Distinct Licensing of IL-18 and IL-1b Secretion in
Response to NLRP3 Inflammasome Activation
Rebecca L. Schmidt1, Laurel L. Lenz1,2*
1Integrated Department of Immunology, National Jewish Health, Denver, Colorado, United States of America, 2Departments of Immunology and Microbiology,
University of Colorado - Denver School of Medicine, Denver, Colorado, United States of America
Inflammasome activation permits processing of interleukins (IL)-1b and 18 and elicits cell death (pyroptosis). Whether these
responses are independently licensed or are ‘‘hard-wired’’ consequences of caspase-1 (casp1) activity has not been clear.
Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes
by a ‘‘non-canonical’’ stimulus, the secreted Listeria monocytogenes (Lm) p60 protein. Primed murine dendritic cells (DCs)
responded to p60 stimulation with reactive oxygen species (ROS) production and secretion of IL-1b and IL-18 but not
pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1b, but did not impair IL-18 secretion. Furthermore, DCs
from caspase-11 (casp11)-deficient 129S6 mice failed to secrete IL-1b in response to p60 but were fully responsive for IL-18
secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1b by NLRP3
Citation: Schmidt RL, Lenz LL (2012) Distinct Licensing of IL-18 and IL-1b Secretion in Response to NLRP3 Inflammasome Activation. PLoS ONE 7(9): e45186.
Editor: David M. Ojcius, University of California Merced, United States of America
Received May 23, 2012; Accepted August 14, 2012; Published September 18, 2012
Copyright: ? 2012 Schmidt, Lenz. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants AI065638 and AI055701 and a Colorado Bioscience Discovery Grant to LLL. RLS was supported in part by NIH
training grant T32-AI-074491. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Dr. Lenz is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data
* E-mail: LenzL@njhealth.org
Inflammasomes regulate the processing of pro-IL-1b and pro-
IL-18 by caspase-1 (casp1) , as well as inflammatory cell death
(pyroptosis) . Inflammasome activation occurs in response to
pathogen or damage-associated molecular patterns (PAMPs or
DAMPs). In the case of NLRP3 inflammasomes, these factors
include microbial proteins, crystalline urea, RNA, Alum, and ATP
[3,4,5,6,7,8]. The diversity of these activating stimuli implies that
complex regulatory mechanisms govern NLRP3-dependent re-
sponses. Indeed, production of reactive oxygen species (ROS) and
modification of the thioredoxin interacting protein, TXNIP, have
been shown to cooperatively ‘‘license’’ NLRP3 inflammasomes to
process IL-1b . Recent findings further suggest that casp8 or
casp11 can impact the response of NLRP3 inflammasomes to
certain pathogen-derived ‘‘non-canonical’’ stimuli [10,11]. It is not
known whether ROS also participate in responses to such stimuli.
Moreover, it remains unclear whether processing of IL-18 also
requires ROS production or might instead be regulated by distinct
ROS-independent licensing mechanisms.
Various members of the IL-1 cytokine family exert pro- or anti-
inflammatory effects [12,13]. Indeed, IL-18 and IL-1b act through
distinct cell surface receptors and have distinct consequences
during microbial infections [13,14,15,16]. In some settings IL-18
can even counteract in vivo effects of IL-1b [12,15,17,18]. Such
findings suggest that tailoring the ratio of IL-1b versus IL-18 that is
processed by inflammasomes might permit fine-tuning of in-
flammatory responses and influence infection outcomes. Yet, it is
unknown whether activated NLRP3 inflammasomes can be
differentially licensed to process IL-1b versus IL-18.
Lm is a bacterial pathogen that activates NLRP3 inflamma-
somes during infection [19,20]. As a facultative intracellular
pathogen, Lm can replicate both within the cytosol of host cells
and extracellularly. The LLO hemolysin is required for Lm access
and growth in the cytosol of many cultured mammalian cell types
and for Lm virulence in vivo . LLO also permits activation of
NLRP3 inflammasomes in cultured cells [19,22]. However, the
specific Lm factors responsible for triggering NLRP3 inflamma-
some activation have previously remained undefined.
The Listeria protein p60 is abundantly secreted and essential for
Lm virulence in vivo [23,24,25]. The p60 protein comprises an N-
terminal portion with two LysM domains flanking an SH3 domain
and a C-terminal portion with a repeat region and a NLPC/p60
domain . The p60 NLPC/p60 domain shares homology with
the Bacillius subtilis LytF endopeptidase and is predicted to mediate
cleavage ofpeptide cross-links
[23,25,27]. LysM domains are widely distributed in bacteria and
plants and in several cases have been shown to bind carbohydrates
[28,29,30,31,32,33]. Bacterial SH3 domains similarly contribute
to protein-protein or protein-glycan interactions [32,33].
Recently, we showed that the Lm p60 protein acts on DCs to
indirectly stimulate NK cell activation . The activation of NK
cells by p60 was an IL-18-dependent process, suggesting a role for
inflammasome activation [23,27]. Here, we mapped a region of
the p60 protein that stimulates bone marrow-derived DCs
(BMDCs) to secrete IL-18 and IL-1b by activating NLRP3
inflammasomes. Peptide derivatives of p60 elicited ROS pro-
duction and stimulated NLRP3-dependent secretion of IL-1b and
in bacterial peptidoglycan
PLOS ONE | www.plosone.org1 September 2012 | Volume 7 | Issue 9 | e45186
IL-18, but did not induce pyroptosis. In addition, we found that
production and secretion of IL-1b, but not IL-18, required ROS
production and did not occur in cells deficient for caspase-11.
