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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\u2212\/\u2212 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. 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