Eryptosis as protection factor against malaria

Abstract

Over the past centuries malaria, a dreadful pathogen born blood related disease has been the major cause of mortality among mankind. As time passed the pathogens coevolved with their hosts, simultaneously manipulating the hosts’ defence mechanisms by intruding and mimicking various host related metabolic and signaling pathways. In the course of their parasitic effect they modified themselves to proliferate and destruct the host to death. The present strategy for controlling and curing infectious diseases has targeted various metabolic or enzymatic systems within the parasite. The most severe drawback of this method of controlling the diseases has led to the development of parasitic resistance and consequent relapse of once-contained infectious diseases amidst the host. I intended for a novel drug discovery paradigm in order to prevent the pathogen related resistance focusing on identifying and targeting host factors essential for pathogen entry, survival and proliferation. The innovative methods involve stimulation of the infected erythrocytes and recognition by the spleen macrophages to get rid of the pathogen and prevent the further course of the disease. In case of malaria the pathogen, Plasmodium, enters erythrocytes and thus escapes recognition by the immune system. The pathogen induces oxidative stress to the host erythrocyte, which triggers eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are identified by macrophages which engulf and degrade the eryptotic cells. To the extent that infected erythrocytes undergo eryptosis prior to exit of Plasmodia and subsequent infection of other erythrocytes, the premature eryptosis may protect against malaria. Accordingly, any therapeutical intervention accelerating suicidal death of infected erythrocytes has the potential to foster elimination of infected erythrocytes, delay the development of parasitemia and favorably influence the course of malaria. Eryptosis is stimulated by a wide variety of triggers including osmotic shock, oxidative stress, energy depletion and a wide variety of xenobiotics. Diseases associated with accelerated eryptosis include sepsis, haemolytic uremic syndrome, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency and Wilson’s disease. Among the known stimulators of eryptosis, paclitaxel, chlorpromazine, cyclosporine, curcumin, azathioprine, amiodarone, anandamide, PGE2 and lead have indeed been shown to favourably influence the course of malaria. Moreover, sickle-cell trait, beta-thalassemia trait, glucose-6-phosphate dehydrogenase (G6PD)-deficiency and iron deficiency confer some protection against a severe course of malaria. The therapeutic agents were chosen on the basis of their proved eryptotic activity, which allowed rapid identification of new and existing licensed drugs for host mediated antimalarial therapy. Importantly, counteracting Plasmodia by inducing eryptosis is not expected to generate resistance of the pathogen, as the proteins involved in suicidal death of the host cell are not encoded by the pathogen and thus cannot be modified by mutations of its genes. The present studies have been conducted to investigate whether the subcutaneous administration of azathioprine or aurothiomalate may modify the course of malaria and survival of Plasmodium berghei -infected mice and further to examine if intraperitoneal administration of amiodarone influence the course of malaria. In vitro infection of human erythrocytes with Plasmodium falciparum increased annexin V binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content in vitro parasitemia at (1 μM) concentration. Administration of azathioprine significantly decreased the parasitemia of circulating erythrocytes and increased the survival of Plasmodium berghei infected mice (from 0% to 77% 22 days after infection). Exposure to aurothiomalate significantly decreased the in vitro parasitemia of P. falciparum-infected human erythrocytes without influencing the intraerythrocytic DNA/RNA content. Administration of sodium aurothiomalate in vivo (daily 10 mg/kg b.w. s.c. from the 8th day of infection) enhanced the percentage of phosphatidylserine exposing infected and noninfected erythrocytes in blood. All non-treated mice died within 30 days of infection. Aurothiomalate-treatment delayed the lethal course of malaria leading to survival of more than 50% of the mice 30 days after infection. The in vitro infection of human erythrocytes with P. falciparum (strain BinH) increased annexin V-binding, an effect significantly augmented by amiodarone (10 µM). Amiodarone further significantly decreased intraerythrocytic DNA/RNA content (≥ 5 µM) and in vitro parasitemia (≥ 1µM). Following infection of mice with Plasmodium berghei ANKA by intraperitoneal injection of parasitized murine erythrocytes (1x106) amiodarone (intraperitoneal 50 mg/kg b.w) significantly decreased the parasitemia and increased the survival of P. berghei infected mice (from 0% to 70% 26 days after infection). Moreover, treatment with amiodarone significantly increased the percentage of PS-exposing infected erythrocytes

In conclusion azathioprine, aurothiomalate and amiodarone stimulate the erythrocytic machinery responsible for the eryptosis following infection with Plasmodium. The exhilaration of eryptosis precedes the full intraerythrocytic maturation of the pathogen, thus preventing the further lethal course of the disease and fosters host survival during malaria. The revelations defend that the stimulation of eryptosis in infected erythrocytes is a host dependent mechanism to combat against infection. The experimental results not only justify that the stimulation of eryptosis in infected erythrocytes is not only a host dependent defence mechanism but also a novel approach to prevent the chances of resistance in plasmodia. This method of identifying new host mediated antimalarial agents may be combined with the regular antimalarial agents with host directed drug therapy and increase the efficacy in eliminating the invaded and invading pathogen, thus control the resurgence of once contained disease.

