A icle
T iple RNA-Seq Re eals Syne gy in a Human Vi us-
Fungus Co-in ec ion Model
G aphical Abs ac
Highligh s
dT iple RNA-seq measu es gene exp ession o co-in ec ed
immune cells
dGene co ela ion ne wo ks e eal di e en hub gene se s
unde co-in ec ion
dCo-in ec ion exp ession includes syne gies and in e e ences
be ween hos and pa hogens
dMolecula basis o i al/ ungal pulmona y in ec ion has
po en ial o he clinic
Au ho s
Bas ian Seelbinde , Julia Walls abe,
Lo ha Ma ischen, ...,
Alexande J. Wes e mann,
Sascha Scha
¨uble, Jue gen Loe le
Co espondence
[email p o ec ed]e
In B ie
Seelbinde e al. demons a e
simul aneous sequencing o RNA isola ed
om human immune cells in ec ed wi h
Aspe gillus umiga us and
cy omegalo i us, wo pulmona y
pa hogens egula ly a ec ing he lungs o
immunosupp essed pa ien s. They
de ec cha ac e is ic gene exp ession
pa e ns o single and co-in ec ions and
e eal syne gis ic i ulence s a egies
be ween he wo pa hogens.
Seelbinde e al., 2020, Cell Repo s 33, 108389
No embe 17, 2020 ª2020 The Au ho s.
h ps://doi.o g/10.1016/j.cel ep.2020.108389 ll
A icle
T iple RNA-Seq Re eals Syne gy
in a Human Vi us-Fungus Co-in ec ion Model
Bas ian Seelbinde ,
1,11
Julia Walls abe,
2,11
Lo ha Ma ischen,
2,11
Es he Weiss,
2
Sebas ian Wu s e ,
2,3
Lukas Page,
2
Claudia Lo
¨ le ,
2
Lydia Busseme ,
2
Anna-Lena Schmi ,
2
Thomas Wol ,
1
Jo
¨ g Linde,
4
Luka Cicin-Sain,
5,6
Jenni e Becke ,
7
Ul ich Kalinke,
7
Jo
¨ g Vogel,
8,9
Gianni Panagio ou,
1,10
He mann Einsele,
2
Alexande J. Wes e mann,
8,9,11
Sascha Scha
¨uble,
1,11
and Jue gen Loe le
2,11,12,
*
1
Sys ems Biology and Bioin o ma ics, Leibniz Ins i u e o Na u al P oduc Resea ch and In ec ion Biology – Hans Kno
¨ll Ins i u e (HKI), 07745
Jena, Ge many
2
Uni e si y Hospi al W€
u zbu g, Medical Hospi al II, WU
¨4i, 97080 W€
u zbu g, Ge many
3
The Uni e si y o Texas MD Ande son Cance Cen e , Depa men o In ec ious Diseases, In ec ion Con ol and Employee Heal h, Hous on,
TX 77030, USA
4
F ied ich-Loe le -Ins i u , Fede al Resea ch Ins i u e o Animal Heal h, Ins i u e o Bac e ial In ec ions and Zoonoses, 07743 Jena, Ge many
5
Depa men o Vaccinology and Applied Mic obiology, Helmhol z Cen e o In ec ion Resea ch, Hanno e -B aunschweig Si e, 38124
B aunschweig, Ge many
6
Clus e o Excellence RESIST (EXC 2155), Hanno e Medical School (MHH) B aunschweig, 38124 B aunschweig, Ge many
7
Ins i u e o Expe imen al In ec ion Resea ch, TWINCORE–Cen e o Expe imen al and Clinical In ec ion Resea ch, a join en u e be ween
he Hanno e Medical School and he Helmhol z Cen e o In ec ion Resea ch, Clus e o Excellence RESIST (EXC 2155), Hanno e Medical
School (MHH), 30625 Hanno e , Ge many
8
Ins i u e o Molecula In ec ion Biology (IMIB), Uni e si y o W€
u zbu g, 97080 W€
u zbu g, Ge many
9
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), 97080 W€
u zbu g, Ge many
10
Depa men o Medicine and S a e Key Labo a o y o Pha maceu ical Bio echnology, Uni e si y o Hong Kong, Hong Kong S.A.R., China
11
These au ho s con ibu ed equally
12
Lead Con ac
*Co espondence: loe le [email protected]
h ps://doi.o g/10.1016/j.cel ep.2020.108389
SUMMARY
High- h oughpu RNA sequencing (RNA-seq) is ou inely applied o s udy di e se biological p ocesses; how-
e e , when pe o med sepa a ely on in e ac ing o ganisms, sys emic noise in insic o RNA ex ac ion, lib a y
p epa a ion, and sequencing hampe s he iden i ica ion o c oss-species in e ac ion nodes. He e, we
de elop iple RNA-seq o simul aneously de ec ansc ip omes o monocy e-de i ed dend i ic cells
(moDCs) in ec ed wi h he equen ly co-occu ing pulmona y pa hogens Aspe gillus umiga us and human
cy omegalo i us (CMV). Compa ing exp ession pa e ns a e co-in ec ion wi h hose a e single in ec ions,
ou da a e eal syne gis ic e ec s and mu ual in e e ences be ween hos esponses o he wo pa hogens.
Fo example, CMV a enua es he ungus-media ed ac i a ion o p o-in lamma o y cy okines h ough NF-kB
(nuclea ac o kB) and NFAT (nuclea ac o o ac i a ed T cells) cascades, while A. umiga us impai s i al
clea ance by coun e ac ing i al nucleic acid-induced ac i a ion o ype I in e e on signaling. Toge he , he
analy ical powe o iple RNA-seq p oposes molecula hubs in he di e en ial moDC esponse o ungal/ i al
single in ec ion o co-in ec ion ha con ibu e o ou unde s anding o he e iology and, po en ially, clea ance
o pos - ansplan in ec ions.
INTRODUCTION
Allogenic s em cell ansplan a ion (alloSCT) has ad anced he
he apy o hema ological malignancies and is po en ially cu a i e
o a spec um o nonmalignan hema ological diso de s (Singh
and McGui k, 2016). The i s success ul solid o gan ansplan a-
ion (SOT) ook place in 1954; oday, ansplan s a is ics a e
s eadily inc easing, wi h o e 36,500 o gan ansplan s in he
Uni ed S a es in 2018 (based on OPTN da a as o Janua y 9,
2019; Ha ison e al., 1956). Reduced-in ensi y condi ioning e-
gimes, no el he apeu ic s a egies o comba g a - e sus-
hos disease, and ailo ed suppo i e ca e ha e imp o ed al-
loSCT and SOT ou comes. Howe e , oppo unis ic in ec ions
a e s ill a clinical challenge and a majo sou ce o pos - ansplan
complica ions (Ullmann e al., 2016).
In asi e aspe gillosis (IA), p edominan ly causing pulmona y
in ec ions, is esponsible o signi ican pos - ansplan
mo bidi y, mo ali y, and inc emen al cos bu dens (D gona
e al., 2014). In addi ion, human cy omegalo i us (CMV)-associ-
a ed in ec ions, including CMV pneumonia, emain he mos
common in ec ious complica ions in alloSCT ecipien s (Ca-
ma go and Komandu i, 2017), and CMV i emia is associa ed
Cell Repo s 33, 108389, No embe 17, 2020 ª2020 The Au ho s. 1
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wi h inc eased ea ly o e all mo ali y a e alloSCT (G een
e al., 2016). CMV-disease- ela ed mo ali y has signi ican ly
declined wi h imp o ed p ophylac ic medica ion, PCR-based
diagnos ics, and p e-emp i e an i i al ea men , ye indi ec
CMV e ec s con inue o ad e sely impac alloSCT ou comes
(de la Ca
´ma a, 2016;Dua e and Lyon, 2018). No ably, CMV in-
ec ions pose an independen isk ac o o de elopmen o IA in
alloSCT ecipien s (Ga cia-Vidal e al., 2008;Ma e al., 2002),
and in asi e mycoses a e a equen cause o mo ali y in pa-
ien s su i ing CMV disease (de la Ca
´ma a, 2016;Nichols
e al., 2002).
