Efficiency and Mechanism Evaluation of Magnolia officinalis Water Extract in Preventing Gastric Ulcer.

In this study we aimed at demonstrating the ability of Magnolia officinalis water extract to ameliorate gastric ulcers in in vitro and in vivo experiments. The gastric mucosa epithelial cell line, RGM 1, was pretreated with Magnolia officinalis water extract (0, 0.1, 1, 2, 5, or 10 mg/ml) and cultured in DMEM/F12 medium (pH 7.4) for 2 h and then in DMEM/F12 medium (pH 4.0) for 10 min. Magnolia officinalis water extract protected the cell viability and decreased reactive oxygen species formation by the acidic medium. In the in vivo experiment, Magnolia officinalis water extract (100 mg/kg) was administrated daily for 28 days in ICR mice via oral gavage, and then Shay's ulcer surgical method was performed to induce gastric ulcers. We analyzed the pH value of stomach acid and the pathological section, inflammation, and cannabinoid receptor type 2 (CB2) cDNA levels of the stomach. Magnolia officinalis water extract not only enhanced the pH value of stomach acid but also ameliorated the ulcer index and inflammation and increased CB2 expression effectively. These results suggest that Magnolia officinalis water extract might be used to decrease the incidence of gastric ulcer.

Toll-like Receptor-Mediated Immunomodulation of Th1-Type Response Stimulated by Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2).

PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4+ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1ß and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

Antibody Evaluation and Mutations of Antigenic Epitopes in the Spike Protein of the Porcine Epidemic Diarrhea Virus from Pig Farms with Repeated Intentional Exposure (Feedback).

The feedback strategy, or controlled exposure of pig herd to the porcine epidemic diarrhea virus (PEDV), significantly decreased losses during a severe outbreak in late 2013 in Taiwan. However, some pig farms still suffered from recurrent outbreaks. To evaluate the association between antibody titers and clinical manifestations, sera and colostra were analyzed from one pig farm that employed the feedback strategy. Furthermore, spike (S) gene full sequences from six positive samples of two farms with and without using feedback were compared to investigate the evolution of PEDV variants circulating in pig herds. The results in this study showed that high PEDV antibody titers do not correlate with the high rate of protection from PEDV infection. In addition, repeated feedback generated the emergence of PEDV variants with unique substitutions of N537S and Y561H in the COE domain and S769F in the SS6 epitopes. These mutations indicated the pathogenetic evolution of PEDV strains existing in the cycle of the feedback method. A very strict biosecurity practice to block the routes of pathogen transfer should be followed to achieve successful control of PEDV infections in pig herds.

Single Nucleotide Polymorphisms of Immunity-Related Genes and Their Effects on Immunophenotypes in Different Pig Breeds.

Enhancing resistance and tolerance to pathogens remains an important selection objective in the production of livestock animals. Single nucleotide polymorphisms (SNPs) vary gene expression at the transcriptional level, influencing an individual's immune regulation and susceptibility to diseases. In this study, we investigated the distribution of SNP sites in immune-related genes and their correlations with cell surface markers of immune cells within purebred (Taiwan black, Duroc, Landrace and Yorkshire) and crossbred (Landrace-Yorkshire) pigs. Thirty-nine SNPs of immune-related genes, including 11 cytokines, 5 chemokines and 23 Toll-like receptors (TLRs) (interferon-α and γ (IFN-α, γ), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), Monocyte chemoattractant protein-1 (MCP-1) and TLR3, TLR4, TLR7, TLR8, and TLR9) were selected, and the percentages of positive cells with five cell surface markers of CD4, CD8, CD80/86, MHCI, and MHCII were analyzed. There were 28 SNPs that were significantly different among breeds, particularly between Landrace and Taiwan black. For instance, the frequency of SNP1 IFN-α -235A/G in Taiwan black and Landrace was 11.11% and 96.15%, respectively. In addition, 18 SNPs significantly correlated with the expression of cell surface markers, including CD4, CD8, CD80/86, and MHCII. The percentage of CD4+ (39.27%) in SNP33 TLR-8 543C/C was significantly higher than those in A/C (24.34%), at p < 0.05. Together, our findings show that Taiwan black pigs had a unique genotype distribution, whereas Landrace and Yorkshire had a more similar genotype distribution. Thus, an understanding of the genetic uniqueness of each breed could help to identify functionally important SNPs in immunoregulation.

Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response.

The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163+ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.

Molecular characterization of cobia (Rachycentron canadum) CD4 homologues revealed the first evidence of soluble CD4 in fish.

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia.

Persistent Infection with Chicken Anemia Virus in 3-Week-Old Chickens Induced by Inoculation of the Virus by the Natural Route.

Naturally acquired chicken anemia virus (CAV) infection in chickens frequently occurs from 3 weeks of age onward after maternally derived antibodies have decayed. The oral inoculation of older chickens with CAV was reported to have negative effects on cell-mediated immune function, and pathological changes were identified. To date, there has been no complete illustration of an immunological and persistent infection. To understand the pathogenesis of persistent CAV infection, an immunological study of CAV-infected 3-week-old specific pathogen-free (SPF) chickens was carried out by different routes of inoculation. The weight, packed cell volumes, and organ samples were obtained at 7, 14, 21, and 28 days postinfection (dpi). Here, we compared hematological, immunological, and sequential pathological evaluations and determined the CAV tissue distribution in different organs. Neither a reduction in weight gain nor anemia was detected in either the inoculated or the control group. The immune-pathological changes were investigated by evaluating the body and thymus weight ratio and specific antibody titer. Delayed recovery of the thymus corresponding to a low antibody response was detected in the orally inoculated group. This is different from what was found in chickens intramuscularly infected with the same dose of CAV. The CAV remaining in a wide range of tissues was examined by viral reisolation into cell culture. The absence of the virus in infected tissues was typically found in the intramuscularly inoculated group. These chickens were immediately induced for a protective antibody response. A few viruses replicating in the thymus were found 21 dpi due to the regression in the antibody titer in the orally inoculated group. Our findings support that a natural infection with CAV may lead to the gradual CAV viral replication in the thymus during inadequate antibody production. The results clearly confirmed that virus-specific antibodies were essential for viral clearance. Under CIA-risk circumstances, administration of the CAV vaccine is important for achieving a sufficient protective immune response.

Molecular cloning of IL-6, IL-10, IL-11, IFN-ɤ and modulation of pro- and anti-inflammatory cytokines in cobia (Rachycentron canadum) after Photobacterium damselae subsp. piscicida infection.

Photobacterium damselae subsp. piscicida (P. damselae subsp. piscicida) is the agent of Photobacteriosis, a serious fish disease that produces an acute infection and high mortality in farmed cobia. It has been proved that regulation of pro- and anti-inflammatory cytokines play a central role in initiation of proper inflammatory responses against bacterial infection. Here we have analyzed the expression of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8 and IFN-ɤ) and anti-inflammatory cytokines (IL-10 and IL-11) in spleen and head kidney during acute P. damselae subsp. piscicida infection of cobia. Our data revealed that cytokines tested showed distinct patterns of expression. While TNF-α and IL-8 showed a decay pattern of expression, IL-1ß response was quite late. Moreover, P. damselae subsp. piscicida infection induced the simultaneous expressions of pro-inflammatory (IL-6, IFN-ɤ) and anti-inflammatory (IL-10, IL-11) cytokines. Together these results indicate the innate immunity of cobia is actively suppressed by P. damselae subsp. piscicida.

Docosahexaenoic acid and phosphatidylserine improves the antioxidant activities in vitro and in vivo and cognitive functions of the developing brain.

Fish oil during early postnatal period may modulate the impact of oxidative stress in the developing brain and thus improve memory and cognitive behaviour. This study investigated the impacts of docosahexaenoic acid (DHA, C22:6, n-3) and/or phosphatidylserine (PS) on antioxidant activities in vitro, and the beneficial effects of feeding with DHA and/or PS on antioxidant activities in brain and liver tissues and on the cognitive functions of the developing brain. Results indicated that DHA and/or PS significantly enhanced antioxidant activities and increased cell viabilities in vitro. Feeding with DHA and/or PS supplementation not only significantly improved escape latency of animals, but it also improved the oxidative parameters in the brain, enhanced glutathione peroxidase activity as well as reduced nitric mono-oxide levels in the liver. DHA and PS may serve to protect cells from oxidative stress and further improve learning and memory ability in vivo.

