Immunity

Article

The NLRP3 Inflammasome Mediates

In Vivo Innate Immunity to Influenza A Virus
through Recognition of Viral RNA
Irving C. Allen,1 Margaret A. Scull,2,3 Chris B. Moore,1 Eda K. Holl,2 Erin McElvania-TeKippe,2 Debra J. Taxman,2
Elizabeth H. Guthrie,1 Raymond J. Pickles,2,3 and Jenny P.-Y. Ting1,2,*
1Lineberger Comprehensive Cancer Center
2Department of Microbiology and Immunology
3Cystic Fibrosis/Pulmonary Research and Treatment Center

The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
*Correspondence: panyun@med.unc.edu
DOI 10.1016/j.immuni.2009.02.005

SUMMARY the RLHs (RIG-I-like helicases), TLRs (toll-like receptors), and

NLR (nucleotide-binding domain and leucine-rich-repeat-
The nucleotide-binding domain and leucine-rich- containing) proteins, have been identified and shown to be acti-
repeat-containing (NLR) family of pattern-recogni- vated in response to viral pathogens (Akira et al., 2006; Ting
tion molecules mediate host immunity to various et al., 2008). The individual members of these families can be
pathogenic stimuli. However, in vivo evidence for the distinguished by ligand specificity, cellular localization, and acti-
involvement of NLR proteins in viral sensing has not vation of unique downstream signaling pathways. The strategy
of employing multiple families of PRRs affords the host a high
been widely investigated and remains controversial.
degree of functional redundancy and provides multiple mecha-
As a test of the physiologic role of the NLR molecule nisms to optimally respond to a diverse range of pathogens
NLRP3 during RNA viral infection, we explored the (Kawai and Akira, 2007).
in vivo role of NLRP3 inflammasome components The NLR proteins are emerging as a major route by which the
during influenza virus infection. Mice lacking Nlrp3, innate immune system responds to microbial pathogens.
Pycard, or caspase-1, but not Nlrc4, exhibited dra- Substantial evidence suggests that the NLR proteins serve as
matically increased mortality and a reduced immune intracellular mediators of PAMP-initiated host-cell signaling,
response after exposure to the influenza virus. although the exact mechanisms underlying NLR responses to
Utilizing analogs of dsRNA (poly(I:C)) and ssRNA pathogens are not completely understood. One of the funda-
(ssRNA40), we demonstrated that an NLRP3-medi- mental reactions of the innate immune response to viral infection
ated response could be activated by RNA species. is the processing and release of proinflammatory cytokines,
including the regulation and release of IL-1b. It has been demon-
Mechanistically, NLRP3 inflammasome activation
strated that the NLR protein NLRP3, together with its adaptor
by the influenza virus was dependent on lysosomal
protein, PYD- and CARD-domain-containing protein (PYCARD)
maturation and reactive oxygen species (ROS). Inhi- (which is also known as Apoptotic Speck protein containing
bition of ROS induction eliminated IL-1b production a CARD [ASC]), regulates IL-1b maturation through the formation
in animals during influenza infection. Together, these of a biochemical complex called the inflammasome (Agostini
data place the NLRP3 inflammasome as an essential et al., 2004). This inflammasome regulates the activation of cas-
component in host defense against influenza infec- pase-1 and subsequent cleavage of the IL-1b and IL-18 precur-
tion through the sensing of viral RNA. sors into their functional forms, which are then released from the
cell. In addition to NLRP3, other NLR proteins, such as NLRC4
and NLRP1, also cause caspase-1 activation and IL-1b produc-
INTRODUCTION tion (Mariathasan and Monack, 2007).
Although the function of the NLRP3 inflammasome in medi-
Innate immune responses to influenza and other pathogens ating responses to bacterial pathogens has been studied by
typically involve a highly conserved host-cell signaling mecha- several groups, the in vivo function of this important signaling
nism designed to protect the host and get rid of harmful complex during the course of viral infection is not well under-
microbes. Recognition of conserved microbial structures, known stood. In vitro transfection of DNA from viruses, bacteria, or
as pathogen-associated molecular patterns (PAMPs) within cells mammalian sources into macrophages activates the NLRP3 in-
of a myeloid origin is a hallmark feature of adequate host immu- flammasome, but this result would suggest a global response
nity. PAMPs are recognized by pattern-recognition receptors or to DNA rather than an antiviral response (Muruve et al., 2008).
sensors (PRRs), which subsequently initiate signaling cascades One report has shown that a synthetic analog of dsRNA, poly(I:C)
resulting in the production of proinflammatory cytokines and activates IL-1b through the NLRP3 pathway (Kanneganti et al.,
type-I interferons. Thus far, three families of PRRs, including 2006). However, another group failed to replicate this finding

556 Immunity 30, 556–565, April 17, 2009 ª2009 Elsevier Inc.
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NLRP3 Inflammasome Mediates Immunity to Influenza

Figure 1. Characterization of Influenza Virus A/PR/8/34 Pathogenicity and Immune Response in Mice Deficient in Inflammasome Signaling
Pathways
(A and B) The survival of Pycard / and Casp1 / mice was measured and compared to wild-type (A) and Myd88 / (B) mice (*p < 0.05; log rank). Mock-
inoculated (n = 7); influenza-infected wild-type (n = 19); Myd88 / (n = 11); Pycard / (n = 12); and Casp1 / (n = 7).
(C) Lungs were harvested 3 days after infection. Sections through the main bronchiole of the left lobe were stained with H&E.
(D) Histological scoring of H&E-stained lung sections as shown in (C) (*p < 0.05; **p < 0.01; mean ± SEM). Wild-type (n = 8); Myd88 / (n = 7); Pycard / (n = 6); and
wild-type mock-infected (n = 4). Results are representative of at least two independent experiments.

