Host Genetic Background Strongly Affects Pulmonary microRNA Expression before and during Influenza A Virus Infection.

Ma ch 2017 | Volume 8 | A icle 2461 O iginal esea ch published: 21 Ma ch 2017 doi: 10.3389/ immu.2017.00246 F on ie s in Immunology | www. on ie sin.o g Edi ed by: Wenzhe Ho, Temple Uni e si y School o Medicine, USA Re iewed by: Dane Pa ke , Columbia Uni e si y, USA Debash ee Cha e jee, Memo ial Sloan Ke e ing Cance Cen e , USA *Co espondence: F ank Pessle [email p o ec ed], [email p o ec ed] †P esen add ess: Ma hias P eusse, Depa men o Molecula Bac e iology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many Special y sec ion: This a icle was submi ed o Mic obial Immunology, a sec ion o he jou nal F on ie s in Immunology Recei ed: 03No embe 2016 Accep ed: 20Feb ua y2017 Published: 21Ma ch2017 Ci a ion: P eusseM, Schugha K and Pessle F (2017) Hos Gene ic Backg ound S ongly A ec s Pulmona y mic oRNA Exp ession be o e and du ing In luenza A Vi us In ec ion. F on . Immunol. 8:246. doi: 10.3389/ immu.2017.00246 hos gene ic Backg ound s ongly a ec s Pulmona y mic o na exp ession be o e and du ing in luenza a Vi us in ec ion Ma hias P eusse1,2†, Klaus Schugha 3,4,5 and F ank Pessle 1,2,6* 1 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, Hanno e , Ge many, 2 Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 3 Depa men o In ec ion Gene ics, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 4 Uni e si y o Ve e ina y Medicine Hanno e , Hanno e , Ge many, 5Depa men o Mic obiology, Immunology and Biochemis y, Uni e si y o Tennessee Heal h Science Cen e, Memphis, TN, USA, 6 Cen e o Indi idualised In ec ion Medicine, Hanno e , Ge many Backg ound: Exp ession o hos mic oRNAs (miRNAs) changes ma kedly du ing in lu- enza A i us (IAV) in ec ion o na u al and adap i e hos s, bu hei ole in gene ically de e mined hos suscep ibili y o IAV in ec ion has no been explo ed. We, he e o e, compa ed pulmona y miRNA exp ession du ing IAV in ec ion in wo inb ed mouse s ains wi h di e en ial suscep ibili y o IAV in ec ion. esul s: miRNA exp ession p o iles we e de e mined in lungs o he mo e sus- cep ible s ain DBA/2J and he less suscep ible s ain C57BL/6J wi hin 120h pos in ec ion (hpi) wi h IAV (H1N1) PR8. E en he miRNomes o unin ec ed lungs di e ed subs an ially be ween he wo s ains. A e a pe iod o ela i e quiescence, majo miRNome ep og amming was de ec ed in bo h s ains by 48 hpi and inc eased h ough 120 hpi. Dis inc g oups o miRNAs egula ed by IAV in ec ion could be de ined: (1) miRNAs (n=39) whose exp ession co ela ed wi h hemagglu inin (HA) mRNA exp ession and ep esen ed he gene al esponse o IAV in ec ion indepen- den o hos gene ic backg ound; (2) miRNAs (n=20) whose exp ession co ela ed wi h HA mRNA exp ession bu di e ed be ween he wo s ains; and (3) ema kably, miR-147-3p, miR-208b-3p, miR-3096a-5p, miR-3069b-3p, and he miR-467 am- ily, whose abundance e en in unin ec ed lungs di e en ia ed nea ly pe ec ly (a ea unde he ROC cu e>0.99) be ween he wo s ains h oughou he ime cou se, sugges ing a pa icula ly s ong associa ion wi h he di e en ial suscep ibili y o he wo mouse s ains. Exp ession o subse s o miRNAs co ela ed signi ican ly wi h pe iphe al blood g anulocy e and monocy e numbe s, pa icula ly in DBA/2J mice; miR-223-3p, miR-142-3p, and miR-20b-5p co ela ed mos posi i ely wi h hese cell ypes in bo h mouse s ains. Highe abundance o an iapop o ic (e.g., miR-467 amily) and lowe abundance o p oapop o ic miRNAs (e.g., miR-34 amily) and hose egula ing he PI3K-Ak pa hway (e.g., miR-31-5p) we e associa ed wi h he mo e suscep ible DBA/2J s ain. conclusion: Subs an ial di e ences in pulmona y miRNA exp ession be ween he wo di e en ially suscep ible mouse s ains we e e iden e en be o e in ec ion, bu e ol ed 2 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 inT ODUcTiOn mic oRNAs (miRNAs) a e sho egula o y RNA molecules ha occu in mos euka yo ic cells and may ha e p o ound e ec s on a b oad ange o biological p ocesses, including apop osis, de elop- men , di e en ia ion, p oli e a ion, and immune esponses (1–4). I , he e o e, comes as no su p ise ha majo changes in miRNA exp ession and ac