Article
Epigenetic imprinting by commensal
probiotics inhibits the IL-23/IL-17 axis in an
in vitro model of the intestinal mucosal
immune system
Darab Ghadimi,*,1 Ulf Helwig,† Juergen Schrezenmeir,‡ Knut J. Heller,* and Michael de Vrese*
*Department of Microbiology and Biotechnology, Max Rubner-Institute, Kiel, Germany; †Partnership for Internal Medicine,
Oldenburg, Germany; and ‡Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institute, Karlsruhe, Germany
RECEIVED JUNE 11, 2011; REVISED MAY 30, 2012; ACCEPTED MAY 31, 2012. DOI: 10.1189/jlb.0611286
ABSTRACT
The pathophysiology of IBD is characterized by a com-
plex interaction between genes and the environment.
Genetic and environmental differences are attributed to
the heterogeneity of the disease pathway and to the
epigenetic modifications that lead to altered gene ex-
pression in the diseased tissues. The epigenetic ma-
chinery consists of short interfering RNA, histone modi-
fications, and DNA methylation. We evaluated the ef-
fects of Bifidobacterium breve (DSMZ 20213) and LGG
(ATCC 53103), as representatives of commensal probi-
otics on the expression of IL-17 and IL-23, which play
an important role in IBD, and on the epigenetic machin-
ery in a 3D coculture model composed of human intes-
tinal HT-29/B6 or T84 cells and PBMCs. The cells were
treated with LPS in the presence or absence of bacte-
ria for 48 h, and the expression of IL-17, IL-23, and
CD40 at the mRNA and protein levels was assessed
using TaqMan qRT-PCR and ELISA, respectively. West-
ern blotting was used to assess the expression of the
MyD88, the degradation of IRAK-1 and I␬B␣, the expres-
sion of the NF-␬B p50/p65 subunits, the p-p38 MAPK
and p-MEK1, as well as histone modifications. NF-␬B
activity was assessed by NF-␬B-dependent luciferase
reporter gene assays. The accumulation of Ac-H4 and
DNA methylation was quantitatively assessed using col-
orimetric assays. B. breve and LGG diminished the
LPS-induced expression of IL-17, IL-23, CD40, and his-
tone acetylation, while slightly enhancing DNA methyl-
ation. These effects were paralleled by a decrease in
Abbreviations: 2/3D⫽two/three-dimensional, Ac⫽acetylated,
ATCC⫽American Type Culture Collection, CBP⫽CREB-binding protein,
CD⫽Crohn’s disease, DSMZ⫽German Collection of Microorganisms and
Cell Cultures, GM⫽gentamycin, H3/H4⫽histone 3/4, IBD⫽inflammatory
bowel disease, IEC⫽intestinal epithelial cell, LAB⫽lactic acid bacteria,
LGG⫽Lactobacillus rhamnosus GG, MD2⫽myeloid differentiation
protein 2, MEK1⫽MAPK/ERK kinase 1, MRS⫽de Man, Rogosa, Sharpe
broth, OD600, OD450, and OD600⫽OD at 600, 450, and 570 nm, re-
spectively, p⫽phosphorylated, qRT-PCR⫽quantitative real-time
RT-PCR, SI⫽stimulation index, TEER⫽transepithelium electrical resis-
tance, TSA⫽trichostatin A, UC⫽ulcerative colitis, WST-1⫽water-
soluble tetrazolium
0741-5400/12/0092-895 © Society for Leukocyte Biology
the nuclear translocation of NF-␬B, as demonstrated by
a decrease in the expression of MyD88, degradation of
IRAK-1 and I␬B␣ expression of the nuclear NF-␬B p50/
p65 subunits, p-p38 MAPK and p-MEK1, and NF-␬B-de-
pendent luciferase reporter gene activity in LPS-stimu-
lated cells. B. breve and LGG may exert their anti-in-
flammatory effects in the gut by down-regulating the
expression of the IBD-causing factors (IL-23/IL-17/
CD40) associated with epigenetic processes involving
the inhibition of histone acetylation and the optimal en-
hancement of DNA methylation, reflected in the limited
access of NF-␬B to gene promoters and reduced NF-
␬B-mediated transcriptional activation. We describe a
new regulatory mechanism in which commensal pro-
biotics inhibit the NF-␬B-mediated transcriptional acti-
vation of IBD-causing factors (IL-23/IL-17/CD40),
thereby simultaneously reducing histone acetylation
and enhancing DNA methylation. J. Leukoc. Biol. 92:
895–911; 2012.
Introduction
It has been suggested recently that the IL-23/IL-17 axis is in-
volved in IBD [1– 4]. Inhibiting IL-23 expression may have a
therapeutic potential in IBD [4, 5], as it regulates the Th1/
Th17 response in UC and CD [2– 4]. An elevation of IL-23 is
also associated with pathogen bacteria-induced gastritis [6].
In addition to IL-17 and IL-23, CD40 costimulatory protein
is an important IBD-causing factor [7]. With respect to the
cellular sources of these IBD-causing factors during IBD and
in the in vitro model of intestinal mucosal immune systems, it
can be concluded from previous and recent studies that IL-23
can be produced preferentially by IECs [8] and only to a
lesser extent by immune cells, such as PBMCs, under stimula-
tion with LPS and inflammation conditions [9, 10]. Consider-
ing the major source of IL-17, Th17 cells and PBMCs are the
pivotal basis of IL-17 in physiologic and pathologic conditions
1. Correspondence: Department of Microbiology and Biotechnology, Max
Rubner-Institute, Hermann-Weigmann-Strasse 1, 24103 Kiel, Germany.
E-mail: darab.ghadimi@mri.bund.de
Volume 92, October 2012 Journal of Leukocyte Biology 895
[11, 12]. More recently, however, intestinal Paneth cells and
colonic epithelial cells have been reported to express mRNA
levels of IL-17A and IL-17F, respectively [13]. These data,
therefore, suggest that there is an extracellular cross-talk be-
tween IECs and PBMCs in the production of IL-17 and IL-23
under aberrant inflammation conditions and stimulation with
LPS, as IL-23 is reported to be sufficient for differentiation of
human CD4⫹ T cells to Th17 cells and their marked produc-
tion of IL-17 [11], which enhances the inflammatory responses
of the IECs to LPS in vitro—a potential mechanism by which
IL-17 causes mucosal damage by CD [2]. With respect to
source of CD40, it is well known that CD40 can be produced
by IECs and immune cells, such as PBMCs. For example, LPS
induces CD40 expression in human PBMCs through the activa-
tion of NF-␬B [14, 15]. Furthermore, CD40 expression was
demonstrated not limited to immune system cells but was also
expressed in human colon primary tumor samples and human
colon cell lines [16]. Although carcinoma intestinal cell lines
do not represent the complex function of IEC in IBD, similar
to mucosal tissue of IBD patients and freshly isolated human
IECs, they express at least two major hallmarks of IBD, namely
TLR4 and CD40 [7].
Recent studies indicate that the epigenetic machinery-de-
pendent regulation of mucosal cytokines plays an important
role in the modulation of IBD [17]. Interactions with bacteria
and their outer membrane (endotoxin, LPS component) have
been shown to affect histone acetylation, the phosphorylation
and methylation states of the TLR4 genes, and the secretion of
IL-8 in host cells [18]. For instance, Schmeck et al. [19] re-
ported that intracellular Listeria induces the Ac-H4, the
p-Ac-H3 at the IL-8 promoter, and the recruitment of the his-
tone acetylase and the transcription CREB in endothelial cells.
Clinical impacts have also been shown for Legionella pneumo-
phila and Moraxella catarrhalis, both of which induce the aber-
rant expression of proinflammatory cytokines [20] by enhanc-
ing MAPK/NF-␬B activity and reducing histone deacetylase activ-
ity [21]. Nonpathogenic, Gram-negative bacteria induce the
recruitment of NF-␬B to the IL-6 gene promoter in IECs via mod-
ulation of histone acetylation [22]. Furthermore, Hamon and
Cossart [23] reported that the activation of the p38 MAPK path-
way by LPS-stimulated TLR4 induces the p-Ac-H3, which is crucial
for recruiting NF-␬B to the promoters of certain genes, such as
IL-12. Additionally, the in vivo association among histone acetyla-
tion, the NF-␬B induction of inflammatory gene expression, and
IBD has been reported recently [24].
With respect to the LAB belonging to the natural intestinal
microflora, a recently published study indicates that Lactobacil-
lus plantarum inhibits LPS-induced IL-23 secretion by Caco-2
cells [8]. However, a role for the epigenetic machinery in me-
diating the influence of probiotic bacteria on the reduction of
the IL-23/IL-17 axis has not been investigated previously.
Therefore, we were interested in understanding if the epige-
netic machinery might be involved in the in vitro IL-23/IL-17-
inhibiting potential of commensal probiotics. We evaluated the
effects of B. breve and LGG on the LPS/TLR4/NF-␬B intracel-
lular signaling cascades and on histone modifications using an
in vitro 3D coculture model that uses two well-studied IEC
lines—HT-29/B6 and T84 —and PBMCs as the original source
896 Journal of Leukocyte Biology Volume 92, October 2012
of intestinal Th17 cells under inflammatory conditions. This
3D coculture model may mimic the in vivo situation, in which
beneficial commensal probiotics interact with the host im-
mune cells via enterocytes. With respect to cytokine expression
and inflammatory responses, previous in vitro and in vivo stud-
ies have demonstrated that compared with conventional 2D
cell cultures, scaffold-based, 3D-cultured cells (e.g., PBMCs)
may be more physiologically relevant based on their superior
ability to emulate the in vivo environment [25]. The levels of
cell proliferation and cytokine secretion in the in vitro scaf-
fold-based, 3D-cultured PBMCs are well correlated with the
extent of inflammatory responses observed in vivo [26]. Addi-
tionally, the 3D cultures have significantly higher expression
levels of key genes that function in cytokine activity, cell
growth, and differentiation regulation [27] and interestingly,
exhibit different LPS receptor expression, LPS-induced cyto-
kine responses, and CD14/MD2 expression [28]. Indeed, it
has been reported that signal transduction in 3D coculture
differs from that in 2D coculture. Additionally, 3D coculture
environments promote polarity and differentiation in normal
epithelial cells and enhance cell biological activities as com-
pared with 2D substrates [29]. Furthermore, it has been sug-
gested that the 3D in vitro cell culture model of PBMCs is a
valuable tool complementary to other models for studying the
cells and mediators involved in the immunological events that
lead to the pathologically relevant morphological changes ob-
served during infection [30]. For these reasons, we investi-
gated the effects of the LAB in this enterocyte-PBMC coculture
model using 2D and 3D PBMC cultures.
MATERIALS AND METHODS
Propagation of bacteria
B. breve (DSMZ 20213, Braunschweig, Germany) and LGG (ATCC 53103)
were used in this study. The bacteria were propagated according to previ-
ously published procedures [31]. Briefly, using 0.02% inoculums from bac-
terial stocks stored at ⫺80°C in 30% glycerol, the bacteria were grown an-
aerobically in a modular atmosphere-controlled system-VA500 workstation
with an airlock (Don Whitley Scientific, UK) in MRS broth medium (Lacto-
bacilli MRS, Merck, Darmstadt, Germany) at 37°C for 12 h. The Escherichia
coli TG1 strain (Cat. #BU-00035, Maxim Biotech, S. San Francisco, CA,
USA) was grown at 37°C in Luria broth. The CFU/ml of the bacterial cul-
tures was estimated by measuring the OD600 of a 1:6 dilution and using an
experimentally derived conversion factor for each strain. The conversion
factors were determined by plating stationary-phase cells and determining
the number of CFU/ml/OD600 U for each strain. The liquid cultures were
centrifuged at 4°C and 5000 g for 10 min, using appropriate volumes to
yield the necessary CFU. After washing three times with endotoxin-free
DPBS (BioWhittaker, Lonza, Basel, Switzerland), the bacterial pellets were
diluted to 1 ⫻ 107 CFU in 50 ␮l of the appropriate cell culture medium.
These bacteria samples were added to the HT-29/B6 or T84 cells in the 3D
coculture cells to yield a 10:1 bacteria:cell ratio. The bacterial suspensions
were also plated on agar plates. The colonies were counted to confirm the
accuracy of their concentrations and the viability of the bacteria. For the
experiments with heat-killed B. breve, the liquid culture was heated to 95°C
for 30 min.
We initially included B. breve and LGG strains as representatives of Gram-