Ample e idence indica es ha CMV al e s he human immune
esponse o escape hos su eillance and es ablish la en pe sis-
ence (Cheung e al., 2009;Hahn e al., 1998;Kaminski and Fish-
man, 2016;Ko enko e al., 2000;Taylo -Wiedeman e al., 1991).
Se e al p o eins encoded by CMV b oadly modula e he magni-
ude and quali y o hos immune cell unc ions (Mille -Ki ell and
Spa e , 2009). Fo example, CMV-sec e ed immunosupp essi e
cy okine homologs inhibi dend i ic cell ma u a ion and su i al
as well as dend i ic cell-media ed T-helpe (Th) cell ac i a ion
and Th1 di e en ia ion (Chang e al., 2004;Ra e y e al.,
2004), which a e c ucial mechanisms o linking inna e and adap-
i e immuni y o ungal pa hogens. Vice e sa, Aspe gillus umi-
ga us, he mos equen cause o IA, supp esses human T cell
ac i a ion in esponse o CMV (S anzani e al., 2005). Howe e ,
he molecula e en s unde lying he di e en ial impac o CMV
and A. umiga us on human mononuclea cell unc ions and he
ecip ocal immune de ense in co-in ec ions a e la gely unex-
plo ed (G ow e al., 2002;Ma ino e al., 2009;Mikulska e al.,
2009;Solak e al., 2013;Up on e al., 2007).
High- h oughpu RNA sequencing (RNA-seq) has g ea ly
ad anced ou unde s anding o in ec ions (Saliba e al., 2017;
Colgan e al., 2017) and e en allows simul aneous s udies o
hos and pa hogen ansc ip omes (Wes e mann e al., 2012).
These ‘‘dual RNA-seq’’ app oaches concu en ly isola e hos
and pa hogen RNA, con e i in o cDNA lib a ies o sequencing,
and sepa a e he ansc ip omes a he compu a ional le el by
mapping sequencing eads o he espec i e e e ence ge-
nomes. To da e, dual RNA-seq has been applied o di e se
in ec ion models o s udy i ulence mechanisms o —and
mammalian immune esponses o— i al (Ju anic Lisnic e al.,
2013;Wesolowska-Ande sen e al., 2017), bac e ial (Wes e -
mann e al., 2017), and ungal (Wol e al., 2018) pa hogens as
well as euka yo ic pa asi es (Choi e al., 2014;Pi man e al.,
2014). Howe e , mul i-o ganism RNA-seq has no p e iously
been applied o co-in ec ion se ings.
He e, we ad anced he concep o mul i-o ganism RNA-seq
by de eloping iple RNA-seq, which we applied o human
monocy e-de i ed dend i ic cells (moDCs) challenged wi h he
wo pulmona y pa hogens CMV s ain TB40 and A. umiga us.
The iden i ied modula ions o he hos ’s immunological s a e
upon single in ec ion and co-in ec ion we e independen ly
alida ed by low cy ome y and mul iplex cy okine sec e ion
assays. Ou collec i e indings sugges unique in e depen-
dencies o CMV and A. umiga us du ing co-in ec ion ha add
o ou molecula unde s anding o he syne gy be ween CMV
in ec ion and he de elopmen o in asi e mold in ec ions in
immunocomp omised pa ien s.
RESULTS
T iple RNA-Seq o Vi al/Fungal Co-in ec ions o
Simul aneously S udy Hos and Pa hogen Gene
Exp ession
In ec ion assays o human immune cells wi h ei he CMV o
A. umiga us we e p e iously es ablished (Paijo e al., 2016;
Lo he e al., 2014). He e, we buil upon hese p o ocols and
challenged moDCs wi h A. umiga us ge m ubes o CMV, ei he
sepa a ely (single in ec ion) o in combina ion (co-in ec ion). Hos
cell iabili y (Figu e S1A), in ec ion a es (Figu es S1B and S1C),
and mo phology (Figu e S1D), analyzed by low cy ome y and
luo escence mic oscopy, demons a ed sus ained in ec ions
wi h he pa hogens in bo h single- and co-in ec ion se ings.
No p ominen changes in ungal mo phology o in ec ion a e
we e obse ed be ween he wo in ec ion se ings (Figu es
S1C and S1D). In con as , we obse ed an inc eased i us in ec-
ion a e in he p esence o A. umiga us as compa ed o CMV
single in ec ion (Figu e S1B).
As a p e equisi e o mul i-o ganism RNA-seq analysis, se e al
pa ame e s—including lysis condi ions, mul iplici ies o in ec-
ions (MOIs), and e icien emo al o ibosomal RNA ( RNA)—
need o be empi ically de e mined o a gi en in ec ion sys em.
He e, we i s es ablished hese pa ame e s o single-in ec ion
se ings o moDCs wi h ei he CMV o A. umiga us (Figu es
S2A–S2C; Da a S1). Applica ion o dual RNA-seq o he esul ing
RNA samples led o he de ec ion o he p esumed in ec ion-
speci ic hos exp ession pa e ns (Figu es S2D and S2E),
including an induc ion o p o-in lamma o y hos ma ke genes
IL1A/B,CCL3, and TLR2 upon A. umiga us in ec ion (B aedel
e al., 2004;Lass-Flo
¨ l e al., 2013;Walsh e al., 2005) and
IFNB and CCL2 ac i a ion a e CMV in ec ion (Loewendo
and Benedic , 2010;McNab e al., 2015). In addi ion, al e ed
ungal exp ession le els in he p esence o moDCs (Figu e S2D),
e.g., o he glio oxin mRNA (gliF) ha encodes a myco oxin
known o be p oduced du ing hos in ec ion, as well as induced
exp ession o immunomodula o y i al mRNAs (Figu e S2E),
u he suppo ed he eliabili y o ou app oach.
To cha ac e ize all h ee ansc ip omes in sequen ial o simul-
aneous co-in ec ion se ings, we expanded he me hod owa d
iple RNA-seq (Figu e 1A). MoDCs we e ei he i s in ec ed wi h
CMV and, a e 4.5 h, addi ionally wi h A. umiga us, o i s in-
ec ed wi h A. umiga us ollowed by CMV challenge 2 h la e ,
o simul aneously in ec ed wi h bo h pa hogens. Since dual
RNA-seq da a indica ed ha majo gene exp ession changes
occu ed du ing he i s hou s a e ungal and i al exposu e,
co-in ec ion samples we e ha es ed a e 9 h and subjec ed
o iple RNA-seq. Co-in ec ion da a we e compa ed wi h an-
sc ip ome da a om single in ec ions (ha es ed in pa allel wi h
co-in ec ions) and wi h da a om unin ec ed moDCs o
A. umiga us mono-cul u es.
We ex apola ed sequencing dep h equi emen s o iple
RNA-seq o su icien ly co e all h ee ansc ip omes. Since
dual RNA-seq indica ed hos ansc ip ome co e age o be
a e limi ing (Figu es S2A and S2B), we inc eased he
sequencing dep h o iple RNA-seq by 5- old, yielding 15
million non- ibosomal human eads, a h eshold abo e which
u he inc eases in sequencing dep h ha e diminishing e u ns
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Figu e 1. T iple RNA-Seq Ou line
(A) T iple RNA-seq pipeline. Se up and ime ame o con ols, single in ec ion, and co-in ec ion a e indica ed; n = 4.
