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)-alpha and tumour necrosis factor (TNF)-alpha 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-alpha, TNF-alpha, 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.     

Porcine circovirus type 2 displays pluripotency in cell targeting

Porcine circovirus type 2 (PCV2) is the causative agent of a multifactorial disease associated with immunocompromisation and co-infections. In vivo, viral DNA and antigens are found in monocytic, epithelial and endothelial cells. Of these, PCV2 replication has only been studied in monocytic cells, in which little or no replication was identified. Accordingly, PCV2 infection was studied in the endothelial cell line PEDSV.15, aortic endothelial cells, gut epithelial cells, fibrocytes and dendritic cells (DC). In all cells except DC PCV2 replication was detectable, with an increase in the levels of capsid and replicase protein. Variations in endocytic activity, virus binding and uptake did not relate to the replication efficiency in a particular cell. Furthermore, replication did not correlate to cell proliferation, although a close association of viral proteins with chromatin in dividing cells was observed. No alteration in the division rate of PCV2-infected cultures was measurable, relating to replicase expression in only a small minority of the cells. In conclusion, the broad cell targeting of PCV2 offers an explanation for its widespread tissue distribution.     

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single‐stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll‐like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin‐6 (IL‐6) and tumour necrosis factor‐α secretion, but not interferon‐α or IL‐12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte‐derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus‐induced immunosuppression observed in infected pigs.     

Enhanced replication of porcine circovirus type 2 (PCV2) in a homogeneous subpopulation of PK15 cell line

Post-weaning multi-systemic wasting syndrome (PMWS) has emerged as a major disease that poses a significant threat to the economics of global swine industry. Porcine circovirus type 2 (PCV2) is the causal agent of PMWS in pigs. Currently, the prevention of PCV2 infection based on vaccines is limited, and the available vaccines are either killed viral vaccines or recombinant protein based vaccines and not cost effective. The PK-15 cells, which is widely used for PCV2 propagation, is not efficient and heterogeneous in terms of permissivity to viral infection. In order to acquire a homogeneous porcine kidney cell line that can reliably produce PCV2 in high titers, cell clones that show high- (PK15-C1) or low-permissive (PK15-A2) phenotype to PCV2 infection were derived from heterogeneous PK15 parent cells by limiting dilution and cell cloning. Maximum virus titers in PK15-C1, PK15-A2 and PK15 parent cells were 10(8), 10(2) and 10(5) tissue culture infective dose 50 (TCID 50)/ml, respectively. The viral proteins of PCV2 were produced and accumulated faster in PK15-C1 cells than those in PK15 parent cells. These results indicate that PK15-C1 cell clone is more permissive to PCV2 infection than PK15 parent cells and thus will be useful for PCV2 replication in vitro, as well as, vaccines, diagnostic and research applications on PCV2.     

Inhibition of porcine circovirus type 2 replication in mice by RNA interference

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS) for which no antiviral treatment is available. To exploit the possibility of using RNA interference (RNAi) as a therapeutic approach against the disease, plasmid-borne short hairpin RNAs (shRNAs) were generated to target the PCV2 genome. Transfection of these shRNAs into cultured PK15 cells caused a significant reduction in viral RNA production that was accompanied by inhibiting viral DNA replication and protein synthesis in infected cells. The effect was further tested in vivo in a mouse model that has been developed for PCV2 infection. Mice injected with shRNA before PCV2 infection showed substantially decreased microscopic lesions in inguinal lymph nodes compared to controls. In situ hybridization and immunohistochemical analyses showed that shRNA caused a significant inhibition in the level of viral DNA and protein synthesis detected in the lymph nodes of the treated mice relative to the controls. Taken together, these results indicate that shRNAs are capable of inhibiting PCV2 infection in vitro as well as in vivo and thus may constitute an effective therapeutic strategy for PCV2 infection.     

Porcine circovirus type 2 explores the autophagic machinery for replication in PK-15 cells

Porcine circovirus type 2 (PCV2), an important pathogen of pigs, causes lymphoid depletion in infected tissues most probably by inducing apoptosis although the precise pathogenesis of PCV2-associated diseases remains unknown. We speculate whether autophagy, another cellular response to stress or infections by bacterial or viral pathogens, is involved in PCV2 infection. Here, we provide the first evidence that PCV2 could trigger autophagosome formation and enhance autophagic flux in PK-15 cells, most likely by its capsid protein. Using activators or inhibitors including siRNA targeting atg5, autophagy was found to enhance viral replication and capsid protein expression. These results suggest that PCV2 might employ the autophagy machinery to enhance its replication in host cells, thus raising the possibility of targeting autophagic pathway as a potential antiviral strategy against PCV2 infection.     

