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Veterinary Microbiology 136 (2009) 266–276 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus Ching-Hsing Liu a, Hso-Chi Chaung a,*, Hsiu-luan Chang b, Yu-Tang Peng a, Wen-Bin Chung a,* a b Department of Veterinary Medicine, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan, ROC Department of Animal Science, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan, ROC A R T I C L E I N F O A B S T R A C T Article history: Received 12 July 2008 Received in revised form 11 November 2008 Accepted 24 November 2008 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 PRRSVchallenged 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-1b and IL-6 by AMs of PRRSV-infected pigs. Infection of pigs with PRRSV also resulted in an increased secretion of IL-1b 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. ß 2008 Elsevier B.V. All rights reserved. Keywords: Porcine reproductive and respiratory syndrome virus Toll-like receptors Cytokines 1. Introduction Porcine reproductive and respiratory syndrome (PRRS) is one of the diseases that causes significant economic losses to the swine industry worldwide. This syndrome is characterized by abortion in pregnant sows and gilts, and respiratory distress with increased susceptibility to secondary infection in piglets and growing pigs (Zimmerman et al., 2006). The causative agent, PRRS virus (PRRSV), was first isolated from pigs in The Netherlands in 1991 (Wensvoort et al., 1991). PRRSV is an enveloped, positivesense, single-stranded RNA virus, classified in the family * Corresponding authors. Tel.: +886 8 7740 292; fax: +886 8 7740 292. E-mail addresses: [email protected] (H.-C. Chaung), [email protected] (W.-B. Chung). 0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.11.016 Arteriviridae (Zimmerman et al., 2006). Field observations showed that farms infected with PRRSV exhibit an increased incidence of secondary viral and bacterial infections (Zimmerman et al., 2006). These field observations have suggested that PRRSV infection might induce immunosuppression in pigs; however, this hypothesis has not been fully confirmed. Some experiments have demonstrated an increased severity of the disease in pigs with a combined infection of PRRSV and secondary microbes, while other experiments have failed to yield the same pattern (Wills et al., 2000; Thanawongnuwech et al., 2000, 2004; Zimmerman et al., 2006). The specific interactions between PRRSV and the secondary invaders are unclear and remain to be elucidated. PRRSV has been shown to induce minimal or no TNFa and IFN-a secretion in pig lungs, while a consistent and substantial production of IL-1 and IL-10 were C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 noticed during infection (van Reeth et al., 1999; van Gucht et al., 2003; Thanawongnuwech et al., 2004). Pigs infected with PRRSV have been reported to exhibit an enhanced production of IL-1, IL-6 and TNF-a in response to lipopolysaccharides (LPS), and the overproduced proinflammatory cytokines were associated with the severity of clinical signs (van Gucht et al., 2003). The mechanism of the synergy occurring between PRRSV and LPS in the induction of proinflammatory cytokine expression is unclear. Recognition of LPS by the innate immune system involves a set of receptor complexes including Toll-like receptor 4 (TLR4), MD2, CD14 and LPS-binding protein (Miyake, 2004). Both CD14 and LPSbinding protein were increased significantly during PRRSV infection; however, the level of TLR4 expression had not been examined (van Gucht et al., 2005). TLRs are a key component of the host innate recognition system. Each TLR family detects distinct microbial pathogenassociated molecular patterns (PAMPs) and triggers the activation of specific signaling pathways, leading to the transcription of inflammatory and anti-inflammatory cytokines (Krishnan et al., 2007). Specifically, TLR2 recognizes lipoteichoic acid (LTA), lipoproteins, and peptidoglycan from gram-positive bacteria (Tötemeyer et al., 2003). TLR3 is engaged in the recognition of double-stranded RNA from viral origin and synthetic double-stranded RNA, polyriboinosinic polyribocytidylic acid, polyIC (Matsumoto et al., 2004). Single-stranded RNA is the ligand recognized by TLR7 and TLR8. TLR9 recognizes unmethylated bacterial CpG DNA motifs (Krishnan et al., 2007). As mentioned above, TLR4 responds to LPS, the main component of the outer membrane of gram-negative bacteria. The determination of TLR4 expression patterns and their association with cytokine production and clinical signs in pigs challenged with PRRSV and LPS will likely lead to a more thorough understanding of the pathogenesis of PRRSV. There are many reports describing the induction of innate immune responses via stimulation of various TLRs in virally infected