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

Monocyte-derived macrophages (Mfs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mf differentiation in this paradigm. Here, we investigate Mf heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mfs. 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 Mf subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mfs. 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+ Mfs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mfs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mf differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mfs, thereby highlighting pro-resolving Mf differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation. Overall design: Neurosphere-derived glial cells were obtained as previously described in John K Mich et al., (Elife, 2014). Briefly, total intestines from E14.5 C57BL/6J mice were digested with collagenase D (0.5 mg/ml; Roche) and DNase I (0.1mg/ml; Roche) in DMEM/F-12, GlutaMAX, supplemented with 1% HEPES, streptomycin/penicillin and 0.1% ß-mercaptoethanol (Gibco) for approximately 30 min at 37°C under gentle agitation. Cells were cultured for 1 week in a CO2 incubator at 37°C in DMEM/F-12, GlutaMAX, streptomycin and penicillin and 0.1% ß-mercaptoethanol (Gibco) supplemented with B27 (Gibco), 40 ng/ml EGF (Gibco) and 20 ng/ml FGF (Gibco). After 1 week of culture, cells were treated with 0.05% trypsin (Gibco), transferred into PDL (Sigma-Aldrich) coated plates and cultured in DMEM supplemented with 10% FBS, 1% HEPES, glutamine, streptomycin and penicillin and 0.1% ß-mercaptoethanol (Gibco) until confluence. EGCs were activated with LPS (100ng/ml; Sigma) for 12 hours. RNA was isolated using RNeasy Micro Kit according to manufacturer's instructions. cDNA indexed library was prepared the Illumina TruSeq RNA library Kit and sequencing was performed on the Illumina NextSeq500 platform at the nucleomics core at VIB, Leuven. The reads were preprocessed by trimming for quality and to remove adapters (cutadapt 1.15 and FastX 0.0.14) followed by filtering for quality (FastX 0.0.14 and ShortRead 1.40.0) and removal of contaminants (bowtie 2.3.31). The reads were then mapped against the reference genome GRCm3882 using STAR (2.5.2b). Further filtering, sorting and indexing was done using samtools 1.5 and counts file is prepared and differential expression analysis is performed using EdgeR.