Acetic acid bacteria (AAB), a group in the family Acetobacteraceae
comprising gram-negative bacteria, are present everywhere in our environment [1]. The oxidization capability of ethanol to produce
acetic acid has enabled us to create many kinds of traditional or industrial fermented foods
all over the world, such as kombucha, vinegars, and nata de coco. It has been well known
that foods fermented by using AAB have health-protecting or helth-improving effects
(antibacterial, antioxidant, anti-diabetic or anti-tumor effects) [2] and people have recently been getting more and more conscious of the
benefits. Among their benefits of AAB or their products, the anti-allergic effects have
attracted our interests now. As chronic inflammatory diseases, allergic diseases such as
food allergy, anaphylaxis, allergic rhinitis, and atopic dermatitis have spread worldwide,
especially in westernized countries, but effective treatments have remained to be
elucidated. On the other hand, early life microbial exposure to bacteria decreases the risks
of children and infants being affected by allergic diseases. It was suggested by the
significant increase in patients with allergic diseases, that has been thought to be the
result of epigenetic changes derived from changes in lifestyles of present-day children, who
have less chance to interact with diverse microbial environments [3]. There are several reports indicating the effects of AAB on
amelioration of allergic inflammatory responses [4,5,6,7].The administration of heat-killed Gluconacetobacter hansenii GK-1, a
strain of AAB, has also been proven to improve nasal rubbing counts in a pollen allergy mice
model [4]. The candidate G. hansenii
GK-1 components exhibiting the effect has been thought to be the lipopolysaccharide (LPS)
fraction binding to the TLR4 (Toll-like recepter 4) receptor on innate cells [5]. In addition, a randomized double-blind
placebo-controlled study of ingestion of G. hansenii GK-1 itself suggested
that G. hansenii GK-1 relieved daily nasal discomfort in Japanese subjects
[6]. Lipopolysaccharides (LPS) major outer membrane
components of the cell wall of gram-negative bacteria, have been reported to modulate
allergic immune responses via LPS tolerance evoked by long-term exposure to LPS [7, 8]. However, in
spite the reported benefits of AAB with respect to the improvement of allergic symptoms, the
accurate effective component and underlying mechanism remains unknown, although AAB could
play a role in ameliorating Th2 immune responses in allergic diseases.Thus, we analyzed the capability of different extracts obtained by preparation process for
LPS from G. hansenii GK-1 to inhibit the interleukin (IL) 4 production of
immune cells purified from food-allergic enteropathy model mice [9], which produce excessively IL-4, when they are stimulated with an
allergen, ovalbumin (OVA). The present study found that a hot water extracted fraction of
G. hansenii GK-1, which had low LPS activity, was a major component in
the AAB to show function in inhibiting the IL-4 production of innate immune cells stimulated
with OVA, leading to the promotion of Foxp3+CD4+T cell induction. The
LPS fraction lacked O-antigen and showed weaker biological activities.
MATERIALS AND METHODS
Mice
BALB/c mice (CLEA Japan, Inc., Tokyo, Japan), OVA23-3 mice, recombination-activating-gene
(RAG)-2-deficient OVA23-3 mice (R23-3 mice), and RAG-2-deficient DO11.10 mice (RD10 mice)
were used as experimental animals. OVA23-3 and R23-3 mice were donated by S. Habu (Tokai
University School of Medicine) [10] and were bred
by Sankyo Labo Service Corp. Inc. (Tokyo Japan). All mice were bred in a specific
pathogen-free; SPF environment at The University of Tokyo and maintained using γ-ray
irradiated feed (CE-2, CLEA Japan, Inc., Tokyo, Japan) and sterilized and deionized water.
In the experiment, 7-week-old mice with matching genders were used. All experiments were
performed in accordance with the guideline of The University of Tokyo. In the experiment,
the mice ingested freely a feed comprising an egg-white diet which protein source consists
of egg-white (an EW diet, Funabashi Farm, Chiba, Japan). A casein diet (CN diet, Funabashi
Farm), the protein source of which was cow casein, was used as a control for the EW
diet.
Preparation of extracts of G. hansenii GK-1
Extracts from G. hansenii GK-1 (FI, FII, FIII, and FIV) were prepared in
accordance with a previously reported procedure to purify LPS from Gram-negative bacteria
[11] and were provided by Kewpie Corporation,
Tokyo, Japan. The procedure is shown in Fig.
