Production of CXC and CC chemokines by human antigen-presenting cells in response to Lassa virus or closely related immunogenic viruses, and in cynomolgus monkeys with lassa fever.

International audienceThe pathogenesis of Lassa fever (LF), a hemorrhagic fever endemic to West Africa, remains unclear. We previously compared Lassa virus (LASV) with its genetically close, but nonpathogenic homolog Mopeia virus (MOPV) and demonstrated that the strong activation of antigen-presenting cells (APC), including type I IFN production, observed in response to MOPV probably plays a crucial role in controlling infection. We show here that human macrophages (MP) produce large amounts of CC and CXC chemokines in response to MOPV infection, whereas dendritic cells (DC) release only moderate amounts of CXC chemokines. However, in the presence of autologous T cells, DCs produced CC and CXC chemokines. Chemokines were produced in response to type I IFN synthesis, as the levels of both mediators were strongly correlated and the neutralization of type I IFN resulted in an inhibition of chemokine production. By contrast, LASV induced only low levels of CXCL-10 and CXCL-11 production. These differences in chemokine production may profoundly affect the generation of virus-specific T-cell responses and may therefore contribute to the difference of pathogenicity between these two viruses. In addition, a recombinant LASV (rLASV) harboring the NP-D389A/G392A mutations, which abolish the inhibition of type I IFN response by nucleoprotein (NP), induced the massive synthesis of CC and CXC chemokines in both DC and MP, confirming the crucial role of arenavirus NP in immunosuppression and pathogenicity. Finally, we confirmed, using PBMC samples and lymph nodes obtained from LASV-infected cynomolgus monkeys, that LF was associated with high levels of CXC chemokine mRNA synthesis, suggesting that the very early synthesis of these mediators may be correlated with a favourable outcome

The exonuclease domain of Lassa virus nucleoprotein is involved in antigen-presenting-cell-mediated NK cell responses

International audienceLassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE: The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a recombinant Lassa virus in which the exonuclease functions have been abolished and demonstrated that NK cells are strongly activated and presented effective functions. These results show that the strategy developed by Lassa virus to evade innate immunity is also effective on NK cells, explaining the weak NK cell activation observed with the wild-type virus. By providing a better understanding of the interactions between Lassa virus and the host immune system, these results are important for the field of arenavirus biology and may be useful for a vaccine approach against Lassa fever

NK cells are strongly activated by Lassa and Mopeia virus-infected human macrophages in vitro but do not mediate virus suppression.

International audienceLassa virus (LASV) and Mopeia virus (MOPV) are closely related Arenaviruses. LASV causes hemorrhagic fever, whereas MOPV is not pathogenic. Both viruses display tropism for APCs such as DCs and macrophages. During viral infections, NK cells are involved in the clearance of infected cells and promote optimal immune responses by interacting with APCs. We used an in vitro model of human NK and APC coculture to study the role of NK cells and to characterize their interactions with APCs during LASV and MOPV infections. As expected, NK cells alone were neither infected nor activated by LASV and MOPV, and infected DCs did not activate NK cells. By contrast, LASV- and MOPV-infected macrophages activated NK cells, as shown by the upregulation of CD69, NKp30, and NKp44, the downregulation of CXCR3, and an increase in NK-cell proliferation. NK cells acquired enhanced cytotoxicity, as illustrated by the increase in granzyme B (GrzB) expression and killing of K562 targets, but did not produce IFN-γ. Contact between NK cells and infected macrophages and type I IFNs were essential for activation; however, NK cells could not kill infected cells and control infection. Overall, these findings show that MOPV- as well as pathogenic LASV-infected macrophages mediate NK-cell activation

Exonuclease domain of the Lassa virus nucleoprotein is critical to avoid RIG-I signaling and to inhibit the innate immune response

International audienceLassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of retinoic acid-inducible gene I (RIG-I) signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection

Enquête sérologique pour le virus West Nile dans l’avifaune de la Dombes (Ain) en 2001 : absence de mise en évidence d’une circulation virale