hosts. Elevated expression of TLR4 in nasal-associated lymphoid tissue has been noted during the acute stage of foot-and-mouth disease in cattle (Zhang et al., 2006). Induction of TLR4 expression with an enhanced production of IFN-b and IL-6 by hepatitis C virus-infected B cells has been reported (Machida et al., 2006). TLR7 and 8 have been shown to be involved in innate immune responses to human parechovirus (Triantafilou et al., 2005). There were increased levels of TLR2, 3, 4, 7 and 8 mRNA in PBMCs of children with acute rotavirus diarrhea (Xu et al., 2006). However, there have been few studies investigating TLR expression patterns by lymphoid tissues or cells of PRRSV-infected pigs (Sang et al., 2008). To gain insight into the pathogenic mechanism of PRRSV, this study was conducted in order to investigate the expression of TLR mRNA and cytokines by pigs infected with PRRSV. Discussion on the association between the expression of TLRs and cytokines and their contribution to the induction of clinical symptoms is also presented. 267 2. Materials and methods 2.1. Virus preparation The PRRSV HF6-2, a third passage of a Taiwanese field isolate (North American genotype) obtained in 2004, was grown and propagated in porcine alveolar macrophages (AMs). AMs used for viral isolation were obtained by lung lavage from SPF pigs as previously described (Chang et al., 2008). The titer of virus stock was 1 107 TCID50/ml as determined by cytopathic effect (CPE) in AMs. 2.2. Animals and experimental design Fifteen, cross-bred (Landrace Yorkshire Duroc) pigs were early weaned at the age of 10 days from 3 litters at a commercial pig farm. Pigs were transferred to an experimental unit with positive pressure at the University Animal Research Center. Sera were collected from pigs every week to monitor the antibodies against PRRSV and porcine circovirus type 2 (PCV2) using a virus neutralization test and an indirect immunofluorescence test, respectively. Pigs were used after two consecutive tests showing no antibodies to PRRSV or PCV2. No PRRSV, PCV2, pseudorabies virus, classical swine fever virus or parvovirus were detected from the buffy coats of all pigs as assayed by polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR) before the experimental challenges of the pigs (Chung et al., 2005). Pigs were randomly divided into four groups at 8 weeks of age. The number of pigs and the treatment for each group were as follows: Group 1 (n = 4): Pigs were intranasally challenged with 1 ml of PRRSV in each nostril for 3 consecutive days. Pigs were necropsied at 7 days postfirst inoculation. Group 2 (n = 3): Pigs were intranasally challenged with 1 ml of culture supernatants (mockinfected) in each nostril for 3 consecutive days. Pigs were necropsied at 7 days post-first inoculation. Group 3 (n = 4): Pigs were intranasally challenged with 1 ml of PRRSV in each nostril for 3 consecutive days. Pigs were necropsied at 14 days post-first inoculation. Group 4 (n = 4): Pigs were intranasally challenged with 1 ml of culture supernatants (mock-infected) in each nostril for 3 consecutive days. Pigs were necropsied at 14 days post-first inoculation. The viral inoculum contained 1 106 TCID50/ml of PRRSV. For each pig, body temperatures were measured and clinical signs were recorded at 1 week before challenge, the day of challenge, and every day after challenge until the end of the experiment. During the necropsies, peripheral blood mononuclear cells (PBMCs), bronchoalveolar lavage cells (BALCs), tracheobronchial lymph nodes and spleens were collected for further examination. Standard pathological and microbiological examinations were conducted for each pig at necropsy. 2.3. Preparations of PBMCs Blood samples from pigs were collected from the jugular veins into blood collection tubes containing EDTA before the necropsies. PBMCs were isolated by density gradient centrifugation using Histopaque (Sigma–Aldrich, 268 C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 St. Louis, MO, USA). Cells were washed three times in phosphate buffered saline (PBS), counted, and resuspended in RPMI-1640 medium at a concentration of 2 106 cell/ ml. Cells were also stained and subjected to differential counting based on cell morphology. 2.4. Preparation of BALCs At necropsy, BALCs were collected by bronchoalveolar lavage from pigs as previously described (Chang et al., 2008). The BALCs were isolated from bronchoalveolar lavage fluids by centrifugation. The pellets were washed twice in PBS, counted, and resuspended in RPMI-1640 medium at a concentration of 2 106 cells/ml. Differential counting was performed on stained cytospin slides of the BALCs. 