Malaria is a life-threatening disease caused by the Plasmodium
]. Globally, approximately 216 million cases of malaria were reported in 2016 with an estimate of 445,000 deaths [1
]. Upon parasite (called sporozoite) transmission to humans through the bites of infected mosquitoes, the parasites distribute via the bloodstream to the liver. Within the liver, each sporozoite multiplies into thousands of merozoites that reach back into the bloodstream, where they infect red blood cells for further replication. Parasite replication occurs in a cyclic fashion within red blood cells. During this erythrocytic cycle, the parasite degrades hemoglobin into amino acids and heme inside the digestive vacuole, where the heme monomer is further oxidized into a toxic inert biocrystalline form called malarial pigment or hemozoin (HZ) [2
]. Upon red blood cell rupture, HZ as well as other parasite toxins including Plasmodium
DNA and glycosylphosphatidylinositol are released into circulation and recognized by pattern recognition receptors expressed on phagocytes and other immune cells in the blood and tissues [5
]. This erythrocytic cycle is responsible for most of the pathological symptoms of malaria such as fever through the induction of a pro-inflammatory pyrogenic immune response [6
Among the phagocytes, neutrophils are the first line of defense to respond to pathogens by generating reactive oxygen species, and antimicrobial peptides and proteases, or by neutrophil extracellular trap (NET) formation [7
]. Studies have linked neutrophil activation and circulating NETs to the pathogenesis of malaria, including parasite sequestration in the microvasculature and endothelial dysfunction, resulting in impaired tissue perfusion and organ dysfunction [9
]. A previous report demonstrated the presence of NETs in children with uncomplicated falciparum malaria with parasites trapped within NETs [12
] and in malaria patients with severe disease [13
]. However, there is a paucity of data on the direct interaction of HZ crystals and neutrophils, but the severity of malaria is associated with the clearance capacity of circulating HZ crystals by neutrophils [15
]. HZ can interact with serum/plasma molecules such as proteins, lipids, and DNA, even before they encounter immune cells [16
]. Whether human plasma can affect the HZ crystal clearing capacity by neutrophils is currently not known. We hypothesized that human plasma proteins would impair the ability of neutrophils to internalize HZ crystals.
2. Material and Methods
2.1. Isolation of Human Blood Neutrophils
Blood from human healthy individuals was collected in S-Monovette with lithium heparin (Sarstedt, Germany), and plasma was separated and neutrophils were isolated using standard dextran sedimentation followed by Ficoll–Hypaque density centrifugation procedures [18
]. Neutrophils were suspended in Roswell Park Memorial Institute (RPMI) medium (0.5 × 105
cells/200 µL or 2.5 × 105
cells/mL) and seeded into 96-well or 24-well plates in a 5% carbon dioxide atmosphere at 37 °C for 30 min before stimulation. The study to obtain whole blood samples from healthy volunteers was approved by the local Ethical Review Board of the Medical Faculty at the Hospital of the Ludwig-Maximilians-University (LMU) Munich. Informed consent was obtained from all subjects.
2.2. Fluorescence Microscopy of Hemozoin Uptake
Human blood neutrophils were cultured ex vivo in the presence or absence of synthetic HZ (50 and 100 µg/mL, Invivogen, San Diego, CA, USA) in RPMI medium with or without 10%, 30%, or 50% human plasma in 8-well chamber slides (7.5 × 105 cells/well, Nunc Lab-Tek, Sigma-Aldrich, Germany) for 1, 2, and 18 h. After incubation, cells were fixed using 4% paraformaldehyde for 10 min at room temperature, washed twice with Dulbecco’s Phosphate-Buffered Saline (D-PBS), and stained with phalloidin green for 40 min (165 nM, Sigma-Aldrich, indicates actin filaments). After membrane staining, cells were mounted with 4′,6-Diamidin-2-phenylindol (DAPI) (Sigma-Aldrich, indicates cell nuclei). The uptake of HZ by neutrophils was visualized using a Leica TL Light-emitting diodes (LED) fluorescence or confocal microscope (Leica, Wetzlar, Germany).
2.3. Uptake of Hemozoin in Neutrophils Using Flow Cytometry
Human blood neutrophils were cultured ex vivo in 96-well plates (0.5 × 105 cells/200 μL) in the presence or absence of HZ (50 and 100 µg/mL) or silica crystals (200 μg/mL, Sigma-Aldrich, Germany) in RPMI medium without or with 10%, 30%, or 50% human plasma for 1, 2, and 18 h. In some experiments, cytochalasin D (10 µM, Sigma-Aldrich) was used to block phagocytosis of HZ crystals. To look at the effect of plasma proteins, HZ crystals were pre-incubated with or without fibrinogen (0.5 mg/mL, Sigma-Aldrich), albumin (3.25 mg/mL, Bethyl Labs, Montgomery, AL, USA), or Ringer’s solution (30%, negative control, Fresenius Kabi, Germany) for 30 min prior to stimulation with neutrophils. After stimulation, culture supernatants were collected and stored at −20 °C until further use, and cells harvested to quantify the percentage of cells that had internalized HZ crystals (HZ crystal+ neutrophils) were determined by flow cytometry using the BD FACSCalibur flow cytometer and FlowJo v7 software (Tree Star, Ashland, OR, USA).
