Ma ch 2018 | Volume 9 | A icle 4001
O iginal esea ch
published: 01 Ma ch 2018
doi: 10.3389/ immu.2018.00400
F on ie s in Immunology | www. on ie sin.o g
Edi ed by:
Massimo Gadina,
Na ional Ins i u e o A h i is and
Musculoskele al and Skin Diseases,
Uni ed S a es
Re iewed by:
Robson Cou inho-Sil a,
Uni e sidade Fede al do Rio de
Janei o, B azil
Cinzia Fionda,
Sapienza Uni e si à di Roma, I aly
*Co espondence:
Ma ga ida Sa ai a
[email p o ec ed]
†P esen add ess:
Guilhe mina M. Ca iche,
Ins i u e o In ec ion Immunology,
TWINCORE, Cen e o Expe imen al
and Clinical In ec ion Resea ch, a
Join Ven u e be ween he Medical
School Hanno e (MHH) and he
Helmhol z Cen e o In ec ion
Resea ch (HZI), Hanno e , Ge many;
Valen ine Mu igneux,
UQ Diaman ina Ins i u e, T ansla ional
Resea ch Ins i u e, Uni e si y o
Queensland, B isbane,
QLD, Aus alia
‡These au ho s ha e con ibu ed
equally o his wo k.
§Co-senio au ho s.
Special y sec ion:
This a icle was submi ed o
In lamma ion,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 11Augus 2017
Accep ed: 13Feb ua y2018
Published: 01Ma ch2018
Ci a ion:
Ca dosoA, Gil Cas oA, Ma insAC,
Ca icheGM, Mu igneuxV, Cas oI,
CumanoA, Viei aP and Sa ai aM
(2018) The Dynamics o In e leukin-
10-A o ded P o ec ion
du ing Dex an Sul a e
Sodium-Induced Coli is.
F on . Immunol. 9:400.
doi: 10.3389/ immu.2018.00400
The Dynamics o in e leukin-10-
a o ded P o ec ion du ing Dex an
sul a e sodium-induced coli is
Ana Ca doso1,2,3,4,5,6,7, An onio Gil Cas o6,7‡, Ana Ca a ina Ma ins1,2‡,
Guilhe mina M. Ca iche1,2†, Valen ine Mu igneux8,9†, Isabel Cas o6,7, Ana Cumano3,4,5,
Paulo Viei a3,4,5§ and Ma ga ida Sa ai a1,2*§
1 i3S – Ins i u o de In es igação e Ino ação em Saúde, Po o, Po ugal, 2 IBMC – Ins i u o de Biologia Molecula e Celula ,
Uni e sidade do Po o, Po o, Po ugal, 3 Depa men o Immunology, Uni é Lymphopoièse, Ins i u Pas eu , Pa is, F ance,
4 Uni e si y Pa is Dide o , So bonne Pa is Ci é, Cellule Pas eu , Pa is, F ance, 5 INSERM U1223, Pa is, F ance, 6 ICVS,
Uni e si y o Minho, B aga, Po ugal, 7 ICVS/3B’s – PT Go e nmen Associa e Labo a o y, B aga, Po ugal, 8 Depa men o
Immunology, Uni é In ég i é du génome, immuni é e cance , Ins i u Pas eu , Pa is, F ance, 9 Depa men o Genomes and
Gene ics, Uni é In ég i é du génome, immuni é e cance , Ins i u Pas eu , Pa is, F ance
In lamma o y bowel disease encompasses a g oup o ch onic-in lamma o y condi ions
o he colon and small in es ine. These condi ions a e cha ac e ized by exace ba ed
in lamma ion o he o gan ha g ea ly a ec s he quali y o li e o pa ien s. Molecula
mechanisms coun e ac ing his hype in lamma o y s a us o he gu o e s a egies o
he apeu ic in e en ion. Among hese egula o y molecules is he an i-in lamma o y
cy okine in e leukin (IL)-10, as shown in mice and humans. Indeed, IL-10 signaling,
pa icula ly in mac ophages, is essen ial o in es inal homeos asis. We sough o
in es iga e he empo al p o ile o IL-10-media ed p o ec ion du ing chemical coli is and
which we e he unde lying mechanisms. Using a no el mouse model o inducible IL-10
o e exp ession (pMT-10), desc ibed he e, we show ha mice p econdi ioned wi h IL-10
o 8days be o e dex an sul a e sodium (DSS) adminis a ion de eloped a milde coli ic
pheno ype. In IL-10-induced coli ic mice, Ly6C cells isola ed om he lamina p op ia
showed a dec eased in lamma o y p o ile. Because ou mouse model leads o ansc ip-
ion o he IL-10 ansgene in he bone ma ow and ele a ed se ic IL-10 concen a ion,
we in es iga ed whe he IL-10 could imp in immune cells in a long-las ing way, hus
con e ing sus ained p o ec ion o coli is. We show ha his was no he case, as IL-10-
a o ded p o ec ion was only obse ed i IL-10 induc ion immedia ely p eceded DSS-
media ed coli is. Thus, despi e he p o ec ion a o ded by IL-10 in coli is, no el s a egies
a e equi ed, speci ically o achie e long-las ing p o ec ion.
Keywo ds: in e leukin-10, mac ophages, in lamma ion, coli is, he apy
inT ODUcTiOn
In lamma o y bowel disease (IBD) comp ises a complex g oup o in lamma o y condi ions o he
gas oin es inal ac (1) a ec ing an inc easing numbe o pa ien s wo ldwide (2–4). Bo h o ms
o IBD, C ohn’s disease (CD) and ulce a i e coli is (UC), esul om al e a ions in he immune
homeos asis o he in es inal issue leading o local uncon olled in lamma ion (5, 6). The gu is a
e y pa icula si e in e ms o immune epe oi e and egula ion, as e en in homeos a ic condi ions
cons an exposu e o an igens occu s (7). Thus, he main enance o in es inal homeos asis, p ima ily
2
Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is
F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400
ca ied ou by in es inal mac ophages, equi es a cons an and
ine- uned balance be ween he s a e o ole ance and in lam-
ma ion (8). In he gu en i onmen , mac ophages encoun e a
ple ho a o s imuli, om die a y an igens o commensal bac e ia,
ye , due o hei unique issue-speci ic cha ac e is ics, emain
ole an (9). In he p edisease s age, he epi helial o mucosal
ba ie s become comp omised allowing bac e ia om he lumi-
nal side o in ade he lamina p op ia o he gu (10). This e en
igge s an acu e in lamma o y esponse due o he ac i a ion o
immune cells by di ec con ac wi h bac e ial p oduc s (10). The
induced in lamma ion esul s ei he in elimina ion o he o eign
bac e ial incu sion o in an exace ba ed immune esponse ha
can esul in issue damage. The damage caused by de egula ed
in lamma ion will pe pe ua e he ac i a ion o e ec o cells and
ul ima ely lead o he clinical onse o IBD (10, 11).
