Post-translational inhibition of IP-10 secretion in IEC by probiotic bacteria: impact on chronic inflammation.

Abstract

Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).
VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF(DeltaARE) and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells.
We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.

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Post-Translational Inhibition of IP-10 Secretion in IEC by
Probiotic Bacteria: Impact on Chronic Inflammation
Gabriele Ho ¨rmannsperger1, Thomas Clavel1, Micha Hoffmann1, Caroline Reiff2, Denise Kelly2, Gunnar
Loh3, Michael Blaut3, Gabriele Ho ¨lzlwimmer4, Melanie Laschinger5, Dirk Haller1*
1Chair for Biofunctionality, ZIEL-Research Center for Nutrition and Food Science, Technische Universita ¨t Mu ¨nchen, Freising-Weihenstephan, Germany, 2Rowett Institute
of Nutrition and Health, Aberdeen University, Aberdeen, United Kingdom, 3Gastrointestinale Mikrobiologie, Deutsches Institut fu ¨r Erna ¨hrungsforschung, Potsdam-
Rehbru ¨cke, Nuthetal, Germany, 4Helmholz-Zentrum Mu ¨nchen, Institute for Pathology, Mu ¨nchen-Neuherberg, Germany, 5Department of Surgery, Technische Universita ¨t
Mu ¨nchen, Munich, Germany
Abstract
Background: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the
context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular
mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).
Methodology/Principal Findings: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible
protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory
components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription
at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell
transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export
machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein
stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal
or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding,
the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the
inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-
10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNFDAREand IL-102/2mice
revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation.
Consistent with reduced tissue pathology in IL-102/2mice, IP-10 protein expression was reduced in primary epithelial cells.
Conclusions/Significance: We demonstrate segment specific effects of probiotic intervention that correlate with reduced
IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein
in IEC to be the molecular mechanism underlying the anti-inflammatory effect.
Citation: Ho ¨rmannsperger G, Clavel T, Hoffmann M, Reiff C, Kelly D, et al. (2009) Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria:
Impact on Chronic Inflammation. PLoS ONE 4(2): e4365. doi:10.1371/journal.pone.0004365
Editor: Baohong Zhang, East Carolina University, United States of America
Received September 8, 2008; Accepted December 17, 2008; Published February 6, 2009
Copyright: ? 2009 Ho ¨rmannsperger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Financial support for this study was obtained from the European Nutrigenomics Organisation, contract nr. FP6-506360 and the Bundesministerium fu ¨r
Bildung und Forschung, AZ0313814. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: haller@wzw.tum.de
Introduction
Inflammatory bowel diseases (IBD) are spontanously relapsing,
immunologically mediated disorders of the gastrointestinal tract.
The incidence and prevalence of the two main idiopathic
pathologies of IBD, Crohn’s disease (CD) and ulcerative colitis
(UC), are rising globally in parallel to the progression of the
industrialisation. More that 3.5 million people suffer from IBD in
the United States and Europe [1], but although extensive research
was performed in this field, the initial trigger is still unknown and
efficient therapies are not available yet. Genetical susceptibility of
the host [2] and the intestinal microbiota both were found to play
a pivotal role in the onset and perpetuation of IBD. Enhanced
numbers of mucosa associated bacteria and decreased microbiota
diversity are associated with these diseases [3]. In contrast to the
fact, that IBD is the result of an overreaction of the intestinal
immune system towards intestinal microbes, clinical studies
showed that oral uptake of specific probiotic bacterial strains like
VSL#3 [4,5] or Escherichia coli Nissle 1917 [6,7] resulted in
attenuation of IBD disease severity [8]. VSL#3, a mixture of eight
different lactic acid bacteria (Lactobacillus (L.) acidophilus, L.
bulgaricus, L. casei, L. plantarum, Streptococcus thermophilus, Bifidobacter-
ium (B.) breve, B. infantis, B. longum) was effective in the prevention
and in the maintenance treatment of pouchitis and UC [9–14].
Apart from its protective effect in clinical studies, VSL#3 was
shown to reduce experimental colitis in IL-10-deficient (IL-102/2)
mice. Treatment of IL-102/2
histopathology, normalization of colonic function, barrier integrity
and mucosal cytokine production [15]. Furthermore, VSL#3 was
shown to induce protective heat-shock-proteins in intestinal
mice resulted in decreased
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epithelial cells (IEC) [16] or proliferation of IL-10-dependent
TGFb-bearing regulatory T-cells in Th1-dependent murine colitis
[17]. However, there is no study showing protective effects of
VSL#3 in the context of CD or experimental ileitis, suggesting
disease-and intestinal segment-specific effects of VSL#3. Exten-
sive progress has been made in understanding probiotic effects of
VSL#3 in the context of IBD but the molecular mechanisms as
well as strain-specificity remain to be elucidated.
IEC are crucial for maintaining intestinal homeostasis [18] and
failure to control inflammatory processes at the epithelial cell level
may critically contribute to the disease pathogenesis. The intestinal
epithelium must interact with and adapt to the constant changing
environment by processing the combined biological information
from luminal enteric bacteria and host-derived signals [19]. IEC
react on bacterial as well as immune-derived pro-inflammatory
signals by secreting cytokines and chemokines like interleukin 6
(IL-6) and interferon c-induced protein 10 (IP-10) to activate and
attract Th1-immune cells and phagocytic cells to the site of
infection. Cytokine and chemokine levels were shown to be
strongly elevated in inflamed intestinal regions of IBD patients
[20]. Experimental studies confirmed that IP-10 plays an
outstanding role in uncontrolled disease development as the
blockade of IP-10 by an anti-IP-10 antibody was sufficient to
decrease disease severity in IL-102/2mice. This effect was shown
to be due to reduced Th1 cell generation in inductive sites and
reduced recruitment of Th1 effector cells to the colon [21].
Another experimental study revealed that anti-IP-10 therapy
attenuatesmurine acquired
(MAIDS) colitis through the blockade of Th1-cell trafficking and
the reduction of IEC apoptosis [22]. Although the importance of
IP-10 as a pro-inflammatory mediator in IBD has been clearly
demonstrated, the molecular mechanisms underlying the regula-
tion of IP-10 expression in IEC during disease development are
not well understood. Interestingly, recent studies revealed that
probiotics are able to regulate chemokine expression in IEC.
Although E. coli strain Nissle 1917 and VSL#3 were shown to
reduce TNF-or Salmonella dublin-induced IL-8 chemokine expres-
sion in IEC [23,24], the molecular mechanisms of target-specific
inhibition of IEC activation through probiotic bacteria under
conditions of chronic intestinal inflammation are not understood.
