Lactobacillus brevis G-101 ameliorates colitis in mice by inhibiting NF-κB, MAPK and...

ORIGINAL ARTICLE

Lactobacillus brevis G-101 ameliorates colitis in mice byinhibiting NF-jB, MAPK and AKT pathways and bypolarizing M1 macrophages to M2-like macrophagesS.-E. Jang1,2, S.R. Hyam1, M.J. Han2, S.-Y. Kim3, B.-G. Lee3 and D.-H. Kim1

1 Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul, Korea

2 Department of Food and Nutrition, Kyung Hee University, Seoul, Korea

3 R&D center, CTOBIO Inc., Gyeonggi-do, Korea

Keywords

colitis, IL-10, Lactobacillus brevis G-101,

macrophage polarization, NF-kB.

Correspondence

Dong-Hyun Kim, Departments of Life and

Nanopharmaceutical Sciences and Pharma-

ceutical Science, College of Pharmacy, Kyung

Hee University, 1, Hoegi, Dongdaemun-ku,

Seoul 130-701, Korea.

E-mail [email protected]

2013/0437: received 5 March 2013, revised

28 May 2013 and accepted 31 May 2013

doi:10.1111/jam.12273

Abstract

Aim: We isolated Lactobacillus brevis G-101 from kimchi lactic acid bacteria

(LAB) strains, which induced IL-10 expression in lipopolysaccharide (LPS)-

stimulated peritoneal macrophages. To evaluate the inflammatory effect of

G-101, we examined its inhibitory effect in 2,4,6-trinitrobenzene sulfonic acid

(TNBS)-induced colitic mice.

Materials and Results: The colitic mice were prepared by intrarectal injection

of TNBS. We measured intestinal mucosal cytokines by enzyme-linked

immunosorbent assay; activation of transcription factors, by immunoblotting;

and macrophage polarization markers, by real-time polymerase chain reaction.

Of 200 LAB strains tested, Lact. brevis G-101 showed most potent activity for

induction of IL-10 expression in LPS-stimulated peritoneal macrophages.

However, it significantly inhibited the expression of TNF-a, IL-1b and IL-6 and

the phosphorylation of IRAK1 and AKT, and activated NF-jB and MAPKs.

Treatment with TNBS caused colon shortening; increased myeloperoxidase

activity; and increased IL-1b, IL-6 and TNF-a expression in mice. Oral

administration of Lact. brevis G-101 significantly inhibited these activities.

Lactobacillus brevis G-101 inhibited TNBS-induced IRAK-1 phosphorylation

and NF-jB activation, as well as the expression of COX-2 and iNOS.

Lactobacillus brevis G-101 inhibited the expression of M1 macrophage markers,

but increased the expression of M2 macrophages in the colons of TNBS-treated

mice.

Conclusions: Lactobacillus brevis G-101 may improve colitis by inhibiting the

IRAK1/NF-jB, MAPK and AKT pathways and by polarizing M1 macrophages

to M2-like macrophages.

Significance and Impact of the Study: These results suggest that IL-10

expression-inducing LAB can ameliorate colitis by inhibiting NF-jB activation

and macrophage polarization.

Introduction

Acute and chronic inflammations are highly regulated

immune processes characterized by the release of cyto-

kines, chemokines and growth factors and by the trans-

migration of inflammatory cells, such as neutrophils,

monocytes and lymphocytes, from the blood to the

affected tissue (Collins et al. 1995; Johnson and Koval

2009). Acute inflammation is a normal and helpful

response to injury. However, chronic inflammation is

persistent and excessive. This inflammatory response

causes progressive damages to the body, leading to a vari-

ety of diseases, such as colitis, rheumatoid arthritis and

even cancer. Of these inflammatory mediators, pro-

inflammatory cytokines such as tumour necrosis factor

(TNF)-a and interleukin (IL)-1b are activated through

888 Journal of Applied Microbiology 115, 888--896 © 2013 The Society for Applied Microbiology

Journal of Applied Microbiology ISSN 1364-5072

nuclear factor-kappaB (NF-jB), and they also activate

NF-jB (Collins et al., 1995; Baldwin 1996). However,

anti-inflammatory cytokine IL-10 downregulates NF-jB-activated inflammatory pathway. These inflammatory

reactions mainly proceed via signalling pathways through

toll-like receptors (TLRs) and/or cytokine receptors

(David et al. 2010). Among this family of receptors,

TLR4, which is associated with the activation of transcrip-

tion factor NF-jB via IL-1 receptor-associated kinases

(IRAKs), may serve as the primary mediator of LPS

signalling (Chow et al. 1999; Cario and Podolsky 2000).