1A and the preparation process for each sample and the resulting products were as
follows: For FI, after culturing, the bacteria were centrifuged, washed, sterilized by
heating at 80°C for 1 min, and freeze-dried (killed bacteria). For FII, FI was extracted
with hot water at 90°C for 20 min (hot water extract). For FIII, FI was treated with the
hot phenol-water method [11], and the aqueous layer
obtained was ultra-filtrated and designated as FIII (phenol extract). For FIV, FIII was
further purified by repeating extraction with the hot phenol-water method and
ultrafiltration 3 times. LPS of Escherichia coli (0111:B4, Standard;
Invivogen, Carlsbad, CA, USA) was used as a control. The LPS contents were measured with
Limulus ES-II Single Test Wako (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). The
structure of LPS was analyzed by Tris-glycine SDS-PAGE analysis [12, 13]. The SDS-PAGE profile of
density gradient fractions was visualized by periodic acid-silver staining (Silver Stain 2
Kit for electrophoresis, FUJIFILM Wako Pure Chemical Corp.) [14].
Fig. 1.
Preparation procedure of each fraction extracted from G. hansenii
GK-1 acetic acid bacteria. (A) Preparation procedures for FI, FII, FIII, and FIV.
(B) Endotoxin activity of each extract of G. hansenii GK-1 (FI,
FII, FIII, and FIV) and that of E. coli (O111:B4) analyzed by
Limulus test. Each value is indicated as the average of three independent tests.
LPS: lipopolysaccharide.
Preparation procedure of each fraction extracted from G. hansenii
GK-1 acetic acid bacteria. (A) Preparation procedures for FI, FII, FIII, and FIV.
(B) Endotoxin activity of each extract of G. hansenii GK-1 (FI,
FII, FIII, and FIV) and that of E. coli (O111:B4) analyzed by
Limulus test. Each value is indicated as the average of three independent tests.
LPS: lipopolysaccharide.
Culture medium
To prepare complete RPMI medium, RPMI 1640 (10.2 g, Nissui Pharmaceutical Co., Ltd.,
Tokyo, Japan) was dissolved to 1 L of distilled water and sterilized. L(+)-glutamine
(0.03%, FUJIFILM Wako Pure Chemical Corp.), sodium hydrogen carbonate (0.2%, FUJIFILM Wako
Pure Chemical Corp.), penicillin G potassium (100 U/mL, Meiji Seika Pharma, Tokyo, Japan),
streptomycin (100 µg/mL, Meiji Seika Pharma), 2-mercaptoethanol (50 µM, FUJIFILM Wako Pure
Chemical Corp.) were further added (serum-free RPMI). Fetal calf serum (FCS; Gibco,
Carlsbad, CA, USA) was added to the serum-free RPMI to 10% (complete RPMI).
Cell preparation and culture
Mice were euthanized by cervical dislocation by experts, and spleen and mesenteric lymph
nodes (MLNs) were removed. The tissues were placed on dishes with complete RPMI on ice for
further analysis. To examine the responses of MLN cells, spleen cells, and
CD4+T cells to extracts of G. hansenii GK-1, MLN cells were
isolated by treatment with 1 mg/mL collagenase (FUJIFILM Wako Pure Chemical Corp.) and
1 mg/mL DNase I (Roche, Mannheim, Germany) in a tube with complete RPMI at 37°C and
stirred for 70 min. To prepare a single-cell suspension, the MLN cells were further passed
through a 100 µm cell strainer (Falcon, Corning, Corning, NY, USA), washed twice with, and
then re-suspended in complete RPMI. Spleen cells were isolated by hemolysis with
ammonium-chloride-potassium lysis buffer and washed with complete RPMI twice.
CD4+T cells (1×105 cells/well) prepared from MLNs or spleen cells
with a MACS cell separation system (Miltenyi biotec, Bergisch Gladbach, Germany) and
mitomycin C-treated spleen cells as antigen-presenting cells (APCs; 4×105
cells/well) were dispensed in a 96-well flat-bottomed plate (Falcon). To examine IL-4
production by spleen cells or CD4+T cells and APCs, OVA (Sigma-Aldrich, St.
Louis, MO, USA) at final concentrations of 0, 0.25, and 1 mg/mL and each extract at final
concentrations of 0, 0.05, 0.2, 1, and 5 µg/mL were added to the culture wells. The final
liquid volume was adjusted to 200 µL/well, the cells were cultured at 37°C in a 5%
CO2 incubator for 24 hr, and the supernatant was collected. To examine IL-6
production, spleen cells from BALB/c mice (2×106 cells/well) were incubated
with FIV or E. coli LPS (final concentrations, 0, 0.5, 1, 2, 5, and
10 µg/mL). Polymyxin B (PMB, InvivoGen) was diluted to the appropriate concentration and
added to the wells of the spleen cell culture with FIV (10 ng/mL) or E.
coli LPS (200 ng/mL). After culture for 24 hr, the supernatant was collected to
analyze IL-4 production.