Over the past centuries malaria, a dreadful pathogen born blood related disease has been the major cause of mortality among mankind. As time passed the pathogens coevolved with their hosts, simultaneously manipulating the hosts’ defence mechanisms by intruding and mimicking various host related metabolic and signaling pathways. In the course of their parasitic effect they modified themselves to proliferate and destruct the host to death. The present strategy for controlling and curing infectious diseases has targeted various metabolic or enzymatic systems within the parasite. The most severe drawback of this method of controlling the diseases has led to the development of parasitic resistance and consequent relapse of once-contained infectious diseases amidst the host. I intended for a novel drug discovery paradigm in order to prevent the pathogen related resistance focusing on identifying and targeting host factors essential for pathogen entry, survival and proliferation. The innovative methods involve stimulation of the infected erythrocytes and recognition by the spleen macrophages to get rid of the pathogen and prevent the further course of the disease. In case of malaria the pathogen, Plasmodium, enters erythrocytes and thus escapes recognition by the immune system. The pathogen induces oxidative stress to the host erythrocyte, which triggers eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are identified by macrophages which engulf and degrade the eryptotic cells. To the extent that infected erythrocytes undergo eryptosis prior to exit of Plasmodia and subsequent infection of other erythrocytes, the premature eryptosis may protect against malaria. Accordingly, any therapeutical intervention accelerating suicidal death of infected erythrocytes has the potential to foster elimination of infected erythrocytes, delay the development of parasitemia and favorably influence the course of malaria. Eryptosis is stimulated by a wide variety of triggers including osmotic shock, oxidative stress, energy depletion and a wide variety of xenobiotics. Diseases associated with accelerated eryptosis include sepsis, haemolytic uremic syndrome, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency and Wilson’s disease. Among the known stimulators of eryptosis, paclitaxel, chlorpromazine, cyclosporine, curcumin, azathioprine, amiodarone, anandamide, PGE2 and lead have indeed been shown to favourably influence the course of malaria. Moreover, sickle-cell trait, beta-thalassemia trait, glucose-6-phosphate dehydrogenase (G6PD)-deficiency and iron deficiency confer some protection against a severe course of malaria. The therapeutic agents were chosen on the basis of their proved eryptotic activity, which allowed rapid identification of new and existing licensed drugs for host mediated antimalarial therapy. Importantly, counteracting Plasmodia by inducing eryptosis is not expected to generate resistance of the pathogen, as the proteins involved in suicidal death of the host cell are not encoded by the pathogen and thus cannot be modified by mutations of its genes. The present studies have been conducted to investigate whether the subcutaneous administration of azathioprine or aurothiomalate may modify the course of malaria and survival of Plasmodium berghei -infected mice and further to examine if intraperitoneal administration of amiodarone influence the course of malaria. In vitro infection of human erythrocytes with Plasmodium falciparum increased annexin V binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content in vitro parasitemia at (1 μM) concentration. Administration of azathioprine significantly decreased the parasitemia of circulating erythrocytes and increased the survival of Plasmodium berghei infected mice (from 0% to 77% 22 days after infection). Exposure to aurothiomalate significantly decreased the in vitro parasitemia of P. falciparum-infected human erythrocytes without influencing the intraerythrocytic DNA/RNA content. Administration of sodium aurothiomalate in vivo (daily 10 mg/kg b.w. s.c. from the 8th day of infection) enhanced the percentage of phosphatidylserine exposing infected and noninfected erythrocytes in blood. All non-treated mice died within 30 days of infection. Aurothiomalate-treatment delayed the lethal course of malaria leading to survival of more than 50% of the mice 30 days after infection. The in vitro infection of human erythrocytes with P. falciparum (strain BinH) increased annexin V-binding, an effect significantly augmented by amiodarone (10 µM). Amiodarone further significantly decreased intraerythrocytic DNA/RNA content (≥ 5 µM) and in vitro parasitemia (≥ 1µM). Following infection of mice with Plasmodium berghei ANKA by intraperitoneal injection of parasitized murine erythrocytes (1x106) amiodarone (intraperitoneal 50 mg/kg b.w) significantly decreased the parasitemia and increased the survival of P. berghei infected mice (from 0% to 70% 26 days after infection). Moreover, treatment with amiodarone significantly increased the percentage of PS-exposing infected erythrocytes

In conclusion azathioprine, aurothiomalate and amiodarone stimulate the erythrocytic machinery responsible for the eryptosis following infection with Plasmodium. The exhilaration of eryptosis precedes the full intraerythrocytic maturation of the pathogen, thus preventing the further lethal course of the disease and fosters host survival during malaria. The revelations defend that the stimulation of eryptosis in infected erythrocytes is a host dependent mechanism to combat against infection. The experimental results not only justify that the stimulation of eryptosis in infected erythrocytes is not only a host dependent defence mechanism but also a novel approach to prevent the chances of resistance in plasmodia. This method of identifying new host mediated antimalarial agents may be combined with the regular antimalarial agents with host directed drug therapy and increase the efficacy in eliminating the invaded and invading pathogen, thus control the resurgence of once contained disease.