All IRAK members form multimeric receptor complexes

(Takeda and Akira 2004). Phosphorylated IRAK-1

activates a multimeric protein complex composed of

TRAF6, TAK1, TAB1 and TAB2, leading to activation of

NF-jB and mitogen-associated MAPK pathways, as well

as induction of pro-inflammatory cytokines. Regulating

expression of these inflammatory mediators can therefore

be beneficial in decreasing inflammatory diseases, such

as colitis and arthritis (Campieri and Gionchetti 1999;

Kawaguchi et al. 2011; Paradkar et al. 2004).

Lactic acid bacteria (LAB) are safe micro-organisms

that improve disturbances of the indigenous microbiota

(Campieri and Gionchetti 1999; Perdigon et al. 1991;

Collins and Gibson 1999), possess antidiabetic effects

(Tabuchi et al. 2003), inhibit carcinogenesis (Perdigon

et al. 1991), have anticolitic effects (Campieri and Gion-

chetti 1999; Daniel et al. 2006; Peran et al. 2007a) and

induce nonspecific activation of the host immune system

(Perdigon et al. 1991). Lactobacillus casei inhibits the

expression of inflammatory cytokines in dextran sulfate

sodium (DSS)-induced colitic mice (Chung et al. 2007).

Lactobacillus casei, Lact. acidophilus, Lact. sontoryeus, Bifi-

dobacterium lactis and Bif. longum show intestinal anti-

inflammatory activity in TNBS-induced colitic animals

(Peran et al. 2007b; Lee et al. 2009, 2010). Nevertheless,

the anticolitic effects of LAB inducing IL-10 expression in

macrophages have not been thoroughly examined.

We isolated Lact. brevis G-101, a strain that induced

IL-10 expression most potently, from 200 kimchi LAB

strains. To evaluate the inflammatory effect of G-101, we

examined its inhibitory effects in 2,4,6-trinitrobenzene

sulfonic acid (TNBS)-induced colitic mice.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), sodium

thioglycollate, tetramethyl benzidine, TNBS, hexadecyl

trimethyl ammonium bromide and radio-immunoprecip-

itation assay (RIPA) lysis buffer were purchased from

Sigma Co. (St Louis, MO, USA). The protease inhibitor

cocktail was purchased from Roche Applied Science

(Mannheim, Germany). Enzyme-linked immunosorbent

assay (ELISA) kits were from Pierce Biotechnology, Inc.,

(Rockford, IL, USA). Antibodies were purchased from

Santa Cruz Biotechnology (Santa Cruz, CA, USA). The

enhanced chemiluminescence (ECL) immunoblot system

was from Pierce Co. (Rockford, IL, USA).

Bacterial strains and growth conditions

Two hundred strains of LAB were isolated from kimchi

using MRS agar, this included 85 Lactobacillus sakei

strains, 37 Leuconostoc mesenteroides strains, 45 Lactoba-

cillus plantarum strains, 21 Lactobacillus curvatus strains,

nine Lactobacillus brevis strains and three Lactobacillus

pentosus strains. Isolated LAB strains were indentified

using the Gram stain kit (BioMerieux, Grenoble, France)

following the protocol developed by Jung et al. (2012).

Additional enzyme activities and biochemical characteris-

tics were determined using the API 20E test strips (Bio-

Merieux, Seoul, Korea). Lactobacillus brevis G-101 was

acid tolerant (can survive for 12 h at pH 4). The 16S

ribosomal DNA was amplified by PCR using 27F, 1492R

primer, followed by purification using the purification kit

(Bionics Inc., Seoul, Korea), and the purified 16S ribo-

somal DNA was sequenced using ABI 3730XL DNA

analysis.

