Article

Bacillus subtilis-specific poly- -glutamic acid regulates development pathways of naive CD4+ T cells through antigen-presenting cell-dependent and -independent mechanisms

Department of Anatomy and Cell Biology, Institute of Biomedical Science, Hanyang University, Sungdong-Gu, Seoul, Korea.
International Immunology (Impact Factor: 2.54). 07/2009; 21(8):977-90. DOI: 10.1093/intimm/dxp065
Source: PubMed
ABSTRACT
Peripheral naive CD4+ T cells selectively differentiate to type 1 Th, type 2 Th and IL-17-producing Th (Th17) cells, depending on the priming conditions. Since these subsets develop antagonistically to each other to elicit subset-specific
adaptive immune responses, balance between these subsets can regulate the susceptibility to diverse immune diseases. The present
study was undertaken to determine whether poly-γ-glutamic acid (γ-PGA), an edible and safe exopolymer that is generated by
microorganisms such as Bacillus subtilis, could modulate the development pathways of Th subsets. The presence of γ-PGA during priming promoted the development of Th1 and Th17 cells but inhibited development of Th2 cells. γ-PGA up-regulated the expression of T-bet and ROR-γt, the master genes of Th1 and Th17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of Th2 cells. γ-PGA induced the expression of IL-12p40, CD80 and CD86 in dendritic cells (DC) and macrophages in a Toll-like receptor-4-dependent
manner, and the effect of γ-PGA on Th1/Th2 development was dependent on the presence of antigen-presenting cells (APC). Furthermore, γ-PGA-stimulated DC favored the
polarization of naive CD4+ T cells toward Th1 cells rather than Th2 cells. In contrast, γ-PGA affected Th17 cell development, regardless of the presence or absence of APC. Thus, these data demonstrate that γ-PGA has the potential
to regulate the development pathways of naive CD4+ T cells through APC-dependent and -independent mechanisms and to be applicable to treating Th2-dominated diseases.

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International Immunology, Vol. 21, No. 8, pp. 977–990
doi:10.1093/intimm/dxp065
ª The Japanese Society for Immunology. 2009. All rights reserved.
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Bacillus subtilis-specific poly-g-glutamic acid
regulates development pathways of naive CD4
1
T cells through antigen-presenting cell-dependent
and -independent mechanisms
Sunghoon Kim
1,
*, Jun Young Yang
2,
*, Kyuheon Lee
1
, Kyu Heon Oh
2
, Mia Gi
2
, Jung Mogg Kim
3
,
Doo Jin Paik
1
, Seokmann Hong
2
and Jeehee Youn
1
1
Department of Anatomy and Cell Biology, Institute of Biomedical Science, Hanyang University, 17 Haengdang-dong,
Sungdong-gu, Seoul 133-791, Korea
2
Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul
143-747, Korea
3
Department of Microbiology, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Seoul, Korea
Keywords: antigen-presenting cells, Bacillus subtilis, poly-c-glutamic acid, T
h
Abstract
Peripheral naive CD4
1
T cells selectively differentiate to type 1 T
h
, type 2 T
h
and IL-17-producing T
h
(T
h
17) cells, depending on the priming conditions. Since these subsets develop antagonistically to
each other to elicit subset-specific adaptive immune responses, balance between these subsets can
regulate the susceptibility to diverse immune diseases. The present study was undertaken to
determine whether poly-g-glutamic acid (g-PGA), an edible and safe exopolymer that is generated by
microorganisms such as Bacillus subtilis, could modulate the development pathways of T
h
subsets.
The presence of g-PGA during priming promoted the development of T
h
1 and T
h
17 cells but inhibited
development of T
h
2 cells. g-PGA up-regulated the expression of T-bet and ROR-gt, the master genes
of T
h
1 and T
h
17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of
T
h
2 cells. g-PGA induced the expression of IL-12p40, CD80 and CD86 in dendritic cells (DC) and
macrophages in a Toll-like receptor-4-dependent manner, and the effect of g-PGA on T
h
1/T
h
2
development was dependent on the presence of antigen-presenting cells (APC). Furthermore, g-PGA-
stimulated DC favored the polarization of naive CD4
1
T cells toward T
h
1 cells rather than T
h
2 cells. In
contrast, g-PGA affected T
h
17 cell development, regardless of the presence or absence of APC. Thus,
these data demonstrate that g-PGA has the potential to regulate the development pathways of naive
CD4
1
T cells through APC-dependent and -independent mechanisms and to be applicable to treating
T
h
2-dominated diseases.
