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In elec in 3 is dispensable o
esis ance agains a mycobac e ial
in ec ion in zeb a ish (Danio e io)
Ma kus J. T. Ojanen1,2, Me i I. E. Uusi-Mäkelä1, Sanna-Kaisa E. Ha jula1, Anni K. sa alah i1,
Kaisa E. Oksanen1, Niklas Kähkönen3, Juha A. E. Mää ä3, Vesa P. Hy önen
3,
Ma ko Pesu
2,4 & Mika Räme 1,5,6,7
Tube culosis is a mul i ac o ial bac e ial disease, which can be modeled in he zeb a ish (Danio
e io). Abdominal ca i y in ec ion wi h Mycobac e ium ma inum, a close ela i e o Mycobac e ium
ube culosis, leads o a g anuloma ous disease in adul zeb a ish, which eplica es he di e en phases
o human ube culosis, including p ima y in ec ion, la ency and spon aneous eac i a ion. He e,
we ha e ca ied ou a ansc ip ional analysis o zeb a ish challenged wi h low-dose o M. ma inum,
and iden i ied in elec in 3 (i ln3) among he highly up- egula ed genes. In o de o cla i y he in
i o signi icance o I ln3 in immuni y, we c ea ed nonsense i ln3 mu an zeb a ish by CRISPR/Cas9
mu agenesis and analyzed he ou come o M. ma inum in ec ion in bo h zeb a ish emb yos and adul
ish. The lack o unc ional i ln3 did no a ec su i al o he mycobac e ial bu den in he zeb a ish.
Fu he mo e, emb yonic su i al was no a ec ed when ano he mycobac e ial challenge esponsi e
in elec in, i ln1, was silenced using mo pholinos ei he in he WT o i ln3 mu an ish. In addi ion,
M. ma inum in ec ion in dexame hasone- ea ed adul zeb a ish, which ha e lowe ed lymphocy e
coun s, esul ed in simila bac e ial bu den in bo h WT ish and homozygous i ln3 mu an s. Collec i ely,
al hough i ln3 exp ession is induced upon M. ma inum in ec ion in zeb a ish, i is dispensable o
p o ec i e mycobac e ial immune esponse.
Tube culosis is an epidemic mul i ac o ial disease caused by Mycobac e ium ube culosis1. The suscep ibili y o
ube culosis depends on he M. ube culosis s ain and on a numbe o hos - ela ed ac o s such as en i onmen-
al condi ions, o he unde lying diseases as well as gene ic a ia ion2–4. C i ical genes o he adap i e immuni y
equi ed o he mycobac e ial immune esponse such as in e e on gamma (IFNG)5,6 and in e leukin 12 (IL12)7,8
we e iden i ied al eady in he 1980’s and 1990’s, espec i ely. The impo ance o hese genes has la e been e i ied
in human ube culosis pa ien s9 and by using expe imen al gene knockou mouse models o ube culosis10–13.
Mo e ecen ly, pa e n ecogni ion ecep o (PRR) gene polymo phisms o Toll-like ecep o s (TLRs)14–16 and
C- ype lec ins17,18, ha e been associa ed wi h M. ube culosis suscep ibili y, delinea ing also he cen al ole o he
inna e immuni y in con olling he mycobac e ial in ec ion.
Lec ins a e ca bohyd a e-binding p o eins impo an o nume ous biological p ocesses such as in acellula
glycop o ein sec e ion, leukocy e a icking and mic obial ecogni ion19,20. Consequen ly, lec ins ac as ecog-
ni ion molecules inside cells, on he cell su ace and in ex acellula luids20. In elec ins (ITLNs) a e a dis inc
amily o lec ins, which we e i s iden i ied in Xenopus lae is21 and we e la e ound in a numbe o cho da es
including human, mouse and zeb a ish (Danio e io)22–25. Al hough ITLN unc ion has been linked o a numbe
o p ocesses such as i on abso p ion26, me abolic diso de s27 as well as cance de elopmen 28,29, hei exac bio-
logical unc ions a e elusi e. Sugges ing a ole o ITLNs in he immune esponse, i ln gene exp ession is highly
up- egula ed upon a bac e ial in ec ion in ish25,30–32. Mo eo e , human ITLN1 (also known as Omen in) has been
1Labo a o y o expe imen al immunology, BioMedi ech ins i u e and acul y o Medicine and Li e Sciences,
Uni e si y o ampe e, ampe e, inland. 2Labo a o y o immuno egula ion, BioMedi ech ins i u e and acul y o
Medicine and Li e Sciences, Uni e si y o ampe e, ampe e, inland. 3Labo a o y o P o ein Dynamics, BioMedi ech
ins i u e and acul y o Medicine and Li e Sciences, Uni e si y o ampe e, ampe e, inland. 4Depa men o
De ma ology, ampe e Uni e si y Hospi al, ampe e, inland. 5Depa men o Pedia ics, ampe e Uni e si y
Hospi al, ampe e, inland. 6Depa men o child en and Adolescen s, Oulu Uni e si y Hospi al, Oulu, inland.
7PeDeGO Resea ch Uni and Medical Resea ch cen e Oulu, Uni e si y o Oulu, Oulu, inland. co espondence and
eques s o ma e ials should be add essed o M.R. (email: [email p o ec ed])
Recei ed: 9 Oc obe 2018
Accep ed: 7 Decembe 2018
Published: xx xx xxxx
OPEN
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shown o bind o he Mycobac e ium bo is Bacillus Calme e-Gué in (BCG)33, and mo e speci ically o exocyclic
1,2-diol glycan epi opes ha a e exp essed selec i ely on mic obial su aces34.
The impo ance o ITLNs o immuni y in i o, howe e , is less clea . P e iously, Voeh inge e al., used ans-
genic mice wi h lung-speci ic ITLN1 and ITLN2 o e -exp ession o s udy he e ec s o hese p o eins in he
mouse in ec ion models o he pa asi e Nippos ongylus b asiliensis and he M. ube culosis bac e ium35. In hese
se ings, he au ho s could no de ec enhanced pa hogen clea ance in he I ln ansgenic mice. In con as , a so
called “na u al dele ion” o he I ln2 gene has been p e iously associa ed wi h a highe suscep ibili y agains he
pa asi e T ichinella spi alis in a C57BL/10 mouse s ain36. Recen ly, an I ln1 knockou mouse s ain was c ea ed
o s udy in lamma o y bone diseases37. In he a o emen ioned s udy, he lack o I ln1 was associa ed wi h a p oin-
lamma o y pheno ype cha ac e ized by ele a ed TNF and IL6 le els in bone issue and in se um, and was shown
o esul in os eopo osis37.
