Lipoteichoic acid fromLactobacillusplantarum elicits boththeproductionof Interleukin-23p19and suppressionofpathogen-mediated Interleukin-10 inTHP-1cellsHan Geun Kim1, Min Geun Gim1, Joo Yun Kim1, Hyun Jin Hwang2, Min Seok Ham2, Jung Min Lee1,Thomas Hartung3, Jung Woo Park4, Seung Hyun Han5 & Dae Kyun Chung1,2

1School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Suwon, Korea; 2RNA Inc., #308 College of Industry, Kyung

Hee University, Suwon, Korea; 3EU Joint Research Centre, European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy;4Department of Biological Sciences, University of Ulsan, Ulsan, Korea; and 5Humoral Immunology Section International Vaccine Institute,

SNU Research Park, Seoul, Korea

Correspondence: Dae Kyun Chung, School

of Biotechnology and Institute of Life Science

and Resources, Kyung Hee University, Suwon,

449-701, Korea. Tel.: 182 31 201 2465; fax:

182 31 202 8333;

e-mail: dkchung@khu.ac.kr

Received 26 January 2006; revised 08

September 2006; accepted 09 October 2006.

First published online 21 December 2006.

DOI:10.1111/j.1574-695X.2006.00175.x

Editor: Artur Ulmer

Keywords

Lactobacillus plantarum ; Lipoteichoic acid;

Interleukin-23; Interleukin-10.

Abstract

In this study, the stimulatory effects of different lactic acid bacteria strains, and

their subcellular fractions, on the THP-1 cell line were evaluated. Lactobacillus

plantarum was found in particular to induce high levels of IL-23p19 mRNA, but it

moderately induced TNF-a production. IL-10 production was not entirely affected

by L. plantarum stimulation. When subcellular fractions of L. plantarum were

used to treat THP-1 cells, IL-23p19 mRNA expression was enhanced in a dose-

responsive manner, specifically by lipoteichoic acid (LTA). The cotreatment of

THP-1 cells by both L. plantarum and Staphylococcus aureus LTA resulted in

decreased IL-10 production when compared with cells treated by S. aureus LTA

alone. Taken together, these data suggest that LTA isolated from L. plantarum

elicits stimulatory effects upon the expression of IL-23p19 and inhibitory effects

on pathogen-mediated IL-10 production.

Introduction

Lactic acid bacteria (LAB) are known for their health-

promoting effects such as the nonspecific enhancement of

the immune system, protection against intestinal infection,

the reduction of serum cholesterol level, and their antiox-

idant properties (Meydani & Ha, 2000; Von der Weid et al.,

2001; Matsuguchi et al., 2003). Several strains of LAB also

have effects upon the production of cytokines, such as IL-12,

IL-10, TNF-a, TGF-b, IL-8 and RANTES, and upon cell

proliferation in human intestinal epithelial cells (Hessle

et al., 1999; Kato & Yokokura, 1999; Wallace et al., 2003).

Interleukin (IL)-23 is a heterodimeric cytokine formed by

complexes of p19 (Oppmann et al., 2000) and a subunit of

IL-12, p40 (Trinchieri, 2003). IL-23 has biological functions

that are similar to IL-12, such as interferon-g production

from CD41 T cells, antitumor effects and antimetastatic

activity. Some previous reports also suggest that IL-23

induces a more robust and sustained cytotoxic T lympho-

cyte and Th1 immune response than IL-12 (Belladonna

et al., 2002; Lo et al., 2003; Trinchieri, 2003; Ha et al., 2004).

Furthermore, IL-23N220L, an N-glycosylation mutant of

this protein that shows reduced expression of excess p40 but

has no inhibitory effects upon IL-23 levels, has been

proposed as an effective adjuvant of a DNA vaccine for

antigen-specific T cell immunity (Ha et al., 2004). However,

our current understanding of the biology of IL-23 is still

considerably less than IL-12, which to-date has been studied

more extensively.

The human cytokine IL-10 is secreted from macrophages

by stimulation of several endogenous and exogenous factors

such as endotoxin, TNF-a, catecholamines and cAMP-ele-

vating drugs (Meisel et al., 1996; Platzer et al., 2000; Riese

et al., 2000; Asadullah et al., 2003). The principal immuno-

logical role of IL-10 is the regulation of the Th1/Th2 balance

and this molecule promotes the development of the Th2

cytokine pattern by inhibiting IFN-gamma production of T

lymphocytes, via the suppression of IL-12 synthesis in

accessory cells (Asadullah et al., 2003). Moreover, IL-10

represses both proinflamatory cytokine production and the

antigen-presenting capacity of macrophages and dendritic

cells (De Waal Malefyt et al., 1991). IL-10 overexpression is

FEMS Immunol Med Microbiol 49 (2007) 205–214 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

also considered to be responsible for immune deviation

towards Th2, which is a hallmark of atopic disorders such as

atopic dermatitis and allergic asthma (Asadullah et al., 2003).

