CNL, a ricin B-like lectin from mushroom Clitocybe nebularis, induces maturation and activation of dendritic cells via the toll-like receptor 4 pathway

Abstract

A novel lectin, isolated from the basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen-presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1 to 10 μg/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48 hr resulted in extensive up-regulation of co-stimulatory molecules CD80 and CD86, as well as of the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a T helper type 1-polarized response in naive CD4⁺ CD45RA⁺ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as interleukin-6, interleukin-8 and tumour necrosis factor-α. By using a specific Toll-like receptor 4 (TLR4) signalling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 nuclear factor-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in nuclear factor-κB activity, with less effect on the induction of p38 MAPK signalling than of lipopolysaccharide-matured DCs. The CNL had the ability to activate human DCs in such a way as to subsequently direct T helper type 1 T-cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies with aims such as to strengthen anti-tumour immune responses.

Introduction

Mushrooms have long been recognized for their medicinal value, particularly in traditional medicine. Much attention has been paid to their potential therapeutic effects and there is increasing interest in discovering new functional compounds in mushrooms. Besides important medicinal characteristics, such as antitumour, antiviral, hypocholesterolaemic and hepatoprotective activities,^1,2 mushroom extracts and mushroom-derived compounds have also been demonstrated to possess immunomodulatory properties, mainly because of the β-glucans that are one of the hydrophilic constituents of mushrooms. They have been shown to exert a stimulatory effect on the maturation and activation of dendritic cells (DCs).³ We have reported previously that Clitocybe nebularis, a widespread gilled fungus, possesses anti-proliferative activity against human leukaemic T cells.⁴ Recently, our group isolated a novel lectin from the basidiomycete mushroom Clitocybe nebularis, designated C. nebularis lectin (CNL). This homodimeric lectin with 15 900 molecular weight subunits belongs to a ricin B-like lectin protein family and displays high affinity for N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc) and human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates. We have also shown that CNL possesses immunomodulatory properties as it exhibited inhibitory action on leukaemic T-cell lines.⁴

Dendritic cells are the most potent antigen-presenting cells in the human body. They are specialized, in the peripheral tissues, in uptake and processing of antigens that are presented on their surface to responding T cells after migration to secondary lymphoid organs during the process of maturation.^5,6 Dendritic cells possess great phenotypic, morphological and functional plasticity, and are therefore able to control both immunity and tolerance, depending on their activation status, and the microenvironment in which they act.⁷ Fully activated DCs, in contrast to immature or semi-mature DCs, express high levels of co-stimulatory molecules and pro-inflammatory cytokines, including interleukin-12 (IL-12) p70, and are critical in initiating effective CD4⁺ T-cell responses. These are characterized by predominantly interferon-γ (IFN-γ) -producing T helper type 1 (Th1) T cells that are able to support and maintain cellular immune responses and effectively fight pathological states such as cancer or invading pathogens.^8,9

The interplay between target glycans and lectins is crucial for the function of DCs, because recognition and regulation of the immune response involves a number of lectins. Moreover, glycosylation on the surface of DCs varies, depending on the environment and the functional state, generating different signals by altered glycans.¹⁰ For instance, proteins involved in Toll-like receptor (TLR) -mediated activation of DCs contain several N-linked glycosylation sites and several studies have been conducted on the functional roles of these glycans. It was shown that certain glycosylations are crucial for the receptor function of TLR3, TLR4 and MD-2 involved in TLR-mediated signal transduction.^11–13 These glycans could be the ligands for lectins, which could elicit or regulate a response of DCs by binding to such glycosylated receptors.

As lectins can elicit various physiological effects by binding to glycosylated cell receptors, the response of human monocyte-derived DCs to exogenously applied CNL was investigated to evaluate its immunomodulatory action on DCs.

Materials and methods

Preparation of CNL from C. nebularis

The CNL was isolated from fruiting bodies of the basidiomycete fungus C. nebularis using serial carbohydrate affinity chromatography on lactosyl- and glucosyl-Sepharose. In the first step, mushroom extract was loaded onto a lactosyl-Sepharose column. Bound proteins were eluted with 0·01 m NaOH, neutralized with 2 m Tris–HCl buffer, pH 6·5, and in the second step, were applied to a glucosyl-Sepharose column. The unbound fractions containing CNL were pooled and dialysed against Dulbecco's phosphate-buffered saline (DPBS). Endotoxin level in the sample was determined using a Limulus amoebocyte lysate test kit, according to the manufacturer's protocol (Charles River Inc., Wilmington, MA).

