83. Reilly N et al Am J Physiol Regul Integr Comp Physiol 2007 E-pub

Reilly et al - 1

PROBIOTICS POTENTIATE IL-6 PRODUCTION IN IL-1β-TREATED CACO-2

CELLS THROUGH A HEAT SHOCK-DEPENDENT MECHANISM

Natasha Reilly1, Vitaliy Poylin1, Michael Menconi1, Andrew Onderdonk2,

Stig Bengmark3, and Per-Olof Hasselgren1

Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School,

Boston, MA1; Department of Pathology, Brigham Women’s Hospital, Harvard Medical

School, Boston, MA2; Liver Institute, University College, London University, United

Kingdom3

Running title: Probiotics and enterocyte IL-6 production

Supported in part by NIH grant R01 DK60546 (POH)

Address for correspondence: Per-Olof Hasselgren, M.D., Department of Surgery, Beth

Israel Deaconess Medical Center, 330 Brookline Avenue ST919, Boston, MA 02215; Tel

(617) 667-1810; Fax (617) 667-1819; e-mail [email protected]

of 42Articles in PresS. Am J Physiol Regul Integr Comp Physiol (July 18, 2007). doi:10.1152/ajpregu.00770.2006

Copyright © 2007 by the American Physiological Society.

mailto:[email protected]

Reilly et al - 2

ABSTRACT

Interleukin-6 (IL-6) may exert anti-inflammatory and protective effects in intestinal

mucosa and enterocytes. The influence of probiotics on mucosal and enterocyte IL-6

production is not known. We tested the hypothesis that the probiotic bacteria

Lactobacillus paracasei and Lactobacillus plantarum regulate IL-6 production in

intestinal epithelial cells. Cultured Caco-2 cells were treated with 1 ng/ml of IL-1β in the

absence or presence of different concentrations of Lactobacillus paracasei or

Lactobacillus plantarum followed by measurement of IL-6 production. The role of heat

shock response was examined by determining the expression of hsp70 and hsp27, by

down-regulating their expression with siRNA, or by treating cells with quercetin.

Treatment of the Caco-2 cells with IL-1β resulted in increased IL-6 production,

confirming previous reports from this laboratory. Probiotics alone did not influence IL-6

production but addition of probitoics to IL-1β-treated cells resulted in a substantial

augmentation of IL-6 production. Treatment of the Caco-2 cells with live Lactobacillus

paracasei increased cellular levels of hsp70 and hsp27 and the potentiating effect on IL-6

production was inhibited by quercetin and by hsp70 or hsp27 siRNA. Results suggest that

probiotics may enhance IL-6 production in enterocytes subjected to an inflammatory

stimulus and that this effect may, at least in part, be heat shock-dependent.

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INTRODUCTION

The intestinal mucosa is an active participant in the inflammatory response to injury,

sepsis, and endotoxemia and becomes the site of cytokine production during these

conditions (10,21,35). In previous studies, we have been particularly interested in

mucosal production of IL-6 and have found evidence that mucosal IL-6 levels are

increased during endotoxemia and sepsis in mice (23,48). Although multiple cell types

may contribute to increased IL-6 production in the mucosa during inflammation,

experiments in IL-1β-treated cultured intestinal epithelial cells (22,30) suggest that the

enterocyte is an important source of IL-6 during inflammation.

IL-6 is a pleiotropic cytokine that can have both pro- (28,32,46) and anti-

inflammatory properties (51). Previous studies provided evidence that IL-6 may exert

protective effects in various tissues during inflammation caused by injury and sepsis

(2,40). In recent experiments in our laboratory, treatment of cultured enterocytes with IL-

6 prevented cell death caused by hyperthermia (11), further supporting the concept that

IL-6 can have beneficial and protective effects. Interestingly, in previous experiments we

found that induction of the heat shock (stress) response potentiated the effect of IL-1β on

IL-6 production in cultured enterocytes (29,34). In other studies we found that induction

of the stress response in mice resulted in potentiated IL-6 production in intestinal mucosa

during sepsis and endotoxemia (47). Because these effects of the stress response were

associated with improved intestinal integrity (45), it is possible that increased mucosal

IL-6 levels, at least in part, are responsible for the beneficial effects of the heat shock

response during sepsis and endotoxemia.

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Multiple studies, both in humans and experimental animals, suggest that so called

probiotics exert protective effects in intestinal mucosa during various inflammatory

conditions (3,4,8,9,13-15). In addition, recent studies suggest that probiotics may reduce

the incidence of postoperative complications, in particular infectious complications, in

patients undergoing major surgical procedures (36). It should be noticed that although

multiple experimental studies support the concept that probiotics may be beneficial in the

treatment of patients with inflammatory diseases of the gut, additional controlled studies

are needed to more definitively identify the role of probiotics in the management of these

patients. Probiotics are living bacteria with low or no pathogenicity that exert beneficial

effects on the health of the host (13,15,38). Different mechanisms are probably involved

in the beneficial effects of probiotics, including inhibited mucosal and enterocyte

production of pro-inflammatory cytokines (18,19), inhibited activation of NF-kB (26,31),

and stimulated production of the antimicrobial peptide human beta defensin-2 in

intestinal epithelial cells (49). The influence of probiotics on enterocyte IL-6 production

is not known but considering our previous observation that IL-6 exerts protective effects

in the mucosa and enterocyte (11,45,47), the present experiments were performed to test

the hypothesis that probiotics may stimulate IL-6 production in intestinal epithelial cells.

