Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

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 This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). ll OPEN ACCESS 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 2Cell Repo s 33, 108389, No embe 17, 2020 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS (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 4Cell Repo s 33, 108389, No embe 17, 2020 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS 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) 6Cell Repo s 33, 108389, No embe 17, 2020 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS 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). 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Cell Repo s 33, 108389, No embe 17, 2020 15 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS 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 e3 Cell Repo s 33, 108389, No embe 17, 2020 A icle ll OPEN ACCESS 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 Cell Repo s 33, 108389, No embe 17, 2020 e4 A icle ll OPEN ACCESS 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 e5 Cell Repo s 33, 108389, No embe 17, 2020 A icle ll OPEN ACCESS 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). Cell Repo s 33, 108389, No embe 17, 2020 e6 A icle ll OPEN ACCESS