Published December 12, 2014
Effects of Lactobacillus plantarum 2142
and sodium n-butyrate in lipopolysaccharide-triggered inflammation:
Comparison of a porcine intestinal epithelial cell line and primary hepatocyte
monocultures with a porcine enterohepatic co-culture system1,2
O. Farkas,*3 G. Mátis,† E. Pászti-Gere,* O. Palócz,* A. Kulcsár,† J. Petrilla,† Gy. Csikó,*
Zs. Neogrády,† and P. Gálfi*
*Department of Pharmacology and Toxicology, Faculty of Veterinary Sciences,
Szent István University, István u. 2, Budapest 1078, Hungary; and †Department of Physiology
and Biochemistry, Faculty of Veterinary Sciences, Szent István University; István u. 2, Budapest 1078, Hungary
ABSTRACT: This study was based on our previously
developed double-layered enterohepatic co-culture system, composed of nontumorigenic porcine intestinal
epithelial cell line (IPEC-J2) and primary culture of porcine hepatocytes. The anti-inflammatory effect of spent
culture supernatant of Lactobacillus plantarum 2142
(Lp2142; 13.3%) and sodium n-butyrate (2 mM) was
tested on IPEC-J2 and hepatocyte monocultures as well
as on the gut–liver co-culture. To mimic inflammation,
lipopolysaccharide (LPS; 1 and 10 μg/mL) was applied.
Production of IL-8 and IL-6 was measured as a marker
of inflammatory responses. The paracellular permeability
of the intestinal epithelium was also monitored by fluoresceinisothiocyanate-labeled dextran 4 assay. Significant
increase of IL-8 concentration was observed in the
IPEC-J2 monoculture (P < 0.01) while the level of IL-6
was not changed following LPS treatment. Concentration
of IL-8 and IL-6 was grown significantly in hepatocyte
monocultures (P < 0.05 and P < 0.001) as well as in the
co-culture after 10 μg/mL LPS treatment (P < 0.001 and
P < 0.001). One microgram per milliliter LPS caused
elevated IL-8 level in the co-culture (P < 0.001) and in
the hepatocyte monoculture (P < 0.01), while it caused
increased IL-6 level only in the hepatocytes (P < 0.001).
Production of IL-8 was significantly decreased by butyrate in case of 1 μg/mL as well as 10 μg/mL LPS exposure in the co-culture (P < 0.001). Application of butyrate also reduced IL-6 level in the co-culture after 10 μg/
mL LPS treatment (P < 0.01). Lactobacillus plantarum
2142 decreased IL-8 level after incubation with 1 μg/
mL LPS (P < 0.001), while in case of 10 μg/mL LPS
treatment only a marginal lowering in IL-8 (P = 0.064)
release was measured. The IL-6 concentration was significantly reduced (P < 0.01 in case of 1 μg/mL LPS
treatment) by Lp2142 in the co-culture. Contrarily, the
elevated IL-8 and IL-6 level of hepatocytes has not been
reduced in case of either butyrate or Lp2142 addition.
The enterohepatic co-culture model offers a possibility
for fast and reliable screening of new candidates against
enteric inflammation, which are of special interest in porcine medicine and health management. According to our
results, Lp2142 and butyrate both seem to be effective as
anti-inflammatory agents in LPS-triggered inflammatory
response, tested in the gut–liver co-culture model.
Key words: butyrate, enterohepatic co-culture, inflammation, intestinal
porcine jejunal epithelial cell line J2, Lactobacillus plantarum, porcine/pig
© 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:3835–3845
doi:10.2527/jas2013-7453
INTRODUCTION
1The
research described here was supported by the Hungarian
Scientific Research Fund (OTKA grant no. 100701).
2We are indebted to Jody Gookin (Department of Clinical Sciences,
College of Veterinary Medicine, North Carolina State University, Raleigh,
NC) for providing the IPEC-J2 cells. We thank for Tamás Reibling for
providing the animals used for hepatocyte isolation.
3Corresponding author: Farkas.Orsolya@aotk.szie.hu
Received November 29, 2013.
Accepted June 13, 2014.
The intestinal epithelium serves as a primary physical barrier against invading bacteria, toxins, and various chemical agents; however, it also plays an active
role in the maintenance of absorption and immune
response (Pitman and Blumberg, 2000). Gut-derived
infections may be triggered by the lipopolysaccharide
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Farkas et al.
(LPS) release from gram-negative bacteria. Effective
treatment of LPS-provoked intestinal inflammation is
essential in modern livestock animal herding, and it may
be improved by the application of newly developed alternatives such as probiotics in porcine medicine as well
(Rakhshandeh and de Lange, 2012).
Several cell culture models were established to mimic
in vivo conditions of the gut enabling studies on LPSevoked intestinal and systemic inflammatory responses. In
cultures, prepared from specific cell types, however, lacks
the metabolic complexity of interactions among various
tissues and organs. A new, enterohepatic co-culture system
has been developed by our research group (Paszti-Gere
et al., 2014), composed of noncancerous porcine intestinal epithelial cell line (IPEC-J2) and primary porcine
hepatocytes. This model was found to be a suitable tool
to describe the crosstalk along the gut–liver axis in vitro
and for the investigation of gut-related inflammatory processes by mimicking in vivo enterocyte–hepatocyte communication in pigs. Our further aim was to investigate the
anti-inflammatory effect of certain probiotic microbes and
their single microbial products (i.e., butyrate) on IPEC-J2
and hepatocyte monocultures as well as on the gut–liver
co-culture exposed to LPS treatment. Proinflammatory
cytokine production was monitored as a marker of inflammatory response, and the paracellular permeability of the
intestinal epithelium was also assessed by fluoresceinisothiocyanate-labeled dextran 4 assay. Our co-culture model
can be applied for fast and reliable screening of new alternatives against enteric inflammation, which are of special
interest in porcine medicine and health management.
MATERIALS AND METHODS
The Porcine Intestinal Epithelial
Cell Line and Culture Conditions
The nontransformed IPEC-J2 cell line, originally isolated from jejunal epithelia of a neonatal unsuckled piglet (Schierack et al., 2006), was a kind gift of J. Gookin,
Department of Clinical Sciences, College of Veterinary
Medicine, North Carolina State University, Raleigh,
NC. The IPEC-J2 cells were grown and maintained in
complete medium, which consisted of a 1:1 mixture of
Dulbecco’s Modified Eagle’s Medium and Ham’s F-12
Nutrient Mixture (plain medium) supplemented with
5% fetal bovine serum (FBS), 5 μg/mL insulin, 5 μg/
mL transferrin, 5 ng/mL selenium, 5 ng/mL epidermal
growth factor, and 1% penicillin–streptomycin (all from
Fisher Scientific, Fisher Scientific, Loughborough, UK).
Cells were grown at 37°C in a humidified atmosphere
of 5% CO2. Cell cultures were tested by PCR and they
were found to be free of Mycoplasma contamination.
