Peroxisome proliferator-activated receptor alpha and gamma agonists differently regulate classical and alternative macrophage activation

Immunome abolism 2015; 2: 1–11 abe an al e na i e mac ophage ac i a ion such as ib osing diseases. Keywo ds: Mac ophage pola iza ion, pe oxisome p oli e a o -ac i a ed ecep o , ib osis DOI 10.1515/immun-2015-0001 Recei ed Ma ch 2, 2015; accep ed Ma ch 23, 2015 1 In oduc ion Mac ophages a e impo an e ec o cells in in lamma o y esponses. They a e in ol ed in bac e ial in ec ions and au oimmune diseases bu also in he low g ade in lamma ion associa ed wi h obesi y and me abolic synd ome. Mac ophages also ha e homeos a ic unc ions and hey ac i a e healing p ocess associa ed wi h in lamma ion and issue inju y [1-3]. To achie e hese mul i o m asks, mac ophages a e p og ammed o display dis inc pheno ypes in esponse o en i onmen al signals [2-5]. Fo example, Th1 cy okine in e e on γ (IFNγ) and oll-like ecep o 4 (TLR4) ligand lipopolysaccha ide (LPS) igge classical, i.e. ype M1, ac i a ion o mac ophages, which is ypical o bac e ial in ec ions and au oimmune eac ions [6]. On he o he hand, Th2 cy okines in e leukin (IL) 4 and 13 igge al e na i e, i.e. ype M2, ac i a ion, which is associa ed wi h wound healing bu also wi h he abe an immune esponses associa ed wi h o e - ac i a ion o ib oblas s and subsequen ib osis [6,7]. Classical ac i a ion o mac ophages leads o he elease o p oin lamma o y and an imic obial ac o s including IL-6, IL-12, umou nec osis ac o (TNF) and ni ic oxide (NO) p oduced by inducible ni ic oxide syn hase (iNOS) pa hway. On he o he hand, al e na i ely ac i a ed mac ophages exp ess ac o s ha ha e been associa ed wi h ecogni ion o ex acellula pa hogens and/o s imula ion o ib oblas s. Some o Abs ac : Pe oxisome p oli e a o -ac i a ed ecep o (PPAR) agonis s, ib a es and hiazolidinediones, a e commonly used d ugs in he ea men o dyslipidemia and diabe es. Thei a ge s, PPARα and PPARγ, ha e also been shown o ha e a ole in he egula ion o in lamma o y esponses linking me abolism and in lamma ion. In he p esen s udy we in es iga ed he e ec s o PPAR agonis s on mac ophage ac i a ion. In addi ion o he p oin lamma o y classical ac i a ion, we also ocused on in e leukin (IL) 4 and 13 -induced al e na i e ac i a ion which is a signi ican mac ophage pheno ype in issue epai ing p ocesses and in ib osing diseases. PPARα agonis s GW7647 and eno ib a e as well as PPARγ agonis GW1929 inhibi ed lipopolysaccha ide-induced classical mac ophage ac i a ion and p oduc ion o he cha ac e is ic bioma ke s o his pheno ype, i.e. IL-6 and ni ic oxide, in mu ine J774 mac ophages. Rema kably, he PPARα agonis s also inhibi ed IL-4 and IL-13 –induced exp ession o al e na i e ac i a ion ma ke s a ginase-1, izz1 and mannose ecep o 1 whe eas he PPARγ agonis GW1929 enhanced hei exp ession in J774 mac ophages. The PPARα agonis s GW7647 and eno ib a e also a enua ed he p oduc ion o al e na i e ac i a ion ma ke s chemokine (C-C mo i ) ligand 13 and pla ele - de i ed g ow h ac o in human THP-1 mac ophages. The p esen indings show ha PPARα and PPARγ agonis s di e en ly egula e classical and al e na i e mac ophage pheno ypes. Fu he mo e, PPARα ac i a ion was in oduced as a no el concep o down- egula e al e na i e mac ophage ac i a ion indica ing ha PPARα agonis s ha e he apeu