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Bioactive Constituents, Metabolites, and Functions

Exopolysaccharides from Lactobacillus plantarum NCU116 enhances colonic mucosal homeostasis by controlling epithelial cells differentiation and c-Jun/Muc2 signaling Xingtao Zhou, Ke Zhang, Wucheng Qi, YuJia Zhou, Tao Hong, Tao Xiong, Mingyong Xie, and Shao-Ping Nie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03939 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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Journal of Agricultural and Food Chemistry

Exopolysaccharides from Lactobacillus plantarum NCU116 enhances colonic mucosal homeostasis by controlling epithelial cells differentiation and c-Jun/Muc2 signaling Xingtao Zhou , Ke Zhang, Wucheng Qi, YuJia Zhou, Tao Hong, Tao Xiong, Mingyong Xie, Shaoping Nie* State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East Road, Nanchang, Jiangxi 330047, China Correspondence to Professor Shaoping Nie Phone and fax: +86 791-88304452. E-mail: [email protected].

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Abstract

2

Probiotic lactobacilli and their exopolysaccharides (EPS) are thought to modulate

3

mucosal homeostasis ， however, its mechanisms remain elusive. Thus, we tried to

4

clarify the role of exopolysaccharides from Lactobacillus plantarum NCU116

5

(EPS116) in the intestinal mucosal homeostasis. Our results indicated that EPS116

6

regulated the colon mucosal healing and homeostasis, enhanced the goblet cell

7

differentiation and promoted the expression of Muc2 gene in vivo and in vitro.

8

Further

9

phosphorylation of transcription factor c-Jun, and facilitated it binding to the

experiments

showed

that

EPS116

promoted

the

expression

and

10

promoter of Muc2. Moreover, knocking down c-Jun or inhibiting its function in

LS

11

174T cells treated with EPS116 led to decreased expression of Muc2，implied that

12

EPS116 promoted the colonic mucosal homeostasis and Muc2 expression via c-Jun.

13

Therefore, our study uncovered a novel model that EPS116 enhanced colon mucosal

14

homeostasis by controlling the epithelial cells differentiation and c-Jun/Muc2

15

signaling.

Keywords: exopolysaccharides, mucosal homeostasis, epithelial cell differentiation, Muc2, c-Jun

2


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16

Introduction

17

Probiotic lactobacilli can be regarded as parts of the natural human microbiota,

18

and have been associated with modulating the mucosal homeostasis and the immune

19

system

20

by lactobacilli awaits further characterization, although in recent years, one of its

21

ingredients, exopolysaccharide, has been demonstrated to act a significant role in

22

regulating the intestinal barrier, homeostasis and immunity [5-7].

[1-4].

However, the molecular mechanisms underlying these protective effects

23

The intraluminal microbiota and systemic tissue are isolated by the intestinal

24

epithelium, but it is not the first line of defense [8].This function belongs to the colonic

25

mucus layers. Mucin is the main component of mucus, an O-glycosyl glycoprotein

26

that protects the intestinal epithelial surface from food particle wear, intracavitary

27

digestive enzymes and pathogen attack

28

closely packed inner layer composed of Muc2, which together make up the colonic

29

mucus

30

epithelium, thus the host is almost inaccessible to bacteria, whereas the outer layer

31

hosts the symbiotic bacterial communities [12, 13]. If the lining of the colonic mucus is

32

damaged or penetrated by bacteria, the epithelial cells will be invaded by a large

33

number of bacteria. The inflammation caused by a large number of bacteria in close

34

contact with the host tissue has all the characteristics of ulcerative colitis and can

35

develop into colon cancer. Knock out Muc2 in mice to eliminate mucus, causing the

36

bacterial invasion, colitis, and cancer [14, 15].

[11].

[9, 10].

A less organized outer layer and a

The inner mucus layer isolates the commensal bacteria from the host

3



37

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The goblet cells from the colonic crypts constitute the inner mucus layer by [16].

38

producing and secreting Muc2 in response to stimuli

The goblet cells on the

39

surface of the colon continually biosynthesize and release Muc2 to form the skeleton

40

of the intestinal mucus, and maintain the mucus layers [17-19].

41

Transcription factor activating protein-1 (AP-1) is composed of dimer made up

42

of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) [20, 21]. AP-1 is

43

involved in several key biological processes, including apoptosis, cell growth, and

44

inflammation, and modulates the expression of various genes, such as Muc2 [20, 22, 23].

