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The anti food allergic activity of sulfated polysaccharide from Gracilaria lemaneiformis is dependent on immunosuppression and inhibition of p38 MAPK Qingmei Liu, Yang Yang, Soheila J. Maleki, Marcos J. C. Alcocer, Shasha Xu, Chaolan Shi, Min-Jie Cao, and Guang-Ming Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01086 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Journal of Agricultural and Food Chemistry

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The anti food allergic activity of sulfated polysaccharide from

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Gracilaria lemaneiformis is dependent on immunosuppression and

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inhibition of p38 MAPK

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Qing-Mei Liu a, Yang Yang a, Soheila J. Maleki b, Marcos Alcocer c, Sha-Sha Xu a, Chao-Lan Shi a,

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Min-Jie Cao a, Guang-Ming Liu a*

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a

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for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou

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Road, Xiamen, 361021, Fujian, P.R. China

College of Food and Biological Engineering, Fujian Collaborative Innovation Center

10

b

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Center, 1100 Robert E. Lee Boulevard, New Orleans, LA 70124, USA

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c

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LE125RD, UK

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Running title: Anti-allergic activity of sulfated polysaccharide from Gracilaria lemaneiformis

U.S. Department of Agriculture, Agriculture Research Service, Southern Regional Research

School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough,

15 16

*Corresponding author:

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Guang-Ming Liu, PhD

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College of Food and Biological Engineering, Jimei University

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Xiamen, 361021, P.R. China

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Tel: +86-592-6180378

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Fax: +86-592-6180470

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Email: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT: Polysaccharides from Gracilaria lemaneiformis in particular possess

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various bioactive functions, but their anti-allergic activity remains incompletely

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defined. Sulfated polysaccharide from Gracilaria lemaneiformis (GLSP) was

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obtained

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column chromatography. BALB/c mice, RBL-2H3 and KU812 cells were used for

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verifying the anti food allergic activity of GLSP. According to the results of mice

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experiment, GLSP was able to alleviate allergy symptoms, to reduce TM-specific IgE

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and IgG1, to suppress Th2 cell polarization, and to promote the function of regulatory

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T (Treg) cells. In addition, GLSP had the ability to inhibit the function of RBL-2H3

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cells. Furthermore, GLSP inhibited the activation of KU812 via suppression of p38

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mitogen-activated protein kinase (MAPK). In conclusion, immunosuppression as well

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as the reduction in the level of p38 MAPK may contribute to GLSP putative activity

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against food allergy. GLSP may be used as a functional food component for allergic

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patients.

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KEYWORDS: sulfated polysaccharide from Gracilaria lemaneiformis; food allergy;

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immunosuppression; p38 MAPK.

by

water

extraction,

ethanol

precipitation

40

2


followed

by

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INTRODUCTION

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Food allergy is a rapidly growing public health concern because of its increasing

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prevalence and life-threatening potential. Food allergy is a kind of hypersensitivity

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caused by food containing some kinds of allergen, and manifests as a variety of

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symptoms that affects nearly 8% of children and 5% of adults worldwide, with

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growing evidence of an increase in prevalence. 1 Shellfish allergy in the Asia-Pacific

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region in particular ranks among the highest in the world and is the most common

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cause of food-induced anaphylaxis in the region.2 In China, 16.7% of the rural

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population is sensitized to shellfish,3 in which tropomyosin (TM) is the major

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allergen.4 TM have been previously demonstrated to be a potent allergen in crab

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which could cause adverse hypersensitivity in a mouse model.5

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After activation by allergens, the subset of CD4+ T-lymphocytes could produce a

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spectrum of cytokines such as interleukin (IL)-4, IL-5, and IL-13 that induce serum

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immunoglobulin E (IgE) production, which in turn mediate the clinical symptoms.6

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The IgE immunoglobulins then attach specifically to Fc epsilon region Receptor 1

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(FcεR1) membrane receptors of mast cells or basophils. Mast cells and basophils are

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key effector cells in allergy, once activated they can release cytokines, chemokines,

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proteases, leukotrienes and bioactive polyamines.7, 8 The activation of mast cells and

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basophils involves a complex network of signal transduction pathways and molecules,

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including various tyrosine kinases, Akt, and mitogen-activated protein kinases

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(MAPKs).9 MAPKs signaling cascades are important in the differentiation, activation,

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proliferation, degranulation, and migration of various immune cells, including 3



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basophils and mast cells.10 In addition, regulatory T cells (Treg) are known to

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alleviate clinical symptoms of hypersensitivity to dietary food allergen by modulating

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the priming of allergen-specific T and B cell responses during oral sensitization and

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mast cell degranulation.11

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Indigenous people from southeast of China whilst eating shellfish simultaneously

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ingest marine algae products. In traditional Chinese medical literature, the marine

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algae are described as improver of digestion, enhancer of immunity amongst others.

