LACTOBACILLUS PARACASEI SUBSPECIES PARACASEI B21060 SUPPRESSES HUMAN
T CELL PROLIFERATION
Ilaria Peluso, Daniele Fina, Roberta Caruso, Carmine Stolfi, Flavio Caprioli, Massimo Claudio
Fantini, *Giorgio Caspani, *Enzo Grossi, ** Laura Di Iorio, ** Francesco Maria Paone, Francesco
Pallone, Giovanni Monteleone. Dipartimento di Medicina Interna, Università Tor Vergata, Rome,
Italy; * Bracco, SpA, Milan, Italy; ** Cattedra di Pediatria, Università Tor Vergata, Rome, Italy.
Short Title: L. paracasei-mediated blockade of human T cell growth
Key words: Lactobacilli, T cells, Foxp3, MCT-1, IBD.
Acknowledgements: This work received support from the “Fondazione Umberto di Mario”, Rome,
Ministero dell’ Istruzione, dell’ Università e della Ricerca, No. 2004065777-004, Ministero della
Salute, No. 6AC/F7.
Address for correspondence
Giovanni Monteleone
Dipartimento di Medicina Interna
Università Tor Vergata
Via Montpellier, 1
00133 Rome,
Italy
Phone +39.06.72596158
Fax +39.06.72596391
Email: [email protected]
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01172-06 IAI Accepts, published online ahead of print on 22 January 2007
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ABSTRACT
Recent studies have shown that probiotics are beneficial in T cell-mediated inflammatory diseases.
The molecular mechanism by which probiotics work remains elusive, but accumulating evidence
indicates that probiotics can modulate immune cell responses. Since T cells express receptors for
bacterial products/components, we examined whether different strains of lactobacilli directly
regulate the functions of human T cells. CD4+ T cells were isolated from blood and intestinal
lamina propria (LP) of normals and patients with inflammatory bowel disease (IBD). Mononuclear
cells (MC) were also isolated from Peyer’s patches (PP). Cells were activated with anti-
CD3/CD2/CD28 in the presence or absence of Lactobacillus (L.) paracasei subspecies (subsp.)
paracasei B21060, L. paracasei subsp. paracasei F19 or L. casei subsp. casei DG. Cell
proliferation and death, Foxp3, intracellular pH, and cytokine production were evaluated by flow
cytometry. We showed that L. paracasei subsp. paracasei B21060 but neither L. paracasei subsp.
paracasei F19 nor L. casei subsp. casei DG inhibited blood CD4+ T cell growth. This effect was
associated with no change in cell survival, expression of Foxp3, and production of IFN-γ, IL-4, IL-5
and IL-10. L. paracasei subsp. paracasei B21060-mediated blockade of CD4+T cell proliferation
required a viable bacterium and was associated with decreased MCT-1 expression and low
intracellular pH. L. paracasei subsp. paracasei B21060 also inhibited the growth of PPMC, normal
and IBD CD4+ LP lymphocytes without affecting cytokine production. Data show that L. paracasei
subsp. paracasei B21060 blocks T cell growth, thus suggesting a mechanism by which these
probiotics could interfere with T cell-driven immune responses.
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INTRODUCTION
Human intestine surface is home to a complex and abundant bacterial flora, that plays an
important role in the maintenance of the health and well-being of the host (28). The luminal
microflora promotes normal gastrointestinal functions, protects against pathogenic bacteria, and
exerts beneficial effects on systemic metabolism (28). Additionally, the indigenous commensals
play a decisive role in shaping and maintaining intestinal immune homeostasis (28).
