Characterization and immunoenhancement activities of Eucommia ulmoides polysaccharides

Accepted Manuscript Title: Characterization and Immunoenhancement activities of Eucommia Ulmoides polysaccharides Author: Haibo Feng Jing Fan Zhenhui Song Xiaogang Du Ying Chen Jishuang Wang Guodong Song PII: DOI: Reference:

S0144-8617(15)00944-3 http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.079 CARP 10381

To appear in: Received date: Revised date: Accepted date:

23-7-2015 8-9-2015 23-9-2015

Please cite this article as: Feng, H., Fan, J., Song, Z., Du, X., Chen, Y., Wang, J., and Song, G.,Characterization and Immunoenhancement activities of Eucommia Ulmoides polysaccharides, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterization and Immunoenhancement activities of Eucommia Ulmoides

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polysaccharides

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Haibo Feng a,1* , Jing Fan b,1, Zhenhui Song a , Xiaogang Du c , Ying Chen a, Jishuang Wang a,

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Guodong Song a

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a Department of Veterinary Medicine, Southwest University, Rongchang, Chongqing 402460, PR

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China

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b Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu, Sichuan, 610051, PR

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China

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c Applied Biophysics and Immune Engineering Laboratory, College of Life and Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, PR China

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1These authors contributed equally to this work.

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∗Corresponding author. tel/fax:+86-23-46751732.

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E-mail address: [email protected].

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14 Abstract

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The aim of this study is to investigate the physicochemical properties, monosaccharide

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composition and immunomodulatory effects of Eucommia ulmoides Oliv. polysaccharide. The

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average molecular weight (Mw) of EUPS was 11.4632 ·· 10 Da. The monosaccharide

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showed that EUPS could significantly enhance the FMDV-specific IgG, IgG1, IgG2a, and IgG2b

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antibody titers and T cell proliferation. Together, these results suggest that EUPS is a strong

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immunostimulant.

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components of EUPS was glucose, fructose, mannose, fucose, galactose and arabinose with a relative mass of 36.6%, 16.6 %, 14.2%, 15.7 %, 9.5% and 7.4 %, respectively. In in vitro experiments, EUPS (1.2 µg/mL to 75 µg/mL) treatment of dendritic cells (DC) increased their surface expression of MHC I/II, CD80, CD40, and CD86 and indicated that EUPS induced DC maturation. Furthermore, EUPS also significantly enhanced lymphocyte proliferation and significantly enhanced cytokine (IL-4 and IFN-γ) production. In in vivo experiments, our data

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Keywords: Eucommia ulmoides Oliv. polysaccharide; adjuvant; lymphocyte proliferation;

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cytokines; dendritic cell

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1 Introduction Eucommia ulmoides Oliv. (EU) has been used as a traditional Chinese medicine for at least

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2000 years and has been anecdotally shown to have with a wide range of pharmacological

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activities (Liu, 2013). EU known as “Du-Zhong”, is obtained from the stem bark of a 15 to 20

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year-old Eucommia ulmoides Oliv. tree and was recorded in ancient Chinese medical texts as

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"Shennong's Herba." This ancient remedy was considered to be a nourishing herb in China, and

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was commonly used as a tonic to strengthen muscles and bones and nourish the kidneys and liver.

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This traditional Chinese medicine was also used as a diuretic and as a treatment for a variety of

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pathological conditions, including rheumatism, arthritis, lumbago, and recurrent miscarriage (Liu,

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et al., 2011; Meng, et al., 2007; Gong, et al., 2010). Pharmacological experiments have shown that

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EU extracts possess anti-inflammatory, analgesic, antioxidant, anti-aging, anti-bacterial and

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anti-fungal activities (Yuan, et al., 2013; Liu, et al., 2010; Kim, et al., 2004; Zhang and Ravipati, et

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al., 2013). It was also been reported that hot water extracts from EU have anti-complement

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activity in hemolytic assays and can significantly improve the serum antibody titers of chickens

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receiving the Newcastle disease vaccine (Xin, et al., 2009; Zhu, et al., 2008).

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In the past few years, monomeric compounds have been isolated from EU, including

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et al. 2010).

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polysaccharide, lignans, iridoids, guttapercha, flavonoids, amino acids, iridoids, saccharides, as well as other trace elements (Xin, et al., 2007; Luo, et al., 2010; He, et al., 2014). Recently, many researchers have demonstrated that polysaccharides fraction derived from EU have various biological activities. For example, EUPS could function as anti-oxidants to scavenge DPPH

free-radical and reduce ferric in vitro (Sun, et al. 2014; Gong, et al., 2008). Moreover, EUPS could act as a regulator of hyperglycemia via decreasing the blood glucose in alloxan-induced diabetic mice (Liu,

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In this study, we evaluated the in vitro effects of Eucommia ulmoides Oliv. polysaccharide

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(EUPS) on dendritic cell (DC) maturation, lymphocyte proliferation and cytokine secretion. At the

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same time, we measured the in vivo effects of EUPS on specific antibody production and

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lymphocyte proliferation. The purpose of this research was to characterize the physicochemical

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properties, monosaccharide composition of EUPS, and evaluate the in vitro and in vivo

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immunoenhancement properties of EUPS, which offer a theoretical basis for the development of a

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novel polysaccharide immunopotentiator.

