Lactobacillus casei CRL 431 administration decreases inflammatory cytokines in a diet-induced obese mouse model

Accepted Manuscript Lactobacillus casei CRL 431 administration decreases inflammatory cytokines in a diet induced obese mouse model Ivanna Novotny Núñez, Carolina Maldonado Galdeano, Alejandra de Moreno de LeBlanc, Gabriela Perdigón PII:

S0899-9007(15)00074-X

DOI:

10.1016/j.nut.2015.02.006

Reference:

NUT 9466

To appear in:

Nutrition

Received Date: 3 December 2014 Revised Date:

4 February 2015

Accepted Date: 17 February 2015

Please cite this article as: Núñez IN, Galdeano CM, de Moreno de LeBlanc A, Perdigón G, Lactobacillus casei CRL 431 administration decreases inflammatory cytokines in a diet induced obese mouse model, Nutrition (2015), doi: 10.1016/j.nut.2015.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

1

Lactobacillus casei CRL 431 administration decreases inflammatory cytokines in a

2

diet induced obese mouse model

3 Probiotic decreases inflammation in obesity model

RI PT

4 5

Ivanna Novotny Núñez1, Carolina Maldonado Galdeano1,2., Alejandra de Moreno de

7

LeBlanc1and Gabriela Perdigón1,2*

SC

6

8 1

Centro de Referencia para Lactobacilos (CERELA-CONICET). Chacabuco 145, San

M AN U

9 10

Miguel de Tucumán (T4000ILC) Tucumán. Argentina.

11

2

12

Farmacia. Universidad Nacional de Tucumán, Argentina.

Cátedra de Inmunología. Instituto de Microbiología. Facultad de Bioquímica, Química y

TE D

13

INN, CMG and AdMdL carried out the microbiological work, the animal studies

15

and the immunological determinations. GP conceived of the study. CMG, AdMdL and GP

16

designed the experiments. INN, CMG and AdMdL performed the statistical analyses and

17

prepared the figures. INN, CMG, AdMdL and GP wrote the draft of the manuscript. All

18

authors read and approved the final version of the manuscript.

20 21

AC C

19

EP

14

Word count: 6934 Figures: 4 Tables: 1

22

*Corresponding

author: Dr. Gabriela Perdigón, Chacabuco 145, San Miguel de Tucumán

23

(T4000ILC) Tucumán. Argentina. Fax +54 381 400 5600, email:

24

[email protected]

ACCEPTED MANUSCRIPT

ABBREVIATIONS

26

Lc: Lactobacillus Casei CRL 431

27

FM: Fermented milk

28

HFD: High fat diet

29

N: Non-obese group

30

O: Obese group

31

PBS: Phosphate saline solution

32

MRS: Mann-Rogosa-Sharp

33

RCA: Reinforced Clostridial agar

34

TLR: Toll like receptor

35

LMC: liver mononuclear cells

36

RBC: red blood cell

37

FITC: fluorescein isothiocyanate

38

PE: Phycoerythrin

39

APC: Allophycocyanin

40

MCP-1: Monocyte chemoattractant protein-1

43 44 45 46

SC M AN U

TE D

EP

42

AC C

41

RI PT

25

ACCEPTED MANUSCRIPT

ABSTRACT

48

Objectives: Obesity is a chronic disease associated with an inflammatory process in which

49

cytokines play an important role. Probiotic microorganisms have been associated with

50

modulation of the host immune system. The aim of the present work was to evaluate the

51

influence of the probiotic bacterium Lactobacillus (L.) casei CRL 431 on the cytokine

52

response in a model of mice under high-fat diet.

53

Methods: BALB/c mice received conventional-balanced-diet or high-fat-diet. The test

54

groups received milk, milk fermented by L. casei (FM) or L. casei as suspension in the

55

drinking water. Pro-inflammatory and regulatory cytokine producer cells were evaluated in

56

the small intestine, liver and the cytokine levels in the intestinal fluids. The percentages of

57

immune cell as macrophages (F4/80), NKT, CD4+, CD8+ populations were determined in

58

the liver. Adipocytes were also isolated and cultured to evaluate cytokines and the

59

chemokine MCP-1 produced by them.

60

Results: The administration of probiotic L. casei CRL 431 exerted an anti-inflammatory

61

response in mice under high-fat diet, evidenced mainly by decreasing pro-inflammatory

62

cytokines, such as IL-6, IL-17 and TNF-α. Probiotic administration was also associated

63

with less immune infiltrating cells in the liver of mice that received high-fat diet and

64

decreased secretion of MCP-1 by the adipocytes. This last observation could be associated

65

with less macrophage accumulation in the adipose tissues, which is characteristic in obese

66

host and contributes to maintain the inflammatory response in this organ. The results

67

obtained show an anti-inflammatory effect of L. casei CRL 431 when is administered as a

68

supplement of the high-fat diet in a mouse model.

69

Keywords: Cytokines, Probiotic, Diet induced obesity, Adipocytes, Small intestine, Liver

AC C

EP

TE D

M AN U

SC

RI PT

47

ACCEPTED MANUSCRIPT

Introduction

71

Obesity is defined as a chronic disease characterized by an excessive accumulation of fat or

72

adipose tissue hypertrophy. Obesity has become a serious public health problem that is

73

increasing and has reached epidemic proportions worldwide [1]. It has multifactorial origin

74

and is strongly associated with metabolic syndrome, causing different diseases, such as

75

cardiovascular diseases, type 2 diabetes, sleep apnea, among others [2, 3]. Obesity is

76

considered an inflammatory process in which the adipose tissue plays an important role [4]

77

Poor eating habits and lifestyles are associated with obesity-related diseases. Nowadays,

78

people tend to choose some dietary supplements that besides its nutritional properties can

79

exert some effect on the health. In this context, the products containing probiotic

80

microorganisms are often included in the daily die.

81

. Probiotics are defined as live microorganisms which, when administered in adequate

82

amounts confer a health benefit on the host [5]. The capacity of certain probiotic

83

microorganisms or fermented products to exert a beneficial balance in the gut microbiota,

84

improve the immunological status of the host, and modulate the cytokine release in the

85

lamina propria of the small intestine was also described [6-8]. The beneficial effect of

86

certain probiotics on body-weight and some immunological and metabolic parameters were

87

evaluated in animal models of obesity, diabetes and hyperlipidaemia [9-11]. It was also

88

reported that probiotic administration to obese hosts improves the gut microbiota [12, 13],

89

modulate genes associated with metabolism and inflammation in the liver and adipose

90

tissue [14], and improves symptoms associated to metabolic syndrome [15].