For macrophage experiment, Lact. brevis G-101 was

anaerobically grown at 37°C in MRS broth without shak-

ing, collected by centrifugation (10 000 g for 30 min)

and washed twice with saline. The resulting pellet was

suspended in phosphate-buffered saline. The cell suspen-

sion (5 ml) was placed in a 50-ml centrifuge tube, heated

in boiling water bath for 10 min and used for experi-

ments on its effect in LPS-stimulated peritoneal macro-

phages.

For mouse experiment, Lact. brevis G-101 was grown

to an optical density between 3 and 4 at 600 nm (early

stationary phase), harvested by centrifugation (10 000 g

for 30 min) and washed with phosphate-buffered saline

(PBS). The collected cells (1 9 108 and 1 9 109 CFU)

suspended in 50 mmol l�1 NaHCO3 buffer containing

1% glucose were orally administered to mice (Lee et al.

2009).

Animals

Male ICR mice (5 weeks old, 20–25 g) were sup-

plied from Orient Experimental Animal Breeding Center

(Seoul, Korea). The mice were housed six per cage,

allowed access to water and standard laboratory

chow (Orient Experimental Animal Breeding Center)

ad libitum and maintained at 20–22°C, 50 � 10%

Journal of Applied Microbiology 115, 888--896 © 2013 The Society for Applied Microbiology 889

S.-E. Jang et al. Lactobacillus brevis G-101 inhibits colitis

humidity and a 12-h diurnal light cycle (lights on

07:00–19:00 h) prior to testing. All experiments were

performed in accordance with the NIH and Kyung Hee

University guidelines for Laboratory Animals Care and

Use and approved by the Committee for the Care and

Use of Laboratory Animals in the College of Pharmacy,

Kyung Hee University.

Isolation and culture of peritoneal macrophages

Mice were intraperitoneally injected with 2 ml of 4%

thioglycolate solution and sacrificed 4 days after injection,

and the peritoneal cavities were swilled with 10 ml of

RPMI 1640 (Joh et al. 2011). The peritoneal lavage fluids

were centrifuged at 300 g for 10 min, and the cells

(2 9 107 cells) were resuspended in RPMI 1640 (5 ml)

and plated. After incubation at 37 °C for 2 h, the cells

were washed three times and nonadherent cells were

removed by aspiration. The cells were cultured in 12-well

plates at 37°C in RPMI 1640 with 10% FBS (1% antibi-

otic-antimycotic; Life Technologies, Grand Island, NY,

USA). The attached cells (1 9 106 cells per well) were

used as peritoneal macrophages. To examine the anti-

inflammatory effects of Lact. brevis G-101 (1 9 103,

1 9 104 and 1 9 105 CFU per well), peritoneal macro-

phages were incubated in the absence or presence of

Lact. brevis G-101 with LPS for 24 h.

Preparation of experimental colitic mice

Male ICR mice were randomly divided into five groups:

normal and TNBS-induced colitic groups treated with or

without Lact. brevis G-101 or mesalazine (10 mg kg�1).

Each group is consisted of 6 mice. TNBS-induced colitis

was induced by the administration of 2�5% (w/v) TNBS

solution (100 ll) in 50% ethanol into the colon of

lightly anesthetized mice via a thin round-tip needle

equipped with a 1-ml syringe (Joh et al. 2011). The nor-

mal group was treated with vehicle alone. The needle

was inserted so that the tip was 3�5–4 cm proximal to

the anal verge. To distribute the agents within the entire

colon and caecum, mice were held in a vertical position

for 30 s after the injection. If an animal quickly excreted

the TNBS–ethanol solution, it was excluded from the

remainder of the study. Lactobacillus brevis G-101

[(1 9 108 or 1 9 109 CFU) per mouse] was orally

administered once a day for 3 days after TNBS treat-

ment. The mice were anaesthetized with aether and sac-

rificed 20 h after the final administration of LAB. The

colon was quickly removed, opened longitudinally and

gently cleared of stool by PBS. Macroscopic assessment

of the disease grade was scored according to a previously

reported scoring system (0, no ulcer and no inflamma-

tion; 1, no ulceration and local hyperaemia; 2, ulceration

without hyperaemia; 3, ulceration and inflammation at

one site only; 4, two or more sites of ulceration and

inflammation; 5, ulceration extending more than 2 cm)

(Lee et al. 2011), and the colon tissue was then used for

immunoblot and enzyme-linked immunosorbent assay

(ELISA) analysis.

