RESEARCH ARTICLE Open Access
Assessmen o global DNA me hyla ion in
pe iphe al blood cell subpopula ions o ea ly
heuma oid a h i is be o e and a e me ho exa e
Ma ía C. de And es
1
, E a Pe ez-Pampin
1
, Manuel Calaza
1
, F ancisco J. San acla a
1
, Ignacio O ea
1
,
Juan J. Gomez-Reino
1,2
and An onio Gonzalez
1*
Abs ac
In oduc ion: DNA me hyla ion is an epigene ic mechanism egula ing gene exp ession ha has been insu icien ly
s udied in he blood o heuma oid a h i is (RA) pa ien s, as only T cells and o al pe iphe al blood mononuclea
cells (PBMCs) om pa ien s wi h es ablished RA ha e been s udied and wi h con lic ing esul s.
Me hod: Fi e majo blood cell subpopula ions: T, B and NK cells, monocy es, and polymo phonuclea leukocy es,
we e isola ed om 19 ea ly RA pa ien s and 17 heal hy con ols. Pa ien samples we e aken be o e and 1 mon h
a e he s a o ea men wi h me ho exa e (MTX). Analysis included DNA me hyla ion wi h high-pe o mance
liquid ch oma og aphy-elec osp ay ioniza ion- andem mass spec ome y-selec ed eac ion moni o ing
(HPLC-ESI-MS/MS-SRM) and exp ession le els o se en me hyla ion-speci ic enzymes by quan i a i e polyme ase
chain eac ion(qPCR).
Resul s: Disease-modi ying an i- heuma ic d ug (DMARD)-naï e ea ly RA pa ien s showed global DNA
hypome hyla ion in T cells and monocy es, oge he wi h a lowe exp ession o DNA me hyl asn e ase 1 (DNMT1), he
main enance DNA me hyl ans e ase, which was also dec eased in B cells. Fu he mo e, signi ican ly inc eased
exp ession o en-ele en ansloca ion1 (TET1), TET2 and TET3, enzymes in ol ed in deme hyla ion, was ound in
monocy es and o TET2 in T cells. The e was also modes dec eased exp ession o DNMT3A in B cells and o g ow h
a es and DNA-damage-inducible p o ein 45A (GADD45A) in T and B cells. T ea men wi h MTX e e ed
hypome hyla ion in T cells and monocy es, which we e no longe di e en om con ols, and inc eased global
me hyla ion in B cells. In addi ion, DNMT1 and DNMT3A showed a end o e e sion o hei dec eased exp ession.
Conclusions: Ou esul s con i m global DNA hypome hyla ion in pa ien s wi h RA wi h speci ici y o some blood cell
subpopula ions and hei e e sal wi h me ho exa e ea men . These changes a e accompanied by pa allel changes in
he le els o enzymes in ol ed in me hyla ion, sugges ing he possibili y o egula ion a his le el.
In oduc ion
Epigene ics has become an a ea o in e es o he s udy o
heuma oid a h i is (RA) [1]. I e e s o s able bu e e s-
ible changes in gene exp ession ha a e he i able h ough
cell di isions bu do no in ol e DNA a ian s. They
main ain he cellula pheno ypes acqui ed du ing de elop-
men and di e en ia ion, and e lec physiological changes
and en i onmen al s ess. This so o cellula memo y is
used o p omo e adap i e pheno ypic changes ha esul
in inc eased i ness, bu when abe an hey could p o-
mo e o pe pe ua e a disease s a us. Epigene ic mecha-
nisms include DNA me hyla ion, his one modi ica ions,
mic oRNA, o he non-coding RNA and nucleosome posi-
ioning. Cu en in e es is based on indings indica ing
ha epigene ic changes can become bioma ke s o di e -
en ia e pa ien s om heal hy con ols and o sepa a e
pa ien subg oups on p ognosis, o on esponse o ea -
men , as well as o se e as new a ge s o ea men [2].
In addi ion, changes in DNA me hyla ion o blood cells
ha e been cha ac e ized as media o s o gene ic isk in RA
and a e o in e es o unde s and disease pa hogenesis [3].
* Co espondence: [email p o ec ed]
1
Labo a o io de In es igacion 10 and Rheuma ology Uni , Ins i u o de
In es igación Sani a ia-Hospi al Clínico Uni e si a io de San iago, T a esia de
Choupana, s/n, 15706 San iago de Compos ela, Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2015 de And es e al.
Open Access
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de And es e al. A h i is Resea ch & The apy (2015) 17:233
DOI 10.1186/s13075-015-0748-5
DNA me hyla ion is he mos widely s udied and well-
cha ac e ized epigene ic change [4]. I happens mainly as
5-me hylycy osine (5mC) a C–phospha e–Gdinucleo-
ides (CpG) by he enzyma ic ans e o a me hyl g oup
om S-adenosyl-L-me hionine (SAM). In he bulk gen-
ome CpG a e a e and highly me hyla ed, bu in clus e s
o CpG dinucleo ides called CpG islands hey a e usually
non-me hyla ed. When CpG islands a gene p omo e s
a e me hyla ed hey a e associa ed wi h long- e m silen-
cing o gene exp ession. In con as , a iable and issue-
speci ic me hyla ion akes place wi h p e e ence ou side
CpG islands [4]. Main enance o he me hyla ed s a us
h ough mi osis equi es a speci ic DNA me hyl ans e -
ase (DNMT), DNMT1, which ecognizes hemime hyla ed
DNA sequences and me hyla es he new DNA s and.