91

Probiotic supplementation to the diet could be a possible alternative to combat the obesity

92

and other disorders associated to it, especially due to the anti-inflammatory effect exerted

93

by these microorganisms [16, 17].

AC C

EP

TE D

M AN U

SC

RI PT

70

ACCEPTED MANUSCRIPT

94

Lactobacillus (L.) casei CRL 431 is a probiotic bacterium which effects on the intestinal

95

immune system have been extensively studied using murine models [18-21].

96

Recently, we evaluated the effect of the administration of L. casei

97

supplementation for a high-fat diet in a mouse model [22]. It was demonstrated that L. casei

98

CRL 431 administration as suspension or as fermented milk (FM) decreased the body

99

weight and biochemical parameters in blood which are associated to the metabolic

100

syndrome. This beneficial effect was associated to the improvement of gut microbiota by

101

increasing bifidobacteria and by avoiding the decrease of bacteroides. Both bacterial

102

populations were found diminished in mice receiving high-fat diets [23] The histology of

103

liver and small intestine, affected by the high fat diet intake, were also improved in mice

104

that received L. casei CRL431 [22].

105

The aim of the present work was to evaluate the influence of the probiotic bacterium L.

106

casei CRL 431 and its FM on the cytokine response (in small intestine, liver and

107

adipocytes) when are administered as diet supplements for mice under a high-fat diet

108

(HFD). We hypothesized that the pro-inflammatory cytokines, which are increased during

109

the diet-induced obesity, can be modulated by the probiotic administration.

AC C

EP

TE D

M AN U

SC

RI PT

CRL 431 as a

ACCEPTED MANUSCRIPT

Material and Methods

111

Bacterial strain and fermented milk

112

L. casei CRL 431 was obtained from the CERELA Culture Collection (San Miguel de

113

Tucumán, Argentina). Overnight cultures were grown at 37°C in 5ml sterile Mann-Rogosa-

114

Sharp (MRS) broth (Britania, Buenos Aires, Argentina). The cells were harvested by

115

centrifugation at 5000 g for 10 min, washed three times with fresh phosphate saline

116

solution (PBS) and then resuspended in 5 ml of sterile 10% (wt/vol) non-fat milk. This

117

bacterial suspension was diluted 1:30 in water and administered ad libitum to the mice. The

118

final concentration of probiotic bacteria was 2±1x108 CFU/ml, a concentration used

119

previously by our group [18, 24]. This count was periodically controlled at the beginning of

120

the administration and each 24 h to avoid modifications of more than one logarithmic unit.

121

To obtain the fermented milk (FM), non-fat dried milk (Svelty, Nestlé Argentina S. A.) was

122

rehydrated and autoclaved (115ºC for 15 min). Overnight cultures of Lc CRL 431 grown in

123

MRS broth were used to inoculate (2% vol/vol) sterile milk, and then was incubated at

124

37ºC during 24 h. Final concentration of the probiotic bacteria in the fermented milk was

125

4±2×108 CFU/ml.This fermented product was prepared each two days and the microbial

126

concentration was monitored.

SC

M AN U

TE D

EP

AC C

127

RI PT

110

128

Experimental groups. Protocol to induce obesity with a high fat diet and sampling

129

procedure

130

Mice were provided for CERELA (San Miguel de Tucumán, Argentina) from a closed

131

random bred colony. The experimental protocol using mice was performed in three

132

independent experiments. Twenty four female BALB/c mice (five weeks old, weighting

133

25±2 g) were used for each trial, and considering the 3 repetitions, the total number of mice

ACCEPTED MANUSCRIPT

used was 72. Mice were divided into two groups: I) Non-obese group (N) fed ad-libitum

135

with conventional balanced diet (45% carbohydrates, 32% fat, 23% proteins, 6% raw fiber,

136

10% total minerals, 1.3% Ca, 0.8% P, 12% moisture and vitamins provided by ACA

137

Nutrition Animal (San Nicolas, Buenos Aires, Argentina), and II) Mice under a HFD (Diet-

138

induced Obese group (O)). The HFD was made in the laboratory using the same

139

conventional balanced diet added with bovine lard and sugar (both for human consumption,

140

purchased in the supermarket). The HFD contained 43.4% of the conventional balanced

141

diet, 43.4% of bovine lard, and 13.2% of sugar; its caloric contribution was 18.6 kcal/d

142

[22]. Each group was divided in three sub-groups according to the addition of the dietary

143

supplements. The total number of mice evaluated for each group was 9 (3 in each repetition

144

of the assay).

145

I) Non obese group had a control (NC) in which animals received conventional balanced

146

diet and water ad-libitum during 2 months, and 3 test subgroups that received conventional

147

balanced diet supplemented daily with milk (N + milk), fermented milk (N + FM) or a

148

suspension of L. casei CRL 431 in the drinking water (N + Lc), ad-libitum. II) Diet-

149

induced obese group had a control (OC) with animals that received HFD and water ad-

150

libitum during 2 months, and 3 test sub-groups that received HFD supplemented with milk

151

(O + milk), fermented milk (O + FM) or the suspension of L. casei (O + Lc) during all the

152

experiment. The administration of the supplements was ad-libitum. The volume consumed

153

was measured daily in each cage, and considering the number of mice, each mouse

154

consumed an average of 3-4 ml of liquid per day. The bottles containing diet supplements

155

were replaced daily to maintain the quality of them.

156

Mice were maintained in a room with a 12-h light/dark cycle at 20±2°C. They were

157

weighed each two days. Mice of each control and test group were sacrificed by cervical

AC C

EP

TE D

M AN U

SC

RI PT

134

ACCEPTED MANUSCRIPT

dislocation at day 60th and small intestine, liver and adipose tissue from each mouse were

159

removed for further studies. .All animal protocols were preapproved by the Animal

160

Protection Committee of CERELA (CRL-BIOT-LI-2010/1A) and all experiments comply

161

with the current laws of Argentina.

