Temporal expression patterns of the melatoninergic system in the human thymus of children

Tempo al exp ession pa e ns o he mela onine gic sys em in he human hymus o child en I an C uz-Chamo o 1 , 2 , Nu ia Ál a ez-Sánchez 1 , C is ina Escalan e-Andicoechea 1 , An onio Ca illo-Vico 1 , 2 , Amalia Rubio 1 , 2 , Juan Miguel Gue e o 1 , 2 , 3 , Pa ocinio Moline o 1 , 2 , 4 , Pa icia J. La done 1 , 2 , * , 4 ABSTRACT Objec i es: To ob ain g ea e knowledge o he ex a-pineal sou ces o mela onin du ing de elopmen , he amoun o indolamine and he exp ession le els o he las wo enzymes in ol ed in i s biosyn hesis, A ylalkylamine N-ace yl ans e ase (AANAT) and ace ylse o onin O- me hyl ans e ase (ASMT), we e analyzed in he human hymus om child en om h ee di e en age g oups ( om days o yea s). The mela onin memb ane and nuclea ecep o exp ession le els also we e s udied. Me hods: Quan i a i e e e se ansc ip ase PCR and wes e n blo we e pe o med o in es iga e he ecep o and enzyme exp ession le els. The esul s we e examined and co ela ed wi h he ages o he hymuses. Resul s: We ound high le els o indolamine in he hymuses o newbo ns (younge han 1 mon h), which dec eased du ing de elopmen ; hymuses om he mon hs ( om 2 o 11 mon hs) and yea s ( om 1 o 12 yea s) g oups showed lowe le els. A simila decline was also obse ed in he mRNA o he AANAT enzyme and he exp ession le els o mela onin ecep o s. Howe e , ASMT exp ession was exac ly he opposi e, wi h low le els in he newbo n g oup and highe le els in he yea s g oup. Ou esul s show ha he hymic syn hesis o mela onin occu s e y ea ly in childhood. Addi ionally, his is he fi s epo ha is ocused on mela onin ecep o s exp ession in he human hymus. Conclusion: Conside ing he limi ed mela onin syn hesis pe o med by he newbo n pineal gland, we sugges ha he high le els o mela onin ound in human hymus in his expe imen al g oup a ise om syn hesis in he issue i sel , which could be con ibu ing o he immune e ficiency a he hymic le el. Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Keywo ds Mela onin; Thymus; AANAT; ASMT; Mela onin ecep o ; Nuclea ecep o ROR-alpha 1. INTRODUCTION Ex a-pineal mela onin syn hesis is no a no el y; many cells and is- sues om he immune sys em ha e and/o p oduce mela onin. This is he case in he hymus, which has been desc ibed as a issue wi h high quan i ies o mela onin [1]. I s biosyn hesis om he amino acid yp ophan equi es ou enzyma ically ca alyzed s eps [2] ha a e pe o med by yp ophan hyd oxylase (TPH), a oma ic amino acid deca boxylase (AADC), a ylalkylamine N-ace yl ans e ase (AANAT), and hyd oxyindole-O-me hyl ans e ase (HIOMT), which is now called N-ace ylse o onin-O-me hyl ans e ase (ASMT). The las wo s eps ha e been p oposed as limi ing s eps o mela onin p oduc ion [3].In gene al, mela onin om a pineal o igin ac s as a egula ho mone ha eaches a ge cells h ough he blood s eam, bu ex a-pineal mela onin appea s o play a mo e impo an unc ion in he issues whe e i is p oduced [4]; hus, i can be e ained in hese si es. In ac , many ex a-pineal issues ha e highe mela onin concen a ions han hose ound in plasma, and his amoun is no gene ally eleased in o he ci cula ion [5]. Mela onin p oduc ion in he pineal gland ollows a hy hmic p ofile, wi h high noc u nal le els and low diu nal le els. I s sec e ion no only fluc ua es h oughou he day bu also h oughou li e. In ac , one o he main ac o s ha influences i s sec e ion is age. I has been obse ed ha a e c ossing he placen a, ma e nal mela onin ac s as one o he main signals o se ing he biological clock o he e us, since no syn hesis o mela onin akes place du ing he p ena al pe iod. A e bi h, mela onin syn hesis unde he con ol o he ci cadian hy hm is no disce nible un il he eigh h week o li e [6], a e which i appea s o de elop apidly un il eaching i s maximum noc u nal le els be ween 3 and 7 yea s o age [7]. Du ing he pe iod o ime ha elapses om bi h o he acquisi ion o a hy hmic mela onin syn hesis p ofile in humans, his defici is sol ed by he p onounced ci cadian 1 Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e sidad de Se illa, A da. Manuel Siu o S/N, 41013, Se ille, Spain 2 Depa amen o de Bioquímica Médica y Biología Molecula e Inmunología, Uni e sidad de Se illa, A da. Sanchez Pizjuan S/N, 41009, Se ille, Spain 3 Depa amen o de Bioquímica Clínica, Hospi al Uni e si a io Vi gen del Rocío, A da. Manuel Siu o S/N, 41013, Se ille, Spain 4 These wo au ho s should be conside ed join senio au ho s. *Co esponding au ho . Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e sidad de Se illa, A da. Manuel Siu o s/n, Edificio IBiS, 41013, Se illa, Spain. Fax: þ34954907048. E-mail: [email p o ec ed] (P.J. La done). Recei ed June 14, 2019  Re ision ecei ed July 12, 2019  Accep ed July 21, 2019  A ailable online 24 July 2019 h ps://doi.o g/10.1016/j.molme .2019.07.007 O iginal A icle MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com 83 hy hms associa ed wi h mela onin p esen in b eas milk. A e pu- be y, mela onin concen a ions show a ma ked noc u nal d op ha g adually con inue h oughou li e; mela onin concen a ions appea o be se e ely a enua ed bu no absen in cen ena ian indi iduals [8]. Thus, pineal and plasma mela onin le els all wi h ad anced age; howe e , li le is known abou indolamine le els in ex a-pineal issues h oughou li e. In he case o he hymus, mos o he wo k pe o med in oden s indica es ha aging dec eases he p oduc ion o mela onin, al hough in a s udy pe o med by ou colleagues [9], hey hypo hesized ha he e is some esis ance by he hymus o main ain i s an ioxidan capaci y. In humans, he e was an in e es ing s udy pe o med in elde ly people ha showed a dec ease in mela onin and AANAT le els in e y old pa icipan s [10]. Howe e , he e is no e idence ega ding wha happens o he mela onine gic sys em in he hymus du ing childhood. Many o he physiological ac ions o mela onin ha e been desc ibed o be pe o med h ough binding o nuclea and memb ane ecep o s, as well as h ough ecep o -independen pa hways. The memb ane e- cep o s a e called MT 1 and MT 2 , and hey a e pa o he G p o ein- coupled ecep o amily [11]. Howe e , he ecen demons a ion o a ully unc ional mi ochond ial GPCR signaling pa hway ac i a ed by mela onin in he b ain [12] leads us o econside ha G p o ein- coupled ecep o s a e no only associa ed wi h he plasma mem- b ane. Among he nuclea binding si es o mela onin, also known as ROR o phan ecep o s, h ee sub ypes ( a , b , g ) and ou splicing a ian s o he a -sub ype a e included [13]. This ecep o ’s amily has al eady been cha ac e ized in he hymus om se e al species, including mouse [14] and a [15,16]; a p esen , he e is no e idence o mela onin binding si es in he human hymus o how hey a e influenced by age. To assess whe he he hymic mela onine gic sys em shows changes ela ed o de elopmen , we s udied he enzyme exp ession equi ed o mela onin syn hesis as well as he nuclea and memb ane mela onin ecep o s in no mal human hymuses om child en o di e en ages. The esul s we e co ela ed wi h he ages o he issues. He e, we de e mined bo h he exp ession o he enzyma ic machine y o he local p oduc ion o mela onin as well as he exp ession o i s specific ecep o s in all s udied ages. 2. MATERIALS AND METHODS 2.1. Thymus samples Thymic issues om newbo ns o 12-yea -old child en we e ob ained a e ca diac su ge y p ocedu es (congeni al hea diseases). The hymus agmen s ha made access o he hea mo e di ficul du ing su ge y we e emo ed, insed in no mal saline solu ion and ozen a 