Abstract

   The efficacy of polysaccharides is widespread, especially in immune
   regulation. However, the genetic basis of the changes in
   polysaccharides regulating immunity is unclear. To obtain genome-wide
   insights into transcriptome changes and regulatory networks, we
   designed a polysaccharide formula, comprising lentinan, pachymaran and
   tremelia, to increase the availability of their optimized active sites.
   In this case, we focused on a model of immunosuppression to investigate
   genes by digital gene expression (DGE) tag profiling in T and B cells.
   These genes were further validated by qRT-PCR and Western blot
   experiments. Consequently, polysaccharide formula treatment helped to
   recover the expression of immune-related genes, including CADM1, CCR2,
   IGLL1, LIGP1, and FCGR3, FCGR2 in B cells, as well as S100A8, S100A9,
   ChIL3, MMP8 and IFITM3 in T cells. These results suggest that treatment
   with polysaccharides improves the immunity of immunosuppressive mice by
   regulating genes associated with T and B cell functions.

Introduction

   Numerous studies have shown the potency of polysaccharide biological
   activities, especially in enhancing immunity^[40]1,[41]2.
   Polysaccharide treatment not only activates macrophages, lymphocytes,
   lymphoid factors-activated killer cells, toxic T cells, and dendritic
   cells (DC) but also promotes the production of cytokines, activates the
   complement system and accelerates the production of
   antibodies^[42]3–[43]6. Among all polysaccharides, those derived from
   fungus have a wide range of applications^[44]7,[45]8. For instance,
   polyporus polysaccharides can inhibit bladder cancer cells^[46]9,
   pachymaran can treat tumors in combination with
   cyclophosphamide(CTX)^[47]10, and Hericium polysaccharides can
   alleviate inflammation and ulcers in the digestive tract^[48]11.

   The function of fungal polysaccharides is potent but unique, due to the
   various junctions between monosaccharides that result in the chemical
   diversity of these molecules. Different polysaccharides vary in some of
   their characteristics, including formula weight, branching degree,
   viscosity, and chain conformation, and these properties strongly affect
   the biological activities of polysaccharides^[49]12–[50]14.
   Additionally, the effect of polysaccharides is closely related to their
   active components. The main component of polysaccharides is dextran,
   which is divided into alpha and beta. Alpha glucan is made up of
   starch, which does not possess immunocompetence in medicine. Beta
   glucan, which is mainly composed of glucans, such as β-1-3D, β-1-4D,
   β-1-6D and so on. Beta glucan has proven good effects on tumors,
   hepatitis and diabetes^[51]15.

   For these reasons, some polysaccharides are combined, and the effect is
   better than that of a single polysaccharide, and the pharmacological
   effects of these polysaccharides are synergistic^[52]16.

   In a preliminary study^[53]17, we found that the formula of multiple
   polysaccharides was more potent to regulate immunologic activities than
   each polysaccharide separately. This formula comprised lentinan,
   pachymaran and tremella, but the mechanisms underlying such effects on
   immunity remain unclear. To further elucidate the mechanisms underlying
   the effects of this polysaccharide formula on the immune system, we
   used immunosuppressive mice and digital gene expression tag profiling
   to identify genes differentially regulated upon treatment with
   polysaccharides. We further performed qRT-PCR and Western blotting to
   confirm the differential expression at both transcript and protein
   levels. The identified genes enabled us to hypothesize mechanisms
   underlying polysaccharide treatment and provided a basis for improving
   immune functions by using the formula of polysaccharides.

Results and Discussion

The effects of polysaccharides on immune functions in immunosuppressive mice

   The effects of the formula of polysaccharides on immune performance in
   immunosuppressive mice were investigated, exposuring to
   cyclophosphamide(CTX) supplemented or no with the polysaccharides.

   As shown in Fig. [54]1, the NK cell cytotoxicity in these mice was
   remarkably reduced by CTX (p < 0.05). In contrast, NK cell cytotoxicity
   in the polysaccharide-treated group was significantly higher than that
   in the immunosuppressive group (p < 0.05). The effect of
   polysaccharides on phagocytosis by peritoneal macrophages is shown in
   Fig. [55]2, indicating that the phagocytosis of peritoneal macrophages
   was markedly inhibited by CTX (p < 0.05). The phagocytosis index of the
   polysaccharide-treated group was significantly higher than that of the
   immunosuppressive group (p < 0.05).

Figure 1.

   [56]Figure 1
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   Effects of the polysaccharide formula on killing activity of NK cells
   in immunosuppressive mice. YAC-1 cells were stained with CFSE as target
   cells, the dead cells were stained with PI, and CFSE^+PI^+ (Q2) were
   considered killed cells. The figure shows that the compound
   polysaccharide could improve the killing activity of NK cells.
   *p < 0.05.

Figure 2.

   [58]Figure 2
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   Effects of the polysaccharide formula on macrophage phagocytosis in
   immunosuppressive mice. The activation of peritoneal macrophage
   phagocytosis showed with microspheres labeled with CFSE. The first peak
   on the left shows that the macrophages phagocytosed one microsphere,
   the second peak shows the phagocytosis of two microspheres, and so on.
   The figure shows that the compound polysaccharide could improve
   macrophage phagocytosis. *p < 0.05.

