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www.impac jou nals.com/onco a ge / Onco a ge , Vol. 7, No. 34
Myeloid cell exp essed p op o ein con e ase FURIN a enua es
in lamma ion
Zuze Ma inez Co do a1, Anna G önholm1, Ville Ky ölä2, Valen ina Ta e ni i3,
Sanna Hämäläinen1, Saa a Ai omäki1, Wilhelmiina Niininen1, Ilkka Jun ila4,5,
An i Ylipää2, Ma i Nyk e 2 and Ma ko Pesu1,6
1 Immuno egula ion, Ins i u e o Biosciences and Medical Technology (BioMediTech), Uni e si y o Tampe e, Tampe e, Finland
2 Compu a ional Biology, Ins i u e o Biosciences and Medical Technology (BioMediTech), Uni e si y o Tampe e, Tampe e,
Finland
3 Depa men o Food, En i onmen al and Nu i ional Sciences (DeFENS), Di ision o Food Mic obiology and Biop ocessing,
Uni e si à degli S udi di Milano, Milan, I aly
4 School o Medicine, Uni e si y o Tampe e, Tampe e, Finland
5 Fimlab Labo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland
6 Depa men o De ma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland
Co espondence o: Ma ko Pesu, email: [email p o ec ed]
Keywo ds: cy okine, FURIN, LysM, mac ophage, TGF-β1, Immunology and Mic obiology Sec ion, Immune esponse, Immuni y
Recei ed: Feb ua y 25, 2016 Accep ed: July 22, 2016 Published: Augus 05, 2016
Abs Ac
The p op o ein con e ase enzyme FURIN p ocesses imma u e p o-p o eins
in o unc ional end- p oduc s. FURIN is up egula ed in ac i a ed immune cells and i
egula es T-cell dependen pe iphe al ole ance and he Th1/Th2 balance. FURIN also
p omo es he in ec i i y o pa hogens by ac i a ing bac e ial oxins and by p ocessing
i al p o eins. He e, we e alua ed he ole o FURIN in LysM+ myeloid cells in i o.
Mice wi h a condi ional dele ion o FURIN in hei myeloid cells (LysMC e- u ( l/ l))
we e heal hy and showed unchanged p opo ions o neu ophils and mac ophages.
Ins ead, LysMC e- u
( l/ l)
mice had ele a ed se um IL-1β le els and educed numbe s
o splenocy es. An LPS injec ion esul ed in accele a ed mo ali y, ele a ed se um p o-
in lamma o y cy okines and up egula ed numbe s o p o-in lamma o y mac ophages.
A genome-wide gene exp ession analysis e ealed he o e exp ession o se e al
p o-in lamma o y genes in es ing FURIN-de icien mac ophages. Mo eo e , FURIN
inhibi ed Nos2 and p omo ed he exp ession o A g1, which implies ha FURIN
egula es he M1/M2- ype mac ophage balance. FURIN was equi ed o he no mal
p oduc ion o he bioac i e TGF-β1 cy okine, bu i inhibi ed he ma u a ion o he
in lamma ion-p o oking TACE and Caspase-1 enzymes. In conclusion, FURIN has an
an i-in lamma o y unc ion in LysM+ myeloid cells in i o.
In oduc Ion
The componen s o he inna e immuni y implemen
he ecogni ion and elimina ion o mic obes h ough a
complex machine y in ol ing ac o s ha equi e a pos -
ansla ional p o eoly ic ac i a ion s ep o pe o m hei
e ec o unc ions. The unc ional ma u a ion o do man
p o-p o eins is ca alyzed by he p op o ein con e ase
sub ilisin/kexin (PCSK) enzymes (PCSK1-2, FURIN,
PCSK5-7, memb ane-bound ansc ip ion ac o si e 1,
PCSK9) [1]. The i s se en PCSKs ope a e a a simila
a ge si e consis ing o basic amino acids [K/R)-(X)
n-(K/R)↓, wi h n being 0, 2, 4 o 6 and X any amino
acid], which esul s in a signi ican deg ee o unc ional
edundancy in a ge ecogni ion and p ocessing in i o.
Howe e , humans bea ing mu a ions in he PCSK genes
as well as PCSK-de icien expe imen al animal models
ha e gene-speci ic pheno ypes, which a gues o subs a e
speci ici y in i o [2].
FURIN was he i s disco e ed and is hus he
mos s udied con en ional PCSK enzyme [3]. FURIN
is ubiqui ously exp essed, and in cells i ca alyzes
he ma u a ion o i s a ge s in he sec e o y pa hway,
endosomes and on he cell su ace. Due o i s widesp ead
exp ession, FURIN has a ple ho a o epo ed a ge s
including cy okines, chemokines and g ow h ac o s as
Resea ch Pape : Immunology
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well as o he p o eases like ma ix me allop o einases. In
mice, he exp ession o Fu in is essen ial o emb yonic
de elopmen , which has imposed limi a ions o ou
unde s anding o i s cell- ype speci ic unc ion in i o
[3, 4]. Howe e , he pheno ypes o issue-speci ic Fu in
de icien mice ha e demons a ed ha FURIN canno be
compensa ed o by o he PCSK enzymes in endo helial
cells (Tie2C e) o in T lymphocy es (CD4C e) [5, 6].