Assay of myeloperoxidase activity in colon

The colons were homogenized in 10 mmol l�1 potassium

phosphate buffer (pH 7�0) containing 0�5% hexadecyl tri-

methyl ammonium bromide, and then centrifuged for

30 min (20 000 g; 4°C). The supernatant was used as a

crude enzyme solution. An aliquot (50 ll) of the superna-tant was added to a reaction mixture of 1�6 mmol l�1

tetramethyl benzidine and 0�1 mmol l�1 H2O2 and

incubated at 37°C. The absorbance of the reaction mixture

was obtained at 650 nm over time. Myeloperoxidase activ-

ity was defined as the quantity of enzyme degrading

1 lmol ml�1 of peroxide at 37°C and expressed in unit

per mg protein (Mullane et al. 1987). The protein content

was assayed by the Bradford method (Bradford 1976).

ELISA and immunoblot analysis in peritoneal

macrophages

For the assay of cytokines, the peritoneal macrophages

(5 9 105 cells) were stimulated with LPS (50 lg ml�1;

Invitrogen, CA, USA) for 30 min and 20 h in the pres-

ence or absence of Lact. brevis G-101 (1 9 103, 1 9 104

Table 1 Primers of polarization markers for Real-time polymerase chain reaction

Molecule Forward primer sequence Reverse primer sequence

TNF-a 5′-TCTTCTCATTCCTGCTTGTGG-3′ (21mer) 5′-GGTCTGGGGCATAGAACTGA-3′ (20mer)

IL-1b 5′-AACCTGCTGGTGTGTGACGTTC-3′ (22mer) 5′-CAGCACGAGGCTTTTTTGTTGT-3′ (22mer)

IL-10 5′-ATGCTGCCTGCTCTTACTGACTG-3′ (23mer) 5′-CCCAAGTAACCCTTAAAGTCCTGC-3′ (24mer)

Arginase I 5′-CAGAAGAATGGAAGAGTCAG-3′ (20mer) 5′-CAGATATGCAGGGAGTCACC-3′ (20mer)

Arginase II 5′-TGATTGGCAAAAGGCAGAGG-3′ (20mer) 5′-CTAGGAGTAGGAAGGTGGTC-3′ (20mer))

CD206 5′-CAGCGGTTGGCAGTGGA-3′ (17mer) 5′-CAGCTGATGGACTTCCTGGTAAG-3′ (23mer)

b-actin 5′-GTGCTATGTTGCTCTAGACT-3′ (20mer) 5′-CACAGGATTCCATACCCAAG-3′ (20mer)


Lactobacillus brevis G-101 inhibits colitis S.-E. Jang et al.

and 1 9 105 CFU per well), lysed ice-cold RIPA lysis

buffer containing 1% protease inhibitor cocktail and 1%

phosphatase inhibitor cocktail and centrifuged at 2000 g

for 10 min. In addition, colons were homogenized and

lysed with ice-cold RIPA lysis buffer. Then the lysates

were centrifuged (15 000 g, 4°C) for 15 min, and the

supernatant was transferred to 96-well ELISA plates, and

the cytokines (IL-10, TNF-a, IL-1b, IL-6) were measured

by ELISA kits according to the manufacture’s protocol.

For the immune blotting, the lysates of macrophages

and colon tissues were separated by 10% SDS–PAGE and

transferred onto polyvinylidene difluoride membranes.

The membranes were blocked with 5% nonfat dried-milk

proteins in 0�05% PBST, then probed with COX-2, iNOS,

p-IRAK-1, IRAK-1, p65, p-p65, EKR, p-ERK, p38, p-p38,

JNK, p-JNK, AKT, p-AKT or b-actin antibody. After

washing with PBST, proteins were detected with HRP-

conjugated secondary antibodies for 50 min. Bands were

visualized with enhanced chemiluminescence reagent

(Joh et al. 2011).

