Murine gamma-herpesvirus 68 causes severe large-vessel arteritis in mice lacking interferon-gamma responsiveness: a new model for virus-induced vascular disease.

Fundamental issues remain unresolved regarding the possible contribution of viruses to vascular pathology, as well as the role of the immune system in regulating these processes. Here we demonstrate that infection of mice with gamma-herpesvirus 68 (gammaHV68) provides a novel model for addressing these issues. Interferon-gamma receptor-deficient (IFNgammaR-/-) mice died weeks to months after gammaHV68 infection from a severe large-vessel panarteritis. GammaHV68-infected B cell-deficient and normal weanling mice exhibited milder large-vessel arteritis. Immunohistochemical analyses demonstrated gammaHV68 antigen in arteritic lesions and revealed a striking tropism of gammaHV68 for smooth muscle cells. These studies demonstrate that IFN-gamma is essential for control of chronic vascular pathology induced by gammaHV68 and suggest gamma-herpesviruses as candidate etiologic agents for human vasculitis.

Murine cytomegalovirus inhibits interferon gamma-induced antigen presentation to CD4 T cells by macrophages via regulation of expression of major histocompatibility complex class II-associated genes.

CD4 T cells and interferon gamma (IFN-gamma) are required for clearance of murine cytomegalovirus (MCMV) infection from the salivary gland in a process taking weeks to months. To explain the inefficiency of salivary gland clearance we hypothesized that MCMV interferes with IFN-gamma induced antigen presentation to CD4 T cells. MCMV infection inhibited IFN-gamma-induced presentation of major histocompatibility complex (MHC) class II associated peptide antigen by differentiated bone marrow macrophages (BMMphis) to a T cell hybridoma via impairment of MHC class II cell surface expression. This effect was independent of IFN-alpha/beta induction by MCMV infection, and required direct infection of the BMMphis with live virus. Inhibition of MHC class II cell surface expression was associated with a six- to eightfold reduction in IFN-gamma induced IAb mRNA levels, and comparable decreases in IFN-gamma induced expression of invariant chain (Ii), H-2Ma, and H-2Mb mRNAs. Steady state levels of several constitutive host mRNAs, including beta-actin, cyclophilin, and CD45 were not significantly decreased by MCMV infection, ruling out a general effect of MCMV infection on mRNA levels. MCMV effects were specific to certain MHC genes since IFN-gamma-induced transporter associated with antigen presentation (TAP)2 mRNA levels were minimally altered in infected cells. Analysis of early upstream events in the IFN-gamma signaling pathway revealed that MCMV did not affect activation and nuclear translocation of STAT1alpha, and had minor effects on the early induction of IRF-1 mRNA and protein. We conclude that MCMV infection interferes with IFN-gamma-mediated induction of specific MHC genes and the Ii at a stage subsequent to STAT1alpha activation and nuclear translocation. This impairs antigen presentation to CD4 T cells, and may contribute to the capacity of MCMV to spread and persist within the infected host.

A severe combined immunodeficient (SCID) mouse model for infection with Entamoeba histolytica.

We used severe combined immunodeficient (SCID) mice to study resistance to invasive infection with Entamoeba histolytica. Seven of seven SCID mice developed liver abscesses when challenged intrahepatically with virulent HM1:IMSS strain E. histolytica trophozoites. Only one of seven similarly challenged immunocompetent congenic C.B-17 mice developed an abscess. Adoptive transfer of polyclonal rabbit anti-E. histolytica antiserum, but not preimmune rabbit serum, completely protected 7 of 12 SCID mice from intrahepatic challenge with ameba. These results demonstrate that lymphocyte-based immunity is important in protection against amebic liver abscess, and that anti-E. histolytica antibody can protect against amebic infection in this system. The SCID mouse may provide a powerful model for studying the components of protective immunity to invasive amebiasis.

Antibody inhibits defined stages in the pathogenesis of reovirus serotype 3 infection of the central nervous system.