The toll-like receptors (TLRs) are a family of vertebrate

receptors that are homologous to the drosophila toll recep-

tors (Dabbagh & Lewis, 2003). The associated signaling

pathways that are initialized by TLRs, from the recognition

of conserved products of microorganisms, cause maturation

of antigen-presenting cells (APC), which then generate pro-

inflammatory cytokines such as IL-12, IL-23 and IL-27, and

promote Th1 cell proliferation (Dabbagh & Lewis, 2003;

Trinchieri, 2003; Ha et al., 2004).

In our current study, we examined the effects of LTA,

which is one of subfractions from Lactobacillus plantarum,

on the production of IL-23p19 and IL-10 mRNAs. Our

results indicate that L. plantarum LTA has different induc-

tion and suppression effects upon a number of cytokine

productions when compared with LTA isolated from the

pathogenic bacterium Staphylococcus aureus.

Materials and methods

Cell line and reagents

The human monocyte-like cell line THP-1 was obtained

from the Korean Cell Line Bank (KCLB, Korea) and main-

tained in RPMI 1640 with 10% FBS (JBI, Korea) at 37 1C in

5% CO2. The THP-1 cells were placed in 12-well plates, and

they were stimulated with heatkilled bacteria or subfractions

from L. plantarum for 4 h or 24 h. Mouse monoclonal anti-

human TLR2 (TL2.1; IgG2a) and mouse monoclonal anti-

human TLR4 (HTA 125; IgG2a) antibodies were purchased

from Santa Cruz Biotechnology Inc., and mouse monoclo-

nal anti-human CD14 (clone #134620; IgG1) was purchased

from R&D Systems. Anti-human TLR1 (GD2.F4; IgG1), and

all isotype-matched control antibodies (IgG1 and IgG2a)

were purchased from eBioscience.

Bacterial strains

The bacterial strains used for the stimulation of THP-1 cells

were Lactobacillus bulgaricus (KCTC 3188), Lactobacillus

casei ssp. casei (KCTC 3109), Lactococcus lactis ssp. cremoris

(ML 4), Enterococcus faecalis (KCTC 3195), Lactobacillus

helveticus (KCTC 3545), Leuconostoc mesenteroides (KCTC

3100), Staphylococcus aureus (KCTC 1621), L. plantarum

(KCTC 10887BP) and Escherichia coli DH5a. L. bulgaricus,

L. casei, L. helveticus, Leu. mesenteroides and L. plantarum

were cultured with MRS broth at 37 1C for 18 h, and

Leuconostoc cremoris and Enterococcus faecalis were cultured

with M17 broth containing 5% glucose at 30 1C for 18 h.

Staphylococcus aureus was amplified with BHI broth

containing 0.5% glucose and 0.5% cystein at 37 1C for 24 h,

and Escherichia coli DH5a was cultured with LB broth

at 37 1C for 18 h.

Preparation of subcellular fractions

Bacterial subcellular fractions were prepared by the method

of Roman Dziarski et al. with minor modifications (Ro-

senthal & Dziarski, 1994). Briefly, bacteria were harvested in

exponential growth phase at an OD (600 nm) of 0.6 and

quickly chilled in an ice-ethanol bath until the temperature

dropped to 4 1C. Pellets were suspended in saline, and the

suspension was boiled at 100 1C for 20 min. Heatkilled

bacterial cells were suspended with ice cold water and mixed

with 0.1 mm glass beads until total disruption of cells was

achieved. Unbroken cells were sedimented by centrifugation

(1500 g, 4 1C) for 10 min, and then the supernatant was

prepared as crude extract. To collect cell walls from the

supernatant, the crude extract was centrifuged for 30 min at

6500 g at 4 1C followed by washing with water three times.

The precipitated cell walls were incubated with RNase

(100 mg mL�1) and DNase (50mg mL�1) for 18 h, and then

trypsin (200mg mL�1) was added and incubated for another

18 h at 37 1C. The cell walls were sedimented by centrifuga-

tion at 6500 g and 4 1C for 30 min followed by washing with

water four times, and the purified cell walls were lyophilized.

One gram of cell walls was incubated with 5% trichloroace-

tic acid (TCA) for 18 h at 22 1C, and sedimented by

centrifugation at 6500 g for 30 min. Teichoic acid (TA) was

recovered from the supernatants by TCA extraction with

ethyl ether. At the same time, the sediment from TCA

extraction was washed three times with water and three

times with acetone, and dried to make peptidoglycan

powder. Genomic DNA was isolated from L. plantarum with

a genomic DNA prep kit (Promega corporation), and then

purified by phenol extraction. Briefly, prepared genomic

DNA was incubated with 100mg mL�1 RNase for 1 h at 37 1C

water bath, and then 100mg mL�1 of proteinase K was added

and incubated for another 1 h in 50 1C water bath. DNA

samples were extracted with equal volumes of (25 : 24 : 1)

phenol:chloroform:isoamyl alcohol. Extracted DNA was

then precipitated with 0.75 volume of room-temperature

isopropanol for 10 min followed by washing with 70%

ethanol. After resuspension of the pellet with distilled

endotoxin-free water, the removal of contamination was

confirmed by agarose gel electrophoresis. Highly purified

LTAs from L. plantarum (pLTA) and S. aureus (aLTA) were

prepared by butanol extraction followed by purification

using octyl-sepharose and DEAE–sepharose chromatogra-

phy as described previously (Morath et al., 2001).

