Abstract
Phagocytosis is a distinguished immunological process that assists the immune system in recognizing and responding effectively to eliminate foreign and self-pathogenic molecules. Improving the overall understanding of this immune mechanism during malaria infection is imperative. The full understanding of how this mechanism eradicates malaria parasites remains unclear, especially for Plasmodium falciparum. Which is an area requiring further broad investigations. In this context, previous studies have shown that various factors such as phagocyte cell subclasses, plasma protein molecules and Plasmodium evasion tactics influence the phagocytic process differently. However, the mechanisms underlying phagocytic activity during Plasmodium falciparum infection are still ambiguous. In this review, we aim to describe essential immunological aspects and the state of current knowledge on phagocytic activities in Plasmodium falciparum infection. We highlight the significant involvement of distinct active cells that induce phagocytosis. In addition, we discuss the implications and therapeutic approaches related to the effective phagocytic activity.
Background
Plasmodium falciparum is considered one of the most significant causative agents of severe malaria and complicated inflammatory symptoms with life-threatening consequences in malaria-endemic regions, mostly in sub-Saharan Africa 1. Plasmodium falciparum is one of five malaria species that infect humans and are transmitted strictly by anopheles mosquitoes and involves complex cellular and biochemical events 2. The malaria parasite undergoes various stages during the life cycle, including the initial stages of the parasite forms (sporozoites) upon entering the human body followed by the asexual phases (merozoites, early ring stages, and late trophozoites), which alter host cell morphology and physiology. The sexual stages (gametocytes) affect erythroblast transmission and sequestration potentially causing anaemia 3,4 Understanding the immunological interactions and invasion mechanisms of Plasmodium falciparum is crucial for developing effective prophylactic and therapeutic strategies against malaria 5.
 A review of the immune response mechanisms in Plasmodium falciparum is paramount due to the malaria infection seriousness, the complexity of immune interactions and its implications for parasite survival and spreading in the host 6,7. Research has indicated that certain immune preventive mechanisms (i.e. Phagocytosis, Cellular Cytotoxicity) play a vital role in the abatement of infected erythrocytes (IEs) prevalence and pathological complications from Plasmodium falciparum infection. This highlighted the importance and consistency of these protective mechanisms for eliminating malaria infection 8,9. Additionally, understanding the host immune mechanisms associated with malaria infection, such as the selective phagocytosis process and the involvement of adaptive immune cells may contribute to the development of new vaccine strategies by improving the protective immunity against malaria infection 10.
 Phagocytosis is a fundamental process responsible for maintaining immune homeostasis, inducing inflammation and pathogen clearance. It is essential for keeping the human body healthy, by engulfing and eliminating the pathogenic molecules, apoptotic cells, infected cells and microorganisms 11. The phagocytosis mechanism involves cellular uptake mediated by specialized phagocytic cells like dendritic cells (DCs), macrophages (MØs), monocytes, and neutrophils with contributions of other plasma molecules for effective phagocytic activity 12. The process involves various biological steps including pathogen molecule recognition, the creation of early and late phagosome vesicles and the formation of phagolysosomes. The phagocytosis process can be classified as either opsonic or non-opsonic depending on how phagocyte cells recognize and interact with a target molecule 13. Opsonic phagocytosis involves the interaction of host promoting proteins (opsonins) such as the antibodies, the complement fragments and collectin molecules with targets, facilitating their recognition by phagocyte cells through specific receptors such as Fcγ and complement receptors. In the non-opsonic class, cellular engagement occurs when phagocytes capture pathogens or IEs directly without prior coating by opsonins 14.
  The phagocytic mechanism involved in eliminating Plasmodium falciparum infections is highly dynamic and incorporates complex interactions of specific immune cells with the Plasmodium parasite. These particular interactions include features that remain poorly understood 15. Recent research has applied simple models to examine phagocytic activity selectively during malaria infection, which assisted in enhancing the overall understanding of phagocytosis 16. So far, some peripheral blood mononuclear cells (PBMC) are thought to serve as phagocytes and are involved in combating the Plasmodium falciparum parasite, while the specific roles conducted by these cells remain unclear. In addition, a few in-vivo studies indicated that there are still major questions that need to be addressed regarding the functional basis of these cells in the phagocytotic mechanism, particularly their role in the eradication of Plasmodium-infected erythrocytes (IEs)17,18.
