RESEARCH ARTICLE Open Access
Lactobacillus rhamnosus L34 and Lactobacillus
casei L39 suppress Clostridium difficile-induced
IL-8 production by colonic epithelial cells
Prapaporn Boonma1, Jennifer K Spinler2,3, Susan F Venable2,3, James Versalovic2,3and Somying Tumwasorn4*
Background: Clostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as C.
difficile-associated disease (CDAD).With increased severity and failure of treatment in CDAD, new approaches for
prevention and treatment, such as the use of probiotics, are needed. Since the pathogenesis of CDAD involves an
inflammatory response with a massive influx of neutrophils recruited by interleukin (IL)-8, this study aimed to investigate
the probiotic effects of Lactobacillus spp. on the suppression of IL-8 production in response to C. difficile infection.
Results: We screened Lactobacillus conditioned media from 34 infant fecal isolates for the ability to suppress C.
difficile-induced IL-8 production from HT-29 cells. Factors produced by two vancomycin-resistant lactobacilli, L.
rhamnosus L34 (LR-L34) and L.casei L39 (LC-L39), suppressed the secretion and transcription of IL-8 without
inhibiting C. difficile viability or toxin production. Conditioned media from LR-L34 suppressed the activation of
phospho-NF-κB with no effect on phospho-c-Jun. However, LC-L39 conditioned media suppressed the activation
of both phospho-NF-κB and phospho-c-Jun. Conditioned media from LR-L34 and LC-L39 also decreased the
production of C. difficile-induced GM-CSF in HT-29 cells. Immunomodulatory factors present in the conditioned
media of both LR-L34 and LC-L39 are heat-stable up to 100°C and > 100 kDa in size.
Conclusions: Our results suggest that L. rhamnosus L34 and L. casei L39 each produce factors capable of
modulating inflammation stimulated by C. difficile. These vancomycin-resistant Lactobacillus strains are potential
probiotics for treating or preventing CDAD.
Keywords: Lactobacillus, Clostridium difficile, Probiotic, IL-8, Anti-inflammatory
Clostridium difficile is a gram-positive, spore-forming
anaerobe that causes antibiotic-associated diarrhea, colitis,
and pseudomembranous colitis in humans [1,2]. C. diffi-
cile-associated disease (CDAD) is acquired in association
with the disruption and alteration of the gut microbiota
. The frequency and severity of primary CDAD are
increasing; as well as recurrent cases and infections refrac-
tory to standard antibiotic therapy . C. difficile toxins,
toxin A (TcdA, 308 kDa) and toxin B (TcdB, 270 kDa), are
the main virulence factors contributing to intestinal tissue
damage and severe inflammation . Both toxins disrupt
the actin cytoskeleton and tight junctions of intestinal
epithelial cells [6,7], and cause apoptotic and necrotic cell
death [8,9]. C. difficile toxins, TcdA and TcdB, induce the
release of chemokines, like IL-8, from intestinal epithelial
cells [10-12]. In addition to IL-8, TcdA induces human
epithelial cells to secrete other CXC chemokines, in-
cluding growth-related oncogene (GRO)-α and neutro-
phil activating protein-78 (ENA-78), along with the CC
chemokine, monocyte chemoattractant protein (MCP)-1
. The disruption of tight junctions is thought to enable
TcdA and TcdB to enter the laminar propria and sub-
mucosa to induce immune cells to secrete chemokines,
proinflammatory cytokines, and mediators which promote
proinflammatory and cytotoxic effects [4,13]. Proinflam-
matory cytokines, especially IL-1β, also act on epithelial
cells to increase IL-8 secretion and upregulate intercel-
lular adhesion molecule-1 (ICAM-1) expression, leading
* Correspondence: somying.T@chula.ac.th
4Department of Microbiology, Faculty of Medicine, Chulalongkorn University,
Full list of author information is available at the end of the article
© 2014 Boonma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Boonma et al. BMC Microbiology 2014, 14:177
to increased ICAM-1 and neutrophil CD11/CD18 receptor-
A prominent feature of CDAD results from the release
of IL-8 from intestinal epithelial cells that causes a massive
influx of neutrophils into the colonic mucosa [4,5,15].
