Immunomodulatory Potential of Probiotic Lactobacillus casei

In Vitro Investigation of the Immunomodulatory Potential
of Probiotic Lactobacillus casei
Thirugnanam Karthikeyan • Mariappan Pravin • Velusamy Shanmuganathan Muthusamy •
Rajaganapathy Bharathi Raja • Baddireddi Subhadra Lakshmi
Published online: 19 December 2012
Ó Springer Science+Business Media New York 2012
Abstract The current study investigated the immuno-
modulatory potential of ethyl acetate soluble supernatant of
Lactobacillus casei (LC-EAS) in vitro. The effect of
LC-EAS on nitric oxide release was analyzed in RAW
264.7 cells, wherein, an inhibition in nitric oxide produc-
tion through suppression of inducible nitric oxide synthase
mRNA expression was observed. Evaluation of LC-EAS
on LPS-induced peripheral blood mononuclear cells
showed a down-regulation in TNF-a and IL-6 genes and an
upregulation of IL-10. An inhibition in the protein
expression of NF-jB, ERK1/2 and STAT3 phosphorylation
confirms the immunomodulatory potential of LC-EAS. The
effect of LC-EAS on in vitro intestinal epithelial cells was
investigated using HT-29 human colon adenocarcinoma
cancer cells. LC-EAS exhibited an inhibition of NF-jB and
ERK1/2 phosphorylation, whereas STAT3 phosphorylation
was unregulated. To evaluate the downstream target of
STAT3 upregulation, expression of the intestinal trefoil
factor TFF3 which is a NF-jB regulator and STAT3
downstream target was studied. LC-EAS was observed to
elevate TFF3 mRNA expression. Overall the study shows
that the anti-inflammatory potential of LC-EAS is through
inhibition of NF-jB in different cell types.
Keywords Probiotics Á NF-jB Á TFF3 Á Lactobacillus
casei Á PBMCs Á HT-29 Á Immunomodulation
Introduction
Probiotics are viable, nonpathogenic microorganisms
(bacteria or yeast) that are able to reach the intestine in
sufficient numbers to confer benefits to the host [1].
Modulation of host immunity is one of the most commonly
purported benefits of consumption of probiotics. The non-
pathogenic Gram-positive commensal Lactobacilli found
in human and mouse gastrointestinal tracts have been
considered as probiotics with beneficial health effects,
including enhanced lymphocyte proliferation [2], innate
and acquired immunity [3] along with anti-inflammatory
activity [4]. In clinical practice, commercial probiotics
have been used as therapeutic agents to ameliorate diarrhea
[5]. The genera most commonly used in probiotic prepa-
rations are Lactobacillus, Streptococcus, Bifidobacterium,
Lactococcus and some fungal strains [4]. Inflammation
initiated by invasion of pathogens or cell injury is a normal
physiological and immune response in the host. Gram-
negative bacteria establish infection by adhering to the
host, wherein the lipopolysaccharide (LPS) presents in the
outer membrane of the bacteria, plays a key role in
mounting infection, leading to an immune response, and
development of a strong inflammatory disorder. LPS is a
potent inducer of monocytes and macrophages, which are
key mediators of the innate immune response. Stimulation
of cells by LPS leads to a cascade of intracellular signaling
events that ultimately result in secretion and production of
cytokines and other inflammatory mediators constituting
the pro-inflammatory response [6]. The normal inflamma-
tory response involves activation of several different cel-
lular components, such as neutrophils, macrophages, and
lymphocytes. In particular, macrophages play an important
role in the modulation of inflammation and immune
response to maintain a defensive reaction [7]. During an
T. Karthikeyan Á M. Pravin Á V. S. Muthusamy Á
R. Bharathi Raja Á B. S. Lakshmi (&)
Tissue Culture and Drug Discovery Lab, Centre for
Biotechnology, Anna University, Chennai 600 025, Tamilnadu,
India
e-mail: lakshmibs@annauniv.edu
123
Probiotics & Antimicro. Prot. (2013) 5:51–58
DOI 10.1007/s12602-012-9122-y