Epidemiological s udies ha e shown ha he e iology o IBD
is mul i ac o ial, wi h gene ic p edisposi ion, dys unc ional in es-
inal ba ie and imbalances o he mic obiome all con ibu ing
o his condi ion (12–15). Genome-wide associa ion s udies
e ealed ha he main gene ic al e a ions associa ed wi h IBD
a e ound in genes encoding p o eins linked o inna e o adap-
i e immuni y, such as he nucleo ide-binding oligome iza ion
domain-con aining p o ein 2, Janus kinase (JAK) 2, and umo
nec osis ac o supe amily 15 (16–18). O he al e a ions a e
associa ed wi h molecules in ol ed in leukocy e a icking,
egula ion o ba ie unc ion and sec e ion o de ensins (17).
Two epo s associa e loss-o - unc ion mu a ions in in e leukin
(IL)-10 o IL-10R subuni s wi h se e e IBD (19, 20). These mu a-
ions esul in se e e en e ocoli is, wi h onse be o e one yea o
age, and un esponsi eness o immunosupp essi e he apies. The
only a ailable he apy o hese pa ien s is immune econs i u-
ion wi h hema opoie ic s em cells (21–23). Al hough comple e
loss-o - unc ion mu a ions in IL-10 and IL-10R s ongly co ela e
wi h IBD, hey ha e an ex emely low occu ence a e (19, 24).
The mos equen mu a ions a ec ing he IL-10 genes associa ed
wi h IBD a e in ac single-nucleo ide polymo phisms associa ed
wi h low exp ession o his molecule (25). Howe e , ha bo ing
such mu a ions does no always ansla e in low se um le els
o IL-10 (23) du ing he disease s age. This is likely due o he
signi ican inc ease on he numbe o IL-10-p oducing myeloid
cells in CD pa ien s (26–29), o he ex en ha ele a ed se um
le els o IL-10 co ela e wi h disease ac i i y in CD (30–32).
The ole o IL-10 in in es inal in lamma ion is also seen in
he mouse model, as IL-10-de icien mice de elop mic obiome-
dependen spon aneous en e ocoli is (33). Fu he mo e, mice
wi h mac ophage es ic ed IL-10R de iciency also de elop a
spon aneous coli ic p o ile (34), s essing he c i ical ole o he
monocy e/mac ophage axis in he immunologic e en s leading o
IBD. In e es ingly, i has been shown, in a model o in ec ion ha
IL-10 can exe a di ec e ec on monocy es/mac ophages subse s,
leading o changes in hei in lamma o y p o ile and su i al (35).
Mo eo e , IL-10 has been shown o con e p o ec ion om hype -
in lamma o y s a es by he induc ion o he JAK1/STAT3 signaling
pa hway ha supp esses exp ession o p oin lamma o y media o s
and ac i a es exp ession o an i-in lamma o y genes (36).
Taking in o accoun he esul s ob ained in mu ine models
o IL-10 pe u ba ion, he gene ic co ela ion es ablished in
humans, and he an i-in lamma o y p ope ies o IL-10, his
cy okine eme ged as a e y p omising candida e o IBD he apy.
Howe e , in IBD pa ien s IL-10-based he apy has no esul ed in
subs an ial clinical imp o emen s (37). The main ca ea s in hese
clinical ials we e he subcu aneous ou e o adminis a ion
and he concen a ion o he ecombinan molecule ha did no
ensu e ha IL-10 le els eached he mucosal si es, poin ing ou
he impo ance o no el-locally a ge ed he apeu ic s a egies.
Fu he mo e, IL-10 adminis a ion o IL-10-de icien mu ine
models only p o ec ed om coli is i adminis e ed be o e disease
es ablishmen (38).
In his s udy, we epo a no el mouse model o IL-10 o e -
exp ession ( he pMT-10 mouse) and use i o be e explo e
he mechanisms o immune egula ion elici ed by IL-10 in he
con ex o in es inal in lamma ion. We show ha a sho pe iod o
IL-10 o e exp ession p io o he induc ion o coli is amelio a es
he disease ou come, despi e he p esence o CD11b+ Ly6C+
cells in he gu , p e iously associa ed wi h he de elopmen o
de imen al in lamma ion. As compa ed o con ol animals ha
do no o e exp ess IL-10, Ly6C cells isola ed om he gu lamina
p op ia o coli ic pMT-10 mice showed a dec eased in lamma o y
p o ile. Thus, we p opose ha IL-10 o e exp ession impai ed he
esponse o hese cells o he s imulus. In addi ion o he local
e ec o IL-10 in con olling exace ba ed immune esponses, ou
model allows o he s udy o IL-10 in imp in ing de no o gene -
a ed and ci cula ing monocy es. This is because, cons an IL-10
exp ession is ound in speci ic issues, in pMT-10 mice, culmina -
ing in a sys emic e ec . The e o e, IL-10 is likely o a ec o he
impo an compa men s, such as he bone ma ow (BM) and
spleen. IL-10-a o ded p o ec ion was only seen i IL-10 igge -
ing immedia ely p eceded dex an sul a e sodium (DSS)-induced
coli is, hus calling o no el s a egies ha sus ain he e ec o
IL-10 o o e long-las ing p o ec ion.
