Gu Mic obes
ISSN: (P in ) (Online) Jou nal homepage: www. and online.com/jou nals/kgmi20
Mi ochond ial dys unc ion-associa ed mic obio a
es ablishes a ansmissible e ac o y esponse o
an i-TNF he apy du ing ulce a i e coli is
Ainize Peña-Cea a, Jani e Cas elo, Jose Luis La ín, Monika Gonzalez-Lopez,
Miguel Angel Pascual-I oiz, Miguel Fue es, Vi ginia Gu ié ez de Juan, Lau a
Bá cena, I zia Ma ín-Ruiz, Aize Pellón, I a xe Seoane, Diego Ba iales,
Ainhoa Palacios, Asie Fullaondo, Iago Rod íguez-Lago, Ma ía L. Ma inez-
Chan a , Ana Mª A ansay, Hec o Rod iguez, Juan Angui a & Le icia Abecia
To ci e his a icle: Ainize Peña-Cea a, Jani e Cas elo, Jose Luis La ín, Monika Gonzalez-Lopez,
Miguel Angel Pascual-I oiz, Miguel Fue es, Vi ginia Gu ié ez de Juan, Lau a Bá cena, I zia
Ma ín-Ruiz, Aize Pellón, I a xe Seoane, Diego Ba iales, Ainhoa Palacios, Asie Fullaondo,
Iago Rod íguez-Lago, Ma ía L. Ma inez-Chan a , Ana Mª A ansay, Hec o Rod iguez, Juan
Angui a & Le icia Abecia (2023) Mi ochond ial dys unc ion-associa ed mic obio a es ablishes a
ansmissible e ac o y esponse o an i-TNF he apy du ing ulce a i e coli is, Gu Mic obes,
15:2, 2266626, DOI: 10.1080/19490976.2023.2266626
To link o his a icle: h ps://doi.o g/10.1080/19490976.2023.2266626
© 2023 The Au ho (s). Published wi h
license by Taylo & F ancis G oup, LLC.
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Mi ochond ial dys unc ion-associa ed mic obio a es ablishes a ansmissible
e ac o y esponse o an i-TNF he apy du ing ulce a i e coli is
Ainize Peña-Cea a
a,b
*, Jani e Cas elo
a
, Jose Luis La ín
a,c
, Monika Gonzalez-Lopez
a
, Miguel Angel Pascual-
I oiz
a
, Miguel Fue es
d
, Vi ginia Gu ié ez de Juan
a
, Lau a Bá cena
a
, I zia Ma ín-Ruiz
a
, Aize Pellón
a
*,
I a xe Seoane
a
, Diego Ba iales
a
, Ainhoa Palacios
a
, Asie Fullaondo
e
, Iago Rod íguez-Lago
,
Ma ía L. Ma inez-Chan a
a,g
, Ana Mª A ansay
a,g
, Hec o Rod iguez
a
, Juan Angui a
a,h
, and Le icia Abecia
a,b
a
CIC bioGUNE, Basque Resea ch and Technology Alliance (BRTA), De io, Spain;
b
Depa men o Immunology, Mic obiology and Pa asi ology,
Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Bilbao, Spain;
c
Applied Ma hema ics Depa men , NEIKER-
Basque Ins i u e o Ag icul u al Resea ch and De elopmen , Basque Resea ch and Technology Alliance (BRTA), De io, Spain;
d
Animal Heal h
Depa men , NEIKER-Basque Ins i u e o Ag icul u al Resea ch and De elopmen , Basque Resea ch and Technology Alliance (BRTA), De io,
Spain;
e
Depa men o Gene ics, Physical An h opology and Animal Physiology, Uni e si y o he Basque Coun y (UPV/EHU), Bilbao, Spain;
Depa men o Gas oen e ology, Hospi al de Galdakao, Galdakao, Spain;
g
CIBERehd, ISCIII, Mad id, Spain;
h
Ike basque, Basque Founda ion o
Science, Bilbao, Spain
ABSTRACT
An i-TNF he apy can induce and main ain a emission s a us du ing in es inal bowel disease.
