International Immunology, Vol. 21, No. 8, pp. 977–990
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 CD41
T cells through antigen-presenting cell-dependent
and -independent mechanisms
Sunghoon Kim1,*, Jun Young Yang2,*, Kyuheon Lee1, Kyu Heon Oh2, Mia Gi2, Jung Mogg Kim3,
Doo Jin Paik1, Seokmann Hong2and Jeehee Youn1
1Department of Anatomy and Cell Biology, Institute of Biomedical Science, Hanyang University, 17 Haengdang-dong,
Sungdong-gu, Seoul 133-791, Korea
2Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul
3Department of Microbiology, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Seoul, Korea
Keywords: antigen-presenting cells, Bacillus subtilis, poly-c-glutamic acid, Th
Peripheral naive CD41T cells selectively differentiate to type 1 Th, type 2 Thand 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-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 Thsubsets.
The presence of g-PGA during priming promoted the development of Th1 and Th17 cells but inhibited
development of Th2 cells. g-PGA up-regulated the expression of T-bet and ROR-gt, the master genes
of Th1 and Th17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of
Th2 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 Th1/Th2
development was dependent on the presence of antigen-presenting cells (APC). Furthermore, g-PGA-
stimulated DC favored the polarization of naive CD41T cells toward Th1 cells rather than Th2 cells. In
contrast, g-PGA affected Th17 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
CD41T cells through APC-dependent and -independent mechanisms and to be applicable to treating
Upon antigen challenge, naive T cells undergo selective de-
velopment pathways to become effector cells. CD4+Thef-
fector cells can be divided into distinct subsets, based on
the profile of cytokines they produce and their functional
capabilities (1, 2). The Th1 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 Th2 subset
produces IL-4, -5, -6, -10 and -13, which are important for
clearing extracellular microbes (4). Factors that control dif-
ferentiation into Th1 or Th2 cells include cytokine milieu, to
which naive T cells and antigen-presenting cells (APC) are
exposed during priming. IL-12 and IFN-c favor Th1 differenti-
ation and inhibit the emergence of Th2 cells. In contrast, the
presence of IL-4 early during priming stimulates the devel-
opment of Th2 cells and represses the production of Th1
cells (5). Recently, IL-17-producing Th (Th17) cells have
beenidentified as constitutinga distinctTh
*These authors are co-first authors
Correspondence to: J. Youn; E-mail: email@example.com, and S. Hong;
Received 24 April 2008, accepted 5 June 2009
Transmitting editor: K. YamamotoAdvance Access publication 26 June 2009
by guest on November 21, 2015
population that differs from Th1 and Th2 cells (6). Besides IL-
17 (or IL-17A), Th17 cells secrete IL-17F, -6, -21 and -22. The
in vitro development of Th17 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
factor (GM-CSF) (8). Therefore, the function of Th17 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 Th subsets has been
generally accepted to be responsible for the susceptibility
to various immune diseases. Th1 cell predominance over
Th2 cells can be a cause of organ-specific autoimmune dis-
eases, such as type 1 diabetes, Crohn’s disease and rheu-
matoid arthritis, whereas Th2-dominated responses take
subjects susceptible to asthma and allergic disorders (9–
12). Furthermore, evidence debating such a Th1/Th2 para-
digm shows, in addition to Th1 cells, that Th17 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 Th 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 Th effector subsets. To this end,
murine naive CD4+T cells were forced to polarize under op-
timal conditions for Th1, Th2 and Th17 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 Th1/Th2 cell development via an APC-
dependent pathway, and it regulates Th17 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 Th2
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,
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
Reagents and antibodies
OVA peptide323–339(ISQAVHAAHAEINEAGR) was synthesized
by the Peptron (Daejeon, Korea). CpG oligodeoxynucleotide
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?1c-PGA solution was <0.01 EU ml?1
and <10 pg ml?1when 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),
streptavidin-TC (all from BD Biosciences), anti-CD80-biotin
(Caltag) and anti-IFN-c-FITC
Generation of bone marrow-derived dendritic cells
Bone marrow-derived dendritic 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 subsequently erythrocytes were removed using ACK
lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 2 mM EDTA).
The cells were washed with PBS and cultured 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?1IL-4 (all from R&D Systems).
The BMDC were harvested and stimulated for 16 h with
5 lg ml?1CpG, 2 lg ml?1LPS or 1 mg ml?1c-PGA, fol-
lowed by FACS.
c-PGA effects on Thcell differentiation
by guest on November 21, 2015
to c-PGA. Our results also suggest that a putative receptor
for c-PGA might be expressed by CD4+T cells, which can
directly trigger APC-independent pathways of adaptive im-
Molecules regulating Th1/Th2 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 Th2
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 Th1 responses and down-regula-
tion of Th2 responses might have the therapeutic potential
to treat diseases whose pathogenesis largely relies on anti-
gen-specific Th2 responses, such as asthma and atopic der-
matitis. One issue with which to be concerned is that, in
addition to Th1/Th2 regulation, c-PGA favors the polarization
of CD4+T cells toward Th17 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
Th17 cell promotion could be beneficial in treating estab-
lished allergic asthma.
