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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.
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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;
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
Hindawi Publishing Corporation
BioMed Research International
Volume 2016, Article ID 8404156, 8 pages
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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  UmL−1 penicillin and streptomycin, at an
initial concentration of 6cellsmL−1,at
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. (ATCCTIB-) was
cultured at a concentration of 5×10
5cellsmL−1 in RPMI
(Gibco) medium supplemented with % fetal bovine serum
and  UmL−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
Salmonella enterica serovar Typhi ATCC  was cul-
.% NaCl solution and diluted to reach 𝐴600 =.,which
corresponds to 8CFUmL−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 HystopaqueSigma.  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
5cellsmL−1 and the cells were then
grown in RPMI supplemented with % fetal bovine serum
at Cand%CO
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 IntraPrepKit (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 Kaluzaowanalysissoware.
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 (9CFUmL−1)andincubatedfor
h at 
PBS, inoculated with S. Typhi  at a concentration of
8CFUmL−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
% adherence = CFUnal/CFUinitial ∗ 100.Jcellswere
stimulated with  𝜇LL. plantarum Lp (9CFUmL−1)and
LPS ( ngmL−1)andincubatedforhat%CO
2and C.
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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
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 Crohns 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
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
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 (. ngmL−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-
S. Typhi
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.
(±. ngmL−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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%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
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
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
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
TLR-4-PE HT-29
mean fluorescence intensity
a, b
S. Typhi
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 strains capacity to inhibit the inammatory stim-
ulus in a J macrophage cell line. Secretion of TNF-𝛼,
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
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- (± pgmL−1), which was reversed by treat-
ment with strain Lp (± pgmL−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,
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[TNF-𝛼] (ng·mL−1)
A, B A, B
[IL-1] (ng·mL−1)
[IL-17] (ng·mL−1)
CD86-APC fluorescence intensity
Isotype control
Unstimulated cell
J774CD86+ mean fluorescence intensity
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.
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% PBMC expressing CD4/CD25
A, B
[IL-10] (ng·mL−1)
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
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
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
 production in PBMCs. e IL- level from the Lp/LPS
treated group also was signicantly increased as compared to
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
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.
[] L. M. T. Dicks and M. Botes, “Probiotic lactic acid bacteria in
the gastro-intestinal tract: health benets, safety and mode of
action,Benecial Microbes,vol.,no.,pp.,.
[] S. Fijan, “Microorganisms with claimed probiotic properties: an
overview of recent literature,International Journal of Environ-
mental Research and Public Health,vol.,no.,pp.,
ment: the international scientic association for probiotics and
prebiotics consensus statement on the scope and appropriate
use of the term probiotic,Nature Reviews Gastroenterology and
Hepatology, vol. , no. , pp. –, .
and M. Principi, “Intestinal microbiota: the explosive mixture
at the origin of inammatory bowel disease?” Wo rld Jo u r n al of
Gastrointestinal Pathophysiology,vol.,no.,pp.,.
[] J. Plaza-Diaz, C. Gomez-Llorente, L. Fontana, and A. Gil,
“Modulation of immunity and inammatory gene expression
in the gut, in inammatory diseases of the gut and in the liver
by probiotics,World Journal of Gastroenterology,vol.,no.,
pp. –, .
[] R.J.SiezenandJ.E.T.Vlieg,“Genomicdiversityandversa-
tility of Lactobacillus plantarum, a natural metabolic engineer,
[] C. B¨
auerl, M. Llopis, M. Antol´
ın et al., “Lactobacillus paracasei
and Lactobacillus plantarum strains downregulate proinam-
mucosa,Genes & Nutrition,vol.,no.,pp.,.
vitro growth inhibition of Helicobacter pylori by lactobacilli
belonging to the Lactobacillus plantarum group,Letters in
Applied Microbiology,vol.,no.,pp.,.
