Artur Summerfield

The pig: a model for human infectious diseases

An animal model to study human infectious diseases should accurately reproduce the various aspects of disease. Domestic pigs (Sus scrofa domesticus) are closely related to humans in terms of anatomy, genetics and physiology, and represent an excellent animal model to study various microbial infectious diseases. Indeed, experiments in pigs are much more likely to be predictive of therapeutic treatments in humans than experiments in rodents. In this review, we highlight the numerous advantages of the pig model for infectious disease research and vaccine development and document a few examples of human microbial infectious diseases for which the use of pigs as animal models has contributed to the acquisition of new knowledge to improve both animal and human health.

Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties

Despite the central role that dendritic cells (DC) play in immune regulation and antigen presentation, little is known about porcine DC. In this study, two sources of DC were employed. Bone marrow haematopoietic cell‐derived DC (BM‐DC) were generated using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) in the presence or absence of tumour necrosis factor‐α (TNF‐α). Monocyte‐derived DC (Mο‐DC) were generated with GM‐CSF and interleukin‐4 (IL‐4). In both systems, non‐adherent cells developed with dendritic morphology, expressing high levels of major histocompatibility complex (MHC) class II. The presence of TNF‐α increased the BM‐DC yield, and enhanced T‐cell stimulatory capacity. Both BM‐DC and Mο‐DC expressed the pan‐myeloid marker SWC3, as well as CD1 and CD80/86, but were also CD14+ and CD16+. The CD16 molecule was functional, acting as a low‐affinity Fc receptor. In contrast, the CD14 on DC appeared to differ functionally from monocyte CD14: attempts to block CD14, in terms of lipopolysaccharide (LPS)‐induced procoagulant activity (PCA), failed. The use of TNF‐α or LPS for DC maturation induced up‐regulation of MHC class II and/or CD80/86, but also CD14. Allogeneic mixed leucocyte reactions and staphylococcal enterotoxin B antigen presentation assays demonstrated that these DC possessed potent T‐cell stimulatory capacity. No T helper cell polarization was noted. Both the BM‐DC and the Mο‐DC induced a strong interferon‐γ and IL‐4 response. Taken together, porcine DC generated in vitro possess certain characteristics relating them to DC from other species including humans, but the continued presence of CD14 and CD16 on mature and immature porcine DC was a notable difference.

Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

ABSTRACT Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.

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.

Classical Swine Fever Virus Interferes with Cellular Antiviral Defense: Evidence for a Novel Function of Npro

ABSTRACT Classical swine fever virus (CSFV) replicates efficiently in cell lines and monocytic cells, including macrophages (MΦ), without causing a cytopathic effect or inducing interferon (IFN) secretion. In the present study, the capacity of CSFV to interfere with cellular antiviral activity was investigated. When the porcine kidney cell line SK-6 was infected with CSFV, there was a 100-fold increased capacity to resist to apoptosis induced by polyinosinic-polycytidylic acid [poly(IC)], a synthetic double-stranded RNA. In MΦ, the virus infection inhibited poly(IC)-induced alpha/beta IFN (type I IFN) synthesis. This interference with cellular antiviral defense correlated with the presence of the viral Npro gene. Mutants lacking the Npro gene (ΔNpro CSFV) did not protect SK-6 cells from poly(IC)-induced apoptosis, despite growth properties and protein expression levels similar to those of the wild-type virus. Furthermore, ΔNpro CSFV did not prevent poly(IC)-induced type I IFN production in MΦ but rather induced type I IFN in the absence of poly(IC) in both MΦ and the porcine kidney cell line PK-15, but not in SK-6 cells. With MΦ and PK-15, an impaired replication of the ΔNpro CSFV compared with wild-type virus was noted. In addition, ΔNpro CSFV, but not wild-type CSFV, could interfere with vesicular stomatitis virus replication in PK-15 cells. Taken together, these results provide evidence for a novel function associated with CSFV Npro with respect to the inhibition of the cellular innate immune system.

Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS)

The composition of peripheral blood leukocyte populations was studied following experimental PCV2-infection in 3-week-old piglets. Four of 10 PCV2-infected piglets developed clinical and pathological symptoms consistent with postweaning multisystemic wasting syndrome (PMWS) between 14 and 21 days post-inoculation (p.i.), and were characterised as PMWS-affected. Only these four PMWS-affected piglets, but neither the non-symptomatic infected nor control animals, developed a clear leukopenia. Kinetic analysis demonstrated a clear loss of both CD21(+) B and CD3(+) T lymphocytes in the PMWS-affected piglets. By CD3/CD4/CD8 triple labelling, the influence of PCV2 infection on all T cell sub-populations was discernible. A loss of CD3(+)CD4(+)CD8(+) memory/activated Th lymphocytes was particularly notable. However, all T lymphocyte sub-populations-CD3(+)CD4(+)CD8(+) memory Th, CD3(+)CD4(+)CD8(-) nai;ve Th, CD3(+)CD4(-)CD8(+) Tc and CD3(+)CD4(-)CD8(-) gammadelta TCR(+) lymphocytes-were susceptible to PCV2 infection-induced lymphopenia. CD3(-)CD4(-)CD8(+) NK cells were also depleted in the PMWS-affected animals, but granulocytes and monocytes were less affected. In conclusion, PCV2 infection induces primarily a lymphopenia, but only in animals which subsequently develop PMWS. The lymphopenia can be identified early p.i., particularly with the B lymphocytes. Memory/activated Th lymphocytes might be affected more than the other T cell sub-populations, but as time progressed a collapse of both T and B cell populations was clear.

Dendritic Cells Harbor Infectious Porcine Circovirus Type 2 in the Absence of Apparent Cell Modulation or Replication of the Virus

ABSTRACT Dendritic cells (DCs) play crucial roles in innate and adaptive immune responses, rendering them critical targets for virus infections. Porcine circovirus type 2 (PCV2) is associated with the development of postweaning multisystemic wasting syndrome (PMWS) in piglets. We demonstrate here that 80 to 90% of monocyte-derived and bone marrow-derived DCs interact with PCV2 similar to the early stages of an infection. There was no evidence for virus replication, but the virus did persist in DCs without loss of infectivity nor the induction of cell death. This could reflect an abortive infection, but there was no evidence of virus uncoating—the infectivity remained intact for at least 5 days. Alternatively, the results may reflect DC endocytosis of antigenic material. However, there was no modulation of DC surface major histocompatibility complex class I and class II, CD80/86, CD25, CD16, or CD14. Furthermore, infected DC did not transmit virus to syngeneic T lymphocytes, even when the latter were activated. Such coculture did not induce PCV2 replication or death of the lymphocytes or DCs. These results demonstrate that PCV2 can persist in DCs in the absence of virus replication or degradation. Such a silent virus infection presents a novel mechanism of not only immune evasion but also escaping the DC degradation pathway. Because of their migratory capacity, infection of DCs thus provides a potent vehicle for transport of the virus throughout the host without the need for replication. In addition, the lymphopenia seen in PMWS is not a direct effect of the virus on lymphocytes but would require additional events, as proposed by others.

In Vitro Induction of Mucosa-Type Dendritic Cells by All-Trans Retinoic Acid

Efficient induction of mucosal immunity usually employs nasal or oral vaccination while parenteral immunization generally is ineffective at generating mucosal immune responses. This relates to the unique ability of resident mucosal dendritic cells (DC) to induce IgA switching and to imprint mucosa-specific homing receptors on lymphocytes. Based on the well-established plasticity of the DC system, this study sought to investigate whether peripheral DC could be modulated toward “mucosa-type” DC by treatment with immunomodulatory, and therefore potentially adjuvant-like, factors. In this study, we show that monocyte-derived DCs pretreated with the vitamin A derivative all-trans retinoic acid (RA) indeed acquired several attributes characteristic of mucosal DC: secretion of TGF-β and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in cocultured lymphocytes. Addition of a TGF-β-neutralizing Ab to cocultures significantly inhibited α4β7 integrin, but not CCR9 mRNA expression by the lymphocytes. Both α4β7 integrin and CCR9 mRNA expression, but not IgA production, were suppressed in the presence of a RA receptor antagonist. None of the observed effects on the lymphocytes were influenced by citral, a retinal dehydrogenase inhibitor, arguing against a role for de novo-synthesized RA. Collectively, our findings identified a novel role for RA as a mucosal immune modulator targeting DC. Our results further demonstrate that DC can act as efficient carriers of RA at least in vitro. Consequently, RA targeting of DC shows potential for promoting vaccine-induced mucosal immune responses via a parenteral route of immunization.

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.

Chicken cells sense influenza A virus infection through MDA5 and CARDIF-signaling involving LGP2

ABSTRACT Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-β (chIFN-β) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-β levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-β promoter, the silencing of endogenous chLGP2 reduced chIFN-β induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.