Immunomodulatory Activity of Ganoderma atrum Polysaccharide on

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Immunomodulatory activity of Ganoderma atrum polysaccharide on purified T lymphocytes through Ca2+/CaN and MAPK pathway based on RNA-seq Quan-Dan Xiang, Qiang Yu, Hui Wang, Ming-Ming Zhao, Shi-Yu Liu, Shaoping Nie, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 13 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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Journal of Agricultural and Food Chemistry

Immunomodulatory activity of Ganoderma atrum

1

2

polysaccharide on purified T lymphocytes through Ca2+/CaN

3

and MAPK pathway based on RNA-seq

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Quan-Dan Xiang#, Qiang Yu#, ∗, Hui Wang&, Ming-Ming Zhao#, Shi-Yu Liu#,

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Shao-Ping Nie#, Ming-Yong Xie#, ∗

7

#

8

Nanchang 330047, China

9

&

10

State Key Laboratory of Food Science and Technology, Nanchang University,

Institute of Life Science & College of Life Sciences, Nanchang University,

Nanchang 330031, China

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∗Corresponding to: Professor Ming-Yong Xie, PhD; Associate professor Qiang Yu,

13

PhD.

14 15

State Key Laboratory of Food Science and Technology, Nanchang University, 235

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Nanjing East Road, Nanchang 330047, China

17 18

Tel.&Fax: +86 791-83969009 (M. Y. XIE); +86 791-88304452 (Q. YU)

19

E-mail: [email protected] (M. Y. XIE); [email protected] (Q. YU)

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ACS Paragon Plus Environment


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ABSTRACT

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Our previous study has demonstrated that Ganoderma atrum polysaccharide

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(PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how

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PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed

24

to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes,

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and further elucidate the underlying mechanism based on RNA-seq. Our results

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showed that PSG-1 promoted T lymphocytes proliferation and increased the

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production of IL-2, IFN-γ and IL-12. Meanwhile, RNA-seq analysis found 394

28

differentially expressed genes. KEGG pathway analysis identified 20 significant

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canonical pathways and 7 biological functions. Furthermore, PSG-1 elevated

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intracellular Ca2+ concentration and calcineurin (CaN) activity, and raised the p-ERK,

31

p-JNK and p-p38 expression levels. T lymphocytes proliferation and the production of

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IL-2, IFN-γ and IL-12 were decreased by the inhibitors of calcium channel and

33

MAPKs. These results indicated that PSG-1 possesses immunomodulatory activity on

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purified T lymphocytes, in which Ca2+/CaN and MAPK pathway play essential role.

35 36

KEYWORDS: Ganoderma atrum polysaccharide; purified T lymphocytes; RNA-seq;

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Ca2+/CaN pathway; MAPK pathway.

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INTRODUCTION

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T cells are adaptive immunity cells, and play an irreplaceable role in immune

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response.1 IL-2, IFN-γ and IL-12 are immunomodulatory cytokines and T cells

41

growth factors that participate in T cell proliferation, differentiation, activation and

42

immunomodulation. As early as the 1980s, Mossman2 and Bob Coffman3 had

43

discovered two subsets of T cells and proposed that Th1 cells mainly produce IL-2

44

and IFN-γ, while Th2 cells mainly produce IL-4, IL-5, and IL-13. Subsequently, it

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was found that IL-12, IFN-γ induce naïve T cell differentiation into Th1 cells, which

46

involve in cellular immunity and clear intracellular pathogens.4, 5 IL-4 favors naïve T

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cells differentiated into Th2 cells, which involves in humoral immunity.4, 5

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Ca2+ has been described as a second signal messenger in various cells of the

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immune system,6 which participates in cell activation,7 and function.8 The continuous

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elevation of intracellular Ca2+ is essential for the expression of T cell activation

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genes.9 Ca2+ channel also involves in the expression of many cytokines, once Ca2+

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channel is impaired, it will lead to a strongly reduced secretion of several cytokines

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such as IL-2, IL-4 and IFN-γ in T cells.8 Calcineurin (CaN) is a Ca2+-activated

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serine/threonine phosphatase, which is a critical enzyme in T cells signal

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transduction.10 It has been demonstrated that Ca2+ linking with CaN results in

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activating immune response in B and T cells.11

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Mitogen-activated protein kinases (MAPKs) are signaling regulators that are

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responsible for T cell survival, differentiation and function, which include three

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classic subfamilies: the extracellular signal–regulated kinases (ERKs), c-Jun

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N-terminal kinases (JNKs) and p38 MAPKs in mammalian cells.12, 13 ERKs function

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in control of cell division, JNKs are pivotal regulators of transcription, and p38