  In this review, the recent findings concerning the phagocytic activities in Plasmodium falciparum infection, the contributions of phagocyte subpopulations and host factors that enhance phagocytosis mechanisms and their associations with parasite clearance are reviewed. Additionally, we highlighted the common parasite tactics to evade phagocytosis and the host immune response. Also, we tried to outline the clinical implications of phagocytosis in malaria vaccine development to explore and improve our understanding of phagocytic activities against Plasmodium falciparum infection.
Vital phagocyte cells in Plasmodium falciparum infection.
Phagocytic responses to Plasmodium falciparum infection involve the participation of key immune cells with intricate immunomodulatory mechanisms. Some studies have shown that phagocyte-specific subpopulations and parasite ligands adjust the effectiveness of the phagocytic interaction with IEs 19.   
Distinct immune cells carry out substantial assignments in the phagocytosis of Plasmodium falciparum-infected erythrocytes (IEs) or blood-stage merozoites. Depending on cell composition and heterogeneity the peripheral blood monocytes can be categorized into three subclasses: classical (CD14+CD16), intermediate (CD14+CD16+), and non-classical monocytes (CD14dimCD16+) 20,21 (Figure 2). The intermediate and non-classical monocytes have been recognized as the efficient cells in both phagocytosis classes to Plasmodium falciparum-IEs, which is attributed due to the distinct expression levels of certain cell receptors (e.g. CD36, TLR 2 and TLR4) 22,23. In addition, a few investigations revealed that classical monocytes and neutrophils play important roles as dominant phagocytic cells involved in the opsonic phagocytosis (OP) process 24. With low concentrations of anti-malaria antibodies, the classical monocytes (CD14+CD16) provide the main principal roles as dominant phagocytic cells involved in the opsonic forms through FcγRIIA receptor 25. While, at high antibody levels, the neutrophils are the dominant phagocytic cells for blood-stage merozoites through the interaction of FcγRIIIB receptors acting synergistically with FcγRIIA.  Meanwhile, the protection from febrile malaria is also connected to the opsonic phagocytosis activity of neutrophils 26. So far, the heterogeneity in neutrophil cells as well as other phagocytic cells has been identified and the particular function of neutrophils in phagocytic mechanisms against IEs and merozoites is still not well understood 19.
 Data characterizing the roles of the tissue MØs subclasses (M1, M2) in the phagocytosis mechanism of Plasmodium falciparum-IEs remain largely unclear. A few studies investigated MØs phenotypes in complicated malaria, reported that M1 (CD68+, CD40+) has a potential contribution to phagocytic function in Plasmodium falciparum infection peculiarly in lung tissues by studying proinflammatory cytokines (i.e. interferon-gamma (IFN-γ), Interleukin 6 (IL-6), Tumor necrosis factor (TNF) and Interleukin 10 (IL-10) 27. Additionally, the MØs generated from murine bone marrow have been shown to exhibit remarkable phagocytic activity against early and late parasite stages, which are accompanied by the induction of inflammatory mediators 28. Based on the previous evidence, these cells play a key role in the phagocytosis of IEs and Plasmodium falciparum parasites.
Immune Receptors Interaction and Plasmodium falciparum.
 A phagocytic assembly mechanism plays a critical role in the effective response of the immune system to a foreign invader. Several stages are involved in the process, including immune recognition, signalling, phagosome formation and phagolysosome maturation, which are essential for effective immune responses 13. The antigen-presenting cells (APCs) such as the Dendritic cells (DCs) and Macrophages (MØs) play an effective role in phagocytosis by sensing Plasmodium falciparum sporozoites (SPZs), followed by increasing the expression of cell activation markers and reducing the motility of cells upon phagocytosis29. Plasmodium falciparum proteins and molecules are key players in immune interactions, due to their direct contribution in the remodelling of host cells. The Plasmodium helical interspersed sub-telomeric (PHIST) proteins including PF3D7_1372300 interact with erythrocyte membrane protein 1 (PfEMP1) and alter the interacted cells 30,31. Thus, Plasmodium falciparum molecules modulate immune sensing cells (DCs, monocytes and MØs) and impact the proinflammatory response and the consequences of malaria infection.
