THE NLR ADAPTOR ASC/PYCARD REGULATES DUSP10, MAP kinase (MAPK) AND

CHEMOKINE INDUCTION INDEPENDENT OF THE INFLAMMASOME*

Debra J. Taxman1, Elizabeth A. Holley-Guthrie

1, Max Tze-Han Huang

1, Chris B. Moore

1,3, Daniel

T. Bergstralh1,4

, Irving C. Allen1, Yu Lei

2, Denis Gris

1 and Jenny Pan-Yun Ting

1

From 1Department of Microbiology and Immunology and 2NC Oral Health Institute; Lineberger

Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599 3Current Address: GlaxoSmithKline, Virology, Research Triangle Park, North Carolina, 27709

4Current Address: The Wellcome Trust / Cancer Research UK Gurdon Institute, Tennis Court Road,

Cambridge, UK, CB2 1QN

Running head: ASC mediates MAPK induction by P. gingivalis

Address correspondence to: Jenny Pan-Yun Ting, Ph.D, Fax: 919-966-8212; Tel: 919-966-5538; E-mail:

panyun@med.unc.edu

ASC/PYCARD is a common adaptor for a

diverse set of inflammasomes which activate

caspase-1, most prominently the NLR-based

inflammasome. Mounting evidence indicates

that ASC and these NLRs also elicit non-

overlapping functions, but the molecular

basis for this difference is unclear. To address

this, we performed microarray and network

analysis of ASC shRNA knockdown cells. In

pathogen-infected cells, an ASC-dependent

interactome is centered on the MAP kinase

(MAPK), ERK, and on multiple chemokines.

ASC did not affect expression of MAPK, but

affected its phosphorylation by pathogens

and TLR agonists via suppression of the dual-

specificity phosphatase, DUSP10/MKP5.

Chemokine induction, DUSP function and

MAPK phosphorylation were independent of

caspase-1 and IL-1. MAPK activation by

pathogen was abrogated in Asc-/-

but not

Nlrp3-/-

, Nlrc4-/-

, or Casp1-/-

macrophages.

These results demonstrate a function for ASC

that is distinct from the inflammasome in

modulating MAPK activity and chemokine

expression, and further identifies DUSP10 as

a novel ASC target.

Secretion of proinflammatory cytokines

and chemokines by macrophages in response to

pathogens is an important innate immune event

orchestrated by a complex signaling network.

Pathogenic signaling leads to the formation of an

intracellular protein complex termed the

inflammasome. A conventional inflammasome

is composed of caspase-1, which promotes

cleavage and maturation of the inflammatory

cytokines IL-1 and IL-18, ASC (apoptotic

speck protein, also known as TMS1 or pycard),

and a nucleotide-binding domain leucine-rich

repeat/NBD-LRR protein (NLR) (1). ASC

serves as a bridge between caspase-1 via CARD-

CARD interactions, and NLRs via pyrin/pyrin

interactions (2,3). Mutations in several NLRs are

associated with inflammatory diseases,

underscoring their importance in innate

immunity (4,5). More recently, AIM2 and RIG-I

have also been identified in separate

inflammasome complexes that rely on the ASC

adaptor (6-10).

Association of specific NLRs within the

inflammasome may be dictated by the type and

dose of infectious microorganism, whereas ASC

is thought to assume a broader role as an NLR

adaptor. For example, ASC and Nlrc4 each have

defined roles for caspase-1 activation and cell

death by Salmonella typhimurium (11,12), while

ASC and caspase-1 are required for T cell

activation and protective immunity against flu

challenge (13), however each of these processes

are independent of Nlrp3. Moreover, both

Francisella tularensis-mediated IL-1

processing and sensitivity to lethal doses of

lipopolysaccharide (LPS) are ASC-dependent,

but Nlrc4 and Nlrp3 independent (11,12). The

pyrin and HIN domain-containing protein,

AIM2 recognizes cytosolic DNA within an

NLR-free ASC inflammasome, further

supporting a mechanism for ASC independent of

NLRs (6-9). Thus, ASC may play a role as a

common downstream factor for different sets of

NLRs and may also function within an

inflammasome complex exclusive of NLR

family proteins.

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.221077The latest version is at JBC Papers in Press. Published on April 12, 2011 as Manuscript M111.221077

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Additional evidence suggests that ASC

function extends beyond the NLR/caspase-1

inflammasome. ASC was identified by its

unique ability to condense into cytosolic speck

structures and induce apoptosis in tumor cell

lines (14). It also is silenced in certain cancers

(15). Moreover, a caspase-independent type of

necrosis induced by high dose Shigella (≥50

MOI) (16), Neisseria gonorrhoeae (17) or

Porphyromonas gingivalis (Pg) (18) is ASC-

dependent. Asc-/- mice exhibit increased

susceptibility to Mycobacterium tuberculosis

without reduction in IL-1, implying additional

ASC function that is distinct from cytokine

cleavage (19). Two recent studies of antigen-

induced murine arthritis show dependence on

Asc, but caspase-1, NLRP3, and NLRC4

independence (20,21). A requirement for Asc,

but not Nlrp3 or Caspase-1, was also recently

demonstrated for antigen-specific humoral

immunity after vaccination with MF59-

adjuvented influenza (22). ASC has been

proposed to regulate cytokine transcription

through activation of NF-B (23,24). AP1,

STAT3, ISGF3 and NF-AT have also been

identified as transcriptional ASC targets in a

reconstituted cell system with exogenously

expressed ASC and a chimeric CARD12/NOD2

protein (25). Given the complexity of

inflammatory signaling it is likely that additional

signaling pathways contribute to inflammasome-

independent ASC function in macrophages.

One crucial feature of pathogenic

signaling is the activation of the mitogen-

activated protein kinase kinase kinases

(MAP3Ks), a family of signaling proteins that

regulate a variety of physiological processes

including proliferation, cell death, stress

response, and differentiation. The MAP3Ks act

as nodes in the toll-like receptor (TLR) signaling

cascade for both the NF-B and MAPK

pathways (26,27). Transcription factors

downstream of these signaling pathways then

collaborate to regulate the expression of immune

and inflammatory mediators. By contrast, the

role of components of the inflammasome

complex in MAP3K signaling has not been

directly explored. MAPK is at the center of

many innate immune responses, thus the link

between ASC and MAPK is an important topic

to explore.

Porphyromonas gingivalis (Pg) is a

gram negative oral pathogen associated with

chronic adult periodontal disease. Pg surface

components including LPS, fimbriae, and

hemagglutinin B induce host inflammatory

responses that result in breakdown of

periodontal ligaments and destruction of the

local alveolar bone (28-30). Although

periodontal disease is localized to the tissues

surrounding the tooth, Pg infection predisposes

people to more serious systemic conditions such

as cardiovascular disease and delivery of

preterm infants (29,31,32). Recently, we showed

that during Pg infection, ASC exhibits

inflammasome-independent functions, including

TNF- and NF-B activation (23). We therefore

elected to use this pathogen to reveal new ASC

functions that might be separable from that of

the inflammasome. Results described herewithin

provide a novel microbial pathogen-induced

mechanism for ASC in activating chemokine

expression through MAPK activation

independent of the conventional caspase-1

inflammasome.

