Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells

Abstract

Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of
Lactobacillus plantarum
Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by
Salmonella
Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-
α
, IL1-
β
, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4
+
CD25
+
T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells.
L. plantarum
Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases.

Full text

Research Article
Immunomodulatory Effects of Lactobacillus plantarum Lp62 on
Intestinal Epithelial and Mononuclear Cells
Thalis Ferreira dos Santos,1Tauá Alves Melo,2Milena Evangelista Almeida,2
Rachel Passos Rezende,2and Carla Cristina Romano2
1State University of Feira de Santana, Transnordestina Avenue S/N, 44030-900 Feira de Santana, BA, Brazil
2State University of Santa Cruz, Highway Ilh´
eus-Itabuna, km 16 S/N, 45662-900 Ilh´
eus, BA, Brazil
Correspondence should be addressed to Carla Cristina Romano; romanocc@uol.com.br
Received  March ; Revised  June ; Accepted  June 
Academic Editor: David Bernardo
Copyright ©  alis Ferreira dos Santos et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Probiotic lactic acid bacteria are known for their ability to modulate the immune system. ey have been shown to inhibit
inammation in experiments with animal models, cell culture, andclinical tr ials. e objective of this study was to elucidate the anti-
inammatory potential of Lactobacillus plantarum Lp, isolated from cocoa fermentation, in a cell culture model. Lp inhibited
IL- production by Salmonella Typhi-stimulated HT- cells and prevented the adhesion of pathogens to these epithelial cells.
e probiotic strain was able to modulate TNF-𝛼, IL-𝛽, and IL- secretion by J macrophages. J activation was reduced by
coincubation with Lp. PBMC culture showed signicantly higher levels of CD+CD+Tlymphocytesfollowingtreatmentwith
Lp. Probiotics also induced increased IL- secretion by mononuclear cells. L. plantarum Lp was able to inhibit inammatory
stimulation in epithelial cells and macrophages and activated a tolerogenic prole in mononuclear cells of healthy donors. ese
results indicate this strain for a possible application in the treatment or prevention of inammatory diseases.
1. Background
Probiotics are dened as live microorganisms which, when
administered in adequate amounts, promote benecial eects
on the host’s health. Microbial genera commonly associated
with probiotic eects usually have the ability to restore the
balance of microbiota, regulate intestinal trac, produce
short-chain fatty acids, and compete with pathogens for
adhesion sites. Other properties, such as immune modulation
and production of specic bioactive substances, are restricted
to some strains. Traditionally, probiotics are used to treat or
prevent the imbalance of the intestinal microbiota caused by
pathogens and/or resulting from antibiotic therapy. However,
new approaches have demonstrated the potential of these
microorganisms as adjuncts to the treatment or prevention
of intestinal and extraintestinal chronic diseases [–].
Inammatory bowel diseases (IBD) have increased espe-
cially in western countries. Despite being considered to
be caused by multifactorial conditions, the gut microbial
population plays a central role in the development of IBD in
genetically susceptible individuals []; therefore, therapeutic
approaches that modify the local microbiota are very attrac-
tive. In this context, probiotics can stimulate the immune
system, resulting in modulation of inammatory mediators
that are responsible for the maintenance of the pathological
process or directing the innate and adaptive responses in a
regulatory sense [].
L. plantarum is a Gram-positive rod-shaped bacterium
found in a wide variety of niches such as vegetables, meat,
sh, and the gastrointestinal tract. Due to its ubiquity and
importance in various fermentation processes, it was the
rst species of the genus Lactobacillus to have its genome
sequenced. Further sequencing revealed considerable genetic
diversity among strains isolated from dierent environments,
which explains the high adaptability of these lactic acid
bacteria []. A number of studies prove the applicability
of various strains of L. plantarum as probiotic. e v
strain, used in an already marketed probiotic, reduced in vitro
expression of proinammatory genes in a culture model of
colonic mucosa []. In addition to anti-Helicobacter pylori
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BioMed Research International
Volume 2016, Article ID 8404156, 8 pages
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BioMed Research International
activity [], it was also able to improve the symptoms of
irritable bowel syndrome in a clinical study using  patients
[]. L. plantarum Lp showed strong immunoregulatory
capacity in a murine colitis model induced by TNBS [],
and the WCFS strain was eective in generating regulatory
T cells in healthy individuals [].