Real-time polymerase chain reaction (RT-PCR)

Total RNAs were extracted from the colon tissues with

the RNeasy Mini kit (Qiagen, Hilden, Germany), and

first-strand cDNA synthesis for arginase (ARG) I, II,

TNF-a, IL-1b, IL-10, CD206 and b-actin was performed

using reverse transcriptase (Takara, Shiga, Japan) accord-

ing to the manufacturer’s protocol. Real-time PCRs were

performed on the Rotor-Gene Q� (Qiagen) using DNA

150 60

40

20

0

60 30

20

10

0

40

20

0

100

50

0NOR

IL-1

0 (p

g m

l–1 )

IL-6

(pg

ml–

1 )T

NF

-α (

pg m

l–1 )

IL-1

β (p

g m

l–1 )

LPS G3 G4 G5 NOR LPS G3 G4 G5

NOR LPS

#

#

#

#

*

**

* * *

* * *

* *

p-p65

p65

p-c-jun

c-jun

LPS – +G3 G4 G5 G3 G4 G5– – – + + +

– –LAB

0·8

0·6

0·4

Inte

nsity

(%

)

0·2

0

#

#

**

** *

*

**

**

β-Actin

G3 G4 G5 NOR LPS G3 G4 G5

(a)

(b)

a b

c d

Figure 1 The effect of Lactobacillus brevis

G-101 on the expression of IL-10, IL-6, IL-1b

and TNF-a and the activation of NF-jB in

LPS-stimulated peritoneal macrophages. The

peritoneal macrophages (1 9 106 per well)

were treated with 50 ng ml�1 LPS in the

absence or presence of G-101 (1 9 103,

1 9 104, 1 9 105 CFU per well) for 20 h. (a)

Levels of IL-10 (a), TNF-a (b), IL-1b (c), and

IL-6 (d) in culture supernatants were measured

by ELISA. (b) NF-jB (p65 and p-p65), AP1

(c-jun and p-c-jun) and b-actin were analysed

by immunoblotting. NOR, normal control; LPS,

treated with LPS alone; G3, treated with

1 9 103 CFU of Lact. brevis G-101 and LPS;

G4, treated with 1 9 104 CFU of Lact. brevis

G-101 and LPS; G5, treated with 1 9 105 CFU

of Lact. brevis G-101 and LPS. Lact. brevis G-

101 heated in boiling water bath for 10 min

was used. Enzyme activity values are the

mean � SD (n = 3). #Significantly different

compared with normal control group

(P < 0�05). * Significantly different compared

with group treated with LPS alone (P < 0�05).( ) p-p65/p65; ( ) p-c-jun/c-jun.



polymerase (Takara) and SYBR Green I (Qiagen) in a

reaction volume of 20 ll. Primers used for real-time PCR

are listed in Table 1. The normalized expression of the

target gene, with respect to b-actin, was computed for all

samples using Microsoft Excel, as previously reported

(Kim et al. 2012),

Statistical analysis

All data are expressed as the mean � standard

deviation (SD), with statistical significance analysed using

one-way ANOVA followed by a Student–Newman–Keulstest (P < 0�05).

0NOR

NOR

TNBS

TNBS

G8 G9 M10

NOR TNBS G8 G9G8

G9

M10

NOR TNBS G8 G9 M10

M10

NOR

#

** *

***

#

#

#

*

* **

* *

TNBS G8 G9 M10

0

0

0

Wei

ght c

hang

e (%

)M

PO

act

ivity

(µU

nit m

g–1 )

Col

on le

ngth

(cm

)M

acro

scop

ic s

core

5

–5

–10

–15

10

3

6

9

2

4

6

8

10

1

2

3

4

5(a) (e)

a

b

c

d

e

(b)

(c)

(d)

Figure 2 The effect of Lactobacillus brevis G-101 on macroscopic disease (a), colon length (b), colonic myeloperoxidase activity (c), body weight

(d) and colonic histology (e) in TNBS-induced colitic mice. TNBS, except in the normal control group (NOR, treated with vehicle alone), was intra-

rectally administered in TNBS, G8, G9 and M10 groups. The test agents (TNBS, vehicle alone; G8, 1 9 108 CFU per mouse of Lact. brevis G-101

with TNBS; G9, 1 9 109 CFU per mouse of Lact. brevis G-101 with TNBS; M10, 10 mg kg�1 mesalazine with TNBS) were orally administered for

3 days after TNBS treatment. The mice were anesthetized and sacrificed 20 h after the final administration of LAB. All values are the mean � SD

(n = 6). #Significantly different compared with normal control group (P < 0�05). * Significantly different compared with group treated with TNBS

alone (P < 0�05).