45

Although goblet cell and Muc2 plays critical roles in intestinal mucosal

46

homeostasis [16, 19, 24]. It is hardly known about its response to Lactobacilli and its EPS.

47

This study was to investigate how EPS116 impact Muc2 expression and the goblet

48

cells differentiation in the colon of mice with colitis. RNAi and ChIP analysis were

49

performed to uncover the potential molecular mechanisms.

50

MATERIALS AND METHODS

51

Materials

52

Cell culture products, Puromycin, Penicillin and streptomycin were from

53

Solarbio Inc (Beijing, China). Reverse transcription PCR and Realtime-qPCR kits

54

were from Takara Bio (Dalian, China). SP600125 (c-Jun inhibitor)

55

MedChem Express (Princeton, NJ). DSS (MW 40–50 kD) was from MP Biomedicals

56

(Santa Ana, California). TRIzol reagent was derived from Thermo Fisher Scientific

57

(Waltham, MA), Lactobacillus plantarum NCU116, DH5a, Stabl3 and pLKO.pig 4


[25]

was from

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58

vector were stored in our lab.

59

Preparation of EPS116

60

The extraction method of the exopolysaccharides from Lactobacillus

61

plantarum NCU116 (EPS116) was described as previously[26]. Our published work

62

showed that the purified EPS116 was homogenous, its MW was estimated to be about

63

3.84×105 Da, and the content of polysaccharide and protein in EPS116 was 83.7%

64

and 15.1% respectively[7,

65

glucosamine, glucuronic acid, mannose, and galactosamine with a molar ratio of

66

4:1.4:2:9.6:1.

67

Cells and culture conditions

26].

Moreover, EPS116 was composed of glucose,

68

Human colon cancer cell line LS174T was kindly provided by Stem Cell Bank,

69

Chinese Academy of Sciences. Because of its high Muc2 expression characteristic,

70

LS174T cell provides an excellent model for the study of Muc2 expression in vitro [23,

71

27].

72

heat-inactivated fetal bovine serum with penicillin and streptomycin in a 5% CO2

73

atmosphere at 37°C.

74

Animal model of colitis

LS174T cells maintained in Eagle's Minimum Essential Medium containing 10%

75

All animal experiments were performed under the program and license

76

(SYKX-2015-0001) approved by the Animal Care Review Committee of Nanchang

77

University, China. C57BL/6 mice (male, 18–22 g, 8 weeks old), from Slac Jingda

78

Laboratory Animal Co. Ltd (Changsha, China). Mice were randomly classified into 4 5



79

groups according to their weight. The standard chow was provided to the healthy

80

control mice, while the other groups (n = 10 mice) received 4% DSS (inflammatory

81

bowel disease (IBD) model

82

EPS116 (dissolved in 100 μL Phosphate-buffered saline) by gavage for 7 consecutive

83

days. The nutrient components of the standard chow were 20% protein, 6% fat, 45%

84

carbohydrate, and 2.5% minerals.

85

Morphological Studies

[28])

in drinking water, and were given 0/80/160 mg/kg

86

Mouse colonic tissues were fixed in 10% buffered formalin and embedded in

87

paraffin, sectioned at 4 μm. Dewaxed and rehydrated sections were stained with

88

hematoxylin and eosin (H&E), or with Alcian blue/Periodic acid Schiff (Ab/PAS).

89

All sections were assessed with an optical microscope (Olympus ix53, Olympus

90

Corporation, Japan) in a blinded manner.

91

Immunofluorescence

92

Paraffin-embedded tissues Carnoys-fixed were deparaffinized and rehydrated.

93

Then antigen retrieval was conducted. Immunostaining was carried out with

94

anti-E-cad and anti-Muc2 primary antibodies (Servicebio Inc., Wuhan, China), anti

95

-KLF4 (Boster-Bio, Wuhan, China) followed by the incubation with a FITC

96

conjugated or Alexa Fluor 568-conjugated secondary antibodies, and DAPI were used

97

for DNA counter-stain. Images were acquired using an inverted fluorescence

98

microscope (Olympus ix53, Olympus Corporation, Japan) with the Zeiss LSM image

99

software and ImageJ software. 6


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Reverse Transcription-quantitative PCR (RT-qPCR) [26].

101

RNA preparation and RT-qPCR were conducted as mentioned before

In

102

short, total RNA was isolated using TRIzol reagent. The integrity, concentration and

103

purity of RNA were checked by RNA formaldehyde denaturing gel electrophoresis

104

and microspectrophotometer (Nanodrop2000, Thermo Fisher Scientific), respectively.

