Visualizing the spatial organization of monocytes, interstitial macrophages, and tissue-specific macrophages in situ

Tissue-resident mononuclear phagocytes (MPs) are an abundant cell population whose localization in situ reflects their identity. To enable assessment of their heterogeneity, we developed the red/green/blue (RGB)-Mac mouse based upon combinations of Cx3cr1 and Csf1r reporter transgenes, providing a complete visualization of their spatial organization in situ. 3D-multi-photon imaging for spatial mapping and spectral cytometry employing the three markers in combination distinguished tissue-associated monocytes, tissue-specific macrophages, and three subsets of connective-tissue-associated MPs, including CCR2+ monocyte-derived cell, CX3CR1+, and FOLR2+ interstitial subsets, associated with distinct sub-anatomic territories. These populations were selectively reduced by blockade of CSF1, CSF2, CCR2, and CX3CR1 and efficiently reconstitute their spatial distribution after transient myelo-ablation, suggesting an autonomous regulatory environment. Our findings emphasize the organization of the MP compartment at the sub-anatomic level under steady-state conditions, thereby providing a holistic understanding of their relative heterogeneity across different tissues. [Display omitted] •Tissue-resident macrophage subsets are associated with distinct sub-anatomic territories•Tissue-resident macrophage subsets harbor distinct molecular dependencies•The RGB-Mac mouse discriminates three common interstitial macrophage subsets across tissues Petit et al. show that the different mononuclear phagocyte subsets in non-lymphoid organs are spatially organized according to distinct sub-territories and depend on different molecular regulators. This spatial mapping provides a better understanding of the diversity of the mononuclear phagocyte system across different tissues.