Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock

Article

Enhanced Cellular Polysulﬁdes Negatively Regulate

TLR4 Signaling and Mitigate Lethal Endotoxin Shock

Graphical Abstract

Authors

Tianli Zhang, Katsuhiko Ono,

Hiroyasu Tsutsuki, Hideshi Ihara,

Waliul Islam, Takaaki Akaike,

Tomohiro Sawa

Correspondence

sawat@kumamoto-u.ac.jp

In Brief

Zhang et al. developed potent persulﬁde

donors consisting of sulfane sulfur atoms

stabilized by N-acetyl-L-cysteine (NAC

polysulﬁdes) via disulﬁde bonds at both

sides. Strong anti-inﬂammatory activity of

NAC polysulﬁdes was demonstrated in

cultured macrophage models and a

mouse endotoxin shock model.

Highlights

d

Cell-permeable, thiol-activable polysulﬁde donors are

developed

Polysulﬁde donor treatment increases intracellular

polysulﬁde levels

Polysulﬁde donor treatment desensitizes macrophages to

TLRs signaling

Mice are protected from lethal endotoxin shock by

polysulﬁde donor treatment

Zhang et al., 2019, Cell Chemical Biology 26, 1–13

May 16, 2019 ª 2019 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2019.02.003

Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

Cell Chemical Biology

Article

Enhanced Cellular Polysulﬁdes

Negatively Regulate TLR4 Signaling

and Mitigate Lethal Endotoxin Shock

Tianli Zhang, Katsuhiko Ono, Hiroyasu Tsutsuki, Hideshi Ihara, Waliul Islam, Takaaki Akaike, and Tomohiro Sawa¹^,⁴^,*

1

2

1

3

1

Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Chuo-ku, Kumamoto

60-8556, Japan

8

²Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan

³Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,

Aoba-ku, Sendai 980-8575, Japan

⁴Lead Contact

*Correspondence: sawat@kumamoto-u.ac.jp

https://doi.org/10.1016/j.chembiol.2019.02.003

SUMMARY

et al., 2017; Numakura et al., 2017; Ono et al., 2017; Peng

et al., 2017). Hydropersulﬁdes/polysulﬁdes are highly potent an-

Cysteine persulﬁde and cysteine polysulﬁdes are tioxidants and hence protect cells from injury induced by reac-

cysteine derivatives having sulfane sulfur atoms tive oxygen species (ROS) (Ida et al., 2014; Kunikata et al.,

2

n

017). CysS[S] H were recently demonstrated to be critical com-

bound to cysteine thiol. Accumulating evidence has

suggested that cysteine persulﬁdes/polysulﬁdes are

abundant in prokaryotes and eukaryotes and play

important roles in diverse biological processes such

as antioxidant host defense and redox-dependent

signal transduction. Here, we show that enhance-

ment of cellular polysulﬁdes by using polysulﬁde

donors developed in this study resulted in marked in-

ponents in mitochondrial sulfur respiration (Akaike et al., 2017).

Also, hydropersulﬁde/polysulﬁde moieties occurring on protein

thiols may be involved in redox-dependent regulation of protein

functions (Millikin et al., 2016). We previously reported that the

transsulfuration enzymes cystathionine b-synthase and cysta-

thionine g-lyase can produce CysSSH from cystine via C-S

bond cleavage (Ida et al., 2014). Cysteinyl-tRNA synthetases

(CARS) were recently found to act as potent CysSSH-producing

hibition of lipopolysaccharide (LPS)-initiated macro- enzymes by using cysteine as a substrate (Akaike et al., 2017).

phage activation. Polysulﬁde donor treatment Furthermore, CARS can catalyze direct incorporation of CysSSH

strongly suppressed LPS-induced pro-inﬂammatory into proteins via de novo protein synthesis (Akaike et al., 2017).

Persulﬁde/polysulﬁde donors that can increase intracellular

responses in macrophages by inhibiting Toll-

like receptor 4 (TLR4) signaling. Other TLR signaling

stimulants—including zymosan A-TLR2 and

poly(I:C)-TLR3—were also signiﬁcantly suppressed

by polysulfur donor treatment. Administration of

polysulﬁde donors protected mice from lethal

endotoxin shock. These data indicate that cellular

persulﬁde/polysulﬁde levels by donating their sulfur atoms to

endogenous acceptor thiols become powerful chemical tools

for understanding the physiological and pathological roles of

persulﬁde species in biological systems. Powell et al. (2018)

recently reported that N-acetyl-L-cysteine (NAC) conjugated to

Bpin through disulﬁde bonding can act as a prodrug for a persul-

ﬁde donor (BDP-NAC). BDP-NAC can release free NAC hydro-

polysulﬁdes negatively regulate TLR4-mediated

pro-inﬂammatory signaling and hence constitute a (Powell et al., 2018). Powell and colleagues also demonstrated

persulﬁde (NAC-SH) in response to hydrogen peroxide exposure

potential target for inﬂammatory disorders.

that BDP-NAC treatment protected cells from cell death caused

by hydrogen peroxide, possibly by producing antioxidant NAC-

SH in cells (Powell et al., 2018). Their study, however, did not

provide details of the metabolism of BDP-NAC and NAC-SH

in cells, particularly the effects of BDP-NAC treatment on the

INTRODUCTION

Cysteine persulﬁde (CysSSH) and cysteine polysulﬁdes intracellular persulﬁde/polysulﬁde metabolome. Zheng et al.

CysS[S]

H, n > 2) are cysteine derivatives having sulfane sulfur (2017) developed a persulﬁde prodrug that generated a hydrox-

atoms bound to cysteine thiol (Fukuto et al., 2018; Ida et al., ymethyl persulﬁde via esterase-dependent activation. They also

014; Ono et al., 2014; Sawa et al., 2018). Accumulating evi- reported that such a persulﬁde-generating prodrug exhibited

(

n

2

dence has suggested that cysteine persulﬁdes/polysulﬁdes are potent cardioprotective effects in a murine model of myocardial

abundant in prokaryotes and eukaryotes in diverse forms such ischemia-reperfusion injury with a bell-shaped therapeutic pro-

as monomeric amino acid hydropersulﬁdes/polysulﬁdes ﬁle (Zheng et al., 2017). Kang et al. (2018) reported another class

(

sulﬁdes/polysulﬁdes (GS[S]

CysSSH, CysS[S]

n

H), reduced L-glutathione (GSH) hydroper- of persulﬁde precursors that produce hydropersulﬁdes via pH-

À

n

H), and protein persulﬁde/polysul- or F -mediated desilylation of O-silyl mercaptan-based sulfur-

ﬁde residues (Akaike et al., 2017; Ida et al., 2014; Kunikata containing molecules. Artaud and Galardon (2014) developed

Cell Chemical Biology 26, 1–13, May 16, 2019 ª 2019 Elsevier Ltd.

1

Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

A

B

OH

O

OH

O

OH

O

^N^a^N^O2

NaHS

HN

S

(

S)n

SH

HN

S

NO

HN

_H+

O

S

NH

O

HO

NAC +

NAC alone NAC +

NaNO₂

NaNO

NaHS

+

2

C

UV (254 nm)

D

NAC + NaNO₂

5

4

3

2

1

000

0

+

NaHS

NAC + NaNO₂

NAC alone

5

200 mAU

2

20

270

320

370

420

470

0

10

15

20

25

Time (min)

Wavelength (nm)

E

F

OH

H

3

C

O

4000

oxNAC

O

C

NAC-S1

NAC-S2 NAC-S3

NAC-S4

(S)n

3000

2000

S

NH

HN

C

O

HO

CH

3

1000

n = 0, oxNAC

1

2

3

4

, NAC-S1

, NAC-S2

, NAC-S3

, NAC-S4

0

-

1000

0

5

10

15

20

25

30

35

Retention time (min)

G

H

I

UV (210 nm)

40 mAU

20 mAU

40 mAU

10

15

20

25

30

10

15

20

25

30

10

15

20

25

30

Retention time (min)

+

[M+H ] = 389.0

+

[M+H ] = 357.0

[

M+H ] = 325.1

5

x 10⁴counts

_x₁₀4 counts

300

₅_x₁₀4 counts

5

200

300

400

500

m/z

200

400

500

200

300

400

500

m/z

Figure 1. Synthesis and Puriﬁcation of NAC Polysulﬁdes

(

A) Reaction scheme of the synthesis of NAC polysulﬁdes.

B) Appearance of the solutions of reduced NAC (left), reduced NAC reacted with NaNO

C) HPLC chromatograms for reduced NAC (bottom), reduced NAC reacted with NaNO

2

(center), and reduced NAC reacted with NaNO

2

and NaHS (right).

and NaHS (top),

2

(middle), and reduced NAC reacted with NaNO

2

detected at 254 nm.

(

legend continued on next page)

2

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disulﬁde precursors that spontaneously rearrange in neutral pH sulfur numbers, probably because of sulfur-sulfur bond rear-

buffer to produce hydropersulﬁdes. By using hydropersulﬁde- rangements during lyophilization. Thus, we used NAC-S1 and

generating molecules, Fukuto and colleagues investigated the NAC-S2 in the following experiments. We also used oxidized

physicochemical and some biochemical properties of hydroper- NAC (oxNAC) as a control reagent that lacks a sulfane sul-

sulﬁde species (Bianco et al., 2016; Millikin et al., 2016). We pre- fur atom.

viously reported that GS[S]

reductase (GR)-mediated reduction of oxidized GSH polysul- oxNAC, NAC-S1, and NAC-S2 were freely soluble in neutral

ﬁdes (GS[S] SG, n > 1) (Ida et al., 2014). Strong ROS-scavenging aqueous buffers at approximately up to 30 mM at room

activity of GS[S] H was demonstrated by using this GS[S] SG- temperature.