Induction of regulatory T cells (Tregs)
The prepared MLNs cells from R23-3 or RD10 mice were dispensed into 96-well plates at
2.5×105 cells/well and incubated for 72 hr in 5% fetal calf serum (FCS)-RPMI
medium in a 5% CO2 incubator at 37°C in the presence of the following materials
at their final concentrations: OVA (0.25 mg/mL, Sigma-Aldrich), TGF-β (2 ng/mL, R&D
Systems, Minneapolis, MN, USA), retinoic acid (1 μM, FUJIFILM Wako Pure Chemical Corp.),
and rIL-2 (2 µg/mL, eBioscience, Waltham, MA, USA). After culturing, the collected cells
were used for analysis by flow cytometry to analyze Foxp3 expression and the supernatant
was collected to analyze IL-4 production.
Analysis of IL-4, IFN-γ, and IL-6 production
A sandwich ELISA was used for analysis of cytokine concentration in culture supernatants.
Rat anti-mouse IL-4 (11B11, BD Pharmingen, San Jose, CA, USA), IFN-γ (R4-6A2, BD
Pharmingen), or IL-6 (MP5-20F3, BD Pharmingen) antibodies in NaHPO3 buffer were
coated onto 96-well polystyrene plates (Invitrogen/Thermo Fisher Scientific K.K., Tokyo,
Japan) and incubated overnight at 4°C. After blocking with 3% BSA in phosphate-buffered
saline (PBS) plus 0.05% Tween®20 (PBS–Tween, Sigma Aldrich), samples diluted
with PBS–Tween were added to the wells and incubated for 2 hr at room temperature. After
washing, biotinylated rat anti-mouse IL-4 (BVD6-24G2, BD Pharmingen), anti-mouse IFN-γ
(XMG1.2, BD Pharmingen), or anti-mouse IL-6 (MP5-32C11, BD Pharmingen) antibodies were
added, and the samples were incubated for further 2 hr at room temperature. After washing,
alkaline phosphatase-labeled streptavidin (BD Pharmingen) diluted with PBS–Tween was added
and the samples were incubated for 1 hr at room temperature. After a final washing, an
enzyme substrate buffer solution, 0.1% p-nitrophenylphosphate, (Tokyo Chemical Industry,
Tokyo, Japan) in a diethanolamine buffer at pH 9.8, was added to the plates, and the
absorbance value of the solution in each well was recorded using an ELISA plate reader at
405 nm. The antibody titer was expressed as the optical density. The absorbance was
calculated with reference to standard curves for each recombinant cytokine: mouse rIL-4
(BD Pharmingen, Cat No. 554434), mouse rIFN-γ (BD Pharmingen, Cat No. 551216), or mouse
rIL-6 (R&D Systems).
Flow cytometry
PBS containing 1% FCS and 0.1% sodium azide (FUJIFILM Wako Pure Chemical Corp.) was used
as the FACS buffer. After collecting the cultured cells for each well and washing them
with FACS buffer, rat anti-mouse CD16/CD32 antibody (2.5 µg/mL of FACS buffer, BioLegend,
San Diego, CA, USA) was added to the cells, and the mixture was incubated at 4°C for
15 min. After washing, 30 µL of FITC-labeled anti-CD4 antibody (H129.19, BD Pharmingen)
was added, and the mixture was allowed to stand in a dark place at 4°C for 20 min. After
washing with FACS buffer, Foxp3 molecule was stained by using Foxp3 Staining Buffer Set
(Invitrogen) in according with the manufacturing procedure. Briefly, the cells were washed
with 1×permeabilization buffer (PB, 10 times diluted with Milli-Q). Rat anti-mouse
CD16/CD32 antibody diluted with PB (2.5 µg/mL) was added, and the mixture was reacted at
4°C for 15 min. After washing with PB, APC-labeled anti-Foxp3 antibody (FJK-16s,
eBioscience) diluted with PB was added for staining, and the mixture was incubated in the
dark at 4°C for 30 min. After washing, the cells were suspended in FACS buffer and
measured using FACSVerse (BD Biosciences, Franklin Lakes, NJ, USA). FlowJo (Tree Star,
Inc., Ashland, OR, USA) was used for the analysis.
Statistical analysis
Student’s t-test (Excel ver. 16.52) was used for comparisons between two groups in the
statistical analysis. For the multigroup comparisons, Dunnett’s test was performed using
the statistical analysis software R.
RESULTS
Lower LPS activities for each extract of G. hansenni than LPS of E. coli.
(0111:B4).
Each extract from G. hansenii GK-1 (FI, FII, FIII, and FIV) was prepared
in accordance with a procedure for purification of LPS from Gram-negative bacteria [11]. The procedure is shown in Fig. 1A, and the detailed preparation process for each sample and
the resulting products are described in the MATERIALS AND METHODS section. The activity of
the LPS of FIII (103.0 mg/one gram of biomass powder) was 40 to 300 times that of FI
(1.8 mg/g) or FII (1.0 mg/g) (Fig. 1B). In FI
and FII, the levels of LPS activity were extremely lower. To obtain FIV, the LPS of FIII
was further purified by extraction with the hot phenol-water method and ultrafiltration 3
times. The purity of FIV (96.6%) was calculated as the ratio of the weight of the
remaining component of the LPS fraction obtained as the FIV fraction, which was calculated
by subtracting the amount of the other components (protein, 6.6 µg; nucleic acid, 27.7 µg)
from the weight of the LPS fraction (1,000 µg) relative to the weight of the LPS fraction
(1,000 µg). The LPS activity of FIV (258.0 mg/g) calculated by the Limulus test was higher
than that of FIII (103.0 mg/one gram of biomass powder), but was lower than that of the
LPS preparation from E. coli (489.0 mg/g).