e probiotic characteristics of each isolated strain are
specic. Dierent species or variants within the same species
can interact with the local microbiota and the host immune
system in particular ways. Consequently, the use of Lacto-
bacillus species as a probiotic needs careful selection to clarify
their potential, mechanisms, and technological properties.
L. plantarum Lp was isolated from a batch of fermenting
cocoa beans and identied by S rDNA gene sequencing
(GenBank access number KU). Its probiotic potential
was attested previously in a study that evaluated its anti-
inammatory capacity in a colitis model induced by acetic
acid in mice []. However, strain Lp was administered in a
pool of other strains, making it dicult to establish the role of
each microorganism in the observed eect. In this study, we
sought to rene this research, by endeavoring to propose a
possible in vitro anti-inammatory mechanism. Strain Lp
modulated the inammatory response in epithelial cells by
preventing S. Typhi adhesion, inhibited macrophage activa-
tion and thereby decreased the levels of cytokines involved
in IBD pathogenesis, and, nally, increased IL- levels in
mononuclear cells of healthy donors.
2. Materials and Methods
2.1. Cell Strains. HT- cells, a cell line derived from human
colon adenocarcinoma, were cultured in -well plates, in
DMEM (Gibco) supplemented with % fetal bovine serum
(Gibco) and  U⋅mL−1 penicillin and streptomycin, at an
initial concentration of 6cells⋅mL−1,at
∘Cand%CO
2.
e cultures were maintained for d until the experiment
day, and, during that period, the medium was replaced every
two days.
e macrophage cell line JA. (ATCCTIB-) was
cultured at a concentration of 5×10
5cells⋅mL−1 in RPMI
(Gibco) medium supplemented with % fetal bovine serum
and  U⋅mL−1 streptomycin and penicillin, for  d in %
CO2and ∘C atmosphere, and the medium was replaced
every two days until the experiment day.
Before inoculating microorganisms in the cell cultures,
the medium was replaced with no added antibiotic.
2.2. Microorganisms. L. plantarum Lp was cultured in MRS
medium (HiMedia) for  h at ∘C. e culture was then
washed twice in .% NaCl solution and used at a titer of
1×10
9CFU⋅mL−1.
Salmonella enterica serovar Typhi ATCC  was cul-
turedinTrypticSoyBrothmedium(HiMedia)forhat
∘C,whilestirringatrpm.eculturewaswashedwith
.% NaCl solution and diluted to reach 𝐴600 =.,which
corresponds to 8CFU⋅mL−1.
2.3. Separation of Peripheral Blood Mononuclear Cells
(PBMCs). Ten healthy donors were selected for blood
collection. e group was composed of six men and four
women, average age  years. Each individual took part in
the study by signing the free informed consent term. e
collection of blood from healthy donors was approved by the
local ethics committee on human research (access number
), in accordance with guidelines established by the
National Health Council. Blood was collected from donors
in heparinized tubes and peripheral blood mononuclear cells
were separated using HystopaqueSigma.  mL Hystopaque
and  mL blood were added to a conical tube. Aer
centrifuging at  ×g for  min, the mononuclear cells
were collected and washed with RPMI. e concentration
was adjusted to 5×10
5cells⋅mL−1 and the cells were then
grown in RPMI supplemented with % fetal bovine serum
at ∘Cand%CO
2.