Cell preparation and culture

Buffy coats from the venous blood of normal healthy volunteers were obtained from the Blood Transfusion Centre of Slovenia, according to institutional guidelines. Peripheral blood mononuclear cells were isolated using Lympholyte^®-H (Cedarlane Laboratories, Ontario, Canada). The cells were washed twice with DPBS, and used as a source for immunomagnetic isolation of CD14-positive cells (CD14 Microbeads, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). These cells (purity of CD14⁺ cells was always greater than 95%, as determined by flow cytometry) were cultured in RPMI-1640 (Cambrex, Verviers, Belgium) medium, supplemented with 10% fetal bovine serum, gentamicin (50 μg/ml; Gibco, Paisley, UK), 800 U/ml of recombinant human granulocyte–macrophage colony-stimulating factor (rhGM-CSF) and 1000 U/ml of rhIL-4 (both Peprotech EC, London, UK). On day 2, half of the medium was exchanged with starting quantities of rhGM-CSF and rhIL-4. After 5 days, non-adherent immature DCs were harvested and characterized by flow cytometry as CD1a^hi, CD80⁻, CD83⁻, CD86^low and HLA-DR^low. For maturation of controls, DCs were activated with 20 ng/ml bacterial lipopolysaccharide (LPS) along with the addition of 800 U/ml rhGM-CSF, and left for 48 hr. To study the immunomodulatory effect of CNL, the lectin was added to immature DC cultures for 48 hr at concentrations of 0·1, 1 and 10 μg/ml.

The T cells were purified from human buffy coats. Whole CD4⁺ T cells were obtained by positive selection using CD4 microbeads (Miltenyi Biotec GmbH). The purity of CD4⁺ cells was always > 95% as determined by flow cytometry. Naive CD4⁺ CD45RA⁺ were isolated using the naive CD4⁺ T-cell isolation kit from Miltenyi Biotec, following strictly the manufacturer's protocol. The purity of naive CD4⁺ T cells was always > 98%.

Allogeneic T-cell proliferation

Dendritic cells obtained after 7 days of culture (either immature, mature or treated with different concentrations of CNL, as described above) were washed twice in DPBS and incubated with mitomycin C (Sigma Aldrich, St. Louis, MO) to block their proliferation. When using immature DCs, after day 5 the cells were cultured for 2 more days using 800 U/ml rhGM-CSF and 1000 U/ml rhIL-4. Purified whole CD4⁺ T cells were used as responders. The assays were carried out in 96-well plates, with a total volume of 200 μl per well. CD4⁺ responder cells were added at 1 × 10⁵ per well, and stimulator DCs at 1 × 10⁴ per well. On day 4 of culture the wells were pulsed with 1 μCi/well [³H]thymidine (Perkin Elmer, Boston, MA) and proliferation was measured by [³H]thymidine incorporation after 18 hr by liquid scintillation counting.

Flow cytometry analysis

The levels of membrane markers were determined by flow cytometry using fluorescence-labelled antibodies. Non-adherent cells were harvested on day 5 or 7 and collected by centrifugation. Antibody was added and the cells were incubated at room temperature for 15 min in the dark. They were then washed twice with DPBS and resuspended in 2% paraformaldehyde. The following monoclonal antibodies were used: FITC-labelled anti-CD1a, anti-CD14, anti-CD40, anti-CD80, anti-CD4, FITC-labelled anti-CD83, FITC-labelled anti-CD86 and phycoerythrin-labelled anti-HLA-DR (all from Biolegend, San Diego, CA). For isotype controls an FITC-IgG1 and phycoerythrin-IgG2a cocktail was used (Biolegend). Results are expressed as mean fluorescence intensity values after subtracting the mean fluorescence intensity obtained with the control antibody.

Quantification of cytokine production

The BD Human Cytometric Bead Array (BD Biosciences, San Jose, CA) was used to assay the protein levels of IL-1β, IL-6, IL-8, IL-12p70 and tumour necrosis factor-α (TNF-α) in the cell culture supernatant, according to the manufacturer's protocol. Dendritic cells were differentiated and either left untreated or were activated with 20 ng/ml LPS or various concentrations (0·1, 1 or 10 μg/ml) of CNL. After 48 hr of stimulation, cells were centrifuged and the supernatant was used for further analysis. In an appropriate assay tube, 50 μl Capture Beads, 50 μl Detection Reagent and 50 μl of the sample (fivefold or 10-fold dilution) were mixed and incubated for 3 hr at room temperature, protected from light. Samples were then washed with 1 ml Wash Buffer and centrifuged at 1200 g for 5 min. Supernatant was carefully aspirated and discarded from each assay tube. The bead pellet was resuspended by adding 300 μl Wash Buffer. Flow cytometry was performed using a FACSCalibur system (Becton Dickinson, Inc., San Jose, CA). A standard curve was prepared by serial dilution of standards and used for determining the cytokine concentrations in supernatants.

