The Impact of PAD4-dependent Neutrophil Extracellular Trap Formation on the Early Development of Intestinal Fibrosis in Crohn's Disease.

BACKGROUND AND AIMS: During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a PAD4-dependent manner, aggravating tissue injury and remodelling. In this study, we investigated the potential pro-fibrotic properties and signalling of NETs in Crohn's disease (CD). METHODS: NETs and activated fibroblasts were labelled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analysed by bulk RNA-sequencing to uncover cell signalling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-kB pathway was evaluated by transfection of CCD-18Co fibroblasts with NF-kB-luciferase reporter plasmid, incorporating C29 to block TLR2 signalling. A chronic DSS mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl). RESULTS: Immunofluorescence showed spatial co-localisation of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of TLR-signalling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in NF-kB expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significantly reduction in collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis. CONCLUSIONS: NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-kB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.

IL-1R signaling drives enteric glia-macrophage interactions in colorectal cancer.

Enteric glia have been recently recognized as key components of the colonic tumor microenvironment indicating their potential role in colorectal cancer pathogenesis. Although enteric glia modulate immune responses in other intestinal diseases, their interaction with the colorectal cancer immune cell compartment remains unclear. Through a combination of single-cell and bulk RNA-sequencing, both in murine models and patients, here we find that enteric glia acquire an immunomodulatory phenotype by bi-directional communication with tumor-infiltrating monocytes. The latter direct a reactive enteric glial cell phenotypic and functional switch via glial IL-1R signaling. In turn, tumor glia promote monocyte differentiation towards pro-tumorigenic SPP1+ tumor-associated macrophages by IL-6 release. Enteric glia cell abundancy correlates with worse disease outcomes in preclinical models and colorectal cancer patients. Thereby, our study reveals a neuroimmune interaction between enteric glia and tumor-associated macrophages in the colorectal tumor microenvironment, providing insights into colorectal cancer pathogenesis.

Intercellular interaction between FAP+ fibroblasts and CD150+ inflammatory monocytes mediates fibrostenosis in Crohn's disease.

Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.

Single cell RNA sequencing reveals endothelial cell killing and resolution pathways in experimental malaria-associated acute respiratory distress syndrome.

Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.

Magnetic resonance imaging as a non-invasive tool to assess gastric emptying in mice.

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 mm3 ; 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 mm3 ; n = 14 vs diabetic: 26 ± 5 mm3 ; n = 18; p = 0.014) but not at t = 120 min (control: 21 ± 3 mm3 ; n = 13 vs diabetic: 18 ± 5 mm3 ; 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.

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.