Wen-Bin Chung

Phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus

It is well documented that there is a delay in the development of effective immunity to porcine reproductive and respiratory syndrome virus (PRRSV) in infected and vaccinated pigs. This suggests that PRRSV might possess some inherent properties to evade host defense mechanisms during the early stage of infection. Dendritic cells (DCs) play a crucial role in the activation and control of T-cells in response to viral antigens. In this study, we investigated the phenotypic and functional property changes of bone marrow-derived immature DCs (BM-imDCs) that take place after infection by PRRSV. Results showed that BM-imDCs were permissive to PRRSV infection, as productive replication took place in these cells. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 is observed at 48 h following infection. Also at 48h following PRRSV infection, a significant increase of IL-10 secretion by BM-imDCs was noticed. Results suggest that the inhibited expression of MHC I and the enhanced secretion of IL-10 by BM-imDCs after PRRSV infection might be among the strategies used by the virus to evade the host immune defenses.

Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus

Field observations have suggested that porcine reproductive and respiratory syndrome virus (PRRSV) predispose pigs to secondary infections. The interaction between PRRSV and the secondary invaders has not yet been well elucidated. In this study, we investigated the mRNA expression of Toll-like receptors (TLR) in lymphoid organs and cells, and cytokine secretions by alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs) in response to pathogen-associated molecular patterns (PAMPs) in PRRSV-challenged pigs. TLR mRNA expressions were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and cytokine concentrations were determined using commercially available ELISA kits. PRRSV infection led to significantly increased secretions of IL-1beta and IL-6 by AMs of PRRSV-infected pigs. Infection of pigs with PRRSV also resulted in an increased secretion of IL-1beta by AMs in response to lipoteichoic acid (LTA) stimulation, and IL-6 by PBMCs in response to lipopolysaccharide (LPS) and LTA stimulation. Infection of pigs with PRRSV tended to up-regulate the mRNA expression of TLR2, 3, 4, 7 and 8 in at least one of the lymphoid tissues and cells. Further research is required to demonstrate the association between the enhanced expressions of the specific TLRs and the increased susceptibility to secondary agents with more severe clinical outcomes in PRRSV-infected pigs.

Toll-like receptor expressions in porcine alveolar macrophages and dendritic cells in responding to poly IC stimulation and porcine reproductive and respiratory syndrome virus (PRRSV) infection

Antigen-presenting cells play critical roles in recognizing, presenting and processing antigens and consequently induce adequate immune response for defending infections. The immature DCs (imDCs) and mature DCs (mDCs) were obtained from in vitro differentiation of bone marrow haematopoietic cells. Results showed that poly IC stimulation down-regulated the expressions of TLR7 and TLR8 in alveolar macrophages (AMs) and imDCs. The release of IL-12 was inhibited from imDCs and mDCs in response to poly IC. Porcine reproductive and respiratory syndrome virus (PRRSV)-infection inhibited TLR3 and TLR7 expressions in AMs and imDCs at 6h post-infection (PI); both of expressions were then restored at 24h PI in both types of cells while they exhibited up-regulated IL-10 and IL-12 expression at 24h PI. Hence, the differential TLR expression patterns in porcine AMs and DCs in discrimination of the imitated viral dsRNA or PRRSV infection may determine their cytokine expressions and thus affect the resulting immune responses.

Immunomodulatory effects of β-glucans on porcine alveolar macrophages and bone marrow haematopoietic cell-derived dendritic cells

The immunopharmacological activities of beta-glucans with a backbone of beta-1,3/beta-1,6-linkages associated with anti-tumor, anti-viral, bacterial and fungal infections have been well documented. Dectin-1, a specific pattern recognition receptor for beta-1,3/beta-1,6-glucans, is expressed mainly on phagocytes, especially macrophages and dendritic cells (DCs). In this study, the encoding nucleotide for the carbohydrate-recognition domain (CRD) of porcine dectin-1 was sequenced for the first time, and the immunomodulatory functions of a synthetic particulate beta-glucan (p-beta-glucan) were examined. Results showed that p-beta-glucan significantly enhanced cell activity and phagocytosis in porcine alveolar macrophages (AMs), immature DCs (imDCs) and mature DCs (mDCs), in a similar way to zymosan. Zymosan enhanced dectin-1/TLR2/TLR4 expression and TNF-alpha/IL-10 production in all of three types of cell, whereas p-beta-glucan increased dectin-1/TLR4 and TNF-alpha/IL-12 production in AMs but inhibited IL-10 in mDCs. These results indicate that the complex collaborating interactions between dectin-1 and TLRs in the recognition of beta-1,3/beta-1,6-glucans with different structural features may direct different cellular responses.

Modulations of phenotype and cytokine expression of porcine bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus.

Phenotypic and functional property changes of bone marrow-derived immature dendritic cells (BM-imDCs) after porcine reproductive and respiratory syndrome virus (PRRSV) infection have been detailed in a previous report. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 were observed in BM-imDCs after the exposure to PRRSV. In this study, we further investigate the expression of surface phenotypes of BM-imDCs in relation to their infection status. Exposure of PRRSV to BM-imDCs resulted in a down-regulated expression of MHC I and an up-regulated expression of CD80/86 in infected cells, as demonstrated by significant alterations in both percentage of expressing cells and mean fluorescence intensity (MFI) in PRRSV-positive cells. A significant suppression in MFI of MHC I and an increase in percentage of cells expressing CD80/86 were observed in noninfected bystander cells. We also demonstrated that exposure of BM-imDCs to PRRSV resulted in a significantly increased secretion of IL-1, IL-6, IL-8, IL-10 and IFN-gamma but not IL-12 or TNF-alpha. In addition, the PRRSV infection modulates cytokine expressions of BM-imDCs through their response to microbial pathogen-associated molecular patterns. These results will prove helpful in clarification of the factors that mediate host defense against PRRSV, as well as the possible interaction mechanisms between PRRSV and other microbes in the pathogenesis of PRRSV infection in pigs.