(Muruve et al., 2008). Thus, the role of NLRP3 and the inflamma- lysosomal enzymes such as cathepsin B, and reactive oxygen
some during viral infection remains unresolved. Equally impor- species (ROS). This report demonstrates that the NLRP3 inflam-
tant, the majority of NLR characterization has been based on masome is an essential component of the in vivo host immune
in vitro or ex vivo data generated from human cell lines and response to viral infection in a model system that is physiologi-
primary mouse cells. Although these studies have provided cally relevant to human disease.
a wealth of information, the underlying relevance of these
proteins in actual pathogenesis and host defense against patho-
gens in vivo has been much less defined. RESULTS
To assess the contribution of NLR inflammasomes in viral
pathogenesis, we assessed the host response to the influenza Loss of NLR Inflammasome Activity Alters Survival
A virus. Influenza infection results in a highly contagious respira- and Inflammation in Response to the Influenza Virus
tory illness leading to substantial morbidity and occasionally To assess the in vivo physiological contribution of NLR and TLR
death. Annual epidemics typically affect 5%–15% of the popula- signaling pathways in response to influenza virus infection, we
tion and are thought to result in 250,000–500,000 deaths annu- utilized gene-deletion mice in an influenza A/PR/8/34 virus infec-
ally. Of the three types of influenza viruses, influenza A viruses tion model. Previous studies utilizing mouse primary macro-
are the most virulent to humans and are capable of infecting phages have demonstrated that NLR components are necessary
multiple mammalian and avian species. Human influenza A for IL-1b release in response to influenza virus infection in culture
viruses can be further divided into different serotypeson the (Kanneganti et al., 2006). However, the in vivo physiological
basis of the antibody response to the viral surface glycoproteins, relevance of these findings has yet to be explored extensively.
hemagglutinin (HA) and neuraminidase (NA). The influenza A To assess the contribution of NLR inflammasomes in mouse
virus genome is composed of eight individual strands of ssRNA, survival and inflammation, we assessed mice deficient in either
which encode a total of 11 different proteins. the NLR adaptor protein ASC (Pycard / ) or caspase-1
In this report, we show that the NLRP3 inflammasome has (Casp1 / ). Mice lacking these inflammasome components
a profound influence on in vivo host immune response and showed significantly increased mortality after influenza infection
survival after airway infection with a mouse-adapted influenza (Figure 1A). As a comparison and as a control, we included the
A virus. Utilizing in vivo challenges with a synthetic analog of Myd88 / mice. Previous studies have demonstrated that
dsRNA, we demonstrated a possible mechanism underlying TLR3, 7, 8, and 9 represent the subset of TLRs that recognize
the NLRP3-associated innate immune response to viruses viral nucleic acids and mediate the induction of type-I IFN (Kawai
involved in the recognition of viral RNA. Furthermore, we and Akira, 2007; Koyama et al., 2007). TLR7, 8, and 9 are depen-
extended the relevancy of these findings to human cells by es- dent upon the TLR adaptor protein MyD88 for proper signal
tablishing that ssRNA, dsRNA, and influenza A virus-mediated transduction. However, mouse survival was only moderately
IL-1b release by human monocytes was dependent on the decreased for Myd88 / animals, suggesting that removal of
NLRP3 inflammasome and relied on intact lysosomal function, MyD88 and the disruption of the associated TLR7, 8, and 9

Immunity 30, 556–565, April 17, 2009 ª2009 Elsevier Inc. 557
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NLRP3 Inflammasome Mediates Immunity to Influenza

A B signaling cascades only modestly affect mouse survival

(Figure 1B).
To assess the extent of inflammation, we euthanized mice
3 days post-inoculation (dpi) and harvested the lungs for
histology (Figure 1C). We observed a marked increase in airway
inflammation, characterized by enhanced macrophage and
neutrophil influx into the airways, 3 dpi. in wild-type and
Myd88 / mice as compared to the mock-inoculated wild-
C type. In contrast, the infected Pycard / and Casp1 / (not
shown) mice demonstrated substantially reduced airway inflam-
mation. Histology scoring, by a blinded reviewer, confirmed the
significant attenuation of airway inflammation in the Pycard /
D E mice (Figure 1D). Together, these data demonstrate that both
Pycard and Casp1, but not Myd88, are required for mouse
survival and mediate airway inflammation in response to influ-
enza virus infection.