i i y ha e been shown o occu as pa o he hos cell esponse o in ec ion by a a ie y o i uses (5–7). Inc easing a en ion has been paid o he ole(s) o miRNAs in in luenza i us in ec ion, wi h mos s udies ha ing ocused on in luenza A i us (IAV). S udies ha e in ol ed cells o issues om na u al hos s, such as humans (8–10), bi ds (11, 12), pigs (13), and macaques (14) and mice as adap i e hos s (15, 16). Majo insigh s ha e been gained by compa ing di e ences in he miRNome esponses o IAV s ains o di e en ial pa hogenici y (14–17) o by compa - ing e ec s o di e en hos backg ounds (16). The impo ance o some o he di e en ially exp essed miRNAs has also been alida ed expe imen ally [e.g., Buggele e al. (18)], hus unde sco - ing ha miRNAs may subs an ially in luence he cou se o IAV in ec ion (19). Howe e , in spi e o his e e g owing in e es in he in e ac ions be ween IAV and he hos miRNome, he e ha e been no s udies in es iga ing di e ences in he miRNA esponse in hos s o well cha ac e ized di e en ial gene ic suscep ibili y o IAV. This is all he mo e su p ising since ecen wo k wi h inb ed mouse s ains o di e en ial suscep ibili y o IAV in ec ion clea ly showed ha he ansc ip omic (mRNA) esponse o IAV is o a g ea ex en de e mined gene ically (20, 21). I is he e o e qui e plausible ha majo gene ic di e ences migh also mani es a he le el o he miRNome’s esponse o IAV in ec ion and ha he desc ip ion o his miRNome will enable iden i ica ion o egula- o y miRNA–mRNA ne wo ks ha a e c i ical o success ul hos de ense agains IAV in ec ion and, pe haps, ela ed i uses. Based on a well-es ablished mouse model o IAV in ec ion, and using deep sequencing o small RNAs (22), we ha e he e o e used wo inb ed mouse s ains o well cha ac e ized di e en gene ic suscep ibili y o IAV in ec ion o iden i y hos s ain-speci ic di e ences in he hos miRNA esponse o his i us. We ind subs an ial di e ences in he miRNA popula ions o he wo s ains, bo h a baseline and du ing in ec ion, and an o e all mo e apid ep og amming in he mo e suscep ible DBA/2J s ain. The esul s sugges ha hos - s ain-speci ic di e ences in abundance and egula ion o miRNAs p o ide a link o hos suscep ibili y o IAV in ec ion. MaTe ials anD MeThODs a ailabili y o Da a Small RNA sequencing da a, a miRNA exp ession able, and ables wi h exp ession di e ences and co ela ions wi h hema ological pa ame e s in pe iphe al blood, including adjus ed p alues, a e a ailable in he Gene Exp ession Omnibus eposi o y (h p:// www.ncbi.nlm.nih.go /geo/que y/acc.cgi?acc=GSE89064). Da a ables con ain he 473 miRNAs ha passed p e-analy ic il e ing. Columns wi hin he able wi h exp ession di e ences (GSE89064_edgeR_miR.xls) ep esen he e ec o in ec ion o mock ea men compa ed o un ea ed mice, di e ences be ween in ec ion and mock ea men a each ime poin , and di e ences be ween mouse s ains a e mock ea men , in ec ion, o wi hou ea men . All esul s we e ob ained wi h he edgeR package. Da a able (GSE89064_miRNA_ s_blood_spea man_co ela ions. xlsx) con ains Spea man co ela ion coe icien s o all miRNAs wi h pe iphe al blood indices. s a emen on e hics app o al All animal wo k was conduc ed acco ding o he na ional guidelines o he animal wel a e law o he Fede al Republic o Ge many and app o ed by he “Niede sächsisches Landesam ü Ve b auche schu z und Lebensmi elsiche hei , Oldenbu g, Ge many” (Pe mi Numbe s: 3392 42502-04-13/1234 and 33.19- 42502-04-13/1234). Vi us O iginal s ocks o mouse adap ed A/Pue o Rico/8/34 (H1N1, PR8) i us we e ob ained om S e an Ludwig (Uni e si y o Müns e ) (23). Vi us s ocks we e p opaga ed in he cho ioal- lan oic ca i y o 10-day-old pa hogen- ee emb yona ed chicken eggs o 48h a 37°C as desc ibed p e iously (24). animal P ocedu es In ec ions we e essen ially ca ied ou as desc ibed p e iously (20, 25). B ie ly, emale 12- o 13-week-old C57BL/6J and DBA/2J mice (n=5–6 pe ime poin and ea men ) we e anes he ized by in ape i oneal injec ion o 10μl/g body weigh o a s ock solu ion o 0.5 ml ke amine (50 mg/ml, In esa A zneimi el GmbH, F eibu g, Ge many), 0.5ml 2% xylazine hyd ochlo ide (Baye Heal h-Ca e, Le e kusen, Ge many), and 9 ml s e ile NaCl 0.9% (Del a-Selec GmbH, D eieich, Ge many). Fo