The double-edged effect of hydroxychloroquine on human umbilical

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The double-edged effect of hydroxychloroquine on human umbilical cordderived mesenchymal stem cells treating lupus nephritis in MRL/lpr mice Shaoshan Mai, Lin Zou, Xiaoyan Tian, Xiao Liao, Yizhao Luan, Xing Han, Yuling Wei, Yue Wu, Shengnan Kuang, Yang Yang, Jie Ma, Qi Chen, and Junqing Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01146 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Molecular Pharmaceutics

The double-edged effect of hydroxychloroquine on human umbilical cord-derived mesenchymal stem cells treating lupus nephritis in MRL/lpr mice Shaoshan Mai1, Lin Zou2,3,4, Xiaoyan Tian1, Xiao Liao5, Yizhao Luan9, Xing Han2, Yuling Wei1,6, Yue Wu7, Shengnan Kuang1,8, Yang Yang1, Jie Ma1, Qi Chen1, Junqing Yang1*

1Department of Pharmacology, Chongqing Medical University, the Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing 400016, China 2Center for Clinical Molecular Medicine, Children's Hospital, Chongqing Medical University, Chongqing 400014, China 3The Pediatrics Research Institute, Ministry of Education Key Laboratory of Child Development and Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China 4Chongqing Engineering Research Center of Stem Cell Therapy, Chongqing 400014, China 5Department of laboratory, Children’s Hospital of Chongqing Medical University, Chongqing40014, China 6Department of Pharmacy, Chongqing Hospital of traditional Chinese Medicine 400011, China 7Department of Neurosurgery, 1st Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China 8Department of Pharmacy, People's Hospital of Rongchang District, Chongqing 402460, China 9State Key Lab of Ophthalmology, Guangdong Provincial Key Lab of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510000, China *

Corresponding author:

Prof. Junqing Yang, Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China; Tel: +86-23-68485161; Fax: +86-23-68485161; E-mail: [email protected] Shaoshan Mai and Lin Zou are co-first authors. The authors declared that they have no conflicts of interest to this work.

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1

Abstract

2

Hydroxychlorquine(HCQ)

3

mesenchymal stem cells (UC-MSCs) were used to treat systemic lupus

4

erythematosus(SLE)，respectively. However, the effect of HCQ on

5

UC-MSCs in lupus nephritis (LN) has not been investigated. In this

6

study, HCQ and UC-MSCs were used in MRL/lpr mice. Surprisingly,

7

although the treatment of both HCQ and UC-MSCs could ameliorate

8

renal damage separately, the presence of HCQ decreased unexpectedly

9

the therapeutic effects of UC-MSCs through interfering expression of

10

IFN-γ. However, HCQ-pretreated UC-MSCs showed significant

11

improvements of renal morphology and function more rapidly than that

12

of UC-MSCs and HCQ alone. To test the role of HCQ in UC-MSCs,

13

MRL/lpr mice and SLE patients’ peripheral blood were used in vivo and

14

vitro. Results showed that after administration of UC-MSCs pretreated

15

by HCQ, CXCR3 expression in renal tissues, serum IL-2 and IgM levels

16

decreased significantly, and serum IL-10 level increased significantly.

17

HCQ pretreatment caused a significant decrease of TNF-α and MCP-1

18

secretion and an increase of IL-1β and CXCL10 release from UC-MSCs.

19

Our results indicate that HCQ play a double-edged role in UC-MSCs. It

20

is necessary for clinical treatment to pre-evaluated concomitant

21

application of UC-MSCs with HCQ. More importantly, the alterative

22

expression of IFN-γ, the improvement of migration ability of UC-MSCs,

and

human

umbilical


cord-derived

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23

the regulation of Th1/Th2 balance, and the changes of antibodies

24

secretion in B cell might be involved in its mechanisms.

25

Key words: lupus nephritis, human umbilical cord-mesenchymal stem

26

cells, hydroxychloroquine, pretreatment, systemic lupus erythematosus

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44



45

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Introduction:

46

Systemic lupus erythematosus (SLE) is a chronic autoimmune

47

inflammatory disease characterized by variability in clinical manifestation

48

and multi-organ involvement. Lupus nephritis (LN) is the most frequent

49

and severe complication of SLE, and can lead to permanent renal damage

50

and chronic kidney disease 1. The renal involvement is more frequent in

51

children than in adults. According to statistics, 60-80% of children with

52

SLE have urinary or renal function abnormalities in the early disease

53

course2. In terms of therapy, immunosuppression with cyclophosphamide,

54

mycophenolate

55

combination with high-dose glucocorticoids, has changed LN from being

56

a serious disease associated with 5-year survival of 20% to a chronic

57

autoimmune illness associated with long-term survival (10-year survival

58

of up to 90%)3. Meanwhile, it is also noteworthy that wider use of older

59

drugs like hydroxychloroquine (HCQ), the prognosis of SLE has greatly

60

improved.

mofetil(MMF),

leflunomide

and

azathioprine,

in

61

HCQ were historically prescribed to patients with mild to moderately

62

severe SLE, especially those with skin manifestations. The Canadian

63

HCQ Study Group examined the effects of HCQ on renal damage and

64

provided an early evidence that HCQ treatment might reduce nephritic

65

flares4. The investigators of the LUMINA (Lupus in Minorities: Nature

66

Versus Nurture) study reported that application of HCQ was associated


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67

with a reduced risk of developing renal damage in patients with SLE5.

68

Some researchers also demonstrated that renal damage was less frequent

69

in HCQ users compared with nonusers (28.4% versus 42.8%)6.

