Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells - PubMed (original) (raw)

Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells

Jennifer Martinez et al. Proc Natl Acad Sci U S A. 2011.

Abstract

The recognition and clearance of dead cells is a process that must occur efficiently to prevent an autoimmune or inflammatory response. Recently, a process was identified wherein the autophagy machinery is recruited to pathogen-containing phagosomes, termed MAPLC3A (LC3)-associated phagocytosis (LAP), which results in optimal degradation of the phagocytosed cargo. Here, we describe the engagement of LAP upon uptake of apoptotic, necrotic, and RIPK3-dependent necrotic cells by macrophages. This process is dependent on some members of the classical autophagy pathway, including Beclin1, ATG5, and ATG7. In contrast, ULK1, despite being required for autophagy, is dispensable for LAP induced by uptake of microbes or dead cells. LAP is required for efficient degradation of the engulfed corpse, and in the absence of LAP, engulfment of dead cells results in increased production of proinflammatory cytokines and decreased production of anti-inflammatory cytokines. LAP is triggered by engagement of the TIM4 receptor by either phosphatidylserine (PtdSer)-displaying dead cells or PtdSer-containing liposomes. Therefore, the consequence of phagocytosis of dead cells is strongly affected by those components of the autophagy pathway involved in LAP.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The uptake of dead cells induces LC3-associated phagocytosis. (A) Internalization of Alexa Fluor-594 zymosan (red), apoptotic cells, necrotic cells, or RIPK3-necrotic cells (SytoRed) and association with GFP-LC3 in primary macrophages cells was followed by time-lapse video for 2 h, 1 frame/5 min. Untreated and rapamycin-treated (200 nM) macrophages were captured at 24 h (representative frames are shown; n = 4). (B) Time course of translocation of GFP-LC3 to a phagosome containing zymosan, apoptotic cells, necrotic cells, or RIPK3-necrotic cells is shown. Error bars represent SD of 4 independent experiments. (C) Macrophages were fed with apoptotic (Left) or necrotic (Right) cells for 2 h and analyzed by electron microscopy.

Fig. 2.

Fig. 2.

Components of the autophagy pathway induce LC3 translocation to the phagosome upon uptake of dead cells. (A) GFP-LC3+ macrophages were transfected with Scrambled or ATG5 siRNA oligonucleotides. At 24 h after transfection, cells were treated with 200 nM rapamycin or fed with apoptotic cells (SytoRed). GFP-LC3 puncta was assessed at 24 h, and translocation of GFP-LC3 to the dead cell-containing phagosome was followed by time-lapse microscopy for 3 h (Left panels). (B) LysM-Cre− ATG7f/f GFP-LC3+ (ATG7+/+) and LysM-Cre+ ATG7f/f GFP-LC3+ (ATG7−/−) macrophages were treated with 200 nM rapamycin or fed with apoptotic cells (SytoRed). GFP-LC3 puncta was assessed at 24 h, and translocation of GFP-LC3 to the dead cell-containing phagosome was followed by time-lapse microscopy for 3 h (Left panels). (C) GFP-LC3+ macrophages were transfected with Scrambled or Beclin1 siRNA oligonucleotides. At 24 h after transfection, cells were treated with 200 nM rapamycin or fed with apoptotic cells (SytoRed). GFP-LC3 puncta was assessed at 24 h, and translocation of GFP-LC3 to the dead cell-containing phagosome was followed by time-lapse microscopy for 3 h (Left panels). (D) ULK1+/+ GFP-LC3+ and ULK1−/− GFP-LC3+ macrophages were treated with 200 nM rapamycin or fed with apoptotic cells (SytoRed). GFP-LC3 puncta was assessed at 24 h, and translocation of GFP-LC3 to the dead cell-containing phagosome was followed by time-lapse microscopy for 3 h (Left panels). (A_–_D) (Right panels) The percentage of GFP-LC3+ dead cell-containing phagosomes (n > 100/group) was obtained from three independent time-lapse videos of A_–_D (3 h each). The average number of GFP-LC3+ puncta per cell (n > 25 cells/group) was obtained from confocal images of macrophages treated with rapamycin for 24 h. Error bars represent SD.

Fig. 3.

Fig. 3.

LC3-associated phagocytosis facilitates phagosome maturation and subsequent degradation of engulfed dead cells. (A) LysM-Cre− ATG7f/f GFP-LC3+ (ATG7+/+, Upper panels) and LysM-Cre+ ATG7f/f GFP-LC3+ (ATG7−/−, Lower panels) macrophages were preloaded with LysoTracker Red and fed with mCerulean-Spectrin murine embryonic fibroblasts induced to undergo apoptosis. Internalization, GFP-LC3 translocation, and phagosomal maturation were followed at 3-min intervals for 18 h (representative images are shown; n > 30/group). Time (in minutes) post uptake is indicated in the top left corner of each panel. (B) Time course of degradation of internalized apoptotic cells, as measured by the disappearance of mCerulean fluorescence, is shown. Error bars represent means ± SD of five independent experiments. (C) WT, LysM-Cre+ ATG7f/f (ATG7−/−), and ULK1−/− macrophages were fed with apoptotic (Apop), necrotic (Nec), or RIPK3-necrotic (R3-Nec) cells; supernatant cultures were analyzed at 18 h by Luminex technology for IL-1β (Left) and IL-6 (Right). Error bars represent SD of three independent experiments.

Fig. 4.

Fig. 4.

LC3-associated phagocytosis of dead cells is triggered by engagement of the PtdSer receptor TIM4. (A) TIM4+/+ GFP-LC3+ (Upper panels) and TIM4−/− GFP-LC3+ (Lower panels) macrophages were fed with Alexa Fluor-594 zymosan (red), apoptotic cells, necrotic cells, or RIPK3-necrotic cells (SytoRed), and internalization and translocation of GFP-LC3 to the dead cell-containing phagosome was followed by time-lapse video for 18 h, 1 frame/5 min (representative frames are shown; n = 4). (B) TIM4+/+ GFP-LC3+ (Upper panels) and TIM4−/− GFP-LC3+ (Lower panels) macrophages were fed with 100% PC or 70% PC:30% PS liposomes (Texas Red), and internalization/translocation of GFP-LC3 to the liposome-containing phagosome was followed by time-lapse video for 12 h, 1 frame/5 min (representative frames are shown; n = 3).

Fig. 5.

Fig. 5.

LAP is required for cell-corpse clearance and phagosomal maturation in the adult C. elegans hermaphrodite germ line. (A) YFP–actin-expressing C. elegans strains were knocked down for ced-1 or bec-1 using RNAi and analyzed 24 h post L4/adult molt by differential interference contrast (DIC) and epifluorescence microscopy (Left). Arrowheads indicate apoptotic germ cells or protein around the apoptotic germ cell. Quantification represents germ-cell corpses and YFP–actin halos around apoptotic cells in the indicated RNAi-treated nematodes (Right). Error bars represent SD of n > 12 animals. (B) YFP-RAB-5–expressing C. elegans strains were knocked down for ced-1 or bec-1 using RNAi and analyzed 24 h post L4/adult molt by DIC and epifluorescence (Left). Arrowheads indicate apoptotic germ cells or protein around apoptotic germ cell. Quantification represents germ-cell corpses and YFP-RAB-5 halos around apoptotic cells in the indicated RNAi-treated nematodes (Right). Error bars represent SD of n > 12 animals.

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