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2 Materials and methods

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2.1 Mice Institute of Cancer Research (ICR) female mice (5 weeks old, Grade II, weighing 18–22 g)

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were purchased from Sichuan Laboratory Animal Center (SLAC) Co. Ltd. (Sichuan, China). A

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total of 50 mice were used in this experiment and kept in stainless steel cages in an air-conditioned

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room with hygenic saw dust bedding. Feed and water were supplied ad libitum. The temperature

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was maintained at 22 ± 1℃ and in a normal light/dark (12/12h) cycle. In this study, all of the

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animal procedures and care conformed to the internationally accepted principles as found in the

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Guidelines for Animal Laboratory Studies issued by the government of China.

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2.2 Materials

Purified EUPS (≥97% purity) was obtained from Tian Qi Biotechnology co. Ltd. Shanxi,

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China. The polysaccharide preparation ( ≥ 97% purity) was purified using a standard

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high-performance liquid chromatography-refractive index detection method. RPMI-1640, fetal

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bovine serum, benzylpenicillin, streptomycin, NaHCO3, and HEPES were purchased from Life

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Technologies

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granulocyte-macrophage colony stimulating factor (rmGM-CSF) and interleukin-4 (IL-4) were

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Husbandry Industry Co., Ltd. (Lanzhou, Gansu, China) Fluorescent labeled anti-mouse

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monoclonal antibodies (CD4-FITC, CD80-PE, CD40-FITC, MHC-II-PE and CD86-PE) were

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obtained from eBioscience (San Diego, CA, USA).

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2.3. Extraction and purification of polysaccharide

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(Carlsbad,

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Corporation

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California,

USA).

Recombinant

murine

obtained from PeproTech, Inc. (Rocky Hill, N.J. USA) and both of these cytokines were prepared at 10 ng/mL in RPMI-1640. Concanavalin A (ConA) was used as a T-cell mitogen, lipopolysaccharide (LPS) was used as a B-cell mitogen, and both were purchased from Sigma Chemical Co. (Saint Louis, Missouri, USA). Cell counting kit-8 (CCK-8) was obtained from the Beyotime Institute of Biotechonology (Haimen, Jiangsu, China). An FMDV O-serotyped inactivated vaccine was acquired from Lanzhou Veterinary Research Institute China Animal

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The dry powder of stem bark of Eucommia ulmoides Oliv. was boiled in water three times

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under reflux, and the aqueous extract was filtered three times using filter paper. The filtrate was

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centrifuged at 5000× g for 15 min, and the ethanol was added to the supernatant and made to a

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working concentration (v/v) of up to 85%, the mixture was precipitated at 4℃ for 12 h. The

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resulting precipitate was collected using centrifugation 5000× g for 15 min, washed three times

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with 70% ethanol, and lyophilized with a pharmaceutical freeze dryer ( Scientz-50F, Ningbo,

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China). Finally, the Eucommia ulmoides Oliv. polysaccharide (EUPS) was obtained.

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EUPS was dissolved in distilled water, and purified with seriatim using the Sevag method to

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eliminate the protein, and then dialyzed against distilled water for 72 h. The non-dialyzable phase

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was re-collected, and then precipitated with 85% ethanol for 12 h at 4℃. The resulting precipitate

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was centrifuged at 5000 × g for 10 min, after washing three times with 70% ethanol, and dried to a

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consistent weight. Subsequently, the precipitate was permeated through a macroporous adsorption

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resin (D101) to eliminate the pigment, and DEAE Sephadex™ A-25 to separate it from the other

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carbohydrates. The eluates were collected and lyophilized to produce the total EUPS fraction.

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2.3. Characterization of EUPS

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2.3.1 Physicochemical property analysis

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The physicochemical properties of EUPS were determined by the following

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methods: solubility test, color observation, α-naphthol test, iodination test,

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phenol---sulfuric acid test, uronic acid carbazole reaction, FeCl3 test, fehling’s

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reaction, Coomassie brilliant blue reaction, and full wavelength scanning ( Wu, et

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standard (Filisetti-Cozzi & Carpita, 1991).

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2.3.3. Infrared spectroscopy analysis

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al., 2011).

2.3.2. Analysis contents of carbohydrate, protein and uronic acid Total sugar content contents were measured using the Anthrone-sulfuric acid method, with

glucose as a standard (Dubois, et al., 1956). The protein contents were assayed using the

Bradford’s method (Bradford, 1976), using bovine serum albumin as a standard. The uronic acid

contents were determined using the carbazole–sulfuric acid method, with glucuronic acid as a

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The organic functional groups of the EUPS were investigated using infrared (IR) spectra,

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recorded within the range of 4000 cm-1 to 400 cm-1 by a FT-IR spectroscope (FTIR-8400S,

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Shimadzu Co., Japan). The purified EUPS was dried and ground with KBr powder and pressed

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into pellets for FT-IR analysis.

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2.3.4. SEM analysis The polysaccharide powder was directly scattered on the sample table, and then examined

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using an SEM system (Jeol, JSM-7500F, Tokyo, Japan) with 5 kV accelerating voltage, as well as

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image magnifications of 500×, 3000× and 5000×.