inflammatory response, activated macrophages secrete
nitric oxide (NO) via the inducible isoforms of NO syn-
thase (iNOS) and large amount of pro-inflammatory
mediators and cytokines, TNF-a, and IL-6, etc. IL-10 is an
anti-inflammatory cytokine with pleiotropic effects in
immunoregulation and inflammation. IL-10 can block
NF-jB activity and is involved in the regulation of the
JAK-STAT signaling pathway. The binding of interleukin
6 family cytokines (including IL-6, oncostatin M, and
leukemia inhibitory factor) to the gp130 receptor normally
leads to balanced signaling through SHP2/Erk and STAT1/
3 pathways [8].
Nuclear transcription factor kappa-B (NF-jB) is a critical
key transcriptional factor that expresses the genes involved
in inflammation. The main inducible form is a heterodimer
consisting of p50/p65 subunit. NF-jB is present in the
cytoplasm as an inactive complex associated with an inhib-
itory protein called IjB. Various internal or external stimuli
cause the dissociation of NF-jB/IjB complex through the
phosphorylation and degradation of IjB by cytoplasmic IjB
kinase (IKK) [9, 10]. The intestine synthesizes products that
tend to limit inflammation and improve the integrity of the
epithelial barrier. One such group of products is the trefoil
factor family (TFF), members of which are synthesized by
goblet cells throughout the large and small intestine and acts
predominantly in stabilizing the mucous layer that overlies
the intestinal epithelium [11]. TFFs reduce the recruitment of
inflammatory cells into the intestine, and the expression of
adhesion molecules that are needed to drive such recruitment
[12]. Therefore, TFFs are a group of factors which allow the
healthy gut to restrain inflammation and live harmoniously
with its abundant flora. The trefoils are key downstream
targets of the SHP2/ERK and STAT signaling pathways
[8, 13]. A down-regulation of ubiquitin system-associated
genes has been reported to play a crucial role in the modu-
lation of pro-inflammatory pathways in intestinal epithelial
cells by Lactobacillus casei (LC) [14]. Alison et al. [15] have
observed that L. casei specifically targets the stability of
IjBa, the specific NF-jB inhibitor, thereby shutting down
this major pro-inflammatory pathway [15]. L. casei has been
observed to mediate an induction of higher level of anti-
inflammatory interleukins such as in IL-4 and IL-10 which
would contribute toward regulating the pro-inflammatory
effect of TNF-a, IL-1b, and IL-6 [16].
Thepresentstudyaimstoinvestigatetheanti-inflammatory
potential of the ethyl acetate soluble culture supernatant of
probiotic bacterium L. casei (LC-EAS). L. casei is a probiotic
bacterium and has been proven effective in improving murine
chronic IBD and is associated with down-regulation of pro-
inflammatory cytokines in lamina propria mononuclear cells
[17]. The anti-inflammatory effect of LC-EAS and its mech-
anism of action are investigated using peripheral blood
mononuclear (PBMC) cells, RAW 264.7 and HT-29 cells as
in vitro models for lymphocytes, macrophages, and intestinal
cells, respectively.
Materials and Methods
Chemicals and Reagents
Cell culture reagents and supplements were purchased from
Life Technologies Inc. (Gaithersburg, MD, USA). Dul-
becco’s Modified Eagle medium (DMEM) and Roswell Park
Memorial Institute (RPMI 1640) were obtained from GIB-
CO, BRL (Carlsbad, CA, USA). Primers (TNFa, iNOS,
IL-10, IL-6, TFF3, and GAPDH) and antibodies (Anti-NF-
jB, IjBa, ERK1/2, STAT3, and b-Actin) were obtained
from Santa Cruz Biotechnology (Santa cruz, CA). All the
other chemicals were obtained from Sigma Aldrich, USA.
Bacterial Culture and Supernatant Preparation
Lactobacillus casei ATCC 334 was incubated in Lacto-
bacillus MRS broth at 37 °C for 24 h, diluted in MRS
broth, and incubated at 37 °C to reach log phase with the
optical density determined as 0.5 at A600 [3]. L. casei was
filtered, and the supernatant recovered from the MRS
culture broth and extracted using hexane and ethyl acetate.
The hexane and ethyl acetate extracts were concentrated
using rota evaporator and drug dilutions (concentrations
ranging from 100, 50, 25, 10, 5, and 1 lg) were prepared in
DMSO.
Cell Lines and Culture Conditions
RAW 264.7 murine monocyte/macrophage cell line and
HT-29 human colon adenocarcinoma cell lines were pur-
chased from NCCS, Pune, India. Human PBMCs were
isolated from healthy volunteers by mutual consent. RAW
264.7 and HT-29 cells were cultured in Dulbecco’s Mod-
ified Eagle’s medium (DMEM), and Human PBMCs were
cultured in Roswell Park Memorial Institute (RPMI 1640)
with 10 % FBS, 2 mM L-glutamine and 1 % Penicillin/
Streptomycin (pen-strep) under a fully humidified atmo-
sphere, 5 % CO2 at 37 °C and subcultured every 3 days.
For experimental conditions, all the cells were grown in
DMEM without FBS and without phenol red. In all the
experiments, untreated and vehicle treated cells were
included as controls.
Isolation of PBMC
Heparinized venous blood was taken from 4 healthy human
volunteers with their mutual consent for the experiment.
52 Probiotics & Antimicro. Prot. (2013) 5:51–58
123

