nature immunology VOLUME 13 NUMBER 4 APRIL 2012
The initiation of innate immune responses depends on the detec-
tion of pathogen-associated molecular patterns by several classes of
germline-encoded pattern-recognition receptors, including Toll-like
receptors (TLRs), RIG-I-like receptors, Nod-like receptors (NLRs)
and sensors of DNA1,2. After stimulation with a pathogen-associated
molecular pattern, these pattern-recognition receptors trigger
activation of the transcription factor NF-κB, type I interferons and
inflammasome signaling pathways, which leads to the production
of proinflammatory cytokines and induction of subsequent adap-
tive immune responses. Whereas TLR3 recognizes viral double-
stranded RNA in endosomes and triggers a signaling pathway
mediated by the adaptor TRIF, the RNA helicases RIG-I and Mda5
function as cytoplasmic sensors of RNA and activate the mito-
chondrial signaling adaptor MAVS (VISA, IPS-1 or Cardif) after
ligand recognition1,2. Studies have shown that RNA polymerase III
can serve as an intracellular sensor of viral DNA by transcribing
viral AT-rich double-stranded DNA into double-stranded RNA,
which in turn stimulates RIG-I and initiates the MAVS-dependent
signaling cascade3,4. Furthermore, IFI16 and DDX41 function
as cytosolic sensors of DNA and interact with the membrane-
associated adaptor STING to activate the type I interferon signal-
ing pathway5,6. The key adaptors TRIF, MAVS and STING of both
RNA and DNA sensors need the kinase TBK1 to activate the tran-
scription factor IRF3, which leads to the induction of type I inter-
Although type I interferon is required for viral clearance, aberrant
production of type I interferon (including IFN-α and IFN-β) can have
a pathological role in autoimmune disorders. Thus, tight regulation
of type I interferon signaling is critical for maintaining the homeo-
stasis of both innate and adaptive immunity. NLRs represent a large
family of cytosolic pattern-recognition receptors that share a typical
nucleotide-binding-and-oligomerization domain (Nod), a leucine-rich
repeat (LRR) region and a variable amino-terminal effector domain.
Many NLRs have been studied extensively as pattern-recognition recep-
tors that trigger relevant signaling pathways after encountering their
pathogen-associated molecular pattern or sensing a danger signal1,2. In
addition, NLRs can function as negative regulators. NLRX1 has been
found to inhibit the type I interferon signaling pathway by binding to
MAVS7,8, whereas NLRC5 has a critical role in the negative regulation
of intracellular antiviral responses via interaction with RIG-I and Mda5
(refs. 9,10). Although NLRP4 has been reported to negatively regulate
NF-κB signaling and autophagic processes through interactions with
the kinase IKK and beclin-1, respectively11,12, its role in the regulation
of type I interferon signaling and antiviral immunity remains unknown.
In this study we report that NLRP4 served as a negative regulator of the
type I interferon signaling pathway by targeting TBK1. NLRP4 recruited
the E3 ubiquitin ligase DTX4 for Lys48 (K48)-linked polyubiquitination
and degradation of TBK1. Our findings identify the NLRP4-DTX4 axis
as an additional signaling cascade for TBK1 degradation to maintain
immune homeostasis during antiviral innate immunity.
1The Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA. 2State Key Laboratory of Pharmaceutical Biotechnology, Department of
Biochemistry, Nanjing University, Nanjing, China. 3Center for Inflammation and Epigenetics, The Methodist Hospital Research Institute, Houston, Texas, USA.
4Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA. 5Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, Texas, USA. 6Department of Pathology and Department of Immunology, Baylor College of Medicine, Houston, Texas, USA.
7These authors contributed equally to this work. Correspondence should be addressed to R.-F.W. (firstname.lastname@example.org).
