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.

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. 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. 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 Mϕs subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo 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.

Background: Methods to study gastric emptying in rodents are time consuming or terminal, preventing repetitive assessment in the same animal. Magnetic resonance imaging (MRI) is a non-invasive technique increasingly used to investigate gastrointestinal function devoid of these shortcomings. Here, we evaluated MRI to measure gastric emptying in control animals and in two different models of gastroparesis.Methods: Mice were scanned using a 9.4 Tesla MR scanner. Gastric volume was measured by delineating the stomach lumen area. Control mice were scanned every 30 min after ingestion of a 0.2 g meal and stomach volume was quantified. The ability of MRI to detect delayed gastric emptying was evaluated in models of morphine-induced gastroparesis and streptozotocin-induced diabetes.Key Results: Magnetic resonance imaging reproducibly detected increased gastric volume following ingestion of a standard meal and progressively decreased with a half emptying time of 59 ± 5 min. Morphine significantly increased gastric volume measured at t = 120 min (saline: 20 ± 2 vs morphine: 34 ± 5 mm 3 ; n = 8-10; p < 0.001) and increased half emptying time using the breath test (saline: 85 ± 22 vs morphine: 161 ± 46 min; n = 10; p < 0.001). In diabetic mice, gastric volume assessed by MRI at t = 60 min (control: 23 ± 2 mm 3 ; n = 14 vs diabetic: 26 ± 5 mm 3 ; n = 18; p = 0.014) but not at t = 120 min (control: 21 ± 3 mm 3 ; n = 13 vs diabetic: 18 ± 5 mm 3 ; n = 18; p = 0.115) was significantly increased compared to nondiabetic mice. Conclusions and Inferences:Our data indicate that MRI is a reliable and reproducible tool to assess gastric emptying in mice and represents a useful technique to study gastroparesis in disease models or for evaluation of pharmacological compounds.

Background Recurrent episodes of intestinal inflammation and tissue remodeling progressively result in fibro-stenosis and bowel obstruction in Crohn’s disease (CD). This irreversible end-stage complication is the main indication for surgical intervention but is often associated with postoperative recurrence of inflammation and repeated tissue resection. Methods To understand transmural inflammation-induced fibro-stenosis, full-thickness ileum from CD patients (n=10) undergoing ileocecal resection were profiled using single-cell RNA sequencing (scRNA seq) on 10x Genomics platform. Our tissue sampling strategy aimed at recapitulating different disease stages from the same patient including normal ileum, chronic active inflammation and stenotic ileum, allowing a better definition of cell heterogeneity, inter-cellular communication, and tissue organization in CD. Healthy ileum from CRC patients (n=5) undergoing right hemicolectomy were included as external control. Flow cytometry (FACS) was carried out to confirm scRNA seq findings. For functional validation, intestinal fibroblasts were co-cultured with NicheNet-predicted cytokines and FACS-sorted myeloid supernatants. Finally, the chronic dextran sulfate sodium (DSS)-induced colitis model was used to validate the transcription regulation in activated fibroblasts. Results Using scRNA seq, we identified a specific subset of activated fibroblasts during chronic inflammation and fibrosis. FACS showed increasing FAP+ fibroblasts in both inflamed and stenotic ileum, compared to control ileum and proximal ileum (p&lt;0.01). Computational methods predicting ligand-receptor signaling suggest that fibroblast activation is mostly mediated by myeloid cell-derived inflammatory signals resulting in collagen deposition and tissue fibrosis. Intestinal fibroblasts co-cultured with inflammatory monocyte supernatant and predicted ligands expressed higher protein levels of FAP, COL3A1 and showed signs of EMT transformation. Furthermore, stimulated intestinal fibroblasts secreted higher levels of ECM proteins, including COL1 (p&lt;0.001), and COL3A1 (p&lt;0.005). After inhibiting an EMT-related transcription factor identified in the activated fibroblasts, collagen expression and extracellular matrix accumulation were decreased in fibroblasts. Finally, inhibition of EMT transformation in chronic DSS colitis resulted in reduced ECM deposition, compared to vehicle mouse (p&lt;0.05). Conclusion Our findings suggest that inflammatory monocytes mediate local activation of fibroblasts promoting excessive collagen deposition. We furthermore show that targeting activated fibroblasts was able to reduce tissue remodeling and may therefore prevent fibrostenosis in CD.

Enteric glial cells (EGCs) have been recently recognized as key components of the colonic tumor microenvironment (TME) indicating their potential role in colorectal cancer (CRC) pathogenesis. Although EGCs modulate immune responses in other intestinal diseases, their interaction with the CRC immune cell compartment remains unclear. Through a combination of single-cell and bulk RNA sequencing, both in CRC murine models and patients, we found that EGCs acquire a reactive and immunomodulatory phenotype that drives tumor-associated macrophage (TAM) differentiation. Tumor-infiltrating monocytes direct CRC EGC phenotypic and functional switch via IL-1R signaling pathway. In turn, tumor EGCs promote monocyte differentiation towards pro-tumorigenic SPP1+ TAMs via secretion of IL-6. Finally, the distinct tumor EGC phenotype correlates with worse disease outcome in patients suffering from CRC. Our study reveals a previously unexplored and crucial neuroimmune interaction between EGCs and TAMs in the colorectal TME, providing important insights into CRC pathogenesis.