Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation[S]

Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages.

Supplementary Figure S III. Examples of lysosomal synapses that are not penetrated by biotin-Alexa546. J774 macrophages were incubated overnight with biotin-fluorescein-dextran to deliver the dextran to lysosomes. Cells were then incubated with streptavidin-labeled apoptotic 3T3 L1 adipocytes (UV treated) for 90 min followed by a 30 sec treatment with 200 µM biotin-Alexa546 to mark extracellular streptavidin labeled structures. Next, 200 µM biotin was applied for 10 min to block any remaining free streptavidin. Cells were then fixed and permeabilized to remove unbound biotin-fluorescein-dextran from the lysosomes. Representative images of biotin-fluorescein-dextran (A), biotin-Alexa546 (B) and merged images superimposed on phase contrast image (C). Arrows indicate regions of lysosomal exocytosis that do not co-localize with biotin-Alexa546 (green, shown in detail in the inset).
Supplementary Figure S IV. Cell death induced by pyroptosis leads to macrophage lysosome exocytosis, but it does not occur in macrophages incubated with viable adipocytes. (A-C) RAW264.7 macrophages were incubated overnight with biotin-fluoresceindextran to deliver the dextran to lysosomes. 3T3 L1 adipocytes were incubated with 10 ng/ml lipopolysaccharide for 4 hr followed by a 2 hr incubation with 10 µM nigericin to induce pyroptosis. The adipocytes were rinsed extensively. Macrophages were then incubated with streptavidin-labeled pyroptotic adipocytes for 90 min followed by a 30 sec treatment with 200 µM biotin-Alexa546 to mark extracellular streptavidin labeled structures. Next, 200 µM biotin was applied for 10 min to block any remaining free streptavidin. Cells were then fixed and permeabilized to remove unbound biotin-fluorescein-dextran from the lysosomes. Representative image of biotin-fluorescein-dextran (A), biotin-Alexa546 (B) and a merged image superimposed on phase contrast image (C). Co-localization of the fluorescein and Alexa546 signals (yellow) demonstrates that lysosomal contents are delivered to extracellular compartments (arrow and inset). (D-E) J774 cells were incubated overnight with biotinfluorescein-dextran to deliver the dextran to lysosomes. Cells were then incubated with either TNF-α induced apoptotic (D) or streptavidin-labeled viable (E) 3T3 L1 adipocytes for 90 min. Cells were then fixed and permeabilized to remove unbound biotin-fluorescein-dextran from the lysosomes. (F) Quantification of the intensity of biotin-fluorescein bound to streptavidin-labeled adipocytes. Data are from three independent experiments. Error bar represents the SEM. *** P ≤ 0.001 student's t test. (G) Percentage of positive propidium iodide stained 3T3 L1 adipocytes after having their surface labeled with NHS-Biotin (DMSO diluted) followed by streptavidin; UVirradiated and 25 nM TNF-α treated adipocytes were used as a positive control of cell death. (H) J774 macrophages were incubated with live 3T3 L1 adipocytes labeled with CypHer 5E, a pH sensitive fluorophore, and Alexa488, a pH insensitive fluorophore. Ratiometric image shows regions at the interface between macrophages and the adipocytes that present a neutral pH.
Supplementary Figure S V. Macrophage phagocytosis is not affected by bafilomycin or protease inhibitor treatment. J774 cells were incubated with Fluoresbrite YG latex beads for 90 min in the presence or absence of 2 µM bafilomycin A1 (A), or in the presence or absence of protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic and aminopeptidases (1:800 dilution) (B). Neither bafilomycin A1 nor protease inhibitor cocktail inhibit macrophage phagocytosis of beads. Error bars represents the SEM. n.s. not significant.

Supplementary Movie S I. Time-lapse ratiometric live cell imaging reveals a neutral pH at regions of interaction between macrophages and live adipocytes.
J774 cells were incubated for 30 min with live 3T3 L1 adipocytes labeled with CypHer 5E, a pH sensitive fluorophore, and Alexa488, a pH insensitive fluorophore. The pH value within each pixel was determined by comparison with ratio images obtained in calibration buffers. The data set was acquired for 20 min with 1 min between frames.
Supplementary Movie S II. Time-lapse ratiometric live cell imaging reveals dynamics of extracellular compartments. J774 cells were incubated for 30 min with UV-induced apoptotic 3T3 L1 adipocytes labeled with CypHer 5E, a pH sensitive fluorophore, and Alexa488, a pH insensitive fluorophore. The pH value within each pixel was determined by comparison with ratio images obtained in calibration buffers. The data set was acquired for 20 min with 1 min between frames. The acidic regions are transient, dissipating and reforming as the sub-compartments formed at the macrophage-adipocyte interface open and close. Some portions of the adipocyte were internalized and can be seen moving as independent vesicles.