positive commensal probiotics and E. coli TG1 as a Gram-negative non-LAB
strain and internal control to confirm that the observed effects are specific
for commensal probiotics. In the cases in which only one LAB strain was
tested, B. breve was preferentially used, as it releases metabolites that exert
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 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics
an anti-inflammation effect capable of crossing the intestinal barrier [32],
and the anti-inflammatory capacity of live and dead B. breve and its soluble
factors has been tested in vitro in mucosal DCs and in vivo. In a murine
model of trinitrobenzene sulfonic acid-induced colitis, B. breve alleviated
inflammation, as shown by a statistically significant decrease in the proin-
flammatory cytokines IL-23 and IL-6 [33, 34]. Furthermore, it has been
shown that another Bifidobacteria, Bifidobacterium infantis, suppresses proin-
flammatory IL-17 production in murine experiments in vivo [12].
Cell culture
Polarized HT-29/B6 and T84 cells. Model intestinal epithelia were pre-
pared using HT-29/B6 or T84 cells, each plated separately at 1 ⫻ 105
cells/cm2 on Transwell-COL inserts (Corning, Kaiserslautern, Germany), as
described previously [35, 36]. The cells were grown in RPMI-1640 culture
medium supplemented with 5% v/v FCS from Gibco (Invitrogen,
Karlsruhe, Germany) and maintained at 37°C in a humidified 5% CO2 in-
cubator. The HT-29/B6 and T84 cells used in this study were assessed peri-
odically with a Mycoplasma detection kit (Roche Applied Science, Manheim,
Germany) and found to be free of mycoplasma. To determine qualitatively
if the HT-29/B6 and T84 cells had reached confluence and formed tight
junctions, the TEER across the monolayer was monitored using an Epithe-
lial Voltohmmeter electrode and Millicell-Electrical Resistance System (Mil-
lipore, Schwalbach, Germany). At the beginning of the experiments, the
TEER of all epithelial cells was measured, and for all experiments, only
intact, polarized cell monolayers exhibiting a net resistance of ⱖ600 ⍀ ⫻
cm2 (HT-29/B6 cells) or ⱖ1300 ⍀ ⫻ cm2 (T84 cells) were used. The T84
cells were found to be more potent than the HT-29/B6 cells at increasing
their tight junctions in monolayer culture, as revealed by the increased
TEER. The main reasons for selecting these two cell lines are that first, the
HT-29/B6 grow as highly differentiated, polarized epithelial monolayers
and possess most of the features of native intestinal epithelia necessary for
maintenance of intestinal barrier function [35]. Secondly, the T84 cells are
crypt-like cells that have been widely used for studying secretion, transcellu-
lar trafficking, inflammation [36], and the LPS-mediated TLR/NF-␬B sig-
naling pathway [37].
The 2D and 3D PBMC culture models. Human PBMCs were prepared
according to previously published procedures [31]. Briefly, venous whole
blood from healthy subjects was drawn into heparinized vacutainers and
diluted 1:1 with pyrogen-free physiological serum (0.9% NaCl). The PBMCs
were then isolated by density gradient centrifugation (1.077 g/ml) (Lym-
phoprep, Axis Shield PoC AS, Oslo, Norway) and washed twice in endotoxin-
free DPBS without Ca2⫹ and Mg2⫹ containing 5% FBS (Gibco, Invitrogen).
All of the PBMCs were ⬎95% viable immediately after purification, as as-
sessed by the microscopic examination of trypan blue exclusion.
To prepare the 3D cell culture model, freshly isolated PBMCs (34,000
cells/200 ␮l/scaffold) were cultured in antibiotic-free RPMI-1640 culture
medium in 12-well 3D insert scaffolds, according to the manufacturer’s sug-
gested protocol (3D Biotek, Brunswick, NJ, USA).
The enterocyte-PBMC coculture model. To establish the 3D coculture
model, confluent HT-29/B6 or T84 monolayers (2⫻106 cells/ml), grown
on the filter membranes of 12-well Transwell-COL inserts were cocultured
with 2D (1.7⫻105 cells/well) and 3D (34,000 cells/200 ␮l/scaffold) PBMCs
that were placed in the lower compartment of the coculture wells, i.e., the
bottom of the culture plates (Fig. 1). This environment simulates the lumi-
Transwell insert
Upper compartment HT-29/B6 or T84 cells
• • • • • •
Microporous membrane
Lower compartment
• • • •• • • 3D Insert scaffold containing 3D PBMCs
www.jleukbio.org
nal exposure of the intestinal epithelium to bacteria, the intestinal barrier
function by the epithelial cells, and the translocation of the membrane-
permeable bacterial components and signaling components from the epi-
thelial cells to the subepithelial space containing the immunocytes. It fur-
thermore enables the interaction between the PBMCs and epithelial cells
via soluble, membrane-permeable substances, e.g., cytokines.
Assessing the effect of the LAB on cytokine
expression
Dose- and time-response experiments. To evaluate the impact of the dif-
ferent concentrations of B. breve on the secretion of the IL-17 and IL-23
proinflammatory cytokines, the upper compartment bathing the filter-
grown HT-29/B6 and T84 intestinal epithelial monolayers was filled with
medium alone (control) or medium containing increasing concentrations
of B. breve (2⫻106, 5⫻106, 1⫻107, 2⫻107, and 4⫻107 CFU/ml, correspond-
ing to bacteria:cell ratios of 1, 2.5, 5, 10, and 20, respectively) in the ab-
sence or presence of basolateral-applied LPS (100 ng/ml) and incubated
for 48 h.
For the time-response analysis, the cells were incubated with 2 ⫻ 107
CFU/ml B. breve in the absence or presence of basolateral-applied LPS
(100 ng/ml) for 6, 12, 24, 48, and 96 h in a similar subset of experiments.
After incubation, the supernatants from the lower compartments were
collected and centrifuged at 400 g for 10 min at 4°C to remove cells, and
the expression of the IL-17 and IL-23 proteins was assessed by specific
ELISA kits, as indicated below in “Determination of cytokine and CD40
protein levels”.
Assessing the differences in the 2D and 3D PBMCs cultured separately
or in coculture with T84 cells and ascertaining the site of LPS action. To
investigate which part of the system was responsible for the secretion of
IL-17 and IL-23 and whether the expression of IL-17 differs between the
normal and scaffold-based, 3D-cultured PBMCs, 2D PBMCs (1.7⫻105
cells/ml in 96-well cell culture microplates) and scaffold-based 3D PBMCs
(34,000 cells/200 ␮l/scaffold) were cultured separately in the absence of
T84 cells and treated without or with LPS (100 ng/ml) for 48 h in the ab-
sence or presence of B. breve at a bacteria:cell ratio of 10:1. Following this
incubation, the cell-free supernatants were analyzed for IL-17 and IL-23 by
ELISA.
Furthermore, to ascertain the site of LPS action, HT-29/B6 and T84
cells (2⫻106 cells/ml) grown in the 12-well Transwell-COL inserts were
treated with 100 ng/ml LPS from E. coli 026:B6 (Sigma, Taufkirchen, Ger-
many) [38], which was added to the upper or lower compartment for 48 h
in the absence or presence of B. breve at a bacteria:cell ratio of 10:1 (which
was added to the upper compartment). Following incubation, the cell-free
supernatants from the upper and lower compartments were analyzed for
IL-23 by ELISA.
Based on our preliminary dose- and time-response experiments, the fol-
lowing parameters were chosen for the experiments: a bacteria:cell ratio of
10:1, a 48-h incubation period, and 100 ng/ml LPS [38]. When the epithe-
lial cells reached confluence and formed tight junctions, the upper com-
partment containing the filter-grown epithelial monolayers was filled with
medium alone or medium containing 2 ⫻ 107 CFU/ml (corresponding to
a 10:1 bacteria:cell ratio) B. breve, heat-killed B. breve, LGG, or E. coli TG1.
LPS was added to the lower compartment of this transwell coculture sys-
tem, which contained the 3D insert scaffolds with the 3D culture of the
Figure 1. Schematic illustration of the 3D
coculture models showing the experimental
setup of the transwell system used to assess
LPS-mediated inflammation. The transwell
inserts on which HT-29/B6 or T84 cells were
cultured were inserted into multiple wells
containing the PBMC-seeded 3D insert scaf-
folds (placed on the bottom of the culture
plates).
Volume 92, October 2012 Journal of Leukocyte Biology 897
PBMCs placed at the bottom of the wells. After 48 h incubation, the super-
natants from the upper and lower compartments were collected and centri-
fuged at 1200 g for 10 min at 4°C. The cell-free supernatants were then
sterilized by passage through a 0.2-␮m pore filter (Millipore, Germany),
and a 100-␮l aliquot of each cell culture was used for cell viability assays.
The remainder of the cell culture supernatants was stored at ⫺80°C for the
analysis of cytokine and CD40 protein expression. The monolayer HT-
29/B6 and T84 cells and the 3D PBMCs were harvested for assays to deter-
mine mRNA expression, histone modifications, proliferation, NF-␬B activ-
ity, and NF-␬B-dependent luciferase reporter gene activity, as indicated be-
low. In some experiments, 10 ng/ml of a TLR4 antagonist (Cat. # tlrl-rslps,
InvivoGen, Toulouse, France) was added to the lower compartments as a
control and to block the TLR4/MD2 complex pathway. Based on the prod-
uct data sheet, this TLR4/MD2 complex inhibitor is guaranteed to block
the LPS-dependent TLR4/MD2 complex pathway by at least two distinct
mechanisms. The main mechanism consists of direct competition between
the underacylated LPS and hexa-acylated LPS for the same binding site on
MD2, and the secondary mechanism involves the ability of the underacy-
lated LPS:MD2 complexes to inhibit the function of the hexa-acylated en-
dotoxin:MD2 complexes via TLR4.
The viability of the bacteria was determined by bacterial plate counts at
the time of addition and after incubation. During the coincubation, LGG
and E. coli TG1 were able to survive, and they seemed to tolerate aerobic
conditions for up to 48 h rather well. However, B. breve tended to be less
tolerant to aerobic conditions, and a significant (P⬍0.05) decrease in the
B. breve population was observed (from 6.88⫾0.33 log10 to 4.82⫾0.24 log10
CFU/ml; P⬍0.05). For this reason, we also included the counts of the dead
bacteria in our experiments.
RNA extraction, semi-qRT-PCR, and TaqMan
qRT-PCR
Semi-qRT-PCR analysis. The expression of IL-17 and IL-23 was screened
initially using semi-qRT-PCR. Total RNA was isolated from the HT-29/B6
and T84 cells and PBMCs with an RNeasy mini kit (Qiagen, Hilden, Ger-
many) and treated with DNA-free (Ambion, HC, UK). The quality and
quantity of the RNA samples were assessed by spectrophotometry, conven-
tional gel electrophoresis, and Agilent RNA 600 Nano Assays in a 2100 bio-
analyzer (Agilent Technologies, Santa Clara, CA, USA) with the corre-
sponding software (version A.01.16). Total RNA (2 ␮g) was reverse-tran-
scribed to cDNA using the Superscript first-strand synthesis system for RT-
PCR, according to the manufacturer’s recommendations (Invitrogen). The
PCR amplification was performed using Platinum PCR Supermix (Invitro-
gen) on a T1 thermal cycler (Biometra, Germany). The primer pairs for
␤-actin (internal control) and for IL-23 and IL-17 were used as described
previously [8, 39]. The PCR amplification with all primers was performed
for 44 cycles of 95°C for 45 s, 56°C for 45 s, and 72°C for 1 min, followed
by a final extension at 72°C for 10 min. After PCR amplification, 15 ␮l of
the products was separated by electrophoresis in a 1.5% agarose gel con-
taining 10 ␮g/ml ethidium bromide and visualized with a UV transillumi-
nator.
TaqMan qRT-PCR analysis. To further examine the cytokine expression
profile, qRT-PCR was performed using the TaqMan system (Applied Biosys-
tems, Darmstadt, Germany). The expression levels of IL-23, IL-17, and
GAPDH (internal reference) were measured by PCR using TaqMan gene
expression assays. A total of 1 ␮g RNA was transcribed to cDNA using
TaqMan reverse transcription reagents, and TaqMan qRT-PCR was per-
formed in 50 ␮l vol for IL-23, IL-17, and GAPDH using the TaqMan Uni-