MaTe ials anD MeThODs
e hics s a emen
In Po ugal, all animal expe imen s we e pe o med in s ic
acco dance wi h ecommenda ions o he Eu opean Union
Di ec i e 2010/63/EU and p e iously app o ed by Po uguese
Na ional Au ho i y o Animal Heal h–Di eção Ge al de
Alimen ação e Ve e iná ia (DGAV). Mice we e eu hanized by
CO2 inhala ion wi h e o s o minimize su e ing.
In F ance, all animal p ocedu es we e app o ed by he Pas eu
Ins i u e Sa e y Commi ee and conduc ed acco ding o F ench
and Eu opean Communi y Ins i u ional guidelines.
animals
The s udy in ol ed he use o he ollowing 7–14-week-old emale
mice: wild- ype C57BL/6j, pMT-10-IL-10 inducible mice, and
pMT-10 c ossed wi h IL-10Rα-de icien mice (39) (pMT-10.
IL-10Rα−/−). Food was adlibi um o all animals.
gene a ion o pMT-10 Mice
pMT10 mice we e gene a ed by A. Gil Cas o and Paulo Viei a.
Mouse IL-10 cDNA was cloned in o he p169ZT ec o , which
FigU e 1 | A no el mouse model o inducible in e leukin (IL)-10 exp ession: pMT-10 mice. (a) Schema ic ep esen a ion showing he a ge ing ec o and inse ion
si e. (B) Kine ics o IL-10 o e exp ession in he se um a di e en ime poin s pos Zn adminis a ion and Zn wi hd awal. pMT-10 mice we e ed wi h no mal
(pMT-10-Zn) o Zn-en iched (pMT-10+Zn) wa e and a he indica ed ime poin s blood was ha es ed and he amoun o IL-10 in se um measu ed by
immunoassay. (c) qRT-PCR iden i ied CD45− TER119− cell subse s om skin, bone ma ow, and small in es ine (SI) as he main p oduce s o IL-10 in pMT-10 mice
ed o 8days wi h Zn-en iched wa e . In bo h (B,c), each poin o ba ep esen s he mean±SEM o h ee independen mice. Da a we e analyzed wi h
(B) wo-way analysis o a iance (Sidak’s mul iple compa isons es ) o (c) S uden ’s - es , *p<0.05; **p<0.01; ***p<0.001.
3
Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is
F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400
ca ies a sheep me allop o ein (MT) 1a p omo e , a β-globin
splice si e and a SV40 polyadenyla ion (polyA) signal. The esul -
ing ec o —pMT-10 (see Figu e 1A)—was hen injec ed in o
C57BL/6j eggs and ansgenic ounde s we e iden i ied by PCR
using MT and IL-10-speci ic p ime s. IL-10 o e exp ession was
induced by eeding he mice a 2% suc ose solu ion wi h 50mM
o zinc (Zn) sul a e.
Dss-induced coli is
Mice we e ed o 8days wi h 3% DSS (TdB consul ancy) in he
d inking wa e , and we e moni o ed, daily, o weigh loss and
disease p og ession. Coli is p og ession was measu ed by he
Disease Ac i i y Index (DAI), as p e iously desc ibed [Table 1
(40)].
assessmen o in es inal in lamma ion
Mice we e eu hanized on day 8 pos -DSS adminis a ion o
ea lie i he symp oms o clinical disease (signi ican weigh
loss o dia hea) became appa en . Samples om colons we e
immedia ely ixed in 4% pa a o maldehyde. Then, 5µm pa a in-
embedded sec ions we e s ained wi h hema oxylin and eosin, and
in lamma ion was assessed in a blinded ashion using a p e iously
desc ibed sys em [Table2 (41)]. Samples we e g aded semiquan-
i a i ely om 0 o 3 o he ou ollowing c i e ia: (i) deg ee
o epi helial hype plasia and goble deple ion; (ii) leukocy e
in il a ion in he lamina p op ia; (iii) a ea o issue a ec ed; and
(i ) he p esence o ma ke s o se e e in lamma ion such as c yp
abscesses submucosal in lamma ion and ulce s. Fo each sample,
c i e ia sco es we e added o gi e an o e all in lamma ion sco e
o 0–12.
cy okine Quan i ica ion
In e leukin-10 concen a ion in he se um was quan i ied using a
comme cially a ailable ELISA ki (R&D sys ems).
P epa a ion o cell suspensions
Isola ion o non-hema opoie ic cells (CD45− TER119−) o hema -
opoie ic (CD45+) BMcells ollowed s anda d p o ocols. B ie ly,
hema opoie ic BMcells we e ex ac ed by lushing he emu s
and ibias wi h 2 mL o HBBS epea edly. To ob ain he BM
non-hema opoie ic cells, he bone agmen s we e incuba ed in
RPMI medium wi h Libe ase TL (0.5mg/ml; Roche) o 30min
a 37°C. To help dissocia ion o non-hema opoie ic cells om he
bone, a e each incuba ion pe iod, he emu s and ibias we e
lushed wi h RPMI. We epea ed his s ep h ee imes a e which
we lushed he bones one las ime, ha es ed he cell suspen-
sions and added 1 olume o RPMI con aining 10% FCS. Small
in es ine (SI), non-hema opoie ic cells we e isola ed as p e iously
desc ibed (42). Skin non-hema opoie ic cells we e isola ed om
ea samples. Samples we e ha es ed and he epide mis exposed
by sepa a ing he ex e nal laye s. Epide mis was incuba ed o
45min a 37°C wi h Libe ase HL (0.5mg/ml; Roche) and DNase I
(1U/mL; In i ogen). A e 45min, non-hema opoie ic cells we e
dissocia ed om he issue by mechanical dis up ion, collec ed,
washed in HBSS con aining 10% FCS and eco e ed. A he
end, non-hema opoie ic cells we e so -pu i ied by excluding all
TaBle 2 | Pa ame e s o his ological analysis o coli is se e i y.
sco e epi helial
hype plasia
and goble
deple ion
leukocy e
in il a ion in
he Lamina
P op ia
a ea
a ec ed
Ma ke s
o se e e
in lamma ion
0 None None/ a e None None
1 Minimal Inc ease 1/3 Minimal
2 Mild Con luen 2/3 Inc eased
3 Ma ked T ansmu al All Con luen
The inal sco e is ob ained by he sum o indi idual sco es. Ma ke s o se e e
in lamma ion included ulce a ion and c yp abscesses.
