Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs

14 Pages·2016·1.02 MB·English

by  Aimee L. Benjamin

Benjaminetal.BMCGenomics (2016) 17:258
DOI10.1186/s12864-016-2565-x
RESEARCH ARTICLE Open Access
Differential responsiveness of Holstein
and Angus dermal fibroblasts to LPS
challenge occurs without major differences
in the methylome
Aimee L. Benjamin1, Benjamin B. Green2, Brian A. Crooker3, Stephanie D. McKay1 and David E. Kerr1*
Abstract
Background: We have previously found substantial animal-to-animal and age-dependent variation in theresponse
ofHolsteinfibroblastcultures challenged with LPS. Toexpand on this finding,fibroblast cultures were established
from dairy (Holstein) and beef(Angus) cattle and challenged with LPS to examine breed-dependent differences in
theinnate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for
expression differenceswas examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis.
Results: TheHolsteinbreeddisplayedamorerobustresponsetoLPSthantheAngusbreedbasedonRNA-Seq
analysisofcultureschallengedwithLPSfor0,2,and8h.Severalimmune-associatedgeneswereexpressedatgreater
levels(FDR<0.05)inHolsteinculturesincludingTLR4atalltimepointsandanumberofpro-inflammatorygenessuch
asIL8,CCL20,CCL5,andTNFfollowingLPSexposure.Despiteextensivebreeddifferencesinthetranscriptome,MIRA-
Sequnveiledrelativelysimilarpatternsofgenome-wideDNAmethylationbetweenbreeds,withanoverall
hypomethylationofgenepromoters.However,byexaminingthegenomein3Kbwindows,49regionsofdifferential
methylationwerediscoveredbetweenHolsteinandAngusfibroblasts,andtwooftheseregionsfellwithinthe
promoterregion(-2500to+500bpofthetranscriptionstartsite)ofthegenesNTRK2andADAMTS5.
Conclusions:FibroblastsisolatedfromHolsteincattledisplayamorerobustresponsetoLPSincomparisontocultures
fromAnguscattle.Differentselectionstrategiesandmanagementpracticesexistbetweenthesetwobreedsthatlikely
giverisetogeneticandepigeneticfactorscontributingtothedifferentimmuneresponsephenotypes.
Keywords:Innateimmunity,DNAmethylation,RNA-Seq,MIRA-Seq
Background prevalence in beef cows reported a moderate incidence
The innate immune response plays a critical role in with a surprising lack of Escherichia coli induced mas-
pathogen detection and the resulting inflammatory titis [2, 3]. This pathogen is a major cause of mastitis in
response that arises to contain and eliminate foreign in- dairy animals and thus differences in susceptibility to E.
vasions into host tissues. Infection of the mammary coli infection may exist between dairy and beef breeds.
glandresultinginmastitis isanimportant and wellchar- Bovine mammary infections due to E. colitypically cause
acterized disease of dairy cattle, such as the Holstein an acute response with inflammation ranging from mild
breed [1]. However, the incidence and severity of this and quickly resolving, to severe forms that can lead to
disease in beef cattle has received little attention al- sepsis, shock, and even death [4]. The response is initi-
though two recent studies that examined mastitis ated following recognition of E. coli lipopolysaccharide
(LPS) by the extracellular Toll-like receptor 4 (TLR4),
which is expressed on multiple cell types within the
*Correspondence:[email protected] mammarygland[5].
1DepartmentofAnimalandVeterinarySciences,UniversityofVermont,Terrill
Hall,570MainStreet,Burlington,VT05405,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2016Benjaminetal.OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Benjaminetal.BMCGenomics (2016) 17:258 Page2of14
While there are multiple factors that can influence the Holstein (dairy) and Angus (beef) cows with LPS and
inflammatory response to mammary gland infection, it then used RNA-seq to examine global gene expres-
has been suggested that host factors play the largest role sion differences. We also employed MIRA-seq to
in determining the severity of E. coli mastitis [6]. These examine if global differences in the methylome could
hostfactorsincludeage,stageoflactation,vaccinationhis- explain differences in the transcriptome.
tory, as well as genetic, and possibly epigenetic mecha-
nisms all contributing to the magnitude and resolution of Results
the host response. Identification of genetic or epigenetic FibroblastIL-8response
factorscontrollingtheinflammatoryresponsemayleadto Dermal fibroblasts (DF) isolated from Holstein and
enhanced selection or management strategies to produce Angus heifers were used to investigate differences in
dairy animals that have reduced collateral damage from the innate immune response between dairy and beef
mastitis. For example, genetic polymorphisms within breeds. Fibroblasts isolated from Holsteins produced
pathogen recognition receptors or signaling pathway in- considerably higher levels of IL8 (P<0.01) in response
termediateshavebeenshowntoleadtodifferencesindis- to a 24 h LPS (100ng/ml) challenge (Fig. 1a) when
ease susceptibility [7, 8]. Epigenetic mechanisms, such as compared to Angus cultures (703±86 pg/ml vs. 322
DNAmethylationorhistonemodificationscan alsoinflu- ±46 pg/ml, respectively). A similar pattern was
ence gene expression resulting in phenotypic variation in observed following IL1B (1ng/ml) treatment (Fig. 1a),
immune response [9]. For example, human intestinal epi- with Holstein DF having a greater magnitude of IL8
thelial cells have increased DNA methylation in theTLR4 response than Angus DF (1355±160 pg/ml vs. 639±
gene promoter that reduces its expression and leads to a 80 pg/ml, respectively). There was no detectable IL8
reducedresponsivenesstoLPS[10]. in media collected from un-stimulated Holstein or
Selective breeding of cattle has been employed for Angus fibroblasts.
