Gene expression profiles in Drosophila phagocytes after incubation with apototic cell fragments

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signaltransduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells that stimulated phagocytes find thereafter and is thus considered as a mechanism to prime phagocytes in innate immunity. S2 cells, a cell line established from Drosophila embryos, were maintained at 25 °C in Schneider's Drosophila medium (Life Technologies Japan, Tokyo, Japan) containing 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. To prepare apoptotic cell fragments, S2 cells were incubated in the presence of cycloheximide (1.5 μg/ml) for 24 h to induce apoptosis. The cells were then centrifuged at 300 × g for 3 min at room temperature, and the resulting supernatants were collected. They were re-centrifuged at 1500 × g for 3 min at 4 °C, and the supernatants were further centrifuged at 3500 × g for 3 min at 4 °C. The resulting precipitates were recovered, suspended with Schneider's Drosophila medium with 1% heat-inactivated FBS, microscopically determined for the number of membranous particles using a hemocytometer, and used as the fragments of apoptotic S2 cells. Semi-confluent S2 cells maintained in Schneider's Drosophila medium with 1% heat-inactivated FBS were supplemented with the apoptotic cell fragments, at a responder/effector ratio of 1 : 25. The mixtures, together with phagocyte cultures with no fragments added as a negative control, were incubated at 25 °C for 1h, and cells were detached from culture containers by the treatment with 0.25% (w/v) trypsin and 0.02% (w/v) EDTA. The recovered cells were collected by centrifugation at 300 × g for 3 min at room temperature, washed twice with PBS, and used as phagocytes stimulated by apoptotic cell fragments, or an untreated control, in the preparation of RNA for a microarray analysis.