Two o he enzymes o he same amily, DNMT3A and
DNMT3B, ha e been cha ac e ized as equi ed o de
no o me hyla ion du ing de elopmen o in esponse o
en i onmen al s imuli o o he s ess. Ac i e deme hyla-
ion depends on he ac ion o he en-ele en ansloca ion
(TET) (TET1, 2 and 3) enzymes [5]. These enzymes p o-
mo e DNA deme hyla ion by con e ing 5mC o 5-
hyd oxyme hylcy osine (5hmC), which hey u he
oxidize in o 5- o mylcy osine and 5-ca boxylcy osine. The
g ow h a es and DNA-damage-inducible p o ein 45A
(GADD45A) enzyme could also con ibu e o deme hyla-
ion [6], especially in au oimmune diseases [7, 8].
Global DNA hypome hyla ion has been ound in se -
e al in lamma o y and au oimmune diseases whe e i en-
ails abe an exp ession o genes and ibosomal RNAs
p obably implica ed in hei pa hology [9, 10]. O he
changes associa ed wi h DNA hypome hyla ion as genome
ins abili y and mu a ions, o use o c yp ic p omo e s ha e
no been desc ibed in he au oimmune diseases. In RA,
DNA hypome hyla ion has been ho oughly demons a ed
in ib oblas -like syno ial (FLS) cells [11–14]. Hypome hy-
la ion a speci ic CpG si es in FLS is associa ed wi h o e -
exp ession o genes ha a e keys o he disease p ocess.
The consequences o DNA hypome hyla ion in FLS ha e
been highligh ed by he ac i a ed pheno ype ha no mal
FLS acqui e a e d ug (5-azacy idine)-induced deme hyla-
ion [11]. Howe e , many aspec s o DNA me hyla ion in
RA emain incomple ely explo ed o a e con o e sial.
Fo example, he e a e con lic ing epo s ega ding
changes in DNA me hyla ion o blood cells [15, 16], o
he exp ession o me hyl ans e ase enzymes [11, 13, 16].
Also, we do no know he me hyla ion s a us o o he im-
po an playe s in RA beyond FLS and blood T cells, he
only cell popula ions s udied o da e [11–14, 17]. In
addi ion, all he p e ious s udies ha e analyzed samples
om es ablished RA pa ien s and could e lec e ec s o
ea men o o disease e olu ion. In his espec , he e is
a epo indica ing ha me ho exa e (MTX) e e s DNA
hypome hyla ion in in lamma o y a h i is [18], in spi e o
MTX inhibi ion o SAM syn hesis [19, 20]. Finally, some
p e ious s udies ha e e alua ed global DNA me hyla ion
wi h echniques ha a e insensi i e and unable o dis in-
guish 5mC om 5hmC, which has di e en unc ional im-
plica ions. These design and echnical issues could ha e
con ibu ed o some o he p e ious disco dan esul s.
The e o e, we aimed o add ess some o hese ques ions
wi h disease-modi ying an i- heuma ic d ug (DMARD)-
naï e ea ly RA pa ien s, be o e and a e ecei ing MTX
and using sensi i e and accu a e echnology [21, 22].
Me hods
Pa ien s and con ols
New consecu i e pa ien s a i ing o he Rheuma ology
Uni and ul illing he 2010 Ame ican College o
Rheuma ology/Eu opean League Agains Rheuma ism
(ACR/EULAR) classi ica ion c i e ia o RA we e included
om Ap il 2011 o Feb ua y 2012 [23]. All had clinical
symp oms o less han 2 yea s o e olu ion compa ible
wi h hei classi ica ion as ea ly RA. Clinical da a and
blood samples we e aken be o e and 1 mon h a e s a -
ing ea men wi h MTX. Clinical ollow-up was done
wi h comple e independence om his s udy. Gende and
age-ma ched heal hy con ols we e ec ui ed a he same
ime. All pa ien s and con ols we e o Caucasian Spanish
ances y. The E hics Commi ee o Clinical Resea ch o
Galicia app o ed his s udy, and w i en in o med consen
was ob ained om all pa icipan s.