Our results suggest the potential of c-PGA reacting as an
adjuvant for Th1 and Th17 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 Th2 responses, yet they have
little capacity to stimulate Th1 responses. Therefore, it
appears mandatory to develop new adjuvants that are less
toxic and more effective in Th1 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
Previously, fermented soybeans have been shown to
possess anti-tumor and immunomodulatory activities (35,
36). In particular, among products from the soybean fer-
mentation process, a fructose polymer named levan was
found to be a potent inducer of IL-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 an approach for the prevention of aller-
gic disorders. The latter paper reported that c-PGA did
not mediate such effects, which conflicts with our results.
This discrepancy may arise from differences of experimen-
tal systems including dose and molecular weight of
In summary, we demonstrated for the first time that c-PGA
is a novel regulator of selective development pathways of Th
subsets through APC-dependent and -independent mecha-
nisms. Our findings suggest that this aspect of c-PGA may
be applicable to preventing or treating Th2 response-related
Seoul R&BD Program (10580).
We thank Dr Moon Hee Sung for providing c-PGA, Dr Chul Hoon
Lee for valuable discussion and Ms Jocelyn Graf for editorial
bone marrow-derived dendritic cells
FMS-like tyrosine kinase 3 ligand
granulocyte–macrophage colony-stimulating factor
pathogen-associated molecular patterns
pattern recognition receptors
transforming growth factor
tumor necrosis factor
yellow fluorescence protein
1 Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and
Coffman, R. L. 1986. Two types of murine helper T cell clone. I.
Definition according to profiles of lymphokine activities and
secreted proteins. J. Immunol. 136:2348.
2 Abbas, A. K., Murphy, K. M. and Sher, A. 1996. Functional
diversity of helper T lymphocytes. Nature 383:787.
3 Hsieh, C. S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., O’Garra, A.
and Murphy, K. M. 1993. Development of TH1CD4+Tcells through
IL-12 produced by Listeria-induced macrophages. Science
4 Glimcher, L. H. 2001. Lineage commitment in lymphocytes:
controlling the immune response. J. Clin. Invest. 108:25.
5 Fiorentino, D. F., Bond, M. W. and Mosmann, T. R. 1989. Two types
of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits
cytokine production by Th1 clones. J. Exp. Med. 170:2081.
6 Harrington, L. E., Hatton, R. D., Mangan, P. R. et al. 2005.
Interleukin 17-producing CD4+effector T cells develop via
a lineage distinct from the T helper type 1 and 2 lineages. Nat.
7 Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M. and
Stockinger, B. 2006. TGFb in the context of an inflammatory
cytokine milieu supports de novo differentiation of IL-17-producing
T cells. Immunity 24:179.
8 Kolls, J. K. and Linden, A. 2004. Interleukin-17 family members
and inflammation. Immunity 21:467.
9 Christen, U. and von Herrath, M. G. 2004. Manipulating the type 1
vs. type 2 balance in type 1 diabetes. Immunol. Res. 30:309.
10 Strober, W., Lu ´dvı ´ksson, B. R. and Fuss, I. J. 1998. The
pathogenesis of mucosal inflammation in murine models of
inflammatory bowel disease and Crohn disease. Ann. Intern.
11 Zheng, D. H. and Flavell, R. A. 1997. The transcription factor
GATA-3 is necessary and sufficient for Th2 cytokine gene
expression in CD4 T cells. Cell 89:587.
12 Umetsu, D. T., McIntire, J. J., Akbari, O., Macaubas, C. and
DeKruyff, R. H. 2002. Asthma: an epidemic of dysregulated
immunity. Nat. Immunol. 3:715.
13 Nakae, S., Nambu, A., Sudo, K. and Iwakura, Y. 2003.
Suppression of immune induction of collagen-induced arthritis in
IL-17-deficient mice. J. Immunol. 171:6173.
c-PGA effects on Thcell differentiation 989
by guest on November 21, 2015
14 Cua, D. J., Sherlock, J., Chen, Y. et al. 2003. Interleukin-23 rather
than interleukin-12 is the critical cytokine for autoimmune
inflammation of the brain. Nature 421:744.
15 Ogawa, A., Andoh, A., Araki, Y., Bamba, T. and Fujiyama, Y. 2004.
Neutralization of interleukin-17
sodium-induced colitis in mice. Clin. Immunol. 110:55.
16 Sung, M. H., Park, C., Kim, C. J., Poo, H., Soda, K. and Ashiuchi,
M. 2005. Natural and edible biopolymer poly-gamma-glutamic
acid: synthesis, production, and applications. Chem. Rec.
17 Shih, I. L. and Van, Y. T. 2001. The production of poly-(gamma-
glutamic acid) from microorganisms and its various applications.