2.5. Stimulation of AMs and PBMCs with PRRSV or microbial PAMPs BALCs and PBMCs from mock- and PRRSV-infected pigs were dispensed onto 6-well tissue culture plates (Nunc, Roskilde, Denmark) at a density of 4 106 cells in 2 ml of RPMI-1640 containing 10% fetal bovine serum. Cells were treated with LTA from Staphylococcus aureus (10 mg/ml; Sigma–Aldrich), LPS from Escherichia coli 055:B5 (1 mg/ml; Sigma–Aldrich), polyIC (50 mg/ml; Amersham Biosciences, Piscataway, NJ, USA), PRRSV (0.1 TCID50/cell) or they remained untreated (control) as the following procedures. BALCs and PBMCs were first treated with tissue culture supernatants (for LTA-, LPS-, polyIC-treated and untreated control cells) or PRRSV (for PRRSV treated cells) and incubated in a 5% CO2 incubator at 37 8C for 2 h. For BALCs cultures, the supernatants and nonadherent cells were then discarded and the adherent cells, which represented the alveolar macrophages, were washed twice with RPMI medium. Complete monolayer cell sheets were obtained for both PRRSV- and mockinfected AMs after the washing procedures. For PBMC cultures, both adherent and nonadherent cells were used for the study. Therefore, the washes of these cells were conducted carefully by gently removing the supernatants only after the cells had settled. Stimulation of AMs and PBMCs by PAMPs was then conducted by adding LTA, LPS, polyIC or culture medium (for untreated control and PRRSV treated cells) to each corresponding well, followed by culture in a 5% CO2 incubator at 37 8C for 48 h. Supernatants were then colleted from the cell cultures for the determination of cytokine concentrations by ELISA. To investigate the TLR expressions, AMs and PBMCs were collected from extra untreated control groups before the PAMPs challenges and used for RNA extraction. 2.7. RNA extraction Total RNA were extracted from tracheobronchial lymph nodes, spleens, AMs and PBMCs using TRIzol1 reagent (Invitrogen, Carlsbad, CA, USA) and according to the manufacturer’s instructions. The final ethanol-washed RNA pellets were air dried and then dissolved in 20 ml diethyl pyrocarbonate (DEPC; Sigma, St. Louis, MO, USA)treated autoclaved water. The concentration of isolated RNA was quantified at OD260, and the purity was checked by calculation of the OD260/OD280 ratio using a spectrophotometer. The total RNA in each sample was adjusted to a final concentration of 200 mg/ml. 2.8. Semi-quantitative RT-PCR Reverse transcription and PCR reactions were performed in a GeneAmp 2700 thermal cycler (Applied Biosystems, Singapore). A total of 1 mg of RNA isolated from each organ or cell was reverse transcribed using the oligo (dT)18 primers (Promega, Madison, WI, USA) and AMV reverse transcriptase (Promega, Madison, WI, USA), essentially following the manufacturer’s recommended procedure. The resultant cDNA was then subjected to PCR. The primer sequences, amplicon sizes, number of PCR cycles used for the amplification, and sequence of primers from which they are derived are summarized in Table 1. To ensure that PCR products were quantified during the exponential phase of the amplification, the PCR conditions and the appropriate numbers of cycle for amplification within the linear range were determined by a preliminary experiment (data not shown). Amplification reactions were performed in a final reaction volume of 25 ml, consisting of 2 ml of cDNA, 0.25 ml of hot start Taq (final concentration of 0.05 U/ml; Promega), 2.5 ml of 10 PCR buffer (Promega), 0.5 ml of dNTP, one set of primers (0.5 ml each, final concentration of 0.2 mM), and 18.75 ml of H2O. All PCR reactions consisted of a denaturating cycle at 94 8C for 10 m, followed by an amplification profile of the appropriate cycles (Table 1) with an initial denaturating cycle at 94 8C for 1 m, followed by annealing at 53 8C for 30 s and extension at 72 8C for 1 m, and a final extension step at 72 8C for 10 m. The amplified PCR products (5 ml of 25 ml total reaction volume) were analyzed by electrophoresis on 1.8% agarose gel (Seakem1, Rockland, ME, USA) and subjected to ethidium bromide staining. Gels were scanned on a densitometer (Bio imaging systems; Syngene, Cambridge, UK) and analyzed using the software provided (Bio imaging systems; Syngene, Cambridge, UK). The amount of TLR mRNA in a sample is expressed as a ratio relative to the amount of mRNA for the housekeeping gene b-actin. 