Blood cell subpopula ions
Double g adien sepa a ion by cen i uga ion was used
o isola e g anulocy es and mononuclea cells wi h
His opaque®-1077 and His opaque®-1119 (Sigma-Ald ich,
S Louis, MO, USA) om 20 mL o EDTA an i-
coagula ed blood as desc ibed [24]. Mononuclea cells
o ming he bu y coa o e he 1077 laye we e ac ion-
a ed by immune-magne ic posi i e selec ion in ou sub-
popula ions CD56+ (na u al kille (NK) cells), CD14+
(monocy es), CD19+ (B lymphocy es) and CD3+ (T lym-
phocy es). The MACS® sys em (Mil enyi Bio ek Be gisch
Gladbach, Ge many) was used o NK cells and BD
IMag™cell sepa a ion sys em (BD Biosciences, San Jose,
CA, USA) o he o he subpopula ions. Pu i y o he
isola ed cells was con olled by luo escence-ac i a ed
cell so ing analysis on a FACScan™cy ome e wi h Cell-
Ques P o So wa e (BD Biosciences) as double-s ained
cells wi h an i-CD45 PE and subpopula ion-speci ic
an ibodies labeled wi h FITC (an i-CD14, CD19, CD3
o he popula ions pu i ied wi h hese an ibodies and
an i-CD15 o g anulocy es) and as CD56
+
and CD3
−
cells o he NK cells. All an ibodies we e om BD
Biosciences. Pu i y o isola ed cell subpopula ions
anged om 90 o 99 %. Pu i ied cells we e p ocessed
wi h he illus a™ ipleP ep Ki (GE Heal hca e. Li le
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 2 o 9
Chal on , UK) acco ding o he manu ac u e ’sins uc-
ions o ob ain genomic DNA and o al RNA.
Global DNA me hyla ion
5mC ela i e le els we e quan i ied ollowing enzyma ic
hyd olysis o genomic DNA as desc ibed [21]. Th ee
s anda ds we e included in each analysis. They we e
iden ical DNA sequences excep o he inclusion o 100
% unmodi ied cy osines, 100 % 5mC o 100 % 5hmC, e-
spec i ely (Zymo Resea ch, I ine, CA, USA). Samples
and s anda ds, 0.1–1μg, we e hyd olyzed o hei com-
ponen nucleosides be o e analysis by incuba ion wi h
DNA Deg adase Plus (Zymo Resea ch) o 2 h a 37 °C.
Sepa a ion and quan i ica ion o he nucleosides was
done by high-pe o mance liquid ch oma og aphy-
elec osp ay ioniza ion- andem mass spec ome y-
selec ed eac ion moni o ing (HPLC-ESI-MS/MS-SRM)
in he API 4000 LC/MS/MS Sys em (AB Sciex, F aming-
ham, MA, USA) including a ZORBAX Eclipse XDB-C18
column (Agilen Technologies, San a Cla a, CA, USA)
and a iple quad upole mass spec ome e . This sys em
allows he accu a e and sensi i e di e en ia ion o 5mC
om he ou deoxy ibonucleosides, he ou ibonucleo-
sides and 5hmC [21]. Quan i ica ion was exp essed as
he a io o 5mC o o al cy osine (5mC/5mC+C).
DNA me hyl an e ase exp ession analysis
To al RNA om each cell subpopula ion was immedia ely
e e se- ansc ibed wi h a ian myeloblas osis i us e e se
ansc ip ase (P omega, Madison, WI, USA) and andom
p ime s. Gene exp ession o DNMT1,DNMT3A,
DNMT3B,TET1,TET2,TET3 and GADD45A was quan i-
ied wi h quan i a i e polyme ase chain eac ion (qPCR).
P ime s we e designed wi h he p ime Exp ess 3.0 so -
wa e (Applied Biosys ems, Fos e Ci y, CA, USA) (Table
S1 in Addi ional ile 1). Fi e housekeeping genes we e ini-
ially es ed (RPL13A,GAPDH,B2M,18S and TBP)as e-
po ed [25]. Those showing s able exp ession (TBP and
18S o all cell subpopula ions excep g anulocy es, whe e
only TBP showed s able le els) in qBase [26] we e used as
e e ence. Quan i ica ions we e pe o med in iplica e 10
μl eac ions con aining 1 μlcDNA,5μlRT
2
SYBR G een
qPCR Mas e mix (Qiagen, Venlo, The Ne he lands), and
250 nM o each p ime in a Ro o -Gene™6000 (Co be
Li e Science, Venlo, The Ne he lands) he mocycle wi h
ini ial ac i a ion a 95 °C o 10 minu es, ollowed by a
wo-s ep p og am o 95 °C o 15 seconds and 60 °C o 60
seconds o 45 cycles (gain = 8). S anda ds we e included
in each un o in e - un calib a ion. Speci ici y o he PCR
eac ions was con i med by mel ing cu e analysis o he
p oduc s as well as by size e i ica ion by DNA elec o-
pho esis. T ans o med exp ession da a we e analyzed wi h
qBase a e adjus ing o ampli ica ion e iciency o each
ansc ip [26].
S a is ical analysis
Da a analysis was pe o med wi h S a is ica 7.0 (S a So ,
Tulsa, OK, USA). Non-no mali y o he a iables was
co ec ed wi h loga i hm o powe ans o ma ions. Di -
e ences be ween pa ien s and con ols we e e alua ed
using main e ec s analysis o a iance wi h co a ia es
(ANCOVA). Co a ia es we e sex and age. Wi hin-
pa ien compa isons, be o e and a e MTX ea men ,
we e done wi h pai ed-samples es s.
