MOLECULAR AND CELLULAR BIOLOGY, Sept. 2009, p. 4841–4851
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 17
Alpha Interferon Induces Long-Lasting Refractoriness of JAK-STAT
Signaling in the Mouse Liver through Induction of USP18/UBP43?
Magdalena Sarasin-Filipowicz,1,2† Xueya Wang,1† Ming Yan,3Francois H. T. Duong,1Valeria Poli,4
Douglas J. Hilton,5Dong-Er Zhang,3and Markus H. Heim1,2*
Department of Biomedicine, University Hospital Basel, CH-4031 Basel, Switzerland1; Division of Gastroenterology and Hepatology,
University Hospital Basel, CH-4031 Basel, Switzerland2; Moores UCSD Cancer Center, Department of Pathology and
Division of Biological Sciences, University of California San Diego, La Jolla, California 920933; Department of
Genetics, Biology and Biochemistry, University of Turin, 10126 Turin, Italy4; and Division of Molecular Medicine,
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade,
Parkville, Victoria 3050, Australia5
Received 19 February 2009/Returned for modification 15 March 2009/Accepted 18 June 2009
Recombinant alpha interferon (IFN-?) is used for the treatment of viral hepatitis and some forms of cancer.
During these therapies IFN-? is injected once daily or every second day for several months. Recently, the
long-acting pegylated IFN-? (pegIFN-?) has replaced standard IFN-? in therapies of chronic hepatitis C
because it is more effective, supposedly by inducing a long-lasting activation of IFN signaling pathways. IFN
signaling in cultured cells, however, becomes refractory within hours, and little is known about the pharma-
codynamic effects of continuously high IFN-? serum concentrations. To investigate the behavior of the IFN
system in vivo, we repeatedly injected mice with IFN-? and analyzed its effects in the liver. Within hours after
the first injection, IFN-? signaling became refractory to further stimulation. The negative regulator SOCS1
was rapidly upregulated and likely responsible for early termination of IFN-? signaling. For long-lasting
refractoriness, neither SOCS1 nor SOCS3 were instrumental. Instead, we identified the inhibitor USP18/
UBP43 as the key mediator. Our results indicate that the current therapeutic practice using long-lasting
pegIFN-? is not well adapted to the intrinsic properties of the IFN system. Targeting USP18 expression may
allow to exploit the full therapeutic potential of recombinant IFN-?.
Since their discovery in 1957, type I interferons (IFNs) have
become valuable and widely used clinical drugs (13, 20). Alpha
IFN (IFN-?) is used for the treatment of chronic hepatitis B
and hepatitis C and of some forms of cancer, whereas IFN-? is
effective for treating multiple sclerosis. With an estimated 3%
of the global population affected, chronic hepatitis C (CHC)
represents a major health concern since it can lead to liver
cirrhosis and hepatocellular carcinoma (27). Treatment of
CHC with recombinant IFN-?-2a or IFN-?-2b injected three
times a week achieved sustained virological response (SVR) in
15 to 20% of patients. A major improvement of the SVR to 35
to 40% was observed by the addition of the broad-spectrum
antiviral agent ribavirin (39). The introduction of a pegylated,
long-acting form of IFN-?-2 (pegIFN-?-2) further increased
the SVR to 50 to 55% of patients (12, 37). The reasons for the
improved efficacy of pegIFN-? are not known, but it is as-
sumed that the constant high serum concentrations achieved
with pegIFN-? provide for uninterrupted antiviral activity
through a permanent stimulation of the IFN signaling path-
ways, whereas the serum concentrations of standard IFN-?
(with an elimination half-life of 4 to 10 h) decline below phar-
macologically active levels in the second half of each 48 h
dosing interval (4, 54). There is, however, no experimental
evidence for the hypothesis that continuously high IFN-? con-
centrations achieve a better activation of the IFN-?-induced
antiviral effector systems. In fact, there is evidence against this
hypothesis provided by cell culture experiments.
It has been known for many years that cultured cells become
refractory to IFN within hours and remain unresponsive for up
to 3 days (26). Maximal activation of the IFN signaling path-
ways is observed within the first 2 h of IFN treatment. Contin-
uous exposure to IFN results in a “desensitization” character-
ized by a return to pretreatment levels of IFN-stimulated gene
(ISG) transcription. Moreover, during the 48 to 72 h after the
initial IFN-? stimulation of the cells, any further IFN treat-
ment fails to reinduce the transcription of ISGs. At present, it
is not known, whether refractoriness also occurs during IFN
therapies in patients. Such knowledge would be important for
a rational design of IFN therapies. Dosing intervals shorter
than the period of refractoriness would strongly reduce the
efficacy of the administered IFN. Likewise, the use of modified
IFN-? with a prolonged serum half-life such as pegIFN-? with
the aim to achieve constant (peg)IFN-? serum concentrations
would not increase efficacy either, if the target cells remain
unresponsive during most of the dosing interval. Admittedly,
the clinical experience showing an improved therapeutic re-
sponse rate with pegIFN-? argues against the occurrence of
desensitization in patients. To investigate whether the liver
becomes refractory to IFN-? in vivo, we analyzed IFN-? sig-
naling in mice repeatedly injected with IFN-?.
Type I IFNs (IFN-? and IFN-?) exert their effects through
the JAK-STAT signaling pathway (9). Upon binding of IFN-?
* Corresponding author. Mailing address: Department of Biomedi-
cine, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Swit-
zerland. Phone: 41 61 265 33 62. Fax: 41 61 265 23 50. E-mail: markus
† M.S.-F. and X.W. contributed equally to this study.
?Published ahead of print on 29 June 2009.
to its cell surface receptor (IFNAR), the receptor-associated
tyrosine kinases JAK1 and TYK2 become activated and phos-
phorylate tyrosines on the cytoplasmic tails of IFNAR chains 1
and 2. The phosphorylated receptors provide specific docking
sites for STAT1, -2, and -3. STATs are activated at the
receptor-kinase complex by tyrosine phosphorylation (17,
50). Activated STATs dissociate from the receptor and
translocate to the nucleus, where they act as transcription
factors binding to specific regions in the promoters of ISGs
(19). In response to IFN-?, STAT1-STAT2 heterodimers
combine with IFN regulatory factor 9 (IRF9) to form the
transcription complex ISGF3, which binds to IFN-stimu-
lated response elements (ISREs) within the promoters of
ISGs (15). IFN-? also activates homo- and heterodimers of
STAT1 and STAT3, which bind to gamma-activated se-
quence (GAS) response elements (8).
The activation of the JAK-STAT pathway is tightly con-
trolled by several negative regulatory mechanisms. SOCS1 and
SOCS3 prevent STAT activation by inhibiting JAKs (25). Fur-
ther downstream, the protein inhibitor of activated STAT 1
binds to hypomethylated STAT dimers and inhibits STAT-
DNA interaction (30, 40, 49). STATs are deactivated by the
nuclear phosphatase TC-PTP, followed by nuclear export (53).
