Janua y 2018 | Volume 9 | A icle 311
O iginal esea ch
published: 29 Janua y 2018
doi: 10.3389/ immu.2018.00031
F on ie s in Immunology | www. on ie sin.o g
Edi ed by:
Alexand e Co hay,
Oslo Uni e si y Hospi al, No way
Re iewed by:
Ana Rosa Pé ez,
Consejo Nacional de In es igaciones
Cien í icas y Técnicas (CONICET),
A gen ina
*Co espondence:
Concha Nie o
[email p o ec ed]
†These au ho s ha e con ibu ed
equally o his wo k.
Special y sec ion:
This a icle was submi ed o
Molecula Inna e Immuni y,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 26July2017
Accep ed: 04Janua y2018
Published: 29Janua y2018
Ci a ion:
Nie oC, B agadoR, MunicioC,
Sie a-Fila diE, AlonsoB,
Esc ibeseMM, Domínguez-And ésJ,
A da ínC, Cas illoA, VegaMA,
Puig-K öge A and Co bíAL (2018)
The Ac i in A-Pe oxisome
P oli e a o -Ac i a ed Recep o
Gamma Axis Con ibu es o he
T ansc ip ome o GM-CSF-
Condi ioned Human Mac ophages.
F on . Immunol. 9:31.
doi: 10.3389/ immu.2018.00031
The ac i in a-Pe oxisome
P oli e a o -ac i a ed ecep o
gamma axis con ibu es o he
T ansc ip ome o gM-csF-
condi ioned human Mac ophages
Concha Nie o1*, Ra ael B agado2, C is ina Municio3, Elena Sie a-Fila di1, Bá ba a Alonso1,
Ma ía M. Esc ibese1, Jo ge Domínguez-And és4, Ca los A da ín4, An onio Cas illo5,6,
Miguel A. Vega1, Amaya Puig-K öge 3† and Angel L. Co bí1†
1 Cen o de In es igaciones Biológicas, Consejo Supe io de In es igaciones Cien í icas (CSIC), Mad id, Spain, 2 Ins i u o de
In es igación Sani a ia, Fundación Jiménez Díaz, Mad id, Spain, 3 Ins i u o de In es igación Sani a ia G ego io Ma añón,
Hospi al Gene al Uni e si a io G ego io Ma añón, Mad id, Spain, 4 Cen o Nacional de Bio ecnología, Consejo Supe io de
In es igaciones Cien í icas (CSIC), Mad id, Spain, 5 Ins i u o In es igaciones Biomédicas “Albe o Sols” (IIBM), and Cen o
Mix o Consejo Supe io de In es igaciones Cien í icas y Uni e sidad Au ónoma de Mad id (ICSIC-UAM), Mad id, Spain,
6
Unidad de Biomedicina (Unidad Asociada al CSIC), IIBM-Uni e sidad Las Palmas de G an Cana ia (ULPGC), and Ins i u o
Uni e si a io de In es igaciones Biomédicas y Sani a ias (IUIBS), Uni e sidad Las Palmas de G an Cana ia (ULPGC),
Las Palmas de G an Cana ia, Spain
GM-CSF p omo es he unc ional ma u a ion o lung al eola mac ophages (A-MØ),
whose di e en ia ion is dependen on he pe oxisome p oli e a o -ac i a ed ecep o
gamma (PPARγ) ansc ip ion ac o . In ac , blockade o GM-CSF-ini ia ed signaling
o dele ion o he PPARγ-encoding gene PPARG leads o unc ionally de ec i e A-MØ
and he onse o pulmona y al eola p o einosis. In i o, mac ophages gene a ed in
he p esence o GM-CSF display po en p oin lamma o y, immunogenic and umo
g ow h-limi ing ac i i ies. Since GM-CSF up egula es PPARγ exp ession, we hypo he-
sized ha PPARγ migh con ibu e o he gene signa u e and unc ional p o ile o human
GM-CSF-condi ioned mac ophages. To e i y his hypo hesis, PPARγ exp ession
and ac i i y was assessed in human monocy e-de i ed mac ophages gene a ed in
he p esence o GM-CSF [p oin lamma o y GM-CSF-condi ioned human monocy e-
de i ed mac ophages (GM-MØ)] o M-CSF (an i-in lamma o y M-MØ), as well as in
ex i o isola ed human A-MØ. GM-MØ showed highe PPARγ exp ession han M-MØ,
and he exp ession o PPARγ in GM-MØ was ound o la gely depend on ac i in A.
Ligand-induced ac i a ion o PPARγ also esul ed in dis inc ansc ip ional and unc-
ional ou comes in GM-MØ and M-MØ. Mo eo e , and in he absence o exogenous
ac i a ing ligands, PPARγ knockdown signi ican ly al e ed he GM-MØ ansc ip ome,
causing a global up egula ion o p oin lamma o y genes and signi ican ly modula ing
he exp ession o genes in ol ed in cell p oli e a ion and mig a ion. Simila e ec s we e
obse ed in ex i o isola ed human A-MØ, whe e PPARγ silencing led o enhanced
exp ession o genes coding o g ow h ac o s and chemokines and down egula ion
o cell su ace pa hogen ecep o s. The e o e, PPARγ shapes he ansc ip ome o
GM-CSF-dependen human mac ophages (in i o de i ed GM-MØ and ex i o isola ed
A-MØ) in he absence o exogenous ac i a ing ligands, and i s exp ession is p ima ily
2
Nie o e al. PPARγ T ansc ip ome in GM-CSF-Condi ioned Human Mac ophages
F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 9 | A icle 31
egula ed by ac i in A. These esul s sugges ha ac i in A, h ough enhancemen o
PPARγ exp ession, help mac ophages o swi ch om a p oin lamma o y o an an i-
in lamma o y pola iza ion s a e, hus con ibu ing o limi issue damage and es o e
homeos asis.