Results

Lactobacillus brevis G-101 induces IL-10 expression

in LPS-stimulated peritoneal macrophages

We measured the ability of 200 LAB strains isolated from

kimchi to induce IL-10 expression in LPS-stimulated peri-

toneal macrophages. LPS treatment significantly reduced

IL-10 expression, but induced the expression of pro-

inflammatory cytokines TNF-a, IL-1b and IL-6 (Fig. 1).

Of these LAB strains (heat-treated), Lact. brevis G-101

induced IL-10 expression reduced by LPS most potently

(data not shown). Furthermore, Lact. brevis G-101 also

inhibited LPS-induced NF-jB and AP1 activation. Treat-

ment with Lact. brevis G-101 (1 9 105 CFU ml�1) in

LPS-stimulated peritoneal macrophages significantly

reversed IL-10 expression to 93% of normal control

group.

Anti-inflammatory effect of Lactobacillus brevis G-101

in TNBS-induced colitic mice

We examined the ability of Lact. brevis G-101 to inhibit

colitis induced by intrarectal injection of TNBS. Oral

administration of TNBS induced loss of body weight and

severe inflammation, and manifested in the form of

shortened, thickened and erythematous colons (Fig. 2).

Histological examination of the TNBS-treated colon

showed massive bowel oedema, dense infiltration of the

superficial layers of the mucosa and epithelial cell disrup-

tion due to large ulcerations. Lactobacillus brevis G-101

treatment inhibited body weight reduction, colon short-

ening and inflammation and thickening on the third day

after TNBS treatment. Lactobacillus brevis G-101 treat-

ment inhibited TNBS-induced MPO activity, a represen-

tative inflammatory marker. The efficacy of Lact. brevis

G-101 was comparable to that of mesalazine, a commer-

cial drug.

TNBS also increased the activation of NF-jB, MAPKs

and AKT, as well as the expression of COX-2 and iNOS

(Fig. 3). Lactobacillus brevis G-101 blocked the induction

of p-IRAK-1, p-p65, p-p38, p-ERK, p-JNK and p-AKT by

TNBS. Lactobacillus brevis G-101 also inhibited TNBS-

induced iNOS and COX-2 expression. Lactobacillus brevis

G-101 (1 9 109 CFU) was comparable to that of mesal-

azine (10 mg kg�1). We also measured the levels of the

pro-inflammatory cytokines, namely TNF-a, IL-1b, IL-6and IL-10, in the colon of TNBS-induced colitic mice by

ELISA (Fig. 4). TNBS increased the protein expression of

IL-1b, IL-6 and TNF-a by 5�6-, 4�0- and 10�5-folds,respectively; however, it reduced IL-10 expression. Lacto-

bacillus brevis G-101 treatment increased anti-inflamma-

tory cytokine IL-10 expression and reduced the expression

of the pro-inflammatory cytokines, namely IL-1b, IL-6

p-IRAK1 p-Akt

Akt

p-ERK

ERK

p-JNK

JNK

p-p38

p38

IRAK1

p-p65

p65

COX-2

iNOS

TNBSLAB

5

#

##

# #

###*

*

** *

**

* * * ** * ** * * * ** *

4

3

2

1

0

Inte

nsity

(%

)

Inte

nsity

(%

)

0

2

4

6

––

++ G8 G9 M10

+ + + TNBSLAB

––

++ G8 G9 M10

+ + +

β-Actin β-Actin

(a) (b)


G-101 on NF-jB (A), MAPK and AKT

activation (B) in TNBS-induced colitic mice.

