INFECTION AND IMMUNITY, Mar. 2005, p. 1452–1465
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 3
Escherichia coli Nissle 1917 Distinctively Modulates T-Cell Cycling and
Expansion via Toll-Like Receptor 2 Signaling
Andreas Sturm,1* Klaus Rilling,1Daniel C. Baumgart,1Konstantinos Gargas,2Tay Abou-Ghazale ´,2
Ba ¨rbel Raupach,3Jana Eckert,4Ralf. R. Schumann,4Corinne Enders,5Ulrich Sonnenborn,5
Bertram Wiedenmann,1and Axel U. Dignass1
Departments of Hepatology and Gastroenterology, Campus Virchow Clinic,1and Institute of Microbiology and Hygiene,4
Charite ´-Universita ¨tsmedizin Berlin, Department of Surgery, DRK Clinics Westend,2and Max Planck Institute for
Infection Biology, Department of Cellular Microbiology,3Berlin, and Ardeypharm GmbH, Biological Research
Received 21 June 2004/Returned for modification 22 July 2004/Accepted 28 September 2004
Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment
of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the
present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood
and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with
E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin
B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and
apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting,
and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and
expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli
Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter
PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression,
contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression
of interleukin-2 (IL-2), tumor necrosis factor ?, and gamma interferon but increased IL-10 production in PBT.
Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT prolif-
eration by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound
T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal
inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa
and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious
antigens in order to maintain immunological homeostasis.
Although the pathogenesis of inflammatory bowel disease
(IBD) is not completely understood, the contributions of ge-
netic and environmental factors are increasingly evident. Re-
cently, a major role in the initiation and perpetuation of
chronic IBD has been attributed to the luminal bacterial flora
(40, 56). Interestingly, recent studies have demonstrated that
bacterial products can directly activate T cells via Toll-like
receptors (TLRs) even in the absence of antigen-presenting
cells or costimulatory molecules (7, 32, 50), suggesting a link
between the luminal bacterial flora and the immune system
(19). Furthermore, in IBD, tolerance of the intestinal immune
system against the physiological microflora is lost (14), and this
breakdown is supposed to contribute to intestinal inflamma-
tion and may explain the beneficial effects of antibiotic therapy
in IBD patients.
Escherichia coli strains play a pivotal role within the unique
intestinal microecological system, which consists of an enor-
mous variety and quantity of different microorganisms. These
microorganisms can be found as physiological constituents of
the intestinal microflora both in healthy individuals and under
pathological conditions in the course of various gastrointesti-
nal diseases. Several lines of evidence suggest that E. coli is
involved in the pathogenesis of IBD (23). E. coli strains iso-
lated from Crohn’s disease lesions have been demonstrated to
adhere to and to disrupt the intestinal barrier (12, 22). Fur-
thermore, distinct E. coli genotypes are associated with chronic
or early recurrent ileal lesions (43). In addition, E. coli-specific
antibodies and the number of E. coli organisms in areas of
intestinal inflammation are significantly elevated in IBD pa-
The probiotic E. coli strain Nissle 1917 has been reported to
maintain remission of ulcerative colitis and pouchitis (35–37,
52) and to prevent colitis in different murine models of colitis
(57). Although E. coli Nissle 1917 was originally isolated in
1917, the underlying mechanism of its beneficial effect in var-
ious intestinal diseases still remains elusive.
T cells play a major role in the pathogenesis of IBD (18).
They must respond to antigens in a selective and balanced
fashion that allows them to mount an effective response by
progressing through the cell cycle, expanding, and finally un-
dergoing apoptosis once the antigen has been cleared (4, 38,
39). This task is particularly challenging for T cells exposed to
antigens that may be present in a variety of types and in large
numbers, as in the gastrointestinal tract. In the intestinal mu-
* Corresponding author. Mailing address: Department of Hepatol-
ogy and Gastroenterology, Campus Virchow Clinic, Charite ´-Universi-
ta ¨tsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.
Phone: 49 30 450 553022. Fax: 49 30 450 553929. E-mail: andreas.sturm
cosa T cells are required to maintain a state of immunological
tolerance toward a myriad of dietary and bacterial antigens
(15, 31, 49). To accomplish this critical assignment, intestinal T
cells cycle differently from systemically circulating T cells, like
peripheral blood T cells (PBT) (68), which encounter a com-
pletely different antigen repertoire (55). Whereas cell cycling
and death are usually tightly balanced (63), many diseases are
based on either uncontrolled cell cycling or impaired apopto-
sis, leading to unrestrained cell proliferation and, thus, cancer
or autoimmune disorders such as IBD.
The aim of our study was to analyze the functional biological
activities of E. coli Nissle 1917 on peripheral and mucosal T
cells and thus to characterize the underlying mechanism of
action. This was achieved by analyzing the effect of E. coli
Nissle 1917 on peripheral blood and mucosal T-cell cycling,
expansion, and apoptosis and by evaluating the expression of
cell cycle regulatory molecules and cytokine secretion. Our
results reveal that E. coli Nissle 1917 inhibits expansion of
naive and memory PBT but not lamina propria T cells (LPT).
This distinct reaction of circulating and tissue-bound T cells
towards E. coli Nissle 1917 may explain the beneficial effect of
probiotics in intestinal inflammation, where PBT are recruited
from the circulation into the intestinal mucosa. Thus, E. coli
Nissle 1917 may downregulate the expansion of newly re-
cruited T cells into the mucosa and limit intestinal inflamma-
tion, whereas the insensitivity of LPT toward probiotic stimu-
lation seems to avoid destabilization of the mucosal immune
defense and may, therefore, contribute to maintenance of in-
testinal immunological homeostasis.
MATERIALS AND METHODS
Reagents and antibodies. Human CD3 monoclonal antibody (MAb) (clone
OKT3; Janssen-Cilag, Neuss, Germany) and CD2 MAb (clones T112and T113;
generously provided by Ellis Reinherz, Boston, Mass.) were used for T-cell
activation. For stimulation of mouse PBT, functional grade purified anti-mouse
CD3e (clone 145-2C11; eBioscience, San Diego, Calif.) was used. Fluorescein
isothiocyanate (FITC)-conjugated anti-cyclin B1, bromodeoxyuridine (BrdU),
CD28-FITC, ?/? T-cell receptor (TCR ?/?)-phycoerythrin (PE), TCR ?/?-FITC,
CD69-FITC, and PE-labeled anti-active caspase 3 were purchased from BD
Pharmingen (Heidelberg, Germany). CD3-PE, CD45RO-PE, and CD45RA-
FITC-labeled MAbs were obtained from DAKO (Hamburg, Germany). Second-
ary FITC-labeled goat anti-mouse was purchased from Biosource (Solingen,
Germany). A Vybrant CFDA SE (carboxyfluorescein diacetate succinimidyl es-
ter) cell tracer kit was obtained from Molecular Probes (Eugene, Oreg.). Pro-
pidium iodide (PI) was purchased from Calbiochem (Schwalbach, Germany). All
protease and phosphatase inhibitors used for Western blotting were purchased
from Sigma-Aldrich (Taufkirchen, Germany). The antibodies against human
caspase 3, DNA fragmentation factor, poly(ADP-ribose) polymerase, retinoblas-
toma (Rb) protein, cyclin A, cyclin D2, p21, p27, and p53 were purchased from
BD Pharmingen. Antibodies against human Bax, Bcl-2, cytochrome c, and E2F-1
were obtained from Santa Cruz Biotechnologies (Heidelberg, Germany). The
cytometric bead array kit was purchased from BD Pharmingen. Highly purified
lipopolysaccharide (LPS), generated from E. coli Nissle 1917, was provided by U.