2.4. Cytometry. For cytometric analysis, the cells were
washed with PBS ( rpm,  min). To detect internal
antigens, the cells were permeabilized using formaldehyde/
saponin-based permeabilization IntraPrepKit (Beckman-
Coulter). e macrophage lineage JA. was externally
labeled with anti-CD-APC and anti-CD-FITC. e HT-
 line was externally and internally labeled with anti-TLR-
-PE and anti-TLR-FITC. Mouse IgG conjugated to FITC,
PE, or APC was used as isotype control. PBMCs were
externally labeled with anti-CD-FITC, anti-CD-PE, and
intracellular anti-Foxp-PE staining. Analyses were made
in FC Beckman-Coulter cytometer. Data were processed
using the Kaluzaowanalysissoware.
2.5. ELISA. Aer bacterial cell coculture assays, the super-
natants were collected for cytokine quantication by ELISA.
e sandwich-ELISA procedures were performed according
to the manufacturer’s instructions. Kits for measurement
of IL-, IL-, IL-𝛽,IL-,IFN-𝛾,TNF-𝛼, and IL- were
obtained from PeproTech, Brazil.
2.6. Coculture Assays. An HT- cell culture was inoculated
with L. plantarum Lp (9CFU⋅mL−1)andincubatedfor
h at 
∘Cand%CO
2.en,thewellswerewashedwith
PBS, inoculated with S. Typhi  at a concentration of
8CFU⋅mL−1,andincubatedforh.Inparallel,S. Typ hi
 and Lp were added to HT- culture for  h, simulta-
neously. Aer cell-bacteria interaction, the supernatants were
collected for cytokine assay. HT- cells were treated with
trypsin-EDTA solution .%, for cell detachment. e plates
were incubated for  minutes at ∘C and then the trypsin
was inactivated with fetal bovine serum. e cells were
washed with RPMI and sent to ow cytometry. e eect of
nonviable Lactobacillus plantarum Lp cells was also tested.
Accordingly, a bacterial cell suspension was inactivated by
heating at ∘C for  minutes. Cell viability was tested
by plating on MRS medium. In addition, the proportion
of adhering S. Typhi related to the initial inoculum was
assessed by serial dilution and plating on MacConkey agar
andtheadherencepercentagewascalculatedbytheformula
% adherence = CFUnal/CFUinitial ∗ 100.Jcellswere
stimulated with  𝜇LL. plantarum Lp (9CFU⋅mL−1)and
LPS ( ng⋅mL−1)andincubatedforhat%CO
2and ∘C.

BioMed Research International 
PBMC cultures were similarly challenged, but the samples
were incubated for  h. Supernatants were collected and
J cells were detached by using cold RPMI. e cells were
processed and analyzed by ow cytometry.
2.7. Data Analysis. e data shown represent the mean ±
SD of the triplicate from three independent experiments.
e statistical dierence between the media (ANOVA) was
assessed using GraphPad Prism . soware.
3. Results and Discussion
e gastrointestinal tract mucosa is home to a diverse and
large population of microorganisms. e epithelial layer and
mucosa-associated immune system should be regulated in
order to tolerate the resident microbiota and food antigens
andsimultaneouslyremainreadytorespondtoinvasionof
enteric pathogens. Accordingly, imbalance in the axis toler-
ance versus response leads to the development of a state of
chronic intestinal inammation, including ulcerative colitis
(UC) and Crohn’s disease (CD). Despite their peculiarities,
inammatory bowel diseases (IBD) are characterized by loss
of epithelial barrier integrity, changes in expression level and
spatial location of innate receptors, and increased production
of proinammatory cytokines []. In view of their eects on
the immune response, probiotics have been used eectively
in the treatment of gastrointestinal tract disorders. As the
immune system is complex and compartmentalized, each
probiotic strain interacts in a particular way, resulting in a
specic response. In this study, we aimed at determining
the anti-inammatory eect of the L. plantarum Lp strain,
testing its activity in vitro in a cell culture model.