105

RT-PCR was performed by Reverse transcription Kit (Takara Bio, China).

106

The mRNA level was checked by qPCR with the SYBR Premix Ex Taq™ II

107

(Takara Bio, Dalian, China). Table 1 showed the specific qPCR primers for target

108

genes. Changes in mRNA levels of target genes were quantified relative to the

109

housekeeping genes (mouse beta-actin or human GAPDH), and expressed as the fold

110

of the control. The qPCR was conducted as follows: 95°C for 20 s, 58.5°C for 20 s

111

and 72°C for 25 s, 40X.

112

Immunoblot analysis

113

The samples were incubated with specific antibodies at room temperature for 2

114

hours. The antibodies were as follows: anti-c-Jun, anti-c-Jun (phospho S63),

115

anti-Muc2 (Abcam, Cambridge, MA), anti-lamin A and anti-GAPDH (Boster-Bio,

116

Wuhan, China). The signals of Immunoblot analysis were determined and quantified

117

by Gel Doc XR+ system (Bio-Rad Laboratories, Hercules, CA).

118

Construction of c-Jun shRNA vector

119

The pLKO.pig plasmid was digested with restriction endonuclease AgeI-HF

120

and EcoRI-HF (New England Biolabs, Ipswich, Ma), and tie off c-Jun targeting 7



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shRNA to produce recombinant plasmids. shRNA target sequences for human c-Jun

122

as follows: CGGACCTTATGGCTACAGTAA, ATTCGATCTCATTCAGTATTA

123

and GAACAGGTGGCACAGCTTAAA. The recombinant vectors were confirmed

124

by DNA sequencing.

125

Generation of c-Jun deficient LS 174T cells

126

To established c-Jun knockdown LS 174T cells, we used the perviously [26].

127

published method

128

into 293t cells by ViaFect™ Transfection Reagent (Promega, Fitchburg, WI).

129

Afterthat the lentiviral particles were secreted and collected, and infected with LS

130

174T cells. Stable integrations were obtained by the incubation within DMEM media

131

plus 2 µg/mL puromycin for 72 h. The knockdown efficiency of c-Jun was measured

132

by Immunoblot analysis.

133

Chromatin immunoprecipitation (ChIP) assay

134

Briefly, the recombinant shRNA plasmids were transfected

ChIP was conducted according to the previously published method

[7]

with

135

minor modifications. Briefly, LS 174T cells were cross-linked with formaldehyde and

136

sonicated to generate DNA fragments. Lysates were incubated with anti-c-Jun (Abcam,

137

Cambridge, MA) or IgG control. ChIP-DNA was analyzed by qPCR using the primers

138

for transcription factor binding site of the Muc2 promoter (forward primer:

139

5'-CCACATTTGGACCAACACAGGA-3'; reverse primer: 5' -AGGAGCCCTGTC

140

TGAGGTTACA-3')

141

56°C for 15 seconds, and 72°C for 25 seconds, 40 cycles.

[29].

The PCR program was as follows: 95°C for 15 seconds,

8


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Statistical analysis

143

Statistics were performed by software GraphPad prism 8. ANOVA was used to

144

compare data among different groups. Dunnett's multiple comparisons test and

145

Tukey's multiple comparisons test were applied. A p-value < 0.05,* indicates

146

statistically significant results. Means ± SEM was shown to present results from two

147

independent animal experiments or from at least three in vitro experiments.

148

Results:

149

EPS116 modulated colon mucosal healing and homeostasis

150

To examine whether EPS116 actively participates in pathophysiological

151

processes, especially, homeostasis in the colon, we generated the DSS-induced colitis

152

mice and measured the impacts of EPS116 on the colon. As shown in Figure 1,

153

EPS116 significantly improved colon length, the ratio of colon length/weight, crypt

154

depth, mucosal thickness, and histopathological damage (histological score)

155

compared with DSS mice. It is noteworthy that the treatment of EPS116 had a

156

dose-dependent effect on the improvement of colitis and colon mucosal homeostasis.

157

EPS116 enhanced goblet cell differentiation in the DSS-induced experimental

158

colitis mice

159

To estimate the effects of EPS116 on goblet cells differentiation, we labeled the

160

glycoproteins in the secretory granules of goblet cells by staining intestinal sections

161

with Alcian blue-periodic acid-Schiff. As shown in figure 2a and 2b, the number of

162

Alcian blue–periodic acid-Schiff positive cells was decreased, and the quantification 9



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163

of goblet cells showed a significant decline of goblet cell number in the mice with

164

colitis compared with the healthy control. While the gavage of EPS116 remarkably

165

increased the number of goblet cells in the colon.