Za ¨hringer (Forschungszentrum Borstel, Germany); synthetic bacterial lipopro-
teins (BLPs; Pam3-Cys-Ser-Lys-Lys-Lys-Lys-OH) and human serum albumin
were generously provided by A. Zychlinsky (Max Planck Institute for Infection
Biology, Department of Cellular Microbiology) (1); and immunostimulatory
DNA sequences (5?-TsCsgsTsCsgsTsTsTsTsgsTsCsgsTsTsTsTsgsTsCsgsTsT-3?)
were purchased from TIB MolBiol (Berlin, Germany). The Limulus amebocyte
lysate for detecting gram-negative bacterial endotoxin was obtained from Cam-
brex (East Rutherford, N.J.).
Preparation of T lymphocytes. Peripheral blood mononuclear cells (PBMC)
from healthy volunteers were isolated from heparinized venous blood by using
Ficoll-Hypaque density gradients. For isolation of PBT, PBMC were incubated
for 30 min at 4°C with magnetically labeled CD19, CD14, and CD16 antibodies
directed against B lymphocytes, monocytes, and neutrophils, respectively (Milte-
nyi Biotec Inc., Bergisch-Gladbach, Germany). T cells were then collected by
using a magnetic cell sorting system (MACS; Miltenyi Biotec Inc.). LPT were
isolated from surgical specimens obtained from patients admitted for bowel
resection for malignant and nonmalignant conditions of the large bowel, includ-
ing colon cancer and benign polyps as previously described (69). Briefly, the
dissected intestinal mucosa was freed of mucus and epithelial cells in sequential
washing steps with dithiothreitol and EDTA and digested overnight at 37°C with
collagenase and DNase. Mononuclear cells were separated from the crude cell
suspension by layering on a Ficoll-Hypaque density gradient. For LPT purifica-
tion, macrophage-depleted lamina propria mononuclear cells were incubated for
30 min at 4°C with magnetically labeled beads as described above and collected
by negative selection by using the MACS system. As assessed by flow cytometry,
the purified PBT and LPT populations contained ?99 and ?92% CD3?cells,
respectively. For isolation of naive (CD45RA?) and memory (CD45RO?) PBT,
PBMC were incubated for 30 min at 4°C with magnetically labeled CD14, CD16,
and CD19 antibodies in combination with CD45RO antibodies to deplete for
CD45RA?PBT or with CD45RA antibodies to negatively select the CD45RO?
population, respectively. LPT were 91% CD45-RO?, and PBT were 54% naive
T cells (CD45-RA?). The CD45RO-depleted PBT population was ?95%
CD45RA?and less than 5% CD45RO?, while the CD45RA-depleted PBT
population was ?95% CD45RO?and less than 5% CD45RA?. Further char-
acterization of the cell populations revealed that freshly isolated PBT were 48%
CD4?, 30% CD8?, and 0% CD69?. Freshly isolated LPT were 54% CD4?, 26%
CD8?, and also 0% CD69?.
Cells were cultured in complete medium (RPMI 1640, 10% fetal calf serum
[FCS], 1.5% HEPES buffer; Biowhittaker, Taufkirchen, Germany) containing 0,
2.5, 5, 10, 25, and 50% (vol/vol) E. coli Nissle 1917-conditioned medium (E. coli
Nissle 1917-CM) in a humidified incubator containing 5% CO2, alone or in the
presence of cross-linked plate-bound anti-CD3 MAb (OKT3; 10 ?g/ml) or sol-
uble anti-CD2 MAb pairs (T112and T113; 1:1000).
E. coli culture and generation of conditioned medium. E. coli Nissle 1917-CM
was generated as described by Yan and Polk (79). Briefly, E. coli Nissle 1917-
layered beads (provided by Ardeypharm GmbH) were incubated for 16 h at 37°C
in Luria-Bertani broth. The bacterial culture was then harvested by centrifuga-
tion at 1,000 ? g for 15 min. The supernatant was discarded, and the bacterial
pellet washed twice in phosphate-buffered saline (PBS) and then resuspended in
T-cell medium (RPMI, 10% FCS, 1.5% HEPES) without antibiotics. After 2 h at
37°C and 5% CO2, the culture was centrifuged at 1,000 ? g, and the supernatant
was recovered and sterile filtered through a 0.22-?m-pore-size syringe-driven
filter. The same method was used to generate conditioned media of E. coli strains
PZ 840, PZ 873, PZ 915, and DSM 498. In some experiments, E. coli Nissle
1917-CM was boiled for 1 h or frozen for 1 week at ?80°C, and 10% FCS and
1.5% HEPES were added after the medium was chilled on ice or thawed.
Additionally, heat-inactivated E. coli Nissle 1917 was prepared by resuspending
bacteria in PBS and inactivating them for 1 h at 65°C. The bacteria were then
washed twice and resuspended in complete medium. Proper inactivation was
controlled by inoculation of heat-inactivated E. coli Nissle 1917 into Luria-
Bertani broth and incubation for 24 h, demonstrating no bacterial growth. As
determined by using a Limulus amebocyte lysate, the E. coli Nissle 1917-CM
contained 1,300 ? 109 endotoxin units/ml.
Flow cytometry. Cell fluorescence was measured with a FACSCalibur (Becton
Dickinson, Heidelberg, Germany) flow cytometer at excitation wavelengths of
488 and 633 nm with band-pass filters optimized for individual fluorochromes.
Flow cytometry data were analyzed by using the CellQuest software program
Analysis of cell cycle phase distribution. Flow cytometry was performed after
staining for DNA content and cyclin B1 essentially as previously described (68).
Briefly, cells were washed twice with PBS, adjusted to 106cells/sample and fixed
in 90% methanol at ?20°C. After fixation, cells were washed and incubated for
45 min at 4°C with a cyclin B1 FITC-conjugated MAb. After the final wash, cells
were resupended in PBS and 5 ?l of RNase (0.6 ?g/ml, 30 to 60 Kunitz units;
Sigma-Aldrich), incubated at 37°C for 15 min, and then chilled on ice. A total of
125 ml of PI (200 ?g/ml) was added prior to analysis by flow cytometry. Each
analysis was performed on at least 25,000 events.