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MAPKs may be concerned with diseases like asthma and autoimmunity. ERKs, 3



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known as physiological function kinases, are activated by mitogens and growth

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factors and are required for regulating cell proliferation and Th2 cell differentiation.14,

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15

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production,16 JNK3 results in preventing neuronal apoptosis in response to excitotoxic

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stress,17 p38 is specifically activated by pathway that results in IL-2 secretion in T

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cells18 and is necessary for IL-12 and IFN-γ production.19

JNK1 and JNK2 are essential for naïve CD4+ T cell differentiation and cytokine

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RNA-seq, also called whole transcriptome sequencing,20 is a recently developed

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technology that uses deep-sequencing technology to obtain gene information of the

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transcriptome.21 Specifically, RNA-seq is used to analyze the structure of

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transcriptome, which is able to extract a wide range of gene ontology,22,

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fusion,24 the polymorphism of coding sequence25 and the function of the non-coding

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sequences.26 In addition, RNA-seq can also be used for alternative splicing

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quantitative analysis27 and capturing the dynamic changes of the transcriptome.28

23

gene

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Ganoderma atrum has been safely used as ingredient of traditional medicine and

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functional food for thousands of years in oriental countries.29 Recently, a

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polysaccharide, named PSG-1 with a purity of > 99.8%, was extract from Ganoderma

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atrum in our laboratory. PSG-1 was a homogeneous protein-bound polysaccharide,

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which contained 10.1% protein and 17 general amino acids, and the molecular weight

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was about 1013 kDa.30 Monosaccharide composition analysis showed that PSG-1 was

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composed of glucose (72.5%), mannose (8.3%), galactose (5.7%) and glucuronic acid

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(13.5%).31 Based on methylation and GC-MS analysis, the main linkage type of

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PSG-1 was 1,3-linked-Glcp, T-Glcp, 1,3,6-Glcp, 1,4-Galp , 1,6-Glcp , 1,2-Manp,

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1,4-GalpA, 1,4-Manp and 1,4,6-Glcp.30 Our recent studies have demonstrated that

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PSG-1 possesses a variety of biological functions, for example, anti-tumor,32

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immunomodulatory,33

protection,34

cardiovascular 4


chemoprotective35

and

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hypoglycemic activities.36 Among them, of note is that PSG-1 has immunomodulatory

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effect on spleen lymphocytes.37 However, the experimental conclusion was derived

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from the mix lymphocytes, which could not illustrate the immunomodulatory effect of

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PSG-1 on purified lymphocytes. Therefore, in the present study, we first tried to

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purify T lymphocytes, then screen the key signal transduction pathways in purified T

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lymphocytes stimulated by PSG-1 via RNA-seq, and further investigate the

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underlying mechanism involved in the immunomodulatory activity of PSG-1 on

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purified T lymphocytes.

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MATERIALS AND METHODS

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Materials and Reagents. Pan T cell isolation kit II was from Miltenyi Biotec

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(Bergisch Gladbach, Rheinisch-Bergischer Kreis, Germany). Enzyme linked

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immunosorbent assay (ELISA) kits were from Wuhan Boster Biological Technology

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Co. (Wuhan, Hubei, China). Cell counting kit-8 was from Dojindo (Kumamoto,

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Japan). All antibodies used in flow cytometry were purchased from BD Biosciences

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(San Jose, CA, USA). The calcineurin assay kit was from Genmed Scientifics, Inc.

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(Shanghai, China). Antibodies against p44/42 MAPK (ERK1/2), SAPK/JNK

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(JNK1/2),

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phospho-SAPK/JNK (p-JNK1/2), phospho-p38 (p-p38) were purchased from Cell

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Signaling Technology (Beverly, MA USA). The related secondary antibodies and

107

β-actin were purchased from Zhong Shan Golden Bridge Biological Technology Co.

108

Ltd (Beijing, China). PD98059, SP600125, SB203580, Fluo-3/AM, RIPA buffer,

109

BCA protein assay kit, and BeyoECL plus ECL chemiluminescence kit were

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purchased from Beyotime Biotechnology (Nanjing, Jiangsu, China). Verapamil was

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purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 medium,

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hypotonic lysis buffer, and bovine serum albumin were purchased from Solarbio

p38

MAPK,

phospho-p44/42

MAPK

5


(ERK1/2)

(p-ERK1/2),


113

Science and Technology Co. Ltd. (Beijing, China).

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T lymphocytes purification. Female Balb/c mice, 6-8 weeks of age, 18−20 g of

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weight, were purchased from Hunan Slac Laboratory Animal Center, Chinese

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Academy of Sciences (Changsha, Hunan, China) certificate no. SCXK (Xiang)

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2016-0002]. All mice used in this study were cared for in accordance with the

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Guidelines for the Care and Use of Laboratory Animals published by the United

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States National Institutes of Health (NIH Publication 85-23, 1996), and all

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experimental procedures were approved by the Animal Care Review Committee,

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Nanchang University.