TNBS, except in the normal control group

(NOR, treated with vehicle alone), was

intrarectally administered in TNBS, G8, G9

and M10 groups. The test agents (TNBS,

vehicle alone; G8, 1 9 108 CFU per mouse of

Lact. brevis G-101 with TNBS; G9, 1 9 109

CFU per mouse of Lact. brevis G-101 with

TNBS; M10, 10 mg kg�1 mesalazine with

TNBS) were orally administered for 3 days

after TNBS treatment. The mice were

anesthetized and sacrificed 20 h after the

final administration of Lact. brevis G-100. The

amounts of proteins were measured by

immunoblotting. All values are the




with group treated with TNBS alone

(P < 0�05). ( ) p-IRAK/IRAK; ( ) p-p65/p65;

( ) COX-2/b-actin; ( ) iNOS/b-actin;

( ) p-Akt/Akt; ( ) p-ERK/ERK; ( ) p-JNK/JNK;

( ) p-p38/p38.



and TNF-a. Treatment with Lact. brevis G-101

(1 9 109 CFU per mice) inhibited the expression of these

cytokines by 89, 69 and 73%, respectively, but reversed

IL10 expression to 89% of the normal control group.

We measured the effect of Lact. brevis G-101 on the

expression of TNF-a, IL-1b, IL-10, ARG I, ARG II and

CD206, which are the markers of M1/M2 macrophages

(Ambarus et al. 2012), in TNBS-induced colitic mice by

real-time PCR (Fig. 5). Treatment with TNBS increased

the expression of ARG II, IL-1b and TNF-a, which are the

markers of M1 macrophages, but reduced the expression

of ARG I, CD206 and IL-10 expression. Treatment with

Lact. brevis G-101 in TNBS-treated mice blocked the

expression of the M1 macrophage markers, namely ARG 1,

TNF-a and IL-1b, but increased the expression of the M2

macrophage markers, namely ARG II, IL-10 and CD206.

50

#

#

##

*

** *

* *

*

** *

* *

200

150

100

50

0

40

30

20

10

0NOR

IL-1

0 (p

g m

g–1 )

IL-6

(pg

mg–

1 )

IL-1

β (p

g m

g–1 )

TN

F-α

(pg

mg–

1 )

TNBS G8 G9 M10

NOR TNBS G8 G9 M10

NOR TNBS G8 G9 M10

NOR TNBS G8 G9 M10

200 30

20

10

0

150

100

50

0

(a) (b)

(c) (d)

Figure 4 The effect of Lactobacillus brevis G-101 on the expression of anti-inflammatory cytokine IL-10 (a) and pro-inflammatory cytokines TNF-a

(b), IL-1b (c), and IL-6 (d) in TNBS-induced colitic mice. TNBS, except in the normal control group (NOR, treated with vehicle alone), was intra-

rectally administered in TNBS, G8, G9 and M10 groups. The test agents (TNBS, vehicle alone; G8, 1 9 108 CFU per mouse of Lact. brevis G-101

with TNBS; G9, 1 9 109 CFU per mouse of Lact. brevis G-101 with TNBS; M10, 10 mg kg�1 mesalazine with TNBS) were orally administered

for 3 days after TNBS treatment. The mice were anesthetized and sacrificed 20 h after the final administration of Lact. brevis G-101. Colonic cyto-

kine levels were measured by ELISA. All values are the mean � SD (n = 6). #Significantly different compared with normal control group (P < 0�05).* Significantly different compared with group treated with TNBS alone (P < 0�05).

0TN

F-α

(fo

ld c

hang

e)

IL-β

(fo

ld c

hang

e)

IL-1

0 (f

old

chan

ge)

AR

G II

(fo

ld c

hang

e)

CD

206

(fol

d ch

ange

)A

RG

I (f

old

chan

ge)

0

30

20

10

0

1

2

0

1

2

0

1

2

5

10

15

20#

#

#

#

#

#

*

*

**

*

**

*

*

**

**

**

*

0

5

10

15

20(a) (b)

(c) (d)

(e) (f)


G-101 on the expression of macrophage

polarization markers in TNBS-induced colitic

mice. TNBS, except in the normal control

group (NOR, treated with vehicle alone), was

intrarectally administered in TNBS, G8, G9

and M10 groups. The test agents (TNBS,

vehicle alone; G8, 1 9 108 CFU per mouse of

Lact. brevis G-101 with TNBS; G9, 1 9 109

CFU per mouse of Lact. brevis G-101 with

TNBS; M10, 10 mg kg�1 mesalazine with

TNBS) were orally administered for 3 days

after TNBS treatment. The mice were

anesthetized and sacrificed 20 h after the

final administration of Lact. brevis G-101.

