Research Article Porcine Reproductive and Respiratory ...downloads.hindawi.com/journals/mi/2014/403515.pdf · translation of pro-IL- [ ]. TLRs and RIG-I like receptors (RLRs) are

Research ArticlePorcine Reproductive and Respiratory SyndromeVirus Induces IL-1120573 Production Depending onTLR4MyD88 Pathway and NLRP3 Inflammasome inPrimary Porcine Alveolar Macrophages

Jing Bi12 Shuang Song1 Liurong Fang1 Dang Wang1 Huiyuan Jing1 Li Gao1 Yidong Cai1

Rui Luo1 Huanchun Chen1 and Shaobo Xiao13

1 State Key Laboratory of Agricultural Microbiology College of Veterinary Medicine Huazhong Agricultural UniversityWuhan 430070 China

2Department of Immunology and Aetology College of Basic Medicine Hubei University of Chinese MedicineWuhan 430065 China

3 Laboratory of Infectious Diseases College of Veterinary Medicine Huazhong Agricultural University 1 Shizishan StreetWuhan 430070 China

Correspondence should be addressed to Shaobo Xiao xiaoshaobomailhzaueducn

Received 10 February 2014 Revised 23 April 2014 Accepted 23 April 2014 Published 21 May 2014

Academic Editor Vera L Petricevich

Copyright copy 2014 Jing Bi et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industryworldwide since the late 1980s Previous studies have reported that PRRSV infection induced the production of IL-1120573 However thecellular sensors and signaling pathways involved in this process have not been elucidated yet Here we studied the mechanismsresponsible for the production of IL-1120573 in response to highly pathogenic PRRSV Upon PRRSV infection of primary porcinealveolar macrophages both mRNA expression and secretion of IL-1120573 were significantly increased in a time- and dose-dependentmanner We also investigated the role of several pattern-recognition receptors and adaptor molecules in this response and showedthat the TLR4MyD88 pathway and its downstream signaling molecules NF-120581B ERK12 and p38 MAPKs were involved inIL-1120573 production during PRRSV infection Treatment with specific inhibitors or siRNA knockdown assays demonstrated thatcomponents of the NLRP3 inflammasome were crucial for IL-1120573 secretion but not for IL-1120573 mRNA expression FurthermoreTLR4MyD88NF-120581B signaling pathway was involved in PRRSV-induced expression of NLRP3 inflammasome componentsTogether our results deciphered the pathways leading from recognition of PRRSV to the production and release of IL-1120573 providinga deeper knowledge of the mechanisms of PRRSV-induced inflammation responses

1 Introduction

IL-1120573 a potent proinflammatory cytokine produced pre-dominantly by monocytes macrophages and lymphocytesplays a central role in regulating immune and inflammatoryresponses [1 2] Production and release of IL-1120573 are inducedby awide variety of stimuli includingmicrobial infection andtissue injury Unlike other proinflammatory cytokines IL-1120573 lacks a signal peptide The production and secretion ofbiologically active IL-1120573 involves at least two separate

signaling cascades [3 4] In the first cascade pattern recog-nition receptors (PRRs) in host cells detect microorganismsand induce transcription of an inactive 31-kDa precursortermed pro-IL-1120573 In the second signaling cascade the for-mation of a large multimeric protein complex known as theinflammasome activates the intracellular cysteine proteasecaspase 1 and subsequently cleaves pro-IL-1120573 into a 17-kDamature IL-1120573 [5] The inflammasome complex is typicallycomposed of three components (i) a nucleotide-bindingoligomerization domain (NOD-) like receptor (NLR) (ii)

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 403515 14 pageshttpdxdoiorg1011552014403515

2 Mediators of Inflammation

apoptosis-associated speck-like protein containing a CARD(ASC) and (iii) caspase 1 [6] Several inflammasomes havebeen described of which the NLRP3 inflammasome is thebest characterized one [7ndash9] NLRP3 contains a C-terminalleucine-rich repeat domain a central nucleotide bindingdomain and an N-terminal PYD The PYD domain ofNLRP3 recruits ASC via a homotypic interaction with thePYD domain Oligomerization of NLRP3ASC leads to therecruitment of procaspase 1 to generate enzymatically activecaspase 1 which in turn processes IL-1120573 [9]

A large number of viruses have been reported to inducethe production of IL-1120573 in infected cells [10] However differ-ent viruses induce the expression of pro-IL-1120573 and process itto its mature form through different signaling pathways andsignaling molecules For example hepatitis C virus inducesIL-1120573 transcription through the TLR7-mediated MyD88 sig-naling pathway and activates the NLRP3 inflammasome topromote IL-1120573 secretion [11] Vaccinia virus and murinecytomegalovirus infection stimulate the formation of theabsent in melanoma 2 (AIM2) inflammasome to activatecaspase 1 the maturation of IL-1120573 [12] Respiratory syncytialvirus (RSV) is mainly recognized by TLR2 and TLR4 toinduce IL-1120573 transcription through the NF-120581B signalingpathway and then the NLRP3 inflammasome processes pro-IL-1120573 to its active form [13]

Porcine reproductive and respiratory syndrome (PRRS)is an economically important swine disease characterized bysevere reproductive failure in sows and respiratory distressin piglets and growing pigs [14 15] The causative agentPRRSV is a single-stranded positive-sense RNA virus classi-fied within the family Arteriviridae [16] Since its emergencein the late 1980s PRRS has been a threat to the globalswine industry causing high economic losses [17] PRRSVinfection in swine can cause severe interstitial pneumonia[18] suggesting that the inflammatory response plays animportant role in infection and pathogenesis of PRRSVIndeed Thanawongnuwech et al reported that the expres-sions of IL-1120573 IL-8 and TNF-120572 were significantly elevated inPRRSV-infected pigs [19] while Qiao et al found that IL-1120573and TNF-120572 were significantly upregulated in PAMs infectedwith the highly pathogenic PRRSV strain HN07-1 comparedto the classical PRRSV strain BJ-4 [20] Furthermore Lunneyet al found that serums IL-1120573 and IL-8 levels significantlycorrelated with persistent infection of PRRSV [21] Thesefindings indicated that PRRSV infection significantly inducesIL-1120573 expression and IL-1120573 is closely related to PRRSVpathogenesis

Although it is clear that PRRSV infection results in IL-1120573 production in vivo and in vitro the mechanism of IL-1120573processing has not been elucidated yet In this study we ana-lyzed the underlyingmechanisms utilized by porcine alveolarmacrophages (PAMs) the target cells of PRRSV infectionin vivo to detect PRRSV and induce IL-1120573 expressionOur results revealed that PRRSV-activated IL-1120573 productiondepended on the TLR4MyD88 pathway and downstreamsignaling molecules NF-120581B ERK12 and p38 MAPKs More-over we found that theNLRP3ASCcaspase 1 inflammasomeis required for IL-1120573 secretion but not for IL-1120573 transcriptionduring PRRSV infection

2 Materials and Methods

21 Virus and Cells PRRSV strain WUH3 [22] originallyisolated from the brains of pigs with ldquohigh fever syndromerdquoin China in 2006 and identified as a highly pathogenic type2 (North American) PRRSV was used in this study PrimaryPAMs were obtained by postmortem lung lavage of 4-week-old pigs from a PRRSV-negative herd as described previously[23] The animals were housed and handled under con-ditions approved by the Huazhong Agricultural UniversityAnimal Ethics Committee (approval number HZAU-AEC-30102922) The isolated PAMs were cultured in RPMI-1640medium (Life Technology NY USA) Marc-145 cells a mon-key kidney cell line highly permissive for PRRSV infectionwere cultured in DMEM with 10 heated-inactivated FBS025 120583gmL Fungizone 100UmL penicillin 10 120583gmL strep-tomycin sulfate and 5120583gmL gentamicin in a humidified37∘C 5 CO

2incubator PRRSV was amplified in PAMs and

viral titers were determined by titration on Marc-145 cells

22 Reagents and Antibodies LPS polymyxin B (PMB)MAPK inhibitors (SP600125 SB202190 and U0126) NF-120581B inhibitor (BAY11-7082) and caspase 1 inhibitor (Boc-D-CMK) were purchased from Sigma-Aldrich (St Louis MOUSA) SP600125 SB202190 U0126 BAY11-7082 and Boc-D-CMK were dissolved in DMSO and APDC were dissolvedin water prior to use Antibodies specific for p38 ERK12and their phosphorylated forms (p-p38 and p-ERK12) werepurchased from Cell Signaling Technology (Beverly MAUSA) Antibody against porcine IL-1120573 was obtained fromRampD (Minneapolis MN USA) The anti-120573-actin antibodyHRP-conjugated goat anti-mouse IgG goat anti-rabbit IgGand donkey anti-goat IgG were purchased from Sigma-Aldrich (St LouisMO USA) AmAbA2F1 used for detectionof PRRSV nonstructural protein 2 (Nsp2) was produced fromhybridoma cells derived fromSp20myeloma cells and spleencells of BALBc mice immunized with recombinant Nsp2protein of PRRSV strain WUH3 [24]