ic po en ial in condi ions associa ed wi h Resea ch A icle Open Access © 2015 E ja-Leena Paukke i e al., licensee De G uy e Open. This wo k is licensed unde he C ea i e Commons A ibu ion-NonComme cial-NoDe i s 3.0 License. E ja-Leena Paukke i, An i Peku inen, Ee a Moilanen Pe oxisome p oli e a o -ac i a ed ecep o α and γ agonis s di e en ly egula e classical and al e na i e mac ophage ac i a ion *Co esponding au ho : Ee a Moilanen: The Immunopha macology Resea ch G oup, Uni e si y o Tampe e School o Medicine and Tampe e Uni e si y Hospi al, Tampe e, Finland, E-mail: ee a.moi[email p o ec ed] E ja-Leena Paukke i, An i Peku inen: The Immunopha macology Resea ch G oup, Uni e si y o Tampe e School o Medicine and Tampe e Uni e si y Hospi al, Tampe e, Finland B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM 2   E ja-Leena Paukke i e al. hese ac o s a e a ginase 1, chemokine (C-C mo i ) ligand 13 (CCL13), pla ele -de i ed g ow h ac o (PDGF), mannose ecep o 1 (m c-1) and, in mu ine mac ophages, ound in in lamma o y zone 1 ( izz1), o ins ance [4,8- 10]. Likewise, also in clinical se ings, al e na i ely ac i a ed mac ophages ha e been linked o de ence agains ex acellula pa hogens, and o esolu ion o in lamma ion, wound healing and ib osis [11-13]. In addi ion o IL-4 and IL-13, pe oxisome p oli e a o - ac i a ed ecep o γ (PPARγ) ligands ha e been ound o pola ize mac ophages owa ds he al e na i e pheno ype [14,15]. PPARs a e nuclea ecep o s ha ha e a c ucial physiological ole in he egula ion o ene gy homeos asis. PPARγ egula es he impo o glucose in o cells, and PPARγ agonis s hiazolidinediones (TZDs) a e widely used as pha macological agen s in he ea men o ype 2 diabe es. Ano he iso ype o PPARs, PPARα is linked o he egula ion o lipid me abolism. Syn he ic ligands include ib a es, which a e used o ea dyslipidemia. As compa ed o PPARγ agonis s, less is known abou he possible in ol emen o PPARα agonis s in he egula ion o immune and in lamma o y esponses [16-19] and hei e ec s on al e na i e ac i a ion o mac ophages emains unknown. In he p esen s udy, we aimed o compa e he e ec s o PPARα and PPARγ agonis s on classical and al e na i e ac i a ion o mac ophages. We used LPS o a combina ion o IL-4 and IL-13 o induce mac ophage pola iza ion o classical o al e na i e pheno ype, espec i ely. The esul s show ha PPARα and PPARγ agonis s di e en ly egula e mac ophage pheno ypes, and in oduce PPARα ac i a ion as a no el concep o down- egula e al e na i e mac ophage ac i a ion. 2 Ma e ials and me hods 2.1 Ma e ials Reagen s we e ob ained as ollows: GW1929 and GW7647 om Toc is Bioscience (B is ol, UK), abbi polyclonal β-ac in, a ginase 1 and STAT6 an ibodies and goa HRP- conjuga ed an i- abbi and donkey HRP-conjuga ed an i- goa polyclonal an ibodies om San a C uz Bio echnology Inc. (San a C uz, CA, USA). pSTAT6 (Ty 641) an ibody was om Calbiochem (Me ck Millipo e, Bille ica, MA, USA) and PPARα and PPARγ an ibodies om Alexis Biochemicals (Enzo Li e Sciences, Lausen, Swi ze land). Recombinan mouse IL-4 and IL-13 and human IL-4 we e om R&D Sys ems (Minneapolis, MN, USA). O he eagen s we e om Sigma-Ald ich Co. (S . Louis, MO, USA) unless o he wise s a ed. 2.2 Cell cul u e Mu ine J774A.1 mac ophages (Ame ican Type Cul u e Collec ion, Manassas, VA, USA) we e cul u ed a 37°C in 5% CO2 a mosphe e in