Recently, ubiquitin-specific peptidase 18 (USP18/UBP43) has
been described as negative regulator in type I IFN signaling.
USP18/UBP43 was originally identified as a protease cleaving
ubiquitinlike modifier ISG15 from target proteins but was re-
cently found to play a negative regulatory role independently
of its ISG15-deconjugating ability (31, 33). By the use of mo-
lecular, biochemical, and genetic approaches, Malakhova et al.
demonstrated that UBP43 specifically binds to the IFNAR2
receptor subunit and inhibits the activity of receptor-associated
JAK1 by blocking the interaction between JAK1 and the IFN
receptor (35). UBP43-deficient mice show a severe phenotype
characterized by brain cell injury, poly(I-C) hypersensitivity,
and premature death (36, 46). Interestingly, they are resistant
to otherwise fatal cerebral infections with lymphocytic chorio-
meningitis virus and vesicular stomatitis virus (45) and have
substantially lower hepatitis B virus DNA levels in a mouse
model of acute hepatitis B (22). Importantly, USP18/UBP43 is
elevated in livers of future nonresponders to pegIFN-? therapy
(5, 47). Moreover, USP18/UBP43 silencing in cells with a rep-
licating chimeric HCV genome results in deregulation of
STAT1 signaling and potentiation of IFN?s ability to inhibit
HCV-RNA replication (43).
To investigate the sensitivity of the liver during prolonged
exposure to therapeutic concentrations of IFN-?, we treated
mice repeatedly with subcutaneous (s.c.) injections of IFN-?
and investigated IFN-? signaling in liver extracts. We report
here that liver cells in vivo become refractory within hours
after the first injection of IFN-?. The negative regulators of
IFN-? signaling, SOCS, were found to be responsible for the
early inhibition of STAT phosphorylation within the first 2 to
4 h but not for the long-term refractoriness. Rather, a long-
lasting upregulation of USP18/UBP43 was found to cause un-
responsiveness to prolonged IFN-? exposure. In the absence of
USP18/UBP43, even a strong upregulation of SOCS1 did not
prevent activation of STAT1 and STAT2.
Taken together, our results demonstrate a refractoriness of
IFN-? signaling in vivo, and indicate that USP18/UBP43 plays
a crucial role in the long-term desensitization of this signal
transduction pathway in the mouse liver. Our findings have
implications for the treatment of patients with CHC. Strategies
aimed at restoring sensitivity to IFN-?, by targeting the up-
regulation of USP18/UBP43 in liver cells, could increase the
efficacy of IFN-? therapies.
MATERIALS AND METHODS
Animals. C57BL/6 mice were obtained from BRL (Biological Research Lab-
oratories, Fu ¨llinsdorf, Switzerland), interleukin-10 (IL-10)-deficient mice and
Alb-Cre [strain B6.Cg-Tg(Alb-cre)21Mgn/J] transgenic mice were obtained from
Jackson Laboratory, Bar Harbor, ME. STAT3lox/loxmice and SOCS3lox/loxmice
were described previously (2, 6). STAT3lox/loxand SOCS3lox/loxmice were crossed
to Alb-Cre transgenic mice to generate AlbCre?STAT3lox/loxand AlbCre?
SOCS3lox/loxconditional knockout (KO) mice, respectively. All transgenic mice
were viable and fertile. AlbCre?STAT3lox/loxand AlbCre?SOCS3lox/loxlitter-
mates were used as negative controls in the experiments. The generation of
SOCS1?/?IFN-??/?and IFN-??/?mice was described previously (1, 7, 52), as
was the generation of UBP43?/?mice on a FVB background (23, 46). Genotyp-
ing for the Cre transgene was performed by PCR using the nucleotides Cre-1
(5?-CACCATTGCCCCTGTTTCACTATC-3?) and Cre-2 (5?-GCCAGGCGTT
TTCTGAGCATAC-3?). Genotyping for the IL-10-deficient mice was performed
by PCR using the nucleotides IL-10-1 (5?-GCCTTCAGTATAAAAGGGGGA
CC-3?), IL-10-2 (5?-GTGGGTGCAGTTATTGTC TTCCCG-3?), and IL-10-
Neo (5?-AATCCATCTTGTTCAATGGCCGATC-3?). STAT3lox/loxgenotyping
was performed by using the primers APRF 11 Up (5?-CACCAACAC ATGCT
ATTTGTAGG-3?), APRF 11 Down (5?-CCTGTCTCTGACAGGCCATC-3?),
and APRF 14 Down (5?-GCAGCAGAATACTCTACAGCTC-3?). SOCS3lox/lox
genotyping was performed with SR221 (5?-GAGTTTTCTCTGGGCGTCCTCC
TAG-3?) and SR222 (5?-TGGTACTCGCTTTTGGAGCTGAA-3?). The ani-
mals were maintained on a 12-h day and 12-h night schedule with ad libitum
access to food and drinking water. Mice were bred in a specific-pathogen-free
environment. Procedures with the animals were conducted with the approval of
the Animal Care Committee of the Canton Basel-Stadt, Switzerland. All
UBP43?/?animals used in the studies were handled in accordance with guide-
lines of The Scripps Research Institute, and the procedures were approved by the
Institutional Animal Care and Use Committee of the institute.
Six- to eight-week-old male animals were used for all experiments. The ani-
mals were anesthetized with isofluorane before blood was dawn from the tail
vessels. The animals were euthanized by CO2narcosis. The resected liver lobes
were immediately frozen in liquid nitrogen and kept at ?70°C until further
processing; one lobe of liver was frozen in TRIzol for RNA isolation. The s.c.
injections with phosphate-buffered saline (PBS) or mIFN-? were performed
between 8:00 a.m. and 5:00 p.m. Recombinant mIFN-? was purchased from
CalBiochem (Juro Supply GmbH, Switzerland). Pegylated human IFN-?-2b was
provided by Essex Chemie AG, Lucerne, Switzerland. PBS was obtained from
the University Hospital Basel. Mouse IL-10 monoclonal antibody was from
Pierce (Perbio Science Switzerland SA, Lausanne, Switzerland) and was injected
intraperitoneally at a dose of 100 ?g 30 min prior to the mIFN-? injections.
ELISA. To isolate serum from mIFN-?- or PBS-injected C57/BL6 mice, 20 to
30 ?l of blood from mouse tail was collected at different time points, kept for 10
min at room temperature and for 30 min at 4°C, and then centrifuged at 2,500 ?
g for 20 min at 4°C. The supernatant was again spun at 1,500 ? g for 10 min at
4°C. For measurement of mIFN-?, the serum was diluted 1:100 in dilution buffer,
and an enzyme-linked immunosorbent assay (ELISA) was performed using a
mouse interferon ELISA kit (Pierce/Perbio Science Switzerland SA, Lausanne,
Switzerland) according to the manufacturer’s instructions.