RNA preparation and real-time PCR

Total RNA was extracted from THP-1 cells with a guanidium

thiocyanate-acid phenol-chloroform method. cDNA was

FEMS Immunol Med Microbiol 49 (2007) 205–214c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

206 H.G. Kim et al.

synthesized with Improm-IITM reverse transcription system

(Promega corporation) according to the manufacturer’s

instructions. To quantify the cytokine mRNA, real-time

PCR amplification was carried out using the ABI prism

7000 sequence detection system (Applied BioSystems, USA),

and the PCR products were detected using SYBR Green. The

primers used for cytokine detection were as follows: IL-23

p19 forward, 50-GCTGAACAGAGAGAATCAGG-30; IL-

23p19 reverse, 50-CTTTTTGCCT-TCTTCTCAAA-30; IL-10

forward, 50-ATGTTCTTTGGGGAGCCAA-30; IL-10 reverse,

50-GTAGCAGTTAGGAAGCCCAA-30; TNF-a forward, 50-

CTCTTCTGGCTCAAAAAGA-GAATT-30; TNF-a reverse,

50-AGGCCCCAGTTTGAATTCTT-30. Cytokine expressions

were normalized using glyceraldehydes-3-phospate dehy-

drogenase (GAPDH).

Establishment of dominant-negative MyD88transfectants and Transient transfection

Dominant negative (dn) MyD88 was manufactured by the

method of Masuo Hosokawa et al. with minor modifications

(Wang et al., 2002). cDNA for dnMyD88 (amino acids

156–296) that contains toll/interleukin-1 receptor domain

was amplified from mRNA obtained from THP-1 cells by

reverse transcription polymerase chain reaction (RT-PCR)

and cloned into pCMV-Tag 2A (Stratagene, USA). THP-1

cells were transfected with the expression vector with

WelFec-QTM transfection reagent (JBI, Korea) according to

the manufacturer’s instructions. At 36 h after the transfec-

tion, cells were used for further analysis. PCR primers used

for RT-PCR were as follows: dnMyD88 forward, 50-GCTAG-

CATGCCTGAGCGTTTCG-30; dnMyD88 reverse, 50-

GGATCCTCAGGGCAGGGACAA-30.

ELISA assay

After stimulation, cell supernatants were removed and

assayed for cytokine production by standard sandwich

ELISA. The TNF-a ELISA was performed using monoclonal

mouse IgG1, clone #28401 for capture and the biotinylated

human TNF-a specific polyclonal goat IgG followed by

streptavidin HRP for detection. ELISA was developed using

o-phenylenediamine as a substrate, and OD was determined

at a wavelength of 450 nm using a 550-nm reference

wavelength. All values were interpolated from either a log-

log or a four-parameter fit of a curve generated from

appropriate standards.

Statistical analysis

All experiments were performed at least three times. The

data shown are representative results as the means� the SD

of triplicate experiments. An unpaired t test was used to

determine the significance between two groups of the data.

Results

The preparations of major subcellular fractions

When studies are performed with gram-positive bacterial

subcellular fractions, it is important to exclude any potential

contaminants, especially endotoxin. This is because human

monocytes are highly responsive to endotoxin. Therefore, we

removed all endotoxin contaminants from prepared L. plan-

tarum subcellular fractions. The LTA preparation was purified

with octyl-sepharose followed by DEAE-sepharose chromato-

graphy, and then a Limulus Amebocyte Lysate (LAL) test and

silver staining were performed to confirm the absence of

endotoxin contamination (Fig. 1a). Prepared peptidoglycan

was repurified with polymyxin B-agarose, and then THP-1

cells were treated with repurified peptidoglycan in the pre-

sence or absence of 500mg mL�1 polymyxin B for 4 h followed

by examination of TNF-a production (Fig. 1b). The genomic

DNA prepared from L. plantarum was treated with phenol

extraction following RNase treatment. Purity of DNA fraction

was confirmed by agarose gel electrophoresis (Fig. 1c), and the

examination of TNF-a production after the stimulation of

THP-1 cells in the presence or absence of polymyxin B (Fig.

1b). The purity of all subcellular fractions from L. plantarum

was tested with a LAL test kit (Cambrex Bio Science, Walkers-

ville). Major fractions such as LTA, peptidoglycan, and DNA

showed high purity, as we expected (Table 1). Especially, LTA

from L. plantarum contains 0.0285 endotoxin unit per mL

(EU mL�1), which means no endotoxin contamination in the

sample. LTA from S. aureus used in this experiment also

showed high purity.

