transcriptional profiling of leucocyte count variation...

Research ArticleTranscriptional Profiling of Leucocyte Count Variation fromPorcine Peripheral Blood Reveals Differential Gene Expression

Adeyinka Abiola Adetula 12 Xiangdong Liu12 Thuy Nhien Tran Thi12

Ali Akbar Bhuiyan12 Xiaoyong Du123 Mengjin Zhu12 Xinyun Li 12 Shuhong Zhao12

YunlongMa 12 and HaiyanWang 123

1Key Lab of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education Huazhong Agricultural UniversityWuhan 430070 China2e Cooperative Innovation Center for Sustainable Pig Production Huazhong Agricultural University Wuhan 430070 China3e College of Informatics Huazhong Agricultural University Wuhan 430070 China

Correspondence should be addressed to Yunlong Ma YunlongMamailhzaueducnand Haiyan Wang wanghaiyanmailhzaueducn

Received 6 February 2018 Revised 8 September 2018 Accepted 15 October 2018 Published 18 November 2018

Academic Editor Jerome Moreaux

Copyright copy 2018 AdeyinkaAbiolaAdetula et alThis is an open access article distributed under theCreative CommonsAttributionLicensewhichpermits unrestricteduse distribution and reproduction in anymedium provided the original work is properly cited

Leucocytes have tremendous health-check importance related to the individual antiviral capacity of pigs and other mammalsHowever the molecular mechanism of the immune response of blood leucocytes in pigs is not completely known This studyinvestigated the leucocyte-count variation before and after poly IC stimulation in a DurocndashErhualian F2 population Pigs withincreased and decreased differences in leucocyte counts were coded as increased responder (IR) and decreased responder (DR)respectively Then we used microarray technology to compare the gene-expression profiles of both groups of pigs Transcriptomicanalysis identified 129 differentially expressed genes (DEGs) in IR pigs and 136DEGs inDR pigs Forty-one commonDEGs showedthat both groups had similar expression patterns of immune responses These results illustrated a differential expression in bothgroups Furthermore qPCR experiment was performed to verify the differential-expression profile Functional annotation of theDEGs indicated that both IR and DR pigs were similar in several biological processes including innate immune response and alsoexhibited distinct differences in biological processes molecular function and pathways These results provided insights into themechanism underlying the antiviral capacity of pigs Trial registration number is CAS Registry Number 24939-03-5

1 Introduction

Leucocytes are nucleated blood cells whose rapid translationof mRNA is regulated by signaling events transduced tothe cell surface antigen so as to interact with proteinsother cells and extracellular matrices Leucocytes are criticalin normal pathophysiological processes and acute phaseconditions such as physiologic and metabolic changes thatoccur in response to generalized acute infections traumasevere inflammatory processes tissue injury and autoim-mune diseases [1] Leucocytes generated from multipotentself-renewing progenitor cells develop from mesodermalhemangioblast cells [2] Immunologists have recognizedleucocyte count and leucocyte differential count as keydiagnostic measurements because of their sharp increase

during acute infections [3] Therefore a complete blood celltest often includes a measurement of the level of leucocytes orwhite blood cells Peripheral blood contains polymorphonu-clear leucocytes that penetrate blood vessel endothelium toenter the area of inflammation where they are essential indestroying the action and increasing the power of resistanceagainst any infection repair and regeneration of tissues [2 4]Alteration in several immune functions assessed in vitro hasbeen monitored with blood leucocytes obtained from pigand human as key diagnostic measurements during acuteinfections [3 5ndash7] The characterization of leucocyte surfaceantigens by monoclonal antibodies and other molecularstudies has determined the cell lineages and blood leucocytesubsets implicated in the immune response [8] Increasinglyinvestigators have explored the possibility of characterizing

HindawiBioMed Research InternationalVolume 2018 Article ID 1496536 11 pageshttpsdoiorg10115520181496536

2 BioMed Research International

the mechanism of diseases by using the subpopulations ofleucocytes in vivo thereby enabling enhanced investigationsof the immune response to various porcine infections suchas Actinobacillus pleuropneumoniae African swine fevervirus (ASFV) classical swine fever virus (CSFV) porcinereproductive and respiratory syndrome virus (PRRSv) andAujeszky disease virus [9ndash16] Strikingly leucocyte differen-tial count in domestic pig has been associated with multiplechromosomal regions but specific locigenes that can par-tially explain the variation between individuals have not beenidentified [17ndash19]

Increased leucocytes are typically found in pigs withconditions associated with viral respiratory tract infectionafter parainfluenza-3 (PI3) virus [20] ASFV infection leadsto serious changes in the concentration of leucocytes detected2ndash3 d after infection [21] PRRSv infection caused a hugeacute drop in total leucocyte counts that affect all PBMCpopulations by 2 d after infection in pregnant gilts [22]To these infectious diseases the resistance of an individualresulted from both innate and acquired immunity Thecapacity of innate immunity or acquired immunity is moreor less controlled by genes [23ndash25] In pigs genetic studieshave revealed several genes that participate in the resistanceto diseases [26] including single-gene porcine ryanodinereceptor (ryr1) regulating malignant hyperthermia [27] andporcine factor H [28] Nevertheless the identities of the mostregulators of leucocytes related traits and their direct targetsare largely unknown despite their biological functions

To define the molecular mechanisms of general gene-expression profiles or changes during rapid leucocytes turn-overs in pig genome we adapted techniques ofmicroarray forgenetic profiling of pigs with the increased and decreased leu-cocyte counts before and after polyinosinicpolycytidylic acid(poly IC) stimulation Poly IC is a synthetic double-strandedRNA that is used experimentally to model viral infections invivo [29] together with the microarray technology that rep-resents a profiling strategy that allows the detection andmea-sure of various responses by a multitude of gene probes setsWe have identified a number of previously uncharacterizedgenes that appear to be expressed in swine blood leucocyteswhile simultaneously establishing the dominantly expressedgenes of the increased and decreased leucocyte phenotypesas well as immune components involved such as influenzaA pathway cytosolic DNA-sensing pathway chemokine sig-naling pathway and cytokine-cytokine receptor interactionwhich are important in the molecular pathways that regulatewhite cells andor hematopoietic stem cell function in normaland pathologic conditions

2 Materials and Methods

21 Ethics Approval All experimental procedures and ani-mal care activities were strictly conducted in accordancewith the guidelines established for the care and use oflaboratory animals of the Standing Committee of HubeiPeoplersquos Congress (No 5) approved by The Scientific EthicsCommittee ofHuazhongAgriculturalUniversity (HZAUSW-2013-014) Therefore all efforts were made to minimizesuffering

22 Animals Experimental design details of the infectionand blood collection procedures are described in [30] Thepigs used in the study were from a DurocndashErhualian F2population and comprised 392 F2 offspring derived from51 F1 and 26 F0 parents In short 8 Duroc boars weremated to 18 Erhualian sows Then 13 F1 boars and 38 F1sows were chosen and mated to produce 392 F2 animalsThe six animals used in the present study were selectedbased on the two-tailed value of leucocyte counts beforeand after poly IC stimulation All animals are fed in thesame conditions and free of virus infections such as PRRSvMycoplasma hyopneumoniae and swine influenza virus At35 d pigs were intramuscularly infected with poly IC (CASRegistry Number 24939-03-5 Hangzhou Meiya Pharmacy)at a dose of 05mgkg Blood samples were taken at 33 and35 d after poly (IC) infection Leucocytes levels on these dpiwere measured using a Japanese photoelectric MEK-8222Kautomatic 5 classification blood analyzer (Nihon kohdenTokyo Japan)

23 Phenotypic Data The statistical significance of the phe-notypic differences between the treatment and control groupswas first examined by assuming unequal variances Studentrsquost-test The residuals obtained from the linear model werefurther used as a target variable for analysis using t-test andlm functions in the R environment