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then suspended with RPMI 1640 medium. Single cell suspensions were prepared by

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filtering the suspension through a sterile sieve mesh. Red blood cells were lysed with

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a hypotonic lysis buffer. The cells were washed twice with cold phosphate-buffered

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saline (PBS, pH7.2), and adjusted to the concentration of 5 × 106 cells/mL in RPMI

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1640 medium with 10% fetal bovine serum, following by incubating in cell culture

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dishes for 4 h. The suspended cells were the spleen lymphocytes. Then T lymphocytes

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were isolated by using Pan T cell isolation kit II according to the manufacturer’s

129

instruction. According to the flow cytometry, the purity of T cells was higher than

130

90%. The survival rate was higher than 95% as detected by trypan blue.

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Cytokine assay. T lymphocytes (density of 1 × 106 cells/mL) were cultured with 160

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µg/mL PSG-1 and/or 5 µg/mL ConA, without PSG-1 as the control group. The cells

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were incubated in 6-well culture plates, after 48 h the levels of cytokines in the

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supernatant were detected by ELISA kits according to the manufacturer’s instructions.

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Proliferation assay. T lymphocytes (5×105 cells/well) were incubated with medium

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alone as the negative control. Cells were pretreated with inhibitors for 30 min,

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including ERK inhibitor PD98059, JNK inhibitor SP600125, p38 inhibitor SB203580,

The spleens were removed aseptically and cut into pieces and

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and calcium channel inhibitor verapamil. Then cells were cultured with 160 µg/mL

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PSG-1 or 5 µg/mL ConA in 96-well plate. After 48 h, T lymphocyte proliferation was

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measured by CCK-8 kit. Each well was added with 10 µL of the CCK-8 solution and

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incubated for 3 h, then the absorbance was measured at 450 nm by microplate reader

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(Bio-Rad Laboratories, Hercules, CA, USA).

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Intracellular calcium concentration measurement. 1 × 106 cells/mL of T

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lymphocytes were incubated in 6-well plate with PSG-1 at the concentration of 160

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µg/mL. After 48 h the cells were collected and washed twice with PBS, then cultured

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with the fluorescent probe Fluo-3/AM at 37 °C for 60 min. The cells were washed

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with PBS, and Ca2+ concentration was analyzed by BD FACSCalibur™ flow

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cytometry (BD Biosciences, San Jose, CA, USA).

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Measurement of CaN activity. The cells (1 × 106 cells/well) were cultured in 6-well

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plate and stimulated with PSG-1 at the concentration of 160 µg/mL for 48 h. CaN

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activity was measured using the CaN activity assay kit. Briefly, the cells were treated

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by cell lysis buffer (reagent B) provided in the kit following the manufacturer’s

153

instructions. The optical density at 660 nm was determined for each sample using a

154

microplate reader.

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RNA extraction and cDNA synthesis. T lymphocytes (1 × 107 cells/mL) were

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cultured in tissue culture flasks with PSG-1 (160 µg/mL) for 48 h. Cells were

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harvested, total RNA was extracted using TRIzol® reagent (Life Technologies,

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Waltham, MA, USA), and reversed transcribed into cDNA using Prime

159

Script RT reagent Kit (TaKaRa Biotechnology, Dalian, Liaoning, China) according to

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the manufacturer’s protocol.

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Quantitative real-time PCR. Target gene was determined using the Quant Studio™

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7 Real-Time PCR System (Life Technologies, Waltham, MA, USA). Relative 7



163

expression of target gene was analyzed by qPCR using SYBR Premix Ex Taq II kit

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(TaKaRa Biotechnology, Dalian, Liaoning, China). For PCR, samples were heated to

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95 °C for 5 min, denatured at 95 °C for 15 s, annealed temperature was 60 °C for 15 s,

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72 °C for 20 s, and cycled 40 times. The primer sequences were listed in Table S1. To

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determine the relative expression compared to the group without PSG-1 stimulated, Ct

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values were normalized to β-actin, and relative expression was calculated using the

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the ∆∆Ct method.