The mammalian reoviruses provide a model for studying specific aspects of the immunopathogenesis of viral infection. We have used two serotype 3 reoviruses to define stages in the pathogenesis of central nervous system (CNS) infection at which a mAb specific for the reoviral cell attachment protein sigma 1 (sigma 1mAbG5) acts to protect mice against lethal disease. sigma 1mAbG5 administered either before or at the time of footpad inoculation with reovirus T3D prevented entry of T3D into the CNS. sigma 1mAbG5 also inhibited the spread of reovirus T3C9 from the gastrointestinal tract to the CNS after peroral inoculation with T3C9. These effects occurred in the absence of a significant effect of sigma 1mAbG5 on primary replication in skeletal muscle (T3D) or the gastrointestinal tract (T3C9). sigma 1mAbG5 administered after T3D had reached the spinal cord inhibited subsequent spread of infectious virus from spinal cord to brain. Even after direct intracerebral inoculation of T3D, sigma 1mAbG5 prevented both growth in the brain and spread of infectious virus from brain to eye, spinal cord, and muscle. Treatment with sigma 1mAbG5 after intracerebral inoculation with T3D prevented neuronal necrosis and resulted in a delayed and topographically restricted inflammatory response. We detected no antibody-resistant T3D variants in vivo after treatment with sigma 1mAbG5. We conclude that systemic IgG does not play a significant role at the primary site of infection with reoviruses, while it clearly acts to prevent infection of the CNS and extension of infection with the CNS. Further study will be directed to defining what components of the immune system do act at primary sites of infection, and to defining the mechanisms by which antibody acts at defined stages in pathogenesis.

Novel cell type-specific antiviral mechanism of interferon gamma action in macrophages.

Interferon (IFN)-gamma and macrophages (Mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-gamma mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mphi (BMMphi). IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold). Although initial STAT-1alpha activation by IFN-gamma was equivalent in MEF and BMMphi, microarray analysis demonstrated that IFN-gamma regulates different sets of genes in BMMphi compared with MEFs. IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-gamma action, which differed in MEF and BMMphi. In BMMphi, IFN-gamma reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-gamma on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-gamma had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo.

Mechanisms responsible for neuroattenuation of herpes simplex virus (HSV) have been defined previously by studies of mutant viruses in cultured cells. The hypothesis that null mutations in host genes can override the attenuated phenotype of null mutations in certain viral genes was tested. Mutants such as those in infected cell protein (ICP) 0, thymidine kinase, ribonucleotide reductase, virion host shutoff, and ICP34.5 are reduced in their capacity to replicate in nondividing cells in culture and in vivo. The replication of these viruses was examined in eyes and trigeminal ganglia for 1-7 d after corneal inoculation in mice with null mutations (-/-) in interferon receptors (IFNR) for type I IFNs (IFN-alpha/betaR), type II IFN (IFN-gammaR), and both type I and type II IFNs (IFN-alpha/beta/gammaR). Viral titers in eyes and ganglia of IFN-gammaR-/- mice were not significantly different from congenic controls. However, in IFN-alpha/betaR-/- or IFN-alpha/beta/gammaR-/- mice, growth of all mutants, including those with significantly impaired growth in cell culture, was enhanced by up to 1,000-fold in eyes and trigeminal ganglia. Blepharitis and clinical signs of infection were evident in IFN-alpha/betaR-/- and IFN-alpha/beta/gammaR-/- but not control mice for all viruses. Also, IFNs were shown to significantly reduce productive infection of, and spread from intact, but not scarified, corneas. Particularly striking was restoration of near-normal trigeminal ganglion replication and neurovirulence of an ICP34.5 mutant in IFN-alpha/betaR-/- mice. These data show that IFNs play a major role in limiting mutant and wild-type HSV replication in the cornea and in the nervous system. In addition, the in vivo target of ICP34.5 may be host IFN responses. These experiments demonstrate an unsuspected role for host factors in defining the phenotypes of some HSV mutants in vivo. The phenotypes of mutant viruses therefore cannot be interpreted based solely upon studies in cell culture but must be considered carefully in the context of host factors that may define the in vivo phenotype.

Interferon gamma regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels.

To define immune mechanisms that regulate chronic and latent herpesvirus infection, we analyzed the role of interferon gamma (IFN-gamma) during murine cytomegalovirus (MCMV) infection. Lethality studies demonstrated a net protective role for IFN-gamma, independent of IFN-alpha/beta, during acute MCMV infection. Mice lacking the IFN-gamma receptor (IFN-gammaR-/-) developed and maintained striking chronic aortic inflammation. Arteritis was associated with inclusion bodies and MCMV antigen in the aortic media. To understand how lack of IFN-gamma responses could lead to chronic vascular disease, we evaluated the role of IFN-gamma in MCMV latency. MCMV-infected IFN-gammaR-/- mice shed preformed infectious MCMV in spleen, peritoneal exudate cells, and salivary gland for up to 6 mo after infection, whereas the majority of congenic control animals cleared chronic productive infection. However, the IFN-gammaR was not required for establishment of latency. Using an in vitro explant reactivation model, we showed that IFN-gamma reversibly inhibited MCMV reactivation from latency. This was at least partly explained by IFN-gamma- mediated blockade of growth of low levels of MCMV in tissue explants. These in vivo and in vitro data suggest that IFN-gamma regulation of reactivation from latency contributes to control of chronic vascular disease caused by MCMV. These studies are the first to demonstrate that a component of the immune system (IFN-gamma) is necessary to regulate MCMV-associated elastic arteritis and latency in vivo and reactivation of a herpesvirus from latency in vitro. This provides a new model for analysis of the interrelationships among herpesvirus latency, the immune system, and chronic disease of the great vessels.