 Plasmodium falciparum molecules trigger immune recognitions via antigenic molecules that interact with specific immune sensing receptors, promoting the upregulation of a cellular immune responsive state, which involves epigenetic modifications, signalling pathway activation, and proinflammatory cytokine production. 15,32. Glycosylphosphatidylinositol (GPI) anchor and hemozoin of Plasmodium falciparum stimulate innate immune responses via Toll-like receptor (TLR) family to activate proinflammatory status in MØs and DCs through highly immune-specific signalling 33. Plasmodium falciparum-erythrocyte membrane protein 1 (PfEMP1) has a significant impact on immune sensation and recognition, leading to immune system components intervention encounter malaria infection 34. The early recognition of infected RBCs (IEs) and malaria molecules occurs through phagocytic cells and other antigen-presenting cells, that induce the immune responsive state on these cells characterized by trimethylation of histone h3 at lysine 4 (H3K4me3) modification at gene promoters enhances transcription of immunoregulatory gene expression (e.g. CgTLRs) and facilitates MyD88-dependent signalling 26.
A critical step in malaria pathogenesis and cerebral malaria development involves the interaction between intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR), which governs the binding between Plasmodium falciparum-infected erythrocytes and endothelial cells 35. The characteristics of Plasmodium falciparum-infected red blood cells (IEs) were investigated, and researchers found that the immune interaction takes place through the lipid phosphatidylserine (PS) surface on IEs and the CD36 binding receptor on phagocytes 36. Research has demonstrated that IEs and Plasmodium falciparum hemozoin stimulate peripheral blood mononuclear cells (PBMCs) through TLR interactions (Figure 1), which can recognize IEs and Plasmodium falciparum molecules through cyclic guanosine‒adenosine monophosphate synthase (cGAS) 37. Other in vitro experiments have shown that phagocytes recognize IEs through neoantigens inserted during Plasmodium falciparum advancement expanding phagocytic activities, particularly the contribution of fragment crystallizable (Fc) receptors 38. The parasite also stimulates the production of autoantibodies and the activation of complement system molecules, notably complement factor 3 (C3) for better identification of Plasmodium pathogens 39. The recognition mechanisms of Plasmodium by phagocytes may take different approaches but share a common purpose in parasite molecule identification.
 The interactions of phagocytic cells with IEs and parasite particles occur in particular forms during malaria infection. Phagocytic cells such as monocytes and macrophages interact with infected erythrocytes and parasite particles via opsonic and non-opsonic phagocytosis, which aids in parasite clearance and immune response regulation. Studies have shown that opsonic antibodies bind to late-stage Plasmodium falciparum-infected erythrocytes, promoting their phagocytic uptake through Fcγ receptors, followed by IE ingestion and destruction within phagolysosomes and the production of nitric oxide and oxygen radicals 17,40,41. In addition, phagocytic cells interact with non-opsonized infected erythrocytes through the CD36 receptor, implicating scavenger receptors in the clearance of ring-stage Plasmodium falciparum 42. The phagocytosis process against IEs aims to eliminate these cells and reutilize the nutrient components from these cells. In such ways, the MØs are mainly responsible for this task by reducing parasitemia and enhancing the host overall survival 43.Phagocytic cells in the spleen interact with infected erythrocytes preferentially the immature forms, which results in splenic trapping through cell pitting and destruction of both infected and uninfected erythrocytes 44,45. These findings highlight the substantial roles of innate immunity cells in malaria and the complex interactions between phagocytic cells and infected erythrocytes or parasite particles.
 Phagocytosis-induced immune signalling in Plasmodium falciparum infection involves the activation of specific cell signalling pathways, including protein kinase C (PKC), ras - extracellular regulated kinases (RAS-ERK), Ca+2 and nuclear factor kappa B (NF-κB), which contribute substantially to the response toward the different stages of parasite and IEs. 15. These signalling events are elicited by several cellular receptor-mediated phagocytic process (i.e. TLR, CD36, FcγR, CR3 and mannose receptors), (Table 1) 46. These receptors provide an integrated task for the phagocytic process, but their full roles in Plasmodium falciparum infection are still not well understood.