Experimental Procedures

Generation of cell lines, isolation of mouse

macrophages and cell culture- THP1 monocytic

cells (ATCC) were cultured in RPMI, 10% FCS.

ShASC#1, shASC#2, and shASC#1mut THP1

cell lines were generated using retroviral vectors

and have been characterized previously (18,23).

DUSP10-expressing THP1 cells were generated

using the full-length ORF cloned into lentiviral

vector pLex-JRed (catalog #OHS4493-

98905681; Open Biosystems). A control empty

vector, pLex-EV, was generated by digesting

pLex-DUSP10 with Xho I to remove the

DUSP10 cDNA and religating the empty vector.

DUSP10 shRNA plasmid pLKO-shDUSP10 was

obtained from the TRC collection (Open

Biosystems). Lentivirus was packaged in 293T

cells using vectors pMD2.G and psPAX2

(Addgene plasmids 12259 and 12260) as

described (33). Cells were selected with 1 g/ml

puromycin for 2 weeks, and JRed expression

confirmed by FACS. Asc-/- and Nlrc4-/- mice

were obtained from Dr. Vishva Dixit at

Genetech; Nlrp3-/- mice from Millenium Inc.;

Casp1-/- mice from Dr. Richard Flavell, Yale

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University (12); and MyD88-/- mice from Dr.

Shizuo Akira. All mice were backcrossed for a

minimum of nine generations to C57BL/6 mice.

Bone marrow derived macrophages were

harvested and cultured in DMEM 10% fetal calf

serum, M-CSF without replating for 6-7 days.

Cells were maintained at low density and serum-

starved 16 h prior to infection. Where indicated,

cells were stimulated with 10 MOI Pg, 0.5 MOI

E. coli, or pharmacological agents detailed in

Supplementary Table 1.

Bacterial culture- Pg strain A7436 was cultured

anaerobically, and E. coli strain DH5

aerobically until late exponential phase (OD 0.8

to 1.2 at 660 nm). Aliquots were stored in media

containing 20% glycerol at -80C and used

within 3-4 months of preparation. Replating of

frozen cultures confirmed accuracy of bacterial

counts to within 2-3 CFU.

Microarray analysis- RNA was isolated using

Qiagen RNeasy columns following 2 h infection

with Pg. Two-color microarray analysis was

performed at the Duke Microarray facility using

34,000 spot custom chips based on the version 3

Human oligo set (Operon). All samples were

compared to a universal control created by

pooling RNA over a timecourse of LPS

treatment. Gene lists were generated from the

averages from 3 independent experiments using

GeneSpring 7.0. To identify Pg-regulated genes

the following criteria were used: minimum raw

signal of 50 in the universal control and 100 in

at least one condition; and ≥5-fold difference

between expression in control vs Pg-infected

cells. The criteria for ASC-dependent genes was

as follows: minimum raw signal of 60 in the

universal control and 100 in at least one

condition; ≥3-fold difference between

expression in Pg-infected THP1 vs shASC#1

cells and shASC#1mut vs shASC#1 cells; and

≤50% difference between Pg-infected THP1 and

shASC#1mut cells. Genes were further analyzed

in GeneSpring 11 for statistical validation using

modernized normalization methods, as presented

in Supplementary Tables 3 and 4 (Lanes 5-7).

Genes which passed the filtering schemes were

uploaded into the Ingenuity Pathways analysis

application, which overlaid them onto a global

molecular network developed from information

within the Ingenuity Pathways Knowledge Base.

Each network was algorithmically generated

based on the connectivity of genes. P values,

which indicate the likelihood that the same

number of genes taken from a random set would

appear in the network, were calculated using

Fischer's exact test.

Real time PCR and PCR-based expression

profiling- Realtime PCR was performed as

described (23) using the primers listed in

Supplementary Table 2. Mouse RNA was

quantified using Taqman® Assays on Demand

(Applied Biosystems). Values represent

averages ±SD of triplicates for RNA isolated on

different days unless otherwise stated. All values

were standardized to 18s rRNA expression.

PCR-based expression profiling was performed

according to manufacturer recommendation

using equal amounts of RNA from six mice with

the Mouse Inflammatory Cytokines and

Receptors array (SABiosciences). Values were

standardized to an average of 5 housekeeping

genes.

Assessment of RNA stability and de novo mRNA

synthesis- To assess RNA stability, cells were

treated for 2 h with LPS, washed to remove the

LPS, and treated with 50 M 5,6-dichloro-1-β-

-ribofuranosylbenzimidazole (DRB; Sigma) for

30 min. RNA was quantified by realtime PCR

over a timecourse as described above. To assess

de novo transcription, RNA was isolated from

nuclear extracts using the PARIS kit (Ambion)

and assessed by realtime PCR using primers that

target nascent transcripts. Efficient separation of

nuclei was confirmed by the absence of GAPDH

on Western blots.

Antibody array- Cell culture supernatants were

collected 24 h following exposure to Pg and

applied to RayBio® Human Cytokine Antibody

Array 5 glass slides according to manufacturer's

protocols. The signal strength for each cytokine

was normalized to the average signal strength of

spiked, internal controls. Data for each treatment

group were assessed as change compared to

uninfected samples.

ELISA Analysis- Supernatants were assessed 18-

24 h following stimulation using human ELISA

Sets for TNF and IL-1 (BD Biosciences),

DuoSets for CCL3 and CCL20(R&D Systems)

or the human quantikine ELISA kit for IGF-1

(R&D Systems). Samples were assayed within

linear range.

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Western and IP/IB analyses- Cells were washed

in 1X PBS and lysed for 20 min in ice-cold 1X

Lysis Buffer (20 mM Tris, pH 7.5, 150 mM

NaCl, 1 mM EDTA, 1 mM EGTA, 1% triton X)

supplemented with Complete EDTA-free

Protease Inhibitor and PhosSTOP Phosphatase

Inhibitor cocktail (Roche). Lysates were

centrifuged for 10 min and supernatants boiled

for 5 min in 1/3 volume 3X SDS Sample Buffer

(187.5 mM Tris, pH 6.8, 6% SDS, 30% glycerol,

150 mM DTT, 0.03% bromphenol blue).

Immunoblots were processed using Abs sc-7383

for p-ERK, equal volumes of sc-93 (ERK1) and

sc-154 (ERK2) for total ERK, sc-474 for JNK,

sc-1615 for actin (Santa Cruz Biotechnology),

#4668 for pJNK (Cell Signaling Technology),

and MAB374 for GAPDH (Millipore). For

caspase-1 IP/western analyses, cells were plated

at 107/ml and lysed following infection by

addition of 0.1% NP-40 and 1X Complete

EDTA-free Protease Inhibitors (Roche). 1 ml of

supernatant was recovered and incubated

overnight with 25 l anti-caspase-1 (sc-515,

Santa Cruz Biotechnology) and 30 l protein

A/G UltraLink resin (Thermo Scientific).

Immunoprecipitates were washed and then

boiled in 3X SDS Sample Buffer. Immunoblots

were performed using antibody IMG-5028

(Imgenex).