Neutrophil-derived inflammatory mediators exert toxic
effects on epithelial cells, causing congestion and edema
of the mucosa and epithelial cell damage [14,16]. IL-8 is a
major CXC chemokine that mediates neutrophil recruit-
ment, activation, and adhesion. IL-8 potency relies on high
binding affinity for neutrophil surface receptors, CXCR1
and CXCR2; which activate chemotaxis [17,18] and ro-
bust effector functions [19,20], and trigger the upregula-
tion of adhesion molecules CD11/CD18 that facilitate
transendothelial migration and subsequent tissue infil-
CDAD is most often associated with the disruption of
a healthy intestinal microbiome after the administration
of antibiotics . Potential biotherapies for the treatment
and prevention of CDAD are probiotics. In particular,
candidate probiotics are capable of enhancing microbiome
stability, interfering with pathogen activity, modulating the
immune system, and are intrinsically resistant to broad-
spectrum antibiotics [22,23]. Meta-analyses of randomized
controlled trials [24,25] support the efficacy of probiotic
Saccharomyces boulardii and Lactobacillus rhamnosus GG
in the prevention of CDAD. Placebo-controlled clinical tri-
als have shown that lyophilized S. boulardii decreased diar-
rhea associated with β-lactam antibiotics , and when
used in combination with metronidazole or vancomycin, S.
boulardii significantly reduced recurrent episodes in pa-
tients with CDAD [27,28]. The probiotic effects of S. bou-
lardii resulted from a secreted protease that interferes with
receptor binding of C. difficile toxins A and B to brush
borders of human colonic epithelial cells  and the
downstream modulation of host mitogen-activated protein
kinase (MAPK) signaling pathway . In vitro studies
with probiotic bacteria show that Lactobacillus delbrueckii
ssp. bulgaricus B-30892 inhibits the cytotoxic effects and
adhesion of C. difficile to Caco-2 cells , while specific
strains of Bifidobacterium spp. and Lactobacillus spp. have
antagonistic effects on the production of C. difficile toxins
A and B .
In the present study, we hypothesized that specific
strains of Lactobacillus species isolated from healthy
hosts produce factors that suppress toxigenic C. diffi-
cile-induced IL-8 production. We investigated the pro-
biotic effect of 34 Lactobacillus infant-fecal isolates on
the suppression of IL-8 production from colonic epithe-
lial cells stimulated by C. difficile. Conditioned media of
two vancomycin-resistant isolates, L. rhamnosus L34
and L. casei L39, suppressed IL-8 production at the level
of transcription without inhibiting C. difficile growth or
toxin production. Large (>100 kDa) heat-stable, soluble
factors are suggested to be responsible for the observed
Human-derived Lactobacillus spp. produce factors that
inhibit IL-8 and GM-CSF production by C. difficile-stimulated
colonic epithelial cells
Lactobacillus conditioned media (LCM) from thirty-four
human-derived Lactobacillus isolates (Additional file 1)
were screened for the ability to suppress pro-inflammatory
cytokine production from C. difficile-stimulated colonic
epithelial cells. LCM from 3 of 34 Lactobacillus isolates, L.
rhamnosus L34 (LR-L34), L. rhamnosus L35 (LR-L35), and
L. casei L39 (LC-L39), significantly suppressed IL-8 pro-
duction by approximately 50% or greater when compared
to media control (Figure 1A). These three IL-8-suppressing
isolates did not stimulate IL-8 production when co-
cultured with HT-29 cells in the absence of C. difficile (data
not shown). Only a single isolate of L. casei was included
in this study (Additional file 1), and the third L. rhamnosus
isolate in this collection, strain L31, did not suppress IL-8
in the same assay (Figure 1A). In a previous study , we
sequenced and compared the genomes of L. rhamnosus
L31, L34, and L35. Due to the fact that LR-L34 and LR-
L35 were isolated from the same host, had similar colony
morphology, were nearly identical at the nucleotide level
and similarly genetically distinct from strain LR-L31, we
determined that they were most likely independent isolates
of the same strain. Therefore, the remaining experiments
in this study were conducted excluding LR-L35.
LCM from LR-L34 and LC-L39 were screened for
effects on fourteen additional cytokines in a multi-plex
luminex assay. In addition to IL-8, C. difficile stimulated
GM-CSF production, but affected no other cytokines
tested (Additional file 2). GM-CSF is an important pro-
inflammatory cytokine that allows neutrophils to persist at
sites of inflammation [34,35] and enhances the chemo-
tactic response of neutrophils to IL-8 . In addition
to IL-8, LCM from both anti-inflammatory lactobacilli
strains significantly inhibited the production of GM-CSF
(Figure 1B). LCM from a different strain of a matched spe-
cies of L. rhamnosus, strain L31, not only had no effect on
IL-8 production, but also did not inhibit GM-CSF produc-
tion in the same assay (Additional file 2).