2.9. Statistic analysis 2.6. Cytokine measurement The concentrations of IL-1b, IL-6, IL-8, and TNF-a in the culture supernatants were measured using commercially available ELISA kits (DuoSet1 ELISA development system, R&D, USA) according to the manufacturer’s instruction. Data were analyzed using the GLM procedures of SAS software (Version 9.1.2, SAS Institute Inc., Cary, NC, USA). Least-square means were used for treatment comparisons if a significant F static was detected by ANOVA. Data is expressed as means standard errors (S.E.). A significance C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 269 Table 1 Primers used in reverse transcription polymerase chain reaction (RT-PCR). Gene specificity Primer sequences 0 0 Size (bps) No. of PCR cycles Accession no.a 106 30 AB085935 TLR2 F5 -GTGCTTTCCGAGAACTTTGT-3 R50 -GCAGAATGAGGATGGCG-30 TLR3 F50 -TCCAACTAACAAACCAGGC-30 R50 -ACATCCTTCCACCATCT-30 186 30 Unpublished data TLR4 F50 -AAGGTTATTGTCGTGGTGT-30 R50 -CTGCTGAGAAGGCGATAC-30 179 30 AB188301 TLR7 F50 -GTGGACTGCACAGACAAG-30 R50 -GGGCATTATAACAACGAGGG-30 655 30 AF245702 TLR8 F50 -CCGCACTTCGCTATCTAAAC-30 R50 -GAAAGCAGCGTCATCATCAA-30 792 35 AB092975 TLR9 F50 -AGATGTTTGCTCGCCT-30 R50 -GGACACTCGGCTATGGA-30 308 30 AB071394 b-Actin F50 -ACCACTGGCATTGTCA-30 R50 -CTCCTGCTCGAAGTCC-30 237 30 U07786 a Sequence of primers from which they are derived. Sequence data was obtained from GenBank. level of p < 0.05 was used and a trend was assumed at 0.05 p < 0.1. 3. Results 3.1. Clinical signs and pathology after PRRSV infection Clinical signs of PRRS such as fever, rough hair, anorexia and dyspnea were first observed at 6 days post-infection (dpi) and became increasingly severe until the end of the experiment for the PRRSV-infected pigs. Typical interstitial pneumonia, characterized by alveolar septa thickening by mononuclear cells especially macrophages, hyperplasia of type II pneumocytes, and accumulation of macrophages, degenerating cells, and proteinaceous fluid in alveolar spaces, was noticed in the infected pigs. PRRSV were detected from the mixed homogenates of tracheobronchial lymph nodes, spleen and lungs from challenged pigs by virus isolation and RT-PCR. No PRRSV, clinical signs, or pathological changes were detected in mock-infected pigs. 3.2. Changes of PBMC and BALC subpopulations after PRRSV infection No significant changes in the percentages of lymphocytes and monocytes in PBMCs were observed in PRRSV-infected pigs at 7 dpi. An increased percentage of monocytes (21.5 0.6), along with a decreased percentage of lymphocytes (78.5 0.6) in PBMCs were noticed in PRRSV-infected pigs at 14 dpi, as compared with mock-infected pigs (11.3 0.7 and 88.7 0.7 for monocytes and lymphocytes, respectively). A decreased percent of macrophages (53.0 5.4) along with an increased percent of lymphocytes (45.5 5.4) in BALCs were observed in PRRSV-infected pigs at 7 dpi, as compared with mock-infected pigs (71.3 4.6 and 27.3 4.7 for macrophages and lymphocytes, respectively). A decreased percent of macrophages (44 4.3) along with an increased percent of lymphocytes (54.5 4.1) in BALCs were also observed in PRRSV-infected pigs at 14 dpi, as compared with mock-infected pigs (71.3 1.8 and 26.8 1.4 for macrophages and lymphocytes, respectively). There were no significant changes in the percent of neutrophils in the BALCs among the different groups. 3.3. Expression of TLRs mRNA in tracheobronchial lymph nodes and spleen There was a tendency of interaction (p = 0.053) between PRRSV infection and dpi for TLR3 expression in tracheobronchial lymph nodes, such that the level of TLR3 mRNA was significantly (p < 0.05) lower in PRRSV-infected pigs 7 dpi than in pigs 14 dpi, but did not differ between mock-infected pigs necropsied at 7 and 14 dpi (Fig. 1). The expression of TLR7 mRNA was significantly (p < 0.05) higher in the tracheobronchial lymph nodes of PRRSVinfected pigs at 14 dpi than that of pigs at 7 dpi and that of the uninfected controls. TLR3 mRNA expression in the spleens were tended to be enhanced by PRRSV infection regardless of the time of infection (1.16 vs. 0.75 for infected and uninfected pigs, respectively; p = 0.085). A similar result was observed for TLR8 (1.58 vs. 1.16 for infected and uninfected pigs, respectively; p = 0.075). These data demonstrate that there is a delay before an increase in the expression of TLR3 and 7 mRNA in tracheobronchial lymph nodes, and a tendency for constitutively elevated expressions of TLR3 and 8 mRNA in the spleens of PRRSV-infected pigs during the experimental period of 14 days. 3.4. Expression of TLRs mRNA in alveolar macrophages and peripheral blood mononuclear cells The expression of TLR2 mRNA in alveolar macrophages from PRRSV-infected pigs at 7 dpi was higher than that of uninfected pigs, a difference not considered significant (p = 0.13); interestingly, the TLR2 mRNA was significantly (p < 0.05) higher than that of PRRSV-infected pigs at 14 dpi (Fig. 2). The mRNA levels of TLR2 and 8 in PBMCs of PRRSVinfected pigs at 7 dpi were significantly (p < 0.05) higher than those of mock-infected pigs and those of PRRSV- 270 C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 Fig. 1. Toll-like receptors (TLRs) mRNA expression in tracheobronchial lymph nodes and spleens of porcine reproductive and respiratory syndrome virus (PRRSV)-infected and mock-infected (uninfected) pigs. Pigs were infected (&) with PRRSV or remained uninfected (&) and were necropsied at 7 and 14 days post-infection (dpi). The TLR mRNA levels in each organ were determined by semi-quantitative RT-PCR and expressed as the ratio of TLR/b-actin RTPCR product band intensity. 