c-Jun pathway

Journal Pre-proof Erythromycin reverses cigarette smoke extract-induced corticosteroid insensitivity by inhibition of the JNK/c-Jun pathway Yan-Fei Bin, Nan Ma, Yan-Xiu Lu, Xue-Jiao Sun, Yi Liang, Jing Bai, Jian-Quan Zhang, Mei-Hua Li, Xiao-Ning Zhong, Zhi-Yi He PII:

S0891-5849(19)30762-2

DOI:

https://doi.org/10.1016/j.freeradbiomed.2019.11.020

Reference:

FRB 14491

To appear in:

Free Radical Biology and Medicine

Received Date: 15 May 2019 Revised Date:

14 November 2019

Accepted Date: 15 November 2019

Please cite this article as: Y.-F. Bin, N. Ma, Y.-X. Lu, X.-J. Sun, Y. Liang, J. Bai, J.-Q. Zhang, M.H. Li, X.-N. Zhong, Z.-Y. He, Erythromycin reverses cigarette smoke extract-induced corticosteroid insensitivity by inhibition of the JNK/c-Jun pathway, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/j.freeradbiomed.2019.11.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

cigarette somking

P38MAPK ERK1/2

P

c-Jun gene

P

Erythromycin

JNK

c-Jun Protein

c-Jun targeted gene （such as IL-8）

Corticosteroid Insensitivity

1

Erythromycin Reverses Cigarette Smoke Extract-Induced

2

Corticosteroid Insensitivity by Inhibition of the JNK/c-Jun Pathway.

3

Yan-Fei Bin1*, Nan Ma1*, Yan-Xiu Lu1*, Xue-Jiao Sun1, Yi Liang1, Jing Bai1,

4

Jian-Quan Zhang1, Mei-Hua Li1, Xiao-Ning Zhong1, Zhi-Yi He1.

5 6

1

7

Guangxi Medical University, Nanning, Guangxi, 530021, China.

Department of Respiratory and Critical Medicine, The First Affiliated Hospital of

8 9

* contributed equally to this work.

10 11

Corresponding author address: Zhi-Yi He, Department of Respiratory and Critical

12

Care Medicine, The First Hospital of Guangxi Medical University, No 6 Shuangyong

13

Road,

14

+86-771-5608132. E-mail: [email protected].

15 16 17 18 19 20 21

Nanning,

Guangxi,

530021,

China.

Tel:

+86-771-5356702;

Fax:

22

Abstract:

23

Corticosteroid insensitivity is a feature of airway inflammation in chronic obstructive

24

pulmonary disease (COPD). Erythromycin exhibits anti-inflammatory activity in

25

COPD, but the concrete mechanism is still unclear. This study aimed to investigate

26

the effects of erythromycin on corticosteroid sensitivity in peripheral blood

27

mononuclear cells (PBMCs) and U937 cells (a human monocytic cell line). PBMCs

28

were collected from non-smokers, healthy smoker volunteers, and COPD subjects.

29

U937 cells were incubated with or without erythromycin and stimulated with TNF-α

30

in the presence or absence of cigarette smoke extract (CSE). The dexamethasone (Dex)

31

concentration required to achieve 50% inhibition of TNF-α-induced interleukin (IL)-8

32

production

33

(MAPK)/Activator protein-1 (AP-1) pathway was also evaluated. Erythromycin

34

improved corticosteroid sensitivity in PBMCs obtained from COPD patients and

35

CSE-treated U937 cells. This improvement in corticosteroid sensitivity was associated

36

with reduced c-Jun expression, which resulted from the inhibition of P38

37

Mitogen-activated protein kinase (P38MAPK), extracellular signal-regulated protein

38

kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) phosphorylation. Erythromycin

39

had no effects on the phosphorylated and total protein expression levels of P38MAPK

40

and ERK; however, it induced inhibition of the phosphorylated and total protein

41

expression levels of JNK. This study provides evidence that erythromycin restores

42

corticosteroid sensitivity in PBMCs and U937 cells. JNK inhibition by erythromycin

43

restores corticosteroid sensitivity via the inhibition of c-Jun expression. Thus,

was

determined

and

the

mitogen-activated

protein

kinase

44

JNK/c-Jun is a potential novel therapeutic target for COPD.

45 46

Abbreviations: AP-1, activator protein-1; COPD, chronic obstructive pulmonary

47

disease; CSE, cigarette smoke extract; ELISA, enzyme-linked immunosorbent assay;

48

ERK, Extracellular signal-regulated protein kinase; GR-α, Glucocorticoid receptor-α;

49

GRE, glucocorticoid response elements; JNK, c-Jun N-terminal kinase; MAPKs,

50

mitogen-activated protein kinases; NF-κB, Nuclear factor kappa B; PBMCs,

51

peripheral blood mononuclear cells; P38MAPK, P38 Mitogen-activated protein

52

kinase; SiRNA, Small interfering RNA

53 54 55

Keywords: Chronic obstructive pulmonary disease; Erythromycin; Corticosteroid

56

insensitivity; Mitogen-activated protein kinases; Activator protein-1.

57 58 59 60 61 62 63 64 65

Running head: Erythromycin restores corticosteroid sensitivity.

66

Introduction:

67

Chronic obstructive pulmonary disease (COPD) is characterized by enhanced

68

chronic inflammatory response in the airways and lungs to inhaled noxious particles

69

or gases[1]. Corticosteroids are currently the mainstream treatment for COPD;

70

however, corticosteroid resistance in COPD results in ineffective inhibition of airway

71

inflammation and limited effects on disease progression in addition to increasing the

72

risk of serious pneumonia[2-4].

73

The anti-inflammatory molecular mechanism of corticosteroids occurs through

74

switching-off of pro-inflammatory transcription factors, such as nuclear factor kappa

75

B (NF-κB) and activator protein-1 (AP-1), which are usually activated by oxidative

76

stress; thus, resulting in switching-on of multiple inflammatory genes[5, 6].