For the experiments, IPEC-J2 cells between passages
42 and 48 were seeded onto 6-well Transwell Polyester
membrane inserts (Corning Inc., Corning, NY), the latter
coated with 8 μg/cm2 rat tail collagen type I (Sigma-Aldrich,
Steinheim, Germany), at a density of 1.5 × 105 cells/mL
(the volume of complete medium was 1.5 mL on the apical
side and 2.5 mL on the basolateral side per well according
to the manufacturer’s instructions). Cells were allowed to
adhere for 24 h before being washed and re-fed every other
day until confluence was reached. Transepithelial electrical resistance (TER) measurement of monolayers was performed on alternate days after seeding, from d 5 to 21 of
culture, using an EVOM Epithelial Tissue Volt/Ohmmeter
(World Precision Instruments, Berlin, Germany).
Bacterial Strains and Spent Culture Supernatants
Lactobacillus plantarum 2142 (Lp2142; culture collection of the Institute of Dairy Microbiology,
Agricultural Faculty of Perugia University, Perugia,
Italy), L. plantarum 299v (culture collection of University
Utrecht, Department of Pathology, Faculty of Veterinary
Medicine, Utrecht University, Utrecht, Netherlands),
and Lactobacillus casei Shirota (culture collection of
University Utrecht, Department of Pathology, Faculty
of Veterinary Medicine, Utrecht University, Utrecht,
Netherlands) were grown in DeMan, Rogosa, and Sharpe
(MRS) broth. Bacillus licheniformis (Norel Animal
Nutrition, Madrid, Spain) was cultured in lysogeny broth
(10 g peptone, 5 g yeast extract, and 10 g NaCl suspended
in distilled water) at 37°C. Inoculation was accomplished
with a stationary culture of probiotical strain (1% inoculum) and the bacteria were grown for 24 h at 37°C. They
were subcultured at least twice before experiments. Spent
culture supernatants (SCS) were prepared by centrifugation of the bacterial suspension (with final bacterial concentrations of 109 cfu/mL) at 3,000 × g at 5°C for 10 min.
Centrifuged culture supernatant was then passed through
a sterile 0.22 μm pore size filter unit.
Sodium n-butyrate was purchased from SigmaAldrich (Steinheim, Germany), and it was diluted in cell
culture medium at a concentration of 2 mM.
Treatment of the Porcine Intestinal
Epithelial Cell Line Monoculture
Before treatment, the confluent monolayers of
IPEC-J2 cells were washed with plain medium. The applied LPS was derived from Salmonella enterica serovar
Typhimurium (Sigma-Aldrich, Steinheim, Germany).
Lipopolysaccharide solutions were prepared freshly before each experiment and added in plain medium at 1
and 10 μg/mL on the apical side of the IPEC-J2 layer
(Paszti-Gere et al., 2014). To monitor the potential ben-
Inflammation on pig enterohepatic co-culture
eficial effect of selected probiotics, IPEC-J2 cells were
treated with certain SCS (13.3 % vol/vol) simultaneously
with LPS administration. Previous experiments showed
that the maximal protection against 1 mM H2O2–triggered inflammation was achieved using 13.3 % vol/vol
of SCS derived from several lactobacilli (Paszti-Gere
et al., 2012b). Applied concentration of sodium n-butyrate (Sigma-Aldrich, Steinheim, Germany) was 2 mM
(Paszti-Gere et al., 2013). After the incubation with LPS
(1 h), cells were washed with plain medium and cultured
for additional 1 h before being subjected to the PCR procedure. Twenty-four hours after LPS and SCS/butyrate
treatment, ELISA was performed. Measurements of TER
were performed both before and after the LPS treatment.
Paracellular Permeability Assessment
The IPEC-J2 cells were plated to confluence on
6-well polyester membrane inserts without collagen
coating and they were allowed to form differentiated
monolayers. Lipopolysaccharide was added at 1 or 10
μg/mL concentration for 1 h and TER measurements
were performed before 4 and 24 h after LPS administration. In parallel to LPS, fluorescein isothiocyanatedextran 4 kDa (FD4; obtained from Sigma-Aldrich,
Munich, Germany) was added at 1 mg/mL to the apical
compartment of IPEC-J2 cell monoculture with different incubation times (4 and 24 h). Changes in paracellular permeability were also quantified with application
of Lp2142 SCS at 13.3% and sodium n-butyrate at 2 mM
simultaneously with LPS treatment. Samples of media
from the basolateral chambers were collected and tracer
concentration was quantified by fluorescence at 485 nm
excitation and 535 nm emission (Victor X2 2030 fluorometer; PerkinElmer, Waltham, MA).
Isolation and Cultivation of Hepatocytes
Hepatocyte isolation was performed according to a
modified protocol of (Puviani et al., 1998; Meng et al.,
2010). Livers were obtained from clinically healthy male
pigs of the Large White breed, weighing approximately
15 kg (Dunahyb, Szekszárd, Hungary). All procedures
were conducted in accordance with international and
national laws and institutional guidelines and approved
by the Local Animal Test Committee of the Faculty of
Veterinary Science, Szent István University, Budapest,
Hungary (permission number: 22.1/912/003/2009).
Animals were anesthetized by intramuscular application of the combination of xylazine (2 mg/kg BW) and
ketamine (20 mg/kg BW). After aseptic opening of the
abdominal cavity by a midline incision, the liver was
excised and the caudate lobe was isolated and exsanguinated by 300 mL chilled EDTA-containing buffer. The
3837
perfusion was continued with 300 mL prewarmed (37°C)
EDTA-free buffer solution. Finally, 100 mL of this buffer,
supplemented with 100 mg collagenase Type IV and 4.8
mM CaCl2, was applied and recirculated to disintegrate
the hepatocytes. Before application for perfusion, all
solutions were oxygenated with carbogen (95% O2 and
5% CO2 at 1 L/min). Velocity of the perfusion was set
to 100 mL/min. After collagenase digestion, the capsule
was disrupted and the digested parenchyma was filtered
through a nylon mesh with 100 μm pore size (Millipore,
Volketswil, Switzerland) to eliminate cell aggregates.
Hepatocyte-enriched fractions were isolated and
washed by low-speed centrifugation (50 × g for 75 s and at
room temperature) 3 times in Williams’ Medium E supplemented with 10,000 IU/mL penicillin, 10 mg/mL streptomycin, 2 mM glutamine, 10% FBS, and 0.2 IU/mL insulin.
Cell viability, assessed by trypan blue exclusion, exceeded
90% in all preparations. Yield of hepatocytes was determined by cell counting in a Bürker chamber and cell concentration was adjusted to 4 × 105/mL. Hepatocytes (1.5
mL cell suspension/well) were seeded on 6-well Costar
TC cell culture dishes (well diameter: 34.8 mm; Corning
International, Corning, NY) and coated with collagen Type
I (10 μg/cm2) according to the manufacturer’s instructions.
Cell cultures were incubated at 37°C in humid atmosphere
with 5% CO2 and culture medium was changed 4 h after
plating. Confluent monolayer of hepatocytes was gained
after a 24-h incubation.