70

Additionally, the researchers found that those who received HCQ had a

71

lower SLE disease activity scores, lower frequency of World Health

72

Organization class IV glomerulonephritis, and lower glucocorticoid doses

73

than those patients not taking HCQ. Studies also showed HCQ treatment

74

was association with lower damage accrual rates7. HCQ is well-tolerated

75

medications with good safety profiles. Gastrointestinal symptoms are the

76

most common adverse effect. However, the retinal toxic effect is an

77

uncommon side effect. Retinopathy is the result of disrupted metabolism

78

of the retinal pigmented epithelium and the degeneration of

79

photoreceptors8. The guidelines for management of SLE recommend the

80

ophthalmological examinations if the patient has visual impairment

81

during HCQ treatment, or if the patient is receiving high doses (>5.0

82

mg/kg daily) of HCQ or continuous treatment beyond 5 years9. Moreover,

83

HCQ is safe and effective for pregnant patients with lupus. Studies

84

showed that pregnant women with SLE who stopped using HCQ had

85

worse disease activity and higher glucocorticoid requirements than those

86

continued taking HCQ

87

should

88

milligram-per-kilogram basis, the safety profile of HCQ is equivalent in

be

10

. Thus, researchers recommended that HCQ

maintained

during

pregnancy.


Additionally,

on

a


Page 6 of 45

89

adult and pediatric patients with SLE11. However，the mechanisms of

90

action of HCQ in ameliorating renal function remain unclear. Studies

91

showed some underlying benefits like suppression of autoantibody

92

presentation, blockade of Toll-like receptor signaling, antiproliferative

93

effects, reducing cytokine and prostaglandin synthesis, and decreasing

94

leukocyte activation12, 13. As result of this, thanks to its far more potential

95

benefits than potential risks for patients with SLE and lupus nephritis,

96

HCQ is recommended to be used throughout the diseases process of SLE,

97

and its adult dosing ranges from 200 mg to 400 mg per day (6.5mg/kg)14.

98

Recently, the development of discoveries about mesenchymal stem

99

cells and induced pluripotent stem cells (iPS) has greatly contributed to

100

stem cell therapy for the treatment of clinical autoimmune diseases.

101

Mesenchymal stem cells (MSCs) are multipotent stromal cells readily

102

isolated from a variety of tissues, including umbilical cords, bone marrow,

103

adipose tissue, and dental pulp, etc. Their differentiation capacity

104

contributes to biological niche in wound healing, as they can home to

105

sites

106

immunoregulatory functions of MSCs promote exploration of immune

107

diseases therapies like treating lupus. Early evidences demonstrated that

108

transplantation

109

significantly inhibit the autoimmune progression in MRL/lpr mice15.

110

Some researchers suggested that allogenic bone marrow MSCs

of

injury,

of

initiate

human

tissue

bone

repair

marrow

and

regeneration.

MSCs(BM-MSCs)


The

can

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111

transplantation may be a feasible and safe salvage therapy in refractory

112

SLE mice and patients16. The reason of therapeutic effects is that MSCs

113

affected B cell receptor according to activating both follicular and

114

marginal zone B cells which depended on IFN-γ and cell contact17.

115

Another explanation is that TGF-β produced by MSCs could reduce

116

inflammatory cytokine-induced inducible NO synthase expression, which

117

means, the property of TGF-β is promoting immune responses in the

118

presence of MSCs18. In clinical experiments, UC-MSCs transportation

119

contributed to amelioration of disease activity, serologic changes, and

120

stabilization of proinflammatory cytokines in SLE patients19,

121

explanation of therapies is that MSCs secrete a dynamic assortment of

122

anti-inflammatory molecules, trophic factors, and bioactive cytokines.

123

Also, MSCs may repair the damaged tissue by means of homing to local

124

sites and interacting with the local stimuli, such as inflammatory

125

cytokines and the hypoxia condition which could stimulate MSCs to

126

produce a large amount of growth factors （e.g. vascular endothelial

127

growth factor）for tissue regeneration21, 22. In recent years, many studies

128

showed the ability of MSCs whether suppress or enhance the immune

129

response depended on their type, intensity, inflammatory stimuli, etc23, 24.

130

MSCs have plastic capacity that should be harnessed in appropriate ways.

131

Some researchers studied the effects on autoimmune responses in

132

lymphocyte. They joint use of MSC and five agents-cyclosporine,


20

. The


133

tacrolimus, rapamycin, mycophenolate acid (MPA), and dexamethasone

134

(DEX). Their data showed that rapamycin antagonized the inhibitory

135

effect of MSCs, whereas MPA promoted it and DEX did not modify it25.

136

In another research reported that pre-stimulation of MSCs with IFN-γ

137

enhanced their capacity to inhibit Th1 inflammatory responses, which

138

diminished mucosal damage in experimental colitis26. Moreover,

139

MSCs pretreated with IL-1β enhanced the ability to recruit monocytes

140

and granulocytes in vitro27. MSCs pretreated with IL-1α effectively

141

promoted the growth of the mouse prostate cancer cell line RM-1 in

142

vivo28. Some researchers also found that pre-treatment with IL-6

143

significantly abolished the ability of MSCs to promote gastric epithelial

144

cell proliferation and migration29.

145

HCQ and MSCs have abilities to control the lupus activity,

146

respectively. However, whether HCQ combined use with MSCs can

147

improve the effects of MSCs on LN? Whether the effects of MSCs

148

pretreated with HCQ are better than MSCs alone on immunomodulation

149

effects? Thus far, there is not relevant report in this field.

150

The aim of this study is to observe the effect of HCQ on human

151

MSCs treating LN in MRL/lpr mice as well as its possible mechanism.