77

Corticosteroids diffuse across the cell membrane, binding to and activating the

78

glucocorticoid receptor (GR) in the cytoplasm. GR recruitment of histone

79

deacetylase-2 (HDAC2) to the activated inflammatory gene complex by activated GR

80

results in effective suppression of activated inflammatory genes within the nucleus[5,

81

7].

82

Studies have explored the mechanisms of corticosteroid resistance in chronic

83

airway inflammatory diseases. It has been well established that phosphoinositide

84

3-kinase (PI3K-δ)/Akt activation induced by oxidative stress, which leads to reduced

85

HDAC2 expression and activity, is associated with corticosteroid insensitivity[8].

86

Some drugs such as nortriptyline and theophylline can restore corticosteroid

87

sensitivity via inhibition of PI3K-δ signaling and restoration of HDAC2 expression

88

and activity levels[9, 10]. Moreover, GR-α phosphorylation on serine 211 or 226[11,

89

12], or increased GR-β expression caused by oxidative stress is another important

90

cause of corticosteroid resistance[13, 14]. However, corticosteroid insensitivity in

91

COPD may be caused by multiple molecular mechanisms. Another potential

92

mechanism of oxidative stress-induced corticosteroid resistance in COPD occurs

93

through mitogen-activated protein kinases (MAPKs) / Activator protein-1 (AP-1).

94

MAPKs include P38 Mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal

95

kinase (JNK), and extracellular signal-regulated kinase (ERK); three distinct

96

stress-activated protein kinase pathways[15]. The MAPK pathway is an intracellular

97

signaling pathway that can be activated by a variety of extracellular stimuli such as

98

oxidative stress induced by cigarette smoke, which enhances the expression of

99

pro-inflammatory factors[16, 17]. AP-1 is a redox-sensitive pro-inflammatory

100

transcription factor comprising c-Jun and c-Fos, and it is primarily regulated by

101

MAPK pathways and post-translational modification via phosphorylation[18, 19].

102

AP-1 over-expression is associated with corticosteroid resistance in asthma.

103

Overexpressed AP-1 interacts with the GR and prevents its binding to glucocorticoid

104

response elements (GREs) and other transcription factors[20, 21]. Increased JNK and

105

c-Jun phosphorylation has been demonstrated in corticosteroid-resistant asthma[22].

106

However, there is little research on increased MAPKs/AP-1 activity in COPD patients

107

with corticosteroid resistance.

108

As corticosteroid therapy is ineffective and has adverse effects, novel and

109

effective anti-inflammatory approaches to COPD are needed. Macrolides such as

110

erythromycin have shown promising anti-inflammatory effects[23], and erythromycin

111

is being used clinically for successful treatment of diffuse panbronchiolitis[24].

112

Previous studies demonstrated that low-dose erythromycin reduced acute exacerbation

113

of COPD and neutrophil elastase level in sputum[25, 26]. Previous studies have also

114

shown that macrolides can inhibit AP-1 activity and reduce IL-8 level in bronchial

115

epithelial cells under oxidative stress[27-29]; however, the explicit anti-inflammatory

116

mechanism of erythromycin is not yet clear.

117

In this study, we hypothesized that erythromycin could reverse corticosteroid

118

insensitivity induced by cigarette smoke via down-regulation of MAPKs/AP-1

119

activity. First, we observed the effect of erythromycin on corticosteroid sensitivity in

120

peripheral blood mononuclear cells (PBMCs) obtained from COPD patients. Then we

121

attempted to elucidate the molecular mechanism of erythromycin in the MAPK/AP-1

122

pathway and corticosteroid insensitivity in U937 cells. We further explored the

123

mechanism of the anti-inflammatory effect of erythromycin and its potential for

124

medicinal application.

125 126 127 128 129 130 131

132

Methods:

133

Isolation and culture of PBMCs from subjects and treatment

134

Peripheral venous blood (50 ml) was drawn from eight healthy subjects, eight

135

healthy smoker subjects, and eight COPD patients. The characteristics of these

136

subjects are presented in Table 1. PBMCs were isolated by Ficoll-Hypaque density

137

gradient centrifugation, as in our previous study[30]. COPD patients who received

138

oral erythromycin, clarithromycin, nortriptyline, corticosteroids, and theophylline

139

were excluded.

140

PBMCs obtained from COPD subjects were divided into two groups; one group

141

received pre-treatment with 10 μg/ml erythromycin (Sigma-Aldrich, Poole, UK) for

142

2h, and the concentration of erythromycin was selected according to our previous

143

study[30]. The cells were incubated with different dexamethasone (Dex)

144

concentrations (10-12 to 10-6 M) (Sigma-Aldrich, USA) for 2 h before stimulation with

145

5 ng/ml TNF-α (PeproTech, USA) for 8 h. Supernatants were harvested, IL-8 level

146

was measured by enzyme-linked immunosorbent assay (ELISA), and cells were

147

collected to extract proteins for Western Blotting.

148 149

This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (2016-KY-143).

150 151 152 153

U937 cell culture and treatment The human monocytic cell line, U937 (human histiocytic lymphoma cell line,

154

TCHu1593.2, Shanghai Cell Bank, Chinese Academy of Sciences), were cultured at

155

37℃ and 5% CO2 in RPMI 1640 medium (Gibco, Shanghai, China) containing 10%

156

heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Solarbio,

157

Beijing, China) at 37℃ in a 5% CO2 humidified atmosphere.

158

U937 cells were seeded in 48-well plates (4×105/ml) and pre-treated with or

159

without erythromycin (10 μg/ml). MAPK inhibitors, SP600125 (10-2 M) and

160

SB203580 (10-6 M), were purchased from Selleckchem (Houston, USA). SCH772984

161

(10-4 M) was obtained from Targetmol (USA) and applied for 2 h. The cells were

162

stimulated with CSE for 2 h and incubated with different Dex concentrations (10-12 to

163

10-6 M) (Sigma-Aldrich) for 2 h before stimulation with 5 ng/ml TNF-α (PeproTech,

164

USA) for 8 h. The IL-8 level in the supernatants was measured using ELISA, and

165

cells were collected to extract proteins for Western Blotting.