FURIN’s egula o y ole is also implica ed in
mul iple human pa hologies. Fo example, FURIN
p ocesses he be a-sec e ase enzyme in Alzheime ’s
disease, SNPs in he Fu in gene a e associa ed wi h blood
p essu e le els, and ele a ed FURIN exp ession p omo es
me as a ic ac i i y in a ious cance ypes, and he p o ein
is ound in ad anced a he oscle o ic plaques [7-10].
FURIN is an impo an modula o o he T-cell-dependen
adap i e immuni y; i becomes up egula ed by T-cell-
ecep o -media ed signaling and h ough he IL-12/STAT4
pa hway in T helpe ype 1 cells [11, 12]. A condi ional
dele ion o FURIN in T cells esul s in he abe an
pola iza ion o T helpe cells, a lack o a p o ec i e
cell-media ed hos -de ense as well as he spon aneous
de elopmen o au oimmuni y in aging animals due o
a b eakage in pe iphe al CD4+Foxp3+ T- egula o y-
cell-dependen immune ole ance [6, 13]. Consequen ly,
a ge ing he ac i i y o FURIN/PCSK has been epo ed
o be bene icial o he expe imen al ea men o , o
example, malignancies and heuma oid a h i is [9, 14-16].
P e ious epo s ha e also implica ed a ole o
PCSK enzymes in spleen [17]. FURIN is exp essed in
splenic ed pulp, a zone en iched wi h mac ophages,
which egula e ex amedulla y myelopoiesis, he emo al
o senescen ed cells, he c oss-p esen a ion o an igens
as well as ole ance o sel -an igens [18-20]. In addi ion,
PCSK1 de icien mice show a ma ked diso ganiza ion
o he ma ginal zone and ed pulp [21]. In addi ion o
he cellula p o-p o eins also he componen s o se e al
in ec ious agen s, including he en elopes o he HI and
In luenza i uses as well as he oxins o Bacillus an acii
and Pseudomonas ae uginosa equi e a PCSK-dependen
p o eolysis s ep o exe hei pa hogenic unc ion [22-24].
The e o e, inhibi o s can p o ec he hos om in ading
PCSK-dependen pa hogens and se e as adju an s o
an ibio ics [25]. As he inna e immune sys em o ms
he i s line o de ense, a ge ing PCSKs speci ically in
myeloid cells could be a po en and well- ole a ed s a egy
o block in ec ions. Howe e , he consequences o PCSK
inhibi ion speci ically in he cells o he inna e immune
sys em in i o ha e emained ambiguous. To add ess his
conund um we ha e he e cha ac e ized a no el issue-
speci ic knock-ou mouse model, in which FURIN is
dele ed in Lysozyme M posi i e cells, i.e. chie ly in
ac i a ed mac ophages and g anulocy es (LysMC e- u ( l/
l)) [26].
esul s And dIscussIon
lysMc e- u
( l/ l)
mice ha e a educed numbe
o splenocy es and ele a ed le els o he p o-
in lamma o y IL-1β cy okine in hei se um
P e ious s udies using ge m-line dele ions and
siRNA ha e demons a ed ha PCSK1 and PCSK7
modula e he sec e ion p ocess o inna e cy okines [21]
and escue uns able MHCI molecules on dend i ic cells,
espec i ely [27]. O he s and we ha e u he shown
ha FURIN is up egula ed in he LPS ac i a ed CD14+
cells [10], and in he plasma o ch onic yphoid ca ie s
[28]. To di ec ly add ess whe he FURIN exp ession in
myeloid cells egula es immuni y in i o, we gene a ed
a mouse model wi h a condi ional dele ion o FURIN
in LysM+ cells. LysMC e- u ( l/ l) mice we e bo n unde
no mal Mendelian a ios, and unlike T-cell speci ic
CD4C e- u ( l/ l) mice [6], hey did no show age- ela ed
heal h p oblems such as in lamma o y bowel disease
(supplemen a y Figu e S1A, B). No ably, in con as
o he epo edly la ge spleens o PCSK1 KO mice
[21] LysMC e- u ( l/ l) animals had signi ican ly educed
numbe s o splenocy es compa ed o con ols, which could
indica e ha FURIN egula es hema opoiesis in LysM+
cells, o cellula homeos asis in he spleen (Figu e 1A,
supplemen a y Figu e S1C, D). The mechanism(s) leading
o he educ ion in splenocy e numbe s in LysMC e- u ( l/
l) animals emains unclea , bu he lowe ed FURIN le els
in he bone ma ow (ca. 60% less mRNA exp ession,
supplemen a y Figu e S2A) could dis up he delica e
mic oen i onmen by educing he p o eoly ic ma u a ion
o hema opoie ic g ow h ac o s o ex acellula ma ix
ecep o s [29].