The Nlrp3 Inflammasome Is Required for Mouse Survival

and Inflammation after Influenza Infection
The above data suggest that proper activation of an NLR inflam-
F G masome is necessary for host survival and for mediating the
innate immune response after influenza virus infection. It is well
established that the NLR protein, NLRP3, can form an inflamma-
some complex with PYCARD and caspase-1 in response to
a plethora of stimuli (Eisenbarth et al., 2008; Li et al., 2008;
Martinon et al., 2006; Sutterwala et al., 2006). In addition to
NLRP3, other NLRs such as NLRC4 have been suggested to
form an NLRP3-independent inflammasome that functions in
IL-1b maturation in response to Salmonella typhimurium and
Pseudomonas aeruginosa infection (Mariathasan et al., 2004;
Miao et al., 2008). After influenza inoculation, all animals demon-
Figure 2. The Nlrp3 Inflammasome Is Required for Survival and strated a decrease in body weight (Figure S1 in the Supplemental
Mediates Airway Inflammation and Viral Clearance after Pulmonary Data). However, as seen in Figure 2A, a dramatic decrease in
Challenge with Influenza Virus survival was observed in Nlrp3 / mice after virus infection,
(A) The survival of Nlrp3 / and Nlrc4 / mice was measured and compared to and no differences in mortality were observed between Nlrc4 /
that of the wild-type (**p < 0.01; log rank). Wild-type mock-infected (n = 7);
and wild-type mice. Interestingly, the removal of Nlrp3 produced
wild-type infected (n = 19); Nlrp3 / infected (n = 16); and Nlrc4 / infected
(n = 7). All survival experiments were performed together with those in Figure 1, a more dramatic effect on animal survival than the removal of
therefore, the wild-type controls are identical to those shown in Figure 1. Pycard or caspase-1, suggesting the possibility that Nlrp3 might
(B) Histological scoring of H&E-stained lung sections as shown in (C) (*p < 0.05; exert biologic effects in addition to caspase-1 activation;
**p < 0.01; mean ± SEM). however, this remains to be explored.
(C) Lungs were harvested 3 dpi. Sections through the main bronchiole of the As observed with the Pycard / animals, Nlrp3 / mice show
left lobe were stained with H&E.
a significant decrease in airway inflammation after influenza
(D) Bronchoalveolar lavage (BAL) was performed, and the total number of cells
present in the bronchoalveolar lavage fluid (BALF) was calculated (*p < 0.05;
infection. Histology scoring revealed no increase in inflammation
**p < 0.01; mean ± SEM). in mock-infected animals, and only a modest increase was
(E) BALF cellular composition was determined via differential staining, and observed in the Nlrp3 / mice, whereas a significant increase
subpopulations were assessed via FACS. Wild-type influenza-infected (n = 8); in airway inflammation was observed in the Nlrc4 / and wild-
Nlrp3 / (n = 5); Nlrc4 / (n = 3); Pycard / (n = 6); wild-type mock-infected type animals (Figure 2B). Histopathology assessments revealed
(n = 4). Results are representative of three independent experiments.
a mild increase in airway inflammation in virus-infected Nlrp3 /
(F) The number of monocytes, neutrophils, and lymphocytes was determined
mice compared to mock-infected mice, but they revealed an
via differential staining of BALF cells (*p < 0.05; mean ± SEM). Wild-type influ-
enza-infected (n = 8); Nlrp3 / (n = 4); Nlrc4 / (n = 4); wild-type mock-infected exacerbated increase in Nlrc4 / and wild-type animals 3 dpi
(n = 5). (Figure 2C). To further characterize the composition of the cell
(G) Influenza viral titers were determined by standard plaque assay either 3 or populations infiltrating the airways during viral infection, we har-
7 dpi (*p < 0.05; mean ± SEM). Wild-type mock-infected, day 3 (n = 3); vested bronchoalveolar lavage fluid (BALF) and assessed cellu-
wild-type influenza-infected, day 3 (n = 9); Nlrp3 / influenza-infected, day 3 larity. Consistent with the histology findings, Nlrp3 / animals
(n = 9); wild-type mock-infected day 7 (n = 4); wild-type influenza-infected,
demonstrated a significant decrease in total BALF cellularity
day 7 (n = 6); and Nlrp3 / influenza-infected, day 7 (n = 7).
when they were compared to either the Nlrc4 / or wild-type
animals (Figure 2D). Analysis of BALF cellularity indicates that
macrophages and neutrophils represent the bulk of infiltrating
cells in response to influenza. Differential profiling revealed no

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NLRP3 Inflammasome Mediates Immunity to Influenza

A B difference was observed between wild-type and Nlrp3 / mice

(Figure 2G). However, by 7 dpi. the Nlrp3 / mice demonstrated
approximately 2-fold higher viral titer in the lungs than did the
wild-type animals (Figure 2G), providing evidence for a defect
in viral clearance in the absence of Nlrp3.

Nlrp3 Inflammasome Components Are Expressed

in the Lung and Myeloid Cells during Influenza Infection
To assess the relevance of Nlrp3 and Pycard in a panel of mouse
C D cells, we assayed expression by quantitative RT-PCR in primary
myeloid, lymphoid, and airway epithelial cells (Thompson et al.,
2006; Willingham et al., 2007). Under naive conditions, both
Nlrp3 and Pycard genes were highly expressed in primary
macrophages, and nominal expression was detected in T and
B lymphocytes (Figures 3A and 3B). In the mouse airway epithe-
lial cells (MAEC), only a low level of Pycard was detected, and
Nlrp3 was not found (Figures 3A and 3B).
E To characterize the effects of viral infection on gene transcrip-
tion in these differentiated cell types, we challenged bone-
marrow-derived macrophages (BMMs) and MAECs with
influenza A/PR/8/34. No significant change in either Nlrp3 or
Nlrc4 transcription was observed after viral challenge in the
BMMs; however, a significant increase in IL-1b mRNA expres-
sion was detected (Figure 3C). In contrast, under naive condi-
tions, Nlrp3 expression was low in the MAECs. After influenza
infection, an increase in Nlrp3 transcription was observed,
whereas no significant changes were found in Nlrc4 and Il-1b
transcription (Figure 3D).
It should be noted that ex vivo cultured mouse BMMs were
refractory to influenza virus infection, as shown by the precipi-
tous drop in viral titer after exposure (Figure S2A). As a result
of the lack of a productive infection, we often observed inconsis-
Figure 3. Nlrp3 Inflammasome Components Are Highly Expressed tent IL-1b cytokine expression at the MOIs used in this study. For
in Myeloid Cells and in the Lungs during Influenza A Virus Infection this reason, we did not pursue additional experiments with
(A and B) Pycard (A) and Nlrp3 (B) gene expression was assessed in primary primary mouse macrophages. Even though IL-1b expression
mouse bone-marrow-derived macrophages (Macs), B cells, T cells, mouse
was inconsistent, at MOIs greater than 10 we were able to
airway epithelial cells (MAECs), and mouse embryonic fibroblasts (MEFs) by
RT-PCR; samples were normalized to 18 s and standardized to expression
observe an increase in IFN-b at 12 hr, and we observed no differ-
in MEFs. Each cell line was assessed in triplicate, and data are representative ences between the genotypes (Figure S2B), indicating that the
of three independent experiments. genes studied did not affect IFN-b production.
(C and D) IL-1b, Nlrp3, and Nlrc4 transcripts were assessed by RT-PCR, To further assess the contribution of the NLRP3 inflamma-
normalized to 18 s, and standardized to naive levels after virus infection in some in vivo, we sought to assess changes in mRNA expression
primary mouse bone-marrow-derived macrophages (BMMs) (C) and MAECs
of inflammasome components and IL-1b over a time course of
(D) (*p < 0.05; mean ± SEM).
influenza virus infection. Total lung RNA was extracted from
(E) Changes in mRNA expression of inflammasome components and IL-1b
were assessed in the lungs over the course of influenza virus infection tissue homogenates, and gene expression was assessed for
(*p < 0.05; mean ± SEM). All experiments are representative of at least two Nlrp3, Pycard, Il-1b, and caspase-1 (Figure 3E). A significant
independent experiments with 3–5 mice per group. increase in gene transcription was observed 3 dpi for all four as-
sessed mRNAs. This was followed by a significant reduction of
significant differences in the percent cellular composition of the all mRNAs except caspase-1 by day 7 (Figure 3E). These results
BALF between genotypes (Figure 2E); however, the Nlrp3 / indicate that influenza induced a substantial increase in all
mice demonstrated an overall decrease in the number of mono- components of the Nlrp3 inflammasome in the lungs and infil-
cytes and granulocytes (Figure 2F). Together, this indicates that trating cells of inoculated animals 3 dpi and also indicate that
the Nlrp3 inflammasome is an essential mediator of in vivo viral infection profoundly affects the inflammasome genes in
inflammatory responses to influenza virus. a way that alters their biologic function.
We have observed that Nlrp3 / animals show reduced To determine the in vivo contribution of the macrophages in
immune responses after influenza challenge. To determine the lungs of mice after virus infection, we harvested lungs 3 dpi
whether this reduction resulted in a defect in viral clearance, and visualized cells containing viral antigen by IHC and confocal
we harvested lungs from wild-type and Nlrp3 / mice 3 and microscopy. The primary cells targeted in vivo in the mouse are
7 dpi. During the early stages of infection (3 dpi), all influenza- the airway epithelial cells (Figures S3A–S3C), and 15% of macro-
challenged mice demonstrated an increase in viral titer, and no phages are the second largest population (Figures S3D–S3F).

