ARTICLE
Received 28 Dec 2014 | Accepted 24 Feb 2015 | Published 7 Apr 2015

DOI: 10.1038/ncomms7748

OPEN

Reduced Tyk2 gene expression in b-cells due tonatural mutation determines susceptibility tovirus-induced diabetes
Kenichi Izumi1,2,3,*, Keiichiro Mine1,*, Yoshitaka Inoue1, Miho Teshima1, Shuichiro Ogawa1, Yuji Kai1,
Toshinobu Kurafuji1, Kanako Hirakawa1, Daiki Miyakawa1, Haruka Ikeda1, Akari Inada4, Manami Hara5,
Hisakata Yamada6, Koichi Akashi2, Yoshiyuki Niho2, Keisuke Ina7, Takashi Kobayashi8, Yasunobu Yoshikai6,
Keizo Anzai3, Teruo Yamashita9, Hiroko Minagawa9, Shuji Fujimoto1, Hironori Kurisaki1, Kazuya Shimoda10,
Hitoshi Katsuta1 & Seiho Nagafuchi1

Accumulating evidence suggests that viruses play an important role in the development ofdiabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in
restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have notbeen identiﬁed. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present
in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 geneon the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes.
Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2
promoter activity, and leads to decreased expression of interferon-inducible genes, resulting
in signiﬁcantly compromised antiviral response. Tyk2-mutant pancreatic b-cells areunresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could
be achieved by b-cell-speciﬁc Tyk2 gene expression. Thus, reduced Tyk2 gene expression inpancreatic b-cells due to natural mutation is responsible for susceptibility to virus-induced
diabetes.

1 Department of Medical Science and Technology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. 2 Department ofMedicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. 3 Department of Hepatology,
Diabetes and Endocrinology, School of Medicine, Saga University, Saga 849-8501, Japan. 4 Department of Diabetes and Genes, Graduate School of Medical
Sciences, Kyushu University, Fukuoka 812-8582, Japan. 5 Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA. 6 Division of Host
Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
7 Department of Molecular Anatomy, Faculty of Medicine, Oita University, Oita 879-5593, Japan. 8 Department of Infectious Diseases Control, Faculty of
Medicine, Oita University, Oita 879-5593, Japan. 9 Department of Microbiology and Medical Zoology, Aichi Prefectural Institute of Public Health, 7-6 Nagare,
Tsujimachi, Kita-ku, Nagoya, Aichi 462-8576, Japan. 10 Division of Gastroenterology and Hematology, Department of Internal Medicine, Faculty of Medicine,
University of Miyazaki, Miyazaki 889-1692, Japan. * These authors contributed equally to this work. Correspondence and requests for materials should be
addressed to S.N. (email: nagafu_s@med.kyushu-u.ac.jp).

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D

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

iabetes mellitus is on the rise worldwide, and is associatedwith improvement in socioeconomic conditions, increasing wealth, higher caloric and fat intake and lower
physical activity1. Accumulating evidence has also suggested theassociation of environmental factors such as toxins and viruses
with diabetes2,3. From the middle of the last century, it has beenrecognized that a number of viruses are closely associated withthe onset of diabetes in man and animals4,5. Recent human type 1
diabetes and viral infection research has suggested the closest linkwith enteroviruses6–9, which belong to the picornavirus group. In
experimental animals, diabetes was described to be induced byencephalomyocarditis (EMC) virus (also a member of thepicornavirus group), cytomegalovirus, mengovirus and kilham
rat virus10. Among these viruses, EMC virus-induced diabetes has
provided an excellent animal model for studying virus-induced
diabetes, and the pathogenesis of EMC virus-induced diabetes inmice has been extensively studied10–12. Development of diabetes
induced by EMC virus has been shown to be inﬂuenced by thestrain and challenge dose of the virus, and also host factors
including sex, immunoprotective function, inﬂammatory
responses with macrophages, cytokines, chemokines, chemical
mediators and genetic background10–12.
Yoon et al. successfully isolated the highly diabetogenic Dvariant of EMC (EMC-D) virus and the nondiabetogenic B strainof the EMC (EMC-B) virus by the plaque clone puriﬁcation
method13. Even diabetogenic EMC-D virus induces diabetes only
in a limited number of mouse strains, such as DBA, SJL andSWR mice, while A/J and BALB/c mice are moderately
susceptible10,12,14–17. C57BL/6, CBA, AKR and C3H/He mice
are all resistant to EMC-D virus-induced diabetes10,12,14–17.
It was reported that a single autosomal-recessive gene, which isinherited in a Mendelian manner, controls susceptibility to thevirus18. However, the susceptibility gene(s) to EMC virus-induced
diabetes in the highly sensitive mice has not been identiﬁed inspite of initial recognition of strain-speciﬁc susceptibility to EMC
virus-induced diabetes more than 40 years ago16,19.
It is thought that innate immunity, such as interferon (IFN),
macrophages and early inﬂammatory responses, most likely
determines outcomes after EMC-D virus infection, since EMC-D
virus-induced diabetes develops within 4 days after infection, andinfection to T-lymphocyte-deﬁcient or B-lymphocyte-deﬁcient
mice did not make the mice more susceptible to virus-induced
diabetes15,17. Recent advances in the ﬁeld of innate immunity
have elucidated the signiﬁcance of pattern recognition receptors
directed against pathogen-associated molecular patterns20. These
include Toll-like receptors (TLR), intracellular helicase such as
melanocyte differentiation-associated protein 5 (MDA5; or IFN
induced with helicase C domain 1 (IFIH1)) for picornavirus,
retinoic acid inducible gene I for paramyxovirus and intracellular
DNA receptors20–22. McCartney et al.23 reported that MDA5 andTLR3 are both required to activate IFN-dependent antiviral
responses, in preventing EMC-D virus-induced diabetes in mice.
In addition, IFN receptor (IFNR)-associated molecules including
Tyrosine kinase 2 (Tyk2), Janus kinase 1 (Jak1), Signal
Transducers and Activator of Transcription (Stat) 1 and Stat2
are all involved in IFNR-mediated downstream signalling,
operating IFN-dependent antiviral responses (Fig. 1).
These observations taken together suggest that molecules
involved in innate immunity could serve as candidate genes that
determine the susceptibility of sensitive strains of mice to virusinduced diabetes. Interestingly, deﬁciency of the Tyk2 gene results
in a reduced antiviral response24. In addition, the human TYK2
gene was mapped to the possible type 1 diabetes susceptibility
locus25.
We report here that novel Tyk2 gene mutations are present invirus-induced diabetes-sensitive strains of mice, associated with2

signiﬁcantly reduced Tyk2 gene expression along with extensively
deteriorated antiviral activity in b-cells, leading to the increased
susceptibility to virus-induced diabetes. Thus, reduced Tyk2 gene
expression in pancreatic b-cells due to natural mutation is
responsible for susceptibility to virus-induced diabetes.
Results
Tyk2 KO mice and virus-induced diabetes. We carried out
intraperitoneal challenge with 1.0  103 plaque-forming unit of
EMC-D virus to Tyk2 gene knockout (Tyk2 KO) and wild-type(Tyk2 WT) male mice with virus-induced diabetes-resistant
C57BL/6J (B6) background, and measured blood glucose levels.
As shown in Fig. 2a, Tyk2 KO mice intraperitoneally injected with
EMC-D virus exhibited high blood glucose levels exceeding
14 mmol l  1 on the fourth day after infection and the high blood
glucose levels persisted thereafter. As controls, we also used a
nondiabetogenic strain of EMC-B virus, and found that the virus
did not induce diabetes even in Tyk2 KO mice. Intravenous
injection of the virus induced diabetes as well as intraperitoneal
challenge, while oral administration of the virus could not induce
diabetes (Supplementary Fig. 1). Thus, it was concluded thatTyk2 KO mice had high susceptibility to EMC-D virus-induced
diabetes, challenged by intraperitoneal route. We also conﬁrmed
low circulating insulin and high glucose levels in Tyk2 KO mice
infected with EMC-D virus along with the reduced insulin content of the pancreas (Fig. 2b). It was concluded that insulinproducing capability of pancreatic b-cells of Tyk2 KO mice
infected with EMC-D virus were signiﬁcantly decreased. The
virus titres in the pancreas of Tyk2 KO mice showed longer
persistence and was at a signiﬁcantly higher level from 1 to 5 days
after infection in comparison with Tyk2 WT mice, with B60-folddifferences between the two groups on day 5 (Fig. 2c), suggestingthat increased proliferation of the EMC-D virus in the pancreasof Tyk2 KO mice might lead to the extensive damage of thepancreatic b-cells.
Importance of Tyk2 gene expression in b-cell. In order to clarify
whether expression of Tyk2 is important in immunocompetentcells or in parenchymal cells, we set up a splenic chimera mousesystem. The irradiated Tyk2 KO recipient mice that were transferred spleen cells derived from either WT donor mice or Tyk2
KO mice developed diabetes 7 days after EMC-D virus infection,
whereas the irradiated WT recipient mice received spleen cells
derived from either WT donor mice or Tyk2 KO mice did not
develop diabetes (Fig. 2d). All irradiate diabetic Tyk2 KO micethat received WT or KO spleen cells died by 14 days after
infection, while WT mice receiving either WT or Tyk2 KO mice
derived spleen cells could escape from the development of diabetes (Fig. 2d), and could survive until 3 weeks after infection.
Irradiated mice presented deteriorated general condition, andtended to succumb to EMC-D virus infection. Taken overall,these results indicate that Tyk2 expression in parenchymal cells,
not in splenic immune cells, is important to resist against EMC-D
virus-induced diabetes.
To determine the signiﬁcance of Tyk2 gene expression inpancreatic b-cells among parenchymal cells, we generated
transgenic (Tg) B6 mice that have mouse insulin promoter(MIP) driving Tyk2 transgene vector (MIP-Tyk2)26. As shown in
Fig. 2e, while Tyk2 KO mice developed hyperglycaemia, MIPTyk2 Tg Tyk2 KO mice could maintain normal blood glucose
levels. Consistent with this, pancreatic islets of MIP-Tyk2 Tg Tyk2
KO mice remained histologically intact with minimal destructive
changes after viral infection compared with extensive damaged
islets in Tyk2 KO mice (Fig. 2f). These results suggested that Tyk2