23 Viral Plaque Assay Plaque assay was essentially per-formed as described previously [25] Briefly 95 confluentMarc-145 cells grown in six-well tissue culture plates wereinfected for 1 h with 10-fold serial dilutions (1000 120583L) ofPRRSV-containing samples After three washes with PBS(pH 74) cells were overlaid with 18 (wv) Bacto agarmixed 1 1 with 2timesDMEM containing 005mgmL neutralred Plaques were counted 4 days postinfection The averageplaque number and standard deviations were calculated fromthree independent experiments Virus titers were expressedas PFUmL

24 Immunofluorescence Assay (IFA) The infected PAMswere fixed permeabilized and subsequently inoculated withthe mAb A2F1 against PRRSV Nsp2 followed by immunos-taining with a FITC-anti-mouse IgG (Sigma St Louis MO)Cellular nuclei were counterstained with 1 120583gmL of DAPI for5min After washing with PBS cells were examined under anLSM510metaconfocal laser scanningmicroscope (Carl ZeissGottingen Germany)

Mediators of Inflammation 3

25 Western Blotting Cells were lysed in 2 SDS proteinsample buffer and subjected to 12 acrylamide SDS-PAGEThe separated proteins were electroblotted onto a nitrocel-lulose membrane Western blotting was carried out with theindicated primary antibody and HRP-conjugated antibodySignals were developed using SupersignalWest Pico Luminolkit (Thermo)120573-Actin was detected with an anti-120573-actinmAbto demonstrate equal protein sample loading

26 siRNA and Cell Transfection The siRNAs targeting theporcine RIG-I TLR1 TLR2 TLR3 TLR4 TLR5 TLR6TLR7 TLR8 TLR9 MyD88 TRIF NLRP3 and ASC weresynthesized by GenePharma (China) and the sequencesare listed in Table S1 available online at httpdxdoiorg1011552014403515 (supplementary materials) Transienttransfection of siRNA was performed using Lipofectamine2000 (Invitrogen) according to the manufacturerrsquos instruc-tions The amount of siRNA used for transfection wasoptimized in preliminary experiments and no significantcellular toxicity was observed

27 RNA Extraction and Real-Time RT-PCR Total cellularRNAwas extracted from the treated PAMs using anRNApreppure cell kit (Qiagen Valencia CA USA) RNA (1 120583g) wasreverse transcribed in a 20 120583L reaction mixture The cDNAproduct was amplified in a 20120583L reactionmixture containingSYBR Green real-time PCR Master Mix (Roche BaselSwitzerland) Gene-specific primers for real-time RT-PCRare listed in Table S2 (supplementary materials) Each cDNAsample was performed in triplicate PCR amplifications wereperformed using a Roche LightCycler 480 real-time System(Roche Basel Switzerland) Gene expression was measuredas a relative quantity as described previously [26]

28 Measurement of the Secreted IL-1120573 Protein The secretedIL-1120573 in cell supernatants was measured using a commercialsandwich ELISA kit (RampD) according to the manufacturerrsquosinstructions

29 Statistical Analyses All experiments were performed atleast three times Data are presented as mean plusmn standarddeviation (SD) A 119875 value less than 005 was consideredsignificant and a 119875 value less than 001 was considered highlysignificant

3 Results

31 Infection Kinetics of the Highly Pathogenic PRRSV StrainWUH3 in PAMs The highly pathogenic PRRSV strainWUH3 used in this study was isolated in Marc-145 cells [22]amonkey kidney cell line highly permissive for PRRSV infec-tion in vitro Because PAMs are the target cells of PRRSVinfection in vivo PRRSV strainWUH3 was adapted in PAMsfor three passages The infection kinetics of the adaptedPRRSV in PAMs were measured by indirect IFA with mAbagainst PRRSV Nsp2 and plaque assay to titrate virus con-tained in the supernatant of infected cells At 24 h postinfec-tion nearly 50 PAMs were infected and the infected cells

became much more at 36 h postinfection Visible cytopathiceffect (CPE) appeared at 48 h postinfection (Figure 1(a))Plaque assays also showed that the viral titers graduallyincreased with the progress of PRRSV infection (Figure 1(b))reaching 10639 PFUmL at 48 h postinfection indicating thatthe adapted PRRSV can efficiently infect PAMs and spread toneighboring cells Consequently the PAMs-adapted PRRSVwas used for subsequent experiments

32 PRRSV Infection Induces IL-1120573 mRNA Expression andRelease in PAMs Previous studies revealed that PRRSVinfection induces porcine IL-1120573 expression in vivo and invitro [19ndash21 27] and the highly pathogenic PRRSV which hasemerged in China and South East Asia since 2006 [28 29]induced stronger IL-1120573 than the classical PRRSV strains [20]To investigate the expression kinetics of IL-1120573 after PRRSVinfection primary PAMs were infected with the PAMs-adapted PRRSV strainWUH3 at a MOI of 01 Real-time RT-PCR and ELISA were performed to detect IL-1120573 mRNA incells and the secreted IL-1120573 protein in supernatants respec-tively As shown in Figure 2(a) PAMs infected with PRRSVexhibited significantly increased expression of IL-1120573 mRNAat 6 h postinfection and increased at a steady-state level withmaximal production at 36 h postinfection Also significantlyincreased IL-1120573 release was detected upon PRRSV infectionand the secreted IL-1120573 was further increased as the infectionprogressed peaking at later time points (48 h) (Figure 2(b))When PAMs were infected with PRRSV at increasing MOIsIL-1120573 mRNA expression and secretion were increased in adose-dependent manner (Figures 2(c) and 2(d))

33 PRRSV-Induced IL-1120573 Production Depends on TLR4-MyD88 Signaling During virus invasion the ldquofirst signalrdquoresults from the activation of PRRs by pathogen associatedmolecular patterns (PAMPs) leading to transcription andtranslation of pro-IL-1120573 [30] TLRs and RIG-I like receptors(RLRs) are the two main types of PRRs involved in theinduction of the innate immune response against RNAviruses [31] To date at least 9 members of the porcine TLRfamily have been identified In order to investigate the roleof TLR and RLR signaling pathways in PRRSV-induced IL-1120573 production specific siRNA targeting RIG-I or each TLRwas designed and synthesized The knockdown efficiencyof siRNA against these receptors was confirmed by real-time PCR (Figure S1A-J) (supplementary materials) NextPAMs were transfected with specific siRNAs or negativecontrol siRNA (psiNegative) followed by PRRSV infectionAs shown in Figures 3(a) and 3(b) knockdown of TLR4significantly decreased PRRSV-induced IL-1120573mRNA expres-sion and secretion while no appreciable change was observedin cells transfectedwith siRNA targeting other tested receptormolecules compared to cells transfected with psiNegativeThese results suggest that TLR4 may contribute to PRRSV-induced IL-1120573 production Because LPS is the agonist ofTLR4 before drawing this conclusion we have to rule out thepossibility that a contaminating LPS in virus stocks may beinvolved in these responses To this end we first measuredthe level of LPS in virus stocks by Limulus assay and no


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Figure 1 Infection kinetics of the highly pathogenic PRRSV strain WUH3 in PAMs (a) PAMs were infected with the adapted PRRSV (3rdpassages) at an MOI of 01 Cells were fixed and permeabilized in cold methanol at different time point (12 24 36 and 48 h) postinfectionImmunofluorescence assays were performed to analyze the replication of PRRSV by detecting the nonstructural protein Nsp2 (greenfluorescence) DAPI (410158406-diamidino-2-phenylindole) was used to stain the nuclei (b) PAMs were infected with the adapted PRRSV at aMOI of 01 Supernatants were collected at different time point (12 24 36 and 48 h) postinfection for plaque assay to determine viral titers

detectable amount of LPSwas observed Chemical polymyxinB (PMB) can bind lipid A domain of LPS and is always usedto inhibit LPS-mediated inflammatory response [32] Thuswe tested the effect of PMB on PRRSV-induced IL-1120573 pro-duction As shown in Figure 4(a) treatment with 20120583gmLof PMB did not significantly affect PRRSV proliferation ThemRNA expression and secretion of IL-1120573 induced by PRRSVinfection were also not inhibited by PMB while the sametreatment significantly abrogated the IL-1120573 production byLPS (Figures 4(b) and 4(c)) Together these results indicatedthat the TLR4 signaling is really involved in PRRSV-inducedIL-1120573 production

It is well known that two adaptor molecules TRIF andMyD88 are recruited by TLR4 to mediate its downstreamsignaling [33] To further identify which adaptor is utilizedby PRRSV to induce IL-1120573 production we also used siRNAto knock down endogenous expression of MyD88 or TRIFas described previously [34] As shown in Figures 3(c) and3(d) compared to the negative control siRNA knockdown ofMyD88 significantly reduced PRRSV-induced IL-1120573 mRNAexpression and secretion in PAMs but no appreciable change

after knockdown of TRIFThese results indicated thatMyD88is the key adaptor downstream of TLR4 responsible forPRRSV-induced IL-1120573 production