RESULTS

Identification of Pg interactomes by

microarray and bioinformatics. We selected Pg

for this study because a previous study

suggested that it activates ASC-mediated

functions that are distinct from IL-1 processing

and inflammasome activation (23). To identify

biological pathways activated by Pg, RNA from

untreated THP1 cells and cells treated with 10

MOI Pg for 2 h was assessed using human

genome microarray chips comprising over

35,000 oligonucleotide probes and representing

approximately 25,000 unique genes (Operon

Biotechnologies, Inc). Using GeneSpring 7.0

microarray analysis software, a list was

compiled of approximately 150 genes that Pg

modulates by ≥5-fold (Supplementary Table 3).

Additional statistical analysis using GeneSpring

11.0 verified the inclusion of the majority of the

genes in this table (Columns 5-7). Unbiased

software analysis was performed to identify

interactomes of genes that encode physically or

functionally interacting proteins (Ingenuity

Systems). Interactomes are networks of genes

that are interconnected based on information

derived from the literature, a textbook, or

canonical knowledge. Three primary networks

of Pg-modulated genes were identified. The first

interactome encompasses the inflammatory

cytokine TNF-. TNFA -

160 fold by Pg infection (Fig. 1A). Other genes

in the TNF- interactome were modulated from

5-319 fold by Pg. The extremely low p value of

this interactome (P < 10-46) suggests very high

likelihood of functionality. Consistently, TNF-

pathways are known to contribute to Pg-

associated pathogenesis during periodontitis

(34,35). The second interactome (Fig. 1B; P <

10-46) encompasses IL1Bwhich itself is

activated more than 200-fold. Both interactomes

show high induction for multiple chemokines.

The third interactome (P < 10-41) is unique in

that its central molecule, NF-B, does not appear

to be significantly regulated at the

transcriptional level but was identified based on

the genes that it is known to modulate (Fig. 1C).

This is consistent with the role of the NF-B

members as early signaling transcriptional

regulators (36). Induction of the transcript for

the CARD-containing serine/threonine kinase

RIPK2 by 15-17-fold (Fig. 1A) could potentially

contribute to the enhanced NF-B pathway

activity by Pg (37,38). The identification of each

of these Pg interactomes is consistent with

known biological effects of Pg in the activation

of cellular signaling pathways toward

inflammation.

Identification of an ASC-dependent Pg

interactome. ASC is an adaptor molecule

important in the induction of apoptosis and

inflammatory response (2,11,15). Our previous

studies demonstrate that in addition to regulating

IL-1 processing through caspase-1 activation,

ASC regulates the transcription of a panel of

cytokines including IL-6, IL-8, IL-10 and TNF-

(23). Because induction of these cytokines is

thought to be NLR and caspase-1 independent,

their regulation by ASC implies ASC-mediated

functions that are distinct from the

inflammasome. For example, several reports

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have shown that TNF expression is not altered

by the deletion of Nlrp3 (12,39). Thus we

reasoned that Pg represents a good system to

reveal differences between ASC-associated

inflammasome-dependent and inflammasome-

independent signaling.

To identify additional genes and

pathways regulated by ASC, two different ASC

knockdowns were utilized with reduced ASC

expression. The lentiviral shRNA construct

shASC#1 caused approximately 90%

knockdown and shASC#2 approximately 70%

knockdown when compared to controls,

including non-transfected THP1 cells, and cells

bearing an empty vector (EV) or a mutated

target site (shASC#1mut) (23) (Fig. 1D). The

shRNAs reduced IL-1 cytokine induction,

verifying that the knockdowns reduce ASC

function as previously observed (Fig. 1E).

Microarray analysis was performed to identify

genes that were differentially regulated by ASC.

Approximately 80 genes were modulated by ≥3-

fold in shASC#1 cells as compared to control

cells (Supplementary Table 4). This set of ASC-

dependent genes was evaluated by Ingenuity

Pathways Analysis and an ASC-dependent

interactome was identified (Fig. 1F, P < 10-42).

As confirmation of the technology, ASC was

reduced 8.8 to 12.6-fold in shASC cells. Several

chemokines, including CCL3, CCL3L1, and

CCL4 were reduced in the absence of ASC,

while CXCL10 was increased approximately 4-

fold. NF-B was also identified as an ASC-

dependent regulator of these genes, consistent

with our previous findings linking ASC-

dependent gene expression to NF-B activation

(23). Since the expression and activity of NF-B

and several chemokines within this interactome

are also highly regulated by Pg (Fig. 1A-C), the

role of ASC may extend to many of the same

pathways that Pg induces. All of the genes

within the ASC-dependent interactome could be

interconnected to the signaling molecule

MAPK1/ERK2, albeit expression of the latter,

like NF-B, was not affected by ASC. Because

MAPKs are primarily regulated post-

transcriptionally, the central position of MAPK1

within this interactome provides novel insight

into potential post-transcriptional mechanisms of

ASC function.

Assessment of ASC-dependent cytokine

and chemokine expression. To verify ASC-

dependent chemokine expression, RNA levels in

ASC knockdown THP1 cells were measured

following a timecourse of infection with Pg.

Consistent with the microarray results, TNFA

was induced by Pg in control cells, with a peak

at 2-4 h post infection, and this induction was

reduced in the ASC knockdown cells (Fig. 2A).

CCL3, CCL4, and CXCL3 each showed a similar

pattern of ASC-dependent RNA expression (Fig.

2B-D). These findings support the identification

of the ASC interactome and show a role for ASC

in Pg-dependent induction of chemokines.

Regulation of mRNA expression can

occur either at the level of transcription or RNA

stability. To distinguish these possibilities, RNA

stability was examined following LPS treatment

using DRB to block de novo transcription (Fig.

2E). The RNA decay profiles were similar in

shASC#1mut and shASC#1 cells, indicating that

regulation of RNA stability is not a major

determinant for ASC-dependent differences in

chemokine expression. Conversely, assessment

of de novo mRNA synthesis using nuclear

extracts and primers that target unspliced

message indicates that regulation is primarily

transcriptional (Fig. 2F).

To further verify a role for ASC in

chemokine induction by Pg we performed

protein array analysis using antibody chips

comprising 79 cytokines, chemokines and

growth factors (RayBiotech, Inc). Supernatants

were assayed from shASC#1mut and shASC#1

cells before and following 24 h infection with

Pg. Six proteins were differentially induced by

Pg in shASC#1mut vs shASC#1 cells (Fig. 3A).

This includes IL-1 which is a known target for

ASC regulation via cleavage by caspase-1

within the inflammasome complex (2). TNF-

was also identified, further confirming our

realtime results. The chemokines from Figs. 2B-

D either were not detected at high levels (for

CCL4), or were not included on this array (for

CCL3 and CXCL3). However, the chemokines,

GRO/GRO-, CCL13 and CCL20 were

identified, further supporting a broad role for

ASC in regulating chemokine expression. The

latter proteins are not thought to be regulated by

caspase-1, suggesting a potential role for ASC in

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caspase-1-independent regulation of chemokine

expression.

