An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Infection.

An Ad anced Human In es inal Cocul u e Model Re eals Compa men alized Hos and Pa hogen S a egies du ing Salmonella In ec ion Leon N. Schul e, a,b Ma hias Schweinlin, c Alexande J. Wes e mann, a,d Ha sha a dhan Janga, b Sa a C. San os, a Silke Appenzelle , e Heike Walles, ,g Jö g Vogel, a,d Ma co Me zge c,g a Ins i u e o Molecula In ec ion Biology (IMIB), Uni e si y o Wü zbu g, Wü zbu g, Ge many b Ins i u e o Lung Resea ch, Philipps Uni e si y, Ma bu g, Ge many c Depa men o Tissue Enginee ing and Regene a i e Medicine, Uni e si y Hospi al Wü zbu g, Wü zbu g, Ge many d Helmhol z Ins i u e o RNA-Based In ec ion Resea ch (HIRI), Helmhol z Cen e o In ec ion Resea ch (HZI), Wü zbu g, Ge many e Comp ehensi e Cance Cen e Main anken, Uni e si y o Wü zbu g, Wü zbu g, Ge many Co e Facili y Tissue Enginee ing, Uni e si y o Magdebu g, Magdebu g, Ge many g F aunho e Ins i u e o Silica e Resea ch ISC, T ansla ional Cen e o Regene a i e The apies TLC-RT, Wü zbu g, Ge many Leon N. Schul e and Ma hias Schweinlin con ibu ed equally o his a icle. Au ho o de was de e mined bo h alphabe ically and in o de o inc easing senio i y. ABSTRACT A majo obs acle in in ec ion biology is he limi ed abili y o ecapi u- la e human disease ajec o ies in adi ional cell cul u e and animal models, which impedes he ansla ion o basic esea ch in o clinics. He e, we in oduce a h ee- dimensional (3D) in es inal issue model o s udy human en e ic in ec ions a a le el o de ail ha is no achie ed by con en ional wo-dimensional monocul u es. Ou model comp ises epi helial and endo helial laye s, a p ima y in es inal collagen sca - old, and immune cells. Upon Salmonella in ec ion, he model mimics human gas o- en e i is, in ha i es ic s he pa hogen o he epi helial compa men , an ad an- age o e exis ing mouse models. Applica ion o dual ansc ip ome sequencing o he Salmonella-in ec ed model e ealed he communica ion o epi helial, endo helial, monocy ic, and na u al kille cells among each o he and wi h he pa hogen. Ou e- sul s sugges ha Salmonella uses i s ype III sec e ion sys ems o manipula e STAT3- dependen inflamma o y esponses locally in he epi helium wi hou accompanying al e a ions in he endo helial compa men . Ou app oach p omises o e eal u he human-specific in ec ion s a egies employed by Salmonella and o he pa hogens. IMPORTANCE In ec ion esea ch ou inely employs in i o cell cul u es o in i o mouse models as su oga es o human hos s. Di e ences be ween mu ine and hu- man immuni y and he low le el o complexi y o adi ional cell cul u es, howe e , highligh he demand o al e na i e models ha combine he in i o-like p ope ies o he human sys em wi h s aigh o wa d expe imen al pe u ba ion. He e, we in- oduce a 3D issue model comp ising mul iple cell ypes o he human in es inal ba ie , a p ima y si e o pa hogen a ack. Du ing in ec ion wi h he oodbo ne pa hogen Salmonella en e ica se o a Typhimu ium, ou model ecapi ula es human disease aspec s, including pa hogen es ic ion o he epi helial compa men , he eby de ia ing om he sys emic in ec ion in mice. Combina ion o ou model wi h s a e-o - he-a gene ics e ealed Salmonella-media ed local manipula ions o human immune esponses, likely con ibu ing o he es ablishmen o he pa ho- gen’s in ec ion niche. We p opose he adop ion o simila 3D issue models o in ec- ion biology, o ad ance ou unde s anding o molecula in ec ion s a egies em- ployed by bac e ial pa hogens in hei human hos . KEYWORDS Salmonella, gene exp ession, in ec ious disease Ci a ion Schul e LN, Schweinlin M, Wes e mann AJ, Janga H, San os SC, Appenzelle S, Walles H, Vogel J, Me zge M. 2020. An ad anced human in es inal cocul u e model e eals compa men alized hos and pa hogen s a egies du ing Salmonella in ec ion. mBio 11:e03348-19. h ps://doi.o g/ 10.1128/mBio.03348-19. Edi o Julian Pa khill, Depa men o Ve e ina y Medicine Copy igh © 2020 Schul e e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional license. Add ess co espondence o Leon N. Schul e, [email p o ec ed], Jö g Vogel, [email p o ec ed], o Ma co Me zge , [email p o ec ed]. This a icle is a di ec con ibu ion om Jö g Vogel, a Fellow o he Ame ican Academy o Mic obiology, who a anged o and secu ed e iews by And eas Bäumle , Uni e si y o Cali o nia, Da is, and Pe a De sch, Uni e si y o Müns e . Recei ed 23 Decembe 2019 Accep ed 10 Janua y 2020 Published RESEARCH ARTICLE Hos -Mic obe Biology c ossm Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 ®mbio.asm.o g 1 18 Feb ua y 2020 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om En e obac e iaceae a e majo commensals o he human gu mic oflo a, bu ce ain membe s o his bac e ial amily, pa icula ly Esche ichia,Salmonella,Shigella, and Ye sinia species, cause a ange o di e en in ec ions ha sum up o millions o cases annually (1). O hese la e pa hogens, Salmonella en e ica se o a Typhimu ium (hence o h S. Typhimu ium) is a majo esea ch model o bac e ial i ulence s a egies and hos de ense mechanisms du ing en e ic in ec ions. Hos cell in ec ion by S. Typhimu ium depends on he conce ed ac i i y o e ec o p o eins encoded on dedica ed genomic i ulence loci, e e ed o as Salmonella pa hogenici y islands (SPIs) (2, 3). The wo majo SPIs (SPI1 and SPI2) addi ionally encode s uc u al componen