GR system (Ida et al., 2014). Lyophilized NAC polysulﬁdes were stable during storage in a

n

H can be formed by glutathione

NAC polysulﬁdes were highly water soluble. For instance,

n

ꢀ

One unique characteristic of persulﬁdes/polysulﬁdes is that freezer (À30 C) without detectable decomposition for more

they can donate sulfane sulfur atoms to acceptor thiols via sulfur than 1 year. The stabilities of NAC polysulﬁdes in physiological

transfer reactions (Fukuto et al., 2018; Ono et al., 2014). Thus, saline were also determined as a function of temperature and in-

related to this ﬁnding, we hypothesized that appropriate persul- cubation periods. NAC polysulﬁdes were stable during incuba-

ꢀ

ﬁdes/polysulﬁdes may act as persulﬁde/polysulﬁde donors in tion at 37 C for 72 h in physiological saline (data not shown). After

ꢀ

biological systems. In this study, we synthesized NAC polysul- 12 days of incubation at 37 C, the concentration of NAC-S2 had

ﬁdes and examined their ability to act as persulﬁde/polysulﬁde decreased slightly to 85%, whereas oxNAC and NAC-S1

ꢀ

donors in vitro, in cultured cells, and in vivo. We investigated sul- showed no detectable decrease (data not shown). At 5 C, no

fur transfer reactions between NAC polysulﬁdes and acceptor detectable loss of NAC polysulﬁdes (oxNAC, NAC-S1, NAC-

thiols in detail by means of mass spectrometry (MS)-based sulfur S2) occurred during up to 12 days of incubation (data not shown).

metabolomic analysis. We studied the biological effects of NAC

polysulﬁdes on innate immune responses by using lipopolysac- Sulfane Sulfur Transfer from NAC Polysulﬁdes to

charide (LPS)-activated cultured macrophages and a mouse Reduced GSH

endotoxin shock model. Our data showed that NAC polysulﬁdes To study sulfane sulfur transfer from NAC polysulﬁdes to

can act as potent persulﬁde/polysulﬁde donors in biological sys- acceptor thiols, we investigated the reactions of NAC polysul-

tems. In view of the strong anti-inﬂammatory activities of NAC ﬁdes with reduced thiols in vitro by using GSH as a biologically

polysulﬁdes demonstrated in this study, persulﬁde/polysulﬁde relevant thiol model. When 100 mM oxNAC was reacted with

donors may become therapeutic agents applicable for inﬂam- GSH (10 or 100 mM) in 100 mM sodium phosphate buffer (pH

ꢀ

mation-associated diseases including sepsis.

7.4) at 37 C, oxNAC concentration showed no apparent change

during an incubation period of up to 60 min (data not shown). In

contrast, in the presence of GSH, concentrations of NAC-S1 and

NAC-S2 decreased rapidly (Figures 2A and 2B). NAC-S1

decreased to 80% and 25% of the initial concentration at

RESULTS

Preparation of NAC Polysulﬁdes

We successfully prepared NAC polysulﬁdes having different 30 min after incubation in the presence of 10 and 100 mM

numbers of sulfane sulfur atoms by reacting NAC with sodium ni- GSH, respectively (Figure 2A). The decrease in NAC-S2 in the

2

trite (NaNO ) and sodium hydrogen sulﬁde (NaHS) in aqueous presence of GSH occurred more quickly; only 20% and 5% of

media (Figure 1A). Formation of S-nitroso-NAC in the ﬁrst step NAC-S2 remained in the presence of 10 and 100 mM GSH,

was suggested by the appearance of a characteristic reddish respectively, after 30 min of incubation (Figure 2B).

color and a high-performance liquid chromatography (HPLC)

Our MS-based assignment of the reaction products indicated

peak having a UV spectrum consistent with S-nitroso-NAC re- the formation of various products in the reactions between NAC

ported previously (Figures 1B–1D) (Shishido and de Oliveira, polysulﬁdes and GSH (Figure S1). The reaction products found

2

000). Addition of NaHS resulted in the disappearance of the include NAC polysulﬁdes with different sulfur numbers (oxNAC,

S-nitroso-NAC peak and the appearance of several new peaks NAC-S1, NAC-S2); mixed disulﬁdes (GS-NAC), trisulﬁdes (GS-

Figure 1C). These new peaks were determined to be NAC poly- S-NAC), and tetrasulﬁdes (GS-SS-NAC) of GSH and NAC;

(

sulﬁdes having different numbers of sulfane sulfur atoms by reduced NAC and NAC hydropolysulﬁdes (NAC-SH, NAC-

means of MS (Figures 1E–1I). NAC polysulﬁdes were puriﬁed SSH); GSH hydropersulﬁdes/polysulﬁdes (GSSH, GSSSH); and

by means of reversed-phase chromatography on the basis of oxidized GSH (GSSG, GSSSG) in the reactions between NAC-

their different hydrophobicities (Figure 1F). After fractionation S1 or NAC-S2 and GSH (Figures 2C, 2D, and S1). The occur-

n

and lyophilization of the polysulﬁdes, the purity of each NAC pol- rence of both reduced and oxidized forms of GS[S] SG clearly

ysulﬁde was determined to be higher than 95% as determined by suggests that NAC polysulﬁdes can donate their sulfane sulfur

chromatograms at 210 nm (Figures 1G–1I). Although NAC-S3 atoms to the acceptor GSH via a sulfur transfer from an NAC pol-

and NAC-S4 were obtained as a single HPLC peak, lyophilized ysulﬁde. We also detected hydrogen sulﬁde (H

2

S), sulﬁte, and

products showed mixtures of NAC polysulﬁdes with various thiosulfate in the reactions between NAC polysulﬁdes and GSH

(

D) UV spectrum of the peak indicated by the arrow in (C).

E) Structures of NAC polysulﬁdes.

2

F–I) Separation of the crude reaction mixture by using a C18 column (F). The reaction mixture (1.5 mL) consisting of NAC, NaNO , and NaHS was applied to a

preparative C18 column. Peaks corresponding to NAC polysulﬁdes were obtained and subjected to lyophilization. HPLC chromatograms (upper panel) and mass

spectra (lower panels) of oxNAC (G), NAC-S1 (H), and NAC-S2 (I) after puriﬁcation.

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A

C

D

1

20

00

oxNAC

NAC-S1

NAC-S2

GG SS -- NN AA CC

NN AA CC - -SS 11

NN AA CC - -SS 22

8

6

4

2

0

GG SS - -NN AA CC

GG SS - -SS - -NN AA CC

GG SS -- SS -- NN AA CC

GS-SS-NAC

GG SS - -SS SS - -NN AA CC

NAC-HPE N- AA MC

NAC-S- HN PA EC -- AS MH

NAC-SS N- HA PC E- S- AS MH

NAC-S- HN PA EC - A- SMH

NAC-SS N- HA PC E- S- AS MH

0

15

30

45

60

Time (min)

B

GS-HPE-AM

GS-HPE G- AS MH

GSS-HP GE S- AS MH

GSSS-H GP SE S- AS MH

GG SS SS GG

GSH

GSS-HP GE -SA SMH

GSSS-H GP SE S- AS MH

GG SS SS GG

1

20

00

8

6

4

2

0

GG SS SS SS GG

Bis-S-HPE -HA MS

Bis-S-HPE- HA MS

2

^S^O3

SO₃

thiosulfate-HPE-AM

S O

thiosulfate-HP ES - AO M

2

3

2

3

0

50

100

0

50

100

0

15

30

45

Time (min)

60

Concentration (µM)

Figure 2. Reactions of NAC Polysulﬁdes with GSH In Vitro

(

A and B) Time-dependent alterations in NAC-S1 (A) and NAC-S2 (B) concentrations in the presence of GSH. NAC polysulﬁdes (100 mM) were incubated in the

ꢀ

presence of 10 mM GSH (open circles) or 100 mM GSH (closed circles) in 100 mM NaPB (pH 7.4) at 37 C for the indicated time periods. Residual amounts of NAC

polysulﬁdes were determined by means of LC-MS/MS.

ꢀ

(

C and D) Product analyses for the reactions between NAC polysulﬁdes and GSH. NAC-S1 (C) and NAC-S2 (D) at 100 mM were reacted with 100 mM GSH at 37 C

for 30 min, followed by terminating the reactions by adding b-(4-hydroxyphenyl)ethyl acetamide (HPE-IAM). Reaction products were determined and quantiﬁed

by means of LC-MS/MS. Data are means ± SD (n = 3).

(

Figures 2C and 2D). These products may be derived from the re- larly, NAC-S1 treatment increased intracellular NAC-S1 levels

actions between hydropersulﬁdes/polysulﬁdes (e.g., NAC-SSH) (Figure 3B). These data suggest that NAC polysulﬁdes were

and reduced thiols and subsequent auto-oxidation (Figure S2A). incorporated into cells when added to the culture medium.

Figure S2A shows the possible reactions involved in the forma- NAC-S2 treatment also increased intracellular NAC-S2 (Fig-

tion of such diverse products, with NAC-S2 used as an example. ure 3C), although the increase was quite small compared with

In these reactions, sulfane sulfur atoms in GSH persulﬁdes/ that observed with oxNAC and NAC-S1 treatment (Figures 3A

polysulﬁdes likely derive from the sulfane sulfur atoms of NAC and 3B). This result may be due to the fact that incorporated

polysulﬁdes. To conﬁrm this expectation, we investigated the NAC-S2 rapidly reacts with cellular thiols such as cysteine and

reaction of GSH with NAC-S2 labeled with the sulfur isotope GSH to donate its sulfane sulfur atoms, as observed in the

3

4

34

S at sulfane sulfur atoms (NAC-[ S]2). Figure S2B clearly in vitro experiments. This explanation is supported by the ﬁnding

shows that GSSH and GSSSH formed in the reaction between that NAC-S2 treatment markedly increased intracellular hydro-

3

4

NAC-[ S]2 and GSH were labeled with S.