The genome o he zeb a ish was assembled o he i s ime in 2002 and he p e alen 11 h assembly
(GRCz11) is an in aluable ool o esea ch using zeb a ish as a disease model38. O e 70% o human genes ha e
a leas one zeb a ish o hologue and o his eason, he zeb a ish immune sys em is highly simila compa ed
o humans38. In ac , mos o he human immune cell popula ions such as T- and B-cells39–41, neu ophils and
mac ophages42, dend i ic cells43 as well as he complemen sys em44 and immunoglobulins45,46, a e ound in he
zeb a ish. Impo an ly, zeb a ish can be modi ied gene ically wi h he clus e ed egula ly in e spaced sho pal-
ind omic epea s (CRISPR)/CRISPR-associa ed 9 (Cas9) mu agenesis47,48, which allows disease modeling using
e e se gene ics, al hough some genes appea di icul o a ge success ully49.
A Mycobac e ium ma inum in ec ion o zeb a ish is nowadays a commonly used model o s udying ube cu-
losis in bo h la ae and adul ish50,51. Compa ed o se e al o he ube culosis models, he mycobac e ial model
o zeb a ish is conside ed sa e, cos -e ec i e and e hical52,53. Mo e impo an ly, M. ma inum is closely ela ed o
M. ube culosis, and he wo bac e ial species ha e compa able pa hogenic cha ac e is ics in he na u al hos s;
mac ophage media ed in acellula mul iplica ion as well as he o ma ion o g anuloma s uc u es54–56. The la al
model enables s udying speci ically he inna e immuni y57,58, whe eas he adul zeb a ish model allows s udying
also componen s o he adap i e immune sys em in bo h an acu e mycobac e ial in ec ion59 as well as du ing
mycobac e ial la ency56,60.
In o de o iden i y candida e genes associa ed wi h he hos esponse agains mycobac e ia, we conduc ed a
gene exp ession mic oa ay in M. ma inum in ec ed adul zeb a ish. He e, we iden i ied a zeb a ish ITLN o ho-
logue i ln3 among he genes ha we e mos induced upon in ec ion. In o de o gain mo e insigh s in o he unc-
ion o ITLNs, we used CRISPR/Cas9 mu agenesis o c ea e knockou i ln3 mu an zeb a ish lines, and used he
zeb a ish M. ma inum in ec ion model o de e mine he in i o signi icance o I ln3 in a mycobac e ial in ec ion.
Resul s
Genome-wide gene exp ession mic oa ay analysis o M. ma inum in ec ed adul zeb a ish. In
o de o iden i y genes in ol ed in he hos immune esponse agains mycobac e ia, we used he zeb a ish M.
ma inum in ec ion model and conduc ed a genome-wide gene exp ession analysis using he mic oa ay pla o m.
To his end, we in ec ed wild- ype (WT) AB zeb a ish wi h M. ma inum (20 CFU; SD 6 CFU) and isola ed hei
o gan blocks (includes all he o gans o he abdominal ca i y) o a ansc ip omic analysis a 14 days pos in ec-
ion (dpi). F om a o al o 43603 p obes used in he analysis, we ound 93 p obes, co esponding o 70 genes, ha
we e up- egula ed and 26 p obes, co esponding o 21 genes, ha we e down- egula ed (log2 old change >
3
)
compa ed o he mock- ea ed (PBS) con ols (Supplemen a y Table1). Fu he e alua ion o he up- egula ed
p obes wi h a GO illa gene on ology (GO) en ichmen analysis61,62 e ealed 22 en iched (p < 0.001) p ocesses
including esponse o ca bohyd a es (GO:0009743), choles e ol homeos asis (GO:0042632) and an igen p ocess-
ing and p esen a ion (GO:0019882) (Supplemen a y Table2). Among he up- egula ed genes we ound i e genes;
si:busm1-194e12.11 (mhc2 amily gene), a achidona e 5-lipoxygenase b, andem duplica e 3 (alox5b.3), zgc:113912
(mhc2 amily gene), CD59 molecule (cd59) and si:busm1-194e12.12 (mhc2 amily gene) wi h well-known immu-
nological unc ions in an igen p ocessing, in lamma ion and in he egula ion o he complemen sys em (Fig.1A,
Supplemen a y Table1). O he 21 down- egula ed genes, i e we e associa ed wi h he immune esponse; CD58
molecule (cd58), myeloid-speci ic pe oxidase (mpx), complemen ac o b-like (c bl), immuno esponsi e gene 1, like
(i g1l) and si:busm1-266 07.1 (mhc2 amily gene) (Fig.1A, Supplemen a y Table1). In e es ingly, app oxima ely
38% o he up- egula ed p obes i.e. pa albumin 1 (p alb1), alpha- opomyosin ( pma), oponin I, skele al, as 2b,
andem duplica e 2 ( nni2b.2) and myosin, hea y polypep ide 1.1 (myhz1.1) we e ela ed o muscle associa ed bio-
logical p ocesses including muscle con ac ion (GO:0006936), muscle sys em p ocess (GO:0003012) and myo i-
b il assembly (GO:0030239) (Supplemen a y Tables1 and 2). The GO-analysis o he down- egula ed p obes also
showed a signi ican en ichmen o ano he 22 p ocesses including esponse o ex e nal bio ic s imulus (GO:
0043207) and choles e ol biosyn he ic p ocess (GO:0006695) and immunological p ocesses, such as esponse o
o he o ganism (GO:0051707), esponse o bac e ium (GO:0009617) and he induc ion o bac e ial agglu ina ion
(GO:0043152) (Supplemen a y Table2).
