Oral administration with chitosan hydrolytic products modulates mitogen-induced and antigen-specific immune responses in BALB/c mice

Article abstract of DOI:10.1016/j.ijbiomac.2019.02.068

The aim of this study was to investigate whether oral administration in BALB/c mice with chitosan hydrolytic products including chitosan hydrolysate, LMWC and a chitooligosaccharide mixture (oligomixture), modulates mitogen-induced and antigen-specific immune responses. A water-soluble chitosan hydrolysate was obtained via cellulase degradation of chitosan, and a LMWC and the oligomixture were separated from this hydrolysate. In non-immunized mice, both the chitosan hydrolysate and oligomixture significantly increased the phagocytic activity of peritoneal macrophages. Three chitosan hydrolytic products significantly increased the mitogen-induced proliferation of splenocytes and Peyer's patch (PP) lymphocytes. LMWC and oligomixture up-regulated IFN-γ secretion, and induced predominantly Th1 cytokine secretion in splenocytes. In antigen-specific immunity, similar effects of the chitosan hydrolytic products were observed on augmenting ovalbumin (OVA)-, as well as mitogen-, induced proliferation of splenocytes harvested from OVA-immunized mice. Interestingly, oligomixture was the most potent chitosan hydrolytic product to elicit OVA-specific IgM, IgG, and IgA production, while LMWC was the most potent one to elevate splenic IFN-γ production and IFN-γ/IL-4 (Th1/Th2) ratio. These results provide the distinct immunomodulatory properties of chitosan hydrolytic products in response to mitogens and specific antigen, paving the way for further development and application of dietary chitosan hydrolytic products against immune disorders and infection.

Full text of DOI:10.1016/j.ijbiomac.2019.02.068

Accepted Manuscript
Oral administration with chitosan hydrolytic products modulates
mitogen-induced and antigen-specific immune responses in
BALB/c mice
Shun-Hsien Chang, Guan-James Wu, Chien-Hui Wu, Chung-
Hsiung Huang, Guo-Jane Tsai
PII:
S0141-8130(18)35839-2
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.02.068
BIOMAC 11722
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
30 October 2018
22 January 2019
11 February 2019
Please cite this article as: S.-H. Chang, G.-J. Wu, C.-H. Wu, et al., Oral administration with
chitosan hydrolytic products modulates mitogen-induced and antigen-specific immune
responses in BALB/c mice, International Journal of Biological Macromolecules,
https://doi.org/10.1016/j.ijbiomac.2019.02.068
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ACCEPTED MANUSCRIPT
Oral administration with chitosan hydrolytic products modulates
mitogen-induced and antigen-specific immune responses in BALB/c mice
b
c
a
a
Shun‐Hsien Chang , Guan-James Wu , Chien-Hui Wu , Chung-Hsiung Huang and
a,d,*
Guo-Jane Tsai
a
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan
Department of Food Science, National Penghu University of Science and
b
Technology, Penghu, Taiwan
c
Department of Seafood Science, National Kaohsiung University of Science and
Technology, Kaohsiung, Taiwan
d
Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University,
Keelung, Taiwan
*
*
2
Corresponding Author:
Dr. Guo-Jane Tsai, Department of Food Science, National Taiwan Ocean University,
Pei-Ning Road, Keelung, 202, Taiwan, ROC; Tel: +886-2-2462-2192#5150; Fax:
+886-2-2462-7954; E-mail: b0090@mail.ntou.edu.tw
Abbreviations:
GM-CSF, granulocyte macrophage colony stimulating factor; IFN-γ, interferon-γ;
IL-1, interleukin-1; IL-4, interleukin-4; IL-6, interleukin-6; TNF-α, tumor necrosis
factor-α; NO, nitric oxide; LMWC, low-molecular-weight chitosan; LPS,
lipopolysaccharides; ConA, concanavalin; OVA, ovalbumin