Lp was isolated from cocoa pulp during seed fermen-
tation. is strain was originally tested in a fermented milk
drink containing other isolates from the same environment
and was able to reverse chemically induced colitis in a
nonisogenic animal model. However, the process of stan-
dardization, quality control, and industrial-scale production
of multistrain probiotic formulations is quite laborious, so
we prefer to focus studies on the strain with the most
promising results. Initially, the Lp anti-inammatory eect
wastestedontheHT-intestinalepithelialcelllineand
the pathogenic bacterium S. Typhi  was used as an
inammatory stimulus. For this approach, the probiotic
bacteria were added before adding the pathogen, or both were
added simultaneously to the cell culture. Aer incubation, IL-
 production and the expression of Toll-like receptors  and 
were evaluated. We also quantied pathogen adhesion to the
epithelial cell in all treatments.
L. plantarum Lp signicantly reduced IL- production
byHT-cells.Incomparisonwiththecontrol(.ng⋅mL−1),
which was only S. Typhi-stimulated, there was an approx-
imately -fold reduction in both groups, treated with the
probiotic prior to addition or simultaneously to the pathogen
challenge (. ng⋅mL−1). When the epithelial cell culture
was stimulated with the probiotic alone, there was no sig-
nicant cytokine production. Additionally, heat-inactivated
Lp anti-inammatory activity was investigated and it
was observed that this group showed no decrease in IL-
1.0
0.9
0.8
0.7
0.6
0.5
0.020
0.015
0.010
0.005
0.000
a
b
b
c
Medium
S. Typhi
Lp62
Lp62/S. Typhi
Lp62/S. Typhi (S)
Lp62 (HI)
Lp62 (HI)/S. Typhi
[IL-8] (ng·mL−1)
F : Quantication of IL- secreted by HT- in culture
supernatant. HT- cells were treated with Lp and S. Typh i.
Levels of IL- secreted into the culture medium were measured.
Unstimulated cultures or cultures stimulated only with Lp or S.
Typ hi 𝑖were used as controls. S: inoculated simultaneously; HI:
heat-inactivated. aSignicant dierence from the medium (without
any stimulation). bSignicant dierence from S. Typhi-stimulated
group. cSignicant dierence from Lp/S. Typhi; 𝑃 < 0.05.
(±. ng⋅mL−1), detected by ELISA (Figure ). In accordance
with these data, adherence of S. Typhi  to HT- cells was
statistically reduced in the groups treated with the probiotic
Lp (Figure ), showing that its anti-inammatory action, in
this model, may be related to the probiotic ability to prevent
contact of the epithelial cell with the pathogen or competition
for adhesion sites. Interestingly, the group treated with heat-
inactivated probiotics had a higher percentage of pathogens
attached to epithelial cells compared to other groups treated
with probiotics, although it was signicantly lower when
compared to the control treated only with S. Typhi. is is
probably the reason why this treatment has been unable to
reduce IL- levels.
IL- is a chemokine that has chemoattractant activity,
leading neutrophils to the site of the inammatory stimulus.
Like TNF-𝛼and IL-, it is expressed at high levels in the
colonic mucosa of IBD patients []. e ability of probiotics
to reduce in vitro IL- levels is well documented and serves
as one of the basic parameters in the selection of probiotic
bacteria with this potential. Ren et al. [] observed a
decrease in IL- produced by Caco- cells prestimulated by L.
plantarum and challenged with Salmonella Typhimu rium . In
line with our ndings, the probiotic caused strong inhibition
of pathogen adhesion. e heat inactivation also led to
loss of the anti-inammatory eect. Carey and Kostrzynska
[] reported that preincubation with Lactobacillus and Bi-
dobacterium supernatantwasabletoinhibitIL-secretion
by epithelial cells challenged with S. Typhimurium. e
eect was lost when probiotics were inactivated by heat. As
in the present study, these observations suggest that some

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40
30
20
10
0.8
0.6
0.4
0.2
0.0
%S. Typhi adherence to HT-29 cells
S. Typhi
Lp62/S. Typhi
Lp62/S. Typhi (S)
Lp62 (HI)/S. Typhi
∗
∗
∗
F : Percentage of S. Typhi adherence to HT- cells. HT-
cells were treated with Lp, and then S. Typhi was added to the
culture. Aer incubation, the probiotic ability to inhibit pathogen
binding to the epithelial cell was measured. e percentage of S.
