Vibrio cholerae cytolysin (VCC) kills target eukaryotic cells by forming transmembrane oligomeric β-barrel pores. Once irreversibly converted into the transmembrane oligomeric form, VCC acquires an unusual structural stability and loses its cytotoxic property. It is therefore possible that, on exertion of its cytotoxic activity, the oligomeric form of VCC retained in the disintegrated membrane fractions of the lysed cells would survive within the host cellular milieu for a long period, without causing any further cytotoxicity. Under such circumstances, VCC oligomers may potentially be recognized by the host immune cells. Based on such a hypothesis, in the present study we explored the interaction of the transmembrane oligomeric form of VCC with the monocytes and macrophages of the innate immune system. Our study shows that the VCC oligomers assembled in the liposome membranes elicit potent proinflammatory responses in monocytes and macrophages, via stimulation of the toll-like receptor (TLR)2/TLR6-dependent signalling cascades that involve myeloid differentiation factor 88 (MyD88)/interleukin-1-receptor-associated kinase (IRAK)1/tumour-necrosis-factor-receptor-associated factor (TRAF)6. VCC oligomer-mediated proinflammatory responses critically depend on the activation of the transcription factor nuclear factor-κB. Proinflammatory responses induced by the VCC oligomers also require activation of the mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase, which presumably acts via stimulation of the transcription factor activator protein-1. Notably, the role of the MAPK p38 could not be documented in the process.

INTRODUCTION

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytolytic protein toxin [13]. It is produced by many pathogenic strains of the Gram-negative bacterium V. cholerae, the causative agent of the severe diarrhoeal disease cholera [4,5]. VCC is known to exert cytotoxic activity against a wide range of target eukaryotic cells [1,2,610]. The cytotoxic activity of VCC is commonly attributed to its ability to form transmembrane oligomeric pores in the target cells [11,12]. Previous structural studies have characterized VCC as a β-barrel, pore-forming protein toxin (β-PFT) [11,12]. Consistent with the β-PFT mode of action [1316], VCC is secreted by the bacterium as water-soluble monomeric molecules, which, on interaction with the target host cells, assemble into transmembrane heptameric β-barrel pores, thus leading to the colloid–osmotic lysis of the target cells [1722]. Conversion of the water-soluble monomeric form of VCC into a transmembrane oligomeric structure is considered to be the most critical event in the toxin's mode of action [2326]. Transition of the VCC monomers into the oligomeric state in the membrane lipid bilayer is an irreversible process [23]. Once converted into the transmembrane oligomeric assembly, VCC oligomers do not dissociate into the monomeric state. Moreover, VCC oligomers formed in the membrane lipid bilayer display remarkable stability in terms of resistance to denaturant- and heat-induced unfolding, as well as resistance to proteolytic degradation [23,27]. Interestingly, VCC oligomers extracted from the membrane lipid bilayer do not display any cytolytic activity [28]. It appears, therefore, that the cytolytically active monomeric form of VCC may represent a transient state of the toxin, which in the process of conferring its cytotoxic response converts into the oligomeric assembly state with distinct structural and functional properties. Consistent with its remarkable physicochemical stability, it is possible that, on cytolysis of the target cells, an oligomeric form of the toxin present in the disrupted membrane fractions may potentially survive in the cellular niche within the host system. It would therefore be logical to speculate that such an oligomeric form of VCC could be considered as a potential target of the host immune system.

Previous studies have indicated a differential interaction of the monomeric and oligomeric forms of VCC with the host immune cells, e.g. a VCC monomer has been shown to induce apoptotic responses in the mouse peritoneal cavity (PerC) B-1a cells [29]. In contrast, a VCC oligomer, extracted from the membrane lipid bilayer via detergent solubilization, could not display any apoptogenic activity in the mouse PerC B-1a cells. Rather, it has been shown to trigger IgA expression, a signature of the mucosal immune responses [29]. In separate studies, a VCC oligomer, but not the monomeric form of the protein, has been shown to possess the ability to elicit up-regulation of co-stimulatory molecules such as CD86 on mouse macrophages [27]. Therefore, it appears that the oligomeric form of VCC has the ability to trigger activation of the host immune cells, without causing cytotoxic cell-killing effects. However, interaction of the transmembrane oligomeric form of VCC with the host immune cells has been explored only to a limited extent. In particular, it has not been explored whether the transmembrane oligomeric form of VCC possesses any ability to trigger the proinflammatory responses in the host innate immune cells, in the absence of its cytotoxic responses.

In the present study, we explored the interactions of the transmembrane oligomeric form of VCC with the monocytes and macrophages of the innate immune system in terms of triggering proinflammatory responses. We also characterized the signalling mechanism(s) associated with the cellular responses elicited by these innate immune cells in response to the VCC oligomeric form. Our study showed that the transmembrane oligomeric form of VCC elicited potent proinflammatory responses in RAW 264.7 (murine macrophage cell line), THP-1 (human monocyte cell line) and human peripheral blood mononuclear cells (PBMCs), as well as in human monocytes purified from the PBMCs. VCC oligomers, incorporated into the membrane lipid bilayer of the liposome vesicles, triggered profound production of the proinflammatory mediators such as nitric oxide (NO) and tumour necrosis factor-α (TNFα), as well as interleukin (IL)-6, in these cells, without causing any significant cytotoxicity. Our data showed that the toll-like receptors (TLRs), TLR2 and TLR6, acted as the essential receptors to mediate direct recruitment of the VCC oligomers by the TLR2/TLR6 heterodimeric complex. The data further showed that the VCC oligomer-induced, proinflammatory responses were critically dependent on the activation of the TLR2/TLR6-mediated downstream signal-transduction cascade(s) that involved MyD88/IRAK1/TRAF6 (myeloid differentiation factor 88/IL-1-receptor-associated kinase 1/TNF-receptor-associated factor 6). As a consequence of the TLR signalling, VCC oligomer stimulation resulted in the activation of the transcription factor NF-κB (nuclear factor-κB), whereas blockade of NF-κB activation suppressed the proinflammatory responses triggered by the VCC oligomers. VCC oligomer-induced, proinflammatory responses were also found to be critically dependent on the activation of the mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase (JNK), which presumably acted via stimulation of the transcription factor activator protein 1 (AP-1). Notably, the proinflammatory responses generated in the monocytes/macrophages in response to the transmembrane oligomeric form of VCC did not require activation of the MAPK family member p38.

EXPERIMENTAL

Preparation of the transmembrane oligomeric form of VCC

Recombinant VCC was purified following a previously described method [24,30]. Asolectin–cholesterol liposomes (1:1 w/w) were prepared as described in Paul and Chattopadhyay [24].

The transmembrane oligomeric form of VCC was prepared by incubating the purified form of monomeric VCC with freshly prepared asolectin–cholesterol liposomes of 1:2 (w/w) protein:lipid ratio. The mixture was incubated at room temperature for 3–4 h. The unbound protein was removed by ultracentrifugation at 105000 g for 20 min at 4°C. The pellet thus obtained was washed twice with PBS (20 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4). The final pellet of proteoliposomes containing the transmembrane oligomeric form of VCC in the asolectin–cholesterol liposomes was suspended in PBS. The protein concentration in the proteoliposomes was estimated using the Bradford reagent (Sigma-Aldrich) [31], with BSA as the standard, and was analysed by SDS/PAGE and Coomassie staining as described below. SDS/PAGE showed ≥98% conversion of the VCC monomer into the oligomeric form.

Analysis of the VCC oligomers by SDS/PAGE and Coomassie staining

Oligomeric assembly of VCC incorporated into the asolectin–cholesterol liposome membranes was incubated in an SDS/PAGE sample buffer containing 1% SDS for 15 min at a specific temperature in the range 20–100°C, and the samples were analysed by conventional reducing SDS/PAGE and Coomassie staining. The oligomeric state of VCC remained stable in the presence of SDS, even at a temperature as high as 70°C (Figure 1).

Transmembrane oligomeric form of VCC

Figure 1
Transmembrane oligomeric form of VCC

(A) Transmembrane oligomeric assembly of VCC showed heat and SDS stability to a remarkable extent. VCC oligomers, incorporated into the asolectin–cholesterol liposomes, were subjected to reducing SDS/PAGE and Coomassie staining after incubation with SDS/PAGE sample buffer for 15 min at a specified temperature. The oligomeric assembly remained stable up to a temperature as high as 70°C. Molecular mass standards are shown, and marked in lane M. (B) MTT-based assay showed that the transmembrane oligomeric form of VCC, incorporated into the asolectin–cholesterol liposomes, did not induce any prominent decrease in the viability of the RAW 264.7 mouse macrophages and THP-1 human monocytes. In contrast, a cytolytically active monomeric form of VCC induced a marked decrease in cell viability. Cells treated with PBS were considered as blanks, whereas cells treated with an equivalent amount of liposomes were designated as the control.