Introduction
Upon antigen challenge, naive T cells undergo selective de-
velopment pathways to become effector cells. CD4
+
T
h
ef-
fector cells can be divided into distinct subsets, based on
the profile of cytokines they produce and their functional
capabilities (1, 2). The T
h
1 subset of CD4
+
T cells secretes
IFN-c and tumor necrosis factor (TNF)-b, which activate cell-
mediated immune responses optimally suited for immunity
to intracellular pathogens (3). In contrast, the T
h
2 subset
produces IL-4, -5, -6, -10 and -13, which are important for
clearing extracellular microbes (4). Factors that control dif-
ferentiation into T
h
1orT
h
2 cells include cytokine milieu, to
which naive T cells and antigen-presenting cells (APC) are
exposed during priming. IL-12 and IFN-c favor T
h
1 differenti-
ation and inhibit the emergence of T
h
2 cells. In contrast, the
presence of IL-4 early during priming stimulates the devel-
opment of T
h
2 cells and represses the production of T
h
1
cells (5). Recently, IL-17-producing T
h
(T
h
17) cells have
been identified as constituting a distinct T
h
effector
*These authors are co-first authors
Correspondence to: J. Youn; E-mail: jhyoun@hanyang.ac.kr, and S. Hong;
E-mail: shong@sejong.ac.kr
Received 24 April 2008, accepted 5 June 2009
Transmitting editor: K. Yamamoto Advance Access publication 26 June 2009
by guest on November 21, 2015http://intimm.oxfordjournals.org/Downloaded from
Page 1
population that differs from T
h
1 and T
h
2 cells (6). Besides IL-
17 (or IL-17A), T
h
17 cells secrete IL-17F, -6, -21 and -22. The
in vitro development of T
h
17 cells is promoted by the pres-
ence of transforming growth factor (TGF)-b and IL-6 in the
priming conditions and is negatively regulated by IFN-c and
IL-4 (7). IL-17 acts on a variety of cells resident in the local
tissue thus stimulating the secretion of other pro-inflamma-
tory mediators, such as IL-8, TNF, monocyte chemoattractant
protein-1 and granulocyte-macrophage colony-stimulating
factor (GM-CSF) (8). Therefore, the function of T
h
17 cells col-
lectively contributes to neutrophilia, the clearance of several
pathogens and in vivo expansion of hemopoietic progenitors.
In light of each subset-specific outcome of immune
responses, biased development of T
h
subsets has been
generally accepted to be responsible for the susceptibility
to various immune diseases. T
h
1 cell predominance over
T
h
2 cells can be a cause of organ-specific autoimmune dis-
eases, such as type 1 diabetes, Crohn’s disease and rheu-
matoid arthritis, whereas T
h
2-dominated responses take
subjects susceptible to asthma and allergic disorders (9–
12). Furthermore, evidence debating such a T
h
1/T
h
2 para-
digm shows, in addition to T
h
1 cells, that T
h
17 lineage is im-
plicated in the pathogenesis of rheumatoid arthritis, multiple
sclerosis and inflammatory bowel disease (13–15). In this re-
spect, agents that can regulate the selective development
pathways of T
h
subsets have therapeutic importance in
treating diverse immune diseases.
Poly-c-glutamic acid (c-PGA) is an anionic polymer that
is composed of
D- and L-glutamic acid units connected by
c-amide linkages between a-amino and c-carboxylic acid
groups (16). c-PGA is mainly produced by Bacillus subtilis
during fermentation processes of soybeans and not present
in humans. Unlike other pathogen-associated molecular pat-
terns (PAMP), c-PGA retains water soluble, biodegradable,
edible and non-toxic properties, raising interest in a broad
range of industries including food and cosmetics (17). Fur-
thermore, recent reports have indicated immune modulatory
activities of c-PGA or its derivatives. For instance, oral
administration of high molecular mass c-PGA (2000 kDa) en-
hanced NK cell-mediated anti-tumor activity in mice bearing
MHC class I-deficient tumors (18). Nanoparticles, composed
of c-PGA and
L-phenylalanine ethylester, served as an adju-
vant, which was able to activate dendritic cells (DC) and en-
hance adaptive immune responses against antigens carried
by the nanoparticles (19). These investigations not only sug-
gest that c-PGA may be potentially useful as effective ther-
apy for tumor and infectious diseases but also that the
action of c-PGA on immune cells may be more diverse than
the above investigations have shown.
