Preferential promotion of apoptosis of monocytes by Lactobacillus casei rhamnosus soluble factors
LcrS 5-30 induces TGF-β production and promotes TGF-β-independent apoptosis
apoptosis of monocytes and THP-1 cells via expressions of mRNAs, including Bcl-2 and Bax,
and proteins, including cytochrome c, caspase 9 and caspase3, by a mitochondrial pathway.
Inhibition of LPS-induced inflammatory cytokines after treatment with LcrS5-30
The apoptosis-inducing effects on pro-inflammatory cells by LcrS5-30 implied its potential
for regulating the secretions of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α).
Lymphocytes, monocytes and THP-1 cells were treated with E. coli-derived LPS and LcrS5-30,
and the changes of inflammatory cytokines secretions were evaluated by ELISA (Fig. 7). As
expected, LPS-treated cells showed elevated levels of inflammatory cytokines. However,
cytokines expressions were markedly reduced after simultaneous incubation with LcrS5-30
(P<0.01, Fig. 7). These experiments suggested that LcrS5-30 was capable of downregulating the
secretion of pro-inflammatory cytokines.
LcrS5-30 induces TGF-β production and promotes TGF-β-independent apoptosis
TGF-β1 is a key regulatory cytokine involved in anti-inflammation that counteracts IL-1 and
TNF-α and modulates cellular functions, such as homing, cellular adhesion, chemotaxis and
T-cell homeostatic regulation (Luethviksson and Gunnlaugsdottir, 2003).In contrast to the
pro-inflammatory cytokines described above, dramatic increases of TGF-β1 levels were observed
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following treatment with LcrS5-30 (Fig. 8A).
TGF-β regulates a wide array of biological functions, including apoptosis (Heldin et al.,
1997). TGF-β production was increased by LcrS5-30 treated THP-1 cells. It was reasonable to
postulate that apoptosis might be triggered by TGF-β rather than LcrS5-30. To confirm this
assumption, a pharmacological inhibitor, SB431542, was applied. The percentages of apoptotic
cells in 10 ng/ml and 1 ng/ml TGF-β exposed preparations were 18.9 ± 0.44% and 16.57 ± 0.67%,
respectively (Fig. 8B). Treatments with 12.5 μg/ml and 25μg/ml of LcrS5-30 induced dissimilar
levels of apoptosis of THP-1 cells, which were 26.35 ± 0.77% and 74.96 ± 0.38%, respectively.
One ng/ml TGF-β and 12.5 μg/ml LcrS5-30 induced apoptosis were SB431542 sensitive.
Nevertheless, 25μg/ml of LcrS5-30 induced dramatic changes in the levels of apoptosis of THP-1
cells, and SB431542 could not reverse this phenomenon (Fig. 8B). Collectively, these results
indicated that LcrS5-30 was capable of inducing TGF-β-independent cell death.
104
DISCUSSION
In the present study, several Lactobacillus strains were investigated for their differential
capabilities to promote apoptosis of THP-1 cells. Lcr exhibited more potent apoptosis inducing
capability than the L. casei and the L. rhamnosus strains. Probiotics, as preventative or
therapeutic agents against IBD, are an attractive, alternative approach for the attenuation of
mucosal inflammation. Many clinical studies during recent decades demonstrated that probiotic
species possessed beneficial effects for IBD (Miele et al., 2009; Bibiloni et al., 2005; Kruis et al.,
2004). However, inconsistency among some results might have resulted from differences in
probiotic species or strains. These inconsistencies have drawn attention to understanding the
mechanisms of probiosis by specific strains for possible clinical applications.
Initial characterization of the apoptosis inducing factor(s) in LcrS employed MWCO
ultrafiltration. The apoptosis inducing factor(s) were present in the 5-30 kDa fraction. Several
studies have described apoptosis promotion via factor(s) produced by probiotics, such as for
human breast cancer cells by fermented soy milk (Chang et al., 2002), human γδ T cells by E.
coli Nissle 1917 supernatant (Guzy et al., 2008) and for human myeloid leukemia-derived cells
by L. reuteri (Iyer et al., 2008). The identities of these factor(s) have not been determined. The
105
Lcr-derived apoptosis-inducing factor(s) are small heat stable proteins or peptides components, based on results of heat inactivation and protease treatment experiments.