Intracellular flow cytometry staining

Profiles of cytokine secretion by naive CD4⁺ T cells at the single cell level were determined by staining the cells intracellularly for various cytokines and analysis by flow cytometry. Freshly isolated, naive CD4⁺ T cells were co-cultured with either immature, LPS-activated, or CNL-activated DCs for 5 days in a 1 : 20 DC : T-cell ratio. Before DC : T-cell co-cultures, the DCs were always washed twice with DPBS. Afterwards, the cells were collected, washed twice with DPBS and rested overnight in complete medium. Cytokine secretion was then induced by 50 ng/ml PMA and 500 ng/ml ionomycin. After 3 hr, brefeldin A was added to the medium at a concentration of 10 μg/ml for another 3 hr. At the end of stimulation, cells were washed twice and fixed with 4% paraformaldehyde, then washed and permeabilized with 0·1% Triton X-100 solution for 10 min. The permeabilized cells were incubated in PBS containing 3% BSA for 30 min to prevent non-specific binding. They were stained intracellularly using FITC anti-IFN-γ and phycoerythrin anti-IL-4 (both from Invitrogen, Carlsbad, CA) and analysed by flow cytometry.

To determine the activation status of nuclear factor-κB (NF-κB), and of p38 mitogen-activated protein kinase (MAPK) after stimulation with CNL, we used fluorescently conjugated antibodies against responding phosphorylated epitopes and flow cytometry techniques as described elsewhere.¹⁴ The DCs were activated with either CNL (10 μg/ml) or LPS (20 ng/ml) for 30 min. Afterwards, paraformaldehyde was added directly to the still warm culture medium to freeze the cell state and avoid protein degradation or further signalling events. After 30 min at room temperature, the fixed cells were washed twice in PBS and permeabilized with ice-cold methanol for 10 min at 4°. The cells were then washed twice with PBS, incubated in staining media for 45 min, and stained with Alexa Fluor 488-conjugated anti-pp38 MAPK (pT180/pY182) (BD Biosciences Pharmingen, San Diego, CA) and Alexa Fluor 488-conjugated p-NF-κB p65 (p-ser536) (Cell Signaling Technology, Beverly, MA) for 45 min. After staining, the cells were washed twice in PBS and analysed on a FACSCalibur system (Becton Dickinson, Inc.).

Apoptosis studies

We validated any potential toxicity of the lectin from C. nebularis used in our study by FACS, using an Apoptosis Detection Kit (Sigma) according to the manufacturer's protocol. The percentages of live cells, dead cells and cells early in the apoptotic process were determined by staining with annexin V FITC conjugate and propidium iodide.

Use of specific inhibitors

To evaluate the importance of the TLR4 activating pathway for DC maturation with CNL, two specific inhibitors were used. Specific TLR4 signalling inhibitor ¹⁵ CLI-095 (or TAK-242) (from Invivogen, San Diego, CA) was used at 1 μm concentration. Before activation with either CNL (10 μg/ml) or LPS (20 ng/ml), the DCs were incubated with CLI-095 for 6 hr, as recommended by the manufacturer. For inhibition of Myd88 signalling molecule, a Myd88 inhibitory peptide (Invivogen) was used at 25 μm concentration, according to the manufacturer's protocol. Before activation, the DCs were incubated with an Myd88 inhibitory peptide (Invivogen) for 6 hr, as recommended by the manufacturer. The DCs were then activated with LPS or CNL for 48 hr. Their activation status was determined by measuring the expression of co-stimulatory molecules CD80 and CD86 by FACS.

TLR4 reporter assay

To study and confirm the signalling of CNL via the TLR4 activating pathway, we used the HEK-Blue™ human TLR4 cell reporter assay (Invivogen), according to the manufacturer's protocol. The reporter cell line is designed by co-transfection of the hTLR4, MD-2/CD14 co-receptor genes and a secreted embryonic alkaline phosphatase reporter gene into HEK293 cells. Stimulation of TLR4 induces the production of secreted embryonic alkaline phosphatase. The HEK-Blue™ human TLR4 cells were washed twice with DPBS and seeded in a 96-well plate at a density of 25 000 cells per well in 200 μl of growth medium (high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml Normomicin™ and 2 mm l-glutamine). Afterwards, the HEK-Blue™ human TLR4 cells were either left non-treated (n.t.) or treated with LPS (20 ng/ml) or various concentrations of CNL (0·1, 1 and 10 μg/ml) for 24 hr. On the second day, 20 μl HEK-Blue™ human TLR4 cell supernatant was collected and added to 180 μl resuspended QUANTI-Blue™ reagent and incubated at 37° for 3 hr. Finally, secreted embryonic alkaline phosphatase levels were determined by reading the optical density (OD) at 655 nm.