Figure 1
Transmembrane oligomeric form of VCC

(A) Transmembrane oligomeric assembly of VCC showed heat and SDS stability to a remarkable extent. VCC oligomers, incorporated into the asolectin–cholesterol liposomes, were subjected to reducing SDS/PAGE and Coomassie staining after incubation with SDS/PAGE sample buffer for 15 min at a specified temperature. The oligomeric assembly remained stable up to a temperature as high as 70°C. Molecular mass standards are shown, and marked in lane M. (B) MTT-based assay showed that the transmembrane oligomeric form of VCC, incorporated into the asolectin–cholesterol liposomes, did not induce any prominent decrease in the viability of the RAW 264.7 mouse macrophages and THP-1 human monocytes. In contrast, a cytolytically active monomeric form of VCC induced a marked decrease in cell viability. Cells treated with PBS were considered as blanks, whereas cells treated with an equivalent amount of liposomes were designated as the control.

Cell lines and culture conditions

Murine macrophage cell line RAW 264.7 and human monocytic cell line THP-1 were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml of penicillin and 100 units/ml of streptomycin (all from Invitrogen Life Technologies). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Isolation of human PBMCs and purification of monocytes from human PBMCs

Work with human blood has been approved by the Institutional Bioethics Committee of IISER, Mohali.

Human PBMCs were isolated following the method described by Sakharwade et al. [32]. Monocytes were isolated from whole human PBMCs by depletion of non-monocytes (using the human Monocyte Isolation Kit II; Miltenyi Biotec), as described in the manufacturer's protocol.

Assay of cell viability

RAW 264.7 cells or THP-1 cells (105 cells) were seeded in 100 μl of Phenol Red-free RPMI-1640 medium supplemented with 10% FBS, and the indicated treatments. Cells were incubated at 37°C for 24 h, and the cell viability was monitored using the MTT-based cell growth determination kit (Sigma-Aldrich) as per the manufacturer's instruction. The precipitated MTT formazan was dissolved in acidified propanol (0.1 M HCl in propan-2-ol). Absorbance was recorded at 570 nm on an iMark Microplate Absorbance Reader (Bio-Rad Laboratories). Cells that had not been treated served as the positive control for 100% viability, whereas media without cells were taken as the negative control corresponding to 0% viability.

Assay of NO production

Production of NO in the cell culture supernatant was monitored by estimating nitrite, a stable degradation product of NO, using a Griess colorimetric assay (Sigma-Aldrich). Cells were plated at a density of 1×106 cells/ml in Phenol Red-free RPMI-1640 medium containing 10% FBS, and grown at 37°C in 5% CO2. After the treatment, culture supernatants were collected and mixed with an equal volume of modified Griess reagent, and incubated in the dark at room temperature for 15 min. The absorbance at 540 nm was recorded using an iMark Microplate Absorbance Reader. The nitrite concentrations were calculated using the standard curve generated with sodium nitrite solutions of known concentrations. For the assay of NO production, RAW 264.7 cells were treated with 10 μg/ml of VCC oligomer for 24 h, unless mentioned otherwise.

TNFα and IL-6 measurements

RAW 264.7 mouse macrophages, THP-1 human monocytes and human PBMCs were plated at the density of 1×106 cells/ml, and treated with 10 μg/ml of VCC oligomers, unless mentioned otherwise. The amounts of TNFα and IL-6 in the cell culture supernatants were measured using the TNFα and IL-6 BD OptEIA™ ELISA sets (BD Biosciences) according to the manufacturer's instructions. For the assay of TNFα production by RAW 264.7, THP-1 and human PBMCs (or human PBMC-derived monocytes), 8, 4 and 8 h of treatment were followed, respectively, unless mentioned otherwise. For the assay of IL-6 secretions, 24 h of treatment were followed, unless mentioned otherwise.

Gene expression analysis

RAW 264.7 and THP-1 cells were plated at a density of 2×106 cells/ml in 2 ml of complete medium, and subjected to treatment as indicated. In all the experiments, 10 μg/ml of VCC oligomer was used, unless mentioned otherwise. After the treatment, RNA was isolated using a Nucleo-pore RNA sure mini kit (Genetix Biotech) according to the manufacturer's instructions. The cDNA was synthesized from the isolated RNA using a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR (RT-PCR) was carried out using a Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific) on an Eppendorf Mastercycler EP Realplex Thermal Cycler following the manufacturer's instructions. Primer sequences for the target genes were obtained from the primer bank [33], and synthesized using material from Integrated DNA Technologies. The RT-PCR data were analysed by the comparative CT method [34].

Cell-surface expression analysis of TLRs

For cell-surface expression analysis of TLRs on treatment with the transmembrane oligomeric form of VCC, RAW 264.7 and THP-1 cells, plated at a density of 1×106 cells/ml, were treated with the VCC oligomer (10 μg/ml), incorporated into the asolectin–cholesterol liposomes, for 24 h. Treatment with liposomes served as the control. After incubation, the cells were harvested and washed with PBS, and then with ice-cold PBS containing 0.1% NaN3 and 1% BSA (FACS buffer). In the case of RAW 264.7, the cells were first incubated with mouse Fc Block (BD Biosciences) for 15 min at 4°C, and then centrifuged and re-suspended in FACS buffer. Cells were stained using FITC-, allophycocyanin (APC)- and phycoerythrin (PE)-conjugated, anti-TLR antibodies and incubated for 1 h. FITC-conjugated anti-mouse/human CD282 (TLR2) and PE-conjugated anti-human CD286 (TLR6) were from BioLegend, and APC-conjugated anti-mouse TLR6 was from R&D Systems.

After antibody staining, cells were washed and re-suspended in FACS buffer, and analysed on a BD FACSCalibur flow cytometer (BD Biosciences) using the program BD CellQuest Pro (http://www.bdbiosciences.com). Histogram plots were processed using the FLOWJO software (http://www.flowjo.com).

Blocking of TLRs with neutralizing antibodies

THP-1 cells or monocytes isolated from human PBMCs were plated in 96-well microtitre plates at a density of 1×106 cells/ml. Cells were subjected to pre-treatment with 5 μg/ml of anti-TLR-neutralizing antibodies (antibodies against human TLR1 and TLR6 were from Invivogen; antibody against human TLR2 was from Biolegend) for 1 h, and subsequently treated with 10 μg/ml of VCC oligomer or the corresponding liposome control. After the treatment, cell culture supernatants were collected for estimation of TNFα production.

The siRNA knockdown of TLR2 and TLR6

TLR2- and TLR6-specific siRNAs were obtained as ON-TARGET plus SMARTpool siRNAs (Dharmacon GE Healthcare). The non-targeting siRNA control (scrambled siRNA) used in our study was the ON-TARGET plus NON-targeting Pool (Dharmacon GE Healthcare). THP-1 cells were transiently transfected with the specified siRNA using the DharmaFECT 1 transfection reagent (Dharmacon GE Healthcare) according to the manufacturer's instruction. Surface expression of TLR2 and TLR6 on the siRNA-transfected THP-1 cells was monitored at 24, 36, 48 and 60 h post-transfection, using the flow cytometry-based assay, as described above.

To monitor the effect of siRNA knockdown on the VCC oligomer-mediated TNFα secretion by the THP-1 cells, cells were transfected with TLR2 siRNA, TLR6 siRNA or scrambled siRNA. Then 48 h post-transfection, the cells were treated with VCC oligomer (10 μg/ml), incorporated into the asolectin–cholesterol liposomes, and incubated further for 4 h to monitor TNFα secretion in the cell culture supernatant.

Experiment with the chemical inhibitors

The chemical inhibitors, PDTC (ammonium pyrrolidinedithiocarbamate), SP600125, SB202190 and IRAK-1/4 inhibitor 1, and dexamethasone were from Sigma-Aldrich; MLN4924 was from R&D Systems; JNK-IN-8 and VX 745 were from Santa Cruz Biotechnology. In all the experiments with the chemical inhibitors, cells were pre-treated with them for 1 h.

Preparation of cell lysates

For preparation of whole cell lysate, cells (approximately 107 cells), treated with VCC oligomer (10 μg/ml) that was incorporated into an asolectin–cholesterol liposome, were washed twice with ice-cold PBS; the cell pellet was re-suspended in 100 μl of lysis buffer (50 mM Tris/HCl, pH 8.0, containing 150 mM NaCl, 0.1% Triton X-100, 0.1% SDS) supplemented with 1% mammalian protease inhibitor cocktail (Sigma-Aldrich), and incubated on ice for 10 min. The cells were disrupted by sonication on ice, and the cell lysates were collected by centrifugation at 24000 g for 20 min and at 4°C.

For cytosolic and nuclear extract preparation, the cell pellet (about 107 cells) obtained after a PBS wash was re-suspended in 200 μl of buffer A (10 mM Hepes, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 0.05% Igepal CA-630, pH 7.9), and incubated in ice for 10 min. Samples were then centrifuged at 800 g for 10 min at 4°C; the supernatant was collected as a cytosolic fraction. The pellet was re-suspended in 100 μl of buffer B (5 mM Hepes, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and 26% glycerol, pH 7.9) containing 300 mM NaCl and 0.1% protease inhibitor cocktail. A nuclear extract was obtained after sonication, and subsequent centrifugation at 24000 g for 20 min and at 4°C.

Protein concentrations in all the fractions were measured using the Bradford assay [31], with BSA as standard.

