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
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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
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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.
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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.
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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|.
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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.
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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,
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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. The unding bodies had
no ole in he design o he s udy and collec ion, analysis, and
in e p e a ion o da a and in w i ing he manusc ip .
eFe ences
1. Sonkoly E, S ahle M, Pi a csi A. mic oRNAs and immuni y: no el playe s in
he egula ion o no mal immune unc ion and in lamma ion. Semin Cance
Biol (2008) 18:131–40. doi:10.1016/j.semcance .2008.01.005
2. Co des KR, S i as a a D. mic oRNA egula ion o ca dio ascula de elop-
men . Ci c Res (2009) 104:724–32. doi:10.1161/CIRCRESAHA.108.192872
3. Agos ini M, Tucci P, S eine JR, Shalom-Feue s ein R, Rouleau M, Abe dam
D, e  al. mic oRNA-34a egula es neu i e ou g ow h, spinal mo phology, and
unc ion. P oc Na l Acad Sci U S A (2011) 108:21099–104. doi:10.1073/pnas.
1112063108
4. Abe W, Nasu K, Nakada C, Kawano Y, Mo iyama M, Na aha a H. miR-196b
a ge s c-myc and Bcl-2 exp ession, inhibi s p oli e a ion and induces apop o-
sis in endome io ic s omal cells. Hum Rep od (2013) 28:750–61. doi:10.1093/
hum ep/des446
5. Chang ST, Thomas MJ, So a P, G een RR, Pale mo RE, Ka ze MG. Nex -
gene a ion sequencing o small RNAs om HIV-in ec ed cells iden i ies
phased mic oRNA exp ession pa e ns and candida e no el mic oRNAs di e -
en ially exp essed upon in ec ion. MBio (2013) 4:e549–612. doi:10.1128/mBio.
00549-12
6. Qi Y, Li Y, Zhang L, Huang J. mic oRNA exp ession p o iling and bioin o -
ma ic analysis o dengue i us in ec ed pe iphe al blood mononuclea cells.
Mol Med Rep (2013) 7:791–8. doi:10.3892/mm .2013.1288
7. Zhang X, Dauche M, A mis ead D, Russell R, Ko ilil S. mic oRNA exp ession
p o iling in HCV-in ec ed human hepa oma cells iden i ies po en ial an i- i al
a ge s induced by in e e on-alpha. PLoS One (2013) 8:e55733. doi:10.1371/
jou nal.pone.0055733
8. Buggele WA, Johnson KE, Ho a h CM. In luenza A i us in ec ion o human
espi a o y cells induces p ima y mic oRNA exp ession. J Biol Chem (2012)
287:31027–40. doi:10.1074/jbc.M112.387670
9. Lam WY, Yeung AC, Ngai KL, Li MS, To KF, Tsui SK, e  al. E ec o
a ian in luenza A H5N1 in ec ion on he exp ession o mic oRNA-141
in human espi a o y epi helial cells. BMC Mic obiol (2013) 13:104.
doi:10.1186/1471-2180-13-104
10. Song H, Wang Q, Guo Y, Liu S, Song R, Gao X, e al. Mic oa ay analysis
o mic oRNA exp ession in pe iphe al blood mononuclea cells o c i -
ically ill pa ien s wi h in luenza A (H1N1). BMC In ec Dis (2013) 13:257.
doi:10.1186/1471-2334-13-257
11. Wang Y, B ahmaksha iya V, Zhu H, Lupiani B, Reddy SM, Yoon BJ, e al.
Iden i ica ion o di e en ially exp essed miRNAs in chicken lung and achea
wi h a ian in luenza i us in ec ion by a deep sequencing app oach. BMC
Genomics (2009) 10:512. doi:10.1186/1471-2164-10-512
12. Wang Y, B ahmaksha iya V, Lupiani B, Reddy SM, Soibam B, Benham AL,
e al. In eg a ed analysis o mic oRNA exp ession and mRNA ansc ip ome
in lungs o a ian in luenza i us in ec ed b oile s. BMC Genomics (2012)