To measure the mouse IL-10 level, the serum was diluted 1:4 in dilution buffer
and ELISA was performed by using a Quantakine mouse IL-10 immunoassay
(R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instruc-
Protein preparation and Western blot analysis. Portions (30 to 50 mg) of liver
tissue were homogenized in a buffer containing 100 mM NaCl, 50 mM Tris (pH
7.5), 1 mM EDTA, 0.1% TX-100, 10 mM NaF, 1 mM phenylmethylsulfonyl
fluoride, 1 mM vanadate, and 1? protease inhibitor cocktail tablets (Roche
Diagnostics GmbH, Mannheim, Germany). Samples were kept at 4°C for 30 min
and centrifuged for 5 min at 15,000 rpm at 4°C. The protein concentration was
determined by using a Lowry protein assay (Bio-Rad Laboratories AG, Reinach,
Then, 10 to 20 ?g of total protein from mouse liver lysates was loaded for
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and trans-
4842SARASIN-FILIPOWICZ ET AL.MOL. CELL. BIOL.
ferred onto a nitrocellulose membrane (Schleicher & Schuell, Switzerland). The
membranes were blocked in 3% bovine serum albumin–3% milk–0.1% Triton
X-100 for 1 h, washed with Tris-buffered saline–Tween 20, and incubated with
the primary antibody overnight at 4°C.
Proteins were detected with primary antibody specific to phospho-STAT1 (Tyr
701) (catalog no. 9171) and phospho-STAT3 (Tyr 705) (catalog no. 9131; Cell
Signaling, Bioconcept, Allschwil, Switzerland) and phospho-STAT2 (Tyr 689)
(catalog no. 07-224; Upstate, Lake Placid, NY). STAT1 p84/p91 (sc-346), STAT2
(sc-950), and STAT3 (sc-482) were purchased from Santa Cruz (LabForce AG,
Nunningen, Switzerland). Mouse monoclonal STAT1-Ab (carboxy terminus; cat-
alog no. 610186) was from Transduction Laboratories, BD Biosciences, Phar-
mingen. Anti-SOCS-1 (ab3691) was purchased from Abcam, Cambridge, United
Kingdom. Anti-?-actin was from Sigma-Aldrich Chemie GmbH, Steinheim, Ger-
many. Blot-FastStain was obtained from Geno Technology, Inc. (Cell Concepts
GmbH, Umkirch, Germany).
After three washes with Tris-buffered saline–Tween 20, the membranes were
incubated with anti-rabbit antibody-horseradish peroxidase conjugate and anti-
mouse antibody-horseradish peroxidase conjugate obtained from Cell Signaling
(Bioconcept, Allschwil, Switzerland), and signals were detected with SuperSignal
West Pico chemiluminescent substrate (Pierce/Perbio Science Switzerland SA).
Alternatively, signals were detected by using an Odyssey infrared imaging system
from Li-Cor after incubation with infrared fluorescent secondary goat anti-
mouse (IRDye 680) or anti-rabbit (IRDye 800) antibodies (both from Li-Cor
Biosciences) for 1 h at room temperature. The infrared image was obtained in a
single scan, and the signal was quantified by using the integrated intensity.
Electrophoretic mobility shift assay (EMSA). Nuclear extracts from 150 to 200
mg of liver tissue were prepared as previously described (16). Nuclear extract (5
to 10 ng of protein sample) aliquots (2 ?l) were incubated with a32P-radiola-
beled mutated serum-inducible element oligonucleotide designated m67-hSIE
with the sequence 5?-CATTTCCCGTAATCAT-3? for STAT1 and STAT3 or the
ISRE probe derived from the IFN-stimulated gene 15 promoter (ISRE-015) with
the sequence 5?-GAAAGGGAAACCGAACTGAAGC-3? (18). For supershift
experiments, 1 ?l of antibody specific for STAT1, STAT2, or STAT3 was added
to the gel shift incubation reactions.
The samples were loaded on a 5% nondenaturing polyacrylamide gel, and
electrophoresis was performed for 4 h at 400 V at 4°C. The gel was dried and
visualized by autoradiography.
RNA isolation and Northern blot analysis. RNA was purified using TRIzol
(TriReagent) provided by the Molecular Research Center, Inc. (Lucerna Chem
AG, Lucerne, Switzerland). RNA was divided into aliquots and stored at ?75°C.
The denatured RNA was separated on a 1.2% agarose formaldehyde MOPS gel
and transferred to a Hybond-N? nylon membrane (Amersham Pharmacia Bio-
tech Europe GmbH, Du ¨bendorf, Switzerland) by capillary diffusion using 20?
SSC buffer (1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The mem-
branes were hybridized to32P-labeled SOCS1 and SOCS3 probes at 65°C for
overnight in a Quickhyb oven (Stratagene Europe, Switzerland) and washed
twice with 2? SSC–0.2% SDS at 42°C for 15 min and then twice with 0.2?
SSC–0.1% SDS at 42°C for 10 min. The results were visualized by autoradiog-
Real-time quantitative reverse transcription-PCR (RT-PCR). RNA was puri-
fied from frozen liver tissue (?20 mg) with NucleoSpin RNA II kit (Macherey-
Nagel, Du ¨ren, Germany) according to the manufacturer’s instructions. RNA was
divided into aliquots and stored at ?75°C. RNA was reverse transcribed by
Moloney murine leukemia virus reverse transcriptase (Promega Biosciences,
Inc., Wallisellen, Switzerland) in the presence of random hexamers (Promega)
and deoxynucleoside triphosphate. The reaction mixture was incubated for 5 min
at 70°C and then for 1 h at 37°C. The reaction was stopped by heating at 95°C for
5 min. SYBR-PCR was performed based on SYBR green fluorescence (SYBR
green PCR master mix; Applied Biosystems, Foster City, CA). The ?CTvalue
was derived by subtracting the threshold cycle (CT) value for mouse ribosomal
protein L19 (mRPL19), which served as an internal control, from the CTvalues
for SOCS1, SOCS3, protein kinase R (PKR), and USP18, respectively. The
primers were designed across exon-intron junctions to prevent influence from
genomic DNA amplification. The primers were 5?-ATCCGCAAGCCTGTG
ACTGT-3? and 5?-TCGGGCCAGGGTGTTTTT-3? for mRPL19 and 5?-GT
GGTTGTGGAGGGTGAGATG-3? and 5?-GGGATGAGGTCTCCAGCC
A-3? for mSOCS1. The primers were 5?-CCTTTCTTATCCGCGACAGC-3?
and 5?-CGCTCAACG TGAAGAAGTGG-3? for mSOCS3. Primers were 5?-
CGTGCTTGAGAGGGTCATTTG-3? and 5?-GGTCCGGAGTCCACAACT
TC-3? for mUSP18 and 5?-AAGAGCCCGCCGAAAACT-3? and 5?-AGCCA
CTGAATGTAGATGTGACAAC-3? for mPKR.
All reactions were run in duplicate by using an ABI 7000 sequence detection
system (Applied Biosystems). The mRNA expression level between T0 and Tn
was expressed as an n-fold increase according to the formula 2?CT(T0)??CT(Tn).