Luminal bacteria can also drive pathologic inflammatory responses. This occurs in patients
with Crohn’s disease (CD) and patients with ulcerative colitis (UC), the major inflammatory bowel
diseases (IBD) in man. Indeed, recent insights into the nature of these diseases suggest that in IBD,
the tissue damage is driven by a dysregulated T cell-mediated immune response that is directed
against normal constituents of the gut microflora, and that manipulation of luminal bacteria helps
limit the mucosal inflammation (38, 35, 31, 4, 8, 32). In this context, it has been shown that feeding
non-pathogenic bacteria, in the form of probiotics, is beneficial in the prevention and treatment of
intestinal inflammation both in humans and animal models of IBD (10, 11, 12, 7, 1). The molecular
mechanism underlying the anti-inflammatory action of probiotics is not however known. While
effects on alteration of the gut flora, intestinal barrier integrity, and production of antimicrobial
compounds have been described, probiotics could also interfere with the mucosal immune response
(32). This is substantiated by the demonstration that probiotics may enhance counter-regulatory
mechanisms and regulate the synthesis of inflammatory cytokines (7, 2, 34, 18). Moreover, recent
data suggest that specific lactobacilli strains might induce the expression of mu-opioid and
cannabinoid receptors in intestinal epithelial cells (30).
Immune cells respond to signals from the microbial environment via pattern recognition
receptors, which include Toll-like receptors (TLRs) (27, 16). Recent studies have demonstrated that
TLRs can be expressed by T cells, raising the possibility that bacteria-driven signals can modulate
immune responses via direct effects on T cells (17, 20). Indeed, engagement of TLR2 on T
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regulatory cells (Treg) enhance their growth (37). Moreover, LPS, which acts through TLR4, and
flagellin, that signals through TLR5, increase the suppressive functions of Treg (3, 5). Based upon
these findings, we sought to determine whether probiotics directly modulate the type and extent of
T cell responses. To this end, we examined the effect of probiotics containing distinct lactobacillus
strains on human peripheral and mucosal T cell functions.
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MATERIALS AND METHODS
Samples and preparation of T lymphocytes
Peripheral blood mononuclear cells (PBMC) were isolated from enriched buffy coats of
healthy volunteer donors and used to purify CD4+ T cells by the CD4 multi-sort magnetic
microbeads kit (Miltenyi Biotec, Bologna, Italy). The remaining CD4-negative fraction of PBMC
was used to purify CD8+ T cells by using CD8 multi-sort magnetic beads (Miltenyi Biotec). Cell
purity was routinely evaluated by flow cytometry, and ranged between 96% and 98%.
Intestinal mucosal samples were taken from 3 patients with CD and 1 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 UC patient the disease was
substantial. All patients were receiving corticosteroids (CS) at the time of surgery. Mucosal samples
were also taken from macroscopically and microscopically unaffected colonic areas of 5 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
hours at 37°C with collagenase (all reagents were from Sigma-Aldrich, Milan, Italy). Lamina
propria mononuclear cells (LPMC) were separated from the crude cell suspension by layering on a
Percoll solution. For CD4+ LPT purification, LPMC were incubated for 30 minutes at 4°C with
CD4-magnetically labelled microbeads. The purity of CD4+ LPT populations
was >92%.
Mononuclear cells (MC) were isolated from freshly obtained Peyer’s patches (PP) taken from 4
children with irritable bowel syndrome undergoing colonoscopy for recurrent abdominal pain, as
previously described (25, 22). The study received approval by the local ethical committee.
