Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination
Han et al. Respiratory Research
Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination
SeungHye Han 1
Jacob A. Jerome 1
Alyssa D. Gregory 0
Rama K. Mallampalli 0 1 2
0 Department of Medicine, Division of Pulmonary , Allergy , and Critical Care Medicine, University of Pittsburgh , 15213 Pittsburgh, PA , USA
1 Department of Medicine, The Acute Lung Injury Center of Excellence, University of Pittsburgh , Pittsburgh, PA , USA
2 Medical Specialty Service Line, Veterans Affairs Pittsburgh Healthcare System , Pittsburgh, PA , USA
Background: Cigarette smoke suppresses innate immunity, making smokers more susceptible to infection. The NLRP3 inflammasome is a multi-protein complex that releases interleukin (IL) -1β and IL -18. These cytokines are critical for a timely host response to pathogens. Whether cigarette smoke affects NLRP3 protein levels, and its ability to form an inflammasome, is not known. Methods and results: Using the human monocyte THP1 cell line and C57BL/6 mice, we show that cigarette smoke decreases NLRP3 levels in cells by increasing ubiquitin-mediated proteasomal processing. Half-life of NLRP3 is shortened with the exposure to cigarette smoke extract. Cigarette smoke extract reduces cellular NLRP3 protein abundance in the presence of lipopolysaccharide, a known inducer of NLRP3 protein, thereby decreasing the formation of NLRP3 inflammasomes. The release of IL-1β and IL-18 by inflammasome activation is also decreased with the exposure to cigarette smoke extract both in THP1 cells and primary human peripheral blood macrophages. Conclusions: Cigarette smoke extract decreased NLRP3 protein abundance via increased ubiquitin-mediated proteasomal processing. The release of IL-1β and IL-18 is also decreased with cigarette smoke extract. Our findings may provide mechanistic insights on immunosuppression in smokers and unique opportunities to develop a strategy to modulate immune function.
NLRP3; Cigarette smoke; Ubiquitin
Nucleotide-binding oligomerization (NOD) -like
receptors (NLRs) are cytosolic pattern recognition receptors
responsible for detecting pathogen- or danger-associated
molecular patterns (PAMPs or DAMPs), and are a
critical surveillance system for innate immunity. Most NLRs
share common structural characteristics: a C-terminal
leucine-rich repeat domain that recognizes PAMPs or
DAMPs, a central NOD domain, and a variable
Nterminal effector domain . They are categorized into
five families based on their N-terminal domains. NLRP3
(NALP3) has a pyrin N-terminal domain which binds
with the adaptor protein, ASC, to recruit pro-caspase-1
(p45), forming a multi-protein complex termed the
inflammasome. Upon inflammasome activation, active
caspase-1 (p20 or p10) is cleaved and pro-inflammatory
cytokines such as interleukin (IL) -1β and -18, which
play an important role in host defense against infection,
are subsequently released.
Cigarette smoking has been known to increase
susceptibility to infection likely from dysregulation of immune
function , but the precise underlying molecular
mechanisms remain unclear. A previous study showed that
cigarette smoke alone does not induce secretion of
IL1β, an inflammasome cytokine, in human monocyte
THP1 cells . On the other hand, NLRP3 appears to be
required for bronchoalveolar secretion of IL-1β in
response to cigarette smoke in an in vivo murine model
. However, it is not known whether cigarette smoke
affects NLRP3 cellular concentrations or how it interacts
with other pathogens to affect cellular protein levels.
This question is important, as the identification of an
effect, and underlying mechanism, could provide us a
therapeutic target to control dysregulated immune
function in smokers.
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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The aim of our study was to investigate the molecular
basis for effects of cigarette smoke extract (CSE) on the
NLRP3 inflammasome, a component also activated by
lipopolysaccharide (LPS). We investigated if CSE
interacts with LPS and modulates NLRP3 inflammasome
activity and cytokine release. To this end, we utilized
human monocyte THP1 cells and primary human
peripheral blood macrophages, and C57BL/6 mice to assess
the in vivo effects of cigarette smoke.