80  C un il RNA and p o ein ex ac ion was pe o med. The s udy ollowed he Helsinki Decla a ion o medical esea ch in ol ing human subjec s. Legal ep esen a i es o he pa ien s signed he w i en in o med consen o pa icipa e in he esea ch. The s udy was app o ed by he E hics Commi ee o he Vi gen del Rocio Hospi al in Se ille, Spain, on Feb ua y 11 h o 2010 (Ac n o 03/2010). The six y- one (61) samples used in his s udy we e g ouped in o he ollowing 3 g oups o pa ien s: newbo ns o days (younge han 1 mon h), mon hs (younge han 1 yea ), and yea s (olde han 1 yea ). All he samples’ cha ac e is ics a e desc ibed in Table S1. 2.2. Measu emen o hymus mela onin le els The issues we e weighed, homogenized in PBS, and cen i uged a 3000 g o 10 min; he supe na an s (500 m L) we e kep o mela onin de e mina ion. The mela onin quan i y in he issue was es ima ed using an ELISA ki (IBL, Hambu g, Ge many) acco ding o he manu- ac u e ’s ecommenda ions. 2.3. RNA isola ion, e e se ansc ip ion, and eal- ime PCR RNA was ex ac ed om he o gans using he T iPu e Isola ion Reagen (Roche, Mannheim, Ge many) acco ding o he manu ac u e ’s in- s uc ions. Single-s and cDNA was syn hesized om 3 m g o RNA using he T ansc ip o Fi s S and cDNA Syn hesis Ki (Roche, Man- nheim, Ge many). Real- ime PCR was pe o med on a Ligh Cycle 480 (Roche) using he Ligh Cycle Ò480 SYBR G een I Mas e (Roche, Mannheim, Ge many). The p ime sequences a e de ailed in Table S2. All PCR eac ions included nega i e con ols in which he empla e cDNA was omi ed. The exp ession le el o each gene was no malized o ha o b -ac in, and he ela i e gene exp ession was calcula ed using he 2  DD C me hod. 2.4. Wes e n blo The issue was homogenized and lysed a 4  Cinlysisbu e con aining 50 mM T is, pH 8; 137 mM NaCl; 10% glyce ol; and 40% Nonide wi h p o ease inhibi o cock ail (SigmaeAld ich, S . Louis, MO, USA). The p o ein con en o he lysa es was quan ified using he B ad o d me hod [17]. Aliquo s con aining 75 m go p o ein we e dena u ed a 85  C o 5 min in Laemmli’sBu e (SigmaeAld ich), subjec ed o SDSePAGE and ans e ed o a PVDF memb ane. The memb anes we e blocked wi h T is-bu e ed salinee0.05% Tween 20 (TBST) con aining 5% non a d y milk o 1 h a RT. The blo s we e hen incuba ed o e nigh wi h p ima y an ibodies agains human MT 1 (Mel1aR, N-20, sc13179) [San a C uz Bio echnology, San a C uz, CA] a a dilu ion o 1:200, human MT 2 (Mel1bR, G-20, sc-28453) [San a C uz Bio echnology, San a C uz, CA] a a dilu ion o 1:200, and human HIOMT (an i-ASMT/ HIOMT, LS-C156543) [LSBiO] a a dilu ion o 1:500. The blo s we e hen washed 3 imes wi h TBST be o e incuba ion o 1 h wi h seconda y an ibodies linked o ho se adish pe oxidase (an i-Rabbi IgG HRP W401; an i-Mouse IgG HRP W402; and an i-Goa IgG HRP V8051) [P omega]. The bound ho se adish pe oxidase was isual- ized using he Wes e n Blo ing Luminol Reagen sc-2048 (San a C uz Bio echnology, San a C uz, CA). The bands ob ained in he blo s we e scanned and analyzed using a ChemiDoc-I Imaging Sys em. The amoun o p o ein loaded in each lane was con olled by immunoblo ing wi h he monoclonal an i-GAPDH an ibody MAB374 (Millipo e) a a dilu ion o 1:1000. 2.5. S a is ical analysis All esul s a e epo ed as he mean SEM. Da a we e analyzed wi h he SPSSÒ 24.0 so wa e. K uskaleWallis es was used o de e - mine he o e all di e ences be ween g oups. Spea man co ela ion coe ficien was used o explo e possible associa ions be ween a i- ables. Values o p 0.05 we e conside ed s a is ically significan . 