(B) Pe cen ages o sequencing eads ha mapped o he anno a ed e e ence genome o he h ee s udied o ganisms. Reads pe o ganism we e u he assigned o
he indica ed ansc ip classes. A u, Aspe gillus umiga us; moDC, monocy e-de i ed dend i ic cell; CMV, cy omegalo i us; mi oRNA, mi ochond ial RNA; miRNA,
mic oRNA; lncRNA, long noncoding RNA; snoRNA, small nucleola RNA; snRNA, small nuclea RNA; ncRNA, noncoding RNA; miscRNA, miscellaneous RNA.
Cell Repo s 33, 108389, No embe 17, 2020 3
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(Ching e al., 2014;Liu e al., 2014). Be ween 67% and 95% o
he quali y- il e ed eads (Da a S1;Da a S2) we e success ully
aligned o he e e ence genomes. While he as amoun o
eads mapped o he human genome, A. umiga us con ibu ed
up o 24% and CMV con ibu ed up o 1.6% o mapped eads
in single-in ec ion and co-in ec ion samples (Figu e 1B). As ex-
pec ed, he majo i y o human (55%) and ungal (55%–75%)
eads de i ed om mRNA. Albei ha ibosomal deple ion was
less e icien o he ungal han o he human ansc ip ome,
RNA-de i ed eads did no exceed 25% in he A. umiga us
da a subse , allowing o di e en ial gene exp ession analyses.
O he noncoding RNA classes we e adequa ely ep esen ed in
he wo euka yo ic ansc ip omes, whe eas CMV-anno a ed
genes all encode mRNAs (Figu e 1B).
Collec i ely, hese da a con i med he high echnical quali y o
he iple RNA-seq da a, wi h ela i e p opo ions o hos - o-
pa hogen ead a ios and assignmen o majo RNA classes
ma ching he p edic ions ex apola ed om he dual RNA-seq
pilo (Da a S1).
T ansc ip omes du ing Co-in ec ion Di e Globally om
Single In ec ions
Al hough p incipal-componen analysis (PCA) o moDC an-
sc ip omes e ealed no ime-poin -speci ic seg ega ion (Fig-
u e 2A), dis inc clus e s we e ob ained o single-in ec ion
condi ions wi h A. umiga us o CMV and co-in ec ion. Simila ly,
A. umiga us samples ailed o clus e acco ding o in ec ion ime
poin ye globally di e ed be ween ungal mono-cul u e and (co-)
in ec ion samples (Figu e 2B). Finally, CMV ansc ip ome p o-
iles o med clus e s o single in ec ion and co-in ec ion despi e
he compac size o he CMV genome (166 genes; Figu e 2C). In
he ollowing, due o he absence o ime-poin -speci ic seg ega-
ion, we dis ega d empo al in o ma ion and analyzed in ec ion
samples based on e iology (i.e., unin ec ed, single i al o single
ungal in ec ion, and co-in ec ion).
The iple RNA-seq app oach po en ially educes sys emic
noise ha may impede c oss-species gene exp ession co ela-
ions when sepa a ely sampling hos and pa hogen ansc ip-
omes (Wes e mann e al., 2017). To globally in es iga e in e -
species co-exp ession, we calcula ed node be weenness
and deg ee, wo me ics o quan i y co-exp ession ne wo k
complexi y and iden i y hub genes (Figu e 3;Da a S3). Focusing
on inna e immuni y-associa ed e ec s, we es ic ed he
ne wo k analysis o human genes con ained wi hin Inna eDB
(B eue e al., 2013). The i s wo ne wo ks depic ed in Figu e 3
show hos -pa hogen gene-gene co ela ions p esen in he in e -
sec ion o he espec i e single- and co-in ec ion ne wo ks,
whe eas he emaining ne wo ks e e o co ela ions in he se
di e ences be ween in ec ion se ings. Co-in ec ion ne wo ks
sha ed only ew c oss-species co ela ions wi h single-in ec ion
ne wo ks, whe eas co-in ec ion exclusi e co ela ions exhibi ed
a high deg ee o connec i i y and we e mos ly based on posi i e
co ela ions. Ve y ew co ela ions we e speci ic o single
A. umiga us in ec ion, bu ungus-hos connec i i y inc eased
du ing co-in ec ion, sugges ing he ungus o adap o—and
maybe bene i om— he p esence o CMV. In con as , he
single CMV in ec ion ne wo k consis ed o many unique, mos ly
posi i e, in e -species co ela ions. This implies a specialized
CMV esponse o human a ge cells la gely igno ing he p es-
ence o he ungus.
C oss-species exp ession co ela ion analysis can pinpoin
hos ac o s wi h po en ial as u u e bioma ke s o d ug a ge s.
CXCL11, o example, showed conside able ne wo k impo -
ance speci ically du ing co-in ec ion wi hou any ob ious co e-
la ions in single-in ec ion se ings (Da a S3). Simila ly, TNF
showed high node deg ee and be weenness, speci ically du ing
co-in ec ion, bu occupied a hub posi ion also in he in e sec ion
co ela ion ne wo k o ungal single- and co-in ec ion (Da a S3).
This hin s owa d a gene ally impo an ole o umo nec osis
ac o (TNF) in he esponse o A. umiga us, ega dless o he
addi ional p esence o CMV. RELA, in con as , possesses cen-
al ne wo k impo ance in he in e sec ion o CMV single- and
co-in ec ion (Da a S3), sugges ing a pa icula ele ance o his
ac o in he immune esponse agains CMV.
Di e en ial Gene Exp ession P o iles in he Th ee
In e ac ing O ganisms
Rela i e o unin ec ed moDCs, he e was an inc eased numbe o
di e en ially exp essed human genes upon co-in ec ion as
compa ed o ei he single in ec ion (Figu e S3A). Mo eo e , we
iden i ied speci ic moDC gene se s wi h dis inc exp ession
pa e ns be ween, bu low a iance wi hin, di e en in ec ion e i-
ologies (Figu e 4A). Fo example, TNF,IL1A,andCXCL8 we e
speci ically up egula ed upon he sensing o A. umiga us as p e-
iously epo ed (Balloy e al., 2008;Ca ey-Ca e al., 2017;Ca -
ey e al., 2015;Co ez e al., 2006;Meh ad e al., 1999;Roilides
e al., 1998). On he o he hand, induc ion o IFNB (encoding a
i s -line de ense ype I in e e on [IFN] o CMV in ec ion; Ma shall
and Geballe, 2009), CXCL10,andCXCL11 (associa ed wi h im-
mune clea ance in CMV i emia; Chee an e al., 2003;Knoblach
e al., 2011;Mu ayama e al., 2012) was speci ic o CMV in ec ion.
The majo i y o A. umiga us genes di e en ially exp essed in
he p esence o moDCs compa ed wi h he ungal mono-cul u e
we e sha ed be ween single in ec ion and co-in ec ion (Fig-
u e S3B). Fo example, he ca 1 mRNA ha encodes a ungal
ca alase o b eak down hos -de i ed hyd ogen pe oxide was
equally highly exp essed by A. umiga us du ing single- and
co-in ec ion. This sugges s ha moDCs we e he p ima y d i e
o ungal ansc ip ional ep og amming. This no wi hs anding, a
g ea e o al numbe o ungal genes we e egula ed du ing
single in ec ion han du ing co-in ec ion, he eby de ining an
A. umiga us gene se whose egula ion migh be dispensable
o in ec ion in he p esence o CMV. CMV exp ession analysis,
on he o he hand, e ealed la gely o e lapping se s o di e en-
ially exp essed i al genes du ing single in ec ion and co-in ec-
ion (Figu e S3B).
