The role of the endocytic and autophagic molecular machineries in the removal of apoptotic cells

Abstract

Every day the human body turns over billions of cells ensuring the disposal of unwanted targets that die by apoptosis. The prompt and efficient removal of apoptotic cells by cell line (vascular SMC). The maturation of phagosomes containing dying cells was compared with the processing of phagosomes loaded with IgG-opsonized particles, which are internalized via Fcγ-receptors and are the best characterized phagocytic model. At the present work, we provide evidence that the nature of the cargo modulates the phagocytic response, since phagosomes carrying apoptotic particles reach the lysosomes with a delay when compared to those containing IgG-opsonized particles. Furthermore, for the first time, we have identified some canonical autophagy effectors in phagolysosome formation, suggesting that LC3-Associated Phagocytosis (LAP), a process involved in phagosome maturation, implies more than the phagosomal recruitment of LC3 (Sanjuan et al., 2007). Indeed, experiments performed in bone marrow-derived macrophages from p62-KO mice clearly suggest that p62, despite not being required for LC3 recruitment, is important for phagolysosome biogenesis. In summary, this data will improve our knowledge on the molecular machinery and mechanisms involved in efferocytosis. In the end, we hope to contribute to a better understanding of efferocytosis and the ways to modulate this process, which could culminate with the discovery of therapies that may benefit patients with atherosclerosis and other type of diseases in which efferocytosis is not efficient.phagocytes, referred as to efferocytosis, plays an essential role during development, tissue repair and in the maintenance of homeostasis, triggering an immunological tolerance (Henson and Hume, 2006). On the other hand, defective clearance promote dying cell accumulation, converting harmless apoptotic cells into a risky secondary necrotic state that, eventually, expose self-antigens, which has been linked to the onset of several human disorders, including autoimmunity and chronic inflammatory diseases, such as atherosclerosis (Elliott and Ravichandran, 2010). Atherosclerosis remains the biggest cause of mortality and disabilities worldwide, especially in developing countries. The formation of the atheroma starts with the retention of low-density lipoproteins (LDL) inside the wall of blood vessels, where they become subjected to several chemical modifications. These modified-LDL induce the recruitment of monocyte-derived macrophages, which internalize the deposited fatty material. Over time, these lipid-loaded macrophages are no longer able to process the cholesterol, forming foam-cells that eventually undergo apoptosis. In early stages of atherogenesis, efferocytosis is very efficient; however in advanced lesions this process somehow fails, triggering an inflammatory response that, in turn, recruits more cells, including neighboring smooth muscle cells (SMC). Besides macrophages, SMC, the major cell type in the blood vessels wall, play an essential role by dealing with the dying cell accumulation, thus preventing atheroma progression (Moore and Tabas, 2011). Although many efforts have been done to understand the machinery involved in the recognition of apoptotic cells by phagocytic cells (receptors and ligands), as well as the immune response elicited, very little is known about the intracellular transport of phagosomes containing apoptotic cells and its subsequent digestion into phagolysosomes, the final degradative compartment of the host cell (Hochreiter-Hufford and Ravichandran, 2013). Beyond that, C. elegans has been the model organism in studies of engulfment and degradation of apoptotic cells, which reinforce the need to have more information about the development of this process in mammalian systems. Thus, it is crucial to our understanding, to figure out the causes of the inefficient efferocytosis and how it contributes to the pathogenesis of certain diseases. In this thesis, we have performed a detailed study on the maturation of phagosomes containing human aged red blood cells, our apoptotic cell model, using a mammalian phagocytic