in anasal in ec ion, a i al dose o 2×103 ocus o ming uni s ( u) in a o al olume o 20μl s e ile PBS was used. Du ing he in ec ion p ocedu e, mice we e held in he up igh posi ion and addi ional anes he ic was einjec ed as needed. Mock ea men was iden ical o he eal anes hesia/in ec ions excep ha ehicle only (s e ile PBS), no con aining i us, was used o in anasal ins illa ion. Mice we e weighed on day 0 jus be o e induc ion o anes hesia and on each subsequen day. Mice we e killed by CO2 asphyxia ion a 6, 12, 18, 24, 48, and 120h wi h espec o in ec ion o mock ea men . Un ea ed mice we e used as =0h con ol. Lungs we e emo ed by cu ing he i e main b onchi u he h oughou in ec ion and could in pa be a ibu ed o di e ences in pe iphe al blood leukocy e popula ions. Thus, pulmona y miRNA exp ession bo h be o e and du ing IAV in ec ion is in pa de e mined gene ically and con ibu es o suscep ibili y o IAV in ec ion in his mu ine hos , and likely in humans. Keywo ds: bioma ke s, gene ics, in ec ion, in luenza i us, lung, mi na, mouse, suscep ibili y 3 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 and we e washed in RNAla e RNA S abiliza ion Reagen (Qiagen Inc., Venlo, Ne he lands), immedia ely a e emo al. A e ans- e in o a new ube con aining 2ml RNAla e , lungs we e s o ed o e nigh a 4°C and hen a −20°C un il u he use. hema ological Measu emen s An analysis o he hema ological da a om his ime cou se expe imen has been published sepa a ely (25). B ie ly, 50 μl o whole blood we e ob ained by ca diac punc u e and hen analyzed wi h a Ve Scan HM5™ (Abaxis, Union Ci y, CA, USA) hema ology analyze . na isola ion Lungs we e homogenized in 4ml RLT bu e (Qiagen) con aining 40μl β-me cap oe hanol and s o ed in 450μl aliquo s a −80°C. A e hawing, 450μl o his suspension was mixed wi h 700μl Qiazol (Qiagen), and all u he s eps o o al RNA isola ion we e pe o med wi h he miRNeasy ki (Qiagen) acco ding o he manu ac u e ’s ecommenda ions. small na sequencing Fi e samples pe condi ion we e submi ed o small RNA sequenc- ing. Sequencing was pe o med using he T uSeq™ Small RNA Sequencing Ki , Ve sion o Janua y 2011 (Illumina, Inc., San Diego, CA, USA). App oxima ely 1,250ng o inpu RNA was used and he syn hesized cDNA was ampli ied wi h 15 PCR cycles. cDNA lib a y agmen s wi h a leng h o abou 150 n we e sepa a ed by gel elec opho esis and hen isola ed om he co esponding excised gel slice. Fou o i e biological eplica es pe condi ion we e analyzed. Use o he Illumina molecula ba code allowed sequenc- ing o eigh samples pe lane. To educe ba ch e ec s, samples om he same ime poin and mouse s ain we e ne e un on he same lane. Sequencing was done using he HiSeq 2500 (Illumina Inc.) sequence . The T uSeq™ Small RNA Sequencing Ki is speci ic o miRNAs and o he small RNAs ha ha e a 3′OH g oup esul ing om enzyma ic clea age by RNA p ocessing enzymes. Quan i a i e eal- ime Pc ( T-qPc ) Quan i a i e eal- ime PCR was pe o med wi h a Ligh Cycle 480® (La Roche AG, Basel, Swi ze land) in 96-well pla es in 20μl eac ion olumes, using 3ng cDNA pe eac ion o miRNAs (miSc ip Re e se T ansc ip ion Ki , miSc ip SYBR G een PCR Ki ; Qiagen) o 15ng cDNA pe eac ion o de ec ion o IAV hemagglu inin (HA) mRNA. Aliquo s om he same RNA samples as o he sequencing expe imen we e used, wi h i e biological eplica es o each condi ion. HA exp ession was no malized agains he mean o he C alues o Ac b and Rpl4 mRNAs. C alues o he miRNAs we e no no malized agains an in e nal con ol. Da a analysis Illumina sequencing da a we e p ep ocessed using he CLC Genomics Wo kbench 6.04 (CLC Bio, Camb idge, MA, USA) and he esul ing sequences we e anno a ed using miRBase 19. Two addi ional downs eam and ups eam bases as well as wo misma ches we e allowed. 