24 Microarray Design and Hybridization Based on leuco-cyte count recorded at 33 and 35 d after poly (IC) infectiona total of six pigs were divided into two groups and codedas increased responders (IR) and decreased responders (DR)Whole blood samples were collected from each of the six pigs4 h after poly IC treatment on day 35 and at day 33 as anunstimulated control The whole blood samples were usedfor the microarray experiments GeneChip Porcine GenomeArrays (Affymetrix) were used to determine gene expressionlevels in the whole blood samples before and after poly ICtreatment of the six pigs RNA labeling and hybridizationwere performed by a commercial Affymetrix array ser-vice (GeneTech Biotechnology Limited Company ShanghaiChina) This methodology was described in our previousstudy [31] All raw probe-microarray data were normalizedby the Robust Multichip Average method from packages ofBioconductor (httpwwwbioconductororg) implementedin the R environment [32ndash34] The model matrix for differ-entially expressed genes (DEGs) identification was defined ina linear format and the model scenario included the samefactors in the linear model for leucocyte count phenotypeanalysis The linear models for microarray data (LIMMA)[35] were used to identify the DEGs To determine thestatistical significance of the DEGs the cutoff for P values wasset to (001) adjusted P values (01) and fold change ge (20)Furthermore a two-way hierarchical clustering analysis wasperformed to identify the DEGs patterns according to [35]

25 Gene Annotation and Pathway Analysis The Affymetrixporcine genome microarray annotation was initially per-formed using the annotation file given by Affymetrix Inc(httpswwwaffymetrixcomindexaffx) Each probe set was

BioMed Research International 3

Table 1 Known immune regulatory DEGs of IR and DR

DEGs Category Probe-sets ID Gene Symbol Log2 FC Reference

IR-Upregulated

Ssc162341S1 at TCN1 31 [43]Ssc189271S1 at MS4A8B 26 [31]Ssc91171S1 at S100A12 23 [44]Ssc63691A1 at CSF1 22 [45]

IR-Downregulated Ssc267091S1 at GPR183 -20 [46]

DR-UpregulatedSsc128291A1 at TNFSF10 35 [47]Ssc260051S1 at ZBP1 33 [48]Ssc82611A1 at CYP2C9 32 [49]

DR-Downregulated Ssc166711S1 at TGFBI -39 [50]

annotated using themethod previously described by [36]Thedifferentially expressed transcripts were further comparedwith the official homologous human gene symbols Usingbioinformatics resource tools in the DAVID website v68[37 38] differentially expressed genes (DEGs) were uploadedto determine the regulated pathways and biological functionsignificantly associated with the gene lists Therefore thisstatistical test assesses the proportion of genes that mapto a particular function or pathway in the IR and DRphenotypes We focused on the most affected pathwaysbiological molecular and cellular functions that belongedto the two phenotypes gene symbols were used in thesubsequent functional analysis

26 Validation of DEGs by qPCR Quantitative real time-PCR(qPCR) was performed to quantify the expression levels of14 DEGs (ISG20 RSAD2 TLR4 S100A12 S100A9 S100A8SH2DIA TNK2 MX1 MX2 OAS1 PLSCR1 tripartite motif(TRIM) 26 and STAT1) One microgram of total RNAobtained from all biological replicates (3 by 3 replicates of IRand DR before and after poly IC stimulation) was reverse-transcribed using EasyScript one-step gDNA Removaland cDNA Synthesis SuperMix (TansGen Biotech BeijingChina) following manufacturerrsquos protocols for the two-stepqPCR assays Quantitative PCR was then performed by usingSYBRqPCRMix (Aid labBiotechnologiesCo Ltd China) inBio-Rad thermal cycler CFX-384 real-time system Detailsof genes annotations and primer set validated by qPCR areincluded in Supplementary File 1 Following amplificationthe differences in the Ct values of the control and experimen-tal samples were used to determine the relative expression ofthe gene in each sample All Ct values were normalized using120573-actin gene [39 40]The relative gene expression levels werecalculated using 2minus(ΔΔCT) method [41 42] The correlationbetween themicroarray and qPCR results for the gene set wasthen performed for each replicate and the statistical signif-icance of the correlations determined The log2 fold-changeof microarray versus qPCR log2 fold-change was graphed todetermine the quality of correlation a slope of one woulddefine perfect correlation The strength of the relationshipwas quantified by Pearsonrsquos coefficient computation

3 Results

31 Poly IC-Stimulated Leucocyte-Count Variation Potentimmune stimulator led to significant pronounced differences

in leucocyte counts Overall mean leucocyte counts werefound to have a huge variation before and after poly ICstimulation (p lt 005) (Figure 1(a)) Before stimulation theoverall mean leucocyte-count values (Mean plusmn SEM) were2115 plusmn 127 after stimulation overall mean leucocyte-countvalues were 1910 plusmn 115 (n = 277) (Figure 1(a)) However themean leucocyte-count values of IR and of DR were calculatedfrom the two-tailed critical value of leucocyte counts after4 h poly IC treatment The mean leucocyte-count valuesfor IR group were 1890 plusmn 1091 before treatment and aftertreatment were increased to 4220 plusmn 2436 (Figure 1(b)) Bycontrast the DR leucocyte-count values were 1423 plusmn 822before treatment and after treatment were reduced to 630 plusmn364 (Figure 1(c)) Increased leucocyte countmay also explaintheir ability to fight back and persist in response to viral infec-tion (Figure 1(b)) whereas a significant decrease in leucocytecount suggested that poly IC temporarily disrupts bonemarrow function where leucocytes are made (Figure 1(c))The differences between the two groups were statisticallysignificant (p lt 001) in the IR and DR groups respectively

32 Transcriptome Clustering and Altered DEGs between IRand DR To understand further the patterns of differentialgene expression and also to know whether the poly ICstimulation effected transcriptionally in the selected pigstwo-way hierarchical clustering was conducted with DEGsfor the construction of heat-map The heat-map illustratesgene expression values between the IR (Figure 2(a)) and DR(Figure 2(b)) The patterns of gene expression indicate thatall the samples clustered into two types indicating pre- andpost-poly IC stimulation Thus to compare temporarily thetranscriptome altering between the IR and DR pigs post-poly IC treatment we further analyzed the DEGs in bothgroups and asked how many of the DEGs overlapped andorwere differentially expressed specifically in each phenotype(Figure 2(c)) 129 DEGs were found in the IR group and 136DEGs in theDR group (Supplementary File 2) NotablymanyDEGs have been reported to be involved in the immuneregulatory processes (Table 1) A total of 35 upregulated and 6downregulated overlappingDEGswere found in both groupsThe IR has 14 and 74 unique upregulated and downregulatedDEGs respectively and the DR has 78 and 17 unique upreg-ulated and downregulated DEGs respectively (Figure 2(c)Supplementary File 2) Following those findings more DEGs


33 days 35 days0

10

50

40

30

20

WBC33daysWBC35days

Method

Cou

nt (

L

)

(a)

0

10

20

30

40

50

60

IR1 IR2 IR3

IRBS IRAS

Leuc

ocyt

e cou

nt (

L

)

(b)

0

2

4

6

8

10

12

14

16

18

DR1 DR2 DR3

DRBS DRAS

Leuc

ocyt

e cou

nt (

L

)

(c)

Figure 1 Changes in (a) poly IC-induced peripheral blood leucocyte (b)The leucocyte count changes in IR (IR1 IR2 and IR3) IRBSIRASrepresent IR before and after poly IC stimulation (c) Leucocyte count changes in DR (DR1 DR2 and DR3) DRBSDRAS represent DRbefore and after poly IC stimulation Error bars indicate standard error of mean (SEM)

were downregulated in the IR pigs than in the DR pigsthereby indicating that IR pigs showed less responsiveness atthe transcription level than did the DR pigs