The Hos Response o Co-in ec ion Sugges s Syne gy
among A. umiga us and CMV
Close inspec ion o moDC exp ession da a iden i ied a subse o
key immune pa hways whose ac i i y di e ed ma kedly be ween
in ec ion se ings, including Toll-like ecep o (TLR) signaling, nu-
cleic acid sensing, and C- ype lec in ecep o signaling (Fig-
u e S4). In co-in ec ed moDCs, exp ession le els o AIM2 and
CCL5, ac o s in ol ed in cy osolic DNA sensing (Figu e S5)
and wi h known oles in he de ense agains bo h ungal and i al
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A
B
C
Figu e 2. PCA Iden i ies Co-clus e ing T ansc ip omes
(A–C) PCA o all h ee o ganisms a e median-by- a io no maliza ion. Ellipses depic 95% con idence in e als o condi ions. PCA o genes associa ed wi h (A)
Homo sapiens, (B) Aspe gillus umiga us, o (C) cy omegalo i us (CMV) e e ence genome. Le : PCAs o all condi ions. Righ : (A) in ec ion condi ions g ouped
independen o in ec ion ime poin , (B) da a o single in ec ion and co-in ec ion g ouped and compa ed o single-cul u e A. umiga us, and (C) da a o single
in ec ion e sus co-in ec ion independen o ime poin . Abb e ia ions a e as explained in he Figu e 1 legend.
Cell Repo s 33, 108389, No embe 17, 2020 5
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Figu e 3. In e species Co ela ion Ne wo ks Pinpoin Syne gies du ing Co-in ec ion
Ne wo ks indica e signi ican co ela ions (edges: p < 0.05; | ho| R0.95; ed indica es posi i e co ela ion; and blue indica es nega i e co ela ion) o gene
exp ession alues (nodes: g een ep esen s Homo sapiens, yellow indica es Aspe gillus umiga us, and ed indica es cy omegalo i us [CMV]) be ween
(legend con inued on nex page)
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in ec ions (Huang and Le i z, 2000;Man e al., 2016;Tyne e al.,
2005), ma ched hei cumula i e induc ion a e single in ec ions
(Figu e 4A). Howe e , a numbe o immune- ela ed moDC genes
did no display such addi i e exp ession pa e ns. Fo ins ance,
he exp ession o nuclea ac o kB (NF-kB)-dependen genes
was mainly d i en by A. umiga us h ough TNF signaling (Fig-
u e 4B). Co-in ec ion wi h CMV, howe e , educed he exp es-
sion o hose genes as compa ed o ungal single in ec ion.
Simila exp ession pa e ns we e de ec ed o IL10 and IL1B.
Vice e sa, RIG-I and ZBP1 signaling (p e iously associa ed
wi h ype I IFN esponses; Onomo o e al., 2010;Yang e al.,
2020), as well as exp ession o IFNB,CXCL10, and TLR3, dis-
played s ong ac i a ion upon CMV single in ec ion, which was
coun e ac ed by he addi ional p esence o A. umiga us (Fig-
u e 4B). The gene o apop osis-associa ed speck-like p o ein
(ASC), which is c i ical o hos cell su i al upon i al in ec ion
(Kuma e al., 2013), was highly exp essed in unin ec ed and
CMV-in ec ed moDCs. In con as , exp ession o ASC was
down egula ed in he p esence o A. umiga us, he eby mi o -
ing he exp ession pa e n o Dec in-1, encoding an impo an
ungal in ec ion senso (Taylo e al., 2007). This poin s a a
dispa a e ole o ASC in esponse o i al and ungal in ec ions,
sugges ing an inhibi ing e ec upon ungal in ec ion ha is domi-
nan o e he induc ion upon i al challenge. This hypo hesis was
u he suppo ed by he ac ha IL1B, despi e i s unc ional
connec ion o ASC (Ma inon and Tschopp, 2007), was no co-
exp essed wi h ASC. Finally, cGAS and STING, bo h encoding
ecep o s o o eign nucleic acids, and STING also unc ioning
as an IFN-s imula ing ac o associa ed wi h he i al glycop o-
ein US9 (Choi e al., 2018), we e induced upon single CMV in ec-
ion bu down egula ed upon co-in ec ion (Figu e 4B).
Taken oge he , hese exp ession da a suppo he exis ence
o wo dis inc hos esponse pa e ns o A. umiga us o CMV
in ec ion (Figu e 4C). Exp ession o IL1B, IL10, and NF-kB-asso-
cia ed genes peaked in A. umiga us-in ec ed moDCs, whe eas
cGAS, STING, and RIG-I signaling and exp ession o IFNB,
CXCL10,TLR3,and ZBP1 showed maximal exp ession upon
CMV single in ec ion. Rela i e o hei induc ion upon he espec-
i e single in ec ions, exp ession le els o all hose hos genes
d opped in co-in ec ed cells, sugges ing mu ual in e e ing e -
ec s be ween he wo hos esponses wi h possibly syne gis ic
e ec s o he wo pa hogens.
Independen Valida ion o Exp ession Changes
Quan i a i e eal- ime PCR measu emen o genes om he
h ee o ganisms ha we e di e en ially exp essed in he iple
RNA-seq da a suppo ed he sequencing-de i ed exp ession
changes (Figu e 5A; Figu e S5). Fo ins ance, di e en ial exp es-
sion o human ZBP1, ungal ca 1, and he mRNA o he i al
en elope glycop o ein UL4 in single in ec ion and co-in ec ion
se ings could be con i med in his way (Figu e 5A).
We nex aced he exp ession o selec ed hos ac o s, which
we e called as di e en ially exp essed in he RNA-seq analysis,
on he p o ein le el. Fo ins ance, as shown by low cy ome y o
an ibody-s ained moDCs, exp ession o he su ace ma ke
CCR7 was highes a e co-in ec ion, which was in line wi h
mRNA le els in he iple RNA-seq da ase (Figu e S6). Addi ion-
ally, al e ed sec e ion le els o key cy okines p oduced om
genes ha showed pa hogen-speci ic exp ession pa e ns
we e con i med by mul iplex ELISA (Figu e 5B; Figu e S7). Fo
example, he sec e ed le els o in e leukin (IL)-1band IFN-b
di e ed be ween ungal and i al in ec ions (Figu e 5B), echoing
he di e en ial exp ession o hei cogna e mRNAs in he
sequencing da a.
Al oge he , hese esul s unde pin he sequencing da a,
con i m some o he key ansc ip omic changes in he in ec ed
hos cells o ex end o he p o ein le el, and p o ide u he sup-
po o he no ion ha pulmona y pa hogens di e en ially a ec
hei hos a a global le el du ing co-in ec ion (Reese e al., 2016).
DISCUSSION
C oss-kingdom in e ac ions in polymic obial in ec ions a e
inc easingly ecognized as c ucial i ulence de e minan s ha
shape he ou come o li e- h ea ening in ec ious diseases (A a-
ni is and Mylonakis, 2015;Be ge on e al., 2017). In addi ion o
di ec physical in e ac ion and in e -kingdom signaling, al e ed
immunopa hology is conside ed o os e co-in ec ions (A ani is
and Mylonakis, 2015). Speci ically, i al pa hogens associa ed
wi h long- e m pe sis ence such as CMV ha e e ol ed an
a mamen a ium o coun e -s a egies o shape-shi he hos
en i onmen , allowing o immune su eillance e asion and
es ablishmen o la en in ec ion (F eeman, 2009;Pica da and
Benedic , 2018). Al hough e idence is limi ed, p e ious s udies
epo ed b oad al e a ions o hos immuni y elici ed by CMV in
immunocomp omised pa ien s, en ailing he p edisposi ion o
subsequen oppo unis ic ungal diseases (Yong e al., 2018).
To imp o e ou unde s anding o his co-occu ence, we he e
se ou o dissec he molecula in e play o A. umiga us, CMV,
and hei sha ed hos cells du ing co-in ec ion.