Im Verlauf der letzten Jahrhunderte war Malaria, ein schreckliches Pathogen aus der Reihe der blutsverwandten Krankheiten die Hauptursache für die Sterblichkeit der Menschheit. Mit der Zeit entwickelten sich die Pathogen gemeinsam mit ihren Wirten weiter und manipulierten dabei die Verteidigungsmechanismen des Wirts, indem sie in verschiedene wirtsspezifische Metabolismen und Signalwege eindrangen und sie imitierten. Im Verlauf ihrer parasitären Auswirkung modifizierten sie sich selbst, um zu proliferieren und den Wirt zu töten. Die gegenwärtige Strategie, um infektiöse Krankheiten zu kontrollieren und zu heilen, hat auf verschiedene metabolische oder enzymatische Systeme innerhalb des Parasiten abgezielt. Die schwerwiegendste Einschränkung von dieser Art, die Krankheiten zu kontrollieren, hat zu der Entwicklung von parasitärer Resistenz geführt und konsequentem Rückfall der once-contained infektiösen Krankheiten inmitten des Wirts. Wir zielten auf ein Entwicklungsmodell für eine neuartige Substanz ab, um die Resistenz des Pathogens zu verhindern, indem wir uns darauf konzentrierten, die Wirtsfaktoren, die für den Befall, das Überleben und die Proliferation des Pathogens essentiell sind, zu identifizieren und auf sie abzuzielen. Die innovativen Methoden beinhalten Stimulation der infizierten Erythrozyten und Erkennung der Makrophagen der Milz, um das Pathogen zu entfernen und den weiteren Krankheitsverlauf zu verhindern. Im Fall von Malaria befällt das Pathogen, Plasmodium, Erythrozyten und entgeht somit der Erkennung durch das Immunsystem. Das Pathogen induziert oxidativen Stress in der Wirtserythrozyte, was Eryptose, d.h. Suizid von Erythrozyten, auslöst. Eryptose ist gekennzeichnet durch Zellschrumpfung, Ausstülpung der Membran und Aktivierung der Membranphospholipide mit Oberflächenexpression von Phosphatidylserin. Erythrozyten, die Phosphatidylserin freilegen, werden von Makrophagen identifiziert, die die eryptotischen Zellen einhüllen und abbauen. Insofern infizierte Erythrozyten vor dem Austritt von Plasmodia und folglicher Infektion anderer Erythrozyten Eryptose begehen, vermag die vorzeitige Eryptose vor Malaria zu schützen. Dementsprechend hat jegliche therapeutische Intervention, die den suizidalen Tod von infizierten Erythrozyten beschleunigt, das Potenzial, die Elimination von infizierten Erythrozyten zu fördern, die Entwicklung des Parasitenwachstums zu verzögern und den Verlauf von Malaria günstig zu beeinflussen. Eryptose wird von einer umfangreichen Vielfalt von Auslösern stimuliert, darunter osmotischem Schock, oxidativem Stress, Energiemangel und einer großen Anzahl von Xenobiotika. Krankheiten, die mit beschleunigter Eryptose in Zusammenhang stehen, beinhalten Sepsis, hämolytisch-urämisches Syndrom, Malaria, Sichelzellanämie, ß-Thalassämie, Glucose-6-Phosphat-Dehydrogenase (G6PDH)-Mangel, Phosphatmangel, Eisenmangel und Morbus Wilson. Unter den bekannten Stimulanzien von Eryptose wurde für Paclitaxel, Chlorpromazin, Cyclosporin, Curcumin, Azathioprin, Amiodaron, Anandamid, PGE2 und Blei in der Tat gezeigt, dass sie den Verlauf von Malaria günstig beeinflussen. Darüber hinaus verleihen die Eigenschaften von Sichelzellen und ß-Thalassämie, Glucose-6-Phosphat-Dehydrogenase (G6PDH)-Mangel und Eisenmangel einen gewissen Schutz vor einem schweren Verlauf von Malaria. Die therapeutischen Mittel wurden auf der Basis ihrer überprüften eryptotischen Aktivität ausgewählt, was eine schnelle Identifikation neuer und existierender zugelassener Arzneimittel für eine wirtsvermittelte Antimalaria-Therapie ermöglicht. Es ist wichtig, dass nicht erwartet wird, dass beim Bekämpfen von Plasmodia über eine Induktion der Eryptose eine Resistenz des Pathogens entwickelt wird, weil