IFN-gamma action in the media of the great elastic arteries, a novel immunoprivileged site.

Infection of medial smooth muscle cells with gamma-herpesvirus 68 (gammaHV68) causes severe chronic vasculitis that is restricted to the great elastic arteries. We show here that persistence of disease in the great elastic arteries is (a) due to inefficient clearance of viral infection from this site compared with other organs or other vascular sites, and (b) associated with failure of T cells and macrophages to enter the virus-infected elastic media. These findings demonstrate immunoprivilege of the media of the great elastic arteries. We found that IFN-gamma acted on somatic cells during acute infection to prevent the establishment of medial infection and on hematopoietic cells to determine the severity of disease in this site. The immunoprivileged elastic media may provide a site for persistence of pathogens or self antigens leading to chronic vascular disease, a process regulated by IFN-gamma actions on both somatic and hematopoietic cells. These concepts have significant implications for understanding immune responses contributing to or controlling chronic inflammatory diseases of the great vessels.

Unraveling immunity to gamma-herpesviruses: a new model for understanding the role of immunity in chronic virus infection.

Murine gamma-herpesvirus 68 (gammaHV68) infection is a new model for understanding how immunity and chronic gamma-herpesvirus infection inter-relate. gammaHV68 is closely related to the human Epstein-Barr virus and Kaposi's sarcoma herpesvirus and is associated with tumors, vasculitis of the great elastic arteries and splenic fibrosis. Advances in the past year have provided an even stronger foundation for believing that gammaHV68 infection of normal and mutant mice will become the pre-eminent animal model for understanding gamma-herpesvirus pathogenesis and immunity. gammaHV68 latency has been characterized employing new assays for quantitating cells carrying the gammaHV68 genome and cells that reactivate gammaHV68 and for detecting the presence of preformed infectious virus in tissues. These advances have fostered the first steps towards a molecular definition of gammaHV68 latency. It appears that gammaHV68 shares latency programs with human gamma-herpesviruses - including the loci for gene 73, v-bcl-2 and the viral homolog of the G-protein coupled receptor. This provides candidate antigens for analysis of the role of T and B cells in regulating latency. Multiple cellular reservoirs for gammaHV68 latency were uncovered with the demonstration that gammaHV68 latently infects macrophages in addition to B cells. A critical role for B cells in regulating the nature of gammaHV68 latency was discovered and the mechanism was shown to be via alteration of the efficiency of reactivation. Studies of the response of CD4(+) and CD8(+) cells during acute and chronic gammaHV68 were performed. These new studies provide key building blocks for further development of this novel and interesting model system.

Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.

A general association of human and primate lymphotropic herpesviruses (gamma-herpesviruses) with the development of lymphomas, as well as other tumors, especially in immunocompromised hosts, has been well documented. The lack of relevant small animal models for human gamma-herpesviruses has impeded progress in understanding the role of these viruses in the development of chronic disease. Recent research characterizing infection of inbred strains of mice with a murine gamma-herpesvirus, gamma-herpesvirus 68 (gammaHV68), is providing insights into viral and host factors involved in the establishment and control of chronic gamma-herpesvirus infection.

Animal models of infection-mediated vasculitis: implications for human disease.

The human vasculitides including Takayasu's arteritis are idiopathic syndromes for which both autoimmune and infectious etiologies have been proposed. Although proof of a correlation between infection and human vasculitis would aid in patient management, it is difficult to confirm causality. To study infection-mediated vascular disease, different animal models have been developed. Infections with the bacteria C. pneumoniae, an RNA virus, and herpesviruses all cause vascular pathology and will be reviewed here. Many aspects of the human diseases are recapitulated in these models, so further animal studies may help elucidate mechanisms of infection-mediated vasculitis. Such results may improve management, and potentially, prevention of these important human diseases. Importantly, the animal models provide an opportunity to define how the immune and inflammatory processes function in the great vessels and the molecular basis for the selectivity of certain viral infections for the great elastic arteries.

Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter.

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Latency, without persistence, of murine cytomegalovirus in the spleen and kidney.

