Laurence Guzylack-Piriou

Type-A CpG oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferon-α, tumour necrosis factor-α and interleukin-12

Natural interferon‐producing cells (NIPC), also referred to as immature plasmacytoid dendritic cells (PDC), constitute a small population of leucocytes secreting high levels of type I interferons in response to certain danger signals. Amongst these signals are those from DNA containing unmethylated CpG motifs. The present work demonstrated that the CpG oligonucleotides (CpG‐ODN) 2216, D32 and D19 induce high amounts of interferon‐α (IFN‐α), tumour‐necrosis factor‐α (TNF‐α) and interleukin (IL)‐12 in porcine peripheral blood mononuclear cells (PBMCs). Swine workshop cluster 3 (SWC3)1ow CD4high cells, with high IL‐3‐binding activity, representing NIPC, were the exclusive cytokine‐producing cells responding to the CpG‐ODN. These cells did not express CD6, CD8 or CD45RA. Importantly, monocyte‐derived DC did not respond to CpG‐ODN by secretion of IFN‐α or TNF‐α or by the up‐regulation of costimulatory molecule expression. CpG‐ODN up‐regulated MHC class II and CD80\86 expression on the NIPC, but were unable to promote NIPC survival. Interestingly, certain CpG‐ODN, incapable of inducing NIPC to secrete IFN‐α or up‐regulate MHC class II and CD80\86, did promote NIPC viability. Taken together, the influence of CpG‐ODN on porcine NIPC, monocytes and myeloid DCs relates to that observed with their human equivalents. These results represent an important basis for the application of CpG‐ODN as adjuvants for the formulation of novel vaccines and demonstrate the importance of the pig as an alternative animal model for this approach.

Porcine peripheral blood dendritic cells and natural interferon‐producing cells

Peripheral blood contains two major particular infrequent dendritic cells (DC) subsets linking the innate and specific immune system, the myeloid DC and plasmacytoid DC equivalent to the natural interferon‐producing cells (NIPC). The functional characterization of these cells demands large volumes of blood, making a large animal model more appropriate and beneficial for certain studies. Here, two subsets of porcine blood mononuclear cells expressing swine workshop cluster 3 (SWC3, a SIRP family member), are described and compared to monocytes. The blood DC specialized in T‐cell stimulation were major histocompatibility complex (MHC) class II+, CD80/86+, CD1+/–, CD4−, and in contrast to monocytes CD14−. A CD16− and a CD16+ subset could be discriminated. Granulocyte–macrophage colony‐stimulating factor and interleukin‐3 were survival factors for this DC subset, and culture induced an up‐regulation of MHC class II and CD80/86. The second subset described, are porcine NIPC, typically CD4++, MHC class IIlow, CD80/86low, CD1−, CD8−/low, CD16−/low and CD45RA−/low. Porcine NIPC had high interleukin‐3 binding capacity, and survived in response to this cytokine. Their unique function was strong interferon type I secretion after virus stimulation. Both subsets were endocytically active when freshly isolated, and down‐regulated this activity after in vitro maturation. Taken together, the present report has delineated porcine blood DC and NIPC, permitting a more detailed understanding of innate immune defences, particularly in response to infections.

Subset-dependent modulation of dendritic cell activity by circovirus type 2

Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single‐stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon‐producing cells (NIPCs)], as well as DC precursors. This PCV2–DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon‐α/tumour necrosis factor‐α (IFN‐α/TNF‐α), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non‐macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG‐oligonucleotide (CpG‐ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2‐induced inhibition of the IFN‐α and TNF‐α normally produced following interaction with CpG‐ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.

Plasmacytoid dendritic cell activation by foot-and-mouth disease virus requires immune complexes

Natural IFN‐producing cells (NIPC), also called plasmacytoid dendritic cells, represent an essential component of the innate immune defense against infection. Despite this, not much is known about the pathways involved in their activation by non‐enveloped viruses. The present study demonstrates that the non‐enveloped foot‐and‐mouth disease virus (FMDV) cannot stimulate IFN‐α responses in NIPC, unless complexed with FMDV‐specific immunoglobulins. Stimulation of NIPC with such immune complexes employs FcγRII ligation, leading to strong secretion of IFN‐α. In contrast to the stimulation of NIPC by many enveloped viruses, FMDV induction of IFN‐α production requires live virus. It is necessary for the virus to initiate its replicative cycle. Moreover, it is an abortive replication, as witnessed by the decrease of dsRNA levels and viral titers with time post infection. Sensitivity of the NIPC stimulation to wortmannin and chloroquin, but not leupeptin, indicates an essential role for the pre‐lysosomal stage endosomal compartment. In conclusion, the present study demonstrates that immune complexes provide the means for a non‐interferogenic virus to induce IFN‐α responses by NIPC. This indicates an important link between NIPC and antibodies in immune responses against non‐enveloped viruses such as FMDV.

Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of Npro

ABSTRACT Pestiviruses prevent alpha/beta interferon (IFN-α/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral Npro nonstructural protein. Npro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire Npro gene resulted in attenuation in pigs. In order to elaborate on the role of the Npro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in Npro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of Npro. We describe two mutations in Npro that eliminate Npro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for Npro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-α/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-α/β induction is not a prerequisite for CSFV virulence.