s o ype III sec e ion sys ems (T3SSs) ha deli e he i ulence e ec o cock ail in o he hos cy osol. While he SPI1 T3SS and associa ed e ec o s media e epi helial cell in asion (4), in acellula su i al is p omo ed by i ulence genes associa ed wi h he SPI2 clus e (5). Hos cell manipula ions media ed by SPI1 and SPI2 e ec o s include ea angemen s o he ac in cy oskele on, manipula ion o phagosomal ma u a ion, and sub e sion o hos immuni y pa hways (3). The immune esponse o S. Typhimu ium has been in es iga ed ex ensi ely. The inna e immune sys em elies on a a ie y o pa e n ecogni ion ecep o s (PRRs), which sense conse ed pa hogen-associa ed molecula pa e ns (PAMPs), such as bac e ial cell wall componen s o flagellin, o elici p oinflamma o y ansc ip ional esponses. In he in es ine, Toll-like ecep o 5 (TLR5) ecognizes Salmonella flagellin, which ac i a es cy okine and chemokine p oduc ion o he ec ui men and ac i a ion o p o essional immune cells, such as NK cells, T cells, and monocy es (6). These cells, in u n, espond by p oducing, e.g., gamma in e e on (IFN- ␥ ) and in e leukin-6 (IL-6), which ac i a e he Janus kinase/signal ansduce and ac i a o o ansc ip ion (JAK/STAT) pa hway on a a ie y o a ge cells o p omo e an imic obial de ense and changes o he cellula su i al and me abolic p og ams (7, 8). O he majo cy okines p oduced by he ac i- a ed epi helium and p o essional immune cells include IL-1, which p omo es NF- ␬ B- dependen immune gene exp ession (9), and IL-8, which unc ions as a majo chemoa - ac an o bac e ium-engulfing neu ophils (10). Recen ly, long noncoding RNAs (lncRNAs) we e also implica ed in he hos esponse o Salmonella in ec ion (11, 12). NeST lncRNA, o ins ance, p o ec s om Salmonella-induced le hali y in mice by p omo ing IFN- ␥ exp ession (13). S. Typhimu ium, in u n, may pa ially e ade his hos de ense h ough i s acul a i e in acellula li es yle and adap a ion o—and e en exploi a ion o — he inflamma o y milieu (14, 15). Responses o Salmonella in ec ions o he human gu necessa ily equi e a fine- uned in e play be ween he gu mucosa, he ascula endo helium, and he cells o he gu -associa ed immune sys em (16). These complex in e ac ions ha e emained di ficul o mimic in a human cell cul u e se ing. The cu en unde s anding o hos sub e sion by S. Typhimu ium and he coun e measu es aken by he immune sys em was la gely deduced om s udies wi h immo alized cell lines o mouse models, i.e., in ec ion models wi h inhe en s eng hs and weaknesses. Cell line monocul u es ha e p o en o be in aluable ools o e eal disc e e molecula and cellula mechanisms, bu hey ine i ably neglec he complex di ision o labo and h ee-dimensional (3D) fine s uc u e wi hin he inflamed issue. In addi ion, c i ical cell componen s, such as pe iphe al immune cells, which a ec he in ec ion p ocess, a e o en missing. Likewise, mice ha e been an impo an model o s udy Salmonella in ec ions on a whole-sys em le el, bu he e a e p o ound di e ences in an imic obial immuni y and Salmonella pa hogenesis be ween oden s and humans (17–19). Fo example, while S. Typhimu- ium induces sel -limi ing gas oen e i is in immunocompe en humans, i causes sys emic in ec ions and e en sepsis in mice (6). The e o e, o be e unde s and he molecula mechanisms unde lying in es inal Salmonella in ec ion in a human se ing, mo e ailo ed models a e needed. Sophis ica ed human h ee-dimensional (3D) in i o models ha e ecen ly ga ne ed much a en ion o scien is s in academia, p oduc de elope s in indus y, egula o y au ho i ies, and socie y in gene al (20, 21). Examples a e p ima y o ganoid cul u es, o a ing-wall essel app oaches, and T answell-like cocul u e se ings (21). Such models Schul e e al. ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 2 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om can deli e impo an in o ma ion abou d ug oxici y and he mode o ac ion, pa ho- physiology, no mal biological issue unc ion, o immune esponses (22, 23), p io o in i o and clinical ex apola ion. O en, howe e , he a ailable models omi impo an cellula componen s, use a ificial cellula g ow h ma ices, o a e di ficul o s anda d- ize. To add ess he pa icula p oblem o a ificial ma ices o suppo cell g ow h, issue models based on ecellula ized collagen sca olds, comp ising mul iple cocul u ed cell ypes o mo e accu a ely mimic he epi helial ba ie s, we e de eloped (20). So a , howe e , such models ha e neglec ed he ascula immune cell componen and ha e no been widely adop ed in in ec ion esea ch. He e, we p esen an ad anced human in es inal ba ie model based upon a ecellula ized po cine collagen sca old ha was in ec ed wi h he bac e ial model pa hogen S. Typhimu ium. This cocul u e model, encompassing bo h he endo helial and he epi helial in es inal ba ie s as well as a na u al collagen ma ix and p o es- sional immune cells, allowed us o s udy ecip ocal hos and pa hogen cell adap a ions du ing acu e S. Typhimu ium in ec ion. We show ha in his model sys em, in con as o small-animal models, S. Typhimu ium in ec ion is es ic ed o he epi helial laye and does no sp ead in o he ascula compa men , he eby mimicking human disease. Dual ansc ip ome sequencing (dual RNA-seq), which comp ehensi ely p ofiles hos and pa hogen gene exp ession du ing bac e ial in ec ions, has been success ully applied