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2.3.5. Monosaccharide composition analysis

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The monosaccharide composition of EUPS was determined using gas chromatography (GC)

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analysis. EUPS (20 mg) was hydrolyzed with 2 M trifluro acetic acid (TFA) at 100℃ for 6 h to

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hydrolyze and release the component monosaccharides. The digested solution was evaporated

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until dry, and 60 mg of NaBH4 and 4 mL of distilled water were added to the dried solids.

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Subsequently, the mixture was incubated with acetic acid for 40 min for acidification. The mixture

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was then evaporated at 60℃ until it was a dry solid. Four mL of 0.1% HCl-MeOH (V/V) was

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added to the dried solids, and the mixture was evaporated to a dry solid again. The dried products

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were obtained for acetylation (Feng, et al., 2015).

The acetylation was carried out using 1:1 pyridine-acetic anhydride in a water bath at 85℃ for

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1 h. The monosaccharide composition of EUPS was determined using the GC-MS alditol acetates

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of standard monosaccharides (D-xylose, D-fructose, D-glucose, D-galactose, L-rhamnose,

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D-arabinose, and D-mannose) with inositol as the internal standard. Each monosaccharide was

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prepared and subjected to GC-MS analysis, separately, but using the same procedure. The

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(GPC) on a Sephadex G-100 column (60 cm × 1.6 cm). The column was eluted using ultrapure

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water and the flow rate was 0.6 ml/min. The average molecular weight was detected using a

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calibration curve, which was established using the Dextran standards (known molecular weights:

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11600, 48600, 80900, 147600, 273000, 667800, 1185000, and 2150000).

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2.4. In vitro test

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2.4.1 Cytotoxicity analysis of splenic lymphocytes

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operation was performed using the following conditions: the column temperature was initially

120℃ for 4 min, increased to 200℃ at a rate of 5℃/min, held for 4 min, and then increased to

250℃ at a rate of 5℃/min; the inlet temperature was 250℃; the detector temperature was 280℃; The N2 carrier gas rate was 20 mL/min (Feng, et al., 2015). 2.3.6. Molecular weight determination of EUPS. The average molecular weight of EUPS was determined using gel permeation chromatography

Page 5 of 25

ICR mice were sacrificed and their spleens were collected and pressed against a steel mesh

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with the flat surface of a syringe plunger. Single splenocyte suspensions were prepared by

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passing the spleen tissue through a fine steel sieve (200 mesh) and the red blood cells were lysed

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by ACK lysis buffer. The splenocytes were washed three times by phosphate buffered saline

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(PBS)(PH7.4). Cells were counted on a hemocytometer and viability was determined using the

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trypan blue dye exclusion technique. In all cases, cell viability exceeded 95%. The resultant cells

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were diluted to 2.5×106 cells/ml with complete medium and cultured in 96-well plates.

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The maximum concentration at which EUPS is not cytotoxic to splenocytes was determined by

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using the CCK-8 assay. Single splenocyte suspensions were added onto 96-well plates to a final

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volume of 100 µL in each well, and 100 µL of EUPS, at different concentrations (from 1.2 to 1200

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µg/mL) was added, in quadruplicate. The plate was incubated at 37℃ with 5% CO2. After 68 h,

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20 µL of WST-8 was added into each well and sequentially incubated for 4 h. The optical density

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(OD) values at 450 nm were determined using a microplate reader (Synergy HT, Bio-TEK). When

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the OD of cells treated with polysaccharide was not significantly lower than that of the cells

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treated with only RPMI-1640, the polysaccharide concentration was not considered to be

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cytotoxic.

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2.4.2 Splenic lymphocyte proliferation assay

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One hundred µL of the splenocyte cell suspension was seeded onto a 96-well plate. Some

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was calculated according to the following equation: SI = the absorbance value for the

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stimulation-cultures divided by the absorbance value for non-stimulated cultures.

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2.4.3 IL-4 and IFN-γ production by splenocytes in vitro

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wells were treated with 100 µL of EUPS (final concentration at 1.2-300 µg/mL ), 100 µL of EUPS plus ConA or LPS (final concentration 5 µg/mL) or RPMI-1640 medium to a final concentration of 200 µL/well. All treatments were done in quadruplicate. The plates were

incubated at 37℃ in 5% CO2 for 68 h. The amount of proliferation was determined using the

CCK-8 assay according to the manufacturer's instructions. The optical density (OD) at 450 nm

was determined using a microplate reader (Synergy HT, Bio-TEK). The stimulation index (SI)

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The single splenocyte suspension was prepared as described above and seeded onto 96-well

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flat-bottom microtiter plates at a concentration of 2.5×106 cells/mL at 100 µL per well. Serial

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dilutions of EUPS (final concentration 9.4, 18.8, 37.5, 75, 150, and 300 µg/mL, respectively) or

Page 6 of 25

serial dilutions of EUPS plus ConA (final concentration 5µg/mL) were added (100 µL per well),

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and the plates were incubated at 37℃ in 5% CO2 for 48 h. After incubation, the culture

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supernatant was collected and the levels of IL-4 and IFN-γ were measured by ELISA (R&D

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Systems Inc., Minneapolis, USA) according to the manufacturer’s instructions.