Dulbecco’s modi ied Eagle’s medium wi h Ul aglu amine1 (Lonza G oup L d, Basel, Swi ze land) supplemen ed wi h 10% hea -inac i a ed oe al bo ine se um (Lonza G oup L d), 100 U/ml penicillin, 100 µg/ml s ep omycin and 250 ng/ml ampho e icin B (In i ogen Co., Ca lsbad, CA, USA) and ha es ed wi h ypsin-EDTA (In i ogen Co.). Cells we e seeded on 24-well pla es o RNA ex ac ion, ni i e and ELISA measu emen s and o p epa a ion o cell lysa es o wes e n blo ing and on 96-well pla es o XTT es . Con luen cul u es we e exposed o esh cul u e medium con aining he compounds o in e es . PPAR agonis s we e added o he medium oge he wi h LPS o a combina ion o IL-4 and IL-13 unless o he wise s a ed. Human THP-1 p omonocy es (Ame ican Type Cul u e Collec ion) we e cul u ed a 37°C in 5% CO2 a mosphe e in RPMI 1640 (Lonza G oup L d) adjus ed o con ain 2 mM ʟ-glu amine (Lonza G oup L d), 10 mM HEPES (Lonza G oup L d), 4.5 g/l glucose (Sigma-Ald ich Co.) and 1.5 g/l bica bona e (Lonza G oup L d) and supplemen ed wi h 10% hea -inac i a ed oe al bo ine se um (Lonza G oup L d), 100 U/ml penicillin, 100 µg/ ml s ep omycin, 250 ng/ml ampho e icin B (In i ogen Co.), and 0.05 mM 2-me cap oe hanol (Sigma-Ald ich Co.). The cells we e di e en ia ed by adding pho bol es e 12-O- e adecanoylpho bol-13-ace a e (100 nM) (Sigma- Ald ich Co.) a he ime o seeding on 24-well pla es. Se en y- wo hou s a e he seeding he cul u es we e con luen and hey we e exposed o esh cul u e medium con aining he compounds o in e es . PPAR agonis s we e added o he medium oge he wi h LPS o a combina ion o IL-4 and IL-13 unless o he wise s a ed. Cell iabili y a e ea men wi h combina ions o LPS o cy okine mix u e and he es ed compounds was assessed by modi ied XTT es (Cell p oli e a ion Ki II, Roche Diagnos ics, Mannheim, Ge many) acco ding o he manu ac u e ’s ins uc ions. 2.3 Ni i e de e mina ion NO p oduc ion was de e mined by measu ing he accumula ion o ni i e, a s able me aboli e o NO in aqueous condi ion, in o he cul u e medium. The cul u e medium was collec ed a indica ed ime poin s and ni i e was measu ed by he G iess eac ion [20]. B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM PPARα agonis s supp ess al e na i e mac ophage ac i a ion   3 2.4 P epa a ion o cell lysa es o wes e n blo ing A indica ed ime poin s, he cells we e apidly washed wi h ice-cold phospha e-bu e ed saline and solubilized in cold lysis bu e con aining 10 mM T is- base, pH 7.4, 5 mM EDTA, 50 mM NaCl, 1% T i on X-100, 0.5 mM phenylme hylsulphonyl luo ide, 1 mM sodium o ho anada e, 20 μg/ml leupep in, 50 μg/ml ap o inin, 5mM NaF, 2 mM sodium py ophospha e and 10 μM n-oc yl-β-D-glucopy anoside. A e incuba ion on ice o 15 minu es, lysa es we e cen i uged (13 400 g, 4°C, 10 min), supe na an s we e collec ed and mixed 3:1 wi h SDS sample bu e (62.5 mM T is–HCl, pH 6.8, 10% glyce ol, 2% SDS, 0.025% b omophenol blue and 5% β-me cap oe hanol). The samples we e s o ed a -20°C un il analysed. An aliquo o he supe na an was used o de e mine p o ein concen a ion by he Coomassie blue me hod [21]. 