Received 23 December 2000/Returned for modification 7 February
2001/Accepted 15 March 2001
We investigated whether certain strains of lactic acid bacteria
(LAB) could antagonize specific T-helper functions in vitro and thus
have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both
interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular,
Lactobacillus paracasei (strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the
potential to induce and suppress Th1 functions, respectively, we
addressed the impact of this bacterium on the outcome of
CD4+ T-cell differentiation. For this purpose, bacteria
were added to mixed lymphocyte cultures where CD4+ T-cells
from naive BALB/c mice were stimulated weekly in the presence of
irradiated allogeneic splenocytes. In these cultures, L. paracasei NCC2461 strongly inhibited the proliferative activity of CD4+ T cells in a dose-dependent fashion. This was
accompanied by a marked decrease of both Th1 and Th2 effector
cytokines, including gamma interferon, IL-4, and IL-5. In contrast,
IL-10 was maintained and transforming growth factor 
(TGF-) was
markedly induced in a dose-dependent manner. The bacteria were not
cytotoxic, because cell viability was not affected after two rounds of
stimulation. Thus, unidentified bacterial components from L. paracasei NCC2461 induced the development of a population of
CD4+ T cells with low proliferative capacity that produced
TGF- and IL-10, reminiscent of previously described subsets of
regulatory cells implicated in oral tolerance and gut homeostasis.
 |
INTRODUCTION |
The immunological properties of
lactic acid bacteria (LAB) have raised a lot of interest in recent
years due to their immune-stimulating properties (32).
Several strains of LAB were reported to display stimulatory properties
on cells of the innate immune system in vitro, including macrophages
(24) and NK cells (14, 25). Immune
stimulation in vitro was characterized by the induction of
proinflammatory cytokines, such as interleukin-12 (IL-12) (20, 23, 33) and tumor necrosis factor alpha (15, 16).
This increase of innate immune functions was mirrored in vivo using animal models (39-42) and in humans given fermented milk
products containing probiotics (46, 47). It has therefore
been proposed that LAB could be used as nonspecific adjuvants of innate
immune responses to increase early defense mechanisms in response to gastrointestinal infections.
Innate immune responses not only serve as an early line of defense
against invading pathogens but also are crucial for the development of
subsequent acquired immune responses that are orchestrated by
CD4+ T cells (31). Murine CD4+ T
lymphocytes can be classified into several subsets depending on the
type of cytokines they produce. Originally, two major subsets of
effector CD4+ T cells were described as the Th1 and Th2
subsets. Th1 and Th2 cells produce mainly high levels of gamma
interferon (IFN-
) and IL-4/IL-5 respectively and carry out distinct
key regulatory functions in an immune response (35).
Th1-derived cytokines mediate principally the cell-mediated immune
functions, such as trigger killing of intracellular parasites by
macrophages, whereas Th2 cytokines mostly favor the generation of
humoral responses dominated by immunoglobulin E that are required for
elimination of helminth infections (reviewed in reference
36). In the mouse, when these two types of responses are
strongly polarized, they are by and large mutually exclusive and
regulate each other through feedback loops mediated by regulatory
cytokines, such as IL-12 and IL-10 (5, 8, 29). The balance
between the two types of responses is considered important in
maintaining homeostasis of the host, since a number of diseases that
have been associated with an exaggerated Th1 or Th2 response are linked
to abnormal production of these cytokines. This balance is thought to
be maintained by specialized subsets of regulatory cells that produce
suppressive cytokines such as IL-10 and transforming growth factor (TGF-) (2, 12). The signals that drive the
differentiation of naive CD4+ T cells toward distinct
effector or regulatory phenotypes have been extensively studied. Murine
naive CD4+ T cells that are primed by antigen-presenting
cells and antigen in the presence of IL-12 preferentially develop
toward the Th1 phenotype (26), whereas the presence of
IL-4 favors Th2 differentiation (38). While the early
sources of IL-4 remain somewhat controversial (4), it
appears that early IL-12 production stems from components of the innate
immune system, such as macrophages stimulated by pathogenic
gram-positive bacteria (22). Interestingly, the genetic background of the responding CD4+ T cells determines the
default pathway toward either phenotype if no exogenous factors are
added during the priming phase (21).