Modulation of dendritic cell differentiation and cytokine secretion by the hydatid cyst fluid of Echinococcus granulosus

Chronic infection by Echinococcus granulosus results in establishment of fluid-filled cysts (hydatid cysts) in liver or lungs of infected hosts, which can escape destruction by the host immune system for long periods. This study explores the modulation by hydatid cyst fluid of the in vitro human monocyte to dendritic cell (DC) transition induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Addition of the fluid to adherent peripheral blood monocytes cultured in GM-CSF/IL-4 stimulates release of prostaglandin E2 (PGE2) and IL-6. Exposure of differentiating DC to the fluid during the 7-day culture in GM-CSF/IL-4 impairs their subsequent ability to secrete IL-12, IL-6 or PGE2 in response to lipopolysaccharide (LPS) stimulation. This inhibition is not dependent on the initial release of PGE2. The presence of hydatid cyst fluid also modulates the phenotype of the cells generated during culture, resulting in increased CD14 expression and decreased expression of CD1a. Finally, hydatid fluid can stimulate predifferentiated DC to mature, as evidenced by release of IL-12 and IL-6, and by up-regulation of class II major histocompatibility complex and CD86. The possible role of dendritic cell modulation in regulating the host immune response to hydatid cysts is discussed.     

Blood dendritic cells and DC-poietins in systemic lupus erythematosus

Dendritic cells (DCs) control immunity and tolerance. Hence, we surmised that systemic lupus erythematosus (SLE), a systemic autoimmune disease with autoreactive T and B cells, might be due to alterations in DC homeostasis. Taken together, our results demonstrate profound alterations of DCs and DC-poietins homeostasis in SLE. Elevated levels of interferon- (IFN) in serum of SLE patients coexist with decreased numbers of cells producing IFN-, i.e., plasmacytoid dendritic cells (PDCs). Decreased numbers of circulating DCs correlate with increased levels of soluble tumor necrosis factor (TNF) receptors, thus suggesting the potential role of TNF pathway in the observed DC alterations. Finally, increased FMS-like tyrosine kinase 3-ligand (FLT3-L) and its correlation with soluble TNF receptors suggest a physiologic response to compensate low DC numbers. Although IFN- remains at the center of immunologic aberrations in SLE, it remains to be determined whether increased shedding of soluble TNF receptors could also be ascribed to IFN-.     

Development of a Plexor real-time PCR assay for the detection of porcine circovirus type 2

A novel, real-time PCR system for the detection of porcine circovirus type 2 (PCV2) was developed. The system employed Plexor technology and detected 10⁸-10¹ copies per reaction of PCV2 DNA within a recombinant plasmid. The examination of clinical material showed consistent diagnostic sensitivity when samples contained more than 10³ viral copies per reaction. Specificity of Plexor real-time PCR was confirmed using the porcine viruses PCV1, PRRSV, CSFV, TTSuV1 and TTSuV2 employing the melting curve analysis of PCR products. The low values of coefficient of variation in the intra- (1.74%) and inter-assay (2.41%) analysis suggested that the assay was a highly reproducible. The Plexor real-time PCR was compared with three other real-time PCR systems (SYBR Green, TaqMan, LUX) with conclusion that it can be used as a method of choice for the detection and quantitation of PCV2.     

Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors.

Peripheral blood mononuclear cells from patients with human immunodeficiency virus (HIV) infection exhibit a progressively marked decrease in the production of virus-induced interferon (IFN)-alpha, a finding that correlates with and is highly predictive of disease progression and opportunistic infections. The major IFN-alpha producing population has recently been defined as the precursor to type 2 dendritic cells (pDC2) or plasmacytoid DC (pDC). Using four-color flow cytometry, we have enumerated the pDC2 vs non-IFN-alpha producing myeloid DC1 in peripheral blood from HIV-infected patients and healthy controls and related these values to CD4 cell numbers, viral load, and functional activity. The patients had reductions in the numbers of both pDC2 (lin-/HLA-DR+/CD123(bright)) and DC1 (lin1-/HLA-DR+/CD123(dim)/CD11c+), both at an absolute level and as a percentage of cells. The decreases were most evident in patients with decreased CD4 levels. Viral load correlated with the functional frequency of the IFN producing cells but not with absolute pDC2 levels. Using intracellular flow cytometric analysis for IFN-alpha, the patients were demonstrated to have fewer pDC2, as well as a lower percentage of responding cells among those remaining. We conclude that deficient production of IFN-alpha by pDC2 from HIV-infected patients results from both selective loss of these cells and their qualitative dysfunction. Given the central role of DC, and in particular, DC2, in linking innate and adaptive immune responses, these qualitative and quantitative changes in pDC2 are likely to be key contributors to HIV pathogenesis.     

