Lactobacillus paracasei subsp. paracasei B21060 Suppresses Human T-Cell Proliferation

Samples and preparation of T lymphocytes.Peripheral blood mononuclear cells were isolated from enriched buffy coats from healthy volunteer donors and used to purify CD4⁺ T cells by the CD4 multisort magnetic microbeads kit (Miltenyi Biotec, Bologna, Italy). The remaining CD4-negative fraction of peripheral blood mononuclear cells was used to purify CD8⁺ T cells by using CD8 multisort magnetic beads (Miltenyi Biotec). Cell purity was routinely evaluated by flow cytometry and ranged between 96 and 98%.

Intestinal mucosal samples were taken from three patients with CD and one patient with UC undergoing resection for a chronic disease unresponsive to medical treatment. In CD patients the primary site of disease was the terminal ileum and right colon, while in the UC patient the disease was substantial. All patients were receiving corticosteroids at the time of surgery. Mucosal samples were also taken from macroscopically and microscopically unaffected colonic areas of five patients undergoing colectomy for colon cancer. The dissected intestinal mucosa was freed of mucus and epithelial cells in sequential washing steps with dithiothreitol and EDTA and then digested for 4 h at 37°C with collagenase (all reagents were from Sigma-Aldrich, Milan, Italy). Lamina propria (LP) mononuclear cells were separated from the crude cell suspension by layering them on a Percoll solution. For CD4⁺ LP lymphocyte purification, LP mononuclear cells were incubated for 30 min at 4°C with CD4-magnetically labeled microbeads. The purity of CD4⁺ LP lymphocyte populations was >92%. Mononuclear cells were isolated from freshly obtained Peyer's patches (PP) taken from four children with irritable bowel syndrome undergoing colonoscopy for recurrent abdominal pain, as previously described (22, 25). The study received approval by the local ethical committee.

Cell culture.Cells were cultured in RPMI 1640 containing 10% fetal bovine serum and standard supplements but no antibiotics (all from Sigma-Aldrich) in 96-multiwell U-bottom plates (10⁵ cells/well/200 μl), with or without anti-CD3/CD2/CD28-bound beads, according to the manufacturer's instructions (Myltenyi Biotec). L. paracasei subsp. paracasei F19 (SIFFRA, Florence, Italy) (14), L. paracasei subsp. paracasei B21060 (Bracco SpA, Milan, Italy) (23), and L. casei subsp. casei DG (Sofar; Trezzano Rosa, Milan, Italy) were provided by the companies as lyophilized products and stored at 4°C until used. Probiotics were dissolved in 1 ml of culture medium just prior to being used, and their concentration and viability were determined by fluorescence-activated cell sorting analysis using SYBR-green and propidium iodide (PI) (13). Probiotics were used at the initial concentration of 10⁴ to 10⁷ bacteria/ml, and analysis of their propagation showed a 100-fold increase in the number of both L. paracasei subsp. paracasei B21060 and L. casei subsp. casei DG at day 3 of culture. In contrast, at the same time point, the number of L. paracasei subsp. paracasei strain F19 increased of nearly 50 times. The amount of d,l-lactate produced by lactobacilli was evaluated in 72-h culture supernatants by spectrophotometry using a commercially available kit (Raisio, Rome, Italy).

To examine the effect of factors secreted by L. paracasei subsp. paracasei B21060 on T-cell function, L. paracasei subsp. paracasei B21060 (10⁶ cells/ml) was resuspended in RPMI 1640 containing no antibiotics and incubated for 72 h at 37°C. The bacterial culture supernatant was then recovered by centrifugation at 1,000 × g for 15 min, filtered through a 0.22-μm-pore-size syringe-driven filter (conditioned medium), and either kept at −80°C until used or boiled for 20 min at 100°C. T cells were cultured in RPMI plus 10% fetal bovine serum containing 0, 12.5, 25, and 50% (vol/vol) freshly prepared or boiled conditioned medium in the presence of anti-CD3/CD2/CD28 beads. In addition, cultures of activated CD4⁺ T cells were added with heat-killed L. paracasei strain B21060 (10⁶ cells/ml) that was prepared by boiling the probiotics for 20 min at 100°C. To ascertain the efficiency of such a treatment, an aliquot of the heat-treated probiotics was resuspended in phosphate-buffered saline, and the viability was determined as indicated above. According to this procedure, cells were considered dead only if they were stained with PI but were SYBR-green negative. To examine the effect of lactate on T-cell growth, graded doses of exogenous d,l-lactate (5 to 50 μg/ml; Sigma-Aldrich) were added to cultures of activated CD4⁺ T cells. To track the proliferation, CD4⁺ or CD8⁺ T cells were incubated in 0.2 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Milan Italy) at 37°C for 30 min. After 3 days culture, fluorescence was detected, and the proportion of cells undergoing divisions was determined.