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NLRP3 Inflammasome Mediates Immunity to Influenza

A B survival after influenza virus infection. We next sought to assess

changes in serum and BALF cytokines known to play roles in
host immune responses to viruses. A significant increase in
IL-1b was detected in the serum and BALF from wild-type
animals. This increase was dependent on Nlrp3 and Pycard
but independent of Nlrc4 (Figures 4A and 4B). To demonstrate
C D that this lack of IL-1b was due to reduced post-translational
processing by the Nlrp3 inflammasome, we assessed lung
transcription of Il1b and observed significant increases in IL-1b
transcription in all mouse lines regardless of genotype
(Figure S4). As previously mentioned, IL-18 has also been shown
to be a critical mediator of influenza infection, and its maturation
E F also relies on NLR inflammasome activation. A significant
increase in serum IL-18 was detected in the wild-type mice
(Figure 4C). However, as with IL-1b, IL-18 was significantly
reduced to mock-infected amounts in mice lacking either Nlrp3
or Pycard (Figure 4C). KC, MIP2a, TNFa, IL-6, IFNg, and IL-12,
like IL-1b, facilitate a wide spectrum of immunologic functions
G H during respiratory virus infection. To evaluate these cytokine
concentrations in the lungs, we assessed the BALF supernatant
by ELISA. A significant decrease in MIP2a was also observed in
the Nlrp3 / and Pycard / animals when they were compared
to wild-type animals (Figure 4D), and these results correlated
with the decreased cellularity observed in the BALF
I J
(Figure 2D). Nlrp3 / mice also showed a modest decrease in
KC (Figure 4E). Pycard / mice demonstrated a statistically
significant decrease in TNFa, and such a decrease was not
observed in the Nlrp3 / animals (Figure 4F). The observed
attenuation in TNFa in the Pycard / animals was observed in
other assays and may reflect a previously-reported inflamma-
some-independent role for Pycard in the control of TNFa
(Taxman et al., 2006). No significant differences were observed
Figure 4. Mice Lacking Components of the Nlrp3 Inflammasome in IL-6, IFNg, or IL-12p40 after influenza infection among the
Demonstrate Significantly Altered Levels of Proinflammatory
different mouse strains (Figures 4G–4I). Because of the
Cytokines after Pulmonary Challenge with Influenza
(A and B) IL-1b was assessed in serum (A) and BALF (B) from wild-type mock- increased mortality observed in the Nlrp3 / mice, a subset of
infected, wild-type influenza-infected, Nlrp3 / , Nlrc4 / , and Pycard / moribund animals was harvested 7 dpi. BALF IL-6 concentra-
mice, 3 days after infection. tions were significantly elevated in the Nlrp3 / mice compared
(C–I) Serum levels of IL-18 were also determined in these animals (C). In addi- to the wild-type animals (Figure 4J). Although unexpected, this
tion to IL-1b and Il-18, MIP2a (D), KC (E), TNFa (F), IL-6 (G), IFNg (H), and IL- observation is consistent with clinical findings in humans, where
12p40 (I) were also assessed (*p < 0.05; mean ± SEM). Mock, n = 5; Nlrp3 / ,
influenza-infected individuals show increased IL-6 associated
n = 6; Nlrc4 / , n = 6; Pycard / , n = 9; wild-type, n = 5.
(J) Serum levels of IL-6 were determined 7 days after infection in wild-type
with increased morbidity (Lee et al., 2007).
mock-infected, wild-type influenza-infected, and moribund Nlrp3 / mice
(*p < 0.05; mean ± SEM). Mock, n = 3; Nlrp3 / , n = 6; wild-type, n = 3. All NLRP3 Inflammasome Activation in Response
data are representative of at least two independent experiments. to Influenza Is Dependent upon Lysosome Maturation
and ROS
However, a majority of macrophages and other leukocytes For clinical relevance, the above study of mice required verifica-
present in the lungs of infected mice were IHC negative (Figures tion in human cells. Airway epithelial cells are the predominant
S3G–S3I). Substantially smaller populations of polymorphonu- cell type infected by influenza, and previous studies have
clear cells and alveolar epithelial cells (<1%) were also infected demonstrated that these cells contribute to innate immune
with virus (not shown). Together, these data suggest that airway responses (Kato and Schleimer, 2007). Primary human airway
epithelial cells and macrophages are the likely cell types respon- epithelial cell cultures (HAE) were infected with the human
sible for mediating the innate immune response during the initial influenza virus A/Victoria /3/75 (H3N2), and the supernatant
stages of influenza virus infection. was harvested from either the apical or basolateral layer as previ-
ously described (Thompson et al., 2006). After infection, we
NLRP3 Inflammasome Components Are Required observed robust viral replication in the HAE cultures (data not
for Influenza-Induced Proinflammatory Mediator shown) and detected increased IL-1b concentrations in superna-
Production tant from the apical (Figure 5A) but not the basolateral layer. To
The above experiments indicate Nlrp3 / and Pycard / mice expand upon these findings, we infected a human nasal airway
demonstrate attenuated airway inflammation and reduced epithelial cell line, JME, and observed increased NLRP3 and