It is not known if murine cytomegalovirus (MCMV) establishes a state of molecular latency independent of low-level persistent infection. The presence of low levels of infectious MCMV distinguishes persistence from molecular latency. Thus, the distinction between persistence and latency has depended on the sensitivity of plaque assays for detecting low levels of infectious virus in tissue of previously infected mice. To determine whether MCMV establishes molecular latency or remains persistent, we developed two assays for detecting low levels of MCMV in tissue. Using prolonged in vitro culture of virus with either mouse embryonic fibroblasts or the murine 3T12 fibroblast cell line, we reproducibly detected a single PFU of MCMV. Inclusion of undiluted sonicated tissue in this assay decreased sensitivity by up to 100-fold. However, sensitivity was improved to 1 PFU of MCMV when sonicated tissue was appropriately diluted. Severe combined immunodeficient (SCID) mice were also used to detect MCMV in sonicated tissue. Infection of SCID mice with a single PFU of MCMV killed two of eight SCID mice, and the 50% lethal dose of MCMV in SCID mice was 2 to 3 PFU. Applying these two methods, we detected infectious virus in 0 of 34 spleens, 1 of 34 kidneys, and 0 of 37 salivary glands from latently infected mice. Spleens and kidneys assessed for persistent virus contained MCMV DNA by PCR and reactivated after 10 to 50 days in explant cultures. Latently infected kidney cells reactivated after adoptive transfer to SCID mice. Quantitation of the MCMV genome by PCR showed that latently infected spleens without detectable infectious MCMV contained about 3,000,000 copies of the MCMV genome. These results demonstrate that MCMV latency in spleen and kidney exists in the absence of low-level persistent infection. Use of assays with defined sensitivity for detection of MCMV in tissue provides a basis for evaluation of cytomegalovirus gene expression in the spleen and kidney during molecular latency.

The T-cell-independent role of gamma interferon and tumor necrosis factor alpha in macrophage activation during murine cytomegalovirus and herpes simplex virus infections.

We defined the normal and innate (without functional B or T cells) inflammatory response to infection with mouse cytomegalovirus (MCMV) or herpes simplex virus (HSV). Intraperitoneal infection with MCMV or HSV induced an inflammatory infiltrate consisting largely of macrophages (M phi) in both normal CB17 and severe combined immunodeficient (SCID) mice (lacking functional B or T cells). M phi from infected mice were activated as shown by (i) spread morphology, (ii) increased expression of major histocompatibility complex (MHC) class II, MHC class I, and intercellular adhesion molecule-1 molecules, and (iii) downregulation of M phi-specific cell surface protein F4/80. In vivo administration of neutralizing antibodies specific for gamma interferon (IFN gamma) or tumor necrosis factor alpha (TNF alpha) inhibited MHC class II induction on infiltrating M phi in both normal and CB17 SCID mice. Anti-TNF alpha decreased the number of M phi in virus-induced inflammatory exudates. The MCMV titer increased in the spleen and liver of IFN gamma-depleted SCID mice, while TNF alpha depletion increased only splenic titers. MCMV-induced pathology was also increased in spleens of IFN gamma- and TNF alpha-depleted SCID mice. We conclude that (i) M phi activation is a prominent part of inflammatory responses to herpesvirus infection and (ii) IFN gamma and TNF alpha play a critical role in both virus-induced M phi activation and control of herpesvirus growth independent of T and B cells. This suggests that IFN gamma- and TNF alpha-mediated M phi activation is an important aspect of innate immunity to viral infection. As the M phi may be involved in MCMV latency, IFN gamma- and TNF alpha-dependent M phi activation during primary infection may be relevant to establishment of viral latency.

SCID mice as models for parasitic infections.

Mice with severe combined immunodeficiency (SCID mice) have become a favored model system for the study of many parasitic diseases. In this review, Samuel Stanley Jr and Herbert Virgin IV provide a brief overview of the biology of the SCID mouse, and review some examples of how the SCID mouse model has been applied to the study of the immunology of a number of different parasitic diseases.

Suppression of the immune response to Listeria monocytogenes. I. Immune complexes inhibit resistance.

Murine infection with the facultative intracellular pathogen Listeria monocytogenes was studied as a model of cell-mediated immunity. Overwhelming lethal infection with Listeria developed when mice were given a secondary challenge with a protein antigen at the same time as Listeria infection. Three days after infection, mice immunized with ovalbumin and then challenged with Listeria and ovalbumin had up to 2.1(10)6 times as many viable Listeria organisms in their spleens as mice challenged only with Listeria. The suppressive effect of secondary challenge with a protein antigen was found to be antigen-specific, transient, and not dependent on route of challenge with either Listeria or ovalbumin. Secondary challenge with a protein antigen suppressed both the primary and the secondary responses to Listeria. Immune serum, or affinity-purified antibody, was found to transfer the suppressive effect to nonimmune mice. These findings demonstrate that acute formation of immune complexes causes a transient state of high susceptibility to infection with an intracellular pathogen. Possible mechanisms for, and implications of, these findings are discussed.