The aim of our study was to analyze the molecular mechanisms of
strain-specific effects of VSL#3 on IP-10 production in activated
IEC. Furthermore, the effect of VSL#3 on TNF-induced ileitis in
heterozygous TNFDAREmice and on colitis in IL-102/2mice was
investigated in the context of IP-10 expression in IEC.
immunodeficiency syndrome
Materials and Methods
Cell culture
The small intestinal epithelial cell line Mode-K [25] (passage
10–25) as well as the human embryonic kidney epithelial cell line
HEK293 [26] was grown in a humidified 5% CO2 atmosphere at
37uC to confluency in 6, 12 or 24 well tissue culture plates (Cell
Star, Greiner bio-one, Frickenhausen, Germany). TLR2 deficient
HEK293 cells were bought from InvivoGen (Cat.Nr.: 293-null)
and HEK293 cells expressing TLR2 were also bought from
InvivoGen (Cat.Nr.: 293-mtlr2). The Mode-K cell culture media
was Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal calf serum (FCS), 1,0% Glutamine and 0,8% antibiotic
antimycotic (Invitrogen, Carlsbad, USA). Glutamine was omitted
for the cultivation of HEK293 cells and Blasticidin was added for
the cultivation of the stably transfected HEK293 cells. Cell culture
media was changed prior to stimulation.
Cell culture stimulation and bacterial treatment
Confluent Mode-K cell monolayers were stimulated with TNF
(10 ng/ml) (R&D Systems Europe), brefeldin A (0,5 mM) (Calbio-
chem), lactacystin (22 mM) (Biomol), NH4Cl (20 mM) (Sigma), 3-
methyladenine (5 mM) and/or VSL#3 bacteria (VSL#3 mixture
or single strains, generous gift from Dr. DeSimone, L’Aquila, Italy)
(moi 20) for 24 h if not otherwise indicated. Viability of cells after
brefeldin A treatment (6h) (93% of viable cells, data not shown)
was analysed by a WST assay (Roche). VSL#3 derived L. casei and
L. plantarum 299v were grown anaerobically (Anaerogen, Oxoid) at
37uC in MRS Broth (Fluka, Heidelberg) containing 0,05% L-
cysteine (Roth). E. coli strain Nissle 1917 (a generous gift from Dr.
Sonnenborn, Ardeypharm GmbH, Herdecke, Germany) was
grown aerobically in LB-media. Bacteria were centrifuged
(4500 g, 10 min) and resuspended in DMEM. L. casei was heat-
killed (30 min, 90uC), fixed (fL.c) (3 hours, 5% formaldehyde, 4uC)
or lysed by lysozyme (Sigma, 50 mg/ml) in filter sterilized 10 mM
Tris buffer (pH 8). Fixed bacteria were washed three times with
sterile PBS before IEC stimulation. For cell surface treatment,
bacteria were incubated with Phospholipase A (2 mg/ml) (Fluka),
Trypsin (2 mg/ml) (Roth) or Proteinase K (50 mg/ml) (Roth) in
filter-sterilized Tris buffer (50 mM Tris, 0,1 M NaCl, pH 8, 1h,
37uC) in a shaker.
Western blot
Purified primary IEC or Mode-K cells were lysed in Laemmli
buffer and 50 mg of protein were subjected to electrophoresis on
10% or 15% SDS-PAGE gels. Anti IP-10 (R&D Systems Europe,
Arlington), anti-IkB, anti-phospho-RelA (Cell Signaling, Beverly,
MA), anti-ubiquitine (Cell Signaling, Beverly, MA), anti-DsRed
(clontech) and anti-ß-actin-antibody (ICN, Costa Mesa, CA) were
used to detect immunoreactive IP-10, IkB, phospho-RelA, DsRed,
ubiquitine and ß-actin, using an enhanced chemoluminescence
light-detecting kit (GE, Arlington Heights, IL).
ELISA
IP-10 and IL-6 concentrations were determined in IEC
supernatants using the appropriate ELISA kits (R&D Systems
Europe) according to the manufacturer’s instructions.
Chromatin Immunoprecipitation (ChIP)
Mode-K cells in 75 cm2flasks were pre-incubated with L. casei
for 1 h and stimulated with TNF (10 ng/ml) for 2 h. Cells were
fixed by formaldehyde fixation (1%) and nuclear extraction as well
as chromatin immunoprecipitation were performed using a ChIP-
kit (Active Motif, Carlsbad, CA, USA) according to the
manufacturer’s instructions. Immunoprecipitation was carried
out over night at 4uC with an anti-NFkappaB p65 antibody (Cell
Signaling, Beverly, MA). DNA/protein/antibody-complexes were
collected with salmon sperm saturated protein A/G agarose for
30 min and washed three times in high salt buffer followed by
three more washes in no salt buffer. DNA was released from the
immune complex by heating and subsequent proteinase K
treatment. DNA was then extracted by phenol-chloroform and
eluted in water. The input control for the PCR was DNA from
total nuclear extract. PCR was performed with total DNA (input
control, 1 ml) and immunoprecipitated DNA (1 ml) using the
followingIP-10promoter-specific
CACGCTTTG, 59-GTCCTGATTGGCTGACT. The length of
the amplified product was 186 bp. PCR products (10 ml) were
subjected to electrophoresis on 2% agarose gels.
primers59-AACAGCT-
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Transfection
Mode-K cells (luciferase assay) or HEK293 cells (IP-10
overexpression assay) (50% confluent, 24-well plate) were
transfected using FuGENE (Roche, Mannheim, Germany)
according to the manufacturer’s instructions (24 h). A mixture of
23 ml DMEM (Invitrogen), 1.5 ml FuGENE and 0.5 mg of the
appropriate plasmid (pGL3-basic-IP10 (p-IP-10), pGL3-basic (ctr-
vector), pIP-10-DsRed, pDsRed-Monomer-C1 (ctr-DsRed)) was
added to every well and incubated for 6h.
The pGL3-basic-IP10 plasmid was created by cloning a murine
IP-10 promoter region (nucleotides 2269 to 228 relative to the
start point of transcription) into the XhoI and BglII sites of the
pGL3-basic vector (Promega, Madison, WI), placing the promoter
directly upstream of the firefly luciferase coding sequence.