3. RESULTS 3.1. Mela onin con en in he human hymus is highe in newbo ns Ci cula ing mela onin le els in he blood o mammals eaches con- cen a ions up o 0.5 nM [18], while ex a-pineal concen a ions a y depending on he issue. To de e mine he mela onin con en s in he hymus samples om child en and o in es iga e whe he hese change wi h age, indolamine le els we e measu ed in issues om he h ee age g oups (Figu e 1). The highes mela onin le els we e ound in he hymuses om newbo ns (app oxima ely 40 pg/mg o issue). This concen a ion dec eased by hal in he hymuses om child en unde 1 O iginal A icle 84 MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com yea old, al hough s a is ically significan di e ences we e only ound be ween he days and yea s g oups (p 0.05). 3.2. The exp ession o he mela onin biosyn he ic machine y in he human hymus om child en changes wi h age Mela onin syn hesis is de i ed om he amino acid yp ophan and is usually ound in issues wi h AANAT and ASMT ac i i y [2].Tode e mine heexp essiono AANAT and ASMT mRNAs in human hymus om child en, cDNAs om he h ee g oups o pa ien s (days, mon hs and yea s) we e subjec ed o eal- ime quan i a i e PCR. The assay e ealed ha AANAT mRNA exp ession was significan ly down egula ed wi h age, wi h he highes le els o his ansc ip being measu ed in he g oup o neona es (Figu e 2A). This dec ease was also suppo ed by he nega i e co ela ion ( ho ¼0.4989; p 0.001) be ween AANAT exp ession and pa ien age (Figu e 2B). In con as , a significan inc ease in ASMT mRNA exp ession was de ec ed in he hymuses o pa ien s olde han 1 yea (Figu e 3A) when compa ed wi h he 12 mon hs-younge pa ien s (p 0.05). This inc ease was also obse ed a he ansla ional le el (Figu e 3B). In wes e n blo s, a well-defined band a app oxima ely 52 kDa co esponding o ASMT was ound in he h ee age g oups, bu he exp ession was highe in he eldes hymuses when compa ed wi h he wo o he g oups (p 0.001). 3.3. The hymus no only syn hesizes mela onin bu also esponds o i The ansc ip ional and ansla ional exp ession o mela onin ecep o s was de e mined using qPCR and wes e n blo analysis, espec i ely. The esul s e ealed ha mRNAs o he MT1,MT2 (Figu e 4A1 and B1), and ROR/RZR ecep o s (Figu e 5) we e significan ly educed (mo e han 60%) in he hymuses om he mon hs and yea s g oups on compa ison o newbo ns. These dec eases we e also obse ed a ansla ional le el o he memb ane ecep o s (Figu e 4A2 and B2), bu hese esul s we e only s a is ically significan o sub ype 1 (MT1). 3.4. The mela onin e ec o sys em dec eases h oughou childhood The a ia ion in mRNA le els o mela onin ecep o s h oughou childhood was suppo ed by s a is ical analyses using he nonpa a- me ic Spea man es , which showed a nega i e co ela ion be ween mela onin ecep o mRNA exp ession and he age o he hymus (Figu e 6). The memb ane ecep o s showed a s ong nega i e co - ela ion (p 0.001, Figu e 6A,B), whe eas in he case o nuclea ecep o s ROR a 1 and ROR a 4, he nega i e co ela ion was mode a e bu also significan (p 0.01 and p 0.001, espec i ely, Figu e 6C,D), indica ing ha exp ession o he mela onin e ec o sys em is highe in newbo ns and dec eases h oughou childhood. Addi ionally, o explo e whe he his exp ession could also be asso- cia ed wi h he mela onin con en s in he hymus, we calcula ed he co ela ion coe ficien s; acco dingly, only RORA1 and RORA4 exp es- sions we e sligh ly co ela ed (p 0.05) wi h in a- hymic mela onin le els (Figu e 7). 