The present study was undertaken to determine whether
B. subtilis-derived c-PGA is able to regulate the selective
development pathway of T
h
effector subsets. To this end,
murine naive CD4
+
T cells were forced to polarize under op-
timal conditions for T
h
1, T
h
2 and T
h
17 cell development in
the presence of c-PGA. The profiles of development were
evaluated in terms of cytokine production and subset-
specific master gene expression. We demonstrated that
c-PGA regulates T
h
1/T
h
2 cell development via an APC-
dependent pathway, and it regulates T
h
17 cell development
via APC-dependent and -independent pathways. Thus, our
results provide evidence of a novel action of c-PGA on the
immune system that has therapeutic potential to treat T
h
2
subset-related diseases.
Methods
Mice
Six-to-eight-week-old C57BL/6 mice were purchased from
the Orient-bio Laboratory (Seoul, Korea), and C3H/HeN and
C3H/HeJ strains were purchased from SLC Inc (Shizuoka,
Japan). Homozygous ovalbumin (OVA) peptide
323–339
-
specific TCR-transgenic OT-II mice on C57BL/6 background
were purchased from the Jackson Laboratory. IL-12p40-
yellow fluorescence protein (YFP) reporter (hereafter yet40)
mice were backcrossed seven generations to C57BL/6 and
intercrossed to derive homozygous knockin mice (20, 21).
All mice were kept under specific pathogen-free conditions
at the animal facility of Hanyang University and Sejong Uni-
versity. All animal experiments conducted in this study were
carried out in accordance with guidelines provided by both
universities.
Reagents and antibodies
OVA peptide
323–339
(ISQAVHAAHAEINEAGR) was synthesized
by the Peptron (Daejeon, Korea). CpG oligodeoxynucleotide
(5#- TCCATGACG TTCCTGACGTT-3#) was manufactured by
Geno Tech (Daejeon, Korea) and LPS derived from Escherichia
coli 026:B6 was purchased from Sigma. c-PGA was donated
by Bioleaders (Daejeon, Korea). c-PGA was dissolved in
a neutral pH buffer, diluted in PBS and used at 1 mg ml
1
of final concentrations, except concentrations as specifically
indicated. The levels of endotoxin and peptidoglycan con-
tained in the 1 mg ml
1
c-PGA solution was <0.01 EU ml
1
and <10 pg ml
1
when measured by Limulus amebocyte
lysate assays (Cambrex Bio Science) and silkworm larvae
plasma assays (Wako Pure Chemicals), respectively. The
mAbs and reagents used for surface and intracellular FACS
were as follows: anti-CD4-PerCP, anti-IL-4-PE, anti-IL-17-PE,
anti-CD11c-PE, anti-CD11b-PE, anti-CD11b-tri-color (TC),
anti-CD11c-APC, biotinylated anti-CD86, streptavidin-PE,
streptavidin-TC (all from BD Biosciences), anti-CD80-biotin
(Caltag) and anti-IFN-c-FITC and anti-TLR4-PE (eBio-
science).
Generation of bone marrow-derived dendritic cells
Bone ma rrow-derived dend ritic cells (BMDC) were gener-
ated from the bone marrow cells as described (22). In
brief, bone marrow cells were flushed with complete
RPMI-1640 medium from femurs and tibiae of yet40 mice
and s ubsequently er ythrocytes were removed using ACK
lysis b uffer ( 0.15 M NH
4
Cl, 10 mM KHCO
3
,2mMEDTA).
The ce lls were washed with PBS and cul tured for 5–8 days
in the medium supplemented with either 100 ng ml
1
FMS-like tyrosine kinase 3 ligand (Flt3L) or 40 ng ml
1
GM-CSF plus 10 ng ml
1
IL-4 (all from R&D Systems).
The BMDC were harvested and stimulated for 16 h with
5 lgml
1
CpG, 2 lgml
1
LPS or 1 mg ml
1
c-PGA, fol-
lowed by FACS.
978 c-PGA effects on T
h
cell differentiation
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to c-PGA. Our results also suggest that a putative receptor
for c-PGA mi ght be expressed by CD 4
+
T cel ls, which can
directly trigger APC-independent pathways of adaptive im-
mune regulation.
Molecules regulating T
h
1/T
h
2 polarization pathways have
been implicated in therapeutic intervention in many immune
diseases. A well-established example comes from bacterial
CpG DNA. The therapeutic effects of CpG on allergies have
been demonstrated by the selective down-regulation of T
h
2
type responses in murine models of asthma and in human
PBMC from atopic patients (31–33). The effects of c-PGA on
selective up-regulation of T
h
1 responses and down-regula-
tion of T
h
2 responses might have the therapeutic potential
to treat diseases whose pathogenesis largely relies on anti-
gen-specific T
h
2 responses, such as asthma and atopic der-
matitis. One issue with which to be concerned is that, in
addition to T
h
1/T
h
2 regulation, c-PGA favors the polarization
of CD4
+
T cells toward T
h
17 cells. It was shown recently that
although IL-17 was essential during antigen sensitization to
establish allergic asthma, in sensitized mice it attenuated
the allergic response by inhibiting DC and chemokine syn-
thesis (34). In this context, the characteristic of c-PGA on
T
h
17 cell promotion could be beneficial in treating estab-
lished allergic asthma.