Antibodies and reagents
Antibodies against ubiquitin and IL-1β were obtained
from Cell Signaling Technology (Danvers, MA).
Leupeptin, ATP, and antibodies against β-actin and GAPDH were
acquired from Sigma-Aldrich (St. Louis, MO). NLRP6
antibodies were purchased from Abcam (Cambridge,
MA). NLRP3 antibodies were from Adipogen (San Diego,
CA). Antibody against ASC was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Protein A/G agarose
beads, pcDNA3.1D TOPO cloning kits, LPS, and
antibodies targeting the V5 tag were purchased from Thermo
Fisher Scientific (Waltham, MA). IL-18 antibodies were
acquired from MBL International (Woburn, MA).
Caspase-1 antibodies were obtained from R&D Systems
(Minneapolis, MN). Cycloheximide (CHX) was purchased
from Enzo Life Sciences (Farmingdale, NY). MG-132 was
purchased from Ubiquitin-Proteasome Biotechnologies
(Aurora, CO). CSE in a vacuum sealed bottle was
purchased from Murty Pharmaceuticals (Lexington, KY).
Human monocyte THP1 cells were purchased from
Sigma-Aldrich. Human peripheral blood macrophages
and human macrophage cell culture medium were
purchased from Celprogen (Torrance, CA). The clonal
primary macrophages were derived from human peripheral
blood, and confirmed positive for Mcl-1, CD4, CD14,
CD206, CD11b/CR3, CD2, and CD19 expression per the
company. RPMI 1640 medium was purchased from
Thermo Fisher Scientific. Fetal bovine serum (FBS) was
purchased from Gemini (Sacramento, CA). THP1 cells
were cultured in RPMI 1640 medium supplemented with
10% FBS. Human peripheral blood macrophages were
cultured in human macrophage cell culture medium
supplemented with 10% FBS. For the half-life
experiments, CHX was used at a concentration of 40 μg/mL in
fresh medium without FBS supplement, avoiding the
possible breakdown of CHX when it is mixed with an
alkaline substance (i.e., nicotine from CSE). For analysis of
secreted proteins, the medium was removed from
treated cells with the same total cell number and
medium volume per well and precipitated with
trichloroacetic acid (TCA). The precipitated pellet was
then mixed with sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS-PAGE) sample loading buffer
and analyzed by immunoblotting.
RNA was isolated from THP1 cells using the RNeasy
Mini Kit from Qiagen (Valencia, CA) per the protocol
supplied in the kit. The concentration of each RNA
sample was measured, followed by conversion to cDNA
using the High-Capacity RNA-to-cDNA kit from
Thermo Fisher Scientific. Real-time PCR was carried out
in a C1000 Thermal Cycler from Bio-Rad (Hercules,
CA) using SYBR Select Master Mix from Thermo Fisher
Scientific per the included protocol. The primers used
were NLRP3 (5′-ATGAGTGCTGCTTCGACATC-3′,
5′-TTGTCACTCAGGTCCAGCTC-3′), and GAPDH
Immunoprecipitation and immunoblotting
Cells were collected in lysis buffer (0.25% Triton X-100 in
PBS and 1:1000 protease inhibitor mixture) and sonicated
for 12 s, followed by centrifugation at 16,100 × g for
10 min. The cell lysate was then incubated and rotated
with 5 μL of anti-ubiquitin antibody at room temperature
for 1 h. Each sample was then incubated with 30 μL of
protein A/G agarose beads and rotated overnight at 4 °C. After
incubation overnight, the beads were spun down at 0.1 × g
for 3 min and then washed with lysis buffer a total of 3
times. SDS-PAGE sample loading buffer was added to the
beads and they were boiled for 5 min before immunoblot
analysis. Immunoblotting was carried out as follows; Equal
amounts of protein in sample loading buffer were
separated by gel electrophoresis, and transferred onto
nitrocellulose membranes . Restore PLUS Western Blot
Stripping Buffer from Thermo Fisher Scientific (Waltham,
MA) was used to reprobe membranes to detect multiple
Cloning and mutagenesis
Human NLRP3 cDNA was cloned into a
pcDNA3.1D/V5His vector provided in the pcDNA3.1D TOPO cloning kit.