33 Gene Expression Verification of DEGs with qPCR Atotal of 14 genes of biological interest identified by themicroarray were verified by qPCRThe tested genes encodedenzymes involved in interferon-stimulation transcriptionfactors transmembrane and receptor binding regulation ofubiquitin-protein ligase cellular activity and lymphocyte-activating from different gene families and molecules playinga role in the immune response of IR and DR Relative expres-sion of 6 upregulated genes (ISG20 RSAD2 TLR4 S100A12S100A9 and S100A8) was significantly reduced after polyIC stimulation in the IR Downregulated gene (SH2DIA)expression was reduced after poly IC stimulation and also

downregulated (TNK2) gene expression increased after polyIC stimulation in the IR (Figure 3(a)) Eight upregulatedgenes namely MX1 MX2 RSAD2 ISG20 OAS1 PLSCR1STAT1 and TLR4 were significantly reduced before poly ICstimulation and increased after poly IC stimulation in theDRUpregulated gene (TRIM26) expression was significantlyincreased before poly IC stimulation and reduced afterpoly IC stimulation in DR (Figure 3(b)) Additionally wefound strong agreement between qPCR and microarray forthe fourteen genes verified The results were presented infitted line plot and correlation output suggests a positivelinear relationship between qPCR and microarray log2 fold-change The value of R2 the coefficient of determinationwas 086 and the linear regression model Y = (12505x ndash03326) and the Pearson correlation was 092 close to oneAll genes expression profiling experiments were consistent


IR_2245

IR_2278

IR_2152

IR_3169

IR_3287

IR_3255

Stimulated Unstimulated

Color Key IR

500

300

100

0

Cou

nt

minus1 0 1

Row Z-Score

(a)

DR

DR_3055

DR_3229

DR_3230

Stimulated

DR_2007

DR_2039

DR_2204

Unstimulated

Color Key

0

minus1 0 1

Row Z-Score

150

50C

ount

(b)

DR_Up

IR_Up DR_Down

IR_Down

(c)

Figure 2 Two-way hierarchical clustering and Venn diagram of overlapped DEGs by poly IC stimulation Gene expression values in (a) IRpigs and (b) DR pigs The columns within the heat-map represent samples (pigs ID) while the rows represent genes The color scheme red(high) expression and green (low) expression The color legend at the top-left of the figure indicates the fold change in gene expression (c)The Venn diagram of DEGs in the two groups

with the expression profiles determined frommicroarray data(Figure 3(c))

34 Comparative Functional Annotation of Poly IC InducedDEGs between IR and DR Consider a total of 265 DEGsin the IR and DR phenotypes (Log2 FC ge 2 and le-20)and 201 orthologues of human genes were annotated inthe DAVID (Database for Annotation Visualization andIntegrated Discovery) database which revealed 20 significantfunctional annotation clusters between the IR and DR (plt 005) The most significant clusters were presented inTable 2 The functional annotation clusters in the IR and DRwere related to innate immunity innate immune responseantiviral defense immunity and defense response to thevirus but differ in the negative regulation of viral genome

replication (Table 2) Furthermore the DEGs of IR andDR in biological processes (BP) molecular functions (MF)and cellular components (CC) were revealed based on theGO categories in DAVID When considering the commonlyshared biological processes between IR and DR phenotypesthese include innate immune response immune responsedefense response response to external stimulus immuneeffector process and viral life cycle (p lt 005 and count ge4) (Figure 4(a) Supplementary File 3) For the GOmolecularfunction only four terms were common between the IR andDR includingmetal ion binding ion binding cation bindingand zinc ion binding whereas the majority of the MF termswere related to the DR background (Figure 4(b) Supple-mentary File 3) In the cellular component category DRwas distributed mostly in extracellular exosome extracellular


Table 2 Major functional annotation clusters in poly IC-induced leucocyte responders based on fold change (FC) and (p lt 001)

GO name Gene count Fold change GenesExpressed in increased leucocyteresponders

Expressed in decreased leucocyteresponders

Innate immunity 10 1820 DDX58 SH2D1A CFB RSAD2 TLR4S100A12 ISG20

DDX58 CFB RSAD2 TLR4 OAS1MX1 MX2 ISG20

Innate immune response 14 718 DDX58 SH2D1A S100A8 S100A9RSAD2 TLR4 TNK2 S100A12 ISG20

DDX58 PTK2B TRIM26 RSAD2TLR4 OAS1 MX2 ISG20 TEC

Immunity 10 1076 DDX58 SH2D1A CFB RSAD2 TLR4S100A12 ISG20


Negative regulation of viralgenome replication 6 2247 - PLSCR1 ISG15 RSAD2 OAS1

MX1 ISG20

Defense response to virus 9 668 IFIT3 PLSCR1 IFIT2 ISG15 RSAD2OAS1 MX1 MX2 ISG20

PLSCR1 ISG15 RSAD2 OAS1MX1 ISG20

Antiviral defense 6 910 DDX58 RSAD2 ISG20 DDX58 RSAD2 OAS1 MX1 MX2ISG20

0000

0200

0400

0600

0800

1000

1200

1400

1600

1800

Fold

chan

ge

IRBSIRAS

ISG

20

RSA

D2

TLR4

S100

A12

S100

A9

S100

A8

SH2D

1A

TNK

2

lowastlowastlowast lowastlowastlowast lowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowast

(a)

Fold

chan

ge

0000

2000

4000

6000

8000

10000

12000

DRBSDRAS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast



MX

1

MX

2

TRIM

26

RSA

D2

ISG

20

OA

S1

PLSC

R1

STAT

1

TLR4

(b)

minus5minus4minus3minus2minus1

012345

minus3 minus2 minus1 1 2 3 4

Mic

roar

ray

(Log

2 Fo

ld C

hang

e)

qPCR (Log 2 Fold Change)

２2= 086

y = 12505x - 03326

Pearson R = 092Plt 000001

0

(c)

Figure 3 Microarray fold change verification with qPCR (a b) Fold change of 14 genes of IR and DR before and after stimulation codedas IRBSIRAS and DRBSDRAS respectively Data are reported for three technical replicates in all biological samples Error bars indicatestandard error of the mean (SEM) Each histogram represents the level of the target gene relative expression level Asterisks symbolized Pvalue significance (p le 005 (lowast) p le 001 (lowastlowast) p le 0001 (lowast lowast lowast) and p le 00001 (lowast lowast lowastlowast) based on assuming unequal variances Studentrsquost-test (c) qPCR validated 14 genes A line plot of qPCR log2 (FC) and microarray log2 (FC) data of 14 genes The linear relationship betweenthe qPCR and microarray log2 (FC) data based on the R2 value and the line linear regression (Y)


0 5 10 15 20 25 30innate immune response

immune responsedefense response

positive regulation of response to stimulusresponse to external stimulus

immune effector processregulation of innate immune response

regulation of response to stressviral life cycle

regulation of immune response

Gene count

Comparative GO biological process distribution

DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count

Comparative GO molecular function distribution

DRIR

(b)

Figure 4 Commonly enriched gene ontology (functional classification) of the DEGs by poly IC stimulation in IR and DR (a) ComparativeGO biological process distribution of the DEGs by poly IC stimulation in the IR and DR pigs (b) Comparative GO molecular functiondistribution of DEGs by poly IC stimulation in the IR and DR pigs