Cell surface staining for phenotypic analysis. Cell phenotype was analyzed by
using CD45RA-PE-, CD45RO-FITC-, and CD3-PE-conjugated MAbs (all from
Dako). The background level of immunofluorescence was determined by incu-
bating cells with FITC- or PE-conjugated mouse immunoglobulin G. After a
30-min incubation on ice, cells were washed twice in 1% bovine serum albumin-
PBS and fixed in 1% paraformaldehyde. Each analysis was performed on at least
Analysis of cell division. Analysis of cell division by dye dilution was per-
formed by using a Vybrant CFDA SE cell tracer kit (Molecular Probes). Cells
VOL. 73, 2005 E. COLI NISSLE 1917 AND T CELLS1453
were washed twice with cold PBS, resuspended in PBS with 5 ?M CFDA SE per
106cells, and incubated for 15 min at 4°C in the dark. The staining was quenched
by adding 5? cell culture medium containing 10% FCS. After staining, cells were
cultured alone (unstimulated) or with soluble anti-CD2 MAb pairs (T112and
T113; 1:1000) or cross-linked plate-bound anti-CD3 MAb (OKT3; 10 ?g/ml),
each with CD28 (5 ?g/ml) and interleukin-2 (IL-2; 20 U/ml). After 4 days, cells
were harvested, washed twice in cold PBS, fixed with 1% paraformaldehyde, and
analyzed by flow cytometry.
Measurement of DNA synthesis time, potential doubling times, G2/M phase
enter fractions. For the determination of S phase duration and potential dou-
bling times, cells were grown for 3 days with or without the respective stimuli and
then incubated for 60 min with 20 ?M BrdU, which was then replaced by
thymidine. At designated time points, cells were harvested and fixed with 90%
methanol. The BrdU-labeled nuclei were then stained with an FITC-conjugated
MAb against BrdU (BD Pharmingen). The nuclei were also stained with PI
following the protocols described above. The denaturation of DNA, allowing
antibody binding to the incorporated BrdU, was achieved by an acid treatment
according to a previously described protocol (68). Mathematical analysis was
performed according to the method of Begg et al. (5) and White et al. (78). The
movement of BrdU-labeled cells across S phase relative to the position of G1and
G2?M (relative movement [RM]) was calculated by the following equation:
RM?t? ? ?FS?t?? FG1?t??/?FG2?M?t?? FG1?t??
where FG1is unlabeled G1mean red fluorescence, FG2?Mis unlabeled G2?M
mean red fluorescence, and FSis the mean red fluorescence of the BrdU-labeled
cells at time t. S phase duration (TS) was calculated as the time for one unit of
relative movement. The potential doubling time was computed by the following
with v defined as ln[1 ? flu(t)/1 ? fld(t)/2], where flu(t) is the fraction of labeled,
undivided cells at time t and fld(t) is the fraction of labeled, divided cells at time t.
Determination of apoptosis and cell death. To determine the number of
apoptotic and necrotic cells, cells were stained with MAb against annexin V (BD
Pharmingen), to detect externalization of phosphatidylserine, and PI, to detect
necrotic cells. Cells were cultured as described, harvested at the respective time
points, stained with FITC-labeled annexin V and PI, and analyzed by flow
cytometry by using the CellQuest software program (BD Pharmingen). A min-
imum of 15,000 cells was analyzed in each case.
Western blotting. For immunoblotting cells were washed in PBS and lysed in
cell lysis buffer (1% Triton X-100, 0.5% NP-40, 0.1% sodium dodecyl sulfate,
0.5% sodium deoxycholate, 5 mM EDTA, 50 mM protease and phosphatase
inhibitor cocktails, 1 mM phenylmethylsulfonyl fluoride, 100 ?g of trypsin-chy-
motrypsin inhibitor per ml, 100 ?g of chymostatin per ml in PBS). The concen-
tration of proteins in each lysate was measured by using a Bio-Rad protein assay
(Mu ¨nchen, Germany). Equivalent amounts of protein (10 ?g) were fractionated
on a 10 to 20% Tris-glycine gel and electrotransferred to a 0.2-?m-pore-size
nitrocellulose membrane (Invitrogen, Karlsruhe, Germany). Membranes were
blocked with 5% milk in 0.1% Tween 20-PBS (Fisher Scientific, Schwerte, Ger-
many), followed by incubation for 60 min at room temperature with the indicated
primary antibody (all primary antibodies were used at a concentration of 2 mg
per ml of 5% milk in 0.1% Tween 20-PBS). The membranes were washed six
times with 0.1% Tween 20-PBS and then incubated for 1 h with the appropriate
horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnol-
ogy), washed, and incubated with the chemiluminescent substrate (Perkin-Elmer
Life Sciences, Rodgau-Ju ¨gesheim, Germany) for 5 min. The membranes were
then exposed to X-ray film (Amersham, Freiburg, Germany).
Measurement of cytokine secretion. To determine cytokine secretion, 105PBT
were cultured for 48 h with or without anti-CD3 MAb and incubated in the
presence or absence of 0, 10, or 25% (vol/vol) E. coli Nissle 1917-CM. The
supernatant was then collected, and cytokine secretion was determined by a
cytometric bead array, performed according to the manufacturer’s instructions
(BD Pharmingen). Briefly, bead populations with distinct fluorescence intensi-
ties, coated with capture antibodies specific for tumor necrosis factor ? (TNF-?),
gamma interferon (IFN-?), IL-10, and IL-2 proteins, were mixed with PE-
conjugated detection antibodies and incubated with recombinant standards or
test sample to form sandwich complexes. Following the acquisition of sample
data by flow cytometry, the cytokine concentrations were calculated by using
cytometric bead array analysis software (BD Pharmingen).
Mice. Mice were bred under specific pathogen-free conditions at the Max
Planck Institute for Infection Biology, Department of Cellular Microbiology.
Mice were housed in filter-top cages and provided with sterile water and food ad
libitum. TLR-2?/?mice were described previously (71, 77).
Statistical analysis. Statistical analysis was performed by using a paired Stu-
dent’s t test. Results are expressed as mean ? standard error of the mean (SEM),
and significance was inferred with P values ?0.05.
E. coli Nissle 1917-CM inhibits PBT cell cycle progression.
E. coli Nissle 1917 has beneficial effects in T-cell-mediated
intestinal diseases such as ulcerative colitis or pouchitis (35–37,
52). However, the underlying mechanisms still remain unclear.
We were therefore interested in the effects of E. coli Nissle
1917 on T-cell function. Culture of T cells in the presence of
living E. coli Nissle 1917 without antibiotics resulted in signif-
icant bacterial overgrowth and complete T-cell death within
12 h (data not shown). We therefore generated E. coli Nissle
1917-CM as described in Materials and Methods to examine
the effect of factors secreted by E. coli Nissle 1917 on cell cycle
progression of PBT. Immediately after isolation, PBT con-
tained ?98% cells in G0/G1phase. When PBT were stimulated
by anti-CD3 MAb for 3 days, the number of cells in the G0/G1
phase dropped to 71% ? 3.5%, while those in the S and G2/M
phases increased to 29% ? 2.1% (Fig. 1A). When 10, 25, or
50% (vol/vol) E. coli Nissle 1917-CM was added to the cell
culture, the number of cycling cells was significantly reduced
with only 21% ? 1.2%, 14% ? 1.6%, and 9% ? 1.1%, respec-
tively, of the cells in S and G2/M phase (Fig. 1A). The inhib-
itory effect of E. coli Nissle 1917-CM on PBT cell cycle pro-
gression was observed within 48 h of cell culture and started to
be detectable in concentrations as low as 2.5% E. coli Nissle
1917-CM (data not shown). To test if this effect is specific for
E. coli Nissle 1917, conditioned media from the nonprobiotic
E. coli strains DSM 498, PZ 840, PZ 873, and PZ 915 were
generated as described in Material and Methods and added to
anti-CD3-activated T-cell cultures. As shown in Fig. 1B, con-
ditioned media from other E. coli strains also inhibited PBT
cell cycle progression. Suppression of cell cycling by E. coli
Nissle 1917-CM was reversed when the conditioned medium
was washed out and replaced by medium that had not been in
contact with E. coli Nissle 1917 (data not shown). In order to
confirm that the inhibitory effect of E. coli Nissle 1917-CM on
PBT cell cycle progression was mediated by soluble factors
secreted by E. coli Nissle 1917 into the culture medium, PBT
were activated in the presence of heat-inactivated E. coli Nissle
1917 microorganisms. Under these conditions, neither cell cy-
cle entry nor progression through the cell cycle was affected
(data not shown).