many generations to produce breeds differing in RNA-Seq and MIRA- Seq analysis was performed on
economically important traits such as enhanced milk eight cultures (n=4/breed) randomly selected from the
production in dairy breeds, and greater feed efficiency pool of animals. These cultures were revived simultan-
and meat quality in beef breeds [11, 12]. Cattle eously from cryopreservation and prepared for a second
breeding has even resulted in sub-species (Bos taurus challenge with LPS and IL1B. TheHolstein cultures pro-
and Bos indicus) that differ markedly in resistance to duced approximately twice (P<0.01) as much IL8 pro-
disease such as that caused by tick infestation [13, tein in response to LPS compared to the Angus cultures
14] or challenge with Theileria annulata [15]. Results (Fig. 1b). Similarly, differential IL8 responses were ob-
from these studies suggest that B. taurus animals de- served following IL1B exposure, with Holstein cultures
velop a greater inflammatory response to pathogens exhibiting a hyper-responsive phenotype in IL8 protein
than do breeds of the B. indicus subspecies. Within production(Fig. 1b).
the B. taurus sub-breed, studies directly comparing
the inflammatory response to mammary infection be- RNA-SeqanalysisofHolsteinandangusLPSresponse
tween dairy and beef breeds have not been con- Total RNA samples for RNA-Seq analysis were obtained
ducted. To further investigate potential differences in from the four Holstein and four Angus cultures follow-
immune responses between dairy and beef animals, ing LPS exposure for 0, 2, and 8 h. These 24 samples
we challenged dermal fibroblasts obtained from generated approximately 54 million reads per sample
A B
**
2000 2000
Holstein ** Holstein
Angus Angus
1500 1500
8 (pg/ml) 1000 ** 8 (pg/ml) 1000 **
IL IL
500 500
0 0
LPS IL1B LPS IL1B
Fig.1IL8proteinproductionfromHolsteinandAngusfibroblasts.aIL8productionbydermalfibroblastsisolatedfrom19-montholdHolstein(n=5)
andAngus(n=12)heifersandchallengedfor24hwithLPS(100ng/ml)orIL1B(1ng/ml).bEightcultures(n=4/breed)wererandomlychosenfor
RNA-seqandMIRA-seqanalysisandlevelsofIL8proteinfollowingasecond24hchallengewitheitherLPS(100ng/ml)orIL1B(1ng/ml)onthese8
culturesareshown.TherewasnodetectableIL8productionfromun-treatedculturesofbothbreeds.Valuesaremean±SEM.*indicatesP<0.05,**
indicatesP<0.01
Benjaminetal.BMCGenomics (2016) 17:258 Page3of14
following quality control. Alignment to the UMD v3.1 various innate immune-associated genes in the LPS sig-
bovine genome resulted in 95 % of the reads falling naling pathway and those coding for response proteins
within alignment parameters (see Methods), thus an (Table 1). There were temporal differences in the induc-
average of 51 million reads per sample. Under our defin- tion of these genes following LPS exposure for 2 or 8 h.
ition of expression (CPM>1 in at least 50 % of the sam- Early induced genes included several pro-inflammatory
ples), there were 20,356 targets detected at hour 0, chemokines and cytokines such IL8, IL1A, TNF, chemo-
which defined the core bovine fibroblast transcriptome kine (C-X-C) motif ligand (CXCL) 2, and Interferon regu-
under basal conditions. At hours 2 and 8 following LPS latory factors (IRF) 1 and 5. Following 8 h of LPS
treatment, there were 21,411 and 21,590 targets de- exposure, several Type I IFN-related genes showed
tected, respectively. By combining breeds, and compar- greater expression compared to hour 0, while TNF ex-
ing basal gene expression to time points following LPS- pression hadreturnedtobasallevels.
