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Hemozoin and Malaria : a love-hate affair

July 5, 2021 - 17:31 -- Pierre Lutgen

Mutaz Akkawi, Pascal Gisenya, Jérôme Munyangi, Pierre Lutgen

…or the key of malaria transmission.

Already 25 years ago it was known that malaria pigment (hemozoin, Hz, biocrystallized heme) is not an inert material. Crude pigment, as present in infected erythrocytes and shed after schizont rupture, may be considered the 'natural diet' ingested by macrophages in infected blood. Hemozoin is a powerful source of radicals, necrosis factor-alpha, interleukins 1 and 6.

Arese P, Schwarzer E. Malarial pigment (haemozoin): a very active 'inert' substance. Ann Trop Med Parasitol. 1997 Jul;91(5):501-16. doi: 10.1080/00034989760879. PMID: 9329987.

Most drug discovery efforts of pharmaceutical companies, and academic research sponsored by them, have focused on the asexual erythrocyte stages that cause the acute symptoms of malaria. Inhibited sporogony or killed gametocytes confer no direct benefit to a person infected by malaria.

A review paper tries to understand the love-hate relationship between Plasmodium parasites and heme, and to clarify several long-standing riddles about heme production and utilization by parasites.
Paul A. Sigala and Daniel Goldberg. Peculiarities and Paradoxes of Plasmodium Heme Metabolism,. Annu. Rev. Microbiol. 2014. 68: 259–78

Free heme is an endogenous danger signal that provokes innate immunity. A recent study from China shows that different heme concentrations have different effects on the production of antibodies in rats and mice. This finding not only suggests that free heme can affect adaptive immune responses to an antigen by a mode of concentration thresholds (hormesis). It might also explain why hemozoin plays different roles in sporozoites, gametocytes and merozoites. The heme biosynthesis pathway is not essential and even redundant in blood stages, it is absolutely required for parasite transmission to mosquitoes.

Li, Guofu Xue, Haiyan, Impact of heme on specific antibody production in mice: promotive, inhibitive or null outcome is determined by its concentration. Heliyon 2017, May 3(5) e 00303

Our understanding of heme acquisition mechanisms and the role of heme biosynthesis in different stages of Plasmodium parasite development has evolved considerably in recent years. Parasites do not require heme biosynthesis for blood-stage growth, it is even eliminated by biocrystallization. However parasite heme biosynthesis is essential for mosquito and liver stage infections. Available evidence suggests that synthesis of heme by malaria parasites is a cooperative activity between the apicoplast and mitochondrion.

Sato S, Clough B, Coates L, Wilson RJ. Enzymes for heme biosynthesis are found in both the mitochondrion and plastid of the malaria parasite Plasmodium falciparum. Protist. 2004 Mar;155(1):117-25.

Heme is an ancient biological cofactor required by nearly all organisms for cellular viability. By virtue of their intraerythrocytic lifestyle, Plasmodium parasites interact with heme as both a toxic by-product of large-scale hemoglobin catabolism and as an essential cofactor for their own metabolism.

Goldberg DE, Sigala PA. Plasmodium heme biosynthesis: To be or not to be essential? PLoS Pathog. 2017;13(9):e1006511. Published 2017 Sep 28. doi:10.1371/journal.ppat.1006511
Rathnapala UL, Goodman CD, McFadden GI. A novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite. PLoS Pathog. 2017;13(6):e1006396

The hormetic effect of heme may also explain why young gametocytes of stade I-IV hide in the bone marrow to avoid the flood of hemozoin in the peripheral blood. They only re-enter the peripheral blood at stage V seven days later when the hemozoin storm is over and they are than ready for the transmission to the mosquito. But while hiding in the bone marrow they are not dormant. They accumulate around erythroblastic islands. Gametocytes can fully develop inside the erythroblasts in vitro and in vivo. Infection of erythroblasts by gametocytes delays erythroid differentiation, thereby allowing gametocyte maturation to coincide with the release of their host cell from the bone marrow. This maturation process interfers with erythropoiesis and contributes to anemia.

Gaëlle Neveu, Cyrielle Richard, Dominique Mazier, Plasmodium falciparum sexual parasites develop in human erythroblasts and affect erythropoiesis. Blood (2020) 136 (12): 1381–1393.

