Download Expression of Toll-like receptor mRNA and cytokines in pigs infected

Veterinary Microbiology 136 (2009) 266–276
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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,
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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-
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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)
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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.
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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.
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