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NLRP3 Inflammasome Mediates Immunity to Influenza

A B C PYCARD expression (Figure 5B) and increased IL-1b release

(Figure 5C). In addition to the JME cell lines, A/Victoria also
induced a dramatic increase in IL-1b in a human monocytic
cell line (THP-1) (Figure 5D). These data indicate that human cells
of both monocytic and epithelial origin are contributors of IL-1b
during influenza virus infection.
To verify that influenza-virus-mediated IL-1b production in
D E THP-1 is dependent upon NLRP3 inflammasome components,
we utilized small heteroduplex RNA (shRNA) knockdown to
assess the contribution of NLRP3 and PYCARD. An shRNA
against the third putative inflammasome component, TUCAN
or CARDINAL (CARD8), was also targeted by shRNA. Successful
RNA reduction is shown in Figures S5A–S5C. These cells were
challenged with A/Victoria/3/75, and cell free supernatants
F G were collected over a 24 hr time-course. No differences in viral
titer were detected between the different knockdown and wild-
type cell lines 24 hr after infection (Figure S5D). When cytokine
was measured, a significant increase in IL-1b was observed in
the wild-type THP-1 cells and cells transfected with a scrambled
shRNA targeting sequence (shmut) 24 hr after infection, and this
increase was significantly attenuated in cells containing shRNA
H I targeting PYCARD and NLRP3, but not in those containing
shRNA targeting CARD8 (Figure 5E; Figure S5E). The role of
CARD8, a gene found in humans but not mice, has not been
previously studied by gene reduction. These data suggest that
the CARD8 protein may not be important for NLRP3 inflamma-
some function induced by influenza virus. To confirm the speci-
ficity of shNLRP3 in IL-1b production, we also assessed TNFa
J release after influenza infection. As seen in Figure S5F, amounts
of TNFa were not reduced in cell lines with shNLRP3 but were
modestly reduced in cells with shASC, consistent with the
mouse data described earlier. Finally, we demonstrate that
ZVAD-CHO, a caspase inhibitor, and YVAD-CHO, a specific
peptide inhibitor of caspase-1, both attenuated IL-1b amounts
in a dose-dependent fashion after influenza virus infection
Figure 5. NLRP3 Inflammasome Activation in Response to the Influ- (Figure 5F).
enza Virus Is Dependent upon Lysosomal Maturation and ROS
To further explore the mechanism underlying IL-1b release in
Production in Human Cells
(A) IL-1b levels were determined in basolateral and apical washes from human response to influenza infection, we used pharmacological antag-
airway epithelial cells challenged with influenza A/Victoria/3/75 (Victoria) 48 hr onist to block specific signaling pathways. Lysosomal degrada-
after infection (*p < 0.05; mean ± SEM). tion of particulate danger-associated molecular patterns
(B) Human nasal airway epithelial cell lines (JME) were infected with A/Victoria/ (DAMPs) has been shown to activate the NLRP3 inflammasome
3/75, and changes in NLRP3 and PYCARD mRNA expression were observed. (Hornung et al., 2008), but this has not been assessed in the
Data were normalized to 18 s and compared to the expression in similarly
context of viral infection. To test this, we utilized bafilomycin A
treated human type II alveolar epithelial-like cell lines (A549).
(C) IL-1b was measured in the supernatants from A/Victoria/3/75-infected JME
to block lysosomal acidification via inhibition of the vacuolar H+
cells 24 hr after infection. ATPase system. The addition of bafilomycin A at concentrations
(D) Human THP-1 monocyte cell lines were infected with A/Victoria/3/75, and used previously to inhibit DAMP signaling (Hornung et al.,
IL-1b was measured in the supernatant over a 24 hr time course. 2008) completely abolished influenza-induced IL-1b release
(E) Human THP-1 cells were infected with lentivirus containing shRNA for (Figure 5G), which suggests a critical role for lysosomes in
PYCARD (shASC), NLRP3 (shNLRP3) or CARD8 (shTUCAN) or for a mutated
virus-mediated NLRP3 inflammasome function. A specific lyso-
sh target sequence (shmut). IL-1b was assessed in cell-free supernatants
24 hr after infection (*p < 0.05; mean ± SEM).
somal cysteine proteinase, cathepsin B, has been associated
(F) The effect of ZVAD-CHO and Ac-YVAD-CHO on IL-1b release was deter- with NLRP3-mediated cell death and IL-1b in response to
mined in THP-1 cells after influenza infection (*p < 0.05; mean ± SEM). nonviral signals (Willingham et al., 2007; Hornung et al., 2008).
(G–I) IL-1b release by THP-1 cells after influenza infection was also assessed Utilizing the cathepsin B-specific inhibitor, Ca-074-Me, we
after treatments with bafilomycin A (G), CA-074-Me (50 mM) and Cathepsin observed a significant attenuation in IL-1b release after influenza
K Inhibitor I (Cat K) (10 mM) (H), and (2R, 4R)-4-aminopyrrolidine-2-4-dicarbox- challenge (Figure 5H). This suggests the importance of cathepsin
ylate (APDC) (100 mM) and N-acetyl-L-cysteine (NAc)(50mM)(*p < 0.05; mean ±
B in NLRP3-mediated response to influenza virus but also
SEM) (I). All data are representative of at least two independent experiments.
(J) The in vivo effect of NAc (250 mg/kg) on BALF IL-1b after influenza infection suggests the involvement of additional lysosomal processes.
was determined. (*p < 0.05; mean ± SEM). NAc mock, n = 1; NAc influenza, This decrease was not observed with specific inhibitors for
n = 5, vehicle Influenza, n = 3. cathepsin K (Figure 5H). The release of lysosomal products

