Unit: Neuro-Immuno Virology

Director: Monique Lafon

Our group is studying the interactions of neurotropic viruses with the nervous system (NS) and the immune system to identify the molecular basis of neuronal survival. An in vivo approach consists in studying the mechanisms developed by pathogenic strains of rabies virus and West Nile virus to subvert the host surveillance. An in vitro approach consists in listing and subsequently characterizing the neuron genes utilized by pathogenic viruses to subvert the T cell response and to promote intrinsic neuronal survival.

Host defence mechanisms help the organism to clear most viral infections after a few days. Nevertheless, this is not always the case since some viruses have elaborated strategies to subvert host defences and thereby promote their own multiplication or establish latency. Among viral infections of the NS, HSV-1 and rabies virus are examples of viral infections which are partially -or not at all- cleared by the immune response, whereas West Nile virus escapes the immunosurveillance in only rare occasions. After primary infection, HSV-1 establishes a latent infection in sensory neurons. Latency, which is characterized by the absence of production of infectious viruses despite the production of viral genome and some viral proteins, settles in the presence of surrounding CD8+ T cells. Reactivation of HSV-1 from latency is stimulated by external stimuli (UV, stress) and in immunocompromised patients. West Nile virus is a mosquito-borne flavivirus that causes lethal encephalitis in aged and immunocompromised patients. In contrast of HSV-1 or West Nile virus, rabies virus, which is transmitted by bites of rabid animals, totally evades the control of the immune system and the issue of rabies encephalitis is irremediably fatal. Rabies virus has selected not only mechanisms which contribute to evade the immune response but also mechanisms which protect neurons from virus-mediated burden. These three viruses are taken as models of three different types of virus/host interactions leading to different levels of neuroprotection.

A- Viruses and Cell cultures

In vitro analysis are performed with 1) human neuroblastoma : SK-N-SH or neuronally differentiated SK-N-SH /RA cells obtained after trans-retinoic acid (RA) treatment (Wainwright, 2001), 2) human post mitotic neurons NT2-N (Pleasure, 1992) obtained from the NT-2 precursor Ntera-2D1 (Stratagene, La Jolla CA, USA) and differentiated according to a modification of the original procedure (Cheung, 1999) and 3) mixed cultures containing human neurons and astrocytes NT2-N/A (PHOTO 1). NT2-N/A are obtained from Ntera-2D1 by a particular protocol of differentiation (Sandhu, 2002). Human post-mitotic neurons (NT2-N) and co-cultured neurons and astrocytes (NT2-N/A) are a model of choice to analyze the interactions between human astrocytes and neurons in vitro. As the rabies virus infects only neurons, these co-culture experiments will allow us to study the interactions between rabies virus-infected neurons and surrounding non-infected astrocytes (Christophe Préhaud and Mireille Lafage).

Experiments are performed with neuroprotective and non-neuroprotective rabies virus mutants.

In vivo analysis are performed by injecting mice in the hind limbs with mouse NS adapted neurovirulent strains of rabies virus or West Nile virus.

B- Transcriptomes of human neurons infected by rabies virus or HSV-1

An extensive search for neuronal genes the transcription of which is up regulated during rabies virus and HSV-1 infection was performed by transcriptome analysis using Affymetrix U133A and U133B micro arrays. Messenger RNAs were prepared from synchronized cultures of human post mitotic neurons NT2-N either non-infected or infected with rabies virus or HSV-1. Comparative analysis has shown that rabies virus triggers a strong IFN-β and CCL5 mRNA expression in humans neurons followed by the increase of transcripts of several interferon-inducible genes such as CXCL-10, OAS, IRF7, MXA, GBP... These results were confirmed by PCR and by immunohistochemistry (PHOTO 2 CXCL10 in neurons) or ELISA (IFN-β). In contrast to rabies virus, none of these genes was up regulated by HSV-1 infection. In the SN rabies virus infects neurons exclusively. How non-infected astrocytes sense the infection of neurons by rabies virus is currently under investigation by comparing the transcriptomes after rabies virus infection in cultures of neurons, NT2-N, and in co-cultures neurons/astrocytes NT2-N/A. (Christophe Préhaud, Françoise Mégret, Mireille Lafage, Monique Lafon in collaboration with the Alsace-Lorraine Genopole, Louis Pasteur University in Strasbourg, and Lars Rogge from the Pasteur Institute).