34

persulﬁde/polysulﬁde levels of cysteine and GSH (Figures 3E,

F, 3H, 3I, and 3L). We also found that NAC-S2 treatment signif-

icantly increased H S (Figure 3J) and thiosulfate (Figure 3Q)

levels in cells. We detected mixed sulﬁdes (GS-[S] -NAC; n =

3

Effects of NAC Polysulﬁde Treatment on the

Intracellular Sulfur Metabolome

2

n

We then examined whether NAC polysulﬁdes could be incor- 0–2) in cells treated with NAC-S2 (Figures 3M–3O). However,

porated into cells and then act as sulfur donors to increase those levels were very small compared with those of cysteine

intracellular polysulﬁde levels. We used cells of the mouse or GSH hydropersulﬁdes/polysulﬁdes. When cells were incu-

macrophage-like cell line RAW264.7 because we wanted to bated with NAC-S2 for 6 h, cellular levels of hydropersulﬁdes/

clarify whether intracellular polysulﬁde levels affect the macro- polysulﬁdes decreased (Figure S3B). Mechanisms or factors

phage’s responses to immunological stimuli. RAW264.7 cells associated with this time-dependent decrease in intracellular

were treated with NAC polysulﬁdes in the absence (Figure S3A) polysulﬁde levels are currently not known and warrant further

or presence (Figure 3) of LPS. Intracellular sulfur species were investigation. Similar responses to NAC polysulﬁde treatment

quantiﬁed by means of liquid chromatography-tandem MS were found for cells that did not receive LPS (Figure S3A).

(LC-MS/MS). Figure 3A indicates that treatment of cells with Reduced NAC, even at 5 mM, did not cause marked increases in

oxNAC resulted in a marked increase in oxNAC in cells. Simi- intracellular polysulﬁde levels (Figure S3A). This result suggests

4

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Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

A

D

G

1

40

20

00

10000

1

60

8

000

120

6

8

6

4

2

0

8

4

0

4000

2000

0

Additives

LPS

B

E

H

6

5

4

3

00

1

60

20

5

4

3

2

1

0

8

4

0

200

100

0

Additives

LPS

C

F

I

7

6

5

4

3

2

1

0

350

300

250

200

7

6

5

4

3

2

1

0

1

50

00

50

0

Additives

LPS

P

J

M

1

4

2

0

8

6

4

2

0

.6

.5

1

200

000

8

6

4

2

00

0

0.4

0.3

0.2

0.1

0

Additives

LPS

K

N

Q

8

6

00

0

.4

.3

.2

8

6

4

2

0

400

0.1

200

0

Additives

LPS

O

L

1.4

1

2

1.2

0

8

6

4

2

0

1

0

.8

.6

.4

0.2

0

Additives

LPS

(

legend on next page)

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that NAC-S2 can act as a very effective sulfur donor in cell cul- points (Figures S4D–S4F). These data suggest that NAC-S2

ture systems. treatment differently affected the phosphorylation network

When cells were treated with NAC-S2 labeled with S at sul- downstream of LPS-Toll-like receptor 4 (LPS-TLR4) signaling

fane sulfur atoms, intracellular hydropersulﬁdes/polysulﬁdes and that inhibition of the IkB kinase (IKK)/NF-kB axis may

34

(

2

e.g., GSSH and GSSSH), H S, and thiosulfate were labeled contribute to suppression of TNF-a production caused by

3

4

with S (Figure S2C). This result indicates that the sulfur transfer NAC-S2 treatment (Scheme S1). Note that reduced NAC at con-

from NAC-S2 to acceptor thiols is responsible for the increased centrations up to 5 mM did not affect cytokine production by

persulﬁde/polysulﬁde levels in cells.

LPS-stimulated cells (Figures S5A and S5B). Under those condi-

After 1 h of incubation, NAC-S1 treatment resulted in a small tions, no statistical increase in persulﬁdes and polysulﬁdes was

increase in hydropersulﬁdes/polysulﬁdes of cysteine and GSH found in LPS-stimulated RAW264.7 cells (Figures S5C–S5J).

as well as H

bation periods (6 h) resulted in marked increases in polysulﬁde inﬂammatory cytokine, interferon-b (IFN-b) (Figure 5). We found

levels in cells treated with NAC-S1 (Figure S3B). that LPS-induced IFN-b production was signiﬁcantly inhibited

2

S and thiosulfate (Figure 3). However, longer incu-

LPS stimulation also induced production of another pro-

We also examined the effects of NaHS treatment on cellular by treatment not only with NAC-S2 but also with NAC-S1 (Fig-

polysulﬁde levels. We found that NaHS treatment increased ure 5A). After 6 h of incubation, cell viability was slightly but

levels of hydropersulﬁdes/polysulﬁdes of cysteine and GSH, signiﬁcantly reduced by NAC-S2 treatment (Figure 5B). Even

but this effect was only marginal compared with that caused if we consider this cell viability reduction, NAC-S2 treatment

by NAC-S2 (Figures 3E, 3F, 3H, 3I, and 3L). In the case of strongly suppressed IFN-b production by LPS-stimulated mac-

NaHS treatment, no further increase in intracellular polysulﬁde rophages. IFN-b released extracellularly can then activate signal

levels was observed even after 6 h of incubation (Figure S3B). transducer and activator of transcription 1 (STAT1) signaling,

We expect that, collectively, NAC polysulﬁdes, particularly which may lead to production of the inﬂammatory mediator nitric

NAC-S2, will be useful as efﬁcient sulfur donors capable of oxide (NO) via expression of inducible nitric oxide synthase

increasing intracellular polysulﬁde levels and that this result (iNOS), in an autocrine and/or paracrine manner (Scheme S1).

may be achieved simply by adding the polysulﬁdes to the culture As Figures 5C–5F illustrate, all IFN-b downstream responses

media.

were markedly inhibited by NAC-S1 and NAC-S2. In our sepa-

rate experiments, STAT1 phosphorylation induced by extracellu-

larly added IFN-b was also strongly inhibited by NAC-S1 and

NAC-S2 (Figure S6A). These data suggest that NAC polysulﬁdes

can suppress IFN-b-dependent inﬂammatory responses by in-

Suppression by NAC Polysulﬁde Treatment of Pro-

inﬂammatory Responses in Macrophages Stimulated

with LPS

As Figure 4A illustrates, RAW264.7 cells produced appreciable hibiting both IFN-b production and STAT1 phosphorylation. We

amounts of the pro-inﬂammatory cytokine tumor necrosis factor also studied the effects of NAC polysulﬁdes on the transcription

alpha (TNF-a) in response to LPS stimulation for 3 h. Statistically of IFN-b by measuring IFN-b mRNA. As Figure S6B shows, NAC-

signiﬁcant, marked inhibition of TNF-a production was found for S2 treatment reduced the level of IFN-b mRNA by approximately

cells after treatment with NAC-S2 (Figures 4A and S4A). Under 50%, but NAC-S1 did not affect the transcription of IFN-b. This

the current experimental conditions, cell viability was not result suggests that NAC polysulﬁdes may exert their inhibitory

affected by NAC-S2 treatment (Figure 4B). These data indicate actions on IFN-b production during transcription and translation

that pro-inﬂammatory responses of macrophages may be and that NAC-S2 can inhibit both processes.

strongly suppressed by NAC-S2 treatment. TNF-a production

We then examined whether NAC polysulﬁdes affect signaling

by LPS-stimulated macrophages was not affected by oxNAC, of other TLRs. RAW264.7 cells were stimulated with zymosan

NAC-S1, or NaHS under the present experimental conditions A and polyinosinic-polycytidylic acid (poly(I:C)) to activate

(

Figure 4A). As Figures 4C and 4D show, LPS stimulation induced TLR2 signaling (Young et al., 2001) and TLR3 signaling (Jiang

phosphorylation of IkBa and nuclear factor kB (NF-kB). We et al., 2005), respectively. As Figure 6A shows, NAC-S2 treat-

found that NAC-S2 treatment signiﬁcantly inhibited phosphory- ment signiﬁcantly reduced TNF-a production in cells stimulated

lation of IkBa (Figures 4C and S4B) and NF-kB p65 (Figures 4D with zymosan A and poly(I:C). These TLR ligands activated NF-

and S4C) in LPS-stimulated macrophages. LPS stimulation kB phosphorylation that was suppressed by NAC-S2 treatment

also induced a transient increase in the phosphorylation of extra- (Figures 6B and 6C).

cellular signal-regulated kinase (ERK), p38 mitogen-activated

protein (MAP) kinase, JNK, and Akt at 1 h after stimulation, after Therapeutic Potential of NAC Polysulﬁdes against

which those phosphorylation proﬁles returned to baseline levels Lethal Endotoxin Shock

(

Figure 4E). We found that high phosphorylation levels of ERK, Macrophage activation in response to LPS is an important innate

p38 MAP kinase, JNK, and Akt were maintained in cells treated immune response that helps eradication of Gram-negative bacte-

with NAC-S2 (Figure 4E). Similar effects of NAC-S2 treatment on ria. However, when host individuals are exposed to continuous

the phosphorylation of IkBa and ERK were found at early time LPS or large quantities of LPS, macrophages are overactivated

Figure 3. Metabolomic Determination of Intracellular Sulfur Species in RAW264.7 Cells Treated with NAC Polysulﬁdes

Cells stimulated with LPS (100 ng/mL) were treated with 0.5 mM NAC polysulﬁdes or NaHS for 1 h. Intracellular sulfur species were identiﬁed by means of LC-MS/

MS as described in the STAR Methods. Controls were cells not treated with LPS or additives. Sulfur species analyzed were (A) oxNAC, (B) NAC-S1, (C) NAC-S2,

(

D) cysteine, (E) CysSSH, (F) CysSSSH, (G) GSH, (H) GSSH, (I) GSSSH, (J) H

Q) thiosulfate. Data are means ± SD (n = 3).

2

S, (K) GSSG, (L) GSSSG, (M) GS-NAC, (N) GSS-NAC, (O) GSSS-NAC, (P) sulﬁte, and

(

6

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Figure 4. Effects of NAC Polysulﬁdes on

A

B

Innate Immune Responses of RAW264.7

Cells Stimulated by LPS

9

6

3

000

0

*

150

100

50

(A) Production of TNF-a from LPS-stimulated

RAW264.7 cells and its suppression by NAC-

S2. RAW264.7 cells were stimulated with LPS

(

100 ng/mL) for 3 h in the absence or presence of

0

0.5 mM NAC polysulﬁdes or NaHS. TNF-a in cul-

ture supernatants was measured by means of

ELISA.

Additives

!" #" $" %" &" '"

Additives

LPS 3 h

(

B–D) Cell viability (B) of RAW264.7 cells cultured

under the same conditions as those for (A). Phos-

phorylation of IkBa (phospho-IkBa) (C) and of NF-

kB p65 (phospho-NF-kB p65) (D) in RAW264.7

cells after LPS stimulation. Representative west-

ern blotting images (upper panels) and their

quantitative data (lower panels). Controls were

cells not treated with LPS or additives. Data are

means ± SD (n = 3). *p < 0.05; **p < 0.01.