To assess the functional relevance of the cell cycle inhibitory
effect of E. coli Nissle 1917-CM, we performed a stathmoki-
netic analysis to determine the effect of E. coli Nissle 1917 on
PBT cell cycle traverse times. This was accomplished by using
BrdU incorporation to model and measure cell cycle kinetics.
The S phase traverse times of PBT activated by CD3 signaling
and cocultured with E. coli Nissle 1917-CM was substantially
slower than that of PBT activated in the absence of E. coli
Nissle 1917 (Table 1). Calculation of potential doubling times
(Tpot) showed that in the absence of E. coli Nissle 1917-CM,
PBT required 24.7 ? 2.4 h to double when activated by anti-
CD3 MAb, whereas in the presence of 10 or 25% E. coli Nissle
1454STURM ET AL.INFECT. IMMUN.
1917-CM, anti-CD3-activated PBT needed 37.0 ? 4.9 and 88.2
? 9.6 h, respectively, to double their cell populations (Table 1).
Modulation of PBT cell cycle regulators by E. coli Nissle
1917. Having shown that E. coli Nissle 1917-CM modulates
PBT cell cycle kinetics, we next investigated how key regula-
tory molecules responsible for not only initiating and advanc-
ing but also inhibiting the cell cycle are modulated by E. coli
Nissle 1917-CM. Western blot analysis showed that in PBT
cyclin D2, the main regulator of the G1phase, was upregulated
by anti-CD3 activation and that this upregulation was pro-
foundly reduced by E. coli Nissle 1917-CM (Fig. 2). The anal-
ysis of the Rb protein, which is essential for G1/S phase tran-
sition, showed that an increase in Rb protein phosphorylation
was marked in CD3-activated PBT but, again, strongly inhib-
ited by E. coli Nissle 1917-CM (Fig. 2). The functional rele-
vance of this effect was confirmed when we showed that E2F-1
and cyclin A, both downstream and dependent on Rb protein
phosphorylation, were also downregulated by E. coli Nissle
1917-CM (Fig. 2). We also analyzed cyclin B1, responsible for
FIG. 1. E. coli Nissle 1917-CM inhibits cell cycle progression of PBT. (A) E. coli Nissle 1917-CM dose dependently decreases the number of
CD3-activated PBT in the S and G2/M phase. (B) CM (50% [vol/vol]) from the E. coli strains DSM 498, PZ 840, PZ 873, and PZ 915 decrease
the number of CD3-activated PBT in the S and G2/M phase. Freshly isolated PBT were cultured without stimulation (unstim) or in the presence
of cross-bound CD3 MAbs (stim) and in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM stimulated for 3 days as described in
Materials and Methods. Each phase of the cell cycle was assessed by measuring DNA content by PI staining followed by flow cytometry. The graphs
in panel A are representative of seven different experiments, and the graphs in panel B are representative of three different experiments. EcN, E.
coli Nissle 1917.
TABLE 1. DNA synthesis and potential doubling times of anti-
CD3-activated PBT are increased by E. coli Nissle 1917-CM
% (Vol/vol) E. coli
S phase traverse
14.2 ? 0.8
21.4 ? 2.1b
39.6 ? 5.6b
24.7 ? 2.4
37.0 ? 4.9b
88.2 ? 9.6b
aValues are means ? standard deviation (three samples per group).
bP ? 0.05 versus 0% E. coli Nissle 1917-CM.
VOL. 73, 2005E. COLI NISSLE 1917 AND T CELLS1455
leading the cells to mitosis after they passed the restriction
point at the G1/S interphase. To exactly localize the cyclin B1
increase within the cell cycle, DNA content was also deter-
mined by PI staining. As seen for early cell cycle regulators, E.
coli Nissle 1917-CM dose dependently inhibited anti-CD3-in-
duced expression of B1 (Fig. 3). To complement the analysis of
cell cycle promoters, we determined the expression levels of
key cell cycle inhibitors. Interestingly, even high doses of E. coli
Nissle 1917-CM failed to increase protein expression levels of
p21 or p53 (Fig. 3).
Effect of E. coli Nissle 1917 on activation-induced cell death
in PBT. Since cell cycling and apoptosis are intimately linked
(29, 75) and both features determine the life span of a cell, we
next analyzed whether E. coli Nissle 1917-CM induces or mod-
ifies T-cell death. When PBT were stimulated with anti-CD3
MAb for 3 days, the proportion of apoptotic cells increased
from 2% ? 0.9% to 5.8 ? 1.6% (data not shown). When
purified and anti-CD3 MAb-activated PBT were cultured for 2
days in the presence of 0, 10, 25 or 50% (vol/vol) E. coli Nissle
1917-CM, the rates of apoptosis and necrosis did not increase,
as determined by annexin V and PI staining (Fig. 4). Cell death
may be initiated by the triggering of various death pathways by
E. coli Nissle 1917 and then discontinued for unknown reasons.
We therefore analyzed the effect of E. coli Nissle 1917-CM on
key regulators of the intrinsic and extrinsic death pathways.
However, even high doses of E. coli Nissle 1917-CM did not
alter Bcl-2, Bax, or cytochrome c expression or induce caspase
3, caspase 8, or FLIP cleavage (data not shown).
Effect of E. coli Nissle 1917 on PBT expansion. The integra-
tion of all the cell cycle parameters studied above determines
the intrinsic capacity of a T-cell population to divide and ex-
pand in response to receptor-mediated activation. To address
the question of whether E. coli Nissle 1917 influences this
capacity, we determined the number of cell divisions in CFDA
SE-labeled PBT, activated in the presence or absence of E. coli
Nissle 1917-CM. After 4 days of stimulation with CD3, CD28,
and IL-2, four cell divisions containing 48% of the original
population were seen in PBT (Fig. 5). In contrast, when 10%
(vol/vol) E. coli Nissle 1917-CM was added, only 3% of equally
activated PBT expanded and generated at most three daughter
cell populations. This effect was augmented when the percent-
age of E. coli Nissle 1917-CM was increased to 25 or 50%
(vol/vol), resulting in an almost complete depression of PBT
expansion (Fig. 5).