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2.4.4 Generation of immature DCs

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The femurs and tibiae of ICR mice were removed and trimmed of surrounding muscle tissue.

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Intact bones were washed twice in PBS. Bone marrow cells were flushed from the mouse femurs

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and tibiae, and erythrocytes were lysed using ACK lysis buffer. DCs were grown at a starting

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concentration of 1 ×105/mL in round-bottomed 24-well plates (Costar) in RPMI-1640

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supplemented with 10% fetal calf serum, IL-4 (10 ng/mL), GM-CSF (10 ng/mL), penicillin (100

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IU/mL), streptomycin (100 IU/mL), kanamycin (100 µg/mL), L-glutamine (2 mL), and NaHCO3

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(1.2%). Cells were incubated at 37℃in 5% CO2. After 24 h, the non-adherent cells and culture

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medium was removed and replaced with fresh medium. The culture medium was replenished with

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an equal volume of fresh medium every two days.

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2.4.5 Cytotoxicity analysis of immature DCs

The concentration at which EUPS was not cytotoxic to DCs was determined using the CCK-8

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assay. EUPS was diluted with RPMI-1640 (1.2 to 1200 µg/mL). DCs numbers were counted on a

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hemocytometer and viability was determined using the trypan blue dye exclusion technique. In all

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control cells, the treatment was not considered to be cytotoxic to DCs.

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2.4.6 Analysis DCs' key surface molecules by flow cytometry (FCM)

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cases, cell viability exceeded 95%. The DC suspension was adjusted to 1×106/mL and seeded onto a 96-well plate. Once the DCs grew into monolayers, 100 µL of each EUPS dilution was added to the wells, each concentration in quadruplicate. The plates were incubated at 37℃ in 5% CO2.

After 68 h, 20 µL of WST-8 was added to each well and incubated for 4 h. The OD value at 450

nm for each well was measured using a microplate reader (Synergy HT, Bio-TEK). When the OD value of the polysaccharide treated cells was not significantly lower than that of the untreated

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The cultured DCs were treated with 1.2 µg/mL, 4.7µg/mL, 18.8µg/mL, or 75µg/mL of EUPS

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or LPS (10 ng/L) for 24 h. The suspensions were blocked with 2 µL of Fcγ mAb (0.5 µg/mL) at

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4 °C for half an hour. The cells were washed with PBS twice, incubated with isotype control

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antibodies, or stained with anti-CD86-PE, anti-CD40-PE, or anti-CD80-PE, and then washed

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twice with PBS twice. The cells were fixed with 4% paraformaldehyde and their fluorescence

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intensities were measured on the FACS Calibur and analyzed by Cell Quest Pro software (BD

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Biosciences, USA).

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2.5. In vivo test

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2.5.1 Animals and immunization

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Fifty female ICR mice were randomly divided into five groups (n = 10 each): a FMDV group,

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a EUPS group, a FMDV plus EUPS group, a FMDV plus alum group, and a naive group. On day

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0 and 14, the mice were immunized subcutaneously in the hind limb with different vaccine

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formulations: (1) 200 µL FMDV vaccine alone, (2) 0.5 mg EUPS alone, (3) 200 µL FMDV

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vaccine plus 0.5 mg EUPS, (4) 200 µL FMDV vaccine plus 200 µg alum, and (5) 200 µL saline.

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2.5.2 Antigen-specific antibody Detects

Serum samples were collected 14 d after the second vaccination to detect the level of specific

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FMDV-IgG titer and IgG isotypes using an indirect enzyme-linked immunosorbent assay as

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previously described (Feng, et al., 2013). Briefly, the wells of a polyvinyl 96-well microtitre plate

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were coated with FMDV solution, 100 µL per well, and the plate was incubated at 4 ℃ for 18 h.

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After washing three times, the wells were blocked using skimmed milk (5%) and incubated at

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37 ℃ for 1 h. After three washes, the serum samples was subsequently seeded into each well,

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100 µL per well, and then the mixture was incubated for 1 h at 37 ℃ . Horseradish

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at 450 nm. The data are expressed as the mean sample ODs minus the mean control ODs.

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2.5.3 Splenocyte proliferation assay

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peroxidase–conjugated antibodies against IgG, IgG2b, IgG1, or IgG2a were added to each well

and incubated for 1 h at 37oC. After washing, the peroxidase activity was measured as follows: 37.5 µL of the substrate solution ( 10 mg of O-phenylenediamine and 37.5 µL of 30% H2O2 added

to 25 mL of 0.1 M citrate–phosphate buffer, pH 5.0) was added into each well, and the mixture

was incubated at 37 ℃ for 15 min. The reaction was terminated using 2 M H2SO4. The optical density (OD) was detected using a microplate reader (Model 680, Bio-Rad) set to read absorbance

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The spleens of the FMDV-immunized ICR mice were collected on day 14 after the second

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immunization. Single splenocyte suspensions were prepared and a splenocyte proliferation assay

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was constructed as described previously (Feng, et al., 2014). Cell numbers were counted on a

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hemocytometer and viability was determined using the trypan blue dye exclusion technique. In all

Page 8 of 25

cases, cell viability exceeded 95%. The amount of T cell proliferation was measured using a

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CCK-8 assay according to the manufacturer's instructions. The ODs were measured using a

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microplate reader (Model 680, Bio-Rad) set to read absorbance at 450 nm. The data are expressed

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as the stimulation index (SI), which was calculated based on the following formula: SI = the

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absorbance value for the antigen stimulated cultures divided by the absorbance value for the

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unstimulated cultures.