152

Materials and methods

153

Animal and experimental protocol

154

Thirty-eight female MRL/lpr mice (7-9 weeks old), weighing 21.7 ± 1.2 g


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155

(mean ± SD), were purchased from Medical Animal Experimental Center

156

of Nanjing Military Region of China. Six female C57BL/6 mice (7-9

157

weeks old), weighing 19.5 ± 0.9 g (mean ± SD), were purchased from

158

Changzhou Cavens Laboratory Animal Co. Ltd. The mice were

159

maintained in a specific pathogen-free Laboratory Animal Institute of

160

Chongqing Medical University. The MRL/lpr mice were randomly

161

divided into the following eight groups: lupus nephritis group (LN) (n=4),

162

mycophenolate

163

hydroxychloroquine-treated group (HCQ) (n=6), human umbilical cord

164

mesenchymal stem cells-treated group (MSC) (n=4), HCQ- and

165

MSC-treated group（HCQ+MSC）(n=5) and MSC pretreated with HCQ

166

(HCQ-MSC) group (n=5). UC-MSCs (5×105 once) were injected into

167

mice via tail vein in 15th week and 17th week, respectively. HCQ

168

(40mg/kg) and MMF (100mg/kg) were intragastrically administered to

169

mice from 15th week to 27th week14,

170

(Ctrl-MSC) group (n=3) and conditional culture medium (CM) group

171

(n=5) were injected with UC-MSCs or conditional culture medium via

172

tail vein in 17th week and 23th week, respectively. The C57BL/6 mice

173

were injected with 0.9% saline water via tail vein in 17th week and 23th

174

week. The Institutional Animal Ethics Committee of Chongqing Medical

175

University approved all the animal experimental procedures. The research

176

was also conducted in accordance with the guide for the Care and Use of

mofetil-treated

group

(MMF)

(n=6),

30

. The mice in control MSC



177

Laboratory Animals promulgated by the United States National Institutes

178

of Health.

179

Reagents

180

Hydroxychloroquine sulfate tablets (Plaquenil) were purchased from

181

Sanofi-Synthelabo Co., Ltd. Mycophenolate mofetil (Cell Cept) were

182

purchased from Roche Co., Ltd. Hydroxychloroquine and PKH26 Red

183

Fluorescent Cell Linker Kits ( Sigma-Aldrich), Braford protein

184

quantitation Assay kit and CCK8 kit (KeyGEN, Nanjing, China), mouse

185

anti-dsDNA kit (mlbio, Shanghai, China), methenamine silver stain

186

(Solarbio, China), mouse IFN-γ ELISA kit (USCN, Wuhan, China),

187

anti-Interferon gamma antibody (Abcam), anti-CXCR-3 antibody (Santa),

188

human IL-1β ELISA kit, human TGF-β1 ELISA kit, human TGF-β1

189

ELISA kit and human MCP-1 ELISA kit (Joyee Biotechnics Co., Ltd,

190

China), human IP-10 ELISA kit(Ray Biotech), mouse IgM ELISA kit,

191

mouse IL-2 ELISA kit, mouse IL-10 ELISA kit (eBioscience)

192

MSCs culture

193

UC-MSCs were derived from Wharton’s jelly of umbilical cords that

194

obtained from Chongqing Engineering Research Center of Stem Cell

195

Therapy at affiliated Children Hospital of Chongqing Medical University.

196

The present study was approved by Chongqing Medical University Ethics

197

Committee and Affiliated Children Hospital of Chongqing Medical

198

University Ethics Committee. Growth medium consisted of α-MEM


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199

medium (Gibco) with Ultra GROTM nutritional additives (Helios,

200

Germany). Cultures were maintained in a humidified atmosphere with 5%

201

CO2 at 37oC. The medium was replaced, and non-adherent cells were

202

removed after 2 days. When cultures were 60–80% confluent, the cells

203

were dissociated with 0.25% Trypsin-EDTA (Sigma-Aldrich) and

204

transferred to new cell culture flasks (Corning, USA) for further

205

expansion. After passage 4, cells were used for treatment. The UC-MSCs

206

were characterized for expression of specific cell surface markers,

207

immune activity and differentiation to osteogenic, adipogenic, and

208

chondrogenic cells, as reported in supporting information of our previous

209

study31.

210

Pretreatment of UC-MSCs with HCQ

211

UC-MSCs (1.5×106) were cultured in 2 cultural flasks(Corning, USA).

212

The culture medium of the UC-MSCs was discarded and replaced with

213

fresh culture medium containing 60µM of HCQ (Sigma-Aldrich). After

214

48 h, the UC-MSCs were used for the following experiments.

215

UC-MSCs conditional medium

216

When the cell count of UC-MSCs pretreated with HCQ in T75 culture

217

vessel reached to 1.5×106, the cells were washed 3 times by D-Hanks

218

and re-cultured by 12ml α-MEM medium. The subsequent α-MEM

219

medium for the next 24h was used as the conditional medium. For in

220

vivo experiments, 12ml medium was divided into 3 parts, 4ml of this



221

medium was concentrated (4000g×30min) using a 3000 Da centrifugal

222

concentrating filter (Millipore, USA) to 250µl31.

223

Peripheral blood mononuclear cells (PBMC) preparation

224

Blood samples of SLE patients (juvenile and teenager) were collected in

225

sterile Vacutainer tubes containing 100 U/ml of heparin. After a

226

maximum storage period of 1 h at room temperature, blood was diluted

227

1:1 in RPMI 1640. Then, to obtain PBMC by following the protocol of

228

gradients of Ficoll-Hypaque (Tiangen, China).

229

Renal function assessments of mice

230

Mice urinary protein was measured once every 3 weeks. Twenty-four-

231

hour urine was collected using metabolism cages every 3 weeks. Urinary

232

protein concentration was measured by coomassie brilliant blue. The

233

protocol followed the instructions of Braford protein quantitative Assay

234

kit. Peripheral blood serum was collected from the eyes once every 4

235

weeks. Anti-dsDNA antibodies were analyzed by enzyme-linked

236

immunosorbent assay (ELISA).

237

Histopathology and immunohistochemistry test

238

The kidneys were fixed with 10% neutral-buffered formalin fixative

239

overnight at 4oC then rinsed for 2 hours in distilled H2O, then, kept in 70%

240

ethanol until ready to be embedded in paraffin. For histopathological

241

examination, 4-µm-thick paraffin sections were stained with hematoxylin

242

and eosin (HE). To observe the situation of glomerular basal membrane,


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243

paraffin sections were stained with periodic acid silver methenamine

244

(PASM).

