BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

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BBA - Molecular Basis of Disease

journal homepage: [Link]/locate/bbadis

Hydrogen sulfide attenuates oxidative stress-induced NLRP3 inflammasome

activation via S-sulfhydrating c-Jun at Cys269 in macrophages
Zhe Lina, Naila Altafa, Chen Lia, Mei Chena, Lihong Pana, Dan Wanga, Liping Xiea, Yuan Zhengb,
⁎
Heling Fub, Yi Hanc, Yong Jia,
a
Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for
Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
b
Animal Core Facility of Nanjing Medical University, Nanjing 211166, China
c
Departments of Geriatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China

A R T I C LE I N FO A B S T R A C T

Keywords: Oxidative stress and inflammation are closely related to cardiovascular diseases. Although hydrogen sulfide
Oxidative stress (H2S) has been shown to have powerful anti-oxidative and anti-inflammatory properties, its role in macrophage
Hydrogen sulfide inflammation was poorly understood. The aim of this study was to investigate the role of H2S in the regulation of
S-sulfhydration macrophage NLRP3 inflammasome activation. We reported here that H2S attenuated hydrogen peroxide (H2O2)-
NLRP3 inflammasome
induced NLRP3 inflammasome activation, which led to caspase-1 activation and IL-1β production in macro-
Macrophages
phages. Moreover, H2S exerted its protective effects by lowering the generation of mitochondrial reactive oxygen
species (mtROS). Mechanistically, S-sulfhydration of c-Jun by H2S enhanced its transcriptional activity on SIRT3
and p62, which contributed to the decrease of mtROS production. S-sulfhydration sites are investigated by site
directed mutagenesis. Findings showed that S-sulfhydrated c-Jun exerted its protective influences via a c-Jun
Cys269-dependent manner. Moreover, the protective effects of H2S were absent in macrophage from SIRT3
knockout mice. In conclusion, these results demonstrate that H2S attenuates oxidative stress-induced mtROS
production and NLRP3 inflammasome activation via S-sulfhydrating c-Jun at cysteine 269 in macrophages.

1. Introduction [7,12]. Macrophage infiltration and oxidative stress play a vital role in
the progression of cardiovascular diseases [4,13–16].
Cardiovascular diseases and their consequences are the most serious The NLRP3 inflammasome is composed of the NLRP3(NACHT, LRR
health problems worldwide [1]. In general, oxidative stress and in- and PYD domains-containing protein 3), the adaptor protein ASC
flammation are closely related to cardiovascular diseases, including (apoptosis-associated speck-like protein containing a CARD), and cas-
atherosclerosis, ischemia-reperfusion injury, cardiomyopathy, and pase-1 [17]. The activation of caspase-1 by NLRP3 inflammasome
heart failure [2,3]. Moreover, the concept of oxidative stress and contributes to the processing and secretion of the proinflammatory
chronic inflammation as part of the pathophysiology of cardiovascular cytokines IL-1β and IL-18, which contribute to the so-called sterile in-
diseases has been accepted [4]. The excessive increase in reactive flammation response [18,19]. To date, several studies have indicated
oxygen species (ROS) generation is pivotal in the progression of car- that NLRP3 inflammasome is activated through three possible path-
diovascular diseases [5,6]. Monocytes/macrophages are involved in the ways, including reactive oxygen species (ROS), intracellular potassium
pathogenesis of atherosclerosis, aortic aneurysm, and heart diseases (K+) concentration, and the disruption of the lysosomal membrane
[7–11]. Additionally, monocytes/macrophages have been found to be [20,21]. Macrophage NLRP3 inflammasome activation has been sug-
diagnostic, prognostic, or therapeutic targets in cardiovascular diseases gested to be involved in cardiovascular diseases [22–28].

Abbreviations: ANOVA, analysis of variance; BMM, bone marrow-derived macrophages; BSA, bovine serum albumin; CVD, cardiovascular diseases; DAPI, 4′,6-diamidino-2-pheny-
lindole; DMSO, dimethyl sulphoxide; DHE, dihydroethidium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; H2S, Hydrogen sulfide; H2O2, Hydrogen peroxide; HRP, horseradish peroxidase; IL-1β, interleukin-1β; M-CSF, macrophage colony-stimulating factor; MMTS, methyl metha-
nethiosulfonate; mtROS, mitochondrial reactive oxygen species; NLRP3, NLR family pyrin domain containing 3; PBS, phosphate buffered solution; p62, sequestosome 1; PMA, 4β-
phorbol-12-myristate-13-acetate; PVDF, polyvinylidene fluoride; RIPA, radio immunoprecipitation assay; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate poly-
acrylamide gel electrophoresis; SIRT3, Sirtuin 3
⁎
Corresponding author at: Key Laboratory of Cardiovascular and Cerebrovascular Medicine, Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative
Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing, 211166, Jiangsu, China.
E-mail address: yongji@[Link] (Y. Ji).

[Link]
Received 17 March 2018; Received in revised form 18 May 2018; Accepted 28 May 2018
0925-4439/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Lin, Z., BBA - Molecular Basis of Disease (2018), [Link]
Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