Further characterization of isolates LR-L34 and LC-
L39 showed that they are resistant to both vancomycin
and metronidazole (both with MIC>256 μg/mL), two
drugs commonly used to treat C. difficile infection in
humans [37,38]. The anti-inflammatory effect of soluble
factors produced by LR-L34 and LC-L39 is not unique to
HT-29 cells as LCM from both strains also suppresses IL-
8 production greater than 50% from human colonocytes
(Caco-2) stimulated by C. difficile (Additional file 3). Try-
pan blue dye exclusion indicated that HT-29 and Caco-2
Boonma et al. BMC Microbiology 2014, 14:177
Page 2 of 11
cell viability (>90%) was not compromised by the presence
of any LCM tested (data not shown). Furthermore, neither
viability of (Additional file 4) nor toxin production by
(Additional file 5) C. difficile was negatively affected by
the presence of LCM. All further in vitro experimenta-
tion was carried out using the C. difficile-HT-29 cell
L. rhamnosus L34 and L. casei L39 affect IL-8 gene
transcription through decreased activity of NF-κB and c-Jun
The effects of soluble factors produced by LR-L34 and LC-
L39 on IL-8 transcription were determined by IL-8 gene-
specific quantitative RT-PCR. C. difficile-stimulated HT-29
cells were treated with LCM for 4 h prior to total RNA
isolation. When compared to media control, treatment
with LCM from LR-L34 or LC-L39 resulted in an
approximate 0.5-fold down-regulation (p-value < 0.001)
of IL-8 mRNA concentration relative to gapdh (Figure 2).
Transcriptional regulation of IL-8 is mediated via NF-
κB and AP-1 by the upstream phosphorylation of subunits
p65 and c-Jun, which result in the respective activation of
NF-κB and AP-1 and subsequent downstream transcrip-
tion of IL-8 [39-42]. To determine whether or not LR-L34
or LC-L39 produce factors that affect activation of NF-
κB or c-Jun, C. difficile-stimulated HT-29 cells were
treated with LCM from either LR-L34 or LC-L39 for 15
and 30 min, then were assayed for effects on phosphory-
lated NF-κB (p-NF-κB) and c-Jun (p-c-Jun) concentrations
by western blot. LCM from LR-L34 decreased p-NF-κB
by 47.93% at 30 min only (p-value <0.001, Figure 3A),
Medium ControlLR-L31 LR-L34 LR-L35LC-L39
Lactobacillus Conditioned Media
Medium Control LR-L31
Lactobacillus Conditioned Media
Figure 1 Infant feces-derived Lactobacillus spp. produce factors that suppress pro-inflammatory cytokine production by C.
difficile-stimulated HT-29 intestinal epithelial cells. LCM from specific strains of human-derived lactobacilli were found to significantly inhibit
IL-8 production by HT-29 cells stimulated with C. difficile. Cells were stimulated with C. difficile in the presence of LCM for 24 h. (A) IL-8 production
was monitored by ELISA, and (B) GM-CSF production was monitored by a Luminex premixed cytokine assay by Millipore. The results were from
three independent experiments in triplicate for figure (A) and one experiment in triplicate for figure (B) and are expressed as the mean±SEM,
**p-value< 0.01 as compared to medium control.
Boonma et al. BMC Microbiology 2014, 14:177
Page 3 of 11
and did not affect p-c-Jun at either time point (Figure 3B).
A 15 min treatment with LC-L39 LCM resulted in de-
creased concentrations of p-NF-κB (18.62%, p-value <0.01,
Figure 3A) and p-c-Jun (38.19%, p-value <0.001, Figure 3B)
while a 30 min treatment resulted in a 43.19% decrease in
p-NF-κB (p-value <0.001, Figure 3A).
The anti-inflammatory factor(s) produced by L. rhamnosus
L34 and L. casei L39 is heat-stable and greater than
100 kDa in size
Heat tolerance and size prediction of anti-inflammatory
factors in LCM from LR-L34 and LC-L39 were charac-
terized as follows. LCM from LR-L34 and LC-L39 were
heated to 100°C for 15 min, 30 min, and 1 h and then
tested for the ability to suppress C. difficile-induced IL-8
production in HT-29 cells. The ability to suppress IL-8
production in this assay was retained at all time points
(Figure 4A). Size fractionation of LR-L34 and LC-L39
LCM was performed using 30 kDa and 100 kDa Amicon®
Centrifugal Filters and filtrates containing factors <30 kDa
and <100 kDa did not suppress IL-8 activity, while frac-
tions containing factors >100 kDa retained the anti-IL-8
activity in each case (Figure 4B).
Standard therapy for treating CDAD with metronidazole
and vancomycin is effective, but the association of these
drugs with high relapse rates represents a major health
problem [43-45]. Recent reports estimate the rate of re-
currence after an initial episode of CDAD to be 13-38%
[46-49]. Standard therapy for recurrent CDAD has been
modified to include probiotics like L.rhamnosus GG, L.
plantarum 299v, or S. boulardii which reduce the rate of
recurrence around 20-30% [28,50,51]. Health promoting
effects of lactobacilli include the stabilization of indigen-
ous microbial populations, protection against intestinal
infection, modulation of the immune system, and effects
on gene expression in the human mucosa [52-54]. In
addition, a recent systematic review and meta-analysis
indicated that probiotics given concurrently with antibi-
otics reduced the risk of antibiotic-associated diarrhea
and C. difficile infection . A key characteristic of the
pathophysiology of CDAD is an inflammatory response
with a marked neutrophil accumulation resulting from
secreted IL-8 [14-16,21]. Lactobacilli are known to in-
hibit IL-8 production by intestinal epithelial cells stimu-
lated with bacterium-derived LPS  but have not yet
been associated with the modulation of C. difficile-in-
duced IL-8 production.
