Interrelationship of Peritoneal Fluid and Mesothelial Macrophages and Species-Specific Pathways in Their Maintenance and Repopulation 2306939

Abstract Introduction The peritoneal cavity is a clinically important immune niche that regulates serosal organ homeostasis and contributes to diseases such as peritoneal metastasis. It comprises a mesothelial surface and a fluid compartment, both enriched in macrophages. In mice, peritoneal macrophages include fluid large cavity macrophages (LCMs) and mesothelial macrophages (Mes.Macs), with mature LCMs defined by GATA6 and CD73 and thought to be replenished from monocytes through a linear LYVE1+ converting stage. However, how this paradigm applies to humans is unclear. Human peritoneal macrophages are predominantly LYVE1+ but lack GATA6 and CD73. Whether this reflects incomplete maturation, or a distinct differentiation pathway remains unknown, undermining the interpretation of murine models of human peritoneal disease. Methods Using spectral flow cytometry, fate mapping, bone marrow chimeras, and 3D imaging, we redefined peritoneal macrophage differentiation and cross-compartmental dynamics. Results We identified two parallel monocyte-LCM differentiation trajectories, overturning the prevailing linear model. A minor pathway (∼20% of monocytes) proceeds through a LYVE1+ converting stage and matures into CD73+ LCMs in a Zeb2-165 kb enhancer-dependent manner. In contrast, the dominant pathway in mice, absent in humans, bypasses the LYVE1+ stage, exhibits early CD73 expression, and directly generates GATA6+ CD73+ mature LCMs independent of the Zeb2-165 kb enhancer. We further show that fluid monocytes, but not LCMs, can differentiate into Mes.Macs in a Talin1-dependent manner, including tumor-associated Mes.Macs. Conclusion These findings redefine peritoneal macrophage ontogeny, reveal species-specific differentiation programs, and demonstrate dynamic exchange between fluid and mesothelial compartments. This work provides a framework for translating murine peritoneal immunity to human biology and highlights fluid-derived macrophages as key contributors to surface-associated pathologies such as peritoneal metastasis. Funding Source NIH/NIAID; NIH/NCI Topic Categories Hematopoiesis and Immune System Development (HEM)