Resul s
Cha ac e is ics o pa ien s, con ols and he isola ed
blood cell subpopula ions
A o al o 19 consecu i e pa ien s wi h RA s a ing
MTX ea men we e ec ui ed (Table 1). The ac ion o
men (73.7 %) was highe han he ypical o pa ien s wi h
RA. Symp oms had s a ed a median o 6.0 mon hs be-
o e MTX ea men wi h only h ee pa ien s su passing
he yea since he i s symp oms and none wi h mo e
han 20 mon hs o e olu ion. These pa ien s had no e-
cei ed DMARDs be o e s a ing MTX. All had al eady
been ea ed wi h low-dose me hylp ednisolone (4–10
mg/day) o a median o 23 days be o e s a ing MTX
(in e qua ile ange (IQR) 12–33.5 days). Ac i i y o RA
was mode a e in mos pa ien s wi h mean disease ac i -
i y sco e in 28 join s (DAS28) o 4.6. Only wo o he pa-
ien s showed e osions on adiog aphic explo a ion in
spi e o he p esence o an i-ci ullina ed pep ide an i-
bodies (ACPA) o heuma oid ac o (RF) posi i i y in
mo e han hal o hem (63.2 % se oposi i e). The ini ial
dose o MTX was 10 mg/week excep o one pa ien
who ecei ed 15 mg/week. A second sample om each
pa ien was aken 1 mon h a e s a ing MTX ea men
o assess he e ec on DNA me hyla ion and exp ession
o DNMTs, TETsandGADD45A. All pa ien s we e in
MTX mono he apy a ha ime and wi hou change in
MTX o me hylp ednisolone doses. Al hough 1 mon h
is oo ea ly o assessing esponse o MTX, a dec ease o
RA ac i i y was al eady e iden in mos pa ien s
(Table 1). Response o MTX was assessed a 6-mon h
ollow-up. A ha ime, 15 pa ien s emained in mono-
he apy wi h MTX, 12 o hem showed good esponse
acco ding o he EULAR c i e ia [27], one showed a
mode a e esponse and wo we e non- esponde s. O
he ou pa ien s no emaining on MTX mono he apy
a 6 mon hs, wo we e on a di e en DMARD due o in-
e icacy o MTX and wo had in e up ed MTX due o
ad e se e ec s. A o al o 17 heal hy con ols ma ched
o age and sex we e ec ui ed and s udied.
Th ee subpopula ions o blood cells monocy es, B and
T lymphocy es we e isola ed om he 19 pa ien s and
17 heal hy con ols. G anulocy es and NK cells we e also
isola ed in he i s eigh pa ien s and eigh con ols bu
no pu sued u he because no di e ences we e
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 3 o 9
obse ed. The esul s om hese wo blood subpopula-
ions will no be p esen ed in de ail. Pu i y o he iso-
la ed subpopula ions anged om 90 o 99 %.
Di e ences in global DNA me hyla ion be ween pa ien s
wi h RA and con ols
Le els o global 5mC we e simila o hose p e iously
epo ed [21, 22]. Compa ison o he global 5mC le el
be ween pa ien s and con ols was done wi h he sam-
ples o RA pa ien s be o e MTX ea men . Main e ec s
ANCOVA wi h sex and age as co a ia es was used o
hese compa isons. T lymphocy es showed signi ican
DNA hypome hyla ion in ea ly RA pa ien s compa ed
wi h heal hy subjec s (Fig. 1a; mean = 3.89 %, 95 % con-
idence in e al (CI) = 3.80–3.99 s.4.15 %, 95 % CI =
3.96–4.38, espec i ely, P= 0.011). The mul i a ia e ana-
lysis also showed signi ican DNA hypome hyla ion in he
monocy es o RA pa ien s (Fig. 1c; mean = 3.96 %, 95 %
CI = 3.87–4.07 s.4.13, 95 % CI = 3.98–4.33, espec i ely,
P= 0.047). Al hough signi ican , hese di e ences we e
small. No signi ican di e ences we e de ec ed in any o
he o he blood cell subpopula ions: B lymphocy es, NK
cells and polymo phonuclea (PMN) cells (Fig. 1b and no
shown). No signi ican co ela ion be ween he o al me h-
ylp ednisolone dose ecei ed un il blood d awing and
le els o 5mC in any o he i e blood subpopula ions we e
obse ed in RA pa ien s (no shown). In he same assay,
5hmC was also de e mined wi hou any signi ican di e -
ence be ween pa ien s and heal hy con ols, showing e y
low le els in all cell subpopula ions (a ound 0.02 % o o al
cy osine) as is cha ac e is ic o mos adul issues [21, 28].
DNMT1,DNMT3A,DNMT3B,TET1,TET2,TET3 and
GADD45A ela i e exp ession was de e mined by qPCR
analysis in each o he blood cell subpopula ions. A signi i-
can dec ease in pa ien s wi h RA was obse ed o some
enzymes: DNMT1 exp ession was dec eased in T cells
(Fig. 2a; mean = 5.6, 95 % CI = 4.3–7.2 s. 10.0, 95 %
CI = 7.7–13.1; P= 0.0022), B cells (Fig. 2b; mean = 3.4,
95 % CI = 2.6–4.3 s. 6.0, 95 % CI = 4.6–7.4; P=0.00046)
and monocy es (Fig. 2c; mean = 5.6, 95 % CI = 4.5–6.7 s.
