IFN-γ production depends on IL-12 and IL-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1-4). Severe dengue infection in humans is characterized by thrombocytopenia, increased vascular permeability, hemorrhage and shock. However, there is little information about host response to DENV infection. Here, mechanisms accounting for IFN-γ production and effector function during dengue disease were investigated in a murine model of DENV-2 infection. IFN-γ expression was greatly increased after infection of mice and its production was preceded by increase in IL-12 and IL-18 levels. In IFN-γ(-/-) mice, DENV-2-associated lethality, viral loads, thrombocytopenia, hemoconcentration, and liver injury were enhanced, when compared with wild type-infected mice. IL-12p40(-/-) and IL-18(-/-) infected-mice showed decreased IFN-γ production, which was accompanied by increased disease severity, higher viral loads and enhanced lethality. Blockade of IL-18 in infected IL-12p40(-/-) mice resulted in complete inhibition of IFN-γ production, greater DENV-2 replication, and enhanced disease manifestation, resembling the response seen in DENV-2-infected IFN-γ(-/-) mice. Reduced IFN-γ production was associated with diminished Nitric Oxide-synthase 2 (NOS2) expression and NOS2(-/-) mice had elevated lethality, more severe disease evolution and increased viral load after DENV-2 infection. Therefore, IL-12/IL-18-induced IFN-γ production and consequent NOS2 induction are of major importance to host resistance against DENV infection.     

IFN-γ Receptor-Deficient Donor T Cells Mediate Protection from Graft-versus-Host Disease and Preserve Graft-versus-Tumor Responses after Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. It has been previously reported that lung GVHD severity directly correlates with the expansion of donor Th17 cells in the absence of IFN-γ. However, the consequence of Th17-associated lung GVHD in the presence of IFN-γ has not been well characterized. In the current study, T cells from IFN-γ receptor knockout (IFN-γR(-/-)) mice, capable of producing IFN-γ but unable to signal in response to IFN-γ, have been used to elucidate further the role of IFN-γ in GVHD. We found the transfer of donor T cells from either IFN-γR(-/-) or IFN-γ knockout (IFN-γ(-/-)) mice resulted in significant increases in donor Th17 cells in the lung. Marked increases in IL-4-producing Th2 cells infiltrating the lungs were also observed in the mice of donor IFN-γR(-/-) T cells. Notably, despite the presence of these cells, these mice did not show the severe immune-mediated histopathological lung injury observed in mice receiving donor IFN-γ(-/-) T cells. Increases in lung GVHD did occur in mice with donor IFN-γR(-/-) T cells when treated in vivo with anti-IFN-γ demonstrating that the cytokine has a protective role on host tissues in GVHD. A survival benefit from acute GVHD was also observed using donor cells from IFN-γR(-/-) T cells compared with control donors. Importantly, tumor-bearing mice receiving IFN-γR(-/-) T cells versus wild-type donor T cells displayed similar graft-versus-tumor (GVT) effects. These results demonstrate the critical role of IFN-γ on host tissues and cell effector functions in GVHD/GVT.     

Long‐term infection of adult mice with murine polyomavirus following stereotaxic inoculation into the brain

Murine polyomavirus is used in various models of persistent virus infection. This study was undertaken to assess the spatial and temporal patterns of MPyV infection in the brains of immunocompetent (BALB/c) and immunocompromised (KSN nude) mice. MPyV was stereotaxically microinfused into the brain parenchyma, and the kinetics of infection were examined by quantitative PCR. In BALB/c mice, the amount of viral DNA in the brain peaked at 4 days p.i. and then rapidly diminished. In contrast, MPyV DNA levels increased up to 4 days and then gradually decreased over the 30‐day observation period in the brain of KSN mice. In both mouse strains, viral DNA was readily detected around the sites of inoculation from 2 to 6 days p.i., and continued to be detected for up to 30 days p.i. In addition, MPyV infection did not lead to a drastic induction of innate immune response in the brains, nor did MPyV‐inoculated mice show any signs of disease. These results indicate that MPyV establishes an asymptomatic long‐term infection in the mouse brain.     

T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.     