7 dpi LN (or spleen) and 14 dpi LN (or spleen) represent tracheobronchial lymph nodes (or spleen) collected at 7 and 14 dpi, respectively. Data are presented as means standard errors. Different letters indicate significant (p < 0.05) differences of mean values among groups in tracheobronchial lymph nodes (A and B) and in spleens (a and b). infected pigs at 14 dpi. The expression of TLR4 mRNA by PBMCs of PRRSV-infected pigs at 7 dpi was also significantly (p < 0.05) higher than those of mock-infected pigs. The expression of TLR7 mRNA in PBMCs of PRRSV-infected pigs at 7 dpi tended toward an increase compared to that of mock-infected pigs (p = 0.08) at the same time point. The data demonstrate that infection of pigs with PRRSV results in elevated expressions of TLR2, 4 and 8 mRNA, and a tendency towards an increased level of TLR7 mRNA in PBMCs at 7 dpi. 3.5. Cytokine expression of alveolar macrophages after PRRSV infection or PAMP stimulation or in a combination Alveolar macrophages were collected from PRRSV- and mock-infected pigs, and the cells were treated with PRRSV, C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 271 Fig. 2. Toll-like receptors (TLRs) mRNA expression in AMs and PBMCs of porcine reproductive and respiratory syndrome virus (PRRSV)-infected (&) and mock-infected (uninfected; &) pigs. Pigs were infected with PRRSV or remained uninfected, and cells were collected at necropsies conducted at 7 and 14 days post-infection (dpi). The TLR mRNA levels in AMs and PBMCs were determined by semi-quantitative RT-PCR and are expressed as the ratio of TLR/bactin RT-PCR product band intensity. 7 dpi AM (or PBMC) and 14 dpi AM (or PBMC) represent AMs (or PBMC) collected at 7 and 14 dpi, respectively. Data are presented as means standard errors. Different letters indicate significant (p < 0.05) differences of mean values among groups in AMs (A and B) and in PBMCs (a and b). various PAMPs, or they remained untreated. Culture supernatants were then colleted from the cell cultures for the determination of cytokine concentrations by ELISA. The concentrations of IL-1b in the culture supernatants of untreated AMs from PRRSV-infected pigs at 7 and 14 dpi were significantly (p < 0.05) elevated compared to those of mock-infected pigs (Fig. 3). The expressions of IL-1b in LTA or PRRSV-stimulated AMs from PRRSV-infected pigs at 14 dpi were significantly (p < 0.05) higher than those of similarly treated AMs from mock-infected pigs. They were also significantly (p < 0.05) higher than those of nonstimulated AMs from both PRRSV- and mock-infected pigs. The concentrations of IL-6 in the un-stimulated AMs from PRRSV-infected pigs at 7 dpi were significantly (p < 0.05) 272 C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 Fig. 3. Cytokine production by alveolar macrophages (AMs) following stimulation with lipoteichoic acid (LTA), lipopolysaccharide (LPS), polyriboinosinic polyribocytidylic acid (polyIC) or porcine reproductive and respiratory syndrome virus (PRRSV) from PRRSV-infected (&) and mock-infected (uninfected; &) pigs. Pigs were infected with PRRSV and AMs were collected at necropsies conducted at 7 and 14 days post-infection (dpi). The collected AMs were treated with LTA, LPS, polyIC or PRRSV and incubated for 48 h. Cytokine concentrations in the supernatants of stimulated AMs were determined by ELISA. CTL, LTA, LPS, IC, PRRSV represent untreated control, LTA, LPS, polyIC, and PRRSV treatments, respectively. Data are presented as means standard errors. Different letters (a–e) indicate significant (p < 0.05) differences in means among groups. higher than those from mock-infected pigs. Levels of IL-6 in the LTA or LPS stimulated AMs from PRRSV-infected pigs at 7 dpi were significantly (p < 0.05) higher than those of AMs treated similarly from mock-infected pigs, but insignificantly different from those of untreated AMs from PRRSV-infected pigs. A similar result was observed for LTAtreated AMs from PRRSV-infected pigs at 14 dpi. PRRSVinfected pigs appeared to have a reduced expression of IL-8 by AMs in response to PAMPs as compared with mockinfected pigs. C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 The data demonstrate that PRRSV infection in pigs induces elevated secretions of IL-1b and IL-6 to the surrounding culture supernatants of AMs. PRRSV infection also enhanced the production of IL-1b by AMs in response to LTA stimulation. 