Interferon (IFN) regulatory factor 7 (IRF7) plays a key role in the production of IFN‐α in response to viral infection, and phosphorylation at IRF7 C‐terminal serine sites is prelude to its function. However, phosphatases that negatively regulate IRF7 phosphorylation and activity have not been reported. In this study, we have identified a conserved protein phosphatase 1 (PP1)‐binding motif in human and mouse IRF7 proteins, and shown that PP1 physically interacts with IRF7. Exogenous expression of PP1 subunits (PP1α, β, or γ) ablates IKKε‐stimulated IRF7 phosphorylation and dramatically attenuates IRF7 transcriptional activity. Inhibition of PP1 activity significantly increases IRF7 phosphorylation and IRF7‐mediated IFN‐α production in response to Newcastle disease virus (NDV) infection or Toll‐like receptor 7 (TLR7) challenge, leading to impaired viral replication. In addition, IFN treatment, TLR challenges and viral infection induce PP1 expression. Our findings disclose for the first time a pivotal role for PP1 in impeding IRF7‐mediated IFN‐α production in host immune responses.

No description available.

Epstein-Barr Virus (EBV) latent infection is associated with a variety of lymphomas and carcinomas. Interferon (IFN) Regulatory Factors (IRFs) are a family of transcription factors, among which IRF7 is the “master” regulator of type I IFNs (IFN-I) that defends against invading viruses. Robust IFN-I responses require a positive feedback loop between IRF7 and IFN-I. In recent years, we have discovered that IRF7 is significantly induced and activated by the principal EBV oncoprotein--Latent Membrane Protein 1 (LMP1); however, IRF7 fails to trigger robust IFN-I responses in EBV latency. We believe this intriguing finding is critical for EBV latency and oncogenesis, yet the underlying mechanism of this paradoxical phenomenon remains unclear. It is well known that tyrosine phosphorylation of most components of the IFN-I Jak-STAT pathway is essential for its signaling transduction. Thus, we have performed phosphotyrosine proteomics. We have found that the IFN-I Jak-STAT pathway is inactive due to the attenuated STAT2 activity, whereas the IFN-II Jak-STAT pathway is constitutively active, in EBV latency. We further confirmed these results by immunoblotting. This pilot study provides valuable information for the critical question regarding how the IRF7-mediated IFN-I response is evaded by EBV in its latency, and will prompt us to elucidate the underlying mechanisms.

No description available.

Les pDCs répondent aux infections virales par la production d'IFN-α. L'élimination du virus de l'hépatite C (VHC) chez plus de 50% des patients infectés par le traitement à l'IFN-alpha suggère que les pDCs jouent un rôle majeur dans le contrôle de l'infection VHC. Les pDCs exposées aux hépatocytes infectés par VHC, produisent beaucoup d'IFN-α. Néanmoins, en dépit de cette production par TLR7 par les pDCs, VHC continue à se répliquer dans le foie infecté. J'ai approfondi les connaissances des mécanismes moléculaires d'exploration des particules de VHC et des cellules infectées par le VHC par les pDCs. J'ai ciblé ma recherche sur le contact des particules de VHC avec la surface des pDCs, sur la voie de signalisation de déclenchée, sur leur effet sur la production des IFNs et des cytokines proinflammatoires et sur la différenciation cellulaire. Nos résultats suggèrent que le virus associé aux cellules signalise dans les pDCs via un mécanisme dépendant de l'endocytose et d'IRF7 mais pas de la voie NF-κB. En dépit de l'induction d'IFN-α, le VHC associé aux hépatocytes n'induit une réponse pleinement fonctionnelle des pDCs. Les particules virales de VHC inhibent, via la fixation de la glycoprotéine E2 aux CLRs, la production d'IFN-α et d'IFN-λ dans les pDCs exposées aux hépatocytes infectés par VHC et induisent dans les pDCs, une phosphorylation rapide d'Akt et Erk1/2, d'une manière similaire au crosslinking de BDCA-2 ou DCIR. Ainsi, le blocage de BDCA-2 et de DCIR avec des fragments Fab des anticorps monoclonaux préserve la capacité des pDCs à produire des IFNs de type I et III en présence des particules virales. / Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-alpha), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of infected patients by treatment with IFN-alpha suggests that pDCs play an important role in the control of HCV infection. pDCs exposed to HCV infected hepatoma cells produce large amounts of IFN-alpha. However, despite large amounts of Toll-like receptor 7-mediated IFN-α, produced by pDCs, HCV still replicates in infected liver. During my PhD training, I went into in depth to understand the molecular mecanisms used by HCV particles and HCV infected hepatocytes to explore pDCs. I focused my research on the binding of HCV particles with the pDC surface, on the triggered downstream signaling pathway, on the cellular differentiation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-kappaB pathway. In spite of IFN-alpha induction, cell-associated HCV does not induce a full functional response of pDCs. HCV particles inhibit, via binding of E2 glycoprotein to CLRs, production of IFN-α and IFN-λ in pDCs exposed to HCV-infected hepatocytes, and induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of monoclonal antibodies preserves the capacity of pDCs to produce type I and III IFNs in the presence of HCV particles.