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Recently, the role of marine algae as anti-allergic agents has been demonstrated in

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vitro and in vivo studies.12 It was shown that Laminaria japonica polysaccharides can

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significantly inhibit airway inflammation of asthmatic mice, adjust the balance of

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cytokines, and improve the pulmonary histopathological condition.13 Ishihara14 had

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reported that porphyran from red algae Porphyra tenera and P. yezoensis were capable

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to inhibit the contact hypersensitivity reaction by decreasing the IgE serum level in

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BALB/c mice. Our previous studies also demonstrated that sulfated polysaccharide

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purified from Porphyra haitanensis (PHPS) which composed mainly of galactose and

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sulfate had an anti-allergic activity by suppressing Th2 immune response in the

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TM-sensitized mice.15 The Rhodophyta Gracilaria lemaneiformis has similar

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polysaccharide content and more affordable compare with Porphyra haitanensis.

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Several biological activities have been attributed to Gracilaria lemaneiformis

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amongst them antitumor, antiviral, hypoglycemic, and immunomodulatory effects.16-18

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However, to the best of our knowledge, there are no studies describing the

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anti-allergic activity of the sulfated polysaccharides from G. lemaneiformis. 4


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In this study, the structural characteristics of sulfated polysaccharide isolated from

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Gracilaria lemaneiformis (GLSP) were described. The anti-allergic activity of GLSP

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was evaluated employing the TM-sensitized mice and cellular models. Moreover, the

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key functional group of GLSP was shown to possess an anti-allergic activity, and the

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effects of GLSP on signaling pathway molecules were further investigated in

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basophils.

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MATERIALS AND METHODS

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Regents and materials. DEAE-cellulose 52 was obtained from Whatman (New

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York, USA). Imject Alum was purchased from Thermo Fisher Scientific Inc (Waltham,

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MA, USA). RPMI 1640, minimum essential medium with Eagle’s salts (EMEM),

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penicillin, streptomycin solution, and fetal calf serum (FCS) were purchased from

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Invitrogen (New York, USA). Anti-dinitrophenyl (DNP)-IgE was obtained from

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Sigma (Louis, MO, USA), and DNP-BSA was purchased from Biosearch (Petaluma，

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CA). Human IgE, anti- human IgE, and goat anti mouse IgE, IgG1, IgG2a antibodies

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were purchased from Abcam (Cambridge, UK). Fluorescein isothiocyanate

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(FITC)-conjugated CD63 was obtained from BD pharmingen (San Diego, CA).

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ELISA kit of histamine was purchased from IBL (Hamburg, Germany). Human

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phospho-MAPK array and ELISA kits of IL-4, IL-13, interferon (IFN)-γ,

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transforming growth factor (TGF)-β, and mouse mast cell proteinase (mMCP)-1 were

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purchased from R&D Systems (Minneapoils, MN, USA).

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Gracilaria lemaneiformis was provided by the Third Institute of Oceanography, 5



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State Oceanic Administration, Xiamen, China.

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Cells. The rat mast cell line (RBL-2H3) and human basophil cell line (KU812)

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were obtained from the American Type Culture Collection (ATCC, Manassas, VA,

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USA). The RBL-2H3 and KU812 cells were grown in EMEM and RPMI 1640,

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respectively, and supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL

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penicillin, and 100 µg/mL streptomycin at 37℃ in a humidiﬁed incubator with a 5%

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CO2/95% air atmosphere (Forma 3111, Thermo, Waltham, MA, USA).

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Mice. Female BALB/c mice, 4-6 weeks of age, were obtained from the Animal

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Center of Xiamen University (China). Mice were housed in a Specific Pathogen Free

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(SPF) environment maintained at 22±1°C with a relative humidity of 55±10%, and

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experiments were performed in conformity with the laws and regulations for

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treatment of live animals in Jimei University (Xiamen, China), SCXK 2012-0005.