RI PT

158

162

Detection of cytokine positive cells and TLR-5 expressing cells in the small intestine and

164

liver by immunohistochemistry

165

Cells positive for TNF-α, IFN-γ, IL-10, IL-6, IL-17 and TLR-5 were studied in the lamina

166

propria of the small intestine and in the liver by indirect immunofluorescence assay

167

following the technique described by Perdigón et al. [19]. For the small intestine,

168

representative portions (3-5 cm of length) were taken from the different sections

169

(duodenum, jejunum, and ileum). Rabbit anti-mouse TNF-α, IFN-γ, IL-10, IL-6 (ProSci

170

Inc. Poway CA, USA), goat anti-mouse IL-17 (BD Bioscience, San Diego, USA) or rabbit

171

anti-mouse TLR-5 (Santa Cruz Biotechnology, CA, USA) polyclonal antibodies were used

172

as primary antibodies and goat anti-rabbit or rabbit anti-goat antibodies conjugated with

173

fluorescein isothiocyanate (FITC; Jackson Immuno Research Laboratories Inc., West

174

Grove, USA) were used as secondary antibodies.

175

The number of fluorescent cells was counted by two researchers (two individual blinded

176

counts per sample) and the results were expressed as the number of positive cells for each

177

cytokine in ten fields of vision as seen at 1000X using a fluorescence light microscope

178

(Carl Zeiss, Germany).

AC C

EP

TE D

M AN U

SC

163

179 180

Determination of the cytokines levels in the small intestinal fluids

ACCEPTED MANUSCRIPT

Small intestine (duodenum, jejunum and ileum) was removed and the intestinal content was

182

collected by washing with 1 ml of PBS and immediately centrifuged at 5000 g for 15 min at

183

4 °C. The supernatant was separated and stored at -20 °C (or -80°C if they would be not

184

analysed within the next seven days) until determination of cytokine (IFN-γ, IL-10, IL-6)

185

concentrations using BD OptEIA cytokine ELISA set (BD Bioscience, San Diego, USA).

186

The results were expressed as the concentration of each cytokine in the intestinal fluids (pg

187

/ ml).

188

SC

RI PT

181

Isolation of liver mononuclear cells (LMC)

190

The liver of each mouse was removed aseptically and placed in sterile tubes containing 1

191

ml of DMEM High Glucose (Gibco, Invitrogen) following the technique described by

192

Soulard et al.[25]. The samples were homogenised under sterile conditions using a metal

193

mesh. Then, they were centrifuged at 2500 g for 10 min and resuspended in Percoll (Sigma,

194

St. Louis, USA) 35% in DMEM, and centrifuged at 2500-2800 g for 20 min. The resulting

195

pellets were lightly mixed with sterile red blood cell (RBC) lysing buffer (Sigma, St. Louis,

196

USA) for 2-4 min. Hemolysis was stopped with the addition of PBS, the samples were

197

centrifuged again at 1800 g for 10 min, and the pellets resuspended in 2 ml of sterile PBS.

198

The cell concentration was adjusted to 3x106 cells / ml, counting the viable cells by trypan

199

blue exclusion method in Neubauer chamber.

TE D

EP

AC C

200

M AN U

189

201

Determination of macrophages (F4/80+), and T populations (CD4+, CD8+ and NKT

202

cells) in liver

203

Mononuclear cells obtained from the liver of each mouse (3x106 cells / ml) were incubated

204

in darkness with the appropriate monoclonal antibodies: Peridinin Chlorophyll Protein

ACCEPTED MANUSCRIPT

Complex (PerCP-Cy5.5)-conjugated BM8 (anti-mouse F4/80 antigen. Pan Macrophage

206

Marker, e-Bioscience, San Diego, USA) for macrophages, Fluorescein Isothiocyanate

207

(FITC) labelled rat anti-mouse CD8 alpha Phycoerythrin (PE) labelled rat anti-mouse

208

CD4,and Allophycocyanin (APC)-conjugated anti-mouse NK1.1 (BD Biosciences

209

Pharmingen, San Diego, USA) for CD8+, CD4+ and NKT cells. Cells were then analysed

210

with a FACS Calibur flow cytometer (BD Bioscience, San Diego, CA, USA) equipped with

211

excitation laser source at 488 nm and 635 nm. Samples were run through the flow

212

cytometer, and 500,000 events were analysed for each sample with FCS Express 4Flow

213

Cytometer (De Novo Software, Glendale, CA, USA).

214

Using the forward- and side-scatter (FSC and SSC) properties of macrophages and

215

lymphocytes in laser light, two gates were drawn. The percentages of F4/80+ was

216

determined in both gates and the percentages of CD4+, CD8+ and NK1.1+ cells in the gate

217

corresponded to lymphocytes.

218

M AN U

SC

RI PT

205

Isolation and culture of adipocytes to determine cytokines and the monocyte

220

chemoattractant protein-1 (MCP-1)

221

Adipose tissues were obtained from mesenteric and visceral fat of each mouse, removed

222

aseptically, weighed to standardize the amount extracted and placed in sterile tubes

223

containing 1 ml of DMEM medium, as was previously described by Ishii et al. [26] and

224

Parra et al. [27]. The samples were mechanically disaggregated, and then were subjected to

225

enzymatic digestion in 1 ml of DMEM medium supplemented with collagenase type I

226

(Sigma Aldrich, St. Louis, USA) at a concentration of 2 mg / ml, and were stirred with

227

magnetic bars for 1 hour at 37 ° C. All these procedures were made under sterile

228

conditions. Subsequently, the products of enzyme digestion (adipocytes) were centrifuged

229

at 1000 g for 10 min. The adipocytes were placed in sterile culture dishes and incubated at

230

37 °C and 5% CO2 for 24 hours. After this incubation, the culture supernatants were

AC C

EP

TE D

219

ACCEPTED MANUSCRIPT

collected and stored at -20 °C (or -80°C if they would be not analysed within the next seven

232

days) until cytokine determinations.

233

The concentration of different cytokines (IL-6, IL-10, IFN-γ and TNF-α) and the

234

chemokine MCP-1 were measured by ELISA using commercial sets (BD OptEIA ELISA

235

sets, BD Biosciences Pharmingen, San Diego, CA, USA). The results were expressed as the

236

concentration of each cytokine and chemokine in the culture supernatants (pg/ml).

RI PT

231

SC

237 Statistical analysis

239

For each trial, test and control groups contained 3 animals that were sacrificed after 2

240

month of experiment. Each experiment was repeated 3 times. Statistical analyses were

241

performed using MINITAB 15 software (Minitab Inc., State College, Pennsylvania, USA).

242

Comparisons were accomplished by an ANOVA general linear model followed by a

243

Tukey’s post-hoc test, and P < 0.05 was considered significant. No significant differences

244

were observed between the three independent replicates; results from three replicates were

245

combined and the comparisons were obtained from 9 animals (N=9).