Results

CNL causes extensive up-regulation of DC maturation markers

The immunomodulatory effect of CNL was determined by studying its effects on the mature DC phenotype 48 hr after its addition to immature DC cultures. Dendritic cells were differentiated from human peripheral blood monocytes for 5 days and either left untreated or activated with LPS or CNL (0·1, 1·0 or 10·0 μg/ml). Forty-eight hours after the addition of CNL, a dose-dependent increase (Fig. 1a) in expression of both CD80 and CD86 was observed, as well as of HLA-DR. At the highest concentration (10 μg/ml), CNL up-regulated the expression of CD80, CD86 and HLA-DR by approximately twofold, eightfold and threefold, respectively (Fig. 1b). Additionally, to see whether the immunostimulatory activity of CNL product depends in any way on the potential endotoxin content, we performed a protein denaturation experiment by incubating the samples, together with LPS-controls, at high temperature (100° for 2 hr). While the immunogenic activity of LPS was not affected, or was even slightly enhanced, by this treatment, that of CNL was completely prevented (Fig. 1c), confirming the absence of endotoxins in the CNL stock used in our studies. Furthermore, to evidently confirm the absence of endotoxin contamination, we tested our lectin stock samples for the presence of LPS using the Limulus amoebocyte lysate test, which showed them to be negative in all cases.

Figure 1.

Clitocybe nebularis lectin (CNL) causes a dose-dependent increase in the expression of mature dendritic cell (DC) phenotype. Dendritic cells were prepared as described in the Materials and methods. Immature DCs (iDCs) were either treated or untreated with various concentrations of CNL (0·1, 1 and 10 μg/ml) for 48 hr. For positive controls, the DCs were stimulated with 20 ng/ml of lipopolysaccharide (LPS) for the same period of time. (a) CNL causes a dose-dependent increase in expression of co-stimulatory molecules CD80 and CD86, as well as in the expression of HLA-DR. The results represent mean ± SD of mean fluorescence intensity values of three independent experiments. (b) Flow cytometric analysis of co-stimulatory molecules CD80 and CD86, HLA-DR, as well as the expression of CD83 is shown for iDCs, CNL-stimulated DCs (10 μg/ml) and LPS-stimulated DCs (20 ng/ml). The dashed line represents isotypic controls and the solid line represents the depicted surface marker. One representative experiment is shown out of three performed. (c) Stocks of CNL, as well as LPS, were either left intact or thermally treated at 100° for 2 hr. Afterwards, the same stocks were used for stimulation of iDCs in a 48-hr culture period, as described in the Materials and methods. After 48 hr, the DCs from various cultures were stained for the expression of activation markers CD80 and CD86. Results are depicted as mean ± SD of three independent experiments. Significance between expression of individual markers between individual samples was calculated using Student's unpaired t-test (ns – non-significant; *P ≤ 0·05; **P ≤ 0·01).

Activation of DCs with CNL induces the production of certain pro-inflammatory cytokines

Next, we wanted to determine the effect of CNL on the ability of DCs to produce various pro-inflammatory cytokines during their maturation. Dendritic cells, differentiated from human monocytes for 5 days, were thoroughly washed and stimulated with CNL (0·1, 1·0 or 10 μg/ml). After 48 hr, supernatants from the DC cultures were collected and analysed for levels of IL-1β, IL-6, IL-8, IL-12p70 and TNF-α. At 10 μg/ml, CNL significantly induced the production of IL-6, IL-8 and TNF-α (Fig. 2), compared with non-treated controls. All three cytokines were induced at a level similar to that observed with LPS (TNF-α at 310 ± 16 pg/ml for CNL and 453 ± 34 pg/ml for LPS; IL-6 at 11 456 ± 1234 pg/ml and 9037 ± 512 pg/ml for CNL and LPS, respectively; IL-8 at 23 905 ± 1595 pg/ml and 22 713 ± 2168 pg/ml for CNL and LPS, respectively). Although an increase was observed for IL-12p70 levels (28 ± 5 pg/ml), it was about half that induced by LPS (69 ± 14 pg/ml). The production of IL-1β was similar to that of untreated control immature DCs (34 ± 8 and 30 ± 5 pg/ml for CNL and immature DCs).