Keywo ds: ansc ip ion ac o , mac ophage, pe oxisome p oli e a o -ac i a ed ecep o , in lamma ion, inna e
immuni y
inT ODUcTiOn
Tissue- esiden mac ophages in homeos asis, as well as monocy e-
de i ed mac ophages wi hin in lamed issues, exhibi a huge
unc ional di e si y which de i es om hei exquisi e sensi-
i i y o ex acellula cues (1, 2). GM-CSF and M-CSF d i e
mac ophage di e en ia ion and su i al (3). Howe e , M-CSF
is equi ed o he gene a ion o mos issue mac ophages (4, 5)
while GM-CSF is needed o de elopmen and main enance o
pulmona y al eola mac ophages (A-MØ) (6). Besides i s ole
in myeloid cell di e en ia ion, GM-CSF is a cen al media-
o o issue in lamma ion (7) and i s neu aliza ion has been
p oposed as a he apeu ic s a egy o in lamma o y diso de s
(8). As a consequence, bo h colony-s imula ing ac o s p o-
mo e he gene a ion o unc ionally dis inc mac ophages (9):
GM-CSF-condi ioned human monocy e-de i ed mac ophages
(GM-MØ) p oduce la ge amoun s o p oin lamma o y cy o-
kines in esponse o s imula ion, whe eas M-CSF-dependen
monocy e-de i ed mac ophages (M-MØ) p ima ily p oduce
an i-in lamma o y ac o s upon ac i a ion (9–11). A he an-
sc ip ional le el, while GM-MØ a e cha ac e ized by he exp es-
sion o a “P oin lamma o y gene se ” (11–13) also de ec ed in
mac ophages unde in lamma o y condi ions in i o, M-MØ
speci ically exp ess an “An i-in lamma o y gene se ” and esem-
ble mac ophages om homeos a ic/an i-in lamma o y se ings
(14, 15). In e es ingly, he GM-MØ-speci ic gene signa u e is c i i-
cally de e mined by ac i in A bo h in i o (15) and in i o (11).
In his ega d, we ha e p e iously demons a ed ha GM-MØ
p oduce la ge amoun s o ac i in A, a membe o he TGFβ amily
(16, 17) ha egula es in lamma o y esponses (18), modula es
cy okine elease (19, 20) and myeloid cell di e en ia ion (21),
and whose unc ional blockade in GM-MØ skews cells owa d
he acquisi ion o an an i-in lamma o y signa u e (11).
The con ibu ion o GM-CSF o di e en ia ion o lung mac-
ophages elies on he GM-CSF-dependen exp ession o pe oxi-
some p oli e a o -ac i a ed ecep o gamma (PPARγ) (22–24), a
nuclea ecep o ha egula es gene ansc ip ion h ough ligand
binding (25–28), an agonism o o he ansc ip ion ac o s
(e.g., NFκB, AP-1) (29, 30) and ec ui men o ep esso com-
plexes in he absence o ligands (31). As a c i ical egula o o
in lamma o y p ocesses (32–34), PPARγ inhibi s human and
mu ine mac ophage esponses o p oin lamma o y s imuli
(35, 36), con ibu es o IL-4-d i en pola iza ion o human and
mu ine mac ophages (37, 38) and de e mines he acquisi ion o
he me abolic disease-speci ic pheno ype o human mac ophages
(39). In human cells, he ubiqui ously exp essed PPARγ1 de i es
om he PPARG1 and PPARG3 mRNA splicing iso o ms, while
he PPARG2 mRNA iso o m codes o PPARγ2, whose exp ession
is es ic ed o adipocy es (40). Mouse A-MØ exhibi much highe
exp ession o PPARγ han o he mac ophages in he s eady-s a e
(34), and i s GM-CSF-dependen exp ession is essen ial o hei
di e en ia ion and ma u a ion om e al monocy es (24). In ac ,
PPARγ exp ession in A-MØ is los in GM-CSF-de icien mice and
in pa ien s wi h pulmona y al eola p o einosis (PAP), a pa hol-
ogy de i ed om a de ec i e exp ession o ac i i y o GM-CSF
(23, 41) and associa ed wi h supp essed ac i in A exp ession (42).
Howe e , i is cu en ly unknown whe he PPARγ is equi ed o
main enance o A-MØ h oughou adul li e (43).
Upon issue inju y, monocy e-de i ed mac ophages modula e
in lamma ion and also p omo e issue epai . In he speci ic case
o lung in lamma ion, monocy e-de i ed mouse A-MØ a e he
majo d i e s o ib osis and become simila o issue- esiden
A-MØ o e ime (44). Since GM-CSF-condi ioned monocy e-
de i ed human mac ophages exhibi po en p oin lamma o y
unc ions upon s imula ion (9, 11), and in spi e o he in insic
an i-in lamma o y unc ions o PPARγ, we hypo hesized ha
PPARγ migh con ibu e o he gene signa u e and unc ional
p o ile o human GM-CSF-condi ioned mac ophages. To add ess
his hypo hesis, we e alua ed he ex en o he PPARγ con ibu ion
o he gene signa u e and unc ional p o ile o human GM-CSF-
dependen mac ophages. We now epo he ac i in A-dependen
exp ession and ac i i y o PPARγ in GM-CSF-condi ioned human
mac ophages, and demons a e ha PPARγ displays pola iza ion-
dependen ac i i ies and signi ican ly shapes he gene signa u e o
p oin lamma o y monocy e-de i ed GM-MØ and human A-MØ
in he absence o exogenous ligands. The ac i in A-dependen
exp ession o PPARγ in GM-MØ and in A-MØ also sugges s a
ole o ac i in A in p omo ing in lamma ion esolu ion.
eXPe iMenTal P OceDU es
gene a ion o human Monocy e-De i ed
Mac ophages InVi o and Ex Vi o
isola ion o a-MØ
Bu y coa s we e ob ained om heal hy blood dono s, as anony-
mously p o ided by he Comunidad de Mad id blood Bank.