FIG. 2. Differential expression of cell cycle regulators in PBT fol-
lowing culture with E. coli Nissle 1917-CM. Immunoblot analysis shows
downregulated expression of cyclin D2, Rb protein phosphorylation
(pRb), E2F-1, and cyclin A following culture with E. coli Nissle
1917-CM as outlined in Materials and Methods. In contrast, the ex-
pression of cell cycle inhibitors p21 and p53 is not altered by E. coli
Nissle 1917-CM. Freshly isolated PBT were cultured with anti-CD3
MAbs in the presence of 0, 10 and 25% (vol/vol) E. coli Nissle
1917-CM for 3 days, after which expression of cell cycle regulators was
assessed by Western blotting. Each panel is representative of four
different experiments. EcN, E. coli Nissle 1917.
FIG. 3. Culture with E. coli Nissle 1917-CM reduces the expression of cyclin B1 in PBT. Flow cytometric analysis shows decreased cyclin B1
expression in the G2/M phase of CD3-activated PBT compared to control cells following culture with E. coli Nissle 1917-CM. Freshly isolated PBT
were cultured with anti-CD3 MAbs in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 3 days, after which cyclin B1 expression
and DNA content were examined by flow cytometry. The figure is representative of seven different experiments. EcN, E. coli Nissle 1917.
1456 STURM ET AL.INFECT. IMMUN.
Modulation of LPT cell cycle and apoptosis by E. coli Nissle
1917. Our data clearly demonstrate that E. coli Nissle 1917
substantially inhibits cell cycle entry and progression of anti-
CD3-activated PBT in a dose-dependent manner. Thus, we
were interested to evaluate the effects of E. coli Nissle 1917 on
freshly isolated LPT, a population of effector-memory cells
(55) that shows unique T-cell features with respect to cell cycle
and apoptosis (47, 68). When LPT were stimulated by anti-
CD3 or anti-CD2 MAb for 3 days, 87% ? 3.8% and 85% ?
2.9% of the cells remained in G0/G1phase, respectively,
whereas 13% ? 1.2% and 15% ? 2.0% of the cells entered the
cell cycle, respectively (Fig. 6A). The number of cells resting in
G0/G1or cycling in S/G2/M phase remained essentially identi-
cal when 10, 25 or 50% (vol/vol) E. coli Nissle 1917-CM was
added to the cell culture (Fig. 6). The unresponsiveness of LPT
toward E. coli Nissle 1917-CM was confirmed when cyclin B1
expression was measured in anti-CD3- and anti-CD-2-acti-
vated LPT (data not shown). We then determined the effect of
E. coli Nissle 1917 on LPT apoptosis by using the same meth-
ods as described for PBT. When LPT were stimulated with
anti-CD3 or anti-CD2 MAb for 3 days, the proportion of
apoptotic cells markedly increased from 8% ? 2.5% to 28% ?
5.1% and 24% ? 4.7%, respectively (data not shown). Com-
parable to results with PBT, the number of apoptotic or ne-
crotic cells was not altered by adding 10, 25, or 50% (vol/vol)
E. coli Nissle 1917-CM to the cell culture (data not shown). To
complete the analysis of E. coli Nissle 1917-CM-induced effects
on LPT cell cycling and apoptosis, we assessed the effect of E.
coli Nissle 1917-CM on LPT expansion. As determined by
CFDA SE, when LPT were stimulated with CD3, CD28, and
FIG. 4. E. coli Nissle 1917-CM does not induce T-cell apoptosis or necrosis in PBT. Analysis of annexin V and PI levels revealed that E. coli
Nissle 1917-CM does not induce cell death in anti-CD3-activated PBT. Freshly isolated PBT were cultured with anti-CD3 MAb in the presence
of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 2 days, after which apoptosis and necrosis were assessed by annexin V and PI staining,
respectively. The figure is representative of three different experiments showing similar results. EcN, E. coli Nissle 1917.
FIG. 5. Expansion of CD3-activated PBT in the presence of E. coli Nissle 1917-CM. CD3 activation induces the generation of at least four cell
divisions comprising 48% of the original population in PBT. In contrast, when 10% (vol/vol) E. coli Nissle 1917-CM was added, only 23% of
similarly activated PBT expanded and generated at most three daughter cell populations. The addition of 25 or 50% E. coli Nissle 1917-CM nearly
completely eliminated PBT expansion. The first peak to the right represents the undivided cell population. The numbers indicate the percentages
of divided cells distributed in the subsequent peaks. Freshly isolated PBT were incubated with 5 ?M CFDA SE and then cultured with anti-CD3
and anti-CD28 MAbs and IL-2 in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 4 days, and cell divisions were determined
by flow cytometry. The figure is representative of four different experiments. EcN, E. coli Nissle 1917.
VOL. 73, 2005E. COLI NISSLE 1917 AND T CELLS 1457
IL-2, 30 to 35% of the cells expanded, generating one daughter
cell population (Fig. 7). In contrast to results with PBT, this
number remained constant in the presence of 0, 10, 25 and
50% (vol/vol) E. coli Nissle 1917-CM (Fig. 7).
The failure of LPT to show different patterns to progress,
expand, or undergo apoptosis following E. coli Nissle 1917
treatment may be due to either their residing in the interstitial
matrix of the lamina propria or their effector-memory pheno-
type. To address this point, PBT were fractionated into naı ¨ve
CD45RA?and effector-memory CD45RO?populations and
stimulated with anti-CD3 in the absence or presence of 25%
(vol/vol) E. coli Nissle 1917-CM. As shown in Fig. 8, 4.1% of
CD45RA?and 3.2% of CD45RO?T cells reached the G2/M
phase at day 3. When 25% (vol/vol) E. coli Nissle 1917-CM was
FIG. 6. E. coli Nissle 1917-CM does not inhibit cell cycle progression of LPT. The number of anti-CD3- or anti-CD2-stimulated LPT in the S
and G2/M phase remains unchanged, regardless whether 0, 10, 25 or 50% (vol/vol) E. coli Nissle 1917 was added. Freshly isolated LPT were
stimulated for 3 days with anti-CD3 or anti-CD2 MAbs and cultured in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM. Each
phase of the cell cycle was assessed by measuring DNA content by PI staining followed by flow cytometry. The graphs are representative of four
different experiments. EcN, E. coli Nissle 1917.
FIG. 7. Expansion of LPTs in the presence of E. coli Nissle 1917-CM. CD3- and CD2-activated LPT generated one daughter cell population,
comprising about 30% of the cell population. LPT expansion remained unchanged in the presence of 10, 25 and 50% (vol/vol) E. coli Nissle
1917-CM. The first peak to the right represents the undivided cell population. The numbers indicate the percentages of divided cells distributed
in the subsequent peaks. Freshly isolated LPT were incubated with 5 ?M CFDA SE and then cultured with anti-CD3 and anti-CD28 MAbs and
IL-2 or with anti-CD2 and anti-CD28 MAbs and IL-2 in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 4 days, and cell
divisions were determined by flow cytometry. The figure is representative of four different experiments. EcN, E. coli Nissle 1917.
1458 STURM ET AL.INFECT. IMMUN.
added to the cell culture, the number of cells in the G2/M
phase dropped significantly in both groups (Fig. 8).
Modulation of cytokine expression by E. coli Nissle 1917.