8.4, 95 % CI = 7.4–9.4; P= 0.00020); DNMT3A was de-
c eased, al hough less ma kedly, in B cells (Fig. 2d;
mean = 27.5, 95 % CI = 17.9–42.2 s. 46.9, 95 % CI =
32.4–67.9; P= 0.044); no di e ence was ound in he ex-
p ession o DNMT3B in any o he blood cell subpopula-
ions (no shown); and GADD45A exp ession was also
mode a ely educed in T cells (Fig. 2e; mean = 12.2, 95 %
CI = 8.7–17.3 s. 20.0, 95 % CI = 13.9–28.8; P= 0.048)
and in B cells (Fig. 2 ; mean = 9.6, 95 % CI = 5.8–45.9 s.
18.3, 95 % CI = 12.9–25.9; P= 0.021). On he con a y,
he ela i e exp ession o TET enzymes was inc eased in
some cell popula ions. Monocy es showed he la ges in-
c eases, wi h he h ee TET enzyme genes showing highe
ela i e le els o exp ession in RA pa ien s han in con-
ols. TET1 and TET3 showed he mos ma ked di e ence
be ween RA pa ien s and con ols (TET1, Fig. 3a; mean =
2.9, 95 % CI = 2.1–4.8 s. 1.9, 95 % CI = 1.6–2.3; P=
0.0044; and TET3, Fig. 3c; mean = 2.0, 95 % CI = 1.7–2.3
s. 1.35, 95 % CI = 1.15–1.6; P= 0.0014), bu also TET2
(Fig. 3b; mean = 2.5, 95 % CI = 1.9–3.2 s. 1.8, 95 % CI =
1.5–2.2; P= 0.019) was inc eased in monocy es o he pa-
ien s wi h RA. TET2 was also modes ly inc eased in he
T cells o RA pa ien s in ela ion wi h he heal hy con ols
(Fig. 3d; mean = 3.4, 95 % CI = 2.4–5.2 s. 2.2, 95 % CI =
1.8–3.0; P= 0.045). No o he di e ences we e de ec ed.
Inc ease o global DNA me hyla ion a e MTX ea men
Global DNA me hyla ion was analyzed 1 mon h a e
s a ing ea men wi h MTX. Compa ison o he pe cen -
age o 5mC in each o he i e blood cell subpopula ions
wi h es s o dependen a iables showed signi ican in-
c eases a e ea men in h ee o hem: T cells ha
showed he mos signi ican inc ease in global DNA 5mC
(Fig. 4a; mean = 4.21 %, 95 % CI = 4.03–4.43 a e 1
mon h o ea men ; P= 0.0014), B cells (Fig. 4b; 4.09
%, 95 % CI = 3.94–4.28 a e ea men s. 3.94 %, 95 %
Table 1 Cha ac e is ics o he pa ien s wi h ea ly RA and o he
heal hy con ols included in he s udy
RA pa ien s Heal hy con ols
Numbe 19 17
Female, (%) 5 (26.3) 6 (35.3)
Age a diagnosis, median (IQR) 61.6 (51–65) 58 (39–64)
Symp oms o MTX, median
mon hs (IQR)
6.0 (2.2–7.3)
RF (%) 11 (57.9)
ACPA (%) 11 (57.9)
E osi e a h i is (%) 2 (10.5)
Smoking (%) 8 (42.1)
Me hylp ednisolone, median
mg/day (IQR)
4(4–8)
Me o h exa e, median mg/week (IQR) 10 (10–10)
CRP (mg/L), median (IQR)
Baseline 15.3 (3.9–23.2)
1 mon h 5.2 (1.2–9.4)
ESR, median (IQR)
Baseline 28.0 (6.0–38.0)
1 mon h 14.0 (6.5–24.0)
DAS28, mean ± SD
Baseline 4.6 ± 1.6
1 mon h 3.2 ± 1.5
RA heuma oid a h i is, IQR in e qua ile ange, MTX me ho exa e, RF
heuma oid ac o , ACPA an i-ci ullina ed pep ide an ibody, CRP C- eac i e
p o ein, ESR e y h ocy e sedimen a ion a e, DAS28 disease ac i i y ac o
in 28 join s, SD s anda d de ia ion
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 4 o 9
CI = 3.82–4.09 be o e MTX; P= 0.018) and monocy es
ha showedabo de lineinc ease(Fig.4c;4.09%;95%
CI = 4.00–4.21 a e 1 mon h o ea men ; P=0.045).
Howe e , some pa ien s did no ollow his end o e-
co e y (Fig. 4). No di e ences in 5mC we e de ec ed in
NK o PMN cells be ween baseline and a e 1 mon h
wi h MTX. No de ec able a ia ion in global 5hmC
le els was obse ed (da a no shown).