3.5. The exp ession o genes associa ed wi h mela onin syn hesis and signaling co ela e in he human hymus The dynamic changes in he mela onine gic sys em in he na u al cou se o de elopmen we e analyzed again using he nonpa ame ic Spea man es (Table 1), which showed s ong co ela ions ( ho >0.8, p 0.001) be ween MT1,MT2, and AANAT gene exp ession. This co ela ion was also posi i e and s a is ically signifi- can be ween he nuclea ecep o s RORA1 and RORA4 ( ho ¼0.642, p0.001). Addi ionally, he nuclea ecep o s also showed a mod- e a e co ela ion ha was significan wi h AANAT,MT1, and MT2 Figu e 2: AANAT is down egula ed in he hymus du ing de elopmen . A) AANAT mRNA exp ession in hymus samples om newbo n (named “days”, whi e ba ), younge han 1- yea -old (named “mon hs”, g ay ba ) and younge han 12-yea -old (named “yea s”, black ba ) pa ien s was analyzed by qRT-PCR. The 2  DD C equa ion was applied o calcula e he ela i e exp ession; alues we e no malized o b -ac in exp ession. The da a ep esen he mean SEM (n ¼45). *p 0.05 and **p 0.01. B) Co ela ion analysis be ween AANAT mRNA exp ession and he age (days) o he human hymus. ***p 0.001. Figu e 1: Mela onin in he human hymus du ing childhood. Mela onin con en s we e measu ed by ELISA in issue homogena es om child en a se e al de elopmen s ages. The da a ep esen he mean SEM (n ¼34). *p 0.05. MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com 85 Figu e 3: ASMT is up egula ed in he hymus o child en. A) ASMT mRNA exp ession on hymus samples om newbo n (named “days”, whi e ba ), younge han 1-yea -old (named “mon hs”, g ay ba ) and younge han 12-yea -old (named “yea s”, black ba ) pa ien s was analyzed by qRT-PCR. The 2  DD C equa ion was applied o calcula e he ela i e exp ession; he alues we e no malized o b -ac in exp ession. The da a ep esen he mean SEM (n ¼48). *p 0.05. B) P o ein ex ac s om he hymus we e analyzed by wes e n blo o ASMT exp ession a se e al ages using GAPDH as a loading con ol. The quan ified esul s (on he op) a e exp essed as he op ical densi y o he ASMT signal a e no maliza ion o GAPDH (ba g aph). The images on he bo om a e ep esen a i e o fi e independen expe imen s. ***p 0.001. Figu e 4: Exp ession o memb ane mela onin ecep o s in human hymus du ing childhood. MT1 (A1) and MT2 (B1) mRNA exp ession in hymus samples om newbo n (whi e ba ), younge han 1-yea -old (g ay ba ) and younge han 12-yea -old (black ba ) pa ien s was analyzed by qRT-PCR. The 2- DD C equa ion was applied o calcula e he ela i e exp ession; he alues we e no malized o B-ACTIN exp ession. The da a ep esen he mean SEM (n ¼46). *p 0.05; **p 0.01; and ***p 0.001. P o ein ex ac s om he hymus we e analyzed by wes e n blo o he exp ession o MT1 (A2) and MT2 (B2) a se e al ages using GAPDH as a loading con ol. The op ical densi ies o he signal om bo h ecep o s a e no maliza ion o GAPDH a e shown on he op. The images on he bo om a e ep esen a i e o fi e independen expe imen s. ***p 0.001. O iginal A icle 86 MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com exp ession. In e es ingly, only he RORA1 sub ype was posi i ely co ela ed wi h ASMT exp ession (p 0.05). 4. DISCUSSION The leading si e o T-cell de elopmen in he human body is he hymus; consequen ly, his is one o he o gans in ol ed in he gen- e a ion and p ese a ion o he adap i e immune sys em [19]. In e ms o he main cellula subpopula ions, he human hymus de elops ully be o e bi h and begins o a ophy a e pube y, when he numbe o hymocy es dec eases and he hymic s oma is g adually eplaced by adipose a h oughou a li e ime. Howe e , acco ding o e idence, esidual T lymphopoiesis con inues un il adul hood [20]. I is well- documen ed ha mela onin influences bo h he mo phology and unc ion o his i al o gan. The fi s s udy ha showed a connec ion be ween he hymus and mela onin was pe o med almos 50 yea s ago, when a pinealec omy caused a educ ion in he size o he hymus om 130 o 70 mg in mice [21]. In 1975, o he au ho s also showed a loss in he p oli e a ion o hymic cells a e pinealec omy [22]. How- e e , se e al s udies ha e shown ha he age- ela ed hymic in olu ion can be delayed o e en e e ed. In his sense, he g a ing o a pineal gland om young animals in o an old one o he adminis a ion o mela onin ( he pineal sec e o y p oduc ) main ained hymus