Interplay between Plasmodium falciparum haemozoin and l-arginine: implication for nitric oxide production

CellMask™ Plasma Membrane Stain and DAPI were obtained from Molecular Probes/Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Protein Extraction Buffer (RIPA Buffer) and protease inhibitor cocktail were obtained from Axxora (USA). Polyclonal anti-iNOS antibody was obtained from Cell Signaling (Danvers, MA, USA). Mouse monoclonal anti-actin antibody, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, and goat anti-mouse antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Filtration supplies were from Millipore^® (Milan, Italy). Unless otherwise stated, chemical reagents were obtained from Sigma-Aldrich (Milan, Italy), while cell culture media, serum and supplements, from EuroClone (Milan, Italy).

Immortalized mouse C57Bl/6 bone marrow derived macrophages (BMDM) from wild type lineage were generated and maintained in the growth medium, as previously reported [25, 26]. The basic composition of the growth medium used for BMDM consisted in Dulbecco’s minimal essential medium, DMEM, which contains 0.084 g/l of l-Arg, supplemented with l-glutamine (2 mM), HEPES (20 mM, pH 7.3), and 10% heat-inactivated fetal bovine serum (FBS) (South America origin EU Approved).

Mycoplasma-free P. falciparum parasites (strain 3D7) were maintained in vitro as previously described [27, 28]. For malaria cultures, fresh erythrocytes where obtained from AVIS Comunale Milano with the consent of healthy donors. The parasites were kept in culture at 5% haematocrit (human type 0 + erythrocytes) at 37 °C in RPMI 1640 medium supplemented with 10% heat-inactivated 0 + human plasma, 20 mM HEPES, pH 7.4, in a standard gas mixture consisting of 1% O₂, 5% CO₂, 94% N₂.

The methodology to isolate HZ has been standardized, applied and characterized in our previous report [28], with some modifications. Briefly, HZ was isolated using a LS column (MACS, Miltenyi^®) from P. falciparum cultures the day after parasitaemia reached > 5% trophozoites. Cultures at low hematocrit (~ 0.5%) were placed onto the column, and the free-HZ was isolated. By using the MACS-LS and low haematocrit, only free HZ was collected and not the parasitized erythrocytes. HZ was pelleted, resuspended in PBS, finely dispersed with a 1-ml syringe, and stored at 4 °C. To remove free haem and contaminating erythrocytes, HZ was extensively washed with PBS and hypotonic solution. The absence of free haem was confirmed by reading the OD at 405 nm (Soret band) in the supernatants obtained after HZ washing. Also, all HZ preparations were endotoxin-free as checked by the Limulus amoebocyte lysate-based assay, as previously described [28].

Stocks of HZ were made by pooling different preparations. Being HZ a crystal of haem molecules bound together, its molar concentration was calculated as haem equivalent, referring to the molecular weight of haem, as previously reported [28]. Briefly, aliquots from HZ pools were diluted in 1 M NaOH, in order to obtain the heme monomers in solution. A standard curve of hemin was also prepared in NaOH 1 M. The samples were read spectrophotometrically at OD 405 nm (Synergy 4, Biotek^® microplate reader) and the concentration of haem monomers in the HZ samples was extrapolated from the standard curve. The doses of HZ used for in vitro stimulations were chosen as relevant to biological conditions, and calculated considering a parasitaemia of 1–4% and the iron content of trophozoites, as reported earlier [28‐30].

BMDMs were incubated with HZ (100 µM) in a 35-mm glass bottom tissue culture dish (MatTek) for 24 h. Cell membranes and DNA were stained with CellMask (red) and DAPI (blue), respectively. Confocal reflection microscopy (to visualize HZ phagocytosed by BMDM) combined with fluorescence microscopy was used as described in detail in [26], on a Leica TCS SP2 AOBS confocal laser-scanning microscope.