AC C

EP

TE D

M AN U

238

ACCEPTED MANUSCRIPT

246

Results

247 Fermented milk or L. casei administration as suspension modulate the number of

249

cytokine and TLR-5 positive cells in the lamina propria of the small intestine

250

No significant differences in the number of IFN-γ+ cells in the small intestine were

251

observed between the different studied groups (Fig. 1A).

252

The number of TNF-α+ cells increased significantly (P<0.05) in non-obese mice that

253

consumed milk as regard to the NC. The other diet supplements did not modify the number

254

of TNF-α+ cells. In mice under a HFD, the number of TNF-α+ cells were significantly

255

increased in control mice (OC) and in the mice from O + milk and O + Lc groups,

256

compared to the NC group. However, this cytokine positive cells in the small intestine of

257

mice from O + FM and O + Lc groups were significantly lower (P<0.05) than in the mice

258

from OC group (Fig. 1B).

259

No significant changes were observed in the number of IL-6, IL-10 and IL-17 positive cells

260

in the small intestine of non-obese mice that received probiotic compared to the control

261

(NC). The numbers of IL-6+, IL-10+ and IL-17+ cells were significantly increased

262

(P<0.05) in the animals from OC group compared to the NC group. Probiotic

263

administration to mice under a HFD (O + FM and O + Lc) decreased significantly (P<0.05)

264

the number of cells positive for these cytokines compared to OC group (Fig. 1). Figures 1G

265

to 1K show examples of this diminution obtained for IL-6+ cells in one representative

266

mouse from each group. IL-6+ cells were maintained increased in O + milk group, without

267

significant differences with the OC mice (Fig. 1C and 1H). As regard to the IL-17

AC C

EP

TE D

M AN U

SC

RI PT

248

ACCEPTED MANUSCRIPT

production, the mice from O + Lc group decreased significantly (P<0.05) the counts of this

269

cytokine producing cells, reaching values even lower than to the NC group (Fig. 1D).

270

The number of TLR-5+ cells did not show significant differences between the mice under a

271

HFD, received or not dietary supplements (OC, O + milk, O + FM or O + Lc), and the NC

272

group (Fig. 1E).

RI PT

268

273

Effect of milk, fermented milk or L. casei administration on the release of cytokines to

275

the small intestine fluid

276

Fig 2 shows the concentration of cytokines IFN-γ, IL10 and IL6 evaluated in the small

277

intestine fluid. IFN-γ increased significantly (P<0.05) in non-obese mice given any of the

278

dietary supplement (Fig. 2A), and IL-10 concentrations increased significantly in mice

279

from N + milk group, both compared to NC group. IFN-γ and IL-10 increased significantly

280

(P<0.05) in OC mice compared to the NC group (Fig. 2A and 2C). No significant

281

differences were observed in the IL-6 concentrations in the intestinal fluid from mice of

282

these groups (Fig. 2B). FM administration to mice under a HFD (O + FM) increased

283

significantly (P<0.05) the levels of IFN-γ, compared to their respective controls. IL-6

284

increased significantly (P<0.05) in mice under a HFD given FM compared to both controls

285

and the other test groups (Fig. 2B). IL-10 concentrations increased significantly (P<0.05) in

286

the fluid of the mice under a HFD that received probiotic (O + FM and O + Lc), compared

287

to the OC group, and these levels were also significant increased (P<0.05) compared to the

288

NC group (Fig. 2C).

AC C

EP

TE D

M AN U

SC

274

289 290

Effect of probiotic and HFD administration on the immune cells in the liver

ACCEPTED MANUSCRIPT

Mononuclear cells were isolated from the liver of the mice and analysed by flow cytometry.

292

The addition of any supplement to the diet of non-obese mice did not modify the number of

293

evaluated immune cells in the liver, maintaining values similar to NC (data not shown). The

294

percentage of NKT cells (recognized by the antibody anti-NK1.1) decreased in the livers of

295

mice under a HFD compared to the NC group, being the O + FM group which increased

296

significantly (P<0.05) the percentage of these cells in the liver compared to the OC, O +

297

milk and O + Lc, reaching values of NC group.

298

The percentage of CD8+ cells did not vary significantly between the mice under a HFD or

299

between these mice and those from the NC group.

300

The study of CD4+ cells showed a significantly increased percentage (P<0.05) in the liver

301

of mice under a HFD compared to NC group. Mice that received HFD and probiotic

302

administration decreased significantly (P<0.05) the percentage of these cells compared to

303

OC group, but without reach the NC values (table 1).

304

The percentage of macrophages (F4/80 + cells) decreased significantly (P<0.05) in OC

305

group compared to the NC group, and the three dietary supplementations (O + milk, O +

306

FM and O + Lc) were able to increase these values, reaching levels similar to those of the

307

CN group (Table 1).

SC

M AN U

TE D

EP

AC C

308

RI PT

291

309

Probiotic administration influences the number of cytokine positive cells in the liver of

310

diet induced obese mice

311

Significant differences were not observed in the number of IFN-γ and IL-10 positive cells

312

in the liver of non-obese mice that received probiotic administration compared to the NC

313

group. Milk supplementation increased the number of IL6+ and TNF-α+ cells in non-obese

314

mice, whereas the administration of L. casei suspension increased IL-17+ cells and also

ACCEPTED MANUSCRIPT

IL6+ cells (Fig. 3). Cytokine positive cells were increased in the OC group in relation to the

316

NC group. None of probiotic dietary supplements were able to restore the normal values

317

(NC group) of IFN-γ+ and IL-6+cells (Fig. 3A); however mice that received milk or FM

318

decreased significantly (P<0.05) the number of IL-6+ cells to the OC group The

319

administration of L. casei suspension to mice under a HFD (O + Lc) maintained

320

significantly increased (P<0.05) the number of IL-6+ cells in the liver, even higher than in

321

OC mice (Fig. 3B). Mice under a HFD that received probiotic were able to decrease

322

significantly (P<0.05) the counts of both TNF-α+ and IL-17+ cells, reaching the values

323

observed in the NC group (Fig. 3C and 3D). The number of IL-10+ cells decreased

324

significantly (P<0.05) in mice under a HFD given milk or FM compared to OC group;

325

however this cytokine was kept increased in the mice from O + Lc group (Fig. 3E).

326

The analysis of TLR-5 expression in liver did not show any significant different between

327

groups of mice that received conventional balanced diet or HFD or between the test groups

328

given special supplements and the respective controls (Data not shown).