Analysis of cytokine production using variousbacterial strains in THP-1 cells

Nine different heat-killed bacterial strains were examined

for their ability to induce cytokine production in THP-1

cells (Fig. 2). Lactobacillus casei and L. plantarum signifi-

cantly induced IL-23p19 mRNA, whereas Enterococcus fae-

calis, Escherichia coli DH5a and S. aureus induced high level

of TNF-a transcription. Some LAB, such as L. bulgaricus, L.

helveticus and Leu. mesenteroides, as well as Escherichia coli

DH5a, induced IL-10 production in THP-1 cells. In parti-

cular, L. plantarum was a highly potent inducer of IL-23p19

mRNA, but it did not induce IL-10 production in THP-1

cells. Hence, we selected L. plantarum and examined its

ability to induce cytokine production in more detail.

LTA isolated from L. plantarum shows the highestIL-23p19 mRNA induction activity in THP-1 cells

IL-23 is a heterodimeric cytokine composed of p19 and p40,

one of a subunit of IL-12. To examine the inducing activity

of those subunits, we quantified the mRNA levels of the

FEMS Immunol Med Microbiol 49 (2007) 205–214 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

207Lactobacillus plantarum LTA on cytokine production

IL-12 and IL-23 subunits in THP-1 cells exposed to LTA

isolated from either L. plantarum (pLTA) or S. aureus

(aLTA). Transcript levels of the p35, p40 and p19 subunits

were then measured by real-time PCR. Each of these subunit

mRNA levels were found to be higher in pLTA treated cells

than in aLTA treated cells, and IL-23p19 mRNA production

was observed to be the most highly inducible species when

compared with the other subunits (Fig. 3a). Therefore, we

chose a p19 subunit for the following experiment as well as

TNF-a and IL-10.

In order to determine the components of L. plantarum

that had cytokine-inducing activities, THP-1 cells were

stimulated with subcellular fractions of L. plantarum. Lacto-

bacillus plantarum extracts were separated into six fractions:

crude extract, cell wall, peptidoglycan , lipoteichoic acid

(LTA), teichoic acid (TA) and DNA. Each of these fractions

was then added to the THP-1 culture medium at a concen-

tration of 10 mg mL�1 and the cytokine mRNA levels were

measured by real time PCR after 24 h of incubation.

Although the cell wall, LTA and TA fractions were each

found to significantly increase IL-23p19 mRNA production,

the LTA fraction showed the highest IL-23p19-inducing

activity (Fig. 3b). In contrast, peptidoglycan and DNA

showed only slight induction of IL-23p19 mRNA. IL-10

mRNA was found to be remarkably induced by crude extract

and DNA. From our six L. plantarum subfractions, all except

DNA and peptidoglycan significantly increased TNF-a pro-

duction in THP-1 cells. DNA and peptidoglycan were again

found to be inconsiderable inducers, in this case of TNF-aproduction.

pLTA, highly purified from L. plantarum inducedIL-23p19 and TNF-a production, but not IL-10production

To examine the effects of pLTA on cytokine production in

more detail, THP-1 cells were stimulated with pLTA as

a dose- or time-dependent manner. aLTA isolated from

S. aureus was used as a control to compare the differences

between pLTA and aLTA for cytokine production. Treatment

of THP-1 cells with pLTA resulted in a dose-dependent

induction in IL-23p19 transcription. In contrast, expression

of IL-10 was almost entirely unaffected by pLTA treatment

(Fig. 4a). Compared with pLTA treatment, aLTA displayed a

1 2 3

Lane 1: High molecular weightmarker (Lambda DNA-HindIIIdigested molecular marker).

Lane 2: DNA preparation is both degraded as well as contaminated with RNA.

Lane 3: High molecular weightgenomic NDA freeof RNAcontamination after RNasetreating followed by phenolextraction.

BSA ng well−1

2 20 200 2000Size

marker pLTA aLTA pTA pDNA

untreatment Polymyxin B 0.5 mg/ml

8

7

6

5

4

3

2

1

0LTA TA DNA PGN

TN

F-α

ng/m

l

TN

F-α

ng/m

l

200

150

50

0

100

LPS

(a)

(b)

(c)

Fig. 1. The confirmation of the purity of the major subcellular fractions.

(a) 20mg of pLTA, aLTA, pTA and pDNA were subjected to 12% sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed

by silver staining. Bovine serum albumin (BSA) was used as a protein

standard. (b) THP-1 cells were incubated with 10 mg mL�1 of LTA, TA,

DNA. Peptidoglycan, and 0.5mg mL�1 lipopolysaccharide in the presence

or absence of polymyxin B (0.5 mg mL�1) for 4 h, and the TNF-a produc-

tion was examined by ELISA. (c) Lactobacillus plantarum genomic DNA

was repurified by phenol extraction to remove RNA or protein contam-

ination, and separated by agarose gel electrophoresis.