Hydrogen sulfide (H2S) is a gaseous transmitter and plays important subcloned into luciferase reporter vector pGL4 (Promega, Madison, WI,
roles in several diseases [29,30]. H2S is a biologically active gas that is USA) by GENEWIZ. pGL4.74 (Promega, Madison, WI, USA) vector was
synthesized naturally by three enzymes, cystathionine γ-lyase (CSE), also purchased from Promega. After incubation with 5 ng/mL PMA for
cystathionine β-synthetase (CBS) and 3-mercaptopyruvate sulfur- 24 h, THP-1 macrophages were transfected with indicated plasmids by
transferase (3-MST) [31]. In addition, H2S acts as an endogenous sca- using Lipofectamine 3000 reagent (Invitrogen) according to manufac-
venger for reactive oxygen species [32–34]. Furthermore, H2S physio- turer's recommendations and the method as previously described [37].
logically modifies protein cysteines to form S-sulfhydration, which Cells were cultured for 24 h after transfection. The luciferase activity
appears to be a physiologic posttranslational modification for proteins was measured by the dual luciferase assay system (Promega, Madison,
[29,35]. Our previously studies have demonstrated that H2S has pow- WI, USA) according to the manufacturer's instructions. Data were nor-
erful antioxidative and anti-inflammatory properties in several patho- malized by the activity of Renilla luciferase. Mcrophages were trans-
logical process, including myocardial fibrosis, endothelial dysfunction, fected with NLRP3 siRNA (sense 5′-GUUUGACUAUCUGUUCUdTdT-3′
diabetes-accelerated atherosclerosis, myocardial hypertrophy, and tes- [41]; GenePharma, Shanghai, China) using Lipofectamine 3000 (In-
ticular dysfunction [36–40]. vitrogen, Carlsbad, CA, USA) according to the manufacturer's instruc-
In this study, exogenous treatment with H2O2, a ROS-generating tions.
agent, was used to mimic oxidative stress in macrophages. We de-
monstrated that H2S attenuated H2O2-induced NLRP3 inflammasome 2.3. Western blotting
activation in macrophages THP-1. H2O2-induced NLRP3 inflammasome
activation led to caspase-1 activation and IL-1β production. We further RIPA lysis buffer (Biyuntian, Hangzhou, China) containing protease
demonstrated that NLRP3 bound to ROS-generated mitochondria in inhibitor cocktail (ThermoFisher, Waltham, MA, USA) was used to
macrophages exposed to H2O2. However, pretreatment with H2S re- obtain whole-cell lysates from macrophages. Protein concentrations
duced these above effects of H2O2 on macrophages. In addition, we were quantified using BCA Protein Assay Kit (Pierce, Rockford, IL,
found that H2S enhanced c-Jun transcriptional activity directly via S- USA). Equal amounts of protein were loaded into SDS-PAGE and
sulfhydration of c-Jun protein at cysteine 269. The increased c-Jun transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA).
transcriptional activity contributed to the increasing expression of Membranes were incubated with rabbit antibodies to caspase-1 (#3866,
SIRT3 and p62, which exhibited remarkable antioxidant and anti-in- CST, Danvers, MA, USA), IL-1β (#ab45692, Abcam, Cambridge, MA,
flammatory effects in macrophages. This study deepens our under- USA), CBS (#ab135626, Abcam), CSE (#ab151769, Abcam), NLRP3
standing of the protective effects of H2S via c-Jun S-sulfhydration in (#13158, CST), SIRT3 (#2627, CST), and GAPDH (#AP0063, Bioworld,
macrophages. Nanjing, China) as well as mouse antibodies to caspase-1 p20 (#AG-
20B-0048, AdipoGen, San Diego, CA, USA), 3-MST (#sc-374,326, Santa
2. Materials and methods Cruz Biotechnology, Santa Cruz, CA, USA), and p62 (#MB9009,
Bioworld). All blots were overlaid with appropriate HRP-conjugated
2.1. Cell culture and treatment secondary antibodies and visualized by enhanced chemiluminescence
substrate (Bio-Rad, Hercules, CA, USA).
THP-1 human monocyte cells obtained from Cell Bank at the
Chinese Academy of Sciences were cultured in 25 mM HEPES-buffered 2.4. Immunofluorescent staining and confocal microscopy
RPMI 1640 (Life Technologies, Rockville, MD, USA) containing 10%
FBS (Life Technologies, Rockville, MD, USA) and 50 μM β-mercap- THP-1 monocyte cells were seeded on glass slides (NEST, Wuxi,
toethanol (Life Technologies, Rockville, MD, USA). THP-1 cells were China) and incubated with 5 ng/mL PMA for 24 h. Then, the cells were
differentiated into macrophages by incubation with 5 ng/mL 4β- treated as described above. After the treatment, the cells were washed
phorbol-12-myristate-13-acetate (PMA; Biyuntian, Hangzhou, China) twice with sterile PBS and fixed with ice-cold methanol, permeabilized
for 24 h. RPMI 1640 complete medium was replaced before addition of with 0.01% Triton X-100 and blocked in 3% BSA. The cells were then
H2S donor, NaHS (100 μM; Sigma-Aldrich, St Louis, MO, USA). After 4 h incubated overnight with primary antibodies, including anti-NLRP3
treatment, cells were incubated in freshly prepared medium containing (#AG-20B-0014, AdipoGen, San Diego, CA, USA), anti-caspase-1
H2O2 (250 μM; Sigma-Aldrich, St Louis, MO, USA) for a further 4 h. (#3866, CST), and anti-c-Jun (#9165, CST). For experiments that used
Mitochondria-targeted antioxidant Mito-TEMPO was purchased from mitotracker, cells were stained with 5 μM MitoTracker Deep Red FM
Abcam (ab144644, Cambridge, MA, USA). Cathepsin B inhibitor CA- (#M22426, Life Technologies) for 20 min prior to methanol fixation.
074Me was purchased from Bachem (4027913, Bubendorf, Secondary fluorescent antibodies (Alexa-488, or -594; Life
Switzerland). P2X7 receptor inhibitor A438079 was purchased from Technologies) were added for 1 h and DAPI (#sc-24,941, Santa Cruz)
Selleck Chemicals (S7705, Shanghai, China). For cell experiments, these was used for nuclear counterstaining. Samples were imaged through
reagents were dissolved in dimethyl sulfoxide (DMSO) and were used at confocal microscope (Zeiss LSM 410, Oberkochen, Germany) and
the desired concentration (indicated in figure legends). quantified using Image-Pro Plus analysis software.