Pathologically, IBD is characterized by a high density of mucosal cells within the
inflamed tissues that mainly consist of activated T cells, peripheral blood neutrophils and
monocytes/macrophages (Lügering et al., 2006). Extensive studies in recent years have shown
that activation and increased survival time of leucocytes might contribute to the severity of
intestinal inflammation and clinical relapses in both CD and UC (Itoh et al., 2001).
Activation-induced cell death (AICD) is an important mechanism to limit the number of active monocytes
and lymphocytes, and to terminate an immune response. LcrS5-30 was effective for promoting
lymphocyte/ monocyte/ THP-1 cell apoptosis in both dose- and time-dependent manners.
Despite a paucity of data regarding probiotics for promoting apoptosis of immune effector
cells as an alternative IBD therapy, experiments with γδ T cell going through programmed cell
death by E. coli Nissle and human myeloid leukemia-derived cells proceeding through
TNF-induced apoptosis by L. reuteri provided a rational basis for an apoptosis-inducing strategy for
IBD therapy. Our study with monocytes showed that 12.5 μg/ml and 25 μg/ml of LcrS5-30 could
disrupt the mitochondrial membrane potential, and apoptotic cell death was induced within 3 hrs
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following exposure to 25 μg/ml of LcrS5-30. Moreover, the increase of the Bax/Bcl-2 ratio, release
of cytochrome c, and activation of caspase-9 and caspase-3, showed that LcrS5-30 is capable of
inducing monocytes apoptosis via a mitochondrial pathway.
An anti-TNF antibody, infliximab, is an FDA-approved treatment for CD and UC. Several
studies demonstrated that infliximab induced monocytes apoptosis (Lügering et al., 2001) and a
loss of CD68+ monocytes, as well as CD4+ and CD8+ T lymphocytes, in the lamina propria
(Baert et al., 1999). Also, infliximab exerted killing activity on human peripheral blood T cells by
as much as 50.6% after 18 h culture with 5 μg/mlinfliximab (Sabatino et al., 2004). In our study,
LcrS5-30 promoted apoptosis of lymphocytes, monocytes and THP-1 cells by as much as 93%,
97% and 74%, respectively. The induction of apoptosis did not require the Fas/FasL signal
transduction pathway. Rather, it involved upregulation of Bax/Bak followed by mitochondrial
release of cytochrome c (Sabatino et al., 2004). Interestingly, the extents of immune effector cells
apoptosis induced by infliximab and probiotic Lcr were similar. Also, given the safety history of
probiotics, Lcr could be a useful adjunctive treatment for IBD patients.
The loss of intestinal epithelial cell (IEC) function, and subsequent changes in epithelial
tight junction protein expressions and IEC apoptosis are also critical components for the initiation
107
and perpetuation of IBD (Xavier and Podolsky, 2007). Probiotics have been investigated for
protective effects by regulating IEC survival for treating and preventing intestinal inflammation.
In vitro results indicated that LGG reduced intestinal epithelial apoptosis by upregulating the
expressions of anti-apoptotic and cytoprotective genes (Lin et al., 2008). Also, soluble factors
from LGG stimulated anti-apoptotic Akt activation and prevented cytokine-mediated apoptosis
(Yan et al., 2007). In this study, LcrS5-30 was quite unique in that it promoted apoptosis of
monocyte-like cells, but not of IEC. These results are consistent with previous studies in which
probiotics could protect intestinal integrity by promoting survival of IEC.
LcrS5-30 possessed potent inhibitor(s) for LPS induced pro-inflammatory cytokines, such
as IL-1β, IL-6, TNF-α, and chemokine, IL-8. The suppression of IL-1β, IL-6, TNF-α and IL-8
suggested that LcrS5-30 might concomitantly suppress lymphocyte/monocyte/THP-1 chemotaxis
and cellular activation. TNF-α is a crucial proinflammatory cytokine in various inflammatory
disorders. Blocking of TNF-α is efficient for the treatment of patients with CD and UC (Rutgeerts
et al., 2004). Interestingly, studies have indicated that apoptosis was indeed linked to the clinical
efficacies of various anti-TNF agents. TNF-α inhibitors, such as infliximab and adalimumab,
have shown clinical efficacy for IBD and exerted strong apoptotic effects (Baert et al., 1999).