Suppression of IL-4 production stimulated by OVA by the hot water extract fraction of
G. hansenii GK-1 (FII) in spleen cells isolated from food-allergic model, OVA23-3
mice
We first analyzed the effect of each fraction (FI to FIII) extracted from G.
hansenii GK-1 on Th2-type responses in food-allergic model mice, OVA23-3.
Significant amounts of IL-4 and IFN-γ were produced by spleen cells purified from OVA23-3
mice, when they were stimulated with OVA (1 mg/mL). The IL-4 production was ameliorated by
addition of 0.05–1 µg/mL of FII; the level of inhibition was highest and most stable at
0.05 µg/mL of FII; the inhibitory effect was lost at higher than 5 µg/mL (Fig. 2A). IFN-γ production seemed to be inhibited at lower concentrations of FII (Fig. 2B). However, although Fig. 2B shows representative data of several repetitions of our
experiments, we could not reproduce the same results for the inhibition of IFN-γ
production at the same concentration of FII. These results indicated that FII did not have
the ability to suppress the IFN-γ production of spleen cells. A lower OVA level
(0.25 mg/mL) also promoted the IL-4 production of spleen cells of OVA23-3 mice, and the
production was inhibited by addition of FII without affecting IFN-γ, as shown in
Supplementary Fig. 1; however, the inhibitory effect of FII was lower under culture
condition in which spleen cells were stimulated with a low concentration of OVA
(0.25 mg/mL), compared with when they were stimulated with a higher concentration of OVA
(1 mg/mL). Therefore, we added OVA at a concentration of 1 mg/mL for further culture
analyses.
Fig. 2.
The hot water extract of G. hansenii GK-1 (FII) inhibited the IL-4
production of spleen cells, but not that of ovalbumin (OVA)-specific
CD4+T cells, purified from food-allergic model mice. (A, B) Spleen cells
of OVA23-3 mice or (C, D) spleen CD4+T cells of R23-3 mice and
antigen-presenting cells prepared from spleens of BALB/c mice were stimulated with
G. hansenii GK-1 fractions (FI, FII, and FIII; final
concentrations 0.05, 0.2, 1, and 5 µg/mL) and OVA (final concentration 1 mg/mL). The
cells were cultured for 24 hr, and (A, C) IL-4 and (B, D) IFN-γ production in the
collected culture supernatant was measured by ELISA. The cells of 4 mice were
pooled, cultured and measured for 3 wells under each condition. Significance was
determined by Dunnett’s test for each G. hansenii GK-1 fraction vs.
without each G. hansenii GK-1 fraction (*p<0.05, **p<0.01,
#p<0.1). The data are representative of two independent
experiments.
The hot water extract of G. hansenii GK-1 (FII) inhibited the IL-4
production of spleen cells, but not that of ovalbumin (OVA)-specific
CD4+T cells, purified from food-allergic model mice. (A, B) Spleen cells
of OVA23-3 mice or (C, D) spleen CD4+T cells of R23-3 mice and
antigen-presenting cells prepared from spleens of BALB/c mice were stimulated with
G. hansenii GK-1 fractions (FI, FII, and FIII; final
concentrations 0.05, 0.2, 1, and 5 µg/mL) and OVA (final concentration 1 mg/mL). The
cells were cultured for 24 hr, and (A, C) IL-4 and (B, D) IFN-γ production in the
collected culture supernatant was measured by ELISA. The cells of 4 mice were
pooled, cultured and measured for 3 wells under each condition. Significance was
determined by Dunnett’s test for each G. hansenii GK-1 fraction vs.
without each G. hansenii GK-1 fraction (*p<0.05, **p<0.01,
#p<0.1). The data are representative of two independent
experiments.R23-3 mice have only OVA-specific CD4+T cells, not B cells nor
CD8+T cells, as a responder to OVA stimulation [15]. In spleen cells of OVA23-3 mice, cells that play a role in the IL-4
inhibitory effect of FII were suggested to be either OVA-specific CD4+T cells
or other innate immune cells (APCs), or both. To clarify which cells responded to FII in
OVA23-3 mice, we used a CD4+T cell culture system in which OVA-specific
CD4+T cells purified from R23-3 mice were cultured with mitomycin C-treated
spleen cells from BALB/c mice as APCs and OVA as an antigen. In this culture system, APCs
hardly responded to any stimulatory materials. None of the three extracts (FI, FII, FIII)
showed significant inhibitory function with respect to IL-4 or IFN-γ production by immune
cells. (Fig. 2C and 2D).These results indicated that unlike the case of the spleen cell culture, FII extracted
from G. hansenii GK-1 did not show an inhibitory effect on IL-4
production in CD4+T cell-culture system, suggesting that FII acted on innate
immune cells as APCs. FII may affect OVA-activated CD4+T cells differently via
APCs depending on its concentration, as indicated by lower levels (0.05–0.2 µg/mL) of it
suppressing IL-4 production more effectively than higher levels (1–5 µg/mL).