Table 3 Pathway regulated by DEGs of IR and DR

GO accession KEGG pathway Gene count Genes

ssc05164 Influenza A 9 DDX58 RNASEL TNFSF10 IRF7RSAD2 TLR4 OAS1 STAT1 MX1

ssc05162 Measles 7 DDX58 TNFSF10 IRF7 TLR4 OAS1STAT1 MX1

ssc05168 Herpes simplex infection 6 DDX58 RNASEL IRF7 OAS1 STAT1DAXX

ssc04623 Cytosolic DNA-sensing pathway 4 DDX58 IRF7 TREX1 ZBP1ssc05160 Hepatitis C 5 DDX58 RNASEL IRF7 OAS1 STAT1ssc05161 Hepatitis B 5 DDX58 PTK2B IRF7 TLR4 STAT1ssc04062 Chemokine signaling pathway 5 PTK2B CCL8 GNB4 PAK1 STAT1

ssc04060 Cytokine-cytokine receptorinteraction 5 ACVR1B TNFSF10 IL1RAP CCL8

IL13RA1

ssc04622 RIG-I-like receptor signalingpathway 3 DDX58 ISG15 IRF7

vesicle extracellular organelle and extracellular regionwhereas no annotation information was found for the IR(Supplementary File 3)

Based on conserved orthologues defined by the KyotoEncyclopedia of Genes and Genomes (KEGG) the DEGs ofIR and DRwere assigned to one ormore conserved biologicalpathways The pathways in KEGG and related genes foundin DR were listed in Table 3 whereas no pathway annotationrecord was found for the IR The analysis identified ninepathways in DR including influenza A measles herpessimplex infection cytosolic DNA-sensing pathway hepatitisC hepatitis B chemokine signaling pathway and cytokine-cytokine receptor interaction pathway (Table 3 Supplemen-tary File 4) The pathway of influenza A was the mostenriched pathway related to the upregulated genes of DRUp- and downregulated genes of DR including RNASELTNFSF10 OAS1 ZBP1 MX1 CCL8 GNB4 PAK1 STAT1and IL13RA1 were also enriched in the chemokine signalingand cytokine-cytokine receptor interaction pathways exceptfor upregulated ISG15 in the RIG-I-like receptor signaling

pathway The DR had more DEGs related to the affectedpathways when compared with IR Table 4 shows the DEGsbetween IR and DR in the affected pathways

4 Discussion

41 DEGs Specific to the IR and DR Pigs In the DEGs specificto the IR pigs 88 genes were detected many of these geneswere significantly enriched and some of them had a putativefunctional role relevant to innate immune response andleucocyte migration involved in inflammatory response Weselected four genes that play roles in the cellular process andsignaling Among cellular process genes a set of calcium-binding proteins were identified in the IR including S100A12S100A9 and S100A8 These genes encode S100 proteins Inthe IR group S100A12 S100A9 and S100A8 were highlyexpressed before poly IC stimulation and significantly lowerexpression was observed after stimulation These calcium-binding protein genes considered markers at the site ofinflammation as previously reported in humans [51] and pigs


Table 4 Differentially expressed genes in the affected pathwaysNegative fold change values indicate downregulation in response topoly IC stimulation

Fold changeIR vs DR

Gene IR DRDDX58 263 399RNASEL - 211TNFSF10 - 345IRF7 192 299RSAD2 204 530TLR4 218 356OAS1 - 302STAT1 - 238DAXX 210 266MX1 - 285TREX1 186 234ZBP1 - 329ACVR1B 295 309IL1RAP 228 295CCL8 - -269PTK2B 193 216GNB4 - 250PAK1 - 220IL13RA1 - 217ISG15 - 233

[31 52] play a major role during infection Among signalinggenes SH2D1A encodes SH2 domain-containing protein 1Aof 128 amino acid (aa) residues (Genbank NC 0104615)Reference [53] found that SH2D1A interacts with signalinglymphocytic activation molecule (SLAM) thereby demon-strating its contribution to the regulation of a transmembraneprotein that is expressed on the surface of activated T andB cells The expression of SH2D1A reduced after stimulationcompared with control in IR we speculate that it mayparticipate in activating the host immune response duringinfection

Considering the 95 DEGs specific to the DR pigs all thegenes that fell within this category had defined functionsNevertheless we noticed six genes including TRIM26 OAS1STAT1 PLSCR1 MX1 and MX2 whose functional roles canregulate host immune responses TRIM26 which encodeda member of the TRIM family implicated in innate andadaptive immunity [54 55] was significantly lower after polyIC stimulation compared with control in the DR pigs OAS1belongs to the OAS family of proteins known to be activesynthetases that synthesize 21015840-51015840-linked oligoadenylates inresponse to viral infections thereby affecting an early step ofthe viral replication cycle [56 57] The expression of OAS1was significantly higher after poly IC stimulation in DR pigswhen compared with control For STAT1 a critical regulatorof the interferon signaling pathway [58 59] its expressionwassignificantly higher after poly IC stimulation in the DR com-paredwith control Phospholipid scramblase 1 gene (PLSCR1)

was highly significant after poly IC stimulation comparedwith control Studies in human reported PLSCR1 gene in theregulation of phosphatidylserine distribution of erythrocytes[60]MoreoverMX1 andMX2 geneswere highly expressed inDR pigs after poly IC stimulation MX dynamin-like GTPase1 (MX1) was implicated in some viral replications includingCSFV [61] and pig-original bovine viral diarrhea virus 2(BVDV-2) [62] Additionally myxovirus (influenza virus)resistance 2 gene (MX2) which encodes interferon (IFN)-inducible proteins plays a critical role in the antivirus state[63 64] Similarly [6] reported the expression of MX1 andSTAT1 geneswere highly expressed shortly after experimentalinfluenza A virus (IAV) infection in circulating leucocytes

42 Biological Processes Found in the IR and DR PhenotypesFor the common biological processes involved in the IR andDR phenotypes innate immune response immune responsedefense response response to external stimulus and immuneeffector process were the most enriched biological functionsfound in the IR and DR phenotypes Among these biologicalprocesses upregulated genes (radical S-adenosyl methioninedomain containing 2 interferon stimulated exonuclease gene20 andDExDH-box helicase 58)were significantly enrichedChemokine (C-Cmotif) ligand 8 SH2 domain contained 1Atyrosine kinase non-receptor 2 and interleukin 7 receptorwere overrepresented downregulated genes regulating thebiological function of the IR and DR phenotypes Addi-tionally DR phenotype had more transcriptional genes thandid the IR in most of the common biological pathwaysthereby suggesting that the former created more effectivehost immune responses than did the latter Moreover IR andDR phenotypes differ in several biological defenses such ascell motility and cellular protein modification process beingfound in IR phenotype whereas the regulation of molecularfunction positive regulation of cellular metabolic processand response to cytokine are event-activated for the hostimmune responses in DR phenotype

43 Pathway Regulated by the DEGs in the DR Pigs Inthe IR pigs no pathways were detected However severalpathways related to antiviral responses were found in theDR pigs such as influenza A chemokine signaling path-way cytokinendashcytokine receptor interaction and RIG-I-likereceptor signaling pathway

Research reports indicate that the influenza A pathwaywas known to activate cellular signal transduction pathwayssuch as NF-120581B signaling PI3KAkt pathway MAPKpathwayPKCPKR signaling and TLRRIG-I signaling cascades [65]These pathways are important for viral entry viral replicationviral propagation and apoptosis these pathways are involvedin antagonizing the host antiviral response Previous studieshave suggested the involvement of various DExDH-boxRNA helicase such as DDX58 (also known as RIG-I) in theinitiation of innate immune responses [66 67] In this studyDDX58 (DExDH-box helicase 58) was the upregulated geneof IR and DR pigs and are often seen in the affected pathwaysthereby suggesting that DDX58 plays an essential role ininitiating an antiviral response [68] Another important gene


that corresponds to this pathway was upregulated gene of DRpigs (RNASEL) a principal mediator of the IFN-inducibleantiviral state that can determine the survival of animalsinfected with highly pathogenic viruses [69 70] Based onthose findings influenza A pathway is crucial for leucocyte-related traits

