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Am J Physiol Regul Integr Comp Physiol 293: R1169-R1179, 2007. First published July 18, 2007; doi:10.1152/ajpregu.00770.2006
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INFLAMMATION AND CYTOKINES

Probiotics potentiate IL-6 production in IL-1beta-treated Caco-2 cells through a heat shock-dependent mechanism

Natasha Reilly,1 Vitaliy Poylin,1 Michael Menconi,1 Andrew Onderdonk,2 Stig Bengmark,3 and Per-Olof Hasselgren1

1Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; 2Department of Pathology, Brigham Women's Hospital, Harvard Medical School, Boston, Massachusetts; and 3Liver Institute, University College, London University, United Kingdom

Submitted 5 November 2006 ; accepted in final form 13 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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-1beta in the absence or presence of different concentrations of L. paracasei or L. plantarum followed by measurement of IL-6 production. The role of heat shock response was examined by determining the expression of heat shock protein 70 (hsp70) and hsp27, by downregulating their expression with small interfering RNA (siRNA), or by treating cells with quercetin. Treatment of the Caco-2 cells with IL-1beta resulted in increased IL-6 production, confirming previous reports from this laboratory. Probiotics alone did not influence IL-6 production, but the addition of probitoics to IL-1beta-treated cells resulted in a substantial augmentation of IL-6 production. Treatment of the Caco-2 cells with live L. 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.

intestine; inflammation; cytokines

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-1beta-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 proinflammatory (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-1beta 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.

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 proinflammatory cytokines (18, 19), inhibited activation of NF-{kappa}B (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-1beta on IL-6 production in cultured enterocytes and that this response, at least in part, is mediated by the heat shock response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 DMEM (Gibco-BRC, Grand Island, NY) supplemented with 10% FBS, 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 six-well tissue culture plates. Cells were grown for 72 h to ~80–90% confluence before use. Cells were treated with 1 ng/ml of human recombinant IL-1beta (Biosource International, Camarillo, CA) or 1 ng/ml of human recombinant TNF-{alpha} (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. To examine the influence of probiotics on cytokine production, live or heat-inactivated Lactobacillus paracasei subsp. paracasei F19 (Belgian Coordinated Collection of Microorganisms, BCCM, deposition number LMG P-17806) or Lactobacillus plantarum 2362 (BCCM deposition number LMG P-20606) (both strains obtained from Medipharm, Kagerod, Sweden and Des Moines, IA) 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 cultured for 3 wk in transwell bicameral chambers to induce full differentiation, as described in detail previously (24).

Cell transfections. To examine the role of heat shock protein 70 (hsp70) and hsp27, the expression of these genes was silenced by transfecting cells with appropriate small interfering RNAs (siRNAs). Caco-2 cells were transfected with predesigned siRNA against hsp70 (sc-29352) or hsp27 (sc-29350). Other cells were transfected with nonspecific (scrambled) siRNA. All siRNA duplexes and reagents were from Santa Cruz Biotechnology (Santa Cruz, CA) and were used according to 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, Lactobacillus 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 g for 3 min), washed in 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 the RESULTS either alone or in combination with IL-1beta. In some experiments, bacteria were heat-inactivated in a water bath at 80°C for 10 min or sonicated with four 20-s bursts before they were added to the Caco-2 cells. The heat inactivation was confirmed by culture to make certain the preparations were sterile. 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 Escherichia coli bacteria were tested, DH5{alpha} E. coli were first cultured in Trypticase soy broth at 37°C, washed, and grown again at 37°C under anaerobic 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 blot analysis. Western blot analysis 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 was removed by centrifugation at 3,800 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 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 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 1:2,000 dilution of mouse anti-human hsp70 (Santa Cruz Biotechnology) or a 1:2,000 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 1:5,000. 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 ± SE. ANOVA followed by Tukey's test was used for statistical comparisons. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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-1beta, live L. paracasei or live L. plantarum, either alone or in combinations. Treatment of the Caco-2 cells with IL-1beta (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-1beta (data not shown), supporting the interpretation that the increased levels of IL-6 in the culture medium of IL-1beta-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-1beta 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-1beta-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 sixfold (compared with the IL-6 production in cells treated with IL-1beta alone) and of L. plantarum approximately twofold. Because the effect on IL-6 production was most pronounced for L. paracasei, this Lactobacillus strain was used in subsequent experiments.


Figure 1
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  		Fig. 1. The effects of IL-1beta, 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-1beta, 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-1beta, 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 expressed as means ± SE with n = 3 in each group. *P < 0.05 vs. control (untreated cells); {dagger}P < 0.05 vs. cells treated with IL-1beta alone by ANOVA. Identical experiments were performed three times with almost identical results.

 
As previous reports have suggested 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-1beta-treated Caco-2 cells was similar after treatment with live and heat-inactivated L. paracasei.

