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
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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. ®
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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 ®
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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. ®
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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.
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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
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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
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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
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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. ®
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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. A.J.W. pe o med expe imen s
and da a analysis and pa icipa ed in manusc ip w i ing and supe ision. H.J. and S.C.S.
pe o med expe imen s. S.A. pe o med bioin o ma ics analysis. H.W. pa icipa ed in
s udy design, unding acquisi ion, and supe ision. J.V. and M.M. pa icipa ed in man-
usc ip w i ing, unding acquisi ion, s udy design, and supe ision.
REFERENCES
1. Pe i WA, Mille M, Binde HJ, Le ine MM, Dillingham R, Gue an RL, J .
2008. En e ic in ec ions, dia hea, and hei impac on unc ion and de el-
opmen . J Clin In es 118:1277–1290. h ps://doi.o g/10.1172/JCI34005.
2. Fab ega A, Vila J. 2013. Salmonella en e ica se o a Typhimu ium skills
o succeed in he hos : i ulence and egula ion. Clin Mic obiol Re
26:308–341. h ps://doi.o g/10.1128/CMR.00066-12.
3. LaRock DL, Chaudha y A, Mille SI. 2015. Salmonellae in e ac ions wi h
hos p ocesses. Na Re Mic obiol 13:191–205. h ps://doi.o g/10.1038/
n mic o3420.
4. Pa el JC, Galan JE. 2005. Manipula ion o he hos ac in cy oskele on by
Salmonella—all in he name o en y. Cu Opin Mic obiol 8:10–15.
h ps://doi.o g/10.1016/j.mib.2004.09.001.
5. Jennings E, Thu s on TLM, Holden DW. 2017. Salmonella SPI-2 ype III
sec e ion sys em e ec o s: molecula mechanisms and physiological
consequences. Cell Hos Mic obe 22:217–231. h ps://doi.o g/10.1016/j
.chom.2017.07.009.
6. Kees a-Gounde AM, Tsolis RM, Bäumle AJ. 2015. Now you see me, now
you don’ : he in e ac ion o Salmonella wi h inna e immune ecep o s.
Na Re Mic obiol 13:206–216. h ps://doi.o g/10.1038/n mic o3428.
7. Doding on DW, Desai HR, Woo M. 2018. JAK/STAT—eme ging playe s in
me abolism. T ends Endoc inol Me ab 29:55–65. h ps://doi.o g/10
.1016/j. em.2017.11.001.
8. Qi Y-F, Huang Y-X, Wang H-Y, Zhang Y, Bao Y-L, Sun L-G, Wu Y, Yu C-L,
Song Z-B, Zheng L-H, Sun Y, Wang G-N, Li Y-X. 2013. Elucida ing he
c oss alk mechanism be ween IFN-gamma and IL-6 ia ma hema ical
modelling. BMC Bioin o ma ics 14:41. h ps://doi.o g/10.1186/1471-2105
-14-41.
9. Cohen P. 2014. The TLR and IL-1 signalling ne wo k a a glance. J Cell Sci
127:2383–2390. h ps://doi.o g/10.1242/jcs.149831.
10. Russo RC, Ga cia CC, Teixei a MM, Ama al FA. 2014. The CXCL8/IL-8 chemo-
kine amily and i s ecep o s in inflamma o y diseases. Expe Re Clin
Immunol 10:593–619. h ps://doi.o g/10.1586/1744666X.2014.894886.
11. Munschaue M, Vogel J. 2018. Nuclea lncRNA s abiliza ion in he hos
esponse o bac e ial in ec ion. EMBO J 37:e99875. h ps://doi.o g/10
.15252/embj.201899875.
12. Imamu a K, Takaya A, Ishida Y᎑I, Fukuoka Y, Taya T, Nakaki R, Kakeda M,
Imamachi N, Sa o A, Yamada T, Onoguchi᎑Mizu ani R, Akizuki G, Tanu T,
Tao K, Miyao S, Suzuki Y, Nagahama M, Yamamo o T, Jensen TH,
Akimi su N. 2018. Diminished nuclea RNA decay upon Salmonella
in ec ion up egula es an ibac e ial noncoding RNAs. EMBO J 37:e97723.
h ps://doi.o g/10.15252/embj.201797723.