E hical app o als o all blood sou ces and p ocesses used in his
s udy we e app o ed by he Cen o de In es igaciones Biológicas
E hics Commi ee. All expe imen s we e ca ied ou in acco d-
ance wi h he app o ed guidelines and egula ions. Human
PBMCs we e isola ed om bu y coa s o e a Lymphop ep™
g adien (#1114545, Axis-Shield PoC AS) acco ding o s anda d
p ocedu es. Monocy es we e pu i ied om PBMCs by magne ic
cell so ing using human CD14 mic obeads (#130-050-201,
Mil enyi Bio ech). Monocy es (95% CD14+ cells) we e cul u ed
3
Nie o e al. PPARγ T ansc ip ome in GM-CSF-Condi ioned Human Mac ophages
F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 9 | A icle 31
a 0.5 × 106 cells/ml o 7 days in RPMI 1640 (#21875-034,
Gibco) supplemen ed wi h 10% inac i a ed e al cal se um
(FCS) (#S1810-500, Biowes ) (comple e medium), a 37°C in a
humidi ied a mosphe e wi h 5% CO2, and con aining 1,000U/ml
human GM-CSF (#11343125, Immuno ools GmbH) o 10ng/ml
human M-CSF (#11343115, Immuno ools GmbH), o gene a e
GM-MØ o M-MØ, espec i ely. Cy okines we e added e e y
2 days. Blocking an i-ac i in A Ab (100 ng/ml) (#MAB3381,
clone 69403, R&D Sys ems) o he inhibi o s o ALK4, ALK5,
and ALK7, SB431542 (10µM) (#S4317, Sigma-Ald ich) o A-83
(1µM) (#2039, Toc is) we e added e e y 24h. Finally, pola ized
mac ophages we e ea ed wi h ul apu e Esche ichia coli 0111:B4
s ain LPS (10 ng/ml) (# l l-3pelps, In i ogen) o 14–16 h.
Exposu e o ecombinan human ac i in A (25ng/ml) (#120-14P,
P ep o ech) was done o 24h (monocy es and THP-1 cells) o
48h (M-MØ). The acu e monocy ic leukemia cell line THP-1,
ob ained om ATCC® (#TIB-202™), was cul u ed in comple e
medium a 37°C in a humidi ied a mosphe e wi h 5% CO2. A-MØ
we e ob ained om pa ien s unde going b onchoal eola la age
(BAL) ollowing he Fundación Jiménez Díaz Medical E hics
commi ee p ocedu es and a e w i en in o med consen om
all subjec s, in acco dance wi h he Decla a ion o Helsinki. BAL
p ocedu e was pe o med wi h a lexible b onchoscope wi h a o al
olume o 200ml o s e ile iso onic saline solu ion a 37°C. BAL
luid ac ions we e main ained a 4°C and cellula deb is emo ed
using a 40µm cell s aine (45). BAL cells we e washed wi h PBS,
cen i uged and esuspended in comple e medium con aining
100 U/mL penicillin and 100µg/mL s ep omycin (#15140-122,
Gibco), 50 µg/ml gen amicin (#G1397, Sigma-Ald ich), and
2.5 µg/ml ampho e icin B (#A2942, Sigma-Ald ich). The cells
we e seeded a 6–8×105 cells pe well in 12-well pla es o 1h and
washed ex ensi ely o emo e non-adhe en cells. Finally, 2ml o
comple e medium wi h an ibio ics was added o each well and he
adhe en cells incuba ed o 16–18h be o e ans ec ion. Mo e
han 95% o adhe en BAL cells we e iden i ied as mac ophages
acco ding o mo phology and pheno ypic analysis.
gene a ion o Mu ine Bone
Ma ow-De i ed Mac ophages InVi o
All expe imen s on mice we e conduc ed acco ding o he Spanish
and Eu opean egula ions on ca e and p o ec ion o labo a o y
animals and we e app o ed by he Cen o de In es igaciones
Biológicas animal acili y and he Consejo Supe io de In es-
igaciones Cien í icas E hics Commi ee. Bone ma ow-de i ed
GM-MØ o M-MØ we e ob ained by lushing he emu s o
6–10-week-old C57BL/6 mice (p o ided by he Animal acili y
a he Cen o de In es igaciones Biológicas), and cul u ing cells
du ing 7days in DMEM (#41966-029, Gibco) supplemen ed wi h
10% FCS and 50mM 2-ME, con aining ei he mu ine GM-CSF
(1,000U/ml) (#315-03, P eP o ech) o human M-CSF (25ng/ml)
(#11343115, Immuno ools GmbH), espec i ely (46, 47). Cy o-
kines we e added e e y 2days.
Flow cy ome y
Mouse monoclonal an ibodies speci ic o human CD14 (Alexa
Fluo -647-labeled an ihuman CD14, #301818, clone M5E2,
Biolegend) and human CD163 (Pe CP-labeled an ihuman CD163,
#333625, clone GHI/61, Biolegend) we e used. Iso ype-ma ched
Pe CP-labeled Mouse IgG1 (κ Iso ype C l An ibody, #400147,
clone MOPC-21, Biolegend) and Alexa Fluo -647 Mouse IgG2a
(κ Iso ype C l An ibody, #400234, clone MOPC-173, Biolegend)
we e included as nega i e con ols.
