Abstract
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) affords stem cell protection and links microbes to intestinal epithelial regeneration. We investigated whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules. To assess crypt survival, a clonogenic microcolony assay was performed with 15 Gy of X-ray irradiation. The fractional crypt survival rate (46.0 ± 15.5% vs. 24.7 ± 9.2%, p < 0.01) and fractional EdU-positive crypt survival rate (29.8 ± 14.5% vs. 9.79 ± 4.37%, p = 0.015) were significantly decreased in the NOD2−/− mice compared with the wild-type (WT) mice at 3.5 days after irradiation. To evaluate intestinal epithelial regeneration capability, organoid reconstitution assays were performed. Small bowel crypts of the WT and NOD2−/− mice were isolated and seeded into Matrigel for 3D culture. In the organoid reconstitution assays, the number of organoids formed did not differ between the NOD2−/− and WT mice. Organoid formation ability was also assessed after exposure to 5 Gy irradiation. Organoid formation ability was significantly decreased in the NOD2−/− mice compared with the WT ones after exposure to 5 Gy irradiation (33.2 ± 5.9 vs. 19.7 ± 8.8/well, p < 0.01). NOD2 supports crypt survival after potentially lethal irradiation damage and is associated with intestinal epithelial regeneration.
Highlights
Crohn’s disease (CD) is a chronic, disabling disease of the gastrointestinal tract and its pathogenesis is still not completely understood
We investigated whether nucleotide-binding oligomerization domain 2 (NOD2) status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules
Because of the high sensitivity of intestinal stem cells (ISCs) in the small intestine to ionizing radiation and self-renewal capacity, the mouse small intestinal crypts were used as a standard model system for the assessment of in vivo crypt microcolony assay
Summary
Crohn’s disease (CD) is a chronic, disabling disease of the gastrointestinal tract and its pathogenesis is still not completely understood. NOD2 mutations in human cells lead to decreased NF-kB activation and reduced expression of pro-inflammatory cytokines, such as interleukin (IL)-8, IL-1β, and tumor necrosis factor-alpha (TNF-α) in response to MDP [4]. This may seem counterintuitive, as CD is generally associated with an increased expression of pro-inflammatory cytokines. Whether NOD2 is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules is unclear. We investigated whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules
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