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ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

IFN–α/βEMCVIFNAR–2

IFNAR–1

EMCV receptor

Jak1
dsRNA

Endosome

dsRNA

ssRNA

CARD

TLR3

TLR7,8Stat1

RNA helicase

Tyk2

Anti– viral proteins

Stat2
MDA5
(IFIH1)

IRF1,2,3
ATF–2
NFkB
PRD

EMCV: encephalomyocarditis virus
IRF: interferon-releasing factor
PRD: positive regulatory domain
CARD: caspase recruitment domain
TLR: toll–like receptor
MDA: melanocyte differenciation antigen(IFIH1: interferon induced with helicase C domain 1)
IFN: interferon

ISRE

ISGs
(PKR, 2–5AS,
Mx1, and so on)

Jak1: Janus kinase 1
Tyk2: Tyrosine kinase 2
Stat: Signal transducers and activator or transcription
IFNAR: IFN-α/β receptor
ISRE: IFN-stimulated response element
ISGs: IFN-stimulated genes
PKR: RNA-dependent protein kinase
2–5AS: 2’ ,5’ -oligoadenylate synthetase
Mx1: IFN-induced GTP-binding protein Mx1

Figure 1 | Type 1 interferon (IFN-a/b) signalling pathway. Tyk2 and Jak1 are reciprocal IFN receptor-associated molecules, mediating the downstream
signal to induce ISGs to resist against viral infection (modified from ref. 3, Fig. 5.3).

gene expression in pancreatic b-cells is crucial for providing
resistance against EMC-D virus-induced diabetes.
Biological significance of IFN-a. EMC-D virus-infected Tyk2
KO mice produced higher levels of IFN-a than Tyk2 WT mice inboth serum (Fig. 3a) and pancreas (Fig. 3b). Since IFN- or IFNinducible poly I:C was reported to modify the outcome ofexperimental virus-induced diabetes27,28, we further examined
the biological significance of increased IFN-a. We initially
examined the outcome of intraperitoneal adoptive transfer of
104 units IFN-a and found that transferred IFN-a appeared in the
blood as early as 2 or 3 h after injection (Supplementary Fig. 2a).
The IFN transfer did induce elevation of IFN-stimulated genes(ISGs) of the pancreatic islet-b cells (Supplementary Fig. 2b),
suggesting that IFN-a transfer is effective to induce IFN-mediatedresponses in the islet-b cells. Treatment with IFN transfer alone
did not affect the glucose levels in noninfected mice (Fig. 3c),suggesting that elevated IFN alone did not operate to damage
pancreatic b-cells. Moreover, a high dose of IFN transfer did notalter the outcome of virus-induced diabetes in mice infected with
EMC-D virus (Fig. 3d). These observations, taken together,suggest that enhanced production of IFN in Tyk2 KO mice was
induced by strong stimulation for the innate immune system toproduce IFN, due to extensive proliferation of EMC-D virus(Fig. 2c), and also indicate that the increased IFN-a in Tyk2 KO
mice did not possess any biological significance to destroy b-cells
or to resist against virus-induced diabetes.
Tyk2 gene mutations in susceptible mice. Since Tyk2 gene KOmice were highly susceptible to EMC-D virus-induced diabetes as
described above, we screened for mutations of the Tyk2 genein virus-induced diabetes-sensitive DBA/2J (DBA), SJL/J (SJL)
and SWR/J (SWR) mice as well as resistant B6, C3H/HeJ andmoderately susceptible A/J, and BALB/cJ mice. We found Tyk2
gene mutations at the promoter region (  678_  674 5A45T,
 713T4C,  735 C4T,  919 G4T,  938_  930 del 9T,
 998 G4C,  1010T4C,  1015 C4T,  1219A4G) fromtranscription start point at exon 1 and exons 9592A4G and

10642A4G only in virus-induced diabetes-sensitive SJL andSWR mice (Table 1). All virus-induced diabetes-resistantand moderately susceptible strains lack Tyk2 gene mutations.
Interestingly, highly virus-induced diabetes-susceptible DBA mice
have the WT Tyk2 gene (Table 1).
Significance of the mutated Tyk2 gene in virus-induced diabetes.
Tyk2
In orderorder toto determine
determinethethesignificancesignificanceof themutationof Tyk2
Inof themutationof thegene,we
wegenerated
generatedcongenicmice
a mutatedgene,congenic
mice
that that
had hada mutated(MT)(MT)
Tyk2
Tyk2 haplotype
gene resistantwith resistantB6 genetic
background.
We
haplotypegene with
B6 genetic
background.
We also
also backcrossed
SJL mice
to obtain
congenic
backcrossed
B6 toB6SJLtomiceto obtain
congenic
mice micewith with
susTyk2 gene.
susceptible
backgroundpossessing
WT Tyk2
gene. TheThe
ceptible
SJL SJL
backgroundpossessing
the WT
Tyk2 gene,congenic B6
B6 mice,mice, carrying
carryingthe
the MT
MT Tyk2
gene, presented
presented highhighcongenic
sensitivity toto EMC-D
sensitivity
EMC-D virus-induced
virus-induced diabetes
diabetes (Fig.
(Fig. 4A)4a) associatedassociatedwith extensive
mice
with
extensive β-cellb-cell damage
damage (Fig.
(Fig. 4B),4b), while
while congenic
congenic SJL/J
SJL/J mice
Tyk2
with theWTWT
gene
showedresistanceprowithTyk2
gene
showedresistance(Fig.
(Fig. 4C),4c), thus
thus proTyk2 geneviding evidence
evidence thatthat thethe MT
MT Tyk2
for deterviding
gene isis responsible
responsible fordetermining susceptibility
to EMC-D
miningsusceptibility to
EMC-D virus-induced
virus-induced diabetes.
diabetes.
Tyk2 WT
Immunohistochemicalstudy
studyrevealed
revealedthat
thatTyk2
WT micemice had
had
Immunohistochemical
mild CD45
positive leukocyte
mildCD45-positive
leukocyte infiltration
infiltration with
with increased
increased IFN-α
IFNa
and upregulation
at 3histocompatibility
days after infectionin theandupregulation ofofclassclassI MHC
I majorcomplexislets. Those
inflammatory
responses
rapidlysubsided
at 5 days(MHC)
in 3 days
after infectionin the islets.
Those
inflammatory
after infection,
preserving
intactislet
β-cells preserving(Supplemresponses
rapidlysubsided almostin 5 days
after
infection,
entary Fig.
3a). islet
In contrast,
mice Fig.
developed
almostintactb-cellsTyk2-mutated(Supplementary3a). Insignificant
contrast,
CD45 positivemiceleukocyte
infiltration
withCD45-positive
increased IFN-αand
Tyk2-mutateddeveloped
significant
leukoupregulationof class
MHC at 3 IFNa
days afterand persisted,cyte
infiltration
withI increased
and infection
upregulationof class I
leadinginto 3thedays
extensiveof pancreaticas wellas
MHC
after destruction
infection andpersisted,β-cells
leading
to theTyk2 KO destruction
mice (Supplementary
3A),
suggesting
extensiveof pancreatic Fig.
b-cells
as wellas Tyk2 thatKO
inflammatory
response Fig.
in the3a),pancreatic
islets thatafter inflammatory
EMC-D virus
mice(Supplementarysuggesting
infection inmay
enhanceisletcell damage
in addition
toresponse
thepossibly
pancreatic
islets the
after
EMC-D
virus infection
mayvirus-induced
β-cellthe
lysis.
Productionof IFN-β
was observed
onlypossibly
enhanceisletcell damage
in addition
to viruson 5 daysb-cell
afterlysis.
infectionin the of
wide
range
pancreaticcells
inducedProduction
IFN-b
wasofobserved
only
on in
5
Tyk2 after
KO mice(Supplementary
3A).
pancreasbutdays
infectionin the wideFig.
range
ofThus,pancreatic
cellstissue
in Tyk2
not infiltrating
leukocytes Fig.
seemedproduceboth tissue,
IFN-αbutandnot-β.
KOmice (Supplementary3a). toThus,
pancreas
Tyk2 KOseemedInterestingly,
MIP-Tyk2
Tg miceboth
producedin -b.
theinfiltrating
leukocytes,
to produce
IFN-aIFN-α&&&
Tyk2ingene
islets throughout
theKO
study
period,Tg
possibly
due to highInterestingly,
Tyk2
MIP-Tyk2
mice produced
IFN-a
the
expression
in pancreatic β-cells,
and minimal
leukocyte
islets&&&&&&&&&&
&&&
&&&&&
&&
infiltrationspossibly
on 5 days
after infection,
escaping
the virus&&&&,
becauseof high Tyk2
genefrom
expression
in