34 NF-120581B ERK12 and p38 Mediate PRRSV-Induced IL-1120573Thedownstream signaling of all TLR receptors involves threemajor signaling pathways MAPKs interferon regulatoryfactors (IRFs) andNF-120581B [35] Previous studies revealed thatPRRSV infection inhibited activation of IRFs [36ndash41] whilesignaling of NF-120581B and MAPK was activated [39 42ndash45]Thus we further investigated the role of NF-120581B and MAPKsin PRRSV-induced IL-1120573 production in PAMs To this endPAMs were infected with PRRSV followed by treatment witha specific NF-120581B inhibitor (BAY11-7082) at different doses(1 2 5 10 and 20120583M) As shown in Figures 5(a) and 5(b)cells treated with BAY11-7082 exhibited a decreased ability toupregulate IL-1120573 mRNA expression and protein secretion ina dose-dependent manner after PRRSV infection indicatingthat NF-120581B is required for IL-1120573 production during PRRSVinfection


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Figure 2 PRRSV infection increases IL-1120573 mRNA expression and secretion in PAMs (a b) PAMs were mock infected or infected withPRRSV at a MOI of 01 Cells and supernatants were collected separately at the indicated time points and subjected to real-time RT-PCR(a) and ELISA (b) to analyze the expression of IL-1120573 (c d) PAMs were infected with PRRSV at different doses (001 01 02 and 05MOI)Cells and supernatants were collected separately at 36 h postinfection for analysis by IL-1120573-specific real-time RT-PCR (c) and ELISA (d)Themock-infected cells were used as negative controls lowast119875 lt 005 and lowastlowast119875 lt 001 compared with the mock-infected cells

Previous studies have demonstrated that PRRSV couldactivate p38 MAPK and ERK12 signaling [42 44] andp38 MAPK and ERK12 are downstream signaling of TLR4receptors [35] To examine the role of the MAPK signalingcascade in the regulation of IL-1120573 during PRRSV infectionPAMs were treated with SB202190 U-0126 and SP600125specific inhibitors of MAPK p38 ERK12 and JNK respec-tively at different doses As shown in Figures 6(a) and 6(b)treatment with the JNK inhibitor SP600125 had no effecton IL-1120573 expression in PRRSV-infected PAMs but the IL-1120573 mRNA and protein expression were reduced following adose-dependent increase of the ERK12 inhibitor U-0126 andthe p38 inhibitor SB202190 indicating that the MAPK p38and ERK12 appear to be involved in PRRSV-induced IL-1120573 production To further confirm these results we detectedthe p38 MAPK and ERK12 phosphorylation As shown inFigure 6(c) knockdown of TLR4 and MyD88 could inhibitPRRSV-induced p38 MAPK and ERK12 phosphorylation

These results further confirmed that the ERK12 and p38 areinvolved in PRRSV-induced IL-1120573 production

35 The NLRP3 Inflammasome Is Required for PRRSV-Medi-ated IL-1120573 Maturation and Secretion The second signal forIL-1120573 production is the inflammasome complex assembly andsubsequent caspase-1 activation [3 5] The NLRP3 inflam-masome is the most characterized inflammasome complexand involved in IL-1120573 maturation during infections withseveral viruses [46] To investigate the role of the NLRP3inflammasome in PRRSV-mediated production of IL-1120573specific siRNAs targeting the porcine NLRP3 inflamma-some receptor (NLRP3) and adaptor (ASC) were designed(Table S1) (Supplementary Materials) The reduced expres-sion levels of NLRP3 and ASC in PAMs treated with specificsiRNAs were confirmed by real-time PCR (Figures S1K andL) (supplementary materials) Silencing NLRP3 or ASCin PAMs significantly decreased IL-1120573 secretion following


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Figure 3 PRRSV-induced IL-1120573 production depends on TLR4-MyD88 pathway (a b) PAMs were transfected with 80 nM of psiNegativepsiRIG-I and psiTLR1-9 respectively At 24 h after transfection cells weremock infected or infected with PRRSV at aMOI of 01The cells andsupernatants were harvested separately 36 h later and subjected to porcine IL-1120573-specific real-time RT-PCR (a) and ELISA (b) respectively(c d) PAMs were transfected with 80 nM of psiMyD88 and psiTRIF respectively At 24 h after transfection cells were mock infected orinfected with PRRSV at a MOI of 01 The cells and supernatants were harvested separately 36 h later and analyzed by IL-1120573-specific real-timeRT-PCR (c) and ELISA (d) respectively lowast119875 lt 005 and lowastlowast119875 lt 001 compared with cells transfected with psiNegative followed by PRRSVinfection

PRRSV infection (Figure 7(a)) while no appreciable changewas observed in IL-1120573 mRNA (Figure 7(b)) and pro-IL-1120573protein expression (Figure 7(c)) compared to cells transfectedwith psiNegative

To further investigate whether caspase 1 activation isrequired for IL-1120573 release during PRRSV infection PAMswere infected with PRRSV followed by treatment with aspecific caspase 1 inhibitor (Boc-D-CMK) at different doses(5 10 25 50 and 100 120583M) As shown in Figure 7(d) cellstreated with Boc-D-CMK exhibited a decreased ability toupregulate IL-1120573 production in a dose-dependent mannerafter PRRSV infection Based on these results we concludedthat NLRP3ASCcaspase 1 inflammasome is indispensable

for efficient IL-1120573 secretion but not for its mRNA expressionduring PRRSV infection

36 TLR4MyD88NF-120581B Signaling Pathway Is Involved inPRRSV-Induced Expression of NLRP3 Inflammasome Compo-nents Having deciphered the signaling pathways involvedin PRRSV-induced IL-1120573 production we further evaluatedthe relationship between TLR4MyD88NF-120581B signalingand NLRP3 inflammasome Because no antibody againstporcine NLRP3 ASC and caspase 1 can be available weanalyzed the mRNA expression of these molecules afterinterfering with specific siRNA or inhibiting with specificinhibitors for TLR4MyD88NF-120581B signaling As shown in


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Figure 4 PMB has no effect on PRRSV proliferation or PRRSV-induced IL-1120573 production (a) PAMs were infected with the adapted PRRSVat aMOI of 01 followed by treatment with polymyxin B (0 5 10 and 20120583gmL) in the absence of serum for 36 h Supernatants were collectedat different time point (12 24 36 and 48 h) postinfection for plaque assay to determine viral titers (b c) PAMs were infected with the adaptedPRRSV at a MOI of 01 or treated with LPS (1120583gmL) followed by treatment with polymyxin B (0 5 10 and 20 120583gmL) in the absence ofserum for 36 h The cells and supernatants were then harvested separately and analyzed by real-time RT-PCR and ELISA respectively

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Figure 5 NF-120581B is required for PRRSV-induced IL-1120573 production (a b) PAMs were mock infected or infected with PRSSV at a MOI of01 followed by treatment with NF-120581B inhibitor (1 2 5 10 and 20120583M) or DMSO vehicle in the absence of serum for 36 h The cells andsupernatants were then harvested separately and analyzed by real-time RT-PCR (a) and ELISA (b) respectively lowast119875 lt 005 and lowastlowast119875 lt 001compared with DMSO-treated cells plus PRRSV infection


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Figure 6 MAPK p38 and ERK12 are critical for PRRSV-induced IL-1120573 production in PAMs Experiments were performed similar to thosedescribed in Figure 3 except that ERK12 inhibitor (1 2 and 5 120583M) p38 MAPK inhibitor (01 02 and 05 120583M) or JNK inhibitor (1 2 and5 120583M)was used Cells and supernatants were then harvested and analyzed by IL-1120573-specific real-time RT-PCR (a) and ELISA (b) respectivelylowastlowast119875 lt 001 compared with DMSO plus PRRSV infection

Figures 8(a) and 8(b) knockdown of TLR4 and MyD88by siRNA significantly decreased PRRSV-induced mRNAexpression of NLRP3 ASC and caspase 1 Similar resultscould be observed when PAMs were treated with NF-120581B-specific inhibitor BAY11-7082 after PRRSV infection(Figures 8(c)ndash8(e)) Based on these data we concludedthat TLR4MyD88NF-120581B signaling pathway is involved inPRRSV-induced expression of NLRP3 ASC and caspase 1

4 Discussion

Interstitial pneumonia is a condition characteristic in pigsafter PRRSV infection [18]More recentlyMorgan et al com-pared the pathogenesis of three different European PRRSV

strains and proposed that the increased clinical and patho-logical effect of the highly pathogenic PRRSV strain is morelikely to be caused by an enhanced inflammatory immuneresponse rather than higher levels of virus replication [47]Similarly previous study also demonstrated that the highlypathogenic North American type PRRSV which emergedin China and South East Asia induced stronger proinflam-matory responses than the classical North American typePRRSV [20] These observations highlight the importantrole of inflammatory response in infection and pathogenesisof PRRSV Thus analyses of the underlying mechanismsresponsible for inflammatory responses may contribute to adeeper understanding of the infection and pathogenesis ofPRRSV IL-1120573 is a key proinflammatory cytokine and plays