versal PCR Master Mix containing AmpliTaq Gold polymerase. The real-
time reactions had a final volume of 50 ␮l containing 25 ␮l TaqMan Uni-
versal PCR Master Mix, 22.5 ␮l TaqMan assays-on-demand primer/probe
mixture (GAPDH: Hs99999903_m1; IL-23: Hs01629265_sl; IL-17:
Hs99999082_ml), and 2.5 ␮l ddH2O containing cDNA corresponding to
10 –20 ng RNA (depending on the target gene) and run on an ABI PRISM
7000 sequence detection system (all reagents were purchased from Applied
Biosystems). The results were analyzed with the SDS software package ver-
sion 2.1. The TaqMan qRT-PCR was performed in at least three indepen-
898 Journal of Leukocyte Biology Volume 92, October 2012
dent experiments. Each TaqMan assay included a fixed standard, a no-tem-
plate control, and a cDNA sample and was run in triplicate for each sam-
ple. The data for each sample were normalized to the GAPDH mRNA level
present in each sample and normalized again between the samples to the
levels of the IL-23 and IL-17 mRNA present in the control, and then the
relative expression levels were determined.
DNA methylation and histone modification assays
In the 3D coculture models, the HT-29/B6 and T84 cells were initially in-
cubated with 2 ⫻ 107/ml B. breve for 30, 60, and 120 min in the absence or
presence of 100 ng/ml LPS, which was added to the lower compartment
(basolateral surface). After incubation, cell lysates were prepared, and the
histone modifications, including the global Ac-H4 and the p-Ac-H3 on Ser-
10/Lys-14, were assessed by Western blot analysis performed according to a
standard protocol (Invitrogen). Briefly, protein samples (50 ␮g/lane) were
mixed with an equal volume of protein sample buffer (NuPAGE LDS) and
incubated at 85°C for 2 min. After incubation, the samples were subjected
to SDS-PAGE (Precast, NuPAGE Novex, 4 –12% Bis-Tris) at 110 V for 80
min and 130 V for 10 min. After electrophoresis, the proteins were trans-
ferred for 1.5 h at 400 mA in transfer buffer (NuPAGE transfer buffer)
onto a 0.2-␮ PVDF membrane, which was blocked in blocking buffer (TBS)
with 5% BSA for 30 min at room temperature, followed by repeated wash-
ing. The membranes were then incubated overnight at 4°C and incubated
with anti-Ac-H4 (Lys8; 1:200), -H4, -p-Ac-H3 (Ser10; 1:1000), or -H3 anti-
bodies (Cell Signaling Technology, Beverly, MA, USA) for 1 h at room
temperature. After incubation, the blots were washed four times in washing
buffer (TBS containing 0.1% Tween 20 and 5% nonfat dry milk) and then
incubated with the secondary HRP-linked antibody (1:2000; Acris, Hidden-
hausen, Germany) in blocking buffer for 1 h at room temperature. Finally,
the membranes were developed with ECL Plus Western blotting detection
reagents (Amersham), and the images were recorded with a cooled charge-
coupled device camera (Fuji Film, Japan). In addition, a similar subset of
experiments was performed to evaluate the epigenetic imprinting effects of
B. breve and LGG during long-term incubation. The HT-29/B6 and T84
cells were incubated for 48 h with the potent histone deacetylase inhibitor
TSA [40] (0.01 ng/ml; Th. Geyer, Hamburg, Germany), the histone
deacetylase inducer GM (0.2 mM) [41], B. breve, or LGG, all of which were
added to the medium in the upper compartments (apical surface) in the
presence of LPS, which was added to the medium in the lower compart-
ments (basolateral surface). After incubation, the p-Ac-H3 and the Ac-H4
were assessed as indicated above. Additionally, for the quantitative eval-
uations, cell lysates were prepared, and the accumulation of acetylated
histones and methylated DNA was quantitatively assessed using a
EpiQuik Global Histone H3/H4 Acetylation Assay Kit and a Methylamp
Global DNA Methylation Quantification Ultra Assay Kit (Epigentek
Group, Farmingdale, NY, USA), respectively, according to the manufac-
turer’s instructions.
Assessment of the upstream activators of the NF-␬B
pathway, transfection, and the NF-␬B-dependent
luciferase reporter gene assay
Aliquots of the cell lysates obtained after 2- and 48-h incubation periods
were also analyzed by immunoblotting for the degradation of IRAK-1 and
I␬B␣, the expression of MyD88 and nuclear NF-␬B p50/p65 subunits, and
the p-p38 MAPK and p-MEK1 using anti-IRAK-1, anti-MyD88, anti-p50, anti-
p65, anti-p-p38, anti-p38, anti-p-MEK1 (Ser 298), anti-I␬B␣ (Ser32/36; 5A5;
1:2000), I␬B␣ (112B2; 1:1000), and rabbit anti-␤-actin mAb (Cell Signaling
Technology).
For the transcription activity assays, confluent HT-29/B6 and T84 mono-
layer cells were transiently transfected for 16 h with a NF-␬B-dependent lu-
ciferase reporter gene (Ad5␬B-LUC; Vector Biolabs, Philadelphia, PA,
USA) in serum-free medium (Gibco, Invitrogen) at different MOIs, as de-
scribed previously [36]. Ad5␬B-LUC, consisting of three consensus NF-␬B-
binding sites, was linked to luciferase. After transfection, the medium was
removed, and the cells were rinsed with PBS. Fresh complete medium was
www.jleukbio.org
 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics
added before the stimulation. The apical surface of the cells was then
treated with B. breve or LGG (bacteria:cell ratios 0, 5, 10) for 24 h in the
presence or absence of 100 ng/ml LPS, which was added to the medium in
the lower compartments (basolateral surface). The cell extracts from the trans-
fected epithelial cells were prepared with an enhanced luciferase assay kit (BD
PharMingen, Heidelberg, Germany), according to the manufacturer’s
instructions. The NF-␬B-dependent luciferase activity was measured on a
GENios microplate luminometer (Tecan, Crailsheim, Germany), and the
results were normalized to the extract protein concentrations as measured
with a Bio-Rad protein assay kit. The NF-␬B-dependent luciferase assays
were performed in triplicate with cell extracts from at least three separate
experiments, and each result is expressed as the mean value of the three
independent experiments ⫾ sem.
Determination of cytokine and CD40 protein levels
The levels of the IL-17, IL-23, and CD40 proteins in the cell-free superna-
tants were quantified using specific human IL-17, IL-23, and CD40 ELISA
development kits (PeproTech, Hamburg, Germany; eBioscience, NatutTec,
Frankfurt, Germany; and Abnova, Heidelberg, Germany, respectively). The
detection limit of the assay was 4 pg/ml for IL-17, 16 pg/ml for IL-23, and
⬍1 pg/ml for CD40. The OD values of the samples were OD450 and OD570
on an ELISA plate reader (Molecular Devices, Munich, Germany). Each
ELISA was performed in triplicate with cell-fee supernatants from at least
three separate experiments, and each result is expressed as the mean value
of three independent experiments ⫾ sem.
Cell viability and proliferation assays
In all experiments, the cell viability and cytotoxicity were determined
routinely using the trypan blue exclusion and WST-1 assays, which were
conducted according to the manufacturer’s instructions (Roche Applied
Science). Cell proliferation was evaluated by measuring DNA synthesis
using a cell proliferation Biotrak ELISA kit (GE Healthcare, Munich,
Germany) as described previously [42]. Proliferation is expressed as the
SI, which was calculated as the ratio of the mean OD of the stimulated:
unstimulated cultures, and a proliferative response with an SI ⬎2 was
regarded as positive.
Statistical analysis
The data were analyzed by ANOVA, and the comparisons among the
groups were performed using the Kruskall-Wallis ANOVA and the
Mann-Whitney U-test. All of the experimental data are expressed as the
mean ⫾ sem. The data were considered statistically significant when P ⱕ
0.05.
RESULTS
B. breve inhibits IL-23/IL-17 secretion in a dose- and
time-dependent manner
As shown in Fig. 2A and B, B. breve inhibited the secretion of
basal and LPS-induced IL-17 and IL-23 in the 3D coculture
model constructed with the HT-29/B6/T84 IEC line and PBMCs.
In the case of IL-17 and IL-23 basal levels, a significant decrease
was only observed at 2 ⫻ 107 CFU/ml (10:1 bacteria:cell ratio) in
both cell lines. In the presence of LPS, however, significant de-
creases in the IL-23 and IL-17 levels were observed at 1 ⫻ 107,
2 ⫻ 107, and 4 ⫻ 107 CFU/ml (5:1, 10:1, and 20:1 bacteria:cell
ratios) with a maximal effect observed at 2 ⫻ 107 CFU bacteria/
ml. These results are in agreement with a previous study showing
that anti-inflammatory capacity of B breve and Streptococcus thermo-
philus conditioned media was retained after transepithelial trans-
www.jleukbio.org
port, under basal, and more extensively, under inflammatory con-
ditions after LPS challenge [32].
As maximum, significant decrease in the basal and LPS-in-
duced IL-23 and IL-17 levels was observed at 2 ⫻ 107 CFU/ml
(corresponding to a 10:1 bacteria:cell ratio), this concentra-
tion was used for all subsequent experiments. Although delete-
rious effects on the cells were not observed with a higher dose
of bacteria (4 ⫻ 107 CFU/ml), this high dose of bacteria did
not inhibit the basal cytokines secretion relative to the 10:1
bacteria:cell ratio. This dose was also used for LGG and E. coli
TG1, although the optimal doses of these three strains needed
to modulate cytokine production are likely different. Fig. 2C
and D shows that B. breve inhibited the basal and LPS-induced
secretion of IL-23 and IL-17 in the lower compartments in a
time-dependent manner.
Markedly, in the presence of LPS, IL-17 and IL-23 produc-
tion was inhibited significantly as early as 24 h, with maximal
inhibitory effect after 48 h of culture, and lasted for 96 h. As
the maximal, significant decreases in the cytokine levels were
observed at 48 h (P⬍0.05), this incubation time was used for
all of the subsequent experiments.
The differences between the 2D and 3D PBMCs
cultured separately or in coculture with T84 cells and
ascertaining the site of action of LPS
To determine which part of the system was responsible for the
secretion of IL-17/IL-23, and if there was a difference in the
IL-17 expression between the conventional (2D) - and 3D-cul-
tured PBMCs, we separately cultured 2D and 3D PBMCs with-
out IECs. As shown in Fig. 2E, LPS significantly enhanced the
production of IL-17 and IL-23 in the 2D-cultured PBMCs.
However, the basal secretion of IL-17 in the 3D-cultured
PBMCs was significantly higher than that observed in the con-
ventional 2D PBMCs. These data imply that the PBMCs pro-
duce IL-17 and IL-23 in response to LPS and that the IECs are
not the only source of IL-23.
Moreover, to determine the site of LPS action, the apical
versus basolateral LPS-stimulating activity was tested in polar-
ized HT-29/B6 and T84 cells grown on Transwell filters. In
this model, the total amounts of IL-17 and IL-23 secreted into
the medium of the upper and lower compartments were mea-
sured after apical and basolateral LPS stimulation, respectively.
We observed that the basal secretion of IL-17 by the HT-29/B6
and T84 cells was negligible, and its level was always lower
than the detection limit indicated by the manufacturer. LPS
applied apically or basolaterally was also not able to induce
IL-17 in the HT-29/B6 and T84 cells. Similarly, as shown in
Fig. 2F, LPS added to the medium in the upper compartments
(apical surface) did not significantly affect the IL-23 levels in
the supernatants from the upper and lower compartments. In
contrast, when LPS was added to the medium in the lower
compartments (basolateral surface), a significant induction of
IL-23 release was observed in the supernatants from the upper
and lower compartments. However, the basolateral secretion
was significantly greater than the apical secretion upon baso-
lateral LPS stimulation (Fig. 2F). These results describe the
rationale for choosing basolateral LPS stimulation in subse-
quent experiments and confirm previous studies with animal
Volume 92, October 2012 Journal of Leukocyte Biology 899
 A B
T84 cells, B.breve T84 cells, B.breve
180 250 T84 cells, LPS+ B.breve
T84 cells, LPS+B.breve
HT-29/B6 cells, B.breve HT-29/B6 cells, B.breve
160 HT-29/B6 cells, LPS+B.breve HT-29/B6 cells, LPS+ B.breve

