Il 22

Murine small intestinal and colonic organoids were cultured as described previously^{7 (link)}. At day 5 (SI) or day 3 (colon) organoids were stimulated with either 500 U/ml of IFNβ (PBL Assay Science), 5 ng/ml of IL-22 (Biolegend) or left unstimulated. For RNA extraction and immunofluorescence, the organoids were stimulated for 24 or 48 h. For detection of STAT3 phosphorylation, organoids were stimulated for 2h. For human organoids, pinch biopsies were obtained from the colon of adult IBD patients undergoing surveillance colonoscopy using 2.8 mm standard biopsy forceps, after approval by the New York University School of Medicine Institutional Review Board. All tissue was isolated from non-inflamed regions of the colon. Intestinal fragments were incubated in Gentle Cell Dissociation Reagent (Stemcell Technologies) on ice for 30 minutes followed by vigorous pipetting to isolate crypts. Crypt were embedded in Corning Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning), and cultured with human IntestiCult Organoid Growth Medium (Stemcell Technologies). The culture medium was changed every 2–3 days. Human organoids were stimulated with 500 U/ml IFNβ (R&D systems) or left unstimulated for 48 h.

Blood was obtained from the retro-orbital sinus, and serum was collected after blood coagulation. Wound edge tissue was collected in RIPA buffer and homogenized in the FastPrep instrument (Fastprep 24, Lysing Matrix D, MP Bio) per manufacturer’s protocol. The Bradford assay was performed to measure protein concentration of each sample. Serum and tissue IL-17A, IL-22, and IL-23 ELISAs were performed per the manufacturer’s instructions (Biolegend). Recombinant IL-17A, IL-22, or IL-23 was used as a positive control (Biolegend).

CD4⁺ T cells were purified from the spleens using CD4⁺ T cell isolation kit from Miltenyi Biotech. For cytokine analysis, 1×10⁶ cells/ml were stimulated with plate-bound anti-CD3ε (2 µg/ml) and soluble anti-CD28 (1 µg/ml) for 3 days and then rested in IL-2 (20 µg/ml) for 4 days. At day 7, cells were restimulated with plate-bound anti-CD3ε (2 µg/ml) and soluble anti-CD28 (1 µg/ml) for 24–48 hrs as previously described (Chen, Yang et al. 2008 (link)). Supernatants were analyzed for IL-17A (Biolegend), IL-21 (eBioscience), and IL-22 (Biolegend) production by ELISA.