1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)

siNegative siNLRP3 siASC

MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)

Figure 7 PRRSV-induced IL-1120573 secretion requires NLRP3 inflammasome (andashc) PAMs were transfected with 80 nM of psiNegativepsiNLRP3 or psiASC respectively At 24 h posttransfection cells weremock infected or infectedwith PRRSVat aMOI of 01The supernatantsand cells were harvested separately 36 h later and analyzed by IL-1120573 ELISA (a) and real-time RT-PCR (b) respectively ForWestern blotting todetect pro-IL-1120573 cells were harvested at 24 h postinfection by using polyclonal antibody against pro-IL-1120573 (c) (d) PAMs were mock infectedor infected with PRRSV at a MOI of 01 followed by treatment with caspase 1 inhibitor (5 10 25 50 and 100120583M) or DMSO vehicle in theabsence of serum for 36 hThe supernatants were then harvested and analyzed by IL-1120573-specific ELISA lowast119875 lt 005 and lowastlowast119875 lt 001 comparedwith DMSO-treated cells plus PRRSV infection

a very important role in shaping the inflammatory responseagainst pathogens [2] In this study we investigated themechanisms particularly the cellular sensors and signalingpathways responsible for the transcription and secretion ofIL-1120573 in response to highly pathogenic PRRSV infection inprimary PAMs the target cells of PRRSV infection in vivoOur results clearly showed that PRRSV infection significantlyinduced IL-1120573 production and processing in primary PAMsin a manner that was dependent on TLR4MyD88 signalingand NLRP3 inflammasome activation respectively

The production and release of IL-1120573 are tightly regu-lated at several levels the transcription of the gene andsynthesis of immature pro-IL-1120573 protein the proteolyticprocessingcleavage of pro-IL-1120573 into the mature form of

IL-1120573 and secretion of mature IL-1120573 into the extracellu-lar milieu [2 3] IL-1120573 gene transcription and translationare triggered by most viruses through a variety of PRRstranscription factors and cytoplasmic signals [3 48] Inthis study we for the first time identified TLR4 as the keyreceptor and MyD88 as the key adaptor to mediate PRRSV-induced IL-1120573 transcription TLR4 mainly recognizes LPSderived from the outer membrane of Gram-negative bacte-ria However TLR4 is also involved in the recognition ofviral envelope proteins including those of RSV vesicularstomatitis virus Ebola virus and mouse mammary tumorvirus [49ndash53] Ebola virus glycoprotein interacts with TLR4to induce proinflammatory cytokines [51] RSV F protein isa TLR4 agonist and activates the innate immune response


lowastlowast

siNegative

NLRP3 ASC Caspase 16

5

4

3

2

1

0psiTLR4 siNegative siTLR4 siNegative siTLR4

MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1

siNegative siMyD88 siNegative siNegative

MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)

Figure 8 TLR4MyD88NF-120581B signaling pathway is involved in PRRSV-induced expression of NLRP3 inflammasome components (a b)PAMs were transfected with 80 nM of psiNegative psiTLR4 or psiMyD88 respectively At 24 h posttransfection cells were mock infected orinfected with PRRSV at a MOI of 01 The cells and supernatants were harvested separately 36 h later and analyzed by NLRP3ASCcaspase1 specific real-time RT-PCR (cndashe) PAMs were mock infected or infected with PRSSV at a MOI of 01 followed by treatment with NF-120581Binhibitor (1 2 5 and 10120583M) orDMSO vehicle in the absence of serum for 36 hThe cells were then harvested to analyse themRNA expressionof NLRP3 (c) ASC (d) and caspase 1 (e) respectively by real-time RT-PCR lowast119875 lt 005 and lowastlowast119875 lt 001 compared with DMSO-treated cellsplus PRRSV infection


via TLR4 [52] Furthermore infection with RSV results inincreased expression of TLR4 mRNA protein and increasedTLR4 membrane localization [49] Interestingly PRRSVinfection also induced TLR4 mRNA expression in the brainand respiratory tract of pigs [54] Thus it is possible thatTLR4 senses one or more proteins encoded by PRRSV toinduce an inflammatory response Identifying the PRRSVprotein(s) that binds to TLR4 is of interest and requiresfurther investigation

In this study we also demonstrated that NF-120581B p38and ERK12 were required for PRRSV-induced IL-1120573 tran-scription and secretion These results are expected sinceboth NF-120581B and MAPK are downstream molecules in theTLRMyD88 signaling pathway It has also been reportedthat PRRSV infection could induce the activation of the NF-120581B signaling pathway [43] however the responsible TLRtriggered by PRRSV to activate NF-120581B signaling had notbeen identified Based on our results we speculate thatPRRSV induces the activation of both NF-120581B signalingand IL-1120573 production via TLR4MyD88 signaling AlthoughMAPK has been demonstrated to be involved in virus-induced IL-1120573 production different MAPKs were utilized bydifferent viruses For example herpes simplex virus 1 has beenreported to induce IL-1120573 production through the p38 MAPKsignaling pathway while ERK12 and JNK signaling pathwayswere utilized byHIVTATprotein to activate the transcriptionof IL-1120573 [55 56] Previous studies have demonstrated thatPRRSV infection could activate MAPK p38 ERK12 andJNK in PAMs and Marc-145 cells [42 44 45] Howeveronly ERK12 and p38 MAPK pathways were associated withPRRSV-induced IL-1120573 expression in our present study andTLR4MyD88 signaling plays an important role in ERK12and p38 phosphorylation Based on this result and combinedwith the role of TLR4MyD88NF-120581B in IL-1120573 expression weconclude that the TLR4MyD88 pathway and its downstreamsignaling molecules NF-120581B p38 and ERK12 were requiredfor PRRSV-induced IL-1120573 production

The proteolytic processing of pro-IL-1120573 is mediated bythe inflammasome complex At least nine inflammasomecomplexes have been described to date [57] In addition toNLR inflammasomes (NLRP1 NLRP3 NLRP6 NLRP12 andNLRC4) there are four additional inflammasomes (AIM2RIG-I IFI6 and PYRIN) which form by non-NLR sensorproteins [57] Work over the last years has identified theNLRP3 and RIG-I inflammasomes as key regulators ofRNA virus-induced IL-1120573 production [8 57] The NLRP3inflammasome is well characterized and many viruses suchas adenovirus influenza A virus HIV encephalomyocarditisvirus and RSV are known to promote IL-1120573 productionby activating the NLRP3 inflammasome [13 58ndash60] TheRIG-I inflammasome has dual functions in inducing IL-1120573production [61ndash63] The first function is to sense cytosolicviral infection and activate NF-120581B via MAVS and a com-plex of the adaptor CARD9 and Bcl-10 resulting pro-IL-1120573production the second function is to bind ASC and therebytrigger caspase-1-dependent inflammasome activation andIL-1120573 generation via a NLRP3-independent mechanism [6163] However the dual functions of RIG-I do not appearto play a role in PRRSV-induced IL-1120573 production because

neither pro-IL-1120573mRNA expression nor mature IL-1120573 secre-tion was influenced during PRRSV infection in RIG-I knock-down cells This result is consistent with our previous studyin which we demonstrated that PRRSV infection interferedwith the RIG-I signaling pathway [39] Unlike the RIG-Ithe NLRP3 inflammasome is required for PRRSV-inducedIL-1120573 production However the NLRP3 inflammasome onlycontributed to IL-1120573 secretion but not to its transcriptionduring PRRSV infection It is uncertain whether otherinflammasomes also function in the secretion of IL-1120573 inPRRSV-infected cells thus this possibility cannot be excludedand warrant further study

Until now the precise mechanism that initiates activationof the NLRP3 inflammasome and the subsequent activationof caspase 1 is not fully understood By analyzing the rela-tionship of NLRP3 expression and NLRP3 inflammasomeactivation Bauernfeind et al found that NLRP3 expressionlevel is a limiting factor for NLRP3 inflammasome activationand NLRP3 expression is tightly regulated by TLR-mediatedNF-120581B signal [64] Indeed there exist NF-120581B and AP1binding sites in the NLRP3 promoter region [13 65] Thuscrosstalks between TLRs and NLRP3 inflammasome havebeen proposed to be essential for the fine regulation of virus-induced IL-1120573 production A body of evidence implicatesthat TLRs and NF-120581B play critical role in the primingactivation of NLRP3 inflammasome [13 57 65 66] TLR4agonist significantly upregulatedNLRP3 expression via a NF-120581B dependent manner in murine macrophages [67] NLRP3expression was significantly increased via TLR2MyD88NF-120581B signaling during RSV infection [13] Because the anti-body against porcine NLRP3 is not available we did notinvestigate the NLRP3 protein expression in PRRSV-infectedPAMs However our data showed that PRRSV infectionsignificantly upregulated mRNA expressions of NLRP3 andASC as well as caspase 1 Furthermore knockdown ofTLR4 or MyD88 or inhibition with NF-120581B-specific inhibitoralso decreased expressions of NLRP3 ASC and caspase 1after PRRSV infectionThus TLR4MyD88NF-120581B signalingpathway is involved in PRRSV-induced pro-IL-1120573 andNLRP3expression (signal 1) andNLRP3 inflammasome activation isinvolved in PRRSV-induced IL-1120573 maturation and secretion(signal 2)