The mRNA expression levels of the transcripts were calculated relative to
mRPL19 from the ?CTvalues using the formula 2??CT.
Pharmacokinetics of mouse IFN-?. We first studied the
pharmacokinetics of s.c. administered mIFN-? by measuring
the mIFN-? serum concentration in mice at different time
points after injection (Fig. 1). A single dose of 1,000 IU/g of
body weight resulted in a fast increase of mIFN-? from basal
4.3 ? 0.07 (mean ? the standard error of the mean [SEM]) to
13.1 ? 0.41 ng/ml within 60 min, followed by a decline to
pretreatment levels 8 h after the injection (Fig. 1A). The serum
mIFN-? half-life was estimated as 4 to 5 h. To achieve con-
stantly elevated serum mIFN-? concentrations like those ob-
tained with human pegIFN-?, we used a priming dose of 1,000
IU/g, followed by repeated injections of 300 IU/g, and this
approach led to IFN-? serum concentrations oscillating be-
tween 6 and 14 ng/ml for up to 16 h (Fig. 1B).
IFN-? signaling in the mouse liver. To study the kinetics of
IFN-? induced activation of the JAK-STAT pathway, we sac-
rificed mice at different time points after mIFN-? injections
and analyzed the activation of pathway components in extracts
of resected livers. The first applied mIFN-? injection scheme
FIG. 1. Pharmacokinetics of mIFN-? injection into mice. (A) Se-
rum concentrations of mIFN-? after s.c. injection of a single dose of
1,000 IU/g. The profile was obtained from two C57/BL6 mice receiving
mIFN-? by s.c. injection. The peak of serum concentration was
reached 1 h after the injection. After 8 h, the concentration of mIFN-?
returned to pretreatment levels. The error bars represent the SEM.
(B) Serum concentrations of mIFN-? after multiple injections. Two
C57/BL6 mice were injected s.c. with 1,000 IU of mIFN-?/g as a
priming dose and four times with 300 IU/g as a maintenance doses
every 3 h. Sera were collected immediately before each injection and
one h after each injection. The black arrows indicate time points of
mIFN-? injections. The error bars represent the SEM.
VOL. 29, 2009IN VIVO REFRACTORINESS OF IFN-? SIGNAL TRANSDUCTION4843
was based on the use of two doses of 1,000 IU/g given at an 8-h
interval (Fig. 2D). In this setting, the second injection was
given at the time point when serum mIFN-? returned to base-
line levels (see Fig. 1A); this approach simulates the clinical
setting of treatment of patients with CHC with standard
IFN-?, where IFN-? serum concentrations decline below phar-
macologically active levels in the second half of each 48-h
dosing interval (4, 54). Analysis of the response to mIFN-?
revealed a strong phosphorylation of STAT1 within 30 min
after the first injection (Fig. 2A). STAT1 activation reached its
maximum after 1 to 2 h and then declined within 4 h. The
second injection at 8 h induced very little STAT1 phosphory-
lation, although the amount of STAT1 in the liver was strongly
and persistently elevated in response to the first mIFN-? in-
jection (Fig. 2A). Moreover, in an independent long-term ex-
periment with seven injections given every 8 h, we did not
observe restoration of IFN-? sensitivity for up to 48 h (data not
shown). To control for circadian variations and stress, mice
were injected at the same time points with PBS (Fig. 2A).
Although there was some variation in STAT1 expression dur-
ing the experiment, the levels were much lower than those
induced by mIFN-?. As expected, PBS did not induce STAT1
phosphorylation. Treatment with mIFN-? also resulted in
STAT3 phosphorylation in liver cells. The maximal activation
occurred at 1 to 2 h and, in contrast to STAT1, the activation
pattern was similar after the second injection. There was no
upregulation of STAT3 expression after mIFN-? treatment
These results were confirmed by EMSA. Phosphorylated
STATs form dimers that can translocate into the nucleus and
FIG. 2. IFN-? induces refractoriness of JAK-STAT signaling in the mouse liver. (A) C57/BL6 mice were treated with two injections of mIFN-?
(1,000 IU/ml) or PBS, according to the time course indicated in panel D. IFN-?-induced tyrosine phosphorylation of STAT1 and STAT3 was
assessed by immunoblotting of liver whole-cell extracts. Blots were stripped and reprobed for total STAT1 and STAT3 and for actin as a loading
control. (B) DNA binding of STAT1 and STAT3 homo- and heterodimers upon stimulation with mIFN-?. Nuclear extracts of liver cells obtained
from two-dose mIFN-? injected mice were analyzed in EMSAs with the SIE-m67 oligonucleotide probe. Nuclear extract from mouse liver 1 h after
mIFN-? stimulation was used as a positive control (lane A). Antisera specific to STAT1 (lane B) and STAT3 (lane C) were used to shift the STAT1
homodimers, STAT3 homodimers, and STAT1-STAT3 heterodimers (all marked with arrows). (C) DNA binding of ISGF3 in liver cells after
two-dose mIFN-? treatment. Liver nuclear extracts of C57/BL6 mice were analyzed in EMSAs using ISRE oligonucleotide probes. Nuclear extract
from mouse liver 1 h after mIFN-? stimulation was used as a positive control (lane A), and shifts of the ISGF3 band were performed by using
antiserum specific for STAT1 (lane B) and STAT2 (lane C). (D) Schematic diagram showing the time course of the experiment with two injections
of mIFN-?. The interval between the injections is 8 h and the vertical bars in the diagram indicate time points of animal sacrifice and liver resection.
4844 SARASIN-FILIPOWICZ ET AL.MOL. CELL. BIOL.
bind specific response elements such as the GAS element. The
first injection of mIFN-? induced a strong STAT1 homodimer
gel shift using the m67-SIE oligonucleotide (a GAS element),
whereas the second injection had little effect (Fig. 2B). STAT3
homodimers were strongly induced after both the first and the
second injections. IFN-? also induces ISGF3, a heterotrimeric
transcription factor composed of activated STAT1 and STAT2,
and IRF9. Similarly to STAT1 homodimer formation, ISGF3
activity was induced only after the first, but not after the
second injection of mIFN-? (Fig. 2C). Taken together, these
data indicate that IFN-? induced JAK-STAT signaling in
the mouse liver is transient and, furthermore, refractory to
a second mIFN-? dose applied 8 h after the first dose, at a
time point when serum concentrations of mIFN-? have re-
turned to pretreatment levels.
We hypothesized that this long-lasting refractoriness of the
signal transduction pathway could explain a limited success of
anti-HCV therapies where standard IFN-? injected 48 h after
the previous dose encountered still refractory signaling compo-
nents. Since pegIFN-?s with their long half-life are therapeuti-
cally more potent, we next analyzed whether the continuous pres-
ence of high serum concentrations of IFN-? prevented the
induction of refractoriness. Pegylated mIFN-? is not available,
but since human pegIFN-?-2b was reported to exert antiviral
effects in SCID mice infected with Modoc virus (a flavivirus)
(28), we investigated whether human pegIFN-? induces
STAT1 phosphorylation in the mouse liver. We injected mice
s.c. with pegIFN-?-2b in doses ranging from 2 to 2,000 ?g/kg
and found no STAT1 activation within 6 h of treatment (Fig.
