Abstract
Objective
Malaria remains an important life-threatening disease that provokes a complex immune response, marked by an initial immune boost followed by long-term suppression, warranting further study. One of the manifestations of compromised immune response is co-infections, which are frequently reported in malaria patients and post-malaria convalescent individuals. Monocyte motility is a crucial step in immune cell recruitment, but this process is not fully efficient in malaria. Malarial pigment hemozoin, avidly phagocytosed by monocytes, inhibits important monocyte functions through lipoperoxidation.
Methods
4-Hydroxynonenal (4-HNE) adducts were detected on primary human monocytes by fluorescent microscopy, and CCR2 and TNFR1/2 receptors by flow cytometry. A two-dimensional migration microfluidic assay was applied for studying monocyte motility.
Results
We show here that, following hemozoin ingestion, monocyte motility is inhibited. This is accompanied by a 2.4 ± 0.3-fold increase of the adducts of the lipoperoxidation product 4-HNE with monocyte proteins. Reduction of cell directional motility by 3.2 ± 0.6 times in response to MCP-1 and by 3.8 ± 1.1 times in response to TNF-α is observed alongside a decrease in CCR2 expression by 55 ± 8%, TNFR1 (CD120a) expression by 79 ± 4% and TNFR2 (CD120b) expression by 58 ± 7%.
Conclusions
The low availability and potential malfunction of these important chemotactic receptors could be proposed as an additional mechanism for the poor immune response in malaria. Therapeutic relevance could be found in interventions aimed at regulating damaged or downregulated receptors and the application of antioxidants or other reagents to contrast protein addition of free 4-HNE.
Significance statement
The study deepened our knowledge about lipid peroxidation processes, which are related to infectious diseases and immunity. Observed impaired immune motility and collapse of receptors could explain immunosuppression manifestations and co-infections in malaria and other diseases accompanied by oxidative stress.
Introduction
Malaria is still a very important deadly infectious disease, with recent increases in case numbers in several countries (World Health Organization 2023), which have suffered humanitarian and health emergencies due to natural disasters, the recent pandemic and global (geo)political instability. Hemozoin (HZ) is a malarial pigment formed by the Plasmodium parasite during the digestion of hemoglobin in infected red blood cells (RBCs). Oxidatively harmful heme molecules are clustered into HZ nanocrystals. HZ accumulates progressively in the growing parasite and is released in the circulation along with multiple merozoites, which are prompted to invade next RBCs. As circulating phagocytes, monocytes are able to actively capture infected RBCs or HZ, which is released from destroyed RBCs during reinfection. This process has been demonstrated in both clinical malaria cases and malaria models (Metzger et al. 1995, Nguyen et al. 1995, Gallo et al. 2012, Birhanu et al. 2017, Mihu et al. 2024). Numerous experimental techniques have been developed to visualize and quantify phagocytosed HZ: microscopy, luminescence (Schwarzer et al. 1994), NMR (Di Gregorio et al. 2020), fluorescent-phase contrast flow cytometry (Rebelo et al. 2013), etc. When studied in vitro, experimental models of malarial phagocytosis were established, and the phagocytosed HZ was dosed in similar concentrations as HZ observed in malaria patients in the circulating monocytes (Lyke et al. 2003, Moiz & Ali 2016). During HZ phagocytosis, strong production of reactive oxygen species (ROS) has been observed (Jaramillo et al. 2005, Barrera et al. 2011) due to the oxidative burst generated by monocytes (Dupré-Crochet et al. 2013). Once inside the phagocytes, HZ continues to produce ROS and non-enzymatically triggers lipid peroxidation (Schwarzer et al. 1996, Schwarzer et al. 2015). One highly active final product of lipid peroxidation is 4-hydroxynonenal (4-HNE) (Uchida 2003, Poli et al. 2008), which can covalently modify lysines, cysteines and histidines in proteins. In malaria, 4-HNE was first described in 1996 (Schwarzer et al. 1996), and knowledge about its role in malaria pathogenesis has been steadily accumulating (Schwarzer et al. 2015, Na-Ek & Punsawad 2020, Schwarzer & Skorokhod 2024).
Functionally, both in clinical and in malaria models, immune cells show strong initial activation by malarial pathogen-associated molecular patterns (PAMPs, which include HZ) producing ROS, cytokines, chemokine, etc. (Jaramillo et al. 2005, Barrera et al. 2011). Consecutively, the activation was followed by long-term immune inhibition (Calle et al. 2021). More important cellular immunity processes, which were described to be inhibited, include the ability to perform repeated phagocytosis and antigen expression (Schwarzer et al. 1998), incomplete dendritic cell differentiation and functionality (Skorokhod et al. 2004, Urban & Todryk 2006, Bujila et al. 2016), immune cell transendothelial migration inhibition (Skorokhod et al. 2014) and others. Several mechanisms associated with oxidative damage have been proposed (Jaramillo et al. 2005, Olivier et al. 2014, Schwarzer et al. 2015), but additional mechanisms are needed to fully explain all observed functional effects.
Immune receptors play a pivotal role in initiating and propagating numerous immune responses, and their proper expression and functionality are essential for immune cell functions, including cell motility, chemotaxis and diapedesis. Issues with chemokine and cytokine receptors, particularly regarding attractants, may be the missing element in explaining the impaired motility of monocytes in malaria. Numerous chemoattractants that target specific receptors, such as chemokines CCL2 (MCP-1), CXCL8 (IL-8), CCL5 (RANTES), complement proteins, TNF-α, formyl peptide FMLP, leukotrienes (LTB4), platelet-activating factor and others, may drive the motility of immune cells (David & Kubes 2019, Dostert et al. 2019, El Sayed et al. 2022, Hall et al. 2024).
