Immunomodula o y Response o Toll-like Recep o Ligand−Pep ide
Conjuga es in Food Alle gy
Jo ge Losada Méndez, F ancisca Paloma es, F ancisca Gómez, Ped o Ramí ez-López,
Ja ie Ramos-So iano, Ma ia Jose To es, C is obalina Mayo ga,*and Ja ie Rojo*
Ci e This: ACS Chem. Biol. 2021, 16, 2651−2664
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ABSTRACT: Co alen conjuga ion o alle gens o oll-like ecep o (TLR) agonis s appea s o be a powe ul s a egy o he
de elopmen o sa e y compounds o alle gen-specific immunomodula o y esponse owa d ole ance in alle gy. In his wo k, we
ha e syn hesized wo amily o ligands, an 8-oxoadenine de i a i e as a ligand o TLR7 and a py imido[5,4-b]indole as a ligand o
TLR4, bo h conjuga ed wi h a T-cell pep ide o P u p 3 alle gen, he lipid ans e p o ein (LTP) esponsible o LTP-dependen
ood alle gy. These conjuga es in e ac wi h dend i ic cells, inducing hei specific ma u a ion, T-cell p oli e a ion, and cy okine
p oduc ion in peach alle gic pa ien s. Mo eo e , hey inc eased he T eg-cell equencies in hese pa ien s and could induce he IL-10
p oduc ion. These ou comes we e ema kable in he case o he TLR7 ligand conjuga ed wi h P u p 3, opening he doo o he
po en ial applica ion o hese alle gen−adju an sys ems in ood alle gy immuno he apy.
■INTRODUCTION
Food alle gy (FA) is cu en ly a bu den o he Heal h Sys ems
mainly in wes e n Eu opean coun ies whe e he p e alence is
inc easing wi h plan o igin as he main igge s in bo h adul
and adolescen popula ions.
1
Lipid ans e p o eins (LTPs)
a e he main alle gens ela ed o plan FA in Medi e anean
popula ion al hough hey a e inc easingly being obse ed in
o he Eu opean coun ies.
2,3
Al hough some pa ien s can
selec i ely eac o a single LTP ( equen ly P u p 3, om
peach), hese p o eins a e conside ed panalle gens, wi h
pa ien s sensi ized o LTPs om diffe en alle genic sou ces
ha can be axonomically un ela ed inc easing he complexi y
o hei clinical managemen .
4
The high numbe o plan oods
in ol ed, independen ly o he se e i y o he eac ions, has an
impo an impac on he quali y o li e o pa ien s because hey
will equi e e y es ic i e die s.
A p esen , alle gen-specific immuno he apy (AIT) ep e-
sen s he unique app oach able o modi y he disease-inducing
ole ance by he immunological modula ion om he ype 2
pa e n o a ype 1 o egula o y (T eg) esponse.
5,6
Howe e ,
con en ional app oaches using alle genic ex ac s ha e
impo an d awbacks in e ms o efficacy, sa e y, du a ion,
and pa ien compliance.
7
The e o e, no el accines ha
o e come such incon eniences a e in demand.
The imp o emen o he efficacy would no only depend on
he alle gen bu also on he adju an s used in he accine
composi ion.
8
The main ole o hese adju an s adminis e ed
oge he wi h he alle gens has shown o enhance he Th1 and
T eg esponse by diffe en mechanisms.
9
Among he adju an s,
sequences con aining inflamma o y dange signals, such as
“pa hogen-associa ed molecula pa e ns”(PAMPs) p esen in
mic oo ganisms, can elici he elease o inflamma o y
cy okines and chemokines ha ini ia e he de ensi e o he
inna e immune esponse.
10,11
This effec is induced h ough
he in e ac ion wi h cellula ecep o s as pa e n- ecogni ion
Recei ed: Oc obe 1, 2021
Accep ed: Oc obe 27, 2021
Published: No embe 11, 2021
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ecep o s
12
ha include a wide a ie y o amilies as C- ype
lec ins (CLRs), oll-like ecep o s (TLRs), among o he s.
13
TLRs a e a ype I ansmemb ane ecep o s in ol ed a he
beginning o he inna e immune esponse.
14
Ten unc ional
TLRs (TLR1−10) a e p esen in humans,
15
each o hem
ecognizing selec i ely diffe en PAMPs, usually biomac omo-
lecules. Some TLRs can be conside ed as e y in e es ing
candida es o p oduce a s ong Th1 (nonalle gic) esponse,
coun e ac ing he unc ion and cy okine p oduc ion o he Th2
cells om alle gic pa ien s.