Howe e , up o 30% o pa ien s do no espond o his he apy by mechanisms ha a e
unknown. He e, we show ha he absence o MCJ, a na u al inhibi o o he espi a o y chain
Complex I, induces gu mic obio a changes ha a e c i ical de e minan s o he lack o
esponse in a mu ine model o DSS-induced in lamma ion. Fi s , we ound ha MCJ exp es-
sion is es ic ed o mac ophages in human colonic issue. The e o e, we demons a e by
ansc ip omic analysis o colon mac ophages om DSS-induced mice ha MCJ-de iciency is
linked o he exp ession o genes belonging o he FcγR signaling pa hway and con ains an
an i-TNF e ac o y gene signa u e iden i ied in ulce a i e coli is pa ien s. The gu mic obial
composi ion changes obse ed upon DSS ea men in he MCJ-de icien mice e ealed he
inc eased p esence o speci ic coli ogenic membe s, including Ruminococcus gna us and
Oscillospi a, which could be associa ed wi h he non- esponse o TNF inhibi o s. Fu he , we
show ha he p esence o a mic obio a associa ed esis ance o ea men is dominan and
ansmissible o esponsi e indi iduals. Collec i ely, ou indings unde sco e he c i ical ole
played by mac ophage mi ochond ial unc ion in he gu ecological niche ha can subs an-
ially a ec no only he se e i y o in lamma ion bu also he abili y o success ully espond
o cu en he apies.
ARTICLE HISTORY
Recei ed 8 Ma ch 2023
Re ised 22 Sep embe 2023
Accep ed 29 Sep embe 2023
KEYWORDS
IBD; mic obio a; complex I;
mi ochond iopa hy; FcγR
signaling; an i-TNF he apy
CONTACT Juan Angui a [email p o ec ed] CIC bioGUNE, Basque Resea ch and Technology Alliance (BRTA), De io, Spain; Le icia Abecia
[email p o ec ed] Depa men o Immunology, Mic obiology and Pa asi ology, Uni e si y o he Basque Coun y (UPV/EHU), Bilbao, Spain
*P esen add ess: Cen e o Hos -Mic obiome In e ac ions, Facul y o Den is y, O al & C anio acial Science, King’s College London, SE1 9RT London, UK.
Supplemen al da a o his a icle can be accessed online a h ps://doi.o g/10.1080/19490976.2023.2266626.
GUT MICROBES
2023, VOL. 15, NO. 2, 2266626
h ps://doi.o g/10.1080/19490976.2023.2266626
© 2023 The Au ho (s). Published wi h license by Taylo & F ancis G oup, LLC.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License (h p://c ea i ecommons.o g/licenses/by-nc/4.0/), which
pe mi s un es ic ed non-comme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. The e ms on which his a icle has been
published allow he pos ing o he Accep ed Manusc ip in a eposi o y by he au ho (s) o wi h hei consen .
In oduc ion
Ulce a i e coli is (UC) is a ch onic, he e ogeneous
and se e e in lamma o y disease ha p ima ily a ec s
he gas oin es inal ac . Al hough i s e iology
emains unknown, e idence sugges s a complex in e -
play be ween mic obio a, he immune sys em, hos
gene ics and en i onmen al ac o s. Whils he e is no
cu e o UC, an i- umo nec osis ac o (TNF) agen s
a e he mos e ec i e o induce and main ain
a emission s a us in in es inal bowel disease (IBD)
pa ien s as hey a ge he excessi e p oduc ion o
TNF, a key p oin lamma o y cy okine p oduced
mainly by ac i a ed immune cells, imp o ing he
quali y o li e o pa ien s. Howe e , he e a e IBD
pa ien s ha a e e ac o y o an i-TNF ea men
and hey can be ca ego ized in o wo g oups; hose
who ne e espond o an i-TNF induc ion he apy
(p ima y ailu e), and 50% o hose who ini ially
espond o an i-TNF ea men bu subsequen ly
lose esponse o e ime (seconda y ailu e).
1
Fu he mo e, he ea men poses isks as se ious
ad e se e ec s including se e e in ec ions ha e been
epo ed.
2
The e o e, he iden i ica ion o key ac o s
ha could allow clinicians o iden i y pa ien s who will
espond o an i-TNF he apy seems a p io i y o p o-
mo e pe sonalized ea men o IBD pa ien s.
Recen ly, a s udy o he UC mucosal ansc ip-
ome e ealed a dec ease in mi ochond ial elec on
anspo chain Complex I ac i i y, which was
ound o co ela e wi h disease se e i y and ea -
men esponse in bo h adul and pedia ic
pa ien s.