Immunoprecipitation and immunoblotting

Human- and mouse-specific antibodies used for the immunoprecipitation experiments and immunoblot analysis were obtained from different sources. Antibodies for inducible NO synthase (iNOS)/NOS2, TLR1, TLR2, TLR6, inhibitor of NFκB (IκB-α), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin and lamin B1 were from Santa Cruz Biotechnology; antibodies for NF-κB/p65, TRAF6, MyD88, IRAK1 and phospho-IκB-α (pSer32/Ser36) were from Sigma-Aldrich; antibodies for c-Jun, c-Fos, phospho-SAPK/JNK (Thr183/Tyr185) and SAPK/JNK were from Cell Signaling Technology; antibody for NF-κB/p50 was from BioLegend. Rabbit IgG isotype control was obtained from Santa Cruz Biotechnology. Horse radish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG secondary antibodies were from Sigma-Aldrich.

For immunoprecipitation experiments, 0.5 μg of antibody was added to the cell lysates (containing 500 μg of protein), and samples were incubated for 3–4 h at 4°C. Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) were then added to the mixtures, and incubated overnight at 4°C. The beads were washed five times with the lysis buffer, boiled in SDS/PAGE sample buffer for 10 min, separated on a 10% SDS/PAGE gel, and the proteins were transferred on to a PVDF membrane and analysed by immunoblotting.

The immunoblots were developed using the ECL Western blotting detection kit and the images acquired using ImageQuant LAS 4010 (both from GE Healthcare Life Sciences).

Structural models

Models of the monomeric and oligomeric forms of VCC (see Supplementary Figure S9) were generated based on the Protein Data Bank (PDB) co-ordinates 1XEZ and 3O44, respectively. Structural coordinates were obtained from the PDB server (http://www.pdb.org). The model for the transmembrane oligomeric form of VCC was generated using the OPM server found online (http://opm.phar.umich.edu/server.php). Protein structural models were rendered using PyMOL (PyMOL Molecular Graphics System–http://pymol.org).

Statistical analysis

Results are expressed as means±S.E.M. Statistical analysis was done using Student's two-sided t-test, and the differences were considered statistically significant at P<0.05. The P values are indicated as follows: *P<0.05, **P<0.01, ***P<0.001.

RESULTS

Transmembrane oligomeric form of VCC does not induce cytotoxicity in monocytes and macrophages

As reported earlier [24], the cytolytically active, water-soluble monomeric form of VCC is converted into the transmembrane oligomeric pore in contact with the membrane lipid bilayer of the target eukaryotic cells; this causes colloid–osmotic lysis of the target cells. Conversion of the VCC monomers into the transmembrane oligomeric structure could also be mimicked in synthetic liposome vesicles [24]. Accordingly, in the present study, the transmembrane oligomeric form of VCC was generated in the asolectin–cholesterol liposomes (see Figure 1A). Consistent with earlier reports, VCC oligomers incorporated into the asolectin–cholesterol liposomes showed remarkable stability in displaying resistance towards SDS- and heat-induced dissociation/denaturation into the monomeric state (see Figure 1A). The oligomeric assembly of VCC remained intact even in the presence of 1% SDS, when treated at temperatures as high as 60–70°C for 15 min (see Figure 1A). The transmembrane oligomeric form of VCC was tested for its ability to trigger any cytotoxic response in RAW 264.7 mouse macrophage and THP-1 human monocyte cell lines. An MTT-based assay was used to monitor the viability of the cells on treatment with the VCC oligomers. VCC oligomer (10 μg/ml) treatment for 24 h resulted in only about a 20% decrease in viability of the RAW 264.7 cells, whereas almost no loss of cell viability was noticed in the case of the THP-1 cells (see Figure 1B). In contrast, the VCC monomer (1 μg/ml) treatment caused a decrease in viability of both cell types tested of >90% (see Figure 1B). These data confirmed that the VCC oligomer preparation did not possess any significant cytotoxicity, whereas the monomeric form of VCC was found to induce potent cytotoxic response in both the RAW 264.7 and the THP-1 cells.

Transmembrane oligomeric form of VCC induces potent proinflammatory responses in macrophages/monocytes in terms of production of NO, TNFα and IL-6

VCC oligomers induce NO production in RAW 264.7 murine macrophages via iNOS up-regulation

Enhanced NO production via up-regulation of iNOS activity is considered to be a hallmark of macrophage proinflammatory responses [3537]. Therefore, to explore its interactions with the macrophages, we first tested the ability of the transmembrane oligomeric form of VCC to evoke NO production in the RAW 264.7 macrophage cell line. VCC oligomers were found to trigger increased NO production by the RAW 264.7 macrophages in a concentration- and time-dependent manner, when tested over the protein concentration range 1–10 μg/ml for a period of 0–24 h (Figures 2A and 2B). The maximum NO production was observed for treatment with 10 μg/ml of VCC oligomer at 24 h. Treatment of cells with the VCC oligomers in the presence of polymyxin B (2.5 μg/ml), a potent agent that binds and inactivates endotoxin, did not significantly affect NO production (Figure 2C). These results confirmed that the production of VCC oligomer-induced NO by RAW 264.7 cells was not the result of possible endotoxin contamination in the VCC oligomer preparation.

Transmembrane oligomeric form of VCC-triggered NO production in RAW 264.7 mouse macrophage cells via up-regulation of iNOS expression
Figure 2
Transmembrane oligomeric form of VCC-triggered NO production in RAW 264.7 mouse macrophage cells via up-regulation of iNOS expression

(A) VCC oligomers, incorporated into the asolectin–cholesterol liposomes, induced NO production in the RAW 264.7 cells in a concentration-dependent manner. Cells were treated with the VCC oligomers for 24 h, and assayed for NO production. Cells treated with liposomes alone, corresponding to the highest concentration of the VCC oligomer preparation, were taken as the control. Results shown are means±S.E.M., and represent the average from three independent experiments. (B) VCC oligomers, incorporated into the asolectin–cholesterol liposomes, induced NO production in RAW 264.7 cells in a time-dependent manner. The time course is shown of NO production in RAW 264.7 cells induced by treatment with VCC oligomers (10 μg/ml; grey bars) incorporated into the asolectin–cholesterol liposomes. Cells treated with liposomes alone served as the control (open bars). Results shown are means±S.E.M. and represent the average from three independent experiments. (C) Polymyxin B pre-treatment did not affect VCC oligomer-induced NO production, whereas LPS-mediated NO generation was significantly blocked by polymyxin B. Polymyxin B (2.5 μg/ml) was added to RAW 264.7 cells 30 min before the treatment with VCC oligomers, and cells were assayed for NO production. Cells treated with Escherichia coli LPS (1 μg/ml) served as the positive control, whereas cells treated with liposomes alone were taken as the negative control. Grey bars represent cells without polymyxin B pre-treatment; open bars represent cells with polymyxin B pre-treatment. Results are expressed as means±S.E.M., and represent the average from three independent experiments. The data suggested that VCC oligomer-induced NO production was not the result of endotoxin contamination in the preparation. (D) VCC oligomer-triggered, transcriptional up-regulation of iNOS mRNA in RAW 264.7 cells. A time-dependent increase in the mRNA expression of iNOS in response to the VCC oligomers in RAW 264.7 cells was determined using a semi-quantitative RT-PCR-based assay. Fold changes at particular time points were calculated for both VCC oligomers (grey bars) and the respective liposome control (open bars) compared with the cells treated with just PBS. All quantifications were normalized to the housekeeping gene RPL13. Results are expressed as means±S.E.M. (E) VCC oligomers induced increased expression of iNOS in RAW 264.4 cells, as probed by immunoblotting with anti-iNOS antibody. RAW 264.7 cells were incubated with the transmembrane oligomeric form of VCC; cell lysates were prepared and analysed by immunoblotting using anti-iNOS/NOS2 antibody. Immunoblotting for β-actin in the cell lysate was used as the loading control. The result shown represents three independent experiments. (F) The inhibition of iNOS by dexamethasone blocked VCC oligomer-induced NO production in RAW 264.7 cells. Cells were pre-treated with dexamethasone, followed by treatment with the VCC oligomers, and the cells were subsequently assayed for NO production. Results shown are means±S.E.M., and represent the average from three independent experiments. Cells treated with liposomes alone served as the control.