Because, in a recent study, treatment of cultured enterocytes with probiotics resulted in

heat shock response (31), an effect that may be caused by a soluble factor released from

probiotics (43), we also examined the potential role of heat shock proteins for IL-6

production in cultured enterocytes treated with probiotics. Results suggest that probiotics

can potentiate the effect of IL-1β on IL-6 production in cultured enterocytes and that this

response, at least in part, is mediated by the heat shock response.

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MATERIAL AND METHODS

Cell cultures

Caco-2 cells, a human colon adenocarcinoma cell line that displays enterocyte-like

features in culture (33), were obtained from American Type Culture Collection

(Rockville, MD). Cells were grown at 37°C in 5% CO2 in Dulbecco’s modified Eagel’s

medium (DMEM; Gibco-BRC, Grand Island, NY) supplemented with 10% fetal bovine

serum, nonessential amino acids, 6 mM glutamine, 10 mM HEPES, 10 µg/ml

apotransferrin, 1 mM pyruvate, 24 mM NaHCO3, 100 U/ml penicillin, and 100 µg/ml

streptomycin. Cells, between passages 5 and 25, were seeded at a density of 100,000

cells/cm2 onto 6-well tissue culture plates. Cells were grown for 72 h to approximately

80-90% confluence before use. Cells were treated with 1 ng/ml of human recombinant

IL-1β (Biosource International, Camarillo, CA) or 1 ng/ml of human recombinant TNFα

(Biosource International) for 20 h and IL-6 and IL-8 production was determined by

measuring cytokine levels in the medium as described below. Untreated Caco-2 cells

served as controls. In order to examine the influence of probiotics on cytokine

production, live or heat-inactivated Lactobacillus paracasei subsp. paracasei F19 (L.

paracasei, Belgian Coordinated Collection of Microorganisms, BCCM, deposition

number LMG P-17806) or L. plantarum 2362 (BCCM deposition number LMG P-20606)

(both strains obtained from Medipharm Inc., Kagerod, Sweden and Des Moines, Iowa,

USA) were added to the culture medium at concentrations described below. In some

experiments, quercetin (Sigma, St Louis, MO) was added to the culture medium at a

concentration of 100 µM. Although most experiments were performed in 80-90%

confluent Caco-2 cells, in a control experiment we used Caco-2 cells that had been

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cultured for 3 weeks in transwell bicameral chambers to induce full differentiation as

described in detail previously (24).

Cell transfections

In order to examine the role of hsp70 and hsp27, the expression of these genes was

silenced by transfecting cells with appropriate siRNA’s. Caco-2 cells were transfected

with pre-designed siRNA against hsp70 (sc-29352) or hsp27 (sc-29350). Other cells were

transfected with non-specific (scrambled) siRNA. All siRNA duplexes and reagents were

from Santa Cruz Biotechnology (Santa Cruz, CA) and were used according the

manufacturer’s protocol.

Preparation of bacteria

The probiotic bacteria L. paracasei and L. plantarum were cultured in De Man-

Rogosa-Sharpe (MRS) broth under anaerobic conditions at 37°C. In other experiments, L.

jensenii (Gram-positive bacteria that are part of the normal vaginal flora) were cultured

under identical conditions and were used instead of L. paracasei and L. plantarum. After

culture, bacteria were collected by centrifugation (8,000 x g for 3 min), washed in

phosphate buffered saline (PBS), pH 7.4, and resuspended in DMEM cell culture

medium. Bacteria were added to the cultured Caco-2 cells at concentrations (determined

as colony forming units, cfu) described in Results either alone or in comination with IL-

1β. In some experiments, bacteria were heat-inactivated in a water bath at 80°C for 10

min or sonicated with four 20 sec bursts before they were added to the Caco-2 cells. The

heat-inactivation was confirmed by culture to make certain the preparations were sterile.

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In other experiments, the medium from cultured L. paracasei was passed through a 0.22-

µm filter (Millipore, Bedford, MA) and aliquots of the filtered medium were added to the

Caco-2 cells.

When the effects of Eschericia coli (E. coli) bacteria were tested, DH5α E. coli were

first cultured in Trypticase soy broth at 37°C, washed, and grown again at 37°C under

unaerobic conditions in MRS broth. The bacteria were then washed, resuspended in

DMEM cell culture medium and added to the Caco-2 cells.

Determination of IL-6 and IL-8

IL-6 and IL-8 levels were determined in cell culture medium by commercially

available ELISA kits (Endogen, Woburn, MA). The limit of detection as described by the

manufacturer was 1 pg/ml for both assays.

Western blotting

Western blotting was performed to determine cellular levels of hsp70 and hsp27.

After incubation, cells were lysed in 300 µl of lysis buffer (0.5 M Tris HCl, pH 7.4, 1.5

M NaCl, 10% NP-40, 10 mM EDTA, and 2.5% deoxycholic acid). Cell debris were

removed by centrifugation at 3,800 x g for 7 min. The supernatant (whole cell lysate) was

stored at -80°C until further analysis.