unc ion and cellula i y [23,24]. Mo eo e , indolamine imp o es he p oli e a i e Figu e 5: The human hymus exp esses nuclea mela onin ecep o s. RORA1 (A) and RORA4 (B) mRNA exp ession in hymus samples om newbo n (whi e ba ), younge han 1- yea -old (g ay ba ) and younge han 12-yea -old (black ba ) pa ien s was analyzed by qRT-PCR. The 2  DD C equa ion was applied o calcula e he ela i e exp ession; he alues we e no malized o b -ac in exp ession. The da a ep esen he mean SEM (n ¼60). **p 0.01 and ***p 0.001. Figu e 6: The mela onin ecep o sys em co ela es in e sely wi h age. Co ela ion analysis be ween age (days) and mRNA exp ession (2  DD C ) o MT 1 (A), MT 2 (B), RORA1 (C), and RORA4 (D) in he human hymus. Each do ep esen s one hymus, and he s aigh -line ep esen s he bes -fi line ob ained by linea eg ession analysis. (n ¼46 o MT1 and MT2;n¼58 o RORA1; and n ¼60 o RORA4). MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com 87 capaci y and deg ee o DNA syn hesis in lymphocy es om he hymus in old a s [25]. The e o e, he e is no doub abou he ela ionship be ween he hymus and mela onin, which is ein o ced by he p es- ence o mela onin ecep o s in his immune o gan om mice and a s [14,15]. Howe e , un il now, he e was no e idence ega ding he exp ession o hese ecep o s in human hymus. Na anjo e al. de ec ed g ea quan i ies o mela onin as well as he exp ession and ac i i y o he AANAT and ASMT enzymes in human hymuses in adul s [1]. Ou s udy is he fi s o e alua e he en i e mela onine gic sys em in he human hymus in child en o se e al ages. Ou esul s show ha he mela onin biosyn he ic machine y in he human hymus is highly exp essed om he beginning o bi h. We de ec ed he mRNA and p o ein exp ession o bo h enzymes in hymuses in newbo ns ( om 6 o 19 days o age) as well as high le els o mela onin. Al hough we canno ule ou ha pa o his mela onin comes om he ci cula ion, based on he high le els o bo h enzymes in he hymuses om he younges age g oup, i is logical o hink ha i s main sou ce is local syn hesis. Taking in o accoun ha pineal mela onin syn hesis is minimal o he fi s six weeks o li e [26], he hymus could show a compensa o y inc ease in mela onin p oduc ion, as has been p e i- ously shown [16]. The ole o highe mela onin p oduc ion in he hu- man hymus a ea ly s ages migh be ela ed wi h wo main unc ions: on one hand by imp o ing he mo phology and hymic unc ion and, on he o he , by p o ec ing agains he oxida i e damage in he issue [27]. Acco ding o he fi s hypo hesis, some au ho s s a e ha he hymic unc ion g adually dec eases in hymopoie ic unc ion as om yea 1 o li e, coinciding wi h he dec ease in hymic epi helial space [28].In ac , a e y ecen a icle showed ha he hymic index o weigh a io (cm3/Kg) was la ges a ea ly in ancy (1e8 weeks) and smalles a 1 yea o age [29]. These findings would ma ch wi h he highe le els o mela onin ound in he younges hymuses. Fu he mo e, since new- bo ns a e especially suscep ible o oxida i e s ess [30], ano he possible ole o mela onin a such an ea ly age would be i s cy op o- ec i e abili y [31]. In ac , he ecen e idence abou mela onin is mainly syn hesized in he mi ochond ia om se e al issues lead us o hink ha he hymus would no be an excep ion [32], especially i we conside ha in euka yo es, mi ochond ia a e he majo sou ce o eac i e oxygen species (ROS) and hey equi e specific onsi e p o- ec ion [32]. Ano he ac o con empla e is he po en ial e ec o su ge y on inc easing local mela onin syn hesis as pa o he acu e inflamma o y esponse [33], which inc eases TNF and leads o he ansc ip ion and ac i a ion o AANAT. Acco ding o his, he p esence o mela onin migh help o p o ec hymocy es agains an oxida i e and inflamma o y damage. Howe e , his e ec would no seem explain he di