Quan i a i e eal Time T-Pc
To al RNA was ex ac ed using he o al RNA and p o ein isola-
ion ki (Mache ey-Nagel). RNA samples we e e o ansc ibed
wi h he High-Capaci y cDNA Re e se T ansc ip ion ki (AB),
and indi idually ampli ied cDNA was quan i ied using he
Uni e sal Human P obe Roche lib a y (Roche Diagnos ics).
Oligonucleo ides o selec ed genes we e designed acco ding o
he Roche so wa e o quan i a i e eal- ime PCR (qRT-PCR),
and hei sequence is indica ed in Table S1 in Supplemen a y
Ma e ial. qRT-PCR was pe o med on a Ligh Cycle ® 480 (Roche
Diagnos ics). Assays we e made in iplica es, and esul s we e
no malized acco ding o he exp ession le els o TBP mRNA o /
and GAPDH mRNA ( o qRT-PCR) o o he mean o he exp es-
sion le el o endogenous e e ence genes HPRT1, TBP and RPLP0
( o mic o luidic gene ca ds). Resul s we e exp essed using he
ΔΔCT (cycle h eshold) me hod o quan i ica ion.
elisa
Mac ophage supe na an s we e es ed o he p esence o cy okines
using comme cially a ailable ELISA se s o human TNFα (BD
Op EIA Human TNF ELISA se , #555212, BD Biosciences),
CCL2 (BD Op EIA Human MCP-1 ELISA se , #555179, BD Bio-
sciences), IL-10 (ELISA MAX S anda d se , #430601, BioLegend),
IL-6 (ELISA MAX S anda d se , #430501, BioLegend), and ac i in
A (DuoSe , #DY338, R&D Sys ems), ollowing he p o ocols sup-
plied by he manu ac u e s.
cell T ans ec ion and epo e gene
assays
HEK293-Tcells, p o ided by he Cell cul u e acili y a he Cen o
de In es igaciones Biológicas, we e ans ec ed wi h an exp es-
sion ec o o PPARγ2 (pBABE-PPARγ2, Addgene) o an emp y
ec o using Supe ec ans ec ion eagen (#301305, Qiagen).
Human GM-MØ o M-MØ (1 × 106 cells) we e ans ec ed
using he Human Mac ophage Nucleo ec o ® Ki (#VPA-1008,
Lonza) wi h 1µg o PPAR epo e DNA mix u e (#CCS-3026L,
Cignal PPAR Repo e assay ki , Qiagen). This mix u e con ains
a PPAR- esponsi e i e ly luci e ase cons uc and a cons i u-
i ely exp essing Renilla luci e ase (40:1) The PPAR-dependen
cons uc encodes he i e ly luci e ase gene unde he con ol
o a minimal CMV p omo e and andem epea s o he PPAR
esponsi e elemen (PPRE). Fi e ly and Renilla luci e ase ac i i-
ies we e de e mined by using he Dual-Luci e ase® Repo e
Assay Sys em (#E1910, P omega).
Wes e n Blo assay
Cell lysa es (40µg) and nuclea ex ac s (30µg) we e subjec ed o
SDS-PAGE and ans e ed on o an Immobilon poly inylidene
di luo ide memb ane (Millipo e, Bed o d, MA, USA). A e
4
Nie o e al. PPARγ T ansc ip ome in GM-CSF-Condi ioned Human Mac ophages
F on ie s in Immunology | www. on ie sin.o g Janua y 2018 | Volume 9 | A icle 31
blocking he unoccupied si es wi h 5% non a d y milk, p o ein
de ec ion was ca ied ou wi h a goa polyclonal agains PPARγ2
(G-18, #sc-22020, San a C uz Bio echnology), a goa a ini y pu i-
ied polyclonal an ibody agains Sp1 (PEP2, #sc-59-G, San a C uz
Bio echnology), o a monoclonal an ibody agains GAPDH (6C5,
#sc-32233, San a C uz Bio echnology), and using he Supe Signal
Wes Pico Chemiluminescen sys em (#34081, The mo Fishe
Scien i ic).
small in e e ing ibonucleic acid (si na)
T ans ec ion
To silence PPARG gene exp ession, human GM-MØ, M-MØ
(1×106 cells) o A-MØ (6–8×105 cells) we e ans ec ed wi h
a PPARG-speci ic siRNA (siPPARG) (50nM) (#s10888, The mo
Fishe Scien i ic), using HiPe Fec ans ec ion eagen (#301705,
Qiagen). A nega i e con ol siRNA om he same company was
used as a ans ec ion con ol (siCon ol) (#4390843, The mo
Fishe Scien i ic). A e 6h o ans ec ion, cells we e allowed o
eco e om ans ec ion in RPMI 1640 medium wi h 10% FCS
and he cells we e ea ed wi h GW7845 (1µM) (kindly p o ided
by Jon Collins, Glaxo Smi hKline, USA) o DMSO o 18–24h
be o e assessing o PPARγ ma ke s.