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 17
on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om
13. Gomez JA, Wapinski OL, Yang YW, Bu eau J-F, Gopina h S, Monack DM,
Chang HY, B ahic M, Ki kegaa d K. 2013. The NeST long ncRNA con ols
mic obial suscep ibili y and epigene ic ac i a ion o he in e e on-
gamma locus. Cell 152:743–754. h ps://doi.o g/10.1016/j.cell.2013.01
.015.
14. Ha aga A, Ohlson MB, Mille SI. 2008. Salmonellae in e play wi h hos
cells. Na Re Mic obiol 6:53–66. h ps://doi.o g/10.1038/n mic o1788.
15. Ri e a-Cha ez F, Baumle AJ. 2015. The py omaniac inside you: Salmo-
nella me abolism in he hos gu . Annu Re Mic obiol 69:31–48. h ps://
doi.o g/10.1146/annu e -mic o-091014-104108.
16. Pa el S, McCo mick BA. 2014. Mucosal inflamma o y esponse o Salmo-
nella yphimu ium in ec ion. F on Immunol 5:311. h ps://doi.o g/10
.3389/fimmu.2014.00311.
17. Zschale J, Schlo ke D, A nhold J. 2014. Di e ences in inna e immune
esponse be ween man and mouse. C i Re Immunol 34:433–454.
18. Mes as J, Hughes CCW. 2004. O mice and no men: di e ences be ween
mouse and human immunology. J Immunol 172:2731–2738. h ps://doi
.o g/10.4049/jimmunol.172.5.2731.
19. Cobu n B, G assl GA, Finlay BB. 2007. Salmonella, he hos and disease:
a b ie e iew. Immunol Cell Biol 85:112–118. h ps://doi.o g/10.1038/sj
.icb.7100007.
20. Go don S, Daneshian M, Bouws a J, Caloni F, Cons an S, Da ies DE,
Dandeka G, Guzman CA, Fabian E, Hal ne E, Ha ung T, Hasiwa N,
Hayden P, Kanda o a H, Kha e S, K ug HF, Kneue C, Leis M, Lian G, Ma x
U, Me zge M, O K, P ie o P, Robe s MS, Roggen EL, T alau T, an den
B aak C, Walles H, Leh C-M. 2015. Non-animal models o epi helial
ba ie s (skin, in es ine and lung) in esea ch, indus ial applica ions and
egula o y oxicology. Al ex 32:327–378. h ps://doi.o g/10.14573/al ex
.1510051.
21. Ba ila J, C abbé A, Yang J, F anco K, Nydam SD, Fo sy h RJ, Da is RR,
Ganga aju S, O CM, Coyne CB, Bissell MJ, Nicke son CA. 2018. Modeling
hos -pa hogen in e ac ions in he con ex o he mic oen i onmen :
h ee-dimensional cell cul u e comes o age. In ec Immun 86:e00282
-18. h ps://doi.o g/10.1128/IAI.00282-18.
22. Baske e DA, Clewell H, Kimbe I, Rossi A, Blaauboe B, Bu ie R,
Daneshian M, Eskes C, Goldbe g A, Hasiwa N, Ho mann S, Jawo ska J,
Knudsen TB, Landsiedel R, Leis M, Locke P, Maxwell G, McKim J, McVey
EA, Ouéd aogo G, Pa lewicz G, Pelkonen O, Roggen E, Ro ida C, Ruhdel
I, Schwa z M, Schepky A, Schoe e s G, Skinne N, T en z K, Tu ne M,
Vanpa ys P, Yage J, Zu lo J, Ha ung T. 2012. A oadmap o he
de elopmen o al e na i e (non-animal) me hods o sys emic oxici y
es ing. Al ex 29:3–91. h ps://doi.o g/10.14573/al ex.2012.1.003.
23. Leis M, Hasiwa N, Ro ida C, Daneshian M, Baske e D, Kimbe I, Clewell
H, Goch T, Goldbe g A, Busque F, Rossi A-M, Schwa z M, S ephens M,
Taalman R, Knudsen TB, McKim J, Ha is G, Pamies D, Ha ung T. 2014.
Consensus epo on he u u e o animal- ee sys emic oxici y es ing.
Al ex 31:341–356. h ps://doi.o g/10.14573/al ex.1406091.
24. Wes e mann AJ, Ba quis L, Vogel J. 2017. Resol ing hos -pa hogen
in e ac ions by dual RNA-seq. PLoS Pa hog 13:e1006033. h ps://doi.o g/
10.1371/jou nal.ppa .1006033.