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Abstract
The aim of this study was to investigate whether oral administration in BALB/c
mice with chitosan hydrolytic products including chitosan hydrolysate, LMWC and a
chitooligosaccharide mixture (oligomixture), modulates mitogen-induced and
antigen-specific immune responses. A water-soluble chitosan hydrolysate was
obtained via cellulase degradation of chitosan, and a LMWC and the oligomixture
were separated from this hydrolysate. In non-immunized mice, both the chitosan
hydrolysate and oligomixture significantly increased the phagocytic activity of
peritoneal macrophages. Three chitosan hydrolytic products significantly increased
the mitogen-induced proliferation of splenocytes and Peyer’s patch (PP) lymphocytes.
LMWC and oligomixture up-regulated IFN-γ secretion, and induced predominantly
Th1 cytokine secretion in splenocytes. In antigen-specific immunity, similar effects of
the chitosan hydrolytic products were observed on augmenting ovalbumin (OVA)-, as
well as mitogen-, induced proliferation of splenocytes harvested from
OVA-immunized mice. Interestingly, oligomixture was the most potent chitosan
hydrolytic product to elicit OVA-specific IgM, IgG, and IgA production, while
LMWC was the most potent one to elevate splenic IFN-γ production and IFN-γ/IL-4
(Th1/Th2) ratio. These results provide the distinct immunomodulatory properties of
chitosan hydrolytic products in response to mitogens and specific antigen, paving the
way for further development and application of dietary chitosan hydrolytic products
against immune disorders and infection.
Keywords: antibody; chitosan hydrolysate; immunomodulation; lymphocyte;
phagocytic activity
1

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1
. Introduction
Innate immunity is a nonspecific defense mechanism that acts as the host’s first
line of defense against infection [1] and is rapidly initiated upon pathogen infection.
Activated phagocytes, such as macrophages, neutrophils, and dendritic cells, recognize
and eliminate pathogens, either by attacking or engulfing larger pathogens [2]. These
cells are also important mediators in the activation of the adaptive immune system [3],
which will respond more vigorously following repeated exposure to the same
pathogen/antigen. The main weapons of this immune system are antibodies, secreted
by B lymphocytes and circulating in the blood (humoral immunity), and T
lymphocytes, able to either destroy directly infected cells or activate macrophages via
the secretion of IFN-γ to kill pathogens (cell-mediated immunity) [4].
Gut-associated lymphoid tissue (GALT) is one of the largest lymphoid organs in
the body, containing up to 70% of the body’s immunocytes, and plays an important
role in the immune system [5, 6]. Typical GALT structures can be seen in Peyer’s
patches (PPs), the aggregated lymphoid follicles in the small intestinal mucosa. PPs
are covered in M (microfold) cells, which lack microvilli and facilitate antigen
translocation and the presentation of luminal antigens to sub-epithelial professional
immune cells [7]. After antigen presentation, lymphocytes in PPs differentiate into
effector lymphocytes and B cells to produce immunoglobulin A (IgA) with the activity
to neutralize pathogen/antigen [8].
Chitosan, a partially deacetylated chitin [poly-β-(1→4) N-acetyl-D-glucosamine]
has immune-enhancing activity, as it stimulates macrophages to produce IL-1, IL-6,
TNF-α, NO, and GM-CSF [9, 10]. These functions are initiated when chitosan binds to
the specific receptors on macrophages [11-13]. Both the chemical structure and the
molecular size of chitosan may affect this binding efficacy and modulate the host
immune responses [14, 15].
2

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Although chitosan has strong biological functions, its water-insolubility makes it
disadvantageous as an immunotherapeutic agent. Water-soluble chitooligomers —
hexamers of N-acetyl chitohexaose and chitohexaose in particular — have attracted
much attention for their immune-enhancing effects [16, 17]. However, the very high
cost of these hexamers limits their application. In our previous study, a water-soluble
chitosan hydrolysate with immuno-activity was obtained by cellulase degradation of
chitosan [18]. A LMWC (20 kDa) and a chitooligosaccharide mixture [designated as
oligomixture, containing sugars with a degree of polymerization (DP) of 1–6] isolated
from this chitosan hydrolysate were shown to have different stimulatory effects on the
cell proliferation and IgM secretion of human hybridoma HB4C5 cells [18]. Also, in the
RAW 264.7 murine macrophage cell line, application of this oligomixture enhanced NO
production, markedly suppressed superoxide production, stimulated phagocytic activity,
and promoted the expression of dendritic cell surface markers, while application of
LMWC produced the opposite effect [13].
In the present study, chitosan hydrolytic products including the chitosan
hydrolysate and its two components, LMWC and oligomixture, were orally
administered to BALB/c mice, and their immunomodulatory activities on
mitogen-induced and antigen-specific immune responses were evaluated. Changes in
splenocyte and PPs lymphocyte cell proliferation, phagocytic activity of peritoneal
macrophages, serum antibodies, and splenocyte cytokine secretion were monitored in
mice, which had received orally administered chitosan hydrolytic products with or
without OVA immunization.
2
. Materials and Methods
2
.1. Reagents and animals
LPS, ConA, OVA, 3-(4,5-dimethylthiazol-2y1)-2,5-diphenyltetrazolium bromide
3