Therefore, the IP-10 promoter region was amplified from Mode-
K cell genomic DNA via pfu (QIAGEN, Hilden) PCR. The sense
primerwas59-GCGCTCGAGCTCAAACAGCTCAC-39
italic portion indicates the XhoI restriciton site) and the reverse
primerwas59-GCGAGATCTTCGAGTGCCGGCTG-39(theitalic
portionindicatestheBglIIrestrictionsite).Afterdigestionandligation
ofPCRproductandvector,therecombinantplasmidwasverifiedby
DNA sequencing (QIAGEN Sequencing Service, Hilden). The
PGL3-basic-vector was used as a transfection control.
The vector pIP-10-DsRed contains a cytomegalovirus (CMV)
promoter sequence driving the expression of the chemokine IP-10,
which is N-terminally fused to Red Fluorescent Protein (DsRed). A
0.3 kb cDNA of murine IP-10, covering the whole open reading
frame, was amplified from the Mode-K cell cDNA pool by pfu
(QIAGEN, Hilden) PCR using primers designed according to the
IP-10 cDNA sequence (GenBank Accession No. NM_021274.1).
The sense primer for IP-10-DsRed was 59-GCGCTCGAGATAT-
GAACCCAAGTGCTGCCG-39 (the italic portion indicates the
XhoI restriction site and bold letters indicate the start codon). The
antisense primer was 59-GCGCCCGGGAATTAAGGAGCCCTT-
TTAGACCT-39 (the italic portion indicates the XmaI site; stop
codon in bold letters). The PCR product was digested and cloned
into the XhoI and XmaI sites of pDsRed-Monomer-C1 (Clontech,
Mountain View, CA). The recombinant plasmid was verified by
DNAsequencing (QIAGEN
pDsRed-Monomer-C1 (constantly expressing dsRed) was used as
a transfection control.
(the
Sequencing Service,Hilden).
Luciferase assay
Transfected Mode-K cells were stimulated with TNF or TNF
and L. casei for 24 h. Cells were lysed in 30 ml lysis buffer
(Promega) and cell debris were separated by high speed
centrifugation. Supernatants (25 ml) were mixed with Reagent A
(PJK, Kleinblittersdorf) and the firefly luminescence was measured
at 550 nm. A volume of 100 ml of Reagent B (PJK, Kleinblitters-
dorf) was added and renilla luminescence was measured at
480 nm. Relative luciferase activity was calculated in percentages:
((firefly/renilla)sample/(firefly/renilla)/control)6100.
RNA isolation, reverse transcription and real-time PCR
RNA from Mode-K cells or isolated primary IECs was
extracted using Trizol Reagent (Invitrogen, Karlsruhe, Germany)
according to the manufacturer’s instructions. Extracted RNA was
solved in 20 ml water containing 0.1% diethyl-pyrocarbonate.
RNA concentration and purity (A260/A280ratio) was determined
by spectrophotometric analysis (ND-1000 spectrophotometer,
NanoDrop Technologies, Willigton, USA). Reverse transcription
was performed using 1 mg total RNA. Real-time PCR was
performed using 1 ml cDNA in a Light CyclerTM system (Roche
Diagnostics, Mannheim, Germany) as previously described [27].
Primer
CATCGCT and reverse 59-CATCTACGGGCAGTGG, IP-10;
sense 59-TCCCTCTCGCAAGGAC and reverse 59-TTGGC-
TAAACGCTTTCAT;18S;
CCAAGGAA and reverse 59-GCTGGAATTACCGCGGCT.
The amplified product was detected by the presence of a SYBR
green fluorescent signal. Melting curve analysis was used to
document amplicon specificity and crossing points (Cp) were
determined. Relative induction of gene mRNA expression was
calculated according to the 22DDCp[28] method and normalized
to the expression of 18S. Data were expressed as fold change
against untreated cells or wildtype mice. Presence or absence of
TLR2 transcripts in the transfected HEK293 cells was proven by
running a 2% Agarose gel with amplicons (321 bp) generated by
reverse transcription of mRNA isolated of the HEK293 cells
followed by real-time PCR.
sequenceswere: TLR2;sense59-TGGGGGTAA-
sense59-CGGCTACCACAT-
T cell transmigration assay
Murine splenocytes were isolated from a fresh spleen (mouse
genotype: C57Bl/6/N) and activated with 2,5 mg/ml Concavalin
A (Sigma) in RPMI (+10%FCS) at 37uC and 5% CO2 for
16 hours. Cells were then centrifuged and about 16108cells were
resuspended in 1 ml RPMI/25 mM Hepes. The cell suspension
was then given onto 5 ml NycoPrep 1,077 A (AXIS-SHIELD,
Oslo via Progen) and centrifuged (600 g, 20 min). The activated T
lymphoblasts which accumulate at the interphase between media
and NycoPrep were taken and resuspended in 30 ml washing
buffer (RPMI/5%FCS/25mM Hepes). The cells were washed
three times (250 g, 10 min) and resuspended to a final cell number
of 26106cells/ml transmigration media (RPMI/5%FCS/25mM
Hepes). The activated murine T lymphoblasts were seeded on top
of transwell filters (5 mm pore size) at a concentration of 26105
cells/ml transmigration media. The lower chamber was filled with
500 ml of transmigration medium and 100 ml of pure cell culture
media or cell culture media from stimulation experiments with
Mode-K cells (control, TNF, TNF/fL.c) or TNF-conditioned
media supplemented with either a neutralizing anti-IP-10 antibody
(30 mg/ml) (R&D Systems) or a control goat-IgG (30 mg/ml)
(Dianova). The assay was then incubated for 2 hours (37uC/5%
CO2) and the number of transmigrated cells in the lower chamber
was analyzed using a CellCounter (Axiovision). The assay was
performed in triplicates and the number of transmigrated cells at
three representative regions (roi) of each single triplicate was
measured.
Co-immunoprecipitation
Mode-K cells (6-well plates) were stimulated with TNF alone or
TNF and L. casei for 6 h. Cells were lysed in 200 ml of 16 lysis
buffer (Cell Signaling, Beverly, MA) supplemented with PMSF
(1 mM). Cell debris were removed by centrifugation (1400 g,
10 min) and supernatants were incubated with anti-IP-10-
antibodies (R&D Systems Europe) (3h, 4uC) in a shaker. Samples
were then incubated over night at 4uC in a shaker together with
13 ml of protein A/G beads (Santa, Cruz, Europe), which were
previously washed twice with 1xlysis buffer (Cell Signaling,
Beverly, MA). Beads were collected by centrifugation (5 min,
8000 g), washed twice with 1xlysis buffer and resuspended in 50 ml
Laemmli buffer for subsequent Western blot analysis.