Table 1. LAL test to confirm the contamination of endotoxin

Subfractions pPGN pCX pCW pTA pDNA pLTA aLTA

OD 405 nm 0.0173 0.272 0.2375 0.2667 0.0249 0.0205 0.027

EU mL�1 0.0216 0.567 0.4932 0.1852 0.0379 0.0285 0.042

LAL, Limulus Amebocyte Lysate; pPGN, peptidoglycan isolated from Lactobacillus plantarum; pCX, crude extract fraction of L. plantarum; pCW, cell wall

fraction of L. plantarum; pTA, teichoic acid isolated from L. plantarum; pDNA, genomic DNA fraction of L. plantarum; pLTA, lipoteichoic acid isolated

from L. plantarum; aLTA, lipoteichoic acid isolated from S. aureus.

FEMS Immunol Med Microbiol 49 (2007) 205–214c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

208 H.G. Kim et al.

step-wise dose-dependent increase in IL-10 production and

induced the highest levels of IL-23p19 at 0.2mg mL�1, and

then declined (Fig. 4b). aLTA significantly induced TNF-a

production by as much as eight-fold more than pLTA

treatment (Fig. 4c). The differences in cytokine produc-

tion between pLTA and aLTA were made apparent by the

0

10

20

30

Un-treated

Lb.B

ulgaricus

Lb. casei

L. cremoris

E. F

aecalis

Lb. helveticus

Leu.m

esenteroide

Lb. plantarum

S. aureus

E. coliD

H5a

rel.

mR

NA

IL-23p19 IL-10 TNF-α

****

*** *

Fig. 2. Cytokine production from THP-1 cells induced by various bacteria strains. THP-1 cells were incubated with 1� 108 cells of bacteria mL�1 of 10

different strains. The cytokine mRNA from the total RNA was measured by real-time PCR after 24 h of incubation. The experiment was repeated three

times with similar results, and the averages and the SD values are shown. �and ��, Po 0.05, and Po 0.01, respectively.

0

50

100

150

200

250

untreated

Crude

extract

Cell w

all

PG

N

LTA

TA

DN

Are

l. m

RN

A

0

50

100

150

200

250

rel.

mR

NA

IL- 23p 19 IL- 10 TNF-α

*

*

*

**

** *

*****

IL- 12p 35 IL- 12p 40 IL- 23p19

un-treated pLTA aLTA

*

*

(b)

(a)

Fig. 3. Cytokine production from THP-1 cells by Lactobacillus plantarum subcellular fractions. (a) THP-1 cells (1� 106 cells well�1) were stimulated with

10 1C of pLTA or aLTA per mL, and 1�108 cells of live L. plantarum. At 4 h after stimulation, total RNA was extracted from culture cells. The amount of

subunit level was measured by real-time PCR. (b) THP-1 cells (1� 106 cells well�1) were stimulated with subcellular fractions isolated from L. plantarum.

Each bacterial fraction was added to the culture medium at 10mg mL�1, and the cytokine production from the total RNA was measured by real-time PCR

after 24 h of incubation. The experiment was repeated three times with similar results, and the averages and the SD values are shown. �, ��, and ���,

Po 0.05, Po 0.01, and Po 0.002, respectively.

FEMS Immunol Med Microbiol 49 (2007) 205–214 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

209Lactobacillus plantarum LTA on cytokine production

time-course treatment of LTAs. IL-23p19 production was

more responsive to pLTA than to aLTA (Fig. 5a), whereas

IL-10 and TNF-a production were more responsive to aLTA

than to pLTA (Fig. 5b and c). Interestingly, pLTA did not

affect to IL-10 production. These results suggest that pLTA

may induce a Th1-type response.

pLTA inhibited aLTA-induced IL-10 production,but showed a synergistic effect on IL-23p19

To examine the effects of pLTA on the aLTA-mediated

cytokine production, THP-1 cells were stimulated with

pLTA and/or aLTA, and then real-time PCR or ELISA was

performed in order to determine cytokine production.

Compared with 10 mg mL�1 of pLTA or aLTA treatment

alone, IL-23p19 production was induced after cotreatment

with 10 mg mL�1 aLTA and increased dose of pLTA (Fig. 6a).

The cotreatment of 10 mg mL�1 pLTA and increased dose of

aLTA did not affect TNF-a production compared with aLTA

treatment alone (Fig. 6b), but IL-10 production was down-

regulated by pLTA-aLTA cotreatment. In particular, the

pretreatment of 10 mg mL�1 pLTA for 24 h followed by aLTA

restimulation at the indicated concentrations significantly

down-regulated IL-10 production (Po 0.05) compared

with aLTA treatment alone (Fig. 6c). These results suggested

that highly purified LTA from LAB might have the ability to

induce Th1 type response by inhibition of pathogen-

induced Th2-type response associated with atopic disorder.