5′ and 3′ ma u e sequences (including a ian s) we e used o u he analysis. All subsequen analyses we e done using he R en i onmen and p og amming code (26). All p alues we e adjus ed o alse disco e y a e (FDR). Two samples we e excluded be o e sequencing due o poo RNA quali y/quan i y, and h ee samples we e excluded a e sequencing due o low ead numbe s. Thus, small RNA sequenc- ing da a o 125 o he 130 o iginally submi ed samples (96%) we e a ailable o analysis, amoun ing o i e biological eplica es o mos condi ions, bu only ou o he ollowing condi ions: un ea ed (0h), mock ea ed (6h), and in ec ed (18hpi) DBA/2J mice, and mock ea ed (6 and 120h) C57BL/6J mice. A o al o 1,276 miRNAs we e de ec ed. Only he 476 anno a ed sequences ha we e de ec ed a a le el o >1 coun pe million (CPM) in >5 samples we e included in subsequen analyses. This did no change he numbe o o al eads app eciably. Th ee miRNAs wi h s ong ou lie samples we e emo ed because o an SD/median a io >2.5 (calcula ed wi h cpm alues o mouse s ain and ime pos ea men ). Line plo s o hese miRNAs did no show any ele an change in exp ession (da a no shown). Di e en ially exp essed miRNAs we e iden i ied using gene alized linea models con ained in he edgeR package (27). Fo exp ession changes wi hin a ime cou se o di e ences be ween mock ea ed and in ec ed mice, a old change (FC) o 1.5 and p alue o 0.05 we e used as cu o . Di e en ial miRNA exp ession in esponse o IAV in ec ion was de ined as ollows: (1) signi ican (p≤ 0.05) wi h espec o he un ea ed con ol ( =0h) and o he mock- ea ed mice a he same ime poin and (2) a ≥1.5 FC di e ence om he un ea ed mice. miRNAs ha we e di e en ially exp essed due o he in ec ion p ocedu e, excluding he i us, we e de ined as ollows: signi ican egula ion a e mock ea men and in ec ion wi h espec o =0h, bu no signi ican exp ession di e ence be ween in ec ed and mock samples om he same ime poin (p≥0.05). A FC cu o o ≥1.5 be ween un ea ed and mock- ea ed mice was used. To compa e absolu e miRNA abundance in DBA/2J s C57BL/6J mice, we used miRNAs ha we e di e en ially exp essed in esponse o in ec ion in a leas one mouse s ain and di e ed be ween he mouse s ains, using he mo e igo ous FC cu o o 2 and p≤0.01. Fu he mo e, we de ined a miRNA subse wi h no signi ican ly di e en abundance be ween bo h mouse s ains a all es ed ime poin s ( a io≤1.5; p≥0.1), bu di e en ial exp ession a e IAV in ec ion in ei he mouse s ain. Clus e s using k-means o longi udinal da a we e calcula ed using he kml package (28). G aphs we e made using la ice (29), gplo s (30), o ggplo 2 (31). Func ional P edic ion mic oRNAs ha we e ound o di e be ween he wo mouse s ains we e analyzed using h ee di e en app oaches. F om he signi ican ly en iched pa hways, we emo ed hose ha did no di e om pu a i e pa hways o miRNAs wi h simila abundance in bo h mouse s ains. Fo he i s app oach, we used he DIANA mi Pa h 2 online ool (32) o anno a ed miRNA con ained in miRBase 18. As se ings, we used mic oT-CDS o a ge p edic ion wi h a h eshold o 0.8. Resul s we e isualized wi h a p alue h eshold o 0.05 using he op ion “pa hways union.” This op ion shows he con ibu ion o each miRNA o FigU e 1 | Weigh loss and exp ession o in luenza a i us (iaV) hemagglu inin (ha) m na. Weigh loss and HA mRNA exp ession h oughou he 5-day ime cou se a e mock ea men o in ec ion wi h IAV s ain PR8 as ou lined in Sec ion “Ma e ials and Me hods.” (a) Weigh loss, exp essed as he mean pe cen age o body weigh measu ed a =0h be o e adminis a ion o anes hesia. No mice had o be killed because o >30% weigh loss. Da a a e based on he mice ha we e sac i iced a he 120h ime poin (n=5–6 pe g oup). (B) Rela i e quan i ica ion o IAV HA mRNA in mouse lung by RT-qPCR in he 5-day ime cou se shown in panel A. dC e e s o C e e ence−C a ge mRNA, whe e C e e ence is he a i hme ic mean o he C alues o β-ac in and Rpl4. As expec ed, HA mRNA was no de ec ed in he mock- ea ed mice (da a no shown). Da a a e based on i e mice pe g oup excep o DBA/2J a 18h (n=4). Solid lines, in ec ion; in e up ed lines, mock ea men . Le panels, DBA/2J s ain; igh panels, C57BL/6J s ain. The colo ed a eas a ound he lines indica e SD. No e ha he x-axes o he panels a e based on di e en scales. *p≤0.05 o di e ence wi h espec o =0h; ‡p≤0.05 o di e ence be ween mock- ea ed and in ec ed mice a he gi en ime poin (Tukey’s es ). This igu e is adap ed om wo a icles published by BioMed Cen al (21, 25), which desc ibe o he aspec s o his ime cou se expe imen . 