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

Tyk2+/+ + EMC-B (n=7)
Tyk2–/– + EMC-B (n =11)
Tyk2+/+ + EMC-D (n=10)
Tyk2–/– + EMC-D (n =11)

25

**

**

20

**

30
**

** **

**

**

15
10
5
0

0

1

2

3

4 5 6 7 8
Days after infection

120%

**
†

20
10
5
0

1

2

3

60%
*

40%

**

**

0

1

2

3

4

5

**

20
15
10
0
0

1

2

3

4

**

0

3
5
Days after infection

**

100
10

1

2

3
4
Days after infection

5

Tyk2–/–+ MIP-Tyk2

**

1,000

Tyk2–/–**

5 6 7 8 9 10 11 12 13 14
Days after infection

Tyk2+/+

Tyk2+/+ + EMC-D (n=5)
Tyk2–/– + EMC-D (n=5)**
Virus titre****
Tyk2+/+ (n=7)
Tyk2+/– (n=14)
Tyk2–/– (n=8)
Tyk2+/– + MIP-Tyk2 (n=8)
Tyk2–/– + MIP-Tyk2 (n=8)

25Days after infection

1

5 6 7 8 9 10 11 12 13 14
Days after infection

520%

10,000

4

30

80%

** ‡

15

35

100%

0%

Tyk2+/+ (n=7)
Tyk2–/– (n=7)
Tyk2+/+ (n=7)
Tyk2–/– (n=7)

25

0

10 11 12

Tyk2+/+ + EMC-D (n=5)
Tyk2–/– + EMC-D (n=5)

140%Insulin (%)

9

Blood glucose(mmol l–1)

Blood glucose(mmol l–1)

30

Tyk2+/+
Tyk2+/+
Tyk2–/–
Tyk2–/–

35

Blood glucose(mmol l–1)

35

Figure 2 | Development of diabetes in EMC-D virus-infected Tyk2 KO mice. (a) Blood glucose levels in WT (Tyk2 þ / þ ) and Tyk2 KO (Tyk2  /  ) micewith B6 genetic background, which was used throughout the study except indicated, infected with EMC-D or EMC-B virus. (b) Insulin content of thepancreas from Tyk2 þ / þ and Tyk2  /  mice infected with EMC-D virus. (c) Virus titres in the pancreas from Tyk2 þ / þ and Tyk2  /  mice infectedwith EMC-D virus. (d) Blood glucose levels of lethally irradiated Tyk2 þ / þ or Tyk2  /  mice reconstituted with Tyk2 þ / þ or Tyk2  /  spleen cells
infected with EMC-D virus. The irradiated Tyk2 WT recipient mice that were transferred spleen cells derived from either WT donor mice or Tyk2 KO mice
(Tyk2 þ / þ ooTyk2 þ / þ , or Tyk2 þ / þ ooTyk2  /  ), or the irradiated KO recipient mice received spleen cells derived from either WT donor mice or
Tyk2 KO mice (Tyk2  /  ooTyk2 þ / þ or Tyk2  /  ooTyk2  /  ). wdied by 10 days after infection, zdied by 14 days after infection. (e) Blood glucose
levels in MIP-Tyk2 Tg Tyk2  /  (Tyk2  /  þ MIP-Tyk2) mice infected with EMC-D virus. (f) Histopathology of pancreatic islets from Tyk2  /  , Tyk2 þ /þ and MIP-Tyk2 Tg Tyk2  /  (Tyk2  /  þ MIP-Tyk2) mice infected with EMC-D virus. Scale bar, 50 mm. In a,d,e, animals with blood glucose levels
exceeding 14 mmol l  1 were diagnosed as diabetic. All data represent the mean±s.e.m. Statistical significance (*Po0.05, **Po0.01) in a,d,e wasdetermined by Kruskal–Wallis test with Bonferroni’s post hoc analysis; in b,c by two-tailed Student’s t-test in comparisons with Tyk2 þ / þ , respectively.

induced islet
β-cellanddamage(Supplementary
Fig. 3a). on
As 5shownpancreaticb-cells,
minimalleukocyte infiltrations
days
Tyk2-deficientislet
in Supplementary
Fig. 3b, transplanted(KO)b-cellisletsafter
infection, escaping
from the virus-induced
to the renal
capsule of Tyk2
did become
infected withdamage(Supplementary
Fig.WT3a).mice
As shown
in Supplementary
intraperitoneallyinjected
EMC-D
virus
and
were
Fig. 3b, transplanted Tyk2-deficient (KO) islets to destroyed
the renal
without of
insulinglucagon(right),comparedwithnoninfectedcapsuleTyk2 orWT
mice did
becomeinfectedwithintrapertransplanted
isletsEMC-D
with positiveand destroyed
glucagon staining
itoneally
injectedvirus insulin
and were
without(left), suggestingthat(right),
EMC-Dcomparedvirus infection
reach systemicallyinsulinor glucagonwith noninfectedtranstarget organs
and positive
destroyedthemandwhen
the target
organ(left),wasplantedislets withinsulinglucagonstainingsusceptible
to
the
viral
infection.
suggesting that EMC-D virus infection reach systemically target
organs and destroyed them when the target organ was susceptible
to the viral infection.
Low Tyk2 gene expression in mutated mice. Since we showed
the significance of mutations of the Tyk2 gene in the susceptibility
to virus-induced diabetes, we next investigated whether mutations of the Tyk2 gene affect expression level of the Tyk2 gene. As
shown in Fig. 5a, the expression of the Tyk2 gene was almost
absent in homozygous Tyk2 gene-mutated (Tyk2mt/mt) cells, as
4

low as Tyk2 KO (Tyk2  /  ) cells in all cell types studied,including mouse embryonic fibroblasts (MEFs), splenocytes andpancreatic b-cells. Restoration of b-cell-specific Tyk2 gene
expression was achieved by MIP-Tyk2 transgene (Fig. 5a). In cells
from heterozygous (Tyk2 þ /  , Tyk2 þ /mt) mice, Tyk2 expression
was moderately conserved (Fig. 5a). In addition, significantly
reduced Tyk2 promoter activity associated with Tyk2 genemutations at the promoter region was confirmed (Fig. 5b). These
results suggest that the Tyk2 MT gene observed in SJL/J mice wasclosely associated with a significant decrease in Tyk2 gene
expression level, possibly due to reduced promoter activity caused
by multiple mutations at the Tyk2 promoter region (Table 1).
Since Tyk2-deficient mice produced high level of IFN (Fig. 2), we
studied the IFN responses by IFN-inducible poly I:C treatment,
coxsackie B4 virus, which belongs to a picorna virus group as well
as EMC-D virus, infection, and herpes simplex virus (HSV), as arepresentative enveloped DNA virus, infection in various genotypes of mice, such as B6, SJL and Tyk2-mutated/B6. As a result,
we found that treatment of poly I:C induced rapid IFN response

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**

60
40
40
20
20
0
0

**
**
0
0

1
2
3
4
1Days after2
3
4
infection
Days after infection

Pancreas
30 Pancreas **
30
25
**
25
20

**
**
5
5

0 0
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 11
12 12
13 13
14 14

**

Days after IFN
Dayswithout
after IFN
treatment
infection
treatment without infection

Tyk2+/+ + EMC-D (n=5)
Tyk2+/+
Tyk2–/– + EMC-D (n=5)
Tyk2–/– + EMC-D (n=5)

20
15
15
10
10
5
5
0
0

0
0

1
2
3
4
1Days after2
3
4
infection
Days after infection

IFN-α 2,500 IUi.p. x 5 days
35 IFN-α 2,500 IUi.p. x 5 days
35
30
Tyk2–/– (n=5)
30
Tyk2–/– +IFN–α25(n=5) (n=5)
Tyk2–/– +IFN–α (n=5)
25
20
20
15
15
10
10
5
5
0
0

5
5

35
35
30

IFN-α 5,000 IU i.p. x 5 days
IFN-α 5,000 IU i.p. x 5 days

30
25
25
20
20
15
15
10
10
5
5
0

Tyk2–/– + EMC-D (n=5)
Tyk2–/– + EMC-D (n=5)+
IFN-α (n=5)
Tyk2–/–+ EMC-D+ IFN-α (n=5)

0

0 0
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 11
12 12
13 13
14 14

100
80
80
60

Tyk2+/+ + EMC-D (n=5)
Tyk2+/+ + EMC-D (n=5)
Tyk2–/–+ EMC-D (n=5)**** Tyk2–/–

**
**

Blood
Blood
glucoseglucose(mmol(mmoll–1)l–1)

Serum
120 Serum
120
100

Blood
Blood
glucoseglucose(mmol(mmoll–1)l–1)

IFN-α
IFN-α
(IU(IU
ml–1
ml)–1)IFN-α
IFN-α(IU(IU
ml–1
ml)–1)