25. Thane R, Goldmann O, Beineke A, Medina E. 2017. Hos -inhe en
a iabili y influences he ansc ip ional esponse o S aphylococcus
au eus du ing in i o in ec ion. Na Commun 8:14268. h ps://doi.o g/
10.1038/ncomms14268.
26. Kusmie ek M, He o en AK, Becks e e M, Nuss AM, De sch P. 2019.
Disco e ing Ye sinia-hos in e ac ions by issue dual RNA-Seq. Me hods
Mol Biol 2010:99–116. h ps://doi.o g/10.1007/978-1-4939-9541-7_8.
27. Nuss AM, Becks e e M, Pimeno a M, Schmühl C, Opi z W, Pisano F,
He o en AK, De sch P. 2017. Tissue dual RNA-seq allows as disco e y o
in ec ion-specific unc ions and ibo egula o s shaping hos -pa hogen
ansc ip omes. P oc Na l Acad SciUSA114:E791–E800. h ps://doi.o g/
10.1073/pnas.1613405114.
28. Luissin AC, Pa kos CA, Nus a A. 2016. Inflamma ion and he in es inal
ba ie : leukocy e-epi helial cell in e ac ions, cell junc ion emodeling,
and mucosal epai . Gas oen e ology 151:616–632. h ps://doi.o g/10
.1053/j.gas o.2016.07.008.
29. Papen o K, Said N, Welsink T, Lucchini S, Hin on JCD, Vogel J. 2009.
Specific and pleio opic pa e ns o mRNA egula ion by A cZ, a con-
se ed, H q-dependen small RNA. Mol Mic obiol 74:139–158. h ps://
doi.o g/10.1111/j.1365-2958.2009.06857.x.
30. Wes e mann AJ, Fö s ne KU, Amman F, Ba quis L, Chao Y, Schul e LN,
Mülle L, Reinha d R, S adle PF, Vogel J. 2016. Dual RNA-seq un eils
noncoding RNA unc ions in hos -pa hogen in e ac ions. Na u e 529:
496–501. h ps://doi.o g/10.1038/na u e16547.
31. Mai J, Vi ue A, Shen J, Wang H, Yang XF. 2013. An e ol ing new
pa adigm: endo helial cells—condi ional inna e immune cells. J Hema-
ol Oncol 6:61. h ps://doi.o g/10.1186/1756-8722-6-61.
32. A ianand MK, Ca ey DR, Fi zge ald KA. 2017. Immunobiology o long
noncoding RNAs. Annu Re Immunol 35:177–198. h ps://doi.o g/10
.1146/annu e -immunol-041015-055459.
33. Schul e LN, Be ams W, S ielow C, Schmeck B. 2019. ncRNAs in inflam-
ma o y and in ec ious diseases. Me hods Mol Biol 1912:3–32. h ps://doi
.o g/10.1007/978-1-4939-8982-9_1.
34. Palme AD, Kim K, Slauch JM. 2019. PhoP-media ed ep ession o he
SPI1 T3SS in Salmonella en e ica se o a Typhimu ium. J Bac e iol 201:
e00264-19. h ps://doi.o g/10.1128/JB.00264-19.
35. G oisman EA. 2001. The pleio opic wo-componen egula o y sys em
PhoP-PhoQ. J Bac e iol 183:1835–1842. h ps://doi.o g/10.1128/JB.183.6
.1835-1842.2001.
36. Lee EJ, G oisman EA. 2010. An an isense RNA ha go e ns he exp es-
sion kine ics o a mul i unc ional i ulence gene. Mol Mic obiol 76:
1020–1033. h ps://doi.o g/10.1111/j.1365-2958.2010.07161.x.
37. Masse E, Vande pool CK, Go esman S. 2005. E ec o RyhB small RNA on
global i on use in Esche ichia coli. J Bac e iol 187:6962–6971. h ps://
doi.o g/10.1128/JB.187.20.6962-6971.2005.
38. Padalon-B auch G, He shbe g R, Elg ably-Weiss M, Ba uch K, Rosenshine
I, Ma gali H, Al u ia S. 2008. Small RNAs encoded wi hin gene ic islands
o Salmonella yphimu ium show hos -induced exp ession and ole in
i ulence. Nucleic Acids Res 36:1913–1927. h ps://doi.o g/10.1093/na /
gkn050.