Figure 2.

Clitocybe nebularis lectin (CNL) increases the production of certain pro-inflammatory cytokines by treated dendritic cells (DCs). The DCs were prepared from human peripheral blood monocytes as described in the Materials and methods. Stimulation of immature DCs (iDCs) with CNL was performed at various concentrations (0·1, 1 and 10 μg/ml) for 48 hr. Afterwards, the supernatants from DC cultures were taken and quantitatively analysed for the presence of interleukin-1β (IL-1β), IL-6, IL-8, IL-12p70 and tumour necrosis factor-α (TNF-α) as described in the Materials and methods. Results are depicted as mean ± SD values of individual cytokine in pg/ml of three independent experiments. Significance of various cytokine levels in culture supernatants, between cultures of immature DCs and DCs stimulated with individual concentrations of CNL, was determined using Student's unpaired t-test (ns – non-significant; **P ≤ 0·01; ***P ≤ 0·001).

Dendritic cells activated with CNL possess increased allostimulatory capacity and induce Th1-type CD4⁺ T-cell responses

To analyse the functional aspect of CNL-activated DCs we examined the potential of treated DCs to stimulate the proliferation of allogeneic whole CD4⁺ T cells, as well as to see how such DCs polarize responding naive CD4⁺ CD45RA⁺ T cells. Dendritic cells were prepared as described in the Materials and methods section, activated with various concentrations of CNL and used as stimulators in subsequent functional assays. As determined by liquid scintillation counting, treatment of DCs with CNL extensively and dose-dependently increased their allostimulatory potential already at lower doses (Fig. 3a), compared with immature DCs [34 155 ± 3159 and 11 534 ± 2810 counts per minute (c.p.m.), for 0·1 μg/ml CNL-treated and non-treated DCs, respectively]. At the highest concentration of CNL used for DC activation (10 μg/ml), the allostimulatory capacity of such DCs was comparable with that of LPS-activated DCs (50 553 ± 6567 and 57 831 ± 6950 c.p.m., for 10 μg/ml CNL-treated and LPS-treated DCs).

Figure 3.

Clitocybe nebularis lectin (CNL) increases the allostimulatory potential of treated dendritic cells (DCs) and endows DCs with the potential to induce T helper type 1-polarized immune responses. The DCs, whole CD4⁺ and naive CD4⁺ CD45RA⁺ T cells were isolated and prepared as described in the Materials and methods. (a) Immature DCs (iDCs) were either left untreated or treated with various concentrations of CNL (0·1, 1 and 10 μg/ml) or lipopolysaccharide (LPS; 20 ng/ml) and further used in allogeneic co-cultures with whole CD4⁺ T cells. After 4 days, the co-cultures were pulsed with [³H]thymidine for 18 hr and proliferation was measured by liquid scintillation counting on day 5. Treatment of DCs with CNL resulted in a dose-dependent increase in their allostimulatory capacity. Results are depicted as mean ± SD of counts per minute (c.p.m.) of four independent experiments. Significance of differences between immature DCs and DCs treated with individual concentrations of CNL was calculated using Student's unpaired t-test (**P ≤ 0·01). (b) Variously treated DCs (0·1, 1 and 10 μg/ml of CNL or 20 ng/ml LPS) were used in 5-day co-cultures with naive CD4⁺ CD45RA⁺ T cells in 1 : 20 DC : T-cell ratio. After 5 days, cells positive for interferon-γ (IFN-γ) and interleukin-4 (IL-4) were determined by intracellular cytokine staining with anti-IFN-γ and anti-IL-4 antibody and analysed by flow cytometry. Numbers in quadrants represent the percentage of positive cells. Results shown are from one representative experiment out of four performed.

We isolated naive CD4⁺ CD45RA⁺ T cells to perform co-cultures with various DC stimulators, as described in the Materials and methods. After 5 days of DC–T-cell co-cultures, the T cells were collected and stained intracellularly for both IFN-γ and IL-4. Whereas non-treated immature DCs induced T-cell populations producing very little IFN-γ or IL-4, CNL treatment of DCs (dose-dependently, in terms of concentration of CNL used for activation of stimulators) induced a significant percentage of IFN-γ-producing T cells. In this manner, DCs that were activated with 10 μg/ml CNL, induced a percentage of IFN-γ-producing T cells that was similar to that induced by DCs, that were activated with LPS (Fig. 3b).