Site directed mutagenesis of NLRP3 was performed using
the QuikChange II XL kit from Agilent Technologies
(Santa Clara, CA) as previously described .
2.5 × 106 Human peripheral blood macrophages were
suspended in 100 μL of 20 mM HEPES in PBS and were
mixed with 4 μg of either WT NLRP3-V5 or K689R
NLRP3-V5 plasmid DNA in a cuvette. The cells were
nucleofected using the Y-010 protocol on an Amaxa
Nucleofector II machine (Basel, Switzerland). After
transfection, 1 mL of 10% FBS human macrophage cell
culture medium was added to each cuvette. The samples
were then transferred to 6-well plates containing 1 mL
of 10% FBS human macrophage cell culture medium, for
a total of 2 mL of culture medium. The cells were grown
until they reached approximately 50% confluency (~48–
72 h) before half-life experiments were initiated.
Male C57BL/6 mice ranging from 8 to 12 weeks of age
were purchased from the Jackson Laboratory (Bar Harbor,
ME) and exposed to 4 non-filtered cigarettes (University
of Kentucky research cigarettes, Lot number 1R5F), 5 days
per week, for a total of 6 months. The mice were
deposited in a smoking chamber which allows the restrained
mice to get direct cigarette smoke exposure towards their
nose . A mouse was exposed to 8.32 mg total
particulate matter per day via the targeted delivery system .
Age-matched littermates were used as controls and were
exposed to filtered air. After 6 months, the mice were
sacrificed by administration of CO2. Following euthanasia,
the lungs were immediately extracted and frozen in liquid
nitrogen for storage at -80 °C. The lungs were then
homogenized in lysis buffer (1% Triton X-100 in PBS and
1:1000 protease inhibitor mixture), before analysis via
SDS-PAGE and immunoblotting. The protocol described
was approved by the University of Pittsburgh Institutional
Animal Care and Use Committee (Protocol #: 12101008).
A Mann-Whitney U test or a Kruskal-Wallis equality of
populations rank test were used to compare
experimental groups. We employed non-parametric methods as
our sample sizes were relatively small to check a normal
sample distribution. All analyses were performed
twotailed, using Stata Statistical Software: Release 13.0
(StataCorp. 2013. College Station, TX: StataCorp LP).
Cigarette Smoke Extract (CSE) decreases NLRP3 protein
NLRP3 protein levels selectively decreased after CSE
exposure in a dose-dependent fashion in human monocyte
THP1 cells (Fig. 1a). We chose 16 h for the duration of
CSE exposure as the effect was maximized after 12–16 h
(data not shown). Other components of the inflammasome
Fig. 1 Cigarette Smoke Extract (CSE) decreases NLRP3 abundance. a THP1 cells (total 3 × 106 cells) were treated with the indicated concentrations of
CSE for 16 h. Cell lysates (Lys) and culture medium (supernatants, Sup) were collected to measure protein levels of NLRP3, NLRP6 (negative control),
ASC, Caspase-1, IL-1β, IL-18 and β-actin/GAPDH (loading control) by immunoblotting. Membranes were stripped to detect multiple different proteins.
Immunoblot shown is representative of four independent experiments. The lysate blots are from two blots that are loaded at the same time from
identical cell lysates. NLRP3, NLRP6 and β-actin are obtained from one, and p45, ASC, and GAPDH are from another blot. For supernatant blots, NLRP3,
NLRP6, and p20 are from the same blot, and IL-1β and IL-18 are from a different blot. b THP1 cells were treated with the indicated concentrations of
CSE for 16 h before RNA isolation. Shown is the NLRP3 mRNA expression fold changes determined by qRT-PCR in a box plot. Data are representative of
four independent experiments. P value was determined by a Kruskal-Wallis test. c Whole lung lysates from non-smoked or smoked C57BL/6 mice were
immunoblotted for NLRP3, NLRP6, and β-actin protein. The relative densitometries of NLRP3 protein for the immunoblots are shown in the right panel.