induced innate immune response, 624 transcripts were A comparison between Holstein and Angus cultures
revealed to have differential gene expression (FDR< revealed 844, 968, and 730 differentially expressed (DE)
0.05; CPM>1; FC≥2) with 470 up- and 154 down- genes(FDR<0.05; CPM>1;FC≥2)athours 0,2,and8-
regulated at hour 2 post-LPS (Additional file 1). At hour post LPS, respectively (Fig. 2; Additional file 3). Of these
8 post-LPS, 331 transcripts were discovered having dif- genes, 369, 517, and 477, respectively, had higher ex-
ferential gene expression compared to hour 0, of which, pression levels in Holstein cultures compared to Angus
250 were up- and 81 were down- regulated (Additional cultures. Pathway enrichment analysis was completed
file 2). LPS treatment of the fibroblast cultures induced with DAVID and genes with differential expression
Table1AverageresponsetoLPSfromcombinedHolsteinandAngusculturesmeasuredbyincreasesinexpressionofimmune-
associatedgenescomparedtohour0post-LPS
Genesymbol GeneName FoldChangea
Transcriptionandactivationpathways Hour2 Hour8
BIRC3 BaculoviralIAPrepeat-containingprotein3 7.3 4.2
NFKBIA NuclearfactorofkappalightpolypeptidegeneenhancerinB-cellsinhibitor,alpha 24.3 9.3
NFKBIZ Nuclearfactorofkappa-Binhibitorzeta 11.7 3.2
NFKB2 NuclearfactorofkappalightpolypeptidegeneenhancerinB-cells2 -- 2.8
NFKBID NuclearfactorofkappalightpolypeptidegeneenhancerinB-cellsinhibitor,delta 2.3 --
Cytokines,chemokines,
andgrowthfactors
CCL2 Chemokine(C-Cmotif)ligand2 14.9 14.6
CCL5 RANTES 49.6 132.8
CCL20 Chemokine(C-Cmotif)ligand20 1102.1 3147.3
CXCL2 Chemokine(C-X-Cmotif)ligand2 67.9 31.6
CXCL6 Chemokine(C-X-Cmotif)ligand6 12.3 45.0
IL1A Interleukin1,alpha 9.2 5.7
IL6 Interleukin6 88.2 124.1
IL8 Interleukin8 325.7 387.8
SAA3 SerumAmyloidA3 65.1 1145.6
TNF TumorNecrosisFactor,alpha 87.4 --
TypeIIFN-relatedgenes
IRF1 Interferonregulatoryfactor1 12.3 4.2
IRF5 Interferonregulatoryfactor5 3.0 --
ISG15 Ubiquitin-likemodifier -- 308.3
MX2 Myxovirus(influenzavirus)resistance2 -- 156.6
OAS1 2'–5'-oligoadenylatesynthetase1 -- 128.7
OAS2 2'–5'-oligoadenylatesynthetase2 -- 65.6
aDataobtainedbyRNA-Seqandpresentedasfoldinductionoftheindicatedgeneateither2or8hpost-LPSincomparisontoexpressionlevelsat0hpost-LPS.
AllfoldchangesshownareFDR<0.05;FC>2;andCPM>1.--indicatesFDR>0.05,FC<2,orCPM<1
Benjaminetal.BMCGenomics (2016) 17:258 Page4of14
genes that are involved in the innate immune response.
A
TLR4, NOD1, and TRAF1, genes involved withthe recog-
6 nitionofpathogenassociatedmolecularpatternsandacti-
Hour 0
ge 4 vationofNFκBpathway,weremoreexpressedinHolstein
an cultures. Chemokines such as chemokine (C-C motif)
h
d c 2 ligand(CCL)2,5,and20,andIL8thatrecruitmonocytes,
ol Tcells, lymphocytes, and neutrophils to sites of infection
ogf2 0 also had higher expression levels in Holstein fibroblasts.
l -2
In addition to genes related to the innate immune re-
-4 sponse,differenceswereobservedingenesthathavebeen
implicated in DNA methylation, such as the family of
DNAmethyltransferase(DNMT)genesandubiquitin-like
0 5 10 15
with PHD and RING finger domains 1 (UHRF1) [16, 17].
log CPM
2 Expression of DNMT3A and 3B, which are involved with
B de novo DNA methylation, were similar between the
breeds. However, expression ofDNMT1, which is respon-
Hour 2
6 sible for maintenance of DNA methylation patterns fol-
lowingDNAreplication,was1.6-foldhigherathour0and
e 4
g
n 1.5-fold higher at hours 2 and 8 in Angus cultures com-
a
d ch 2 pared to Holstein cultures. Angus cultures also had 2.3–,
ol 2.0-,and2.0-foldhigherexpressionofUHRF1at0,2,and
ogf2 0 8hpost-LPStreatment(Table2).Interestingly,greaterac-
l -2
tivation of a potentially anti-inflammatory signaling path-
-4 way in Angus cultures was noted by the 3.1-fold higher
expressionofthegeneencodingchemokine(C-X-Cmotif)
0 5 10 15
receptor 4 (CXCR4) at hour 0 compared to Holsteins.
log2 CPM WhileCXCR4expressionwasnotdifferentathours2and
C 8 post-LPS, the sole ligand of this receptor, CXCL12, was
6 expressed5.7–,7.4-,and6.5-foldhigherinAnguscultures
Hour 8 athour0andat2and8hpostLPSstimulation.
e 4
g
n
a
ch 2
d RT-qPCRconfirmationofRNA-Seq
ol
gf2 0 Several immune-response associated genes were selected
o for RT-qPCR confirmation of expression differences ob-
l
-2 served between Holstein and Angus cultures following
LPS treatment as determined by RNA-Seq. TLR4, IL8,
-4
CCL20,CCL5, and TNFshowed similar expression levels
0 5 10 15 inRT-qPCRanalysisincomparisontotheRNA-Seqdata
log2 CPM set. Basal expression of IL8,CCL20,CCL5, and TNF was
Fig.2ScatterplotsofRNA-Seqanalysis.Eightfibroblastcultures(fourof similar between the breeds. However, by 2 h after LPS
eachbreed)wereusedinRNA-Seqtoexploregeneexpression stimulation, marked differences were observed between
differences.ScatterplotsofindicesanalyzedfromRNA-Seqdata Holstein and Angus expression of IL8,TNF, CCL20, and
weregeneratedforexpressionlevel(log2CPM)anddifferential CCL5, with a 3.0–, 24.3-, 4.9-, and 3.9- fold higher ex-
expression(log FoldChange)at0h(a),2h(b),and8h(c)following
2 pression in Holstein fibroblasts compared to Angus
LPSexposure.Positivefoldchangevaluesindicatehigherexpressionin
Holsteincultureswhilenegativefoldchangevaluesshowhigher fibroblasts (Fig. 3 b, c, d, and e). A similar pattern was
expressioninAnguscultures.ReddotsdenoteFDR<0.05 observedat 8h, with Holstein cultureshaving higher ex-
pression in the following genes: IL8 (13.1-fold), TNF
between the breeds at 2 h post-LPS were involved with (13.0-fold),CCL20(35.3-fold), andCCL5(17.8-fold). LPS
the MAPK (P=0.029) and TGF-beta (P=0.031) signaling exposure did not induceTLR4 expression in either breed;
pathways. By 8 h post-LPS, the NOD-like (P=0.003), however, Holstein cultures consistently expressed higher
Toll-like (P=0.02), and RIG-I-like (P=0.02) receptor sig- levels of TLR4 compared to Angus cultures (Fig. 3a; 4.5–,
naling pathways were represented in our set of DE genes 5.7-,and3.1-foldhigherathours0,2,and8,respectively).