To further verify these findings,

supernatants were collected from control and

ASC knockdown lines 24 h following infection

with Pg and assessed by ELISA. Levels of

secreted TNF-, CCL3, and CCL20 were

reduced in shASC#1 cells, with somewhat less

reduction in shASC#2 cells suggesting dose-

dependent regulation (Fig. 3B-D). IGF-1

expression was not ASC-dependent,

demonstrating specificity of this effect (Fig. 3E).

These findings further support ASC-dependent

chemokine expression suggested by the

interactome described in Fig. 1F.

MAPK activation is ASC dependent in

THP1 cells. MAPKs are activated by

phosphorylation by upstream kinases (26,27). To

provide support for the central position of

MAPK1/ERK2 within the ASC interactome

(Fig. 1F), we assessed phosphorylation levels of

ERK in ASC knockdown cells following Pg

infection. Consistent with the microarray results,

total expression of ERK1 and ERK2 was not

significantly different in control shASC#1mut vs

shASC#1 knockdown cells and was not

regulated by Pg infection. Levels of phospho-

ERK (p-ERK) induction peaked at

approximately 60-90 minutes for both cell lines,

but were quantitatively reduced in ASC

knockdown cells over a 120 min timecourse of

infection (Fig. 4A). These findings are

consistent with a role for ASC in ERK

activation.

To determine whether effects of ERK

activation by ASC have broad significance

during microbial infection, control and ASC

knockdown cells were treated for 60 min with E.

coli and a variety of agents known to activate

cells through different TLRs (Fig. 4B). For each

of these treatments, levels of p-ERK were

reduced in the absence of ASC. These findings

indicate that in addition to Pg, ASC mediates

ERK activation through E. coli and a variety of

TLR agonists.

ERK is one of three related MAPK

pathways activated in stimulated cells. To test

whether the JNK and p38 pathways also display

reduced activation in ASC knockdown cells,

phosphoblots were repeated. Levels of phospho-

p38 in THP1 cells were too low to measure

accurately (data not shown). However, similar to

p-ERK, levels of p-JNK activation by Pg were

delayed and reduced in ASC knockdown cells,

indicating that the role of ASC in MAPK

activation extends to other MAPK pathways

(Fig. 4C).

To determine whether reduction in ERK

and JNK activation might explain the reduced

chemokine levels in ASC knockdown THP1

cells, ELISA assays were repeated in the

presence of specific inhibitors of ERK and JNK

pathways (Fig. 4D). Levels of CCL3 and CCL20

following Pg infection were reduced by both

inhibitors, either alone or in combination, while

the carrier, DMSO, did not affect expression.

This effect was specific because levels of IGF-1

were unchanged (Fig. 4E). These results confirm

the importance of the ERK and JNK pathways in

chemokine induction by Pg.

MAPK activation and chemokine

induction by Pg in primary mouse macrophages

is ASC-dependent. To confirm our findings in a

primary cell system, macrophages were

harvested from wildtype C57BL/6 mice and

matched Asc-/- mice and infected with Pg over a

timecourse. Levels of p-ERK activation were

nearly eliminated in macrophages from Asc-/-

mice, while levels of p-JNK and p-p38 were

modestly reduced (Fig. 5A-C). These results

confirm the role for ASC in Pg-induced MAPK

phosphorylation in a primary cell system.

To determine whether reduced MAPK

activity in mouse macrophages correlates with

reduced chemokine activation, RNA was pooled

from six WT and Asc-/- mice following Pg

infection and assessed by PCR-based expression

profiling. Several chemokines were identified

(Fig. 5D). This list of candidate genes includes

Ccl3, which was decreased, and Cxcl10 which

was increased both in the RNA from pooled

Asc-deficient mouse macrophages and THP1

shASC#1 cells (Fig. 1F). Other chemokines

identified in the two model system differed,

mostly likely explained by differences between

species and the transformed or nontransformed

state of the cells. Nonetheless, the regulation of

these chemokines would be consistent with our

overall finding that ASC modulates chemokine

expression.

To verify Asc-dependent expression of

chemokines in mouse macrophages, Taqman®

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PCR was performed before and following 2h Pg

infection and fold induction was calculated.

Consistent with the above results, Asc was

required for high level inducbility of Ccl3,

Ccl19 and Ccl25, while Cxcl10 was increased in

the absence of Asc (Fig. 5E). Ccl3 expression

was dependent upon ERK, JNK and p38 (Fig.

5F), verifying an essential role for MAPKs in

chemokine expression in primary mouse

macrophages.

The dual-specificity phosphatase

DUSP10/MKP5 negatively regulates MAPK

phosphorylation and chemokine activation by

Pg. The modulation of multiple MAPKs by ASC

suggests that ASC might lie upstream of a

common regulator of MAPKs. Examination of

ASC-dependent genes identified by microarray

analysis (Supplementary Table 4) revealed that

DUSP10/MKP5 is approximately 3.5-fold higher

in shASC-containing cells. The dual-specificity

phosphatases (DUSPs) are known to negatively

regulate the activity of multiple MAPKs through

dephosphorylation (40,41). To verify the effect

of ASC on DUSP10 expression, realtime PCR

analysis was performed. DUSP10 levels were

increased in both ASC knockdown lines relative

to the controls (Fig. 6A, top panel). As a control,

levels of transcript for MAPK1/ERK2 were

ASC-independent (Fig. 6A, bottom panel).

Increased DUSP10 in ASC knockdown cells

was observed over a timecourse of infection

with Pg in THP1 cells (Fig. 6B) and mouse

macrophages (Fig. 6C). Because DUSP10 is

known to correlate negatively with the activity

of MAPKs (42), these findings are consistent

with the reduced post-transcriptional activation

of MAPKs in ASC knockdown cells following

Pg stimulation and provide a potential

mechanism for diminished MAPK

phosphorylation.

To directly assess the role of DUSP10 in

MAPK phosphorylation, DUSP10 was

exogenously expressed in THP1 cells (Fig. 6D).

Cells were infected with Pg, and MAPK

phosphorylation levels were measured by

immunoblotting. ERK phosphorylation was

significantly reduced and JNK phosphorylation

nearly eliminated in pLex-DUSP10 cells as

compared to non-transfected THP1 cells and the

empty vector control, pLex-EV (Fig. 6E). These

results verify that DUSP10 can negatively

regulate MAPK phosphorylation following Pg

infection. To determine whether increased

DUSP10 expression can regulate chemokine

induction, supernatants from pLex-EV control

cells and pLex-DUSP10 cells were collected

following infection with Pg and assessed by

ELISA. Levels of secreted CCL3 and CCL20

were ablated in pLex-DUSP10 cells (Fig. 6F).

These results suggest a potential mechanism for

reduced MAPK activation and chemokine

activation in ASC knockdown cells through the

increased expression of DUSP10.

To further examine the capacity of

DUSP10 to negatively regulate ERK

phosphorylation, double knockdown THP1

shRNA cell lines were produced with reduced

ASC and DUSP10 expression (Fig. 6G). Cells

expressing the control vector, pLKO showed

ASC-dependent pERK and pJNK

phosphorylation, however in cells containing

shDUSP10, MAPK phosphorylation was no

longer dependent on ASC (Fig. 6H). These

results suggest that DUSP10 expression is

required for ASC-mediated ERK

phosphorylation.