For Western blot analysis, aliquots of the cell lysates containing 50 µg of protein, as

determined by using the Bicinchoninic Acid (BCA) Protein Assay (Pierce, Rockford, IL),

were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% sodium

dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol) for 7 min and then separated

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by electrophoresis on a 10% Tris-glycine gradient gel (Novex, San Diego, CA). A protein

ladder (See-Blue Standard, Novex) was included as a molecular weight marker. The

proteins were transferred to nitrocellulose membranes (Xcell II Blot Module, Novex).

The membranes were blocked with 5% nonfat dried milk in Tris-buffered saline, pH 7.6,

containing 1% Tween-20 for 60 min followed by incubation with a 1:2000 dilution of

mouse anti-human hsp70 (Santa Cruz Biotechnology) or a 1:2000 dilution of rabbit anti-

human hsp27 antibody as primary antibodies. A goat anti-mouse IgG (Promega,

Madison, WI) or a goat anti-rabbit IgG (Santa Cruz Biotechnology) secondary antibody

was used at a dilution of 1:5000. The blots were then incubated in enhanced

chemiluminescence reagent (ECL, Amersham Life Sciences, Buckingham, UK) and

exposed on radiographic film (X-Omot AR, Eastman-Kodak, Rochester, NY).

Determination of mRNA levels

For determination of IL-6 mRNA levels, RNA was extracted and real-time PCR was

performed as described in detail previously (50).

Statistics

Experiments were performed at least three times to ensure reproducibility of results.

The results are reported as means ± SEM. Analysis of variance followed by Tukey’s test

was used for statistical comparisons. p<0.05 was considered statistically significant.

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RESULTS

In the present study, we examined the effects of two intestinal Lactobacillus strains

because recent reports suggest that several species of Lactobacillus are probiotics that

have protective immunomodulating properties (1,14,27), are able to prevent adhesion of

pathogenic bacteria to the intestinal wall (44), and can preserve mucosal integritiy (17).

In initial experiments, we determined IL-6 production in cultured Caco-2 cells treated

with IL-1β, live L. paracasei or live L. plantarum, either alone or in combinations.

Treatment of the Caco-2 cells with IL-1β (1.0 ng/ml) for 20 h resulted in increased IL-6

production (Fig 1A), confirming previous reports from our laboratory (29,30,34). In

separate experiments we found that cellular levels of IL-6 remained constant during

incubation for 20 h in the absence or presence of IL-1β (data not shown), supporting the

interpretation that the increased levels of IL-6 in the culture medium of IL-1β-treated

Caco-2 cells reflected increased production of IL-6 rather than increased transport of

cellular IL-6 into the medium. This was also supported by previous experiments in our

laboratory in which treatment of cultured Caco-2 cells with IL-1β resulted in increased

IL-6 mRNA levels in addition to increased amounts of the cytokine in the incubation

medium (29,30,34). Treatment of the Caco-2 cells with L. paracasei (108 bacteria/ml) or

L. plantarum (108 bacteria/ml) alone did not influence IL-6 production. In contrast, when

the bacteria were added to IL-1β-treated cells, the IL-6 production was substantially

increased, consistent with a synergistic (potentiating) effect of the probiotics (Fig 1A).

The potentiating effect of L. paracasei was approximately 6-fold (compared with the IL-

6 production in cells treated with IL-1β alone) and of L. plantarum approximately 2-fold.

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Because the effect on IL-6 production was most pronounced for L. paracasei, this

Lactobacillus strain was used in subsequent experiments.

Because previous reports suggest that some (but not all) effects of probiotics can be

exerted by both live and heat-inactivated bacteria (18,49), we next compared the effects

of live and heat-inactivated L. paracasei on IL-6 production in Caco-2 cells. As shown in

Fig 1B, the potentiating effect on IL-6 production in IL-1β-treated Caco-2 cells was

similar after treatment with live and heat-inactivated L. paracasei.

In order to test whether the potentiating effect of L. paracasei on IL-6 production

was caused by a product secreted from the bacteria, we next treated Caco-2 cells with

medium from cultured L. paracasei. Result from that experiment showed that medium

from cultured L. paracasei did not exert a potentiating effect on IL-6 production in IL-

1β-treated Caco-2 cells (Fig 2A). Taken together with the results in Fig 1, these

observations suggest that a factor present in live and heat-inactivated L. paracasei

potentiates IL-6 production in IL-1β-treated Caco-2 cells. In order to determine whether

this factor requires intact bacterial cell walls to be effective, we next compared the effect

of live intact L. paracasei with that caused by sonicated L. paracasei. Similar to intact L.

paracasei, sonicated L. paracasei potentiated the effect of IL-1β and the effect of

sonicated bacteria was of the same magnitude as that of intact bacteria (Fig 2B). Notably,

in this experiment, IL-6 production in Caco-2 cells treated with L. paracasei alone (third

bar in Fig 2B) was higher than in the preceding experiments (compare with Fig 1A and

B). This result probably reflected a day-to-day variability with regards to absolute IL-6

levels and illustrates the importance of including all study groups simultaneously when

experiments are performed. Importantly, although the basal levels of IL-6 were somewhat

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different, the potentiating effect of L. paracasei on IL-6 production was seen also in this

experiment.