200
140

120

IL-23 (pg/ml)
IL-17(pg/ml)

§
150
100
§
§
80 § §
§ 100 §

60 *
* *
40 50
*
20

0 0
0 2x10*6 5x10*6 1x10*7 2x10*7 4x10*7 0 2x10*6 5x10*6 1x10*7 2x10*7 4x10*7
--------------------------------------------------------------------- ----------------------------------------------------------------------
B.breve concentrations(CFU/ml) B.breve concentrations(CFU/ml)

C D
T84 cells, B.breve T84 cells, B.breve
T84 cells, Control T84 cells, Control
280 400
T84 cells, LPS T84 cells, LPS
T84, LPS+B.breve T84 cells, LPS+ B.breve
HT-29/B6 cells, B.breve HT-29, B.breve
240
HT-29/B6 cells, Control HT-29/B6 cells, Control
320
HT-29/B6 cells, LPS HT-29/B6 cells, LPS
200 HT-29/B6 cells, LPS+B.breve HT-29/B6 cells, LPS+B.brve
IL-23(pg/ml)
IL-17(pg/ml)

240
160
§ §
§
§
120 §
§ 160

80

80
**
40 **
* **

0 0
6 12 24 48 96 6 12 24 48 96

Incubation periods (h) Incubation periods(h)

E F
1500 1200
IL-23 in 2D PBMCs Apical,T84 cells
IL-17 in 2D PBMCs Apical, HT-29/B6 cells
1250 IL-17 in 3D PBMCs 1000 *ϕ Basolateral, T84 cells
* Basolateral, HT-29/B6 cells
Cytikines (pg/ml)

1000 800
*
IL-23 (pg/ml)

*
§ϕ
750 § 600

* §
__#__
500 400 §
§ §
§ §
§
§
ϕ § § §
250 200
§ *

0 0
Control LPS TLR4 antagonist B.breve
______________________ ol
Contr PS(ap
ic) aso) c)
aso
)
pic
)
aso
) _______
apic baso
api
L S(b ist( nist(b nist(a nist(b LPS
LPS LP agon g o a g o g o
n t nta 4 a n t nt a +B.breve(apic)
4a 4a 4a
T L R TLR ) + T L R + T L R
p i c s o )
S(a PS(ba
LP L

900 Journal of Leukocyte Biology Volume 92, October 2012 www.jleukbio.org

 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics

and human IECs showing that administration of LPS to the
basolateral surface of rat IEC-6 cells disrupted the barrier func-
tion of tight junction, in terms of TEER, whereas LPS adminis-
tered to the apical surface altered neither the barrier function
nor the localization of 7H6 antigen in rat IEC-6 cells [43].
This is a result of the fact that the expression of TLR4, its co-
receptor MD2, or both, which are necessary for LPS respon-
siveness in IECs, is polarized to the basolateral membrane in
IECs. Hence, LPS sensing and LPS responsiveness occur along
on the basolateral membrane of polarized IECs in culture,
such as the human colonic adenocarcinoma T84 cell line [37,
44]. Moreover, bacterial LPS, added to the apical surface of
polarized IECs, has previously been shown to be transported
from the apical to the basolateral pole of the epithelium [45].
Finally, when Caco-2 cells were cocultured in the presence of
PBMCs, apical stimulation of polarized Caco-2 cells with LPS
had no effect on permeability of Caco-2 cells, as well as on ba-
solateral secretion of proinflammatory cytokines, even at con-
centrations as high as 1 ␮g/ml [14].
B. breve and LGG inhibit the LPS-induced expression
of IL-17 and IL-23 mRNA and protein in the 3D
coculture model constructed with IECs and PBMCs
In the 3D coculture system constructed with HT-29/B6 or T84
cells and PBMCs, LPS, added only to the lower compartments,
significantly induced the secretion of IL-17 in the lower com-
partments when compared with the control. B. breve and LGG
significantly inhibited the basal and LPS-induced secretion of
IL-17 (Fig. 3A). As it is known that only Th17 cells produce
IL-17, we measured the IL-17 concentration in the cell-free
supernatants of the lower compartments, where IL-17 may
be generated from naïve T cells upon LPS stimulation of the
PBMCs. Fig. 3A also shows that the IL-17 concentrations in the
3D cocultures of HT-29/B6 cells and PBMCs are relatively dif-
ferent from those observed in the 3D cocultures of T84 cells
and PBMCs, although this was not statistically significant.
These differences may reflect the physiological differences in
the response of the HT-29/B6 and T84 cell lines to the treat-
ments [36] and/or to the PBMC-derived mediators. With re-
gard to IL-23 secretion, the profiles of the IL-23 levels were
similar to those observed for IL-17 (Fig. 3B). These data show
that polarized HT-29/B6 and T84 cells preferentially and con-
stitutively secreted basal IL-23 into the basolateral side in com-
parison with the apical side, and IL-23 concentrations in baso-
lateral media were significantly higher than that observed in
the apical side, suggesting no potential flow of medium be-
tween the apical and basolateral compartments. However, ba-
solateral-to-apical differences in IL-23 production were dimin-
ished by LPS, alone or in combination with E. coli TG1. In
contrast, B. breve and LGG inhibited the basal and LPS- in-
duced secretion of IL-23 into upper and lower compartments
and notably, retained the basolateral-to-apical differences in
IL-23 production. This may simply reflect the greater barrier
integrity of cells, which strongly blocks any potential flow of
medium between the apical and basolateral compartments. To
confirm that the observed effects were specific for B. breve and
LGG, we included a Gram-negative non-LAB strain, E. coli TG1,
as an internal control. E. coli TG1, given alone, had no effect
but resulted in an additive increase in IL-17 secretion in com-
bination with LPS, overall, in accordance with a previous study
[46], showing that E. coli TG1 as a proinflammatory control
strain stimulated bone marrow-derived DCs to produce large
amounts of proinflammatory cytokines. E. coli TG1 did not in-
hibit IL-23/IL-17 expression but rather, additively aggravated
the LPS-induced IL-23/IL-17 expression in the HT-29/B6 and
T84 cells. Therefore, the subsequent experiments were con-
ducted without this strain.
Like IL-23, B. breve and LGG diminished LPS-stimulated
CD40 protein expression (Fig. 3C). As IECs produce IL-23 in
response to LPS via the TLR4/MD2 complex [2, 37, 47], we