Mycobac e ial in ec ion up- egula es i ln3 exp ession in bo h zeb a ish emb yos and
adul ish. P e ious s udies in se e al animal models ha e shown he exp ession o he In elec in (ITLN)
gene o be induced upon a bac e ial in ec ion25,30,32. Acco dingly, he exp ession o he zeb a ish i ln3
(ENSDARG00000003523) was inc eased on a e age 3.3- old (log2 change) upon a M. ma inum in ec-
ion in ou mic oa ay analysis (Fig.1A, Supplemen a y Table1). In con as , wo o he i ln genes; i ln2
(ENSDARG00000036084) and i ln2-like (ENSDARG00000093796) we e down- egula ed compa ed o he
PBS con ols (−3.5 and −3.2 log2 old change, espec i ely) (Fig.1A, Supplemen a y Table1), sugges ing a
di e se egula ion o i ln genes in he M. ma inum in ec ed zeb a ish. Since bo h ENSDARG00000036084 and
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Figu e 1. Zeb a ish in elec in genes a e di e en ially exp essed upon M. ma inum in ec ion. (A) A genome-wide
gene exp ession mic oa ay was conduc ed in adul WT AB zeb a ish injec ed wi h M. ma inum (20 CFU; SD 6
CFU) (n = 2) o PBS (n = 3). A e age nume ical esul s (log2) o each p obe in bo h in ec ed ish (y-axis) and PBS
con ols (x-axis) a e shown. Up- and down- egula ed ansc ip s (log2 old change
3
) in he o gan blocks a e
shown in g ey, and he common immunological genes a e anno a ed. Two i ln3 p obes as well as i ln2 and i ln2-like
p obes a e highligh ed. (B–E) The exp ession o zeb a ish i ln genes (i ln1, i ln2, i ln2-like and i ln3) was measu ed
wi h qPCR in he o gan blocks o he M. ma inum in ec ed (6 CFU; SD 3 CFU) and PBS injec ed adul WT e46
zeb a ish a 1 (n = 12 and n = 4, espec i ely) and 6 dpi (n = 12 and n = 8, espec i ely) as well as 4 (n = 12 and
n = 11, espec i ely) and 9 wpi (n = 12 and n = 10, espec i ely). (F–I) The exp ession o i ln1, i ln2, i ln2-like and
i ln3 was de e mined wi h qPCR in he M. ma inum (39 CFU; SD 47 CFU) in ec ed WT AB emb yos (n = 5 a all
imepoin s) and in PBS injec ed con ols (n = 5 a all imepoin s) a 1–7 dpi. No e he di e en scales o he y axes
in B-I. Gene exp essions we e no malized o ee 1a1l1 exp ession and a ge genes we e un once in he qPCR
analyses. A wo- ailed Mann-Whi ney es was used in he s a is ical compa ison o di e ences in B–I.
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ENSDARG00000093796 sha e he same gene name, i ln2, in Ensembl genome b owse , ENSDARG00000093796
is e e ed o as i ln2-like h oughou he ex .
To con i m he di e en ial exp ession pa e n o he i ln amily membe s in he zeb a ish mycobac e ial in ec-
ion and o s udy he kine ics o he hos esponse mo e ca e ully, we analyzed i ln1 (ENSDARG00000007534),
i ln2, i ln2-like and i ln3 gene exp ession om he abdominal ca i y o gan blocks o M. ma inum in ec ed (6
CFU; SD 3 CFU) WT e46 backg ound adul zeb a ish wi h qPCR a 1 and 6 dpi, as well as a 4 and 9 weeks pos
in ec ion (wpi) (Fig.1B–E). In line wi h ou mic oa ay da a, i ln3 was signi ican ly induced a 4 wpi (3.8- old,
P = 0.002) and 9 wpi (5.9- old, P = 0.003) (Fig.1E), whe eas i ln2 was down- egula ed compa ed o he PBS con-
ols bo h a 6 dpi (0.3- old, P = 0.019) and 4 wpi (0.2- old, P < 0.001) (Fig.1C). No signi ican di e ences in he
ela i e mRNA exp ession le els o he i ln1 (Fig.1B) o i ln2-like (Fig.1D) genes we e obse ed be ween in ec ed
and he PBS injec ed adul ish a any o he measu ed ime poin s.
Nex , we in ec ed WT AB zeb a ish emb yos wi h mycobac e ia and pe o med an exp ession analysis o
he i ln genes by qPCR. He e, M. ma inum (39 CFU; SD 47 CFU) was mic oinjec ed in o he yolk sac o he
emb yos and he gene exp ession was quan i ied daily be ween 1 and 7 dpi (Fig.1F–I). In he mycobac e ia
in ec ed emb yos we de ec ed he up- egula ion o bo h i ln1 (1.8 o 6.4- old, P = 0.008–0.016) (Fig.1F) and i ln3
(1.8 o 111.4- old, P = 0.008–0.032) (Fig.1I) s a ing a 2 dpi and con inuing un il 7 dpi, as well as he induc ion
o i ln2 a 7 dpi (21.6- old, P = 0.008) (Fig.1G) and i ln2-like (Fig.1H) be ween 4 and 7 dpi (20.7 o 76.1- old,
P = 0.008–0.032) compa ed o he PBS con ols. Also, in line wi h p e ious epo s sugges ing ha o he in ec-
ious diseases up- egula e ITLN exp ession, a signi ican induc ion o i ln3 exp ession (8.4- old a 7hpi; 11.8- old
a 18hpi; 5.5- old a 24hpi; 4.4- old a 48hpi, P = 0.002 in all compa isons) was obse ed in S ep ococcus pneumo-
niae (T4 se o ype) in ec ed (296 CFU; SD 32 CFU) emb yos (Supplemen a y Figu e1).
In o de o unde s and he in ec ion-inducible na u e o he zeb a ish i ln genes a s eady s a e, we quan i ied
he ela i e mRNA le els o i ln1, i ln2, i ln2-like and i ln3 in he li e , spleen, kidney and in es ine o unchal-
lenged WT e46 zeb a ish by qPCR (Fig.2A–D). He e, we ound ha i ln2 exp ession was es ic ed o he in es-
ine (Fig.2B), whe eas i ln3 showed he highes ela i e exp ession in he li e and he highes o e all exp ession
compa ed o he housekeeping gene (euka yo ic ansla ion elonga ion ac o 1 alpha 1, like 1; ee 1a1l1) (Fig.2D).