Figure 2
Transmembrane oligomeric form of VCC-triggered NO production in RAW 264.7 mouse macrophage cells via up-regulation of iNOS expression

(A) VCC oligomers, incorporated into the asolectin–cholesterol liposomes, induced NO production in the RAW 264.7 cells in a concentration-dependent manner. Cells were treated with the VCC oligomers for 24 h, and assayed for NO production. Cells treated with liposomes alone, corresponding to the highest concentration of the VCC oligomer preparation, were taken as the control. Results shown are means±S.E.M., and represent the average from three independent experiments. (B) VCC oligomers, incorporated into the asolectin–cholesterol liposomes, induced NO production in RAW 264.7 cells in a time-dependent manner. The time course is shown of NO production in RAW 264.7 cells induced by treatment with VCC oligomers (10 μg/ml; grey bars) incorporated into the asolectin–cholesterol liposomes. Cells treated with liposomes alone served as the control (open bars). Results shown are means±S.E.M. and represent the average from three independent experiments. (C) Polymyxin B pre-treatment did not affect VCC oligomer-induced NO production, whereas LPS-mediated NO generation was significantly blocked by polymyxin B. Polymyxin B (2.5 μg/ml) was added to RAW 264.7 cells 30 min before the treatment with VCC oligomers, and cells were assayed for NO production. Cells treated with Escherichia coli LPS (1 μg/ml) served as the positive control, whereas cells treated with liposomes alone were taken as the negative control. Grey bars represent cells without polymyxin B pre-treatment; open bars represent cells with polymyxin B pre-treatment. Results are expressed as means±S.E.M., and represent the average from three independent experiments. The data suggested that VCC oligomer-induced NO production was not the result of endotoxin contamination in the preparation. (D) VCC oligomer-triggered, transcriptional up-regulation of iNOS mRNA in RAW 264.7 cells. A time-dependent increase in the mRNA expression of iNOS in response to the VCC oligomers in RAW 264.7 cells was determined using a semi-quantitative RT-PCR-based assay. Fold changes at particular time points were calculated for both VCC oligomers (grey bars) and the respective liposome control (open bars) compared with the cells treated with just PBS. All quantifications were normalized to the housekeeping gene RPL13. Results are expressed as means±S.E.M. (E) VCC oligomers induced increased expression of iNOS in RAW 264.4 cells, as probed by immunoblotting with anti-iNOS antibody. RAW 264.7 cells were incubated with the transmembrane oligomeric form of VCC; cell lysates were prepared and analysed by immunoblotting using anti-iNOS/NOS2 antibody. Immunoblotting for β-actin in the cell lysate was used as the loading control. The result shown represents three independent experiments. (F) The inhibition of iNOS by dexamethasone blocked VCC oligomer-induced NO production in RAW 264.7 cells. Cells were pre-treated with dexamethasone, followed by treatment with the VCC oligomers, and the cells were subsequently assayed for NO production. Results shown are means±S.E.M., and represent the average from three independent experiments. Cells treated with liposomes alone served as the control.

Up-regulation of iNOS is known to mediate increased NO production in macrophages in response to proinflammatory mediators [3537]. We therefore explored whether a VCC oligomer could trigger up-regulation of iNOS gene expression as well as increased iNOS protein expression in RAW 264.7 cells. We examined the iNOS gene expression profile in RAW 264.7 cells in response to VCC oligomer treatment, by using a semi-quantitative RT-PCR-based assay. VCC oligomer triggered up-regulation of iNOS gene expression, starting at the time point 4 h, whereas iNOS gene expression peaked at 12 h (Figure 2D). Western blot analysis of the VCC oligomer-treated RAW 264.7 cells also confirmed a time-dependent increase in iNOS protein expression when examined over a period of 0–24 h (Figure 2E). Finally, we confirmed the direct involvement of iNOS gene up-regulation in increased NO production by RAW 264.7 macrophages in response to VCC oligomer treatment. For this, the cells were pre-treated with dexamethasone before VCC oligomer treatment. Dexamethasone is a synthetic glucocorticoid that is known to inhibit iNOS gene expression [38]. Pre-treatment of RAW 264.7 cells with dexamethasone (1–10 μM) for 1 h resulted in a concentration-dependent reduction in NO production in response to the VCC oligomer treatment (Figure 2F). Taken together, our data established that the transmembrane oligomeric form of VCC triggered NO production via up-regulation of iNOS expression in the RAW 264.7 macrophage cell line.

VCC oligomers trigger TNFα production in RAW 264.7 murine macrophages, THP-1 human monocytes and human PBMCs

In response to the inflammatory stimulus, cells of monocytic lineage are known to produce proinflammatory cytokines such as TNFα [36,39,40]. Therefore, we assessed the ability of the VCC oligomers to initiate proinflammatory responses in terms of triggering TNFα production in monocytes and macrophages. We observed that the VCC oligomer treatment resulted in an up-regulation of TNFα gene expression in both RAW 264.7 and THP-1 cells, as estimated by the semi-quantitative RT-PCR-based assay (see Supplementary Figures S1A and S1B). Next, we examined TNFα production in the RAW 264.7 mouse macrophage cell line (Figure 3A), THP-1 human monocytic cell line (Figure 3B) and human PBMCs (see Supplementary Figure S2A) on treatment with the transmembrane oligomeric form of VCC. For assessment of TNFα production, cells were treated with VCC oligomers, supernatants were collected over a period of 4–24 h of treatment and TNFα secretion was estimated by ELISA. VCC oligomers were found to trigger profound TNFα release in all three monocyte/macrophage cell types tested. Consistent with the earlier reports [32], TNFα production was found to be less pronounced in the case of the THP-1 monocytic cell line and in human PBMCs compared with that of the RAW 264.7 macrophage cell line. This was consistent with the fact that the monocytic cells exhibit fewer proinflammatory responses compared with the macrophages.

Transmembrane oligomeric form of VCC triggered TNFα and IL-6 production in RAW 264.7 mouse macrophages and THP-1 human monocytes
Figure 3
Transmembrane oligomeric form of VCC triggered TNFα and IL-6 production in RAW 264.7 mouse macrophages and THP-1 human monocytes

VCC oligomers in the asolectin–cholesterol liposomes induced secretion of (A) TNFα in the culture supernatant of RAW 264.7 cells, (B) TNFα in the culture supernatant of THP-1 cells, (C) IL-6 in the culture supernatant of RAW 264.7 cells and (D) IL-6 in the culture supernatant of THP-1 cells. Cells were treated with the VCC oligomers (grey bars), liposome control (black bars) and PBS blank (open bars). Cell culture supernatants were collected after incubation for the indicated time periods, and were estimated for TNFα and IL-6 levels. Results of the TNFα measurements are means±S.E.M., and represent the average from three independent experiments. Results of the IL-6 assay are the means±S.E.M., and represent at least two independent experiments.

Figure 3
Transmembrane oligomeric form of VCC triggered TNFα and IL-6 production in RAW 264.7 mouse macrophages and THP-1 human monocytes

VCC oligomers in the asolectin–cholesterol liposomes induced secretion of (A) TNFα in the culture supernatant of RAW 264.7 cells, (B) TNFα in the culture supernatant of THP-1 cells, (C) IL-6 in the culture supernatant of RAW 264.7 cells and (D) IL-6 in the culture supernatant of THP-1 cells. Cells were treated with the VCC oligomers (grey bars), liposome control (black bars) and PBS blank (open bars). Cell culture supernatants were collected after incubation for the indicated time periods, and were estimated for TNFα and IL-6 levels. Results of the TNFα measurements are means±S.E.M., and represent the average from three independent experiments. Results of the IL-6 assay are the means±S.E.M., and represent at least two independent experiments.

VCC oligomers induce release of IL-6 in RAW 264.7 murine macrophages, THP-1 human monocytes and human PBMCs

To explore the ability of the VCC oligomers to evoke proinflammatory responses, we monitored production of another proinflammatory cytokine IL-6 [41] from the monocytes and macrophages. VCC oligomer treatment triggered up-regulation of IL-6 gene expression in both RAW 264.7 and THP-1 cells, as monitored by the semi-quantitative RT-PCR-based assay (see Supplementary Figures S1C and S1D). Moreover, it resulted in a profound release of IL-6 from the RAW 264.7 murine macrophages (Figure 3C), THP-1 human monocytes (Figure 3D) and human PBMCs (see Supplementary Figure S2B). When monitored over a period of 4–24 h, the transmembrane oligomeric form of VCC, at a concentration of 10 μg/ml, elicited IL-6 release from all these cell types in a time-dependent manner. The extent of IL-6 secretion was found to be more pronounced in RAW 264.7 macrophages and human PBMCs, compared with the THP-1 monocytic cells.

Altogether, our results confirmed that the transmembrane oligomeric form of VCC could trigger potent proinflammatory responses in terms of NO, TNFα and IL-6 production in monocytes and macrophages.

Transmembrane oligomeric form of VCC elicits proinflammatory responses in macrophages and monocytes via activation of TLR2/TLR6-dependent pathways

Pathogens and pathogen-associated molecules are recognized by immune cells via a specific set of pattern recognition receptors (PRRs) [4244]. TLRs are the archetypal PRRs involved in initiating diverse cellular responses in the host immune cells on recognition of the pathogen-associated molecules, and they do so via activation of distinct intracellular signalling cascades [4547]. Given the potential of the VCC oligomer to trigger potent proinflammatory responses in monocytes/macrophages, we explored the potential involvement of the TLRs in the mechanism.

First, a semi-quantitative RT-PCR-based assay was performed to evaluate the gene expression profile of cell surface TLRs such as TLR1, TLR2, TLR4, TLR5 and TLR6 in the RAW 264.7 macrophages treated with VCC oligomer. VCC oligomer induced up-regulation of TLR2 and TLR6 gene expression in the RAW 264.7 cells (see Supplementary Figure S3A), whereas expression of TLR1, TLR4 and TLR5 (see Supplementary Figure S3A) was not found to be significantly increased. A time-course study showed the maximum up-regulation of TLR2 and TLR6 genes after 4 h of treatment was with the VCC oligomer. VCC oligomer treatment also resulted in increased surface expressions of TLR2 and TLR6 on the RAW 264.7 (see Supplementary Figure S3B) and THP-1 (see Supplementary Figure S3C) cells, as monitored by the flow cytometry-based assay. TLR2 is known to mediate intracellular signalling cascades in response to various bacterial components by forming heterodimers with either TLR1 or TLR6 [48,49]. As the VCC oligomer up-regulated expression of TLR2 and TLR6, it appeared from our data that the TLR2/TLR6 hetero-dimer might be acting as the functional receptor complex mediating the proinflammatory signals in response to VCC oligomer in the RAW 264.7 macrophages and THP-1 mono-cytes.