Treatment of Hepatocyte Monocultures
Twenty-four hours after isolation, culture medium
of hepatocytes was changed to serum-free Williams’
Medium E and treated with LPS (1 and 10 μg/mL) and
SCS for 1 h. Medium was collected and hepatocytes
were harvested after a 24-h incubation.
Preparation of Double-Layered Co-culture System
The IPEC-J2 cells were used for experiments 21 d
after plating when a confluent monolayer was formed
expressing high TER values (>1,200 Ω). A doublelayered co-culture system (24 h after hepatocyte culturing) was prepared in 6-well plates by placing inserts
with confluent IPEC-J2 layer over the confluent layer of
primary hepatocytes. The volume of the applied culture
medium was 1.5 mL on the apical side (serum-free plain
medium for IPEC-J2) and 2.5 mL on the basolateral side
(Williams’ Medium E). After 1 h adaptation, LPS (1 and
10 μg/mL) was added either with Lp2142 SCS or with
sodium n-butyrate to the apical side. After 1 h further incubation, medium from the apical side was replaced by
serum-free plain medium. Enterocytes and hepatocytes
were harvested after 24 h and kept separate.
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Farkas et al.
Quantitative Real-Time PCR
After 1 h LPS treatment and 1 h further incubation,
culture medium was removed and 1 mL of ice-cold
TRIzol reagent (Invitrogen, Carlsbad, CA) was added
to the monoculture of IPEC-J2 cells. Samples were collected and kept at –80°C until further processing. Total
RNA was isolated from the cells according to the manufacturer’s instructions. To prevent DNA contamination, the isolated RNA (2 μg) was treated with AMP-D1
DNase I (Sigma Aldrich, Steinheim, Germany). Quantity
and A260:A280 and A260:A230 ratios of the extracted
RNA were determined using a NanoDrop ND-1000
Spectrophotometer (Thermo Scientific, Wilmington,
DE). Quality and quantity control of the isolated RNA
was performed both before and after the deoxyribonuclease treatment.
Synthesis of the first strand of cDNA from 1 g of total RNA was achieved using RevertAid H Minus First
Strand cDNA Synthesis Kit (Fermentas, St. Leon-Roth,
Germany) according to the manufacturer’s recommendations, using the random hexamer as a priming method. Quantitative real-time PCR (qRT-PCR) was performed using the iQ SYBR Green Supermix kit (BioRad,
Hercules, CA) on the MiniOpticon System (BioRad). The
cDNA was diluted 5-fold before equal amounts were
added to duplicate qRT-PCR reactions. Tested genes of
interest were IL-8, TNF-α, and TLR-4 (Toll-like receptor
4). Hypoxanthine phosphoribosyl transferase (HPRT)
and Cyclophilin-A (CycA) were used as reference genes.
The primer sequences were the following: IL-8 forward
5′-AGAGGTCTGCCTGGACCCCA-3′ and reverse
5′-GGGAGCCACGGAGAATGGGT-3′
(Paszti-Gere
et al., 2012a), TNF-α forward 5′-TTCCAGC­TGGCC­
CCTTGAGC-3′ and reverse 5′-GAGGGCATTG­
GCATACCCAC-3′ (Hyland et al., 2006), TLR-4 forward 5′-CTCTGCCTTCACTACAGAGA-3′ and reverse
5′-CTGAGTCGTCTCCAGAAGAT-3′ (Moue et al., 2008),
HPRT forward 5′-GGACTTGAATCATGTTTGTG-3′
and reverse 5′-CAGATGTTTCCAAACTCAAC-3′
(Hyland et al., 2006), and CycA forward 5′-GCG­
TCTCCTTCGAGCTGTT-3′ and reverse 5′-CCAT­
TATGGCGTGTGAAGTC-3′ (Nygard et al., 2007). For
each PCR reaction, 2.5 μL cDNA was added directly to a
PCR reaction mixture, set to a final volume of 25 μL, containing 1x concentrated iQ SYBR Green Supermix and
0.2 μM of the appropriate primers. The thermal profile for
all reactions was 3 min at 95°C and then 40 cycles of 20 s
at 95°C, 30 s at 60°C, and 30 s at 72°C. At the end of each
cycle, the fluorescence monitoring was set for 10 s. Each
reaction was completed with a melting curve analysis to
ensure the specificity of the reaction. To determine the efficiencies of the PCR reactions, standard curves were obtained for each target and reference gene, using serial dilutions of a reference cDNA. Real-time PCR efficiencies
(E) were calculated according to the equation E = 10 (–1/
slope). To determine the stability of the reference genes,
the geNorm (version 3.5; geNorm: downloaded from
http://medgen.ugent.be/~jvdesomp/genorm/) was used.
Cytokine Measurement by EnzymeLinked Immunosorbent Assay
After treatment, IPEC-J2 monocultures, hepatocyte
monocultures, and double-layered co-cultures were incubated for 24 h. Culture media were collected, centrifuged (245 × g for 10 min at room temperature), and diluted (5-fold) to measure the IL-8 concentrations. Level
of IL-8 and tumor necrosis factor α (TNF-α) secretion
(pg/mL) was determined by porcine-specific ELISA Kits
(Invitrogen) according to the manufacturer’s instructions.
Concentration of IL-6 was also determined by porcinespecific IL-6 ELISA Kit (Abcam, Cambridge, UK).
Statistical Analysis
Relative gene expression levels of the genes of
interest were calculated by the Relative Expression
Software Tool (REST) 2009 software (Qiagen GmbH,
Hilden, Germany). Statistical analysis of other data
was performed with R 2.14.0 software (Foundation for
Statistical Computing, Vienna, Austria). Differences
were considered significant if the P-value was <0.05.
One-way ANOVA and Tukey’s honest significant difference method, as post hoc tests, were performed to test
the differences between treatment groups.
RESULTS
Preliminary Anti-inflammatory Activity Test of Spent
Culture Supernatants and Sodium n-Butyrate
To select the most promising probiotic strains, prior test of anti-inflammatory activity of bacterial SCS
(13.3 % vol/vol) and sodium n-butyrate (2 mM) was
performed. Relative expression of IL-8 gene was determined in case of IPEC-J2 cells exposed to LPS (10 μg/
mL), and increased expression of IL-8 after 1 h LPS treatment was observed. After simultaneous treatment with
LPS and Lp2142 SCS, the relative gene expression of
IL-8 was significantly decreased (P < 0.05). Supernatant
of L. plantarum 299v and L. casei Shirota did not reduce
the IL-8 mRNA level significantly compared to the LPStreated controls. Incubation of IPEC-J2 cells with the
SCS of B. licheniformis resulted significant upregulation
of IL-8 gene. The IL-8 mRNA level was almost 40-fold
(39.2 ± 13.02) of the untreated control cells and 12-fold
of the LPS-treated samples. Spent culture supernatant of
B. licheniformis per se caused also a significant increase
Inflammation on pig enterohepatic co-culture
in the relative gene expression level of IL-8. There was
no significant change in the IL-8 mRNA level after simultaneous treatment of enterocytes with LPS and growth
media of probiotic bacteria. Sodium n-butyrate exposure (2 mM) led to significant decrease of IL-8 relative
gene expression. Significant reduction in the TNF-α level
was observed when Lp2142 SCS was added to IPEC-J2
cells exposed to 10 μg/mL LPS (P < 0.05). Incubation
cultured enterocytes with sodium n-butyrate (2 mM) also
decreased relative expression of TNF-α significantly (P <
0.05). No significant difference was found in TNF-α expression after treatment of SCS from L. plantarum 299v
and L. casei Shirota, respectively. Bacillus licheniformis
SCS caused a substantial increase in TNF-α relative gene
expression level per se and in LPS-treated samples. There
was no alteration in the mRNA level of TLR-4 after exposed to 10 μg/mL LPS for 1 h.
