Periodic Reporting for period 4 - BoneMalar (Mechanisms of bone marrow sequestration during malaria infection)
Berichtszeitraum: 2020-12-01 bis 2022-05-31
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. 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.
The major focus of the proposed research is to investigate the mechanism of bone marrow adherence and endothelial transmigration in malaria parasites. 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 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. 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.
The major focus of the proposed research is to investigate the mechanism of bone marrow adherence and endothelial transmigration in malaria parasites. 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.
We have demonstrated that gametocytes, the only stage capable of malaria transmission to the mosquito vector, develop in the extravascular environment of the human bone marrow. Preliminary studies in the mouse model have confirmed this finding and suggest existence of an asexual parasite reservoir in the bone marrow. In this innovative multidisciplinary proposal, we aimed to investigate the host pathogen interactions at the interface between infected red blood cell and bone marrow vasculature. Specifically, we focused on the following questions: how do parasites home to bone marrow? What are the changes in the bone marrow endothelium upon infection? How do parasites adhere with and transmigrate across the vascular endothelium in the bone marrow?
In aim 1 we proposed to define the specificity of parasite sequestration and endothelial activation in bone marrow. We have concluded several studies to address this aim. Specifically, we have used stage-specific parasite reporter lines and quantified their distribution across tissues, including bone marrow in the mouse model, demonstrating gametocyte enrichment in bone marrow, spleen and liver. We have also measured the homing of individual parasite stages to specific tissues, including bone marrow and spleen. These experiments further confirmed that bone marrow and spleen are the major sequestration sites for P. berghei gametocytes, in particular the extravascular environment. They also demonstrated that ring and merozoite stages are the stages preferentially homing to bone marrow. Indeed, a subset of merozoites were found within bone marrow resident erythroid cells, demonstrating their homing and subsequent invasion in bone marrow. This work has been published in 2018 (De Niz et al, Sci Adv, 2018). In a next series of experiments, we have performed a systematic study to investigate parasite infection in blood and haematopoietic tissues of spleen and bone marrow, in the rodent malaria model Plasmodium berghei. Specifically, we have used a combination of flow cytometry and single cell RNAseq to quantify the distribution of parasite stages and host cell types across the three locales and over time. These studies defined the host response upon parasite infection and parasite transcriptional signatures according to host cell. Specifically, we defined a host cell tropism for parasite invasion, metabolism and stage conversion in P. berghei. This work has been published in 2022 (Hentzschel et al, Sci Adv, 2022).
In aim 2 we proposed to define the signature of vascular activation in bone marrow upon malaria infection. In the mouse model, we have been able to visualize the extent of vascular leakage across tissues using fluorescent dextran, and demonstrated that it is limited to bone marrow, spleen and liver. This work has been published (De Niz et al., Sci Adv 2018). In in vitro assays with P. falciparum we were able to identify a series of candidate antigens on the gametocyte-infected RBC surface, most of them shared with asexual parasites. This work is also published (Dantzler et al., Sci Trans Med, 2019). Using the mouse model P. berghei we have also concluded a study focusing on parasite vascular sequestration and host responses, revealing a link between sequestration in adipose tissue, activation of the adipokine leptin and cerebral malaria. This work was performed in collaboration with Prof. Jay Mitchell and published recently (Mejia et al., Sci Adv 2021).
In aim 3 we proposed to elucidate mechanisms by which malaria parasites enter and exit the bone marrow parenchyma. We defined a series of mobility phenotypes of mature gametocytes in bone marrow and spleen, including their deformability and passage across the endothelial barrier. We also demonstrated that specific receptor-ligand interactions are required for merozoite homing to bone marrow, in particular P-selectin. This work has been published (De Niz et al., Sci Adv 2018).
Finally, we have published a review article (Venugopal et al., Nat Rev Micro 2020), providing the first overview of the new paradigm of haematopoietic infection in malaria.
In aim 1 we proposed to define the specificity of parasite sequestration and endothelial activation in bone marrow. We have concluded several studies to address this aim. Specifically, we have used stage-specific parasite reporter lines and quantified their distribution across tissues, including bone marrow in the mouse model, demonstrating gametocyte enrichment in bone marrow, spleen and liver. We have also measured the homing of individual parasite stages to specific tissues, including bone marrow and spleen. These experiments further confirmed that bone marrow and spleen are the major sequestration sites for P. berghei gametocytes, in particular the extravascular environment. They also demonstrated that ring and merozoite stages are the stages preferentially homing to bone marrow. Indeed, a subset of merozoites were found within bone marrow resident erythroid cells, demonstrating their homing and subsequent invasion in bone marrow. This work has been published in 2018 (De Niz et al, Sci Adv, 2018). In a next series of experiments, we have performed a systematic study to investigate parasite infection in blood and haematopoietic tissues of spleen and bone marrow, in the rodent malaria model Plasmodium berghei. Specifically, we have used a combination of flow cytometry and single cell RNAseq to quantify the distribution of parasite stages and host cell types across the three locales and over time. These studies defined the host response upon parasite infection and parasite transcriptional signatures according to host cell. Specifically, we defined a host cell tropism for parasite invasion, metabolism and stage conversion in P. berghei. This work has been published in 2022 (Hentzschel et al, Sci Adv, 2022).
In aim 2 we proposed to define the signature of vascular activation in bone marrow upon malaria infection. In the mouse model, we have been able to visualize the extent of vascular leakage across tissues using fluorescent dextran, and demonstrated that it is limited to bone marrow, spleen and liver. This work has been published (De Niz et al., Sci Adv 2018). In in vitro assays with P. falciparum we were able to identify a series of candidate antigens on the gametocyte-infected RBC surface, most of them shared with asexual parasites. This work is also published (Dantzler et al., Sci Trans Med, 2019). Using the mouse model P. berghei we have also concluded a study focusing on parasite vascular sequestration and host responses, revealing a link between sequestration in adipose tissue, activation of the adipokine leptin and cerebral malaria. This work was performed in collaboration with Prof. Jay Mitchell and published recently (Mejia et al., Sci Adv 2021).
In aim 3 we proposed to elucidate mechanisms by which malaria parasites enter and exit the bone marrow parenchyma. We defined a series of mobility phenotypes of mature gametocytes in bone marrow and spleen, including their deformability and passage across the endothelial barrier. We also demonstrated that specific receptor-ligand interactions are required for merozoite homing to bone marrow, in particular P-selectin. This work has been published (De Niz et al., Sci Adv 2018).
Finally, we have published a review article (Venugopal et al., Nat Rev Micro 2020), providing the first overview of the new paradigm of haematopoietic infection in malaria.
The work supported by this grant established a new paradigm in malaria research, namely the infection of hematopoietic niche as major reservoir for malaria parasites. Most significant achievements include:
- Identification of the hematopoietic niche as major reservoir for malaria transmission stages in human and rodent parasites
- Identification of an asexual replication cycle in the hematopoietic niche
- Identification of motile gametocytes that are capable of endothelial transmigration
- First dual single cell RNAseq study in the malaria field
- First characterisation of immunogenic parasite antigens on the gametocyte-infected red blood cell surface
- Identification of the hematopoietic niche as major reservoir for malaria transmission stages in human and rodent parasites
- Identification of an asexual replication cycle in the hematopoietic niche
- Identification of motile gametocytes that are capable of endothelial transmigration
- First dual single cell RNAseq study in the malaria field
- First characterisation of immunogenic parasite antigens on the gametocyte-infected red blood cell surface