Uncovering the role of host peroxisomal functions in Plasmodium liver stage infection

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011Malaria, the world’s leading tropical parasitic disease, is caused by protozoan parasites of the genus Plasmodium. During its life cycle, Plasmodium inhabits an insect vector and a vertebrate host. Liver infection in the vertebrate host is the asymptomatic obligatory step before the onset of malaria disease. Cellular and molecular interactions between host and parasite play a key role in the establishment of susceptibility to malaria infection, and so the identification of relevant host factors is crucial for the rational development of new antimalarial strategies. We hypothesized that peroxisomes-less Plasmodium may have acquired host-dependency at the level of liver peroxisomes, and that it can take advantage of host cell peroxisomal functions and metabolites during liver stage. The myriad pathways in which peroxisomes are involved and their abundance in mammalian livers seems to place these organelles in a privileged position to be exploited in the context of intracellular parasitism. Live fluorescence microscopy and flow cytometry of DsRedlabeled peroxisomes revealed that the intracellular presence of Plasmodium can alter the dynamic properties of the host peroxisomal population. We then focused on the two major mammalian peroxisomal functions, fatty acid β-oxidation and detoxification of reactive oxygen species. Impairment of fatty acid β-oxidation by a drug inhibitor, knockdown of β- oxidation enzymes and overexpression of a key peroxisomal thiolase showed that a hostfactor dependency does exist and that it is important for both cell invasion and subsequent parasite development. This is probably tied to the parasite’s metabolic requirements for membrane biosynthesis during these processes. Catalase inhibition and knockdown of other peroxisomal peroxidases showed that this antioxidant network does not play a strong role in Plasmodium infection, but fluorescence microscopy revealed that the peroxisomal marker enzyme catalase may be recruited by the parasite to complement the functions of its own antioxidant systems in the maintenance of redox homeostasis during liver stage.A malária constitui a principal doença parasitária tropical no mundo, sendo causada por protozoários do género Plasmodium. O ciclo de vida deste parasita inclui dois hospedeiros: um insecto vector e um vertebrado. A infecção do fígado do hospedeiro vertebrado é uma etapa obrigatória e precede a manifestação clínica da doença. As interacções celulares e moleculares entre parasita e hospedeiro têm um papel determinante no estabelecimento da susceptibilidade à infecção e, portanto, a identificação de factores do hospedeiro relevantes para o desenrolar da infecção é essencial numa perspectiva de desenvolvimento de novas estratégias anti-maláricas. No âmbito deste trabalho formulámos a hipótese de que o parasita causador da malária, o qual é desprovido de peroxissomas, poderá, ao longo da evolução, ter adquirido a capacidade de subverter as funções e/ou metabolitos peroxissomais do hospedeiro vertebrado. De facto, a diversidade de vias metabólicas em que os peroxissomas estão envolvidos, bem como a sua abundância no fígado, levantam a questão da importância destes organelos num contexto de parasitismo intracelular. Começámos por mostrar que a presença de Plasmodium pode alterar as propriedades dinâmicas da população peroxissomal da célula hospedeira. Focámo-nos, então, nas duas principais funções dos peroxissomas, a β-oxidação de ácidos gordos e a degradação de espécies reactivas de oxigénio. Bloqueio da β-oxidação através de um inibidor ou por silenciamento da expressão de enzimas-chave desta via metabólica, bem como sobreexpressão de uma importante tiolase peroxissomal, permitiu-nos demonstrar que existe de facto uma dependência entre parasita e hospedeiro e que a β-oxidação peroxissomal é importante tanto para a invasão da célula hospedeira como para o subsequente desenvolvimento do parasita. Este efeito está provavelmente associado às necessidades lipídicas do parasita, nomeadamente para a síntese de membranas durante ambos os processos. Por outro lado, inibição da catalase e silenciamento da expressão de outras peroxidases peroxissomais revelou que esta rede antioxidante não tem um papel crucial na infecção por Plasmodium. Curiosamente, experiências de microscopia de fluorescência sugerem que a catalase do hospedeiro poderá ser recrutada pelo parasita, o que poderá constituir um mecanismo de homeostase durante a infecção hepática

Loss of endothelial membrane KIT ligand affects systemic KIT ligand levels but not bone marrow hematopoietic stem cells

A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow

Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr+ perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis

Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge

Environmental signals rather than layered ontogeny imprint the function of type 2 conventional dendritic cells in young and adult mice

Conventional dendritic cells (cDC) are key activators of naive T cells, and can be targeted in adults to induce adaptive immunity, but in early life are considered under-developed or functionally immature. Here we show that, in early life, when the immune system develops, cDC2 exhibit a dual hematopoietic origin and, like other myeloid and lymphoid cells, develop in waves. Developmentally distinct cDC2 in early life, despite being distinguishable by fate mapping, are transcriptionally and functionally similar. cDC2 in early and adult life, however, are exposed to distinct cytokine environments that shape their transcriptional profile and alter their ability to sense pathogens, secrete cytokines and polarize T cells. We further show that cDC2 in early life, despite being distinct from cDC2 in adult life, are functionally competent and can induce T cell responses. Our results thus highlight the potential of harnessing cDC2 for boosting immunity in early life.</p

Single-cell multi-omics identifies chronic inflammation as a driver of TP53-mutant leukemic evolution

Understanding the genetic and nongenetic determinants of tumor protein 53 (TP53)-mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 ‘multihit’ HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53-mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53-heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53-mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukemia, and are of broad relevance to other cancer types

Ezh2 and Runx1 Mutations Collaborate to Initiate Lympho-Myeloid Leukemia in Early Thymic Progenitors.

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs

The road not taken: lineage bias and restriction of haematopoietic stem and progenitor cells

The mammalian haematopoietic system is a progressively lineage-restricted branching hierarchy that replenishes mature blood cells throughout the life of an organism. Self-renewing haematopoietic stem cells (HSCs) reside at the apex of the hierarchy and give rise to multipotent progenitors that undergo increasingly restricted lineage commitments, until they finally assume a single cell fate at the exclusion of all others. Several competing models of the haematopoietic hierarchy have been proposed, and it is yet unclear if all mechanisms of lineage commitment in definitive haematopoiesis are identical between foetal liver (FL) and adult bone marrow (BM). Herein we explored early lineage fate decisions in the first branches of the lymphoid-primed multipotent progenitor (LMPP) model of haematopoiesis. We prospectively isolated the earliest known immune-restricted progenitor from early FL and yolk sac, prior to the establishment of definitive haematopoiesis in the embryo. These progenitors express early lymphoid-affiliated genes – recombination-activating gene 1 (Rag1), interleukin-7 receptor (Il7r), and Fms-like tyrosine kinase 3 (Flt3) – and were shown to be equivalent to the adult BM LMPP compartment, having robust combined lympho-myeloid potentials but little or no megakaryocyte/erythroid potentials. Moreover, the physiological contribution of this immune-restricted progenitor to embryonic myelopoiesis was conclusively demonstrated, supporting the relevance of the LMPP model of the haematopoietic hierarchy in early embryonic lymphoid commitment. In other studies pertaining to the myeloid/megakaryocyte/erythroid branch of the LMPP model, we characterised the function of individual platelet-biased and platelet-restricted HSCs in adult BM. Based on our findings we propose a revised model of the LMPP hierarchy, with the addition of a primitive megakaryocyte-restricted pathway of HSC differentiation. In contrast, we found that platelet-biased reconstitution behaviour is very rare in FL HSCs, but is promptly acquired by FL-derived adult HSCs. Therefore, the megakaryocyte-restricted differentiation branch proposed might be exclusive to adult haematopoiesis. </p