o in ec ed cell line-based, wo-dimensional (2D) monocul u es ( e iewed in e e ence 24) and mouse models o in ec ion (25–27). Fo he fi s ime, we he e applied dual RNA-seq o a 3D issue model o cha mRNA and noncoding RNA exp ession changes in he communica ing, pu ified hos cell ypes (in es inal epi helial cells [IECs], endo helial cells, monocy es, NK cells) and in Salmonella. Ou da a se s de e mined STAT3 signaling o be a cen al hos pa hway a ge ed by he pa hogen. Using CRISPR/Cas9-edi ed IECs and Salmonella i ulence mu an s, we show ha he T3SS- dependen manipula ion o STAT3 locally changes he inflamma o y milieu o he benefi o he pa hogen bu lea es he basola e al milieu unal e ed. Thus, 3D in ec ion models may e eal compa men alized pa hogen s a egies no isible in con en ional human cell cul u es. Ou dual RNA-seq da a may se e he communi y as an impo an esou ce o p io i izing Salmonella i ulence ac o s o u he in es iga ion and o defining cell ype-specific exp ession signa u es o pa hogenic a ack a he in es inal ba ie . RESULTS An enginee ed human in es inal issue model o s udy Salmonella in ec ion. Due o he exis ing limi a ions in he cu en ly a ailable 3D in i o cul u e models, in ec ion s udies wi h human-pa hogenic bac e ia ypically neglec he issue mic o- s uc u e a he p ima y si e o in ec ion. Wi h ega d o he in es inal ba ie , i s majo cons i uen s a e he epi helial lining and he unde lying collagen sca old o he lamina p op ia, ha bo ing blood essels o nu ien exchange and immune cell ec ui men (28). To model he in es inal ba ie in a commonly used T answell-like se ing, we fixed adecellula ized po cine small in es inal submucosa (SIS) collagen sca old in o a cell c own o ob ain wo sepa a ed compa men s (Fig. 1A). The apical compa men was popula ed wi h a human in es inal epi helial cell (IEC) line (Caco-2) and ma u ed in o a igh epi helial lining. The basola e al su ace o he ma ix was popula ed wi h p ima y human mic o ascula endo helial cells, and he unde lying sepa a ed cul u e compa - men was supplemen ed wi h pe iphe al blood leukocy es as a p oxy o he ascula immune sys em (Fig. 1A). To confi m he sui abili y o ou model o in ec ion s udies, we conduc ed a pilo expe imen wi h a S. Typhimu ium s ain cons i u i ely exp essing he g een fluo es- cen p o ein (GFP) (29) and acked he bac e ia wi hin he issue cons uc . Fluo es- cence mic oscopy analysis o c oss sec ions isualized epi helial and endo helial cell monolaye s, sepa a ed by he collagen sca old, as well as Salmonella-in ec ed cells wi hin he epi helium, bu no he endo helium (Fig. 1B o D). Flow cy ome y o in ec ed models iden ified a Salmonella-posi i e subpopula ion o epi helial bu no A Cocul u e Model Mimicking Human In es inal In ec ion ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 3 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om endo helial cells (Fig. 2A; see also Fig. S1A in he supplemen al ma e ial), and assays coun ing he numbe s o colony o ming uni s (CFUs) e ealed he s e ili y o he basola e al cul u e medium (Fig. S1B). In line wi h hese esul s, no indica ion o in ec ion o basola e al leukocy es was ob ained (Fig. S1C). These esul s confi m Salmonella o be unable o c oss he epi helium. The fluo escence signal in ensi y emi ed by in aded epi helial cells inc eased o e ime, indica i e o Salmonella in a- cellula eplica ion (Fig. 2B) a a a e compa able o p e ious findings om a 2D Caco-2 in ec ion model (30). Fu he mo e, an inc ease in he pe cen age o in aded cells o e ime indica ed sp eading o he in ec ion wi hin he epi helium (Fig. 2C). Despi e he absence o bac e ial ansmission ac oss he epi helial ba ie , he endo helial cell compa men esponded o he in ec ion by elease o he majo phagocy e a ac an IL-8 (Fig. 2D). Thus, ou issue model success ully ecapi ula es an epi helially e ained Salmonella in ec ion and immune signaling ac oss he in es inal ba ie and he eby esembles human disease, which usually in ol es gas oen e i is, bu no sys emic in ec ion, as is obse ed in mice. P ocessing o Salmonella-in ec ed in es inal issue models o ansc ip omics. We sough o u ilize ou new model o gain an imp o ed unde s anding o he ep og amming o hos immuni y by S. Typhimu ium du ing in ec ion. To his end, in ec ions we e ca ied ou o 24 h wi h GFP-posi i e Salmonella applied o he apical compa men ollowed by fluo escence-ac i a ed cell so ing (FACS)-based sepa a ion BC D BC D A B C D FIG 1 Cons uc ion o he in es inal issue model and expe imen al layou . (A) (Le ) Illus a ion o a c oss-sec ion h ough a cell c own de ice wi hin a cul u e dish, wi h he collagen memb ane being fixed be ween ou e and inne me al ings o c ea e he apical and basola e al compa men s. (Righ ) Schema ic ep esen a ion o he enginee ed in es inal ba ie and expe imen al se up. The epi helium and endo helium a e sepa a ed by a collagen laye (SIS, small in es inal submucosa), and leukocy es a e supplied in o he basola e al compa men . In ec ion is igge ed by addi ion o GFP-posi i e Salmonella in o he apical compa men . (B) Fluo escence mic oscopy analysis o a c oss sec ion h ough a Salmonella-in ec ed in es inal ba ie model (24 h pos in ec ion; MOI, 10). The epi helium and endo helium a e isualized by pCK and CD31 s aining (g een), espec i ely. Nuclei a e s ained wi h DAPI (blue). Salmonella a e s ained wi h an an i-LPS an ibody and a e shown in ed. (C) Magnifica ion showing he epi helial laye wi h Salmonella-in ec ed cells (a ows). (D) Magnifica ion o he endo helial cell laye . Ba s, 100 ␮ m (B) o 20 ␮ m (C, D). Schul e e al. ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 4 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om o Salmonella-in aded epi helial cells (GFP posi i e) and nonin aded bys ande epi he- lial cells (GFP nega i e), RNA ex ac ion, RNA deple ion, and dual RNA-seq (Fig. 3A). To ollow he p opaga ion o he immune esponse ac oss he in es inal ba ie , cells o he endo helial lining (CD31 ⫹ ), monocy es (CD14 ⫹ ), and NK cells (CD56 ⫹ ) we e FACS pu ified om he same models and hei ansc ip omes we e sequenced. The co e- sponding cell ypes om unin ec ed models se ed as hos con ols and he bac e ial inoculum se ed as he Salmonella p ein ec ion e e ence. P incipal-componen analysis (PCA) o hos cell RNA-seq da a ( ow Z-sco es) (Fig. 3B) e ealed ha samples p ima ily clus e ed acco ding o cell ype a he han ea men (in ec ed e sus nonin ec ed). In line wi h his obse a ion, specific exp ession signa u es we e e ealed o IECs, endo helial cells, monocy es, and NK cells (Fig. 3C). Inspec ion o he de ec ed hos ansc ip classes (Fig. 3D) p o ed he in ended deple ion o RNAs ac oss all cell ypes. The mRNA ac ion occupied ⬃88% o all mapped eads, ollowed by small nucleola RNAs (snoRNAs; 3.9%), small nuclea RNAs (snRNAs; 3.3%), and lncRNAs (2.3%). In he ollowing, we ocus on egula ed mRNAs and lncRNAs on he hos side. Gene ally, he hos esponse o Salmonella in ec ion (bo h he numbe o egula ed ansc ip s and hei median old change in exp ession) was highe in cells o he basola e al compa men (endo helial cells, monocy es, NK cells) han in cells o he in ec ed epi helium (Fig. 3E and F). This confi ms he sensing o he apically e ained in ec ion by he ascula componen s o he issue cons uc , as seen in Fig. 2D. In e es ingly, he o e lap among he in ec ion- egula ed hos genes be ween he di e en cell ypes was small (Fig. 3F), p obably eflec ing he non edundan unc ions o IECs, endo helial cells, monocy es, and NK cells du ing bac e ial in ec ion. Cell ype-specific signa u es o he ascula immune esponse. To cha ac e ize he na u e o he espec i e esponses by he ou in e ac ing hos cell ypes, we closely inspec ed mRNA and lncRNA exp ession changes a e in ec ion. Fi s , we sough o cha ac e ize he p opaga ion o he esponse o ou issue model o in ec ion ac oss he A Epi helium GFP con ol 0 h 1 h 4 h 8 h 24 h PE mui leh odnE 0,0 0,5 1,0 1,5 2,0 2,5 3,0 0 4 8 12162024 old change hou s p.i. in acellula eplicaon 0,00 5,00 10,00 15,00 0 5 10 15 124 % in ec ed cells hou s p.i. in ec i y BCBasola e al IL-8 D 0 500 1000 1500 2000 2500 pg IL8 / ml - + + + + + - S. Tm hou s p.i.hou s p.i. 3 2.5 2 1.5 1 0.5 0 2.7 ± 0.5 5.7 ± 2.1 11.9 ±11.2 10.2 ±1.5 10.6 ±1.2 FIG 2 Tempo al analysis o S. Typhimu ium in ec ion o he issue model. (A) Rep esen a i e FACS sca e plo s showing mock- and S. Typhimu ium-in ec ed (MOI, 10) epi helial ( op ow) and endo helial (bo om ow) cells in he ed (cellula au ofluo escence) and g een (GFP-exp essing Salmonella) channels o e he cou se o 24 h. (B) Quan ifica ion o he old inc ease in he geome ic mean g een fluo escence in ensi y compa ing he S. Typhimu ium-in ec ed samples pos in ec ion (p.i.) o he con ol a 0 h. (C) Quan ifica ion o he pe cen age o in ec ed (GFP-posi i e) cells a 1 and 24 h pos in ec ion. (D) Quan ifica ion o IL-8 cy okine le els in he basola e al compa men upon mock con ol o S. Typhimu- ium (S. Tm) ea men a he indica ed ime poin s ia ELISA. A Cocul u e Model Mimicking Human In es inal In ec ion ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 5 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om in es inal ba ie . Despi e he absence o Salmonella ansmission in o he ascula compa men , endo helial cells up egula ed ( old change [FC] ⱖ2; alse disco e y a e [FDR] ⬍0.05) 344 mRNAs and down egula ed (FC ⱕ0.5; FDR ⬍0.05) 392 mRNAs upon apical in ec ion (Fig. 4A). In monocy es, which, in conjunc ion wi h lymphocy es, such as NK o T cells, unc ion o o ches a e he pe iphe al inflamma o y esponse, 427 mRNAs we e up egula ed and 448 mRNAs we e down egula ed compa ed o hei egula ion in he mock-in ec ed con ols (Fig. 4B). In NK cells, which, besides hei cy o oxic p ope ies, p o ide an imic obial cy okine signals o igge an ibac e ial esponses and an igen p esen a ion, 372 mRNAs we e up egula ed and 37 we e down egula ed upon in ec ion (Fig. 4C). Du ing an acu e esponse o in ec ion, he endo helium unc ions o ansmi he local immune ac i a ion signals in o he bloods eam o igge a sys emic esponse (31). In line wi h his, among he 10 mos highly induced mRNAs we e hose encoding p oinflamma o y cy okines and immune cell- ec ui ing chemokines, such as IL-6, CXCL6, and CXCL3L1 (Fig. 4A). Simila ly, monocy es up egula ed mRNAs encoding majo p oinflamma o y chemokines and sys emically ac ing cy okines, such as CXCL5, CXCL3, IL-1 ␣ , and IL-1 ␤ (Fig. 4B). Among he op induced mRNAs in NK cells we e hose encoding neu ophil a ac an IL-8 (CXCL8), endo helial cell a achmen p o ein TNFSF4, o he IL-1 ␣ /IL-1 ␤ decoy p o ein IL-1 ecep o 2 (IL-1R2) (Fig. 4C), sugges ing a i al in ol emen o NK cells