The CCR2 receptor is one of the main receptors involved in monocyte chemotaxis (El Sayed et al. 2022, Hall et al. 2024), with a rapid turnover following chemoattractant internalization (Volpe et al. 2012). Chemokine receptors activation, and a series of downstream Rho-GTPases, regulate directional migration, modulating polarization, actin cytoskeleton remodeling and additional receptor expression in healthy conditions (Petrie et al. 2009, David & Kubes 2019). Dysregulation or abnormal surface expression of CCR2 is believed to strongly affect the monocyte response during chemotaxis.
TNF receptors are involved in chemotaxis, as their ligands TNF-α and the TNF family member CD95L also function as chemoattractants, as shown in vivo (Issekutz & Issekutz 1993, Issekutz 1995, Dostert et al. 2019), even these receptors are primarily crucial for inducing and amplifying the overall cellular response to TNF-α (Dostert et al. 2019). Thus, the dysfunction of TNF receptors can lead to a downregulation of the cellular immune response.
This study is based on the experimental observation of lipid peroxidation and motility loss in human monocytes by HZ phagocytosis. For the first time, the downregulation of the receptors CCR2, TNFR1 and TNFR2 is shown. We discuss putative pathways that connect lipoperoxidation with receptor expression and motility, but further research is needed to fully understand the molecular mechanisms.
Materials and methods
Reagents
Unless otherwise stated, reagents were obtained from Merck (Germany).
Primary human cells
Cell donors were healthy volunteers who provided informed written consent in accordance with the Declaration of Helsinki, following approval from and in compliance with the local Research Ethics Committee of the University of Torino (Italy).
Monocyte preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from freshly collected blood as described (Skorokhod et al. 2004). Starting from the isolated PBMCs, monocytes were subsequently enriched to >85% purity as confirmed by flow cytometry (FACS) analysis performed by a BD FACSCalibur cytofluorograph (Becton–Dickinson) by CD14 and MHC class II expression. Monocyte enrichment was performed by negative selection with Dynabeads CD2 and CD19 (Thermo Fisher Scientific, Dynal Biotech, Norway), following the manufacturer’s instructions. Specifically, 1 mL of the manufacturer’s bead solution was applied to 4 × 108 PBMCs for each type of Dynabeads. After enrichment, monocytes were washed twice and suspended in macrophage serum-free medium (M-SFM; Thermo Fisher Scientific, Invitrogen, USA) at 3 × 107 cells/ml for migration assays or for plating at 2 × 107 cells/well in six-well plates (Becton–Dickinson Falcon, USA) for 4-HNE–protein conjugate identification. The plates were incubated in a humidified CO2/air incubator at 37 °C for 30 min, nonadherent cells were removed by three washes and fresh M-SFM was added. We used serum-free medium (M-SFM), which was better suited for our experiment for at least two reasons. First, we used the non-serum medium to avoid heterogeneity or undesirable monocyte activation due to primary serum. Second, this medium does not significantly activate monocytes in short time periods, up to 24–48 h. This medium generally maintains the viability and numerous characteristics of monocytes without the need to add specific growth/stimulation factors.
Preparation, quantification and opsonization of HZ, latex beads and RBCs
HZ was isolated before each experiment using a Percoll gradient from the supernatants of synchronous parasite cultures after schizont rupture, as detailed elsewhere (Skorokhod et al. 2014). It was then quantified by luminescence assay (Schwarzer et al. 1994) and opsonized with fresh human serum, as previously described (Skorokhod et al. 2014). The opsonized HZ batches were assessed for the presence of lipopolysaccharide (LPS) using two assays: E-Toxate assay from Merck, Sigma-Aldrich (Limulus amebocyte lysate; gel solidification assay, sensitivity threshold 0.05–0.1 EU/mL), and the Limulus amebocyte lysate kinetic chromogenic assay (Lonza, European Endotoxin Testing Service, sensitivity threshold 0.005 EU/mL). In the HZ batches used in this study, the LPS levels consistently remained below the detection limits of both assays. For opsonizing latex beads (bioinert phagocytosis control), equal volumes of the manufacturer’s latex bead suspension (Merck, Sigma-Aldrich, Germany), phosphate-buffered saline (PBS; 150 mM NaCl, 10 mM Na2HPO4/NaH2PO4, pH 7.4) and fresh human serum were mixed and incubated at 37 °C for 30 min. We also used RBCs as phagocytosis control, which contain the same quantity of heme corresponding to the quantity of heme in HZ. To opsonize RBCs, freshly drawn heparinized human blood was centrifuged at 1,180 g for 5 min at room temperature and the white blood cells and platelets were removed by aspiration. The isolated RBCs were washed twice with PBS supplemented with 2 mM glucose (PBS-G), resuspended to a 33% hematocrit with an anti-D IgG (Rhophylac, ZLB Behring SpA, Italy) diluted 1:64 (vol/vol) in PBS-G and incubated for 30 min at 37 °C. After opsonization, the RBCs were washed twice and resuspended in PBS-G for the phagocytosis assay.