In order to further characterize the influence of L. paracasei on IL-6 production in

the Caco-2 cells, we examined the effects of different concentrations of the bacteria

added to the enterocytes as well as the time course for the potentiating effects. When

different concentrations (107 – 109 bacteria/ml) of L. paracasei were added to IL-1β-

treated Caco-2 cells, the maximum effect on IL-6 production was noticed for 108

bacteria/ml (Fig 3A). None of the concentrations tested here influenced IL-6 production

when added alone to the Caco-2 cells. The potentiating effect of L. paracasei on IL-6

production in IL-1β-treated Caco-2 cells was noticed already after 3 h, at which time

point the IL-6 production was increased approximately 2-fold, and increased

progressively to be approximately 4-fold after 20 h (Fig 3B).

In order to test whether the potentiating effects of L. paracasei on IL-6 production in

IL-1β-treated Caco-2 cells noticed here are specific for probiotics, we next treated Caco-

2 cells with L. jensenii. These bacteria are Gram-positive (similar to L. paracasei and L.

plantarum) but non-probiotic bacteria that are commonly found in the normal vaginal

flora. When cultured Caco-2 cells were treated with L. jensenii (108 bacteria/ml), the IL-

1β-induced IL-6 production was potentiated to a similar degree as was noticed after

treatment of the Caco-2 cells with L. paracasei or L. plantarum. In this experiment, IL-6

production was 3.0 ± 0.8 pg/ml in IL-1β treated Caco-2 cells and 14.9 ± 1.2 pg/ml in

Caco-2 cells treated with IL-1β + L. jensenii. Treatment of the Caco-2 cells with L.

jensenii alone did not result in IL-6 production. Thus, not only probiotic Gram-positive

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bacteria but other Gram-positive bacteria as well may potentiate IL-6 production in IL-

1β-treated cultured enterocytes.

We next examined whether gram-negative bacteria can also potentiate the effects of

IL-1β on IL-6 production in cultured Caco-2 cells. This was done by treating cultured

Caco-2 cells with the E. coli strain DH5α. Although IL-6 production was somewhat

higher in Caco-2 cells treated with both E. coli and IL-1β than in Caco-2 cells treated

with IL-1β alone, this difference was much smaller than the approximately 6-fold

potentiating effect noticed in L. paracasei-treated cells in the same experiment (Fig 4A).

We next determined whether L. paracasei can influence IL-6 production in Caco-2 cells

stimulated with a cytokine other than IL-1β. Treatment of the Caco-2 cells with TNFα

did not result in measurable IL-6 production and this unresponsiveness to TNFα was not

influenced by L. paracasei (Fig 4B).

In order to test whether L. paracasei can potentiate the production of cytokines other

than IL-6, we examined IL-8 production in cultured Caco-2 cells. Similar to previous

reports (39), treatment of the Caco-2 cells with IL-1β resulted in a robust increase in IL-8

production (Fig 4C). L. paracasei alone or in combination with IL-1β did not influence

IL-8 production in the cultured Caco-2 cells.

Most of the experiments in the present study were performed in 80-90% confluent

Caco-2 cells, similar to previous reports from our laboratory in which the regulation of

IL-1β-induced IL-6 production was examined (29,30). In another study from our

laboratory, this model was validated by showing that IL-1β increased IL-6 production in

fully differentiated Caco-2 cells grown on filters in transwell bicameral chambers (24).

Here, we performed a control experiment using the same cell culture system as described

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in detail previously (24) to test whether L. paracasei can potentiate IL-1β-induced IL-6

production in fully differentiated Caco-2 cells. This also allowed us to treat the Caco-2

cells with IL-1β from the basal chamber and with L. paracasei in the apical chamber,

mimicking the situation in vivo when enterocytes are exposed to bacteria from the

intestinal lumen and to circulating cytokines from the blood stream. When this

experimental approach was used, IL-6 production (determined in the basal chamber) in

IL-1β-treated Caco-2 cells was potentiated by 57% by the addition of L. paracasei (108

bacteria/ml) to the apical chamber (IL-6 levels being non-detectable in control cells, 11.5

pg/ml after treatment with IL-1β alone for 20 h and 18.1 pg/ml after treatment with IL-1β

and L. paracasei; means from two individual experiments). In a separate experiment, L.

paracasei alone did not influence IL-6 production in fully differentiated Caco-2 cells.

Although the effect of L. paracasei on IL-6 production in IL-1β-treated fully

differentiated Caco-2 cells noticed here was less pronounced than in 80-90% confluent

Caco-2 cells, the results suggest that L. paracasei can influence the response to IL-1β

with regards to IL-6 production in fully differentiated Caco-2 cells as well.

The next set of experiments was designed to elucidate the mechanism of the

potentiating effect of L. paracasei on enterocyte IL-6 production. Because Petrof et al

(31) reported recently that treatment of cultured mouse colonocytes with probiotics

induced increased expression of the heat shock proteins hsp25 and hsp72 (corresponding

to human hsp27 and 70, respectively) and because we found previously that induction of

the stress response by hyperthermia or treatment with proteasome inhibitors potentiated

the IL-6 production in IL-1β-treated Caco-2 cells (29,34), we examined the role of the

heat shock response in the potentiated IL-6 production noticed here after treatment with

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L. paracasei. First, we tested whether treatment of the Caco-2 cells with L. paracasei

induced a heat shock response. As seen in Fig 5A and B, treatment of the Caco-2 cells

with live L. paracasei resulted in increased levels of both hsp70 and hsp25 with the most

pronounced effect seen for hsp70. Treatment of the cells with IL-1β did not result in

changes in the heat shock protein concentrations and the combined treatement with IL-1β

and L. paracasei did not influence the changes induced by L. paracasei alone.