Es ablishmen o he T iple RNA-Seq App oach
To s udy he complex in e play o di e en pa hogen classes
wi h hei human hos and among each o he , we implemen ed
he p e iously p oposed concep o mul i-o ganism iple RNA-
seq (Wes e mann e al., 2017). We selec ed moDCs—being a
he bo de o inna e and adap i e immuni y—as a hos model
o es ablish his echnology o a a ie y o bo h biological and
echnical aspec s. Myeloid p ecu so s o m a ese oi o la en
CMV in ec ion and hei di e en ia ion in o dend i ic cells can
igge i us eac i a ion (Hahn e al., 1998;Ja is and Nelson,
2002), while moDCs ep esen a well-s udied su oga e model
in he con ex o ungal in ec ion (Hsieh e al., 2017;Mezge
e al., 2008;Mo on e al., 2011). Addi ionally, moDCs can be
gene a ed in la ge quan i ies by well-s anda dized p o ocols
o he up on op imiza ion o in ec ion and RNA p ocessing
o ganisms. The as e isk indica es ha co ela ions be ween CMV and A. umiga us we e emo ed. Node be weenness and deg ee a e measu es o ne wo k
complexi y. Highe deg ee indica es inc eased (local) connec i i y. Highe be weenness indica es densi y. Co esponding boxplo s show ne wo k-wise median
(cen e line), con idence in e al (boxes), and quan ile (25% and 75%) alues. # Genes and # Co ela ions display numbe s o genes and co ela ion ypes,
espec i ely.
Cell Repo s 33, 108389, No embe 17, 2020 7
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Figu e 4. De ailed Di e en ial Gene Exp ession Analysis o Monocy e-De i ed Dend i ic Cells
(A) Genes selec ed o high a iance ac oss all condi ions and low a iance wi hin indi idual condi ions ega dless o in ec ion ime poin . Le : di e en ially
exp essed genes (DEGs) wi h dis inc exp ession pa e ns ac oss expe imen al condi ions (no s imula ion, single in ec ion, and co-in ec ion). Righ : examples o
inna e immune- ele an genes allowing o clea sepa a ion o in ec ion ypes (in e ed om inna eDB; h ps://www.inna edb.com/).
(B and C) Selec ion o hos ac o s in ol ed in monocy e-de i ed dend i ic cell esponse o cy omegalo i us (CMV), Aspe gillus umiga us in ec ion, o
co-in ec ion is depic ed based on he clus e ing o simila exp ession p o iles (B) and in opological con ex (C). Topological analyses a e based on Kyo o
Encyclopedia o Genes and Genomes e e ence signaling maps (Figu e S4). Abb e ia ions a e as explained in he Figu e 1 legend.
8Cell Repo s 33, 108389, No embe 17, 2020
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT o RESOURCE SOURCE IDENTIFIER
An ibodies
an i-human CD40-PE-Vio770 ( ecombinan
human IgG1, REA ini y)
Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-110-948, Clone: monoclonal
REA733; RRID:AB_2658003
an i-human CD80-APC ( ecombinan human
IgG1, REA ini y)
Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-117-719, Clone: monoclonal
REA661; RRID:AB_2751414
an i-human CD209-VioBlue( ecombinan
human IgG1, REA ini y)
Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-110-456, Clone: monoclonal
REA617; RRID:AB_2656252
an i-human CCR7-VioBlue( ecombinan
human IgG1, REA ini y)
Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-117-353, Clone: monoclonal
REA546; RRID:AB_2733933
an i-human TLR2-PE-Vio770 ( ecombinan
human IgG1, REA ini y)
Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-099-022, Clone: monoclonal
REA109; RRID:AB_2656975
an i-human TLR3-APC (mouse IgG1k) Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-096-885, Clone: monoclonal
TLR3.7; RRID:AB_2660004
Bac e ial and Vi us S ains
human CMV s ain TB40/E-mNeonG een Helmhol z Cen e o In ec ion
Resea ch, B aunschweig, Ge many
Kasmapou e al., 2017
h ps://www.helmhol z-hzi.de
human CMV s ain TB40/SE Uni e si y F eibu g, Medical Cen e ,
Ins i u e o Vi ology, F eibu g,
Ge many; Sampaio e al., 2017
h ps://www.uniklinik- eibu g.de/de.h ml
Biological Samples
Human pe iphe al enous blood om heal hy
adul dono s o gene a ion o monocy e-de i ed
dend i ic cells
Paijo e al., 2016;Sallus o and
Lanza ecchia, 1994
N/A
Chemicals, Pep ides, and Recombinan P o eins
RNAp o ec Cell Reagen QIAGEN, Hilden, Ge many Ca #76526
Viobili y 405/520 Fixable Dye Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-109-814
iTaq Uni e sal SYBRG een Supe mix Bio-Rad Labo a o ies GmbH,
Feldki chen, Ge many
Ca #1725124
Recombinan human IL-4, p emium g ade Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-093-922
Recombinan human GM-CSF,p emium g ade Mil enyi Bio ec, Be gisch Gladbach,
Ge many
Ca #130-093-866
C i ical Comme cial Assays
RiboPu e-Yeas Ki The mo Fishe Scien i ic, Wal ham,
MA, USA
Ca #AM1926
DNase I, RNase ee (1.000 U) The mo Fishe Scien i ic, Wal ham,
MA, USA
Ca #EN0521
M-MLV e e se ansc ip ase In i ogen, Ca lsbad, CA, USA Ca #28025013
cDNA Fi s S and Syn hesis Ki The mo Fishe Scien i ic, Wal ham,
MA, USA
Ca #K1612
Ribo-Ze o Gold RNA emo al ki (human,
mouse, a ) (24 eac ions)
Illumina, San Diego, CA, USA Ca #MRZG12324
P oca aPlex 16-plex Immunoassay (IFN-a, IFN-b,
IFN-g, IL-1a,IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12p70,
IL-17A, IL-23, CXCL10, CXCL11, CCL5, TNF-a)
The mo Fishe Scien i ic, Wal ham,
MA, USA
Ca #PPX-16, cus omized
(Con inued on nex page)
e1 Cell Repo s 33, 108389, No embe 17, 2020
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Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
Deposi ed Da a
T iple RNA-seq, A. umiga us, CMV and H. sapiens h ps://www.ncbi.nlm.nih.go /geo/ GSE134344
Dual RNA-seq A. umiga us and H. sapiens h ps://www.ncbi.nlm.nih.go /geo/ GSE135450
Dual RNA-seq CMV and H. sapiens h ps://www.ncbi.nlm.nih.go /geo/ GSE136217
Expe imen al Models: O ganisms/S ains
Aspe gillus umiga us (wild ype) ATCC ATCC: 46645
Aspe gillus umiga us dToma o ATCC; Lo he e al., 2014 ATCC: 46645
Aspe gillus umiga us GFP ATCC; Lo he e al., 2014 ATCC: 46645
Oligonucleo ides
See Table S1 o p ime sequences (ALAS1, IL15,
CCR7, ZBP1, VEGFA, IFNG, TLR3, CD40, GliT, GliZ,
Il 3, Ca 1, S bA, UL4, UL8, UL20, US16, UL9, US9)
This pape N/A
So wa e and Algo i hms
CellQues P o So wa e (Bec on & Dickinson) h ps://www.bd.com/en-us Bec on & Dickinson, F anklin Lakes,
NJ, USA
FACSDi a So wa e h ps://www.bd.com/en-us Bec on & Dickinson, F anklin Lakes,
NJ, USA
FlowJo So wa e, 10 h ps://www. lowjo.com;
h ps://www.bd.com/en-us
T ees a / Bec on & Dickinson, Ashland,
OR, USA
FCS Exp ess So wa e, 7 h ps://deno oso wa e.com De No o So wa e, Pasedena, CA, USA
NIS Elemen s Imaging so wa e, 5.02.00 h ps://www.mic oscope.