Pulse-chase experiment
Mode-K cells (6-well plates) in DMEM (0,5% FCS) were
stimulated with TNF during the pulse period (S35Methionine/
Cysteine Labeling Mix, 25 mCi/ml (Perkin Elmer)) (3h). After the
pulse period, the cells were washed three times with 16PBS and
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Figure 1. L. casei selectively inhibits TNF-induced secretion of chemotactically functional IP-10. Mode-K cells were stimulated for 24 h
with (A) VSL#3 (moi 20) or (B) L. casei (moi 20) with or without TNF (10 ng/ml) activation for 24 h. The concentration of IP-10 and IL-6 in cell culture
supernatants was measured by ELISA. Bars represent mean values (+/2 SD) of triplicate samples. (C) 100 ml DMEM or cell culture supernatant from a
24 h stimulation experiment with Mode-K cells (control, TNF (10 ng/ml), TNF+L. casei (moi 20)) were added to 500 ml of transmigration medium. The
amount of transmigrated cells per region of interest was determined by axiovision cell counting. Pictures show representative sections of
transmigrated cells of three independent experiments and bars represent mean values (+/2 SD) of triplicate samples.
doi:10.1371/journal.pone.0004365.g001
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Figure 2. The inhibition of TNF-induced IP-10 secretion is mediated by a surface protein of L. casei. (A) Mode-K cells were stimulated for
24 h with L. casei, Lactobacillus plantarum 299v or E. coli Nissle 1917 (moi as indicated) with or without TNF (10 ng/ml) activation for 24 h. The
concentration of IP-10 and IL-6 in cell culture supernatants was measured by ELISA. Bars represent mean values (+/2 SD) of triplicate samples. (B)
Mode-K cells were stimulated with L. casei (moi 20) killed by formaldehyde fixation (fix), lysozyme (lys)-digestion or heat (heat)-treatment with or
without TNF (10 ng/ml) activation for 24 h. (C) L. casei was treated with trypsin (try), proteinase K (pK) or phospholipase A (pA) followed by
formaldehyde-fixation (fL.c). (D) TLR2 deficient as well as TLR2 expressing HEK293 cells (absence/presence of TLR2 in the cells was analysed by real-
time PCR) were stimulated for 24 h with TNF (10 ng/ml) and L. casei (moi 20). IP-10 concentration was measured in the culture supernatants by ELISA.
The bars represent mean values (+/2 SD) of triplicate samples.
doi:10.1371/journal.pone.0004365.g002
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Figure 3. L. casei-induced inhibition of IP-10 secretion is mediated by a post-transcriptional mechanism (A) Mode-K cells were
stimulated with TNF (10 ng/ml) and L. casei (moi 20) for 15 and 30 min and TNF-induced proteasomal degradation of IkB and (B) TNF-induced
activation of RelA was analyzed by Western Blot in two independent experiments. (C) TNF-induced recruitment of NFkB RelA to the IP-10 promoter
was analyzed in a ChIP experiment using an anti-RelA antibody after pre-incubation of Mode-K cells with L. casei (moi 20) (1h) and subsequent
stimulation with TNF (10 ng/ml) for 2 h. (D) Control (ctr-vector)- and IP-10 reporter (p-IP-10)-plasmid transfected Mode-K cells were stimulated with
TNF (10 ng/ml) with or without L. casei (moi 20) and intracellular luciferase activity was measured by a luminescence assay after 24 h. (E) Mode-K cells
were stimulated with TNF (10 ng/ml) and/or L. casei (Moi 20) for 6 h followed by mRNA isolation, RT-PCR and qPCR. (F) Mode-K cells were stimulated
with TNF (10 ng/ml) alone and together with L. casei (moi 20) for 24 h. Intracellular accumulation of IP-10 was analyzed by Western blot and the
shown figure is representative for more than three independent experiments.
doi:10.1371/journal.pone.0004365.g003
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either lysed in 200 ml of 1xlysis buffer (Cell Signaling, Beverly,
MA) supplemented with PMSF (1 mM) or they underwent a chase
period of 3h with or without stimulation with L. casei. After the
chase period, cells were washed three times with 16PBS and lysed.
Co-immunoprecipitation for IP-10 was performed as described
and beads were resuspended in 20 ml of Laemmli buffer for
subsequent electrophoresis on a 15% SDS gel. After drying the gel
it was placed on a Kodak Storage Phosphor screen (Amersham
bioscience) in a cassette (Amersham Bioscience) over night. The
Storage Phosphor screen was then scanned by a Typhoon TRIO+
scanner (Amersham, Bioscience).
Animals
Conventionally raised TNFDAREmice (a generous gift from
Kollias G., Institute for Immunology, Biomedical Sciences
ResearchCenter ‘‘Al. Fleming’’,
background and SPF-raised IL-102/2
background as well as wildtype C57BL/6 and 129 SvEv mice
were fed 1,36109cfu of VSL#3 bacteria (a generous gift from Dr.
DeSimone, VSL#3 pharma, Italy) in 13,2% (w/v) gelatine, 20%
(w/v) glucose in water every weekday for 15 and 21 weeks starting
at the age of three weeks. Placebo fed mice were used as controls,
respectively. The gelatine was prepared freshly every third day.
Mice were killed at the age of 18 and 24 weeks followed by
sampling of gut content and IEC isolation. Sections of the distal
ileum, cecal tip and distal colon were fixed in 10% neutral
buffered formalin (Sigma Aldrich). Fixed tissues were hematoxylin-
and eosin-stained and embedded in paraffin. Histology scoring
was performed by blindly assessing the degree of lamina propria
mononuclear cell infiltration, crypt hyperplasia, goblet cell
depletion and architectural distortion, resulting in a score from 0
(not inflamed) to 12 (inflamed), as previously described [29].
Animal use was approved by the institution in charge (approval
no. 55.2-1-S4-2531-74-06 and 32-2347/4+63).
Greece)
mice on 129 SvEv
onC57BL/6
Isolation of primary mouse intestinal epithelial cells
Primary IEC were purified as previously described [27]. Briefly,
either ileal or cecal and colonic tissue was cut into pieces and
incubated (37uC, 15 min) in Mode-K cell culture media
supplemented with 1 mM dithiothreitol (Roth, Karlsruhe, Ger-
many). The tissue/IEC suspensions were filtered, centrifuged
(7 min, 300 g, RT) and cell pellets were resuspended in DMEM
containing 5% fetal calf serum. The remaining tissue was
incubated in 30 ml PBS (10 min, 37uC) containing 1.5 mM
EDTA (Roth, Karlsruhe, Germany). After filtration, the tissue was
discarded and the cell suspension from this step was centrifuged as
above. Finally, primary IEC were purified by centrifugation
through a 20%/40% discontinuous Percoll gradient (GE Health-
care, Uppsala, Sweden) at 600 g for 30 min. Primary IEC were
collected in lysis buffer for subsequent Western blot analysis or in
trizol for subsequent RNA isolation. Purity of IEC was confirmed
by anti-CD3+-Western blot analysis.