3
In addi ion, Mo awea e al. s a ed ha
hos mi ochond ia-mic obio a c oss alk was dis-
u bed in IBD.
4
Consequen ly, mi ochond ial dys-
unc ion and mic obial composi ion migh play an
impo an ole in he esponse o he apy.
The e o e, mic obial-hos mi ochond ia in e ac-
ions seem o be a miles one in he de elopmen
o e ec i e he apies o UC pa ien s.
Abe an immune esponses agains bac e ia a e
ex ensi ely desc ibed in he pa hogenesis o UC.
Du ing UC, mac ophages a e ac i a ed by anslo-
ca ed pa hogenic bac e ia and a e associa ed wi h
he inc eased p oin lamma o y s a us cha ac e is ic
o he disease.
5
In his ega d, me hyla ion-
con olled J p o ein (MCJ, Dnajc15), a na u al
nega i e egula o o mi ochond ial espi a ion
ha inhibi s complex I ac i i y o he elec on
anspo chain egula ing oxida i e phospho yla-
ion and ATP p oduc ion,
6,7
has been linked o
mac ophages. De iciency o MCJ in bone ma ow-
de i ed mac ophages leads o he up egula ion o
he umo nec osis ac o α-con e ing enzyme
(TACE) inhibi o , issue inhibi o o me allop o-
einase 3 (TIMP-3), which inhibi s TNF shedding
om he plasma memb ane.
8
As mac ophages a e
pi o al o coo dina ing p ocesses in he gu and
a signi ican in il a ion is p oduced du ing coli is,
we in es iga ed he exp ession le els o bo h genes
in colon samples om UC pa ien s epo ing MCJ
down egula ion and TIMP3 up egula ion.
9
These
esul s led us o s udy he impac o MCJ de iciency
in acu e
9
and ch onic
10
expe imen al coli is and a e
in line wi h pa ame e s dis up ed du ing IBD ac i -
i y in indi iduals su e ing mic obial dysbiosis.
11
In
addi ion, we ha e encen ly epo ed ha he in lu-
ence o MCJ de iciency on mic obial composi ion
has a de imen al e ec on he se e i y o UC.
12
Because o he in ima e ela ionship ha is
epo ed o exis be ween mi ochond ial dys unc-
ion and he e icacy o cu en he apies, pa icu-
la ly an i-TNF ea men s, as well as he ole played
by mac ophages du ing UC, we ha e now in es i-
ga ed he e ec o mi ochond ial dys unc ion on he
colon esponse o an i-TNF he apy. Ou esul s
show ha he mi ochond ial elec on anspo
chain Complex I ac i i y is de e minan on he
esponse o an i-TNF he apy. Impo an ly, ou
esul s link mi ochond ial dys unc ion wi h
a mic obio a composi ion leading o a lack o he -
apeu ic e icacy ha is dominan and ans e able o
o he wise esponsi e indi iduals. These da a high-
ligh he ele ance o he physiological esponse o
in es inal mac ophages o main ain a homeos a ic
ecological niche ha a ec s bo h he se e i y o gu
in lamma ion and he esponse o cu en he apies.
Resul s
MCJ de iciency p e en s a p o ec i e esponse o
an i-TNF agen s du ing expe imen al coli is
Mi ochond ia dys unc ion, including he educ ion
o complex I ac i i y in ac i e pa ien s,
3
has been
2A. PEÑA-CEARRA ET AL.
ela ed o disease se e i y and a e ac o y esponse
o ea men du ing UC. We ha e shown ha he
absence o he complex I nega i e egula o , MCJ,
esul s in inc eased DSS-induced pa hology.
9
In
o de o e alua e he impac o mi ochond ial dys-
unc ion on he esponsi eness o an i-TNF he -
apy du ing he disease, we e alua ed he esponse
o MCJ-de icien mice o In liximab (IFX, an i-
TNF) ea men . MCJ de iciency diminished he
he apeu ic e icacy o he in liximab biosimila ,
as e idenced by he absence o a signi ican
imp o emen in body weigh (Figu e 1(a)) and
his ological sco es (Figu e 1(b)), in con as o
WT mice ha esponded success ully o he ea -
men . Mo eo e , TACE le els wi hin he colon
only inc eased in ea ed WT mice independen ly
o TNF (Figu e 1(c)) and in il a ed mac ophage
numbe s ha did no a y (Figu e S1a,b). TACE
ac i i y is esponsible o he inal elease o ma u e
TNF p o ein om cells. MPO le els we e signi i-
can ly inc eased (p ≤ .05) in bo h MCJ KO g oups
compa ed o WT g oups independen ly o ea -
men (Figu e S1c).