Phagocytosis of HZ, latex beads and RBCs
Monocytes suspended in M-SFM were fed with i) HZ at 25 fmol heme/cell, ii) latex beads at 6,400 beads/cell or iii) RBCs at 12 RBCs for monocyte for 3 h before the start of two-dimensional (2D) migration assay or FACS analysis. Alternatively, adherent monocytes were cultured at 2 mln cells/well in six-well plates in the presence of 25 fmol HZ heme/cell. After 30 min, the first HZ crystals were observed inside the monocyte and phagocytosis was completed after 3 h incubation at 37 °C. Note that the amount of HZ was previously calibrated to match the intracellular HZ levels typically observed in clinical malaria cases. After phagocytosis, the cells were washed with RPMI 1640 to remove any non-ingested hemozoin (HZ). Three hours after HZ phagocytosis or control cell treatments, adherent cells were either permeabilized for microscopic analysis of 4-HNE adducts or detached and permeabilized in suspension for FACS analysis of 4-HNE adducts.
Apoptosis test
Apoptosis of monocytes was tested by FACS before and after the phagocytosis or experimental treatments, following the manufacturer’s specifications (Annexin V-FITC Apoptosis Detection Kit; Merck, Sigma-Aldrich). The kit uses annexin V conjugated with fluorescein isothiocyanate (FITC) to label phosphatidylserine sites on the membrane surface and propidium iodide to label the cellular DNA in necrotic cells where the cell membrane has been totally compromised. The monocyte preparations with the percentage of apoptotic cells over 3% were excluded from the study.
Monocyte 2D chemotaxis assay
Two-dimensional chemotaxis/migration assay was performed in microfluidic μ-Slide Chemotaxis from ibidi (Germany) (Zengel et al. 2011, Elgamoudi Bassam & Korolik 2022). Monocytes were suspended in M-SFM and i) left unfed (control), or ii) fed with latex beads (control meal), or iii) fed with RBCs, or iv) fed with opsonized HZ or v) treated with 10 μM 4-HNE (Biomol, Germany). After 1 h phagocytosis/treatment in suspension, 2 × 105 monocytes were seeded in the central channel of the μ-Slide to adhere and complete phagocytosis during a further 2 h at 37 °C. Non-ingested HZ, latex beads, RBCs, free 4-HNE and nonadherent cells were then removed by three washes with M-SFM, and adherent cells were kept in M-SFM. In some preparations, cytochalasin B was added at 25 μg/mL 30 min before the chemoattractants to block actin reorganization. The assay was started (t = 0) by loading cell culture medium without or with 100 ng/mL MCP-1 (R&D Systems, USA) or 10 ng/mL TNF-α (200 U/mL, Peprotech, USA) in the lateral compartment of the μ-Slide to create a chemotactic gradient. Migration of cells was assessed by microscopy at 0, 30 and 120 min. The assay allowed detection of cell movements of ≥2 μm (detection limit). Motility in the absence of external chemoattractants was determined as the number of monocytes that i) migrated in random directions or ii) did not migrate. This test was used to exclude cell preparations that were completely incapable of even random migration. Indeed, the cells that were unable to exhibit random motility also failed to respond to chemoattractant stimuli.
In the presence of chemoattractants, motility was recorded as the number of monocytes that i) migrated toward the chemotactic attractant (positive chemotaxis), ii) migrated randomly away from the chemotactic attractant or iii) did not migrate. Migration index was calculated for moving cells as the ratio of directionally migrated cells toward attractant versus randomly migrated cells after 120 min. Cell movement was detected by an inverted microscope (Leica DM IRB; Leica Microsystems, Germany) equipped with a 100 oil planar apochromatic objective with 1.32 numerical aperture, a Leica camera (DFC 420 C) and a Leica DFC software, version 3.3.1.
Assessment of 4-HNE adducts by fluorescence microscopy
For the detection of 4-HNE adducts, the adherent cells were analyzed under conditions similar to those of cells initiating chemotactic motility (M-SFM medium and MCP-1 pre-stimulation for 30 min). Adherent monocytes were washed with PBS at 37 °C, fixed and permeabilized in the dish as follows. Initially, after the PBS was removed, cells were fixed in 3% methanol-free formaldehyde solution (molecular biology and histology grade) in PBS for 15 min at room temperature and washed two times with PBS. Then, cells were permeabilized by 0.1% Triton X-100 in PBS for 15 min at room temperature, washed two times with PBS and then incubated with blocking solution PBS containing 1% BSA for 30 min at room temperature. Then, cells were incubated with primary mouse monoclonal antibodies anti-HNE adducts (provided by Koji Uchida) diluted 1:50 from original stock 50 μg/mL for 1 h at room temperature, washed with PBS-BSA 1% and incubated with secondary FITC-conjugated goat anti-mouse IgG secondary antibodies (dilution 1:300 for 1 h at room temperature from manufacturer’s stock solution 1 mg/mL, Merck-Millipore). After final washing with PBS-BSA 1%, the signal from adherent labeled cells was detected by fluorescent inverted microscope (Leica DM IRB; Leica Microsystems, Germany) equipped with a 100 oil planar apochromatic objective with 1.32 numerical aperture, Leica camera (DFC 420 C) and Leica DFC software, version 3.3.1.