13:278. doi:10.1186/1471-2164-13-278
13. Sko gaa d K, Ci e a S, Vasby D, Podolska A, B eum SO, Du wald R, e al.
Exp ession o inna e immune genes, p o eins and mic oRNAs in lung issue
o pigs in ec ed expe imen ally wi h in luenza i us (H1N2). Inna e Immun
(2013) 19(5):531–44. doi:10.1177/1753425912473668
14. Li Y, Li J, Belisle S, Baskin CR, Tumpey TM, Ka ze MG. Di e en ial mic oRNA
exp ession and i ulence o a ian, 1918 easso an , and econs uc ed
1918 in luenza A i uses. Vi ology (2011) 421:105–13. doi:10.1016/j.
i ol.2011.09.011
15. Li Y, Chan EY, Li J, Ni C, Peng X, Rosenzweig E, e al. mic oRNA exp ession
and i ulence in pandemic in luenza i us-in ec ed mice. J Vi ol (2010)
84:3023–32. doi:10.1128/JVI.02203-09
16. Peng X, G alinski L, Fe is MT, F ieman MB, Thomas MJ, P oll S, e  al.
In eg a i e deep sequencing o he mouse lung ansc ip ome e eals di e -
en ial exp ession o di e se classes o small RNAs in esponse o espi a o y
i us in ec ion. MBio (2011) e00198-11. doi:10.1128/mBio.00198-11
17. Lo eday EK, S in i V, Diede ich S, Pasick J, Jean F. Tempo al- and s ain-spe-
ci ic hos mic oRNA molecula signa u es associa ed wi h swine-o igin H1N1
and a ian-o igin H7N7 in luenza A i us in ec ion. J Vi ol (2012) 86:6109–22.
doi:10.1128/JVI.06892-11
18. Buggele WA, K ause KE, Ho a h CM. Small RNA p o iling o in luenza A
i us-in ec ed cells iden i ies miR-449b as a egula o o his one deace y-
lase 1 and in e e on be a. PLoS One (2013) 8:e76560. doi:10.1371/jou nal.
pone.0076560
19. Wol S, Wu W, Jones C, Pe wi asa i O, Mahalingam S, T ipp RA. mic oRNA
egula ion o human genes essen ial o in luenza A (H7N9) eplica ion. PLoS
One (2016) 11:e0155104. doi:10.1371/jou nal.pone.0155104
20. Albe s R, S i as a a B, Wu H, Viegas N, Ge e s R, Klawonn F, e al. Gene
exp ession changes in he hos esponse be ween esis an and suscep ible
inb ed mouse s ains a e in luenza A in ec ion. Mic obes In ec (2010)
12:309–18. doi:10.1016/j.micin .2010.01.008
21. P eusse M, Tan awy MA, Klawonn F, Schugha K, Pessle F. In ec ion- and
p ocedu e-dependen e ec s on pulmona y gene exp ession in he ea ly
phase o in luenza A i us in ec ion in mice. BMC Mic obiol (2013) 13:293.
doi:10.1186/1471-2180-13-293
22. P eusse M, Della Be a C, Pessle F. P o iling o miRNA exp ession: RNA
sequencing in he spo ligh . In: Yue J, Lu L, edi o s. miRNAs and Human
Diseases. Ke ala, India: Resea ch Signpos (2013). p. 313–35.