The induction of type I interferon (IFN) and subsequent activation of interferon stimulated genes (ISGs) represent a first line of defense against viral infection. Typically type I IFN signaling leads to the phosphorylation of the STAT1 and STAT2 transcription factors (TFs) which then form a trimetric complex with IRF-9 and translocate to the nucleus to induce ISG expression. However, the results of this study showed that IFN-mediated upregulation of the ISG Oas1b, the product of which confers resistance to flavivirus induced disease, can be induced in a STAT1-independent manner. Since numerous ISGs have antiviral functions, many viruses have evolved strategies to disrupt the type I IFN-signaling pathway. In cases when STAT1 activation is blocked by a viral infection, STAT1-independent upregulation of ISGs provides an additional strategy for the cell to mount an effective antiviral response. Infection of mouse embryofibroblasts (MEFs) with West Nile virus (WNV) induced the production of IFN beta and STAT1 and STAT2 phosphorylation but blocked nuclear translocation and binding of these TFs to the promoters of the ISGs, Oas1a, Oas1a, Irf7 and Irf1. However, each of these antiviral ISGs was efficiently upregulated in infected cells and IRF-9 was shown to be crucial for the upregulation of Oas1a, Oas1b and Irf-7. IRF-3 or IRF-7 was needed to maintain the upregulation of these genes at later times of infection. In contrast, the upregulation of Irf1 by WNV infection did not depend on the tested IRFs but was reduced by inhibition of the p38 or NF-kappa B pathways. Although Irf1 mRNA was efficiently upregulated in WNV-infected cells IRF-1 protein synthesis was blocked. The precise mechanism of the IRF-1 translational suppression is not yet known, but the suppression was shown not to be due to increased proteasomal degradation of IRF-1 nor to alternative splicing of Irf1 mRNA. Preliminary results suggest miRNAs may play an indirect role in regulating IRF-1 translation. The results of this study expand knowledge about the strategies evolved by viruses to evade host cell antiviral responses and also provide valuable insights about alternative mechanisms utilized by the host cell to counteract viral infections.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22833号 / 医博第4672号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 濵﨑 洋子, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les cellules dendritiques plasmacytoïdes sont les productrices majeures d’IFN de type I dans l’organisme humain. Durant les infections virales chroniques, telles que l’infection par le Virus de l’Hépatite C, les pDCs sont fonctionnellement altérées. L’efficacité dans plus de 50% des cas du traitement par IFN-α, utilisé jusqu’à récemment, suggère que la modulation de la fonction des pDCs serait une cible intéressante pour le traitement HCV. Les pDCs reconnaissent l’ARN du HCV par les récepteurs Toll-like, et disposent de plus d’un set de récepteurs dits régulateurs qui régulent la production d’IFN-I. L’activation de ces RR inhibe la production d’IFN-I par les pDCs stimulées par les agonistes de TLR7/9. Nous montrons ici que la glycoprotéine d’enveloppe E2 du HCV est un nouveau ligand des RR BDCA2 et DCIR des pDCs, et que cette liaison est responsable de l’inhibition d’IFN-I via l’activation de la voie de signalisation BCR-like. Nous avons ensuite voulu restaurer la production d’IFN-I dans les pDCs en ciblant les kinases décrites de la voie BCR-like, Syk et Mek. En inhibant Syk, l’IFN-I n’a été que partiellement restauré par les concentrations subliminales de l’inhibiteur; les concentrations élevées de cet inhibiteur ont bloqué la production d’IFN-I, suggérant l’implication de Syk dans la voie TLR7/9 comme montré pour l’activation des TLR dans les macrophages. En inhibant MEK, la restauration d’IFN-I est efficace. Les mécanismes de cette restauration sont explorés. Le ciblage pharmacologique de la signalisation BCR-like constituerait une nouvelle approche intéressante pour étudier les mécanismes de modulation de l’activation des pDCs dans les conditions physiopathologiques. / Plasmacytoid dendritic cells are major producers of type I IFN in human organism. During chronic viral infections, such as Hepatitis C Virus infection, pDCs are functionally impaired. More than 50% efficiency of IFN-α treatment, until recently used, suggested that modulation of pDC function could be an important target for HCV treatment. pDCs recognize HCV RNA by Toll-like receptors, and dispose of a set of so-called regulatory receptors that regulate IFN-I production. Crosslinking of these RR such as BDCA-2 and ILT7 has been shown to inhibit IFN-I production by pDCs stimulated with TLR7/9 agonists. In this work we show that HCV envelope glycoprotein E2 is a novel ligand of pDC RR, BDCA-2 and DCIR, and that this binding is responsible for IFN-I inhibition via the activation of the BCR-like pathway. Then we assayed to restore IFN-I in pDCs with crosslinked RR by targeting well-known kinases of BCR-like pathway, Syk and Mek. When inhibiting Syk, IFN-I was only partially restored by subliminal concentrations of Syk inhibitor; high concentrations of Syk inhibitor effectively blocked IFN-I production, suggesting involvement of Syk in the TLR7/9 pathway as it was already demonstrated in TLR activation in macrophages. When inhibiting MEK, the restoration of type I IFN was effective. The underlying mechanisms leading to the restoration are further explored. Pharmacological targeting of BCR-like signaling may constitute an attractive new approach to study mechanisms of modulation of pDC activation in pathophysiological conditions.