BMDM were seeded and incubated overnight (96-well plates, 1 × 10⁵ cells/well). Next day, the cells were primed with 12.5 U/ml Type I Interferon-gamma (IFN-γ) for 2 h, or left untreated, and then stimulated with different concentrations of HZ (12.5, 25, 50, 100 µM). Muramyldipeptide (MDP) (2 µg/ml) or lipopolysaccharide (LPS) (200 ng/ml) were used as positive controls. After 24 h of incubation, the supernatants were collected and used for NO determination by the Griess reagent. These supernatants are referred as “24 h”. Fresh medium was then added and an extra incubation time of 24 h was carried out. The “24 h + 24 h” supernatants were collected and assayed for NO content. In some experiments, fresh medium was added again, further 24 h incubation was performed and the “24 h + 24 h + 24 h” supernatants collected for NO measurement by Griess reagent.

BMDM were seeded as explained above. Medium was replaced with fresh standard medium or with medium in which l-Arg was added to obtain ten-fold concentration of l-Arg (840 mg/l) (l-Arg 10×) with respect to the l-Arg content (84 mg/l) of the standard media formulation (DMEM). Respective media (standard medium or l-Arg 10×) were used for all the experimental steps described below. Cells were primed for 2 h with 12.5 U/ml IFN-γ, and then treated with medium, HZ (25 or 100 µM), MDP (2 µg/ml), or LPS (200 ng/ml) for 24 h or 24 h + 24 h. At each time point, the production of NO was determined by the Griess reagent.

To measure NO production, the indirect measure of the accumulation of nitrite in the cell supernatants was evaluated by the Griess reagent, a standard assay largely used in different models [28, 31‐33]. The Griess reagent consists of a mixture of equal parts of Reagent A (1% [w/v] sulphanilamide), and Reagent B (0.1% [w/v] naphthylethylenediamine dihydrochloride, and 2.5% [w/v] phosphoric acid). One hundred microliters of supernatants were mixed with an equal volume of Griess mixture and after 10 min of incubation at room temperature, the nitrite levels were measured by a spectrophotometer (Synergy 4 microplate reader, Biotek^®, GE) at 540 nm. To determine the levels of nitrite, a standard curve of NaNO₂ was prepared in parallel to the samples.

Medium alone or in the presence of HZ (200 µM) was incubated overnight at 37 °C under an atmosphere of 5% CO₂. HZ-treated medium was centrifuged (3000 rpm, 10 min), and supernatants were filtered (0.20 µm/PSFV). The presence of free haem was excluded spectrophotometrically, as described above. The HZ-treated medium will be referred as HZ-conditioned medium (HZ-CM). The medium alone (untreated conditioned medium, U-CM) received the same handling as the HZ-CM.

BMDM were seeded and incubated overnight, as described above. The medium was then replaced with U-CM or HZ-CM. Then, both priming with IFN-γ and the treatments with HZ (25 or 100 µM), or MDP (1 or 2 µg/ml) were performed in U-CM or HZ-CM. The plates were incubated for 24 h and the levels of nitrite were determined in the supernatants by the Griess reagent.

BMDM were seeded in 96-well plates (1 × 10⁵ cells/well), at 37 °C under an atmosphere of 5% CO₂. After 24 h, cells were primed or not with IFN-γ 12.5 U/ml for 2 h, and then stimulated with medium, HZ (25 or 100 μM), MDP (2 μg/ml), or tert-butyl hydrogen peroxide (t-BHP, 10 µM). After 0.5, 18 or 48 h, cells were washed with PBS, and then the levels of reactive oxygen species (ROS) were measured by treating stimulated BMDM for 15 min with 50 µl of 2′7′-dichlorofluorescein diacetate (H₂DCFDA, 15 µM). Then, the cells were washed with PBS, and 200 µl PBS were added to each well for 30 min; 150 µl of supernatants were transferred into a flat-bottom black plate, and ROS were measured, and recorded as fluorescence units (FU) (Ex/Em:485 nm/535 nm; Synergy 4, Biotek^®).