Bioresour. Technol. 79:207.
18 Kim, T. W., Lee, T. Y., Bae, H. C. et al. 2007. Oral administration of
high molecular mass poly-gamma-glutamate induces NK cell-
mediated antitumor immunity. J. Immunol. 179:775.
19 Uto, T., Wang, X., Sato, K. et al. 2007. Targeting of antigen to
dendritic cells with poly (gamma-glutamic acid) nanoparticles
induces antigen-specific humoral and cellular immunity. J.
20 Im, W., Kim, H., Yun, D. et al. 2005. Cytokine reporter mouse
system for screening novel IL12/23 p40-inducing compounds.
Mol. Cells 20:288.
21 Reinhardt, R. L., Hong, S., Kang, S. J., Wang, Z. E. and Locksley,
R. M. 2006. Visualization of IL-12/23p40 in vivo reveals immunos-
timulatory dendritic cell migrants that promote Th1 differentiation.
J. Immunol. 177:1618.
22 Gilliet, M., Boonstra, A., Paturel, C. et al. 2002. The development
of murine plasmacytoid dendritic cell precursors is differentially
regulated by FLT3-ligand and granulocyte/macrophage colony-
stimulating factor. J. Exp. Med. 195:953.
23 Fortier, A. H., Hoover, D. L. and Nacy, C. A. 1982. Intracellular
replication of Leishmania tropica in mouse peritoneal macro-
phages: amastigote infection of resident cells and inflammatory
exudate macrophages. Infect. Immun. 38:1304.
24 Berrington, W. R. and Hawn, T. R. 2007. Mycobacterium
tuberculosis, macrophages, and the innate immune response:
does common variation matter? Immunol. Rev. 219:167.
25 Nomura, F., Akashi, S., Sakao, Y. et al. 2000. Cutting edge:
endotoxin tolerance in mouse peritoneal macrophages correlates
with down-regulation of surface Toll-like receptor 4 expression.
J. Immunol. 164:3476.
26 Bettelli, E., Carrier, Y., Gao, W. et al. 2006. Reciprocal de-
velopmental pathways for the generation of pathogenic effector
TH17 and regulatory T cells. Nature 441:235.
27 Korn, T., Bettelli, E., Gao, W. et al. 2007. IL-21 initiates an
alternative pathway to induce proinflammatory Th17 cells. Nature
28 Akira, S. and Takeda, K. 2004. Toll-like receptor signaling. Nat.
Rev. Immunol. 4:499.
29 Trinchieri, G. 2003. Interleukin-12 and the regulation of innate
resistance and adaptive immunity. Nat. Rev. Immunol. 3:133.
30 Schnare, M., Barton, G. M., Holt, A. C., Takeda, K., Akira, S. and
Medzhitov, R. 2001. Toll-like receptors control activation of
adaptive immune responses. Nat. Immunol. 2:947.
31 Kline, J. N., Waldschmidt, T. J., Businga, T. R. et al. 1998.
Modulation of airway inflammation by CpG oligodeoxynucleotides
in a murine model of asthma. J. Immunol. 160:2555.
32 Bohle, B., Jahn-Schmid, B., Maurer, D., Kraft, D. and Ebner, C.
1999. Oligodeoxynucleotides containing CpG motifs induce IL-12,
IL-18 and IFN-gamma production in cells from allergic individuals
and inhibit IgE synthesis in vitro. Eur. J. Immunol. 29:2344.
33 Parronchi, P., Brugnolo, F., Annunziato, F. et al. 1999. Phosphor-
othioate oligodeoxynucleotides promote the in vitro development
of human allergen-specific CD4+T cells into Th1 effectors. J.
34 Schnyder-Candrian, S., Togbe, D., Couillin, I. et al. 2006.
Interleukin-17 is a negative regulator of established allergic
asthma. J. Exp. Med. 203:2715.
35 Takahashi, C., Kikuchi, N., Katou, N., Miki, T., Yanagida, F. and
Umeda, M. 1995. Possible anti-tumour-promoting activity of
components in Japanese soybean fermented food, Natto: effect
on gap junctional intercellular communication. Carcinogenesis
36 Cohen, L. A., Zhao, Z., Pittman, B. and Scimeca, J. 2003. Effect
of soy protein isolate and conjugated linoleic acid on the
growth of Dunning R-3327-AT-1 rat prostate tumors. Prostate
37 Xu, Q., Yajima, T., Li, W., Saito, K., Ohshima, Y. and Yoshikai, Y.
2006. Levan (beta-2, 6-fructan), a major fraction of fermented
soybean mucilage, displays immunostimulating properties via
Toll-like receptor 4 signaling: induction of interleukin-12 pro-
duction and suppression of T-helper type 2 response and
immunoglobulin E production. Clin. Exp. Allergy 36:94.
c-PGA effects on Thcell differentiation
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