Received 24 October 2011; accepted 13 January 2012; published online 4 March 2012; doi:10.1038/ni.2239
NLRP4 negatively regulates type I interferon
signaling by targeting the kinase TBK1 for
degradation via the ubiquitin ligase DTX4
Jun Cui1–3,7, Yinyin Li1,3,4,7, Liang Zhu1, Dan Liu5, Zhou Songyang5, Helen Y Wang1,3 & Rong-Fu Wang1,3,6
Stringent control of the type I interferon signaling pathway is important for maintaining host immune responses and homeostasis,
yet the molecular mechanisms responsible for its tight regulation are still poorly understood. Here we report that the pattern-
recognition receptor NLRP4 regulated the activation of type I interferon mediated by double-stranded RNA or DNA by
targeting the kinase TBK1 for degradation. NLRP4 recruited the E3 ubiquitin ligase DTX4 to TBK1 for Lys48 (K48)-linked
polyubiquitination at Lys670, which led to degradation of TBK1. Knockdown of either DTX4 or NLRP4 abrogated K48-linked
ubiquitination and degradation of TBK1 and enhanced the phosphorylation of TBK1 and the transcription factor IRF3. Our results
identify a previously unrecognized role for NLRP4 in the regulation of type I interferon signaling and provide molecular insight
into the mechanisms by which NLRP4-DTX4 targets TBK1 for degradation.
© 2012 Nature America, Inc. All rights reserved.
VOLUME 13 NUMBER 4 APRIL 2012 nature immunology
NLRP4 negatively regulates type I interferon signaling
To identify possible roles for members of the NLR family in antiviral
immunity, we transfected HEK293T human embryonic kidney cells
(293T cells) with an IFN-β luciferase reporter and the internal control
renilla luciferase, as well as expression vectors containing candidate
genes encoding NLRs, then treated the cells intracellularly for 24 h with
the synthetic RNA duplex poly(I:C) to trigger type I interferon signaling;
this identified NLRP4 as an inhibitor of activation of the IFN-β
luciferase reporter (Fig. 1a). Pancreas, testis, placenta and spleen had
high expression of human NLRP4 mRNA (Supplementary Fig. 1a). We
readily detected NLRP4 protein in 293T cells, THP-1 human monocytes
and BxPC-3 human pancreatic cells (Supplementary Fig. 1b). As IFN-β
activation requires coordination between the activation of NF-κB and
that of IRF3, we used an interferon-stimulated response element (ISRE)
luciferase reporter (which requires activation by IRF3 only) to evaluate
whether the inhibition of type I interferon by NLRP4 was dependent on
its inhibitory effect on NF-κB signaling. Intracellular poly(I:C)-induced
activity of the ISRE luciferase reporter was potently inhibited by NLRP4
(Fig. 1a), which suggested that NLRP4 directly inhibits IFN-β activation
by blocking IRF3 signaling. We obtained similar results with 293T-TLR3
cells (293T cells that express TLR3) treated with poly(I:C) (Fig. 1b) or
293T cells treated with poly(dA:dT) (Fig. 1c) or infected with vesicular
stomatitis virus tagged with enhanced green fluorescent protein
(VSV-eGFP; Fig. 1d), when we transfected the cells with increasing
amounts of expression vector for NLRP4. These results suggested that
NLRP4 is a negative regulator of the type I interferon signaling pathway.
To determine how NLRP4 inhibits the type I interferon signaling, we
assessed the phosphorylation of IRF3 in 293T cells expressing NLRP4
together with RIG-I, Mda5, MAVS or TRIF and found that NLRP4
potently inhibited the phosphorylation of endogenous IRF3 induced by
these innate immune receptors and adaptors (Fig. 1e). As activation of
IFN-β is also associated with the translocation of IRF3 from the cyto-
plasm into the nucleus, we examined the translocation of endogenous
IRF3 in cells with or without expression of GFP-tagged NLRP4. In
cells transfected with empty vector, IRF3 rapidly translocated from the
cytoplasm to the nucleus after intracellular treatment with poly(I:C).
In contrast, IRF3 was retained in the cytoplasm of cells expressing
GFP-tagged NLRP4 after stimulation (Fig. 1f). These results suggested
that NLRP4 inhibits the activation of type I interferon induced by
stimulation with double-stranded RNA and DNA or viral infection by
blocking the phosphorylation and translocation of IRF3.
Knockdown of NLRP4 enhances IFN-b and antiviral responses
We next determined whether specific knockdown of endogenous
NLRP4 would increase IFN-β expression under physiological condi-
tions. We used an NLRP4-specific small interfering RNA (siRNA) and
two NLRP4-specific lentivirus short hairpin RNA (shRNA) constructs
to knock down the expression of NLRP4. All three efficiently inhibited
the expression of transfected and endogenous NLRP4 in 293T cells and
THP-1 cells (Fig. 2a and Supplementary Fig. 2a–c). We next assessed
the effects of NLRP4 knockdown on the activation of type I interferon.
