Wang, Y. et al. Rig-I−/− mice develop colitis associated with downregulation of G alpha i2. Cell Res. 17, 858-868

Department of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Cell Research (Impact Factor: 12.41). 11/2007; 17(10):858-68. DOI: 10.1038/cr.2007.81
Source: PubMed
ABSTRACT
RIG-I (retinoid acid-inducible gene-I), a putative RNA helicase with a cytoplasmic caspase-recruitment domain (CARD), was identified as a pattern-recognition receptor (PRR) that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I(-/-) mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I(-/-) mice are viable and fertile. Histological analysis shows that Rig-I(-/-) mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer's patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation of T-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein alpha i2 subunit (G alpha i2) in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated G alpha i2 expression. Moreover, G alpha i2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of G alpha i2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of G alpha i2 and disturbed T-cell homeostasis.

Full-text

Available from: Xue-Song Liu, Feb 11, 2015
Cell Research | www.cell-research.com
Colitis and downregulation of Gαi2 in Rig-I
-/-
mice
858
npg
ORIGINAL ARTICLE
Rig-I
-/-
mice develop colitis associated with downregula-
tion of Gαi2
Yi Wang
1*
, Hong-Xin Zhang
1*
, Yue-Ping Sun
1
, Zi-Xing Liu
1
, Xue-Song Liu
1
, Long Wang
2,3
, Shun-Yuan Lu
2,3
, Hui
Kong
3
, Qiao-Ling Liu
3
, Xi-Hua Li
1
, Zhen-Yu Lu
1
, Sai-Juan Chen
2
, Zhu Chen
2
, Shi-San Bao
4
, Wei Dai
5
, Zhu-Gang
Wang
1,2,3,4
1
Department of Medical Genetics, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;
2
State Key Laboratory
of Medical Genomics, Rui-Jin Hospital Afliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;
3
Shanghai Research Center for Model Organisms, Shanghai 201203, China;
4
Department of Pathology, University of Sydney, Sydney,
2570 NSW, Australia;
5
Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA
Introduction
RIG-I (retinoid acid-inducible gene-I) was originally
found to be upregulated in differentiating NB4 cells that
are induced by all-trans retinoic acid (ATRA) [1, 2]. It is
a member of the DExD/H box protein family containing
a C-terminal helicase domain and two tandem caspase-
recruitment domains (CARDs) [3]. Recent studies have
shown that RIG-I plays a crucial part in guarding against
viral invasion as an intracellular molecular sensor. RIG-I
binds to synthetic dsRNA or viral dsRNA through its heli-
case domain, inducing a conformational change that allows
the N-terminal CARD domains to recruit the downstream
signaling protein MAVS (also called IPS-1, VISA, and
Cardif). The interaction between RIG-I and MAVS triggers
both NF-κB and IRF3 signaling pathways, leading to the
activation of the IKK and TBK-1/IKKε kinase complexes,
and, subsequently, the induction of IFN-β. Hepatitis C virus
(HCV) can interrupt RIG-I signaling to IRF-3 and NF-κB
through the cleavage of MAVS by its NS3/4 protease [4-
11]. The RIG-I-mediated antiviral activity is negatively
Cell Research (2007) 17:858-868.
© 2007 IBCB, SIBS, CAS All rights reserved 1001-0602/07 $ 30.00
www.nature.com/cr
npg
RIG-I (retinoid acid-inducible gene-I), a putative RNA helicase with a cytoplasmic caspase-recruitment domain
(CARD), was identied as a pattern-recognition receptor (PRR) that mediates antiviral immunity by inducing type I
interferon production. To further study the biological function of RIG-I, we generated Rig-I
-/-
mice through homologous
recombination, taking a different strategy to the previously reported strategy. Our Rig-I
-/-
mice are viable and fertile. His-
tological analysis shows that Rig-I
-/-
mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate
sodium-induced colitis. Accordingly, the size and number of Peyers patches dramatically decreased in mutant mice. The
peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naïve T cells,
indicating an important role for Rig-I in the regulation of T-cell activation. It was further found that Rig-I
deciency leads
to the downregulation of G protein αi2 subunit (Gαi2) in various tissues, including T and B lymphocytes. By contrast,
upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated Gαi2 expression. Moreover,
Gαi2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these ndings
suggest that Rig-I has crucial roles in the regulation of Gαi2 expression and T-cell activation. The development of colitis
may be, at least in part, associated with downregulation of Gαi2 and disturbed T-cell homeostasis.
Keywords: Rig-I knockout mice, colitis, Peyers patches, T-cell homeostasis, Gαi2 expression
Cell Research (2007) 17:858-868. doi: 10.1038/cr.2007.81; published online 25 September 2007
*These authors contributed equally to this work.
Correspondence: Zhu-Gang Wang
Tel/Fax: +86-21-6445799
E-mail: zhugangw@shsmu.edu.cn
Received 22 January 2007; revised 1 April 2007; accepted 9 May 2007;
published online 25 September 2007
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Yi Wang et al.
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regulated by the A20 protein [12]. It has been shown that
RIG-I, but not the TLR system, has an essential role in the
antiviral response in vivo in various cells except pDCs [13].