3A). Because human pegIFN-? did not stimulate JAK-STAT
signaling in the mouse liver, we applied the second mIFN-?
injection scheme, in which mice were injected with 1,000 IU of
FIG. 3. Refractoriness of JAK-STAT signaling in the presence of continuously elevated mIFN?. (A) C57/BL6 mice were treated with human
pegIFN-?-2b s.c. for 1 h at increasing doses (lanes 1 to 5) and for up to 6 h with 2,000 ?g/kg (body weight) (lanes 7 to 11). Injection of mIFN-?
served as positive control (lane 6). (B) Schematic diagram showing the time course of the multiple injection experiment. Mice were injected with
1,000 IU of mIFN-?/g as a priming dose, followed by four injections of 300 IU of mIFN-?/g as maintenance doses. The vertical bars in the diagram
indicate the time points of animal sacrifice and liver resection. (C) The phosphorylation of STAT1, STAT2, and STAT3 was assessed by Western
blotting with extracts of mouse liver after mIFN-? treatment. Blots were stripped and re blotted for total STAT1, STAT2, and STAT3 and stained
with Blot-FastStain as a loading control. (D) DNA binding of ISGF3 in liver cells after continuous mIFN-? treatment. Liver nuclear extracts of
C57/BL6 mice were analyzed in EMSAs using ISRE oligonucleotide probes. Extract from mouse liver 1 h after mIFN-? stimulation was used as
a positive control (lane A), and shifts of the ISGF3 band were performed by using antibody specific for STAT1 (lane B) and STAT2 (lane C).
(E) SOCS1 and SOCS3 mRNA expression level in the continuous presence of mIFN-?. Total RNA from mIFN-?-injected mice was prepared and
subjected to Northern blot analysis for SOCS1 and SOCS3 mRNA expression. Equal loading of the gel was verified by ethidium bromide staining
and comparing the intensities of the 28S rRNA and 18S rRNA bands. (F) SOCS1 protein expression was determined by Western blotting with
whole-cell extracts of mouse liver after mIFN-? treatment.
VOL. 29, 2009 IN VIVO REFRACTORINESS OF IFN-? SIGNAL TRANSDUCTION 4845
mIFN-?/g as a priming dose, followed by four injections of 300
IU of mIFN-?/g as maintenance doses (Fig. 3B). With this
regimen, steadily elevated mIFN-? concentrations were main-
tained for up to 16 h mimicking the pharmacokinetics of
pegIFN-? in patients (Fig. 1B). Mice were sacrificed at differ-
ent time points (Fig. 3B), and IFN-? signaling was analyzed
with Western blots and EMSAs. There was strong but transient
phosphorylation of STAT1 and STAT2 1 h after initial injec-
tion of mIFN-?, but subsequent injections failed to induce
further STAT1 phosphorylation, although STAT1 expression
was highly upregulated (Fig. 3C). Accordingly, the ISGF3 gel
shift signal was only detectable at 1 h after the initial injection
(Fig. 3D). In contrast to STAT1 and STAT2, activation of
STAT3 was prolonged in the continuous presence of mIFN-?
(Fig. 3C). In conclusion, IFN-? treatment of mice induced a
strong initial activation of STAT1 and STAT2, followed by a
rapid inhibition of signaling and a persistent refractoriness also
in the continuous presence of IFN-?.
Negative regulation of IFN-? signaling by SOCS1 and SOCS3
in the mouse liver. Within hours, IFN-? induces the transcrip-
tional upregulation of SOCS1 and SOCS3, two negative regula-
tors of the JAK-STAT pathway that are instrumental for the
termination of STAT phosphorylation at the receptor-kinase
complex (25). We therefore tested whether the long-term refrac-
toriness of the IFN signal transduction system in mouse liver is
due to a continuous high-level expression of SOCS proteins.
SOCS1 mRNA was detectable at 1 and 3 h, but not during later
time points (Fig. 3E) despite the continuously high serum con-
was barely detectable at later time points (Fig. 3F). Induction of
SOCS3 showed a different pattern. In the continuous presence of
high mIFN-? levels, SOCS3 mRNA expression was induced after
3 h and remained substantially elevated for an extended period of
time (Fig. 3E). The observed SOCS3 upregulation could be
caused by the prolonged STAT3 activation, because STAT3 is a
transcriptional inducer of the SOCS3 gene (3). Since SOCS3 is
known to inhibit IFN-?-induced STAT1 phosphorylation (51),
the prolonged in vivo refractoriness of the IFN system might be
caused by the observed SOCS3 induction.
Role of IL-10, STAT3, SOCS3, and SOCS1 in the long-term
refractoriness of IFN-? signal transduction. Because signaling
through the IFN-? receptor-kinase complex is inhibited by
SOCS3, the signals that maintain high STAT3 phosphorylation
and SOCS3 expression cannot be transmitted through the IFN
receptor but rather have to be derived from a cytokine recep-
tor that is independent of SOCS3. IL-10 is an attractive can-
didate, because it is a strong activator of STAT3 and inducer of
SOCS3 and, importantly, the IL-10 receptor is not inhibited by
SOCS3 (55). Furthermore, IL-10 inhibits expression of IFN-?
induced genes by suppressing STAT1 phosphorylation in
monocytes (21) and attenuates IFN-?-induced STAT1 phos-
phorylation in the mouse liver (48). We therefore measured
mouse IL-10 serum levels upon a single injection or multiple
injections of mIFN-? and indeed found strong induction of
IL-10 (Fig. 4A). After a single mIFN-? injection, the IL-10
serum concentrations were transiently elevated (Fig. 4A, left
panel), but in the setting of multiple injections with the result-
ing constantly elevated serum IFN-? concentration, the IL-10
levels remained high (Fig. 4A, right panel). This indicates that
the IFN-? induced pathways that lead to elevated serum IL-10
do not become refractory.
To clarify the role of IL-10 in the observed refractoriness of
IFN-? signaling, we used IL-10-deficient mice and injected
them with two doses of mIFN-? given 8 h apart (Fig. 4B, upper
panel). STAT1 activation in these mice was assessed 1 h after
the first and the second injections (Fig. 4B, lower panel). While
the first mIFN-? injection induced a strong phospho-STAT1
signal after 1 h, the second injection 8 h later had little effect
on STAT1 phosphorylation (time point 9 h) both in the wild-
type (WT) and the IL-10-deficient mice. These results were
confirmed in WT mice injected with neutralizing IL-10 anti-
body 30 min prior to mIFN-? injections (data not shown). This
indicates that refractoriness of IFN-? signaling is not IL-10
dependent. Of note, there was no decrease of STAT3 phos-
phorylation in the IL-10-deficient mice compared to the WT
mice. We conclude that the elevated serum IL-10 concentra-
tions induced by mIFN-? injections are not required for the
prolonged activation of STAT3 and do not cause refractori-
To test whether the activation of STAT3 or the upregu-
lation of SOCS3 are required for the induction of the re-
fractory state, we used hepatocyte-specific STAT3- and SOCS3-
deficient mice. Toward this aim, we crossed STAT3lox/loxmice (2)
and SOCS3lox/loxmice (6) with albumin-Cre transgenic mice (42).