 The engulfment process of infected erythrocytes and parasite molecules involves dynamic mechanisms and complex components. This step represents the substantial stage in the clearance of Plasmodium parasites. Several factors contribute to this unique immune mechanism, including curved membrane-protein complexes (CMCs) and actin polymerization, which reduce the bending energy and promote quicker engulfment 47. Plasmodium-infected erythrocytes undergo oxidative changes leading to hemichrome formation, band 3 aggregation and deposition of complements and immunoglobulins (i.e. IgG) to increase phagocytic capacity 48. Then opsonized infected erythrocytes interact with Fc receptor-mediated phagocytosis, enhancing inflammasome activation and proinflammatory cytokine secretion, whereas later parasite forms are recognized by non-opsonic receptors CD36 49. Once ingested, parasites and infected cells are encased in phagolysosomes, where they are subjected to a hostile environment that facilitates their destruction through enzymes and toxic substances 40. These enzymes neutralize the parasite by breaking down proteins and other Plasmodium components. While all these complementary mechanisms of phagocytosis help maintain low parasitemia and prevent malaria complications, the complexity of the Plasmodium falciparum life cycle and its ability to evade immune responses pose significant challenges to developing effective treatments and vaccines.
Host factors influencing phagocytosis.
The most abundant plasma proteins influencing phagocytosis in malaria infection include complement proteins, IgG Immunoglobulin isotypes, collectins and other protein complexes. These proteins are pivotal in promoting phagocytosis against Plasmodium falciparum to reinforce the phagocytic activity of immune cells. While these proteins play an important role in the immune response against Plasmodium falciparum, variations in their levels are related to malaria severity and outcomes. 50.
 The complement system is involved in phagocytosis through secreted proteins, which may strengthen the phagocytosis of IEs 51. Complement particularly (C3) fragments perform an essential duty in opsonizing infected and uninfected erythrocytes, causing severe malarial anaemia (SMA) via erythrocyte phagocytosis 52. Defects in complement regulatory proteins such as complement regulatory 1(CR1) contribute to increased complement activation and augmentation of deposition on cells resulting in more phagocytic activities 53. Additionally, the role of immunoglobulins is reported to significantly assist in phagocytosis. Antibody-dependent phagocytosis of IEs and uninfected erythrocytes is stated to play an efficient role in the protection and elimination of malaria pathogens 54. IgG specifically subclass (IgG3 and IgG1) is a potent naturally occurring antibody, that shown to have unique implications and correlation with opsonic phagocytic activity and it found to promote distinct phagocytic effects due to the specific IgG binding features toward malaria antigens on both infected erythrocytes cell surface or parasite antigenic surface (Figure 1b)55. Immunoglobulin G (IgG) with antigenic specificity toward merozoite antigens was identified to have an explicit connection with increasing phagocytosis activity against IEs 56.
 Furthermore, Studies have shown that certain merozoite proteins on IEs such as erythrocyte-binding antigens (i.e. 140 and 175) can promote antibody-dependent phagocytosis of Plasmodium falciparum-infected erythrocytes, support malaria protection by targeting merozoite antigens on ring-stage parasites 54. Additionally, several research highlighted the contributions of other plasma proteins to the phagocytic mechanism, that involves effectively in the acute inflammatory response. Soluble CD163 and CD14 are abundant plasma proteins that have been shown to bolster phagocytosis, correlating to malaria severity and facilitating the immune response 57. Moreover, plasma proteins like C-reactive protein (CRP) and lipid-binding protein (LBP) assist phagocytosis by opsonizing pathogens, thus playing an important role in the inflammatory immune response to malaria 50.
 The role of fibrinogen and platelets demonstrated in removing parasite hemozoin and increasing phagocytic activity 58. Collectins such as Mannose-binding lectin (MBL) play a key role in innate immune recognition of glycosylated parasite-derived proteins and induction of phagocytosis against IEs and merozoite engulfment, aiming for more effective malaria clearance 59. These findings emphasized the participation of auxiliary proteins in phagocytosis enhancement over infected red blood cells efficiently, through targeting Plasmodium falciparum particles.
Immune Evasion strategies and phagocytosis.