The Institutional biosafety and ethical committee approved
the studies, and the subjects gave written informed consent.
Mononuclear cells were isolated in a Ficoll–Hypaque
(Pharmacia, Piscataway, NJ) density gradient using stan-
dard procedures which separated PBMCs from whole
blood. The buffy coat containing PBMCs was removed
carefully following centrifugation and washed twice in
RPMI medium containing 10 % FCS. Cells were counted
and assessed for viability using MTT assay.
Cell Proliferation Assay
The peripheral blood mononuclear cells were separated as
described previously. In brief, 200 lL of PBMCs were
cultured in triplicates in a 96-well U-bottom microtiter
plates containing 2 9 105
cells/well in RPMI medium
containing 10 % FCS, and 1 lg/mL of PHA (phytohem-
agglutinin). The cells were incubated for 24 h with dif-
ferent concentrations of LC-EAS. The cell proliferation
was measured in a liquid scintillation counter after the cells
were pulsed with 1 lCi/well of [3
H] thymidine for the last
16 h of incubation. The results are expressed in terms of
percent inhibition with respect to untreated control.
Experiments were done thrice in triplicates. Results are the
mean ± SD when compared with untreated control group.
Assessment of Cytotoxicity by Lactate Dehydrogenase
(LDH) Release Assay
The cytotoxicity of LC-EAS was assayed by measuring the
release of the stable cytosolic enzyme, lactate dehydroge-
nase (LDH). After incubation with various concentrations
of LC-EAS for 24 h, MTT reagent was added and incu-
bated for 4 h. To this 100 lL of DMSO was added and the
plates read at 490 nm on a scanning multi-well spectro-
photometer. Triton X was used as positive control and the
untreated cells as negative control.
Measurement of Nitrite by Griess Reaction
RAW 264.7 cells were treated with LC-EAS in the pres-
ence or absence of LPS (2 lg/mL). After 24 h, nitrite
production in the culture supernatants was assessed through
Griess reaction by measurement of the total amount of
inorganic nitric oxide (NO); 100 lL aliquots were removed
from the medium and incubated with an equal volume of
Griess reagent (1 % sulfanilamide and 0.1 % naphthyl
ethylenediamine dihydrochloride in 2.5 % H3PO4), for
10 min at room temperature, and absorbance was measured
at 540 nm in an ELISA reader. Nitrite concentration was
calculated with reference to a standard curve obtained
using NaNO2.
Measurement of Cytokine mRNA by Semi-quantitative
RT-PCR
PBMCs, macrophages (RAW 264.7 cells), and HT-29 cells
were incubated at required time points with optimized
doses of LC-EAS, and cells were immediately homoge-
nized using TRI reagent (SIGMA, UK), and RNA isolated
by phenol–chloroform extraction. The aqueous phase
containing RNA was precipitated by adding equal volume
of isopropyl alcohol. The RNA obtained was then con-
verted to cDNA by reverse transcription using MMLV
reverse transcriptase enzyme and subjected to PCR with
specific primers. PCR products were run on 1.2 % agarose
gels, stained with ethidium bromide and photographed.
PCR products were analyzed on ethidium bromide stained
agarose gels.
Analysis of NF-jB Signaling by Western Blotting
PBMCs and HT-29 cells were treated with optimized doses
of LC-EAS for the required time points. The lysis of cells
was done using lysis buffer [50 mM Tris–HCL (pH 7.5),
150 mM NaCL, 1 % Nonidet p-40, 2 mM EDTA, 1 mM
EGTA, 1 mM NaVO3, 10 mM NAF, 1 mM Dithiothreitol,
25 lg/mL Aprotinin, 1 mM Phenylmethylsulfonyl fluoride,
25 lg/mL Leupeptin] and kept on ice for 30 min. The cell
lysates were centrifuged at 14,000 rpm at 40 °C for 20 min
and the supernatant stored at -70 °C. Protein concentra-
tion was measured by Bradford’s method. Aliquots of the
lysates (125 lg of protein) were separated on a 10 % SDS-
polyacrylamide gel and transferred onto a nitrocellulose
membrane with glycine transfer buffer [192 mM Glycine,
25 mM Tris–HCL (ph 8.8), 20 % methanol (v/v)]. After
blocking the nonspecific site with 5 % nonfat dried milk in
PBS, the membrane was incubated with specific primary
and secondary antibody. The chromogenic substrate NBT/
BCIP was used for visualization.
Calculation of IC50
The IC50 is the 50 % inhibitory concentration of the extract
and is determined from the dose–response curve.
Statistical Analysis
All data are expressed as mean ± SD. The statistical sig-
nificance between means of the independent groups was
analyzed using one-way ANOVA followed by Dunnett’s
post hoc for other parameters, and p value of less than 0.05
was considered to be statistically significant.
Probiotics & Antimicro. Prot. (2013) 5:51–58 53
123

