Kenneth C. McCullough

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.

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)1owu2003CD4high 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 CD8086 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 CD8086, 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.

Synthetic Virus-Like Particles Target Dendritic Cell Lipid Rafts for Rapid Endocytosis Primarily but Not Exclusively by Macropinocytosis

DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20–30 nm) virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.

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.

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.

The porcine dendritic cell family.

n Abstractn n Considering the pivotal roles played by dendritic cells (DCs) in both innate and adaptive immune responses, advances in the field of porcine immunology DC biology have recently progressed rapidly. As with the more extensively studied murine and human DCs, porcine DC can be generated from bone marrow haematopoietic cells or monocytes, and have been analysed in various immunological and non-immunological tissues. Both conventional DC (cDC) and plasmacytoid DC (pDC) have been characterized. The function of porcine monocyte-derived DC has not only been characterized in terms of antigen presentation and lymphocyte activation, but also their response to various ligands of pattern recognition receptors. These have been characterized in terms of the induction of DC maturation and pro-inflammatory, Th1-like or Th2-like cytokines secretion. Porcine pDC most effectively sense virus infections and are characterized by their capacity to produce large quantities of IFN-α and the pro-inflammatory cytokines TNF-α, IL-6 and IL-12. As such, the DC family as a whole is a powerful ally in the host battle against pathogen attack. Nevertheless, DC in particular tissue environments or under particular stimuli can down-regulate immune response development. This is not only important for preventing over-activation of the immune system and also for ensuring tolerance against self or “friendly” substances including food components, but may also be used as a mechanism of pathogens to evade immune responses.n n

The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies

Monoclonal antibodies (MAbs) were used to delineate the antigenic relationship between the three morbillivirus types: measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). Panels of six to 31 MAbs against the haemagglutinin (H), fusion (F), nucleocapsid protein (NP), phosphoprotein (P) and matrix (M) proteins of MV and the H, F, NP and P proteins of CDV were employed. Nine strains of MV, three strains of CDV and four strains of RPV were examined by radioimmunoprecipitation assay and immune fluorescence for reactivity with the heterologous MAbs. Overall, the NP and in particular the F proteins of the morbilliviruses showed a high degree of epitopic homology; the P and M proteins showed a partial epitopic homology, with the greatest variation between the M proteins of CDV and MV; the H proteins showed a low degree of epitopic homology and then only between MV and RPV. These data indicate that the major cross-protecting antigen in heterotypic vaccination amongst morbilliviruses is the F antigen. The epitopic relationships found between morbilliviruses as identified by the MAbs were classified as follows. (i) Group-specific epitopes were present on all strains of the three morbillivirus types. (ii) Group-cross-reactive epitopes were present on only some of the strains from each morbillivirus type (these epitopes identified the presence of intratypic strain variation in all proteins of all three virus types). (iii) Type-specific epitopes, i.e. MV unique or CDV unique, were found only on the homologous morbillivirus type. (iv) CDV-RPV intertypic and MV-RPV intertypic epitopes were, respectively, epitopes shared by CDV and RPV but not with any MV strain, and epitopes shared by MV and RPV but not with any CDV strain. These cross-reactivities and type-specific reactions were obtained with the internal viral proteins (M, P and NP). The epitopes of the F proteins were mainly group-specific and no CDV-RPV or MV-RPV intertypic epitopes were found. The epitopes of the H protein were either type-specific or MV-RPV intertypic. These data support the proposed evolutionary relationship between the morbilliviruses.

Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA

Porcine circovirus type 2 (PCV2) infection of natural interferon producing cells (NIPCs) impairs the induction of interferon (IFN)‐α and tumour necrosis factor (TNF)‐α by cytosine‐phosphorothioate‐guanine (CpG) oligodeoxynucleotides (ODNs), thereby preventing both their autocrine maturation and the paracrine maturation of myeloid dendritic cells (DCs). The present study shows that the PCV2‐mediated inhibition of NIPCs was mediated by viral DNA, although it was independent of virus replication. The inhibitory effect of PCV2 DNA was more diversified than if it had simply targeted CpG‐ODN‐induced cytokines (IFN‐α, TNF‐α, interleukin‐6, IL‐12). A broad spectrum inhibition was noted, affecting responses induced by toll‐like receptor (TLR)‐7 and TLR9 agonists, as well as viruses including pseudorabies virus, transmissible gastroenteritis virus and classical swine fever virus. From these results, it would appear that PCV2 DNA can induce a dominant negative signal influencing independent pattern recognition receptor‐induced activation cascades. Despite a concomitant internalization of PCV2 DNA and CpG‐ODNs, no colocalization was observed, indicating that PCV2 DNA and CPG‐ODNs may not target the same receptor. This study describes a novel modulation of the innate immune response, which would render the host more susceptible to secondary or concomitant microbial infections.