  Plasmodium falciparum and infected erythrocytes employ diverse tactics to evade phagocytosis and immune defense network. These adaptations allow the parasite to manipulate the host immune response, leading to persistent infections and severe malaria complications. Plasmodium falciparum expresses different sorts of proteins on the parasite surfaces and infected erythrocytes (IEs), Plasmodium falciparum immune evasion mechanisms have long been linked to these proteins 60. The entire strategies of Plasmodium falciparum immune evasion are still not fully understood but their action ways can be categorized into three mainlines 61. The class of tactics that are dependent primarily on Plasmodium falciparum self-antigen, also it is known as surface antigen variation.  Furthermore, some tactics rely upon the interaction with host cells and molecules interaction, while some other strategies target the immune system and manipulate host immune response 62.
The selective antigen expression strategy of Plasmodium falciparum is critical for immune evasion as well as adapting to the immune environment during the early stages of infection. Plasmodium falciparum-infected erythrocytes (IEs) can express surface antigens like RIFIN to interact with inhibitory receptors (e.g. LILRB1, LILRB2 and LAIR1) to suppress immune cell activation and utilize multiple evasion mechanisms 60. Furthermore, Plasmodium falciparum through the selective antigens expression technique upregulates a specific variant antigen (VAR) that binds to chondroitin sulfate A (CSA). The VAR2CSA proteins help the parasite to avoid macrophage phagocytosis and natural killer (NK) cell-mediated killing, ensuring parasite survival and infection persistence 61.
One of the key evasion mechanisms of Plasmodium falciparum is recruiting the complement regulatory molecules such as factor H (FH) on IE or parasite surfaces to inhibit complement activation and subsequent lysis 63,64. This draws attention to the dual role of the complement system in Plasmodium falciparum infection (Figure 3). Additionally, Plasmodium falciparum is capable of interacting with plasma proteins like vitronectin (VTN) through surface proteins (e.g. SERA5 and SE36) at late infection stages, thereby preventing phagocytosis of the parasite and IEs 65. Plasmodium falciparum through the glycoconjugate binding employs carbohydrate-mediated interactions with certain erythrocyte antigens (e.g. ABO and Duffy), to enhance its adhesion and evade immune responses within the bloodstream 66. Finally, Plasmodium falciparum is capable of evading phagocytosis by inducing Kupffer cell apoptosis and interfering with macrophage phagocytic functions via hemozoin 67,68. These immune evasion strategies present significant challenges for vaccine success. Although understanding of Plasmodium falciparum immune evasion strategies is substantial, it is also vital to understand how host-parasite interactions work to develop a more effective vaccine strategy.
Therapeutic potential of phagocytosis in Plasmodium falciparum infection.
   Malaria vaccination is may reliant on enhancing the phagocytosis process, which augments host immune responses to infection through both opsonin and non-opsonin dependent mechanisms. The central role of phagocytosis in vaccine-induced immunity is to expedite parasite antigen uptake 6,69.
 Opsonic phagocytosis can contribute vastly to protective immunity to malaria, particularly through antibodies that target merozoite antigens. Vaccination with the NTS-DBL1-α domain of PfEMP1 can generate naturally acquired antibodies as same as in immunized individuals in endemic areas and provoke opsonic phagocytosis to IEs 70. Researchers emphasized the importance of DNA vaccination versus Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) can trigger specific neutralizing antibodies and T-cell boost, drawing attention to the promising strategy in malaria vaccine progression 71. Also, vaccines targeting merozoite proteins on the erythrocyte cell surface can enhance phagocytic responses to prevent clinical malaria caused by Plasmodium falciparum and control parasitemia 54. Additionally, the sporozoite chemoprophylaxis regimens are able to generate a strong effect of T-cell and antibody responses, potentially enhancing vaccine efficacy by targeting phagocytic responses against malaria infection 72. Studies have indicated that vaccine development focused on phagocytic responses to Plasmodium falciparum infection can be supported through the use of a topical adjuvant with TLR7 agonist such as imiquimod to enhance humoral immunity 73. A liposomal adjuvant system was found to encourage robust antibody and CD8+ T-cell responses against Plasmodium falciparum, indicating the potential role of phagocytic tactics in malaria vaccine progress 74. Studies have shown that children with higher opsonic phagocytosis activity are at a significantly lower risk of febrile malaria, as demonstrated by elevation levels of opsonizing antibodies like IgG1 and IgG3 69,75. Furthermore, the approach of stimulating the phagocytic response in vaccine development aims to induce protective immunity by neutralizing parasite infectivity and has been investigated using a synthetic vaccine against Plasmodium falciparum that targets the dominant epitope (NANP)3 on parasite 76. These advancements underscore the need for more investigation into the phagocytic approach for powerful vaccine development upon Plasmodium falciparum.