Results
Extraction and Preliminary Screening
The culture supernatant of L. casei was subjected to
sequential extraction [18] using hexane and ethyl acetate
and concentrated using rota evaporator. The extracts were
reconstituted using DMSO below 0.02 %. Analysis of the
extracts for their inhibitory effect on mitogen-induced
lymphocyte proliferation showed the ethyl acetate extract
to exhibit 80 ± 2.5 % inhibition (LC-EAS) in comparison
with hexane (20 ± 3 %) at their maximum concentration
(100 lg/mL) (data not shown).
Dose Response Analysis of Bioactive Extract
on Mitogen-induced Lymphocyte Proliferation
The bioactive extract LC-EAS was subjected to PHA-
induced PBMC proliferation, to identify the minimum dose
required for maximum inhibitory activity, at 24 h measured
using [3
H] thymidine uptake assay. A significant increase
in proliferation of PBMCs was observed upon induction
with PHA which was observed to be considerably inhibited
by LC-EAS in a dose-dependent manner. The IC50 of
LC-EAS was found to be 10 lg/mL (Fig. 1) and was used
for further studies.
Effect of LC-EAS on Cell Cytotoxicity
To confirm that the suppressive effect of LC-EAS on
lymphocyte proliferation is not due to cytotoxicity, the
supernatants were assayed for the enzyme lactate dehy-
drogenase (LDH). The cell cytotoxicity of LC-EAS was
studied at doses ranging from 100 to 1 lg/mL by means of
LDH release at 24 h. The results clearly show the LC-EAS
to be nontoxic even at a higher dose of 100 lg/mL (Fig. 2).
Effect of LC-EAS on Nitric Oxide Production
and iNOS mRNA Expression in RAW 264.7 Cells
Nitric oxide (NO) synthesis was determined by Griess
reaction using nitrite as an indicator of NO [19]. The effect
of LC-EAS on pro-inflammatory mediator, nitric oxide
release was assessed on RAW 264.7 cells after induction
with 2 lg/mL LPS. A significant decrease in NO produc-
tion (Fig. 3a) and a decrease in the mRNA expression of
inducible nitric oxide synthase (iNOS) (Fig. 3b) were
observed upon treatment with 10 lg/mL of LC-EAS.
Effect of LC-EAS on Pro-inflammatory Cytokine
and NF-jB Signaling in PBMC’s
Inhibition of pro-inflammatory cytokines is one mechanism
by which autoimmune and inflammatory disorders can be
regulated [20]. PBMCs were induced with 2 lg/mL LPS at
varying time points (6, 12, 24 h). The expression levels of
pro-inflammatory cytokines, TNFa, and IL-6 were
observed to be reduced upon treatment with 10 lg/mL
LC-EAS (Fig. 4). An upregulation in the mRNA expres-
sion of the TH2 anti-inflammatory cytokine IL-10 was
observed on treatment with LC-EAS (Fig. 4). To under-
stand the mechanism by which LC-EAS regulates lym-
phocyte proliferation and inflammatory mediators, the
effect of 10 lg/mL LC-EAS was analyzed on phosphory-
lated forms of ERK1/2, STAT3, NF-jB, and IjBa. Cells
treated with LC-EAS after LPS induction showed a
Fig. 1 Dose response analysis of LC-EAS on mitogen-induced
lymphocyte proliferation. Dose response study was performed on
mitogen-induced PBMC with varying concentrations (100, 50, 25, 10,
5, and 1 lg/mL) of LC-EAS. The IC50 dose was found to be 10 lg/
mL. The results are expressed in terms of percent inhibition with
respect to control. Experiments were done in triplicates and repeated
three times. Results are the mean ± SD when compared with
untreated control group
Fig. 2 Cytotoxic effect of LC-EAS on PBMCs. Dose response study
was done at different concentrations ranging from 100, 50, 25, 10, 5,
and 1 lg/mL. After 24 h, cytotoxicity was assessed based on the
lactate dehydrogenase released and the absorbance read at 490 nm.
Triton X served as the positive control and untreated cells as negative
control (NC). The results are expressed as % of cytotoxicity over
untreated cells. Data presented as mean ± SD from three separate
experiments for each data point
123