TaBle 1 | Disease Ac i i y Index (DAI) pa ame e s.
sco e Weigh loss s ool consis ency Bleeding
0 No loss No mal No blood
1 1–5% Mild so B own colo
2 5–10% Ve y so Reddish colo
3 10–20% Dia hea Bloody s ool
4>20% G oss bleeding
DAI is ob ained by he sum o each indi idual sco e.
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Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is
F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400
CD45+ TER119+ cells (Figu e S1A in Supplemen a y Ma e ial).
Cell suspensions o all o he o gans we e ob ained by mechanical
dis up ion.
Lamina p op ia leukocy es (LPLs) we e p epa ed as p e iously
desc ibed (43). B ie ly, LPLs we e ha es ed, dissocia ed and
esuspended in Hank’s Balanced Solu ion (HBSS) supplemen ed
wi h 1% e al cal se um (FCS; Gibco). To isola e LPLs, he colon
was lushed wi h phospha e-bu e ed saline (PBS; Gibco), opened
and cu in o 1cm pieces. To elimina e epi helial cells hese ag-
men s we e incuba ed a 37°C in Ca- and Mg- ee PBS con aining
10% FCS and 5.0mM EDTA unde s ong agi a ion o 30min.
Fo LPL isola ion, he emaining agmen s we e incuba ed in
RPMI medium wi h Libe ase TL (0.5mg/ml; Roche) o 30min
a 37°C. To comple e he diges ion, he suspension was epea edly
passed h ough a 10ml sy inge o 5min, il e ed h ough a 40μm
cell s aine (BD Bioscience) and collec ed by cen i uga ion. The
cell pelle was esuspended in 44% Pe coll (GE Heal hca e), laid
o e 67% Pe coll, and cen i uged a 600g o 20min a 20°C.
Cells a he in e ace we e collec ed, washed in HBSS con aining
1% FCS and eco e ed.
an ibodies
An ibodies we e conjuga ed o luo och omes (FITC, PE, PECy7,
APC, APCCy7, Paci ic Blue, and BV711) and we e speci ic o he
ollowing mouse an igens: CD3 (145-2C11; Biolegend), CD11b
(M1/70; Sony), CD11c (HL3; Biolegend), CD19 (6D5; Sony),
CD45.2 (104; Biolegend), Ly6C (Hk1.4; eBioscience), Ly6G
(RB6-8C5; BD Pha mingen), CD45 (30F11; Sony), and TER119
(Te 119; BD Pha mingen).
cell so ing and Mul iplex eal Time-Pc
analysis
CD45− TER119− o CD45+ cells we e so -pu i ied based on he
exp ession o CD45 and TER119 using an A ia so e (BD). Dead
cells we e elimina ed by exclusion wi h p opidium iodide (PI).
mRNA om so ed cells was ex ac ed using RNeasy Mic o ki
(Qiagen) and con e ed in o cDNA by e e se ansc ip ion wi h
P imeSc ip RT Reagen ki (Taka a, Clone ech). qRT-PCR was
pe o med using Taqman p ime s (see Table S1 in Supplemen a y
Ma e ial o e e ences) and Taqman Uni e sal Mas e Mix
(Applied Biosys ems). qRT-PCR eac ions we e pe o med on a
ABI 7300 he mocycle (Applied Biosys ems).
Lamina p op ia leukocy es we e FACS-so pu i ied based on
he exp ession o CD45.2, CD11b and Ly6C, using an A ia III
so e (BD). Cells exp essing CD3, CD19, CD11c, and Ly6G we e
excluded. Dead cells we e elimina ed by exclusion wi h PI. CD45.2+
CD11b+ Ly6C+ cells we e so ed di ec ly in o a mix o 9µl o
CellsDi ec One-S ep qRT-PCR ki (Li e Technologies), con ain-
ing a mix u e o dilu ed p ime s (0.05× inal concen a ion, see
Table S1 in Supplemen a y Ma e ial o e e ences). P eampli ied
cDNA (18 cycles), was ob ained acco ding o he manu ac u e ’s
ins uc ions and was dilu ed 1:5 in TE bu e (pH=8; Ambion).
The sample mix u e was as ollows: dilu ed cDNA (2.9µl), Sample
Loading Reagen (0.32µl; Fluidigm), and Taqman Uni e sal PCR
Mas e Mix (3.5µl; Applied Biosys ems). The assay mix u e was
as ollow: Assay Loa ing Reagen (Fluidigm) and Taqman Mix. A
48×48 Dynamic A ay in eg a ed luidic ci cui (IFC; Fluidigm)
was p imed wi h con ol line luid, and he chip was loaded wi h
assays and samples wi h and X IFC Con olle (Fluidigm). The
expe imen s we e un on a BioMa k HD (Fluidigm) o 40 cycles.
Gene exp ession was no malized o Hp and assessed by he
2ΔC me hod.
s a is ical analysis
S a is ical analysis was pe o med wi h he S uden ’s - es o wo-
way analysis o a iance as indica ed in he igu e legends. The
analysis was pe o med wi h P ism So wa e (G aphPad). G aphs
con aining e o s ba s show means±SEM. S a is ical signi icance
is ep esen ed as ollows: *p<0.05, **p<0.01, and ***p<0.001.
esUlTs
gene a ion o a no el Mouse Model o
il-10 O e exp ession
To s udy he biological impac o IL-10 o e exp ession in di -
e en se ings, we enginee ed a no el mouse model o allow
o inducible IL-10 exp ession, he pMT-10 mouse (44). Fo
his, a cons uc con aining he IL-10 cDNA unde he con ol
o he inducible sheep MT p omo e was in oduced in he
genome o wild- ype BL/6 mice (Figu e1A). Whole genome
sequencing e ealed a single inse ion o he ansgene in ch o-
mosome 10, be ween posi ions 78.813.656 and 78.992844bp
(Figu e1A). We es ima ed, by qRT-PCR, he numbe o cop-
ies o he ansgene o be 50–100 (da a no shown). The MT
p omo e is ac i a ed in he p esence o 50mM o Zn in he
o ganism, adminis e ed in he d inking wa e . Kine ic analysis
o IL-10 in he se um o pMT-10 mice ed wi h Zn-en iched
wa e showed a apid inc ease o ci cula ing IL-10 (Figu e1B).