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. Results from that experiment showed that medium from cultured L. paracasei did not exert a potentiating effect on IL-6 production in IL-1beta-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-1beta-treated Caco-2 cells. 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-1beta, 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. 1, A and B). This result probably reflects a day-to-day variability with regard 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 different, the potentiating effect of L. paracasei on IL-6 production was seen also in this experiment.


Figure 2
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  		Fig. 2. The effects of IL-1beta, 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-1beta, 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-1beta, intact or sonicated L. paracasei alone, or in combination, as indicated in the figure and at concentrations identical to those used in A, followed by measurement of IL-6 in the culture medium. Results are expressed as means ± SE with n = 3 in each group. *P < 0.05 vs. control; {dagger}P < 0.05 vs. IL-1beta alone by ANOVA. Identical experiments were performed three times with almost identical results.

 
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-1beta-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-1beta-treated Caco-2 cells was noticed already after 3 h, at which time point the IL-6 production was increased approximately twofold and increased progressively to be approximately fourfold after 20 h (Fig. 3B).


Figure 3
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  		Fig. 3. A: effect of different concentrations of L. paracasei on IL-6 production in Caco-2 cells cultured in the absence of presence of IL-1beta. Cultured Caco-2 cells were treated for 20 h with different concentrations of L. paracasei alone or together with 1 ng/ml of IL-1beta 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-1beta-treated Caco-2 cells. Cells were treated with 1 ng/ml of IL-1beta or IL-1beta + 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 expressed as means ± SE with n = 3 in each group. Identical experiments were performed three times with almost identical results.

 
To test whether the potentiating effects of L. paracasei on IL-6 production in IL-1beta-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 nonprobiotic 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-1beta-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-1beta-treated Caco-2 cells and 14.9 ± 1.2 pg/ml in Caco-2 cells treated with IL-1beta + 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 bacteria but other gram-positive bacteria as well may potentiate IL-6 production in IL-1beta-treated cultured enterocytes.

We next examined whether gram-negative bacteria can also potentiate the effects of IL-1beta on IL-6 production in cultured Caco-2 cells. This was done by treating cultured Caco-2 cells with the E. coli strain DH5{alpha}. Although IL-6 production was somewhat higher in Caco-2 cells treated with both E. coli and IL-1beta than in Caco-2 cells treated with IL-1beta alone, this difference was much smaller than the approximately sixfold 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-1beta. Treatment of the Caco-2 cells with TNF-{alpha} did not result in measurable IL-6 production and this unresponsiveness to TNF-{alpha} was not influenced by L. paracasei (Fig. 4B).


Figure 4
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  		Fig. 4. A: effects of L. paracasei and E. coli on IL-6 production in IL-1beta-treated Caco-2 cells. Cells were treated for 20 h with 1 ng/ml of IL-1beta, 108/ml of L. paracasei, or 108/ml of DH5{alpha} E. coli, either alone or in combinations, as indicated in the figure, followed by measurement of IL-6 in the culture medium. B: effects of L. paracasei on IL-6 production in IL-1beta- and TNF-{alpha}-treated Caco-2 cells. Cultured Caco-2 cells were treated for 20 h with 1 ng/ml of IL-1beta or 1 ng/ml of TNF-{alpha} alone or in combination with 108/ml of L. paracasei. C: effect of IL-1beta and L. paracasei on IL-8 production in cultured Caco-2 cells. Cells were treated for 20 h with 1 ng/ml of IL-1beta 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 expressed as means ± SE with n = 3 in each group. *P < 0.05 vs. control; {dagger}P < 0.05 vs. IL-1beta alone by ANOVA. Identical experiments were performed three times with almost identical results.

 
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-1beta resulted in a robust increase in IL-8 production (Fig. 4C). L. paracasei alone or in combination with IL-1beta 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-1beta-induced IL-6 production was examined (29, 30). In another study from our laboratory, this model was validated by showing that IL-1beta 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 in detail previously (24) to test whether L. paracasei can potentiate IL-1beta-induced IL-6 production in fully differentiated Caco-2 cells. This also allowed us to treat the Caco-2 cells with IL-1beta 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 bloodstream. When this experimental approach was used, IL-6 production (determined in the basal chamber) in IL-1beta-treated Caco-2 cells was potentiated by 57% by the addition of L. paracasei (108 bacteria/ml) to the apical chamber (IL-6 levels were nondetectable in control cells, 11.5 pg/ml after treatment with IL-1beta alone for 20 h and 18.1 pg/ml after treatment with IL-1beta and L. paracasei; means are provided 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-1beta-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-1beta 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 hsp70, 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-1beta-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 L. paracasei. First, we tested whether treatment of the Caco-2 cells with L. paracasei induced a heat shock response. As seen in Fig. 5, A 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-1beta did not result in changes in the heat shock protein concentrations and the combined treatment with IL-1beta, and L. paracasei did not influence the changes induced by L. paracasei alone.