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

Days after infection
aftertransfer
infectionwith Days
adoptiveof IFN
with adoptive transfer of IFN
Figure 3 | Lack of biological importance of produced IFN-a in Tyk2 KO mice. (a) IFN-a level in the serum. (b) IFN-a level in the pancreas. IFN-a
Figure 3 |inLack
biologicalof WT
produced
IFN-a
Tyk2onKO(a) IFN-alevel inwith
the EMC-D
serum. (b)in the using
pancreas.
IFN-a
produced
Tyk2ofKO(Tyk2  /importance ) and Tyk2
(Tyk2 þ/ þ )inmice
B6mice.
background,
infectedvirusIFN-awerelevelmeasured
ELISA.
produced
Tyk2 KO(Tyk2
 /IFN-a
 ) andTyk2 WTþ//þ )andmice
onþB6
infected
with Animals
EMC-D virus
were measured
usingexceeding
ELISA.
(c) Blood in
glucoselevelsafter
treatmentin (Tyk2
Tyk2 
Tyk2
/ þbackground,mice without
infection.
with blood
glucose levels
1 were diagnosed(c)mmol
Bloodl glucose
levels afterasIFN-atreatment
in Tyk2
 / levels
 andfollowing
Tyk2 þ /intraperitoneal
þ mice without
infection.
Animals
glucoseexceeding days,
diabetic.
(d) Blood
glucoseinjectionof highdose with
IFN-ablood(5,000IU) forlevels
five consecutive
14
 1 were diagnosed as diabetic. (d) Blood glucose±
intraperitoneal
of or
high
dose IFN-a
for five consecutive
days,
14
mmol
l
after EMC-D virus infection. All data represent the mean levels
s.e.m.following
Statisticalsignificance injection(*Po0.05**Po0.01)
in (5,000
a,b wasIU)
determinedby two-tailed
after
EMC-D
virus
All data represent
theBonferroni’s
mean±s.e.m.
Statisticalsignificance (*Po0.05 or **Po0.01) in a,b was determined by two-tailed
Student’s
t-test;in infection.
c,d by Kruskal–Wallis
test withpost
hoc analysis.
Student’s t-test; in c,d by Kruskal–Wallis test with Bonferroni’s post hoc analysis.

Table 1 | Mutations of the Tyk2 gene in virus-induced
Table
1 | Mutations ofmice.
the Tyk2 gene in virus-induced
diabetes-susceptible
diabetes-susceptible mice.
Susceptibility ofStrainSusceptibility
StrainEMC-Dvirus- of
EMC-D virusinducedDM
induced DM
Resistant
C57BL/6J
Resistant
C57BL/6J
C3H/HeJ
C3H/HeJ
Moderately
BALB/cJModerately
BALB/cJ
susceptible
susceptible
A/J
A/J
Highly susceptible DBA/2J
Highly susceptible DBA/2J
SJL/J
SJL/J
SWR/J
SWR/J

Promoter
Promoterregion*region*
WT
WT
WT
WT
WT
WT
WT
MT
MT
MT

Exon 7
Exon 79592A4G
9592A4G(H234R)(H234R)
WT
WT
WT
WT
WT
WT
WT
MT
MT
MT

Exon 8
Exon 810642A4G
10642A4G(K355E)(K355E)
WT
WT
WT
WT
WT
WT
WT
MT
MT
MT

DM, diabetes mellitus; EMC-D, encephalomyocarditis virus D strain; MT, mutated type; WT, wild
type.
DM, diabetes mellitus; EMC-D, encephalomyocarditis virus D strain; MT, mutated type; WT, wild
*Mutationsof Tyk2 gene promoter region (MT) within  1,300 bp upstream from transcription
type.
start point were 678_
674 5A45T,
713T4C,C4T,
 919 G4T,
938_  930
*Mutationsof Tyk2
gene 
promoterregion(MT)within7351,300bp upstream
fromtranscriptiondel 9T,
 998
 1010T4C,
 1015
 1219A4G.
start
point
wereG4C,
 678_
 674 5A45T,
 C4T,
713T4C, 735 C4T,  919 G4T,  938_  930del 9T,  998 G4C,  1010T4C,  1015 C4T,  1219A4G.

(Supplementary Fig. 4a) in all strains of mice studied. Coxsackie(Supplementary
Fig.also4a) enhanced
in all strains
mice studied.
B4 virus infection
IFNofproductionin Coxsackie
all strains
B4 virus infection
IFNinfection
production
in allinduced
strains(Supplementary
Fig.also4b),enhanced
while HSV
scarcely(Supplementary
Fig. 4b), whileFig.
HSV
scarcelyinducedIFN response (Supplementary4c),infection
suggestingthat enveloped
IFN response(Supplementary4c), suggestingthat envelopedDNAvirus may
be a poor IFN Fig.
inducer,
at least in thosestrains of
DNA studied.
virus may be a poor IFN inducer, at least in those strains ofmice
mice studied.

Tyk2 gene mutation reduced antiviral effect. In order to assess
Tyk2 geneantiviral
effect.
In orderassesswhetherthemutation
deletion reduced
or mutationof theTyk2gene toleads
towhetherthe deletionoforthemutationof thegeneprotective
leads toreduced expression
Tyk2 gene
andTyk2
reducedreducedgene
and reduced
protective
capabilityexpression
dependent ofon theIFNTyk2
againstEMC-Dvirus-induced
diacapability
dependentthe
onexpression
IFN againstlevels
EMC-D
virus-induced
diabetes,
we evaluatedof theTyk2 gene, Jak1
betes,and
we evaluatedthe expressionof the Tyk2
gene, Jak1
gene
ISGs in pancreaticb-cells,levels
splenocytes
and embryonic
gene
and ISGsin pancreatic
splenocytes
andstudied
embryonic
fibroblasts(mEFs)
with IFNb-cells,
treatment.
We first
thefibroblasts(mEFs)of with
firstderived
studiedfrom
theexpression levels
Tyk2 IFN
and treatment.
Jak1 genes We
in cells
expression
levels of Tyk2
and Jak1mice.
genesIt inwas
cellsfoundderivedTyk2gene-deficientor -mutatedthatfromtheTyk2 gene-deficientor Tyk2-mutatedmice.
It was foundexpression
levels of thegene inTyk2-mutatedmicethat
weretheasexpression
levelsof theKO
Tyk2
genebefore
in Tyk2-mutatedmice
were aslow as thoseof Tyk2
mice
and after IFN
treatment
low as6a).
those
Tyk2
KO themiceexpression
before andafter
treatment
(Fig.
We of
also
studiedlevelof IFNthe Jak1
gene,(Fig. 6a).
also studied
the expressionof the with
Jak1 gene,
which
is We
a signaltransduction
molecule levelassociatedcytowhich isdomain
a signaloftransduction
molecule
associatedwithcytoplasmic
the IFN receptor,
as wellas Tyk2(Fig.
1).
plasmic domain
the gene
IFN wasreceptor,
as well as but
Tyk2
(Fig.
1).
Expressionof theofJak1
not suppressed
wellmainExpressionof thewell maintainedin Tyk2
KOJak1or geneTyk2 was
MT notmicesuppressed(Fig. 6a), butsuggestingthetained in Tyk2
KOsupplemental
or Tyk2 MT role
micein(Fig.
6a), suggestingalternative
and/or
Tyk2-deficient
micetheinalternative and/or
supplemental
in Tyk2-deficient
mice byinmediatingdownstream
signal roletransduction
stimulated
24,29. We
mediatingdownstream
signal thetransduction
byIFN
further analysed
expression stimulated
levels of ISGs
IFN24,29.byWeanalysed
thestimulation,
expression expression
levels of of
ISGs
inducedtypefurther
I IFN. Before
IFN-b
all
inducedtype Iabsent
IFN. Before
IFN-b
stimulation,all
ISGs wasbyalmostin all cell
types
even fromexpression
Tyk2 þ / þ ofWT
/ þ WT
ISGs (Fig.
was almost
absent inIFN
all celltypes evenfrom Tyk2
mice6b). Following
stimulation,
expressionof þISGs
wasmice (Fig. 6b).
Following
IFN from
stimulation,
expression
ISGs wassignificantly
increasedin cells
WT mice(Fig. 6b).ofConsistentsignificantly
increasedin cells
from
WTgene,mice the(Fig.expression
6b). Consistent
with the expression
levelsof theTyk2levels/ þ WTwith
the wereexpression
the Tyk2 þgene,
the expressionof
ISGshigh inlevels
cells offrom
mice, whilelevelstheyþ / þ WT mice, while theyof ISGs
were highin cells
fromfrom
Tyk2Tyk2
weremarkedly
reducedin cells
KO mice and Tyk2 MT
were markedly
cellsfrom Tyk2
KO miceand Tyk2
MT
mice(Fig. 6b), reducedwith lowinbut
distinct
expressionof ISGs,
possibly
miceto(Fig.
6b), with lowJak1-mediatedbut distinct expressionof ISGs,
possiblydue
complementary
partial signaltransductiondue
to complementary
Jak1-mediatedpartialsignal transduction
as describedabove (Fig.
6b). MIP-Tyk2
transgenesignificantly
as described above (Fig. 6b). MIP-Tyk2 transgene significantly

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Publishers Limited. All rights reserved.

& 2015 Macmillan Publishers Limited. All rights reserved.

