Enteric glial cells favour accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation

ObjectiveMonocyte-derived macrophages (M{varphi}s) are crucial regulators during muscularis inflammation. However, it is unclear which microenvironmental factors are responsible for monocyte recruitment and neurotrophic M{varphi} differentiation in this paradigm. Here, we investigate M{varphi} heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue protective M{varphi}s. DesignSingle cell RNA sequencing was performed on immune cells from the muscularis of wild-type and CCR2-/- mice at different timepoints after muscularis inflammation. CX3CR1GFP/+ and CX3CR1CreERT2 R26YFP mice were analyzed by flow cytometry and immunofluorescence. The transcriptome of enteric glial cells (EGCs) was investigated using PLPCreERT2 Rpl22HA mice. In addition, we assessed the effect of supernatant from neurosphere-derived EGCs on monocyte differentiation based on the expression of pro- and anti-inflammatory factors. ResultsMuscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving M{varphi} subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo M{varphi}s, which were both derived from CCR2+ monocytes. Interestingly, we found that EGCs were able to sense damage to the muscularis to stimulate monocyte recruitment and differentiation towards pro-resolving M{varphi}s via CCL2 and CSF1, respectively. ConclusionOur study provides a comprehensive insight into pro-resolving M{varphi} differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and M{varphi}s, thereby highlighting pro-resolving M{varphi} differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.