Assessment of 4-HNE adducts by FACS
The method of detection of 4-HNE adducts by FACS was successfully developed in 2005 and widely applied (Skorokhod et al. 2005, Gallo et al. 2018, Skorokhod et al. 2021) after the availability of monoclonal antibodies anti-HNE adducts (Toyokuni et al. 1995, Waeg et al. 1996). In this procedure, the cells in suspension were fixed in 3% formaldehyde solution (molecular biology and histology grade) in PBS for 15 min at room temperature, washed two times with PBS, permeabilized by 0.1% Triton X-100 in PBS for 15 min at room temperature, washed two times with PBS and incubated with PBS-BSA 1% for blocking for 30 min at room temperature. Consequently, the cells were incubated with primary mouse monoclonal antibodies anti-HNE adducts (provided by Koji Uchida) diluted 1:100 from original stock 50 μg/mL for 1 h at room temperature, washed with PBS-BSA 1% and incubated with secondary FITC-conjugated goat anti-mouse IgG secondary antibody (for 1 h at 1:1,000 dilution from manufacturer’s stock solution 1 mg/mL, Merck-Millipore). After final washing with PBS-BSA 1%, the signal was detected by a FACSCalibur cytofluorograph (Becton–Dickinson).
Assessment of receptor expression by FACS
Receptor expression was assessed on the surface of non-permeabilized monocytes. Cells from each donor were gently detached by scraping, washed in 1% PBS-BSA and separately incubated with primary mouse antibodies against CCR2 (Thermo Fisher Scientific, Invitrogen), TNF receptor 1 (TNFR1, TNFR/CD120a, ImmunoTools GmbH, Germany) and TNF receptor 2 (TNFR2, Thermo Fisher Scientific, Invitrogen) for 1 h at room temperature. After washing with PBS-BSA 1%, a secondary anti-mouse FITC-conjugated IgG antibody (1:1,000 dilution, Merck-Millipore) was applied for 1 h at room temperature. Control cells were incubated with isotype control antibodies from the same manufacturer as of the secondary antibodies (Merck, Millipore) to account for background signal.
Statistical analysis
The nonparametric Mann–Whitney U test was employed to assess the significance of the differences between the group means (IBM SPSS Statistics 18; IBM, USA). Unless otherwise stated, P values of 0.05 or lower were considered statistically significant. Standard error (SE) bars represent the level of uncertainty in the group means.
Results
To investigate whether HZ phagocytosis induces 4-HNE production in monocytes, 4-HNE conjugates with cellular proteins were assessed using microscopy (Fig. 1) and flow cytometry (Fig. 2). Morphologically, cytoskeletal reorganization begins in control cells after stimulation with MCP-1 for 30 min, as seen by the polarization of folds on the cell surface (Fig. 1, upper left panel). 4-HNE–protein adducts were not detectable on control cells, with only minimal background signal (Fig. 1, upper right panel). In contrast, HZ was clearly visible inside the monocytes as brownish-black conglomerates (Fig. 1, lower left panel) and it induced abundant production of 4-HNE, detected as 4-HNE–protein adducts (Fig. 1, lower right panel). This is visible after cell permeabilization as a green signal distributed throughout the entire cell with slight accumulation close to phagocytosed HZ in upper left part of the cell (Fig. 1, lower right panel). Note, in this study for the first time, 4-HNE-adducts were measured in total monocyte proteins after permeabilization of adherent cells.
HZ phagocytosis induces 4-HNE production in primary human monocytes. Monocytes were isolated from healthy donors and exposed to HZ. After 3 h phagocytosis, non-phagocytosed HZ was removed by washing the cells and the cells were stimulated using MCP-1 for 30 min. 4-HNE–protein adducts were detected with monoclonal anti-4-HNE adduct antibodies (right column panels) in control cells (upper panels) and HZ-fed cells (lower panels) after permeabilization of adherent cells. Images were acquired using a Leica DR LMB microscope with a 100× oil planar apochromatic objective (numerical aperture 1.32), a Leica DFC420 C camera and a Leica software version 3.3.1. Representative image from 50 cells analyzed for each of three monocyte donors.
Citation: Redox Experimental Medicine 2025, 1; 10.1530/REM-24-0017
HZ phagocytosis induces 4-HNE–protein adducts detected by FACS. Monocytes were fed with HZ, latex beads or RBC or treated with 4-HNE (10 μM) or kept unfed and untreated. 4-HNE–protein adducts were detected by FACS after cell permeabilization. The mean values ± SEs of monocytes from five donors are plotted. Significances of differences are signed * for P < 0.05 versus any of three controls: unfed monocytes (CTRL), latex beads-fed monocytes (LATEX) and RBC-fed monocytes (RBC).
Citation: Redox Experimental Medicine 2025, 1; 10.1530/REM-24-0017
In addition to microscopic visualization in cell adhesion, 4-HNE adducts of cellular proteins were quantified by FACS analysis in monocyte suspensions after cell permeabilization. A similar trend was observed as in adherent monocytes: HZ induced 4-HNE production, which was assessed as 4-HNE protein conjugates (Fig. 2), at significantly higher levels than in control cells (2.4 ± 0.3 times versus untreated control monocytes; 2.5 ± 0.5 times versus latex beads-fed monocytes; 1.9 ± 0.5 times versus RBC-fed monocytes). The levels of 4-HNE adducts were comparable to those generated by 10 μM externally added free 4-HNE (Fig. 2).