We identified two Lactobacillus isolates from a library
of infant feces-derived Lactobacillus spp. that can signifi-
cantly suppress IL-8 production by C. difficile-stimulated
HT-29 cells. IL-8 suppression by LR-L34 and LC-L39 did
not result from negative effects on C. difficile viability as
l o r t noCm u i deM
Fold Change in IL-8 Gene Expression (LCM/Medium Control)
Lactobacillus Conditioned Media
Figure 2 Lactobacillus soluble factors suppress IL-8 transcription in HT-29 cells. IL-8 gene expression was determined in C. difficile-stimulated
HT-29 cells after 4 h incubation with medium control or LCM from either LR-L34 or LC-L39. Quantitative real-time PCR was conducted with primers
specific to IL-8 and GAPDH transcripts. Gene expression data were normalized to housekeeping gene, GAPDH. Fold change ratios of IL-8 (LCM
strain/medium control) from one experiment in triplicate were calculated, and results represent the mean±SEM, ***p-value<0.001 as compared to
Boonma et al. BMC Microbiology 2014, 14:177
Page 4 of 11
colony counts from the co-culture supernatants of either
C. difficile alone, or in combination with MRS, or LCM
from LR-L34 or LC-L39, were not significantly different
(Additional file 4). Trejo et al.  demonstrated that
spent culture supernatants of B. bifidum 5310 and L. plan-
tarum 83114 diminished the production of toxin A and
toxin B by C. difficile ATCC 9689 and clinical isolate 117.
IL-8 suppression by LR-L34 and LC-L39 seems not to re-
sult from attenuated toxin production and endocytosis by
HT-29 cells. No significant difference of intracellular toxin
concentrations in C. difficile and HT-29 cells was seen
by LCM treatment as compared to medium control
(Additional file 5). However, we cannot exclude the pos-
sibility that LCM of LR-L34 and LC-L39 interfere with
toxin self-cleavage in the endosome prior to entering
the cytosol which induces several downstream inflamma-
tory consequences . It is likely that C. difficile-secreted
toxins were endocytosed by HT-29 cells as a very small
amount of toxin was present in tissue culture medium at
24 h of the co-culture assay.
Normalized Protein Concentrations (p-NF-kB/NF-kB)
Normalized Protein Concentrations (p-c-Jun/c-Jun)
Figure 3 Human-derived Lactobacillus spp. suppress activation of C. difficile-induced transcription factors. Concentrations of activated
NF-κB (A) and c-Jun (B) were determined by western blot on whole cell lysates of HT-29 cells stimulated with C. difficile with or without medium
control or LCM treatment. Concentrations were measured at 15 and 30 min using antibodies corresponding to p-NF-κB p65, NF-κB p65, β-actin,
p-c-Jun, and c-Jun. Relative protein concentrations were determined by densitometry, and activated transcription factors were normalized to their
non-activated counterpart (p-NF-κB p65 (Ser 536) to NF-κB p65; p-c-Jun to c-Jun). The results were from three independent experiments in
duplicate and are expressed as the mean± SEM, *p-value <0.05 and **p-value <0.01.
Boonma et al. BMC Microbiology 2014, 14:177
Page 5 of 11
LR-L34 and LC-L39 suppressed IL-8 gene transcrip-
tion at 4 h after co-culture with HT-29 cells which is in
agreement with a report of Imaoka et al.  for B. bifi-
dum strain Yakult. The IL-8 gene promoter contains bind-
ing sites for transcription factors such as NF-κB and AP-1
, which control the transcription of IL-8 in C. difficile-
stimulated HT-29 colonic epithelial cells [40,60]. LCM
from LR-L34 decreased p-NF-κB, and did not affect p-c-
Jun while LC-L39 LCM decreased both of p-NF-κB and
p-c-Jun, with greater effects on pNF-κB. Modulation of
pro-inflammatory signaling pathways by probiotic bacteria
and yeasts have been demonstrated previously. Ma et al.
 reported that L. reuteri inhibited TNF-induced IL-8
production in both T84 and HT-29 intestinal epithelial
cells by inhibiting nuclear translocation of NF-κB. S.
boulardii modifies host cell pro-inflammatory signaling
pathways during bacterial infection by blocking the acti-
vation of NF-κB and MAPK [62-64]. Our data showed
that C. difficile stimulated GM-CSF production in addition
to IL-8 and conditioned media generated from LR-L34
and LC-L39 can also suppress GM-CSF production.