As expec ed, FURIN was almos comple ely
(>95% less mRNA) absen om bo h he neu ophils and
mac ophages o LysMC e- u ( l/ l) mice (supplemen a y
Figu e S2B, C). A low cy ome ic analysis demons a ed
ha he lack o FURIN did no signi ican ly al e he
p opo ions o mac ophages (F4/80+CD11b+) and
neu ophils (G 1+CD11b+) in he pe i oneal ca i y
(Figu e 1B), spleen (supplemen a y Figu e S3A) o bone
ma ow (supplemen a y Figu e S4A). We nex analyzed
whe he dele ing FURIN in myeloid cells imposed
seconda y e ec s on lymphoid cells, and ound ha he
g oss numbe s o splenic CD3+ T and B220+ B cells we e
una ec ed (supplemen a y Figu e S3B). Ins ead, he e
was a small, bu signi ican educ ion in he numbe s
o CD3+CD4+CD8- T helpe cells in he spleen while
he p opo ion o CD3+CD4-CD8+ cy o oxic T cells was
ele a ed (supplemen a y Figu e S3C). The lowe a io o
splenic CD4+/CD8+ T cells has p e iously been associa ed
wi h in lamma ion [30], bu he quan i ica ion o CD62L+
T cells did no e eal ma ked di e ences in he numbe s
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o memo y T cells be ween LysMC e- u
( l/ l)
and li e ma e
con ol animals (supplemen a y Figu e S3C). Collec i ely,
he analyses o di e en cell compa men s indica e ha
FURIN exp ession in LysM+ cells is no essen ial o
he de elopmen o neu ophils o mac ophages, bu i
p omo es he de elopmen o splenocy es and may also
indi ec ly a ec he CD4+/CD8+ T cell a io in he spleen.
We ha e p e iously demons a ed ha dele ing
FURIN in T cells esul s in he up egula ion o bo h he
Th1 and Th2 signa u e cy okines in se um [6]. To add ess
how FURIN exp ession in LysM+ cells con ols he
p oduc ion o inna e cy okines in s eady s a e we measu ed
he le els o p o-in lamma o y IL-1β, TNF-α, IL-6 and
MCP-1 cy okines as well as he an i-in lamma o y IL-10 in
se um (Figu e 1C). These da a demons a ed ha FURIN
exp ession in myeloid cells a enua es he p oduc ion o
p o-in lamma o y IL-1β, which is indica i e o an au o-
in lamma o y pheno ype [31, 32].
Genome-wide nA mic oa ay analysis e eals
he up egula ion o se e al p o-in lamma o y
genes in FURIN de icien mac ophages
Al hough p op o ein con e ases p ocess hei
a ge molecules pos - ansla ionally, dele ing PCSKs
also esul s in abe an gene exp ession signa u es, which
gi es clues abou he mechanism o he ac ions o PCSKs
in a gi en cell ype [6, 12, 33]. To add ess how FURIN
egula es he gene ic signa u e in mac ophages we p o iled
hei gene exp ession using qRT-PCR and mic oa ay
analyses. Simila ly o human CD14+ monocy es [10]
FURIN was e icien ly up egula ed in mouse pe i oneal
mac ophages ha we e ac i a ed wi h LPS+/- IFNγ in
i o, (supplemen a y Figu e S2B). The lack o FURIN
in endo helial cells (Tie2C e) causes he compensa o y
exp ession o o he biochemically edundan PCSKs
[5], which in e e es wi h he in e p e a ion o FURIN’s
biological signi icance. Howe e , we obse ed ha
dele ing FURIN in mac ophages did no a ec he
exp ession o o he PCSK enzymes (supplemen a y Figu e
S2D). These da a indica e ha he LysMC e-media ed
dele ion can be eliably used o assess he speci ic ole o
FURIN in LysM+ cells.
In o de o cha ac e ize he FURIN-dependen
global gene exp ession pa e ns we pe o med a genome-
wide mic oa ay analysis using es ing pe i oneal
mac ophages om LysMC e- u ( l/ l) and li e ma e WT
animals (Figu e 2A, 2B). FURIN de icien pe i oneal
mac ophages displayed a ep oducible up egula ion o
many genes exp essed in ac i a ed mac ophages, such
Figu e 1: P opo ions o splenocy es, pe i oneal mac ophages, neu ophils and se um cy okines in s eady s a e
lysMc e- u ( l/ l) mice. A. To al numbe s o li e splenocy es om LysMC e- u ( l/ l) (KO) and WT li e ma e con ol mice we e
calcula ed using ypan blue s aining (n = 7/geno ype, 6-8 weeks o age). b. Flow cy ome ic analyses o F4/80+CD11b+ (mac ophages)
and G 1+CD11b+ (neu ophils) pe i oneal cells we e pe o med om LysMC e- u ( l/ l) and WT li e ma e con ol mice (n = 4/geno ype, 6-8
weeks o age). c. Le els o se um cy okines om s eady s a e LysMC e- u ( l/ l) and WT li e ma e con ol mice (n = 6/geno ype, 6-8 weeks
old mice). Plo s ep esen a e age ± SEM. S a is ics we e calcula ed wi h he wo- ailed unpai ed S uden ’s - es .
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as Se pinb1a, Se pinb2, Hca 2 , Eg 1, Il6, Il1β, P gs2,
Ccl2, Ccl7 and C5a 1 [34-38]. In addi ion, we obse ed
an enhanced exp ession o Dusp6 and Fcg 1, which a e
down egula ed in al e na i ely ac i a ed mac ophages
[39]. In con as , among he down egula ed genes o
FURIN de icien mac ophages, we de ec ed o example
A 7 whose dele ion is associa ed wi h he cons i u i e
ac i a ion o mac ophages [40]. FURIN is induced by i s
subs a e TGF-β1 [41]. Acco dingly, he exp ession o
Ccnd1 was enhanced in he absence o FURIN, simila ly
o wha was seen in TGF-β1 null cells [42]. These da a
sugges ha FURIN has an in insic inhibi o y unc ion on
he exp ession o genes ha associa e wi h he ac i a ion
o p o-in lamma o y M1 ype mac ophages [43].