T ansc ip ional analysis o colon mac ophages
om MCJ-de icien mice shows o e lapping signa-
u es o a e ac o y esponse o ea men .
We ha e shown ha MCJ is exp essed weakly in
he in es inal issue bo h in mice and humans,
9
and
ha mac ophages a e, oge he wi h CD8 T cells, he
cells o he immune sys em wi h he highes exp es-
sion le els o he p o ein.
6,8
We, he e o e, de e -
mined he exp ession o MCJ in in lamed and
heal hy colon issue om IBD pa ien s by immuno-
luo escence s aining. The ansmemb ane sca enge
ecep o CD163 was also used o iden i y he mac o-
phage popula ion wi hin he issue. MCJ was eadily
de ec ed in bo h diseased (Figu e 2(a)) and adjacen ,
heal hy colon issue in colocaliza ion wi h CD163.
These da a indica e ha he impac o MCJ in IBD
pa ien s is p ima ily associa ed wi h mac ophages.
We hen examined he ole o MCJ in he
immune esponse du ing he disease. We
a
b c
WT MCJ-KO
IFX-
IFX+
WT
MCJ KO
WT
MCJ KO
IFX- IFX+
0
10
20
30
40
**
***
IFX- IFX+
0
100
200
300
*
**
Figu e 1. In i o an i-TNF he apeu ic esponse. WT and MCJ-KO mice we e ea ed he i s 6 d wi h DSS and in liximab (IFX) was
adminis a ed o ally om day 3 o day 6, ollowed by 3 d o eco e y pe iod. All g oups a e posi i e o DSS. (a) Weigh loss (%) (n = 8
mice pe g oup a minimum). S a is ical di e ences (P alue < .05) we e obse ed be ween WT IFX- and WT IFX+ om day 3 onwa d
(as e isks below WT IFX+ line) and be ween MCJ KO IFX+ and WT IFX+ a day 3 and om day 5 onwa d (as e isks abo e WT IFX- line).
(b) His ological sco es and ep esen a i e images o colon issue (scale ba size, 100 µm). (c) TACE ac i i y in colonic p o ein ex ac s
(R u/mg p o ein). Fo s a is ical analysis wo-way ANOVA was pe o med. Wi hin boxplo s, as e isks abo e boxes e sus con ol
geno ype (IFX+ e sus IFX-) and as e isks abo e line e sus di e en geno ypes in he same expe imen al g oup. (W _i xn and MCJ
KO_IFXn: DSS posi i e and IFX nega i e; WT_IFXp and MCJ KO_IFXp: DSS and IFX posi i e).
GUT MICROBES 3

pe o med a ansc ip ome analysis o colon
mac ophages unde DSS-induced coli is. P incipal
componen analysis showed changes acco ding o
he disease s a us o he animals (Figu e 2(b)). The
analysis o he 50 mos egula ed genes showed
di e en pa e ns o exp ession associa ed wi h
he p esence/absence o MCJ. MCJ-de icien
mac ophages exp essed 305 up egula ed genes
compa ed o WT con ols. Among hem, he e
we e se e al con i med suscep ibili y- ela ed
genes o UC such as I 5, Tn s 9, Fcg 2b Slc11a1,
I gal, Gp 65, Cd40, and Lsp1, sugges ing a ele an
ab
c
MCJ KO
Mice Up (305)
MCJ KO
Mice Down (34)
UC gene signa u e
Up (3600)
UC gene signa u e
Down (1696)
Mo i P alue
SpiB 1e-16
E g 1e-9
El 5 1e-8
Runx 1e-6
d
e
MCJ KO DSS+
WT DSS+
MCJ KO DSS-
WT DSS-
MCJ KO Mice
Up (305)
An i-TNF (50)
Co icos e oid
(115)
S gn
Clec4e
P gs2
T em1
Clec4d
Fp 2
Gp 84
Il1b
Osm
Ch onic in lamma ion
MCJ CD163 MCJ-CD163
MCJ CD163 MCJ-CD163
Heal hy adjacen
Figu e 2. T ansc ip omic analysis o in es inal issue mac ophages. (a) Immuno luo escence o MCJ ( ed) and CD163 (g een) in
in lamed and heal hy adjacen colon issue om IBD pa ien s. Colocaliza ion is ep esen ed in yellow. (b) P incipal componen analysis
showing di e ences be ween WT and MCJ-de icien DSS posi i e colon mac ophages´ ansc ip omes. (c) HOMER iden i ied se e al
ansc ip ion ac o s en iched in DSS-induced MCJ-KO colon mac ophages. (d) enn diag am ep esen ing 339 di e en ially exp essed
genes ound in colon mac ophages due o MCJ-KO and a human co e ec al UC gene exp ession signa u e consis ing o 5296 genes.