Our results suggest the potential of c-PGA reacting as an
adjuvant for T
h
1 and T
h
17 response augmentation. On the
other hand, we found that the ability of c-PGA to activate
DC seemed to be less potent than LPS and CpG. In fact,
many potent adjuvants including CFA and LPS induce se-
vere local reactions and were excluded from clinical use for
humans, despite a potent activity of immune modulation.
Aluminum phosphate or hydroxide are relatively safe and
currently used as predominant adjuvants in humans. These
compounds are able to induce T
h
2 responses, yet they have
little capacity to stimulate T
h
1 responses. Therefore, it
appears mandator y to develop new adjuvants that are less
toxic and more effective in T
h
1 response induction. In this re-
spect, despite less potent activity, edible and non-toxic fea-
tures of c-PGA could contribute to the usefulness of c-PGA
as an immunomodulatory agent retaining minimized side
effects.
Previously, fermented soyb eans have been shown to
possess anti -tumor a nd i mmunomod ulator y ac tivitie s (35,
36). In particular, among products from the soybean fer-
mentation process, a fructose polymer named levan was
found to be a potent induc er of I L-12p40 and TNF- a pro-
duction by macrophages in vitro via TLR-4 (37). Moreover,
levan suppressed allergic inflammation with IgE produc-
tion, thus offering a n approach for the prevention of al ler-
gic diso rders. The latter paper reporte d that c-PGA did
not mediate such effects, whic h conflicts with our results.
This discrepancy may arise from differences of experi men-
tal systems including dose and molecular weight of
c-PGA.
In summary, we demonstrated for the first time that c-PGA
is a novel regulator of selective development pathways of T
h
subsets through APC-dependent and -independent mecha-
nisms. Our findings suggest that this aspect of c-PGA may
be applicable to preventing or treating T
h
2 response-related
diseases.
Funding
Seoul R&BD Program (10580).
Acknowledgements
We thank Dr Moon Hee Sung for p roviding c-PGA, Dr Chul Hoon
Lee for valuable discussion and Ms Jocelyn Graf for editorial
assistance.
Abbreviations
APC antigen-presenting cells
BMDC bone marrow-derived dendritic cells
DC dendritic cells
Flt3L FMS-like tyrosine kinase 3 ligand
GM-CSF granulocyte–macrophage colony-stimulating factor
b
2
m b
2
microglobulin
OVA ovalbumin
PAMP pathogen-associated molecular patterns
c-PGA poly-c-glutamic acid
PRR pattern recognition receptors
TC tri-color
TGF transforming growth factor
T
h
17 IL-17-producing T
h
TLR Toll-like receptor
TNF tumor necrosis factor
Treg regulatory T
YFP yellow fluorescence protein
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    • "Thus, we examined whether TLR4 is responsible for γPGA-mediated basophil reduction using TLR4-mutant C3H/HeJ and TLR4-sufficient C3H/HeN mice; as expected, γPGA treatment did not diminish the basophil population in C3H/HeJ mice unlike in C3H/HeN mice, suggesting that basophil reduction by γPGA is mediated through the TLR4 pathway (Fig 2D). Moreover, because DCs are known to initiate γPGA-mediated immune responses [17, 19], we analyzed the effect of γPGA on DCs. A single in vivo injection of γPGA up-regulated IL12 production and the expression of MHC class II molecules and costimulatory molecules such as CD86 in DCs (Fig 2E) . "
    [Show abstract] [Hide abstract] ABSTRACT: Recent studies have demonstrated that Bacillus subtilis-derived poly-gamma glutamic acid (γPGA) treatment suppresses the development of allergic diseases such as atopic dermatitis (AD). Although basophils, an innate immune cell, are known to play critical roles in allergic immune responses and repeated long-term administration of γPGA results in decreased splenic basophils in an AD murine model, the underlying mechanisms by which γPGA regulates basophil frequency remain unclear. To investigate how γPGA modulates basophils, we employed basophil-mediated Th2 induction in vivo model elicited by the allergen papain protease. Repeated injection of γPGA reduced the abundance of basophils and their production of IL4 in mice, consistent with our previous study using NC/Nga AD model mice. The depletion of basophils by a single injection of γPGA was dependent on the TLR4/DC/IL12 axis. CD1d-dependent Vα14 TCR invariant natural killer T (iNKT) cells are known to regulate a variety of immune responses, such as allergy. Because iNKT cell activation is highly sensitive to IL12 produced by DCs, we evaluated whether the effect of γPGA on basophils is mediated by iNKT cell activation. We found that in vivo γPGA treatment did not induce the reduction of basophils in iNKT cell-deficient CD1d KO mice, suggesting the critical role of iNKT cells in γPGA-mediated basophil depletion at the early time points. Furthermore, increased apoptotic basophil reduction triggered by iNKT cells upon γPGA stimulation was mainly attributed to Th1 cytokines such as IFNγ and TNFα, consequently resulting in inhibition of papain-induced Th2 differentiation via diminishing basophil-derived IL4. Taken together, our results clearly demonstrate that γPGA-induced iNKT cell polarization toward the Th1 phenotype induces apoptotic basophil depletion, leading to the suppression of Th2 immune responses. Thus, elucidation of the crosstalk between innate immune cells will contribute to the design and development of new therapeutics for Th2-mediated immune diseases such as AD.