heal hca e.nikon.com
Nikon Ins umen s, Ams e dam,
Ne he lands
Rh ps://c an. -p ojec .o g/ 3.5.1
ea u eCoun s h ps://c an. -p ojec .o g/ Rsub ead 1.28.0
DESeq h ps://bioconduc o .o g 1.30.0
DESeq2 h ps://bioconduc o .o g 1.18.1
limma oom h ps://bioconduc o .o g 3.34.6
edgeR h ps://bioconduc o .o g 3.20.7
geo2RNaseq h ps://singula i y-hub.o g/accoun s/
login/?nex =/collec ions/4387;
h ps://bi bucke .o g/Xen ics/
geo2 naseq/s c/mas e /
0.9.12
Fas QC h ps://www.bioin o ma ics.
bab aham.ac.uk/p ojec s/ as qc/
0.11.8
T immoma ic h p://www.usadellab.o g/cms/?
page= immoma ic
0.36
HiSa 2 h ps://daehwankimlab.gi hub.io/
hisa 2/
2.1.0
SAM ools h ps://www.h slib.o g/ 1.7
Mul iQC h ps://mul iqc.in o/ 1.5
Jupy e h ps://jupy e .o g/ 4.4.0
Tidy e se h ps://c an. -p ojec .o g/ 1.2.1
Mag i h ps://c an. -p ojec .o g/ 1.5.0
Reshape2 h ps://c an. -p ojec .o g/ 1.4.4
ggplo 2 h ps://c an. -p ojec .o g/ 3.1.1
Ggpub h ps://c an. -p ojec .o g/ 0.2.5
Ggsci h ps://c an. -p ojec .o g/ 2.9
Dply h ps://c an. -p ojec .o g/ 0.7.6
Tidy h ps://c an. -p ojec .o g/ 1.3.1
S ing h ps://c an. -p ojec .o g/ 1.3.0
(Con inued on nex page)
Cell Repo s 33, 108389, No embe 17, 2020 e2
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RESOURCE AVAILABILITY
Lead Con ac
Fu he in o ma ion and eques s o esou ces and eagen s should be di ec ed o and will be ul illed by he Lead Con ac , Jue gen
Loe le ([email p o ec ed]).
Ma e ials A ailabili y
All p ima y ma e ial gene a ed in his s udy will be made a ailable upon eques ollowing publica ion. A comple ed Ma e ials T ans e
Ag eemen migh be necessa y, especially i he e is po en ial o comme cial applica ion.
Da a and Code A ailabili y
All p ima y sequencing da a and p ocessed da a desc ibed in his manusc ip ha e been deposi ed in he NCBI Gene Exp ession
Omnibus unde he accession numbe s GEO: GSE134344, GSE135450 and GSE136217.
Code o p ep ocessing RNA sequencing da a and analysis is deposi ed and a ailable a h ps://gi hub.com/SchSascha/
manusc ip _ ipleRNAseq.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Heal hy blood dono s we e exclusi ely in he age be ween 18-59 yea s and o he wise excluded om p o iding blood samples. Bo h
sexes we e included in he s udy equally and a andom; he sex o he blood dono s was anonymised and ep esen s he no mal
dis ibu ion wi hin his popula ion.
E hics S a emen
The p ocessing o human pe iphe al enous blood om heal hy adul dono s was app o ed by he E hical Commi ee o he Uni e -
si y Hospi al W€
u zbu g (#302/12).
METHOD DETAILS
P ima y cell isola ion and di e en ia ion
To gene a e moDCs, monocy es we e isola ed om leuko educ ion sys em chambe s con aining blood om heal hy olun ee s and
s anda d densi y-g adien cen i uga ion ollowed by posi i e selec ion using magne ic-ac i a ed cell so ing (CD14 Mic oBeads, hu-
man, Mil enyi Bio ec). Monocy es we e cul u ed in CellGenix GMP dend i ic cell medium (se um- ee, CellGenix) supplemen ed wi h
120 mg gen amicin (Re obacin, Me ck) in 24-well pla es wi h 1 310
6
cells/ml. Cells we e di e en ia ed o 6 days by addi ion o
1,000 U/ml g anulocy e mac ophage-colony s imula ing ac o (Mil enyi Bio ec) and 1,000 U/ml in e leukin (IL)-4 (Mil enyi Bio ec)
(Mezge e al., 2008;Paijo e al., 2016;Sallus o and Lanza ecchia, 1994).
Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
Anno a ionDbi h ps://bioconduc o .o g 1.44.0
o g.Hs.eg.db h ps://bioconduc o .o g 3.10.0
se Rank h ps://c an. -p ojec .o g/ 1.1.0
Pa h iew h ps://bioconduc o .o g 1.26.0
Phea map h ps://c an. -p ojec .o g/ 1.0.12
ig aph h ps://c an. -p ojec .o g/ 1.2.5
W i eXLS h ps://c an. -p ojec .o g/ 5.0.0
co plo h ps://c an. -p ojec .o g/ 0.84
Rcolo B ewe h ps://c an. -p ojec .o g/ 1.1-2
en ichR h ps://c an. -p ojec .o g/ 2.1.0
cowplo h ps://c an. -p ojec .o g/ 1.1.0
O he
H. sapiens e e ence genome h ps://www.ncbi.nlm.nih.go / GRCH 38 89
A. umiga us A 293 e e ence genome h p://www.aspe gillusgenome.o g/ s03-m05- 09
CMV e e ence genome h ps://www.ncbi.nlm.nih.go / EF999921.1
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Pulmona y pa hogens
Repo e s ain human CMV TB40/E-mNeonG een and CMV TB40/SE (non- luo escen ) we e gene a ed acco ding o published
p o ocols (Kasmapou e al., 2017;Paijo e al., 2016;Sampaio e al., 2017). A. umiga us (ATCC 46645) ge m ubes we e p epa ed
o e nigh in RPMI (In i ogen) and used o dual o iple RNA-seq. Fo low cy ome y and mic oscopy (see below), dToma o- o
GFP-exp essing A. umiga us ge m ubes we e used.
moDC in ec ion assays
P ima y human cells we e in ec ed wi h CMV a a mul iplici y o in ec ion (MOI) 3 and wi h subsequen cen i ugal enhancemen a 300
xg o 30 min. A. umiga us ge m ubes, which ep esen an in asi e immunogenic mo pho ype o he ungus, we e added a MOI 0.5.
Fo co-in ec ions, pa hogens we e added simul aneously o subsequen ly (Figu e 1A). A e ha es ing, cells we e cen i uged and
pelle s collec ed in HBSS (In i ogen) o RNAp o ec Cell Reagen (QIAGEN), and cell- ee supe na an s we e s o ed a 80C.
Fo low cy ome y and mic oscopy, (see below) moDCs we e in ec ed wi h CMV o 24 h be o e adding ge m ubes, because
de ec ion o NeonG een luo escence in in ec ed cells equi es su icien eplica ion o he i us.
Flow cy ome y analysis
CMV and A. umiga us in ec ion a es we e de e mined by low cy ome y, measu ing luo escen signals o moDCs posi i e o
mNeonG een (CMV) o GFP (A. umiga us), espec i ely (Figu es S1B and S1C). Viabili y o moDCs was de e mined by s aining
wi h Viobili y 405/520 Fixable Dye (Mil enyi Bio ec), de ining nega i e popula ions as iable. Addi ionally, cells we e s ained wi h
an i-CD40-PE-Vio770 (REA733, Mil enyi Bio ec), an i-CD80-APC (REA661, Mil enyi Bio ec), an i-CD209-VioBlue, (REA617, Mil enyi
Bio ec), an i-CCR7-VioBlue (REA546, Mil enyi Bio ec), an i-TLR2-PE-Vio770 (REA109, Mil enyi Bio ec) o an i-TLR3-APC (TLR3.7,
Mil enyi Bio ec). Mean luo escence in ensi ies o hese su ace ma ke s we e measu ed and iso ypes sub ac ed. Da a
we e acqui ed on a FACSCalibu using CellQues So wa e (Bec on & Dickinson) o FACSCan o II using FACSDi a so wa e
(Bec on & Dickinson). Da a analysis was done wi h FlowJo (T ees a /Bec on & Dickinson, e sion 10) o FCS Exp ess 7 (De No o
So wa e).