2.5 Wes e n blo ing P io o wes e n blo ing, samples we e boiled o 10 minu es and 20 μg o p o ein was loaded pe lane on 8% o 10% SDS-polyac ylamide gels and sepa a ed by elec opho esis. P o eins we e ans e ed o Hybond enhanced chemiluminescence ni ocellulose memb ane (GE Heal hca e, Li le Chal on , Buckinghamshi e, UK). A e he ans e , he memb ane was blocked in TBS/T (20 mM T is-base pH 7.6, 150 mM NaCl, 0.1% Tween-20) con aining 5% o non- a d y milk o 5% bo ine se um albumin a oom empe a u e o 1 h and incuba ed wi h he p ima y an ibody in he blocking solu ion a 4 °C o e nigh . The memb ane was washed wi h TBS/T, incuba ed wi h he seconda y an ibody in he blocking solu ion a oom empe a u e o 1 hou and washed. Bound an ibody was de ec ed using Supe Signal Wes Pico o Du a chemiluminescen subs a e (Pie ce, Rock o d, IL, USA) and ImageQuan LAS 4000 mini imaging sys em (GE Heal hca e). The chemiluminescen signal was quan i ied wi h ImageQuan TL 7.0 image analysis so wa e (GE Heal hca e). 2.6 RNA ex ac ion and quan i a i e eal- ime PCR A he indica ed ime poin s, cul u e medium was emo ed and o al RNA ex ac ion o he cells was ca ied ou wi h GenElu e™ Mammalian To al RNA Minip ep ki (Sigma- Ald ich Co.). 100 ng o o al RNA was e e se- ansc ibed o cDNA using TaqMan Re e se T ansc ip ion eagen s and andom hexame s (Applied Biosys ems, Fos e Ci y, CA, USA). cDNA ob ained om he e e se ansc ip ion eac ion was dilu ed 1:20 wi h RNase- ee wa e and was subjec ed o quan i a i e PCR using TaqMan Uni e sal PCR Mas e Mix and ABI P ism 7000 sequence de ec ion sys em (Applied Biosys ems). P ime s and p obes o a ginase 1 and glyce aldehyde- 3-phospha e dehyd ogenase (GAPDH) we e op imized acco ding o he manu ac u e ’s ins uc ions in TaqMan Uni e sal PCR Mas e Mix P o ocol pa numbe 4304449 e ision C and we e as ollows: 5’-TCCAAGCCAAAGTCCTTAGAGATTAT-3’, 5’-CGTCATACTCTGTTTCTTTAAGTTTTTCC-3’, 5’-CGCCTTTCTCAAAAGGACAGCCTCGA-3’ ( o wa d and e e se p ime s and p obe o mouse a ginase 1) and 5’-GCATGGCCTTCCGTGTTC-3’, 5’-GATGTCATCATACTTGGCAGGTTT-3’, 5’-TCGTGGATCTGACGTGCCGCC-3’ ( o wa d and e e se p ime s and p obe o mouse GAPDH). The exp ession o mouse PPARα, PPARγ, izz1 and m c-1 and human CCL13 and PDGF mRNA was measu ed by using TaqMan® Gene Exp ession Assay (Mm00440939_m1, Mm01184322_m1, Mm00445109_m1, Mm00485148_m1, Hs01033504_g1, Hs00966522_m1, Applied Biosys ems). PCR eac ion pa ame e s we e as ollows: incuba ion a 50°C o 2 min, incuba ion a 95°C o 10 min, and he ea e 40 cycles o dena u a ion a 95°C o 15 s and annealing and ex ension a 60°C o 1 min. Each sample was de e mined in duplica e. The ela i e mRNA le els we e quan i ied and compa ed using he ela i e s anda d cu e me hod as desc ibed in Applied Biosys ems Use Bulle in numbe 2 (a ginase 1) o ΔC me hod (TaqMan® Gene Exp ession Assays). 2.7 Enzyme-Linked Immunoso ben Assay (ELISA) Cul u e medium samples we e kep a -20°C un il assayed. The concen a ions o IL-6 and CCL13 in cul u e medium we e de e mined by ELISA acco ding o he manu ac u e ’s ins uc ions using eagen s om R&D Sys ems Eu ope (Abingdon, UK). 2.8 S a is ics Resul s a e exp essed as mean + s anda d e o o mean (SEM). When indica ed, s a is ical signi icance was calcula ed by analysis o a iance ollowed by Dunne ’s mul iple compa isons es o by unpai ed es wi h Welch co ec ion. All he s a is ical analyses we e pe o med B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM 4   E ja-Leena Paukke i e al. using G aphPad InS a e sion 3.10 o Windows (G aphPad So wa e, San Diego, CA, USA). Di e ences we e conside ed signi ican a p<0.05. E hical app o al: The conduc