DNA isolation from gut content and bacteria-specific PCR
Gut content DNA was extracted from 200 mg of content using
the QIAamp DNA Stool Mini Kit (Qiagen) according to the
manfacturer’s instructions. Streptococcus thermophilus (S. thermophilus)
specific PCR of the 16S–23S rRNA gene spacer region was
performed as described previously [30].
Immunohistochemical labeling
The cecum was dissected and immediately immersed in Tissue
Tek OCT compound (Agar Scientific) and frozen in liquid
nitrogen. Samples were stored under liquid nitrogen until
analysed. 8 mm sections were cut using a Leica CM1950 cryostat,
picked up onto polylysine coated slides and air dried at room
temperature for 45 minutes. Sections were fixed in 4%parafor-
maldehyde 0.1M phosphate buffer (pH7.4) for 5 minutes before
being washed with 6 changes of PBS (pH 7.4) over 20 minutes.
Slides were then incubated with 0.1% Triton6100 (Sigma) in PBS
for 3 minutes before being washed as above. Fc receptors
were blocked (Fcc III/II receptor from BD Pharminogen) and
slides incubated in 10% BSA 5% normal donkey serum in PBS for
2 hrs at room temperature. Blocking buffer was removed by
capillary action and the slides incubated over night at 4uC in either
IP-10 goat polyclonal IgG (Santa Cruz G-15 sc-14641) or control
goat IgG (Santa Cruz sc-2028) in 2% BSA. The slides were
washed with 6 changes of PBS over 1 hour. Alexa Fluor 488
donkey anti goat IgG (Invitrogen Molecular Probes) was applied to
the sections and incubated for 30 minutes at room temperature.
The slides were washed as above, counter stained with DAPI and
mounted in vectashield (Vector laboratories). Sections were viewed
on a Zeiss Axioskop microscope using a FITC and DAPI filter set
and imaged using an QIMAGING camera.
Statistical analysis
Data were expressed as mean of triplicates +/2 standard
deviation. Statistical tests were performed using two-tailed Student
test for the in vitro experiments. The in vivo feeding studies were
analysed using rank sum test. Differences were considered
significant if values were ,0.05 (*) or ,0.01 (**).
Results
VSL#3-derived L. casei specifically inhibits TNF-induced
secretion of chemotactically functional IP-10
To investigate the effects of VSL#3 on pro-inflammatory IP-10
expression in IEC, we stimulated Mode-K cells with VSL#3
bacteria (Moi 20) in the presence or absence of TNF, a potent
inducer of IP-10 expression. The concentration of secreted IP-10
in the cell culture supernatant was analyzed by ELISA. VSL#3
did not induce IP-10 expression. In contrast, VSL#3 significantly
reduced TNF-induced IP-10 but not IL-6 expression (Figure 1A,
suggesting an IP-10 specific mechanism for the inhibitory function
of IP-10. Besides, the stimulation of IEC with VSL#3 bacteria
alone induced IL-6 expression. A series of additional experiments
with the eight VSL#3 single bacterial strains (Moi 20) revealed
that one of the eight strains, L. casei, exhibits analogous effects on
IP-10 and IL-6 expression as the complex mixture VSL#3
(Figure 1B). L. casei was therefore identified as the effective
bacterial strain in the probiotic mixture concerning the observed
cytokine expression profile.
Figure 4. The inhibition of IP-10 secretion is independent of the signal-specific activation of IEC. (A) Mode-K cells were stimulated for
24 h with IFNc (100 ng/ml) and L. casei (moi 20). Cell culture supernatants were analyzed by ELISA and (B) intracellular IP-10 protein levels by Western
blot. (C) Mode-K cells were transfected with an IP-10 overexpression (pIP-10-DsRed)- or a ctr-DsRed-plasmid (transfection control) for 6 h and were
then treated with L. casei for 24 h. IP-10 levels in the supernatants were determined by ELISA and intracellular DsRed-tagged IP-10 (IP-10-DsRed) or
DsRed levels (transfection control) were determined by Western blot. Bars in A/C represent mean values (+/2 SD) of triplicate samples and all figures
are representative for three independent experiments.
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To analyze whether the observed inhibition of IP-10 has any
functional consequence, we performed a T cell transmigration
assay with Mode-K culture supernatants after a 24h stimulation
experiment. Figure 1C shows that TNF-conditioned media
increases the transmigration of activated T cells whereas
supernatants of IEC that were costimulated with L. casei did not
exert this chemotactic effect. To show that the reduced
transmigration rate in the TNF/L. casei–conditioned media is
due to the inhibition of IP-10 secretion by the probiotic bacteria,
we used an IP-10 neutralizing antibody in the TNF-conditioned
media. We demonstrated that the neutralization of IP-10 in this
supernatant was sufficient to reduce the transmigration of T cells
analogous to the effect of L. casei. These results clearly support the
functional importance of the observed inhibition of IP-10 secretion
in IEC by L. casei.
Inhibition of TNF-induced IP-10 expression in IEC is
mediated by a cell surface protein of L. casei
Further analysis of other clinically relevant probiotic strains,
Lactobacillus plantarum 299v and E. coli Nissle 1917, revealed that the
inhibition of TNF-induced IP-10 secretion is dose-dependent and
a unique feature of L. casei (Figure 2A). To investigate whether the
inhibition of TNF-induced IP-10 expression was due to an active
interaction between live L. casei and IEC, we inactivated L. casei by
formaldehyde fixation, lysozyme lysis or heat treatment and
subsequently stimulated Mode-K cells. Fig. 2B shows that fixed
bacteria significantly inhibited TNF-induced IP-10 expression.
However, lysis as well as heat-inactivation of L. casei resulted in
complete loss of the observed inhibitory effect, suggesting a heat-
labile surface structure of L. casei as the active bacterial component.
To characterize this component in more detail, we treated L. casei
with phospholipase A or the peptidases trypsine or proteinase K.