between the two breeds. Table 2 lists several of the DE Ingeneral,geneexpressionvaluesdeterminedbyRT-qPCR
Benjaminetal.BMCGenomics (2016) 17:258 Page5of14
Table2Differentialgeneexpression(foldchange)of To determine differential methylation rates between
immune-associatedgenesathours0,2,and8post-LPStreatment Holstein and Angus fibroblasts, the genome was divided
offibroblastscollectedfromHolsteinanimalsascomparedto into 3Kb windows, and then the read count in each win-
fibroblastscollectedfromAngusanimals dow was compared. Analysis of the 3Kb windows re-
Gene Hour0 Hour2 Hour8 vealed 49 regions that had different levels of methylation
Innateimmunerelatedgenes between Holstein and Angus cultures based on the fol-
CCL2 -- -- 2.9 lowing thresholds: FDR<0.1; CPM>1; FC≥2 (Table 3).
Ofthesedifferentially methylated regions (DMR), 24 had
CCL5 -- -- 5.7
higher ratesofmethylation inculturesfromHolsteinan-
CCL20 -- -- 8.6
imals, while 25 displayed higher methylation levels in
CCL26 -- 2.8 3.6
Angus fibroblasts. Of the 24 regions with higher methy-
CX3CL1 -- -- 14.8 lation in Holstein, 14 were found partially within an an-
CXCL12 −5.7 −7.4 −6.5 notated gene while 10 were not. Within the 25 DMR
CXCL16 -- -- 2.3 with higher methylation levels in Angus cultures 13 fell
CXCR4 −3.1 -- -- within annotated genes while the remaining 12 regions
did not. Only two of the DMR (greater methylation in
HMGB1 −1.9 −1.9 −1.8
Angus cultures) were located within the defined pro-
IFIH1 -- -- 3.6
moter regions of genes: a disintegrin and metalloprotease
IL1R1 -- 1.5 -- with thrombospondin motifs 5 (ADAMTS5) and neuro-
IL8 -- -- 4.5 trophic tyrosine kinase, receptor type 2 (NTRK2). These
IRF5 2.4 2.4 2.6 DMRs were not reflected by differences in expression of
either gene between the Holstein and Angus cultures at
MX2 -- -- 14.0
hours0,2,and8post-LPS(Table3).ADMRspanningin-
NOD1 -- 2.4 --
tron 3 and exon 4 of the agouti signaling protein (ASIP)
TLR2 −3.5 −2.8 --
gene had greater methylation in the Angus cells and was
TLR4 3.0 4.0 2.5 associated with a marked increase in its expression in
TLR6 1.8 -- 2.5 thesecellsatalltimepoints.
TNF -- 6.9 --
InfluenceofDNAmethylationongeneexpression
TNFSF18 6.5 6.0 5.1
The genome-wide association between gene expression
TNFSF4 4.4 -- 2.6
and methylation levels within a gene body or gene pro-
TRAF1 2.4 2.4 --
moter region was investigated by plotting MIRA-Seq
TRAIP -- −2.7 -- values for a gene body or gene promoter against RNA-
DNAMethylationassociatedgenes Seq values for 8 h post-LPS. As data from the two
DNMT1 −1.6 −1.5 −1.5 breeds appeared similar, the combined data points from
both the Holstein and Angus cultures were binned as ei-
DNMT3A -- -- --
ther low or high, with a gating value of reads per kilo-
DNMT3B -- -- --
base per million matched reads (RPKM) =5for RNA-seq
UHRF1 −2.3 −2.0 −2.0
or RPKM=0.5 for MIRA-Seq. Whentherelationshipbe-
DataobtainedbyRNA-SeqandpresentedasfoldchangebetweenHolsteinand tween methylation levels in either the gene promoter re-
Anguscultures.PositivevaluesindicatehigherexpressioninHolsteinculturesas
gion or the gene body and that gene’s subsequent
comparedtoAnguscultures.AllpresentedvaluesindicateFDR<0.05withan
averageCPM>1,while--notesavalueswitheitherFDR>0.05oranaverage expression level was analyzed, it revealed the two values
CPM<1
weresignificantlydependentupononeanother(P<0.001;
O.R. = 0.83; 95 % C.I. = 0.76–0.89 and P<0.001;
are in agreement with the transcriptomic results from O.R. = 1.26; 95 % C.I. = 1.14–1.40), for gene pro-
RNA-Seqdata. moter (Fig. 4a) and gene body (Fig. 4b), respectively.