166

Transcription factor KLF4 (Kruppel-like factor 4) plays a key role in the [30].

167

development and differentiation of goblet cells in the intestine

168

checked the expression of KLF4 in the mice colon. Compared with the DSS group,

169

KLF4 protein markedly increased in the colon of the mice administrated EPS116

170

(Figure 2c and 2d). Thus, our data indicated that EPS116 promoted goblet cell

171

differentiation in the colon of mice with colitis.

172

EPS116 increased Muc2 expression in the colon of colitis Mice

173

Therefore, we

The major secretory products of goblet cells are the mucin glycoproteins, whose [16].

174

main component is Muc2

Herein, we detected the expression of mucin genes,

175

such as Muc2, Muc5AC, Muc5B and Muc6. Muc2 mRNA levels significantly

176

increased in the colon of the mice with EPS116 exposure other than the DSS group,

177

while the expression of Muc5AC, Muc5B or Muc6 was insignificantly changed

178

(Figure 3a).

179

To further measure the protein expression of Muc2, we stained colon samples

180

with an antibody of E-Cad (red), Muc2 (green) and DAPI (blue). As shown in figure

181

3b and 3c, compared with the DSS group, Muc2 protein obviously increased in the

182

colon of the mice treated with EPS116 (increased 44.3% and 86.3% by 80 and 160

183

mg/kg of EPS116 treatments, respectively). 10


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184


EPS116 increased Muc2 expression in vitro

185

To gain more insight of EPS116 on goblet cell function, we detected the

186

expression changes of MUC2 induced by EPS116 in the human goblet cell line

187

LS174T. Our results showed that a high dose of EPS116 remarkably upregulated the

188

expression (mRNA and protein) of Muc2 in LS174T cells (Figure 4).

189

EPS116 modulated the expression and phosphorylation of c-Jun in LS 174T cells

190

AP-1(composed of c-Jun, JunB, JunD or c-Fos), Foxa1 and STAT3 regulate the [23, 29, 31].

191

expression of various genes, such as Muc2

To clearer understand the

192

mechanism by which EPS116 controls Muc2 expression, we sought to seek which

193

transcription factor was activated by EPS116 and subsequently modulated the

194

expression of Muc2. Hence we checked the expression of c-Jun, Foxa1 and STAT3,

195

and found that only c-Jun was significantly increased (Figure 5a). Further

196

experiments showed that the protein and phosphorylation level of c-Jun was also

197

upregulated by EPS116 (Figure 5b and 5c).

198

EPS116 induced c-Jun binding to the promoter of Muc2 gene

199

To further detect whether EPS116-mediated activated AP-1 bonded to the

200

promoter of Muc2 gene, we conducted ChIP assay to check the efficiency of c-Jun

201

binding to the promoter of Muc2. By comparison with medium, c-Jun binding to the

202

promoter of Muc2 was dramatically increased in LS174T cells under EPS116

203

treatment (Figure 5e).

204

Activation of Muc2 expression by EPS116 required c-Jun 11



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205

Increased phosphorylation level of c-Jun and its binding to the promoter of

206

Muc2 in LS174T cells under EPS116 treatment signaled that c-Jun might be involved

207

in the modulation of the expression of Muc2 by EPS116. To further test this

208

hypothesis, we used RNAi and c-Jun inhibitor (SP600125) to interrupt the function of

209

c-Jun. Our experiments indicated that SP600125 decreased the expression of Muc2 in

210

LS174T cells under EPS116 treatment (Figure 6a, 6b and 6c). Next, RNAi was

211

carried out to knockdown c-Jun in LS174T cells and validated it via Immunoblot

212

analysis (Figure 6d). We detected the impact of EPS116 on the expression of Muc2 in

213

c-Jun knockdown LS174T cells. Our data showed that c-Jun deficient interrupt the

214

increased expression of Muc2 in LS174T cells by EPS116, compared to LS174T cells

215

with empty vector or wild type LS174T cells (Figure 6e and 6f).

216

Discussion

217

Many of the well-characterized probiotic strains are lactobacilli; they are

218

associated with various health benefits, such as the maintenance of mucosal

219

homeostasis and modulation of the immune system, which can potentially improve

220

human intestinal health[32]. Researchers have demonstrated that EPS produced by gut

221

microbiota could also modulate intestinal barrier function, mucosal homeostasis, and

222

immunity

223

mucosal healing and homeostasis. EPS116 alleviate colitis through promoting the

224

colonic mucosal healing in DSS induced IBD (inflammatory bowel disease) mice.