39. P ei e V, Si ka A, Tome R, Tedin K, B inkmann V, Vogel J. 2007. A small
non-coding RNA o he in asion gene island (SPI-1) ep esses ou e mem-
b ane p o ein syn hesis om he Salmonella co e genome. Mol Mic obiol
66:1174–1191. h ps://doi.o g/10.1111/j.1365-2958.2007.05991.x.
40. Chao Y, Papen o K, Reinha d R, Sha ma CM, Vogel J. 2012. An a las o
H q-bound ansc ip s e eals 3=UTRs as a genomic ese oi o egu-
la o y small RNAs. EMBO J 31:4005–4019. h ps://doi.o g/10.1038/emboj
.2012.229.
41. Ma H, Han P, Ye W, Chen H, Zheng X, Cheng L, Zhang L, Yu L, Wu X, Xu
Z, Lei Y, Zhang F. 2017. The long noncoding RNA NEAT1 exe s an ihan-
a i al e ec s by ac ing as posi i e eedback o RIG-I signaling. J Vi ol
91:e2250-16. h ps://doi.o g/10.1128/JVI.02250-16.
42. Ko YP, Flick MJ. 2016. Fib inogen is a he in e ace o hos de ense and
pa hogen i ulence in S aphylococcus au eus in ec ion. Semin Th omb
Hemos 42:408–421. h ps://doi.o g/10.1055/s-0036-1579635.
43. Hannemann S, Gao B, Galan JE. 2013. Salmonella modula ion o hos cell
gene exp ession p omo es i s in acellula g ow h. PLoS Pa hog
9:e1003668. h ps://doi.o g/10.1371/jou nal.ppa .1003668.
44. Hannemann S, Galan JE. 2017. Salmonella en e ica se o a -specific an-
sc ip ional ep og amming o in ec ed cells. PLoS Pa hog 13:e1006532.
h ps://doi.o g/10.1371/jou nal.ppa .1006532.
45. G i enniko S, Ka in E, Te zic J, Mucida D, Yu G-Y, Vallabhapu apu S,
Schelle J, Rose-John S, Che ou e H, Eckmann L, Ka in M. 2009. IL-6 and
S a 3 a e equi ed o su i al o in es inal epi helial cells and de elop-
men o coli is-associa ed cance . Cance Cell 15:103–113. h ps://doi
.o g/10.1016/j.cc .2009.01.001.
46. Hodge DR, Hu EM, Fa a WL. 2005. The ole o IL-6 and STAT3 in
inflamma ion and cance . Eu J Cance 41:2502–2512. h ps://doi.o g/10
.1016/j.ejca.2005.08.016.
47. Leona d F, Collno EM, Leh CM. 2010. A h ee-dimensional cocul u e o
en e ocy es, monocy es and dend i ic cells o model inflamed in es inal
mucosa in i o. Mol Pha m 7:2103–2119. h ps://doi.o g/10.1021/
mp1000795.
48. Higbee RG, Bye s AM, Dhi V, D ake D, Fahlenkamp HG, Gangu J,
Kachu in A, Kachu ina O, Leis i z D, Ma Y, Meh a R, Mishkin E, Mose J,
Mosque a L, Nguyen M, Pa khill R, Pawa S, Poisson L, Sanchez-Schmi z
G, Schanen B, Singh I, Song H, Tapia T, Wa en W, Wi man V. 2009. An
immunologic model o apid accine assessmen —a clinical ial in a
es ube. Al e n Lab Anim 37(Suppl 1):19–27. h ps://doi.o g/10.1177/
026119290903701S05.
49. Fo bes e JL, Goulding D, Vallie L, Hannan N, Hale C, Picka d D, Muk-
hopadhyay S, Dougan G. 2015. In e ac ion o Salmonella en e ica se o-
a Typhimu ium wi h in es inal o ganoids de i ed om human induced
plu ipo en s em cells. In ec Immun 83:2926–2934. h ps://doi.o g/10
.1128/IAI.00161-15.
50. Co JY, Ma gale -Ca alà M, Li X, Mah AT, Kuo CJ, Monack DM, Amie a MR.
2019. Con olling epi helial pola i y: a human en e oid model o hos -
Schul e e al. ®
Janua y/Feb ua y 2020 Volume 11 Issue 1 e03348-19 mbio.asm.o g 18
on Ma ch 11, 2020 a GESELLSCHAFT FUR BIOTECHNO-h p://mbio.asm.o g/Downloaded om
pa hogen in e ac ions. Cell Rep 26:2509–2520.e4. h ps://doi.o g/10
.1016/j.cel ep.2019.01.108.