[] P. Ducrott´
e, P. Sawant, and V. Jayanthi, “Clinical trial: Lacto-
bacillus plantarum v (DSM ) improves symptoms of
irritable bowel syndrome,World Journal of Gastoenterology,
[] R. K. Duar y, M. A. Bhausaheb, V. K. B atish, and S. Grover, “Anti-
inammatory and immunomodulatory ecacy of indigenous
probiotic Lactobacillus plantarum Lp in colitis mouse model,
Molecular Biology Reports,vol.,no.,pp.,.
[] M. J. Smelt, B. J. de Haan, P. A. Bron et al., “e Impact of
Lactobacillus plantarum WCFS teichoic acid D-alanylation
on the generation of eector and regulatory T-cells in healthy
mice,PLoS ONE,vol.,no.,articlee,.
[] T. F. Dos Santos, Probiotic potential of lactic acid bacteria isolated
from cocoa beans fermentation in southern Bahia, Brazil [M.S.
thesis], Santa Cruz State University, Biology Department, , action=&co obra=.
[] M. Z. Cader and A. Kaser, “Recent advances in inammatory
bowel disease: mucosal immune cells in intestinal inamma-
[] C. Banks, A. Bateman, R. Payne, P. Johnson, and N. Sheron,
“Chemokine expression in IBD. Mucosal chemokine expression
is unselectively increased in both ulcerative colitis and Crohn’s
disease,Journal of Pathology,vol.,no.,pp.,.
[] D.-Y. Ren, C. Li, Y.-Q. Qin et al., “Lactobacilli reduce chemokine
IL- product ion in response to TNF-𝛼and Salmonellachallenge
of caco- cells,BioMed Research International,vol.,Article
ID ,  pages, .
[] C. M. Carey and M. Kostrzynska, “Lactic acid bacteria and bi-
dobacteria attenuate the proinammatory response in intesti-
nal epithelial cells induced by salmonella enterica serovar
typhimurium,Canadian Journal of Microbiology,vol.,no.,
pp. –, .
[] J. G. Magalhaes, I. Tattoli, and S. E. Girardin, “e intestinal
epithelial barrier: how to distinguish between the microbial
ora and pathogens,Seminars in Immunology,vol.,no.,pp.
–, .
[] H. Karlsson, C. Hessle, and A. Rudin, “Innate immune re-
sponses of human neonatal cells to bacteria from the normal
BioMed Research International
gastrointestinal ora,Infection and Immunity,vol.,no.,
[] M. Schenk and C. Mueller, “Adaptations of intestinal macropha-
ges to an antigen-rich environment,Seminars in Immunology,
[] J. Rugtveit, A. Bakka, and P. Brandtzaeg, “Dierential distribu-
tion of B. (CD) and B. (CD) costimulatory molecules
on mucosal macrophage subsets in human inammatory bowel
disease (IBD),Clinical and Experimental Immunology,vol.,
[] M. Hausmann, S. Kiessling, S. Mestermann et al., “Toll-like
receptors  and  are up-regulated during intestinal inamma-
tion,Gastroenterology, vol. , no. , pp. –, .
[] S. Pathmakanthan, C. K. F. Li, J. Cowie, and C. J. Hawkey, “Lac-
tobacillus plantarum v: benecial in vitro immunomodu-
lation in cells extracted from inamed human colon,Journal
of Gastroenterology and Hepatology,vol.,no.,pp.,
[] J. A. Pe˜
na and J. Versalovic, “Lactobacillus rhamnosus GG
decreases TNF-𝛼production in lipopolysaccharide-activated
murine macrophages by a contact-independent mechanism,
Cellular Microbiology,vol.,no.,pp.,.
[] M. Matsumoto and Y. Benno, “Anti-inammatory metabolite
production in the gut from the consumption of probiotic yogurt
containing Bidobacterium animalis subsp. lactis LKM,
Bioscience, Biotechnology and Biochemistry,vol.,no.,pp.
–, .