To confirm intracellular uptake of HZ by neutrophils, human neutrophils were cultured with or without HZ (50 and 100 µg/mL) in RPMI medium for 2 h and then stained with the pHrodo red acetoxymethyl (AM) intracellular pH indicator (Thermo Fisher Scientific, Germany) for flow cytometry analysis as per the manufacturer’s protocol. An increase in the mean fluorescence intensity (MFI) of pHrodo red indicates an intracellular pH drop following HZ uptake.
2.4. Preparation and Stimulation of Neutrophils with Platelet-Poor Plasma
Human blood was drawn and standard dextran sedimentation performed to remove red blood cells. The top layer was removed and transferred into a 15 mL FALCON tube prior to centrifugation at 1500 g for 15 min. Using a new transfer pipette, the top layer was transferred into a new 15 mL FALCON tube and centrifuged at 1500 g for 15 min. After centrifugation, the top ¾ of plasma was removed using a transfer pipette and transferred into a new 15 mL FALCON tube. Isolated human blood neutrophils were then cultured in the presence or absence of synthetic HZ (50 and 100 µg/mL) in RPMI medium with or without 10%, 30%, or 50% human platelet-poor plasma for 2 h. After stimulation, neutrophils were either stained for fluorescence microscopy or harvested for flow cytometry to determine the percentage of HZ crystal+ neutrophils.
2.5. Morphological and Ultrastructural Analysis by Electron Microscopy
Blood neutrophils from healthy individuals were isolated and cultured (6.4 × 105 cell/mL) ex vivo in the presence or absence of HZ (50 and 100 µg/mL) in RPMI medium with or without 30% human plasma for 2 h, and processed for scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
For SEM, the samples were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 at 4 °C for 24 h. Afterwards, the samples were adhered to poly-l-lysine-coated glass slides, dehydrated with increasing concentrations of ethanol (30% to 100%), followed by critical point drying in carbon dioxide to remove any water trace. Samples were then mounted on a stub and coated with gold. The analysis was carried out on a FEI Scios SEM (Hillsboro, OR, USA).
For TEM, the samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C for 24 h. Post-fixation was performed using a solution of 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 10.0 mM CaCl2 in 0.1 M cacodylate buffer for 1 h. Afterwards, samples were dehydrated in an increasing acetone gradient (30% to 100%) and embedded in Polybed 812 resin. Ultrathin sections were obtained, mounted on copper grids, and stained with uranyl acetate and lead citrate. Analysis was performed on a JEOL JM 1400 TEM (Jeol, Tokyo, Japan).
2.6. Statistical Analysis
Statistical analysis was performed using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA). Data were compared either by one-way ANOVA with Tukey’s post-hoc test to calculate significance between three or more groups, or two-way ANOVA with Bonferroni’s comparison post-hoc test was carried out when using two parameters with multiple groups. Data are presented as mean ± SD. Differences were considered significant if p < 0.05. ns indicates not significant. Sample sizes are indicated in each corresponding figure legend.
We hypothesized that human plasma would alter the ability of neutrophils to internalize malaria-related HZ crystals. Indeed, our ex vivo data revealed that the HZ internalization by neutrophils occurred via endocytosis into phagosomes as well as into vesicles/vacuoles (Figure 5
E). Interestingly, plasma proteins including fibrinogen impaired this uptake process, whereas the presence of platelets enhanced it. These findings highlight the importance of factors regulating neutrophil endocytosis in vivo that are frequently ignored in ex vivo studies.