23. Blazejewska P, Koscinski L, Viegas N, Anhlan D, Ludwig S, Schugha K.
Pa hogenici y o di e en PR8 in luenza A i us a ian s in mice is de e -
mined by bo h i al and hos ac o s. Vi ology (2011) 412:36–45. doi:10.1016/j.
i ol.2010.12.047
24. Wilk E, Schugha K. The mouse as model sys em o s udy hos -pa hogen
in e ac ions in in luenza A in ec ions. Cu P o oc Mouse Biol (2012)
2:177–205. doi:10.1002/9780470942390.mo110173
25. P eusse M, Schugha K, Wilk E, Klawonn F, Pessle F. Hema ological
pa ame e s in he ea ly phase o in luenza A i us in ec ion in di e en ially
suscep ible inb ed mouse s ains. BMC Res No es (2015) 8:225. doi:10.1186/
s13104-015-1195-8
26. Zhang J, Zhao F, Yu X, Lu X, Zheng G. mic oRNA-155 modula es he p oli -
e a ion o ascula smoo h muscle cells by a ge ing endo helial ni ic oxide
syn hase. In J Mol Med (2015) 35:1708–14. doi:10.3892/ijmm.2015.2181
27. Robinson MD, Mcca hy DJ, Smy h GK. edgeR: a bioconduc o package o
di e en ial exp ession analysis o digi al gene exp ession da a. Bioin o ma ics
(2010) 26:139–40. doi:10.1093/bioin o ma ics/b p616

17
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
28. Genolini C, Falissa d B. KmL: k-means o longi udinal da a. Compu S a .
(2010) 25:317–28. doi:10.1007/s00180-009-0178-4
29. Sa ka D. La ice: Mul i a ia e Da a Visualiza ion wi h R. Pa is: Sp inge
(2008).
30. Wa nes GR, Bolke B, Bonebakke L, Gen leman R, Liaw WHA, Lumley T,
e al. gplo s: Va ious R P og amming Tools o Plo ing Da a. R package (2013).
A ailable om: h p://c an. -p ojec .o g/web/packages/gplo s/index.h ml
31. Wickham H. ggplo 2: Elegan G aphics o Da a Analysis. Sp inge (2009).
32. Vlachos IS, Kos oulas N, Ve goulis T, Geo gakilas G, Reczko M, Ma agkakis
M, e  al. DIANA miRPa h .2.0: in es iga ing he combina o ial e ec o
mic oRNAs in pa hways. Nucleic Acids Res (2012) 40:W498–504. doi:10.1093/
na /gks494
33. Mic oCosm. Mic oCosm Ta ge s Ve sion 5. En igh Lab (2011). A ailable
om: h p://www.ebi.ac.uk/en igh -s /mic ocosm/h docs/ a ge s/ 5/
34. Be el D, Wilson M, Gabow A, Ma ks DS, Sande C. The mic oRNA.o g
esou ce: a ge s and exp ession. Nucleic Acids Res (2008) 36:D149–53.
doi:10.1093/na /gkm995
35. Wang X. miRDB: A mic oRNA a ge p edic ion and unc ional anno a ion
da abase wi h a wiki in e ace. RNA (2008) 14(6):1012–7. doi:10.1261/
na.965408
36. Ke esz M, Io ino N, Unne s all U, Gaul U, Segal E. The ole o si e accessibili y
in mic oRNA a ge ecogni ion. Na Gene (2007) 39:1278–84. doi:10.1038/
ng2135
37. Huang Da W, She man BT, Lempicki RA. Sys ema ic and in eg a i e analysis
o la ge gene lis s using DAVID bioin o ma ics esou ces. Na P o oc (2009)