16
In his sense, s udies pe o med
wi h agonis s o hese TLRs as adju an s ha e been explo ed in
he amewo k o he design o new accines o
immuno he apies o alle gic diseases.
17−19
F om hem,
syn he ic TLR4 agonis s ha e p og essed o be used in
AIT.
20
In his con ex , he use o he agonis o TLR4
monophospho yl-lipid A (MPLA), a less oxic de i a i e o
LPS, oge he wi h g ass pollen alle gens, has been shown o be
a boos e o AIT, inducing he IFNγp oduc ion and educing
he IgE le els in alle gic pa ien s.
21,22
In addi ion, he
conjuga ion o MPLA o o albumin (OVA) p o ein has been
epo ed o p omo e dend i ic cell (DC) ma u a ion and
induce a Th1 esponse.
15,23
A u he small-scale in i o s udy
in alle gic pa ien s has iden ified MPLA as po en ia ing
alle goid esponses in AIT.
24
On he o he hand, se e al
TLR9 agonis s ha e been assessed in combina ion wi h an
alle gen in clinical ials o AIT, demons a ing a s ong
capaci y o induce Th1 esponse and consequen ly p o iding
benefi when adminis e ed as an adju an o AIT.
25
TLR9
ac i a ion has been shown o be capable o p oducing a Th1
esponse wi h IFNγp oduc ion and IgE syn hesis inhibi ion
using modified oligodeoxy ibonucleo ides con aining CpG
mo i s in a FA animal model
9
and by he co-adminis a ion
o chenopodium album alle gens and CpG in alle gic hini is
pa ien s.
26,27
Rega ding TLR7 agonis s, he e a e ew s udies
indica ing hei po en ial used in AIT,
28,29
despi e imidazo-
quinoline compounds (imiquimod and esiquimod), TLR7
agonis s ha e consis en ly demons a ed a capaci y o e e se
Th2 esponses in a ou o an an i-alle gic Th1 esponse and
IL-10 p oduc ion.
30,31
In addi ion, one s udy u ilizing nano-
pa icles wi h an OVA pep ide in he p esence/absence o
imidazoquinoline compound demons a ed he p oduc ion o
ole ogenic DCs and he induc ion o T egs capable o
supp essing he esponse o ood challenge.
32
Al hough TLR7
agonis s a e u ilized in clinical se ings,
29,33
he e a e ew
s udies highligh ing hei effec s in modi ying human ood
alle gic esponses.
Despi e ecen ad ances in he use o agonis s in modula ing
he TLR4 o TLR7 o induce a Th1 esponse,
20
hese s udies
ha e ce ain limi a ions such as he sho du a ion o he
egimens s udied, he le el o alle gen doses e alua ed, o he
low immunomodula o y p ofile o TLR agonis s. The e o e, i
is needed o op imize he adju an ed-alle gen immuno he apy
egimens and o define immunological p ope ies o TLR
ligands, which ac as agonis s enhancing he ole ance esponse
o ood alle gens.
He e, based on p e ious p eceden s and aking in o
conside a ion he syn he ic accessibili y o agonis s o u he
unc ionaliza ion, we ha e ocused on TLR4 and TLR7 ligands
(TLR4lig and TLR7lig) o add ess ou alle gen conjuga es wi h
he aim o de elop compounds o AIT in FA. We ha e
add essed he immunological esponse induced by compounds
ha include, besides he TLR4 o TLR7 agonis s, a syn he ic
pep ide o he alle genic epi ope P u p 3 (Pp3), TLR4lig-Pp3,
and TLR7lig-Pp3, espec i ely, in cells om LTP alle gic
pa ien s and analyzed he ype o esponse in o de o assess
he modula o y capaci y o hese ligands. Ou findings indica e
ha his app oach could be conside ed an in e es ing syn he ic
s a egy o he de elopmen o new accines o FA
immuno he apy.
■RESULTS AND DISCUSSION
Syn hesis o he TLR4 Agonis (TLR4lig). We ha e
ocused ou effo s o p epa e a TLR4 agonis based on
py imido[5,4-b]indole de i a i es. In he cou se o a high-
h oughpu sc eening o iden i y ac i a o s o inna e
immuni y,
34
a se ies o py imido[5,4-b]indoles we e ecen ly
disco e ed by Co am as selec i e TLR4 agonis s.