Mic oscopy
Mo phology and luo escence signals o CMV (mNeonG een) o A. umiga us (dToma o) we e analyzed in 48-well cell cul u e pla es
using a Nikon Eclipse Ti mic oscope (Nikon) wi h an Okolab incuba o se a 37C. Images we e ob ained a 20- old magni ica ion and
p ocessed using NIS Elemen s Imaging so wa e (Nikon, e sion 5.02.00).
Es ablishmen o op imized RNA-seq condi ions o single in ec ions
As a p e equisi e o mul i-o ganism RNA-seq analysis in ou in ec ion model, lysis condi ions should be su icien ly ha sh o dis up
cellula memb anes o all in e ac ing o ganisms, bu su icien ly mild o main ain high RNA in eg i y. Addi ionally, mul iplici ies o
in ec ion (MOIs) mus be adjus ed o ensu e homogeneous co e age o indi idual ansc ip omes in he esul ing sequencing
da a. Tha is, he ela i e p opo ions o ansc ip omes in isola ed RNA samples should ma ch he a io o he espec i e genome
sizes. The e o e, we i s e alua ed yield and quali y o RNA isola ed om moDCs in ec ed wi h ei he A. umiga us o CMV a di e en
ime poin s and MOIs, and obse ed high RNA in eg i y (RIN > 7) o all condi ions (Figu es S2A and S2B).
While highly abundan in any o ganism (> 90% o o al cellula RNA), ibosomal RNA ( RNA) p o ides li le in o ma i e alue abou
cellula physiology. The e o e, ibosomal ansc ip s a e ypically deple ed om sequencing lib a ies, ei he by ac i e RNA pull-ou
(e.g., Ribo-Ze o echnology) o en ichmen o polyadenyla ed ansc ip s. In p inciple, bo h op ions a e sui able o ou in ec ion
model, since mRNAs o all h ee in e ac ing o ganisms a e polyadenyla ed. We ound ha bo h app oaches e icien ly deple ed
human ibosomal eads (Figu e S2C). Howe e , o e ain po en ially in e es ing non-polyadenyla ed ansc ip s (e.g., mic oRNAs
[miRNAs], small nucle(ol)a RNAs [snRNAs, snoRNAs] and polyA- long noncoding RNAs [lncRNAs]; Figu e S2C), we employed
Ribo-Ze o echnology o u he RNA-seq expe imen s.
A e RNA emo al om single-in ec ion samples, cDNA lib a ies we e p epa ed and sequenced o shallow dep h (5-8 million
eads/lib a y) o ini ial quali y assessmen . Ob ained sequencing eads aligned o hei pa en al e e ence genome wi h li le
c oss-mapping obse ed (Figu es S2A and S2B). In ac , he as majo i y o c oss-mapped eads de i ed om mi ochond ial genes
p esen in bo h Aspe gillus and human cells, and all o hese eads we e emo ed om u he analyses. As expec ed, he ungal- o-
human ead a io in Aspe gillus-in ec ed samples inc eased wi h MOI. In line wi h he high p opo ions o ungal eads (20% o o al
mapped eads), human exon co e age was he a e-limi ing ac o (Figu e S2A), hus a o ing he low-dose in ec ions (MOI 0.5) o
u he expe imen s. While he as majo i y o eads in CMV-in ec ed moDCs mapped o he human genome, i al ead p opo ion
and exon co e age inc eased ime-dependen ly, indica i e o i al eplica ion (Figu e S2B).
E en wi h he low sequencing dep h used in his pilo expe imen , he induc ion o ma ke genes o human dend i ic cell ac i a ion
and pa hogenici y- ela ed ungal genes was de ec ed (Figu e S2D). Fo example, up egula ion o IL-1, CCL3, and TLR2 indica ed
ac i a ion o well-desc ibed p o-in lamma o y cascades in moDCs in ec ed wi h Aspe gillus (B aedel e al., 2004;Lass-Flo
¨ l e al.,
2013;Walsh e al., 2005). Fungal cells also showed ele a ed exp ession o genes o oxic molecules such as he glio oxin GliF (La ge
´,
1999). Simila ly, exp ession p o iles du ing i us in ec ion e lec ed expec ed pa e ns (Figu e S2E) as CMV-in ec ed moDCs up egu-
la ed IFN-gand CCL2 (Loewendo & Benedic , 2010;McNab e al., 2015), whe eas i al gene exp ession was gene ally induced o e
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ime (Figu e S2E displays i al exp ession a wo la e s ages ela i e o an ea ly in ec ion s age) indica ing ac i e p oli e a ion (Dunn
e al., 2003;Mou a si e al., 2002;Wie z e al., 1996).
RNA ex ac ion, RNA deple ion, cDNA lib a y p epa a ion, and iple RNA-seq
Fo ex ac ion o human, ungal and i al RNA, RiboPu e RNA Pu i ica ion Ki yeas (The mo Fishe Scien i ic) was used acco ding o
he manu ac u e ’s ins uc ions. To al RNA was ea ed wi h 0.13 U/ml DNase I (The mo Fishe Scien i ic) o 30 min a 37C o emo e
con amina ing genomic DNA. The in eg i y o DNase- ea ed RNA was assessed on a bioanalyze (Agilen ). All samples had RNA
in eg i y numbe s (RIN) R7.0. Whe e explici ly indica ed (as polyA
+
), o al RNA was di ec ly con e ed in o i s -s and cDNA using
an oligo(dT)
25
p ime , agmen ed ( ou 30 s ul asound pulses), and p ocessed as ou lined below. O he wise, ibosomal ansc ip s
we e ac i ely emo ed using Ribo-Ze o Gold RNA emo al ki s (human, mouse, a ) (Illumina) ollowing manu ac u e ’s ins uc ions
o 500 ng DNase- ea ed RNA as inpu o RNA deple ion.
The cDNA lib a ies o Illumina sequencing we e gene a ed by Ve is Bio echnologie AG, F eising-Weihens ephan, Ge many, wi h
RNA- ee RNA shea ed ia ul asound sonica ion ( ou 30 s pulses, 4C) o gene a e 200- o 400-nucleo ide agmen s, on a e age.
F agmen s < 20 nucleo ides we e emo ed using Agencou RNAClean XP ki s (Beckman Coul e Genomics), and Illumina T uSeq
adap e s we e liga ed o he 30ends o emaining agmen s. Fi s -s and cDNA syn hesis was pe o med using M-MLV e e se an-
sc ip ase (NEB) wi h 30adap e s used as p ime a ge si es. Fi s -s and cDNA was pu i ied, and 50Illumina T uSeq sequencing
adap e s we e liga ed o 30ends o an isense cDNA. Resul ing cDNA was PCR-ampli ied o abou 10 o 20 ng/ml using high- ideli y
DNA polyme ase. T uSeq ba code sequences we e included in 50and 30T uSeq sequencing adap e s. The cDNA lib a ies we e pu-
i ied using Agencou AMPu e XP ki s (Beckman Coul e Genomics) and analyzed by capilla y elec opho esis (Shimadzu Mul iNA
mic ochip).