The highly purified LPS fraction from G. hansenii GK-1 (FIV) biologically functioned
but weakly affected spleen cells of OVA23-3 mice
G. hansenii GK-1 has been reported to function in suppressing
development of pollen allergy, and the LPS was suggested to function through TLR4 [4, 5]. However,
the function of FIII, which corresponded to the crude LPS fraction, was lower than that of
FII in inhibiting IL-4 production by spleen cells of OVA23-3 mice (Fig. 2A). This result might have been caused by insufficient
purification of the LPS fraction (FIII). To confirm whether the LPS fraction of G.
hansenii GK-1 showed an inhibitory effect on IL-4 production, we examined the
activity of FIV in a spleen cell culture system. Because FIV inhibited the IL-4 production
of spleen cells by preliminary investigation, we directly compared its effect with that of
LPS purified from E. coli (InvivoGen). While the LPS from E.
coli tended to ameliorate the IL-4 production of spleen cells from OVA23-3 mice
at concentrations of more than 50 ng/mL when they were stimulated with OVA, similar
results were obtained in the case of FIV, but at concentrations of 200 ng/mL and less than
10 ng/mL (Fig. 3A). The inhibitory effects on IL-4 of each LPS from E. coli (200
ng/mL) or G. hansenii GK-1(10 ng/mL) tended to be abrogated by the
addition of the proper concentration of PMB (100 ng/mL, Supplementary Fig. 2).
Fig. 3.
Purified LPS from G. hansenii GK-1 (FIV) inhibited the IL-4
production of spleen cells from food-allergic model mice, but did not induce IL-6
production of BALB/c spleen cells.
(A) OVA23-3 spleen cells were stimulated with LPS fraction extracted from FIV or
E. coli LPS (final concentrations, 0.1, 0.5, 2, 10, 50, and 200
ng/mL) and OVA (final concentration 1 mg/mL). The cells were cultured for 24 hr, and
the production of IL-4 in the collected supernatant was measured by ELISA. (B)
BALB/c mouse spleen cells were stimulated with FIV and E. coli LPS
(final concentrations, 0.5, 1, 2, 5, and 10 µg/mL). The cells were cultured for 24
hr, and the production of IL-6 in the collected supernatant was measured by ELISA.
The cells were pooled for 3 mice, cultured and measured in 3 wells per each
condition. Significance was determined by Dunnett’s test for each sample
concentration vs a sample concentration of 0 ng/mL or 0 µg/mL (*p<0.05,
**p<0.01, ***p<0.001, #p<0.1). LPS: lipopolysaccharide; OVA:
ovalbumin; N.D.: not detected. The data are representative of two independent
experiments.
Purified LPS from G. hansenii GK-1 (FIV) inhibited the IL-4
production of spleen cells from food-allergic model mice, but did not induce IL-6
production of BALB/c spleen cells.(A) OVA23-3 spleen cells were stimulated with LPS fraction extracted from FIV or
E. coli LPS (final concentrations, 0.1, 0.5, 2, 10, 50, and 200
ng/mL) and OVA (final concentration 1 mg/mL). The cells were cultured for 24 hr, and
the production of IL-4 in the collected supernatant was measured by ELISA. (B)
BALB/c mouse spleen cells were stimulated with FIV and E. coli LPS
(final concentrations, 0.5, 1, 2, 5, and 10 µg/mL). The cells were cultured for 24
hr, and the production of IL-6 in the collected supernatant was measured by ELISA.
The cells were pooled for 3 mice, cultured and measured in 3 wells per each
condition. Significance was determined by Dunnett’s test for each sample
concentration vs a sample concentration of 0 ng/mL or 0 µg/mL (*p<0.05,
**p<0.01, ***p<0.001, #p<0.1). LPS: lipopolysaccharide; OVA:
ovalbumin; N.D.: not detected. The data are representative of two independent
experiments.LPS from E. coli induced strong production of IL-6 from BALB/c spleen
cells, while FIV induced only a minimal amount, with it rather inhibiting the IL-6
production of spleen cells from OVA23-3 mice (Fig.