Indeed, as soon as day 3 a e IL-10 induc ion, he le els o
his cy okine in he se um we e e y high (7–12 ng/ml)
FigU e 2 | Dex an sul a e sodium (DSS)-induced pa hology is amelio a ed
by p eexposu e o in e leukin (IL)-10. (a) BL/6, pMT-10, and pMT-10.
IL-10Rα−/− mice we e ed o 8days wi h no mal (BL/6-Zn, pMT-10-Zn, and
pMT-10.IL-10Rα−/− Zn, espec i ely) o Zn-en iched (BL/6+Zn, pMT-
10+Zn, and pMT-10.IL-10Rα−/−+Zn) wa e , ollowed by 8days o 3% DSS
adminis a ion also in he d inking wa e . (B,F) Disease p og ession based on
DAI pa ame e s was egis e ed e e y day o 8days. Each poin ep esen s
he mean±SEM o h ee o i e independen mice, in wo independen
expe imen s. (c) Colon leng h measu emen a day 8 o DSS adminis a ion.
(D) Rep esen a i e H&E-s ained sec ions o la ge bowel a 40× magni ica ion
(scale ba =200µm). (e) Coli is sco es de i ed om e alua ion o colon and
cecum om ei he g oup. Each do ep esen s one independen animal;
ep esen ed is also mean±SEM. Da a we e analyzed wi h (B,F) wo-way
analysis o a iance (Sidak’s mul iple compa isons es ) o (c,e) S uden ’s
- es . (B) # compa e BL/6-Zn agains BL/6+Zn; ^ compa e BL/6-Zn
agains pMT-10-Zn; • compa e BL/6-Zn agains pMT-10+Zn; ∅ compa e
BL/6+Zn agains pMT-10-Zn; x compa e BL/6+Zn agains pMT-10+Zn,
* compa e pMT-10-Zn agains pMT-10+Zn. One symbol, p<0.05; wo
symbols, p<0.01; h ee symbols, p<0.001.
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(Figu e1B). Mo eo e , suspending Zn adminis a ion led o
a sha p d op in IL-10 in se a in only 24h, o below de ec-
ion le els in only 48h (Figu e1B). As expec ed, ci cula ing
IL-10 was unde ec able in pMT-10 mice ed wi h no mal wa e
(Figu e1B). T ansc ip ional analysis o di e en o gans and
cellula compa men s o induced pMT-10 mice e ealed ha
he exp ession o he exogenous IL-10 cDNA was es ic ed
o CD45−TER119− cells om he SI, skin and, o a less ex en ,
BM (Figu e1C). IL-10 induc ion was no de ec ed in he o he
o gans analyzed (li e , spleen, kidney, cho oid plexus, lung,
and colon) no in hema opoie ic cells isola ed om he BM
(Figu e S1B in Supplemen a y Ma e ial). Thus, he pMT-10
mouse model allows o imely con olled IL-10 o e exp es-
sion in speci ic ana omic loca ions, accompanied by a s ong
inc ease o he le els o his cy okine in he se um.
Dss-induced coli is is amelio a ed in
il-10 P eexposed Mice
Despi e he clea link be ween low le els o IL-10 and suscep-
ibili y o coli is in human (45) as well as in mouse models (33,
34), adminis a ion o IL-10 o ea his condi ion showed only
limi ed e ec s (37). A possible eason may be he poo acces-
sibili y o IL-10 o he si e o in lamma ion. In his con ex , and
in iew o he high exp ession seen in he SI o induced mice, he
pMT-10 mouse model o e s an oppo uni y o u he add ess
he e ec s o IL-10 exp ession in he gu in he con ex o coli is.
Fo his, we used he DSS expe imen al model, a highly eliable
and ep oducible way o causing UC-like symp oms in he mouse
model by inducing acu e in lamma ion wi h he ec ui men o
in lamma o y cells (46).
We in es iga ed he impac o IL-10 o e exp ession p io o
DSS-induced coli is. Fo his, pMT-10 mice we e induced o
o e exp ess IL-10 o 8days, be o e ini ia ion o DSS adminis-
a ion (Figu e2A). As con ols, non-induced pMT-10 o BL6
mice ed wi h con ol o Zn-en iched wa e we e used. In ou
expe imen al se ing, wild- ype BL/6 mice s a ed o show signs
o disease om days 4 o 5 a e adminis a ion o 3% DSS in
he d inking wa e (Figu e2B). Con ol pMT-10 mice showed
a p og ession o he DAI e y simila o BL/6 mice (Figu e2B).
As compa ed o pMT-10 o BL6 ed wi h con ol wa e , mice
p econdi ioned wi h IL-10 showed signi ican ly lowe DAI
a e day 5 (Figu e2B) indica ing ha IL-10 con e ed pa ial
p o ec ion. Zn adminis a ion o BL6 mice p io o DSS, esul ed
in pa ial p o ec ion. Indeed, by day 7 o DSS adminis a ion,
a signi ican ly lowe DAI was obse ed in BL/6 mice ed wi h
Zn-en iched wa e as compa ed o BL6 con ol (Figu e 2B).
Ne e heless, he maximal p o ec ion was obse ed o pMT-10
mice o e exp essing IL-10, which sugges s a syne gis ic e ec
o IL-10 and Zn in he amelio a ion o he disease (Figu e2B).