Development of a blocking ELISA for detection of serum neutralizing antibodies against porcine circovirus type 2

A monoclonal antibody (Mab)-based blocking ELISA was developed for the detection of serum neutralizing antibodies to porcine circovirus type 2 (PCV2). The Mab with neutralizing activity, which was produced by immunizing a recombinant capsid protein of PCV2 expressed in insect cells, was used as the detector antibody. The assay was evaluated in comparison with a serum neutralization assay, and its sensitivity and specificity were determined to be 98.8% and 88.5%, respectively. A significant positive correlation was found between results of the blocking ELISA and the serum neutralization assay (r = 0.9381). The assay was verified by testing experimental and commercial pig sera. A longitudinal antibody profile showed that serum neutralizing antibodies were detected 2 weeks after vaccination and that the detection rate reached 100% at 4 weeks. The serum neutralizing antibody profile showed a decrease from the age of 4 to 13 weeks, and seroconversion after 13 weeks in pigs from a commercial pig farm. Additionally, the positive detection rate in 703 sera collected from nine commercial pig farms was 73%. This report demonstrates that the assay is a simple, specific, sensitive and convenient method for epidemiological surveys and evaluations of serum neutralizing antibodies against PCV2.     

Circumventing pyroptosis via hyperactivation shapes superior immune responses of human type 2 dendritic cells compared to type 3 dendritic cells

Exploiting inflammasome activation in dendritic cells (DCs) is a promising approach to fight cancer and to augment adjuvant-induced immune responses. As inflammasome formation is typically accompanied by pyroptosis, hyperactivation—defined as inflammasome activation in the absence of pyroptosis—represents a mechanism of circumventing cell death of DCs while simultaneously benefitting from inflammasome signaling. We previously demonstrated a unique specialization for inflammasome responses and hyperactivation of human cDC2 among all human DC subsets. As recent investigations revealed heterogeneity among the human cDC2 population, we aimed to analyze whether the two recently identified cDC2 subpopulations DC2 and DC3 harbor similar or different inflammasome characteristics. Here, we report that both DC2 and DC3 are inflammasome competent. We show that DC3 generally induce stronger inflammasome responses, which are associated with higher levels of cell death. Although DC2 release lower levels of inflammasome-dependent IL-1β, they induce stronger CD4⁺ T cell responses than DC3, which are predominantly skewed toward a T_H1/T_H17 phenotype. Thus, mainly DC2 seem to be able to enter a state of hyperactivation, resulting in enhanced T cell stimulatory capacity.     

Effects of dexamethasone on the pathogenesis of porcine circovirus type 2 infection in piglets

To investigate the effect of immunosuppression on porcine circovirus type 2 (PCV2) infection, hysterectomy-produced, colostrum-deprived piglets were inoculated with the virus by the intranasal or intraperitoneal route, with or without dexamethasone (DEX) treatment. Eleven piglets aged 8 days were divided into four groups, namely group A (four animals given PCV2), B (three given PCV2 with DEX), C (two given sterile medium with DEX) and D (two given sterile medium). No significant clinical signs were observed. Histopathological and immunohistochemical examination revealed granulomatous inflammation and PCV2 antigen in the lymphoid tissues of group B piglets, but not in the other three groups. Flow cytometric analysis of peripheral blood lymphocytes showed a reduced number of CD4+ T cells in DEX-treated piglets (groups A and C). No differences between groups were observed in respect of the number of B cells, serum IgG concentration, or PCV2 antibody titre. These results indicate that DEX influenced the pathogenic effects of PCV2 infection in lymphoid organs, and that suppression of cell-mediated immunity may play a role in the aetiology of postweaning multisystemic wasting syndrome.     

Engagement of BDCA-2 blocks TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) are a functionally distinct lineage of dendritic cells characterized by the release of large amounts of type I interferon (IFN I). IFN I release is efficiently triggered by viral infection and modulates several aspects of immune reactions including the activation of cytotoxic mechanisms finalized to the elimination of infected cells. In this study, we report that TLR7 and TLR9 ligands can induce the secretion of biologically active TNF-related apoptosis-inducing ligand (TRAIL) by PDC. Accordingly, PDC supernatant is endowed with TRAIL-mediated cytotoxic activity when tested on a TRAIL-sensitive Jurkat cell line. TRAIL production is only partially dependent on the autocrine production of IFN I as documented by the use of a blocking anti-IFNRA antibody and the stimulation with exogenous IFN I. Importantly, both TRAIL secretion and cytotoxic activity of PDC supernatants are completely abolished by BDCA2 ligation. These results provide further insights into the biological role of BDCA-2 and document a negative regulatory pathway of PDC cytotoxic activity that may be relevant in pathological situations such as tumors and autoimmune diseases.