down-regulation in the expression of ERK1/2, STAT3, and
NF-jB, whereas an upregulation of IjBa was observed
(Fig. 5).
Effect of LC-EAS on TFF3 mRNA Level Expression
and NF-jB Signaling in HT-29 Cells
In this study, HT-29 cells were induced with 2 lg/mL LPS
prior to treatment with LC-EAS at various time points
(6, 12, 24 h). A significant increase in the expression of
TFF3 mRNA expression was observed after 12 h treatment
(Fig. 6). The intestinal epithelial cells possess a TFF3
regulated NF-jB signaling in which ERK1/2 and STAT3
play a diverse role in regulating intestinal inflammation.
HT-29 cells were induced with 2 lg/mL LPS for 6, 12, and
24 h. LC-EAS treated HT-29 cells showed a down-regu-
lation of ERK1/2 and NF-jB, whereas STAT3 expression
was observed to be upregulated (Fig. 7).
Discussion
Probiotics have long been used as an alternative to tradi-
tional medicine with the aim of maintaining intestinal
homeostasis and preventing disease. Foods for human
consumption containing lactic acid bacteria include fer-
mented milks, cheese, wine, fruit juice, and sausages.
Single and mixed cultures of live microorganisms are used
in probiotic preparations [4]. Probiotic bacteria have the
ability to protect the intestine from pathological damage by
stimulating the immune system. We have investigated the
key components involved in the regulation of anti-inflam-
matory potential of probiotic Lactobacillus casei. Several
studies have evaluated the anti-inflammatory potential of
compounds by studying their effect on lymphocyte prolif-
eration in the presence of allogenic cells, mitogens, and
specific antigens [20]. In the present study, the ability of
LC-EAS on proliferation of mononuclear cells, crucial for
generation of effective immune response, was analyzed in
Fig. 3 a Effect of LC-EAS on LPS-induced nitric oxide (NO)
production in RAW 264.7 cell line. RAW 264.7 cells were induced
with LPS (2 lg/mL) followed by treatment with LC-EAS for 24 h.
b Analysis of LPS (2 lg/mL) induced iNOS expression in RAW
264.7 macrophages at different time points (6, 12, 24 h). Lane 1
100 bp marker, Lane 2 control, Lane 3 LPS (6 h), Lane 4 LPS (12 h),
Lane 5 LPS (24 h), Lane 6 LPS ? LC-EAS (6 h), Lane 7 LPS ? LC-
EAS (12 h), Lane 8 LPS ? LC-EAS (24 h). GAPDH was considered
as internal control. c Densitometric analysis for the quantitative
analysis of iNOS mRNA expression
Fig. 4 a Analysis of LC-EAS on LPS-stimulated pro-inflammatory
cytokine expression in PBMCs. PBMCs were induced with LPS
(2 lg/mL) for various time points (6, 12, 24 h), and the inhibitory
effect of LC-EAS on pro-inflammatory cytokine expression (TNFa,
IL-10, IL-6) was studied by RT-PCR analysis. GAPDH was
considered as internal control. Lane 1 100 bp marker, Lane 2 control,
Lane 3 LPS (6 h), Lane 4 LPS (12 h), Lane 5 LPS (24 h), Lane 6
LPS ? LC-EAS (6 h), Lane 7 LPS ? LC-EAS (12 h), Lane 8
LPS ? LC-EAS (24 h). b Densitometric analysis for the quantitative
analysis of mRNA expression
123