[7, 33, 34].

Our data indicated that EPS116 were able to modulate the colon

225

The mammalian intestinal epithelium is continuously renewed from the

226

proliferative intestinal stem cells and transit amplifying cells at the crypt base every

227

3~7 days

228

cells, including enterocytes, enteroendocrine cells, goblet cells, and Paneth cells

[35].

The intestinal epithelium consists of four major types of differentiated

12


[36].

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229

The characteristics of the inflammatory bowel disease ulcerative colitis are a

230

reduction in goblet cells and a decrease in the mucus layer [37, 38]. Ren, C et al. proved

231

that Lactic acid bacteria might affect the intestinal barrier function by regulating

232

goblet cells

233

transcription factor KLF4 and the number of goblet cells in the mice with colitis,

234

which indicated that EPS116 might regulate the intestinal stem cells to differentiate

235

into goblet cell in mice colon through KLF4.

[39].

Our experiment showed that EPS116 increased the expression of

236

Mucus is produced by the goblet cells and classical contains several

237

components. One of these, the mucins, gives the mucus its gel-like characterization

238

[40].

239

and Muc6

240

expression in the colon of mice with colitis. These results indicated that EPS116

241

promoted the colon mucosal healing and homeostasis via modulating Muc2 gene.

The typically mucins secreted by the goblet cells are Muc2, Muc5AC, Muc5B [19, 41].

Our experiments showed that EPS116 only increased Muc2

242

Next, we investigated a potential mechanism of the relation between EPS116

243

and Muc2 gene in vitro. High dose of EPS116 remarkably increased the expression of

244

Muc2 in the human goblet cell line LS174T, which was similar with the results of

245

Shikha Bhatia et al. They demonstrated that Galacto-oligosaccharides increased the

246

expression of Muc2 in goblet cells[42]. The expression of total c-Jun and

247

phosphorylation of c-Jun (Ser63) was markedly increased in LS174T cells, while

248

Foxa1 and STAT3 were almost unchanged, which suggested that EPS116 activated

249

transcription factor c-Jun. Next we wanted to know whether EPS116 promotes the

250

binding of c-Jun to the promoter of Muc2 gene, then induced the transcription of

251

Muc2 gene. ChIP assay showed that EPS116 promoted the binding of c-Jun to the

252

promoter of Muc2 gene. RNAi and inhibitor experiments showed that knocking down

253

c-Jun or inhibiting its function in LS174T cells under EPS116 treatment decreased 13



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254

the expression of Muc2. Hence, the above experimental results confirm our

255

hypothesis. Collectively, EPS116 promoted the colon mucosal healing and

256

homeostasis through increasing the expressions of Muc2, and c-Jun played a role as a

257

key signaling molecule in the process. Recent studies have shown that activated c-Jun

258

acted as a key role in epithelial protection and intestinal homeostasis

259

induced the transcription of genes involving apoptotic and propagation. Meanwhile, it

260

also promoted the expression of some genes, which significantly promote the

261

restitution of intestinal mucus

262

Herein, we inferred that the transcription factor c-Jun activated by EPS116 increased

263

Muc2 expression in goblet cells of mice colon.

[23].

[43].

c-Jun

Those researches were in consistent with our data.

264

EPS116 was showed as a new healthy product for keeping intestinal health that

265

played a role in the activity of regulating intestinal mucus (Figure 1 and 2). In order

266

to further develop EPS116 as a healthy product modulating intestinal mucosal

267

homeostasis, we should better understand the mechanisms by which EPS116

268

modulate the intestinal mucosal homeostasis. According to the data of this study, a

269

model that EPS116 modulated the intestinal mucosal homeostasis was put forward

270

(Figure 7). Firstly EPS116 induced the expression and phosphorylation of c-Jun, then

271

the activated c-Jun translocated into the nucleus and bound to the promoter of Muc2,

272

subsequently promoted Muc2 expression, which in turn improved the intestinal

273

mucosal homeostasis. However, this phenomenon was reversed after knocking down

274

c-Jun or inhibiting its function in LS174T cells with EPS116 treatment. Herein the

275

hypothesis that the EPS116 up-regulated Muc2 via c-Jun phosphorylation and

276

activation in goblet cells were confirm. Furthermore, our data showed that EPS116

277

increased the expression of KLF4 and the number of goblet cells in the mice with

278

colitis, which indicated that EPS116 might regulate intestinal stem cells to 14


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279

differentiate into goblet cells via activating transcription factor KLF4, subsequently

280

maintained the intestinal mucosal homeostasis. Hence, our study revealed that

281

EPS116 modulated the colon mucosal homeostasis by controlling the epithelial cells

282

differentiation and c-Jun/Muc2 signaling.