3B). This may show another effect of FIV as a food compound to regulate immune
responses. However, because the biological activity of G. hansenii GK-1
LPS was affected by the storage period and was easily lost, the IL-4-supressive activity
of FIV was suggested to be less stable than that of FII. This shows that major components
of G. hansenii GK-1 extracts inducing IL-4 inhibition via immune cells
are possibly components other than the purified LPS fraction (FIV), although the LPS
fraction itself does possess the biological activity. In addition, the differences in
optimal concentration and stability in exhibiting IL-4 inhibitory effects between LPS from
E. coli and the LPS fraction of G. hansenii GK-1 (FIV)
were considered to be dependent on the differences in their structures.
Addition of FII to the Treg induction culture promotes Foxp3 molecule expression in
CD4+T cells of food-allergic model, R23-3 model mice
We clarified that both FII and FIV extracts from G. hansenii GK-1 had
the capability to inhibit the IL-4 production of OVA-specific spleen cells. This
inhibitory effect on IL-4 production may play a role in inducing Tregs in allergies,
because Foxp3+ expression is regulated by IL-4 treatment [16]. In R23-3 mice, an EW (egg-white) -diet significantly induced
weight loss, a representative factor of the induction of enteropathy, and in this weight
loss, IL-4-producing OVA-specific CD4+ T cells in MLNs have been reported to
play a critical role in this weight loss and intestinal inflammation [17]. In contrast to EW-fed R23-3 mice, EW-fed RD10 mice
(RAG-2-deficient OVA-T cell receptor genes transgenic mice similar to R23-3 mice but used
as tolerance-inducing model mice) did not exhibit weight loss [15]. Furthermore, in R23-3 mice that were fed the EW diet and showed
excessive IL-4 inflammatory responses, Treg induction was strongly obstructed for 7 days
after the start of EW-feeding compared with RD10 mice [15]. In addition, MLNs play an important role in Treg induction [18].Therefore, in this study, we analyzed whether the LPS fraction (FIV) from G.
hansenii GK-1 could ameliorate obstructed Treg induction in MLNs of EW-fed
R23-3 mice. Our FACS strategy is shown in Fig.
4A. EW-feeding induced significant weight loss in R23-3 mice compared with RD10 mice
(Supplementary Fig. 3), as reported previously [15]. The lower level (5.27%) of Treg induction in MLNs cells purified from R23-3
mice fed with the EW diet for 7 days showed significant (p<0.05) recovery as a result
of the addition of FII (6.24%; Fig. 4B, right),
but not of FIV. Under the same culture conditions, the level of Tregs in MLNs
CD4+Tcells of EW-diet fed RD10 mice was not inhibited (53. 3%), and the rate
was not improved by the addition of FII (52.3%; Fig.
4B, left). While the addition of FII to the culture did not promote Treg
development in CN-fed mice, FIV tended to increase the rate of Tregs in CN-fed RD10 mice
(p<0.101) but not in CN-fed R23-3 mice, suggesting that FIV showed function in inducing
Tregs under normal conditions that was independent of its IL-4-related function, when they
were stimulated with OVA (1 mg/mL). Strong IL-4 production in the culture supernatant was
observed in MLNs cells of EW-fed R23-3 mice, and the production tended to be inhibited by
the addition of FII (Fig 4C, R23-3, p<0.191).
On the other hand, no IL-4 was detected in the culture supernatants of MLNs cells from
EW-fed RD10 mice and CN-fed the mice (Fig 4C,
RD10), when they were stimulated with OVA (1 mg/mL).
Fig. 4.
The hot water extract from G. hansenii GK-1 (FII) induced
Foxp3+T cells in mesenteric lymph node (MLN) cells of R23-3 mice with
severe inflammation caused by EW-feeding.
(A) Representative plots of a scheme examining the Foxp3+T cells. (B)
Rates of Foxp3+T cells in CD4+T cells from MLN cells of RD10
(left) and R23-3 (right) mice fed with the EW-diet for 7 days (severe inflammation
phase). MLN cells were incubated under Foxp3+T cell induction conditions
stimulated with OVA (1 mg/mL), rIL-2, TGF-β, and retinoic acid, in the presence or
absence of FII or FIV. The collected cells were analyzed by flow cytometry. (C) IL-4
production in the culture supernatant was analyzed by ELISA. 〇 indicates the value
of each well, and the horizontal line indicates the mean of each group. The cells of
3 mice were pooled, cultured and analyzed for 3 wells for each condition.
Significance was determined by Student’s t-test (*p<0.05, #p<0.1).
The data are representative of two independent experiments.