ed esea ch is no ela ed o ei he human o animals use. 3 Resul s 3.1 J774 mac ophages exp ess PPARs and can ep esen classical and al e na i e ac i- a ion pheno ypes J774 mac ophages we e ound o exp ess PPARα and PPARγ as de ec ed by wes e n blo ing and RT-qPCR (Figu e 1). As shown in Figu es 2 and 3, he ac i a ion o he mac ophages wi h LPS inc eased he exp ession o classical ac i a ion ma ke s including IL-6 and inducible ni ic oxide syn hase (iNOS) whe eas s imula ion wi h a combina ion o IL-4 and IL-13 induced he exp ession o al e na i e ac i a ion ma ke s a ginase 1, izz1 and m c-1. The le els o a ginase 1, izz1 and m c-1 mRNA we e highes a 36 h, 12 h and 12 - 24 h, espec i ely, a e he onse o he in e leukin s imula ion (Figu e 2). 3.2 PPAR agonis s educed he le els o classical ac i a ion ma ke s a e LPS-s imu- la ion, bu had di e ing e ec s on al e na- i e ac i a ion PPARα agonis s GW7647 and eno ib a e and PPARγ agonis GW1929 a enua ed classical mac ophage ac i a ion as shown by educed iNOS exp ession (Figu e 3a) and NO and IL-6 p oduc ion (Figu e 3b) in J774 mac ophages exposed o LPS. The agonis s did no al e he le els o classical ac i a ion ma ke s in he absence Figu e 1 The exp ession o PPARα and PPARγ in J774 mac ophages. (a) P o eins we e ex ac ed and PPARα and PPARγ p o ein exp ession was de e mined by wes e n blo ing. The gels shown a e ep esen a i es o h ee o he s wi h simila esul s. (b) To al RNA was ex ac ed and he le els o PPARα and PPARγ mRNA we e analysed by RT-qPCR. The esul s we e no malized agains GAPDH mRNA and he le el o PPARα mRNA was compa ed o ha o PPARγ (n=6). Figu e 2 Time-dependen exp ession o al e na i e ac i a ion ma ke s in J774 mac ophages. The cells we e s imula ed wi h IL-4 and IL-13 and incuba ions we e e mina ed a indica ed ime poin s and o al RNA was ex ac ed. A ginase 1 (a), izz1 (b) and m c-1 (c) mRNA we e de e mined by RT-qPCR. The esul s we e no malized agains GAPDH mRNA. Resul s ep esen he mean ± SEM (n=4). B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM PPARα agonis s supp ess al e na i e mac ophage ac i a ion   5 and eno ib a e also dec eased he exp ession o a ginase 1 p o ein (Figu e 7). 3.3 PPARα agonis s educed he le els o al e na i e ac i a ion ma ke s also in human THP-1 mac ophages Be o e e alua ing he e ec s o PPARα agonis s on al e na i e ac i a ion in human mac ophages, we examined CCL13 and PDGF mRNA ansc ip ion ollowing IL-4 s imula ion, as ma ke s o al e na i e ac i a ion [3,22]. In THP-1 mac ophages IL-4-enhanced CCL13 and PDGF exp ession, and hei mRNA le els con inued o inc ease a leas un il 36 hou s a e he addi ion o IL-4 (Figu e 8). GW7647 and eno ib a e clea ly dec eased IL-4–induced CCL13 exp ession as shown by educed cellula le els o CCL13 mRNA and dec eased amoun s o CCL13 p o ein in he cul u e medium (Figu e 9a-b). GW7647, and o a lesse o LPS o a ec cell iabili y a he concen a ion used as de e mined by XTT es (da a no shown). To in es iga e i PPARs a e able o induce al e na i e ac i a ion in mac ophages, we es ed he e ec s o he PPAR agonis s on he exp ession o a ginase 1. As expec ed, he PPARγ agonis GW1929 inc eased he exp ession o a ginase 1 mRNA when measu ed a e 24 h incuba ion. In con as , he PPARα agonis s GW7647 and eno ib a e dec eased he le els o a ginase 1 (Figu e 4). Nex , we es ed he e ec