5 Conclusion

To conclude we have uncovered the pathways involved in therecognition of PRRSV to the production and release of IL-1120573 PRRSV-induced IL-1120573 production is tightly regulated atthe levels of transcription translation and posttranslationalprocessing The TLR4MyD88 pathway and its downstreamsignaling molecules (NF-120581B p38 and ERK12) play majorroles in PRRSV-induced IL-1120573mRNA expression and pro-IL-1120573 production while the NLRP3 inflammasome is requiredfor the processing of pro-IL-1120573 and pro-IL-1120573 secretionDuring preparing this paper Zhang et al reported thatectopic expression of PRRSV-encoded small envelope proteinE an ion channel-like protein triggers the activation ofinflammasomes [68] Whether or not other PRRSV-encodedproteins are involved in inflammasome activation and


IL-1120573 production and which inflammasome(s) is activated byPRRSV or its encoded protein(s) remain undetermined Dis-section of these issues is important for better understandingof PRRSV-induced inflammation responses

Conflict of Interests

The authors declare that there are no conflicts of interest inthis study

Authorsrsquo Contribution

Jing Bi and Shuang Song made equal contributions to thework

Acknowledgments

This work was supported by the National Basic ResearchProgram (973) of China (2014CB522703) the National Nat-ural Sciences Foundation of China (31225027 31201890 and31172326) the Research Fund for the Doctoral Programof Higher Education of China (20120146110016) and theFundamental Research Funds for the Central Universities(2013PY043)

References

[1] S Bose and A K Banerjee ldquoInnate immune response againstnonsegmented negative strand RNA virusesrdquo Journal of Inter-feron and Cytokine Research vol 23 no 8 pp 401ndash412 2003

[2] C A Dinarello ldquoBiologic basis for Interleukin-1 in diseaserdquoBlood vol 87 no 6 pp 2095ndash2147 1996

[3] C Eder ldquoMechanisms of interleukin-1120573 releaserdquo Immunobiol-ogy vol 214 no 7 pp 543ndash553 2009

[4] G Lopez-Castejon and D Brough ldquoUnderstanding the mecha-nism of IL-1120573 secretionrdquo Cytokine and Growth Factor Reviewsvol 22 no 4 pp 189ndash195 2011

[5] O Takeuchi and S Akira ldquoPattern recognition receptors andinflammationrdquo Cell vol 140 no 6 pp 805ndash820 2010

[6] K Schroder and J Tschopp ldquoThe inflammasomesrdquoCell vol 140no 6 pp 821ndash832 2010

[7] S L Cassel S C Eisenbarth S S Iyer et al ldquoTheNalp3 inflam-masome is essential for the development of silicosisrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 26 pp 9035ndash9040 2008

[8] AMGram J Frenkel andM E Ressing ldquoInflammasomes andviruses cellular defence versus viral offencerdquo Journal of GeneralVirology vol 93 no 10 pp 2063ndash2075 2012

[9] I C Allen M A Scull C B Moore et al ldquoThe NLRP3 inflam-masome mediates in vivo innate immunity to influenza A virusthrough recognition of viral RNArdquo Immunity vol 30 no 4 pp556ndash565 2009

[10] C A Dinarello ldquoInterleukin-1 in the pathogenesis and treat-ment of inflammatory diseasesrdquo Blood vol 117 no 14 pp 3720ndash3732 2011

[11] D Burdette A Haskett L Presser S McRae J Iqbal and GWaris ldquoHepatitis C virus activates interleukin-1120573 via caspase-1-inflammasome complexrdquo Journal of General Virology vol 93no 2 pp 235ndash246 2012

[12] V A K Rathinam Z Jiang S N Waggoner et al ldquoThe AIM2inflammasome is essential for host defense against cytosolicbacteria and DNA virusesrdquo Nature Immunology vol 11 no 5pp 395ndash402 2010

[13] J Segovia A Sabbah V Mgbemena et al ldquoTLR2MyD88NF-120581B pathway reactive oxygen species potassium efflux activatesNLRP3ASC inflammasome during respiratory syncytial virusinfectionrdquo PLoS ONE vol 7 no 1 Article ID e29695 2012

[14] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[15] X J Meng ldquoEmerging and re-emerging swine virusesrdquo Trans-boundary and Emerging Diseases vol 59 no 1 pp 85ndash102 2012

[16] S Dea C A Gagnon H Mardassi B Pirzadeh and D RoganldquoCurrent knowledge on the structural proteins of porcine repro-ductive and respiratory syndrome (PRRS) virus comparisonof the North American and European isolatesrdquo Archives ofVirology vol 145 no 4 pp 659ndash688 2000

[17] E J Neumann J B Kliebenstein C D Johnson et alldquoAssessment of the economic impact of porcine reproductiveand respiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[18] KD Rossow ldquoPorcine reproductive and respiratory syndromerdquoVeterinary Pathology vol 35 no 1 pp 1ndash20 1998

[19] RThanawongnuwech BThacker P Halbur and E LThackerldquoIncreased production of proinflammatory cytokines followinginfection with porcine reproductive and respiratory syndromevirus andMycoplasma hyopneumoniaerdquo Clinical and DiagnosticLaboratory Immunology vol 11 no 5 pp 901ndash908 2004

[20] S Qiao L Feng D Bao et al ldquoPorcine reproductive and res-piratory syndrome virus and bacterial endotoxin act in synergyto amplify the inflammatory response of infectedmacrophagesrdquoVeterinary Microbiology vol 149 no 1-2 pp 213ndash220 2011

[21] J K Lunney E R Fritz J M Reecy et al ldquoInterleukin-8interleukin-1120573 and interferon-120574 levels are linked to PRRS virusclearancerdquo Viral Immunology vol 23 no 2 pp 127ndash134 2010

[22] B Li L Fang Z Xu et al ldquoRecombination in vaccine andcirculating strains of porcine reproductive and respiratorysyndrome virusesrdquo Emerging Infectious Diseases vol 15 no 12pp 2032ndash2035 2009

[23] M F de Abin G Spronk M Wagner M Fitzsimmons J EAbrahante and M P Murtaugh ldquoComparative infection effi-ciency of Porcine reproductive and respiratory syndrome virusfield isolates onMA104 cells and porcine alveolarmacrophagesrdquoCanadian Journal of Veterinary Research vol 73 no 3 pp 200ndash204 2009

[24] DWang L Cao Z Xu et al ldquoMiR-125b reduces porcine repro-ductive and respiratory syndrome virus replication by nega-tively regulating the NF-120581B Pathwayrdquo PLoS ONE vol 8 no 2Article ID e55838 2013

[25] Y Fang R R R RowlandM Roof J K Lunney J Christopher-Hennings and E A Nelson ldquoA full-length cDNA infectiousclone of North American type 1 porcine reproductive andrespiratory syndrome virus expression of green fluorescentprotein in the Nsp2 regionrdquo Journal of Virology vol 80 no 23pp 11447ndash11455 2006

[26] X Xu C H Woo R R Steere et al ldquoEVI1 acts as an induciblenegative-feedback regulator of NF-KappaB by inhibiting p65acetylationrdquo Journal of Immunology vol 188 no 12 pp 6371ndash6380 2012


[27] Y LiuW Shi E Zhou et al ldquoDynamic changes in inflammatorycytokines in pigs infected with highly pathogenic porcinereproductive and respiratory syndrome virusrdquo Clinical andVaccine Immunology vol 17 no 9 pp 1439ndash1445 2010

[28] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 article e526 2007

[29] Y Feng T Zhao T Nguyen et al ldquoPorcine respiratory andreproductive syndrome virus variants Vietnam and China2007rdquo Emerging Infectious Diseases vol 14 no 11 pp 1774ndash17762008

[30] M G Netea C A Nold-Petry M F Nold et al ldquoDifferentialrequirement for the activation of the inflammasome for pro-cessing and release of IL-1120573 in monocytes and macrophagesrdquoBlood vol 113 no 10 pp 2324ndash2335 2009

[31] S Jensen and A R Thomsen ldquoSensing of rna viruses a reviewof innate immune receptors involved in recognizing RNA virusinvasionrdquo Journal of Virology vol 86 no 6 pp 2900ndash2910 2012

[32] K Inden J Kaneko A Miyazato et al ldquoToll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidinof Staphylococcus aureusrdquo Microbes and Infection vol 11 no 2pp 245ndash253 2009

[33] T Kawai and S Akira ldquoTLR signalingrdquo Cell Death and Differ-entiation vol 13 no 5 pp 816ndash825 2006