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(MTT), peroxidase-conjugated goat anti-mouse IgG, and peroxidase-conjugated goat
anti-mouse IgM were purchased from Sigma Chemical Co. (Gillingham, UK). Goat
anti-mouse IgA, peroxidase-conjugated goat anti-mouse IgA, mouse IgM, and mouse
IgG were purchased from Rockland (Gilbertsville, PA, USA). Goat anti-mouse IgM,
and goat anti-mouse IgG (H + L) were purchased from Pierce (Rockford, IL, USA).
Penicillin-streptomycin solution (100X) and bovine serum albumin (BSA) were
purchased from Invitrogen Corp. (Carlsbad, CA, USA). Fetal calf serum (FCS) was
purchased from PAA Lab. (Linz, Austria). Enriched RPMI 1640-Dulbecoo’s-Ham’s F12
(eRDF) was purchased from Kyokuto Pharmaceutical Kogyo Company (Tokyo, Japan).
Saccharomyces cerevisiae BCRC 21607 was obtained from the Bioresource Collection
and Research Center (Hsinchu, Taiwan). BALB/c mice were obtained from the Animal
Center at the College of Medicine, National Taiwan University (Taipei, Taiwan).
2
.2. Preparation of chitosan:
Chitin was isolated from shrimp (Solenocera prominentis) shells using the methods
reported by Tsai and Su [19]. The isolated chitin powder was further deacetylated with
5
3
0% NaOH in a 140°C oil bath for 1 h to obtain chitosan with a molecular weight of
00 kDa and a 95% degree of deacetylation (DD 95), which were evaluated using
size-exclusion high-performance liquid chromatography (SE-HPLC) [20] and a colloid
titration method [21], respectively.
2
.3. Production of chitosan hydrolysate and component separation.
According to the protocol of Wu and Tsai [18], 100 g DD95 were added to a 5 L
fermenter (CMCF-5, Chin Chi Co., Taipei, ROC) containing 2000 mL acetic
acid-bicarbonate buffer (pH 5.2), followed by the addition of cellulase (10 U/mL). After
digestion at 55°C and rotation at 125 rpm for 9 h, the hydrolysate was neutralized and
centrifuged (12,000 × g, 30 min). The supernatant (designated as chitosan hydrolysate
in this study) was added to an equal volume of methanol and separated into two
fractions: the chitooligosaccharide mixture (designated as oligomixture) in the upper
fraction and LMWC in the precipitate [18]. The chitosan hydrolysate, LMWC, and
4

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oligomixture were lyophilized and stored at 25°C prior to use. The molecular weight
and deacetylation degree of LMWC was 20 kDa and 83%, respectively, which were
analyzed by using size-exclusion high-performance liquid chromatography (SE-HPLC)
[
20] and a colloid titration method [21], respectively. The oligomixture was composed
of chitooligosaccharides with DP of 1-6, and the weight percentages for these sugars
were 36.8%, 9.5%, 2.7%, 2.1%, 16.3% and 32.6%, respectively [18]. All samples were
confirmed to be endotoxin-free using a Pyrotell® Limulus Amebocyte Lysate kit
(Associates of Cape Cod Inc., E. Falmouth, MA, USA) [13].
2
.4. Animals and experimental design
Conventional female BALB/c mice (4 weeks old) were obtained from the Animal
Center at the College of Medicine, National Taiwan University (Taipei, Taiwan). The
mice were housed six to a cage and fed ad libitum. The cages were kept in a humidity,
light and temperature-controlled environment. Approval for the present study was
obtained from the National Taiwan Ocean University Institutional Animal Care, and the
mice were handled and euthanized according to its guidelines.
The mice were fed a normal diet for 1 week, and were then divided into four
groups, each consisting of 10 mice, and each control or experimental compound was
administered orally as follows: 0.5 mL saline (for control group), chitosan hydrolysate,
LMWC, or oligomixture at a dosage of 500 mg/kg body weight (for experimental group)
6
times per week for 4 weeks via a gastric tube. Sera were collected in the 2nd and 4th
weeks for antibody measurement. Animals were euthanized on dry ice and peritoneal
macrophages were collected. The spleen and PP, as a systemic and intestinal immune
barometer, respectively, and were removed and processed, as reported in section 2.5.2.
Another animal feeding experiment was conducted to determine specific
immune-modulation, in which mice from both the control group and experimental
groups were immunized via intraperitoneal injection of 0.2 mL OVA solution (50 μg/mL)
in the 2nd week. A boosting injection was given in the 4th week. Sera were collected in
the 5th week for OVA-specific antibody measurement. The mice were euthanized 1
week after the boosting injection, and the splenocytes and PP cells were collected for
proliferation assay.
2
.5. Preparation of peritoneal macrophages.
The murine peritoneal macrophages were collected based on the method described
5