C- Rabies virus and neuroprotection

Highly pathogenic strains of rabies virus are neuroprotective. Neurons infected with the highly pathogenic strains of rabies virus do not encounter death. This paradox is observed in vivo and in vitro and in different types of cells (Baloul and Lafon, 2003, Lay et al 2003, Préhaud et al, 2003, Thoulouze M-I et al, 1997, 2003). Bcl-2 over-expression may contribute to rabies virus mediated cell survival both in vitro and in vivo (Thoulouze et al, 2003 and Baloul and Lafon, submitted) PHOTO 3. A more extensive search of early survival genes was performed by transcriptome analysis using Affymetrix U133A and U133B microarrays. Messenger RNAs were prepared from synchronized cultures of human post mitotic neurons NT2-N infected with neuroprotective and non-neuroprotective rabies virus mutants. Comparative analysis identifies a few genes which are differentially transcribed by neuroprotective rabies virus. Over expression of these genes was confirmed by RT-PCR. Involvement of two of these genes in neuron survival is currently under investigation (Christophe Préhaud, Françoise Mégret in collaboration with the Alsace-Lorraine Genopole, Louis Pasteur University in Strasbourg, and Lars Rogge from the Pasteur Institute).

The molecular basis of neuronal survival following infection are also analysed by comparing the activation pathways triggered in human neuroblastoma cell lines (SK-N-SH/RA) after infection with a neuroprotective and a non neuroprotective rabies virus strain. Several pathways, including phosphatidylinositol-3-OH-kinase (PI3K)-AKT or ERK (extra cellular signal-regulated kinase) are involved in neuronal survival (Brunet A et al, 2001, Hetman et Gozdz, 2004). We are currently testing the hypothesis that strains of neuroprotective virus may activate one of these pathways or inhibit some pathways known to be implicated in apoptosis such as SAPK (stress-activated protein kinases: p38 MAPK and JNK). (Michèle Roa and Monique Lafon).

D- Immunosubversion and infection of NS by neuronotropic viruses

D.1 Fas-L and rabies virus

We are searching for the immunosubversive molecules which rabies virus and HSV-1 up regulate to escape the immune response. NS infection by rabies virus escapes the control of protective T cells. We found that migratory protective T cells died of apoptosis shortly after their entry into the NS. We demonstrated that death of T cell results of an over expression of Fas-L by rabies virus infected neurons. Migratory T cells expressing Fas received a death signal through the ligation of Fas-L expressed by rabies virus infected neurons. In mice lacking Fas-L, (gld mice) rabies virus neuroinvasion was reduced and disease was less severe. In the meantime, death of T cells decreased in gld mice compared to T cell death observed in mice expressing Fas-L. These data demonstrated that fatal rabies virus triggers early Fas-L expression leading to the destruction of migratory T cells by the Fas/Fas-L apoptosis pathway. These data indicate that up regulation of Fas-L is one of the mechanisms which contribute to rabies virus immunosubversion (Lafon et al, 2004, Baloul et al, 2004).

In addition to the up regulation of Fas-L, we observed that rabies virus also up regulated HLA-G, and one member of the B and T lymphocytes attenuators (BTLA).