283

In a word, we have proved that EPS116 was able to modulate the colon

284

mucosal healing and homeostasis, which might be caused by controlling the epithelial

285

cells differentiation and c-Jun/Muc2 signaling.

286

Abbreviations

287

(AP-1), Activator protein 1; (ChIP), Chromatin immunoprecipitation;

288

(DAI), disease activity index; (DAPI), 40, 6-diamidino-2-phenylindole;

289

(DSS), dextran sodium sulfate; (EPS), exopolysaccharides;

290

(EPS116), exopolysaccharides from Lactobacillus plantarum NCU116;

291

(IBD), inflammatory bowel disease; KLF4 (Kruppel-like factor 4);

292

(LAB), lactic acid bacteria.

293

Author Contributions

294

X.M.Y., S.P. N., X.T.Z. conceived the study; X.T.Z., W.C.Q. performed the

295

experiments; X.T.Z., S.P.N., K. Z. wrote and revised the manuscript； T.H., Y.J. Z.,

296

T.X. contributed technical assistance.

297

Funding

298

Grant support is from the National Natural Science Foundation of China for

299

Distinguished Young Scholars (31825020), the National Key Technology R & D

300

Program of China (2012BAD33B06), the Natural Science Foundation of Jiangxi 15



301

Province (20192BAB204024), the Outstanding Science and Technology Innovation

302

Team Project in Jiangxi Province (2016RCYTB0030).

303

Notes

304

No competing financial interest was disclosed.

305

REFERENCES

306

1.

307 308

van Baarlen, P., J. M. Wells, and M. Kleerebezem, Regulation of intestinal homeostasis and immunity with probiotic lactobacilli. Trends Immunol, 2013. 34(5): p. 208-15.

2.

Khailova, L., D. N. Frank, J. A. Dominguez, and P. E. Wischmeyer, Probiotic administration

309

reduces mortality and improves intestinal epithelial homeostasis in experimental sepsis.

310

Anesthesiology, 2013. 119(1): p. 166-77.

311

3.

312 313

Selle, K. and T. R. Klaenhammer, Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health. FEMS Microbiol Rev, 2013. 37(6): p. 915-35.

4.

Bron, P. A., P. van Baarlen, and M. Kleerebezem, Emerging molecular insights into the

314

interaction between probiotics and the host intestinal mucosa. Nature Reviews Microbiology,

315

2012. 10(1): p. 66-U90.

316

5.

Kook, S. Y., Y. Lee, E. C. Jeong, and S. Kim, Immunomodulatory effects of

317

exopolysaccharides produced by Bacillus licheniformis and Leuconostoc mesenteroides

318

isolated from Korean kimchi. Journal of Functional Foods, 2019. 54: p. 211-219.

319

6.

Matsuzaki, C., A. Hayakawa, K. Matsumoto, T. Katoh, K. Yamamoto, et al.,

320

Exopolysaccharides Produced by Leuconostoc mesenteroides Strain NTM048 as an

321

Immunostimulant To Enhance the Mucosal Barrier and Influence the Systemic Immune

322

Response. Journal of Agricultural and Food Chemistry, 2015. 63(31): p. 7009-7015.

323

7.

Zhou, X., W. Qi, T. Hong, T. Xiong, D. Gong, et al., Exopolysaccharides from Lactobacillus

324

plantarum NCU116 Regulate Intestinal Barrier Function via STAT3 Signaling Pathway. J

325

Agric Food Chem, 2018. 66(37): p. 9719-9727.

326

8.

Ambort, D., M. E. Johansson, J. K. Gustafsson, H. E. Nilsson, A. Ermund, et al., Calcium and

327

pH-dependent packing and release of the gel-forming MUC2 mucin. Proc Natl Acad Sci U S

328

A, 2012. 109(15): p. 5645-50.

329

9.

Ambort, D., M. E. Johansson, J. K. Gustafsson, A. Ermund, and G. C. Hansson, Perspectives

330

on mucus properties and formation--lessons from the biochemical world. Cold Spring Harb

331

Perspect Med, 2012. 2(11).

332

10.

333 334

2014. 63(2): p. 216-7. 11.