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by Lewis, et al. [22]. Briefly, a 5 cm incision was made in the abdominal wall of the
euthanized mice, followed by injection of 3 mL Hanks balanced salt solution (HBSS)
containing 1X penicillin-streptomycin solution. Peritoneal exudates were harvested
from peritoneum lavage. The cells were washed twice with HBSS, re-suspended in
eRDF supplemented with 10% FCS, and incubated at 37°C for 1 h. The adherent cells
were used in the phagocytic activity assay.
2
.5.1. Phagocytic activity of macrophages.
The phagocytic activity of peritoneal macrophages was evaluated using the
suspension assay as described by Bin-Hafeez, et al. [23], with minor modifications.
6
Briefly, a 0.5 mL aliquot of cells (2 × 10 cells/mL) was mixed with 0.5 mL of 10%
7
FCS opsonized yeast (Saccharomyces cerevisiae) cells (2 × 10 cells/mL). The mixture
was incubated at 37°C for 1 h. After incubation, 50 μL of this mixture were smeared on
a glass slide, air dried, and stained with Giemsa stain. The macrophages that
phagocytized yeast cells were counted using a light microscope. Two hundred cells from
each animal were examined for each assay.
2
.5.2. Lymphocyte proliferation assay.
The murine spleen and PP cells were suspended in 5 mL eRDF with 10% FCS
added. After the addition of an equal volume of Ficoll-paque (Amersham Pharmacia
Biotech AB, Uppsala, Sweden) and centrifugation (400 xg, 20 min), the white opaque
mononuclear cell fraction from the interface between the medium and the Ficoll-paque
was collected, and washed 3 times with 5 mL eRDF medium (containing 10% FCS).
6
One hundred microliters of the splenocyte and PP lymphocyte cell suspensions (2 × 10
cells/mL) were added to the wells of microtiter plates, followed by the addition of 20 L
of Con A (15 g/mL), LPS (30 g/mL), or OVA (100 g/mL). After incubation at 37°C
for 48 h, the viable cells were measured via MTT assay [21]. The proliferation index
was calculated as follows.
Proliferation index = ( A sample / Acontrol ) × 100 %
A sample: absorbance of cell from mice receiving sample in MTT assay.
Acontrol: absorbance of cell from control mice in MTT assay.
2
.5.3. Determination of cytokines by ELISA.
The splenocytes were cultured in eRDF medium containing 10% FCS with/without
6