2.2. Transient transfection and dual-luciferase reporter assay 2.5. Measurement of ROS and mtROS formation

Human c-Jun (Gene ID: 3725) cDNA was purchased from GENEWIZ, Superoxide production in cells was detected by dihydroethidium
Inc. (Suzhou, China). Human c-Jun cDNA was cloned into pcDNA3.1 by (DHE; Vigorous, Beijing, China) assay as the manufacturer's instruc-
GENEWIZ. Single mutation at cysteine-99 (C99A), cysteine-269 tions. Briefly, macrophages were treated as described above, after
(C269A), or cysteine-320 (C320A) in c-Jun was conducted by which the cells were washed twice with PBS and incubated with 5 μM of
GENEWIZ. pAP1-TA-luc vector is a luciferase reporter construct with DHE for 30 min and washed twice with PBS. Fluorescence was mea-
multiple AP-1 (TGACTAA) response elements and purchased from sured with a Nikon TE2000 Inverted Microscope and quantified using
Biyuntian (Hangzhou, China). The response elements of c-Jun in SIRT3 Image-Pro Plus analysis software. Mitochondrial ROS were measured
and p62 genes promoter were predicted via using JASPAR databases using MitoSOX (#M36008, Molecular Probes, Carlsbad, CA, USA).
(Supplementary data 1). The wild type and deletion of the c-Jun re- Briefly, macrophages were treated as above described, after which the
sponse element in the SIRT3 and p62 promoter were purchased from cells were washed twice with PBS and loaded with 5 μM of MitoSOX
GENEWIZ, Inc. (Suzhou, China). SIRT3-WT (−500–−1), and 100 nM MitoTracker Green FM (#M7514, Molecular Probes) for
SIRT3Δ−337–−325, p62-WT (−500–−1), and p62Δ−389–−377 were 20 min. Fluorescence was measured through confocal microscope (Zeiss

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Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

LSM 410) and quantified using Image-Pro Plus analysis software. committees of Nanjing Medical University. Written informed consent
was obtained from all volunteers.
2.6. Modified biotin switch assay of S-sulfhydration
2.10. Statistical analysis
Modified biotin switch assay was performed as described by Ju et al.
[42]. Briefly, cells were lysed in HEN buffer (250 mM HEPES (pH 7.7), All data are presented as means ± s.e.m. as indicated. Statistical
0.1 mM neocuproine, and 1 mM EDTA) supplemented with 150 μM analysis was performed by a two-tailed unpaired Student's t-test or one-
deferoxamine, 1% NP-40, and protease inhibitor cocktail (Thermo- way ANOVA (analysis of variance), followed by the post hoc Tukey's
Fisher). Samples were sonicated and centrifuged at 16,000 g for 15 min test. For all tests, P-values lower than 0.05 were considered statistically
at 4 °C. Protein concentrations of the supernatant samples were quan- significant.
tified using BCA Protein Assay Kit (Pierce). Lysates were diluted to
reach 2 mg/mL final protein concentration and were added to blocking 3. Results
buffer (HEN buffer adjust to 2.5% SDS and 20 mM MMTS (methyl
methanethiosulfonate)) at 50 °C for 30 min with frequent vortexing. 3.1. Effects of H2S on NLRP3 inflammasome activation of macrophages
The MMTS was then removed by acetone and the proteins were pre- exposed to H2O2
cipitated at −20 °C for 20 min. Proteins were resuspended in HENS
buffer (HEN buffer containing 1% SDS) and 4 mM biotin-HPDP (N-[6- IL-1β, which is synthesized mainly by monocytes and macrophages,
(biotinamido)hexyl]-3′-(2′-pyridyldithio) propinamide). After incuba- is a powerful mediator of inflammatory response to sterile insults. The
tion for 2 h at 25 °C, biotinylated proteins were purified by streptavidin- stimulation of H2O2 on macrophages cells induced the transcription of
agarose beads (ThermoFisher), which were then washed with HENS proinflammatory mediators [46]. To determine whether H2O2 induces
buffer. The biotinylated proteins were eluted in 2× Laemmeli sample NLRP3-mediated IL-1β production in macrophages, we ascertained
buffer and subjected to western blotting analysis with anti-c-Jun anti- NLRP3-inflammasome activation of macrophages exposed to H2O2. The
body. The used chemicals were purchased from Sigma-Aldrich (St IL-1β production and caspase-1 activation were analyzed by western
Louis, MO, USA). blotting and we found that macrophages (human primary macrophages
and THP-1-derived macrophages) responded to H2O2 with a significant
2.7. Real-time PCR increasing of IL-1β production and caspase-1 activation (Fig. 1A and
Supplementary Fig. 1A). However, IL-1β production and caspase-1 ac-
Total RNA was extracted using TRIzol reagent (Takara, Dalian, tivation were reduced by pretreatment of macrophages with NaHS (H2S
China). Total RNA was reverse transcribed to cDNA by using the donor; Fig. 1A and Supplementary Fig. 1A). Next, we measured the co-
PrimeScript RT Master Mix Kit (Takara, Dalian, China). Real-time PCR localization of caspase-1 and NLRP3 in the presence or absence of H2S.
was carried out with the resulting cDNAs in triplicate using SYBR Green Findings showed an increased co-localization of caspase-1 and NLRP3
remix (Takara, Dalian, China) and the ABI 7500 Real-Time PCR System in macrophages (human primary macrophages and THP-1-derived
(Applied Biosystems, Foster City, CA, USA). GAPDH was used for nor- macrophages) exposed to H2O2, while the effect was again rescued by
malization. Primers' sequences are obtained from PrimerBank (pga. H2S (Fig. 1B and Supplementary Fig. 1B). Moreover, the protein levels
[Link]/primerbank). (PrimerBank ID: SIRT3, 157671925c1; of H2S-producing enzymes CBS, CSE, and 3-MST were measured and
p62, 214830450c1; GAPDH, 378404907c1) in supplementary data 1. findings showed that the expression of H2S-producing enzymes were
decreased in H2O2-treated macrophages (Supplementary Fig. 1C). The
2.8. Animals and murine macrophage cells culture decreased IL-1β production and caspase-1 activation in H2S-pretreated
macrophages might be dependent on NLRP3 inflammasome activation.
SIRT3−/− mice were the gift of Professor Hongliang Li (Wuhan To test this hypothesis, the NLRP3 was knocked down in THP-1-derived
University, Wuhan, China) [37]. All animal experiments were approved macrophages by short interfering RNA (siRNA, Supplementary Fig. 1D).
by the Committee on Animal Care of Nanjing Medical University. Results showed that IL-1β production and caspase-1 activation from the
Peritoneal macrophages were harvested from the peritoneal cavity with THP-1-derived macrophages with NLRP3 knockdown was substantially
a cold RPMI 1640 medium. After centrifugation at 1200 rpm for 10 min diminished in response to H2O2 with or without H2S pretreatment
at 4 °C, the cell pellet was washed once and resuspended in a complete (Supplementary Fig. 1E). Recent studies have indicated that oxidative
medium: 25 mM HEPES-buffered RPMI 1640 (Life Technologies, stress-induced mtROS generation leads to NLRP3 inflammasome for-
Rockville, MD, USA) containing 10% FBS (Life Technologies, Rockville, mation and activation in macrophages [47]. Thus, THP-1-derived
MD, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin [38]. macrophages were pretreated with the P2X7 antagonists A438079, ca-
Bone marrow-derived macrophages (BMM) were obtained by treating thepsin B inhibitor CA-074Me, or Mito-TEMPO prior to stimulation
bone marrow cells with macrophage colony-stimulating factor (M-CSF). with H2O2. Surprisingly, when macrophages were pretreated with Mito-
Briefly, mice were sacrificed, bone marrow was isolated from the hind TEMPO, but not with A438079 or CA-074Me, we observed decreased
legs. The bones were flushed with RPMI 1640 medium using a 5-mL IL-1β production and caspase-1 activation in macrophages exposed to
syringe and a 25-gauge needle. Bone marrow cells were isolated and H2O2 (Supplementary Fig. 2A). Therefore, H2O2-induced NLRP3 in-
cultured in complete RPMI 1640 media containing recombinant murine flammasome activation was independent of P2X7 receptor, or the ca-
M-CSF (10 ng/mL, Peprotech, Rocky Hill, NJ, USA) for 5–7 days thepsin B activation, but dependent on the mitochondrial ROS gen-
[43,44]. eration in macrophages.