Thus, RIG-I is thought to be the third pattern-recognition
receptor in addition to TLRs and NLRs. Moreover, RIG-I
can also regulate the transcription of many genes including
interferon-γ stimulated gene 15 in MCF-7 cells and COX-2
in endothelial cells [14,15].
Human inammatory bowel disease (IBD), including
Crohns disease and ulcerative colitis, is a multifactor
disease that is manifested by cellular inammation and
intestinal tissue damage. It is also characterized by the dys-
function of mucosal immunity, including the malfunction
of T cells and aberrant cytokine production. However, the
precise mechanism that underlies colitis remains unclear.
Studies on mice with targeted disruption of the genes that
regulate the T-cell immune responses have shed light on the
development of colitis. For example, mice with targeted dis-
ruption of Gαi2 develop colitis with 100% penetrance [16,
17]. Gαi2 is regarded as one of the candidate genes for IBD
in human, because genetic linkage studies have mapped the
Gαi2 gene within an IBD susceptible locus at chromosome
3p21 [18]. Heterotrimeric G proteins consist of α, β and γ
subunits. Gαi2 is one of the Gα subunits, which has a role
in a large variety of cellular and metabolic processes [19-
23]. Furthermore, a decreased number of Peyers patches
was also observed in Gαi2
-/-
mice [16,17,19]. It is important
to emphasize that Peyers patches provide the rst line of
defense against pathogen invasion in the intestine. Mice
with Gαi2 ablation exhibit an accelerated transition from
double-positive to single-positive thymocytes, leading to
selective CD4
+
T-cell disorders. Increases in effector T
cells and decreases in naïve T cells were also observed in
the spleen of Gαi2
-/-
mice [24,25]. These data suggest that
Gαi2 is associated with the induction of colitis and that it
is a negative regulator of the T-cell response.
To further
investigate
the biological function of RIG-I, we
generated Rig-I-decient (Rig-I
-/-
) mice through homolo-
gous recombination, taking a different strategy to the previ-
ously reported strategy [13]. We found that Rig-I
-/-
mice are
viable and fertile. They develop a colitis-like phenotype and
increased susceptibility to dextran sulfate sodium (DSS)-
induced colitis, which is accompanied by a decrease in the
size and number of Peyers patches, abnormal activation
of peripheral T cells and downregulation of Gαi2. These
ndings reveal a novel role for Rig-I in the regulation of
intestinal mucosal immunity and Gαi2 expression.
Materials and Methods
Generation of Rig-I knockout mice
A Rig-I targeting vector was designed to delete the 6.4-kb frag-
ment that contains exons 4 to 8, which encodes part of the CARD
domain 2, and that contains the A and B motifs of the RNA helicase
domain. The targeting construct was electroporated into ES cells.
After double selection with G418 and GANC, the resistant clones
were genotyped using Southern blotting. Two correctly recombined
ES cell clones were used to create Rig-I mutant mice through blas-
tocyst microinjection. The Rig-I
+/-
mice were generated by crossing
chimeras with wild-type 129S1 mice. They were genotyped by
PCR using two primer pairs in one reaction, which allows the am-
plication of wild type and targeted alleles. The primers used for
genotyping were 5′-GCCTAGCTAGCCAAAGTAACAC-3′ and
5′-GCAGCGCATCGCCTTCTATC-3′ for the targeted allele, and
5′-CACAGTTGCCTGCTGCTCAT-3 and 5′-CAGGAAGAGC-
CAGAGTGTCAGAAT-3′ for the wild-type allele. PCR was run for
30 cycles at 94 °C for 30 s, 58 °C for 90 s, 72 °C for 90 s, and a nal
extension at 72 °C for 10 min after an initial denaturation at 94 °C
for 5 min. The Rig-I mutant mice were bred in specic pathogen-free
conditions and maintained in a 129S1 background.
Northern blot analysis
Mouse primary embryonic broblasts (MEFs) were isolated from
13.5 dpc embryos and plated at a concentration of 2×10
6
cells in
10-cm dishes. The cells were treated with 1000 U/ml murine IFN-β
for 24 h. Total RNA was extracted using the TRIZOL reagent (In-
vitrogen, Carlsbad, CA, USA) and analyzed with northern blotting,
using a 477-bp Rig-I cDNA fragment (477 bp, composed of exons
11 to 14) as a probe.
Western blot analysis
Cell suspensions from the spleen were prepared by passing tis-
sues through a cell strainer. 10
6
splenocytes were stimulated by LPS
(20 µg/ml) for 72 h. Cell lysates were prepared by adding 0.6 ml of
RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS in PBS) with freshly supplemented protease inhibitors (100 µg/
ml PMSF, 50 KIU/ml aprotinin, 1 mM sodium orthovanadate). In
addition, cell lysates were also prepared from the colons and in-
testines of mice at 8 weeks of age as described above. Cell lysates
(50 µg) from cells or tissues were electrophoresed and transferred
to PVDF membrane (BioRad, Hercules, CA, USA) according to
the manufacturers protocol. The protein blots were blocked with
5% non-fat milk in TBS, then incubated overnight with antibodies
to RIG-I (1:2000 dilution in the blocking buffer), to Gαi2 (1:1000)
(Santa Cruz Biotechnology, Inc. CA, USA), or to α-tubulin (1:10 000)
(KPL, Gaithersburg, MD, USA). After washing, the membrane was
incubated with horseradish peroxidase (HRP) conjugated anti-rab-
bit IgG (1:10 000, Sigma Chemical Co., St Louis, MO, USA). The
blots were immersed in chemiluminescence luminol reagent (Pierce
Chemical Co., Rockford, IL, USA) and exposed to X-ray lm (East-
man Kodak, Rochester, NY, USA).