Typhiadherence was calculated in relation to the initial inoculum 1×
108.S:simultaneouslyinoculated;HI:heat-inactivated. ∗Signicant
dierence in relation to S. Typhi-stimulated group (𝑃 < 0.05).
factor released by metabolically active probiotic bacteria is
responsible for the observed eect. We may thus emphasize
thattheinhibitionofpathogenadhesioncancontributetothe
anti-inammatory action.
No changes were detected in TLR expression in
any experimental group. Interestingly, TLR- intracellular
expression was found to be increased in Lp-treated groups
but did not dier signicantly from the S. Typhi-stimulated
control. When probiotic and pathogen were given simultane-
ously, there was a signicant increase in receptor expression
(Figure ). LPS is a TLR- agonist. Under stimulation, the
receptor triggers transcription of proinammatory genes. In
the intestinal mucosa, the receptors that recognize microbe-
associated molecular patterns are expressed at low levels
to avoid overstimulation and thus chronic inammation.
Alternatively, these receptors are expressed in a compart-
mentalized way, like TLR-, which recognizes agellin and
is expressed basolaterally and is activated only if the colonic
mucosa is invaded []. Despite its anti-inammatory prole,
Lp was able to raise TLR- expression; however, it was
detectable only internally. According to Karlsson et al. [],
L. plantarum canberecognizedbyTLR-,but,inour
experiments, we believe that it was not able to activate the
downstream route that leads to the production of proin-
ammatory cytokines such as IL-. However, we did not
investigate other products of TLR- activation in this cell
model.
Macrophages located in the intestinal lamina itself rep-
resent the major reservoir of these cells in the human body.
ey are adapted to eciently remove any pathogen that
triestocrossthemucosa,whilemaintaininghomeostasisof
60
40
20
0
TLR-4-PE HT-29
mean fluorescence intensity
a
aa
a, b
Medium
S. Typhi
Lp62
Lp62/S. Typhi
Lp62/S. Typhi (S)
F : TLR- expression in HT- cells. HT- cells were
stimulated with Lp and then challenged with S.Typhi.Inparallel,
the eect of simultaneous (S) addition of the two microorganisms
was tested. HT- cells were labeled internally with anti-TLR-
and analyzed by ow cytometry. aStatistically dierent from the
medium (unstimulated cell). bStatistically dierent from the S.
Typhi-stimulated group; 𝑃 < 0.05.
the intestinal environment []. Considering that changes
in the phenotypic and functional prole of these cells have
implications in IBD pathogenesis, we decided to evaluate
the Lp strain’s capacity to inhibit the inammatory stim-
ulus in a J macrophage cell line. Secretion of TNF-𝛼,
IL-𝛽,IL-,andIL-wasmeasuredinthecellculture
supernatant and surface CD expression was evaluated by
ow cytometry. J cell stimulation with LPS increased
TNF-𝛼IL- secretion  and  times, respectively (Figures
(a) and (b)). Simultaneous cell challenge with Lp and
LPS signicantly decreased the secretion of these cytokines
relative to the LPS control. Lp was also able to stimu-
late TNF-𝛼release, but . times less than LPS-stimulated
cell. Despite showing similar performance, with decreased
secretioncomparedtotreatmentwithLp,IL-showed
no statistical dierence between the groups. Likewise, the
dierent treatments did not alter IL- levels secreted by
J cells. Interestingly, LPS stimulated the release of high
levels of IL- (± pg⋅mL−1), which was reversed by treat-
ment with strain Lp (± pg⋅mL−1) (Figure (c)). A small
but signicant dierence was detected in the costimulatory
molecule CD expression on the J macrophage sur-
face (Figure (d)). While incubation with LPS increased its
expression, Lp or Lp/LPS groups showed a reduction of
activated macrophages.