The ability to suppress GM-CSF of these lactobacilli
potentially enhances their anti-inflammatory effects on
C. difficile infection.
No Heat0.25 h 0.5 h 1.0 h
<30 kDa >30 kDa<100 kDa >100 kDa
Figure 4 IL-8 suppression by LCM after heat treatment and size fractionation. Soluble factors in LCM from LR-L34 and LC-L39 were assayed
for heat stability and size prediction. LCM from L. rhamnosus L34 and L. casei L39 was either heated to boiling for various time points (A) or size
fractionated by filtration (B) and the effects on IL-8 production by C. difficile-stimulated HT-29 cells was evaluated by ELISA. The results were from
three independent experiments in triplicate and are expressed as the mean± SEM, **p-value<0.01 and ***p-value <0.001.
Boonma et al. BMC Microbiology 2014, 14:177
Page 6 of 11
Probiotic factors can vary from organism to organism in
regards to activity, size, and stability. Sougioultzis et al.
 reported that S. boulardii probiotic yeast produce a
heat stable, <1 kDa soluble, anti-inflammatory compound
that blocks NF-κB activation and NF-κB-mediated IL-8
gene expression in both HT-29 colonic epithelial cells and
THP-1 monocytes. L. rhamnosus GG secretes p40 (40 kDa)
and p75 (75 kDa) proteins that activate EGFR and down-
stream PI3K/Akt and PKC signaling that modulate intes-
tinal epithelial cell survival and growth . Castagliuolo
et al.  reported that a 54-kDa protease of S. boulardii
can inhibit the effect of C. difficile toxins A and B in HT-29
cells. L. reuteri ATCC PTA 6475 produces the small mol-
ecule, histamine, which inhibits TNF production in THP-1
monocytes via PKA and ERK signaling . Active sub-
stances in LCM of LR-L34 and LC-L39 were found to be
heat-stable and >100 kDa. Although LR-L34 and LC-L39
also inhibit TNF production by THP-1 monocytes, they do
not produce histamine (data not shown). Therefore the
TNF-suppressive mechanism of action of these lactobacilli
must be different from L. reuteri, and further investigation
is needed to characterize the soluble factors produced
by these specific Lactobacillus isolates. The discovery of
vancomycin-resistant LR-L34 and LC-L39 with IL-8-
suppressing ability in this study offers us potential pro-
biotic strains for combating CDAD. These strains may
harbor other probiotic properties, including the ability to
replenish normal microbiota, a key factor for treating
CDAD as shown by the recent successes with intestinal
microbiome transplant therapies [68,69].
We have demonstrated the probiotic effect of two
vancomycin-resistant strains, L. rhamnosus L34 and L.
casei L39, on the suppression of IL-8 production from C.
difficile-stimulated colonic epithelial cells. These strains
suppressed IL-8 production by inhibiting activation of
transcription factors for IL-8 gene expression without
inhibiting C. difficile growth or toxin production. Our data
also suggest that heat-stable, >100 kDa factors are respon-
sible for IL-8 suppression. These vancomycin-resistant
Lactobacillus strains are potential probiotics for treating
or preventing CDAD.
Bacterial strains and culture conditions
Thirty-four Lactobacillus spp. isolated from infant feces
were analyzed in this study (Additional file 1). All lacto-
bacilli were routinely cultured in an anaerobic chamber
(Concept Plus, Ruskinn Technology, UK) (10% CO2,
10% H2, and 80% N2) for 24 h at 37°C in de Man,
Rogosa, Sharpe (MRS) medium (Oxoid, England).
A C. difficile isolate, designated strain B2-CU-0001-54
was obtained from feces of an infected patient positive
for C. difficile toxins A and B by VIDAS® Clostridium
difficile A & B (Biomérieux, France) at the Department
of Microbiology, Faculty of Medicine, Chulalongkorn
University. This strain is positive for TcdA and TcdB as
determined by PCR for toxin A and B genes  and
the reactivity with mouse anti-TcdA and anti-TcdB mono-
clonal antibodies (Meridian Life Science, Inc.). C. difficile
B2-CU-0001-54 was routinely cultured anaerobically on
Brucella agar (Oxoid, England) at 37°C for 48 h. Cells were
harvested, re-suspended in McCoy’s medium, and adjusted
to a McFarland 6 standard (1.8×109cells/mL) prior to co-
culture with HT-29 cells. This study was approved by the
Ethics Committee of Faculty of Medicine, Chulalongkorn
University, Bangkok, Thailand (COA no.617/2011, IRB
no.246/54). Written informed parental consent for fecal
samples was obtained from participants.