However, another anti-TNF agent, etancercept, did not establish a positive influence on the
108
course of CD (Van den Brande et al., 2003) due to a failed apoptosis inducing ability in
monocytes and lymphocytes. Our results indicated that LcrS5-30 possessed both TNF-α
suppression and apoptosis promotion capabilities, as seen with infliximab.
TGF-β is a potent anti-inflammatory cytokine and has a vital role for suppressing the
activation and proliferation of inflammatory cells (Letterio and Roberts, 1998). Also, TGF-β has
been implicated for an essential role in disease remission by promoting the maturation of
intestinal epithelial cells and for healing wounds and ulcers (Kader et al., 2005). L. paracasei has
been reported to induce populations of regulatory CD4+ T cells, which produce high levels of
modulatory cytokines, IL-10 and TGF-β (von der Weid et al., 2001). Lactobacilli modulate
cytokine production in bone-marrow-derived dendritic cells with a net effect of altering overall
cytokine profiles in a species-dependent manner (Christensen et al., 2002). One study also
showed that pediatric IBD patients in remission compared to those with active disease had higher
levels of TGF-β1 (Kader et al., 2005). In this study, because LcrS5-30 could induce high TGF-β1
production, but not IL-10 (data not shown), by lymphocytes, monocytes, and THP-1 cells implied
that LcrS5-30 altered the balance between pro-inflammatory and anti-inflammatory cytokines, and
highlights their important immunomodulatory roles in inflammatory diseases.
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TGF-β can trigger apoptosis in myeloid leukemia cells (Heldin et al., 1997). SB-431542,
a specific inhibitor of TβRI, inhibits the TGF-β-induced apoptosis in several cell types by
blocking TGF-β signaling. Upregulated TGF-β production promotes monocytes apoptosis and
contributes to the prevention of tissue injury. It was important to confirm the consequences of
TGF-β production in regulating the apoptosis of monocytes following LcrS5-30 treatment.
SB-431542 significantly blocked TGF-β1- and 12.5μg/ml - induced apoptosis. The higher dose of
LcrS5-30 induced a significant amount of apoptosis in THP-1 cells, but this was inefficiently
abrogated by SB 431542. Thus, LcrS5-30 could trigger TGF-β-independent apoptosis.
In conclusion, probiotic Lcr produces heat-stable molecules with a MW range of 5-30
kDa, primarily proteins, which promoted lymphocyte, monocyte and THP-1 cell apoptosis
without affecting intestinal epithelial cells. LcrS5-30 triggered apoptosis of immune cell in vitro by
a mitochondrial pathway, but not via the TGF-β signaling pathway. LcrS5-30 also inhibited
LPS-induced inflammatory cytokines in activated immune cells. This investigation demonstrated a
role for LcrS5-30 in promoting apoptosis of immune cells and suggests the possibility of a
probiotics-based regimen for prevention of IBD.
110
FIGURES AND LEGENDS
A.
111
B.
112
Figure 1. Strain-dependent promotion of apoptosis of THP-1 cells in the absence or
presence of probiotic-derived secreted factors.
LcrS significantly increased the numbers of Annexin V positive cells, but fewer were found for L.
casei and L. rhamnosus. Apoptotic THP-1 cells following exposure to 25μg/ml probiotic
supernatants were determined by double staining with Annexin V-FITC and Propidium iodide
(PI). Cells that were Annexin V positive and PI negative were early apoptosis cells. Cells positive
both for Annexin V and PI represented cells in late apoptosis. Results representative of typical
donors (panel A) and mean ± standard error of the mean (panel B) for 3 different experiments.
Control = complete RPMI cell culture medium. MRS=bacterial culture medium. 1 μM Taxol was
a positive control. ***P< 0.001 for change versus LcrS.
113
114
A.
B.
Figure 2. Partial characterization of factors promoting THP-1 apoptosis. THP-1 cells were
stimulated with 25μg/ml of 4 different fractions (panel A). THP-1 cells were stimulated with
LcrS5-30 that had either been boiled for 30 mins or treated with 1mg/ml trypsin or proteinase K.
After 24 h stimulation, apoptotic THP-1 cells following exposure to 25μg/ml LcrS5-30 were
determined by double staining with Annexin- V FITC and PI (panel B). Each bar represents mean
± standard error of the mean of 3 individual experiments. ***P< 0.001 for change versus control.