2.9. Human macrophage culture 3.2. Effects of H2S on mitochondrial ROS production of macrophages
exposed to H2O2
Human peripheral blood monocytes were isolated from each subject
by density gradient centrifugation and cultured as our described pre- To determine whether the protective role of H2S against NLRP3
viously [45]. Briefly, human peripheral blood monocytes were differ- inflammsome activation is related to reduction of mitochondrial ROS
entiated into macrophages using human macrophage colony stimu- (mtROS), mitochondrial redox status was monitored by the mitochon-
lating factor (M-CSF, 1 ng/mL, Peprotech, Rocky Hill, NJ, USA) for drial superoxide-sensitive dye MitoSOX. The fluoroprobe MitoSOX was
5 days. The investigation conforms to the principles outlined in the introduced for selective detection of superoxide in the mitochondria of
Declaration of Helsinki. The study protocol was approved by the ethics live cells and was validated with confocal microscopy by measuring the

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Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

Fig. 1. Protective effects of H2S on H2O2-induced NLRP3 inflammasome activation in macrophages. (A) and (B) Human primary macrophages were pretreated with
NaHS (100 μM; H2S donor) for 4 h before H2O2 (250 μM) for a further 4 h. (A) Representative western blotting and quantification of caspase-1, caspase-1 p20, pro-IL-
1β, and IL-1β protein expression. Data are means ± s.e.m., n = 4, *P < 0.05 (one-way ANOVA, post hoc Tukey). (B) Intracellular co-localization of caspase-1 and
NLRP3 in the presence and absence of H2S was examined by confocal microscopy. Representative data were shown from three independent experiments (n = 3).
Scale bars, 10 μm.

Fig. 2. Effects of H2S on cellular ROS and mitochondrial ROS production of macrophages exposed to H2O2. Human primary macrophages were pretreated with NaHS
(100 μM; H2S donor) for 4 h before H2O2 (250 μM) for a further 4 h. (A) Mitochondrial ROS was measured using MitoSOX. Mitochondrial localization of MitoSOX
signal was confirmed by co-localization with MitoTracker Green. Representative data were shown from three independent experiments (n = 3). Scale bars, 10 μm.
(B) Mitochondrial recruitment of NLRP3 was examined by using MitoTracker Deep Red stain and multicolor confocal immunofluorescence microscopy.
Representative data were shown from three independent experiments (n = 3). Scale bars, 10 μm.

mtROS. MitoSOX fluorescence colocalized with the mitochondria as Furthermore, cellular redox status was monitored by the superoxide-
detected using the probe MitoTracker Green. As expected, treatment of sensitive dye dihydroethidium (DHE). THP-1-derived macrophages re-
macrophages (human primary macrophages and THP-1-derived mac- sponded to H2O2 with a significant rise in cellular ROS formation and
rophages) with H2O2 resulted in mtROS generation as demonstrated by this rise was reduced by pretreatment of cells with H2S donor (Sup-
an increasing in MitoSOX fluorescence; however, pretreatment with plementary Fig. 2C).
H2S attenuated mtROS generation (Fig. 2A and Supplementary Fig. 2A).
Moreover, recruitment of NLRP3 to mitochondrial was detected with
3.3. H2S S-sulfhydrated c-Jun at cysteine-269 and reduced mtROS
MitoTracker Deep Red stain and multicolor confocal immuno-
production
fluorescence microscopy. The results exhibited an increased recruit-
ment of NLRP3 by mitochondrial in macrophages (human primary
Previous studies have manifested that H2S increases activator pro-
macrophages and THP-1-derived macrophages) exposed to H2O2, while
tein 1 (AP-1) binding activity with SIRT3 promoter and SIRT3 sup-
the effect was also rescued by H2S (Fig. 2B and Supplementary Fig. 2B).
presses NLRP3 inflammasome activation in association with decreased