273 3.6. Cytokine expression of PBMCs after PRRSV infection or PAMP stimulation or in a combination Treatments and the determination of subsequent cytokine secretions were similarly conducted for PBMCs Fig. 4. Cytokine production by peripheral blood mononuclear cells (PMBCs) following stimulation with lipoteichoic acid (LTA), lipopolysaccharide (LPS), poly IC, or porcine reproductive and respiratory syndrome virus (PRRSV) from PRRSV-infected (&) and mock-infected (uninfected; &) pigs. Pigs were infected with PRRSV and PBMCs were collected at necropsies conducted at 7 and 14 days post-infection (dpi). The collected PBMCs were treated with LTA, LPS, polyriboinosinic polyribocytidylic acid (polyIC) or PRRSV and incubated for 48 h. Cytokine concentrations in the supernatants of stimulated PBMCs were determined by ELISA. CTL, LTA, LPS, IC, PRRSV represent untreated control, LTA, LPS, polyIC, PRRSV treatments, respectively. Data are presented as means standard errors. Different letters (a–f) indicate significant (p < 0.05) differences in means among groups. 274 C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 as described previously for AMs. The concentrations of IL-6 in LPS or LTA treated PBMCs from PRRSV-infected pigs at 14 dpi were significantly (p < 0.05) higher than those of identically treated PBMCs from mock-infected pigs (Fig. 4). They were also significantly higher than those of untreated PBMCs from mock- or PRRSV-infected pigs. IL-8 expressions in LTA or LPS treated PBMCs from PRRSV-infected pigs at 7 dpi were significantly (p < 0.05) lower than those of similarly treated PBMCs obtained from mock-infected pigs. However, the amount of IL-8 detected in the culture supernatants of LPS or polyIC-treated PBMCs from PRRSVinfected pigs at 14 dpi were significantly (p < 0.05) higher than those of similarly treated PBMCs from mock-infected pigs. The data demonstrate that PRRSV infection in pigs induces an increased expression of IL-6 by PBMCs in response to LPS and LTA stimulation. 4. Discussion PRRSV infection-induced elevated production of IL-1b and IL-6 by AMs, as detected in the culture supernatants, confirming constitutive secretions of IL-1b and IL-6 by AMs harvested from PRRSV-infected pigs. The increased mRNA expression of TLRs, especially TLR2 and TLR4 in PBMCs from PRRSV-infected pigs at 7 dpi but not at 14 dpi, suggests that these TLRs might up-regulate secretion of proinflammatory cytokines from pigs exposed to secondary bacterial infections. It is therefore conceivable that the exaggerated production of proinflammatory cytokines via the activation of the TLR pathway might contribute to the exacerbated clinical signs in a combined infection of PRRSV and secondary invaders. This is supported by the increased production of IL-6 by PBMCs, and IL-1b by AMs from PRRSV-infected pigs in response to LTA or LPS. These in vitro data confirm previous finding that RRRSV infection in pigs enhances the production of IL-1 and IL-6 in response to LPS inoculation (van Gucht et al., 2003, 2005). The increased expression and regulatory response of TLR2 and to a lesser extent TLR4, on monocytes have been reported in patients with gram-positive and gram-negative sepsis (Armstrong et al., 2004). Thus, the increased expression levels of TLR2 and 4 mRNA by PBMCs might be one of the possible mechanisms that contribute to the enhanced cytokine secretions and the associated severe clinical signs in PRRSV-infected pigs in response to LPS. A primary PRRSV infection in pigs has been demonstrated to exacerbate clinical outcomes of secondary bacterial infections including Streptococcus suis, Salmonella choleraesuis and Mycoplasma hyopneumoniae (Wills et al., 2000; Thanawongnuwech et al., 2000, 2004). However, the exact mechanisms of interaction between PRRSV and the secondary bacterial invaders are still unclear. Recognition of pathogens requires TLR-mediated signals to initiate innate and subsequent adaptive immunity. Conceivably, the modulated expressions of TLRs following PRRSV infection in pigs along with the specific PAMPs presented by the secondary pathogens would both significantly influence the outcomes of disease in the combined infection. Chronic hepatitis caused by hepatitis C virus (HCV) has been reported to predispose patients to secondary viral and bacterial infections (Dolganiuc et al., 2006). TLR-mediated innate immunity has also been suggested to be a potential factor in HCV pathogenesis. However, discrepant results have been reported regarding the TLR expression patterns in patients with chronic HCV infection; Atencia et al. (2007) demonstrated significant down-regulation of TLR3, TLR7 and RIG-I mRNA in HCV patients, while in contrast, Dolganiuc et al. (2006) showed an up-regulated expression of most TLRs examined, including TLR3 and TLR7, in monocytes and lymphocytes of patients with chronic HCV infection. TLR2 has also been suggested to mediate the exaggerated lung inflammation during coinfection of mice with influenza virus and Streptococcus pneumoniae (Seki et al., 2004). However, experiments using TLR2 knockout mice