BMDM were plated in 6-well plates (3 × 10⁶ cells/well), at 37 °C under an atmosphere of 5% CO₂. At the end of an overnight incubation, the cells were primed or not with IFN-λ for 2 h and then treated with medium, HZ (25 or 100 μM), or MDP (5 μg/ml). The plates were incubated for 6 h, 24 h, or 24 h + 24 h. After each time point, cells were washed several times in ice-cold PBS, lysed in RIPA buffer (50 mM TRIS–HCl [pH 7.5], 105 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] and 2 mM ethylenediamine tetraacetic acid [EDTA]) and protease inhibitor cocktail, and incubated on ice for 15 min. The lysate was centrifuged at 15,000 rpm for 15 min at 4 °C. Equal amounts of proteins of each sample supernatant (50 µg), determined by Bradford method [34], were separated by electrophoresis on 7.5% SDS–polyacrylamide gel (SDS-PAGE). Electrophoresed proteins were transferred onto PVDF membranes (Bio-Rad, Richmond, CA, USA) with the Bio-Rad Transfer Blot Apparatus at 150 mA for 16 h at 4 °C using 25 mM Tris HCl, 190 mM glycine, 20% methanol, and 0.05% SDS as transfer buffer. The membranes were then blocked for 2 h and 30 min in blocking buffer (TTBS: 0.5% Tween-Tris-buffered-saline) with 5% bovine serum albumin (SIGMA, St. Louis, MO, USA)]. The blots were incubated with the specific primary antibody (mouse polyclonal anti-iNOS antibody), and mouse monoclonal anti-Actin antibody for 16 h at 4 °C in blocking buffer, washed 6 times for 5 min each in TTBS and incubated with the proper secondary horseradish peroxidase (HRP)-linked IgG antibodies (donkey anti-rabbit and goat anti-mouse), at room temperature in blocking buffer. After 1 h incubation, the membranes were washed and incubated again in TTBS, and finally developed with the ECL western blotting detection reagents (GE Healthcare Life Sciences, UK) following the instructions of the manufacturer. Immunoreactive proteins were visualized by autoradiography on Amersham Hyperfilm™ ECL™ (GE Healthcare Life Sciences, UK).

Quantitative analyses of immune reactive proteins were performed by digital scanning (CAMAG VideoStore 3.00.0.06 serial n° 0607C003 and VideoScan TLC/HPTLC Evaluation Software version 1.01.00 serial n° 0607D003).

A quantitative analysis of l-Arg in the medium was done through the liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique described elsewhere [35]. Briefly, standard medium (MED) or medium seven times more concentrated in l-Arg than standard medium (MED l-Arg 7×) were prepared as controls. A final concentration of 200 μM of HZ was prepared as HZ-CM in standard culture medium or in MED l-Arg 7× (HZ-CM l-Arg 7×). l-Arg (40 ng/ml) was used as control.

The time retention values, converged in the peak areas (area), were evaluated for each sample. Area values were determined for an internal control of l-Arg (40 ng/ml), medium (untreated conditioned medium, U-CM), medium treated with HZ 100 μM (haemozoin-conditioned medium, HZ-CM), medium enriched with l-Arg (U-CM l-Arg 7×), or HZ-CM enriched with l-Arg (HZ-CM l-Arg 7×). Before the chromatographic analyses, samples were prepared and incubated at 37 °C for 24 h under an atmosphere of 5% CO₂. Next, media were centrifuged and filtered (0.20 µm/PSFV). The area corresponding to the content of l-Arg exhibited by each treatment was retrieved. The mobile phases used are displayed in Table 1.

The instrument conditions were as follows: capillary temperature 275 °C; spray voltage 5.00 kV, sheath gas flow rate 60 (arb); auxiliary gas flow rate 15 (arb). Numbers in columns (“Phase A” and “Phase B”) represent the percentage (%) of the respective eluent.

Differences between groups were analyzed by one-way or two-way ANOVA analysis and post-hoc multiple comparisons tests (Bonferroni, Sidak, Tukey), using the software GraphPad Prism 6. Data are representative of at least three independent experiments in triplicate.

BMDM were treated for 24 h with HZ, which was readily phagocytosed, as evaluated by confocal microscopy (Fig. 1). Priming of BMDM with IFN-γ, shown to be required for the production of nitric oxide (NO) upon HZ stimulation, was always performed before treatment with different concentrations of HZ [28]. Detectable levels of NO were observed at 24 or 48 h but not at 6 h of incubation, therefore the majority of the experiments were done at 24 h. The nitrite levels released into the supernatants were evaluated as a measure of NO production. Figure 2a shows that doses of HZ up to 25 µM linearly increased NO production, but, unexpectedly, higher doses, from 50 to 100 µM, induced an inverse dose–response.

To exclude that the decrease in nitrite levels was due to a possible oxidation of nitrite to nitrate by HZ, in some experiments, all nitrate were converted into nitrite and the total nitrite levels in the supernatants were measured using the Griess reagent. The results were comparable to those obtained measuring only nitrite (Additional files 1, 4), indicating that the lower amount of nitrite observed with high doses of HZ was not due to the oxidation of nitrite to nitrate.

It has been shown that ROS can modulate NO production [36], and that both HZ [37] and βH, the synthetic HZ, induce ROS [38]. Thus, to investigate whether oxidative stress could be involved in the lower levels of NO found in the supernatants of BMDM treated with increasing concentrations of HZ, ROS release by primed or unprimed BMDM was tested. The lowest (25 µM) and the highest dose (100 µM) of HZ were chosen from the previous experiments. In addition, cells treated with medium only or activated by control agents such as MDP, a bacterial cell walls component [39], or tert-butyl hydrogen peroxide (t-BHP) were included. Tert-butyl hydrogen peroxide was used as a positive control since it is an organic peroxide, able to induce ROS formation to levels similar to hydrogen peroxide, but with less cellular toxicity [40]. As shown in Fig. 2b, at 0.5 h HZ induced low but significant levels of ROS only at 100 µM, but not at 25 µM. At 18 h, HZ induced a dose-dependent increase of ROS. This activity was independent of priming, as there were no differences in unprimed or primed cells. In addition, MDP or t-BHP stimulated the production of ROS in the same manner at 18 h (Fig. 2b). After 48 h incubation, the levels of ROS induced by all stimuli did not change significantly compared to 18 h, indicating that ROS produced from activated BMDM reached a plateau concentration already at 18 h. Collectively, these data indicated that, at all time points, HZ induced ROS in a dose-dependent manner. This result does not reflect the inverse dose–response of NO production induced by HZ, reported in Fig. 2a.

It has been described that free haem can compromise the transport of l-Arg in the erythrocytes [23]. To exclude a possible involvement of free haem in the regulation of NO production, HZ was extensively washed and the presence of free haem, as well as of haemoglobin, was measured and excluded in any preparations used.

Since l-Arg is a substrate also for Arginase-1, the activation of this enzyme by HZ was also excluded. The levels of urea, the main product of arginase activity, in cellular extracts from macrophages treated with HZ were comparable to those observed in untreated cells (Additional files 2, 4). In addition, in murine macrophages, arginase-1 is known to be regulated by Th2 cytokines, such as IL-4, IL-10, and IL-13 [41‐43], whereas IFN-γ is needed for iNOS up-regulation. In our mouse macrophages model IFN-γ was used for iNOS induction and thus for NO release from treated macrophages.

To investigate whether HZ, according to the concentration used, induces iNOS expression in BMDM, the levels of iNOS were assessed at different time points by western blotting. As expected, unprimed macrophages did not express iNOS at any time point tested, while primed cells expressed low levels of iNOS even in the absence of a stimulus (Fig. 3). Primed macrophages treated for 6 h with HZ at 25 or 100 µM expressed high iNOS levels, comparable to those induced by MDP. The levels of iNOS peaked at 6 h, with a progressive decrease at the additional time points. At 24 h and at 24 h + 24 h, after washing and medium replacement, HZ induced iNOS in BMDM only at the dose of 100 μM. Levels of iNOS expression in cells treated with MDP were higher than medium at both 6 and 24 h, but disappeared at 24 h + 24 h. Overall, the iNOS expression in BMDM stimulated with HZ followed a kinetic similar to that induced by MDP (i.e. decrease in iNOS expression over time) and was dose-dependent. This is at variance with what observed for the NO levels and indicates that the production of NO by BMDM stimulated with HZ does not reflect the levels of iNOS.