STAT1-independent control of a neurotropic measles virus challenge in primary neurons and infected mice

Neurons are chiefly nonrenewable; thus, cytolytic immune strategies to clear or control neurotropic viral infections could have lasting neurologic consequences. IFN-γ is a potent antiviral cytokine that is critical for noncytolytic clearance of multiple neurotropic viral infections, including measles virus (MV); however, the downstream pathways through which IFN-γ functions in neurons have not been defined. Unlike most cell types studied to date in which IFN-γ affects gene expression via rapid and robust activation of STAT1, basal STAT1 levels in primary hippocampal neurons are constitutively low, resulting in attenuated STAT1 activation and consequently slower kinetics of IFN-γ-driven STAT1-dependent gene expression. Given this altered expression and activation of STAT1 in neurons, we sought to determine whether STAT1 was required for IFN-γ-mediated protection from infection in neurons. To do so, we evaluated the consequences of MV challenge of STAT1-deficient mice and primary hippocampal neurons explanted from these mice. Surprisingly, the absence of STAT1 did not restrict the ability of IFN-γ to control viral infection either in vivo or ex vivo. Moreover, the canonical IFN-γ-triggered STAT1 gene expression profile was not induced in STAT1-deficient neurons, suggesting that IFN-γ regulates neuronal STAT1-independent pathways to control viral replication.     

T cell-derived interferon-γ is required for host defense to Toxoplasma gondii

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter "GREVEN" mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4⁺, CD8⁺, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifng^fl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Indoleamine 2,3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.     

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.     

共表达猪繁殖与呼吸综合征病毒GP5基因及猪γ-干扰素基因的腺病毒载体的构建与鉴定

利用PCR方法分别扩增猪繁殖与呼吸综合征病毒全长GP5基因（E蛋白）,EMCV的核糖体介入位点（IRES）序列及猪γ-干扰素（IFN-γ）基因全长序列,序列测定正确后用DNA重组法将三者串联后插入pAdenoVator-CM V5-IRES-GFP穿梭质粒中,形成的穿梭质粒plRES-GP5-IFN-γ用PmeⅠ线性化后,与腺病毒骨架载体pAdEasy-1共转化感受态大肠埃希氏菌BJ5183,经同源重组,构建成含有GP5基因和IFN-γ基因的重组腺病毒载体,pacⅠ酶切线性化充分暴露反向末端重复序列后,脂质体转染HEK293A细胞,借助GFP的表达可以在转染后的2～3天观察到包装病毒rAdeno-GP5-IFN-γ产生,7～10天出现病毒蚀斑。经PCR法及酶切证实各中间过程载体及最终的包装病毒中携带有目的基因,western-blot证实两基因在腺病毒中得到了表达。大肠杆菌内同源重组法能有效和较为方便的构建出含有目的基因的腺病毒载体rAdeno-GP5-IFN-γ,重组子能够在HEK293细胞中稳定扩增,病毒包装的成功为进一步研究PRRSVE蛋白的免疫效果及IFN-γ的作用奠定了基础。     

Synergistic roles of eukaryotic translation elongation factors 1Bγ and 1A in stimulation of tombusvirus minus-strand synthesis

Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic translation elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3' end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic translation elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for translation factors during TBSV replication are separate from their canonical roles in host and viral protein translation.     

Susceptibility to acute mouse adenovirus type 1 respiratory infection and establishment of protective immunity in neonatal mice

There is an incomplete understanding of the differences between neonatal immune responses that contribute to the increased susceptibility of neonates to some viral infections. We tested the hypothesis that neonates are more susceptible than adults to mouse adenovirus type 1 (MAV-1) respiratory infection and are impaired in the ability to generate a protective immune response against a second infection. Following intranasal infection, lung viral loads were greater in neonates than in adults during the acute phase but the virus was cleared from the lungs of neonates as efficiently as it was from adult lungs. Lung gamma interferon (IFN-γ) responses were blunted and delayed in neonates, and lung viral loads were higher in adult IFN-γ(-/-) mice than in IFN-γ(+/+) controls. However, administration of recombinant IFN-γ to neonates had no effect on lung viral loads. Recruitment of inflammatory cells to the airways was impaired in neonates. CD4 and CD8 T cell responses were similar in the lungs of neonates and adults, although a transient increase in regulatory T cells occurred only in the lungs of infected neonates. Infection of neonates led to protection against reinfection later in life that was associated with increased effector memory CD8 T cells in the lungs. We conclude that neonates are more susceptible than adults to acute MAV-1 respiratory infection but are capable of generating protective immune responses.     