The DAI encompasses h ee sco es, one o which is he weigh
loss. Rela i ely o con ol animals, bo h pMT-10 and BL/6 mice
ed wi h Zn p io o DSS adminis a ion showed less weigh loss
(Figu e S2 in Supplemen a y Ma e ial). pMT-10 mice p eexposed
o IL-10 showed he leas educ ion in colon leng h as compa ed
o all con ol g oups (pMT-10 o BL/6 ed wi h con ol wa e ,
and BL/6 ed wi h Zn-en iched wa e – Figu e2C), in line wi h
maximal p o ec ion being con e ed by IL-10. His ologic analysis
o he o gan, comp ising he analysis o in lamma o y in il a es,
a chi ec u al dis o ion (c yp sho ening and b anching) and

FigU e 3 | Ly6C+ cells p eexposed o in e leukin (IL)-10 e eal a less in lamma o y p o ile upon DSS-induced coli is han hose p eexposed o Zn. (a) pMT-10 o
BL/6 mice we e ed wi h Zn-en iched wa e o 8days, ollowed by 4days o 3% DSS adminis a ion. (B) A he end o he DSS ea men , Lamina p op ia
leukocy es (LPLs) we e isola ed and Ly6C+ cells so -pu i ied. Shown is he ga ing s a egy o Ly6C+ cells pu i ica ion. (c) So -pu i ied Ly6C+ cells (n=25 cells)
we e analyzed by qRT-PCR o a o al o 22 genes using he BioMa k HD sys em. Samples we e no malized o Hp exp ession. Rep esen ed is he exp ession
hea map compiling he genes which exp ession was de ec ed in ei he mouse g oup. Each hea map ec angle ep esen s he mean o gene exp ession ob ained o
cells isola ed om i e independen mice. (D) The equency o he di e en leukocy e subse s was de e mined upon s aining o LPLs o Ly6C+ cell so ing. Each
do ep esen s one independen animal; ep esen ed is also mean±SEM. Da a we e analyzed wi h S uden ’s - es , *p<0.05.
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ulce a ion, showed an imp o emen in pMT-10 mice p eexposed
o IL-10 as compa ed o BL/6 p eexposed o Zn (Figu es2D,E).
Howe e , despi e a educ ion in he his ological sco e o some
pMT-10 mice p eexposed o IL-10, he o e all g oup did no
each s a is ical signi icance when compa ed o he o he con ol
g oups (Figu es2D,E).
Thus, ou da a showed ha IL-10 o e exp ession p io o
in es inal insul a o ded a signi ican deg ee o p o ec ion
om DSS-induced coli is. Addi ionally, ou da a also sugges
a syne gis ic e ec o IL-10 and Zn in he amelio a ion o he
disease. To in es iga e his issue, we epea ed he expe imen
using pMT-10 mice c ossed wi h IL-10Rα−/− mice. Since pMT-
10.IL-10Rα−/− double mu an mice a e un esponsi e o IL-10,
he e ec s obse ed would only be due o Zn adminis a ion. In
hese mice, we obse ed an accele a ed disease p og ession upon
DSS adminis a ion, wi h an ele a ed DAI sco e as ea ly as day
3, in line wi h he known ole o IL-10 in con olling he disease
(Figu e2F). In hese mice, Zn adminis a ion did no con e p o-
ec ion agains DSS-induced coli is (Figu e2F). Taken oge he ,
ou indings suppo he no ion ha he p o ec ion con e ed by
Zn equi es IL-10 signaling.
P eexposu e o il-10 P omo es a Mo e
con olled in lamma o y esponse
P e ious s udies ha e shown ha monocy es and mac ophages
a e he majo e ec o subse s o colonic in lamma ion (34, 47).
Mice wi h mac ophage-speci ic IL-10R de iciency de elop a
spon aneous coli ic p o ile, emphasizing he impo ance o IL-10
in egula ing he mac ophage esponse o p e en uncon olled
in lamma ion (34). Thus, we nex in es iga ed whe he IL-10
amelio a ed DSS-induced coli is by es ic ing he monocy e/
mac ophage esponse. Conside ing ha Zn adminis a ion also
imp o ed he ou come o DSS-induced coli is in BL/6 mice, we
compa ed he ansc ip ional p o ile o monocy es/mac ophages
om BL/6 o pMT-10 mice p eexposed o Zn and subjec ed o
DSS adminis a ion o 4days. We chose his ime poin , since
signs o coli is induced by DSS in bo h BL/6 and pMT-10 mice
only become ob ious a e day 4 o DSS adminis a ion. Thus,
BL/6 and pMT-10 mice we e ed wi h Zn-en iched wa e o
8days and hen ecei ed DSS o 4days (Figu e3A). A his ime
poin , Ly6C+ cells om each mouse om he di e en g oups
we e FACS pu i ied (Figu e3B). Exp ession o 22 genes (Table
S1 in Supplemen a y Ma e ial) associa ed wi h he uncon olled
FigU e 4 | The in e leukin-10 p o ec ion con e ed agains DSS-induced coli is is no long las ing. (a) pMT-10 mice we e ed wi h con ol (pMT-10-Zn) o
Zn-en iched (pMT-10+Zn) wa e o 8days, ollowed by a 7- o 21-day es ing pe iod whe e only no mal wa e was a ailable, and by 8days o 3% DSS.
(B) Disease p og ession based on Disease Ac i i y Index (DAI) pa ame e s was egis e ed e e y day o 8days. (c) Colon leng h measu emen a day 8 o DSS
adminis a ion. (D) Rep esen a i e H&E-s ained sec ions o la ge bowel a 40× magni ica ion (scale ba =200µm). (e) Coli is sco es de i ed om e alua ion o colon
and cecum om bo h g oups. Each do ep esen s one independen animal, in wo independen expe imen s; ep esen ed is also mean±SEM. Da a we e analyzed
wi h (B) wo-way analysis o a iance (Sidak’s mul iple compa isons es ) o (c,e) S uden ’s - es .
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immune esponse de eloped in IBD we e analyzed by mul i-
plex RT-PCR. All samples, om bo h g oups, exp essed h ee
house-keeping genes (Hp , Ac b, and Gapdh). O he 22 genes
analyzed, we ailed o de ec exp ession o 9 (Il4, Il9, Il12α, Il12β,
Il13, Il17, Il23, I nγ, and Cx3cl1) in Ly6C+ cells isola ed om he
lamina p op ia in bo h g oups. We de ec ed exp ession o he 10
emaining genes in Ly6C+ cells, in Zn- ed BL/6 and pMT-10 mice
a e DSS adminis a ion (Figu e3C), bu no exp ession in he
absence o insul (da a no shown). Thus, Ly6C+ cells al e hei
exp ession p o ile in esponse o DSS insul . Mos in e es ingly,
on day 4 pos -DSS adminis a ion, Ly6C+ cells isola ed om
induced pMT-10 mice p esen ed an o e all less in lamma o y
p o ile han hose isola ed om BL/6 mice (Figu e3C). In he
case o Tn α and Cd86, he di e ences obse ed be ween he wo
mouse g oups we e s a is ically signi ican (Figu e3C). In all,
hese indings sugges ha exposu e o IL-10 be o e DSS induc-
ion ac s by p e en ing an in lamma o y p o ile in Ly6C+ cells.
