Mireille Lafage

Virus Infection Switches TLR-3-Positive Human Neurons To Become Strong Producers of Beta Interferon

ABSTRACT To study the capacity of human neurons to mount innate immunity responses to viral infections, we infected cells of a human postmitotic neuron-derivative cell line, NT2-N, with rabies virus (RABV) and herpes simplex type 1 (HSV-1). Changes in neuronal gene expression were analyzed by use of Affymetrix microarrays. Applying a twofold cutoff, RABV increased the transcription of 228 genes, and HSV-1 increased the transcription of 263 genes. The most striking difference between the two infections concerns genes involved in immunity. These genes represent 24% of the RABV-upregulated genes and only 4.9% of the HSV-1-upregulated genes. Following RABV infection, the most upregulated genes belong to the immunity cluster and included almost exclusively genes for beta interferon (IFN-β) primary and secondary responses as well as genes for chemokines (CCL-5, CXCL-10) and inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha, interleukin 1 alpha). In contrast, HSV-1 infection did not increase IFN-β gene transcripts and triggered the production of only IL-6 and interferon regulatory factor 1 mRNAs. The microarray results were confirmed by real-time PCR, immunocytochemistry, and enzyme-linked immunosorbent assay. Human neurons were found to express Toll-like receptor 3. They produced IFN-β after treatment with poly(I:C) but not with lipopolysaccharide. Thus, human neurons can mount an innate immunity response to double-stranded RNA. These observations firmly establish that human neurons, in absence of glia, have the intrinsic machinery to sense virus infection.

The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA.

Inflammation is an important factor in the pathogenesis of neurodegenerative diseases, such as Alzheimers disease or multiple sclerosis, and during microbial infections of the nervous system. Glial cells were thought to be the main contributor for cytokine and chemokine production and Toll-like receptor (TLR) expression in the brain. Here, we report that human neurons express TLR-3, a major receptor in virus-mediated innate immune response. We established that these cells can mount a strong inflammatory response characterized by the expression of inflammatory cytokines (TNF-α, IL-6), chemokines (CCL-5 and CXCL-10), and antiviral molecules (2′5′OAS and IFN-β) after treatment with dsRNA—a by-product of viral infection and ligand of TLR-3. This work firmly establishes that human neurons, in absence of glia, have the intrinsic machinery to trigger robust inflammatory, chemoattractive, and antiviral responses.

Modulation of HLA-G Expression in Human Neural Cells after Neurotropic Viral Infections

ABSTRACT HLA-G is a nonclassical human major histocompatibility complex class I molecule. It may promote tolerance, leading to acceptance of the semiallogeneic fetus and tumor immune escape. We show here that two viruses—herpes simplex virus type 1 (HSV-1), a neuronotropic virus inducing acute infection and neuron latency; and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection—upregulate the neuronal expression of several HLA-G isoforms, including HLA-G1 and HLA-G5, the two main biologically active isoforms. RABV induces mostly HLA-G1, and HSV-1 induces mostly HLA-G3 and HLA-G5. HLA-G expression is upregulated in infected cells and neighboring uninfected cells. Soluble mediators, such as beta interferon (IFN-β) and IFN-γ, upregulate HLA-G expression in uninfected cells. The membrane-bound HLA-G1 isoform was detected on the surface of cultured RABV-infected neurons but not on the surface of HSV-1-infected cells. Thus, neuronotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially. HLA-G may therefore be involved in the escape of certain viruses from the immune response in the nervous system.

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.

Detrimental Contribution of the Immuno-Inhibitor B7-H1 to Rabies Virus Encephalitis

Rabies virus is the etiological agent of an acute encephalitis, which in absence of post exposure treatment is fatal in almost all cases. Virus lethality rests on its ability to evade the immune response. In this study, we analyzed the role of the immuno-inhibitory molecule B7-H1 in this virus strategy. We showed that in the brain and spinal cord of mice, rabies virus infection resulted in significant up-regulation of B7-H1 expression, which is specifically expressed in infected neurons. Correlatively, clinical rabies in B7-H1−/− mice is markedly less severe than in wild-type mice. B7-H1−/− mice display resistance to rabies. Virus invasion is reduced and the level of migratory CD8 T cells increases into the nervous system, while CD4/CD8 ratio remains unchanged in the periphery. In vivo, neuronal B7-H1 expression is critically depending on TLR3 signaling and IFN-β, because TLR3−/− mice—in which IFN-β production is reduced—showed only a limited increase of B7-H1 transcripts after infection. These data provide evidence that neurons can express the B7-H1 molecule after viral stress or exposure to a particular cytokine environment. They show that the B7-H1/PD-1 pathway can be exploited locally and in an organ specific manner—here the nervous system—by a neurotropic virus to promote successful host invasion.