    Full-text · Article · Apr 2016 · PLoS ONE
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    • "co-stimulatory molecules [5] . CD11c + splenic DCs and bone marrow-derived DCs express IL12p40, CD40, and CD86 in response to cPGA stimulation. "
    [Show abstract] [Hide abstract] ABSTRACT: Bacillus subtilis-derived poly-γ-glutamic acid (γPGA) stimulates dendritic cells (DCs) to produce IL12, leading to CD4+ T cell differentiation toward the Th1 phenotype, but DCs consist of heterogeneous subpopulations with a variety of immune functions. Among these, natural killer dendritic cells (NKDCs) play an important role in anti-tumor immune responses. Herein, we demonstrate the role of NKDCs in γPGA-meditated anti-tumor immune responses. NK1.1+ CD11c+ NKDCs were stimulated upon γPGA stimulation in vitro and in vivo to up-regulate lymphocyte activation markers, MHC class I and II, and co-stimulatory molecules. In particular, NKDCs were activated by γPGA to produce IFNγ and TNFα, like NK cells, as well as IL12, like DCs, implying that NKDCs have unique and multifunctional roles. Importantly, NKDCs stimulated by γPGA conferred stronger anti-tumor effects in mice and showed increased cytotoxicity against various tumor cell lines in vitro. In conclusion, NKDCs are one of the key players in anti-tumor immunity induced by γPGA.
    Full-text · Article · Dec 2013 · Biochemical and Biophysical Research Communications
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    • "Nanoparticles, composed of í µí»¾-PGA and L-phenylalanine ethylester, served as an adjuvant, which was able to activate dendritic cells (DC) and enhance adaptive immune responses against antigens carried by the nanoparticles [17]. Bacillus-derived í µí»¾-PGA regulates the developmental pathways of naive CD4 T cells through antigen-presenting cell-dependent and -independent mechanisms [18]. Moreover , recently, Lee et al. reported that Bacillus-derived í µí»¾-PGA attenuates allergic airway inflammation through a toll-like receptor-4-dependent pathway in a murine model of asthma [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Poly- γ -glutamic acid ( γ -PGA), naturally secreted from various strains of Bacillus, has anti-inflammatory activity. In inflammatory bowel disease (IBD), inflammation is promoted and sustained by angiogenesis; however, the role played by γ -PGA in this condition is unclear. Therefore, we evaluated γ -PGA effects on angiogenesis and inflammation in a dextran sulfate sodium- (DSS-) induced mouse colitis model. Experimental colitis was induced in male C57BL/6 mice by administering 3% DSS. Disease activity index (DAI), histopathological scores, microvascular density, myeloperoxidase activity, and VEGF-A and VEGFR2 expression were compared among control mice, DSS-treated mice, and mice receiving 3% DSS along with γ -PGA at 50 mg/kg body weight per day or 3% DSS with γ -PGA at 200 mg/kg body weight per day. We found that γ -PGA significantly attenuated weight loss, DAI, and colon shortening. γ -PGA also significantly reduced histopathological evidence of injury. Moreover, γ -PGA significantly attenuated DSS-induced blood vessel densities. Furthermore, γ -PGA attenuated DSS-induced expression of VEGF-A and its receptor, VEGFR2. In addition, γ -PGA treatment led to reduced recruitment of leukocytes to the inflamed colon. Therefore, our results indicate that γ -PGA has potential application in conditions marked by inflammatory-driven angiogenesis and mucosal inflammation.
    Full-text · Article · Dec 2013 · Mediators of Inflammation
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