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addition of LPS (5 g/mL), Con A (2.5 g/mL), or OVA (100 g/mL) at 37°C for 48 h.
The amounts of IFN-γ and IL-4 in the supernatant of splenocyte cultures were
quantified using their respective DuoSet ELISA Development kits (R&D Systems,
Minneapolis, MN, USA) as directed by the manufacturer.
2
.5.4. Determination of antibodies by ELISA.
The total antibodies or OVA-specific antibodies in sera were quantified according
to the method of Wu and Tsai [18], with minor modification. Two hundred microliters
3
of 10 -fold-diluted goat-anti-mouse antibodies or 20 μg/mL OVA in 50 mM carbonate
coating buffer (pH 9.6) were added to the wells of the microtiter plate and incubated
overnight at 4°C. After washing with phosphate buffered saline (PBS; 0.2 g KH2PO4,
1
.15 g Na2HPO4, 0.2 g KCl, and 8 g NaCl in 1 L of deionized water) containing 0.05%
Tween-20 (PBST), 100 µL of the sample serum were added to the well. The plates were
incubated at 37°C for 1 h, washed, and 200 µL of peroxidase-labeled goat anti-mouse
antibodies were added to each well (1:3000 dilution with PBST plus 1% BSA). After
incubation at 37°C for 1 h, the plates were washed again, 100 µL of ABTS (from an
assay kit obtained from KPL, Gaithersburg, Germany; prepared by mixing equal
volumes of solutions A and B, as described in the manufacturer’s instructions) were
added and the plates were incubated at 37°C for 15 min. Finally, 100 µL of 1.5% oxalic
acid was added to each well and the absorbance was measured at 410 nm.
2
.6. Statistical analysis.
The results are presented as the mean ± SD. Data were analyzed using the
statistical analysis system program. Statistical comparisons were made using ANOVA
and Duncan’s tests. Differences of p ＜ 0.05 were considered statistically significant.
3
. Results and Discussion
3
.1. In vivo immunomodulatory effects of chitosan hydrolytic products on phagocytic
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activity of macrophages and lymphocyte responses
During the 4 weeks of the oral administration experiment, the body weights of all
mice slightly increased, and the gains in body weight were not significantly different
between the experimental groups and the control (Fig. 1A). After the oral administration
of chitosan hydrolytic products for 4 weeks, the percentage of peritoneal macrophages
with phagocytic activity were 20.6 ± 5.9, 8.1 ± 4.0, and 14.5 ± 4.1% from hydrolysate,
LMWC, and oligomixture, respectively, while that of the control group was 6.1 ± 1.6%.
Chitosan hydrolysate and oligomixture increased the phagocytic activity of
macrophages (Fig. 1B).
Macrophages, which act as phagocytic, microbicidal, and tumoricidal cells, play a
critical role in the host’s first line of defense against infections. In a previous study,
chitosan hydrolysate and its oligomixture component were demonstrated to enhance NO
production in IFN-γ-treated murine RAW264.7 macrophages in vitro [13]. In the
present study, the chitosan hydrolysate and oligomixture were further demonstrated to
enhance the phagocytic activity of peritoneal macrophages in mice.
(A)
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(
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Fig. 1. Chang et al.
In addition to phagocytic activity of macrophages, lymphocyte proliferative
responses also correlate with the resistance against infection. Therefore, we investigated
the lymphoproliferative responses of splenocytes and PP lymphocytes harvested from
chitosan hydrolytic products-treated mice in response to the mitogens for T-cell (ConA)
and B-cell (LPS). The chitosan hydrolysate, LMWC, and oligomixture significantly
increased the proliferation index of splenocytes stimulated by the mitogens (Fig. 2A),
and significantly increased the proliferation index of PP lymphocytes stimulated by Con
A (Fig. 2B). James and Zeitz [24] indicated that lymphocytes in the intestinal mucosa
first interact with antigens in the organized lymphoid tissues (PPs and lymphoid
follicles in the colon), and after maturation they migrate through the mesenteric lymph
nodes to reach systemic circulation. Porporatto, et al. [25] demonstrated the presence of
chitosan in PPs after oral administration, and they further showed that chitosan in PPs
can up-regulate the major histocompatibility complex (MHC) class II molecules of
antigen-presenting cells (APCs). In consistent with the above studies, our results further
indicate that chitosan hydrolytic products are potential to up-regulate T-cell immune
responses in PP and T-cell and B-cell immunity in the spleen, indicating that the
immune cells regulated by hydrolytic products at the intestine potentially migrate by
circulation to the spleen to further modulate systemic immunity. Maeda and Kimura
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[
26] reported that S180-bearing mice treated with LMWCs (21 and 46 kDa) and
chitooligosaccharides showed increased antitumor activity and induced activation of
macrophages through the production of cytokines such as IFN-g, IL-12, and IL-18 from
intestinal intraepithelial lymphocytes and splenic lymphocytes. However, larger MW
chitosans (130 or 650 kDa) had no such effect. This suggests a possible mechanism by
which the chitosan hydrolytic products administered to the gastrointestinal tract were
able to elicit widespread systemic responses, as indicated by the increasing proliferative
index of splenocytes and PP cells in the present study.
(A)
Spleen
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Peyer's patch
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ConA
Mitogen
Fig. 2. Chang et al.
It has been reported that chitin could be served as a mitogen to induce the
differentiation of B lymphocytes into antibody-forming cells in vitro [27]. Recently,
chitin, chitosan and their derivatives have been utilized as adjuvants to enhance
antibodies production [28-30]. As antibody production represents an end-point affecting
response of T lymphocyte-mediated antigen recognition, we further investigated
whether oral administration with chitosan hydrolytic products influence the levels of
antibody production. We used ELISA to measure the total levels of IgG, IgA, IgM, and
IgE in sera in the 2nd and 4th weeks of administration, as shown in Fig. 3. The levels of
IgA, IgG, and IgM were significantly increased by administration of oligomixture.
LMWC increased the IgG and IgM levels, while the hydrolysate increased the IgM
level compared with the control. Based on the results of B-cell proliferation and
antibody production, chitosan hydrolytic products, especially oligomixture, are
promising to up-regulate humoral immunity. Noticeably, the level of IgE production
was not influenced by chitosan hydrolytic products (Fig. 3), indicating a limited risk of
chitosan hydrolytic products to induce type 1 hypersensitivity [31].
In general, naive T cells recognize antigen in the context of MHC molecules via
TCR complex and develop into armed affecting T cells which can largely be divided
into T helper (Th) cells. Among the subsets of Th cells, Th1 cells secrete IFN-γ and IL-2
to augment cell-mediated immune responses. In contrast, Th2 cells uniquely secrete
IL-4, IL-5, and IL-6 to help the antibodies production by B cells, and mediate the
11