O no e, he equency o in lamma o y mac ophages ec ui ed
o he in lamed gu was simila be ween he wo g oups, and he
same was ue o CD3 Tcells and CD19 Bcells, showing ha
IL-10 o e exp ession does no impac he ec ui men o immune
cells o he gu (Figu e3D).
il-10 P o ec ion agains Dss-induced
coli is is no long las ing
In ou mouse model, IL-10 is also o e exp essed in he BM and
is ound a high le els in he se um, possibly c ea ing an an i-
in lamma o y en i onmen ha could p econdi ion de no o
gene a ed o ci cula ing monocy es. In his se ing, he ci cula -
ing monocy es could hus be educa ed o be less esponsi e once
ec ui ed o he colon du ing DSS-induced coli is. To s udy his
possibili y, we combined a pe iod o IL-10 o e exp ession wi h
a es ing pe iod o 7 o 21 days p io o DSS adminis a ion
(Figu e4A). As shown in Figu e1B, he le els o IL-10 e u n
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o basal ones as soon as 24 h a e he zinc adminis a ion is
suspended. Thus, a e a 7-day es , he ci cula ing le els o IL-10
a e no malized. Fu he mo e, we chose he 21days ime-poin ,
because p e ious epo s showing ha esiden in es inal mac-
ophages ha e a li e span o app oxima ely 3weeks (48). A e
7-day es pe iod, he p o ec ion in DAI a o ded by p e ious
IL-10 exposu e was pa ly los (Figu e4B), being less p onounced
han when no es ing was pe o med and only obse ed a la e
ime-poin s. A e he 21-day es ing pe iod, he p o ec ion in
DAI o pMT-10 p e iously o e exp essing IL-10 was comple ely
abla ed (Figu e4B). Fu he mo e, independen ly o he es ing
pe iod, no di e ences we e obse ed be ween he expe imen al
g oups in wha conce ned colon leng h (Figu e4C) o his ology
(Figu es 4D,E). Thus, we conclude ha IL-10 o e exp ession,
o e a pe iod o 8days, does no con e long-las ing p o ec ion
agains in es inal in lamma ion.
DiscUssiOn
Despi e he ac ha IBD is a ea able condi ion, he e a e many
limi a ions o he he apeu ic app oaches cu en ly a ailable
(5). Majo obs acles in his con ex a e he he e ogenei y o he
disease, which implies ha dosage and schedule may di e ac oss
disease condi ions, and he equi emen o a localized ac ion o
he he apeu ic agen . In iew o he s ong immune componen
associa ed wi h disease de elopmen , i is no su p ising ha
he apeu ic manipula ions o he immune esponse ha e been
widely sough app oaches o ackle IBD. Indeed, a commonly
used he apy o IBD is he adminis a ion o an i- umo nec osis
ac o alpha (TNF-α) an ibodies (49). Howe e , in line wi h he
abo e men ioned limi a ions, up o one- hi d o IBD pa ien s do
no espond o his he apy, and hose who espond e en ually
de elop some deg ee o in ole ance o he medica ion (50). In
his con ex , se e al animal models o IBD, bo h spon aneous
and expe imen ally induced (such as DSS), we e de eloped o
in es iga e he ole o a ious ac o s on he pa hogenesis o he
disease and o e alua e he di e en he apeu ic op ions. A mol-
ecule ha has been widely s udied in he con ex o IBD is IL-10.
This cy okine keeps in es inal in lamma ion in check by exe ing
a di ec e ec on monocy e/mac ophage popula ions (34). Thus,
i is no su p ising ha IL-10-based he apies ha e been es ed in
IBD. Howe e , bo h in human (51–54) and mouse models (38),
adminis a ion o IL-10 did no signi ican ly imp o e in es inal
in lamma ion, pe haps in pa due o he ac ha adminis e ed
IL-10 did no each he in lamed issue.
In his s udy, we epo a no el ansgenic mouse model o
inducible IL-10 o e exp ession, he pMT-10 mice, in which
high IL-10 ansc ip ion is obse ed in he in es ine, skin and
BM. Upon induc ion o he ansgene, high le els o IL-10 a e
de ec ed also in he se um. Taking ad an age o his no el mouse
model, we in es iga ed he dynamics o IL-10 a o ded p o ec ion
du ing DSS-induced coli is. We ound ha induc ion o IL-10
p io o DSS adminis a ion impac ed he p og ession o coli is.
We show ha a sho pe iod o IL-10 o e exp ession be o e he
induc ion o coli is amelio a ed he disease ou come, despi e he
p esence o CD11b+ Ly6C+ cells in he gu , p e iously associa ed
wi h he de elopmen o de imen al in lamma ion. Howe e ,
in compa ison o con ol animals ha do no o e exp ess IL-10,
Ly6C cells isola ed om he lamina p op ia o coli ic pMT-10
mice showed a dec eased in lamma o y p o ile. Thus, we hypo h-
esize ha IL-10 o e exp ession impai s he esponse o hese cells
o he insul , ea i ming bo h he c i ical ole o hese cells on
in es inal in lamma ion (47) and ha o IL-10 in egula ing hei
in lamma o y esponses (34). In line wi h a p e iously desc ibed
p o ec i e ole o Zn in he con ex o in es inal in lamma ion
(55, 56), we show some e ec o Zn in educing he se e i y o
coli is, which occu ed bo h la e and o a lowe ex en han ha
obse ed o he combined condi ion Zn+IL-10. In addi ion, he
p o ec i e e ec o Zn ailed o o e come he exace ba ed coli is
obse ed in mice ha did no espond o endogenous IL-10. The
mechanis ic bases unde lying he p o ec ion con e ed by Zn
alone emain unknown. One possibili y is ha Zn may con ibu e
o diminish he amoun o ee adical species gene a ed du ing
acu e coli is which con ibu e o p o ein, DNA chain and lipid
damage (57). In any case, as IBD pa ien s o en p esen a Zn de i-
ciency and espond well o Zn supplemen a ion he apy (58), he
exploi a ion o combined IL-10 and Zn he apies may be wo h
conside ing. In line wi h his, he bene i s o combined Zn and
an i-TNF he apy we e p e iously desc ibed (55).