The hot water extract from G. hansenii GK-1 (FII) induced
Foxp3+T cells in mesenteric lymph node (MLN) cells of R23-3 mice with
severe inflammation caused by EW-feeding.(A) Representative plots of a scheme examining the Foxp3+T cells. (B)
Rates of Foxp3+T cells in CD4+T cells from MLN cells of RD10
(left) and R23-3 (right) mice fed with the EW-diet for 7 days (severe inflammation
phase). MLN cells were incubated under Foxp3+T cell induction conditions
stimulated with OVA (1 mg/mL), rIL-2, TGF-β, and retinoic acid, in the presence or
absence of FII or FIV. The collected cells were analyzed by flow cytometry. (C) IL-4
production in the culture supernatant was analyzed by ELISA. 〇 indicates the value
of each well, and the horizontal line indicates the mean of each group. The cells of
3 mice were pooled, cultured and analyzed for 3 wells for each condition.
Significance was determined by Student’s t-test (*p<0.05, #p<0.1).
The data are representative of two independent experiments.
Lack of O-antigen in the LPS fraction (FIV) of G. hansenii GK-1
Because the biological activity of FIV was shown to be lower than that of E.
coli (0111:B4; Fig. 3), to clarify
structural differences in LPS between G. hansenii GK-1 and E.
coli (0111:B4), we conducted a Tris-glycine SDS-PAGE analysis and visualized
the SDS-PAGE profile of density gradient fractions by periodic acid oxidized silver
staining (Supplementary Fig. 4). The SDS-PAGE profile from LPS of E. coli
showed a clear ladder pattern of O-antigens, whereas that from G.
hansenii GK-1 lacked the pattern of O-antigens, although lipooligosaccharide
(LOS) was confirmed by a clear band, suggesting that the LPS of G.
hansenii GK-1 was comprised by only the LOS structure without O-antigen.
Endotoxin activity was two times stronger in LPS from E. coli
(489.0 mg/g) compared with that of FIV (258.0 mg/g; Fig. 1B). Therefore, cytotoxic effects of the LPS fraction of G.
hansenii GK-1 might be lower than that of LPS from E. coli,
because without O-antigen, cognate interaction and stimulation of signal transduction with
innate immune cells may be weakened in the LPS of G. hansenii GK-1 [19, 20].
DISCUSSION
In this study, we found that the hot water extract (FII) of G. hansenii
GK-1, not the LPS fraction (FIV) had the ability to inhibit the IL-4 production of spleen
cells from food-allergic model mice, leading to the promotion of
Foxp3+CD4+T cell induction in MLNs, which has a substantial role in
food-allergic intestinal inflammation [15]. In the
LPS fraction from G. hansenii GK-1 (FIV), the ability to induce
Foxp3+T cells was proven to be exhibited under normal conditions. Although FIV
could inhibit the IL-4 production of spleen cells, the biological activity was suggested to
be weaker than those of FII and the LPS from E. coli. used in our study.
The ability of FII from G. hansenii GK-1 suggested the possibility of using
the extracts in ameliorating food-allergic reactions. It is possible to provide some
scientific and underlying evidence for its effects from clinical observations of G.
hansenii GK-1: it reduced nasal discomfort in Japanese subjects [6], and it decreased rubbing counts in pollen-allergic
rhinitis model mice [4].The existence of effector-memory T cells and its strong Th2 responses has been known to
prevent the differentiation of naïve T cells into Foxp3+T cells. [16, 21].
Therefore, under severe allergic conditions, as in EW-fed R23-3 mice, the induction and
activation of Foxp3+T cells is suggested to be restricted. In EW-fed R23-3 mice,
EW-feeding for 28 days fully induced Foxp3+T cells, but they were observed to be
only slightly induced by day 7 of feeding [15].
However, FII promoted Foxp3+T cells induction from MLNs cells purified from the
model mice under the severe conditions of 7 days of feeding with the EW diet. Therefore,
even if the increase in the rate of Foxp3+T cells seemed to be small (5.27→6.24%,
Fig. 4), we thought that the potential of FII to
ameliorate allergic responses was rather high. The target cells of FII from G.
hansenii GK-1 were suggested to be innate immune cells, not CD4+T
cells, because the IL-4 levels in the supernatants of CD4+T cells cultured with
inactivated APCs plus OVA were not affected by the addition of FII. However, it remains
unknown whether IL-4 was produced by innate immune cells as well as CD4+T cells
in the spleen cells and how the extracts inhibit IL-4 production via innate immune cells in
the model mice.Examination of the FII used in the analysis for Supplementary Fig. 4, which was another lot
of the FII used in the analyses for Fig. 2 and
Supplementary Fig. 1, revealed that FII was mainly consisted of nucleic acids (65.5 mg /
gram of biomass powder; QuantiFluor dsDNA system, Promega, Madison, WI, USA) and other
components suggested to be proteins (29.3 mg/g), in addition to a small LPS fraction
(5.2 mg/g activity as measured by Limulus ES-2 Single Test WAKO). These results suggested
that contrary to our prediction that LPS, as a major component of AAB, has activity that
suppresses allergic symptoms, components other than the LPS fraction of G.
