Articles

RNA viruses promote activation of the NLRP3

inflammasome through a RIP1-RIP3-DRP1
signaling pathway
Xiaqiong Wang1,4, Wei Jiang1,4, Yiqing Yan1, Tao Gong1, Jiahuai Han2, Zhigang Tian1,3 & Rongbin Zhou1,3

The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the
mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine
© 2014 Nature America, Inc. All rights reserved.

kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus–induced activation of the NLRP3 inflammasome. Infection with an
RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to
mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove
the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3–dependent necrosis. Together
our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus–induced activation of the NLRP3 inflammasome
and establish a direct link between inflammation and cell-death signaling pathways.

The Nod-like receptor NLRP3, together with the adaptor ASC and detects viral infection remains unclear, in contrast to the mechanisms
caspase-1, can form a protein complex called the ‘NLRP3 inflam- known for the recognition of DNA viruses by AIM2.
masome’, which is responsible for the maturation and secretion of The serine-threonine kinase RIP3 (RIPK3) is a member of the RIP
proinflammatory cytokines, such as interleukin 1β (IL-1β) and family that functions as a key participant in programmed necrosis
IL-18 (refs. 1,2). The NLRP3 inflammasome is activated by a wide by forming a necrosome protein complex with RIP1 (refs. 19–21).
range of stress signals that derive not only from pathogens but In addition to their central role in necrosis, the necrosome com-
also from environmental stress, metabolic dysregulation and tissue ponents RIP1 and RIP3 have been proposed to be involved in the
damage. So, in addition to its critical role in host defense against activation of inflammasomes22–26. When inhibitory factors of
pathogens, the NLRP3 inflammasome is involved in the progres- necrosomes, such as members of the IAP family or caspase-8, are
sion of various inflammatory human diseases, such as gout and inhibited, RIP1 and RIP3 can promote spontaneous activation of the
type 2 diabetes 1–3. Although evidence has shown that potassium inflammasome22–24. In addition, the activation of inflammasomes
npg

efflux, mitochondria dysfunction and lysosomal disruption are induced by infection with Yersinia pestis depends on RIP1 but not
involved in activation of the NLRP3 inflammasome 4–8, the pre- RIP3 (refs. 25,26), which suggests that the role of RIP1 in bacteria-
cise mechanisms and signaling pathway of this activation remain induced activation of inflammasomes is independent of necrosomes.
poorly understood. In mice with mutant tyrosine-phosphatase Ptpn6, RIP1 drives autoin-
As a crucial component of the innate immune system, the inflam- flammation by promoting IL-1α production without the involvement
masome serves an important role in host defense by recognizing of RIP3 or the inflamamsome27. Thus, although more and more evi-
viral infection and triggering responses from the innate immune dence suggests a close connection between inflammation and necro-
system9–11. Some DNA viruses, such as vaccinia virus and mouse sis, it is still unclear whether the RIP1-RIP3 complex can contribute
cytomegalovirus, can stimulate activation of the AIM2 inflamma- to the activation of inflammasomes in physiological or pathological
some via direct binding between double-stranded DNA and AIM2 conditions. In addition, although the mechanism by which the RIP1-
(ref. 12). The NLRP3 inflammasome can recognize both RNA viruses RIP3 complex induces necrosis is emerging, how this complex signals
and some DNA viruses, including adenovirus, influenza virus, Sendai activation of the inflammasome and inflammation is unclear. In this
virus and vesicular stomatitis virus (VSV)9,13–15. Although published study, we found that the RIP1-RIP3 complex was involved in RNA
reports have suggested that NLRP3 detects viral infection through ion virus–induced activation of the NLRP3 inflammasome. Infection with
flux generated by the virus-encoded M2 ion channel or through the an RNA virus initiated assembly of the RIP1-RIP3 complex, which
recognition of viral RNA10,14,16–18, the mechanism by which NLRP3 activated the GTPase DRP1 and promoted mitochondrial damage,

1Institute of Immunology and CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center, University of Science and
Technology of China, Hefei, China. 2State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Innovation Center for Cell Biology, Xiamen University,
Xiamen, Fujian, China. 3Innovation Center for Cell Biology, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China. 4These authors contributed
equally to this work. Correspondence should be addressed to R.Z. (zrb1980@ustc.edu.cn) or Z.T. (tzg@ustc.edu.cn).

Received 8 August; accepted 23 September; published online 19 October 2014; doi:10.1038/ni.3015

1126 VOLUME 15  NUMBER 12  DECEMBER 2014  nature immunology

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production of reactive oxygen species (ROS) and activation of the NLRP3-dependent manner (Fig. 1e). Similarly, IL-1β production
NLRP3 inflammasome. induced by VSV, Sendai virus or influenza virus was significantly
impaired in Rip3−/− macrophages compared with that in wild-type
RESULTS cells (Fig. 1f), which suggested a role for RIP3 in RNA virus–induced
RIP3 promotes RNA virus–induced NLRP3 activation activation of the inflammasome. IL-1β secretion induced by DNA
To assess the role of RIP3 in inflammasome activation, we treated viruses such as adenovirus or herpes simplex virus (HSV) was also
bone marrow–derived macrophages (BMDMs) from RIP3-deficient NLRP3 dependent (Fig. 1e). However, in contrast to the induction of
(Ripk3−/−; called ‘Rip3−/−’ here) mice with classic NLRP3 agonists, its production by RNA viruses, IL-1β production induced by adeno-
including ATP, monosodium urate (MSU), aluminium sulfate (alum) virus or HSV was RIP3 independent (Fig. 1f).
and nigericin. In these conditions, IL-1β secretion was similar in We next investigated the role of RIP3 in activation of the NLRP3
Rip3−/− cells and Rip3+/+ cells (Fig. 1a). Similarly, RIP3 deficiency had inflammasome during in vivo infection with RNA viruses. Infection
no effect on activation of the AIM2 inflammasome by treatment with with influenza virus, VSV or HSV resulted in a higher concentration
the synthetic double-stranded DNA poly(dA:dT) (Fig. 1a), which of IL-1β and IL-18 in the bronchoalveolar lavage fluid (BALF) of wild-
suggested that RIP3 was not involved in inflammasome activation type (Rip3+/+) mice than did mock infection (Fig. 2a,b). Infection
induced by classic agonists of NLRP3 or AIM2. When we investi- with influenza virus or VSV induced a lower concentration of
gated whether RIP3 was involved in virus-induced activation of the IL-1β and IL-18 in the BALF of Rip3−/− mice than in that of wild-type
inflammasome, we found that VSV-induced secretion of IL-1β was mice, while the concentration of IL-1β and IL-18 induced by infec-
suppressed in BMDMs from both Nlrp3−/− mice and Rip3−/− mice tion with HSV was not significantly different in Rip3−/− mice versus
compared with its secretion in wild-type cells (Fig. 1b). The secretion wild-type mice (Fig. 2a,b); this suggested that RIP3 was required
of IL-18 and cleavage of caspase-1 were also reduced in Nlrp3−/− and for RNA virus–induced activation of the NLRP3 inflammasome
Rip3−/− BMDMs compared with that in wild-type cells (Fig. 1c,d), in vivo. However, the concentration of IL-6, an inflammasome-
© 2014 Nature America, Inc. All rights reserved.

