Mice deficient in STAT1 but not STAT2 or IRF9 develop a lethal CD4+ T-cell-mediated disease following infection with lymphocytic choriomeningitis virus

Interferon (IFN) signaling is crucial for antiviral immunity. While type I IFN signaling is mediated by STAT1, STAT2, and IRF9, type II IFN signaling requires only STAT1. Here, we studied the roles of these signaling factors in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild-type (WT) mice and mice lacking either STAT2 or IRF9, LCMV infection was nonlethal, and the virus either was cleared (WT) or established persistence (STAT2 knockout [KO] and IRF9 KO). However, in the case of STAT1 KO mice, LCMV infection was lethal and accompanied by severe multiorgan immune pathology, elevated expression of various cytokine genes in tissues, and cytokines in the serum. This lethal phenotype was unaltered by the coabsence of the gamma interferon (IFN-γ) receptor and hence was not dependent on IFN-γ. Equally, the disease was not due to a combined defect in type I and type II IFN signaling, as IRF9 KO mice lacking the IFN-γ receptor survived infection with LCMV. Clearance of LCMV is mediated normally by CD8(+) T cells. However, the depletion of these cells in LCMV-infected STAT1 KO mice was delayed, but did not prevent, lethality. In contrast, depletion of CD4(+) T cells prevented lethality in LCMV-infected STAT1 KO mice and was associated with a reduction in tissue immune pathology. These studies highlight a fundamental difference in the role of STAT1 versus STAT2 and IRF9. While all three factors are required to limit viral replication and spread, only STAT1 has the unique function of preventing the emergence of a lethal antiviral CD4(+) T-cell response.     

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.     

Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection

Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4⁺ T cell responses, and the expansion of naïve CD8⁺ T cells into primary CD8⁺ T cells specific for LCMV GP_33–41 was relatively normal. In contrast, the expansion of the LCMV NP₃₉₆-specific CD8⁺ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8⁺ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8⁺ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8⁺ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8⁺ T cell responses.     

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-alphabeta) in the control of MCMV replication, with a prominent role of IFN-beta. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-alphabeta receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-alphabeta signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-alphabeta-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-alphabeta stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.     

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

The kinetics of CD8 T cell IFN-gamma responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-alphabeta and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-gamma response detectable in serum samples and spleen and liver homogenates. Production of IFN-gamma was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-gammadelta T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-gamma induction, and viral peptide stimulation enhanced CD8 T cell IFN-gamma expression in culture. The IFN-gamma response was associated with IL-18 and IFN-alphabeta expression. Furthermore, both factors augmented peptide-driven IFN-gamma production in culture, and mice lacking IL-18 or IFN-alphabeta functions had reduced day 4 IFN-gamma. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.     

Reciprocal immunomodulation in a schistosome and hepatotropic virus coinfection model

Human coinfection with the helminth parasite Schistosoma mansoni and hepatitis B and hepatitis C viruses is associated with increased hepatic viral burdens and severe liver pathology. In this study we developed a murine S. mansoni/lymphocytic choriomeningitis virus (LCMV) coinfection model that reproduces the enhanced viral replication and liver pathology observed in human coinfections, and used this model to explore the mechanisms involved. Viral coinfection during the Th2-dominated granulomatous phase of the schistosome infection resulted in induction of a strong LCMV-specific T cell response, with infiltration of high numbers of LCMV-specific IFN-gamma-producing CD8+ cells into the liver. This was associated with suppression of production of the Th2 cytokines dominant during S. mansoni infection and a rapid increase in morbidity, linked to hepatotoxicity. Interestingly, the liver of coinfected mice was extremely susceptible to viral replication. This correlated with a reduced intrahepatic type I IFN response following virus infection. Schistosome egg Ags were found to suppress the type I IFN response induced in murine bone marrow-derived dendritic cells by polyinosinic-polycytidylic acid. These results suggest that suppression of the antiviral type I IFN response by schistosome egg Ags in vivo predisposes the liver to enhanced viral replication with ensuing immunopathological consequences, findings that may be paralleled in human schistosome/hepatotropic virus coinfections.     

Role of an intact splenic microarchitecture in early lymphocytic choriomeningitis virus production

An acute infection with lymphocytic choriomeningitis virus (LCMV) is efficiently controlled by the cytotoxic-T-cell (CTL) response of the host, and LCMV titers in the spleen and peripheral solid organs usually fall sharply after day 4 to 6 postinfection. Surprisingly, infection of immunodeficient recombination-activating gene 2-deficient (RAG2-/-) mice with 5 x 10(2) PFU of LCMV-WE causes about 80-fold-lower LCMV titers in the spleen on day 4 postinfection compared with C57BL/6 control mice. This could not be attributed to NK cell activity, since common gamma-chain-deficient RAG2-/- mice lacking NK cells show low LCMV titers comparable to those for RAG2-/- mice. Furthermore, the reduced early LCMV production in spleens could not be explained by an enhanced gamma interferon production in RAG2-/- mice. Analysis of mutant mice exhibiting various defects in the splenic microarchitecture, including (i) tumor necrosis factor alpha-negative (TNF-alpha-/-), lymphotoxin alpha-negative (LTalpha-/-), B-cell-deficient muMT mice, (ii) immunoglobulin M-negative mice, and (iii) RAG2-/- mice reconstituted with wild-type versus TNF-alpha-/- LTalpha-/- B cells, revealed a clear correlation between an intact splenic marginal zone, rapid early replication of LCMV in the spleen, and efficient CTL induction. These results suggest that by the preferential infection of the highly organized splenic microarchitecture, LCMV seems to successfully exploit one of the key elements in the chain of the adaptive immune system. Not only does the early tropism of LCMV for the splenic marginal zone trigger a potent immune response, but at the same time the marginal zone may also become a target of early CTL-mediated immunopathology that impairs immune responsiveness.     

CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.     

Role of specific innate immune responses in herpes simplex virus infection of the central nervous system

Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.     

Absence of cross-presenting cells in the salivary gland and viral immune evasion confine cytomegalovirus immune control to effector CD4 T cells

Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.     

An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity

Type III IFNs (IFN-lambda/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-lambda was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Ralpha-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR(-/-)) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA(-/-) and IFNAR(-/-) mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-alphabeta expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-lambda expression, whereas IL-28Ralpha signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Ralpha only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-lambda and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.     

Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria

It is known that host cells can produce type I IFNs (IFN-alphabeta) after exposure to conserved bacterial products, but the functional consequences of such responses on the outcome of bacterial infections are incompletely understood. We show in this study that IFN-alphabeta signaling is crucial for host defenses against different bacteria, including group B streptococci (GBS), pneumococci, and Escherichia coli. In response to GBS challenge, most mice lacking either the IFN-alphabetaR or IFN-beta died from unrestrained bacteremia, whereas all wild-type controls survived. The effect of IFN-alphabetaR deficiency was marked, with mortality surpassing that seen in IFN-gammaR-deficient mice. Animals lacking both IFN-alphabetaR and IFN-gammaR displayed additive lethality, suggesting that the two IFN types have complementary and nonredundant roles in host defenses. Increased production of IFN-alphabeta was detected in macrophages after exposure to GBS. Moreover, in the absence of IFN-alphabeta signaling, a marked reduction in macrophage production of IFN-gamma, NO, and TNF-alpha was observed after stimulation with live bacteria or with purified LPS. Collectively, our data document a novel, fundamental function of IFN-alphabeta in boosting macrophage responses and host resistance against bacterial pathogens. These data may be useful to devise alternative strategies to treat bacterial infections.     

Protection from lethal infection by adoptive transfer of CD8 T cells genetically engineered to express virus-specific innate immune receptor

CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3zeta cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.     

IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation

Cytokines that are induced by infection may contribute to the initiation of immune responses through their ability to stimulate dendritic cells (DCs). In this paper, we have addressed the role of IL-15 in DC activation, investigating its expression by DCs in response to three different signals of infection and examining its ability to stimulate DCs. We report that the expression of both IL-15 and the IL-15 receptor alpha-chain are increased in splenic DCs from mice inoculated with dsRNA (poly(I:C)), LPS, or IFN-alphabeta, and in purified murine splenic DCs treated with IFN-alphabeta in vitro. Furthermore, IL-15 itself was able to activate DCs, as in vivo or in vitro exposure of splenic DCs to IL-15 resulted in an up-regulation of costimulatory molecules, markedly increased production of IFN-gamma by DC and an enhanced ability of DCs to stimulate Ag-specific CD8(+) T cell proliferation. The magnitude of all of the IL-15-induced changes in DCs was reduced in mice deficient for the IFN-alphabeta receptor, suggesting a role for IFN-alphabeta in the stimulation of DCs by IL-15. These results identify IL-15 as a stimulatory cytokine for DCs with the potential for autocrine activity and link its effects to expression of IFN-alphabeta.     

West Nile virus noncoding subgenomic RNA contributes to viral evasion of the type I interferon-mediated antiviral response

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.     

ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.     

Interferon Regulatory Factor-1 Protects from Fatal Neurotropic Infection with Vesicular Stomatitis Virus by Specific Inhibition of Viral Replication in Neurons

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1^−/− mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1^−/− mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.     

Type I IFN modulates host defense and late hyperinflammation in septic peritonitis

TLRs are considered important for the control of immune responses during endotoxic shock or polymicrobial sepsis. Signaling by TLRs may proceed through the adapter proteins MyD88 or TIR domain-containing adaptor inducinng IFN-beta. Both pathways can lead to the production of type I IFNs (IFN-alphabeta). In the present study, the role of the type I IFN pathway for host defense and immune pathology in sepsis was investigated using a model of mixed bacterial peritonitis. Systemic levels of IFN-alphabeta protein were markedly elevated during septic peritonitis. More detailed analyses revealed production of IFN-beta, but not IFN-alpha subtypes, and identified CD11b+ CD11c- macrophage-like cells as major producers of IFN-beta. The results further demonstrate that in IFN-alphabeta receptor I chain (IFNARI)-deficient mice, the early recruitment of neutrophils to the infected peritoneal cavity was augmented, most likely due to an increased local production of MCP-1 and leukotriene B4. In the absence of IFNARI, peritoneal neutrophils also exhibited enhanced production of reactive oxygen intermediates and elevated expression of Mac-1. Conversely, administration of recombinant IFN-beta resulted in reduced leukotriene B4 levels and decreased peritoneal neutrophil recruitment and activation. Analysis of the cytokine response to septic peritonitis revealed that IFNARI deficiency strongly attenuated late, but not early, hyperinflammation. In accordance with these findings, bacterial clearance and overall survival of IFNARI(-/-) mice were improved. Therefore, the present study reveals critical functions of the type I IFN pathway during severe mixed bacterial infections leading to sepsis. The results suggest that type I IFN exerts predominantly adverse effects under these conditions.