245

To detect the expression of IFN-γ and CXCR3, paraffin sections were

246

first incubated with normal serum matched to the species for secondary

247

antibodies. After incubation with the primary antibodies (rabbit

248

anti-IFN-γ antibody 1:100 or goat anti-CXCR3 1:50) sections were

249

incubated with HRP conjugated goat anti-rabbit IgG (for rabbit

250

anti-IFN-γ antibody) or donkey anti-goat IgG (for goat anti-CXCR3) for

251

30 min at room temperature. After washing in PBS, the reaction products

252

were visualized after incubation with DAB.

253

Images were obtained on an Olympus BX51 microscope with 20 visual

254

field in every section. Sections were calculated optical density value by

255

using image-pro Plus software.

256

Scores of lymph node in Mice

257

Guidelines for scoring lymph nodes were as follows: score 0, negative;

258

score 1, milder enlargement on one side; score 2, milder enlargement on

259

bilateral lymph nodes; score 3, enlargement (not influence action); score

260

4, positive (large/firm, and influence action)32.

261

Measurement of spleen index in mice

262

After fasting 12 hours, bodies were weighted, then the animals were

263

sacrificed, and the spleen were dissected and weighted. The spleen index

264

was calculated by using the formula: Spleen index= [spleen weight/final



265

body weight].

266

Cell Counting Kit-8 (CCK-8) assay

267

UC-MSCs at a density of 1×106 cells/ml were seeded in 96-well plates

268

with 0.1ml cultural medium at different gradient concentration of HCQ

269

and incubated for 24-168 hours in a humidified atmosphere with 5% CO2

270

at 37oC after application of CCK-8 kit, and values of OD450 for each

271

group were read by the microplate reader according to manufacturer’s

272

protocol.

273

Enzyme-Linked Immunosorbent Assay (ELISA) analysis

274

The concentrations of human TNF-α, MCP-1, IL-1β, TGF-β and

275

CXCL10 in UC-MSCs culture medium and mice IL-10, IL-2 and IgM in

276

mice serum were measured by ELISA kit according to the manufacturers’

277

instruction. The optical density (OD) was measured using a detector

278

(Bio-Rad, USA) at 450 nm wavelength.

279

Quantitative real-time RT-PCR test

280

Total RNA of UC-MSCs was isolated using TRIzol (Invitrogen, USA).

281

The cDNA synthesis kit (Takara, Japan) was used to generate cDNA.

282

IL-1β gene expression analysis was determined by quantitative real-time

283

RT-PCR using the SYBR Green Master mix (Takara, Japan) and a q-PCR

284

System (Bio-Rad, USA). The results were analyzed using the 2-∆∆CT

285

method with normalization against it sinner control glyceraldehyde

286

3-phosphate dehydrogenase (GAPDH) gene expression (n = 3 for each


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287

group).

The

288

5’-CAGAAGTACCTGAGCTCGCC-3’ and the reverse primer sequence

289

was 5’-AGATTCGTAGCTGGATGCCG-3’, which resulted in a PCR

290

product of 153 bp. The GAPDH forward primer sequence was

291

5’-CAGCGACACCCACTCCTCCACCTT-3’ and the reverse primer

292

sequence

293

resulted in a PCR product of 124 bp.

294

Transwell Migration Assay

295

UC-MSCs with the presence or absence of HCQ pretreatment after

296

PKH26 Red Fluorescent stained at a density of 1×106 cells/ml in 0.1ml

297

α-MEM medium with Ultra GROTM nutritional additives were added into

298

the upper chamber of a 6.5mm diameter transwell insert with a pore size

299

of 8µm (BD, USA). The lower chamber in 24-well plates contained SLE

300

patients’ peripheral blood mononuclear cell (PBMC) (1×106/well) in

301

0.6ml of IMDM medium with 10% serum of SLE patients (the details of

302

SLE patients were shown in Supple. Table 1). After incubation at 37oC

303

and 5% CO2 for 24 hours, the upper surface of the membrane was

304

scraped gently to remove nonmigratory cells. Cells which were on the

305

lower surface of the membrane and migrated into the lower compartment

306

of the chamber were fixed in 4% paraformaldehyde for 10 minutes. The

307

number of migrating cells was quantified using image pro-plus 6.0 in five

308

random morphologic fields per well, averaged the values, and was

was

utilized

IL-1β

forward

primer

sequence

5’-CATGAGGTCCACCACCCTGTTGCT-3’,


was

which


309

multiplied by the ratio of per fluorescence microscopic field area to

310

bottom area per 24 wells. Finally, its proportion in total number of

311

UC-MSCs (1×105) seeded in the upper chamber was considered as the

312

migration rate.

313

Cell Scratch Experiment

314

UC-MSCs with the presence or absence of HCQ pretreatment at a density

315

of 1×105 cells/well were seeded in six-well plates and grown up to 90%

316

confluent. After starvation at the α-MEM medium for 12 hours, straight

317

line scratches were created with sterile tips in the cell plate, and then cells

318

were gently washed 3 times by D-Hanks, then, adding α-MEM medium

319

with Ultra GROTM nutritional additives. Cell movement and scratch width

320

changes were observed at 0 and 24 hours, respectively. After the scratch

321

experiments, the cells of per well were observed under an inverted

322

microscope. The average gaps of migration distance were measured using

323

the edge method. Each scratch edge was measured by the software

324

Image-Pro plus 6.0. The migration distance was calculated according to

325

the following formula: average gaps = edge distance (0h) - edge distance

326

(24h).

327

Microarray analysis

328

To explore the changes of IFN-γ and CXCR3, we retrieved the previous

329

researches, and included the datasets from J Liu’s(supplementary)33, Kae

330

Teramoto’s34 (reference) and Paulson J’s (reference, GSE27346) studies.


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331

The visualization was realized using R software.

332

Statistical data analysis

333

All results were presented as mean ± SEM of data from at least three

334

separate experiments. Differences between two groups were determined

335

by unpaired Student’s t-test if the variance was normally distributed.