166 167 168

CSE preparation

169

CSE was prepared according to our previous study[31]. Ten full-strength

170

burning cigarettes (Changsha, China) without filters were continuously pumped with

171

a 50 ml syringe apparatus. The smoke slowly dissolved in 20 ml of RPMI 1640. Each

172

cigarette yielded five draws of the syringe (up to the 50-ml mark), and it took

173

approximately 10-15 s to complete each individual draw. Then the pH of CSE was

174

adjusted to 7.4, The CSE solution was filtered twice through a 0.22 μm filter

175

membrane and was used within 2 h. The concentration of CSE samples was adjusted

176

to achieve an optical density of 0.25 that stimulated cells without inducing cell death.

177 178 179

Cell viability assay

180

Cell viability was assessed using the cell counting kit 8 (CCK-8; Dojindo

181

Molecular Technologies, Japan) according to the manufacturer’s instructions. The

182

U937 cells (3×103/well) were incubated with different concentrations of erythromycin,

183

CSE, JNK inhibitor (SP600125), P38MAPK inhibitor (SB203580), and ERK inhibitor

184

(SCH772984) in 96-well plates for different time periods (12 h and 24 h), and then 10

185

ml of CCK-8 solution was added to the culture medium. The cells were further

186

incubated for 2 hours at 37℃, and the optical density was measured at 450 nm and

187

was considered the indirect index of relative cell viability. The results are presented in

188

the supplemental figure.

189

.

190 191 192

Small interfering RNA (siRNA) transfection experiments

193

U937 cells (5×104 cells/well) were cultured in RPMI 1640 medium (Gibco,

194

Shanghai, China) containing 10% FBS. Silencing of target genes was achieved via

195

lentiviral transduction of specific siRNA vectors obtained from GeneChem (Shanghai,

196

China). U937 cells were transfected in triplicate with 4.0 µg of c-Jun-specific or

197

control siRNA (GeneChem, Shanghai, China) for 16 h using the Lenti-KDTM EasyI

198

RNAi according to the manufacturer’s instructions (GeneChem, Shanghai, China).

199

The processes of transduction and the establishment of stable cell lines were

200

performed according to the manufacturer’s instructions. Transfection efficiency was

201

detected by Western Blotting.

202 203 204

Total RNA isolation and Real-Time Polymerase Chain Reaction (RT-PCR)

205

Total RNA was extracted from U937 cells using the TRIzol reagent (TaKaRa,

206

Dalian, China). The primer sequences are listed in Table 2. The quality and quantity

207

of total RNA were analyzed with a spectrophotometer. RNA was reverse transcribed

208

into cDNA using the PrimeScriptTM RT reagent kit with the gDNA Eraser (TaKaRa,

209

Dalian, China), and mRNA was prepared using kits. RT-PCR was performed using

210

SYBR® Premix Ex TaqTM II (Takara, Dalian, China). Procedures were performed

211

according to the manufacturers’ instructions.

212 213 214

Western Blot analysis

215

PBMCs and U937 cells were intervened and then collected in 10 ml centrifuge

216

tubes by centrifugation at 1000 rpm/min for 5 min at 4℃. The supernatant was

217

discarded, and 1 ml PBS suspension cells were centrifuged at 1000 rpm/min for 5 min

218

and washed twice at 4℃ to remove the impurities.

219

Proteins were extracted from PBMCs and U937 cells using Radio-Immuno

220

Precipitation Assay buffer supplemented with a phosphatase and protease inhibitor

221

cocktail (Asvio Technology, Guangzhou, China). Protein concentrations were

222

quantified with the bicinchoninic acid assay kit (Beyotime, Shanghai, China)

223

according to the manufacturer’s instructions. Further, 20 μg of the sample was

224

electrophoresed on a 10% SDS-polyacrylamide gel, electroblotted onto nitrocellulose

225

membranes (Millipore, USA), and blocked with 5% bovine serum albumin dissolved

226

in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. The

227

membranes were incubated overnight at 4°C with primary antibodies (Cell Signaling

228

Technology, Boston, USA) against c-Jun (#9165) (1:1000), Phospho-c-Jun (Ser73)

229

(#3270)

230

Phospho-P38MAPK (Thr180/Tyr182) (#4511) (1:1000), P44/42MAPK (Erk1/2)

231

(#4695) (1:1000), Phospho-P44/42MAPK (Erk1/2) (Thr202/Tyr204) (#4370)

232

(1:1000), SAPK/JNK (#9252) (1:1000), and Phospho-SAPK/JNK (Thr183/ Tyr185)

233

(#4668) (1:1000). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

234

antibody (dilution 1:1000, PeproTech, USA) was used as a control to validate protein

235

loading. The goat-anti-rabbit IgG secondary antibody (1:10000, LI-COR Biosciences,

236

USA) was incubated for 1 h at room temperature, and blots were visualized with

237

enhanced chemiluminescence (Pierce Biotechnology). Membranes were analyzed

238

using the Odyssey imaging system (LI‐COR Biotechnology, Lincoln, NE, USA).

(1:1000),

239 240 241

ELISA for IL-8

c-Fos

(#2250)

(1:1000),

P38MAPK

(#8690)

(1:1000),

242

IL-8 levels in supernatants of cultured PBMCs and U937 cells were measured in

243

triplicate by ELISA using specific kits according to the manufacturer’s instructions

244

(Cusabio, Wuhan, China). Dex-IC50 was calculated with Prism 6.0 (GraphPad

245

Software Inc., USA). The X axis represents the concentration of Dex, and the Y axis

246

represents the inhibition rate of IL-8 at different concentrations of Dex. Data were

247

expressed as dose-effect curves.

248 249

Data analysis

250

Results are presented as means ± SD. Statistical analyses were carried out using

251

SPSS for Windows (version 16.0.0; SPSS, Chicago, IL, USA). One-way ANOVA,

252

Bonferroni post hoc correction (α = 0.0167), and Tukey test were conducted to

253

evaluate significant differences in the data. The Kolmogorov–Smirnov test was

254

conducted to determine the normality of the data. When the distribution was not

255

normal, Mann–Whitney post hoc test was conducted to compare the differences

256

among the groups. Statistical significance was set at P < 0.05.