the presence of the mitogen PHA (Fig. 1). Pro-inflamma-
tory cytokines like TNFa and IL-6 play an important role
in the inflammatory response by responding to a wide
variety of invasive stimuli, and its overproduction has been
shown to induce the production of various inflammatory
mediators such as iNOS and nitric oxide [6]. There is a
large body of evidence to suggest that NO is involved in
several inflammatory disorders. To study the role of nitric
oxide, RAW 264.7 cell line was used, as macrophages are
the earliest trigger in immune response. Significant
decrease in the LPS-induced NO production was observed
on treatment with LC-EAS at 24 h (Fig. 3a) which could
be due to suppression of iNOS, as evidenced by RT-PCR
studies. Similarly, treatment with LC-EAS showed a down-
regulation in the expression of iNOS from 6 h in com-
parison with LPS-treated control cells (Fig. 3b). Nitric
oxide appears to mediate or augment the synthesis of TNFa
and chemokines in macrophage cells. They also activate
the inflammatory cellular mediators and induce NF-jB
signaling that in turn modulates important cellular events
including gene expression, DNA damage, and cellular
proliferation contributing to various inflammatory
disorders. RT-PCR analysis revealed LC-EAS’s capability
to down-regulate LPS-stimulated production of TNFa and
IL-6 up to 24 h and an upregulation in the mRNA
expression of the anti-inflammatory cytokine IL-10
(Fig. 4).
NF-jB is a transcription factor that plays a crucial role
in immunity, inflammation, cell proliferation, and apopto-
sis. Activation of NF-jB occurs mainly via IjB kinase
(IKK)—mediated phosphorylation of inhibitory molecules
such as IjBa [21]. TNFa-mediated NF-jB activation
occurs via ERK dependant pathway in the nucleus. ERK is
involved in LPS-induced NO production, iNOS expression,
and TNFa secretion [22]. STAT3 interacts with nuclear
factor-jB (NF-jB) at multiple levels and is activated by
several NF-jB regulated gene products, including IL-6.
These transcription factors regulate a multitude of genes
essential for STAT3 activation, thereby promoting
inflammation [23]. Hence, it was crucial to understand the
role of LC-EAS on NF-jB signaling. Treatment for
LC-EAS to LPS-stimulated PBMCs showed an upregula-
tion of IjBa (Fig. 5) at 6 h and down-regulation of NF-jB,
ERK1/2, and STAT3 expressions (Fig. 5).
TFF3, an intestinal trefoil factor, modulates NF-jB and
acts as a novel negative regulatory molecule in intestinal
epithelial cells [24]. HT-29 cell line, originating from
human colon adenocarcinoma, shows many characteristic
features of the intestinal epithelium and hence was used as
Fig. 5 a Analysis of LC-EAS on NF-jB signaling. PBMCs were
stimulated with LPS (2 lg/mL) prior to LC-EAS treatment. Phos-
phorylation of NF-jB, IjBa, STAT3, and ERK1/2 was assessed by
Western blot. Lane 1 control, Lane 2 LPS (6 h), Lane 3 LPS (12 h),
Lane 4 LPS (24 h), Lane 5 LPS ? LC-EAS (6 h), Lane 6 LPS ? LC-
EAS (12 h), Lane 7 LPS ? LC-EAS (24 h). b-actin served as an
internal control. b Densitometric analysis for the quantitative analysis
of protein expression
Fig. 6 a Analysis of LC-EAS on LPS-stimulated TFF3 expression in
HT-29 cells. HT-29 cells were induced with LPS (2 lg/mL) for
various time points (6, 12, 24 h), and the inhibitory effect of LC-EAS
on TFF3 was studied by RT-PCR. Lane 1 100 bp marker, Lane 2
control, Lane 3 LPS (6 h), Lane 4 LPS (12 h), Lane 5 LPS (24 h),
Lane 6 LPS ? LC-EAS (6 h), Lane 7 LPS ? LC-EAS (12 h), Lane 8
LPS ? LC-EAS (24 h). GAPDH was considered as internal control.
b TFF3 gene expression was quantified using densitometry
123