Neutrophils play an important role in the pathogenesis of malaria via processes including Plasmodium
parasite killing and NET formation [11
]. Recent reports have shown that heme, a known malaria danger-associated molecular pattern released during parasite egress, robustly induces NETs but not infected red blood cells, merozoites, and digestive vacuoles containing HZ, in tumor necrosis factor (TNF)-α-primed human neutrophils [14
]. We found that human blood neutrophils internalized HZ in phagosomes and vesicles/vacuoles, which triggered morphological abnormalities without leading to cell membrane rupture and NET release. This suggests that although HZ does not directly induce NETs [14
], they are known to significantly contribute to immune activation in other immune cells [5
]. In contrast, very small nanoparticles (10 to 40 nm in size) and larger crystalline particles such as monosodium urate, calcium phosphate, cholesterol, and calcium oxalate crystals can induce mixed lineage kinase domain-like protein (MLKL)-driven neutrophil necroptosis and NET formation [19
]. However, it is possible that neutrophils such as monocytes and macrophages remain viable after ingestion of HZ crystals, and that lysosome formation and acidification is normal [23
], although HZ degradation might be impaired due to the inability of the lysosome to depolymerize HZ crystals. Thus, HZ can reside in these cells for long periods of time, but repeated phagocytosis or oxidative burst for further Plasmodium
parasite killing is impaired [4
], suggesting a state of sequestration of activated neutrophils [26
]. Unresponsiveness of neutrophils in malaria accounts for increased susceptibility toward bacterial co-infections [27
The process of endocytosis is characterized by polymerization of actin filaments and fusion of phagosomes with lysosomes to form phagolysosomes in macrophages [29
] and human monocytes [30
]. Unlike macrophages and monocytes, neutrophils do not form classical phagolysosomes and instead contain a large number of preformed granules that can rapidly fuse with phagosomes upon internalization of pathogens or larger amounts of particles [31
]. Our data showed that human neutrophils ingested larger HZ crystal masses via direct uptake (phagocytosis) into phagolysosomes, whereas single and smaller HZ crystals might be internalized into vesicles/vacuoles via a different endocytotic uptake mechanism known as pinocytosis, due to the small size of HZ (0.1–1 µm). Previous studies have shown that pinocytosis does not require actin-dependent engulfment of small particles, for instance, zymosan, nanoparticles, or latex beads, by neutrophils [32
], macrophages and endothelial cells [33
], and non-phagocytic cells [34
]. However, further studies are needed to confirm the clearance of HZ by neutrophils via pinocytosis.
The role of HZ-binding proteins in the recognition, immune modulation and physiological clearance of HZ in neutrophils remains to be elucidated. We report for the first time that human plasma from healthy individuals, specifically fibrinogen, impairs the ability of neutrophils to ingest HZ but not silica crystals ex vivo. This is in line with previous reports showing that blood proteins such as apolipoprotein E, serum amyloid A, LPS binding protein, complement factor H, albumin, and fibrinogen that were found to be elevated in malaria individuals are able to bind to HZ [16
]. Hence, HZ-binding proteins alter the recognition of HZ as a danger signal for neutrophil clearance. The in vivo relevance of these findings remains to be proven, as the hematin-core crystal in HZ may remain shielded from serum proteins by the surrounding membranes/lipids [26
It is known that circulating neutrophils and platelets interact during infection including malaria, inflammation, and thrombosis, and that they can modulate each other’s functions [35
]. In malaria patients, circulating platelets and platelet-bound neutrophils are reduced, hence these complexes are either lost, the neutrophils migrate to tissues, or they form NETs [13
]. Our ex vivo data showed that the ability of neutrophils to clear HZ crystals even further decreases in platelet-poor plasma compared to normal human plasma. This may imply that neutrophils require platelets for HZ clearance. Previous reports have shown that activated platelets can initiate or amplify various neutrophil responses including phagocytosis, production of oxygen radicals, and NET formation. Such responses are initiated either by a direct contact or by the release of soluble mediators such as chemokine (C-C motif) ligand 5 (CCL5) and platelet factor 4 [37
]. In addition, platelet interactions enhance the phagocytic capacity of neutrophils towards various bacteria in vitro [39
]. Conversely, neutrophils can also release soluble mediators such as cathepsin G and elastase that augment platelet responses by activation of protease-activated receptors on platelets [42
]. However, further studies are needed to investigate the crosstalk between HZ-mediated platelet activation and HZ crystal uptake by neutrophil.
Limitations of our study are that we lack access to plasma from malaria-infected patients to investigate the impact of malaria-related plasma proteins on the phagocytic capacity of neutrophils to ingest HZ. As mentioned above, many plasma proteins that can bind to HZ have been identified in malaria patients [16
], and it is possible that besides fibrinogen, other plasma proteins may alter the uptake of HZ by neutrophils. Furthermore, the role of HZ in modulation of host innate and inflammatory responses has been investigated using different HZ preparation protocols. HZ can be synthesized from hematin (sHZ) or natural HZ (nHZ), or digestive vacuoles containing hemozoin can be purified from infected red blood cells in culture [27
]. We used synthetic HZ and not nHZ or digestive vacuoles of Plasmodia
for our ex vivo cell culture experiments. Although sHZ and nHZ crystals are similar in size, and capable of inducing the same level of inflammation, sHZ with a smaller or larger crystal size may differently affect the function of neutrophils.