TE D

M AN U

SC

RI PT

315

329

Probiotic administration modifies the amounts of certain cytokine produced by

331

adipocytes

332

Fig4 shows results from the study of different cytokines and the chemokine MCP-1 in the

333

supernatant obtained from cultures of adipocytes. The most remarkable differences between

334

the diet supplementations were obtained for IL-6, TNF-α and MCP-1, which increased

335

significantly (P<0.05) in the culture supernatant of adipocytes isolated from OC mice

336

compared to the ones isolated from NC group (Fig. 4A, 4B and 4D). The adipocytes

337

obtained from mice under a HFD receiving probiotics (O + FM or O + Lc) showed lower

AC C

EP

330

ACCEPTED MANUSCRIPT

concentrations of IL-6 and MCP-1 in the culture supernatant compared to the adipocytes

339

from OC group (Fig. 4A and 4D). The levels of TNF-α remained high in the culture

340

supernatant of adipocytes isolated from mice under a HFD (receiving or not the dietary

341

supplement) compared to the NC group (Fig. 4B). IL-10 levels were also significantly

342

lower (P<0.05) in the adipocyte culture supernatants from O + Lc group, compared to the

343

other groups that received HFD (Fig 4C). It was also observed that non-obese mice

344

receiving either of the dietary supplements increased significantly (P<0.05) the

345

concentration of MCP-1 in the supernatant of adipocyte cultures, compared to the NC

346

group (Fig. 4D).

M AN U

SC

RI PT

338

347 348

Discussion

349

In the present work, in a model of mice under HFD, we analysed the modifications of the

351

cytokine profiles in the small intestine, liver and adipose tissue, exerted by the

352

administration of the probiotic strain L. casei CRL431 as suspension or contained in a FM,

353

considering the positive effect observed previously in the body weight and clinical

354

parameters in mice that received probiotic [22]. In that previous work, it was observed that

355

mice given balanced diet increased a 50 ± 2 % the live body during the 2 months of the

356

experiment and the administration of L. casei decreased this body weight gain at 32 ± 4 %,

357

independent if the diet supplementation was as suspension or as FM. Mice that received

358

HFD increased more than 100% the body weight at the end of the experiment (2 months);

359

however the administration of L. casei as a supplement of the HFD decreased significantly

360

the body weight, especially when the probiotic was given in the FM (75 ± 2 % of body

361

weight gain). In the present work, the effect of L. casei CRL 431 as a diet supplement in

AC C

EP

TE D

350

ACCEPTED MANUSCRIPT

animals that received simultaneously HFD was evaluated and compared with the effect

363

exerted in mice given a balanced diet. Even when some effects were observed in non-obese

364

mice that received the probiotic under study, the most relevant effects were obtained in the

365

diet induced obese mice and this can be explained because the parameters selected to be

366

analysed were those reported modified by the HFD. The results at the intestinal level

367

showed that mice under a HFD increased the production of different pro-inflammatory

368

cytokines such as TNF-α, ΙL-6, IL-17 and also the regulatory cytokine IL-10. These

369

increases in the OC group could be related to the development of an inflammatory state

370

associated with the metabolic syndrome, as was also demonstrated in other obesity

371

associated pathologies such as diabetes type 2 and arteriosclerosis [28-30]. These results

372

agree with those reported by Khaodhiar et al., who observed increased levels of TNF-α and

373

IL-6 in the serum of obese individuals in relation to the non-obese[31]. With regard to IL-

374

17, Ahmed and Gaffen suggested that the obesity predisposes to the generation of a Th17

375

response[32], and it was also demonstrated that obesity promotes the selective expansion of

376

Th17 cells [33]. T cells of the obese mice are expanded and produce progressively more IL-

377

17 than the cells from lean mice, in an IL-6-dependent process. The increases observed for

378

IL-10+ cells in the small intestine of mice from OC group could be due to the own body

379

response to diminish the inflammatory state caused by obesity. In this sense, it was reported

380

that dendritic cells of obese patients produced a significant increase of IL-10 compared to

381

lean patients [34].

382

The diminution of the inflammatory state in the small intestine of mice under a HFD that

383

received FM or L. casei suspension was associated to the decrease in the number of the

384

cells positive for pro-inflammatory cytokines, which was accompanied by counts of IL-10+

AC C

EP

TE D

M AN U

SC

RI PT

362

ACCEPTED MANUSCRIPT

cells similar to the ones obtained in non-obese animals (Fig 1), and increased levels of this

386

cytokine in the intestinal fluid (Fig. 2). However, with the current study, we cannot

387

distinguish what cell population is implicated in the production of IL-10. It is possible that,

388

in addition to dendritic cells (explained above), T reg lymphocytes are involved in this anti-

389

inflammatory response, as was reviewed by Materese et al. [35].These results agree with

390

those performed by Satoh-Asahara et al., where the increases in the IL-10 levels had been

391

considered as an advantage due to their anti-inflammatory capabilities [36].

392

TLR5 was also evaluated, since recent studies showed that TLR5-deficient mice presented

393

hyperphagia, dyslipidemia, hypertension, insulin resistance and increased adiposity, which

394

conduced to the development of the obesity and the metabolic syndrome [37] TLR-5

395

recognizes flagellin, which is the major protein component of flagella in both Gram (+) and

396

Gram (-) bacteria [38]. TLR-5 is expressed in intestinal epithelial cells and in

397

immunological cells [39] [37]. At difference of the results obtained by other authors, our

398

obesity model did not associate with decreased TLR-5 expression (in small intestine or

399

liver). This fact may be due to the degree of obesity reached in the two months of

400

experiment.

401

Considering that probiotic supplementation to mice given HFD was related to less influx of

402

mononuclear cells in the liver [22], it was also evaluated if L. casei supplementation could

403

modulate the influx of some immune cells such as macrophages (F4/80+), CD4+, CD8+

404

and NKT cells in this organ. The study of CD4+ and CD8+ cells showed significant

405

increases in diet-induced obese mice compared to the NC group. However, mice under a

406

HFD that received FM or L. casei suspension showed significantly lower T cell infiltration,

407

especially for CD4+ cells, than OC. This fact could be related with a decrease in the

408

inflammation observed (Table 1). NKT cells, whose function is to balance the production

AC C

EP

TE D

M AN U

SC

RI PT

385

ACCEPTED MANUSCRIPT

of pro- and anti-inflammatory cytokines, decreased in the livers of OC mice, and increased

410

significantly only in mice under HFD that received FM. These results were similar to those

411

obtained by other authors, which showed that a probiotic mixture (VSL #3) improved

412

significantly the depletion of the hepatic NKT cells caused by obesity induced by a HFD in

413

a murine model [11]. The number of macrophages (F4/80+) was also evaluated in the liver

414

and showed a significant decrease in the OC group compared to the NC group; however,

415

the three dietary supplements increased these cells, recovering the values observed in the

416

NC group.