Immunity 30, 556–565, April 17, 2009 ª2009 Elsevier Inc. 561
 Immunity
NLRP3 Inflammasome Mediates Immunity to Influenza

A B Figure 6. The NLRP3 Inflammasome Is Required for

Airway Inflammation Induced by Nucleic Acid Analogs
(A) Lungs were harvested 24 hr after poly(I:C) challenge.
Sections through the main bronchiole of the left lobe were
stained with H&E.
(B) Histological scoring of H&E-stained lung sections as shown
in (A) (*p < 0.05; **p < 0.01; mean ± SEM).
(C) BALF IL-1b was determined after poly(I:C) challenge in
wild-type, Nlrp3 / , and mock-challenged wild-type mice
C D (*p < 0.05; mean ± SEM).
(D) The effect of PYCARD (shASC) and NLRP3 (shNLRP3)
shRNA knockdown on IL-1b release in response to poly(I:C)
in THP-1 cells was assessed (*p < 0.05; mean ± SEM).
(E) The effect of Ac-YVAD-CHO on IL-1b release was deter-
mined in THP-1 cells after poly(I:C) challenge (*p < 0.05;
mean ± SEM).
(F) IL-1b secretion stimulated by ssRNA40 was assessed after
E F the shRNA knockdown of PYCARD (shASC) and NLRP3
(shNLRP3) (*p < 0.05; mean ± SEM).
(G–I) IL-1b release following poly(I:C) stimulation in THP-1 cells
was assessed after treatments with bafilomycin A (100 nM) (G),
CA-074-Me (50 mM) (H), and (2R, 4R)-4-aminopyrrolidine-2-4-
dicarboxylate (APDC) (100 mM) and N-acetyl-L-cysteine (NAc)
(50 mM) (*p < 0.05; mean ± SEM) (I).

G H
inhibited the generation of IL-1b in the BALF after
influenza infection (Figure 5J). In addition to NAc,
bafilomycin A and CA-074-ME were also assessed;
however, the pharmacokinetics for each of these
compounds were not optimized for our in vivo
assessments (data not shown). Collectively, these
data suggest that NLRP3 inflammasome activation
in response to the virus occurs when the cell senses
I alterations of lysosomal content and ROS induction
in the cytosol after virus infection by using path-
ways similar to those activated by DAMPs.

Nlrp3 Mediates Airway Inflammation

and IL-1b Induction by the Viral RNA
Analogs poly(I:C) and ssRNA40
The data presented thus far demonstrate that Nlrp3
mediates in vivo airway inflammation in response to
influenza virus infection in mice and inflammasome
into the cytosol has been demonstrated to promote the genera- formation in both mice and humans. To further explore the mech-
tion of ROS, and recent data have suggested that ROS genera- anism underlying NLRP3 activation, we examine the in vivo
tion by asbestos, monosodium urate and ATP is a necessary immune response to the viral RNA analog, poly(I:C). To assess
step in inflammasome activation (Dostert et al., 2008). To test whether poly(I:C) is important for pulmonary inflammasome acti-
whether this occurs during influenza virus infection, we treated vation, mice were challenged intranasally with poly(I:C), and the
THP-1 cells with the ROS inhibitors N-acetyl-L-cysteine (NAc) resultant airway inflammation was characterized. Poly(I:C)
or (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC) (Dos- induced moderate airway inflammation in the wild-type mice,
tert et al., 2008). Our data demonstrate attenuated IL-1b produc- and inflammation was significantly reduced in the Nlrp3 /
tion from cells treated with either ROS inhibitor (Figure 5I), animals (Figure 6A). Nlrp3 / mice exposed to poly(I:C) showed
suggesting that ROS also contributes to NLRP3 inflammasome a significant reduction in the overall amount of airway inflamma-
activation in response to influenza. tion (Figure 6B) and BALF IL-1b concentration (Figure 6C).
Influenza infection in vivo results in a substantial increase in To verify this result in human cells, we utilized the shRNA
ROS production in the airways of humans and mice. To assess knockdown cell lines described above and demonstrate that
the physiological relevance of the in vitro ROS inhibitor findings, poly(I:C)-induced IL-1b is dependent on PYCARD and NLRP3
we treated mice with the ROS inhibitor NAc via intranasal admin- (Figure 6D). To confirm the role of caspase-1 in this mechanism,
istration as previously described (Springer et al., 2007). Similar to we exposed cells to the caspase-1 inhibitor yVAD-CHO, which
the monocyte cell line findings, treatment with NAc completely significantly attenuated poly(I:C)-mediated IL-1b release