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Purification of polysaccharide from Gracilaria lemaneiformis. Polysaccharides

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from Gracilaria lemaneiformis were extracted and purified according to the

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procedures described by Shi.15 Briefly the algae was extracted in 95℃ water, ethanol

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precipitated, and fractionated in an anion exchange column (DEAE-cellulose 52). The

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carbohydrate content of the polysaccharide was monitored by the anthrone-sulfuric

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acid method.19 The endotoxin was removed from the purified polysaccharide fraction

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using Detoxi Endotoxin Removing Gel (Pierce, Rockford, USA). The endotoxin

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content of the polysaccharide fraction was determined to be < 0.03 EU/mg as

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measured by the Limulus amebocyte lysate assay (Associates of Cape Cod, New York,

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USA). 6


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Characterization

of

polysaccharide

from

Gracilaria

lemaneiformis.

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Carbohydrate was quantified by the anthrone-sulfuric acid method using galactose as

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the standard.19 The 3,6-anhydrogalactose, sulfate and protein contents were

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respectively determined as the described by Shi.15 The molecular weight distribution

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of the polysaccharide was determined by high performance gel-permeation

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chromatography (HPGPC) (Agilent-1100, Santa Clara, USA) equipped with a

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ZORBAX Eclipse XDB-C18 column (250 mm ×4.6 mm, column temperature 30 °C).

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The monosaccharide composition of GLSP was determined after acid hydrolyses of

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the polysaccharide by ion chromatography (IC) analysis (ICS-3000, Dionex,

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Sunnyvale, CA, USA) in a CarboPac PA20 column (3×150 mm), using 0.25 M NaOH

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at a flow rate of 0.5 mL/min. The infrared spectra of the polysaccharide fraction was

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collected in a Fourier transformed infrared spectrometer (FTIR) (VECTOR-22,

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BRUKER) in the wave number range of 4000-500 cm−1 using the KBr-disk method.

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Food allergy model. The purification of TM from mud crab (Scylla paramamosain) 20

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was carried out as described previously

Mice were sensitized and monitored as

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described by Yamaki.21 Briefly, four groups of mice experiment were established to

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investigate the anti food allergic activity of GLSP, and the schemes were PBS group,

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TM group, GLSP-preventive group, and GLSP-treatment group, respectively.

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Twenty-four mouse were randomized to the four groups. For the sensitization, mice

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were immunized with 50 µg of TM in 150 µL of phosphate-buffered saline (PBS)

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emulsified with Imject Alum by intraperitoneal injection on days 0 and 14. Then mice

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were challenged 6 times from day 28 to 38 by gavage with 10 mg TM in 200 µL of 7



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PBS every other day for establishing the food allergy model. In the preventive

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protocol, GLSP (5 mg/mouse in 200 µL of PBS) was orally administered daily from

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the day before the 1st challenge to the day before sacrifice (from day 27 to day 40)

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(regarded as GLSP-preventive group). And in the treatment protocol, GLSP (5

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mg/mouse in 200 µL of PBS) intake daily from mice already challenged with TM 3

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times (from day 33 to day 40) to evaluate the therapeutic effect of GLSP on food

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allergy (regarded as GLSP-treatment group). Meanwhile, the negative control mice

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(PBS group) were immunized with 150 µL PBS at day 0 and 14, and given

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intragastrically of 200 µL PBS at day 28, 30, 32, 34, 36, and 38.

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Anaphylactic symptoms. Anaphylactic symptoms and diarrhea rates (the

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proportion of diarrheal mice in each group) were scored for 1 h after each challenge.22

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Rectal temperatures were measured for 0-1 h after the 6th challenge using a rectal

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temperature

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concentrations of histamine and mMCP-1 in serum obtained for 1 h after the 6th

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challenge was determined using commercially available ELISA kits following the

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manufacturer's instructions. All mice were sacrificed on day 41, the levels of serum

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TM-specific IgE, IgG1, and IgG2a were measured using ELISA as described

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previously.23 Furthermore, western blot analysis of pooled serum TM-specific was

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performed as described by Yamaki.22

electronic

thermometer

(Prosper,

Shanghai,

China).

And

the

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Mesenteric lymph node (MLN) cells restimulation for cytokines measurement.