Considering the above mentioned results, Lp2142
SCS and sodium n-butyrate were selected as test compounds in the following experiments, including monoand co-culture studies.
Studies on the Porcine Intestinal
Epithelial Cell Line Monoculture
Stimulation of IPEC-J2 monoculture with LPS (1 and
10 μg/mL; 1 h) increased IL-8 release into the basolateral compartment (P = 0.002 and P < 0.001, respectively).
Besides LPS treatment, enterocytes were exposed simultaneously to probiotic Lp2142 SCS or sodium n-butyrate,
respectively. At 2 mM sodium n-butyrate application, concentration of IL-8 was significantly decreased compared to
both 1 and 10 μg/mL LPS-treated enterocytes (P = 0.007
and P < 0.001, respectively). Supernatant of Lp2142 also
decreased IL-8 concentration compared to the LPS-treated
enterocytes by attenuating inflammatory effect of LPS (P
< 0.001 and P < 0.001, respectively). The effect of Lp2142
SCS was independent on the LPS concentration in the 1
to 10 μg/mL range (see Fig. 1). Level of IL-6 remained
under the detection limit in case of control cells and this
phenomenon did not changed after LPS treatment. Level of
TNF-α was also determined, but TNF-α protein could be
not detected in LPS-treated IPEC-J2 cells after 24 h.
Partial disruption of IPEC-J2 monolayer integrity as
a result of LPS treatment (1 and 10 μg/mL; 1 h) was observed when measured by the paracellular transport of FD4
tracer. After 4 h incubation (Fig. 2), the FD4 fluorescence
intensity on the basolateral chamber was significantly increased in the LPS-treated cultures compared to the untreated samples (1 μg/mL LPS treatment: P = 0.011 and 10
μg/mL LPS treatment: P < 0.001). After 24 h (see Fig. 3),
the FD4 fluorescence intensity increased only on the basolateral chamber of the higher dose of LPS treatment (1 μg/
mL LPS treatment: P = 0.711 and 10 μg/mL LPS treatment:
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Figure 1. Concentration of IL-8 in the basolateral medium of intestinal
porcine jejunal epithelial cell line J2 monocultures exposed to lipopolysaccharide (LPS) treatment (at 1 and 10 μg/mL; 1 h). Effect of Lactobacillus
plantarum 2142 and butyrate on the IL-8 protein levels (n = 3/group; **P <
0.01; ***P < 0.001). Data are shown as means + SEM. CTR = control;
LPS1 = 1 μg/mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM sodium n-butyrate; LP = 13.3% Lactobacillus plantarum.
P = 0.041). This LPS-elevated basolateral FD4 fluorescence intensity was not altered significantly by simultaneous 1 h treatment of IPEC-J2 cells with Lp2142 SCS in addition to LPS administration. Similar results were obtained
for FD4 values after incubation with LPS (10 μg/mL) and
sodium n-butyrate at the same time. Transepithelial electric resistance values were also measured before and after
LPS treatment to check the integrity of polarized IPEC-J2
monolayer. It was ascertained that neither LPS nor test
compounds (Lp2142 SCS and sodium n-butyrate) significantly affect TER values (data not shown).
Studies on Hepatocyte Monoculture
Significant increase in IL-8 concentration was observed in the hepatocyte monoculture medium 24 h after incubation with 1 and 10 μg/mL LPS, respectively, compared
to untreated cells (P = 0.008 and P = 0.011, respectively).
Addition of sodium n-butyrate to the culture medium did
not reduce the IL-8 level either in case of 1 μg/mL or in
case of 10 μg/mL LPS treatment. Lactobacillus plantarum
2142 SCS did not decrease IL-8 concentration after incubation with LPS (see Fig. 4). Moreover, Lp2142 SCS and
10 μg/mL LPS treatment caused a significant increase of
IL-8 compared to the 10 μg/mL LPS exposure (P < 0.05).
Significant increase of IL-6 level was measured in the
hepatocyte monoculture medium 24 h after incubation with
1 and 10 μg/mL LPS, respectively (P < 0.001 in each case).
Sodium n-butyrate addition did not reduce the IL-6 level
either in case of 1 μg/mL or in case of 10 μg/mL LPS treatment. The same effect could be observed after Lp2142 SCS
addition (see Fig. 5). The simultaneous administration of
butyrate and 10 μg/mL LPS caused significant increase in
IL-6 level compared to the LPS (10 μg/mL)-treated cells.
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Farkas et al.
Figure 2. Penetration of the fluorescein isothiocyanate-dextran 4 kDa
from the apical to the basolateral compartment of intestinal porcine jejunal
epithelial cell line J2 cells as a result of lipopolysaccharide (LPS) treatment (at 1 and 10 μg/mL; 1 h treatment time; detection after 4 h). Effect of
Lactobacillus plantarum 2142 and butyrate on the paracellular permeability
(n = 6/group; *P < 0.05; ***P < 0.001). Data are shown as means + SEM.
CTR = control; LPS1 = 1 μg/mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM
sodium n-butyrate; LP = 13.3% Lactobacillus plantarum.
Studies on the Double-Layered Co-culture
To check the integrity of the double-layered co-cultures, TER values of the IPEC-J2 cells were measured.
Co-culture experiments were performed with confluent
polarized IPEC-J2 cells with high TER values after 24 h
of co-cultivation. Transepithelial electric resistance of
control and 1 μg/mL LPS-exposed cells were 1,360 ±
467 and 1,329 ± 477 Ω, respectively. Transepithelial
electric resistance value of 10 μg/mL LPS-treated cells
was 1,572 ± 486 Ω, proving that integrity of the polarized IPEC-J2 monolayer was not altered.
After incubation with LPS (1 and 10 μg/mL), IL-8
protein concentration was increased in the culture media
in the basolateral compartment of the co-culture (P < 0.001
and P < 0.001, respectively). Spent culture supernatant of
Lp2142 per se caused a significant IL-8 release into the
culture medium (P = 0.031). Concentration of IL-8 after
sodium n-butyrate treatment was significantly decreased
in case of 1 μg/mL as well as 10 μg/mL LPS treatment
(P < 0.001 and P < 0.001, respectively). Lactobacillus
plantarum 2142 reduced IL-8 protein level after incubation with 1 μg/mL LPS (P < 0.001), while in case of
10 μg/mL LPS treatment, only a marginal decrease in IL-8
release was measured (P = 0064; see Fig. 6).