To check functional consequences of HZ phagocytosis for monocytes, we assessed their capacity to migrate. In Fig. 3, the ‘migration index’, which characterizes the cells’ ability to move toward a chemotactic stimulus, is shown for two important attractants: MCP-1 and TNF-α. Following HZ phagocytosis, monocytes exhibited a significantly reduced motility capacity: the migration index decreased by 3.2 ± 0.6 times for MCP-1 and by 3.8 ± 1.1 times for TNF-α compared to unfed human monocytes (Fig. 3). Cells treated with 10 μM 4-HNE showed a similar inhibition of migration capacity. Cytochalasin B, used in some preparations as a positive inhibitory control to block actin polymerization, led to a 90% ± 7% inhibition of cell motility in MCP-1 gradient.
Directional monocyte migration is decreased in HZ-fed and 4-HNE-treated cells. The chemoattractants MCP-1 and TNF-α were applied and cells were quantified for directional or random migration during 120 min of chemotaxis. Migration index was calculated for moving cells as the ratio of directionally migrated cells toward attractant versus randomly migrated cells after 120 min (for details see the paragraph above ‘Monocyte 2D chemotaxis assay’). Migration index for any condition was normalized, setting the migration index of corresponding unfed monocyte =1 (CTRL). The mean values ± SEs of at least 100 analyzed monocytes per experimental condition, from one representative donor out of the three analyzed, are plotted. Significances of differences are signed * for P < 0.05 versus any of tree controls: un-fed monocytes (CTRL), latex beads fed monocytes (LATEX) and red blood cell-fed monocytes (RBC).
Citation: Redox Experimental Medicine 2025, 1; 10.1530/REM-24-0017
The expression of receptors CCR2, TNFR1 and TNFR2, which mediate chemotaxis in response to MCP-1 and TNF-α, respectively, was measured 3 h after HZ phagocytosis, before exposing the cells to MCP-1 or TNF-α gradient. This was done to assess the baseline conditions of monocytes for initiating directional migration. Two parameters were analyzed by FACS: mean receptor expression in all monocytes, reported as mean fluorescence intensity (MFI, Fig. 4, panels A, B, C), and the rate of cells with high receptor expression (Fig. 4, panels D, E, F). HZ phagocytosis induced a significant decrease in the expression of CCR2 by 55 ± 8% (Fig. 4, panel A), of TNFR1 by 79 ± 4% (Fig. 4, panel B) and of TNFR2 by 58 ± 7% (Fig. 4, panel C). The number of cells expressing CCR2 was not significantly changed by HZ (−26 ± 8%, Fig. 4, panel D), the decrease was similar for TNFR1 (−20 ± 3%, Fig. 4, panels E), but much more pronounced for TNFR2, with reductions of 41 ± 11% (Fig. 4, panel F). This indicates that, in the case of MCP-1 and TNFR1, receptor downregulation occurred significantly as MFI in all examined cells. In addition, for the TNFR2 receptor, downregulation was observed not only across all cells, but it was more pronounced within a subpopulation of cells that showed a marked loss of surface receptors. Similar effects were observed for monocytes with 10 μM external 4-HNE treatments (Fig. 4).
Decrease of CCR2, TNFR1 and TNFR2 surface expression on monocytes after HZ phagocytosis or 4-HNE treatment. Primary human monocytes were fed with HZ or treated with 10 μM 4-HNE, and receptor expression was measured by FACS. MFI and the rate of cells highly positive for receptor expression are presented. The mean values ± SEs of monocytes from five donors are plotted. Significances of differences are signed * for P < 0.05 and ** for P < 0.1 versus any of tree controls: unfed monocytes (CTRL), latex beads fed monocytes (LATEX) and red blood cell-fed monocytes (RBC). The shares of highly CCR2-, TNFR1- and TNFR2-expressing subpopulations in unfed control monocytes were 75.5 ± 11.8%, 67.25 ± 7.9 and 79.75 ± 5.1%, respectively.
Citation: Redox Experimental Medicine 2025, 1; 10.1530/REM-24-0017
Apoptosis was excluded as a cause of 4-HNE production and functional changes, as apoptosis level was monitored in all experimental conditions for all monocyte subsets, and the percentage of annexin V-positive cells (moderate apoptosis) was under 3%.
Discussion
In malaria, where an impaired immune response to secondary infections has been observed (Lyke et al. 2005), the inhibition of monocyte functions (Calle et al. 2021) may help to explain the clinical manifestations, that nevertheless require further investigation at molecular level. Some affected monocyte functions in malaria are already known, including the inhibition of monocyte motility (Nielsen et al. 1986). Reduced transendothelial migration and chemotaxis have been linked to disruption in cytoskeletal organization, attributed to lipoxidation-dependent modifications in functional proteins such as actin and coronin, both crucial for cytoskeleton organization (Skorokhod et al. 2014), and provoked by HZ phagocytosis. However, the expression and function of chemoattractant receptors in this context have not yet been addressed. In this study, we examined monocyte migration capacity toward MCP-1 and TNF-α, and the surface expression of CCR2 and TNF receptors on monocytes in a malaria model. As a consequence of HZ phagocytosis, we observed impaired directed migration and decreased expression of CCR2, TNFR1 and TNFR2 (Figs 3 and 4). The migration index for HZ-fed monocytes decreased by 3–4 times compared to control unfed monocytes (Fig. 3). To exclude the influence of the physical presence of HZ cargo on monocyte motility, a similar load of chemically inert latex beads was used and analyzed as a control. The motility of latex-fed monocytes was similar to that of unfed monocytes (Fig. 3), likely due to the nature of monocytes, which can perform chemotaxis and transendothelial migration after massive phagocytosis in vivo. Another control condition involved RBC-fed monocytes, designed to mimic the presence of heme in HZ-fed monocytes. Healthy monocytes were able to promptly digest the engulfed RBCs, and their motility characteristics were similar to those of unfed monocytes (Fig. 3). Regarding the chemoattractant MCP-1, which is one of the most widely encountered chemoattractants in vivo, HZ-fed monocytes exhibited inadequate motility with MCP-1 (Fig. 3). It is important to note that the chemoattractant properties of TNF-α are not widely studied, as TNF-α is not the most potent attractant for immune cells (Dostert et al. 2019). Our novel data on TNF-α as an attractant (Fig. 3) are highly significant to the overall immune response, as TNF-α is associated with cerebral malaria and malaria anemia, both of which are potentially life-threatening complications of malaria infection (Lucas et al. 1997, Postma et al. 1999). In addition, it was recently hypothesized that TNF-α is an important link in the increasing coexistence of malaria and type 2 diabetes in a malaria endemic population (Ademola et al. 2023), where lipid peroxidation and 4-HNE could be mechanistically involved.