Most of the studies on mechanisms of in vitro CD4+ T-cell
differentiation mentioned above have made use of peptide-specific T-cell receptor transgenic naive CD4+ T cells. A simpler
method to study the mechanisms of CD4+ T cell
differentiation has been the mixed lymphocyte reaction (MLR), where
purified CD4+ T-cell responders from naive mice are mixed
with allogeneic irradiated whole splenocytes as accessory cells
(17). These MLR studies have allowed study of the
proliferative capacity and cytokine production profiles of distinct
murine CD4+ T-cell subsets distinguished on the basis of
cell surface markers and to address the importance of different types
of accessory cells in this process (17, 18). Because LAB
strongly induce innate IL-12 in accessory cells (20, 23,
33), we have used this MLR system to evaluate the impact of LAB
on the subsequent generation of Th1 and Th2 effector functions.
| |
MATERIALS AND METHODS |
Mice and bacteria.
Female 6-week-old BALB/c and C57BL/6 mice
were purchased at Iffa-Crédo (L'Abresle, France) and were
maintained under specific-pathogen-free conditions in our animal
facilities at the Nestlé Research Center, Lausanne, Switzerland,
and in accordance with the ethical regulations of the Veterinary
Service of the Canton de Vaud, Switzerland. Mice were sacrificed before
8 weeks of age by cervical dislocation under 3% Isoflurane anesthesia
(Abbot SA) for sampling of spleens.
All strains of lactobacilli were cultured in MRS broth without acetate
at 37°C under anaerobic conditions. L. johnsonii NCC533, L. gasseri NCC2493, and L. paracasei NCC2461 were
originally isolated from human feces. L. acidophilus NCC90
was originally provided by The University of Piacenza. L. casei strain Shirota was isolated from a commercial product
(Yakult, Japan). L. casei strain GG was obtained from Valio
(Finland). All bacteria were harvested by centrifugation
(3,000 × g for 15 min) at stationary growth phase.
Pelleted bacteria were then washed three times in phosphate-buffered saline (PBS) and diluted to a final working concentration of
109 CFU/ml in complete medium, i.e., RPMI 1640 medium
containing 10% inactivated fetal calf serum (FCS), 2 mM
L-glutamine, 0.05 mM 2-mercaptoethanol, 100 U of penicillin
per ml, and 100 mM Streptomycin (all reagents from Life Technologies
AG). This stock suspension was aliquoted and stored at 
80°C. One
fresh aliquot was thawed for every new experiment to avoid variability
in the cultures between experiments.
Stimulation of splenocytes with LAB.
Whole splenocytes were
obtained from BALB/c or C57BL/6 mice by homogenizing spleens with a
tissue grinder. After elimination of erythrocytes, spleen cells were
washed three times in ice-cold Hanks' balanced salt solution (Life
Technologies AG) containing 5% FCS and were resuspended at
107 cells/ml in complete medium. Cells
(106/well) were cultured in 96-well plates in the presence
or absence of various concentrations of bacteria (concentrations are
indicated in the figure legends). Lipopolysaccharide (LPS)
(Escherichia coli serotype O55:B5; Sigma) was added at 1 µg/ml as a positive control culture for IL-10 production. After
24 h of culture, the supernatants were subjected to IL-12p40 and
IL-10 quantification by enzyme-linked immunosorbent assay (ELISA).
MLR.
CD4+ T cells were purified from
erythrocyte-depleted spleens of BALB/c mice using an anti-CD4
monoclonal antibody coupled to MACS microbeads (Myltenyi Biotec,
Bergisch Gladbach, Germany) as specified by the manufacturer. Cell
purity was verified by flow cytometric analysis and exceeded 90%.
Purified CD4+ T cells (105 cells/well) were
mixed with irradiated (3,000 rads) allogeneic splenocytes
(106 cells/well) from C57BL/6 mice in 200 µl of complete
medium in round-bottom 96-well plates. Cultures were incubated for 7 days at 37°C in a 5% CO2 incubator under 80% humidity.
After this primary culture, CD4+ T cells were washed,
purified again using the MACS system, and restimulated for another 7 days with freshly isolated irradiated C57BL/6 splenocytes in a
secondary culture under conditions identical to these in the primary
culture. During the primary and secondary weekly stimulations, the
cultures contained either medium alone, LPS (1 µg/ml), blocking
monoclonal antibody (MAb) to IL-4 (clone 1D11; Pharmingen), or bacteria
added at concentrations of 107 or 106 CFU/ml.
