Investigating colonic macrophage heterogeneity in steady-state, inflammation, and resolution

Abstract

Macrophages play multiple key homeostatic roles throughout the intestine. However, dysregulated macrophages are considered main drivers of gastrointestinal diseases such as inflammatory bowel disease (IBD), a relapsing remitting disorder. Whether these distinct roles are performed by discrete subsets of macrophages or if tissue resident macrophages change their behaviour in the context of inflammation or resolution is unclear. To determine the nature of macrophages in these different contexts, I used single cell RNA sequencing (scRNA-seq), multi-parameter flow cytometry, immunofluorescent microscopy, and genetic fate-mapping to characterise the macrophage compartment in steady-state, and during colonic inflammation resolution in mice.

This revealed that there are at least two phenotypically and anatomically distinct populations of tissue resident macrophages: CD11c+CD163– and CD11c–CD163+. A combination of genetic fate-mapping techniques showed that these macrophage subsets have differential replenishment kinetics, with CD163+ macrophages being more long-lived than their CD11c+ macrophage counterparts.

It was unclear how acute inflammation would impact the macrophage subsets, and whether it would induce persistent effects on the intestinal macrophage compartment following resolution. To investigate this, I established a mouse model of resolving colitis. With a combination of flow cytometry and immunofluorescence staining, I found both classical monocytes and the macrophage subsets to accumulate in experimental colitis. During inflammation resolution, monocyte numbers subsided, and the macrophage compartment appeared to return to baseline. Cx3cr1-based fate mapping revealed that high proportions of the macrophage subsets were replenished during colitis, however many managed to survive the injury. To determine the potential differential effects of inflammation on the macrophages which had persisted throughout, versus those that had been recruited following the initiation of inflammation, I used a combination of scRNAseq and Cx3cr1-based fate mapping. This revealed hundreds of persistent transcriptional changes in the macrophages compartment in resolution. Interestingly, resident-macrophages fate-mapped from health through to resolution appear to have more restricted plasticity, with far fewer transcriptional changes when compared to controls, than with their more recently differentiated counterparts.

My findings reveal that the colonic macrophage compartment is composed of transcriptionally, phenotypically and anatomically discrete population of macrophages. These macrophage subsets have distinct replenishment kinetics and are differentially affected by inflammation with some functionally changed even following resolution. These findings have important implications for understanding the mechanisms which cause aberrant macrophage function in gastrointestinal disease.