This indicates a strong inverse relationship between
MIRA-Seq methylation in either the gene promoter or gene
The 8 DNA samples (4 per breed) processed for MIRA- body and the level of gene expression. Analysis of
Seq analysis resulted in approximately 44 million reads the 0- and 2-h time points following LPS treatment
per sample following quality control. Alignment to the gave similar results (data not shown).
bovine UMD v3.1 reference genome yielded 97 % of Levels of DNA methylation were also measured, as
reads falling within alignment parameters (see Methods) RPKM, in gene promoters, gene bodies, or intergenic re-
and an average of 43 million mapped reads per sample. gions (Fig. 4c). Data from un-treated cultures of the 4
Benjaminetal.BMCGenomics (2016) 17:258 Page6of14
A TLR4 B IL-8 C TNF
0 Holstein 0 Holstein ** 0 Holstein
Angus
Angus Angus
**
Ct 10 (4.*5 ) (5*.7 ) (3.1) Ct 10 (3.0) (13.1) Ct 10 (24.3)
delta delta delta (13**. 0)
20 20 20
30 30 30
0 2 4 6 8 0 2 4 6 8 0 2 4 6 8
Hours Post LPS Hours Post LPS Hours Post LPS
D CCL20 E CCL5
0 Holstein 0 Holstein *
Angus Angus
*
10 (35.3) 10
elta Ct (4.9) elta Ct (3.9) (17.8)
d 20 d 20
30 30
0 2 4 6 8 0 2 4 6 8
Hours Post LPS Hours Post LPS
Fig.3SelectedgeneexpressionbyHolsteinandAngusfibroblastsinresponsetoLPS.DifferencesinexpressionofTLR4(a)IL8(b)TNF(c)CCL20
(d)andCCL5(e)betweenHolsteinandAngusfibroblastculturesathours0,2,and8post-LPSexposureweredeterminedbyRT-qPCR.Valuesare
expressedasdeltaCt,orthedifferenceincyclestothreshold(Ct)betweenthegeneofinterestandtheendogenousgenecontrolbeta-actin
(ACTB).Folddifferences(2-ΔΔCt)inexpressionbetweenHolsteinandAngusculturesareindicatedinparentheses.Allvaluesaremean±SEM.
(n=4/group).*P<0.05and**P<0.01(t-test)
Holstein and 4 Angus were combined for this analysis, determined by a lower milk somatic cell count. This re-
as there were no significant breed differences. Levels of duced response was accompanied by similar bacterial
methylation in all three regions were significantly differ- clearance rates between the phenotypes, suggesting the
ent (P<0.05), with gene promoters having the lowest heightened response of the high responding animals did
and intergenic regions having the highest levels of not offer any advantage in resolution of the infection.
methylation. There is limited data to suggest that a high response
phenotype is characteristic of dairy in comparison to beef
Discussion breedsofcattle[13–15]andthus,inthecurrentstudy,we
Variation exists between cows in their innate immune re- have expanded our use of the fibroblast model to explore
sponse to mastitis. A greater understanding of the basis differences between a dairy breed (Holstein) and a beef
for this variation could facilitate new breeding, selection, breed(Angus)intheirinnateresponsestoLPSexposure.
ormanagementpracticestodevelopanimalswithsuperior Previous studies have looked at differences in the in-
ability to contain and eliminate pathogens from the nor- nate immune response between dairy breeds (Holstein
mally sterile mammary gland. We have previously used and Jersey) following an intra-mammary challenge with
dermal fibroblasts to model this animal variation within S. aureus [20] or E. coli [21]. While there was high inter-
groups of Holstein cows [18, 19]. In these studies we animal variation in milk levels of IL8 and TNF during
found substantial differences between dairy cows in the the challenge,theoverallresponsesweresimilarbetween
magnitude of their fibroblasts’ response following treat- Holstein and Jersey animals. This suggested that the in-
ment with LPS or PAM2CSK4 that are ligands for TLR4 nate immune response is conserved between these two
and TLR2/6, respectively. Intra-mammary bacterial chal- dairy breeds. Likewise, an intravenous LPS challenge
lengeswerealsoconductedongroupsoflactatinganimals comparing beef breeds (Angus and Romosinano) re-
from these studies that had been classified as having low vealed small differences in the serum response profiles
or high responding phenotypes. Low responding animals of TNF and IL1B [22]. Each of these studies examined
developedareducedinflammatoryresponse characterized differences within dairy or within beef breeds, while few
by less leakage of bovine serum albumin (BSA) into milk, studies have explored differences in innate immune re-
as well as a less severe reduction in milk quality, as sponses between a dairy and a beef breed. In one such
Benjaminetal.BMCGenomics (2016) 17:258 Page7of14
Table3Differentiallymethylated3KbregionsbetweenHolsteinandAngusculturesasdeterminedbyMIRA-Seqandgeneexpression
dataforannotatedgenes
RNA-Seqfoldchangec
FCa Chr Genomiccoordinates Geneb Hour0 Hour2 Hour8
−2.9 1 8811701–8814700 ADAMTS5 -- -- --
4.2 1 4686501–4689500 LOC100848874 -- -- --
2.0 1 8325701–8328700 LOC526789 -- -- --