Regulation of cytokine production in THP-1 cellsstimulated by bacterial cells and LTA from eitherL. plantarum or S. aureus

To examine whether bacterial cells can stimulate the expres-

sion of TLRs, we stimulated THP-1 cells with various

0

50

100

150

200

250

0 0.2 2 5 10

0 0.2 2 5 10

Lb. plantarum LTA µg mL−1

LTA µg mL−1

IL- 23p19 IL- 10

0

20

40

60

80

100

S. aureus

rel.

mR

NA

/GA

PD

Hre

l. m

RN

A/G

AP

DH

IL- 23p19 IL- 10

0

50

100

150

200

250

0 µgmL−10.2 µgmL−12 µgmL−1 5 µgmL−110 µgmL−1

TN

F-α

ng

mL−1

pLTA aLTA

(a)

(b)

(c)

Fig. 4. Cytokine production from THP-1 cells stimulated with pLTA or

aLTA. THP-1 cells were stimulated with pLTA (a) or aLTA (b) as indicated

concentration for 4 h. IL-23p19 and IL-10 mRNA production was

determined by real-time PCR from total RNA. (c) TNF-a production was

measured by ELISA from THP-1 cells stimulated with pLTA or aLTA as

indicated concentration for 4 h. The experiment was repeated three

times with similar results, and the averages and the SD values are shown.

0

50

100

150

200

250

0 h 1 h 4 h 6 h 12 h 24 h

0 h 1 h 4 h 6 h 12 h 24 h

0 h 1 h 4 h 6 h 12 h 24 h

IL-2

3p19

rel

. mR

NA

/GA

PD

H

pLTA 10 aLTA 10

0

50

100

150

200

250

TN

F-

α ng

mL−1

pLTA 10 aLTA 10

0

100

200

300

400

500

600

700

IL-1

0 p

g m

L−1

pLTA 10 aLTA 10

(a)

(b)

(c)

Fig. 5. Time course of cytokine induction by pLTA or aLTA treatment. (a)

THP-1 cells were stimulated with 10 mg mL�1 pLTA or aLTA for indicated

time points. The amount of IL-23p19 mRNA was determined by real-time

PCR from total RNA. TNF-a (b) and IL-10 (c) production from THP-1 cells

stimulated with 10 mg mL�1 of pLTA or aLTA for indicated time points was

examined by ELISA. Data (mean � SD) of three independent experi-

ments are presented.

FEMS Immunol Med Microbiol 49 (2007) 205–214c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

210 H.G. Kim et al.

bacteria and measured the mRNA levels of TLR1 through

TLR10. All bacterial strains used in this experiment induced

TLR2 expression (Fig. 7a); however, there were slight

differences between strains (Fig. 7b). To examine whether

TLRs mediate LAB-induced cytokine production, we per-

formed neutralization experiments with anti-TLRs or anti-

CD14 antibodies. THP-1 cells were incubated with anti-

TLR1, anti-TLR2, anti-TLR4 or anti-CD14 for 30 min

before stimulation of heat-killed L. plantarum for 24 h.

Results demonstrated that preincubation with anti-TLR1,

anti-TLR2 and anti-CD14 antibodies inhibited production

of cytokines, including IL-23p19, TNF-a and IL-10 (Fig. 7c),

indicating that L. plantarum induced cytokine production

through TLR1-TLR2 signaling including CD14.

To investigate if pLTA and aLTA have different require-

ments for TLR stimulation, we examined the need for TLR1,

TLR2, TLR4 and CD14. Treatment with antibodies to TLR1,

TLR2, or CD14 reduced the TNF-a production by THP-1

cells in response to pLTA (Fig. 8a). Similarly, antibodies to

TLR1, TLR2 or CD14 significantly reduced the aLTA-induced

TNF-a production (Fig. 8b). Anti-TLR2 and anti-CD14

inhibited production of IL-23p19 in response to pTLA

treatment, and IL-10 production, was also inhibited by anti-

TLR2 or anti-CD14 in THP-1 cells stimulated by aLTA (Fig.

8c). To show the involvement of the TLR2 signaling pathway

in more detail, dnMyD88-expressing vector was transiently

transfected into THP-1 cells. At 36 h following transfection,

the expression of dnMyD88 was confirmed by Western

blotting, and these cells showed no increase in either IL-

23p19 or IL-10 production in response to pLTA or aLTA,

respectively (Fig. 8d). These results suggested that both types

of LTA share several additional receptor molecules required

for the stimulation of THP-1 cells.

Discussion

LTA is a cell wall component of gram-positive bacteria. LTA

is considered to be analogous to the lipopolysaccharide of

gram-negative bacteria, and shares many of its biochemical

and physiological properties (Ginsburg, 2002). (Morath

et al.(2001) has reported that the pro-inflammatory prop-

erty of LTA is dependent upon its D-alanine content . LTA

from L. plantarum Dlt-mutant containing 1.1% D-Ala sub-

stitutions showed an increased ability to stimulate IL-10 and

a decreased ability to induce IL-12 in comparison to wild-

type LTA (41% of D-Ala substitution). This resulted in the

induction of Th2 type response (Grangette et al., 2005).