After the secondary culture, a fraction of the live CD4+ T
cells were analyzed by flow cytometry for memory markers as characterized by low expression of MEL-14 (CD62L) and high expression of CD44 (Pharmingen). The remainder of the cells were washed, purified
one last time by MACS, and stimulated for 48 h in medium alone in
the presence of fresh irradiated C57BL/6 splenocytes. After this time,
supernatants were collected and frozen at 20°C until analyzed by
ELISA. To measure cell proliferation, cells were pulsed for a further
16 h with 1 µCi of [methyl-3H] thymidine
(Amersham, Zürich, Switzerland). The cells were then harvested on
nitrocellulose filters (Packard, Zürich, Switzerland), and bound
[methyl-3H]thymidine was measured by
scintillation counting (TopCount; Packard).
ELISA.
Cytokines were detected in culture supernatants by
sandwich ELISA. IFN- was detected using R4-6A2 and biotinylated
XMG1.2 as coating and detecting MAbs, respectively. For IL-4 detection, 1D11 and biotinylated 24G2 were used; for IL-5, TRFK4 and biotinylated TRFK5 were used; for IL-10, 16E3 and biotinylated 2A5 were used (all
MAbs from Endogen, Woburn, Mass.). Briefly, coating antibodies were
incubated on Maxisorp ELISA plates (Life Technologies AG, Basel,
Switzerland) at 5 µg/ml in PBS overnight at room temperature. After
four washes in PBS containing 0.05% Tween 20, the wells were blocked
for 1 h at room temperature with PBS containing 20% FCS and
0.05% Tween 20. After further washing, supernatants were added at a
dilution of 1:2 and the samples were serially diluted in culture medium
and incubated in the presence of negative (normal medium) and positive
(recombinant standard; Pharmingen) controls for 4 h at room
temperature. After further washing, secondary biotinylated MAbs were
added at a concentration of 0.5 to 2 µg/ml and the samples were
incubated for 1 h at room temperature. The wells were then washed,
1 µg of horseradish peroxidase-labeled streptavidin (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added, and the wells were
incubated for 30 min. Plates were washed one last time and incubated
with TMB microwell substrate (Kirkegaard & Perry). The reactions were
stopped with 1 M phosphoric acid. Optical densities were read at 450 nm. TGF- and IL-12p40 were quantified using the Quantikine
anti-human and Quantikine M ELISA kits, respectively from R&D Systems
(Abingdon, United Kingdom). Cytokine levels were extrapolated from the
standard curve calculated from dilutions of the recombinant cytokines.
Statistics.
All experiments were carried out at least three
times, and cultures were performed in triplicate wells. Values and
error bars in the graphics represent the mean ± standard error of
the mean (SEM). In Table 1, the SEM is not shown and did not exceed
12% of the means. When necessary, Student's paired t test
was performed on the data to assess significant differences
(P < 0.05).
| |
RESULTS |
Distinct strains of lactobacilli rapidly induce different levels of
IL-12 and IL-10 protein synthesis in murine spleen cells.
There is
evidence that nonpathogenic gram-positive bacteria may be strong IL-12
inducers in mononuclear adherent cells while gram-negative bacteria are
more efficient IL-10 inducers (19). Several strains of
lactobacilli (LAB) were tested for their capacity to induce the
secretion of IL-12 and IL-10 after 24 h of culture with BALB/c
splenocytes. All tested strains induced IL-12p40, although at very
different levels (Fig. 1A). It appeared
that L. paracasei NCC2461 was the strongest IL-12p40 inducer
(>160 pg/ml at 107 CFU/ml). Surprisingly, we observed that
all LAB strains also induced substantial IL-10 synthesis (Fig. 1B),
albeit somewhat lower levels than those of IL-12p40. L. paracasei strain NCC2461 and L. casei strain GG induced
the largest amounts of IL-10 (>60 pg/ml at 107 CFU/ml).