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MLNs were isolated from mice and minced in RPMI 1640 medium with 10% fetal

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calf serum and 1% penicillin-streptomycin solution at day 41. Cells were cultured in 8


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24-well plates and re-stimulated with the antigen TM (10 µg/mL) for 3 days. Then the

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cell supernatants were collected for measuring the concentrations of IL-4, IL-13,

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IFN-γ, TGF-β using ELISA kits following the manufacturer's instructions. And the

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cell precipitate were collected for mRNA preparation and real-time quantitative PCR

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(QPCR) for transcription factors according to the previous description.24

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RBL-2H3 assay. The release of β-hexosaminidase and histamine by RBL-2H3 cell

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was measured as a model of IgE-mediated mast cell allergic reaction, using the

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sensitization of anti-DNP-IgE and the stimulation of DNP-BSA.24, 25 The cytokines

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(IL-4 and TNF-α) were evaluated as described previously.24 In order to evaluate the

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role of sulfate in cell degranulation, the sulfate of GLSP was removed by heating

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GLSP as pyridinium salt in DMSO.26 The sulfate content of GLSP was measured by

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barium chloride-gelatin method.15

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Flow cytometry assay. To examine the effects of GLSP on the expression of

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activation-linked antigens of basophils, flow cytometry experiments were performed.

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Human basophil KU812 cells (1×106 cells) pretreated with or without GLSP (100-200

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µg/mL) for 24 h were incubated with 10 µg/mL of human IgE for 3 h and stimulated

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with 20 µg/mL of anti-IgE for 30 min in tyrode’s buffer. Then, cells were washed and

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stained with FITC-conjugated CD63 for 20 min at 4°C. Guava easyCyte 6-2L system

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and GuavaSoft 3.1.1 software (Millipore, Massachusetts, USA) were used for the

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signal-colour flow cytometry analysis. IgE-induced upregulation of CD63 was

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calculated from mean fluorescence intensities (MFIs) obtained with stimulated,

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unstimulated cells and GLSP pretreated cells. 9



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Proteome ProfilerTM array. KU812 cells after the same treatment with flow

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cytometry assay were lysed and relative phosphorylation levels of all three major

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families of MAPK (ERK1/2, p38α/β/δ/γ, JNK1/2/3/pan) and Akt 1/2/3/pan, RSK1/2,

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GSK-3α/β, MSK2, MEK3/6, HSP27 as well as p70 S6 kinase were determined by

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human phospho-MAPK array according to the manufactures’ instructions.

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Statistical analysis. Data were presented as means ± SD. Differences between

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means were analyzed by ANOVA of Duncan test. A p value of less than 0.05 was

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considered statistically significant. In the animal model study samples from individual

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mice were processed and analyzed separately. Each experiment was repeated at least 3

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times.

205 206 207

RESULTS Composition

of

polysaccharide

from

Gracilaria

lemaneiformis.

A

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polysaccharide fraction from Gracilaria lemaneiformis was purified as single

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chromatography peak and called hereafter as GLSP. The fraction contained

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3,6-anhydrogalactose, sulfate and protein at levels of 8.23%, 11.26%, and 0.98%,

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respectively. The average molecular weight of GLSP was 152,481 Da as determined

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by HPLC analysis (data not shown). According to the description by Sun,27 as shown

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in IC analysis results, the peak at 31.963 min was the galactose in standard substances

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(gray line in Fig. 1a), and the strong peak at 31.963 min content of 87.49% peak area

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was shown in GLSP sample (red line in Fig. 1a) . The FTIR spectrum of the sample

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was shown in Fig. 1b. GLSP exhibited absorption peaks at 3289 cm-1, 2920 cm-1and 10


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1070 cm-1, which were characteristic absorptions of -OH, C-H and C-O,

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respectively.28 The IR absorption at 931cm-1 had the characteristic absorption of

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3,6-anhydrogalactose. Infrared spectroscopy provided useful information for the

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position of sulfate groups of polysaccharide. The IR spectra show an absorption band

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at 1260 cm-1 and 850 cm-1, indicated the presence of the total sulfate ester.29

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The attenuation of anaphylaxis by GLSP in TM-sensitized mice. To test the

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effect of GLSP on food allergy, the animal experiment was implemented according to

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the protocol shown in Fig. 2a. Daily dose of GLSP moderately decreased the scores of

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the anaphylactic response (Fig. 2b) and diarrhea rates (Fig. 2c) for 1 h after the

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3rd-6th challenge in both GLSP-preventive and GLSP-treatment groups. The average

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rectal temperature of TM group mice was 3 °C less than PBS group mice for 1 h after

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the 6th challenge. Hypothermia induced was remitted by the administration of GLSP

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in GLSP-preventive mice (p

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