lysMc e- u
( l/ l)
mice show inc eased le hali y,
up egula ed p o-in lamma o y cy okines and
ele a ed numbe s o ly6-c+ mac ophages a e
an LPS injec ion
Bo h he high IL-1β le els in se um and he
esul s om he mic oa ay analysis poin ed o a p o-
in lamma o y pheno ype in LysMC e- u ( l/ l) mice. To
es i FURIN in LysM+ cells also es ains in lamma o y
esponses in i o we subjec ed mice o lipopolysaccha ide
(LPS) igge ed in lamma ion. LysMC e- u ( l/ l) and
li e ma e WT mice we e i s challenged wi h a single
i.p. injec ion o 25 mg/kg (LPS) and moni o ed o 72
hou s (Figu e 3A). We obse ed a signi ican ly highe
mo ali y in LysMC e- u ( l/ l) mice 24 hou s pos -injec ion
(62% mo ali y in KO s. 38% mo ali y in WT) (p =
Figu e 2: Genome-wide gene exp ession analyses in es ing wild ype and FURIN de icien pe i oneal mac ophages.
A. The 19 mos di e en ially (log old change > |1| and mul iple es ing co ec ed p- alue < 0.05) exp essed genes be ween wild ype and
FURIN de icien uns imula ed pe i oneal mac ophages. Fo isualiza ion pu poses, he mean o each gene’s exp ession has been sub ac ed
om indi idual exp ession alues o highligh he di e ences be ween he samples. The ed and g een colo s indica e an induc ion o
supp ession o gene exp ession, espec i ely, ela i e o he mean ac oss samples. The igu e shows wo biological eplica es o bo h
geno ypes. b. A sca e plo o he mean gene exp ession ac oss eplica es o wild ype and FURIN de icien uns imula ed pe i oneal
mac ophages. Selec ed genes wi h a log old change > |1| ha e been highligh ed. The o ange lines show he limi whe e he log old change
equals 1 o -1.
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0.0109), which indica es an inc eased sensi i i y o a p o-
in lamma o y s imulus.
A lowe dose o LPS (100 µg/kg) was hen used
o e alua e how he myeloid-cell-exp essed FURIN
egula es cy okine le els in he se um and he gene a ion/
mig a ion o p o-in lamma o y F4/80+CD11b+Ly6C+Ly6G-
monocy es/mac ophages, which a e cha ac e ized by
accele a ed in lamma o y e ec o unc ions including
he high p oduc ion o IL-6, IL-1β, TNF-α, NOS2 and
CCL2 [44, 45]. LPS-injec ed LysMC e- u ( l/ l) mice had
signi ican ly enhanced le els o he p o-in lamma o y
cy okines TNF-α and IL-6 1 and 3 hou s pos -in ec ion,
espec i ely, whe eas he p oduc ion o an i-in lamma o y
IL-10 was educed a he 3-hou ime-poin (Figu e 3B-
3D).
In addi ion, a 3 hou s pos -injec ion he e
was a signi ican augmen a ion in he p opo ion o
F4/80+CD11b+Ly6C+Ly6G- M1 ype mac ophages in
he spleen (Figu e 3E). A qRT-PCR analysis on ex i o
ac i a ed FURIN KO mac ophages u he showed an
up egula ion in he exp ession o Nos2 (M1 ma ke gene)
upon an LPS/IFN-γ s imulus, whe eas he IL-4 induced
exp ession o A g1 (M2 ma ke gene) was clea ly educed
[43] (Figu e 3F). P e iously, he p ominence o he M1
o e he M2 mac ophage pheno ype has been associa ed
wi h an inc eased suscep ibili y o a sep ic shock [46, 47],
as was obse ed in LPS-challenged LysMC e- u
( l/ l)
mice.
PCSKs con ol he quan i y and ac i a ion o he
human Toll-like ecep o 7 (TLR7) hos esponses by
di ec p o eolysis [48]. We nex es ed i he exp ession
le els o LPS/TLR4-dependen genes a e a ec ed by he
FURIN de iciency in pe i oneal mac ophages. Fi s , he
ime cou se changes in he mRNA exp ession o he Il1b,
Tn a, Il6 and Il10 genes indica ed an up egula ed base
line exp ession bu oughly simila dynamics in esponse
o LPS in FURIN de icien mac ophages compa ed o
Figu e 3: LPS-challenged LysMC e- u ( l/ l) mice exhibi highe mo ali y, accele a ed in lamma ion and an up egula ed
p o-in lamma o y mac ophage esponse. A. Kaplan Meie su i al cu e o LysMC e- u ( l/ l) and WT li e ma e con ol mice (n
= 8/geno ype, 6-8 weeks o age, all male) a e he in ape i oneal injec ion o a single le hal dose o LPS (25 mg/kg). LysMC e- u ( l/ l)
mice exhibi ed a signi ican ly highe mo ali y han WT li e ma e con ols du ing he i s 24 h. b.-d. Le el o se um cy okines TNF-α,
IL-6, IL-10 in LysMC e- u ( l/ l) and WT li e ma e con ols mice (n = 4/geno ype, 6-8 weeks o age) a e an in ape i oneal injec ion wi h
NaCl (0.9%) o 100 µg/kg o LPS (plo s ep esen a e age ± SEM). S a is ics we e calcula ed wi h he wo- ailed unpai ed S uden ’s - es .
e. Flow cy ome ic analysis o a splenic F4/80+CD11b+Ly6C+Ly6G- cell popula ion in LysMC e- u ( l/ l) mice and WT li e ma e con ol
mice (n = 3/geno ype, 6-8 weeks o age) injec ed wi h NaCl (0.9%) o LPS (100 µg/kg) a 0, 1h and 3h. The a ows indica e he ga ing
s a egy. (Plo s ep esen a e age ± SEM). S a is ics we e calcula ed using he wo- ailed unpai ed S uden ’s - es . F. Nos2 and A g1 mRNA
exp ession was assessed by quan i a i e RT-PCR in wild ype and FURIN de icien pe i oneal mac ophages ea ed wi h LPS+IFN-γ (1 µg/
ml+20 ng/ml) o IL-4 (50 ng/ml). One ep esen a i e expe imen ou o h ee independen expe imen s is shown.