−3.4 2 88463334–88466333 -- -- -- --
−3.4 2 88469334–88472333 -- -- -- --
2.6 2 18491734–18494733 OSBPL6 -- 1.4 1.5
2.0 5 116626706–116629705 FBLN1 -- -- --
2.3 6 5529182–5532181 --- -- -- --
−8.3 7 18532846–18535845 LOC100337044 -- -- --
2.9 8 23178787–23181786 LOC100300143 -- -- --
−2.6 8 34929387–34932386 -- -- -- --
−2.7 8 78669587–78672586 -- -- -- --
−2.8 8 12568687–12571686 -- -- -- --
−4.0 8 79335787–79338786 NTRK2 -- -- --
−2.6 8 98801087–98804086 -- -- -- --
−2.6 9 20444051–20447050 -- -- -- --
−2.7 9 93571151–93574150 -- -- -- --
−4.0 9 88292251–88295250 ULBP1 -- -- --
−2.7 10 32127701–32130700 -- -- -- --
2.1 12 51081922–51084921 -- -- -- --
−2.5 13 64236797–64239796 ASIP −14.0 −15.2 −13.4
2.8 13 48576797–48579796 LRRN4 -- -- --
2.6 14 36500947–36503946 --- -- -- --
2.1 15 53581057–53584156 LOC100336675 -- -- --
−2.3 15 9012857–9015856 -- -- -- --
−7.5 15 27535857–27538856 -- -- -- --
−2.8 15 55516557–55519556 SERPINH1 -- -- --
−2.4 17 7224294–7227293 MAB21L2 -- -- --
2.2 17 17977294–17980293 MAML3 -- -- --
−2.8 18 60220698–60223697 LOC100848332 -- -- --
−3.6 18 63217498–63220497 LOC783134 -- -- --
3.4 18 33452798–33455797 -- -- -- --
2.4 19 42556475–42559474 -- -- -- --
2.3 19 62858675–62861674 AXIN2 -- -- --
−2.4 19 43381475–43384474 CNTNAP1 -- 1.8 2.0
−3.6 19 23655275–23658274 HIC1 -- 1.7 --
2.2 19 42109475–42112474 KRTAP9-2 -- -- --
2.4 19 62705675–62708674 RGS9 2.0 2.1 2.5
4.8 23 49777993–49780992 --- --- --- ---
2.8 24 59112931–59115930 CCBE1 −2.3 −2.2 −1.8
2.4 24 62388931–62391930 --- --- --- ---
2.5 25 27266601–27269600 --- --- --- ---
Benjaminetal.BMCGenomics (2016) 17:258 Page8of14
Table3Differentiallymethylated3KbregionsbetweenHolsteinandAngusculturesasdeterminedbyMIRA-Seqandgeneexpression
dataforannotatedgenes(Continued)
2.4 26 23658031–23661030 C26H10orf26 --- --- 1.4
−2.4 26 16776031–16779030 LOC100848660 --- --- ---
3.2 27 23720367–23723366 --- --- --- ---
−2.4 X 63726195–63729194 LOC100848206 2.4 2.6 2.3
2.1 X 111529495–111532494 LOC516666 --- --- ---
2.1 X 128195395–128198394 --- --- --- ---
−2.6 X 55935095–55938094 --- --- --- ---
aDataobtainedbyMIRA-Seqandpresentedasfolddifferenceinreadcountofmethylatedregions.Positivefoldchangeindicateshighermethylationlevelsin
HolsteincultureswhilenegativevaluesarehighermethylationinAnguscultures
bDMRswithanassociatedgeneindicatethatsomeportionofthe3Kbregionfallswithinanannotatedgene,while--indicatesDMRsthatareintergenic.Bolded
genenamesindicatethatthediscoveredDMRfellwithinthepromoterregionofthatgene(-2500to+500bpfromgenetranscriptionstartsite)
cPositivevaluesindicategreaterexpressioninHolsteinculturesascomparedtoAngus
study, skin biopsies were collected from Holstein (B. may suggest the Holstein animals will detect and respond
taurus) and Brahman (B. indicus) animals following an to Gram-negative bacteria more vigorously than Angus
experimental tick challenge to compare localized innate animals. Additionally, several subunits of the NFKB tran-
immune responses at tick-attachment sites [14]. Micro- scription factor complex were differentially expressed be-
array analysis of the biopsies revealed that Holstein tween breeds. Specifically, Holstein cultures had greater
animals had greater expression of many innate immune expression of RELB (1.8-fold) at hours 0 and 2, and
response-associated genes in comparison to the Brahmans. NFKB1 (1.6-fold) and NFKB2 (1.8-fold) at hour 8 post
It was suggested that the greater expression of chemokine LPS treatment. This transcription factor is central to in-
and cytokinesat tick-attachment sitesmightfacilitatefeed- nate immune signaling pathways leading from a diverse
ing by the tick, thereby leading to the lower resistance to array of pattern recognition receptors including the TLR
tick infestations observed in the Holstein breed. Similarly, and NOD receptor families [25]. Once activated, NFKB
Holstein and Sahiwal (B. indicus) calves were used to ex- promotes the transcription of chemokine and cytokine
plore breed differences in the inflammatory response [15]. genes associated with the innate immune system. While
Inthatstudy,calvesfrombothbreedsunderwentanexperi- the activity of NFKB is carefully regulated under normal
mental challenge with Theileria annulata, the protozoan conditions, excessive production of pro-inflammatory cy-
parasite that causes bovine theileriosis. Holstein calves ex- tokinescanleadtoitsfurtheractivationwhichmaycreate
periencedsevere clinicalsymptomsfollowingthe challenge achronicinflammatorycondition[26].Thehigherexpres-
while Sahiwal calves seemed better able to control the in- sion of both TLR4 and the various subunits of NFKB
flammatoryresponse.Furthermore,wehavepreviouslypre- complex in the Holstein fibroblasts would enable the
senteddatafromarelatedexperiment,nowbeingprepared greaterresponsivenesstoLPSobservedinthisbreed.
