Genetic dissection of the molecular mechanisms of phagocytosis of apoptotic cells by drosophila macrophages

Lead Research Organisation: MRC Cell Biology Unit

Abstract

In all multicellular organisms, excess cells are being produced that need to be deleted for development to proceed. These cells are eliminated by apoptosis a suicidal form of genetically programmed cell death. Although much progress has been made in understanding the molecular mechanisms of apoptosis, those underlying phagocytosis, the process by which specialized cells such as macrophages (white blood cells) remove dying cells, are far less understood. Phagocytosis also plays a critical role in innate immunity where it represents the first line of host defence against invading microorganisms such as bacteria and tissue debris that are removed by this process. Failure to clear dying cells can result in autoimmunity, and may promote the development of highly malignant tumours, while failure to clear microorganisms can result in septicemia (blood infection), a critical and often fatal condition. Thus understanding the molecular mechanisms of phagocytosis is crucial.|We use the fruitfly, Drosophila melanogaster, to dissect the molecular mechanisms of phagocytosis of dying cells by embryonic macrophages using genetics and cell biology approaches. As in human, fly macrophages also serve to protect the organism against microbial infection. Our work on croquemort, a fly homologue of mammalian CD36 and that of others on draper, a fly homologue of mammalian CD91, all of which are receptors that have been implicated in phagocytosis of dying cells have already revealed conservation in the molecular mechanisms of phagocytosis across evolution. Thus understanding this process in the fruitfly model system is likely to provide insight into human innate immunity and autoimmunity, and ultimately to impact on human health.

Technical Summary

Apoptosis is a genetically programmed cell death that allows homeostasis and tissue remodelling during development of multicellular organisms. The final step in apoptosis is phagocytosis or the engulfment of apoptotic cells by specialized cells, such as macrophages. Phagocytosis of apoptotic cells triggers anti-inflammatory signals, playing an important role in the resolution of inflammation. Failure to clear apoptotic cells might contribute to autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), and promote the development of highly malignant tumours. Phagocytosis is also critical in limiting infection by clearing pathogens that accumulate during infection. Our laboratory uses Drosophila melanogaster, as a genetically tractable model organism to dissect the molecular mechanisms of phagocytosis. We focus our studies on the phagocytosis of apoptotic cells by embryonic macrophages, which has the advantages of eliminating the needs for experimental infection by injection or wounding and allows us to study phagocytosis in vivo in the whole organism in its physiological condition. We previously characterized Croquemort (Crq), a CD36-related receptor expressed in embryonic macrophages, and demonstrated its genetic requirement for phagocytosis of apoptotic corpses by these cells. These data highlighted conservation in the mechanisms of phagocytosis of apoptotic cells in Drosophila, as mammalian CD36 also participate in this process, and promoted Drosophila as a suitable model system. To identify new components of this phagocytic machinery, we conducted a genetic screen and identified 13 deficiency mutants with phagocytosis defects, two of which we have now fully characterized, pallbearer and undertaker, and identified the corresponding genes (Silva et al., in press, Immunity 2007; Cuttell et al. submitted manuscript). We also performed a genome-wide RNAi screen (Drosophila RNAi Screening Center, Harvard Medical School, Boston, MA, USA) for genes required for phagocytosis of apoptotic corpses by Schneider S2 cells, a Drosophila blood cell line, and identified 211 candidate genes (manuscript in preparation). We continue using both genetics and cell biology approaches to study the role of crq, pallbearer and undertaker, as well as genes that fall into their pathways. We started delineated the Pallbearer pathway by finding several components of the E3-ubiquitin ligase complex including pallbearer that control efficient phagocytosis of apoptotic corpses. We will further address the potential role of mammalian homologues of our newly characterized genes in collaboration with our colleagues at the MRC Centre for Inflammation Research in Edinburgh, and their possible roles in phagocytosis of pathogens with colleagues in the USA. We anticipate that this will ultimately help us better our understanding of the molecular mechanisms of phagocytosis both during development and in innate immunity.