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
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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
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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.
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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.
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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.
REFERENCES
[1] Na anjo, M.C., Gue e o, J.M., Rubio, A., La done, P.J., Ca illo-Vico, A.,
Ca ascosa-Salmo al, M.P., e al., 2007. Mela onin biosyn hesis in he hymus
o humans and a s. Cellula and Molecula Li e Sciences 64:781e790.
[2] Sugden, D., 1989. Mela onin biosyn hesis in he mammalian pineal gland.
Expe ien ia 45:922e932.
[3] Ceinos, R.M., Chansa d, M., Re el, F., Calga i, C., Miguez, J.M.,
Simonneaux, V., 2004. Analysis o ad ene gic egula ion o mela onin syn-
hesis in Sibe ian hams e pineal emphasizes he ole o HIOMT. Neu osignals
13:308e317.
[4] Ca illo-Vico, A., Cal o, J.R., Ab eu, P., La done, P.J., Ga cia-Mau ino, S.,
Rei e , R.J., e al., 2004. E idence o mela onin syn hesis by human lym-
phocy es and i s physiological significance: possible ole as in ac ine, au o-
c ine, and/o pa ac ine subs ance. The FASEB Jou nal 18:537e539.
[5] Venegas, C., Ga cia, J.A., Escames, G., O iz, F., Lopez, A., Doe ie , C., e al.,
2012. Ex apineal mela onin: analysis o i s subcellula dis ibu ion and daily
fluc ua ions. Jou nal o Pineal Resea ch 52:217e227.
[6] Se on-Fe e, M., To es-Fa an, C., Fo celledo, M.L., Valenzuela, G.J., 2001.
The de elopmen o ci cadian hy hms in he e us and neona e. Semina s in
Pe ina ology 25:363e370.
[7] Ka asek, M., 2004. Mela onin, human aging, and age- ela ed diseases.
Expe imen al Ge on ology 39:1723e1729.
[8] Mag i, F., Sa a, S., Cinche i, W., Guazzoni, V., Fio a an i, M., C a ello, L.,
e al., 2004. Quali a i e and quan i a i e changes o mela onin le els in
physiological and pa hological aging and in cen ena ians. Jou nal o Pineal
Resea ch 36:256e261.
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 89
[9] Sanchez-Hidalgo, M., de la Las a, C.A., Ca ascosa-Salmo al, M.P.,
Na anjo, M.C., Gomez-Co e a, A., Caballe o, B., e al., 2009. Age- ela ed
changes in mela onin syn hesis in a ex apineal issues. Expe imen al
Ge on ology 44:328e334.
[10] Pal se , M.A., Polyako a, V.O., K e noy, I.M., Ande son, G., K e naia, T.V.,
Linko a, N.S., e al., 2016. Mo pho unc ional and signaling molecules o e lap
o he pineal gland and hymus: ole and significance in aging. Onco a ge 7:
11972e11983.
[11] Jocke s, R., Delag ange, P., Duboco ich, M.L., Ma kus, R.P., Renaul , N.,
Tosini, G., e al., 2016. Upda e on mela onin ecep o s: IUPHAR Re iew 20.
B i ish Jou nal o Pha macology 173:2702e2725.
[12] Suo u, Y., Li, W., Jean-Alphonse, F.G., Jia, J., Kha a , N.K., Li, J., e al., 2017.
Dual ole o mi ochond ia in p oducing mela onin and d i ing GPCR signaling o
block cy och ome c elease. P oceedings o he Na ional Academy o Sciences
o he Uni ed S a es o Ame ica 114:E7997eE8006.
[13] Eme , M., Ozcan, H., Ozel, L., Yayla, M., Halici, Z., Hacimu uoglu, A., 2016.
A e iew o mela onin, i s ecep o s and d ugs. Eu asian Jou nal o Medicine
48:135e141.
[14] Ca illo-Vico, A., Ga cia-Pe ganeda, A., Naji, L., Cal o, J.R., Rome o, M.P.,
Gue e o, J.M., 2003. Exp ession o memb ane and nuclea mela onin e-
cep o mRNA and p o ein in he mouse immune sys em. Cellula and Mo-
lecula Li e Sciences 60:2272e2278.