Minimum inhibitory concentration assay
The minimum inhibitory concentration of vancomycin on
LR-L34 and LC-L39 was determined by the broth micro-
dilution procedure as previously described . Briefly,
100 μl of lactobacilli (final concentration 1 × 106CFU/mL
or 1 × 105CFU/well) were inoculated into the wells of a
96 well plate containing 100 μl of vancomycin in serial
2-fold dilutions from 256 to 0.125 μg/mL, then were cul-
tured anaerobically for 48 h. Optical density was measured
at 600 nm using a microplate reader Multiskan® EX
(Thermo Scientific, USA). The results were compared
with growth control (lactobacilli alone) and the endpoint
of MIC is the concentration where no growth or a reduc-
tion in growth by 90%, is observed.
Preparation of Lactobacillus conditioned media
LCM were prepared as previously described . Briefly,
24 h cultures were adjusted to an OD6000.1 and incubated
anaerobically for 48 h. Supernatants were collected, filtered
with 0.22 μm Millex-GV Filter Units (Millipore, USA), and
500 μL aliquots were concentrated by Eppendorf Vacufuge®
vacuum concentrator (Eppendorf North America, USA) at
60°C for 2.5 h. Pellets were resuspended in an equal vol-
ume of McCoy’s 5a modified medium (Gibco-Invitrogen,
Carlsbad, CA, USA) or Eagle’s Minimal Essential Medium
(Gibco-Invitrogen, USA) and stored at −20°C until further
Cell lines and culture conditions
Human colonic epithelial cells, HT-29 or Caco-2, were ob-
tained from the American Type Culture Collection (ATCC
HTB-38 and HTB-37, respectively; Manassas, VA, USA).
HT-29 cells were maintained in McCoy’s 5a modified
medium supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (Gibco-Invitrogen, USA) at 37°C under
5% CO2for 48 h. Caco-2 cells were maintained in Eagle’s
Minimal Essential Medium supplemented with 20% (v/v)
Boonma et al. BMC Microbiology 2014, 14:177
Page 7 of 11
heat-inactivated fetal bovine serum at 37°C under 5%
CO2for 72 h. Adherent cells were detached with 0.25%
(v/v) Trypsin (Gibco-Invitrogen, USA) in 1 mM EDTA
(Gibco-Invitrogen, USA) and resuspended in their re-
spective medium. Resuspensions of each cell type were
used in subsequent C. difficile co-culture assays.
LCM treatment and C. difficile co-culture with colonic
Colonic epithelial cells were treated with LCM and stim-
ulated to produce IL-8 by co-culture with C. difficile.
HT-29 (2.0 × 104cells) or Caco-2 cells (5.0 × 104cells)
were pre-incubated for 24 h in a 96-well format as de-
scribed above. LCM (5% v/v) was added with or without
the subsequent addition of viable C. difficile B2-CU-0001-
54 (6.0 × 106CFU/well with HT-29 cells or 4.5 × 107CFU/
well with Caco-2 cells) and co-incubated for an additional
24 h. Cell culture supernatants were collected by centri-
fugation (125 × g, 4°C for 7 min) and stored at -20°C
until further use.
Effects of LCM on C. difficile viability
To ensure IL-8 suppression did not result from the antag-
onism of C. difficile growth by soluble factors in LCM, C.
difficile B2-CU-0001-54 was assayed for viability after co-
incubation with LCM and HT-29 cells. Briefly, co-culture
supernatants were serially diluted and cultured anaerobic-
ally on Brucella agar (Oxoid, England) at 37°C for 48 h.
Counts of isolated C. difficile B2-CU-0001-54 colonies
from co-culture assays with and without LCM treatment
Effects of LCM on C. difficile toxins in the co-culture assay
To determine whether Lactobacillus spp. produce factors
that suppress C. difficile toxin secretion, intracellular and
secreted toxin concentrations from co-culture assays were
determined as previously described  with the following
modifications. Secreted toxin was measured from LCM-
treated, C.difficile-stimulated HT-29 cell supernatants,
which were concentrated 10-fold by speed vacuum drying
and spotted onto polyvinylidene fluoride (PVDF) mem-
branes (Bio-Rad, Philadelphia, USA). Blocked membranes
were incubated in succession with mouse anti-TcdA or
anti-TcdB monoclonal antibodies (Meridian Life Science,
Inc.), biotinylated goat anti-mouse IgG, and extravidin-
alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA).
The presence of toxin was determined colorimetrically
with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-
phosphate (NBT/BCIP) solution (Sigma-Aldrich, St. Louis,
MO, USA). Intracellular toxin concentrations were deter-
mined from LCM-treated, C.difficile-stimulated HT-29 cell
pellets. Pellets from 20 ml of cell culture were washed,
resuspended in 2 ml cell culture medium, and lysed by
sonication (Vibra-Cell™, Sonics & Materials, Inc., USA).
Cell lysates were collected, 100 μl spotted onto PVDF
membranes, and analyzed for the presence of toxin as
described above. Toxin concentrations were calculated by
ChemiDoc™ XRS (Bio-Rad, Philadelphia, USA).