Con e sely, i ln1 was exp essed in all o he s udied issues wi h he second highes o e all exp ession le els
(Fig.2A), while i ln2-like was p ima ily exp essed in he zeb a ish kidney and he in es ine (Fig.2C). These esul s
a e in line wi h a p e ious qPCR analysis o he i ln gene amily membe s in unchallenged adul zeb a ish25.
C ea ing i ln3 mu an zeb a ish using CRISPR/Cas9 mu agenesis. The ype II CRISPR/Cas sys-
em is an in aluable echnology o a ge ed genome edi ing63,64, and o da e i has been u ilized in a numbe o
model o ganisms. We and o he s ha e used he CRISPR/Cas9 mu agenesis me hod success ully in he zeb a -
ish47,49,65,66. He e, we used he CRISPR/Cas9 me hod o c ea e zeb a ish ca ying nonsense i ln3 mu a ions o
ou in i o s udies (Fig.3). To his end, we iden i ied a unc ional gRNA a ge ing he second exon o he i ln3
gene wi h an a e age mu agenesis e iciency o 39.5% (Fig.3A,B). A e an ou c oss o pa en al mu a ion ca -
ie s (F0-gene a ion) wi h WT TL zeb a ish, we obse ed wo ge m-line ansmi ed ameshi mu a ions in
he F1-p ogeny co esponding o a o al loss o i e base pai s (−5 bp; loss o GCATC) and o a o al gain o
eigh base pai s (+8 bp; loss o GGAGCATC and gain o TGCTAGGTAAGTATCA) a he a ge loci (Fig.3C).
Analyses wi h he T ansla e ool (Expasy; SIB, Swiss Ins i u e o Bioin o ma ics)67 o bo h he −5 bp and +8 bp
mu a ions con i med he dis up ed eading ames om amino acids 47 and 45 onwa ds esul ing in p ema u e
s op-codons a e 79 and 71 amino acids, espec i ely (Fig.3C). These wo di e en i ln3 null mu an zeb a ish
lines we e named i ln3u a145 (−5 bp mu a ion) and i ln3u a148 (+8 bp mu a ion). qPCR analysis o uninjec ed and
M. ma inum in ec ed (422 CFU; SD 221 CFU, 2 wpi) adul zeb a ish e ealed diminished i ln3 ansc ip le els in
he homozygous i ln3u a145/u a145 ( esidual exp ession less han 1%, P < 0.001) and i ln3u a148/u a148 mu an s ( esidual
exp ession less han 0.1%, P < 0.001) compa ed o he WT con ols (Supplemen a y Figu e2), sugges ing ha he
indel-mu a ions lead o he nonsense-media ed RNA decay o he mu an mRNAs68. Fu he mo e, he inhe i ance
o he mu a ions ollowed Mendelian a ios o bo h o he mu an lines, and he homozygous i ln3u a145/u a145
and i ln3u a148/u a148 mu an s did no show any de elopmen al de ec s no pheno ypical di e ences compa ed o
hei WT siblings (Supplemen a y Figu e3).
Figu e 2. Exp ession o zeb a ish i ln genes in adul zeb a ish issues. Rela i e exp ession o (A) i ln1, (B)
i ln2, (C) i ln2-like and (D) i ln3 was measu ed wi h qPCR in he unin ec ed adul WT e46 zeb a ish li e
(n = 10), spleen (n = 10), kidney (n = 10) and he in es ine (n = 10). No e he di e en scales o he y axes. Gene
exp essions we e no malized o ee 1a1l1 exp ession and a ge genes we e un once in he qPCR analyses.
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Nonsense mu a ion in i ln3 does no a ec hos esis ance agains M. ma inum in zeb a ish emb yos.
The up- egula ion o he exp ession o he i ln3 gene in a M. ma inum in ec ion sugges s a possible ole o I ln3
in he hos immuni y agains mycobac e ial in ec ions. To es i he esis ance owa ds a mycobac e ial in ec ion
is al e ed in homozygous i ln3 mu an emb yos, we i s in ec ed M. ma inum (40 CFU; SD 30 CFU) in o he
yolk sac o he ungeno yped F2-p ogeny o he e ozygous i ln3u a145/+ and i ln3u a148/+ zeb a ish and ollowed hei
su i al un il 7 days pos e iliza ion (dp ) (Fig.4A,B). Pos -expe imen geno yping e ealed an a e age su i al
o 47% in he i ln3u a145 backg ound emb yos and 48% in he emb yos wi h he i ln3u a148 backg ound. Howe e ,
any signi ican di e ences in he su i al be ween he homozygous and he e ozygous i ln3 mu an s o WT ish
could no be obse ed in ei he i ln3u a145 (Fig.4A) o i ln3u a148 ish lines (Fig.4B) be o e 7 dpi (7 dp ). Nex , we
quan i ied he mycobac e ial bu den in he emb yos ha had su i ed by qPCR using p ime s o M. ma inum
in e nal ansc ibed space (MMITS)56 (Fig.4C). The M. ma inum quan i ica ion e ealed bac e ial copy numbe
medians (log10) o 4.18 and 4.15 in 100 ng o zeb a ish DNA in he i ln3u a145 and i ln3u a145 WT g oups, espec-
i ely. Compa ably, he e ozygous i ln3u a145/+ and i ln3u a148/+ ish had copy numbe medians o 4.02 and 4.22 (in
100 ng o zeb a ish DNA, log10), espec i ely, and he homozygous i ln3u a145/u a145 and i ln3u a148/u a148 mu an s
3.53 and 4.25 (in 100 ng o zeb a ish DNA, log10). Thus, he e we e no s a is ically signi ican di e ences in he
mycobac e ial bu dens be ween he di e en geno ypes in nei he i ln3u a145 no i ln3u a148 zeb a ish.
