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

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory 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. Results showed that 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+ MhcIIhi and Timp2+ MhcIIlo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206+ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mφs and EGC proliferation during muscularis inflammation. 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. Cells isolated from the muscularis of the small intestine of the mice (WT or CCR2 KO) 24 hours or 72 hours after intestinal manipulation were sorted for cd45+ cells. Cells were loaded onto 10x chip targeting the recovery of 1000 cells. Single cell RNA sequencing libraries were prepared using 10x genomics platform according to manufacturer’s instructions (Single cell 3’ solution). Sequencing libraries were loaded at 1.4 pM on an Illumina NextSeq500 with NextSeq 500/550 Mid Output v2 kit (150 cycles) (Illumina). Cell Ranger software (v3.0.2) (10x Genomics) was used to demultiplex Illumina BCL files to FASTQ files (cellranger mkfastq) to perform alignment (to mouse GRCm38/mm10 genome), filtering, UMI counting and to produce gene-barcode matrices. Library preparation, sequencing and sample demultiplexing was done at the Genomics Platform of the GIGA Institute (Liège University, Belgium). Counts from the data of naive mice, 24 hour post intestinal manipulation and 72 hour post intestinal manipulation were concatenated and analysed using Seurat R package. Cells with less than 200 gene and 10% percent of mitochondrial genes were removed from the analysis.