4
Figure 2. Effect of B. breve on IL-17 and IL-23 secretion in HT-29/B6 or T84 cells and PBMCs cultured either in 3D coculture models or sepa-
rately. (A–D) The dose- and time-dependent effects of B. breve on IL-23/IL-17 production by the 3D coculture models constructed with HT-29/B6
or T84 cells and PBMCs. The HT-29/B6 or T84 cells (2⫻106 cells/ml) were cultured in triplicate in 12-well Transwell-COL inserts, and freshly iso-
lated PBMCs (34,000 cells/200 ␮l/scaffold) were cultured in 12-well 3D insert scaffolds and placed on the bottom of the culture plates (lower
compartment). The upper compartment that bathes the filter-grown HT-29/B6 or T84 epithelial monolayers, was filled with medium alone or me-
dium containing increasing concentrations of B. breve in the absence or presence of basolateral-applied LPS (100 ng/ml) for 48 h. The cell-free
supernatants from the lower compartments were harvested, and the (A) IL-17 or (B) IL-23 concentrations were measured in triplicate by specific
ELISA. In the same set of experiments, the cells were cultured with 2 ⫻ 107 CFU/ml (a bacteria:cell ratio of 10:1) B. breve in the absence or pres-
ence of basolateral-applied LPS (100 ng/ml) for the indicated incubation periods, and the concentrations of IL-17 (C) and IL-23 (D) in the cell-
free supernatants of the lower compartments were measured as indicated above. *Significantly different when compared with control cell cultures
(in the case of HT-29/B6 and T84 cells) without addition of LPS, P ⬍ 0.05; §significantly different when compared with control cell cultures (in
the case of HT-29/B6 and T84 cells) with addition of LPS, P ⬍ 0.05. (E) Separately and without HT-29/B6 or T84 cells, the 2D PBMCs (1.7⫻105
cells/ml in 96-well cell culture microplates) and scaffold-based 3D PBMCs (34,000 cells/200 ␮l/scaffold) were treated without or with 100 ng/ml
LPS for 48 h in the absence or presence of B. breve at a bacteria:cell ratio of 10:1. Following the incubation, the cell-free supernatants were ana-
lyzed for IL-17 and IL-23 expression by ELISA. (F) To determine the site of LPS action, HT-29/B6 and T84 cells (2⫻106 cells/ml) were grown in
12-well Transwell-COL inserts and then stimulated for 48 h with 100 ng/ml LPS on the apical (upper compartment) or basolateral (lower com-
partment) pole in the absence or presence of B. breve at a bacteria:cell ratio of 10:1, which was added to the medium of the upper compartments.
Following the incubation, the cell-free supernatants from the upper and lower compartments were analyzed for IL-23 expression by ELISA. Each
ELISA was run in triplicate with cell-free supernatants from at least three separate experiments, and each result is expressed as the mean value of
three independent experiments ⫾ sem. *P ⬍ 0.05 versus control (in the case of F, it means significant for HT-29/B6 and T84 cells);
§P ⬍ 0.05 versus LPS alone (in the case of F, it means significant for HT-29/B6 and T84 cell). ␸P ⬍ 0.05 versus apical levels (in the case of F, it means
significant for HT-29/B6 and T84 cells); #. P ⬍ 0.05 vs. 2D cultured PBMCs; apic, Apical (upper compartment of the filters); baso, basolateral (lower
compartment of the filters).

www.jleukbio.org Volume 92, October 2012 Journal of Leukocyte Biology 901

A B
700 1400
HT-29/B6 cells AP, HT-29 cells
T84 cells + BL, HT-29/B6 cells
600 + 1200 AP,T84 cells +
BL, T84 cells +

500 1000 +
+
+
*

IL-23 (pg/ml)
IL-17(pg/ml)

400
* 800
* *
**
300 600
§
§
§ § § §
200 400
φ §
φ § §
§ § §
200 φ φ* φ§ φ§
100 φ φ * §
* § § * * §
* * * * * * * *
0 0
ve G 1 ol
LP .brev
S e G ve GG
1
G1 is
t
TG
l S ntr
LP .bre LG .bre
ve

tro TG
G

LG
1

st +L +T on
G

n Co
LG
re

ni
Co B B
T

B S S g
go
.b

S+

LP LP ta
S+

S+
B

an
ta
LP

LP
LP
S+

an

R 4
LP

L
4

+T
R
TL

PS
S+

L
LP

C D
* 1000
IL-23 in HT-29/B6 cells
600 AP, HT-29/B6 cells
BL,HT-29/B6 cells * * CD40 in HT-29/B6 cells
*
Basolateral concentrations (pg/ml)

AP, T84 cells IL-17 in HT-29/B6 cells

500 * BL,T84 cells 800 * IL-23 in T84 cells
* CD40 inT84 cells
IL-17 in T84 cells
CD40 (pg/ml)

400
600 *
*
300 § * §
§ §
§ § 400 §
§ § §
200 § §
§ §
§ 200
100 § * *
§§

0 0
rol LPS
t
ev
e G ve G ol S
ev
e
ev
e
t nis LG br
e LG nt r LP
Con go .br B. Co B.
br br
B.
t a B +
4a
n + PS d
ed
S L ille ill
LR LP t -k -k
S +T H ea at
LP he
S+
LP

Figure 3. Effect of LAB on IL-17, IL-23, and CD40 production in response to LPS in the coculture models constructed with HT-29/B6 or T84
cells in the upper compartment and 3D PBMCs in the lower compartments. The HT-29/B6 or T84 cells (2⫻106 cells/ml) were cultured in 12-well
Transwell-COL inserts, and freshly isolated PBMCs (34,000 cells/200 ␮l/scaffold) were cultured in 12-well 3D insert scaffolds and placed on the
bottom of the culture plates. LPS (100 ng/ml) was added to the culture medium of the lower compartments for 48 h in the absence or presence
of B. breve, heat-killed B. breve, or LGG (added to the culture medium of the upper compartment) at a bacteria:cell ratio of 10:1. As a control, 10
ng/ml of the TLR4/MD2 complex antagonist was added to the lower compartments. After the incubation, the supernatants from the upper and
lower compartments were harvested and analyzed by ELISA for the secretion of (A) IL-17 in the lower compartments IL-23 (B) and CD40 (C) in
the upper and lower compartments. In the case of heat-killed B.breve, IL-17, IL-23, and CD40 were assessed only in the lower compartments (D).
Each ELISA was performed in triplicate with cell-free supernatants from at least three separate experiments, and each result is expressed as the
mean value of three to five (in the case of A and B) independent experiments ⫾ sem. ␸P ⬍ 0.05 versus apical; *P ⬍ 0.05 versus control; §P ⬍
0.05 versus LPS alone; ⫹P ⬍ 0.05 versus TG1 alone. AP, Apical (upper compartment of the filters); BL, basolateral (lower compartment of the
filters).

902 Journal of Leukocyte Biology Volume 92, October 2012 www.jleukbio.org

 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics

used a TLR4 antagonist to block the TLR4/MD2 pathway and protein expression of IL-17, IL-23, and CD40 into the baso-
measured the protein levels of the inflammation markers IL- lateral compartments of 3D coculture models constructed
17, IL-23, and CD40 in the upper and lower compartments. with HT-29/B6 or T84 cells and PBMCs. In the case of the
Fig. 3A–C shows that the TLR4 antagonist largely abolished heat-killed B.breve, we measured IL-17, IL-23, and CD40 only
the LPS-stimulated production of these inflammation markers. in the lower compartments, as B.breve releases metabolites
Previous studies have revealed that probiotic bacteria and their that can cross the intestinal barrier under normal or inflam-
components inhibit LPS/TLR4/NF-␬B pathway-mediated aber- matory conditions. Furthermore, its soluble factors usually
rant inflammation [32, 48, 49]; however, anaerobic B. breve was retain their anti-inflammatory properties after transepithe-
not able to cope with the aerobic conditions of the cell cul- lial transport across intestinal cell monolayers, mainly in
ture. Therefore, we included heat-killed B. breve to determine inflammatory conditions [32].
whether the effects of the live B. breve to lower aberrant in- Furthermore, our preliminary, semi-qRT-PCR results demon-
flammation could also be mediated by dead B. breve. Fig. 3D strated that B. breve and LGG inhibited the LPS-stimulated ex-
shows that the heat-killed B. breve inhibited LPS-stimulated pression of IL-23 in the HT-29/B6 and T84 monolayers, as

A B
LPS+B. breve

10 HT-29/B6 cells
LPS+ LGG

T84 cells
B. breve

* *
Control

8
LGG

TaqMan real-time RT-PCR

LPS

* *
(Relative to GAPDH)
IL-23 mRNA

IL-23 in HT-29/B6 cells

4 §

§
§
§
2
IL-23 in T84 cells §
§
* * * *
0
Control LPS B.breve LGG B.breve LGG
---------------------
LPS
IL-23 in 3D PBMCs