Lamikanra AA, Theron M, Kooij TWA, Roberts DJ (2009) Hemozoin (Malarial Pigment) Directly Promotes Apoptosis of Erythroid Precursors. PLoS ONE 4(12): e8446. https://doi.org/10.1371/journal.pone.0008446

Over the years it became evident that not only intravenous artesunate often causes hemolysis, but also ACT therapy. Hemolysates increase erythropoiesis in the bone marrow and this provides the large supply of hemoglobulin needed by young gametocytes. In a recent large-scale clinical trial in Maniema, RDC, it was found that Artemisia afra void of artemisinin causes less hemolysis than Artemisia annua rich in artemisinin. This may explain why Artemisia afra rapidly and completely eliminates gametocytes and consequently blocks transmission to mosquitoes.

Jérôme Munyanga, Lucile Cornet-Vernet, Michel Idumbo, Chen Lu, Pierre Lutgen, Christian Perronne, Nadège Ngombe, Jacques Bianga, Bavon Mupenda, Paul Lalukala, Guy Mergeai, Dieudonné Mumba, Melissa Towler, Pamela Weathers. Artemisia annua and Artemisia afra tea infusions vs. artesunate-amodiaquine (ASAQ) in treating Plasmodium falciparum malaria in a large scale, double blind, randomized clinical trial. Phytomedicine, 2019, 57, 49-56

A large amount of hemolysates swimming in the peripheral blood will enhance gametocytogenesis.

Schneweis S, Maier WA, Seitz HM. Haemolysis of infected erythrocytes--a trigger for formation of Plasmodium falciparum gametocytes? Parasitol Res. 1991;77(5):458-60.

Addition of red cell lysate has been shown to increase the rate of gametocytogenesis.
R Carter, LH Miller, Evidence of environmental modulation of gametocytogenenis in Plasmodium falciparum in continuous culture. Bulletin WHO, 1979, 57, 37-52

Silica present in the trichomes of Artemisia plants as phytoliths may play an unsuspected role. Neutrophils are extremely efficient phagocytes, which also engulf hemozoin, removing it from the peripheral blood circulation. A recent study from Brazil has shown that neutrophils have a two times higher capacity to take up silica particles of the same size as hemozoin particles. The administration of Artemisia infusions containing silica (not of organic solvent extracts) will lead in the peripheral blood to a much longer presence of hemozoin able to kill stade V gametocytes by ROS.

Lautenschlager SOS, Kim T, Bidóia DL, Nakamura CV, Anders HJ, Steiger S. Plasma Proteins and Platelets Modulate Neutrophil Clearance of Malaria-Related Hemozoin Crystals. Cells. 2019;9(1):93. Published 2019 Dec 30.
Zimmerman BT, Canono BP, Campbell PA. Silica decreases phagocytosis and bactericidal activity of both macrophages and neutrophils in vitro. Immunology. 1986 Dec;59(4):521-5

Another strange phenomenon should deserve more attention. In 1997 it was discovered that chloroquine not only inhibits the hemozoin formation, but also depolymerizes hemozoin. Chloroquine directly interacts with purified hemozoin and results in its breakdown with concomitant release of heme from the pigment. This will reduce the hemozoin load in peripheral blood during gametocyte maturation in the bone marrow. After release of the stage V gametocyte, they will find a field free of the oxidative stress caused by hemozoin and the mosquito will at the same time suck a blood rich in heme which is needed for development of gametocytes into gametes. Heme is essential for gamete development in the mosquito.

Pandey AV, Tekwani BL. Depolymerization of malarial hemozoin: a novel reaction initiated by blood schizontocidal antimalarials. FEBS Lett. 1997 Feb 3;402(2-3):236-40.

Long enduring parasitemias of P. falciparum are of considerable epidemiological importance and may be responsible for a large part of the transmission of this species in certain endemic areas.

Geoffrey M. Jeffery, Don E. Eyles. Infectivity to Mosquitoes of Plasmodium falciparum as Related to Gametocyte Density and Duration. The American Journal of Tropical Medicine and Hygiene, 1955, 4, 781 – 789

Therefore, the impact of strategies aiming to control asexual parasites needs to be re-examined. Inefficient strategies might be predicted to increase and not suppress malaria transmission.

R. E. Sinden. Asexual blood stages of malaria modulate gametocyte infectivity to the mosquito vector – possible implications for control strategies. Parasitology. Volume 103, Issue 2. October 1991, pp. 191-196

In a paper published in 2013, doi.org/10.1371/journal.pmed.1001564, the WorldWide Antimalarial Resistance Network (WWARN) DP Study Group expresses its concern on the effect of resistance to ACTs and malaria transmission.