115
116
A.
B.
C.
117
Figure 3. LcrS5-30 promoted apoptosis of monocytes, but not intestinal epithelial cells. THP-1
cells (panels A and B) and HT-29 cells (panel C) were treated with varying concentrations of
LcrS5-30 or 10 μM Taxol as a positive control for 24h. TUNEL staining was observed by light
microscope. Arrows indicate representative apoptotic cells (panel A). The percentage of cells that
underwent apoptosis from a representative experiment (panel B). Apoptotic HT-29 cells exposed
to LcrS5-30 were determined by Annexin V-FITC and PI double staining followed by flow
cytometric analysis (panel C). All experiments were performed on at least 3 separate occasions.
***P< 0.001 for change versus control.
118
119
A.
B.
Figure 4. Incubation with LcrS5-30 increased the numbers of Annexin V positive cells in a
time- and dose-dependent manner. Lymphocytes (■), monocytes (■) or THP-1 cells (□) (1×106
per test) were collected after incubation with LcrS5-30 for varying times (panel A) and for doses
ranging from 3.125 to 25 μg/ml (panel B). Cell apoptosis was determined by double staining with
Annexin V-FITC and PI. Results are mean ± standard error of the mean from triplicate cultures.
*P< 0.01 for change versus control.
120
121
A.
B.
Figure 5. Cytofluorometric analysis of mitochondrial membrane potential (ΔΨm). One
representative analysis of ΔΨm in monocytes after stimulation with varying concentrations of
LcrS5-30 (panel A). Respective percentages of cells with depolarized mitochondria (%ΔΨm) are
indicated in the upper box of each group (7.66, 11.59, 13.71 and 56.43%, respectively). Thus, a
decrease in ΔΨm corresponds to an increase in percent of monocytes %ΔΨm. JC-1 red
fluorescence/JC-1 green fluorescence ratio for lymphocytes, monocytes and THP-1 with
depolarized ΔΨm, with or without the LcrS5-30, for varying times (panel B). *P< 0.01 for change
versus control.
122
123
A.
B.
C.
Figure 6. LcrS5-30 causes decreased Bcl-2 expression, increased Bax and caspase 9
expressions, cytochrome c (Cyt c) release and caspase 3 activation. Monocytes and THP-1
cells (1×106) were treated with or without 20μg/ml of LcrS5-30 for indicated times. mRNA was
detected by RT-PCR (panel A), and the protein levels were determined by Western blot (panel B).
Both the mRNA and protein expressions of actin were internal controls. Cells were incubated
with FITC-conjugated anti-active caspase-9 antibody and analysis by flow cytometry (panel C).
Monocytes and THP-1 cells exhibiting positive intracellular active caspase-9 fluorescence were
enumerated, and the results are expressed as a percentage of the total number of cells analyzed.
Results are mean ± standard error of the mean of 3 individual experiments. *P< 0.01 for change
versus control.
124
125
A.
B.
126
C.
D.
Figure 7. Inhibition of cytokine production by LcrS5-30 by lymphocytes, monocytes and
THP-1 cells. Cell culture bioassay were performed by stimulating lymphocytes (■), monocytes
(■) or THP-1 cells (□) with E.coli-derived LPS and 25 μg/ml of LcrS5-30. Human IL-1β (panel A),
IL-6 (panel B), IL-8 (panel C) and TNF-α (panel D) cytokine were determined in culture
supernatants by sandwich ELISA following cell culture. Each bar represents mean ± standard
error of the mean of 3 individual experiments. *P< 0.01 for change versus LPS.
127
128
A.
B.
Figure 8. LcrS5-30 induced TGF-β1 production and promoted TGF-β independent apoptosis.
Human TGF-β1 quantities were determined by TGF-β1 specific ELISA in culture supernatants following lymphocytes (■), monocytes (■) or THP-1 cells (□) culture (panel A). THP-1 cells were treated with TGF-β1 or LcrS5-30 in the absence or presence of 1μg/ml of TGF-β inhibitor, SB431542, for 24 h (panel B). Cell apoptosis was determined by double staining with Annexin V-FITC and PI. T Results are mean ± standard error of the mean from triplicate cultures. *P< 0.01 for change versus LPS. *** P< 0.001 for change versus 1ng/ml SB431542 versus the respective control.
129
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