Figure 4. Semi-qRT-PCR and TaqMan qRT-PCR analysis of IL-23 and

IL-17 in 3D PBMCs IL-17 mRNA expression in response to B. breve and LGG. The coculture
model was constructed with polarized monolayers of filter-grown HT-
29/B6 or T84 cells in the upper compartments and 3D PBMCs placed on
the bottom of the culture plates in the lower compartment of the filters.
β-actin B. breve and LGG (bacteria:cell ratio of 10:1) were added to the medium
of the upper compartments in the absence or presence of 100 ng/ml
LPS, which was added to the medium of the lower compartments. After a
C 48-h incubation, total RNA was extracted and reverse-transcribed to
cDNA, and the mRNA levels of IL-17, IL-23, and ␤-actin were analyzed
IL-23 in 3D PBMCs cocultured with HT-29/B6
IL-17 in 3D PBMCs cocultured with HT-29/B6
initially by semi-qRT-PCR using specific primers for IL-17, IL-23, and ␤-ac-
6 IL-23 in 3D PBMCs cocultured with T84 cells tin. After PCR amplification, 15 ␮l of the products was separated by elec-
IL-17 in 3D PBMCs cocultured with T84 cells trophoresis in a 1.5% agarose gel containing 10 ␮g/ml ethidium bromide
and visualized with a UV transilluminator (A). One representative result
Cytokines mRNA levels
(Relative to GAPDH)

*
from three independent experiments is shown. The IL-23 and IL-17
*
4 * mRNA levels were also analyzed in more detail by TaqMan qRT-PCR (B
*
* TaqMan real-time RT-PCR and C). The data for each sample are normalized to the GAPDH mRNA
* level present in each sample, normalized again between the samples to
the levels of IL-23 and IL-17 mRNA present in the control, and the rela-
§ tive expression levels are shown. Each TaqMan assay was performed in
§ §
2
§ §
§ triplicate for each sample. The data shown are the means ⫾ sem of three
§ §
independent experiments. *P ⬍ 0.05 versus control; §P ⬍ 0.05 versus LPS
alone.

0 * * * * * * *
Control LPS B.breve LGG B.breve LGG
-----------------------
LPS

www.jleukbio.org Volume 92, October 2012 Journal of Leukocyte Biology 903

A B

T84 cells HT-29/B6 cells

Time [min] - 30 60 120 Time [min] - 30 60 120
LPS +B. breve -p- Ac- H3 LPS+B. breve -p- Ac- H3

LPS -p- Ac- H3 LPS -p- Ac- H3

B. breve -p- Ac- H3 B. breve -p- Ac- H3

- H3
- H3

LPS +B. breve - Ac- H4 LPS+B. breve - Ac- H4

- Ac- H4 - Ac- H4
LPS LPS
B. breve - Ac- H4 B. breve - Ac- H4
- H4 - H4

C D
-p-Ac-H3
-p-Ac-H3
-H3
-H3
-Ac-H4 -Ac-H4

-H4
-H4
- + - - - - GM - + - - - - GM
- - + - - - TSA - - + - - - TSA
- - - + - + LPS - - - + - + LPS
- - - - + + B. breve - - - - + + LGG

E
50 HT-29/B6 cells
F
Histone H3 acetylation[ng/mg protein]

60 HT-29/B6 cells
T84 cells T84 cells * *
*
DNA metylation[ng/mg protein]

40 ** * 50

§ § §
§ § §
40
30 *
* * 30

20 §
§
§ § 20
* *
10
* * * 10

*
0
0 Control TSA GM LPS B.breve L.GG LPS+ LPS+
Control TSA GM LPS B.breve L.GG LPS+ LPS+
B.breve L.GG
B.breve L.GG

904 Journal of Leukocyte Biology Volume 92, October 2012 www.jleukbio.org

 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics

well as IL-17 and IL-23 in the 3D PBMCs (Fig. 4A). The IL-23
and IL-17 mRNA levels were also analyzed using TaqMan qRT-
PCR, which is more reliable and sensitive. As shown in Fig. 4B,
B. breve and LGG significantly inhibited the LPS-stimulated
IL-23 mRNA levels in the HT-29/B6 and T84 cells. Further-
more, B. breve and LGG inhibited the IL-23 and IL-17 mRNA
levels in the LPS-treated 3D PBMCs when cocultured with HT-
29/B6 or T84 cells (Fig. 4C).
B. breve and LGG decrease the global Ac-H4 and the
p-Ac-H3 at Ser-10/Lys-14 and enhance DNA
methylation
To evaluate the levels of histones and their acetylated forms in
the 3D coculture experiments, the T84 and HT-29/B6 mono-
layers were incubated initially with 2 ⫻ 107/ml B. breve, which
was added to the medium in the upper compartments (apical
surface) or to the medium in the lower compartments (baso-
lateral surface) in the presence of LPS for 30, 60, and 120
min, and the cell lysates were subjected to Western blot analy-
sis. As shown in Fig. 5A (T84 cells) and B (HT-29/B6 cells),
LPS alone induced the global Ac-H4 and the p-Ac-H3 at Ser-
10/Lys-14 in a time-dependent manner. The maximum in-
crease in the Ac-H4 and the p-Ac-H3 occurred at 120 min. B.
breve inhibited the LPS-induced Ac-H4 and p-Ac-H3. Addition-
ally, to evaluate these effects in more detail and to assess the
epigenetic imprinting effects of B. breve and LGG during long-
term incubation, we next incubated the HT-29/B6 and T84
cells for 48 h with the histone deacetylase inducer GM, the
potent histone deacetylase inhibitor TSA, B. breve, or LGG in
the presence of LPS. Fig. 5C and D shows that GM inhibited
the global Ac-H4 and the p-Ac-H3 at Ser-10/Lys-14. TSA in-
duced the global Ac-H4 and the p-Ac-H3 at Ser-10/Lys-14. LPS
alone induced the global Ac-H4 and the p-Ac-H3 at Ser-10/
Lys-14. B. breve and LGG (Fig. 5C and D, respectively) inhib-
ited the LPS-induced global Ac-H4 and p-Ac-H3 at Ser-10/
Lys-14 in the T84 cells incubated for 48 h. Further, to confirm
these results, the accumulation of acetylated histones and
methylated DNA was also quantitatively assessed in the cell ly-
sates. As shown in Fig. 5E, quantitative analysis of these cell
lysates revealed that TSA and LPS enhanced the accumulation
of Ac-H4, whereas GM decreased the accumulation of Ac-H4.
B. breve significantly decreased the accumulation of Ac-H4 in
the untreated and LPS-treated HT-29/B6 and T84 cells,
whereas LGG significantly inhibited only the LPS-induced ac-
cumulation of Ac-H4. Finally, Fig. 5F shows that LPS decreased
DNA methylation as compared with the control. B. breve and
LGG slightly enhanced the DNA methylation in the untreated
cells and significantly restored LPS-reduced DNA methylation.
B. breve and LGG inhibit LPS-induced NF-␬B
transcription activity
As histone acetylation mediates the bacteria-, LPS-, TLR4-, and
cytokine-mediated induction of the NF-␬B-dependent tran-
scription activity, causing the aberrant expression of inflamma-
tory genes [50 –54], the activation of NF-␬B, through its classi-
cal pathways, was evaluated by determining the key signaling
molecules that are generally used to mediate the activation of
the transcription factors NF-␬B, MAPK, and NF-␬B-dependent
luciferase reporter gene activity. As shown in Fig. 6A and B,
Western blot analysis and quantitative densitometry of the blot
indicated that LPS alone induced the expression of MyD88,
the degradation and disappearance of IRAK1 and I␬B␣, the
expression of the nuclear NF-␬B p50/p60 subunits, and the
p-p38 MAPK and p-MEK1, which were correlated with in-
creased NF-␬B-dependent luciferase gene reporter activity (Fig.
6D). B. breve inhibited the LPS-induced expression of the nu-
clear NF-␬B p50/p65 subunits and MyD88, increased the level
of the IRAK1 and I␬B␣ proteins by decreasing LPS-induced
IRAK1/I␬B␣ degradation, and abolished the LPS-induced
p-p38 MAPK and p-MEK1 (Fig. 6A and B). Additionally, as B.
breve and LGG were able to inhibit the hyper-Ac-H3/H4 in the
T84 cells in the 3D cocultures with PBMCs during long-term
(48-h) incubation of the cells with LPS, we also analyzed the
cell lysates following the 48-h incubation period by immuno-
blotting of the above signaling molecules in HT-29/B6 and
T84 cells. Fig. 6C shows that LGG inhibited the LPS-induced
expression of MyD88 and the nuclear NF-␬B p50/p65 sub-
units, the degradation of the I␬B␣ and IRAK-1 proteins, and
the p-p38 MAPK and p-MEK1 in HT-29/B6 and T84 cells.
Consistent with the inhibition of the LPS-induced NF-␬B activ-
ity, Fig. 6D shows that B. breve and LGG dose-dependently re-
pressed the LPS-induced NF-␬B-dependent luciferase activity in
the HT-29/B6 and T84 cells, reaching a maximum attenuation
at a dose of 10:1 bacteria/cell. The TLR4 antagonist abolished
the NF-␬B-dependent luciferase activity in the unstimulated
and LPS-stimulated cells. The TLR4 antagonist-mediated inhi-
bition of the basal NF-␬B-dependent luciferase activity may be

4
Figure 5. Effect of B. breve and LGG on histone modifications and DNA methylation in response to LPS in the 3D coculture models constructed
with polarized monolayers of Transwell filter-grown IECs in upper compartments and 3D PBMCs in lower compartments, placed on the bottom of
culture plates. T84 cells (A) or HT-29/B6 cells (B), at a density of 2 ⫻ 106 cells/ml, were treated initially for 30, 60, and 120 min with B. breve
added to the medium of the upper compartments (apical surface; bacteria:cell ratio of 10:1) in the presence of 100 ng/ml LPS, which was added
to the medium of the lower compartments (basolateral surface). After incubation, the cell lysates from the T84 and HT-29/B6 cells were prepared
and subjected to Western blotting to analyze the levels of global H4, Ac-H4, global H3, and p-Ac-H3. To evaluate these effects in more detail and
to assess the epigenetic imprinting effects of B. breve and LGG during long-term incubation, the HT-29/B6 and T84 cells were next incubated for
48 h with 0.2 mM GM (a histone acetylation inhibitor), 0.01 ng/ml TSA (a potent histone acetylation inducer), and B. breve or LGG (bacteria:cell
ratio of 10:1) in the presence of 100 ng/ml LPS added to the medium of the lower compartments. After the incubation, the prepared cell lysates
were subjected to Western blot analysis to assess the effects of B. breve (C) and LGG (D) on the Ac-H3 and the p-Ac-H3 in the LPS-stimulated cells.
The data are representative of three independent experiments. To confirm these results, the accumulation of Ac-H4 (E) and methylated DNA (F)
were also quantitatively assessed in the prepared cell lysates, as described in Materials and Methods. Each value represents the mean ⫾ sem of four
independent experiments performed in duplicate. *P ⬍ 0.05 versus control; §P ⬍ 0.05 versus LPS alone.