5 Conclusions

We investigated the transcriptome of leucocyte variation inperipheral blood of pigs induced by poly IC The microarrayanalysis identified many DEGs that were involved in theimmune defense responses of both the IR and DR pigsalthough the two groups showed different immune responsesafter dsRNA stimulation Overall our study will enhance theunderstanding of themolecular basis for the antiviral capacityof pigs and potentially important genes for disease resistancebreeding

Data Availability

All data are publicly available and consent for use of all datais available

Conflicts of Interest

The authors hereby declare no conflicts of interest regardingthe publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (31501922 31361140365) National HighTechnology Plan of China (2013AA102502) National PigIndustry Technology System Project (CARS-35) Da BeiNong Group Promoted Project for Young Scholar of HZAU(2017DBN019) and the Fundamental Research Funds for theCentral Universities of China (2662018JC033)

Supplementary Materials

Supplementary File 1 list of the differentially expressed genes(DEGs) analyzed in qPCR and primers used for their ampli-fication Supplementary File 2 differential gene expressionof IR and DR pigs Supplementary File 3 comparative func-tional classification of differentially expressed genes (DEGs)in IR and DR pigs Supplementary File 4 pathway in the DRpigs (Supplementary Materials)

References

[1] A A Sinha M T Lopez and H O Mcdevitt ldquoAutoimmunediseases the failure of self tolerancerdquo Science vol 248 no 4961pp 1380ndash1388 1990

[2] K RogersBlood physiology and circulationTheRosen Publish-ing Group 2010

[3] P Brodin andMM Davis ldquoHuman immune system variationrdquoNature Reviews Immunology vol 17 no 1 pp 21ndash29 2017

[4] J Parkin and B Cohen ldquoAn overview of the immune systemrdquoe Lancet vol 357 no 9270 pp 1777ndash1789 2001

[5] M Pomorska-Mol I Markowska-Daniel and K Kwit ldquoIm-mune and acute phase response in pigs experimentally infectedwith H1N2 swine influenza virusrdquo FEMS Immunology amp Medi-cal Microbiology vol 66 no 3 pp 334ndash342 2012

[6] L Brogaard P M H Heegaard L E Larsen et al ldquoLate regula-tion of immune genes andmicroRNAs in circulating leukocytesin a pig model of influenza A (H1N2) infectionrdquo ScientificReports vol 6 2016

[7] O Ramilo W Allman W Chung et al ldquoGene expressionpatterns in blood leukocytes discriminate patients with acuteinfectionsrdquo Blood vol 109 no 5 pp 2066ndash2077 2007

[8] L Piriou-Guzylack and H Salmon ldquoMembrane markers of theimmune cells in swine An updaterdquoVeterinary Research vol 39no 6 article no 54 2008

[9] G D Appleyard S E Furesz and B N Wilkie ldquoBlood lym-phocyte subsets in pigs vaccinated and challenged with Acti-nobacillus pleuropneumoniaerdquo Veterinary Immunology andImmunopathology vol 86 no 3-4 pp 221ndash228 2002

[10] J E Butler M Sinkora N Wertz W Holtmeier and C DLemke ldquoDevelopment of the neonatal B and T cell repertoire inswine Implications for comparative and veterinary immunol-ogyrdquo Veterinary Research vol 37 no 3 pp 417ndash441 2006

[11] W-H Feng M B Tompkins J-S Xu et al ldquoThymocyte andperipheral blood T lymphocyte subpopulation changes in pig-lets following in utero infection with porcine reproductive andrespiratory syndrome virusrdquo Virology vol 302 no 2 pp 363ndash372 2002

[12] TG KimmanA T J Bianchi GWensvoort T GMDe Bruinand C Meliefste ldquoCellular immune response to hog choleravirus (HCV) T cells of immune pigs proliferate in vitro uponstimulation with live HCV but the E1 envelope glycoprotein isnot a major T-cell antigenrdquo Journal of Virology vol 67 no 5 pp2922ndash2927 1993

[13] L Lopez Fuertes N Domenech B Alvarez et al ldquoAnalysis ofcellular immune response in pigs recovered from porcine res-piratory and reproductive syndrome infectionrdquo Virus Researchvol 64 no 1 pp 33ndash42 1999

[14] C L V Martins M J P Lawman T Scholl C A Mebus and JK Lunney ldquoAfrican swine fever virus specific porcine cytotoxicT cell activityrdquoArchives of Virology vol 129 no 1-4 pp 211ndash2251993

[15] T Pauly K Elbers M Konig T Lengsfeld A Saalmuller andH-J Thiel ldquoClassical swine fever virus-specific cytotoxic Tlymphocytes and identification of a T cell epitoperdquo Journal ofGeneral Virology vol 76 no 12 pp 3039ndash3049 1995

[16] M Susa M Konig A Saalmuller M Reddehase and H ThielldquoPathogenesis of classical swine fever B-lymphocyte deficiencycaused by hog cholera virusrdquo Journal of Virology vol 66 pp1171ndash1175 1992

[17] G Reiner R Fischer S Hepp T Berge F Kohler and HWillems ldquoQuantitative trait loci for white blood cell numbersin swinerdquo Animal Genetics vol 39 no 2 pp 163ndash168 2008

[18] S Yang J Ren X Yan et al ldquoQuantitative trait loci for porcinewhite blood cells and platelet-related traits in a white duroc timesErhualian F2 resource populationrdquoAnimal Genetics vol 40 no3 pp 273ndash278 2009

[19] Y-F Gong X Lu Z-P Wang et al ldquoDetection of quantitativetrait loci affecting haematological traits in swine via genomescanningrdquo BMC Genetics vol 11 article no 56 2010


[20] A Leusink-Muis R T Broeke G Folkerts F De Clerck andF P Nijkamp ldquoBetamethasone prevents virus-induced airwayinflammation but not airway hyperresponsiveness in guineapigsrdquo Clinical amp Experimental Allergy Reviews vol 29 no 2 pp82ndash85 1999

[21] Z Karalyan H Zakaryan H Arzumanyan et al ldquoPathologyof porcine peripheral white blood cells during infection withAfrican swine fever virusrdquo BMC Veterinary Research vol 8article no 18 2012

[22] A Ladinig W Gerner A Saalmuller J K Lunney C Ashleyand J C S Harding ldquoChanges in leukocyte subsets of pregnantgilts experimentally infected with porcine reproductive andrespiratory syndrome virus and relationships with viral loadand fetal outcomerdquoVeterinary Research vol 45 no 1 article no128 2014

[23] M Clapperton S C Bishop and E J Glass ldquoInnate immunetraits differ betweenMeishan and Large White pigsrdquo VeterinaryImmunology and Immunopathology vol 104 no 3-4 pp 131ndash144 2005

[24] A L Vincent B JThacker P GHalburM J Rothschild andEL Thacker ldquoIn vitro susceptibility of macrophages to porcinereproductive and respiratory syndrome virus varies betweengenetically diverse lines of pigsrdquo Viral Immunology vol 18 no3 pp 506ndash512 2005

[25] K Wimmers K G Kumar K Schellander and S PonsuksilildquoPorcine IL12A and IL12B gene mapping variation and evi-dence of associationwith lytic complement and blood leucocyteproliferation traitsrdquo International Journal of Immunogeneticsvol 35 no 1 pp 75ndash85 2008

[26] H N Kadarmideen ldquoBiochemical ECF18R and RYR1 genepolymorphisms and their associations with osteochondral dis-eases and production traits in pigsrdquo Biochemical Genetics vol46 no 1-2 pp 41ndash53 2008

[27] J Fujii K Otsu F Zorzato et al ldquoIdentification of a mutationin porcine ryanodine receptor associatedwithmalignant hyper-thermiardquo Science vol 253 no 5018 pp 448ndash451 1991