in uning he ascula inna e immune esponse. The ACIEC Endo Mo NK mock In + In - mock In . mock In . mock In . B 2D o 3D PCA Endo Mono NK IEC PC2 -3 -2 -1 0 1 2 3 PCA PC1 DEndo Mo IEC NK EF 0% 20% 40% 60% 80% 100% mRNA snRNA lncRNA o he Read dis ibuon snoRNA IEC Endo Mo NK In + In - 1000 100 10 1 0.1 0.01 old-change FACS Dual RNA-Seq: •IECs (GFP+) •IECs (GFP-) •Endo helium •Monocy es •NK cells FIG 3 O e iew o RNA-seq o he samples and compa ison o he global hos esponses. (A) Expe i- men al scheme. The indica ed cell ypes we e pu ified om he issue model by FACS and sepa a ely analyzed by RNA-seq. IECs we e sepa a ed in o GFP-posi i e (GFP ⫹ ;Salmonella-in ec ed) and GFP- nega i e (GFP ⫺ ; nonin ec ed) popula ions and subjec ed o dual RNA-seq analysis. (B) PCA o RNA-seq lib a ies, based on ow Z-sco es. (C) Hea map ep esen a ion o hie a chical clus e ing esul using RNA-seq da a ables om all cell ypes ( ow Z-sco es, colo coded acco ding o he key p o ided a he bo om). In , da a ob ained om Salmonella-in ec ed models; In ⫹ and In ⫺ , GFP-posi i e and -nega i e epi helial cells, espec i ely, om Salmonella-challenged models. (D) A e aged dis ibu ion o RNA-seq eads om all lib a ies o e he main RNA classes. (E) Do -plo ep esen a ion o gene exp ession changes (ⱖ2- old up o down compa ed o he le el o exp ession by he mock- ea ed con ols) a e aged ac oss bo h eplica es and o he indica ed condi ions. (F) Venn diag am depic ing he o e lap o egula ed genes o which he esul s a e shown in panel E be ween he di e en cell ypes. IEC, in es inal epi helial cells; Endo, mic o ascula endo helial cells; Mo o Mono, monocy es; NK, na u al kille cells. Schul e e al. ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 6 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om 0 10 20 30 40 50 mock in . old-change 0 5 10 15 mock in . old-change 0 10 20 30 mock in . old-change 0 1 2 3 4 5 mock in . old-change 0 10 20 30 40 50 mock in . old-change 0 2 4 6 8 mock in . old-change 0 2 4 6 8 mock in . old-change AEndo helium mRNAs BMonocy e mRNAs CNK cell mRNAs Endo helium lncRNAs Monocy e lncRNAs NK cell lncRNAs Validaon Validaon 344 392 LIF DRAXIN CX3CL1 PLA1A CSF2 IL6 CXCL6 NOD2 CCL20 SAA1 HIST1H2BI RHAG SCPEP1 PDK4 CRISP2 EFEMP1 RAMP3 CD34 AGT GIMAP7 31 25 AL031316.1 MSC-AS1 AC025580.2 AC023157.3 LUCAT1 AL132780.1 LINC01137 AC020916.1 AL021937.4 AL137145.1 LINC01876 LINC01116 AC024896.1 LINC02035 AL357507.1 AC019163.1 LINC01094 AC125807.2 AC018616.1 LINC01235 427 448 TCN2 MPEG1 CD36 RNASE1 FUCA1 F13A1 CLEC10A SIGLEC1 COLEC12 FCN1 29 47 MIR3945HG AL031316.1 MSC-AS1 AC083837.2 AC087645.2 AC007384.1 AC025580.2 AC003101.1 MIAT ADORA2A-AS1 AC100830.1 LINC01504 TSC22D1-AS1 AC093227.1 LINC00847 LINC01715 ZBTB11-AS1 AL022311.1 AC007563.2 AL021937.4 CXCL5 CA12 CXCL1 MT1G IL1B IL1R2 IL1A MT1H MT2A CXCL3 372 37 APOBEC3C P4HA1 JAML KIAA1191 PASK WDR73 ALAD PFKFB4 HK2 SLC47A1 30 9 MIAT AC007952.4 LINC00996 LINC02390 AP002433.1 AL157935.1 AZIN1-AS1 AL137145.1 H1FX-AS1 AC020916.1 FP236383.1 AC078777.1 AL035071.1 LINC02453 AL035701.1 CR381653.1 DARS-AS1 AC078883.1 XIST AL021918.3 CXCL8 PID1 ZFY TEX14 TNFSF4 IL1R2 ILDR2 F11R ZNRF1 SYPL1 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 0.001 0.01 0.1 0.2 0.5 1 2 5 10 100 1000 0.001 0.01 0.1 0.2 0.5 1 2 5 10 100 1000 0 2 4 6 mock in . old-change 0 1 2 3 4 5 mock in . old-change 0 1 2 3 4 5 mock in . old-change CSF2 MSC-AS1 LUCAT1 old-change Validaon old-change IL6 CA12 MSC-AS1 MiR3945HG old-change old-change CXCL5 0 2 4 6 8 mock in . old-change 0 2 4 6 mock in . old-change AC007952.4 MIAT old-change old-change CXCL8 PID1 mock in . mock in . mock in . mock in . mock in . mock in . FIG 4 Hos gene exp ession changes in he ascula compa men . (A) Hea map ( wo expe imen al eplica es, R1 and R2) showing changes in mRNA (le ) and lncRNA (middle) exp ession in endo helial cells upon apical Salmonella in ec ion (24 h; MOI, 10) om ha ob ained by mock ea men o he in es inal issue cons uc . The esul s o he op 10 up- and down egula ed genes a e shown in magnifica ion o he igh o each hea map, and he genes a e labeled by name. Fold changes in exp ession a e colo coded acco ding o he key p o ided below panel C. (Righ ) Valida ion o he induced exp ession changes o selec ed mRNAs and lncRNAs upon in ec ion by qRT-PCR. Combined esul s (mean ⫾SD) om h ee independen expe imen s a e shown. (B) Same as panel A bu o monocy e exp ession da a. (C) Same as panel A bu o NK-cell da a. A Cocul u e Model Mimicking Human In es inal In ec ion ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 7 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om induc ion o IL-8 was iden ified o be he common denomina o o endo helial, monocy ic, and NK-cell esponses (Fig. S2). O e all, only a ew RNAs we e induced in mo e han one cell ype (Fig. S2), illus a ing he ex ensi e di ision o labo du ing inna e immune esponses o Salmonella. No ewo hy was he finding ha he esponse o in ec ion by all h ee basola e al cell ypes included he di e en ial exp ession o dozens o lncRNAs which make up a class o ansc ip s wi h eme ging unc ions in e eb a e immuni y (32, 33). Regula ion o selec ed lncRNAs in all cell ypes could be confi med by quan i a i e eal- ime PCR (qRT-PCR) analysis (Fig. 4A o C). These measu emen s also confi med MSC-AS1 o be asha ed lncRNA ma