We ha e also compa ed exp ession o DNMT1,
DNMT3A,DNMT3B,GADD45A,TET1,TET2 and TET3
in he i e cell subpopula ions a e MTX ea men wi h
hei baseline le els. No clea changes we e obse ed
(Fig. 5). Only DNMT1 showed a end o inc eased le els
a e ea men in T cells and monocy es ha was nea
signi ican (P= 0.06 and 0.07, espec i ely). A no sig-
ni ican end o an inc ease o DNMT3A in B cells (P=
0.06) was also obse ed. No simila end was ound o
any o he o he enzymes. On he con a y, he di e -
ences wi h heal hy con ols ha we e p esen be o e
ea men we e s ill p esen a e 1 mon h on MTX ex-
cep o TET2 in T cells ha was no longe signi ican ly
di e en . In pa icula , he TET enzyme exp ession in
monocy es showed he same le el o di e ence wi h
heal hy con ols han be o e ea men (TET1 P
be o e
=
Fig. 1 Di e ences in global DNA me hyla ion be ween heal hy con ols (HC) and ea ly heuma oid a h i is (RA) pa ien s. The pe cen age o
5-me hylcy osine (5mC) o e he o al con en o cy osine in o al DNA o aT cells, bB cells, and cmonocy es is shown. Each do ep esen s a
subjec . Ho izon al ba s wi h an emp y squa e a e means. Di e ences in (a)and(c) we e signi ican , P<0.05
Fig. 2 Exp ession o me hyla ion enzymes in blood cells o heal hy con ols (HC) and ea ly heuma oid a h i is (RA) pa ien s. No malized ela i e
exp ession o DNMT1 ob ained by quan i a i e polyme ase chain eac ion (qPCR) in aT cells, bB cells, and cmonocy es, DNMT3A in B cells (d)
and o GADD45A in T cells (e) and B cells ( ) is shown. O he con en ions a e as in Fig. 1. All hese compa isons we e signi ican . No compa ison
in o he cells subpopula ion and none o he DNMT3B analyses we e signi ican
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 5 o 9

0.037, P
a e
= 0.039; TET2 P
be o e
= 0.019, P
a e
= 0.013;
and TET3 P
be o e
=0.0014,P
a e
=0.0033),aswellas he
exp ession o GADD45A in T and B cells (T cells P
be o e
=
0.048, P
a e
=0.049;BcellsP
be o e
=0.021,P
a e
=0.041).
The e o e, TET enzyme gene exp ession in monocy es
and GADD45A in T and B cells we e no signi ican ly
modi ied by his ea men .
Discussion
Ou esul s suppo he in ol emen o dec eased DNA
me hyla ion in RA and i s e e sal in esponse o MTX.
They also widen he spec um o changes in me hyla ion
and in he enzymes egula ing his p ocess in RA, as well
as p o ide cell-speci ic esul s. In pa icula , his is he
i s ime ha changes in B cells and in monocy es ha e
Fig. 3 Di e ences be ween heal hy con ols (HC) and ea ly heuma oid a h i is (RA) pa ien s in en-ele en ansloca ion (TET) enzymes exp ession.
No malized ela i e exp ession o aTET1,bTET2, and cTET3 in monocy es; and o dTET2 in T cells. Con en ions a e as in Fig. 1. All di e ences
we e signi ican a P< 0.05
Fig. 4 Inc eased global DNA me hyla ion in heuma oid a h i is (RA) pa ien s a e 1 mon h on me ho exa e (MTX). The pe cen age o
5-me hylcy osine (5mC) o e he o al con en o cy osine in o al DNA o aT cells, bB cells, and cmonocy es is shown. Each pai o do s
joined by dashed lines ep esen s a subjec be o e and a e MTX. All changes we e signi ican wi h P<0.05
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 6 o 9
been desc ibed and ha he main me hyla ion- ela ed
enzymes ha e been s udied in he blood o RA pa ien s.
The esul s suppo he implica ion o DNA hypome hy-
la ion in RA and highligh he in e ac ion be ween MTX
and DNA me hyla ion ha could con ibu e o i s he a-
peu ic e icacy in RA.
A couple o p e ious s udies ha e analyzed whole
pe iphe al blood mononuclea cells (PBMCs) wi h dis-
co dan esul s [15, 16]. One o hese s udies ound
global DNA hypome hyla ion in PBMCs wi h an
enzyme-linked immunoso ben assay (ELISA) ki o
ques ionable accu acy gi en he low pe cen age o
5mC (1.22 % in con ols) [16] in compa ison wi h
ou esul s, o he esul s in RA T cells [17], and in
o he cells and issues [21, 22]. The second s udy did
no ind di e ences in DNA me hyla ion be ween
PBMCs o RA disco dan monozygo ic wins a 1505
CpG si es analyzed by bead a ay [15]. Howe e , hese
si es we e no andomly dis ibu ed and hei ep e-
sen a i eness o global DNA me hyla ion is unce ain,
and could be insensi i e o de ec he small di e ence
we ha e obse ed. The mos speci ic s udy o da e
has analyzed PBMCs in wo subse s, T cells and non-
T cells, wi h a HPLC me hod [17]. The T cells
showed global DNA hypome hyla ion, whe eas he
non-T cells did no . These esul s a e in ag eemen
wi h ou obse a ions.