Mic oa ay analysis
Global gene exp ession analysis was pe o med on RNA
ob ained om h ee independen samples o GM-MØ ha
had been ans ec ed wi h siPPARG o siCon ol o 48 h,
and using a whole human genome mic oa ay om Agilen
Technologies (Palo Al o, CA, USA). Only p obes wi h signal
alues >60% quan ile in a leas one condi ion we e conside ed
o he di e en ial exp ession and s a is ical analysis. S a is ical
analysis o di e en ial gene exp ession was ca ied ou using
empi ical Bayes mode a ed es implemen ed in he limma
package1 and using pai ed - es . All he abo e p ocedu es
we e coded in R.2 Mic oa ay da a we e deposi ed in he
Gene Exp ession Omnibus3 unde accession no. GSE88768.
The di e en ially exp essed genes we e analyzed o anno-
a ed gene se s en ichmen using he online ool ENRICHR4
(48, 49). En ichmen e ms we e conside ed signi ican when
hey had a Benjamini-Hochbe g-adjus ed p alue<0.05. Fo
gene se en ichmen analysis (GSEA) (50), he p e iously
de ined “P oin lamma o y gene se ” and “An i-in lamma o y
gene se ” (12), which con ain he op and bo om 150 p obes
om he GM-MØ e sus M-MØ limma analysis o he mic o-
a ay da a in GSE68061 ( anked on he basis o he alue o he
s a is ic), we e used.
s a is ical analysis
S a is ical analysis was pe o med using pai ed S uden ’s - es ,
and p<0.05 was conside ed signi ican (*p<0.05, **p<0.01,
and ***p<0.001).
1 h p://www.bioconduc o .o g
2 h p://www. -p ojec .o g
3 h p://www.ncbi.nlm.nih.go /geo
4 h p://amp.pha m.mssm.edu/En ich /
esUlTs
PPa γ ac i a ion has Di e en
T ansc ip ional and Func ional Ou comes
in human gM-MØ and M-MØ
To ini ially assess he PPARγ ac i a ion-dependen ansc ip ional
p o ile o GM-MØ and M-MØ, bo h human mac ophage sub ypes
we e exposed o 24h o he PPARγ agonis GW7845 and he
exp ession o he GM-MØ-speci ic “P oin lamma o y gene se ”
and M-MØ-speci ic “An i-in lamma o y gene se ” (de i ed om
he da a con ained in he Gene Exp ession Omnibus GSE68061)
(11, 12) was de e mined. PPARγ ac i a ion up egula ed he pa a-
digma ic PPARγ a ge genes CD36 and FABP4, and down egu-
la ed FLT1 and CSF1 exp ession, in bo h mac ophage sub ypes
(Figu e1A). Howe e , GW7845 down egula ed IL6, IL10, CCL2,
HAMP, and CCR2 and enhanced THBS1, exclusi ely in GM-MØ
(Figu e1A). These GW7845- igge ed gene exp ession changes
we e dependen on PPARγ ac i a ion as hey we e signi ican ly
impai ed upon siRNA-media ed knockdown o PPARG mRNA
(Figu es1B,C). Speci ically, PPARG mRNA knockdown inhibi ed
he GW7845-media ed modula ion o CD36 and CSF1 exp ession
in M-MØ (Figu e1B) and signi ican ly impai ed he GW7845-
media ed modula ion o CD36, CSF1, FLT1, CCL2, CCR2, IL10,
and HAMP in GM-MØ (Figu e1C). Analogous indings we e
obse ed in mu ine bone ma ow-de i ed mac ophages, whe e
Ppa γ ac i a ion modi ied he exp ession o a common se o
genes in bo h mac ophage sub ypes bu signi ican ly diminished
he exp ession o Cs 1 and Cc 2 only in GM-MØ (Figu e 2).
The e o e, al hough PPARγ ac i a ion al e s he exp ession o
known PPARγ a ge s in bo h GM-MØ and M-MØ, i also p o-
mo es human mac ophage sub ype-dependen ansc ip ional
changes because he exp ession o CCR2, IL10, CCL2, and HAMP
is down egula ed by GW7845 only in p oin lamma o y GM-MØ.
To de e mine whe he he dis inc ansc ip ional e ec s o
PPARγ ac i a ion in GM-MØ and M-MØ had a unc ional co ela e,
he LPS-induced cy okine-p oducing abili y o bo h mac ophage
sub ypes was e alua ed in he p esence o GW7845. As expec ed,
LPS s imula ion o GM-MØ caused he p e e en ial p oduc ion
o he p oin lamma o y cy okines TNFα and IL-6, whe eas LPS-
s imula ed M-MØ p ima ily eleased IL-10 (9–11) (Figu e1D). In
line wi h he ansc ip ional esul s, GW7845 signi ican ly educed
he LPS-induced p oduc ion o TNFα and IL6 om GM-MØ, bu
had no e ec on he LPS-induced cy okine elease om M-MØ
(Figu e1D). Impo an ly, he inhibi o y e ec o GW7845 on he
LPS-induced TNFα p oduc ion o GM-MØ was PPARγ-dependen ,
as i was educed upon PPARγ knockdown (Figu e1E). The e o e,
agonis -media ed ac i a ion o PPARγ exclusi ely modula es he
LPS-induced cy okine p oduc ion om p oin lamma o y human
monocy e-de i ed GM-MØ, u he a guing o a pola iza ion-
dependen e ec o PPARγ in human mac ophages.