Histological assessment of colitis
Colons of wild type and Rig-I
-/-
mice at 8 weeks of age were
collected and xed with 10% formalin for sectioning, followed by
hematoxylin-and-eosin staining. Histological assessment was per-
formed in a double-blind fashion. In brief, scores were determined
as follows [26-28]: 0, normal morphology; 1, focal inammatory
cell inltrate around the crypt base; 2, diffuse inltration of inam-
matory cells around the crypts or erosion/destruction of the lower
one-third of the glands; 3, erosion/destruction of the lower two-thirds
of the glands or loss of all the glands but with the surface epithelium
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Colitis and downregulation of Gαi2 in Rig-I
-/-
mice
860
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remaining; and 4, loss of all the glands and epithelium. Each eld
was assigned a grade of 0 to 4.
Induction and assessment of DSS-induced colitis
Wild type and Rig-I
-/-
mice at 8 weeks of age were divided into
two groups, receiving either water alone (control) or 3% (w/v) DSS
(40 000–50 000 MW; Sigma Chemical Co., St Louis, MO, USA) in
water for 5 days. The mice were checked each day for the develop-
ment of colitis by monitoring their body weight and diarrhea. The
degree of diarrhea was scored as follows: 0, normal; 2, loose stool;
and 4, watery diarrhea [29, 30]. All mice were sacriced after the
experiment and the colons were taken for histological analysis.
The methods and histological scores used were same as described
above.
Analysis of apoptosis
Peyer’s patches were collected from wild type and Rig-I
-/-
mice and
xed in 10% neutral buffered formalin. Parafn-embedded Peyers
patches were sectioned at 5 µm and apoptosis was investigated us-
ing the in situ TUNEL assay kit (Promega Co., Madison, WI, USA)
according to the manufacturers instructions. Apoptosis was also
analyzed by ow cytometry. Peyers patches were washed three
times in cold PBS, and cell suspensions were obtained by passing
tissue through a 200-µm nylon mesh. The Peyers patch lymphocytes
(1×10
6
) were stained with Annexin-V-FITC (Becton Dickinson,
San Jose, CA, USA), PI and B220-APC (Becton Dickinson Im-
munocytometry, San Jose, CA, USA) antibodies for 30 min at 4°C.
The analysis was performed on a FACSCalibur machine (Becton
Dickinson, Franklin Lakes, NJ, USA) and the data obtained were
processed with CellQuest software (Becton Dickinson, San Jose,
CA, USA).
Analysis of T-cell subsets
Splenic lymphocytes from wild type and Rig-I
-/-
mice were ob-
tained by passing tissue through a 200-µm nylon mesh. Erythrocytes
were lysed using a lysis buffer. Cells were counted and stained with
uorochrome-conjugated antibodies specic for CD4, CD8, CD44
and CD62L (eBioscience, San Diego, CA, USA) for 30 min. Flow
cytometry analysis was performed as described above.
Semi-quantitative RT-PCR and real-time PCR
Total RNA was extracted with the Trizol reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturers protocol. For
semi-quantitative RT-PCR and real-time PCR, RNA was treated with
RNase-free DNase, and then 1 mg total RNA was reverse-transcribed
according to the standard protocol (TaKaRa Shuzo, Ltd, Kyoto). The
primers used were as follows: 5′-TTGGCCGCTCACGAGAATA-3′
and 5′-GCTGACCACCCACATCAAACA-3′ for Gαi2; 5′-TCTTT-
GCTGACCTGCTGGATT-3′ and 5′-TTCGAGAGGTCCTTTTCAC-
CA-3′ for HPRT; and 5′-AACGAGCGGTTCCGATGCCCTGAG-3′
and 5′-TGTCGCCTTCACCGTTCCAGTT-3 for β-actin. Semi-
quantitative RT-PCR consists of 25-30 cycles at 94 °C for 30 s, 58 °C
for 30 s, 72 °C for 30 s and a nal extension at 72 °C for 10 min
after an initial denaturation at 94°C for 5 min. Real-time PCR was
performed using an ABI9700 PCR machine (Applied Biosystems,
Foster City, CA, USA) for 40 cycles at 94 °C for 15 s, 58 °C for
15 s, 72 °C for 30 s and a nal extension at 72 °C for 10 min after
an initial denaturation at 94 °C for 5 min.
Purication of T cells and B cells from the spleen
Splenocytes of wild type and Rig-I
-/-
mice were collected as de-
scribed above. Splenic T cells labeled with CD3-PE were sorted out
with a MoFlo high-speed cell sorter (DakoCytomation, Denmark).
Splenic B cells were isolated using a Dynabeads
®
Mouse pan B
(B220) (Dynal Biotech, Lake Success, NY, USA).
NB4 cell culture and treatment with ATRA
Retinoid acid-sensitive NB4 cells were cultured in RPMI 1640
medium (GIBCO, Grand Island, NY, USA), containing 10% FCS,
100 µg/ml streptomycin and 100 U/ml penicillin in an incubator at
37 °C. The cells were treated with 1 µM ATRA for 0, 24, 48 and 72
h, then the cells were lysed and total RNA was extracted for semi-
quantitative RT-PCR.