The release of cytokines is crucial for the differentiation of T
cells. Having demonstrated that E. coli Nissle 1917 substan-
tially inhibits PBT cell cycling, we were interested whether E.
coli Nissle 1917 modulates cytokine release patterns in T cells.
PBT were isolated and activated with anti-CD3 MAb in the
absence or presence of 10 and 25% (vol/vol) E. coli Nissle
1917-CM. After 3 days, the supernatants were collected, and
cytokine expression was assessed by using a cytometric bead
array assay. As depicted in Fig. 9, E. coli Nissle 1917-CM
significantly inhibited IL-2, TNF-?, and IFN-? production (P
? 0.01) in a dose-dependent manner. In contrast to the pro-
found downregulation of proinflammatory cytokines, the ex-
pression of IL-10 was markedly upregulated by E. coli Nissle
1917-CM (P ? 0.01) (Fig. 9).
Inhibiting costimulatory molecules by E. coli Nissle 1917.
Our data presented so far demonstrate that E. coli Nissle 1917
inhibits T-cell cycling early in the cell cycle. The response of a
cell upon antigen presentation and its subsequent activation
and cycling depends on cell activation via the TCR. In addi-
tion, costimulatory molecules such as CD2 and CD28 are cru-
cial to fully stimulate a T cell and avoid anergy (59). To inves-
tigate whether E. coli Nissle 1917 alters T-cell activation and
expression of costimulatory molecules, PBT were activated via
the CD3 pathway in the absence or presence of 25 and 50%
(vol/vol) E. coli Nissle 1917-CM, and the distributions of
CD69, CD2, and CD28 as well as TCR ?/? and TCR ?/? were
determined by flow cytometry. When PBT were activated by
anti-CD3 MAbs, 35% ? 3.1% of the cells were CD69 positive
after 24 h. When 25 or 50% (vol/vol) E. coli Nissle 1917-CM
was added to the culture medium, the number of CD69-posi-
tive cells dropped significantly to 12% ? 2.9% and 7% ? 1.9%,
respectively (P ? 0.05). In addition, as assessed by flow cytom-
etry, CD2 expression dropped by 46% ? 5.9% (P ? 0.01), and
CD28 expression decreased by 58% ? 4.7% (P ? 0.01). We
also determined the effect of E. coli Nissle 1917 on TCR ?/?
and TCR ?/? cell populations. When PBT were activated by
anti-CD3 MAb in the absence of E. coli Nissle 1917-CM, 94%
of the cell population contained TCR ?/? and 6% contained
TCR ?/? chains. Interestingly, when 50% (vol/vol) E. coli
FIG. 8. E. coli Nissle 1917-CM reduces cell cycling of CD45RA?PBT and CD45RO?PBT. The number of CD3-activated PBT in the S and
G2/M phase at 3 days dropped comparably in CD45RA?and CD45RO?PBT. Freshly isolated PBT were sorted in CD45RA?PBT and CD45RO?
PBT and cultured with anti-CD3 MAbs in the presence of 0 and 25% (vol/vol) E. coli Nissle 1917-CM for 3 days, after which cyclin B1 expression
and DNA content were examined by flow cytometry. The figure is representative of three different experiments. EcN, E. coli Nissle 1917.
VOL. 73, 2005E. COLI NISSLE 1917 AND T CELLS 1459
Nissle 1917-CM was added to the cell culture, the TCR ?/?-
positive cell fraction increased to 16% of the cells analyzed
(data not shown).
Effect of pathogen-associated molecular patterns on PBT
cell cycle. Having demonstrated that E. coli Nissle 1917-CM
inhibits cell cycle progression of activated PBT, we were next
interested to determine which mechanism mediates this effect.
We therefore first boiled and froze the E. coli Nissle 1917-CM
at ?80°C for a prolonged time. As depicted in Fig. 10, the
inhibitory effect of E. coli Nissle 1917-CM on T-cell cycling was
FIG. 9. Distinct cytokine expression profiles induced by E. coli Nissle 1917-CM in PBT. E. coli Nissle 1917-CM dose dependently and
significantly inhibited IL-2, TNF-?, and IFN-? production in PBT, while IL-10 production was markedly upregulated. ? Freshly isolated PBT (105
cells) were cultured for 3 days without stimulation or in the presence of anti-CD3 MAb and 0, 10, and 25% (vol/vol) E. coli Nissle 1917-CM.
Supernatants were then collected, and cytokine secretion was analyzed by using a cytometric bead array assay. Each bar represents mean ? SEM
of three experiments. *, P ? 0.05 for change versus CD3 with 0% E. coli Nissle 1917-CM. EcN, E. coli Nissle 1917.
FIG. 10. Effect of pathogen-associated molecular patterns on PBT cell cycle progression. E. coli Nissle 1917-CM inhibits PBT cell cycling of
anti-CD3-activated PBT, regardless whether the CM is frozen at ?80°C or heat inactivated as outlined in Materials and Methods. When E. coli
Nissle 1917 was heat inactivated before the T-cell medium was added, cell cycling was not impaired. Whereas E. coli Nissle 1917 LPS and CpG
DNA did not modulate PBT cycling, BLPs mimicked the effect of E. coli Nissle 1917-CM and substantially decreased PBT cycling. Freshly isolated
PBT were stimulated for 3 days with anti-CD3 MAb and cultured in the presence of the respective substances as described in Materials and
Methods. The percentages of cells in the S/G2/M phase was assessed by measuring DNA content by PI staining followed by flow cytometry. Each
bar represents the mean ? SEM of three to four experiments. *, P ? 0.05 for change versus control (0% E. coli Nissle 1917-CM). EcN, E. coli
1460STURM ET AL.INFECT. IMMUN.
preserved. This indicates that proteins are not likely responsi-
ble for the inhibitory effect of E. coli Nissle 1917 on the T-cell
cycle. We therefore analyzed the effect of bacterial products
such as LPS, BLPs, or immunostimulatory DNA (CpG DNA)
on the T-cell cycle. These pathogen-associated molecular pat-
terns (PAMP) have been demonstrated to activate the innate
immune response and to mediate their effects through differ-
ent TLRs (10). To investigate whether these PAMPs mimic the
effect of E. coli Nissle 1917-CM, PBT were stimulated for 3
days with anti-CD3 MAbs in the presence or absence of LPS
generated from E. coli Nissle 1917 (2 ?g/ml), BLP (100 ng/ml),
or CpG DNA (2 ?g/ml), and the number of cycling cells was
assessed by measuring their DNA content. As shown in Fig. 10,
whereas LPS and CpG DNA did not influence PBT cycling,
BLPs mimicked the effect of E. coli Nissle 1917-CM and sub-
stantially decreased PBT cycling (P ? 0.05) (Fig. 10). BLPs
signals through TLR-2, and just recently TLR-2 expression has
been described on activated T cells (32). We therefore isolated
PBT from wild-type and TLR-2 knockout mice and activated
the cells in the presence of 0, 10, and 25% (vol/vol) E. coli
Nissle 1917-CM and BLPs (100 ng/ml). As depicted in Fig. 11,
comparable to results with human PBT, E. coli Nissle 1917-CM
dose dependently inhibited cell progression into the S or G2/M
phase. In contrast, when PBT from TLR-2-deficient mice were
cultured in the presence of 0, 10, and 25% (vol/vol) E. coli
Nissle 1917-CM, the number of cycling cells remained essen-
tially unchanged (Fig. 11). In addition, by demonstrating that
BLPs inhibit cell cycle progression of wild-type but not TLR-
2-deficient PBT, we confirm the specificity of this pathway and
provide evidence that BLPs play a crucial role in the modula-
tion of T-cell cycle progression by binding to TLR-2 (Fig. 11).