4 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 he pa hways en ichmen , showing only miRNAs whose a ge s a e signi ican ly (p≤0.05) en iched o ha pa hway. F om he signi ican pa hways, we emo ed hose wi h a 1.5- old lowe numbe o signi ican miRNAs o he same pa hway in he con ol g oup o miRNAs wi h simila abundance be ween he wo mouse s ains. Fo he second s a egy, we used pu a i e miRNA a ge s ha we e ound in a leas h ee o ou da abases (Mic oCosm, PITA, miRanda and miRDB) [Mic oCosm (33); miRanda (34); miRDB (35); PITA (36)]. Ta ge s ha we e sha ed by a leas wo miRNAs we e chosen o unc ional analysis. Using he R packages “goseq” and “GO.db,” we iden i ied signi ican ly en iched GO Te ms using pu a i e a ge s o all 473 de ec ed miRNAs as backg ound. O he signi ican ly en iched e ms o hos s ain-dependen miRNAs, we chose only hose which had a leas 1.5- old mo e a ge s o a pa hway, compa ed o he con ol g oup wi h simila miRNA abundance, o ha we e no ound in he con ol g oup. Pu a i e miRNA a ge s o s ep 2 we e analyzed using he online ool DAVID (Da abase o Anno a ion, Visualiza ion and In eg a ed Disco e y) Bioin o ma ics Resou ces 6.7 (37, 38) and he Mus musculus genome as backg ound. Fo unc ional obse a ions, we used de aul se ings. Resul s o he DAVID ool included a Func ional Anno a ion Table wi h unc ional e ms o each gene; a Func ional Anno a ion Cha ha calcula es he en ichmen o each e m and a Func ional Anno a ion Clus e ing ha clus e s Te ms ha a e ela ed. O he Func ional Anno a ion Table, we added he op ions old en ichmen and FDR. Func ional Anno a ion Cha s we e downloaded and loaded in o he R p og am o allow di ec compa isons o di - e en en ichmen ables. To iden i y unc ions o miRNAs wi h di e en abundance in DBA/2J and C57BL/6J mice, only hose signi ican ly en iched e ms (FDR ≤ 0.05) we e chosen ha we e ound o miRNAs wi h di e en , bu no o miRNAs wi h simila abundance in bo h mouse s ains o we e a leas 1.5- old mo e highly en iched. O he s a is ical analyses Fo pai wise compa isons o miRNA o mRNA C alues, we used Tukey’s Hones Signi ican Di e ences Tes o homogeneous a iances. We used a signi icance h eshold o p≤0.05. Recei e ope a ing cha ac e is ic (ROC) cu e analysis was pe o med using he R package ROCR (39) and coun s pe million (cpm) da a. esUlTs B iske P og ession o in ec ion and Mo e P onounced Weigh loss in he DBa/2J Mouse s ain Highe weigh loss, compa ed o less suscep ible mouse s ains, is a hallma k o he high suscep ibili y o DBA/2J mice o IAV in ec ion. Consis en wi h ou p e ious obse a ions (40), ma ked weigh loss was obse ed a e in ec ion o his s ain, which eached a maximum o app oxima ely 25% by day 5 (Figu e1A). As shown p e iously (40), weigh loss o he less suscep ible s ain, C57BL/6J, was less p onounced, as e lec ed in a me e 5–10% educ ion by day 4–5. The e was no signi ican weigh loss among he mock- ea ed mice be ween day 1 and 5. Quan i a i e eal- ime PCR e ealed a b isk ise o mRNA encoding IAV HA in lungs o bo h mouse s ains a e in ec ion (Figu e1B). HA mRNA was de ec ed a low le els as ea ly as 6hpi in bo h s ains, ollowed by a apid ise ha peaked a 48 and 120hpi in DBA/2J and C57BL/6J mice, espec i ely. HA mRNA le els we e signi ican ly highe in DBA/2J han FigU e 2 | global mi na exp ession changes h oughou he 120h ime cou se o in luenza a i us in ec ion. Values o all 473 miRNAs ha passed p eanaly ical il e ing a e shown in panels (a,B) ( u quoise lines, mock ea men ; ed lines, in ec ion). (a,B) change in exp ession (log2); FC was calcula ed wi h he R package edgeR (27). (a) DBA/2J s ain; (B) C57BL/6J s ain. (c) Mul idimensional scaling based on he exp ession da a o all 473 de ec ed miRNAs (edgeR package). The da a, as well as he da a shown in Figu es3–6, a e based on i e mice pe condi ion, excep ha ou mice we e a ailable o un ea ed (0h), mock ea ed (6h), and in ec ed (18h) DBA/2J mice, and o mock ea ed (6 and 120h) C57BL/6J mice. 5 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 in C57BL/6J. This di e ence was signi ican as ea ly as 12hpi wi h a a io DBA/2J o C57BL/6J o 3.6:1 and inc eased o a cons an a io o abou 23 o 24:1 be ween 18 and 48 hpi, which diminished ma kedly by 120 hpi due o a decline in HA ansc ip le els in he DBA/2J s ain by his ime poin . Thus, he in ec