A Gammaherpesvirus Cooperates with Interferon-alpha/beta-Induced IRF2 to Halt Viral Replication, Control Reactivation, and Minimize Host Lethality

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship

Host immunity and murine gammaherpesvirus 68 infection

Herpesviruses are large highly prevalent DNA viruses. They are some of the commonly occurring chronic infections of human beings. Herpesviruses infect hosts causing modest to inconsequential disease before establishing life-long latent infection in hosts. Although latent infection is largely restricted for expression of viral genes, herpesviruses often undergo reversion to lytic cycle with gene expression in a phase known as reactivation. High levels of herpesviruse reactivation and associated pathogenesis is observed in immunodeficient patients. In this body of work we have studied the infection of a gammaherpesvirus. Gammaherpesvirus latency and reactivation are primarily associated with several cancers in immunocompromised patients. Hence it is important to understand immune responses which control gammaherpesviruses reactivation and pathogenesis. Here we have used a murine gammaherpesvirus (murine gammaherpesvirus 68 or MHV68) to study roles of secreted glycoproteins called interferons in controlling infection. MHV68 is closely related to the human gammaherpesviruses which are hard to study owing to their species specificity. Usage of MHV68 helps us study a gammaherpesvirus interaction with vertebrate immune system in vivo. We identified that antiviral type I interferons (comprising of interferon alpha and beta) regulate MHV68 replication in vivo, in vitro and prevents latent virus from reactivation from peritoneal cells and splenocytes of mice by directly inhibiting MHV68 M2gene expression. Functional type I interferons induce signaling pathway which upregulate expression of several genes including transcription factors called interferon regulatory factors (IRF). We observed that type I interferon induced IRF2 directly binds a promoter region on M2gene which is a reactivation associated gene and represses M2 transcription and associated viral reactivation. A mutant virus which is unable to bind IRF2 in M2 promoter displayed increased replication, reactivation and M2 gene expression in vivo which results in increased host lethality in immunocompromised mice. Hence MHV68 cooperates with antiviral effects of the interferon system to facilitate maintenance of latency and minimize host lethality long term. We further identified that although type I interferons significantly control MHV68 infection in vitro and in vivo, none of the effects are mediated by crucial interferon stimulated genes PKR and RNAseL. These interferon stimulated genes are considered to be among the first line of defense against any viral replication. Our investigations revealed that like the other human herpesviruses including the gammaherpesviruses, MHV68 also inhibits PKR-mediated antiviral response. Hence we have shown here a complicated relationship between a murine gammaherpesvirus and host type I interferon system. During replication, MHV68 inhibits type I interferon mediated antiviral response by inhibiting PKR-response; but to facilitate establishment and maintenance of long-term latency MHV68 cooperates with different antiviral responses mediated by type I interferons. These data can be used as valuable model to further our knowledge about the human gammaherpesviruses and better understanding of therapeutic design for patients suffering from herpesvirus reactivation associated diseases. Lastly, gammaherpesviruses establish latency for the life of the host. We wanted to study the effects of latency on other host responses and chose to study development of non-viral tumors in MHV68 infected mice. We identified that MHV68 latent infection changes host response (sometimes to facilitate and sometimes to inhibit) against foreign tumors introduced in mice. Human beings are infected with several herpesviruses and other chronic infections. It is of crucial importance to understand if these change the host response to tumors. Our data sheds lights on some new concepts regarding the relationship between gammaherpesvirus latency and non-viral tumors

TNF Signaling Dictates Myeloid and Non-Myeloid Cell Crosstalk to Execute MCMV-Induced Extrinsic Apoptosis

Cytomegaloviruses all encode the viral inhibitor of caspase-8-induced apoptosis (vICA). After binding to this initiator caspase, vICA blocks caspase-8 proteolytic activity and ability to activate caspase-3 and/or caspase-7. In this manner, vICA has long been known to prevent apoptosis triggered via tumor necrosis factor (TNF) family death receptor-dependent extrinsic signaling. Here, we employ fully wild-type murine cytomegalovirus (MCMV) and vICA-deficient MCMV (∆M36) to investigate the contribution of TNF signaling to apoptosis during infection of different cell types. ∆M36 shows the expected ability to kill mouse splenic hematopoietic cells, bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC). Antibody blockade or genetic elimination of TNF protects myeloid cells from death, and caspase-8 activation accompanies cell death. Interferons, necroptosis, and pyroptotic gasdermin D (GSDMD) do not contribute to myeloid cell death. Human and murine fibroblasts or murine endothelial cells (SVEC4-10) normally insensitive to TNF become sensitized to ∆M36-induced apoptosis when treated with TNF or TNF-containing BMDM-conditioned medium. We demonstrate that myeloid cells are the natural source of TNF that triggers apoptosis in either myeloid (autocrine) or non-myeloid cells (paracrine) during ∆M36 infection of mice. Caspase-8 suppression by vICA emerges as key to subverting innate immune elimination of a wide variety of infected cell types

Multiple Autonomous Cell Death Suppression Strategies Ensure Cytomegalovirus Fitness

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochon-drial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease en-hances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and-independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host

Fra-2-expressing macrophages promote lung fibrosis in mice

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with Type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections and Fra-2 transgenic mice (Fra-2tg) exhibit spontaneous lung fibrosis. Here we show that Bleomycin-induced lung fibrosis is attenuated upon myeloid-inactivation of Fra-2 and aggravated in Fra-2tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is up-regulated in three lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2-dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine pro-fibrotic activity of macrophages. Importantly, ColVI knock-out mice (KO) and ColVI-KO bone marrow chimeras are protected from Bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2tg mice and in the Bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and co-localize in lung macrophages. Therefore, the Fra-2/ColVI pro-fibrotic axis is a promising biomarker and therapeutic target for lung fibrosis, and possibly other fibrotic diseases

mAb therapy controls CNS‐resident lyssavirus infection via a CD4 T cell‐dependent mechanism

Abstract Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre‐symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti‐lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11‐dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell‐dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ‐binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization‐independent T cell‐mediated effects, even against an established CNS infection by a lethal neurotropic virus