFU of either MHV68-Cre or MHV68-WT virus intranasally. On day 16 post infection, splenocytes were analyzed by limiting dilution analyses to determine (A) the frequency of virus capable of establishing latency and (B) the frequency of virus capable of reactivating from latency.</p

An inducible system to study M2 mediated signaling events.

<p>Doxycycline inducible M2 expressing cell lines were generated using the M12 murine B lymphoma cell line. C30p is the parental cell line stably carrying the TetON regulator plasmid, which is used as a negative control. C30bulk refers to the bulk inducible cells prior to limiting dilution analysis. DS10, DS14 and DS15 are individual clones of M2 inducible cell lines. (A) Representative bright-field and mCherry fluorescence images of the same field of DS10 cells that were either left uninduced (top panel) or treated with doxycycline for 48 hours (bottom panel). (B) The indicated cell lines were either left uninduced or induced with doxycycline for 48 hours. Whole cell lysates were harvested and 40 µg protein was resolved by SDS-PAGE followed by immunoblotting for M2, IRF4 and β-actin using antibodies described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#s4" target="_blank">Materials and Methods</a>. (C) Supernatants from 2×10⁶ cells that were either doxycycline induced for 24 hours, or left uninduced, were analyzed for IL-10 secretion by ELISA. Data shown is a representative figure from multiple experiments showing similar results.</p

Tyrosines 120 and 129 of M2 are indispensable for M2 mediated NFAT activation and IRF4 expression.

<p>(A) 3×10⁶ of the indicated cell types were nucleofected with 10 µg of the NFAT reporter plasmid s using Ingenio electroporation solution as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#ppat-1003858-g002" target="_blank">Figure 2A</a>. After transfection, the cells were divided into two reactions - one half was left untreated and the other half received doxycycline for 48 hours prior to quantitating luciferase activity. Each condition was performed in triplicate. Luciferase activity is represented as fold over uninduced ± SEM. (B) Whole cell lysates from the indicated cell lines were analyzed for expression of M2, IRF4 and β-actin as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#ppat-1003858-g001" target="_blank">Figure 1B</a>.</p

Inhibition of NFAT pathway in primary murine B cells leads to reduced IRF4 expression and IL-10 levels correlating with changes in B cell surface phenotype.

<p>(A–E) Primary murine B cells were isolated as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#s4" target="_blank">materials and methods</a>, transduced with retroviruses expressing either M2 or M2stop. At the time of transduction, half of the cells received 500 ng/mL CsA and the other half was left untreated. (A) The cells were analyzed for Thy1.1 expression by flow cytometry on days 2 to 5 post-transduction. Cells were first gated on live cells and then gated on Thy1.1 and plotted as frequency of cells that were positive for Thy1.1. (B) Supernatants were collected from the cells used in (A) and analyzed for IL-10 levels by ELISA. (C and D) Whole cell lysates from cells in replicate wells as in (A) were harvested for analyzed for IRF4, NFATc1, NFATc2 and β-actin expression by immunoblotting. 10 µg protein was loaded per well. (E) Gated live cells from the indicated conditions were analyzed for their expression of the surface markers B220, CD138, MHC-IA^b, IgD, CD23 and CD25. Representative histograms are from day 4 post-transduction depicting one of triplicate samples per condition.</p

Tyrosine 129 of the Murine Gammaherpesvirus M2 Protein Is Critical for M2 Function <i>In Vivo</i>

<div><p>A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes – predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe <i>in vitro</i> and <i>in vivo</i> studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells <i>in vitro</i>, only Y129 is critical for reactivation from latency and plasma cell differentiation <i>in vivo</i>.</p></div