o PPAR agonis s on IL-4 + IL-13 -induced al e na i e pheno ype in J774 cells. The selec i e PPARγ agonis GW1929 inc eased IL-4 + IL-13 -induced p o ein exp ession o a ginase 1, and mRNA le els o izz1 and m c-1, when de e mined a e 24 hou incuba ion (Figu e 5). In con as , he PPARα agonis s dec eased IL-4 + IL-13 -induced le els o a ginase 1, izz1 and m c-1 mRNA, when de e mined a e 24 hou incuba ion (Figu e 6). In line wi h he esul s a mRNA le el, GW7647 Figu e 3 PPARα and PPARγ agonis s supp essed classical mac ophage ac i a ion. (a) Cells we e ea ed wi h he PPARα agonis s GW7647 o eno ib a e o wi h he PPARγ agonis GW1929 and s imula ed wi h LPS. A e 24 h incuba ion, p o eins we e ex ac ed and iNOS exp ession was analysed by wes e n blo ing. β-ac in was used as a loading con ol. (b) Cells we e ea ed wi h he PPARα agonis s GW7647 o eno i- b a e o wi h he PPARγ agonis GW1929 and s imula ed wi h LPS. A e 24 h incuba ion, ni i e accumula ed in o he cul u e medium was measu ed by G iess eac ion as a ma ke o NO p oduc ion. IL-6 in he cul u e medium was measu ed by ELISA. Resul s ep esen he mean + SEM (n=4). ** = p<0.01 as compa ed o cells cul u ed wi h LPS alone. B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM 6   E ja-Leena Paukke i e al. ex end eno ib a e, also dec eased PDGF mRNA le els in THP-1 cells (Figu e 9c). 3.4 PPAR agonis s did no a ec he phos- pho yla ion o STAT6 STAT6 has been shown o be an impo an cy osolic media o o ansc ip ional e ec s o IL-4 and IL-13. The e o e we in es iga ed he e ec s o PPAR agonis s on STAT6 phospho yla ion (i.e. ac i a ion). Cos imula ion wi h IL-4 and IL-13 induced he phospho yla ion o STAT6 peaking a 30 minu es a e addi ion o IL-4 and IL-13 in J774 mac ophages (Figu e 10a). As seen in Figu e 10b-c, nei he eno ib a e no GW7647 modi ied he phospho yla ion o STAT6 in J774 o THP-1 mac ophages. 4 Discussion The p esen indings in oduce PPARα agonis s as compounds able o down- egula e al e na i e mac ophage ac i a ion, while PPARγ agonis s enhanced he al e na i e pheno ype o mac ophages. Fu he , bo h PPARα and PPARγ agonis s a enua ed he classical mac ophage ac i a ion. The al e na i e ac i a ion o adipose issue mac ophages has been p oposed o p o ec lean people om insulin esis ance and o he obesi y-linked me abolic mani es a ions. Thus, PPARγ agonis s and o he compounds ha pola ize mac ophages owa ds he Figu e 5 PPARγ agonis GW1929 enhanced he exp ession o al e na- i e ac i a ion ma ke s in J774 mac ophages. Cells we e ea ed wi h GW1929 and s imula ed wi h IL-4 and IL-13 o 24 hou s. (a) P o eins we e ex ac ed and he le els o a ginase 1 we e analysed by wes e n blo ing. β-ac in was used as a loading con ol. (b-c) To al RNA was ex ac ed and subjec ed o RT-qPCR. The esul s we e no malized agains GAPDH mRNA. Resul s ep esen he mean + SEM (n=4). ** = p<0.01 as compa ed o cells cul u ed wi h IL-4 and IL-13. Figu e 4 PPARα agonis s supp essed, bu PPARγ agonis s enhanced he exp ession o he classical ma ke o al e na i e ac i a ion, i.e. a ginase 1, in J774 mac ophages. Cells we e ea ed wi h he PPARα agonis s GW7647 o eno ib a e o wi h he PPARγ agonis GW1929 o 24 hou s. To al RNA was ex ac ed and a ginase 1 mRNA was de e mined by RT-qPCR. The esul s we e no malized agains GAPDH mRNA. Resul s ep esen he mean + SEM (n=4). * = p<0.05 and ** = p<0.01 