These results emphasize the importance of LTA composition

in the proinflammatory or anti-inflammatory properties of

gram-positive bacteria. As shown in Fig. 3a, the peptidogly-

can fraction of L. plantarum only moderately increased the

cytokine production in THP-1 cells, which is consistent with

0

50

100

150

200

250

0 µg mL 0.01 0.1µg mL 1µg mL 10 µg mL

TN

F-a

ng

mL

aLTA onlypLTA 10µg mL +aLTA cotreatmentpLTA10 µg mL (-20 h pretreatment->aLTA 6 h stimulation

0

50

100

150

200

250

300

350

400

0.01 0.1µg mL 1µg mL 10 µg mL

IL-1

0 pg

mL

aLTA only

pLTA10+aLTA cotreatmentpLTA10(-24 h prestimulation)->aLTA 6 h stimulation

*

0 µg mL

µg mL

µg mL

(b)

(a)

(c)

0

400

800

1200

1600

un

-tre

ate

d

aL

TA

10

aL

TA

10

+p

LT

A0

.2

aL

TA

10

+p

LT

A2

aL

TA

10

+p

LT

A5

aL

TA

10

+p

LT

A1

0

aL

TA

10

+p

LT

A2

0

pL

TA

10

rel.

mR

NA

/GA

PD

H IL- 23p19 pf

pf

pfpf

Fig. 6. Synergistic or inhibitory effect of cytokine production in THP-1

cells by pLTA and aLTA cotreatment. THP-1 cells were costimulated with

10 mg mL�1 aLTA and 0.2–20 mg mL�1 pLTA for 4 h, and the amount of IL-

23p19 mRNA was examined by real-time PCR from total RNA The

statistical significances were represented by the comparison between

pLTA treatment only and cotreatment of pLTA and aLTA (a). THP-1 cells

were preincubated with 10mg mL�1 pLTA or medium for 24 h, and

restimulated with 0.01–10 mg mL�1 aLTA for 4 h. THP-1 cells were also

costimulated with 10 mg mL�1 pLTA and 0.01–10 mg mL�1 aLTA for 4 h.

TNF-a (b) and IL-10 (c) production was determined from culture super-

natants by ELISA. The experiment was repeated three times, and data are

represented as means� SDs. �, Po 0.05.

FEMS Immunol Med Microbiol 49 (2007) 205–214 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

211Lactobacillus plantarum LTA on cytokine production

another recent report showing that peptidoglycan is recog-

nized by a nucleotide-binding oligomerization domain

(NOD) but not TLR2 (Travassos et al., 2004). Peptidoglycan

was recognized by the LRR region of NOD, and the Rip2

adaptor molecule is associated with the CARD domain of

NOD, resulting in the activation of NF-kB (Chin et al., 2002;

Kobayashi et al., 2002; Inohara & Nunez, 2003). Bacterial

unmethylated DNA (CpG ODN) binds to TLR9 and initi-

ates signal transduction cascades in a MyD88–dependent

manner. Recently, CpG ODN was used as vaccine adjuvant

(Ulevitch, 2004). Our experiments using subcellular frac-

tions, including LTA, peptidoglycan, DNA from L. plantar-

um, demonstrate that IL-23p19 and TNF-a were

significantly induced by LTA. Although all subcellular frac-

tions were able induced cytokine production, IL-10 produc-

tion was entirely unaffected by LTA (Fig. 3b). These results

suggest that pLTA has a potent Th1-inducing activity.

IL-23 is a heterodimeric cytokine composed of p19 and

p40, one of subunits of IL-12 (Trinchieri, 2003). Many

reports have characterized the role of the IL-12 subunits,

p35 and p40, but very few studies have investigated p19.

Recent studies have shown that IL-23 and IL-12 have similar

biological functions (Ha et al., 2004), and our experiments

showed that THP-1 cells stimulated with whole cell bacteria

or LTAs induced higher levels of IL-23p19 than other

subunits such as IL-12p35 and IL-12p40 (Fig. 3a). Hence,

0

50

100

150

200

250

Un-

trea

ted

S. c

rem

oris

E. F

aeca

lis

Leu.

m

esen

tero

ide

Lb. p

lant

arum

S. a

ureu

s

TLR

2 m

RN

A/G

AP

DH

0

5

10

15

20

25

30

35

rel.m

RN

A

IL-23p19 TNF-α IL-10

L. plantarum − + + + + + + +

IgG1 Antibody − − αTLR1 αTLR2 αTLR4 αCD14 IgG2a

**

*****

**

*****

***

**

**

GAPDH

TLR 1

TLR 2

TLR 3

TLR 4

TLR 5

TLR 6

TLR 7

TLR 8

TLR 9

TLR 10

(a)