Classical Swine Fever Virus Npro Limits Type I Interferon Induction in Plasmacytoid Dendritic Cells by Interacting with Interferon Regulatory Factor 7

ABSTRACT Viruses are detected by different classes of pattern recognition receptors that lead to the activation of interferon regulatory factors (IRF) and consequently to the induction of alpha/beta interferon (IFN-α/β). In turn, efficient viral strategies to escape the type I IFN-induced antiviral mechanisms have evolved. Previous studies established that pestivirus Npro antagonizes the early innate immune response by targeting the transcription factor IRF3 for proteasomal degradation. Here, we report that Npro of classical swine fever virus (CSFV) interacts also with IRF7, another mediator of type I IFN induction. We demonstrate that the Zn-binding domain of Npro is essential for the interaction of Npro with IRF7. For IRF3 and IRF7, the DNA-binding domain, the central region, and most of the regulatory domain are required for the interaction with Npro. Importantly, the induction of IRF7-dependent type I IFN responses in plasmacytoid dendritic cells (pDC) is reduced after wild-type CSFV infection compared with infection with virus mutants unable to interact with IRF7. This is associated with lower levels of IRF7 in pDC. Consequently, wild-type but not Npro mutant CSFV-infected pDC show reduced responses to other stimuli. Taken together, the results of this study show that CSFV Npro is capable of manipulating the function of IRF7 in pDC and provides the virus with an additional strategy to circumvent the innate defense.

Plasmacytoid Dendritic Cells Migrate in Afferent Skin Lymph

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.

Innate immune responses against foot-and-mouth disease virus: current understanding and future directions.

Foot-and-mouth disease (FMD) represents one of the most economically important diseases of farm animals. The basis for the threat caused by this virus is the high speed of replication, short incubation time, high contagiousness, and high mutation rate resulting in constant antigenic changes. Thus, although protective immune responses against FMD virus (FMDV) can be efficacious, the rapidity of virus replication and spread can outpace immune defence development and overrun the immune system. FMDV can also evade innate immune responses through its ability to shut down cellular protein synthesis, including IFN type I, in susceptible epithelial cells. This is important for virus evolution, as FMDV is quite sensitive to the action of IFN. Despite this, innate immune responses are probably induced in vivo, although detailed studies on this subject are lacking. Accordingly, this interaction of FMDV with cells of the innate immune system is of particular interest. Dendritic cells (DC) can be infected by FMDV and support viral RNA replication, and viral protein synthesis but the latter is inefficient or abortive, leading most often to incomplete replication and progeny virus release. As a result DC can be activated, and particularly in the case of plasmacytoid DC (pDC), this is manifest in terms of IFN-alpha release. Our current state of knowledge on innate immune responses induced by FMDV is still only at a relatively early stage of understanding. As we progress, the investigations in this area will help to improve the design of current vaccines and the development of novel control strategies against FMD.

FcγRII-dependent sensitisation of natural interferon-producing cells for viral infection and interferon-α responses

Natural interferon‐producing cells (NIPC), also called plasmacytoid dendritic cells, are the most potent producers of IFN‐α in response to viral and bacterial components,s serving an important function in innate immune defences. The present work demonstrates that NIPC responsiveness can be primed by immunisation, increasing their capacity to produce IFN‐α after viral infection. NIPC isolated from pigs immunised against classical swine fever virus (CSFV), a member of the Flaviviridae, were more receptive to viral infection and produced higher levels of IFN‐α than NIPC from immunologically naive animals. This sensitisation of NIPC was maintained for at least 8 months after immunisation. IFN‐α production was dependent on live virus and required replication, and the “immune” NIPC responded to lower infectious doses of virus. Co‐localisation of the virus with FcγRII (CD32) in polarised structures was observed with “immune” NIPC only. This FcγRII‐dependent virus capture and sensitisation of NIPC, evidently mediated through cytophilic CSFV‐specific antibodies, was inhibited by non‐specifically aggregated immunoglobulin as well as by pre‐formed virus‐antibody complexes. In conclusion, these results demonstrate that NIPC not only represent a major player in innate immunity but are also functionally linked to the immunological memory of the adaptive immune system.

Hemagglutinin-Dependent Tropism of H5N1 Avian Influenza Virus for Human Endothelial Cells

ABSTRACT Although current H5N1 highly pathogenic avian influenza viruses (HPAIV) are inefficiently transmitted to humans, infected individuals can suffer from severe disease, often progressing rapidly to acute respiratory distress syndrome and multiorgan failure. This is in contrast with the situation with human influenza viruses, which in immunocompetent individuals usually cause only a respiratory disease which is less aggressive than that observed with avian H5N1 viruses. While the biological basis of inefficient transmission is well documented, the mechanisms by which the H5N1 viruses cause fatal disease remain unclear. In the present study, we demonstrate that human pulmonary microvascular endothelial cells (hPMEC) had a clearly higher susceptibility to infection by H5N1 HPAIV than to infection by human influenza viruses. This was measurable by de novo intracellular nucleoprotein production and virus replication. It was also related to a relatively higher binding capacity to cellular receptors. After infection of hPMEC, cell activation markers E-selectin and P-selectin were upregulated, and the proinflammatory cytokines interleukin-6 and beta interferon were secreted. H5N1 virus infection was also associated with an elevated rate of cell death. Reverse genetics analyses demonstrated a major role for the viral hemagglutinin in this cell tropism. Overall, avian H5N1 viruses have a particular receptor specificity targeting endothelial cells that is different from human influenza viruses, and this H5N1 receptor specificity could contribute to disease pathogenesis.