e ences ound among he h ee g oup o ages since all samples we e ob ained om he su gical in e en ion. AANAT mRNA exp ession le els dec eased o e he cou se o g ow h, bo h in he mon hs ( om 2 o 11 mon hs o age) and yea s ( om 1 o 12 yea s) g oups. In ac , he exp ession o his enzyme is in e sely co ela ed wi h he age o he hymuses. In con as , he exp ession pa e n o ASMT was exac ly he opposi e, wi h he highes le els ound in he yea s g oup. The unexpec ed esul o high le els o AANAT and low le els o ASMT in he newbo n samples migh sugges ha ASMT would no play an impo an ole in he hymic syn hesis o mela onin du ing he ea ly days o bi h, wi h AANAT being p ima ily esponsible o mela onin le els in he o gan. As he hymus ma u es, AANAT le els dec ease, and ASMT le els ise ins ead. This inc ease in ASMT exp ession could be a mechanism o o e come he dec ease in AANAT so as o main ain su ficien mela onin le els in he o gan. Ano he possibili y, albei a - e ched, is ha he las wo enzymes in ol ed in mela onin syn hesis a e in e sed, such ha he ASMT enzyme ac s fi s and he AANAT a e wa ds. This al e na e pa hway has been obse ed in plan s [34] and i is wo hy o u he in es iga ion. We ound ha mela onin ecep o exp ession suppo s he e ec s o mela onin p oduced locally. The p esence o mela onin ecep o s in he hymus o se e al species had been p e iously epo ed [14,16,35]. Howe e , his is he fi s desc ip ion o he exp ession o a mela onin e ec o sys em in human hymus samples. We de ec ed mRNA and p o ein exp ession o bo h memb ane ecep o s sub ypes. The highes le els we e ound in he days g oup bu he exp ession dec eased h oughou childhood. The same mRNA exp ession pa e n was obse ed o he nuclea ecep o s (ROR a 1 and ROR a 4 a ian s), while he highes le els we e obse ed in newbo ns. The e o e, we ound Figu e 7: Nuclea mela onin ecep o exp ession co ela es posi i ely wi h hymic mela onin con en s. Co ela ion analysis be ween he mela onin con en s and he mRNA exp ession (2  DD C )o RORA1 (A) and RORA4 (B) in he human hymus. Each do ep esen s one hymus, and he s aigh -line ep esen s he bes -fi line ob ained by linea eg ession analysis. (n ¼32 o RORA1 and n ¼33 o RORA4). Table 1 eSpea man’s ank co ela ion coe ficien s be ween he mRNA exp ession o mela onin syn hesis and signaling genes. Genes AANAT ASMT MT1 MT2 RORA1 RORA4 AANAT 10.1363 0.8775*** 0.8892*** 0.4219** 0.3751* ASMT 10.0407 0.177 0.3399* 0.205 MT1 1 0.8676*** 0.4318** 0.4469** MT2 1 0.4284** 0.3434* RORA1 1 0.642*** RORA4 1 *p 0.05, **p 0.01, ***p 0.001. O iginal A icle 88 MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com ha he exp ession o he MT 1 ,MT 2 , and ROR a ecep o s in he hymus dec eased wi h age. In line wi h his, an age-dependen dec ease in MT 1 and MT 2 ecep o exp ession was also epo ed in se e al issues by Sanchez-Hidalgo e al. [15] and la e by Hill e al. [36]; hey sug- ges ed ha his educ ion led o he ea ly onse o senescence. In ou s udy, he co ela ion coe ficien s showed ha he exp ession o memb ane ecep o s is in e sely ela ed wi h he age o he hymuses, bu he exp ession o nuclea ecep o s appea s o also be associa ed wi h mela onin le els. Al hough a co ela ion does no indica e cau- sali y, in his case, i is no illogical o hink ha mela onin le els may influence he exp ession o he nuclea ecep o ha has al eady been desc ibed o di ec ly in e ac wi h i so as o exe i s ansc ip ional unc ions [37]. We we e unable o find a ela ionship be ween he mela onin con en in he hymus and he o he genes in he mela o- nine gic sys em, which could be due o he educed sample size o p oblems wi h he mela onin quan ifica ion echnique. A logical bu no less in e es ing finding om ou s udy was he highly s a is ically significan associa ion be ween he exp