3
Ou o 305 genes up egula ed in coli is induced MCJ-de icien mice colon mac ophages, 183 we e sha ed wi h he human UC gene
signa u e. (e) enn diag am showing sha ed genes be ween up egula ed genes due o MCJ-KO in DSS-induced mice colon
mac ophages and pa ien s e ac o y o an i-TNF and co icos e oid ea men s.
4A. PEÑA-CEARRA ET AL.
ole o mi ochond ia dys unc ion in immune cells
such as mac ophages du ing he cou se o he
disease.
The HOMER package iden i ied a se o an-
sc ip ion ac o s pu a i ely esponsible o he
exp ession changes in mac ophages om DSS-
induced coli is animals.
13
The analysis o he
genes up egula ed in MCJ-de icien mac ophages
compa ed o WT mice iden i ied se e al ansc ip-
ion ac o s (Figu e 2(c), Table S2). Two o hem
(SpiB and El 5) a e ela ed o Fcg 2b exp ession,
while Runx is in ol ed in I 5 exp ession and E g in
he exp ession o Slc11a1 and Tn s 9. Mo eo e ,
El 5 and SpiB possess mo i s ha egula e Il1b
exp ession, while E g showed a mo i ha egula es
he exp ession o Tn , Il10 and Tg bi. On he o he
hand, E g and Runx may egula e Cxcl3 exp ession
and he e o e be in ol ed in leucocy e ec ui men .
In o de o disce n whe he ansc ip ional pa -
e ns associa ed wi h mi ochond ial dys unc ion in
mu ine colon mac ophages a e ela ed o he dis-
ease in humans, UC ansc ip omes om human
ec al samples we e compa ed o expe imen al coli-
is mac ophage ansc ip omes. Ou o he 305
up egula ed genes in mac ophages isola ed om
he colons o MCJ-de icien mice wi h induced
expe imen al coli is compa ed o DSS+ con ols
(WT), 183 we e sha ed be ween bo h coho s
(mouse model and pa ien s) (Figu e 2(d), Table
S3), sugges ing a c i ical ole o mi ochond ial
unc ion du ing disease p og ession.
A subse o genes ob ained om ansc ip ional
analyses we e linked o ea men esponse and shi s
in mic obial composi ion. In his ega d, he 305
up egula ed genes due o MCJ-de iciency in mac o-
phages om expe imen al coli is mice we e com-
pa ed o he lis o genes p oposed o iden i y
pa ien s ha a e e ac o y o TNF blockade,
14
and
co icos e oids.
3
S ikingly, 18 genes we e sha ed wi h
hese 2 gene signa u es (Figu e 2(e)), in which T em1
and Osm, connec ed o dec eased esponsi eness o
an i-TNF he apy, we e sha ed be ween he h ee
gene signa u es. Besides, he high exp ession o ac i-
a ing Fcg 1 and Fcg 3 ound in in lamma o y MCJ-
de icien mac ophages, which a e cell su ace glyco-
p o eins ha bind o he Fc po ion o IgG an ibodies,
and he high le els o colonic IgG ound (Figu e 3( )),
sugges ed ha he MCJ-de icien geno ype migh be
associa ed wi h esis ance o TNF blockade.
Func ional anno a ion en ichmen analysis using
ToppGene, ToppClus e , and ClueGO mapped
g oups o ela ed genes o immunological
p ocesses.