[34] S Song J Bi D Wang et al ldquoPorcine reproductive and res-piratory syndrome virus infection activates IL-10 productionthrough NF-KappaB and p38 MAPK pathways in porcine alve-olarmacrophagesrdquoDevelopmental ampComparative Immunologyvol 39 no 3 pp 265ndash272 2013

[35] H Kumar T Kawai and S Akira ldquoToll-like receptors andinnate immunityrdquo Biochemical and Biophysical Research Com-munications vol 388 no 4 pp 621ndash625 2009

[36] L K Beura S N Sarkar B Kwon et al ldquoPorcine reproduc-tive and respiratory syndrome virus nonstructural protein 1120573modulates host innate immune response by antagonizing IRF3activationrdquo Journal of Virology vol 84 no 3 pp 1574ndash15842010

[37] G Calzada-Nova W M Schnitzlein R J Husmann and F AZuckermann ldquoNorth American porcine reproductive and res-piratory syndrome viruses inhibit type I interferon productionby plasmacytoid dendritic cellsrdquo Journal of Virology vol 85 no6 pp 2703ndash2713 2011

[38] Z Chen S Lawson Z Sun et al ldquoIdentification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcinereproductive and respiratory syndrome virus infected cells nsp1function as interferon antagonistrdquo Virology vol 398 no 1 pp87ndash97 2010

[39] R Luo S Xiao Y Jiang et al ldquoPorcine reproductive andrespiratory syndrome virus (PRRSV) suppresses interferon-120573production by interfering with the RIG-I signaling pathwayrdquoMolecular Immunology vol 45 no 10 pp 2839ndash2846 2008

[40] D Patel Y Nan M Shen K Ritthipichai X Zhu and Y-JZhang ldquoPorcine reproductive and respiratory syndrome virusinhibits type I interferon signaling by blocking STAT1STAT2nuclear translocationrdquo Journal of Virology vol 84 no 21 pp11045ndash11055 2010

[41] D Yoo C Song Y Sun Y Du O Kim and H-C Liu ldquoMod-ulation of host cell responses and evasion strategies for porcinereproductive and respiratory syndrome virusrdquo Virus Researchvol 154 no 1-2 pp 48ndash60 2010

[42] J Hou L Wang R Quan et al ldquoInduction of interleukin-10 isdependent on p38 mitogen-activated protein kinase pathway inmacrophages infected with porcine reproductive and respira-tory syndrome virusrdquo Virology Journal vol 9 article 165 2012

[43] S-M Lee and S B Kleiboeker ldquoPorcine arterivirus activates theNF-120581B pathway through I120581B degradationrdquo Virology vol 342no 1 pp 47ndash59 2005

[44] Y J Lee and C Lee ldquoStress-activated protein kinases areinvolved in porcine reproductive and respiratory syndromevirus infection and modulate virus-induced cytokine produc-tionrdquo Virology vol 427 no 2 pp 80ndash89 2012

[45] S Yin Y Huo Y Dong et al ldquoActivation of c-Jun NH(2)-terminal kinase is required for porcine reproductive and res-piratory syndrome virus-induced apoptosis but not for virusreplicationrdquo Virus Research vol 166 no 1-2 pp 103ndash108 2012

[46] C S Yang D M Shin and E K Jo ldquoThe role of NLR-relatedprotein 3 inflammasome in host defense and inflammatorydiseasesrdquo International Neurourology Journal vol 16 no 1 pp2ndash12 2012

[47] S B Morgan S P Graham F J Salguero et al ldquoIncreasedpathogenicity of European porcine reproductive and respi-ratory syndrome virus is associated with enhanced adaptiveresponses and viral clearancerdquoVeterinaryMicrobiology vol 163no 1-2 pp 13ndash22 2013

[48] L R Watkins M K Hansen K T Nguyen J E Lee and S FMaier ldquoDynamic regulation of the proinflammatory cytokineinterleukin-1120573 molecular biology for non-molecular biolo-gistsrdquo Life Sciences vol 65 no 5 pp 449ndash481 1999

[49] MMMonick T O Yarovinsky L S Powers et al ldquoRespiratorysyncytial virus up-regulates TLR4 and sensitizes airway epithe-lial cells to endotoxinrdquo Journal of Biological Chemistry vol 278no 52 pp 53035ndash53044 2003

[50] P Georgel Z Jiang S Kunz et al ldquoVesicular stomatitis virusglycoprotein G activates a specific antiviral Toll-like receptor 4-dependent pathwayrdquoVirology vol 362 no 2 pp 304ndash313 2007

[51] O Atsushi P M Pitha A Yoshimura and R N HartyldquoInteraction between Ebola virus glycoprotein and host Toll-like receptor 4 leads to induction of proinflammatory cytokinesand SOCS1rdquo Journal of Virology vol 84 no 1 pp 27ndash33 2010

[52] P Rallabhandi R L Phillips M S Boukhvalova et al ldquoRes-piratory syncytial virus fusion protein-induced Toll-like recep-tor 4 (TLR4) signaling is inhibited by the TLR4 antago-nists rhodobacter sphaeroides lipopolysaccharide and eritoran(E5564) and requires direct interaction with MD-2rdquoMbio vol3 no 4 Article ID e00218-12 2012

[53] J C Rassa J LMeyers Y Zhang R Kudaravalli and S R RossldquoMurine retroviruses activate B cells via interaction with toll-like receptor 4rdquo Proceedings of the National Academy of Sciencesof the United States of America vol 99 no 4 pp 2281ndash22862002

[54] J C Miguel J Chen W G van Alstine and R W JohnsonldquoExpression of inflammatory cytokines and Toll-like recep-tors in the brain and respiratory tract of pigs infected withporcine reproductive and respiratory syndrome virusrdquo Veteri-nary Immunology and Immunopathology vol 135 no 3-4 pp314ndash319 2010

[55] S HuW S Sheng S J Schachtele and J R Lokensgard ldquoReac-tive oxygen species drive herpes simplex virus (HSV)-1-inducedproinflammatory cytokine production by murine microgliardquoJournal of Neuroinflammation vol 8 article 123 2011

[56] M K Mamik S Banerjee T F Walseth et al ldquoHIV-1 and IL-1120573 regulate astrocytic CD38 through mitogen-activated protein


kinases and nuclear factor-120581B signaling mechanismsrdquo Journalof Neuroinflammation vol 8 article 145 2011

[57] M Lamkanfi and V M Dixit ldquoModulation of inflammasomepathways by bacterial and viral pathogensrdquo Journal of Immunol-ogy vol 187 no 2 pp 596ndash602 2011

[58] A U Barlan T M Griffin K A Mcguire and C M WiethoffldquoAdenovirus membrane penetration activates the NLRP3inflammasomerdquo Journal of Virology vol 85 no 1 pp 146ndash1552011

[59] J V Rajan D Rodriguez E A Miao and A Aderem ldquoTheNLRP3 inflammasome detects encephalomyocarditis virus andvesicular stomatitis virus infectionrdquo Journal of Virology vol 85no 9 pp 4167ndash4172 2011

[60] A Pontillo L T Silva T M Oshiro C Finazzo S Crovella andA J S Duarte ldquoHIV-1 induces NALP3-inflammasome expres-sion and interleukin-1120573 secretion in dendritic cells from healthyindividuals but not from HIV-positive patientsrdquo AIDS vol 26no 1 pp 11ndash18 2012

[61] H Poeck M Bscheider O Gross et al ldquoRecognition of RNAvirus by RIG-I results in activation of CARD9 and inflamma-some signaling for interleukin 1Β productionrdquoNature Immunol-ogy vol 11 no 1 pp 63ndash69 2010

[62] H Poeck and J Ruland ldquoFrom virus to inflammation mecha-nisms of RIG-I-induced IL-1120573 productionrdquo European Journal ofCell Biology vol 91 no 1 pp 59ndash64 2012

[63] F L van de Veerdonk M G Netea C A Dinarello and LA B Joosten ldquoInflammasome activation and IL-1120573 and IL-18processing during infectionrdquo Trends in Immunology vol 32 no3 pp 110ndash116 2011

[64] F G Bauernfeind G Horvath A Stutz et al ldquoCutting edgeNF-120581B activating pattern recognition and cytokine receptorslicense NLRP3 inflammasome activation by regulating NLRP3expressionrdquo Journal of Immunology vol 183 no 2 pp 787ndash7912009

[65] M M Rahman and G McFadden ldquoMyxoma virus lacking thepyrin-like protein M013 is sensed in human myeloid cells byboth NLRP3 and multiple toll-like receptors which indepen-dently activate the inflammasome and NF-ΚB innate responsepathwaysrdquo Journal of Virology vol 85 no 23 pp 12505ndash125172011

[66] Y He L Franchi and G Nunez ldquoTLR agonists stimulateNLRP3-dependent IL-1beta production independently of thepurinergic P2X7 receptor in dendritic cells and in vivordquo Journalof Immunology vol 190 no 1 pp 334ndash339 2013

[67] Y Qiao P Wang J Qi L Zhang and C Gao ldquoTLR-inducedNF-kappaB activation regulates NLRP3 expression in murinemacrophagesrdquo FEBS Letters vol 586 no 7 pp 1022ndash1026 2012