which suggested a role for RIP3 in promoting VSV-induced independent cytokine, was similar in Rip3−/− mice and wild-type mice
activation of the NLRP3 inflammasome. To exclude the possibility in all infection conditions (Fig. 2c). Together these results indicated
that the impaired IL-1β secretion in Rip3−/− cells might have been involvement of RIP3 in RNA virus–activated activation of the NLRP3
due to less cell death, we investigated whether RIP3 deficiency inflammasome.
affected cell death induced by viral infection. Infection with VSV
induced minimal death of both wild-type cells and Rip3−/− cells RNA virus–induced NLRP3 activation depends on RIP1
(Supplementary Fig. 1a). In addition, VSV-induced production of In programmed necrosis, activation of RIP3 is initiated by assembly of
interferon-β was not affected by RIP3 deficiency (Supplementary the necrosome with RIP1 (refs. 19,20). To address whether RIP1 was
Fig. 1b), which suggested a specific role for RIP3 in activation of the required for virus-induced activation of the NLRP3 inflammasome,
NLRP3 inflammasome. we knocked down expression of RIP1 mRNA in THP-1 human macro­
We further investigated whether RIP3 was involved in activation phages with short hairpin RNA (shRNA) constructs. VSV-induced
of the NLRP3 inflammasome induced by other viruses. Consistent secretion of IL-1β and cleavage of caspase-1 were reduced in THP-1
with published reports10,14,15, RNA viruses, such as Sendai virus and cells in which RIP1 was silenced compared with that in cells in which
influenza virus, stimulated IL-1β secretion in macrophages in an it was not silenced (Fig. 3a,b). Similarly, knockdown of Rip1 mRNA

a b c d WT Rip3–/– Nlrp3–/–
Rip3+/+ WT WT ** VSV
Rip3–/– Rip3–/– Rip3–/– ** 0 3 5 0 3 5 0 3 5
npg

(MOI)
4 NS 800 Nlrp3–/– ** 300 Nlrp3
–/–
NS IL-1β
3
NS
600
** **
IL-1β (ng/ml)

IL-1β (pg/ml)

IL-18 (pg/ml)

** SN
200 **
NS p20
2 400 **
NS 100 Pro-IL-1β
1 200
ND Input
0 0 0 Pro-casp1
0 3 5 0 3 5
S

SU

um

in

)
dT
U

AT

ic

VSV (MOI) VSV (MOI)

M

Al

er

A:
ig

(d
N

ly

e f
Po

Nlrp3+/+ Rip3+/+
1,000 Nlrp3
–/–
Rip3–/– NS
1,000
Figure 1  Role of RIP3 in RNA virus–induced activation of the 800
**
** 800 NS
**
IL-1β (pg/ml)

NLRP3 inflammasome. (a) Secretion of IL-1β by LPS-primed Rip3+/+

IL-1β (pg/ml)

600 ** 600
or Rip3−/− BMDMs left unstimulated (US) or stimulated for 30 min
with ATP or nigericin or for 4 h with MSU, alum or poly(dA:dT). 400 ** 400 *
ND, not detectable. (b,c) Secretion of IL-1β (b) and IL-18 (c) by * *
200 200
LPS-primed wild-type (WT), Rip3−/− or Nlrp3−/− BMDMs infected for
6 h with various doses of VSV (multiplicity of infection (MOI), 0 0
I

SV

SV
U

horizontal axis). (d) Immunoblot analysis of IL-1β and cleaved

Fl

Fl
VS

Se

Ad

VS

Se

Ad
H

caspase-1 (p20) in culture supernatants (SN) of LPS-primed

wild-type, Rip3−/− or Nlrp3−/− BMDMs infected for 6 h with various doses (above lanes) of VSV, and immunoblot analysis of the precursors of IL-1β
(pro-IL-1β) and caspase-1 (pro-casp1) in lysates of those cells (Input). (e,f) Secretion of IL-1β by LPS-primed Nlrp3+/+ or Nlrp3−/− BMDMs (e) or
Rip3+/+ or Rip3−/− BMDMs (f) left uninfected (UI) or infected for 6 h with VSV or adenovirus (AdV) or for 24 h with Sendai virus (SeV), influenza
virus (Flu) or HSV. NS, not significant (P > 0.05; unpaired t-test (a) or two-way analysis of variance (ANOVA) (f)); *P < 0.05 and **P < 0.001
(two-way ANOVA). Data are from three independent experiments with biological duplicates in each (a–c,e,f; mean and s.e.m. of n = 6 (duplicates in
three experiments)) or are representative of at least three independent experiments (d).

nature immunology  VOLUME 15  NUMBER 12  DECEMBER 2014 1127

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Figure 2  Role of RIP3 in virus-induced a Rip3

Rip3
–/–
+/+
b Rip3
Rip3
–/–
+/+
c Rip3
Rip3
+/+
–/–
activation of the NLRP3 inflammasome in vivo. 300 1,000
* 800 * NS
Concentrations of IL-1β (a), IL-18 (b) and IL-6 (c) 800

IL-1β (pg/ml)

IL-18 (pg/ml)
600

IL-6 (pg/ml)
in BALF from Rip3+/+ or Rip3−/− mice (n = 11 per 200
* 600 NS
genotype) left uninfected or infected with NS 400
* NS
400
influenza virus, VSV or HSV, assessed 3 d after 100 NS
200 200
infection. NS, not significant; *P < 0.001
0 0 0
(two-way ANOVA). Data are from three UI Flu VSV HSV UI Flu VSV HSV UI Flu VSV HSV
independent experiments (mean and s.e.m.).

by small interfering RNA (siRNA) in BMDMs inhibited VSV-induced RIP1-RIP3 complex, although MLKL was essential for RNA virus–
IL-1β secretion but did not affect the priming effects of the Toll-like induced necrosis, which suggested that the RIP1-RIP3 complex induced
receptor 2 (TLR2) agonist Pam3CSK4 (Fig. 3c and Supplementary cell death and inflammation via distinct downstream pathways.
Fig. 1c,d). Furthermore, inhibition of RIP1’s kinase activity with
specific inhibitors of RIP1 (Nec-1 or Nec-1s)28 also suppressed The RIP1-RIP3 complex promotes mitochondrial damage
VSV-induced IL-1β secretion, while the control inhibitor Nec-1i Mitochondrial dysfunction is involved in activation of the NLRP3
and the indoleamine 2,3-deoxygenase inhibitor 1-MT had no effect inflammasome induced by various agonists, including RNA
(Fig. 3d). These results indicated that RIP1 was important for RNA viruses4–6,31–33. Because mitochondrial fission has been proposed to
virus–induced activation of the NLRP3 inflammasome. Consistent be downstream of the RIP1-RIP3 complex in promoting necrosis 30
with that, infection with VSV promoted binding of RIP1 to RIP3 and because ROS produced by damaged mitochondria are important
in BMDMs (Fig. 3e), which suggested that viral infection initiated regulators of RNA virus–induced activation of the NLRP3 inflamma-
formation of the RIP1-RIP3 complex. These results indicated that for- some34,35, we investigated whether RIP1 or RIP3 was involved in RNA
© 2014 Nature America, Inc. All rights reserved.