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con ols (Figu e 4A). Likewise, a ocused analysis o
TLR-associa ed mRNAs using mic oa ay da a showed
only minimal di e ences be ween he dynamic exp ession
pa e ns o WT and FURIN KO mac ophages (Figu e 4B).
Howe e , he dynamic exp ession o se e al o he p o-
in lamma o y genes, including T em1, Nos2, Il15, Il33,
and Il12 b1, was inhibi ed by FURIN in LPS ac i a ed
mac ophages (Figu e 4C) [49-52]. These indings we e
accompanied by he ep ession o he genes Ch25h, Ol 1
and A g1, which a e ypically induced in al e na i ely
ac i a ed mac ophages [38]. Finally, we e alua ed he
exp ession o p o- and an i-in lamma o y cy okines in
pe i oneal mac ophages ha we e s imula ed wi h a ious
TLR ligands and IFN-γ (supplemen a y Figu e S5). These
esul s show ha he an i-in lamma o y unc ion o FURIN
canno be a ibu ed o a speci ic s imulus, bu can be seen
in TLR2, TLR4 and TLR7/8 ac i a ed mac ophages.
Collec i ely, hese da a sugges ha FURIN is dispensable
o he immedia e TLR4 esponses, bu unde sco e he
impo ance o FURIN as an an i-in lamma o y ac o in
bo h es ing and ac i a ed mac ophages.
FURIN de icien mac ophages sec e e less
bioac i e TGFβ-1 cy okine bu show ele a ed
exp ession le els o he ac i a ed TNF-α
Con e ing Enzyme (TACE) and Caspase-1 p20
The p oduc ion o se e al cy okines sec e ed by
mac ophages, including TNF-α, IL-1β and TGF-β1, is
dependen on p o eoly ic p ocessing in he cell [53].
Speci ically, TGF-β1 is ini ially p oduced as an inac i e
Figu e 4: LPS-induced gene exp ession dynamics in wild ype and FURIN de icien pe i oneal mac ophages. A. mRNA
exp ession le els we e de e mined using quan i a i e RT-PCR, and he no malized exp ession in he uns imula ed WT sample was a bi a ily
se o 1. Cu es show changes in he exp ession o Il1b, Tn a, Il6 and Il10 mRNAs in WT and FURIN KO pe i oneal mac ophages ha
we e le uns imula ed o we e s imula ed wi h LPS (1µg/ml) o 1-24h as indica ed. The housekeeping gene 18S was used o no malize
he gene exp ession. (The igu e shows one ep esen a i e expe imen o wo independen expe imen s ± SEM). b. Mic oa ay analysis o
he TLR signaling pa hway genes. Log2 old changes o all genes in bo h WT and FURIN KO pe i oneal mac ophages we e compu ed
o each ime poin wi h an LPS (1µg/ml) s imula ion, espec i e o he uns imula ed condi ion. A each ime poin , he a io o he old
changes illus a es he deg ee o di e ence in he LPS esponse o e ime. The igu e shows he a e age o wo independen expe imen s.
The ed colo indica es an inc ease and he g een a dec ease in he log2 old change di e ence. c. Genome wide analysis o he genes wi h
he s onges di e en ial esponse o a LPS s imula ion be ween WT and FURIN KO pe i oneal mac ophages. A selec ion o di e en ially
esponding genes showing g ea es di e ences be ween a ios a one o se e al ime poin s a e p esen ed. Da a we e p ocessed as in B.
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p o-cy okine, which is con e ed in o an ac i e ac o ia
a complex pos - ansla ional cascade in ol ing a clea age
s ep ca alyzed by FURIN [6, 54]. Fu he , unde mined
TGF-β1 signaling has been associa ed wi h an impai ed
ansi be ween he mac ophage (M1/M2) pheno ypes,
sus ained in lamma ion and delayed wound healing [55].
Also, TNF-α Con e ing Enzyme (TACE), which eleases
soluble TNF-α om i s memb ane-bound p ecu so , is
p o eoly ically ac i a ed by a FURIN-like p op o ein
con e ase [56] whose dele ion esul s in he de elopmen
o an an i-in lamma o y pheno ype in mac ophages [57].
In addi ion, he ac i a ion/deac i a ion o he Caspase-1
cascade, which di ec ly p ocesses IL-1β, plays an
impo an ole in he dynamics o mac ophage pola iza ion
[58]. P e ious da a also imply a unc ional connec ion
be ween PCSK ac i i y and IL-1β; he p o eoly ic clea age
o he an h ax oxin by FURIN ac i a es Caspase-1 in
mac ophages [59] and high le els o IL-1β ha e been
obse ed in an expe imen al model o a h i is in mice ha
we e ea ed wi h a FURIN inhibi o [60].