The si e o he bac e ial injec ion can a ec he immune esponse in he emb yos50. The e o e, we nex ea ed
he ungeno yped F2-p ogeny o i ln3u a145/+ and i ln3u a148/+ zeb a ish by injec ing M. ma inum in o he blood ci -
cula ion alley o 2-day-old emb yos. In hese ish, he M. ma inum in ec ion (46 CFU; SD 31 CFU) was no able
o cause any mo ali y p io o he expe imen al end-poin o 5 dpi (7 dp ). Howe e , his allowed us o quan i y
he M. ma inum bu den in all o he in ec ed emb yos a he end-poin (Fig.4D). He e, he bac e ial copy numbe
medians (log10) in 100 ng o zeb a ish DNA we e 3.61 (WT i ln3u a145), 3.83 (WT i ln3u a148), 3.63 (i ln3u a145/+),
3.77 (i ln3u a148/+), 3.75 (i ln3u a145/u a145) and 3.79 (i ln3u a148/u a148). Simila ly o he yolk sac in ec ion, mycobac e-
ial quan i ica ion did no e eal any di e ences be ween he indi iduals o he di e en geno ypes in ei he he
i ln3u a145 o he i ln3u a148 zeb a ish backg ound. No ewo hy, we also in ec ed he ungeno yped F2-p ogeny o
i ln3u a145/+ and i ln3u a148/+ zeb a ish wi h S. pneumoniae (se o ypes 1 and T4, blood ci cula ion alley in ec ion a
2 dp ) and ollowed he su i al o he ish o 5 dpi69. The e was no di e ence be ween WT emb yos and he i ln3
mu an s (Supplemen a y Figu e1).
Dele e ious mu a ions may lead o gene ic compensa ion, which in u n can a ec he obse ed pheno ype
in gene knockou models70. To add ess his, we used a mo pholino-oligonucleo ide o silence i ln1 in ou i ln3
mu an zeb a ish oge he wi h he yolk sac mycobac e ial in ec ion o zeb a ish emb yos. In o de o ensu e
e icien e mina ion o ansla ion in all o he ou zeb a ish i ln1 ansc ip s, we a ge ed he second exon (E2)
Figu e 3. Gene a ion o i ln3u a145 and i ln3u a148 mu an zeb a ish lines using CRISPR-Cas9 mu agenesis. (A) An
app op ia e guide RNA (gRNA) a ge si e was iden i ied in he second exon o i ln3. (B) 2.5% aga ose TAE gel
elec opho esis was pe o med o e alua e occu ence o a ge si e mu a ions in zeb a ish. The in i o CRISPR/
Cas9 mu agenesis e iciency was es ima ed wi h he T7EI assay in he gRNA and Cas9 mRNA injec ed emb yos.
The size o he uninjec ed WT con ol PCR p oduc is 210 bp, whe eas he PCR p oduc s o he mu a ed
emb yos a e pa ially clea ed a he a ge si e. The clea age e iciency was calcula ed om he band in ensi ies
and he mu agenesis e iciency calcula ed acco ding o he ollowing o mula: % mu agenesis = 100 × (1 –
(1- ac ion o clea age)1/2)64. GeneRule 50 bp DNA Ladde (#SM0373, The mo Fische Scien i ic) was used as
a molecula weigh ma ke (MW). Gel image is c opped o exclude po ions ha do no con ain expe imen al
samples. (C) gRNA a ge si es we e sequenced om F1-gene a ion mu an zeb a ish and wo ameshi
mu a ions (-5 bp dele ion, i ln3u a145 and +8 bp inse ion, i ln3u a148) de ec ed, leading o unca ed p o ein
p oduc s o 79 and 71 amino acids, espec i ely.
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Figu e 4. The lack o i ln3 does no a ec he su i al o he mycobac e ial bu den o M. ma inum in ec ed
zeb a ish emb yos. (A,B) M. ma inum (40 CFU; SD 30 CFU) was injec ed in o he yolk sac o he WT (i ln3u a145)
(n = 31), i ln3u a145/+ (n = 74), i ln3u a145/u a145 (n = 22), WT (i ln3u a148) (n = 19), i ln3u a148/+ (n = 27) and i ln3u a148/
u a148 (n = 16) zeb a ish emb yos a 0 dp and he su i al eco ded un il 7 dpi. A log- ank (Man el-Cox) es
was used o he s a is ical compa ison o di e ences. The da a was collec ed om a single expe imen . (C)
Mycobac e ial bu den was measu ed by qPCR om he yolk sac in ec ed WT (i ln3u a145) (n = 17), i ln3u a145/+
(n = 32), i ln3u a145/u a145 (n = 10), WT (i ln3u a148) (n = 9), i ln3u a148/+ (n = 15) and i ln3u a148/u a148 (n = 7) emb yos
ha we e ali e a 7 dpi. (D) M. ma inum (46 CFU; SD 31 CFU) was injec ed in o he blood ci cula ion alley o he
WT (i ln3u a145) (n = 29), i ln3u a145/+ (n = 77), i ln3u a145/u a145 (n = 36), WT (i ln3u a148) (n = 31), i ln3u a148/+ (n = 57)
and i ln3u a148/u a148 (n = 19) zeb a ish emb yos a 2 dp and he M. ma inum bu den quan i ied a 5 dpi. Bac e ial
load is ep esen ed in panels C and D as bac e ial copies (log10) in 100 ng o zeb a ish DNA. A wo- ailed Mann-
Whi ney es was used in he s a is ical compa ison o di e ences in C and D.