La mucoviscidose est une pathologie pulmonaire causée par un dysfonctionnement du canal CFTR et caractérisée par un mucus visqueux, une susceptibilité accrue aux infections chroniques et une inflammation excessive. Une première partie de ma thèse a eu pour objectif d’étudier les mécanismes inflammatoires impliqués dans le développement de la pathologie. Nous avons plus particulièrement analysé le rôle de l’IL-1β et de l’IL-17 dans la réponse à l’infection par Pseudomonas aeruginosa, dans le modèle murin ΔF508 de mucoviscidose. La seconde partie de ma thèse a porté sur l’étude de la malaria pulmonaire et cérébrale, une complication létale de l’infection à P. falciparum. Nous avons mis en évidence l’importance de trois voies d’activation des lymphocytes T CD8+ cytotoxiques dans le développement de la neuropathologie induite par Plasmodium berghei ANKA chez la souris : la protéine PKC-θ, la sous-unité β2 du récepteur à l’IL-12 et le récepteur des IFN de type I, mais qui ne semblent pas impliquées dans l’inflammation pulmonaire associée. / Cystic fibrosis is a pulmonary pathology, caused by the CFTR channel dysfunction, and characterized by high mucus viscosity, increased sensitivity to chronic infections and excessive inflammation. The aim of my thesis was first to study the inflammatory mechanisms involved in this lung pathology. Indeed, we analyzed the role of IL-1β and IL-17 in response to Pseudomonas aeruginosa infection, in the ΔF508 mouse model of cystic fibrosis. In the second part of my thesis, I studied pulmonary and cerebral malaria, a lethal complication of P. falciparum infection. We showed the importance of three pathways implicated in cytotoxic CD8+ T lymphocytes activation during the Plasmodium berghei ANKA-induced neuropathology development in mice: PKC-θ protein, β2 subunit of IL-12 receptor and type I IFN receptor, which did not seem essential for the associated lung inflammation.

No description available.