35
In hese
s udies, he agmen s o hese molecules ha we e implica ed
di ec ly in he in e ac ion wi h he TLR4 ecep o we e
iden ified (Figu e 1).
F om hese s udies, he benzyl ing o he py imido[5,4-
b]indole appea ed as he mos con enien si e o s uc u e
modifica ion wi hou al e ing he binding p ope ies o he
molecule, a ac confi med la e on by he same g oup.
36
Conside ing his p eceden , we decided o modi y he C8
posi ion o he py imido[5,4-b]indole ing o he in oduc ion
o a maleimide g oup, equi ed o he pep ide (alle gen)
conjuga ion ia a hiol−ene eac ion (compound 11,Scheme
1). We ca ied ou he syn he ic s a egy depic ed in Scheme 1,
using he C8 posi ion o he py imido[5,4-b]indole con en-
ien ly de i a ized.
B iefly, compound 3was syn hesized by b omina ing he
comme cially a ailable 2-aminobenzoni ile wi h NBS and
ace oni ile as desc ibed in he li e a u e.
37
Then, he N-
alkyla ion eac ion o ob ain compound 4was op imized using
mo e ene ge ic condi ions (hea ing a 75 °C in DMF) and
wi h an excess o e hylb omoace a e (6 equi ), he yield being
no ably imp o ed (up o 63%). In e media es 5−7we e
syn hesized acco ding o he li e a u e p ocedu es.
36
Then, he
o ma ion o he hioe he 8wi h he ee ca boxylic acid by
eac ion o 7wi h b omoace ic acid was di ec ly achie ed in
quan i a i e yield. The nex s ep consis ed o he amida ion
eac ion o he ca boxylic acid 8wi h cyclohexylamine using
HATU as coupling agen , yielding amide 9in high yield.
Compound 9was submi ed o an Ullmann- ype coupling,
ca alyzed by Cu(I), p o iding he aniline de i a i e 10. The
las s ep o he syn he ic ou e was he in oduc ion o a
maleimide g oup using 3-maleimidop opionic acid,
38
wi h
HATU as he coupling agen , o gi e he maleimide de i a i e
11, eady o be conjuga ed wi h he selec ed alle gen.
Figu e 1. Py imido[5,4-b]indole de i a i e, a ligand o TLR4, and
egions ha influence he IFN ype I p omo ion: he N-3 posi ion
(yellow egion) mus include a hyd ophobic g oup, p e e ably an
unsubs i u ed phenyl ing; he indole N-5 posi ion (blue egion) has
o be unsubs i u ed; and he e minal amide ( ed egion) equi ed a
highly hyd ophobic subs i uen such as a cyclohexyl esidue.
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Syn hesis o he TLR7 Agonis (TLR7lig). Fo TLR7,
he e we e some examples o small molecules de eloped as
agonis s. In 2002, i was shown ha imidazoquinolines we e
capable o ac i a ing TLR7;
39
howe e , i was no un il 2004
ha he iden ifica ion o he na u al ligand o TLR7, guanine
and u idine- ich single-s anded RNAs, was epo ed.
40
Wi hin
he amily o imidazoquinolines (Figu e 2), Imiquimod (12)is
a d ug app o ed o ex e nal geni al wa s caused by human
papilloma i us in ec ion.
41
Adenine de i a i es we e iden ified
as IFN induc o s.
42
La e , 9-benzyl-2-(2-me hoxye hoxy)-8-
oxoadenine (13) was epo ed as a TLR7 agonis (Figu e
2).
43,44
The e o e, all hese s uc u es could be conside ed as
candida es o alle gy accine adju an s due o hei abili y o
modula e he Th1/Th2 immune esponse.
Recen ly, a TLR 7 agonis was conjuga ed wi h a pep ide
de i ed om OVA,
45
showing ha he conjuga e p omo ed he
DC ma u a ion h ough he p oduc ion o IL-12p40 and
CD86, as well as he T-cell p oli e a ion. Isobe’s g oup
epo ed ha some modifica ions in he N-9 posi ion o he
a yl agmen o compound 14 (Figu e 2) had no any
significan effec s on hei biological ac i i y.