4:44–57. doi:10.1038/np o .2008.211
38. Huang Da W, She man BT, Lempicki RA. Bioin o ma ics en ichmen ools:
pa hs owa d he comp ehensi e unc ional analysis o la ge gene lis s. Nucleic
Acids Res (2009) 37:1–13. doi:10.1093/na /gkn923
39. Sing T, Sande O, Bee enwinkel N, Lengaue T. ROCR: isualizing clas-
si ie pe o mance in R. Bioin o ma ics (2005) 21:3940–1. doi:10.1093/
bioin o ma ics/b i623
40. S i as a a B, Blazejewska P, Hessmann M, B ude D, Ge e s R, Mauel S, e al.
Hos gene ic backg ound s ongly in luences he esponse o in luenza a i us
in ec ions. PLoS One (2009) 4:e4857. doi:10.1371/jou nal.pone.0004857
41. Zhang XB, Song L, Wen HJ, Bai XX, Li ZJ, Ma LJ. Up egula ion o mic oRNA-31
a ge ing in eg in alpha5 supp esses umo cell in asion and me as asis by
indi ec ly egula ing PI3K/AKT pa hway in human gas ic cance SGC7901
cells. Tumou Biol (2016) 37:8317–25. doi:10.1007/s13277-015-4511-y
42. He meking H. The miR-34 amily in cance and apop osis. Cell Dea h Di e
(2010) 17:193–9. doi:10.1038/cdd.2009.56
43. Lize M, Pila ski S, Dobbels ein M. E2F1-inducible mic oRNA 449a/b sup-
p esses cell p oli e a ion and p omo es apop osis. Cell Dea h Di e (2010)
17:452–8. doi:10.1038/cdd.2009.188
44. Zheng GX, Ra i A, Gould GM, Bu ge CB, Sha p PA. Genome-wide impac
o a ecen ly expanded mic oRNA clus e in mouse. P oc Na l Acad Sci U S A
(2011) 108:15804–9. doi:10.1073/pnas.1112772108
45. Olsen CW, Keh en JC, Dybdahl-Sissoko NR, Hinshaw VS. bcl-2 al e s
in luenza i us yield, sp ead, and hemagglu inin glycosyla ion. J Vi ol (1996)
70:663–6.
46. Wu ze WJ, Planz O, Eh ha d C, Gine M, Silbe zahn T, Pleschka S, e al.
Caspase 3 ac i a ion is essen ial o e icien in luenza i us p opaga ion.
EMBO J (2003) 22:2717–28. doi:10.1093/emboj/cdg279
47. Isken B, Genzel Y, Reichl U. P oduc i i y, apop osis, and in ec ion dynamics
o in luenza A/PR/8 s ains and A/PR/8-based easso an s. Vaccine (2012)
30:5253–61. doi:10.1016/j. accine.2012.05.065
48. Bha naga N, Li X, Padi SK, Zhang Q, Tang MS, Guo B. Down egula ion o
miR-205 and miR-31 con e s esis ance o chemo he apy-induced apop osis
in p os a e cance cells. Cell Dea h Dis (2010) 1:e105. doi:10.1038/cddis.
2010.85
49. Saini S, Yamamu a S, Majid S, Shah ya i V, Hi a a H, Tanaka Y, e  al.
mic oRNA-708 induces apop osis and supp esses umo igenici y in enal can-
ce cells. Cance Res (2011) 71:6208–19. doi:10.1158/0008-5472.CAN-11-0073
50. Wang LP, Ma XQ, Cai JC. [Clinicopa hological signi icance and unc ion o
miR-135b in he occu ence and de elopmen o gas ic cance ]. Zhonghua Yi
Xue Za Zhi (2012) 92:3269–73. doi:10.3760/cma.j.issn.0376-2491.2012.46.009
51. Ma J, Cheng L, Liu H, Zhang J, Shi Y, Zeng F, e al. Genis ein down- egula es
miR-223 exp ession in panc ea ic cance cells. Cu D ug Ta ge s (2013)
14:1150–6. doi:10.2174/13894501113149990187
52. Zhang WG, Chen L, Dong Q, He J, Zhao HD, Li FL, e al. mmu-miR-702
unc ions as an an i-apop o ic mi on by media ing ATF6 inhibi ion in mice.
