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

Abstract Objective Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which microenvironmental factors are responsible for monocyte recruitment and neurotrophic Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue protective Mφs. Design Single cell RNA sequencing was performed on immune cells from the muscularis of wild-type and CCR2 -/- mice at different timepoints after muscularis inflammation. CX3CR1 GFP/+ and CX3CR1 CreERT2 R26 YFP mice were analyzed by flow cytometry and immunofluorescence. The transcriptome of enteric glial cells (EGCs) was investigated using PLP CreERT2 Rpl22 HA 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. Results Muscularis 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 Mφ subpopulations during the resolution of muscularis inflammation, i.e . Cd206 + MhcII hi and Timp2 + MhcII lo Mφs, 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 Mφs via CCL2 and CSF1, respectively. Conclusion Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.