ke o immune ac i a ion in endo helial cells and monocy es. Toge he , hese esul s demons a e ex ensi e ewi ing o he coding and noncoding ansc ip omes o key human cell ypes in ol ed in ascula immune ac i a ion du ing in es inal Salmonella in ec ion. Hos -pa hogen ansc ip omics o he in ec ed epi helium. Dual RNA-seq simul- aneously eco ds he gene exp ession o a bac e ium and i s mammalian hos , which allowed us o s udy ecip ocal hos -pa hogen adap a ions du ing he epi helially e ained S. Typhimu ium in ec ion wi hin FACS-sepa a ed IECs. Mapping o RNA-seq eads om he bac e ial inpu and epi helial mock-in ec ed con ol lib a ies confi med almos exclusi e alignmen o he bac e ial o human e e ence genome, espec i ely (Fig. 5A). Wi h ega d o he in ec ed samples, in he in aded (GFP-posi i e) bu no in he bys ande (GFP-nega i e) epi helial cells, ⬃1% o he o al eads mapped o he Salmonella genome (Fig. 5A), e i ying success ul sepa a ion o in ec ed om nonin- ec ed hos cells a he cell so ing s ep. Compa ison o hese in acellula Salmonella ansc ip omes o p e iously eco ded exp ession da a o in acellula Salmonella wi hin human 2D monocul u es (30) by PCA e ealed a seg ega ion acco ding o monocy ic/mac ophage and epi helial cell lineages (Fig. 5B). Salmonella genes p e e - en ially exp essed du ing epi helial cell (bu no monocy e) in ec ions we e en iched o Gene On ology (GO) e ms ela ing o ni ogen compound me abolism (Fig. 5C). Thus, ou in es inal human issue in ec ion model ecapi ula es an epi helial cell-adap ed Salmonella gene exp ession p og am, a guing ha he in aepi helial en i onmen d i es Salmonella gene exp ession la gely independen ly o he p esence o absence o addi ional hos cell ypes in he cul u e. Compa ison o eads om in aepi helial Salmonella o hose om he bac e ial inpu sample e ealed he up egula ion o 527 Salmonella mRNAs and he down egu- la ion o 145 Salmonella mRNAs (Fig. 5D). Hos cell in asion by Salmonella equi es he ac i a ion o genes encoded by he SPI1 locus (4), whe eas in acellula su i al depends on he exp ession o genes encoded by SPI2 (5). The swi ch om SPI1 o SPI2 gene exp ession in ol es he PhoP/Q wo-componen sys em (34), ac i a ion o which is he e o e necessa y o in acellula su i al (35). Acco dingly, in acellula Salmonella up egula ed he exp ession o genes belonging o he PhoP egulon and SPI2-encoded genes and down egula ed he exp ession o SPI1 genes compa ed o he gene exp es- sion o he bac e ial inpu (Fig. 5E). Hos cell manipula ion by Salmonella occu s h ough e ec o p o eins sec e ed h ough he T3SS encoded on SPI1 and SPI2. In line wi h he ac i a ion o SPI2, exp ession o SPI2 T3SS-associa ed e ec o s, whe he encoded on SPI2 i sel o wi hin he co e genome (excep o SseG), was up egula ed in Salmonella inside flow-so ed IECs (Fig. 5F). On he o he hand, SPI1-associa ed e ec o s we e la gely down egula ed (Fig. 5F). Dual RNA-seq also cap u es he exp ession o bac e ial noncoding ansc ip s, pa icula ly he class o small noncoding RNAs (sRNAs). P e iously, we ha e unco e ed sRNA exp ession pa e ns du ing he in acellula phase o he Salmonella in ec ion cycle (30). Confi ming ha he bac e ial exp ession pa e ns de ec ed in FACS-en iched GFP-posi i e IECs indeed eflec in acellula Salmonella ansc ip ome signa u es, wo PhoP-ac i a ed sRNAs, PinT and AmgR (30, 36), we e highly induced compa ed o hei exp ession in he bac e ial inoculum (Fig. 5D). Addi ionally, ou da a e eal he egu- la ion o dozens o u he sRNAs in in acellula Salmonella (Fig. 5D; Table S1). Fo Schul e e al. ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 8 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om 0,1 1 10 100 old-change SPI1 effec o s SPI2 effec o s 100 10 1 0.1 sseJ si B pipB sp C sseF sopD2 si A sp B sseI sspH2 sseG sopA g gE sopE2 a A sopD sipC sipB sipA sopE SPI1 SPI1 PhoP PhoP SPI2 SPI2 0.01 0.1 1 10 100 1000 Regulons F old-change 1000 100 10 1 0.1 0.01 PhoP SPI2SPI1 R1 R2 R1 R2 R1 R2 Salmonella egulons E PhoQ PhoP SPI2SPI1 (PinT) PinT Is E 5S (bac e ial) U6 (human) DapZ In R 80 98 88 78 in i o- induced in i o- ep essed [n ] G ARead dis ibuon 0% 20% P_1 P_2 k_1 k_2 s_1 s_2 s_1 s_2 0 % 100 % 1.3 % 1.1 % Salmonella Human Inpu Mock GFP + GFP - PC1 PC2 PC3 B CD Salmonella mRNAs Salmonella sRNAs 527 145 pagC pspA as sseB_1 uxuA pspC mg B SL1344_1190 SL1344_1345 sodCI p gH lgK in F cheW in A in E laG in G in C o gAa R1 R2 R1 R2 23 19 AmgR PinT STnc510 S yR-3 RyhB RUF_341c.10 Is E RybA Is C STnc800 zipA-leade S yR-29 STnc3680 S yR-55 STnc1460 pk3 DapZ SLnc0014 Gc B In R R1 R2 R1 R2 0.001 0.01 0.1 0.2 0.5 1 2 5 10 100 1000 I II THP-1_R1 THP-1_R2 dTHP-1 HEK Caco-2_R1 Caco-2_R2 HeLa_R2 HeLa_R1 HeLa_R3 THP-1_R1 THP-1_R2 dTHP-1 HEK Caco-2_R1 Caco-2_R2 HeLa_R2 HeLa_R1 HeLa_R3 FIG 5 Gene exp ession o in aepi helial Salmonella. (A) P opo ion o bac e ial eads in he bac e ial inpu sample, mock- ea ed in es inal epi helial cells (IECs), bys ande IECs (GFP nega i e), and in aded IECs (GFP posi i e). (B) (Le ) PCA o Salmonella in acellula ansc ip omes om in ec ed THP1 monocy es and mac ophages (dTHP1) o he indica ed epi helial cell ypes. Caco-2 cell eplica es R1 and R2 a e om his s udy; he es a e om a p e ious s udy (30). (Righ ) Z-sco e hea map showing Salmonella gene exp ession clus e s in he PCA da a ( o he indica ed cell ypes). Gene clus