Gene (2013) 531:235–42. doi:10.1016/j.gene.2013.09.005
53. Fan N, Wang J. mic oRNA 34a con ibu es o i us-media ed apop osis
h ough binding o i s a ge gene Bax in in luenza A i us in ec ion. Biomed
Pha maco he (2016) 83:1464–70. doi:10.1016/j.biopha.2016.08.049
54. B ogaa d L, Heegaa d PM, La sen LE, Mo ensen S, Schlegel M, Du wald
R, e  al. La e egula ion o immune genes and mic oRNAs in ci cula ing
leukocy es in a pig model o in luenza A (H1N2) in ec ion. Sci Rep (2016)
6:21812. doi:10.1038/s ep21812
55. Ri e a A, Ba T, Rais M, Engelmann F, Messaoudi I. mic oRNAs egula e
hos immune esponse and pa hogenesis du ing in luenza in ec ion in hesus
macaques. Vi al Immunol (2016) 29:212–27. doi:10.1089/ im.2015.0074
56. Izumi B, Nakasa T, Tanaka N, Nakanishi K, Kamei N, Yamamo o R, e al.
mic oRNA-223 exp ession in neu ophils in he ea ly phase o seconda y
damage a e spinal co d inju y. Neu osci Le (2011) 492:114–8. doi:10.1016/j.
neule .2011.01.068
57. K ame NJ, Wang WL, Reyes EY, Kuma B, Chen CC, Ramak ishna C, e al.
Al e ed lymphopoiesis and immunode iciency in miR-142 null mice. Blood
(2015) 125:3720–30. doi:10.1182/blood-2014-10-603951
58. Sha ma A, Kuma M, Aich J, Ha iha an M, B ahmacha i SK, Ag awal A, e al.
Pos ansc ip ional egula ion o in e leukin-10 exp ession by hsa-miR-106a.
P oc Na l Acad Sci U S A (2009) 106:5761–6. doi:10.1073/pnas.0808743106
59. Zhang N, Be an MJ. Dice con ols CD8+ T-cell ac i a ion, mig a ion,
and su i al. P oc Na l Acad Sci U S A (2010) 107:21629–34. doi:10.1073/
pnas.1016299107
60. Zhu D, Pan C, Li L, Bian Z, L Z, Shi L, e al. mic oRNA-17/20a/106a modula e
mac ophage in lamma o y esponses h ough a ge ing signal- egula o y
p o ein alpha. J Alle gy Clin Immunol (2013) 132:426–436.e428. doi:10.1016/j.