an in vitro model. Activation of NF-jB by TFF3 is com-
posed of homodimers of p65 subunits, whereas activation
by TNFa consists of homodimers of p65 complexed with
p50. These differences are accompanied by varying effects
of TNFa and TFF3 on IjBa degradation. TFF3 causes only
partial degradation of this inhibitor, which is rapidly re-
synthesized, while TNFa causes a more profound degra-
dation of the inhibitor [11]. Previous studies [23, 25, 26]
have shown that TFF3 is expressed in goblet cells and are
secreted on intestinal lumen under normal circumstances
and target intestinal epithelial cells. In contrast to TNF,
TFF3 induces upregulation of Twist protein in intestinal
epithelial cells. Thus, TFF3 probably plays an important
role in maintaining Twist protein in intestinal epithelial
cells, which may contribute to the down-regulation of
inflammatory mediators in vivo [24]. Also, TFF3 acts as
bridging molecule between inflammatory and cell survival
pathways, with increased TFF3 activating Akt/PI3K
through phosphorylation of EGFR [27]. Since NF-jB
generally acts downstream of Akt/PI3K pathway, TFF3
could be playing a homeostatic role in maintaining intes-
tinal mucosa. An upregulation of TFF3 was observed upon
treatment of HT-29 human colon cells with LC-EAS
(Fig. 6). The expression of ERK1/2 and NF-jB was
observed to be down-regulated upon treatment with
LC-EAS (Fig. 7), whereas STAT3 was found to be
upregulated in a time-dependent manner (Fig. 7). Timothy
et al. [8] have reported that an intestinal deficiency of
activated STAT3 leads to a down-regulation of TFF3
which might be expected to predispose the intestine toward
ulceration and inflammatory bowel disease. In summary,
the present study demonstrated the immunomodulatory
effect of LC-EAS. In monocytes and lymphocytes, the anti-
inflammatory activity of LC-EAS was observed to be
mediated through down-regulation of nitric oxide release,
down-regulation of iNOS, TNFa, IL-6 (mRNA expression)
and ERK1/2, STAT3 and NF-jB (protein expression). An
upregulation in the mRNA expression of IL10 and the
protein expression of IjBa was also observed. In intestinal
epithelial cells, the effect of LC-EAS was regulated
through STAT3 dependent TFF3 signaling of NF-jB.
References
1. Christina LO, Wallace KM (2010) Probiotic bacteria and intes-
tinal epithelial barrier function. Am J Physiol Gastrointest Liver
Physiol 298:807–819
2. Kirjavainen PV, El-Nezami HS, Salminen SJ, Ahokas JT, Wright
PF (1999) The effect of orally administered viable probiotic and
dairy lactobacilli on mouse lymphocyte proliferation. FEMS
Immunol Med Mic 26:131–135
3. Fang Y, Brent PD (2002) Probiotic bacterium prevents cytokine-
induced apoptosis in intestinal epithelial cells. J Biol Chem
227:50959–50965
4. Maldonado GC, Moreno De, de LeBlanc A, Vinderola G, Bibas
B, ME Perdigo’nG (2007) Proposed model: mechanisms of
immunomodulation induced by probiotic bacteria. Clin Vaccine
Immunol 14:485–492
5. Fang HW, Fang SB, Chiang Chiau JS, Yeung CY, Chan WT,
Jiang CB, Cheng ML, Lee HC (2010) Inhibitory effects of Lac-
tobacillus casei subsp. rhamnosus on Salmonella lipopolysac-
charide induced inflammation and epithelial barrier dysfunction
in a co-culture model using Caco-2/peripheral blood mononuclear
cells. J Med Microbiol 59:573–579
6. Gayathri B, Manjula N, Vinaykumar KS, Lakshmi BS, Bala-
krishnan A (2007) Pure compound from Boswellia serrata extract
exhibits anti-inflammatory property in human PBMCs and mouse
macrophages through inhibition of TNFa, IL-1b, NO and MAP
kinases. Int Immunopharmacol 7:473–482
7. Kang SR, Han DY, Park KI, Park HS, Cho YB, Lee HJ, Lee WS,
Ryu CH, Ha YL, Lee DH, KimJA KimGS (2011) Suppressive
effect on lipopolysaccharide-induced proinflammatory mediators
by Citrus aurantium L. in macrophage RAW 264.7 cells via NF-
jB signal pathway. Evid Based Compl Alt 10:1–12
8. Timothy CW, James RG (2002) Inflammation intersection: gp130
balances gut irritation and stomach cancer. Nat Med 8:1080–1082
9. Baeuerle PA, Baltimore D (1996) NF-jB: ten years after. Cell
87:13–20
10. Pahl HL (1999) Activators and target genes of Rel/NF-jB tran-
scription factors. Oncogene 18:6853–6866
11. Kim EB (2005) A new twist on trefoils. Focus on ‘‘TFF3 mod-
ulates NF-jB and a novel regulatory molecule of NF-jB in
Fig. 7 a Analysis of LC-EAS on STAT3, ERK1/2, and NF-jB.
Antibodies recognizing phosphorylated STAT3, ERK1/2, NF-jB, and
b-actin were used to perform Western blotting. HT-29 cells were
stimulated with LPS (2 lg/mL) prior to LC-EAS treatment. Lane 1
control, Lane 2 LPS (6 h), Lane 3 LPS (12 h), Lane 4 LPS (24 h),
Lane 5 LPS ? LC-EAS (6 h), Lane 6 LPS ? LC-EAS (12 h), Lane 7
LPS ? LC-EAS (24 h). b-actin served as an internal control.
b Protein expression was assessed by densitometry
123