hansenii GK-1 functioned in vitro as major and stable components
that inhibited the IL-4 production of spleen cells from food-allergic model mice, although
in vivo, they may affect immune cells in cooperation with each other.LPS constitutes the outer membrane of Gram-negative bacteria and is composed of three
units: a hydrophilic polysaccharide, O-antigen, and lipid A, which known as the hydrophobic
domain. It generally functions as a strong immunomodulatory component by causing
proinflammatory responses in target cells. In the present study, we focused on LPS of AAB in
reducing allergic responses [3, 6] and examined the effects of the LPS fraction of G.
hansenii GK-1 on the regulation of IL-4 responses by using food-allergic model
mice. Although the LPS fraction of G. hansenii GK-1 (FIV) had the ability
to ameliorate the IL-4 production of OVA-activated spleen cells from OVA23-3 mice (Fig. 3), we thought that the activity was less stable
than for FII or LPS from E. coli. This is because the addition of PMB only
weakly abrogated the LPS activity inhibiting IL-4 production (Supplementary Fig. 2) and the
activity of FIV declined 1–2 months after preparation, even when preserved at −20°C. Unlike
the LPS from E. coli used in this study (Fig. 3, Supplementary Fig. 2), the LPS fraction of G. hansenii
GK-1 (FIV) was shown to be a form lacking O-antigen as a structure confirmed by the SDS-PAGE
profile in this experiment (Supplementary Fig. 4). LPS without O-antigen is one of the
mutant forms of LPS classified based on the absence or presence of O-antigen. The absence of
O-antigen in the LPS of G. hansenii GK-1 may the cause of the unstable
inhibitory effect on IL-4 production by spleen cells. Actually, the structure of the
O-antigen sugar moiety has been reported to affect proinflammatory cytokine production
(IL-6) and to determine the course and severity of urinary tract infections [19], and it has also been reported to affect IFN-γ
production in human NK cells [20]. Regarding
relationships between function of FIV and its structure, the endotoxin activities of FIV
were lower compared with those of LPS from E. coli. We also observed
significant IL-6 production in spleen cells from BALB/c mice stimulated with LPS from
E. coli, but the IL-6 production in these cells was inhibited by FIV.
Therefore, the present study suggests that the difference between the severe inflammatory
activities of LPS and its inhibitory function may be due to structural characteristics
related to the absence of O-antigen in the LPS of G. hansenii GK-1. This
characteristic of the LPS would enable us to use extracts of G. hansenii
GK-1 for improving allergic immune responses more safely than other Gram-negative bacteria,
even if other Gram-negative bacteria, such as E. coli, have similar
abilities to inhibit IL-4 production (Fig. 3A).
Reports indicating the improvement of pollen-allergic rhinitis by injection with G.
hansenii GK-1 suggested that a main source of the activity may be the LPS
fraction [4, 5].
Although our results suggest a weak effect of the LPS fraction (FIV) of G.
hansenii GK-1 in inducing Foxp3+T cells under strong IL-4 conditions,
the possibility remains that the G. hansenii GK-1 LPS fraction may function
in the mitigation of the symptoms under the appropriate conditions [22].Excess IL-4 production has been reported to prevent the expression of Foxp3 on
CD4+T cells [16]. In this study, it
remains to be clarified in detail whether the inhibitory effect of FII of G.
hansenii GK-1 directly promotes the differentiation of CD4+T cells
into regulatory T cells. Compared with spleen cells of EW-fed R23-3 mice, the spleen cells
of the RD10 mice, which exhibit tolerant characteristics when fed the EW diet, maintained a
higher level of Foxp3 expression in CD4+T cells (>52% in FII/RD10 mice) under
lower IL-4 conditions in the culture supernatant (Fig.
4). Therefore, we thought that the increase in the rate of Foxp3-expressing
CD4+T cells was caused by the addition of FII of G. hansenii
GK-1 and its IL-4 inhibitory effect, although further studies are needed. However, in the
EW-fed RD10 mice, we should have analyzed Foxp3+CD4+T cell induction
by suppressing IL-4 production during an earlier period of EW-feeding, in which spleen cells
would positively proliferate and produce IL-4 by stimulation with OVA. This is because we
previously reported that RD10 mice fed an EW diet for 7 days acquired tolerance [15], and this might have prevented us from observeing the
inhibitory activity of FII clearly.Inhibition of IL-4 production has been thought to play an important role in the regulation
of different allergic symptoms. The cytotoxicity of FII would be low, because
CD4+T cells of RD10 mice could differentiate into
Foxp3+CD4+T cells for 72 hr of incubation without dying, as shown in
Fig. 4. Therefore, although we could not observe
any effect of FII on Th1 responses, we think that G. hansenii GK-1, mainly
through FII, can possibly contribute to the suppression of IL-4 production in different
clinical allergic cases. Furthermore, the structural analysis using FIV urged us to suggest
that G. hansenii GK-1 and its extracts will contribute to improving
allergic responses as a safe and new types of food components from Gram-negative
bacteria.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.