e s (I and II) disc imina ing epi helial om monocy e/mac ophage in acellula Salmonella ansc ip omes a e highligh ed. (C) Magnifica ion o clus e I om panel B ( igh ) wi h en iched GO e ms. (D) Hea map ( wo expe imen al eplica es, R1 and R2) showing changes in mRNA exp ession (le ) and sRNA exp ession ( igh ) in Salmonella inside in ec ed epi helial cells (24 h) om he exp ession o he inpu bac e ial con ol. The esul s o he op 10 up- o down egula ed genes a e shown in magnifica ion o he igh o each hea map, and he genes a e labeled by name. Fold changes in exp ession a e colo coded acco ding o he key p o ided on he le . (E) (Top) Schema ic illus a ion o he swi ch om SPI1 (in asion) o SPI2 (in acellula su i al) gene exp ession upon in acellula ac i a ion o he PhoP/Q wo-componen sys em. (Bo om) Box plo depic ing he egula ion o genes belonging o he SPI1, PhoP, and SPI2 egulons in in acellula Salmonella compa ed o he Salmonella inoculum. (F) Fold changes in he le els o mRNAs encoding Salmonella e ec o s sec e ed h ough he SPI2 o SPI1 T3SS ( he mean ⫾SD was calcula ed om bo h RNA-seq eplica es). (G) No he n blo alida ion o he di e en ial exp ession o Salmonella sRNAs p io o in ec ion (inoculum) o 24 h a e in ec ion (MOI, 10 o 100). Radioac i e signals we e absen om mock-in ec ed con ol samples (mock), suppo ing he specifici y o he selec ed DNA p obes o he in ended Salmonella ansc ip s and excluding c oss- eac i i y wi h human RNAs. n , numbe o nucleo ides. A Cocul u e Model Mimicking Human In es inal In ec ion ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 9 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om concen a ion, 20 ␮ g/ml) in o he apical compa men a 1 h pos in ec ion. A he ime poin s a e in ec ion indica ed abo e (see ex and figu es in Resul s), epi helial and endo helial cells we e collec ed di ec ly om he sca old by ypsin-Accu ase cell de achmen solu ion ea men upon 2 washes wi h PBS. Leukocy es we e collec ed di ec ly om he basola e al compa men and u he pu ified by cell so ing (see below). Fo CFU assays, Caco-2 cells we e lysed wi h PBS con aining 0.01% T i on X-100. The lysa es we e se ially dilu ed in PBS and pla ed on o LB aga , ollowed by o e nigh incuba ion a 37°C. Con ol samples we e mock ea ed (mock ea men was same ea men used o he in ec ed samples bu wi h he addi ion o s e ile medium ins ead o he bac e ial suspension). CRISPR/Cas9-based genome edi ing. A syn he ic DNA segmen (Me abion; see Table S2 in he supplemen al ma e ial) was cloned in o he BbsI si e o he pX458 CRISPR ec o ( om he F. Zhang lab [74] h ough Addgene) o exp ession o a guide RNA a ge ing he STAT3 coding sequence. Caco-2 cells we e ans ec ed wi h 1 ␮ g o plasmid DNA using he Lipo ec amine 2000 eagen (The mo Fishe ) acco ding o he manu ac u e ’s ins uc ions. A 24 h a e ans ec ion, single ans ec ed (GFP-posi i e) cells we e spo ed in o 96-well pla es ha had been p efilled wi h comple e medium con aining 100 ␮ g/ml o he No mocin an ibio ic mix u e (In i ogen), using a FACSA ia III cell so e (BD) wi h a 100- ␮ m nozzle size. Du ing clonal expansion in he wells o he 96-well pla e, esh medium was added e e y 5 days. Knockou success was e alua ed by PCR amplifica ion o he STAT3 coding sequence om genomic DNA and confi med by Sange sequencing (Seqlab GmbH, Gö ingen, Ge many). Quan i a i e eal- ime PCR. qRT-PCR analyses we e ca ied ou using a Powe SYBR g een RNA- o-C 1-s ep ki (The mo Fishe ) acco ding o he manu ac u e ’s ins uc ions and a Quan S udio3 eal- ime PCR machine (Applied Biosys ems). RNA was ex ac ed using he TRIzol eagen (The mo Fishe ) me hod. To emo e genomic DNA, he ex ac ed nucleic acids we e incuba ed wi h DNase I (The mo Fishe ) and an RNase inhibi o (P omega) o 30 min a 37°C and subsequen ly ex ac ed wi h phenol-chlo o o m- isoamyl alcohol (Sigma-Ald ich), ollowed by p ecipi a ion wi h 30:1 e hanol–5 M sodium ace a e. The qRT-PCR p ime s a e lis ed in Table S2. Fold changes based on h eshold cycle (C T ) alues we e calcula ed using he 2 ⫺ΔΔCT me hod (75), and human U6 snRNA was used as an in e nal e e ence. Immunos aining and flow cy ome y. Cells collec ed om he ba ie model we e analyzed using a FACSCalibu o a FACSA ia III de ice (BD). To pu i y monocy es and NK cells om he collec ed leukocy es, he cells we e s ained wi h an i-CD14-FITC (ca alog numbe 11-0149-42; The mo Fishe ) and an i-CD56-allophycocyanin (ca alog numbe 17-0567-41; The mo Fishe ) an ibodies in PBS, 0.1% FCS and so ed using a FACSA ia III de ice (100- ␮ m nozzle, single-cell pu i y se ing). FCS3.0 files we e analyzed using Flowing so wa e (h p://flowingso wa e.b k.fi/). Dual RNA-seq and compu a ional analyses. Fo RNA-seq analysis, cellula RNA was ex ac ed using ami Vana RNA isola ion ki (The mo Fishe ) acco ding o he o al RNA isola ion p o ocol supplied wi h he ki . RNA was deple ed using a Ribo-Ze o gold (epidemiology) ki (Illumina). Lib a ies we e gene a ed and sequenced on a Nex Seq 500 pla o m a Ve is Bio ech (F eising, Ge many) as p e iously desc ibed (30). Demul iplexed eads we e mapped o he GRCh38 human e e ence anno a ion using he CLC Genomics Wo kbench (Qiagen) wi h s anda d se ings (misma ch cos ⫽2, inse ion cos ⫽3, dele ion cos ⫽3, leng h ac ion ⫽0.8, simila i y ac ion ⫽0.8). The da a ables ob ained we e fil e ed o genes wi