With these bacteria, detectable amounts of IL-10 were still measured at
106 and 105 CFU/ml whereas no or low levels of
IL-10 were measured at these doses with the other strains. Cultures
containing 1 µg of LPS per ml produced on average 520 ± 100 pg
of IL-10 per ml in their supernatants, while the IL-12p40 level
remained low to undetectable (<5 pg/ml).
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FIG. 1.
Induction of IL-12p40 (A) and IL-10 (B) by various
strains of lactobacilli. Among the bacterial strains used were L. johnsonii NCC533, L. acidophilus NCC90, L. gasseri NCC2493, L. paracasei NCC2461, and two strains
of L. casei, Shirota and GG. Bacteria were added to spleen
cultures at concentrations of 107 CFU/ml (black boxes),
106 CFU/ml (grey boxes), or 105 CFU/ml (white
boxes). Error bars indicate the SEM of triplicate cultures.
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Because IL-12 and IL-10 have mutually antagonistic functions (5,
8, 29), we were interested in determining whether, among the LAB
strains tested, those inducing high levels of IL-10 would be poor IL-12
inducers and vice versa. This appeared not to be the case, since
L. paracasei NCC2461 triggered the highest production of
both IL-12 and IL-10 whereas L. casei Shirota, a weak IL-10
inducer, was also a poor IL-12 inducer. Therefore, there did not appear
to be a pattern of reciprocal IL-12 and IL-10 induction among the LAB
strains tested but, rather, a parallel induction of both cytokines.
A similar pattern of IL-12 and IL-10 synthesis in response to these
various LAB strains was found in spleen cells from C57BL/6 mice (data
not shown), suggesting that induction of IL-12 and IL-10 by LAB was not
dependent on the genetic background of the accessory cells.
L. paracasei NCC2461 inhibits CD4+ T-cell
proliferation.
Because L. paracasei NCC2461 was the
most effective inducer of IL-12 and IL-10, this strain was used
throughout the following experiments. MLR of purified CD4+
T cells from naïve BALB/c mice were maintained by weekly
restimulation with irradiated allogeneic splenocytes from C57BL/6 mice.
During the 2 first weeks of the cultures, the cells were primed in
medium alone or in the presence of various concentrations of L. paracasei NCC2461, 1 µg of LPS per ml, or 10 µg of blocking
MAb against IL-4 per ml. LPS was used as a positive control for rapid
induction of elevated amounts of IL-10 in these cultures. The anti-IL-4 MAb was added to block endogenous production of IL-4, which has been
shown in another system to prevent the default differentiation of
BALB/c CD4+ T cells toward the Th2 phenotype and to induce
a switch to the Th1 phenotype (38). After the 2-weekly
priming cultures, CD4+ T cells were restimulated one last
time with irradiated allogeneic splenocytes in medium alone and
proliferation was measured. In contrast to cells primed in the presence
of medium alone, which proliferated vigorously, we observed a marked
(P < 0.05) inhibition of CD4+ T-cell
proliferation when L. paracasei NCC2461 was added at
107 CFU/ml to the priming cultures (Fig. 2). This
inhibition was still observed (P < 0.05) when the
bacteria were added at 106 CFU/ml, but to a lesser degree.
When LPS and anti IL-4 MAb were added to the priming cultures, cell
proliferation was also strongly decreased (Fig.
2).
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FIG. 2.
Inhibition of CD4+ T-cell proliferation by
L. paracasei NCC2461. CD4+ T cells were primed
in medium alone () or in the presence of L. paracasei
NCC2461 (107 or 106 CFU/ml), LPS (1 µg/ml),
or a blocking MAb against IL-4 (1D11; 10 µg/ml). Proliferation was
measured 48 h after the third stimulation. Error bars indicate the
SEM of triplicate cultures.
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Since proliferation was low in wells containing L. paracasei
NCC2461, LPS, and anti-IL-4 MAb, we examined the viability of the cells
grown under these conditions. After 4 and 7 days following the second
stimulation, the cells were stained with trypan blue to assess
viability and with a CD4-specific MAb to quantify the proportion of
CD4+ T cells by fluorescence-activated cell sorting. When
cells were cultured in medium alone, the total number of cells
decreased and the proportion of CD4+ T cells increased
(Table 1). This showed that there was an
expansion of CD4+ T cells concomitantly with a loss of
viable irradiated splenocytes in these cultures. In wells containing
L. paracasei NCC2461, LPS, and anti-IL-4 MAb, there was a
minor but measurable increase in the relative proportion of
CD4+ T cell in the wells. However, because the total number
of cells tended to decrease, the actual number of CD4+ T
cells increased only a little in these wells. This demonstrated that
CD4+ T cells did expand somewhat after 2 weeks of culture
in the presence of L. paracasei NCC2461 and that they were
viable at this stage. Furthermore, 7 days after the second stimulation,
virtually all (>95%) live CD4+ T cells in the different
culture conditions displayed a memory phenotype (MEL-14lo
CD44hi), suggesting that the bacteria did not prevent
T-cell priming (data not shown).