as compa ed o un ea ed cells. B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM PPARα agonis s supp ess al e na i e mac ophage ac i a ion   7 al e na i e pheno ype may be bene icial in obesi y ela ed me abolic diso de s [23,24]. In con as , compounds able o inhibi al e na i e mac ophage pheno ype may ha e a he apeu ic e ec in ib osing diseases, which a e complica ed wi h abe an ly enhanced ac i a ion o al e na i e mac ophages [25-27]. Al e na i ely ac i a ed mac ophages ac i a e ib oblas s o p oli e a e, mig a e o he ib osing a ea and o p oduce ex acellula ma ix componen s esul ing in ib oblas -popula ed g anula ion issue [28]. Howe e , he mechanisms how his occu s a e no known in de ail. Acco ding o p e ious epo s, sys emic scle osis (SSc) is an example o he diseases whe e mac ophages induce p o ib o ic ac ion o ib oblas s. Ho me and He zog showed in 2010 ha mos o he mac ophages in he Figu e 6 PPARα agonis s supp essed he exp ession o al e na i e ac i a ion ma ke s in J774 mac ophages. Cells we e ea ed wi h GW7647 o eno ib a e and s imula ed wi h IL-4 and IL-13 o 24 hou s. To al RNA was ex ac ed and a ginase 1 (a), izz1 (b) and m c-1 (c) mRNA was de e mined by RT-qPCR. The esul s we e no malized agains GAPDH mRNA. Resul s ep esen he mean + SEM (n=4). * = p<0.05 and ** = p<0.01 as compa ed o cells cul u ed wi h IL-4 and IL-13. Figu e 7 PPARα agonis s supp essed a ginase 1 p o ein exp es- sion in J774 mac ophages. Cells we e ea ed wi h GW7647 (a) o eno ib a e (b) and s imula ed wi h IL-4 and IL-13 o 24 hou s. P o eins we e ex ac ed and he le els o a ginase 1 we e analysed by wes e n blo ing. β-ac in was used as a loading con ol. Resul s ep esen he mean + SEM (n=4). ** = p<0.01 as compa ed o cells cul u ed wi h IL-4 and IL-13. B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM 8   E ja-Leena Paukke i e al. lung issue o pa ien s wi h SSc-associa ed in e s i ial lung disease display ma ke s o al e na i e mac ophage ac i a ion [27]. Also in ano he epo , m c-1 exp ession was ele a ed in pe iphe al blood mononuclea cells (PBMCs) om pa ien s wi h limi ed cu aneous SSc and pulmona y hype ension when compa ed o heal hy con ols [25]. Addi ionally, pa ien s wi h ei he limi ed o di use cu aneous SSc ha e been epo ed o ha e ele a ed se um le els o al e na i e mac ophage- ela ed cy okines IL-4, IL-10 and IL-13 [25,26]. IL-13 ecep o an agonis s also a enua ed skin and lung ib osis in expe imen al models. Acco dingly, ea men wi h IFN-γ (which d i es mac ophages owa ds M1 pheno ype) has been shown o imp o e skin sco es in pa ien s wi h SSc [31]. Mos in e es ingly, an a ginase inhibi o , pi enidone, has been p o en o be bene icial in pa ien s wi h idiopa hic pulmona y ib osis when compa ed o placebo in wo mul icen e s udies [32]. Pi enidone was app o ed o he ea men o idiopa hic pulmona y ib osis in Eu ope Figu e 8 Time-dependen exp ession o al e na i e ac i a ion ma ke s in THP-1 cells. The cells we e s imula ed wi h IL-4 and incuba ions we e e mina ed and o al RNA was ex ac ed a indica- ed ime poin s. CCL13 (a) and PDGF (b) mRNA was de e mined by RT-qPCR. The esul s we e no malized agains GAPDH mRNA. Resul s ep esen he mean ± SEM (n=4). Figu e 9 PPARα agonis s supp essed he exp ession o al e na i e ac i a ion ma ke s in THP-1 cells. (a, c) Cells we e ea ed wi h GW7647 o eno ib