[28] J H Jansen K Hoslashgasen and A M Groslashndahl ldquoPorcine mem-branoproliferative glomerulonephritis type II an autosomalrecessive deficiency of factor HrdquoVeterinary Record vol 137 no10 pp 240ndash244 1995

[29] K H Benam R Villenave C Lucchesi et al ldquoSmall airway-on-a-chip enables analysis of human lung inflammation and drugresponses in vitrordquo Nature Methods vol 13 no 2 pp 151ndash1572016

[30] X Liu J Huang S Yang et al ldquoWhole blood transcriptomecomparison of pigs with extreme production of in vivo dsRNA-induced serum IFN-ardquo Developmental amp Comparative Immu-nology vol 44 no 1 pp 35ndash43 2014

[31] H Chen C Li M Fang et al ldquoUnderstanding Haemophilusparasuis infection in porcine spleen through a transcriptomicsapproachrdquo BMC Genomics vol 10 article no 64 2009

[32] R C Team A language and environment for statistical com-puting R Foundation for statistical computing Vienna Austria2016

[33] W Huber A Von Heydebreck H Sultmann A Poustka andM Vingron ldquoVariance stabilization applied to microarray datacalibration and to the quantification of differential expressionrdquoBioinformatics vol 18 supplement 1 pp S96ndashS104 2002

[34] L Gautier L Cope B M Bolstad and R A Irizarry ldquoAffymdashanalysis of Affymetrix GeneChip data at the probe levelrdquo Bio-informatics vol 20 no 3 pp 307ndash315 2004

[35] R C GentlemanV CareyWHuber R Irizarry and R DudoitBioinformatics andComputational Biology SolutionsUsing R andBioconductor Springer New York NY USA 2005

[36] S Tsai J P Cassady B A Freking D J Nonneman G ARohrer and J A Piedrahita ldquoAnnotation of the Affymetrix1porcine genomemicroarrayrdquoAnimal Genetics vol 37 no 4 pp423-424 2006

[37] D W Huang B T Sherman and R A Lempicki ldquoSystematicand integrative analysis of large gene lists using DAVID bioin-formatics resourcesrdquo Nature Protocols vol 4 no 1 pp 44ndash572009

[38] D W Huang B T Sherman and R A Lempicki ldquoBioin-formatics enrichment tools paths toward the comprehensivefunctional analysis of large gene listsrdquo Nucleic Acids Researchvol 37 no 1 pp 1ndash13 2009

[39] J Wang Y Wang H Wang X Hao Y Wu and J GuoldquoSelection of reference genes for gene expression studiesin porcine whole blood and peripheral blood mononuclearcells under polyinosinicPolycytidylic acid stimulationrdquo Asian-Australasian Journal of Animal Sciences vol 27 no 4 pp 471ndash478 2014

[40] D L Foss M J Baarsch and M P Murtaugh ldquoRegulationof hypoxanthine phosphoribosyltransferase glyceraldehyde-3-phosphate dehydrogenase and 120573-actin mRNA expression inporcine immune cells and tissuesrdquo Animal Biotechnology vol9 no 1 pp 67ndash78 1998

[41] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCTmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[42] T D Schmittgen and K J Livak ldquoAnalyzing real-time PCR databy the comparative CT methodrdquo Nature Protocols vol 3 no 6pp 1101ndash1108 2008

[43] G Reiner F Dreher M Drungowski et al ldquoPathway dereg-ulation and expression QTLs in response to Actinobacilluspleuropneumoniae infection in swinerdquo Mammalian Genomevol 25 no 11-12 pp 600ndash617 2014

[44] X Li J Tang J Xu et al ldquoThe inflammation-related geneS100A12 is positively regulated by CEBPΒ and AP-1 in pigsrdquoInternational Journal of Molecular Sciences vol 15 no 8 pp13802ndash13816 2014

[45] K A Sauter L A Waddell Z M Lisowski et al ldquoMacrophagecolony-stimulating factor (CSF1) controls monocyte produc-tion and maturation and the steady-state size of the liver inpigsrdquo American Journal of Physiology-Gastrointestinal and LiverPhysiology vol 311 no 3 pp G533ndashG547 2016

[46] C Liu X V Yang J Wu et al ldquoOxysterols direct B-cellmigration through EBI2rdquoNature vol 475 no 7357 pp 519ndash5232011

[47] N Schlichting T Dehne K Mans et al ldquoSuitability of porcinechondrocyte micromass culture to model osteoarthritis invitrordquo Molecular Pharmaceutics vol 11 no 7 pp 2092ndash21052014

[48] L Xie L Fang D Wang et al ldquoMolecular cloning and func-tional characterization of porcine DNA-dependent activator ofIFN-regulatory factors (DAI)rdquo Developmental amp ComparativeImmunology vol 34 no 3 pp 293ndash299 2010

[49] H A Thorn A Lundahl J A Schrickx P A Dickinson andH Lennernas ldquoDrug metabolism of CYP3A4 CYP2C9 andCYP2D6 substrates in pigs and humansrdquo European Journal ofPharmaceutical Sciences vol 43 no 3 pp 89ndash98 2011

[50] H Karring Z Valnickova I B Thoslashgersen et al ldquoEvidenceagainst blood derived origin for transforming growth factor


beta induced protein in corneal disorders caused by mutationsin the TGFBI generdquo Molecular Vision vol 13 pp 997ndash10042007

[51] D Foell M Frosch C Sorg and J Roth ldquoPhagocyte-specificcalcium-binding S100 proteins as clinical laboratorymarkers ofinflammationrdquoClinica Chimica Acta vol 344 no 1-2 pp 37ndash512004

[52] H Chen L Cheng S Yang et al ldquoMolecular characterizationinduced expression and transcriptional regulation of porcineS100A12 generdquoMolecular Immunology vol 47 no 7-8 pp 1601ndash1607 2010

[53] J Sayos C Wu M Morra et al ldquoThe X-linked lymphopro-liferative-disease gene product SAP regulates signals inducedthrough the co-receptor SLAMrdquo Nature vol 395 no 6701 pp462ndash469 1998

[54] S De Jong K R VanEijk DW L H Zeegers et al ldquoExpressionQTL analysis of top loci from GWAS meta-analysis highlightsadditional schizophrenia candidate genesrdquo European Journal ofHuman Genetics vol 20 no 9 pp 1004ndash1008 2012

[55] F Zhang Z Zhang X Yan et al ldquoGenome-wide associationstudies for hematological traits in Chinese Sutai pigsrdquo BMCGenetics vol 15 article no 41 2014

[56] H Kristiansen H H Gad S Eskildsen-Larsen P Despresand R Hartmann ldquoThe oligoadenylate synthetase family anancient protein family withmultiple antiviral activitiesrdquo Journalof Interferon amp Cytokine Research vol 31 no 1 pp 41ndash47 2011

[57] K Thavachelvam H H Gad M S Ibsen et al ldquoRapid uptakeand inhibition of viral propagation by extracellular OAS1rdquoJournal of Interferon ampCytokine Research vol 35 no 5 pp 359ndash366 2015

[58] J E Darnell Jr I M Kerr and G R Stark ldquoJak-STAT pathwaysand transcriptional activation in response to IFNs and otherextracellular signaling proteinsrdquo Science vol 264 no 5164 pp1415ndash1421 1994

[59] L C Platanias ldquoMechanisms of type-I- and type-II-interferon-mediated signallingrdquo Nature Reviews Immunology vol 5 no 5pp 375ndash386 2005

[60] N Arashiki and Y Takakuwa ldquoMaintenance and regulation ofasymmetric phospholipid distribution in human erythrocytemembranes Implications for erythrocyte functionsrdquo CurrentOpinion in Hematology vol 24 no 3 pp 167ndash172 2017