Construction of pGL3-Gαi2 and pcDNA3.1-Rig-I
A 1.2-kb fragment of mouse Gαi2 promoter was isolated from
mouse genomic DNA by PCR using 5′-AGCCATCCCTCCC-
GCCCCCCATTT-3′ as the forward primer and 5′-TCTCAGGTCC-
GCAGTTCCGAGCGA-3′ as the reverse primer. The PCR product
obtained was cloned into pGL3-Basic (Promega Co., Madison,
WI, USA), using the XhoI and SacI sites. Full-length Rig-I cDNA
was also amplied by PCR using the forward primer 5′-ACTGCG-
GCCGCCCCACTTCGTTCATCTCTG-3′ and the reverse primer
5′-ACTGGGTACCACGGACATTTCTGCAGGATC-3′. The PCR
product was sequenced and cloned to pcDNA3.1 at the NotI and
KpnI sites. PCR amplication conditions consisted of 30 cycles at
94 °C for 30 s, 60 °C for 90 s, 68 °C for 180 s and a nal extension
at 72 °C for 10 min after an initial denaturation at 94 °C for 5 min.
The construction was sequenced to verify that the proper sequence
was amplied.
Luciferase assay
NIH3T3 cells were seeded at a concentration of 8 ×10
4
/ml in 12-
well plates. The cells were co-transfected with the Gαi2 luciferase
reporter construct (300 ng), Renilla luciferase and pcDNA3.1-Rig-I
with increasing amounts (0, 300, 500 and 700 ng), or pcDNA3.1
(to keep the total amount of plasmids constant) using the SuperFect
Transfection Reagent (Qiagen GmbH, Germany). In a standard
assay, each transfection was tested in duplicate according to the
protocol of the dual-luciferase reporter assay system kit (Promega
Co., Madison, WI, USA).
Statistical analysis
In vivo experiments were performed using 5-10 mice per group.
Each in vitro experiment was performed in triplicate and repeated
three times. All values were expressed as the mean ± SD. Student’s
two-tailed t-test was used to analyze the signicance among different
groups. A p-value of less than 0.05 was considered to be signicant
and is shown by an asterisk.
Results
Targeted disruption of Rig-I
Intracellular viral infection is detected by the cytoplas-
mic RNA helicase RIG-I, which highlights a novel role for
CARD-containing proteins in coordinating immune and
apoptotic responses [31-34]. To further investigate the func-
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Yi Wang et al.
861
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tion of RIG-I, we disrupted 5 (4 to 8 exons) out of 18 exons
of the Rig-I gene. In the mutant allele, exons 4 to 8 were
replaced by the neo cassette (Figure 1A). G418 and GANC
double resistant ES clones were examined for recombina-
tion by PCR. The correctly recombined ES clones were
further conrmed by Southern blotting (Figure 1B). Two
targeted ES clones were identied. Mice with wild type
and the targeted allele were identied using PCR, which
exhibited different bands of 1.58 kb (wild-type allele) and
2.84 kb (knockout allele) (Figure 1C). The disruption of
Rig-I was conrmed by northern blotting with a Rig-I probe
(Figure 1D) and western blotting using a polyclonal anti-
body to Rig-I (Figure 1E), respectively. It was found that
Rig-I can be induced by IFNβ and LPS in various tissues
and cell types [15,35], as shown in wild-type MEFs treated
with IFNβ and splenocytes treated with LPS (Figure 1D
and 1E). By contrast, no signals were detected by northern
and western blotting in Rig-I
-/-
MEFs and splenocytes even
Figure 1 Targeted disruption of Rig-I. (A) Rig-I targeting strategy. Mouse genomic Rig-I contains 18 exons. The start codon and
stop codon are indicated as and , respectively. The targeting vector was designed to delete a 6.4-kb fragment containing exons
4 to 8, which encode part of CARD domain 2, and containing the A and B motifs of the RNA helicase domain. The 3′ probe used
for southern blotting (—) and two primer pairs (arrows) for PCR genotyping are indicated. The respective sizes of the wild type and
targeted bands hybridized with the 3′ probe in southern blotting upon BamHI (shown as B) digestion, and of the PCR fragments
amplied from wild type and mutant alleles are indicated. (B) Two recombined ES cell clones show the expected bands as detected
by Southern blot analysis. (C) PCR using mouse tail DNA as a template and two primer pairs in one reaction shows three differ-
ent genotypes. (D) Northern blot analysis of Rig-I in MEFs. Total RNA from wild type, Rig-I
+/-
and Rig-I
-/-
MEFs treated with or
without 1000 U/ml IFN-β for 6 h was extracted and subjected to northern blot analysis using an HindIII fragment of Rig-I cDNA
(477 bp, composed of exons 11 to 14). The same membrane was re-hybridized with an 18S probe as a control. Note that no signal
was detected in Rig-I
-/-
MEFs with or without treatment with IFN-β. (E) Western blot analysis of Rig-I expression in wild type and
Rig-I
-/-
splenocytes with or without treatment with LPS (20 µg/ml) was performed using the polyclonal antibody raised in mice by
immunizing mice with a glutathione S-transferase (GST)-RIG-I fusion protein encompassing the full-length human RIG-I. The same
membrane was blotted again with antibody to β-actin. As reported by others, Rig-I can be induced by LPS in various tissues as well
as splenocytes, while no bands were visualized in Rig-I
-/-
splenocytes even after treatment with LPS.