To determine how DUSP10 expression

is regulated, THP1 cells were treated with a

panel of inhibitors for specific MAPK pathways.

Inhibition of ERK caused increased DUSP10

expression with the greatest increase achieved

using a combination of ERK and JNK inhibitors

(Fig. 6I, top panel). The converse results were

observed for TNFA as expected (bottom panel).

Inhibition of p38 did not affect DUSP10 nor

TNFA expression. Since the p38 inhibitor

SB203580 also can potently inhibit RIPK2,

these results could suggest that RIPK2 is not

involved in the short term regulation of DUSP10

in THP1 cells (43). For primary mouse

macrophages, all three inhibitors affected

Dusp10 expression, though the greatest

enhancement of expression was observed

following inhibition of either JNK or ERK and

p38/RIPK2 in combination (Fig. 6J). These

findings suggest that a negative feedback loop

occurs in which DUSP10 regulates MAPK

activation, and MAPKs in turn regulate DUSP10

expression. The complement of the specific

MAPKs involved appears to vary for THP1 cells

vs mouse macrophages. However, a similar

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pattern of reciprocal regulation occurs in each

system.

Induction of chemokines and ERK

phosphorylation by Pg is inflammasome

independent. ASC is a central component of the

inflammasome that is responsible for processing

of mature IL-1 from pro-IL-1 (2,11). To test

the possibility that reduction in chemokine

levels in ASC knockdown cells is due to

autocrine activation by secreted IL-1, THP1

cells were treated with the IL-1 receptor

antagonist Kineret® under conditions shown

previously to block IL-1 signaling (16,23).

Realtime PCR analysis (Fig. 7A) and ELISA

assay (Fig. 7B) showed that Kineret® did not

affect levels of induction of a panel of

chemokines by Pg. The caspase-1 inhibitor

YVAD-cmk reduced the secretion of mature IL-

1 as expected, but did not affect levels of

CCL3 or CCL20 (Fig. 7B). Levels of activation

of p-ERK following Pg infection also remained

high in the presence of either Kineret® (Fig. 7C)

or YVAD (Fig. 7D), further ruling out a

significant contribution for IL-1/caspase-1

signaling in ASC-dependent ERK activation by

Pg. These findings suggest that the induction of

MAPKs and chemokines by Pg is IL-1 and

caspase-1 independent, and furthermore suggest

that the reduced chemokine induction in ASC

knockdown cells is not explained by autocrine

IL-1 signaling or signaling through a caspase-1

containing inflammasome complex.

To directly test whether caspase-1

activation is dependent on DUSP10 expression,

immunoprecipitation/immunoblot experiments

were performed to assess levels of cleaved

caspase-1 following Pg infection (Fig 7E).

Activation of caspase-1 was ablated in shASC#1

cells as expected (lane 6 vs lane 5). However,

expression of DUSP10 did not reduce caspase-1

activation following Pg infection (lane 8 vs lane

7). These findings suggest that the regulation of

MAPK signaling by DUSP10 constitutes a

distinct ASC-dependent activity that is

independent of caspase-1 and the conventional

inflammasome. Further studies will be necessary

to determine whether the inflammasome

independence extends to other genes and

pathways within the ASC-dependent

inflammasome identified in Figure 1F.

Macrophages from wild-type B6 and

gene deletion mice were tested to confirm

inflammasome independence of MAPK

activation in a primary cell system. Following

Pg infection, phosphorylation of p-ERK was

dramatically reduced in the positive control,

macrophages from MyD88 -/- mice. This is

expected due to the prominent role of TLR/IL-

1R in this pathway (26,27) (Fig. 7F). However

an examination of macrophages from Casp1-/-,

Nlrp3/-, and Nlrc4-/- mice show that these genes

do not affect ERK activation. The persistence of

ERK activation in the Nlrp3-/- and Nlrc4-/-

macrophages shows independence from Nlrp3

and Nlrc4 inflammasome function. The use of

Casp1-/- mice most clearly indicates that p-ERK

activation is independent of the caspase-1

inflammasome. These findings further argue for

a role for ASC-dependent MAPK activation that

is exclusive of the caspase-1 inflammasome.

DISCUSSION

Using a combination of gene profiling

analysis and bioinformatics we identified several

pathways activated by Pg. The finding that Pg

activates NF-B and TNF-related signaling is

consistent with other microarray studies of Pg

infection (28,34). Our further assessment of

ASC-modulated genes revealed several

chemokines that are ASC-dependent. These

findings are novel, and led us to the

identification of MAPK1/ERK2 as a potential

ASC-regulated protein within a complex

interactome of chemokines, signaling molecules,

and transcriptional regulators. MAPK regulation

was not transcriptional, but rather at the level of

phosphorylation. This work demonstrates the

power of combining microarray technology with

software-based network analysis to study cell

signaling pathways. In this instance the relevant

proteins are regulated post-transcriptionally and

would therefore be missed by microarray

analysis alone.

Western analysis confirmed the role of

MAPKs in ASC-mediated signaling in THP1

cells and Asc-/- mice following Pg infection.

ASC also was important in the activation of

ERK in response to E. coli, and to agonists of

TLR2, 4, and 5. The contribution of MAPKs to

the activation of chemokines by Pg correlates

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with these results and provides one potential

functional outcome for reduced MAPK

activation in ASC deficient cells. The

chemokines identified and the dependency on

specific MAPKs in each model system had

partial overlap. Differences in the two model

system might be explained by human vs mouse

differences, the transformed vs nontransformed

state of the cell, or perhaps by differences in the

stage of differentiation; however, the

fundamental finding of ASC-dependent MAPK

activation and chemokine induction was

conserved.

Our results identify DUSP10 as a key

regulator in ASC-dependent MAPK activation.

The DUSPs function by reversing the tyrosine

and serine/threonine phosphorylation of the

MAPKs that occurs upon activation (41).

DUSP10 expression was enhanced in the

absence of ASC. Furthermore, exogenous

expression of DUSP10 reduced ERK

phosphorylation and chemokine induction, and

conversely, reduction of DUSP10 by RNA

interference reversed the ASC-dependent ERK

phosphorylation. Interestingly, DUSP10

expression also was regulated reciprocally by

the MAPKs in a classic negative feedback loop.

The reciprocal regulation of DUSP10 and

MAPKs could explain the profound effect on the

expression of chemokines within the ASC-

dependent interactome.

It is noteworthy that a connection

between ASC and the transcription factor AP1

has recently been established using a

reconstituted cell system that is engineered to

respond to the bacterial cell wall component

muramyl dipeptide (25). AP1 is among a large

number of transcriptional regulators that are

downstream targets of MAPKs (26). The

regulation of AP1 described in the former study

appears is at the level of transcription, whereas

our results reveal a post-transcriptional

regulatory mechanism. Further studies will be

necessary to define effects of ASC-dependent

DUSP repression and MAPK activation on AP1

activity. ASC-dependent post-transcriptional

activation of AP1 could provide a

complementary mode of controlling AP1

activity. Given the broad role of MAPKs in a

number of biological pathways, it is likely that

ASC-dependent MAPK activation controls the

activity of multiple additional transcription

factors and pathways.