C

D

Additives

LPS

Additives

-

+

2

LPS

-

+

Time (h) 0 1

Phospho-

2

3 1

2

3

1

3

1

2

3

1

2

3

Time (h)

0

3

6

3

6

3

6 3 6 3 6

(

kDa)

(

kDa)

65

40

Phospho-

NF- B p65

I

B

42

Actin

65

NF- B p65

(

E) Effects of NAC polysulﬁde treatments on the

*

phosphorylation status of ERK, p38 MAP kinase,

JNK, and Akt in RAW264.7 cells stimulated with

LPS. At the indicated time points, lysates were

prepared from RAW264.7 cells stimulated with

**

*

1

00 ng/mL LPS in the presence of 0.5 mM NAC

polysulﬁdes or NaHS. Representative western

blotting images are shown.

Time (h)

LPS

0

-

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Time (h)

LPS

0 3 6 3 6 3 6 3 6 3 6

+

-

+

Additives

E

mice survived 96 h after LPS administra-

tion (p = 0.007 versus LPS + saline group,

Figure 7C). This ﬁnding clearly suggested

that NAC-S2 treatment effectively pro-

tected mice from lethal endotoxin shock

in vivo. In sharp contrast, NaHS treatment

failed to protect mice from endotoxin

shock but rather signiﬁcantly exacerbated

the disorder (p = 0.0059 versus LPS + sa-

line group, Figure 7D); all mice died within

Additives

LPS

-

+

Time (h)

0

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

4

4 kDa

Phospho-ERK

ERK

42 kDa

4

4 kDa

42 kDa

4

3 kDa

0 kDa

Phospho-p38

p38

4

48h aftertreatmentwithNaHS(Figure7D).

We then investigated cytokine produc-

5

4

4 kDa

6 kDa

Phospho-JNK

JNK

tion in this animal model. Serum samples

obtained from mice given no LPS con-

tained no detectable levels of TNF-a,

but LPS administration induced a marked

increase in serum TNF-a (Figure 7E). As

an important ﬁnding, NAC-S2 treatment

signiﬁcantly reduced TNF-a levels in

5

4 kDa

4

6 kDa

6

0 kDa

Phospho-Akt

Akt

6

to produce excess amounts of pro-inﬂammatory cytokines serum compared with the result obtained for the saline-treated

(

a cytokine storm), which ﬁnally leads to lethal endotoxin shock group (Figure 7E). We also examined the production of INF-b in

Cavaillon, 2018; Kuzmich et al., 2017). We used a mouse endo- this animal model, but the serum levels were below the detection

toxin shock model to see whether NAC polysulﬁdes suppress limit even after LPS administration (data not shown). As an inter-

LPS-induced macrophage activation in vivo and protect mice esting ﬁnding, NAC-S2 treatment signiﬁcantly increased levels

from lethal endotoxin shock. In the present experimental setting, of persulﬁdes, polysulﬁdes, and thiosulfate in the lungs of

8

0% of mice died within 96 h after receiving intraperitoneal LPS mice compared with saline treatment (Figure S7A). This result

20 mg/kg body weight) (Figure 7A). Treatment with NAC polysul- suggests that NAC polysulﬁdes can act as persulﬁde and

(

ﬁdesstarted 30 min after LPS administration. As Figure 7A shows, polysulﬁde donors in vivo. Different responses to NAC-S2 treat-

oxNAC treatment did not rescue mice from endotoxin shock. ment were observed in the liver (Figure S7B). Additional careful

NAC-S1 treatment improved survival to 40%, but the effect did determination of the pharmacokinetics of NAC polysulﬁde

not reach statistical signiﬁcance (Figure 7B). In the NAC-S2 treat- administration to mice is ongoing. These data suggest that

ment group, the survival rate was markedly improved; 90% of NAC polysulﬁdes, particularly NAC-S2, can exert a protective

Cell Chemical Biology 26, 1–13, May 16, 2019

7

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Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

A

B

6

h

24 h

*

1

0000

140

120

100

80

*

1

20

00

8

6

4

2

000

0

*

80

60

6

0

4

2

0

40

20

0

Additives

LPS (6 h)

LPS

LPS (6 h)

LPS (24 h)

C

D

**

*

**

Additives

Time (h)

P-STAT1

0

3 6 3 6 3 6 3 6 3 6

9

1 kDa

8

4 kDa

1 kDa

9

Time (h)

0

3

6

3

6

3

6

3

6

3

6

STAT1

84 kDa

Additives

LPS

E

F

LPS (24 h)

3

2

1

0

**

Additives

**

0

iNOS

Actin

130 kDa

42 kDa

Additives

LPS (24 h)

LPS

Figure 5. Suppression by NAC Polysulﬁdes of IFN-b-Dependent Pro-inﬂammatory Responses in RAW264.7 Cells Stimulated with LPS

A) Production of IFN-b from LPS-stimulated RAW264.7 cells and its suppression by NAC polysulﬁdes. RAW264.7 cells were stimulated with LPS (100 ng/mL) for

h in the absence or presence of 0.5 mM NAC polysulﬁdes or NaHS. IFN-b levels in culture supernatants were measured by means of ELISA.

B) Viability of RAW264.7 cells cultured under various conditions for 6 h (left panel) and 24 h (right panel). Cells were treated with 100 ng/mL LPS in the presence of

.5 mM NAC polysulﬁdes or NaHS. Controls were cells not treated with LPS or additives. Data are means ± SD (n = 3).

(

6

(

0

(

C) Western blotting of phosphorylation of STAT1 in RAW264.7 cells after LPS stimulation.

D) Quantitative data for results in (C).

E) Effects of NAC polysulﬁde treatment on iNOS expression by RAW264.7 cells stimulated with LPS. Expression of iNOS by cells 24 h after LPS stimulation was

determined by means of western blotting. Representative western blotting image (left panel) and its quantitative data (right panel).

F) Nitrite levels in culture supernatants of RAW264.7 cells. Nitrite levels were determined for culture supernatants obtained from RAW264.7 cells at 24 h after LPS

(

stimulation by means of the Griess assay. Controls were cells not treated with LPS or additives.

Data are means ± SD (n = 3). *p < 0.05; **p < 0.01.

effect against lethal endotoxin shock by means of their anti- during storage, even in aqueous media. This characteristic will

inﬂammatory functions, possibly through suppression of LPS- save researchers the trouble of preparing fresh solutions just

induced inﬂammatory responses.

before each experiment. Second, one can easily prepare NAC

34

polysulﬁdes that are labeled with S at sulfane sulfur moieties

34

DISCUSSION

by using S-sulﬁde as the starting material. Transfer of sulfur

34

from S-labeled NAC polysulﬁdes and cellular acceptor thiols

In this study, we describe the preparation, physicochemical can thus be determined by monitoring isotope-labeled sulfur

properties, sulfur-donating capabilities, and biological activities metabolomic data in complex biological milieu (e.g., Figure S2).

of NAC polysulﬁdes. NAC polysulﬁdes can easily be prepared Furthermore, researchers can study the metabolism of persul-

by reacting NAC and sulﬁdes in acidic media containing nitrite ﬁdes/polysulﬁdes, with a particular focus on sulfane sulfur

34

(Figure 1). NAC polysulﬁdes may have certain advantages as atoms, by detecting sulfur metabolites labeled with S (Fig-

persulﬁde/polysulﬁde donors. First, NAC polysulﬁdes are stable ure S2C). Third, decomposition metabolites that are derived

8

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A

B

C _P_o_l_y_I_:_C

Zymosan A

Additives

+

1

2000

0000

*

Additives

Phospho-

NF- B p65

Phospho-

NF- B p65

8

6

4

2

000

0

*

NF- B p65

1

.5

1

*

0

.5

0

1

2

3

4

5

6

7

8

9

Additives

Poly I:C

Stimulant

Zymosan A

+

Zymosan A

Poly I:C

Figure 6. Effects of NAC-S2 on TLR2 and TLR3 Signaling in RAW264.7 Cells

RAW264.7 cells were stimulated with poly(I:C) (25 mg/mL) or zymosan A (100 mg/mL) for 3 h in the absence or presence of 0.5 mM NAC polysulﬁdes. (A) Production

of TNF-a from zymosan A- and poly(I:C)-stimulated RAW264.7 cells and its suppression by NAC-S2. TNF-a in culture supernatants was measured by means of

ELISA. Phosphorylation of NF-kB p65 (phospho-NF-kB p65) in RAW264.7 cells treated with zymosan A (B) or poly(I:C) (C). Representative western blotting

images (upper panels) and the related quantitative data (lower panels). Controls were cells not treated with ligands or additives. Data are means ± SD (n = 3).

*

p < 0.05; **p < 0.01.

from NAC polysulﬁdes, including NAC itself, are expected to be

biologically inert. Thus, one can expect that NAC polysulﬁdes

may be suitable for therapeutic purposes as prototype drugs if

they have beneﬁcial effects.

GS-[S]

n

-NAC + NAC / GS[S] H + RS-NAC (Equation 4)

(n = 1,2)

The reaction between NAC-SSH and GSH, however, resulted

in the formation of GSSSH (Equation 5, Figure S2A):

Our sulfur metabolomic analyses revealed that NAC polysul-

ﬁdes can donate their sulfane sulfur atoms to acceptor thiols

such as GSH, which would result in the production of hydroper-

NAC-SSH + GSH / NAC + GSSSH

(Equation 5)

Additional reactions between GS[S]

H (Figure S2A). We detected appreciable amounts of

S1–S3). Reactions between NAC polysulﬁdes and reduced thiols thiosulfate in the reaction of NAC polysulﬁdes and GSH, which

n

H and reduced thiols pro-

sulﬁdes/polysulﬁdes in vitro and in cells (Figures 2, 3, and duced HS

n

suggests that H

2 2

S was oxidized under the current conditions

such as GSH can be initiated by a nucleophilic attack by reduced

thiols, which would lead to formation of mixed sulﬁdes and

to form thiosulfate (Figure S2A). We also detected GSSG in the

NAC hydropersulﬁdes/polysulﬁdes (NAC-SH, NAC-SSH), as ex- reactions between NAC polysulﬁdes and GSH (Figures 2C and

pressed by Equations (1)–(3) in the case of NAC-S2 and GSH 2D). GSSG can be formed according to Equation (6) and others.