[] S. Latvala, T. E. Pietil¨
a, V. Veckman et al., “Potentially probiotic
bacteria induce ecient maturation but dierential cytokine
production in human monocyte-derived dendritic cells,Wo rld
Journal of Gastroenterology, vol. , no. , pp. –, .
[] A. Geremia, P. Biancheri, P. Allan, G. R. Corazza, and A. Di
Sabatino, “Innate and adaptive immunity in inammatory
bowel disease,Autoimmunity Reviews,vol.,no.,pp.,
[] J. L. Coombes and K. J. Maloy, “Control of intestinal home-
ostasis by regulatory T cells and dendritic cells,Seminars in
Immunology, vol. , no. , pp. –, .
[] H.Dong,I.Rowland,L.V.omas,andP.Yaqoob,“Immuno-
modulatory eects of a probiotic drink containing Lactobacillus
casei Shirota in healthy older volunteers,European Journal of
[] S. Sierra, F. Lara-Villoslada, L. Sempere, M. Olivares, J. Boza,
and J. Xaus, “Intestinal and immunological eects of daily oral
administration of Lactobacillus salivarius CECT to healthy
[] J. Ma˜
e, E. Pedrosa, V. Lor´
en et al., “A mixture of Lactobacillus
plantarum CECT  and CECT  enhances systemic
immunity in elderly subjects. A dose-response, double-blind,
placebo-controlled, randomized pilot trial,Nutricion Hospita-
... The microorganism Lactiplantibacillus plantarum Lp62 has had its anti-inflammatory potential verified in an animal model of experimental colitis and in an in vitro cellular model, inhibiting the inflammation after the challenge of macrophages, lymphocytes, and epithelial cells with Salmonella enterica (Dos Santos et al. 2016). ...
... The cultures initially contained 10 6 cell/mL and were maintained for seven days to reach postconfluence. The culture medium was replenished every other day (Dos Santos et al. 2016). ...
The present work aimed to produce a cupuassu juice (Theobroma grandiflorum) fermented by the probiotic bacterium Lactiplantibacillus plantarum Lp62 and to analyze its antioxidant potential, antimicrobial activity and resistance to biological barriers. The fermented beverage showed an increase in the content of phenolics, flavonoids and antioxidant potential. The culture showed antagonistic activity against pathogens, but this result was not observed when the juice was tested. The probiotic strain remained viable under refrigeration, even in an acidified environment, and survived simulated gastrointestinal transit in vitro. L. plantarum Lp62 showed 30% adherence to HT-29 intestinal cells and proved to be safe in terms of antibiotic resistance and production of virulence factors. Fermentation increased the functional characteristics of cupuassu juice. This drink proved to be a good vehicle for the delivery of the probiotic bacteria L. plantarum Lp62.
... Contrasting to membrane cholesterol, sphingolipids act on epithelial defense against the invasive pathogen by enhancing the NOD2-mediated human beta-defensin 2 (hBD-2) response [80]. The probiotic Lactobacillus plantarum Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated IECs and prevented the adhesion of pathogens to the cells [57]. The treatment of probiotics before and after infection having different effects on the Salmonella-induced IL-8 response in IECs suggests the critical timing of probiotic supplementation [25]. ...
... The probiotic Lactobacillus plantarum ZS2058 significantly reduced the pathogenicity of Salmonella colitis by promoting the colon IL23/IL-22 axis in the mouse [36]. Lactobacillus plantarum Lp62 was able to suppress IL-17, IL1-β, and TNF-α production in LPS-stimulated J774 macrophages [57]. ...