Another aspect of malaria pathogenesis is redox imbalance, particularly lipid peroxidation, which is strongly evident in malaria (Prasannachandra et al. 2006, Aguilar et al. 2014, Schwarzer et al. 2015, Na-Ek & Punsawad 2020). It is important to note that, in malaria, beyond HZ and other PAMPs, many antimalarial drugs also exhibit pro-oxidative properties (Kavishe et al. 2017, Berneburg et al. 2022, Skorokhod et al. 2023b ), further contributing to oxidative stress in both malaria patients and convalescents (Nsonwu-Anyanwu et al. 2019, Gomes et al. 2022). The lipid peroxidation product 4-HNE has been detected to modify functional enzymes such as eNOS and cytochromes P450 (Negre-Salvayre et al. 2022, Skorokhod et al. 2023a , Skorokhod et al. 2024), and to be implicated in disease pathogenesis (Poli et al. 2008, Sutti & Albano 2022). In addition, 4-HNE modifications in immune cell receptors can lead to dysfunction and compromised cell functionality (Skorokhod et al. 2021, Cervellati et al. 2023). In this study, we showed for the first time the presence of 4-HNE adducts in permeabilized monocytes using microscopy. In control, non-fed cells, only traces of signal were detected, indicating the minimal physiological presence of 4-HNE adducts with the proteins (Fig. 1). Instead, in HZ-laden cells, a strong green FITC signal from anti-HNE adducts antibodies was observed. The nonenzymatic production of 4-HNE by HZ was confirmed by observing 4-HNE adducts accumulated near HZ deposits within the cell (Fig. 1).
In this study, the most important finding is the observed decrease in CCR2, TNFR1 and TNFR2 receptors following HZ phagocytosis (Fig. 4). In control conditions (unfed, latex-fed and RBC-fed monocytes), receptor expression, although variable between conditions and donors, was statistically the same across all control groups (Fig. 4). HZ treatment resulted in a decrease of receptor expression as measured by MFI on each cell (Fig. 4A, B, C) and a near loss of receptors in a subpopulation of monocytes, when analyzing the rate of highly positive cells (Fig. 4D, E, F). This impact was not significant for the CCR2 receptor (Fig. 4A), likely due to rapid receptor recycling, and was marginally significant (P < 0.1) for TNFR1 and TNFR2 (Fig. 4B and C). Nevertheless, the damage to TNF receptors appears to be the most pronounced, warranting further studies to elucidate the exact mechanisms of this receptor depletion. We believe that this effect is linked to 4-HNE production in monocytes induced by HZ, as 4-HNE accumulates in HZ-fed cells and replicates the HZ effect on receptors (Fig. 4) when added exogenously in plausible concentrations observed after HZ phagocytosis.
The exact causality of the observed events is still to be experimentally elucidated. We are reasoning about the molecular explanations of the observed experimental data; therefore, some hypotheses regarding the underlying mechanisms are proposed below. These ideas need to be carefully explored in further experimental studies.
HZ phagocytosis by primary human monocytes is accompanied by a strong and long-lasting oxidative burst that gives rise to ROS from the very beginning of phagocytosis up to 2 h (Schwarzer et al. 1992), with subsequent increase of lipid peroxides and their reactive product 4-HNE for at least 12 h. Similar pro-oxidant changes can be provoked by supplementing phorbol myristate acetate (PMA) to monocytes (Schwarzer et al. 1992). PMA induces both ROS generation and TNF p75 receptor shedding in human immortalized monocytes (Zhang et al. 2001). In that study, the authors experimentally recapitulated receptor shedding with H2O2 supplementation and hypothesized the modification of a thiol group of a cysteine residue in the prodomain of the inactive TNF-converting enzyme (TACE), resulting in the activation of TACE and the shedding of TNFR. Cysteine residues in proteins are preferred covalent ligands for 4-HNE elicited by the peroxide treatment. The binding of the nine carbon atom long 4-HNE to the TACE N-terminal prodomain cysteine might provoke a conformational change and detachment from the catalytic domain, leading to the activation of TACE and the shedding of surface TNFR. This molecular mechanism would perfectly fit with the observation after hydroperoxide or PMA exposure of the monocytic cell line. We suppose the TACE activation by 4-HNE binding as likely molecular mechanism for the immediate downregulation of membrane-bound TNFRs shortly after HZ phagocytosis or supplementation of exogenous 4-HNE. Whether other cysteine-rich domains of TACE are modified by 4-HNE and whether the modifications are functionally meaningful still need to be assessed in oxidatively stressed cells that show high secretion rates of TNFRs and proinflammatory cytokines.