Investigation of Beta-Glucans Binding to Human/Mouse Dectin-1 and Associated Immunomodulatory Effects on Two Monocyte/Macrophage Cell Lines

Dectin‐1, a specific pattern recognition receptor for β‐1,3/β‐1,6‐glucans, is expressed mainly on phagocytes. Human dectin‐1 (hDectin‐1) and mouse dectin‐1 (mDectin‐1) were separately expressed on HEK293 cell surfaces for examination of the binding abilities of a synthetic particulate β‐glucan (pβG), a product extracted from Saccharomyces cerevisiae, in this study. The binding of zymosan‐FITC to hDectin‐1 and mDectin‐1 was inhibited by pβG at similar concentrations for 50% inhibition of binding (IC50). However, the kinetics of the time course and dose response to zymosan stimulation observed for U937 and J774A.1 differed. Superoxide anion production was increased in U937 but reduced in J774A.1 when cells were treated with pβG, zymosan, or laminarin, whereas ovalbumin endocytosis was enhanced in U937 and J774A.1 treated either with pβG, zymosan, laminarin, or barley‐glucan. These results indicate that the binding affinity of pβG to hDectin‐1 is similar to the binding affinity to mDectin‐1, and that stimulation by pβG as well as various forms of β‐1,3‐glucans on U937 and J774A.1 resulted in upregulation of cell activity and ovalbumin endocytosis. Additionally, other coreceptors on U937 and J774A.1 may be involved in directing different responses to superoxide anion production in these two types of cells. These results will likely contribute to further investigations on identifying the biological forms of β‐glucans capable of binding its specific receptor as the effective immunomodulators.

Decay of Maternally Derived Antibodies and Seroconversion to Respiratory Viral Infection in Pig Herds

This study investigated the decay of maternally derived antibodies to pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) in the pig herds in Taiwan. Additionally, the serological profiles of these viral pathogens were examined in a cross-sectional study of six pig populations. The observed half lives of maternally derived serum neutralization antibodies against PRV and PRRSV were 2.0 and 3.0 weeks, respectively. The estimated times to seronegativity to PRV and PRRSV were 12.2 and 10.4 weeks, respectively. At one week of age, over 80% of pigs were positive for PCV2 and hemagglutinin 1 (H1) SIV antibodies, but antibody decreased to undetectable levels at 11 and 5 weeks of age, respectively. Except for SIV infection, which probably exhibited sporadic, mild to moderate outbreaks, the most common sequence of seroconversion to the viral respiratory infections in the six pig herds appear to be in an order of PCV2, followed by PRRSV and PRV. The results of this study provide basic data needed to design effective vaccination programs and intervention strategies for controlling swine respiratory diseases.

Expression profile of Toll-like receptor mRNA in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Field and experimental studies have shown that co-infection of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) increases the severity of the disease. The present study investigates the mRNA expression profile of Toll-like receptors (TLRs) in pigs co-infected with PRRSV and PCV2. SPF pigs were infected with PRRSV, PCV2 or in a combination of both. The mRNA expression levels of TLRs and related cytokines in peripheral blood mononuclear cells (PBMCs) of pigs were determined by quantitative real-time RT-PCR. The mRNA expression profiles of TLRs by PBMCs from pigs co-infected with PRRSV and PCV2 displayed two distinct patterns: an increased expression profile for TLRs2, 4 and 8, and a decreased expression profile for TLRs3, 7 and 9. An up-regulated expression of IL-1β and IL-10 mRNA and a down-regulated expression of INF-α and TNF-α mRNA in PBMCs of co-infected pigs were also observed.

Expression of Toll-like receptor signaling-related genes in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) have been shown to develop more severe diseases than pigs infected with PRRSV or PCV2 only. The underlying interaction mechanisms between the two viruses in developing the disease are unclear. The present study investigates the mRNA expression of Toll-like receptor (TLR) signaling-related molecules in peripheral blood mononuclear cells from pigs infected with PRRSV or PCV2 or both. The mRNA expression levels were determined by quantitative real-time RT-PCR. Co-infection of pigs with PRRSV and PCV2 resulted in a negatively synergistic effect on the mRNA expression of the negative regulators of TLR, including A20, Bcl-3, IRAK-M, MKP-1, SARM1 and SIGIRR, as well as the TLR downstream transcription factors IRF-1 and IRF-3. A positively synergistic effect of a combined infection of PRRSV and PCV2 on the CD14 mRNA expression was also observed.

Leukotriene C4 release and gene expressions of IL-8 and MCP-1 in porcine alveolar epithelial type II cells.

Leukotrienes (LT) and chemokines are important chemotactic compounds in regulating the recruitment and activation of immune cells during pulmonary inflammatory reactions. Results showed that LTC4 release by porcine alveolar epithelial type II cells (AEC IIs) is significantly enhanced by either LTB4 or LPS stimulation. The basal level of IL-8 gene expression in AEC IIs was only 1/3 of that observed in alveolar macrophages (AMs) while AEC IIs expressed a higher basal level of monocyte chemotactic peptide-1 (MCP-1) and also in response to LPS stimulation than do AMs. The increasing basal and LT-induced MCP-1 gene expressions after 8h of incubation were observed in AEC IIs but decreased in AMs. These findings suggest that AEC IIs play an important role in initial inflammatory reactions of the lung by releasing LTC4, and that they also modulate later inflammatory reactions, evidenced by consistent elevation of MCP-1 gene expression after and during exogenous challenge in pigs.