With the ISRE luciferase reporter assay, we found that knockdown of
NLRP4 resulted in much more activity of the ISRE luciferase reporter
triggered by poly(I:C), intracellular poly(I:C), poly(dA:dT) or VSV-eGFP
in 293T cells or 293T-TLR3 cells (Fig. 2b). To further demonstrate the
effects of NLRP4 knockdown on the expression of interferon-responsive
genes, we knocked down NLRP4 in THP-1 cells and then infected the
cells with VSV-eGFP; we found that infection with VSV-eGFP resulted in
much higher expression of IFNB mRNA and IFN-β protein in cells trans-
fected with NLRP4-specific siRNA than in those transfected with siRNA
with a scrambled sequence (Fig. 2c). Consistent with that, knockdown of
NLRP4 also resulted in higher expression of several interferon-stimulated
genes, including ISG15, IFIT2 (which encodes ISG-56), IFIT1 (which
encodes ISG-54) and CCL5, after infection with VSV-eGFP (Fig. 2d). We
obtained similar results with human peripheral blood mononuclear cells
(PBMCs) transfected with NLRP4-specific siRNA or scrambled siRNA
(Fig. 2e). These results suggested that knockdown of NLRP4 enhanced
IFN-β activation and the expression of interferon-stimulated genes.
To demonstrate a link between the enhanced type I interferon
response and antiviral immunity in cells in which NLRP4 was knocked
down, we knocked down NLRP4 expression in THP-1 cells and then
infected the cells with VSV-eGFP at a multiplicity of infection (MOI) of
1 or 10. Knockdown of NLRP4 rendered the cells resistant to viral infec-
tion and resulted in considerably fewer GFP+ (virus-infected) cells than
among cells treated with scrambled siRNA (Fig. 2f). Flow cytometry
Figure 1 NLRP4 negatively regulates the
type I interferon signaling pathway.
(a–d) Luciferase activity in 293T cells (a,c,d)
or 293T-TLR3 cells (b) transfected with plasmid
encoding a luciferase reporter for IFN-β (IFN-β–luc)
or ISRE (ISRE-luc; 100 ng each), together with
empty vector (no wedge) or an expression vector
for NLRP4 (0, 50 and 100 ng; wedge), followed
by no treatment (control (Ctrl)) or treatment with
intracellular (IC) poly(I:C) (1 µg/ml; a), poly(I:C)
(10 µg/ml; b), poly(dA:dT) (1 µg/ml; c) or VSV-eGFP
(MOI, 0.01; d); results are presented relative to renilla luciferase activity. (e) Immunoblot analysis (IB) of total and phosphorylated (p-) IRF3 in 293T
cells transfected with various combinations (above lanes) of plasmid for Flag-tagged RIG-I, Mda5, MAVS or TRIF plus vector for HA-tagged NLRP4,
probed with antibodies (α-) along left margin. (f) Fluorescence microscopy of IRF3 in 293T cells transfected with empty vector (EV) or vector for
GFP-tagged NLRP4, then left untreated (top row) or treated with intracellular poly(I:C). DAPI, DNA-intercalating dye. Original magnification, ×40.
*P < 0.05, **P < 0.01 and ***P < 0.001, versus cells with the same treatment without NLRP4 expression (two-tailed Student’s t-test). Data are
representative of three independent experiments (mean and s.d. in a–d).
© 2012 Nature America, Inc. All rights reserved.
Cell culture and reagents. HEK293T, THP-1, RAW264.7 and BxPC-3 cells
(American Type Culture Collection) were maintained in DMEM (Mediatech)
or RPMI-1640 medium (Invitrogen) containing 10% heat-inactivated FCS.
Mouse embryonic fibroblasts were prepared from embryos of C57BL/6
mice at day 15 and were cultured in DMEM supplemented with 10% FBS as
described29. Buffy coats of blood from healthy donors (from the Gulf Coast
Regional Blood Center) were used for isolation of PBMCs by density-gradient
centrifugation with Lymphoprep (Nycomed Pharm). The use of PBMCs was
in accordance with institutional guidelines and approved protocols by Baylor
College of Medicine and The Methodist Hospital Research Institute. Poly
(I:C), poly(I:C)-LyoVec and Poly(dA:dT) were from Invivogen.