Initial studies of Asc-/- mouse

macrophages failed to show a difference in

MAPK activation (11). Differences between

these findings and ours could reflect the use of

different stimuli to activate p-ERK, or different

methods of macrophage isolation or culture.

MAPKs can be readily activated by a wide array

of stimuli. In our protocol, macrophages were

plated for at least 6 days without disruption

because MAPKs can be easily induced by

changes in adherence. There are multiple types

of proliferative stimuli and pathways that can

lead to MAPK activation (41), and for this

reason we have serum-starved the primary cell

macrophages to reduce background stimulation

from serum components. Serum starvation is one

major difference between our study and earlier

studies, and is typically required to see ERK

activation.

Classically, ASC acts within an

inflammasome complex that also contains

caspase-1 and one of several different NLR

proteins (1) or the HIN domain protein AIM2

for cytosolic DNA viruses (6-9). In contrast to

IL-1 processing, activation of MAPK and

chemokines via an ASC-dependent pathway did

not require caspase-1. Furthermore, exogenous

DUSP10 expression reduced MAPK

phosphorylation and chemokine activation

without affecting caspase-1 processing. ERK

phosphorylation was not reduced in Casp1-/-,

Nlrp3-/- or Nlrc4-/- mice. These findings suggest

that the MAPK pathway of chemokine

activation is caspase-1 and NLR-independent,

providing a novel inflammasome-independent

function for ASC. ASC also is required for

caspase-independent activation of necrosis (16-

18) and for antigen-induced arthritis (20,21).

Previously we showed that NF-B activation by

Pg is caspase-1 independent (23). The present

study provides an additional caspase-

independent mechanism of ASC that could

explain ASC functionality in the absence of the

inflammasome.

In summary, ASC has important roles in

both inflammasome-dependent and

inflammasome-independent signaling cascades.

We have revealed a novel mechanism for ASC

in the activation of MAPKs that is regulated

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through DUSP10 suppression, and a seminal

result of this activation is the alteration of

chemokines necessary for host response to

microbial pathogens.

REFERENCES

1. Pedra, J. H., Cassel, S. L., and Sutterwala, F. S. (2009) Curr Opin Immunol 21, 10-16

2. Martinon, F., Burns, K., and Tschopp, J. (2002) Mol Cell 10, 417-426

3. Srinivasula, S. M., Poyet, J. L., Razmara, M., Datta, P., Zhang, Z., and Alnemri, E. S.

(2002) J Biol Chem 277, 21119-21122

4. Ting, J. P., Kastner, D. L., and Hoffman, H. M. (2006) Nat Rev Immunol 6, 183-195

5. Becker, C. E., and O'Neill, L. A. (2007) Semin Immunopathol 29, 239-248

6. Hornung, V., Ablasser, A., Charrel-Dennis, M., Bauernfeind, F., Horvath, G., Caffrey, D.

R., Latz, E., and Fitzgerald, K. A. (2009) Nature 458, 514-518

7. Fernandes-Alnemri, T., Yu, J. W., Datta, P., Wu, J., and Alnemri, E. S. (2009) Nature

458, 509-513

8. Burckstummer, T., Baumann, C., Bluml, S., Dixit, E., Durnberger, G., Jahn, H.,

Planyavsky, M., Bilban, M., Colinge, J., Bennett, K. L., and Superti-Furga, G. (2009) Nat

Immunol 10, 266-272

9. Roberts, T. L., Idris, A., Dunn, J. A., Kelly, G. M., Burnton, C. M., Hodgson, S., Hardy,

L. L., Garceau, V., Sweet, M. J., Ross, I. L., Hume, D. A., and Stacey, K. J. (2009)

Science 323, 1057-1060

10. Poeck, H., Bscheider, M., Gross, O., Finger, K., Roth, S., Rebsamen, M.,

Hannesschlager, N., Schlee, M., Rothenfusser, S., Barchet, W., Kato, H., Akira, S.,

Inoue, S., Endres, S., Peschel, C., Hartmann, G., Hornung, V., and Ruland, J. Nat

Immunol 11, 63-69

11. Mariathasan, S., Newton, K., Monack, D. M., Vucic, D., French, D. M., Lee, W. P.,

Roose-Girma, M., Erickson, S., and Dixit, V. M. (2004) Nature 430, 213-218

12. Sutterwala, F. S., Ogura, Y., Szczepanik, M., Lara-Tejero, M., Lichtenberger, G. S.,

Grant, E. P., Bertin, J., Coyle, A. J., Galan, J. E., Askenase, P. W., and Flavell, R. A.

(2006) Immunity 24, 317-327

13. Ichinohe, T., Lee, H. K., Ogura, Y., Flavell, R., and Iwasaki, A. (2009) J Exp Med 206,

79-87

14. Masumoto, J., Taniguchi, S., Ayukawa, K., Sarvotham, H., Kishino, T., Niikawa, N.,

Hidaka, E., Katsuyama, T., Higuchi, T., and Sagara, J. (1999) J Biol Chem 274, 33835-

33838

15. Conway, K. E., McConnell, B. B., Bowring, C. E., Donald, C. D., Warren, S. T., and

Vertino, P. M. (2000) Cancer Res 60, 6236-6242

16. Willingham, S. B., Bergstralh, D. T., O'Connor, W., Morrison, A. C., Taxman, D. J.,

Duncan, J. A., Barnoy, S., Venkatesan, M. M., Flavell, R. A., Deshmukh, M., Hoffman,

H. M., and Ting, J. P. (2007) Cell Host Microbe 2, 147-159

17. Duncan, J. A., Gao, X., Huang, M. T., O'Connor, B. P., Thomas, C. E., Willingham, S.

B., Bergstralh, D. T., Jarvis, G. A., Sparling, P. F., and Ting, J. P. (2009) J Immunol 182,

6460-6469

18. Huang, M. T., Taxman, D. J., Holley-Guthrie, E. A., Moore, C. B., Willingham, S. B.,

Madden, V., Parsons, R. K., Featherstone, G. L., Arnold, R. R., O'Connor, B. P., and

Ting, J. P. (2009) J Immunol 182, 2395-2404

by guest on February 1, 2019http://w

ww

.jbc.org/D

ownloaded from

11

19. Mayer-Barber, K. D., Barber, D. L., Shenderov, K., White, S. D., Wilson, M. S.,

Cheever, A., Kugler, D., Hieny, S., Caspar, P., Nunez, G., Schlueter, D., Flavell, R. A.,

Sutterwala, F. S., and Sher, A. J Immunol 184, 3326-3330

20. Kolly, L., Karababa, M., Joosten, L. A., Narayan, S., Salvi, R., Petrilli, V., Tschopp, J.,

van den Berg, W. B., So, A. K., and Busso, N. (2009) J Immunol 183, 4003-4012

21. Ippagunta, S. K., Brand, D. D., Luo, J., Boyd, K. L., Calabrese, C., Stienstra, R., Van de

Veerdonk, F. L., Netea, M. G., Joosten, L. A., Lamkanfi, M., and Kanneganti, T. D. J