If the heat shock response induced by L. paracasei participates in the regulation of

IL-6 production in IL-1β-treated Caco-2 cells, inhibition of the heat shock response

should prevent the potentiation of IL-6 production. When cells were treated with

quercetin, a substance known to inhibit the induction of the heat shock response (25), the

potentiating effect of L. paracasei on IL-6 production was abolished (Fig 5C). Although

this result lends strong support to the notion that the effect of live L. paracasei observed

in the present study is mediated by the heat shock response, one potential pitfall of the

experiment using quercetin is that the drug may have non-specific effects other than

inhibition of the heat shock response. In addition, it is not known from this experiment

which specific heat shock protein that is involved in the effect of L. paracasei.

To address these questions, we next silenced the hsp70 and hsp27 genes by

transfecting cells with siRNA’s against hsp70 and hsp25 mRNA. Treatment of Caco-2

cells with hsp70 siRNA resulted in a substantial reduction of hsp70 levels and prevented

the increase in hsp70 levels induced by live L.paracasei (Fig 6A). Measurement of IL-6

production in the same cells showed that the potentiating effect of L. paracasei was

substantially reduced after treatment with siRNA (from an approximately 3-fold increase

in IL-6 production to an approximately 50% increase over the IL-6 production caused by

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IL-1β alone) (Fig 6B). When the corresponding experiments were performed in Caco-2

cells treated with siRNA against hsp27 mRNA, the L. paracasei-induced increase in

hsp27 levels was blocked (Fig 7A) and the potentiating effect of L.paracasei on IL-6

production was abolished (Fig 7B). Note that in this experiment, IL-6 production in

untreated control cells was higher than in most of the other experiments in this study.

Although we do not have a definitive explanation for this finding, it is consistent with a

day-to-day variation of basal IL-6 production commonly seen in the current experimental

model. This is why it is important to include all treatment groups and control group

simultaneously in each expeiment as was done in the present study. Taken together, the

results in Fig 6 and 7 suggest that both hsp70 and hsp27 participate in the potentiating

effect of L.paracasei on IL-6 production in IL-1β-treated Caco-2 cells.

In order to further assess the role of the heat shock response in the potentiating effect

of L. paracasei on IL-6 production, two additional experiments were performed. Because

we found in initial experiments, that both heat-inactivated and sonicated L. paracasei

potentiated IL-6 production in IL-1β-treated Caco-2 cells (see Fig 1B and 2B), we tested

whether heat-inactivated and sonicated L. paracasei induced a heat shock response

(similar to live L. paracasei). Surprisingly, when cultured Caco-2 cells were treated for

20 h with heat-inactivated or sonicated L. paracasei under identical experimental

conditions as used for the experiments shown in Fig 1B and 2B, hsp27 and hsp70 levels

as determined by Western blotting followed by densitometry were not increased (data not

shown). In the same experiment, treatment of the Caco-2 cells with live L. paracasei

resulted in a 48% and 44% increase in hsp27 and hsp70 levels, respectively, confirming

the results shown in Fig 5A and 5B. These observations suggest that additional

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mechanisms (in addition to induction of the heat shock response) may be involved in the

potentiating effects of L. paracasei on IL-6 production in IL-1β-treated Caco-2 cells, at

least in the potentiating effects caused by heat-inactivated and sonicated L. paracasei.

In a second experiment designed to further assess the role of the heat shock response

in the potentiating effect of L. paracasei on IL-6 production, we compared the early time

courses for L. paracasei-induced heat shock response and L. paracasei-induced

potentiation of IL-6 expression in IL-1β-treated Caco-2 cells. In this experiment, IL-6

mRNA levels were determined by real-time PCR because we hypothesized that IL-6

mRNA levels would be influenced by IL-1β and L. paracasei at early time points.

Treatment of the Caco-2 cells with IL-1β alone resulted in an early increase in IL-6

mRNA levels noticed already after 1 h and present up to 3 h after addition of IL-1β (Fig

8). In additional experiments, IL-6 mRNA levels had returned to basal levels after 20 h

(data not shown), a time point at which IL-6 protein levels were increased. These

observations are in line with previous reports from our laboratory showing an early

upregulation (within 1 h) of IL-6 mRNA levels in IL-1β-treated Caco-2 cells (29,30) and

support the concept that IL-1β-induced IL-6 production is regulated at the transcriptional

level. Importantly, in the present experiments, addition of live L. paracasei to the Caco-2

cells resulted in a potentiation of the IL-1β-induced IL-6 mRNA levels with an almost 5-

fold potentiation noticed after 1 h and an approximately 10-fold potentiation noticed after

3 h (Fig 8). Western blotting followed by densitometry showed a 23%, 51%, and 20%

increase in hsp27 levels in L. paracasei-treated Caco-2 cells at 1 h, 2 h, and 3 h,

respectively (means from 2 experiments). The corresponding figures for hsp70 levels

were 20%, 57%, and 33%. Thus, treatment of Caco-2 cells with live L. paracasei resulted

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in an early heat shock response that was present when an early potentiation of IL-6

mRNA expression was noticed. Although not conclusive, the results are supportive of a

role of the heat shock response in the potentiating effects of live L. paracsei on IL-6

production in IL-1β-treated Caco-2 cells.