257 258 259

Results:

260

1. Erythromycin improved corticosteroid sensitivity and decreased c-Jun levels in

261

PBMCs obtained from COPD patients.

262

To examine the effect of erythromycin on corticosteroid sensitivity, PBMCs were

263

collected from healthy subjects, healthy smoking subjects, and COPD patients (none

264

of the COPD patients were active smokers). The corticosteroid effect was assessed as

265

the Dex concentration necessary to induce 50% inhibition of TNF-α-induced IL-8

266

production in PBMCs (Dex-IC50). The log (Dex-IC50) value for PBMCs obtained

267

from COPD patients was significantly greater than that for PBMCs obtained from

268

healthy subjects and smokers (6.00±5.71×10-8 M VS 4.73±3.48×10-10 M and

269

5.81±2.97×10-10 M, P＜0.05) (Figure 1A), indicating that PBMCs show corticosteroid

270

resistance in COPD. However, there was a significant improvement in corticosteroid

271

sensitivity of PBMCs obtained from COPD patients who received pre-treatment with

272

erythromycin (6.00±5.71×10-8 M VS 4.00 ±3.42×10-9 M, P＜0.05) (Figure 1A).

273

We examined the effect of erythromycin on AP-1 protein expression in PBMCs.

274

Western blot was used to determine AP-1 expression (Figure 1B-D). Compared with

275

healthy subjects and smoker subjects, the expression of c-Jun was significantly

276

elevated in PBMCs obtained from COPD patients (Figure 1C). Meanwhile, c-Fos

277

showed no differences among PBMCs obtained from healthy volunteers, healthy

278

smoking volunteers, and COPD patients (Figure 1D). Erythromycin reduced c-Jun

279

expression but had no effect on c-Fos (Figure 1B-D).

280 281

These results suggest that erythromycin can improve corticosteroid sensitivity and induce a decrease in c-Jun in PBMCs obtained from COPD patients.

282 283

2. Erythromycin restored corticosteroid sensitivity and decreased CSE-induced c-Jun

284

protein expression in U937 cells.

285

To demonstrate that erythromycin restores corticosteroid sensitivity, U937 cells

286

were pre-treated with or without erythromycin for 2 h before exposure to CSE for 2 h.

287

The cells were incubated with different Dex concentrations (10-12 to 10-6 M) for 2 h

288

before stimulation with 5 ng/ml TNF-α for 8 h. Compared with the control group, the

289

IC50 values for Dex were significantly higher in the CSE group (3.07±1.65×10-7 M

290

VS 7.63±2.67×10-9 M, P ＜ 0.05) (Figure 2A), and erythromycin restored

291

corticosteroid sensitivity in U937 cells (2.52±1.38×10-8 M VS 3.07±1.65×10-7 M, P＜

292

0.05) (Figure 2A). Compared with the control group, the IL-8 level was significantly

293

increased in the CSE group (104.4 ± 14.0 pg/ml VS 494.5 ± 38.8 pg/ml, P＜0.05),

294

and erythromycin (10 μg/ml) inhibited IL-8 release from U937 cells (283.8 ± 53.9

295

pg/ml VS 494.5 ± 38.8 pg/ml, P＜0.05) (Figure 2B).

296

Compared with the control group, the c-Jun and c-Fos mRNA levels were

297

significantly increased in U937 cells exposed to CSE for 8 h (Figures 2C and D).

298

Pre-treatment with erythromycin for 2 h significantly suppressed the c-Jun and c-Fos

299

mRNA expressions levels in U937 cells, with no significant difference from that in

300

the control group (Figures 2C and D). The c-Jun protein level was significantly

301

increased after exposure to CSE for 8 h, while the c-Fos level showed no change

302

(Figure 2E-G). In addition, erythromycin significantly suppressed c-Jun expression

303

but had no effect on c-Fos protein expression induced by CSE (Figures 2E-G).

304

Meanwhile, the expression of p-c-Jun was significantly elevated by CSE, while

305

erythromycin had no effect on the p-c-Jun protein expression (Figures 2H and I).

306

We also investigated the effects of different erythromycin concentrations on c-Jun

307

expression. c-Jun protein levels were elevated with an increase in erythromycin

308

concentration

309

anti-inflammatory effects after an increase in its dose.

310 311

(Figures 2J and K), suggesting that erythromycin did not exert any

This result shows that erythromycin can improve corticosteroid sensitivity and reduce c-Jun expression in U937 cells exposed to CSE.

312 313

3. Erythromycin and MAPK inhibitors decreased IL-8 expression and improved

314

corticosteroid sensitivity.

315

The potential mechanisms underlying improved corticosteroid sensitivity after

316

treatment with MAPK inhibitors were investigated. The effect of MAPK inhibitors on

317

corticosteroid resistance in U937 cells was assessed. The JNK inhibitor (SP600125,

318

10-2 M), P38MAPK inhibitor (SB203580, 10-6 M), and ERK1/2 inhibitor (SCH772984,

319

10-4 M) were used to test effects of CSE-induced corticosteroid resistance in U937

320

cells. U937 cells were incubated with or without erythromycin or MAPK inhibitors

321

for 2 h and exposed to CSE for 2 h before incubation with different Dex

322

concentrations (10-12 to 10-6 M) for 2 h before stimulation with 5 ng/ml TNF-α for 8 h.

323

Compared with CSE exposure alone, all MAPK inhibitors reversed corticosteroid

324

resistance in U937 cells (Figure 3A) and suppressed CSE-induced IL-8 production

325

(Figure 3B). Dex-IC50 did not show any difference between treatment with

326

erythromycin and MAPK inhibitors (Figure 3A).

327 328 329

These data suggest that MAPK inhibitors can improve corticosteroid sensitivity and alleviate CSE-induced inflammation in U937 cells.

330

4.

331

expressions.