In o de o ind ou i FURIN egula es he
p o eoly ic ma u a ion o he a o emen ioned in lamma o y
cy okines in mac ophages we analyzed he sec e ion o
bioac i e TGF-β1 as well as he p o eoly ic ac i a ion o
TACE and Caspase-1. Fi s , FURIN KO mac ophages
we e ound o sec e e signi ican ly lowe le els o
bioac i e TGF-β1 in ELISA analyses han cells om wild-
ype con ols, whe eas he Tg b1 mRNA le els we e no
a ec ed (Figu e 5A, supplemen a y Figu e S6). In con as ,
we ound ha he lack o FURIN up egula ed he ma u e
TACE p o ein in LPS-s imula ed pe i oneal mac ophages
(Figu e 5B), which is in line wi h he ele a ed TNF-α
p oduc ion in LPS-challenged LysMC e- u ( l/ l) mice in
i o (Figu e 3B). These da a also indica e ha FURIN is
no he bona- ide PCSK ha ac i a es TACE, and sugges
ha ano he mac ophage-exp essed and LPS-induced
PCSK enzyme, such as PCSK6 o PCSK7 (supplemen a y
Figu e S2D), could be mo e impo an o he ma u a ion
o TACE in i o [61]. Finally, an analysis o Caspase-1
p ocessing also showed a highe p oduc ion o Caspase-
1p20 in FURIN de icien mac ophages (Figu e 5C).
These da a a e consis en wi h he obse ed highe le els
o se um IL-1β and e eal a no el p o eoly ic egula o
(FURIN) o his key p o-in lamma o y cy okine.
The analyses o p o eoly ically ac i a ed
mac ophage cy okines hus showed ha a FURIN
de iciency educes he p oduc ion o an i-in lamma o y
TGF-β1 bu up egula es he ac i a ion o he TACE
Figu e 5: FURIN de icien mac ophages p oduce less bioac i e TGF-β1 and show hype ac i a ion o TACE and
Caspase-1 p20. A. The p oduc ion o bioac i e TGF-β1 was measu ed in he supe na an s o uns imula ed wild ype and FURIN de icien
pe i oneal mac ophages using an enzyme linked immunoso ben assay (ELISA) (n = 3/geno ype). Plo s ep esen a e age ± SEM. S a is ics
we e calcula ed wi h he wo- ailed unpai ed S uden ’s - es . b. WT and FURIN de icien pe i oneal mac ophages we e le uns imula ed o
we e s imula ed wi h LPS (1 µg/ml) o 5-30 minu es as indica ed. Ma u e Tumo Nec osis Fac o -α-Con e ing Enzyme (TACE) (93 kDa)
and β-Ac in (43 kDa) we e de ec ed by wes e n blo ing. No malized (TACE/Ac in) le els a e p esen ed in he uppe panel. Shown is one
ep esen a i e expe imen ou o h ee eplica es wi h simila esul s. c. FURIN de icien o li e ma e wild ype bone ma ow mac ophages
(n = 2/geno ype) we e le uns imula ed o we e s imula ed wi h ul apu e LPS (500 ng/ml) o 2 hou s, hen ATP (1mM) was added o he
cells o 15 min. P o-Caspase (55 kDa), Caspase-1 p20 (20 kDa) and ac in (43 kDa) we e de ec ed wi h wes e n blo ing.
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and Caspase-1 enzymes. These indings oge he wi h
he inhe en up egula ion o p o-in lamma o y mRNAs
collec i ely con ibu e o he p o-in lamma o y pheno ype
o LysMC e- u ( l/ l) mice. Unde s anding he unde lying
mechanism(s) o abe an cy okine exp essions by
FURIN de icien mac ophages clea ly equi es u he
s udies, bu a leas educed TGFβ-1 ma u a ion is likely
o play a ole [62]. In conclusion, ou da a indica e ha
inhibi ing FURIN speci ically in inna e immune cells
could s eng hen hos esponses. This, oge he wi h he
plausible educ ion in he ac i a ion o pa hogens, could
be bene icial o ea ing and p e en ing PCSK-dependen
in ec ions.
MA e IAls And Me hods
Expe imen al animals
Mice bea ing loxed u alleles [4] we e backc ossed
six imes wi h C57BL/6 mice. LysMC e mice wi h he
C57BL/6 backg ound we e pu chased om Taconic.
LysMC e mice we e b ed wi h u
( l/ l)
animals o gene a e
myeloid-speci ic FURIN knockou mice LysMC e-
u ( l/ l). Mice we e housed unde pa hogen- ee s anda d
condi ions. All mouse expe imen s we e pe o med in
acco dance wi h he Na ional Animal Expe imen Boa d,
Finland, (Pe mi # ESAVI/2837/04.10.07/2015).
Isola ion, cul u e and ex i o ac i a ion o
mac ophages om he mouse pe i oneal ca i y
and o neu ophils om he bone ma ow
Pe i oneal cells we e ex ac ed om he pe i oneal
ca i y and cul u ed ollowing a p e iously published
p o ocol [63]. A e 1 h incuba ion WT and FURIN
de icien pe i oneal mac ophages we e s imula ed wi h
LPS (1 µg/ml, E. coli 0127:B8 se o ype; Sigma-Ald ich,
S Louis, MO, USA), TLR ligands (Zymosan: 10 µg/ml,
R848: 1 µg/ml; bo h om In i oGen, San Diego, CA,
USA) and/o cy okines (IFN-γ: 20 ng/ml, IL-4: 50 ng/ml;
Pep oTech, Rocky Hill, NJ, USA). A e s imula ion, he
cells we e sc aped o dislodge hem and collec ed o RNA
ex ac ion (RNAeasy, Qiagen, Düsseldo , Ge many).