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The current study was undertaken to investigate the role of macrophages as a cellular immune function against immunization with whole sonicated Listeria monocytogenes antigens (WSLMAgs) and the effect of probiotics. A preparation of WSLMAgs containing whole L. monocytogenes cell, after two subcutaneous immunization of BALB/c mice with 0.5ml WSLMAgs (0.5mg/ml) at an interval of two weeks. The bacterial identification was conducted by a conventional culture method using Listeria selective media PALCAM and confirmed by Polymerase Chain Reaction (PCR), As well as, the immunohistochemical and pathological change of it was studied in vivo by inoculating mice with pre-challenge WSLMAgs and post-challenge with virulent L. monocytogenes (1×108 CFU/mL). The results revealed the cellular immune function against pre- and post-immunization in spleen organ via lymphocytic hyperplasia in white pulp and coalescence of lymphoid follicles and marker F4/80+ show the immune-positive cells in aggregation adjacent to lymphoid follicle or focal aggregation of macrophages between follicles. In conclusion, the effectiveness of sonicated L. monocytogenes pre and post-immunization then challenge with virulent L. monocytogenes in the induction of cellular immune response, might serve as an immunization platform for applicants.
Les bactéries lactiques présentent un grand intérêt économique de par leur large utilisation dans l’industrie agroalimentaire. Parmi elles, Streptococcus thermophilus (ST) est d’une importance majeure puisqu’elle est la plus utilisée après Lactococcus lactis, pour la fabrication de produits laitiers fermentés et de fromages. De plus, cette bactérie est le seul streptocoque à bénéficier du statut GRAS (Generally Recognized As Safe). Outre son intérêt en industrie laitière, ST présente des effets bénéfiques sur la santé intestinale de l’Homme. Bien que ces effets soient largement documentés, le statut probiotique de ST reste encore à conforter. C’est pourquoi des études sont actuellement menées afin de sélectionner des souches de ST à fort potentiel probiotique. Parmi les critères importants figurent leur capacité à survivre aux conditions drastiques du tube digestif (TD) et leur capacité à adhérer aux cellules intestinales. Dans cette optique, les objectifs de cette thèse étaient d’étudier, dans un premier temps, la capacité d’adhésion in vitro de la souche ST LMD-9 à différentes lignées cellulaires intestinales d’origine humaine et d’évaluer la survie de cette souche au stress biliaire. Afin de mettre en évidence un rôle potentiel de certaines protéines de surface dans ces deux processus, trois mutants issus de cette souche et inactivés dans les gènes prtS (protéase pariétale), srtA (sortase A) et mucBP (protéine de liaison aux mucines), ont été inclus dans cette étude. Dans un second temps, l’impact de l’adhésion de LMD-9 a été analysé, d’une part sur l’expression de gènes codant certaines mucines dans les cellules eucaryotes, et d’autre part sur l’expression de gènes qui seraient spécifiquement induits durant le processus d’adhésion, ceci en utilisant la technologie R-IVET (Recombinase-based In Vivo Expression Technology). Les résultats obtenus ont permis de montrer que la souche LMD-9 était capable de survivre jusqu’à une concentration de 3 mM en sels biliaires et que les protéines de surface PrtS, SrtA et MucBP seraient impliquées dans la résistance à ce stress. Nos résultats ont également montré que LMD-9 adhérait aux trois différentes lignées cellulaires, suggérant ainsi que la souche pourrait interagir avec les différentes mucines qu’elle peut rencontrer dans le TD. De plus, l’implication de certaines protéines de surface dans l’adhésion de LMD-9 s’est avérée dépendante des caractéristiques de ces lignées, qu’il s’agisse de cellules entérocytaires (Caco-2) ou productrices de mucus (HT29-MTX et HT29-CL.16E). Concernant l’impact de l’adhésion de la souche LMD-9 sur l’expression des gènes MUC2 et MUC5AC, aucun effet sur le taux de transcrits n’a été observé dans nos conditions expérimentales. Par ailleurs, nos résultats ont permis, pour la première fois, d’identifier les gènes spécifiquement induits dans la souche LMD-9 durant l’adhésion aux cellules épithéliales. Nous avons ainsi montré que l’adhésion de la souche LMD-9 ne dépend pas uniquement des protéines de surface, mais d’autres fonctions et voies métaboliques seraient également impliquées. Ce travail de thèse contribue ainsi à apporter de nouvelles connaissances liées (i) au choix du modèle cellulaire dans les études d’adhésion bactérienne in vitro, (ii) à l’aptitude de la souche LMD-9 à survivre au stress biliaire en faisant intervenir certaines protéines de surface et (iii) à la compréhension des mécanismes moléculaires de l’adhésion de LMD-9 aux cellules épithéliales intestinales