Docosahexaenoic acid and phosphatidylserine supplementations improve antioxidant activities and cognitive functions of the developing brain on pentylenetetrazol-induced seizure model.

Epilepsy provoked by pentylenetetrazol (PTZ) is caused by an abnormal excitatory postsynaptic potential, which results in increased production of reactive oxygen species, and finally reducing cognitive functions. The objective of this study was to investigate the effects of dietary supplementation with DHA and PS, administered either alone or in combination, on oxidative stress and behavioral and cognitive spatial memory in neonatal rats with PTZ-induced epileptic seizure. In this study, rat pups received repetitive doses of PTZ for induction of epileptic seizure and docosahexaenoic acid (DHA, C22:6, n-3) and phosphatidylserine (PS) were orally administrated alone or together to the PTZ-induced epileptic animals daily for 36 d. The spatial memory, nitric mono-oxide (NO) production, and enzymatic activities of superoxide dismutase (SOD) and catalase in brain and liver tissues were determined. PTZ administration significantly reduced the cell numbers in the hippocampus, shortened the escape latency in the safe target region, decreased activities of SOD and catalase, but increased NO content in both brain and liver tissues, while DHA and PS significantly extended the escape latency, reversed the oxidative parameters observed in the brain, and enhanced SOD activity in the liver. Dietary supplementation with DHA and PS may protect brain tissue from the oxidative stress caused by epileptic seizures and could serve to improve learning and memory ability in vivo.

Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens.

Flagellin, a bioactive Toll-like receptor (TLR) 5 ligand, may trigger the innate immunity that in turn is important for subsequent adaptive immune responses. In the present study, the adjuvant effects of the monomeric and polymeric forms of Salmonella flagellin (mFliC and pFliC, respectively) were examined in specific-pathogen free (SPF) chickens immunized intramuscularly (i.m.) or intranasally (i.n.) with formalin-inactivated avian influenza virus (AIV) H5N2 vaccines. Results showed that mFliC cooperating with the 64CpG adjuvant significantly induced influenza-specific antibody titers of plasma IgA in the i.m.-vaccinated animals. The nasal IgA levels in the i.n.-mFliC-coadministrated AIV vaccinated chickens were significantly elevated compared to levels observed in the control group (H5N2 vaccine alone). The pFliC cooperating with the 64CpG adjuvant significantly enhanced cell proliferation of splenocytes in the i.m.-vaccinated animals. TLR3 and TLR5 expressions were activated by flagellin stimulation in vitro and in vivo. These results suggest that flagellin can be used as an adjuvant in an AIV H5N2 vaccine, especially for mucosal immunity.

Immunoadjuvant efficacy of plasmids with multiple copies of a CpG motif coadministrated with avian influenza vaccine in chickens.

Unmethylated CpG motifs are capable of evoking a range of immunostimulatory effects in vertebrates and have tremendous potential to be used as therapeutic agents and adjuvants. This particular type of CpG motif has been demonstrated to be an excellent immune adjuvant mediated by Toll-like receptor 9 (TLR9) in various mammalian vaccines; however, only a few studies confirm its efficacy in avian vaccines. In the present study, immunomodulatory activities of plasmids with various copy numbers of a CpG motif were evaluated in chickens inoculated with an avian influenza vaccine. Results showed that the plasmid with 64 copies of the CpG motif (64CpG-plasmid) significantly enhanced the mRNA expressions of interferon-gamma (IFN-γ), TLR3 and TLR7 in chicken splenocytes compared to plasmids with lesser copies of the CpG motif in vitro. Chickens inoculated with the H5N2 avian influenza inactivated vaccines (V52) coadministrated with the 64CpG-plasmid (V52-64CpG) showed significant increments of hemagglutination inhibition (HI) titers, peripheral blood mononuclear cell (PBMC) proliferation, and mRNA expressions of IFN-α, IFN-γ, TLR3, TLR7 and TLR21 in splenocytes as compared to those of chickens inoculated with V52 alone, V52 adjuvanted with aluminum gel (V52-gel), or with V52-gel plus vector. Additionally, following challenge with a highly virulent H5N1 strain, a higher survival rate (100%) was observed in chickens inoculated with V52-64CpG as compared to those that received V52-gel (80%) or PBS (0%). The 64CpG-plasmid significantly enhanced chicken immunity in vitro and in vivo; thus it can be a potent adjuvant in an avian influenza vaccine for chickens.