Intestinal macrophages are adapted to maintain local
homeostasis, even in a complex and potentially activating
molecule-rich environment. However, in the inamed mu-
cosa, for example, in patients with CD and UC, macrophages
exhibit an altered phenotype characterized by high expres-
sion of costimulatory molecules such as CD and CD,

BioMed Research International 
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15
10
5
0
[TNF-𝛼] (ng·mL−1)
A
A, B A, B
Medium
LPS
Lp62
Lp62/LPS
(a)
1.0
0.9
0.8
0.7
0.6
0.5
0.05
0.04
0.03
0.02
0.01
0.00
[IL-1] (ng·mL−1)
A
B
B
Medium
LPS
Lp62
Lp62/LPS
(b)
1.4
1.2
1.0
0.8
0.6
0.08
0.06
0.04
0.02
0.00
[IL-17] (ng·mL−1)
Medium
LPS
Lp62
A
BB
Lp62/LPS
(c)
4
3
2
1
0
Cell
LPS
Lp62
Lp62/LPS
A
B
B
250
200
150
100
50
0
100101102103
CD86-APC fluorescence intensity
Isotype control
Unstimulated cell
LPS
Lp62
Lp62/LPS
J774CD86+ mean fluorescence intensity
(d)
F : J macrophages stimulated with Lp and/or LPS: J macrophages were stimulated with LPS and Lp for  hours. e levels
of IL-, TNF-𝛼, and IL- were measured in culture supernatant by ELISA ((a), (b), and (c), resp.). e CD expression was analyzed by ow
cytometry (d). ASignicant dierence compared to unstimulated cells (culture medium). BSignicant dierence from the control stimulated
with LPS only; 𝑃 < 0.05.

BioMed Research International
8
6
4
2
0
% PBMC expressing CD4/CD25
Medium
LPS
Lp62
Lp62/LPS
B
A, B
(a)
[IL-10] (ng·mL−1)
2.5
2.0
1.5
1.0
0.5
0.0
Cell
LPS
Lp62
Lp62/LPS
A
B
B
(b)
F : CD+CD+T lymphocytes and IL- secretion in PBMC treated with Lp. Cultures of peripheral blood mononuclear cells were
challenged with LPS and Lp. e proportion of CD+CD+cells was determined by ow cytometry (a). IL- production was examined
in the culture supernatant by ELISA (b). AStatistical dierence compared to the control without stimulation. BStatistical dierence from the
control only stimulated with LPS; 𝑃 < 0.05.
as well as the innate receptors TLR- and TLR-, specialized
in detecting bacterial antigens [, ]. In this context, these
cells become potent producers of proinammatory cytokines
such as IL-𝛽,TNF-𝛼, IL-, and MCP-. Trials with murine
and human cells have shown that probiotics can prevent or
reverse the functional change of macrophages, characteristic
of chronic inammatory diseases. According to Pathmakan-
than et al. [], L. plantarum v reduced the secretion
of TNF-𝛼and IL-𝛽in mucosal mononuclear cells from
IBD patients stimulated with E. coli or Salmonella Dublin
andincreasedtheIL-levels.TNF-𝛼production is also
aectedbytheLPS-stimulatedmacrophageRAWand
treated with Lactobacillus rhamnosus GG []. Matsumoto
and Benno [] found that metabolites released in the
stools of patients fed with yoghurt containing Bidobacterium
animalis LKM were able to reverse the inammation
causedbyLPSinJcells.eeectofprobioticbacteriaon
antigens presenting cells such as macrophages and dendritic
cells is strain-dependent, since they also may be able to
upregulate the production of costimulatory molecules and
proinammatory cytokines []. IL induces neutrophil
recruitment to the inamed site and triggers the release of
inammatory cytokines in macrophages. However, its role
in inducing colitis remains uncertain, as it even presents
a protective activity in the gut, depending on the model
studied. e main source of this cytokine is  cells;
however, the innate immunity cells, including macrophages,
can produce it []. Here, we observe that Lp modulated
IL- secretion in J macrophages. Further studies are
needed to determine the impact of this probiotic on IL-
 production in the in vivo colitis model. In the present
study, we speculate that L. plantarum Lp was capable
of limiting J macrophage activation and consequently
preventing proinammatory cytokine secretion, contributing
to the maintenance of local homeostasis.