Lastly, when L. paracasei NCC2461 was added to purified
CD4+ T cells grown on plastic-bound anti-CD3 (i.e., in the
absence of accessory cells) as described previously (9),
there was no significant inhibition of cell proliferation (data not
shown). This further shows that the bacteria were not toxic to growing CD4+ T cells and also implies that the suppressive effects
of L. paracasei NCC2461 required the presence of accessory cells.
L. paracasei NCC2461 inhibits the secretion of Th1 and
Th2 cytokines by CD4+ T cells.
Since L. paracasei NCC2461 inhibited CD4+ T cell proliferation,
we wanted to investigate whether production of effector cytokines was
also affected. Cytokine levels were measured 48 h after the third MLR
restimulation. When cells were differentiated in medium alone, they
secreted large amounts of effector cytokines, including IFN-, IL-4,
and IL-5 (Fig. 3). When they were
differentiated in the presence of 107 CFU of L. paracasei NCC2461 per ml, there was a sharp decrease in the
amounts of these three cytokines. However, IFN- was produced at
normal levels in the presence of 106 CFU of L. paracasei NCC2461 per ml, while IL-4 was partially suppressed
(P < 0.05) and IL-5 remained undetectable. Thus, the presence of L. paracasei NCC2461 during the differentiation
of CD4+ T cells had a negative impact on both Th1 and Th2
effector cytokines, but there appeared to be a dose threshold for the
suppression of IFN- and IL-4 in this system. Addition of LPS during
differentiation suppressed all three cytokines quite effectively
(P < 0.05). Addition of the blocking anti-IL-4 MAb
increased (P > 0.05) IFN- levels but strongly
inhibited (P < 0.05) IL-4 and slightly suppressed (P > 0.05) IL-5 secretion in the restimulation
cultures. Hence, LPS had an impact similar to the high concentrations
of L. paracasei NCC2461 in these cultures whereas anti IL-4
antibodies exclusively decreased the secretion of Th2 cytokines.
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FIG. 3.
Inhibition of Th1 and Th2 effector cytokines by L. paracasei NCC2461 in MLR. CD4+ T cells were primed in
medium alone () or in the presence of L. paracasei NCC2461
(107 or 106 CFU/ml), LPS (1 µg/ml), or a
blocking anti-IL-4 MAb (1D11; 10 µg/ml). Cytokine levels were
measured 48 h after the third stimulation. Error bars indicate the
SEM of triplicate cultures.
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L. paracasei NCC2461 maintains IL-10 and induces
TGF- secretion by CD4+ T cells.
To determine
whether the inhibitory properties of L. paracasei NCC2461
could be due to the concomitant induction of a suppressive phenotype,
we measured the production of IL-10 and TGF- in these cultures. When
CD4+ T cells were differentiated in medium alone, they
secreted high levels of IL-10 but no or low levels of TGF- (Fig.
4). Unlike all other cytokines tested
(Fig. 3), the addition of 107 CFU of L. paracasei NCC2461 per ml had no measurable inhibitory impact on
IL-10 secretion (Fig. 4). In addition, there was a dose-dependent induction of TGF-. Addition of LPS partially (P > 0.05) suppressed IL-10 secretion and induced intermediate levels
of TGF- in these cultures. Anti-IL-4 MAbs markedly inhibited IL-10
secretion (P < 0.05) but had no impact on TGF-
production. Hence, these data show that after 2 weeks of allogeneic
splenocyte stimulation in the presence of L. paracasei
NCC2461, effector CD4+ T cells produced predominantly IL-10
and TGF- while cells stimulated in medium alone produced IFN-,
IL-4, IL-5, and IL-10.