Subsequent bacterial stimulation of Mode-K cells revealed that
both peptidases completely reversed the effect of L. casei on TNF-
induced IP-10 expression. In contrast, phospholipase A treatment
did not affect the inhibitory effects of L. casei (Figure 2C). These
results demonstrated that the inhibition of TNF-activated IP-10
expression by L. casei is likely mediated through a cell surface
protein. Consistently, the inhibition of TNF-induced IP-10
expression was not dependent on the pattern recognition receptor
TLR2 (Figure 2D). Of note, the inhibition of IP-10 was dependent
upon the presence of L. casei during TNF stimulation, since the
preincubation of IEC with L. casei did not result in an inhibition of
subsequently TNF-induced IP-10 expression (data not shown).
L. casei induces post-transcriptional inhibition of TNF-
induced IP-10 expression in IEC
We were next interested in the molecular mechanism
underlying the observed inhibitory effect of L. casei on IP-10
expression. Since the transcription factor NFkB plays an
important role in the induction of IP-10 expression [31], we
investigated whether NFkB signaling might be impaired by
stimulation of IEC with L. casei. As shown in Figure 3A, TNF-
induced IkB degradation as well as TNF-induced RelA phos-
phorylation (Figure 3B) was not inhibited by L. casei. In addition,
ChIP analysis revealed that TNF-induced recruitment of RelA to
the IP-10 promoter was not affected by the probiotic bacteria
(Figure 3C). To investigate whether the activation of any other
relevant transcription factor was blocked by L. casei, we transfected
Mode-K cells with an IP-10-promoter reporter gene construct and
observed that TNF-induced IP-10 promoter-dependent luciferase
expression was not inhibited by L. casei (Figure 3D). Consistent
with unaffected IP-10 promoter activity, TNF-induced increase of
IP-10 mRNA levels was not inhibited by L. casei (Figure 3E). In
contrast to the elevated IP10 transcript level, Western blot analysis
revealed that intracellular IP-10 protein was almost completely lost
after 24 hours of costimulation with L. casei (Figure 3F).
Inhibition of IP-10 secretion is independent of the signal-
specific activation of IEC
We next performed a stimulation experiment with IFNc,
another potent inducer of IP-10 expression. As shown in
Figure 4, IFNc-induced IP-10 secretion (Figure 4A, ELISA
analysis) and intracellular IP-10 protein expression (Figure 4B,
Western blot analysis) was completely inhibited by L. casei. These
results were analogous to the above described results observed
under TNF-stimulation, suggesting that the inhibitory effects of L.
casei were TNF-independent. To further characterize the signal-
specificity of the inhibitory effect, we next performed a stimulation
experiment with HEK293 cells that were transfected with a
murine IP-10 overexpression plasmid resulting in constitutive
expression of the chemokine. As shown in Figure 4C, L. casei
significantly reduced intracellular and secreted levels of IP-10 in
this experimental setup, whereas the production of the Ds-Red-
control protein was not affected by the probiotic treatment. This
result showed that the inhibitory effect of L. casei is indeed
independent of the signal-specific activation pathways for IP-10,
supporting our finding that the observed inhibition is mediated by
a post-transcriptional mechanism.
L. casei inhibits IP-10 secretion and triggers subsequent
intracellular degradation of the chemokine
We next investigated whether L. casei had an inhibitory effect on
IP-10 protein translation. As shown in Figure 1, general protein
translation and secretion was not impaired by the probiotic
bacteria as indicated by normal or even increased secretion of
other cytokines/chemokines like IL-6 (Figure 1A/B) or MIP-2
(data not shown). Western blot analysis of intracellular IP-10
protein levels revealed that TNF-induced IP-10 protein expression
was not inhibited by L. casei at early time points (3h after
costimulation). In contrast, the loss of intracellular IP-10 protein
was detectable after 6 to 24 h of costimulation although it was not
Figure 5. Degradation of IP-10 protein is the result of an IP-10 specific secretional blockade. (A) Mode-K cells were stimulated for 1, 3 or
24 h with TNF (10 ng/ml) alone or together with L. casei (moi 20) and intracellular as well as secreted levels of IP-10 were analyzed by Western Blot or
ELISA analysis. Bars in A represent mean values (+/2 SD) of triplicate samples. The shown figure is representative for three independent experiments.
(B) Mode-K cells were stimulated with TNF (10 ng/ml) during a 3h pulse period (S35-labelled cysteine/methionine, 25 mCi) followed by a 3 h chase
period in DMEM supplemented with L. casei (moi 20) or not. Subsequent immunoprecipitation for IP-10 was followed by protein separation on a 15%
SDS gel. Intracellular levels of S35-labelled IP-10 were then made visible by a Phosphoimager plate. The shown figure is representative for two
independent experiments. (C) Mode-K cells were stimulated with TNF (10 ng/ml) alone or together with L. casei in the presence or absence of
brefeldin A (0,5 mM). The amount of intracellular IP-10 after 6 h of stimulation was analyzed by Western blot. (D) Mode-K cells were stimulated for 6 h
with TNF (10 ng/ml) alone or with L. casei (moi 20) before lysis and subsequent immunoprecipitation using an anti-IP-10 antibody was performed.
Western blot was performed to investigate the presence of IP-10 and ubiquitine in the precipitated fractions (single experiment). (E) Mode-K cells
were stimulated with IFNc (100 ng/ml) or TNF (10 ng/ml) alone or together with L. casei (moi 20) in the presence of lactacystin or NH4Cl for 24h and
intracellular IP-10 was analyzed by Western blot. The shown figures are representative for three independent experiments.
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secreted into the cell culture supernatant (Figure 5A). This result
demonstrated that initial TNF-induced IP-10 translation is not
targeted by the probiotic bacteria suggesting that the observed loss
of IP-10 protein is due to post-translational degradation. To
further analyse the fate of initially produced IP-10 protein in more
detail, we performed a S35pulse-chase labelling experiment.
Interestingly, IP-10 immunoprecipitation experiments showed that
TNF-induced S35-IP-10 protein, that was produced during the
initial 3h pulse period, was absent in Mode-K cells after the
subsequent 3h chase period, demonstrating the functionality of
the secretion system. In contrast, TNF-induced S35-IP-10 was still
detectable intracellularly after the chase period in Mode-K cells
that were costimulated during the chase period (Figure 5B). This
result clearly showed that L. casei prevents the secretion of IP-10
protein. However, this finding was in sharp contrast to the
observation that IP-10 protein is lost intracellularly at later time
points instead of intracellular accumulation as it would be
expected to be the result of a secretional blockade. To further
analyze this discrepancy, we performed TNF and L. casei
c(o)stimulation experiments in the presence of brefeldin A, a
known inhibitor of vesicular transport from the endoplasmic
reticulum to the golgi network. Surprisingly, we found that the
inhibition of the protein export machinery by brefeldin A resulted
in complete loss of intracellular IP-10 protein analogous to the
results described above in the costimulation experiments with L.
casei (Figure 5C). This result confirms that indeed, a secretional
blockade of IP-10 results in subsequent loss of intracellular IP-10
protein, suggesting the initiation of degradative mechanisms as a
consequence of initial IP-10 accumulation.