   The effects of polysaccharides on lymphocytes were further
   investigated. As shown in Fig. [60]3A,B, the proportion of CD3^+ (T
   cell) and CD19^+ (B cell) spleen lymphocytes was notably unbalanced in
   mice treated with CTX (p < 0.01), with the proportion of B lymphocytes
   significantly reduced (p < 0.01) and the proportion of T lymphocytes
   significantly increased (p < 0.05). As lymphocytes were markedly
   inhibited by CTX, a compensatory activation of T and B lymphocytes to
   supplement the lost quantity was observed: CD3^+CD69^+ (activated T
   cells) and CD19^+CD69^+ (activated B cells) cells were significantly
   increased (p < 0.01). However, these changes were significantly
   reversed in mice fed the formula of polysaccharides (p < 0.05).

Figure 3.

   [61]Figure 3
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   Effect of the polysaccharide formula on lymphocytes. (A and B)
   Proportion and activation of T and B cells. Lymphocytes isolated from
   the spleen were stained with CD3-PEcy7, CD19-PE and CD69-FITC, and
   analyzed by flow cytometry. CD3^+ (P4) were considered T cells, CD19^+
   (P3) were considered B cells, CD3^+CD69^+ (Q2) were considered
   activated T cells, and CD19^+CD69^+ were considered activated B cells
   (Q2-1). (C) Cytokines in peripheral blood, *P < 0.05. (D) Antibody in
   serum and the small intestine, *p < 0.05.

   The effects of polysaccharides on peripheral blood cytokines are shown
   in Fig. [63]3C. The peripheral blood cytokines were significantly
   reduced after treatment with CTX (p < 0.05). TNF-α and IFN-γ in serum
   were notably improved in the polysaccharide-treated group when compared
   with those in the immunosuppressive group (p < 0.05).

   The concentration of IgA in the serum and sIgA in the small intestine
   was significantly reduced by CTX (p < 0.05). In the
   polysaccharide-treated group, the levels of IgA in the serum and sIgA
   in the small intestine were notably improved when compared with those
   in the immunosuppressive group (p < 0.05). (As shown in Fig. [64]3D).

   These experiments demonstrate that treatment with CTX suppresses the
   immune response, as reflected in the functional decline of macrophages,
   NK cells, T cells and B cells, which was consistent with the findings
   of previous studies^[65]18. Feeding the formula of polysaccharides
   improved the functions of peritoneal macrophages, NK cells, T cells and
   B cells in immunosuppressed mice.

Ingestion of polysaccharides reverses the decrease in immune gene expression

   To obtain genome-wide insight into the transcriptome changes in
   polysaccharides that regulate immunity, we sorted two major immunocytes
   for DGE experiments. The purities of the sorted T and B cells for the
   DNA library preparation and expression of sequencing are shown in
   Fig. [66]4(A,B). Figure [67]4C,a shows that treatment with CTX provokes
   a broad decrease in a number of genes expressed in B (Fig. [68]4C,a)
   and T (Fig. [69]4D,a) lymphocytes and increases the expression of few
   genes only. Compared with no treatment, treatment with polysaccharides
   seemed to increase gene expression at both low and high doses in
   immunosuppressed mice. The top ten genes that were less down-regulated
   in mice fed polysaccharides upon treatment with CTX (reversion of
   phenotype) are presented in Fig. [70]4C,D. Particularly, the expression
   of genes 24108/Ubiquitin, 12259/C1qa, 170741/Pilrb-1, 16316/Igll1,
   110454/Ly6a, 60440/Ligp1, 58860/Adamdec1, 246256/Fcgr2, 54725/Cadm1,
   12772/Ccr2 and 14131/Fcgr3 was markedly decreased in B lymphocytes
   (Fig. [71]4C,b and c), while the compound polysaccharides
   dose-dependently reversed this down-regulation. In addition, the
   expression of 20202/S100a9, 20201/S100a8, 12655/Chil3, 17394/Mmp8,
   66141/Ifitm3, 76905/Lrg1, 20862/Stfa2, 24728/Oas2 and 245195/Retnlg in
   T lymphocytes was significantly decreased by CTX injection, and the
   ingestion of polysaccharides partially reversed this effect (see
   Fig. [72]4D,b and c).

Figure 4.

   [73]Figure 4
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   Analysis of the polysaccharide formula on genes in T and B cells in
   immunosuppressed mice by DGE. (A,B) Lymphocytes from the spleen
   isolated by microbeads were stained with CD3-PE, and B220-PE (B cells)
   to test the purity. The figure shows that the isolated cells are pure.
   (C,a) Quantitative statistics of differentially expressed genes in B
   cells, the red shows up-regulated genes, and the green shows
   down-regulated genes. PL is the low dose group of polysaccharides with
   200 mg/kg/bw and PH is the high dose of polysaccharide with
   400 mg/kg/bw. (b) Hierarchical clustering of differential gene
   expression in B cells. The clustered map accorded by the log2 of
   significant difference multiples between the groups. Each row
   represents a gene and each column represents the comparison between the
   two groups, the red shows up-regulated genes, and the green shows
   down-regulated genes, the deeper the color the higher the gene
   expression. The gene ID is shown on the right, and the enrichment and
   the function of the displayed genes are shown, with references of the