22.9 kb
1 2
B
3
4 5
6 7 8
9
10
Xh
Xb
11
12
B
13
14 15
16
17
18
1.58 Kb
E
B
Xh
N
9
10
3
2.2 Kb
2.5 Kb
9.9 Kb
Xh
Xb B
B
B
1
2
3
9
10
11
12
13
14
15
1617
18
2.84 Kb
3′-probe
2.84 Kb
1.58 Kb
22.9 Kb
9.9 Kb
51 65 95 120
wt +/- -/-
- + - + - +
IFNβ
Rig-I
18 S
wt -/-
- + - +
LPS
Rig-I
β-actin
+/- +/+ -/- +/- +/- +/+ +/+ +/+ +/- +/+ -/- -/-
Wild type
allele
Targeting
vector
Targeting
allele
A
B
D
C
E
hsv-TK PGK
Neo PGK
Neo PGK
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Colitis and downregulation of Gαi2 in Rig-I
-/-
mice
862
npg
after IFNβ and LPS treatment, respectively. The mutant
mice develop normally and they are fertile.
Rig-I
-/-
mice develop colitis with increased susceptibility to
DSS-induced colitis
It was observed that the body weight of Rig-I
-/-
mice
progressively decreased from 3 months of age on compared
with that of the wild type (data not shown). Rig-I
-/-
mice at
8 weeks of age displayed a colitis-like phenotype (Figure
2A); the incidence was around 70% (data not shown). After
treatment with DSS, Rig-I
-/-
mice exhibited more severe
damage and inammatory inltration in the mucosa of the
colon than was observed in wild-type mice (Figure 2A).
The histological scores were signicantly increased in
Rig-I
-/-
mice (Figure 2B). More body weight loss and higher
faecal scores were also observed in Rig-I
-/-
mice after DSS
treatment (Figure 2C and 2D). These ndings indicate that
Rig-I
-/-
mice are much more susceptible to DSS-induced
colitis than wild-type mice.
Decrease in number and size of Peyer’s patches in Rig-I
-/-
mice
Since the disruption of Rig-I in mice leads to the devel-
opment of a colitis-like phenotype and increased suscepti-
bility to DSS-induced colitis, and the development of colitis
in Gαi2
-/-
mice is accompanied by fewer Peyers patches,
we also checked the number and size of Peyers patches
in Rig-I
-/-
mice. It was found that the number and size of
Peyers patches were signicantly reduced in Rig-I
-/-
mice
compared with wild-type mice (Figure 3A and 3B). The
Figure 2 Rig-I
-/-
mice exhibit colitis and are susceptible to DSS-induced colitis. (A) Histological analysis of colons from wild type
and Rig-I
-/-
mice with or without treatment with DSS (200×). Wild type and Rig-I
-/-
mice of 8 weeks of age were administered with
3% DSS in drinking water. The mice were sacriced on day 5 and the colons were analyzed. More severe damage and inammatory
inltration can be observed in the colon mucosa of Rig-I
-/-
mice compared with wild-type mice. (B) Histological score of colitis in
wild type and Rig-I
-/-
mice. (C) The body weights of the wild type and Rig-I
-/-
mice were monitored everyday. The values for body
weight are expressed as a percentage of body weight on day 0. Asterisks indicate signicant differences between groups (p < 0.05).
(D) Diarrhea in wild type and Rig-I
-/-
mice upon treatment with DSS was monitored everyday. Asterisks indicate signicant differ-
ences between groups (p < 0.05). Five mice were used for each group.
DSS (-) DSS (+)
0 1 2 3 4 5
0 1 2 3 4 5
Days post-challenge
Days post-challenge
*
*
*
Wt
-/-
wt
-/-
*
*
wt
-/-
wt
-/-
p=0.0041*
p=0.0052*
DSS (-) DSS (+)
0
-1
-2
-3
-4
-5
-6
Loss of body weight (%)
6
5
4
3
2
1
0
Faecal scores
5
4
3
2
1
0
Pathological scores
A B
C D
*
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Yi Wang et al.
863
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reduced size and number of Peyer’s patches raise the pos-
sibility of increased apoptosis in Rig-I
-/-
Peyers patches.
As expected, a signicant increase in apoptotic cells was
detected by in situ TUNNEL (Figure 3C), and was further
conrmed by Annexin-V ow cytometry in B220
+
cells in
the Peyers patches decient for Rig-I
(Figure 3D
and 3E).
Abnormal splenic T-cell subsets in Rig-I
-/-
mice
It is known that the development of colitis is associ-
ated with abnormal T-cell activation [36,37]. For this
reason, we compared the proportion and total number
of peripheral T-cell subsets in adult (6–8 weeks of age)
Rig-I
-/-
mice with wild-type mice. The total numbers of
splenic CD4
+
and CD8
+
T cells were found to be similar
between wild type and Rig-I
-/-
mice (data not shown).