Biol Chem

22. Ellebedy, A. H., Lupfer, C., Ghoneim, H. E., Debeauchamp, J., Kanneganti, T. D., and

Webby, R. J. Proc Natl Acad Sci U S A 108, 2927-2932

23. Taxman, D. J., Zhang, J., Champagne, C., Bergstralh, D. T., Iocca, H. A., Lich, J. D., and

Ting, J. P. (2006) J Immunol 177, 4252-4256

24. Stehlik, C., Fiorentino, L., Dorfleutner, A., Bruey, J. M., Ariza, E. M., Sagara, J., and

Reed, J. C. (2002) J Exp Med 196, 1605-1615

25. Hasegawa, M., Imamura, R., Motani, K., Nishiuchi, T., Matsumoto, N., Kinoshita, T.,

and Suda, T. (2009) J Immunol 182, 7655-7662

26. Banerjee, A., and Gerondakis, S. (2007) Immunol Cell Biol 85, 420-424

27. Krishnan, J., Selvarajoo, K., Tsuchiya, M., Lee, G., and Choi, S. (2007) Exp Mol Med 39,

421-438

28. Zhou, Q., and Amar, S. (2007) J Immunol 179, 7777-7790

29. Darveau, R. P., Tanner, A., and Page, R. C. (1997) Periodontol 2000 14, 12-32

30. Zhang, P., Martin, M., Michalek, S. M., and Katz, J. (2005) Infect Immun 73, 3990-3998

31. Gibson, F. C., 3rd, and Genco, C. A. (2007) Curr Pharm Des 13, 3665-3675

32. Lin, D., Moss, K., Beck, J. D., Hefti, A., and Offenbacher, S. (2007) J Periodontol 78,

833-841

33. Moore, C. B., Guthrie, E. H., Huang, M. T., and Taxman, D. J. Methods Mol Biol 629,

141-158

34. Milward, M. R., Chapple, I. L., Wright, H. J., Millard, J. L., Matthews, J. B., and Cooper,

P. R. (2007) Clin Exp Immunol 148, 307-324

35. Boyce, B. F., Li, P., Yao, Z., Zhang, Q., Badell, I. R., Schwarz, E. M., O'Keefe, R. J., and

Xing, L. (2005) Keio J Med 54, 127-131

36. Hayden, M. S., and Ghosh, S. (2008) Cell 132, 344-362

37. Moreira, L. O., El Kasmi, K. C., Smith, A. M., Finkelstein, D., Fillon, S., Kim, Y. G.,

Nunez, G., Tuomanen, E., and Murray, P. J. (2008) Cell Microbiol 10, 2067-2077

38. Tigno-Aranjuez, J. T., Asara, J. M., and Abbott, D. W. Genes Dev 24, 2666-2677

39. Hsu, L. C., Ali, S. R., McGillivray, S., Tseng, P. H., Mariathasan, S., Humke, E. W.,

Eckmann, L., Powell, J. J., Nizet, V., Dixit, V. M., and Karin, M. (2008) Proc Natl Acad

Sci U S A 105, 7803-7808

40. Chi, H., Barry, S. P., Roth, R. J., Wu, J. J., Jones, E. A., Bennett, A. M., and Flavell, R.

A. (2006) Proc Natl Acad Sci U S A 103, 2274-2279

41. Jeffrey, K. L., Camps, M., Rommel, C., and Mackay, C. R. (2007) Nat Rev Drug Discov

6, 391-403

42. Theodosiou, A., Smith, A., Gillieron, C., Arkinstall, S., and Ashworth, A. (1999)

Oncogene 18, 6981-6988

43. Argast, G. M., Fausto, N., and Campbell, J. S. (2005) Mol Cell Biochem 268, 129-140

by guest on February 1, 2019http://w

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FOOTNOTES

*This work was supported by RO1-DE016326 (JT). We thank Drs. Richard Flavell, Vishva M. Dixit,

Fayyaz Sutterwala, Shizuo Akira, and Millenium Pharmaceuticals for supplying gene deficient mice.

Lentiviral packaging vectors pMD2.G and psPAX2 were kindly provided by Dr. Didier Trono (Addgene).

The abbreviations used are: MAPK, MAP kinase; ASC, apoptotic speck protein; NLR, nucleotide-binding

domain leucine-rich repeat /NBD-LRR protein; LPS, lipopolysaccharide; Pg, Porphyromonas gingivalis;

MAP3K, mitogen-activated protein kinase kinase kinase; TLR, toll-like receptor; DRB, 5,6-dichloro-1-β-

-ribofuranosylbenzimidazole; DUSP, dual-specificity phosphatase

FIGURE LEGENDS

Fig. 1. Network analysis of Pg-stimulated genes in control and ASC knockdown cells. (A-C)

Interactomes identified by Ingenuity analysis of the GeneSpring 7.0 microarray genes in Supplementary

Table 3. Values represent fold stimulation for Pg-stimulated vs control THP1 cells. Positive values and

red color represent increase in Pg-stimulated cells, while green color and negative values represent

decrease. Bold lines represent physical interactions between proteins and dotted lines represent functional

interactions. (A) Cell to Cell Signaling Interactome, P<10E-46; (B) Cellular Growth and Proliferation

Interactome, P<10E-46; (C) Gene Expression Interactome, P< 10E-41. (D) Assessment of ASC

knockdown in shASC THP1 cell lines. Control cells include THP1 and THP1 expressing an empty vector

(EV) or a mutated target site (shASC#1mut). ASC knockdown cell lines, shASC#1 and #2, encode

different shRNAs for ASC. ASC expression was measured by realtime PCR and normalized to an average

of 100 in control cell lines. Data represent averages ±SD for 3 independent experiments. (E) IL-1 ELISA

of supernatants from control and ASC knockdown cells following Pg infection. Data represent averages

±SD for 3 independent experiments. (F) Ingenuity analysis of genes differentially activated by Pg in the

presence and absence of ASC. The “Inflammatory and Immunological Disease” Interactome depicted

(P<10E-42) was based on GeneSpring 7.0 microarray values from Supplementary Table 4. Values

represent fold stimulation for Pg-infected shASC#1 vs shASC#1mut cells. Negative values and green

color represent decrease in shASC#1 cells, while positive values and red color represent increase.

Fig. 2. Chemokine transcription is reduced in shASC-containing THP1 cell lines. (A) TNFA expression

over a timecourse of infection with Pg. Realtime PCR values were normalized to 1 in uninfected THP1

cells. Representative of 3 independent experiments. (B-D) Expression of CCL3, CCL4, and CXCL3as

measured by realtime PCR. Data were normalized to 100 in Pg-induced THP1 cells. N.D., not detectable.

Data represent averages ±SD for at least 3 independent experiments. (E) RNA decay following DRB

treatment as measured by realtime PCR. Starting values were normalized to 100. Data represent averages

±SD and are representative of 3 independent experiments. (F) Realtime PCR of nascent transcripts in

control and shASC cells. Data were normalized to 1 in uninfected cells. Representative of 3 independent

experiments.