International audienceBy the end of 2000, the West Nile (WN) virus re-emerged in France after 30 years of silence. Migratory birds coming from sub-saharan regions may be responsible for this new introduction in the Camargue delta. The current environmental conditions, favouring vectors development, are thought to play a crucial role in the occurrence of WN outbreaks and the spread of the virus. Numerous aquatic migratory bird species stop off north from Lyon, France, in "The Dombes" wetlands. This continental area, located along a migratory corridor, displays some of the environmental determinants of a WN entry point. That’s why we serologically evaluated the WN virus circulation in resident birds. The sentinel birds involved in this study live in close contact with birds migrating from Africa to Northern Europe. The sentinels were sampled from October 2001 to February 2002, after the migration period. Three hundred and sixty four (364) sera sampled from ducks, coots, pigeons, turtle doves, jackdaws, a buzzard and a white stork were analysed by ELISA. None of these sera was found to be positive for WN. The monitoring network set for this preliminary investigation may be repeated for future studies focused on this area prone to the introduction of avian zoonosis.La fièvre West Nile (WN) a émergé de nouveau en France fin 2000 après 30 ans de silence. Les oiseaux migrateurs venant des zones sub-sahariennes pourraient être responsables de cette nouvelle introduction en Camargue. Les conditions environnementales actuelles, favorables au développement des vecteurs, semblent jouer un rôle déterminant dans l’apparition d’épisodes ou l’installation du virus. De nombreuses espèces d’oiseaux migrateurs aquatiques s’arrêtent aussi au nord de Lyon, dans les étangs de la Dombes. Cette région, située le long d’un couloir de migration emprunté par les oiseaux, présente des conditions environnementales favorables à l’introduction du WN. Nous avons initié une approche destinée à évaluer la présence du virus chez les oiseaux vivant dans cette région d’introduction potentielle. Les animaux sentinelles recrutés pour cette étude partagent ce territoire avec les oiseaux migrant d’Afrique vers l’Europe du Nord. L’étude a été réalisée entre septembre 2001 et février 2002. Un total de 364 sérums, obtenus d’Anatidés, Rallidés, Columbidés, Corvidés, Accipitridés et Ciconidés ont été sondés par ELISA. La circulation du virus WN n’a pas été mise en évidence lors de cette enquête. Cette exploration préliminaire a permis d’assembler un système de collecte qui pourra être reconduit dans le futur dans cette région sujette à l’introduction de zoonoses aviaires

Enquête sérologique pour le virus West Nile dans l’avifaune de la Dombes (Ain) en 2001 : absence de mise en évidence d’une circulation virale

International audienceBy the end of 2000, the West Nile (WN) virus re-emerged in France after 30 years of silence. Migratory birds coming from sub-saharan regions may be responsible for this new introduction in the Camargue delta. The current environmental conditions, favouring vectors development, are thought to play a crucial role in the occurrence of WN outbreaks and the spread of the virus. Numerous aquatic migratory bird species stop off north from Lyon, France, in "The Dombes" wetlands. This continental area, located along a migratory corridor, displays some of the environmental determinants of a WN entry point. That’s why we serologically evaluated the WN virus circulation in resident birds. The sentinel birds involved in this study live in close contact with birds migrating from Africa to Northern Europe. The sentinels were sampled from October 2001 to February 2002, after the migration period. Three hundred and sixty four (364) sera sampled from ducks, coots, pigeons, turtle doves, jackdaws, a buzzard and a white stork were analysed by ELISA. None of these sera was found to be positive for WN. The monitoring network set for this preliminary investigation may be repeated for future studies focused on this area prone to the introduction of avian zoonosis.La fièvre West Nile (WN) a émergé de nouveau en France fin 2000 après 30 ans de silence. Les oiseaux migrateurs venant des zones sub-sahariennes pourraient être responsables de cette nouvelle introduction en Camargue. Les conditions environnementales actuelles, favorables au développement des vecteurs, semblent jouer un rôle déterminant dans l’apparition d’épisodes ou l’installation du virus. De nombreuses espèces d’oiseaux migrateurs aquatiques s’arrêtent aussi au nord de Lyon, dans les étangs de la Dombes. Cette région, située le long d’un couloir de migration emprunté par les oiseaux, présente des conditions environnementales favorables à l’introduction du WN. Nous avons initié une approche destinée à évaluer la présence du virus chez les oiseaux vivant dans cette région d’introduction potentielle. Les animaux sentinelles recrutés pour cette étude partagent ce territoire avec les oiseaux migrant d’Afrique vers l’Europe du Nord. L’étude a été réalisée entre septembre 2001 et février 2002. Un total de 364 sérums, obtenus d’Anatidés, Rallidés, Columbidés, Corvidés, Accipitridés et Ciconidés ont été sondés par ELISA. La circulation du virus WN n’a pas été mise en évidence lors de cette enquête. Cette exploration préliminaire a permis d’assembler un système de collecte qui pourra être reconduit dans le futur dans cette région sujette à l’introduction de zoonoses aviaires