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responses against extracellular parasites [32].
To investigate the effect of chitosan hydrolytic products on the production of
IFN-γ and IL-4, the splenocytes were cultured with or without mitogen for 48 h, and the
level of cytokine secretion in supernatants were measured by ELISA. The levels of
spontaneous secretion of IFN-γ and IL-4 were very low although both LMWC and
oligomixture statistically increased IFN-γ secretion (Fig 4A & 4B).
Both IFN-γ and IL-4 secretion by splenocytes in response to ConA were markedly
augmented in all experimental groups (Fig 4A & 4B). The IFN-γ/IL-4 ratio for mice that
received with hydrolysate, LMWC, and oligomixture were 2.5 ± 0.3, 5.0 ± 0.1, and 10.2
±
1.0, respectively, which were significant higher than that of the control (1.3 ± 0.1)
Fig. 4C). These results demonstrated that oral administration of chitosan hydrolytic
products would modulate Th1/Th2 immune balance toward Th1 polarization.
(
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nd
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Fig. 3 Chang et al
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(A)
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3
.2. Oral administration of chitosan hydrolytic products modulated antigen-specific
and mitogen-induced immune responses in OVA-immunized mice.
Chitosan has been reported as an effective adjuvant by changing the permeability of
epithelia for increasing the uptake of antigens and the contact with the immune system
11, 33]. As the vaccine adjuvant, chitosan could increase cell-mediated immunity and
[
circulating antibody production [9, 28]. Water-soluble chitooligosaccharides and
N-acetyl chitooligosaccharides—chitohexaose and N-acetyl chitohexaose in
particular—have also been considered as immunopotentiators [17, 34]. Although many
studies have investigated the modulatory effects of chitosan on antigen-specific immune
responses, chitosan was applied as vaccine adjuvants in most of these studies. In the
present study, we addressed whether oral administration with chitosan hydrolytic
products affects antigen-specific immune responses in OVA-immunized BALB/c mice.
As shown in Fig. 5A, the proliferation index of splenocytes co-cultured in vitro
with OVA for the group that received LMWC was significantly higher than that of the
control (Fig. 5A). All samples of hydrolysate, LMWC, and oligomixture significantly
increased the ConA- and LPS-stimulated splenocyte proliferation in OVA-immunized
mice; the LMWC group in particular showed markedly enhanced splenocyte
proliferation compared with the control group (Fig. 5A). The in vitro proliferation
indexes of PP cells co-cultured with OVA from the groups receiving hydrolysate and
oligomixture were significantly higher than that of the control (Fig. 5B). Compared with
the proliferation index of PP B cells (stimulated by LPS) without OVA immunization
(
Fig. 2B), the proliferation of PP B cells markedly increased after OVA immunization
Fig. 5B). Although the reason for the differences in enhancement profiles between the
(
splenocytes and the PP cells is presently unclear, we are confident that oral
administration of the chitosan hydrolytic products activates the splenocytes by
increasing their proliferation with or without antigen immunization.
The modulatory effects of chitosan hydrolytic products on the OVA-specific
humoral immune responses of BALB/c mice were also evaluated (Fig. 6). The
OVA-specific IgM (Fig. 6A), IgG (Fig. 6B), and IgA levels (Fig. 6C) were significantly
increased for the group that received oligomixture compared to those of the control. The
OVA-specific IgE levels were similar for all groups (Fig. 6D). Moreover, the levels of
IgG subclasses, including both IgG1 and IgG2a, were also measured. The level of IgG1
in the group that received LMWC was significantly decreased; while the level of IgG2a
in the group that received oligomixture was significantly increased (Fig. 7).
1
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Spleen
a
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Oligomixture
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Hydrolysate
LMWC
Oligomixture
b
bc
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c
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bc
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bcab
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LPS
ConA
OVA
Mitogen
Fig. 5 Chang et al.
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(A)
(B)
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Fig. 6 Chang et al.