die Proteine, die beim suizidalen Tod der Wirtszelle beteiligt sind, nicht durch das Pathogen kodiert werden und damit durch Mutationen seiner Gene nicht verändert werden können. Die bisherigen Studien wurden durchgeführt, um zu untersuchen, ob die subkutane Verabreichung von Azathiopron oder Aurothiomalat den Verlauf von Malaria und das Überleben von Mäusen, die mit Plasmodium berghei infiziert wurden, modifizieren kann und weiter, um zu untersuchen, ob intraperitoneale Verabreichung von Amiodaron den Verlauf von Malaria beeinflussen kann. In vitro-Infektion humaner Erythrozyten mit Plasmodium falciparum erhöhte die Annexin V-Bindung und verminderte anfänglich den forward scatter.Effekte, die durch Azathioprin signifikant erhöht werden konnten. Bei höheren Konzentrationen verminderte Azathioprin signifikant den intraerythrozytären DNA/RNA-Gehalt (1 µM) und Parasitenwachstum in vitro (1 µM). Verabreichung von Azathioprin verminderte signifikant die Infektionsrate von zirkulierenden Erythrozyten und erhöhte das Überleben von Mäusen, die mit Plasmodium berghei infiziert wurden (von 0% bis zu 77% 22 Tage nach der Infektion). Einwirkung von Aurothiomalat verminderte signifikant das Parasitenwachstum in vitro von humanen Erythrozyten, die mit P. falciparum-infiziert wurden, ohne den intraerythrozytären DNA/RNA-Gehalt zu beeinflussen. Die Verabreichung von Natriumaurothiomalat in vivo (täglich 10 mg/kg KG s.c. ab dem 8. Tag der Infektion) erhöhte den prozentualen Anteil von Phosphatidylserin-exprimierenden infizierten und nicht-infizierten Erythrozyten im Blut. Alle nicht-behandelten Mäuse starben innerhalb von 30 Tagen nach Infektion. Behandlung mit Aurothiomalat verzögerte den tödlichen Verlauf von Malaria und führte zum Überleben von über 50 % der Mäuse 30 Tage nach Infektion. Die in vitro-Infektion humaner Erythrozyten mit P. falciparum (Linie BinH) erhöhte die Annexin V-Bindung, ein Effekt der durch Amiodaron (10 µM) signifikant verstärkt wurde. Amiodaron verminderte außerdem signifikant den intraerythrozytären DNA/RNA-Gehalt (≥ 5 µM) und Parasitenwachstum in vitro (≥ 1µM). Nachdem Mäuse über eine intraperitoneale Injektion von parasitierten murinen Erythrozyten (1x106) mit Plasmodium berghei ANKA infiziert worden waren, verminderte Amiodaron (intraperitoneal 50 mg/kg KG) signifikant die Infektionsrate und erhöhte das Überleben von Mäusen, die mit P. berghei infiziert wurden (von 0% bis zu 70% 26 Tage nach der Infektion). Darüber hinaus erhöhte die Behandlung mit Amiodaron signifikant die Prozentzahl von Phosphatidylserin-exprimierenden infizierten Erythrozyten. Zusammenfassend stimulieren Azathioprin, Aurothiomalat und Amiodaron den erythrozytären Mechanismus, der für Eryptose infolge von Infektion mit Plasmodium verantwortlich ist. Die Auslösung von Eryptose geht der vollständigen intraerythrozytären Reifung des Pathogens voran, verhindert somit den tödlichen Verlauf der Krankheit und fördert das Überleben des Wirts während Malaria. Die Erkenntnisse sprechen dafür, dass die Stimulation von Eryptose in infizierten Erythrozyten ein wirtsabhängiger Mechanismus ist, um die Infektion zu bekämpfen. Die experimentellen Ergebnisse rechtfertigen, dass die Stimulation von Eryptose in infizierten Erythrozyten nicht nur ein wirtsabhängiger Abwehrmechanismus ist, sondern auch eine neuartige Herangehensweise, um die Resistenzmöglichkeiten in Plasmodia zu verhindern. Diese Methode, neue wirtsvermittelte Antimalariamittel zu identifizieren, kann mit den regulären Antimalariamitteln, die auf den Wirt zielen, kombiniert werden und die Effizienz bei der Elimination der eingedrungenen und eindringenden Pathogene erhöhen und damit das Wiederaufleben einer once contained Erkrankung zu kontrollieren.