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

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

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

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 â recombination-activating gene 1 (Rag1), interleukin-7 receptor (Il7r), and Fms-like tyrosine kinase 3 (Flt3) â 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

Autophagy preserves hematopoietic stem cells by restraining mTORC1-mediated cellular anabolism

Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained mTOR (mammalian target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. mTOR inhibition by rapamycin treatment in vivo was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting mTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation

DNMT1 deficiency impacts on plasmacytoid dendritic cells in homeostasis and autoimmune disease

Dendritic cells (DCs) are heterogeneous immune regulators involved in autoimmune diseases. Epigenomic mechanisms orchestrating DC development and DC subset diversification remain insufficiently understood but could be important to modulate DC fate for clinical purposes. By combining whole-genome methylation assessment with the analysis of mice expressing reduced DNA methyltransferase 1 levels, we show that distinct DNA methylation levels and patterns are required for the development of plasmacytoid (pDC) and conventional DC subsets. We provide clonal in vivo evidence for DC lineage establishment at the stem cell level, and we show that a high DNA methylation threshold level is essential for Flt3-dependent survival of DC precursors. Importantly, reducing methylation predominantly depletes pDC and ameliorates systemic lupus erythematosus in an autoimmunity mouse model. This study shows how DNA methylation regulates the production of DC subsets and provides a potential rationale for targeting autoimmune disease using hypomethylating agents