Neu ophils we e isola ed om he bone ma ow o
LysMC e- u ( l/ l) and wild- ype mice wi h he An i-Ly-6G
Mic oBead Ki (Mil enyi Bio ec No den AB, Lund, SE)
and we e le uns imula ed o we e s imula ed wi h 100
µM o MLP o 1 hou a 37 °C.
Flow cy ome ic analyses
Pe i oneal, splenic and bone ma ow cells we e
analyzed using low cy ome y (BD FACSCan o™ II)
and he FlowJo so wa e (T ees a Inc, Ashland, OR,
USA). An i-CD16/CD32 (Clone: 93) (eBioscience,
San Diego, CA, USA) was used o block Fc ecep o s
and cell popula ions we e su ace s ained ollowing he
eBioscience FACS p o ocol wi h Pe CP-Cy5.5- labeled
an i- F4/80 (BM8), PE-labeled an i-G -1 (RB6-8C5), PE-
Cy7-labeled an i-CD11b (M1/70), FITC-labeled an i-B220
(RA3-6B2), APC-labeled an i-CD3, PE-Cy7 labeled an i-
CD4 (RM4-5), APC-H7 labeled an i-CD8 (53-6.7), PE-
labeled an i-CD62L (MEL-14), PE-labeled an i-Ly-6G
(RB6-8C5), APC labeled an i-Ly-6C (HK1.4), PE-Cy7-
labeled an i-CD11c (N418), FITC-labeled an i-CD11b
(M1/70) (eBioscience, San Diego, CA, USA).
Cy okine measu emen s
Se um cy okines we e measu ed in bo h s eady
s a e and LPS injec ed mice. B ie ly, LysMC e- u ( l/ l)
and WT li e ma e mice (6-8 weeks o age) we e injec ed
in ape i oneally wi h NaCl (0.9%) o LPS (100 µg/kg,
E. coli 111:B4 se o ype; Sigma-Ald ich) and se um was
collec ed a he 0, 1, 3 h ime poin s. The se um cy okines
TNF-α, IL-6, MCP-1 and IL-10 we e quan i ied using
he BD™ Cy ome y Bead A ay Mouse In lamma ion
Ki (Ca alog # 552364) acco ding o he manu ac u e ’s
ins uc ions (BD Biosciences, F anklin Lakes, NJ, USA).
IL-1β le els we e measu ed wi h mouse IL-1 be a ELISA
Ready-SET-Go!® (eBioscience, San Diego, CA, USA)
(Ca alog # 88-7013-22). Bioac i e TGF-β1 was measu ed
in he supe na an s o cul u ed pe i oneal mac ophages
isola ed om LysMC e- u ( l/ l) and WT li e ma e mice
using Human/Mouse TGF be a 1 ELISA Ready-SET-Go!
(2nd Gene a ion) (Ca alog # 88-8350-76) (eBioscience,
San Diego, CA, USA).
LPS induced endo oxemia
LysMC e- u
( l/ l)
and WT li e ma e mice (6-8 weeks
o age) we e injec ed in ape i oneally wi h a le hal dose o
LPS (25 mg/kg, E. coli 0111:B4 se o ype; Sigma Ald ich,
S Louis, MO, USA) o wi h NaCl (0.9%) as a con ol.
Su i al o he mice was moni o ed o 72 h a 3-hou
in e als.
Quan i a i e eal- ime polyme ase chain analyses
The mRNA exp ession in s imula ed and non-
s imula ed pe i oneal mac ophages was assessed using
qRT-PCR. B ie ly, o al RNA was ex ac ed using he
RNeasy ki (Qiagen, Düsseldo , Ge many), quan i ied
using a NanoD op ND-2000 (The mo Fishe Scien i ic,
Massachuse s, USA) and ansc ibed in o cDNA wi h he
iSc ip Selec cDNA Syn hesis Ki (Bio-Rad, He cules,
CA, USA). Gene exp ession le els we e examined using
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he Bio-Rad CFX96 Real-Time Sys em and he Sso Fas
E a G een Supe mix (Bio-Rad, He cules, CA, USA). The
p ime s used o quan i a i e eal- ime PCR a e shown
in he supplemen a y Table S1. Gene exp ession was
no malized o 18s le els; he no malized h eshold cycle
(C ) alues we e sub ac ed om he a ge C alues o
each sample (del aC ). The ela i e le els o he a ge
mRNA we e calcula ed as 2-del adel aC .