Effects of LCM on cytokine production by ELISA and
Supernatants from co-culture assays were tested for the
effects of soluble factors produced by Lactobacillus on
IL-8 production and other related cytokines. IL-8 con-
centrations in LCM-treated, C. difficile-stimulated HT29
or Caco-2 cell co-culture supernatants were measured
using a Human CXCL8/IL-8 ELISA kit (R&D Systems,
Minneapolis, MN) according to the manufacturer’s in-
structions. To determine whether Lactobacillus spp. can
modulate cytokines other than IL-8 in this assay, cell su-
pernatants from C. difficile-stimulated HT-29 cells in the
presence or absence of LCM were screened by a Human
Cytokine/Chemokine-Premixed 14-plex kit (Millipore,
Billerica, MA) in a Luminex 100 system (Luminex Cor-
poration, Austin, TX) for quantification of the analytes
GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-10, IL-12 (p70), IL-13, MCP-1, and TNF-α. Analytes
at concentrations exceeding the minimum detectable dose
were evaluated and raw data were obtained with Master-
Plex CT version 22.214.171.124 and analyzed with MasterPlex QT
version 126.96.36.199 (Hitachi MiraiBio, San Francisco, CA).
Analysis of IL-8 gene transcription by qPCR
The effects of LCM on the transcription of IL-8 were
determined by qPCR as previously described  with
the following modifications. HT-29 cells (8.0×105cells)
were pre-incubated in a 24-well format as outlined
above. Cells were LCM-treated (5% v/v) with or without
the subsequent addition of viable C. difficile B2-CU-
0001-54 (2.4×108CFU/well) at 37°C, 5% CO2for 4 h.
Total RNA was extracted from HT-29 cells using TRIzol
reagent (Invitrogen, USA) according to the manufac-
turer’s instructions. Synthesis of cDNA was completed
using the SuperScript® VILO™ cDNA Synthesis kit (Invi-
trogen), and qPCR was performed in a LightCycler® 2.0
(Roche, Germany) for 45 cycles of: 15 s at 94°C, 25 s at
60°C, and 25 s at 72°C. The amplified product was de-
tected using Light Cycler® Fast Start DNA MasterPLUS
SYBR Green I (Roche, Germany) at 530 nm. The following
primers were used to amplify cDNA fragments: IL-8 for-
ward primer (5′-ACACTGCGCCAACACAGAAATTA-
3′), IL-8 reverse primer (5′-TTTGCTTGAAGTTTCA
CTGGCATC-3′); GAPDH forward primer (5′-GCAC
CGTCAAGGCTGAGAAC-3′), GAPDH reverse primer
(5′-ATGGTGGTGAAGACGCCAGT-3′). IL-8 gene ex-
pression, relative to GAPDH, was calculated according
to the 2-ΔΔCpmethod .
Boonma et al. BMC Microbiology 2014, 14:177
Page 8 of 11
Examination of cell signaling pathways by quantitative
Changes in the NF-κB signaling pathway coordinating
with LCM treatment were analyzed by western blot as
previously described  with minor modifications. LCM-
treated, C. difficile-stimulated HT-29 cells were lysed in a
radioimmunoprecipitation assay (RIPA) buffer (50 mM
TrisHCl, pH7.4, 150 mM NaCl, 10% NP-40, 0.5% Na-
DOC, 0.1% SDS). Protein concentrations from cell lysates
were measured using the Pierce® BCA protein assay kit
(Pierce Biotechnology, Illinois, USA). Cell extracts were
fractionated by 10% sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE), transferred onto
PVDF membranes (Bio-Rad, Philadelphia, USA), and
blocked (10% non-fat milk in TBST (50 mM Tris, pH 7.5,
0.15 M NaCl, 0.05% Tween 20). Blocked membranes were
incubated with mouse antibodies against NF-κB (p65),
phospho-NF-κB (p65), c-Jun, phospho-c-Jun and β-actin
(Santa Cruz Biotechnology, California, USA), washed with
TBST and treated with horseradish peroxidase-labeled
goat anti-mouse secondary antibodies for 1 h. Peroxidase
signals were detected and imaged by ChemiDoc™ XRS
(Bio-Rad, Philadelphia, USA).
Characterization of IL-8 suppressive factors in LCM
Lactobacillus anti-inflammatory factors responsible for
suppressing IL-8 production from C. difficile-stimulated
colonic epithelial cells were assessed for thermal stabil-
ity and size estimation. Heat stability was assessed by
heating LCM to 100°C for 15 min, 30 min, and 1 h. The
sizes of active factors present in LCM were estimated by
30 kDa and 100 kDaAmicon® Ultra-4 Centrifugal Filters
(Merck, Massachusetts, USA). After each treatment, LCM
was tested for the ability to suppress IL-8 production
from C. difficile-treated HT-29 cells using techniques
All experiments were performed at least in triplicate and
the results were reported as mean ±standard deviation
or standard error of mean (SEM). The data were ana-
lyzed in Microsoft Excel using the Student’s t-test with
one-tailed distribution and considered statistically sig-
nificant at a p-value ≤ 0.05, unless otherwise stated.