Small and middle T antigens contribute to lytic and abortive polyomavirus infection.

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was less than 10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.     

Impact of host responses on control of hepatitis C virus infection in Chinese blood donors

A study was undertaken to explore the molecular mechanisms underlying control of HCV infection in blood donors in China. Factors including clinical information, anti-HCV reactivity (S/CO), IFN-α and IFN-γ, viral loads and genotypes were correlated with 160 index plasma samples at three statuses of 45 recovered, 76 chronic or 39 false positive anti-HCV reactive blood donors. The spontaneous recovery rate was 37.2%. Viral loads of 76 HCV plasmas ranged between 59.8 IU/ml and 2.43 × 10⁶ IU/ml (median 3.67 × 10⁴ IU/ml). Genotypes 1, 2, 3 and 6 of 63 HCV strains were identified phylogenetically. Recovered donors were significantly younger (p = 0.002) and had lower level IFN-γ (p = 0.001) than chronically HCV infected donors. Circulating levels of IFN-α and IFN-γ were higher in those with low viral load and were low in middle or high viral load samples. The ratio of IFN-α to IFN-γ (IFN-α/γ) was significantly positively correlated with viral load (p = 0.037), and viral load was inversely correlated with IFN-γ in chronic HCV infection regardless of genotype. The study revealed clearly different relationships between IFN-α and IFN-γ in relation to viral load in HCV. A novel measure of IFN-α/γ ratio could be a new approach to evaluate long term outcome of HCV infection.     

IFN-gamma mediates enhancement of HIV replication in astrocytes by inducing an antagonist of the beta-catenin pathway (DKK1) in a STAT 3-dependent manner

Typically, IFN-γ is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-γ induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-γ to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-γ relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous β-catenin signaling, a pathway that is a potent inhibitor of HIV replication, IFN-γ diminished β-catenin signaling in astrocytes by 40%, as evaluated by both active β-catenin protein expression and β-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-γ-mediated inhibition of β-catenin signaling was dependent on its ability to induce an antagonist of the β-catenin signaling pathway, Dickkopf-related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-γ to enhance HIV replication in astrocytes. These data demonstrated that IFN-γ induces HIV replication in astrocytes by antagonizing the β-catenin pathway. To our knowledge, this is the first report to point to an intricate cross-talk between IFN-γ signaling and β-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface.     

Transgenic Expression of IFN-γ in the Eye as a Model for Studying Ocular Inflammatory Disease

To study the effects of IFN-γ on intraocular inflammation we used mice with transgenic expression of IFN-γ in the retina of the eye. These transgenic mice (rhoγ) were challenged intraocularly with soluble and cellular antigens and then studied for development of uveitis and delayed-type hypersensitivity reaction after a second intracutaneous challenge with the same antigen. Further experiments tested the influence of IFN-γ on infection with herpes simplex (HSV) by intraocular virus inoculation in rhoγ mice and rhoγ mice crossed with MHC class I or class II deficient mice. Pathology was studied by morphology immunocytochemistry and electron microscopy. Growth of HSV was assessed by plaque assay on Vero cells. Rhoγ transgenic mice produced IFN-γ in the photoreceptors of the retina and secreted the cytokine into the vitreous of the eye. These animals suffered from cellular infiltration of the eyes, cataracts, and degeneration of the photoreceptors. In contrast to control mice, transgenic mice had increased uveal inflammation and developed delayed-type hypersensitivity after intraocular challenge with alloantigens such as allogeneic splenocytes and inactivated virus. Rhoγ mice also experienced protection from infection with herpes simplex virus-1 (HSV-1) and HSV-2, suffering a less severe course of disease than control mice. Cytokine-induced protection was not based on viral replication block and did not require simultaneous expression of MHC class I and class II. The rhoγ transgenic mouse provided a model suitable for testing mechanisms of IFN-γ action in the eyein vivo.The surprising results offer new arguments in the evaluation of cytokine effects.