The pMT-10 mouse model allows o local, as well as sys emic,
IL-10 o e exp ession. The ac ha we de ec ed inc eased an-
sc ip ion o he IL-10 ansgene in he BM and ele a ed le els o
se ic IL-10 led us o hypo hesize ha p eexposu e o IL-10 migh
induce long-las ing ansc ip ional changes in ci cula ing mono-
cy es, o example h ough epigene ic imp in ing. I his we e he
case, we migh be able o educa e hese cells o gain long-las ing
ole ance o DSS-induced coli is. Ou da a ob ained a e a 1- o
3-week es pos -IL-10 exposu e show ha ecen IL-10 exposu e
is equi ed o maximal p o ec ion. Thus, he p o ec i e e ec s o
IL-10 we e no sus ained o e ime, implying ha IL-10 p esence
a he ime o insul is necessa y o p e en coli is. The e o e,
inducing IL-10 exp ession in ou mouse model a he beginning
o disease would be o in e es . Un o una ely, we we e unable
o explo e his possibili y because he Zn necessa y o ac i a e
he ansgene p ecipi a es in he p esence o DSS, when bo h a e
p o ided in he d inking wa e . In addi ion, we we e unable o
induce high le els o IL-10 exp ession in he se um o mice ed
wi h a Zn-en iched die and e hical issues p e en ed us om
a emp ing o induce sus ained high le els o IL-10 by equen
ga age o i.p. injec ions o Zn-con aining p epa a ions.
In conclusion, we he ein p esen a no el mouse model o
inducible IL-10 o e exp ession. We also show he po en ial o his
model o he s udy o he IL-10 biology in he speci ic se ing o
DSS-induced coli is. Ou da a u he suppo he p o ec i e ole
o IL-10 in in es inal in lamma ion, showing ha his cy okine
delays disease p og ession e en when deli e ed be o e DSS
adminis a ion. Howe e , he e ec is no long-las ing, which
calls o al e na i e app oaches o p e en IBD.
eThics sTaTeMenT
In Po ugal, all animal expe imen s we e pe o med in s ic
acco dance wi h ecommenda ions o he Eu opean Union
Di ec i e 2010/63/EU and p e iously app o ed by Po uguese
9
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Na ional Au ho i y o Animal Heal h–Di eção Ge al de
Alimen ação e Ve e iná ia (DGAV). Mice we e eu hanized by
CO2 inhala ion wi h e o s o minimize su e ing. In F ance, all
animal p ocedu es we e app o ed by he Pas eu Ins i u e Sa e y
Commi ee and conduc ed acco ding o F ench and Eu opean
Communi y Ins i u ional guidelines. Mice we e eu hanized by
CO2 inhala ion wi h e o s o minimize su e ing.
aUThO cOnT iBUTiOns
AC, AM, and GC pe o med he expe imen s. AGC and PV made
he pMT-10 mice. VM sequenced he genome o he pMT-10
mouse. AC, AM, AGC, IC, AC, PV, and MS planned he expe i-
men s and analyzed da a. AC, AGC, PV, and MS w o e he a icle.
acKnOWleDgMenTs
We a e g a e ul o D . We ne Mülle (Facul y o Biology,
Medicine and Heal h, Uni e si y o Manches e , Uni ed Kingdom)
o p o iding he IL-10R-de icien mice. We also hank he excel-
len suppo gi en by he animal house wo ke s a IBMC-i3S.
FUnDing
We acknowledge he Po uguese Founda ion o Science and
Technology (FCT) o p o iding a PhD g an o AC (SFRH/
BD/84704/2012). This a icle is a esul o he p ojec No e-01-
0145-FEDER-000012—S uc u ed p og am on bioenginee ed
he apies o in ec ious diseases and issue egene a ion, suppo ed
by No e Po ugal Regional Ope a ional P og amme (NORTE
2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen ,
h ough he Eu opean Regional De elopmen Fund (FEDER).
The MS lab is also inanced by a FCT-ANR g an (FCTANR/
BIM-MEC/0007/2013). This wo k was also backed by he COST
Ac ion BM1404 Eu opean Ne wo k o In es iga o s T igge ing
Explo a o y Resea ch on Myeloid Regula o y Cells (h p://www.
mye-euni e .eu), which is suppo ed by he Ho izon 2020—EU
F amewo k P og am Resea ch and Inno a ion P og amme.
MS is a FCT Associa e In es iga o . AGC lab: This wo k was
de eloped unde he scope o he p ojec NORTE-01-0145-
FEDER-000013, suppo ed by he No he n Po ugal Regional
Ope a ional P og amme (NORTE 2020), unde he Po ugal
2020 Pa ne ship Ag eemen , h ough he Eu opean Regional
De elopmen Fund (FEDER); by he p ojec NORTE-01-0145-
FEDER-000023, suppo ed by he No he n Po ugal Regional
Ope a ional P og amme (NORTE 2020), unde he Po ugal
2020 Pa ne ship Ag eemen , h ough FEDER; and by FEDER,
h ough he Compe i i eness Fac o s Ope a ional P og amme
(COMPETE), and by Na ional unds, h ough he Founda ion o
Science and Technology (FCT), unde he scope o he p ojec
POCI-01-0145-FEDER-007038. PV is unded by ANR, h ough
he p ojec MYELOTEN (ANR-13-ISV1-0003-01).
sUPPleMenTa Y MaTe ial
The Supplemen a y Ma e ial o his a icle can be ound online a
h p://www. on ie sin.o g/a icles/10.3389/ immu.2018.00400/
ull#supplemen a y-ma e ial.
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