To investigate the inverse relationship between the doses of HZ and NO production, primed BMDM were treated with medium, different concentrations of HZ, or MDP for 24 h. At the end of the incubation, the supernatants were collected and used for NO determination. Fresh medium was then added to both stimulated and unstimulated cells. One or two extra incubation times of 24 h each where then carried out, the supernatants recovered and assayed for NO content. As depicted in Fig. 4, the levels of NO released during the first 24 h were inversely related to the HZ dose, whereas NO induced by MDP was significantly higher than medium. Surprisingly, and opposite to what observed after the first 24 h, the levels of NO detected in the 24 h + 24 h supernatants increased considerably compared to the first 24 h and were proportional to the dose of HZ used. Moreover, in the supernatants recovered at the end of the following 24 h of incubation (i.e. 24 h + 24 h + 24 h), the levels of NO were lower than at 24 h + 24 h, but retained a direct proportionality with the dose of HZ. The release of NO by cells treated with MDP at both time points was comparable to that at 24 h. It seems thus that medium replacement after the first 24 h of stimulation of HZ restores the dose–response for NO production, which seems to peak after 24 h + 24 h and decreases in the following 24 h.

The observation that media replacement after 24 h restored the dose-dependent production of NO by HZ-treated BMDM and that iNOS was not a limiting factor led to the hypothesis that the availability of l-Arg, the substrate of iNOS, could be low or reduced in the groups where HZ was also present. To verify this hypothesis, the experiments were repeated by stimulating BMDM using an l-Arg-enriched medium in which l-Arg was added to reach final concentration ten-fold higher than that of the standard medium (l-Arg 10×). The levels of NO were evaluated both after 24 h and after medium change at 24 h + 24 h (Fig. 5a). In all cases, the stimulation of primed BMDM with medium, HZ (25 or 100 µM), MDP, or LPS led to an increase of NO production. Using medium with l-Arg 10×, a further increase of NO production was seen in all groups, and, in particular, in the sample treated with 100 µM of HZ, which did not induce NO in the standard medium. The dose of HZ was positively correlated with the production of NO, and opposite to what observed in normal medium conditions. Therefore, it appears that an excess of l-Arg in the medium restores the production of NO induced by high HZ doses (100 µM), indicating a possible interaction between l-Arg and HZ.

To investigate the ability of HZ to directly reduce l-Arg availability, a HZ treated-conditioned medium (HZ-CM) was prepared, as described in the “Methods” section. Untreated conditioned medium (U-CM) was also prepared. BMDM were treated with different concentrations of HZ or MDP either in U-CM or HZ-CM. The production of NO by both doses of HZ or MDP was abolished when the stimuli were prepared in HZ-CM, but not in U-CM (Fig. 5b). Being l-Arg the substrate of iNOS, these results suggested that HZ might interact with l-Arg, present in the culture medium, depleting the medium of the substrate for iNOS, thus reducing NO production.

To measure the effective presence of l-Arg, U-CM and HZ-CM were analysed by LC–MS/MS. A control of l-Arg (40 ng/ml) was run on the column (Table 2), exhibiting a peak area of 384,603. The peak area of U-CM, which contains 40 ng/ml of l-Arg, was 501,886. In contrast, in the HZ-CM no l-Arg was detected (Table 2). To better quantify the amount of l-Arg that interacted with HZ, a preparation of HZ-CM and U-CM was made using a medium containing l-Arg 7 times more concentrated than in normal medium (HZ-CM l-Arg 7× and U-CM l-Arg 7×, respectively). The samples were then run in a LC–MS/MS column and it was found that the peak area exhibited by U-CM l-Arg 7× was 1.7 times higher than HZ-CM l-Arg 7×.