We employed immunoprecipitation-based assays to confirm TLR2/TLR6 heterodimer complex formation in monocytes/macrophages in response to VCC oligomer exposure. When RAW 264.7 cells treated with VCC oligomer were immunoprecipitated with anti-TLR2 or anti-TLR6 antibody, we observed enhanced association of TLR6 or TLR2, respectively (Figure 4A). In contrast, no such association of TLR1 with TLR2 was noticed under identical experimental conditions (Figure 4A). A similar amount of association between TLR2 and TLR6 was documented in THP-1 cells on treatment with VCC oligomer (Figure 4A). Notably, immunoprecipitation with anti-TLR2 and anti-TLR6 showed a marked association of VCC with TLR2/TLR6 in RAW 264.7 and THP-1 cells (Figure 4A), thus suggesting a direct interaction of the VCC oligomer with the TLR2/TLR6 heterodimeric receptor complex. It is important to mention that immunoprecipitation with a control isotype IgG, followed by immunoblotting with anti-VCC, could not detect any VCC signal, thus ruling out the possibility of any non-specific association of VCC during the immunoprecipitation assay (see Supplementary Figure S3D).

Role of TLR2/TLR6 in the VCC oligomer-mediated, proinflammatory responses

Figure 4
Role of TLR2/TLR6 in the VCC oligomer-mediated, proinflammatory responses

(A) Association of the VCC oligomer with the TLR2/TLR6 heterodimer. RAW 264.7 and THP-1 cells were treated with VCC oligomer for 1 h and cell lysates were prepared. Samples were normalized for the protein content, immunoprecipitated (IP) with anti-TLR2 or anti-TLR6 antibodies, and subjected to immunoblotting (IB) for TLR1, TLR2, TLR6 and VCC as indicated. Cells treated with liposome alone served as the control. The data indicated a significant extent of increased association between TLR2 and TLR6 on stimulation with the VCC oligomer. No significant association was noticed between TLR1 and TLR2. The data also showed association of VCC oligomer with the TLR2/TLR6 complex. Results represent three independent experiments. (B) Neutralization of TLR2 and TLR6 suppressed VCC oligomer-induced TNFα secretion from THP-1 human monocytic cells (left panel) and monocytes purified from human PBMCs (right panel). Cells were treated with anti-TLR1-, anti-TLR2- or anti-TLR6-neutralizing antibodies, and were subsequently incubated with VCC oligomer incorporated into the asolectin–cholesterol liposome. Cell culture supernatants were collected and analysed for TNFα production. Cells treated with liposomes alone were taken as the negative control, whereas treatments with VCC oligomer without anti-TLR pre-treatment were considered as the positive control. The results shown are means±S.E.M., and represent the average from three independent experiments. (C) The siRNA-mediated knockdown of TLR2 and TLR6 suppressed VCC oligomer-induced TNFα secretion from THP-1 human monocytic cells. Cells were transfected with the TLR2 siRNA, TLR6 siRNA or scrambled siRNA. Subsequently, the cells were treated with the VCC oligomer incorporated into the asolectin–cholesterol liposomes (grey bars), and monitored further for TNFα secretion. Open bars represent the treatment with liposomes alone. Treatment with VCC oligomer with no siRNA pre-treatment was considered as the positive control. Results shown are means±S.E.M., and represent the average from three independent experiments.

Figure 4
Role of TLR2/TLR6 in the VCC oligomer-mediated, proinflammatory responses

(A) Association of the VCC oligomer with the TLR2/TLR6 heterodimer. RAW 264.7 and THP-1 cells were treated with VCC oligomer for 1 h and cell lysates were prepared. Samples were normalized for the protein content, immunoprecipitated (IP) with anti-TLR2 or anti-TLR6 antibodies, and subjected to immunoblotting (IB) for TLR1, TLR2, TLR6 and VCC as indicated. Cells treated with liposome alone served as the control. The data indicated a significant extent of increased association between TLR2 and TLR6 on stimulation with the VCC oligomer. No significant association was noticed between TLR1 and TLR2. The data also showed association of VCC oligomer with the TLR2/TLR6 complex. Results represent three independent experiments. (B) Neutralization of TLR2 and TLR6 suppressed VCC oligomer-induced TNFα secretion from THP-1 human monocytic cells (left panel) and monocytes purified from human PBMCs (right panel). Cells were treated with anti-TLR1-, anti-TLR2- or anti-TLR6-neutralizing antibodies, and were subsequently incubated with VCC oligomer incorporated into the asolectin–cholesterol liposome. Cell culture supernatants were collected and analysed for TNFα production. Cells treated with liposomes alone were taken as the negative control, whereas treatments with VCC oligomer without anti-TLR pre-treatment were considered as the positive control. The results shown are means±S.E.M., and represent the average from three independent experiments. (C) The siRNA-mediated knockdown of TLR2 and TLR6 suppressed VCC oligomer-induced TNFα secretion from THP-1 human monocytic cells. Cells were transfected with the TLR2 siRNA, TLR6 siRNA or scrambled siRNA. Subsequently, the cells were treated with the VCC oligomer incorporated into the asolectin–cholesterol liposomes (grey bars), and monitored further for TNFα secretion. Open bars represent the treatment with liposomes alone. Treatment with VCC oligomer with no siRNA pre-treatment was considered as the positive control. Results shown are means±S.E.M., and represent the average from three independent experiments.

We confirmed the role of TLR2 and TLR6 in VCC oligomer-mediated, proinflammatory responses by using anti-TLR-neutralizing antibodies. Pre-treatment with both anti-TLR2- and anti-TLR6-neutralizing antibodies caused more than a 5-fold reduction in TNFα production from the THP-1 monocytes on VCC oligomer treatment (Figure 4B, left panel). In contrast, the anti-TLR1-neutralizing antibody was found to block TNFα secretion only marginally (approximately 1.4-fold reduction) (Figure 4B, left panel). Anti-TLR2- and anti-TLR6-neutralizing antibodies also supressed TNFα secretion by VCC oligomer-treated monocytes purified from human PBMCs (Figure 4B, right panel).

In addition we used an siRNA-mediated knockdown strategy to establish the involvement of TLR2 and TLR6 further in the generation of the VCC oligomer-induced, proinflammatory response. First, knockdown of either TLR2 or TLR6 in the THP-1 cells by siRNA was verified by monitoring the cell-surface expression profile of the specific TLRs, using the flow cytometry-based assay (see Supplementary Figure S4). Knockdown of either TLR2 or TLR6 resulted in a profound decrease in TNFα secretion by THP-1 cells, on VCC oligomer treatment (Figure 4C).

Altogether, these results confirmed that the VCC oligomer activated the TLR2/TLR6 heterodimeric receptor complexes on monocytes/macrophages, and elicited proinflammatory responses in these cells via activation of the TLR2/TLR6-dependent pathways.

VCC oligomers activate MyD88, IRAK1 and TRAF6

Activation of TLRs is known to initiate downstream signalling pathways via recruitment of MyD88, which in turn activates IRAK1 and TRAF6 [46,50]. Therefore, to obtain detailed insights into the implications of the VCC oligomer-induced TLR2/TLR6 activation for the downstream signalling pathways, we wanted to explore the involvement of MyD88, IRAK1 and TRAF6 in the process.

A semi-quantitative RT-PCR assay showed that the VCC oligomers up-regulated MyD88 gene expression in RAW 264.7 cells (see Supplementary Figure S5A, left panel). Western blot analysis also confirmed increased expression of MyD88 in response to VCC oligomers, in both RAW 264.7 macrophages and THP-1 monocytes (Figure 5A). More importantly, VCC oligomers were found to trigger enhanced association of MyD88 with TLR2, as detected by the co-immunoprecipitation-based assay (Figure 5B). Our data therefore suggested that the VCC oligomers could engage the TLR2/TLR6 complex to initiate MyD88-dependent signalling in RAW 264.7 macrophages as well as in THP-1 monocytes. Consistent with such a notion, VCC oligomer exposure resulted in an increased expression of IRAK1 and TRAF6 in RAW 264.7 cells and THP-1 monocytes (Figure 5C and see Supplementary Figure S5A). Critical involvement of IRAK activation in the process could also be confirmed by the observation that the VCC oligomer-induced, proinflammatory responses (in terms of NO, TNFα and IL-6 production) in RAW 264.7 and THP-1 cells were suppressed on IRAK inhibitor treatment (Figure 5D).

