Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells
Introduction
For centuries, mushrooms have been abundant sources of bioactive compounds for treatment of various diseases [1], [2], [3], [4], [5]. Mushrooms display in vivo and in vitro immunomodulatory activity, in particular since fungal compounds exhibit anti-tumour activity, based on modulation of the immune system. Immunomodulatory activity is demonstrated for crude fungal extracts and isolated compounds like polysaccharides, polysaccharopeptides, polysaccharide-proteins and proteins from the fruiting body, spores, mycelia, and culture medium of various mushrooms (reviewed in [6]). The major immunomodulating activity of these bioactive compounds includes their mitogenicity, potential stimulation of hematopoietic stem cells and activation capacity of immune effector cells like human peripheral blood mononuclear cells (hPBMC) [6], [7], [8], [9].
hPBMC represent a heterogeneous population of immune cells including B cells, T cells, monocytes, NK cells and various granulocytes [10]. In a close interplay, these cells orchestrate innate and acquired immune responses that might either be enhanced or reduced by the addition of mushroom compounds to these hPBMC cultures. When addition of fungal compounds results in altered differentiation or functional responses of hPBMC cultures in vitro, this would signify the ability of these fungal compounds to induce systemic immune responses that modulate disease resistance.
One approach to evaluate immunomodulating activity is to determine the capacity of fungal compounds to influence the cytokine production by hPBMC. Cytokines are soluble glycoproteins that are crucial in the induction and regulation of immune responses. Changes in cytokine levels can result in various pathological conditions and cause disturbances in the cytokine-mediated interplay between innate and acquired immune responses [11]. Although unstimulated hPBMC do not produce much cytokines in vitro, various stimuli cause preferential stimulation of T cells or monocytes in hPBMC. Phorbol Myristate Acetate plus calcium ionophore (PMA/Ca-I), and Concanavalin A (ConA) are widely used to activate T cells, however, both result in a different effector action. Lectins, carbohydrate-binding (glycol-)proteins which potentially link to cell surface glycoproteins, such as ConA, are T cell activators by binding to particular sugar residues on the TCR and CD3 proteins in the absence of antigen-presenting cells [12]. In contrast, PMA mimics diacylglycerol and activates protein kinase C and thus eventually T cells [13], [14]. Lipopolysaccharide (LPS) strongly activates macrophages and monocytes, and induces cytokines such as IL-12 and TNF-α both in vivo and in vitro [15], [16].
The ability of fungal compounds, like fungal immunomodulatory proteins (FIPs), to alter the cytokine response has been described earlier [17]. Hsu et al. reported the purification of FIP-Vvo and suggested that the immunomodulatory effects might be due to cytokine regulation of hPBMC. In addition, the proteins were capable of agglutinating rat red blood cells and were able to stimulate proliferation [18]. FIPs are classified into a distinct family since they are a group of fungal proteins defined by amino acid sequence similarity and their actions on immunological responses [19], [20], [21]. FIPs have been isolated and purified from G. lucidum [19], F. velutipes [20], V. volvacea [18], Ganoderma tsugae [17], and V. volvacea [22] and are designated as LZ-8, Fve, Vvo, Gts, and Vvl, respectively. All of these studies were either performed with mice, or assessed the immunomodulating activity as effects on the proliferation of hPBMC.
The aim of this study was to assess the bioactivity of fungal proteins and polysaccharides by determining which of the yielded proteins or polysaccharides displayed immunomodulating activity. As proposed in the literature, the immunomodulating activity was assessed by measuring the cytokines IFN-γ, IL-4, IL-10, IL-12 and TNF-α in the culture supernatants of freshly isolated hPBMC cultures. hPBMC were used unstimulated or were stimulated with PMA/Ca-I, ConA or LPS. Another assessment of the bioactivity of the fungal proteins was done by determining the carbohydrate-binding specificity in a hemagglutination test using rabbit red blood cells (RRBC). In addition, the difference in hemagglutination activity of untreated and trypsin treated RRBC gave an indication of which type of proteins are present in the different protein fractions.
Section snippets
Micro-organism and media
Mother spawn of the mushroom strains Agaricus blazei M7700, Coprinus comatus M8102, Flammulina velutipes M4600, F. velutipes M4622, Ganoderma lucidum M9720, Grifola frondosa M9821, and Volvariella volvacea M6100 were commercially obtained from Mycelia (SacO2, Combiness, Belgium). Lentinus edodes S-9-2 was commercially obtained from Fungisem S.A. (Autol, Spain) and Pleurotus ostreatus 2222 was a kind gift of the Instituto Nacional de Engenharia Tecnologia e Inovação (INETI, Lisboa, Portugal).
Analysis of the protein crude extracts by SDS-PAGE gels
The protein extracts were analyzed by SDS-PAGE (15% w/v). As shown in Fig. 1, all extracts contain a diversity of proteins. The majority of extracts contain 12–17 kDa proteins, which is in the molecular weight range of known FIPs [18], [20], [26]. In addition, most extracts contain higher molecular weight proteins which is in line with higher molecular weights of identified lectins [27].
Cytokine production in the absence of mushroom extracts
Fig. 2 shows the cytokine production of the negative control (hPBMC culture with medium only) and the
Discussion
This study showed that mushroom mycelia can be cultured efficiently in selected media and, by using specific isolation procedures, high yield production of polysaccharides and proteins can be obtained. These polysaccharides and proteins display defined immunomodulatory activity as shown by the modulation of cytokine production in in vitro hPBMC cultures. Some of these cytokines are involved in T cell subset induction and activation and thus potentially able to modulate disease induction and
Acknowledgements
We like to thank J. Bastiaans for her technical input. We thank Prof. Dr. J.H.W.M. Rombout and Dr. J.J. Mes for critically reviewing the manuscript.
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Both authors contributed equally to this work..