However, the naïve T cells dened as CD44
low
CD62L
high
were markedly decreased (p = 0.001), whereas the per-
centages of CD44
high
CD62L
low
effector T cells (p = 0.008)
and CD44
high
CD62L
high
memory T cells (p = 0.019) were
signicantly increased in the CD4
+
splenic compartment of
Figure 3 Regression of Peyers patches in Rig-I
-/-
mice. (A) The number of Peyers patches decreased sharply in Rig-I
-/-
mice com-
pared with wild-type mice (10 mice per each group). (B) The size of Peyers patches (as indicated by circles) in the intestines of
Rig-I
-/-
mice decreased signicantly (6×, n = 10). (C) In situ TUNNEL analysis shows increased apoptotic cells in Peyers patches
of Rig-I
-/-
mice (400×). Circles indicate the site of Peyers patches in the transverse section of intestines. (D) Apoptotic cells were
analyzed by ow cytometry after annexin V staining. As shown in (D) and (E), a dramatic increase in apoptotic cells among the
B220
+
population derived from Rig-I
-/-
Peyers patches can be observed (n = 5).
wt
p=0.04*
wt -/-
wt wt -/- -/-
wt -/-
15
10
5
0
peyer’s patches/mouse
13.3%
33.9%
Annexin V
B220
B220
B220
PI
-/-
Annexin V
p=0.00097*
35
30
25
20
15
10
5
0
Apoptotic cells (%)
wt -/-
D
E
A
B
C
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Colitis and downregulation of Gαi2 in Rig-I
-/-
mice
864
npg
Rig-I
-/-
mice
(Figure 4A and 4B). For CD8
+
splenic compart-
ments, the percentage of CD44
low
CD62L
high
naive T cells
was decreased (p = 0.006), accompanied by an increased
percentage of CD44
high
CD62L
low
effector T cells (p =
0.009), while CD44
high
CD62L
high
memory T cells remained
unchanged (Figure 4C and 4D). These ndings suggest that
the deletion of Rig-I in mice leads to the abnormal activa-
tion of peripheral T cells.
Reduced expression of Gαi2 in Rig-I
-/-
mice
Previous data have shown that the induction of colitis
in Gαi2
-/-
mice is associated with the regression of Peyers
patches and disorder of T-cell subsets [16]. The phenotype
observed in Rig-I
-/-
mice is to some degree similar to that
observed in Gαi2
-/-
mice, suggesting that Gαi2 and Rig-I
may function in the same signaling pathway in the de-
velopment of colitis. To address this hypothesis, we rst
compared Gαi2 expression levels between wild type and
Rig-I
-/-
mice. Interestingly, we found that the expression of
Gαi2 was signicantly reduced in the various tissues tested
by real-time PCR (Figure 5A) and western blotting (Figure
5B) in Rig-I
-/-
mice. We then isolated T and B cells from
the spleen and checked the difference of Gαi2 expression
between wild type and Rig-I
-/-
cells. At the transcriptional
level, it was found that Gαi2 expression was repressed in
both B and T lymphocytes in the absence of Rig-I (Figure
5C and 5D). These data suggest that Rig-I may play a part
in the regulation of Gαi2 expression, and that the develop-
Figure 4 Hyperactivation of peripheral T cells in Rig-I
-/-
mice. Splenocytes from wild type and Rig-I
-/-
mice were stained with CD4,
CD8, CD62L and CD44 antibodies conjugated with uorescence, and the results were analyzed by ow cytometry. To identify naïve
T cells (dened as CD44
low
CD62L
high
), memory T cells (dened as CD44
high
CD62L
high
) and effector T cells (CD44
high
CD62L
low
),
three-color ow cytometry was performed. (A, B) In CD4
+
cells, the percentages of memory T cells and effector T cells signicantly
increased in the absence of Rig-I, while naïve T cells decreased by more than 50% compared with the wild type. The p-values that
are labeled indicate there are signicant differences between groups (n = 5). (C, D) In CD8
+
cells, and in the CD4
+
cohort, a marked
decrease in naïve T cells and an increase in effector T cells were found. However, memory T cells in CD8
+
cells remained unchanged.
The data shown are representative of three independent experiments.
p=0.001*
p=0.019*
p=0.008*
wt
-/-
wt
-/-
p=0.009*
p=0.006*
Naïve T Memory T Effector T
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
Percentage (%)
Percentage (%)
Naïve T Memory T Effector T
wt
-/-
wt
-/-
24.7%
52.6%
7.2%
35.6%
24.1%
20.6%
26.6%
9.87%
38.26%
25.4%
2.13%
46.99%
CD44
CD44
CD44
CD44
CD62L
CD62L
CD62LCD62L
CD62L
CD8
CD4
CD62L
A B
C D
Page 7
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Yi Wang et al.
865
npg
ment of colitis and regression of Peyers patches in Rig-I
-/-
mice may be caused by the downregulation of Gαi2.
Rig-I regulates the transcriptional activity of the Gαi2
promoter
It is well known that Rig-I is induced in NB4 cells by
treatment with ATRA [1]. But can the upregulation of
endogenous Rig-I by ATRA increase Gαi2 expression?
To this end, we tested the Gαi2 expression in NB4 cells
treated with ATRA. As expected, the induction of Rig-I
was paralleled with increased expression of Gαi2 (Figure
6A), further demonstrating an important role for Rig-I in
the regulation of Gαi2 transcription. To further test the
possibility of whether Rig-I regulates Gαi2 promoter ac-
tivity, we constructed a Gαi2 promoter/luciferase reporter
construct and co-transfected NIH3T3 cells with increasing
doses of the Rig-I expression vector. As shown in Figure
6B, Rig-I indeed activates Gαi2 promoter activity in a dose-
dependent manner. Thus, we may conclude that Rig-I has
a crucial role in the normal transcription of Gαi2.