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Cell culture
Cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS) and standard
supplements but no antibiotics (all from Sigma-Aldrich) in 96 multiwells U bottom (105 cells
/well/200µl), with or without anti-CD3/CD2/CD28-bound beads, according to the manufacturer’s
instructions (Myltenyi Biotec). L. paracasei subspecies paracasei F19 (SIFFRA, Florence, Italy)
(14), L. paracasei subsp. paracasei B21060 (Bracco SpA, Milan, Italy) (23), and L. casei
subspecies 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 culture
medium just prior to being used, and their concentration and viability were determined by FACS
analysis using SYBR-green and propidium iodide (PI) (13). Probiotics were used at the initial
concentration of 104-10
7 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 subspecies casei DG at
day 3 of culture. By contrast, at the same time point, the number of L. paracasei strain subspecies
paracasei F 19 increased of nearly 50 times. The amount of D-L Lactate produced by lactobacilli
was evaluated in 72 hours-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 hours at 37°C. The bacterial culture supernatant was
then recovered by centrifugation at 1,000 x g for 15 minutes, 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
minutes at 100 °C. T cells were cultured in RPMI plus 10% FBS containing 0, 12.5, 25, and 50%
(vol/vol) freshly prepared or boiled conditioned medium in the presence of anti-CD3/CD2/CD28
beads. Additionally, 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 minutes at 100°C. To
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ascertain the efficiency of such a treatment, an aliquot of the heat-treated probiotics was
resuspended in PBS and the viability was determined as indicated above. According to this
procedure, cells were considered as 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
(Sigma-Aldrich, 5-50 µg/ml) 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 minutes. 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
indicated above, and the fraction of Annexin V (AV) and PI-positive cells was evaluated by flow
cytometry using a commercially available kit (Beckmann Coulter, Milan, Italy).
Cell phenotype analysis
Anti-CD4 FITC, anti-CD8 APC (both from Beckmann 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.
Cytokines assays
IFN-γ, 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 2µM 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-
carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Sigma-Aldrich) at 37°C for 30 minutes as
previously described (24). Then mean intensity green and red fluorescences were detected by flow
cytometry, and pH was calculated using a standard curve with potassium phosphate buffers (pH
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ranging from 6 to 8). Experiments were performed in the presence of 10µM nigericin (Sigma-
Aldrich).
Western blotting
MCT-1 was analyzed in total extracts of CD4+ T cells using a goat anti-human MCT-1
antibody (ABCAM plc, Cambridge, UK) (final dilution 1:3000) followed by a HRP-peroxidase-
conjugated rabbit anti-goat IgG (Dako SpA, Milan, Italy) (final dilution 1:30000). The reaction was
detected with a sensitive enhanced chemiluminescence kit (West DURA, Pierce, Rockford, IL,
USA). After the analysis of MCT-1, blots were stripped and incubated with a mouse anti-human β-
actin antibody (final dilution 1: 5000, Sigma-Aldrich) followed by a goat anti-mouse antibody
conjugated to HRP-peroxidase (1: 30000 dilution) and detection by chemiluminescence.
Statistics
Two way Analysis of Variance (ANOVA) followed by pairwise multiple comparison
procedures (Student-Newman-Keuls Method) and paired Student’s t-test were performed. Data are
expressed as mean ± standard error mean (SEM).
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RESULTS
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
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).
By 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 anti-mitogenic 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.
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 10
5 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 vs 16 ± 2 and 14 ± 3% in the presence of
104 or 10
5 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 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-positive-CD4+CD25+ cells in response to a variety of stimuli,
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including bacterial products (9). Therefore, we examined whether the anti-proliferative 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 anti-
mitogenic 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 anti-mitogenic
effects also in cultures of CD8+ T cells, that do not contain Treg (Fig. 2B).
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 anti-proliferative 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).
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 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 up-regulation was evident at 24 hours, reached the maximal induction at 48 hours, and
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then declined (Fig. 4). L. paracasei subsp. paracasei B21060 inhibited MCT-1 expression and this
was evident at each time point (Fig. 4A).
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). As lactic acid can modulate T cell growth, we also evaluated
the level of D-L- lactate in culture supernatants of Lactobacilli. Notably, after 72 hours 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). Additionally, we
showed that the addition of 10 and 20 µg/ml exogenous D-L- lactate to CD4+ T cell cultures
markedly inhibited the cell growth (Fig. 4C), while 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 a massive cell death (not shown). Overall these findings
suggest that the anti-mitogenic 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-labelled 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 anti-proliferative 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
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were also treated with boiled conditioned medium. Both the freshly prepared and boiled conditioned
media dose-dependently inhibited the growth of activated CD4+T cells (Fig. 5).