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RNA-seq analysis. Library preparation and sequencing were performed at Shanghai

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Personal Biotechnology Co., Ltd. (Shanghai, China). Total RNA sample libraries

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were extracted using the Illumina TruSeq Stranded mRNA LT sample preparation kit

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(Illumina Inc., San Diego, CA, USA), according to the standard manufacturer's

174

instructions. rRNA was removed from the total RNA by ribosomal RNA removal kit

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(Illumina Inc., San Diego, CA, USA), the purified mRNA was denatured and diluted

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to 0.1 µg to 0.2 µg of total RNA before interrupted producing libraries with an insert

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size between 200 and 300 bp. cDNA was then synthesized from RNA using Super

178

Script II Reverse Transcriptase (Invitrogen Corp., Carlsbad, California, USA), the

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first strand of cDNA was synthesized with hexahedron random primers and reverse

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transcriptase, and the second strand cDNA was synthesized by using the first strand of

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cDNA as template. In the second strand of cDNA synthesis, dTTP was replaced by

182

dUTP, thus maintain the strandedness of the library. After the library was constructed,

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the fragmented library was enriched by PCR amplification, and then the library was

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selected according to the size of the fragment. The size of the library was 300 - 400 bp.

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Then 3’-adenylation, adaptor ligation and libraries were subjected to 15 cycles of

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PCR to generate RNA-Seq libraries ready for sequencing. Before sequencing

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RNA-Seq libraries were qualified by the Agilent 2100 Bioanalyzer with the high 8


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sensitivity DNA kit (Agilent Technologies, Palo Alto, California, USA).

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Quantification of libraries for clustering was performed using the KAPA library

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quantification kit - Illumina/Universal (KAPA Biosystems, Wilmington, MA, USA)

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and Quant Studio™ 7 Real-Time PCR System (Life Technologies, Waltham, MA,

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USA). Sequencing was performed using the Next-Generation Sequencing (NGS)

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technology, based on Illumina NextSeq500 (NextSeq control software v1.2/Real Time

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Analysis v2.1) platform. The library was diluted and denatured according to the

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effective concentration of the library and the number of library required and

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sequencing was carried out to produce single-end 76 bp reads using NextSeq500 High

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Output reagent kit (Illumina Inc., San Diego, CA, USA). FastQC was used to quality

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control, quality score Q > 30 of reads were used to downstream analysis. HTSeq

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0.6.1p2 was used to count the read counts for each gene in the ensembl annotation.

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Bioinformatics analysis. For differentially expressed genes analysis, we used the

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DESeq (version 1.18.0) to detected the differential expression (n=3) with the gene

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symbol annotation (FDR< 0.05, fold change > 2). In addition, principle component

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analysis (PCA) was carried out on the genes significantly expressed between all

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different groups. Enrichment analysis of differentially expressed genes was conducted

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utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway

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enrichment analysis. KEGG, which is a database resource for understanding

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high-level functions and utilities of the biological system from molecular-level

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information and the significantly and differentially expressed genes with fold changes

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information included in the KEGG pathway database. We counted the number of

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differentially expressed genes at different levels of KEGG pathway, and determined

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the metabolic pathways and signaling pathways.

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Western blot analysis. The whole cells were lysed using RIPA buffer containing the 9



213

protease inhibitor. The concentration of protein samples were measured with the

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enhanced BCA protein assay kit. Then protein samples were denatured at 95 °C for 5

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min, loaded onto SDS-PAGE and transferred to the PVDF membrane (Millipore Co.,

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Belford, MA, US). The membrane was sealed with 5% bovine serum albumin (BSA)

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and incubated with primary antibody overnight at 4 °C and followed by incubation

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with HRP conjugated anti-rabbit antibody. Then the protein was detected by using

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Molecular Imager ChemiDoc™ XRS Imaging System (Bio-Rad Laboratories,

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Hercules, CA, USA), band density was normalized to β-actin, and bands were

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quantified with the image analysis software Quantity One 4.6.2.

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Statistical analysis. Data are expressed as means ± SD. The statistical analysis was

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used SPSS 19.0 software. A p-value of P < 0.05 was considered to be statistically

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significant.

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RESULTS

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Effect of PSG-1 on the proliferation and cytokines production of T lymphocytes.

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To test the effect of PSG-1 on the proliferation of T lymphocytes, cell proliferation

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was measured by CCK-8 kit. As Figure 1A showed that PSG-1 in combine with

229

ConA, T lymphocytes proliferation was significantly (P < 0.01) increased, indicating

230

that PSG-1 synergized with ConA to promote the proliferation of T lymphocytes.

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When PSG-1 was used alone to stimulate T lymphocytes, the proliferation was also

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significantly (P < 0.05) enhanced, demonstrating that PSG-1 could directly induced T

233

lymphocytes proliferation. To investigate the effect of PSG-1 on the level of

234

cytokines, the cells were cultured with PSG-1 (160 µg/mL) and/or ConA (5 µg/mL).