have showed that TLR2 does not contribute to the host immune response to postinfluenza pneumococcal pneumonia (Dessing et al., 2007). The above information indicates that the interaction between the primary and secondary pathogens leading to the induction of diseases in the host is clearly a complex process, and interpretation of data to demonstrate any association between TLR expression and disease progression should therefore be conducted with caution. PRRSV infection tended to induce increased expressions of TLR7 mRNA in tracheobronchial lymph nodes of pigs at 14 dpi. An increased expression of TLR8 mRNA in PBMCs of PRRSV-infected pigs was also observed at 7 dpi. PRRSV is a single-stranded RNA virus, thus, it is able to be recognized by TLR7 and TLR8. TLR7 has been reported to recognize Epstein-Barr virus, vesicular stomatitis virus, and influenza virus (Lund et al., 2004; Martin et al., 2007). The recognition of these viruses by TLR7 expressed on plasmacytoid dendritic cells and B cells mediates the subsequent cytokine production and activation of cells (Lund et al., 2004). The up-regulated mRNA expressions observed in tracheobronchial lymph nodes for TLR7 at 14 dpi and in PBMCs at 7 dpi imply that these TLRs may be involved in the immune responses and pathogenesis of PRRSV infection. During viral infection, double-stranded RNA is produced and recognized by TLR3. As an arterivirus, replication of PRRSV yields partially double-stranded, replicative intermediates and thus can be recognized by TLR3 (Snijder and Meulenberg, 1998). Sang et al. (2008) reported that there was an increased expression of TLR3 gene in AMs of 2-week-old pigs congenitally infected with PRRSV. In this study, the expression of TLR3 mRNA in PRRSV-infected pigs at 14 dpi was higher than that of pigs at 7 dpi, but was not different from that of mock-infected pigs. The activation of TLR3 induces the transcription of a set of genes encoding for antiviral mediators including, interferon a/b (IFN a/b) and inflammatory cytokines (Matsumoto et al., 2004). Recent studies have shown that TLR3mediated immune responses can have both beneficial and harmful effects for the host. Influenza A virus infection increased the expression of TLR3, which thereby mediated the release of inflammatory cytokines that causes acute pneumonia (Le Goffic et al., 2006). However, TLR3deficient mice produced significantly lower levels of inflammatory mediators and also a lower number of CD8+ T lymphocytes in the lungs relative to wild-type C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 mice. This pattern led to a reduced number of lung lesions and higher survival rate in these mice, although with higher viral burdens (Le Goffic et al., 2006). Respiratory syncytial virus (RSV) infection also induced an increased expression of TLR3 on the epithelial cells of lungs. Rudd et al. (2006) demonstrated that TLR3 is necessary for the regulation of pathogenic responses in the lung, but is not required for effective clearance of virus. In the absence of TLR3, RSV induced an increased expression of T helper 2 cytokines, IL-5 and IL-13, which cause mucus overproduction, known to be one of the pathological symptoms of the disease (Rudd et al., 2006). Similarly, to achieve successful control of a complex disease like PRRS, the host innate immune system needs to be first activated to an appropriate and nonpathogenic immune level. The delayed increase in the expressions of TLR3 and TLR7 mRNA in the tracheobronchial lymph nodes of PRRSV-infected pigs might, therefore, be one of the regulation mechanisms for the host to properly maintain the optimal and the least pathogenic response to PRRSV infection. On the other hand, it might be one of the immune evasion mechanisms of PRRSV for its initial survival in the host. The similar mRNA expression profiles of TLR3 and TLR7 observed in tracheobronchial lymph nodes during the experimental period of 14 days period raises the possibility of a coordinated regulation of TLR3 and TLR7 expressions by an unidentified mechanism. Reports have demonstrated that coordinated activation of multiple innate immune molecules occur in the host and/or cell in order to regulate immune responses. TLR3, 7, 8 and 9 are localized intracellularly and can discriminate virus-derived molecular patterns from self-nucleic acid (Krishnan et al., 2007). Wang et al. (2006) has shown that physical interactions among TLRs results in inhibitory effects of TLR8 on TLR7 and TLR9, and TLR9 on TLR7, but not vice versa. The inhibitory interactions among these three TLRs may allow the host to maintain the appropriate immune response. Treatments of naive B cells with Epstein-Barr virus (EBV) and UVinactivated virus have been reported to up-regulate the expression of TLR7 and down-regulate the expression of TLR9 (Martin et al., 2007). The