L. paracasei subsp. paracasei B21060 suppresses the proliferation of Peyer’s patches (PP) and
intestinal lamina propria 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 PPMC as the number of cells we isolates 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). Production of IL-10 was augmented by activation of PPMC, but again this
synthesis was not increased by L. paracasei subsp. paracasei B21060 (Fig. 6D).
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-D). In addition, the significant increase in the percentage of Foxp3-positive
cells induced by anti-CD3/CD2/CD28 (P<0.05) was not modified by L. paracasei subsp. paracasei
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B21060 (Fig. 7E). Subsequently, we assessed the anti-mitogenic effect of L. paracasei subsp.
paracasei B21060 on LP CD4+ T cells isolated from IBD patients. As shown in figure 8, the
proliferation of CD4+T cells isolated from the gut of 3 patients with CD and 1 patient with UC was
significantly inhibited by L. paracasei subsp. paracasei B21060 (P<0.05).
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Discussion
This study was undertaken to examine whether different strains and subspecies of
Lactobacilli modulate the functions of CD4+ T cells. We show that L. paracasei subsp. paracasei
B21060 directly inhibits the in vitro proliferation of human CD4+ T cells, consistent with the
demonstration that T cells are able to directly respond to bacteria (17, 20). Our study also reveals
that the L. paracasei subsp. paracasei B21060-mediated anti-mitogenic effect is specific, as two
other different Lactobacilli or Lactobacillus subsp., namely L. paracasei subsp. paracasei F19 and
L. casei subsp. casei DG, did not suppress CD4+ T cell growth. This effect would not seem to rely
simply on a different propagation of the Lactobacilli or Lactobacillus subsp. in our system, as the
number of L. paracasei subsp. paracasei B21060 was similar to that of L. casei subsp. casei DG at
any time point during the culture. Therefore, our data further support earlier observations that
different probiotics strains/subspecies may have distinct regulatory properties and exert variable
effects on the course of inflammatory diseases (32, 34). It is also known that Lactobacilli or
Lactobacillus subsp. may differently modulate specific immune responses depending on the cell
system considered. For example, Lactobacilli are powerful inducers of Th1-type cytokines, such as
IL-12 and TNF-α, in blood cells, while they down-regulate TNF-α when used in ex vivo organ
cultures of IBD mucosal explants (2, 21, 15). While our study was ongoing, Sturm et al (36)
demonstrated that conditioned medium of E.coli strain Nissle 1917 inhibited the growth of blood
and mucosal T cells, and that such an effect was associated with decreased synthesis of IFN-γ, and
high IL-10 secretion. This later finding well fits with data of other studies that have linked the
beneficial effect of probiotics with the induction of IL-10 in experimental models of IBD (7).
However, no increase in IL-10 was seen in our cell cultures added with L. paracasei subsp.
paracasei B21060. Moreover, L. paracasei subsp. paracasei B21060 did not alter the percentage of
Foxp3-positive cells, thus suggesting that the anti-proliferative effect of L. paracasei subsp.
paracasei B21060 does not rely on the expansion of counter-regulatory mechanisms.
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A surprising finding of our study is that the CD4+ T cell growth arrest induced by L.
paracasei subsp. paracasei B21060 was associated with no change in the production of Th1/Th2
cytokines. These immunomodulatory effects of L. paracasei subsp. paracasei B21060 resemble
those exerted by inhibitors of the monocarboxylate transporter MCT-1, given that such compounds
suppress T cell growth without affecting cytokine production (24). Therefore, we extended our
analysis by examining the effect of L. paracasei subsp. paracasei B21060 on MCT-1. By Western
blotting we show that L. paracasei subsp. paracasei B21060 inhibits the expression of MCT-1, and
consequently reduces the intracellular pH in CD4+ T cells. Although, no functional experiment was
carried-out to mechanistically link the down-regulation of MCT-1 and reduction of intracellular pH
with the cell growth arrest, it is noteworthy that the intracellular pH is crucial in controlling cell
growth, and that acidification of the intracellular compartment leads to cell growth arrest (33). In
line with this, we show that the amount of D-L lactate made by L. paracasei subsp. paracasei
B21060 was greater than that produced by the other two lactobacilli, and that the addition of
exogenous lactate to CD4+ T cell cultures caused a marked inhibition of CD4+ T cell proliferation.