Discussion
DExD/H box proteins are putative RNA helicases that
are characterized by their ability to unwind dsRNA with
intrinsic ATPase activity [38]. They have been implicated
in a number of cellular processes that involve the altera-
tion of secondary RNA structures such as translation ini-
tiation, nuclear and mitochondrial splicing, and ribosome
and spliceosome assembly [39]. RIG-I encodes a member
of the DExD/H box family proteins. Human RIG-I is
located on chromosome 9p12 and encodes a 925-amino-
acid protein that contains the RNA helicase-DEAD box
motif. It is highly conserved from Caenorhabditis elegans
to mammals and is expressed ubiquitously in the human
and mouse. Originally, RIG-I was found to be induced in
the acute promyelocytic leukemia cell line NB4 during
ATRA-induced cell differentiation, suggesting that RIG-I
might be an important mediator in the ATRA-signaling
pathway [1]. Most importantly, RIG-I was recently iden-
tied as an essential regulator for virus-induced antiviral
Figure 5 Expression of Gαi2 is reduced in Rig-I
-/-
tissues or cells. (A) Total RNA from the tissues of wild type and Rig-I
-/-
mice
was extracted and reversely transcribed to cDNA. The expression of Gαi2 was analyzed by real-time PCR. The data from real-time
PCR were normalized to an internal control and plotted relative to the level of Gαi2 in the tissues of wild-type mice. Among the
tissues tested, Gαi2 expression was reduced by 30 to 50% in the spleen, lung and kidney, while a mild reduction in the other tis-
sues is shown. (B) Western blot analysis shows that Gαi2 is highly expressed in wild-type intestines and colons. However, Gαi2 is
dramatically reduced in Rig-I decient intestines and colons.
α-Tubulin was blotted as a loading control. (C, D) The expression of
Gαi2 in sorted T and B lymphocytes was analyzed at the transcriptional level. Splenic T cells were enriched by sorting CD3-PE
staining cells. B cells were puried by Dynabeads
®
Mouse pan B (B220) (Dynal Biotech, Lake Success, NY, USA). Both real-time
PCR (C) and semi-quantitative RT-PCR (D) show decreased expression of Gαi2 in splenic B and T lymphocytes. β-actin in (D)
serves as an internal control.
Brain Heart Lung Stomach Spleen Liver Kidney
intestine colon
wt -/- wt -/-
1.2
1.0
0.8
0.6
0.4
0.2
0
Ratio (ko/wt)
Gαi2
α-tubulin
Gαi2
β-actin
Gαi2
β-actin
wt -/-
B cells T cells
B cell
T cell
0.6
0.5
0.4
0.3
0.2
0.1
0
Ratio (ko/wt)
A C
B
D
Page 8
Cell Research | www.cell-research.com
Colitis and downregulation of Gαi2 in Rig-I
-/-
mice
866
npg
immunity, capable of sensing intracellular viral dsRNA,
which transduces signals through an adaptor protein
(MAVS/IPS-1/VISA/Cardif), leading to the activation of
IRF-3 and NF-κB, and augmenting interferon production
in response to viral infection [4-11]. The in vivo importance
of Rig-I in antiviral defense was further demonstrated in
Rig-I-decient mice, in which exons 8 to 10 of Rig-I were
deleted, by showing the cell-type-specic requirements
for Rig-I in the antiviral response [13]. Unfortunately,
Rig-I knockout mice generated by Akira’s group mostly
died during embryogenesis, and a few newborn mice died
within 3 weeks after birth owing to extensive apoptosis
in the fetal liver. In this study, we also generated Rig-I
knockout mice, in which 5 exons (exons 4 to 8) encoding
part of CARD domain 2, and the A and B motifs of the
RNA helicase domain (aa141-405) were replaced with a
Neo cassette by homologous recombination. Disruption
of Rig-I was demonstrated by northern blot analysis using
the 3′-end fragment of Rig-I cDNA (477 bp, composed of
exons 11 to 14) as a probe, and by western blot analysis
using the polyclonal antibody specic to RIG-I. Unlike the
previous study [13], our homozygous Rig-I knockout mice
were viable and fertile. The genotype distribution in the lit-
termates obtained from crossing heterozygote mice follows
a Mendelian pattern of inheritance. MEFs derived from our
Rig-I
-/-
mice also showed a compromised antiviral response,
similar to the previous report (unpublished data). However,
the Rig-I
-/-
mice displayed signicant age-dependent loss
of body weight. Extensive pathological analysis showed
that 70% of adult Rig-I
-/-
mice spontaneously developed
a colitis-like phenotype with increased susceptibility to
DSS-induced colitis. The different outcomes between our
mutant mice and that reported by Kato et al. [13] likely
result from the disruption of different regions of the Rig-I
gene. In fact, this kind of phenomenon is not uncommon
in mouse mutagenesis studies. A typical example emerged
from the comparison of the phenotypes represented by three
different Prnp
-/-
mice [40]. This could be explained by the
different truncated proteins that are expressed after genomic
modication, although they may be expressed at very low
levels. In our case, this possibility could not be excluded,
but northern or western blotting detected no signals that
correspond to truncated Rig-I messages or proteins. The
Rig-I
-/-
mice we generated survived and displayed a coli-
tis-like phenotype, providing us an alternative model for
studying the mechanisms that underlie inammatory bowel
disease, including colitis.