417

To evaluate if the modifications in the immune populations observed in the liver could be

418

related with the inflammatory state of this organ, cytokine positive cells were analysed in

419

the hepatic tissues. The results obtained were similar to the ones observed in the small

420

intestine samples with increases in the number of pro-inflammatory cytokines

421

accompanying with higher amounts of IL-10+ cells. The anti-inflammatory effect observed

422

in the liver of mice that received the probiotic administration was associated with increases

423

of IL-10+ cells (Fig. 3). It is also important to note that the inflammatory markers were

424

higher in the liver than in the small intestine, when compared non-obese mice and mice

425

under HFD. This last group duplicated or even increased more than twice the number of

426

cytokine positive cells in the liver compared to mice that received conventional balanced

427

diet. This anti-inflammatory response in the liver from mice under a HFD that received

428

probiotic can be also related to the improvement in the liver histology reported previously

429

in these animals [22]. The adipocytes isolated from mice of the different control and test

430

groups were analysed. Results showed that adipocytes from OC mice produce higher

431

secretion of pro-inflammatory cytokines compared to the adipocytes from NC mice. As was

432

observed in the other organs studied, the increases of pro-inflammatory cytokines were

AC C

EP

TE D

M AN U

SC

RI PT

409

ACCEPTED MANUSCRIPT

accompanied by increases in the secretion of the regulatory cytokine IL-10 by the

434

adipocytes. The obesity is associated with macrophage accumulation in adipose tissue [40],

435

the secretion of MCP-1 by the adipocytes was also evaluated, showing that adipocytes from

436

OC mice increased significantly the release of this chemokine compared to the non-obese

437

mice. The results obtained from mice under a HFD that received probiotic showed a

438

reduction in the secretion of IL-6 and MCP-1 by the adipocytes, maintaining a regulated

439

immune response, through the secretion of IL-10 (Fig. 4).

440

The results obtained with the different cytokines analysed in the small intestine, liver and

441

adipocytes led us to conclude that the administration of the probiotic strain L. casei CRL

442

431 to mice under a HFD induced an anti-inflammatory response, evidenced mainly by the

443

decrease of pro-inflammatory cytokines, such as IL-6, IL-17 and TNF-α. Most of the

444

parameters analysed did not show significant differences associated to the form of

445

administration for this probiotic, as a suspension or as a FM. Probiotic administration was

446

also related with less immune infiltrating cells in the liver and a lower secretion of MCP-1

447

by the adipocytes, associated with less accumulation of macrophages in the adipose tissue.

TE D

M AN U

SC

RI PT

433

EP

448 Conclusion

450

The results obtained in the present work showed that probiotic supplementation to hosts

451

susceptible to develop obesity can be effective, and that this effect is mainly related to the

452

anti-inflammatory capacity of the selected probiotic strain.

453

AC C

449

454 455

Conflict of Interest

ACCEPTED MANUSCRIPT

456

The authors declare no conflict of interest.

457 Acknowledgements

459

This work had been financially supported by (Consejo Nacional de Investigaciones

460

Científicas y Técnicas) and CIUNT (Universidad Nacional de Tucumán), Argentina.

AC C

EP

TE D

M AN U

SC

RI PT

458

ACCEPTED MANUSCRIPT

461

References

462 1.

464

WHO:

Obesity

and

overweight.

2012,

Available

from

http://www.who.int/mediacentre/factsheets/fs311/en.

RI PT

463

465

2.

Haslam DW, James WP: Obesity. Lancet; 2005, 366(9492):1197-1209.

466

3.

Romero-Corral A, Montori VM, Somers VK, Korinek J, Thomas RJ, Allison TG, Mookadam F, Lopez-Jimenez F: Association of bodyweight with total mortality and

468

with cardiovascular events in coronary artery disease: a systematic review of cohort

469

studies. Lancet; 2006, 368(9536):666-678. 4.

471 472

Trayhurn P, Wood IS: Adipokines: inflammation and the pleiotropic role of white adipose tissue. Br J Nutr; 2004, 92(3):347-355.

5.

M AN U

470

SC

467

FAO/WHO: Evaluation of health and nutritional properties of powder milk and live

473

lactic acid bacteria. Food and Agriculture Organization of the United Nations and

474

World Health Organization Expert Consultation Report 2001, Available from

475

www.fao.org/es/ESN/probio/probio.htm .

476

6.

Maldonado Galdeano C, Novotny Nunez I, de Moreno de LeBlanc A, Carmuega E, Weill R, Perdigon G: Impact of a probiotic fermented milk in the gut ecosystem and

478

in the systemic immunity using a non-severe protein-energy-malnutrition model in

479

mice. BMC Gastroenterol; 2011, 11:64.

480

7.

TE D

477

Maassen CB, van Holten-Neelen C, Balk F, den Bak-Glashouwer MJ, Leer RJ, Laman JD, Boersma WJ, Claassen E: Strain-dependent induction of cytokine

482

profiles in the gut by orally administered Lactobacillus strains. Vaccine; 2000,

483

18(23):2613-2623. 8.

485

Christensen HR, Frokiaer H, Pestka JJ: Lactobacilli differentially modulate

expression of cytokines and maturation surface markers in murine dendritic cells. J

486 487

AC C

484

EP

481

Immunol; 2002, 168(1):171-178.

9.

Hamad EM, Sato M, Uzu K, Yoshida T, Higashi S, Kawakami H, Kadooka Y,

488

Matsuyama H, Abd El-Gawad IA, Imaizumi K: Milk fermented by Lactobacillus

489

gasseri SBT2055 influences adipocyte size via inhibition of dietary fat absorption in

490

Zucker rats. Br J Nutr; 2009, 101(5):716-724.

ACCEPTED MANUSCRIPT

491

10.

Yadav H, Jain S, Sinha PR: Antidiabetic effect of probiotic dahi containing

492

Lactobacillus acidophilus and Lactobacillus casei in high fructose fed rats.

493

Nutrition; 2007, 23(1):62-68. 11.

496

insulin resistance by increasing hepatic NKT cells. J Hepatol; 2008, 49(5):821-830. 12.