While the definition of probiotics is one of live microorganisms, recent studies have
demonstrated that bacterial DNA could be responsible for some beneficial effects of probiotics
(29). However, our data indicate that the L. paracasei subsp. paracasei B21060-
immunomodulatory effects require the presence of a viable bacterium, as heat-killed L. paracasei
subsp. paracasei B21060 did not inhibit the growth of CD4+ T cells. The results presented herein
suggest that the L. paracasei subsp. paracasei B21060-induced anti-proliferative effect is mediated
by secretion of lactic acid.
In conclusion, our study shows that L. paracasei subsp. paracasei B21060 blocks the
proliferation of T lymphocytes, and this occurs in cultures of CD4+ T cells isolated from the
normal and inflamed intestinal LP, as well as in cultures of PP cells. It is thus tempting to speculate
that L. paracasei subsp. paracasei B21060 might affect both the inductive and effector phases of
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the human mucosal T cell response. However, additional in vivo studies will be necessary to
ascertain if results from these in vitro experiments can be translated to the clinical practice, and
examine whether L. paracasei subsp. paracasei B21060 can interfere with T cell-driven immune
responses.
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FIGURE LEGEND
Figure 1. L. paracasei subsp. paracasei B21060 suppresses the growth of activated blood CD4+ T
cells. (A) CFSE-labeled CD4+ T cells (5 x 105 cells/ml) were stimulated with anti-CD3/CD2/CD28
(ACT) in presence or absence of L. paracasei subsp. paracasei B21060 (P) (1 x 106 cells/ml) or L.
paracasei subsp. paracasei F19 (F19) (1 x 106 cells/ml) or L. casei subsp. casei DG (DG) (1 x 10
6
cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry. Data
indicate mean ± SEM of 6 different experiments.
B. Effect of L. paracasei subsp. paracasei B21060 on CD4+ T cell death. Cells were cultured as
indicated in A, and the percentage of cell death was assessed by FACS analysis of AV and/or PI-
positive cells. Data indicate mean ± SEM of 3 separate experiments.
Figure 2. A. Percentage of Foxp3-positive 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) (1 x 106 cells/ml), L. paracasei subsp. paracasei F19 (F19) (1 x 10
6
cells/ml), or L. casei subsp. casei DG (DG) (1 x 106 cells/ml). After 3 day culture, Foxp3 was
analyzed by flow cytometry. Data indicate mean ±SEM of 4 separate experiments. B. L. paracasei
subsp. paracasei B21060 inhibits the proliferation of activated blood CD8+ T cells. CFSE-labeled
CD8+ T cells (5 x 105 cells/ml) were either left unstimulated (UNST) or stimulated with anti-
CD3/CD2/CD28 (ACT) in presence or absence of L. paracasei subsp. paracasei B21060 (P) (1 x
106 cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry.
Data indicate mean ± SEM of 3 different experiments.
Figure 3. CD4+ T cells (5 x 105 cells/ml) were either left unstimulated (UNST) or stimulated with
anti-CD3/CD2/CD28 (ACT) in presence or absence of L. paracasei subsp. paracasei B21060 (P) (1
x 106 cells/ml), L. paracasei subsp. paracasei F19 (F19) (1 x 10
6 cells/ml), or L. casei subsp. casei
DG (DG) (1 x 106 cells/ml). After 3 days, the cell-free culture supernatants were collected and
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analyzed for the content of IFN-γ (A), IL-4 and IL-5 (B) and IL-10 (C) by flow cytometry. Data are
expressed as pg/ml and indicate the mean ± SEM of 5 separate experiments.