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Compared with the control group, PSG-1 increased the secretion of IL-2, IFN-γ and

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IL-12, when combined with ConA, the production of three cytokines were

237

dramatically (P < 0.01) increased (Figure 1B, C and D), but has no effect on the 10


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production of IL-4 (data not shown).

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RNA-seq and bioinformatics analysis. In order to give further insights into the

240

underlying molecular mechanisms involve in the immunomodulatory activity of

241

PSG-1 on T lymphocytes, RNA-seq was performed. Differentially expressed genes

242

analysis showed 394 genes were differentially expressed in a significant manner

243

(FDR< 0.05, fold change > 2) in PSG-1 stimulated T lymphocytes, of which the up

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and down regulated genes were 283 and 111, respectively. The top 50 up- and

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down-regulated genes were listed in Table S2 and Table S3. Principle component

246

analysis (PCA) on the complete transcriptome of T lymphocytes in the PSG-1

247

treatment group (P group) and control group (C group), demonstrated that two

248

different groups can be distinguished, illustrating that P and C cluster separately

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(Figure 2A). Moreover, KEGG pathway enrichment analysis identified significant

250

enrichment of differentially expressed genes involved in 152 canonical pathways.

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Figure 2B showed the top 20 significantly enriched canonical pathways. The analysis

252

also demonstrated significant enrichment of functions associated with diseases, and

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the top 7 biological functions were showed in Figure 2C.

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To certify the RNA-seq results, we randomly selected 9 differential expression

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genes that were up or down regulated in PSG-1 stimulated T lymphocytes and

256

measured gene expression by RT-qPCR. The result showed that the tendency of

257

RT-qPCR and RNA-seq were the same (Figure 2D).

258

PSG-1 stimulated Ca2+/CaN pathway in T lymphocytes. After exposure to PSG-1

259

(160 µg/mL) for 48 h, Ca2+ concentration and CaN activity were measured by flow

260

cytometry and CaN activity assay kit. We found that PSG-1 increased the level of

261

Ca2+ (Figure 3A), and markedly (P < 0.01) enhanced CaN activity (Figure 3B). In

262

addition, canonical pathway analysis demonstrated that calcium pathway (P < 0.01) 11



263

was significantly associated with immunomodulatory activity of PSG-1 in T

264

lymphocytes (Figure 2C). Heatmap (Figure 3C) demonstrated 7 up-regulated genes

265

and 3 down-regulated genes associated with calcium pathway.

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Effect of calcium channel inhibitor on proliferation and cytokines production

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induced by PSG-1 in T lymphocytes. As shown in the Figure 4A, when pretreated T

268

lymphocytes with verapamil for 30 min, and stimulated with PSG-1 (160 µg/mL) for

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another 48 h, the proliferation of T lymphocytes were significantly suppressed. In

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addition, Figure 4B/C/D showed that in the presence of PSG-1 and ConA, the

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production of IL-2, IFN-γ and IL-12 were 2801.56, 8029.83 and 2563.66 pg/mL,

272

respectively. When pretreated with verapamil (40 µM), the levels significantly

273

reduced to 337.7, 3831.84 and 634.75 pg/mL, respectively, which were much lower

274

than the group that PSG-1 combined with ConA.

275

PSG-1 activated MAPKs in T lymphocytes. To assess the possible involvement of

276

three MAPK families: ERK, JNK and p38 MAPK in PSG-1 stimulated T lymphocytes

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immunomodulation, we used western blot analysis to detect the expression of ERK,

278

p-ERK, JNK, p-JNK, p38 and p-p38 in T lymphocytes after 48 h exposure to PSG-1.

279

The data showed that all proteins expression were increased compared with control

280

group (Figure 5A/C/E). When treated with PD98059, SP600125 or SB203580, the

281

ratio of p-ERK/ERK, p-JNK/JNK and p-p38/p-38 were dramatically (P < 0.01)

282

decreased compared with the group that stimulated with PSG-1 alone (Figure 5B/D/F).

283

Moreover, the heatmap (Figure 5G) identified 14 differentially expressed gene

284

involved in MAPK pathway, of which the up- and down-regulated genes were 12 and

285

2, respectively.

286

Effect of MAPKs inhibitors on proliferation and cytokines production induced

287

by PSG-1 in T lymphocytes. To investigate the role of MAPK families in T 12


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lymphocyte proliferation, we exposed T lymphocytes to the inhibitors of ERK, JNK

289

and p38 MAPK for 30 min, respectively. The inhibitors significantly suppress T

290

lymphocyte proliferation, of which the inhibitor of p38 (SB203580) suppressed T

291

lymphocytes proliferation most (Figure 6A). To determine the role of MAPKs in the

292

regulation of IL-2, IFN-γ and IL-12 secretion in PSG-1-stimulated T lymphocytes, we

293

pretreated T lymphocytes with PD98059, SP600125, and SB203580 for 30 min,

294

followed by exposure to PSG-1 and ConA for 48 h, and found that all the inhibitors

295

markedly reduced the production of IL-2, IFN-γ and IL-12 (Figure 6B/C/D).