497 498 499 500

Arora T, Singh S, Sharma RK: Probiotics: Interaction with gut microbiome and antiobesity potential. Nutrition; 2013, 29(4):591-596.

13.

RI PT

495

Ma X, Hua J, Li Z: Probiotics improve high fat diet-induced hepatic steatosis and

da Silva ST, Dos Santos CA, Bressan J: Intestinal microbiota, relevance to obesity and modulation by prebiotics and probiotics. Nutr Hosp; 2013, 28(4):1039-1048.

14.

SC

494

Park DY, Ahn YT, Park SH, Huh CS, Yoo SR, Yu R, Sung MK, McGregor RA, Choi MS: Supplementation of Lactobacillus curvatus HY7601 and Lactobacillus

502

plantarum KY1032 in diet-induced obese mice is associated with gut microbial

503

changes and reduction in obesity. PLoS One; 2013, 8(3):e59470.

504

15.

M AN U

501

Chen J, Wang R, Li XF, Wang RL: Bifidobacterium adolescentis supplementation

505

ameliorates visceral fat accumulation and insulin sensitivity in an experimental

506

model of the metabolic syndrome. Br J Nutr; 2012, 107(10):1429-1434. 16.

508 509

Wan YM, Zhu YQ, Xia B, Luo J: Treating TNBS-induced colitis in rats with

TE D

507

probiotics. Turk J Gastroenterol; 2011, 22(5):486-493. 17.

Chaves S, Perdigon G, de Moreno de LeBlanc A: Yoghurt consumption regulates the immune cells implicated in acute intestinal inflammation and prevents the

511

recurrence of the inflammatory process in a mouse model. J Food Prot; 2011,

512

74(5):801-811. 18.

514

in the gut and in mucosal immune stimulation. J Appl Microbiol; 2004, 97(4):673-

515 516

681.

19.

517

Perdigon G, Maldonado Galdeano C, Valdez JC, Medici M: Interaction of lactic

acid bacteria with the gut immune system. Eur J Clin Nutr; 2002, 56 Suppl 4:S21-

518 519

Galdeano CM, Perdigon G: Role of viability of probiotic strains in their persistence

AC C

513

EP

510

26. 20.

Galdeano CM, Perdigon G: The probiotic bacterium Lactobacillus casei induces

520

activation of the gut mucosal immune system through innate immunity. Clin

521

Vaccine Immunol; 2006, 13(2):219-226.

ACCEPTED MANUSCRIPT

522

21.

Galdeano CM, de Moreno de LeBlanc A, Vinderola G, Bonet ME, Perdigon G:

523

Proposed model: mechanisms of immunomodulation induced by probiotic bacteria.

524

Clin Vaccine Immunol; 2007, 14(5):485-492.

525

22.

Nunez IN, Galdeano CM, de LeBlanc Ade M, Perdigon G: Evaluation of immune response, microbiota, and blood markers after probiotic bacteria administration in

527

obese mice induced by a high-fat diet. Nutrition; 2014, 30(11-12):1423-1432.

528

23.

RI PT

526

Cani PD, Neyrinck AM, Fava F, Knauf C, Burcelin RG, Tuohy KM, Gibson GR, Delzenne NM: Selective increases of bifidobacteria in gut microflora improve high-

530

fat-diet-induced diabetes in mice through a mechanism associated with

531

endotoxaemia. Diabetologia; 2007, 50(11):2374-2383. 24.

Castillo NA, Perdigon G, de Moreno de Leblanc A: Oral administration of a

M AN U

532

SC

529

533

probiotic Lactobacillus modulates cytokine production and TLR expression

534

improving the immune response against Salmonella enterica serovar Typhimurium

535

infection in mice. BMC Microbiol; 2011, 11:177.

536

25.

Soulard V, Roland J, Sellier C, Gruner AC, Leite-de-Moraes M, Franetich JF, Renia L, Cazenave PA, Pied S: Primary infection of C57BL/6 mice with Plasmodium

538

yoelii induces a heterogeneous response of NKT cells. Infect Immun; 2007,

539

75(5):2511-2522.

540

26.

TE D

537

Ishii-Yonemoto T, Masuzaki H, Yasue S, Okada S, Kozuka C, Tanaka T, Noguchi M, Tomita T, Fujikura J, Yamamoto Y et al: Glucocorticoid reamplification within

542

cells intensifies NF-kappaB and MAPK signaling and reinforces inflammation in

543

activated preadipocytes. Am J Physiol Endocrinol Metab; 2010, 298(5):E930-940. 27.

545 546

during high-fat feeding in mice. J Nutr Biochem; 2008, 19(2):109-117.

28.

547

Yudkin JS, Yajnik CS, Mohamed-Ali V, Bulmer K: High levels of circulating

proinflammatory cytokines and leptin in urban, but not rural, Indians. A potential

548

explanation for increased risk of diabetes and coronary heart disease. Diabetes Care;

549 550

Parra P, Bruni G, Palou A, Serra F: Dietary calcium attenuation of body fat gain

AC C

544

EP

541

1999, 22(2):363-364. 29.

Pickup JC, Chusney GD, Mattock MB: The innate immune response and type 2

551

diabetes: evidence that leptin is associated with a stress-related (acute-phase)

552

reaction. Clin Endocrinol (Oxf); 2000, 52(1):107-112.

ACCEPTED MANUSCRIPT

553

30.

Pickup JC, Mattock MB, Chusney GD, Burt D: NIDDM as a disease of the innate

554

immune system: association of acute-phase reactants and interleukin-6 with

555

metabolic syndrome X. Diabetologia; 1997, 40(11):1286-1292.

556

31.

Khaodhiar L, Ling PR, Blackburn GL, Bistrian BR: Serum levels of interleukin-6 and C-reactive protein correlate with body mass index across the broad range of

558

obesity. JPEN J Parenter Enteral Nutr; 2004, 28(6):410-415. 32.

561

Rev; 2010, 21(6):449-453. 33.

562 563

Winer S, Paltser G, Chan Y, Tsui H, Engleman E, Winer D, Dosch HM: Obesity

SC

560

Ahmed M, Gaffen SL: IL-17 in obesity and adipogenesis. Cytokine Growth Factor

predisposes to Th17 bias. Eur J Immunol; 2009, 39(9):2629-2635. 34.

O'Shea D, Corrigan M, Dunne MR, Jackson R, Woods C, Gaoatswe G, Moynagh

M AN U

559

RI PT

557

564

PN, O'Connell J, Hogan AE: Changes in human dendritic cell number and function

565

in severe obesity may contribute to increased susceptibility to viral infection. Int J

566

Obes (Lond); 2013. 35.