ion e ol ed mo e b iskly and peaked soone in he mo e suscep ible DBA/2J s ain, wi h di e ences mani es - ing as ea ly as 12hpi. global changes in mi na exp ession h oughou he Time cou se Eigh y-eigh pe cen (10.7 million on a e age) o all ob ained sequences co esponded o anno a ed miRNAs. A o al o 473 miRNA species passed p e-analy ic da a p ocessing and we e selec ed o subsequen analyses. In bo h mouse s ains, analysis o he 473 selec ed miRNAs (Figu es2A,B) e ealed a FigU e 3 | Po en ial con ibu ions o in il a ing leukocy es o pulmona y mi nome ep og amming. (a) Line plo s isualizing he associa ions be ween miRNA abundance and di ec ion o egula ion o miRNAs egula ed in esponse o in luenza A i us (IAV) in ec ion (115 di e en miRNA species, FC ≥|1.5|, FDR adjus ed p alue≤0.05 in ei he mouse s ain, see Figu e4). The clus e s we e iden i ied wi h he k means o longi udinal da a (kml) package o R (28) using he FC (log2) da a o IAV in ec ed mice compa ed o un ea ed ( =0h) mice. (B) Co ela ion o miRNA exp ession wi h pe iphe al blood leukocy e pa ame e s. Using da a om in ec ed and con ol (unin ec ed) mice, exp ession (cpm) o 473 miRNAs was co ela ed wi h hema ological pa ame e s o he same mice. Signi ican co ela ions (Spea man co ela ion; ρ ≥|0.5| and p≤0.05) a e indica ed in di e en colo s acco ding o mouse s ain: ed, signi ican co ela ion in DBA/2J only; blue, signi ican co ela ion in C57BL/6J only; iole , signi ican co ela ion in bo h s ains. The g een smoo hing line was gene a ed using he R unc ion smoo h.spline (s a s package) using de aul se ings. WBC, whi e blood cells; LYM, LYM%, absolu e and ela i e lymphocy e coun s; MON, MON%, absolu e and ela i e monocy e coun s; GRA, GRA%, absolu e and ela i e g anulocy e coun s. 6 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 7 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 na ow ange in miRNA exp ession FC du ing he ime cou se. Acco dingly, mul idimensional scaling (MDS, Figu e 2C) e ealed ha samples om mock- ea ed and in ec ed mice a any ime poin be ween 6 and 24h o med wo igh clus e s (one o each mouse s ain) wi h he espec i e unin ec ed ( =0h) mice. In he DBA/2J mice, an appa en weak up and down egula- ion o some miRNAs due o he mock ea men was seen a 6h (Figu es2A,B), bu his was no s a is ically signi ican , and he e was no appa en endency owa d a mock e ec in he C57BL/6J s ain. Thus, he in ec ion/anes hesia p ocedu e did no induce signi ican changes in miRNA popula ions in ei he mouse s ain. O no e, a ma ked di e ence in miRNomes be ween he s ains was appa en e en in he unin ec ed lungs (Figu e2C). These s ain-speci ic di e ences domina ed he global di e ences in miRNomes h oughou he ime cou se, which emained ela- i ely cons an . In bo h mouse lines, line plo s (Figu es 2A,B), MDS (Figu e 2C), and Euclidian dis ance dissimila i y analysis (EDDA, da a no shown) consis en ly indica ed a global miRNA ep og amming beginning a 48hpi, ollowed by a p onounced u he inc ease a 120hpi. The o e all mean FC was cons an wi hin he i s 24 h and inc eased in DBA/2J and C57BL/6J mice a e 48h and 120h, espec i ely. As indica ed by MDS and con i med using EDDA, miRNome ep og amming a 48 hpi was g ea e in he DBA/2J han he C57BL/6J s ain, whe eas he wo s ains eached a simila o e all deg ee o ep og amming by 120 hpi. No ably, HA mRNA exp ession in DBA/2J mice dec eased be ween 48 and 120hpi (Figu e1B), bu miRNome ep og amming inc eased u he du ing he same ime ame. The e o e, hos miRNA ep og amming did no seem o be coupled di ec ly o i al HA RNA exp ession. To es whe he he e was any associa ion be ween he ini ial exp ession le el o he in ec ion- egula ed miRNAs and he di ec- ion o hei egula ion, he 115 in ec ion- egula ed miRNAs (di - e en ially exp essed in DBA/2J o C57BL/6J mice wi h espec o mock-in ec ed mice; FC ≥|1.5|, FDR adjus ed p alue≤0.05) we e g ouped in o h ee clus e s acco ding o baseline exp es- sion le el and di ec ion o egula ion (Figu e 3A). Indeed, a nega i e co ela ion be ween ini ial exp ession and di ec ion o egula ion was iden i ied in bo h mouse s ains: down egula ed miRNAs ended o ha e a high baseline exp ession le el, in e me- dia