It should be noted that hsp27 and hsp70 were upregulated earlier (1-3 h) in the

current study than in the study by Petrof et al (31) where hsp25 and hsp72 were increased

12 h after treatment of cultured young adult mouse colon (YAMC) cells with the

probiotic formulation VSL#3. The reasons for the different time course of heat shock

protein induction between our study and that of Petrof et al (31) are not known but may

be differences in cell types being used or different probiotics used for treatment of the

cells.

DISCUSSION

In the present study, the probiotic bacterium L. paracasei potentiated the effects of

IL-1β on IL-6 production in cultured Caco-2 cells. In addition, treatment of the Caco-2

cells with live L. paracasei increased the expression of hsp70 and hsp27 and silencing of

these genes with siRNA or treatment of the cells with quercetin prevented the effects of

live L. paracasei suggesting that the potentiating effect of L. paracasei on IL-6

production was, at least in part, regulated by the heat shock response. Because heat-

inactivated and sonicated L. paracasei also potentiated the IL-6 production in IL-1β-

treated Caco-2 cells, without inducing a heat shock response, multiple mechanisms in

addition to the heat shock response are probably involved in the potentiating effect of

probiotics on enterocyte IL-6 production.

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It should be noted that most experiments in the present study were performed in 80-

90% confluent Caco-2 cells rather than in fully differentiated enterocytes. This

experimental design was chosen to make comparisons possible with multiple previous

studies in which we examined IL-6 production in approximately 80-90% confluent Caco-

2 cells, including studies in which the potentiating effects of the heat shock response on

IL-6 production were determined (29,30,34). Importantly, we and others found recently

that although the absolute amounts of IL-6 produced from the apical and basolateral

membranes in fully differentiated cultured intestinal epithelial cells may be different,

treatment of the cells with IL-1β and other cytokines induced qualitatively similar

responses at the apical and basolateral membranes (24,41). In a control experiment

performed in the current study, L. paracasei potentiated the IL-1β-induced IL-6

production in fully differentiated Caco-2 cells cultured in a transwell bicameral system

(although to a lesser degree than noticed in 80-90% Caco-2 cells), further validating the

use of 80-90% confluent Caco-2 cells for the study of mechanisms involved in the

regulation of IL-6 production by probiotics.

Although beneficial effects of probitotics have been reported in several recent

clinical studies in the treatment of various inflammatory bowel diseases (3,8,9,13-15) and

as preoperative treatment of patients undergoing major surgical procedures (4,36),

additional well-controlled clinical studies are needed to further define the role of

probiotics in the management of inflammatory bowel disease. Probiotics probably exert

beneficial effects through multiple mechanisms. In general, probiotics may provide

benefits by bacterial interference with intestinal pathogens (5,37) and by direct

interaction with cells in the intestinal mucosa (18,19,26,31,49). In previous studies,

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Reilly et al - 19

treatment of cultured enterocytes with probiotics resulted in inhibited activity of the

transcription factor NF-kB (26,31), upregulated expression of the antimicrobial peptide

human beta defensin-2 (49), improved epithelial barrier function (19), prevention of

cytokine-induced apoptosis (52) and reduced production of the pro-inflammatory

cytokine IL-8 (39). The apparently contradictory results in the study by Ma et al (18), i.e.,

reduced IL-8 production in stimulated enterocytes treated with probiotics, and the results

in the present report of unchanged IL-8 production in L. paracasei-treated Caco-2 cells,

may reflect different stimuli used to induce IL-8 production [TNFα in the study by Ma et

al (18) and IL-1β in the present study], different types of probiotics [L. reuteri in the

study by Ma et al (18) versus L. paracasei in the present study] and different enterocyte

cell lines [T84 and HT-29 cells in the study by Ma et al (18) and Caco-2 cells in the

present report].

An additional potential mechanism by which probiotics may provide mucosal

protection, perhaps secondary to induction of the heat shock response, is increased

production of the anti-inflammatory cytokine IL-10 (45). Interestingly, recent studies

suggest that local delivery in the gut of IL-10 by genetically manipulated Lactococcus

lactis may be beneficial in the treatment of patients with Crohn’s disease (6,42).

In the present study, we found a novel mechanism by which probiotics may exert

beneficial effects, i.e., potentiated IL-6 production by enterocytes subjected to an

inflammatory stimulus. This observation is important because there are multiple lines of

evidence suggetsing that IL-6 has mainly anti-inflammatory and protective effects in the

intestinal mucosa. For example, in previous studies we found that heat shock-induced

potentiation of mucosal IL-6 production in endotoxemic mice prevented mucosal injury

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Reilly et al - 20

(45,47). In other studies, IL-6 exerted cell protective effects in cultured enterocytes by

inducing thermotolerance (11). Studies from other laboratories as well support the

concept that IL-6 may act as an anti-inflammatory cytokine controlling both local and

systemic inflammatory responses (2,40,51).