336

Comparisons among three or more groups were conducted using one-way

337

ANOVA. Statistical analyses were performed using SPSS 17.0. The value

338

of p < 0.05 was considered statistically significant.

339

Results

340

Pretreatment of HCQ enhances the improvement of UC-MSCs on

341

renal function in MRL/lpr mice

342

MRL/lpr mice, which showed Fas-mediated apoptosis, were animal

343

models for autoimmune disease of SLE and LN. Although the therapeutic

344

effect of either HCQ or MSCs alone in LN was well established，it was

345

not known whether the HCQ could cause an effect on MSCs therapy.

346

Therefore, we observed the effect of combined use of MSCs with HCQ

347

and MSCs pretreated with HCQ on lupus nephritis in mice.

348

At first, we added graded amount of HCQ to UC-MSCs, which

349

determined the best concentration of HCQ to pretreatment with

350

UC-MSCs. According to UC-MSCs pretreated with HCQ in gradient

351

concentration and different time, the results showed that the proliferation

352

of UC-MSCs were significant inhibited when the concentration of HCQ



353

was 180µM.

An enormous amount of cell died in the concentration,

354

while the inhibitory effect was not obvious at the concentration below

355

180µM. After seven days, the OD values were decreased correspondingly

356

and consistently, the reason of which was the cells growth reached their

357

plateau (Supple. Fig1A). Determination of changes of expression of

358

inflammatory cytokines (TNF-α, ΜCP-1, IL-1β and TGF-β1) contributed

359

to determine the best concentration and action period (Supple. Fig1B-D).

360

SLE-like autoimmune disorders usually appear at age 7-9 weeks in

361

MRL/lpr mice. UC-MSCs were injected into mice via tail vein in 15th

362

week and 17th week, respectively. HCQ and MMF were intragastrically

363

administered to mice from 15th week to 27th week (The details of

364

procedure in “material and methods” and Fig1A). We set the group of

365

MMF which is the recommended drug of remedy for LN as a positive

366

control. To explore the effects of treatment interventions in autoantibody

367

and renal function, we determined the anti-dsDNA in serum.

368

Compared with normal control group, the levels of anti-dsDNA were

369

constantly increased in peripheral blood of MRL/lpr mice from 14th to

370

27th weeks. The levels of anti-dsDNA were significantly decreased in 18th

371

week in MMF group, MSC group and HCQ-MSC group and continued to

372

have statistical significance with control group till sacrificed, while in

373

22nd week the HCQ group and HCQ+MSC group began to decrease

374

significantly. Compared with MSC group, the level of anti-dsDNA was


Page 18 of 45

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375

significantly higher in HCQ+MSC group during 18th-27th week. However,

376

from the 22nd week on, the level of anti-dsDNA in HCQ-MSC group was

377

significant lower than that in MSC group (Fig1B).

378

As expected, MRL/lpr mice showed an obvious renal impairment.

379

The levels of urine protein were time-dependently increased from 14th

380

week to 27th week in LN group. The levels of urine protein were

381

significant decreased in MMF group and HCQ-MSC group compared

382

with LN group during the 20 weeks. Furthermore, the level of urine

383

protein in HCQ-MSC group was significant lower than that in MSC

384

group. However, the level of urine protein of HCQ+MSC group was

385

significant higher than that of MSC group and HCQ group from 20th

386

week on, and until the 27th week, was lower than that of LN group

387

(Fig1C).

388

In addition, histological analysis with HE and PASM staining

389

revealed pathological changes in kidney. Compared with normal group,

390

an amount of inflammatory cells infiltration in renal interstitial (Fig1D,

391

HE, LN, 200×, black arrow), thrombus deposition in kidney tubules

392

(Fig1D, HE, LN, 400×, red arrow), vacuolar degeneration in renal

393

interstitial and tubules (Fig1D, HE, LN, 400×, black arrow) and most

394

tissues necrosis were shown in LN group. In MMF group, the size of

395

renal glomerulus was normally, a few kidney tubules were slight swelling,

396

and a small number of inflammatory cells were gathered in several renal



397

interstitials. A few kidney tubules swellings and mild inflammatory cells

398

infiltration were observed in HCQ group. The size of glomerular capsule

399

and the number of cells in tubules were normally in MSC group and

400

HCQ-MSC group. However, numbers of inflammatory cells were

401

infiltrated in renal interstitial, which presented histopathology symptom

402

of nephritis in HCQ+MSC group (Fig1D, HE). According to PASM

403

staining, vacuolar degeneration (Fig1D, PASM, LN, black arrow),

404

glomerulus and renal tubules swelling, glomerular basal membrane

405

thickened, and crescentic glomerulus (Fig1D, PASM, LN, red arrow)

406

were shown in LN group. The capsular space of glomerulus shrunk,

407

glomerular basal membrane thickened, and renal tubules obvious

408

swelling were also found in HCQ+MSC group. The impairment of renal

409

glomerulus and renal tubules were improved in the other groups (Fig1D,

410

PASM).

411

HCQ promotes effects of UC-MSCs on lymph node scores and

412

spleen index in mice

413

Lymph nodes are the indispensable parts of immune system. To a certain

414

extent, the size of lymph node can reflect organic autoimmune response.

415

Compared with normal group, the scores of lymph nodes were

416

significantly higher in LN group. The scores were significantly

417

decreased in MMF group and HCQ+MSC group compared with LN

418

group. The tendency of amelioration was shown in HCQ-MSC group


Page 20 of 45

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419

and MSC group (Fig2A). However, there were no significant differences

420

among group HCQ, HCQ+MSC and MSC versus HCQ-MSC. Spleen is

421

one of important organs in immune system, which plays a crucial role in

422

body immune response. SLE will lead to spleen swelling. The body

423

weights of mice from 10th week to 27th week were measured, and the

424

weights when they were sacrificed were to calculate the spleen index

425

(Supple.Fig2). Under macroscopic observation, the spleen of LN group

426

mice was shown obvious swelling and in dark red without luster. The

427

spleen index in LN group was significant higher than that of normal

428

group. Compared with LN group, the sizes and the indexes of spleen in

429

the other groups were decreased (Fig2B).