46
Scheme 1. Syn hesis o Py imido[5,4-b]indole De i a i e 11
a
a
Reagen s and condi ions: (a) e hylb omoace a e, K2CO3, DMF, 75 °C, (63%); (b) KO Bu, THF, <30 °C (39%); (c) PhNCS, E OH, eflux
(93%); (d) ace yl chlo ide, E OH, eflux (72%); (e) B CH2COOH, KOH, E OH, (quan .); ( ) cyclohexylamine, HATU, TEA, DMF,
(quan .); (g) CuI, NaN3, NaAsc, DMEN, DMSO/H2O, 90 °C (72%); (h) 3-maleimidop opionic acid, HATU, DIPEA, DMF, (73%).
Figu e 2. S uc u e o TLR7 ligands desc ibed in he li e a u e.
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We we e inspi ed by hese p eceden s conce ning small
ligands as agonis s o TLR7, and ou syn he ic s a egy was
ocused on he in oduc ion o a TEG-based linke in he
benzyl esidue a he N-9 posi ion o compound 14 o
conjuga e he alle gen (Scheme 2). This space should p o ide
enough sepa a ion be ween he ligand and he alle gen o a oid
in e e ences du ing he binding o he TLR7 ecep o h ough
he adenine moie y.
The syn he ic ou e o syn hesize compound 21 is depic ed
in Scheme 2. 4-B omome hylbenzoic acid was ea ed wi h
hionyl chlo ide o ob ain he co esponding acyl chlo ide,
which was immedia ely eac ed wi h he amino linke 15,
47
unde anhyd ous condi ions, yielding he amide 16. This
in e media e was ob ained as a mix u e o Cl- and B -
de i a i es in he benzylic me hylene due o undesi ed pa ial
chlo ina ion by hionyl chlo ide ea men ; howe e , his did
no in e e e wi h he syn he ic pa hway. The nex s ep was he
N-alkyla ion o 2-bu oxy-9H-pu in-6-amine 17, syn hesized
acco ding o he li e a u e,
46
wi h 16 in he p esence o
po assium ca bona e o ob ain compound 18 in 69% yield.
Then, he elec ophilic a oma ic subs i u ion wi h elemen al
b omine in chlo o o m p o ided he 8-b omina ed de i a i e
19. B omine a posi ion C-8 was hyd olyzed unde high-ene gy
condi ions wi h efluxing o mic acid o gi e compound 20 in
mode a e yield. Finally, his compound was conjuga ed wi h
p op-2-yn-1-yl-3-maleimidop opanoa e,
18
ia Cu(I) ca alyzed
azide−alkyne 1,3 dipola cycloaddi ion (CuAAC) o p o ide
he maleimide de i a i e 21 in excellen yield.
TLR7 and TLR4 Alle gen Conjuga ion (TLR7lig-Pp3
and TLR4lig-Pp3). Once TLR4 and TLR7 ligands 11 and 21,
espec i ely, we e p epa ed con enien ly unc ionalized wi h a
maleimide g oup, we affo ded he conjuga ion o he alle gen
o hese adju an s. The alle gen (Pp3),
48
whose sequence is
SSNGIRNVNNLARTPDRQAC, was based on he egion 26−
Scheme 2. Syn hesis o Adenine De i a i e 21
a
a
Reagen s and condi ions: (a), SOCl2, 2-(2-(2-(2-azidoe hoxy)e hoxy)e hoxy)e han-1-amine (15), DCM, eflux (60%); (b) K2CO3, DMF, 65 °C
(69%); (c) B 2, CHCl3, (66%); (d) HCO2H, eflux (47%); (e) p op-2-yn-1-yl-3-maleimidop opanoa e, CuB , Ten aGel-TBTA esin, ACN/
DMSO, (94%).
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46 o he P u p 3 p o ein wi h an addi ional e minal cys eine.
This pep ide, conjuga ed wi h a glycodend ime , was
p e iously es ed using in i o and in i o expe imen s o
induce ole ance agains peach alle gy.
18
The co alen conjuga ion o Pp3 o 11 and 21 was achie ed
h ough he click hiol−ene eac ion. The condi ions o his
conjuga ion we e op imized o ob ain he co esponding
conjuga es di ec ly in 30 min (moni o ed by RP-HPLC)
combining he maleimide de i a i e and he pep ide in an
equimola a io in dime hylsul oxide (DMSO)/phospha e
buffe ed saline (PBS). The co esponding conjuga es
TLR4lig-Pp3 (1) and TLR7lig-Pp3 (2) we e ob ained in high
yield and pu i y. These conjuga es we e success ully cha ac-
e ized by MS spec ome y (Figu e 3).