55

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

25
** **

**

20

*
*

InsulinHE

Insulin

15

Day 3

10
5
0

1

2

3

4

5 6 7 8 9 10 11 12 13 14
Days after infection
Day 5

0

SJL/J background

35
30

*

*

**

*

*

**

25

Day 7

Blood glucose (mmol l–1)

HE
Tyk2mt/mt (n =7)
Tyk2+/mt (n =7)
Tyk2+/+ (n =7)

Tyk2mt/mt

20
15
10
Tyk2mt/mt (n =5)
Tyk2+/mt (n =7)
Tyk2+/+ (n =7)

5
0

0

1

2

3

4

Day 10Blood glucose (mmol l–1)

30

Tyk2+/+**Day 0

C57BL/6J background** ** **
**
**
**
**

35

5 6 7 8 9 10 11 12 13 14
Days after infection

Figure 4 | Signiﬁcance of the mutated Tyk2 gene in congenic mice. (a) Blood glucose levels of B6 mice with Tyk2 þ / þ , heterozygous mutated Tyk2
(Tyk2 þ /mt) and homozygous mutated Tyk2 gene (Tyk2mt/mt) infected with EMC-D virus. (b) Histopathology of pancreatic islets from Tyk2 þ / þ or
Tyk2mt/mt mice with B6 background infected with EMC-D virus. Scale bar, 50 mm. (c) Blood glucose levels of in Tyk2 þ / þ , Tyk2 þ /mt, Tyk2mt/mt genecarrying congenic mice, with SJL genetic background, infected with EMC-D virus. In a,c, animals with blood glucose levels exceeding 14 mmol l  1 werediagnosed as diabetic. All data represent the mean±s.e.m. Statistical signiﬁcance (*Po0.05, **Po0.01) in a,c was determined by Kruskal–Wallis test with
Dunnett’s post hoc analysis.

restored the decreased expression of Tyk2 in bb-cells, but not in
splenocytes or mEF cells derived from Tyk2-deﬁcient mice
(Fig. 6b). These results suggest that the expression of Tyk2 is
involved in mediating the IFN signalling pathway and theinduction of ISGs. Finally, we assessed the ability of IFN-b
treatment to inhibit virus-induced cell damage in vitro. The
inhibition of virus-induced cell death on stimulation with IFN-b
was signiﬁcantly reduced in mEF or islet cells derived from Tyk2mutated mice, to levels as low as Tyk2 KO mice (Fig. 6c). In
addition, it should be noted that after treatment with a highconcentration of IFN, Tyk2 MT mEF cells could restore thereduced antiviral activity, while Tyk2 MT b-cells sustained
deteriorated the capability to resist against virus-induced cell
death (Fig. 6c), indicating that b-cells were more sensitive thanmEF cells in decreased IFN-dependent antiviral responses
because of Tyk2 gene mutation. The ﬁnding was consistent
with the in vivo experiments above in EMC-D virus-infected Tyk2
KO mice, which showed the ineffectiveness of an increased IFN
level to rescue virus-induced b-cell damage, and failed to preventthe development of diabetes (Fig. 2a). Moreover, we could also
show that b-cell-speciﬁc Tyk2 gene expression using Tyk2 MTmice crossed with MIP-Tyk2 Tg mice (MIP-Tyk2 Tg Tyk2mt/mt)
showed resistance to EMC-D virus-induced diabetes (Fig. 6d),
similar to MIP-Tyk2 Tg Tyk2 KO mice, conﬁrming the importantrole of intact Tyk2 gene expression in pancreatic b-cells in theprevention of EMC virus-induced diabetes.
6

Discussion
In the present study, we could, for the ﬁrst time to the best of ourknowledge, identify the natural susceptibility gene as Tyk2 to
EMC virus-induced diabetes in mice. Tyk2 gene mutation indiabetes-sensitive SJL and SWR mice is closely associated withsigniﬁcantly reduced Tyk2 gene expression including that inpancreatic b-cells. It was suggested that the decreased Tyk2 gene
expression was due to decreased Tyk2 promoter activity; however,other mechanisms may also be involved in the extensive
suppression of Tyk2 gene expression in these mutated mice. In
addition, the importance of intact Tyk2 gene expression inpancreatic b-cells in the resistance against EMC-D virus-induced
diabetes was revealed by the MIP-Tyk2 transgenic mice crossedwith Tyk2 KO or Tyk2-mutated mice. The observation isconsistent with a previous report that showed that the pancreaticb-cell-speciﬁc suppression of cytokine response including IFN
leads to high sensitivity to coxsackie B4 virus infection30,
indicating that the appropriate cytokine response in pancreaticb-cells is most important to resist against virus-induced diabetes.
It should be noted that the Tyk2-deﬁcient mice infected with
EMC-D virus produced a high level of IFN but failed to rescue themice from developing diabetes because high level of IFN could
reverse the antiviral response in ﬁbroblasts24, but not pancreaticb-cells as shown by this study, suggesting the b-cell-speciﬁc
unresponsiveness to a high level of IFN is critical in losing thecapability to evade from virus-infected cell damage.

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ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

Splenocyte

60
40
20
0

Ty

Ty

k2

80

Control

Tyk2+/+

Tyk2mt/mt

+/
m
t
m
t/m
t
Ty

k2

–/
–

k2

**

Ty

+/
–

**
k2

Ty

Ty

+/
+

*

k2

Relative expressionk2**

P=0.0002

100

mEF

1
0.8
0.6
0.4
0.2
0
Ty

**

**

Relative activity (%)

+/
+
k2
+
Ty /–
–/
–
+ k2–
M
IP /–
-T
Ty yk
k2 2
Ty +/m
k2
t
m
t/m
t

**

**

**

k2
+
Ty /+
k2
+
k2
Ty /–
–/
–
+ k2–
M
IP /–
-T
Ty yk2k2
Ty +/m
k2
t
m
t/m
t

**

Ty

k2
Ty

**

**

120

25
20
15
10
5
0
Ty

**

~

Relative expression

β-cell
60
58
56
4
2
0

Figure 5 | Expression of Tyk2 protein and gene in Tyk2 gene-mutated mice. (a) Relative expression levels of the Tyk2 gene were measured by
qRT–PCR (see Methods). Pancreatic b-cells, splenocytes and MEF cells were isolated from B6 mice with genotypes as indicated in the ﬁgure.
(b) Luciferase assay using genes possessing Tyk2 þ / þ or Tyk2mt/mt promoter region. All data represent the mean±s.e.m. Statistical signiﬁcance(*Po0.05, **Po0.01) was determined by two-tailed Student’s t-test in comparisons with Tyk2 þ / þ , respectively.

Although this study could clarify the genetic cause of theelevated susceptibility to the highly diabetogenic strain of EMC-D
virus, these mice are still resistant to non-diabetogeneic EMC-B
virus (Fig. 2a)13. Clinical outcomes of infectious diseases are
dependent on many factors, including pathogenicity of thepathogen and host resistance factors such as immunologicalresistance, age, sex and stochastic elements. Interestingly, thedifferent diabetogenic potential of different strains of EMC
viruses has been noted to be dependent on the genetic variation31.
Although diabetogenic EMC-D virus and nondiabetogenic EMCB virus could not be distinguished by either neutralization assay
or competitive radioimmunoassay, only one amino-acid change,
induced by only one nucleotide substitution, is critical to acquire
diabetogenicity of the EMC virus32. These beautiful studies
suggested that a single point mutation in a potential diabetogenic
virus can easily acquire higher diabetogenicity in the natural state.
To prove the causality of the pathogen in inducing an
infectious disease, historically and ideally, ‘Koch’s postulate’should be fulﬁlled33. The one organism–one disease paradigm iscentral to Koch’s postulate, in which it is essentially required toshow that an isolated pathogen can cause the disease inexperimental animals33. However, this rule cannot be applied to
all microbe-induced diseases. With respect to virus-induced
diabetes, many viruses have been noted to be associated withthe onset of diabetes3,6–9,34. However, distinguishing between
whether the pathogen was a causal factor or was only acoincidental co-infection is difﬁcult3,10. In addition, whencumulative environmental insults lead to the development ofdiabetes35, virus infection may serve as one of the many risk
factors that may contribute to diabetes development. Accordingly,
only a few reports have proved the diabetogenicity of virus
isolates from humans36,37. Both viruses were isolated by using SJL
mice36,37, and these studies appear to fulﬁll Koch’s postulate.
Since SJL mice carry the susceptible Tyk2 gene mutation as shownby this study, the SJL mouse model may be an appropriate animal
assay system to simulate human virus-induced diabetes
susceptibility.
Among the susceptible strains of mice, DBA/2 mice, which arealso highly susceptible to EMC-D virus-induced diabetes, lackTyk2 gene mutation, indicating that genes other than Tyk2 may
be responsible for increased susceptibility to virus-induced
diabetes. Interestingly, it was reported that polymorphisms of