VCC oligomer-induced up-regulation of MyD88 expression in RAW 264.7 and THP-1 cells

Figure 5
VCC oligomer-induced up-regulation of MyD88 expression in RAW 264.7 and THP-1 cells

(A) Cells were treated with VCC oligomer incorporated into asolectin–cholesterol liposomes, and cell lysates were probed for a MyD88 protein expression profile by immunoblotting. Immunoblotting for β-actin in the cell lysates served as the loading control. (B) The VCC oligomer triggered increased association of MyD88 with TLR2 in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomer, in asolectin–cholesterol liposomes, for 1 h; cell lysates were prepared and protein concentrations normalized. TLR2/MyD88 complex formation was assessed by immunoprecipitation (IP) with anti-TLR2 followed by immunoblotting (IB) with anti-MyD88. Immunoblotting with anti-TLR2 was used as the loading control. Cells treated with liposomes alone served as the control. Immunoprecipitation with a control isotype IgG followed by immunoblotting with anti-MyD88 could not detect any MyD88 signal, thus ruling out the possibility of any non-specific association of MyD88 during the immunoprecipitation assay (see Supplementary Figure S5B). (C) VCC oligomer-induced up-regulation of IRAK1 and TRAF6 expression in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomer and incorporated into the asolectin–cholesterol liposomes, and cell lysates were probed for an IRAK1 and TRAF6 protein expression profile by immunoblotting. Immunoblotting for β-actin in the cell lysates served as the loading control. (D) IRAK inhibition blocked VCC oligomer-induced, proinflammatory responses in RAW 264.7 and THP-1 cells. Cells were pre-treated with the IRAK inhibitor, followed by treatment with the VCC oligomers. Subsequently, the cells were analysed for NO, TNFα and IL-6 production in the cell culture supernatants.

Figure 5
VCC oligomer-induced up-regulation of MyD88 expression in RAW 264.7 and THP-1 cells

(A) Cells were treated with VCC oligomer incorporated into asolectin–cholesterol liposomes, and cell lysates were probed for a MyD88 protein expression profile by immunoblotting. Immunoblotting for β-actin in the cell lysates served as the loading control. (B) The VCC oligomer triggered increased association of MyD88 with TLR2 in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomer, in asolectin–cholesterol liposomes, for 1 h; cell lysates were prepared and protein concentrations normalized. TLR2/MyD88 complex formation was assessed by immunoprecipitation (IP) with anti-TLR2 followed by immunoblotting (IB) with anti-MyD88. Immunoblotting with anti-TLR2 was used as the loading control. Cells treated with liposomes alone served as the control. Immunoprecipitation with a control isotype IgG followed by immunoblotting with anti-MyD88 could not detect any MyD88 signal, thus ruling out the possibility of any non-specific association of MyD88 during the immunoprecipitation assay (see Supplementary Figure S5B). (C) VCC oligomer-induced up-regulation of IRAK1 and TRAF6 expression in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomer and incorporated into the asolectin–cholesterol liposomes, and cell lysates were probed for an IRAK1 and TRAF6 protein expression profile by immunoblotting. Immunoblotting for β-actin in the cell lysates served as the loading control. (D) IRAK inhibition blocked VCC oligomer-induced, proinflammatory responses in RAW 264.7 and THP-1 cells. Cells were pre-treated with the IRAK inhibitor, followed by treatment with the VCC oligomers. Subsequently, the cells were analysed for NO, TNFα and IL-6 production in the cell culture supernatants.

VCC oligomers trigger activation of NF-κB to mediate proinflammatory responses in RAW 264.7 macrophages and THP-1 monocytes

TLR/MyD88-dependent signalling pathways are known to generate proinflammatory responses in monocytes and macrophages via activation of the transcription factor NF-κB [46,5153]. Therefore, to explore the ability of VCC oligomer to activate NF-κB, we first examined the mRNA expression profile of NF-κB family members [RelA(p65), RelB, c-Rel, p50 and p52] in RAW 264.7 macrophages in response to the transmembrane oligomeric form of VCC. Semi-quantitative RT-PCR data showed that the VCC oligomer triggered up-regulation of all the NF-κB family members, whereas the most profound up-regulation was observed for RelA gene expression (Figure 6A).

VCC oligomers triggered activation of NF-κB

Figure 6
VCC oligomers triggered activation of NF-κB

(A) VCC oligomer triggered transcriptional up-regulation of NF-κB subunits: mRNA expression profile of NF-κB subunits in response to the VCC oligomer treatment (for 4 h) in RAW 264.7 cells. Cells were treated with VCC oligomers incorporated into the asolectin–cholesterol liposomes, and the target mRNA expressions were assessed using semi-quantitative RT-PCR. Fold changes were calculated for both the VCC oligomers (grey bars) and the respective liposome control (open bars) compared with the cells treated with just PBS. All quantifications were normalized to the housekeeping gene RPL13. Results are expressed as means±S.E.M. and represent the average from three independent experiments. (B, C) Nuclear translocation of NF-κB subunits p65 (RelA) and p50 triggered by the VCC oligomers in RAW 264.7 (B) and THP-1 (C) cells. Cells were treated with VCC oligomers in asolectin–cholesterol liposomes, and nuclear extracts were prepared and probed for NF-κB p65 and p50 by immunoblotting. Immunoblotting for lamin B was used as the loading control. (D) VCC oligomers triggered phosphorylation of IκB, as well as a decrease in the level of total IκB in RAW 264.7 cells. Cells were treated with VCC oligomer generated in asolectin–cholesterol liposome, and the cell lysate was analysed for IκB (total) and phospho-IκB by immunoblotting. GAPDH was also probed as the loading control.

Figure 6
VCC oligomers triggered activation of NF-κB

(A) VCC oligomer triggered transcriptional up-regulation of NF-κB subunits: mRNA expression profile of NF-κB subunits in response to the VCC oligomer treatment (for 4 h) in RAW 264.7 cells. Cells were treated with VCC oligomers incorporated into the asolectin–cholesterol liposomes, and the target mRNA expressions were assessed using semi-quantitative RT-PCR. Fold changes were calculated for both the VCC oligomers (grey bars) and the respective liposome control (open bars) compared with the cells treated with just PBS. All quantifications were normalized to the housekeeping gene RPL13. Results are expressed as means±S.E.M. and represent the average from three independent experiments. (B, C) Nuclear translocation of NF-κB subunits p65 (RelA) and p50 triggered by the VCC oligomers in RAW 264.7 (B) and THP-1 (C) cells. Cells were treated with VCC oligomers in asolectin–cholesterol liposomes, and nuclear extracts were prepared and probed for NF-κB p65 and p50 by immunoblotting. Immunoblotting for lamin B was used as the loading control. (D) VCC oligomers triggered phosphorylation of IκB, as well as a decrease in the level of total IκB in RAW 264.7 cells. Cells were treated with VCC oligomer generated in asolectin–cholesterol liposome, and the cell lysate was analysed for IκB (total) and phospho-IκB by immunoblotting. GAPDH was also probed as the loading control.

Among the NF-κB family members, the p65/p50 heterodimer represents the classic NF-κB heterodimer acting as the potent activator of gene expression, leading to generation of proinflammatory responses [46]. The p65/p50 heterodimer is retained in the cytoplasm in its inactive form as a complex with IκB. In response to the extracellular proinflammatory stimulus, IκB is phosphorylated and degraded via activation of signalling cascades involving TLR/MyD88/IRAK1/TRAF6. This results in the release of a functional p65/p50 complex, and its subsequent nuclear translocation [46]. Consistent with such a classic mechanism of NF-κB activation, VCC oligomer treatment triggered nuclear translocation of both p65 and p50 subunits in RAW 264.7 macrophages as well as in THP-1 monocytes (Figures 6B and 6C). Accordingly, we also observed increased phosphorylation of IκB, along with a decrease in the level of total IκB, in response to VCC oligomer exposure in RAW 264.7 cells (Figure 6D). Taken together, these data confirmed activation of NF-κB by the VCC oligomer.

Finally, we confirmed direct involvement of NF-κB activation in generating proinflammatory responses evoked by the VCC oligomer. For this, we monitored the effect(s) of the NF-κB inhibitor MLN4924 [54] on NO, TNFα and IL-6 production by RAW 264.7 cells, as well as TNFα and IL-6 production by THP-1 cells on VCC oligomer treatment. Pre-treatment with MLN4924 resulted in a decreased nuclear translocation of NF-κB(p65) in both RAW 264.7 and THP-1 cells, on activation by the VCC oligomer (Figure 7A). As shown in Figures 7B–7D, MLN4924 drastically suppressed proinflammatory responses, in terms of NO, TNFα and IL-6 production, by the VCC oligomer-treated RAW 264.7 macrophages and THP-1 monocytes. Similar results were also obtained when tested with another NF-κB inhibitor, PDTC [55] (see Supplementary Figure S6). Pre-treatment with MLN4924 resulted in decreased iNOS, TNFα and IL-6 mRNA levels in the RAW264.7 cells, on VCC oligomer treatment (Figures 7B–7D, right panels). These data therefore supported the notion that the VCC oligomer-induced proinflammatory responses did indeed involve NF-κB-mediated transcriptional activation of proinflammatory mediators such as NO, TNFα and IL-6.