Figure 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 presence or absence of L. paracasei subsp.
paracasei B21060 (P) (1 x 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 in A, and
the intracellular pH was assessed by flow cytometry. Data indicate mean ± SEM of 3 different
experiments. C. Representative CFSE dilution profiles of CD4+ T cells stimulated with anti-
CD3/CD2/CD28 (ACT) in presence and/or absence of the specified doses of exogenous D-L
Lactate for 72 hours. One of two representative experiments is shown.
Figure 5. A. Representative dot plots of SYBR-green (SYBR) and propidium iodide (PI) of L.
paracasei subsp. paracasei B21060. The lactobacilli were stained before (L. paracasei subspecies
paracasei B21060= P) or after heating at 100 °C for 20 minutes (heat-killed L. paracasei
subspecies paracasei B21060= HKP). Numbers indicate the percentage of bacteria in the designated
gates. Viable bacteria are PI-negative and SYBR-positive, while 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. The 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 x 105 cells/ml) were stimulated with anti-CD3/CD2/CD28 (ACT) in
presence or absence of live L. paracasei subsp. paracasei B21060 (P) (1 x 106 cells/ml) or heat-
killed L. paracasei subsp. paracasei B21060 (1 x 106 cells/ml) (1 x 10
6 cells/ml). After 3 days, the
percentage of proliferating cells was evaluated by flow cytometry. Data indicate mean ± SEM of 3
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 x 105 cells/ml) were stimulated with anti-CD3/CD2/CD28 (ACT) in presence or absence of either
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freshly prepared or boiled conditioned medium, at the indicated dilutions. After 3 days, the
percentage of proliferating cells was evaluated by flow cytometry. Data indicate mean ± SEM of 3
different experiments.
Figure 6. L. paracasei strain B21060 suppresses the growth of activated Peyer’ patches
mononuclear cells (PPMC) without affecting the synthesis of cytokines. PPMC (5 x 105 cells/ml)
were stimulated with anti-CD3/CD2/CD28 (ACT) in presence or absence of L. paracasei subsp.
paracasei B21060 (P) (1 x 106 cells/ml). After 3 days, the percentage of CFSE-labeled proliferating
cells (A), and the content of IFN-γ (B), IL-4 and IL-5 (C), and IL-10 (D) in the culture supernatants
were evaluated by flow cytometry. Data indicate the mean ± SEM of 4 separate experiments.
Figure 7. L. paracasei strain B21060 suppresses the growth of normal intestinal lamina propria
CD4+ T cells, without affecting the cytokine synthesis. CFSE-labeled CD4+ T cells (5 x 105
cells/ml) were stimulated with anti-CD3/CD2/CD28 (ACT) in presence or absence of L. paracasei
subsp. paracasei B21060 (P) (1 x 106 cells/ml). After 3 days, the percentage of proliferating cells
(A), the content of IFN-γ (B), IL-4 and IL-5, (C), and IL-10 (D) in the culture supernatants, and the
percentage of Foxp-3-positive cells were evaluated by flow cytometry. Data indicate mean ±SEM
of 5 separate experiments.
Figure 8. L. paracasei subsp. paracasei B21060 suppresses the growth of IBD intestinal lamina
propria CD4+ T cells. CFSE-labeled CD4+ T cells (5 x 105 cells/ml), isolated from 3 patients with
Crohn’s disease (CD) and one patient with ulcerative colitis (UC), were stimulated with anti-
CD3/CD2/CD28 (ACT) in presence or absence of L. paracasei subsp. paracasei B21060 (P) (1 x
106 cells/ml). After 3 days, the percentage of proliferating cells was evaluated by flow cytometry.
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