The host must meet the challenging task of avoiding an
overly aggressive response to the 1014microorganisms consti-
tuting the intestinal microflora while also mounting an effective
immune response to intestinal pathogens and preventing bac-
terial spread within the organism (9). This generally tightly
regulated balance between tolerance and immunity is dis-
turbed in IBD, where insufficiently characterized environmen-
tal factors are assumed to induce and facilitate mucosal inflam-
mation in a genetically susceptible host (18, 61, 62). Although
the pathogenesis of IBD is not fully understood, it is widely
accepted that a disturbed T-cell function plays a crucial role in
the initiation and perpetuation of IBD (18). Whereas modu-
lation of a deregulated immune response is a fundamental
basis in the treatment of IBD, other contributors to disease
initiation and perpetuation such as the intestinal microflora
have been largely neglected (60). Recently, probiotic bacteria
have been used in the treatment of IBD and have been shown
to exert significant beneficial effects in ulcerative colitis and
pouchitis (20, 21, 35, 36, 46, 52). The aim of this study was,
therefore, to extensively investigate the effects of E. coli Nissle
1917 on peripheral and mucosal T cell function.
Proliferation of T cells in response to antigen stimulation is
necessary to expand the T-cell pool, generate effector cells,
and, thus, mount an effective immune response (25). We there-
fore assessed whether E. coli Nissle 1917 affects the initiation
and progression of T cells through the cell cycle. Culture of
activated PBT with E. coli Nissle 1917-conditioned T-cell me-
dium strongly inhibited cell cycle entry and progression. Re-
cently, it has been shown that the probiotic E. coli strain Laves
FIG. 11. E. coli Nissle 1917-CM does not inhibit cell cycle progression in TLR-2 deficient mice. E. coli Nissle 1917-CM dose dependently
decreases the number of CD3-activated PBT in the S and G2/M phase in wild-type mice but not TLR-2?/?littermates. Freshly isolated PBT were
stimulated for 3 days with mouse anti-CD3 MAb and cultured in the presence of 0, 10, and 25% (vol/vol) E. coli-CM and BLPs (100 ng/ml). Each
phase of the cell cycle was assessed by measuring DNA content by PI staining followed by flow cytometry. The graphs are representative of two
different experiments. EcN, E. coli Nissle 1917.
VOL. 73, 2005 E. COLI NISSLE 1917 AND T CELLS1461
1913 ameliorates experimental colitis in mice and reduces the
proliferative response of lymphocytes to specific bacterial an-
tigens (33). This is in accordance with our finding that cell cycle
progression was also inhibited by other E. coli strains and
suggests that the capability to modulate T-cell cycling is shared
by different E. coli strains.
Cell cycling is the result of traversing through individual
cycle phases, and the time a cell spends in each phase deter-
mines how quickly a T cell reaches mitosis and finally divides
(26). Activation of PBT with anti-CD3 MAb leads to a short S
phase, resulting in a 24-h potential doubling time. When PBT
were activated in the presence of E. coli Nissle 1917-CM, the
time spent in the S phase increased significantly, resulting in a
potential doubling time of up to 88 h when 25% (vol/vol) E.
coli Nissle 1917-CM was added. These results demonstrate that
E. coli Nissle 1917-CM significantly reduces the capacity of
PBT to expand following antigen stimulation, resulting in an
attenuated immune response.
To understand the molecular mechanisms underlying the
effect of E. coli Nissle 1917 on cell cycle control, we measured
expression levels of the key promoters responsible for the
progression of distinct phases of the cell cycle (cyclin D2, Rb
protein, E2F-1, cyclin A, and cyclin B1) (63), as well as relevant
inhibitors of T-cell cycling (p21 and p53) (4, 64). The delayed
cycling of PBT in the presence of E. coli Nissle 1917 after
stimulation with CD3 MAb coincided with a lack of upregula-
tion of cyclin D2. Furthermore, E. coli Nissle 1917 reduced
phosphorylation of Rb protein and thus the release of pocket
proteins such as E2F oncogene products (76). This step would
propel the cells beyond the restriction point at the G1/S inter-
phase, the so-called point of no return, where cell cycling is
independent from the initial activation signal and can only be
terminated by apoptosis (8, 67). Consequently, cell cycle pro-
moters of later cell cycle phases that depend on the phosphor-
ylation Rb protein were also downregulated by E. coli Nissle
1917. Although cell cycle progression was markedly decreased
by E. coli Nissle 1917, p21 and p53 were not upregulated,
indicating that no endogenous inhibitor can be assigned re-
sponsibility for this effect.
Apoptosis of T cells preserves immunological homeostasis
and tolerance by countering the profound changes in the num-
ber of T cells stimulated by diverse antigens (75). To prevent
autoimmunity or malignancy, the cell death program is initi-
ated to terminate cell function once the antigen has been
cleared or when cell proliferation is uncontrolled (39). Rheu-
matoid arthritis and IBD are characterized by an impaired
apoptosis of T cells, and drugs that induce apoptosis of T cells
or macrophages, like the anti-TNF-? antibody infliximab, play
an important role in treating these diseases (17, 41, 72, 73).
However, the immunosuppression induced by these drugs is
sustained and causes an increased rate of opportunistic infec-
tions (11, 17, 27, 28, 53). E. coli Nissle 1917-CM did not induce
apoptosis of T cells, demonstrating that E. coli Nissle 1917
selectively reduces the expansion of T cells by inhibiting their
proliferating capacity but not by inducing their death. Since the
full capacity to cycle was restored when E. coli Nissle 1917-CM
was removed, our findings demonstrate that the immunosup-
pressive effect of E. coli Nissle 1917 can be eliminated and is
reversible, unlike drugs that induce T-cell apoptosis.
LPT represent a highly specialized population of immune
cells dedicated to maintaining immunological homeostasis by
establishing a state of tolerance to dietary and bacterial anti-
gens and eliminating dangerous non-self antigens (14, 44, 66).
Local homeostasis requires a tight regulation of LPT re-
sponses, including proliferation, clonal expansion, cytokine
production, and achieving a balance between cell death and
survival (4, 42). Although E. coli Nissle 1917-CM significantly
suppresses PBT expansion, cell cycling of mucosal T cells was
not restrained, indicating that the effect of E. coli Nissle 1917
on T-cell cycling is distinct in different T-cell populations. E.
coli Nissle 1917-CM caused a comparable reduction of cell
cycle progression in CD3-activated CD45RA?and CD45RO?