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2.6. Statistical analysis

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Data analysis was performed with SPSS software (SPSS, Version 11.5, SPSS Inc., Chicago, IL)

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Values are expressed as mean ± standard deviation (S.D.). Duncan and LSD’s multiple range test

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were used to determine the difference among groups. P＜0.05 were considered to be statistically

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significant.

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3 Results

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3.1. Characterization of EUPS

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3.1.1 Physicochemical property analysis

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The physicochemical properties of EUPS are as follows. The color of the EUPS

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was brown, and it was water soluble. The results of the carbazole (· ),

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phenol---sulfuric acid (+), α-naphthol (+), iodination (· ), fehling’s (· ), and FeCl3 tests

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(· ), confirmed that the EUPS was composed of polysaccharides but did not contain

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Briefly, the average EUPS contained 97.4% of total carbohydrate, using the Anthrone-sulfuric acid

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method. The proteins content was 2.1%, using bovine serum albumin as a standard, and the

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polysaccharide samples were not contaminated with uronic acid. The average molecular weight of

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EUPS was 11.4632 ·· 10 Da, according to the calibration curve with standard dextrans. The

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component monosaccharides released from the EUPS were hydrolyzed with TFA and GC, and

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uronic acid, reducing sugar, starch, or polyphenol. The results of Coomassie brilliant blue (+), and the full wavelength scanning (+) analysis indicated that the

EUPS contained proteins.

3.1.2 Composition and characterization of polysaccharides The monosaccharide composition and chemical characteristics of EUPS were evaluated.

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analysis of hydrolysates was performed using precolumn-derivatization techniques with

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aldononitrile acetate. EUPS contained neutral monosaccharide composition of glucose, fructose,

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mannose, fucose, galactose and arabinose with a relative mass of 36.6%, 16.6 %, 14.2%, 15.7 %,

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9.5% and 7.4 %, respectively.

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3.1.3 FT-IR spectroscopy

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The FT-IR spectra of the EUPS in the range of 4000 to 400 cm−1 are illustrated in Fig.1. The

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typical major broad stretching peak around 3447.35 cm-1 corresponded to the specific absorption

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of the O-H stretch, and the stretching peak at around 2939.14 cm-1 corresponded to the C-H stretch.

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The absorption peak in the region of 1637.37 cm-1 was attributed to the C=O asymmetric stretch.

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The absorption peak at 1405.88 cm− was attributed to the COO– asymmetric stretch. In the region

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of 1000-1200 cm-1, EUPS had a characteristic absorption band which was dominated by ring

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vibrations overlapped with the (C-O-C) glycosidic band vibrations and (C-OH) stretching

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vibrations of side groups. The absorption peaks at around 1118.54 cm-1, 1153.24 cm-1 and 1263.77

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cm-1 revealed that EUPS was a pyranose sugar. The absorptions peak at 890.79 cm-1 indicated that

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the polysaccharide contained a β-type glycosidic bond in its structure.

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Fig. 1. FTIR spectra of EUPS.

3.1.4 SEM analysis

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The SEM photographs of the EUPS are shown in Fig. 2. Fig. 2A, B and C show that EUPS

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was sponge-like, and that its surface exhibits a rough surface with pores and crevices on it. The

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size distribution of the EUPS powder was not within a narrow range, and the particle size was not

Page 10 of 25

uniform. In summary, the EUPS were qualitatively identified by comparing their micrographs with

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the standards.

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Fig 2 (A)

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Fig 2 (B)

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Fig 2 (C)

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Fig. 2. Scanning electron micrographs of the four polysaccharides. (A) SMP-1 (500×), (B) SMP-1 (3000×), (C) SMP-2 (5000×)

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3.2 Experiment in vitro

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3.2.1 EUPS cytotoxicity to splenic lymphocytes and DCs in vitro The cytotoxicity of EUPS to splenic lymphocytes and DCs was evaluated using the CCK-8

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assay. Our data showed that the highest concentration of EUPS that was not cytotoxic to splenic

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lymphocytes was 300 µg/mL (Table 1). Nine concentrations of EUPS (1.2, 2.4, 4.8, 9.4, 18.8, 37.5,

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75, 150, and 300 µg/mL) were tested in this experiment. The results of the cytotoxicity experiment

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are shown in Table 1, and the highest concentration at which EUPS was not cytotoxic to DCs was

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150 µg/mL. Four concentrations of EUPS (1.2, 4.8, 18.8, and 75 µg/mL) were tested in this

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experiment.