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 ß-1,3/ß-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 ß-glucan (pßG), a product extracted from Saccharomyces cerevisiae, in this study. The binding of zymosan-FITC to hDectin-1 and mDectin-1 was inhibited by pßG 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 pßG, zymosan, or laminarin, whereas ovalbumin endocytosis was enhanced in U937 and J774A.1 treated either with pßG, zymosan, laminarin, or barley-glucan. These results indicate that the binding affinity of pßG to hDectin-1 is similar to the binding affinity to mDectin-1, and that stimulation by pßG as well as various forms of ß-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 ß-glucans capable of binding its specific receptor as the effective immunomodulators.

Association of interleukin-10 polymorphisms with cytokines in type 2 diabetic nephropathy.

OBJECTIVE: Interleukin-10 (IL-10) polymorphic variants are linked with cytokine production and are involved in many chronic inflammatory diseases, including type 2 diabetes mellitus (T2DM). We investigated the hypothesis that IL-10 promoter polymorphisms may be associated with cytokine expressions involved in the progression of diabetic nephropathy (DN). STUDY DESIGN: A total of 72 Taiwanese subjects were included in this study; along with a control group, patients had a diagnosis of DN lasting ≥2 years, and patients had a diagnosis of T2DM with normal renal functions lasting ≥5 years. Their IL-10 and tumor necrosis factor-α (TNF-α) genotyping and association of blood chemistry, plasma IL-10, TNF-α, and monocyte chemoattract protein-1 (MCP-1), and urinary MCP-1 were investigated. RESULTS: The IL-10-(-592) genotype exhibited significant association with cytokine expressions in DN: significantly higher TNF-α and lower plasma IL-10 levels were observed in IL-10-(-592)AA, whereas a higher urine MCP-1 level was found in Taiwanese patients with the IL-10-(-592)CC genotype. CONCLUSIONS: IL-10-(-592) promoter polymorphisms may influence IL-10 and MCP-1 production, which may be an indicator of nephropathy risk in Taiwanese T2DM patients.

Adjuvant effects of chicken interleukin-18 in avian Newcastle disease vaccine.

Interleukin-18 (IL-18) can induce interferon-gamma (IFN-gamma) production and promote Th1 immunity, and hence, it modulates immune functions. In the present study, the in vitro and in vivo immunomodulatory activities of full length or mature chicken IL-18 expressed in a prokaryotic expression system (pCHIL18-F and pCHIL18-M, respectively) and chicken IL-18 expressed in a eukaryotic expression system (euCHIL18) were examined. Results showed that pCHIL18-F, pCHIL18-M and euCHIL18 significantly enhanced IFN-gamma mRNA expression in chicken splenocytes, which successfully increased IFN-gamma-induced nitric oxide (NO) synthesis by macrophages. Vaccination with cell-cultured Newcastle disease vaccine (NDTC) co-administrated with pCHIL18-F, pCHIL18-M or euCHIL18 resulted in significant increments of hemagglutination inhibition (HI) titers, cell proliferation of peripheral blood mononuclear cells (PBMC), and ratios of CD8(+) to CD4(+) in chickens compared with inoculation of PBS or NDTC alone. Thus, full length and mature chicken IL-18 expressed using a prokaryotic system and using a eukaryotic system showed equivalent in vitro and in vivo biological activities, and all forms effectively enhanced cell-mediated and humoral immunity, suggesting possible future use as a potential adjuvant in chicken NDTC vaccine production.

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 beta-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.

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.

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.