After 1 μg/mL LPS incubation, no significant difference in IL-6 level was detected in the basolateral compartment of the co-culture (see Fig. 7). Higher LPS concentration (10 μg/mL) caused increased IL-6 level (P <
0.001) in the basolateral culture media. Supernatant of
Lp2142 and sodium n-butyrate decreased IL-6 protein
level after incubation with 10 μg/mL LPS (P = 0.004
and P = 0.009, respectively).
Figure 3. Penetration of the fluorescein isothiocyanate-dextran 4 kDa
from the apical to the basolateral compartment of intestinal porcine jejunal epithelial cell line J2 cells as a result of lipopolysaccharide (LPS) treatment (at 1
and 10 μg/mL; 1 h treatment time; detection after 24 h). Effect of Lactobacillus
plantarum 2142 and butyrate on the paracellular permeability (n = 6/group;
*P < 0.05). Data are shown as means + SEM. CTR = control; LPS1 = 1 μg/
mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM sodium n-butyrate; LP = 13.3%
Lactobacillus plantarum.
DISCUSSION
Intestinal epithelial cells are active participants in the
gut immune response. They can mediate gut-derived systemic inflammatory processes through the production of
proinflammatory cytokines, such as IL-6 and IL-8, that are
crucial for the recruitment and activation of various immune cells (Pie et al., 2007). Lipopolysaccharide-evoked
inflammation is associated with weakened gut barrier integrity via enhancement of paracellular permeability to
macromolecules, causing increased absorption of certain
toxins as well as possible invasion of pathogenic bacteria
(Hanson et al., 2011). The elimination of LPS-provoked inflammation is of key importance in pig health management
(Rakhshandeh and de Lange, 2012; Mani et al., 2013).
In vitro models are ethically justified alternatives to
study gastrointestinal diseases. Most of the current intestinal models involve tumorigenic cell lines, which may
have less extrapolability to in vivo situation compared
to nontransformed cells. However, IPEC-J2, a noncarcinogenic swine jejunal epithelial cell line, was developed (Schierack et al., 2006) and it is increasingly used
as a porcine 3-dimensional (3D) small intestinal model.
Functional 3D gut models cultured on membrane inserts
with apical and basolateral compartments are capable of
exploring changes in inflammatory parameters and screening positive effects of probiotic bacteria or other components in vitro (Cencic and Langerholc, 2010). Substances,
including not only absorbed but nascent compounds such
as enterocyte-derived inflammatory mediators, travel first
from the small intestine via the portal vein to the liver.
Hepatic responses to the intestinal mediator release cover
enhanced production of proinflammatory cytokines and
acute-phase proteins (Morgan, 1997; Renton, 2004). Choi
Inflammation on pig enterohepatic co-culture
Figure 4. Concentration of IL-8 in the culture medium of primary hepatocyte monocultures exposed to lipopolysaccharide (LPS) treatment (at 1 and 10
μg/mL; 1 h). Effect of Lactobacillus plantarum 2142 and butyrate on the IL-8
protein levels (n = 4/group; *P < 0.05; **P < 0.01). Data are shown as means +
SEM. CTR = control; LPS1 = 1 μg/mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM
sodium n-butyrate; LP = 13.3% Lactobacillus plantarum.
et al. (2004) created a double-layered co-culture system
with dual compartments comprising monolayers of cultured human hepatocellular carcinoma (HepG2) cells and
human colorectal adenocarcinoma (Caco-2) cells grown
on a semipermeable membrane insert.
An animal-originated co-culture model, composed of
nontransformed intestinal and hepatic cells, was not available up to the present. A novel double-layered porcine
enterohepatic co-culture system consisting of IPEC-J2
intestinal epithelial cells and primary hepatocytes was
previously established to study LPS-triggered inflammatory responses. The model was characterized earlier by
measuring the TER of IPEC-J2 cells and by confirmation of hepatocellular albumin production (Paszti-Gere
et al., 2014). Lipopolysaccharide-induced inflammatory
response increased IL-8 mRNA level and protein release
in the IPEC-J2 monoculture as well as in the co-culture.
Due to the widespread appearance of resistance
amongst human pathogenic bacteria, there is a growing interest to replace the application of antibiotics by different
natural alternatives, such as probiotics. Recently, numerous
beneficial effects of probiotics in treatment of intestinal infections, disorders, and diseases have been reported.
Various Bacillus species are commercially available
in probiotic products in both human and veterinary medicine (Hong et al., 2005), but their mechanism of action
has not been yet fully understood. Several studies have
been completed in pigs using the BioPlus1 2B supplement (containing B. licheniformis and Bacillus subtilis spores; CHR Hansen, Hørsholm, Denmark), which
demonstrated improved sow and piglet performance
(Alexopoulos et al., 2004). Enterocytes pretreated with
B. licheniformis and co-cultured with S. enterica serovar
Typhimurium demonstrated that B. licheniformis inhibited S. enterica stimulated IL-8 secretion (Skjolaas et al.,
3841
Figure 5. Concentration of IL-6 in the culture medium of primary hepatocyte monocultures exposed to lipopolysaccharide (LPS) treatment (at 1 and 10
μg/mL; 1 h). Effect of Lactobacillus plantarum 2142 and butyrate on the IL-6
protein levels (n = 3/group; ***P < 0.001). Data are shown as means + SEM.
CTR = control; LPS1 = 1 μg/mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM
sodium n-butyrate; LP = 13.3% Lactobacillus plantarum.
2007). Contrarily, treatment of LPS-triggered IPEC-J2
cells with B. licheniformis SCS performed by us was not
effective to reduce IL-8 mRNA level (data not shown).
Ambiguous results have been obtained with the potential probiotic Lactobacillus reuteri, a porcine-derived
strain that was reported to have no effect on the IL-8
response of cultured enterocytes to Salmonella enterica
serovar Typhimurium infection (Skjolaas et al., 2007).
However, 3 human-derived L. reuteri strains were reported to decrease enteral IL-8 production after LPS exposure in vitro (Liu et al., 2010).
In the present study, lactobacilli were proved to be
effective in the treatment of intestinal inflammation on
our in vitro models. We demonstrated that SCS (13.3
% vol/vol) of Lp2142 was effective in decreasing LPSinduced IL-8 mRNA level in IPEC-J2 monoculture.
Suppression of IL-8 protein release into the basolateral
media after LPS exposure was observed not only on intestinal monoculture but on the gut–liver co-culture as
well. These results are in good agreement with a recent
study, revealing that Lp2142 as well as its SCS reduced
growth of Salmonella enterica serovar Typhimurium,
synthesis of IL-8 and heat shock protein 70 in undifferentiated crypt-like and differentiated villus-like Caco-2
cells (Nemeth et al., 2006).