ely up egula ed miRNAs an in e media e baseline exp ession le el, and s ongly up egula ed miRNAs he lowes . The h ee clus e s con e ged owa d 120hpi, a a poin co esponding o app oxima ely i e no malized and log2 ans o med eads (log2 coun s pe million; cpm). Thus, he di ec ion o egula ion o he miRNAs depended on hei ini ial abundance (exp ession le el). These obse a ions a e consis en wi h a model in which miR- NAs wi h high baseline exp ession a e p edominan ly exp essed in esiden lung cells, bu miRNAs o low ini ial abundance a e mainly exp essed in immune cells and hei abundance inc eases du ing in ec ion due o hei immig a ion in o he lung combined wi h he co esponding changes in hei ac i a ion s a e. To assess he con ibu ions o changes in immune cell popula- ions o changes in miRNA exp ession, we quan i ied co ela ions be ween pe iphe al blood leukocy e pa ame e s and exp ession o each o he miRNAs (Figu e3B). O e all, highe numbe s o signi ican co ela ions we e measu ed in DBA/2J mice [compa e ed (DBA/2J) and and blue (C57BL/6J) ci cles in Figu e3], which was pa icula ly p onounced in o al leukocy e (WBC) and monocy e coun s. Absolu e g anulocy e numbe s and miRNA exp ession co ela ed mos signi ican ly in bo h mouse s ains (23 miRNAs wi h signi ican co ela ions; iole ci cles in Figu e3), wi h miR-223-3p, miR-142-3p, and miR-20b-5p co ela ing he mos posi i ely in bo h mouse s ains ([ρDBA/2J+ρC57BL/6J]/2>0.7). The e was only a negligible co ela ion (one miRNA in DBA/2J mice) wi h absolu e lymphocy e coun s. To es he abo e s a ed hypo hesis ha pulmona y miRNAs exp essed in in il a ing immune cells a e ini ially exp essed a low le el in he lung, we asked whe he he e was an associa ion be ween miRNA co ela- ion wi h pe iphe al blood indices and ini ial miRNA abundance. Indeed, miRNAs ha co ela e posi i ely (ρ≥0.2) wi h MON, GRA, GRA/LYM, and GRA% had a lowe ini ial abundance (≥1.5- old lowe ; p alue≤0.01, Wilcoxon es ) han miRNAs ha co ela e nega i ely (ρ≤−0.2). Taken oge he , hese esul s sugges ha exp ession o pulmona y miRNAs du ing he i s 120h o in ec ion may be in pa explained by in il a ion wi h myeloid immune cells, pa icula ly in he mo e suscep ible DBA/2J s ain. Mouse s ain-speci ic Di e ences in mi nome egula ion a e iaV in ec ion As men ioned abo e, 115 miRNAs we e signi ican ly egula ed a e in ec ion. The majo i y (75%) we e up egula ed and only abou 25% we e down egula ed. O no e, he numbe o di e en ially exp essed miRNAs and hei FC di ec ion was simila in bo h mouse s ains (Figu e4), i.e., miRNAs ha we e up egula ed in one s ain, we e also up egula ed in he o he , and ice e sa. In bo h s ains, a i s signi ican esponse o he in ec ion was appa en a 48hpi, wi h a sligh ly highe numbe o di e en ially exp essed miRNAs and highe FC alues being obse ed in he DBA/2J s ain han in he C57BL/6J s ain. An a leas wo old g ea e induc ion in C57BL6/J mice was de ec ed in eigh miRNAs (miR-190a-3p, miR-449a-5p, miR-449a-3p, miR-449c-5p, miR-3096a-5p, miR-3096b-5p, miR-3096b-3p, and miR-669c-5p). Consis en wi h MDS and EDDA, he wo s ains eached a simila o e all deg ee o ep og amming by 120hpi, bu di e ences a he ea lie ime poin s we e g ea es a 48h. In a nex s ep, we ied o iden i y miRNAs ha migh con- ibu e o he di e ences in miRNomes be ween he wo mouse s ains e ealed in Figu e2 and, hus, migh a ec hos s ain suscep ibili y. Abundances o a subse o 37 in ec ion- egula ed miRNAs di e ed (FC ≥|2|, FDR adjus ed p alue ≤ 0.01) be ween he wo mouse s ains a any o he es ed ime-poin s (Figu e 5), 22 o which we e mo e abundan in DBA/2J and 15 mo e abundan in C57BL/6J mice. Six o hese (16%) we e signi ican ly in ec ion- egula ed in DBA/2J only and 10 (27%) in C57BL/6J only. Pulmona y exp ession o eigh miRNAs di e ed be ween he wo mouse s ains e en be o e in ec ion. Exp ession o 10 miRNAs di e ed signi ican ly be ween he wo s ains be ween 6 and 18hpi, exp ession o 13 miRNAs a 24hpi, exp ession o 20 miRNAs a 48hpi, and exp ession o 23 FigU e 4 | mi nas egula ed du ing in luenza a i us (iaV) in ec ion. Hea map showing all 115 miRNAs ha we e di e en ially exp essed a e IAV in ec ion (see Ma e ials and Me hods) o DBA/2J