To evaluate the effect of lactobacilli on cell survival, CD4⁺ T cells were cultured as described above, and the fraction of annexin V (AV)- and PI-positive cells was evaluated by flow cytometry using a commercially available kit (Beckman Coulter, Milan, Italy).

Cell phenotype analysis.Anti-CD4 fluorescein isothiocyanate and anti-CD8 APC (both from Beckman Coulter), anti-Foxp3 APC (Società Italiana Chimici, Florence, Italy), and control isotype antibodies were used for analysis of relative antigens according to the manufacturer' instructions.

Cytokine assays.Gamma interferon (IFN-γ), interleukin-4 (IL-4), IL-5, and IL-10 were analyzed in cell culture supernatants by flow cytometry using a commercially available kit (Bender, Vienna, Austria).

pH measurements.Cells were incubated in RPMI with 2 μM 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Sigma-Aldrich) at 37°C for 30 min as previously described (24). The mean intensity green and red fluorescence levels were then detected by flow cytometry, and the pH was calculated by using a standard curve with potassium phosphate buffers (pH ranging from 6 to 8). Experiments were performed in the presence of 10 μM nigericin (Sigma-Aldrich).

Western blotting.MCT-1 was analyzed in total extracts of CD4⁺ T cells using goat anti-human MCT-1 antibody (final dilution, 1:3,000; ABCAM plc, Cambridge, United Kingdom), followed by horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin G (final dilution, 1:30,000; Dako SpA, Milan, Italy). The reaction was detected with a sensitive enhanced chemiluminescence kit (West DURA; Pierce, Rockford, IL). After the analysis of MCT-1, blots were stripped and incubated with mouse anti-human β-actin antibody (final dilution, 1:5,000; Sigma-Aldrich), followed by goat anti-mouse antibody conjugated to horseradish peroxidase (1:30,000 dilution) and detection by chemiluminescence.

Statistics.Two-way analysis of variance, followed by pairwise multiple comparison procedures (the Student-Newman-Keuls method) and the paired Student t test, was performed. The data are expressed as means ± the standard errors of the mean (SEM).

L. paracasei subsp. paracasei B21060 inhibits blood CD4⁺ T-cell proliferation.Culture of CD4⁺ T lymphocytes with anti-CD3/CD2/CD28 resulted in a significant increase in the percentage of proliferating cells (Fig. 1A, P < 0.001). The addition of L. paracasei subsp. paracasei B21060 (10⁶ cells/ml) to such cultures significantly inhibited the cell growth (P < 0.01). In contrast, equivalent concentrations of L. paracasei subsp. paracasei F19 or L. casei subsp. casei DG did not inhibit cell proliferation (Fig. 1A). To exclude that the antimitogenic effect of L. paracasei subsp. paracasei B21060 was secondary to changes in cell viability, we evaluated the fraction of AV- and/or PI-positive cells in the same cultures. Figure 1B shows that L. paracasei subsp. paracasei B21060 did not augment the percentage of AV- and/or PI-positive cells.

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FIG. 1.

L. paracasei subsp. paracasei B21060 suppresses the growth of activated blood CD4⁺ T cells. (A) CFSE-labeled CD4⁺ T cells (5 × 10⁵ cells/ml) were stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml) or L. paracasei subsp. paracasei F19 (F19) (10⁶ cells/ml) or L. casei subsp. casei DG (10⁶ cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry. The data indicate mean ± the SEM of six different experiments. (B) Effect of L. paracasei subsp. paracasei B21060 on CD4⁺ T-cell death. Cells were cultured as indicated for panel A, and the percentage of cell death was assessed by fluorescence-activated cell sorting analysis of AV- and/or PI-positive cells. The data indicate means ± the SEM of three separate experiments.