The short chain fatty acid butyrate, released by probiotic bacteria, is one of the major end products of the
anaerobic microbial fermentation of carbohydrates in
the large intestine of monogastric mammals. Due to its
numerous beneficial properties such as health improvement and enhancement of growth performance in pigs
(Galfi and Bokori, 1990), butyrate is of special interest as
feed additive, mainly applied as its sodium salt. Butyrate
can act due to its selective antibacterial effect on enteral
3842
Farkas et al.
Figure 6. Concentration of IL-8 in the basolateral medium of intestinal
porcine jejunal epithelial cell line J2 primary hepatocyte co-cultures exposed to
lipopolysaccharide (LPS) treatment (at 1 and 10 μg/mL; 1 h treatment). Effect
of Lactobacillus plantarum 2142 and butyrate on the IL-8 protein levels (n =
4/group; ***P < 0.001; #P = 0.064). Data are shown as means + SEM. CTR =
control; LPS1 = 1 μg/mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM sodium
n-butyrate; LP = 13.3% Lactobacillus plantarum.
Figure 7. Concentration of IL-6 in the basolateral medium of intestinal
porcine jejunal epithelial cell line J2 primary hepatocyte co-cultures exposed to
lipopolysaccharide (LPS) treatment (at 1 and 10 μg/mL; 1 h treatment). Effect
of Lactobacillus plantarum 2142 and butyrate on the IL-6 protein levels (n =
3/group; **P < 0.01; ***P < 0.001). Data are shown as means + SEM. CTR =
control; LPS1 = 1 μg/mL LPS; LPS10 = 10 μg/mL LPS; SB = 2 mM sodium
n-butyrate; LP = 13.3% Lactobacillus plantarum.
pathogens, modulation of proliferation, and differentiation
and function of the gastrointestinal epithelium and by enhancing the barrier function and immune response of the
gut (Guilloteau et al., 2009). Orally administered sodium
butyrate exerts anti-inflammatory action as well and was
described to attenuate inflammation and mucosal lesions
in experimental acute ulcerative colitis (Vieira et al., 2012).
Anti-inflammatory effect was observed by us in this
study on IPEC-J2 monocultures after simultaneous LPS
and sodium n-butyrate (2 mM) treatment; that is, reduced IL-8 mRNA level and protein concentration were
detected. Attenuation of LPS-induced cytokine response
by butyrate treatment has been already observed in some
other cases as well. Sodium n-butyrate (1 and 3 mM, respectively) significantly reduced IL-8 protein concentration in the culture medium of LPS-stimulated HT-29
colon adenocarcinoma cells (Lee et al., 2005). Short
chain fatty acids including butyrate diminished TNF-α
and nitrogen monoxide production by LPS-stimulated
neutrophils, possibly mediated by its histone deacetylase activity (Vinolo et al., 2011). The effect of short
chain fatty acids on activation of nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB), a
pivotal transcription factor involved in the expression
of proinflammatory genes, was also investigated, where
propionate and butyrate attenuated the activation of NFκB by LPS.
Interleukin-6 is also an important factor involved in
the regulation of the acute-phase response to injury and
infection. Increase of IL-6 concentration in the co-cultures was detected by us after 10 μg/mL LPS treatment,
and attenuation of inflammatory response was observed
in case of Lp2142 SCS as well as butyrate addition. It
could be stated that the same trend could be observed that
in case of IL-8. Nevertheless, the level of IL-6 was more
difficult to follow up. In case of IPEC-J2 monoculture
samples, detection of IL-6 was not efficient. To mimic
intestinal inflammation, differentiated Caco-2 cells were
treated with a cocktail of proinflammatory substances (IL1, TNF-α, interferon γ, and LPS) by Sergent et al. (2010).
Among other parameters, IL-8 and IL-6 were quantified
by ELISA. They found that nontreated enterocytes constitutively secreted IL-8, whereas IL-6 secretion was barely
detectable. Level of IL-8 in inflamed cells was about
100-fold of IL-6. Devriendt et al. (2010) observed IL-6
secretion in IPEC-J2 cells after stimulation by different
enterotoxic Escherichia coli strains, but it seemed to express rather apically than basolaterally. In the same study,
TNF-α was measured both from the apical and basolateral
medium by ELISA. They observed no TNF-α secretion
after stimulation of the IPEC-J2 cells either with the different bacterial strains or in the control cells.
Epithelial Toll-like receptor (TLR) expression has
been described as being fundamental in the host defense
to bacterial challenges (Abreu, 2010). Arce et al. (2010)
have studied TLR-1, TLR-2, TLR-3, TLR-4, TLR-6, TLR-8,
TLR-9, and TLR-10 expression in swine jejunal IPEC-J2
and porcine ileal epithelial cells after LPS treatment.
Expression values of TLR were in general (except to TLR8 and TLR-10) higher in IPI-2I cells than in IPEC-J2 cells.
Their hypothesis was that this could represent a major in
vivo response in ileum than in jejunum when the gut is
exposed to bacterial wall component (such as LPS from
Salmonella enterica serovar Choleraesuis used in this
study). This result agrees with those found in human epithelial cell lines, which show a low level expression of
TLR-4 explained by the intestinal epithelial cells relative
resistance to the permanent exposure to gram-negative
Inflammation on pig enterohepatic co-culture
commensal bacteria (Abreu et al., 2001). Burkey et al.
(2007) used the expression of mRNA in pigs to determine
the constitutive expression of mRNA for TLR among tissues in healthy swine. They found that all tissues, except
for the jejunum, had greater TLR-4 expression when compared to the liver (reference point) and TLR-4 mRNA tended to be decreased in the colon and spleen by Salmonella
enterica serovar Choleraesuis (P < 0.1). Burkey et al.
(2009) described that TLR-2, TLR-4, and TLR-9 are constitutively expressed in vitro in IPEC-J2 cells. Expression of
TLR-4 was largely unaffected by LPS, Salmonella enterica serovar Typhimurium, or Salmonella enterica serovar
Choleraesuis after direct apical exposure of the model
swine jejunal epithelium. Lipopolysaccharide (10 μg/mL;
1 h treatment) did not change the relative expression of
TLR-4 in our measurements. The experiments were repeated under different conditions (LPS derived from E. coli
and 3 h incubation after treatment, respectively) but no difference in the TLR-4 expression was detected compared to
the untreated controls (unpublished data).
More than one hundred studies from the past 10 yr
were reviewed by Hur et al. (2012) on therapeutic approaches for treating inflammatory bowel diseases (IBD).
These studies suggest that the anti-inflammatory effects
exhibited by natural products are mainly caused by their
ability to modulate cytokine production. Similarly to
the human IBD, according to our results, probiotics and
butyrate may be useful tools in the treatment of enteral
inflammations in veterinary medicine as well.
In the study of Miyauchi et al. (2009), tight junction
barrier impairment was induced by TNF-α in the human epithelial Caco-2 cells and evaluated by measuring
the TER values. The effects of 4 probiotic lactobacilli
strains on TNF-α–induced changes in TER and the level
of IL-8 were studied. Their results showed that pretreatment with certain bacteria significantly ameliorated the
TNF-α–induced decrease in TER and IL-8 production;
however, the effects of other bacteria were rather weak.