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Table 1

The highest non-toxic concentrations of EUPS on splenocytes and dendritic cells (DC) isolated from mice Concentrations

of

EUPS

（µg/mL)

(OD Values)

(OD Values)

d

0.0302±0.061e 0.0327±0.032e

0.2312±0.004a

0.1043±0.016d

0.2929±0.048cd

0.1701±0.036a

0.3326±0.026c

0.2287±0.035 ab

37.5

0.2831±0.035 c

0.2513±0.041b

18.8

0.2681±0.047b

0.3554±0.063d

9.4

0.2558±0.030b

0.3122±0.039 c

4.7

0.2593±0.009 b

0.2997±0.025c

2.4

0.2485±0.024a

0.2436±0.048b

1.2

0.2415±0.024a

0.1923±0.033a

Cell control

0.2361±0.023a

0.1832±0.023 a

300 150

Ac ce p

75

te

0.2301±0.042a

600

311

Proliferation of DCs

0.1922±0.041e

1200

310

Proliferation of splenocytes

M

308

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301

Note: Data within a column without the same superscripts (a–e) differ significantly (P< 0.05).

3.2.2 Effects of EUPS on splenocyte proliferation in vitro

312

The effect of a single treatment of EUPS on splenocyte proliferation is shown in Fig. 3A. The

313

SI for the proliferation of the EUPS-treated cells was significantly higher than the untreated group.

314

(P＜0.05). Stimulation of lymphocytes with EUPS and ConA (EUPS concentration between 18.8

315

to 300 µg/mL) dramatically increased proliferation (Fig.3.B), while treatment with EUPS plus

Page 12 of 25

LPS (EUPS at 9.4 to 300 µg/mL) caused significantly higher proliferation than the cells treated

317

with only LPS (P＜0.05; Fig.3.C).

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320 321

Fig. 3. Effects of the EUPS on splenocyte proliferation in vitro. Single splenocytes were isolated from ICR mice

322

and cultured with EUPS (1.2-300 µg/mL), EUPS plus Con A (5µg/mL) or EUPS plus LPS (5µg/mL) for 68 h.

Page 13 of 25

323

Amount of proliferation was measured using the CCK-8 assay, as described in the methods. The stimulation index

324

is presented as mean ± S.D.. Significant differences are designated as different letters (P< 0.05).

325

3.2.3 Effects of EUPS on cytokine production of lymphocyte in vitro The amount of IL-4 produced by EUPS-treated lymphocytes was higher than untreated cells (P

327

＜0.05), when concentrations of EUPS were 18.8 to 37.5 µg/mL (Fig.4.A). When lymphocytes

328

were treated with EUPS and ConA, (EUPS 9.4 µg/mL to 300 µg/mL) the production of IL-4 was

329

significantly higher than in ConA-treated cells (P＜0.05; Fig.4.B). EUPS (18.8-75 µg/mL)

330

induced more production of IFN-γ than untreated cells (P＜0.05; Fig.4.C). EUPS (37.5 µg/mL to

331

300 µg/mL) plus ConA increased the production of IFN-γ compared to ConA treatment alone (P＜

332

0.05; Fig.4.D).

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These results indicate that EUPS can significantly stimulate lymphocytes to secret the Th1-type

334

cytokine IFN-γ and the Th2- type cytokine IL-4 and suggests that EUPS can augment both Th1

335

and Th2 responses simultaneously.

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339 340

Fig. 4. Effects of the EUPS on IL-4 and IFN-γ production by splenocytes in vitro. IL-4 (A, B) and IFN-γ (C, D)

341

production by ICR mouse splenocytes stimulated with EUPS alone, EUPS plus ConA, or EUPS plus LPS.

Page 15 of 25

342

Splenocytes were isolated from ICR mice and incubated with EUPS (1.2~300µg/mL) and EUPS plus Con A

343

(5µg/mL) for 48 h. The concentrations of IFN-γ and IL-4 in supernatants were determination by ELISA. The

344

values are presented as mean ± S.D.. Significant differences are designated as different letters (P< 0.05).

345

3.2.4 The effect of EUPS on the expression of DCs' key surface molecules in vitro The effect of EUPS on the maturation of DCs from bone marrow cells was also evaluated.

347

DCs were incubated with four concentrations EUPS (1.2, 4.7, 18.8, and 75 µg/mL) for 24 h, and

348

the expression of surface molecules (MHC-II, CD80, CD86 and CD40) was measured by flow

349

cytometry. As shown in Table. 2, EUPS increased the expression MHC-II, CD80, CD86 and CD40

350

by DC cells compared to untreated cells. The data suggested that EUPS can activate DCs and

351

induce DC maturation.

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Table 2

353

Effects of EUPS on the expression of CD40, CD80, CD86, MHCⅡby DCs as measured by flow cytometry

LPS

38.795±0.549

4.7µg/mL

b

31.377±0.891

1.2µg/mL c

28.579±0.318

Cell Control c

24.637±0.872d

CD40

46.817±0.159

CD80

94.716±1.763 a

81.312±1.272 bc 84.139±0.863 b 73.215±0.925 c 71.467±9.307 c 69.358±0.546c

CD86

93.175±1.549 a

85.321±0.357 b 86.732±1.169 b 79.332±0.345bc 72.563±0.421 c 71.134±0.648 c

MHCⅡ 73.393±0.233 a

59.959±0.363 b 661.67±0.863 b 554.682±0.239 bc 51.288±0.507 c 50.263±0.765 c

d

36.354±0.464

18.8µg/mL bc

Note: Data within a row without the same superscripts (a–d) differ significantly (P< 0.05).