Interestingly, in the present study, L. paracasei and L. plantarum did not influence

IL-6 production in the cultured Caco-2 cells under basal conditions but only in cells that

were treated with IL-1β. This observation supports clinical observations that probiotics

provide beneficial effects in the setting of intestinal inflammation (3,4,8,9,13-15). A lack

of effect of probiotics on basal cytokine production in enterocytes has been reported by

others as well (18,19).

In the present study, both live and heat-inactivated L. paracasei enhanced IL-6

production in IL-1β-stimulated enterocytes. In contrast, treatment of the cells with culture

medium from the probiotics did not influence IL-6 production. These observations

suggest that the effects of L. paracasei noticed in the present study were caused by a

factor present in the bacteria, possibly in the bacterial wall, rather than by a secreted

product. In previous reports, the effects of probiotics in cultured intestinal epithelial cells

were induced by live (18) or inactivated bacteria (49) or by secreted product(s) present in

the culture medium of the probiotics (31,43). These apparently conflicting results may

reflect differences in probiotic bacteria as well as different intestinal epithelial cell lines

being used in previous studies. It is also possible that different effects of probiotics are

caused by different mechanisms. Regardless, the observations suggest that probiotics can

exert their biological effects by various cell-associated and secreted factors.

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Reilly et al - 21

Induction of the heat shock response by probiotics, as observed in the present study,

is similar to a recent report by Petrof et al (31) in which treatment of cultured mouse

colonic epithelial cells with the probiotics VSL #3 resulted in increased expression of

hsp72 and 25. In that study (31), evidence was found that inhibition of the chymotrypsin-

like activity of the proteasome may be a mechanism by which probiotics induce the heat

shock response. Although we did not examine the effect of L. paracasei on Caco-2 cell

proteasome activity in the current report, in recent experiments we found that treatment

of cultured Caco-2 cells with proteasome inhibitors resulted in a heat shock response (34)

similar to the effects of L. paracasei noticed here. Induction of the heat shock response

by proteasome inhibition was reported in a number of other cell types as well

(7,16,20,53).

Our results from experiments in which treatment of the Caco-2 cells with quercetin

or hsp70 or hsp27 siRNAs blocked the L. paracasei-induced potentiation of IL-6

production strongly suggest that the heat shock response was involved in the regulation

of IL-6 production. The observation that the heat shock response regulates IL-6

production in stimulated enterocytes is in line with previous reports from this laboratory

in which the heat shock response was induced by other mechanisms. Thus, in previous

experiments we found that when the heat shock response was induced in cultured Caco-2

cells by hyperthermia, the IL-1β-induced IL-6 production as well as the expression of IL-

6 mRNA were upregulated (29). Further support for a role of the heat shock response in

enterocyte IL-6 production was found in subsequent experiments in which treatment of

cultured Caco-2 cells with proteasome inhibitors induced a heat shock response and

potentiated IL-6 production (34). In additional experiments, induction of the heat shock

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Reilly et al - 22

response in vivo by hyperthermia or treatment with sodium arsenite augmented mucosal

IL-6 production in endotoxemic mice (47). Similar to the results in the present study, heat

shock by itself did not influence enterocyte or mucosal IL-6 production in our previous

studies but augmented IL-6 production that was already increased by endotoxemia in

mice (47) or treatment of Caco-2 cells with IL-1β (29,34). Although the mechanisms by

which the heat shock response potentiates IL-6 production in stimulated enterocytes are

not fully known at present, we recently found evidence that upregulated expression and

activity of the transcription factor C/EBPβ may play an important role (12).

In conclusion, the present study provides the first evidence that the probiotic

bacterium L. paracasei may enhance IL-6 production in enterocytes subjected to an

inflammatory stimulus and that the effect of live L. paracasei is, at least in part, heat

shock-dependent. Because other studies have shown that IL-6 has anti-inflammatory and

protective effects in the intestinal mucosa, the present results offer a novel mechanism by

which probiotics may exert some of their beneficial effects although additional

experiments will be needed to define the role of IL-6 in cell protective effects provided

by probiotics.

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Reilly et al - 23

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LEGENDS TO FIGURES

Fig 1 The effects of IL-1β, L. paracasei and L. plantarum on IL-6 production in cultured

Caco-2 cells. (A) Cultured Caco-2 cells were treated for 20 h with 1 ng/ml of IL-1β,

108/ml of L. paracasei or L. plantarum alone or in combinations as indicated in the figure

followed by measurement of IL-6 levels in the culture medium. (B) Cultured Caco-2 cells

were treated for 20 h with 1 ng/ml of IL-1β, live or heat-inactivated L. paracasei (108

bacteria/ml) alone or in combinations as indicated in the figure followed by measurement

of IL-6 levels in the culture medium. Results are means ± SEM with n=3 in each group.

*p<0.05 vs control (untreated cells); +p<0.05 vs cells treated with IL-1β alone by

ANOVA. Identical experiments were performed three times with almost identical results.