3
O e iew o he ClueGo-de i ed immune
sys em- ela ed pa hways (Figu e 3(a,b)) compa ing
gene exp ession based on MCJ de iciency du ing
DSS-induced coli is showed 46% en ichmen o he
Fc gamma ecep o (FCGR) signaling pa hway, ol-
lowed by myeloid cell (13,1%), mac ophage (6,7%)
and dend i ic cell ac i a ion (6,7%), egula ion o
adap i e immune esponse (6,7%) and leukocy e di -
e en ia ion (6,7%). The P alues o he op speci ic
biological p ocesses and pa hways we e ob ained as
an ou pu om ToppGene. A mo e de ailed
ToppClus e pa hways analysis ou pu showed genes
ela ed o immunological and biological p ocesses
(Figu e 3(c,d)). MCJ-de icien mice p esen ed highe
exp ession o genes in ol ed in immune sys em ac i-
a ion and he e o e, highe p oduc ion o cy okines
and chemokines, and highe ansendo helial mig a-
ion o leukocy es. Impo an ly, IgA p oduc ion was
up egula ed in MCJ-de icien coli is mice.
Fu he mo e, he s udy o mic obio a–hos in e ac-
ions along he DSS-induced coli is pe iod showed
dis inc immune esponse kine ics. Li e bac e ia and
immunoglobulin G (IgG)-coa ed ecal bac e ia we e
quan i ied by low cy ome y. S a ing a day 2 a e
he ini ia ion o DSS ea men , he pe cen age o li e
bac e ia was simila in all expe imen al g oups
(Figu e 3(e)). MCJ-de icien DSS-induced mice
exhibi ed highe IgG-coa ed ecal bac e ia han WT
mice inc easing g adually un il day 8, being signi i-
can ly highe om day 6 (p = 0.022) o day 8 (p =
0.011) (Figu e 3( )). These da a migh indica e ea lie
mic obial-hos c oss- alk and a as e ac i a ion o he
immune sys em. O e all, hese da a sugges ha MCJ
modi ies he kine ics o he immune esponse.
We hen sough o ind co ela ions be ween
genes exp essed in colon mac ophages and speci ic
mic obial ope a ional axonomic uni s (OTUs).
We analyzed he mic obio a composi ion in MCJ-
de icien and WT mice, bo h unde homeos a ic
and DSS-induced pa hology. As expec ed,
9
MCJ
de iciency esul ed in signi ican changes in mic o-
bio a composi ion (Figu e S2(a,b)).
Figu e 4 shows he 50 s onges co ela ions
be ween genes di e en ially exp essed due o MCJ
de iciency in mac ophages in il a ed du ing colon
in lamma ion and OTUs p esen in he 4
GUT MICROBES 5
b
c
a
d
FcγR signalling
pa hway (46,7%)
Myeloid dend i ic cell ac i a ion (6.7%)
Mac ophage ac i a ion (6.7%)
Alpha-be a T cell p oli e a ion (6.7%)
Posi i e egula ion o leukocy e di e en ia ion (6.7%)
Regula ion o adap a i e immune esponses (6.7%)
TLR7 signalling pa hway (6.7%)
Myeloid cell ac i a ion in ol ed in immune esponses (13.1%)
Up
Down
Regula ion o mic o ubule polyme iza ion (50%)
Chape one co ac o -dependen e olding (50%)
NOD-like ecep o s.
In acellula senso s
o PAMPs
Mi ochond ial
espi a ion
Cy okines
Cell adhesion and
mig a ion
Immune sys em
e
02468
0
50
100
150
Days
% Li e bac e ia
*
DSS
02468
0
20
40
60
Days
% IgG
*
*
DSS
WT DSS+
MCJ KO DSS+
WT DSS-
MCJ KO DSS-
Figu e 3. Func ional anno a ion en ichmen analysis o in lamed in es inal mac ophages acco ding o MCJ le els. RNA-seq da a
analysis shows 305 up egula ed and 34 down egula ed genes in MCJ-KO mice ea ed wi h DSS compa ed o con ol coli is mice (FDR
<0.05 and old change ≥1.5). ClueGO cha s ela ed o immunological unc ion e eals (a) up egula ion o FCγR signaling pa hway
(46,7%) in MCJ-KO coli is mice compa ed o WT coli is (Log2 > 2, P alue < 0.05) and (b) down egula ion o chape one co ac o
e olding (50%) and mic o ubule polyme iza ion (50%). De ailed unc ional anno a ion en ichmen analyses o (c) 305 up egula ed
and (c) 34 down egula ed genes in MCJ-KO coli is mice using ToppGene, ToppClus e and Cy oscape a e ep esen ed. (c) Pa hways
ela ed o up egula ed genes; immune sys em (pu ple), cy okines (o ange), cell adhesion and mig a ion (yellow) and o he pa hways
(g een). (d) Biological p ocesses en iched by down egula ed genes; mi ochond ial espi a ion (blue), cy okines (o ange), NOD-like
ecep o s, he in acellula senso s o PAMPS (pu ple) and hea acclima ion ( ed). (e) Fecal li e bac e ia pe cen age (n = 3) and ( ) ecal
IgG-coa ed bac e ia pe cen age ob ained by low cy ome e (n = 3). Fo s a is ical analysis wo-way ANOVA was used.