361

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355

75µg/mL a

M

(% positive cells).

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352

362

compared with those immunized with FMDV + alum or FMD vaccine alone (P < 0.05). In

363

addition, all of the IgG subclasses tended to be higher in mice that had been treated with EUPS

364

plus FMD vaccine compared to mice that had been treated with only the FMD vaccine (Fig. 5B, C,

365

D). The data suggest that EUPS significantly promoted FMDV-specific antibody production in the

366

immunized mice.

356 357 358 359 360

3.3 Experiment in vivo

3.3.1 The effect of EUPS on the humoral response in vivo To confirm the effect of EUPS on the humoral immune response, serum was collected from each

animal on day 14 after the boost immunization, and the amount of FMDV-specific IgG, IgG1, IgG2b, and IgG2a antibodies was measured using an indirect ELISA. As shown in Fig 5A, the FMDV-specific IgG level was dramatically increased in the mice immunized with FMDV+EUPS

Page 16 of 25

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Fig. 5. Effects of the EUPS as adjuvant on antibody response in vivo. The serum sample from ICR mice were

370

collected for ELISA on day 14 after the second immunization. The FMDV-specific IgG(A), IgG1(B), IgG2a(C),

371

and IgG2b(D) were measured by ELISA as described in Materials and methods section. The values are presented

372

as mean±standard deviation (n = 10). P < 0.05.

373

3.3.2 Effects of EUPS on T cells proliferation in vivo

375 376 377 378

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The effects of EUPS on T cell proliferation in the immunized mice are listed in Fig.6. The SI of

the proliferation in the FMD vaccine + EUPS group was significantly higher than those in the

FMDV vaccination group, FMD vaccine + alum group, EUPS groups, naive groups, and BSA

groups, respectively (P < 0.05). EUPS as an adjuvant can cause significantly higher levels of T cell proliferation compared to FMDV vaccination alone.

Page 17 of 25

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Fig. 6. Effects of EUPS as adjuvant on T cell proliferation in vivo. T cells were isolated from ICR mice in all

381

groups on day 14 after the boost. The levels of T cell proliferation was measured by the CCK-8 method as

382

described in Materials and methods section, and expressed as SI. The values are presented as mean±standard

383

deviation (n = 10). P < 0.05.

384

4. Discussion

DCs and T cells are the most important cellular components of the cellular immune response.

386

As professional antigen presenting cells, DCs have a central role in initiating T-cell responses

387

against pathogens, and only the mature DCs can directly activate the adaptive immune response

388

393 394

maturation. For example, Pueraria lobata polysaccharide can promote the expression of (CD40,

395

CD86 MHCⅠ/II) on DCs, and increased production of IL-12, IL-1β, and TNF-α of DCs. Those

396

results suggested that PLP activates murine DCs and leads to maturation (Kim, et al., 2013). Meng

397

et al. (2011) have reported that Laminaria japonica polysaccharides (LJP) enhanced expression of

398

key membrane molecules (CD80, CD86, CD40, CD83, and MHC II) on DCs, suggesting that LJP

399

has a strong ability to induce the maturation of DCs. In our study, EUPS activated DCs and

389 390 391 392

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Ac ce p

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380

(Chakraborty, et al., 2014; Yong, et al., 2009). Mature DCs have a high capacity to prime T cells,

process and uptake antigen, and are characterized by their high expression of surface MHC II,

CD40, CD80, and CD86. The expression of specific co-stimulatory molecules (CD80/CD86 and

CD40) on DCs is correlated with T lymphocyte differentiation and the upregulation of CD80,

CD86 and CD40 typically results in the reinforcement of T-cell activity (Zhang, et al., 2013; Zou,

et al., 2011). Many researchers have demonstrated that plant polysaccharides can induce DCS

Page 18 of 25

induced DC maturation in vitro, characterized by a higher expression of MHC II molecules, CD40,

401

CD80, and CD86. Lymphocyte proliferation is a direct measure of immune function (Actor, 2014). It is

403

generally known that T and B lymphocytes are both crucial for the regulation of the immune

404

response. T lymphocytes mainly mediate cellular responses and can undergo mitosis after

405

incubation with ConA in vitro. B lymphocytes mainly initiate the humoral immune response and

406

can proliferate while cultured with LPS (Marits, et al., 2014; Bishop, et al., 2001). Abula et al.

407

(2011) confirmed that Siberian solomonseal rhizome polysaccharide (SRPS) could promote

408

lymphocyte proliferation levels in vitro, and co-administering the SRPS with the ND vaccine in

409

chickens significantly promoted the levels of lymphocyte proliferation, suggesting that SRPS

410

enhanced cellular immunity in vivo. Wang and Meng et al. (2013) reported that Cordyceps

411

militaris polysaccharide (CMP) could stimulate lymphocyte proliferation in vitro. CMP act as an

412

adjuvant for ND vaccine in chickens could dramatically improve lymphocyte proliferation in vivo.