430

HCQ decreases serum IFN-γ content as well as kidney IFN-γ

431

expression in mice

432

Some researchers have demonstrated that MSCs has immunosuppressive

433

effect in the presence of IFN-γ, TNF-α, IL-1α, or IL-1β by upregulating

434

the expression of iNOS (in mouse cells) or IDO (in human cells)35.We

435

compared the content of serum IFN-γ of normal mice and that of MRL/lpr

436

mice in 6th weeks and 24th weeks by analyzed public data, the date

437

showed the level of serum IFN-γ of

438

control(Figure3A). As a result of this, we focused on IFN-γ to explore

439

why therapeutic effect of the combined use of HCQ and MSCs was

440

decreased compared with that of exclusive use of HCQ and MSCs. Our

were higher than that of normal



441

results showed that content of serum IFN-γ was constantly increased in

442

mice of LN group. In 22nd week, the content of serum IFN- γ was

443

significant lower in MMF group than in LN group. After two weeks of

444

administration, from 17th week on, the content of serum IFN- γ was

445

significant lower in HCQ+MSC group than in LN group and MSC group.

446

However, until 27th week, the content of serum IFN- γ was higher in

447

HCQ+MSC group than in MSC group. Compared with HCQ group, the

448

content of serum IFN- γ in HCQ+MSC group was significant lower in

449

17th week and higher from 22nd week (Fig3B). The expression of IFN-γ in

450

kidney corresponded with the content of serum IFN- γ. In 27th week, the

451

arrowhead indicated IFN- γ expression was mainly gathering in renal

452

tubules. Compared with LN group, the expression of IFN-γ in kidney was

453

decreased in HCQ, MSC and HCQ+MSC groups. But, the expression of

454

IFN-γ in kidney was no obviously difference among MSC group, HCQ

455

group and HCQ+MSC group (Fig3C).

456

UC-MSCs pretreated by HCQ can not protect LN mice only via

457

paracrine activity

458

MSCs can generate the therapeutic action through cell engraftment

459

differentiation to directly repair the injured tissues, or through paracrine

460

to facilitate self-healing of tissues. In previous study, we have evidenced

461

that UC-MSCs protect LPS-induced ALI mice via paracrine activity31.

462

To explore whether paracrine activity of UC-MSCs pretreated by HCQ


Page 22 of 45

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463

directly

caused

immunosuppression

in

MRL/lpr

464

concentrated-conditioned medium from pretreatment UC-MSCs was

465

used to treat MRL/lpr mice (Fig4A). Neutrophils and eosinophils

466

infiltration were shown in kidney of CM group mice (Fig4B, black

467

arrow). The capsular space of glomerular shrunk, glomerular basal

468

membrane thickened, renal tubules obvious swelling and necrosis were

469

presented in renal issues of CM group mice (Fig4C). The results

470

demonstrated that the conditional medium of UC-MSCs could not

471

ameliorate the renal injury in MRL/lpr mice. The level of serum

472

anti-DNA in CM group was significant higher than in Ctrl-MSC group

473

in 27th week (Fig4D). The level of urine protein in CM group was

474

significant higher than in Ctrl-MSC group from 20th week to 27th week

475

(Fig.4E). The spleen was slight swelling in CM group (Fig4G). However,

476

the lymph scores had no significant differences (Fig4F). The results

477

indicated that some soluble cytokines secreted by UC-MSCs were not

478

directly responsible for MRL/lpr mice treatment.

479

HCQ pretreatment improves the migration ability of MSCs

480

To explore the possible mechanism of UC-MSCs pretreated with HCQ in

481

ameliorating lupus nephritis, cell scratch and transwell assays were used

482

to compare the migration capacity of UC-MSCs and UC-MSCs pretreated

483

with HCQ（Figure5）. Results showed that scratch healed slower as shown

484

by shorter healed gaps in UC-MSCs group than that in HCQ -MSCs


mice,

the


485

group in 12 hours after the scratch test(Fig5A). Consistently, transwell

486

assay also revealed that the migration rate of UC-MSCs pretreated with

487

HCQ was significantly increased compared with that of UC-MSCs,

488

confirming that the migration capacity toward SLE patients’ lymphocytes

489

of UC-MSC pretreated with HCQ was improved in vitro (Fig5B. The

490

details of patients were shown in Suppl. Table1). The change of

491

UC-MSCs migration toward lymphocytes suggests that the UC-MSCs

492

secrete potent chemotactic factor. Therefore, production of leukocyte

493

chemokines by UC-MSCs was tested in the present study. Some

494

researchers had showed that MSCs express a broad range of mRNAs

495

encoding cytokines, chemokines and their receptors. Also, they evidenced

496

that CXCL12, CX3CL1, and CXCL10 lead to significant MSC

497

migration36,

498

UC-MSCs pretreated with HCQ was increased compared with UC-MSCs

499

(Fig5E). An extensive microarray analysis of glomerular gene expression

500

in lupus MRL/lpr mice suggested an important role for the CXCR3

501

pathway in lupus nephritis34. We used the DEGs from Kae Teramoto’s34

502

and Paulson J’s (reference) study to analyze, which showed CXCR3 was

503

upregulated in kidney of

504

experimental results showed that CXCR3, a chemokine receptor of

505

CXCL10, was up-regulated of renal issues in LN group mice.

506

Interestingly, the expression of CXCR3 in renal issues of HCQ-MSC

37

. Similarly, we found CXCL10 (IP-10) secreted from

MRL/lpr

mice(Figure5C). Also, our


Page 24 of 45

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507

group was significantly decreased (Fig5D).