Erythromycin and MAPK inhibitors decreased c-Jun protein and mRNA

332

The effects of SP600125 (10-2M), SB203580 (10-6M), and SCH772984 (10-4M)

333

on c-Jun protein and mRNA expressions in U937 cells were assessed. When U937

334

cells were incubated with CSE for 8 h, c-Jun protein and mRNA levels were

335

significantly increased. On incubation with erythromycin, SP600125, SB203580, and

336

SCH772984 for 2 h before stimulation with CSE, c-Jun protein and mRNA levels

337

were significantly decreased compared with the levels observed after CSE stimulation

338

alone (Figures 4A-C). c-Jun mRNA and protein levels did not show any difference

339

between treatment with erythromycin and MAPK inhibitors (SP600125, SB203580,

340

and SCH772984) (Figures 4A-C). c-Jun levels in CSE-exposed U937 cells pre-treated

341

with erythromycin or MAPK inhibitors were not significantly different from those in

342

the control group (Figures 4A-C).

343

These results suggest that c-Jun expression is regulated by MAPKs.

344 345

5. c-Jun knockdown decreased the IL-8 level and reversed corticosteroid insensitivity

346

in U937 cells.

347

To determine the role of c-Jun in corticosteroid resistance in U937 cells, we

348

performed c-Jun knockdown by RNA interference and Western Blotting was used to

349

verify the efficiency of RNA interference. c-Jun protein levels were reduced by

350

approximately 80% after transfection with c-Jun siRNA (Figures 5A and B). The cells

351

were exposed to CSE for 8 h, and the effect of Dex (10-7 M) on TNF-α–induced IL-8

352

was assessed by ELISA. Compared with the CSE group, IL-8 expression was

353

significantly decreased in c-Jun knockdown CSE-exposed cells (469.5 ± 38.5 pg/ml

354

VS 304.8 ± 30.9 pg/ml, P＜0.05) (Figure 5C). The percent inhibition of IL-8 was

355

significantly higher in c-Jun knockdown CSE-exposed cells than in the CSE group at

356

Dex (10-7 M) (54.1 ± 0.01% VS 41.6 ± 0.03%, P=0.01) (Figure 5D). These results

357

showed that knockdown of c-Jun in U937 cells stimulated with CSE did not cause

358

corticosteroid resistance and increased expression of IL-8.

359 360

These data established that c-Jun plays a critical role in the mechanism of corticosteroid resistance in U937 cells.

361 362

6. Erythromycin inhibited the JNK/c-Jun pathway activity.

363

To confirm the impact of erythromycin on the expression and activity of MAPKs,

364

U937 cells were pre-treated with or without erythromycin or MAPK inhibitors for 2 h

365

before incubation with CSE for 8 h. The mRNA and phosphorylated and total protein

366

levels of MAPKs were assessed. We found that the mRNA and total protein levels of

367

P38MAPK (Figures 6A-D) and ERK1/2 (Figures 6E-H) did not change after

368

incubation with CSE for 8 h, but the phosphorylated protein levels of P38MAPK

369

(Figure 6D) and ERK1/2 (Figure 6H) were significantly increased after exposure to

370

CSE. SB203580 significantly decreased the phosphorylated protein expression level

371

of P38MAPK, and SCH772984 decreased the phosphorylated protein expression level

372

of ERK (Figures 6D and H). However, erythromycin failed to decrease the mRNA

373

and phosphorylated and total protein levels of P38MAPK and ERK1/2 (Figures 6A, C,

374

E, and G). The mRNA and phosphorylated and total protein levels of JNK were

375

significantly increased after stimulation with CSE for 8 h (Figures 6I-L). These values

376

were significantly decreased in CSE-exposed U937 cells pre-treated with

377

erythromycin or SP600125 compared to U937 cells treated with CSE alone (Figures

378

6I-L). Additionally, the corresponding values in CSE-exposed U937 cells pre-treated

379

with erythromycin or SP600125 were not significantly different from those in the

380

control group (Figures 6I-L).

381 382

These data suggest that erythromycin decreased c-Jun expression by inhibiting JNK activity.

383 384 385

Discussion:

386

Corticosteroids are ineffective in suppressing airway and lung inflammation in

387

COPD due to corticosteroid resistance. Previous studies have demonstrated that

388

PBMCs obtained from COPD patients are less responsive to corticosteroids and that

389

resistance can be reversed by macrolide antibiotics[30, 32]. However, the

390

anti-inflammatory mechanism of macrolide antibiotics remains unclear. This study

391

reinforces the notion that erythromycin improves corticosteroid sensitivity in PBMCs

392

obtained from COPD patients and in U937 cells. We found that c-Jun plays essential

393

roles in corticosteroid resistance and erythromycin-mediated restoration of

394

corticosteroid sensitivity via a decrease in its expression. We focused on the upstream

395

members of the c-Jun pathway and demonstrated that erythromycin decreases c-Jun

396

expression by down-regulating JNK activity.

397

Oxidative stress induced by cigarette smoke is a major cause of chronic airway

398

inflammation in COPD patients. AP-1 is a pro-inflammatory transcription factor, and

399

its structure is a homodimer and heterodimer containing Fos, Jun, activating

400

transcription factor (ATF), and MAF protein families. It is activated by oxidative

401

stress, which enhances the expression of inflammatory factors such as IL-8 and

402

TNF-α[33, 34]. Previous studies reported that over-expression of AP-1 might be a

403

mechanism of corticosteroid insensitivity in asthma as AP-1 binds GR and prevents

404

its interaction with GRE and other transcription factors[21]. There is little data on the

405

role of AP-1 in corticosteroid resistance in COPD patients. Our previous study

406

showed corticosteroid resistance in PBMCs obtained from COPD patients and

407

reversal of corticosteroid resistance by erythromycin through restoration of HDAC2

408

expression via inhibition of PI3K-δ activity[30]. However, inhibition of PI3K-δ

409

activity does not completely restore corticosteroid sensitivity, and erythromycin may

410

be a multi-target anti-inflammatory drug. Our other study also demonstrated that

411

erythromycin inhibited H2O2-induced activity of AP-1 in human bronchial epithelial

412

cells[27]. Therefore, we postulated that erythromycin improves corticosteroid

413

sensitivity probably via a decrease in AP-1 expression.