51. Yin YB, Zhou DG. 2018. O ganoid and en e oid modeling o Salmonella
in ec ion. F on Cell In ec Mic obiol 8:257. h ps://doi.o g/10.3389/ cimb
.2018.00257.
52. Bein A, Shin W, Jalili-Fi oozinezhad S, Pa k MH, Son heime -Phelps A,
To aglie i A, Chalkiadaki A, Kim HJ, Ingbe DE. 2018. Mic ofluidic o gan-
on-a-chip models o human in es ine. Cell Mol Gas oen e ol Hepa ol
5:659–668. h ps://doi.o g/10.1016/j.jcmgh.2017.12.010.
53. Sale no-Goncal es R, Fasano A, Sz ein MB. 2011. Enginee ing o a mul-
icellula o gano ypic model o he human in es inal mucosa. Gas oen-
e ology 141:e18–e20. h ps://doi.o g/10.1053/j.gas o.2011.04.062.
54. Ku z JR, Goggins JA, McLachlan JB. 2017. Salmonella in ec ion: in e play
be ween he bac e ia and hos immune sys em. Immunol Le 190:
42–50. h ps://doi.o g/10.1016/j.imle .2017.07.006.
55. Ke neis S, Bogdano a A, K aehenbuhl JP, P ingaul E. 1997. Con e sion
by Peye ’s pa ch lymphocy es o human en e ocy es in o M cells ha
anspo bac e ia. Science 277:949–952. h ps://doi.o g/10.1126/science
.277.5328.949.
56. Blanco LP, DiRi a VJ. 2006. Bac e ial-associa ed chole a oxin and GM1
binding a e equi ed o anscy osis o classical bio ype Vib io chole ae
h ough an in i o M cell model sys em. Cell Mic obiol 8:982–998.
h ps://doi.o g/10.1111/j.1462-5822.2005.00681.x.
57. Jaslow SL, Gibbs KD, F icke WF, Wang L, Pi man KJ, Mammel MK,
Thaden JT, Fowle VG, Hamme GE, El enbein JR, Ko DC. 2018. Salmo-
nella ac i a ion o STAT3 signaling by Sa A e ec o p omo es in acel-
lula eplica ion and p oduc ion o IL-10. Cell Rep 23:3525–3536. h ps://
doi.o g/10.1016/j.cel ep.2018.05.072.
58. Lu R, Wu S, Zhang Y-G, Xia Y, Zhou Z, Ka o I, Dong H, Bissonne e M, Sun
J. 2016. Salmonella p o ein A A ac i a es he STAT3 signaling pa hway
in colon cance . Neoplasia 18:307–316. h ps://doi.o g/10.1016/j.neo
.2016.04.001.
59. Kuhn KA, Manie i NA, Liu TC, S appenbeck TS. 2014. IL-6 s imula es
in es inal epi helial p oli e a ion and epai a e inju y. PLoS One
9:e114195. h ps://doi.o g/10.1371/jou nal.pone.0114195.
60. Wes e mann AJ, Ven u ini E, Sellin ME, Fö s ne KU, Ha d W-D, Vogel J.
2019. The majo RNA-binding p o ein P oQ impac s i ulence gene
exp ession in Salmonella en e ica se o a Typhimu ium. mBio 10:e02504
-18.
61. Rad ke AL, Wilson JW, Sa ke S, Nicke son CA. 2010. Analysis o in e ac-
ions o Salmonella ype h ee sec e ion mu an s wi h 3-D in es inal
epi helial cells. PLoS One 5:e15750. h ps://doi.o g/10.1371/jou nal.pone
.0015750.
62. Schweinlin M, Wilhelm S, Schwedhelm I, Hansmann J, Rie sche R,
Ju owich C, Walles H, Me zge M. 2016. De elopmen o an ad anced
p ima y human in i o model o he small in es ine. Tissue Eng Pa C
Me hods 22:873–883. h ps://doi.o g/10.1089/ en.TEC.2016.0101.
63. Nicke son CA, Goodwin TJ, Te longe J, O CM, Buchanan KL, Uicke WC,
Emami K, LeBlanc CL, Ramamu hy R, Cla ke MS, Vande bu g CR, Ham-
mond T, Pie son DL. 2001. Th ee-dimensional issue assemblies: no el
models o he s udy o Salmonella en e ica se o a Typhimu ium pa ho-
genesis. In ec Immun 69:7106–7120. h ps://doi.o g/10.1128/IAI.69.11
.7106-7120.2001.