[15] Sanchez-Hidalgo, M., Gue e o Mon a ez, J.M., Ca ascosa-Salmo al
Mdel, P., Na anjo Gu ie ez Mdel, C., La done, P.J., de la Las a
Rome o, C.A., 2009. Dec eased MT1 and MT2 mela onin ecep o exp es-
sion in ex apineal issues o he a du ing physiological aging. Jou nal o
Pineal Resea ch 46:29e35.
[16] Jimenez-Jo ge, S., Jimenez-Caliani, A.J., Gue e o, J.M., Na anjo, M.C.,
La done, P.J., Ca illo-Vico, A., e al., 2005. Mela onin syn hesis and
mela onin-memb ane ecep o (MT1) exp ession du ing a hymus de elop-
men : ole o he pineal gland. Jou nal o Pineal Resea ch 39:77e83.
[17] B ad o d, M.M., 1976. A apid and sensi i e me hod o he quan i a ion o
mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-dye binding.
Analy ical Biochemis y 72:248e254.
[18] Acuna-Cas o iejo, D., Escames, G., Venegas, C., Diaz-Casado, M.E., Lima-
Cabello, E., Lopez, L.C., e al., 2014. Ex apineal mela onin: sou ces, egula ion,
and po en ial unc ions. Cellula and Molecula Li e Sciences 71:2997e3025.
[19] Fe ando-Ma inez, S., Ruiz-Ma eos, E., Dudako , J.A., Vela di, E., G illa i, J.,
K eil, D.P., e al., 2015. WNT signaling supp ession in he senescen human
hymus. The Jou nals o Ge on ology Se ies A Biological Sciences and Medical
Sciences 70:273e281.
[20] Polyako a, V.O., Linko a, N.S., K e noy, I.M., Kha inson, V., 2011. Func ional
uni y o he hymus and pineal gland and s udy o he mechanisms o aging.
Bulle in o Expe imen al Biology and Medicine 151:627e630.
[21] Vaughan, M.K., Rei e , R.J., 1971. T ansien hype ophy o he en al p os a e
and coagula ing glands and accele a ed hymic in olu ion ollowing pinealec omy
in he mouse. Texas Repo s on Biology and Medicine 29:579e586.
[22] Csaba, G., Ba a h, P., 1975. Mo phological changes o hymus and he hy oid
gland a e pos na al ex i pa ion o pineal body. Endoc inologia Expe imen alis
9:59e67.
[23] Tian, Y.M., Li, P.P., Jiang, X.F., Zhang, G.Y., Dai, Y.R., 2001. Reju ena ion o
degene a i e hymus by o al mela onin adminis a ion and he an agonis ic
ac ion o mela onin agains hyd oxyl adical-induced apop osis o cul u ed
hymocy es in mice. Jou nal o Pineal Resea ch 31:214e221.
[24] Mocchegiani, E., San a elli, L., Tibaldi, A., Muzzioli, M., Bulian, D., Cip iano, K.,
e al., 1998. P esence o links be ween zinc and mela onin du ing he
ci cadian cycle in old mice: e ec s on hymic endoc ine ac i i y and on he
su i al. Jou nal o Neu oimmunology 86:111e122.
[25] El-Sokka y, G.H., Rei e , R.J., Abdel-Gha a , S., 2003. Mela onin supple-
men a ion es o es cellula p oli e a ion and DNA syn hesis in he splenic and
hymic lymphocy es o old a s. Neu oendoc inology Le e s 24:215e223.
[26] Kennaway, D.J., S amp, G.E., Goble, F.C., 1992. De elopmen o mela onin
p oduc ion in in an s and he impac o p ema u i y. The Jou nal o Clinical
Endoc inology and Me abolism 75:367e369.
[27] Rei e , R.J., Mayo, J.C., Tan, D.X., Sainz, R.M., Ala o e-Jimenez, M., Qin, L.,
2016. Mela onin as an an ioxidan : unde p omises bu o e deli e s. Jou nal
o Pineal Resea ch 61:253e278.