Treatment of clinical Plasmodium falciparum malaria with sulfadoxine-pyrimethamine and amodiaquine is associated with increased post-treatment gametocyte carriage.
Ahmad, A., Prom, A., Bradley, J. et al. Gametocyte carriage after seasonal malaria chemoprevention in Plasmodium falciparum infected asymptomatic children. Malar J 20, 169 (2021).

As artesunate monotherapy causes recrudescence after a few weeks, one may wonder what effect this has on gametocyte carriage. In a trial the PCR-corrected cure rate by day 14 was 92% (46 of 50 patients), but it dropped to 72% (36 of 50 patients) by day 28.
Steffen Borrmann, Ayola A. Adegnika, Short-Course Artesunate Treatment of Uncomplicated Malaria. Journal of Antimicrobial Chemotherapy (2002) 50, 751–754

Residual merozoites on day 7, 14 or 28 after any antimalarial treatment may still undergo gametocytogenesis. It is thus essential that all merozoites are removed from the blood.
CJ Ngwa, G Pradel. The Biology of Malaria Gametocytes. Book, 2016. Doi: 10.5772/65464

Several papers indicate that piperaquine compared to other antimalarials leads to a high level of recrudescence and a higher gametocytogenesis. Gametes are even generated by piperaquine in asymptomatic patients with low asexual parasite densities.

Reuling IJ, van de Schans LA, Sauerwein RW, Bousema T. A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model. Elife. 2018 Feb 27;7:e31549.

Pasay CJ, Rockett R, Sekuloski S, et al. Piperaquine Monotherapy of Drug-Susceptible Plasmodium falciparum Infection Results in Rapid Clearance of Parasitemia but Is Followed by the Appearance of Gametocytemia. J Infect Dis. 2016;214(1):105-113.

Ryan Farid, Matthew W. Dixon, Leann Tilley, James S McCarthy, Initiation of gametocytogenesis at very low parasite density in Plasmodium falciparum infection, The Journal of Infectious Diseases, Volume 215, Issue 7, 1 April 2017, Pages 1167–1174,

Another reason why gametocytes hide in the bone marrow, is because they find much higher concentrations of hypoxanthine in the bone marrow, 4 times higher than in peripheral blood. Hypoxanthine is commonly a required reagent in malaria parasite cultures, since Plasmodium falciparum requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism.

King ME, Honeysett JM, Howell SB. Regulation of de novo purine synthesis in human bone marrow mononuclear cells by hypoxanthine. J Clin Invest. 1983;72(3):965-970.
Berman P.A., Human L. (1991) Hypoxanthine Depletion Induced by Xanthine Oxidase Inhibits Malaria Parasite Growth in Vitro. In Man VII. Advances in Experimental Medicine and Biology, vol 309A. Springer, Boston, MA

The hemozoin carried by the gametocytes is not carried inside, but on the outside in Granham bodies in the form of whorls. Whatever the mechanism, storing hemozoin in Granham bodies outside the cytoplasm of the parasite could provide intraerythrocytic sexual forms of Plasmodium falciparum additional protection.
Orjih Au, Hemozoin accumulation in Granham bodies of Plasmodium falciparum gametocytes. Parasitol Res 2012 111, 2353-9

Similar granula of hemozoin are found in Schisostoma worms. When these iron-containing granules are degraded this results in the accumulation of a large amount of iron in the vitelline cells and eggs of developed Schistosoma japonicum. So far the only function of Hz that has been reported is to detoxify the free heme in blood-feeding parasites, such as Plasmodium, Schistosome. Hz has been considered a disposal product during the digestion of red blood cells. These studies on Granham bodies and Schistosoma granules suggest a new role for Hz in iron transport.
Sun, J., Hu, W. & Li, C. Beyond heme detoxification: a role for hemozoin in iron transport in S. japonicum . Parasitol Res 112, 2983–2990 (2013). https://doi.org/10.1007/s00436-013-3470-8

The sexual differenciation of asexual trophozoites into gametocytes starts by some trophozoites which are highly loaded in hemozoin. Hemozoin's accumulation during the sexual differentiation phase may thus serve a useful role in the mosquito, perhaps as a source of some essential products and heme.
Ghazi A. Jamjoom. Evidence for a role of hemozoin in metabolism and gametocytogenesis. MalariaWorld Journal, 2017, 8:10. ISSN 2214-4374

The importance of gametocyte iron metabolism is best documented by the fact that transmission is inhibited by iron chelators. This is consequently a target for new transmission blocking strategies.
Patricia Ferrer, Joel Vega-Rodriguez, Antimalarial Iron Chelator FBS0701 Blocks Transmission by Plasmodium falciparum
Gametocyte Activation Inhibition. ASM Journals. Antimicrobial Agents and Chemotherapy. 2015 Vol. 59, No. 3

Hemozoin has been implicated in the modulation of immune responses during malaria infection. Dendritic cells and macrophages are the major source of IL-12. Hemozoin also promotes immunoglobulin G2a antibody responses.

Hemozoin induces dendritic cells to produce IL-12 (after incubation of DC cells with HZ)
Coban C, Ishii KJ, Sullivan DJ, Kumar N. Purified malaria pigment (hemozoin) enhances dendritic cell maturation and modulates the isotype of antibodies induced by a DNA vaccine. Infect Immun. 2002;70(7):3939-3943. doi:10.1128/IAI.70.7.3939-3943.2002

Other authors find that malarial patients produce a significant amount of anti-haemozoin IgM antibodies that can influence the progression of the disease by inhibiting the production of cytokines.
Biswas S, Karmarkar MG, Sharma YD. Antibodies detected against Plasmodium falciparum haemozoin with inhibitory properties to cytokine production. FEMS Microbiol Lett. 2001 Jan 15;194(2):175-9. doi: 10.1111/j.1574-6968.2001.tb09465.x. PMID: 11164304.

Many antibodies interact with heme. This interaction with heme is a strong predictor of different molecular and functional qualities of antibodies and may lead to a considerable broadening of the repertoire of recognized antigens

Lecerf, M., Kanyavuz, A., Rossini, S. et al. Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities. Commun Biol 4, 391 (2021). https://doi.org/10.1038/s42003-021-01931-7
Hadzhieva M, Vassilev T, Bayry J, Kaveri S, Lacroix-Desmazes S, Dimitrov JD. Relationship between natural and heme-mediated antibody polyreactivity. Biochem Biophys Res Commun. 2016 Mar 25;472(1):281-6. doi: 10.1016/j.bbrc.2016.02.112. Epub 2016 Feb 27. PMID: 26926563.

A Bulgarian study showed that upon heme binding, immunoglobulin G gained an ability to interact with previously unrecognized bacterial antigens and intact bacteria. IgG-heme complexes had an enhanced ability to trigger complement-mediated bacterial killing. It was also shown that heme, bound to immunoglobulins, acted as a cofactor in redox reactions.
Dimitrov JD, Roumenina LT, Vassilev TL. Antibodies use heme as a cofactor to extend their pathogen elimination activity and to acquire new effector functions. J Biol Chem. 2007 Sep 14;282(37):26696-26706.

Strange is also the finding that bites of mosquitoes have a strong negative impact on the production of gametocytes, suggesting that parasites are investing prefentially in asexual rather than gametocyte stages. A simultaneous increase of the IgE titers was observed.
Michael J. Donovan, David L. Sacks, Shaden Kamhawi, Uninfected Mosquito Bites Confer Protection against Infection with Malaria Parasites, Infection and Immunity, 2020, Vol. 75, No. 5
Megan B. Vogt, Anismrita Lahon, Mosquito saliva alone has profound effects on the human immune system. PLOS 2018 https://doi.org/10.1371/journal.pntd.0006439
Lawaly R, Konate L, Marrama L, et al. Impact of mosquito bites on asexual parasite density and gametocyte prevalence in asymptomatic chronic Plasmodium falciparum infections and correlation with IgE and IgG titers. Infect Immun. 2012;80(6):2240-2246. doi:10.1128/IAI.06414-11

The persistence of hemozoin for more than 200 days in liver and spleen after clearance of Plasmodium infections may have a long lasting effect on immunoglobulins.
Frita R, Carapau D, Mota MM, Hänscheid T. In Vivo Hemozoin Kinetics after Clearance of Plasmodium berghei Infection in Mice. Malar Res Treat. 2012;2012:373086. doi: 10.1155/2012/373086. Epub 2012 Apr 11. PMID: 22567535; PMCID: PMC3337493.