D-2 HLA-G and rabies virus and HSV-1

HLA-G is a non-classical HLA class I antigen with reduced polymorphism. HLA-G mediates destruction of T and NK cells by ligation with different receptors: the inhibitory receptor KIR2DL4 expressed on NK, the ILT2 inhibitory receptors expressed on T and NK cells and the CD8 molecules expressed by T cells. HLA-G may contribute to tumour evasion and foetal semi-allograft tolerance. In vitro in human NT2-N cultures we observed that rabies virus up regulates the expression of HLA-G in rabies virus infected neurons as well as in neighboured non-infected neurons. HLA-G over expression in non-infected cells was also observed in the mixed human neuron/astrocyte cultures NT2-N/A, in which the astrocytes which are resistant to rabies virus infection up regulated HLA-G when cultivated with infected neurons. This strongly suggests that rabies virus infection can create a generalized immunosubversive environment surrounding the infected neurons as stealth against the immune response (Monique Lafon Françoise Mégret, Christophe Préhaud, Mireille Lafage, Michèle Roa in collaboration with Nathalie Rouas-Freiss and Philippe Moreau Unité d'Immuno-Hématologie CEA/Hôpital Saint-Louis, Paris, directed by Pr Edgardo Carosella).

D-3 BTLA and rabies virus

BTLA act as strong inhibitors of T and B cell activation and can be regarded as contributors of the immune tolerance, tumour immune evasion and regulation of the inflammatory responses (Greenwald R et al 2005). We observed that rabies virus infection up regulates the expression of BTLA in vitro and in vivo. BTLA mRNAs were increased in NT2-N and NT2-N/A cultures by rabies virus infection. Infected neurons of mouse CNS express BTLA in the early stages of the CNS invasion. The role of BTLA in rabies immunoevasion is currently under investigation. (Monique Lafon, Mireille Lafage, Myriam Lucia Velandia,, Françoise Mégret, Christophe Préhaud, in collaboration with Pr Heinz Wiendl, Würzburg University, Germany)

D-4 West Nile infection and NK

West Nile virus, a mosquito-borne flavivirus is the etiological agent of West Nile fever and causes lethal meningo-encephalitis in aged and immunocompromised patients. Innate immunity and protection conferred by NK decreased in aged patients. We are investigating the role of innate immunity in NS, and of NK in particular in the control of SN invasion by West Nile virus.

(Thérèse Couderc, Anne-Claire Brehin, Monique Lafon in collaboration with Philippe Despres from Pasteur Institute)

E - Inflammation and neuroprotection

Following the entry of pathogens in the NS, the factors that orientate the innate immune response to immunopathology or neuroprotection are not very well defined. Inflammation in the brain may have deleterious effects. Nevertheless, IL-6 is also a neuroprotective agent that improves survival in various stress conditions, including traumatic brain injury. Similarly, TNF-α can protect neurons against direct metabolic excitotoxic and oxidative stress.

During rabies virus infection, the NS produces several types of chemokines, such as MCP-1 or CXCL-10 and cytokines including TNF-α  IL-1β and IL-6 as well as the chemokine receptors CCR1, 2, 5, 8 and CXCR3. In addition, transcriptional analysis indicates that human neurons infected by rabies virus are strong producers of IFN-β and chemokines. In vivo, mice lacking the TNFR type 1 (P55 TNFR KO mice) show delayed morbidity and reduced mortality compared to wild-type mice when infected with the rabies virus (Camelo S et al 2000). The fact that mice deficient in inflammatory pathways died later than immunocompetent mice indicates that NS invasion is delayed when inflammation is reduced. This suggests that the strong inflammatory reaction triggered by the rabies virus infection may in fact promote neuroinvasiveness of rabies virus. Search for viral molecules which may modulate inflammation and neuroprotection is in progress (Monique Lafon).

Photo 1: Mixed cultures of human post-mitotic neurons and astrocytes (Christophe Prehaud)

Photo 2: Rabies virus infected human post-mitotic neurons NT2-N produce CXCL10 chemokine (green). Nuclei are stained in blue (Hoechst). (Mireille Lafage)

Photo 3: Expression of Bcl-2 (orange) by rabies virus-infected neurons in the CNS of rabies virus-infected NS. Nuclei are stained in blue. Fragmentized nuclei correspond to nuclei of apoptotic migratory lymphocytes (Leila Baloul)


Keywords: neurons, NT-2N, rabies virus, HSV-1, West Nile virus, HLA-G, BTLA, Fas-L, micro-arrays, Neurology, Virology, Immunology

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