Levels of colonic TNF-a (a), IL-1b (b), IL-10 (c),

ARG I (d), ARG2 (e) and CD206 (f) were

measured by real-time PCR. All values are the




with group treated with TNBS alone

(P < 0�05).



Discussion

IBD does not progress significantly in germ-free animals

(Chandran et al. 2003), indicating that intestinal microfl-

ora, which comprise approx. 1000 different bacterial spe-

cies (Benno et al. 1993; Chandran et al. 2003), may play

an important role in initiating and perpetuating colonic

inflammation. Of intestinal microbiota, gram-negative

bacteria, including Enterobacteriaceae, are significantly

increased in the colitic patients, as well as in mice treated

with colitic inducers such as TNBS and dextran sulfate

sodium. These gram-negative bacteria produce LPS, a

bacterial endotoxin. The LPS activate the biosynthesis of

diverse mediators of inflammation, such as TNF-a,IL-1b, and IL-6, via a Toll-like receptor (TLR) 4-linked

NF-jB, MAPK, and AKT pathways in macrophages

(Aderem and Ulevitch 2000). LPS reduces IL-10 expres-

sion. To improve IBD, inclusion of dietary ingredients

regulating LPS signalling, such as b-sitosterol and VSL#3,

has recently gathered attention (Sartor 2004; Lee et al.

2012). Of them, LAB suppress the growth of pathogens,

improve gut microbiota disturbance (Perdigon et al.

1991; Campieri and Gionchetti 1999), and improve IBD

(Campieri and Gionchetti 1999; Chung et al. 2007; Peran

et al. 2007a; Lee et al. 2009, 2010). These LAB strains

inhibit the expression of pro-inflammatory cytokines by

regulating NF-kB activation [Lee et al. 2009, 2010]. How-

ever, IL-10 expression-inducing LAB was not studied

thoroughly.

Therefore, in the present study, we screened for IL-10

expression-inducing LAB among the LAB strains isolated

from kimchi. Then, we selected Lact. brevis G-101 (heat-

treated), which significantly increased IL-10 expression in

LPS-stimulated macrophages. Lactobacillus brevis G-101

inhibited the expression of the pro-inflammatory cyto-

kines, namely, TNF-a, IL-1b, and IL-6, and the activation

of their transcription factors NF-jB and AP1. Lactobacil-

lus brevis G-101 ameliorated inflammatory markers

(shortening of the colon, increase of MPO activity and

pro-inflammatory cytokines, and activation of NF-jB) in

TNBS-induced colitic mice, as reported previously (Lee

et al. 2009, 2010). Furthermore, Lact. brevis G-101 inhib-

ited the phosphorylation of MAPKs, which is activated by

TAK1 in TLR4/NF-jB pathway, and AKT, which is phos-

phorylated by MyD88-PI3K signalling, in TNBS-induced

colitic mice. These results suggest that Lact. brevis G-101

may inhibit the inflammation by regulating the signalling

pathway of upstream molecule(s) of IRAK1 such as

TLR4. Additionally, G-101 polarized TNBS-induced M1

macrophages, which express TNF-a, IL-1b, and ARG I, to

M2 macrophages, which express IL-10, ARG II and

CD206. G-101 may be able to reverse a series of molecular,

cellular and immunological responses observed during

the inflammation process in vivo and then polarize LPS-

stimulated macrophages to M2 macrophages.

Conclusions

Based on these findings, Lact. brevis G-101, which

induces IL-10 expression in LPS-stimulated macrophages,

may be able to improve colitis by inhibiting TLR-4-linked

NF-jB, MAPK and AKT signalling pathways and polariz-

ing M1 macrophages to M2 macrophages,

Declaration of interest

The authors report no conflicts of interest.

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