Stimulation of DCs with CNL does not cause DC apoptosis

Next, it was important to see how CNL affects DCs in terms of cytotoxicity, over a longer culture period. For this purpose, we cultured fully differentiated immature DCs with the highest concentration of CNL, which was used in our experiments, for a period of 72 hr. After 5 days of differentiation from monocytes, the DCs were either left untreated or were stimulated with LPS or CNL. Even at 10 μg/ml, the addition of CNL to DC cultures did not result in any significant increase in the number of dead or apoptotic cells compared with either non-treated or LPS-stimulated control DCs (Fig. 4).

Figure 4.

Stimulation of dendritic cells (DCs) with Clitocybe nebularis lectin (CNL) does not cause their apoptosis. Dendritic cells were prepared as described in the Materials and methods. Immature DCs were either left untreated (n.t.) or stimulated with the highest concentration of CNL used in all experiments (10 μg/ml) or with lipopolysaccharide (LPS; 20 ng/ml). After 72 hr, the DCs were stained with annexin and propidium iodide and the samples were analysed by flow cytometry for the detection of early apoptotic and dead cells. Results shown are representative of three independent experiments. Numbers in quadrants represent the percentage of annexin-positive or propidium iodide-positive cells.

Activation of DCs with CNL proceeds via the TLR4 pathway, predominantly activating the transcription factor NF-κB

To gain an insight into molecular and signalling events of CNL-induced DC activation, we used specific inhibitors of the Myd88 and TLR4 pathways, as described in the Materials and methods. We measured the ability of CNL to up-regulate both CD80 and CD86 co-stimulatory molecules after blockade of the TLR4 intracellular domain, using a specific inhibitor CLI-095. The same experiment was performed for blockade of Myd88 signalling transducer, using a Myd88 inhibitory peptide. Blockade of either TLR4 or Myd88 completely prevented the immunostimulatory effect of CNL on expression of CD80 and CD86 (Fig. 5a).

Figure 5.

Activation of dendritic cells (DCs) with Clitocybe nebularis lectin (CNL) proceeds via the Toll-like receptor 4 (TLR4)/Myd88 pathway and primarily activates nuclear factor-κB (NF-κB). Dendritic cells were prepared as described in the Materials and methods and either left untreated (n.t.) or treated with 10 μg/ml CNL or 20 ng/ml lipopolysaccharide (LPS). (a) Immature dendritic cells (iDCs) pre-incubated with peptide inhibitor of Myd88 signalling protein or with CLI-095, a small molecule-specific inhibitor of TLR4 intracellular domain or left untreated. Afterwards, the DCs were exposed to either CNL or LPS for 48 hr. The cells were then collected and analysed for surface expression of CD80 and CD86. Results are shown as mean ± SD of mean fluorescence intensity values for specific surface marker as depicted. (b) Immature DCs were left untreated or activated with 10 μg/ml of CNL or 20 ng/ml LPS for 10 min. Afterwards, the cells were immediately fixed with 4% PFA and permeabilized using ice-cold methanol, as described in the Materials and methods. The DCs were then stained with Alexa Fluor 488-conjugated, anti-p-NF-κB-p65 (p-Ser536) or with anti-p-p38 mitogen-activated protein kinase (MAPK; p-T180/p-Y182) monoclonal antibodies. Samples were analysed by flow cytometry and the results are depicted as mean ± SD of mean fluorescence intensity ratio between individual samples and their isotypic controls. (c) Confirmation of signalling via TLR4 was determined using the HEK-Blue™ hTLR4 reporter assay. The HEK-Blue™ human TLR4 cells were seeded in a 96-well and either left non-treated (n.t.) or treated with LPS (20 ng/ml) or various concentrations of CNL (0·1, 1 or 10 μg/ml). The activation of TLR4 by LPS (positive control) or CNL was evaluated by determining the secreted embryonic alkaline phosphatase release into culture supernatant via measurement of optical density at 655 nm, as described in the Materials and methods. Significance between individual pairs was determined using Student's unpaired t-test (ns – non-significant; **P ≤ 0·01).

NF-κB and p38 MAPK are two important signalling elements involved in TLR4-dependent DC activation, and it is relevant to know whether they are involved in CNL-induced DC activation. The DCs were therefore exposed to either LPS or CNL (10 μg/ml) for 30 min and then stained intracellularly for the presence of phosphorylated p65 NF-κB subunit and phosphorylated p38 MAPK. Whereas LPS induced similar activation of NF-κB and p38MAPK, CNL induced activation mainly of NF-κB and caused approximately twofold lower activation of p38 MAPK than LPS (Fig. 5b).

Finally, to confirm the involvement of the TLR4 activating pathway in the immunostimulatory activity of the CNL, we performed a TLR4 cell reporter assay using commercially available HEK-Blue™ human TLR4 cells, co-transfected with TLR4 and a secreted embryonic alkaline phosphatase, which is secreted from the reporter cell line if activated with TLR4 agonists. We stimulated the HEK-Blue™ human TLR4 cell line with LPS for positive control, along with various concentrations of CNL (0·1, 1 and 10 μg/ml) or left them non-treated (only culture medium) for negative control. According to the OD measurements of secreted embryonic alkaline phosphatase levels, as expected, the non-treated cells did not show activation of TLR4 (Fig. 5c). Stimulation of HEK-Blue™ human TLR4 cell line with LPS resulted in OD values of 2·682 ± 0·242. Stimulation of reporter cell line with CNL resulted in a dose-dependent increase in activation of TLR4. The OD values for CNL were 0·345 ± 0·032, 0·811 ± 0·087 and 2·141 ± 0·085 for stimulation with 0·1, 1 and 10 μg/ml of CNL, respectively. The OD value of non-treated TLR4 reporter cells was 0·097 ± 0·042. These results confirm that CNL extensively and significantly induces the activation of the TLR4 pathway in a dose-dependent manner (Fig. 5c).

Discussion

In the present study, we have evaluated the immunomodulatory effects of CNL, a novel lectin isolated from the basidiomycete C. nebularis, on human DCs. In the nanomolar range (10 μg/ml CNL equals 63.3 nm), it induces extensive phenotypic as well as functional maturation of DCs, which are subsequently capable of stimulating efficient T-cell responses. Lectins have previously been reported to exert immunomodulatory effects on DCs. A galactose-N-acetyl-d-galactosamine inhibitable lectin (Gal-lectin) from Entamoeba histolytica has been shown to induce maturation and activation of DCs as well as Th1 cytokine production.¹⁶ Gal-lectin also activated macrophages in which the lectin increased gene transcription and surface expression of TLR2.¹⁷ In addition, N-acetyl-d-galactosamine binding mistletoe lectin 3 from mistletoe (Viscum album) has also been shown to induce the maturation of DCs.^18,19

Dendritic cells are situated at centre stage in the modulation of immune responses,^5,20,21 especially when considering the adaptive branch of immunity, and are extremely potent modulators of T-cell immune responses, both in terms of tolerance and immunity. To acquire their immunostimulatory potential, immature DCs must undergo a full maturation process as a response to ‘danger signals’ that consist of various bacterial and viral molecular patterns that bind to and activate receptors such as TLRs and nucleotide oligomerization domain-like receptors.²² Through maturation, DCs acquire distinctive morphology with numerous cytoplasmic processes called dendrites through which they enlarge their surface and ensure enhanced DC-T-cell contact. Maturation of DCs is further characterized by increased cell surface expression of MHC class II and co-stimulatory molecules, along with induced production of pro-inflammatory cytokines such as IL-12 and TNF-α. In this manner they acquire the ability to prime a strong stimulation of T lymphocytes for proliferation and differentiation towards various effector T-cell lineages.^5,6,21,23

When CNL was added to immature DC cultures, it induced an extensive increase in DC maturation marker CD83 (Fig. 1b). When used at 10 μg/ml, it greatly increased the surface expression of co-stimulatory molecules CD80 and CD86, as well as antigen-presenting molecules HLA-DR (Fig. 1a,b). This activity was dose-dependent (Fig. 1a) and was complemented by an extensive increase in the allostimulatory capacity of CNL-treated DCs. Interestingly, a notable increase in the ability of treated DCs to stimulate the proliferation of responding CD4⁺ T cells was already observed when CNL was used at 0·1 μg/ml (Fig. 3a). The reason for this, in comparison with the milder induction of co-stimulatory molecules at similar CNL concentrations, most probably resides in mechanisms of subsequent DC–T-cell interactions. The increased HLA-DR expression and low induction of CD80/CD86 observed when DCs were stimulated with the lowest CNL dose, probably suffice for a threshold of initial T-cell activation strong enough to achieve increased expression of CD40 ligand on T cells, which would subsequently further activate the DCs and strengthen the stimulatory circuit.^24,25