335 336

Johansson, M. E., H. Sjovall, and G. C. Hansson, The gastrointestinal mucus system in health and disease. Nat Rev Gastroenterol Hepatol, 2013. 10(6): p. 352-61.

12.

337 338

McGuckin, M. A. and S. Z. Hasnain, There is a 'uc' in mucus, but is there mucus in UC? Gut,

Donaldson, G. P., S. M. Lee, and S. K. Mazmanian, Gut biogeography of the bacterial microbiota. Nat Rev Microbiol, 2016. 14(1): p. 20-32.

13.

Wlodarska, Marta, Chengwei Luo, Raivo Kolde, Eva d'Hennezel, John W. Annand, et al., 16


Page 16 of 29

Page 17 of 29


339

Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses

340

Inflammation. Cell Host & Microbe, 2017. 22(1): p. 25-+.

341

14.

342 343

Velcich, A., W. Yang, J. Heyer, A. Fragale, C. Nicholas, et al., Colorectal cancer in mice genetically deficient in the mucin Muc2. Science, 2002. 295(5560): p. 1726-9.

15.

Morampudi, V., U. Dalwadi, G. Bhinder, H. P. Sham, S. K. Gill, et al., The goblet

344

cell-derived mediator RELM-beta drives spontaneous colitis in Muc2-deficient mice by

345

promoting commensal microbial dysbiosis. Mucosal Immunol, 2016. 9(5): p. 1218-33.

346

16.

Pelaseyed, T., J. H. Bergstrom, J. K. Gustafsson, A. Ermund, G. M. Birchenough, et al., The

347

mucus and mucins of the goblet cells and enterocytes provide the first defense line of the

348

gastrointestinal tract and interact with the immune system. Immunol Rev, 2014. 260(1): p.

349

8-20.

350

17.

351 352

Johansson, M. E. and G. C. Hansson, The goblet cell: a key player in ischaemia-reperfusion injury. Gut, 2013. 62(2): p. 188-9.

18.

Sovran, B., L. M. Loonen, P. Lu, F. Hugenholtz, C. Belzer, et al., IL-22-STAT3 pathway plays

353

a key role in the maintenance of ileal homeostasis in mice lacking secreted mucus barrier.

354

Inflamm Bowel Dis, 2015. 21(3): p. 531-42.

355

19.

356

New developments in goblet cell mucus secretion and function. Mucosal Immunol, 2015. 8(4):

357 358

p. 712-9. 20.

359 360

21.

Fabienne, B., E. Emilie, Z. Kazem, P. Marc, and J. E. Isabelle, The AP-1 transcriptional complex: Local switch or remote command? Biochim Biophys Acta Rev Cancer, 2019.

22.

363 364

Schonthaler, H. B., J. Guinea-Viniegra, and E. F. Wagner, Targeting inflammation by modulating the Jun/AP-1 pathway. Ann Rheum Dis, 2011. 70 Suppl 1: p. i109-12.

361 362

Birchenough, G. M., M. E. Johansson, J. K. Gustafsson, J. H. Bergstrom, and G. C. Hansson,

Madrigal, P. and K. Alasoo, AP-1 Takes Centre Stage in Enhancer Chromatin Dynamics. Trends Cell Biol, 2018. 28(7): p. 509-511.

23.

Song, S., J. C. Byrd, N. Mazurek, K. Liu, J. S. Koo, et al., Galectin-3 modulates MUC2 mucin

365

expression in human colon cancer cells at the level of transcription via AP-1 activation.

366

Gastroenterology, 2005. 129(5): p. 1581-91.

367

24.

368 369

2012. 15(1): p. 57-62. 25.

370 371

Hansson, G. C., Role of mucus layers in gut infection and inflammation. Curr Opin Microbiol, Yoshimura, K., H. Aoki, Y. Ikeda, K. Fujii, N. Akiyama, et al., Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase. Nat Med, 2005. 11(12): p. 1330-8.

26.

Zhou, X., T. Hong, Q. Yu, S. Nie, D. Gong, et al., Exopolysaccharides from Lactobacillus

372

plantarum NCU116 induce c-Jun dependent Fas/Fasl-mediated apoptosis via TLR2 in mouse

373

intestinal epithelial cancer cells. Sci Rep, 2017. 7(1): p. 14247.

374

27.

Bu, X. D., N. Li, X. Q. Tian, and P. L. Huang, Caco-2 and LS174T cell lines provide different

375

models for studying mucin expression in colon cancer. Tissue & Cell, 2011. 43(3): p.

376

201-206.

377

28.