Meaning o global DNA hypome hyla ion in au o-
immune diseases has been bes s udied in sys emic lupus
e y hema osus (SLE) CD4 T cells. These cells show global
and si e-speci ic hypome hyla ion ha is associa ed wi h
inc eased exp ession o immune esponse genes, many o
hem in he in e e on (INF) signaling pa hway, o e ex-
p ession o ibosomal RNA, modi ica ions o imp in ing,
and eac i a ion o endogenous e o i us [8–10, 29]. All
hese changes could con ibu e o he b eakdown o im-
mune ole ance and o ch onic in lamma ion. Suppo o
his causal ole has been p o ided by he d ug-induced
SLE ha ollows ea men wi h hyd alazine o
p ocainamide, which dec ease DNA me hyla ion, and by
animal s udies [8–10]. Al hough some o he abo e-
men ioned changes could be SLE-speci ic, i is likely ha
DNA hypome hyla ion in RA T cells has a simila ole. In
his ega d, he e is al eady e idence showing ha speci ic
CpG si es a TNFSF5 a e simila ly hypome hyla ed in T
cells o SLE and RA [30]. In addi ion, a ecen la ge s udy
in RA has ound e idence o me hyla ion changes in
PBMCs as possible media o s in gene ic suscep ibili y [3].
As al eady men ioned, global DNA me hyla ion has
no been p e iously analyzed in monocy es o RA pa-
ien s. In SLE, monocy es sha e se e al hypome hyla ed
si es wi h CD4 T cells, al hough hey we e mo e nume -
ous and p ominen ly hypome hyla ed in T cells [29]. In
RA monocy es, he me hyla ion le el a a CpG si e in
he IL6 p omo e ha was hypome hyla ed in RA
PBMCs was in e sely co ela ed wi h in e leukin (IL)-6
lipopolysaccha ide (LPS)-induced exp ession [31]. I can
only be p esumed ha changes in me hyla ion as his
one could also con ibu e o he disease p ocess.
DNMT1 was he mos ma kedly and consis en ly de-
c eased DNA me hyl ans e ase in ou s udy. This esul
is in con as wi h a p e ious epo ha ound inc eased
DNMT1 exp ession in PBMCs o RA pa ien s [16], bu i
is in ag eemen wi h mul iple obse a ions in o he
au oimmune diseases [8, 9, 15]. In addi ion, impai ed
DNMT1 unc ion has a causal ole in global DNA hypo-
me hyla ion and au oimmuni y as demons a ed by d ug-
induced SLE [9]. In RA, s udies o DNMT1 ha e been
much mo e limi ed and es ic ed o FLS in cul u e. They
show compa able le els o he obse ed in os eoa h i is
(OA) FLS, bu DNMT1 exp ession is ma kedly down egu-
la ed by incuba ion wi h small amoun s o in lamma o y
cy okines [11, 13]. These p e ious epo s sugges possible
mechanisms o he dec eased DNMT1 exp ession and in-
dica e ha down egula ion o DNMT1 is e y likely a
majo ac o in DNA hypome hyla ion. Howe e , co el-
a ion be ween DNMT1 le els and DNA hypome hyla ion
was no p esen in B cells in ou s udy. The lack o
Fig. 5 La ges changes in exp ession o me hyla ion enzymes a e 1 mon h on me ho exa e (MTX). No malized ela i e exp ession o aDNMT1
in T cells, and bin monocy es, and cDNMT3A in B cells, be o e and a e 1 mon h on MTX is shown. Figu e con en ions a e as in Fig. 4. The
h ee changes showed P< 0.1, bu none was signi ican
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 7 o 9
hypome hyla ion in B cells was especially ema kable be-
cause hey we e he only cell ype showing dec eased ex-
p ession o DNMT1 and DNMT3A.No educ iono
DNMT3A le els has been p e iously desc ibed in RA FLS
o in SLE T cells [13, 32–34], which a e he wo cell ypes
ha ha e been s udied. In u n, no di e ences in
DNMT3B ha e been desc ibed in ag eemen wi h ou
nega i e esul s ega ding his enzyme [13, 32–34].
In addi ion o he changes in he enzymes ha me hy-
la e DNA, ou s udy is he i s add essing he DNA
deme hylases in an au oimmune disease. Exp ession o
he genes o he h ee TET enzymes we e inc eased in
he monocy es o RA pa ien s, and TET2 was also in-
c eased in T cells. These esul s could con ibu e o-
ge he wi h he dec ease in DNMT1 o he global
hypome hyla ion obse ed in he pa ien s. O possible
ele ance o RA is ha he TET3 locus has been associ-
a ed wi h SLE suscep ibili y in Asians [35], and ha
TET1 egula es ansc ip ion and p ocessing o IL1βand
o he p o-in lamma o y genes in expe imen s wi h cell
lines [36]. P e iously, GADD45A was he main known
ac i e DNA deme hylase [6], howe e his ole seems o
co espond o he TET enzymes [5]. Howe e , in e es in
GADD45A has emained in he au oimmune diseases
because i is o e exp essed in CD4 T cells o SLE
pa ien s, co ela ing wi h global and si e-speci ic DNA
hypome hyla ion in hese cells [7, 8]. In con as ,
GADD45A exp ession was diminished in ou analysis o
RA pa ien s, bo h in T and B cells. The obse ed dec ease
is no cong uen wi h global DNA hypome hyla ion in
T cells gi en i s p oposed deme hyla ing unc ion, bu
GADD45A has o he unc ions and one o hem could
ha e a ole he e: i s ole as inhibi o o T cell ecep o
(TCR) signaling [37].