The IFN-? signal transduction pathway was assessed in these
mice by using the two-dose mIFN-? injection setting (Fig. 4B,
upper panel). Neither the deletion of STAT3 nor the deletion of
SOCS3 could restore responsiveness to the second injection of
mIFN-? (Fig. 4C), arguing against a substantial role of STAT3
and SOCS3 as mediators of IFN-? refractoriness.
SOCS1 mRNA and protein levels were elevated only at early
time points of the 16-h kinetic analysis (Fig. 3B, E, and F); it
was therefore unlikely that this negative regulator mediates
long-term refractoriness. We nevertheless wanted to directly
test if long-term refractoriness is mediated by SOCS1. Mice
deficient in SOCS1 are not viable due to hypersensitivity to
IFN-? (52). We therefore analyzed the role of SOCS1 in
SOCS1?/?/IFN-??/?mice (and IFN-??/?mice as controls)
using the two-dose mIFN-? injection setting (Fig. 4B, upper
panel). IFN-??/?mice showed similar pSTAT1 signaling in
response to mIFN-? if compared to WT mice and, as expected,
the deletion of SOCS1 did not prevent refractoriness in re-
sponse to a second injection of mIFN-? (Fig. 4D).
Prolonged upregulation of USP18/UBP43 is responsible for
IFN-? refractoriness. Recently, USP18/UBP43 emerged as an
important negative regulator in type I IFN signaling (31, 33, 35,
43). We therefore measured USP18 mRNA levels in livers of
WT, IL-10-deficient, and hepatocyte-specific STAT3- and
SOCS3-deficient mice after two injections of mIFN-?. In all of
these mouse strains, USP18 mRNA was strongly upregulated
1 h after the first injection and also 1 h after the second
injection (time point 9 h) (Fig. 5A). In the setting of repeated
injections (Fig. 3B), leading to constantly elevated serum
mIFN-? concentrations, USP18 mRNA was upregulated 1 h
after the first injection and remained induced more than five-
fold at all later time points for up to 16 h (data not shown).
This expression profile suggests an involvement of USP18/
UBP43 in the induction and maintenance of a refractory state
of the IFN-? signaling pathway in the liver.
4846SARASIN-FILIPOWICZ ET AL.MOL. CELL. BIOL.
We therefore assessed IFN-? signaling in livers of USP18/
UBP43-deficient mice (36, 46). In the two-dose injection set-
ting, UBP43?/?mice showed a 2.2-fold stronger STAT1 phos-
phorylation after the first injection of mIFN-? than WT
controls (Fig. 5B and C). This finding is consistent with previ-
ous in vitro findings in mouse embryonic fibroblasts lacking
UBP43 and in human hepatoma Huh7.5 cells where silencing
of USP18/UBP43 leads to prolonged responsiveness to IFN-?
and enhanced antiviral efficacy against HCV (35, 43). Impor-
tantly, UBP43?/?mice were responsive also to the second
injection of mIFN-? and showed significant increase of
pSTAT1 signals at 9 h if compared to the 8-h time point (Fig.
5B and C). The pSTAT1 signals were increased at all time
points in UBP43?/?mice, again supporting the important neg-
ative regulatory role of UBP43 in IFN-? signaling. Of note, the
pSTAT1 signal at 9 h was not as strong as at 1 h. We conclude
that UBP43 is not the only mediator of long-term refractori-
ness. However, UBP43 is a very important component of long-
term refractoriness, because the second dose of mIFN-? can-
not only induce a pSTAT1 signal that is significantly stronger
than at time point 8 h, but is as strong as the pSTAT1 signal
observed after a first injection in a WT mouse (Fig. 5B and C;
FIG. 4. IL-10, STAT3, SOCS3, and SOCS1 are not responsible for refractoriness of IFN-?-induced JAK-STAT signaling. (A) In the left panel,
injection of a single s.c. dose of mIFN-? (1,000 IU/g) induces a transient increase in IL-10 serum concentrations. The level of mouse IL-10 as
determined by ELISA is shown. In the right panel, repeated injections of 300 IU of mIFN-?/g every 4 h after an initial priming dose of 1,000 IU/g
induce constantly high mouse IL-10 serum concentrations. During mIFN-? maintenance doses, the concentration of IL-10 stays at levels of ?40
pg/ml (black triangles). As a negative control, the level of serum IL-10 was determined in a mouse multiply injected with PBS (see the graph with
black squares). (B) STAT1 activation in response to two injections of mIFN-? in IL-10-deficient mice. The upper panel shows a schematic
representation of the experimental design with two mIFN-? injections performed 8 h apart. Animals were sacrificed at time points 0 (control) and
1 h and 9 h (1 h after the second injection). Immunoblotting for pSTAT1/STAT1, pSTAT3/STAT3, and actin is shown for C57BL/6 WT mice and
for IL-10?/?animals. (C) The upper panel shows results for STAT activation in response to two injections of mIFN-? in mice with liver-specific
KO of STAT3 or SOCS3. Immunoblotting for pSTAT1/STAT1, pSTAT3/STAT3, and actin is shown for WT C57BL/6 mice and for SOCS3lox/lox
Cre?and STAT3lox/loxCre?animals. The lower panel presents the results of quantitative RT-PCR analysis of SOCS3 mRNA expression in the livers
of WT, SOCS3lox/loxCre?, and STAT3lox/loxCre?animals at time points 0, 1 h, and 9 h (1 h after the second injection). The data are plotted as the
amount of SOCS3 mRNA relative to mRPL19 mRNA and are means (with the SEM) of two animals per time point for the KO mice and four
animals per time point for the WT mice. (D) STAT activation in response to two injections of mIFN-? in mice deficient for SOCS1. Immuno-
blotting for pSTAT1/STAT1 and actin is shown for IFN-??/?mice and for IFN-??/?SOCS1?/?mice.
VOL. 29, 2009 IN VIVO REFRACTORINESS OF IFN-? SIGNAL TRANSDUCTION4847
compare the time point 1 h in the WT mice to the time point
9 h in the UBP43?/?mice). Supporting the role of UBP43 in
long-term refractoriness, we observed a persistent phosphory-
lation of STAT1 and STAT2 for up to 13 h in UBP43?/?mice
that were repeatedly injected with mIFN-? (Fig. 5D; see Fig.
3B for experimental setup). We conclude that IFN-? refracto-
riness is associated with the presence of USP18/UBP43 in the
liver and that absence of USP18/UBP43 allows for a substan-
tial stimulatory effect of the second mIFN-? dose, as well as of
maintenance doses in the setting of repeated injections.