jaci.2013.02.005
61. Zheng Y, Chen KL, Zheng XM, Li HX, Wang GL. Iden i ica ion and bioin o -
ma ics analysis o mic oRNAs associa ed wi h s ess and immune esponse
in se um o hea -s essed and no mal Hols ein cows. Cell S ess Chape ones
(2014) 19:973–81. doi:10.1007/s12192-014-0521-8
62. Liu XF, Wang RQ, Hu B, Luo MC, Zeng QM, Zhou H, e al. mic oRNA miR-
15a con ibu es abno mal immune esponse in myas henia g a is by a ge ing
CXCL10. Clin Immunol (2016) 164:106–13. doi:10.1016/j.clim.2015.12.009
63. Sami M, Vaas L, Pessle F. mic oRNAs in he hos esponse o i al
in ec ions o e e ina y impo ance. F on Ve Sci (2016) 3:86. doi:10.3389/
e s.2016.00086
64. Choi EJ, Kim HB, Baek YH, Kim EH, Pascua PN, Pa k SJ, e al. Di e en ial
mic oRNA exp ession ollowing in ec ion wi h a mouse-adap ed, highly
i ulen a ian H5N2 i us. BMC Mic obiol (2014) 14:252. doi:10.1186/
s12866-014-0252-0
65. Fos e PS, Plank M, Collison A, Tay HL, Kaiko GE, Li J, e al. The eme ging
ole o mic oRNAs in egula ing immune and in lamma o y esponses in he
lung. Immunol Re (2013) 253:198–215. doi:10.1111/im .12058
66. Ma ce B, Che alie B, Luxa di G, Co aux C, Za agosi LE, Cibois M, e al.
Con ol o e eb a e mul iciliogenesis by miR-449 h ough di ec ep ession o
he Del a/No ch pa hway. Na Cell Biol (2011) 13:693–9. doi:10.1038/ncb2241
67. Wang L, Fu C, Fan H, Du T, Dong M, Chen Y, e  al. miR-34b egula es
mul iciliogenesis du ing o gan o ma ion in zeb a ish. De elopmen (2013)
140:2755–64. doi:10.1242/de .092825
68. I a ani J, Mel ille GN, Ho s mann G. T acheob onchial clea ance in heal h
and disease: wi h special e e ence o in e cilia y luid. Ciba Found Symp
(1978) 54:235–52.
69. Liu G, F igge i A, Yang Y, Pa k YJ, Tsu u a Y, Ab aham E. miR-147, a
mic oRNA ha is induced upon oll-like ecep o s imula ion, egula es
mu ine mac ophage in lamma o y esponses. P oc Na l Acad Sci U S A (2009)
106:15819–24. doi:10.1073/pnas.0901216106
70. Baue n eind F, Riege A, Schildbe g FA, Knolle PA, Schmid-Bu gk JL,
Ho nung V. NLRP3 in lammasome ac i i y is nega i ely con olled by miR-
223. J Immunol (2012) 189:4175–81. doi:10.4049/jimmunol.1201516
71. Chen Q, Wang H, Liu Y, Song Y, Lai L, Han Q, e al. Inducible mic oRNA-223
down- egula ion p omo es TLR- igge ed IL-6 and IL-1be a p oduc ion in
mac ophages by a ge ing STAT3. PLoS One (2012) 7:e42971. doi:10.1371/
jou nal.pone.0042971
72. Kelada S, Se hupa hy P, Okoye IS, Kis asis E, Czieso S, Whi e SD, e al. miR-
182 and miR-10a a e key egula o s o T eg specialisa ion and s abili y du ing
18
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
schis osome and Leishmania-associa ed in lamma ion. PLoS Pa hog (2013)
9:e1003451. doi:10.1371/jou nal.ppa .1003451
73. Xue F, Li H, Zhang J, Lu J, Xia Y, Xia Q. miR-31 egula es in e leukin 2 and
kinase supp esso o as 2 du ing T cell ac i a ion. Genes Immun (2013)
14:127–31. doi:10.1038/gene.2012.58
74. Eh ha d C, Wol T, Pleschka S, Planz O, Bee mann W, Bode JG, e  al.
In luenza A i us NS1 p o ein ac i a es he PI3K/Ak pa hway o media e
an iapop o ic signaling esponses. J Vi ol (2007) 81:3058–67. doi:10.1128/
JVI.02082-06
75. Cao W, Yang W, Fan R, Li H, Jiang J, Geng M, e al. miR-34a egula es cis-
pla in-induce gas ic cance cell dea h by modula ing PI3K/AKT/su i in
pa hway. Tumou Biol (2014) 35:1287–95. doi:10.1007/s13277-011-1171-7
76. Zhang DG, Li WZ, Wang GF, Su Y, Zeng J, Zhang C, e al. He e ologous
SH3-p85be a inhibi s in luenza A i us eplica ion. Vi ol J (2010) 7:170.
doi:10.1186/1743-422X-7-170
77. Lu X, Masic A, Liu Q, Zhou Y. Regula ion o in luenza A i us induced
CXCL-10 gene exp ession equi es PI3K/Ak pa hway and IRF3 ansc ip ion
ac o . Mol Immunol (2011) 48:1417–23. doi:10.1016/j.molimm.2011.
03.017
78. Tian GP, Chen WJ, He PP, Tang SL, Zhao GJ, L YC, e al. mic oRNA-467b
a ge s LPL gene in RAW 264.7 mac ophages and a enua es lipid accumula-
ion and p oin lamma o y cy okine sec e ion. Biochimie (2012) 94:2749–55.
doi:10.1016/j.biochi.2012.08.018
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