296

DISCUSSION

297

Polysaccharides

are

natural

polymer

compounds

bounded

together

by

298

monosaccharide unit and their derivatives.38 They are not only the structure of cells

299

and energy substances,39 but also biological substances with a variety of physiological

300

functions, of which immunomodulation is the most important and fundamental

301

function.40 Spleen is the largest peripheral lymphoid organ, whose significant immune

302

function is based on various immune cells inside, including lymphocytes,

303

macrophages, and dendritic cells.41 T lymphocytes account for about 35% of

304

lymphocytes, and their primary role is to regulate the immune response caused by

305

antigens and eliminate intracellular microbes.42 Zhong et al. showed that Ganoderma

306

polysaccharide B (GL-B) could significantly increase IFN-γ mRNA expression in T

307

lymphocytes.29 In our previous study, we have found that PSG-1 has

308

immunomodulatory effect on spleen lymphocytes, as evidenced by increased the

309

intracellular Ca2+ concentration and CaN activity, activated NFAT activity, and

310

induced IL-2 production.37 Moreover, PSG-1 increased the numbers of CD4+ T

311

lymphocytes and the ratio of CD4+/CD8+, and restored the IL-2, INF-γ, IL-10 levels

312

of spleen lymphocyte in cyclophosphamide-induced mice.35 However, it lacks of 13



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313

study about the immunomodulatory effect of PSG-1 on purified T lymphocytes. T

314

lymphocytes play an important role in immune diseases.43 It is possible to fully

315

understand the corresponding links of the immune response through exploration of T

316

lymphocytes function, which has great significance for researching immune response

317

of the body. To demonstrate the effect of PSG-1 on the proliferation of T lymphocytes,

318

we measured the proliferation by CCK-8 kit. Results showed that PSG-1 dramatically

319

stimulated T lymphocytes proliferation. Moreover, in the presence of stimulus such as

320

ConA favors the proliferation of T lymphocytes. As intercellular signaling proteins,

321

cytokines have important functions in the control of homeostasis in organism.37 It is

322

generally

323

immunomodulation.44 Liu et al. have indicated that Ganoderma lucidum

324

polysaccharides encapsulated in liposome significantly promoted splenocytes

325

proliferation, secretion of IL-6, IFN-γ, IL-4, TNF-α, and activation of CD3+CD4+ and

326

CD3+CD8+ T lymphocytes.45 To investigate whether PSG-1 has immunomodulatory

327

effect on T lymphocytes, we measured the levels of cytokines produced by T

328

lymphocytes and found that PSG-1 promoted the expression of IL-2, IFN-γ and IL-12,

329

but there was no effect on IL-4 expression. The results noted above were consistent

330

with our previous findings, indicating that PSG-1 has an immunomodulatory effect on

331

T lymphocytes. However, the specific mechanism was still unclear.

332

known

that

cytokine

environment

plays

an

vital

role

in

RNA-seq has been considered as a highly efficient, widely used, and conventional

333

molecular biology method to obtain transcriptome information.21,

334

RNA-seq was performed to illustrate the effect of PSG-1 on the differentially

335

expressed genes in T lymphocytes, trying to globally screen the signal transduction

336

pathway of PSG-1 stimulated T lymphocytes. RNA-seq analysis identified 283

337

up-regulated genes, 111 down-regulated genes. Furthermore, KEGG pathway analysis 14


46

Therefore,

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338

illuminated significant enrichment canonical pathways and biological functions that

339

were significantly enriched with the differentially expressed genes obtained from the

340

RNA-seq analysis. From the results we could find that the top 7 significant

341

enrichment biological functions were associated with diseases. Besides, 10 out of the

342

top 20 significantly enriched canonical pathways were related to immunity, and

343

calcium and MAPK pathway were two significant pathways in PSG-1 stimulated T

344

lymphocytes. Thus, we focused on these two pathways to further investigate the

345

immunomodulatory effect of PSG-1 on T lymphocytes.