Figure 5
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  		Fig. 5. Influence of IL-1beta and L. paracasei on cellular levels of hsp70 (A) and hsp27 (B). Cultured Caco-2 cells were treated for 20 h with 1 ng/ml of IL-1beta or 108/ml of L. paracasei alone or in combination. Hsp70 and hsp27 levels were determined by Western blot analysis. beta-Actin levels were determined as loading controls. The experiment was repeated three times with almost identical results. B: effect of quercetin on L. paracasei-induced potentiation of IL-6 production in IL-1beta-treated Caco-2 cells. Cells were treated for 20 h with 1 ng/ml of IL-1beta or 108/ml L. paracasei in the absence or presence of 100 µM quercetin. Results are expressed as means ± SE with n = 3 in each group. *P < 0.05 vs. corresponding control group; {dagger}P < 0.05 vs corresponding IL-1beta group by ANOVA. Identical experiments were performed three times with almost identical results.

 
If the heat shock response induced by L. paracasei participates in the regulation of IL-6 production in IL-1beta-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 nonspecific effects other than inhibition of the heat shock response. In addition, it is not known from this experiment which specific heat shock protein 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 threefold increase in IL-6 production to an ~50% increase over the IL-6 production caused by IL-1beta 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 Figs. 6 and 7 suggest that both hsp70 and hsp27 participate in the potentiating effect of L. paracasei on IL-6 production in IL-1beta-treated Caco-2 cells.


Figure 6
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  		Fig. 6. The influence of reduced hsp70 levels on the L. paracasei-induced potentiation of IL-6 production in IL-1beta-treated Caco-2 cells. A: Hsp70 levels in cultured Caco-2 cells transfected with hsp70 siRNA or control (nonspecific) RNA, as described in MATERIALS AND METHODS and subsequently treated with IL-1beta or L. paracasei alone or in combination. B: effects of IL-1beta and L. paracasei on IL-6 production in Caco-2 cells transfected with hsp70 siRNA or nonspecific RNA. Results are expressed as means ± SE with n = 3 in each group. *P < 0.05 vs. control; {dagger}P < 0.05 vs. corresponding IL-1beta group by ANOVA. Identical experiments were performed 3 times with almost identical results.

 

Figure 7
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  		Fig. 7. The influence of reduced hsp27 levels on the L. paracasei-induced potentiation of IL-6 production in IL-1beta-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 METHODS and subsequently treated with IL-1beta or L. paracasei alone or in combination. B: effects of IL-1beta and L. paracasei on IL-6 production in Caco-2 cells transfected with hsp27 siRNA or control RNA. Results are means ± SE with n = 3 in each group. *P < 0.05 vs. control; {dagger}P < 0.05 vs. corresponding IL-1beta group by ANOVA. Identical experiments were performed three times with almost identical results.

 
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-1beta-treated Caco-2 cells (see Figs. 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 Figs. 1B and 2B, hsp27 and hsp70 levels, as determined by Western blot analysis 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. 5, A and B. These observations suggest that additional 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-1beta-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-1beta-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-1beta and L. paracasei at early time points. Treatment of the Caco-2 cells with IL-1beta 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-1beta (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-1beta-treated Caco-2 cells (29, 30) and support the concept that IL-1beta-induced IL-6 production is regulated at the transcriptional level. Importantly, in the present experiments, the addition of live L. paracasei to the Caco-2 cells resulted in a potentiation of the IL-1beta-induced IL-6 mRNA levels with an almost fivefold potentiation noticed after 1 h and an approximately tenfold potentiation noticed after 3 h (Fig. 8). Western blot analysis followed by densitometry showed a 23%, 51%, and 20% increase in hsp27 levels in L. paracasei-treated Caco-2 cells at 1, 2, 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 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-1beta-treated Caco-2 cells.


Figure 8
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  		Fig. 8. The effects of IL-1beta 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-1beta, L. paracsei either alone or in combination. Untreated cells served as control. IL-6 mRNA levels were determined by real-time PCR. Results are means ± SE with n = 6 in each group. *P < 0.05 vs. control at the corresponding time point; +P < 0.05 vs. IL-1beta alone at the corresponding time point by ANOVA.

 
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), in which hsp25 and hsp72 were increased 12 h after treatment of cultured young adult mouse colon 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 the treatment of the cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, the probiotic bacterium L. paracasei potentiated the effects of IL-1beta 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-1beta-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.

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 ~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-1beta 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-1beta-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 probiotics 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, treatment of cultured enterocytes with probiotics resulted in inhibited activity of the transcription factor NF-{kappa}B (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 proinflammatory cytokine IL-8 (39). The apparently contradictory results in the study by Ma et al. (18), that is, 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 [TNF-{alpha} in the study by Ma et al. (18) and IL-1beta in the present study], different types of probiotics [L. reuteri in the study by Ma et al. (18) vs. 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, that is, 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 (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-1beta. 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-1beta-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.

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-1beta-induced IL-6 production and 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 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-1beta (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/EBPbeta 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.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Institutes of Health Grant R01-DK60546 (to P.-O. Hasselgren).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Address for correspondence: P.-O. Hasselgren, Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave., ST919, Boston, MA 02215 (e-mail: phasselg{at}bidmc.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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