PPa γ is P e e en ially exp essed by
P oin lamma o y gM-csF-Dependen
human Mac ophages
Gi en he di e en e ec o PPARγ on GM-MØ and M-MØ,
we nex de e mined PPARγ exp ession and unc ion in bo h
FigU e 1 | Pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARγ) media es he di e en ial e ec o GW7845 on he gene and cy okine p o ile o human
GM-CSF-condi ioned monocy e-de i ed mac ophages (GM-MØ) and M-CSF-dependen monocy e-de i ed mac ophages (M-MØ). (a) Exp ession o he indica ed
genes in GM-MØ and M-MØ exposed o 24h o ei he GW7845 (GW, 1µM) o ehicle (DMSO), as de e mined by quan i a i e eal- ime PCR assay using
mic o luidic gene ca ds. Resul s a e indica ed as he exp ession o each gene a e GW7845 ea men ela i e o i s exp ession in he p esence o DMSO. Each
expe imen was pe o med in iplica e, and mean and SEM o h ee independen expe imen s is shown (*p<0.05; **p<0.01; ***p<0.001). (B,c) Exp ession o
he indica ed genes in M-MØ (B) o GM-MØ (c) ans ec ed wi h ei he siPPARG o siCon ol (siC), and ea ed wi h GW7845 (1µM) o DMSO o 24h. Rela i e
mRNA exp ession indica es he exp ession o each gene in he di e en condi ions and ela i e o i s exp ession in DMSO- ea ed siC- ans ec ed cells (a bi a ily se
o 1). (Le panels) PPARG mRNA exp ession in siPPARG- ans ec ed cells ela i e o he PPARG mRNA le el in siC- ans ec ed cells (a bi a ily se o 1). Mean and
SEM o ou independen expe imen s a e shown (*p<0.05; **p<0.01; ***p<0.001). (D) TNFα, IL-10, and IL-6 p oduc ion in LPS- ea ed (24h) GM-MØ and
M-MØ ha had been p eexposed (4h) o DMSO o GW7845 (GW, 1µM). Resul s indica e he concen a ion o each cy okine o each condi ion ela i e o he
cy okine le els de ec ed in cells ea ed wi h DMSO and LPS (a bi a ily se o 1). Mean and SEM o h ee independen expe imen s a e shown (*p<0.05;
***p<0.001). (e) TNFα p oduc ion in LPS- ea ed (24h) un ans ec ed (−), siCon ol- ans ec ed o siPPARG- ans ec ed GM-MØ ha had been p eexposed (4h)
o DMSO o GW7845 (GW, 1µM). Resul s indica e he concen a ion o TNFα o each condi ion ela i e o he cy okine le els de ec ed in un ans ec ed cells
ea ed wi h DMSO and LPS (a bi a ily se o 1). Mean and SEM o ou independen expe imen s a e shown (*p<0.05).
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FigU e 2 | Di e en ial e ec o pe oxisome p oli e a o -ac i a ed ecep o gamma ac i a ion on mouse GM-CSF-condi ioned bone ma ow-de i ed mac ophages
(GM-MØ) and M-CSF-dependen bone ma ow-de i ed mac ophages (M-MØ). Rela i e exp ession o he indica ed genes in mu ine bone ma ow-de i ed GM-MØ
(a) and M-MØ (B) exposed o ei he DMSO o GW7845 (1µM) o 24h, as de e mined by quan i a i e eal- ime PCR. Resul s a e exp essed as he exp ession o
each gene in he p esence o GW7845 ela i e o i s exp ession in he p esence o ehicle (DMSO). Mean and SD o h ee independen expe imen s is shown
(*p<0.05).
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human mac ophage sub ypes. T ans ec ion o a PPRE epo e
cons uc in bo h mac ophage sub ypes e ealed ha global
PPAR-dependen ansc ip ional ac i i y is highe in GM-MØ
han in M-MØ (Figu e3A), hus sugges ing ha GM-MØ a e
endowed wi h a s onge PPARγ-dependen ansc ip ional
ac i i y. Rega ding exp ession, GM-MØ con ained highe le els
o PPARG1/3 (encoding he ubiqui ous PPARγ1 iso o m) and
PPARG2 (coding o he PPARγ2 iso o m) mRNAs han M-MØ
(Figu e 3B). In ac , he adipocy e- es ic ed PPARG2 mRNA
(40) was ba ely de ec able in M-MØ (Figu e3B). The p e e en ial
exp ession o he PPARγ2-encoding mRNA was also obse ed in
mu ine bone ma ow-de i ed GM-MØ, whe eas mouse M-MØ
exhibi ed signi ican ly highe Ppa g1 exp ession han mouse
GM-MØ (Figu e3C), in ag eemen wi h a p e ious epo (51)
and in line wi h he dis inc gene p o iles o monocy e-de i ed
human M-MØ and bone ma ow-de i ed mouse M-MØ (13, 52).
Kine ic analysis e ealed ha PPARG2 mRNA is up egula ed
in human monocy es exposed o GM-CSF o 3, 5, and 7days
(Figu e 3D). Al hough PPARG1 is exp essed a highe le els
han PPARG2 mRNA (25- old app ox.), PPARγ2 p o ein could
be de ec ed in whole cell and nuclea ex ac s om GM-MØ
(Figu es3E,F). The e o e, GM-CSF-condi ioned p oin lamma-
o y human mac ophages exhibi a highe exp ession o
PPARγ (PPARγ1 and PPARγ2) han M-CSF-condi ioned an i-
in lamma o y human mac ophages.
ac i in a con ols PPa γ exp ession in
gM-csF-Dependen Mac ophages
The GM-CSF-dependen exp ession o PPARγ is essen ial o
he di e en ia ion o A-MØ (24). Since p oin lamma o y human
GM-MØ pola iza ion is dependen on he au oc ine/pa ac ine
ac ion o ac i in A (11), we nex ques ioned whe he ac i in A
con ibu es o he p e e en ial exp ession o PPARG mRNAs in
GM-MØ. Ac i in A signi ican ly ele a ed PPARG1/3 and PPARG2
mRNA le els in M-MØ, monocy es and THP-1 myeloid cells
(Figu e 4A). Mo eo e , inhibi ion o ac i in A-ini ia ed Smad
signaling by ei he SB431542 (Figu e4B) o A-83 (Figu e4C),
o blockade o ac i in A wi h an an i-ac i in neu alizing an i-
body (Figu e4D), signi ican ly educed PPARG1/3 and PPARG2
mRNA le els in GM-CSF-dependen p oin lamma o y GM-MØ.
