, For the irradiation-injury studies, organoids (in vitro XRT) received
, Mice were euthanized 4 h after treatment. Imaging Detection. For confocal laser-scanning microscopy, the organoids or the tissue was fixed and embedded in agarose or in OCT compound 4583 (Sakura) as already described (37). Sections of 150 ?m (agarose) or 10 ?m (OCT) were obtained by using an HM650V vibratome (Thermo Fisher) or a CM3050S cryostat (Leica), respectively. For antigen retrieval, slides were steamed for 20 min in 10 mM citrate buffer (pH 6) and cooled for 30 min
, Cell Signaling Technology
, Asp175), chicken anti-GFP, issue.1, p.500
, :200; Abgent; AP1802a), rabbit anti-p65 (1:100; Abcam; 7970), mouse anti-Olfm4 (1:200, Abcam; ab13970), rabbit anti-LC3B
, Thermo Fisher), and corresponding secondary antibodies (Alexa Fluor 488, 568, or 647; Thermo Fisher) were used. DNA was stained by DAPI (1 ?g/mL; Thermo Fisher). The sections were mounted with ProLong Gold Antifade Reagent (Thermo Fisher). Images were acquired using an IX-81 (Olympus) or Opterra swept-field confocal microscope (Bruker) equipped with thermic chamber and CO 2 control, p.568
, Flow Cytometric Analysis and Sorting above; the dye was added after dissociation) or live imaging. For total ROS, CellROX Deep Red (2.5 ?M, 30 min at 37°C; Thermo Fisher) and ROSstar 550 (2.5 ?M, 30 min at 37°C; Li-Cor) were used. For mtROS, MitoSOX Red (5 ?M for 30 min at 37°C, Measurement of Intracellular ROS. Total intracellular ROS and mitochondrial ROS were measured by flow cytometry (cf
, Mitochondrial Superoxide indicator; Molecular Probes) was used. For live imaging, DNA was stained with Hoechst, vol.33342
, Thermo Fisher) and cells were washed with DMEM FluoroBrite (Gibco). Resveratrol (40 ?M
, Sigma) was used as an antioxidant agent and directly added to the culture media just before irradiation and during subsequent culture
, Live organoids were incubated for 30 min at 37°C in the dark with 50 nM MitoTracker Deep Red and/or 50 nM LysoTracker (both from Thermo Fisher). DNA staining and washes were performed as described above. Bafilomycin A1 (Sigma) was added to the culture media for organoid-formation efficiency tests at a concentration of 100 nM as determined by a dose-response study
, Identification of stem cells in small intestine and colon by marker gene Lgr5, Nature, vol.449, pp.1003-1007, 2007.
, Distinct levels of radioresistance in Lgr5 + colonic epithelial stem cells versus Lgr5 + small intestinal stem cells, Cancer Res, vol.77, pp.2124-2133, 2017.
, Lgr5 + stem cells are indispensable for radiation-induced intestinal regeneration, Cell Stem Cell, vol.14, pp.149-159, 2014.
, The colonic crypt protects stem cells from microbiota-derived metabolites, Cell, vol.165, pp.1708-1720, 2016.
, Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis, Cell, vol.118, pp.229-241, 2004.
, Lipopolysaccharide from crypt-specific core microbiota modulates the colonic epithelial proliferation-to-differentiation balance, MBio, vol.8, pp.1680-1697, 2017.
URL : https://hal.archives-ouvertes.fr/pasteur-01680337
, A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis, J. Immunol, vol.190, pp.3541-3551, 2013.
, The cytosolic bacterial peptidoglycan sensor Nod2 affords stem cell protection and links microbes to gut epithelial regeneration, Cell Host Microbe, vol.371, pp.792-798, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01950138
, NOD2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation, Nat. Med, vol.16, pp.90-97, 2010.
, Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry, Nat. Immunol, vol.11, pp.55-62, 2010.
, Autophagy in immunity and inflammation, Nature, vol.469, pp.323-335, 2011.
, The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation, Nature, vol.531, pp.523-527, 2016.
, Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1? production, Nature, vol.456, pp.264-268, 2008.
, Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche, Nature, vol.459, pp.262-265, 2009.
, pH-dependent internalization of muramyl peptides from early endosomes enables Nod1 and Nod2 signaling, J. Biol. Chem, vol.284, pp.23818-23829, 2009.
, Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury, Cancer Lett, vol.327, pp.48-60, 2012.
, Interplay between metabolic identities in the intestinal crypt supports stem cell function, Nature, vol.543, pp.424-427, 2017.
, A dual role for receptor-interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy, J. Biol. Chem, vol.287, pp.25565-25576, 2012.
, Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts, Nature, vol.469, pp.415-418, 2011.
, Niche-independent high-purity cultures of Lgr5 + intestinal stem cells and their progeny, Nat. Methods, vol.11, pp.106-112, 2014.
, Effect of resveratrol on mitochondrial function: Implications in parkin-associated familiar Parkinson's disease, Biochim. Biophys. Acta, vol.1842, pp.902-915, 2014.
, Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection, J. Biol. Chem, vol.278, pp.8869-8872, 2003.
, Intrinsic autophagy is required for the maintenance of intestinal stem cells and for irradiation-induced intestinal regeneration, Cell Rep, vol.20, pp.1050-1060, 2017.
, mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake, Nature, vol.486, pp.490-495, 2012.
, Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival, Proc. Natl. Acad. Sci. U.S.A, vol.112, pp.7148-7154, 2015.
, Two faces of the coin: Minireview for dissecting the role of reactive oxygen species in stem cell potency and lineage commitment, J. Adv. Res, vol.14, pp.73-79, 2018.
, The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy, Mol. Biol. Cell, vol.19, pp.2092-2100, 2008.
, The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nod1 and Nod2 in an autophagy-independent manner, Immunity, vol.39, pp.858-873, 2013.
, Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease, International IBD Genetics Consortium (IIBDGC), vol.491, pp.119-124, 2012.
, DNA double-strand breaks: Signaling, repair and the cancer connection, Nat. Genet, vol.27, pp.247-254, 2001.
, CARD15/NOD2 is required for Peyer's patches homeostasis in mice, PLoS One, vol.2, p.523, 2007.
, A global double-fluorescent Cre reporter mouse, Genesis, vol.45, pp.593-605, 2007.
, Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection, Gastroenterology, vol.145, pp.1347-1357, 2013.
, In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker, Mol. Biol. Cell, vol.15, pp.1101-1111, 2004.
, Depletion of murine intestinal microbiota: Effects on gut mucosa and epithelial gene expression, PLoS One, vol.6, p.17996, 2011.
, Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland, J. Pathol, vol.228, pp.300-309, 2012.
, Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment, Nat. Protoc, vol.6, pp.1221-1228, 2011.