508

HCQ pretreatment enhances effect of UC-MSCs of ameliorating

509

lupus nephritis in MRL/lpr mice through altering the serum levels of

510

IL-10，，IL-2 and IgM

511

The respective contributions of the Th1 and Th2 cytokines to the

512

pathogenesis of LN was still a matter of debate. Because several Th2

513

cytokines, e.g.IL-10, were known to promote antibody production by B

514

cells. However, immune responses involving the Th1 phenotype have

515

also been found in the murine model of lupus38. Our experimental data

516

showed that IL-2 related Th1 was decreased in serum of MRL/lpr after

517

treatment with UC-MSCs pretreated with HCQ and individual HCQ

518

(Fig6A). The typical pathology of lupus nephritis was immune complex

519

deposition. IgM, one of secretion antibody from B cell, could reflect the

520

process of lupus nephritis. The levels of serum IgM was significant

521

decreased in HCQ-MSC group compared with that in LN group (Fig6B).

522

Discussion

523

Lupus nephritis (LN) is one of the major complications of SLE. Onset

524

in childhood and adolescent occur in 10%-15% of all cases of SLE39.

525

Therefore, juvenile LN is a problem that cannot be ignored. The

526

preclinical and clinical researches have indicated that exclusive use of

527

HCQ or MSCs can improve renal function and disease remission.

528

However, there is no reported that the effect of the combined use of HCQ



529

with MSCs on lupus nephritis. By detecting anti-DNA antibody, urine

530

protein, pathological issues, lymph node and spleen index which are

531

reflection of immune active, our results indicate the therapeutic effect of

532

the combined use of HCQ with MSCs on lupus nephritis in mice is not as

533

good as that of exclusive use of HCQ and MSCs, respectively. It is

534

commonly known that MSCs have dual regulation effects in immune

535

system. Microenvironment, thereby, determines the immunoregulatory

536

effects of these cells35. Some researchers reported that IFN-γ plays a

537

crucial role in activation of T cells and NK cells and in inhibiting B cells

538

activity as well40. The immunosuppressive ability of MSCs was not innate,

539

but, rather, was induced by the proinflammatory cytokines like IFN-γ in

540

combination with TNF-α, IL-1α, or IL-1β. These cytokines made a

541

dramatic upregulation of iNOS, several leukocyte chemokines and

542

attracted immune cells, including T cells, B cells, and antigen-presenting

543

cells, to MSCs where had high levels of NO. Then, MSCs could suppress

544

immune cell function25. Hence, we predicted that the reason of the

545

therapeutic effect of the combined use of MSCs with HCQ decreased was

546

relevant to the sort of cytokines. It is reported that HCQ can inhibit the

547

production of TNF-α, IL-6, TNF-γ and IL-1β in PBMC41. It is not clear

548

whether HCQ inhibits a series of cytokines and further leads to

549

microenvironment changes in vivo of mice, and then, blocks the MSCs

550

effects. In the present study, we analyzed data of MRL/lpr mice in


Page 26 of 45

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551

publication and determined the level of IFN-γ in peripheral blood of mice

552

and the expression of IFN-γ in renal issues of mice. Our experimental

553

results showed that the level of IFN-γ of peripheral blood in HCQ+MSC

554

group was lower than MSC group, and that the changes of IFN-γ

555

expression in renal issues were similar with serum IFN-γ. These results

556

indicated that the changes of microenvironment in mice may result in

557

increasing immune response of MSCs. Interestingly, the result was

558

consistent with the evidence that antigen process relied on a certain

559

concentration of IFN-γ and took advantage of ‘double-edged’ of MSCs to

560

cure cancer42. Our experimental results suggested that HCQ and MSCs, to

561

certain extent, were incompatible in improving renal function. However,

562

the dual roles of MSCs may regulate immune responses under different

563

circumstances. The presence or absence of IFN-γ determines whether the

564

recruited leukocytes are inhibited. Also, it plays a critical role in

565

triggering MSC-mediated immunosuppression43. Therefore, HCQ act as a

566

switch trigger in immunomodulation of MSCs. Determining the level of

567

IFN-γ in serum before administration of HCQ and MSCs is an important

568

implication for current clinical application of MSCs.

569

Recent studies demonstrated that exposure of MSCs to inflammatory

570

signals significantly potentiated the immunosuppressive effects of MSCs

571

on T cells, monocytes and dendritic cells44. Fortunately, due to their

572

plasticity, the ‘educated’ MSCs will be used in clinical treatment better.



573

Recently, Tim Both et al. found that HCQ decreases human

574

MSC-derived osteoblast differentiation and mineralization in vitro45.

575

This study focused on the potential differentiation ability of MSCs.

576

Which means, they explored the direct effects of HCQ on human

577

MSC-derived osteoblast activity. However, our study concentrated on

578

the immunomodulatory ability of MSCs that had not differentiated or

579

prepared for changing into other cells. Interesting, there is no relevant

580

reported that whether effects of UC-MSCs pretreated with HCQ on

581

improvement of renal morphology and function onsets are better than

582

that of MSCs alone. Therefore, in this study, we found that the renal

583

pathomorphology impairment was significant ameliorate in HCQ-MSC

584

group and that the levels of serum anti-dsDNA and urine protein was

585

decreased faster in HCQ-MSC group than MSC group. Our results

586

indicated UC-MSCs pretreated with HCQ have more advantages in

587

controlling nephritis symptom and improving renal function impairment

588

than UC-MSCs alone.

589

To explore how UC-MSCs pretreated with HCQ contribute to tissue

590

regeneration and immune modulation, we focused on the effect of

591

paracrine secretion. Our previous study had evidenced UC-MSCs protect

592

LPS-induced ALI mice via paracrine activity paracrine31. Similarly, if

593

we find the relevant factors, we could use factors or antibody to cure LN.