P value was determined by a Mann-Whitney test
such as pro-caspase-1 (p45) and ASC were not changed
after CSE exposure. It has been reported that oligomeric
NLRP3 inflammasome particles are released from
macrophages after activation of the inflammasome . To
exclude the possibility that cellular levels of NLRP3 protein
decreased due to the secretion of NLRP3 protein as
extracellular oligomeric complexes, we measured the abundance
of secreted proteins in the culture medium (supernatant)
by immunoblotting. Minimal levels of NLRP3 protein were
detected without significant changes after CSE exposure
(Fig. 1a). Neither active caspase-1 (p20) nor cytokines such
as IL-1β and IL-18 were detected in supernatants, which is
consistent with prior studies . Cell viability was not
significantly different with CSE exposure (mean viability of
89% ± standard deviation (S.D.) of 6%, p = 0.30 by
KruskalWallis test). The steady-state mRNA expression of NLRP3,
however, tended to increase after CSE exposure (Fig. 1b).
This suggests that the CSE-induced change in protein
levels is mainly mediated by post-translational regulation,
and the increase in mRNA expression of NLRP3 is likely a
compensatory response to the decreased NLRP3 protein
levels. In order to assess in vivo effects of CSE, we
measured NLRP3 protein levels in mouse lung lysates. The
amount of NLRP3 protein was also selectively reduced in
lung tissue from mice that were exposed to cigarette smoke
for 6 months, compared with those from non-smoking
control mice (Fig. 1c).
CSE shortens NLRP3 half-life via increased
ubiquitinmediated proteasomal degradation
Overall cellular ubiquitination was increased in cells
with CSE exposure in a dose-dependent manner (Fig. 2a).
We have previously showed that NLRP3 protein is
degraded via the ubiquitin proteasome system . To
determine whether CSE increases NLRP3 ubiquitination,
we measured the interaction between NLRP3 protein
and ubiquitin by co-immunoprecipitation both at
baseline and after CSE exposure. The NLRP3-ubiquitin
interaction was increased by more than 100% after CSE
exposure when densitometrically controlled for input
loading (Fig. 2b). CSE induced degradation of NLRP3
protein was inhibited by MG132, a proteasome inhibitor,
but not by a lysosomal inhibitor, leupeptin, confirming
that the effect of CSE on NLRP3 protein occurs through
Fig. 2 CSE triggers NLRP3 protein degradation via the ubiquitin proteasome system. a Above: Overall abundance of ubiquitin (arrows) increases with
CSE exposure. THP1 cells were incubated with the indicated concentrations of CSE for 20 h. Below: Protein levels of ubiquitin, NLRP3, NLRP6, GAPDH,
and β-actin were determined by immunoblotting. Membranes were stripped to detect multiple proteins. Ubiquitin and GAPDH are from the same
blot, and NLRP3, NLRP6, and β-actin are from a separate blot. b THP1 cells were incubated with or without CSE at 80 μg/mL for 17 h. Cells were lysed
and immunoblotted for NLRP3 protein (left panel, input; cell lysates). In the right panel, ubiquitin was immunoprecipitated, followed by NLRP3
immunoblotting. Modestly increased intensity of several bands (arrows) with CSE exposure are observed, and corrected for input the results are shown
graphically (bottom). The bar graph represents mean ± S.D. from two independent experiments. c THP1 cells were incubated with or without CSE at
80 μg/mL, and with or without MG-132 at 20 μM or leupeptin at 40 μM for 16 h. As MG-132 is water insoluble, dimethyl sulfoxide (DMSO) was used as
a vehicle. Control was normalized by the amount of DMSO used (0.1%). Immunoblotting was performed to determine NLRP3, NLRP6, and β-actin
protein levels. The blots shown are representative of two to three independent experiments
the ubiquitin proteasome system (Fig. 2c). The NLRP3
protein mass was reduced by 55% ± 27% with CSE
exposure, and the CSE-induced reduction was not changed
significantly in the presence of DMSO (54% reduction ±
S.D. of 41%, p = 0.77 by Mann-Whitney U test).