The LPS-conferred inflammation resulted in increased rate of paracellular FD4 transport of IPEC-J2
cells suggesting that LPS alone could deteriorate cell
monolayer integrity (Paszti-Gere et al., 2014). It was
found in our study that the protective effect of Lp2142
and sodium n-butyrate was not significant in restoration
of weakened barrier integrity of IPEC-J2 cells. These results are concordant with our cytokine levels measured
in IPEC-J2 monoculture: there was no significant interaction between reduction of IL-8 level and changes in
TER/FD4 values; that is, changes in inflammatory cytokine levels do not result in changes in barrier functions.
Direct LPS treatment affected the IL-8 and IL-6 secretion of hepatocytes significantly; however, enteral
cytokine production could also alter the function of liver
cells. Hence, protective effect of Lp2142 and butyrate
3843
on LPS-induced inflammatory cytokine production was
monitored not only on IPEC-J2 monocultures but on primary hepatocytes as well as on the newly established enterohepatic co-culture system. Treatment of primary hepatocyte monocultures with Lp2142 SCS did not result
in reduced IL-8 and IL-6 concentration of the culture medium. Contrarily, the anti-inflammatory effect of Lp2142
was observed when IPEC-J2 cells were treated apically
in the co-culture. The same positive effect was noticed
when co-cultures were treated with LPS (1 and 10 μg/mL)
and 2 mM sodium n-butyrate at the same time. In contrast,
butyrate treatment on monoculture of hepatocytes did not
suppress the LPS-evoked increased IL-8 protein release.
Butyrate administered with LPS (10 μg/mL) evocated
increased IL-6 level, while simultaneous Lp2142 SCS
and LPS treatment caused increased IL-8 level in the
hepatocytes. Supernatant of Lp2142 per se also caused
significant increase in IL-8 level on hepatocyte monocultures (data not shown). This could be ascribed the immunomodulatory effect of probiotic compounds.
Concerning our results, it can be stated that the influence of anti-inflammatory compounds such as SCS of
Lp2142 and sodium n-butyrate on the release of proinflammatory cytokines such as IL-8 and IL-6 is different
in hepatocyte monoculture compared to the enterohepatic
co-culture. Considering the above mentioned results, it is
important to define that different in vivo processes could
be simulated by the applied in vitro models. In some cases, for example, substantial lesions of the epithelial barrier, hepatocytes could be also directly stimulated by LPS
absorbed from the gut; such in vivo conditions could be
well mimicked by the application of primary hepatocyte
monocultures. Indirect effects of LPS and gut-derived cytokines on the liver—similarly as it happens in vivo—can
be studied properly on the gut–liver co-culture model.
It can be concluded that the tested probiotic
Lactobacillus strain and butyrate both seemed to be effective in reducing proinflammatory cytokine production in LPS-triggered inflammatory response in vitro.
These candidates might be useful tools in the prevention
and treatment of enteral infections and inflammations
caused by enteropathogenic gram-negative bacteria by
improving gut health and can be further applied in veterinary medicine as well. According to our results, the
applied novel gut–liver co-culture system has proven
to be an appropriate in vitro model to study the antiinflammatory efficacy of probiotics and their metabolic
products in LPS-provoked inflammation.
LITERATURE CITED
Abreu, M. T. 2010. Toll-like receptor signalling in the intestinal epithelium: How bacterial recognition shapes intestinal function.
Nat. Rev. Immunol. 10:131–144.
3844
Farkas et al.
Abreu, M. T., P. Vora, E. Faure, L. S. Thomas, E. T. Arnold, and M.
Arditi. 2001. Decreased expression of Toll-like receptor-4 and
MD-2 correlates with intestinal epithelial cell protection against
dysregulated proinflammatory gene expression in response to
bacterial lipopolysaccharide. J. Immunol. 167:1609–1616.
Alexopoulos, C., I. E. Georgoulakis, A. Tzivara, S. K. Kritas, A.
Siochu, and S. C. Kyriakis. 2004. Field evaluation of the efficacy of a probiotic containing Bacillus licheniformis and Bacillus
subtilis spores, on the health status and performance of sows and
their litters. J. Anim. Physiol. Anim. Nutr. (Berl.) 88:381–392.
Arce, C., M. Ramirez-Boo, C. Lucena, and J. J. Garrido. 2010. Innate
immune activation of swine intestinal epithelial cell lines
(IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium. Comp. Immunol. Microbiol. Infect. Dis. 33:161–174.
Burkey, T. E., K. A. Skjolaas, S. S. Dritz, and J. E. Minton. 2007.
Expression of Toll-like receptors, interleukin 8, macrophage
migration inhibitory factor, and osteopontin in tissues from pigs
challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis. Vet. Immunol. Immunopathol. 115:309–319.
Burkey, T. E., K. A. Skjolaas, S. S. Dritz, and J. E. Minton. 2009.
Expression of porcine Toll-like receptor 2, 4 and 9 gene transcripts in the presence of lipopolysaccharide and Salmonella enterica serovars Typhimurium and Choleraesuis. Vet. Immunol.
Immunopathol. 130:96–101.
Cencic, A., and T. Langerholc. 2010. Functional cell models of the
gut and their applications in food microbiology – A review. Int.
J. Food Microbiol. 141(Suppl. 1):S4–S14.
Choi, S. H., M. Nishikawa, A. Sakoda, and Y. Sakai. 2004. Feasibility
of a simple double-layered coculture system incorporating metabolic processes of the intestine and liver tissue: Application
to the analysis of benzo[a]pyrene toxicity. Toxicol. In Vitro
18:393–402.
Devriendt, B., E. Stuyven, F. Verdonck, B. M. Goddeeris, and E. Cox.
2010. Enterotoxigenic Escherichia coli (K88) induce proinflammatory responses in porcine intestinal epithelial cells. Dev.
Comp. Immunol. 34:1175–1182.
Galfi, P., and J. Bokori. 1990. Feeding trial in pigs with a diet containing sodium n-butyrate. Acta Vet. Hung. 38:3–17.
Guilloteau, P., R. Zabielski, J. C. David, J. W. Blum, J. A. Morisset,
M. Biernat, J. Wolinski, D. Laubitz, and Y. Hamon. 2009.
Sodium-butyrate as a growth promoter in milk replacer formula
for young calves. J. Dairy Sci. 92:1038–1049.
Hanson, P. J., A. P. Moran, and K. Butler. 2011. Paracellular permeability is increased by basal lipopolysaccharide in a primary
culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2. Innate Immun. 17:269–282.
Hong, H. A., L. H. Duc, and S. M. Cutting. 2005. The use of bacterial
spore formers as probiotics. FEMS Microbiol. Rev. 29:813–835.
Hur, S. J., S. H. Kang, H. S. Jung, S. C. Kim, H. S. Jeon, I. H. Kim,
and J. D. Lee. 2012. Review of natural products actions on cytokines in inflammatory bowel disease. Nutr. Res. 32:801–816.