In line wi h hese esul s, gene a ion o GM-MØ in he p esence
o A-83 esul ed in signi ican ly educed exp ession o he PPARγ
a ge gene ABCA1, a gene whose exp ession is esponsi e o
PPARγ-LXR ac i a ion in human mac ophages (Figu e 4E).
Fu he , analysis o ex i o isola ed human A-MØ e ealed he
cons i u i e exp ession o ac i in A (Figu e 4F), and ha he
exp ession o PPARG1/3 and PPARG2 mRNA, as well as he
exp ession o he PPARγ a ge ABCA1 mRNA, we e signi ican ly
educed in he p esence o he A-83 Smad signaling inhibi o
(Figu e4G). Al oge he , hese esul s indica e ha ac i in A is a
posi i e egula o o PPARγ exp ession and ac i i y in GM-CSF-
condi ioned mac ophages bo h in i o and in i o.
iden i ica ion o he PPa γ-Dependen
gene P o ile in gM-csF-condi ioned
P oin lamma o y human Mac ophages
Gi en he ansc ip ional e ec s o PPARγ knockdown (Figu e1C),
and o mo e ho oughly add ess he ole o PPARγ in in i o
gene a ed GM-CSF-condi ioned mac ophages, we de e mined
he PPARγ-dependen ansc ip ional p o ile o GM-MØ in
he absence o exogenous agonis s. siRNA-media ed PPARγ
knockdown signi ican ly modi ied he ansc ip ome o GM-MØ,
al e ing he exp ession o 314 p obes (283 anno a ed genes)
(p< 0.003, Table S2 in Supplemen a y Ma e ial). Speci ically,
PPARγ knockdown led o down egula ion o 139 genes and
up egula ion o 144 genes in GM-MØ (Figu e5A). Twen y- i e
pe cen o he genes down egula ed by siPPARG (36 ou o 139)
had been p e iously p edic ed as PPAR a ge s (53), including
20 genes up egula ed by long- e m osigli azone ea men o
human monocy e-de i ed dend i ic cells (54) and wo genes
whose exp ession is also diminished in mouse Ppa γ−/− mac-
ophages (CD36 and GPD1) (24) (Figu e5B). Simila ly, he se o
genes up egula ed upon PPARγ knockdown con ained 19 genes
FigU e 3 | Exp ession o pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARγ) iso o ms in human and mouse GM-CSF-condi ioned mac ophages (GM-MØ)
and M-CSF-dependen mac ophages (M-MØ). (a) Basal PPAR-dependen ansc ip ional ac i i y in GM-MØ and M-MØ. Mean and SEM o he ela i e PPAR-
dependen luci e ase ac i i y (compa ed o Renilla luci e ase ac i i y) o se en independen expe imen s is shown (*p<0.05). (B) PPARG1/3 and PPARG2 mRNA
exp ession le els in GM-MØ and M-MØ, as de e mined by quan i a i e eal- ime PCR (qRT-PCR) and ela i e o TBP mRNA le els. Mean and SEM o h ee
independen expe imen s is shown (*p<0.05; ***p<0.001). (c) Ppa g1 and Ppa g2 mRNA exp ession in bone ma ow-de i ed mu ine GM-MØ and M-MØ, as
de e mined by qRT-PCR and ela i e o Tbp mRNA le els. Mean and SD o h ee independen samples is shown (*p<0.05). (D) PPARG1/3 and PPARG2 mRNA
exp ession le els along GM-MØ and M-MØ di e en ia ion, as de e mined by qRT-PCR and ela i e o TBP mRNA le els. A ep esen a i e expe imen is shown.
(e,F) PPARγ2 p o ein le els in whole cell (e) o nuclea lysa es (Nucl. Ex .) (F) om mock- ans ec ed (C) o human PPARγ2- ans ec ed HEK293Tcells (e) and wo
independen samples o GM-MØ and M-MØ (dono #1 and dono #2). GAPDH (e) and Sp1 (F) p o ein le els we e de ec ed in pa allel as p o ein loading con ols.
The band co esponding o PPARγ2 p o ein is indica ed by an as e isk.
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p edic ed as PPAR a ge s (53) (Figu e5B), including 5 genes
up egula ed by osigli azone in human dend i ic cells (54) and
CCL2 and CCL7, whose o hologous genes a e o e exp essed in
mu ine Ppa γ−/− mac ophages (24). Con e sely, siPPARG down-
egula ed he exp ession o MSR1, whose mouse o holog is o e -
exp essed in Ppa γ−/− mac ophages (24). The PPARγ- egula ed
gene se  also included genes whose exp ession dis inguishes
A-MØ om o he issue- esiden mouse mac ophages (KRT79,
BCAR3, MAFF, WWTR1) (55) o ha e been de ined as human
A-MØ-en iched genes (EDN1, CXCL1, TNFAIP6, IL7R) (56)
(Table S2 in Supplemen a y Ma e ial). The e o e, PPARγ knock-
down in GM-CSF-condi ioned human mac ophages allowed he
iden i ica ion o a la ge se o genes (Table S2 in Supplemen a y
Ma e ial) whose exp ession is speci ically modula ed by PPARγ
in he absence o an exogenous agonis . Besides, and in ag eemen
wi h he di e gen ansc ip ional p o iles o unc ionally simila
human and mouse mac ophages (57), he human mac ophage
PPARγ-dependen gene se in human mac ophages only pa ially
o e laps wi h he lis PPARγ- egula ed genes p e iously iden i-
ied in mouse mac ophages.