These data demonstrate that p60 is a non-canonical microbial
activator of NLRP3 inflammasomes and reveal the existence of
different licensing requirements for processing of IL-18 versus IL-
Materials and Methods
This study was carried out in strict accordance with the
recommendations of the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health, the
Public Health Service Policy on the Humane Care and Use of
Laboratory Animals, and the Association for Assessment and
Accreditation of Laboratory Animal Care. The protocols used
were approved by the Institutional Animal Care and Use
Committee at National Jewish Health (Protocol Permit AS2682-
9-13). All efforts were made to minimize suffering.
B6.NLRP32/2mice originally from Dr. Richard Flavell (Yale
University) were generously provided by Dr. Philippa Marrack
(National Jewish Health). C57BL/6 mice (Jackson labs) and 129S6
mice (Taconic labs) were maintained in our colony. All mice were
housed in the Biological Research Center of National Jewish
Wild type Listeria monocytogenes 10403s and an isogenic strain
with an in-frame deletion of p60 were used in these studies .
Escherichia coli TOP10 cells (Invitrogen) were used for expression of
Accession numbers p60 (NCBI accession ZP_05235088.1).
Production and Purification of Proteins and Synthetic
DNA coding for the mature p60, and the L1S, LysM2, or
NLPC/p60 domain fragments were cloned into the pTrcHis-
TOPO TA cloning vector (Invitrogen) as described previously
. IPTG-induced expression in E.coli was performed as
described . Polymyxin B columns were used as indicated by
the manufacturer (Thermo Scientific) to remove LPS from purified
proteins. In some experiments, purified proteins were boiled at
100uC for 10 minutes to denature protein structure or digested
with Pronase protease mixture (Sigma-Aldrich) to ablate BMDC-
stimulating activity. The LysM1 peptide (STVVVEAGDTLW-
NAAE) was synthesized by Biomatick (Wilmington, DE).
BMDC Culture and Stimulation
BMDCs were cultured and stimulated as described . Cells
were plated for .12 hours in antibiotic-free media, primed for 3
hours with 10 ng/ml ultra-pure LPS, 20 mg/ml polyinosine-
polycytidylic acid (PIC) (Invivogen), 2 ng/ml recombinant mouse
IL-12, or media alone. Purified p60 or L1S protein [30 mg/ml],
synthetic LysM1 peptide [15 mg/ml], aluminum hydroxide (alum)
(Accurate Chemical, Westbury, NY) [500 mg/ml], or 5 mM ATP
(Invitrogen) were used as stimuli. For infection studies, the BMDC
were infected with log phase 10403s wt or Dp60 Lm at MOI of 1,
washed at 1 hpi and treated with 10 mg/ml gentamycin. For
inhibitor studies, diphenyl iodonium chloride (DPI) [10 mM]
(Sigma-Alderich,), Z-VAD-FMK, or Z-WEHD-FMK [10 mM]
(Tocris, Minneapolis, MN) were applied 2 hours after TLR
priming agents and 1 hour prior to protein stimulation.
Western Blot, ELISA and LDH Assay
Lysates from BMDCs treated as above were probed with
polyclonal a-IL-1b (R&D Systems) and monoclonal a-Caspase-11
Cas11.17D9 (Biolegend, San Diego, CA ). Concentrations of
murine IL-1b and IL-18 were measured using commercial ELISA
kits (BD Biosciences) on supernatants collected at the indicated
times. LDH release was measured using the Cytotox96 cytotox-
icity kit as per manufacturer instructions (Promega).
Cell lysates were collected from BMDCs treated as above 6
hours post L1S stimulation. RNA was isolated using the RNeasy
kit (Qiagen) and cDNA was obtained using the IM-Prom-II kit
(Promega) according to the manufacturer’s instructions. Specific
expression primers for IL-1b, IL-18, NLRP3, and GAPDH were
synthesized as previously published [34,35,36,37]. RT-PCR
Primers are listed in Table 1. Quantitative PCR reactions using
iTaq SYBR green supermix (BioRad) were performed and
analyzed using the 7300 Real Time PCR instrument (AB Applied
Biosystems). Data were computed using the 7300 ABS software
(AB Applied Biosystems).
BMDCs were washed in PBS and labeled with 10 mM of the
ROS probe, CM-H2DCFDA (Invitrogen) for one hour. Washed
cells were then treated with p60 protein 620 mg/ml PIC or 2 ng/
ml IL-12 for 4 hours. Live cells were washed in PBS and read at
485/528 nm. Fluorescence was assessed using a BioTek Synergy
plate reader and expressed as arbitrary fluorescence units above
Statistic analysis was performed using Graph Pad Prism 5. P
values were assessed using unpaired, two-tailed Student’s t tests
(a=0.05). In the figures, * denotes P values between 0.05 and
0.01, ** denotes P values between 0.01 and 0.001, and *** denotes
P values , or =0.001. All error bars represent SEM. All
experiments were performed in triplicate. Data shown are
representative of at least three independent experiments.
The Lm p60 Protein Induces IL-18 and IL-1b Secretion
Infection of BMDC by Lm induced secretion of IL-18 and IL-
1b, which was significantly reduced upon infection with Dp60 Lm
(Fig. 1A,B). Lm deficient for p60 are not growth deficient in
Table 1. Primers for Quantitative RT-PCR.
GCCCATCCTCTGTGACTCAT AGGCCAC AGGTATTTTGTCG
Independent Cytokine Responses to NLRP3 Activation
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