Hyland, K. A., D. R. Brown, and M. P. Murtaugh. 2006. Salmonella
enterica serovar Choleraesuis infection of the porcine jejunal
Peyer’s patch rapidly induces IL-1beta and IL-8 expression. Vet.
Immunol. Immunopathol. 109:1–11.
Lee, S. K., T. Il Kim, Y. K. Kim, C. H. Choi, K. M. Yang, B. Chae,
and W. H. Kim. 2005. Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression. Biochem. Biophys. Res. Commun.
337:457–463.
Liu, Y., N. Y. Fatheree, N. Mangalat, and J. M. Rhoads. 2010. Humanderived probiotic Lactobacillus reuteri strains differentially
reduce intestinal inflammation. Am. J. Physiol. Gastrointest.
Liver Physiol. 299:G1087–G1096.
Mani, V., A. J. Harris, A. F. Keating, T. E. Weber, J. C. Dekkers, and
N. K. Gabler. 2013. Intestinal integrity, endotoxin transport and
detoxification in pigs divergently selected for residual feed intake. J. Anim. Sci. 91:2141–2150.
Meng, F. Y., Z. S. Chen, M. Han, X. P. Hu, X. X. He, Y. Liu, W. T. He,
W. Huang, H. Guo, and P. Zhou. 2010. Porcine hepatocyte isolation
and reversible immortalization mediated by retroviral transfer and
site-specific recombination. World J. Gastroenterol. 16:1660–1664.
Miyauchi, E., H. Morita, and S. Tanabe. 2009. Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part by increasing expression of zonula occludens-1 and myosin light-chain
kinase in vivo. J. Dairy Sci. 92:2400–2408.
Morgan, E. T. 1997. Regulation of cytochromes P450 during inflammation and infection. Drug Metab. Rev. 29:1129–1188.
Moue, M., M. Tohno, T. Shimazu, T. Kido, H. Aso, T. Saito, and
H. Kitazawa. 2008. Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally
established porcine intestinal epitheliocyte cell line. Biochim.
Biophys. Acta 1780:134–144.
Nemeth, E., S. Fajdiga, J. Malago, J. Koninkx, P. Tooten, and J. van
Dijk. 2006. Inhibition of Salmonella-induced IL-8 synthesis and
expression of Hsp70 in enterocyte-like Caco-2 cells after exposure
to non-starter lactobacilli. Int. J. Food Microbiol. 112:266–274.
Nygard, A. B., C. B. Jorgensen, S. Cirera, and M. Fredholm. 2007.
Selection of reference genes for gene expression studies in pig
tissues using SYBR green qPCR. BMC Mol. Biol. 8:67.
Paszti-Gere, E., E. Csibrik-Nemeth, K. Szeker, R. Csizinszky, C.
Jakab, and P. Galfi. 2012a. Acute oxidative stress affects IL-8
and TNF-alpha expression in IPEC-J2 porcine epithelial cells.
Inflammation 35:994–1004.
Paszti-Gere, E., E. Csibrik-Nemeth, K. Szeker, R. Csizinszky, O.
Palocz, O. Farkas, and P. Galfi. 2013. Lactobacillus plantarum
2142 prevents intestinal oxidative stress in optimized in vitro
systems. Acta Physiol. Hung. 100:89–98.
Paszti-Gere, E., G. Matis, O. Farkas, A. Kulcsar, O. Palocz, G. Csiko,
Z. Neogrady, and P. Galfi. 2014. The effects of intestinal LPS
exposure on inflammatory responses in a porcine enterohepatic
co-culture system. Inflammation 37:247–260.
Paszti-Gere, E., K. Szeker, E. Csibrik-Nemeth, R. Csizinszky, A.
Marosi, O. Palocz, O. Farkas, and P. Galfi. 2012b. Metabolites
of Lactobacillus plantarum 2142 prevent oxidative stress-induced overexpression of proinflammatory cytokines in IPEC-J2
cell line. Inflammation 35:1487–1499.
Pie, S., A. Awati, S. Vida, I. Falluel, B. A. Williams, and I. P. Oswald.
2007. Effects of added fermentable carbohydrates in the diet on
intestinal proinflammatory cytokine-specific mRNA content in
weaning piglets. J. Anim. Sci. 85:673–683.
Pitman, R. S., and R. S. Blumberg. 2000. First line of defense: The
role of the intestinal epithelium as an active component of the
mucosal immune system. J. Gastroenterol. 35:805–814.
Puviani, A. C., C. Ottolenghi, B. Tassinari, P. Pazzi, and E. Morsiani.
1998. An update on high-yield hepatocyte isolation methods
and on the potential clinical use of isolated liver cells. Comp.
Biochem. Physiol. A Mol. Integr. Physiol. 121:99–109.
Rakhshandeh, A., and C. F. de Lange. 2012. Evaluation of chronic immune
system stimulation models in growing pigs. Animal 6:305–310.
Renton, K. W. 2004. Cytochrome P450 regulation and drug biotransformation during inflammation and infection. Curr. Drug Metab.
5:235–243.
Schierack, P., M. Nordhoff, M. Pollmann, K. D. Weyrauch, S. Amasheh,
U. Lodemann, J. Jores, B. Tachu, S. Kleta, A. Blikslager, K. Tedin,
and L. H. Wieler. 2006. Characterization of a porcine intestinal
epithelial cell line for in vitro studies of microbial pathogenesis
in swine. Histochem. Cell Biol. 125:293–305.
Inflammation on pig enterohepatic co-culture
Sergent, T., N. Piront, J. Meurice, O. Toussaint, and Y. J. Schneider.
2010. Anti-inflammatory effects of dietary phenolic compounds
in an in vitro model of inflamed human intestinal epithelium.
Chem. Biol. Interact. 188:659–667.
Skjolaas, K. A., T. E. Burkey, S. S. Dritz, and J. E. Minton. 2007.
Effects of Salmonella enterica serovar Typhimurium, or serovar
Choleraesuis, Lactobacillus reuteri and Bacillus licheniformis
on chemokine and cytokine expression in the swine jejunal
epithelial cell line, IPEC-J2. Vet. Immunol. Immunopathol.
115:299–308.
3845
Vieira, E. L. M., A. J. Leonel, A. P. Sad, N. R. M. Beltrao, T. F. Costa,
T. M. R. Ferreira, A. C. Gomes-Santos, A. M. C. Faria, M. C. G.
Peluzio, D. C. Cara, and J. I. Alvarez-Leite. 2012. Oral administration of sodium butyrate attenuates inflammation and mucosal
lesion in experimental acute ulcerative colitis. J. Nutr. Biochem.
23:430–436.
Vinolo, M. A., H. G. Rodrigues, E. Hatanaka, F. T. Sato, S. C.
Sampaio, and R. Curi. 2011. Suppressive effect of short-chain
fatty acids on production of proinflammatory mediators by neutrophils. J. Nutr. Biochem. 22:849–855.