the IFIH1 gene, which is an intracellular pathogen recognition
receptor for picornavirus including enteroviruses, operating as an
inducer of IFN production20, is associated with risk for type 1
diabetes38. Moreover, a rare IFIH1 gene variant confer resistanceto type 1 diabetes39. In addition, it was reported that MDA5 andTLR3 are both required to prevent diabetes in mice infected with
EMC-D virus23. These observations taken together suggest that
the MDA5/IFIH1 gene may possibly be a common susceptibility
gene to virus-induced diabetes in humans and mice. In order todevelop mouse models with higher virus-induced diabetes
susceptibility for use as an in vivo assay system to evaluate
diabetogenicity of candidate viruses, the identiﬁcation of genes
associated with high susceptibility to virus-induced diabetes in
DBA/2 mice or in humans including the MDA5/IFIH1 gene needs
further investigation. These studies may in future lead to theidentiﬁcation of the ‘diabetogenic’ virus.
Although many viruses are possibly associated with theinduction of diabetes in humans, accumulating evidence hassuggested that enterovirus group may be a major causal pathogen
in virus-induced diabetes. Therefore, development of vaccines
against diabetogenic enteroviruses will hopefully contribute toprevent a major portion of virus-induced diabetes in humans.
In conclusion, this manuscript reports the identiﬁcation of themurine natural virus-induced diabetes susceptibility gene as Tyk2,
and demonstrates that Tyk2 gene expressed in pancreatic b-cells
controls susceptibility to virus-induced diabetes.
Methods
Virus. EMC-B and -D virus was kindly provided by Dr A.L.Notkins, NIH, USA
and J-W Yoon, University of Calgary, Canada. The viral titre was determined byplaque assay on mEF cells.
Mice. WT of C57BL/6J (B6), C3H/HeJ, BALB/c, A/J, DBA/2J, SJL/J and SWR/J J
mice were purchased from Kyudo (Japan). Tyk2 KO mice, having EMC-D virusinduced diabetes-resistant B6 genetic background, were generated by backcrossing
129/Sv-derived Tyk2 KO mice24 to B6 mice for 12 times. All strains of male mice
aged 6–10 weeks were used throughout the study.
The MIP-Tyk2 transgenic construct was assembled using an 8.5-kb fragmentof the MIP that includes a region from  8.5 kb to  12 bp (relative to thetranscriptional start site), the coding region of Tyk2 (3.6 kb; Clontech, Palo Alto,CA) and a 2.1-kb fragment of the human growth hormone (hGH) gene cassettefor high-level expression (ref. 26). The fragments of MIP and hGH cassette werefrom MIP-EGFP-hGH transgenic construct kindly provided by Dr M. Hara27.
The 14.6-kb MIP-Tyk2-hGH fragment was isolated from the vector by digestion of

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7

Splenocyte

**

**

10
8
6
4
2
0

120

**

0

**
100

Ty
k2
+/
Ty +
k2
+/
Ty –
k2
–/
–
Ty
k2
+/
m
Ty
t
k2
m
t/m
t

**

MEF

**

**

0.4

IFN(–)
IFN(+)

2.5
2
1.5
1
0.5
0

**
*

**
**
*

Tyk2–/– (n =4)
**
**

20

1

Tyk2+/+ (n =3)
Tyk2+/– (n =3)
Tyk2–/– (n =3)
Tyk2+/mt (n =3)
Tyk2mt/mt (n =3)
Tyk2–/– + MIP-Tyk2 (n =3)% Inhibition

60

**

t

t/m

t

–

m

m

+/
k2

**

20

Ty

Ty

k2

–

–/

+/
k2

Ty

Ty

k2

+
Ty

k2

+/

k2
Ty +/m
k2 t
m
t/m

**

**

40

t

–

2
yk
-T

IP

**

**

k2

–/

–

+

M

Ty

–

–/

+/

k2

k2

Ty

**

Ty

2
1.6
1.2
0.8
0.4
0

**

m

m

+/
Ty

k2

k2
Ty

**

Tyk2mt/mt + MIP-Tyk2 (n =7)

25
20
15
**

10

t

**
*

5
0
0

t/m

m

1

2

3

4

5 6 7 8 9 10 11 12 13 14
Days after infection

k2

m

+/
k2

**

Ty

Ty

1000

Tyk2mt/mt (n =7)

30

t

–

**

–/
k2

k2

Ty

+/

–

+
+/

Ty

m

**

**

k2
Ty

t/m

2

t
+/

k2

k2
Ty

IP

-T

Ty

M

100

k2

–/

–

+

m

yk

–
–/

**

IFN(–)
IFN(+)

2.5
2
1.5
1
0.5
0

t

**

10
IFN-β (U/mL)

Blood glucose (mmol l–1)

t

t/m

t

–
k2

–/

–
+/
k2

Ty

+/
k2
Ty

–/
k2
Ty
k2

k2

Ty

Ty

+/

–

**

1

35

Ty

t
t
m
+/

m

k2

k2
Ty

**

**

+

2
yk
-T

Ty

IP

IFN(+)

–

+

M

t/m

–
–/

–
+/

Ty

k2

**

k2

+
+/
k2

**

IFN(–)

Ty

Ty

**

**
+

**

+/

**

k2

**

20
16
12
8
4
0
Ty

**

k2
+
Ty /+
k2
Ty
+
k2
Ty /–
–/
k2
–
+
–/
M
–
IP
-T
y
Ty
k
k2 2
Ty +/m
k2 t
m
t/m
t

*

**

80

0

**

Ty

Ty

Relative
expression

Mx1

12
10
8
6
4
2
0

**

**

Ty

*

**

IFN(–)
IFN(+)

1000

β-cell

MEF
3.5
3
2.5
2
1.5
1
0.5
0

100

IFN-β (U/mL)

0.1

6
5
4
3
2
1
0
Ty

30
25
20
15
10
5
0

k2
+
Ty /+
k2
+
k2
Ty /–
–/
k2
–
+
–/
M
–
IP
-T
Ty yk2k2
Ty +/m
k2
t
m
t/m
t

Relative
expression

2-5AS

Ty

Ty

**
+

**

+/

*

k2

**

Ty

**

20
16
12
8
4
0

10

120

Splenocyte

Ty

**

k2
+
Ty /+
k2
+
k2
Ty /–
–/
k2
–
+
–/
M
–
IP
-T
yk
Ty
2
k2
Ty +/m
k2
t
m
t/m
t

Relative
expression

PKR

20
16
12
8
4
0

Tyk2+/mt (n =4)
Tyk2mt/mt (n =4)

**
**
0 **
0.1

100
β-cell

Tyk2+/+ (n =4)
Tyk2+/– (n =4)

*

**

**
**
**
**

60
40

*

**

*
*
**

80

/+
Ty
k2
+/
–
Ty
k2
–/
–
Ty
k2
m
t/
Ty
k2 +
m
t/m
t

**

**

**
**

**

Ty
k2
+

Relative
expression

2.5
2
1.5
1
0.5
0

0.8

Ty
k2
+
Ty /+
k2
Ty
+/
k2
–
T
–/
yk
–
2–
+
/–
M
IP
-T
Ty yk
k2 2
+
Ty /m
k2 t
m
t/m
t

**

**

**

IFN(–)
IFN(+)

1.2

Ty
k2
+
Ty /+
k2
Ty
+/
k2
–
T
–/
yk
–
2–
+
/–
M
IP
-T
y
Ty k2
k2
Ty mt
k2 /+
m
t/m
t

**

MEF
1.6

25
20
15
10
5
0

**

% Inhibition

β-cell

70
60
50
40
30
20
10
0

Ty
k2
+
Ty /+
k2
Ty
+
k2
Ty /–
–/
k2
–
+
–/
M
–
IP
-T
Ty yk2k2
Ty +/m
k2
t
m
t/m
t

Relative
expression

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

Ty
k2
+
Ty /+
k2
Ty
+
k2
Ty /–
–/
k2
–
+
–/
M
–
IP
-T
Ty yk2k2
Ty mt/
k2 +
m
t/m
t