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o he gene wi h a splice-blocking (SB) mo pholino (Fig.5A). In ou ini ial SB mo pholino i a ion expe imen s,
2.8 ng o i ln1-blocking mo pholino did no e eal any ad e se e ec s on he su i al o on he pheno ype o
unchallenged zeb a ish emb yos wi hin he i s 7 dp . Howe e , lowe WT i ln1 mRNA le els we e obse ed in
he SB mo phan s wi h esidual exp ession o 17.1% a 4 dpi, 21.8% a 5 dpi and 33.9% a 6 dpi compa ed o he
andom con ol (RC) injec ed emb yos (Fig.5B), demons a ing ha his amoun o he SB mo pholino silences
he exp ession o i ln1 e icien ly du ing emb yonic de elopmen . In addi ion, de ec able i ln1 exp ession le els
we e obse ed in he RC mo phan s al eady a 1 dpi, whe eas in he SB mo pholino injec ed emb yos i ln1 exp es-
sion was e iden la e s a ing a 2 dpi based on qPCR (Fig.5B, Supplemen a y Figu e4). Nex , we pe o med
mo pholino-M. ma inum co-injec ions (20 CFU; SD 19 CFU) in o he yolk sac o he un-geno yped F2-p ogeny
Figu e 5. Mo pholino media ed silencing o i ln1 exp ession does no al e he su i al o he WT o i ln3
knockou zeb a ish ina M. ma inum in ec ion. (A) A schema ic ep esen a ion o he e ec s o he mo pholino
media ed silencing o i ln1. A splice si e blocking mo pholino (SB) was used o p e en he no mal splicing
e en be ween exon 2 and exon 3 in i ln1. Mo pholino binding o i s a ge si e leads o an al e na i e splicing
e en ha dele es he s a codon con aining exon 2 om he ansc ip . Consequen ly, his p e en s ansla ion
o he I ln1 p o ein. In o de o quan i y he ela i e amoun o he WT i ln1 ansc ip , qPCR p ime s we e
designed o speci ically ampli y only he WT i ln1 mRNA. (B) WT i ln1 exp ession was quan i ied wi h qPCR
om he i ln1 SB mo pholino (n = 3) and andom con ol mo pholino (RC) injec ed zeb a ish (n = 3) a 1–7
dp . Gene exp ession was no malized o ee 1a1l1 exp ession. All samples we e un once as echnical duplica es.
(C–E) Su i al o he mo pholino and M. ma inum (20 CFU; SD 19 o 13 CFU; SD 10 CFU) co-injec ed
emb yos we e ollowed un il 7 dpi. In panel C, WT (i ln3u a145 and i ln3u a148) emb yos injec ed wi h ei he SB
(n = 47 and n = 53, espec i ely) o RC mo pholino (n = 29 and n = 54) a e shown, whe eas in panels D and
E he i ln3u a145 backg ound (n = 45–73) and i ln3u a148 backg ound emb yos (n = 18–35) injec ed wi h SB
mo pholino a e depic ed, espec i ely. No e ha he SB mo pholino injec ed WT (i ln3u a145) emb yo g oup is
shown in bo h C and D panels in o de o simpli y da a ep esen a ion. The da a in panel C was collec ed om
wo indi idual expe imen s, whe eas o he da a is om a single expe imen . A log- ank (Man el-Cox) es was
used o he s a is ical compa ison o di e ences. MO = mo pholino.
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o i ln3u a145/+ and i ln3u a148/+ zeb a ish (Fig.5C–E) and he F3-p ogeny o WT i ln3u a148 (13 CFU; SD 10 CFU)
(Fig.5C) and ollowed hei su i al up o 7 dpi. The e we e ew dying emb yos among unin ec ed emb yos
upon RC o i ln1 mo pholino injec ion (Supplemen a y Figu e4), whe eas he mo ali y eached 77.8–100% in
he mo pholino-M. ma inum co-injec ed emb yos. No ewo hy, he compa ison be ween he in ec ed RC and
SB mo pholino injec ed WT i ln3u a145 and i ln3u a148 emb yos did no show any di e ences in su i al (Fig.5C).
Mo eo e , inhibi ing i ln1 exp ession in homozygous i ln3u a145/u a145 and i ln3u a148/u a148 mu an s lead o a sim-
ila mo ali y compa ed o he co esponding he e ozygous and WT siblings o he same gene ic backg ound
(Fig.5D,E), indica ing ha he simul aneous lack o i ln1 and i ln3 unc ionali y does no a ec mycobac e ial
esis ance in he zeb a ish emb yo. Consis en ly, we did no de ec any di e ences in he mRNA le els o i ln1, i ln2
and i ln2-like be ween he homozygous i ln3 mu an s and he WT con ols ei he in uninjec ed (4 dp ) o M. ma i-
num (25 CFU; SD 23 CFU, 4 dp /4 dpi) in ec ed emb yos (Supplemen a y Figu e5), sugges ing ha he e is no
ansc ip ional compensa ion by he o he s udied in elec in gene membe s in he i ln3u a145/u a145 and i ln3u a148/u a148
mu an ish. Simila ly, no ansc ip ional compensa ion by i ln1, i ln2 o i ln2-like was obse ed in he adul i ln3 mu an
zeb a ish ei he in s eady s a e o upon M. ma inum in ec ion (Supplemen a y Figu e2).
Adul i ln3 mu an zeb a ish ha e a no mal immune esponse owa ds a M. ma inum in ec ion.
In o de o es he mycobac e ial suscep ibili y o he i ln3 mu an s in adul zeb a ish, we pe o med a low-dose
(48 CFU; SD 5 CFU) mycobac e ial inocula ion in o he abdominal ca i y o he ish and ollowed hei su i al
o up o 24 wpi (Fig.6A,B). A e he ollow-up, an a e age o 67% o he i ln3u a145 backg ound zeb a ish had su -
i ed, co esponding o 74% o he WT, 73% o he i ln3u a145/+ and 59% o he i ln3u a145/u a145 ish. In he i ln3u a148
backg ound ish, a combined su i al pe cen age o 81% was obse ed (78% in he WT, 82% in he i ln3u a148/+
and 84% in he i ln3u a148/u a148 ish). Simila ly o he emb yonic su i al expe imen s, no s a is ically signi ican
di e ences in he su i al be ween he geno ypes we e obse ed.
We and o he s ha e p e iously shown ha he ou come o a mycobac e ial in ec ion in adul zeb a ish depends
no only on he hos geno ype bu also on he in ec ion dose. While, a so called low-dose inocula e can esul in
la ency and a ch onic disease56, a highe dose leads o a as p og essing acu e in ec ion59,71. We hypo hesized
Figu e 6. Adul i ln3 mu an zeb a ish ha e compa able su i al and mycobac e ial bu den compa ed o
WT ish upon M. ma inum in ec ion. (A) The WT (i ln3u a145) (n = 38), il n3u a145/+ (n = 40), il n3u a145/u a145
(n = 38) and (B) WT (i ln3u a148) (n = 38), il n3u a148/+ (n = 38), il n3u a148/u a148 (n = 38) zeb a ish we e in ec ed
wi h M. ma inum (48 CFU; SD 5), and hei su i al ollowed o 24 weeks. A log- ank (Man el-Cox) es
was used o he s a is ical compa ison o di e ences. The da a was collec ed om a single expe imen . (C,D)
The i ln3u a145 and i ln3u a148 backg ound zeb a ish we e in ec ed wi h M. ma inum (422 CFU; SD 221 CFU)
and bac e ial bu den (log10) in 100 ng o zeb a ish DNA de e mined a 2 and 4 wpi om he o gan blocks
(wi hou he kidney). G oup sizes a 2 and 4 wpi, espec i ely, we e as ollows: WT (i ln3u a145) n = 10, n = 8;
il n3u a145/+ n = 12, n = 12; il n3u a145/u a145 n = 12, n = 12; WT (i ln3u a148) n = 9, n = N/A; il n3u a148/+ n = 9, n = 14
and il n3u a148/u a148 n = 8, n = 12. All samples we e un once. A wo- ailed Mann-Whi ney es was used in he
s a is ical compa ison o di e ences. N/A = no ish a ailable o analysis.
