Malaria is a mosquito-borne infectious disease caused by parasites of the Plasmodium (P) genus and transmitted through the bite of infected female Anopheles mosquitos. P falciparum (Pf), the most frequent and lethal P parasite in humans, differs from the others for the unique feature that both asexual and sexual developmental stages occur largely within organs rather than in blood. While homing and maturation of asexual parasites occurs virtually in all organs, human bone marrow (BM) is emerging as a privileged site for the development of the sexual parasites (gametocytes). This supports a key role of the human BM in the host-parasite interplay and in depth investigations are needed to elucidate mechanisms and cellular players involved in the process. Changes in plasticity of the Pf gametocyte infected erythrocytes (GIEs), now addressable by genetic and chemical approaches, is thought to play a significant role. However, to recapitulate in proper experimental systems the complex architecture and cellular heterogeneity of the human BM microenvironment is still a challenging issue.
Aim of this proposal is to combine a BM Humanized Mouse Model with GIEs. For the development of this system we will use a) human erythrocytes infected with wild type Pf transgenic lines whose gametocytes express fluorescent reporter genes at specific developmental stages and b) hybrid bone/BM organoids generated from human osteoprogenitor cells grown in "pellet" culture and then transplanted at heterotopic sites into immunodeficient mice. We also plan to test Pf lines mutated for genes involved in the conversion from rigid immature to more deformable mature GIE and/or chemical compounds able to interfere with this process. Through this project we expect to characterize the role of the human BM microenvironment in the homing and maturation of Pf-GIEs and to provide a model to study the mechanisms involved in the transmission of the Pf from humans to the mosquito vectors.
Reduction of malaria burden is currently based on surveillance of endemic transmission and imported malaria cases, cutting down of Anopheles mosquito vector populations and control of human mobility and of the climate change. Recent studies have prompted renewed attention to the biology of the transmission of Pf from infected humans to the mosquito vector. For this to occur, Pf gametocytes (the sexual stages) have to mature in the human body inside erythrocytes. Sequestration and maturation of the Pf gametocytes, which takes approximately 10 days, has been largely thought to occur in internal organ as only mature gametocytes, the stage responsible for parasite transmission to the Anopheles mosquito vector, are seen in the circulating peripheral blood. Recent evidence has accumulated about an unsuspected role of the human BM, and in particular of its extra-vascular compartment, as a privileged site for the sequestration and maturation of the immature Pf gametocytes (1-5). Consequently, it is emerging the intriguing scenario according to which the BM can represent a "niche" not only for hematopoietic stem cells (hematopoietic niche) and neoplastic stem cells (leukemia niche for leukemia stem cells, metastatic niche for BM metastasis-initiating cancer cells) but also for the malaria parasite (malaria niche). Obviously, homing and adaptation of gametocytes to the BM microenvironment requires fine cellular and molecular interactions with resident cells that are currently largely unknown. The possibility to investigate these interactions at both cellular and molecular levels requires, mainly for ethical reasons, emancipation from the need of patients' samples to seek fundamental mechanisms of the parasite adaptation to the BM microenvironment. However, to date, a model to investigate the role of the bone/BM organ in malaria (and more in general in an infectious disease) does not exist. An animal model combining humanized hematopoiesis with humanized endothelial linings and humanized BM stromal component would be ideal.
The BMHMM, based on the growth of human BM-derived osteoprogenitor cells in "pellet" culture followed by transplantation of the chondroid rudiments in the subcutaneous tissue of immunodeficient mice to generate hybrid bone/BM organoids represent a valid and reliable tool to investigate the interplay (at least in short term) between the sexual stages of Pf and the human BM microenvironment and, more in general, the first step in the development of a fully humanized mouse model of bone-BM organ. In this model, routinely generated in our laboratory (6-9) and already validated for homing, maintenance and proliferation of human cell injected in the mouse venous system such as human CD34 hematopoietic progenitors (7) and human prostate carcinoma and myeloid acuta leukemia cells (unpublished data), endothelial cells and hematopoiesis are of mouse origin, bone and non-hematopoietic BM cells (adipocytes and stroma) are of human origin whereas the immature or mature human GIEs have to be injected into the mouse.
Our preliminary data on homing of Pf-GIEs in the bone/BM organoids are promising. The availability and refinement of such a model through the use WT fluorescent parasite lines, whose gametocytes express fluorescent reporter genes at specific maturation stages (10,11), parasite transgenic lines genetically modified in genes involved in the regulation of the mechanical properties of the gametocytes (available through the collaboration with Dr. Pietro Alano) and, as complementary/alternative activities, chemical compounds affecting the mechanical properties of GIEs, will contribute to a better characterization of the emerging role of the BM as site of homing and maturation of Pf gametocytes. In particular, it will allow us to evaluate how changes in GIEs plasticity affect the ability of immature GIEs to home, survive and mature within the BM microenvironment and the ability of mature GIEs to be released from the BM microenvironment into the circulation. More in general, we feel that this project will provide important updates on the processes involved in the transmission of the parasite from human to the Anopheles mosquito vector.
References
1) Bullettino Reale Accademia Medica 1893;20:151.
2) Trans R Soc Trop Med Hyg 1981;75:103.
3) Malar J 2011;11:285.
4) Blood 2014;123:959.
5) Sci Transl Med 2014 Jul 9;6:244re5.
6) Cell 2007;131:324.
7) Stem Cell Res 2014;12:659.
8) Development 2017;144:1035.
9) Haematologica 2021;106:865.
10) Sci Rep 2015;5:16414.
11) Front Microbiol 2015;6:391.