Periodic Reporting for period 3 - BoneMalar (Mechanisms of bone marrow sequestration during malaria infection)
Période du rapport: 2019-06-01 au 2020-11-30
Plasmodium falciparum causes the most severe form of malaria with almost one million deaths every year, most of them in sub-Saharan Africa. The pathology of the disease can be attributed to the asexual stage of the parasite’s life cycle completed within red blood cells. A small subset of asexual parasites becomes committed to the sexual cycle, and these committed parasites produce sexual progeny, or gametocytes, that develop into mature male and female sexual forms.
Mature gametocytes (termed Stage V) are present in the human blood circulation and responsible for transmission. During the preceding 6-8 days immature gametocyte develop and undergo a morphological transformation (termed Stages I-IV) while sequestering in tissue. The transmission-competent Stage V gametocytes are ingested by the mosquito vector and undergo maturation into gametes and fertilization in the mosquito mid gut. Transmission represents a bottleneck in the parasite’s life cycle and an important target for intervention strategies.
In the human host, tissue sequestration is of fundamental importance for both sexual and asexual red blood cell stage parasites in order to avoid clearance by the spleen. The intravascular space of the microcirculation also provides an ideal nutritive and microaerophilic environment necessary to support rapid parasite proliferation and expansion. Tissue-specific sequestration of asexual parasite stages results in pathology such as cerebral malaria and pregnancy associated
malaria. Sequestration of red blood cells infected with asexual parasites involves endothelial cytoadherence that is the result of specific receptor-ligand interactions. Tissue-specific parasite sequestration and associated pathology have been recapitulated to some extent in the mouse malaria model and in non-human primates.
In a systematic autopsy tissue study that we performed with Prof. Terrie Taylor and colleagues in Blantyre, Malawi, we identified a new sequestration site in the extravascular environment of the human bone marrow (BM) during P. falciparum infection. Preliminary studies in collaboration with Dr. Volker Heussler (University of Berne) have also demonstrated a similar phenotype in the mouse malaria model. These combined data suggested that the BM extravascular environment (and possibly that of the spleen) is reservoir both for asexual parasite replication and development of transmission stages, with potential implications for antimalarial therapy and the emergence of drug resistance. We were also able to provide initial evidence for unique parasite movement and endothelial transmigration behaviour in BM and spleen, suggesting specialized interactions between infected red blood cells (iRBCs) and endothelial cells in these 2 organs.
Pathogenesis and clinical symptoms in malaria infection are mediated by an extensive inflammatory response and vascular dysfunction. We hypothesize that these events are not merely ‘side-effects’ of infection, but rather that the parasite has evolved to purposefully drive and subvert these reactions to support its survival, expansion and ultimately transmission: Initial sequestration of infected RBCs in the microvasculature provides escape from clearance and microaerophilic environment that supports rapid expansion of asexual progeny, which is necessary to generate sufficient numbers of gametocytes (that derive from this progeny at low rates) for successful transmission. Maturation of gametocytes is associated with parasite exit from the intravascular space and into the unique environment of the BM parenchyma, and transmission requires return of mature gametocytes to the blood circulation. These critical processes of adhesion to the luminal surface of the vasculature and egress in and out of the circulation are reminiscent of the normal process of immune cell trafficking during normal protective immune and inflammatory responses. Our own preliminary data, along with existing and underappreciated published data (e.g. biopsy imaging studies) further suggest that specific cell biological remodelling processes involved in immune cell trafficking may be employed for iRBC trafficking. Thus, we hypothesize that both the expansion of asexual parasites (pathology) and the maturation of sexual gametocytes (transmission) are integrally linked as the purposeful subversion of the inflammatory cascade and specific mechanisms that normally drive immune cell trafficking. A corollary to this hypothesis is that keys to both treatment and eradication lie in dissecting such subversion mechanisms, as we propose to do herein.