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ha he e ec s caused by he lack o I ln3 could be mo e p ominen in an in ec ion wi h a highe mycobac e ial
dose. Consequen ly, we in ec ed WT ish as well as he e ozygous and homozygous i ln3 mu an s om bo h he
i ln3u a145 and i ln3u a148 backg ounds wi h a highe M. ma inum dose (422 CFU; SD 221 CFU) and quan i ied he
bac e ial bu den a 2 and 4 wpi (Fig.6C,D). In hese ish, we de ec ed M. ma inum copy numbe medians (log10)
o 4.19 (WT i ln3u a145), 3.76 (i ln3u a145/+), 4.16 (i ln3u a145/u a145), 3.69 (WT i ln3u a148), 3.73 (i ln3u a148/+) and 3.92
(i ln3u a148/u a148) in 100 ng o zeb a ish DNA a 2 wpi and 4.72 (WT i ln3u a145), 4.36 (i ln3u a145/+), 4.60 (i ln3u a145/
u a145), 4.10 (i ln3u a148/+) and 3.72 (i ln3u a148/u a148) a 4 wpi. No ewo hy, no WT i ln3u a148 ish we e a ailable a
4wpi o a bac e ial quan i ica ion. Al oge he , hese da a indica e ha he loss o I ln3 unc ion is dispensable o
he hos esis ance agains abdominal ca i y M. ma inum in ec ion in adul zeb a ish.
Dexame hasone media ed lymphocy e deple ion in i ln3 knockou zeb a ish does no a ec
he su i al o mycobac e ial bu den in a M. ma inum in ec ion. We ha e ecen ly published
a zeb a ish immune-supp ession model o mycobac e ial eac i a ion using o ally adminis e ed dexame ha-
sone60. The dexame hasone ea men dec eases he o al amoun o lymphocy es by an a e age o 36% ( om
a ela i e p opo ion o 19.3% o 12.4%), and consequen ly leads o eac i a ion o he M. ma inum in ec ion.
In u n, a numbe o s udies ha e sugges ed ha I ln3 unc ions in mic obial su eillance and he e o e in he
inna e immuni y23,24,34. In o de o highligh he impo ance o inna e immune mechanisms in he mycobac e ial
de ense, we used he dexame hasone ea men o speci ically deple e he lymphocy e popula ion in he adul
i ln3 mu a ion ca ying zeb a ish lines i ln3u a145 and i ln3u a148, and subsequen ly in ec ed bo h WT and homozy-
gous i ln3 mu an s wi h M. ma inum (47 CFU; SD 4 CFU) (Fig.7A). Expec edly, ou low cy ome ic analysis
demons a ed a signi ican dec ease in he lymphocy e coun s o bo h WT i ln3u a145 and i ln3u a148 ish (31.5%,
P = 0.002 and 23.7%, P = 0.010, espec i ely) as well as he i ln3u a145/u a145 and i ln3u a148/u a148 mu an s (31.5% and
40.5%, P < 0.001 in bo h compa isons) h ee weeks a e ini ia ing he dexame hasone adminis a ion a 2 wpi
(Fig.7B–D). In addi ion, nei he he o al cell coun no he amoun o myeloid cells and blood cell p ecu so s
we e a ec ed by dexame hasone (Supplemen a y Figu e6). We did no de ec any subs an ial mo ali y o ei he
he i ln3u a145 o he i ln3u a148 mu an s o WT ish du ing he i e-week ollow-up pe iod. As is shown in he
Fig.7D,E, he bac e ial amoun s did no di e be ween he g oups; in 100 ng o zeb a ish DNA, mycobac e ial
copy numbe medians (log10) o 2.60 and 2.65 in WT i ln3u a145, 2.55 and 3.10 in i ln3u a145/u a145, 2.87 and 2.43 in
WT i ln3u a148 and 2.25 and 2.91 in i ln3u a148/u a148 zeb a ish we e obse ed a 2 and 4 wpi, espec i ely.
In conclusion, ou da a a e in acco dance wi h p e ious li e a u e on he possible ole o i lns in immuni y, as
he zeb a ish i ln3 is highly induced in a mycobac e ial in ec ion. Howe e , M. ma inum in ec ion expe imen s
using bo h zeb a ish emb yos and adul ish sugges ha i ln3 is dispensable o a p o ec i e mycobac e ial hos
esponse. Mo eo e , i ln1 does no seem o compensa e o he lack o unc ional i ln3 in he emb yonic in ec-
ion model. O no e, unlike has been epo ed o human ITLN1, we we e unable o demons a e di ec binding
o ecombinan I ln3 o mycobac e ia (o S. pneumoniae o Esche ichia coli) in i o (Supplemen a y Figu e7),
which may explain he nonessen ial ole o I ln3 o zeb a ish immuni y in ou models.
Discussion
The gene ics o he hos a ec he ou come o a M. ube culosis in ec ion, i.e. he de elopmen o ac i e ube -
culosis4. Genome-wide exp ession analyses using mic oa ay and RNA sequencing pla o ms a e impo an o
unde s anding complica ed biological p ocesses such as he hos immune de ense agains pa hogens. To da e, a
hand ul o ansc ip ome s udies ha e been done in he zeb a ish M. ma inum in ec ion model using mic oa ay
echnology59,72,73, he digi al gene exp ession (DGE) me hod74 and RNA sequencing75–77. Collec i ely, by using
bo h zeb a ish emb yos and adul ish, hese s udies ha e p o ided impo an insigh s in o he inna e and adap i e
hos esponse agains mycobac e ial in ec ions.