TLRnlig-Pp3 Up ake. We ha e analyzed he le el o up ake
o TLRnlig-Pp3 on monocy e-de i ed DCs (moDCs), using 10
nM o TLR4lig-Pp3 (1) o TLR7lig-Pp3 (2), bo h labeled wi h
Alexa fluo 647, by flow cy ome y and con ocal mic oscopy
(CM).
49
Flow cy ome y measu emen s indica ed ha
TLRnlig-Pp3 we e up aken by moDCs in a ime-dependen
manne , which is as e and highe o TLR4lig-Pp3 compa ed
o TLR7lig-Pp3 (Figu e 4A). Fu he mo e, he do plo s and
CM images showed ha he in e naliza ion o he TLR4lig-Pp3
was isible om he fi s hou o incuba ion, while o he
TLR7lig-Pp3, i appea ed a e 9 h (Figu e 4A,B). The moDC
up ake con inued o inc ease a e 48 h o bo h TLRnlig-Pp3,
achie ing simila pe cen ages o posi i e cells. Addi ionally, he
CM images indica e ha TLRnlig-Pp3 in acellula dis ibu ion
in moDCs was diffe en o each ligand, wi h TLR7lig-Pp3
showing pe inuclea accumula ion, while wi h TLR4lig-Pp3, an
accumula ion nea es o memb ane and cy oplasm was
obse ed (Figu e 4B).
These diffe ences in he in e naliza ion ime and in he
loca ion o he TLRnlig-Pp3 in he moDCs can be due o he
diffe en ial posi ion o hei ecep o s because he exp ession o
TLR4 is a he cell su ace, while TLR7 is a in acellula
compa men s, such as endosomes.
50
These in e naliza ion
pa hways would acili a e ood alle gen up ake o inc ease he
effec i e esponse. The e o e, his TLRnlig-Pp3 could con ib-
u e o he design o efficacious, sho e , and sa e
immuno he apy p o ocols.
51
TLRnlig-Pp3 Induce Changes in he moDC Ma u a-
ional S a us and Cy okines P oduc ion. Du ing he fi s
s eps o he immune esponse, he an igen in e ac s wi h
moDCs inducing hei ma u a ion. The ype o ma u a ion
esponse ha will be cha ac e ized by he exp ession o co-
s imula o y molecules and pa e n o cy okine p oduc ion will
o ches a e he u he immunological esponse media ed by T
helpe lymphocy es.
52
The assessmen o he TLRnlig-Pp3-
induced ma u a ional pheno ypical changes in he exp ession
o ac i a ion/ egula ion (CD83 and PD-L1) and ma u a ion
(CD80 and CD86) cell su ace ma ke s indica ed ha
compa ed o ole an con ols, he e was a significan inc ease
o CD80, CD83, and CD86 in alle gic pa ien s when moDCs
we e s imula ed wi h TLR7lig-Pp3, while only o CD83 wi h
TLR4lig-Pp3 (Figu e 5A,B). The analysis o he effec o he
s uc u es including he TLR4lig (10) and TLR7lig (20) ligands
bu no Pp3 indica ed ha we e also able o s imula e CD80
and CD86, espec i ely, in alle gic pa ien s compa ed o
ole an con ols. This effec is in ag eemen wi h p e ious
Figu e 3. Conjuga ion o Pp3 o maleimide scaffolds 11 and 21 o ob ain he conjuga es TLR4lig-Pp3 (1) and TLR7lig-Pp3 (2), espec i ely ( op)
and hei co esponding ESI-MS spec a and HPLC ch oma og ams (bo om).
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s udies,
53
in which i was shown ha diffe en TLR adju an s
(wi hou alle gen) we e able o modula e he moDC ac i i y/
ma u a ion in bi ch pollen alle gic pa ien s and animal
model.
53
This capaci y o ac i a e he inna e esponse,
enhancing he moDC immunomodula ion, has also been
desc ibed o o he small syn he ic TLR4 and TLR7
ligands.
54,55
These da a sugges ha he changes in he exp ession o
CD83 induced by TLRnlig-Pp3 a e ela ed o he specific
esponse gene a ed by he alle gen pep ide because we ha e
ound diffe ences no only be ween alle gic pa ien s and
ole an con ols bu also be ween he s uc u es wi h
(TLRnlig-Pp3) and wi hou (TLRnlig) pep ide. This confi ms
he esul s o p e ious epo s whe e i has been desc ibed ha
TLR ligands can show diffe en ial exp ession be ween he co-
s imula o y molecules, high CD83 exp ession, and a sligh ly
educed CD80 and CD86 exp ession.