346

Ca2+ is necessary for cell proliferation, activation and functions. T lymphocytes

347

depend on Ca2+ signaling to activate developmental and activation procedures.47 CaN

348

is a kind of calmodulin-dependent enzyme and CaN regulation in vivo is through

349

changing intracellular calcium.48 Ca2+ involves in immune response in T cells mainly

350

through Ca2+/CaN/NFAT pathway, the increasing of intracellular calcium, activates

351

calcineurin and triggers the dephosphorylation of NFAT, and then NFAT translocates

352

into the nucleus, and activates a series of signaling events.49 To explore the role of

353

Ca2+/CaN pathway in PSG-1 stimulated T lymphocytes, we detected Ca2+

354

concentration and CaN activity, and further measured the proliferation and the

355

cytokines production with the pretreatment of calcium channel inhibitor. The results

356

showed that PSG-1 dramatically improved the intracellular Ca2+ concentration and

357

CaN activity, and the calcium channel inhibitor suppressed PSG-1 induced T

358

lymphocyte proliferation and production of IL-2, IFN-γ and IL-12. Moreover,

359

RNA-seq and KEGG pathway analysis significantly highlighted the importance of

360

Ca2+ signaling in immunomodulatory activity of PSG-1 on purified T lymphocytes.

361

These results suggested that PSG-1 exerted immunomodulatory effect on T

362

lymphocytes through Ca2+/CaN signaling pathway. 15



363

MAPKs are evolutionarily conserved enzymes that related to many physiological

364

processes, such as cellular processes, immune responses in mammalian species.13

365

Recently, studies have shown that MAPK inhibitors suppressed the levels of TNF-α

366

protein and TNF-α mRNA expression in macrophages of S-180 tumor-bearing mice.50

367

Our results showed that PSG-1 elevated the levels of MAPK expression. When

368

exposure to inhibitors, the phosphorylation of MAPK were decreased to relatively low

369

levels. To explore whether PSG-1 stimulated T lymphocytes proliferation and the

370

cytokines production through MAPK pathway, we took advantage of ERK, JNK and

371

p38 inhibitors and found that the proliferation of T lymphocytes was inhibited to

372

varying degrees, in which SB203580 has a strongest inhibitory effect. Besides that, all

373

the inhibitors markedly reduced the secretion of IL-2, IFN-γ and IL-12. Furthermore,

374

the heatmap demonstrated the differentially expressed genes involved in MAPK

375

pathway activated by PSG-1. These results suggested that MAPK pathway was

376

involved in the immunomodulatory activity of PSG-1 on purified T lymphocytes.

377

In summary, the present study demonstrated that PSG-1 increased the proliferation

378

and the production of IL-2, IL-12 and IFN-γ in purified T lymphocytes, and

379

elucidated the underlying mechanism that Ca2+/CaN and MAPK pathway played an

380

important role in the immunomodulatory activity of PSG-1 on purified T lymphocytes

381

based on RNA-seq. This study revealed the potential of PSG-1 developed as a novel

382

immunomodulatory agent, and provided a new perspective for the research of

383

bioactive components in functional foods. However, all the experiments were done in

384

vitro, the immunomodulatory mechanism of T lymphocytes in vivo is complex, which

385

needs more detailed investigation.

386

ABBREVIATIONS USED

387

PSG-1, Ganoderma atrum polysaccharide; CaN, calcineurin; IL-2, interleukin-2; 16


Page 16 of 39

Page 17 of 39


388

IL-12, interleukin-12; IFN-γ, Interferon-γ; MAPK, mitogen-activated protein kinases;

389

the extracellular signal–regulated kinases, ERKs; c-Jun N-terminal kinases, JNKs;

390

RNA-seq, RNA sequencing, ConA, concanavalin A.

391

ACKNOWLEDGEMENTS

392

This work was financially supported by the National Natural Science Foundation of

393

China (No. 21265011), Research Project of State Key Laboratory of Food Science

394

and Technology (No. SKLF-ZZA-201611), Natural Science Foundation of Jiangxi,

395

China (No. 20161BAB214161), is gratefully acknowledged.

396

SUPPORTING INFORMATION

397

Table S1. Primer sequences used for real-time qPCR.

398

Table S2. The top 50 up-regulated genes RNA-seq profiling in PSG-1 stimulated T

399

lymphocytes.

400

Table S3. The top 50 down-regulated genes RNA-seq profiling in PSG-1 stimulated T

401

lymphocytes.