a e and s imula ed wi h IL-4 o 24 hou s. To al RNA was ex ac ed and CCL13 (a) and PDGF (c) mRNA was de e mi- ned by RT-qPCR. The esul s we e no malized agains GAPDH mRNA. (b) Cells we e ea ed wi h GW7647 o eno ib a e and s imula ed wi h IL-4. A e 24 h incuba ion, CCL13 accumula ed in o he cul u e medium was measu ed by ELISA. Resul s ep esen he mean + SEM (n=4). ** = p<0.01 as compa ed o cells cul u ed wi h IL-4. B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM PPARα agonis s supp ess al e na i e mac ophage ac i a ion   9 TNF [17,33,34]. In addi ion, PPARγ agonis s ha e been epo ed o shi he pola iza ion o mac ophages owa ds he al e na i e pheno ype [14,15] and ha was also seen in he p esen s udy. In e es ingly, we epo he e ha PPARα agonis s ha e an opposi e e ec on M2 pola iza ion. To ou knowledge, he p esen s udy is he i s epo showing he inhibi o y e ec s o PPARα agonis s on he al e na i e ac i a ion o mac ophages indica ing ha PPARα and PPARγ agonis s di e en ly egula e al e na i e mac ophage ac i a ion. The di e si y o mac ophage pheno ypes has bes been desc ibed in mouse mac ophages. Some o he known ma ke s o al e na i e ac i a ion, e.g. izz1, a e exp essed only in mice [35] and some o he ma ke s, like a ginase 1, a e impo an in i o bu become silenced apidly in ex i o condi ions in human cells [36,37]. Also as epo ed in his s udy, J774 mac ophages inc eased Figu e 10 The e ec s o PPARα agonis s on STAT6 phospho yla ion in J774 and THP-1 cells. (a) Cells we e s imula ed wi h IL-4 and IL-13 and incuba ions we e e mina ed a indica ed ime poin s. P o eins we e ex ac ed and he le els o pSTAT6 we e analysed by wes e n blo ing. To al STAT6 was used as a loading con ol. The gel shown is a ep esen a i e o h ee o he s wi h simila esul s. (b) J774 mac ophages we e p eincuba ed wi h he PPARα agonis s o he ehicle o an hou be o e addi ion o IL-4 and IL-13. A e 30 min incuba ion wi h IL-4 and IL-13 p o eins we e ex ac ed and he le els o pSTAT6 we e analysed by wes e n blo ing. (c) THP-1 cells we e p eincuba ed wi h he PPARα ago- nis s o he ehicle o an hou be o e addi ion o IL-4. A e 30 min incuba ion wi h IL-4, p o eins we e ex ac ed and he le els o pSTAT6 we e analysed by wes e n blo ing. (b,c) To al STAT6 and β-ac in we e used as loading con ols. Resul s ep esen he mean + SEM (n=4). ** = p<0.01 as compa ed o cells cul u ed wi h IL-4 and IL-13 (a,b) o IL-4 (c). in 2011 and may be bene icial also in o he ib osing diseases including SSc. A he momen , he e a e no speci ic ea men s a ge ing he pa hophysiology o he ib osing p ocesses in SSc o ela ed diseases bu mos o he he apies used ac as immunomodula o s, like me ho exa e, cyclophosphamide and aza hiop ine, o asodila o s, like calcium channel blocke s, p os acyclin analogs, elmisa an, phosphodies e ase inhibi o s and endo helin I an agonis s. Howe e , he e icacy o hese d ugs is e y limi ed and be e ea men op ions a e c i ically needed. PPARα agonis s ib a es and PPARγ agonis s TZDs a e in clinical use o he ea men o me abolic diso de s. In line wi h he p esen esul s, bo h ib a es and TZDs ha e also shown o dec ease he p oduc ion o IFNγ o LPS –induced p oin lamma o y media o s ypical o classical mac ophage ac i a ion like iNOS, NO, IL-6 and B ough o you by | Tampe e Uni e si y Lib a y Au hen ica ed Download Da e | 12/1/16 2:18 PM