VCC oligomer-mediated, proinflammatory responses in RAW 264.7 and THP-1 cells required activation of NF-κB

Figure 7
VCC oligomer-mediated, proinflammatory responses in RAW 264.7 and THP-1 cells required activation of NF-κB

(A) NF-κB inhibition by MLN4924 suppressed the VCC oligomer-induced nuclear translocation of the NF-κB subunit p65 in RAW 264.7 and THP-1 cells. Cells were pre-treated with MLN4924, followed by treatment with the VCC oligomers (for 1 h). Subsequently, nuclear extracts were prepared, and probed for NF-κB p65 by immunoblotting. Immunoblotting for lamin B was used as the loading control. VCC oligomer treatment without inhibitor pre-treatment served as the positive control. (BD) NF-κB inhibition by MLN4924 blocked the VCC oligomer-induced, proinflammatory responses in RAW 264.7 and THP-1 cells. Cells were pre-treated with MLN4924, followed by treatment with the VCC oligomers. Subsequently, the cells were analysed for NO [left panel in (B)], TNFα [left and middle panels in (C)] and IL-6 [left and middle panels in (D)] production in the cell culture supernatants. NF-κB inhibition by MLN4924 also blocked the VCC oligomer-induced, up-regulation of iNOS [right panel in (B)], TNFα [right panel in (C)] and IL-6 [right panel in (D)] mRNA levels in RAW 264.7 cells. Cells treated with VCC oligomers, without inhibitor pre-treatment served as the positive controls. Cells treated with liposomes alone served as the negative controls.

Figure 7
VCC oligomer-mediated, proinflammatory responses in RAW 264.7 and THP-1 cells required activation of NF-κB

(A) NF-κB inhibition by MLN4924 suppressed the VCC oligomer-induced nuclear translocation of the NF-κB subunit p65 in RAW 264.7 and THP-1 cells. Cells were pre-treated with MLN4924, followed by treatment with the VCC oligomers (for 1 h). Subsequently, nuclear extracts were prepared, and probed for NF-κB p65 by immunoblotting. Immunoblotting for lamin B was used as the loading control. VCC oligomer treatment without inhibitor pre-treatment served as the positive control. (BD) NF-κB inhibition by MLN4924 blocked the VCC oligomer-induced, proinflammatory responses in RAW 264.7 and THP-1 cells. Cells were pre-treated with MLN4924, followed by treatment with the VCC oligomers. Subsequently, the cells were analysed for NO [left panel in (B)], TNFα [left and middle panels in (C)] and IL-6 [left and middle panels in (D)] production in the cell culture supernatants. NF-κB inhibition by MLN4924 also blocked the VCC oligomer-induced, up-regulation of iNOS [right panel in (B)], TNFα [right panel in (C)] and IL-6 [right panel in (D)] mRNA levels in RAW 264.7 cells. Cells treated with VCC oligomers, without inhibitor pre-treatment served as the positive controls. Cells treated with liposomes alone served as the negative controls.

VCC oligomer-induced, proinflammatory response generation in RAW 264.7 macrophages and THP-1 monocytes does not involve MAPK p38 activation, but critically depends on JNK-dependent activation of AP-1

Apart from the activation of the canonical NF-κB pathway, the TLR/MyD88 signalling pathway has also been shown to activate MAPKs in the generation of the proinflammatory responses [46,56]. Therefore, in the present study, we wanted to explore the role(s) of the MAPKs, p38 and JNK, in the process of VCC oligomer-induced, proinflammatory response generation in RAW 264.7 macrophages and THP-1 monocytes.

A large number of studies have documented critical role(s) of MAPK p38 in mediating proinflammatory responses in macrophages/monocytes in response to the pathogen-derived factors including lipopolysaccharide (LPS) [56,57]. Accordingly, we investigated the implication of MAPK p38 activation for proinflammatory response generation in RAW 264.7 macrophages and THP-1 monocytes, on VCC oligomer treatment. For this, we monitored the effect of the MAPK p38 inhibitor SB202190 [58] on TNFα and IL-6 production by RAW 264.7 macrophages and THP-1 monocytes, in response to stimulation with the VCC oligomer. Our data showed that this did not significantly affect TNFα (Figure 8A) or IL-6 (Figure 8B) production by either RAW 264.7 or THP-1 cells, on treatment with the transmembrane oligomeric form of VCC. In contrast, LPS-induced TNFα (Figure 8A) and IL-6 (Figure 8B) production was found to be significantly inhibited by SB202190 in both cell types studied. Similar results were obtained when tested with another p38 inhibitor, VX 745 [59] (see Supplementary Figure S7). The data therefore suggested that the transmembrane oligomeric form of VCC triggered proinflammatory responses in terms of TNFα and IL-6 secretion from RAW 264.7 and THP-1 cells without critically requiring activation of the MAPK p38-mediated signal transduction pathways.

VCC oligomer-induced, proinflammatory response generation in RAW 264.7 and THP-1 cells did not appear to involve MAPK p38

Figure 8
VCC oligomer-induced, proinflammatory response generation in RAW 264.7 and THP-1 cells did not appear to involve MAPK p38

(A, B) The MAPK p38 inhibitor SB202190 did not affect (A) TNFα and (B) IL-6 production in RAW 264.7 mouse macrophages and THP-1 human monocytes in response to VCC oligomer treatment. The inhibitory effects of SB202190 on LPS-induced TNFα and IL-6 secretion from both cells were also monitored, as a control. Cells treated with liposomes alone served as the negative control.

Figure 8
VCC oligomer-induced, proinflammatory response generation in RAW 264.7 and THP-1 cells did not appear to involve MAPK p38

(A, B) The MAPK p38 inhibitor SB202190 did not affect (A) TNFα and (B) IL-6 production in RAW 264.7 mouse macrophages and THP-1 human monocytes in response to VCC oligomer treatment. The inhibitory effects of SB202190 on LPS-induced TNFα and IL-6 secretion from both cells were also monitored, as a control. Cells treated with liposomes alone served as the negative control.

The TLR/MyD88/IRAK1/TRAF6 signalling cascade is known to activate another MAPK family member, JNK, which in turn stimulates AP-1, a crucial transcription factor known to trigger transcriptional activation of the proinflammatory mediators in macrophages/monocytes [46,56,60]. In the present study, we observed VCC oligomer-induced activation of JNK by augmenting its phosphorylation in both RAW 264.7 and THP-1 cells (Figure 9A). To explore further the ability of the VCC oligomer to activate AP-1, we observed nuclear translocation of the AP-1 subunits c-Jun and c-Fos [61] in RAW 264.7 cells as well as in THP-1 cells, in response to VCC oligomer treatment (Figure 9B). Moreover, the JNK-specific inhibitor, JNK-IN-8 [62], blocked the nuclear translocation of c-Jun in RAW 264.7 cells, on VCC oligomer treatment (Figure 9C), thus confirming the role of JNK-mediated activation of AP-1 in response to VCC oligomer treatment. We further assessed the implication of JNK activation for the proinflammatory responses evoked by the VCC oligomer, using the inhibitor JNK-IN-8. We observed that JNK-IN-8 pre-treatment significantly affected NO (Figure 9D), TNFα (Figure 9E) and IL-6 (Figure 9F) production by RAW 264.7 macrophages, and TNFα (Figure 9E) and IL-6 (Figure 9F) secretion by THP-1 monocytes, on VCC oligomer treatment. Similar results were obtained when tested with another JNK inhibitor, SP600125 (see Supplementary Figure S8). Altogether, these data clearly showed that the transmembrane oligomeric form of VCC could activate MAPK family member JNK, which contributed towards generation of the proinflammatory responses in the macrophages and monocytes, presumably via activation of the transcription factor AP-1.

VCC oligomers induced activation of JNK and AP-1 to mediate proinflammatory responses from RAW 264.7 and THP-1 cells

Figure 9
VCC oligomers induced activation of JNK and AP-1 to mediate proinflammatory responses from RAW 264.7 and THP-1 cells

(A) VCC oligomers triggered phosphorylation of JNK in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomers, and cell lysates were probed for total JNK and phospho-JNK by immunoblotting. GAPDH and β-actin served as loading controls for RAW 264.7 and THP-1 cells, respectively. (B) VCC oligomers triggered nuclear translocation of AP-1 subunits c-Fos and c-Jun in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomers in asolectin–cholesterol liposomes, and nuclear extracts were prepared and probed for c-Fos and c-Jun by immunoblotting. Immunoblotting for lamin B was used as the loading control. (C) The JNK inhibitor JNK-IN-8 suppressed the VCC oligomer-induced nuclear translocation of c-Jun in RAW 264.7 cells. Cells were pre-treated with JNK-IN-8 followed by treatment with the VCC oligomers (for 1 h). Nuclear extracts were prepared and probed for c-Jun by immunoblotting. Immunoblotting for lamin B was used as the loading control. VCC oligomer treatment without inhibitor pre-treatment served as the positive control. (DF) JNK-IN-8 significantly suppressed VCC oligomer-mediated, proinflammatory responses in RAW 264.7 and THP-1 cells. Cells were pre-treated with JNK-IN-8 followed by treatment with the VCC oligomers. Subsequently, the cells were analysed for (D) NO, (E) TNFα and (F) IL-6 production in the cell culture supernatants. Cells treated with VCC oligomer without inhibitor pre-treatment served as the positive control. Cells treated with liposomes alone served as the negative control.