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The potential for the positive manipulation of the gut microbiome through the introduction of beneficial microbes, as also known as probiotics, is currently an active area of investigation. The FAO/WHO define probiotics as live microorganisms that confer a health benefit to the host when administered in adequate amounts. However, dead bacteria and bacterial molecular components may also exhibit probiotic properties. The results of clinical studies have demonstrated the clinical potential of probiotics in many pathologies, such as allergic diseases, diarrhea, inflammatory bowel disease and viral infection. Several mechanisms have been proposed to explain the beneficial effects of probiotics, most of which involve gene expression regulation in specific tissues, particularly the intestine and liver. Therefore, the modulation of gene expression mediated by probiotics is an important issue that warrants further investigation. In the present paper, we performed a systematic review of the probiotic-mediated modulation of gene expression that is associated with the immune system and inflammation. Between January 1990 to February 2014, PubMed was searched for articles that were published in English using the MeSH terms "probiotics" and "gene expression" combined with "intestines", "liver", "enterocytes", "antigen-presenting cells", "dendritic cells", "immune system", and "inflammation". Two hundred and five original articles matching these criteria were initially selected, although only those articles that included specific gene expression results (77) were later considered for this review and separated into three major topics: the regulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver. Particular strains of Bifidobacteria, Lactobacilli, Escherichia coli, Propionibacterium, Bacillus and Saccharomyces influence the gene expression of mucins, Toll-like receptors, caspases, nuclear factor-κB, and interleukins and lead mainly to an anti-inflammatory response in cultured enterocytes. In addition, the interaction of commensal bacteria and probiotics with the surface of antigen-presenting cells in vitro results in the downregulation of pro-inflammatory genes that are linked to inflammatory signaling pathways, whereas other anti-inflammatory genes are upregulated. The effects of probiotics have been extensively investigated in animal models ranging from fish to mice, rats and piglets. These bacteria induce a tolerogenic and hyporesponsive immune response in which many genes that are related to the immune system, in particular those genes expressing anti-inflammatory cytokines, are upregulated. By contrast, information related to gene expression in human intestinal cells mediated by the action of probiotics is scarce. There is a need for further clinical studies that evaluate the mechanism of action of probiotics both in healthy humans and in patients with chronic diseases. These types of clinical studies are necessary for addressing the influence of these microorganisms in gene expression for different pathways, particularly those that are associated with the immune response, and to better understand the role that probiotics might have in the prevention and treatment of disease.
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Inflammatory bowel diseases (IBDs), namely Crohn's disease and ulcerative colitis, are lifelong chronic disorders arising from interactions among genetic, immunological and environmental factors. Although the origin of IBDs is closely linked to immune response alterations, which governs most medical decision-making, recent findings suggest that gut microbiota may be involved in IBD pathogenesis. Epidemiologic evidence and several studies have shown that a dysregulation of gut microbiota (i.e., dysbiosis) may trigger the onset of intestinal disorders such as IBDs. Animal and human investigations focusing on the microbiota-IBD relationship have suggested an altered balance of the intestinal microbial population in the active phase of IBD. Rigorous microbiota typing could, therefore, soon become part of a complete phenotypic analysis of IBD patients. Moreover, individual susceptibility and environmental triggers such as nutrition, medications, age or smoking could modify bacterial strains in the bowel habitat. Pharmacological manipulation of bowel microbiota is somewhat controversial. The employment of antibiotics, probiotics, prebiotics and synbiotics has been widely addressed in the literature worldwide, with the aim of obtaining positive results in a number of IBD patient settings, and determining the appropriate timing and modality of this intervention. Recently, novel treatments for IBDs, such as fecal microbiota transplantation, when accepted by patients, have shown promising results. Controlled studies are being designed. In the near future, new therapeutic strategies can be expected, with non-pathogenic or modified food organisms that can be genetically modified to exert anti-inflammatory properties.