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Fig. 3. H2S S-sulfhydrated the c-Jun at cy-

steine-269 and reduced mtROS production.
(A) Human primary macrophages were pre-
treated with NaHS (100 μM; H2S donor) for
4 h. After that, the cells were collected for
modified biotin switch assay and western
blotting for c-Jun. Representative western
blotting and quantification of S-sulfhydrated
c-Jun and c-Jun protein expression. Data are
means ± s.e.m., n = 4, *P < 0.05 (two-
tailed unpaired Student's t-test). (B) THP-1-
derived macrophages were transfected with
control or wild-type c-Jun plasmids for 24 h
4 following incubation of NaHS (100 μM) for
additional 4 h. After that, the cells were col-
lected for modified biotin switch assay and
western blotting for c-Jun. Representative
western blotting and quantification of S-
sulfhydrated c-Jun and c-Jun protein ex-
pression. Data are means ± s.e.m., n = 6,
*P < 0.05 (two-tailed unpaired student's t-
test). (C) THP-1-derived macrophages were
transfected with control or wild-type c-Jun
plasmids or wild-type c-Jun, C99A mutant,
C269A mutant, and C320A mutant plasmids
for 24 h following incubation of NaHS
(100 μM) for additional 4 h. Representative
western blotting and quantification of S-
sulfhydrated c-Jun and c-Jun protein ex-
pression. Data are means ± s.e.m., n = 4,
*P < 0.05 (one-way ANOVA, post hoc
Tukey). (D) and (E) THP-1-derived macro-
phages were transfected with wild-type c-
Jun, C99A mutant, C269A mutant, and
C320A mutant plasmids for 24 h following
incubation of NaHS (100 μM) for additional
4 h before H2O2 (250 μM) for a further 4 h.
(D) Cellular ROS production was detected by
DHE staining. Scale bars, 100 μm. Data are
means ± s.e.m., n = 5, *P < 0.05 (one-way
ANOVA, post hoc Tukey). (E) Mitochondrial
ROS was measured using fluorescent probe
MitoSOX. Mitochondrial localization of
MitoSOX signal was confirmed by co-locali-
zation with MitoTracker Green.
Representative data were shown from three
independent experiments (n = 3). Scale bars,
10 μm.

mtROS production. We wondered whether H2S mediates the decreased with a significant rise in cellular ROS and mtROS formation, which was
mtROS production by S-sulfhydrating c-Jun (subunit of AP-1) in mac- rescued by H2S treatment (Fig. 3D and E). However, mutation of C269,
rophages. S-sulfhydration is a physiological process wherein H2S at- but not C99 or C320 in the c-Jun protein, reversed the effects of H2S on
taches an additional sulfur to the thiol (–SH) groups of cysteines the reducing of cellular ROS and mtROS production, pointing to the
yielding a hydropersulfide (–SSH). We examined S-sulfhydration of c- critical role of C269 in c-Jun S-sulfhydration. (Fig. 3D and E).
Jun in macrophages exposed to H2S by the modified biotin switch
assay. As expected, S-sulfhydration of c-Jun in macrophages (human 3.4. Effects of S-sulfhydrated c-Jun on NLRP3 inflammasome activation
primary macrophages and THP-1-derived macrophages) markedly in-
creased after H2S treatment (Fig. 3A and Fig. 3B). To identify the S- Since S-sulfhydrated c-Jun reduced mtROS production, we ex-
sulfhydrated cysteine residue of c-Jun, we mutated cysteine-99, cy- amined the influences of S-sulfhydrated c-Jun on NLRP3 inflammasome
steine-269, and cysteine-320 (the only three cysteine residues) in the c- activation. We transfected the wild-type c-Jun or C269A plasmids to
Jun protein. All the mutants contained only a single amino acid mu- THP-1-derived macrophages exposed to H2O2. Results showed that S-
tation (cysteine to alanine) at the designated residue (C99A, C269A, sulfhydration of c-Jun increased by pretreatment with H2S in wild-type
C320A). Mutation of cysteine-269, but not cysteine-99 and cysteine-320 c-Jun-transfected macrophages, while mutation of C269 in the c-Jun
abolished the increased level of c-Jun S-sulfhydration after H2S treat- protein reversed the effect of H2S on c-Jun S-sulfhydration (Fig. 4A).
ment compared with wild-type c-Jun transfection (Fig. 3C and Sup- Furthermore, IL-1β production and caspase-1 activation were reduced
plementary Fig. 3). To determine whether C269 was involved in H2S- by pretreatment of wild-type c-Jun-transfected macrophages with H2S,
reduced mtROS production, DHE and MitoSOX Red assays were per- while these influences of S-sulfhydrated c-Jun were also reversed in
formed. We found that THP-1-derived macrophages responded to H2O2 C269A-transfected macrophages (Fig. 4B). Additionally, we measured

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Fig. 4. Effects of S-sulfhydrated c-Jun on NLRP3 inflammasome activation. THP-1-derived macrophages were transfected with wild-type c-Jun and C269A mutant
plasmids for 24 h. After that macrophages were treated the same as described in Fig. 1. (A) Cells were collected for modified biotin switch assay and western blotting
for c-Jun. Representative western blotting and quantification of S-sulfhydrated c-Jun and c-Jun protein expression. Data are means ± s.e.m., n = 5, *P < 0.05 (one-
way ANOVA, post hoc Tukey). (B) Representative western blotting and quantification of caspase-1, caspase-1 p20, pro-IL-1β, and IL-1β protein expression. Data are
means ± s.e.m., n = 5, *P < 0.05 (one-way ANOVA, post hoc Tukey). (C) Intracellular co-localization of caspase-1 and NLRP3 in the presence and absence of H2S
was examined by confocal microscopy. Representative data were shown from three independent experiments (n = 3). Scale bars, 10 μm. (D) Mitochondrial re-
cruitment of NLRP3 was examined by using MitoTracker Deep Red stain and multicolor confocal immunofluorescence microscopy. Representative data were shown
from three independent experiments (n = 3). Scale bars, 10 μm.