Figure 9
VCC oligomers induced activation of JNK and AP-1 to mediate proinflammatory responses from RAW 264.7 and THP-1 cells

(A) VCC oligomers triggered phosphorylation of JNK in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomers, and cell lysates were probed for total JNK and phospho-JNK by immunoblotting. GAPDH and β-actin served as loading controls for RAW 264.7 and THP-1 cells, respectively. (B) VCC oligomers triggered nuclear translocation of AP-1 subunits c-Fos and c-Jun in RAW 264.7 and THP-1 cells. Cells were treated with VCC oligomers in asolectin–cholesterol liposomes, and nuclear extracts were prepared and probed for c-Fos and c-Jun by immunoblotting. Immunoblotting for lamin B was used as the loading control. (C) The JNK inhibitor JNK-IN-8 suppressed the VCC oligomer-induced nuclear translocation of c-Jun in RAW 264.7 cells. Cells were pre-treated with JNK-IN-8 followed by treatment with the VCC oligomers (for 1 h). Nuclear extracts were prepared and probed for c-Jun by immunoblotting. Immunoblotting for lamin B was used as the loading control. VCC oligomer treatment without inhibitor pre-treatment served as the positive control. (DF) JNK-IN-8 significantly suppressed VCC oligomer-mediated, proinflammatory responses in RAW 264.7 and THP-1 cells. Cells were pre-treated with JNK-IN-8 followed by treatment with the VCC oligomers. Subsequently, the cells were analysed for (D) NO, (E) TNFα and (F) IL-6 production in the cell culture supernatants. Cells treated with VCC oligomer without inhibitor pre-treatment served as the positive control. Cells treated with liposomes alone served as the negative control.

DISCUSSION

The pathogenic functions of the bacterial β-barrel, pore-forming protein toxins (β-PFTs) are commonly attributed to their ability to disrupt the integrity of the target host cell membranes via formation of the transmembrane oligomeric pores [63]. The consequences of membrane pore formation by the β-PFTs are traditionally implicated in processes including direct killing of the target cells via colloid–osmotic lysis, as well as delivery of the toxic entities into the target cells. In a large number of cases, however, bacterial β-PFTs have been shown to trigger multiple cellular responses, the nature and extent of which depend on the particular β-PFT under consideration, and also on the target cell types studied [63,64]. In particular, cross-talk between the β-PFT molecules and the host innate immune cells has received much attention. It has been appreciated that β-PFTs can be recognized as specific pathogen-associated molecular patterns by the PRRs [such as TLRs and Nod-like receptors (NLRs)] present on the innate immune cells, thus triggering various cellular responses that include cytokine signalling and activation of the ‘so-called’ inflammasome complexes [6466], e.g. multiple members of the cholesterol-dependent cytolysin subfamily in the β-PFT family (such as perfringolysin O from Clostridium perfringens, pneumolysin from Streptococcus pneumoniae) act as the TLR4 agonists to activate the mouse macrophages [64,67]. Staphylococcus aureus α-haemolysin, another archetypical bacterial β-PFT, activates the NLRP3 inflammasome in human and mouse monocytic cells [68]. In the same fashion, VCC has previously been tested to evoke any such responses in the host innate immune cells, and it has been observed that VCC triggers potent apoptosis of mouse macrophages in a TLR-independent fashion [8]. In a separate study, it has also been shown that VCC can activate the NLRP3 inflammasome in mouse macrophages [69]. It is important to mention that most of the studies characterizing the interactions of the β-PFTs, including that of VCC, with the host innate immune cells have used the water-soluble, monomeric, cytotoxic form of the toxins. However, structural mechanisms for the β-PFT mode of action indicate that the final oligomeric assembly of the β-PFT molecules, owing to their remarkable structural stability, would be better suited as the target for the host immune system, compared with the monomeric form. Based on such a proposition, in the present study we tested the interaction of the transmembrane oligomeric form of VCC, as a prototype β-PFT family member, with the macrophages and monocytes of the innate immune system, and established the ability of VCC oligomers to elicit proinflammatory responses in macrophages and monocytes, elucidating the detailed pathways involved in such proinflammatory response generation.

Consistent with its property as a cytotoxin, the present study showed potent cell-killing activity of the monomeric form of VCC against the RAW 264.7 mouse macrophages and THP-1 monocytes. In contrast, the transmembrane oligomeric form of VCC did not induce any such cytotoxic activity in RAW 264.7 and THP-1 cells, even at a 10-fold higher protein concentration. Our data showed that the transmembrane oligomeric form of VCC possessed a potent ability to trigger production of the proinflammatory mediators NO, TNFα and IL-6 from the RAW 264.7 mouse macrophage cell lines, THP-1 monocytic cell lines and monocytes derived from human PBMCs. When subjected to VCC oligomer treatment, RAW 264.7 macrophages exhibited transcriptional activation of TLR2 and TLR6, but not of TLR1, TLR4 and TLR5. Consistent with such an observation, the VCC oligomer triggered increased surface expression of TLR2 and TLR6 on RAW 264.7 macrophages and THP-1 monocytes. More importantly, the VCC oligomer induced activation of the TLR2/TLR6-dependent signal transduction pathways in RAW 264.7 and THP-1 cells. It favoured engagement of the TLR2/TLR6 heterodimeric receptor complex, presumably via direct interaction with TLR2 and TLR6. The VCC oligomer-induced activation of the TLR2/TLR6 complex also allowed recruitment of the adaptor protein MyD88, to initiate the downstream signal transduction events via stimulation of IRAK1 and TRAF6. Once the VCC oligomer-mediated activation of the TLR2/TLR6 signalling processes had been established, we then explored the direct involvement of the TLR2/TLR6 receptors in the VCC oligomer-induced, proinflammatory signal generation of the target innate immune cells. For this, we blocked the activation of TLR2 or TLR6 via neutralizing antibodies. We also employed an siRNA-mediated knockdown approach to down-regulate TLR2/TLR6. In both cases, blocking or down-regulation of TLR2/TLR6 resulted in suppression of the proinflammatory responses from the THP-1 monocytes on VCC oligomer treatment. Our data therefore confirmed that the VCC oligomer-mediated, proinflammatory response generation depended critically on TLR2/TLR6-mediated signalling. VCC oligomer-induced, proinflammatory responses were also found to depend on the activation of NF-κB. VCC oligomer treatment was found to trigger activation of NF-κB in RAW 264.7 and THP-1 cells. Moreover, blocking of NF-κB activation resulted in suppression of the proinflammatory responses in these target cells, presumably due to compromised transcriptional activation of the proinflammatory mediators. It also appeared from our data that the MAPK family member, JNK, played a critical role in the process of VCC oligomer-mediated, proinflammatory response generation, presumably via activation of the transcription factor AP-1. Notably, MAPK p38 did not seem to have an essential role in the proinflammatory response generation by the VCC oligomer. This contrasts significantly with some of the previous observations, which show prominent involvement of p38 in the cellular responses triggered by the cytotoxic monomeric form of various β-PFTs, including that by the monomeric form of VCC [64,70]. At present, it remains unclear how VCC oligomer-mediated, proinflammatory response generation in monocytes/macrophages differentially engages the two MAPK family members JNK and p38.

Proinflammatory responses mediated by the transmembrane oligomeric form of VCC have been found to contrast significantly with the potent cytotoxicity caused by the cytolytically active monomeric form of VCC. The present study therefore suggests a potentially distinct outcome of exposing the two VCC forms to the host cells: (i) killing of the target host cells via formation of transmembrane oligomeric pores by the cytolytically active monomeric form of VCC (see Supplementary Figure S9A); and (ii) generation of potent proinflammatory responses via activation of the host immune cells, on being converted into its transmembrane oligomeric state (see Supplementary Figure S9B). Further studies are required to explore the physiological implications of such differential responses generated by the two structural variants of VCC in the context of the V. cholerae pathogenesis process.

AUTHOR CONTRIBUTION

Barkha Khilwani designed experiments, performed experiments and analysed the data. Arunika Mukhopadhaya designed experiments, analysed data and supervised the study. Kausik Chattopadhyay designed experiments, analysed data, supervised the study and wrote the manuscript.

FUNDING

This work was supported by a grant from the Department of Biotechnology (DBT), India (DBT grant BT/PR13350/BRB/10/751/2009) (to K.C.), and also through funding under the Centre of Excellence in the Frontier Areas of Science and Technology programme of the Ministry of Human Resource Development, Government of India, in the area of protein science, design and engineering (to K.C.). We acknowledge the University Grants Commission, India for a Research Fellowship (to B.K.). We also thank the Indian Institute of Science Education and Research Mohali for financial support.

Abbreviations

     
  • β-PFT

    β-barrel, pore-forming protein toxin

  •  
  • AP-1

    activator protein-1

  •  
  • APC

    allophycocyanin

  •  
  • GAPDH

    glyceraldehyde 3-phosphate dehydrogenase

  •  
  • IκB-α

    inhibitor of NFκB

  •  
  • IL

    interleukin

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • IRAK1

    IL-1-receptor-associated kinase 1

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MyD88

    myeloid differentiation factor 88

  •  
  • NF-κB

    nuclear factor-κB

  •  
  • NLR

    Nod-like receptor

  •  
  • NO

    nitric oxide

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PDTC

    ammonium pyrrolidinedithiocarbamate

  •  
  • PE

    phycoerythrin

  •  
  • PerC

    peritoneal cavity

  •  
  • PRR

    pattern recognition receptor

  •  
  • RT-PCR

    real-time PCR

  •  
  • TLR

    toll-like receptor

  •  
  • TNFα

    tumour necrosis factor-α

  •  
  • TRAF6

    TNF-receptor-associated factor 6

  •  
  • VCC

    Vibrio cholerae cytolysin

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