56
We ha e analyzed in alle gic pa ien s he effec o he
inse ion o he Pp3 pep ide conjuga ed o TLR ligands
(TLRnlig-Pp3) compa ed o he effec o including hese
elemen s in a sepa a ed way. Da a showed ha TLR4lig-Pp3 led
o a significan inc ease o CD80, CD83, and PD-L1 compa ed
o he s imula ion wi h TLR4lig plus Pp3 pep ide in a sepa a e
way (Figu e 5A). Fu he mo e, o TLR7lig-Pp3, we obse ed a
significan inc ease o CD83, CD86, and PD-L1 compa ed o
TLR7lig plus Pp3 pep ide in a sepa a e way (Figu e 5B). These
esul s showed ha a mo e efficien immunological esponse is
p oduced when bo h alle gen and adju an in e ac oge he
wi h he an igen-p esen ing cells (APC) as DCs. TLRnlig-Pp3
up ake co ela es wi h unc ional changes in moDCs in
i o.
57,58
The e o e, an efficien an igen p esen a ion equi es
he ac i a ion o co-s imula o y molecules.
59
He e, TLRnlig-
Pp3 ac i a e, egula e, and ma u a e he su ace ma ke
exp ession om moDCs, sugges ing ha he con ac wi h
TLRs is impo an o ini ia ion he immune esponses.
To u he cha ac e ize he esponse pa e n du ing he
moDC ma u a ion ollowing incuba ion wi h TLRnlig-Pp3, we
measu ed he cy okine elease in o he cul u e supe na an .
Ou analysis in alle gic pa ien s compa ed o ole an con ols
de ec ed significan inc eases o IL-5 and IFNγwi h all he
condi ions con aining TLR7lig (TLR7lig, TLR7lig-Pp3, and
TLR7lig plus Pp3 in a sepa a e way), indica ing ha his effec
is a he due o TLRlig (Figu e 5D). In he case o TLR4
(Figu e 5C), only significan inc eases o IL-5 we e obse ed in
alle gic pa ien s compa ed o ole an con ols when
s imula ing wi h TLR4lig-Pp3 and TLR4lig plus Pp3 pep ide
in a sepa a e way. No diffe ences in he induc ion o IL-10
p oduc ion we e obse ed in alle gic pa ien s compa ed o
ole an con ols o any o he ligands assayed (Figu e 5D).
Figu e 4. TLRnlig-Pp3 in e naliza ion and localiza ion. (A) Timing o fluo escence-labeled TLRnlig-Pp3 up ake by moDCs and flow cy ome y plo s
a diffe en ime poin s, showing he TLRnlig-Pp3 pe cen ages on CD86+moDCs. (B) Rep esen a i e con ocal images o moDCs incuba ed wi h
TLRnlig-Pp3 a 10 nM a diffe en ime poin s ( om 1 o 48 h). Da a a e consis en wi h he flow cy ome y analysis showing a high fluo escence
signal (pink) om TLR4lig-Pp3 a 1 h and TLR7lig-Pp3 a 9 h inside he moDCs. The con ocal images a 48 h show diffe en cellula egions. The
sub-memb ane ac in was s ained wi h A o 488-phalloidin (g een) and nuclei wi h Hoechs (blue).
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No diffe ences we e obse ed in he cy okine p oduc ion
a e s imula ion wi h TLRnlig-Pp3 and TLRnlig plus Pp3 in a
sepa a e way. In e es ingly, we obse ed ha TLR7lig-Pp3
induced significan lowe IL-4 and IL-5 le els, while highe
IFNγle els compa ed o he Pp3 only in alle gic pa ien s,
indica ing he specific immunomodula o y capaci y o his
compound.
Al oge he , hese da a indica ed ha TLR7lig-Pp3 s imula-
ion induced moDCs ma u a ion accompanied by a dec ease o
Th2 pa e n (IL-4 and IL-5) and an inc ease o he Th1
cy okine (IFNγp oduc ion).
20,51,60
In ac , he in e ac ion o a
TLR7 ligand wi h i s ecep o igge s he IFNγinduc ion
pa hway as i has been desc ibed p e iously.
61
The e o e, he
effec o TLR7lig-Pp3 in he p oduc ion o ype 1 immune
esponse is essen ial o he immuni y and p e en ion o he
de elopmen o Th2 esponses,
62
making i an a ac i e
compound o AIT.