[28] Haynes, B.F., Sempowski, G.D., Wells, A.F., Hale, L.P., 2000. The human
hymus du ing aging. Immunologic Resea ch 22:253e261.
[29] Moo e,S.E.,Ful o d,A.J.C.,Sosseh,F.,Nshe,P.,Da boe,M.K.,P en ice,A.M.,2019.
Thymic size is inc easedby in ancy, bu no p egnancy, nu i ional supplemen a ion
in u al Gambian child en: a andomized clinical ial. BMC Medicine 17:38.
[30] Chen, Y.C., Tain, Y.L., Sheen, J.M., Huang, L.T., 2012. Mela onin u ili y in
neona es and child en. Jou nal o he Fo mosan Medical Associa ion eTaiwan
yi zhi 111:57e66.
[31] Chaiya i , P., Lueng akoon, K., Wannakasemsuk, W., Vichi ananda, V.,
Klan i , P., Ho mdee, D., e al., 2017. Biological unc ions o mela onin in ela ion
o pa hogenesis o o al lichen planus. Medical Hypo heses 104:40e44.
[32] Tan, D.-X., Rei e , R.J., 2019. Mi ochond ia: he bi h place, ba le g ound and
he si e o mela onin me abolism in cells. Mela onin Resea ch 2:44e66.
[33] Ma kus, R.P., Cecon, E., Pi es-Lapa, M.A., 2013. Immune-pineal axis: nuclea
ac o kappaB (NF-kB) media es he shi in he mela onin sou ce om pine-
alocy es o immune compe en cells. In e na ional Jou nal o Molecula Sci-
ences 14:10979e10997.
[34] Back, K., Tan, D.X., Rei e , R.J., 2016. Mela onin biosyn hesis in plan s:
mul iple pa hways ca alyze yp ophan o mela onin in he cy oplasm o
chlo oplas s. Jou nal o Pineal Resea ch 61:426e437.
[35] Gup a, S., Halda , C., Ahmad, R., 2015. Pho ope iodic egula ion o nuclea
mela onin ecep o RORalpha in lymphoid o gans o a opical oden
Funambulus pennan i: ole in seasonal oxida i e s ess. Jou nal o Pho o-
chemis y and Pho obiology B Biology 142:141e153.
[36] Hill, S.M., Cheng, C., Yuan, L., Mao, L., Jocke s, R., Dauchy, B., e al., 2013.
Age- ela ed decline in mela onin and i s MT1 ecep o a e associa ed wi h
dec eased sensi i i y o mela onin and enhanced mamma y umo g ow h.
Cu en Aging Science 6:125e133.
[37] La done, P.J., Gue e o, J.M., Fe nandez-San os, J.M., Rubio, A., Ma in-
Laca e, I., Ca illo-Vico, A., 2011. Mela onin syn hesized by T lymphocy es as
a ligand o he e inoic acid- ela ed o phan ecep o . Jou nal o Pineal
Resea ch 51:454e462.
[38] Slominski, R.M., Rei e , R.J., Schlab i z-Lou se i ch, N., Os om, R.S.,
Slominski, A.T., 2012. Mela onin memb ane ecep o s in pe iphe al issues:
dis ibu ion and unc ions. Molecula and Cellula Endoc inology 351:152e166.
[39] Vishwas, D.K., Halda , C., 2014. MT1 ecep o exp ession and AA-NAT ac i i y
in lympha ic issue ollowing mela onin adminis a ion in male golden hams e .
In e na ional Immunopha macology 22:258e265.
[40] La done, P.J., Ca illo-Vico, A., Moline o, P., Rubio, A., Gue e o, J.M., 2009.
A no el in e play be ween memb ane and nuclea mela onin ecep o s in
human lymphocy es: significance in IL-2 p oduc ion. Cellula and Molecula
Li e Sciences 66:516e525.
[41] Wee kamp, F., de Haas, E.F., Nabe , B.A., Comans-Bi e , W.M., Boge s, A.J., an
Dongen, J.J., e al., 2005. Age- ela ed changes in he cellula composi ion o he
hymus in child en. The Jou nal o Alle gy and Clinical Immunology 115:834e840.
[42] Odinoko , D., Hamblin, M.R., 2018. 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.
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