Fo sequencing, cDNA lib a ies we e pooled in app oxima ely equimola amoun s. Pools we e size- ac iona ed o 200-600 bp us-
ing di e en ial cleanup wi h Agencou AMPu e ki s (Beckman Coul e Genomics). Aliquo s o cDNA pools we e analyzed by capilla y
elec opho esis (Shimadzu Mul iNA mic ochip). Sequencing was pe o med on a Nex Seq 500 pla o m (Illumina) a Ve is Bio-
echnologie AG, F eising-Weihens ephan, Ge many (single-end mode, 75 cycles).
Quan i a i e e e se ansc ip ion PCR-based alida ion o di e en ial gene exp ession
RNA o un ea ed moDCs o moDCs a e in ec ion wi h CMV, A. umiga us o bo h was e e se ansc ibed in o cDNA using The mo
Fishe cDNA Fi s S and Syn hesis Ki s. P ime s (Sigma-Ald ich) we e designed using P ime -Blas (Ye e al., 2012) and The Aspe -
gillus Genome Da abase (Ce quei a e al., 2014), a oiding he occu ence o a ge sequences in he o he wo o ganisms (Table S1).
Quan i a i e e e se ansc ip ion (qRT)-PCR was conduc ed using SYBRG een Mas e Mix om (BioRad) in a S ep One Sys em
(Applied Biosys ems). P ime speci ici y was con i med by aga ose (Ro h) gel elec opho esis (Se a) o PCR amplicons using
e hidium b omide (The mo Fishe ). Gel images we e documen ed in a Mul i-Image Ligh Cabine (Alpha Inno ech).
Mul iplex cy okine sec e ion assays
Cell cul u e supe na an s we e analyzed by mul iplex cy okine sec e ion assays acco ding o he manu ac u e ’s ins uc ions using
16-plex P oca aPlex Immunoassays (The mo Fishe Scien i ic) including IFN-a, IFN-b, IFN-g, IL-1a, IL-1b, IL-2, IL-6, IL-8, IL-10,
IL-12p70, IL-17A, IL-23, CXCL10, CXCL11, CCL5, and TNF-a.
Cy okines wi h concen a ion le els abo e measu emen ange we e se o 1.05 imes he maximum. Concen a ion le els below
measu emen ange o nega i e concen a ions we e se o 0. Signi ican changes in he concen a ion o cy okines we e de e mined
using pai wise wo-sided Wilcoxon ank-sum es s. P alues o each es we e adjus ed o mul iple es ing using FDR. We ejec ed
he null hypo hesis o FDR < 0.1.
QUANTIFICATION AND STATISTICAL ANALYSIS
RNA-seq da a p ocessing
P ep ocessing o aw eads including quali y con ol and gene abundance es ima ion was done wi h GEO2RNaseq pipeline e sion
0.9.12 in R e sion 3.5.1 (Seelbinde e al., 2019). Quali y analysis was done wi h Fas QC e sion 0.11.8 be o e and a e imming.
Read-quali y imming was done wi h T immoma ic e sion 0.36. Adap o sequences we e emo ed, window size imming pe -
o med (15 nucleo ides, a e age Q < 25) including 50and 30pe -base imming o Q < 3 and emo al o sequences sho e han
30 nucleo ides. The e e ence genome in FASTA o ma was c ea ed by combining e e ences Homo sapiens (GRCH 38 89),
A. umiga us A 293 (s03-m05- 09) and human he pes i us 5 s ain TB30/E clone TB40-BAC4 (EF999921.1). Re e ence anno a ion
was c ea ed by ex ac ing and combining exon ea u es om co esponding anno a ion iles. The e e ence genome was indexed
wi h exon in o ma ion using HiSa 2 e sion 2.1.0. Pai ed-end ead alignmen used HiSa 2 on he c ea ed e e ence genome. Only
conco dan ly aligned pai s o eads we e used. Mapping s a is ics pe o ganism we e calcula ed using he ‘‘calc_ iple_mapping_
s a s’’ unc ion o GEO2RNaseq. SAM ools e sion 1.7 wi h he ‘‘ lags a ’’ subcommand was used o deduce alignmen quali y.
Gene abundance es ima ion was done wi h ea u eCoun s (Rpackage Rsub ead e sion 1.28.0) in pai ed-end mode wi h de aul pa-
ame e s. Mul iQC e sion 1.5 was used o summa ize he ou pu o Fas QC, T immoma ic, HiSa , ea u eCoun s and SAM ools (Da a
S1). In addi ion o he coun ma ix wi h gene abundance o all h ee species, species-speci ic gene coun ma ices we e ex ac ed
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om he comple e coun ma ix. Fo nons a is ical analyses, coun ma ices we e no malized using median-by- a io no maliza ion
(MRN) as desc ibed be o e (Ande s and Hube , 2010). Co ela ion analysis, p incipal componen analysis, and clus e ing we e pe -
o med pe species based on MRN gene-abundance da a. Clus e ing be ween samples used he comple e linkage ( a hes
neighbo ) me hod. Raw iles a e accessible unde he Gene Exp ession Omnibus accession numbe GSE134344, GSE135450
and GSE136217.
Di e en ial gene exp ession analysis
Di e en ial gene exp ession was analyzed by GEO2RNaseq pe species. Pai wise es s we e pe o med be ween con ol, single-
in ec ion, and co-in ec ion g oups wi h and wi hou conside a ion o in ec ion ime. Fou s a is ical ools (DESeq 1.30.0, DESeq2
1.18.1, limma oom 3.34.6 and edgeR 3.20.7) we e used, and p alues we e co ec ed o mul iple es ing using he alse-disco e y
a e me hod q = FDR(p) o each ool. In addi ion, mean MRN, ansc ip s pe kilobase million (TPKM) and eads pe kilobase million
(RPKM) alues we e compu ed pe es pe g oup including co esponding log
2
old-changes. Gene exp ession di e ences we e
conside ed signi ican i hey we e epo ed signi ican by all ou ools (q < 0.01 and |log
2
MRN| R1 o H. sapiens; q < 0.05 o
A. umiga us and CMV).
To compa e RNA-seq and qRT-PCR de i ed ela i e di e ences, exp ession alues we e scaled by he maximum alue pe epli-
ca e. To keep s a is ical analysis compa able, signi icance was assessed based on pai wise es s. Resul ing p alues we e co -
ec ed o mul iple es ing using FDR. Tes s we e pe o med indi idually o RNA-seq and qRT-PCR pe gene (Figu e 5;Figu e S5).
In e species and in aspecies gene exp ession analyses
Abundances o genes wi h nonze o co e age om all h ee species and all samples we e MRN no malized. Spea man’s co ela ions
be ween gene abundances we e calcula ed pe ea men g oup (moDC, moDC + A. umiga us [A u], moDC + CMV, moDC + CMV +
A u) o in ec ion ime 0 h. Only signi ican co ela ions wi h p < 0.01 and absolu e co ela ion R30% we e used o u he analysis.
Rpackage iG aph e sion 1.2.4.1 was used o c ea e, compa e, analyze (node deg ee and be weenness) and plo signi ican
co ela ions as ne wo ks. Node deg ee cen ali y desc ibes he numbe o edges inciden o a node. Node be weenness cen ali y
desc ibes he numbe o sho es pa hs h ough a node o all pai s o nodes. Fo c oss-species analysis, co ela ions be ween genes
o he same species we e igno ed. Fo addi ional immune sys em ele an gene co ela ion analysis, genes om H. sapiens we e
e ained only i hey we e p esen in he cu a ed da abase Inna eDB (h ps://www.inna edb.com/). moDC gene se analysis wi h
dis inc exp ession pa e ns be ween, bu low a iance wi hin di e en in ec ion e iologies yielded 160 genes ha we e u he il e ed
o immune sys em- ele an genes (Figu e 4).
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