64. Song J, Willinge T, Rong aux A, Eynon EE, S e ens S, Manz MG, Fla ell
RA, Galán JE. 2010. A mouse model o he human pa hogen Salmonella
yphi. Cell Hos Mic obe 8:369–376. h ps://doi.o g/10.1016/j.chom.2010
.09.003.
65. Ma hu R, Oh H, Zhang D, Pa k S-G, Seo J, Koblansky A, Hayden MS,
Ghosh S. 2012. A mouse model o Salmonella yphi in ec ion. Cell
151:590–602. h ps://doi.o g/10.1016/j.cell.2012.08.042.
66. Manz MG. 2007. Human-hema o-lymphoid-sys em mice: oppo uni ies
and challenges. Immuni y 26:537–541. h ps://doi.o g/10.1016/j.immuni
.2007.05.001.
67. Hansmann J, G oebe F, Kahlig A, Kleinhans C, Walles H. 2013. Bio eac o s in
issue enginee ing—p inciples, applica ions and comme cial cons ain s.
Bio echnol J 8:298–307. h ps://doi.o g/10.1002/bio .201200162.
68. B icks T, Paullie P, Legend e A, Fleu y M-J, Zelle P, Me lie F, An on PM,
Lecle c E. 2014. De elopmen o a new mic ofluidic pla o m in eg a ing
co-cul u es o in es inal and li e cell lines. Toxicol In Vi o 28:885–895.
h ps://doi.o g/10.1016/j. i .2014.02.005.
69. Maschmeye I, Hasenbe g T, Jaenicke A, Lindne M, Lo enz AK, Zech J,
Ga be L-A, Sonn ag F, Hayden P, Ayehunie S, Laus e R, Ma x U, Ma e ne
E-M. 2015. Chip-based human li e -in es ine and li e -skin co-
cul u es—a fi s s ep owa d sys emic epea ed dose subs ance es ing
in i o. Eu J Pha m Biopha m 95:77–87. h ps://doi.o g/10.1016/j.ejpb
.2015.03.002.
70. Kim HJ, Ingbe DE. 2013. Gu -on-a-chip mic oen i onmen induces hu-
man in es inal cells o unde go illus di e en ia ion. In eg Biol (Camb)
5:1130–1140. h ps://doi.o g/10.1039/c3ib40126j.
71. Saliba AE, Wes e mann AJ, Go ski SA, Vogel J. 2014. Single-cell RNA-seq:
ad ances and u u e challenges. Nucleic Acids Res 42:8845–8860.
h ps://doi.o g/10.1093/na /gku555.
72. G oebe F, Kahlig A, Lo S, Walles H, Hansmann J. 2013. A bio eac o
sys em o in e acial cul u e and physiological pe usion o ascula ized
issue equi alen s. Bio echnol J 8:308–316. h ps://doi.o g/10.1002/bio
.201200160.
73. Schul e LN, Eulalio A, Mollenkop HJ, Reinha d R, Vogel J. 2011. Analysis
o he hos mic oRNA esponse o Salmonella unco e s he con ol o
majo cy okines by he le -7 amily. EMBO J 30:1977–1989. h ps://doi
.o g/10.1038/emboj.2011.94.
74. Ran FA, Hsu PD, W igh J, Aga wala V, Sco DA, Zhang F. 2013. Genome
enginee ing using he CRISPR-Cas9 sys em. Na P o oc 8:2281–2308.
h ps://doi.o g/10.1038/np o .2013.143.
75. Li ak KJ, Schmi gen TD. 2001. Analysis o ela i e gene exp ession da a
using eal- ime quan i a i e PCR and he 2(⫺del a del a C(T)) me hod.
Me hods 25:402–408. h ps://doi.o g/10.1006/me h.2001.1262.
76. Saldanha AJ. 2004. Ja a T ee iew—ex ensible isualiza ion o mic oa ay
da a. Bioin o ma ics 20:3246–3248. h ps://doi.o g/10.1093/bioin o ma ics/
b h349.
77. Kambu o A, Wie ling C, Leh ach H, He wig R. 2009. Consensus-
Pa hDB—a da abase o in eg a ing human unc ional in e ac ion ne -
wo ks. Nucleic Acids Res 37:D623–D628. h ps://doi.o g/10.1093/na /
gkn698.
A Cocul u e Model Mimicking Human In es inal In ec ion ®
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