[68] K Zhang Q Hou Z Zhong et al ldquoPorcine reproductive andrespiratory syndrome virus activates inflammasomes of porcinealveolarmacrophages via its small envelope protein ErdquoVirologyvol 442 no 2 pp 156ndash162 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


apoptosis-associated speck-like protein containing a CARD(ASC) and (iii) caspase 1 [6] Several inflammasomes havebeen described of which the NLRP3 inflammasome is thebest characterized one [7ndash9] NLRP3 contains a C-terminalleucine-rich repeat domain a central nucleotide bindingdomain and an N-terminal PYD The PYD domain ofNLRP3 recruits ASC via a homotypic interaction with thePYD domain Oligomerization of NLRP3ASC leads to therecruitment of procaspase 1 to generate enzymatically activecaspase 1 which in turn processes IL-1120573 [9]

A large number of viruses have been reported to inducethe production of IL-1120573 in infected cells [10] However differ-ent viruses induce the expression of pro-IL-1120573 and process itto its mature form through different signaling pathways andsignaling molecules For example hepatitis C virus inducesIL-1120573 transcription through the TLR7-mediated MyD88 sig-naling pathway and activates the NLRP3 inflammasome topromote IL-1120573 secretion [11] Vaccinia virus and murinecytomegalovirus infection stimulate the formation of theabsent in melanoma 2 (AIM2) inflammasome to activatecaspase 1 the maturation of IL-1120573 [12] Respiratory syncytialvirus (RSV) is mainly recognized by TLR2 and TLR4 toinduce IL-1120573 transcription through the NF-120581B signalingpathway and then the NLRP3 inflammasome processes pro-IL-1120573 to its active form [13]

Porcine reproductive and respiratory syndrome (PRRS)is an economically important swine disease characterized bysevere reproductive failure in sows and respiratory distressin piglets and growing pigs [14 15] The causative agentPRRSV is a single-stranded positive-sense RNA virus classi-fied within the family Arteriviridae [16] Since its emergencein the late 1980s PRRS has been a threat to the globalswine industry causing high economic losses [17] PRRSVinfection in swine can cause severe interstitial pneumonia[18] suggesting that the inflammatory response plays animportant role in infection and pathogenesis of PRRSVIndeed Thanawongnuwech et al reported that the expres-sions of IL-1120573 IL-8 and TNF-120572 were significantly elevated inPRRSV-infected pigs [19] while Qiao et al found that IL-1120573and TNF-120572 were significantly upregulated in PAMs infectedwith the highly pathogenic PRRSV strain HN07-1 comparedto the classical PRRSV strain BJ-4 [20] Furthermore Lunneyet al found that serums IL-1120573 and IL-8 levels significantlycorrelated with persistent infection of PRRSV [21] Thesefindings indicated that PRRSV infection significantly inducesIL-1120573 expression and IL-1120573 is closely related to PRRSVpathogenesis

Although it is clear that PRRSV infection results in IL-1120573 production in vivo and in vitro the mechanism of IL-1120573processing has not been elucidated yet In this study we ana-lyzed the underlyingmechanisms utilized by porcine alveolarmacrophages (PAMs) the target cells of PRRSV infectionin vivo to detect PRRSV and induce IL-1120573 expressionOur results revealed that PRRSV-activated IL-1120573 productiondepended on the TLR4MyD88 pathway and downstreamsignaling molecules NF-120581B ERK12 and p38 MAPKs More-over we found that theNLRP3ASCcaspase 1 inflammasomeis required for IL-1120573 secretion but not for IL-1120573 transcriptionduring PRRSV infection

2 Materials and Methods

21 Virus and Cells PRRSV strain WUH3 [22] originallyisolated from the brains of pigs with ldquohigh fever syndromerdquoin China in 2006 and identified as a highly pathogenic type2 (North American) PRRSV was used in this study PrimaryPAMs were obtained by postmortem lung lavage of 4-week-old pigs from a PRRSV-negative herd as described previously[23] The animals were housed and handled under con-ditions approved by the Huazhong Agricultural UniversityAnimal Ethics Committee (approval number HZAU-AEC-30102922) The isolated PAMs were cultured in RPMI-1640medium (Life Technology NY USA) Marc-145 cells a mon-key kidney cell line highly permissive for PRRSV infectionwere cultured in DMEM with 10 heated-inactivated FBS025 120583gmL Fungizone 100UmL penicillin 10 120583gmL strep-tomycin sulfate and 5120583gmL gentamicin in a humidified37∘C 5 CO

2incubator PRRSV was amplified in PAMs and

viral titers were determined by titration on Marc-145 cells

22 Reagents and Antibodies LPS polymyxin B (PMB)MAPK inhibitors (SP600125 SB202190 and U0126) NF-120581B inhibitor (BAY11-7082) and caspase 1 inhibitor (Boc-D-CMK) were purchased from Sigma-Aldrich (St Louis MOUSA) SP600125 SB202190 U0126 BAY11-7082 and Boc-D-CMK were dissolved in DMSO and APDC were dissolvedin water prior to use Antibodies specific for p38 ERK12and their phosphorylated forms (p-p38 and p-ERK12) werepurchased from Cell Signaling Technology (Beverly MAUSA) Antibody against porcine IL-1120573 was obtained fromRampD (Minneapolis MN USA) The anti-120573-actin antibodyHRP-conjugated goat anti-mouse IgG goat anti-rabbit IgGand donkey anti-goat IgG were purchased from Sigma-Aldrich (St LouisMO USA) AmAbA2F1 used for detectionof PRRSV nonstructural protein 2 (Nsp2) was produced fromhybridoma cells derived fromSp20myeloma cells and spleencells of BALBc mice immunized with recombinant Nsp2protein of PRRSV strain WUH3 [24]

23 Viral Plaque Assay Plaque assay was essentially per-formed as described previously [25] Briefly 95 confluentMarc-145 cells grown in six-well tissue culture plates wereinfected for 1 h with 10-fold serial dilutions (1000 120583L) ofPRRSV-containing samples After three washes with PBS(pH 74) cells were overlaid with 18 (wv) Bacto agarmixed 1 1 with 2timesDMEM containing 005mgmL neutralred Plaques were counted 4 days postinfection The averageplaque number and standard deviations were calculated fromthree independent experiments Virus titers were expressedas PFUmL

24 Immunofluorescence Assay (IFA) The infected PAMswere fixed permeabilized and subsequently inoculated withthe mAb A2F1 against PRRSV Nsp2 followed by immunos-taining with a FITC-anti-mouse IgG (Sigma St Louis MO)Cellular nuclei were counterstained with 1 120583gmL of DAPI for5min After washing with PBS cells were examined under anLSM510metaconfocal laser scanningmicroscope (Carl ZeissGottingen Germany)







3 Results






NSP2 DAPI Merge12

h24

h36

h48

h

(a)

7

6

5

4

3

2

1

012h 24h 36h 48h

log10

of v

irus t

iter (

PFU

mL)

(b)







6h 12h 24h 36h 48h

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV


lowastlowast

lowastlowast

lowastlowast

30

25

20

15

10

5

0

(a)

2500

2000

1500

1000

500

06h 12h 24h 36h 48h

MockPRRSV

IL-1120573

(pg

mL)

lowastlowast

lowastlowast


(b)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A 300

250

200

150

100

50

0

PRRSV infection


lowastlowast


lowastlowast

(c)

1200

1000

800

400

200

0

600

Mock

PRRSV infection


IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowast

(d)






Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV

lowastlowast

50

45

40

35

30

25

20

15

10

5

0

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

(a)

lowastlowast

2000

1800

1600

1400

1200

1000

800

600

400

200

0

MockPRRSV

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

IL-1120573

(pg

mL)

(b)

MockPRRSV

lowastlowast

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

50

45

40

35

30

25

20

15

10

5


(c)

lowastlowast

MockPRRSV


1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

(d)







7

6

5

5

1020

4

3

2

1

0

0

12h 24h 36h 48hlog10

of v

irus t

iter (

PFU

mL)


(a)

60

50

40

30

20

10

05 20100

MockPRRSVLPS


Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(b)

5 20100

MockPRRSVLPS

1600

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)


(c)


140

120

100

80

60

40

0

20


lowast

DMSO DMSO

PRRSV infection

1 2 5 10 20

BAY11-7082 (120583M)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(a)

DMSO DMSO

PRRSV infection


1200

1000

800

400

200

01 2 5 10 20

600

IL-1120573

(pg

mL)

BAY11-7082 (120583M)

(b)



PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results






NSP2 DAPI Merge12

h24

h36

h48

h

(a)

7

6

5

4

3

2

1

012h 24h 36h 48h

log10

of v

irus t

iter (

PFU

mL)

(b)







6h 12h 24h 36h 48h

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV


lowastlowast

lowastlowast

lowastlowast

30

25

20

15

10

5

0

(a)