Fig 2 The effects of IL-1β, medium from cultured L. paracasei, and sonicated L.

paracasei on IL-6 production in cultured Caco-2 cells. (A) Cultured Caco-2 cells were

treated for 20 h with 1 ng/ml of IL-1β, 108/ml of L. paracasei or medium from cultured

L. paracasei followed by measurement of IL-6 levels in the culture medium. (B) Cultured

Caco-2 cells were treated for 20 h with IL-1β, intact or sonicated L. paracasei alone or in

combinations as indicated in the figure and at concentrations identical to those used in

Fig 2A, followed by measurement of IL-6 in the culture medium. Results are means ±

SEM with n=3 in each group. *p<0.05 vs control; +p<0.05 vs IL-1β alone by ANOVA.

Identical experiments were performed three times with almost identical results.

Fig 3 (A) The effect of different concentrations of L. paracasei on IL-6 production in

Caco-2 cells cultured in the absence of presence of IL-1β. Cultured Caco-2 cells were

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Reilly et al - 32

treated for 20 h with different concentrations of L. paracasei alone or together with 1

ng/ml of IL-1β followed by measurement of IL-6 in the culture medium. (B) Time-course

for the potentiating effect of L. paracasei on IL-6 production in IL-1β-treated Caco-2

cells. Cells were treated with 1 ng/ml of IL-1β or IL-1β + 108/ml of L. paracasei for

various periods of time up to 20 h followed by measurement of IL-6 levels in the culture

medium. Results are means ± SEM with n=3 in each group. Identical experiments were

performed three times with almost identical results.

Fig 4 (A) The effects of L. paracasei and E. coli on IL-6 production in IL-1β-treated

Caco-2 cells. Cells were treated for 20 h with 1 ng/ml of IL-1β, 108/ml of L. paracasei, or

108/ml of DH5α E. coli, either alone or in combinations as indicated in the figure,

followed by measurement of IL-6 in the culture medium. (B) The effects of L. paracasei

on IL-6 production in IL-1β- and TNFα-treated Caco-2 cells. Cultured Caco-2 cells were

treated for 20 h with 1 ng/ml of IL-1β or 1 ng/ml of TNFα alone or in combination with

108/ml of L. paracasei. (C) The effect of IL-1β and L. paracasei on IL-8 production in

cultured Caco-2 cells. Cells were treated for 20 h with 1 ng/ml of IL-1β or 108/ml of L.

paracasei alone or in combination as indicated in the figure followed by measurement of

IL-8 levels in the culture medium. Results are means ± SEM with n=3 in each group.

*p<0.05 vs control; +p<0.05 vs IL-1β alone by ANOVA. Identical experiments were


Fig 5 The influence of IL-1β and L. paracasei on cellular levels of (A) hsp70 and (B)

hsp27. Cultured Caco-2 cells were treated for 20 h with 1 ng/ml of IL-1β or 108/ml of L.

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paracasei alone or in combination as indicated in the figure. Hsp70 and hsp27 levels

were determined by Western blotting. β-Actin levels were determined as loading

controls. The experiment was repeated three times with almost identical results. (B) The

effect of quercetin on L. paracasei-induced potentiation of IL-6 production in IL-1β-

treated Caco-2 cells. Cells were treated for 20 h with 1 ng/ml of IL-1β or 108/ml L.

paracasei in the absence or presence of 100 µM quercetin as indicated in the figure.

Results are means ± SEM with n=3 in each group. *p<0.05 vs corresponding control

group; +p<0.05 vs corresponding IL-1β group by ANOVA. Identical experiments were


Fig 6 The influence of reduced hsp70 levels on the L. paracasei-induced potentiation of

IL-6 production in IL-1β-treated Caco-2 cells. (A) Hsp70 levels in cultured Caco-2 cells

transfected with hsp70 siRNA or control (non-specific) RNA as described in Materials

and Methods and subsequently treated with IL-1β or L. paracasei alone or in

combination as indicated in the figure. (B) The effects of IL-1β and L. paracasei on IL-6

production in Caco-2 cells transfected with hsp70 siRNA or non-specific RNA. Results

are means ± SEM with n=3 in each group. *p<0.05 vs control; +p<0.05 vs corresponding

IL-1β group by ANOVA. Identical experiments were performed 3 times with almost

identical results.

Fig 7 The influence of reduced hsp27 levels on the L. paracasei-induced potentiation of

IL-6 production in IL-1β-treated Caco-2 cells. (A) Hsp27 levels in cultured Caco-2 cells

transfected with hsp27 siRNA or control (scrambled) RNA as described in Materials and

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Methods and subsequently treated with IL-1β or L. paracasei alone or in combination as

indicated in the figure. (B) The effects of IL-1β and L. paracasei on IL-6 production in

Caco-2 cells transfected with hsp27 siRNA or control RNA. Results are means ± SEM

with n=3 in each group. *p<0.05 vs control; +p<0.05 vs corresponding IL-1β group by

ANOVA. Identical experiments were performed three times with almost identical results.

Fig 8 The effects of IL-1β and live L. paracasei on IL-6 mRNA levels in cultured Caco-2

Cells. Cells were treated for 1, 2, or 3 h with IL-1β, L. paracsei either alone or in

combination as indicated in the figure. Untreated cells served as control. IL-6 mRNA

levels were determined by real-time PCR. Results are means ± SEM with n=6 in each

group. *p<0.05 vs control at the corresponding time point; +p<0.05 vs IL-1β alone at the

corresponding time point by ANOVA.

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