Single cell RNA-sequencing
Résumé
Rabbits are reliable animal models used in immunological research, in particular to study gut mucosal immunity. The lamina propria, underlying the epithelium in the mucosa, is considered the primary effector site of the intestinal immune response and is composed of a heterogeneous immune cell population. A detailed characterization of the diversity of mononuclear hematopoietic cells (CD45+) in the rabbit caecal lamina propria is lacking, even though this gut segment has a key role in the interaction with the gut microbiota. The present study aimed to fill that void. We isolated caecal lamina propria mononuclear cells by physical, chemical and enzymatic dissociation followed by a purification in a Percoll gradient. Single live CD45+ cells were sorted by flow cytometry (16% of total) and droplet-based single cell RNA-sequencing was performed. Data were analyzed using the Cell Ranger software and the R package Seurat. After applying quality filters, 4 246 cells were kept and the expression of 29 587 genes was detected. 13 cell clusters were identified, with the majority affiliated to B, T, NK and dendritic cells and macrophages based on the expression of canonical markers (JCHAIN, RORA, KIT, NKG7, C1QB, CD14). Our method thus enabled us to clearly identify the main mononuclear immune cell types present in the lamina propria with little contamination with other cell types (e.g. fibroblast). This study provides the first single cell phenotyping data on rabbit caecal mononuclear hematopoietic cells and offers great perspectives for the study of microbiota gut mucosal immunity regulations.