Distinct Lysosome Phenotypes Influence Inflammatory Function in Peritoneal and Bone Marrow-Derived Macrophages

Figure 4

Differential role of the lysosome during activation of the lipotoxic inflammasome in pMACs compared to BMDMs. (a, b) pMACs (a) or BMDMs (b) were preloaded with the indicated doses of palmitate (palm) or with BSA for 2 h followed by BSA or palm ±50 ng/mL LPS for 20 h after which IL-1β in the supernatant was quantified by ELISA. (c, d) pMACs (c) or BMDMs (d) were stimulated with palmitate and LPS in the presence of bafilomycin (BAF, 25 nM) or CAO74-ME (10 μM) and IL-1β release at 20 h was determined by ELISA. (e, f) pMACs (e) or BMDMs (f) were prepared from WT (white bars) or ATG5KO (black bars; ATG5fl/fl X LysM-Cre) mice and were subsequently stimulated with BSA-PBS, BSA-LPS, or palm-LPS for 20 h. IL-1β release was quantified by ELISA. The inset in (e) shows data for BSA-PBS versus BSA-LPS with an adjusted scale. (g) Total cellular protein was isolated from pMACs or BMDMs and expression of ATG5 was determined by western blotting (left panel). As a control for the antibody, protein was also isolated from WT and ATG5KO pMACs and analyzed by western blot (right panel). Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. for BSA-PBS versus palm-LPS; vehicle versus inhibitor; or WT versus KO.

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