Using cycloheximide to inhibit protein synthesis, the
half-life of NLRP3 was also shortened after CSE
exposure (Fig. 3a–b). We previously demonstrated that
Lys689 is a NLRP3 ubiquitination acceptor site . We
transfected primary human peripheral blood
macrophages with wild-type and K689R NLRP3 mutant
plasmids, and measured the half-life of ectopically expressed
NLRP3 protein with or without CSE exposure. The
halflife of K689R NLRP3 mutant was not changed after CSE
exposure, while the wild-type variant had a shortened
half-life similar to the endogenous NLRP3 protein
(Fig. 3c–d). These results suggest that CSE triggers
ubiquitination at the K689 site to accelerate NLRP3
degradation because the K689R mutant exhibited a longer t ½.
In both THP1 cells and primary human peripheral blood
macrophages, the half-life of NLRP3 was slightly longer
(~ 5–6 h in an unchallenged condition) than in U937
cells (~ 4 h in an unchallenged condition) .
CSE attenuates LPS-induced release of IL-1β and IL-18
We previously showed that LPS increases NLRP3
protein levels, thereby increasing the release of IL-1β and
IL-18 in human inflammatory cells when
inflammasomes are activated . Thus, we examined whether
CSE affects NLRP3 inflammasome activation and
subsequent release of cytokines induced by LPS. As before,
the amount of NLRP3 protein modestly increased
with LPS both in THP1 cells and primary human
monocyte-derived macrophages, and CSE alone
produced a reduction in NLRP3 mass (Fig. 4a). Further,
the potent effects of CSE on reduction of NLRP3
mass in cells were maintained despite addition of LPS
to the culture medium (Fig. 4a). LPS prolongs the
half-life of NLRP3 protein by reducing its degradation
. The combinatorial, yet opposite effects of LPS
(inhibiting degradation) and CSE (promoting
degradation) resulted in a 30–40% decrease in NLRP3
protein levels as shown in the lower panels of Fig. 4a.
NLRP3 protein was not released into the culture
medium after CSE exposure. CSE exposure of cells
was sufficient to impair LPS-induced cleavage of
active caspase-1, and thus the release of IL-1β and
ILFig. 3 CSE decreases the half-life of the NLRP3 protein. a THP1 cells were incubated with or without CSE at 80 μg/mL for 18 h prior to CHX exposure
at 40 μg/mL for the indicated time periods. Cells were collected and assayed for NLRP3 and β-actin (loading control) by immunoblotting for a half-life
study. Representative images are shown. b Densitometric plots of NLRP3 protein decay versus time of CHX exposure with best fit lines, depicting the
pooled data mean ± S.D. of three independent experiments. c Primary human macrophages from peripheral blood were transfected with 4 μg of
either WT NLRP3-V5 or the point mutant K689R NLRP3-V5 plasmid. Following transfection, the cells were incubated with or without CSE at 120 μg/mL
for 21 h, prior to CHX exposure at 40 μg/mL at different time points for a half-life study. Cells were collected and assayed for NLRP3-V5 and β-actin by
immunoblotting. d Densitometric plots of adjusted NLRP3 protein decay over time under different conditions were best fitted. The half-life of WT
NLRP3 protein was reduced with CSE exposure, while a K689R NLRP3 mutant was not. Data are mean ± S.D. of two independent experiments
Fig. 4 CSE reduces LPS-induced NLRP3 abundance and release of cytokines. a THP1 cells were incubated with or without 80 μg/mL of
CSE, with or without 400 ng/mL of LPS, for 18 h (four groups; none, CSE, LPS, and LPS + CSE). Human primary macrophages derived from
monocytes were incubated with or without 120 μg/mL of CSE, with or without 200 ng/mL of LPS, for 20 h. Protein levels of NLRP3,
NLRP6, β-actin or GPADH were determined by immunoblotting. Culture medium (supernatants) were also collected for immunoblotting of
NLRP3 and NLRP6. The relative densitometries of NLRP3 protein adjusted for loading control (β-actin or GPADH) are shown in the bottom
panels. Data are mean ± S.D. of two independent experiments. b Equal numbers of THP1 (3 × 106 cells) were plated in equivalent amounts
of culture medium and incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h. Cells were then
exposed to 5 mM of ATP for 15 min. Culture medium was precipitated with TCA for immunoblotting of IL-1β, IL-18, and caspase-1.