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FIG. 4.
L. paracasei NCC2461 does not suppress IL-10
and induces TGF- production by CD4+ T cells in MLR.
CD4+ T cells were primed in medium alone () or in the
presence of L. paracasei NCC2461 (107 or
106 CFU/ml), LPS (1 µg/ml), or a blocking anti-IL-4 MAb
(1D11; 10 µg/ml). Cytokine levels were measured 48 h after the
third stimulation. Error bars indicate the SEM of triplicate
cultures.
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| |
DISCUSSION |
Most reported studies on cytokine modulation by LAB in vitro have
focused on the induction of modulatory cytokines by innate components
of the immune system (15, 16, 20, 23-25, 33). It is well
known that innate immunity plays a crucial role in determining the type
of adaptive immune response that is generated consequently (reviewed in
reference 31). We therefore asked how the induction of
those innate cytokines by LAB could impact on the subsequent
development of CD4+ T-cell functions in vitro. To study the
impact of LAB antigens on CD4+ T-cell priming and
differentiation into effector cells, we have used a previously
described system of MLR (17, 18). In these cultures,
CD4+ T cells from naive BALB/c mice were stimulated
repeatedly on a weekly basis with allogeneic splenocytes. In a
different system that used naive TCR transgenic CD4+ T
cells primed and restimulated with the specific antigen over long
periods, BALB/c CD4+ T cells were reported to default
toward a dominant Th2 phenotype if no exogenous factor was provided in
the priming cultures (21). Because several LAB species
were shown to induce innate IL-12 in macrophages (20, 23,
33), our original aim was to see whether certain strains of LAB
could thereby antagonize the differentiation of BALB/c CD4+
T cells toward their default Th2 phenotype and, instead, induce a
switch toward a dominant Th1 response.
In our hands, both Th1 and Th2 cytokines were produced at high levels
by CD4+ T cells from BALB/c mice stimulated by allogeneic
splenocytes under neutral conditions. This could have been because
splenic CD4+ T cells from naive mice contained a minor
(<5%) population that displayed a memory (MEL-14lo
CD44hi) phenotype. In previous studies showing a default
toward Th2 in the BALB/c background, this subpopulation had been sorted
out (21). Surprisingly, L. paracasei (strain
NCC2461) inhibited the production not only of Th1 cytokines but also of
Th2 cytokines. This suppression correlated with a rapid (probably
innate) induction of the suppressive cytokine IL-10 in murine splenic
cells and with the emergence of a CD4+ T-cell phenotype
characterized by the production of IL-10 and TGF- and low
proliferative capacity. Because we have seen that LPS induced high
levels of innate IL-10 in murine splenocytes, we have used LPS in the
MLR cultures as a positive control for high levels of endogenous IL-10
and suppression of Th proliferation. Indeed, addition of LPS to the
priming cultures had effects similar to those elicited by L. paracasei NCC2461 given at high concentrations (107
CFU/ml), i.e., a strong suppression of effector cytokines and of
proliferation. Addition of a blocking anti-IL-4 MAb also inhibited CD4+ T-cell proliferation and Th2 cytokines (including
IL-10) but did not impair the Th1 response and did not induce TGF-
in the MLR cultures. Thus, as expected from other systems showing that IL-4 is a key cytokine for driving Th2 differentiation
(38), blocking endogenous IL-4 favored the switch to a
dominant Th1 response in these cultures. Altogether, the data showed
that L. paracasei NCC2461 inhibited the function of BALB/c
CD4+ T cells and that this inhibition was not due to a
switch to a Th1 phenotype but, rather, to a general suppressed state
resembling anergy that coincided with a decreased proliferative
capacity and the production of suppressive cytokines.