562 Immunity 30, 556–565, April 17, 2009 ª2009 Elsevier Inc.
Immunity
NLRP3 Inflammasome Mediates Immunity to Influenza

(Figure 6E). To determine whether the resultant NLRP3- and 2008). However, the in vivo importance of NLRX1 remains to
PYCARD-dependent IL-1b release was unique to poly(I:C), we be determined. The involvement of NLRP3 in the macrophage
also stimulated cells with single-stranded GU-rich RNA response to RNA viruses has been controversial. One study
(ssRNA40) complexed with the cationic lipid LyoVec to facilitate has demonstrated the importance of Nlrp3 during viral infection
uptake. A significant NLRP3- and PYCARD-dependent increase in culture (Kanneganti et al., 2006), whereas another group was
in IL-1b release was observed after ssRNA40 stimulation, indi- unable to confirm inflammasome activation after poly(I:C) chal-
cating that NLRP3 and PYCARD mediate responses to ssRNA lenge or infection with the RNA viruses reovirus and vesicular
molecules (Figure 6F). To explore the intracellular pathways stomatitis virus in culture. Although we did not assess reovirus
that lead to NLRP3 activation by RNA analogs, we utilized phar- or VSV, our human monocytic cell line and in vivo mouse data
macological inhibitors as described earlier and show that inflam- clearly show that NLRP3 and PYCARD are indeed required for
masome activation in response to poly(I:C) requires lysosomal IL-1b maturation in response to influenza virus infection as well
maturation, cathepsin B, and the generation of ROS (Figures as to dsRNA analogs and ssRNA. Therefore, our data support
6G–6I). Together, these data suggest that NLRP3 inflammasome the Kanneganti et al. study. However, we had difficulty studying
activation in response to virus is mediated by the recognition of influenza viral infection of mouse macrophages ex vivo because
viral RNA and involves pathways associated with lysosomal of a failure to establish a productive infection. Thus, we have not
maturation and the induction of ROS. precisely duplicated the ex vivo data observed by the first report.
A possible explanation that would reconcile the apparent
DISCUSSION discrepancies in the field is that different NLRs might mediate
responses to different RNA viruses. The precedence for this is
In this report, we assess the in vivo contribution of NLRP3 to host provided by MDA5 and RIG-I, which recognize different types
immune responses to viral infection. NLRP3 forms a multiprotein of dsRNA and show viral specificity. RIG-I responds to RNA
inflammasome complex, which activates caspase-1 and leads viruses, including paramyxoviruses and influenza virus, whereas
to the maturation of several key proinflammatory cytokines, MDA5 is essential for picornavirus recognition (Kato et al., 2008;
such as IL-1b and IL-18. It has been established that NLRP3 Kato et al., 2006). Thus, it is possible that NLRP3 is responsible
responds to both microbe-associated molecules and damage- for dsRNA, ssRNA, DNA, Sendai virus, and influenza virus recog-
associated molecular patterns (DAMPS) such as uric acid, nition and that an unidentified NLR is activated by reovirus and
alum salt, and silica (Dostert et al., 2008; Eisenbarth et al., VSV.
2008; Hornung et al., 2008; Li et al., 2008; Martinon et al., Infection of mice with influenza virus results in a dramatic
2006). In all of these cases, it is clear that NLRP3 promotes increase in morbidity and mortality and mimics many of the path-
inflammation. A key question is whether NLRP3 plays a beneficial ophysiological aspects of the human disease. The current study
or pathogenic role. In this study, we clearly demonstrate that shows that Pycard and Nlrp3 deficiencies reduce inflammation
NLRP3 and other components of the NLRP3 inflammasome in the lung, and this reduced inflammation is correlated with
play a beneficial role in the in vivo innate immune response to increased mortality and a viral clearance defect. Thus, the
influenza infection and are required for normal host immune NLRP3 inflammasome-driven antiviral response is beneficial to
responses to the virus. An examination of the mechanism by the host after influenza infection. Although the precise role for
which NLRP3 mediates host response to an RNA virus demon- both IL-1b and IL-18 in the immune response against viral path-
strates that recognition of RNA analogs or RNA molecules is ogens remains elusive, in vivo assessments of IL-1R1- and
capable of mediating these responses. A further exploration of IL-18-deficient mice have shown that these animals have
the mechanism shows that similar to the activation of NLRP3 reduced acute airway inflammation associated with influenza
by DAMPs such as particulate alum, MSU crystals and silica, virus infection and that both have significantly decreased
activation of the NLRP3 pathway by the influenza virus, and survival (Schmitz et al., 2005). These results are in agreement
more specifically by RNA analogs, is dependent on an intact with our findings. Our data also highlight the observation that
lysosomal pathway and functional lysosomal enzymes such as host response to influenza virus is specific and requires NLRP3
cathepsin B and is also reliant on ROS both in vitro and in vivo. but not NLRC4. It should be noted that a recent study identified
This supports the hypothesis that NLRP3 is activated through a role for ASC and caspase-1, but not Nlrp3, in the initiation of
common intracellular changes caused by either PAMPs or adaptive immunity against influenza virus (Ichinohe et al.,
DAMPs, rather than a model that evokes the direct interaction 2009). Similar to the findings presented here, Ichinohe et al.
of NLRP3 with ligands of diverse molecular structures. demonstrate a role for both ASC and caspase-1 in mouse
The recognition of viral pathogens by cells of the innate survival and in vivo host immune responses to influenza virus
immune system is essential for the initiation of an ensuing inflam- infection. Also similar to our findings, their data suggest that
matory response. The endosomal TLRs TLR3, TLR7, and TLR8 Nlrp3 is responsible for recognition of influenza virus within the
and cytoplasmic RIG-I and MDA-5 are receptors of viral PAMPs alveolar macrophages and dendritic cells in a type I IFN-inde-
and the primary route for the elicitation of host immune pendent manner in culture. Additionally, we have not extensively
responses to viruses through the regulation of type-I interferons. assessed the establishment of CTL and IgA responses in our
Among the NLR family, there are few reports linking these report; however, our work shows that NLRP3 did not affect
proteins to the viral host response or viral pathogenesis. One IFN-g production during viral infection and agrees with their
report identifies endogenous NLRX1 as an inhibitor of the RIG-I observation that this gene does not affect adaptive immunity to
and MDA-5 pathway through interactions with the essential influenza virus. However, Ichinohe et al. fail to establish a role
mitochondrial antiviral signaling adaptor (MAVS) (Moore et al., for Nlrp3 in mouse survival and leukocyte recruitment to the