568 569

Matarese G, Procaccini C, De Rosa V, Horvath TL, La Cava A: Regulatory T cells in obesity: the leptin connection. Trends Mol Med; 2010, 16(6):247-256.

36.

Satoh N, Shimatsu A, Kotani K, Himeno A, Majima T, Yamada K, Suganami T,

TE D

567

570

Ogawa Y: Highly purified eicosapentaenoic acid reduces cardio-ankle vascular

571

index in association with decreased serum amyloid A-LDL in metabolic syndrome.

572

Hypertens Res; 2009, 32(11):1004-1008. 37.

Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S,

EP

573 574

Sitaraman SV, Knight R, Ley RE, Gewirtz AT: Metabolic syndrome and altered gut

575

microbiota in mice lacking Toll-like receptor 5. Science; 2010, 328(5975):228-231. 38.

577

S, Underhill DM, Aderem A: The innate immune response to bacterial flagellin is

578 579

mediated by Toll-like receptor 5. Nature; 2001, 410(6832):1099-1103.

39.

580 581

Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira

AC C

576

Romagne F: Current and future drugs targeting one class of innate immunity

receptors: the Toll-like receptors. Drug Discov Today; 2007, 12(1-2):80-87. 40.

Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW, Jr.:

582

Obesity is associated with macrophage accumulation in adipose tissue. J Clin

583

Invest; 2003, 112(12):1796-1808.

ACCEPTED MANUSCRIPT

584

Figure legends

585 Fig. 1. Effect of high fat diet and probiotic administrations on cytokine positive cells in the

587

small intestine.The numbers of cytokine positive cells (A-E) and TLR-5 expressing cells

588

(F) were determined by indirect immune fluorescence on the small intestine tissue slides of

589

mice from different groups: Non obese control (NC), non-obese mice given milk, FM or L.

590

casei (N + milk, N + FM and N + Lc), control mice under a high fat diet (diet induced

591

obese control, OC) and diet-induced obese mice that received milk, FM or L. casei (O +

592

milk, O + FM and O + Lc). The results were expressed as the number of positive cells per

593

10 fields of vision (1000X). Data correspond to the means ± SD of 9 animals from three

594

separated experiments. * and # represent significant differences (P<0.05) with NC and OC,

595

respectively. Representative figures (100X) of IL-6+ cells from mouse of NC (G), OC (H),

596

O + milk (I), O + FM (J), and O + Lc (K) are shown in the bottom. Arrows show the

597

positive cells (fluorescent cells).

TE D

M AN U

SC

RI PT

586

598

Fig. 2. Effect of high fat diet and probiotic administration on the release of cytokine to the

600

intestinal fluid. Cytokine concentrations were determined in the intestinal fluid of mice

601

from the different control and test groups. Results were expressed as pg/ml of each

602

cytokine. Data correspond to the mean± SD of 9 animals from three separated experiments.

603

*

AC C

604

EP

599

and # represent significant differences (P<0.05) with NC and OC, respectively.

605

Fig. 3. Effect of high fat diet and probiotics on the cytokine positive cells in the liver. The

606

numbers of cytokine positive cells were determined by indirect immunofluorescence on the

607

liver tissue slides of mice from different groups: Non obese control (NC), non-obese mice

ACCEPTED MANUSCRIPT

given milk, FM or L. casei (N + milk, N + FM and N + Lc), control mice under a high fat

609

diet (OC) and diet-induced obese mice that received milk, FM or L. casei (O + milk, O +

610

FM and O + Lc). The results were expressed as the number of positive cells per 10 fields of

611

vision (1000X). Data correspond to the means ± SD of 9 animals from three separated

612

experiments.* and

613

respectively. Representative figures (100X) of TNF-α+ cells from a mouse of NC (A), OC

614

(B), O+FM (D), and O + Lc (E) are shown. Arrows show the positive cells (fluorescent

615

cells).

represent significant differences (P<0.05) with NC and OC,

SC

#

RI PT

608

M AN U

616 617

Fig. 4. Effect of probiotic administration to diet-induced obese mice on the cytokines and

619

MCP-1 production by adipocytes. Adipocytes were isolated from mesenteric and

620

subcutaneous fat of non-obese control (NC), diet-induced obese control mice (OC), and

621

mice under a high fat diet that received milk, FM or L. casei as suspension (O + milk, O +

622

FM or O + Lc). Cytokines and MCP-1 concentrations were determined in the supernatant

623

of adipocyte cultures. Results were expressed as pg/ml. Data correspond to the mean± SD

624

of 9 animals from three separated experiments.

625

(P<0.05) with NC and OC, respectively.

AC C

EP

TE D

618

*

and

#

represent significant differences

ACCEPTED MANUSCRIPT Table 1. Immune populations in liver NKT 80±8 a 63±5 b 61±6 b 88±6 a 64±6 b

CD8+ 3±1 a 5±1 a 6±2 a 3±2 a 5±1 a

CD4+ 21±2 a 63±4 b 48±11b,c 40±13 b 36±2 c

F4/80+ 58±8 a 34±11 b 63±8 a 72±10 a 58±6 a

RI PT

Groups NC OC O+milk O+FM O+Lc

AC C

EP

TE D

M AN U

SC

Mononuclear cells isolated from liver of mice were labeled with the specific antibodies and the percentages of cells positive were analyzed by flow cytometry. Results are expressed as average of the percentage obtained for N=9 mice (from tree independent experiments) ± SD. a,b,c Mean for each population without a common letter differs significantly (P<0.05). NC: non-obese control, OC: obese control, O + milk, O + FM and O + Lc: correspond to groups of mice under high fat diet received milk, fermented milk or a suspension of L. casei, respectively.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights The administration of the probiotic Lactobacillus casei CRL 431 to diet induced obese mice decreased the inflammatory state in the small intestine, liver and adipose tissues. Probiotic administration, especially as fermented milk was associated to decrease of pro-

RI PT

inflammatory cytokines, such as IFN-γ, TNF-α, IL-6 and IL-17 in small intestine and liver of diet induced obese mice.

Probiotic administration was also related with less immune infiltrating cells in the liver of diet induced obese mice.

SC

Lactobacillus casei CRL 431 administered as suspension and mainly as FM was able to reduce the secretion of IL-6 and MCP-1 by the adipocytes isolated from diet induced obese mice,

AC C

EP

TE D

M AN U

maintaining a regulated immune response, through the secretion of IL-10.