Additional file 1: Infant feces-derived Lactobacillus spp. used in this
Additional file 2: Production of cytokines and chemokines by C.
difficile-stimulated HT-29 cells.
Additional file 3: Infant feces-derived Lactobacillus spp. produce
factors that suppress pro-inflammatory cytokine production by C.
difficile-stimulated Caco-2 intestinal epithelial cells. LCM from
human-derived lactobacilli were found to significantly inhibit IL-8
production from Caco-2 cells stimulated with C. difficile. Cells were
stimulated with C. difficile in the presence of LCM for 24 h and IL-8
production was monitored by ELISA. The results were from three
independent experiments in triplicate and are expressed as the mean ±
SEM, **p-value <0.01 as compared to medium control.
Additional file 4: C. difficile viability was not affected by
Lactobacillus conditioned media. IL-8 suppression did not result from LCM
effects on the antagonism of C. difficile growth. C. difficile B2-CU-0001-54
was assayed for viability after co-incubation with LCM and HT-29 cells.
Counts of isolated C. difficile B2-CU-0001-54 colonies from co-culture
assays with and without LCM treatment from three independent
experiments in triplicate were compared and results are reported as
mean ± SD. No significant difference in viability of C. difficile was seen
by LCM treatment as compared to medium control.
Additional file 5: Lactobacillus conditioned media had no effect on
C. difficile toxins in co-culture assay. IL-8 suppression did not result
from attenuated toxin production and endocytosis to HT-29 cells.
Extracellular and intracellular toxin concentrations were determined from
LCM-treated, C. difficile-stimulated HT-29 cells. Cell culture supernatants or
lysates of C. difficile and HT-29 cells were collected, spotted onto PVDF
membranes and assayed in succession with mouse anti-TcdA or
anti-TcdB monoclonal antibodies. The presence of toxin was determined
colorimetrically and toxin concentrations were calculated by ChemiDoc™
XRS (Bio-Rad, Philadelphia, USA). The results were from three independent
experiments in triplicate and are expressed as the mean±SEM. No
significant difference in intracellular toxin concentrations was seen by LCM
treatment as compared to medium control. (A) Whole cell lysates, (B) culture
supernatant of HT-29 cells, (C) the ratio between spot intensity of C. difficile
co-culture with LCM and C. difficile with MRS.
The authors declare that they have no competing interests.
JKS, JV, and ST conceived and designed the study. PB designed and
performed the experiments. SV carried out Luminex assay. JKS, JV, and ST
supervised and provide funding for the research. PB, JKS, and ST wrote the
manuscript. All authors read and approved the final manuscript.
We would like to graciously acknowledge Tor Savidge for his advice,
guidance, and generosity, and extend our gratitude to both the Texas
Children’s Microbiome Center and the Functional Genomics & Microbiome
Core for providing equipment and resources for a fruitful collaboration.
This work was supported by the Thailand Research Fund through the
Royal Golden Jubilee PhD Program (PHD/0295/2550 to PB); the Thai
Government Research Budget for Fiscal year 2009 and 2010; the
Rachadapisek Sompoj research fund, Faculty of Medicine, Chulalongkorn
University (Grant No. RA51/1and RA55/20 to ST). Funding was also provided
in part by the Texas Children’s Microbiome Center, Department of Pathology
at Texas Children’s Hospital; and the National Institutes of Health, National
Cancer Institute (Grant No. U01 CA170930 to JV); by the National Institute of
Diabetes and Digestive and Kidney Diseases (Grant Nos. UH3 DK083990 to JV,
P30 DK56338, and R01 DK065075 to JV); and by the National Center for
Complementary and Alternative Medicine (Grant No. R01 AT004326 to JV).
1Interdisciplinary Program of Medical Microbiology, Graduate School,
Chulalongkorn University, Bangkok, Thailand.2Texas Children’s Microbiome
Center, Department of Pathology, Texas Children’s Hospital, Houston, Texas,
USA.3Department of Pathology & Immunology, Baylor College of Medicine,
Houston, Texas, USA.4Department of Microbiology, Faculty of Medicine,
Chulalongkorn University, Bangkok, Thailand.
Received: 24 February 2014 Accepted: 18 June 2014
Published: 2 July 2014
Boonma et al. BMC Microbiology 2014, 14:177
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Cite this article as: Boonma et al.: Lactobacillus rhamnosus L34 and
Lactobacillus casei L39 suppress Clostridium difficile-induced IL-8
production by colonic epithelial cells. BMC Microbiology 2014 14:177.
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