CD163 and Tim-4 identify resident intestinal macrophages that are spatially regulated by TGF-β.

Macrophages localize in sub-tissular niches associated with their ontogeny and activity. In the intestine, a paradigm has emerged that long-lived macrophages are present in the muscular layer, while highly monocyte-replenished populations are found in the lamina propria (LP). Whether long-lived macrophages are restricted in such a simplified manner has not been well explored. Moreover, the impact of specific gut-associated factors on macrophage identity across intestinal tissue layers is unknown. We generated scRNA-seq data from WT and Ccr2-/- mice to identify phenotypic features of long-lived macrophage populations in distinct intestinal layers and identified CD163 as a marker to distinguish submucosal/muscularis (S/M) from LP macrophages. Challenging the emerging paradigm, long-lived macrophages were found in the LP and S/M, with distinct transcriptomes and responsiveness to proinflammatory stimuli. Employing transgenic mice, we demonstrate a critical role for TGF-β signalling in maintaining the identity of long-lived LP but not S/M macrophages and that macrophage-derived TGF-β1 is required to instruct intestinal macrophage identity after development.