Macrophage source influences peritoneal persistence and immune cell dynamics in assays of reverse cholesterol transport.

Assays to measure macrophage-to-feces reverse cholesterol transport (RCT) in mouse models are well-established, yet the immunologic consequences of injecting allogeneic versus syngeneic macrophages remain undefined in the context of this assay. In this study, we compared primary bone marrow-derived macrophages (BMDMs) and reticulosarcoma-derived J774 macrophages as cholesterol-loaded donor cells in a well-established in vivo RCT assay to determine whether macrophage origin influences host immune activation and cholesterol flux. Following intraperitoneal injection, J774 macrophages exhibited markedly reduced peritoneal persistence relative to BMDMs, with <1% of cells detectable at 24 or 48 hours post-injection, compared to sustained retention of the BMDMs. This was accompanied by distinct immune activation profiles where J774 macrophages induced prolonged innate immune activation, whereas BMDMs elicited coordinated and transient responses involving early B cell activation that resolved by 48 hours. Despite these differences in cellular persistence and immune response, RCT outcomes were comparable between groups under the conditions tested, with no major differences observed in hepatic uptake or fecal excretion of [3H]-cholesterol in BMDM-injected mice at 24 hours compared to J774-injected mice. Mechanistically, these findings suggest that the presence of macrophages to mediate cholesterol efflux in the early steps of RCT likely dominates total RCT measurements, potentially buffering the impact of later differences in macrophage persistence and immune activation. Together, these results demonstrate that macrophage source influences peritoneal persistence and immune cell dynamics, but does not substantially alter RCT outcomes under the conditions tested, underscoring the importance of considering macrophage source when designing and interpreting macrophage-to-feces RCT studies.