(

Figure S2A).

GS[S] H + GSH / H S + GSSG

(Equation 6)

n

2 n

GSH + NAC-SS-NAC / GS-NAC + NAC-SSH (Equation 1)

In our cell culture experiments, we found similar reaction prod-

ucts incells after NACpolysulﬁdetreatment(Figure3). Thisﬁnding

strongly supports the belief that NAC polysulﬁdes can permeate

cell membranes and donate their sulfane sulfurs to acceptor thiols

such as cysteine and GSH that are present in cells, which would

result in marked elevation of endogenous persulﬁde/polysulﬁde

levels. We should note that, in cell experiments, we found appre-

GSH + NAC-SS-NAC / GS-S-NAC + NAC-SH (Equation 2)

GSH + NAC-SS-NAC / GS-SS-NAC + NAC (Equation 3)

In fact, we detected all expected products derived accord- ciable amounts of sulﬁde (H S) andthiosulfate after NAC-S2 treat-

2

ing to Equations (1)–(3) (Figures 2 and S2A). The present mentfor1 h (Figure 3). This resultsuggests the existence in cells of

data showed that the amounts of reaction products in Equa- metabolizing pathways for hydropersulﬁdes/polysulﬁdes to pro-

tion (1) (GS-NAC and NAC-SSH) were higher than those in duce H S and thiosulfate. ETHE1, a mitochondrial-localized

2

Equation (2) and Equation (3) (GS-S-NAC and NAC-SH for enzyme, can catalyze reductive degradation of GSSH to form

Equation 2, GS-SS-NAC and NAC for Equation 3), which sug- GSH and sulﬁte with the use of molecular oxygen as a co-sub-

gests that Equation (1) proceeded more favorably than did strate (Jung et al., 2016; Kabil and Banerjee, 2012; Mineri et al.,

Equation (2) and Equation (3) (Figure 2D), probably because 2008). Sulﬁde:quinone oxidoreductase, also a mitochondrial

NAC-SSH behaves as a better leaving group than NAC-SH enzyme, oxidizes sulﬁde to form sulﬁte, thiosulfate, or sulfate (Hil-

n

and NAC. GS-[S] -NAC (n = 1,2) formed in this way then reacts debrandt and Grieshaber, 2008). Thiosulfate is also formed in the

with reduced thiols to form GS[S]

tion (4) (Figure S2A).

n

H, as expressed in Equa- reaction catalyzed by rhodanese with the use of sulﬁte and GSSH

as substrates(Iciek et al., 2015). Organic hydropersulﬁdes(RSSH)

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A

C

PBS

1

00

0

100

80

60

40

20

0

PBS

8

6

4

2

0

LPS+NAC-S2

0

LPS+saline

0

LPS+saline

LPS+oxNAC

48 72

0

24

96

0

24

48

72

96

Time (h)

E

1

50

B

D

**

1

00

100

80

60

40

20

0

PBS

8

6

4

2

0

100

LPS+NAC-S1

LPS+saline

40

0

LPS+NaHS

24

0

24

48

72

96

Time (h)

0

48

72

96

Time (h)

PBS

LPS+saline

LPS+NAC-S2

Figure 7. Protection of Mice from Lethal Endotoxin Shock by NAC-S2 Treatment

Mice received intraperitoneal LPS (20 mg/kg body weight) or PBS as a negative control. Thirty minutes after LPS administration, vehicle control (saline, A), oxNAC

A), NAC-S1 (B), NAC-S2 (C), or NaHS (D) (each at 120 mmol/kg) was injected intraperitoneally into mice. After 24 h, mice received the same treatment again. PBS

(

control n = 6; each treatment group n = 10. In (E), serum TNF-a levels in mice receiving PBS alone (n = 4), LPS, and saline (n = 6), or LPS and NAC-S2 (n = 6) were

measured. In the case of LPS and saline or NAC-S2 treatment, serum samples were collected 30 h after LPS administration. **p < 0.01.

can reportedly be enzymatically reduced to form corresponding NK-kB, and MKK/MAPK, JNK, and ERK, which activate AP-1

2

thiols (RSH) and H S (Doka et al., 2016). In fact, Nagy and col- (Scheme S1). Our western blotting analyses suggested that the

leagues reported that thioredoxin reductase as well as GR may IKK/NF-kB axis was markedly inhibited by NAC-S2 treatment

act as persulﬁde-reducing enzymes in cells to maintain persulﬁde (Figures 4 and S4), whereas the MKK/MAPK, JNK, and ERK path-

homeostasis (Doka et al., 2016).

ways were instead activated by NAC-S2 treatment (Figure 4E).

Ezerina et al. (2018) recently reported that treatment of adeno- LPS-TLR4-NF-kB signaling was reportedly activated by ROS

carcinoma cell lines with 10 mM reduced NAC resulted in the (Asehnoune et al., 2004). ROS may activate NF-kB signaling

production of intracellular sulfane sulfur species as detected through IKK activation (Gloire et al., 2006) in a direct or an indirect

by using the sulfane sulfur probe SSip-1 DA. In the present study, manner via activation of upstream components of IKK activation

we found that treatment of non-stimulated RAW264.7 cells with such as PKD and SHIP-1 (Nakajima and Kitamura, 2013). As Fig-

reduced NAC at 5 mM resulted in a marked increase in intracel- ure 3 shows, NAC-S2 treatment rapidly increased levels of intra-

lular cysteine (Figure S3A), whereas we did not observe any cellular hydropersulﬁdes/polysulﬁdes, particularly GSSH and

increase in intracellular persulﬁdes and polysulﬁdes under these GSSSH to a signiﬁcant extent. Because of the highly potent anti-

conditions, as determined by means of LC-MS/MS (Figures S3A oxidant properties of hydropersulﬁdes/polysulﬁdes, enhanced

and S5). This result suggests that the metabolism of reduced formation of these compounds by NAC-S2 treatment may result

NAC may vary and depend on cell types, concentrations of in reduced ROS levels in response to LPS stimulation. NAC-S2-

reduced NAC, and incubation periods.

mediated production of hydropersulﬁdes/polysulﬁdes may thus

Innate immunity is the ﬁrst line of defense against pathogenic be a mechanism for reduced TNF-a production through the

invaders (Kawai and Akira, 2007, 2011; Kuzmich et al., 2017). IKK/NF-kB axis. The TLR4 adaptor family MyD88 activates

The TLR family plays an important role in detecting various IRAK (IL-1R-associated kinase), which results in the recruitment

microbial components and in eliciting innate immunity (Kawai of the protein kinase complex involving TAK1 (transforming

and Akira, 2007, 2011; Kuzmich et al., 2017). LPS, a cell wall growth factor-b-activated kinase-1) and TABs (TAK1-binding

component of Gram-negative bacteria, is recognized by TLR4 proteins) (Kawai and Akira, 2007). TAK1 has been suggested to

to activate expression of an array of inﬂammatory cytokine activate two distinct pathways: the IKK-dependent NF-kB activa-

genes (Kawai and Akira, 2007, 2011; Kuzmich et al., 2017). tion pathway and the MKK/MEK-dependent MAPK (ERK, JNK,

TLR4-LPS signal transduction is initiated via the TIRAP-MyD88 p38) pathway (Kawai and Akira, 2007). As mentioned above,

and TRAM-TRIF pathways and leads to expression of inﬂamma- NAC-S2 treatment suppressed the NF-kB pathway and instead

tory cytokines such as TNF-a and IFN-b, as illustrated in Scheme sustained ERK activation (Figure 4). Continued study is war-

S1 (Kawai and Akira, 2007, 2011; Kuzmich et al., 2017). In this ranted to clarify the detailed molecular mechanisms involved in

study, we found that NAC-S2 treatment strongly suppressed NAC-S2-mediated modulation of TLR4 signaling by identifying

production of cytokines (TNF-a and IFN-b) from RAW264.7 target molecules regulated by NAC-S2.

cells stimulated with LPS (Figures 4, 5, S4, and S5). With

In addition to TLR4, TLR2, and TLR3 utilize TAK1 to

regard to TNF-a production, signal pathways downstream of activate NF-kB signaling, thereby leading to the production of

TIRAP-MyD88 include IKK and PI3K/AKT, which activate pro-inﬂammatory cytokines including TNF-a (Kawai and Akira,

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2

007, 2011; Kuzmich et al., 2017). We found that NAC-S2 treat- models for their ability to block TLR4-mediated cytokine produc-

ment strongly inhibited NF-kB activation and TNF-a production, tion, and several have been tested in clinical trials (Kuzmich et al.,

induced by different TLR ligands such as zymosan A, poly(I:C), 2017). The known TLR4 antagonists belong to different classes

and LPS (Figures 4 and 6). Thus, the TAK1-NF-kB pathway of chemical compounds, primarily glycolipids, which mimic the

may be a target for the anti-inﬂammatory actions of NAC-S2.

natural TLR4 ligand lipid A, but also heterocyclic compounds,

We found that IFN-b production was inhibited not only by NAC- peptides, opioids, steroids, taxanes, and others, and these com-

S2but also by NAC-S1 (Figure 5). Such reduced IFN-b production pounds have natural and synthetic origins (Kuzmich et al., 2017).

may be associated with NAC polysulﬁde-mediated inhibition of

In this study, we demonstrated that NAC-S2 treatment pro-

iNOS expression and NO production (Figure 5). IFN-b-induced tected mice from lethal endotoxin shock, with a concomitant

phosphorylation of STAT1, which is essential for iNOS expression reduction in TNF-a production (Figure 7). This result suggests

(

Scheme S1), was also signiﬁcantly inhibited by both NAC-S1 and that persulﬁde/polysulﬁde donors may be applicable as TLR4

NAC-S2 (Figure S6A). One hypothesis for why the TRAM-TRIF/ antagonists that can be used to treat endotoxin shock. Persul-

IFN-b axis is inhibited by NAC-S1 is that TRAM-TRIF-mediated ﬁde/polysulﬁde donors solely or in combination with other