Rega ding TLR4lig-Pp3, i p omo ed a moDC diffe en ial
ma u a ion in alle gic pa ien s wi h a educ ion o Th2
cy okines compa ed o he pep ide, al hough wi hou changes
in he IFNγand IL-10 le els. This sligh esponse in he
p oduc ion o cy okines (IFNγand IL-10) could be associa ed
wi h he ma u a ion s age o he moDCs because he TLR
ligands can influence in a diffe en way he moDC ac i a ion,
an igen p esen a ion, co-s imula ion, and cy okine p oduc-
ion.
53,63
TLRnlig-Pp3 S imula e he Specific P oli e a i e
Response wi h a Th1 P ofile. Ou findings ha he
TLRnlig-Pp3 we e up aken by moDCs, affec ing hei ac i a ion
and ma u a ion, sugges ed ha hey could be in ol ed in he
ollowing s eps o he immunological esponse, whe e ma u e
Figu e 5. TLRnlig-Pp3 change moDC ma u a ion and cy okine p oduc ion. Ba s ep esen median and SEM o he (A,B) MI o he diffe en
su ace ma ke s on moDCs and o he (C,D) cy okine p oduc ion in supe na an s om moDCs assays o Pp3, TLRnlig, TLRnlig-Pp3, and TLRnlig
plus Pp3 o alle gic pa ien s (n= 9, colo ba s) and ole an con ols (n= 9, whi e ba s) a 10 nM. The Mann−Whi ney U es was used o
pai wise compa isons be ween un ela ed g oups and Wilcoxon signed- ank es was used o pai wise compa isons in ela ed samples, showing
significan diffe ences as **,(p< 0.0125 and p< 0.0010, Bon e oni co ec ion, espec i ely). The do ed line ep esen s he MI > 2.
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moDCs p esen he alle gen o Th lymphocy es. To examine
his possibili y, we de e mined lymphocy e p oli e a ion using
homologous p e-s imula ed moDCs as APCs and he p ofile o
he esponse in e ms o cy okine p oduc ion. Diffe en T-
lymphocy es (CD3+T-, CD4+T-, and CD8+T-cells), B-cells
(CD19+), and NK-cells (CD56+) subpopula ions ha e been
epo ed o shape immunological esponses in diffe en ways.
The esul s indica ed ha only TLR7lig-Pp3 led o
significan ly inc eased CD3+T-cell p oli e a ion in alle gic
pa ien s compa ed o ole an con ols, while TLR4lig-Pp3
seemed o s imula e he T-cell p oli e a ion wi hou inducing a
significan diffe ence be ween g oups (Figu e 6A). No
diffe ence in he p oli e a i e esponse was ound o o he
subpopula ions as B-cells and NK-cells wi h any o he TLRnlig-
Pp3 be ween alle gic pa ien s and ole an con ols because he
p oli e a ion o ha cell subpopula ion was nega i e [p o-
li e a ion index (PI) < 2] (Figu e S1).
Mo eo e , in alle gic pa ien s, he e was a significan inc ease
in CD3+and CD4+T-cell p oli e a ion a e s imula ion wi h
TLR4lig-Pp3 e sus TLRlig and e sus TLR4lig plus Pp3
pep ide; addi ionally, TLR7lig-Pp3 induced a specific p oli e -
a i e esponse o CD3+T-cells compa ed o TLR7lig and
TLR7lig plus Pp3 pep ide (Figu e 6A). These esul s sugges
ha he TLRnlig-Pp3 induce a Pp3-specificp oli e a i e
esponse o T-lymphocy es.
Rega ding he cy okine p ofile p oduced du ing he
p oli e a i e esponse, he mos in e es ing esul s indica ed
ha bo h TLRnlig-Pp3 induced a dec ease o IL-4 and IL-5
le els compa ed o Pp3 pep ide (alone), which is significan o
IL-5 wi h TLR4lig-Pp3, and an inc ease o IFNγis significan
only o TLR7lig-Pp3 in alle gic pa ien s (Figu e 6B). These
changes ha a e obse ed only in alle gic pa ien s indica ed
ha he compounds combining he alle genic pep ide and he
adju an had he capaci y o modula e he Th2 esponse in a
specific way.
