Mitochondrial dysfunction-associated microbiota establishes a transmissible refractory response to anti-TNF therapy during ulcerative colitis

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. View supplemen a y ma e ial Published online: 16 Oc 2023. Submi you a icle o his jou nal A icle iews: 2734 View ela ed a icles View C ossma k da a Ci ing a icles: 3 View ci ing a icles Full Te ms & Condi ions o access and use can be ound a h ps://www. and online.com/ac ion/jou nalIn o ma ion?jou nalCode=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 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 . 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