activation of NF-kB by EBV latency membrane protein LMP1 was suspected to suppress the expression of TLR9 (Martin et al., 2007). To regulate the immune responses during Salmonella infections, TLR4 is activated which results in the release of inflammatory mediators, which in turn increased the expression of TLR2 while it suppressed the expression of TLR4 (Tötemeyer et al., 2003). Both TLR3 and TLR7 can mediate the production of type I interferon (Matsumoto et al., 2004; Lund et al., 2004; Zhang et al., 2008). However, in vitro and in vivo studies have shown that PRRSV is a poor inducer of IFN a secretion (van Reeth et al., 1999; Chang et al., 2005). A recent report has revealed that PRRSV infection inhibited the expression of IFN-b mainly by inactivating IFN-b promoter stimulator 1 in the retinoic acid-inducible gene I (RIG-I) signaling pathway. The activation of the TIR domain-containing adaptor inducing IFN-b (TRIF) of TLR3 was only partially inhibited by PRRSV infection (Luo et al., 2008). This, together with our results, indicates that immune responses and pathogenesis of PRRSV infection involve complex interactions between PRRSV and the host immune system. A 275 considerable delay in the initial onset of effective immunity to PRRSV in infected and vaccinated pigs has been reported (Meier et al., 2003). Therefore, there is probably a coordinated regulation of multiple innate immune molecules, such as TLR3, TLR7, RIG-I and other TLRs, interacting with one another to initiate a delayed development of an immune response to PRRSV in pigs. The components of PRRSV contributing to the delayed expression of TLR3 and 7 mRNA are unclear, and warrant further study. It is well known that IL-8 is a potent chemoattractant and activator for neutrophils, lymphocytes and basophils. High concentrations of IL-8 have been detected in BALFs of patients and experimental animals with respiratory viral infections such as RSV (König et al., 1996; Lukashevich et al., 1999). The increased expression of IL-8 is associated with the severe infiltration of neutrophils into the lungs following infection by RSV (König et al., 1996). On the contrary, Lassa virus infection suppresses the expression of IL-8 in human monocytes/macrophages, which coincides with the minimal inflammatory responses observed in patients with Lassa hemorrhagic fever (Lukashevich et al., 1999). In this study, an unchanged or decreased expression of IL-8 by AMs was observed in PRRSV-infected pigs. PRRSV infection in pigs also resulted in a suppressed expression of IL-8 by AMs in response to PAMPs, observations which might explain the lack of a significant increase in the percentage of neutrophils observed in BALCs following PRRSV infection in pigs. RSV infection has been shown to induce increased expression of IL-10, which inhibits the production of IL-8 by human AMs in response to LPS (Panuska et al., 1995). A profound secretion of IL-10 has been reported in PRRSV-infected pigs and primary cultured cells (Thanawongnuwech et al., 2004; Chang et al., 2008). The suppressed IL-8 secretion might therefore be due, at least in part, to the effect of simultaneously increased production of IL-10. A decreased expression of IL-8 in LTA or LPS treated PBMCs from PRRSV-infected pigs at 7 dpi was also noticed; however, the IL-8 levels in LPS or polyIC-treated PBMCs from PRRSV-infected pigs at 14 dpi were increased. An increased expression of IL-8 by PBMCs was also observed in PRRSV-infected pigs at 14 dpi. It has been reported that there was a decreased percentage of IL-8 producing monocytes in PBMCs of piglets congenitally infected with PRRSV (Aasted et al., 2002). In this study, an increased percentage of monocytes in PBMCs were observed in PRRSV-infected pigs at 14 dpi. Therefore, the dynamic changes of IL-8 expression in PBMCs after PRRSV infection might simply reflect the changes in percentages of leukocyte subpopulations in pigs. In summary, this study was able to demonstrate substantially increased secretions of IL-1b and IL-6 by AMs of PRRSV-infected pigs. PRRSV infection also enhanced the production of IL-1b by AMs following LTA stimulation, and also IL-6 by PBMCs in response to LPS and LTA. Infection of pigs with PRRSV tended to induce increased expressions of TLRs 2, 3, 4, 7 and 8 mRNA in at least one of the lymphoid tissues or cells during the 14day period of our experiment. These results suggest that TLR-mediated innate immunity likely plays a critical role in the pathogenesis of PRRSV infection in pigs. 276 C.-H. Liu et al. / Veterinary Microbiology 136 (2009) 266–276 Acknowledgement This research was supported in part by grants NSC 952313-B-020-007-MY3 from the National Science Council of Republic of China. References Aasted, B., Bach, P., Nielsen, J., Lind, P., 2002. 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