402

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23



544

FIGURE CAPTIONS

545

Figure 1. Effect of PSG-1 on the proliferation and cytokines production of T

546

lymphocytes. (A) Effect of PSG-1 on the proliferation of T lymphocytes. T

547

lymphocytes were stimulated with PSG-1 (160 µg/mL) and/or ConA (5 µg /mL) for

548

48 h, and the cells were measured by CCK-8 kit. Data were expressed as means ± SD

549

(n = 12), *P < 0.05, **P < 0.01 versus control group. (B) The level of IL-2. (C) The

550

production of IFN- γ. (D) The level of IL-12.Similar results were obtained in three

551

separate experiments, no statistical significance (ns), *P < 0.05 versus control group;

552

##

553

Figure 2. RNA-seq analysis and pathway analysis in PSG-1 stimulated T

554

lymphocytes. RNA-seq was performed on RNA isolated from T lymphocytes. C

555

represented control group and P represented the group with PSG-1 treatment. (A)

556

Principal component analysis (PCA) performed on whole transcriptome of T

557

lymphocytes. The different shapes in the figure represented different samples, and

558

color represented different groups (n = 3). (B) The top 20 canonical pathways

559

enriched by differentially expressed genes from the RNA-seq analysis comparing with

560

C group and P group highlighted by KEGG pathway analysis. The size of the dot in

561

the figure indicated the number of differentially expressed gene was annotated to the

562

pathway and the color represented P value of the pathway. (C) The top 7 significantly

563

enriched biological functions associated with differentially expressed genes in T

564

lymphocytes. (D) The fold change of relative mRNA expression level of Lcn2, H2-Aa,

565

Arrdc4, Adamtsl4, H2-Eb1, Smyd1, Tmem140, Gpr83 and Ecm1 measured by

566

RNA-seq and qPCR (n = 3).

567

Figure 3. PSG-1 stimulated Ca2+/CaN pathway in T lymphocytes. (A) Effect of

568

PSG-1 on the intracellular Ca2+ concentration in T lymphocytes. The cells were

P < 0.01 versus ConA alone group.

24


Page 24 of 39

Page 25 of 39


569

stimulated with PSG-1 (160 µg/mL) and/or ConA (5 µg/mL). After 48 h the cells

570

were collected and washed twice with PBS, and cultured with the fluorescent probe

571

Fluo-3/AM at 37 °C for 60 min. The Ca2+ level was analyzed by flow cytometry (n =

572

3). (B) Effect of PSG-1 on CaN activity in T lymphocytes. The CaN activity was

573

determined by the CaN activity assay kit (n = 3), *P < 0.05, **P < 0.01 versus control

574

group. (C) Heatmap representation of differentially expressed genes associated with

575

calcium pathway.

576

Figure 4. Effect of calcium channel inhibitor on proliferation and cytokines

577

production induced by PSG-1 in T lymphocytes. (A) The inhibitor of Ca2+ prevented

578

T lymphocytes proliferation. The cells were pretreated with verapamil (40 µM/mL)

579

for 30 min, then cells proliferation were detected by CCK-8 kit (n = 12), *P < 0.05,

580

**P < 0.01 versus control group,

581

C and D) The levels of IL-2, IFN-γ and IL-12 produced by T lymphocytes. The cells

582

from Balb/c mice were pretreated with verapamil (40 µM/mL) for 30 min, and then

583

cells were treated with PSG-1 (160 µg/mL) and/or ConA (5 µg/ml) for 48 h. The data

584

were expressed as mean ± SEM for three separate experiments. ##P < 0.01 vs PSG-1

585

in combination with ConA.

586

Figure 5. PSG-1 activated MAPKs in T lymphocytes. (A, C and E) The

587

immunoblotting bands of p-ERK, ERK, p-JNK, JNK, p-p38 and p38. (B, D and F)

588

Mean ± SED of immunoblotting bands of p-ERK/ERK, p-JNK/JNK, p-p38/p38 (n=3).

589

*P < 0.05 versus control group,

590

heatmap representation of differentially expressed genes associated with MAPK

591

pathway.

592

Figure 6. Effect of MAPKs inhibitors on proliferation and cytokines production

593

induced by PSG-1 in T lymphocytes. (A) The inhibitors of ERK, JNK and p38

##

P < 0.01 versus the group PSG-1 plus ConA. (B,

##

P < 0.01 versus PSG-1 alone group. (G) The

25



Page 26 of 39

594

restrained PSG-1 induced T-cell proliferation. T lymphocytes were pretreated with

595

PD98059, SP600125, or SB 203580 (40 µM/mL) for 30 min, then cells proliferation

596

was detected by CCK-8 kit (n = 12). **P < 0.01 versus control group;

597

versus group that PSG-1 combinated with ConA. (B, C and D) The production of IL-2,

598

IFN-γ and IL-12 in T lymphocytes. The cells (1×106 cells/well) were pretreated with

599

inhibitors for 30 min, and stimulated with PSG-1 (160 µg/mL) in presence ConA (5

600

µg/mL) for 48 h. Similar results were obtained in three separate experiments,

601

0.01 versus the group of PSG-1 plus ConA.

26


##

P < 0.01

##

P

Immunomodulatory Activity of Ganoderma atrum Polysaccharide on

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