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An expert panel was convened in October 2013 by the International Scientific Association for Probiotics and Prebiotics (ISAPP) to discuss the field of probiotics. It is now 13 years since the definition of probiotics and 12 years after guidelines were published for regulators, scientists and industry by the Food and Agriculture Organization of the United Nations and the WHO (FAO/WHO). The FAO/WHO definition of a probiotic-"live microorganisms which when administered in adequate amounts confer a health benefit on the host"-was reinforced as relevant and sufficiently accommodating for current and anticipated applications. However, inconsistencies between the FAO/WHO Expert Consultation Report and the FAO/WHO Guidelines were clarified to take into account advances in science and applications. A more precise use of the term 'probiotic' will be useful to guide clinicians and consumers in differentiating the diverse products on the market. This document represents the conclusions of the ISAPP consensus meeting on the appropriate use and scope of the term probiotic.
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Probiotics are defined as live microorganisms, which when administered in adequate amounts, confer a health benefit on the host. Health benefits have mainly been demonstrated for specific probiotic strains of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, Escherichia coli. The human microbiota is getting a lot of attention today and research has already demonstrated that alteration of this microbiota may have far-reaching consequences. One of the possible routes for correcting dysbiosis is by consuming probiotics. The credibility of specific health claims of probiotics and their safety must be established through science-based clinical studies. This overview summarizes the most commonly used probiotic microorganisms and their demonstrated health claims. As probiotic properties have been shown to be strain specific, accurate identification of particular strains is also very important. On the other hand, it is also demonstrated that the use of various probiotics for immunocompromised patients or patients with a leaky gut has also yielded infections, sepsis, fungemia, bacteraemia. Although the vast majority of probiotics that are used today are generally regarded as safe and beneficial for healthy individuals, caution in selecting and monitoring of probiotics for patients is needed and complete consideration of risk-benefit ratio before prescribing is recommended.
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The probiotic properties of two selected lactobacilli strains were assessed. L. salivarius and L. plantarum displayed higher hydrophobicity (48% and 54%, resp.) and coaggregation ability with four pathogens (from 7.9% to 57.5%). L. salivarius and L. plantarum had good inhibitory effects on S. aureus (38.2% and 49.5%, resp.) attachment to Caco-2 cells. Live lactobacilli strains and their conditioned media effectively inhibited IL-8 production (<14.6 pg/mL) in TNF- α -induced Caco-2 cells. Antibiotic-treated and the sonicated lactobacilli also maintained inhibitory effects (IL-8 production from 5.0 to 36.3 pg/mL); however, the heat-treated lactobacilli lost their inhibitory effects (IL-8 production from 130.2 to 161.0 pg/mL). These results suggest that both the structural components and the soluble cellular content of lactobacilli have anti-inflammatory effects. We also found that pretreatment of Caco-2 cells with lactobacilli inhibited S. typhimurium-induced IL-8 production (<27.3 pg/mL). However, lactobacilli did not inhibit IL-8 production in Caco-2 cells pretreated with S. typhimurium. These results suggest that the tested lactobacilli strains are appropriate for preventing inflammatory diseases caused by enteric pathogens but not for therapy. In short, L. salivarius and L. plantarum are potential candidates for the development of microbial ecological agents and functional foods.
