Fig. 2: Transient communication between the extracellular environment and mature phagolysosomes of pro-inflammatory macrophages. | Nature Communications

Fig. 2: Transient communication between the extracellular environment and mature phagolysosomes of pro-inflammatory macrophages.

From: Macrophages disseminate pathogen associated molecular patterns through the direct extracellular release of the soluble content of their phagolysosomes

Fig. 2: Transient communication between the extracellular environment and mature phagolysosomes of pro-inflammatory macrophages.The alternative text for this image may have been generated using AI.

a BMMØs were pulsed overnight with membrane impermeant fluid-phase tracer AF594 hydrazide (red) and chased to lysosomes over 2 h prior to the phagocytosis of experimental particles labelled with the pH-sensitive fluor CFSE (green) and pH-stable fluor AF647SE (magenta). Sequential images of a phagolysosome containing AF594 hydrazide (by prior fusion with AF594 hydrazide-loaded lysosomes) undergoing transient pH neutralization. AF594 hydrazide fluid-phase fluorophore (red) was lost upon neutralization of the phagolysosome (indicated by the transient increase in CFSE fluorescence on the bead). No increase in cytosolic AF594 fluorescence could be detected following its loss from the phagolysosome indicating extracellular release. b Graphical illustration of the experimental particle assay used to measure the loss of partially digested particles from phagolysosomes. The particles bear self-quenched DQ Green BSA, pHrodoSE and the reference fluor AF647SE. c–e Phagosomal proteolysis and pH were monitored in BMMØs following phagocytosis of experimental particles bearing the quenched DQ Green BSA substrate (green), the pH indicator pHrodoSE (red) and the reference fluorophore AF647SE (magenta). Eructophagy was demonstrated by the simultaneous loss of soluble peptide products of DQ Green BSA (green) and neutralization of the phagolysosome (red), followed by resumption of proteolysis and re-acidification. d DQ Green BSA phagosome and DQ Green cytosol are plotted on the right axis. AF647SE reference and pHRodoSE are plotted on the left axis. e Data are presented as the percentage of phagosomes that underwent ≥1 eructophagy event within the corresponding 10 h period post-phagocytosis. Error bars presented as means ± SEM from n = 3 independent experiments. Images were captured using an IN Cell Analyzer 2000 between 90 min and 3 h postphagocytosis at 37 °C. Time 0 represents an eructophagy event. Scale bars denote 10 μm. Source data are provided as a Source Data file.

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