ession o he mela onin memb ane ecep o s and AANAT; a lowe enzymes le els, he e we e also lowe ecep o s le els. These ecep o s a e la gely esponsible o media ing he downs eam e ec s o mela onin [38], and AANAT is he majo enzyme in mela onin syn hesis [39]. Thus, an inc ease in local syn hesis o mela onin ia AANAT du ing he ea ly age coincides wi h an inc ease in he ac ion o mela onin ia MT 1 and MT 2 ecep o s. The exp ession o he wo ROR a iso o ms s udied was also associa ed wi h he exp ession o AANAT and he memb ane ecep o s, howe e , hese co ela ions we e lowe . This is no he fi s ime ha we sugges in e play be ween nuclea and mela onin ecep o s, as his was p e iously shown in human lymphocy es [40]. Howe e , he way in which hese in e ac ions ake place in he hymus and how hey a e egula ed a e s ill poo ly unde s ood and equi e u he s udies. 5. CONCLUSION The hymic mela onine gic sys em is p esen and especially ac i e om he fi s days o human li e; on excluding ASMT, he hymic mela onine gic sys em is in e sely co ela ed wi h he age o he hu- man hymus. We igno ed whe he hese a ia ions a e ela ed o changes in he composi ion and cellula i y as desc ibed by Wee kamp e al. [41], bu we belie e ha he dec ease in he ecep o s hem- sel es as well as he mela onin con en migh ha e an al e ing e ec on he a chi ec u e o he hymus, consequen ly a ec ing he main unc ion o his o gan. Consis en wi h ou conclusion, Odinoko and Hamblin sugges ha pho o-biomodula ion can e e age-associa ed hymic in olu ion h ough s imula ion o ex a-pineal syn hesis, hus imp o ing he immune unc ion [42]. Al hough addi ional s udies a e necessa y o be e unde s and he ne wo k be ween he neu oendo- c ine and immune sys ems wi hin he human hymus, ou findings sugges ha mela onin locally p oduced may pa icipa e in in a- hymic ma u a ion and T-cell di e en ia ion, which leads o he de elopmen o cell-media ed immuni y in humans. STATEMENT OF ETHICS Legal ep esen a i es o he pa ien s ha e gi en hei w i en in o med consen . FUNDING SOURCES This s udy was suppo ed by he Regional Go e nmen Minis y o Heal h (PI-0209-2010), he PAIDI P og am om he Andalusian Go e nmen (CTS160), and he Andalusian Public Founda ion P og ess and Heal h (PI-0485-2014). ICC ecei ed a p edoc o al ellowship om he FPU p og am (FPU13/01210). NAS was suppo ed by he Ins i u o de Salud Ca los III, Minis e io de Economía y Compe i i idad (RD06/ 0013/0001 and RD12/0043/0012). AUTHOR CONTRIBUTIONS D s La done and Moline o concep ualized and designed he s udy, con ibu ed o he in e p e a ion o he da a analysis, d a ed he ini ial manusc ip , and e ised he manusc ip ; D s C uz-Chamo o, Ál a ez- Sanchez and Ms Escalan e-Andicoechea designed he da a collec ion ins umen s, collec ed da a, ca ied ou he ini ial analyses, and e iewed he manusc ip ; D s Gue e o, Ca illo-Vico, and Rubio co- o dina ed and supe ised da a collec ion and c i ically e iewed he manusc ip o impo an in ellec ual con en ; all au ho s app o ed he final manusc ip as submi ed and ag ee o be accoun able o all aspec s o he wo k. ACKNOWLEDGEMENTS The au ho s hank M . John Leslie B own o co ec ing he language o he manusc ip CONFLICT OF INTEREST The au ho s ha e no conflic s o in e es o decla e. APPENDIX A. SUPPLEMENTARY DATA Supplemen a y da a o his a icle can be ound online a h ps://doi.o g/10.1016/j. molme .2019.07.007. 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Aging o lymphoid o gans: can pho o- biomodula ion e e se age-associa ed hymic in olu ion ia s imula ion o ex apineal mela onin syn hesis and bone ma ow s em cells? Jou nal o Biopho onics 11:e201700282. O iginal A icle 90 MOLECULAR METABOLISM 28 (2019) 83e90 Ó2019 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). www.molecula me abolism.com