The non-opsonizing phagocytosis is increasingly recognized as a critical mechanism in the development of malaria vaccines. There is evidence that CD36 scavenger receptors facilitate the phagocytosis of non-opsonized erythrocytes infected with Plasmodium falciparum gametocytes, indicating the fortune of this pathway in parasite clearance that could be targeted for vaccine advancement 77,78. Research indicates also, that MØs demonstrated improvement in non-opsonic phagocytic activity against infected red blood cells in the presence of interferon-gamma (IFN-γ), which suggests that cytokine modulation might improve vaccination outcomes 79.
  A complete understanding of the phagocytic mechanisms against infected erythrocytes and Plasmodium falciparum molecules and the role of phagocyte cells in malaria elimination is crucial for the malaria therapeutic approaches, which include several clinical challenges and scientific limitations. Phagocytosis of infected and uninfected erythrocytes is essential, while the selective discriminating between cells and intercepting early parasite stages represents a great obstacle to effective phagocytic activity 38,54. Moreover, the limited efficacy of conventional anti-malaria drugs due to increased resistance, inhibitory effects and potential toxicities hinder specific immune cell activities 80,81. Recent studies have pointed to the gap of knowledge regarding the in-depth characterization of phagocytosis complex interactions. That requires more precisely targeted studies, aiming to solve the existing limitations and obstacles to leveraging this distinguishing activity in more effective malaria treatment. 19,82.
 This review focuses on enhancing the understanding of the dynamics of phagocytosis in malaria infection especially against Plasmodium falciparum, which is necessary to develop effective antimalaria vaccines and therapies. By discussing recent and previous findings that are headed to explore the impact of effective phagocyte cells, host factors, Immune evasion, and therapeutic challenges in malaria infection clearance, the collective knowledge gained from these discussions might be useful in developing novel antimalarial therapies and vaccines.
 Subsequent research should explore in more detail the phagocytic mechanism lines on advanced cellular and molecular levels by providing deeper insights into the protective and potential effects of this mechanism. Additionally, the impact of merozoite antigens on the phagocytic process should be more determined by focusing on the potential antigenic targets for controlling parasitemia and preventing clinical malaria. Understanding the molecular pathways involved, particularly that one controls the interaction between phagocyte cells and parasites, which remain inadequately described.  In addition, investigating the specific mechanisms that influence the balance between protection and inflammation is fundamental for developing novel antimalarial therapies.
Conclusion.
The phagocytosis process impacts effectively the overall host protective immune response, which contributes substantially to the elimination of infected cells by Plasmodium falciparum and helps in controlling the malaria infection.  Recognising the importance of the phagocytosis interactions in Plasmodium falciparum infection depends on understanding the roles of different phagocytic cells, host interrelated factors, parasite immune evasion and the complex interactions with infected erythrocytes and parasite particles. The well-immune characterization, determining the mainstream contributors and the impact of Plasmodium immune evasion in phagocytosis is imperative for designing innovative vaccines and new therapeutic approaches. That may offer promising prospects to enhance the immune response, promote efficient removal of Plasmodium falciparum and reduce the consequences of malaria disease through targeting the phagocytic tactics. Exploring phagocytic-specific activities such as opsonins, distinct cell subpopulation involvement and inhibitory molecule interaction represents a great immunological challenge. Thus far, there is a need for more comprehensive studies to grasp the complete spectrum of phagocytic mechanisms involved in combating Plasmodium falciparum infection.
 
Abbreviations
IEs; infected erythrocytes, DCs; dendritic cells, MØs; macrophages, PfEMP1; Plasmodium falciparum erythrocyte membrane protein 1, TLR; Toll-like receptors, FcγR; fragment crystallizable gamma receptor, CD36; Cluster of Differentiation 36, IgG; immunoglobulin G.