We next addressed the question which degradation pathway
might be responsible for the observed loss of IP-10 protein in IEC.
Co-immunoprecipitation analysis revealed that IP-10 was ubiqui-
tinated in the presence of L. casei (Figure 5D) indicating
proteasomal degradation. Since TNF-induced IP-10 expression
depends on proteasomal IkB degradation, we stimulated cells with
IFNc to investigate the effect of proteasomal inhibition on IP-10
degradation. Figure 5E shows that stimulation with the proteaso-
mal inhibitor lactacystin did not rescue IFNc-induced IP-10
protein from degradation. Next, we investigated whether IP-10
protein was degraded through lysosomal pathways. The inhibition
of lysosomal degradation by NH4Cl (Figure 5E) did not prevent
the loss of IP-10 protein, suggesting an additional proteolytic
mechanism for IP-10 degradation.
L. casei mimicks the effects of the inhibition of
autophagic-lysosomal pathways on cytokine/chemokine
expression
To investigate whether autophagic-lysosomal degradation
pathways might play a role in the observed loss of IP-10 protein,
we applied TNF and L. casei to the epithelial cell culture in the
presence of 3-methyladenine (3-MA). Surprisingly, we demon-
strated that the stimulation of IEC with 3-MA, which is a known
inhibitor of vesicle formation in the context of autophagy-
lysosomal degradation, did not prevent the loss of intracellular
IP-10 (Figure 6A, Western blot analysis), but rather mimicked the
effects of L. casei on the TNF-induced cytokine secretion pattern
(Figure 6A, ELISA analysis). Figure 6A shows that 3-MA
decreased the level of IP-10 protein intracellularly as well as in
the cell culture supernatant. In contrast, TNF-induced IL-6
secretion was not inhibited in the presence of 3-MA. These
observations indicated that IP-10 but not IL-6 secretion is
dependent on the formation of secretory vesicles that can be
inhibited by 3-MA. We therefore hypothesize that L. casei impairs
3-MA-dependent vesicular trafficking resulting in disturbed IP-10
secretion and subsequent degradation of IP-10 protein (Figure 6B).
The protective activity of VSL#3 on experimental colitis
is intestinal segment specific and correlates with IP-10
expression in IEC
To evaluate the physiological impact of VSL#3 on experimen-
tal intestinal inflammation, we performed probiotic feeding studies
with heterozygous TNFDAREmice, an animal model for TNF-
induced experimental ileitis [32], and IL-102/2mice, an animal
model for experimental colitis [33]. TNFDAREmice, IL-102/2
mice and wildtype mice were fed VSL#3 (1.36109cfu per
mouse/day) for 15 and 21 weeks post-weaning. Histopathological
analysis of distal ileal sections revealed that VSL#3 did not inhibit
the development of severe ileal inflammation in TNFDAREmice
(Figure 7A). As expected, we found that TNF-induced ileitis
correlates with enhanced IP-10 protein expression in primary ileal
epithelial cells from TNFDAREmice. The absence of T-cell
contaminations in the purified IEC from inflamed mice confirmed
the purity of the epithelial cell isolation (data not shown).
Consistent with the tissue pathology, VSL#3 did not reduce IP-
10 protein expression in primary ileal IEC (Figure 7B). The
presence of VSL#3 bacteria in the intestine was proven by the use
of bacteria-specific PCR on DNA isolated from intestinal contents
(Figure 7C). In contrast to the unresponsiveness of heterozygous
TNFDAREmice to VSL#3 treatment, we found that cecal
(Figure 8A) but not colonic (Figure 8B) inflammation in IL-102/2
mice was significantly reduced by the probiotic mixture, suggesting
intestinal segment specific effects of VSL#3. In consistence with
the results derived from TNFDAREmice, we detected elevated
levels of IP-10 in isolated intestinal epithelial cells of IL-102/2
mice compared to wildtype mice. Although we were not able to
separate cecal and colonic epithelial cells during the isolation
procedure (for the sake of yield), Western blot analysis showed
that VSL#3 clearly reduced the expression of IP-10 even in the
pooled cell population. Real-time PCR analysis of IP-10 mRNA
levels in these primary IECs showed that the observed reduction
of IP-10 protein is not due to reduced levels of IP-10 mRNA
(Figure 8C), supporting our finding that the reduction of IP-10
protein in IEC is the result of a post-transcriptional mechanism
induced by VSL#3. Most importantly, immunohistochemical
staining of IP-10 in cecal tissue sections confirmed that VSL#3
strongly reduced IEC-specific IP-10 expression in the cecum of
IL102/2mice (Figure 8D).
Discussion
The present study shows for the first time bacterial strain-
specific effects of the clinically relevant probiotic mixture VSL#3
on the expression of a pro-inflammatory chemokine in primary
Figure 6. Blockade of 3-MA-dependent vesicular transport by L. casei might induce the degradation of IP-10. (A) Mode-K cells were
stimulated with TNF (10 ng/ml) alone or together with L. casei (moi 20) in the presence or absence of 3-MA for 24 h and the amount of IP-10 in the
cell culture supernatant as well as intracellularly was analyzed by ELISA and Western blot. Figure A is representative for three independent
experiments and bars represent mean values (+/2 SD) of triplicate samples. (B) The scheme illustrates our hypothesis that L. casei might target 3-MA
dependent vesicular transport of IP-10 resulting in a secretional blockade and subsequent degradation of the chemokine.