594

And then, the regulatory and control ability of MSCs will be greatly


Page 28 of 45

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595

improved. In this study, conditional medium with paracrine factors

596

secreted by UC-MSCs pretreated with HCQ was used. However，

597

ironically, the therapeutic effect of conditional medium was not better

598

than that administrated UC-MSCs directly. Our results suggested that

599

secretion factors in vitro secreted by UC-MSCs pretreated with HCQ

600

had not enough ability to cover the renal injury improvement. Which

601

means, for the treatment of LN, UC-MSCs should be in internal and

602

contact with other cells.

603

What could happen after UC-MSCs entering body, how they arrive

604

at the target of kidney, what could be the key points to explain the

605

mechanisms. Thus, the distribution of MSCs after they are transplanted

606

into the body demonstrated in many studies. MSCs labeled with

607

fluorescence were injected via tail vein to mice. These MSCs initially

608

resided in the lungs, then, were detectable at sites of injury or

609

inflammatory according to inflammatory environment46-48. Whether

610

homing or not was the fundamental condition to remedy. Our experiment

611

results evidenced that UC-MSCs pretreated with HCQ had a much faster

612

migrate capacity than UC-MSCs without pretreatment by HCQ. This

613

specific migration of MSCs was reportedly guided by some chemokines.

614

CCR2 has been proved that it can guide the MSCs homing to the heart

615

subjected to ischemia reperfusion49. CXCL16, CXCL11, CXCL13 and

616

CCL22 can enhance the migration of MSCs bidirectionally across



617

human bone marrow endothelial cells50. In the early studies, there was a

618

great deal of attention on chemokines and chemokine receptors in renal

619

injury51. CXCR3 pathway was involved in the development of

620

autoimmune diseases, especially, it created local amplification loops of

621

inflammation in target organs52. CXCR3+ CD4+ T cells were enriched in

622

inflamed kidneys and urine，and provided a new biomarker for acute

623

nephritis flares in SLE patients53. The expression of CXCR3 and its

624

chemokine CXCL10 were high in serum of SLE patients which was

625

relevant to renal injury and immune regulation of Th1 cell54. In the

626

present study，our experimental results showed that the expression of

627

CXCR3 was high in LN group with damaged renal issues. Pretreatment

628

with HCQ caused a significant increase of CXCL10 release from MSCs.

629

MSCs were introduced to the renal with high expression of CXCR3 and

630

further reduced the expression of CXCR3 and decreased the Th1 cells

631

gather.

632

The pathological mechanism of LN was involved in the abnormality

633

of B and T cells, such as imbalance of Th1 and Th2 of T cell subsets led

634

to disorder in immune system. Disordered T cell stimulated B cell

635

secreted overload antibodies, which eventually made the organisms

636

become the battlefield of their own attacks55. Our experimental results

637

also showed that the level of IL-2 in LN group is increasing as well as the

638

level of IgM (data not show) before treatment. However, by comparing


Page 30 of 45

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639

with the expression of relevant factors in normal group, LN group and

640

HCQ-MSC group, we found that MSCs pretreated with HCQ had ability

641

to restructure balance of Th1/Th2 and decrease the level of antibody

642

secreted by B cells.

643

In summary, our experimental results indicate that HCQ play a

644

double-edged role in MSCs on treating LN, as the therapeutic effect of

645

the combined use of HCQ with MSCs on LN in mice is not as good as

646

that of exclusive use of HCQ and MSCs, respectively. Nevertheless,

647

HCQ pretreatment enhanced effects of MSCs on improvement of renal

648

morphology and function onsets. As a result of this, our experimental

649

results provide a fundamental instruction for using of HCQ and MSCs

650

on LN treatment.

651

Conflict of interest

652

The authors declare no conflict of interest.

653

Supporting Information

654

Effect of HCQ stimulation on UC-MSCs proliferation and inflammatory

655

cytokines expression, body weight of mice and clinical patients’ date

656

supplied as Supporting Information.

657

Acknowledgments

658

Professor Junqing Yang and Professor Lin Zou made substantial ACS Paragon Plus Environment


Page 32 of 45

659

contributions to the conception and design the study. Shaoshan Mai

660

participated in discussion, performance of the study and in the manuscript

661

writing. Xiao Liao, Xing Han participated in collecting clinical patients’

662

samples and information. Yizhao Luan searched database and analyzed

663

the public data. Xiaoyan Tian, Yuling Wei, Yue Wu, Shengnan Kuang,

664

Yangyang, Jie Ma and Qi Chen participated in the vivo and vitro

665

experiments

666

This research is partially financial supported by Natural Scientific Founda

667

tion of China (81373444, 81570372), and the Ministry of Science and Te

668

chnology (2016YFA0101300).

669

We have followed these guidelines (“Guidelines for preparing color

670

figures for everyone including the colorblind.” Roskoski R Jr. Pharmacol

671

Res. 2017 Feb 8; 119:240-241. doi: 10.1016/j.phrs.2017.02.005.) either in

672

the methods or figure legend.

and

carried

out

the

673


data

analysis.

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674

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Figure1. Changes of serum anti-dsDNA antibody, urine protein and kidney histopathology of mice. MSCs pretreated with HCQ reduced levels of autoantiboby and improved renal function in MRL/lpr mice，while co-administration of HCQ and MSCs abolished the therapeutic effects of MSCs alone. (A)The administration scheme of MSCs, MMF, HCQ and HCQ-pretreated MSCs. (B) Serum anti-dsDNA antibody levels by ELISA. (C) Urine protein levels of mice. (D) The changes of histopathology of kidney in mice. (Normal, C57BL/6 mice; LN, MRL/lpr mice; MMF(100mg/kg), treatment with mycophenolate mofetil in MRL/lpr mice; MSC, treatment with UC-MSCs in MRL/lpr mice; HCQ(40mg/kg), treatment with hydroxychloroquine in MRL/lpr mice; HCQ+MSC, treatment with hydroxychloroquine and UC-MSCs; HCQ-MSC, treatment with UC-MSCs pretreated with hydroxychloroquine(60µM) in MRL/lpr; mean ± SEM, n=4-6.*P

The double-edged effect of hydroxychloroquine on human umbilical

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