6A. PEÑA-CEARRA ET AL.
expe imen al g oups. Those included associa ions
be ween Oscillospi a and Ruminococcus gna us,
o en inc eased wi h disease ac i i y, and Cs 7 ha
may play a ole in immune egula ion h ough he
hema opoie ic sys em. Akke mansia muciniphila
p esence, o en ega ded as an immunomodula o ,
co ela ed wi h Jak2 exp ession. Jak2 is in ol ed in
he p oduc ion o key p o-in lamma o y cy okines
and consequen ly, Jak inhibi o s a e cu en ly being
in es iga ed as he apeu ic agen s o UC.
g__S ep ococcus
g__Lac obacillus
s__Lysinibacillus bo oni ole ans
g__Pseudomonas
s__Bac e oides acidi aciens
g__Oscillospi a
o__Clos idiales
__Ruminococcaceae
s__Ruminococcus gna us
__S24-7
s__Akke mansia muciniphila
__S24-7
s__Akke mansia muciniphila
g__Bac e oides
o__Clos idiales
g__Co ynebac e ium
s__Akke mansia muciniphila
o__Clos idiaceae
__Co iobac e iaceae
g__Lac obacillus
__En e ococcaceae
g__Ach omobac e
__Clos idiaceae
o__RF32
__En e ococcaceae
__Rikenellaceae
__S24-7
__Clos idiaceae
s__Akke mansia muciniphila
o__Clos idiales
s__Ruminococcus gna us
o__Clos idiales
g__Allobaculum
s__Bac e oides acidi aciens
__S24-7
__Clos idiaceae
g__Bac e oides
o__Clos idiales
__Rikenellaceae
s__Bac e oides acidi aciens
__Lachnospi aceae
__S24-7
g__En e ococcus
o__Clos idiales
g__Bac e oides
g__Odo ibac e
o__Clos idiales
g__Su e ella
Figu e 4. Hea map o Spea man´s ank co ela ion coe icien s. Bac e ial abundances om he 4 expe imen al g oups ha we e no
ea ed wi h an ibio ic and genes modi ied by he le el o MCJ in colon mac ophages upon in es inal in lamma ion (DSS+) we e used.
To ind associa ions hie a chical all agains all associa ion (HAIIA) was pe o med. A. muciniphila co ela es wi h Jak2 and R. gna us
wi h Cs 7. The numbe s indica e he highes co ela ion be ween bac e ia and gene exp ession, ha ing numbe 1 he highes
co ela ion. Red colo illus a es posi i e co ela ion and blue nega i e. In he x axis each column shows a di e en gene and in he
y axis each ow shows di e en bac e ial species; Fi micu es (black on ), P o eobac e ia ( ed), bac e oide es (blue), ac inobac e ia
(g een) and Ve ucomic obia (pu ple).
GUT MICROBES 7
signi icance was assessed by wo-way ANOVA s a-
is ical es de e mined as *P alue < 0.05, **P alue
< 0.01, ***P alue < 0.001, and ****P alue < 0.0001.
Signi icance is ep esen ed by an as e isk o as e -
isks upside he box o indica e di e ences wi hin
he same geno ype in di e en expe imen al con-
di ions, in liximab nega i e (IFX-) e sus in lixi-
mab posi i e (IFX+), and cohoused in liximab
posi i e (Coh) e sus alone in liximab posi i e
(Alone). As e isks abo e he lines indica e di e -
ences be ween he di e en geno ypes in he same
expe imen al g oup.