www.jleukbio.org Volume 92, October 2012 Journal of Leukocyte Biology 905

 p50
A B

LPS+TLR4 antagonist
p65
5
MyD88

β-Actin normalised densitometric units

TLR4 antagonist
IRAK-1

LPS+ B. breve
IkBa
4 * Phospho-p38 MAPK
Phospho-MEK1
*

B. breve
*
Control
* * §
3

LPS
§
§
§ §
§
Phospho-MEK1 2 §
§§

Phospho-p38 MAPK §§ §
1 * §
* §
IkBα

IRAK1 0
e
ntro
l S nis
t
rev ist ve
LP on re
MyD88 Co tago B.b tag
B .b
an an
R4 S+
TL LR4 LP
T
p65 S+
LP
p50
D 4
HT-29/B6 cells
β-actin T84 cells

*
3
*
Luciferase activity

C
LPS+ LGG

( Relative value)
Control

2
LGG

§
LPS

§ §§
§
§
Phospho-MEK1 1
Phospho- p38 MAPK
IkBα **
0
l e 5 10 0 5 10
ev LGG
0
ro /ml) TLR4
---------------------------------
IRAK1 nt br antagonist B.breve LGG
B.
Co 0ng ---------------
HT-29/B6 (1 __________________
[ 10ng /ml ] Bacteria - to- cell ratios
MyD88 ist 0:
1) LPS
g on (1
ta t io [ 100 ng / ml ]
p65 an ra
R4 ell
TL o -c
t
p50 ia-
er
ct
Ba
β-actin
Figure 6. Effect of B. breve and LGG on the LPS-stimulated transcriptional activity of
Phospho-MEK1 NF-␬B. (A) T84 cells were incubated with medium alone (Control) or LPS in the ab-
Phospho-p38 MAPK sence or presence of B. breve at a bacteria:cell ratio of 10:1. The cell lysates obtained
from the 2-h incubation period were analyzed by immunoblotting for the protein ex-
IkBα pression of the nuclear NF-␬B p50/p65 subunits and MyD88, the degradation of I␬B␣
and IRAK-1, and the p-p38 MAPK and p-MEK1. ␤-Actin was included as a standard for
IRAK1 T84 protein loading. The data are representative of three independent experiments. (B)
Quantitative densitometric analysis of the Western blot is presented, with the ␤-actin-
MyD88 normalized densitometric units for each protein plotted against the treatments. The
data are presented as the means ⫾ sem (indicated with error bars) from three inde-
p65
pendent experiments. *P ⬍ 0.05 versus control; §P ⬍ 0.05 versus LPS alone. (C) HT-
p50 29/B6 cells or T84 cells were treated with 100 ng/ml LPS, which was added to the
medium of the upper compartments in the absence or presence of LGG (bacteria:cell
β-actin
ratio of 10:1). The cell lysates from the 48-h incubation period were analyzed by West-
ern blotting for the protein expression of the nuclear NF-␬B p50/p65 subunits and MyD88, the degradation levels of I␬B␣ and IRAK-1, and the p-p38 MAPK
and p-MEK1. ␤-Actin was included as a standard for protein loading. The data are representative of three independent experiments. (D) The effect of B. breve
or LGG on the NF-␬B-dependent promoter luciferase reporter gene activity in the HT-29/B6 and T84 cells, and HT-29/B6 or T84 cells were transiently trans-
fected with the Ad5␬B-LUC vector. The cells were then incubated with various concentrations of B. breve or LGG (bacteria:cell ratios of 0, 5, and 10), which
were added to the medium of the upper compartments in the presence or absence of 100 ng/ml LPS or 10 ng/ml TLR4 antagonist (added to the medium
of the lower compartments). In the nuclear extracts, the NF-␬B-dependent luciferase activity was measured after 24 h using a Tecan GENios microplate
reader, and the results were normalized to the extract protein concentrations. *P ⬍ 0.05 versus control; §P ⬍ 0.05 versus LPS-stimulated cells. The data
(means⫾sem of triplicate determinations) are representative results of three independent experiments.

906 Journal of Leukocyte Biology Volume 92, October 2012 www.jleukbio.org

 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics
a result of the fact that the culture medium is not absolutely
LPS-free.
Cell viability and proliferation assays
Under the conditions studied, we observed that none of the
agents decreased the viability of the HT-29/B6 and T84 mono-
layers or the 3D PBMCs at the concentrations tested. To ex-
clude the possibility that the decreased expression of the IL-
23/IL-17 and the decreased activity of the NF-␬B-dependent
luciferase reporter gene were a consequence of cell death as a
result of exposure to LPS, the TLR4 antagonist, or the tested
bacteria, the viability of the cells was assessed routinely, as
stated previously. Microscopic examination of the cells incu-
bated with bacteria and challenged with agents revealed that
the viability was ⬎93% in all experiments, demonstrating that
the inhibition of IL-23, IL-17, CD40, and the NF-␬B-dependent
luciferase reporter gene by the bacteria and the TLR4 antago-
nist is not a result of cell death (Fig. 7A). Our observations are
supported by reports from other groups indicating that LPS, at
the concentration used in this study, had no cytotoxic effects
on the viability of cell monolayers [55] or PBMCs from
healthy and IBD patients [9, 38, 56], even after culturing for
48 h.
Notably, LPS alone did not decrease, but rather increased,
the cell viability of the HT-29/B6 and T84 monolayers, and
the TLR4 antagonist abolished this effect (Fig. 7A). To deter-
mine whether this pattern of LPS-induced cell viability is asso-
ciated with cell proliferation, we also assessed the proliferation
of the HT-29/B6 and T84 cells. Fig. 7B shows that the LPS-
induced increase in cell viability was related to proliferation
and that the TLR4 antagonist abolished the LPS-stimulated
proliferation. This corresponds with previous studies showing
that LPS induces the proliferation of IEC lines through TLR4-
mediated signaling, which aids in proliferation and protection
against apoptosis and is therefore important for intestinal epi-
thelial repair from injury [57–59].
DISCUSSION
In this study, we used an in vitro 3D coculture model com-
posed of IECs and PBMCs to explore the molecular mecha-
nism by which B. breve and LGG modulate the IL-23/IL-17 axis,
which is involved in IBD. Our experiments demonstrated that
B. breve and LGG inhibited the LPS-stimulated mRNA and pro-
tein expression of the IL-23/IL-17 axis and CD40 in the 3D
coculture models constructed with HT-29/B6 or T84 cells and
PBMCs. Together with previous studies [8, 12], the above re-
sults suggest that B. breve and LGG may exert their anti-inflam-
matory effects on the epithelium by reducing the expression
of the IL-23/IL-17 axis as a hallmark of IBD.
Under the conditions studied, we observed that the effective
inhibition of basal IL-17 and IL-23, as well as maximum inhibi-
tion of LPS-induced IL-23 and IL-17 by B. breve, only occurred
at a bacteria:HT-29/B6 or T84 cell ratio of 10:1. Such a ratio
was used by Foligne et al. [46] for investigating the cytokine
profiles of DCs treated with probiotic bacteria. It is also known
that bifidobacteria can regulate cytokine production in IECs in
www.jleukbio.org
a dose-dependent manner [49]. However, we believe that the
optimum immunomodulating effect by the bacteria at the 10:1
ratio is a result of the average number of PAMP receptors,
such as TLRs and nucleotide binding oligomerization do-
mains, on the cells [58]. High doses of bacteria may lead to
the competitive use of the available receptors and false recep-
tor binding. Therefore, bacteria at high doses failed (in our
hands) to inhibit these cytokines.
It has been reported that histone modifications may under-
lie the chronic inflammation of UC and CD and that higher
histone acetylation levels predispose cells to IBD, likely
through the induction of proinflammatory cytokines [18 –20].
Histone acetylation mediates the bacteria-, LPS-, and cytokine-
induced NF-␬B transcription activity that causes the expression
of inflammatory genes and is correlated with the NF-␬B tran-
scription activity for other genes, including those involved in
the differentiation, proliferation, and activation of cells [51,
52, 54]. LPS exposure leads to the phosphorylation/acetyla-
tion of histones at the promoters of proinflammatory genes in
lung cells [60], macrophages, lymphocytes, and monocytic
cells [61]. Interestingly, in murine macrophages and experi-
mental colitis, LPS strongly stimulates IL-23 expression
through histone acetylation and NF-␬B activation [52]. LPS is
also known to stimulate the in vivo phosphorylation/acetyla-
tion of histones and the recruitment of NF-␬B to the CD40
promoter, all of which are important events for CD40 gene
transcription, indicating that histone acetylation and NF-␬B
activation play critical roles in the transcriptional activation of
the CD40 gene by LPS in immune cells [62]. The LPS-stimu-
lated IL-17 expression in human PBMCs [63] and the in vivo
association among histone acetylation, NF-␬B-induced expres-
sion of inflammatory genes, and IBD in humans have been
reported recently [24]. In this regard, we first aimed to investi-
gate whether the B. breve- and LGG-mediated inhibition of IL-
23/IL-17 expression is also associated with inhibition of his-
tone acetylation in response to LPS. Our experiments revealed
that B. breve and LGG inhibited LPS-induced histone acetyla-
tion in the IECs. LPS-mediated TLR4 signaling occurs via a
cascade of events, including the stimulation of MyD88 and
IRAK-1, which in turn simultaneously activate the IKK com-
plex. These events then lead to the degradation of I␬B␣, the
p-NF-␬B p65/p50 subunits, and NF-␬B and MAPK activity, re-
sulting in p-MEK1 (the upstream kinase of ERK1) and p-p38
MAPK. This ultimately results in aberrant inflammatory re-
sponses by inducing proinflammatory cytokines in vitro and in
vivo [50, 53]. Therefore, we next evaluated the impact of B.
breve and LGG on the LPS/TLR4-mediated intracellular signal-
ing cascades, including signaling molecules that are generally
involved in mediating the activation of the NF-␬B and AP-1
transcription factors, as well as the MAPKs, and assessed their
impact on NF-␬B-dependent luciferase reporter gene activity in
the IECs stimulated with LPS. Our findings were consistent
with the in vitro and in vivo observations that probiotic bacte-
ria inhibit LPS/TLR4/NF-␬B-mediated proinflammatory cyto-
kine expression in colonic epithelial cells and in PBMCs that
are involved in colitis [32, 36, 48, 49, 64 – 67]. Furthermore, B.
breve and LGG inhibited the LPS-induced degradation of
IRAK1 and I␬B␣, the p-p38 MAPK/p-MEK1, and the expres-
Volume 92, October 2012 Journal of Leukocyte Biology 907
 A B 6
Proliferation HT-29/B6 cells
T84 cells