The major focus of the proposed research is to investigate the mechanism of bone marrow adherence and endothelial transmigration in malaria parasites. For this purpose we will test two alternative hypotheses for parasite transmigration from the vasculature into the bone marrow parenchyma (e.g. extravasation) and one hypothesis for transmigration of mature gametocytes from the parenchyma back into the vasculature (e.g. intravasation). The specific aims are as follows: 1) Define specificity of parasite sequestration in the bone marrow, 2) Define the signature of vascular activation in bone marrow upon malaria infection, and 3) Elucidate mechanisms by which malaria parasites enter and exit the bone marrow parenchyma.
Mature gametocytes (termed Stage V) are present in the human blood circulation and responsible for transmission. During the preceding 6-8 days immature gametocyte develop and undergo a morphological transformation (termed Stages I-IV) while sequestering in tissue. The transmission-competent Stage V gametocytes are ingested by the mosquito vector and undergo maturation into gametes and fertilization in the mosquito mid gut. Transmission represents a bottleneck in the parasite’s life cycle and an important target for intervention strategies.
In the human host, tissue sequestration is of fundamental importance for both sexual and asexual red blood cell stage parasites in order to avoid clearance by the spleen. The intravascular space of the microcirculation also provides an ideal nutritive and microaerophilic environment necessary to support rapid parasite proliferation and expansion. Tissue-specific sequestration of asexual parasite stages results in pathology such as cerebral malaria and pregnancy associated
malaria. Sequestration of red blood cells infected with asexual parasites involves endothelial cytoadherence that is the result of specific receptor-ligand interactions. Tissue-specific parasite sequestration and associated pathology have been recapitulated to some extent in the mouse malaria model and in non-human primates.
In a systematic autopsy tissue study that we performed with Prof. Terrie Taylor and colleagues in Blantyre, Malawi, we identified a new sequestration site in the extravascular environment of the human bone marrow (BM) during P. falciparum infection. Preliminary studies in collaboration with Dr. Volker Heussler (University of Berne) have also demonstrated a similar phenotype in the mouse malaria model. These combined data suggested that the BM extravascular environment (and possibly that of the spleen) is reservoir both for asexual parasite replication and development of transmission stages, with potential implications for antimalarial therapy and the emergence of drug resistance. We were also able to provide initial evidence for unique parasite movement and endothelial transmigration behaviour in BM and spleen, suggesting specialized interactions between infected red blood cells (iRBCs) and endothelial cells in these 2 organs.
Pathogenesis and clinical symptoms in malaria infection are mediated by an extensive inflammatory response and vascular dysfunction. We hypothesize that these events are not merely ‘side-effects’ of infection, but rather that the parasite has evolved to purposefully drive and subvert these reactions to support its survival, expansion and ultimately transmission: Initial sequestration of infected RBCs in the microvasculature provides escape from clearance and microaerophilic environment that supports rapid expansion of asexual progeny, which is necessary to generate sufficient numbers of gametocytes (that derive from this progeny at low rates) for successful transmission. Maturation of gametocytes is associated with parasite exit from the intravascular space and into the unique environment of the BM parenchyma, and transmission requires return of mature gametocytes to the blood circulation. These critical processes of adhesion to the luminal surface of the vasculature and egress in and out of the circulation are reminiscent of the normal process of immune cell trafficking during normal protective immune and inflammatory responses. Our own preliminary data, along with existing and underappreciated published data (e.g. biopsy imaging studies) further suggest that specific cell biological remodelling processes involved in immune cell trafficking may be employed for iRBC trafficking. Thus, we hypothesize that both the expansion of asexual parasites (pathology) and the maturation of sexual gametocytes (transmission) are integrally linked as the purposeful subversion of the inflammatory cascade and specific mechanisms that normally drive immune cell trafficking. A corollary to this hypothesis is that keys to both treatment and eradication lie in dissecting such subversion mechanisms, as we propose to do herein.