To investigate whether maintained phosphorylation of STATs
in UBP43?/?mice is reflected by maintenance of IFN-? target
gene induction, we measured SOCS1 and PKR expression in
WT and USP18/UBP43-deficient mice. While in WT mice
SOCS1 and PKR mRNA were induced only in response to the
first injection, UBP43?/?mice were hyper-responsive at 1 h
and, at 9 h, when SOCS1 and PKR mRNA were no longer
inducible in WT mice, their levels even further increased in
UBP43?/?mice (Fig. 5E). Remarkably, despite these high
SOCS1 mRNA levels at 9 h, all IFN-? stimulated STATs
showed a strong phosphorylation (Fig. 5B). Similarly, contin-
uous stimulation with mIFN-? resulted in continuously ele-
FIG. 5. USP18/UBP43-deficient mice remain responsive to IFN-?. (A) Quantitative RT-PCR analysis of USP18 mRNA expression in response
to the first and second injections of mIFN-?. RNA was isolated from the livers of WT, IL-10?/?, SOCS3lox/loxCre?, and STAT3lox/loxCre?animals
at time points 0, 1 h, and 9 h (1 h after the second injection). The data are plotted as the amount of USP18 mRNA relative to mRPL19 and are
means (with the SEM) of two animals per time point for the KO mice and four animals per time point for the WT mice. (B) Activation of the
JAK-STAT signaling pathway in response to the first and second injection of mIFN-? in WT and USP18/UBP43-deficient mice. Phosphorylation
of STAT1, STAT2 (upper band), and STAT3 was assessed by Western blotting with extracts of mouse liver from WT and USP18/UBP43?/?mice.
Blots were stripped and reblotted for total STAT1, STAT2, and STAT3 and for actin as a loading control. (C) Bar diagram of phospho-STAT1
signals in WT and USP18/UBP43-deficient mice in response to the first and second injections of mIFN-?. Phospho-STAT1 and actin signals from
three mice per time point were quantified by using an Odyssey infrared imaging system. The integrated intensities of phospho-STAT1 signals were
divided by the actin values. The y axis displays arbitrary units. Shown are mean values and the 95% confidence intervals. (D) STAT activation in
response to continuous presence of mIFN-? in WT and USP18/UBP43-deficient mice. Phosphorylation of STAT1 and STAT2 (upper band) was
assessed by Western blotting with extracts of mouse liver from WT and USP18/UBP43?/?mice. Blots were stripped and reblotted for total STAT1
and for actin as loading control. (E) Quantitative RT-PCR analysis of SOCS1 and PKR mRNA expression in response to the first and the second
injections of mIFN-? in WT and in USP18/UBP43-deficient mice. Two mice were sacrificed per time point. Shown are mean values (with the SEM)
of the ratio of SOCS1 and PKR mRNA over mRLP19 mRNA. (F) Quantitative RT-PCR analysis of SOCS1 mRNA expression in response to
continuous stimulation with mIFN-? in WT mice and in USP18/UBP43-deficient mice.
4848SARASIN-FILIPOWICZ ET AL.MOL. CELL. BIOL.
vated mRNA levels of SOCS1 in UBP43?/?mice (Fig. 5F). We
therefore conclude that in the absence of USP18/UBP43,
SOCS1 cannot inhibit IFN-?-induced phosphorylation and ac-
tivation of STAT1, STAT2, and STAT3. This provides a strong
argument for the importance of USP18/UBP43 as negative
regulator of IFN responses in the liver.
Desensitization of the IFN signal transduction pathways
during prolonged exposure of cultured cells to IFN-? has been
described more than 20 years ago (26), but very little was
known regarding whether and to what extent IFN refractori-
ness occurs in animals and humans. Infections that activate the
endogenous type I IFN system usually last for several days and
weeks and can even last for years, such as chronic viral hepa-
titis. Intuitively, one would assume that the IFN system re-
mains responsive and effective, at least in all those situations in
which the infection is being cleared. In the present study we
present strong evidence that the IFN-? signaling pathways in
mouse liver become unresponsive within hours after the first
application of mIFN-?, indicating that desensitization may also
occur upon clinical use of IFN and negatively influence the
Refractoriness was observed in mice that received multiple
injections of mIFN-? and had sustained serum IFN-? levels
between 6 and 14 ng/ml, i.e., concentrations that induce a
strong STAT1 activation before the initiation of the refractory
state (see 30-min time points in Fig. 1A and 2A). The repeated
injection scheme was applied to mimic the constant high
pegIFN-? serum levels observed in CHC patients, because
pegylated mouse IFN-? is not available and human pegIFN-?
does not activate the JAK-STAT pathway in mouse liver (Fig.
3A). We cannot formally prove that pegylated mouse IFN-?
would induce a refractory state of the IFN signaling pathway.
However, pegIFN-? binds to the same receptor and uses the
same signaling pathway as unmodified IFN-? and is therefore
very likely to also induce the same negative regulators. Refrac-
toriness was also observed in mice that received two mIFN-?
doses, i.e., a second injection 8 h after the initial dose, and thus
at a time when mIFN-? serum concentrations returned to
pretreatment levels. The refractory state was characterized by
an almost complete inhibition of tyrosine phosphorylation of
STAT1 and STAT2. The residual STAT1 and STAT2 activa-
tion documented by the faint phospho-STAT1 and -STAT2
signals detected in Western blots was not sufficient to induce
target genes, such as SOCS1 and PKR. One possible explana-
tion may be provided by the IFN-?-induced increase of total
STAT1 and, to a lesser extent, STAT2 protein amounts, which
further reduced the ratio of phosphorylated to unphosphory-
lated STATs. The induction pattern of SOCS1 mRNA with a
peak at 1 to 3 h is consistent with its well-known role in the
early negative-feedback regulation of IFN-? signaling. Since
SOCS1 is not expressed to any detectable degree at later time
points (Fig. 3E and F), its involvement in the long-lasting
inhibition of STAT1 and STAT2 phosphorylation is unlikely
and, indeed, SOCS1-deficient mice exhibit refractoriness to a
second dose of mIFN-? (Fig. 4D).
STAT3 can be activated by IFN-? to form transcriptionally
active homodimers or STAT1-STAT3 heterodimers (14). In-
terestingly, STAT3 showed an activation pattern that differed
from STAT1 and STAT2. STAT3 was maximally phosphory-
lated after 1 h and remained activated at most time points
during the course of the multiple injection experiment (Fig.