Jak1

Tyk2

ARTICLE

Figure 6 | Expression of IFN-stimulated genes and antiviral effect in IFN-treated cells. (a) Relative expression levels of the Tyk2 and Jak1 genes measured
using qRT–PCR (See Methods) in IFN-b-stimulated pancreatic b-cells, splenocytes and MEF cells, isolated from various genotypes of mice with B6
background as indicated in the ﬁgure. (b) Relative expression levels of ISGs (PKR, 2–5AS, Mx1) measured by qRT–PCR in IFN-b-stimulated b-cells,
splenocytes and MEF cells, isolated from B6 mice with genotypes as indicated in the ﬁgure. (c) IFN-dependent inhibitory activity against EMC-D
virus-induced cell death in MEF or b-cells derived from mice with genotypes as indicated. (d) Blood glucose levels in MIP-Tyk2 Tg Tyk2mt/mt(Tyk2mt/mt þ MIP-Tyk2) mice infected with EMC-D virus. In d, animals with blood glucose levels exceeding 14 mmol l  1 were diagnosed as diabetic.
In a,b, relative activity of each gene expression before and after IFN-b stimulation was shown. All data represent the mean±s.e.m. Statistical signiﬁcance(*Po0.05, **Po0.01) in a,b were determined by two-tailed Student’s t-test between IFN-b-stimulated each sample and that of Tyk2 þ / þ ; in c by
Kruskal–Wallis test with Dunnett’s post hoc analysis; in d by Kruskal–Wallis test with one-way analysis of variance.
the plasmid construct with HindIII and PvuI and with agarose gel electrophoresis.
The fragment was further puriﬁed using a QIAEXII Gel Extraction Kit (Qiagen,
Valencia, CA). The puriﬁed transgene DNA was microinjected into the pronuclei
of C57BL/6 mice at the Keio University, generating MIP-Tyk2 Tg mice. In order toverify the speciﬁc expression of Tyk2 in pancreatic b-cells, we compared theexpression level of Tyk2 in pancreatic b-cells and splenocytes. The mRNA level ofTyk2 in islet cells was quantiﬁed by quantitative reverse transcription polymerase
chain reaction (qRT–PCR).
In order to transfer Tyk2 mutations in virus-induced diabetes-sensitive SJL miceon a resistant B6 genetic background, male SJL/J mice were serially backcrossedwith female B6 mice for eight times. Similarly, B6 mice with the WT Tyk2 gene
were backcrossed to SJL mice for ﬁve times, generating SJL mice with the WT Tyk2
gene. The heterozygous carriers of the Tyk2 mutation, used as breeders at eachbackcross generation, were identiﬁed by sequencing the genomic DNA isolated
from the tail. Congenic level of the Tyk2 gene, specially around the mutated Tyk2
gene, was studied using 384 single-nucleotide polymorphism (SNP) markers by
Charles River Genetic Testing Services. Overall mean congenic rate was 97.6%.
8Congenic mice carrying mutated Tyk2 gene, SNP marker Chro9-1 at 13259371 bp,
near the Tyk2 gene at 2110408 to 21131275 bp, and Chr9-3 distant from the Tyk2
gene at 23780447 bp, were both from B6 mice. Therefore, at least less than
77851036 bp at near side and 2649292 bp at distant side were successfully replacedby those of B6 mice derived gene. Congenic level of 129/Sv-derived Tyk2 gene KOmice with B6 background was 99.1%.
All of the procedures involving mice were approved by the Keio and Kyushu
University Animal Care and Use Committees.
Infection of mice with EMC-D virus. Mice were intraperitoneally (i.p.), intravenously or orally infected with 0.2 ml PBS containing 1.0  103 plaque-forming unit per
mouse of EMC-D virus. The mice were also intraperitoneally infected with coxsackie
B4 virus and HSV that were clinical isolates derived from the affected patients.
Assessment of diabetes condition and measurement of IFN-a. Blood glucose
was measured by the glucose oxidase method (glutest sensor; Sanwa, Japan), andNATURE COMMUNICATIONS | 6:6748 | DOI: 10.1038/ncomms7748 | www.nature.com/naturecommunications& 2015 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7748

insulin was measured by an enzyme-linked immunosorbent assay (ELISA) commercial kit as a standard (Morinaga, Japan). Serum IFN-a was measured by an
ELISA kit (Cosmo Bio, Japan). Mice with blood glucose levels greater than
14.0 mmol l  1 were diagnosed as diabetic.
Histological and immunohistochemical analyses. Pancreases were removed andrinsed with saline. The tissues were ﬁxed with 10% formalin and embedded inparafﬁn. Tissue sections were cut at a thickness of 3.0 mm and stained withhaematoxylin and eosin. For insulin stain, the parafﬁn-embedded sections were
stained with anti-insulin antibody (Molecular Probes, Eugene, OR). The signals
were detected by a Vectastain avidin–biotin–peroxidase complex system with adiaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA). Immunohistochemical study was also carried out using frozen sections of the pancreas,
followed by the ﬁxation with 4% paraformaldehyde/PBS. To stain insulin,
glucagon, IFN-a, IFN-b, CD45 and MHC class I. For insulin, guinea pig anti-pig
insulin antibody (AbD Serotec, Kidlington, UK)and Alexa Fluor 488 goat antiguinea pig IgG (Life Technologies, USA), for glucagon, rabbit glucagon antibody(Cell Signaling Technology, Danvers, USA) and Alexa Fluor 594 goat anti-rabbit
IgG (Life Technologies), for IFN-a rabbit polyclonal antibody against mouse IFN
alpha (PBL Assay Science, USA) and anti-rabbit IgG(H þ L)F(ab0 )2Fragment (Cell
Signaling Technology), for IFN-b, rabbit anti-IFN-beta (AbD Serotec)and Alexa
Fluor 594 goat anti-rabbit IgG (Life Technologies), for CD45, rat anti-CD45
antibody (Abcam, Cambridge, UK) and Alexa Fluor 488 goat anti-rat IgG(LifeTechnologies), for MHC class I, goat anti-MHC class antibody (Abcam) andAlexa Fluor 594 goat anti-rat IgG (Life Technologies) were used.
Glucose tolerance. The intraperitoneal glucose tolerance test (IPGTT) was
performed in mice after fasting for over 12 h. Blood was obtained from the tip of
the tail. After baseline blood glucose level measurements, animals received an
intraperitoneal injection of 1.5 g glucose per kg body weight. Blood glucose level
was measured at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90 and 120 min.
Measurement of virus replication. The virus concentrations of the pancreatic
tissues from EMC-D virus-infected mice were determined with plaque assay using
mEF cells, as described previously15.
Treatment of mouse with IFN-a or poly I:C. We purchased recombinant mouse
IFN-a (PBL Biochemical Laboratories, Piscataway, NJ) and poly I:C (SigmaAldrich, Saint Louis, MI). We dissolved poly I:C in PBS in various concentrations
and injected mice i.p. We used IFN alpha with Dulbecco’s modiﬁed Eagle medium(DMEM) containing 5% fetal bovine serum and 1% penicillin/streptomycin (PcSM;
Gibco, Carlsbad, CA). When the sample was determined to contain 10 IU of mouse
IFN-a measured by the ELISA method, it was equivalent to 52.4 pg of IFN-a.
Anti-EMC-D virus activity. Antiviral activity was evaluated by CellTiter96
Aqueous (Promega, Madison, WI). mEF cells were cultured in a 96-well culture
plate, 4  105 cells per well, and after 12 h serial dilutions of IFN-b (Sigma-Aldrich)were added. After 12 h, the cells were washed with PBS and cultured with EMC-D
virus media. After an hour, virus media were removed and culture media added.
Twenty-four hours later, 20 ml per well One Solution Reagent was added to themedia and cell viability was determined at 490 nm after a 3-h incubation.
Splenic chimera mice. On day 0, recipient mice were lethally irradiated with
8.0 Gy whole-body irradiation (MBR-1520R-3, Hitachi, Japan) and then the
animals were intravenously injected with 2  107 splenocytes on the same day.
Forteen days later, mice were infected with EMC-D virus.
Transplantation of islets. Islets were isolated from Tyk2 KO or WT mice andtransplanted into the renal capsule of B6 mice.
Genetic variant analysis of the Tyk2 gene at promoter region and exons. The
variations in Tyk2 promoter, Exon7 and Exon8 region, were analysed by cycle
sequencing (BigDye Terminator v1.1 Cycle Sequencing Kit, ABI 3130  l DNA
Sequencer, Applied Biosystems, Carlsbad, CA) according to the manufacturer’s
protocol. We used the following primer sets to amplify the intended region of
genomic DNA: for promoter region, forward 50 -CCAGGGCTTTCTGGTTT
GTA-30 and reverse 50 -CTATCTGGCCCAGGCAATAA-30 ; for Exon7, forward
50 -CAGCTGCTCATCATGTCACC-30 and reverse 50 -AAGCCAGGGTAGCTCAA
GTG-30 ; for Exon8, forward 50 -TGGGCTCAGTTTCCTTCTGT-30 and reverse
50 -GAACAGAGGCACAGCAACTG-30 . We used following primers to sequencefor 238 to  590 (relative to the transcriptional start site) of the promoter region,
50 -CACTCACCGTTTCCGGAGTA-30 ; for  560 to  713, 50 -TCAATTCCCAG
CAACCACAT-30 ; for  793 to  957, 50 -GTGGTTGCACATGCCTGTAA-30 ; for 668 to  876, 50 -CTGTAGACCAGGCTGGCTTC-30 ; for  936 to  1288,
50 -CCAGGGCTTTCTGGTTTGTA-30 ; for  941 to  1346, 50 -CAGCTGAAGGGACAGTCTCA-30 ; for Exon7, 50 -AAGCCAGGGTAGCTCAAGTG-30 ; for
Exon8, 50 -GAACAGAGGCACAGCAACTG-30 .