mation of the RIP1-RIP3 complex promoted activation of the NLRP3 virus–induced mitochondrial damage and ROS production. Similar
inflammasome during infection with an RNA virus. to other NLRP3 agonists4, infection with VSV promoted mitochon-
The RIP1-RIP3 complex induces necrosis through sequential drial fission, induction of ROS and the formation of mitochondrial
activation of the effector MLKL and the phosphatase PGAM5 aggregates in the perinuclear space of wild-type BMDMs (Fig. 4a,b
(refs. 29,30). However, secretion of IL-1β induced by VSV or classic and Supplementary Fig. 1h), and VSV-induced mitochondria
agonists of NLRP3 was normal in MLKL-deficient (Mlkl−/−) BMDMs fission, ROS production and aggregate formation were impaired in
(Fig. 3f,g), although VSV-induced necrosis was blocked in Mlkl−/− Rip3−/− BMDMs (Fig. 4a,b and Supplementary Fig. 1h). Inhibition
BMDMs (Fig. 3h). Similarly, knockdown of PGAM5 mRNA in THP-1 of RIP1 activity with Nec-1s diminished the VSV-induced mitochon-
cells had no effect on activation of the NLRP3 inflammasome induced dria fission, ROS production and aggregate formation in wild-type
by viral infection or other agonists (Supplementary Fig. 1e–g). These BMDMs (Fig. 4c,d and Supplementary Fig. 1i). In contrast,
results indicated that MLKL and PGAM5 were not involved in virus- nigericin-induced mitochondrial ROS production and aggregate
induced activation of the NLRP3 inflammasome downstream of the formation were not affected when RIP1 or RIP3 was inhibited

a Scr shRIP1
b Scr
c Scr
siRip1 (1)
d 1.5 e
shRIP1 siRip1 (2) NS NS
VSV (MOI) 0 3 5 0 3 5 NS VSV
***
IL-1β (ng/ml)

1,000 NS 1.0
5 NS
IL-1β *** *** (MOI) 0 3 5
SN 4 800
***
IL-1β (pg/ml)
IL-1β (ng/ml)
npg

RIP3
p20
3 600 0.5 * IP:RIP1
Pro-IL-1β 2 400 ** RIP1
** **
RIP1 Input 1 200 0
VSV – + + + + + + + + + RIP3
0 0
β-actin 0 3 5 0 3 5 UT MSU VSV Nec-1i (µM) – – 20 40 – – – – – – Input
VSV (MOI) RIP1
Nec-1 (µM) – – – – 20 40 – – – –
Nec-1s (µM) – – – – – – 20 40 – –
1-MT (mM) – – – – – – – – 0.4 0.6
Figure 3  RNA virus–induced activation of the NLRP3 inflammasome
depends on RIP1 but not on MLKL. (a) Immunoblot analysis of IL-1β
MIkI
MIkI
+/+
–/–
MIkI
MIkI
+/+
–/– f
WT
Rip3
–/– g h
1,000 4 140 –/–
and cleaved caspase-1 in supernatants of THP-1 cells stably expressing NS NS MIkI ***
800 120
control shRNA with a scrambled sequence (Scr) or RIP1-specific ***
IL-1β (pg/ml)

IL-1β (ng/ml)

3 NS
Survival (%)

100
shRNA (shRIP1) and infected for 6 h with various doses (above lanes) 600
2 NS
80
NS 60
of VSV (top), and immunoblot analysis of pro-IL-1β, RIP1 and β-actin 400
1 40
(loading control throughout) in lysates of those cells (bottom). 200
20
(b) Secretion of IL-1β by THP-1 cells stably expressing shRNA and 0 0 0
UT VSV zVAD zVAD + VSV
infected with VSV as in a. (c) Secretion of IL-1β by Pam3CSK4-primed
T

SU

um

in
U

AT

ic
M

Al

er

BMDMs transfected with control siRNA with a scrambled sequence (Scr) or

ig
N

Rip1-specific siRNA (either of two constructs, siRip1 (1) or siRip1 (2)) and
left untreated (UT) or stimulated for 4 h with MSU or infected for 6 h with VSV. (d) Secretion of IL-1β by LPS-primed BMDMs pretreated for 1 h with
various inhibitors (below plot) and left uninfected (−) or infected (+) for 6 h with VSV. (e) Immunoblot analysis of RIP1 or RIP3 in immunoprecipitates
(IP) or lysates (Input) of LPS-primed BMDMs infected with for 1 h with various doses of VSV (above lanes). (f,g) Secretion of IL-1β by LPS-primed
Mlkl+/+ or Mlkl−/− BMDMs left untreated or infected with for 6 h VSV (f) or stimulated for 4 h with MSU or alum or for 30 min with ATP or nigericin (g).
(h) Survival of wild-type, Rip3−/− or Mlkl−/− BMDMs treated for 24 h with the caspase inhibitor z-VAD-fmk (zVAD) alone (20 µM) or with z-VAD-fmk
(20 µM) plus VSV (MOI, 3), assessed as the release of lactate dehydrogenase. NS, two-way ANOVA (c,d,g) or unpaired t-test (f); *P < 0.05, **P < 0.01
and ***P < 0.001 (two-way ANOVA (b,c,h) or unpaired t-test (d)). Data are representative of at least three independent experiments (a,e) or are from
three independent experiments with biological duplicates in each (b–d,f–h; mean and s.e.m. of n = 6).

1128 VOLUME 15  NUMBER 12  DECEMBER 2014  nature immunology

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a UT VSV Nigericin b Rip3

+/+ c UT VSV Nigericin d Mock
Rip3–/– Nec-1s

Cells with mito aggregates (%)

Cells with mito aggregates (%)
100 100
+/+ Mock
Rip3 80 80
*
* 60
60

40 40

20 20
Rip3
–/– Nec-1s
0 0

in
T

in

U
U

VS
VS

ic
ic

er
er

ig
ig
MitoTracker DAPI MitoTracker DAPI

N
N
Figure 4  The RIP1-RIP3 complex is required for mitochondrial damage and the translocation of ASC to mitochondria during e 60
Rip3+/+
Rip3–/–
infection with an RNA virus. (a) Confocal microscopy of LPS-primed Rip3+/+ or Rip3−/− BMDMs (left margin) left untreated or
*

Cells with ASC-mito

infected for 2 h with VSV or stimulated for 30 min with nigericin (above images) and then stained with red fluorescent

colocalization (%)
mitochondrial dye MitoTracker Red and with the DNA-binding dye DAPI (blue). Scale bar, 25 µm. (b) Quantification of cells 40
(treated as in a) with mitochondrial aggregates in the perinuclear space (‘mito aggregates’). (c) Confocal microscopy of
LPS-primed BMDMs given no pretreatment (Mock) or pretreated for 1 h with Nec-1s (30 µM) (left margin), followed by 20
treatment (above images) and staining as in a. Scale bar, 25 µm. (d) Quantification of cells treated as in c, assessed as in b.
(e) Quantification of the colocalization of ASC and mitochondria (‘ASC-mito’) as assessed by confocal microscopy of LPS-
primed Rip3+/+ or Rip3−/− BMDMs left uninfected or infected for 2 h with VSV and then stained with antibody to ASC, MitoTracker 0

V
U
Red and DAPI. *P < 0.001 (two-way ANOVA). Data are representative of three experiments (a,c) or are from three independent

VS
experiments with biological duplicates in each (b,d,e; mean and s.e.m. of n = 6).
© 2014 Nature America, Inc. All rights reserved.