We used he adul zeb a ish M. ma inum (ATCC 927) in ec ion model oge he wi h a zeb a ish gene exp es-
sion mic oa ay o iden i y no el candida e genes in a mycobac e ial in ec ion. F om his da a, we iden i ied a
o al o 91 di e en ially exp essed genes (log2 old change >
3
) ha we e linked o 44 en iched p ocesses, includ-
ing genes associa ed wi h he immune esponse. P e ious s udies ha e shown se e al genes o he complemen
sys em (e.g. complemen componen c3b, c3b; complemen componen 6, c6) o be up- egula ed in an in ec-
ion59,72–74,76, whe eas he exp ession o some complemen associa ed genes (e.g. complemen ac o b, c b; mannose
binding lec in, mbl) has been shown o be educed72,74. In line wi h p e ious esul s, we also saw an induc ion o
cd59 ( egula ion o memb ane a ack complex o ma ion) as well as educed exp ession o c bl (componen o he
C3 con e ase). Con e sely, al hough p e ious ansc ip omic s udies ha e shown he induc ion o genes ha a e
in ol ed in neu ophil and mac ophage ela ed unc ions (e.g. mpx and i g1l)73,76, ou da a indica ed
down- egula ion o hese ansc ip s in an in ec ion. In summa y, he a o emen ioned simila i ies and di e ences
be ween hese ansc ip omic s udies can esul om a numbe o ac o s including he de elopmen al s age o
he hos (emb yos s. adul ish), he ime poin s chosen o sample collec ion, he di e en ou comes o an in ec-
ion (ch onic s. acu e), he use o di e en bac e ial s ains (E11, Mma20 o ATCC 927) and doses, and hey can
be due o di e ences in he echnical execu ion o sample p epa a ion and analyses.
In e es ingly, ci ca 38% o he up- egula ed p obes we e ela ed o muscle associa ed biological p ocesses
including muscle con ac ion (GO:0006936), muscle sys em p ocess (GO:0003012) and myo ib il assem-
bly (GO:0030239). Suppo ing he ele ance o his inding, a genome-wide exp ession analysis in he ui ly
D osophila melanogas e iden i ied se e al muscle speci ic genes such as ac in88F (Ac 88F) and opomycin 2
(Tm2) o be induced a e a Pseudomonas ae uginosa in ec ion78. Consis en ly, he down- egula ion o muscle
exp essed genes ( oponin C41C, TpnC41C; glu a hione S-T ans e ase 2, Gs 2) was la e connec ed o an inc eased
suscep ibili y o in ec ion, sugges ing an immunological ole o muscle issue79,80. Al hough he di e en ial
exp ession o muscle speci ic genes can be indi ec ly linked o he immune esponse h ough he egula ion o
o he physiological unc ions, as has been also sugges ed by Cha e jee e al.,81, bo h mouse and zeb a ish muscle
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Acknowledgemen s
This s udy was inancially suppo ed by he Academy o Finland (M.R., 277495, M.P. 295814 and 286477), he
Sig id Juselius Founda ion (M.R.), he Jane and Aa os E kko Founda ion (M.R.), he Compe i i e S a e Resea ch
Financing o he Expe Responsibili y A ea o Tampe e Uni e si y Hospi al (M.R., M.P. 9U047 and 9V049),
he Compe i i e S a e Resea ch Financing o he Expe Responsibili y a ea o Oulu Uni e si y Hospi al (M.R.),
he Tampe e Tube culosis Founda ion (M.O., M.R., S.-K.H., M.P.), he Ci y o Tampe e Science Founda ion (S.-
K.H), he Väinö and Laina Ki i Founda ion (M.O., S.-K.H.), he Finnish Cul u al Founda ion, he Cen al Fund
(S.-K.H.), he Finnish Conco dia Fund (S.-K.H.), he O ion Resea ch Founda ion s (S.-K.H), he Maud Kuis ila
Memo ial Founda ion (M.O.), he Uni e si y o Tampe e Doc o al P og amme in Biomedicine and Bio echnology
(M.O.), he Cance Socie y o Finland (M.P.) and Tays ukisää iö (Tays Suppo Founda ion) (M.P.). We hank he
Tampe e Zeb a ish Co e Facili y, pa ly unded by Biocen e Finland, o main aining and p o iding he zeb a ish.
The use o he acili ies and expe ise o he P o ein Technologies co e acili y o he Uni e si y o Tampe e, a
membe o Biocen e Finland, is also g a e ully acknowledged. We also g ea ly acknowledge Hannaleena Piippo,
Jenna Ilomäki, Leena Mäkinen, Ca ina Bäue lein, Mi ja Niskanen, Juha Saa ike u, Janey Ba on, Ch is ophe
Gaul , Janne Kä nä, Ma ianne Ka lsbe g, Ine He man, Anna G önholm and La i eh Azizi o echnical assis ance
and Jukka Leh iniemi o aking he adul zeb a ish images. In addi ion we hank Henna Myllymäki, Hannu
Tu peinen, Ma aleena Pa ikka and Te o Jä inen o scien i ic ad ice and suppo , as well as Hannah P a and
Helen Coope o p oo - eading his manusc ip .
Au ho Con ibu ions
M.P., V.H. and M.R. p o ided ma e ials and acili ies o he esea ch. M.O., M.U., J.M., V.H., M.P. and M.R.
designed and/o di ec ed he expe imen s. M.O., A.S., N.K., S.-K.H., K.O. and M.U. pe o med he expe imen s.
M.O. and M.U. analyzed he da a. M.O., M.U. and M.R. w o e he pape . All au ho s e iewed and app o ed he
manusc ip .
Addi ional In o ma ion
Supplemen a y in o ma ion accompanies his pape a h ps://doi.o g/10.1038/s41598-018-37678-1.
Compe ing In e es s: The au ho s decla e no compe ing in e es s.
www.na u e.com/scien i ic epo s/
17
Scien i ic RepoR s | (2019) 9:995 | h ps://doi.o g/10.1038/s41598-018-37678-1
Publishe ’s no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.
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