In addition, macrophage migration inhibitory factor (MIF) might have a role in TNFR regulation. MIF plays a key role in the immune response to various pathogens (de Dios Rosado & Rodriguez-Sosa 2011, Sumaiya et al. 2022). In malaria, HZ acquired by monocytes has been identified as a suppressor of MIF, with clinical implications for malaria patients (Awandare et al. 2007). Suppressed MIF has been shown to inhibit TNFR expression, thereby reducing cytokine responsiveness (Toh et al. 2006, Qi-Yu et al. 2007). Thus, HZ or its byproducts impact on MIF (Awandare et al. 2007) and could explain the observed decrease in TNFR in this study.
HZ and 4-HNE-provoked shedding of TNF receptors might have impact in the development of immunity and severity of malaria infections. In experimental models, knocking out TNFR1 resulted in insufficient development of memory responses to blood-stage malaria infection in mice (Li & Langhorne 2000), and levels of soluble TNFR1 and TNFR2 correlated with parasitemia in pregnant women and are considered potential markers for malaria-associated inflammation (Thévenon et al. 2010). TNFR2 binding to membrane standing TNF-α was shown to induce ICAM-1, increased leukocyte binding and cerebral malaria in a malaria model (Lucas et al. 1997), while soluble TNFRs derived from proteolytic cleavage of membrane-standing cellular receptors TNFR1 and TNFR2 may play a role in low-dose TNF-α-mediated protection against cerebral malaria. These cleaved receptors competitively inhibit the interaction of TNF-α with membrane-bound receptors and provide a possible mechanism for regulating the availability of TNF-α (Postma et al. 1999).
In contrast to TNFRs, the constitutively expressed CCR2 in the membrane of monocytes is not shed by proteases but has a rapid turnover after internalization after ligand binding and it is additionally inducible under pro-inflammatory conditions via the transcription factor NF-kappa-B. Our former studies with HZ-laden monocyte-derived DCs had shown that NF-kappa-B is regularly moving to the nucleus under LPS stimulus but was repressed by 4-HNE-dependent induction of PPAR-gamma (Skorokhod et al. 2004). Whether the trans-repression of CCR2 transcription by NF-kappa-B occurs in HZ-fed cells and whether it results in CCR2 loss in the cell membrane remains to be proven.
The early loss of CCR2 after HZ phagocytosis or 4-HNE exposure may indicate that another, transcription-independent, yet undescribed mechanism is responsible for the receptor’s downregulation. The binding of the abundantly secreted MCP-1 due to HZ phagocytosis (Barrera et al. 2011) might explain a quick internalization of CCR2. Inhibition by 4-HNE of CCR2 recycling to the cell surface has to be proven.
CCR2 expression and its response to MCP-1 (CCL2) depend on several cytokines, such as GM-CSF (Sierra-Filardi et al. 2014, Croxford et al. 2015, Hall et al. 2024). Previously, we demonstrated that GM-CSF receptor expression and functionality were impaired by 4-HNE in a malaria model (Skorokhod et al. 2021). During monocyte differentiation by GM-CSF stimulation, 4-HNE-dependent inhibition of the GM-CSF receptor resulted in an insufficient response to GM-CSF. The impaired GM-CSF interaction with its receptor very likely impacts the expression (Fig. 4) and functionality of CCR2 too.
CCR2 and CD11b/CD18 integrins are often associated due to their presence and importance in active monocytes with a pro-inflammatory, actively migrating profile (Schenkel et al. 2004, Konstantin Nissen et al. 2022). CCR2 and TNF receptors act as primary responders to chemotactic gradients, while the monocyte CD11b integrin interacts with endothelial cells during diapedesis (Schenkel et al. 2004, Raveney et al. 2010). Importantly, the CD11b/CD18 integrins have been shown to play a direct role in the interaction between monocytes and hemozoin (HZ) during the recognition and phagocytosis of HZ, naturally enveloped by fibrinogen in patient plasma (Barrera et al. 2011). Interestingly, here, the stimulation of monocytes by HZ through CD11b/CD18 did not increase CCR2. In fact, a decrease in CCR2 after 3 h of phagocytosis was observed (Fig. 4), suggesting strong inhibitory regulation by HZ or 4-HNE.
In addition, the CCR2/CCL2 pathway has been shown to stimulate MIF, which in turn promotes MCP-1 (CCL2) release and contributes to macrophage recruitment (Gregory et al. 2006). Thus, the low CCR2 receptor levels observed here and the potential disruption of the CCR2/CCL2 pathway could be contributing factors in the reduction of MIF and TNFR levels induced by HZ.
Abstracting from the causes that decrease CCR2 on the cell surface, the role of the observed changes in CCR2 in malaria could be very important. CCR2 (CD192) is the receptor for monocyte chemotactic protein-1 (MCP-1), and ligand binding induces chemotaxis of monocytes. Several studies describe CCR2 expression on phagocytes to determine the outcome in malaria infection, underpinning the importance of the described receptor loss by HZ and 4-HNE in monocytes for malaria pathology. Host responses controlling blood-stage malaria include the CCR2-dependent migration of monocytes from bone marrow to spleen. In a mice malaria model, the absence of this receptor results in higher parasitemia (Sponaas et al. 2009). Similarly, high CCR2 expression in a monocyte subpopulation was associated with low parasitemia and CCR2 expression-mediated antiparasitic activity in human falciparum malaria patients (Chimma et al. 2009). In addition, CCR2 was essential to induce efficient adaptive immunity in humans, as shown by the results of anti-malaria vaccination (Bosteels et al. 2021). CCR2 was also required in a mouse malaria model, different from the human malaria by parasite specie, for reestablishing the homeostasis of pulmonary leukocytes during recovery from respiratory distress during acute malaria (Pollenus et al. 2021).