We also established the optimal concentration at which L. paracasei subsp. paracasei B21060 inhibits CD4⁺ T-cell growth. At the initial concentrations of 10⁴ or 10⁵ cells/ml, L. paracasei subsp. paracasei B21060 reduced the percentage of proliferating CD4⁺ T cells (21 ± 3% in the absence of L. paracasei subsp. paracasei B21060 versus 16 ± 2 and 14 ± 3% in the presence of 10⁴ or 10⁵ cells/ml, respectively), but such effects were not significant. A marked bacterial overgrowth and complete CD4⁺ T-cell death were seen in cultures added with 10⁷ cells/ml (not shown). Therefore, in the subsequent experiments L. paracasei subsp. paracasei B21060 was used at 10⁶ cells/ml.

L. paracasei subsp. paracasei B21060 alters neither the percentage Foxp3⁺ T cells nor the synthesis of cytokines.Treg are a subpopulation of CD4⁺ CD25⁺ T lymphocytes that specifically express the forkhead transcription factor forkhead winged helix transcription factor gene (Foxp3) and are highly specialized for the suppression of proliferation of effector T cells (39). In addition to naturally occurring Treg that are produced by the thymus, Treg can arise in the periphery upon conversion of CD4⁺ CD25⁻ T cells into Foxp3⁺ CD4⁺ CD25⁺ cells in response to a variety of stimuli, including bacterial products (9). Therefore, we examined whether the antiproliferative effect of L. paracasei subsp. paracasei B21060 was associated with changes in the percentage of Foxp3-positive cells. Culture of CD4⁺ T cells with anti-CD3/CD2/CD28 significantly augmented the fraction of Foxp-3-positive cells (Fig. 2A, P < 0.01). However, this percentage was not increased further by L. paracasei subsp. paracasei B21060, thus arguing against the hypothesis that the antimitogenic effect of L. paracasei subsp. paracasei B21060 is due to an expansion of Treg. This was substantiated further by the demonstration that L. paracasei strain B21060 exerted antimitogenic effects also in cultures of CD8⁺ T cells that do not contain Treg (Fig. 2B).

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FIG. 2.

(A) Percentage of Foxp3⁺ cells induced from blood CD4⁺ T cells after culture with medium alone (Unst) or anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml), L. paracasei subsp. paracasei F19 (10⁶ cells/ml), or L. casei subsp. casei DG (10⁶ cells/ml). After 3 day culture, Foxp3 was analyzed by flow cytometry. The data indicate means ± the SEM of four separate experiments. (B) L. paracasei subsp. paracasei B21060 inhibits the proliferation of activated blood CD8⁺ T cells. CFSE-labeled CD8⁺ T cells (5 × 10⁵ cells/ml) were either left unstimulated (Unst) or stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry. The data indicate means ± the SEM of three different experiments.

Subsequently, we examined whether L. paracasei subsp. paracasei B21060 modulates the production of CD4⁺ T cell-derived cytokines. Culture of cells with anti-CD3/CD2/CD28 significantly enhanced the synthesis of IFN-γ (Fig. 3A, P < 0.001). However, no further increase was seen when these cell cultures were added with any lactobacilli or Lactobacillus subsp. (Fig. 3A). Similarly, probiotics were not able to alter the secretion of IL-4 and IL-5 (Fig. 3B). Since some probiotics enhance IL-10 synthesis and IL-10 exerts antiproliferative effects on CD4⁺ T cells (7), we also measured IL-10. Activation of cells caused a significant increase in IL-10, but such a synthesis was not modified by probiotics (Fig. 3C).

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FIG. 3.

CD4⁺ T cells (5 × 10⁵ cells/ml) were either left unstimulated (Unst) or stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml), L. paracasei subsp. paracasei F19 (10⁶ cells/ml), or L. casei subsp. casei DG (10⁶ cells/ml). After 3 days, the cell-free culture supernatants were collected and analyzed for the IFN-γ (A), IL-4 and IL-5 (B), and IL-10 (C) contents by flow cytometry. The data are expressed as pg/ml and indicate the means ± the SEM of five separate experiments.

L. paracasei subsp. paracasei B21060 inhibits the expression of monocarboxylate transporter MCT-1.MCT-1, a membrane protein of the MCT family, is involved in the proton-linked transport of lactate, pyruvate, and other monocarboxylates across the plasma membrane (6). Inhibition of MCT-1 in activated T cells blocks the proliferation but not cytokine production (24), thus showing striking similarities with the effect of L. paracasei subsp. paracasei B21060 on CD4⁺ T-cell activation. Therefore, we evaluated the effect of L. paracasei subsp. paracasei B21060 on MCT-1. Western blotting of total extracts revealed that activation of CD4⁺ T cells augmented the expression of MCT-1. This upregulation was evident at 24 h, reached the maximal induction at 48 h, and then declined (Fig. 4). L. paracasei subsp. paracasei B21060 inhibited MCT-1 expression, and this was evident at each time point (Fig. 4A).