Antibodies. Anti-NLRP4 (C-20; sc-50623), anti-IRF3 (sc-9082), anti-GFP (FL;
sc-8334) and anti-ubiquitin (sc-8017) were from Santa Cruz Biotechnology;
horseradish peroxidase–anti-Flag (M2) and anti-β-actin (A1978) were from
Sigma; horseradish peroxidase–anti-hemagglutinin (3F10), horseradish
peroxidase–anti-c-Myc (11814150001) and unlabeled anti-c-Myc (11667203001)
were from Roche Applied Science. Anti-IKKα (IMG-136A) was from Imgenex;
antibody to IRF3 phosphorylated at Ser396 (4947), anti-IKKi (2690) and anti-
TBK1 (3013) were from Cell Signaling Technology.
Transfection and reporter assays. HEK293T cells (2 × 105) were plated
in 24-well plates and transfected, through the use of Lipofectamine 2000
(Invitrogen), with plasmid encoding an NF-κB, IFN-β or ISRE luciferase
reporter (firefly luciferase; 100 ng) and pRL-TK (renilla luciferase plasmid;
10 ng) together with 100 ng plasmid encoding Flag-tagged RIG-I, Mda5, MAVS,
TBK1 or IKKi, and increasing concentrations (0, 100, or 200 ng) of plasmid
encoding HA-NLRP4 or 100 ng plasmid encoding NLRP4 containing the PYD,
Nod or LRR domain. Empty pcDNA3.1 vector was used to maintain equal
amounts of DNA among wells. Cells were collected at 24 h after transfection
and luciferase activity was measured with a Dual-Luciferase Assay (Promega)
with a Luminoskan Ascent luminometer (Thermo Scientific) according to the
manufacturer’s protocol. Reporter gene activity was determined by normaliza-
tion of the firefly luciferase activity to renilla luciferase activity. An Amaxa
nucleofector kit V was used according to the manufacturer’s protocols (Lonza
Amaxa) for transfection of plasmids or siRNAs into THP-1 cells.
Immunoprecipitation and immunoblot analysis. For immunoprecipita-
tion, whole-cell extracts were prepared after transfection or stimulation with
appropriate ligands, followed by incubation overnight with the appropriate
antibodies plus Protein A/G beads (Pierce). For immunoprecipitation with
anti-Flag or anti-hemagglutinin, anti-Flag or anti-hemagglutinin agarose gels
(Sigma) were used. Beads were then washed five times with low-salt lysis
buffer, and immunoprecipitates were eluted with 3x SDS Loading Buffer (Cell
Signaling Technology) and resolved by SDS-PAGE. Proteins were transferred
to nitrocellulose membranes (Bio-Rad) followed by further incubation with
the appropriate antibodies. LumiGlo Chemiluminescent Substrate System
(KPL) was used for protein detection.
Cytokine-release assay. Human IFN-β was detected with ELISA kits accord-
ing to the manufacturer’s protocols (PBL Biomedical Laboratories).
Cycloheximide-chase assay. Cells were treated for various periods of time
with cycloheximide (100 µg/ml) after virus infection, then were collected and
analyzed by immunoblot.
Immunofluorescence staining. Cells in culture plates or chamber slides were
fixed for 20 min at −20 °C with methanol and nonspecific receptors were blocked
with 10% normal goat serum. IRF3 was stained with polyclonal rabbit anti-IRF3
(sc-9082; Santa Cruz Biotechnology), followed by rabbit antibody to Texas red (A-
6399; Invitrogen). Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole;
Invitrogen). Immunofluorescence staining was visualized and cells were photo-
graphed with an Olympus 1X71S1F fluorescence microscope.
Viral infection. VSV-eGFP was provided by S. Balachandran. Cells were
infected at various multiplicities of infection as described10,29.
Statistical analysis. The significance of differences between groups was
assessed with a two-tailed Student’s t-test.
Additional methods. Information on molecular cloning of the gene encoding
full-length human NLRP4 and other related genes, real-time PCR analysis and
knockdown of NLRP4 and DTX4 by RNA-mediated interference is available
in the Supplementary Methods.
© 2012 Nature America, Inc. All rights reserved.