The major focus of the proposed research is to investigate the mechanism of bone marrow adherence and endothelial transmigration in malaria parasites. For this purpose we will test two alternative hypotheses for parasite transmigration from the vasculature into the bone marrow parenchyma (e.g. extravasation) and one hypothesis for transmigration of mature gametocytes from the parenchyma back into the vasculature (e.g. intravasation). The specific aims are as follows: 1) Define specificity of parasite sequestration in the bone marrow, 2) Define the signature of vascular activation in bone marrow upon malaria infection, and 3) Elucidate mechanisms by which malaria parasites enter and exit the bone marrow parenchyma.
In this first project period we have developed a series of tools and focused on aims 1 and 3. The combined data from these activities has been published in De Niz et al, Sci Adv 2018.
With regards to aim 1, we first established the rodent malaria model, Plasmodium berghei to investigate parasite sequestration in the bone marrow. Specifically we used a series of stage-specific reporter parasite lines in combination with transgenic reporter mice and a series of imaging approaches and quantified parasite stage composition across tissues. These experiments demonstrated specific enrichment of immature gametocytes in the bone marrow extravascular space (parenchyma and sinusoids) while asexual parasites were found across various tissues including extravascular niches of bone marrow and spleen. In parallel we further investigated P. falciparum sequestration in the bone marrow of human autopsy cases. These experiments identified a significant reservoir of asexual parasites in the extravascular niche, supporting the data from the rodent malaria model. In a separate study not funded through this award, a similar phenotype was observed with another human malaria parasite, P. vivax in the non-human primate model (Obaldia et al, mBio 2018).
With regards to aim 3, we again made use of the rodent malaria model to first determine which stage predominantly reaches the bone marrow extravascular niche. We were able to show that the majority of parasites reach the extravascular niche as invasive merozoites, however infected RBCs can also pass, in particular when the endothelial barrier becomes leaky during inflammation. Initial experiments also suggested involvement of specific receptor-ligand interactions during merozoite homing and/or transmigration. We also noted efficient transmigration across the bone marrow endothelial barrier of mature gametocytes. These stages exhibited mobile behavior in bone marrow and spleen, similar to leukocytes, that appeared to depend on an active cytoskeleton.
With regards to aim 1, we first established the rodent malaria model, Plasmodium berghei to investigate parasite sequestration in the bone marrow. Specifically we used a series of stage-specific reporter parasite lines in combination with transgenic reporter mice and a series of imaging approaches and quantified parasite stage composition across tissues. These experiments demonstrated specific enrichment of immature gametocytes in the bone marrow extravascular space (parenchyma and sinusoids) while asexual parasites were found across various tissues including extravascular niches of bone marrow and spleen. In parallel we further investigated P. falciparum sequestration in the bone marrow of human autopsy cases. These experiments identified a significant reservoir of asexual parasites in the extravascular niche, supporting the data from the rodent malaria model. In a separate study not funded through this award, a similar phenotype was observed with another human malaria parasite, P. vivax in the non-human primate model (Obaldia et al, mBio 2018).
With regards to aim 3, we again made use of the rodent malaria model to first determine which stage predominantly reaches the bone marrow extravascular niche. We were able to show that the majority of parasites reach the extravascular niche as invasive merozoites, however infected RBCs can also pass, in particular when the endothelial barrier becomes leaky during inflammation. Initial experiments also suggested involvement of specific receptor-ligand interactions during merozoite homing and/or transmigration. We also noted efficient transmigration across the bone marrow endothelial barrier of mature gametocytes. These stages exhibited mobile behavior in bone marrow and spleen, similar to leukocytes, that appeared to depend on an active cytoskeleton.
The De Niz et al publication provides several novel and surprising findings and represents a landmark study in malaria research. In particular the notion of merozoite homing and endothelial transmigration, as well as the observed mobility of mature gametocytes open up new areas for investigation in vitro and in animal models. We have also developed a series of tools, including intravital imaging in the bone marrow on infected mice and a Nanostring expression platform to measure parasite signatures in hist tissues. These and other cutting edge tools will be further applied in the second project period. In this period we will focus on mechanistic questions, in particular host parasite interactions at the endothelial interface and endothelial activation and function in the bone marrow.