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(A)
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Fig. 7. Chang et al.
Due to the distinct effects of LMWC and oligomixture on modulating IgG1 and
IgG2a production, the effects of chitosan hydrolytic products on modulating the
secretion of the IFN-γ (Th1 cytokine, promoting IgG2a production) and IL-4 (Th2
cytokine; promoting IgG1 production) were examined. The spontaneous IFN-γ
production was significantly increased in mice that received oligomixture (Fig. 8A), and
the spontaneous IL-4 production was significantly decreased for all experimental groups
(Fig. 8B).
When splenocytes were co-cultured with OVA or Con A, the IFN-γ secretion was

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significantly enhanced in the groups receiving the LMWC and oligomixture, while IL-4
secretion was significantly inhibited for all experimental groups. Accordingly, the
IFN-γ/IL-4 ratio was significantly increased for all experimental groups (Figs. 8A, B, C).
Cytokines have been reported to affect antibody class switching; for example, IL-4
causes antibody class switch to IgE, IgG1, and IgG4 [35, 36], IFN-γ causes antibody
class switch to IgG2a and IgG3 [37, 38] and TGF-β causes the IgA class switch [39].
The significant increase in IFN-γ production and decrease in IL-4 production upon the
application of the three chitosan hydrolytic products (Fig. 8) may partly explain the
increase in IgG2a in the oligomixture-receiving group and the decrease in IgG1 in the
LMWC-receiving group (Fig. 7). Shibata, et al. [40] have shown that oral
administration of chitin resulted in decreased IL-4 and increased IFN-γ production.
Concordantly, our results show that the immune responses were redirected toward a Th1
response by both LMWC and oligomixture. Chitin and chitosan have been shown to
induce the TGF-β production by PP cells [41], macrophages [25], and platelets [42].
Nonetheless, except for TGF-β, numerous factors, such as CD40L, TLR4 ligand, BAFF
and APRIL, are also known to induce IgA switch [43]. Further comprehensive
investigations are required to elucidate the action mechanism of oligomixture-induced
IgA production.
It has been reported that particulate antigens exhibit dissimilar immunologic
properties with that to soluble antigens. Particulate antigens tend to be selectively
phagocytized by APCs to elicit cellular immune responses. In contrast, soluble antigens
tend to be internalized via pinocytosis to explore humoral immune responses [44]. With
respect to polysaccharide immunomodulators, particulate β-glucan also showed the
capability of inducing Th1 cell activation compared to the soluble one [45]. It is
expectable that chitosan hydrolytic products with higher molecular weight are relative
insoluble than that with lower molecular weight. Therefore, in the antigen- specific
response it is reasonable that oligomixture was more potent to elicit antibody production,
while LMWC was more potent to elevate lymphocyte proliferation and IFN-γ secretion.
On the other hand, it has been reported that LPS and chitosan share a binding site on
monocytes which involves CD14, a co-receptor with TLR4 [46, 47]. Qiao et al. have
substantiated that chitosan oligosaccharides could inhibit binding of LPS to TLR4 and
thus reduce the production of pro-inflammatory mediators [48]. In contrast, Zhang et al.
have demonstrated that chitosan oligosaccharides possessed immune-stimulating
properties by activating TLR4 [49]. Therefore, we suggest that the different impact of
1

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chitosan hydrolytic products on the activation receptor of immune cells may be another
potential mechanism for their distinct immunomodulatory properties. The underlying
mechanistic relationship between the molecular weight, structure, solubility and binding
characteristics of chitosan hydrolytic products with their distinct immunomodulatory
properties is an intriguing issue that warrants further investigations.
2

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(A)
2
500
000
500
000
a
Control
Hydrolysate
LMWC
Oligomixture
2
1
1
b
b
a
c
b
c
5
00
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c
a
a
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b b
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50
a
b
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3
2
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a
c
c
a
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2
1
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1
1
00
80
60
40
20
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a
b
b
8
6
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2
0
0
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c
a
b
b
a
a
b
c
c
c
b b
c
LPS
ConA
OVA
Spontaneous
Fig. 8. Chang et al.
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In summary, oral administration of the chitosan hydrolytic products of LMWC,
and oligomixture modulated mitogen-specific and OVA-specific immune responses.
Especially, oligomixture was more potent to elicit OVA-specific antibody production;
while LMWC was more potent to elevate splenocytes proliferation and splenic IFN-γ
secretion in OVA-immunized mice. Our results provide the distinct
immunomodulatory properties of chitosan hydrolytic products in response to
mitogens and specific antigen, suggesting that dietary chitosan hydrolytic products
may be helpful for the prevention and therapy of immune disorders and pathogen
infection.
Acknowledgements
The financial support of the National Science Council of the Republic of China
(NSC 92-2313-B-019-041) is gratefully acknowledged.
4