the co-localization of caspase-1 and NLRP3 in the wild-type c-Jun- or p62 (Supplementary Fig. 4). Furthermore, we transfected the wild-type
C269A-transfected THP-1-derived macrophages. As expected, pretreat- c-Jun or C269A plasmids to THP-1-derived macrophages exposed to
ment with H2S showed an attenuated co-localization of caspase-1 and H2O2 and explored the nuclear translocation of c-Jun in the wild-type c-
NLRP3 in wild-type c-Jun-transfected macrophages, while this effect of Jun- or C269A-transfected macrophages. The results showed that pre-
S-sulfhydrated c-Jun was again reversed in C269A-transfected macro- treatment with H2S displayed an increased nuclear translocation of c-
phages (Fig. 4C). Similarly, co-localization of NLRP3 with mitochondria Jun in wild-type c-Jun-transfected macrophages, while this effect of S-
was also decreased in wild-type c-Jun-transfected group and increased sulfhydrated c-Jun was again reversed in C269A-transfected macro-
in C269A-transfected group (Fig. 4D). Collectively, these results sug- phages (Fig. 5B). To investigate whether S-sulfhydrated c-Jun could
gested that H2S inhibited the NLRP3 inflammasome activation via S- increase SIRT3 and p62 expression, we examined the expression of
sulfhydration of c-Jun at cysteine-269. SIRT3 and p62 by using real-time PCR and western blotting assays.
Findings showed that the mRNA levels of SIRT3 and p62 increased by
pretreatment with H2S in wild-type c-Jun-transfected macrophages,
3.5. S-sulfhydration of c-Jun enhanced its transcriptional activity on SIRT3 while this effect of S-sulfhydrated c-Jun was reversed in C269A-trans-
and p62 fected macrophages (Fig. 5C). Consistently, the protein levels of SIRT3
and p62 also increased by pretreatment with H2S in wild-type c-Jun-
Previous studies have revealed that SIRT3 and p62 suppress NLRP3 transfected macrophages, while this influence of S-sulfhydrated c-Jun
inflammasome activation in association with mtROS production and was again reversed in C269A-transfected macrophages (Fig. 5D). To
damaged mitochondria clearance, respectively. Additionally, c-Jun is investigate whether the S-sulfhydration of c-Jun could regulate its
an important factor in p62 transcription. As expected, treatment of transcriptional activation, pAP1-TA-luc luciferase reporter plasmids, a
human primary macrophages with H2O2 decreased the expression of vector construct with multiple c-Jun/AP-1 (TGACTAA) response ele-
SIRT3 and p62; however, pretreatment with H2S restored the SIRT3 and ments, was transfected into macrophages. The reporter assays revealed
p62 expression (Fig. 5A). To test whether the upregulated expression of a reduced luciferase activity in macrophages exposed to H2O2; how-
SIRT3 and p62 induced by H2S is independent on its direct mtROS- ever, the activity was reversed by H2S (Fig. 5E). Furthermore, we found
scavenging effect, we treated macrophages with the mitochondria-tar- that the promoters (−500–−1) of human SIRT3 (−337–−325) and
geted antioxidant Mito-TEMPO prior to exposure to H2O2 and measured p62 (−389–−377) contain c-Jun response element by using JASPAR
the expression of SIRT3 and p62. However, results showed that the databases ([Link] (Supplementary data 1). A
pretreatment of Mito-TEMPO failed to increase the levels of SIRT3 and

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Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

Fig. 5. S-sulfhydration of c-Jun enhanced its transcriptional activity on SIRT3 and p62. (A) Human primary macrophages were pretreated with NaHS (100 μM; H2S
donor) for 4 h before H2O2 (250 μM) for a further 4 h. Representative western blotting and quantification of SIRT3 and p62 protein expression. Data are
means ± s.e.m., n = 6, *P < 0.05 (one-way ANOVA, post hoc Tukey). (B-G) THP-1-derived macrophages were treated the same as described in Fig. 4. (B)
Intracellular distribution of c-Jun was determined by confocal microscopy. Representative data were shown from three independent experiments (n = 3). Scale bars,
10 μm. (C) The expression SIRT3 and p62 of mRNA was determined by real-time PCR. Data are means ± s.e.m., n = 4, *P < 0.05 (one-way 7 ANOVA, post hoc
Tukey). (D) Representative western blotting and quantification of SIRT3 and p62 protein expression. Data are means ± s.e.m., n = 5, *P < 0.05 (one-way ANOVA,
post hoc Tukey). (E) Macrophages were transfected with pAP1-TA-luc luciferase vector and pGL4.74 plasmids for 12 h following incubation of NaHS (100 μM) for
additional 4 h before H2O2 (250 μM) for a further 4 h. The luciferase activity was determined using a dual-luciferase reporter assay system. Data are means ± s.e.m.,
n = 7, *P < 0.05 (one-way ANOVA, post hoc Tukey). (F) Macrophages were transfected with wild-type c-Jun or C269A mutant plasmids. After 24 h cells were
transfected with SIRT3-WT or SIRT3-Δ (p62-WT or p62-Δ (G)) luciferase vector and pGL4.74 plasmids for 12 h following incubation of NaHS (100 μM) for additional
4 h before H2O2 (250 μM) for a further 4 h. The luciferase activity was determined using a dual-luciferase reporter assay system. Data are means ± s.e.m., n = 4,
*P < 0.05 (one-way ANOVA, post hoc Tukey).

number of luciferase reporter plasmids containing the wild type and type and SIRT3-WT or p62-WT transfected THP-1-derived macro-
deletion of the c-Jun response elements in the SIRT3 and p62 promoter phages. However, H2S-induced enhancement of SIRT3 or p62 promoter
were constructed. With the deletion constructs, the stimulatory effects activity was abolished in c-Jun-C269A and SIRT3-Δ or p62-Δ trans-
of H2S on SIRT3 or p62 promoter activity were observed in c-Jun-wild fected macrophages (Fig. 5F and G). These suggest that the c-Jun

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Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

Fig. 6. The protective effects of H2S on SIRT3−/− mice macrophage. Peritoneal macrophages and bone marrow-derived macrophages (BMM) were treated the same
as described in Fig. 1. (A) SIRT3−/− bone marrow-derived macrophages were collected for modified biotin switch assay and western blotting for c-Jun. Re-
presentative western blotting and quantification of S-sulfhydrated c-Jun and c-Jun protein expression. Data are means ± s.e.m., n = 5, *P < 0.05 (one-way
ANOVA, post hoc Tukey). (B) Representative western blotting and quantification of caspase-1, caspase-1 p20, pro-IL-1β, and IL-1β protein expression in SIRT3−/−
BMM. Data are means ± s.e.m., n = 5, *P < 0.05 (one-way ANOVA, post hoc Tukey). (C) Intracellular co-localization of caspase-1 and NLRP3 in the presence and
absence of H2S was examined by confocal microscopy in SIRT3−/− peritoneal macrophages. Representative data were shown from three independent experiments
(n = 3). Scale bars, 10 μm. (D) Mitochondrial localization of MitoSOX signal was confirmed by co-localization with MitoTracker Green in SIRT3peritoneal mac-
rophages. Representative data were shown from three independent experiments (n = 3). Scale bars, 10 μm. (E) Mitochondrial recruitment of NLRP3 9 was examined
by using MitoTracker Deep Red stain and multicolor confocal immunofluorescence microscopy in SIRT3−/− peritoneal macrophages. Representative data were
shown from three independent experiments (n = 3). Scale bars, 10 μm.

response elements on the upstream of the SIRT3 and p62 promoters are however, the activation of caspase-1 and production of IL-1β did not
responsible for the effects of H2S on SIRT3 and p62 transcription by S- reduce in SIRT3−/− BMM exposed to H2O2 and H2S pretreatment
sulfhydrating c-Jun. Collectively, these results suggest that H2S upre- (Fig. 6B). Thirdly, the increasing of co-localization of caspase-1 and
gulates the SIRT3 and p62 genes expression via S-sulfhydrating c-Jun NLRP3 in SIRT3−/− peritoneal macrophages exposed to H2O2 also did
and increasing its binding activity with the SIRT3 and p62 promoters. not rescue with H2S pretreatment (Fig. 6C). Fourthly, treatment of
SIRT3−/− peritoneal macrophages with H2O2 resulted in mtROS gen-
eration and pretreatment with H2S also did not attenuate this effect
3.6. The protective effects of H2S on macrophage from SIRT3−/− mice (Fig. 6D). Fifthly, the recruitment of NLRP3 to mitochondrial also dis-
played no difference in SIRT3−/− peritoneal macrophages exposed to
Our results suggest that H2S-regulated SIRT3 plays an important H2O2 in the presence and absence of H2S (Fig. 6E). These results de-
role in the regulation of oxidative stress and inflammation response monstrated that SIRT3 was required for H2S to inhibit excessive oxi-
through S-sulfhydrating c-Jun in macrophages exposed to H2O2 in vitro. dative stress and NLRP3 inflammasome activation in macrophages.
To further confirm the pathophysiological significance of H2S-induced
SIRT3 expression, we examined the protective effects of H2S on mtROS
generation and NLRP3 inflammasome activation in SIRT3−/− mice 4. Discussion
macrophages. Peritoneal macrophages and bone marrow-derived mac-
rophages (BMM) were pretreated with H2S before H2O2 treatment. Cardiovascular risk factors such as hypercholesterolemia, hy-
Firstly, we confirmed that S-sulfhydration of c-Jun still increased by pertension, and diabetes mellitus enhance ROS generation, resulting in
pretreatment with H2S in SIRT3−/− BMM (Fig. 6A). Secondly, oxidative stress [48]. Macrophage, the dominant cell type recruited to

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Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