for full publication, indicating that Holstein heifers have Anappropriateinnateimmuneresponseisalsodependent
greaterserumIL6andTNFlevelsafteranintravenousLPS on timely production of anti-inflammatory factors once the
challenge compared to age-matched Angus heifers [23]. In stimulatory agent has been eliminated. The chemokine re-
ourcurrentstudy,acomparisonofHolsteinandAngus ceptor4(CXCR4)anditssoleligand,CXCL12areimportant
fibroblasts revealed significant breed differences in re- in this regard and their interaction has been shown to sup-
sponse to an LPS challenge, with Holstein cultures press TLR4 signaling and NFKB activation in response to
exhibiting a hyper-responsive phenotype compared to LPS in human embryonic kidney cells [27] and to suppress
thehypo-responsivenessofAnguscultures. LPS-induced IL6 production by murine bone marrow-
derivedmacrophages[28].TheCXCL12chemokinehasthe
Holsteinvs.AngusdifferencesinLPSresponse abilityto activate anti-inflammatorypathwaysinT-cells and
RNA-Seq analysis of LPS-challenged fibroblasts revealed a macrophages and to suppress inflammation [29, 30]. A sin-
largenumberofimmune-associateddifferentiallyexpressed gle injection of a CXCL12 analog administered to mice in
genesbetweenHolsteinandAnguscultures.Manyofthese parallel with LPS was able to decrease the levels of LPS-
genesareknowntoplaycriticalrolesintheresponseto— induced plasma TNF [28]. Within our fibroblasts, Angus
andtherecoveryfrom—bacterialinfectionssuchasE.coli cultures expressed greater levels of CXCR4 and CXCL12
mastitis[6,24].Inparticular,TLR4,whichistheextracellu- prior to LPS treatment. At hours 2 and 8 post-LPS,
lar receptor responsible for recognition of LPS, was CXCL12expressioncontinuedtobehigherinAnguswhile
expressed at higher levels in Holstein cultures before (4.5 the differences in CXCR4 expression disappeared. Due to
fold)andfollowingLPSstimulation(5.7and3.1fold).This theinhibitoryfunctionofCXCR4andCXCL12,thehigher
Benjaminetal.BMCGenomics (2016) 17:258 Page9of14
A Fig.4RoleofDNAmethylationongeneexpressionanddifferential
methylationlevelsduetogenomiclocation.Astherewereno
1000
significanteffectsofbreedonthecorrelationsbetweengenomic
locationofDNAmethylationandsubsequentgeneexpression,data
wascombinedforthe4Holsteinand4Angusculturesinthis
800
analysis.Scatterplotsshowtherelationbetweengeneexpressionat
M) 8hpost-LPStreatmentandlevelsofDNAmethylationatthegene’s
K 600 promoter(a)orgenebody(b)Geneexpressionlevels(RNA-Seq)
P andDNAmethylation(MIRA-Seq)werenormalizedtogenesizeand
R
( presentedasRPKM.cTheaveragemethylationlevelsofcombined
n
ssio 400 HLeotltsetreiningadnednAontegsudsifMfeIrReAn-tSiaelqmliebtrharyileastiobnasleedveulspoasnmgeenaosumreicdrbegyiaon.
e
r one-wayANOVAwithBonferonnipost-test(p<0.05).Allvalues
p
Ex 200 aremean±SEM
e
n
e
G
0 basalexpressionofthesetwogenesinAngusculturesmay
0 1 2 3 4 5 havereducedtheirLPSresponsiveness.
Breed differences in the expression of genes associated
Promoter Methylation (RPKM)
withthemaintenanceofDNAmethylationwerealsoun-
B
covered in RNA-Seq analysis. DNMT1 and UHRF1 were
1000
more highly expressed in Angus cultures both basally
and following LPS exposure. DNMT1 is responsible for
800 maintaining DNA methylation patterns following repli-
cation [16], and UHRF1 interacts with hemimethylated
)
M
DNA and recruits DNMT1 to facilitate methylation of
K 600
P the daughter strand [17]. Greater expression of these
R
( two genes in Angus cultures may indicate this breed is
n
sio 400 better able to maintain methylation levels. Increased
es methylation of gene promoter regions is generally asso-
r
xp ciated with reduced gene transcription and thus could
E 200
e account for the reduced expression of pro-inflammatory
n
e genesinfibroblastsfrom this breed.