Wirtz, S., V. Popp, M. Kindermann, K. Gerlach, B. Weigmann, et al., Chemically induced

378

mouse models of acute and chronic intestinal inflammation. Nat Protoc, 2017. 12(7): p.

379

1295-1309.

380

29.

381 382

Ye, D. Z. and K. H. Kaestner, Foxa1 and Foxa2 control the differentiation of goblet and enteroendocrine L- and D-cells in mice. Gastroenterology, 2009. 137(6): p. 2052-62.

30.

Katz, J. P., N. Perreault, B. G. Goldstein, C. S. Lee, P. A. Labosky, et al., The zinc-finger 17



383

transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon.

384

Development, 2002. 129(11): p. 2619-2628.

385

31.

Wang, R., I. K. Kwon, N. Singh, B. Islam, K. Liu, et al., Type 2 cGMP-dependent protein

386

kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium.

387

Cell Death Differ, 2014. 21(3): p. 427-37.

388

32.

389 390

Honda, K. and D. R. Littman, The microbiota in adaptive immune homeostasis and disease. Nature, 2016. 535(7610): p. 75-84.

33.

Laino, J., J. Villena, P. Kanmani, and H. Kitazawa, Immunoregulatory Effects Triggered by

391

Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host

392

Cells. Microorganisms, 2016. 4(3).

393

34.

Vinderola, G., G. Perdigon, J. Duarte, E. Farnworth, and C. Matar, Effects of the oral

394

administration of the exopolysaccharide produced by Lactobacillus kefiranofaciens on the gut

395

mucosal immunity. Cytokine, 2006. 36(5-6): p. 254-260.

396

35.

397 398

maintenance in homeostasis and disease. Trends Mol Med, 2011. 17(10): p. 584-93. 36.

399 400

Vereecke, L., R. Beyaert, and G. van Loo, Enterocyte death and intestinal barrier van der Flier, L. G. and H. Clevers, Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol, 2009. 71: p. 241-60.

37.

Gersemann, M., S. Becker, I. Kubler, M. Koslowski, G. X. Wang, et al., Differences in goblet

401

cell differentiation between Crohn's disease and ulcerative colitis. Differentiation, 2009.

402

77(1): p. 84-94.

403

38.

Heazlewood, C. K., M. C. Cook, R. Eri, G. R. Price, S. B. Tauro, et al., Aberrant mucin

404

assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation

405

resembling ulcerative colitis. Plos Medicine, 2008. 5(3): p. 440-460.

406

39.

Ren, C. C., J. Dokter-Fokkens, S. F. Lozano, Q. X. Zhang, B. J. de Haan, et al., Lactic Acid

407

Bacteria May Impact Intestinal Barrier Function by Modulating Goblet Cells. Molecular

408

Nutrition & Food Research, 2018. 62(6).

409

40.

410 411

barrier in the gut. Tissue barriers, 2015. 3(1-2): p. e982426-e982426. 41.

412 413

Cornick, Steve, Adelaide Tawiah, and Kris Chadee, Roles and regulation of the mucus Ramanan, Deepshika and Ken Cadwell, Intrinsic Defense Mechanisms of the Intestinal Epithelium. Cell Host & Microbe, 2016. 19(4): p. 434-441.

42.

Bhatia, Shikha, P. Nagendra Prabhu, Ann C. Benefiel, Michael J. Miller, JoMay Chow, et al.,

414

Galacto-oligosaccharides may directly enhance intestinal barrier function through the

415

modulation of goblet cells. Molecular Nutrition & Food Research, 2015. 59(3): p. 566-573.

416

43.

Burger-van Paassen, N., A. Vincent, P. J. Puiman, M. van der Sluis, J. Bouma, et al., The

417

regulation of intestinal mucin MUC2 expression by short-chain fatty acids: implications for

418

epithelial protection. Biochem J, 2009. 420(2): p. 211-9.

419

Figure Captions

420

Figure 1. EPS116 modulated colon mucosal healing and homeostasis. Colon

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length (a, b), the ratio of colon weight/length (c) in the mice after administration of

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4% DSS with various EPS116 concentrations (0, 80, 160 mg EPS116/kg body weight) 18


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for 7 days. (d) Representative photomicrographs of colon (hematoxylin and eosin;

424

light microscopy, magnifications, 200 X). (e) Crypt depth (cd); (f) mucosal thickness

425

(mt). (g) Histological scores of (c).Values represent mean ± SD of the mean; #,

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Exopolysaccharides from Lactobacillus plantarum ... - ACS Publications

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