inflammatory sites, is known to secrete ROS and may be primarily re- sulfhydration. However, mutation of cysteine-269, but not cysteine-99
sponsible for oxidative damage. Numerous stimuli such as oxidized low- and cysteine-320, largely prevented the protective influences of H2S on
density lipoprotein (OxLDL), high glucose, hydrogen peroxide (H2O2), the reducing of ROS and mtROS production exposed to H2S, pointing to
tumor necrosis factor (TNF), and hypoxia can cause oxidative stress and the critical role of C269 in c-Jun S-sulfhydration. After that, we ex-
promote macrophage inflammation, which is associated with the pa- plored the effects of S-sulfhydrated c-Jun on NLRP3 inflammasome
thophysiology of cardiovascular diseases [49–54]. The reactive oxygen activation. As expected, the results showed that S-sulfhydration of c-Jun
species that are generated by mitochondrial respiration, especially cysteine-269 was involved in suppressing NLRP3 inflammasome acti-
H2O2, are potent inducers of oxidative damage [55]. Here, we treated vation by H2S in macrophages exposed to H2O2. Therefore, we de-
macrophages with exogenous H2O2 for mimicking different stimuli-in- monstrated that the H2S inhibited the NLRP3 inflammasome activation
duced oxidative stress in macrophages. Remarkably, H2O2-treated via S-sulfhydration c-Jun at cysteine-269. Taken together, the findings
macrophages showed an increase in oxidative stress and inflammation that mutation of the c-Jun S-sulfhydrated cysteines diminished the
[46,52,56,57]. In the present study, we demonstrated that H2O2 could suppressive influences of H2S on NLRP3 inflammasome activation are
induce the activation of NLRP3 inflammasome, leading to the activa- consistent with the suggestion that there is a relevant link between the
tion of caspase-1 and production of pro-inflammatory cytokines IL-1β in S-sulfhydration of c-Jun and its antioxidative properties by H2S.
macrophages. Moreover, as evident from the NLRP3 siRNA studies, we SIRT3 can enhance antioxidant enzyme activity to exert its ROS-
demonstrated that H2O2-induced IL-1β production and caspase-1 acti- scavenging capacity [37,70]. Recent studies have shown that SIRT3
vation was dependent on NLRP3 inflammasome activation. We also decreases NLRP3 inflammasome activation in association with dimin-
found that the expression of H2S-producing enzymes was decreased in ished mtROS production via activating SOD2 [71,72]. Several groups,
H2O2-treated macrophages, indicating the generation of endogenous including our own, have previously indicated that c-Jun/AP-1 is able to
H2S was decreased. In consideration of the powerful antioxidative and bind with SIRT3 promoter and enhances its expression [37,73,74]. p62
anti-inflammatory properties of H2S, we next put our focus on whether binds polyubiquitinated proteins and damaged organelles and targets
H2S could play a protective role in reducing oxidative stress and in- them to autophagosomal clearance via its ubiquitin association domain
flammation in macrophages exposed to H2O2. Similarly, our data and LC3 binding motif, respectively [75,76]. Additionally, p62 also has
showed that exogenous H2S treatment protected macrophages from antioxidant capacity [77–80]. p62 is recruited to damaged mitochon-
NLRP3 inflamasome activation against H2O2 insult. Recent studies dria and eliminates them, thus contributing to the prevention of ex-
suggest that H2S reduces NLRP3 inflammasome activation in free fatty cessive inflammasome activation [76]. Moreover, c-Jun/AP-1 is an
acids and monosodium urate (MSU) crystals-stimulated macrophages, important factor in p62 transcription [81–83]. The question we posed
or microglia after intracerebral haemorrhage [58–60]. What we ob- here is whether S-sulfhydrated c-Jun could increase SIRT3 and p62
served in macrophages stimulated with H2O2 is that oxidative stress- expression. The findings in this study showed that, via a c-Jun C269-
induced NLRP3 inflammasome activation is dependent on the mi- dependent manner, the S-sulfhydrated c-Jun resulted in increasing
tochondrial ROS generation, but not P2X7 receptor or cathepsin B ac- SIRT3 and p62 expression and protected macrophages against H2O2
tivation. These results are consistent with the study of Castelblanco insult. To further confirm that the upregulated expression of SIRT3 and
et al., which showed that H2S donors inhibited MSU crystal-induced p62 induced by H2S is independent on its direct mtROS-scavenging
mtROS generation [60]. It remains to be further elucidated whether the effect, we treated macrophages with the mitochondria-targeted anti-
protective role of H2S against NLRP3 inflammsome activation is related oxidant Mito-TEMPO prior to exposure to H2O2 and found that the
to mtROS reduction, as the conception that mtROS induces NLRP3- pretreatment of Mito-TEMPO failed to increase the levels of SIRT3 and
dependent inflammasome activation has been established [61,62]. For p62. These results suggested the antioxidant effects of H2S were mainly
example, mitochondrial oxidative stress in lesional macrophages am- dependent on S-sulfhydrated c-Jun in H2O2-treated macrophages.
plifies atherosclerotic lesion development by promoting NF-κB-medi- However, both mRNA and protein expression of SIRT3 was significantly
ated entry of monocytes and other inflammatory processes [63]. Ad- enhanced by H2S in H2O2-exposed macrophages. This phenomenon
ditionally, NLRP3 inflammasome activation by mitochondrial oxidative drove us to focus on SIRT3. Moreover, we found that the influences of
stress in macrophages contributes to the development of angiotensin H2S on mtROS generation and NLRP3 inflammasome activation was
II–induced aortic aneurysm [64]. Interestedly, we observed that gen- diminished in the macrophages of SIRT3−/− mice, suggesting that the
eration of mtROS and recruitment of NLRP3 to mitochondrial was in- protective effects of H2S against oxidative stress and NLRP3 in-
creased in macrophages exposed to H2O2, while these effects of H2O2 flammasome activation partly dependent on SIRT3.
was rescued by H2S treatment. Therefore, for the first time, we de- Taken together, the results showed that H2S is privotal in the reg-
monstrated that H2S protected macrophages from H2O2-induced NLRP3 ulation of oxidative stress and inflammation response through S-sulf-
inflammasome activation via reducing the generation of mtROS. hydrating c-Jun in macrophages exposed to H2O2 (Fig. 7). H2S donors
Our next focus is to explore how H2S reduced the generation of have therapeutic potential for diseases such as cancer, peptic ulcer
mtROS. H2S can cause protein S-sulfhydration (or persulfidation) to
affect their functions [29,65]. Previous studies have demonstrated that
S-sulfhydration of GAPDH affects PSD95 degradation and memory
[66]. And S-sulfhydration of NF-κB inhibits apoptosis [67]. S-sulfhy-
dration of KATP could contribute to H2S-induced vasodilation [68]. H2S
is also involved in endoplasmic reticulum stress via triggering S-sulf-
hydration of PTP1B [69]. In recent years, more and more proteins have
been found to be regulated by S-sulfhydration [65]. Our groups have
previously characterized that H2S increases AP-1 binding activity with
SIRT3 promoter, thus enhancing SIRT3 transcription to attenuate en-
dothelial oxidative stress [37]. Accordingly, we wondered whether H2S
could S-sulfhydrated c-Jun (subunit of AP-1) in macrophages. Inter-
estingly, we found that c-Jun is basically S-sulfhydrated and exogen-
ously applied H2S further enhanced c-Jun S-sulfhydration. To discover
S-sulfhydrated cysteine residues of c-Jun, we mutated cysteine-99, cy-
steine-269, and cysteine-320 (the only three cysteine residues) in c-Jun Fig. 7. The proposed signaling pathway underlying H2S regulation of c-Jun by
protein. We confirmed that cysteine-269 was responsible for c-Jun S- S-sulfhydration in macrophages.

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Z. Lin et al. BBA - Molecular Basis of Disease xxx (xxxx) xxx–xxx

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Conflict of interest
G.S. Abela, L. Franchi, G. Nunez, M. Schnurr, T. Espevik, E. Lien, K.A. Fitzgerald,
K.L. Rock, K.J. Moore, S.D. Wright, V. Hornung, E. Latz, Nlrp3 inflammasomes are
The authors declare that there is no conflict of interest. required for atherogenesis and activated by cholesterol crystals, Nature 464 (2010)
1357–1361.
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B. Friguet, T. Fulop, T. Simmet, K. El Hadri, M. Rouis, Anti-inflammatory and an-
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