PBT, suggesting that the lack of LPT to respond to E. coli
Nissle 1917-CM may not be solely caused by their memory
status. Other factors like the mucosal microenvironment and
the overall activation status may play an essential role. The
unique response pattern of LPT and PBT may provide a pos-
sibility to limit the risk of an impaired mucosal immune re-
sponse to potentially deleterious microbes and to downregu-
late PBT proliferation and expansion when PBT are actively
recruited into the mucosa during intestinal inflammation (6).
During intestinal inflammation, E. coli Nissle 1917 also trans-
locates from the gastrointestinal lumen into the lamina propria
and Peyer’s patches (57, 58) and there may come in contact
with PBT to decrease their expansion. However, migration of
E. coli Nissle 1917 into the lamina propria in the course of
intestinal inflammation is not a prerequisite for orally admin-
istrated probiotics to exert systemic immune-modulatory ef-
fects, since three orally administrated probiotics (Lactobacillus
casei, Lactobacillus gasseri, and Lactobacillus rhamnosus) have
been demonstrated to inhibit lymphoproliferation without in-
flammation in a mouse model (30).
An effective inflammatory immune response initially re-
quires recruitment of immunocompetent cells to the site of
inflammation and subsequently appropriate activation and reg-
ulation (6). Cytokines play a critical role in this setting, since
they regulate the proliferation and differentiation of T cells
and determine the course of an inflammatory process by re-
leasing pro- and anti-inflammatory cytokines. In accordance
with the demonstrated suppression of T-cell cycling, E. coli
Nissle 1917-CM reduced IL-2 secretion of activated PBT, in-
hibiting the capacity of PBT to cycle. The potent proinflam-
matory cytokines TNF-? and IFN-? have been demonstrated
to play a crucial role in the pathogenesis of IBD (18, 54). E. coli
Nissle 1917-CM profoundly reduced the secretion of these
cytokines, indicating that E. coli Nissle 1917-CM has immuno-
modulatory capacities beyond the inhibition of cell cycling. The
beneficial effects of E. coli Nissle 1917 on inflammatory pro-
cesses are further underlined by the upregulation of IL-10
secretion, a potent immunomodulatory cytokine with anti-in-
flammatory properties, which has beneficial effects in treating
mucosal inflammation (13, 45).
The expansion of T cells depends on their activation, which
is achieved by the presentation of antigenic peptides in the
context of the major histocompatibility complex to the TCR
(74). In addition, costimulatory molecules are needed to fully
activate T cells and avoid anergy (42, 59). E. coli Nissle
1917-CM significantly downregulates the expression of CD2
and CD28 and therefore reduces activation of PBT when they
are stimulated by anti-CD3 MAb. This finding indicates that E.
1462STURM ET AL.INFECT. IMMUN.
coli Nissle 1917 exerts its immunomodulatory effects early in
the activation process, an observation further supported by the
fact that E. coli Nissle 1917 downregulates protein expression
of early cell cycle regulators in PBT.
About 95% of PBT have TCRs that are composed of ? and
? polypeptide chains, and 5% of the PBT have a TCR com-
posed of ? and ? chains (2). TCR ?/? cells recognize antigens
only when they are bound to major histocompatibility complex
molecules (3). In contrast, TCR ?/? cells can recognize anti-
gens directly as intact proteins or nonpeptide compounds and
therefore play a crucial role in the immune response to micro-
bial pathogens (16). It has been shown that the number of ?/?
T cells is increased after infection with Escherichia coli in
C3H/HeN mice (48). In this study, we demonstrate for the first
time, that soluble factors released from E. coli Nissle 1917
increase the number of ?/? T cells and therefore increase the
capability of PBT populations to recognize microbial patho-
E. coli Nissle 1917-CM inhibited PBT cell cycling even when
the medium was frozen or boiled, suggesting that this phenom-
enon is not caused by secreted or shed peptides but may be
mediated through PAMPs. Interestingly, CM consisting of
heat-inactivated bacteria did not modulate cell cycling of PBT.
This may be explained by alterations of the bacterial wall or
BLPs during the boiling process of living bacteria. Another
explanation for this observation would be an interruption of
putative BLP modifications in living bacteria that occur before
shedding and that are essential for the binding of BLPs to
TLRs. To determine whether PAMPs may be involved in E.
coli Nissle 1917-mediated effects on T-cell function, the effects
of LPS, BLPs, or immunostimulatory DNA (CpG DNA) on
T-cell function were examined. Interestingly, only BLPs in-
duced a significant downregulation of PBT cell cycling that was
comparable to the effect of E. coli Nissle 1917-CM. BLPs act
mainly through TLR-2 (1) and act as adjuvants for human
T-cell responses (32, 65). Using TLR-2-deficient mice, we
could demonstrate that the suppressing effect of E. coli Nissle
1917-CM on PBT cell cycling is mediated by TLR-2, identify-
ing this pathway as a mediator of E. coli Nissle 1917 signaling
in PBT. This finding is supported by the fact that TLR-2 is
expressed on activated T cells (32). Rachmilewitz and cowork-
ers just published a study that shows that genomic DNA iso-
lated from VSL-3, a probiotic mixture containing viable lyoph-
ilized gram-positive bacteria, and E. coli DH5? ameliorate the
severity of dextran sulfate sodium-induced colitis (51). This
effect was mediated by TLR-9 signaling. Genomic DNA or
CpG DNA is known to signal via TLR-9 (24, 34), which is
expressed on antigen-presenting cells. T cells do not respond to
CpG DNA in the absence of dendritic cells (24), which is in
accordance with the results of our study, where CpG DNA did
not modulate T-cell cycling in a purified T-cell system, thus
demonstrating for the first time a direct interaction of probi-
otics with T cells via TLRs and suggesting different pathways
by which probiotics can mediate their effects.
In conclusion, we demonstrate evidence that E. coli Nissle
1917-CM and BLPs inhibit PBT cell cycling via the TLR-2
receptor pathway. In contrast to PBT cell cycling, LPT cell
cycling was not affected by E. coli Nissle 1917. E. coli Nissle
1917-CM inhibited activation of costimulatory molecules, re-
duced activation of PBT, and increased the proportion of ?/? T
cells. In addition, E. coli Nissle 1917-CM decreased the secre-
tion of proinflammatory cytokines and increased the secretion
of anti-inflammatory cytokines. Although a reduction of PBT
cell cycling was also observed to a lesser extent when condi-
tioned media were generated from other E. coli strains, E. coli
Nissle 1917 is currently the only E. coli strain with clinically
proven efficacy in the treatment of IBD while also fulfilling the
safety requirements necessary in the treatment humans. The
results of this study provide further evidence that probiotic
bacteria broadly influence the human immune system reveal an
underlying mechanism, and may explain the beneficial effects
of probiotic bacteria like E. coli Nissle 1917 in intestinal in-
This work was financially supported by the Deutsche Forschungsge-
meinschaft (grant STU247/2-1 and 247/3-1 to A.S.), ArdeyPharm
GmbH, and the Charite ´ Medical School, Berlin, Germany.
We thank Arturo Zychlinsky for helpful discussion and critical read-
ing of the manuscript, Johanna Harder-d’Heureuse and Diana Metzke
for technical assistance, and U. Zaehringer for providing purified E.
coli Nissle 1917 LPS. We also thank the Department of Surgery, DRK
Clinics Westend, for providing tissue samples.
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