414

We collected PBMCs from non-smokers, healthy smokers, and COPD patients to

415

test our hypothesis and found that PBMCs obtained from COPD patients were less

416

responsive to corticosteroids and that erythromycin improved corticosteroid

417

sensitivity. This result was in accordance with previous research[30] The c-Jun

418

protein level was increased in PBMCs obtained from COPD patients and

419

erythromycin decreased the c-Jun level, but the c-Fos protein level did not show any

420

change in PBMCs obtained from COPD patients and healthy smokers. To explore the

421

molecular mechanism of erythromycin in alleviating glucocorticoid resistance, U937

422

cells were exposed to CSE and corticosteroid insensitivity was induced. We found

423

that pre-treatment with erythromycin reversed corticosteroid insensitivity in U937

424

cells. In addition, similar to PBMCs obtained from COPD patients, CSE-induced

425

c-Jun mRNA, total and phosphorylated protein levels were increased in U937 cells,

426

and erythromycin decreased CSE-induced c-Jun protein and mRNA expression levels.

427

Further, erythromycin suppressed c-Fos mRNA expression but had no effect on c-Fos

428

protein expression in U937 cells. This disparity between the c-Fos protein and mRNA

429

expressions may be due to post-transcriptional gene regulation by mRNA

430

modifications, such as mRNA methylation. This result revealed that erythromycin

431

restored corticosteroid sensitivity via a decrease in the c-Jun level. The effect of

432

different erythromycin concentrations on c-Jun was evaluated. Interestingly,

433

pre-treatment with an increasing erythromycin concentration caused an increase in the

434

expression of c-Jun protein in U937 cells. Thus, we confirmed that this effect is not

435

dose-dependent, which reinforces the observation that only low-dose erythromycin

436

has anti-inflammatory effects.

437

In asthma, a previous study demonstrated that c-Jun activity was not reduced in

438

PBMCs obtained from patients with corticosteroid-resistant asthma despite the

439

administration of high doses of oral glucocorticoids[35]. Although several studies

440

confirmed that oxidative stress induces increased AP-1 levels in COPD, the role of

441

c-Jun in corticosteroid resistance in COPD has not been proven. To confirm the role

442

of c-Jun in corticosteroid resistance in U937 cells exposed to CSE, we knocked down

443

the expression of c-Jun in U937 cells. We found that CSE failed to induce

444

corticosteroid resistance in c-Jun-knockdown U937 cells. However, IL-8 levels were

445

decreased in these cells. These findings revealed that c-Jun is essential for

446

corticosteroid insensitivity in U937 cells and that erythromycin improves

447

corticosteroid sensitivity by inhibiting increased c-Jun expression.

448

The MAPK family includes the following three distinct stress-activated protein

449

kinase pathways: P38MAPK, JNK, and ERK1/2. Phosphorylated MAPKs are the

450

activated forms. It is well known that MAPKs are major targets that become

451

phosphorylated upon oxidative stress and they regulate AP-1 expression and activity

452

through phosphorylation of distinct substrates[18, 19, 36]. MAPKs are directly or

453

indirectly activated by oxidative stress and play a critical role in enhancing chronic

454

airway and lung inflammation[37, 38]. Corticosteroids inhibit the expression of

455

multiple inflammatory genes probably via inhibition of the MAPK signaling

456

pathways[39, 40]. In recent studies, increased P38MAPK activity was found in

457

alveolar macrophages, airway epithelial cells, and CD20+ and CD8+ lymphocytes

458

obtained from COPD patients, and phospho-P38MAPK expression was related to the

459

degree of lung function impairment[41, 42]. Chronic exposure of mice to cigarette

460

smoke activated P38MAPK and induced lung inflammation[43], and treatment with

461

the P38MAPK inhibitor decreased cytokine levels in macrophages[44] and improved

462

the lung function and relieved dyspnea in moderate to severe COPD patients[45]. In

463

addition, nicotine induced IL-8 production through ERK1/2 and JNK activation and

464

ERK1/2 and JNK inhibition significantly decreased IL-8 expression in human

465

bronchial epithelial cells[46]. It has been reported that the P38MAPK inhibitor

466

SB203580 inhibited the P38MAPK activity and reversed corticosteroid resistance in

467

PBMCs via dephosphorylation of the GR on serine226 in severe asthma [47]. It has

468

also been demonstrated that hyper-activation of JNK causes phosphorylation of the

469

GR and defective GR nuclear translocation in corticosteroid-insensitive severe

470

asthma[48]. However, the relationship between MAPKs/c-Jun activity and

471

corticosteroid resistance in COPD is not yet clear. To determine the role of MAPKs in

472

corticosteroid resistance and to identify the MAPK component that regulates c-Jun

473

expression, erythromycin, pan-JNK inhibitor SP600125, P38MAPK inhibitor

474

(SB203580), and ERK inhibitor (SCH772984) were used for pre-treatment of U937

475

cells before exposure to CSE. Our study found that all MAPK inhibitors reversed

476

CSE-induced corticosteroid resistance in U937 cells. All MAPK inhibitors decreased

477

the CSE-induced c-Jun protein and mRNA levels. These results revealed that all

478

MAPK family members are involved in corticosteroid resistance and they regulate

479

c-Jun expression. Taken together, our data demonstrate that MAPK/c-Jun pathway

480

activation appears to play a role in the CSE-mediated corticosteroid resistance in

481

U937 cells.

482

Long-term, low-dose oral erythromycin has been shown to have an

483

anti-inflammatory effect in chronic airway inflammatory diseases. Our study showed

484

that low-dose erythromycin and MAPK inhibitors decreased the c-Jun protein and

485

mRNA levels and reversed CSE-induced corticosteroid resistance in U937 cells.

486

However, it is not yet known which member of the MAPK family is inhibited by

487

erythromycin. Further work is needed to demonstrate how erythromycin mediates the

488

activity of MAPKs. In this part of the study, erythromycin and MAPK inhibitors were

489

used for pre-treatment of U937 cells, followed by incubation with CSE. We observed

490

that the total protein and mRNA levels of ERK1/2 and P38MAPK did not change

491

after exposure to CSE, whereas the phosphorylated protein levels of P38MAPK

492

(Thr180/Tyr182) and ERK1/2 (Thr202/Tyr204) were significantly increased.