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FIG. 4.

L. paracasei subsp. paracasei B21060 inhibits the expression of MCT-1 and reduces the intracellular pH in activated CD4⁺ T cells. CD4⁺ T cells were either left unstimulated (Unst) or stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml). Total extracts were analyzed for MCT-1 by Western blotting. One of three separate experiments is shown. (B) Cells were cultured as indicated for panel A, and the intracellular pH was assessed by flow cytometry. The data indicate means ± the SEM of three different experiments. (C) Representative CFSE dilution profiles of CD4⁺ T cells stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of the specified doses of exogenous d,l-lactate for 72 h. The results for one of two representative experiments is shown.

The inhibition of MCT-1 is followed by an intracellular accumulation of lactate and hence reduction of the intracellular pH (26). Indeed, L. paracasei subsp. paracasei B21060 significantly reduced the intracellular pH (Fig. 4B). Since lactic acid can modulate T-cell growth, we also evaluated the level of d,l-lactate in culture supernatants of Lactobacilli. Notably, after 72 h culture, the production of d,l-lactate by L. paracasei subsp. paracasei B21060 was significantly greater (354 ± 14 μg/ml) than that produced by L. casei subsp. casei DG (254 ± 22 μg/ml) and by L. paracasei subsp. paracasei F19 (165 ± 5 μg/ml) (P < 0.05 and P < 0.01, respectively). In addition, we showed that the addition of 10 and 20 μg of exogenous d,l-lactate/ml to CD4⁺ T-cell cultures markedly inhibited cell growth (Fig. 4C), whereas no inhibitory effect was seen when d,l-lactate was used at 5 μg/ml (not shown). Moreover, incubation of CD4⁺ T cells with concentrations of d,l-lactate greater than 50 μg/ml resulted in massive cell death (results not shown). Overall, these findings suggest that the antimitogenic effect of L. paracasei subsp. paracasei B21060 may be in part mediated by lactate.

The L. paracasei subsp. paracasei B21060-induced inhibition of CD4⁺ T-cell proliferation is mediated by thermostable secreted factor(s).To assess whether the effect of L. paracasei subsp. paracasei B21060 on CD4⁺ T-cell proliferation requires a viable bacterium, CFSE-labeled CD4⁺ T cells were cultured with anti-CD3/CD2/CD28 in the presence or absence of live or heat-killed L. paracasei subsp. paracasei B21060 (Fig. 5A). Figure 5B shows that heat-killed L. paracasei subsp. paracasei B21060 did not inhibit CD4⁺ T-cell proliferation. Then, we evaluated whether the antiproliferative effect of L. paracasei subsp. paracasei B21060 was mediated by soluble factor(s). To begin to address this issue, we used L. paracasei subsp. paracasei B21060-derived conditioned medium. To evaluate whether the effect of such conditioned medium was due to thermolabile molecules, cell cultures were also treated with boiled conditioned medium. Both the freshly prepared and the boiled conditioned media dose dependently inhibited the growth of activated CD4⁺ T cells (Fig. 5).

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FIG. 5.

(A) Representative dot plots of SYBR green (SYBR) and PI of L. paracasei subsp. paracasei B21060. The lactobacilli were stained before (L. paracasei subsp. paracasei B21060 [P]) or after heating at 100°C for 20 min (heat-killed L. paracasei subsp. paracasei B21060 [HKP]). Numbers indicate the percentage of bacteria in the designated gates. Viable bacteria are PI negative and SYBR positive, whereas dead bacteria are PI positive and SYBR negative. Double positivity indicates bacteria with a slightly damaged membrane but still alive. One of three representative experiments is shown. (B) Viable but not heat-killed L. paracasei subsp. paracasei B21060 inhibits the proliferation of activated blood CD4⁺ T cells. CFSE-labeled CD4⁺ T cells (5 × 10⁵ cells/ml) were stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of live L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml) or heat-killed L. paracasei subsp. paracasei B21060 (10⁶ cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry. The data indicate means ± the SEM of three different experiments. (C) Freshly prepared and boiled conditioned media of L. paracasei subsp. paracasei B21060 inhibit the growth of activated blood CD4⁺ T cells. CFSE-labeled CD4⁺ T cells (5 × 10⁵ cells/ml) were stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of either freshly prepared or boiled conditioned medium at the indicated dilutions. After 3 days, the percentage of proliferating cells was evaluated by flow cytometry. The data indicate means ± SEM of 3 different experiments.