IFN-b production requires a longer activation period, such as TLR4 antagonists warrant additional investigation as potential

6 h, compared with that for the TIRAP-MyD88 pathway (Fig- therapeutic agents.

ure 5A). As seen in Figure S3B, a time-dependent increase in In summary, we demonstrated here that NAC polysulﬁdes are

intracellular hydropersulﬁde/polysulﬁde levels was evident for cell permeable and are reduced thiol-activable persulﬁde/

cells treated with NAC-S1. Therefore, hydropersulﬁde/polysul- polysulﬁde donors. NAC polysulﬁdes strongly desensitize

ﬁde levels may become sufﬁcient to inhibit TRAM-TRIF pathways macrophages to TLR-mediated innate immune responses that

even in cells treated with NAC-S1.

accompanies reduced production of pro-inﬂammatory cyto-

NAC polysulﬁdes may exert anti-inﬂammatory effects in kines. NAC polysulﬁdes may thus become powerful chemical

addition to antioxidant actions. Protein thiols are expected to be tools for studying biological processes that are sensitive to

modiﬁed by NAC polysulﬁdes directly or via formation of low-mo- endogenous persulﬁde/polysulﬁde levels and for developing

lecular-weight hydropersulﬁdes/polysulﬁdes such as GSH hydro- strategies to treat pathological conditions associated with dysre-

polysulﬁdes. Inﬂammatory responses including NF-kB activation gulated inﬂammatory responses. Integrated omics approaches

may be modulated through posttranslational thiol modiﬁcations combining genomic, transcriptomic, proteomic, and metabolo-

such as protein sulfhydration (Paul and Snyder, 2012). Stimulator mic analyses may help understanding of the mechanisms of

of interferon genes (STING) is one of the innate immune adaptor how NAC polysulﬁdes modulate cellular responses by inﬂu-

proteins essential for infection-induced production of type I encing thousands of cell signaling processes.

IFNs (Ishikawa et al., 2009). S-Palmitoylation of cysteine at 91

(

Cys91) of STING is critical for STING clustering and activation

Mukai et al., 2016). Haag et al. (2018) recently discovered that

SIGNIFICANCE

(

nitrofuran-type small molecules inhibited S-palmitoylation-

induced STING activation through covalent binding to Cys91 of

STING. This ﬁnding suggests that NAC polysulﬁdes may exert

their inhibitory actions on IFN-b production by modulating STING

activity in a protein S-sulfhydration-dependent manner. Several

S-sulfhydration proteomics methods have been developed,

including a modiﬁed biotin-switch assay (Pan and Carroll, 2013),

tag-switch assay (Ida et al., 2014; Zhang et al., 2014), protein per-

sulﬁde detection protocol (Doka et al., 2016), and polyethylene

glycol-conjugated maleimide-labeling gel shift assay (Akaike

et al., 2017); these techniques may become powerful tools to

identify target proteins as well as signaling cascades affected

by protein S-sulfhydration. Additional study is thus warranted to

determine the effects of NAC polysulﬁde treatment on the S-sulf-

hydrationproteome, andparticularly itsassociationwithLPS-TLR

signaling cascade activity.

Chemical compounds that effectively eliminate (scavengers)

or enhance (donors) endogenous persulﬁdes and polysul-

ﬁdes may become powerful tools for studying the biological

roles of reactive sulfur species. This work reports that

sulfane sulfur atoms stabilized by NAC (NAC polysulﬁdes)

via disulﬁde bonds at both sides can act as potent persulﬁde

and polysulﬁde donors in cells. Mechanistic studies re-

vealed that sulfane sulfur atoms in NAC polysulﬁdes are

readily transferred to acceptor thiols present in cells. The

major ﬁndings of this study are that innate immune re-

sponses in macrophages that are governed by TLR signaling

are highly affected by endogenous persulﬁde/polysulﬁde

levels and that they are strongly suppressed by NAC polysul-

ﬁdes. In addition, NAC polysulﬁdes had anti-inﬂammatory

functions in mice and thus protected mice from lethal endo-

toxin shock. NAC polysulﬁdes developed in this study will be

useful to investigators who are interested in studying the

roles of reactive sulfur species, including regulatory roles

of these species in immune responses.

Although activation of TLR4 signaling is an important response

for eradicating pathogenic invaders, excessive and dysregulated

activation of TLR4 signaling related to LPS overload leads to

endotoxin shock, which results in acute, life-threatening organ

dysfunctions (Cavaillon, 2018; Kuzmich et al., 2017). Endotoxin

shock, with increased LPS levels in blood, overexpression of

pro-inﬂammatory cytokines, activation of the blood coagulation

system, and accumulation of ﬁbrinogen dysfunction products,

causes a breakdown of local and general hemodynamics and

endothelial dysfunction via TLR signaling pathways (Kuzmich

et al., 2017). Various compounds have been tested in animal

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

Cell Chemical Biology 26, 1–13, May 16, 2019 11

Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Animal Models

Bianco, C.L., Chavez, T.A., Sosa, V., Saund, S.S., Nguyen, Q.N.N., Tantillo,

D.J., Ichimura, A.S., Toscano, J.P., and Fukuto, J.M. (2016). The chemical

biology of the persulﬁde (RSSH)/perthiyl (RSS) redox couple and possible

role in biological redox signaling. Free Radic. Biol. Med. 101 , 20–31.

B Cell Lines and Culture Conditions

d METHOD DETAILS

Cavaillon, J.M. (2018). Exotoxins and endotoxins: inducers of inﬂammatory

B Reagents

cytokines. Toxicon 149 , 45–53.

B Preparation of NAC Polysulﬁdes

B Preparation of HPE-AM Adduct Standards

B LC-MS and LC-MS/MS

Doka, E., Pader, I., Biro, A., Johansson, K., Cheng, Q., Ballago, K., Prigge, J.R.,

Pastor-Flores, D., Dick, T.P., Schmidt, E.E., et al. (2016). A novel persulﬁde

detection method reveals protein persulﬁde- and polysulﬁde-reducing func-

tions of thioredoxin and glutathione systems. Sci. Adv. 2 , e1500968.

B Reaction between NAC Polysulﬁdes and GSH

B Cell Treatment

Ezerina, D., Takano, Y., Hanaoka, K., Urano, Y., and Dick, T.P. (2018). N-Acetyl

B Sulfur Metabolomic Analyses

B Cell Viability

2

cysteine functions as a fast-acting antioxidant by triggering intracellular H S

and sulfane sulfur production. Cell Chem. Biol. 25 , 447–459.

B Western Blotting

Fukuto, J.M., Ignarro, L.J., Nagy, P., Wink, D.A., Kevil, C.G., Feelisch, M.,

Cortese-Krott, M.M., Bianco, C.L., Kumagai, Y., Hobbs, A.J., et al. (2018).

Biological hydropersulﬁdes and related polysulﬁdes - a new concept and

perspective in redox biology. FEBS Lett. 592 , 2140–2152.

B Determination of Cytokine Production

B Nitrite Assay

B RT-PCR

B Mouse Endotoxin Shock Model

d QUANTIFICATION AND STATISTICAL ANALYSIS

Furukawa, T., Yahiro, K., Tsuji, A.B., Terasaki, Y., Morinaga, N., Miyazaki, M.,

Fukuda, Y., Saga, T., Moss, J., and Noda, M. (2011). Fatal hemorrhage

induced by subtilase cytotoxin from Shiga-toxigenic Escherichia coli .

Microb. Pathog. 50 , 159–167.

SUPPLEMENTAL INFORMATION

Gloire, G., Legrand-Poels, S., and Piette, J. (2006). NF-k B activation by reac-

tive oxygen species: ﬁfteen years later. Biochem. Pharmacol. 72 , 1493–1505.

Supplemental Information includes seven ﬁgures, one table, and one scheme

and can be found with this article online at https://doi.org/10.1016/j.chembiol.

Haag, S.M., Gulen, M.F., Reymond, L., Gibelin, A., Abrami, L., Decout, A.,

Heymann, M., van der Goot, F.G., Turcatti, G., Behrendt, R., et al. (2018).

Targeting STING with covalent small-molecule inhibitors. Nature 559 ,

2019.02.003.

2

69–273.

ACKNOWLEDGMENTS

Hildebrandt, T.M., and Grieshaber, M.K. (2008). Three enzymatic activities

catalyze the oxidation of sulﬁde to thiosulfate in mammalian and invertebrate

mitochondria. FEBS J. 275 , 3352–3361.

We thank J.B. Gandy for her editing of the manuscript. This work was sup-

ported in part by Grants-in-Aid for Scientiﬁc Research ([A], [B], [C], Innovative

Areas [Research in a Proposed Area], and Challenging Exploratory Research)

from the Ministry of Education, Science, Sports, and Technology (MEXT),

Japan, to H.T. (17K10019), H.I. (16H04674 and 26111011), T.A. (26111008,

Iciek, M., Kowalczyk-Pachel, D., Bilska-Wilkosz, A., Kwiecien, I., Gorny, M.,

and Wlodek, L. (2015). S-Sulfhydration as a cellular redox regulation. Biosci.

Rep. 36 , e00304.

2

6111001, and 16K15208), and T.S. (17K19205 and 18H02098); a grant from

the Japan Agency for Medical Research and Development (AMED) to T.S.

JP18fm0208029); a grant from the Smoking Research Foundation to H.I.; a

Ida, T., Sawa, T., Ihara, H., Tsuchiya, Y., Watanabe, Y., Kumagai, Y.,

Suematsu, M., Motohashi, H., Fujii, S., Matsunaga, T., et al. (2014). Reactive

cysteine persulﬁdes and S -polythiolation regulate oxidative stress and redox

signaling. Proc. Natl. Acad. Sci. U S A 111 , 7606–7611.

(

grant from the Takeda Science Foundation to K.O. and H.T.; and a grant

from the Otsuka Toshimi Scholarship Foundation to T.Z.

Ishikawa, H., Ma, Z., and Barber, G.N. (2009). STING regulates intracellular

DNA-mediated, type I interferon-dependent innate immunity. Nature 461 ,

AUTHOR CONTRIBUTIONS

7

88–792.

T.S. designed the study. T.Z., K.O., H.T., H.I., W.I., and T.A. conducted the ex-

periments. T.Z., K.O., and H.T. analyzed the data. T.S. and T.Z. wrote the

manuscript. T.S. and T.A. edited the manuscript.

Jiang, W., Sun, R., Wei, H., and Tian, Z. (2005). Toll-like receptor 3 ligand at-

tenuates LPS-induced liver injury by down-regulation of toll-like receptor 4

expression on macrophages. Proc. Natl. Acad. Sci. U S A 102 , 17077–17082.

Jung, M., Kasamatsu, S., Matsunaga, T., Akashi, S., Ono, K., Nishimura, A.,

Morita, M., Abdul Hamid, H., Fujii, S., Kitamura, H., et al. (2016). Protein

polysulﬁdation-dependent persulﬁde dioxygenase activity of ethylmalonic

encephalopathy protein 1. Biochem. Biophys. Res. Commun. 480 , 180–186.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: September 12, 2018

Revised: December 3, 2018

Accepted: January 31, 2019

Published: March 7, 2019

Kabil, O., and Banerjee, R. (2012). Characterization of patient mutations in

human persulﬁde dioxygenase (ETHE1) involved in H S catabolism. J. Biol.

2

Chem. 287 , 44561–44567.

Kang, J., Xu, S., Radford, M.N., Zhang, W., Kelly, S.S., Day, J.J., and Xian, M.

(

2018). O /S relay deprotection: a general approach to controllable donors of

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Cell Chemical Biology 26, 1–13, May 16, 2019 13

Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rabbit polyclonal anti-actin antibody

Mouse monoclonal anti-phospho-IkBa

Rabbit monoclonal anti-phospho-STAT1

Rabbit polyclonal anti-STAT1

Sigma-Aldrich

Cat#A2066; RRID: AB_476693

Cat#9246; RRID: AB_2267145

Cat#7649; RRID: AB_11220426

Cat#9172; RRID: AB_10693929

Cat#3033; RRID: AB_331284

Cat#4370; RRID: AB_2315112

Cat#4695; RRID: AB_390779

Cat#4668; RRID: AB_2307320

Cat#9252; RRID: AB_2250373

Cat#4060; RRID: AB_2315049

Cat#4691; RRID: AB_915783

Cat#4511; RRID: AB_2139682

Cat#8690; RRID: AB_10999090

Cat#sc-372; RRID: AB_632037

Clone number: i2G4

Cell Signaling Technology

Santa Cruz Biotechnology

Nakamura et al., 2006

Rabbit monoclonal anti-phospho-NF-kB p65

Rabbit monoclonal anti-phospho-p44/42 MAPK(ERK1/2)

Rabbit monoclonal anti- p44/42 MAPK(ERK1/2)

Rabbit monoclonal anti-phospho-SAPK/JNK

Rabbit polyclonal anti-SAPK/JNK

Rabbit monoclonal anti-phospho-Akt

Rabbit monoclonal anti-Akt

Rabbit monoclonal anti-phospho-p38

Rabbit monoclonal anti-p38

Rabbit polyclonal anti-NF-kB p65 antibody

iNOS-speciﬁc monoclonal antibody

Chemicals, Peptides, and Recombinant Proteins

NAC

Wako Pure Chemical Industries

Sigma-Aldrich

013-05133; CAS: 616-91-1

517-30351; CAS: 16721-80-5

197-02561; CAS: 7632-00-0

263-01491;CAS: 58856-93-2

L2880

NaHS

NaNO

2

Zymosan A

LPS (Escherichia coli 055:B5)

GSH

Sigma-Aldrich

G4251; CAS: 70-18-8

G4376; CAS: 27025-41-8

8234-MB-010

GSSG

Sigma-Aldrich

IFN-b

R&D Systems

Poly I:C

L-Cysteine

HPE-IAM

R&D Systems

4287; CAS: 24939-03-5

10309-12; CAS: 52-90-4

24600; CAS: 53527-07-4

N/A

Nacalai Tesque

Molecular BioSciences

Wintner et al., 2010

Sigma-Aldrich

3

4

Na

3

2

S

15

1

[

C

2

,

N]GSH

683620

Experimental Models: Cell Lines

RAW264.7 cells (male)

RIKEN BioResource Center

Japan SLC Inc.

RCB0535

N/A

Experimental Models: Organisms/Strains

C57BL/6 Mice (male, 9-week-old)

Oligonucleotides

Primer: mouse IFN-b forward:

Sugiyama et al., 2004

Furukawa et al., 2011

N/A

5

’-AAACAATTTCTCCAGCACTG-3’

Primer: mouse IFN-b reverse:

’-ATTCTGAGGCATCAACTGAC-3’

Primer: mouse GAPDH forward:

’- TGAGGCCAGTGCTGAGTATG -3’

Primer: mouse GAPDH reverse:

’- CCTTCCACAATGCCAAAGTT-3’

5

Software

GraphPad Prism 7.0

GraphPad Software, La Jolla, CA, USA

www.graphpad.com

e1 Cell Chemical Biology 26, 1–13.e1–e4, May 16, 2019

Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Tomohiro

Sawa (sawat@kumamoto-u.ac.jp ).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animal Models

C57BL/6 mice (male, 9-week-old) were purchased from Japan SLC Inc. (Shizuoka, Japan) and housed at the Center for Animal

Resources and Development, Kumamoto University. All procedures were approved by the Kumamoto University Ethics Review

Committee for Animal Experimentation (Experiment number F29-194) and were performed to minimize the number of animals

used and their suffering.

Cell Lines and Culture Conditions

Cells of the murine macrophage-like cell line RAW264.7 (gender: male; RIKEN BioResource Center, Tsukuba, Japan) were cultured in

Dulbecco’s Modiﬁed Eagle’s Medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% heat-inactivated fetal

bovine serum (MP Biomedicals, Santa Ana, CA, USA) and 1% penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan) in a 5% CO

ꢀ

2

humidiﬁed incubator at 37 C.

METHOD DETAILS

Reagents

NAC, NaHS, zymosan A, and NaNO

GSSG), and anti-actin antibody (A2066) were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-Cysteine was purchased

from Nacalai Tesque. b-(4-Hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) was purchased from Molecular BioSciences (Boulder,

2

were purchased from Wako. LPS (Escherichia coli 055:B5), GSH, oxidized L-glutathione

(

3

4

CO, USA). Isotope-labeled sodium sulﬁde Na

2

S was prepared according to the literature (Wintner et al., 2010). Anti-phospho-

IkBa (#9246), anti-phospho-STAT1 (#7649), anti-STAT1 (#9172), anti-phospho-NF-kB p65 (#3033), anti-phospho-ERK1/2 (#4370),

anti-ERK1/2 (#4695), anti-phospho-JNK (#4668), anti-JNK (#9252), anti-phospho-Akt (#4060), anti-Akt (#4691), anti-phospho-p38

(

#4511), and anti-p38 (#8690) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NF-kB p65

antibody (C-20, sc-372) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant mouse IFN-b (8234-MB-010)

and poly I:C (4287) were from R&D Systems (Minneapolis, MN, USA). iNOS-speciﬁc monoclonal antibody i2G4 was prepared as

described previously (Nakamura et al., 2006). All reagents were of the highest grade available and used without further puriﬁcation.

Preparation of NAC Polysulﬁdes

NAC polysulﬁdes were prepared by reacting NAC with sulﬁde in the presence of certain oxidants, similar to the protocol used for

preparation of cysteine polysulﬁdes as reported previously (Akaike et al., 2017; Ida et al., 2014; Ono et al., 2017). In brief, NAC

ꢀ

(

60 mM) was reacted with 60 mM NaNO

2

in H

2

O at 37 C for 5 min, followed by addition of NaHS (ﬁnal concentration, 45 mM) and

ꢀ

formic acid (ﬁnal concentration, 0.2%) and another incubation at 37 C for 15 min. The reaction mixture was subjected to preparative

HPLC for puriﬁcation of NAC polysulﬁdes. Preparative HPLC was performed by using the Agilent 1260 Inﬁnity series equipped with a

photodiode array detector and an automated fraction collector (Agilent Technologies, Santa Clara, CA, USA). Samples (1500 ml) were

ꢀ

injected onto a YMC-Triart C18 Plus column (10 3 250 mm; YMC Co., Ltd., Kyoto, Japan) at 35 C. Mobile phases A (H

2

O + 0.1%

formic acid) and B (acetonitrile) were used with a linear gradient from 0.2% to 40% B for 22 min, with the gradient maintained at

0% B for 1 min, after which the gradient was decreased to 0.2% B in 1 min, with a ﬂow rate of 3 ml/min. NAC polysulﬁdes were

4

detected at 210 nm. Formation of NAC polysulﬁdes was determined by means of MS as described below. Peaks corresponding

to NAC polysulﬁdes were collected by using a fraction collector and were subjected to lyophilization. NAC polysulﬁdes labeled

3

with S at sulfane sulfur moieties were prepared by using Na

4

34

2

S instead of NaHS.

Preparation of HPE-AM Adduct Standards

In this study, hydropersulﬁdes/polysulﬁdes were quantiﬁed by means of LC-MS/MS after derivatization with HPE-IAM to form

stable HPE-AM adducts (Akaike et al., 2017). For precise quantiﬁcation, isotope-labeled HPE-AM adducts were spiked into samples.

HPE-AM adducts of cysteine, GSH, their hydropersulﬁdes/polysulﬁdes, and H

2

S were prepared as described previously (Akaike

et al., 2017; Ida et al., 2014; Ono et al., 2017). The same protocol was applied to preparation of NAC adducts. Isotope labeling

15

3

4

13

34

2

C , N]GSH (Sigma-Aldrich), [ S]NAC, and

was performed by using isotope-labeled starting materials including Cys SH, [

3

4

34

S. Cys SH and [ S]NAC were synthesized by using the method reported recently (Ono et al., 2017). HPE-AM adducts of sulﬁte

34

Na

2

ꢀ

and thiosulfate were prepared by reacting 100 mM Na

2

SO

3

or Na

2

S

2

O

3

with 50 mM HPE-IAM in 50 mM Tris-HCl (pH 7.4) at 37 C for

1

5 min. All persulﬁde HPE-AM adducts were puriﬁed by means of reversed-phase HPLC.

Cell Chemical Biology 26, 1–13.e1–e4, May 16, 2019 e2

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LC-MS and LC-MS/MS

NAC polysulﬁdes, HPE-AM adducts, and related molecules were analyzed by means of LC-electrospray ionization (ESI)-MS with the

Agilent 6460 Triple Quadrupole LC/MS system (Agilent Technologies). LC conditions used were as follows: column, YMC-Triart C18

ꢀ

Plus column (2.1 3 50 mm) (YMC Co.); column temperature, 45 C; injection volume, 1 ml; mobile phases: A, 0.1% formic acid, and B,

acetonitrile; gradient (B concentration), 0 min - 1%, 10 min - 80%, 10.5 min - 1%, 15 min - 1%; ﬂow rate, 0.2 ml/min. General

ꢀ

conditions used for ESI-MS were as follows: nebulizer gas, nitrogen delivered at 50 psi; nebulizer gas temperature, 250 C; capillary

voltage, 3500 V; collision gas, G1 grade nitrogen (Taiyo Nippon Sanso Corp., Tokyo, Japan). Multiple reaction monitoring (MRM) was

used to quantify the analytes. Table S1 provides the MRM parameters used in this study.

Reaction between NAC Polysulﬁdes and GSH

NAC polysulﬁdes (100 mM) were reacted with GSH (10 and 100 mM) in 50 mM sodium phosphate buffer (pH 7.5) at 37 C. At various

ꢀ

time points, 5 ml of the reaction mixtures were taken and mixed with 95 ml of 0.1% formic acid to terminate the reactions. NAC

polysulﬁdes that remained in the mixtures were quantiﬁed by means of LC-MS/MS. To identify the products formed in the

reactions between NAC polysulﬁdes and GSH, 5 mM HPE-IAM was added to the reaction mixtures containing NAC polysulﬁdes

and GSH (10 and 100 mM) after 30 min of incubation to derivatize reduced thiols. Products formed were investigated by means of

LC-MS and LC-MS/MS.

Cell Treatment

RAW264.7 cells (male) were plated in 6-, 24-, or 96-well plates (at a density of 1.0 3 10 cells/well, 2.5 3 10 cells/well, and 5.0 3

6

5

4

0 cells/well, respectively) and allowed to grow overnight. On the day of the experiment, cells were treated with LPS (100 ng/ml),

1

or were not treated with LPS, in the presence of NAC polysulﬁdes, NAC, or NaHS as indicated in the ﬁgure legends. To determine

the effect of NAC polysulﬁdes on TLR2 and TLR3 signaling pathways, cells were stimulated with zymosan A (TLR2 ligand, 100 mg/ml)

or poly I:C (TLR3 ligand, 25 mg/ml) in the presence of NAC polysulﬁdes for 3 h. To examine the effect of NAC polysulﬁdes

on IFN-b-induced phosphorylation of STAT1, cells were treated with NAC polysulﬁdes for 3 h and then stimulated with IFN-b

(

100 pg/ml, approximately 120 U/ml) for 10, 30, or 60 min.

Sulfur Metabolomic Analyses

We performed LC-MS/MS-based sulfur metabolomic analysis to quantify various sulfur species present in cells (Akaike et al., 2017;

Ida et al., 2014; Kunikata et al., 2017; Numakura et al., 2017; Ono et al., 2017). In brief, cells treated under certain conditions were

washed once with phosphate-buffered saline (PBS) and were harvested with a methanol solution containing 5 mM HPE-IAM. Cell

ꢀ

suspensions were homogenized with a Bioruptor (Cosmo Bio, Tokyo, Japan), followed by incubation at 37 C for 15 min. After

centrifugation, supernatants were diluted 10-fold with 0.1% formic acid containing known amounts of isotope-labeled standards,

after which they were subjected to LC-MS/MS metabolomic analysis. Precipitates were lysed by sonication with 1% sodium dodecyl

sulfate (SDS) in PBS. Protein concentrations in the precipitates were measured by using the BCA Protein Assay Kit (Wako).

Cell Viability

We determined cell viability by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay based on the

4

mitochondrial reduction of MTT to formazan. RAW264.7 cells were seeded in a 96-well plate at a density of 5 3 10 cells/well. After

overnight culture, cells were treated with LPS (100 ng/ml) and NAC polysulﬁdes. After 3, 6, and 24 h of incubation, culture medium

was replaced with 200 ml of fresh medium. A 7.5 mg/ml solution of MTT in PBS was added into each well (20 ml/well). After incubation

for 1.5 h, 150 ml of culture supernatant was removed from each well and the formazan crystals were lysed by using 100 ml of MTT stop

solution (0.4% HCl, 10% Triton X-100 in isopropanol). After incubation for 12 h, absorbance was measured at 570 nm, with 655 nm as

the reference wavelength, via a microplate reader (Bio-Rad, Hercules, CA, USA).

Western Blotting

Cells were lysed in SDS sample buffer (62.5 mM Tris-HCl pH6.8, 2% SDS, 6% glycerol, 0.005% bromophenol blue, and 2.5%

2

-mercaptoethanol) and then boiled for 5 min. Proteins were separated by using SDS-polyacrylamide gel electrophoresis and

were then transferred to polyvinylidene diﬂuoride membranes (Merck Millipore, Darmstadt, Germany) at 100 V for 1 h. Membranes

were blocked with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) (20 mM Tris pH 7.5, 137 mM

ꢀ

NaCl, and 0.1% Tween 20) for 60 min and then incubated for 1 h at room temperature or overnight at 4 C with the primary antibodies

as indicated. After membranes were washed with TBS-T, they were incubated with horseradish peroxidase (HRP)-conjugated

anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology) at room temperature for 1 h. After samples were washed

with TBS-T, protein bands were detected by using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) with the

luminescent image analyzer ChemiDoc XRS system (Bio-Rad).

Determination of Cytokine Production

Levels of the cytokines TNF-a and IFN-b in culture supernatants were measured by means of the mouse TNF-a ELISA kit (R&D

Systems) and mouse IFN-b ELISA kit (PBL Assay Science, Piscataway, NJ, USA), respectively, according to the manufacturers’

instructions. Absorbance at 490 nm was then measured by using an iMark Microplate Reader (Bio-Rad).

e3 Cell Chemical Biology 26, 1–13.e1–e4, May 16, 2019

Please cite this article in press as: Zhang et al., Enhanced Cellular Polysulﬁdes Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin

Shock, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.003

Nitrite Assay

NO production by RAW264.7 cells was determined by means of the Griess assay (Marzinzig et al., 1997). Cells were treated with LPS

(

100 ng/ml), or were not treated with LPS, in the presence of certain additives such as NAC polysulﬁdes. Culture supernatants (50 ml)

were collected and subjected to the Griess assay. The absorbance of samples at 570 nm was compared with that of the NaNO

standard on a microplate reader (Bio-Rad).

2

RT-PCR

Total RNA was extracted from RAW264.7 cells by means of the RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol.

Total RNA (1 mg) was reverse transcribed by using the ReverTraAce qPCR RT Kit (TOYOBO, Osaka, Japan) according to the

manufacturer’s instructions. cDNA was ampliﬁed by PCR by means of KOD FX polymerase (TOYOBO). The PCR conditions were

ꢀ

as follows: initial denaturation at 94 C for 2 min, 28 cycles at 98 C for 10 s, 50 C for 30 s, and 68 C for 30 s, followed by a ﬁnal

ꢀ

step at 68 C for 5 min. Primers for PCR are given in the Key Resources Table. PCR products were subjected to electrophoresis

on 2% agarose gels, and the bands were stained with ethidium bromide and visualized under UV light.

Mouse Endotoxin Shock Model

The effects of NAC polysulﬁdes on in vivo inﬂammatory responses were studied by using the mouse endotoxin shock model (Kawai

et al., 1999). Male 9-week-old C57BL/6 mice (average body weight about 25 g) received intraperitoneal injections of PBS (0.1 ml,

solvent control) or LPS [20 mg/kg body weight (0.1 ml of 5 mg/ml), from E. coli 055:B5]. Thirty minutes later, mice received intraper-

itoneal injections of physiological saline (0.1 ml, solvent control), NAC polysulﬁdes [120 mmol/kg body weight (0.1 ml of 30 mM)], or

NaHS [120 mmol/kg body weight (0.1 ml of 30 mM)]. An additional treatment was performed 24 h after the ﬁrst treatment. Survival of

mice was monitored at 24-h intervals for 96 h after the LPS injection. Survival curves were constructed by using the Kaplan-Meier

method and GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). To quantify various sulfur species present in tissues,

mice were killed at 27 h after the LPS injection. Livers and lungs were immediately homogenized by using the Polytron homogenizer

PT1200E (Kinematica AG, Lucerne, Switzerland) in a cold methanol solution containing 5 mM HPE-IAM, followed by incubation for

ꢀ

2

0 min at 37 C. After centrifugation, aliquots of the supernatants were diluted 100-fold with 0.1% formic acid containing known

amounts of isotope-labeled internal standards, which were then subjected to LC-MS/MS metabolomic analysis as described above.

Precipitates were lysed by sonication with 1% SDS in PBS. Protein concentrations in the precipitates were measured by using the

BCA Protein Assay Kit (Wako). Blood samples were collected from each group of mice and serum samples were obtained by

centrifugation. The levels of TNF-a and IFN-b in serum samples were determined by means of the mouse TNF-a and IFN-b ELISA

kits (R&D Systems) according to the manufacturer’s instructions as described above.

QUANTIFICATION AND STATISTICAL ANALYSIS

All data are expressed as means ± SD. Data for each experiment were acquired from at least three independent experiments.

Statistical analyses were performed by using Student’s t test, with signiﬁcance set at p < 0.05. Survival curves constructed by using

the Kaplan-Meier method were analyzed by means of the log-rank (Mantel-Cox) test, with signiﬁcance set at p < 0.05.

Cell Chemical Biology 26, 1–13.e1–e4, May 16, 2019 e4

Article Doi

Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock

DOI: 10.1016/j.chembiol.2019.02.003

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Article abstract of DOI:10.1016/j.chembiol.2019.02.003

Full text of DOI:10.1016/j.chembiol.2019.02.003

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