(Fig. 4a–d and Supplementary Fig. 1h,i), which suggested a specific and phosphorylate DRP1 to promote its activation. Furthermore,
role for the RIP1-RIP3 complex in RNA virus–induced mitochon- infection with VSV promoted binding of RIP1 to RIP3 and DRP1
dria damage. Furthermore, VSV-induced colocalization of ASC with in BMDMs (Fig. 5g), which suggested that infection with VSV
mitochondria was inhibited in Rip3−/− BMDMs compared with their induced the formation of a RIP1-RIP3-DRP1 complex. The VSV-
colocalization in wild-type cells (Fig. 4e and Supplementary Fig. 2). induced interaction between RIP1 and DRP1 was blocked in Rip3−/−
Together these results indicated that the RIP1-RIP3 complex was able BMDMs (Fig. 5g), which suggested that RIP3 regulated the interac-
to promote aberrant fission and damage of mitochondria to activate tion between RIP1 and DRP1 during infection with VSV. Together
the NLRP3 inflammasome during infection with an RNA virus. our results indicated that the RIP1-RIP3 complex was able to promote
activation of DRP1 via interaction between RIP1 and DRP1 during
RIP1 and RIP3 induce translocation of DRP1 to mitochondria infection with an RNA virus.
The GTPase DRP1 catalyzes the process of mitochondrial fission.
Because infection with an RNA virus promoted aberrant mitochon- DRP1 is required for RNA virus–induced NLRP3 activation
drial fission, we investigated whether infection with VSV promoted Because the RIP1-RIP3 complex activated DRP1 and promoted its
activation of DRP1. Phosphorylation at Ser616 induces activation of translocation to mitochondria, we next investigated the role of DRP1
DRP1 and its translocation to mitochondria, which then stimulates in RNA virus–induced activation of the NLRP3 inflammasome. The
mitochondrial fission36. We observed that infection with VSV induced VSV-induced formation of mitochondrial aggregates, mitochondrial
phosphorylation of DRP1 at Ser616 in wild-type BMDMs, while infec- fission and ROS production were impaired in BMDMs in which Drp1
npg

tion with adenovirus or treatment with other NLRP3 agonists did not expression was silenced by siRNA compared with those effects in
(Fig. 5a); this suggested that infection with an RNA virus was able to BMDMs in which it was not silenced (Fig. 6a,b and Supplementary
activate DRP1. Moreover, VSV-induced phosphorylation of DRP1 was Fig. 6a,b). Consistent with that impaired mitochondrial damage and
impaired in Rip3−/− BMDMs relative to such phosphorylation in wild- ROS production, VSV-induced production of IL-1β was also impaired
type BMDMs (Fig. 5b). Inhibition of RIP1 kinase activity by Nec-1 or in BMDMs in which Drp1 mRNA was silenced with siRNA compared
Nec-1s inhibited the VSV-induced phosphorylation of DRP1 (Fig. 5c). In with its production in BMDMs in which Drp1 mRNA was not silenced
contrast, VSV-induced phosphorylation of DRP1 was similar in Mlkl−/− (Fig. 6c), while VSV-induced production of tumor-necrosis factor
BMDMs and wild-type BMDMs (Fig. 5d), which suggested that the RNA was not affected (Fig. 6d). Similar to the effect of RIP3 deficiency,
virus–induced activation of DRP1 depended on RIP1 and RIP3 but did inhibition of Drp1 expression with siRNA did not affect the DNA
not depend on the necrosis effector MLKL. Furthermore, VSV-induced virus–induced production of IL-1β (Fig. 6c). In addition, knockdown
translocation of DRP1 to mitochondria in BMDMs was inhibited follow- of Drp1 mRNA had no effect on MSU-, ATP- or nigericin-induced
ing deletion of RIP3 or inhibition of RIP1 by Nec-1s (Supplementary activation of the NLRP3 inflammasome (Supplementary Fig. 6c),
Fig. 3 and Supplementary Fig. 4) but was not inhibited by deletion which suggested a specific role for DRP1 in RNA virus–induced
of MLKL (Supplementary Fig. 5). This suggested that the RIP1-RIP3 activation of the inflammasome. The mitochondrial fission protein
complex was able to activate DRP1 and promote its translocation to mito- Fis1 is an important component of the mitochondrial outer mem-
chondria during infection with an RNA virus. brane that can recruit DRP1 to mitochondria and promote mito-
Next we investigated how RIP1 and RIP3 activated DRP1. When chondrial fission37. Consistent with the effect of silencing Drp1,
overexpressed in HEK293T human embryonic kidney cells, RIP1 we found that silencing of Fis1 with siRNA in BMDMs inhibited
interacted with DRP1, while overexpressed RIP3 did not (Fig. 5e). VSV-induced mitochondrial damage and IL-1β secretion but had no
In an in vitro phosphorylation assay, RIP1 phosphorylated DRP1 at effect on MSU- or nigericin-induced secretion of IL-1β (Fig. 6e,f and
Ser616 (Fig. 5f), which suggested that RIP1 was able to interact with Supplementary Fig. 6d–f).

nature immunology  VOLUME 15  NUMBER 12  DECEMBER 2014 1129

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a b c d e

h)
Ad (0.5 .5

(1 )
(0 h)
Nec-1 – – – – – +

(0

h)

V 5h
SU h)

e h)
+/+ –/–
Rip3+/+ –/–

h)
Rip3 MIkI MIkI

V )
AT ricin

Ad .5
VS (1 h

3
Nec-1s – – – – + –

VS (0.
M (1
N (2

IP

IP
Flag

EV
k
SU

R
1-MT – – – + – – VSV (h) 0 0.5 1 0 0.5 1

P
oc

VSV (h) 0 0.5 1 0 0.5 1

v
v
ig
M
M

Nec-1i – – + – – – DRP1 (eGFP) IP:Flag

p-DRP1 p-DRP1 VSV – + + + + + p-DRP1
p-DRP1 RIP1 (Flag)
DRP1 DRP1 DRP1 RIP3 (Flag)
DRP1 Input
β-actin β-actin β-actin β-actin DRP1 (eGFP)

f g Rip3+/+ Rip3
–/–

3
oc

IP

IP
Flag
Figure 5  The RIP1-RIP3 complex promotes RNA virus–induced activation of DRP1. VSV – + – +