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Figure captions
Fig. 1. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on the body weight (A) and phagocytic activity of peritoneal
macrophages (B) of BALB/c mice. The mice were divided into four groups (10
mice per group) and administrated orally with 0.5 ml saline (for control group),
chitosan hydrolysate, LMWC, or oligomixture at the dosage of 500 mg/kg body
weight (for experimental group) 6 times per week for 4 weeks by gavage. (A)
The body weight of each mouse was recorded per week. (B) After 4 weeks of
treatment, the mice were sacrificed, and the peritoneal macrophages were
harvested for the assay of phagocytic activity as described in the Materials and
methods. Data are expressed as mean with SD (n=10). Differences of p＜0.05
were considered statistically significant.
Fig. 2. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on mitogen-stimulated proliferation of splenocytes (A) and PP (B)
lymphocytes. The mice were treated as described in the Materials and methods.
After 4 weeks of treatment, the mice were sacrificed, and splenocytes and
Peyer’s patch (PP) lymphocytes were prepared and cultured in the absence or
presence of LPS or ConA for 48 h. Cell proliferation was determined by MTT
assay. Data are expressed as mean with SD (n=10). Differences of p＜0.05 were
considered statistically significant.
Fig. 3. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on the levels of IgA(A), IgG(B), IgM(C), and IgE(D) production in
serum of BALB/c mice. The mice were treated as described in the Materials
and methods. After 2 and 4 weeks of treatment, the serum samples were
collected, and the levels of total IgA, IgG, IgM, and IgE production in sera were
determined by ELISA. Data are expressed as mean with SD (n=10). Differences
of p＜0.05 were considered statistically significant.
Fig. 4. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on the secretion of IFN-γ (A) and IL-4 (B), and IFN-γ/IL-4 ratio
by splenocytes. The mice were treated as described in the Materials and
methods. After 4 weeks of treatment, the mice were sacrificed, and splenocytes
were prepared and cultured in the absence or presence of LPS or ConA. After 48
9

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h, the levels of IFN-γ and IL-4 secretion in the supernatants were determined by
ELISA. Further, the ratio of IFN-γ/IL-4 was evaluated. Data are expressed as
mean with SD (n=10). Differences of p＜0.05 were considered statistically
significant.
Fig. 5. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on mitogen- or OVA-induced proliferation of splenocytes (A) and
PP (B) lymphocytes harvested from OVA-immunized mice. The mice were
treated as described in the Materials and methods. After 4 weeks of treatment,
the mice were sacrificed, and splenocytes and Peyer’s patch (PP) lymphocytes
were prepared and cultured in the presence of OVA, LPS or ConA for 48 h. Cell
proliferation index was determined by MTT assay. Data are expressed as mean
with SD (n=10). Differences of p＜0.05 were considered statistically significant.
Fig. 6. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on the levels of OVA-specific IgM(A), IgG(B), IgA(C), and IgE(D)
in sera of OVA-immunized BALB/c mice. OVA-immunized mice were treated
as described in the Materials and methods. After 4 weeks of treatment, the
serum samples were collected, and the levels of OVA-specific IgA, IgG, IgM,
and IgE production in sera were determined by ELISA. Data are expressed as
mean with SD (n=10). Differences of p＜0.05 were considered statistically
significant.
Fig. 7. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on the levels of OVA-specific IgG1(A) and IgG2a(B) in sera of
OVA-immunized BALB/c mice. OVA-immunized mice were treated as
described in the Materials and methods. After 4 weeks of treatment, the serum
samples were collected, and the levels of OVA-specific IgG1 and IgG2a
production in sera were determined by ELISA. Data are expressed as mean with
SD (n=10). Differences of p＜0.05 were considered statistically significant.
Fig. 8. Effects of oral administration of chitosan hydrolysate, LMWC, and
oligomixture on the secretion of IFN-γ (A) and IL-4 (B), and IFN-γ/IL-4 ratio
by splenocytes harvested from OVA-immunized BALB/c mice.
OVA-immunized mice were treated as described in the Materials and methods.
After 4 weeks of treatment, the mice were sacrificed, and splenocytes were
1
0