Three cell deaths and a funeral: macrophage clearance of cells undergoing distinct modes of cell death

Abstract

Macrophage clearance of apoptotic cells has been extensively investigated, but less is known regarding the clearance of cells dying by other forms of programmed cell death, e.g., necroptosis or ferroptosis. Here, we established a model of three different cell deaths using the same cell line and the occurrence of distinct cell death modalities was verified by using the specific inhibitors, zVAD-fmk, necrostatin-1, and ferrostatin-1, respectively. Cell death was characterized by using transmission electron microscopy (TEM), the gold standard for the demarcation of different cell death modalities. Moreover, using annexin V as a probe, we could detect surface exposure of phosphatidylserine (PS) in all three types of cell death, and this was confirmed by using specific anti-PS antibodies. We then co-cultured the cells with human monocyte-derived macrophages and found that cells dying by all three death modalities were engulfed by macrophages. Macrophage clearance of apoptotic cells was more efficient when compared to necroptotic and ferroptotic cells with multiple internalized target cells per macrophage, as shown by TEM. We propose that clearance of dying cells also should be taken into account in the classification of different cell death modalities.

Fig. 1. Three modes of programmed cell death.

(a) Schematic figures showing the cell death pathway for apoptosis, necroptosis, and ferroptosis. The cell death inducers used in the present study are indicated in red and the specific inhibitors are shown in green. (b) Time course of cell death as evidenced by LDH release in Jurkat (apoptosis, ferroptosis) or FADD-DN Jurkat cells (necroptosis). Cell death was induced by the addition of 250 ng/mL Fas antibody, 10 ng/mL TNF-α, or 2 µM RSL3 in the presence or absence of the respective cell death inhibitors, zVAD-fmk (10 µM), Nec-1 (40 µM), or Fer-1 (5 µM). Refer to Figure S1–S3 for additional morphological and biochemical indices of cell death, and Figure S4 for cell death results based on light scatter

Fig. 2. PS exposure in programmed cell death.

a Quantification of annexin V-FITC/PI staining of Jurkat cells or FADD-DN Jurkat cells exposed to 250 ng/mL Fas antibody, 10 ng/mL TNF-α, or 2 µM RSL3 for the indicated time-points. The lower bars (light shading) represent the annexin V-positive/PI-negative cells and the upper bars (dark shading) the annexin V-positive/PI-positive (“double-positive”) cells. Data shown are mean values ± S.D. (n = 3). Statistically significant differences were noted for all the indicated samples versus control, and for the 24 h apoptotic samples versus the 24 h necroptotic and ferroptotic samples. *p < 0.05, ***p < 0.001. b Representative annexin V-FITC/PI results for cells undergoing apoptosis, necroptosis, and ferroptosis. The percentages of cells in each quadrant are shown. Refer to Figure S5 for annexin V-FITC results with/without cell death inhibitors versus staining with anti-PS antibodies, and Figure S6 for results on the (loss of) CD31 expression following cell death induction

Fig. 3. Macrophage engulfment of dying cells.

a Quantification of macrophage engulfment of target cells triggered to undergo apoptosis, necroptosis, or ferroptosis for 3 or 24 h. Macrophages and target cells were co-cultured for 1 h and results were scored as described in Materials and methods. Data shown are mean values ± S.D. (n = 3). Statistically significant differences were noted for the indicated samples versus the respective control (i.e., untreated WT or untreated FADD-DN cells), and for the apoptotic samples versus the necroptotic and ferroptotic samples at 24 h. *p < 0.05, **p < 0.01, ***p < 0.001. Below are representative images of control macrophages (b), or macrophages engaged in the ingestion of TAMRA-labeled apoptotic (c), necroptotic (d), or ferroptotic (e) target cells treated for 24 h with 250 ng/mL Fas antibody, 10 ng/mL TNF-α, or 2 µM RSL3, respectively, prior to macrophage co-culture for 1 h

Fig. 4. Macrophage engulfment of dying cells.

Jurkat cells or FADD-DN Jurkat cells were triggered to undergo three different cell death modalities for 24 h using anti-Fas antibodies, TNF-α, and RSL3, respectively, and were then co-cultured for 1 h with primary human monocyte-derived macrophages. Non-engulfed cells were washed away and macrophages were collected, fixed, and processed for TEM. Shown here are images of control macrophages (a) and macrophages engulfing apoptotic (b), necroptotic (c), or ferroptotic (d) Jurkat cells. Scale bars represent 5, 10, or 20 µm. Note the presence of multiple, largely intact target cells with condensed chromatin in macrophages in (b) as opposed to the uptake of severely fragmented cells in (c) and (d)