Fig. 3. Analysis of secreted cytokine and chemokine levels in control and shASC-containing THP1 lines.

(A) Fold induction of cytokines and chemokines in supernatants 24h following Pg infection. Values

represent ratios of binding on RayBio® Human Cytokine Antibody Array 5 chips for 3-fold or more

difference between shASC #1mut and shASC#1 cells. (B-E) ELISA of TNF-, CCL3, CCL20, and IGF-1

prior to or following 24 h infection with Pg. Data represent averages ±SD for at least 3 independent

experiments.

Fig. 4. MAPK activation is reduced in shASC-containing THP1 cells. (A) Western analysis of p-ERK,

total ERK (ERK1 and ERK2) and GAPDH in shASC#1mut and shASC#1 cells following a timecourse of

infection with Pg. Representative of at least 5 independent experiments. (B) Western analysis of p-ERK

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and GAPDH in shASC#1mut and shASC#1 cells following 60 min treatment with E. coli or TLR agonist.

Representative of 3 independent experiments. P3C, Pam3Cys-Ser-(Lys)4-trihydrochloride. (C) Western

analysis of p-JNK and total JNK in shASC#1mut and shASC#1 cells following a timecourse of infection

with Pg. Representative of at least 3 independent experiments. (D) ELISA of chemokine levels in THP1

cells treated with Pg, DMSO solvent, ERK and/or JNK inhibitor. Data represent average ±SD for at least

3 independent experiments. N.D., not detectable. (E) ELISA of IGF-1 levels in Pg-treated cells following

addition of DMSO, ERK or JNK inhibitor. Data represent averages ±SD for 3 independent experiments.

Fig. 5. MAPK activation and chemokine induction in primary mouse macrophages is Asc-dependent. (A-

C) Western blot of p-ERK, p-JNK and p-p38 in primary mouse macrophages from WT C57BL/6 and Asc-

/- mice following a timecourse of infection with Pg. Blotting for the Asc protein is shown as a verification

of the knockout, and GAPDH as a loading control. Representative of at least 3 independent experiments.

(D) Chemokines modulated in Asc-/- mice as assessed by pathway-focused gene expression profiling of

pooled RNA from six mice. Values represent fold expression for Pg-infected Asc-/- vs WT macrophages.

Negative values represent decrease in Asc-/- macrophages, and positive values increase. A complete list of

genes is provided in Supplementary Table 5. (E) Induction of chemokines following 2h Pg infection as

assessed by Taqman® PCR. Data represent averages ±SD for 4 independent experiments. *, p<0.05; **,

p<0.05 (F) Effects of MAPK inhibitors on Ccl3 expression as determined by Taqman® Assays.

Expression levels were normalized to 100 in control cells. Data represent averages ±SD for 3 independent

experiments.

Fig. 6. Elevated DUSP10 levels negatively regulate Pg-induced chemokine expression. (A) Realtime

analysis of DUSP10 and MAPK1/ERK2 RNA in control and shASC-containing cell lines following 2 h

Pg infection. Expression was normalized to 1 in THP1 cells. Representative of at least 3 independent

experiments (B-C) Realtime PCR of DUSP10 RNA over a timecourse of infection with Pg for (B)

shASC#1mut vs shASC#1 THP1 cells and (C) WT C57BL/6 and Asc-/- murine primary macrophages.

Expression was normalized to 1 in uninfected control cells. Data represent averages ±SD for 3

independent experiments. (D) Realtime PCR of DUSP10 RNA levels in THP1, pLex-EV, and pLex-

DUSP10 cells. Values were normalized to 1 in THP1 cells. Data represent average +SD for three

independent experiments. (E) Western analysis of p-ERK and p-JNK in non-transfected THP1 cells,

pLex-EV- and pLex-DUSP10-containing cells following a timecourse of infection with Pg.

Representative of 3 independent experiments. (F) ELISA of CCL3 and CCL20 in supernatants from cells

expressing pLex-EV or pLex-DUSP10 18 h following infection with Pg. N.D., not detectable. Data

represent averages ±SD for 3 independent experiments. (G) Realtime PCR of ASC and DUSP10 RNA

levels in single or double knockdown THP1 cells created by transduction with lentivirus expressing

shASC#1 or shASC#1mut and shDUSP10 (pLKO-shDUSP10) or an empty vector (pLKO). Values were

normalized to an average of 100 in control cells. Data represent average +SD for three independent

experiments. (H) Western analysis of pERK and pJNK in shASC#1mut- and shASC#1-containing THP1

cells transduced with pLKO control or pLKO-shDUSP10 expressing lentivirus. Representative of 3

independent experiments. (I-J) Realtime PCR analysis of DUSP10 expression following pretreatment

with MAPK inhibitors in (I) THP1 cells and (J) primary mouse macrophages. TNFA is shown as a

control. Expression was normalized to 1 in control cells. Data represent averages ±SD for 3 independent

experiments. *, p<0.05.

Fig. 7. Induction of chemokines and ERK phosphorylation by Pg is IL-1 caspase-1, and NLRP3

independent. (A) Realtime PCR of chemokine RNA in THP1 cells treated with Pg and/or the IL-1

receptor antagonist, Kineret®. Data normalized to an average of 100 in Pg-treated THP1 cells and

represent averages ±SD for 3 independent experiments. N.D., not detectable. (B) ELISA of IL-1, CCL3,

and CCL20 in supernatant from THP1 cells treated with Pg, Kineret®, and/or the caspase-1 inhibitor

YVAD-cmk. (C-D) Western analysis of p-ERK in THP1 cells following a timecourse of infection with

Pg. Kineret® (C) or YVAD-cmk (D) was added as indicated. GAPDH is shown as a loading control.

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Representative of 3 independent experiments. (E) Caspase-1 activation in shASC#1 and pLex-DUSP10

cells following Pg infection. IP/IB was performed in uninfected cells (lanes 1-4) and following 2.5 h Pg

infection (lanes 5-8). An immunoblot for actin in cell lysates is shown as a loading control.

Representative of 3 independent experiments. (F-I) Western blot of p-ERK following a timecourse of

infection with Pg in primary mouse macrophages from WT C57BL/6 vs MyD88-/-, Casp1-/-, Nlrp3-/-, or

Nlrc4-/- mice. GAPDH is shown as a loading control. Representative of 3 independent experiments. (J)

Western blot of p-ERK following a timecourse of infection with Pg in primary mouse macrophages.

Supernatant from Pg-infected WT or ASC-/- mouse macrophages was applied to WT and ASC-/- mouse

macrophages 5 minutes prior to infection.

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Figure 7

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Daniel T. Bergstralh, Irving C. Allen, Yu Lei, Denis Gris and Jenny Pan-Yun TingDebra J. Taxman, Elizabeth A. Holley-Guthrie, Max Tze-Han Huang, Chris B. Moore,

chemokine induction independent of the inflammasomeThe NLR adaptor ASC/pycard regulates DUSP10, MAP kinase (MAPK) and

published online April 12, 2011J. Biol. Chem. 

  10.1074/jbc.M111.221077Access the most updated version of this article at doi:

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  http://www.jbc.org/content/suppl/2011/04/26/M111.221077.DC1

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