h a numbe o eads pe kilobase pe million (RPKM) alue o ⱖ0.5 in bo h sequenced eplica es unde a leas one expe imen al condi ion. Genes exhibi ing old changes in exp ession o ⱖ2o ⱕ0.5 (calcula ed based on RPKMs) in bo h eplica es we e conside ed di e en ially exp essed. Hie a chical clus e ing was pe o med using he Clus e p og am (Michael Eisen lab) wi h he co ela ion (uncen e ed) simila i y me ic and he cen oid linkage clus e ing me hod. Hea maps we e gene a ed using he Ja a T eeView p og am (76). PCA analysis was done in R so wa e using he sc ip p comp (s a s) and he gl package. Ne wo k plo s we e gene a ed wi h Cy oscape so wa e ( e sion 3.7.1). KEGG pa hway analysis and induced ne wo k analysis we e pe o med using he ConsensusPa hDB molecula unc ional in e - ac ion da abase (77). Bac e ial bioin o ma ics analyses (Fig. 5B and C) we e pe o med as ollows. Samples including genes wi h an RPKM o ⬎1 in a leas one sample and a coe ficien o a ia ion o ⬎0.5 we e subjec ed o a 3-dimensional p incipal-componen analysis (wi h he R so wa e sca e plo 3d package, e sion 0.3-41), using log 2 (RPKM) alues as he inpu . Unsupe ised comple e linkage clus e ing (wi h he R so wa e hea map.2 unc ion om he gplo s package, e sion 3.0.1.1) was pe o med on ows and columns using he Euclidian dis ance as a simila i y me ic and log 2 (RPKM) alues as he inpu . Salmonella GO e m en ichmen analysis (Fig. 5C) was pe o med using he ShinyGO ool ( e sion 0.60; h p://bioin o ma ics .sds a e.edu/go/) o he GO e m biological p ocess wi h an FDR cu o o 0.05. Wes e n blo analysis. Fo Wes e n blo analysis, samples we e collec ed in adioimmunop ecipi- a ion assay bu e supplemen ed wi h Laemmli bu e and boiled o 5 min. P o eins we e sepa a ed on 10% polyac ylamide-SDS gels and ans e ed on o ni ocellulose memb anes (ca alog numbe 10600015; Ame sham) by semid y blo ing. P o eins we e de ec ed using an i-STAT3 (ca alog numbe 9139; Cell Signaling), an i-phospho-STAT3 (ca alog numbe 9134; Cell Signaling), and an i-ac in (ca alog numbe sc-1616; San a C uz) p ima y an ibodies, ho se adish pe oxidase-linked seconda y an ibodies, and an enhanced chemiluminescence (ECL) eagen (ca alog numbe RPN2232; Ame sham). Images we e ob ained using an In as Ad anced ECL image sys em. His ology. Tissue samples we e fixed wi h 4% pa a o maldehyde o 1ha 4°C. Samples we e embedded in pa a fin and sec ioned o a hickness o 5 ␮ m wi h a mic o ome (model SM2010 R; Leica). Tissue slices we e fi s depa a finized using he Ro iclea clea ing agen (Ca l Ro h) and ehyd a ed in a g aded se ies o e hanol acco ding o s anda d p o ocols. Cha ac e iza ion o he issue samples was done by immunofluo escence s aining. Fo an igen e ie al, issue slices we e hea p e ea ed a 100°C o 20 min in pH 6 ci a e bu e (Ca l Ro h). A e blocking unspecific binding by PBS wi h 0.3% T i on Schul e e al. ® Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 16 on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om X-100 (Sigma-Ald ich), 5% bo ine se um albumin (BSA; PanReac AppliChem), and 5% donkey se um (Biozol) o 30 min, he slices we e incuba ed wi h p ima y an ibodies a 4°C o e nigh . The ollowing p ima y an ibodies we e used a a 1:100 dilu ion: pan-cy oke a in (pCK; specific o epi helial cells; Dako), CD31 (endo helial cell specific; Abcam), and LPS ( o he de ec ion o Salmonella; Abcam). A e washing, an i-mouse/an i- abbi immunoglobulin-Alexa Fluo 555 and -Alexa Fluo 647 seconda y an ibodies we e added a a dilu ion o 1:400 in an ibody dilu ion solu ion, and he mix u e was incuba ed o 1ha oom empe a u e. Samples we e moun ed using Mowiol moun ing medium wi h DAPI (4=,6-diamidino-2- phenylindole; Sigma-Ald ich) o nuclea s aining. Imaging was achie ed using an in e ed fluo escence mic oscope (Keyence BZ-9000). ELISA. Enzyme-linked immunoso ben assays (ELISAs) we e pe o med using human IL-6 (ca alog numbe 88-7066-86) and IL-8 (ca alog numbe 88-8086-86) Ready-Se -Go ELISA ki s (The mo Fishe ) acco ding o he manu ac u e ’s ins uc ions. The cell cul u e supe na an s we e cen i uged o 1 min a maximal speed o pelle he cell deb is, and samples we e used a a 1:10 (IL-6) o 1:150 (IL-8) dilu ion. The samples we e analyzed using a Tecan Sun ise pla e eade , and absolu e quan ifica ion was achie ed using he cy okine s anda ds supplied wi h he ELISA ki . Da a a ailabili y. RNA-seq da a ha e been uploaded o he NCBI GEO eposi o y (GEO accession numbe GSE136717). SUPPLEMENTAL MATERIAL Supplemen al ma e ial is a ailable online only. FIG S1, PDF file, 0.1 MB. FIG S2, PDF file, 0.2 MB. FIG S3, PDF file, 0.2 MB. FIG S4, PDF file, 0.2 MB. TABLE S1, PDF file, 0.1 MB. TABLE S2, PDF file, 0.1 MB. ACKNOWLEDGMENTS We hank Kon ad Fö s ne o assis ance wi h he bioin o ma ics analyses. This wo k was suppo ed by a g an om he Deu sche Fo schungsgemeinscha (g an DFG GRK 2157, 3D Tissue Models o S udying Mic obial In ec ions by Human Pa hogens, o J.V., M.S., and S.C.S.) and by he on Beh ing-Rön gen-S i ung ( BR p ojec 63-0036, o L.N.S.). We decla e no compe ing in e es s. L.N.S. pe o med expe imen s and da a analysis and pa icipa ed in manusc ip w i ing, unding acquisi ion, s udy design, and supe ision. M.S. pe o med expe imen s and pa icipa ed in s udy design and manusc ip w i ing. 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