Abb e ia ions
DSS Dex an sul a e sodium
FcγR Fc gamma ecep o
HRP Ho se adish Pe oxidase
IBD In lamma o y bowel disease
IFX In liximab
MCJ Me hyla ion-con olled J p o ein
OTU Ope a ional axonomic uni
SCFA Sho -chain a y acid
TACE Tumo nec osis ac o α-con e ing enzyme
TIMP3 Tissue inhibi o o me allop o einase 3
TNF Tumou nec osis ac o
UC Ulce a i e coli is
Acknowledgmen s
We hank Es ibaliz A ondo o echnical suppo .
Disclosu e s a emen
No po en ial con lic o in e es was epo ed by he au ho (s).
Funding
This wo k was suppo ed by g an [RTI2018-096494-B-100 and
PID2021-124328OBI00 o JA] om he Spanish Minis y o
Economy and Compe i i eness co- inanced wi h FEDER unds,
he V G an om GETECCU-MSD (G upo Español de T abajo
en En e medad de C ohn y Coli is ulce osa o LA), Basque
Go e nmen p ojec o heal h [numbe 2015111117 o LA] and
Resea ch Commi ee om OSI Ba ualde-Galdakao (2018-2-2 o
IRL and LA). APC was a ellow o he Uni e si y o he Basque
Coun y (UPV/EHU) and is cu en ly a pos doc o al ellow unded
by he Basque Go e nmen . Suppo was p o ided by he Basque
Depa men o Indus y, Tou ism and T ade (E o ek and Elka ek
P og ams) and he Inno a ion Technology Depa men o Bizkaia
Coun y. CIC bioGUNE hanks MINECO o he Se e o Ochoa
Excellence Acc edi a ion [SEV-2016-0644].
ORCID
Ainize Peña-Cea a h p://o cid.o g/0000-0003-3855-6664
Jani e Cas elo h p://o cid.o g/0000-0002-9628-8242
Monika Gonzalez-Lopez h p://o cid.o g/0000-0003-1506-
0332
Miguel Fue es h p://o cid.o g/0000-0001-5454-0078
I zia Ma ín-Ruiz h p://o cid.o g/0000-0002-9915-8101
Aize Pellón h p://o cid.o g/0000-0003-0415-6566
Ainhoa Palacios h p://o cid.o g/0000-0001-8945-5260
Asie Fullaondo h p://o cid.o g/0000-0001-5387-4762
Iago Rod íguez-Lago h p://o cid.o g/0000-0003-1133-
4578
Ma ía L. Ma inez-Chan a h p://o cid.o g/0000-0002-
6446-9911
Ana Mª A ansay h p://o cid.o g/0000-0002-8271-612X
Juan Angui a h p://o cid.o g/0000-0003-2061-7182
Le icia Abecia h p://o cid.o g/0000-0003-4097-8903
Au ho s’ con ibu ions
Concep ion and design o he s udy (LA), da a collec ion
(APC, JC, MAPI, VGJ, AP, DB, APa and LA), da a analysis
(APC, JLL, MGL, MAPI, MF, IS, AMA, JLL and LA), d a ing
he manusc ip (APC, JA and LA), manusc ip e ision (HR
and MLMC), s a is ical analysis (APC, JLL and LA), ob ained
unding (AF, IRL, JA, and LA), and echnical suppo (LB and
IMR). All au ho s app o ed he inal e sion o publica ion.
Da a a ailabili y s a emen
Raw sequences used o me agenomics analysis we e made
a ailable a Eu opean Nucleo ide A chi e (ENA www.ebi.ac.
uk/ena) unde he p ojec numbe PRJEB33422 o dysbiosis
and PRJEB41595 o in liximab expe imen . Raw sequences
used o pe o m he ansc ip omic analysis we e uploaded o
GEO (Gene Exp ession Omnibus) da abase unde p ojec
accession code GSE135033 (h ps://www.ncbi.nlm.nih.go /
geo/que y/acc.cgi?&acc=GSE135033).
E hics app o al
Animal p o ocols we e app o ed by he Animal Resea ch
E hics Boa d o CIC bioGUNE (Spain; pe mi numbe
CBBA-0615). Collec ion o colon samples om IBD pa ien s
we e app o ed by he Clinical Resea ch E hics Boa d o
Euskadi (CEIC-E; code 16-12).
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