Non-Pathogenic Mopeia Virus Induces More Robust Activation of Plasmacytoid Dendritic Cells than Lassa Virus

Lassa virus (LASV) causes a viral haemorrhagic fever in humans and is a major public health concern in West Africa. An efficient immune response to LASV appears to rely on type I interferon (IFN-I) production and T-cell activation. We evaluated the response of plasmacytoid dendritic cells (pDC) to LASV, as they are an important and early source of IFN-I. We compared the response of primary human pDCs to LASV and Mopeia virus (MOPV), which is very closely related to LASV, but non-pathogenic. We showed that pDCs are not productively infected by either MOPV or LASV, but produce IFN-I. However, the activation of pDCs was more robust in response to MOPV than LASV. In vivo, pDC activation may support the control of viral replication through IFN-I production, but also improve the induction of a global immune response. Therefore, pDC activation could play a role in the control of LASV infection

Production of chemokines by APC infected with recombinant LASV.

<p>The production of CC and CXC chemokines by DC (A, B) and MP (C, D) was assessed after mock infection (white bars), or infection with recombinant wild-type LASV (gray bars) or rLASV NP-D389A/G392A (rNP LASV) (black bars). (A, C) The synthesis of mRNAs was analyzed by RT-qPCR 24 h after infection. (B, D) The protein levels released in the supernatants were quantified by ELISA 24 h (only for CXCL9, 10 and 11) and 48 h after infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 4 (A, C) and 3–7 (B, D) independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (p<0.001).</p

Production of chemokines by MOPV- and LASV-infected DC cocultured with T cells and correlation with type I IFN synthesis.

<p>(A) Levels of CC chemokine and CXCL11 mRNA were quantified relative to GAPDH mRNA levels, after 2 days of coculture of mock- (white bars), MOPV- (gray bars), or LASV- (black bars) infected iDC and autologous T cells. Levels of CXCL10 mRNA were evaluated in iDC/T cell coculture 2 days after each stimulation of the T cells with infected or inactivated virus-stimulated iDC. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± standard error (SE) of 4 independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05) and ** (<i>p</i><0.01). (B) The levels of CC and CXC chemokines were quantified by ELISA in the supernatants after 2 days of coculture of mock- (white bars), MOPV- (gray bars), or LASV- (black bars) infected iDC and autologous T cells. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed in pg/ml, except for CXCL10 expressed in ng/ml. Significant differences are indicated as follows: * (<i>p</i><0.05). (C) Correlation between CCL4, CCL7, CXCL10, and CXCL11 mRNA levels and type I IFN synthesis by MOPV- or LASV-infected iDC cultured with naïve T cells 2 days after infection, represented by a linear regression with a correlation coefficient <i>r</i> and a probability of correlation <i>p</i>.</p

Role of type I IFN in the production of chemokines in MOPV- and LASV-infected iDC/T-cell cocultures.

<p>(A) The synthesis of mRNAs for CCL4, CCL7, CXCL10 and CXCL11 was evaluated in mock- (gray bars) or MOPV- (black bars) infected iDC cocultured with naïve T cells for 2 days in the presence of neutralizing Ab against CD118. (B) The levels of these mRNAs then were quantified 2 days after restimulation with mock- (gray bars) or inactivated MOPV- (black bars) pulsed iDC of T cells previously cocultured with mock- or MOPV-infected iDC in the presence of irrelevant IgG2a or CD118-neutralizing Ab. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 3 independent experiments.</p