[61] Y Zhao D Pang T Wang et al ldquoHumanMxA protein inhibitsthe replication of classical swine fever virusrdquoVirus Research vol156 no 1-2 pp 151ndash155 2011

[62] J Tao J Liao JWang et al ldquoPig BVDV-2 non-structural protein(Npro) links to cellular antiviral response in vitrordquoVirus Genesvol 53 no 2 pp 233ndash239 2017

[63] O Haller P Staeheli and G Kochs ldquoInterferon-induced Mxproteins in antiviral host defenserdquo Biochimie vol 89 no 6-7 pp812ndash818 2007

[64] K Sasaki P Tungtrakoolsub T Morozumi H Uenishi MKawahara and T Watanabe ldquoA single nucleotide polymor-phism of porcine MX2 gene provides antiviral activity againstvesicular stomatitis virusrdquo Immunogenetics vol 66 no 1 pp25ndash32 2014

[65] P Gaur A Munjhal and S K Lal ldquoInfluenza virus and cellsignaling pathwaysrdquo Medical Science Monitor vol 17 no 6 ppRA148ndashRA154 2011

[66] T Kim S Pazhoor M Bao et al ldquoAspartate-glutamate-alanine-histidine box motif (DEAH)RNA helicase a helicases sense

microbial DNA in human plasmacytoid dendritic cellsrdquo Pro-ceedings of the National Acadamy of Sciences of the United Statesof America vol 107 no 34 pp 15181ndash15186 2010

[67] Z Zhang T Kim M Bao et al ldquoDDX1 DDX21 and DHX36helicases form a complex with the adaptor molecule TRIF tosense dsRNA in dendritic cellsrdquo Immunity vol 34 no 6 pp866ndash878 2011

[68] Y-M Loo J Fornek N Crochet et al ldquoDistinct RIG-I andMDA5 signaling by RNA viruses in innate immunityrdquo Journalof Virology vol 82 no 1 pp 335ndash345 2008

[69] A Zhou B A Hassel and R H Silverman ldquoExpression cloningof 2-5A-dependent RNAase a uniquely regulated mediator ofinterferon actionrdquo Cell vol 72 no 5 pp 753ndash765 1993

[70] A Zhou J Paranjape T L Brown et al ldquoInterferon action andapoptosis are defective in mice devoid of 2101584051015840-oligoadenylate-dependent RNase Lrdquo EMBO Journal vol 16 no 21 pp 6355ndash6363 1997

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


the mechanism of diseases by using the subpopulations ofleucocytes in vivo thereby enabling enhanced investigationsof the immune response to various porcine infections suchas Actinobacillus pleuropneumoniae African swine fevervirus (ASFV) classical swine fever virus (CSFV) porcinereproductive and respiratory syndrome virus (PRRSv) andAujeszky disease virus [9ndash16] Strikingly leucocyte differen-tial count in domestic pig has been associated with multiplechromosomal regions but specific locigenes that can par-tially explain the variation between individuals have not beenidentified [17ndash19]

Increased leucocytes are typically found in pigs withconditions associated with viral respiratory tract infectionafter parainfluenza-3 (PI3) virus [20] ASFV infection leadsto serious changes in the concentration of leucocytes detected2ndash3 d after infection [21] PRRSv infection caused a hugeacute drop in total leucocyte counts that affect all PBMCpopulations by 2 d after infection in pregnant gilts [22]To these infectious diseases the resistance of an individualresulted from both innate and acquired immunity Thecapacity of innate immunity or acquired immunity is moreor less controlled by genes [23ndash25] In pigs genetic studieshave revealed several genes that participate in the resistanceto diseases [26] including single-gene porcine ryanodinereceptor (ryr1) regulating malignant hyperthermia [27] andporcine factor H [28] Nevertheless the identities of the mostregulators of leucocytes related traits and their direct targetsare largely unknown despite their biological functions

To define the molecular mechanisms of general gene-expression profiles or changes during rapid leucocytes turn-overs in pig genome we adapted techniques ofmicroarray forgenetic profiling of pigs with the increased and decreased leu-cocyte counts before and after polyinosinicpolycytidylic acid(poly IC) stimulation Poly IC is a synthetic double-strandedRNA that is used experimentally to model viral infections invivo [29] together with the microarray technology that rep-resents a profiling strategy that allows the detection andmea-sure of various responses by a multitude of gene probes setsWe have identified a number of previously uncharacterizedgenes that appear to be expressed in swine blood leucocyteswhile simultaneously establishing the dominantly expressedgenes of the increased and decreased leucocyte phenotypesas well as immune components involved such as influenzaA pathway cytosolic DNA-sensing pathway chemokine sig-naling pathway and cytokine-cytokine receptor interactionwhich are important in the molecular pathways that regulatewhite cells andor hematopoietic stem cell function in normaland pathologic conditions

2 Materials and Methods

21 Ethics Approval All experimental procedures and ani-mal care activities were strictly conducted in accordancewith the guidelines established for the care and use oflaboratory animals of the Standing Committee of HubeiPeoplersquos Congress (No 5) approved by The Scientific EthicsCommittee ofHuazhongAgriculturalUniversity (HZAUSW-2013-014) Therefore all efforts were made to minimizesuffering

22 Animals Experimental design details of the infectionand blood collection procedures are described in [30] Thepigs used in the study were from a DurocndashErhualian F2population and comprised 392 F2 offspring derived from51 F1 and 26 F0 parents In short 8 Duroc boars weremated to 18 Erhualian sows Then 13 F1 boars and 38 F1sows were chosen and mated to produce 392 F2 animalsThe six animals used in the present study were selectedbased on the two-tailed value of leucocyte counts beforeand after poly IC stimulation All animals are fed in thesame conditions and free of virus infections such as PRRSvMycoplasma hyopneumoniae and swine influenza virus At35 d pigs were intramuscularly infected with poly IC (CASRegistry Number 24939-03-5 Hangzhou Meiya Pharmacy)at a dose of 05mgkg Blood samples were taken at 33 and35 d after poly (IC) infection Leucocytes levels on these dpiwere measured using a Japanese photoelectric MEK-8222Kautomatic 5 classification blood analyzer (Nihon kohdenTokyo Japan)

23 Phenotypic Data The statistical significance of the phe-notypic differences between the treatment and control groupswas first examined by assuming unequal variances Studentrsquost-test The residuals obtained from the linear model werefurther used as a target variable for analysis using t-test andlm functions in the R environment

24 Microarray Design and Hybridization Based on leuco-cyte count recorded at 33 and 35 d after poly (IC) infectiona total of six pigs were divided into two groups and codedas increased responders (IR) and decreased responders (DR)Whole blood samples were collected from each of the six pigs4 h after poly IC treatment on day 35 and at day 33 as anunstimulated control The whole blood samples were usedfor the microarray experiments GeneChip Porcine GenomeArrays (Affymetrix) were used to determine gene expressionlevels in the whole blood samples before and after poly ICtreatment of the six pigs RNA labeling and hybridizationwere performed by a commercial Affymetrix array ser-vice (GeneTech Biotechnology Limited Company ShanghaiChina) This methodology was described in our previousstudy [31] All raw probe-microarray data were normalizedby the Robust Multichip Average method from packages ofBioconductor (httpwwwbioconductororg) implementedin the R environment [32ndash34] The model matrix for differ-entially expressed genes (DEGs) identification was defined ina linear format and the model scenario included the samefactors in the linear model for leucocyte count phenotypeanalysis The linear models for microarray data (LIMMA)[35] were used to identify the DEGs To determine thestatistical significance of the DEGs the cutoff for P values wasset to (001) adjusted P values (01) and fold change ge (20)Furthermore a two-way hierarchical clustering analysis wasperformed to identify the DEGs patterns according to [35]