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The intestine and its immune system have evolved to meet the extraordinary task of maintaining tolerance to the largest, most complex and diverse microbial commensal habitat, while meticulously attacking and containing even minute numbers of occasionally incoming pathogens. While our understanding is still far from complete, recent studies have provided exciting novel insights into the complex interplay of the many distinct intestinal immune cell types as well as the discovery of entirely new cell subsets. These studies have also revealed how proper development and function of the intestinal immune system is dependent on its specific microbiota, which appears to have evolutionarily co-evolved. Here we review key immune cells that maintain intestinal homeostasis and, conversely, describe how altered function and imbalances may lead to inflammatory bowel disease (IBD). We highlight the latest developments within this field, covering the major players in IBD including intestinal epithelial cells, macrophages, dendritic cells, adaptive immune cells, and the newly discovered innate lymphoid cells, which appear of characteristic importance for immune function at mucosal surfaces. We set these mucosal immune pathways in the functional context of IBD risk genes where such insight is available. Moreover, we frame our discussion of fundamental biological pathways that have been elucidated in model systems in the context of results from clinical trials in IBD that targeted key mediators secreted by these cells, as an attempt of 'functional' appraisal of these pathways in human disease.
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To date it remains unclear how probiotics affect the immune system. Bacterial envelope components may play an essential role, as these are the first to establish bacterial-host cell interactions. Teichoic acids (TAs), and especially lipoteichoic acids, are the most pro-inflammatory components of the gram-positive bacterial envelope. This effect is dependent on D-alanyl substitution of the TA backbone and interactions with TLR2 on host cells. Although the pro-inflammatory properties of TAs have been established in vitro, it remains unclear how TAs affect immunomodulation in vivo. In this study, we investigated the role of TA D-alanylation on L. plantarum-induced intestinal and systemic immunomodulation in vivo. For this, we compared the effect of L. plantarum WCFS1 and its TA D-Alanylation negative derivative (dltX-D) on the distribution of dendritic cell and T cell populations and responses in healthy mice. We demonstrated that the majority of the L. plantarum-induced in vivo immunomodulatory effects were dependent on D-alanylation (D-Ala), as some L. plantarum WCFS1-induced immune changes were not observed in the dltX-D-treated group and some were only observed after treatment with dltX-D. Strikingly, not only pro-inflammatory immune responses were abolished in the absence of D-Ala substitution, but also anti-inflammatory responses, such as the L. plantarum-induced generation of regulatory T cells in the spleen. With this study we provide insight in host-microbe interactions, by demonstrating the involvement of D-alanylation of TAs on the bacterial membrane in intestinal and systemic immunomodulation in healthy mice.
AIM: To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells (moDCs). METHODS: Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS), respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors. RESULTS: All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp. cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class II and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory (TNF-α, IL-12, IL-6, and CCL20) and Th1 type (IL-12 and IFN-γ) cytokines, while B. breve and L. lactis were also potent inducers of anti-inflammatory IL-10. Mitogen-activated protein kinase (MAPK) p38, phosphatidylinositol 3 (PI3) kinase, and nuclear factor-kappa B (NF-κB) signaling pathways were shown to be involved in bacteria-induced cytokine production. CONCLUSION: Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another.
Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC). The exact cause of IBD remains unknown. Available evidence suggests that an abnormal immune response against the microorganisms of the intestinal flora is responsible for the disease in genetically susceptible individuals. The adaptive immune response has classically been considered to play a major role in the pathogenesis of IBD. However, recent advances in immunology and genetics have clarified that the innate immune response is equally as important in inducing gut inflammation in these patients. In particular, an altered epithelial barrier function contributes to intestinal inflammation in patients with UC, while aberrant innate immune responses, such as antimicrobial peptides production, innate microbial sensing and autophagy are particularly associated to CD pathogenesis. On the other hand, besides T helper cell type (Th)1 and Th2 immune responses, other subsets of T cells, namely Th17 and regulatory T (Treg) cells, are likely to play a role in IBD. However, given the complexity and probably the redundancy of pathways leading to IBD lesions, and the fact that Th17 cells may also have protective functions, neutralization of IL-17A failed to induce any improvement in CD. Studying the interactions between various constituents of the innate and adaptive immune systems will certainly open new horizons in the knowledge about the immunologic mechanisms implicated in gut inflammation.