Immunity 30, 556–565, April 17, 2009 ª2009 Elsevier Inc. 563
 Immunity
NLRP3 Inflammasome Mediates Immunity to Influenza

lungs, which represents a point of contention between this previ- with the ROS inhibitor NAc (250 mg/kg) via intranasal administration (as
ously published work and the data we present here. One obvious previously described by Springer et al., 2007) throughout the course of virus
challenge.
difference between these publications is the subtle differences in
Influenza-inoculated mice were euthanized either 3 or 7 dpi, and serum was
the virus preparation, which may explain some of the opposing collected after cardiac puncture. Total cytokine levels were determined by
phenotypes. ELISA (R&D Biosystems or BD Biosciences). In some cases, bronchoalveolar
Although our in vivo study shows that influenza virus is found lavage (BAL) was performed, and the number of cells present in the BAL fluid
in both airway epithelial cells and macrophages in the mouse, (BALF) was determined with a hemacytometer. A morphology-based differen-
only mouse macrophages express detectable Nlrp3 transcript, tial cell count was conducted on cytospin preparations from the BALF and
and they are thus the likely cell type mediating the beneficial stained with Diff-Quik solution (Sigma). BALF cellularity was also determined
via FACS with antibodies reactive to CD11b, CD11c, GR-1, B220, CD4, and
functions of host protection by Nlrp3. However, in humans we
CD8 according to standard techniques. Centrifuging the remaining BALF
find NLRP3 expression in both airway epithelial cells and macro- removed cells, and cytokine levels in the supernatant were determined.
phages and provide further evidence that NLRP3 and PYCARD For histopathologic examination, lungs were fixed by inflation and immer-
mediate inflammatory cytokine release in cells of human origin. sion in 10% buffered formalin. To evaluate airway inflammation, we subjected
Thus, NLRP3 and PYCARD may play an even more profound fixed lung slices to hematoxylin and eosin (H&E) staining. Evaluators who were
role in host inflammation and protection upon influenza infection blinded to genotype and treatment scored lung sections (0 [none]–3 [extreme])
on the basis mononuclear and polymorphonuclear cell infiltration, perivascular
of humans. Considering the disastrous nature of a possible influ-
and peribronchiolar cuffing, and estimates of the percent of lung involved with
enza pandemic, our study suggests that targeting the NLRP3 the inflammation.
inflammasome for enhanced function could become an impor- For assessment of viral titer, whole lungs were removed, weighed, and
tant therapeutic measure against such an infection. homogenized in PBS. Lung viral titers were determined by standard plaque
assay in the supernatants.
EXPERIMENTAL PROCEDURES
Statistical Analysis
Experimental Animals Data are presented as the mean ± standard error of the mean (SEM). Analysis
All studies were conducted in accordance with the National Institutes of Health of variance (ANOVA) followed by Tukey-Kramer HSD for multiple comparisons
Guide for the Care and Use of Laboratory Animals and the Institutional Animal was performed on complex data sets. Statistical significance for single data
Care and Use Committee guidelines of University of North Carolina, Chapel points was assessed by the Student’s two-tailed t test. Survival curves were
Hill. The generation of mice lacking functional Nlrp3 (Cryopyrin), Nlrc4 generated via the product-limit method of Kaplan and Meier, and comparisons
(ICE-Protease Activating Factor), Apoptotic Speck protein containing a Card were made via the log rank test. In all cases, a p value of less than 0.05 was
(Pycard), Casp1, and Myd88 has been previously described (Adachi et al., considered statistically significant.
1998; Mariathasan et al., 2004; Sutterwala et al., 2006). The Nlrp3 / mice
were originally produced at Millenium Pharmceuticals, were kindly provided
by Dr. Richard Flavell as N5, and were backcrossed for another four genera- SUPPLEMENTAL DATA
tions at UNC-CH, resulting in mice that were backcrossed for a total of nine
generations. Supplemental Data include Supplemental Experimental Procedures and five
figures and can be found with this article online at http://www.immunity.
Virus Propagation com/supplemental/S1074-7613(09)00139-3.
Influenza virus A/PR/8/34 (H1N1) was propagated in the allantoic cavity of
10-day-old embryonated specific-pathogen-free chicken eggs and was ACKNOWLEDGMENTS
mouse adapted by a minimum of six serial passages through mice as previ-
ously described (Cottey et al., 2001). The influenza virus A/Victoria/3/75 The authors thank the Carolina Vaccine Institute for supplying aliquots of the
(H3N2) is a recombinant virus generated from cloned cDNA in 293T cells mouse-adapted influenza A/PR/8/34 virus. We acknowledge Wendy Barclay
and propagated in MDCK cells. Viral titers were determined by standard for critical review and advice on viral propagation and Rob Tarran for providing
plaque assay on confluent monolayers of MDCK cells. the JME cell line. We thank Richard Flavell (Yale University), Vishva M. Dixit
(Genentech, Inc), Fayyaz Sutterwala (the University of Iowa), and Millenium
Influenza Virus Infection of Human Primary Cells and Cell Lines Pharmaceuticals for supplying the Nlrp3 / , Pycard / , Nlrc4 / and
Cells were challenged with A/Victoria (MOI = 1) for 2 hr at 37 C. After incuba- Casp1 / mice. We also thank Willie June Brickey and Sushmita Jha for main-
tion, supernatant was replaced with fresh media, and cells were incubated at taining mouse colonies and providing histology scoring assistance. This work
37 C. Cell-free supernatants were harvested at select time points for viral titer is supported by 3-U54-AI057157-06S1, AI067798, 05-0064, and 1U19-
and cytokine analysis. To assess viral PAMPs, we incubated cells for 24 hr AI077437-01 (J.P.Y.T.). I.C.A. is supported by a National Institutes of Health
in the presence of naked poly(I:C) (10 mg)(Sigma) or ssRNA40 complexed training grant. M.A.S. is a recipient of the George H. Hitchings Fund for Health
with LyoVec (InvivoGen) (10 mg). For pharmacological assessments, cells Research and Science Education of the Triangle Community Foundation.
were treated with bafilomycin D (100–250 nM), Ac-ZVAD-CHO (10–100 mM),
Ac-ZVAD-CHO (10–100 mM), Ca-074-Me (10–50 mM), Cathepsin K Inhibitor I
Received: July 3, 2008
(10–50 mM), N-acetyl-L-cysteine (NAC) (50–100 mM), or (2R, 4R)-4-aminopyr-
Revised: October 17, 2008
rolidine-2-4-dicarboxylate (APDC) (50–100 mM) as previously described (Dos-
Accepted: February 10, 2009
tert et al., 2008; Hornung et al., 2008; Willingham et al., 2007).
Published online: April 9, 2009

In Vivo Influenza A Virus Infection and poly(I:C) Challenge

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