Ferroptosis could come to mind as a process ongoing in observed monocytes with the manifestations presented in this paper. The abundance of heme in HZ, detectable levels of HNE and the collapse of several receptors are manifestations theoretically attributable to ferroptosis. However, we exclude ferroptosis for the following reasons: i) during the observation time of 3 h after treatment with HZ, no signs of apoptosis were detected and ii) under similar experimental conditions, a slight increase in glutathione levels was observed in HZ-fed monocytes with HZ-elicited ROS production and lipoperoxidation (Schwarzer et al. 1999). A hallmark of ferroptosis is decreased GSH levels (Li et al. 2020), while the maintenance of glutathione in its reduced state, together with cellular iron-binding proteins such as ferritin, obviously protects the phagocyte from ferroptosis. This capacity to cope with iron seems plausible for monocytes, the precursor cells of macrophages, which professionally clear blood from aged RBCs, degrading them and storing or recycling the released iron (Recalcati & Cairo 2021). The monocyte itself was shown to engulf 6–8 RBCs in one phagocytic cycle, thus degrading 12–16 fmole of heme to bilirubin and iron during approximately 2 h (Schwarzer et al. 1992). After that, the intact monocyte is ready to start the next phagocytic cycle and is far from being damaged by iron.
A general consideration regarding the impact of the obtained results is that the described impairment of directed cell migration, due to the loss of chemokine receptors on the monocyte surface elicited by HZ phagocytosis and 4-HNE exposure, might interfere with crucial immune functions in vivo. Blood monocytes contribute substantially to immune surveillance by patrolling the microvascular regions and functioning as sentinels of infections (Kamei & Carman 2010). Their receptors are ready to bind to and to be activated by chemokines, followed by the intracellular reconstruction of actin fibers, which prepares the cell for directed motion and diapedesis. In early studies, impaired chemotaxis has been found in monocytes obtained from malaria patients’ blood. (Nielsen et al. 1986). HZ is the natural meal of blood monocytes, and HZ-laden monocytes and granulocytes are normally found in malaria patients (Metzger et al. 1995, Mihu et al. 2024). Thus, phagocytosis of HZ by monocytes or their treatment with 4-HNE, which lowers the expression of receptors that sense chemokine gradients formed by MCP-1 or TNF-α and decrease chemotaxis, as shown here, may plausibly lead to inefficient transendothelial chemotaxis in vivo. The defective monocyte responsiveness may facilitate superinfections and explain the poor adaptive immune response to the malaria parasite, all hallmarks of severe falciparum malaria.
Blood monocytes are not only important cells for the innate immune defense against the malaria parasite, but link innate and adaptive immunity to blood-stage malaria. As precursor cells, monocytes need to extravasate into peripheral tissues to differentiate into tissue macrophages and dendritic cells. The primary site of immune responses against Plasmodium parasites is the spleen. Antigen-presenting cells of the marginal zone of the spleen white pulp, such as monocytes/macrophages and DCs, phagocytose parasitized RBCs and HZ in the red pulp and may initiate adaptive immune responses by migrating toward T-cells deep in the white pulp for T-cell activation. However, histologic assessments of the spleen architecture of adults dying from malaria (Urban et al. 2005) showed HZ-containing macrophages and DCs enriched in the marginal zone. These HZ-laden antigen-presenting cells seemed unable to move along the chemotactic gradient to meet lymphocytes in the white pulp, thus causing inefficient specific immune response.
Therapeutically, interventions aimed at regulating damaged or downregulated receptors could be considered. Although many studies propose intentionally blocking the CCR2–CCL2 axis to reduce inflammatory responses in various diseases or prevent tumor cell extravasation (Feria & Díaz-González 2005, Xu et al. 2021), in the case of malaria we suggest that stimulating CCR2 during post-malaria convalescence could help restore normal immune function. A similar concept has been discussed in neuropsychiatric disorders, where the role of CCL2–CCR2 signaling in affective disorders is ambivalent (Curzytek & Leśkiewicz 2021); in some cases, an elevated CCL2–CCR2 response is beneficial for effective therapy (Edberg et al. 2020). In addition, the use of antioxidants and scavengers for lipid peroxidation products has been proposed to counteract the harmful effects of ROS and lipid peroxidation (Gomes et al. 2022, Abiko et al. 2023), although their clinical efficacy still requires extensive research. Concluding, the oxidative aspects of malaria are crucial to understand its pathogenesis and warrant further study within the field of redox experimental medicine.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
OS conceived the study, performed experiments, analyzed data and wrote the paper. VB performed experiments and analyzed data. EV, DU and KU performed experiments. ES conceived the study, performed experiments, analyzed data and wrote the paper.
Data availability
All data supporting the findings of this study are available within the article.
Acknowledgements
The authors would like to acknowledge Paolo Arese for his scientific inspiration as the founder of the Malaria Research Group at the University of Torino (Italy) and the initiator and founder of the Italian Malaria Network.
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