R
p-DRP1
(a) Immunoblot analysis of phosphorylated (p-) and total DRP1 in LPS-primed wild-type DRP1
BMDMs left untreated (Mock) or stimulated for various times (above lanes) with MSU, RIP1 (Flag)
RIP3 IP:RIP1
nigericin or ATP or infected with various times (above lanes) with adenovirus or VSV. RIP3 (Flag)
(b) Immunoblot analysis of phosphorylated and total DRP1 in LPS-primed Rip3+/+ or RIP1
DRP1 (eGFP)
Rip3−/− BMDMs infected for various times (above lanes) with VSV. (c) Immunoblot
RIP1
analysis of phosphorylated and total DRP1 in LPS-primed BMDMs pretreated for 1 h
with various inhibitors (above blots) and then left uninfected (−) or infected (+) for 30 min RIP3 Input
with VSV. (d) Immunoblot analysis of phosphorylated and total DRP1 in LPS-primed Mlk+/+ or
Mlkl−/− BMDMs infected for various times (above lanes) with VSV. (e) Immunoblot analysis of the interaction of DRP1

RIP1 or RIP3 with DRP1 in immunoprecipitates or lysates of HEK293T cells transfected to express enhanced green
fluorescent protein (eGFP)-tagged DRP1 plus either empty vector (EV) or vector expressing Flag-tagged RIP1 or RIP3 (above lanes). (f) In vitro
phosphorylation assay of DRP1 incubated with purified RIP1 or RIP3 from lysates of HEK293T cells transfected as in e, analyzed by immunoblot with
antibody to DRP1 phosphorylated at Ser616. (g) Immunoblot analysis of DRP1, RIP1 and RIP3 in immunoprecipitates or lysates of LPS-primed Rip3+/+
© 2014 Nature America, Inc. All rights reserved.

or Rip3−/− BMDMs left uninfected (−) or infected (+) for 1 h with VSV. Data are representative of three independent experiments.

Dissipation of the membrane potential by CCCP, a chemical inhib- consistent with a role for DRP1 in CCCP-induced mitochondrial
itor of oxidative phosphorylation, can induce mitochondrial fission fission (Supplementary Fig. 7e,f); this suggested that CCCP acti-
via DRP1 (ref. 38), so to further study the specificity of the RIP1- vated the NLRP3 inflammasome in a DRP1-dependent but RIP3-
RIP3-DRP1 signaling pathway in RNA virus–induced activation independent manner. Together these results indicated that DRP1
of NLRP3, we investigatd the role of RIP3 in CCCP-induced mito- acted downstream of the RIP1-RIP3 complex and promoted RNA
chondria fission. CCCP did not induce phosphorylation of DRP1 at virus–induced activation of the NLRP3 inflammasome.
Ser616, and although CCCP induced IL-1β production and translo- Next we found that knockdown of Drp1 mRNA in BMDMs did
cation of DRP1 to mitochondria in wild-type BMDMs, this was not not suppress VSV-induced necrosis (Fig. 6g). Similarly, knock-
affected by RIP3 deficiency (Supplementary Fig. 7a–c). However, down of Fis1 mRNA in BMDMs had no effect on VSV-induced
CCCP-induced secretion of IL-1β was impaired when Drp1 expres- necrosis (Fig. 6h). Together these results indicated that DRP1 acted
sion was inhibited by siRNA in BMDMs (Supplementary Fig. 7d), downstream of the RIP1-RIP3 complex to promote mitochondrial

a UT VSV Nigericin
b Scr
siDrp1 (1)
c Scr
siDrp1 (1)
d Scr
siDrp1 (1)
siDrp1 (2) siDrp1 (2) siDrp1 (2)
100 * NS 500 NS 1,000 NS NS NS
* *
npg

80 400 * 800
aggregates (%)
Cells with mito

IL-1β (pg/ml)

Scr
TNF (pg/ml)

60 300 600
40 200 400
20 100 200
0 0 0
UT VSV Nigericin UI VSV AdV UI VSV AdV

siDrp1 (1)
e Scr
siFis1 (1) f Scr
siFis1 (1) g Scr
siDrp1 (1) h Scr
siFis1 (1)
siFis1 (2) * siFis1 (2) siDrp1 (2) siFis1 (2)
600 2.5 140 140
* NS
120 NS 120 NS
2.0
IL-1β (ng/ml)
IL-1β (pg/ml)

100 100
Survival (%)
Survival (%)

400 NS
1.5 80 80
NS
60 60 NS
siDrp1 (2) 1.0
200 40 40
0.5
ND 20 20
0 0 0 0
UT VSV UT MSU Nigericin zVAD zVAD + zVAD zVAD +
MitoTracker DAPI
VSV VSV

Figure 6  Role of DRP1 in RNA virus–induced mitochondrial damage and activation of the NLRP3 inflammasome. (a) Confocal microscopy of BMDMs
transfected with control siRNA or Drp1-specific siRNA (left margin) and left untreated or infected for 2 h with VSV or stimulated for 30 min with
nigericin (above images), followed by staining with MitoTracker Red and DAPI. Scale bar, 25 µm. (b) Quantification of cells treated as in a (assessed
as in Fig. 4b). (c,d) Secretion of IL-1β (c) or tumor-necrosis factor (TNF) (d) by LPS-primed BMDMs transfected with siRNA as in a and then left
uninfected or infected for 6 h with VSV or adenovirus. (e,f) Secretion of IL-1β by LPS-primed BMDMs transfected with control siRNA or Fis1-specific
siRNA and left untreated or infected for 6 h with VSV (e) or stimulated for 4 h with MSU or for 30 min with nigericin (f). (g,h) Survival of LPS-primed
BMDMs transfected with siRNA as in a (g) or e,f (h) and then stimulated for 24 h with z-VAD-fmk with or without VSV (as in Fig. 3h), assessed as
release of lactate dehydrogenase. NS, two-way ANOVA; *P < 0.001 (two-way ANOVA). Data are representative of three independent experiments (a) or
are from three independent experiments with biological duplicates in each (b–h; mean and s.e.m. of n = 6).

1130 VOLUME 15  NUMBER 12  DECEMBER 2014  nature immunology

 Articles

a WT b WT c Mock Poly(I:C)
d e Scr f Scr
Rip3–/– Rip3–/– Scr siDrp1 (1) siDrp1 (1)

3 –/–

3 –/–
Nlrp3–/– Nlrp3–/–

3 –/–

3 –/–
shRIP1 siDrp1 (2) siDrp1 (2)
**

lrp

lrp
1.5 600 *

ip

ip
4 *

T
**

W
N

N
R

R
** 600 300
IL-1β ** ** *
** 3

IL-18 (pg/ml)
IL-1β (ng/ml)

IL-1β (ng/ml)

IL-18 (pg/ml)
IL-1β (pg/ml)
1.0 400 SN 400 200
p20 2
0.5 200 200 100
Pro-casp1 1
Input
0 0 0 0 0
US Poly(I:C) US Poly(I:C) Pro-IL-1β US Poly(I:C) US Poly(I:C) US Poly(I:C)

g Scr
siFis1 (1)
h Scr
siFis1 (1)
Figure 7  dsRNA activates the NLRP3 inflammasome in a RIP1–, RIP3– and DRP1-dependent siFis1 (2) siFis1 (2)
manner. (a,b) Secretion of IL-1β (a) and IL-18 (b) by LPS-primed wild-type, Rip3−/− or 1,000 400 **
** **
Nlrp3−/− BMDMs left unstimulated or stimulated for 6 h with poly(I:C). (c) Immunoblot 800 **

IL-18 (pg/ml)
300

IL-1β (pg/ml)
analysis of IL-1β and cleaved caspase-1 in culture supernatants of LPS-primed wild-type, 600
200
Rip3−/− or Nlrp3−/− BMDMs stimulated for 6 h with poly(I:C) (top), and immunoblot analysis 400
of the precursors of IL-1β and caspase-1 in lysates of those cells (bottom). (d) Secretion of 200
100
IL-1β by THP-1 cells stably expressing control shRNA or RIP1-specific shRNA and left
0 0
unstimulated or stimulated for 6 h with poly(I:C). (e–h) Secretion of IL-1β (e,g) or IL-18 (f,h) by US Poly(I:C) US Poly(I:C)
LPS-primed BMDMs transfected with control or Drp1-specific siRNA (e,f) or control or Fis1-specific
siRNA (g,h) and then left unstimulated or stimulated for 6 h with poly(I:C). *P < 0.01 and **P < 0.001 (two-way ANOVA (a,b,e–h) or unpaired t-test (d)).
Data are from three independent experiments with biological duplicates in each (a,b,d–h; mean and s.e.m. of n = 6) or are representative of three
independent experiments (c).
© 2014 Nature America, Inc. All rights reserved.

damage and activation of the NLRP3 inflammasome, while MLKL His, where ‘x’ is any amino acid) family of helicases and has been
acted downstream of RIP1 and RIP3 to induce necrosis; this suggested proposed to sense cytosolic dsRNA and activate the NLRP3 inflamma-
that the RIP1-RIP3 complex used different pathways to signal inflam- some during viral infection40. Similar to results in published reports
mation or necrosis during infection with an RNA virus. in which knockdown of DHX33 mRNA in THP-1 cells resulted in only
slightly decreased production of IL-1β induced by respiratory syncy-
Double-stranded RNA activates NLRP3 via RIP1 and RIP3 tial virus40, we found that shRNA-mediated knockdown of DHX33
Because cytosolic RNA has been proposed to be the trigger of the mRNA in THP-1 cells had only a slight effect on the VSV-induced
NLRP3 inflammasome during infection with an RNA virus14,16,18, we production of IL-1β, while silencing of RIP1 mRNA in these cells
investigated whether this process depends on RIP1 or RIP3. Similar almost completely inhibited the VSV-induced production of IL-1β
to infection with an RNA virus, transfection of poly(I:C), a synthetic (Supplementary Fig. 8d,e); this suggested that DHX33 was not a
analog of double-stranded RNA (dsRNA), induced less secretion of major sensor of viral RNA–induced activation of NLRP3 during viral
IL-1β or IL-18 and cleavage of caspase-1 in Rip3−/− BMDMs than infection. We also assessed the role of TLR3, which senses extracel-
in wild-type BMDMs (Fig. 7a–c). In addition, knockdown of RIP1 lular dsRNA and activates innate immunity41, in RNA virus–induced
mRNA in THP-1 cells inhibited the production of IL-1β induced by activation of the NLRP3 inflammasome. Consistent with published
transfection of poly(I:C) compared with such production in THP-1 reports16, VSV-induced production of IL-1β was not affected by defi-
cells in which RIP1 mRNA was not silenced (Fig. 7d); this suggested ciency in TLR3 (Supplementary Fig. 8f,g). Together these results
that cytosolic RNA–induced activation of the NLRP3 inflammasome suggested that RIG-I, Mda5, DHX33 and TLR3 were not the main
npg

depended on the RIP1-RIP3 complex. Moreover, secretion of IL-1β sensors of RNA involved in RNA virus–induced activation of the
or IL-18 induced by transfection of poly(I:C) into BMDMs was sup- NLRP3 inflammasome.
pressed by siRNA-mediated knockdown of Drp1 or Fis1 mRNA com-
pared with such secretion in BMDMs in which Drp1 or Fis1 mRNA DISCUSSION
was not silenced (Fig. 7e–h).These results indicated that the RIP1- The NLRP3 inflammasome can sense infection with either RNA
RIP3-DRP1 pathway was important for cytosolic RNA–induced acti- viruses or DNA viruses to activate innate immune responses and has
vation of the inflammasome. been proposed as a crucial component of host defense in viral infec-
tion9–11, but the mechanisms by which NLRP3 detects viral infec-
RNA virus activates NLRP3 independently of RNA sensors tion are still unclear. Here we demonstrated a role for the RIP1-RIP3
We next sought to determine what activates the NLRP3 inflamma- complex in RNA virus–induced activation of the NLRP3 inflamma-
some upstream of RIP1 and RIP3 during infection with an RNA virus some. Inactivation of RIP1 or RIP3 severely impaired such activation,
and analyzed the role of known sensors that detect RNA in this proc- including cleavage of caspase-1 and secretion of IL-1β and IL-18,
ess. Two cytosolic sensors of viral RNA, RIG-I and Mda5, induce but had no effect on DNA virus–induced activation of the NLRP3
antiviral innate immunity, but their role in virus-induced activation inflammasome. We also identified DRP1 as the effector downstream
of inflammasomes is controversial15,39. IL-1β production induced by of RIP1-RIP3 that promoted NLRP3 activation. Our results thus indi-
infection with VSV or cytosolic treatment with poly(I:C) was simi- cate that the RIP1-RIP3 complex is an important participant in innate
lar in RIG-I-deficient (Ddx58−/−; called ‘Rig-I−/−’ here) BMDMs and immune and inflammatory signaling pathways during viral infection
Mda5-deficient (Ifih1−/−; called ‘Mda5−/−’ here) BMDMs primed with beyond those previously identified for necrosis.
lipopolysaccharide (LPS) compared with that in their wild-type counter­ Although increasing evidence has suggested that mitochondria
parts (Supplementary Fig. 8a,b), although VSV-induced production serve as a platform for activation of the NLRP3 inflammasome by
of interferon-β was blocked in Rig-I−/− BMDMs (Supplementary producing mitochondrial ROS, lipids or DNA in response to damage
Fig. 8c). DHX33 is a member of DExD/H-box (Asp–Glu–x–Asp or or ‘stress’4–6,32,33, the signaling pathways upstream of mitochondrial

nature immunology  VOLUME 15  NUMBER 12  DECEMBER 2014 1131

 Articles

damage have remained unclear. Our results have demonstrated a role but independent of RIG-I (ref. 15). Our results are consistent with the
for the RIP1-RIP3 complex in RNA virus–induced mitochondrial results of the latter study. These divergent results could be explained
damage, ROS production and activation of the NLRP3 ­inflamamsome. by the use of different stimulation protocols because it is possible that
Our results are consistent with those of published reports showing RIG-I might contribute to induction of the expression of NLRP3 or
that RIP3 signaling is able to promote mitochondrial production of pro-IL-1β by viral infection in unprimed cells. In addition, our results
ROS21,23. In addition, RIP3-dependent activation of the inflamma- indicated minimal roles for the RNA sensors DHX33 and TLR3 in
some in macrophages in which IAP proteins are inhibited can be RNA virus–induced activation of inflammasomes, which suggests that
inhibited by ROS inhibitors22. Such results suggest that RIP1 and RIP3 an undefined sensor of RNA might act upstream of the RIP1-RIP3
function as important signaling molecules upstream of mitochondrial complex to promote activation of the NLRP3 inflammasome.
damage to promote activation of the NLRP3 inflammasome, at least Collectively, our results have shown a role for the RIP1-RIP3
during infection with an RNA virus or when IAP proteins are inhib- complex in RNA virus–induced activation of the NLRP3 inflamma-
ited. However, the signaling pathways upstream of mitochondrial some. This not only improves understanding of the mechanisms by
damage that are involved in activation of the NLRP3 inflammasome which the NLRP3 inflammasome is activated but also establishes a
induced by other agonists need to be further identified. direct link between inflammation and necrosis signaling pathways.
MLKL and PGAM5 have been proposed to be the downstream Our identification of the RIP1-RIP3 complex as a crucial regulator
effectors of the RIP1-RIP3 complex in necrosis 29,30. Here, we found in virus-induced inflammation might provide a potential new tar-
that MLKL and PGAM5 were not involved in RNA virus–induced get for the treatment of inflammatory diseases associated with viral
activation of the NLRP3 inflammasome, although RNA virus–induced infection.
necrosis was indeed blocked in Mlkl−/− macrophages. However, MLKL
and PGAM5 were required for spontaneous activation of the NLRP3 Methods
inflammasome in caspase-8-deficient dendritic cells because inhibi- Methods and any associated references are available in the online
© 2014 Nature America, Inc. All rights reserved.

tion of the expression of MLKL or PGAM5 by siRNA reduced LPS- version of the paper.
induced secretion of IL-1β in caspase-8-deficient dendritic cells22.
Note: Any Supplementary Information and Source Data files are available in the
Such results suggest that RIP1 and RIP3 might promote activation
online version of the paper.
of the NLRP3 inflammasome via different signaling pathways in dif-
ferent ‘stressed’ conditions. The different roles and mechanisms of Acknowledgments
PGAM5 or MLKL in activation of the NLRP3 inflammasome needs We thank J. Tschopp (University of Lausanne) for Nlrp3−/− mice; S. Akira
(Osaka University) for Rig-I−/− mice; V.M. Dixit (Genentech) for Rip3−/− mice;
to be further investigated in the future.
M. Colonna (Washington University) for Mda5−/− mice; Z. Jiang (Peking
Our results have demonstrated that DRP1 acted downstream of University) for Sendai virus and VSV (Indiana strain); and Z. Song (Wuhan
RIP1 and RIP3 to activate the NLRP3 inflammasome by promoting University) for the DRP1 construct. Supported by the National Basic Research
mitochondrial fission during infection with an RNA virus. However, Program of China (2014CB910800), the National Natural Science Foundation of
the roles of DRP1 and mitochondrial fission in inflammasome activa- China (81330078 and 81222040), the Doctoral Fund of the Ministry of Education
of China (20123402120001, 20123402110010), the One Hundred Person Project
tion are complicated. Although nigericin did not induce phosphoryla- (R.Z.) and the Fundamental Research Funds for the Central Universities
tion of DRP1 at Ser616, it still induced the translocation of DRP1 to (R.Z. and W.J.).
mitochondria, which suggests the existence of another mechanism
for the activation of DRP1. Moreover, nigericin-induced mitochon- AUTHOR CONTRIBUTIONS
X.W., W.J., Y.Y. and T.G. performed the experiments; W.J., J.H., Z.T. and R.Z.
drial damage, ROS production and inflammasome activation were
designed the research; X.W., W.J., Z.T. and R.Z. wrote the manuscript; and R.Z. and
normal when Drp1 was silenced, which indicates the existence of Z.T. supervised the project.
other pathways that regulate mitochondrial fission and mitochondrial
npg

damage during activation of the NLRP3 inflammasome induced by COMPETING FINANCIAL INTERESTS
classic agonists. Indeed, DRP1-independent mitochondrial fission The authors declare no competing financial interests.
has been described38,42. Reprints and permissions information is available online at http://www.nature.com/
DRP1 has been proposed to be downstream of PGAM5 in trig- reprints/index.html.
gering necrosis30. After the induction of necrosis by tumor-necrosis
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nature immunology  VOLUME 15  NUMBER 12  DECEMBER 2014 1133

 ONLINE METHODS or poly(dA:dT) (0.5 µg/ml) through the use of Lipofectamine according to the
Mice. Nlrp3−/−, Rip3−/−, Rig-I−/−, Mda5−/−, Mlkl−/− and Tlr3−/− mice have been manufacturer’s protocol (Invitrogen). For viral infection, primed BMDMs or
described44–49. All mice were on a C57BL/6 background except that Rig-I−/− differentiated THP-1 cells were infected for 6 h with VSV or adenovirus or for
mice were on the BALB/c background. The sex-matched littermates of the 24 h with Sendai virus, influenza virus or HSV type 1. If not indicated other-
mutant mice were used as controls. All animal experiments were approved by wise, cells were infected VSV, HSV type 1, influenza virus or Sendai virus at
the Ethics Committee of University of Science and Technology of China. an MOI of 5 or with adenovirus at a dose of 1 × 104 particles/cell. Cell extracts
and precipitated supernatants were analyzed by immunoblot.
Reagents. Nigericin, CCCP (carbonyl cyanide m-chlorophenyl hydra-
zone), MSU, ATP, PMA (phorbol 12-myristate 13-acetate), z-VAD-fmk Enzyme-linked immunosorbent assay. Mouse IL-6 or IL-1β (R&D Systems),
­(benzyloxycarbonyl–Val-Ala-Asp–fluoromethylketone), poly(dA:dT) and human IL-1β (BD Biosciences) or mouse IL-18 (eBioscience) in supernatants
1-MT (1-methyl-[d]-tryptophan) were from Sigma. Nec-1s was from of cell culture or BALF were quantified by enzyme-linked immunosorbent
Biovision. Nec-1i was from Millipore. Nec-1 was from Cayman Chemical. assay according to the manufacturer’s instructions.
Imject-Alum was from Pierce Biochemicals. Poly(I:C), Pam3CSK4 (tripalmitoyl
cysteinyl seryl tetralysine lipopeptide) and ultrapure LPS were from Invivogen. Real time PCR. BMDMs were dissolved in TRIzol reagent (Invitrogen). cDNA
MitoTracker and MitoSOX (fluorogenic dye specifically targeted to mitochon- was synthesized from extracted total RNA with an M-MLV Reverse Transcriptase
dria in live cells) were from Invitrogen. The LDH (lactate dehydrogenase) cyto- kit according to the manufacturer’s protocol (Invitrogen). SYBR Green Premix
toxicity assay kit was from Beyotime. Protein G agarose was from Millipore. (Takara) was used for quantitative PCR with a StepOne Real Time PCR System
Antibody to human pro-IL-1β (60136-1-Ig) and antibody to Fis1 (anti-Fis1; (Applied Biosystems). The gene encoding glyceraldehyde phosphate dehydro-
10956-1-AP) were from Proteintech. Antibody to mouse IL-1β (AF-401-NA) genase was used as an internal control. The sequences of primers for quantita-
was from R&D Systems. Antibodies to mouse caspase-1 (p20) (AG-20B-0042) tive PCR were as follows: interferon-β, 5′-CCAACAAGTGTCTCCTCCAAA
and anti-NLRP3 (AG-20B-0014) were from Adipogen. Anti-β-actin (P30002) TT-3′ (forward) and 5′-GTAGGAATCCAAGCAAGTTGTAGCT-3′
was from Abmart. Anti-PGAM5 (186-200), anti-VSV (V4888) and anti-Flag (reverse); GAPDH, 5′-GGTGAAGGTCGGTGTGAACG-3′ (forward) and
(F2555) were from Sigma. Anti-RIP1 (3493), anti-DRP1 (8570), antibody to 5′-CTCGCTCCTGGAAGATGGTG-3′ (reverse).
© 2014 Nature America, Inc. All rights reserved.

DRP1 phosphorylated at Ser616 (4494) and anti-human caspase-1 (2225)

were from Cell Signaling. Anti-RIP3 (2283) was from ProSci. Anti-DHX33 Transfection and immunoprecipitation. Constructs were transfected into
(sc-137424) and anti-ASC (sc-22514-R) were from Santa Cruz. Antibody to HEK293T cells through the use of polyethylenimine. After 18 h, cells were
human cleaved IL-1β (A5208206) was from Sangon Biotech. collected and resuspended in lysis buffer (50 mM Tris, pH 7.8, 50 mM NaCl,
1% (vol/vol) Nonidet-P40, 5 mM EDTA and 10% (vol/vol) glycerol). Extracts were
Viruses. VSV (Indiana strain) was propagated and amplified by infection of immunoprecipitated with anti-Flag (identified above) and beads and then were
a monolayer of BHK-21 haby hamster kidney cells. HSV type 1 (KOS strain) assessed by immunoblot analysis. LPS-primed BMDMs (1 × 107) were treated for
was propagated on Vero African green monkey kidney cells. Influenza virus 1 h with VSV. BMDMs were then resuspended in lysis buffer and proteins were
(strain A/PR/8/34) and Sendai virus were grown for 2 d at 37 °C in the allantoic immunoprecipitated from extracts with anti-RIP1 (identified above).
cavities of 10-day-old fertile chicken eggs. Viruses in culture supernatants were
quantified by standard plaque assay. Adenovirus was from Beijing Five Plus In vitro phosphorylation of DRP1. Flag-tagged RIP1 or RIP3 was immunopre-
Molecular Medicine Institute. These viruses were stored at −80 °C. cipitated (with M2 (anti-Flag) beads; Sigma) from HEK293T cells transiently
expressing these proteins. eGFP-DRP1 was immunoprecipitated from eGFP-
Viral infection in vivo. Anesthetized mice were challenged by intranasal DRP1–expressing HEK-293T cells with anti-eGFP (M20004; Abmart) and pro-
administration of 3 × 103 plaque-forming units of influenza virus, 1 × 105 tein G agarose beads. After incubation for 4 h at 4 °C, the beads were collected
plaque-forming units of VSV or 1 × 105 plaque-forming units of HSV, each in and washed twice with ice-cold lysis buffer (described above) and once with
50 µl of PBS. At 3 d after infection, BALF was collected and was centrifuged for ice-cold kinase buffer (50 mM Tris·HCl, pH 7.5, 5 mM MgCl2, 0.1 mM sodium
the removal of cells, and cytokines in the supernatant were quantified. orthovanadate, 0.1 mM sodium pyrophosphate, 1 mM NaF and 1 mM PMSF).
Phosphorylation of DRP1 proceeded for 30 min at 37 °C in kinase buffer (final
Generation of THP-1 cells expressing shRNA. shRNA targeting mRNA of volume, 30 ml), ATP and immunoaffinity-purified RIP1 or RIP3. After sepa-
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RIP1, PGAM5 and DHX33 was from Sigma. The protocol for generating THP-1 ration of samples by SDS-PAGE, protein phosphorylation was detected with
cells stably expressing shRNA has been published50. antibody to DRP1 phosphorylated at Ser616 (identified above).

siRNA-mediated gene silencing in BMDMs. BMDMs were plated in 12-well Confocal microscopy. BMDMs were plated on coverslips overnight and
plates (at a density of 4 × 105cells per well) and then were transfected with then were used for stimulation or staining with MitoTracker Red (50 nM)
50 nM siRNA through the use of Lipofectamine RNAiMAX according to the or MitoSOX (5 µM). After being washed three times with Tween-20 in PBS
manufacturer’s guidelines (Invitrogen). siRNA (sequences, Supplementary (PBST), the cells were fixed for 15 min with 4% PFA in PBS and then were
Table 1) was chemically synthesized by GenePharma, and the negative control washed three times with PBST. After permeabilization with Triton X-100 and
siRNA was also from GenePharma. blockade of nonspecific binding with 10% goat serum in PBS, cells were incu-
bated overnight at 4 °C d with primary antibodies (identified above) in 10%
Cell preparation and stimulation. Human THP-1 cells were grown in RPMI- goat serum. After being washed with PBST, cells were incubated for 60 min
1640 medium supplemented with 10% FBS and 50 µM 2-mercaptoethanol. with Alexa Fluor 488–conjugated goat antibody to rabbit immunoglobulin G
THP-1 cells from American Type Culture Collection were routinely tested (A11008; Invitrogen) in 10% goat serum in PBS and were rinsed in PBST.
for contamination by mycoplasma. THP-1 cells were differentiated for 3 h A Zeiss LSM700 was used for confocal microscopy. For quantification of mito-
with 100 nM PMA and then were incubated overnight. BMDMs were derived chondrial aggregates (>3 µm in diameter) in the perinuclear region or colo-
from tibia and femoral bone marrow cells as described 50 and were cultured calization of ASC with mitochondria, scanning fields were randomly selected
in DMEM complemented with 10% FBS, 1 mM sodium pyruvate and 2 mM and at least 100 cells were counted in each slide.
l-glutamine in the presence of culture supernatants of L929 mouse fibroblasts.
For induction of inflammasome activation, 5 × 105 macrophages were plated Statistical analyses. Statistical analyses were made with an unpaired t-test for
overnight in 12-well plates and the medium was changed to Opti-MEM (1% two groups or two-way ANOVA (GraphPad Software) for multiple groups with
FBS) the following morning, then the cells were primed for 4 h with ultrapure all data points showing a normal distribution. Researchers were not blinded to
LPS (100 ng/ml) or Pam3CSK4 (400 ng/ml). After that, the cells were stimulated the genotype of the mice or cells. No exclusion of data points was used. Sample
for 4 h with MSU (150 µg/ml) or Alum (300 µg/ml) or for 30 min with ATP sizes were selected on the basis of preliminary results to ensure an adequate
(2.5 mM) or nigericin (10 µM). Cells were transfected with poly(I:C) (1 µg/ml) power. P values of <0.05 were considered significant.

nature immunology doi:10.1038/ni.3015

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© 2014 Nature America, Inc. All rights reserved.
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doi:10.1038/ni.3015
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