Mic oa ay da a analysis
Two biological eplica es o FURIN KO and
WT pe i oneal mac ophages we e le uns imula ed o
we e s imula ed o 1, 4 and 24 hou s wi h LPS (1 µg/
ml)(E. coli 0127:B8 se o ype; Sigma Ald ich, S Louis,
MO, USA). The samples we e collec ed and he RNA
was isola ed using Qiagen RNeasy on-column DNase
(Qiagen, Düsseldo , Ge many). 50 ng o pu i ied RNA
was ampli ied and dye labeled using Agilen ’s Low Inpu
Quick Amp Labeling ki and RNA Spike In ki (Agilen
Technologies, Cali o nia, USA). Ini ial and pu i ied RNA
con en s along wi h dyed and ampli ied cRNA con en s
we e measu ed wi h a NanoD op ND-2000 (The mo Fishe
Scien i ic, Massachuse s, USA). RNA and cRNA quali y
was inspec ed using he Agilen 2100 Bioanalyze RNA
6000 Nano ki (Agilen Technologies, Cali o nia, USA).
300ng o Cy-3 and 300ng o Cy-5 labeled sample we e
hyb idized oge he on an Agilen Mouse Chip 8x60K
(Design ID 028005) (Agilen Technologies, Cali o nia,
USA) o e nigh a 65°C using he Gene Exp ession
Hyb idiza ion ki . The chips we e washed wi h he Gene
Exp ession Wash Pack acco ding o he ins uc ions. The
chips we e scanned using an Agilen Technologies Scanne
model G2565CA using he scan p o ile Agilen G3_
GX_2Colo . Scan esul s we e con e ed in o nume ical
da a by he Agilen Fea u e Ex ac ion so wa e e sion
10.7.3. The da a was analyzed using he R so wa e
[64]. Raw p obe le el in ensi y alues we e no malized
wi h loess eg ession and quan ile no maliza ion using
R package “limma” o obus ly gene a e sample-wise
compa abili y [65].
Di e en ial gene exp ession was e alua ed
using he log2- ans o med old change di e ence and
s a is ical es ing using he wo-sample S uden ’s - es .
Genes showing an a e age exp ession lowe han he
log2 ans o med in ensi y alue 6 we e il e ed ou as
non-in o ma i e. The Kyo o Encyclopedia o Genes and
Genomes (KEGG) pa hway da abase was used o ob ain
a gene se in ol ed in Toll-like ecep o signaling. [66].
The aw mic oa ay da a a e a ailable a Gene Exp ession
Omnibus (GEO): h p://www.ncbi.nlm.nih.go /geo/que y/
acc.cgi? oken=ililkmqq ql yn&acc=GSE84117.
Wes e n blo analyses
Fo he Caspase-1 expe imen bone ma ow
mac ophages we e cul u ed using a s anda d p o ocol
[63] and we e le uns imula ed o we e s imula ed o
6 hou s wi h ul apu e LPS (E. coli 0111:B4, 500 ng/
ml; In i oGen, San Diego, CA, USA). Cells we e
washed wice wi h PBS and incuba ed wi h ATP
(1mM, Sigma Ald ich, S Louis, MO, USA). Nex , he
cells we e collec ed and lysed in a bu e speci ic o
Caspase-1 (50 mM T is, pH 7.4, 150 mM NaCl, 2 mM
E hylenediamine e aace ic acid (EDTA) pH: 8.2 mM
E hyleneglycol e aace ic acid (EGTA) pH: 7.5, 10%
Glyce ol, 1% T i on X-100, 50 mM Sodium luo ide, 200
μM Sodium anada e) [67]. Fo he TACE-1 expe imen ,
pe i oneal mac ophages we e le uns imula ed o we e
s imula ed wi h LPS o 5-30 minu es (1 µg/ml, E. coli
0127:B8 se o ype; Sigma Ald ich, S Louis, MO, USA).
Nex , he cells we e collec ed and lysed in a bu e speci ic
o TACE (1% T i on X-100, 15 mM NaCl, 50 mM T is
HCL pH 7.4, p o ease inhibi o s cock ail (Roche), 10mM
1,10-phenan h oline) [68]. Equal amoun s o p o eins we e
sepa a ed on 12% polyac ylamide gels and ans e ed
on o ni ocellulose memb anes (Wha man, GE Heal hca e,
Polla ds Wood, UK). The p o eins we e p obed wi h a
p ima y an ibody, ei he an i-ADAM17-cy oplasmic
domain (ab39162; abcam) o an i-caspase-1- /ICE
(AHZ0082; In i ogen), ollowed by a HRP-conjuga ed
seconda y an ibody (RnD sys ems, Minneapolis, MN,
USA). An i-ac in (MAB1501R; Me ck Millipo e,
Bille ica, MA, USA) was used as a loading con ol.
Immunoblo s we e isualized wi h an ECL de ec ion
sys em (GE Heal hca e, Polla ds Woods, UK). Signal
in ensi ies we e analyzed using he NIH ImageJ so wa e.
s a is ical analysis
S a is ical analyses we e pe o med using he
wo- ailed unpai ed S uden ’s - es . P ˂ 0.05 indica es
s a is ical signi icance and he a iabili y is depic ed using
he s anda d e o o he mean (SEM). The Kaplan Meie
es was applied o es ima e su i al in he LPS induced
endo oxemia expe imen .
con en ions and Abb e ia ions
PCSK: P op o ein Con e ase Sub ilisin/Kexin
ype; FBS: Fe al Bo ine Se um; ELISA: Enzyme-Linked
Immunoso ben Assay; CBA: Cy ome ic Bead A ay;
qRT-PCR: quan i a i e Re e se T ansc ip ion Polyme ase
Chain Reac ion; IL: In e leukin; TLR: Toll Like Recep o ;
LPS: Lipopolysaccha ide; R848: Resiquimod; IFN-γ:
In e e on gamma; TNF-α: Tumo Nec osis Fac o
alpha; MCP-1: Monocy e Chemo ac ic P o ein 1; TACE: