GSE167464
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: To induce gene recombination of Rpl22HA in PLP1CreERT2 Rpl22HA transgenic mice at 10-12 weeks of age were injected twice intraperitoneally with 100 µl of 15 mg/ml tamoxifen (Sigma) dissolved in Miglyol 812 (Caesar & Loretz, Hilden, Germany). All animals were sacrificed 3-4 weeks after the first injection. The whole small intestine of each mouse was peeled by removing the mucosa and submucosa in order to obtain preparations of circular and longitudinal smooth muscle layer and adherent myenteric plexus. Next, samples were flash frozen until processing. Immunoprecipitated samples were processed and analyzed according to published protocols4, 5. In brief, mRNA was captured with Dynabeads oligo(dT) (Life Technologies) according to manufacturer's guidelines. A bulk variation of MARSseq6 was used to prepare libraries for RNA-seq. RNA was reversed transcribed with MARSseq barcoded RT primer in a 10 mL volume with the Affinity Script kit (Agilent). Reverse transcription was analyzed by qRT-PCR and samples with a similar CT were pooled (up to eight samples per pool). Each pool was treated with Exonuclease I (NEB) for 30 min at 37°C and subsequently cleaned by 1.2 x volumes of SPRI beads (Beckman Coulter). Subsequently, the cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20mL reaction, incubating for 2 h at 16°C. The product was purified with 1.4 x volumes of SPRI beads, eluted in 8 mL and in vitro transcribed (with the beads) at 37°C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37°C and the amplified RNA (aRNA) purified with 1.2 volumes of SPRI beads. The aRNA was fragmented by incubating 3 min at 70°C in Zn2+ RNA fragmentation reagents (Ambion) and purified with 2 x volumes of SPRI beads. The aRNA was ligated to the MARS-seq ligation adaptor with T4 RNA Ligase I (NEB). The reaction was incubated at 22°C for 2 h. After 1.5 x SPRI cleanup, the ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent) and a primer complementary to the ligated adaptor. The reaction was incubated for 2 min at 42°C, 45 min at 50°C, and 5 min at 85°C. The cDNA was purified with 1.5 x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5mM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kappa Biosystems). The amplified pooled library was purified with 0.7 x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument. RNaseq libraries were sequenced using the Illumina NextSeq 2000. Data analysis was performed by using the UTAP transcriptome analysis pipeline (Kohen et al., 2019). Raw reads were trimmed using cutadapt with the parameters: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a “A–times 2 -u 3 -u -3 -q 20 -m 25). Reads were mapped to the genome (mm10, Gencode annotation version 10.0) using STAR (v2.4.2a) with the parameters –alignEndsType EndToEnd, –outFilterMismatchNoverLmax 0.05, –twopassMode Basic, –alignSoftClipAtReferenceEnds No. The pipeline quantifies the 3' of Gencode annotated genes (The 3' region contains 1,000 bases upstream of the 3' end and 100 bases downstream). UMI counting was done after marking duplicates (in-house script) using HTSeq-count in union mode. Only reads with unique mapping were considered for further analysis, and genes having minimum 5 reads in at least one sample were considered. Gene expression levels were calculated and normalized using DESeq2 (1.26.0)