Inammatory bowel disease is considered to be associ-
ated with a breakdown of tolerance to the resident intestinal
ora [41, 42] and immune activation in the gut-associated
lymphatic tissue (GALT). The GALT consists of Peyers
patches and mesenteric lymph nodes as organized intestinal
lymphoid follicles. Previous studies have shown that a de-
ciency of Peyers patches and mesenteric lymph nodes may
be in part responsible for the development of colitis in mice.
It is known that intraluminal and intestinal wall antigens
have the capacity to induce tolerance toward inammatory
intestinal immune responses. A reduction in the number of
Peyers patches and mesenteric lymph nodes, especially the
loss of normally present regulatory cells (such as dendritic
cells) in these organs, may result in the failure of tolerance
induction in the gut [43]. Therefore, the decrease in Peyers
patches that is due to increased apoptosis is, to some degree,
related to the induction of colitis in Rig-I
-/-
mice.
It has been shown that the disruption of several genes
in mice leads to chronic inammation of the bowel [17,
44-46]. Among them, Gai2-decienct mice display growth
retardation and develop lethal diffuse colitis with clinical
and histopathological features that closely resemble ulcer-
ative colitis in humans [17]. It has also been shown that
Figure 6 Induction of Gαi2 promoter activity by Rig-I. (A) NB4
cells were treated with 1 mm ATRA for the time indicated, and total
RNA was extracted. Induction of RIG-I and Gαi2 expression was
analyzed by semi-quantitative RT-PCR. HPRT served as a control.
The data shown represent one of the three independent experiments.
(B) NIH3T3 cells were co-transfected with the Gαi2 promoter lu-
ciferase reporter construct, Renilla luciferase and pcDNA3.1-Rig-I
with increasing amounts (0, 300, 500 and 700 ng), or pcDNA3.1.
Forty-eight hours after transfection, Gαi2 promoter-driven luciferase
activity was highly activated with increasing amounts of Rig-I-ex-
pressing vector present. The results shown represent one of three
independent experiments.
0 h 24 h 48 h 72 h
Gαi2 promoter activity
Gαi2
RIG-I
HPRT
Basic 0 300 500 700
pcDNA 3.1-Rig-I construct (ng)
16
14
12
10
8
6
4
2
0
Relative Luc activity (
×
10
4
)
A
B
Page 9
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Yi Wang et al.
867
npg
Gai2
-/-
mice exhibit a local increase in memory CD4
+
and
CD8
+
cells that are characterized by increased levels of
CD44 and decreased levels of CD45RB and CD62L, an
increase in pro-inammatory Th1-type cytokines and an
increase in the inltration of activated CD4
+
T cells in the
intestinal mucosa [47, 48]. All of these ndings strongly
suggest that Gαi2-deciency leads to a hyperimmune re-
sponse and that the Gαi2 protein may negatively regulate
T-cell immunity [17, 24, 47].
The regression of Peyers patches and development of
colitis in Rig-I
-/-
mice raise the possibility that Rig-I may
play a part in the regulation of T-cell homeostasis. As ex-
pected, we found that Rig-I deciency leads to an increase
in splenic CD4
+
and CD8
+
effector T cells with a decrease
of naïve T cells, indicating the hyper-activation of effector
T cells in Rig-I
-/-
mice. These data also suggest an important
role for Rig-I in the regulation of T-cell activation.
Gαi2
-/-
mice display colitis with 100% penetrance with
smaller Peyer’s patches and Gαi2
-/-
mice also exhibit
disorders of the T-cell subsets [16]. These observations
suggest that there may be a link between Rig-I and Gαi2.
Therefore, we examined Gαi2 expression in various tissues
of wild type and Rig-I
-/-
mice. As expected, Gαi2 expres-
sion decreased distinctly in many tissues of Rig-I
-/-
mice,
especially in the colons and intestines. On the contrary, up-
regulation of Rig-I in NB4 cells upon treatment with ATRA
is accompanied by elevated Gαi2 expression. Luciferase
assay further demonstrated that Rig-I can markedly activate
Gαi2 promoter activity in a dose-dependent manner. Based
on these ndings, we propose that Rig-I may function as
a positive regulator for Gαi2 transcription.
In this report, we identied a novel role of Rig-I in T-
cell activation and Gαi2 expression by showing the distinct
phenotypes of Rig-I
-/-
mice. The development of colitis in
Rig-I
-/-
mice might be in part associated with the downregu-
lation of Gαi2 and disturbed T-cell homeostasis.
Acknowledgments
This work was partially supported by grants from the
National Natural Science Foundation of China (39925023),
Ministry of Science and Technology (2001CB509901,
2001AA216081), Ministry of Education (00TPJS111) of
China, Science and Technology Commission of Shanghai
Municipality (03DZ14088, 06DZ05907), E-Institutes of
Shanghai Municipal Education Commission (E03003), and
Foundation of Shanghai Jiao Tong University and School
of Medicine (BXJ0