2500

2000

1500

1000

500

06h 12h 24h 36h 48h

MockPRRSV

IL-1120573

(pg

mL)

lowastlowast

lowastlowast


(b)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A 300

250

200

150

100

50

0

PRRSV infection


lowastlowast


lowastlowast

(c)

1200

1000

800

400

200

0

600

Mock

PRRSV infection


IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowast

(d)






Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV

lowastlowast

50

45

40

35

30

25

20

15

10

5

0

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

(a)

lowastlowast

2000

1800

1600

1400

1200

1000

800

600

400

200

0

MockPRRSV

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

IL-1120573

(pg

mL)

(b)

MockPRRSV

lowastlowast

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

50

45

40

35

30

25

20

15

10

5


(c)

lowastlowast

MockPRRSV


1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

(d)







7

6

5

5

1020

4

3

2

1

0

0

12h 24h 36h 48hlog10

of v

irus t

iter (

PFU

mL)


(a)

60

50

40

30

20

10

05 20100

MockPRRSVLPS


Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(b)

5 20100

MockPRRSVLPS

1600

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)


(c)


140

120

100

80

60

40

0

20


lowast

DMSO DMSO

PRRSV infection

1 2 5 10 20

BAY11-7082 (120583M)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(a)

DMSO DMSO

PRRSV infection


1200

1000

800

400

200

01 2 5 10 20

600

IL-1120573

(pg

mL)

BAY11-7082 (120583M)

(b)



PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NSP2 DAPI Merge12

h24

h36

h48

h

(a)

7

6

5

4

3

2

1

012h 24h 36h 48h

log10

of v

irus t

iter (

PFU

mL)

(b)







6h 12h 24h 36h 48h

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV


lowastlowast

lowastlowast

lowastlowast

30

25

20

15

10

5

0

(a)

2500

2000

1500

1000

500

06h 12h 24h 36h 48h

MockPRRSV

IL-1120573

(pg

mL)

lowastlowast

lowastlowast


(b)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A 300

250

200

150

100

50

0

PRRSV infection


lowastlowast


lowastlowast

(c)

1200

1000

800

400

200

0

600

Mock

PRRSV infection


IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowast

(d)






Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV

lowastlowast

50

45

40

35

30

25

20

15

10

5

0

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

(a)

lowastlowast

2000

1800

1600

1400

1200

1000

800

600

400

200

0

MockPRRSV

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

IL-1120573

(pg

mL)

(b)

MockPRRSV

lowastlowast

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

50

45

40

35

30

25

20

15

10

5


(c)

lowastlowast

MockPRRSV


1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

(d)







7

6

5

5

1020

4

3

2

1

0

0

12h 24h 36h 48hlog10

of v

irus t

iter (

PFU

mL)


(a)

60

50

40

30

20

10

05 20100

MockPRRSVLPS


Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(b)

5 20100

MockPRRSVLPS

1600

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)


(c)


140

120

100

80

60

40

0

20


lowast

DMSO DMSO

PRRSV infection

1 2 5 10 20

BAY11-7082 (120583M)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(a)

DMSO DMSO

PRRSV infection


1200

1000

800

400

200

01 2 5 10 20

600

IL-1120573

(pg

mL)

BAY11-7082 (120583M)

(b)



PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















6h 12h 24h 36h 48h

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV


lowastlowast

lowastlowast

lowastlowast

30

25

20

15

10

5

0

(a)

2500

2000

1500

1000

500

06h 12h 24h 36h 48h

MockPRRSV

IL-1120573

(pg

mL)

lowastlowast

lowastlowast


(b)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A 300

250

200

150

100

50

0

PRRSV infection


lowastlowast


lowastlowast

(c)

1200

1000

800

400

200

0

600

Mock

PRRSV infection


IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowast

(d)






Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV

lowastlowast

50

45

40

35

30

25

20

15

10

5

0

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

(a)

lowastlowast

2000

1800

1600

1400

1200

1000

800

600

400

200

0

MockPRRSV

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

IL-1120573

(pg

mL)

(b)

MockPRRSV

lowastlowast

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

50

45

40

35

30

25

20

15

10

5


(c)

lowastlowast

MockPRRSV


1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

(d)







7

6

5

5

1020

4

3

2

1

0

0

12h 24h 36h 48hlog10

of v

irus t

iter (

PFU

mL)


(a)

60

50

40

30

20

10

05 20100

MockPRRSVLPS


Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(b)

5 20100

MockPRRSVLPS

1600

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)


(c)


140

120

100

80

60

40

0

20


lowast

DMSO DMSO

PRRSV infection

1 2 5 10 20

BAY11-7082 (120583M)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(a)

DMSO DMSO

PRRSV infection


1200

1000

800

400

200

01 2 5 10 20

600

IL-1120573

(pg

mL)

BAY11-7082 (120583M)

(b)



PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

MockPRRSV

lowastlowast

50

45

40

35

30

25

20

15

10

5

0

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

(a)

lowastlowast

2000

1800

1600

1400

1200

1000

800

600

400

200

0

MockPRRSV

siNeg

ativ

e

siTLR

1

siTLR

2

siTLR

3

siTLR

4

siTLR

5

siTLR

6

siTLR

7

siTLR

8

siTLR

9

siRIG

-I

IL-1120573

(pg

mL)

(b)

MockPRRSV

lowastlowast

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

50

45

40

35

30

25

20

15

10

5


(c)

lowastlowast

MockPRRSV


1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

(d)







7

6

5

5

1020

4

3

2

1

0

0

12h 24h 36h 48hlog10

of v

irus t

iter (

PFU

mL)


(a)

60

50

40

30

20

10

05 20100

MockPRRSVLPS


Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(b)

5 20100

MockPRRSVLPS

1600

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)


(c)


140

120

100

80

60

40

0

20


lowast

DMSO DMSO

PRRSV infection

1 2 5 10 20

BAY11-7082 (120583M)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(a)

DMSO DMSO

PRRSV infection


1200

1000

800

400

200

01 2 5 10 20

600

IL-1120573

(pg

mL)

BAY11-7082 (120583M)

(b)



PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















7

6

5

5

1020

4

3

2

1

0

0

12h 24h 36h 48hlog10

of v

irus t

iter (

PFU

mL)


(a)

60

50

40

30

20

10

05 20100

MockPRRSVLPS


Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(b)

5 20100

MockPRRSVLPS

1600

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)


(c)


140

120

100

80

60

40

0

20


lowast

DMSO DMSO

PRRSV infection

1 2 5 10 20

BAY11-7082 (120583M)

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

(a)

DMSO DMSO

PRRSV infection


1200

1000

800

400

200

01 2 5 10 20

600

IL-1120573

(pg

mL)

BAY11-7082 (120583M)

(b)



PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PRRSV infection

ERK12 p38 JNK1 2 5

01 02 051 2 5

60

50

40

30

20

10

0

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A




U-0126SB202190SP600125

Inhibitor (120583M)

(a)

PRRSV infection

lowastlowast

lowastlowast

lowastlowast

lowastlowast

1400

1200

1000

800

600

400

200

0

IL-1120573

(pg

mL)

ERK12 p38 JNK1 2 5

01 02 051 2 5

U-0126SB202190SP600125

Inhibitor (120583M)

(b)

siNegative

PRRSV

siTLR4siMyD88

p38

p-p38

ERK12

p-ERK12

120573-Actin

minus minus


minus minus

minus

+ ++

++++

(c)



4 Discussion




1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1200

1000

800

400

200

0

600

IL-1120573

(pg

mL)


MockPRRSV


(a)


MockPRRSV

Relat

ive e

xpre

ssio

n of

IL-1120573

mRN

A

45

40

35

30

25

20

15

10

5

0

(b)

siNegative

PRRSV

siNLRP3siASC

minus minus


minus minus

minus

+ ++

++++

120573-Actin

Pro-IL-1120573

(c)

2000

1600

1200

800

400

0

IL-1120573

(pg

mL)

lowastlowast

lowastlowast

lowastlowast

lowast

PRRSV infection

DMSO DMSO 5 10 25 50 100

Boc-D-CMK (120583M)

(d)






lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

siNegative


5

4

3

2

1


MockPRRSV

Relat

ive e

xpre

ssio

n of

mRN

A

lowastlowast

lowastlowast

(a)

NLRP3 ASC Caspase 1


MockPRRSV

7

6

5

4

3

2

1

0


IL-1120573

(pg

mL)

siMyD88 siMyD88

(b)

PRRSV infection12

10

8

6

4

2

0Relat

ive e

xpre

ssio

n of

NLR

P3 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(c)

PRRSV infection

6

7

8

5

4

3

2

1

0Relat

ive e

xpre

ssio

n of

ASC

mRN

A

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowast

(d)

PRRSV infection

6789

10

543210

Relat

ive e

xpre

ssio

n of

Cas

pase

1 m

RNA

DMSO DMSO 1 2 5 10

BAY11-7082 (120583M)

lowastlowastlowast

(e)








5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusion








Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Porcine Reproductive and Respiratory ...downloads.hindawi.com/journals/mi/2014/403515.pdf · translation of pro-IL- [ ]. TLRs and RIG-I like receptors (RLRs) are