On the way to elucidate the Lp anti-inammatory
prole, its ability to induce a regulatory phenotype in sys-
temic circulation lymphocytes was checked by analyzing the
CD+CD+Foxp+population and the IL- secretion by
peripheral blood mononuclear cells. Lp stimulated PBMC
presented CD+CD+population around %, signicantly
dierent from the unstimulated control and the control
stimulated with LPS alone. Incubating cells with Lp/LPS
increased the percentage of this population signicantly com-
pared to the unstimulated control (Figure (a)). However,
no dierences were found in intracellular staining of Foxp
betweengroups.LpdisplayedtheabilitytoincreaseIL-
 production in PBMCs. e IL- level from the Lp/LPS
treated group also was signicantly increased as compared to
theunstimulatedcontrol.PBMCchallengedwithLPSonly
showed lower IL- secretion compared to the other groups
(Figure (b)).
IL- producing regulatory T cells can be found in the
intestinal mucosa of healthy humans and mice. In studies
involving the transfer of Treg cells, IL- produced by these
cells were able to attenuate colitis []. e ability of some
probiotic strains to activate a regulatory prole is well
documented in clinical trials. According to Dong et al. [],
feeding with L. casei Shirota for  weeks increased the IL-
/IL- ratio in the plasma of healthy individuals and the
expression of CD on T cells was signicantly higher. IL-
serum levels were higher aer consumption of Lactobacillus
salivarius CECT []. Similar to our ndings, the mixture
of L. plantarum CECT  and CECT  was able to
raise the percentage of T lymphocytes CD+CD+and IL-
mucosal levels []. Strain Lp increased the population of
CD+CD+lymphocytes in PBMC culture, but signicant
expression of Foxp was not detected. Treg cells are char-
acterized by CD+CD+expressiononthesurfacebutare
dependent on the Foxp transcription factor to exercise their

BioMed Research International 
function on colonic lamina propria.IncreasedIL-levels
aer treatment with Lp point to a regulatory T cell prole,
but cytokine production by other cells present in the culture
should be considered.
In the intestinal environment, epithelial cells, microor-
ganisms, and immune cell aggregates contribute to maintain-
ing homeostasis. According to the widely accepted model,
epithelial cells are responsible for releasing factors that will
direct the antigen presenting cells to a nonresponsive prole
or activating a regulatory response. e T cells generated
in this environment would be responsible for maintaining
homeostasis by releasing considerable amounts of IL- and
TGF-𝛽. Evidence suggests that the composition of the local
ora is directly correlated to the balance between response
and tolerance. In this sense, probiotics have been eective
in restoring the tolerogenic prole of the intestinal mucosa,
by modulating the activity of the cells that participate in
this process [, , ]. In this paper, the marked anti-
inammatory eect related to the lactic acid bacteria L.
plantarum Lp was observed on intestinal epithelial cells,
macrophage, and lymphocyte. In a cell culture model, this
strain was able to prevent S. Typhi adhesion to epithelial
cells and hence inhibit IL- secretion. A slight decrease in
macrophage activation was also observed which may have
contributed to reducing proinammatory cytokine produc-
tion. Finally, the Lp strain was able to enhance IL-
secretion and increase the CD+CD+cell population. Since
it showed immunomodulatory capacity on the main cells
involved in the intestinal mucosal immunity, Lp is a strong
candidatetoassistintherapyforinammatorydiseases.
4. Conclusions
e results presented in this paper should serve as a basis
for further studies that can investigate the pathways involved
in the Lp anti-inammatory eect. Equally important are
approaches in search of safe use of all the newly discovered
strains, mainly because probiotics are used in the context of
a previously damaged mucosa. Furthermore, in vivo trials are
essential in the study of probiotic action due to particularities
and the high complexity of the intestinal environment.
Competing Interests
e authors declare no competing interests between the
authors and the sponsoring institutions of this research.
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