Attenuation of Rabies Virulence: Takeover by the Cytoplasmic Domain of Its Envelope Protein

Survival of rabies virus–infected neurons depends on a single amino acid in the PDZ-binding site of a viral protein. Tipping the Balance Strains of rabies virus, which infects neurons, may be virulent, in which case the cells survive long enough for the virus to replicate and spread, or they may be attenuated, in which case the infected cells die by apoptosis. Préhaud et al. compared one attenuated and one virulent viral strain and found that a single amino acid change in a region of a viral envelope protein that binds to host cell proteins was sufficient to account for the death or survival of infected cells. The binding properties of the attenuated virus protein were expanded, thereby affecting the balance in the activities of host kinases and phosphatases sufficiently to trigger cell death. These findings may inform strategies to engineer attenuated viruses, which are often used in live vaccines. The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.

Absence of the p55 Kd TNF-α receptor promotes survival in rabies virus acute encephalitis

We investigated the role played by inflammation in acute encephalitis following infection with a neurotropic virus by comparing the disease caused by the CVS strain of rabies virus in C57BL/6 and mice deficient for the p55 Kd TNF-a receptor (p55TNFR 7/7 ). Morbidity (weight loss and paralysis) and mortality of infected mice were associated with viral propagation, cytokine (IL6, IL-10, TNF-a and IFN-g) production, induction of apoptosis and infiltration of inflammatory cells. Mortality occurred later in p55TNFR 7/7 (than in C57BL/6 mice. In contrast, morbidity and the number of cells undergoing apoptosis were similar in C57BL/6 and p55TNFR 7/7 mice.) This suggests that morbidity and mortality are independently regulated and that the death of the animal was not due to CNS apoptosis. Delayed mortality correlated with: a reduction in viral load on day 9 p.i., an increase in IFN-g and IL-10 concentrations and a reduction in inflammatory cell infiltration in the CNS. Thus, these data indicate that CVS infection elicits an inflammatory response within the CNS and suggest that cytokines signaling via the p55 Kd TNF-a receptor is deleterious for the survival of the host. These results strongly suggest that, the modulation of TNF-a and upregulation of IFN-g would be a powerful anti-virus strategy in cases of viral encephalitis. Journal of NeuroVirology (2000) 6, 507 ‐ 518.

The type I interferon response bridles rabies virus infection and reduces pathogenicity.

Rabies virus (RABV) is a neurotropic virus transmitted by the bite of an infected animal that triggers a fatal encephalomyelitis. During its migration in the nervous system (NS), RABV triggers an innate immune response, including a type I IFN response well known to limit viral infections. We showed that although the neuroinvasive RABV strain CVS-NIV dampens type I IFN signaling by inhibiting IRF3 phosphorylation and STAT2 translocation, an early and transient type I IFN response is still triggered in the infected neuronal cells and NS. This urged us to investigate the role of type I IFN on RABV infection. We showed that primary mouse neurons (DRGs) of type I IFN(α/β) receptor deficient mice (IFNAR−/− mice) were more susceptible to RABV than DRGs of WT mice. In addition, exogenous type I IFN is partially efficient in preventing and slowing down infection in human neuroblastoma cells. Intra-muscular inoculation of type I IFNAR deficient mice [IFNAR−/− mice and NesCre (+/−) IFNAR (flox/flox) mice lacking IFNAR in neural cells of neuroectodermal origin only] with RABV reveals that the type I IFN response limits RABV dissemination in the inoculated muscle, slows down invasion of the spinal cord, and delays mortality. Thus, the type I IFN which is still produced in the NS during RABV infection is efficient enough to reduce neuroinvasiveness and pathogenicity and partially protect the host from fatal infection.

Ambivalent Role of the Innate Immune Response in Rabies Virus Pathogenesis

ABSTRACT The neurotropic rabies virus (RABV) has developed several evasive strategies, including immunoevasion, to successfully infect the nervous system (NS) and trigger a fatal encephalomyelitis. Here we show that expression of LGP2, a protein known as either a positive or negative regulator of the RIG-I-mediated innate immune response, is restricted in the NS. We used a new transgenic mouse model (LGP2 TG) overexpressing LGP2 to impair the innate immune response to RABV and thus revealed the role of the RIG-I-mediated innate immune response in RABV pathogenesis. After infection, LGP2 TG mice exhibited reduced expression of inflammatory/chemoattractive molecules, beta interferon (IFN-β), and IFN-stimulated genes in their NS compared to wild-type (WT) mice, demonstrating the inhibitory function of LGP2 in the innate immune response to RABV. Surprisingly, LGP2 TG mice showed more viral clearance in the brain and lower morbidity than WT mice, indicating that the host innate immune response, paradoxically, favors RABV neuroinvasiveness and morbidity. LGP2 TG mice exhibited similar neutralizing antibodies and microglia activation to those of WT mice but showed a reduction of infiltrating CD4+ T cells and less disappearance of infiltrating CD8+ T cells. This occurred concomitantly with reduced neural expression of the IFN-inducible protein B7-H1, an immunoevasive protein involved in the elimination of infiltrated CD8+ T cells. Our study shows that the host innate immune response favors the infiltration of T cells and, at the same time, promotes CD8+ T cell elimination. Thus, to a certain extent, RABV exploits the innate immune response to develop its immunoevasive strategy.

Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus 1 infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG2 alpha, recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1.