413

In our study, EUPS induced the proliferation of lymphocytes compared to the untreated cells. In

414

combination with ConA, EUPS significantly increased lymphocyte proliferation and our data

415

suggest that EUPS acts synergistically with ConA to dramatically increase T lymphocyte

416

proliferation. Furthermore, in EUPS plus LPS treated cells, EUPS synergistically acts with LPS to

417

significantly promote B lymphocyte proliferation. The research results in vivo also showed that the

418

423 424

and transient antibody responses (Wang and Meng, et al., 2013; Abdin, et al., 2013). Type 2 helper

425

T cells (Th2 cells) mediate Th2 responses characterized by the production of IL-4, IL-10, IL-5,

426

and IL-13, and evoke humoral immune responses but relatively weak cellular immune activity.

427

IFN-γ is a Th1 cytokine and has both immunostimulatory and immunomodulatory effects. Most

428

importantly, IFN-γ has been used as an indicator for innate and adaptive immunity against viral,

429

and some bacterial and protozoal infections. IL-4 has many biological activities, including

419 420 421 422

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400

SI in the EUPS adjuvant groups was larger than the SI values of mice in the FMDV vaccination groups.

The production and release of cytokines modulates the functions of the T helper cellular

immune response. Type 1 helper T cells (Th1 cells) mediate Th1 responses and produce IFN-γ, IL-2, TNF-α, and IL-12 that initiate protective immunity against intracellular infectious agents such as viruses, certain bacteria and protozoa. Th1 cells stimulate cellular immunity but only weak

Page 19 of 25

regulation of humoral and adaptive immunity, differentiation of B cells into plasma cells, and

431

activation of T cell and B cell proliferation (Ranasinghe, et al., 2014). Huang et al. (2013)

432

demonstrated that Rehmannia glutinosa polysaccharide (RGP) stimulated T lymphocytes and

433

could significantly up-regulate IFN-γ and IL-2 production in vitro. Wang and Deng et al. (2013)

434

observed that a polysaccharide extracted from Kadsura (KPS) induced significant levels of IL-2,

435

IFN-γ and TNF-α secretion by chicken lymphocytes in vitro. The experimental results indicated

436

that KPS significantly enhanced cytokine secretion. In this study, we evaluated the effect of EUPS

437

on the production of IFN-γ (Th1-type cytokine) and IL-4 (Th2-type cytokine) by T lymphocytes in

438

vitro. The concentrations of IFN-γ and IL-4 produced by splenocyte suspensions were detected by

439

ELISA and the data showed that EUPS enhanced both Th1 and Th2 cytokines production in vitro.

440

This suggests that EUPS can increase the differentiation and proliferation of Th cells, and promote

441

a balanced Th1/Th2 immune response.

The humoral immune response mediated by B-lymphocytes has an important impact on

443

resistance to pathogen infection. The serum antibody level is the crucial marker that reflects the

444

state of humoral immunity (Montomoli, et al., 2011). Wang and Meng, et al., (2013) have reported

445

that two kinds of polysaccharide isolated from Cordyceps militaris (CM) could significantly

446

promote the serum antibody titer of Newcastle disease vaccine in chickens. Yang et al. (2014)

447

have demonstrated that using polysaccharides from Physalis alkekengi L. (PPSB) as an adjuvant in

448

453 454

lymphocyte proliferation either by itself or in combination with ConA and LPS, which could

455

promote the production of IL-4 and IFN-γ by T lymphocytes, and could stimulate DC proliferation

456

and maturation. The in vivo experiments suggested that administration of EUPS could

457

dramatically enhance the FMDV-specific IgG, IgG1, IgG2a, and IgG2b antibody titers and T cell

458

proliferation. These data reveal that EUPS strongly enhances immune responses and that it could

459

be used as a potential adjuvant for vaccine design.

449 450 451 452

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442

Ac ce p

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430

a DNA vaccine can significantly enhance specific antibody titers IgG, IgG2b, and IgG1Our

experimental results revealed that FMDV-specific IgG, IgG1, IgG2b, and IgG2a levels in the FMD

vaccine plus EUPS group were significantly higher than in the other groups. This finding

suggested that EUPS could dramatically promote the FMDV-specific humoral immune response. In summary, immuno-enhancing activities of EUPS were demonstrated using both in vitro and

in vivo methods. The in vitro experiments indicated that EUPS significantly enhanced T and B

Page 20 of 25

460

Acknowledgements The project was supported by the national Natural Science Foundation of China (Project

462

No.31402238) and in part by the Fundamental Research Funds for the Central Universities

463

(Project No. SWU113091) and in part by Foundation of Chengdu University (Project No.

464

2015XJZ07).

465

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ulmoides. International Immunopharmacology, 8, 1222-1230. Zou, Y.X., Meng, J.J., Chen, W.N., Liu, J.L., Li, X., Li, W.W., Lu, C.L., Shan, F.P. (2011).

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11, 1103-1108. Highlights

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1. Chemical properties, monosaccharide composition, scanning electron microscopy (SEM)

587

analyses, and immunoenhancement activities of EUPS were firstly characterized.

588

2. EUPS significantly stimulated proliferation of splenic lymphocyte in vitro.

589

3. EUPS significantly promoted IL-4 and IFN-γ production of T lymphocyte in vitro.

590

4. EUPS could activate DCs and induce them to the maturation in vitro.

591

5. EUPS as adjuvant significantly improved the humoral and cellular immune response in vivo.

an

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Page 25 of 25