493

However, our study showed that erythromycin did not affect the mRNA or the

494

phosphorylated and total protein levels of ERK1/2 and P38MAPK. ERK1/2 and

495

P38MAPK phosphorylation was suppressed by their respective inhibitors. This result

496

confirmed that erythromycin has no effect on ERK1/2 and P38MAPK

497

phosphorylation. The JNK mRNA and the phosphorylated and total protein

498

expression levels were significantly increased in U937 cells exposed to CSE.

499

Pre-treatment with erythromycin significantly inhibited the mRNA as well as the total

500

and phosphorylated JNK expression levels, which is equivalent to the effect of the

501

JNK inhibitor. This suggests that erythromycin can inhibit JNK expression and

502

activity. Based on these results, we conclude that erythromycin reduces c-Jun

503

expression in U937 cells by inhibiting JNK activity.

504

Erythromycin is already being used clinically to reduce the frequency of

505

exacerbations and to improve the quality of life in COPD patients. Our data

506

demonstrate that JNK/c-Jun inhibition is a potential novel strategy for improving

507

corticosteroid sensitivity in COPD. More importantly, we surmise that erythromycin

508

can reverse CSE-induced corticosteroid insensitivity in PBMCs obtained from COPD

509

patients and U937 cells by JNK/c-Jun pathway inhibition.

510 511 512

Authors’ contributions

513

Yanfei Bin and Nan Ma wrote the first draft of the manuscript. Jing Bai, Jianquan

514

Zhang, and Xiaoning Zhong reviewed and edited the manuscript. Xuejiao Sun,Yi

515

Liang, Yanxiu Lu and Meihua Li conducted pleurodesis and statistical analysis. Zhiyi

516

He and Yanfei Bin designed, conducted, supervised, and reviewed the manuscript; and

517

coordinated the study. All authors approved the final version of the manuscript.

518 519 520

Funding:

521

This study was supported by grants from the National Natural Science Foundation of

522

China (81660006 and 81860010) and Natural Science Foundation of Guangxi

523

(2016JJA140287).

524 525 526

Conflict of interest:

527

The authors declare that there are no conflicts of interest.

528 529

Limitations: This study demonstrated that erythromycin could reverse corticosteroid

530

insensitivity in PBMCs obtained from COPD patients and U937 cells. However, our

531

study did not assess whether erythromycin has such an effect on other inflammatory

532

cells, such as neutrophils and lymphocytes. In addition, this study only demonstrated

533

the mechanism of erythromycin for reversing corticosteroid resistance through in vitro

534

cell experiments, but data regarding the precise in vivo concentration of erythromycin

535

is lacking.

536 537 538

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704 705 706

Table 1: Characteristics of the participants Characteristics

Non-smokers

Healthy

COPD patients

smokers Age (years)

55±6

59±5

60±6

Sex M (F)

4 (4)

8 (0)

6 (2)

BMI (Kg/m2)

22.5±1.2

23.1±0.9

20.2±1.1#*

Smoking history (pack-y)

0

25±3

28±2

Smoking status current

0 (0)

8 (0)

0 (8)

FEV1 (%pred)

98.1±5.8

97.3±5.1

49.5±4.9#*

FEV1 (L)

2.8±0.2

2.7±0.3

1.4±0.1#*

FVC (L)

3.4±0.2

3.3±0.2

2.8±0.3#*

FEV1/FVC (%)

81.8±4.6

80.6±4.5

50.1±6.4#*

ICS

0

0

5

(former)

707

Data are expressed as means ± SD, and one-way ANOVA was used for statistical

708

analysis. FEV1: Forced expiratory volume in one second, FVC: forced vital capacity,

709

BMI: Body Mass Index, ICS: Inhaled corticosteroid. #P＜0.05 compared with

710

non-smokers. *P＜0.05 compared with healthy smokers.

711 712 713

714

Table 2: Sequences of PCR primers Gene

Primers

sequence

Product length GAPDH

forward 5’-TGGGCTTCCCAGAAGAGATG-3’ reverse 5’-TGGTGAAGACGCCAGTGGA-3’

c-Jun

forward 5’-AGTCAGGCAGACAGACAGACACA-3’ reverse 5’-GGGCAGTTAGAGAGAAGGTGAAAA-3’

c-Fos

716

88bp

forward 5’-CTAGCCAATGTTGACACAATACCAG-3’ reverse 5’- TGGACCGATATCACGAGCAG-3’

715

131bp

forward 5’-CGTTGGTACAGGGCTCCAGAA-3’ reverse 5’-CTGCCAGAATGCAGCCTACAGA-3’

JNK

105bp

forward 5’-TAAAGCCCATAAGGCCAGAAACTC-3’ reverse 5’-GAAGTCAATGTTAAGCTGCCAAGAA-3’

ERK1/2

96bp

forward 5’-TGGGCTTCCCAGAAGAGATG-3’ reverse 5’-TGAGGAGAGGCAGGGTGAA-3’

P38MAPK

138bp

158bp

717

Figure 1. Erythromycin (EM) improved corticosteroid sensitivity in PBMCs.

718

The Dex-IC50 in PBMCs collected from healthy volunteers (HV), smoker volunteers

719

(SV), and COPD patients.

720

(A) Corticosteroid sensitivity in PBMCs from each group, and the effects of EM on

721

corticosteroid sensitivity in PBMCs obtained from COPD patients. PBMCs were

722

pre-treated with 10 μg/ml EM for 2 h. The cells were treated with dexamethasone

723

(Dex) (10-6 to 10-12 M) for 2 h and then stimulated with TNF-α (5 ng/ml) for 8 h.

724

Corticosteroid sensitivity was measured on the basis of the rate of IL-8 inhibition by

725

Dex. (B and C) c-Jun protein expressions in PBMCs obtained from HV, SV, and

726

COPD patients. (B and D) c-Fos protein expressions in PBMCs obtained from HV,

727

SV, and COPD patients.