intestinal epithelial cells via a mechanism distinct from TNF-a’’.
Am J Physiol Cell Physiol 289:1069–1071
12. Soriano-Izquierdo A, Gironella M, Massaguer A, May FE, Salas
A, Sans M, Poulsom R, Thim L, Pique´ JM, Pane´s J (2004) Trefoil
peptide TFF2 treatment reduces VCAM-1 expression and leu-
kocyte recruitment in experimental intestinal inflammation.
J Leukocyte Biol 75:214–223
13. Rivat C, Rodrigues S, Bruyneel E, Pie’tu G, Robert A, Redeuilh
G, Bracke M, Gespach C, Attoub S (2005) Implication of STAT3
signaling in human colonic cancer cells during intestinal trefoil
factor 3 (TFF3)–and vascular endothelial growth factor–mediated
cellular invasion and tumor growth. Cancer Res 65:195–202
14. Tien MT, Girardin SE, Regnault B, Le Bourhis L, Dillies MA,
Coppeé JY, Bourdet-Sicard R, Sansonetti PJ, Pe´dron T (2006) Anti-
inflammatory effect of Lactobacillus casei on Shigella-infected
human intestinal epithelial cells. J Immunol 176(2):1228–1237
15. Alison T, Elaine VM, Palok A (2010) Probiotic-induced priming
of innate immunity to protect against rotaviral infection. Probi-
otics Antimicrobial Proteins 2(2):90–97
16. Cecilia H, Julio V, Hortensia Z, Susana A, Graciela A (2009)
Lactobacillus casei modulates the inflammation-coagulation
interaction in a pneumococcal pneumonia experimental model.
J Inflamm 6:28
17. Angulo S, Llopis M, Antolıń M, Gironella M, Sans M, Malage-
lada JR, Pique´ JM, Guarner F, Pane´s J (2006) Lactobacillus casei
prevents the upregulation of ICAM-1 expression and leukocyte
recruitment in experimental colitis. Am J Physiol Gastrointest
Liver Physiol 291:1155–1162
18. Hemashenpagam N (2011) Purification of secondary metabolites
from soil actinomycetes. Intl J Microbio Res 3:148–156
19. Chen LG, Yang LL, Wang CC (2007) Anti-inflammatory activity
of mangostins from Garcinia mangostana. Food Chem Toxicol
42:688–693
20. Manjula N, Gayathri B, Vinaykumar KS, Shankernarayanan NP,
Vishwakarma RA, Balakrishnan A (2006) Inhibition of MAP
kinases by crude extract and pure compound isolated from
Commiphora mukul leads to down regulation of TNF-a, IL-1b
and IL-2. Int Immunopharmacol 6:122–132
21. Viatour P, Merville MP, Bours V, Chariot A (2005) Phosphory-
lation of NF-jB and I-jB proteins: implications in cancer and
inflammation. Trends Biochem Sci 30:43–52
22. Schmitt KR, Diestel A, Lehnardt S, Schwartlander R, Lange PE,
Berger F, Ullrich O, Abdul-Khaliq H (2007) Hypothermia sup-
presses inflammation via ERK signaling pathway in stimulated
microglial cells. J Neuroimmunol 189:7–16
23. Yu H, Pardoll D, Jove R (2009) STATs in cancer inflammation and
immunity: a leading role for STAT3. Nat Rev Cancer 9:798–809
24. Ya-Qin Z, Xiao-Di T (2005) TFF3 modulates NF-jB and a novel
negative regulatory molecule of NF-jB in intestinal epithelial
cells via a mechanism distinct from TNF-a. Am J Physiol Gas-
trointest Liver Physiol 289:1085–1093
25. Sands BE, Podolsky DK (1996) The Trefoil peptide family. Annu
Rev Physiol 58:253–273
26. Wright NA, Hoffmann W, Otto WR, Rio MC, Thim L (1997)
Rolling in the clover: trefoil factor family (TFF)-domain pep-
tides, cell migration and cancer. FEBS Lett 408:121–123
27. Bharathi Raja S, Murali MR, Devaraj H, Niranjali SD (2011)
Differential expression of gastric MUC5AC in colonic epithelial
cells: TFF3-wired IL1b/Akt crosstalk-induced mucosal immune
response against Shigella dysenteriae infection. J cell science
125:703–713
123

Immunomodulatory Potential of Probiotic Lactobacillus casei

Recomendados

Recomendados

Más contenido relacionado

La actualidad más candente

La actualidad más candente (20)

Similar a Immunomodulatory Potential of Probiotic Lactobacillus casei

Similar a Immunomodulatory Potential of Probiotic Lactobacillus casei (20)

Más de KarthikeyanThirugnan3

Más de KarthikeyanThirugnan3 (6)

Último

Último (20)

Immunomodulatory Potential of Probiotic Lactobacillus casei