Se e al o he di e ences we obse ed in RA pa ien s
we e e e ed a e 1 mon h o MTX ea men . Fi s , glo-
bal DNA hypome hyla ion in T cells, B cells and mono-
cy es was e e ed in mos pa ien s. These esul s a e
eminiscen o a smalle s udy including pa ien s wi h in-
lamma o y a h i is (RA and pso ia ic a h i is (PsA)) on
long- e m ea men wi h MTX [18]. This ou come is con-
a y o he expec ed gi en he MTX supp ession o SAM
[38], which is he majo sou ce o me hyl g oups o DNA
me hyla ion. Howe e , con ol o in lamma ion by MTX
could lead o educed cell p oli e a ion, which is a sou ce
o SAM consump ion h ough inc eased ecycling o poly-
amines [39], and o e e sal o DNMT1 and DNMT3A
exp ession gi en hei sensi i i y o p o-in lamma o y
cy okines [11, 13]. The wo mechanisms could con ibu e
o he eco e y o DNA me hyla ion.
Conclusions
Ou esul s suppo he implica ion o DNA me hyla ion
in RA and in i s esponse o MTX ea men . They also
widen he spec um o changes and he ypes o cells
ha a e a ec ed by hem: global DNA hypome hyla ion
in T cells and monocy es associa ed wi h a lowe exp es-
sion o DNMT1 and inc eased exp ession o he h ee
TET enzymes in monocy es and TET2 in T cells, o-
ge he wi h dec eased DNMT1 and DNMT3A exp ession
in B cells. Se e al o hese changes we e e e ed a e
MTX ea men , mos ema kably he DNA global hypo-
me hyla ion. The di e ences and changes ound he e in
pe iphe al blood could e lec impo an mechanisms
bo h in disease e olu ion and in i s con ol by MTX and
sugges mul iple new a eas o u u e esea ch.
Addi ional ile
Addi ional ile 1: Table S1. P ime s used o assessing he exp ession
le el o me hyla ion- ele an enzymes by qPCR. (DOC 36 kb)
Abb e ia ions
5hmC: 5-hyd oxyme hylcy osine; 5mC: 5-me hylcy osine; ACPA: an i-ci ullina ed
pep ide an ibody; ACR: Ame ican College o Rheuma ology; ANCOVA: analysis
o a iance wi h co a ia es; CI: con idence in e al; CpG: cy osine-phospha e-
guanine dinucleo ide; DAS28: disease ac i i y sco e in 28 join s;
DMARD: disease-modi ying an i- heuma ic d ug; DNMT: DNA me hyl asn e ase;
EULAR: The Eu opean League Agains Rheuma ism; FLS: ib oblas -like
syno iocy e; GADD45A: g ow h a es and DNA-damage-inducible p o ein 45
alpha; HPLC-ESI-MS/MS-SRM: high-pe o mance liquid ch oma og aphy-
elec osp ay ioniza ion- andem mass spec ome y-selec ed eac ion
moni o ing; IQR: in e qua ile ange; MTX: me ho exa e; NK: na u al kille ;
PBMCs: pe iphe al blood mononuclea cells; PMN: polymo phonuclea ;
qPCR: quan i a i e polyme ase chain eac ion; RA: heuma oid a h i is;
RF: heuma oid ac o ; SAM: S-adenosyl-L-me hionine; SLE: sys emic lupus
e y hema osus; TET: en-ele en ansloca ion.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s
Au ho s’con ibu ions
MCA designed he s udy, pe o med expe imen s, analyzed esul s and w o e
he i s d a o he manusc ip . EP-P ollowed he pa ien s and ec ui ed
pa ien s and con ols. MC con ibu ed o qPCR expe imen s and pe o med
s a is ical analyses. FJS pe o med TET enzyme expe imen s. IO con ibu ed
o DNA me hyla ion quan i ica ion. JJG-R con ibu ed o ec ui men and
ob ained unding. AG designed he s udy, ob ained unding, analyzed esul s
and supe ised he s udy. All au ho s con ibu ed o in e p e a ion o he
esul s and o he inal e sion o he manusc ip ha all ha e app o ed a e
e ising i c i ically o impo an in ellec ual con en .
Acknowledgemen s
We hank Ma ia del Sol Po o-Sil a o he help in ec ui ing he pa ien s and
Ca men Pena-Pena o echnical assis ance.
Funding
The p esen wo k was suppo ed by Fondo de In es igacion Sani a ia o
he Ins i u o de Salud Ca los III (Spain), g an s PI11/01048, PI12/01909 and
RD12/0009/0008 ha a e pa ially inanced by he Eu opean Regional
De elopmen Fund o he Eu opean Union.
Au ho de ails
1
Labo a o io de In es igacion 10 and Rheuma ology Uni , Ins i u o de
In es igación Sani a ia-Hospi al Clínico Uni e si a io de San iago, T a esia de
Choupana, s/n, 15706 San iago de Compos ela, Spain.
2
Depa men o
Medicine, Uni e si y o San iago de Compos ela, Rúa de San F ancisco, s/n,
15782 San iago de Compos ela, Spain.
de And es e al. A h i is Resea ch & The apy (2015) 17:233 Page 8 o 9
Recei ed: 31 Janua y 2015 Accep ed: 10 Augus 2015
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