Cell culture and stimulation. Islet cells40 and splenocytes were isolated fromTyk2 þ / þ , Tyk2 þ /  , Tyk2  /  , Tyk2mt/ þ , Tyk2mt/mt, MIP-Tyk2 Tg mice. The
mEF cells were obtained by homogenizing Tyk2 þ / þ , Tyk2 þ /  , Tyk2  /  ,Tyk2mt/ þ , Tyk2mt/mt mice embryos. All mEF cells were used before the ﬁfth
passage. Islet cells, splenocytes and mEF cells were maintained in DMEM (Gibco),
RPMI medium 1640 (Gibco) and DMEM þ GlutaMAX-1 (Gibco), supplemented
with 10% fetal bovine serum (PAA) and 1% PcSM (Gibco), respectively. Islet cells,
splenocytes and mEF cells were stimulated with IFN-b (500 U ml  1; SigmaAldrich) for 12 h. Islet cells, splenocytes and mEF cells from Tyk2 þ / þ , Tyk2 þ /  ,Tyk2  /  , Tyk2mt/ þ , Tyk2mt/mt mice, and islet cells and splenocytes from MIPTyk2 Tg mice were stimulated with IFN-b (500 U ml  1) for 12 h. After 12 h, thecells were collected, and total cellular RNA was isolated to measure the expressionof ISGs (Pkr, 2–5AS, Mx1) induced by IFN-b stimulation.
RNA extraction, cDNA reverse transcription and qRT–PCR analysis. RNA wasextracted using ISOGEN (Nippon Gene, Japan) according to the manufacturer’s
instructions. The total RNA was reverse-transcribed into cDNA by using a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT–PCR
was carried out by using the ABI 7500 Real-Time PCR system with Power SYBR
green Master Mix (Applied Biosystems). The threshold cycle (Ct) value wasnormalized to that of Glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The
qRT–PCR was performed by using the following primer pairs: for Tyk2, forward
50 -AGCCATCTTGGAAGACAGCAA-30 and reverse 50 -GACTTTGTGTGCGATGTGGAT-30 ; for Jak1, forward 50 -AGCATGGTATCTCTCCTCTCTG-30 andreverse 50 -GATTCGGTTCGGAGCGTACC-30 ; for Pkr, forward 50 -CCTCAGAGA
ACGTGTTTACG-30 and reverse 50 -TCAATTCTGTGTTTCGCTTT-30 ; for 2–5AS,
forward 50 -CCCCATCTGCATCAGGAGGTGGAG-30 and reverse 50 -AAGTCATAATACTTTGTCCAGTAG-30 ; for Mx1, forward 50 -CTGAGATGACCCAGCACCTGAA-30 and reverse 50 -CTCCAGGAACCAGCTGCACTTAC-30 ; for Gapdh,
forward 50 -AGGTCGGTGTGAACGGATTTG-30 and reverse 50 -TGTAGACCATGTAGTTGAGGTCA-30 .
Promoter activity analysis. The PCR-ampliﬁed Tyk2 promoter fragment, either
WT or mutant type sequences, was cloned into a pGL4.17[luc2/Neo] vector(Promega). WT and mutant type Tyk2 promoter fragments were prepared from
C57BL/6J and SJL/J mice, respectively, by PCR ampliﬁcation using forward primer
50 -CCCGGTACCCCTTGGGACCTTTGGAGAAT-30 and reverse primer 50 -CCC
GCTAGCCACTCACCGTTTCCGGAGTA-30 , and then Kpn1 and Nhe1 were cut,leading to a 4,167-bp product. Luciferase assay was conducted by transfection ofvectors to 293T cells with 24-well culture plates. Luciferase activity was measured
after 24-h transfection in 293T cells using a dual luciferase assay kit (Promega). The
experiments were repeated four times.
Statistical analysis. Statistical analyses were performed with the JMP9.0.2 software (SAS, Cary, NC). Differences between the means were assessed by two-tailed
Student’s t-test. Other differences among multiple means were assessed using
several appropriate methods for each experiment, as by Kruskal–Wallis test with
Bonferroni’s post hoc analysis, by Kruskal–Wallis test with Dunnett’s post hoc
analysis or by one-way analysis of variance with Dunnett’s post hoc analysis. Error
bars represent the s.e.m. Null hypotheses were rejected at the 0.05 level.

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Acknowledgements
We thank Itsuka Matsumoto, Ai Umei, Miki Kawano, Makoto Matsuo, Hiroko Hamada,Yumi Kamada, Yasuko Kajiki, Kanade Tokuzumim, Yukari Shimoda, Takamitsu Kasagi,
Airsa Moroishi and Drs Yuji Ueki, Kazunobu Amako, Kaoru Aida and Tetsuro
Kobayashi for their help in conducting this research. We also thank Drs Angela Koh andChiri Nagatsuka for their help in preparing the manuscript. We appreciate the technicalsupport from the Research Support Center, Graduate School of Medical Sciences, Kyushu
University. This work was supported by grants (19209037 and 21659230) from the
Ministry of Education, Culture, Sports, Science and Technology and a grant from the
Ministry of Health, Labor, and Welfare of Japan.

Author contributions
K.I., T.Y., H.M., S.F. and S.N. prepared viruses including EMC-D virus, EMC-B virus,
Coxsackie B4 virus and HSV. K.I., H.Y., Y.Y., K.A., Y.Y. and K.S. are involved in thestudies regarding metabolic and immunologic conditions of virus-induced diabetes inTyk2 KO mice, generating congenic Tyk2 KO mice. Y.Kai. and Y.Kurafuji. conducted theradiation transfer experiments. K.I., A.I. and D.M. did histopathologic and immunohistochemical studies of pancreas tissue. T.K., M.H. and H.K. generated MIP-Tyk2 Tg
mouse and studied the signiﬁcance of beta-cell-speciﬁc restoration of the Tyk2 gene inTyk2 KO and mutated mice. K.M. and S.N. performed the Tyk2-mutated congenic mouseresearch involving susceptibility to virus-induced diabetes, promoter function of mutatedTyk2 gene and IFN-induced antiviral responses in vivo and in vitro. M.T., K.H., H.K. and
H.I. assessed the quantitative gene expressions including Tyk2, Jak1 and interferonstimulated genes. K.I., K.M., K.A., H.K. and S.N. designed the experiments and wrote themanuscript. All authors read, gave comments to and agreed with the manuscript andSupplementary Information.

Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
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How to cite this article: Izumi, K. et al. Reduced Tyk2 gene expression in b-cells due tonatural mutation determines susceptibility to virus-induced diabetes. Nat. Commun.
6:6748 doi: 10.1038/ncomms7748 (2015).
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Supplementary Data

Supplementary,Fig.1
35

Blood Glucose (mmol/L)

30
25

Tyk2 +/+ +EMC-D(PO)
Tyk2 -/- +EMC-D(PO)
Tyk2 +/+ +EMC-D(IV)
Tyk2 -/- +EMC-D(IV)

20
15
10
5
0
0

1

2

3

4

5

6

7

Days After Infection

Supplementary Figure 1. The effect of routes of infection to cause virus-induced
diabetes.
1x103 p.f.u. of EMC-D virus was given to Tyk2 WT (Tyk2+/+) and KO (Tyk2-/-) B6 mice(n=4) by intravenous injection (IV) or oral infection (PO). The blood glucose levels
were followed until determination of the development of diabetes. More than 14mmol/Lblood glucose level was diagnosed as diabetic (red line). (n=4)

1

Supplementary,Fig.2
B

Serum

25

10000,
104

20

1000,
103

Rela=ve,Expression,

IFN-α(IU/ml)

A

15
10

Islet
IFN(9),
IFN(+),

102
100,

10,
1,

5

0.1,

0
0

1

2

3

4

5

6

PKR,

7

295,AS,

Mx1,

IFN-α Time CourseSupplementary Figure 2. Expressions of IFN stimulated genes (ISGs) inpancereatic islets after IFN-α transfer.
IFN-α was intraperitoneally injected to WT B6 mice. (A) Assessment of
the level of IFN-α in the blood after 1x103 untis of IFN-α transfer. (n=5) (B)
Quantification of IFN-stimulated genes (ISGs), such as PKR, 2-5AS and Mx1, using
quantitative PCR in islet cells obtained 3 hrs after 1x104 untis of IFN-α transfer.
Expression levels of those genes before stimulation was used as a standard. (n=5)

2Supplementary Fig. 3. Immunohistochemical study of islets in mice infected with
EMC-D virus.
(A) Immunofluorescence detection of insulin (green), glucagon red), CD45(green),
MHC class I (red), IFN-α (green), and IFN-β (red). Wild type B6, Tyk2 KO (Tyk2-/-)/B6, Tyk2 mutated (Tyk2mt/mt) /B6, and Tyk2 KO (Tyk2-/-) MIP-Tyk2 Tg/B6 mice
were used. All mice were studied after intraperitoneal infection of 1x103 p.f.u. of
EMC-D virus. Scale bar, 50µm. (B) Transplanted islets derived from Tyk2 KO/B6 mice
to renal capsule of WT B6 mice were stained with insulin (green) and glucagon (red), 5
days after intraperitoneal infection with 1x103 p.f.u. of EMC-D virus. Scale bar, 50µm.

3Supplementary,Fig.4
A

30
Tyk2 mt/mt +Poly I:C
B6+Poly I:CSJL+Poly I:C

IFN-α(IU/ml)

25
20
15
10
5
0
12hr

1day

2day

3day

Days After Infection

BCIFN-α(IU/mL)

80

100

Tyk2 mt/mt +CVB4
B6
+CVB4
SJL
+CVB4

Tyk2 mt/mt +HSV
B6
+HSV
SJL
+HSV

80
IFN-α(IU/mL)

100

60
40

60
40
20

20
0

0
0

1

2

3

0

Days After Infection

1

2

3

Days After Infection

Supplementary Figure 4. Induction of IFN-α in various strains of mice as B6, SJL,
and Tyk2 mutated B6 mice.
(A) 250 ug of poly I:C was given intraperitoneally. (B) 1x103 p.f.u. of coxsackie B4
was intraperitoneally injected. (n=3) (C) 1x103 p.f.u. of HSV was intraperitoneallyadministered to the mice. Levels of IFN-α were measured by ELISA. (n=3)

4