Previous studies have suggested that gram-positive bacteria tend to
preferentially induce IL-12 whereas gram-negative bacteria predominantly induce IL-10 in macrophages (19, 20). We
were surprised to detect substantial production of IL-10 in splenocytes cultured with LAB. IL-10 is a suppressive cytokine that inhibits IL-12
production by macrophages and Th1 functions induced by IL-12 and
IFN- (5, 8). Therefore, it appears that LAB, in
particular L. paracasei NCC2461, induced immunoregulatory
cytokines which may have opposite effects on Th1 responses. Although
the innate induction by L. paracasei NCC2461 of both IL-12
and IL-10 seems difficult to reconcile, it is possible that the
residual dominant effect of either cytokine on CD4+ T-cell
differentiation may depend on the genetic background of the responding
T cells. Indeed, earlier studies have shown that Th1 responsiveness to
IL-12 depends on the expression of its receptor 2 chain, which is
switched off upon Th2 differentiation (50). Accordingly,
the IL-12 receptor 2 chain is regulated differently in various
genetic backgrounds (13). The experiments described in
this paper were performed with CD4+ T cells from BALB/c
mice, a mouse strain that rapidly loses surface expression of IL-12
receptor 2 chain after priming (13). Interestingly, the
attenuation of IL-12 receptor expression has been reported to require
the presence of TGF- (10), a cytokine that was markedly induced in our cultures containing L. paracasei NCC2461.
Therefore, our current hypothesis is that the early induction of IL-12
by L. paracasei NCC2461 in accessory cells prevented the
genetically programmed Th2 differentiation of naive BALB/c T cells and,
instead, induced an early differentiation toward a Th1 phenotype.
However, this early Th1 response was subsequently downregulated by
endogenous IL-10 (which blocked further IL-12 secretion) and TGF-
(which downregulated IL-12 responsiveness) in a synergistic manner.
In line with this hypothesis, we observed that the suppression of
IFN- and IL-4 was completely and partially lost, respectively, when
lower concentrations (106 CFU/ml) of bacteria were added to
the cultures, suggesting a threshold in the suppressive effects of the
bacteria. This was perhaps because less TGF- was produced under
these conditions and further strengthens the idea that TGF- may play
a key role in the suppressive effects of L. paracasei
NCC2461. It should be noted, however, that LPS also strongly suppressed
proliferation and IFN- production, although it induced less TGF-
than did 106 CFU/ml of bacteria. However, LPS induced far
stronger production of IL-10 in primary cultures. It therefore remains
likely that LPS and L. paracasei NCC2461 elicited their
suppressive effects by different mechanisms. Addition of blocking
antibodies to IL-10 and/or TGF- in the priming cultures may resolve
the relative importance of these two cytokines in the suppressive
effects elicited by the bacteria or by LPS.
To our knowledge, there have been no previous in vitro data
demonstrating inhibition of Th1 cytokines by LAB in developing CD4+ T cells. Instead, LAB have been shown to promote Th1
functions (3, 19, 20, 23) and decrease Th2 responses
(30, 37, 49) in numerous studies. A few studies have
nevertheless reported induction by LAB of IL-10 (16, 34)
and suppression of Th1-related immunopathologies, such as inflammatory
bowel disease (27, 48). Inflammatory bowel disease had
been associated with elevated levels of Th1 cytokines in the colon
(44) and by a dysregulation in the maintenance of
homeostasis by IL-10 (1) and TGF- (43). Experiments in animal models have shown that the endogenous bacterial flora may contribute to the disease (28, 45), but its
interaction with pathogenic T cells is not understood. It has
nonetheless been suggested that the disease was a consequence of a loss
of T-cell tolerance to resident bacteria (6) and that the
balance between IL-12 and IL-10 played a key role in this process
(7). Recently, it was shown that activation of
CD4+ T cells in the presence of IL-10 contributed to their
differentiation toward a novel phenotype of cells, named Tr1, that
produced high levels of IL-10 and TGF- (12). The same
study showed that Tr1 cells could prevent the onset of colitis in SCID
mice containing CD45RBhi cells (12), probably
by the induction of an anergic state in pathogenic Th1 cells
(11). The existence and specificity of these Tr1
regulatory cells in vivo is not known, but it is conceivable that their
activity may be regulated by components of the enteric bacterial flora.
This paper reports that components from a LAB strain induce a Tr1-like
population that produces substantial levels of TGF- and IL-10 and
provides evidence for the mechanism by which probiotic bacteriotherapy
may prevent intestinal inflammation. This question deserves to be
addressed in vivo using mouse models of colitis.
We gratefully acknowledge the technical assistance of Kim-Yen
Saudan and the critical reading of the manuscript by Nabila Ibnou-Zekri, Dirk Haller, and Pierre Guesry.
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and
Interleukin-10
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Abstract
Introduction