Also, equal numbers of human monocyte-derived macrophages in equal amounts of culture medium were incubated with or without
120 μg/mL of CSE, with or without LPS at 400 ng/mL, for 40 h. Cells were then exposed to 5 mM of ATP for 30 min. Cell lysates and
culture medium were collected for immunoblotting. p45 and p20 are probed in the same blot, but presented in different exposure.
IL-1β and IL-18 are from the same blot
18 both in THP1 cells and primary human
monocytederived macrophages (Fig. 4b).
Our study reveals a unique observation that CSE
decreases NLRP3 protein levels, mediated by increased
ubiquitin proteasomal processing. Further, we
demonstrate that CSE suppresses NLRP3 levels even in the
presence of endotoxin thereby preventing the release of
IL-1β and IL-18, critical cytokines for antimicrobial host
defense. Our findings provide potential mechanistic
insights for smoking-related immunosuppression, and the
results may uncover unique opportunities to develop
therapeutic strategies to modulate cytokine signaling. For
example, small molecules that stabilize NLRP3 protein
levels (e.g. targeting of NLRP3 deubiquitinating enzymes)
might be one opportunity that emerges from the results of
these and other studies.
Cigarette smoke has been known to dysregulate both
innate and adaptive immune function, making smokers
more susceptible to infection with worse outcomes [2, 10].
Specifically, smokers have increased susceptibility to
bacterial pneumonia, tuberculosis, periodontitis and surgical
infections . The function of neutrophils and
macrophages in smokers is defective, and they secrete lower
levels of IL-6 and tumor necrosis factor (TNF) , which
are crucial for early response to pathogens .
In addition, IL-1β and IL-18 are also known to play an
important role in host defense. IL-1β activates the release
of TNF and IL-6, and induces Th17 cell differentiation for
cellular adaptive responses . IL-18 is essential for the
induction of IFN-γ and regulation of Th1 responses .
Both cytokines, IL-1β and IL-18, are synthesized as
premature forms in cells, and cleaved by caspase-1 (p20 or
p10) to be bioactive. Caspase-1 is activated by
multiprotein complexes, inflammasomes, consisting of three
components: a sensor NLR, adaptor ASC, and effector
pro-caspase-1 (p45). The most studied is the NLRP3
inflammasome, which is associated with immune
responses that limit microbial invasion, thereby protecting
hosts . A previous study showed that cigarette smoke
decreases caspase-1 activity when THP1 cells are
stimulated with asbestos . However, it is not known whether
cigarette smoke directly affects NLRP3 protein mass. Our
study shows that CSE decreases the level of NLRP3
protein via increased degradation, most likely increased
ubiquitin-mediated proteasomal processing. The
CSEinduced degradation of NLRP3 was observed despite
addition of LPS, a known inhibitor of NLRP3
ubiquitination that stabilizes the NLRP3 protein . Release of
IL1β and IL-18 was also decreased after CSE exposure, likely
from a decreased amount of activated NLRP3
inflammasome complex, as evidenced by reduced levels of active
caspase-1 (Fig. 4b).
The ubiquitin proteasome system mediates disposal of
the majority of proteins in cells. In lung epithelial cells,
cigarette smoke increases total cellular poly-ubiquitinated
proteins , which is consistent with our findings
(Fig. 2a). CSE also induces the degradation of proteins
involved with cell death and proliferation [18, 19]. Our
studies indicate that CSE also suppresses immune function by
modifying the activity of the ubiquitin proteasome system.