L. paracasei subsp. paracasei B21060 suppresses the proliferation of PP and intestinal LP lymphocytes.PP is one of the major sites where the intestinal immune system interacts and responds to luminal antigens (19). Therefore, we examined whether L. paracasei subsp. paracasei B21060 modulates the response of freshly isolated PP lymphocytes. In these experiments, we used unfractionated PP mononuclear cells (PPMC) since the number of cells we isolated from small endoscopic biopsies was not sufficient to purify CD4⁺ T cells. Activation of PPMC with anti-CD3/CD2/CD28 enhanced the percentage of proliferating cells, and this effect was inhibited by L. paracasei subsp. paracasei B21060 (Fig. 6A). Activated cells produced higher levels of IFN-γ than unstimulated cells (P < 0.01), but such a synthesis was not significantly modified by L. paracasei subsp. paracasei B21060 (Fig. 6B). In contrast, the synthesis of IL-4 and IL-5 was not enhanced by activation of cells, and this was evident regardless of whether cells were left untreated or treated with L. paracasei subsp. paracasei B21060 (Fig. 5C). The production of IL-10 was augmented by the activation of PPMC, but again this synthesis was not increased by L. paracasei subsp. paracasei B21060 (Fig. 6D).

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FIG. 6.

L. paracasei strain B21060 suppresses the growth of activated PPMC without affecting the synthesis of cytokines. PPMC (5 × 10⁵ cells/ml) were stimulated with anti-CD3/CD2/CD28 (ACT) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml). After 3 days, the percentage of CFSE-labeled proliferating cells (A) and the IFN-γ (B), IL-4 and IL-5 (C), and IL-10 (D) contents in the culture supernatants were evaluated by flow cytometry. The data indicate the means ± the SEM of four separate experiments.

Since intestinal LP contains a large population of effector-memory T cells that exhibit marked phenotypic and functional differences with peripheral blood and PP cells (19), we also examined whether L. paracasei subsp. paracasei B21060 affects the proliferation and cytokine production of LP CD4⁺ T lymphocytes. Initially, we evaluated the effects of L. paracasei subsp. paracasei B21060 on the proliferation and cytokine response of normal intestinal LP CD4⁺ cells activated with anti-CD3/CD2/CD28. L. paracasei subsp. paracasei B21060 significantly inhibited the proliferation of activated LP CD4⁺ T cells (Fig. 7A, P < 0.05) but did not alter the production of any cytokine (Fig. 7B to D). In addition, the significant increase in the percentage of Foxp3⁺ cells induced by anti-CD3/CD2/CD28 (P < 0.05) was not modified by L. paracasei subsp. paracasei B21060 (Fig. 7E). Subsequently, we assessed the antimitogenic effect of L. paracasei subsp. paracasei B21060 on LP CD4⁺ T cells isolated from IBD patients. As shown in Fig. 8, the proliferation of CD4⁺ T cells isolated from the guts of three patients with CD and one patient with UC was significantly inhibited by L. paracasei subsp. paracasei B21060 (P < 0.05).

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FIG. 7.

L. paracasei strain B21060 suppresses the growth of normal intestinal LP CD4⁺ T cells without affecting the cytokine synthesis. CFSE-labeled CD4⁺ T cells (5 × 10⁵ cells/ml) were stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml). After 3 days, the percentage of proliferating cells (A); the IFN-γ (B), IL-4 and IL-5, (C), and IL-10 (D) contents in the culture supernatants; and the percentage of Foxp-3-positive cells were evaluated by flow cytometry. The data indicate means ± the SEM of five separate experiments.

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FIG. 8.

L. paracasei subsp. paracasei B21060 suppresses the growth of IBD intestinal LP CD4⁺ T cells. CFSE-labeled CD4⁺ T cells (5 × 10⁵ cells/ml), isolated from three patients with CD and one patient with UC, were stimulated with anti-CD3/CD2/CD28 (Act) in the presence or absence of L. paracasei subsp. paracasei B21060 (P) (10⁶ cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry.