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Hemozoin, sporozoites and immunity.

June 20, 2021 - 09:53 -- Pierre Lutgen

Mutaz Akkawi, Jerome Munyangi, Pierre Lutgen

Already 200 years ago malaria pigment, later called hemozoin, was discovered.
Virchow, R. Zur pathologischen Physiologie des Bluts. Archiv f. pathol. Anat. 1, 547–563 (1847). https://doi.org/10.1007/BF02114475

But the persistence of hemozoin for more than 200 days in liver and spleen after clearance of Plasmodium infections was only described recently.
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.
Martin Olivier, Kristin Van Den Ham Malarial pigment hemozoin and the innate inflammatory response Front. Immunol., 05 February 2014 https://doi.org/10.3389/fimmu.2014.00025

Naturally acquired immunity to falciparum malaria protects millions of people routinely exposed to Plasmodium falciparum infection from severe disease and death. There is no clear concept about how this protection works. There is no general agreement about the rate of onset of acquired immunity or what constitutes the key determinants of protection; much less is there a consensus regarding the mechanism(s) of protection.
Doolan DL, Dobaño C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev. 2009;22(1):13-36. doi:10.1128/CMR.00025-08

Blood-stage parasitemia prevents the development of a second liver-stage Plasmodium infection in the same organism.

Spottiswoode N, Duffy PE, Drakesmith H. Iron, anemia and hepcidin in malaria. Front Pharmacol. 2014;5:125. Published 2014 May 30. doi:10.3389/fphar.2014.00125

A study from Portugal has shown that blood-stage parasitaemia, above a minimum threshold, impairs the growth of a subsequent sporozoite infection of liver cells. Blood-stage parasites stimulate the production of the host iron-regulatory factor hepcidin, which redistributes iron away from hepatocytes, reducing the development of the iron-dependent liver stage.
Portugal S, Drakesmith H, Mota MM. Superinfection in malaria: Plasmodium shows its iron will. EMBO Rep. 2011;12(12):1233-1242. Published 2011 Dec 1. doi:10.1038/embor.2011.213

They were also impressed by the long residence time of hemozoin in the body. Considering that the average life span of a mouse is around 850 days, it is impressive that hemozoin could still be detected in mouse organs after 280 days. This represents approximately a quarter of the life span of a mouse. Assuming that the same happens in humans and considering that in malaria endemic regions, such as sub-Saharan Africa, the same individual is likely to get infected several times throughout life, then it is likely that the amount of hemozoin accumulated in organs is enormous.
Frita, Rosangela, Malaria and tuberculosis co-infection: role for hemozoin immunosuppression. Teses de Doutoramento. Universidade de Lisboa, Faculdade de Medicina 2014, http://hdl.handle.net/10451/11435

A prospective community study in a highly malaria endemic area of Papua New Guinea suggest that concurrent or very recent infections provide protection from superinfecting parasites.
al-Yaman F, Genton B, Reeder JC, Anders RF, Smith T, Alpers MP. Reduced risk of clinical malaria in children infected with multiple clones of Plasmodium falciparum in a highly endemic area: a prospective community study. Trans R Soc Trop Med Hyg. 1997 Sep-Oct;91(5):602-5

In a study in Malawi participants had a median follow-up time of 720 days. Persistent asymptomatic infections were associated with decreased risk of malaria illness in all ages.
Buchwald AG, Sixpence A, Chimenya M, et al. Clinical Implications of Asymptomatic Plasmodium falciparum Infections in Malawi. Clin Infect Dis. 2019;68(1):106-112. doi:10.1093/cid/ciy427

Intraleucocytic hemozoin has been suggested as a measure of disease severity in malaria. A study on 146 children aged 6 months to 14 years in 4 categories--cerebral malaria, mild malaria, asymptomatic malaria and 'no malaria'--in Ibadan, Nigeria, an area of intense malaria transmission in Africa. The proportion of hemozoin-containing monocytes did not differ significantly between the mild malaria, asymptomatic malaria and no malaria groups but the cerebral malaria group had a higher median value than the other 3 groups.
O K Amodu , A Adeyemo, P E Olumese, Intraleucocytic malaria pigment and clinical severity of malaria in children. Trans R Soc Trop Med Hyg. Jan-Feb 1998;92(1):54-6. doi: 10.1016/s0035-9203(98)90952-x.