and/o C57BL/6J mice. FC da a and p alues (FDR adjus ed) ep esen he di e ence be ween in ec ed and mock- ea ed mice. Red: up egula ed; blue: down egula ed. The igh ba indica es log2 coun s pe million (edgeR package) o hese miRNAs in DBA/2J and C57BL/6J mice oge he , anging om low (whi e) and mid (blue) o high (black) abundance. Exp ession o 93 miRNAs changed du ing he in ec ion in he DBA/2J s ain and 95 in he C57BL/6J s ain, co esponding o a o al o 115 egula ed miRNAs. Se en y- h ee o hese we e egula ed in bo h s ains (***p alue≤0.001; **p alue≤0.01; *p alue≤0.05). S a s a e only shown o di e ences be ween in ec ed and mock- ea ed mice and we e also included o signi ican miRNAs wi h an FC <|1.5|. 8 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 FigU e 5 | mi nas ha di e in absolu e abundance be ween DBa/2J and c57Bl/6J mice. Depic ion o 37 miRNAs wi h a di e en ial abundance o ≥|2| and a p alue (FDR adjus ed) o a ≤0.01 be ween DBA/2J and C57BL/6J mice h oughou he ime cou se. Red colo s in he hea map indica e miRNAs ha a e mo e abundan in DBA/2J mice (22 miRNAs), blue colo s indica e miRNAs ha a e mo e abundan in C57BL/6J mice (15 miRNAs). The 115 miRNAs ha a e in ec ion dependen ly egula ed in DBA/2J o C57BL/6J mice (Figu e4) we e used as inpu miRNAs. The blue ba indica es mean miRNA abundance ac oss all samples (log2 cpm). The FC o hese miRNAs a 120hpi is shown using shades o blue (low DBA/2J s C57BL/6J a io, i.e., highe abundance in C57BL/6J mice) and shades o ed (high DBA/2J s C57BL/6J a io, i.e., highe abundance in DBA/2J mice) (***p alue≤0.001; **p alue≤0.01; *p alue≤0.05). S a s we e also included o signi ican miRNAs wi h an absolu e a io <2. 9 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 miRNAs a 120hpi. The miRNAs whose exp ession was lowe / highe in DBA/2J mice in he unin ec ed s a e mainly con inued o be exp essed a a lowe /highe le el a e in ec ion, e en a e 5days. Thus, wo majo pa e ns could be disce ned among he hos s ain-dependen ly egula ed miRNAs: (1) miRNAs ha we e di e en ially exp essed in he wo s ains h oughou he en i e ime cou se, and (2) miRNAs whose di e en ial exp ession mani es ed a he la e ime poin s. Mos miRNAs o g oup wo we e mo e abundan in DBA/2J mice. miR-147-3p cons i u ed an excep ion in ha i was ma kedly o e exp essed in C57BL/6J mice wi hin he i s 24hpi and had a simila exp ession le el as DBA/2J a 48hpi. miR-3096b-3p and miR-3096b-5p showed he s onges hos s ain-dependen di e ence in bo h di ec ions. The DNA In elligen Analysis (DIANA), he Gene On ology ana- lyze o RNA-seq da a (R package goseq), and he Da abase o Anno a ion, Visualiza ion, and In eg a ed Disco e y (DAVID) we e hen used o sea ch o pu a i e mRNA a ge s o miRNAs whose exp ession was di e en be ween he wo mouse s ains (Figu e 5) and he unc ional pa hways pu a i ely associa ed wi h hese mRNAs. Among he pa hways ela ing o immune unc ions, Fc gamma R-media ed phagocy osis, HIF-1 signaling pa hway, posi i e egula ion o p o ein kinase B signaling cascade, 16 P eusse e al. Hos miRNAs in IAV In ec ion F on ie s in Immunology | www. on ie sin.o g Ma ch 2017 | Volume 8 | A icle 246 expe imen al design and p epa a ion o manusc ip . FP: ini ial concep o he s udy, expe imen al design, p epa a ion o he manusc ip , and supe ision o he s udy. acKnOWleDgMenTs The au ho s hank Robe Ge e s and s a membe s, especially Michael Ja ek and Tschong-Hun Im, o he Genome Analysis pla o m o he Helmhol z Cen e o In ec ion Resea ch (HZI) o pe o ming he RNA sequencing and F ank Klawonn (HZI) and Es he Wilk (HZI) o help ul discussion, Mahmoud Bahga - Riad (HZI) o help ul discussion and ins uc ion, Mohamed Sami (TWINCORE Cen e o Expe imen al and Clinical In ec ion Resea ch) o help ul discussion, echnical assis ance, and suppo in li e a u e esea ch, and Anja Ga zemeie (HZI) o echnical assis ance. FUnDing The s udy was suppo ed by in e nal unds om he Helmhol z Associa ion (P og am In ec ion and Immuni y), he Helmhol z Associa ion’s C oss-p og amme Ini ia i e on Pe sonalized Medicine (iMed), and he Helmhol z In e na ional G adua e School o In ec ion Resea ch o MP. 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