BioProject

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction during homeostasis. More...

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction during homeostasis. To experimentally address this concept, we generated intestinal epithelial cell (IEC)-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7?IEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape and proliferation rate remained unchanged in these mice. However, non-apoptotic cell extrusion was increased in Casp3/7?IEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single cell RNA sequences analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome composition and immune regulation. Overall design: Single-cell RNA sequencing was performed on mesenteric lymph nodes (mLN), small intestinal intra-epithelial lymphocytes (SI-IEL), SI lamina propria lymphocytes (SI-LPL), colonic IELs and colonic LPLs isolated from Casp3/7ΔIEC mice and Casp3/7FL/FL littermates. Mesenteric lymph nodes were collected, smashed and single cells were collected by centrifugation. SI-IELs and colon IELs were were isolated from the epithelial layer by treatment with the reducing agent Dithioerythritol (DTE). Single cells were prepared from the SI and colonic lamina propria by enzymatic digestion using collagenases followed by Percoll gradient centrifugation. Finally, fluorescence activated cell sorting (FACS) of live immune cells (CD45+) and live epithelial cells (CD45-) was performed using a BD FACSAria™ cell sorter. After preparation of single cells from the SI-IEL of both Casp3/7ΔIEC mice and Casp3/7FL/FL littermates, 50% live immune cells (expressing CD45) and 50% cells which lack expression of CD45 were sorted for RNA sequencing (samples FGH001 and FGH002). From the lamina propria fraction of both Casp3/7ΔIEC mice and Casp3/7FL/FL littermates, only CD45-expressing cells were sorted for RNA sequencing (samples FGH003 and FGH004). The same principle was applied to the colon IELs and LPLs from both Casp3/7ΔIEC mice and Casp3/7FL/FL littermates. However, for each genotype, the IELs and LPLs were combined into one sample resulting in each sample consisting of 25% CD45+ IELs, 25% CD45- IELs and 50% CD45+ LPLs (samples FGH005 and FGH006). From the mesenteric lymph nodes (mLN) of both Casp3/7ΔIEC mice and Casp3/7FL/FL littermates, only CD45+ immune cells were sorted (samples FGH007 and FGH008) .TotalSeq-A Cell Hashing Antibodies were used to include three replicates (mice) per sample. Less...