25 Gene Annotation and Pathway Analysis The Affymetrixporcine genome microarray annotation was initially per-formed using the annotation file given by Affymetrix Inc(httpswwwaffymetrixcomindexaffx) Each probe set was




IR-Upregulated







3 Results





33 days 35 days0

10

50

40

30

20

WBC33daysWBC35days

Method

Cou

nt (

L

)

(a)

0

10

20

30

40

50

60

IR1 IR2 IR3

IRBS IRAS

Leuc

ocyt

e cou

nt (

L

)

(b)

0

2

4

6

8

10

12

14

16

18

DR1 DR2 DR3

DRBS DRAS

Leuc

ocyt

e cou

nt (

L

)

(c)






IR_2245

IR_2278

IR_2152

IR_3169

IR_3287

IR_3255


Color Key IR

500

300

100

0

Cou

nt

minus1 0 1

Row Z-Score

(a)

DR

DR_3055

DR_3229

DR_3230

Stimulated

DR_2007

DR_2039

DR_2204

Unstimulated

Color Key

0

minus1 0 1

Row Z-Score

150

50C

ount

(b)

DR_Up

IR_Up DR_Down

IR_Down

(c)
















MX1 ISG20




0000

0200

0400

0600

0800

1000

1200

1400

1600

1800

Fold

chan

ge

IRBSIRAS

ISG

20

RSA

D2

TLR4

S100

A12

S100

A9

S100

A8

SH2D

1A

TNK

2


lowastlowast

(a)

Fold

chan

ge

0000

2000

4000

6000

8000

10000

12000

DRBSDRAS

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




MX

1

MX

2

TRIM

26

RSA

D2

ISG

20

OA

S1

PLSC

R1

STAT

1

TLR4

(b)


012345


Mic

roar

ray

(Log

2 Fo

ld C

hang

e)


２2= 086

y = 12505x - 03326


0

(c)









Gene count


DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count


DRIR

(b)









IL13RA1





4 Discussion




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





IR-Upregulated







3 Results





33 days 35 days0

10

50

40

30

20

WBC33daysWBC35days

Method

Cou

nt (

L

)

(a)

0

10

20

30

40

50

60

IR1 IR2 IR3

IRBS IRAS

Leuc

ocyt

e cou

nt (

L

)

(b)

0

2

4

6

8

10

12

14

16

18

DR1 DR2 DR3

DRBS DRAS

Leuc

ocyt

e cou

nt (

L

)

(c)






IR_2245

IR_2278

IR_2152

IR_3169

IR_3287

IR_3255


Color Key IR

500

300

100

0

Cou

nt

minus1 0 1

Row Z-Score

(a)

DR

DR_3055

DR_3229

DR_3230

Stimulated

DR_2007

DR_2039

DR_2204

Unstimulated

Color Key

0

minus1 0 1

Row Z-Score

150

50C

ount

(b)

DR_Up

IR_Up DR_Down

IR_Down

(c)
















MX1 ISG20




0000

0200

0400

0600

0800

1000

1200

1400

1600

1800

Fold

chan

ge

IRBSIRAS

ISG

20

RSA

D2

TLR4

S100

A12

S100

A9

S100

A8

SH2D

1A

TNK

2


lowastlowast

(a)

Fold

chan

ge

0000

2000

4000

6000

8000

10000

12000

DRBSDRAS

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




MX

1

MX

2

TRIM

26

RSA

D2

ISG

20

OA

S1

PLSC

R1

STAT

1

TLR4

(b)


012345


Mic

roar

ray

(Log

2 Fo

ld C

hang

e)


２2= 086

y = 12505x - 03326


0

(c)









Gene count


DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count


DRIR

(b)









IL13RA1





4 Discussion




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



33 days 35 days0

10

50

40

30

20

WBC33daysWBC35days

Method

Cou

nt (

L

)

(a)

0

10

20

30

40

50

60

IR1 IR2 IR3

IRBS IRAS

Leuc

ocyt

e cou

nt (

L

)

(b)

0

2

4

6

8

10

12

14

16

18

DR1 DR2 DR3

DRBS DRAS

Leuc

ocyt

e cou

nt (

L

)

(c)






IR_2245

IR_2278

IR_2152

IR_3169

IR_3287

IR_3255


Color Key IR

500

300

100

0

Cou

nt

minus1 0 1

Row Z-Score

(a)

DR

DR_3055

DR_3229

DR_3230

Stimulated

DR_2007

DR_2039

DR_2204

Unstimulated

Color Key

0

minus1 0 1

Row Z-Score

150

50C

ount

(b)

DR_Up

IR_Up DR_Down

IR_Down

(c)
















MX1 ISG20




0000

0200

0400

0600

0800

1000

1200

1400

1600

1800

Fold

chan

ge

IRBSIRAS

ISG

20

RSA

D2

TLR4

S100

A12

S100

A9

S100

A8

SH2D

1A

TNK

2


lowastlowast

(a)

Fold

chan

ge

0000

2000

4000

6000

8000

10000

12000

DRBSDRAS

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




MX

1

MX

2

TRIM

26

RSA

D2

ISG

20

OA

S1

PLSC

R1

STAT

1

TLR4

(b)


012345


Mic

roar

ray

(Log

2 Fo

ld C

hang

e)


２2= 086

y = 12505x - 03326


0

(c)









Gene count


DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count


DRIR

(b)









IL13RA1





4 Discussion




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



IR_2245

IR_2278

IR_2152

IR_3169

IR_3287

IR_3255


Color Key IR

500

300

100

0

Cou

nt

minus1 0 1

Row Z-Score

(a)

DR

DR_3055

DR_3229

DR_3230

Stimulated

DR_2007

DR_2039

DR_2204

Unstimulated

Color Key

0

minus1 0 1

Row Z-Score

150

50C

ount

(b)

DR_Up

IR_Up DR_Down

IR_Down

(c)
















MX1 ISG20




0000

0200

0400

0600

0800

1000

1200

1400

1600

1800

Fold

chan

ge

IRBSIRAS

ISG

20

RSA

D2

TLR4

S100

A12

S100

A9

S100

A8

SH2D

1A

TNK

2


lowastlowast

(a)

Fold

chan

ge

0000

2000

4000

6000

8000

10000

12000

DRBSDRAS

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




MX

1

MX

2

TRIM

26

RSA

D2

ISG

20

OA

S1

PLSC

R1

STAT

1

TLR4

(b)


012345


Mic

roar

ray

(Log

2 Fo

ld C

hang

e)


２2= 086

y = 12505x - 03326


0

(c)









Gene count


DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count


DRIR

(b)









IL13RA1





4 Discussion




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













MX1 ISG20




0000

0200

0400

0600

0800

1000

1200

1400

1600

1800

Fold

chan

ge

IRBSIRAS

ISG

20

RSA

D2

TLR4

S100

A12

S100

A9

S100

A8

SH2D

1A

TNK

2


lowastlowast

(a)

Fold

chan

ge

0000

2000

4000

6000

8000

10000

12000

DRBSDRAS

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




MX

1

MX

2

TRIM

26

RSA

D2

ISG

20

OA

S1

PLSC

R1

STAT

1

TLR4

(b)


012345


Mic

roar

ray

(Log

2 Fo

ld C

hang

e)


２2= 086

y = 12505x - 03326


0

(c)









Gene count


DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count


DRIR

(b)









IL13RA1





4 Discussion




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









Gene count


DR

IR

(a)

0 5 10 15 20 25 30

metal ion binding

ion binding

cation binding

zinc ion binding

Gene count


DRIR

(b)









IL13RA1





4 Discussion




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Fold changeIR vs DR










5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




5 Conclusions


Data Availability




Acknowledgments




References













































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


transcriptional profiling of leucocyte count variation...

Documents