Previous studies suggest that the downstream products
of NLRP3 inflammasomes such as IL-1β or IL-18 are
associated with the pathophysiology of smoke-driven
chronic obstructive pulmonary disease (COPD) although
direct evidence to link NLRP3 protein with the disease is
sparse. It is possible that the response to cigarette smoke
could differ by cell type, model system, or kinetics. The
level of IL-1β and/or IL-18 was increased in the lungs,
lavage fluid, or sputum of COPD subjects or animals
exposed to cigarette smoke [20–23]. However, cigarette
smoke alone does not secrete IL-1β in THP1 cells ,
and we found that the release of IL-1β and IL-18 is
reduced after CSE exposure in THP1 cells and
monocyte-derived macrophages. Pulmonary cells such as
lung epithelial cells or alveolar macrophages could have
different responses to cigarette smoke exposure in terms
of cellular NLRP3 protein levels, while monocytes or
monocyte-derived macrophages have decreased NLRP3
protein levels with cigarette smoke leading to
immunosuppression. Indeed, we did not observe significant
changes in NLRP3 protein levels in A549 alveolar
epithelial cells and Beas-2B bronchial epithelial cells after
CSE exposure (data not shown). Another possibility is
that increased IL-1β and IL-18 in COPD is mainly
derived from a pathway other than NLRP3 inflammasome
activation. Although IL-18 knockout mice exhibit reduced
pulmonary inflammation and emphysema compared to
wild-type mice after cigarette smoke [21, 24], pulmonary
inflammation occurred independent of NLRP3/caspase-1
axis after four weeks of cigarette smoke exposure . To
clarify these points, further studies are necessary.
Cigarettes smoke is a mixture of more than 4500
chemical compounds . Cigarette smokers inhale and
absorb many toxic compounds both in vaporous and
particulate phases by burning cigarettes. Immune
modulation by cigarette smoke results from the sum of all
CSE compounds over time rather than a single
compound . We used CSE stored in a vacuum sealed
bottle at -80 °C and thereby minimizing the potential
confounding effect that might occur by dissipating
reactive intermediates. It is unclear to what extent the
cigarette smoke in the airway is absorbed into systemic
circulation in a human body. However, the dose range of
CSE in our study seems appropriate to study immunologic
effects based on previous studies [28–30], although the
degree of systemic absorption may differ individually .
In summary, our study demonstrates that CSE induces
degradation of NLRP3 protein via the ubiquitin
proteasome system in human monocytes and macrophages.
This CSE-induced degradation is not prevented by LPS, a
known stimulus of NLRP3; CSE diminished the formation
of NLRP3 inflammasomes and subsequent release of IL-1β
and IL-18. Future studies will be carried out to specifically
explore molecular targets capable of restoring
inflammasome cytokine release and microbial clearance in smokers.
This may serve as a unique opportunity to prevent
smoking-related morbidity and mortality secondary to
CHX: Cycloheximide; COPD: Chronic obstructive pulmonary disease;
CSE: Cigarette smoke extract; DAMP: Danger-associated molecular pattern;
IL: Interleukin; LPS: Lipopolysaccharide; NLR: NOD-like receptor;
NOD: Nucleotide-binding oligomerization; PAMP: Pathogen-associated
molecular pattern; SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis; TCA: Trichloroacetic acid
This work was supported by a Merit Review Award from the US Department
of Veterans Affairs, National Institutes of Health R01 grants HL096376,
HL097376, HL098174, HL081784, 1UH2HL123502, P01HL114453 (to R.K.M.); an
American Heart Association Award 15POST25700096, Breathe Pennsylvania
Lung Health Research Grant (to S.H.); and Flight Attendant Medical Research
Institute (to A.D.G.). The contents presented do not represent the views of
the Department of Veterans Affairs or the United States Government.
Availability of data and material
The data generated or analyzed during this study available from the
corresponding author on reasonable request.
SH designed the study, performed experiments, analyzed the data, and
wrote the manuscript. JAJ performed experiments and assisted with drafting
or revising of the manuscript. ADG performed animal experiments. RKM
supervised the study and critically revised the manuscript. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
The animal experiments were approved by the University of Pittsburgh
Institutional Animal Care and Use Committee.
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