To gain o ma ion on he biological p ocesses signi ican ly
a ec ed a e PPARγ knockdown in human mac ophages, unc -
ional en ichmen analysis was pe o med using GSEA (50).
Con i ming he alidi y o he esul s, PPARγ knockdown led o
a e y signi ican educ ion in he exp ession o genes associ-
a ed wi h he e ms “KEGG_PPAR_Signaling_Pa hway” and
“KEGG_Pe oxisome” (Figu e5C). In line wi h i s known an i-
in lamma o y unc ion (32, 58), educ ion o PPARγ exp ession
FigU e 4 | The ac i in A/Smad signaling pa hway de e mines he di e en ial exp ession o PPARG1 and PPARG2 in human GM-CSF-condi ioned
monocy e-de i ed mac ophages (GM-MØ) and M-CSF-dependen monocy e-de i ed mac ophages (M-MØ). (a) PPARG1 and PPARG2 exp ession in un ea ed o
ac i in A- ea ed M-MØ, monocy es and THP-1 cells. Cells we e ea ed wi h 25ng/ml ecombinan human ac i in A o 24h (monocy es and THP-1 cells) o 48h
(M-MØ). Mean and SEM o h ee independen expe imen s is shown (*p<0.05). (B) PPARG1 and PPARG2 gene exp ession in GM-MØ gene a ed in he p esence
o ei he DMSO o he Smad signaling inhibi o SB431542 (10µM). Mean and SEM o h ee independen expe imen s is shown (*p<0.05; ***p<0.001).
(c) PPARG1 and PPARG2 gene exp ession in GM-MØ gene a ed in he p esence o ei he DMSO o he Smad signaling inhibi o A-83 (1µM) o 1, 3, o 7days.
Mean and SEM o h ee independen s expe imen s is shown (*p<0.05; **p<0.01; ***p<0.001). (D) PPARG1 and PPARG2 gene exp ession in GM-MØ gene a ed
in he p esence o a neu alizing an iac i in A an ibody (α-Ac A) o an iso ype-ma ched an ibody (−). One ep esen a i e expe imen is shown. In (a–D), esul s a e
e e ed o he PPARG1 o PPARG2 mRNA le els de ec ed in un ea ed cells (a bi a ily se o 1). (e) ABCA1 gene exp ession in GM-MØ gene a ed in he p esence
o ei he DMSO o he Smad signaling inhibi o A-83 (1µM) o se en days. Resul s a e e e ed o he ABCA1 mRNA le el in DMSO- ea ed cells. Mean and SEM o
h ee independen s expe imen s is shown (*p<0.05). (F) Ac i in A exp ession le els in se en independen samples o human al eola mac ophages kep in cul u e
o 24h a e isola ion. (g) PPARG1, PPARG2, and ABCA1 mRNA le els in human al eola mac ophages cul u ed o 24h a e isola ion in he p esence o ei he
DMSO o he Smad signaling inhibi o A-83 (1µM). Resul s a e e e ed o he mRNA le els o each gene in DMSO- ea ed cells (a bi a ily se o 1). Means and
SEM o h ee independen s samples is shown (*p<0.05).
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in GM-MØ caused a signi ican inc ease in he exp ession
o genes wi hin he “Hallma k_In lamma o y Response,”
“Hallma k_TNFA signaling ia NFKB,” and “GO_Cellula
Response o IL1” gene se s (Figu e5D). Also in ag eemen wi h
he an i-in lamma o y ac i i y o PPARγ (32, 58), PPARγ knock-
down p omo ed a signi ican global up egula ion o he GM-MØ-
speci ic “P oin lamma o y gene se ” (12) (Figu e5D), and specially
o wo GM-MØ-speci ic genes like ECSCR and HSD11B1 (11)
FigU e 5 | Pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARγ) con ols he global ansc ip ional signa u e o GM-CSF-condi ioned human monocy e-
de i ed mac ophages (GM-MØ). (a) Numbe o anno a ed genes whose exp ession is highe o lowe in siPPARG- ans ec ed han in siCon ol- ans ec ed (siC)
GM-MØ a he indica ed p- alues. (B) Venn diag am analysis o he genes di e en ially exp essed in siPPARG- ans ec ed and siCon ol- ans ec ed GM-MØ
compa ed o expe imen ally e i ied PPARγ a ge genes (PPARγ a ge s) and compu a ionally p edic ed PPAR a ge genes (p edic ed PPAR a ge s), as epo ed in
he PPARgene da abase (53). (c–e) Gene se en ichmen analysis on he “ s a is ic- anked” lis o genes ob ained om he siPPARG-GM-MØ e sus siCon ol-GM-
MØ limma analysis, using he indica ed gene se . In (D), he p e iously de ined GM-MØ-speci ic “P oin lamma o y gene se ” (12) was also used. (F) Exp ession o
he indica ed genes in siPPARG- ans ec ed and siCon ol- ans ec ed (siC) GM-MØ, as de e mined by quan i a i e eal- ime PCR on h ee o i e independen
GM-MØ samples. Resul s a e indica ed as he mRNA le els o each gene in siPPARG- ans ec ed ela i e o he le els in siCon ol- ans ec ed GM-MØ (n=3–5;
*p<0.05; **p<0.01).
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