728

Data are expressed as mean ± SD. Comparisons were performed by one-way ANOVA.

729

Eight samples were included in each group, and experiments were repeated three

730

separate times with similar results. *P＜0.05.

731 732 733

Figure 2. EM restored corticosteroid sensitivity and decreased the expression of

734

CSE-induced c-Jun protein in U937 cells.

735

(A) U937 cells were treated with EM for 2 h before incubation with CSE for 2 h, and

736

then they were incubated with different Dex concentrations (10-6 to 10-12 M) for 2 h

737

before TNF-α induction for 8 h. Corticosteroid sensitivity was measured on the basis

738

of the rate of IL-8 inhibition by Dex. (B) IL-8 levels were measured in U937 cells

739

pre-treated with EM for 2 h followed by incubation with vehicle control or CSE for 8

740

h. IL-8 was detected in the supernatant by ELISA. (C) c-Fos mRNA expression levels

741

were measured in U937 cells pre-treated with EM for 2 h and then the cells were

742

incubated with vehicle control or CSE for 8 h. (D) c-Jun mRNA expression levels

743

were measured in U937 cells pre-treated with EM for 2 h and then the cells were

744

incubated with vehicle control or CSE for 8 h. (E and F) c-Fos protein expressions

745

were measured in U937 cells pre-treated with EM for 2 h and then the cells were

746

incubated with vehicle control or CSE for 8 h. (E and G) c-Jun protein expressions

747

were measured in U937 cells pre-treated with EM for 2 h and then the cells were

748

incubated with vehicle control or CSE for 8 h. (H and I) p-c-Jun protein expressions

749

were measured in U937 cells pre-treated with EM for 2 h and then the cells were

750

incubated with vehicle control or CSE for 8 h. (J and K) c-Jun protein expression was

751

measured in U937 cells incubated with 10, 20, and 40 μg/ml EM for 2 h before

752

incubation with CSE for 8 h.

753

Data are expressed as mean ± SD. Comparisons were performed by one-way ANOVA.

754

Five samples were included in each group, and experiments were repeated three

755

separate times with similar results.*P＜0.05.

756 757 758

Figure 3. EM and MAPK inhibitors decreased IL-8 expression and improved

759

corticosteroid sensitivity.

760

(A) U937 cells were treated with EM or MAPK inhibitors for 2 h followed by

761

incubation with CSE for 2 h. They were then incubated with different Dex

762

concentrations for 2 h before TNF-α induction for 8 h. (B) IL-8 levels were measured

763

in U937 cells pre-treated with EM or MAPK inhibitors for 2 h before incubation with

764

vehicle control or CSE for 8 h. IL-8 was detected in the supernatant by ELISA.

765

Data are expressed as mean ± SD. Comparisons were performed by one-way ANOVA.

766

Five samples were included in each group, and experiments were repeated three

767

separate times with similar results.*P＜0.05.

768 769 770

Figure 4. EM and MAPK inhibitors decreased the c-Jun protein and mRNA

771

expressions.

772

U937 cells were pre-treated with or without EM and MAPK inhibitors for 2 h, and

773

then they were incubated with CSE for 8 h. c-Jun mRNA expression was measured by

774

RT-PCR and c-Jun protein expression was measured by Western Blotting,

775

respectively. (A) c-Jun mRNA expressions were measured in U937 cells pre-treated

776

with or without EM, SCH772984 (10-4 M), SB203580 (10-6 M), and SP600125 (10-2

777

M) for 2 h, and the cells were stimulated with CSE for 8 h. (B and C) c-Jun protein

778

expressions were measured in U937 cells treated with EM, SCH772984 (10-4 M),

779

SB203580 (10-6 M), and SP600125 (10-2 M) for 2 h followed by CSE stimulation for 8

780

h.

781

Data are expressed as mean ± SD. Comparisons were performed by one-way ANOVA.

782

Five samples were included in each group, and experiments were repeated three

783

separate times with similar results.*P＜0.05.

784 785 786

Figure 5. c-Jun knockdown decreased the IL-8 level and reversed corticosteroid

787

insensitivity in U937 cells.

788

(A and B) The effect of c-Jun siRNA on c-Jun protein expression levels was measured

789

by Western Blotting. (C) IL-8 levels were measured in U937 cells after c-Jun

790

knockdown with or without stimulation by CSE. (D) U937 cells were transfected with

791

c-Jun siRNA for 16 h, and corticosteroid sensitivity was measured based on the rate

792

of inhibition of TNF-α–induced IL-8 at 10-7M Dex.

793

Data are expressed as mean ± SD. Comparisons were performed by one-way ANOVA.

794

Five samples were included in each group, and experiments were repeated three

795

separate times with similar results.*P＜0.05. NT (no treatment), CSE (Cigarette

796

smoke extract).

797 798 799

Figure 6. EM inhibited the JNK/c-Jun pathway activity.

800

U937 cells were pre-treated with or without EM and MAPK inhibitors for 2 h, and

801

then they were incubated with CSE for 8 h. MAPK mRNA expression was measured

802

by RT-PCR and MAPK protein expression was measured by Western Blotting,

803

respectively. (A-D) The effect of EM on the expression of P38MAPK mRNA and the

804

phosphorylated and total protein levels. (E-H) The effect of EM on the expression of

805

ERK mRNA and the phosphorylated and total protein levels. (I-L) The effect of EM

806

on the expression of JNK mRNA and the phosphorylated and total protein levels.

807

Data are expressed as mean ± SD. Comparisons were performed by one-way ANOVA.

808

Five samples were included in each group, and experiments were repeated three

809

separate times with similar results. *P＜0.05.

810

. Erythromycin improved corticosteroid sensitivity and decreased c-Jun levels in PBMCs obtained from COPD patients. . Cigarette smoke extract exposure induced corticosteroid insensitivity and increased the level of c-Jun in U937 cells. . Knockdown of c-Jun partially reversed corticosteroid insensitivity in U937 cells. . Erythromycin rescued cigarette smoke extract-induced corticosteroid insensitivity by inhibition of the JNK/c-Jun pathway.