A study from Austria suggests that Kupffer cells control body iron homeostasis by exerting negative regulatory signals toward hepcidin expression. If Kupffer cells are overloaded with hepcidin this control function may be impaired
Theurl M, Theurl I, Hochegger K, Obrist P, Subramaniam N, van Rooijen N, Schuemann K, Weiss G. Kupffer cells modulate iron homeostasis in mice via regulation of hepcidin expression. J Mol Med (Berl). 2008 Jul;86(7):825-35.
Viriyavejakul, P., Khachonsaksumet, V, C. Liver changes in severe Plasmodium falciparum malaria: histopathology, apoptosis and nuclear factor kappa B expression. Malar J 13, 106 (2014)
Pradel, Gabriele & Frevert, Ute. (2001). Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion. Hepatology. 33. 1154 - 1165. 10.1053/jhep.2001.24237.

Kupffer cells (KC) are macrophages. They reside within the lumen of the liver sinusoids and are the first point of contact for incoming pathogen loaded blood. Upon activation KC release various products, including cytokines, nitric oxide and reactive oxygen species. These factors influence neighboring cells, such as hepatocytes.
Bilzer, M., Roggel, F. and Gerbes, A.L. (2006), Role of Kupffer cells in host defense and liver disease. Liver International, 26: 1175-1186. https://doi.org/10.1111/j.1478-3231.2006.01342.x
L. A. Gonçalves, J. Rodo, L. Rodrigues-Duarte, L. V. de Moraes, and C. Penha-Gonçalves, “HGF secreted by activated Kupffer cells induces apoptosis of Plasmodium-infected hepatocytes,” Frontiers in Immunology, vol. 8, 2017.

Already in 1964 it was known that Kupffer cells pick up large amounts of hemozoin when schizonts rupture. Kupffer cells show marked hypertrophy and hyperplasia and are filled with malarial pigment.
Aikawa, Masamichi, Tatiana T. Antonovych. “Electron Microscopic Observations of Plasmodium Berghei and the Kupffer Cell in the Liver of Rats.” The Journal of Parasitology, vol. 50, no. 5, 1964, pp. 620–629.
Nobes MS, Ghabrial H, Simms KM, Smallwood RB, Morgan DJ, Sewell RB. Hepatic Kupffer cell phagocytotic function in rats with erythrocytic-stage malaria. J Gastroenterol Hepatol. 2002 May;17(5):598-605. doi: 10.1046/j.1440-1746.2002.02742.x. PMID: 12084035.

The most generalized feature observed throughout the liver is Kupffer cell hyperplasia with malaria hemozoin deposition. In mouse malaria models, the highest Hz levels are found in the liver compared to other organs including the spleen, and total Hz levels increase with disease severity
Rupani AB, Amarapurkar AD. Hepatic changes in fatal malaria: an emerging problem. Ann Trop Med Parasitol. 2009 Mar;103(2):119-27. doi: 10.1179/136485909X385054. PMID: 19208296.

Kupffer cells have the ability to profoundly affect hepatocyte function, while hepatocytes, in turn, possess the capacity to modify Kupffer cell function. Nitric oxide, produced from the amino acid L-arginine, is a short-lived radical that is a potent mediator of cellular function and cell-cell interaction, and is synthesized by both Kupffer cells and hepatocytes. The hemozoin load in the Kupffer cells will impair this cell-cell interaction
Harbrecht BG, Billiar TR. The role of nitric oxide in Kupffer cell-hepatocyte interactions. Shock. 1995 Feb;3(2):79-87. PMID: 7538434.

Another study concludes that ingestion and metabolism of parasitized erythrocytes by liver macrophages during malaria infection decreases the sporozoite organelle motion.
Bellows CF, Molina RM, Brain JD. Diminished organelle motion in murine Kupffer cells during the erythrocytic stage of malaria. J R Soc Interface. 2011 May 6;8(58):711-9. doi: 10.1098/rsif.2010.0260. Epub 2010 Nov 10.

Intravenous injection of Plasmodium falciparum-derived hemozoin in malaria-free mice induced inflammation.Hemozoin also generates IL-6 and reactive oxygen species (ROS) which migh kill the sporozoite when it passes through the Kupffer cell.
Deroost K, Lays N, Pham T-T, Baci D, Van den Eynde K, et al. (2014) Hemozoin Induces. Hepatic Inflammation in Mice and Is Differentially Associated with Liver Pathology Depending on the Plasmodium Strain. PLoS ONE 9(11): e113519.doi:10.1371/journal.pone.0113519
Lamikanra AA, Theron M, Kooij TWA, Roberts DJ (2009) Hemozoin (Malarial Pigment) Directly Promotes Apoptosis of Erythroid Precursors. PLoSONE 4(12): e8446. doi:10.1371/journal.pone.0008446
Velagapudi, R., Kosoko, A.M. & Olajide, O.A. Induction of Neuroinflammation and Neurotoxicity by Synthetic Hemozoin. Cell Mol Neurobiol 39, 1187–1200 (2019). https://doi.org/10.1007/s10571-019-00713-4

This inflammatory effect may affect the apicoplast of the sporozoites and the merozoites in the liver, leading to a phenomenon known as delayed death. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells which arrest intraerythrocytic development of merozoites after one cycle.
Kennedy K, Cobbold SA, Hanssen E, et al. Delayed death in the malaria parasite Plasmodium falciparum is caused by disruption of prenylation-dependent intracellular trafficking. PLoS Biol. 2019;17(7):e3000376. Published 2019 Jul 18. doi:10.1371/journal.pbio.3000376

Hemozoin is even studied as adjuvant in malaria vaccines. It enhances or modulates immunoglobulins.
Coban C, Yagi M, Ohata K, Igari Y, Tsukui T, Horii T, Ishii KJ, Akira S. The malarial metabolite hemozoin and its potential use as a vaccine adjuvant. Allergol Int. 2010 Jun;59(2):115-24. doi: 10.2332/allergolint.10-RAI-0194. Epub 2010 Apr 24. PMID: 20414048.

In immunized animals, macrophages remove and destroy antibody-coated sporozoites. Sporozoites coated with antibodies degenerate within vacuoles of the macrophages.
Danforth HD, Aikawa M, Cochrane AH, Nussenzweig RS. Sporozoites of mammalian malaria: attachment to, interiorization and fate within macrophages. J Protozool. 1980 May;27(2):193-202

The antibody response to sporozoites of Plasmodium falciparum and the role of these antibodies in protection against malaria have not been systematically investigated. In a prospective study in Thailand, an antibody response to sporozoites was observed only in individuals who developed parasitemia. Antibodies were detected against an epitope in the repeat region of the circumsporozoite (CSP) protein.
Webster HK, Brown AE, Chuenchitra C, Permpanich B, Pipithkul J. Characterization of antibodies to sporozoites in Plasmodium falciparum malaria and correlation with protection. J Clin Microbiol. 1988 May;26(5):923-7.
Brown AE, Webster HK, Pavanand K, Sattabongkot J, Gingrich JB. Comparison of antibody responses to the circumsporozoite protein repeat region and to intact sporozoites during acute falciparum malaria. Trans R Soc Trop Med Hyg. 1989 Mar-Apr;83(2):154-7.

Analysis of serum from eight human volunteers that were immunized via the bites of Plasmodium falciparum infected mosquitoes, revealed five that developed specific antibodies against sporozoites and were completely protected against subsequent challenge. In contrast, in three volunteers not protected, antibodies were below the level of detection. Moreover, these antibodies were detected in the plasma of 83% of the individuals living in a malaria endemic area of Kenya. Furthermore, these antibodies conferred greater inhibition of sporozoites entry into HepG2-A16 cells in vitro.
Nguyen, T.V., Sacci, J.B., de la Vega, P. et al. Characterization of immunoglobulin G antibodies to Plasmodium falciparum sporozoite surface antigen MB2 in malaria exposed individuals. Malar J 8, 235 (2009).

Antibodies generated against Plasmodium falciparum CSP have been shown to block sporozoite invasion of hepatocytes and protection afforded by some vaccins.
Rodríguez-Galán, A., Salman, A.M., Bowyer, G. et al. An in vitro assay to measure antibody-mediated inhibition of P. berghei sporozoite invasion against P. falciparum antigens. Sci Rep 7, 17011 (2017). 5

The University of AlQuds has done extensive work on the inhibition of hemozoin by Artemisia plants and it is planned to further investigate this effect on the sporozoite invasion.
Akkawi M, Jaber S, Abu-Remeleh Q, Engeu OP, Lutgen P (2014). Investigations of Artemisia Annua and Artemisia Sieberi Water Extracts Inhibitory Effects on β-Hematin Formation. Med Aromat Plants 3: 150. doi: 10.4172/2167-0412.1000150

Presently the work of the team of Dominique Mazier, (as posted by the Sorbonne laboratory on internet May 5, 2021) may shed some light on the development of parasites in the liver, in a comparative in vitro assessment of the anti-malarial effect of Artemisia annua and Artemisia afra infusions on the pre-erythrocytic stages of Plasmodium falciparum.