180 5
*

Proliferation (Stimulation Index)

160 *
HT-29/B6 cells *
T84 cells
PBMCs 4
140 *

120
Cell viability (%)

§
§ 3
100 §
§
§
§
80 §
2
60

40 1
§
§
20
* *
0 0
l S st e e t e e ol t t
ev
e e e e
ev
ro S
nt LP ni ev ev is ntr LP nis ev ev nis ev ev
Co go br br on br . br Co go .br .br go .br B.
br
B. . .
g t a B B t a B
B B +
ta ta +B an led a n S e d
an ed an d R4 kil 4 LP ill
4 ill 4 PS lle TL at- LR t-k
R - k R L -k
i
He +T Hea
TL at TL at LP
S
S+
He P S+ he LP
L
S+
LP

Figure 7. Effect of B. breve on cell viability and proliferation in the coculture models constructed with HT-29/B6 or T84 IECs in the upper com-
partment and 3D PBMCs in the lower compartment, placed on the bottom of culture plates. The apical surface of the HT-29/B6 or T84 cells was
incubated with B. breve and heat-killed B. breve (bacteria:cell ratio of 10:1) in the presence or absence of basolaterally applied LPS (100 ng/ml) or
TLR4 antagonist (10 ng/ml) for 48 h. After incubation, the cell-viability percentages of the HT-29/B6 and T84 cells as well as PBMCs (A) and the
proliferation of the HT-29/B6 and T84 cells (B) were assessed by WST-1 assays and the measurement of DNA synthesis, as described in Materials
and Methods. With respect to proliferation, a significant response was observed only when the T84 cells were stimulated with LPS (P⬍0.05). The
TLR4 antagonist, B. breve, and heat-killed B. breve abolished the LPS-induced proliferation (P⬍0.05). Note the absence of a T84 proliferation re-
sponse when the T84 cells were treated with B. breve or heat-killed B. breve. The results are shown as the mean ⫾ sem of four separate
experiments.*P ⬍ 0.05 versus control; §P ⬍ 0.05 versus LPS alone.

sion of the nuclear NF-␬B p50/p65 subunits and MyD88. Ac- quently leading to the inhibition of LPS-induced NF-␬B activa-
cordingly, the B. breve- and LGG-mediated inhibition of the tion and promoter activity [36, 49]. From these results, we can-
LPS-induced histone acetylation was associated with decreased not rule out the possibility that the activation of the IL-23/
expression of the NF-␬B-dependent luciferase reporter gene, IL-17 axis may also be a result of an increase in viability (or
which is reflected by the decreased, LPS-stimulated production proliferation) of cells upon LPS challenge or that the inhibi-
of IL-17, IL-23, and CD40, as well as proliferation of HT-29/B6 tion of the IL-23/IL-17 axis by B. breve involves the attenuation
and T84 cells. These data provide evidence that B. breve and of the LPS-induced proliferation of cells.
LGG repress conserved inflammatory pathways via the inhibi- As there is a reciprocal interplay between histone acetylation
tion of NF-␬B activity and by specifically inhibiting the access and DNA methylation in the regulation of proinflammatory
of NF-␬B to the promoters of cytokine genes, thereby alleviat- cytokines and as LPS induces the proinflammatory IL-1 and
ing inflammatory responses. In agreement with our results, TNF-␣ cytokines by the molecular inhibition of DNA methyl-
others [48, 49] have shown that under inflammatory condi- ation, while enhancing histone acetylation [5], we further in-
tions, the inflammation-alleviating effects of probiotic bacteria vestigated the effects of B. breve and LGG on DNA methylation
are greater than those observed in basal conditions and are in IECs. These bacteria enhanced DNA methylation slightly in
specific for LPS-induced NF-␬B activation in IECs. untreated cells and abolished the LPS-reduced DNA methyl-
As LPS stimulates the proliferation of human IECs [59, 68, ation. The inhibition of the LPS-mediated reduction of DNA
69] through TLR4, which is important for intestinal epithelial methylation by B. breve and LGG may be explained by B. breve/
repair from injury by aiding in proliferation and protecting LGG-mediated folate production [70], which enhances DNA
against apoptosis [68], we also evaluated cell proliferation. In- methylation [71].
deed, the B. breve-mediated inhibition of LPS-induced prolifer- Based on our findings, B. breve and LGG alleviate the patho-
ation of T84 cells, in the absence of cytotoxic effects, corre- genic factors for IBD by lowering LPS-induced NF-␬B activity
lated well with the B. breve-mediated inhibition of LPS-induced and by decreasing LPS-induced histone modifications. The ca-
histone acetylation and NF-␬B-dependent luciferase reporter pacity of these probiotics to attenuate the expression of IL-23,
gene activity. This effect is presumably a result of the inhibi- IL-17, and CD40, which are causes and risk factors of IBD, at
tion of LPS-mediated histone modifications [60 – 62], subse- the epigenetic level may have two explanations. First, B. breve

908 Journal of Leukocyte Biology Volume 92, October 2012 www.jleukbio.org

 Ghadimi et al. Epigenetic regulation of the IBD-causing IL-23/IL-17 axis by commensal probiotics
and LGG repressed LPS-induced histone acetylation and NF-␬B
activity. This may occur as a result of the close and positive
interplay/correlation between histone acetylation/phosphory-
lation status and NF-␬B transcriptional activity. Indeed, NF-␬B
binding to transcriptionally active chromatin is influenced by
the status of histone acetylation/phosphorylation, as well as
the signaling cascades that impact the levels of histone acetyla-
tion and thereby control chromatin remodeling and gene ex-
pression [22, 54]. Second, we observed that B. breve and LGG
inhibited the LPS-induced degradation of IRAK1 and I␬B␣
and interestingly, the p-p38 MAPK and p-MEK1, which are key
direct and indirect upstream activators of the NF-␬B pathway,
and subsequent cytokine gene expression in vitro and in vivo
[50, 53, 57]. Principally, the p38 MAPK and MEK1 inhibitors
block the phosphorylation/acetylation of histones and proin-
flammatory cytokines, whereas LPS stimulates the cytoplasmic-
to-nuclear translocation of NF-␬B by inducing the degradation
of I␬B␣ and p-p38 MAPK and p-MEK1. These activities con-
tribute to the LPS-induced NF-␬B activation that is correlated
with the p-Ac-H3/H4 and the subsequent production of proin-
flammatory cytokines in pathogen bacteria-infected endothelial
cells [19, 22]. Interestingly, our time-course experiment re-
vealed that the B. breve-mediated inhibition of IL-17 and IL-23
is not restricted to the 48-h incubation period but was main-
tained even after 96 h of incubation. This reflects the B. breve/
LGG-mediated inhibition of histone acetylation and slight in-
duction of DNA methylation, as the nature of epigenetic regu-
lation is essentially a long-lasting modulation of gene
regulation. Based on these data, reasonable molecular mecha-
nisms underlying the inhibitory effects of B.breve and LGG on
LPS-induced histone acetylation are the inhibition of the nu-
clear translocation of NF-␬B, achieved by decreasing the ex-
pression of MyD88, the degradation of IRAK-1 and I␬B␣, and
particularly, the expression of the nuclear NF-␬B p50/p65 sub-
units, as well as a decrease in p-p38 MAPK and p-MEK1, all of
which are important and ultimate mediators for LPS-induced
histone acetylation [18 –20, 52, 62, 72]. Indeed, in LPS-stimu-
lated cells, transcription factors, such as NF-␬B, are activated
and translocate to the nucleus, bind DNA, and subsequently
recruit coactivator molecules, such as CBP, p300, general con-
trol nonderepressible 5, and p300/CBP-associated factor.
These coactivator molecules are part of multiprotein com-
plexes that are recruited by DNA-bound transcription factors
to the transcription start site and act as molecular switches.
When transcription factors bind to these coactivator mole-
cules, intrinsic histone acetyltransferase activity is triggered on
these proteins, resulting in acetylation on core histone pro-
teins [24].
Although we did not include pathogenic bacteria in our ex-
periments, it seems plausible that the observed effects of B.
breve and LGG are specific because pathogenic bacteria (e.g.,
Helicobater pylori) have been shown to aberrantly activate the
IL-23/IL-17 axis in H. pylori-related gastritis [1] and induce the
p-Ac-H3/H4 and proinflammatory cytokines (e.g., IL-8) in en-
dothelial cells [18 –20, 73]. Moreover, the molecular induction
of histone acetylation and the inhibition of DNA methylation
lead to a massive increase in the LPS-stimulated IL-1␤ expres-
sion and an increase in the basal production of TNF-␣ [74].
www.jleukbio.org
Considering the functional similarities between the in vivo
situation and the in vitro model used, our experiment may be
regarded as somewhat artificial. For example, the model does
not include the intraepithelial lymphocyte component, the
sensing by the luminal processes of the DCs, or the trafficking
of the bacterial components by the M cells. Therefore, extrap-
olations to in vivo effects must be considered with caution.
However, even more simple in vitro models have been shown
to predict in vivo results [75]. Regardless, these results support
the suggestion that therapeutic interventions aimed at sup-
pressing the IL-23/IL-17 axis may be beneficial in IBD [3, 5,
76] and complement the in vitro and in vivo observations of
the anti-inflammatory effects of the LAB, as B. breve and LGG
attenuated the IL-23/IL-17 axis in the LPS-stimulated IECs.
The reduction in histone modifications coinciding with the
induction of DNA methylation suggests that this anti-inflamma-
tory effect is, to some extent, mediated by epigenetic pro-
cesses.
In conclusion, using a 3D coculture system composed of
IECs and PBMCs as an in vitro model of the intestinal mucosal
immune system, we have described a novel regulatory mecha-
nism by which commensal probiotics inhibit the NF-␬B-medi-
ated transcriptional activation of inflammatory genes (IL-23,
IL-17, and CD40), thereby reducing histone acetylation and
simultaneously enhancing DNA methylation in LPS-treated
IECs.
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KEY WORDS:
bacteria 䡠 histone modification 䡠 inflammation 䡠 cytokines
Volume 92, October 2012 Journal of Leukocyte Biology 911