G
0
0 10 20 30 40 50 Methylationdifferencesbetweenbreeds
The breed comparison also included an examination into
Gene Body Methylation (RPKM)
thepotentialrolethatdifferencesinearlylifeenvironment
C mightplayindeterminingbreeddifferencesinananimal’s
1.0 epigenome. Holstein calves are removed from their dams
within24hof birth,movedtoindividualhousing,andfed
c a milk replacer diet; while Angus calves are allowed to
0.8
nurse from the dam for months and are typically housed
b withtheherdoutonpasture.Theseenvironmentaldiffer-
0.6 ences could affect DNA-methylation patterns and lead to
M
differences in gene transcription in response to immune
K a
P stimulation. The MIRA-Seq analysis of Holstein and
R 0.4
Angus fibroblasts revealed 49 3Kb regions of the genome
that were differentially methylated between breeds
0.2 (Table3).Inaglobalsense,thebreeddifferencewasmin-
imaldespitethehigherexpressionofDNMT1andUHRF1
in Angus cultures. Unexpectedly, genes involved in the
0.0
LPS-response pathway were under-represented in these
Promoter Gene Body Intergenic
49 regions. While it would appear that DNA methylation
does not play a major role in the differential breed re-
sponse to LPS observed in our fibroblast model, MIRA-
Seq is only a moderate-resolution technique and a
Benjaminetal.BMCGenomics (2016) 17:258 Page10of14
potentiallimitationof the current study.TheMIRA tech- young calves to avoid raising high response animals that
nique is based on the high affinity of a two-protein com- aremorelikelytodevelopsevereratherthanmilderforms
plex formed by methyl-CpG-binding proteins (MBD) 2b of coliform mastitis. Alternatively, selective modification
and 3L1, which bind to methylated DNA in a methylated of the Holstein genome with appropriate Angus genes
CpGdensity-dependentmanner[31].Regionsoffragmen- maybeusedtogenerateHolsteinanimalswithareduced,
ted DNA with greater methylated CpG content, such as potentiallybeneficial,innateresponsetoinfection.
CpG islands, will have enhanced affinity for the MBD
complex, allowing for enrichment of these regions in the Conclusions
final analysis, whereas regions with low CpG content will Our previous work on characterizing within-breed vari-
be under-represented. While this allows for a comparison ation in innate immune response has now been ex-
oftheoverallmethylomebetweenindividuals,MIRA-Seqis tended to reveal substantial differences between a dairy
not sensitive enough to detect more subtle, but influential, and a beef breed. Our fibroblast model system indicates
differences in methylation at specific CpG sites. For ex- that divergent selection for dairy or beef character has
ample, differential methylation at a single CpG site in the resultedinsubstantialdifferencesinimmunephenotypes
IL6 gene promoter was attributed to differential serum between these breeds, with dairy animals being more re-
levelsofIL6inpatientssufferingfromrheumatoidarthritis sponsive to LPS than beef animals. Genetic differences
[32]. Patients that were experiencing elevated serum levels between the dairy and beef breeds are likely of key im-
of IL6 had lower methylation rates at a single CpG site in portance,butmanagementdifferencesresultingindiffer-
the gene promoter, suggesting that methylation at this site ing in utero and neonatal environments may lead to
represses IL6 transcription. Subtle differences in CpG epigenetic differences as well. Events such as health of
methylation between Holstein and Angus cultures may the dam during pregnancy or the level of maternal care
havebeenlostinouranalysis.Futurestudiesofmethylation a calf receives in early life may lead to epigenetic modifi-
differences between cattle breeds would benefit from a cations that alter that animal’s innate immune response
technology with greater resolving power such as those phenotype as an adult. Our current studies also provide
basedonsequencingofbisulfiteconvertedDNA[33]. a set of gene expression biomarkers, measurable in der-
mal fibroblasts, which are indicative of an animal’s in-
Identificationofbiomarkerslinkedtophenotypic nateimmuneresponsemagnitude.
responses
Previously, we have demonstrated age-dependent in- Methods
creases in LPS-responsiveness of fibroblasts collected Animalsandexperimentalset-up
from the same animals at 5 and 16 months of age [34]. Femaleanimalsoftwocattlebreedswereusedinthisstudy:
Cultures isolated from young (5-month-old) Holsteins Angus (n=12) and Holstein (n=5). All animals were 19.4
produced considerably lower levels of IL8 following LPS (±0.1) months of age at the time of skin sampling (see
exposure compared to cultures collected from the older below) and were housed and cared for at the University of
(16-month-old) Holsteins. RNA-Seq analysis was com- Minnesota. The Institutional Animal Care and Use Com-
pleted on the young vs. old cultures following LPS treat- mittee at both the University of Vermont and the Univer-
ment, and revealed several pro-inflammatory response sity of Minnesota approved all animal procedures prior to
genes, including IL8, IL6, CCL20,TNF, and CXCL2 that beginningthestudy.
had greater expression in older cultures following LPS
stimulation [35]. Comparing the RNA-Seq data sets Dermalfibroblastisolation
from the previous young vs. older Holstein study, and Skin biopsies were collected from the shoulder area of
the current Holstein vs. Angus study revealed several animals housed at the University of Minnesota as previ-
genes that had similar expression patterns defining a ously described [18] and shipped overnight on ice packs
high vs. low response phenotype. The higher response to the University of Vermont in a transport media con-
phenotype of older animals in the previous study, and sisting of 1X Dulbecco’s PBS (DPBS; Hyclone Laborator-
Holstein animals in the current study, is characterized ies, Logan, UT) with 1X antibiotic cocktail (100 U/mL
by greater expression of TLR4, IL8,CCL2,CCL5,CCL20, penicillin, 100μg/mL streptomycin, and 0.25μg/mL
and TNF. These genes are critical in the recognition of amphotericin B; Hyclone Laboratories). Once received,
and resulting inflammation following infection by Gram- biopsy samples were processed as described [19]. Briefly,
negative bacteria. The identification of this set of bio- fibroblasts were isolated with a 0.5 % collagenase type I
markers from the global transcriptome may allow for enzyme solution (Life Technologies, Grand Island, NY)
greaterprecisioninclassifyingananimal’sinnateimmune and seeded in a 25cm2 flask (Corning Inc., Corning, NY)
response phenotype even at a young age. This has poten- in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone
tialutilityinselectinglowresponsereplacementheifersas Laboratories) with 10 % FBS (Hyclone Laboratories), 1X