Similar mechanisms could account for the induction of Th1 polarization of responding naive T cells by CNL-activated DCs (Fig. 3b). Namely, when measuring the cytokine profile of CNL-treated DCs, even at 10 μg/ml, CNL was unable to induce extensive production of bioactive IL-12p70, a crucial Th1-polarizing cytokine that directs T-cell responses towards IFN-γ⁺ IL-4^low-producing T cells. In this context, when used as stimulators in a co-culture with naive CD4⁺ T cells, the CNL-treated DCs were nevertheless effective in inducing a Th1-type response, similar to that in LPS-stimulated DCs (Fig. 3b). However, although an extremely powerful immunogen, LPS alone is also not a very effective inducer of IL-12 production in DCs,²⁶ but can induce effective Th1-type responses in vitro. As reported by Snijders et al.,²⁶ DCs need two separate signals to induce increased IL-12 production and CD40 ligand expressed in early stages of the immune response by T cells is one of them. In this manner, although at the first sight the immunostimulation of DCs achieved with CNL may not appear adequate for initiating strong cellular immune responses, because of the lower IL-12 production, their stimulatory capacity clearly reaches Th1-polarizing capabilities once they come into contact with T cells (Fig. 3b).

Mature DCs produce several other pro-inflammatory cytokines, in addition to IL-12. We have observed an extensive increase in production of a broader profile of crucial DC-pro-inflammatory cytokines, namely IL-6, IL-8 and TNF-α (Fig. 2), as a result of CNL stimulation.

Many lectins isolated from medicinal mushrooms and other plants have been shown to act in a cytotoxic manner against human tumour cell lines such as H3B, JAr, U937, S-180, HeLa and others.^27–31 It was therefore of importance to evaluate to what extent treatment with CNL induces any potential apoptosis in treated DCs used in our study. Interestingly, even a 48-hr treatment of DCs with the highest concentration of CNL used in our study (10 μg/ml) failed to induce any significant apoptosis of DCs compared with LPS-stimulated DCs, as determined by annexin/propidium iodide staining (Fig. 4).

Although a number of mushroom- and plant-derived proteins have been designated with immunostimulatory function, not many studies have reported on the mechanism of action that enables rapid maturation of cells such as DCs by these proteins. Interesting data were presented by Kim et al.,³² who reported on DC maturation-inducing proteoglycan isolated from Phelinnus linteus that acts via a TLR/NF-κB pathway. As TLRs were a clear target of these immunomodulatory proteins, we set out to determine whether they play a role in CNL-induced maturation of DCs. Using a Myd88 inhibitory peptide that blocks the pathway of certain TLRs, including TLR4, the immunostimulatory potential of CNL was completely reversed in terms of co-stimulatory molecule up-regulation (Fig. 5a). Further, pre-incubating the DCs with a TLR4-specific small molecule inhibitor, CLI-095,¹⁵ before CNL activation, resulted in a similar loss of up-regulation (Fig. 5a).

The involvement of down-stream signalling mediators in the activation of DCs by CNL was further investigated by measuring the activation levels of NF-κB and p38 MAPK following CNL stimulation. The CNL caused an increase in levels of phosphorylated p65 NF-κB subunit, similar to that in LPS-induced signalling (Fig. 5b). The levels of phosphorylated p38 MAPK were, however, lower than those induced by LPS. In DC activation, high p38 MAPK activation is needed for increased IL-12p70 production ³³ and this effect can also be seen by looking at the IL-12p70 production levels induced by either LPS or CNL (Fig. 2). As both LPS and CNL appear to activate DCs via TLR4, it is difficult to speculate for changes in p38 MAPK activation levels. A possible explanation could be that CNL binds to additional surface signalling proteins that are involved in shaping the DC maturation process, leading to differentially activated DCs with lower IL-12p70-producing capacity. However, as stated earlier, LPS also cannot induce extensive IL-12p70 production in DCs,³⁴ yet both LPS-activated and CNL-activated DCs are able to induce Th1-polarized T-cell responses in an allogeneic co-culture (Fig. 3b). Finally, the capacity of CNL to activate the TLR4 pathway was confirmed by performing a TLR4 reporter assay (Fig. 5c). The CNL was shown to extensively and dose dependently activate TLR4 on reporter cell lines and at the highest concentration used (10 μg/ml), CNL was able to induce TLR4 activation to a degree similar to that of LPS (Fig. 5c).

In summary, we report a potent immunostimulatory action on human DCs of the novel lectin CNL, isolated from C. nebularis mushroom. The CNL is a powerful stimulator of DC maturation, thereby endowing these cells with the ability to generate strong Th1-polarized T-cell responses. Furthermore, we show that this activity is mediated in a TLR4/NF-κB fashion. These results provide an important basis for further research into the pharmacological activity of C. nebularis and its components and the potential of C. nebularis for immunotherapeutic purposes such as anti-cancer pharmaceuticals.

Disclosures

The authors have no financial conflict of interest.