(c)

(b)

Fig. 7. THP-1 cells stimulated with bacteria-induced cytokine production through the TLR2 signaling. THP-1 cells were stimulated with

1� 108 cells mL�1 of bacteria for 24 h, and then cDNAs were synthesized with extracted total RNA. The amount of TLR expression was examined with

reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for TLRs (a) and quantitative real-time PCR (b). The primers used in real-

time PCR are: TLR2 forward, 50 ACC CTA GGG GAA ACA TCT CT 30; TLR2 reverse, 50 AGC TCT GTA GAT CTG AAG CAT C 30. (c) THP-1 cells were

pretreated with 10mg mL�1 of anti-TLR1, anti-TLR2, anti-TLR4 and anti-CD14 antibodies for 30 min before 1� 108 cells mL�1 of Lactobacillus plantarum

treatment for 24 h. The level of IL-23p19, TNF-a and IL-10 was determined by real-time PCR. Data are expressed as means� SDs. �� and ���, Po 0.01

and Po 0.001, respectively. Statistical significance between groups was evaluated by ANOVA.

FEMS Immunol Med Microbiol 49 (2007) 205–214c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

212 H.G. Kim et al.

we chose p19 as one of the target cytokines in the present

study in addition to TNF-a, a Th1-inducing proinflamma-

tory cytokine. In addition, we considered the production of

IL-10 because it is a known factor associated with atopic

disorder and an inhibitor for Th1 type response (Asadullah

et al., 2003). In contrast, TNF-a induces Th1 responses, but

the overproduction of TNF-a can induce systemic autoim-

mune disease. Although an appropriate production of IL-10

and TNF-a is essential to the activation of the immune

system, excessive production of these cytokines cause severe

immune diseases. Therefore, the immune regulation with

LAB or their cell wall components is considerable because

they induce moderate cytokine production and inhibit

pathogen-induced cytokine overproduction.

In general, cytokine expression is dependent upon TLR

signaling pathways (Schroder et al., 2003). However, the

mechanism of the association of IL-23p19 production with

TLRs has not previously been reported. In our present study,

LTA from L. plantarum (pLTA) induced less cytokine

production than LTA from S. aureus (aLTA). Compared

with pLTA-induced cytokine production, aLTA-induced IL-

10 and TNF-a production were elevated as much as two-fold

and 9.7-fold, respectively (Figs 4 and 5). Like aLTA, the

signaling transduction following pLTA treatment required

TLR1, TLR2, CD14, and MyD88 recruitment (Fig. 8). These

results suggest that the diversity of cytokine production was

not occurred in signaling pathway. These differences may be

due to structure differences between both LTAs, but we

believe a detailed experiment is necessary to verify this

hypothesis.

In conclusion, L. plantarum seems to be involved in IL-23

mRNA production in THP-1 cells. In particular, pLTA

isolated from L. plantarum was a potent inducer of IL-

23p19 mRNA. In addition, the upregulation of IL-10

production by aLTA is decreased by cotreatment with pLTA.

Both pLTA-mediated IL-23p19 mRNA production and

aLTA-mediated IL-10 production are dependent on TLR1-

TLR2-CD14 and MyD88. Our current findings may indicate

that highly purified LTA from L. plantarum is a good

candidate for therapeutic use as it may reduce IL-10-

mediated atopic dermatitis and may also induce proinflam-

matory cytokines such as IL-23 and TNF-a through the

TLR2 signaling pathway.

(a)

(b)

(c)

(d)

Fig. 8. pLTA- and aLTA-induced cytokine production through TLR1, TLR2

and CD14 dependent signaling. THP-1 cells were treated with anti-TLR1,

anti-TLR2, anti-TLR4, anti-TLR6 or anti-CD14 for 30 min before stimula-

tion with 10mg mL�1 pLTA (a) and 5 mg mL�1 aLTA (b) for 4 h. TNF-aproduction was examined by ELISA. (c) THP-1 cells were pretreated with

anti-TLR2 or anti-CD14 for 30 min, and then stimulated with 10mg mL�1

pLTA or aLTA for 4 h. (d) THP-1 cells were transiently transfected with

dominant negative (dn) MyD88, and stimulated with 10 mg mL�1 pLTA or

aLTA for 4 h. IL-23p19 and IL-10 mRNA production were determined by

real-time PCR from total RNA. Data are expressed as the mean � SDs,

and are representative of three separate experiments. �� and ���,

Po 0.01 and Po 0.001, respectively. Statistical significance between

groups was evaluated by ANOVA.

FEMS Immunol Med Microbiol 49 (2007) 205–214 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

213Lactobacillus plantarum LTA on cytokine production

Acknowledgements

This work was supported by the Korea Research Foundation

Grant funded by the Korean Government (MOEHRD)

(KRF-2004-041-F00076), Republic of Korea.

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