doi:10.1371/journal.pone.0004365.g006
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IEC and IEC lines. VSL#3-derived L. casei was identified to
inhibit TNF-induced IP-10 expression to the same extent as the
complex probiotic mixture. This effect was found to be a unique
feature of VSL#3-derived L. casei as other clinically relevant
probiotic bacteria like L. plantarum 299 and E. coli Nissle 1917 did
not reduce IP-10 secretion. Since formaldehyde fixed L. casei had
the same inhibitory effect on IP-10 secretion as live bacteria, active
interaction between bacteria and IEC is not necessary for the
inhibitory effect, suggesting a bacterial cell surface component as
the effective probiotic structure. In contrast to L. rhamnosus GG, a
probiotic bacteria that was shown to have protective effects on
IEC survival and growth via two secreted proteins [34], we
demonstrated that the inhibition of TNF-induced IP-10 secretion
was due to a heat-labile surface protein of L. casei. The
proteinasous nature of the bacterial structure responsible for the
inhibition of IP-10 was supported by the finding that the effect was
independent of TLR2-dependent signalling mechanisms. Stimu-
lation experiments with IFNc and IP-10 overexpression experi-
ments revealed stimuli-independent mechanisms to underly the
inhibition of IP-10 expression by L. casei. Given the high
physiological relevance of elevated IP-10 expression in inflamed
intestinal tissue regions of IBD patients and animal models of
experimental colitis [20–22], the inhibitory effect on this
chemokine may play an important role in the anti-inflammatory
effects of the probiotic mixture VSL#3 on inflammatory bowel
diseases [9]. We were able to show that IP-10 expression in IEC
correlates with inflammation, as IP-10 expression was elevated in
primary ileal IEC from heterozygous TNFDAREmice as well as in
colonic IEC from IL-102/2mice compared to healthy wildtype
mice. In a healthy host, pathogen-induced secretion of IP-10
triggers enhanced recruitment of effector Th1 cells and monocytes
into the mucosa, resulting in subsequent deletion of the infectious
agent. In the case of IBD, chronically activated IEC steadily
produce high amounts of IP-10 in the absence of any pathogenical
stimuli resulting in constant Th1 and monocyte recruitment. The
presence and activity of these effector cells may lead to the loss of
epithelial cell integrity and tissue damage. In consequence, the
expression of IP-10 in IEC has to be tightly regulated to prevent
infections on the one hand and the development of pathological
conditions like chronic intestinal inflammations on the other hand.
Consistently, IP-10 expression has been shown to be modulated at
various levels. The activation of PPARc by PPARc ligands
inhibited IP-10 expression by reducing IP-10 promoter activity
[35]. In addition, butyrate inhibited IFNc and TNF-induced IP-10
transcription in colonic subepithelial myofibroblasts [36] and
interleukin 10 suppressed LPS-induced IP-10 gene transcription in
peritoneal macrophages [37]. Many studies have analyzed the
transcriptional regulation of cytokines and chemokines like IP-10
but although post-transcriptional and post-translational regulation
of chemokines is suggested to be very important [38], exact
mechanisms are mostly unknown. The pro-inflammatory chemo-
kine interleukin 8 was shown to be upregulated by a TLR5-
mediated, p38-dependent post-transcriptional mechanism [39]
and furthermore, to be processed at the amino terminal end by a
matrix metallo proteinase [40]. In addition, the biosynthesis of
IFNc was shown to be suppressed post-transcriptionally by
the stimulation with lead [41]. Concerning IP-10, complex
post-transcriptional regulatory mechanisms seem to play an
important role. Recent studies revealed regulatory mechanisms
like IP-10 mRNA half-life modulation in monocytes [42] or C-
terminal truncation through furin, a pro-protein convertase,
resulting in another active form of the chemokine [43].
Furthermore, IP-10 was found to be processed C-terminally by
gelatinase B and neutrophil collagenase [40]. In addition, the
activity of IP-10 was shown to be regulated by its oligomerization
status [44]. Our study revealed for the first time that probiotic
bacteria are able to induce post-translational regulation of IP-10
protein in IEC. Decreased IP-10 protein stability was identified as
a new post-translational regulatory mechanism.
L. casei-induced ubiquitination of IP-10 is very likely involved in
the observed degradation of the chemokine although it does not
target the protein for proteasomal degradation. It has already been
shown that stimulation with L. casei has multiple effects on the
expression of genes involved in proteasome and ubiquitination
pathways in IEC [45], but the exact mechanisms are not known
yet. In contrast to lysosomal degradation of LPS-induced TNF
under hypoxia [46], lysosomal pathways were not involved in L.
casei-induced loss of IP-10 protein, suggesting alternative degra-
dation pathways to be involved. The inhibition of protein secretion
through brefeldin A was found to induce degradation of IP-10
instead of intracellular accumulation, as it was shown for other
secretory proteins like makrophage inflammatory protein-2 [47].
This result led to the hypothesis that L. casei might selectively
impair secretion of IP-10 followed by subsequent degradation as a
protective mechanism to prevent protein accumulation. In
contrast to interleukin-6, the secretion of IP-10 was found to be
dependent on tightly regulated vesicular transport. L. casei mimicks
the effect of 3-methyladenine, an inhibitor of vesicular trafficking
and exosome secretion [48], which specifically inhibits IP-10
secretion and induces loss of intracellular IP-10 protein.
Considering the extensive post-translational processing of IP-10,
this inhibitory mechanism might be due to the necessity for
chemokine maturation processes in endo-lysosomal compartments
prior to exocytosis as it has been shown for several other secretory
proteins [49,50]. We therefore hypothesize that L. casei inhibits
vesicle trafficking similar to 3-methyladenine, resulting in impaired
maturation and secretion of IP-10 followed by subsequent
degradation of the chemokine (Figure 6B).
Our in vivo feeding studies revealed that VSL#3 has no effect on
the development of ileal inflammation in heterozygous TNFDARE
mice. This result may indicate that the constant overexpression of
TNF in these mice and the resulting T-cell driven immunopa-
thology constitute such a strong pro-inflammatory stimulus that
probiotic therapy is not sufficient to reduce tissue inflammation.
Apart from that, one could raise the hypothesis that probiotic
therapy is generally less effective in the ileum than in the colon due
to high luminal content passage rates in this intestinal segment.
The failure of VSL#3 in the context of ileitis together with the
observed reduction of cecal inflammation in IL-102/2mice is
consistent with clinical data showing effective probiotic treatment
mostly in the context of UC [9] and pouchitis patients [51]. The
finding that VSL#3 has protective effects on cecal but not on
colonic inflammation clearly reveals intestinal segment specific
effects of the probiotic mixture. The differences in response to
Figure 7. VSL#3 does not reduce experimental ileitis in TNFDAREmice. TNFDARE(n=6) and wt mice (n=5) were fed VSL#3 (1,36109cfu/
mouse/day) or placebo for 15 weeks. Mice were sacrificed at 18 weeks of age and histopathological scoring was performed by blindly assessing the
inflammation between 0 (not inflamed) and 12 (highly inflamed). Figure A shows the median histopathologic score of TNFDAREmice in the study. (B)
Ileal IEC were isolated and pooled proteins (50 mg) of all mice in a group were analyzed for IP-10 expression by Western blot. (C) The presence of
VSL#3 derived S. thermophilus (S.t) in the gut of the TNFDAREmice was examined by bacteria-specific PCR analysis.
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