TLRnlig-Pp3S imula e he T eg Cells and IL-10
P oduc ion. To u he in es iga e he capaci y o TLRnlig-
Pp3- ea ed moDCs o induce T eg cells, we pe o med co-
cul u e expe imen s and analyzed he T eg cell pe cen ages.
The esul s indica ed significan ly highe pe cen ages o T eg
cells and IL-10 p oduc ion in alle gic pa ien s compa ed o
ole an con ols when s imula ed wi h bo h TLRnlig-Pp3,
Figu e 6. Specific lymphocy e p oli e a ion and egula o y esponse in he p esence o bo h TLRnlig-Pp3. Ba s ep esen median and SEM o he
(A) PI o T-cells, (B) o he cy okine p oduc ion in supe na an s om lymphocy e p oli e a ion assay, and (C) o he T eg-cell pe cen ages and IL-
10 p oduc ion o Pp3, TLRnlig, TLRnlig-Pp3, and TLRnlig plus Pp3 o alle gic pa ien s (n= 9, colo ba s) and ole an con ols (n= 9, whi e ba s)
a 10 nM. The Mann−Whi ney U es was used o pai wise compa isons be ween un ela ed g oups, and Wilcoxon signed- ank es was used o
pai wise compa isons in ela ed samples, showing significan diffe ences as ** (p< 0.0125 and p< 0.0010, Bon e oni co ec ion, espec i ely). The
do ed line ep esen s he PI > 2.
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being significan o TLR7lig-Pp3 (Figu e 6C). Mo eo e , hese
effec s we e significan ly highe compa ed o Pp3 pep ide
alone, TLRnlig as well as TLRnlig plus Pp3 only in alle gic
pa ien s excep wi h TLR4lig-Pp3 o he IL-10 p oduc ion
(Figu e 6C). Rega ding he TLR4 ac i a ion, despi e
p oducing IFNγ, hey can induce a low p oduc ion o IL-10,
as has been obse ed in ou expe imen al se up.
53
In con as ,
ega ding TLR7 ac i a ion, i has been desc ibed in alle gic
animal models ha using small molecula weigh compounds,
such as TLR7 ligands, he alle gen-induced Th2 esponses as
well as he ai way inflamma ion and ai way hype - eac i i y can
be supp essed h ough IL-10 sec e ion.
64,65
These da a sugges ed ha TLRnlig-Pp3-ac i a ed moDCs
p omo ed he p esen a ion o he alle gen (Pp3) and gene a ed
aspecific immunological esponse wi h a ype 1/T eg
immunological pa e n and he supp ession o Th2 effec o
cells.
Effec o he Combina ion o Bo h TLRnlig-Pp3 on
Immunological Cells. A e hese esul s, we wonde whe he
he immunological effec would be enhanced by s imula ing
cells simul aneously wi h bo h TLRs, looking o a syne gis ic
effec . I is known ha he use o syn he ic TLR4 and TLR7
ligands as adju an s o accine in in ec ion diseases induced a
apid, sus ained, and b oadly p o ec i e esponses.
54
Mo eo e ,
in a s udy in amoxicillin-induced maculopapula exan hema, i
was shown ha he simul aneous s imula ion wi h TLR2 and
TLR4 agonis s could be c i ical o he induc ion o he specific
immune esponses, inc easing he moDC ma u a ion and T-
cell p oli e a ion and emula ing he immune esponse he
alle gic pa ien s had a he acu e phase o he eac ion a e he
d ug adminis a ion.
66
Recen ly, he dual ole o TLR ligands
Figu e 7. Effec o he combina ion o bo h TLRnlig-Pp3 on immunological cells. Ba s ep esen median and SEM o he (A) MI o he diffe en
su ace ma ke s on moDCs, o he (B) cy okine p oduc ion in supe na an s om moDCs assays, o he (C) PI o T-cells, o he (D) cy okine
p oduc ion in supe na an s om lymphocy e p oli e a ion assay, and o he (E) T eg-cell pe cen ages and IL-10 p oduc ion o combina ion o
bo h TLRnlig-Pp3 in alle gic pa ien s (n= 9, colo ba s) and ole an con ols (n= 9, whi e ba s) a 10 nM. The Mann−Whi ney U es was used
o pai wise compa isons be ween un ela ed g oups, and Wilcoxon signed- ank es was used o pai wise compa isons in ela ed samples, showing
significan diffe ences as ** (p< 0.016, Bon e oni co ec ion, espec i ely). The do ed line ep esen s he MI and he PI > 2, espec i ely.
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