3C). Accordingly, expression of SOCS3, a known target gene
of STAT3, was also upregulated during the entire experiment
(Fig. 3E). Assuming that SOCS3 inhibits IFN-? signaling in the
mouse liver, as has been reported in cultured cells (51), the
continuous activation of STAT3 cannot be due to IFN-?-in-
duced signals. Among other cytokines known to stimulate
STAT3 activation, IL-10 was an attractive candidate, particu-
larly because its receptor-kinase complex is not inhibited by
SOCS3 (55). IFN-? not only exerts direct antiviral effects
against HCV but also plays an important immunomodulatory
role in chronic HCV infection. IL-10 as an immunosuppressive
cytokine is potentially implicated in the treatment outcome in
CHC. For instance, IL-10 production was substantially in-
creased in PBMCs from CHC patients obtained 12 h after the
first injection of IFN-?-2 when in vitro stimulated with lipo-
polysaccharide or the HCV protein NS3 (32). Blocking of the
IL-10 receptor, in turn, was shown to generate favorable T-
helper cell responses in vitro in PBMCs originating from CHC
patients (44). Interestingly, baseline IL-10 levels were signifi-
cantly increased in patients with CHC and no response to
IFN-based treatment compared to responders and healthy
control subjects (38). Likewise, production of IL-10 during
LCMV infection in mice was associated with viral persistence,
and blockade of the IL-10 receptor resulted in viral clearance
(11). Our novel finding of high IL-10 levels in mouse sera in
response to repeated mIFN-? injections was therefore a very
promising candidate mechanism for explaining refractoriness
of IFN-? signal transduction. However, we found induction of
a refractory state in IL-10-deficient mice, indicating that IL-10
is not responsible for IFN-? refractoriness (Fig. 4B). Likewise,
mice with liver-specific deficiency in STAT3 and SOCS3 were
refractory to prolonged mIFN-? stimulation (Fig. 4C), a find-
ing that further argues against an important role of the
STAT3-SOCS3 axis in the induction of IFN-? refractoriness.
USP18/UBP43 was originally identified as a protease cleav-
ing ubiquitinlike modifier ISG15 from target proteins. ISG15 is
an ubiquitinlike protein that conjugates to numerous proteins
in cells treated with IFN-?. The negative regulatory role of
UBP43 in IFN-? signaling was initially thought to be mediated
through its ISG15-deconjugating ability (31, 33). However, ab-
lation of ISG15 did not reverse the IFN-hypersensitive pheno-
type of UBP43?/?mice (24, 41). Moreover, IFN-?-induced
STAT1 phosphorylation and ISG induction were inhibited by
an active site cysteine mutant (c61s) of UBP43, UBP43C61S,
which is no longer enzymatically active (35). Indeed, USP18/
UBP43 blocks JAK1 phosphorylation through a specific inter-
action with the IFNAR2 subunit of the receptor and thereby
attenuates IFN signaling independent of its isopeptidase activ-
ity toward ISG15 (35). USP18/UBP43 is induced by IFN-? (10,
34) and provides a negative-feedback loop that restricts IFN-?
signals. In the liver, USP18/UBP43 shows a low constitutive
expression (31), and we found a strong upregulation of USP18
mRNA after treating mice with s.c. injections of mIFN-? (Fig.
5A). In contrast to SOCS1 with its transient upregulation in
response to the first injection of mIFN-? (Fig. 3E), USP18/
UBP43 was highly induced also 1 h after a second injection of
VOL. 29, 2009IN VIVO REFRACTORINESS OF IFN-? SIGNAL TRANSDUCTION4849
mIFN-? (Fig. 5A) and remained fivefold increased for up to
48 h (data not shown). Since the apparent half-life of USP18
mRNA is 3 to 4 h (29), this prolonged upregulation of USP18/
UBP43 requires continuous transcriptional activation of its
gene, possibly sufficiently induced by the very weak STAT1
activity observed after a second injection of mIFN-? (Fig. 2A).
This would implicate that the USP18 gene promoter is more
sensitive to STAT1 stimulation than promoters of other ISGs,
i.e., of SOCS1 (Fig. 5E). Whatever the mechanism that main-
tains its prolonged upregulation, UBP43 is clearly important
for the induction of IFN-? refractoriness, since USP18/
UBP43-deficient mice remain sensitive to continuous stimula-
tion with mIFN-? (Fig. 5B to F). It is interesting in this context
that USP18 mRNA expression, but not SOCS1 expression, is
increased in the livers of “preactivated” future nonresponders
to pegIFN-? treatment (5, 47). USP18/UBP43 therefore is of
special interest not only as predictor of treatment outcome but
may also be a potentially critical determinant of responses to
pegIFN-? in patients with CHC.
USP18/UBP43 restricts the IFN-?-induced upregulation of
more than 700 genes, among them SOCS1 (56). Silencing of
USP18 in Huh7.5 cells leads to increased cellular protein
ISGylation in response to IFN-? and a general enhancement
of ISG expression (43). Indeed, SOCS1 was highly expressed in
the liver of UBP43?/?mice injected with mIFN-? (Fig. 5E and
F). Interestingly, in UBP43?/?mice SOCS1 expression was
further increased after the second injection of mIFN-?. Despite
the very high expression of SOCS1 at 9 h, the second injection of
mIFN-? induced a strong phosphorylation of STAT1 in
UBP43?/?mice (Fig. 5B and E). Similarly, SOCS1 mRNA was
highly increased in UBP43?/?mice during the entire 13 h of
the experiment with repeated mIFN-? injections, while at the
same time STAT1 phosphorylation was strong (Fig. 5D and F).
These results provide genetic evidence that for a complete
inhibition of IFN-?-induced STAT phosphorylation, SOCS1
requires the presence of USP18/UBP43.
Our results have potentially important consequences for the
treatment of patients with chronic viral hepatitis with recom-
binant IFN-?. If we assume that also the human liver becomes
refractory to IFN-? within hours after the first administration
of recombinant IFN-? and that liver cells remain unresponsive
to further IFN-? stimulation for an unknown time, then the
current practice of injecting pegIFN-? with its very long half-
life would lack a pharmacodynamic rational. The pegIFN-?
effect on its prime target cells, the HCV-infected hepatocytes,
may be restricted to the early phase of the dosing interval,
whereas the prolonged pegIFN-? presence could have un-
wanted secondary effects in other organ systems such as the
central nervous system, the skin, the muscles and the joints.
Although the mechanisms underlying the increased efficacy of
pegIFN-? compared to standard IFN-? remain unsolved, it is
conceivable that the continuously high serum IFN-? concen-
trations obtained with pegIFN-? lead to an activation of IFN-?
signal transduction as soon as hepatocytes recover from their
refractory state. Ideally, choosing a dosing interval for standard
IFN-? that would avoid the refractory period of hepatocytes
and result in a maximal restimulation of the IFN system might
represent a cost-effective strategy and also reduce toxic side
effects of (peg)IFN-? therapies. The results presented here
should therefore motivate an in-depth analysis of the pharma-
codynamic effects of the current pegIFN-? treatments in the
livers of patients with CHC.
This study was supported by Swiss National Science foundation
grant 320000-116106, Swiss Cancer League grant KLS-01832-02-2006,
Krebsliga Basel grant 8/05, and National Institutes of Health grant
HL091549 (D.-E.Z.), as well as by a grant from the Roche Research
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